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Predicting the relative bioavailability of DDT and its metabolites in historically-contaminated soils using a Tenaximproved physiologically-based extraction test (TI-PBET) Chao Li, Hong-Jie Sun, Albert L. Juhasz, Xinyi Cui, and Lena Q. Ma Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b03891 • Publication Date (Web): 30 Dec 2015 Downloaded from http://pubs.acs.org on December 30, 2015
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Predicting the relative bioavailability of DDT and its metabolites in historically-contaminated
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soils using a Tenax-improved physiologically-based extraction test (TI-PBET)
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Chao Li1, Hongjie Sun1, Albert L. Juhasz2, Xinyi Cui1* and Lena Q. Ma1,3*
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Nanjing University, Nanjing 210023, China
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Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095, Australia
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State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment,
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Soil and Water Science Department, University of Florida, Gainesville, FL 32611, USA
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*Corresponding authors, State Key Laboratory of Pollution Control and Resource Reuse,
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School of the Environment; Tel/fax: 011-86-89690631, E-mail:
[email protected];
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[email protected] 18 19
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Abstract
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Due to their static nature, physiologically-based in vitro assays often fail to provide sufficient
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sorption capacity for hydrophobic organic contaminants (HOCs). The addition of a sorption
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sink to in vitro intestinal solution has the potential to mimic dynamic intestinal uptake for
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HOCs, thereby increasing their desorption from soil. However, the effectiveness of sorption
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sinks for improving in vitro assays needs to be compared with in vivo data. In this study,
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Tenax was added as a sorption sink into the physiologically based extraction test (PBET),
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while DDT and its metabolites (DDTr) were investigated as typical HOCs. Tenax added at
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0.01−0.2 g to the PBET intestinal solution sorbed ~100% of DDTr in 6.3-19 mins, indicating
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its ability as an effective sorption sink. DDTr bioaccessibility in six contaminated soils using
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Tenax-improved PBET (TI-PBET; 27−56%) was 3.4-22 fold greater than results using the
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PBET (1.2−15%). In vivo DDTr relative bioavailability (RBA) was measured using a mouse
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model with values of 17.9−65.4%. The inclusion of Tenax into PBET improved the in vivo-in
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vitro correlation from r2=0.36 (slope=2.1 for PBET) to r2=0.62 (slope=1.2 for TI-PBET),
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illustrating that the inclusion of Tenax as a sorption sink improved the in vitro prediction of
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DDTr RBA in contaminated soils.
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INTRODUCTION Dichlorodiphenyltrichloroethane (DDT) and its metabolites (DDTr) are among the
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most widely used chemicals in the world. As hydrophobic organic contaminants (HOCs),
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they are persistent in the environment even though they have been banned from use in
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numerous countries for several decades. It was reported that DDTr concentrations in 1.9% of
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surveyed soils in China exceed the safety limit of 1 mg kg−1, making it the most serious
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legacy organic contaminant in China.1 DDTr act as environmental estrogens by disrupting
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hormone function and causing reproductive impairments, thereby posing a threat to the
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ecosystem as well as human health.2.
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Incidental soil ingestion is an important route of human exposure to DDTr,
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especially for children with high frequency of hand-to-mouth contact.3 However, predicting
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exposure using total contaminant concentration may overestimate the risk via oral ingestion
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due to contaminant bioavailability constraints.4 For incidental soil ingestion pathway, oral
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bioavailability refers to the fraction of DDTr in the soil matrix that reaches the systemic
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circulation following ingestion. As such, to better refine exposure estimates via this pathway,
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DDTr bioavailability should be considered. In vivo methods utilizing swine and mouse
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animal models have been used to measure contaminant bioavailability.4 However, these
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approaches are ethically challenging, expensive and time consuming. Therefore, simple, rapid
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and inexpensive in vitro methods, which measure the contaminant fraction that is mobilized
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in gastrointestinal (GI) fluids and is potentially available for uptake into the systemic
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circulation (i.e., bioaccessible), have been developed as surrogate assays.5−8
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To gain regulatory acceptance as a surrogate measure of contaminant bioavailability, a
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strong correlation between in vivo and in vitro data is necessary. Although in vitro tests have
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been developed to predict the bioavailability of inorganic contaminants such as As, Cd, and
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Pb,9−11 a dearth of information is available regarding the bioavailability and bioaccessibility
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of HOCs in soils. Of the few studies assessing HOCs, poor in vivo-in vitro correlations have
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been observed due partially to the low bioaccessibility values derived for these compounds as
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a consequence of solubility constraints imparted by the in vitro assay.12−15 Contaminant
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absorption in the GI tract is a dynamic process, which can facilitate the desorption of HOCs 4
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from soil. Since most in vitro methods measure the static partitioning of HOCs into GI fluid,
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they often fail to maintain a concentration gradient to facilitate further HOC desorption. Recently, several studies demonstrated that the inclusion of a sorption sink with
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strong affinity for HOCs into GI solution improves bioaccessibility measurement in soil and
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indoor dust. Several materials have been used, including C18 membranes,13 a composite of
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silicone rod and activated carbon,16,17 and Tenax.18,19 The addition of a sorption sink has the
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potential to improve the in vivo-in vitro correlation by simulating the dynamic uptake
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processes of HOCs in the digestive tract, which facilitates mobilization of HOCs from the
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soil matrix. However, only one study to date has compared PAH bioaccessibility (determined
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using C18 membranes incorporated into in vitro solution) and PAH relative bioavailability
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(RBA; swine model) with modest results (r2=0.45)13. As a consequence, further studies are
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needed to determine the effectiveness of adding a sorption sink to in vitro assays to predict
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the RBA of HOCs. Tenax is a porous resin having high affinity and fast sorption rates for HOCs.20
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These properties coupled with its reusability as an economical material makes Tenax an ideal
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sorption sink. In our recent study, Tenax was included in the physiologically based extraction
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test (PBET) to measure PAH bioaccessibility in soils. Inclusion of Tenax resulted in PAH
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bioaccessibility values that were four-fold greater than that measured using the PBET alone.19
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However, the feasibility using Tenax as a sorption sink in the PBET was not assessed against
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an in vivo model due to the analytical complexities associated with the assessment of PAH
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RBA.15
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In the present study, DDTr (i.e., the sum of DDT and its metabolites) were selected
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as the target HOCs, with in vivo DDTr RBA in contaminated soil being measured using a
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mouse model. Unlike the complicated metabolism of PAHs, DDTr can rapidly partition into
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adipose tissue.21 As a result, accumulation of DDTr in adipose tissue may be used as a
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biological endpoint to determine DDTr RBA.14 The aim of this study was to test the
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feasibility of Tenax-improved PBET (TI-PBET) to predict DDTr bioavailability in
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contaminated soils. The specific objectives were to: 1) measure DDTr bioaccessibility in
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contaminated soils using both PBET and TI-PBET; 2) determine DDTr RBA using a mouse 5
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adipose model; and 3) assess the feasibility of TI-PBET to predict DDTr RBA via an in vivo-
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in vitro correlation. It was hypothesized that the addition of Tenax as a sorption sink to the
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PBET would improve the in vivo-in vitro correlation and make it a suitable method to predict
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DDTr RBA in contaminated soils.
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MATERIALS AND METHODS Chemicals. DDT and its metabolites (DDTr) were investigated in this study, including
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1-(2-chlorophenyl)-1-(4-chlorophenyl)-2,2-dichloroethane (o, p’-DDD), p,
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p’-dichlorodiphenyldichloroethane (p, p’-DDD), 1-(2-chlorophenyl)-1-(4-
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chlorophenyl)-2,2-dichloroethylene (o, p’-DDE), p, p’-dichlorodiphenyl- dichloroethylene (p,
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p’-DDE), 1,1,1-trichloro-2-(p-chlorophenyl)-2- (o-chlorophenyl)ethane (o,p’-DDT), and p,
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p’-dichlorodiphenyltrichloroethane (p, p’-DDT). DDTr standards were obtained from J&K
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Scientific (Shanghai, China) with a purity > 98%. Stock solutions were prepared in hexane at
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a concentration of 1000 mg L-1. Tenax TA (60−80 mesh) was purchased from Sigma-Aldrich
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(St. Louis, MO, USA). Before use, Tenax TA beads were extracted three times with
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hexane:acetone (v/v 1:1) in an ultrasonicator for 5 min. Other chemicals and solvents used
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were of analytical grade.
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Soil samples. Six contaminated soils were obtained from various sites in China (n = 3)
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and Australia (n = 3). Soils 1 and 2 were collected from a former DDT production factory,
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which was closed in 1983.22 Soil 3 was collected from a former pesticide factory in Wuhan
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city, Hubei Province, which was closed in 1994. Soils 4−6 were collected from former cattle
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tick dip sites in New South Wales, Australia, where DDT was utilized as a tickicide from the
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early 1950s. Soil samples were air-dried and sieved to obtain
