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Polychlorinated naphthalenes in human milk: Health risk assessment to nursing infants and source analysis
Environment International 136 (2020) 105436
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Polychlorinated naphthalenes in human milk: Health risk assessment to nursing infants and source analysis
T
Cui Lia,b, Lei Zhangc, Jingguang Lic, Yihao Mind, Lili Yanga,b, Minghui Zhenga,b,e, Yongning Wuc, ⁎ Yuanping Yanga,b, Linjun Qina,b, Guorui Liua,b,e, a
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China b College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100190, PR China c China National Center for Food Safety Risk Assessment, Beijing 100022, PR China d College of Science, China Agricultural University, Beijing 100083, PR China e Institute of Environment and Health, Hangzhou Institute for Advanced Study, UCAS, Hangzhou, PR China
A R T I C LE I N FO
A B S T R A C T
Handling Editor: Da Chen
Polychlorinated naphthalenes are teratogenic environmental contaminants. Mother milk is the most important food for nursing infants. The World Health Organization actively promotes breastfeeding for its immunological, psychological, and economic advantages. We firstly measured concentrations of polychlorinated naphthalenes in human milk from 19 provinces in China and estimated their potential health risks to nursing infants and their possible sources. Concentrations ranged from 211.07 to 2497.43 pg/g lipid. The high prevalence of highly toxic hexachlorinated naphthalenes (Hexa-CN66/67) in human milk samples indicated a higher health risk in the sampling areas. Cancer risk posed to nursing infants was not significant, but potential non-carcinogenic adverse health effects were suggested and should be emphasized in some sampling areas. Unintentional emission of polychlorinated naphthalenes from industries that employ thermal processes appears to be the main source for PCNs in human milk in most sampling areas. Correlation analysis also suggested PCNs as impurities in polychlorinated biphenyl mixtures as a previously unrecognized source of polychlorinated naphthalenes in human milk.
Keywords: Polychlorinated naphthalenes (PCNs) Human milk Exposure Risk assessment Source analysis
1. Introduction Persistent organic pollutants (POPs) are highly toxic organic chemicals. The Word Health Organization (WHO) has been carrying out global surveys on POPs in human milk for more than three decades, mainly focusing on polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and DDT (van den Berg, 2017). However, studies on polychlorinated naphthalenes (PCNs) in human milk are very scarce. PCNs with one or more chlorine atoms as substituents on the naphthalene ring have raised concerns due to their potential toxicity. Several PCN congeners have physicochemical and toxic properties similar to those of dioxins, such as a tendency to accumulate in biological tissues (Hayward, 1998; Falandysz, 2003), induce aryl hydrocarbon receptor-mediated responses (Barc and Gregoraszczuk, 2014; Villeneuve, 2000; Blankenship, 2000), and exert adverse health effects (Stragierowicz et al., 2018;
Pardyak, 2016; Galoch et al., 2006; Popp, 1997; Ward, 1996; Li et al., 2011). PCNs have been shown to be potent fetotoxic and teratogenic contaminants in animal experiments (Kilanowicz et al., 2011), and exposure can induce biochemical and histopathologic changes in the liver and thymus (Hootha, 2012). Moreover, occupational exposure to PCNs has been reported to cause liver toxicity (Popp, 1997; Ward, 1996), chloracne, and even death (Hayward, 1998); symptoms of occupational PCN exposure include digestive problems, anorexia, nausea, and vertigo (Hayward, 1998). PCNs have been listed as persistent organic pollutants by the Stockholm Convention in 2015 (UNEP, 2019), with a focus on reducing exposure worldwide. PCNs are ubiquitous in the environment, and have even been found in polar areas (Harner et al., 1998). Environmental PCNs could be ascribed to three categories of sources including synthesis as technical PCNs formulations, impurities in the technical polychlorinated biphenyls (PCBs), and unintentional releases via thermal processes. Global
⁎ Corresponding author at: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China. E-mail address: [email protected] (G. Liu).
https://doi.org/10.1016/j.envint.2019.105436 Received 19 October 2019; Received in revised form 13 December 2019; Accepted 20 December 2019 0160-4120/ © 2019 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/).
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(Thermo Fisher Scientific, Waltham, MA, USA) at 120 °C and 1500 psi. The extraction solvent was a mixture of hexane and dichloromethane (1:1, V:V). The extracts were spiked with a mixture of 1 ng 13C-labeled PCN internal standard (ECN-5102, including CN-27, CN-42, CN-52, CN67, CN-73, and CN-75) and concentrated to dryness for gravimetric lipid determination. The residues were then reconstituted with 5 mL of hexane. Three columns were used: The first column was an acid silica column with an elution of 90 mL of hexane. The second column was a multi-layer silica column with an elution of 90 mL of hexane. The third column was a basic alumina column and the elution was a 100-mL mixture of hexane and dichloromethane (95:5, V:V). Next, the eluted fractions were concentrated to about 20 μL and spiked with 1 ng 13Clabeled injection standard for high-resolution gas chromatography/ high-resolution mass spectrometry analysis. ECN-5102 13C10-labeled PCN internal standard and ECN-5260 13C10-labeled injection standard (including CN-64) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Pesticide residue-grade hexane and dichloromethane were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA).
synthesis of technical PCNs formulations has been estimated at approximately 150,000 metric tons (Falandysz, 1998). Concentration of PCNs as impurities in PCB formulations is estimated at 870 μg/g (Yamashita et al., 2000; Huang, 2015; Domingo, 2004). Unintentional formation and emission from industrial activities, including waste incineration and metallurgical processes, are a growing concern (Liu, 2012; Liu et al., 2014; Jiang, 2015) considering the restriction of PCNs and PCBs as technical products. The massive historical production and releases of PCNs as well as the on-going releases from numerous industrial sources underscore the need to assess human PCN body burdens and their associated health risks. Human exposure to PCNs is inevitable including pathways of air inhalation, dietary intake and so on. The occurrences of PCNs in air, edible biota have been reported (Rotander, 2012; Braune and Muir, 2017; Cui, 2018; Fernandes, 2010; Egeback et al., 2004) and there are thousands of industrial sources of unintentional PCNs and also historically PCBs manufactures in China. Understanding human PCN body burden is significant for their potential health risk assessment. Human milk is considered as one of the core monitoring matrices of the United Nations Environment Programme’s persistent organic pollutants monitoring program with the aim of evaluating the effectiveness of implementing Stockholm Convention. Human milk monitoring can provide exposure information on both the mother and the breastfed infant through a non-invasive method of collection. Mother milk is the most important food for nursing infants. The World Health Organization also actively promotes breastfeeding for its immunological, psychological, and economic advantages, and a feeding mode of exclusive breastfeeding for six months followed by continuous breastfeeding with appropriate complementary foods for up to two years or beyond is recommended for the vast majority of infants (WHO, 2008). If significant PCN present in mother milk, this route of continuous PCN exposure during early life might cause long-term damage. Although a few studies measured PCN levels in human serum and adipose tissues (Park et al., 2010; Horii, 2010; Fromme, 2015; Weistrand and Noren, 1998; Schiavone et al., 2010; Kunisue et al., 2009), data on PCN loads in human milk and their potential health risks to nursing infants are not reported (Lunden and Noren, 1998; Pratt, 2013). In the present study, we measured the concentrations of 75 PCN congeners in 77 pooled human milk samples from 3790 individual samples collected from 19 provinces in China. Analysis was carried out by isotope dilution high-resolution gas chromatography/high-resolution mass spectrometry. To the best of our knowledge, this is the first study to report PCN loads in human milk and assess their potential sources and health risks to nursing infants in China. It was expected that this study could provide important information on PCN internal exposures and potential risks to nursing infants not only for China, but also for other countries by fulfilling the international Stockholm Convention.
2.2. Instrumental analysis Congener-specific analysis of PCNs was carried out by high-resolution gas chromatography combined with high-resolution mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). Gas chromatographic conditions were as follows: the injection volume was 1 μL in splitless injection mode; the carrier gas was helium; the column was a DB-5MS capillary column (60 m × 0.25 mm × 0.25 μm, Agilent Technologies, Santa Clara, CA, USA); the inlet temperature was 260 °C; the carrier gas flow rate was 1 mL/min. The temperature program was 80 °C for 2 min, increased by 20 °C/min to 180 °C and held for 1 min, then increased by 2.5 °C/min to 280 °C and held for 2 min, and finally increased to 290 °C at 10 °C/min and held for 5 min. There were 57 peaks on the column, including 41 single peaks and 16 composite peaks. The calibration curves were obtained from CS1-11 standard solutions ranging from 1 ppm to 1000 ppm. Standard solutions were mixed with PCN-MXA, PCN-MXC, PN-31S, which were obtained from Wellington Laboratories (Ontario, Canada), and ECN-2663, ECN-2664, ECN-2665, ECN-2641, ECN-2653, ECN-2623, ECN-2623, ECN-2620, ECN-2621, which were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Mass spectrometry conditions: ionization mode was electron impact; the source temperature was 280 °C; electron energy was 45 eV; transmission line temperature was 290 °C; reference material was perfluorokerosene; the mass spectrometry acquisition method was selective ion monitoring. To ensure the accuracy of the quantification, the resolution of the mass spectrometer was tuned to about 10,000. The data were processed with TargetQuan software (Thermo Fisher Scientific, Waltham, MA, USA). Statistical analysis of the data was conducted using SPSS version 25 and R version 3.5.1. Half of the limit of detection value was assigned for congeners with concentrations below the limit of detection.
2. Materials and methods 2.1. Sample collection and pretreatment Sample collection was conducted by the China National Center for Food Safety Risk Assessment (CFSA) following the fourth WHO-coordinated survey of human milk for persistent organic pollutants in cooperation with the United Nations Environment Programme (UNEP) (WHO, 2007). A total of 3790 human milk samples were collected between 2017 and 2019 from 77 cities/counties around China. At least 50 individual human milk samples were collected from each city sampling site and more than 30 human milk samples from each county sampling site. Detailed information about the samples is shown in Table S1. All samples were stored in a refrigerator at −20 °C until analysis. Prior to extraction, all samples were freeze-dried for more than 72 h and homogenized. Approximately 1 g dry weight of sample was mixed with 2–3 g of diatomaceous earth, then extracted using ASE350
2.3. Quality assurance and quality control The diatomaceous earth was pre-extracted before sample extraction. One procedural blank sample was analyzed with each batch of samples to evaluate laboratory interference and contamination. Only a small amount of monochlorinated, dichlorinated, and trichlorinated naphthalenes was detected in the blank samples, which was 10% lower than concentrations in the samples. The PCNs concentrations in samples were not corrected using values from blanks. The average recoveries of 13 C10-labeled CN-27, CN-42, CN-52, CN-67, CN-73, and CN-75 were 54–100%, which was satisfactory for the PCN trace analysis (Method, 1625). The limit of detection for each congener was set as three times 2
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Fig. 1. Distribution (a) and concentrations (b) of polychlorinated naphthalenes (PCNs) in human milk from 19 provinces in China.
lowest levels were found in Inner Mongolia. The regional distribution of PCDD/Fs in human is similar to that of PCNs in our study, but the overall concentration of PCDD/Fs is much lower than PCNs. The last study of PCNs in human milk was conducted in Ireland and published in 2013; only 12 PCN congeners were measured, ranging in level from 59 to 168 pg/g lipid (Pratt, 2013). Average concentration of the sum of the 12 congeners in our study was 180.76 pg/g lipid, which is a little bit higher than Ireland. According to comparison with the very limited data, our findings indicate a slightly higher level of PCN exposure through human milk in the Chinese general population. A Swedish study published over two decades ago reported a notable decrease, from 3081 to 483 pg/g lipid, in the concentrations of PCNs in human milk between 1972 and 1992 (Lunden and Noren, 1998). Unfortunately, they did not give an explanation for the decrease. Of note, concentration of PCNs in human milk from Ireland was in good consistent with the decreasing trend found in Sweden but is different in China where we could found concentrations above 2000 pg/g lw in samples collected during 2017 to 2019. This indicated a variability of PCNs contamination in human milk from different countries. Congener profiles of PCNs in human milk samples from 19 provinces in China varied by region (Fig. 2). Some profiles were dominated by dichlorinated and trichlorinated naphthalenes, which accounted for 68.42% of the total, while others were dominated by pentachlorinated and heptachlorinated naphthalenes, which accounted for 31.58% of the total. High concentrations of heptachlorinated naphthalenes were found in Guangdong, Guangxi, and Fujian provinces, accounting for 63%, 38%, and 29% of total PCNs, respectively. Behnisch et al. reported higher bioactivity for tetrachlorinated to octachlorinated naphthalenes
the signal-to-noise ratio. PCN congener peaks were identified by comparing the retention times of target congeners to available standards. The abundance ratio of the target ion and the qualifier ion had to be within 15% of the theoretical value.
3. Results and discussion 3.1. Levels and congener profiles of ∑75PCN in human milk PCNs were detected in all 77 pooled samples analyzed in this study. Fig. 1 shows the distribution and concentrations of ∑75 PCN in human milk from the 19 provinces. Result from one-sample T test (p-value < 0.01) indicated a significant regional variability, which ranged widely from < 300 pg/g lipid in Gansu and Shanxi provinces to > 2000 pg/g lipid in Fujian and Guangdong provinces. Data on PCN levels in human milk are scarce; for comparisons with reports from other regions, we compiled all the available data on PCN loads in human specimens (Table 1). The PCN levels we found were lower than those reported in adipose tissue samples from Canada (Williams et al., 1993), Sweden (Witt and Niessen, 2000), or Italy (Schiavone et al., 2010), and comparable with those in adipose tissue samples from the US (Kunisue et al., 2009). Levels measured in blood samples in Korea (Park et al., 2010) and Germany (Kunisue et al., 2009) were comparable to the levels we recorded, but higher in blood samples from an industrial city in China (Jin, 2019). According to a previous study (Zhang et al., 2016), concentrations of PCDD/Fs in human milk from 16 provinces in China ranged from 24.8 pg/g lipid to 488.8 pg/g lipid and relatively high levels were found in Guangdong and Guangxi provinces while the
Table 1 Levels of polychlorinated naphthalenes in human samples (picograms per gram lipid). Country
Year
Sample type
Congeners
Range/Mean
Reference
Sweden Canada Sweden Sweden Germanya the USA the USA Italy Korea Ireland German China China
1972–1992 1993 1998 1998 2000 2002–2003 2003–2005 2005–2006 2007 2010 2013–2014 2016 2017–2019
human milk adipose adipose liver adipose plasma adipose adipose blood human milk blood blood human milk
TeCN-OCN 4 22 22 – – 38 – TeCN-OCN 12 TeCN-OCN MoCN-OCN MoCN-OCN
483–3081 1020–3650 999–3909 1375–26113 1700–8500 345–1540 21–2500 500–14000 2170 59–168 101–1406 14300–50700 211–2497
Lunden and Noren (1998) Williams et al. (1993) Witt and Niessen (2000) Witt and Niessen (2000) Kunisue et al. (2009) Horii (2010) Kunisue et al. (2009) Schiavone et al. (2010) Park et al. (2010) Pratt (2013) Fromme (2015) Jin (2019) This study
a
Data from Germany, Russia and Kazakhstan; -: without data; Year refers to period of data collection. 3
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toxicities similar to those of dioxins. Thus, we converted the concentrations of PCNs to toxic equivalent quantity concentrations relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using Eq. (1). To the best of our knowledge, only 36 PCN congeners has reported relative potency factors (Falandysz et al., 2014). Therefore, we targeted these 36 congeners during the calculation of toxic equivalent quantity concentrations in this study. The relative potency factors of individual PCN congeners to TCDD are listed in Table S2. The second step is the calculation of intake using Eq. (2). Calculation of subchronic daily intake is recommended for exposure periods between 2 weeks and 7 years (EPA, 1989); in this study, we used a feeding mode of 2 years divided into three stages: 0–6, 7–12, and 13–24 months, using a human milk contact rate of 750, 600, and 500 mL/day, respectively (WHO, 2001; CNS, 2016). The exposure frequency was 360 day/year. Exposure durations of 0.5 year, 0.5 year, and 1 year were used for the exposure periods of 0–6, 7–12, and 13–24 months, respectively. Lipid content in human milk was taken as the mean value of samples from the same province. Body weights (MEPC, 2016) of 7.2, 9.5, and 11.2 kg evaluated in China were used for the exposure durations of 0–6, 7–12, and 13–24 months, respectively. Averaging time was 182.5, 182.5, and 365 days, respectively.
Fig. 2. Congener profiles of polychlorinated naphthalenes in human milk from 19 provinces in China.
in vitro (Behnisch et al., 2003), indicating a possible higher risk in the areas where higher-chlorination congener profiles were found. Some PCN congeners (Tri-CN24/14, Penta-CN51, Hexa-CN66/67, and PentaCN52/60) were abundant in the samples, and Hexa-CN66/67 has been reported to have higher relative potency factor value of 0.002 than other PCN congeners (Falandysz et al., 2014). Histopathologic changes in the liver and thymus of female Harlan Sprague-Dawley rats were observed following exposure to Hexa-CN66/67, as well as significant increases in the activity of the cytochromes CYP1A1 and CYP1A2 (Hooth, 2012). The contribution of Hexa-CN66/67 to the toxic equivalent quantity relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ranged from 49.2% to 88.4% (mean: 79.5%). The highest value (88.4%) was observed in samples from Beijing, followed by 87.9% (Jiangxi province) and 87.3% (Sichuan province). Higher Hexa-CN66/ 67 concentrations in human milk samples may indicate a greater health risk. In previous studies of human-derived specimens, Hexa-CN66/67, Penta-CN52/60, and Penta-CN51 were also found to be the predominant congeners (Schiavone et al., 2010; Lunden and Noren, 1998; Pratt, 2013). We also observed high concentrations of CN74 in several provinces, with an average concentration of 670.08, 409.76, 158.35, and 93.84 pg/g lipid in Guangdong, Guangxi, Fujian, and Zhejiang, respectively. A higher concentration of Hepta-CN73 than Hepta-CN74 in human blood samples had been reported (Park et al., 2010; Fromme, 2015), but we did not find high concentrations of Hepta-CN73 in our human milk samples. One possible explanation for the higher CN74 concentrations may be the higher ratio of CN74 to CN73 in PCBs possibly released from electronic waste recycling processes (Yamashita et al., 2000). PCBs in capacitors and transformers can be released into the air during these recycling processes (Wang, 2011); Guangdong and Zhejiang are areas where electronic waste recycling is conducted in China (Wang, 2011; Ma, 2018).
TEQ∑ PCNs =∑ (Ci × REFi)
(1)
where TEQ is the TCDD toxic equivalency concentration in picogram per gram lipid, Ci is the concentration of the ith PCN congener in picogram per gram lipid, and REFi is the relative potency factor for an individual PCN congener.
Subchronic daily intake =
TEQ∑ PCNs × CR × LC × EF × ED BW × AT
(2)
where CR is the human milk contact rate in milliliters per day, LC is the lipid content in grams per 100 mL, EF is the exposure frequency in days per year, ED is the exposure duration in years, BW is body weight, and AT is the averaging time in days, subchronic daily intake in pg toxic equivalency/kg body weight/day. Fig. 3 shows the calculated subchronic daily intake values, which were 0.27–0.95 pg toxic equivalency/kg body weight/day for infants aged 0–6 months, 0.16–0.57 pg toxic equivalency/kg body weight/day for infants aged 7–12 months, and 0.12–0.41 pg toxic equivalency/kg body weight/day for children aged 13–24 months. WHO recommends that the upper range of the tolerable daily intake of 4 pg toxic equivalency/kg body weight/day should be considered a maximal tolerable intake, with the ultimate goal of reducing intake below 1 pg (WHO, 1998). The subchronic daily intake values for infants aged 0–6 month in Jiangxi, Hunan, Guangdong, and Zhejiang provinces were approximately 0.8 pg toxic equivalency/kg body weight/day. This suggests that reducing of PCNs in human milk is required in these provinces for the health consideration of 0 to 6 month old infant. For risk assessment, we estimated both the cancer risk and chronic hazard index using Eqs. (3) and (4), respectively. The cancer risk was assessed to characterize potential carcinogenic effects and probabilities that an individual would develop cancer over a lifetime of exposure. The chronic hazard index was calculated to characterize potential noncarcinogenic effects. PCNs concentrations were converted to toxic equivalency concentrations, and we also used the related slope factor and chronic reference dose of TCDD for the risk assessment; these were obtained from the US Environmental Protection Agency (EPA, 2019). A value of 6.3 × 103 mg/kg body weight/day and the slope factor of hexachlorodibenzo-p-dioxin were used for cancer risk calculation because of inadequate data for TCDD. The chronic reference dose for TCDD was 7 × 10−7 mg/kg body weight/day.
3.2. PCN health risks to nursing infants To identify potential health risks of PCN exposure through breastfeeding, we conducted an exposure and health risk assessment in this study using a method recommended by the United States Environmental Protection Agency (EPA, 1989), with some modification. To the best of our knowledge, this is the first reported exposure and risk assessment of PCNs in human milk. The first step in exposure quantification is the estimation of exposure concentrations. Risk assessment parameters for PCNs are currently not available because of inadequate epidemiological and toxicological data for the congeners. However, PCN congeners display
Cancer risk = subchronic daily intake × slope factor
Chronic hazard index = 4
SDI RfD
(3)
(4)
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Fig. 3. Subchronic daily intake of polychlorinated naphthalenes in human milk (in picograms toxic equivalency per kilogram body weight per day).
Hunan, Guangdong, Zhejiang, Jiangsu, and Beijing, indicating potential non-cancer health effects. Infants are a vulnerable population, and control of PCN contamination in these areas is required, as well as continuous monitoring.
where SDI is the subchronic daily intake and RfD is the chronic reference dose in milligrams per kilogram body weight per day. Table 2 lists the calculated cancer risk and chronic hazard index. The cancer risk for infants from the 19 provinces was in the range of 7.3 × 10−7 to 6 × 10−6. A risk of 10−6 indicates a probability of one chance in a million of an individual developing cancer. A range of 10−4 to 10−7 was the magnitude recommended by the National Oil and Hazardous Substances Pollution Contingency Plan of the US Environmental Protection Agency (EPA, 1994). The calculated cancer risk indicated no significant cancer risk for infants. The calculated chronic hazard index was in the range of 0.38–1.35 for infants aged 0–6 months, 0.27–0.82 for infants aged 7–12 months, and 0.16–0.58 for children aged 13–24 months. When the hazard index for an exposed individual exceeds 1, there may be concern for potential non-cancer health effects (EPA, 1989). Of note, the chronic hazard index for infants aged 0–6 months exceeded 1 for the samples collected in Jiangxi,
3.3. Possible sources of PCNs Industrial synthesis of PCNs, impurities in technical PCBs, and emissions from multiple thermal related industries are the main sources of PCNs in the environment (Fernandes et al., 2017). Congener profiles of PCNs from different sources show a great variability. Congener profiles from industrial thermal sources are typically dominated by lesschlorinated congeners (Liu et al., 2014; Liu, 2015; Abad et al., 1999; Hu, 2013). Technical synthesis of PCNs was known as Halowaxe (HW) series. Low-chlorinated PCN congeners were dominant in HW1000 and HW1031, whereas high-chlorinated PCN congeners were dominant in
Table 2 Estimated cancer risk and chronic hazard index (CHI) values for nursing infants due to intake of mother milk. Province
TEQ (pg/g lw)
CHI 0–6
Jiangsu Henan Hunan Guangdong Guangxi Shanghai Jiangxi Zhejiang Gansu Guizhou Inner Mongolia Hebei Beijing Fujian Jilin Heilongjiang Shaanxi Liaoning Sichuan
0.18 0.11 0.21 0.19 0.12 0.16 0.28 0.25 0.07 0.09 0.09 0.22 0.31 0.27 0.18 0.09 0.08 0.12 0.10
1.13 0.56 1.32 1.30 0.68 0.89 1.35 1.16 0.38 0.44 0.59 0.99 1.04 0.94 0.49 0.45 0.48 0.65 0.78
Risk 6–12
12–24
0.69 0.34 0.80 0.79 0.41 0.54 0.82 0.70 0.23 0.27 0.36 0.60 0.63 0.57 0.30 0.27 0.29 0.39 0.47
0.49 0.24 0.57 0.56 0.29 0.38 0.58 0.50 0.16 0.19 0.25 0.42 0.44 0.40 0.21 0.19 0.21 0.28 0.33
TEQ: Toxic equivalents. 5
0–6 5.0 2.5 5.8 5.7 3.0 3.9 6.0 5.1 1.7 2.0 2.6 4.4 4.6 4.1 2.2 2.0 2.1 2.9 3.4
6–12 × × × × × × × × × × × × × × × × × × ×
−6
10 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6
3.0 1.5 3.5 3.5 1.8 2.4 3.6 3.1 1.0 1.2 1.6 2.6 2.8 2.5 1.3 1.2 1.3 1.7 2.1
× × × × × × × × × × × × × × × × × × ×
12–24 −6
10 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6
2.1 1.1 2.5 2.5 1.3 1.7 2.6 2.2 7.3 8.4 1.1 1.9 2.0 1.8 9.3 8.4 9.2 1.2 1.5
× × × × × × × × × × × × × × × × × × ×
10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−7 10−7 10−6 10−6 10−6 10−6 10−7 10−7 10−7 10−6 10−6
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which was not realized and expected before.
HW1051, and Tri-CNs and Penta-CNs were major congeners in other Halowaxe series (Yamashita et al., 2003; Noma et al., 2004). Lowerchlorinated PCB mixtures have been shown to contain relatively greater percentages of less-chlorinated naphthalene congeners, while more highly chlorinated PCBs contained greater proportions of more-chlorinated naphthalenes (Yamashita et al., 2000; Huang, 2015). To identify the sources of PCNs in the human milk samples, we conducted Pearson’s correlation matrix analysis to assess the correlation between PCN patterns in human milk and in PCN sources. Principal component analysis can be used to group environmental samples by emission characteristics (Liu et al., 2014; Park et al., 2010), but cannot provide a detailed correlation between groups. We analyzed seven Halowaxe series (HW1000, HW1001, HW1013, HW1014, HW1031, HW1051, and HW1099), 18 PCB mixtures (series of Aroclors, Kanechlors, Clophens, Phenoclors, Sovol, and Chlorofen), and two important thermal processes (municipal waste incineration and iron foundries). Concentrations of PCNs from the three sources were derived from the literature (Yamashita et al., 2000; Liu et al., 2014; Noma et al., 2004). The concentrations of each congener were normalized to the total PCN concentration before data analysis. Fig. 4 shows the correlation between PCNs in human milk samples from the 19 provinces and PCN sources. We found strong correlations between PCNs emitted from thermal processes and PCNs in human milk from most provinces, with the exception of Guangdong, Guangxi, Hunan, Fujian, Zhejiang, and Jiangsu. The HW1000, HW1001, HW1014, and HW1099 PCN products were correlated with PCN levels in samples from several provinces. HW1014 was correlated with milk samples from Jiangsu, Zhejiang, and Hunan provinces, suggesting a higher contribution of industrial synthesis than thermal processes in these provinces. Correlations between PCNs in human milk and PCNs in PCB mixtures were significantly weaker than correlations with thermal processes or industrial synthesis, although PCNs in human milk from Guangdong province correlated well with Phenoclor DP5 and those from Zhejiang province with Phenoclor DP4. Of note, as mentioned above, we observed high chronic hazard index in Zhejiang, Guangodng, Hunan, Jiangxi and Jiangsu province. PCNs in human milk from these provinces were found to have a relatively closer correlation with technical PCNs products (HW1014) or PCNs as impurity of PCBs products (Phenoclor DP5 and Phenoclor DP4). PCN Congeners in these products were dominated by higher chlorinated naphthalenes, which have a relatively higher relative potency factor. This may explain the high chronic hazard index in these provinces. Overall, unintentional emission of polychlorinated naphthalenes from industries thermal processes appears to be the main source for PCNs in human milk in most provinces of China. The correlations of PCNs between human milks and PCNs as impurities in PCB formulations indicated the additional PCNs contamination resulted from the historically production and use of technical PCB formulations,
4. Conclusions Our findings indicate the complexity and regional variability of PCN profiles and sources in human milk. Cancer risk posed to nursing infants was not significant, but potential non-carcinogenic adverse health effects were suggested and should be emphasized in some sampling areas. Unintentional emission of PCNs from industries that employ thermal processes appears to be the main source for PCNs in human milk in most sampling areas. PCNs as impurities in PCB mixtures may be an important and hitherto unrecognized source for PCNs in human milk from province that with large electronic waste recycling activities. We recommend continuous monitoring and reduction of PCNs in the environment to minimize exposure. CRediT authorship contribution statement Cui Li: Investigation, Writing - original draft, Formal analysis, Data curation, Visualization. Lei Zhang: Formal analysis, Validation. Jingguang Li: Conceptualization, Resources, Funding acquisition. Yihao Min: Formal analysis, Writing - review & editing. Lili Yang: Resources, Writing - review & editing. Minghui Zheng: Conceptualization, Supervision, Funding acquisition. Yongning Wu: Conceptualization, Supervision, Funding acquisition. Yuanping Yang: Formal analysis, Writing - review & editing. Linjun Qin: Formal analysis, Writing - review & editing. Guorui Liu: Conceptualization, Supervision, Methodology, Writing - review & editing, Funding acquisition, Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We gratefully acknowledge support from the National Key R&D Program of China (grant no. 2017YFC1600502), Beijing Natural Science Foundation (8182052) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2016038) and CAS Interdisciplinary Innovation Team (JCTD-2019-03). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.envint.2019.105436.
Fig. 4. Correlation between polychlorinated naphthalenes from three potential sources and breast milk samples from 19 provinces in China. (a) thermal processes; (b) industrial synthesis; (c) impurities in polychlorinated biphenyls. 6
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Polychlorinated naphthalenes in human milk: Health risk assessment to nursing infants and source analysis
T
Cui Lia,b, Lei Zhangc, Jingguang Lic, Yihao Mind, Lili Yanga,b, Minghui Zhenga,b,e, Yongning Wuc, ⁎ Yuanping Yanga,b, Linjun Qina,b, Guorui Liua,b,e, a
State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China b College of Resources and Environment, University of Chinese Academy of Sciences, Beijing 100190, PR China c China National Center for Food Safety Risk Assessment, Beijing 100022, PR China d College of Science, China Agricultural University, Beijing 100083, PR China e Institute of Environment and Health, Hangzhou Institute for Advanced Study, UCAS, Hangzhou, PR China
A R T I C LE I N FO
A B S T R A C T
Handling Editor: Da Chen
Polychlorinated naphthalenes are teratogenic environmental contaminants. Mother milk is the most important food for nursing infants. The World Health Organization actively promotes breastfeeding for its immunological, psychological, and economic advantages. We firstly measured concentrations of polychlorinated naphthalenes in human milk from 19 provinces in China and estimated their potential health risks to nursing infants and their possible sources. Concentrations ranged from 211.07 to 2497.43 pg/g lipid. The high prevalence of highly toxic hexachlorinated naphthalenes (Hexa-CN66/67) in human milk samples indicated a higher health risk in the sampling areas. Cancer risk posed to nursing infants was not significant, but potential non-carcinogenic adverse health effects were suggested and should be emphasized in some sampling areas. Unintentional emission of polychlorinated naphthalenes from industries that employ thermal processes appears to be the main source for PCNs in human milk in most sampling areas. Correlation analysis also suggested PCNs as impurities in polychlorinated biphenyl mixtures as a previously unrecognized source of polychlorinated naphthalenes in human milk.
Keywords: Polychlorinated naphthalenes (PCNs) Human milk Exposure Risk assessment Source analysis
1. Introduction Persistent organic pollutants (POPs) are highly toxic organic chemicals. The Word Health Organization (WHO) has been carrying out global surveys on POPs in human milk for more than three decades, mainly focusing on polychlorinated dibenzo-p-dioxins (PCDDs), polychlorinated dibenzofurans (PCDFs), polychlorinated biphenyls (PCBs) and DDT (van den Berg, 2017). However, studies on polychlorinated naphthalenes (PCNs) in human milk are very scarce. PCNs with one or more chlorine atoms as substituents on the naphthalene ring have raised concerns due to their potential toxicity. Several PCN congeners have physicochemical and toxic properties similar to those of dioxins, such as a tendency to accumulate in biological tissues (Hayward, 1998; Falandysz, 2003), induce aryl hydrocarbon receptor-mediated responses (Barc and Gregoraszczuk, 2014; Villeneuve, 2000; Blankenship, 2000), and exert adverse health effects (Stragierowicz et al., 2018;
Pardyak, 2016; Galoch et al., 2006; Popp, 1997; Ward, 1996; Li et al., 2011). PCNs have been shown to be potent fetotoxic and teratogenic contaminants in animal experiments (Kilanowicz et al., 2011), and exposure can induce biochemical and histopathologic changes in the liver and thymus (Hootha, 2012). Moreover, occupational exposure to PCNs has been reported to cause liver toxicity (Popp, 1997; Ward, 1996), chloracne, and even death (Hayward, 1998); symptoms of occupational PCN exposure include digestive problems, anorexia, nausea, and vertigo (Hayward, 1998). PCNs have been listed as persistent organic pollutants by the Stockholm Convention in 2015 (UNEP, 2019), with a focus on reducing exposure worldwide. PCNs are ubiquitous in the environment, and have even been found in polar areas (Harner et al., 1998). Environmental PCNs could be ascribed to three categories of sources including synthesis as technical PCNs formulations, impurities in the technical polychlorinated biphenyls (PCBs), and unintentional releases via thermal processes. Global
⁎ Corresponding author at: State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China. E-mail address: [email protected] (G. Liu).
https://doi.org/10.1016/j.envint.2019.105436 Received 19 October 2019; Received in revised form 13 December 2019; Accepted 20 December 2019 0160-4120/ © 2019 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/).
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(Thermo Fisher Scientific, Waltham, MA, USA) at 120 °C and 1500 psi. The extraction solvent was a mixture of hexane and dichloromethane (1:1, V:V). The extracts were spiked with a mixture of 1 ng 13C-labeled PCN internal standard (ECN-5102, including CN-27, CN-42, CN-52, CN67, CN-73, and CN-75) and concentrated to dryness for gravimetric lipid determination. The residues were then reconstituted with 5 mL of hexane. Three columns were used: The first column was an acid silica column with an elution of 90 mL of hexane. The second column was a multi-layer silica column with an elution of 90 mL of hexane. The third column was a basic alumina column and the elution was a 100-mL mixture of hexane and dichloromethane (95:5, V:V). Next, the eluted fractions were concentrated to about 20 μL and spiked with 1 ng 13Clabeled injection standard for high-resolution gas chromatography/ high-resolution mass spectrometry analysis. ECN-5102 13C10-labeled PCN internal standard and ECN-5260 13C10-labeled injection standard (including CN-64) were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Pesticide residue-grade hexane and dichloromethane were purchased from J.T. Baker Chemical Co. (Phillipsburg, NJ, USA).
synthesis of technical PCNs formulations has been estimated at approximately 150,000 metric tons (Falandysz, 1998). Concentration of PCNs as impurities in PCB formulations is estimated at 870 μg/g (Yamashita et al., 2000; Huang, 2015; Domingo, 2004). Unintentional formation and emission from industrial activities, including waste incineration and metallurgical processes, are a growing concern (Liu, 2012; Liu et al., 2014; Jiang, 2015) considering the restriction of PCNs and PCBs as technical products. The massive historical production and releases of PCNs as well as the on-going releases from numerous industrial sources underscore the need to assess human PCN body burdens and their associated health risks. Human exposure to PCNs is inevitable including pathways of air inhalation, dietary intake and so on. The occurrences of PCNs in air, edible biota have been reported (Rotander, 2012; Braune and Muir, 2017; Cui, 2018; Fernandes, 2010; Egeback et al., 2004) and there are thousands of industrial sources of unintentional PCNs and also historically PCBs manufactures in China. Understanding human PCN body burden is significant for their potential health risk assessment. Human milk is considered as one of the core monitoring matrices of the United Nations Environment Programme’s persistent organic pollutants monitoring program with the aim of evaluating the effectiveness of implementing Stockholm Convention. Human milk monitoring can provide exposure information on both the mother and the breastfed infant through a non-invasive method of collection. Mother milk is the most important food for nursing infants. The World Health Organization also actively promotes breastfeeding for its immunological, psychological, and economic advantages, and a feeding mode of exclusive breastfeeding for six months followed by continuous breastfeeding with appropriate complementary foods for up to two years or beyond is recommended for the vast majority of infants (WHO, 2008). If significant PCN present in mother milk, this route of continuous PCN exposure during early life might cause long-term damage. Although a few studies measured PCN levels in human serum and adipose tissues (Park et al., 2010; Horii, 2010; Fromme, 2015; Weistrand and Noren, 1998; Schiavone et al., 2010; Kunisue et al., 2009), data on PCN loads in human milk and their potential health risks to nursing infants are not reported (Lunden and Noren, 1998; Pratt, 2013). In the present study, we measured the concentrations of 75 PCN congeners in 77 pooled human milk samples from 3790 individual samples collected from 19 provinces in China. Analysis was carried out by isotope dilution high-resolution gas chromatography/high-resolution mass spectrometry. To the best of our knowledge, this is the first study to report PCN loads in human milk and assess their potential sources and health risks to nursing infants in China. It was expected that this study could provide important information on PCN internal exposures and potential risks to nursing infants not only for China, but also for other countries by fulfilling the international Stockholm Convention.
2.2. Instrumental analysis Congener-specific analysis of PCNs was carried out by high-resolution gas chromatography combined with high-resolution mass spectrometry (Thermo Fisher Scientific, Waltham, MA, USA). Gas chromatographic conditions were as follows: the injection volume was 1 μL in splitless injection mode; the carrier gas was helium; the column was a DB-5MS capillary column (60 m × 0.25 mm × 0.25 μm, Agilent Technologies, Santa Clara, CA, USA); the inlet temperature was 260 °C; the carrier gas flow rate was 1 mL/min. The temperature program was 80 °C for 2 min, increased by 20 °C/min to 180 °C and held for 1 min, then increased by 2.5 °C/min to 280 °C and held for 2 min, and finally increased to 290 °C at 10 °C/min and held for 5 min. There were 57 peaks on the column, including 41 single peaks and 16 composite peaks. The calibration curves were obtained from CS1-11 standard solutions ranging from 1 ppm to 1000 ppm. Standard solutions were mixed with PCN-MXA, PCN-MXC, PN-31S, which were obtained from Wellington Laboratories (Ontario, Canada), and ECN-2663, ECN-2664, ECN-2665, ECN-2641, ECN-2653, ECN-2623, ECN-2623, ECN-2620, ECN-2621, which were purchased from Cambridge Isotope Laboratories (Tewksbury, MA, USA). Mass spectrometry conditions: ionization mode was electron impact; the source temperature was 280 °C; electron energy was 45 eV; transmission line temperature was 290 °C; reference material was perfluorokerosene; the mass spectrometry acquisition method was selective ion monitoring. To ensure the accuracy of the quantification, the resolution of the mass spectrometer was tuned to about 10,000. The data were processed with TargetQuan software (Thermo Fisher Scientific, Waltham, MA, USA). Statistical analysis of the data was conducted using SPSS version 25 and R version 3.5.1. Half of the limit of detection value was assigned for congeners with concentrations below the limit of detection.
2. Materials and methods 2.1. Sample collection and pretreatment Sample collection was conducted by the China National Center for Food Safety Risk Assessment (CFSA) following the fourth WHO-coordinated survey of human milk for persistent organic pollutants in cooperation with the United Nations Environment Programme (UNEP) (WHO, 2007). A total of 3790 human milk samples were collected between 2017 and 2019 from 77 cities/counties around China. At least 50 individual human milk samples were collected from each city sampling site and more than 30 human milk samples from each county sampling site. Detailed information about the samples is shown in Table S1. All samples were stored in a refrigerator at −20 °C until analysis. Prior to extraction, all samples were freeze-dried for more than 72 h and homogenized. Approximately 1 g dry weight of sample was mixed with 2–3 g of diatomaceous earth, then extracted using ASE350
2.3. Quality assurance and quality control The diatomaceous earth was pre-extracted before sample extraction. One procedural blank sample was analyzed with each batch of samples to evaluate laboratory interference and contamination. Only a small amount of monochlorinated, dichlorinated, and trichlorinated naphthalenes was detected in the blank samples, which was 10% lower than concentrations in the samples. The PCNs concentrations in samples were not corrected using values from blanks. The average recoveries of 13 C10-labeled CN-27, CN-42, CN-52, CN-67, CN-73, and CN-75 were 54–100%, which was satisfactory for the PCN trace analysis (Method, 1625). The limit of detection for each congener was set as three times 2
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Fig. 1. Distribution (a) and concentrations (b) of polychlorinated naphthalenes (PCNs) in human milk from 19 provinces in China.
lowest levels were found in Inner Mongolia. The regional distribution of PCDD/Fs in human is similar to that of PCNs in our study, but the overall concentration of PCDD/Fs is much lower than PCNs. The last study of PCNs in human milk was conducted in Ireland and published in 2013; only 12 PCN congeners were measured, ranging in level from 59 to 168 pg/g lipid (Pratt, 2013). Average concentration of the sum of the 12 congeners in our study was 180.76 pg/g lipid, which is a little bit higher than Ireland. According to comparison with the very limited data, our findings indicate a slightly higher level of PCN exposure through human milk in the Chinese general population. A Swedish study published over two decades ago reported a notable decrease, from 3081 to 483 pg/g lipid, in the concentrations of PCNs in human milk between 1972 and 1992 (Lunden and Noren, 1998). Unfortunately, they did not give an explanation for the decrease. Of note, concentration of PCNs in human milk from Ireland was in good consistent with the decreasing trend found in Sweden but is different in China where we could found concentrations above 2000 pg/g lw in samples collected during 2017 to 2019. This indicated a variability of PCNs contamination in human milk from different countries. Congener profiles of PCNs in human milk samples from 19 provinces in China varied by region (Fig. 2). Some profiles were dominated by dichlorinated and trichlorinated naphthalenes, which accounted for 68.42% of the total, while others were dominated by pentachlorinated and heptachlorinated naphthalenes, which accounted for 31.58% of the total. High concentrations of heptachlorinated naphthalenes were found in Guangdong, Guangxi, and Fujian provinces, accounting for 63%, 38%, and 29% of total PCNs, respectively. Behnisch et al. reported higher bioactivity for tetrachlorinated to octachlorinated naphthalenes
the signal-to-noise ratio. PCN congener peaks were identified by comparing the retention times of target congeners to available standards. The abundance ratio of the target ion and the qualifier ion had to be within 15% of the theoretical value.
3. Results and discussion 3.1. Levels and congener profiles of ∑75PCN in human milk PCNs were detected in all 77 pooled samples analyzed in this study. Fig. 1 shows the distribution and concentrations of ∑75 PCN in human milk from the 19 provinces. Result from one-sample T test (p-value < 0.01) indicated a significant regional variability, which ranged widely from < 300 pg/g lipid in Gansu and Shanxi provinces to > 2000 pg/g lipid in Fujian and Guangdong provinces. Data on PCN levels in human milk are scarce; for comparisons with reports from other regions, we compiled all the available data on PCN loads in human specimens (Table 1). The PCN levels we found were lower than those reported in adipose tissue samples from Canada (Williams et al., 1993), Sweden (Witt and Niessen, 2000), or Italy (Schiavone et al., 2010), and comparable with those in adipose tissue samples from the US (Kunisue et al., 2009). Levels measured in blood samples in Korea (Park et al., 2010) and Germany (Kunisue et al., 2009) were comparable to the levels we recorded, but higher in blood samples from an industrial city in China (Jin, 2019). According to a previous study (Zhang et al., 2016), concentrations of PCDD/Fs in human milk from 16 provinces in China ranged from 24.8 pg/g lipid to 488.8 pg/g lipid and relatively high levels were found in Guangdong and Guangxi provinces while the
Table 1 Levels of polychlorinated naphthalenes in human samples (picograms per gram lipid). Country
Year
Sample type
Congeners
Range/Mean
Reference
Sweden Canada Sweden Sweden Germanya the USA the USA Italy Korea Ireland German China China
1972–1992 1993 1998 1998 2000 2002–2003 2003–2005 2005–2006 2007 2010 2013–2014 2016 2017–2019
human milk adipose adipose liver adipose plasma adipose adipose blood human milk blood blood human milk
TeCN-OCN 4 22 22 – – 38 – TeCN-OCN 12 TeCN-OCN MoCN-OCN MoCN-OCN
483–3081 1020–3650 999–3909 1375–26113 1700–8500 345–1540 21–2500 500–14000 2170 59–168 101–1406 14300–50700 211–2497
Lunden and Noren (1998) Williams et al. (1993) Witt and Niessen (2000) Witt and Niessen (2000) Kunisue et al. (2009) Horii (2010) Kunisue et al. (2009) Schiavone et al. (2010) Park et al. (2010) Pratt (2013) Fromme (2015) Jin (2019) This study
a
Data from Germany, Russia and Kazakhstan; -: without data; Year refers to period of data collection. 3
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toxicities similar to those of dioxins. Thus, we converted the concentrations of PCNs to toxic equivalent quantity concentrations relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) using Eq. (1). To the best of our knowledge, only 36 PCN congeners has reported relative potency factors (Falandysz et al., 2014). Therefore, we targeted these 36 congeners during the calculation of toxic equivalent quantity concentrations in this study. The relative potency factors of individual PCN congeners to TCDD are listed in Table S2. The second step is the calculation of intake using Eq. (2). Calculation of subchronic daily intake is recommended for exposure periods between 2 weeks and 7 years (EPA, 1989); in this study, we used a feeding mode of 2 years divided into three stages: 0–6, 7–12, and 13–24 months, using a human milk contact rate of 750, 600, and 500 mL/day, respectively (WHO, 2001; CNS, 2016). The exposure frequency was 360 day/year. Exposure durations of 0.5 year, 0.5 year, and 1 year were used for the exposure periods of 0–6, 7–12, and 13–24 months, respectively. Lipid content in human milk was taken as the mean value of samples from the same province. Body weights (MEPC, 2016) of 7.2, 9.5, and 11.2 kg evaluated in China were used for the exposure durations of 0–6, 7–12, and 13–24 months, respectively. Averaging time was 182.5, 182.5, and 365 days, respectively.
Fig. 2. Congener profiles of polychlorinated naphthalenes in human milk from 19 provinces in China.
in vitro (Behnisch et al., 2003), indicating a possible higher risk in the areas where higher-chlorination congener profiles were found. Some PCN congeners (Tri-CN24/14, Penta-CN51, Hexa-CN66/67, and PentaCN52/60) were abundant in the samples, and Hexa-CN66/67 has been reported to have higher relative potency factor value of 0.002 than other PCN congeners (Falandysz et al., 2014). Histopathologic changes in the liver and thymus of female Harlan Sprague-Dawley rats were observed following exposure to Hexa-CN66/67, as well as significant increases in the activity of the cytochromes CYP1A1 and CYP1A2 (Hooth, 2012). The contribution of Hexa-CN66/67 to the toxic equivalent quantity relative to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) ranged from 49.2% to 88.4% (mean: 79.5%). The highest value (88.4%) was observed in samples from Beijing, followed by 87.9% (Jiangxi province) and 87.3% (Sichuan province). Higher Hexa-CN66/ 67 concentrations in human milk samples may indicate a greater health risk. In previous studies of human-derived specimens, Hexa-CN66/67, Penta-CN52/60, and Penta-CN51 were also found to be the predominant congeners (Schiavone et al., 2010; Lunden and Noren, 1998; Pratt, 2013). We also observed high concentrations of CN74 in several provinces, with an average concentration of 670.08, 409.76, 158.35, and 93.84 pg/g lipid in Guangdong, Guangxi, Fujian, and Zhejiang, respectively. A higher concentration of Hepta-CN73 than Hepta-CN74 in human blood samples had been reported (Park et al., 2010; Fromme, 2015), but we did not find high concentrations of Hepta-CN73 in our human milk samples. One possible explanation for the higher CN74 concentrations may be the higher ratio of CN74 to CN73 in PCBs possibly released from electronic waste recycling processes (Yamashita et al., 2000). PCBs in capacitors and transformers can be released into the air during these recycling processes (Wang, 2011); Guangdong and Zhejiang are areas where electronic waste recycling is conducted in China (Wang, 2011; Ma, 2018).
TEQ∑ PCNs =∑ (Ci × REFi)
(1)
where TEQ is the TCDD toxic equivalency concentration in picogram per gram lipid, Ci is the concentration of the ith PCN congener in picogram per gram lipid, and REFi is the relative potency factor for an individual PCN congener.
Subchronic daily intake =
TEQ∑ PCNs × CR × LC × EF × ED BW × AT
(2)
where CR is the human milk contact rate in milliliters per day, LC is the lipid content in grams per 100 mL, EF is the exposure frequency in days per year, ED is the exposure duration in years, BW is body weight, and AT is the averaging time in days, subchronic daily intake in pg toxic equivalency/kg body weight/day. Fig. 3 shows the calculated subchronic daily intake values, which were 0.27–0.95 pg toxic equivalency/kg body weight/day for infants aged 0–6 months, 0.16–0.57 pg toxic equivalency/kg body weight/day for infants aged 7–12 months, and 0.12–0.41 pg toxic equivalency/kg body weight/day for children aged 13–24 months. WHO recommends that the upper range of the tolerable daily intake of 4 pg toxic equivalency/kg body weight/day should be considered a maximal tolerable intake, with the ultimate goal of reducing intake below 1 pg (WHO, 1998). The subchronic daily intake values for infants aged 0–6 month in Jiangxi, Hunan, Guangdong, and Zhejiang provinces were approximately 0.8 pg toxic equivalency/kg body weight/day. This suggests that reducing of PCNs in human milk is required in these provinces for the health consideration of 0 to 6 month old infant. For risk assessment, we estimated both the cancer risk and chronic hazard index using Eqs. (3) and (4), respectively. The cancer risk was assessed to characterize potential carcinogenic effects and probabilities that an individual would develop cancer over a lifetime of exposure. The chronic hazard index was calculated to characterize potential noncarcinogenic effects. PCNs concentrations were converted to toxic equivalency concentrations, and we also used the related slope factor and chronic reference dose of TCDD for the risk assessment; these were obtained from the US Environmental Protection Agency (EPA, 2019). A value of 6.3 × 103 mg/kg body weight/day and the slope factor of hexachlorodibenzo-p-dioxin were used for cancer risk calculation because of inadequate data for TCDD. The chronic reference dose for TCDD was 7 × 10−7 mg/kg body weight/day.
3.2. PCN health risks to nursing infants To identify potential health risks of PCN exposure through breastfeeding, we conducted an exposure and health risk assessment in this study using a method recommended by the United States Environmental Protection Agency (EPA, 1989), with some modification. To the best of our knowledge, this is the first reported exposure and risk assessment of PCNs in human milk. The first step in exposure quantification is the estimation of exposure concentrations. Risk assessment parameters for PCNs are currently not available because of inadequate epidemiological and toxicological data for the congeners. However, PCN congeners display
Cancer risk = subchronic daily intake × slope factor
Chronic hazard index = 4
SDI RfD
(3)
(4)
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Fig. 3. Subchronic daily intake of polychlorinated naphthalenes in human milk (in picograms toxic equivalency per kilogram body weight per day).
Hunan, Guangdong, Zhejiang, Jiangsu, and Beijing, indicating potential non-cancer health effects. Infants are a vulnerable population, and control of PCN contamination in these areas is required, as well as continuous monitoring.
where SDI is the subchronic daily intake and RfD is the chronic reference dose in milligrams per kilogram body weight per day. Table 2 lists the calculated cancer risk and chronic hazard index. The cancer risk for infants from the 19 provinces was in the range of 7.3 × 10−7 to 6 × 10−6. A risk of 10−6 indicates a probability of one chance in a million of an individual developing cancer. A range of 10−4 to 10−7 was the magnitude recommended by the National Oil and Hazardous Substances Pollution Contingency Plan of the US Environmental Protection Agency (EPA, 1994). The calculated cancer risk indicated no significant cancer risk for infants. The calculated chronic hazard index was in the range of 0.38–1.35 for infants aged 0–6 months, 0.27–0.82 for infants aged 7–12 months, and 0.16–0.58 for children aged 13–24 months. When the hazard index for an exposed individual exceeds 1, there may be concern for potential non-cancer health effects (EPA, 1989). Of note, the chronic hazard index for infants aged 0–6 months exceeded 1 for the samples collected in Jiangxi,
3.3. Possible sources of PCNs Industrial synthesis of PCNs, impurities in technical PCBs, and emissions from multiple thermal related industries are the main sources of PCNs in the environment (Fernandes et al., 2017). Congener profiles of PCNs from different sources show a great variability. Congener profiles from industrial thermal sources are typically dominated by lesschlorinated congeners (Liu et al., 2014; Liu, 2015; Abad et al., 1999; Hu, 2013). Technical synthesis of PCNs was known as Halowaxe (HW) series. Low-chlorinated PCN congeners were dominant in HW1000 and HW1031, whereas high-chlorinated PCN congeners were dominant in
Table 2 Estimated cancer risk and chronic hazard index (CHI) values for nursing infants due to intake of mother milk. Province
TEQ (pg/g lw)
CHI 0–6
Jiangsu Henan Hunan Guangdong Guangxi Shanghai Jiangxi Zhejiang Gansu Guizhou Inner Mongolia Hebei Beijing Fujian Jilin Heilongjiang Shaanxi Liaoning Sichuan
0.18 0.11 0.21 0.19 0.12 0.16 0.28 0.25 0.07 0.09 0.09 0.22 0.31 0.27 0.18 0.09 0.08 0.12 0.10
1.13 0.56 1.32 1.30 0.68 0.89 1.35 1.16 0.38 0.44 0.59 0.99 1.04 0.94 0.49 0.45 0.48 0.65 0.78
Risk 6–12
12–24
0.69 0.34 0.80 0.79 0.41 0.54 0.82 0.70 0.23 0.27 0.36 0.60 0.63 0.57 0.30 0.27 0.29 0.39 0.47
0.49 0.24 0.57 0.56 0.29 0.38 0.58 0.50 0.16 0.19 0.25 0.42 0.44 0.40 0.21 0.19 0.21 0.28 0.33
TEQ: Toxic equivalents. 5
0–6 5.0 2.5 5.8 5.7 3.0 3.9 6.0 5.1 1.7 2.0 2.6 4.4 4.6 4.1 2.2 2.0 2.1 2.9 3.4
6–12 × × × × × × × × × × × × × × × × × × ×
−6
10 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6
3.0 1.5 3.5 3.5 1.8 2.4 3.6 3.1 1.0 1.2 1.6 2.6 2.8 2.5 1.3 1.2 1.3 1.7 2.1
× × × × × × × × × × × × × × × × × × ×
12–24 −6
10 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6
2.1 1.1 2.5 2.5 1.3 1.7 2.6 2.2 7.3 8.4 1.1 1.9 2.0 1.8 9.3 8.4 9.2 1.2 1.5
× × × × × × × × × × × × × × × × × × ×
10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−6 10−7 10−7 10−6 10−6 10−6 10−6 10−7 10−7 10−7 10−6 10−6
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which was not realized and expected before.
HW1051, and Tri-CNs and Penta-CNs were major congeners in other Halowaxe series (Yamashita et al., 2003; Noma et al., 2004). Lowerchlorinated PCB mixtures have been shown to contain relatively greater percentages of less-chlorinated naphthalene congeners, while more highly chlorinated PCBs contained greater proportions of more-chlorinated naphthalenes (Yamashita et al., 2000; Huang, 2015). To identify the sources of PCNs in the human milk samples, we conducted Pearson’s correlation matrix analysis to assess the correlation between PCN patterns in human milk and in PCN sources. Principal component analysis can be used to group environmental samples by emission characteristics (Liu et al., 2014; Park et al., 2010), but cannot provide a detailed correlation between groups. We analyzed seven Halowaxe series (HW1000, HW1001, HW1013, HW1014, HW1031, HW1051, and HW1099), 18 PCB mixtures (series of Aroclors, Kanechlors, Clophens, Phenoclors, Sovol, and Chlorofen), and two important thermal processes (municipal waste incineration and iron foundries). Concentrations of PCNs from the three sources were derived from the literature (Yamashita et al., 2000; Liu et al., 2014; Noma et al., 2004). The concentrations of each congener were normalized to the total PCN concentration before data analysis. Fig. 4 shows the correlation between PCNs in human milk samples from the 19 provinces and PCN sources. We found strong correlations between PCNs emitted from thermal processes and PCNs in human milk from most provinces, with the exception of Guangdong, Guangxi, Hunan, Fujian, Zhejiang, and Jiangsu. The HW1000, HW1001, HW1014, and HW1099 PCN products were correlated with PCN levels in samples from several provinces. HW1014 was correlated with milk samples from Jiangsu, Zhejiang, and Hunan provinces, suggesting a higher contribution of industrial synthesis than thermal processes in these provinces. Correlations between PCNs in human milk and PCNs in PCB mixtures were significantly weaker than correlations with thermal processes or industrial synthesis, although PCNs in human milk from Guangdong province correlated well with Phenoclor DP5 and those from Zhejiang province with Phenoclor DP4. Of note, as mentioned above, we observed high chronic hazard index in Zhejiang, Guangodng, Hunan, Jiangxi and Jiangsu province. PCNs in human milk from these provinces were found to have a relatively closer correlation with technical PCNs products (HW1014) or PCNs as impurity of PCBs products (Phenoclor DP5 and Phenoclor DP4). PCN Congeners in these products were dominated by higher chlorinated naphthalenes, which have a relatively higher relative potency factor. This may explain the high chronic hazard index in these provinces. Overall, unintentional emission of polychlorinated naphthalenes from industries thermal processes appears to be the main source for PCNs in human milk in most provinces of China. The correlations of PCNs between human milks and PCNs as impurities in PCB formulations indicated the additional PCNs contamination resulted from the historically production and use of technical PCB formulations,
4. Conclusions Our findings indicate the complexity and regional variability of PCN profiles and sources in human milk. Cancer risk posed to nursing infants was not significant, but potential non-carcinogenic adverse health effects were suggested and should be emphasized in some sampling areas. Unintentional emission of PCNs from industries that employ thermal processes appears to be the main source for PCNs in human milk in most sampling areas. PCNs as impurities in PCB mixtures may be an important and hitherto unrecognized source for PCNs in human milk from province that with large electronic waste recycling activities. We recommend continuous monitoring and reduction of PCNs in the environment to minimize exposure. CRediT authorship contribution statement Cui Li: Investigation, Writing - original draft, Formal analysis, Data curation, Visualization. Lei Zhang: Formal analysis, Validation. Jingguang Li: Conceptualization, Resources, Funding acquisition. Yihao Min: Formal analysis, Writing - review & editing. Lili Yang: Resources, Writing - review & editing. Minghui Zheng: Conceptualization, Supervision, Funding acquisition. Yongning Wu: Conceptualization, Supervision, Funding acquisition. Yuanping Yang: Formal analysis, Writing - review & editing. Linjun Qin: Formal analysis, Writing - review & editing. Guorui Liu: Conceptualization, Supervision, Methodology, Writing - review & editing, Funding acquisition, Project administration. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments We gratefully acknowledge support from the National Key R&D Program of China (grant no. 2017YFC1600502), Beijing Natural Science Foundation (8182052) and the Youth Innovation Promotion Association of the Chinese Academy of Sciences (2016038) and CAS Interdisciplinary Innovation Team (JCTD-2019-03). Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.envint.2019.105436.
Fig. 4. Correlation between polychlorinated naphthalenes from three potential sources and breast milk samples from 19 provinces in China. (a) thermal processes; (b) industrial synthesis; (c) impurities in polychlorinated biphenyls. 6
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