Inverse age-dependent accumulation of decabromodiphenyl ether and other PBDEs in serum from a general adult population

Mercè Garí and Joan O. Grimalt *

Department of Environmental Chemistry, Institute of Environmental Assessment and Water Research (IDAEA). Spanish Council for Scientific Research (CSIC). Jordi Girona, 18. 08034 Barcelona. Catalonia. Spain

* Author for correspondence. Phone: +34 93 4006118; Fax: +34 93 2045904; E-mail:

Keywords

Age-dependence; Catalonia; Decabromodiphenyl ether; Human exposure; Polybromodiphenyl ethers; Serum samples

Abbreviations: AMAP, Arctic Monitoring and Assessment Programme; BDE, brominated diphenyl ether; BMI, body mass index; LD, limit of detection; LQ, limit of quantification; MS, mass spectrometry; NHANES, National Health and Nutrition Examination Survey; NICI, negative ion chemical ionization; PBDE, polybromodiphenyl ether; POP, persistent organic pollutant.

Abstract

Polybromodiphenyl ethers (PBDEs), including the decabromodiphenyl congener (BDE-209), were determined in serum of 731 individuals from a general adult population (18-74 years) collected in 2002 in Catalonia (north-eastern Spain). The BDE-209 was the predominant congener (median 3.7 ng/g lipid) followed by BDE-47 (2.6 ng/g lipid) and BDE-99 (1.2 ng/g lipid). PBDEs in this population (median 15.4 ng/g lipid) ranked among the highest of previously described concentrations in populations in Europe, Asia, New Zealand and Australia, yet it was lower than those found in North American reports. Age was clearly the socio-demographic factor of highest influence on the PBDE distributions. However, unlike usual trends of higher accumulation of POPs through age, the higher concentrations were found in young individuals (< 30 years) rather than in adults (≥ 30 years), with differences of 14%, 31% and 46% in the most abundant congeners (i.e. BDE-209, BDE99 and BDE-47, respectively). This age-dependent distribution of PBDEs (including the case for BDE-209, which is shown for the first time in this study) is explained by the higher and widespread use of these compounds since the 1980s. In view that these compounds remain highly used, this accumulation pattern is likely to evolve, anticipating an increasing level of PBDE concentrations in future general population surveys, yet probably assuming an age-dependent increase pattern. Socio-economic level was also a determinant of BDE-47 concentrations, but only relevant for the least affluent class, suggesting that lifestyle and environmental conditions in the dwelling place may also contribute to exposure. Nonetheless, gender, body mass index, place of birth, parity and education level did not show any statistically significant influence on the observed PBDE distributions.

1. Introduction

Polybromodiphenyl ethers (PBDEs) have been used as flame retardants for some decades in a wide range of products. They have been distributed in three commercial mixtures of congeners with different levels of bromination (penta-BDE, octa-BDE, and deca-BDE), which are named after the dominating homologue group. The pentabrominated formulation was the major source of BDE-47 and BDE-99 congeners and was mainly employed as additive of polyurethane foams in furniture, carpets and bedding. The octabrominated mixture was dominated by BDE-183 followed by BDE-153 and BDE-154, being used in flame-retardant thermoplastics, such as high impact polystyrene. The decabrominated product was essentially composed of decabromodiphenyl ether (BDE-209) and was used predominantly for textiles and plastics for a variety of electronic products, in particular TVs and computers. According to the PBDE global market demands in 2001, the deca-BDE formulation was the dominant one (83%), followed by penta-BDE (11%) and octa-BDE (6%) (Guardia et al., 2006).

PBDEs are semi-volatile and persistent in the environment. They accumulate in lipids and biomagnify through the food web (Johnson-Restrepo et al., 2005). They have been found in various environmental compartments as well as in human fluids and tissues (Hites, 2004). Total PBDE (ΣPBDE) concentrations in human serum have increased in the past decades, as reported in time trend studies conducted in the US (Sjödin et al., 2004) and Europe (Schröter-Kermani et al., 2000; Thomsen et al., 2002), including increases from 0.44 ng/g lipid (1977) to 3.3 ng/g lipid (1999) in Norway (Thomsen et al., 2002). However, as PBDEs are relatively new in the environment, their concentrations are still lower than those of other environmental pollutants of similar chemical properties, such as polychlorobiphenyls (Guvenius et al., 2003), that have been present for a longer period. Increasing concentrations but still lower than other persistent organic pollutants used in the past raise the need for impact assessment studies to examine trends and anticipate future deleterious effects.

Human exposure to PBDEs may occur in many daily life situations. Diet is one of the main sources, e.g. fatty fish (Sjödin et al., 2000) or red meat and poultry (Fraser et al., 2009). Several studies reporting the concentrations of PBDEs in a number of foodstuffs are available (Bocio et al., 2003; Domingo et al., 2006; Gómara et al., 2006; Schecter et al., 2005). The indoor environment and dust ingestion, both at home and in the workplace, is also a substantial contributor to PBDE exposure (Lorber, 2008).

Public health concerns have been expressed regarding the potential exposure of humans to these compounds (Sjödin et al., 2003; Birnbaum and Staskal, 2004), e.g. effects of pre- and post-natal exposure to low PBDE levels on neurodevelopment have been described (Gascon et al., 2011). Accordingly, the production and use of penta- and octa-BDE formulations were banned in the European Union in 2004. Only deca-BDE is still permitted, despite its use in electronic applications was banned in Europe in 2008.

There is now evidence that BDE-209 also accumulates in humans (Antignac et al., 2009; Covaci and Voorspoels, 2005; Fängström et al., 2005; Gómara et al., 2007; Jin et al., 2009; Lunder et al., 2010; Sjödin et al., 2008; Takasuga et al., 2004; Thomas et al., 2006; Uemura et al., 2010; Vizcaino et al., 2011; Weiss et al., 2006; Zhu et al., 2009), including newborns (Vizcaino et al., 2011). However, this compound is rarely measured in human studies from non-exposed populations. Limited information is therefore available on its occurrence and effects in human populations, although its measurement has been recommended (Hites, 2004).

Furthermore, many of the available studies on human PBDE exposure are based on relatively low samples (n < 250), and often only concerning specific collectives (e.g. mothers), thus limiting the possibilities for a comprehensive assessment of the human exposure to these pollutants. The NHANES study conducted in the US population in 2003-04 constitutes an exception (n = 2040), comprising the two genders and a wide age range, although it does not report the decabromodiphenyl ether (BDE-209), which is an important limitation. To the best of our knowledge, there has not been an equivalent study in Europe to date, serving to complete and corroborate it.

The present study investigates PBDE serum levels in a European general population (that of Catalonia, a Mediterranean region in southern Europe) covering both genders, a wide age range and diverse socio-demographic conditions, including body mass index (BMI), parity among women, birthplace, educational level, and social class. Samples were obtained from a general public health survey encompassing 8,400 people, conducted in 2002. Fourteen PBDE congeners including BDE-209, were analysed in a subset of 731 individuals. The present study is therefore the second largest survey in terms of individual PBDE measurements in a human population, and the first to cover specifically BDE-209. The results are then compared with other populations worldwide and discussed in terms of socio-demographic determinants.

2. Materials and methods

2.1. Population and study design

The study sample (n = 731) is based on a public health survey (n = 8,400) that was conducted by the Government of Catalonia in 2002, including a health exam and blood tests (n = 2,100). Catalonia is a Mediterranean region of 32,000 km2 in southern Europe, with a total population of 6.5 million inhabitants as of 2002. Its major economic activities include agriculture and industry.

The survey provided a valuable and representative sample of the general population in terms of age (18-74 years), sex and socio-demographic conditions. Information on body mass index (BMI), cholesterol and triglyceride was obtained from the health exam. Information on the demographic variables, such as age, sex, place of birth, educational level and parity in women was obtained from face-to-face interviews that were conducted between October 2001 and April 2002. Social class was estimated through the household occupational status based on the Spanish Occupational Classification (Grupo de trabajo SEE-SEMFC, 2000). Further details of the study design are available in precedent publications (Juncà et al., 2003; Porta et al., 2010).

2.2. Laboratory analytical methods

A total of 14 PBDE congeners were analysed, as follows: BDE-17 and BDE-28 (tribrominates); BDE-47, BDE-66 and BDE-71 (tetrabrominates); BDE-85, BDE-99 and BDE-100 (pentabrominates); BDE-153, BDE-154 and BDE-138 (hexabrominates); BDE-183 and BDE-190 (heptabrominates); and BDE-209 (decabrominates). The analytical methods and quality control procedures were standard, as described in detail in a precedent publication (Vizcaino et al., 2009). They are summarised next.

Serum samples (1 ml) were introduced into 10-ml centrifuge tubes. A standard solution containing PCB-209 (100 ng/ml; 25 µl) followed by 3 ml of n-hexane and 2 ml of conc. H2SO4 were added. The suspension was mixed in a vortex (ca. 1,500 rpm, 30 s) and centrifuged (ca. 3,500 rpm, 5 min). The supernatant n-hexane layer was transferred into a second centrifuge tube using a Pasteur pipette. Further n-hexane (2 ml) was added to the first tube containing the H2SO4/serum mixture, stirred and then centrifuged. This last step was repeated again yielding a combined extract of 7 ml of n-hexane, to which 2 ml of conc. H2SO4 were added; the suspension was then mixed (vortex mixer, ca 1500 rpm, 60 s), centrifuged (3500 rpm, 10 min), and the supernatant n-hexane was then transferred to a conical bottomed, graduated tube. These n-hexane extracts were reduced to near dryness under a stream of pure nitrogen. Then, the solutions were quantitatively transferred to GC vials using four 25 μl rinses of isooctane. BDE-118 (20 μl) and [13C]-BDE-209 (10 μl) standards were added before injection. The PBDE analysis was performed by gas chromatography (GC, Agilent Technologies 6890N) coupled with mass spectrometry (MS, Agilent Technologies 5975) operating in a negative chemical ionization mode (NICI). The instrument was equipped with a low bleed SGE-BPX5 MS fused silica capillary column (length of 15 m, internal diameter of 0.25 mm, 0.10 μm film thickness).

One procedural blank was included in each sample batch, with either 9, 15 or 19 samples. Method detection limits were calculated at three times the standard deviation of the procedural blank levels. Detection limits ranged between 0.0018 and 0.0089 ng/ml, depending on the BDE congener. PBDE identification was based on retention time and mass spectral information. Quantification was performed by reference to linear calibration curves and correction by the standards (Vizcaino et al., 2009). Average recovery of the PCB-209 standard was 64 ±22% (mean ± standard deviation). Final validation was made by analysis of proficiency testing materials obtained from the Arctic Monitoring and Assessment Program (AMAP Ring Test, 2012). The laboratory participates regularly in the AMAP Ring Test Proficiency Program for POPs in human serum and the results, including BDE-209 concentrations, usually range within 20% of the consensus values.

2.3. Data analysis

Data analysis and graphics were performed using the statistical software R (version 2.15; Vienna, 2012). Statistical analyses were focused on the following congeners: 28, 47, 85, 99, 100, 153, 154 and 209. These compounds were selected because they met detection limits in over 50% of samples. For the samples with non-detected and non-quantified values, a score of one-half the LD and the LQ was assigned, respectively. Serum concentrations were expressed in a lipid-adjusted basis (ng/g lipid) based on levels of cholesterol and triglyceride (Phillips et al., 1989). These levels were determined enzymatically, using Txad-Pap and CIN-UV methods, respectively.

Geometric means (GM) and 95% confidence intervals (CI) have been used for descriptive analysis (Figure 1). For the multivariate regression models, concentrations were transformed into the natural logarithm in order to normalise the distribution of the values and to avoid violating regression assumptions of the normal distribution. The statistical significance of age, sex, place of birth, BMI, educational level and social class, as well as parity in women, were assessed for the distributions of serum PBDE concentrations using multivariate regression models. For these models each of the following variables were categorised into two groups: For age: young (<30 years) and adults (>30 years); for place of birth: Spanish-born (including Catalan-born people) and born in another country; for parity: women without offspring and women with one or more children; for educational level: low education (including individuals without formal education and with primary school studies) and high education (comprising studies at secondary school and university); for social class: I-IV and V (which is the least affluent one). The BMI was kept in the three original groups: normal weight, overweight and obesity. Since all covariates were expressed as factors (dummy variables), the exponentials of the coefficients provided the odds ratios (OR) against the reference categories. Confidence intervals at 95% were used. Robustness checks were made in order to confirm the results of the analyses.

3. Results and discussion

3.1. Socio-demographic characteristics of the studied population

Forty-four percent of the participants were men and fifty-six percent were women (Table 1). The mean age was 45 years (standard deviation = 15), ranging between 18 and 74 years. About 70% of the total population was born in Catalonia, 26% were of other Spanish origin and only 3% were born abroad. The BMI encompassed a large spectrum of cases from underweight (16.9 kg/m2) to obesity (52.7 kg/m2): overweight affected 38% of the sample whereas obesity was found in 20% (Table 1). Concerning parity, 27% of the women had no descendants, 18% had one child and 55% were multiparous. Roughly one third of the studied individuals had a primary school degree, 46% had secondary degree and about 12% had a university degree. Almost half of the study population was classified within the three more affluent social classes (classes I to III), whereas about 8% belonged to the least affluent class (class V) (Table 1). No significant differences in educational or social class characteristics were found between the two gender groups.

3.2. PBDE distributions

The congener BDE-209 was found above the limit of detection in most cases (83% of the cases), followed by congeners BDE-47 and BDE-153 (with 74% and 70%, respectively) (Table 2). BDE congeners 28, 85, 99, 100 and 154 were found above limit of detection in around 50-60% of the samples. The congeners only found above the limit of detection in less than 30% of the serum samples were not included in the calculations of the sections dealing with age, gender, BMI, education or social class dependences.

BDE-209 was the most abundant congener (median 3.7 ng/g lipid; Table 2) accounting for 28% of total PBDEs. BDE-47 and BDE-99 were the second and third predominant congeners, accounting for 18% (median 2.6 ng/g lipid) and 10% (median 1.2 ng/g lipid) of ΣPBDE, respectively. Although BDE-99 was found at higher concentration than BDE-153 (1.2 ng/g lipid vs 0.94 ng/g lipid), it was identified above the limit of detection in less samples (58% versus 70%, respectively) (Table 2). The relatively high proportions of BDE-209, BDE-47 and BDE-99 are consistent with the predominance of the commercial mixtures deca-BDE and penta-BDE in the studied samples. However, in the original composition of penta-BDE, the congener BDE-99 predominates over BDE-47 (Alaee et al., 2003).

The dominance of the BDE-209 congener in PBDE distributions from human populations has also been observed in a study on French women (Antignac et al., 2009), in two studies of Japanese populations (Takasuga et al., 2004; Uemura et al., 2010) and in two studies of Chinese populations (Jin et al., 2009; Zhu et al., 2009). BDE-47 has been observed to predominate the congener distributions of most populations in which BDE-209 was not measured: e.g. studies in North America (Castorina et al., 2011; Johnson et al., 2010; Sandanger et al., 2007; Sjödin et al., 2001, 2008), New Zealand (Harrad and Porter, 2007), Asia (Bi et al., 2006; Lee et al., 2007) and Europe, including studies conducted in Sweden, Norway, Germany and the Faroe Islands (Fängström et al., 2005; Guvenius et al., 2003; Schröter-Kermani et al., 2000; Thomsen et al., 2002). In some cases in which BDE-209 was included, BDE-47 was also found to predominate over the decabromate congener (Gómara et al., 2007; Vizcaino et al., 2011). In a few European studies performed in British (Thomas et al., 2006), Belgian (Roosens et al., 2010) and Greek (Kalantzi et al., 2011) populations, BDE-153 was found to be the dominant PBDE congener. The second of these studies did not include BDE-209. These differences in congener profile may reflect exposure to different commercial PBDE formulations, such as the penta, octa and deca-BDE mixtures mentioned above.

Moreover, environmental processes (Bezares-Cruz et al., 2004; Lacorte et al., 2003) and metabolic transformations (Bartrons et al., 2011, 2012; Robrock et al., 2008) may also influence on the final mixtures of accumulated PBDEs. In this respect, the studied population of Catalonia showed showed a relatively high abundance of BDE-47 and BDE-28 (Table 2), compared to the proportion of BDE-99 in the penta-BDE commercial mixtures (Alaee et al., 2003). Some metabolic or environmental effects (Alaee et al., 2003; Bartrons et al., 2012; Robrock et al., 2008) may be responsible for these enhanced concentrations of the tri- and tetrabromo congeners. Similarly, the high relative proportions of BDE-153 and BDE-154 in the studied population (Table 2), compared to BDE-183, which is the dominant congener in the octa-BDE commercial mixtures (Alaee et al., 2003), may also reflect debromination of more brominated BDE congeners and the bioaccumulation of these hexabromo congeners. In fact, metabolic enrichment of BDE-153 upon exposure to another PBDE, namely BDE-209, has been observed in mammals (e.g. polar bears; Sormo et al., 2006) and fish (Kierkegaard et al., 1999), involving increases of both BDE-153 and BDE-154 in the latter.

3.3. PBDE concentrations

ΣPBDE, encompassing the 14 congeners analysed, ranged between 2.2 ng/g lipid and 150 ng/g lipid (Table 2), with a median value of 15 ng/g lipid (Table 3). BDE-209 concentrations ranged between 0.45–50 ng/g lipid, with median and mean values of 3.7 ng/g lipid and 4.6 ng/g lipid, respectively (Tables 2 and 3). This congener shows the highest concentration (Table 3), which suggests that the deca-BDE commercial mixture is a major source of exposure in the population of the study.

Compared to other European studies, the analysed Catalan population has the highest ΣPBDE concentrations (medians ranging between 2.1 ng/g lipid in Sweden and 5.6 ng/g lipid in the UK), even above other populations studied in Spain (medians 11 and 9.6 ng/g lipid) (Table 3). Part of the difference is due to the high proportion of BDE-209 in the Catalan population. Only two European studies have shown a dominance of BDE-209 in the total BDE mixture: France (median 5.8 ng/g lipid; Antignac et al., 2009) and Belgium (median 11.1 ng/g lipid; Covaci and Voorspoels, 2005) (Table 3); yet in these two cases, particularly in the second, the number of individuals examined is comparatively small and thus any comparison should be done with caution.