PAPER
www.rsc.org/methods | Analytical Methods
Methodology for Hg determination in honey using cloud point extraction and cold vapour-inductively coupled plasma optical emission spectrometry† Fernanda dos Santos Depoi, Fabrina R. S. Bentlin and Dirce Pozebon* Received 27th September 2009, Accepted 27th November 2009 First published as an Advance Article on the web 16th December 2009 DOI: 10.1039/b9ay00189a This paper describes the development of a method for the determination of mercury in honey. Analyte pre-concentration/matrix separation is carried out by cloud point extraction (CPE), while cold vapouroptical emission spectrometry (CV-ICP OES) is used to detect the analyte. A careful analytical work was carried out in order to evaluate precision and accuracy of the method. Mercury was complexed with ammonium diethyldithiophosphate (DDTP) and Triton X-114 was used as surfactant. Parameters such as type and concentration of acid (HCl or HNO3 + KCl), concentrations of complexant, surfactant and reductant (NaBH4), dilution of the surfactant-rich phase and the mass of honey were evaluated. Two procedures of sample preparation were investigated: sonication in presence of L-cysteine and acid decomposition in closed vessel. Sonication was very effective for extraction of inorganic mercury (Hg2+), but it was observed the sample should be decomposed to ensure the quantification of total mercury. Certified dog fish liver, analyte recovery and comparison with an independent technique (cold vapour-inductively coupled plasma mass spectrometry) were used to evaluate the method. Analyte recovery close to 100% was observed when up to 2.0 g of honey were subjected to sonication or up to 1.0 g of honey was decomposed with acid. The enrichment factor (EF) obtained was 13 and the limit of detection (LOD) was 2.2 ng g1. The Hg concentration in the analyzed samples was lower than the LOD.
1. Introduction Besides being a source of nutrients and minerals when used as food, honey can also be seen as an indicator of environmental pollution due to the possible incorporation of toxic elements.1 Most of the elements present in honey come from the soil. They are absorbed by plants through the root, going to the nectar and then to the honey produced by bees. Knowing the concentration of toxic elements in honey is of great interest for quality control or even in relation to the nutritional aspect.2 The concentration of toxic elements such as As, Cd, Sb, Co, Ni, Hg and Pb can vary in honey,which depends on the type of honey (floral) and geological features of the collection site.3 Mines and steelworks, industrial and urban areas or highways in or near the bees forage area can result in an increase of the concentrations of certain elements in honey due to pollution by chemical wastes and exhaust fumes. The honey is a mixture of sugars (27–44% fructose, 22–41% glucose, 2.7–16% maltose and 1.5–3.0% sucrose), proteins, amino acids (up to 0.09%), vitamins and other elements (Na, 0.03 to 0.05%; K, 0.04 to 0.12%; Ca, 0.15 to 0.03%; Mg, 0.05 to 0.08%; Fe, 0.0005 to 0.004% and trace elements, up to 0.01%).4,5 The honey matrix is relatively complex and may interfere with the Universidade Federal do Rio Grande do Sul, Instituto de Qu´ımica, 91501970 Porto Alegre, RS, Brazil. E-mail:
[email protected]; Fax: + 55 33087304; Tel: + 55 51 33087215 † Electronic supplementary information (ESI) available: Fig. S1: Scheme of the sample preparation and CPE procedures. Fig. S2: Influence of the acid medium on the pre-concentration of Hg. Fig. S3: Effect of the surfactant on the pre-concentration of Hg. Fig. S4: Effect of the volume of methanol added to the surfactant-rich phase. See DOI: 10.1039/b9ay00189a
180 | Anal. Methods, 2010, 2, 180–185
determination of trace elements. Techniques such as inductively coupled plasma mass spectrometry (ICP-MS),3 inductively coupled plasma optical emission spectrometry (ICP OES),2,6 atomic absorption spectrometry with electrothermal atomization (ETAAS),7 instrumental neutron activation analysis (INAA)8 and differential pulse anodic stripping voltammetry (DPASV)5 have been used for the determination of trace elements in honey. Among the toxic elements, mercury deserves special attention, especially for its ability to accumulate in various organisms9 and being transformed into organic mercury, usually in the form of methyl mercury (CH3Hg+). The concentration of Hg is very low in honey and usually not detected, excepting the product coming from very specific and contaminated areas.10,11 Few studies were found concerning the determination of Hg in honey. Due to the low concentration of this element and the complexity of the honey matrix, the separation of the matrix and analyte pre-concentration are recommended. The cloud point extraction (CPE) may be suitable for this purpose. In short, the CPE is based on the property that aqueous micellar solutions of nonionic surfactants become turbid when conditions such as temperature, salt and surfactant are modified.12 As a result, the aqueous micellar solution is separated into two isotropic phases: a surfactant-rich (with small volume) and a surfactant-poor (aqueous) with greater volume. One element, when complexed, can be extracted and included in the surfactant-rich phase, thus leading to a pre-concentration and separation of the element. The selection of suitable complexant, pH, ionic strength, surfactant type and concentration, temperature, equilibrium reaction and centrifugation time are parameters that have to be examined to make CPE successful. This journal is ª The Royal Society of Chemistry 2010
Different compounds have been used as complexant of Hg involving CPE: (5-bromo-2-pyridylazo)-5-diethylaminophenol [(5-Br-PADAP)]13 for Hg determination in biological samples by ETAAS; 5-Br-PADAP for Hg determination in water by ICPOES;14 I-methylene blue and pyrrolidine dithiocarbamate (APDC) for Hg determination in seafood by ICP OES;15 and ammonium diethyl dithiophosphate (DDTP) for Hg determination in biological materials by cold vapour generation coupled to atomic absorption spectrometry (CV-AAS).16 DDTP is a very suitable complexing agent because it is stable in acidic medium.17–20 Furthermore, this reagent is sufficiently hydrophobic to be used in CPE. With respect to the surfactant, which is necessary for the formation of micelles, octylphenoxypolyethoxyethanol (Triton X-114, a nonionic surfactant) has been more widely used,15,16,21 mainly because of the low cloud point temperature (between 22 and 25 C) and low cost. It is noteworthy that the CPE has been applied to different types of matrix, but not for honey. The main objective of this research is the development of a method for the determination of Hg in honey. To do this, Hg is pre-concentrated by CPE and then detected by CV-ICP OES (cold vapour generation-inductively coupled plasma optical emission spectrometry). DDPT is used as complexant, while Triton X-114 is used as surfactant. The main objectives include: (i) to obtain the largest enrichment factor as possible, (ii) a viable procedure of sample preparation, and (iii) low reagent consumption and low waste production.
2. Material and methods 2.1.
Instrumentation
An Optima 2000 DV-ICP OES spectrometer (from PerkinElmer) was used. Argon from White Martins/Praxair (Brazil) was used as plasma gas and auxiliary gas, whereas nitrogen with a purity of 99.996% (from White Martins/Praxair, Brazil) was used as purging gas. The main instrumental parameters are resumed in Table 1. A home made system (Fig. 1) was used for mercury vapour generation. The system consists basically of a confluence and a gas liquid separator. For Hg determination by CV-ICP-MS (cold vapour-inductively coupled plasma mass spectrometry), an ELAN DRC II instrument (from PerkinElmer/SCIEX) was employed. For cold vapour generation, the system depicted in Fig. 1 was coupled to Table 1 Instrumental and ICP OES parameters Plasma power/W Plasma gas flow rate/mL min1 Auxiliary gas flow rate/mL min1 Plasma view Injector tube Resolution Spray Chamber Nebulizer gas/L min1 Nebulizer Integration time/s Spectral line/nm Replicates Background correction Signal processing
1500 15 0.2 axial alumina, 2 cm i.d. High Cyclonic 0.75 GemCone (the sample uptake rate was 1 mL min1) automatic, 2.5–5.0 253.652 2 2 points per peak peak area (7 points per peak)
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Fig. 1 Diagram of the system used for cold vapor generation. Ar: carrier gas (argon at a flow rate of 0.6 mL min1), G/L: gas/liquid separator; W: waste; HCl and NaBH4 solutions concentration: 0.1 mol L1 and 0.1% (m/v), respectively. PTFE (0.8 mm i.d.), orange-white Tygon (0.95 mm i.d., for NaBH4 and sample solutions) and red-red Tygon (4.5 mm i.d. for the HCl solution) tubing were used for the propulsion of the solutions. The peristaltic pump of the ICP OES spectrometer was used.
the instrument and used. In this case, the carrier gas was 1.2 L min1. An ultrasonic processor (Unique, Brazil) equipped with a 4 mm diameter-titanium tip, and a heating block (TE-015/1 from Tecnal, Brazil) were used for the homogenization and/or extraction of Hg in honey. A water bath with temperature control (DeLeo, Brazil) was used as a source of heating and assists the CPE, whereas a centrifuge (Fanen, Baby 206, Brazil) was used for the separation of phases. 2.2.
Standards and reagents
All chemicals were of analytical-grade. Water (resistivity of 18.2 MU cm) purified in a Milli-Q system (from Millipore Corp.) was used. Nitric acid (65% m/m), hydrochloric acid (37% m/m), hydrogen peroxide (30% m/m) and methanol, all from Merck were used. Sodium tetrahydroborate (NaBH4 from Vetec, Brazil) was used as reductant of Hg. A NaBH4 solution (1% m/v) was prepared fresh daily by dissolving the solid in 0.1% m/v NaOH (Merck) solution. Solutions with lower NaBH4 concentration were prepared by appropriate dilution of the 1% m/v NaBH4 solution with water. Anhydrous L-cysteine (C3H7NO2S$HCl, from Vetec, Brazil) was used to assist the Hg pre-concentration and/or extraction. A stock solution containing 2.8% (m/v) L-cysteine was prepared in water. Potassium chloride (Across Organics) was used for Hg pre-concentration in presence of HNO3. A stock solution with 0.09% (m/v) of KCl was prepared in water. DDTP [(C2H5O)2P(S)SNH4] from Aldrich and Triton X-114 from Sigma were used for the pre-concentration of Hg. A stock solution with 1.0% (m/v) of DDTP was prepared by the dissolution of the reagent in water. The stock solution of Triton X-114 (10% m/v) was prepared by weighing 5.00 g of the reagent in a polypropylene vial and adding 50 mL of water. This procedure was used because of the difficulty in measuring an exact volume of the viscous reagent. Antifoam Y-30 from SigmaAldrich was used to minimize foam production in the vapour generation system. For direct Hg determination (pneumatic nebulization) using ICP-OES, solutions where prepared in presence of 500 mg L1 of Au (Titrisol, Merck) in order to avoid memory effect. Solutions of Hg2+ were prepared by appropriate dilution of a 1000 mg L1 stock solution of the species (Titrisol, Merck). Methyl mercury chloride (CH3HgCl) from Aldrich, containing 1000 mg L1 of Hg in methanol was used. Intermediate solutions containing 1.0 mg L1 of Hg in the form of Hg2+ or 1.3 mg L1 of Anal. Methods, 2010, 2, 180–185 | 181
Hg in the form of CH3HgCl, both in 1.0% (m/v) HNO3 were prepared just before the calibration procedure. The Hg concentration in the calibration solutions ranged from 0.3 to 10 mg L1 for determination using CPE and CV ICP OES, 1.0 to 10 mg L1 for CV-ICP OES (without Hg pre-concentration), and from 10 to 30 mg L1 for direct (pneumatic nebulization) determination using ICP OES. Samples of bee honey from different sites in the South of Brazil and of different floral origin (eucalyptus, orange, Pluchea sagittalis and field flowers) were analyzed in this work. The reference material DOLT-3 (Dogfish Liver) from the National Research Council of Canada was analyzed for evaluating the accuracy and precision of the method. 2.3.
Samples and sample preparation
The honey samples were slightly heated in a water bath at 40 C for 2 h. After cooling, aliquots containing 0.100–2.00 g of each sample were weighed directly into PTFE flasks, to which 0.5 mL of HNO3 + 0.5 mL of H2O2 were added and the mixture allowed to stand for 12 h. Subsequently, the flasks were closed with screw caps and heated to 100 C in a metal block for 3 h. After cooling to room temperature, the flasks were opened, the resulting solution transferred to graduated polypropylene vials and the volume brought to 25 mL by adding 0.5 mol L1 HCl. The procedure was performed in triplicate for each sample, with three replicates of the blank in parallel. In the procedure of sonication, 0.500–2.00 g of each sample were weighed into polypropylene flask, to which 5.0 mL of 1% (m/v) L-cysteine were added and the mixture sonicated by a probe introduced into the flask. The mixture was sonicated for 30 s and at 70 W of power. Then, the volume was completed to 25 mL by adding 0.5 mol L1 HCl. For mercury recovery tests, aliquots of the solution of Hg2+ or CH3HgCl were added to the sample of honey before decomposition or sonication. For direct Hg determination by CV-ICP OES (without Hg pre-concentration) the sample solutions were diluted in 0.5 mol L1 HCl. 2.4.
Cloud point extraction
Aliquots of 7 mL of sample solution were transferred to graduated polypropylene vials, to which DDTP, L-cysteine and Triton X-114 were added (the optimal concentrations of these reagents in the final mixture were 0.05% m/v, 0.2% m/v and 0.3% m/v, respectively). When HNO3 was used for CPE, 1.0 mL of 0.01% (m/v) KCl was also added to the mixture. Subsequently, the volume of the mixture in the vial was completed to 14 mL with 0.5 mol L1 HCl, the vial was sealed with a plastic film and then the mixture was heated in a water bath at 50 C for 25 min. To accelerate the separation of the phases, the mixture was centrifuged at 2500 rpm for 10 min and then cooled in an ice bath for 15 min. The final volume of the surfactant-rich phase was 250 mL. The surfactantpoor phase was separated by inversion of the vial, and the residual solution was removed with a Pasteur pipette. Subsequently, 100 mL of methanol + 1.0 mL of 0.5 mol L1 HCl were added to the surfactant-rich phase. The Hg present in this solution was then measured by the system shown in Fig. 1. The calibration solutions were subjected to the same treatment as the samples. The overall procedure is resumed in Fig. S1 of supplementary file.† 182 | Anal. Methods, 2010, 2, 180–185
3. Results and discussion 3.1.
Analyte pre-concentration
A solution containing 3 mg L1 of Hg2+ was used to develop the method. Initially, the influence of HCl or HNO3 + KCl on the pre-concentration of the analyte was evaluated. The acidic medium was tested in view of the stability of the complexing agent (DDTP) in this medium, and also because the solution obtained from the sample is usually acid.22 When HNO3 was used alone, no phase separation occurred and then KCl was added as electrolyte to induce the separation of the phases.21 It had already been observed that KCl favored the extraction of mercury in river sediment, possibly by the high affinity of Hg for Cl.23 In the present work, preliminary tests were made with respect to the influence of the KCl concentration on the CPE, whereas the highest Hg2+ signal was observed when the salt concentration was about 0.007% (m/v). The highest sensitivity was observed for 0.5 mol L1 HCl. It was also observed that the signal of the analyte was usually higher in presence of HCl than in presence of HNO3 (see Fig. S2 in supplementary file).† The cloud point temperature increases with the acid concentration increasing, while the DDTP deteriorates.24 This may be the reason for the signal decreasing with the acid concentration increasing. With respect to the ligand (DDTP) concentration, the highest Hg signal was observed when the DDTP concentration was 0.05% (m/v) for both media (Fig. 2). This concentration is ten times lower than that reported in the literature,16 for preconcentration of Hg based on CPE and determination by atomic absorption spectrometry with cold vapor generation (CV AAS). It is observed in Fig. 2 the analyte signal decreases with the DDTP concentration increasing. This may be due to the formation of charged species that can interact with the surfactant, and/or DDTP free molecules can interact with the surfactant and thus compete with the formation of DDTP-Hg molecules.25 With regard to the surfactant concentration, the highest analyte signal was observed for 0.3% (m/v) Triton X-114 in presence of HNO3 and 0.1% (m/v) Triton X-114 in presence HCl (see Fig. S3 in supplementary file).† However, if the surfactant concentration was 0.1% (m/v), the volume of the
Fig. 2 Effect of the ligand (DDTP) concentration on Hg (3.0 mg L1 of Hg2+) pre-concentration. Pre-concentration medium: 0.5 mol L1 HCl or 0.5 mol L1 HNO3 + KCl 0.007% (m/v), and Triton X-114 0.3% (m/v).
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resulting surfactant-rich phase was very small and more difficult to see, making phases separation difficult. Thus, as a compromise condition, the concentration of Triton X-114 was fixed in 0.3% (m/v). It was observed that the signal of Hg decreases with the surfactant concentration increasing, and this was due to the increased volume of surfactant-rich phase, which increased dilution. It is important to mention that, regardless of the surfactant-rich phase volume, 100 mL methanol + 1000 mL of acid solution were added to it. The decrease of analyte signal with increasing concentration of surfactant had also been observed in other studies.
3.2.
Vapour generation
Since the surfactant-rich phase is very viscous and its volume low (250 mL), this was diluted in order to facilitate its transportation through the CV system (Fig. 1), and also reduce foaming generation in the gas/liquid separator. It was observed the Hg emission intensity decreased with the methanol concentration increasing (see Fig. S4 in supplementary file),† because some interactions between the solvent and the reducing agent (NaBH4) can occur. Thus, 100 mL of methanol were added to the surfactant-rich phase in further measurements. The reaction between Hg and NaBH4 is accelerated when in acidic medium. Therefore, the concentration of HCl aspirated and transported through the CV system (Fig. 1) to assist the Hg2+ reduction was also evaluated. It can be seen in Fig. 3 the signal of Hg decreases with the HCl concentration increasing, because of the excess of H2 produced, destabilizing the system and undermining the separation of mercury (Hg0). This effect is more pronounced when the extraction of Hg is performed in medium containing HCl. According to Fig. 4, the signal of Hg increases with the NaBH4 concentration increasing (up to 0.1% m/v), and then decreases. It was observed the reaction medium became turbulent in the presence of higher concentrations of NaBH4, which can influence the kinetics of the Hg2+ reaction reduction and separation of the mercury vapour generated. Thus, the concentration of NaBH4 was set in 0.1% (m/v) for further measurements.
Fig. 3 Effect of the HCl concentration transported trough the CV system. A solution containing 3.0 mg L1 of Hg2+ was used. Pre-concentration medium: 0.5 mol L1 HCl or 0.5 mol L1 HNO3 + KCl 0.007% (m/ v), DDTP 0.05% (m/v), and Triton X-114 0.3% (m/v).
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Fig. 4 Effect of the reductant concentration (NaBH4) on the Hg signal (3.0 mg L1 of Hg2+ was used). Pre-concentration medium: 0.5 mol L1 HCl or 0.5 mol L1 HNO3 + KCl 0.007% (m/v), DDTP 0.05% (m/v), and Triton X-114 0.3% (m/v).
3.3.
Determination of Hg in honey
Two procedures of sample preparation were evaluated: sonication in presence of L-cysteine and decomposition with acid. Although there was good recovery of the Hg2+ added to honey subjected to sonication, the decomposition of the sample with acid was evaluated considering the possible presence of organic species of Hg, especially CH3Hg+. It has been observed by other authors26 that both CH3Hg+ and Hg2+ were extracted to the solution of a biological sample when the mixture was heated and sonicated in presence of L-cysteine. In the present study, a sample of certified dog fish liver was sonicated in presence of L-cysteine and then submitted to CPE, according to the method developed. It is important to mention that the recovery of Hg2+ was very poor when the sample was sonicated in absence of L-cysteine. In presence of L-cysteine, it was observed that the Hg2+ concentration found was in accordance with that indicated on the certificate (obtained by difference between the certified values for total Hg and CH3Hg+), as shown in Table 2. A sample of honey was enriched with CH3Hg+ and then subjected to the same treatment given to the certified sample, and the recovery of CH3Hg+ was low. In summary, the sonication in the presence of L-cysteine was very efficient to extract mercury, but if CH3Hg+ were present this could not be quantitatively determined by means of CPE. Considering the results obtained, the honey samples were then acid digested to ensure the detection and quantification of all mercury present. However, it was found that the recovery of the Hg2+added to the honey sample and subjected to acid digestion and CPE was not detected for the range of sample mass investigated. No turbidity of the solution was observed and no phase separation occurred. Other elements present in honey are free when it is decomposed and can interfere. The L-cysteine was then used as a masking agent,27 and recoveries of about 100% were achieved. It was observed that the concentration of L-cysteine in the sample solution should be in the range of 0.2 and 0.8% (m/v). Additional tests were also conducted to determine whether the L-cysteine was acting as a complexant of Hg, but the cloud point formation did not occur in the absence of DDTP, under the conditions of the developed method. Anal. Methods, 2010, 2, 180–185 | 183
Table 2 Concentrations of Hg species found in sonicated (in presence of L-cysteine) or acid digested samples. CPE is used for analyte pre-concentration prior to the detection by CV-ICP OES. The results are the mean and confidence interval of the mean (n ¼ 3 and a ¼ 0.05) Certified or spiked/mg g1 Procedure Sonication
Sample DOLT - 3 Honey
Hg2+ 1.78 0.250
Acid digestion
DOLT-3 Honey
Hg Total 3.37 0.14 0.250
Found/mg g1 CH3Hg+ 1.59 0.12 0.125
honey is digested and the analyte pre-concentrated. However, the recovery is less than 85% (see A in Fig. 5) if the analyte is measured directly in the sample solution by CV-ICP OES. When the honey sample is sonicated in the presence of L-cysteine (see C and D in Fig. 5), recovery is almost 100% for the full range of sample mass, regardless of the separation of the sample matrix. It is believed that sonication does not destroy the sugars, which masks the interferents. According to the results achieved so far, the decomposition of the sample with subsequent addition of L-cysteine is the most appropriate, given the good recovery of the analyte and the possibility to determine the total Hg. On the other hand, if sonication is used in the presence of L-cysteine, a larger amount of honey sample can be used, improving the LOD. There are no published data in relation to organic mercury in honey, but it must be very low and not detected by the techniques currently available for routine work. Thus, the sonication procedure can be recommended for Hg extraction from honey. 3.4.
Table 3 Conditions for mercury determination in honey using cloud point extraction (CPE) and CV-ICP OES
Sample mass/g DDTP concentration/% (m/v) L-cystein/% (m/v) Triton X-114/% (m/v) Reduction medium HCl/mol L1 Volume of methanol added to the surfactant-rich, phase/mL
2.0 (sonication) 1.0 (acid digestion) 0.05 0.2 0.3 0.1 100
Method parameters
The conditions set for the determination of Hg in honey using CPE and CV-ICP OES are summarized in Table 3, whereas the figures of merit of the proposed method are presented in Table 4. For comparison purposes, additional information is presented in Table 4. The LOD was defined as the concentration equivalent to three times the standard deviation (3s) of ten replicates of the blank, which underwent the same procedure of the samples and calibration solutions. The enrichment factor (EF) was obtained by the ratio of the slopes of calibration curves obtained by CPE CV-ICP OES and CV-ICP OES. It can be seen the EF is higher when the pre-concentration of Hg is made in the presence of HCl.
Fig. 5 Effect of amount of honey mass on Hg recovery. A: Decomposition with acid and direct determination; B: decomposition with acid and CPE; C: sonication and direct determination; D: sonication and CPE. The honey sample was spiked with Hg2+ in order to obtain 10 mg L1 of the element in the sample solution.
Selected conditions
CH3Hg+ undetected 0.055 0.012
Hg total 3.07 0.50 0.249 0.007
The effect of the amount of honey was investigated for both procedures of sample preparation. The maximum mass of sample tested was 2.0 g because of the difficulty of decomposing larger honey mass in closed vessel. According to Fig. 5 (B), the recovery of Hg is about 100% for sample mass up to 1.00 g, when the
Parameter investigated
Hg2+ 1.74 0.02 0.251 0.012
3.5 Samples analysis Table 5 shows the results for the different honey samples that were analyzed. It is observed that, despite the low LOD of the developed method, mercury was not detected in the samples. However, it was detected by CV-ICP-MS, a very sensitive
Table 4 Figures of merit of the method developed for mercury determination in honey using cloud point extraction (CPE) and CV-ICP OES
Medium
HCl HNO3
CV-ICP OES CPE CV-ICP OES CV-ICP OES CPE CV-ICP OES ICP OES
184 | Anal. Methods, 2010, 2, 180–185
Slope of the calibration curve
Enrichment factor (EF)
Limit of detection (LOD)/mg L1
LOD/ng g1
386 5156 283 2524 —
— 13 — 9 —
0.45 0.044 0.40 0.11 6.0
22.5 2.2 20.5 5.5 300
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Table 5 Determination of Hg in honey samples; Hg concentrations found are the mean and confidence interval of the mean (n ¼ 3 and a ¼ 0.05) Botanical origin
a
Eucalyptus Eucalyptus Orange Orange Pluchea sagittalis Pluchea sagittalis Field Flowers Field Flowers a
CV-ICP-MS/ng g1
a
