**Industrial Contaminants and Pesticides in Food Products**

Ruud Peters1, Henry Beeltje2 and Marc Houtzager2 *1RIKILT – Institute of Food Safety, 2TNO Built Environment and Geosciences, Environment, Health and Safety, Netherlands* 

#### **1. Introduction**

68 Pesticides in the Modern World - Risks and Benefits

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Feb. 19, 2007 www.dropdata.org/cocoa/index.htm (The World's Worst Cocoa Problems, March 2011) www.chocolate.gourmetreceipe.com (Chocolate and its In our modern world a large number of man-made chemicals are being used. As a consequence their widespread presence in the environment is becoming increasingly well documented (Vethaak et al, 2002; Peters et al., 2008). They are found in a vast range of consumer products and include plasticizers, emulsifiers, flame retardants, perfluorinated compounds, artificial musks and organotin compounds. While they have undoubtedly improved the quality of our lives, a consequence of their intensive use is a widespread presence in the environment. Human exposure to these compounds may be through contact with consumer products containing such chemicals as additives, but also through food products. Since many of these compounds have a lipophilic nature there is a potential for bio-accumulation through the food chain especially in products with a high fat content. This is reflected in the presence of persistent organic compounds such as organochlorine pesticides and polychlorinated biphenyls that can be found in food products although there use has been seized many years ago. Many of these compounds have also been found in human blood indicating that humans are exposed to these chemicals (CDC, 2001, 2003; Guenther et al., 2002). This exposure may be through different routes. One is the use of these chemicals as additives in consumer products such as carpets, curtains, toys and electronic equipment. The exposure of these chemicals in house dust indicates the potential for human exposure. Another route for human exposure is, of course, through food products. Since many of these compounds have a lipophilic nature, they can be bio-accumulated through the food chain especially in products with a high fat content. This study focused on the presence and concentrations of a number of typical man-made chemicals in food products that many of use daily. The chemicals considered in this study are: brominated flame retardants (BFR's), phthalates, artificial musks, alkylphenols (AP's), organochlorine pesticides (OCP's), polychlorobiphenyls (PCB's), organotin compounds (OT's) and perfluorinated compounds (PFC's).

#### **2. Methods and materials**

#### **2.1 Sampling and sample pre-treatment**

All samples, mostly fresh food products were purchased in regular shops in nine European countries including the Netherlands, the United Kingdom, Germany, Finland, Sweden,

Industrial Contaminants and Pesticides in Food Products 71

**Polychlorinated biphenyls (PCBs): Polybrominated diphenylethers (PBDEs):**  PCB-18 PCB-118 BDE-17 BDE-138 PCB-28/31 PCB-123 BDE-28 BDE-153 PCB-22 PCB-138/158 BDE-32 BDE-154 PCB-41 PCB-141 BDE-35 BDE-156 PCB-44 PCB-149 BDE-37 BDE-166 PCB-49 PCB-151 BDE-47 BDE-181 PCB-52 PCB-153/168 BDE-49/71 BDE-183 PCB-54 PCB-156 BDE-66 BDE-184 PCB-56/60 PCB-157 BDE-75 BDE-190 PCB-64 PCB-167 BDE-77 BDE-191 PCB-70 PCB-170 BDE-85 BDE-196 PCB-74 PCB-177 BDE-99 BDE-197 PCB-87 PCB-180 BDE-100 BDE-206 PCB-90 PCB-183 BDE-119 BDE-207 PCB-99 PCB-187 BDE-126 BDE-209

PCB-104 PCB-189 **Other brominated flame retardents:** 

PCB-110 PCB-199 tetrabromobisphenol-A (TBBPA) PCB-114 PCB-203 hexabromocyclododecane (HBCD)

**Perfluorinated chemicals (PFCs): Artificial musks:**  perfluoro-octanoic acid (PFOA) galaxolide (HHCB) perfluoro-octane sulphonate (PFOS) tonalide (AHTN) perfluoro-octane sulfonamide (PFOSA) musk xylene (MX) perfluoro-nonanoic acid (PFNA) musk ketone (MK)

perfluoro-dodecanoic acid (PFDoA) **Organotin compounds:** 

**Alkylphenols (AP):** di-butyltin (DBT)

nonylphenol isomers (NP) mono-octyltin (MOT) octylphenol isomers (OP) di-octyltin (DOT)

mono-butyltin (MBT)

tri-butyltin (TBT)

Table 1. Chemical parameters determined in this study including abbreviations used in the

**Organochlorine pesticides (OCPs): Phthalates:** 

o,p'-DDD p,p'-DDD o,p'-DDT p,p'-DDT

PCB-101 PCB-188

PCB-105 PCB-194

perfluoro-decanoic acid (PFDA) perfluoro-undecanoic acid (PFUnA)

text and result tables

perfluoro-tetradecanoic acid (PFTrA)

α-hexachlorohexane (α-HCH) di-methyl phthalate (DMP) β-hexachlorohexane (β-HCH) di-ethyl phthalate (DEP) γ-hexachlorohexane (γ-HCH) = lindane di-isobutyl phthalate (DiBP) hexachlorobenzene (HCB) di-butyl phthalate (DBP) α-chlordane benzylbutyl phthalate (BBP) β-chlordane di-(2-ethylhexyl) phthalate (DEHP) o,p'-DDE di-isononyl phthalate (DiNP) p,p'-DDE di-isodecyl phthalate (DiDP)

Spain, Poland, Italy, Estonia and Greece. Samples were sent to the laboratory where laboratory samples were prepared and stored at -18°C until analysis. In general, solid food samples were cut into small pieces and homogenised with a blender. If not the entire sample was used or homogenised, proportional sub-sampling was applied and the collected subsamples were homogenised. Milk was acidified with formic acid and the solid part containing the proteins and fat was separated from the liquid phase. Both parts were stored for analysis. Orange juice was centrifuged and vacuum filtrated and the solid and liquid parts were stored for analysis. A selected number of chemical parameters were determined in each sample, based on expectations and reports in the literature.

#### **2.2 Chemical parameters**

The chemical parameters determined in this study are listed in table 1, including the abbreviations that are used throughout the text and in the result tables. Note that not all parameters are determined in all samples.

#### **2.3 Analytical procedures**

For the determination of the OCP's, PCB's, BFR's, phthalates and artificial musks, a weight sub-sample of the homogenised laboratory sample was mixed with anhydrous sodium sulphate in a mortar and spiked with internal standards. The internal standards used were 13C-labelled standards for PCB's and BFR's, 2D-labelled standards for OCP's and phthalates, and a surrogate standard for the artificial musks. The samples were Soxhlet extracted for 16 hours using a mixture of 10% diethyl ether in hexane. For milk and orange juice a proportional amount of the liquid phase was pre-extracted with hexane and this hexane extract was used in the Soxhlet extraction of the solid part of these samples. Olive oil was directly diluted in hexane. One procedural blank, consisting of 40 g anhydrous sodium sulphate, was included in every batch of 10 samples. All extracts were concentrated to a volume of 50 ml and split into two equal parts of 25 ml. For the determination of the OCP's, PCB's and BFR's, one part of the extract was washed several times with sulphuric acid of increasing concentration to remove the major part of the lipids. The remaining extract was concentrated and purified over a glass chromatographic column packed with florisil and capped with anhydrous sodium sulphate to isolate the fraction containing the OCP's, PCB's, PBDE's and HBCD. The eluent was concentrated to a small volume and a syringe standard (1,2,3,4-tetrachloronaphthalene) was added. This final extract was analysed on an Agilent 6890 series gas chromatograph coupled to an Agilent 5973 mass spectrometer (GC/MS) and equipped with a HP-5-MS, 30 m × 0.25 mm (i.d.), film thickness 0.25 µm, fused silica capillary column. The mass spectrometer was operated in the selected ion monitoring mode and typically two or three characteristic ion masses were monitored for each analyte. The samples were analyzed for the following OCP's; α-, β- and γ-hexachlorohexane (HCH), hexachlorobenzene (HCB), α- and β-chlordane, o,p'-, p,p'-DDE, o,p'-, p,p'-DDD and o,p'-, p,p'-DDT: The following PCB congeners: 18, 28/31, 22, 41/64, 44, 49, 52, 54, 56/60, 70, 74, 87, 90/101, 99, 104, 105, 110, 114, 118, 123, 138, 141, 149, 151, 153/168, 156, 157, 158, 167, 170, 177, 180, 183, 187, 188, 189, 194, 199 and 203: The following PBDE congeners: 17, 28, 32, 35, 37, 47, 49/71, 66, 75, 77, 85, 99, 100, 119, 126, 138, 153, 154, 156, 166, 181, 183, 184, 190, 191, 196, 197, 206, 207 and 209 and HBCD.

For the determination of the phthalates and artificial musks the second part of the extract was purified using a dimethylformamide-hexane partitioning to remove lipids. In this

Spain, Poland, Italy, Estonia and Greece. Samples were sent to the laboratory where laboratory samples were prepared and stored at -18°C until analysis. In general, solid food samples were cut into small pieces and homogenised with a blender. If not the entire sample was used or homogenised, proportional sub-sampling was applied and the collected subsamples were homogenised. Milk was acidified with formic acid and the solid part containing the proteins and fat was separated from the liquid phase. Both parts were stored for analysis. Orange juice was centrifuged and vacuum filtrated and the solid and liquid parts were stored for analysis. A selected number of chemical parameters were determined

The chemical parameters determined in this study are listed in table 1, including the abbreviations that are used throughout the text and in the result tables. Note that not all

For the determination of the OCP's, PCB's, BFR's, phthalates and artificial musks, a weight sub-sample of the homogenised laboratory sample was mixed with anhydrous sodium sulphate in a mortar and spiked with internal standards. The internal standards used were 13C-labelled standards for PCB's and BFR's, 2D-labelled standards for OCP's and phthalates, and a surrogate standard for the artificial musks. The samples were Soxhlet extracted for 16 hours using a mixture of 10% diethyl ether in hexane. For milk and orange juice a proportional amount of the liquid phase was pre-extracted with hexane and this hexane extract was used in the Soxhlet extraction of the solid part of these samples. Olive oil was directly diluted in hexane. One procedural blank, consisting of 40 g anhydrous sodium sulphate, was included in every batch of 10 samples. All extracts were concentrated to a volume of 50 ml and split into two equal parts of 25 ml. For the determination of the OCP's, PCB's and BFR's, one part of the extract was washed several times with sulphuric acid of increasing concentration to remove the major part of the lipids. The remaining extract was concentrated and purified over a glass chromatographic column packed with florisil and capped with anhydrous sodium sulphate to isolate the fraction containing the OCP's, PCB's, PBDE's and HBCD. The eluent was concentrated to a small volume and a syringe standard (1,2,3,4-tetrachloronaphthalene) was added. This final extract was analysed on an Agilent 6890 series gas chromatograph coupled to an Agilent 5973 mass spectrometer (GC/MS) and equipped with a HP-5-MS, 30 m × 0.25 mm (i.d.), film thickness 0.25 µm, fused silica capillary column. The mass spectrometer was operated in the selected ion monitoring mode and typically two or three characteristic ion masses were monitored for each analyte. The samples were analyzed for the following OCP's; α-, β- and γ-hexachlorohexane (HCH), hexachlorobenzene (HCB), α- and β-chlordane, o,p'-, p,p'-DDE, o,p'-, p,p'-DDD and o,p'-, p,p'-DDT: The following PCB congeners: 18, 28/31, 22, 41/64, 44, 49, 52, 54, 56/60, 70, 74, 87, 90/101, 99, 104, 105, 110, 114, 118, 123, 138, 141, 149, 151, 153/168, 156, 157, 158, 167, 170, 177, 180, 183, 187, 188, 189, 194, 199 and 203: The following PBDE congeners: 17, 28, 32, 35, 37, 47, 49/71, 66, 75, 77, 85, 99, 100, 119, 126, 138, 153, 154, 156, 166, 181, 183, 184, 190, 191,

For the determination of the phthalates and artificial musks the second part of the extract was purified using a dimethylformamide-hexane partitioning to remove lipids. In this

in each sample, based on expectations and reports in the literature.

**2.2 Chemical parameters** 

**2.3 Analytical procedures** 

parameters are determined in all samples.

196, 197, 206, 207 and 209 and HBCD.


Table 1. Chemical parameters determined in this study including abbreviations used in the text and result tables

Industrial Contaminants and Pesticides in Food Products 73

With the exception of PCBs and organotin compounds, the results are not corrected for the recovery of the internal standards or spikes since the spikes used were not compound specific and their recovery is only used to evaluate the performance of the method. With each series of ten samples a blank sample was included. For the blank analysis the complete analytical procedure was followed, including all chemicals and solvents, but no sample was added. Blank results were only found for the phthalates DEHP, DIBP and DBP, The results were corrected for these blank values and the detection limits were raised to 10 ng/g for

Organochlorine pesticides (OCPs) include compounds like DDT, lindane, hexachlorobenzene and chlordane, among others. DDT is a well-known agricultural insecticide that has been used extensively on a global basis for over 40 years. Although their manufacture and application are now largely prohibited or restricted in industrialized western countries because of their toxicity and persistence, they can still be found in environmental and biological matrices due to their persistence. Pesticide exposure has been associated with arthritis, diabetes, neurobehavioral changes and DNA damage Cox et al., 2007; Lee et al. 2007; Jurewicz et al., 2008; Rusiecki et al., 2008). The structures of p,p'-DDT

 As a part of its monitoring program, the Food and Drug Administration (FDA) determines the levels of pesticide residues in a wide variety of foods typically consumed by Americans. Over the past ten years, these surveys have detected DDE and other OCPs in a variety of foods including meat, fish and shell fish products, eggs, root vegetables, legumes (beans, peas, and peanuts), some fruits, and leafy greens. In 1999 DDE or DDT were detected in 22% of the 1,040 food items analysed in the FDA Total Diet Study (FDA, 1999). The results for the 2003 Total Diet Study indicate DDT, but mainly DDE in 18% of the various food items in concentrations ranging from 0.1 to 11 ng/g (FDA, 2003). Those for chlordane and lindane range from 0.1 to 3.8 and from 0.1 to 8.4 ng/g product. In general, the concentrations as well as the frequency of detection of OCPs were lower in the 2003 study. The results of the OCPs in this study are presented in table 2 at the end of this section, and graphically in figure 1. OCPs are found in 17 of the 25 samples. The predominant OCPs that are detected are p,p'-DDE and HCB both found in 15 of the 25 samples. In addition o,p'-DDE and cis-chlordane were found in one and two samples respectively. The maximum concentration found for p,p'-DDE was 5.6 ng/g in a sample of pickled herring. The median concentrations for p,p'-DDE and HCB were 0.43 and 0.14 ng/g. Compared to the FDA's Total Diet Study, p,p'-DDE and HCB are found more frequently but in lower concentrations. This compares with a recent study of Schecter et al. who reported p,p'-DDE in 23 out of 31 samples in concentrations ranging from 0.06 to 9.0 ng/g

DIBP and DBP, and to 20 ng/g for DEHP.

and it's breakdown product p,p'-DDE, are shown below.

with a median value of 0.51 ng/g (Schecter et al., 2010).

**3.2 Organochlorine pesticides** 

partitioning the hexane extract is extracted with dimethylformamide (DMF) to isolate the phthalates and artificial musks. After removal of the hexane layer, water is added to DMF fraction and the analytes are re-extracted into fresh hexane. This extract was concentrated and purified over a glass chromatographic column packed with florisil and capped with anhydrous sodium sulphate to isolate the fraction containing the phthalates and artificial musks. The eluent was concentrated to a small volume, the syringe standard was added and this final extract was analysed with the identical GC/MS system as described above. The mass spectrometer was operated in the selected ion monitoring mode and typically two or three characteristic ion masses were monitored for each analyte. The samples were analyzed for the following phthalates; di-methyl- (DMP), di-ethyl- (DEP), di-isobutyl- (DIBP), dibutyl- (DBP), benzylbutyl- (BBP), di-(2-ethylhexyl)- (DEHP), di-isononyl- (DINP) and diisodecyl phthalate (DIDP): The following artificial musks; musk ketone (MK), musk xylene (MX), tonalide (AHTN) and galaxolide (HHCB).

AP's and the brominated flame retardant tetrabromobisphenol-A (TBBPA) were isolated using a steam distillation procedure described by Guenther et al., 2002. In the round bottom flask of the steam distillation apparatus typically 10 g of the laboratory sample was spiked with an internal standard and mixed with 250 ml Milli-Q water to which was added 1 ml of concentrated hydrochloric acid and 20 g of sodium chloride. During the overnight distillation process the organic phenols are isolated in the organic solvent, in this case hexane, in the backflow cooling system of the apparatus. The hexane extract is isolated, dried with anhydrous sodium sulphate and concentrated. Following drying the extract was reduced in volume nearly to dryness under a stream of nitrogen and re-dissolved in 1 ml of methanol. During concentration of the hexane extract care has to be taken to avoid losses of the AP's and rinsing of the glass surfaces with methanol is necessary. The methanol extract is analysed with liquid chromatography coupled with mass spectrometry (LC/MS) in the selected ion monitoring mode.

For the determination of perfluorinated compounds, specifically perfluoro-octanoate (PFOA), perfluoro-octane sulphonate (PFOS) and perfluoro-octane sulphonamide (PFOSA) sub-samples of 5 gram were collected in 50 ml poly-propylene tubes and extracted using acetonitrile. The samples were centrifuged and the clear liquid is decanted and purified over a glass chromatographic column packed with florisil, silica, LC-NH2 and activated carbon. The residue in the poly-propylene tube is extracted two times more and each extract is decanted over the same chromatographic column, thus combining the purified extracts. 0.5 ml octanol is added as a keeper and the extracts are concentrated to a small volume.
