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Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Enantiomer-Selective and Quantitative Trace Analysis of Selected Persistent Organic Pollutants (POP) in Traditional Food from Western Greenland ab
c
ad
Pernilla Carlsson , Dorte Herzke & Roland Kallenborn a
University Centre in Svalbard, Longyearbyen, Norway
b
University of Tromsø, Tromsø, Norway
c
Norwegian Institute for Air Research, FRAM–High North Research Centre on Climate and the Environment, Tromsø, Norway d
Click for updates
Norwegian University of Life Sciences, Ås, Norway Published online: 22 Apr 2014.
To cite this article: Pernilla Carlsson, Dorte Herzke & Roland Kallenborn (2014) Enantiomer-Selective and Quantitative Trace Analysis of Selected Persistent Organic Pollutants (POP) in Traditional Food from Western Greenland, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:9-11, 616-627, DOI: 10.1080/15287394.2014.887425 To link to this article: http://dx.doi.org/10.1080/15287394.2014.887425
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 77:616–627, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.887425
ENANTIOMER-SELECTIVE AND QUANTITATIVE TRACE ANALYSIS OF SELECTED PERSISTENT ORGANIC POLLUTANTS (POP) IN TRADITIONAL FOOD FROM WESTERN GREENLAND Pernilla Carlsson1,2, Dorte Herzke3, Roland Kallenborn1,4 1
University Centre in Svalbard, Longyearbyen, Norway University of Tromsø, Tromsø, Norway 3 Norwegian Institute for Air Research, FRAM–High North Research Centre on Climate and the Environment, Tromsø, Norway 4 Norwegian University of Life Sciences, Ås, Norway
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2
Enantiomeric fractions (EF) are today considered a powerful tool to elucidate selective uptake processes of chiral contaminants in biota. In this study, concentration levels and EF were determined by gas chromatograph–mass spectrometer (GC/MS) for α-hexachlorocyclohexane (α-HCH) and trans-, cis-, and oxychlordane in selected Greenlandic traditional food items, collected at the local market in Nuuk in 2010. The food items selected were raw and smoked fish (salmon and halibut, n = 6), whale meat (n = 8), seal meat (n = 2) and narwhal mattak (skin and blubber, n = 6). The EF were nonracemic ( =0.5) for all samples except for α-HCH in narwhal, trans-chlordane in whale and smoked salmon, and cis- and oxychlordane in seal. The EF for α-HCH were significant for all fish samples, but not for mammalian samples. Data indicate that different uptake and/or transformation mechanisms may be responsible for nonracemic distributions of chiral pesticides in mammals and fish species analyzed. There were no general enantiomer-selective transformation/accumulation trends found for chlordanes. Data indicate that enantiomer-specific properties are an important prerequisite for interaction of chiral contaminant with internal metabolic processes. However, marked differences within these groups were identified. The EF in ringed seals were racemic for most of the analyzed pesticides (i.e., chlordanes). However, narwhal were characterized by nonracemic EF for all chiral pesticides analyzed. Median levels of α-HCH ranged from 2 to 24 ng/g lw and from 15.1 to 626.6 ng/g lw for trans-nonachlor, with lowest levels observed in smoked salmon and highest levels in narwhal mattak. This study confirmed that concentration levels of analyzed pesticides in the investigated food items were below the tolerable daily intake (TDI) threshold.
organisms representing high trophic levels are of particular concern with respect to human exposure risk (AMAP, 2003, 2009a, 2009b). For Arctic indigenous populations, it is stated in various reports that traditional food habits mainly consist of marine fish and mammals, rich in nutrients and essential fatty acids with considerable health benefits (AMAP, 2009a). Since most of the traditional food items are based on lipid-rich tissues from marine mammals of the North, traditional food is often a
Persistent organic pollutants (POP) have been the focus of research with regard to exposure risk and health aspects for humans as well as wildlife for the past five decades. A selection of 39 POP is today regulated globally within the Stockholm POP convention (Stockholm Convention, 2013). Persistent organic pollutants bioaccumulate in lipid-rich tissues and are found in considerable concentrations especially in marine vertebrates, including fish and marine mammals. Thus, food items originating from
Address correspondence to Roland Kallenborn, Norwegian University of Life Sciences, P.O. Box 5003, Christian M. Falsen veg 1, NO-1432 Ås, Norway. E-mail: [email protected] 616
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SELECTED PERSISTENT ORGANIC POLLUTANTS IN FOOD
main source of POP intake. This situation is also called the “Arctic dilemma” (AMAP, 2009b; Deutch et al., 2007). Long-term monitoring programs in the North confirmed that the levels of POP (except polychlorinated biphenyls, PCB) have not decreased in the local food since the 1970s (AMAP, 2009b). However, human exposure to POP has decreased as the intake of traditional food among Inuit in Greenland has significantly diminished during the past two decades. Today intake of traditional food ranges between 25 and 30% of daily energy intake (Deutch et al., 2006, 2007; AMAP, 2009b). The investigation of chiral contaminants, consisting of two (or more) non-superimposable stereoisomers (enantiomers), is today considered a versatile scientific tool for determination of bioavailability and biodegradation processes. The enantiomers of a chiral compound have the same physical–chemical properties but differ in their three-dimensional (stereochemical) structures. The pesticides investigated in the present study were produced and sold as racemates. Based on today’s scientific understanding, enantiomers undergo similar passive uptake/elimination processes. However, biological processes are usually based on interactions with enzymes, hormones, or selective active transport across membranes (carrier molecules), which create a homochiral environment in organisms. All biochemically active biogenic compounds are based on L-amino acids and thus form a homochiral environment where enantiomers display different properties with respect to, among others, activation of enzymatic systems, degradation, and active excretion (Hühnerfuss et al., 1993; Kallenborn and Hühnerfuss, 2001). The determination of enantiomeric fractions (EF) might thus be used as an indicator during thorough investigation of selective uptake, elimination, and/or transformation processes (Eljarrat et al., 2008; Bidleman and Falconer, 1999). Knowledge regarding differences in toxicity and uptake of enantiomers is today actively used to optimize manufacturing of chemicals while minimizing the environmental impact as much as possible (i.e., pesticides) (Kallenborn and Hühnerfuss, 2001;
617
Hallanger, 2010). Enantiomeric signatures have been used earlier as indicators for the origin of air masses and as an indicator for volatilization and exchange processes from water masses to air (Genualdi et al., 2009; Jantunen, 2009; Kallenborn et al, 1991; European Food Safety Authority [EFSA], 2011). Levels of contaminants in food in Greenland have been monitored for decades as a part of the Arctic Monitoring and Assessment Programme (AMAP). Surveillance studies have assessed human exposure to POP (Bjerregaard et al., 2001; Johansen et al., 2004; Deutch et al, 2004, 2006, 2007). The main focus of our study was to examine stereoselective uptake and transformation of chiral chlorinated pesticides in target food items. To assess the effect of smoking processes on EF, both fresh and smoked salmon were acquired. To our knowledge, few data are currently available regarding selective enantiomer distribution of these chiral compounds in processed human food items. This study provides novel information regarding today’s levels of pesticides in traditional Greenlandic food items.
MATERIALS AND METHODS Sample Collection and Preparation Fresh salmon (n = 6), smoked salmon (n = 6) and halibut (n = 6), whale beef/meat, that is, muscle tissue (n = 8), narwhal mattak (Greenlandic traditional food consisting of blubber and parts of the skin; n = 6), and seal beef/meat, that is, muscle tissue (n = 2) were purchased at the local fish market and grocery shops in Nuuk, Greenland, in June 2010. The samples were kept frozen (–18◦ C) and were shipped to Longyearbyen, Svalbard, Norway. For further quantitative and enantiomer selective analysis, frozen samples were transported to the Norwegian Institute for Air Research (NILU) at the Fram Centre in Tromsø, Norway. All samples were kept frozen until sample preparation and analysis. Two replicates were taken from each fish, whale, and seal meat sample. The method for the preparation of the food samples was
Downloaded by [University of California, San Diego] at 12:56 01 February 2015
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described previously (Herzke et al., 2005), and details are found in Carlsson et al. (2013) where the same samples were analyzed for a suite of other POP including PCB. All solvents were of gas chromatography grade from Merck (Darmstadt, Germany). Briefly, samples were dried with sodium sulfate, 8 ng of 13 C-labeled trans-chlordane (purchased from Cambridge Isotope Laboratory [CIL], Andover, MA) was added as internal standard, and samples were cold-extracted with cyclohexane/acetone (50:50 v/v). Lipids were removed with a gel permeation chromatography (GPC) system (Waters Envirogel GPC Cleanup column, column no. 1: 19 × 150 mm, column no. 2: 19 × 300 mm, 15-μm particles of divinylbenzene copolymer SX3) with dichloromethane (DCM) as solvent. The final cleanup step was carried out with Florisil. The amount of extractable organic material (EOM) was determined gravimetrically. Before quantification, octachloronaphthalene (OCN) was added to all samples as recovery standard. The results were not corrected for recovery. Chromatographic Separation and Quantification All samples were analyzed at NILU in Tromsø for a suite of conventional pesticides: α-, β-, and γ-hexachlorocyclohexane (HCH), trans-chlordane (TC), cis-chlordane (CC), trans-nonachlor (TNC), cis-nonachlor (CNC), oxychlordane (OC), and hexachlorobenzene (HCB). Quantification standards were bought from Ultra Scientific, Kingstown, RI. For analyses of these compounds, 1 μl of each sample was injected with a split/splitless injector at 250◦ C (Agilent Technologies) into a gas chromatograph (GC; Agilent7890) connected to a mass spectrometer (MS; Agilent 5973 single quadrupole) operated in single ion monitoring (SIM) mode. The GC was equipped with a 30m DB5-MS column (0.25 mm inner diameter and 0.1 μm film thickness; J&W, Folsom, USA). Helium (6.0 quality, Hydrogas, Porsgrunn, Norway) was used as carrier gas at a flow rate of 1 ml/min. The following temperature program was used: start at 70◦ C, hold for
3 min; 15◦ C/min increase to 180◦ C; 5◦ C/min increase to 280◦ C; and held for 5 min. The mass spectrometer was operated in negative chemical ionisation (NCI) mode, with methane (5.5 quality, Hydro gas Porsgrunn, Norway) as reagent gas. The transfer line temperature was held at 280◦ C and source temperature was set to 220◦ C. The monitored m/z is presented in supplementary material (Table S1). Further details regarding separation and quantification method were previously described (Bustnes et al., 2008; Herzke et al., 2009). Enantiomeric Selective Analyses Samples were analyzed using the same GCMS conditions as for pesticide analyses (see preceding section, Chromatographic Separation and Quantification). An enantiomer-selective capillary column was used for the separation (20% tert-butyldimethylsilyl-β-cyclodextrin as chiral separator, dissolved in 15% phenyl-, 85% methylpolysiloxane; BGB-172 from BGB Analytik AG, Böckten, Switzerland). The following temperature program was applied for splitless injection (1 μl injected): initial temperature 90◦ C (1 min isotherm), 20◦ C/min to 160◦ C, 1◦ C/min increase to 180◦ C (16 min isotherm), 20◦ C/min increase to 240◦ C (13 min isotherm); total length of the enantiomer-selective chromatographic separation was 57 min. All samples were analyzed for enantiomeric distribution of α-HCH and trans-, cis-, and oxychlordane, and EF was calculated using the areas of the respective (+)and (–)-enantiomers in the chromatogram: EF =
[(+)] [(+) + (−)]
(1)
Quality Control One quantification and one reference mass were determined for each target organochlorine (OC) pesticide. A lab blank and a standard reference material (SRM) were analyzed for every 10th sample. The NIST 1945 (whale blubber) was used as SRM. The limit of detection (LOD) was calculated as three times the
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SELECTED PERSISTENT ORGANIC POLLUTANTS IN FOOD
signal-to-noise ratio (3 × S/N) for each compound and the limit of quantification (LOQ) was calculated as 10 times the S/N, or 10 times the lab blank if the target analyte was detected in the lab blank. The LOD for pesticides and HCB ranged between 1 and 71 pg/g wet weight (ww), depending on compound and matrix. A complete overview of the LOD is presented in Table S2 in the supplementary material. The median recovery was 86% (range: 50–127%) for trans-chlordane. As a part of the quality control procedure for the enantiomer selective analyses, the method uncertainty was set at EF ± 2σ (σ = standard deviations in the standard). A racemic distribution was considered: α-HCH: EF = 0.5–0.52; trans- and cischlordane: EF = 0.48–0.52; oxychlordane: EF = 0.49–0.53. The elution order was confirmed by Genualdi et al. (2009) and enantiomer-enriched standards; the (+)enantiomer of each compound was purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). The elution order was: (–)-α-HCH, (+)-α-HCH, (+)-OC, (–)-OC, (+)-TC, (+)-CC, (–)-CC, (–)-TC. Chromatograms regarding the enantiomeric separation can be found in the supplementary material (Figures S1 and S2). Calculation of Daily Intake In this study, daily intake of the food items was calculated based on species composition in the spring diet, according to reported intake in Johansen et al. (2004). The calculated daily intake of POP was estimated using the median pesticide concentrations of each food item in the present study, and multiplied by the reported amount of daily intake of each food item (Johansen et al., 2004)
RESULTS AND DISCUSSION HCB, trans-nonachlor, and cis-nonachlor contributed 60–78% of pesticide burden analyzed in all samples (Figure 1). The relative levels of cis-chlordane decreased from fish samples to mammalian samples, while
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FIGURE 1. Relative distribution of the selected organochlorine pesticides (OCP) based on the median distribution. Abbrevations: HCB = hexachlorobenzene, CNC = cis-nonachlor, TNC = trans-nonachlor, CC = cis-chlordane, TC = transchlordane, OC = oxychlordane, HCH = hexachlorocyclohexane. SmokeS = smoked salmon, SmokeH = smoked halibut, WB = whale beef, NW = narwhal (mattak), SB = seal beef.
relative contribution of oxychlordane to POP burden rose from fish to mammals. Narwhal mattak showed highest levels of all compounds determined, except for trans-chlordane and cis-chlordane, where the highest levels were observed in fresh salmon. Levels and concentrations of the analyzed pesticides in the present study are provided in Table 1. Hexachlorocyclohexanes (HCH) and Hexachlorobenzene (HCB) The levels of HCB in salmon were in agreement with earlier studies from central Greenland (Table 2). HCB levels in halibut (wet weight basis, ww) for the present study were similar to those of Riget et al. (2004) and Vorkamp et al. (2004), who reported 4.2 ng/g ww, even though fresh filet was analysed, whereas smoked halibut samples were quantified in the present study (Table 2). The seal meat in the present study contained more HCB than meat from central western Greenland, but it was in agreement with seal meat from eastern Greenland (Table 2) (Riget et al., 2004). The age of the seals in the present study is not known, but seals from central western Greenland, from which the meat was prepared, were young (around 1 yr), which may explain their lower levels compared to the present study. Regarding HCH, the levels in seal and whale meat in the present study were lower compared to earlier
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TABLE 1. Median Values (ng/g lw) and Concentration Range of Pesticides in the Samples
Salmon SmokeS SmokeH WB NW SB
Oxychlordane
trans-Chlordane
cis-Chlordane
trans-Nonachlor
cis-Nonachlor
9.82 3.35 7.52 45.45 268.74 65.74
16.10 3.91 3.58 2.60 4.83 4.46
36.28 9.25 8.62 20.99 17.15 2.40
68.71 15.05 23.44 189.68 626.60 115.74
32.34 7.38 8.35 73.53 108.65 21.19
5.6–31.1 3.2–12.3 6.5–103.2 16.0–57.3 173.7–527.4 43.3–88.2
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α–HCH Salmon SmokeS SmokeH WB NW SB
3.56 2.01 8.37 9.06 24.00 17.32
8.6–51.1 3.7–7.5 2.5–7.2 1.5–5.3 3.3–8.4 3.4–5.5
β–HCH 2.7–10.4 1.5–2.9 4.9–14.9 6.1–11.1 17.4–66.7 12.4–22.2
1.56 1.91 1.48 24.68 59.96 15.06
18.9–116.9 8.6–17.1 6.0–17.1 7.6–26.0 10.1–30.3 2.0–2.8
γ–HCH 0.6–3.8 1.5–3.0
Journal of Toxicology and Environmental Health, Part A: Current Issues Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uteh20
Enantiomer-Selective and Quantitative Trace Analysis of Selected Persistent Organic Pollutants (POP) in Traditional Food from Western Greenland ab
c
ad
Pernilla Carlsson , Dorte Herzke & Roland Kallenborn a
University Centre in Svalbard, Longyearbyen, Norway
b
University of Tromsø, Tromsø, Norway
c
Norwegian Institute for Air Research, FRAM–High North Research Centre on Climate and the Environment, Tromsø, Norway d
Click for updates
Norwegian University of Life Sciences, Ås, Norway Published online: 22 Apr 2014.
To cite this article: Pernilla Carlsson, Dorte Herzke & Roland Kallenborn (2014) Enantiomer-Selective and Quantitative Trace Analysis of Selected Persistent Organic Pollutants (POP) in Traditional Food from Western Greenland, Journal of Toxicology and Environmental Health, Part A: Current Issues, 77:9-11, 616-627, DOI: 10.1080/15287394.2014.887425 To link to this article: http://dx.doi.org/10.1080/15287394.2014.887425
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Journal of Toxicology and Environmental Health, Part A, 77:616–627, 2014 Copyright © Taylor & Francis Group, LLC ISSN: 1528-7394 print / 1087-2620 online DOI: 10.1080/15287394.2014.887425
ENANTIOMER-SELECTIVE AND QUANTITATIVE TRACE ANALYSIS OF SELECTED PERSISTENT ORGANIC POLLUTANTS (POP) IN TRADITIONAL FOOD FROM WESTERN GREENLAND Pernilla Carlsson1,2, Dorte Herzke3, Roland Kallenborn1,4 1
University Centre in Svalbard, Longyearbyen, Norway University of Tromsø, Tromsø, Norway 3 Norwegian Institute for Air Research, FRAM–High North Research Centre on Climate and the Environment, Tromsø, Norway 4 Norwegian University of Life Sciences, Ås, Norway
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2
Enantiomeric fractions (EF) are today considered a powerful tool to elucidate selective uptake processes of chiral contaminants in biota. In this study, concentration levels and EF were determined by gas chromatograph–mass spectrometer (GC/MS) for α-hexachlorocyclohexane (α-HCH) and trans-, cis-, and oxychlordane in selected Greenlandic traditional food items, collected at the local market in Nuuk in 2010. The food items selected were raw and smoked fish (salmon and halibut, n = 6), whale meat (n = 8), seal meat (n = 2) and narwhal mattak (skin and blubber, n = 6). The EF were nonracemic ( =0.5) for all samples except for α-HCH in narwhal, trans-chlordane in whale and smoked salmon, and cis- and oxychlordane in seal. The EF for α-HCH were significant for all fish samples, but not for mammalian samples. Data indicate that different uptake and/or transformation mechanisms may be responsible for nonracemic distributions of chiral pesticides in mammals and fish species analyzed. There were no general enantiomer-selective transformation/accumulation trends found for chlordanes. Data indicate that enantiomer-specific properties are an important prerequisite for interaction of chiral contaminant with internal metabolic processes. However, marked differences within these groups were identified. The EF in ringed seals were racemic for most of the analyzed pesticides (i.e., chlordanes). However, narwhal were characterized by nonracemic EF for all chiral pesticides analyzed. Median levels of α-HCH ranged from 2 to 24 ng/g lw and from 15.1 to 626.6 ng/g lw for trans-nonachlor, with lowest levels observed in smoked salmon and highest levels in narwhal mattak. This study confirmed that concentration levels of analyzed pesticides in the investigated food items were below the tolerable daily intake (TDI) threshold.
organisms representing high trophic levels are of particular concern with respect to human exposure risk (AMAP, 2003, 2009a, 2009b). For Arctic indigenous populations, it is stated in various reports that traditional food habits mainly consist of marine fish and mammals, rich in nutrients and essential fatty acids with considerable health benefits (AMAP, 2009a). Since most of the traditional food items are based on lipid-rich tissues from marine mammals of the North, traditional food is often a
Persistent organic pollutants (POP) have been the focus of research with regard to exposure risk and health aspects for humans as well as wildlife for the past five decades. A selection of 39 POP is today regulated globally within the Stockholm POP convention (Stockholm Convention, 2013). Persistent organic pollutants bioaccumulate in lipid-rich tissues and are found in considerable concentrations especially in marine vertebrates, including fish and marine mammals. Thus, food items originating from
Address correspondence to Roland Kallenborn, Norwegian University of Life Sciences, P.O. Box 5003, Christian M. Falsen veg 1, NO-1432 Ås, Norway. E-mail: [email protected] 616
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SELECTED PERSISTENT ORGANIC POLLUTANTS IN FOOD
main source of POP intake. This situation is also called the “Arctic dilemma” (AMAP, 2009b; Deutch et al., 2007). Long-term monitoring programs in the North confirmed that the levels of POP (except polychlorinated biphenyls, PCB) have not decreased in the local food since the 1970s (AMAP, 2009b). However, human exposure to POP has decreased as the intake of traditional food among Inuit in Greenland has significantly diminished during the past two decades. Today intake of traditional food ranges between 25 and 30% of daily energy intake (Deutch et al., 2006, 2007; AMAP, 2009b). The investigation of chiral contaminants, consisting of two (or more) non-superimposable stereoisomers (enantiomers), is today considered a versatile scientific tool for determination of bioavailability and biodegradation processes. The enantiomers of a chiral compound have the same physical–chemical properties but differ in their three-dimensional (stereochemical) structures. The pesticides investigated in the present study were produced and sold as racemates. Based on today’s scientific understanding, enantiomers undergo similar passive uptake/elimination processes. However, biological processes are usually based on interactions with enzymes, hormones, or selective active transport across membranes (carrier molecules), which create a homochiral environment in organisms. All biochemically active biogenic compounds are based on L-amino acids and thus form a homochiral environment where enantiomers display different properties with respect to, among others, activation of enzymatic systems, degradation, and active excretion (Hühnerfuss et al., 1993; Kallenborn and Hühnerfuss, 2001). The determination of enantiomeric fractions (EF) might thus be used as an indicator during thorough investigation of selective uptake, elimination, and/or transformation processes (Eljarrat et al., 2008; Bidleman and Falconer, 1999). Knowledge regarding differences in toxicity and uptake of enantiomers is today actively used to optimize manufacturing of chemicals while minimizing the environmental impact as much as possible (i.e., pesticides) (Kallenborn and Hühnerfuss, 2001;
617
Hallanger, 2010). Enantiomeric signatures have been used earlier as indicators for the origin of air masses and as an indicator for volatilization and exchange processes from water masses to air (Genualdi et al., 2009; Jantunen, 2009; Kallenborn et al, 1991; European Food Safety Authority [EFSA], 2011). Levels of contaminants in food in Greenland have been monitored for decades as a part of the Arctic Monitoring and Assessment Programme (AMAP). Surveillance studies have assessed human exposure to POP (Bjerregaard et al., 2001; Johansen et al., 2004; Deutch et al, 2004, 2006, 2007). The main focus of our study was to examine stereoselective uptake and transformation of chiral chlorinated pesticides in target food items. To assess the effect of smoking processes on EF, both fresh and smoked salmon were acquired. To our knowledge, few data are currently available regarding selective enantiomer distribution of these chiral compounds in processed human food items. This study provides novel information regarding today’s levels of pesticides in traditional Greenlandic food items.
MATERIALS AND METHODS Sample Collection and Preparation Fresh salmon (n = 6), smoked salmon (n = 6) and halibut (n = 6), whale beef/meat, that is, muscle tissue (n = 8), narwhal mattak (Greenlandic traditional food consisting of blubber and parts of the skin; n = 6), and seal beef/meat, that is, muscle tissue (n = 2) were purchased at the local fish market and grocery shops in Nuuk, Greenland, in June 2010. The samples were kept frozen (–18◦ C) and were shipped to Longyearbyen, Svalbard, Norway. For further quantitative and enantiomer selective analysis, frozen samples were transported to the Norwegian Institute for Air Research (NILU) at the Fram Centre in Tromsø, Norway. All samples were kept frozen until sample preparation and analysis. Two replicates were taken from each fish, whale, and seal meat sample. The method for the preparation of the food samples was
Downloaded by [University of California, San Diego] at 12:56 01 February 2015
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described previously (Herzke et al., 2005), and details are found in Carlsson et al. (2013) where the same samples were analyzed for a suite of other POP including PCB. All solvents were of gas chromatography grade from Merck (Darmstadt, Germany). Briefly, samples were dried with sodium sulfate, 8 ng of 13 C-labeled trans-chlordane (purchased from Cambridge Isotope Laboratory [CIL], Andover, MA) was added as internal standard, and samples were cold-extracted with cyclohexane/acetone (50:50 v/v). Lipids were removed with a gel permeation chromatography (GPC) system (Waters Envirogel GPC Cleanup column, column no. 1: 19 × 150 mm, column no. 2: 19 × 300 mm, 15-μm particles of divinylbenzene copolymer SX3) with dichloromethane (DCM) as solvent. The final cleanup step was carried out with Florisil. The amount of extractable organic material (EOM) was determined gravimetrically. Before quantification, octachloronaphthalene (OCN) was added to all samples as recovery standard. The results were not corrected for recovery. Chromatographic Separation and Quantification All samples were analyzed at NILU in Tromsø for a suite of conventional pesticides: α-, β-, and γ-hexachlorocyclohexane (HCH), trans-chlordane (TC), cis-chlordane (CC), trans-nonachlor (TNC), cis-nonachlor (CNC), oxychlordane (OC), and hexachlorobenzene (HCB). Quantification standards were bought from Ultra Scientific, Kingstown, RI. For analyses of these compounds, 1 μl of each sample was injected with a split/splitless injector at 250◦ C (Agilent Technologies) into a gas chromatograph (GC; Agilent7890) connected to a mass spectrometer (MS; Agilent 5973 single quadrupole) operated in single ion monitoring (SIM) mode. The GC was equipped with a 30m DB5-MS column (0.25 mm inner diameter and 0.1 μm film thickness; J&W, Folsom, USA). Helium (6.0 quality, Hydrogas, Porsgrunn, Norway) was used as carrier gas at a flow rate of 1 ml/min. The following temperature program was used: start at 70◦ C, hold for
3 min; 15◦ C/min increase to 180◦ C; 5◦ C/min increase to 280◦ C; and held for 5 min. The mass spectrometer was operated in negative chemical ionisation (NCI) mode, with methane (5.5 quality, Hydro gas Porsgrunn, Norway) as reagent gas. The transfer line temperature was held at 280◦ C and source temperature was set to 220◦ C. The monitored m/z is presented in supplementary material (Table S1). Further details regarding separation and quantification method were previously described (Bustnes et al., 2008; Herzke et al., 2009). Enantiomeric Selective Analyses Samples were analyzed using the same GCMS conditions as for pesticide analyses (see preceding section, Chromatographic Separation and Quantification). An enantiomer-selective capillary column was used for the separation (20% tert-butyldimethylsilyl-β-cyclodextrin as chiral separator, dissolved in 15% phenyl-, 85% methylpolysiloxane; BGB-172 from BGB Analytik AG, Böckten, Switzerland). The following temperature program was applied for splitless injection (1 μl injected): initial temperature 90◦ C (1 min isotherm), 20◦ C/min to 160◦ C, 1◦ C/min increase to 180◦ C (16 min isotherm), 20◦ C/min increase to 240◦ C (13 min isotherm); total length of the enantiomer-selective chromatographic separation was 57 min. All samples were analyzed for enantiomeric distribution of α-HCH and trans-, cis-, and oxychlordane, and EF was calculated using the areas of the respective (+)and (–)-enantiomers in the chromatogram: EF =
[(+)] [(+) + (−)]
(1)
Quality Control One quantification and one reference mass were determined for each target organochlorine (OC) pesticide. A lab blank and a standard reference material (SRM) were analyzed for every 10th sample. The NIST 1945 (whale blubber) was used as SRM. The limit of detection (LOD) was calculated as three times the
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SELECTED PERSISTENT ORGANIC POLLUTANTS IN FOOD
signal-to-noise ratio (3 × S/N) for each compound and the limit of quantification (LOQ) was calculated as 10 times the S/N, or 10 times the lab blank if the target analyte was detected in the lab blank. The LOD for pesticides and HCB ranged between 1 and 71 pg/g wet weight (ww), depending on compound and matrix. A complete overview of the LOD is presented in Table S2 in the supplementary material. The median recovery was 86% (range: 50–127%) for trans-chlordane. As a part of the quality control procedure for the enantiomer selective analyses, the method uncertainty was set at EF ± 2σ (σ = standard deviations in the standard). A racemic distribution was considered: α-HCH: EF = 0.5–0.52; trans- and cischlordane: EF = 0.48–0.52; oxychlordane: EF = 0.49–0.53. The elution order was confirmed by Genualdi et al. (2009) and enantiomer-enriched standards; the (+)enantiomer of each compound was purchased from Dr Ehrenstorfer GmbH (Augsburg, Germany). The elution order was: (–)-α-HCH, (+)-α-HCH, (+)-OC, (–)-OC, (+)-TC, (+)-CC, (–)-CC, (–)-TC. Chromatograms regarding the enantiomeric separation can be found in the supplementary material (Figures S1 and S2). Calculation of Daily Intake In this study, daily intake of the food items was calculated based on species composition in the spring diet, according to reported intake in Johansen et al. (2004). The calculated daily intake of POP was estimated using the median pesticide concentrations of each food item in the present study, and multiplied by the reported amount of daily intake of each food item (Johansen et al., 2004)
RESULTS AND DISCUSSION HCB, trans-nonachlor, and cis-nonachlor contributed 60–78% of pesticide burden analyzed in all samples (Figure 1). The relative levels of cis-chlordane decreased from fish samples to mammalian samples, while
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FIGURE 1. Relative distribution of the selected organochlorine pesticides (OCP) based on the median distribution. Abbrevations: HCB = hexachlorobenzene, CNC = cis-nonachlor, TNC = trans-nonachlor, CC = cis-chlordane, TC = transchlordane, OC = oxychlordane, HCH = hexachlorocyclohexane. SmokeS = smoked salmon, SmokeH = smoked halibut, WB = whale beef, NW = narwhal (mattak), SB = seal beef.
relative contribution of oxychlordane to POP burden rose from fish to mammals. Narwhal mattak showed highest levels of all compounds determined, except for trans-chlordane and cis-chlordane, where the highest levels were observed in fresh salmon. Levels and concentrations of the analyzed pesticides in the present study are provided in Table 1. Hexachlorocyclohexanes (HCH) and Hexachlorobenzene (HCB) The levels of HCB in salmon were in agreement with earlier studies from central Greenland (Table 2). HCB levels in halibut (wet weight basis, ww) for the present study were similar to those of Riget et al. (2004) and Vorkamp et al. (2004), who reported 4.2 ng/g ww, even though fresh filet was analysed, whereas smoked halibut samples were quantified in the present study (Table 2). The seal meat in the present study contained more HCB than meat from central western Greenland, but it was in agreement with seal meat from eastern Greenland (Table 2) (Riget et al., 2004). The age of the seals in the present study is not known, but seals from central western Greenland, from which the meat was prepared, were young (around 1 yr), which may explain their lower levels compared to the present study. Regarding HCH, the levels in seal and whale meat in the present study were lower compared to earlier
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TABLE 1. Median Values (ng/g lw) and Concentration Range of Pesticides in the Samples
Salmon SmokeS SmokeH WB NW SB
Oxychlordane
trans-Chlordane
cis-Chlordane
trans-Nonachlor
cis-Nonachlor
9.82 3.35 7.52 45.45 268.74 65.74
16.10 3.91 3.58 2.60 4.83 4.46
36.28 9.25 8.62 20.99 17.15 2.40
68.71 15.05 23.44 189.68 626.60 115.74
32.34 7.38 8.35 73.53 108.65 21.19
5.6–31.1 3.2–12.3 6.5–103.2 16.0–57.3 173.7–527.4 43.3–88.2
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α–HCH Salmon SmokeS SmokeH WB NW SB
3.56 2.01 8.37 9.06 24.00 17.32
8.6–51.1 3.7–7.5 2.5–7.2 1.5–5.3 3.3–8.4 3.4–5.5
β–HCH 2.7–10.4 1.5–2.9 4.9–14.9 6.1–11.1 17.4–66.7 12.4–22.2
1.56 1.91 1.48 24.68 59.96 15.06
18.9–116.9 8.6–17.1 6.0–17.1 7.6–26.0 10.1–30.3 2.0–2.8
γ–HCH 0.6–3.8 1.5–3.0
