Arch. Environ. Contam. Toxicol. 46, 183–188 (2004)
DOI: 10.1007/s00244-003-2138-2
A R C H I V E S O F
Environmental
Contamination
a n d Toxicology
© 2004 Springer-Verlag New York Inc.
Effect of Bile Type on the Bioaccessibility of Soil Contaminants in an In Vitro
Digestion Model
A. G. Oomen, C. J. M. Rompelberg, E. Van de Kamp, D. P. K. H. Pereboom, L. L. De Zwart, A. J. A. M. Sips
Center of Substances and Integrated Risk Assessment, National Institute of Public Health and the Environment, P.O. Box 1, NL-3720 BA Bilthoven,
The Netherlands
Received: 20 September 2002 /Accepted: 4 May 2003
Abstract. Soil ingestion is an important pathway of exposure
for many nonvolatile contaminants for man and in particular
for children. A fraction of the ingested contaminant may not
dissociate from the soil particles during digestion in the gastrointestinal tract, and is thus not available for transport across the
intestinal epithelium. In order to estimate the contaminant
fraction that is mobilized from soil, i.e., the bioaccessible
fraction, several in vitro digestion models have been developed. The currently existing digestion models display many
differences. One aspect that may affect bioaccessibility and
may induce differences between digestion models is the bile
that is used. Often freeze-dried bile of animal origin is preferred to purified bile salts. However, also the animal origin of
bile may give rise to differences in bioaccessibility because bile
composition appears to be species dependent. In the present
study, we compared the bioaccessibility of benzo[a]pyrene,
arsenic, cadmium, and lead of four different soils after digestion with ox bile from two different suppliers, pig bile, and
chicken bile. Bioaccessibility appeared to vary amongst the
different soils and contaminants. Only chicken bile increased
the bioaccessibility of lead and cadmium significantly and
relevantly for one of four soils. For chicken bile, the bioaccessibility of lead was 3–5.5 times greater than for the other bile
types and the bioaccessibility of cadmium was 1.5 times
greater. In all other cases, the bioaccessibility differences were
less than 10%, which is considered irrelevant for risk assessment purposes.
Human risk assessment in The Netherlands for contaminants in
soil, as embedded in the Dutch Soil Protection Act, is mostly
based on studies in which the contaminants are ingested in a
liquid or food matrix. However, a major exposure pathway of
many nonvolatile contaminants is soil ingestion rather than
exposure via the diet (Mushak 1998; Paustenbach et al. 1986;
Swartjes 1999). This is especially true for children due to their
hand-to-mouth behavior (Calabrese et al. 1989; Van Wijnen et
Correspondence to: A. G. Oomen; e-mail: agnes.oomen@rivm.nl
al. 1990). Contaminants in a soil matrix are generally less
bioavailable than contaminants in liquid or food, as tested on
animals (Casteel et al. 1997; Freeman et al. 1994; Ruby et al.
1999). The oral bioavailable fraction of a contaminant is defined here as the fraction that enters the systemic circulation.
Four major processes involved in oral bioavailability for soil
contaminants are: (1) soil ingestion, (2) mobilization of the
contaminants from the soil matrix during gastro-intestinal digestion, which is referred to as bioaccessibility, (3) transportation of bioaccessible contaminants across the intestinal epithelium to reach the portal blood (or lymph) stream, and (4)
first-pass metabolism (only the fraction that passes the liver
without being inactivated and/or excreted is bioavailable).
The bioavailable fraction of a contaminant can spread
through the body by the systemic circulation, and may exert
systemic toxicity.
It is generally assumed that contaminants that are sorbed
to soil particles in the gastro-intestinal tract do not become
fully bioaccessible. The fraction of contaminant that does
not become bioaccessible will not be available for transport
across the intestinal epithelium and therefore will not become bioavailable (Ruby et al. 1999). For metals, limited
bioaccessibility may also be due to the presence of less
soluble metal species. It is likely that risks of soil contaminants are overestimated in many cases due to less bioaccessibility and thus less bioavailability of the contaminants
in a soil matrix with respect to contaminants in a food or
liquid matrix. In recent years, several in vitro digestion
models have been developed as tools to estimate the difference of bioaccessible fractions of contaminants ingested in
liquid/food matrices and soil matrices (Hack and Selenka
1996; Oomen et al. 2002; Rotard et al. 1995; Ruby et al.
1999). The in vitro digestion models simulate the digestion
process of the human gastro-intestinal tract.
The currently existing digestion models have different designs, varying from simple to sophisticated (Oomen et al.
2002). One aspect that differs among digestion models is the
bile that is used. Some models do not introduce bile into the
system, whereas other models use purified, uniform bile salts.
Another category of models uses bile of animal origin. These
differences in bile type may affect the bioaccessibility and may
induce differences among digestion models.
184
Bile may have severe impact on bioaccessibility, which
cannot be accounted for in digestion models without any type
of bile. Bile decreases the surface tension by means of its
surfactant properties (Luner 2000). Surface tension may in turn
be important for the wetting and mobilization of contaminants
from soil (Charman et al. 1997; Porter and Charman 2001).
Furthermore, bile can form complexes with metals (Feroci et
al. 1995; Oomen et al. 2003b) and may create an apolar
environment in the interior of bile salt micelles for hydrophobic
contaminants (Oomen et al. 2000).
Bile salts form in vivo mixed micelles together with
phospholipids, cholesterol, and other fat-soluble compounds
(Friedman and Nylund 1980). Purified, uniform bile salts are
unable to form mixed micelles. In order to have mixed
micelles in chyme, some in vitro digestion models employ
bile extracts of animal origin (Hack and Selenka 1996;
Minekus et al. 1995; Oomen et al. 2002; Rotard et al. 1995;
Ruby et al. 1996). Because human physiology is being
simulated, it would be ideal to use human bile in the in vitro
digestion models. For obvious ethical reasons, human bile
extracts are not available at large scale. A consequent issue
is the choice of the animal species from which bile should be
acquired, since each species has its own bile characteristics.
In addition, only bile from animals that are slaughtered is
commercially available on a larger scale. Most in vitro
digestion models use ox or pig bile, but one of the earliest in
vitro digestion models employed chicken bile (Rotard et al.
1995). The effect of the bile type that is used in in vitro
digestion models on the bioaccessibility of soil contaminants has not yet been studied.
The present study describes a comparison of the bioaccessible fractions that are obtained with different bile types, e.g.,
ox bile from two different suppliers, chicken bile, and pig bile.
The results are discussed with respect to the composition of the
different bile types, which was acquired from the literature.
Different types of contaminants were used in this study because
the relationship between bioaccessibility and bile type may
depend on the contaminant, due to different physicochemical
characteristics of each contaminant. In addition, contaminants
were selected on the basis of their political relevance: soil
ingestion is assumed to be an important pathway of human
exposure to these contaminants, and the incidence rate of the
contaminants in soil is high. The contaminants selected were a
hydrophobic polycyclic aromatic hydrocarbon, benzo[a]pyrene
(B[a]P), the metals cadmium (Cd) and lead (Pb), and the
metalloid arsenic (As).
Materials and Methods
Materials
All laboratory materials for work with metals were rinsed with hydrochloric acid. Afterwards, the material was cleaned carefully with
milliQ water. All chemicals were obtained from Merck, except glucuronic acid (Fluca), lipase (Sigma), and mucin (Roth). Pig and
chicken bile were obtained from Sigma; ox bile from Sigma and ICN.
All bile types were freeze-dried.
A. G. Oomen et al.
Soils and Concentration Levels
Different soils were used for the experiments with B[a]P than for the
experiments with As, Cd, and Pb. OECD-medium, which is standardized soil material according to the Organization for Economic Cooperation and Development (OECD), was spiked with B[a]P at concentrations of one-half and five times the current Dutch Intervention
Value of 40 mg/kg dry matter soil. OECD-medium consists of 10%
dry peat (⬍1 mm), 20% clay, 70% sand, and at most 1% CaCO3, and
is required by the OECD to assess the toxicity of chemicals on
earthworms. In addition, three soil types that are relevant for the Dutch
situation, i.e., silt loam, silty clay loam, and sand, were spiked with
B[a]P at a concentration equivalent to the Intervention Value. These
soils were dried at a temperature between 60°C and 90°C for 16 –18 h.
Subsequently, the soils were crushed to pulverize soil lumps, sieved
through a 1-mm screen, and spiked with a concentrated hexane solution with B[a]P. The hexane was evaporated in the fume hood overnight; and the soils were mixed thoroughly. They were spiked at least
two weeks before the in vitro digestion experiments. The silt loam,
silty clay loam, and sand soils were stored dry. The OECD-medium
and the other soils were treated similarly, except that to OECDmedium 50% by weight of milliQ water was added after spiking.
Flanders soil, Oker 11, Montana 2710, and Montana 2711 soil are
field soils that are historically contaminated with As, Cd, and Pb. The
Montana 2710 and 2711 soils are standard reference materials that are
commercially available at the National Institute of Standards and
Technology (NIST). Montana 2710 soil is certified to contain 626 ⫾
38 mg As, 21.8 ⫾ 0.2 mg Cd, and 5532 ⫾ 80 mg Pb per kg of dry
matter soil. Montana 2711 soil is certified to contain 105 ⫾ 8 mg As,
41.70 ⫾ 0.25 mg Cd, and 1162 ⫾ 31 mg Pb per kg of dry matter soil.
Flanders soil and Oker 11 soil were kindly provided by the Flemish
Institute for Technological Research (Vito), Belgium and the RuhrUniversität Bochum (RUB), Germany, respectively. Concentrations of
As, Cd, and Pb in Flanders soil and Oker 11 soil were determined in
our laboratory as described below. Flanders soil (dry) was found to
contain 55 ⫾ 2 mg As/kg, 14 ⫾ 1 mg Cd/kg, and 612 ⫾ 28 mg Pb/kg.
Oker 11 soil (dry) was found to contain 206 ⫾ 8 mg As/kg, 24 ⫾ 1 mg
Cd/kg, and 5454 ⫾ 230 mg Pb/kg.
In Vitro Digestion Model
The in vitro digestion model that is employed in this study is described
by Oomen et al. (2003a), and has been one of the models that took part
in a round robin study of five different digestion models (Oomen et al.
2002). Figure 1 presents a schematic representation of the in vitro
digestion model. Briefly, saliva, gastric juice, duodenal juice, and bile
are prepared artificially, with the constituents and their concentrations
based on human physiology (Oomen et al. 2003a). The digestion starts
by introducing 9 mL of synthetic saliva (pH 6.5 ⫾ 0.2) to 0.6 g of soil
(dry weight). This mixture is rotated head-over-heels for 5 min at 55
rpm. Subsequently, 13.5 mL of artificial gastric juice (pH 1.07 ⫾ 0.07)
is added and the mixture is rotated for 2 h. Finally, 27 mL of artificial
duodenal juice (pH 7.8 ⫾ 0.2) and 9 mL artificial bile (pH 8.0 ⫾ 0.2)
are added simultaneously and the mixture is rotated for another 2 h. All
digestive juices are heated to 37 ⫾ 2°C, and mixing occurs in a rotator
that is heated to 37 ⫾ 2°C also. At the end of the in vitro digestion
process, the digestion tubes are centrifuged for 5 min at 3000 g,
yielding the synthetic chyme (the supernatant) and the digested soil
(the pellet). The synthetic chyme simulates the juice as present in the
human small intestine. The pH of the chyme is at least 5.5. The
concentration of freeze-dried bile in the bile juice is 6 g/L, resulting in
a bile concentration of 0.9 g/L in chyme.
A control digestion tube without soil was always included for each
series of experiments. The concentration of B[a]P, As, Cd, or Pb never
exceeded the detection limit.
Bile Type and Contaminant Bioaccessibility
185
Fig. 1. Schematic representation of the in vitro digestion procedure
Analysis of B[a]P
Results and Discussion
To determine B[a]P in chyme, 100 ␮L chyme and 200 ␮L milliQ
water were transferred to a cup and mixed by a Vortex shaker for 5 s.
Subsequently, 500 ␮L hexane with benzo[ghi]perylene as the internal
standard (20 ng/mL) was added. B[a]P was extracted from the aqueous
solution in the organic solvent by head-over-heels rotation for 15 min.
Phase separation was obtained at 13000 rpm in an Eppendorf centrifuge for 5 min, and the hexane phase was transferred into another vial.
The hexane was evaporated under a gentle nitrogen stream at 40°C,
upon which 800 ␮L methanol was added to the residue. After thorough
shaking, the samples were analyzed by HPLC with methanol/water
(90/10) as the elution fluid, a Hypersil 5 ODS C18 column, and
fluorescence detection.
Bile Juices
The bile juices that were made with bile from the various
animal origins showed differences in color and transparency.
Bile juice prepared with chicken bile resulted in a greenish,
transparent bile juice. Bile juice with pig bile had a somewhat
yellow/orange, turbid appearance. Bile juice using ox bile
resulted in a yellowish turbid solution for the ox bile from both
manufacturers. These findings suggest that bile from different
animal species has different characteristics.
Percentage Bioaccessible B[a]P, As, Cd, and Pb
Analysis of As, Cd, and Pb
To determine the As, Cd, and Pb in chyme, 0.9 mL chyme was diluted
tenfold by adding 8.1 mL HNO3 (0.1 M). It was analyzed by inductively coupled plasma/mass spectrometry (ICP-MS) (Perkin Elmer,
Elan 6000).
As, Cd, and Pb levels were determined in triplicate in the Flanders
and Oker 11 soil. To that end, milliQ water was added to about 0.5 g
dry matter soil, until a weight of 3 g was reached. The 3-g samples
were diluted with 6 mL milliQ water and 1 mL 65% HNO3, and heated
in microwave pressure vessels (G-ACV-100) in a microwave (CEM
MDS 2000). After dissolution of the contaminants from its matrix, the
samples were centrifuged to remove solid particles and treated in the
way already described for chyme to determine As, Cd, and Pb by
ICP-MS.
Calculation Bioaccessibility
The bioaccessibility was calculated as the percentage of the contaminant mobilized from the soil into chyme after in vitro digestion,
shown in Equation 1:
Bioaccessibility(%) ⫽
contaminant mobilized from soil during digestion (␮g)
⫻ 100%
contaminant present in soil before digestion (␮g)
(1)
Figure 2 presents the percentage bioaccessible B[a]P for digestions with ox bile (from two different suppliers), pig bile, and
chicken bile, with OECD-medium spiked at two levels and
three different spiked soil types, i.e., silt loam, silty clay loam,
and sand. As can be seen in Figure 2, the bioaccessibility of
B[a]P from sand was a factor of four to seven greater than that
of the other soil types.
Figure 3 presents the percentage bioaccessible As, Cd, and
Pb for digestions with ox bile (from two different suppliers),
pig bile, and chicken bile. Digestions were performed with
Flanders, Oker 11, Montana 2710, and Montana 2711 soils.
Similar to the results of B[a]P, the bioaccessible fractions of
As, Cd, and Pb were greatest for a particular soil: Flanders soil.
This indicates that mobilization from soil during digestion is
soil dependent, independently of whether the soil was spiked or
historically contaminated. Furthermore, Figure 3 shows that,
within one soil type, bioaccessibility differed among contaminants. For a discussion on the influence of soils and contaminants on bioaccessibility, and a comparison of digestion models for some of the soils, we refer to Oomen et al. (2002).
Effect of Bile Type
Figures 2 and 3 show that in most cases bile had little effect on
bioaccessibility. A large bile effect was observed in two cases:
186
A. G. Oomen et al.
Fig. 2. Percentages bioaccessible
B[a]P for digestions of five different soil materials with ox bile from
two different suppliers, pig bile,
and chicken bile. The error bars
represent the standard deviation of
the percentages B[a]P that were
bioaccessible of six digestion tubes
Pb and Cd bioaccessibilities of Montana 2711 soil were factors
of 3–5.5 and 1.5 greater, respectively, for digestion with
chicken bile than for digestion with pig or ox bile. The error
bars in Figures 2 and 3 represent the standard deviation of the
percentages bioaccessible contaminants as determined from six
and three different digestion tubes, respectively. The small
standard deviations would lead to statistically significant differences (two-way ANOVA per contaminant with replication,
and Student’s t-test) in bioaccessibility among the bile types for
several soils and contaminants. However, for risk assessment
purposes, only differences in percentage bioaccessibility
greater than 10% are relevant. Consequently, for the contaminants and soils studied here, relevant and significant differences are only observed for Pb and Cd bioaccessibility for
digestion of Montana 2711 soil with chicken bile. Student’s
t-test shows that p ⱕ 0.001 for all combinations of pig and ox
bile with chicken bile for Pb and Cd bioaccessibility from
Montana 2711 soil. This suggests that bile only has considerable effect on the bioaccessibility for a few specific combinations of soil, contaminant, and bile type.
Bile Salt Composition
In Table 1 an overview is presented, as determined by Alvaro
et al. (1986), of the bile salt compositions in the gallbladder of
the chicken, ox, pig, and man. The biliary bile salt compositions for man measured by Wildgrube et al. (1986) and Hay et
al. (1993) are in accordance with the data on human bile salt
composition by Alvaro et al. (1986) and are shown in Table 1
as well. Table 1 shows that the animal species displayed
considerable difference in the type of bile salt, i.e., number and
position of hydroxyl groups, and in the conjugation pattern
with glycine or taurine. Chicken bile consists of 100% taurineconjugated bile salts, whereas the biles of ox, pig, and man
contain 50%, 7%, and 16 –27%, respectively. Taurine-conjugated bile salts are more hydrophilic than glycine-conjugated
bile salts (Alvaro et al. 1986), whereas trihydroxy bile salts are
more hydrophilic than dihydroxy bile salt (dihydroxy bile salts
have “deoxy” in their name) (Alvaro et al. 1986). Chicken bile
consists of approximately 85% dihydroxy bile salts. The percentages for the ox, pig, and man are 21%, 86%, and 53– 65%,
respectively.
Previous experiments have shown that bile has considerable
mobilization capacity for hydrophobic organic compounds and
Pb: The bioaccessibility of PCBs and lindane increased two to
four times when increasing the bile from a digestion without
bile to four times the default bile level (Oomen et al. 2000). For
Pb the bioaccessibility was halved in absence of bile compared
to the default bile level (Oomen et al. 2003b). In addition, it is
known that the different characteristics of bile salts result in
differences in their solubility and wetting capacity (Johnson
1994; Luner 2000). It is therefore remarkable that bile seems to
have little effect on the bioaccessibility in most cases studied
here, despite the differences in bile composition between species. It is also remarkable that a specific combination of a
contaminant (Pb, Cd) and soil (Montana 2711 soil), can result
in different percentage that was bioaccessible for digestions
with chicken bile. The cause and the mechanism behind this
phenomenon are unknown to us.
The choice of the animal species that is used to provide bile
is usually arbitrary. Table 1 shows that not one of the animals
listed displays a bile salt profile similar to that of humans.
Human bile can best be described as an intermediate of ox and
pig biles because the percentage of a specific bile salt in bile is
in most cases between the percentages found for ox bile and pig
bile. The bile salts taurochenodeoxycholic acid and glycodeoxycholic acid are exceptions. The molar percentage of taurine-conjugated bile salts and the percentage of trihydroxy bile
salts for man are greater than those for the pig and smaller than
those for the ox. The present work shows that, in most cases,
the use of chicken, ox, or pig bile does not affect the bioaccesibility. However, chicken bile may occasionally give rise to
considerably greater bioaccessibility percentages. Ox or pig
bile is preferred to chicken bile in in vitro digestion experiments because: (1) Chicken bile may lead to an irregular and
unaccountable bioaccessibility pattern. (2) The composition of
chicken bile is very different from the composition of human
Bile Type and Contaminant Bioaccessibility
187
Fig. 3. Percentages bioaccessible arsenic
(A), cadmium (B), and lead (C) for digestions of four different soil types with ox bile
from two different suppliers, pig bile, and
chicken bile. The error bars represent the
standard deviation of the percentages that
were bioaccessible of three digestion tubes
bile. (3) The percentage of a specific bile salt in human bile is
in almost all cases an intermediate of ox and pig biles. (4) Ox
and pig bile lead to similar percentages that were bioaccessible
for all soils and contaminants tested.
At present, it is too early to conclude from these data that ox
and pig biles will lead to the same bioaccessibility for any test
compound. Similar conclusions that can be drawn for four
different soil types for the hydrophobic organic contaminant
B[a]P, the metals Cd and Pb, and the metalloid As, suggest that
this conclusion might be general.
188
A. G. Oomen et al.
Table 1. Biliary bile salt composition in chicken, ox, pig, and human
Bile salt (%)
Chicken
Reference
n
(Alvaro et al. (Alvaro et al. (Alvaro et al. (Alvaro et al. (Wildgrube et al. (Hay et al.
1986)
1986)
1986)
1986)
1986)
1993)
3
3
3
3
14
10
Ox
Taurohydeoxycholic acid (THDCA)
Taurocholic acid (TCA)
Taurochenodeoxycholic acid (TCDCA)
Taurodeoxycholic acid (TDCA)
Glycohycholic acid (GHCA)
Glycohydeoxycholic acid (GHDCA)
Glycocholic acid (GCA)
Glycochenodeoxycholic acid (GCDCA)
Glycodeoxycholic acid (GDCA)
Molar percentage of tauro-conjugated bile salts
–
15 ⫾ 2
85 ⫾ 8
–
–
–
–
–
–
100
–
38 ⫾ 4
4⫾1
8⫾1
–
–
42 ⫾ 4
3⫾1
6⫾5
50
Pig
4⫾1
–
3⫾1
–
13 ⫾ 2
48 ⫾ 4
1⫾1
31 ⫾ 3
–
7
Man
–
9⫾1
12 ⫾ 2
5⫾1
–
–
26 ⫾ 3
32 ⫾ 3
16 ⫾ 2
26
Man
–
7⫾3
7⫾3
2⫾2
–
–
37 ⫾ 8
33 ⫾ 9
10 ⫾ 6
16
Man
–
12 ⫾ 3
11 ⫾ 3
2⫾1
–
–
31 ⫾ 3
29 ⫾ 2
9⫾3
27
n Represents the number of animals or patients from which bile was obtained and analyzed.
Acknowledgments. The authors are grateful to Dr. Carolien Versantvoort for in-depth discussions.
References
Alvaro D, Cantafora A, Attili AF, Ginanni Corradini S, De Luca C, et
al. (1986) Relationships between bile salts, hydrophobicity and
phospholipid composition in bile of various animal species. Comp
Biochem Physiol 83B:551–554
Calabrese EJ, Barnes R, Stanek III EJ, Pastides H, Gilbert CE, Veneman P, et al. (1989) How much soil do young children ingest: An
epidemiologic study. Regul Toxicol Pharmacol 10:123–137
Casteel SW, Cowart RP, Weis CP, Henningsen GM, Hoffman E,
Brattin WJ, et al. (1997) Bioavailability of lead to juvenile swine
dosed with soil from the smuggler mountain NPL site of Aspen,
Colorado. Fundam Appl Toxicol 36:177–187
Charman WN, Porter CJH, Mithani S, Dressman JB (1997) Physicochemical and physiological mechanisms for the effects of food on
drug absorption: The role of lipids and pH. J Pharm Sci 86:269 –
282
Feroci G, Fini A, Fazio G (1995) Interaction between dihydroxy bile
salts and divalent heavy metal ions studied by polarography. Anal
Chem 67:4077– 4085
Freeman GB, Johnson JD, Liao SC, Feder PI, Davis AO, Ruby MV, et
al. (1994) Absolute bioavailability of lead acetate and mining
waste lead in rats. Toxicology 91:151–163
Friedman HI, Nylund B (1980) Intestinal fat digestion, absorption, and
transport. A review. Am J Clin Nutr 33:1108 –1139
Hack A, Selenka F (1996) Mobilization of PAH and PCB from
contaminated soil using a digestive tract model. Toxicol Lett
88:199 –210
Hay DW, Cahalane MJ, Timofeyeva N, Carey MC (1993) Molecular
species of lecithins in human gallbladder bile. J Lipid Res 34:
759 –768
Johnson LR, Alpers DH, Christensen J, Jacobson ED, Walsh JH
(1994) Physiology of the gastrointestinal tract. Raven Press, New
York
Luner PE (2000) Wetting properties of bile salt solutions and dissolution media. J Pharm Sci 89:382–395
Minekus M, Marteau P, Havenaar R, Huis in ’t Veld JHJ (1995) A
multicompartimental dynamic computer-controlled model simulating the stomach and small intestine. ATLA 23:197–209
Mushak P (1998) Uses and limits of empirical data in measuring and
modeling human lead exposure. Environ Health Perspect 106:
1467–1484
Oomen AG, Sips AJAM, Groten JP, Sijm DTHM, Tolls J (2000)
Mobilization of PCBs and lindane from soil during in vitro digestion and their distribution among bile salt micelles and proteins of
human digestive fluid and the soil. Environ Sci Technol 34:297–
303
Oomen AG, Hack A, Minekus M, Zeijdner E, Cornelis C, Schoeters G,
et al. (2002) Comparison of five in vitro digestion models to study
the bioaccessibility of soil contaminants. Environ Sci Technol
36:3326 –3334
Oomen AG, Rompelberg CJM, Bruil MA, Dobbe CJG, Pereboom
DPKH, Sips AJAM (2003a) Development of an in vitro digestion
model for estimation of bioaccessibility of soil contaminants.
Arch Environ Contam Toxicol 44:281–287
Oomen AG, Sips AJAM, Tolls J, Van den Hoop MAGT (2003b) Lead
speciation in artificial human digestive fluid. Arch Environ Contam Toxicol 44:107–115
Paustenbach DJ, Shu HP, Murray FJ (1986) A critical examination of
assumptions used in risk assessments of dioxin contaminated soil.
Regul Toxicol Pharmacol 6:284 –307
Porter CJH, Charman WN (2001) In vitro assessment of oral lipid
based formulations. Advan Drug Deliv Rev 50:S127–S149
Rotard W, Christmann W, Knoth W, Mailahn W (1995) Bestimmung
der resorptionsverfügbaren PCDD/PCDF aus Kieselrot. UWSF-Z
Umweltchem Ökotox 7:3–9
Ruby MV, Davis A, Schoof R, Eberle S, Sellstone CM (1996) Estimation of lead and arsenic bioavailability using a physiologically
based extraction test. Environ Sci Technol 30:422– 430
Ruby MV, Schoof R, Brattin W, Goldade M, Post G, Harnois M, et al.
(1999) Advances in evaluating the oral bioavailability of inorganics in soil for use in human health risk assessment. Environ Sci
Technol 33:3697–3705
Swartjes FA (1999) Risk-based assessment of soil and groundwater
quality in the Netherlands: Standards and remediation urgency.
Risk Anal 19:1235–1249
Van Wijnen JH, Clausing P, Brunekreef B (1990) Estimated soil
ingestion by children. Environ Res 51:147–162
Wildgrube HJ, Stockhausen H, Petri J, Füssel U, Lauer H (1986)
Naturally occurring conjugated bile acids measured by high-performance liquid chromatography in human, dog and rabbit bile.
J Chromatogr 353:207–213