Food Control 121 (2021) 107586

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Food Control
journal homepage: www.elsevier.com/locate/foodcont

Quantification and risk assessment of carcinogenic polycyclic aromatic

hydrocarbons in retail smoked fish and smoked cheeses
Radu C. Racovita a, Catinca Secuianu a, b, *, Florentina Israel-Roming a, c
a
University “Politehnica” of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Inorganic Chemistry, Physical Chemistry and
Electrochemistry, 1-7 Gh. Polizu St., Bucharest, 011061, Romania
b
Imperial College London, Department of Chemical Engineering, South Kensington Campus, London, SW7 2AZ, United Kingdom
c
University of Agronomic Sciences and Veterinary Medicine, Biotechnology Department, 59 Marasti Blvd., Bucharest, 011464, Romania

A R T I C L E I N F O A B S T R A C T

Keywords: Polycyclic aromatic hydrocarbons (PAHs) are known or potential carcinogens occurring in foodstuffs during
Benzo[a]pyrene smoking processes, among others. The European Commission (EC) has defined clear limits for benzo[a]pyrene
Smoked foodstuffs and the total concentration of benz[a]anthracene, chrysene, benzo[b]fluoranthene, and benzo [a]pyrene –
Gas chromatography-mass spectrometry
known as the PAH4 group – that must not be exceeded by any food product commercialized within the European
QuEChERS protocol
Margin of exposure
Union (EC Regulation No. 835/2011). In this work, we determined the concentrations of 16 PAHs, including the
Incremental lifetime cancer risk PAH4 group, in 17 retail smoked fish and 11 retail smoked cheeses from four and nine producers, respectively.
To this end, we first developed a gas chromatography-mass spectrometry method for identification and quan­
tification of PAHs in smoked food matrices and validated it intra-laboratory according to the guidelines of EC
Regulation No. 836/2011. For the extraction of PAHs from foodstuffs and their further clean-up, we adopted a
Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) protocol, based on partitioning between acetonitrile
and water, followed by dispersive solid-phase extraction using commercially available QuEChERS kits. The
analyses of smoked fish revealed a fairly wide range of PAH concentrations, resulting both from differences in the
type of smoked fish and different producers. Smoked sprat exhibited the highest total PAH concentration (around
100 ng/g), with acenaphthylene, phenanthrene, and fluorene contributing more than half of this amount, while
the lowest PAH content (about 30 ng/g) was observed in smoked trout. None of the analyzed fish samples
exceeded the EC limits for either benzo [a]pyrene or PAH4 in the corresponding product categories. While no
such limits exist for the smoked cheese category, it was found smoked cheeses had much lower PAH levels
compared to smoked fish, around 10 ng/g for ordinary pressed cheeses and 20 ng/g for generically designated
“Brădet” cheeses, irrespective of producer. The majority PAHs in all investigated cheeses were found to be
phenanthrene, naphthalene, and fluorene, while PAH4 accounted for only about 10% of total PAHs. Cancer risk
assessment was performed using both margin of exposure and incremental lifetime cancer risk parameter de­
terminations and none of the samples analyzed showed significant concern.

1. Introduction foods, such as fruit, vegetables, fish and seafood. Furthermore, certain
food processing technologies and home cooking practices (e.g. smoking,
Polycyclic aromatic hydrocarbons (PAHs) are a diverse class of drying, roasting, baking, grilling, barbecuing, and frying) can generate
environmental and food contaminants formed as the result of incom­ additional PAHs in foods (Alexander et al., 2008).
plete pyrolysis of organic matter. Various industrial processes (e.g. PAHs are especially dangerous due to their genotoxic and carcino­
processing of coal, petroleum, and natural gas), as well as other genic effects (Alomirah et al., 2011). In 2008, the European Food Safety
anthropogenic factors (motor vehicle exhaust and cigarettes) and nat­ Authority (EFSA) – Panel on Contaminants in the Food Chain (CONTAM
ural phenomena (forest fires and volcanic eruptions) are responsible for Panel) formulated a list of 16 priority PAHs of particular concern to
the release of PAHs into the environment, from where they can reach human health: benzo [a]pyrene (BaP), chrysene (CHR), benz [a]

* Corresponding author. University “Politehnica” of Bucharest, Faculty of Applied Chemistry and Materials Science, Department of Inorganic Chemistry, Physical
Chemistry and Electrochemistry, 1-7 Gh. Polizu St., Bucharest, 011061, Romania.
E-mail addresses: [email protected], [email protected] (C. Secuianu), [email protected] (F. Israel-Roming).

https://doi.org/10.1016/j.foodcont.2020.107586
Received 7 July 2020; Received in revised form 25 August 2020; Accepted 26 August 2020
Available online 7 September 2020
0956-7135/© 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
R.C. Racovita et al. Food Control 121 (2021) 107586

anthracene (BaA), benzo [b]fluoranthene (BbFA), benzo [k]fluo­ successfully for other food matrices, such as tea (Sadowska-Rociek et al.,
ranthene (BkFA), indeno [1,2,3-cd]pyrene (IP), dibenz [a,h]anthracene 2014), bread (Eslamizad et al., 2016), eggs (Yeudakimau et al., 2013),
(DBahA), benzo [ghi]perylene (BghiP), benzo [j]fluoranthene (BjFA), meat (Kao, Chen, Huang, Chen, & Chen, 2014; Racovita, Secuianu,
cyclopenta [cd]pyrene (CPP), dibenzo [a,l]pyrene (DBalP), dibenzo [a, Ciuca, & Israel-Roming, 2020), and seafood (Johnson, 2012).
e]pyrene (DBaeP), dibenzo [a,i]pyrene (DBaiP), dibenzo [a,h]pyrene In the present work, the occurrence of PAHs in 17 types of smoked
(DBahP), 5-methylchrysene (MCH), and benzo [c]fluorene (BcFL) fish from four different producers and 11 types of smoked cheese from
(Alexander et al., 2008). In addition, the CONTAM Panel recommended nine producers was investigated. The recently demonstrated versatility
that PAH4 (the sum of BaP, CHR, BaA and BbFA) be used as the most of the QuEChERS preparation method for food matrices, in combination
suitable indicator of PAH content in foods (Alexander et al., 2008). with GC-MS analysis, recommended it for implementation in this work,
Consequently, the European Commission (EC) adopted Regulation No. which was intended to fill part of the aforementioned gap in PAH
835/2011 establishing the maximum levels admissible for PAH4 and contamination studies for smoked food products commercially available
BaP in various foodstuffs distributed within the European market (Eu­ on the Romanian market. To our knowledge, only one past study on
ropean Commission, 2011a). Special regulations were also adopted for Romanian smoked fish, namely carp, silver carp, rainbow trout, and
traditionally smoked meat and fishery products from certain European brook trout from three local smokehouses in Brasov county, exists
Union (EU) member states, establishing more permissive limits within (Mihalca et al., 2011), and no previous studies dedicated to PAHs in
their own national markets on a temporary basis (European Commis­ Romanian smoked cheeses. While explicit BaP and PAH4 concentration
sion, 2014). limits have been set for smoked fish at the level of the European common
It is thus essential that adequate analytical methods are continuously market (European Commission, 2011a), no such limits have been
developed and implemented for monitoring PAH content in foodstuffs defined in the European legislation for smoked cheeses. Nevertheless, it
within the European Union and beyond. Recent trends have been is of interest to both food control specialists and the general public to
focused on chromatographic methods, particularly high performance have an overview of PAH contamination levels in this latter category of
liquid chromatography (HPLC) with either ultraviolet–visible (UV–Vis) smoked foodstuffs as well, and several studies from other European
(Plaza-Bolanos, Garrido Frenich, & Martinez Vidal, 2010; Yeudakimau, countries have been published on this topic, including the Czech Re­
Provatas, Perkins, & Stuart, 2013) or fluorescence detection (FLD) public (Suchanova, Hajslova, Tomaniova, Kocourek, & Babicka, 2008),
(Garcia Londono, Reynoso, & Resnik, 2015; Plaza-Bolanos et al., 2010) Poland (Pluta-Kubica et al., 2020), Spain (Guillén & Sopelana, 2004),
and gas chromatography with mass spectrometric detection (GC-MS) Italy (Pagliuca et al., 2003), Slovakia (Michalski & Germuska, 2003),
(Alomirah et al., 2011; Lee, Jeong, Park, & Lee, 2018; Mahugija & Njale, and Switzerland (Bosset et al., 1998).
2018; Malarut & Vangnai, 2018). Such methods have been successfully
employed for PAH assessment in bread (Eslamizad et al., 2016), tea 2. Materials and methods
(Garcia Londono et al., 2015; Sadowska-Rociek, Surma, & Cieslik,
2014), coffee (Orecchio, Paradiso, & Culotta, 2009; Stanciu, Dobrinas, 2.1. Chemicals
Birghila, & Popescu, 2008), vegetables (Soceanu, Dobrinas, Stanciu, &
Popescu, 2014), honey (Dobrinas, Birghila, & Coatu, 2008), smoked Acetonitrile (HPLC grade, min. 99.9%) was purchased from Scharlab
meat (Ledesma, Rendueles, & Diaz, 2014) and smoked fish (Mahugija & S.L. (Spain) and used without further purification. The water used in all
Njale, 2018; Mihalca, Tiţa, Tiţa, & Mihalca, 2011). experiments was from a Milli-Q Reference Water Purification System,
In particular, there is a scarcity of monitoring surveys and risk with a resistivity of 18 MΩ cm. Helium (min. 99.9999%) and nitrogen
assessment studies of PAH contamination of food products sold in (min. 99.996%) were both from Linde GmbH (Germany). QuEChERS
Eastern Europe and the Balkans. So far, these have been limited to extraction cartridges were purchased from Thermo Fisher Scientific Inc.
vegetables (Soceanu et al., 2014), cereal products (Rozentale, Zacs, (USA) and consisted of 50-mL centrifuge tubes containing 6 g anhydrous
Perkons, & Bartkevics, 2017; Zachara, Gałkowska, & Juszczak, 2017), magnesium sulfate and 1.5 g sodium acetate. QuEChERS dSPE purifi­
coffee (Stanciu et al., 2008), fruit juices (Soceanu, Dobrinas, Popescu, & cation kits for fatty samples were purchased from Agilent Technologies
Stanciu, 2011), edible oils (Dost & Ideli, 2012), baby foods (Soceanu, Inc. (USA) and consisted of 15-mL centrifuge tubes containing 400 mg
Dobrinas, & Popescu, 2016), honey and propolis (Dobrinas et al., 2008), PSA (primary-secondary amine), 400 mg C18-silica, and 1200 mg
smoked meat products (Djinovic, Popovic, & Jira, 2008; Reinik et al., anhydrous magnesium sulfate. A JTB-0005 certified reference material
2007; Rozentale et al., 2015; Racovita, Secuianu, Ciuca, & from Ultra Scientific Inc. (now Agilent Technologies Inc.) was used for
Israel-Roming, 2020), smoked fish (Mihalca et al., 2011; Zachara et al., PAH chromatographic characteristic and recovery determinations and
2017), and smoked cheese (Pluta-Kubica, Filipczak-Fiutak, Domagała, consisted of a 16 PAH solution in acetonitrile, including: 20 μg/mL
Duda, & Migdał, 2020). Thus, in the context of food safety, there is an acenaphthene (ANe), 15 μg/mL acenaphthylene (ANy), 0.8 μg/mL
ongoing necessity for further exploration of PAH contamination levels in anthracene (A), 4 μg/mL benz [a]anthracene (BaA), 4 μg/mL benzo [b]
foods, and especially smoked foods, very appreciated by many con­ fluoranthene (BbFA), 4.5 μg/mL benzo [k]fluoranthene (BkFA), 3.5 μg/
sumers. A major element of difficulty in PAH analysis has been the mL benzo [ghi]perylene (BghiP), 5 μg/mL benzo [a]pyrene (BaP), 3.5
isolation, purification, and concentration of PAHs from the food matrix. μg/mL chrysene (CHR), 3.5 μg/mL dibenz [a,h]anthrancene (DBahA), 8
Traditionally, this has been an extremely laborious process, involving μg/mL fluoranthene (FA), 5 μg/mL fluorene (F), 4.5 μg/mL indeno
multi-step sample processing, including saponification, Soxhlet or [1,2,3-cd]pyrene (IP), 20 μg/mL naphthalene (N), 3.5 μg/mL phenan­
pressurized liquid extraction, and clean-up by column chromatography threne (PA), and 8.5 μg/mL pyrene (P). 10-azabenzo [a]pyrene (ABaP)
or solid-phase extraction (SPE) (Plaza-Bolanos et al., 2010). However, was a certified reference material (neat, min. 99.6%, code BCR-092) by
recently, the applicability of the QuEChERS (Quick, Easy, Cheap, the Institute for Reference Materials and Measurements (IRMM) of the
Effective, Rugged and Safe) sample preparation method, commonly Joint Research Centre (Belgium) and was purchased from LGC Limited
employed for pesticide residue analysis in foodstuffs (Anastassiades, (United Kingdom).
Lehotay, Štajnbaher, & Schenck, 2003), has been demonstrated for PAHs
as well (González-Curbelo et al., 2015; Rejczak & Tuzimski, 2015). This 2.2. Smoked food materials and certified reference materials
is a faster and less laborious method, involving only common extraction,
agitation, and centrifugation steps, as well as simpler clean-up by Smoked fish typically consisted of smoked fillets in vacuum-sealed
dispersive solid-phase extraction (dSPE). The method was applied plastic packaging and was acquired from local supermarkets. It was
originally for PAH assessment in fish (Ramalhosa, Paiga, Morais, stored at 4 ◦ C for no more than 1 or 2 days from the moment of purchase
Delerue-Matos, & Pinto Oliveira, 2009), but has since been adapted until use. In total, 17 types of smoked fish from four different producers

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Table 1 analysis.
Producer codes, commercial names, and product codes of smoked fish and
smoked cheeses available on the Romanian market that were investigated in this
2.4. PAH analysis by GC-MS
work.
Smoked product Producer Commercial name of smoked Product The setup used consisted of a Thermo Scientific Focus GC coupled
category code product code
with a Polaris Q ion trap MS. One-microliter sample injections were
Smoked fish 1 Smoked herring fillet 1-a performed by a TriPlus Autosampler, in splitless mode, at an inlet
Smoked fish 1 Hot smoked mackerel 1-b
temperature of 250 ◦ C. The type of column used was TR-5MS from
Smoked fish 1 Smoked Norwegian salmon 1-c
Smoked fish 2 Smoked herring fillet 2-a Thermo Fisher Scientific (polydimethylsiloxane with 5% phenyl groups
Smoked fish 2 Smoked mackerel fillet 2-b as stationary phase, 30 m length, 0.25 mm i. d., 0.25 μm film thickness).
Smoked fish 2 Smoked salmon fillet 2-c Helium served as carrier gas at a constant flow rate of 1 mL/min. The MS
Smoked fish 2 Smoked sprat 2-d transfer line was maintained at 280 ◦ C and the ion source temperature
Smoked fish 2 Smoked Alaska wild salmon 2-e
was 250 ◦ C. The GC oven temperature program was as follows: 1 min at
Smoked fish 3 Smoked herring fillet 3-a
Smoked fish 3 Smoked mackerel fillet 3-b 70 ◦ C; ramp 10 ◦ C/min to 300 ◦ C; constant for 10 min (Racovita et al.,
Smoked fish 3 Smoked cod fillet 3-c 2020). In order to increase sensitivity, all GC-MS measurements were
Smoked fish 3 Smoked sprat 3-d done in single ion monitoring (SIM) mode (Racovita & Jetter, 2016),
Smoked fish 4 Smoked herring fillet 4-a
using m/z ratios characteristic for each PAH (see below).
Smoked fish 4 Smoked mackerel fillet 4-b
Smoked fish 4 Sliced smoked salmon fillet 4-c
Smoked fish 4 Smoked tuna fillet 4-d 2.5. Calculation of BaP toxic equivalent concentration (TEQBaP) and
Smoked fish 4 Cold smoked trout fillet 4-e
chronic daily intake (CDI)
Smoked cheese A Sliced pressed cheese smoked A-1
with natural birch wood
Smoked cheese B Smoked pressed cheese B-1 In order to consider the different carcinogenicities of the 16 PAHs
Smoked cheese C Smoked pressed cheese C-1 quantified for every type of sample into a single, cumulative parameter,
Smoked cheese D Smoked pressed cheese D-1 the BaP toxic equivalent concentrations were calculated according to the
Smoked cheese E Smoked pressed cheese E− 1 ∑
Smoked cheese F Smoked braided cheese F-1
formula: TEQBaP = ni=1 Ci ⋅TEFi (Lee et al., 2018), where Ci is the con­
Smoked cheese G Smoked pressed cheese G-1 centration of PAH i (ng/g) and TEFi is the toxic equivalency factor of the
Smoked cheese G Smoked pressed cheese “Brădet” G-2 same PAH i (Nisbet & LaGoy, 1992).
Smoked cheese H Smoked braided cheese “Brădet” H-1 Daily PAH dietary exposure levels were calculated by dividing the
Smoked cheese H Smoked pressed cheese “Brădet” H-2
total intake level over a lifetime by the average lifespan of a Romanian,
Smoked cheese I Smoked pressed cheese “Brădet” I-1
which is currently 75.3 years (European Commission, 2019a). In the
case of carcinogens, this approach is founded on the assumption that a
were studied, each type being denoted by a combination of number high dose received within a short time is equivalent to a low dose taken
hyphenated to a small letter (e.g. 1-a), where the number identified the constantly in a lifetime (Yoon, Park, Lee, Yang, & Lee, 2007). Thus, the
producer and the letter the specific fish sample. Smoked cheeses span­ chronic daily intake (CDI) due to a certain foodstuff was calculated ac­
ned 11 types from nine producers and were also purchased from local cording to the equation: CDI = TEQBW⋅AT BaP ⋅IR⋅ED
(Lee et al., 2018), where:
supermarkets in vacuum-sealed plastic packaging. They were coded TEQBaP (ng/g) is the BaP toxic equivalent concentration calculated as
using a sequence of capital letter and number (e.g. A-1), the letter shown above, IR (g/day) is the average daily intake of that particular
denoting the producer and the number the specific cheese. They were foodstuff, ED is the exposure duration (considered as an average adult
also stored at 4 ◦ C prior to use for no more than a week after purchase. period of 45 years), BW is the average body weight of Romanians (taken
Table 1 summarizes the details regarding the nature of smoked fish and as 77 kg, i.e. an average between female and male average weights re­
smoked cheese retail products chosen for investigation in this work and ported by World Data) (World Data, 2020), and AT is the average life
their codification. An additional certified reference material consisting expectancy for a Romanian mentioned above (75.3 years). Because
of coconut oil essentially devoid of PAHs (BCR-459, IRMM Belgium) was average daily intake data for smoked fish and smoked cheeses were not
used for recovery determinations of PAHs. available either at national or EU level, IR values were taken as total
average daily intakes of any kind of fish and cheese, at 21.64 g/day
2.3. Sample preparation (European Commission, 2019b) and 8.22 g/day, respectively (European
Commission, 2019b). While this approach constitutes clearly an over­
In a typical experiment, store-purchased smoked foodstuffs were estimation, if the results show little or no risk at such level, they are valid
chopped finely and homogenized in a Zephyr Z-1111-O bowl chopper even more so at lower levels of daily intake.
(Germany), taking care, in the case of fish, to remove by tweezers any
bone fragments present prior to chopping. About 10 g (weighed to a 2.6. Risk assessment via calculation of margin of exposure (MOE) and
precision of ±0.1 mg) of minced sample were transferred into the 50-mL incremental lifetime cancer risk (ILCR)
QuEChERS extraction cartridge containing also 10 mL water and 10 mL
acetonitrile. Samples were stirred vigorously at 2500 rpm using a DLAB Two quantitative parameters were used for risk assessment, namely
MX-S vortex (Dragon Lab, China) for 1 min, then centrifuged for 5 min at the margin of exposure (Alexander et al., 2008) and incremental lifetime
4000 rpm using a Medibas + centrifuge (Auxilab S.L., Spain). 5 mL from cancer risk (United States Environmental Protection Agency, 1991). The
the upper (organic) layer were transferred to a 15 mL dSPE purification former was calculated by dividing the benchmark dose lower confidence
tube. Vortexing and centrifugation were repeated. After settling, 1.5 mL limit (BMDL10), which was established to be 0.34 mg/kg b. w./day by
of sample were transferred to a 2 mL GC amber vial (ISOLAB GmbH, EFSA (Alexander et al., 2008), by the CDI value computed taking into
Germany), 10 μL of internal standard (ABaP) solution of known con­ account the TEQBaP due only to the PAH4 group: MOE = BMDL10/CDI.
centration in acetonitrile were added, and the sample was concentrated According to EFSA, MOE values <104 are interpreted as possible
to dryness under a gentle stream of N2 at 50 ◦ C, before being taken up concern, 104-105 as low concern, >105 as negligible concern as long as
with fresh acetonitrile into 150 μL conical glass inserts (Agilent Tech­ actions are taken to minimize further exposure, while >106 values are
nologies Inc., USA) to a total volume of 100 μL. Finally, 1 μL of this translated as negligible concern.
solution was injected into the GC-MS for qualitative and quantitative ILCR values were computed by multiplying the total CDI with the

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R.C. Racovita et al. Food Control 121 (2021) 107586

Fig. 1. Gas chromatogram obtained in SIM mode for the mixture of 16 PAHs plus 10-azabenzo [a]pyrene internal standard (at 25.87 min). The baseline upshift
between 25.5 and 29 min is due to additional ions with m/z 252 formed through column bleeding.

cancer risk value of BaP determined from animal tests, i.e. 7.3 (mg/kg b. lutions in acetonitrile of the JTB-0005 certified reference mixture of 16
w./day)− 1 (United States Environmental Protection Agency, 1991): PAHs, and the sensitivity was calculated as the slope of the peak area vs.
ILCR = 7.3 × CDI. If ILCR<10− 6 then level of risk is considered concentration line. The limits of detection and quantification were
acceptable and without negative consequences; values in the interval measured for each analyte from the standard deviations of the means of
10− 6-10− 4 denote potential risk, and values greater than 10− 4 corre­ peak areas for a set of 20 blanks (European Commission, 2007), i.e. for
spond to serious risk (United States Environmental Protection Agency, an analyte i: LODi = 3⋅σi/Si and LOQi = 10⋅σi/Si, where σi and Si are the
1991). standard deviation and sensitivity, respectively, for analyte i. Relative
response factors RRFi of each analyte i with respect to the internal
3. Results and discussion standard (ABaP) were calculated using the equation: RRFi = Acii ⋅AcISIS ⋅MMISi ,
where Ai and AIS are the peak areas (in arbitrary units) of analyte i and
3.1. Analytical method validation internal standard, ci and cIS are the concentrations (in μg/mL) of analyte
i and internal standard, and Mi and MIS are the molar weights (in g/mol)
Prior to the analysis of retail smoked foodstuffs, GC-MS method of analyte i and internal standard, respectively. Average recoveries were
performance parameters and analyte characteristics, such as retention determined using three 10-g replicates of the certified reference material
times (RT), relative response factors (RRF), linear ranges, sensitivities BCR-459, which were each spiked with 200 μL of a 10-fold diluted so­
(S), limits of detection (LOD), and limits of quantification (LOQ), were lution of the 16-PAH mix in acetonitrile, prior to QuEChERS extrac­
determined. A typical gas chromatogram recorded for the 16 PAH tion/partitioning with acetonitrile and water and matrix interference
reference mixture + internal standard under the experimental condi­ clean-up by dSPE. Horwitz ratios HORRATR were calculated by
tions described in section 2.4 is shown in Fig. 1. dividing the relative standard deviation of concentrations obtained for
The linear range of each analyte was obtained through serial di­ the spiked samples with the predicted relative standard deviation given

Table 2
Characteristic m/z values monitored in SIM mode, retention times, relative response factors, linear ranges, R2 values for linear ranges, sensitivities, limits of detection
and quantification, and recoveries from spiked samples of the 16 PAHs investigated in this work.
No. PAH m/z value RT RRF Linear range R2 S (mL/ LOD LODa LOQ LOQa Recovery HORRATR
abbreviation monitored (min) (μg/mL) μg) (ng/ (ng/g) (ng/ (ng/g) (%)
mL) mL)

1 N 128 8.19 0.704 10− 3 – 1 0.9999 1.17⋅106 0.244 0.018 0.814 0.059 91.4 0.308
2 ANe 152 11.92 0.598 10− 3 – 1 0.9991 1.23⋅106 0.367 0.028 1.224 0.092 88.4 0.460
3 ANy 153 12.33 0.691 10− 3 – 1 0.9996 1.50⋅106 0.234 0.027 0.779 0.090 58.1 0.280
4 F 165 13.59 0.922 10− 4 – 1 0.9999 1.55⋅106 0.211 0.014 0.703 0.047 98.9 0.543
5 PA 178 15.94 0.730 10− 4 – 1 0.9998 1.25⋅106 0.091 0.006 0.302 0.020 103.4 0.809
6 A 178 16.06 0.059 10− 4–10− 1
0.9899 1.03⋅106 0.373 0.055 1.244 0.183 45.3 0.947
7 FA 202 18.86 0.838 10− 4 – 1 0.9999 1.17⋅106 0.156 0.015 0.520 0.049 71.1 0.284
8 P 202 19.41 0.828 10− 4 – 1 0.9998 1.22⋅106 0.185 0.020 0.616 0.065 62.8 0.604
9 BaA 228 22.34 0.483 10− 4 – 1 0.9950 3.73⋅105 0.674 0.093 2.246 0.309 50.4 0.241
10 CHR 228 22.44 0.692 10− 4 – 1 0.9960 5.78⋅105 0.517 0.070 1.724 0.232 51.5 0.202
11 BbFA 252 24.90 0.687 10− 3 – 1 0.9780 3.18⋅105 0.559 0.058 1.863 0.195 63.8 1.253
12 BkFA 252 24.96 0.780 10− 4 – 1 0.9889 4.29⋅105 0.459 0.061 1.531 0.202 50.5 1.034
13 BaP 252 25.78 0.388 10− 4 – 1 0.9990 3.76⋅105 0.763 0.083 2.544 0.275 61.6 0.470
14 IP 276 29.70 0.271 10− 4 – 1 0.9905 2.40⋅105 0.825 0.154 2.750 0.512 35.8 0.308
15 DBahA 278 29.80 0.502 10− 4 – 1 0.9779 2.23⋅105 0.711 0.124 2.371 0.413 38.3 0.516
16 BghiP 276 30.89 0.711 10− 3 – 1 0.9843 3.34⋅105 0.798 0.104 2.662 0.346 51.2 0.284
a
ng of PAH per g of BCR-459.

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 R.C. Racovita et al.
Table 3
Concentrations of individual PAHs, the PAH4 group, and total PAHs in samples of smoked fish denoted by producer number-product letter code. Values represent averages ± standard deviations (n = 3), in ng PAH per g
fresh weight of smoked fish. Average values below the limit of quantification are reported as “< LOQ”. If below the limit of detection, “n.d.” (i.e. not detected) is reported.
PAH 1-a 1-b 1-c 2-a 2-b 2-c 2-d 2-e 3-a 3-b 3-c 3-d 4-a 4-b 4-c 4-d 4-e

N 4.6 ± 0.4 2.8 ± 0.4 2.6 ± 0.4 5.3 ± 0.4 3.8 ± 0.8 3.6 ± 0.3 8.5 ± 3.1 ± 0.5 5.6 ± 0.6 2.9 ± 0.5 4.1 ± 0.7 7.2 ± 1.9 6.1 ± 0.5 2.7 ± 0.3 3.2 ± 0.6 2.4 ± 0.4 2.6 ± 0.4
2.1
ANe 1.1 ± 0.2 0.6 ± 0.2 0.4 ± 0.1 1.3 ± 0.1 0.8 ± 0.2 0.8 ± 0.2 2.0 ± 0.6 ± 0.2 0.7 ± 0.2 0.6 ± 0.1 0.7 ± 0.2 2.2 ± 0.5 1.2 ± 0.3 0.5 ± 0.2 0.7 ± 0.2 <0.092 0.4 ± 0.2
0.3
ANy 10.3 ± 12.3 ± 8.3 ± 2.4 8.8 ± 1.9 15.1 ± 9.3 ± 2.1 20.4 ± 6.6 ± 1.6 11.7 ± 14.6 ± 11.4 ± 24.9 ± 5.6 15.3 ± 11.1 ± 7.3 ± 2.2 8.1 ± 2.2 5.7 ± 1.5
2.1 3.0 3.5 4.7 2.7 3.2 1.6 3.9 2.9
F 3.1 ± 0.6 4.7 ± 0.6 7.3 ± 1.4 3.3 ± 0.6 3.9 ± 0.7 10.6 ± 13.5 ± 9.7 ± 2.8 2.9 ± 0.4 5.5 ± 0.5 6.9 ± 0.8 15.2 ± 3.2 4.8 ± 0.6 5.1 ± 0.8 7.7 ± 2.5 4.5 ± 1.5 5.1 ± 1.3
2.9 3.1
PA 12.1 ± 7.2 ± 2.7 10.2 ± 12.8 ± 7.4 ± 2.0 9.7 ± 2.3 18.1 ± 8.5 ± 2.2 9.8 ± 1.9 6.3 ± 1.8 9.6 ± 2.1 21.3 ± 3.7 18.5 ± 5.6 ± 1.3 10.8 ± 9.9 ± 2.6 6.5 ± 1.8
2.2 2.1 3.0 3.8 4.0 2.9
A 3.4 ± 1.2 3.3 ± 0.8 1.9 ± 0.5 3.4 ± 0.8 3.6 ± 1.1 3.0 ± 1.2 5.8 ± 2.2 ± 0.5 3.8 ± 1.3 3.4 ± 0.7 2.3 ± 0.5 7.8 ± 2.0 5.1 ± 1.4 3.8 ± 0.7 2.3 ± 0.5 2.2 ± 0.3 1.6 ± 0.5
1.4
FA 2.9 ± 0.7 2.3 ± 0.5 2.5 ± 0.4 2.4 ± 0.4 2.3 ± 0.6 3.6 ± 0.7 6.3 ± 2.4 ± 0.4 2.1 ± 0.3 3.1 ± 0.7 2.0 ± 0.2 6.1 ± 1.5 3.0 ± 0.3 2.2 ± 0.5 2.7 ± 0.5 2.2 ± 0.4 1.5 ± 0.2
1.3
P 2.4 ± 0.5 1.4 ± 0.4 2.0 ± 0.3 1.7 ± 0.3 1.8 ± 0.3 2.2 ± 0.5 5.8 ± 1.8 ± 0.4 1.4 ± 0.5 0.9 ± 0.3 1.2 ± 0.4 6.9 ± 1.2 1.5 ± 0.4 0.8 ± 0.2 1.9 ± 0.3 1.3 ± 0.3 1.5 ± 0.4
0.8
5

BaA 0.8 ± 0.3 0.8 ± 0.2 0.5 ± 0.1 0.8 ± 0.2 0.7 ± 0.2 0.6 ± 0.1 2.4 ± 0.8 ± 0.3 0.5 ± 0.1 0.7 ± 0.3 0.6 ± 0.2 2.6 ± 0.6 0.6 ± 0.2 0.8 ± 0.3 0.9 ± 0.4 0.7 ± 0.2 1.6 ± 0.5
0.5
CHR 1.4 ± 0.3 1.8 ± 0.4 0.9 ± 0.2 1.5 ± 0.3 1.5 ± 0.4 1.7 ± 0.4 6.3 ± 1.9 ± 0.5 1.7 ± 0.3 1.3 ± 0.4 1.4 ± 0.4 6.4 ± 1.2 1.8 ± 0.4 1.1 ± 0.3 1.3 ± 0.5 1.0 ± 0.3 1.9 ± 0.6
0.9
BbFA <0.195 <0.195 n.d. 0.3 ± 0.1 <0.195 n.d. 1.5 ± n.d. n.d. <0.195 <0.195 2.0 ± 0.5 n.d. 0.4 ± 0.2 <0.195 n.d. 0.4 ± 0.2
0.6
BkFA <0.202 <0.202 n.d. <0.202 n.d. n.d. 1.1 ± <0.202 n.d. <0.202 <0.202 1.6 ± 0.5 n.d. 0.4 ± 0.1 <0.202 n.d. <0.202
0.4
BaP 0.3 ± 0.1 0.6 ± 0.1 0.5 ± 0.2 0.3 ± 0.1 0.7 ± 0.2 0.5 ± 0.3 2.2 ± n.d. n.d. 1.1 ± 0.3 1.0 ± 0.3 2.9 ± 0.5 0.4 ± 0.1 0.9 ± 0.2 0.7 ± 0.2 0.5 ± 0.3 0.5 ± 0.2
0.5
IP <0.512 0.6 ± 0.2 <0.512 <0.512 0.6 ± 0.1 <0.512 2.6 ± <0.512 <0.512 1.8 ± 0.4 <0.512 2.8 ± 0.6 <0.512 1.7 ± 0.3 <0.512 <0.512 0.7 ± 0.2
0.5
DBahA 0.5 ± 0.1 <0.413 <0.413 0.5 ± 0.2 <0.413 <0.413 1.4 ± <0.413 n.d. 1.2 ± 0.2 <0.413 1.9 ± 0.4 <0.413 1.2 ± 0.3 0.5 ± 0.1 n.d. 0.5 ± 0.2
0.3
BghiP n.d. 0.4 ± 0.2 n.d. 0.5 ± 0.2 0.4 ± 0.1 <0.346 0.8 ± n.d. n.d. 0.7 ± 0.3 <0.346 1.3 ± 0.3 0.4 ± 0.1 <0.346 <0.346 n.d. <0.346
0.3
PAH4 2.6 ± 0.4 3.3 ± 0.6 2.0 ± 0.4 2.9 ± 0.6 3.1 ± 0.4 2.8 ± 0.5 12.5 ± 2.8 ± 0.4 2.1 ± 0.3 3.2 ± 0.5 3.0 ± 0.6 13.8 ± 2.4 2.8 ± 0.4 3.1 ± 0.4 2.9 ± 0.6 2.2 ± 0.4 4.4 ± 0.7
1.9
Total 43.5 ± 39.2 ± 37.4 ± 43.3 ± 43.0 ± 46.4 ± 98.7 ± 38.2 ± 40.5 ± 44.3 ± 42.1 ± 113.1 ± 59.2 ± 38.6 ± 40.8 ± 33.1 ± 30.8 ±
4.8 4.9 4.5 4.6 5.1 5.5 9.1 4.7 4.2 5.0 3.9 10.6 6.3 4.5 5.5 4.3 3.7

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R.C. Racovita et al. Food Control 121 (2021) 107586

Table 4
Concentrations of individual PAHs, the PAH4 group, and total PAHs in samples of smoked cheese denoted by producer letter-product number code. Values represent
averages ± standard deviations (n = 3), in ng PAH per g fresh weight of smoked cheese. Average values below the limit of quantification are reported as “< LOQ”. If
below the limit of detection, “n.d.” (i.e. not detected) is reported.
PAH A-1 B-1 C-1 D-1 E− 1 F-1 G-1 G-2 H-1 H-2 I-1

N 2.51 ± 0.47 2.64 ± 0.36 2.13 ± 3.02 ± 0.41 2.38 ± 1.71 ± 2.05 ± 4.16 ± 0.60 4.29 ± 0.71 4.54 ± 0.68 4.87 ± 0.82
0.33 0.29 0.23 0.26
ANe 0.23 ± 0.05 0.28 ± 0.08 0.21 ± 0.36 ± 0.10 0.24 ± 0.22 ± 0.18 ± 0.43 ± 0.09 0.43 ± 0.08 0.37 ± 0.06 0.47 ± 0.12
0.04 0.05 0.04 0.05
Any 0.33 ± 0.06 0.34 ± 0.06 <0.090 0.49 ± 0.13 0.37 ± 0.41 ± 0.23 ± 0.72 ± 0.16 0.88 ± 0.26 0.53 ± 0.15 1.02 ± 0.28
0.03 0.08 0.05
F 1.26 ± 0.28 1.44 ± 0.31 1.03 ± 1.59 ± 0.34 0.99 ± 1.08 ± 0.84 ± 2.06 ± 0.38 1.98 ± 0.32 1.66 ± 0.31 2.47 ± 0.44
0.20 0.27 0.22 0.17
PA 3.31 ± 0.57 3.42 ± 0.61 2.86 ± 3.53 ± 0.73 2.09 ± 3.16 ± 2.78 ± 5.94 ± 1.05 4.19 ± 0.84 4.61 ± 0.59 6.43 ± 0.96
0.39 0.26 0.40 0.33
A 0.64 ± 0.22 0.72 ± 0.19 0.58 ± 0.69 ± 0.24 0.33 ± 0.87 ± 0.59 ± 1.53 ± 0.42 1.46 ± 0.42 1.76 ± 0.53 2.11 ± 0.51
0.17 0.13 0.24 0.20
FA 0.87 ± 0.26 0.63 ± 0.17 0.53 ± 0.75 ± 0.16 0.41 ± 0.68 ± 0.54 ± 1.26 ± 0.37 1.17 ± 0.32 1.66 ± 0.40 2.26 ± 0.46
0.11 0.08 0.19 0.13
P 0.68 ± 0.13 0.31 ± 0.08 0.37 ± 0.59 ± 0.14 0.26 ± 0.55 ± 0.46 ± 0.86 ± 0.28 0.88 ± 0.25 0.96 ± 0.23 1.41 ± 0.42
0.08 0.07 0.12 0.11
BaA 0.63 ± 0.18 0.44 ± 0.12 0.37 ± 0.41 ± 0.11 0.42 ± 0.34 ± 0.52 ± 0.94 ± 0.20 1.04 ± 0.23 0.88 ± 0.21 1.12 ± 0.26
0.09 0.08 0.07 0.11
CHR 0.56 ± 0.14 0.29 ± 0.06 0.41 ± 0.32 ± 0.06 0.28 ± 0.31 ± 0.35 ± 0.66 ± 0.17 0.76 ± 0.19 0.81 ± 0.17 0.84 ± 0.22
0.11 0.06 0.10 0.08
BbFA n.d. n.d. n.d. <0.195 n.d. n.d. n.d. <0.195 0.25 ± 0.05 <0.195 0.32 ± 0.10
BkFA n.d. n.d. n.d. n.d. n.d. n.d. n.d. <0.202 <0.202 <0.202 0.21 ± 0.06
BaP 0.32 ± 0.07 n.d. <0.275 0.43 ± 0.12 0.33 ± <0.275 n.d. 0.42 ± 0.17 0.34 ± 0.16 <0.275 0.41 ± 0.11
0.09
IP <0.512 n.d. n.d. <0.512 <0.512 n.d. n.d. <0.512 <0.512 <0.512 <0.512
DBahA <0.413 n.d. n.d. <0.413 n.d. n.d. n.d. <0.413 <0.413 n.d. <0.413
BghiP <0.346 n.d. <0.346 <0.346 <0.346 n.d. n.d. <0.346 <0.346 <0.346 <0.346
PAH4 1.52 ± 0.31 0.73 ± 0.22 0.91 ± 1.23 ± 0.35 1.04 ± 0.82 ± 0.87 ± 2.11 ± 0.43 2.40 ± 0.47 1.93 ± 0.28 2.69 ± 0.34
0.24 0.17 0.26 0.21
Total 11.93 ± 10.51 ± 9.04 ± 12.90 ± 8.62 ± 9.47 ± 8.54 ± 19.92 ± 18.77 ± 18.53 ± 24.59 ±
1.24 1.06 0.83 1.33 0.71 0.85 0.76 2.03 1.90 1.75 2.29

by the modified Horwitz equation (Thompson, 2000). 3.2. PAH levels in smoked fish
Table 2 outlines analyte characteristics under the experimental
conditions described in sections 2.3-2.4. Linear ranges for the 16 ana­ The concentrations of the 16 PAHs are reported for the 17 types of
lytes spanned 3 to 4 orders of magnitude. The narrower linear ranges retail smoked fish in Table 3, along with the sum of concentrations of the
(10− 3 – 1 μg/mL) were obtained for N, ANe, ANy, and BbFA, as well as EC-regulated PAH4 group and the total sum of all PAH concentrations.
for A (10− 4 – 10− 1 μg/mL). For all remaining PAHs, the linear range was Most importantly, none of the smoked fish samples exhibited a BaP
one order of magnitude wider, i.e. 10-4 – 1 μg/mL. Sensitivities for all 16 concentration or PAH4 cumulative concentration above the limits set by
analytes were very high, in the order of 105–106 mL/μg. Consequently, the EC Regulation No. 835/2011, which suggests that producers are
the limits of detection and quantification were quite low, the former probably carefully monitoring PAH levels during their production pro­
being typically below 0.1 ng/g, except for the heavier and late-eluting cesses. It should be noted here that, while for the vast majority of
IP, DBahA, and BghiP, which had LODs slightly above 0.1 ng/g. smoked fish assortments a BaP limit of 2 ng/g and a PAH4 limit of 12 ng/
Average recoveries were greater than 45% for all analytes, again with g have been imposed EU-wide effective September 1, 2014, in the case of
the exception of high molecular weight IP and DBahA, which had smoked sprat higher limits of 5 ng/g BaP and 30 ng/g PAH4 have been
somewhat lower recoveries, in the 35–40% range. Most importantly, defined by the same regulation (European Commission, 2011a), such
recoveries of BaA, CHR, BbFA, and BaP were all within the 50–120% that samples 2-d and 3-d were also within legal limits (Table 3).
range, LODs were all below 0.3 ng/g and LOQs below 0.9 ng/g, as Nevertheless, these two smoked sprat samples showed not only the
stipulated by EC Regulation No. 836/2011 (European Commission, highest levels of PAH4 and BaP in particular, but also the highest total
2011b). Also, in agreement with the same regulation, all Horwitz ratios PAH content per gram of as-sold smoked fish (fresh weight). Some dif­
HORRATR were found to be well below the value of 2, which is ferences were noted between PAH concentrations of different types of
consistent with a satisfactory method precision (European Commission, smoked fish from the same producer, as well as of the same kind of fish
2011b). prepared by different producers, the latter resulting likely from different
As briefly mentioned before, in the smoked fish category, 17 prod­ technological conditions of the industrial smoking process (Due­
ucts from four different producers were investigated, including four dahl-Olesen, Christensen, Højgård, Granby, & Timm-Heinrich, 2010;
kinds each of smoked herring, mackerel, and salmon fillets, two kinds of Hokkanen et al., 2018; Mihalca et al., 2011; Wretling et al., 2010;
smoked sprat, and one each of smoked cod, tuna, and trout fillets Racovita et al., 2020). Total PAH concentration was similar for all fish
(Table 1). In terms of smoked cheeses, the selection comprised 11 va­ types in the case of producers 1–3, but, in the case of producer 4, samples
rieties from nine producers, namely six types of smoked pressed cheese, of tuna and trout fillets (4-d and 4-e) had a lower total PAH content than
three types of “Brădet” pressed cheese, one braided cheese, and one mackerel and salmon fillets (4-b and 4-c), while smoked herring fillets
“Brădet” braided cheese, which is a type of cheese that originated from a (4-a) exhibited by far the highest total concentration. Increases of PAH
small village in Argeş county, Romania, but is nowadays produced on an concentrations between closely related food samples that had been
industrial scale by several national producers and commercially avail­ subjected to identical smoking protocols were correlated with simulta­
able nation-wide. neous increases of sample fat content by some previously published
studies (Pöhlmann, Hitzel, Schwägele, Speer, & Jira, 2013; Simko, 2005;

6
R.C. Racovita et al. Food Control 121 (2021) 107586

Zabik, Booren, Zabik, Welch, & Humphrey, 1996). This could account Table 5
for the observed variation where high fat fish fillets, such as mackerel BaP toxic equivalent concentrations of PAH4 (TEQBaP,PAH4) and of all 16 PAHs
and salmon, showed higher total PAH contents than low fat fish fillets, (TEQBaP,PAH16), chronic daily intakes of PAH4 (CDIPAH4) and of all 16 PAHs
like tuna and trout, but does not explain why herring had even higher (CDIPAH16), margins of exposure (MOE), and incremental lifetime cancer risk
total PAH level in spite of lower fat content in comparison to mackerel, (ILCR) values for the 17 types of smoked fish investigated. Values given repre­
sent averages ± standard deviations (n = 3).
nor the disproportionately higher total PAH, PAH4, and BaP concen­
trations of smoked sprat samples from producers 2 and 3, which had fat Product TEQBaP, TEQBaP, CDIPAH4 CDIPAH16 MOE x ILCR x
code (ng/kg/ (ng/kg/ 10− 6 106
contents only higher to those of trout, tuna, and cod. However, these PAH4 PAH16
(ng/g) (ng/g) day) day)
discussions are limited to the situation where smoking conditions were
the same for all fish types, which is not known for retail products with 1-a 0.39 ± 2.96 ± 0.07 ± 0.50 ± 5.14 3.64
0.10 0.51 0.02 0.09 ± 1.36 ± 0.63
very limited information on their commercial label. Herring fillets from
1-b 0.70 ± 0.83 ± 0.12 ± 0.14 ± 2.90 1.01
producer 4 (4-a) were also considerably richer in total PAHs when 0.10 0.10 0.02 0.02 ± 0.42 ± 0.13
compared to the other three producers (1-a/2-a/3-a), while no sub­ 1-c 0.56 ± 0.61 ± 0.09 ± 0.10 ± 3.62 0.75
stantial differences were noted for either mackerel (1-b/2-b/3-b/4-b) or 0.20 0.20 0.02 0.03 ± 1.30 ± 0.25
2-a 0.42 ± 3.00 ± 0.07 ± 0.50 ± 4.76 3.68
salmon (1-c/2-c/2-e/4-c). No major differences were noted across
0.10 1.01 0.02 0.17 ± 1.15 ± 1.23
different producers for BaP or PAH4 concentrations for a given fish 2-b 0.78 ± 0.92 ± 0.13 ± 0.15 ± 2.58 1.13
species. However, when comparing different species, the average con­ 0.20 0.20 0.03 0.03 ± 0.66 ± 0.25
centration of PAH4 was, as mentioned, highest for sprat (2-d/3-d), 2-c 0.58 ± 0.65 ± 0.10 ± 0.11 ± 3.51 0.79
seconded by trout (4-e), followed by mackerel (1-b/2-b/3-b/4-b) and 0.30 0.30 0.05 0.05 ± 1.83 ± 0.37
2-d 2.65 ± 10.16 ± 0.45 ± 1.71 ± 0.76 12.46
cod (3-c), then herring (1-a/2-a/3-a/4-a) and salmon (1-c/2-c/2-e/4-c),
0.51 1.58 0.09 0.27 ± 0.15 ± 1.94
while the lowest was found for tuna (4-d). Sprat samples aside, the other 2-e 0.10 ± 0.15 ± 0.017 ± 0.026 ± 20.44 0.19
important marker, individual BaP concentration appeared less variable 0.03 0.03 0.005 0.005 ± 6.28 ± 0.04
across fish types, although one group of samples (comprising mackerel, 3-a 0.07 ± 0.14 ± 0.011 ± 0.023 ± 30.21 0.17
0.01 0.02 0.002 0.003 ± 4.71 ± 0.02
salmon, and cod) showed consistently higher BaP content than a second
3-b 1.18 ± 7.44 ± 0.20 ± 1.25 ± 1.71 9.12
group (herring, tuna, and trout). In terms of the composition of the PAH 0.30 1.05 0.05 0.18 ± 0.44 ± 1.28
mixture, three of the tricyclic aromatics (ANy, F, and PA), taken together 3-c 1.07 ± 1.13 ± 0.18 ± 0.19 ± 1.88 1.39
always accounted for more than half of total PAHs, regardless of fish 0.30 0.30 0.05 0.05 ± 0.53 ± 0.37
species or producer. For samples of smoked herring, salmon, tuna, and 3-d 3.42 ± 13.54 ± 0.58 ± 2.27 ± 0.59 16.60
0.51 2.06 0.09 0.35 ± 0.09 ± 2.53
trout, PA was the most abundant PAH, while for sprat, mackerel, and
4-a 0.48 ± 0.58 ± 0.08 ± 0.10 ± 4.23 0.72
cod, ANy had the highest abundance of all PAHs quantified. The only 0.10 0.10 0.02 0.02 ± 0.90 ± 0.13
exception were the samples of smoked salmon from producer 2 (2-c and 4-b 1.03 ± 7.31 ± 0.17 ± 1.23 ± 1.96 8.96
2-e), where fluorene surpassed both PA and ANy. Bicyclic naphthalene 0.20 1.51 0.03 0.25 ± 0.39 ± 1.86
4-c 0.80 ± 3.36 ± 0.13 ± 0.56 ± 2.52 4.12
was typically fourth most abundant, except for herring samples
0.20 0.54 0.03 0.03 ± 0.64 ± 0.66
(1-a/2-a/3-a/4-a) where it surpassed fluorene and most of the mackerel 4-d 0.58 ± 0.63 ± 0.10 ± 0.11 ± 3.49 0.77
samples (1-b/3-b/4-b) in which it was surpassed by anthracene. 0.30 0.30 0.05 0.05 ± 1.81 ± 0.37
4-e 0.72 ± 3.33 ± 0.12 ± 0.56 ± 2.82 4.08
3.3. PAH levels in smoked cheeses 0.21 1.02 0.03 0.17 ± 0.81 ± 1.25

As mentioned before, EC Regulation No. 835/2007 does not set carcinogenicity of all PAHs found in a foodstuff in terms of the BaP toxic
explicit BaP and PAH4 limits for smoked cheeses. In any case, levels of equivalent concentration (TEQBaP) and to assess exposure in the form of
all PAHs were found to be much lower in retail smoked cheeses on the chronic daily intake (CDI). These parameters were computed in two
Romanian market in comparison to smoked fish (Table 4). Cumulative ways: using strictly the PAH4 contributors (TEQBaP,PAH4 and CDIPAH4),
PAH4 concentration had a very narrow range across different producers, as well as using all 16 PAH contributors (TEQBaP,PAH16 and CDIPAH16). If
from 0.73 to 2.69 ng per gram of as-sold smoked cheese, while BaP a certain PAH was found below its limit of quantitation in a given
concentration was found to be consistently below 0.5 ng/g or even not sample, the contribution of that PAH was considered zero when calcu­
quantifiable in half of the samples analyzed, thus raising no concerns. lating the TEQBaP value for that sample. For risk characterization, since
Total PAH concentrations were also found to be about 3–4 times lower there is no BMDL10 reference value available in the literature that en­
than for most fish samples, at roughly 10 ng/g in the case of ordinary compasses all 16 PAHs, CDIPAH4 was used to calculate margins of
pressed cheeses and around 20 ng/g in the case of “Brădet” pressed exposure (MOE); however, the more comprehensive CDIPAH16 was used
cheeses. The latter higher concentrations could potentially be due, in when computing incremental lifetime cancer risk (ILCR) values. The
part, to higher fat contents of “Brădet” cheeses (44–45%) in comparison results obtained are presented in Tables 5 and 6.
to the ordinary pressed or braided cheeses (20–30%). More likely, TEQBaP,PAH4 were generally below 2 ng/g, which is the EC limit for
however, they are due to the fact that smoking procedures of “Brădet” BaP in smoked fish, among other foodstuffs (European Commission,
pressed cheeses demand the use of fir wood and resin for generating 2011a). Non-surprisingly, the only two exceptions were the smoked
smoke, which was shown to yield the highest PAH accumulation levels sprat samples 2-d and 3-d. When considering the equivalent carcino­
in a past study using “Diavoletto” Italian cheese (Pagliuca et al., 2003). genicity of all PAHs, eight smoked fish samples exhibited values TEQBaP,
Irrespective of cheese type or producer, the sum of PA, N, and F con­
PAH16 greater than 2, which included, in addition to the two smoked
centrations accounted for more than half of total PAH content. PA was sprat samples 2-d and 3-d, two smoked herring samples 1-a and 2-a, two
most abundant, followed by N and then by F, for nine of the eleven types smoked mackerel samples 3-b and 4-b, one smoked salmon sample 4-c,
of smoked cheese investigated, while for the remaining two (pressed and the smoked trout sample 4-e (Table 5).
cheese E− 1 and “Brădet” braided cheese H-1) the concentrations of N More relevant than TEQBaP values themselves are the MOE and ILCR
and PA were almost equal and were also followed by that of F. parameters. Using the EFSA-recommended MOE factor, all cheese
samples and almost all fish samples could be classified as causing
3.4. Exposure assessment and risk characterization negligible concern (MOE>106) (Tables 5 and 6). Once again, the only
two exceptions were the smoked sprat samples 2-d and 3-d with MOEs in
As already mentioned, it is customary to ascertain the total

7
R.C. Racovita et al. Food Control 121 (2021) 107586

Table 6 restricted by EC Regulation No. 835/2011 were, in all instances, found

BaP toxic equivalent concentrations of PAH4 (TEQBaP,PAH4) and of all 16 PAHs to be well below the regulated limits for the corresponding product
(TEQBaP,PAH16), chronic daily intakes of PAH4 (CDIPAH4) and of all 16 PAHs categories.
(CDIPAH16), margins of exposure (MOE), and incremental lifetime cancer risk The 11 kinds of smoked cheeses investigated originated from nine
(ILCR) values for the 11 types of smoked cheeses investigated. Values given different producers and included six types of pressed cheese, one braided
represent averages ± standard deviations (n = 3).
cheese and four types of “Brădet” cheese, a high fat pressed cheese very
Product TEQBaP, TEQBaP, CDIPAH4 CDIPAH16 MOE x ILCR appreciated by consumers. PAH concentrations did not vary much be­
code (ng/kg/ (ng/kg/ 10− 7 x 107
PAH4 PAH16
tween cheeses of the same type marketed by different producers, but
(ng/g) (ng/g) day) day)
“Brădet” smoked cheeses consistently showed about twice higher levels
A-1 0.389 ± 0.404 ± 0.0248 ± 0.0258 ± 1.37 1.88 of all PAHs in comparison to the other varieties.
0.072 0.072 0.0046 0.0046 ± 0.26
None of the smoked fish and cheese samples posed significant cancer
±
0.34
B-1 0.047 ± 0.063 ± 0.0030 ± 0.0040 ± 11.36 0.29 risk as assessed by margin of exposure (MOE) and incremental lifetime
0.012 0.012 0.0008 0.0008 ± 2.91 ± cancer risk (ILCR) calculations.
0.06 To the best of our knowledge, this is the first report on PAH
C-1 0.041 ± 0.054 ± 0.0026 ± 0.0034 ± 12.97 0.25
contamination levels of Romanian smoked cheeses.
0.009 0.009 0.0006 0.0006 ± 2.86 ±
0.04
D-1 0.474 ± 0.491 ± 0.0302 ± 0.0313 ± 1.12 2.29 Funding sources
0.121 1.121 0.0077 0.0077 ± 0.29 ±
0.56
This work was supported by a grant of Ministry of Research and
E-1 0.375 ± 0.385 ± 0.0239 ± 0.0245 ± 1.42 1.79
0.090 0.090 0.0058 0.0058 ± 0.34 ± Innovation, CNCS - UEFISCDI, project number PN–III–P1-1.1-PD-2016-
0.42 0934, within PNCDI III.
F-1 0.037 ± 0.054 ± 0.0024 ± 0.0034 ± 14.37 0.25
0.007 0.007 0.0005 0.0005 ± 2.74 ±
0.03
CRediT authorship contribution statement
G-1 0.056 ± 0.068 ± 0.0035 ± 0.0044 ± 9.60 0.32
0.011 0.011 0.0007 0.0007 ± 1.91 ± Radu C. Racovita: Investigation, Conceptualization, Methodology,
0.05 Formal analysis, Data curation, Writing, Project administration, Funding
G-2 0.521 ± 0.551 ± 0.0332 ± 0.0352 ± 1.02 2.57
acquisition, Resources. Catinca Secuianu: Conceptualization, Meth­
0.171 0.171 0.0109 0.0109 ± 0.34 ±
0.80 odology, Validation, Formal analysis, Visualization, Resources, Super­
H-1 0.477 ± 0.505 ± 0.0304 ± 0.0322 ± 1.12 2.35 vision. Florentina Israel-Roming: Supervision.
0.162 0.162 0.0103 0.0103 ± 0.38 ±
0.75
H-2 0.096 ± 0.128 ± 0.0061 ± 0.0082 ± 5.55 0.60 Declaration of competing interest
0.021 0.022 0.0013 0.0014 ± 1.22 ±
0.10
I-1 0.562 ± 0.623 ± 0.0359 ± 0.0398 ± 0.95 2.90 The authors declare that they have no known competing financial
0.113 0.114 0.0072 0.0073 ± 0.19 ± interests or personal relationships that could have appeared to influence
0.53 the work reported in this paper.

the 105–106 interval, which also implies negligible concern when action Acknowledgements
is taken to minimize future exposure (Table 5).
All the cheese samples posed no risk on the basis of the US Envi­ The authors would like to thank Dr. Maria Mihaly (University
ronmental Protection Agency’s ILCR factor as well (Table 6). On the “Politehnica” of Bucharest) for access to instrumentation, certified
contrary, when inspecting the ILCR values calculated, the majority of reference materials provided, and many fruitful discussions.
smoked fish samples placed in the 10− 6 – 10− 4 range, thus being char­
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