Monitoring of the benzene and toluene contents in human milk

Environment International 30 (2004) 397 – 401 www.elsevier.com/locate/envint

Monitoring of the benzene and toluene contents in human milk F. Fabietti a, A. Ambruzzi b, M. Delise a,*, M.R. Sprechini b a

Food Department, Istituto Superiore di Sanita`, viale Regina Elena 299, 00161 Rome, Italy b Children’s Hospital ‘‘Bambino Gesu`’’, salita S. Onofrio, Rome, Italy Received 29 June 2003; accepted 15 September 2003

Abstract Twenty-three samples of human milk collected from the milk bank of a children’s hospital were analysed with a view to monitoring the possible presence of some of the most common aromatic hydrocarbons (benzene and toluene) and to quantify their concentrations. The analysis was carried out by the ‘‘purge and trap’’ technique combined with gas chromatography and with the use of the mass spectrometer as detector. The hydrocarbons themselves were used in a deuterated form as internal standards. The analysis of the data showed the presence of both hydrocarbons, even though their quantity was much lower than that detected in other foods. D 2003 Elsevier Ltd. All rights reserved. Keywords: Human milk; Benzene; Toluene; Gc-mass; Purge and trap

1. Introduction A lot of scientific evidence has shown that human milk benefits the normal growth and development of a new-born baby, thanks to the peculiarity of its nutrients, its enzymatic and hormonal components, its growth and anti-infective factors, that makes it a unique and inimitable nourishment that can be defined a biological system. From a strictly nutritional point of view, human milk provides the new-born baby with almost all the nutrients that he needs, is easily digestible and assimilable and helps to defend the baby from various infections through passive immunization. The psychological aspects of this nourishment are remarkable, in that it strengthens the mother –child relationship, and has a great value for the psychic and neurological development of the new-born baby. Moreover, the preventive role that human milk plays for some pathologies of adult age, such as the development of food allergies, obesity, risk factors for chronic-degenerative diseases of adulthood, should not be underestimated (Uauy and Peirano, 1999; Uauy and Mena, 2001).

* Corresponding author. Fax: +39-6-49902046. E-mail address: [email protected] (M. Delise). 0160-4120/$ - see front matter D 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2003.09.007

The renewed interest of the international scientific research in human milk is motivated by the high number of mothers that breastfeed their babies and by the ever-increasing knowledge on the biological functions of human milk, described as an ideal nourishment for the first 6 months of life, as the American Academy of Paediatrics stated. However, qualified scientists who study environmental pollution have raised the alarm with regard to the contamination of the human body and, in particular, the concentration of xenobiotic substances of various kind in biological fluids (Pratigya et al., 2001). When such substances, that are present in the water, soil, air and foodstuffs, because of their lipophilic nature, enter the human body, they tend to accumulate in fatty tissue to be later processed by metabolic mechanisms and amass in human milk (Rogan et al., 1980). Therefore, human milk could constitute a potential vehicle for many contaminants that could be present in its lipidic fraction (Anderson and Wolff, 2000). In studies carried out in various countries, molecules of polychlorinated biphenyls (PCB), DDT, dibenzodioxins, dibenzofurans, organochlorine pesticides, polycyclic aromatic hydrocarbons (PAH), organic solvents, drugs, makeup, food additives were detected in human milk (Jiyeon et al., 2002; Turusov et al., 2002). Some of these substances can have neurotoxic, immunotoxic and carcinogenic effects, while others can interfere as

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endocrine disruptors, thus causing various pathologies (Babasaheb, 1995). The potential long-term effects of these contaminants have not been totally assessed, but the international interest in them is growing, especially in industrialised countries, where the exposure to some of them through human milk is from 10 to 100 times higher than the maximum acceptable dose (TDI) (Przyrembel et al., 2000). The concentration of possible contaminants in human milk clearly depends on many factors that essentially include life-style, the place in which the wet-nurse spends part of her day and her job. Therefore, the following elements are of primary importance: diet (if it is based on meat, fish or dairy products), smoking habits, the presence of factories, garages (repair shops), workshops, petrol stations near the wet-nurse’s house, prolonged stays in working environments where chemical solvents, reagents and similar products are used. On the strength of this introduction, this study represents a screening in the qualitative and quantitative assessment of the presence of two aromatic hydrocarbons, benzene and toluene, in human milk. These hydrocarbons are diffused everywhere because of pollution that is mostly due to traffic and the wastes of factories which use benzene and toluene as solvents. These two elements develop a toxic action on humans, that is both acute and chronic, and in particular, a prolonged exposure to benzene has proved important in the development of myelofibrosis, a myeloproliferative disease, in which the fibrous tissue replaces the bone marrow. Moreover, the same hydrocarbon may cause forms of leukemia in humans exposed to high concentrations of it (Gist and Burg, 1997). Benzene must be metabolized to exert its toxic effects (Snyder, 1987) through a very complex process that leads to more toxic products; the most important of them are phenol, hydroquinone and catechol (Medinsky et al., 1989). Age can play an important role in metabolism of benzene; during same experiments, the younger animals displayed a greater rate of metabolism and a higher sensibility to toxic effects, rather than the older ones (Snyder and Kocsis, 1975; McMurry et al., 1994). Moreover, fetuses and offspring of pregnant mice exposed to benzene showed long-term functional changes in hematopoiesis (Keller and Snyder, 1986, 1988). There are also studies about skeleton malformations in offspring of benzene exposed animals (Dowty et al., 1976; Murray et al., 1979). Today, we know benzene is an immunotoxicant (Dean et al., 1979; Snyder, 1987; McMurry et al., 1991) causing changes in circulating leucocytes including lymphocytes (Aksoy et al., 1971, 1987). It is also known that benzene has depressive effect on T lymphocytes than on B cell (Popp et al., 1992) and affects both humoral and cellular acquired immunity in animals (Cronkite et al., 1982).

The metabolites of benzene affect the immune system of animals: inhibition of interleukin 2 (IL-2) production (Post et al., 1985), inhibition of maturation and proliferation of B lymphocyte in the marrow, spleen and thymus, but it should be noted that benzene and benzene metabolites at low doses (Pfeifer and Irons, 1981) or on cell-mediated immunity (Aoyama, 1986) can be stimulatory. Because of its known carcinogenic nature, no TDI limits exist for benzene, whereas limits only relative to its concentration in drinking waters exist, which were developed by the WHO as guidelines, and allow a maximum content of 10 Ag kg 1 (European Commission, 1999).

2. Materials and methods Twenty-three samples of human mature milk, collected during the whole day (24 h) from both breasts, were supplied by children’s hospital ‘‘Bambino Gesu`’’ of Rome. Each sample was accompanied by a card with the birth data of the wet-nurse, information about her job, place of residence (classification of the area, nearness to factories, intensity of road traffic), diet, and drugs use. The samples were supplied frozen by the hospital and kept so until the moment of the analysis. The aromatic hydrocarbons were analysed by gas chromatography combined with mass spectrometry, using the purge and trap technique, because of their high volatility. As it is known, the purge and trap technique consists of an extraction, from the matrix, by means of flow nitrogen, of the analytes, that, passing through a specific trap, are adsorped and subsequently removed by desorption at high temperature. By means of cooling with liquid nitrogen, they are concentrated and injected on gas chromatographic column, using the mass spectrometer as detector. Standard solutions of each hydrocarbon in methanol were used to verify the retention times of the peaks and proceed to their identification. Such solutions were diluted in totally outgassed milk to obtain a concentration of about 10 mg kg 1 for each hydrocarbon, suitable for the analysis at the gas chromatograph-mass, using the complete scanning mode (SCAN MODE) and comparing the spectra obtained with those present in the instrument’s library (NBS 75 K). For quantitative analysis, we chose the internal standard method, as we already did in our previous articles (Fabietti et al., 2000, 2001), using the deuterated form of each hydrocarbon. In this way, variations of retention times were avoided, as well as the different response of the instrument, if we had used just one standard. In fact, the retention times of benzene and toluene are almost the same as their deuterated forms and they were quantified by the single ion monitoring method (SIM MODE).

F. Fabietti et al. / Environment International 30 (2004) 397–401

SIM MODE allows the mass spectrometer to detect specific compounds with very high sensitivity. Since the instrument collects only the masses of interest, it responds only to those compounds which possess the selected mass fragments. Therefore, because only a few masses are monitored, the result is an increase in sensitivity, accuracy and precision. Using the ‘‘extract ion’’ technique, it is possible to evaluate the area of the most abundant ions related to the fragmentation of the single compounds. To this end, deuterated benzene and toluene standards, each at a concentration of 10 Ag kg 1 were added to the samples. The ions taken into consideration were: 77– 78– 84 –91 – 92 –98 – 100 m z 1. An aliquot of 5 ml of sample, added of standard as described above, was put into a glass U vessel without filter baffle and connected to the Hewlett-Packard 7695 Purge & Trap apparatus. The operating conditions of the purge and trap analysis are shown in Table 1. For the adsorption, we used a specific BTEX HewlettPackard trap. Injection took place automatically by connection with a Hewlett-Packard 6890 gas chromatograph

Table 1 Operating conditions of purge and trap analysis Purge and trap control method version 1.00 Parameter

Value

Line temperature Valve temperature Mount temperature MCS line temperature Purge ready temperature Purge temperature Turbo cool temperature Sample heater Prepurge time Sample preheat time Sample preheat temperature Purge time Drypurge time MCS desorb temperature GC start option GC cycle time Cryo focuser Cryo standby temperature Cryo focus temperature Cryo inject time Cryo inject temperature Desorb preheat temperature Desorb time Desorb temperature Sample drain Bake time Bake temperature Bake gas bypass Bake gas bypass delay time MCS bake temperature

200 jC 200 jC 40 jC 100 jC 35 jC 30 jC 20 jC Off 0.00 min 5.00 min 90 jC 11 min 2 min 220 jC End of Desorb 0.00 min On 150 jC 150 jC 1.00 min 150 jC 180 jC 8 min 220 jC Off 20 min 225 jC On 0.3 min 180 jC

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equipped with a column Supelco SPB-5, length 60 m, 0.20 mm i.d., film thickness 0.20 Am. Gas chromatographic analytical conditions were:

Initial temperature Final temperature Increment

40 jC for 1 min 200 jC for 5 min 15 jC min 1

The mass spectrometer had a temperature of 280 jC at the source. Parameters for data acquisition were: Group Benzene Toluene

Time interval of sampling (min) 5.00 – 6.00 6.00 – 7.00

Scan rate for sampling (cycles s 1.16 1.16

Each sample was analysed in triplicate. The limit of detection of the method was 0.01 Ag kg for each hydrocarbon analysed.

1

)

1

3. Results and discussion Table 2 shows the results relative to the concentrations of benzene and toluene in Ag kg 1 detected in the samples of human milk analysed in triplicate and the relevant means and standard deviations. The total mean and relevant standard deviation of all the samples analysed are also shown. The analyses of the data shown demonstrated that the concentration of benzene varied from a low of 0.01 Ag kg 1 to a high of 0.18 Ag kg 1, with most of the samples below 0.1 Ag kg 1 and a mean of 0.06 Ag kg 1. The values of the concentrations of toluene presented a greater variability, which went from a low of 0.04 Ag kg 1 to a high 2.54 Ag kg 1 with a mean of 0.76 Ag kg 1. As we had already found out on other matrices in already mentioned studies, the two hydrocarbons were present in all the samples analysed, because of their ubiquitous diffusion, even though in fairly low amounts. It should be observed that almost all the samples with a high concentration of benzene presented as high a concentration of toluene. On the basis of questionnaires filled in by milk donors, which reported information on eating habits, place of residence (urban or rural area, intensity of road traffic), the use of drugs or food supplements, and job, we tried to establish a connection between the highest values found in milk and some of the above-mentioned factors. The questionnaires presented many analogies among them, especially in relation to the diet (mainly meat-based) and the non-use of drugs or other similar products, whereas some differed in terms of location of the house, workplace and intensity of road traffic.

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Table 2 Content of benzene and toluene expressed in Ag kg

1

, mean and standard deviation for each sample

Benzene

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

Toluene

1st test

2nd test

3rd test

Mean

S.D.

1st test

2nd test

3rd test

Mean

S.D.

0.04 0.02 0.09 0.03 0.03 0.09 0.04 0.03 0.02 0.16 0.09 0.11 0.02 0.02 0.05 0.11 0.04 0.01 0.06 0.11 0.10 0.12 0.07

0.03 0.01 0.07 0.02 0.01 0.04 0.07 0.03 0.04 0.21 0.10 0.09 0.02 0.01 0.09 0.09 0.03 0.01 0.07 0.10 0.07 0.10 0.05

0.03 0.02 0.07 0.02 0.02 0.06 0.05 0.03 0.03 0.18 0.11 0.08 0.01 0.02 0.07 0.10 0.04 0.01 0.06 0.09 0.08 0.11 0.07

0.03 0.01 0.07 0.02 0.02 0.06 0.05 0.03 0.03 0.18 0.10 0.09 0.02 0.02 0.07 0.10 0.04 0.01 0.06 0.10 0.08 0.11 0.06

0.01 0.01 0.01 0.004 0.01 0.03 0.02 0.002 0.01 0.03 0.01 0.02 0.01 0.01 0.02 0.01 0.01 0.002 0.01 0.01 0.02 0.01 0.01

0.19 0.04 0.44 0.17 0.34 0.61 0.36 0.23 0.21 2.84 1.27 1.84 0.17 0.08 1.28 2.08 0.76 0.17 1.23 2.06 1.08 0.12 0.07

0.20 0.04 0.37 0.11 0.44 0.65 0.59 0.18 0.16 2.38 1.49 1.61 0.18 0.06 2.14 1.56 0.96 0.11 1.46 1.92 0.94 0.10 0.05

0.21 0.05 0.41 0.12 0.38 0.60 0.53 0.20 0.18 2.40 1.37 1.70 0.16 0.06 1.77 1.72 0.84 0.15 1.32 2.00 1.00 0.10 0.06

0.20 0.04 0.40 0.13 0.38 0.62 0.49 0.20 0.18 2.54 1.37 1.72 0.17 0.07 1.73 1.78 0.85 0.14 1.34 1.99 1.00 0.10 0.06

0.01 0.002 0.04 0.03 0.05 0.03 0.12 0.03 0.03 0.26 0.11 0.12 0.01 0.01 0.43 0.27 0.10 0.03 0.12 0.07 0.07 0.01 0.01

Mean for all the samples S.D. for all the samples

0.06 0.04

0.76 0.76

Mean and standard deviation for all the samples. S.D. = standard deviation.

The highest values of hydrocarbons found in the samples analysed almost always corresponded to samples collected from women that lived in urban or semi-urban or highly trafficked areas. The two hydrocarbons were detected in all the milk samples analysed, even though in significantly lower amounts than those that can be transmitted by other foods. This means that even though the value of human milk in the diet of a new-born baby is of high importance, we do not have to lower our guard with regards to the monitoring of such solvents, in all sectors, so as to reduce their use and ensuing diffusion into the environment to the lowest possible amounts. Therefore, this article aims at being the first in a long line of monitoring studies carried out on this important nourishment, and will be followed by other studies on various xenobiotic elements. This will help provide data concerning the possible contamination of human milk in order to try and grant a safer use of it from the sanitary point of view.

References Anderson HA, Wolff MS. Environmental contaminants in human milk. J Expo Anal Environ Epidemiol 2000;10(6 Pt 2):755 – 60. Aksoy M, Dincol K, Akgun T, Erdem S, Dincol G. Hematological effects

of chronic benzene poisoning in 217 workers. Br J Ind Med 1971; 28:296 – 302. Aksoy M, Ozeris S, Sabuncu H, Inanici Y, Yanardag R. Exposure to benzene in Turkey between 1983 and 1985: a hematological study on 231 workers. Br J Ind Med 1987;44:785 – 7. Aoyama K. Effects of benzene inhalation on lymphocyte subpopulations and immune response in mice. Toxicol Appl Pharmacol 1986;85: 92 – 101. Babasaheb RS. Chemical contaminants in human milk: an overview. Environ Health Perspect 1995;103(6):197 – 205. Cronkite EP, Inoue T, Carsten AL, Miller ME, Bullis JE, Drew RT. Effects of benzene inhalation on murine pleuripotential stem cells. J Toxicol Environ Health 1982;9:411 – 21. Dean JH, Padarathsingh ML, Jerrels TR, Keys L, Northing JW. Assessment of immunobiological effects induced by chemicals, drugs or food additives: II Studies with cyclophosphamide. Drug Chem Toxicol 1979; 2:133 – 53. Dowty BJ, Laseter JL, Storer J. The transplacental migration and accumulation in blood of volatile organic constituents. Pediatr Res 1976; 10:696 – 701. European Commission. Opinion on certain aromatic hydrocarbons present in food. Scientific Committee on Food. 1999;21:1 – 5. January. Fabietti F, Delise M, Piccioli Bocca A. Aromatic hydrocarbon residues in milk: preliminary investigation. Food Control 2000;11:313 – 7. Fabietti F, Delise M, Piccioli Bocca A. Investigation into the benzene and toluene content of soft drinks. Food Control 2001;12:505 – 9. Gist GL, Burg JR. Benzene—a review of the literature from a health effects perspective. Toxicol Ind Health 1997;13(6):661 – 714. Jiyeon Y, Dongchun S, Soungeun P, Yoonseok C, Donghyun K, lkonomou MG. PCDDs, PCDFs and PCBs concentrations in breast milk from two areas in Korea: body burden of mothers and implications for feeding infants. Chemosphere 2002;46(3):419 – 28.

F. Fabietti et al. / Environment International 30 (2004) 397–401 Keller K, Snyder CA. Mice exposed in utero to low concentrations of benzene exhibit enduring changes in their colony forming hematopoietic cells. Toxicology 1986;42:171 – 81. Keller K, Snyder CA. Mice exposed in utero to 20 ppm benzene exhibit alliterated numbers of recognizable hematopoietic cells up to seven weeks after exposure. Fundam Appl Toxicol 1988;10:224 – 32. McMurry ST, Lochmiller RL, Vestey MR, Qualls CW, Elangbam CS. Acute effects of benzene and cyclophosphamide exposure on cellular and humoral immunity of cotton rats, Sigmodon hispidus. Bull Environ Contam Toxicol 1991;46:937 – 45. McMurry ST, Lochmiller RL, Vestey MR, Qualls CW. Cellular immune responses of nutritionally stressed juvenile cotton rats (Sigmodon hispidus) during acute benzene exposure. Arch Environ Contam Toxicol 1994;27(1):14 – 9. Medinsky M, Sabourin PJ, Lucier G, Birnbaum LS, Henderson RF. A physiological model for stimulation of benzene metabolism in rats and mice. Toxicol Appl Pharmacol 1989;99:193 – 206. Murray FJ, John JA, Rampy LW, Kuna RA, Schwetz BA. Embryotoxicity of inhaled benzene in mice and rabbits. Am Ind Hyg Assoc J 1979; 40:993 – 8. Pfeifer RW, Irons RD. Inhibition of lectin-stimulated lymphocyte agglutination and mitogenesis by hydroquinone: reactivity with intracellular sulfhydryl groups. Exp Mol Pathol 1981;35:189 – 98. Popp W, Vahrenholz C, Yaman S, Muller C, Muller G, Schmieding W,

401

Norpoth K, Fahnert R. Investigations of the frequency of DNA strand breakage and crosslinking and sister chromatid exchange frequency in the lymphocytes of female workers exposed to benzene and toluene. Carcinogenesis 1992;13:57 – 61. Post GB, Snyder R, Kalf GF. Inhibition of RNA synthesis and interleukin 2 production in lymphocytes in vitro by benzene and its metabolites, hydroquinone and p-benzoquinone. Toxicol Lett 1985;29:161 – 8. Pratigya G, Dubey JK, Patyal SK, Amit N. Contamination of mothers’ milk with pesticide residues. Pestic Res J 2001;13(2):207 – 12. Przyrembel H, Heinrich HB, Vieth B. Exposition to and health effects of residues in human milk. Adv Exp Med Biol 2000;478:307 – 25. Rogan WJ, Bagniewska A, Damstra T. Pollutants in breast milk. N Engl J Med 1980;302(26):1450 – 3. Snyder CA. Benzene. In: Snyder R, editor. Ethel Browning’s Toxı´city and Metabolism of Industrial Solvents. New York: Elsevier, 1987. pp. 3 – 37. Snyder R, Kocsis JJ. Current concepts of chronic benzene toxicity. Crit Rev Toxicol 1975;3:265 – 88. Turusov V, Rakitsky V, Tomatis L. Dichlorodiphenyl-trichloroethane (DDT): ubiquity, persistence and risks. Environ Health Perspect 2002; 110(2):125 – 7. Uauy R, Mena P. Lipids and neurodevelopment. Nutr Rev 2001;59(8, Part II):S34 – 46. Uauy R, Peirano P. Breast is best: human milk is the optimal food for brain development. Am J Clin Nutr 1999;70(4):433 – 4.