3.2. Identification of Potential Migrants in Packaging Materials
With the aim of identifying potential migrants from the food packaging materials, a non-targeted analysis was performed using GC-MS. Acetonitrile extracts from packaging samples were analyzed.
More than 90 compounds were detected, and NIST/EPA/NIH 11 Mass spectral library (version 2.0) and Wiley RegistryTM 8th (UK) were used for the identification. Moreover, 21 compounds were confirmed by comparison with standards, and the remaining peaks were considered to be tentatively identified.
An in silico model, specifically Toxtree v2.6.13 (Ideaconsult Ltd., Sofia, Bulgaria) software, was used to predict the toxicity of the identified substances. This tool classified the molecules into three classes of toxicity (low, intermediate, high) according to their chemical structure.
Both confirmed and tentatively identified compounds are listed in Table 2
. Considering that, in general, a direct matching factor (SI) and a reverse search matching factor (RSI) of 900 or greater is an excellent match, and 800–900 is a good match, solely compounds with matching factors SI and RSI higher than 800 were included.
Many substances of different chemical nature, including aldehydes, ketones, carboxylic acids, alcohols, and so on, were tentatively identified in the analyzed samples. 2-Heptanone was found only in one sample (LE01), and this compound has been identified in paper and board samples [15
]. Benzaldehyde and benzoic acid are included in the European Union positive list of substances authorized in plastic materials [5
]. They were found in eight (LE01, LE02, LS01, LS02, QF02_P, YN01_P/L, YS01_P/L, FN03_P/L) and three samples (YN01_P/L, YS01_P/L, FN02_P), respectively. Other compounds identified were 4-methyl-2-heptanone, phenylacetaldehyde, 2-nonanone, and 1-dodecanol.
Four diisocyanates, namely 2,6-toluene diisocyanate, 2,4-toluene diisocyanate, isophorone diisocyanate, and 4,4′-diphenylmethane diisocyanate, were detected in several samples, including cheese (QS01_L, QL01_E, QF_02L), yogurt (YN01_L, YS01_L), and dairy desserts (FN02_P, FN03_L) packaging. They are widely used to produce polyurethane adhesives. All of them are classified as III class according to Cramer rules. 1-(2-Methoxypropoxy)-2-propanol was found in two samples (LE02, LS02), and this compound has also been identified in polyurethane adhesives [16
Abietic acid and their derivatives, such as retene, dehydroabietal, methyl dehydroabietate, and dehydroabietic acid, were found in different samples. These compounds have been reported in hotmelt adhesives [17
]. Retene belongs to class III according to Cramer rules and was detected only in one sample (LE01); dehydroabietal was identified in five samples—LE01, LE02, LE03, LS01, and LS02, and methyl dehydroabietate was found in samples LE01, LE02, LE03, LS01, LS02, YN01_L, YS01_L, FN02_P, and both belong to class II. Dehydroabietic acid and abietic acid were detected in three samples, YN01_L, YS01_L, and FN03_L, and presented intermediate and high toxicity, respectively.
Styrene, a monomer widely used in the plastic industry, was identified in eight samples (LS01, LS02, QS01_I, QF02_P, YN01_P, YS01_P, FN01_L, FN03_P/L). 1,2-Diphenylpropane and 1,3-diphenylpropane were identified in samples YN01_P, YS01_P, QS01_I, QF02_P, YN01_P, YS01_P, FN03_P, respectively. These two compounds with high toxicity (Cramer class III) have been reported in polystyrene-based materials—1,2-diphenylpropane, a thermal degradation product of polystyrene, and 1,3-diphenylpropane, an isomer of the styrene dimers [18
]. A metabolite of styrene, specifically styrene-7,8-oxide, was detected in only one sample, QF02_P, and the in vitro studies have shown that this compound is carcinogenic [20
Caprolactam belongs to Cramer class III, and it was identified in eleven samples, comprising milk, cheese, yogurt, and dairy dessert packaging (LE01, LE02, LS01, LS02, QF01, QF02_L, YN01_P/L, YS01_L/P, FN01_P/L, FN02_P, FN03_P/L). It is employed as a monomer in the manufacture of polyamide, and it has been also identified as a residue from printing inks [21
]. A caprolactam cyclic dimer, 1,8-diazacyclotetradecane-2,9-dione, was identified in the sample QF01.
Bis(2-hydroxyethyl) terephthalate, a monomer intermediate in the synthesis of PET, was identified in one sample QS01_P [22
Phthalic anhydride was found in two samples of dairy dessert packaging (FN01_L, FN03_P); this substance is authorized as a monomer and additive in the manufacture of plastic materials. 1,1′-Oxydi-2-propanol was detected in milk packaging (LE01, LE02, LE03, LS01, and LS02); its use is also authorized as an additive and monomer in plastic materials. Both compounds have high toxicity (class III).
Butylated hydroxytoluene (BHT), a synthetic phenolic antioxidant, authorized as an additive in plastic food contact materials with an SML of 3 mg/kg was identified in 13 samples (LE01, LE02, LE03, LS01, LS02, QS01_L/P/I, QL01_I, QF02_L/P, YN01_L, YS01_L, FN01_L, FN02_P/L, FN03_P/L). In addition to its use as a polymeric additive, it is employed as a food additive. In three of the eleven samples, specifically QS01_L, QL01_I, and FN02_L, a metabolite of BHT, namely 2,6-di-tert-butyl-4-methylene-2,5-cyclohexadienone, was detected. 3,5-Di-tert-butyl-4-hydroxybenzaldehyde, another derived metabolite of BHT, was only detected in one sample (FN02_L). The antioxidants, as well as their degradation products or metabolites, have attracted particular attention owing to their potential negative effects as a result of their toxicity [23
Several compounds that could be considered non-intentionally added substances (NIAS) have been identified in the analyzed samples. For example, 2,4-di-tert-butylphenol, a degradation product of the antioxidants Irgafos®
168 and Irganox®
1010, was detected in eleven samples (LE01, LE03, LS01, LS02, QL01_E, QF01, YN01_P/L, YS01_P/L, FN01_P, FN02_L, FN03_L), and 2,6di-tert-butyl-p-benzoquinone, which has also been reported as a degradation product of the antioxidants Irganox 1010, Irgafos 168, and Irganox PS 802, was found in eight samples (LE03, LS02, QS01_L, QF02_L/P, YN01_P/L, YS01_P/L, FN01_P/L, FN03_P/L) [24
]. Both compounds present different toxicity; while the first is classified as class I (low toxicity), the second one is classified as class II (intermediate toxicity). Another degradation product of the antioxidant Irganox®
1010, 7,9-di-tert-butyl-1-oxaspiro[4.5]deca-6,9-diene-2,8-dione, was found in all samples (LE01, LE02, LE03, LS01, LS02, QS01_L, QL01_E, QF01, QF02_L/P, YN01_P/L, YS01_P/L, FN01_P, FN02_L, FN03_P/L), and this compound is classified in class III according to Cramer rules.
3,6,9,12,15-Oxabicyclo(15,3)heneicosa-1(21),17,19-triene-2,16-dione was only found in one sample (FN01_L); this substance has been reported as an antioxidant degradant and belongs to class III according to Cramer rules. Diphenylmethane, which has been described as a monomer degradant, was detected in three samples (YN01_L, YS01_L, FN03_ L) and similarly presents high toxicity [24
In two samples of packaging cheese (QS01_I, QF02_P) and in one sample corresponding to the packaging of dairy desserts (FN03_P), trans-1,2-diphenylcyclobutane was identified. In a study conducted by Lago and Ackerman [24
], this substance has been reported as a monomer byproduct.
Diethylene glycol monoethyl ether was found in milk packaging samples (LE01, LE02, LE03, LS01, LS02); this compound has been described by Bentayeb et al. [25
] in a study in which the authors investigated the set-off phenomenon of photoinitiators in food packaging materials by using direct analysis in real-time coupled to time-of-flight mass spectrometry (DART/TOF-MS). It was tentatively identified as a set-off compound other than photoinitiators.
One of the other print-related compounds identified in some of the samples analyzed (QF01_P, YN01_P, YS01_P, and FN03_P) was 1,1-diphenylethylene, which has been classified as high toxicity substance [24
Benzophenone was detected in six samples—LE01, LE02, LE03, LS01, LS02, and QF02_L/P; this compound has also high toxicity and belongs to class III according to Cramer rules.
Several phthalates, including diethyl phthalate (DEP) (LE01, LE02, LE03, LS01, LS02, QS01_-/P/I, QF02_L/P, YN01_P/L, YS01_P/L, FN01_P/L, FN02_L, FN03_P/L), diisobutyl phthalate (DIBP) (LE01, LE02, LE03, LS01, LS02, QF02_P, YN01_P/L, YS01_P/L, FN01_P/L, FN02_P/L, FN03_P/L), dibutyl phthalate (DBP) (LE01, LE02, LE03, LS01, LS02, YN01_L, YS01_L), and bis(2-ethylhexyl) phthalate (DEHP) (LE03, LS01, QS01_P, QL01_E, QF01, QF02_L YN01_L, YS01_L, FN01_P/L, FN02_P/L, FN03_L), among others, were found in different samples. These compounds, besides plasticizers, can be found in printing inks formulations and also have been employed as solvents to hold color [3
Others common plasticizers, such as acetyltributyl citrate (ATBC) (QS01_L, QL01_E/I, QF01, QF02_L, YN01_P/L, YS01_P/L, FN01_L, FN02_P/L, FN03_L) and diethylhexyl adipate (DEHA) (LE01, LS01, LS02, LE03, YN01_L, YS01_L, FN03_P/L), were detected in different material samples; these substances are authorized as an additive in plastic food contact materials. 2-Ethyl-1-hexanol, the alcoholic component of DEHA, was identified in the sample FN03_P [27
Squalene was detected in eleven substances (LE01, LE02, LS01, LS02, QS01_P/I, QL01_I, QF01, QF02_L/P, YN01_P/L, YS01_P/L, FN01_P/L, FN02_P/L, FN03_P/L). This compound chemically is a hydrocarbon. One of its uses is as a plasticizer. On the other hand, triacetin was identified in three samples (QL01_E, YN01_L, YS01_L), besides its use as a food additive; this compound has been described as an eco-friendly plasticizer [28
Lubricants, such as isopropyl laurate and glyceryl tricaprylate, were found in samples FN02_L and QS01_P, respectively.
Two slip agents—erucamide and hexadecanamide—were identified in six (QS01_L/P, QL01_E/I, QF01, FN01_P, FN02_P/L, FN03_P/L) and seven samples (QS01_P, QL01_E, YN01_L, YS01_L, FN01_P, FN02_L, FN03_L), respectively; both compounds are classified in class III according to Cramer rules.
Other compounds classified in class III (high toxicity), including 2,5-dimethyl-2,5-hexanediol, 1,1,3-trimethyl-3-phenylindan, 2,3-dimethyl-2,3-diphenylbutane, 1-phenylnaphthalene, and (1-methyl-2,2-diphenylcyclopropyl) sulfanylbenzene, were identified in several samples.
3.4. Migrants Concentration in Food and Dietary Exposure Estimation
Foods were grouped in three pools for each subgroup of the population, namely 12–35 months, 3–9 years, and 10–17 years, and they were prepared, as described above. The concentrations of the analytes studied in the food composite samples are presented in Table 4
. Samples were analyzed in duplicate (average ± SD). All of the phthalates studied, that is, DEP, DIBP, DBP, and DEHP, were found in the three pools. In general, the pool that corresponds to the adolescent group was the one that presented the highest values ranging from 0.0317 to 0.1627 µg/g. Among the phthalates, DBP, followed by DEHP, were found at higher concentrations. Van Holderbeke et al. [32
] reported DEHP as the most frequent phthalate in the milk and dairy products group, and moreover, in another work conducted by the same authors [7
], it was found that this food group was the one that contributed the most to the exposure to DBP.
However, the other analytes studied—BP, 1,3-DPP, and DEHT—were found below LOQ in all composite food analyzed samples.
The concentrations of phthalates reported in this work were generally higher than those described by Sakhi et al. [2
] in Norwegian foods and beverages, except in the case of DEHP, in which they found higher values in different samples of cheese. Van Holderbeke et al. [32
] also found higher levels of DEHP in milk and dairy products sold in the Belgian market.
Nevertheless, Jia et al. [31
] reported lower concentrations for DEP (13 µg/kg) and DEHP (57 and 42 µg/kg) in milk and yogurt samples.
Values reported in a TDS carried out in Canada (2013) were lower than those found in our study for DEP, DIBP, and DBP, whereas in the case of DEHP, they found higher concentrations in some of the dairy products [30
Cariou et al. [33
] used GC-MS to determine four phthalates (DIBP, DBP, BBzP, and DEHP) in different food items. The concentrations determined in whole milk were <2.7 ng/g for DIBP, 0.5 ng/g for DBP, and 21.8 ng/g for DEHP, and in concentrated milk samples, the values were 2.9, 0.4, and 25.5 ng/g for DIBP, DBP, and DEHP, respectively. These values were also lower than those reported in the present study.
Exposure to contaminants through the diet is one of the essential elements in risk evaluations. The exposure to the selected chemicals previously identified in the packaging materials was investigated. The dietary exposure (mean and 95th percentile) for the different age groups is summarized in Table 4
Migration from food packaging seems to be one important source of exposure to phthalates. Estimated mean exposure to DBP and DEHP was quite similar; values ranged from 2.42 µg/kg bw per day (pool 10–17 years) to 4.40 µg/kg bw per day (pool 12–35 months) for DBP and from 1.35 µg/kg bw per day (pool 10–17 years) to 4.07 µg/kg bw per day (pool 12–35 months) for DEHP. These values were higher than those calculated for DIBP and DEP. The lowest exposure was found for DEP, and the values varied between 0.472 µg/kg bw per day (pool 10-17 years) and 1.19 µg/kg bw per day (pool 12–35 months). The estimated 95th percentile exposure varied from 6.54 µg/kg bw per day (pool 10–17 years) to 21.3 µg/kg bw per day (pool 12–35 months) for DEHP and from 2.29 µg/kg bw per day (pool 10–17 years) to 6.24 µg/kg bw per day (pool 12–35 months) for DEP. It is interesting to note that, in general, the dietary exposure decreases with increasing age. This can be explained in part due to an increase in body weight with age. Sirot et al. [34
] observed a similar trend in a study on the exposure to acrylamide in the diet of the French population.
For certain phthalates, the EFSA has established tolerable daily intakes (TDI); for those of interest in this study, the TDIs were 0.01 mg/kg bw per day for DBP and 0.05 mg/kg bw per day for DEHP. In the case of DEP, the World Health Organization (WHO) [35
] specifies a TDI of 0.5 mg/kg bw per day (WHO, 2003). Recently, the EFSA Panel on Food Contact Materials, Enzymes, and Processing Aids (CEP Panel) [36
] at the request of the European Commission has updated the establishment of the risk of DBP, BBP, DEHP, DINP, and DIDP. In a draft update published, the CEP panel has re-confirmed the individual TDI derived in 2005 for all the phthalates but also proposes a group-TDI for DEHP, DBP, and BBP and establishes a value of 50 μg/kg bw per day, expressed as DEHP equivalents.
In examining our data (Table 4
) and considering the individual TDI, the estimated exposure for all age groups was below the TDI, except for DBP at the 95th percentile, which exceeded the TDI up to 2.3 times in the 12–35 months age group. In line with this result, Cirillo et al. [37
] studied neonatal exposure to phthalates and bisphenol A and also found that the daily intake of DBP exceeded TDI up to 175%.
In a study carried out by Sakhi et al. [2
], the estimated dietary exposure to phthalates in the Norwegian adult population was examined. The results revealed that DEHP and DiNP presented the highest mean estimated dietary exposure with values ranging from 396 to 436 ng/kg bw/day and from 477 to 494 ng/kg bw/day for DEHP and DiNP, respectively.
Fierens et al. [7
] used a semi-probabilistic modeling approach for dietary phthalate exposure in the Belgian adult population. The phthalates considered in this study were DEP, DBP, BBP, and DEHP. Of all the phthalates investigated, DEHP showed the highest predicted exposure value (1.45 µg/kg bw/day). These results were lower than those reported by Sakhi et al. [2
] for the Norwegian population.
Dietary exposure to DEHP in the Chinese population was investigated by Sui et al. [38
]. The mean dietary exposure values found were 4.51, 3.41, 2.46, and 2.03 µg/kg bw per day for the 2–6 years, 7–12 years, 13–17 years, and ≥ 18 years, respectively. The average value for the 2–6 years group was twice higher than that determined in this work for the 3–9 years group. This difference could be attributed partly to an increase in body weight with age and also due to the food products included in the study of Sui et al. [38
Results from the UK TDS (total diet study) have shown that DEHP is the phthalate that provides the highest exposure, being dairy products, fish, and milk the main contributors for toddler (1.5–2.5 years) subgroup and meat and dairy products for toddler (3.5–4.5 years) subgroup. At 97.5th percentile, the values range from 5.7 to 9.9 µg/kg bw/day for toddlers, between 2.7 and 6.7 µg/kg bw/day for young people, and from 3.4 to 4 µg/kg bw/day for adults [8
Phthalate intake in the Belgian preschool children population was investigated by Sioen et al. [39
]. The dietary exposure was calculated by using the Monte Carlo risk assessment program (MCRA); the authors found that the highest intakes corresponded to DEHP, followed by DiBP and DBP.
In the present study, if we take into account the exposure to DEHP, DBP, and DiBP, a total mean dietary exposure due to the sum of these three phthalates was estimated to be 4.0 µg/kg bw/day (pool 10–17 years), 4.6 µg/kg bw/day (pool 3–9 years), and 6.9 µg/kg bw/day (pool 12–35 months). In all pools, a group-TDI lower than 50 µg/kg bw/day was determined [36
Other substances identified in packaging materials, specifically BP, 1,3-DPP, and DEHT, were also considered in this study. Regarding BP, the average dietary exposure ranged from 0.744 µg/kg bw per day (pool 10–17 years) to 2.25 µg/kg bw per day (pool 12–35 months), in all cases, was lower than the TDI (0.03 mg/kg bw per day) specified by EFSA [6
]. The estimated dietary exposure to DEHT was also below TDI (1 mg/kg bw) in all age groups [40
]. For 1,3-DPP, the estimated dietary exposure ranged from 0.372 µg/kg bw per day (pool 10–17 years) to 1.13 µg/kg bw per day (pool 12–35 months).
Although, in general, low exposure data are obtained; however, it is important to consider that consumers are exposed to different chemicals through the diet from several sources; accordingly, there is combined exposure to multiple chemicals (cumulative exposure) and also exposure to a certain substance from several sources (aggregate exposure) [41
]. Therefore, it is necessary to take into account all these aspects to evaluate the possible effects arising from exposure to multiple chemicals in foods.