L.I. Medved’s Research Center of Preventive Toxicology, Food and Chemical Safety, Ministry of Health, Ukraine (State Enterprise), Kyiv, Ukraine
Abstract. Goal. An analytical review of literature on the current state of the problem of the determination of polycyclic aromatic hydrocarbons (PAHs) in food products and discussion of ways to introduce EU norms into the practice of domestic laboratories that control the content of PAHS in foods.
Key Words: polycyclic aromatic hydrocarbons, benzo[a]pyrene, food products, dispersion solid phase extraction, QuEChERS method.
Introduction. Polycyclic aromatic hydrocarbons (PAHs) constitute a group of approximately 10,000 organic compounds, some of which are found in large quantities in the environment and in food products. PAHs contain condensed aromatic (benzene) rings and do not have any heteroatoms or substituting groups. Every PAH has two or more benzene rings. The PAHs containing up to four condensed benzene rings are known as light PAHs, and those that contain over four condensed benzene rings are referred to as heavy PAHs. The heavy PAHs are more stable and more toxic than light PAHs. PAHs are mostly lipophilic, but some of them (especially light ones) can be quite well soluble in water and are usually classified as persistent organic pollutants (POPs) [1,2].
In the environment, PAHs are formed as a result of natural and anthropogenic processes, mostly in various thermal processes, i.e. as a result of incomplete combustion of carbon-containing materials, such as crude oil and petroleum products, coal, natural gas, wood or waste [3]. Maximum amounts of PAHs are formed when such materials burn at 500–700 °C, e.g. when wood is burning in forest fires or in tobacco smoke [3]. Substantial amounts of PAHs are released in industrial production of coke, iron, aluminium and steel, with exhaust gases of motor vehicles and when engine oils are used. The available evidence demonstrates that PAHs result from the same processes, which lead to formation of dioxins [4].
Humans are exposed to PAHs in various ways. For non-smokers, the main sources of PAH include contaminated food products [5], water and air; the health hazards of smoking may be significant in smokers. Food products may be contaminated both by environmental sources (natural and manmade) and from industrial processing of food raw materials (drying, smoking, roasting), as well as from some home-based cooking practices (grill, charcoal grilling, barbecue, etc.) [1,6]. Fish and seafood can be contaminated with the PAHs present in water or in the atmosphere, as well as through oil spills [7]. Some marine organisms, such as bivalve mollusks, mussels and oysters are known to absorb and accumulate PAHs from contaminated water.
PAH toxicity. Toxic effects of PAHs and their abundance in food products have been assessed by many organizations, including US Environmental Protection Agency (US EPA) [8], International Water Resources Association, International Association of Cancer Registries (IACR) [9], EU’s Scientific Committee on Food (SCF) [10], Joint Expert Committee on Food Additives (JECFA) of FAO/WHO [11], the International Program on Chemical Safety (IPCS) [12] and European Food Safety Authority (EFSA) [13].
US EPA has selected 16 PAHs, most frequently found in samples during environmental monitoring, including acenaphthene, acenaphthylene, anthracene, fluoranthene, fluorene, naphthalene, phenanthrene, pyrene, benzo[a]anthracene, benzo[b]fluoranthene, benzo[к]fluoranthene, benzo[ghi]perylene, benzo[a]pyrene, chrysene, dibenzo[a,h]anthracene and indeno[1,2,3-сd]pyrene [7,14]. This is not the list of the most toxic substances, but rather prioritization of substances based on their abundance balanced with their toxic effects and human exposure potential [7].
Animal studies of individual PAHs, mainly of benzo[a]pyrene, have shown various toxic effects, such as hepatotoxicity, reproductive and developmental toxicity and immunotoxicity. A number of PAHs produce carcinogenic effects in experimental animals, as well as genotoxicity and mutagenicity in in vitro and in vivo testing. In 2012, International Water Resources Association came to the conclusion that one of the best studied PAHs, benzo[a]pyrene, was a human carcinogen [15]. Some other PAHs have also been identified as carcinogens with genotoxic potential. Although, similar to dioxins and polychlorinated biphenyls, PAHs are lipophilic chemicals, they are metabolized and degrade more rapidly in either human bodies or in the environment [16].
EU’s Scientific Committee on Food (SCF) has evaluated toxicities of 33 PAHs [17]. For most PAHs, their carcinogenic and genotoxic potential is a critical factor to evaluate the associated hazards and risks. During their PAH testing in food products, SCF was informed by evaluations of various international review panels and by substances prioritized rather by health hazards than by abundance in food products [7]. In 2002, SCF recommended that 15 PAHs be monitored, including the 8 high-molecular weight PAHs, which are also part of the US EPA list. The Committee suggested using benzo[a]pyrene as a marker of carcinogenic PAHs in food products.
Data review conducted by EU member states and EFSA in 2008 has shown some PAHs, such as chrysene, to be present in some of the food samples where benzo[a]pyrene was not found. In such cases, benzo[a]pyrene may not serve as a marker for PAH contamination of food products [7]. Eight PAHs (PAH8), namely benzo[a]anthracene, benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ghi]perylene, benzo[a]pyrene, chrysene, dibenzo[a,h]anthracene and indeno[1,2,3-cd]pyrene were chosen as the most acceptable indicators of PAH presence in food products. However, compared to the four-substance set (PAH4), the latter including benzo[a]anthracene, benzo[b]fluoranthene, benzo[a]pyrene and chrysene, the PAH8 set did not influence final assessment significantly.
Regulatory limits of PAH in food products and in the environment. Despite the fact that PAH16 (ЕРА) and PAH15 (EU) are very useful for environmental monitoring programs, regulating their levels in food products based on full lists has not been made part of food legislation [7].
US legislation
US Federal Government has set up regulatory standards and guidelines to protect their population from potential health hazards of exposure to PAH in food, water or air.
According to the Safe Drinking Water Act (SDWA), the US ЕРА has set maximum legal limit of benzo[a]pyrene in drinking water at 0.2 µg/L.
A different limit was set by the National Institute for Occupational Safety and Health (NIOSH) and the Occupational Health and Safety Act (OSHA) based on occupational hazards of coal production. OSHA specifies a legal threshold limit value (TLV) of PAH in the air at 0.2 mg/m3 of air, averaged for an 8-hour workplace exposure.
Exposure margins were also set for mineral oil mist according to OSHA and NIOSH: at 5 mg/m3 averaged for an 8-hour exposure period and at 5 mg/m3 averaged for a 10-hour work shift, respectively.
The authors of this article did not find literature evidence of any US legislation concerning MRLs (maximum residues levels) of any PAHs in food products.
EU legislation
The first time when the European Commission established maximum levels for benzo[a]pyrene was in 2005, as a Commission Regulation (EC) No. 208/2005, which amended Commission Regulation (EC) No. 466/2001. Subsequently, maximum levels for benzo[a]pyrene were specified in Commission Regulation (EC) No. 1881/2006, which was “setting maximum levels for certain contaminants in foodstuffs”. Maximum levels for the total of PAH4 (benzo[a]pyrene, benzo[a]anthracene, benzo[b]fluoranthene and chrysene) were incorporated into Commission Regulation (EC) No. 835/2011[18], amending Regulation (EC) No. 1881/2006. New maximum levels for the total of PAH4 were introduced while retaining individual maximum levels for benzo[a]pyrene.
Table
Section 6. Maximum levels for benzo[a]pyrene and the total of benzo[a]pyrene, benzo[a]anthracene, benzo[b]fluoranthene and chrysene in food products, Regulation (EC) No. 835/2011
The approach of the European Commission to establishing maximum levels of marker PAHs in key food products (smoked meat and meat products, smoked fish and smoked fish products, butter and fats and baby foods) is aimed at ensuring that PAH levels in foodstuffs remain at a level that would not cause health problems and at protecting the market from foodstuffs with no detectable benzo[a]pyrene but with other PAHs present [7]. In Ukraine, identical marker PAHs in the same food products are regulated by similarly designed maximum permissible levels (MPLs), as provided by Order No. 368 of the MoH of Ukraine of 13 May 2013.
Council Directive 98/83/EC of 3 November 1998 on the quality of water intended for human consumption [19] has established a maximum limit for benzo[a]pyrene at 0.010 µg/L and a maximum limit for the sum of benzo[b]fluoranthene, benzo[k]fluoranthene, benzo[ghi]perylene and indeno[1,2,3-cd]pyrene at 0.10 µg/L.
Methods of Analysis for PAHs. Commission Regulations (EC) No. 333/2007 and No. 2016/582 specify the requirements to sampling methods and methods of analysis for PAHs in foodstuffs [20,21], the latter methods compliant with Regulation (EC) No. 1881/2006 (see Section 6). The Regulations also establish requirements to the laboratories, which perform the analysis. The principal requirements for analytical laboratories include being accredited for PAH testing by recognized international standards, expertise in specific tests and a continuous participation in interlaboratory tests. The Regulations also cover the sampling procedure to be followed by the Sampling Authorized Person.
Extraction. The methods used to extract PAHs from food products greatly depend on the nature of the food matrix [7]. General approach to extracting PAHs from fatty foods, chiefly from samples of edible oils, includes saponification of lipids with methanolic KOH/NaOH solution with subsequent removal of non-saponified PAH-containing fraction using extraction with n-hexane. Presently, automated and more effective methods of extraction are used, including pressurized liquid extraction (PLE) [22] and microwave-assisted extraction (МАЕ) [23].
Purification of extracts. The extraction solvents used to extract PAHs in automated instruments, e.g. in PLE instruments, are identical to those used for classic extraction methods (i.e. Soxhlet extraction); therefore, PLE essentially is not a selective extraction method [7]. Therefore, an effective clean-up procedure to remove coextractive substances is crucial to PAH detection and assay, since in crude extracts PAHs are present in trace level quantities. Coupled with adsorption chromatography and solid phase extraction (SPE) using silica gel [24], aluminium oxide [25], florisil [26], C18 [27], or styrene copolymer from divinylbenzene [28], gel penetrating chromatography (GPC), by ensuring division according to differences in molecular size, allows obtaining relatively pure extracts from food matrices, avoiding the problems with chromatographic obstacles for most PAHs. However, the multi-step sample preparation usually results in high consumption of harmful organic solvents, high associated costs and long assay times with potentially higher risks for analyte loss and in low precision and accuracy of the test [7]. Merging extraction and clean-up into a single stage, achieved by adding stationary phase (e.g. silica gel or aluminum oxide) directly into the PLE extraction site allows both fat-free extracts and a high percentage of recovery of all PAHs.
Methods for identification and quantification of PAH. Two principal instrument-based methods of analysis are used to assess polycyclic aromatic hydrocarbons in food products, that is, HPLC-FLD and GC-MS. Mass spectrometric methods have become popular due to high selectivity of detection, confirming identities of tested PAHs based on mass-specters of analytes and the possibility to use stable isotope-labelled PAHs as internal standards. The main methods include tandem mass spectrometry (MS/MS) and high-resolution mass spectrometry (HRMS).
As noted above, during PAH extraction from such complex matrices as food products, a substantial portion of other components of the matrix is inevitably co-extracted with target analytes. Oils, wax, essential oils and natural pigments such as carotenoids and chlorophylls are examples of the most typical matrix components found in extracts from samples of plant origin. In animal tissues, lipids are the principal class of coextractable compounds. Effective separation of these substances, which may adversely affect identification and/or quantification of PAHs, is an essential precondition for reliable results.
Usual methods of PAH assays in samples of foods with high levels of fats involve labour- and time-consuming cleanup procedures. In addition to that, there are known analytical problems associated with extractable lipids, which hinder chromatographic separation of PAHs and their detection with mass spectrometry; as a result, identification of PAHs is becoming rather challenging. In this connection, there has always been a need for appropriate methods of sample preparation to detect residues of various xenobiotics. Such methods are expected to be free from the common shortcomings of traditional methods, to be fast and simple to perform, to cost less, to require minimal volumes of solvents and to guarantee high selectivity without the complexity of cleanup procedures. To achieve the goals of the study, the authors have employed the QuEChERS method, as a Quick, Easy, Cheap, Effective, Rugged and Safe analytical method of sample processing to assess for various classes of pesticides [29]. However, in recent years this method is widely used to test not only for pesticides, but also for a large variety of food products and matrices. Several modifications of the QuEChERS method have been suggested to assess PAHs in seafood such as shrimps, scallops, mussels, fish and in meat [30]. Due to differences in the matrix effect in assays of various food products, the applicability of the QuEChERS method must be thoroughly evaluated.
Below, as an example, we present the results of our validation of a GC/MS-based assay for indicator PAHs (benzo[a]pyrene benzo[a]anthracene benzo[b]fluoranthene and chrysene) combined with the QuEChERS procedure, used with samples of fish (Alaska Pollack, Theragra chalcogramma).
Materials and Methods
Reagents
Acetonitrile, analytical grade (HPLC)
Magnesium sulphate anhydrous, analytical grade (Merck)
NaCl, ACS-grade (Merck)
Primary secondary amine (PSA), a sorbent for solid phase extraction (Supelclean PSA)
Sorbent for solid phase extraction (С18), Bondesil C18 Bulk Sorbent, 40 μm, Varian
Materials and Hardware
Centrifuge tubes, 50 mL
Centrifuge tubes, 10 mL
Centrifuge (Universal 320 Hettich Centrifugen to 4000 ref)
Gas chromatograph (Finnigan Trace GC Ultra) with an electron-capture detector (ECD) and a DB-5 capillary column (0.53 mm x 60 m)
Household-grade refrigerator
GC assessment
Separation with GC was performed using a DB-5 capillary column (60 m, internal diameter 0.53 mm, film thickness 0.5 µm) under the following conditions: constant nitrogen flow of 4.0 mL/min; inlet temperature: 270 oC; detector temperature (ECD): 300 oC; injection volume: 1 µL (indivisible); the initial oven temperature of 60 oC is maintained for 1.0 minute with subsequent increase at 100 oC/min to 210 oC, then maintained for 1.0 minute, then at 5 oC/min.
Extraction and cleanup procedure
The schematics of the main stages of QuEChERS assay for PAHs in fish (Alaska Pollack)
Results and discussion.
Figs. 1–3 show typical chromatograms.
Fig. 1. Chromatogram of blank reagents
Fig. 2. Chromatogram of a standard solution of PAH, 25 ppb
1-benz[a]anthracene, 2-chrysene, 3-benzo[b]fluoranthene, 4-benzo[a]pyrene
Fig. 3 Chromatogram of a reference sample of fish with addition of a standard PAH mixture, 25 ppb
1-benz[a]anthracene, 2-chrysene, 3-benzo[b]fluoranthene, 4-benzo[a]pyrene
To extract PAHs from samples of food products, a non-buffer QuEChERS method with acetonitrile, anhydrous magnesium sulphate and sodium chloride is used. In this case, acetonitrile, an organic solvent, provides for high levels of PAH extraction with minimal amounts of coextractive substances. In addition, acetonitrile is a suitable solvent for a GC analysis. To clean-up the resulting extract, a mixture of anhydrous magnesium sulphate and PSA/C18 sorbents is used. Anhydrous magnesium sulphate removes water from the organic phase. The C18 sorbent removes long-chain fatty compounds, sterols and other nonpolar substances. The PSA sorbent is used to remove sugars and fatty acids, organic acids, lipids and some pigments. When PSA is used in combination with C18, additional amounts of lipids and sterols may be removed.
The limit of quantification for PAH. The limit of quantification for each PAH was assessed with processing of samples of Alaska Pollack with a reference solution of test PAHs. The limit of quantification (LOQ) was mainly within the range of 0.1 to 0.8 ng/g of raw weight.
Recovery test. The recovery test was performed by adding test PAHs to reference samples of fish; the analytical procedure was used to test these samples as described above. The recovery of PAHs was from 65 % to 105 %.
Repeatability. Repeatability testing was performed by fortification of six samples of Alaska Pollack with test PAHs at the levels of 0.1 to 2.0 µg/g. All repeat samples (n = 6) were performed, and RSD % values were calculated for each of the PAHs. The method was found to be accurate; RSDr % (CV %) values for all test PAHs were less than 20 %.
Testing of matrix effects. Matrix effects were absent for all test PAHs.
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