Test for bioaccessibility of metals and PAH from soil report · 2006-07-20 · Test for...

Environment Agency Report

July 2005

Test for bioaccessibility of metals and PAH from soil Test selection, validation and application

Test for bioaccessibility of metals and PAH from soil July 2005

Agern Allé 5

DK-2970 Hørsholm, Denmark

Tel: +45 4516 9200

Fax: +45 4516 9292

Dept. fax:

e-mail: [email protected]

Web: www.dhi.dk

Client

Environment Agency

Client’s representative

Sohel Saikat

Project

Test for bioaccessibility of metals and PAH from soil

Project No

52339/06

Date July 2005

Authors

Christian Grøn Approved by

Report CHG LIA LIA

Revision Description By Checked Approved Date

Key words

Bioaccessibility, bioavailability, contaminated soils, metals, cadmium, lead, nickel, polycyclic aromatic hydrocarbons, dibenz(a,h)anthracene, benzo(a)pyrene, validation

Classification

Open

Internal

Proprietary

Distribution No of copies

Environment Agency DHI:

Sohel Saikat CHG, LIA

1 1x2

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CONTENTS

1 PREFACE ..................................................................................................................1-1

2 BIOACCESSIBILITY AND BIOAVAILABILITY IN RISK ASSESSMENT OF CONTAMINATED SOILS ...........................................................................................2-5

2.1 Toxicity.......................................................................................................................2-5 2.2 Bioavailability .............................................................................................................2-6 2.2.1 In vivo bioavailability methods ....................................................................................2-6 2.2.2 Relative bioavailability ................................................................................................2-7 2.2.3 Comparability of in vivo bioavailability methods for soil ..............................................2-8 2.2.4 Application of bioavailability in risk assessment..........................................................2-9 2.3 Bioaccessibility.........................................................................................................2-11 2.3.1 In vitro bioaccessibility test methods.........................................................................2-12 2.3.2 Comparability of in vitro bioaccessibility test methods ..............................................2-15 2.4 In vitro bioaccessibility to in vivo bioavailability correlation .......................................2-15 2.4.1 In vitro bioaccessibility to in vivo bioavailability correlation lead................................2-16 2.4.2 In vitro bioaccessibility to in vivo bioavailability correlation cadmium ........................2-18 2.4.3 In vitro bioaccessibility to in vivo bioavailability correlation nickel .............................2-18 2.4.4 In vitro bioaccessibility to in vivo bioavailability correlation arsenic ...........................2-19 2.4.5 In vitro bioaccessibility to in vivo bioavailability correlation PAH ...............................2-20

3 SELECTION OF METHODS FOR IMPLEMENTATION AND VALIDATION IN DENMARK ...............................................................................................................3-23

3.1 Selection of test conditions and methods .................................................................3-23 3.1.1 Test method principles and segments ......................................................................3-24 3.1.2 Fasted or fed state test conditions............................................................................3-25 3.1.3 Gastrointestinal segments ........................................................................................3-25 3.1.4 Selection of practical test performance.....................................................................3-26 3.1.5 Decision on test method...........................................................................................3-27 3.2 Test quality control ...................................................................................................3-27 3.3 Methods for chemical analysis of soils and test solutions .........................................3-27 3.4 Pretreatment of soils ................................................................................................3-29

4 VALIDATION OF THE INITIALLY SELECTED RIVM METHODS.............................4-33 4.1 Analysis of soils........................................................................................................4-33 4.2 Analysis of test solutions ..........................................................................................4-35 4.3 Test performance validation .....................................................................................4-36 4.4 Correction of relative bioaccessibility of metals for test pH .......................................4-39 4.5 Effects of test conditions upon PAH bioaccessibility .................................................4-42

5 APPLICATION OF THE SELECTED BIOACCESSIBILITY TEST METHODS ..........5-43 5.1 Application of the RIVM methods to Danish soils......................................................5-43 5.2 Application of the methods to soils with internationally documented in vivo

bioavailability data ....................................................................................................5-47 5.2.1 Identification and retrieval of soil samples ................................................................5-47 5.2.2 Soil analysis .............................................................................................................5-48 5.2.3 Application of the RIVM methods to soils with internationally documented in vivo

bioavailability data ....................................................................................................5-49 5.2.4 Application of alternative bioaccessibility test methods.............................................5-57

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5.2.5 Application of the RIVM fasted state stomach segment only method to soils with internationally documented in vivo bioavailability data..............................................5-58

5.2.6 Application of the SBRC method to soils with internationally documented in vivo bioavailability data ....................................................................................................5-61

5.2.7 Comparison of different bioaccessibility test methods applied to soils with internationally documented in vivo bioavailability data..............................................5-64

5.2.8 Application of alternative bioaccessibility test methods to Danish soils.....................5-67

6 CONCLUSIONS AND RECOMMENDATIONS.........................................................6-69

7 REFERENCES.........................................................................................................7-77 Appendix A..................................................................................................................87

Test conditions in mouth and oesophagus segments..................87 Appendix B..................................................................................................................91

Test conditions in stomach segments .........................................91 Appendix c ..................................................................................................................97

Test conditions in intestinal segments.........................................97 Appendix D................................................................................................................103

Practical test performance ........................................................103 Appendix E................................................................................................................109

Quality control requirements .....................................................109 Appendix F................................................................................................................113

Analytical methods applied for soil and test solutions ...............113 Appendix G ...............................................................................................................117

Soil pretreatment methods........................................................117

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LIST OF FIGURES Figure 2-1 Schematic presentation of oral uptake processes 2-6 Figure 2-2 Blood concentration of lead in response to dose administered as contaminated soil or as soluble lead salt

2-9 Figure 2-3 In vitro bioaccessibility of lead from soils against in vivo bioavailability as reported in the literature for

in vitro test methods with a stomach segment only 2-17 Figure 2-4 In vitro bioaccessibility of lead from soils against in vivo bioavailability as reported in the literature for

in vitro test methods with an intestinal segment 2-17 Figure 2-5 In vitro bioaccessibility of cadmium from soils against in vivo bioavailability as reported in the literature

for an in vitro test method with a stomach segment (left) followed by an intestinal segment (right) 2-18 Figure 2-6 In vitro bioaccessibility of arsenic from soils against in vivo bioavailability as reported in the literature

for in vitro test methods with a stomach segment only 2-19 Figure 2-7 In vitro bioaccessibility of arsenic from soils against in vivo bioavailability as reported in the literature

for in vitro test methods with a stomach segment only 2-19 Figure 2-8 In vitro bioaccessibility of benzo(a)pyrene from soils against in vivo bioavailability as reported in the

literature for in an vitro test method with a stomach segment and an intestinal segment 2-20 Figure 3-1 Outline of steps and segments in in vitro bioaccessibility test methods 3-24 Figure 4-1 Metal concentrations in selected soils analyzed after diluted nitric acid digestion and after aqua regia

digestion, both with ICP-OES quantification 4-34 Figure 4-2 Linearity data for RIVM fasted state method with cadmium, lead and nickel 4-38 Figure 4-3 Linearity data for RIVM fed state method with BaP and DBahA 4-39 Figure 4-4 Recovery of cadmium, lead and nickel with the RIVM fasted state test without solid phase added against

pH 4-40 Figure 4-5 Recovery of lead with the RIVM fasted state test against pH including all reference tests from the study

4-41 Figure 5-1 Sampling plan for the Danish field sites 5-44 Figure 5-2 Total soil concentrations obtained in previous studies against total soil concentrations obtained in this

study 5-49 Figure 5-3 In vitro bioaccessibility of cadmium obtained with the RIVM fasted state test method against in vivo

bioavailability obtained with juvenile swine 5-50 Figure 5-4 In vitro bioaccessibility of cadmium obtained with other test methods against in vitro bioaccessibility

obtained with the RIVM fasted state test method 5-50 Figure 5-5 In vitro bioaccessibility of lead obtained with the RIVM fasted state test method against in vivo

bioavailability obtained with juvenile swine, mini pigs, rats and rabbits 5-52 Figure 5-6 In vitro bioaccessibility of lead obtained with other test methods against in vitro bioaccessibility obtained

with the RIVM fasted state test method 5-52 Figure 5-7 In vitro bioavailability of lead obtained with the juvenile swine method against bioaccessibility obtained

with the RIVM fasted state method 5-53 Figure 5-8 In vitro bioaccessibility of nickel obtained with other test methods against in vitro bioaccessibility

obtained with the RIVM fasted state test method 5-55 Figure 5-9 In vitro bioaccessibility of BaP obtained with the RIVM fed state test method against in vivo

bioavailability obtained with mini pigs and with different methods in mice 5-56 Figure 5-10 In vitro bioaccessibility of PAH obtained with another test method against in vitro bioaccessibility

obtained with the RIVM fed state test method 5-57 Figure 5-11 In vitro bioaccessibility of cadmium obtained with the RIVM fasted state stomach segment only test

method against in vivo bioavailability obtained with juvenile swine 5-59 Figure 5-12 In vitro bioaccessibility of cadmium obtained with another test method against in vitro bioaccessibility

obtained with the RIVM fasted state stomach only test method 5-60 Figure 5-13 In vitro bioaccessibility of lead obtained with the RIVM fasted state stomach segment only test method

against in vivo bioavailability obtained with juvenile swine 5-60 Figure 5-14 In vitro bioaccessibility of lead obtained with the RIVM fasted state stomach segment only test method

against in vivo bioavailability obtained with juvenile swine, final pH < 1.8 5-61 Figure 5-15 In vitro bioaccessibility of cadmium obtained with the SBRC test method against in vivo bioavailability

obtained with juvenile swine 5-62 Figure 5-16 In vitro bioaccessibility of cadmium obtained with another test method against in vitro bioaccessibility

obtained with the SBRC test method 5-63

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Figure 5-17 In vitro bioaccessibility of lead obtained with the SBRC test method against in vivo bioavailability obtained with juvenile swine 5-63

Figure 5-18 In vitro bioaccessibility of lead other test against in vitro bioaccessibility obtained with the SBRC test method 5-64

Figure 5-19 In vitro bioaccessibility of cadmium as obtained with the three test methods against in vivo bioavailability as obtained in juvenile swine 5-65

Figure 5-20 In vitro bioaccessibility of lead as obtained with the three test methods against in vivo bioavailability as obtained in juvenile swine or mini pig. 5-66

Figure 6-11 In vitro bioaccessibility of lead as obtained with the three test methods against in vivo bioavailability as obtained in juvenile swine or mini pigs, data with pH after RIVM stomach only below 1.8 5-66

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LIST OF TABLES Table 2-1 Examples of experimental animals and study principles applied to estimate bioavailability of selected soil

contaminants 2-7 Table 2-2 Relative in vivo bioavailability of lead obtained with different experimental animals 2-8 Table 2-3 Relative bioavailability measured in different target organs in vivo in rats and swine and in two different

soil samples 2-8 Table 2-4 Bioaccessibility test methods with in vivo bioavailability data for one or more contaminated soils 2-14 Table 2-5 Bioaccessibilties of lead, cadmium and arsenic from 3 soils as obtained with 3 selected in vitro test

methods 2-15 Table 2-6 In vitro bioaccessibilities of lead from the Bunker Hill soil against the in vivo bioavailabilities measured

in humans 2-18 Table 2-7 Bioavailability and bioaccessibility of PAH from 4 different soils given ordered relative to the most

bioavailable/bioaccessible soil for each method 2-21 Table 3-1 Segments in the three selected in vitro bioaccessibility test methods 3-25 Table 4-1 Data quality objectives for analyses and tests 4-33 Table 4-2 Principles and analytical quality obtained in soil analysis 4-34 Table 4-3 Recovery with the DS 259 method for the certified reference materials NIST 2710 and 2711 4-35 Table 4-4 Principles and analytical quality obtained in test solution (RIVM method) analysis during validation 4-36 Table 4-5 Recovery of metals from soil test solutions prepared with the RIVM fasted state method 4-36 Table 4-6 Test validation for RIVM fasted state applied for the metals lead, cadmium and nickel 4-36 Table 4-7 Precision and recovery for the RIVM fasted state test without soil added for the metals cadmium, lead and

nickel 4-37 Table 4-8 Test validation for RIVM fed state applied for the PAH benzo(a)pyrene (BaP) and dibenz(a,h)anthracene

(DBahA) 4-38 Table 4-9 Recovery for the RIVM fed state test without soil added for the PAH BaP and DBahA 4-39 Table 4-10 Functions for calculation of relative bioaccessibility from absolute bioaccessibility corrected for pH 4-41 Table 4-11 Suggested acceptable final pH intervals in the test according to the RIVM methods with the pH data

from the test application with Danish soils 4-41 Table 4-12 Bioaccessibility of BaP and DBahA with varied intestinal test time and pH adjustment, mean ± standard

deviation 4-42 Table 5-1 Total concentrations and relative bioaccessibilities for two Danish soils as obtained with the RIVM

methods, mean ± standard deviation 5-43 Table 5-2 Total soil concentrations at the Danish field sites 5-44 Table 5-3Relative bioavailabilities at the Danish field sites 5-45 Table 5-4 Texture of the soils from the 7 Danish sites 5-45 Table 5-5 Bioaccessible soil concentrations at the Danish field sites 5-46 Table 5-6 Variability analysis for bioaccessibility at the 7 Danish sites 5-46 Table 5-7 Relative standard deviations for total soil concentrations, bioaccessible soil concentrations and relative

bioaccessibility for the 7 Danish sites 5-47 Table 5-8 Relative standard deviation as obtained by duplicate analysis of randomly selected soil samples 5-49 Table 5-9 In vitro bioaccessibility of cadmium obtained with the RIVM fasted state method in different test series of

this study, mean ± standard deviation 5-51 Table 5-10 Absolute bioaccessibility obtained with the RIVM fasted state test method at RIVM and in this study,

and absolute bioavailability as obtained in humans 5-54 Table 5-11 In vitro bioaccessibility of lead obtained with the RIVM fasted state method in different test series of this

study, mean ± standard deviation 5-54 Table 5-12 In vitro bioaccessibility of nickel obtained with the RIVM fasted state method in different test series of

this study 5-55 Table 6-1 Data on homogeneity and metal concentrations in soils from different sources 5-58 Table 5-14 Relative bioaccessibilities and relative standard deviations for test of 6 Danish soil with three different

test methods, cadmium 5-67 Table 5-15 Relative bioaccessibilities and relative standard deviations for test of 6 Danish soil with three different

test methods, lead 5-68 Table 7-1 Realistic reduction factors for estimation of bioaccessible concentrations from total soil concentrations

based upon data from 7 Danish sites 6-71 Table 7-2 Summary of the applicability of the bioaccessibility test methods for risk assessment of selected soil

contaminants for oral exposure 6-72

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DEFINITIONS AND ABBREVIATIONS

Definitions are in part adapted from: ISO/CD 17924-1 draft standard dated August 11th 2004 on Soil quality – Human exposure assessment – Part 1: Guidance on the applica-tion and selection of physiologically based extraction methods for the estimation of the human bioaccessibility/bioavailability of metals from soil and soil material. Abbreviations are as used in this report related to the definitions. The bioaccessibility test methods and their abbreviations are explained in section 2.3.1 of the report. Bioaccessibility Proportional transfer of a substance from soil or soil material into (human) gastric (and intestinal) juice(-s). Bioavailability (AB) Fraction of a substance present in a soil that becomes available at the systemic circula-tion (blood stream) site of physiological activity. Estimated daily exposure (EDE) The average dose of a chemical estimated to reach the human body per day. Exposure The dose of a chemical that reaches the human body. Ingestion The act of taking substances (e.g. soil and soil material) into the body by mouth. In vitro bioaccessibility test Laboratory method simulating the dissolution of contaminants in the (human) gastroin-testinal system. Maximum contaminant limits (MCL) The highest concentration of a contaminant in a matrix such as soil allowed by regula-tion. No observed adverse effect level (NOAEL) Dose at which no adverse effect on a receptor can be observed. Provisional tolerable weekly intake (PTWI) Designation of the provisional weekly tolerable amount of a substance which can be ta-ken in by a human body in his lifetime through the food chain without effecting human health. Relative bioaccessibility (RAC) The ratio between the amount of a contaminant dissolving in the (human) gastrointesti-nal system when ingested with e.g. soil and the amount dissolved when ingested as in the toxicity experiment underlying the criteria.

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Relative bioavailability (RAF) The ratio between the amount of a contaminant reaching systemic circulation when in-gested with e.g. soil and the same amount obtained when ingested in the toxicity ex-periment underlying the criteria. Tolerable daily intake value (TDI) Designation of the daily tolerable amount of a substance which can be taken in by a human body in his lifetime through the food chain without effecting human health.

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SUMMARY

The effects of soil contaminants upon both the soil environment and humans depend upon the bioavailability of the contaminant from the soil. On the other hand, maximum contaminant limits are generally based upon toxicity studies with animals or uptake studies with humans exposed to the contaminants in their most soluble form. Therefore, there is a need for methods that can estimate the bioavailability of soil contaminants to humans after e.g.: oral exposure.

A variety of in vivo methods have been used for estimation of the bioavailability of soil contaminants in experimental animals, but all are expensive, subject to considerable random and systematic errors and may impose ethical consideration regarding the perti-nence of using experimental animals for the purpose.

As an alternative, a variety of in vitro methods have been introduced to estimate the bio-accessibility of soil contaminants in laboratory tests, e.g.: the dissolution of the con-taminants in the human gastrointestinal tract, as an indicator of in vivo bioavailability. These methods are also subject to random and systematic errors, but are less costly and without the ethical problems. A crucial point for the applicability of the in vitro methods is that they must give bioaccessibilities well correlated to accepted in vivo bioavailabili-ties. In previously published studies, a good correlation has been found for lead and to some degree for arsenic and cadmium between in vitro bioaccessibility and in vivo bioavailability. For nickel and PAH, only very few in vivo bioavailability data have been published.

After a series of studies on bioaccessibility of metals (cadmium, lead and nickel) and PAH (dibenzo(a)pyrene and dibbenz(a,h)anthracene) funded by the Danish Environ-mental Agency, it is recommended to apply the in vitro RIVM fasted state test method for cadmium and the same test method without the intestinal step for lead for quantita-tive estimates of the bioavailability for oral exposure. If the pH values in the test solu-tions are not within specified limits, the test must be repeated with smaller amounts of soil. Furthermore, it is recommended to apply the in vitro fasted state method from RIVM and the RIVM fed state method for qualitative assessments of the bioavailability of nickel and PAH from soil, respectively.

An investigation program including as a minimum testing of 5 soil samples from each site is suggested giving relative bioaccessibilities, “total” concentrations and bioacces-sible concentrations of the contaminants for each sample (site specific application of bioaccessibility).

The general conclusion is that correction of soil cadmium and lead concentrations for relative bioaccessibility in evaluation of compliance with soil quality criteria and cleanup levels based upon reduced bioavailability/bioaccessibility of the contaminants may be recommended in site specific risk assessment approach. Conversely, the data available at present do not allow for general regulation of soil quality criteria and cleanup levels for specific contaminants, soil types or sources. One test can with modi-fications be used for measuring cadmium, lead and nickel bioaccessibility and another but similar test for PAH, but for nickel and PAH, the bioaccessibilities can currently only be used estimates of the relative risk associated with different soils.

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1 PREFACE

Most soil quality criteria and cleanup levels for soil contaminants are based upon toxic-ity studies on oral exposure with soluble, highly bioavailable contaminant forms in-gested with water or with food. When ingested with soil, metals and PAH are likely to be less available than in the toxicity studies. Reduced availability of soil contaminants may reduce the risk at a contaminated site and therefore, the Environmental Agencies and research institutions of several countries have worked to provide methods to assess the soil contaminant availability for human, oral exposure.

This report is a condensation and translation of two reports on bioaccessibility tests and their use in assessment of the risk for human, oral exposure of selected soil contami-nants prepared for the Danish Environmental Protection Agency (DEPA):

• Human bioaccessibility of soil contaminants /1/

• In vivo bioavailability and in vitro bioaccessibility of soil contaminants /2/

Parts of a previous literature review on human bioaccessibility of heavy metals and PAH from soil /3/ and recently published literature has been included in the report.

The report has been prepared for the Environment Agency, United Kingdom under su-pervision of Sohel Saikat and after agreement with the DEPA.

The projects for the DEPA have been followed by Danish expert committees with the following members:

• Irene Edelgaard, DEPA (chairman)

• Poul Bo Larsen, DEPA

• Christina Ihlemann, DEPA

• Anne Krag, County of Funen

• Annette Bech Nielsen, Copenhagen Municipality

• Mariam Wahid, Copenhagen Municipality

• Charlotte Weber, Danish County Association

• Ole Ladefoged, the Danish Veterinary and Food Administration

• Arne Scheel Thomsen, Danish Medical Public Health Office (DMPHO)

• Henrik Hansen, DMPHO

• Anders Carlsen, DMPHO

• John Jensen, the Danish National Environmental Research Institutions


The literature review and its recommendation were discussed with an international ref-erence group:

• Cathy Rompelberg, National Institute for Public Health and the Environment (RIVM), the Netherlands

• Barry Smith, British Geological Survey (BGS), United Kingdom

• Michael Ruby, Exponent Environmental Group, United States

Test methods were made available by:

• Agnes Oomen, RIVM, the Netherlands

• John Drexler, University of Colerado, US

Selected results of the bioaccessibility testing /1;2/ were presented and discussed at workshops arranged by the Environment Agency (Oxford, UK, March 15, 2005) and the Bioaccessibility Research Group of Europe (BARGE, Hørsholm, Denmark, April 19-20, 2005).

Testing of the correlation between in vivo bioavailability and in vitro bioaccessibility of soil contaminants /2/ was possible through the assistance of research groups with access to soils tested in vivo for bioavailability. The following groups made soils samples and data available for the project:

• Stan Casteel, University of Missouri, US

• Michael Ruby, Exponent, US

• Nick Basta, Ohio State University, US

• William Brattin, Syracuse Research Corporation, US

• Agnes Oomen, RIVM, the Netherlands

• Jürgen Wittsiepe, Ruhr-Universität Bochum, Germany

• Eric Weyand, Maple City Research Inc., US

• National Institute of Standards & Technology (commercially available soil refer-ence material with published bioaccessibility test data), US

Furthermore, access was obtained to a soil sample tested for bioavailability of lead to humans by Dr. Mark Maddaloni, now United States Environmental Protection Agency (US EPA).

The report is subdivided into 5 main sections:

• Introduction to the use of bioaccessibility testing of soil contaminants in risk as-sessment


• Selection of methods for implementation and validation in Denmark

• Validation of the selected RIVM methods

• Application of the selected RIVM methods and of alternative methods (RIVM fasted state stomach only and SBRC)

• Conclusions and recommendations

The text is based upon a number of reviews and textbooks that are not explicitly quoted /4-19/, in addition to published methods and studies quoted with precise references.

The emphasis in the Danish projects has been upon the soil contaminants cadmium, lead, nickel (metals), benzo(a)pyrene (BaP) and dibenz(a,h)anthracene (DBahA, poly-cyclic aromatic hydrocarbons, PAH). Information on other soil contaminants has to a lim-ited extent been included in the literature review, but more extensive studies on other con-taminants such as arsenic are available in the literature and in reports, see e.g.: /15;20-25/.



2 BIOACCESSIBILITY AND BIOAVAILABILITY IN RISK ASSESSMENT OF CONTAMINATED SOILS

The highest concentrations of contaminants that are acceptable in soils are generally based upon estimates of human exposure (how large amounts of the contaminant can impact the human via the sum of exposure routes) and of the toxicity of the contami-nants to humans.

2.1 Toxicity

The limit values for soil (the maximum contaminant limits for soil, MCL’s) are gener-ally calculated on the basis of a tolerable daily intake value (TDI) or a provisional, tol-erable weekly intake (PTWI), that can be derived from the no observed adverse effect level (the NOAEL) found in human data or experimental animal data. For genotoxic carcinogens for which no lower threshold for increased risk for cancer risk is assumed, the TDI value is set at a level that corresponds to a tolerable low (negligible) cancer risk level. Examples of applied risk levels are doses comparable to excessive risks of 10-5 or 10-6 i.e.: a calculated hypothetical risk of one extra cancer outcome among 100.00 or 1 million people in a lifetime.

In calculating the tolerable soil exposure estimates, the impact of other sources is taken into account by allocating the total tolerable amount to different exposure routes, e.g.: food, drinking water and soil. The allocation is given as the allocation factor (fal) which is the fraction of TDI that is allowed from soil exposure.

Oral ingestion is one of the most important exposure routes for humans to soil contami-nants /26/, and MCL’s are in most cases developed based upon oral uptake by children /27/. The MCL for soil ingestion is obtained by dividing the TDI (corrected for alloca-tion) with the estimated daily soil exposure (EDE):

MCL (mg contaminant/kg soil) =

TDI (mg contaminant/person/day) x fal/EDE (kg soil/person/day)

In determination of the TDI, data on oral toxicity are primarily considered. Often, these data pertain to animal experiments where the substance is administrated to the animals mixed in the feed or in drinking water (the vehicle or transporter of the contaminant). As an alternative, epidemiological studies relating observed human health effects to re-corded exposures have been used1. The amount of contaminant needed to produce ad-verse health effects in the animal is then recorded. Most toxicological studies report the total ingested amount only and do seldom indicate exact values for the bioavailability of the substances administered.

1 An example is that the US toxicity value for arsenic was developed from epidemiological data on exposure in drinking water and it should be noted,

that water soluble arsenic ingested with drinking water is nearly completely absorbed (i.e.: 80-90%) /30/.


2.2 Bioavailability

When extrapolating from the experimental conditions in toxicity studies to other condi-tions e.g.: to intake of contaminated soil, the approach taken as described requires, that the uptake efficiency is equal for all scenarios, i.e.: that the absolute bioavailability, AB, of the contaminant is constant. The absolute, oral bioavailability can be defined as:

AB = internal dose/external (administered) dose

In words, the absolute, oral bioavailability is the fraction of an orally ingested contami-nant that reaches systemic circulation, i.e.: enters the blood stream, or a defined target organ. The absolute oral bioavailability of a contaminant may range from close to 0 to almost 1 (i.e.: 100%) depending upon the physiochemical form of the contaminant. In this context, the use of the concept of absolute, oral bioavailability rests upon the as-sumption that adverse health effects are systemic and thus triggered by the contaminants reaching the blood stream, i.e.: the internal exposure, as opposed to the external expo-sure measured directly as intake of contaminated medium multiplied by the concentra-tion of the contaminant in the medium, Figure 2-1.

Figure 2-1 Schematic presentation of oral uptake processes

2.2.1 In vivo bioavailability methods A range of different methods are available for in vivo measurement of oral bioavailabil-ity, see e.g.: /3;17/ for recent reviews.

A selection of methods used for determining bioavailability of soil contaminants is given in Table 2-1, see also section 2.2.2 for explanation of the concept of relative bioavailability.

INGESTION

Mouth

Ingestedamount

M

DISSOLUTION

Lumen ofstomach and

small intestine

Bioaccessibleamount

Mb

ABSORPTION

Membranes ofstomach and

small intestine

Absorbedamount

Ma

REDUCTION

Membranesand liver

Bioavailableamount

Md

SOIL

TARGET

Bioaccessibilityfactor

fb

Absorbabilityfactor

fa

Bioavailabilityfactor

fbad = fa x fb x fd

Reductionfactor

fd

Externalexposure

Internalexposure


Table 2-1 Examples of experimental animals and study principles applied to estimate bioavailability of se-lected soil contaminants

Experimen-tal animal

Targets Principles Contaminants Refe-rences

Juvenile swine

Weighted concen-trations in blood and organs, for some contaminants selected organs or blood only

Bioavailability rela-tive to soluble me-tal salt in feed

Lead2 /28/

Mini pigs Concentrations in selected organs, secreted amounts with urine or with faeces

Absolute bioavail-ability or bioavail-ability relative to soluble form added with feed

Lead, nickel, cadmium and PAH

/29/

Sprague-Dawley rats

Blood concentra-tions


Lead and ar-senic

/30/

Sprague-Dawley rats

Weighted concen-trations in blood and organs


Lead /31/

New Zealand white rabbits

Blood Bioavailability rela-tive to soluble me-tal salt in feed

Lead /32/

Humans Blood Absolute bioavail-ability applying iso-tope dilution

Lead /33/

Mice Excreted amounts in urine

Bioavailability rela-tive to extract of soil PAH from same soils in feed

PAH /34;35/

The absolute bioavailability can be measured as the ratio between amounts in the blood of laboratory animals after oral ingestion (uptake of bioavailable fraction) and after in-travenous injection (100% uptake). Alternative and less direct approaches are at hand as e.g.: estimating the absorbed amount of orally ingested contaminant as the amount that is excreted with urine or as the amount not excreted with faeces.

It should be noted that no generally accepted method exists for estimation of the bioavailability of organic contaminants such as PAH from soils due to the complexity imposed by the metabolization of most organic compounds.

2.2.2 Relative bioavailability A more feasible approach is to measure the relative bioavailability or relative absorption fraction (RAF). RAF is obtained as:

RAF = amount taken up from soil matrix/amount of soluble contaminant taken up from the matrix used in the toxicity study

2 Also used for cadmium and arsenic


In words, the relative bioavailability is the ratio between the amount of a contaminant reaching systemic circulation or a defined target organ when ingested with e.g.: soil and the same amount obtained when ingested in the toxicity experiment.

2.2.3 Comparability of in vivo bioavailability methods for soil In vivo bioavailabilities obtained with different experimental animals have been re-ported for one soil sample and for different soils samples from the same 4 sites, Table 2-2.

Table 2-2 Relative in vivo bioavailability of lead obtained with different experimental animals /30;32;36/

Rats Juvenile swine Rabbits One sample Butte MW-1 0.093 - 0.48 Same site, different samples Butte 0.093-0.23 0.19 0.48 Bingham Creek 0.36 0.28-0.31 - Murray 0.41 0.53-0.71 - Joplin 0.34 0.59-0.67 -

Evidently, the bioavailability found for soil lead depends upon the experimental animal used in the study.

The US Environmental Protection Agency (US EPA) recently concluded in a study on stabilization of lead in contaminated soils that the data obtained for relative bioavail-abilities in different experimental animals and in humans did not show a clear correla-tion between the bioavailabilites obtained in the different species /37/.

The effect of selected target organ upon in vivo bioavailability can be seen from the dif-ferent relative bioavailabilities obtained for lead from the same soil samples, Table 2-3. It can be argued that the target should be the organ relevant from a toxicological point of view as e.g.: blood for lead and kidneys for cadmium. Alternatively, lead bioavail-ability can be calculated as a weighted average of blood (weight 3), liver, bone and kid-ney (each weight 1) in order to obtain a robust, concentration independent (see below) relative bioavailability estimate /28/.

Table 2-3 Relative bioavailability measured in different target organs in vivo in rats and swine and in two different soil samples /31;38/

Blood Kidney Liver Bone Rats Joplin 0.34 0.48 0.27 0.34 Juvenile swine Butte 0.22 0.13 0.090 0.13

A linear dose response is generally seen with organs such as liver, kidney or bone as target organs but with blood as the target “organ”, a non linear response has been ob-served for lead with decreasing blood response at increasing exposure dose, Figure 2-2 /28;31/. The uptake of lead in blood is best fitted to an exponential function:


)1(* )*(21

3 dCkb ekkC −−+=

, with k1, k2 og k3 as constants, Cb the blood concentration (µg Pb/L blood), and Cd the administered dose (mg Pb/kg bodyweight/day). It has been suggested that the non-linearity of the blood lead dose response curve is to some extent due to processes in the blood system rather than to non-linear uptake from the gastro-intestinal system, consid-ering also the linear dose response relationship seen for the other organs /39/.

Figure 2-2 Blood concentration of lead in response to dose administered as contaminated soil or as solu-ble lead salt, redrawn from/31/

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As can be seen from Figure 2-2, both the absolute and the relative bioavailability of lead from both soil and soluble reference decrease with increasing dosage, as seen also in other studies, see e.g.: /40/.

In order to account for this effect, it has been suggested to calculate the relative bioavailability of lead as the ratio between the maximum (plateau) blood concentration (k2 in the exponential function) obtained from soil and from the soluble reference /31/.

To illustrate the different in vivo bioavailabilities obtained with different calculation points and methods can be mentioned, that the relative bioavailability of lead from Joplin soil was 0.44 calculated at 60 µg Pb/L blood, 0.32 at 100 µg Pb/L blood and 0.34 calculated from the maximum concentrations /31/.

In summary, the relative bioavailability of e.g.: lead depends upon the experimental animal, the target organ and the calculation method employed.

2.2.4 Application of bioavailability in risk assessment If the relative bioavailability of a contaminant deviates from 1 (~100%) when ingested in soil as compared to ingestion in the toxicity experiments behind the TDI, a correction of the MCL to account for this can be argued for. If a reliable and safe generic RAF


value could be found and agreed upon, this would then result in a proportional change in the MCL:

MCLtrue = MCL/RAF

Alternatively, the concentration held against the MCL is not the total soil contaminant concentration, C, but the concentration of bioavailable soil contaminant, Cb:

Cb = C x RAF

This approach can be applied in site specific risk assessment.

For substances where the critical toxic effect is not systemic toxicity but local toxicity (i.e.: local irritation, intestinal cancers), the toxic effect is considered to be depending upon the concentration in the gastrointestinal tract, and the MCL will be dependent of bioaccessibility rather than the bioavailability.

It should be noted that although most relative bioavailabilities are less than 1 and would result in an increased MCL, RAF values above 1 could be found that would result in a demand for a decreased MCL and thus for increased intervention.

In the US and Canada, the RAF values have been used to increase cleanup levels after risk assessment on a case by case basis. Adjustment of cleanup levels based upon bioavailability studies has been reported from the US for arsenic, lead, mercury, PAH, PCB’s and dioxins /9;10;41/ and from Canada for lead and nickel /42/.

The US EPA allows for using the concept of relative bioavailability in risk assessment /43/, but does not give guidance to the practical implementation yet. Still, according to recent reviews /9/,/10;41/ several state regulatory agencies have issued guidance docu-ments. Adjustment of the bioavailability is an option in the US EPA model for risk as-sessment of lead uptake in children /44/. In vivo data for relative bioavailability are in most cases required to allow the adjustment of lead bioavailability. This reflects the general attitude in the US EPA: that bioavailability based adjustments of maximum con-taminant levels or cleanup levels should be based upon in vivo studies with experimen-tal animals resembling humans, e.g.: with immature or juvenile swine /42/.

A set of general factors to be considered deciding whether to include bioavailability studies at a site has been suggested /5/:

+ limited number of critical contaminants

+ contaminant levels exceeding but close to MCL’s or cleanup goals

+ form of contaminant likely to exhibit low RAF

+ high probability of public and regulatory acceptance of RAF based MCL adjust-ment

+ large soil volumes affected

+ costly cleanup technologies required


+ adequate cleanup technologies not available

+ risk of environmental deterioration due to required cleanup

+ old, weathered contamination (not unambiguous)

- demand for fast intervention

- contaminant species likely to yield high RAF

- soil characteristics likely to yield high RAF

In other words: if an increase in MCL’s is likely to result from a bioavailability study, and if the costs of cleanup are sufficiently large, a bioavailability study is worth consid-ering. It should be noted that site specific RAF data are generally required in the US /9/.

2.3 Bioaccessibility

The bioaccessible fraction of a soil contaminant is the fraction of the total soil concen-tration that can be dissolved in the gastrointestinal tract of the organism in question, compare Figure 2-1. The bioaccessible concentration is considered the upper limit of the contaminant concentration that can reach systemic circulation and thus cause a systemic toxic impact, again compare Figure 2-1, factors fa and fd are in the range 0-1. Corre-sponding to the term relative bioavailability (RAF), the relative bioaccessibility factor (RAC) is obtained as:

RAC = amount dissolved from soil matrix/amount of soluble contaminant dis-solved from the matrix used in the toxicity study

Bioaccessibility is generally measured using an in vitro laboratory tests simulating the conditions in the gastrointestinal tract, but in vivo measurements of soil contaminants in the stomachs and intestines of animals have been reported, see e.g.: /32/.

In Europe, the emphasis in risk assessment is currently on developing in vitro tests for bioaccessibility as an estimate of the bioavailability of soil contaminants /17/. Also, the US EPA is moving towards accepting “validated” in vitro tests for lead.

The rationale behind this is that in vitro tests:

• are faster, less costly and more reproducible than in vivo tests

• yield a conservative estimate of internal exposure

As requirements for bioaccessibility test methods to be applied in risk assessment of contaminated soils it should be considered that the methods are:

• justifiable

• robust

• relevant


With “justifiable” is meant that the test simulate the appropriate processes in the human gastrointestinal tract, i.e.: is based upon human physiology. The physiology of the hu-man gastrointestinal tract has been review elsewhere (see e.g.: /17;45/). For physiologi-cally based test methods, the selection of the segments or segments (mouth, oesophagus, stomach, small intestine, colon etcetera) to include and the conditions in each segment should reflect the selected target (fasted child, fed adult etcetera) and the physiology of contaminant uptake. It is generally acknowledged, that the oral uptake of e.g.: lead and other metals from contaminated soils is occurring in the small intestine /46;47/ leading to the suggestion that physiologically based test methods should include mouth, oe-sophagus, stomach and small intestinal segments.

“Robust” methods can be reapplied at the same laboratory or at another laboratory giv-ing approximately the same result for the same soil, i.e.: the within laboratory and be-tween laboratory variations are sufficiently small. Robustness evaluation should also in-clude that the test can be applied with method detection limit resembling what is required considering the MCL’s in question.

The term “relevant” means that the test yields results that reflect the in vivo bioavailabil-ity of the soil contaminants, i.e.: that there is a linear correlation between the in vivo bioavailability as obtained in experiments with accepted animals (or humans) and the in vitro test results and preferentially, that the in vitro test results are in general equal to or slightly higher than the in vivo bioavailabilities.

The balancing of these three requirements will depend upon an overall technical and po-litical evaluation considering the application of the data in risk assessment, economy and probability of public acceptance.

2.3.1 In vitro bioaccessibility test methods In a recent review /3/, a range of factors for consideration in design of in vitro bioacces-sibility test methods were derived from the physicochemical properties of a range of po-tential soil contaminants:

• low pH dissolution of iron oxyhydroxides and/or disruption of cation exchange complexes (all metals)

• high pH and enzymatic dissolution of soil organic matter (PAH)

• “surfactant” aided dissolution (PAH)

• complex binder aided dissolution (all cationic metals, high chloride important for lead)

• presence of solubility impacting ions such as phosphate (As, Cr and Pb)

• sequential testing (acidic followed by alkaline) with separate release measure-ments in each sequence to avoid errors from dissolution followed by precipitation (all cationic metals)

• aerobic and anaerobic conditions where pertinent (As and Cr)


In the same review /3/, another range of factors for consideration in design of in vitro bioaccessibility test methods were derived from the properties of the human digestion system, if a physiologically based test method is aimed at:

• low pH for dissolution of soil constituents, lower than 2

• acidic digestion time, at the least 3 hours

• subsequent high pH for dissolution of soil constituents, higher than 7

• alkaline digestion time, at the least 10 hours

• additions of enzyme types found in the human gastrointestinal tract

• additions of bile and other chyme constituents capable of dissolving apolar con-taminants and metals

• digestion at 37°C

• optional representation of both aerobic (oxidizing) and anaerobic (reducing) con-ditions

From the published papers on the development bioaccessibility test methods, the review /3/ identified a range of important experimental details for specification in a suitable bioaccessibility test method (first record listed is referenced, most findings published by several authors):

• mixing or stirring rate /48/

• digestion time /48/

• presence of food substitutes such as milk powder /48-50/

• digestion pH, requirement for buffering /30/

• soil particle size /51/

• liquid to solid ratio, L/S > 100 /52/

• presence of organic acids in digestion fluid /53/

• bile amount added /12/

• mucin added /54/

• gastric and intestinal digestion required /54/

• chloride added/55/

Further details with respect to test method conditions are given in section 3.1.


The design of an in vitro bioaccessibility test method thus must be a compromise be-tween a series of factors derived from contaminant chemistry, human digestion physiol-ogy and practical test considerations, in addition to factors derived from the anticipated use of the results in risk assessment of contaminated soils.

At the least 10 different methods for bioaccessibility testing of soil contaminants were identified in a recent review /3/. For 8 of these methods, the relevance of the test results can be evaluated by comparison with in vivo bioavailability data for one or more con-taminated soils, see Table 2-4.

Table 2-4 Bioaccessibility test methods with in vivo bioavailability data for one or more contaminated soils

Method Segments included

Addition of food

Principle Contaminants tested

Refe-rence

PBET Stomach and intestine

None Simplified ba-sed upon hu-man physiology

Lead, arsenic /30/

SBRC Stomach, intestine op-tional

None Simple buffered acid, aiming at robust worst case

Lead, arsenic /56;57/

IVG Stomach or intestine after stomach

Optional Simplified ba-sed upon hu-man physiology

Lead, arsenic and cadmium

/20;38;58/

MB Saliva, stom-ach and in-testine

None Simplified ba-sed upon hu-man physiology

Lead /59/

RIVM Saliva, stom-ach and in-testine

Optional, part of fed state ver-sion of the test for or-ganic con-taminants

Corresponding to human phy-siology

Lead /46;60/

DIN Stomach and intestine, saliva op-tional

Optional Corresponding to human phy-siology

Lead, cad-mium, nickel and PAH

/50/

SHIME Stomach and small intes-tine, colon optional

Optional Corresponding to human phy-siology

Lead /61/

TIM Stomach and intestine

Included Dynamic simu-lation of human physiology

Lead /61/

The details of the different test methods have been summarized previously, see e.g.: /3;19;62/.

It should be noted, that most in vitro bioaccessibility tests do not include the effects of the microbial communities present in the in vivo gastrointestinal system, and do not in-clude the effects of active transport of contaminants from the digestion solution /19/.


2.3.2 Comparability of in vitro bioaccessibility test methods At this point, it should be emphasised that different test methods with different compo-sitions of test solutions will inevitably yield different test results as also demonstrated employing five different test methods with each of three different soils /62/. Still, differ-ent test methods with different test solution compositions may all provide justifiable, robust and relevant test results as defined above, see section 2.3, just with different cor-relations between in vitro bioaccessibility test results and in vivo bioavailability data from animal studies.

In one study /62/, the bioaccessibilities obtained with different test methods for the same soil samples were compared, see Table 2-5.

Table 2-5 Bioaccessibilties of lead, cadmium and arsenic from 3 soils as obtained with 3 selected in vitro test methods /62/

SBRC DIN RIVM Lead Oker 11 0.56 0.16 0.29 Montana 2711 0.90 0.46 0.11 Flanders 0.91 0.31 0.66 Cadmium Oker 11 0.92 0.62 0.51 Montana 2711 0.99 0.45 0.40 Flanders 0.92 0.38 0.78 Arsenic Oker 11 0.11 0.11 0.19 Montana 2711 0.59 0.41 0.59 Flanders 0.50 0.30 0.95

The differences in bioaccessibilities obtained with different methods are obvious from the data in the table. Overall, the simple methods with a stomach segment seem to give higher bioaccessibilities than more physiologically correct methods with intestinal seg-ments included. Still, which method is most “correct” depends upon the criteria set up for an acceptable method, see section 2.3, and in most cases upon the correlation be-tween the in vitro bioaccessibilities and in vivo bioavailabilities for the methods in ques-tion, see section 2.4.

2.4 In vitro bioaccessibility to in vivo bioavailability correlation

In order to be relevant, an in vitro bioaccessibility test method can be expected to pro-vide data correlated to data obtained with in vivo bioavailability data. This approach does have limitations:

• in vivo data for bioavailability may not have been obtained in the same experimen-tal animals as the toxicity data behind the MCL

• in vitro tests for bioaccessibility aim at simulating the dissolution in the human gas-trointestinal tract, whereas the in vivo bioavailability methods are based upon the conditions in the experimental animals used


• in vivo methods include both dissolution of contaminants from soils, uptake through the gastrointestinal walls and any subsequent excretion, whereas the in vitro test in-clude dissolution only

• in vitro test bioaccessibility data depends upon the test method used

• in vivo bioavailability data depends upon the experimental animals, dosages, target organs and calculation methods

• both in vivo and in vitro results are subject to variation due to soil inhomogeneity, method performance variations and for in vivo results in addition to biological vari-ability

In other words: in vitro bioaccessibility test methods and in vivo bioavailability studies will give different results as they are designed to investigate different parts of the oral uptake of contaminants from soils, and the results from both types of studies are associ-ated with variation as well as with systematic and random errors. Still, a valid in vitro bioaccessibility test method should reflect differences in solubility of soil contaminants in the gastrointestinal system and thus relate to an upper limit of the potential for uptake of the contaminants from the soil tested, as compared to other soils and to soluble forms of the contaminant. If the rate limiting step of contaminant uptake is the dissolution from soil, there will be a linear relation between bioaccessibility and bioavailability.

A correlation is expected for in vitro relative bioaccessibility to in vivo relative bioavail-ability rather than to absolute bioavailability. Accordingly, the in vivo data presented in the subsequent chapters and sections are relative bioavailabilities, if nothing else is noted.

2.4.1 In vitro bioaccessibility to in vivo bioavailability correlation lead The correlations in vitro to in vivo for lead reported in the literature are shown for test methods with a stomach segment only in Figure 2-3 and for tests with an intestinal seg-ment in Figure 2-4, see Table 2-1 and Table 2-4 for explanations and references for ap-plied methods.

The published correlations clearly demonstrates, that:

• different bioavailability calculation methods yield different in vitro to in vivo rela-tionships for the same bioaccessibility test method and the same experimental animals (top figures, Figure 2-3 and Figure 2-4)

• different bioaccessibility test methods yield different in vitro to in vivo relation-ships for the same bioavailability method and experimental animal (bottom fig-ures, Figure 2-3)

• bioaccessibility test methods with an intestinal segment yield lower in vitro to in vivo relationships than methods with a stomach segment only for the same bioavailability methods and experimental animas (Figure 2-3 and Figure 2-4)

• some combinations of in vitro bioaccessibility and in vivo bioavailability test methods provide poor correlation (bottom right, Figure 2-4)


It should be noted that the IVG bioaccessibility test method applies an intestinal segment after removing the contaminants dissolved in the stomach segment, and this method is thus expected to provide lower test results than methods that do not use this approach.

Figure 2-3 In vitro bioaccessibility of lead from soils against in vivo bioavailability as reported in the litera-ture for in vitro test methods with a stomach segment only /8;30;63;64/

Figure 2-4 In vitro bioaccessibility of lead from soils against in vivo bioavailability as reported in the litera-ture for in vitro test methods with an intestinal segment /63-66/

Poor in vitro to in vivo correlation was reported for the NIST 2710 certified reference material soil using the mass balance bioaccessibility test method and rats for bioavail-ability studies /59/.

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Table 2-6 demonstrates that in vitro test methods such as RIVM and TIM in fasted state versions could yield bioaccessibility data that for one soil sample corresponded to the in vivo uptake in humans, whereas other methods did show very significant discrepancies, refer to Table 2-4 for explanations and references for applied methods.

Table 2-6 In vitro bioaccessibilities of lead from the Bunker Hill soil against the in vivo bioavailabilities measured in humans /61/

Method Bioaccessibility Absolute bioavailability humans Fasted Fed Fasted Fed PBET 0.13 0.22 RIVM 0.32 0.24 DIN 0.14 0.29 SHIME 0.020 0.24 TIM 0.28 0.038

0.26 0.025

2.4.2 In vitro bioaccessibility to in vivo bioavailability correlation cadmium The correlation in vitro to in vivo for cadmium reported in one published study with a test method with a stomach segment and for a test with an intestinal segment is shown in Figure 2-1, see Table 2-1 and Table 2-4 for explanations and references for applied methods.

Figure 2-5 In vitro bioaccessibility of cadmium from soils against in vivo bioavailability as reported in the lit-erature for an in vitro test method with a stomach segment (left) followed by an intestinal seg-ment (right) /58/

A linear relationship in vitro to in vivo and generally higher bioaccessibility than bioavailability was found for the bioaccessibility test method with a stomach segment, whereas the linear relationship was less evident and the bioaccessibility data lower for test in a subsequent intestinal segment. Again, it should be noted that the IVG bioacces-sibility test method applies an intestinal segment after removing the contaminants dis-solved in the stomach segment, and this method is thus expected to provide lower test results than methods that does not use this approach.

In vitro bioaccessibility and in vivo bioavailability data were also reported for the DIN test against mini pigs /29/, but the set up of the bioavailability study did not allow for calculation of the relative bioavailability of cadmium (no soluble references included) and the data can thus not be evaluated here.

2.4.3 In vitro bioaccessibility to in vivo bioavailability correlation nickel Two sets of in vitro bioaccessibility and in vivo bioavailability data were reported for nickel from soil. Data from the DIN test against mini pigs /29/ did not allow for calcula-


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tion of the relative bioavailability of nickel (no soluble references included) and the data can thus not be evaluated here. For a Canadian study, the data could not be released for publication.

2.4.4 In vitro bioaccessibility to in vivo bioavailability correlation arsenic The correlations in vitro to in vivo for arsenic reported in the literature are shown for test methods with a stomach segment only in and for tests with an intestinal segment in Figure 2-6 and Figure 2-7, see Table 2-1 and Table 2-4 for explanations and references for applied methods.

Figure 2-6 In vitro bioaccessibility of arsenic from soils against in vivo bioavailability as reported in the lit-erature for in vitro test methods with a stomach segment only /20;57/

Figure 2-7 In vitro bioaccessibility of arsenic from soils against in vivo bioavailability as reported in the lit-erature for in vitro test methods with an intestinal segment /20;29/

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It should be noted, that the in vivo data used for correlation with the IVG, PBET and DIN in vitro data were based upon urinary excretion as customary for arsenic studies, whereas the target organs behind the in vivo data used for correlation with the SBRC in vitro data were not stated in the reference.

The tests with a stomach segment only all show linear correlation in vitro to in vivo, SBRC better than IVG stomach better than PBET, but with slightly higher in vivo than in vitro data. The tests with an additional intestinal segment also show linear correlation in vitro to in vivo, PBET better than IVG intestine with very few data for the DIN test method. The in vivo data are higher or slightly higher than the in vitro data for PBET and IVG, respectively.

Again, it should be noted that the IVG bioaccessibility test method applies an intestinal segment after removing the contaminants dissolved in the stomach segment, and this method is thus expected to provide lower test results than methods that do not use this approach.

2.4.5 In vitro bioaccessibility to in vivo bioavailability correlation PAH In vitro bioaccessibility and relative in vivo bioavailability data were reported for the PAH compound benzo(a)pyrene (BaP) as obtained with the DIN test against mini pigs /29/, Figure 2-8. Only a limited number of soil samples were tested (4), and the relative in vivo bioavailability was found as the amount of the compound not excreted with fae-ces. The bioavailabilities must thus be considered an upper limit of the fraction of the compound reaching systemic circulation in the mini pigs.

Figure 2-8 In vitro bioaccessibility of benzo(a)pyrene from soils against in vivo bioavailability as reported in the literature for in an vitro test method with a stomach segment and an intestinal segment /29/

A more detailed analysis /3/ of the in vitro bioaccessibilities and absolute in vivo bioavailabilities (again obtained as upper limits of bioavailability from PAH not ex-creted with faeces) from this study /29/ demonstrated that for 4 of the PAH in all 4 soils and for all of the 12 PAH in one soil, a reasonable in vitro to in vivo correlation was ob-tained, but this was not the case considering all PAH in all 4 soils.

For pyrene, the bioavailability can be estimated from the amount not excreted with fae-ces as mentioned above, but the data in the report fra /29/ also allows for estimation of the bioavailability from the amounts of the metabolite 1-hydroxypyrene excreted with urine, Table 2-7.


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Evidently, the order of the bioavailabilities and bioaccessibilities differ among the methods applied and the compounds considered.

It should be noted, that the variability in the in vivo bioavailability data was consider-able (up to 100% relative standard deviation) in this study and that for one experimental group, the data from one of four animals were omitted from the data treatment due to excessive variation.

Table 2-7 Bioavailability and bioaccessibility of PAH from 4 different soils given ordered relative to the most bioavailable/bioaccessible soil for each method, calculated from /29/

Soil identifi-cation

Bioavailability order calculated from non excreted PAH with fae-ces

Bioavailability order calculated from ex-creted 1-hydroxypyren with urine

Bioaccessibility order

Pyrene BaP Pyrene Pyrene Bruchsal 0.39 0.65 0.73 0.24 Carl 1 0.79 1.00 0.60 0.28 Lothringen 1 0.75 0.86 0.53 0.36 Lothringen 2 1.00 0.85 1.00 1.00



3 SELECTION OF METHODS FOR IMPLEMENTATION AND VALIDATION IN DENMARK

The selection of test methods for use in Denmark included a series of selection steps:

• selection of test conditions (test method and practical performance)

• selection of quality requirements and quality control of test results and analytical procedures

• selection of methods for chemical analysis of soils and digestion solutions

• selection of soil pretreatment procedures

3.1 Selection of test conditions and methods

Based upon the points of importance for selection of bioaccessibility test conditions from details on the human gastrointestinal physiology, section 2.3.1, on the physico-chemical properties of the relevant soil contaminants, section 2.3.1, on the test design requirements, se section 2.3.1 and on the use of the in vitro bioaccessibility test data in risk assessment, section 2.3, the following test details were found to be most important to consider /3/:

• buffered low pH (pH < 2) high chloride gastric segment

• buffered slightly alkaline (pH > 7) phosphate containing intestinal segment

• aerobic followed by anaerobic conditions (stomach and intestine, respectively, op-tional)

• separate assessment of bioaccessibility in the two segments (gastric and gastric fol-lowed by intestinal)

• addition of enzymes, bile and milk powder (or similar food constituent)

• sufficient time in each segment (3 hour in gastric segment, 10 hours in intestinal segment)

• L/S stability (L/S > 100)

• efficient mixing of soil and test solutions

Three different methods are currently widely used for routine testing:

• SBRC (Solubility/Bioavailability Research Consortium method developed from the Physiologically Based Extraction Test, PBET, method) /57;67/

• DIN (Deutsches Institut für Normung) /68/


• RIVM (National Institute for Public Health and the Environment) /60;69/

The SBRC method was originally developed by John Drexler, University of Colorado /36/, is also known as the Drexler or RBALP (relative bioaccessibility leaching proce-dure) method and is available through the Internet /57/. The original PBET method is still used at some European laboratories.

The digestive tract method /70/ was developed into the DIN method /71/, and the group behind the digestive tract method now apply the DIN method /65/.

The RIVM method comprises two versions, a fasted state version for metals such as lead /69/ and a fed state version for organic contaminants such as PAH /60/, both of which are discussed in this report.

None of the three methods satisfy all the above mentioned test requirements, but the subsequent sections present and discus the test conditions in the methods, based upon the references given above, including as appropriate data from published method stud-ies. The discussions have been supported by personal communication with the principal researchers behind the PBET method, Michael Ruby, Exponent Environmental Group, the SBRC method, John Drexler, University of Colorado, and the RVM methods, Agnes Oomen, RIVM, including a visit to RIVM.

3.1.1 Test method principles and segments Studies of bioaccessibility of organic soil contaminants such as dioxins have employed both stomach and intestinal segments /72/. The conditions of this method are close to those employed in the RIVM method. For one soil, the bioaccessibility of PAH in-creased by a factor of above 7 by adding an intestinal segment to a stomach segment /70/. Conversely, the bioaccessibility of lead decreased by a factor of almost 10 after adding an intestinal segment to a simple stomach segment based test /73/. The impact of adding the intestinal segment upon lead bioaccessibility was decreased by adding milk powder as a food surrogate /73/. For the PBET test, a decrease in lead dissolution has been demonstrated during transition from the low pH of the stomach segment to the higher pH of the intestinal segment /74/.

The general steps of the three test methods mentioned above are presented in Figure 3-1 and Table 3-1.

Figure 3-1 Outline of steps and segments in in vitro bioaccessibility test methods

The three methods are all initially based upon human gastrointestinal physiology, con-sidering factors such as composition of digestion juices, relative amounts of juices dur-ing digestion and ratios soil to digestion juices. The link to human physiology is main-tained for the RIVM and DIN methods /69;71/, whereas the SBRC method is simplified with the emphasis on obtaining a good correlation in vitro bioaccessibility to in vivo with the most simple test /75/.

Samplepretreatment

Mouth and oesophagus dissolution

Chemical analysis of soil and solutions

Intestine dissolution

Stomach dissolution

Samplepretreatment

Mouth and oesophagus dissolution

Chemical analysis of soil and solutions

Intestine dissolution

Stomach dissolution


The SBRC method with a stomach segment only is used widely in the US for lead, cadmium and arsenic bioaccessibility testing, and the test data are well correlated to ju-venile swine in vivo bioavailability data, sections 2.4.1, 2.4.2 and /76/. The stomach segment based SBRC test is also recommended for nickel, whereas addition of the op-tional intestinal segment is recommended for chromium and mercury /67/.

Table 3-1 Segments in the three selected in vitro bioaccessibility test methods

Test method SBRC RIVM DIN Fasted Fed Mouth and oesophagus No Yes Yes Optional Stomach Yes Yes Yes Yes Intestine Optional Yes Yes Yes Food addition No No Yes Optional

The RIVM method is described as a ”realistic worst case” test method /69/. In the pres-entation of the RIVM method /69/, it is stated that the small intestinal segment is in-cluded only and not the more distal parts of the intestine, because dissolution of e.g.: lead is primarily occurring in this part of the gastrointestinal system.

3.1.2 Fasted or fed state test conditions The impact of food surrogate addition upon dissolution of metals is inconclusive, see e.g.: /61/. Studies on food addition have shown that the use of cooking oil as a food sur-rogate is technically not feasible /77/, and that addition of milk powder increased the dissolution of e.g.: PAH from one soil in both stomach and intestinal segments by fac-tors 2-4 /70/ and from 5 other soils by factors 5-10 using combined stomach and intesti-nal segments /78/. Furthermore, it has been suggested to add an unsaturated fatty acid to include the effects of mixed bile lipid micelles upon the dissolution of dioxins from soil /72/, but the impact of this was not documented.

The SBRC and the fasted state RIVM tests simulate dissolution in fasted children /75/, /69/, whereas the other RIVM test simulates dissolution in children after a standard meal (fed state) /79/. The RIVM fed state test is designed to include the introduction of a food surrogate (baby food) in the test and to reflect the changes in composition of the digestion juices after introduction of food by e.g.: increase in pH, in bile and in enzyme concentrations /79;80/. The amount and type of food added is adjusted to yield stable high dissolution of the PAH benzo(a)pyrene (BaP) /79/. The DIN method allows for ad-dition of milk powder as a food surrogate without changing the test solutions /68/.

Fasted state conditions were decided upon for test of metal bioaccessibility and fed state for organic contaminants in order to achieve the “realistic worst case” bioaccessibility estimates aimed at for risk assessment of contaminated soils.

3.1.3 Gastrointestinal segments The rationale for selecting gastrointestinal segments for the bioaccessibility tests is pre-sented in Appendices A-C.

It was decided initially to aim at one or more test methods that simulate the physiology of the human digestion including all the relevant segments: mouth/oesophagus, stomach


and small intestine. In order to reduce test costs and because a routine test rather than a research test was aimed at, test solutions were to be analysed after the full digestion pro-cedure only.

The available data does not allow evaluation of the importance of a mouth/oesophagus segment. The conditions should reflect physiologically relevant conditions with slightly higher pH and amylase concentrations in the fed than in the fasted state versions.

The stomach segment should have a final pH of <2 in order to reflect the conditions in the stomach of a fasted child, respectively 2-2.5 in the fed state reflecting the conditions in the stomach of a child after a meal. The chloride concentration should be enhanced from that originating from the acid addition only in order to increase complexation and dissolution of e.g.: lead. Organic acids, serum albumin and pepsin should be added to increase dissolution of soil, contaminants and food components. The test time for the stomach segment should be set to not less than 2 hours and thus high compared to typi-cal physiological conditions and yielding high bioaccessibilities.

The intestinal segment should have an intermediary pH of not less than 5.5 for fasted state and 6.5-7.0 for fed state in order to reduce the precipitation of metals such as lead at fasting conditions and to ensure the most complete dissolution possible of organic contaminants at fed conditions. Organic acids and high concentrations of bile, pan-creatin, lipase and serum albumin should be added to ensure high solubility of both metals and organic contaminants. In the fed state, the concentrations of bile and en-zymes should be increased as appropriate in order to reflect the physiological changes imposed after a meal. A test time in the intestinal segment of not less than 2 hours is suggested with higher test times resembling physiological residence times in the upper part of the small intestine the most.

3.1.4 Selection of practical test performance It was decided to aim at a test with ”all over” mixing at 37°C with e.g.: 10 rpm and en-suring full mixing (no settled solids in test bottles) ensured by visual inspection. Ad-justment of pH should be by addition of test solutions of appropriate pH and sufficient buffer capacity to ensure final pH values in the different segments within acceptable ranges as established elsewhere. Phase separation should not aim at removal of colloids and could be by centrifugation at approximately 3000 g for 5 minutes.

Compatibility of materials and cleaning with the contaminants and concentrations tested should be demonstrated by tests of blank and control samples.

Test L/S should be approximately 100 L/kg (see also appendix C) from considerations respecting practical test performance and requirements for low test analytical detection limits.

Redox conditions are not controlled as long as redox sensitive contaminants are not tested for.

Further details on practical test performance are given in Appendix D.


3.1.5 Decision on test method Based upon evaluation of the test conditions for the three test methods, see sections 3.1.2, 3.1.3 and 3.1.4, it was decided to initially implement and validate the RIVM in vi-tro bioaccessibility test methods in Denmark. The rationale for this decision was, that these tests:

• include all important dissolution processes for soil contaminants in the human gas-trointestinal system

• can be applied with large test series at reasonable costs

• is supported by an active research, development and application environment

• are available in versions fully compatible with fasted and fed state for metals and organic contaminants, respectively

It was considered to modify the RIVM test conditions but initially, this was rejected in order to maintain the benefits of applying the same test in more than one laboratory and one country, and thus enable comparison and sharing of results and know how.

In consequence of the results of the initial validation of the RIVM fasted state test for lead, it was decided to modify the RIVM test to include mouth/oesophagus and stomach segments only (see sections 4.4, 5.2, 5.2.5 and 6), and to compare data obtained with this method with data obtained with the SBRC method, see section 5.2.6.

3.2 Test quality control

Chemical analysis of soil samples and test solution samples must be subject to quality control applying normal tools such as duplicate analysis, blanks, control samples, profi-ciency tests etc., as well as conventional evaluation of analytical quality (analytical de-tection limit, precision, “trueness”) relative to established quality requirements and to statistical stability using control charts.

It should be emphasized here, that the complicated matrices of digestive juices may im-pact recoveries and precisions considerably for both PAH and metals, and the methods applied must thus take this into consideration in method validation, in quality control and in design of quantification procedures employing principles such as standard addi-tion calibration (metals) or external standard curve calibration including the full analyti-cal procedure including extraction (PAH).

The test quality control should include 2 test blanks, 2 synthetic control samples and 1 duplicate test per locality, see also Appendix E for more detailed discussion, but it should be considered to provide access to a stable and homogenous matrix reference material for use as matrix control samples in all test series.

3.3 Methods for chemical analysis of soils and test solutions

For analysis of soils in investigations of contaminated soils, the DEPA requires applica-tion of the Danish Standard Method (DS) 259: digestion with dilute nitric acid, HNO3,


1:1, under elevated temperature and pressure (autoclave) followed by quantification by ICP3 or AAS4 /81/. The method is also presupposed in establishing the Danish soil metal MCL’s. It would be expected that different digestion methods will provide differ-ent recoveries of soil metals and it is generally assumed that digestion according to DS 259 provides only partial recovery.

PAH in contaminated soils shall in Denmark currently be analysed applying a method based upon extraction with a mixture of toluene and aqueous pyrophosphate solution, followed by GC5-MS6 quantification /81/, but two new methods are currently being de-veloped for the DEPA. The published information on analytical methods for soil PAH in a bioaccessibility context does not allow for an evaluation of the impact of any method differences. Still, for PAH analysis the aim is complete extraction of soil PAH (in contrast to e.g.: the DS 259 digestion method for metals) and it would thus be ex-pected that the differences between different methods would be marginal.

There are no specifications of analytical methods that must be used for test solutions as part of bioaccessibility testing of metals or PAH in contaminated soils in Denmark. For groundwater, metal analysis according to the Danish Standards DS 2211 (AAS) or DS 11885 (ICP) would generally be applied, whereas PAH analysis would be done after ex-traction with GC-MS quantification.

The following methods for analysis should be applied, for soils in order to comply with Danish requirements, and for test solutions based upon the methods required for the test methods:

• Metals in soils: digestion of soil sample with nitric acid (DS 259, HNO3, 1:1) fol-lowed by ICP-OES7 analysis with calibration after external standard curve

• Metals in test solutions: dilution of test solutions with nitric acid solution followed by ICP-OES analysis with calibration after standard addition

• PAH in soils: extraction of soil samples with aqueous pyrophosphate solution and dichloromethane followed by GC-MS analysis with calibration after external standard curve and correction for internal deuteriated standards

• PAH in test solutions: extraction of test solution with dichloromethane, solid phase extraction and GC-MS analysis with calibration after external standard curve in test solutions using internal deuteriated standards

The quality objectives for analysis of soils and test solutions are given in Table 4-1, see also Appendix F for more detailed discussion. Requirements for analytical and test de-tection limits are set to 1/10 of the MCL’s in question. Official quality requirements have not been established in Denmark for these analysis and tests, but the data quality objectives set are equivalent to or slightly lower than those given for quality class 3 in the Danish guidelines for the quality of environmental measurements and sampling /82/.

3 Inductively coupled plasma, a multielement method primarily for determination of metals 4 Atomic absorption spectrophotometry, a older method for determination of elements, primarily metals, one by one 5 Gas chromatography, a method for separation of primarily organic compounds 6 Mass spectrometry, a method for quantification and identity control of primarily organic compounds 7 Optical emission spectroscopy, a method for quantification primarily of metals


3.4 Pretreatment of soils

Drying is generally done in order to allow for efficient subsequent grinding and sieving. Volatile samples will disappear during drying, and redox sensitive species may change due to oxidation.

Grinding/milling/crushing is done in order to allow for subsequent homogenization and representative subsampling of test portions. Gentle grinding (mortar or in closed plastic bag) may be sufficient to break clay aggregates. Milling and crushing will also break large particles into smaller fragments and is generally done in mechanical mills etc.

With more efficient drying, grinding and homogenization, more representative test por-tions can be obtained and tests and analyses can accordingly be more precise. Con-versely, the more efficient methods are also less gentle and will impose larger changes in contaminant concentrations and properties, dependent upon the contaminants in ques-tion.

It is thus essential to decide upon an appropriate pretreatment procedure considering also the use of the test results in risk assessment of the contaminated soils.

For pretreatment of soils in Danish investigations on contaminated soils intended for metal analysis, it is required to /81/:

• removal of particles larger than 5-10 mm prior to analysis

and given as options are:

• drying at 40°C

• grinding

• sieving to less than 2 mm

• homogenization

Fractions of large particles higher than 10% removed should be reported. For PAH, manual removal of particles larger than 5-10 mm is the only option available according to the guideline.

The international standard ISO 11464 for physical chemical analysis of soils suggests:

• drying

• removal of large particles

• sieving to <2 mm

• removal of large particles from particles >2 mm

• grinding of clods >2 mm in mortar

• sieving to <2 mm of grinded clods


• homogenization

• subsampling

If the test portion is less than 2 g, the samples should further be:

• milled/crushed

• sieved to <250 µm

In ISO 14507 for analysis of soils for organic contaminants, the suggested procedures are:

• addition of chemical drying agents (e.g.: sodium sulphate (sic.))

• mechanical milling with cooling under liquid nitrogen

As alternative, when milling is not feasible, it is suggested to:

• remove large particles

• grind by hand in mortar

These procedures are time consuming and could impact soil contaminants concentra-tions and properties excessively.

Conventional sample pretreatment at Danish laboratories would be removal of particles larger than 2 mm by hand from samples without drying. Typical test portions for metals would be 1 g and for PAH 50 g.

Sieving to <250 µm particle size is used in the SBRC method, because these small par-ticles adhere to the hands of children and therefore are swallowed by the children. Dan-ish MCL’s are set considering not only ingestion of soil particles adherent to the hands (and other surfaces) but also direct ingestion of small amounts of soils. In response to this risk assessment approach, full soil samples should be tested.

The following procedure was decided upon for pretreatment of soil samples for bioac-cessibility testing of metals (non redox sensitive) and PAH (larger than 4 rings), see also Appendix G for more detailed discussion:

• drying of not les than 250 g of soil at 40°C until constant weight (±5% over 24 hours)

• manual removal of pebbles, twigs etc.

• grinding by hand in mortar to break clay aggregates

• sieving to <2 mm

• repeated grinding and sieving of clods >2 mm if necessary

• homogenization by mixing with a spoon or spatula


• subsampling of test portions

• weighing of all removed fractions for reporting

Sieves of hard plastic (low plasticizer contents) and spoons/spatulas with teflon coating are used.

The procedure is intended for use with soil samples that should be tested for both the metals and the PAH, with the necessary compromises made for that purpose. The pro-cedure is suitable with risk assessment based upon soil ingestion with e.g.: children, but not for risk assessment based upon ingestion of soil particles adhering to hands, vegeta-bles etc, see discussion in Appendix G.

The procedure will not be adequate prior to testing for volatile compounds (mercury, many organic compounds including PAH smaller than 4 rings) and not for soil samples with an anaerobic history.



4 VALIDATION OF THE INITIALLY SELECTED RIVM METHODS

The quality objectives set for the analytical methods and the test methods for this study are presented in Table 4-1, revised as found appropriate during the method implementa-tion, validation and application.

Table 4-1 Data quality objectives for analyses and tests

Matrix Metals PAH Parameter Cadmium Lead Nickel Benzo(a)

pyrene Dibenz(a,h) anthracene

Analysis of soil Relative standard deviation <15% Recovery 90-110% Analytical limit of detection (mg/kg dw)8

0.5 4 3 0.01 0.01

Analysis of test solution Relative standard deviation <10% Recovery 90-110% Analytical limit of detection (µg/L)

5 40 30 0.1 0.1

Test of soil Relative standard deviation <15% Analytical limit of detection (mg/kg dw)

0.5 4 3 0.01 0.01

As an additional requirement, the linear range of the tests should be at the least up to 5 times the MCL for each contaminant to be tested for.

4.1 Analysis of soils

The principles and analytical quality obtained in soil analysis are shown in Table 4-2.

The analytical method for metals in soils is the Danish standard method (DS 259), but the method differs from the method used in most other countries with respect to the di-gestion method, see section 3.3. A selection (16) of soil samples from the Danish field study (section 5.1) was therefore analyzed additionally according to a commonly ap-plied method employing aqua regia digestion but still with ICP-OES quantification,

Figure 4-1.

8 Set to 1/10 of the MCL to be tested against


Table 4-2 Principles and analytical quality obtained in soil analysis

Metals PAH Parameter Cadmium Lead Nickel Benzo(a)


Principles Digestion of soil sample with nitric acid followed by ICP-OES analysis with calibration after external standard curve

Extraction of soil sample with aqueous pyrophos-phate solution and di-chloromethane followed by GC-MS analysis with cali-bration after external stan-dard curve and correction for internal deuteriated standards

Relative standard deviation (%)

10 10 10 12 12

Recovery9 (%)

90-110 95-105 95-105 95-105 -10

Analytical limit of detection (mg/kg dw)

0.1 3 1 0.005 0.005

Figure 4-1 Metal concentrations in selected soils analyzed after diluted nitric acid digestion and after aqua regia digestion, both with ICP-OES quantification

The results according to the Danish DS 259 have a fine linear correlation to those ob-tained after the commonly applied aqua regia method.

At high concentrations, the aqua regia method provide slightly higher results for cad-mium and nickel and at concentrations close to the Danish MCL of 40 mg Pb/kg, the aqua regia method provides slightly higher values for lead. The analytical performance

9 As recovery for reference material certified to this method (metal CRM) or for control samples with added PAH 10 Recovery of dibenzo(a,h)anthracene was not part of the laboratory routine quality control

y = 1.10x - 0.235R2 = 0.9999

0

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was furthermore tested on a matrix soil reference material with certified metal concen-trations, Table 4-3. It should be noted that NIST 2710 was included in soil analysis in the first test series, whereas NIST 2711 was included in the third test series.

Table 4-3 Recovery with the DS 259 method for the certified reference materials NIST 2710 and 2711

NIST 2710, series 1 Pb Cd Ni Certified value (mean mg/kg ± uncertainty)

5532 ± 80 21.8 ± 0.8 14.3 ± 1.0

DS 259 (mg/kg)

5100 20 9.7

Recovery DS 259 (%)

92 92 68

NIST 2711, series 3 Pb Cd Ni Certified value (mean mg/kg ± uncertainty)

1162 ± 31 41.7 ± 0.25 20.6 ± 1.0

DS 259 (mg/kg)

1100 31.0 -11

Recovery DS 259 (%)

95 74 -

Overall, the soil metal concentrations obtained according to the Danish standard will be slightly below concentrations obtained with aqua regia based methods, lead and cad-mium, or well below, nickel, see also the verifications in section 5.2.2 for metals and for PAH and the discussion of test series 3 in chapter 5.2.4. For nickel, the analytical dis-crepancy should be considered evaluating the bioaccessibilities presented in this report.

4.2 Analysis of test solutions

The principles and analytical quality obtained in test solution analysis are shown in Table 4-4.

It should be noted that for lead analysis, it is critical that a procedure with dilution of test solutions with dilute nitric acid is used prior to ICP analysis and not preservation with concentrated nitric acid is followed. Even though the later procedure is standard for many applications, it has been demonstrated to cause low lead results for bioaccessibil-ity test solutions from some soils /2/, probably due to precipitation of humic acid – lead complexes from the test solution caused by the very low pH.

In order to verify the recovery of metals in the test solutions, an experiment with addi-tion of standards to test solutions obtained with RIVM fasted state test of soils sus-pected of being particularly difficult to analyze was performed, Table 4-5. The data demonstrate varying recoveries, for some samples and metals slightly below what was aimed at when defining the quality objectives of the analysis (recovery 90-110%).

11 -: not analyzed


Table 4-4 Principles and analytical quality obtained in test solution (RIVM method) analysis during valida-tion

Metals PAH Parameter Cadmium Lead Nickel Benzo(a)


Principles Dilution of test solutions with nitric acid solution followed by ICP-OES analysis with calibra-tion after standard addition

Extraction of test solution with dichloromethane, solid phase extraction and GC-MS analysis with calibration after external standard curve in test solutions using internal deuteriated stan-dards

Relative standard deviation (%)

0.58 4.5 0 3.3-3.7 2.1-4.1

Recovery (%)

99-100 103-112 99 98-116 99-100

Analytical limit of detection (µg/L)

2 10 20 0.05 0.05

Table 4-5 Recovery of metals from soil test solutions prepared with the RIVM fasted state method

Sample Concentration in test solution Recovery of added metal (mg Pb/L) (mg Cd/L) (mg Ni/L) (% Pb) (% Cd) (% Ni) Bruchsal 1.9 0.32 1.6 90 75 91 8-37926 17 3.2 14 89 98 90 8-122651 17 4.1 14 78 105 92

4.3 Test performance validation

The RIVM fasted state test for bioaccessibility of metals from soil was implemented in the DHI laboratory with normal procedures of check of blanks, recoveries etc. (data not shown) and subsequently subjected to a test validation procedure roughly according to the principles applied for analytical methods /83/, Table 4-6.

Table 4-6 Test validation for RIVM fasted state applied for the metals lead, cadmium and nickel

Cadmium Lead Nickel Analytical detection limit (mg/kg dw) 0.2 2 0.8 Precision total (%RSD) 7.2 20 8.8 Precision between series (%RSD12) 6.9 19 8.4 Precision within series (%RSD) Soil - 5.7 7.4 Soil spiked to 1 x MCL 1.8 5.0 2.5 Soil spiked to 5 x MCL 2.9 7.0 1.2 Recovery (% of added) Soil spiked to 1 x MCL 66 7.3 69 Soil spiked to 5 x MCL 73 5.6 72 Linearity (mg/kg TS) 25 (160) 130

12 RSD: relative standard deviation


The data quality objectives set, chapter 4, were met, except for the too high standard de-viation between series obtained for lead. At this point, the excessive between series RSD was explained by the use of the test solutions with limited stability over a period of 3 days without preparing fresh solutions each day. The test quality data obtained re-sembled those previously obtained for metals with the RIVM fed state method at RIVM /79/.

The low recoveries of metals, in particular of lead, added to soil was further studied us-ing reference tests without solid phase and with wheat flour added as solid phase (the toxicological vehicle for cadmium), Table 4-7. The pH in the test solutions after stom-ach and intestinal segments with spiked soil were approximately 1.5 and 5.50-6.00, re-spectively, and thus not out of the required ranges, see Table 4-11. The data demon-strated that the low lead recovery was mostly caused by soil components, but that a slight reduction (approximately 30%) in lead recovery was seen also for the test system without soil. At this time, this was attributed to test properties reflecting the processes occurring in the human gastrointestinal tract also, and it was expected that this should be addressed by using soil bioaccessibilities relative to reference bioaccessibilities in the test without soil added.

The test linearity was demonstrated up to 4-5 times the relevant MCL with fine linearity for cadmium and nickel but a deviation from linearity (Z test, 95% confidence level /83/) for lead that was explained by the previously mentioned day to day variations (dif-ferent concentrations were tested at different days). The linearity data are presented in Figure 4-2.

Table 4-7 Precision and recovery for the RIVM fasted state test without soil added for the metals cad-mium, lead and nickel

Cadmium Bly Nikkel Precision within series (%RSD) Test spiked to 1 x MCL 1.9 5.0 2.2 Test spiked to 5 x MCL 2.9 3.2 4.3 Wheat flour spiked to 5 x MCL 0.6 1.9 <0.1% Recovery (% of added) Test spiked to 1 x MCL 99 61 99 Test spiked to 5 x MCL 98 80 98 Wheat flour spiked to 5 x MCL 96 74 99


Figure 4-2 Linearity data for RIVM fasted state method with cadmium, lead and nickel

The RIVM fed state test for bioaccessibility of PAH from soil was implemented in the DHI laboratory with normal procedures of check of blanks, recoveries etc. (data not shown) and subsequently subjected to a test validation procedure according to the prin-ciples applied for analytical methods /83/ but less detailed (no between series experi-ments) because of budgetary restraints, Table 4-8.

Table 4-8 Test validation for RIVM fed state applied for the PAH benzo(a)pyrene (BaP) and dibenz(a,h)anthracene (DBahA)

BaP DBahA Analytical detection limit (mg/kg dw) 0.01 0.005 Precision within series (%RSD13) Soil 13 -14 Soil spiked to 1 x MCL 14 15 Soil spiked to 5 x MCL 11 14 Recovery (% of added) Soil spiked to 1 x MCL 84 86 Soil spiked to 5 x MCL 73 78 Linearitet (mg/kg TS) 0.01-50 0.005-50

The data quality objectives set, chapter 4, were met.

The recoveries of PAH added to soil was further studied using reference tests without solid phase and with wheat flour added as solid phase (the toxicological vehicle for BaP), Table 4-9. The number of replicates was not sufficient to allow for calculation of RSD, but the mean relative differences were 3-4%. The data demonstrated that the re-coveries were in the same range (approximately 80%) for the test with and without

13 RSD: relative standard deviation 14 Results below analytical detection limit

0

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Added ug Ni

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added solid phases. The recovery below 100% was attributed to test properties reflect-ing the processes occurring in the human gastrointestinal tract also, and it was expected that this should be addressed by using soil bioaccessibilities relative to reference bioac-cessibilities in the test without soil added.

Table 4-9 Recovery for the RIVM fed state test without soil added for the PAH BaP and DBahA

BaP DBahA Recovery (% of added) Test spiked to 10 x MCL 78 85 Wheat flour spiked to 10 x MCL 74 81

The test linearity was demonstrated up to 500 times the relevant MCL (Z test, 95% con-fidence level /83/), but the linearity data had excessive variation due to analytical prob-lems at that stage of the study. The linearity data are presented in Figure 4-3.

Figure 4-3 Linearity data for RIVM fed state method with BaP and DBahA

4.4 Correction of relative bioaccessibility of metals for test pH

The bioavailability of soil contaminants relative to the bioavailability of the soluble ref-erence form behind the toxicological evaluations is the relevant parameter for including oral bioavailability in risk assessment of soil contaminants, see sections 2.2.2 and 2.2.4. Similarly, the bioaccessibility parameter relevant to risk assessment is the relative bio-accessibility calculated as the ratio of bioaccessibility of the contaminant from the soil sample to the bioaccessibility of the soluble toxicological reference form of the con-taminant in the same test without soil added. During validation of the RIVM test meth-ods, see section 4.3, it was found that the references for all the contaminants of this study could be reference tests without solid phase, as the addition of wheat flour as food surrogate did not impact the recovery significantly, compare Table 4-7 and Table 4-9. Still, the low bioaccessibilities of lead and the varying values of final test pH in the ap-plication study, see section 5.2.3, as well as the low recovery of lead from spiked soil, see Table 4-6, did call for an additional study of the pH effects upon bioaccessibility of metals and in particular of lead.

Lead precipitates at high pH according to:

)()(2 2 sOHPbOHPb→←

−++ +

In pure aqueous solution, precipitation of lead hydroxide starts from pH = 6 at high lead concentrations /84/, but pH for precipitation depends upon both lead concentration,

0

1

2

3

0 1 2 3

Added ug BaP

Fo

und

ug

BaP

0

1

2

3

0 1 2 3

Added ug DBahA

Fo

und

ug

DB

ahA

0

1

2

3

0 1 2 3

Added ug BaP

Fo

und

ug

BaP

0

1

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Added ug DBahA

Fo

und

ug

DB

ahA


presence of metal complex ligands (reduces precipitation) and other anion forming in-soluble lead salts (increases lead precipitation). It should be noted here, that there is cur-rently no consensus on the value of the equilibrium constant (the solubility product) for simple precipitation of lead as lead hydroxide with estimates varying 4 orders of magni-tude (highest 4.2*10-15, lowest 1.4*10-20) /84/.

Accordingly, it is not possible to establish the critical pH for lead precipitation and as-sess the recovery of lead in the complicated digestion juices constituting the test solu-tions in bioaccessibility testing from theory.

A study of the recovery of the metals cadmium, lead and nickel from RIVM fasted state test without solid phase and with pH adjusted with base additions to values between 4.5 and 7 demonstrated a decrease in lead recovery from pH 4.5 to 5, a decrease in cad-mium recovery with pH for values above 5.5 and constant recovery of nickel, Figure 4-4. The pH impact upon reference bioaccessibility or recovery was further analysed by including the recoveries and pH values obtained for all reference tests done through this study, see Figure 4-5 for lead as an example, and the appropriate functions for pH de-pendent calculation of the relative bioaccessibilities were established, Table 4-10.

Figure 4-4 Recovery of cadmium, lead and nickel with the RIVM fasted state test without solid phase added against pH

30

50

70

90

110

4 5 6 7

pH

Bio

acce

ssib

ility

/rec

ove

ry(%

)

Lead Cadmium Nickel

It should be noted that these correction functions were established from tests done with-out solid phases such as soils added. This is in accordance with the use of the relative bioaccessibility data in risk assessment of the contaminants as found in soils, because this specific purpose calls for an estimate of the difference in bioaccessibility from soil and from the soluble reference only. Still, this does not fully address the problems that may arise from a reduction in contaminant dissolution caused by soil constituents that decrease or increase the contaminant dissolution per se, as only the pH effect is cor-rected for.


Figure 4-5 Recovery of lead with the RIVM fasted state test against pH including all reference tests from the study

Table 4-10 Functions for calculation of relative bioaccessibility from absolute bioaccessibility corrected for pH

pH interval I pH interval II Cadmium 4.0≤ pH < 5.5

0.889 ≥5.5

1.46-0.101*pH Lead 4.0≤ pH < 5.2

2.38-0.366*pH ≥5.2 0.483

Nickel 4.0≤ pH < 5.5 0.899

≥5.2 0.949

From the pH data obtained in the application of the RIVM test methods with soils from the Danish sites, see section 5.1, a set of pH interval requirements were established, see Table 4-11.

Table 4-11 Suggested acceptable final pH intervals in the test according to the RIVM methods with the pH data from the test application with Danish soils

Stomach segment Intestinal segment Fasted Fed Fasted Fed Interval of pH initially aimed at

1.2 2.0-2.5 >5.5 6.5-7.0

Mean pH measured ± stan-dard deviation

1.65±0.12 2.21±0.24 6.39±0.28 7.00±0.05

Measured range

1.39-1.83 1.95-2.78 5.82-6.78 6.90-7.13

Suggested acceptable pH ranges

<1.8 2.0-2.5 5.5-7.0 6.8-7.2

The application of the RIVM fasted state test methods to soil samples with in vivo bioavailability data, see section 5.2, did suggest that these intervals were not achieved

y = -36.6x + 238

R2 = 0.7559

y = -1.01x + 54.2

R2 = 0.0093

0

20

40

60

80

100

4 5 6 7

pH

Bio

acce

ssib

ility

/rec

ove

ry o

f le

ad(%

)


for many of the soils tested but apparently, there was no correlation between final pH values and the validity of the lead in vitro to in vivo correlation, see section 5.2.3. Con-sequently, a test method robust enough to maintain acceptable pH ranges in the test segments for the majority of soil samples is required, see also the comparison of test data obtained with different test methods and different final pH values section 5.2.7.

4.5 Effects of test conditions upon PAH bioaccessibility

In order to verify the test conditions in the RIVM fed state bioaccessibility test method for PAH, an experiment was done with varying test pH and test time. A Danish urban soil contaminated with ashes from a porcelain factory was tested according to the stan-dard conditions, with increased test time in the intestinal segment (4 hours against 2 hours standard), with sequential pH increase in a 4 hours intestinal segment and with a lower addition of pH adjustment solution in a 4 hours intestinal segment, see Table 4-12.

Table 4-12 Bioaccessibility of BaP and DBahA with varied intestinal test time and pH adjustment, mean ± standard deviation

BaP DBahA Standard 2 hours, standard pH 0.173 ±0.0004 0.220±0.015 4 hours, standard pH 0.299±0.015 0.350±0.004 2 hours low pH, 2 hours standard pH 0.280±0.021 0.299±0.016 4 hours, low pH 0.306±0.028 0.421±0.033

Increased intestinal test time did increase the dissolution of the PAH in the test, whereas the different pH conditions did have significant (95% confidence level) impact upon the bioaccessibility. Final pH after 4 hours and low pH was approximately 6, for the re-maining conditions approximately 7.

Previously, it was found /85/, that the primary factor for PAH dissolution in the RIVM fed state test was pH, indicative also bile concentration, but not test time. These data were obtained with synthetic samples spiked with PAH solutions (no matrix soil pre-sent). The test method was accordingly revised to apply the high pH also applied in the version used in this study in order to achieve the required “worst case realistic” highest bioaccessibilities for soil PAH.

From the current findings, it must be concluded that the bioaccessibility of the PAH BaP and DBahA from soil can increase further with increased test time. Consequently, there is a need for estimates of the “true” bioaccessibility of soil PAH to allow for proper adjustment of the test conditions.


5 APPLICATION OF THE SELECTED BIOACCESSIBILITY TEST METHODS

The RIVM test methods /46;60/ were applied to a selection of soils from Danish sites and to a series of soils with internationally documented in vivo bioavailability data available. Fasted state conditions were applied for metals and fed state for PAH. The metals included were cadmium, lead and nickel, and the PAH were benzo(a)pyrene (BaP) and dibenzo(a,h)anthracene (DBahA).

The SBRC method and a modified version of the RIVM fasted state method without the intestinal segment were applied to a selection of the soils with documented in vivo metal bioavailability data. The metals included were cadmium and lead.

5.1 Application of the RIVM methods to Danish soils

The calculations of relative bioaccessibilities in this application study were done rela-tive to experimental references with soluble contaminants added.

During test implementation and validation, two Danish soils were tested, Table 5-1. No further information is available for these soils and the data are given as references only.

Table 5-1 Total concentrations and relative bioaccessibilities for two Danish soils as obtained with the RIVM methods, mean ± standard deviation

Cd Pb Ni BaP DBahA Soil close to highway Total concentration (mg/kg dw) 0.34 33 11 0.12 0.018 Relative bioaccessibility (%) 70±4.5 14±3.4 16±2.0 12±2.1 < DL15 Tar contaminated soil Total concentration (mg/kg dw) 0.26 40 14 52 10 Relative bioaccessibility (%) 85±12 6.1±0.4 12±0.7 124±37 152±21

At 7 Danish sites contaminated with one or more of the target contaminants of this study, soil samples were sampled following a sampling plan as shown in Figure 5-1. The samples were taken in the most contaminated depths, in most cases 0.1-0.3 m be-low the surface, using a hand auger, followed by pretreatment and testing as described previously, see chapter 3.

The sampling and test plan was designed for use in site risk assessment in order to allow for separate estimation of site variability and test reproducibility from analysis of the relative ranges, see e.g.: /86/ for the statistical method.

Based upon the total soil concentrations, five of the seven sites would require interven-tion based upon concentrations of one or more contaminants exceeding the intervention values, whereas the remaining two sites would be considered contaminated with con-centrations exceeding soil quality criteria.

15 The bioaccessible concentration was below the analytical detection limit of the test: 0,005 mg/kg dw


Figure 5-1 Sampling plan for the Danish field sites

The total contaminant concentrations from the sites, the relative bioaccessibilities and the bioaccessible concentrations are presented in Table 5-2, Table 5-3 and Table 5-5, re-spectively.

During the bioaccessibility testing, samples from two positions at the metal casting site and from one position from the metal slag site exhibited pH values in the test solutions above 2 in the stomach segment and/or above 7 in the intestinal segment for the fasted state test, and these samples accordingly exhibited lower lead bioaccessibilities than the other samples tested. Still, there was no general correlation between test pH values and lead bioaccessibilities.

Table 5-2 Total soil concentrations at the Danish field sites

Cd Pb Ni BaP DBahA (mean±standard deviation) (mg/kg dw) (mg/kg dw) (mg/kg dw) (mg/kg dw) (mg/kg dw) Danish soil quality criteria 0.5 40 30 0,1 0,1 Danish intervention values 516 400 30 1 1 Area with >100 years of urban history

1.3 ±0.64

680 ±110

10 ±1.3

3.9 ±1.7

0.80 ±0.47

Urban soil close to highway 0.47 ±0.28

73 ±72

10 ±6.1

0.22 ±0.31

0.08 ±0.06

Urban soil close to metal in-dustry

2.2 ±0.60

330 ±110

15 ±2.5

- -

Rural area with fishing net tar-ring

- - - 5.4 ±9.1

0.99 ±1.7

Urban soil with metal slags 28 ±16

3.900 ±1.800

48 ±21

- -

Urban soil with metal casting sand

2.2 ±1.2

710 ±410

69 ±36

- -

Urban soil with ashes from porcelain factory

0.71 ±0.54

160 ±170

11 ±3.7

1.8 ±1.1

0.33 ±0.22

16 The MCL relevant to bioaccessbility (i.e.: based upon human, oral exposure) is given in bold

One location

XX

XX

Two positions

X X

Duplicatesamples

One location

XXXX

XXXX

Two positions

X X

Duplicatesamples


The relative bioaccessibility data demonstrated that almost all contaminants had bioac-cessibilities well below 100%, lowest for nickel and PAH and with the highest site to site variability for lead.

Table 5-3 Relative bioaccessibilities at the Danish field sites

Cd Pb Ni BaP DBahA (mean±standard deviation) (%) (%) (%) (%) (%) Area with >100 years of urban history

54±9.9 78±20 29±4.8 15±3.1 14±3.3

Urban soil close to highway

68±4.5 29±17 19±2.4 38±27 40±24


57±3.6 107±18 16±2.2 - -


- - - 5.7±0.03 12±9.1

Urban soil with metal slags

52±7.1 43±48 22±19 - -


35±13 53±28 32±14 - -


43±5.7 27±10 22±2.5 16±2.1 20±6.0

The soil texture was determined for the 7 sites and the soils classified according to Dan-ish standard methods /87/,/88/ Table 5-4, but no correlation could be found between tex-ture and relative bioaccessibilities.

Table 5-4 Texture of the soils from the 7 Danish sites

Coarse sand

Fine sand Silt Clay Humus

(mean±standard deviation) (%) (%) (%) (%) (%) Area with >100 years of urban history

57±1.2 24±1.5 7.3±0.6 7.3±0.6 4.8±0.3

Loamy sand Urban soil close to highway

37±2.6 36±3.2 13±1.2 13±5.2 1.8±0.4

Sandy loam Urban soil close to metal in-dustry

35±4.9 30±5.5 15±1.0 15±2.9 6.0±2.2

Loam Rural area with fishing net tar-ring

74±6.5 14±1.0 2.0±0 2.0±0 4.1±2.3

Sand Urban soil with metal slags

35±5.3 33±2.1 14±2.0 16±1.0 1.6±0.2

Loam Urban soil with metal casting sand

35±3.1 29±8.1 14±2.3 11±1.7 11±0.8

Sandy loam Urban soil with ashes from porcelain factory

32±2.6 26±14 25±14 13±2.1 5.0±1.9

Sandy loam


The total soil concentrations, C, and the relative bioaccessibilities, RAC, were used to calculate the bioaccessible concentrations, Cba, of the contaminants in the soils, Table 5-5, according to:

(%)100

(%)* RACCCba =

Table 5-5 Bioaccessible soil concentrations at the Danish field sites

Cd Pb Ni BaP DBahA (mean±standard deviation) (mg/kg dw) (mg/kg dw) (mg/kg dw) (mg/kg dw) (mg/kg dw) Danish soil quality criteria 0.5 40 30 0.1 0.1 Danish intervention values 517 400 30 1 1 Area with >100 years of urban history

0.67 ±0.42

500 ±170

3.0 ±0.66

0.53 ±0.26

0.10 ±0.04

Urban soil close to highway 0.22 ±0.22

24 ±19

1.6 ±1.6

0.08 ±0.07

0.02 ±0.02


1.2 ±0.23

360 ±130

2.4 ±0.65

- -


- - - 0.40 ±0.81

0.07 ±0.12

Urban soil with metal slags 17 ±9.2

1.900 ±2.200

12 ±12

- -


0.84 ±0.60

460 ±260

21 ±7.5

- -


0.25 ±0.20

59 ±52

2.4 ±0.98

0.33 ±0.20

0.08 ±0.06

Considering the bioaccessible concentrations of the soil contaminants only, three sites would require intervention, three sites would be considered contaminated and one site would be considered uncontaminated. In this context, the term contaminated is defined as imposing a risk for human, oral exposure.

The contribution to the overall uncertainty from introducing the bioaccessibility correc-tion was estimated based upon the duplicate sampling and testing plan, Table 5-6. The variation analysis demonstrates, as expected, that the primary contribution to data varia-tion is the field variation at either location or position.

Table 5-6 Variability analysis for bioaccessibility at the 7 Danish sites

Mean relative range Cd Pb Ni BaP DBahA (%) (%) (%) (%) (%)

Test variation 13 14 12 11 24 Position variation 13 50 39 46 52 Location variation 37 66 36 24 48

The implications of this can be seen from Table 5-7, where the overall relative standard deviations are given for total soil concentrations, bioaccessible soil concentrations and relative bioaccessibilities for the 7 Danish sites. Evidently, even with the inevitable test variation, the total data variation does not increase considerably.

17 The MCL relevant to bioaccessibility (i.e.: based upon human, oral exposure) is given in bold


Table 5-7 Relative standard deviations for total soil concentrations, bioaccessible soil concentrations and relative bioaccessibility for the 7 Danish sites

Relative standard deviations on: Cd Pb Ni BaP DBahA total concentration /bioaccessible

concentration /relative bioaccessibil-

ity

(%) (%) (%) (%) (%)

Area with >100 years of urban history

50/63/13 16/33/30 12/22/16 43/48/23 60/42/27

Urban soil close to highway 60/100/9,2 98/76/67 60/100/18 140/93/72 68/91/64


28/19/6,8 33/37/11 17/27/15 - -


- - - 170/200/42 170/180/68

Urban soil with metal slags 57/54/15 47/110/95 43/100/67 - -


53/71/37 57/55/56 52/36/35 - -


76/80/16 100/89/42 33/40/13 61/60/15 68/79/23

5.2 Application of the methods to soils with internationally docu-mented in vivo bioavailability data

5.2.1 Identification and retrieval of soil samples Soil samples with internationally documented in vivo bioavailability data were identi-fied partly via the international BioAvailability Research Group Europe (BARGE) that among its members today counts most international (also outside Europe) research groups active within bioaccessibility testing, partly via a search of the scientific litera-ture and published reports.

The samples from the BARGE were identified though personal communication, and the literature search was done at the Technical Knowledge Centre of Denmark with the fol-lowing search profile:

(bioavailability or uptake or oral or absorption)

and

soil

and

(polycyclic aromatic hydrocarbons or PAH or 50-32-8 or 53-70-3 or 7440-43-9 or 7439-92-1 or 7440-02-0)

and

(vivo or human or infant# or animal# or pig# or swine# or rat# or mice or mouse or sheep# or rabbit# or primate# or monkey# or hamster#)

in the on line literature bases:


MEDLINE, BIOSIS og CAPPLUS

with:

>1995

not (vegetable# or crop# or plant# or translocation# or phyto# or dermal)

The literature search thus covered the recent chemical, biological and medical publica-tions on the oral uptake of the specified contaminants (by name or CAS number) from soil, but excluding dermal uptake and plant uptake.

In all 356 titles and abstracts were identified, 82 papers and reports were retrieved lead-ing to the identification of 15 research groups and 3 authorities with relevant, published activities. All were contacted at the least 3 times in order to identify soil samples with in vivo bioavailability data leading to in all:

• 48 samples for lead

• 17 samples for PAH

• 15 samples for cadmium

• 2 samples for nickel

retrieved from totally 8 sources, see chapter 1. One Canadian set of samples with data for nickel were unfortunately not available to the study due to confidentiality restric-tions.

5.2.2 Soil analysis All retrieved soil samples were analysed using the methods previously described, see section 4.1.

Comparison (linear regression) of the soil concentrations obtained in other studies using different methods and those obtained in this study, Figure 5-2, demonstrates acceptable to fine linear correlations (R2 better than 0.875) for all contaminants but nickel (R2 = 0.789) and slightly lower concentrations (by 10-30%) in this study for all contaminants but lead.

The results from the duplicate analysis performed of a random selection of retrieved soils samples, Table 5-8, shows good precision of the analysis and low inhomogeneity of the samples, see also chapter 5.2.4 for variations between soils from different sources.


Figure 5-2 Total soil concentrations obtained in previous studies against total soil concentrations obtained in this study

Table 5-8 Relative standard deviation as obtained by duplicate analysis of randomly selected soil samples

Pb Cd Ni BaP DBahA Number of duplicates 12 12 12 4 4 Relative standard deviation (%) 5.0 9.7 6.2 3.0 13

5.2.3 Application of the RIVM methods to soils with internationally documented in vivo bioavailability data The calculation of relative bioaccessibilities in this application study was done using the correction functions from Table 4-10.

Cadmium In all, 13 samples with cadmium in vivo bioavailability, all obtained for juvenile swine, were retrieved and tested with the RIVM fasted state in vitro bioaccessibility test method, Figure 5-3.

y = 1.06x - 15

R2 = 0.963

0

5000

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0 5000 10000 15000 20000 25000

This study (mg Pb/kg dw)

Pre

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stu

die

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w)

y = 1.31x - 5.2

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This study (mg Cd/kg dw)

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y = 1.20x + 10

R2 = 0.844

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This study (mg Ni/kg dw)

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R2 = 0.906

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0 100 200 300

This study (mg BaP/kg dw)

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y = 1.11x + 1.9

R2 = 0.875

0

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This study (mg DBahA/kg dw)P

revi

ou

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(mg

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ahA

/kg

dw

)

y = 1.06x - 15

R2 = 0.963

0

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0 5000 10000 15000 20000 25000

This study (mg Pb/kg dw)

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y = 1.31x - 5.2

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y = 1.20x + 10

R2 = 0.844

0

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0 20 40 60 80 100

This study (mg Ni/kg dw)

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)

y = 1.17x + 11

R2 = 0.906

0

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0 100 200 300

This study (mg BaP/kg dw)

Pre

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y = 1.11x + 1.9

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0

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0 10 20 30 40 50

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revi

ou

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ies

(mg

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ahA

/kg

dw

)


Figure 5-3 In vitro bioaccessibility of cadmium obtained with the RIVM fasted state test method against in vivo bioavailability obtained with juvenile swine

y = 0.93x + 0.039R2 = 0.635

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 0.2 0.4 0.6 0.8 1 1.2 1.4


In v

itro

bio

acce

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ility

There is a reasonable linear relationship between the in vitro and the in vivo data, and the overall trend is slightly higher bioaccessibilities than bioavailabilities.

For some of the retrieved soils, in vitro bioaccessibility data obtained with other meth-ods (IVG /58/ and DIN /29/) have been available for comparison with the data obtained in this study with the RIVM fasted state test, Figure 5-4.

Figure 5-4 In vitro bioaccessibility of cadmium obtained with other test methods /29;58/ against in vitro bio-accessibility obtained with the RIVM fasted state test method

y = 0.55x + 0.28R2 = 0.747

y = 0.46x + 0.32R2 = 0.532

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4

In vitro bioaccessibility in this study

In v

itro

bio

acce

ssb

ility

in o

ther

stu

die

s

DIN IVG stomach DIN IVG stomach


The data obtained with the three bioaccessibility test methods exhibit reasonable linear correlation with the RIVM fasted state data as the highest for high bioaccessibilities.

A small number of samples (5) have been tested in different series in this study, Table 5-9, where series 1 and 2 are separated by approximately 6 months, and series 2a and 2b are separated by a few weeks.

Table 5-9 In vitro bioaccessibility of cadmium obtained with the RIVM fasted state method in different test series of this study, mean ± standard deviation

Sample Series 1 Series 2a Series 2b Oker 11 0.54±0.006 0.75±0.007 0.72±0.01 Bunker Hill nt18 0.71±0.01 0.72 Urban soil with metal slags 0.69 nt 0.56 Urban soil with metal casting sand 0.26 nt 0.38 Urban soil with ashes from porcelain factory 0.45 nt 0.52

The variations in bioaccessibilities as obtained in different series separated by longer time are considerable, whereas the variations in data obtained within a small time inter-val are also small. It should be noted that the soil sample Oker 11 was tested from two different batches in series 1 and series 2.

Lead In all, 44 samples with lead in vivo bioavailability obtained for juvenile swine, mini pigs, rats (two methods) and rabbits were retrieved and tested with the RIVM fasted state in vitro bioaccessibility test method, Figure 5-5.

Evidently, the bioaccessibility data obtained with the RIVM fasted state method are not correlated to the bioavailability data. Furthermore, the bioaccessibilities were generally much lower than the bioavailabilities.

Separation of bioavailability data obtained with one experimental animal and one method did not improve the correlation, as would also not be expected from Figure 5-5.

The relative bioaccessibilities are corrected for the final pH of the test solution in the in-testinal segment, see section 4.4, but exclusion of soil samples with high pH (> 1.8) in the stomach segment did not improve the correlation.

A number of samples are mine waste and thus not strictly soil samples for which the RIVM method was developed. Still, exclusion of all samples that were not identified as soil samples did not improve the correlation. Here, it should be remembered that some soil samples are contaminated with mine waste and the distinction between soils and mine wastes may thus be somewhat artificial.

For some of the retrieved soils, in vitro bioaccessibility data obtained with other meth-ods (IVG stomach and intestine /58/, DIN /29/), MB /59/, SBRC /39/, PBET /30/) have been available for comparison with the data obtained in this study with the RIVM fasted state test, Figure 5-6.

18 nt: not tested


Figure 5-5 In vitro bioaccessibility of lead obtained with the RIVM fasted state test method against in vivo bioavailability obtained with juvenile swine, mini pigs, rats and rabbits

0

0.2

0.4

0.6

0.8

1

1.2

0 0.2 0.4 0.6 0.8 1 1.2


In v

itro

bio

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Juvenile swine Mini pigs Rats method 1 Rats method 2 Rabbits Humans

The bioaccessibilities obtained with the RIVM fasted state method for lead are corre-lated to the data obtained with the other methods, except for the highest values and the DIN method, but the RIVM data are generally lower than those obtained with the other methods.

Figure 5-6 In vitro bioaccessibility of lead obtained with other test methods /58/, /29/), /59/,/39/ /30/ against in vitro bioaccessibility obtained with the RIVM fasted state test method

0

0.2

0.4

0.6

0.8

1

0.0 0.2 0.4 0.6 0.8 1.0

In vitro bioaccessibility this study

In v

itro

bio

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ssib

ility

oth

er s

tud

ies

SBRC MB DIN IVG stomach PBET


The data included in Figure 5-6 were all, except for the DIN method, obtained with the method versions without an intestinal segment, as those are the versions generally ap-plied. Comparison of the RIVM fasted state method data that does include also an intes-tinal segment with data obtained with versions of the other methods applying their intes-tinal segments (PBET, IVG, MB and DIN) did not provide better correlations (data not shown). Still, the RIVM fasted state method did in reality provide the best correlation to bioavailability data compared to data obtained with other methods in versions with in-testinal segment included (data not shown).

Part of the explanation of the poor correlation between the lead bioaccessibilities ob-tained with the RIVM fasted state method and the juvenile swine bioavailabilities could in part be that the in vivo data may be biased. Figure 5-7 shows the in vivo data plotted against in vitro data, and the plot resembles the blood lead to dose curve shown in Figure 2-2. The blood uptake, section 2.2.3, exponential function do to a reasonable de-gree explain the data variability (R2 = 0.402). Even though this may question the valid-ity of the juvenile swine bioavailability data, it does not justify the very low bioaccessi-bilities obtained for the majority of samples with the RIVM fasted state method.

Figure 5-7 In vitro bioavailability of lead obtained with the juvenile swine method against bioaccessibility obtained with the RIVM fasted state method

For one soil sample tested in this study, the Bunker Hill soil, a set of data is available also with RIVM fasted state data after stomach and after intestinal segments /61/ and also in vivo bioavailability obtained in humans /33/, Table 5-10.

Bioavailability=k1+k2*(1-exp(-k3*bioaccessibility))

0.0 0.2 0.4 0.6 0.8 1.0 1.2

In vitro bioaccessibility

0.0

0.2

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1.2

In v

ivo

bioa

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y


Table 5-10 Absolute bioaccessibility obtained with the RIVM fasted state test method at RIVM /61/ and in this study, and absolute bioavailability as obtained in humans /33/

In vitro RIVM

stomach seg-ment only

In vitro RIVM stomach seg-ment followed by intestinal segment

In vitro RIVM stomach seg-ment followed by intestinal segment, this study

In vivo in hu-mans

Bunker Hill 0.71±0.01 0.32±0.03 0.031 0.26±0.081

Evidently, bioaccessibility of lead is decreased by the addition of the intestinal segment, but in the study done at RIVM not to a value below the in vivo value. In this study, the in vitro value was clearly below the in vivo value. Very careful comparison of the test practical test performance at RIVM and in this study did not reveal any discrepancies that might explain the very large variations in data obtained for the same samples at the two laboratories (RIVM and DHI).

A small number of samples (6) have been tested in different series in this study, Table 5-11, where series 1 and 2 are separated by approximately 6 months, and series 2a and 2b are separated by a few weeks.

Table 5-11 In vitro bioaccessibility of lead obtained with the RIVM fasted state method in different test se-ries of this study, mean ± standard deviation

Sample Series 1 Series 2a Series 2b Oker 11 0.40±0.02 0.19±0.01 0.17±0.003 Bunker Hill nt19 0.063±0.004 0.055 Urban soil with metal slags 0.054 nt 0.13 Urban soil close to highway 0.11 nt 0.18 Urban soil with metal casting sand 0.15 nt 0.11 Urban soil with ashes from porcelain factory 0.13 nt 0.36

The variations in bioaccessibilities as obtained in different series separated by longer time are considerable, whereas the variations in data obtained within a small time inter-val are also small. The variation appears random rather than systematic. It should be noted that the soil sample Oker 11 was tested from two different batches in series 1 and series 2.

Nickel No soil samples could be retrieved with relative in vivo bioavailability data from soil available. A series of soil samples from a Canadian site were not available, and for a se-ries of soil samples from a bioavailability study in mini pigs /29/, no soluble nickel ref-erence was included in the experiments and only absolute bioavailabilites could thus be obtained.

A linear relationship was obtained for the bioaccessibility data from this study against data obtained with the DIN method for the same 6 samples, with highest results seen for the RIVM fasted state test in this study Figure 5-8.

19 nt: not tested


A small number of samples (4) have been tested in different series in this study, Table 5-12, where series 1 and 2 are separated by approximately 6 months, whereas series 2a and 2b are separated by a few weeks.

The variations in bioaccessibilities as obtained in different series separated by longer time are considerable, whereas the variations in data obtained within a small time inter-val are also small. The variation appears random rather than systematic. It should be noted that the soil sample Oker 11 was tested from two different batches in series 1 and series 2.

Figure 5-8 In vitro bioaccessibility of nickel obtained with other test methods /29/) against in vitro bioac-cessibility obtained with the RIVM fasted state test method

y = 0.92x + 0.040R2 = 0.74

0

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0.5

0.0 0.1 0.2 0.3 0.4 0.5


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DIN DIN

Table 5-12 In vitro bioaccessibility of nickel obtained with the RIVM fasted state method in different test se-ries of this study

Sample Series 1 Series 2a Series 2b Oker 11 0.23±0.01 0.26±0.007 0.25±0.01 Urban soil with metal slags 0.24 nt20 0.22 Urban soil with metal casting sand 0.47 nt 0.37 Urban soil with ashes from porcelain factory 0.26 nt 0.23

PAH A series of 4 soil samples with in vivo bioavailability data from a study in mini pigs /29/ were retrieved. Only for benzo(a)pyrene (BaP), a soluble reference was included in the experiments. Dibenzo(a,h)anthracene could thus be evaluated from absolute bioavalabil-ity data only, and the other PAH included in this in vivo study /29/ were not part of the present bioaccessibility study.

Additionally, 13 soil samples were retrieved with in vivo bioavailability data from mice, for 3 samples from excretion of the BaP metabolite 3-hydroxybenzo(a)pyrene (3-OH-

20 nt: not tested


BaP), all with urine excretion data for the pyrene metabolite 1-hydroxypyrene (1-OH-P) and 10 with data for formation of PAH DNA adducts.

At this point, it should be recalled that an accepted and validated method for in vivo de-termination of PAH bioavailability is currently not available.

The in vitro bioaccessibilities obtained for BaP in this study are plotted against all in vivo data in Figure 5-9.

Figure 5-9 In vitro bioaccessibility of BaP obtained with the RIVM fed state test method against in vivo bioavailability obtained with mini pigs and with different methods in mice

0.0

0.2

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0.6

0 0.2 0.4 0.6 0.8 1 1.2


In v

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Mini pigs Mice 3-OH-BaP Mice 1-OH-P Mice DNA adducts

Evidently, there is no general correlation in vitro to in vivo, but a linear correlation is indicated for the four samples tested in mini pigs, and the bioavailabilities were higher than the bioaccessibilities. Here, it should be recalled that the data from the mini pigs are upper limits to PAH bioavailability as they are based upon the amount of PAH not excreted with faeces and thus do not include processes such as metabolization of the PAH in the gut.

The number of data points with bioavailabilities calculated from urinary excretion of 3-OH-BaP is too limited to allow for analysis of the correlation and for the bioavailabili-ties based upon urinary excretion of the pyrene metabolite 1-OH-P, no correlation was seen, refer also to section 2.4.5.

The correlations of the bioaccessibilities of DBahA to in vivo data were not better than for BaP. For this correlation analysis, the in vivo data from mini pigs as absolute bioavailabilities were used based upon an assumption of close to 100% bioavailability of the soluble reference.

The information available on the different soils is too limited to allow for an analysis of the reasons for the poor in vitro to in vivo correlations.


Linear relationships were indicated for the bioaccessibility data from this study against data obtained with the DIN method for the same 4 samples, with highest results seen for the RIVM fasted state test in this study for high bioaccessibilities and lowest for low bioaccessibilities Figure 5-10.

The apparent correlation between DIN and RIVM fed state bioaccessibilities does not support that increasing test time in the intestinal segment and thus the bioaccessibility of PAH, see section 4.5, would provide more relevant in vitro data.

Figure 5-10 In vitro bioaccessibility of PAH obtained with another test method /29/) against in vitro bioac-cessibility obtained with the RIVM fed state test method

y = 0.57x + 0.13R2 = 0.338

y = 0.62x + 0.11R2 = 0.557

0.0

0.1

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0.3

0.0 0.1 0.2 0.3


In v

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BaP DBahA DBahA BaP

No soil samples have been tested for PAH in different series in this study, and repro-ducibility over time of the test can thus not be evaluated.

5.2.4 Application of alternative bioaccessibility test methods Two alternative methods were applied to samples tested in vivo for bioavailability in or-der to identify a test method with a more satisfactory correlation in vitro to in vivo for lead, see section 5.2.3. Among the samples with in vivo data, only samples with data ob-tained in juvenile swine or minipigs were selected for this supplementary correlation study. In addition, 6 soil samples from the Danish sites were selected in order to illus-trate the different bioaccessibilities obtained with different methods for the Danish soils.

The alternative methods selected were a version of the RIVM fasted state bioaccessibil-ity test with the stomach segment only and the SBRC method as published by John Drexler, see section 2.3.1.

The RIVM fasted state stomach segment only was chosen because the high pH of the intestinal segment of the RIVM fasted state method was identified as one of the reasons for the poor in vitro to in vivo correlation for this method, see section 5.2.3.


The SBRC method was chosen because this method is aiming at being a robust, low pH stomach only test with published acceptable in vitro to in vivo correlation (see e.g.: /57/ and /75/). A further advantage is that this method is expected to comply with require-ments set for a bioaccessibility test method to be endorsed by the US EPA for use in site risk assessment in the US.

As stated before, see section 5.2.1, the soil samples with in vivo data were obtained from 8 different sources with several soils obtained as replicates from different sources. In the correlation study with the RIVM methods, see section 5.2.3, as many samples as possible were tested from the same source. In pursuing this objective, the sample amounts were depleted for a range of samples from this single source during the first correlation study. In the correlation study of the alternative methods, it was conse-quently necessary to use soil samples from more sources than in the first correlation study. Table 5-13 demonstrates that the homogeneity of the samples from different sources was comparable, compare also with the relative standard deviations of soil analysis found in series 1 (Table 5-8), and further indicates, that the soil concentrations did not differ with sources.

Table 5-13 Data on homogeneity and metal concentrations in soils from different sources

Source 1 Source 2 Source 3 Source 4 Lead relative standard deviation 3.6% (5)21 3.3% (3) 3.4% (4) 4.2% (2) Cadmium relative standard deviation 2.9%22 (4) 0% (3) 6.0% (4) 5.5% (2) Mean lead concentration difference rela-tive to source 1, first analytical series

-7.9% (3) 1.3% (1) -12% (2) -6.2% (5)

Mean cadmium concentration difference relative to source 1, first analytical series

-8.7% (2) 0% (1) -8.1% (2) -7.4% (5)

The total soil lead concentrations for 6 Danish soil samples were on the average 1.8% higher in the correlation study as compared to previous analytical series, respectively 21% lower for cadmium. Considering also the low recovery of 74% for cadmium from the NIST 2711 certified reference material obtained in this series, see Table 4-3, it is concluded that the cadmium concentrations obtained in the correlation study here was probably biased and too low. Consequently, soil cadmium concentrations from the first analytical series were used for the calculations of cadmium bioaccessibilities in this study.

Also, a relative difference in lead and cadmium from source 1, Table 5-13, as obtained in the first analytical series and in the second could suggests that the procedure for soil samples and analysis should be considered uniform and constant within ±10% only.

5.2.5 Application of the RIVM fasted state stomach segment only method to soils with internationally documented in vivo bioavailability data The RIVM fasted state test method /46/ excluding the intestinal segment and with the amount of soil sample reduced from standard 0.6 g to 0.3 g was used. The liquid to solid ratio of 22.5 for the total mouth/oesophagus and stomach segments of the RIVM test method was maintained.

21 (n): number of duplicate analysis 22 One outlier with excessive variation between duplicate analysis excluded, with the outlier the % RSD was 17


The calculations of relative bioaccessibilities in this correlation study were done relative to experimental references with soluble contaminants added.

Cadmium In all, 12 samples with cadmium in vivo bioavailability for juvenile swine were tested, Figure 5-11.

Figure 5-11 In vitro bioaccessibility of cadmium obtained with the RIVM fasted state stomach segment only test method against in vivo bioavailability obtained with juvenile swine

There is a linear correlation between the in vitro and the in vivo data, and the overall trend is higher bioaccessibilities than bioavailabilities.

For some of the tested soils, in vitro bioaccessibility test data obtained with another method (IVG /58/) have been available for comparison with the RIVM fasted state stomach only data, see Figure 5-12.

The data show linear correlation between the results from the two methods with higher IVG results in the low bioaccessibility range and higher RIVM results in the high range.

No samples have been tested for cadmium bioaccessibility with this method in different series in this study.

y = 0,88x + 0,25R2 = 0,575

0

0,2

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0,8

1

1,2

1,4

0 0,2 0,4 0,6 0,8 1 1,2 1,4


In v

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Figure 5-12 In vitro bioaccessibility of cadmium obtained with another test method /58/ against in vitro bio-accessibility obtained with the RIVM fasted state stomach only test method

Lead In all, 18 samples with lead in vivo bioavailability for juvenile swine were tested, Figure 5-13.

Figure 5-13 In vitro bioaccessibility of lead obtained with the RIVM fasted state stomach segment only test method against in vivo bioavailability obtained with juvenile swine

There is not a linear correlation between the in vitro and the in vivo data.

For 6 of the samples tested, the final pH after stomach test was above the target value of 1.8, see section 4.4. The correlation of the bioaccessibility obtained for the remaining 12

y = 0,47x + 0,28R2 = 0,475

0

0,2

0,4

0,6

0,8

1

1,2

1,4

0 0,2 0,4 0,6 0,8 1 1,2 1,4

In vitro bioaccessibility RIVM fasted state, stomach only

In v

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in o

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stu

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s

IVG stomach

y = 0,48x + 0,11R2 = 0,138

0

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In v

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samples with final pH < 1.8 with in vivo bioavailability is good, Figure 5-14, with over-all higher bioaccessibility than bioavailability in the low bioavailability range and lower bioaccessibility in the high range.

The data suggest that the poor correlation obtained for all soil samples, Figure 5-13, was due to too high pH after the stomach segment and consequently, that a good correlation might be obtained for all samples if the pH was kept below 1.8 by adjustment of the test procedure.

Figure 5-14 In vitro bioaccessibility of lead obtained with the RIVM fasted state stomach segment only test method against in vivo bioavailability obtained with juvenile swine, final pH < 1.8

For some of the tested soils, in vitro bioaccessibility test data obtained with other meth-ods (IVG /58/, DIN /29/, SBRC /89/) have been available for comparison with the RIVM fasted state stomach only data. The data showed no linear correlation between the results obtained with the RIVM fasted state stomach only method and the data ob-tained with the three other in vitro methods, data not shown. The number of data for the RIVM test satisfying the pH < 1.8 requirement was too limited (3) to allow for an evaluation of the correlation with the three other methods.

No samples have been tested for lead bioaccessibility with this method in different se-ries in this study.

5.2.6 Application of the SBRC method to soils with internationally documented in vivo bioavailability data The SBRC test method /57/ with the amount of soil sample reduced from standard 1.0 g to 0.5 g was used. The liquid to solid ratio of 100 for the SBRC test method was main-tained.

The calculations of relative bioaccessibilities in this correlation study were done relative to experimental references with soluble contaminants added.

y = 0,68x + 0,17R2 = 0,758

0

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In v

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Cadmium In all, 12 samples with cadmium in vivo bioavailability for juvenile swine were tested, Figure 5-15.

There is not a linear correlation between the in vitro and the in vivo data, and the overall trend is higher bioaccessibilities than bioavailabilities with most bioaccessibilities in the range 0.8-1.2, even for some soil samples with in vivo bioavailabilities at or below 0.6.

Figure 5-15 In vitro bioaccessibility of cadmium obtained with the SBRC test method against in vivo bioavailability obtained with juvenile swine

For some of the tested soils, in vitro bioaccessibility test data obtained with another method (IVG /58/) have been available for comparison with the RIVM fasted state stomach only data, see Figure 5-16.

The data do not show linear correlation between the results obtained with the two meth-ods, and the SBRC results are in the range 0.8-1 except for one sample.

No samples have been tested for cadmium bioaccessibility with this method in different series in this study.

y = 0,60x + 0,48R2 = 0,407

0

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Figure 5-16 In vitro bioaccessibility of cadmium obtained with another test method /58/ against in vitro bio-accessibility obtained with the SBRC test method

Lead In all, 18 samples with lead in vivo bioavailability for juvenile swine were tested, Figure 5-17.

Figure 5-17 In vitro bioaccessibility of lead obtained with the SBRC test method against in vivo bioavailabil-ity obtained with juvenile swine

There is a linear correlation between the in vitro and the in vivo data and the overall trend is higher bioaccessibilities than bioavailabilities, in particular for low bioavail-abilities.

y = 0,69x + 0,28R2 = 0,630

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y = 0,50x + 0,25R2 = 0,239

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In vitro bioaccessibility this study

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IVG stomach


For some of the tested soils, in vitro bioaccessibility test data obtained with other meth-ods (IVG /58/, DIN /29/, SBRC /89/) have been available for comparison with the RIVM fasted state stomach only data. The data showed linear correlation between the results obtained with the SBRC method in this study and those obtained previously with the same method /89/ with higher bioaccessibilities generally obtained in this study, Figure 5-18. There was no correlation between the SBRC and the data obtained with the IVG stomach in vitro method and the correlation could not be evaluated for DIN (one sample only).

In a section specifying the quality control procedures for the SBRC method /8/, it is re-quired that the test solution lead concentration from test of the NIST 2711 certified ref-erence material should be 9.22±1.50 mg Pb/L. In this study, the test solution concentra-tion from NIST 2711 was 9.7 mg Pb/L (duplicate tests) and thus well within the required range.

Figure 5-18 In vitro bioaccessibility of lead other test /29;58;89/ against in vitro bioaccessibility obtained with the SBRC test method

y = 0,65x + 0,12R2 = 0,502

0

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SBRC IVG G DIN SBRC

No samples have been tested for lead bioaccessibility with this method in different se-ries in this study.

5.2.7 Comparison of different bioaccessibility test methods applied to soils with internationally documented in vivo bioavailability data

Cadmium A comparison of the three bioaccessibility test methods applied to soil samples with in-ternationally documented in vivo bioavailability data from juvenile swine, Figure 5-19, shows that there is a linear correlation between in vitro and in vivo data for the RIVM fasted state (best correlation) and RIVM fasted state stomach only tests but not for SBRC.


For RIVM fasted state stomach only and SBRC both simulating the stomach dissolution only, all samples with in vivo relative bioavailabilities above approximately 0.6 gave in vitro relative bioaccessibilities close to 1, i.e.: complete dissolution of soil cadmium. Also, the in vitro data obtained with these methods were above or much above the in vivo data, whereas the data obtained with the full RIVM fasted state test method were closer to the in vivo data.

Considering also the in vitro to in vivo correlation demonstrated previously for the full set of soil samples with Cd in vivo data, Figure 5-3, with the RIVM fasted state method, this method is considered to provide the best and satisfactory in vitro to in vivo correla-tion for cadmium.

Figure 5-19 In vitro bioaccessibility of cadmium as obtained with the three test methods against in vivo bioavailability as obtained in juvenile swine

Lead A comparison of the three bioaccessibility test methods applied to soil samples with in-ternationally documented in vivo bioavailability data from juvenile swine or mini pigs, Figure 5-20, shows that only the SBRC method provides linear correlation between in vitro and in vivo data for lead.

If only samples with pH after the RIVM stomach segment below the required value of 1.8 are included, both RIVM stomach only and SBRC provides linear correlation with similar correlation coefficients (R2), Figure 5-21, with the RIVM stomach only test giv-ing in vitro data that are on the average slightly closer to the in vivo data.

The full RIVM fasted state test method including the intestinal segment still exhibited a large fraction of samples with apparent discrepancy between in vitro and in vivo data. As the samples with deviating pH after the stomach segment are now excluded and the effect of deviating pH after the intestinal segment is compensated in the calculation of the relative bioaccessibility, see section 4.4, other factors must cause this apparent bias

y = 0,96x - 0,013R2 = 0,646

y = 0,59x + 0,48R2 = 0,402

y = 0,87x + 0,25R2 = 0,586

0

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RIVM RIVM stomach SBRC RIVM SBRC RIVM stomach


of the bioaccessibility data. The reduction in recovery of added lead in the full RIVM test, see section 4.3, suggest that constituents added with the soil (such as e.g.: phos-phate) may have caused the excessively low lead bioaccessibilities of lead from some soil samples.

Figure 5-20 In vitro bioaccessibility of lead as obtained with the three test methods against in vivo bioavail-ability as obtained in juvenile swine or mini pig.

Figure 5-21 In vitro bioaccessibility of lead as obtained with the three test methods against in vivo bioavail-ability as obtained in juvenile swine or mini pigs, data with pH after RIVM stomach only below 1.8

y = 0,69x + 0,28R2 = 0,628

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RIVM RIVM stomach SBRC SBRC

y = 0,68x + 0,17R2 = 0,758

y = 0,69x + 0,23R2 = 0,748

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RIVM RIVM stomach SBRC RIVM stomach SBRC


Considering also the poor in vitro to in vivo correlation demonstrated previously for the full set of soil samples with Pb in vivo data, Figure 5-5, with the RIVM fasted state method, this method is not considered to provide satisfactory in vitro to in vivo correla-tion for lead. The SBRC and the RIVM fasted state stomach only methods are both con-sidered to provide satisfactory in vitro to in vivo correlations, with the RIVM data being closest to in vivo data. Still, this conclusions is based upon the assumption that the RIVM fasted state stomach only can be modified to ensure pH after the stomach seg-ment below 1.8 without changing the overall in vitro data.

5.2.8 Application of alternative bioaccessibility test methods to Danish soils In all 6 soil samples from each of 6 Danish sites have been subjected to the RIVM fasted state, the RIVM fasted state stomach segment only and the SBRC test methods for cadmium and lead, Table 5-14 and Table 5-15.

Table 5-14 Relative bioaccessibilities and relative standard deviations for test of 6 Danish soil with three different test methods, cadmium

RIVM fasted state RIVM fasted state stomach only

SBRC


0.50 1.22 1.16


-23 1.02 1.17

Urban soil close to metal industry

0.64 0.92 0.89


0.56 0.83 0.95

Urban soil with metal cast-ing sand

0.38 0.85 0.91


0.52 1.11 1.09

Overall relative standard deviation (%)

13 7.2 12

Considering the evaluation of the three in vitro methods, see section 5.2.7, the in vitro relative bioaccessibilities obtained with the RIVM fasted state method is evaluated to provide the best estimate, Table 5-14.

In accordance with the findings in section 5.2.7, the cadmium bioaccessibilities ob-tained with the two other methods are higher and probably not a good prediction of cadmium bioavailability from these soils. The test precisions (including sample inho-mogeneity), Table 5-14, were similar for RIVM fasted state and SBRC, but better for RIVM fasted state stomach only.

Considering the evaluation of the three in vitro methods, see section 5.2.7, the in vitro lead relative bioaccessibilities obtained with the RIVM fasted state method is consid-ered to have provided too low values, Table 5-15, in accordance with the findings in section 5.2.7, and the data are probably not good predictions of lead bioavailabilities from these soils.

23 -: bioaccessibility test gave result below test detection limit


Table 5-15 Relative bioaccessibilities and relative standard deviations for test of 6 Danish soil with three different test methods, lead

RIVM fasted state RIVM fasted state stomach only

SBRC


0.88 0.65 0.90


0.18 0.64 0.62

Urban soil close to metal industry

1.08 0.87 0.90


0.13 0.2424 0.76

Urban soil with metal cast-ing sand

0.11 0.2525 0.75


0.36 0.51 0.72

Overall relative standard deviation (%)

14 10 2.2

For the 4 soil samples with test pH in the required range (< 1.8), the RIVM fasted state stomach only is considered to provide the lead relative bioaccessibility values best esti-mating in vivo relative bioavailabilities, whereas the in vitro results for the remaining two samples are probably too low. Finally, the data obtained with the SBRC test method are probably reasonable but high estimates of relative bioavailability of lead from the 6 soils. The test precisions (including sample inhomogeneity), Table 5-14, were similar for the two RIVM methods, but considerably better for the SBRC method.

24 pH after stomach segment above required limit of 1.8 25 pH after stomach segment above required limit of 1.8


6 CONCLUSIONS AND RECOMMENDATIONS

Most quality criteria and cleanup levels (maximum contaminant levels, MCL’s) for soil contaminants are based upon oral exposure and toxic effect studies with contaminants as pure chemical substances ingested with water or with food. When ingested with soil, the bioavailability of substances such as metals and polycyclic aromatic hydrocarbons (PAH) is likely to be different from that in the studies that the MCL’s are based on.

Dissolution of food, soil and contaminants take place throughout the human gastrointes-tinal system. Uptake of the contaminants predominantly takes place in the small intes-tine, where conditions range from the slightly acidic, high chloride gastric conditions just after transit from the stomach to subsequent neutral to slightly alkaline, high phos-phate intestinal conditions. The importance of uptake from the acidic high chloride con-ditions of the stomach and of aerobic/anaerobic processes is not clear. The chemical conditions in the gastrointestinal tract are complicated and vary between individuals of different physiology, age, health etc. and for each individual with parameters such as feeding conditions, activity etc.

All metals can occur in different mineral forms and associations, and with soil constitu-ents depending upon the source of contamination and the weathering of the contami-nated soil, and these differences will impact the bioavailability of the metals from soil. Similarly, PAH is expected to exhibit reduced availability after aging of a PAH con-taminated soil.

Documented and accepted in vivo methods are available for measuring the bioavailabil-ity of lead from soils using e.g.: juvenile swine and the methods have been applied also for cadmium and arsenic. Bioavailability will vary with the experimental animals, the experimental set up and the calculation methods used, and the measurements are associ-ated with the variability inherent in all work with biological systems. No accepted method is available for in vivo measurement of the bioavailability of organic contami-nants such as PAH from soils, primarily because of the problems associated with the metabolization of such compounds during digestion and uptake. A large number of bioavailability in vivo studies with experimental animals have been published, a review of these is outside the scope of the present studies, but reduced bioavailability has been reported for at the least arsenic, cadmium, lead and PAH.

Bioavailability can only be measured using experimental animals with oral uptake physiology resembling that of humans (or in humans) which is costly and associated with ethical concerns. Therefore, simulation of the dissolution of soil contaminants in the human gastrointestinal tract in laboratory tests has been suggested (in vitro) to pro-vide an upper limit of human, oral bioavailability: the bioaccessibility.

Bioaccessibility of the soil contaminants depends upon the contaminant chemistry, the soil properties and the chemical conditions in the gastrointestinal system. Data are available from the open literature on bioaccessibility of soil contaminants, in particular for lead and arsenic, to some degree for cadmium, but very limited for nickel and PAH. The overall picture is that reduced soil bioaccessibility is very likely for cadmium and lead, likely for arsenic, and possible for nickel and PAH. The bioaccessibility of the


contaminants is highly variable even within the same soil type, source type and test, as far as can be concluded from the limited the data available.

A number of different in vitro test methods are available to measure bioaccessibility of soil contaminants, but the results are not generally comparable between methods. Bio-accessibility methods are associated with the variability inherent in all test procedures. The data on the quality of the bioaccessibility test methods are limited but methods of required quality are available or can be made available. A suitable bioaccessibility method simulating human physiology must include all important dissolution processes and emphasise all important test details, most important are:

• buffered low pH (pH < 2) high chloride gastric segment

• buffered slightly alkaline (pH > 7) phosphate containing intestinal segment

• aerobic followed by anaerobic conditions (stomach and intestine, respectively, op-tional)

• separate assessment of bioaccessibility in the two segments (gastric and gastric followed by intestinal, optional, depends upon contaminant)

• addition of enzymes, bile and milk powder (or similar food constituent)

• sufficient reaction time in each segment (3 hour in gastric segment, 10 hours in in-testinal segment)

• L/S stability (L/S > 100)

No currently available method satisfies all these requirements, but several methods will require only limited adjustment in order to do so: PBET (different versions), DIN and RIVM. As an alternative, the robust but simple SBRC method should be considered, but at the expense of the physiological relevance of the test.

It is mandatory and urgent for the future use of bioaccessibility testing of soil contami-nants that one single method or set of methods is agreed upon. Alternatively, a set of methods applicable each to different purposes (e.g.: heavy metals and organic contami-nants) should be the aim. To reduce costs and complexity of testing, the lowest number of tests possible should be aimed at.

For use in Denmark, the first choice has been the RIVM fasted state and fed state test methods for metals and PAH, respectively. This selection of these methods has been based upon the requirement for methods that are, see section 2.3:

• justifiable (simulate relevant processes)

• robust (can be repeated with “the same” result)

• relevant (can be correlated to uptake measured in animals or humans)

The RIVM fasted state method simulates the digestion processes in the mouth, oe-sophagus, stomach and upper small intestine of fasted children and is thus aiming at a precautionary approach (“realistic worst case”) for metal bioaccessibility. The RIVM


fed state method simulates the digestions processes in the mouth, oesophagus, stomach and upper small intestine of fed children and is thus aiming at a precautionary approach (“realistic worst case”) for bioaccessibility of apolar, organic contaminants such as PAH.

Implementation and validation of the tests for cadmium, lead, nickel and the PAH benzo(a)pyrene (BaP) and dibenz(a,h)anthracene (DBahA) at the DHI laboratories have demonstrated that the quality objectives set, section 4, could be met, section 4.3, yield-ing satisfactory test analytical detection limits and linearity, as well as reasonable preci-sion, whereas “trueness” could not be evaluated due to lack of reference materials or in-terlaboratory studies addressing the methods selected. For lead, an unsatisfactory between series and between laboratories precision was obtained. A quality control scheme and a sampling plan enabling assessment of site variability and test precision has been suggested, sections 3.2 and 5.1. During implementation and validation of the test methods, the need for careful evaluation of the quality for both analytical methods, sample preservation and test methods has been demonstrated.

In application of the RIVM test methods for 7 Danish contaminated sites, section 5.1, relative bioaccessibilities well below 100% were found for most of the contaminants tested for. Table 6-1 shows the range of reduction factors for bioaccessible concentra-tions from total concentrations that can be expected based upon the data from the 7 Danish sites. It should be noted that the reduction factor for lead is probably overesti-mated due to the excessively low bioaccessibilities obtained with the RIVM fasted state method for lead, see below in this chapter.

Table 6-1 Realistic reduction factors for estimation of bioaccessible concentrations from total soil concen-trations based upon data from 7 Danish sites

Cd Pb Ni BaP DBahA Range of reduction factors

1-3 (1-4)26 1-10 1-20 1-20

Furthermore, the application of bioaccessibility test to the 7 Danish sites demonstrated that the variability of bioaccessible soil contaminant concentrations was of the same or-der of magnitude as the variability of the total soil concentrations.

In the literature, correlation between in vivo bioavailability data and in vitro bioaccessi-bility data have been demonstrated with some tests for lead, and to some degree for cadmium and arsenic. The correlation for lead reported in the literature is best, if test methods with a stomach segment only are considered. Bioaccessibility will impact hu-man exposure if dissolution of the soil contaminants is rate limiting compared to ab-sorption or if only one fraction (e.g.: mineral species) of the soil contaminant is readily bioaccessible and another fraction that might be 100%, is not. Still, the data material is not sufficient to establish whether, to what degree and for which contaminant bioacces-sibility is rate or dissolution limiting.

The correlation obtained in this study for cadmium RIVM fasted state in vitro bioacces-sibility data and in vivo bioavailability data (published data, soils made available by the scientists responsible) demonstrated a satisfactory correlation, section 5.2.3. For lead,

26 Reduction factor probably overestimated, see text, more realistic factor for these soils may be 1-2


the correlation was not satisfactory (non-linear and low in vitro test data). For nickel and PAH, soil samples with in vivo bioavailability data of accepted quality were not re-trieved and the correlation thus not evaluated.

The poor between laboratory and between series variability, as well as the poor in vitro to in vivo correlation for the RIVM fasted state method lead data were attributed to the high and insufficiently stable pH in the stomach and intestinal segment of the test, as well as to a lead precipitating effect of other soil constituents.

As alternatives, the in vitro to in vivo correlation of a version of the RIVM fasted state test with mouth/oesophagus and stomach segments only (RIVM fasted state stomach only), i.e.: without the intestinal segment, and the SBRC method was studied for cad-mium and lead. For cadmium, the alternative test methods did not provide improved correlation. For lead, the SBRC provided linear in vitro to in vivo correlation with high in vitro data, and the RIVM fasted state stomach only also provided linear correlation with slightly more realistic bioaccessibilities, if soil samples giving too high pH in the test solution were excluded.

For all contaminants except for lead, a linear correlation was found between the RIVM test data and the data obtained with other in vitro methods in other studies, and the RIVM data were in general similar to or higher than published data obtained with other methods. For lead, a linear correlation of RIVM data was found for most methods, but the RIVM data were low compared to the data obtained with other methods. For the al-ternative RIVM fasted state stomach only, the data were linearly correlated to in vitro data obtained with other test methods for cadmium, but not for lead (all samples includ-ing pH outliers). For the alternative SBRC method, the data were just linearly correlated to in vitro data obtained with other test methods for lead, but not for cadmium.

The overall evaluation of the applicability of the bioaccessibility test methods for use in risk assessment of contaminated sites for oral exposure is summarized in Table 6-2. Test methods are evaluated as suitable for quantitative application, if satisfactory test robustness and in vivo correlation has been demonstrated. Test methods are evaluated as suitable for qualitative application, if satisfactory test robustness has been obtained and in vivo data have not been available for correlation.

Table 6-2 Summary of the applicability of the bioaccessibility test methods for risk assessment of selected soil contaminants for oral exposure

Cd Pb Ni BaP DBahA RIVM fasted state

Quantitative applicability

Not applicable Qualitative ap-plicability

Not evaluated

RIVM fed state

Not evaluated Not evaluated Not evaluated Qualitative applicability

RIVM fasted state stomach only

Not applicable Quantitative applicability

Not evaluated Not evaluated

SBRC

Not applicable Quantitative applicability with reserva-tions

Not evaluated Not evaluated


The recommendation of the RIVM fasted state stomach only test is provided that pH af-ter the stomach segment is within the specified range. For soils where this is not the case, alternative versions of the method with smaller amounts of soil, additional pH ad-justment or more efficiently buffered stomach solutions can be applied. With the “reser-vations” in Table 6-2 for lead and the SBRC method is meant that this method is an ac-ceptable alternative, but more conservative (higher bioaccessibilities) than the RIVM fasted state stomach only method..

In risk assessment, it is suggested to evaluate the MCL’s against the bioaccessible con-centrations, Cba, of the contaminants in the soils calculated as:

RACCCba *=

In the equation, C is the total soil contaminant concentration and RAC is the bioaccessi-bility as the fraction of the contaminant in the site soil samples relative to the bioacces-sibility of the contaminant in soluble form comparable to that used in toxicity studies behind the MCL (a toxicological reference).

Reduced bioaccessibility and/or bioavailability have been taken into consideration in site specific regulation of cleanup levels for contaminated sites in the US and Canada, in particular for mine waste and ore processing sites. Endorsement of bioaccessibility tests as part of risk assessment of contaminated soils (oral, human exposure) is under consid-eration in the Environmental Protection Agencies of the US and Denmark, and studies have been initiated by the environmental authorities in the UK, Germany and the NL.

The general conclusion is that correction of soil contaminant concentrations for bioac-cessibility in evaluation of compliance with soil quality criteria and cleanup levels based upon reduced bioavailability/bioaccessibility of the contaminants may be recom-mended in site specific risk approach. Conversely, the data available at present do not allow for general regulation of soil quality criteria and cleanup levels for specific con-taminants, soil types or sources.

As short term recommendations, it is suggested to:

• endorse the use of bioaccessibility testing for those contaminants and those test methods that are robust and exhibit proven correlation between in vivo and in vi-tro data

o purpose: to ensure utilisation of accessible information on contaminant availabil-ity in risk assessment of contaminated soils in order to achieve cost efficient and safe remediation

• prepare guidelines describing test methods to be used, quality control, data quality objectives and practical use in risk assessment

o purpose: to ensure that the tests are applied in a uniform and transparent form with sufficient but not excessive test quality

• establish a national set of reference values of bioaccessibilities for the typical, im-portant sites where availability is expected to be included in risk assessment (oral exposure based)


o purpose: to provide the site investigator with the background for deciding for or against including bioaccessibility test in the study, and the administrator with the background of evaluating the obtained bioaccessibility data

• establish stable and homogenous reference materials certified to the selected test(s) for mandatory use in all in vitro bioaccessibility test series intended for use in risk assessment

o purpose: to enable the laboratories and the data users to evaluate and compare test quality

Selection of contaminants for test endorsement and accompanying measures should take into account the significance of each compound as soil contaminant (toxicity and occur-rence).

As a long term recommendation, it is suggested to perform:

• selection, implementation, validation and interlaboratory comparison of one test method or one set of test methods for bioaccessibility of soil contaminants (Euro-pean or preferentially transatlantic scale, ISO, CEN, BARGE, US EPA, the Envi-ronment Agency, RIVM)

o purpose: to give access to a reliable method or set of methods for testing as common reference and to ensure compliance of all future data

• production of corresponding high quality in vivo bioavailability and in vitro bio-accessibility data for the important contaminants, soil types, sources and specia-tions (European or preferentially transatlantic scale)

o purpose: to produce relative bioavailability versus bioaccessibility “calibration” curves and demonstrate bioaccessibility as rate limiting factor for bioavailability for more contaminants

As research tasks, further refinement of the theory behind implementation of bioacces-sibility and bioavailability in risk assessment of soil contaminants should include:

• identification of in vivo segment of contaminant uptake

o purpose: to enable precise selection of test segment conditions (stomach or stomach and intestine) to be used for bioaccessibility testing of different con-taminants

• evaluation of gut redox conditions and impact upon bioaccessibility

o purpose: to enable selection of aerobic/anaerobic conditions for bioaccessibility testing of redox sensitive species

• description of the mechanisms of uptake, in particular the kinetics of dissolution and absorption in different segments, with different vehicles etc

o purpose: to ensure that the conceptual model of human uptake used is correct and that the bioaccessibility is de facto rate limiting for bioavailability


• development of robust, validated and accepted in vivo methods for measurement of bioavailability in relevant experimental animals for contaminants without such methods available, in particular for organic contaminants such as PAH

o purpose: to enable validation of in vitro bioaccessibility test methods against in vivo data for a broader selection of contaminants



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/72/ Ruby,MV, Fehling,KA, Paustenbach,DJ, Landenberger,BD, Holsapple,MP: Oral Bioaccessibility of Dioxins/Furans at Low Concentrations (50-350 ppt Toxicity Equivalent) in Soil. Environmental Science & Technology 36:4905-4911, 2002

/73/ Hack,A, Kraft,M, Mackrodt,P, Selenka,F, Wilhelm,M: Mobilisierung von Blei und Quecksilber aus real kontaminiertem Bodenmaterial durch synthetische Verdauungssäfte unter besonderer Berücksichtigung des Einflusses von Lebensmitteln. Umweltmedizin in Forschung und Praxis 3:297-305, 1998

/74/ Ruby,MV, Davis,A, Link,TE, Schoof,R, Chaney,RL, Freeman,GB, Bergstrom,PD: Development of an in Vitro Screening Test to Evaluate the in Vivo Bioaccessibility of Ingested Mine-Waste Lead. Environmental Science & Technology 27:2870-2877, 1993

/75/ Kelley ME, Brauning SE, Schoof RA, Ruby MV: Assessing Oral Bioavailability of Metals in Soil. 2002,

/76/ Ruby, M. V. Personal communication. 2003.

/77/ Rotard, W., Christmann, W., Knoth, W, and Mailahn, W. Bestimmung der resorptionsver-füghbaren PCDD/PCDF aus Kieselrot. UWSF-Z Umweltchem Ökotox 7, 3-9. 1995.

/78/ Hack,A, Selenka,F, Wilhelm,M: Mobilisierung von PAK durch synthetische Verdauungssäfte aus dem kontaminierten Bodenmaterial einer Altlastenfläche. Umweltmedizin in Forschung und Praxis 3:275-280, 1998

/79/ Versantvoort, C. H. M., van de Kamp, E., and Rompelberg, C. J. M. Development and applicabil-ity of an in vitro digestion model in assessing the bioaccessibility of contaminants from food. 320102002. 2004. Bilthoven, The Netherlands, RIVM. RIVM report.

/80/ Oomen, A. G. RIVM. 2003.

/81/ Miljøstyrelsen. Prøvetagning og analyse af jord. 13. 1998. København, Miljøstyrelsen. Ve-jledninger.

/82/ Miljø- og Energiministeriet. Bekendtgørelse om kvalitetskrav til miljømålinger udført af akkred-iterede laboratorier, certificerede personer m.v. Bekendtgørelse 637. 30-6-1997.

/83/ Lund, U. O., Andersen, K. J., and Sørensen, P. S. Håndbog i metodevalidering for miljølaborato-rier. 29-12-1994.

/84/ Liu,Q, Liu,Y: Distribution of Pb(II) species in aqueous solution. Journal of Colloid and Interfase Science 268:266-269, 2003

/85/ Versantvoort, C. H. M., van de Kamp, E., and Rompelberg, C. J. M. Development and applicabil-ity of an in vitro digestion model in assessing the bioaccessibility of contaminants from food. 320102002. 2004. Bilthoven, The Netherlands, RIVM. RIVM report.

/86/ Stuckert, K. and Grøn, C. Håndbog i analysekvalitet for laboratoriebrugere. 2001-4. 2001. København, Denmark, Amternes Videncenter for Jordforurening. Teknik og Administration.

/87/ Plantedirektoratet. Fælles arbejdsmetoder for jordbundsanalyser. 1994. Landbrugsministeriet.

/88/ Hansen, H. C. B. Environmental Soil Chemistry. 1998. Royal Veterinary and Agricultural Univer-sity.

/89/ Henningsen, G. M., Weis, Chr. P., Casteel, S. W., Brown, S. L., Hoffman, E., Brattin, W. J., Drex-ler, J., and Christensen, S. Differential Absorption of Lead in 20 Soils from Superfund Mine-Waste Sites: Measures of Bioavailablility Using Juvenile Swine as a Model of Young Children. 1999. Conference on Topics in Toxicology and Risk Assessment.


/90/ Sips, A. J. A. M., Bruil, M. A., Dobbe, C. J. G., van de Kamp, E., Oomen, A. G., Pereboom, D. P. K. H., Rompelberg, C J M, and Zeilmaker, M. J. Bioaccessibility of contaminants from ingested soil in humans. 711701012/2001. 2001. Bilthoven, National Institute of Public Health and the En-vironment. RIVM Report.

/91/ Pine SH, Hendrickson JB, Cram DJ, Hammond GS: Organic Chemistry. 1980,

/92/ Gasser,UG, Walker,WJ, Dahlgren,RA, Borch,RS, Burau,RG: Lead Release from Smelter and Mine Waste Impacted Materials under Simulated Gastric Conditions and Relation to Speciation. Environmental Science & Technology 30:761-769, 1996

/93/ Versantvoort, C H M, Rompelberg, C J M, and Sips, A. J. A. M. Methodologies to study human intestinal absorption. 630030001. 2000. Bilthoven, The Netherlands, National Institute of Public Health and the Environment. RIVM Report.

/94/ Ruby,MV, Davis,A, Kempton,JH, Drexler,JW, Bergstrom,PD: Lead Bioavailability: Dissolution Ki-netics under Simulated Gastric Conditions. Environmental Science & Technology 26:1242-1248, 1992

/95/ Davis,A, Ruby,MV, Bergstrom,PD: Bioavailability of Arsenic and Lead in Soils from the Butte, Montana, Mining district. Environmental Science & Technology 26:461-468, 1992

/96/ Davis,A, Ruby,MV, Goad,Ph, Eberle,S, Chryssoulis,S: Mass Balance on Surface-Bound, Miner-alogic, and Total Lead Concentrations as Related to Industrial Aggregate Bioaccessibility. Envi-ronmental Science & Technology 31:37-44, 1997

/97/ Hamel, S. C. The Estimation of Bioaccessibility of Heavy Metals in Soils using Artificial Biofluids. 1998. The State University of New Jersey.

/98/ Yand,J, Mosby,DE, Casteel,SW, Blanchar,RW: In Vitro Lead Bioaccessibility and Phosphate Leaching as Affected by Surface Application of Phosphoric Acid in Lead-Contaminated Soil. Ar-chives of Environmental Contamination and Toxicology 43:399-405, 2002

/99/ Pierzynski,GM: Past, present, and future approaches for testing metals for environmental con-cerns and regulatory approaches. Commun.Soil Sci.Plant Anal. 29:1523-1536, 1998

/100/ Proctor DM, Hays S, Ruby MV, Liu S, Sjong A, Goodman M, Paustenbach D: Rate of hexavalent chromium reduction by human gastric fluid (Abstract). Presentation at Annual Meeting of the So-ciety of Toxicology, 2002, Nashville, Tennessee, US, 2002

/101/ Ellickson, K. M. The Bioaccessibility of Selected Radionuclides and Heavy Metals: an Investiga-tion of Bioaccessibility, Bioavailability and Natural Soil Characteristics. 2001. The State Univer-sity of New Jersey.

/102/ Ruby,MV: Determining the oral bioavailability of PAHs from soil. Division of Environmental Chem-istry Preprints of Extended Abstracts 37:237-238, 1997

/103/ Oomen,AG, Sips,AJAM, Groten,JP, Sijm,DTHM, Tolls,J: Mobilization of PCBs and Lindane from Soil during in Vitro Digestion and Their Distribution among Bile Salt Micelles and Proteins of Hu-man Digestive Fluid and the Soil. Environmental Science & Technology 34:297-303, 2000

/104/ Oomen, A. G., Tolls, J., Sips, A. J. A. M., and van den Hoop, A. G. T. Lead Speciation in Artificial Human Digestive Fluid. Archives of Environmental Contamination and Toxicology 44, 107-115. 2003.

/105/ Grøn, C. and Andersen, L. Human bioaccessibility of heavy metals and PAH from soil. 2003. Miljøstyrelsen.

/106/ Cave, M. R., Wragg, J., Palumbo, B., and Klinck, B. A. Measurement of the bioaccessibility of arsenic in UK soils. 2002. Nottingham, British Geological Survey.

/107/ Lioy, P. J., Gallo, M. A., Georgopoulos, P., and Roy, A. Comparison of the Bioavailability of Ele-mental Waste Laden Soils using In Vivo and In Vitro Analytical Methodology and Refinement of Exposure/Dose Estimates. 1998. Environmental and Occupational Health Sciences Institute.


/108/ Stumm W, Morgan JJ: Aquatic Chemistry. Wiley-Interscience, 1981,

/109/ Smith, B. and Rawlins, B. G. Review of the bioaccessibility of arsenic in soils using the physio-logically-based extraction test (PBET). Technical Report WP/98/11C. 1998. British Geological Survey.

/110/ Hamel,SC, Buckley,B, Lioy,PJ: Bioaccessibility of Metals in Soils for Different Liquid to Solid Ra-tios in Synthetic Gastric Fluid. Environmental Science & Technology 32:358-362, 1998

/111/ Miljøstyrelsen. Afskæringskriterier for forurenet jord. 425. 1998. København, Miljøstyrelsen. Mil-jøprojekt.

/112/ Wragg, J. and Cave, M. R. In-vitro methods for the measurement of the oral bioaccessibility of selected metals and metalloids in soils: a critical review. 2003. Nottingham, British Geological Survey.

/113/ Proctor DM, Hays S, Ruby MV, Liu S, Sjong A, Goodman M, Paustenbach D: Rate of hexavalent chromium reduction by human gastric fluid (Abstract). Presentation at Annual Meeting of the So-ciety of Toxicology, 2002, Nashville, Tennessee, US, 2002

/114/ Oomen,AG, Hack,A, Minekus,M, Zeijdner,E, Cornelis,C, Schoeters,G, Verstraete,W, van de Wiele,T, Wragg,J, Rompelberg,CJM, Sips,AJAM, van Wijnen,JH: Comparison of Five In Vitro Di-gestion Models To Study the Bioaccessibility of Soil Contaminants. Environmental Science & Technology 36:3326-3334, 2002

/115/ Green-Pedersen, H. and Andersen, K. J. Homogenisering og delprøvetagning af jordprøver til sporelementanalyse. 2002. Miljøstyrelsens Referencelaboratorium for Miljøkemiske Analyser.

/116/ Mercier,G, Duchesne,J, Carles-Gibergues,A: A simple and fast screening test to detect soils pol-luted by lead. Environmental Pollution 118:285-296, 2002

/117/ Wild,SR, Obbard,JP, Munn,CI, Berrow,ML, Jones,KC: The Long-Term Persistence of Polynuclear Aromatic Hydrocarbons (PAHs) in an Agricultural Soil Ammended with Metal-Contaminated Sew-age Sludges. Science of the Total Environment 101:235-253, 1991

/118/ Cousins,IT, Kreibich,H, Hudson,LE, Lead,WA, Jones,KC: PAHs in soils: contemporary UK data and evidence for potential contaminantion problems caused by exposure of samples to laboratory air. Science of the Total Environment 203:141-156, 1997



A P P E N D I C E S

A-86 DHI Water & Environment


A P P E N D I X A

Test conditions in mouth and oesophagus segments



The primary digestive process in mouth and oesophagus is dissolution of starch, a proc-ess that is catalysed by the enzyme α- amylase. No studies have been identified provid-ing information on the importance of this process for bioaccessibility but it has been suggested that a physiologically based test should include all important segments in the human digestion /90/. In the DIN method, it is declared that this segment is of limited importance to the bioaccessibility /50/.

Partial hydrolysis of starch corresponding to the primary digestive process in a mouth and oesophagus segment is also catalysed by warm, dilute hydrochloric acid under con-ditions corresponding to those of the stomach segment of the bioaccessibility test meth-ods /91/.

The SBRC method does not include a mouth/oesophagus segment, the segment is op-tional in the DIN method, and it is integrated into the RIVM methods, Table 3-1. The compositions of the RIVM and DIN mouth/oesophagus test solutions are different with higher concentrations and longer test period for the DIN method. For both methods, pH is 6.5.

A

-90

DH

I Wat

er &

Env

ironm

ent

Pro

ced

ure

S

BR

C

RIV

M

DIN

C

om

men

ts

Fas

ted/

fed

stat

e ve

rsio

ns27

T

est s

olut

ion28

In

orga

nic

cons

titu-

ents

KC

l

896

1500

KS

CN

200

500

N

aH2P

O4

88

8 20

00

N

a 2P

O4

NaC

l

298

1667

NaO

H

72

/-

Na 2

SO

4

570

1833

NaH

CO

3

-/16

94

500

C

aCl 2

500

Org

anic

con

stitu

-en

ts

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a

200

333

α-

Am

ylas

e

145/

290

833

U

ric a

cid

15

33

Gal

acta

ric a

cid

50

/25

2500

pH

6.5 ±

0.2/

6.8±

0.2

6.4

Tes

t con

ditio

ns

Sam

ple

mas

s

0.6

g/0.

4 g

2 g

T

est s

olut

ion

vol-

ume

9

mL/

6 m

L 30

mL

L/S

rat

io

15

15

Tes

t tim

e

5 m

inut

es

30 m

inut

es

T

empe

ratu

re

37

±2°C

37

°C

27

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own

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e co

nditi

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ong

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two

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ion

28 If

not

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ted

othe

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e: m

g/L

used

for

the

test

sol

utio

n


A P P E N D I X B

Test conditions in stomach segments



The primary digestive processes in the stomach are acid dissolution and hydrolysis of proteins and lipids catalysed by a variety of enzymes. The primary factor for metal dis-solution in the stomach segment is pH and chloride concentration, see section 2.3, where the lowest pH and the highest chloride concentration physiologically relevant must be aimed at.

A study of lead dissolution kinetics under simulated stomach conditions has suggested that a test time of 1 hour is adequate for this segment /92/. Fast release of lead from con-taminated soils (66) and wastes (19) was reported simulating gastric conditions over time /92/, with more than 30% of total lead dissolved within 10 minutes for most sam-ples. The study also demonstrated higher bioaccessibility with lower pH and faster re-lease with higher temperatures. Other studies have suggested a test time of 1.5-2 hours as most appropriate for the stomach simulation /32/. A test time of 2 hours has been ap-plied as a realistic upper limit of physiological conditions /93/. Also, the dissolution rate has been demonstrated to be comparatively constant over 2 hours for lead from a mine waste contaminated soil /94/ thus enabling a more robust test. Still, only 25% of the equilibrium concentration of lead was obtained after 2 hours /95/. Lead dissolution after 1 and 2 hours was constant in a study of one soil but for 5 waste incineration slags, 2 hours test time increased the lead dissolution by a factor of up 1.7 /96/. Another study demonstrated a limited increase (factor 1.1-1.2) in lead dissolution from soil increasing the test time from 2 to 6 hours /97/, and a similar small effect for arsenic. A recent study did not demonstrate increased dissolution of BaP, lead, cadmium and arsenic by in-creasing the test time from 2 to 16 hours in the RIVM fed state test /79/.

Studies of stomach processes have shown a 94% replacement of stomach contents within 1 hour of a meal /66/ indicating that a stomach residence time of one hour is a realistic estimate.

Dissolution of lead minerals in the stomach depends on the acid concentration present in the test during dissolution, with low acid concentrations leading to both slower dissolu-tion and lower final dissolved concentration /51/. An average decrease in stomach lead bioaccessibility of 57% was observed with 7 soils impacted by mine wastes when rais-ing the test pH from 1.3 to 2.5, whereas the decrease was smaller for arsenic /30/. The effect of acid concentration in solution is caused by both pH (formation of HSO4

-) and chloride concentration (formation of soluble PbCl+) /32/. Arsenic bioaccessibility from 2 soils was lower by 8 - 25% in the PBET test with pH = 2.5 than with pH = 1.3 but the effect was less than for e.g. lead /30/. A low pH and a high chloride concentration in the stomach solution increase dissolution of minerals (e.g.: iron oxides) /74;94/.

The pH of the fasted stomach is generally below 2 (interval pH = 1 – 4), increases to 4 – 5 after a meal /66/ and decreases to below 2 after approximately 1 hour /68/.

Phosphate in the stomach segment will reduce the dissolution of lead as demonstrated in test of soils with and without added phosphoric acid /98/. It has been demonstrated with sequential extraction that in the presence of phosphate changes, the speciation of lead changes towards less extractable species /99/.

Recent data suggest that both synthetic stomach fluid and human stomach fluid can sig-nificantly reduce toxic and soluble Cr(VI) to less toxic and more insoluble Cr(III) within the time range relevant to stomach transit /100/. Whether the same effect will oc-cur for As(V) is not clarified.


The RIVM method in the fasted state aims at pH = 1.2 corresponding to the conditions in the stomach of a fasted child /90/. The fed state, RIVM method aims at pH = 2-2.5 /79/. For the SBRC method, the test solution pH is 1.5 and for the DIN method, 2.0.

The test solutions of the RIVM methods are more complex than the solutions in the SBRC and DIN methods and are closer to physiological conditions.

In the RIVM methods, organic acids (galactaric and glucuronic acids) are added. This has been shown to increase solubility of e.g.: lead and chromium in the intestinal seg-ment of another test method, but to decrease the dissolution in the stomach segment /101/. For PAH, addition of galactaric acid has been demonstrated to increase soil PAH dissolution by a factor of 5 /70/. In the SBRC method, a high concentration of the amino acid glycine is added to buffer the test solution and to increase metal solubility by com-plexation.

The protein hydrolysing enzyme pepsin is added to high concentrations in the RIVM and DIN methods, but none of the methods apply addition of lipid hydrolysing enzymes in this segment. The RIVM methods add bovine serum albumin in order to keep fatty acids dissolved.

A

-95

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A P P E N D I X C

Test conditions in intestinal segments



Based upon the location of human oral heavy metal uptake in the upper small intestine, see section 2.3, the intestinal test segments of most bioaccessibility test methods simu-late the conditions in this part of the intestine. The primary contaminant dissolution processes in the upper part of the small intestine are dissolution of organic matter due to an increased pH, dissolution of metals due to complexation with e.g.: bile acids and dis-solution of apolar, hydrophobic organic contaminants by bile components with surfac-tant properties. Hydrolysis of proteins (proteases), carbohydrates (amylases) and lipids (lipases) catalysed by enzymes are important digestive processes.

The residence time in the small intestine responsible for the most of soil dissolution and contaminant uptake is generally 3-5 hours (mean 3.5 hours for children) /66/. The maximum residence time in the intestine has been set to 6 hours /68/.

The pH in the upper small intestine has been estimated to 7 /66/, but the typical pH variation is from approximately 4 just after emptying of stomach contents into the intes-tine to 6 – 7,5 after some time /68/.

It has been suggested to test the PAH bioaccessibility employing conditions correspond-ing to the physiology of the small intestine with a 4 hours test period and a test solution of a phosphate buffer, sodium chloride, pig bile and milk powder, but the data have not been published /102/. For dioxins, it has been demonstrated that a simple test solution resulted in bioaccessibilities 50 to 100 times lower than obtained with physiologically based test conditions /77/.

Increasing concentrations of bile have increased dissolution of e.g.: PCB from synthetic soil /103/, and increased (factor 2) solubility of dioxins from slags have been observed with increasing (factor 6) bile concentration /71/.

Organic acids or proteins in intestinal test solution increase lead solubility due to com-plexation but for cationic metals, this effect may be more than counteracted by the solu-bility decreasing effect of the increasing pH (e.g.: 74% reduction of dissolved lead from soil and mine waste upon transfer from the low pH stomach segment to the higher pH and higher protein/organic acid intestinal segment /66/).

The RIVM fasted state method aims at pH>5.5 corresponding to pH in the upper part of the small intestine /69/, whereas the fed state version aims at pH 6.5-7.0. These pH in-tervals were selected in order to ensure high lead bioaccessibility in the fasted state ver-sion and high organic contaminants bioaccessibility in the fed state version. Still, recent studies have demonstrated the highest bioaccessibilities of both lead and PAH at the higher pH of the fed state test, compared to the fasted state test /79/. The SBRC method aims at a pH of 7.0 and the DIN method of 7.5.

The SBRC method is using an optional intestinal test segment with a simple test solu-tion featuring low concentrations of pancreatin and bile.

The RIVM method fasted state uses a higher concentration of sodium bicarbonate than the DIN method in order to obtain the desired pH with just one addition and without need for subsequent pH adjustments /69/. A further increase of the bicarbonate concen-tration is used in the fed state RIVM method in order to ensure the higher pH interval aimed at for this version /79/.


RIVM and DIN methods include additions of freeze dried bile to high concentrations in order to increase dissolution of both metals and organic contaminants /69/.

Sodium dihydrogenphosphate is included in the RIVM method in order to simulate the presence of phosphate in saliva, gastric juice and intestinal juice /104/, and the presence of phosphate will probably reduce the dissolution of metals such as lead /105/.

In the RIVM fed state method, the concentrations of pancreatin and lipase are increased in order to reflect the physiological changes occurring after ingestion of a meal and thus to simulate the concomitant increase in soil, contaminant and food dissolution.

A test time of 2 hours was chosen for the RIVM test as representing “physiologically relevant intestinal residence times” /69/.

A

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03

9000

pH

, duo

dena

l jui

ce

7.

8 ±0.

2/8.

1±0.

2

The

pH

of t

he te

st s

olut

ions

is

not c

ontr

olle

d in

the

DIN

m

etho

d, b

ut th

e so

il so

lutio

n te

xt m

ixtu

re p

H is

con

trol

led

pH

, bile

8.0 ±

0.2

pH

adj

ustm

ent

NaH

CO

3

-/94

11

T

est c

ondi

tions

S

ampl

e m

ass

1.00

± 0

.05

g 0.

6 g/

0.4

g 2

g

Tes

t sol

utio

n vo

l-um

e 95

27

mL

duod

enal

juic

e an

d 9

mL

bile

(Σ36

58.

2)/ 1

2 m

L du

oden

al

juic

e, 6

mL

bile

and

2 m

L so

-di

um b

icar

bona

te s

olut

ion

( Σ38

)

100

mL

(Σ20

0)

L/S

rat

io

95

60 (

Σ 97

)/ 5

0 (Σ

95)

50

(Σ

100)

Tes

t tim

e 4

hour

s 2

hour

s 6

hour

s

Tem

pera

ture

37

±2°C

37

±2°C

37

°C

T

est m

ixtu

re p

H

7.0

± 0.

2 >

5.5/

6.5-

7.0

7.5

In th

e S

BR

C m

etho

d, th

e in

tes-

tinal

pH

is in

itial

ly e

stab

lishe

d by

titr

atio

n to

pH

= 7

.0

35

Bov

ine

seru

m a

lbum

ine,

a b

lood

pro

tein

36

Incl

udin

g pr

evio

us s

egm

ents


A P P E N D I X D

Practical test performance



The practical test performance must be designed to ensure a reproducible and efficient test. Details on practical test performance for the three selected bioaccessibility test methods and important points discussed below.

Mixture of soil and test solutions should be by “all over” or “end over”, as this method is recognized to be more efficient than e.g.: stirring /69/.

Early versions of the SBRC method used a piece of dialysis tube filled with sodium bi-carbonate solution to increase pH from the stomach segment to the intestinal segment /74/, but this procedure has now been changed to a simple titration with base to achieve the pH increase /75/. The DIN method applies addition of sodium bicarbonate combined with titration to achieve the pH increase, whereas the RIVM methods applies addition of buffered solutions in fixed amounts /69;79/. Avoiding the titration step in pH adjust-ment is the most efficient approach, but there is a risk that the correct pH intervals are not achieved.

Materials for and cleaning of sample containers and test bottles must reflect the proper-ties of the contaminants in question. For metals, HDPE (high density polyethylene) has been suggested in the SBRC method /75/, the DIN method requires use of glass with test for organic contaminants and of PE (polyethylene) with test for metals /68/, and the RIVM methods apply polycarbonate bottles for both metals and organic contaminants, excluding phthalates /80/. With dioxins, it has been suggested to further deactivate the glass surfaces exposed to the test solutions in order to further reduce the risk of adsorp-tion of these highly apolar and adsorbable compounds /77/.

All equipment in contact with test solutions in metal bioaccessibility testing must be cleaned with dilute acid, e.g.: hydrochlorid acid, followed by washing with clean labo-ratory water. RIVM applies furthermore a laboratory dishwasher that performs a sterili-zation of the equipment ion order to prevent microbial growth in test solutions /69/.

After incubation, soil and test solution must be separated (phase separation) before analysis of the test solutions and, if pertinent, the solid phase (e.g.: the soil). Centrifuga-tion or filtration with their individual pro’s et contra’s are usually the choice.

For centrifugation, it has been reported that 2500 g for 2 hours is sufficient to remove all visible particles from test solutions, but a combination with membrane filtration (0,45 µm porediameter) has nevertheless been included in this study /77/. With the RIVM method, settling over night and centrifugation at 3000 g for both 5 and 30 min-utes has been demonstrated to give approximately the same bioaccessibilities of lead from soil /69/, and the simple 3000 g centrifugation for 5 minutes is thus applied in the RIVM methods.

Filtration through 0.45 µm cellulose filters has been used as the only phase separation in bioaccessibility testing for arsenic /106/. Centrifugation at 906 g for 10 minutes fol-lowed by 0.45 µm filtration has been applied as well, but the efficiency was not docu-mented /47/.

A comparative study of centrifugation methods demonstrated similar performances of 10 minutes centrifugation at both 5000 g and 7000 g, as well as of centrifugation for 2 hours at 3.000 g /73/. A combination of 10 minutes centrifugation at 7000 g and subse-


quent filtering through 20 µm stainless steel sieve has been suggested for bioaccessibil-ity testing of PAH /70/.

For dioxins, glasfiber and nitrocelluose filters were found suitable (<5% adsorption of dissolved dioxins), whereas teflon filters exhibited high adsorptive losses.

Analytical problems reported for centrifuged, unfiltered test solutions applying ICP-MS were attributed to matrix effects caused by particles /107/, but evaluation of the analyti-cal procedures applied suggests that these problems can be solved applying adequate analytical procedures (e.g.: standard addition).

The inherent difficulty in selecting the proper phase separation conditions is that the fate of colloids is not well defined using the suggested, practical methods. Colloids may have sizes from a few nm to 10 µm and thus end up in both solid and liquid fractions. The boundary between particles and colloids is not well defined and not easy to estab-lish operationally, even though a 10 µm particle size is sometimes used. Traditionally, colloids are defined as particles that do not settle from a solution /108/. Contaminants bound to small colloids are probably more bioaccessible than those bound to large col-loids /109/. Furthermore, we do not have generic information on the degree to which colloids can be taken up by the membranes of the gastrointestinal system.

For these reasons, it is suggested to apply separation techniques that provide visibly par-ticle free test solutions with the highest efficiency possible. Here, centrifugation appears to be the most practical solution.

The DIN method suggest to add a precipitate washing step after centrifugation to achieve full recovery of all solutes /68/, but with 2 g soil with an estimated pore volume of approximately 1 mL and 200 mL test solution, the effect of this would be less than 1% of the total solutes and the effort would hardly be justified by the enhanced recov-ery.

The applied liquid to solid ratio (L/S) of test solution to soil may impact the bioaccessi-bility test result, as a small L/S ratio (5 – 25 L/kg) may yield low results (diffusion limi-tation of dissolution) /66/. It has been demonstrated that L/S ratios over 1000 L/kg had only marginal effects upon bioaccessibilities of 4-5 metals from 2 soils /110/. In the later study, an L/S ratio of 100 L/kg was sufficient to achieve constant bioaccessibility for one of the soils.

An estimate of a physiologically relevant L/S ratio would be 500 L/kg based upon a se-cretion of stomach juice of 100 mL/hour /110/ and a daily soil intake of 0,2 g /111/.

The amount of soil for testing is primarily balanced between the requirement for a small volume of test solution (and thus a small amount of soil with fixed L/S ratio) and an amount of soil large enough to allow for a representative test portion /66/.

In the original version, the PBET test was done anaerobically with argon flushing as the method of test mixing, but this has been proven unnecessary for metals such as lead /112/, but an effect of aerobic versus anaerobic conditions can not be excluded for redox sensitive contaminants such as chromium or arsenic /113/.

A

-107

D

HI W

ater

& E

nviro

nmen

t P

roce

du

re

SB

RC

R

IVM

D

IN

Co

mm

ents

F

aste

d/fe

d37

Mix

ing

30±2

rpm

38 a

ll ov

er

55 r

pm a

ll ov

er

200

spm

39 s

hake

r or

300

rpm

m

agne

tic s

tirre

r (2

cm

mag

net)

Adj

ustm

ent o

f pH

In

the

stom

ach

segm

ent,

pH

ad

just

men

t is

with

test

sol

u-tio

ns a

pplie

d, b

ut if

fina

l pH

in

the

test

sol

utio

n ha

s in

crea

sed

to a

bove

2, t

he te

st m

ust b

e re

peat

ed w

ith s

tepw

ise

addi

-tio

ns o

f hyd

roch

loric

aci

d to

m

aint

ain

low

pH

. In

the

inte

sti-

nal s

egm

ent,

the

test

mix

ture

is

titra

ted

man

ually

with

sod

ium

hy

drox

ide

solu

tion

to p

H =

7.5

±

0.2

pH a

djus

tmen

t is

with

test

sol

u-tio

ns a

pplie

d, b

ut fi

nal p

H in

the

test

sol

utio

n is

con

trol

led

Adj

ustm

ent o

f pH

is c

ontin

uous

w

ith a

n au

totit

rato

r, a

ltern

a-tiv

ely

with

con

tinuo

us, m

anua

l tit

ratio

n

Pha

se s

epar

atio

n F

iltra

tion

thro

ugh

0.45

µm

por

e di

amet

er d

ispo

sabl

e ce

llulo

se

acet

ate

filte

rs

Cen

trifu

gatio

n at

300

0 g40

for

5 m

inut

es

Cen

trifu

gatio

n at

700

0 g

for

10

min

utes

, opt

iona

l filt

ratio

n th

roug

h 20

µm

sta

inle

ss s

teel

si

eve

(org

anic

con

tam

inan

ts)

or

30 µ

m n

ylon

sie

ve (

met

als)

for

rem

oval

of f

loat

ing

part

icle

s

37

Onl

y sh

own

whe

re th

e co

nditi

ons

diffe

r am

ong

the

two

vers

ions

38

rpm

: rou

nds

per

min

ute

39 s

pm: s

trok

es p

er m

inut

e 40

g: g

ravi

ty a

ccel

erat

ion



A P P E N D I X E

Quality control requirements


For bioaccessibility tests, different quality control tools have been applied. Reagent blanks, test blanks, replicate tests, synthetic control samples, matrix control samples and mass balance calculations are most frequently applied tools.

Mass balance calculations include analysis of all test solutions and the tested soil after removal of the test solutions in order to test whether the amount of contaminant added to the test system is fully recovered in test solutions and tested soil, see e.g.: /47/. It should be emphasized here, that the mass balance approach can not be applied if the soil analytical method implies a partial dissolution of soil contaminants only, as is the case for the Danish dilute nitric acid digestion method (DS 259). The reason for this is that the solubility of the soil contaminants in the analytical digestion procedure will most likely change after the test treatment of the soil.

The SBRC method demands, in addition to an extensive quality control program with specified quality requirements also documentation of sample handling and transport in the form of a ”chain of custody report” /75/. The certified reference material NIST 2711 is required as matrix control sample with an experience based test solution lead concen-tration and variation given /75/.

The DIN method refers to generic requirements for quality control of analytical meth-ods, requires reporting of analytical detection limit, and demands furthermore control of the mass balance once for every 20 samples tested /68/.

With respect to the use of the NIST 2711 soil as matrix control sample (metals) in the RIVM fasted state method, it should be emphasized that the laboratory is currently in-vestigating the reasons for inconsistent control results /80/. The control material can thus not be recommended currently for the RIVM test method. For the RIVM fasted state method, test blanks for lead are generally below the analytical detection limit for lead in test solutions with the method applied at RIVM (8 µg Pb/L).

A

-111

D

HI W

ater

& E

nviro

nmen

t P

roce

du

re

SB

RC

R

IVM

D

IN

Co

mm

ents

F

aste

d/fe

d41

Rea

gent

s bl

anks

1

per

reag

ents

pre

para

tion

and

at th

e le

ast 1

per

20

sam

ples

m

ax. 2

5 µg

Pb/

L

Not

req

uire

d N

ot r

equi

red

Tes

t bla

nks

1 pe

r se

ries

and

at th

e le

ast 1

pe

r 20

sam

ples

, max

. 50

µg

Pb/

L

1 pe

r se

ries

Not

req

uire

d

Rep

licat

es

1 pe

r se

ries

and

at th

e le

ast 1

pe

r 10

sam

ples

, max

. 20%

C

V42

All

sam

ples

in tr

iplic

ate

Not

req

uire

d

Con

trol

sam

ples

, sy

nthe

tic

1 pe

r se

ries

and

at th

e le

ast 1

pe

r 20

sam

ples

, rec

over

y 85

-11

5% fo

r le

ad

Not

req

uire

d N

ot r

equi

red

Con

trol

sam

ples

, m

atrix

1

test

of M

onta

na 2

711

stan

-da

rd r

efer

ence

soi

l fro

m N

IST

43

per

50 s

ampl

es, r

ecov

ery

ex-

perie

nce

valu

e 84

-116

% fo

r le

ad

1 te

st o

f Mon

tana

271

1 st

an-

dard

ref

eren

ce s

oil f

rom

NIS

T

per

serie

s fo

r m

etal

s/

none

cur

rent

ly

Not

req

uire

d

Mas

s ba

lanc

e N

ot r

equi

red

For

1 s

ampl

e pe

r se

ries

For

1 p

er 2

0 sa

mpl

es, m

ax.

10%

dev

iatio

n fr

om 1

00%

mas

-s

reco

very

41

Onl

y sh

own

whe

re th

e co

nditi

ons

diffe

r am

ong

the

two

vers

ions

42

Coe

ffici

ent o

f var

iatio

n or

rel

ativ

e st

anda

rd d

evia

tion

43 N

IST

: Nat

iona

l Ins

titut

e of

Sta

ndar

ds a

nd T

echn

olog

y



A P P E N D I X F

Analytical methods applied for soil and test solutions



The three bioaccessibility test methods do apply different methods for determination of soil contaminant concentration. These methods would generally be expected to yield different results for metals /114/ and results different from the Danish DS 259 method. In a published method comparison, nitric acid digestion was found to yield soil concen-trations at 1/3 of the concentrations obtained with hydrofluoric acid digestion /97/. Whether different analytical methods applied as part of the bioaccessibility testing would reduce comparability of data was thus controlled in this study by analysis of a se-lection of soil samples with digestion after DS 259 and with aqua regia, a method ap-plied for soil contaminant routine analysis in most European countries and also in the RIVM and DIN bioaccessibility test methods, see section 4.1.

The three bioaccessibility test methods all apply ICP methods without digestion for de-termination of test solution metals, but digestion with nitric acid has been suggested /107/. The RIVM test method has applied HPLC for PAH analysis for test solutions.

A

-116

D

HI W

ater

& E

nviro

nmen

t P

roce

du

re

SB

RC

R

IVM

D

IN

Co

mm

ents

M

etal

s, s

oil

Dig

estio

n of

sam

ples

with

2:1

H

NO

3/H

2O, f

ollo

wed

by

30%

H

2O2,

and

ICP

ana

lysi

s

Dig

estio

n w

ith a

qua

regi

a (H

NO

3/H

Cl)

follo

wed

by

ICP

-M

S44

ana

lysi

s

Dig

estio

n w

ith a

qua

regi

a fo

l-lo

wed

by

AA

S a

naly

sis

Met

als,

test

sol

u-tio

ns

ICP

of t

est s

olut

ions

D

ilutio

n x

10 o

f tes

t sol

utio

ns in

0,

1 M

HN

O3

and

ICP

-MS

an

alys

is

Dilu

tion

of te

st s

olut

ion

x 5-

10

and

anal

ysis

by

ICP

-MS

or

ICP

O

ES

45

For

ana

lysi

s of

test

sol

utio

ns

with

AA

S, d

iges

tion

is r

equi

red

PA

H, s

oil

Not

spe

cifie

d H

PLC

46 fo

r be

nzo(

a)py

rene

S

oxhl

et e

xtra

ctio

n w

ith h

ex-

ane/

acet

one,

4:1

, fol

low

ed b

y G

C47

-MS

ana

lysi

s or

cor

re-

spon

ding

met

hods

PA

H, t

est s

olut

ions

N

ot s

peci

fied

HP

LC fo

r be

nzo(

a)p

yren

e. F

or

othe

r or

gani

c co

ntam

inan

ts

extr

actio

n w

ith h

exan

e, a

ddi-

tion

of m

etha

nol,

cent

rifug

atio

n,

conc

entr

atio

n by

eva

pora

tion

and

anal

ysis

by

GC

-EC

D48

Ext

ract

ion

of te

st s

olut

ion

with

he

xane

follo

wed

by

addi

tion

of

NaC

l (aq

., sa

t.) a

nd a

ceto

ne,

anal

ysis

by

GC

-MS

or

corr

e-sp

ondi

ng, S

PE

49 n

ot s

uita

ble

due

to in

terf

eren

ce fr

om a

dded

m

ilk p

owde

r

Sap

onifi

catio

n of

milk

pow

der

cons

titue

nts

can

be r

equi

red

to

obta

in fu

ll re

cove

ry o

f PA

H.

Add

ition

of s

atur

ated

sod

ium

ch

lorid

e so

lutio

n an

d ac

eton

e,

as w

ell a

s ce

ntrif

ugat

ion

can

be

requ

ired

to b

reak

sol

vent

to

test

sol

utio

n em

ulsi

ons.

44

MS

: mas

s sp

ectr

omet

ry, a

det

ectio

n m

etho

d us

ed fo

r bo

th m

etal

s an

d or

gani

c co

mpo

unds

45

OE

S: o

ptic

al e

mis

sion

spe

ctro

scop

y, a

mul

tiele

men

t det

ectio

n m

etho

d us

ed p

rimar

ily fo

r m

etal

s 46

HP

LC: h

igh

perf

orm

ance

liqu

id c

hrom

atog

raph

y, a

met

hod

used

for

anal

ysis

(se

para

tion)

prim

arily

of o

rgan

ic c

ompo

unds

47

GC

: gas

chr

omat

ogra

phy,

a m

etho

d us

ed fo

r an

alys

is (

sepa

ratio

n) p

rimar

ily o

f org

anic

com

poun

ds

48 E

CD

: ele

ctro

n ca

ptur

e de

tect

or, a

met

hod

used

for

anal

ysis

(de

tect

ion)

prim

arily

of o

rgan

ic c

ompo

unds

49

SP

E: s

olid

pha

se e

xtra

ctio

n, a

met

hod

used

to c

once

ntra

te a

nd p

urify

ext

ract

s pr

imar

ily o

f org

anic

com

poun

ds a

s pa

rt o

f the

ir an

alys

is


A P P E N D I X G

Soil pretreatment methods



Generally, small particles with their higher contents of clay and organic material exhibit higher concentrations of metals and adsorbable organic contaminants such as PAH /115/. This has been demonstrated also for dioxins with 5 times higher concentrations in the fine particles (<20 µm) than in the total soil sample /77/. Dissolution kinetics will also be faster from small particles due to the larger specific surface, but the equilibrium concentration will not be larger with small particles due to particle size only /71;94/. Higher bioaccessibility has been observed for lead in particles < 125 µm as compared to larger particles /116/.

For sewage sludge, it has been demonstrated that drying at 100°C did not lead to loss of PAH /117/, and that air drying of soil did not result in analyte loss/samples contamina-tion for PAH with more than 4 rings /118/. Consequently, own drying of soil samples at 40°C would probably not impact the concentrations of the PAH benzo(a)pyrene and dibenz(a,h)anthracene (both 5 ring PAH, boiling point 496 and 524°C, respectively). It should be noted that published bioaccessibility studies of organic contaminants applied air drying of soil samples only /70/, /78/, /72/.

A

-120

D

HI W

ater

& E

nviro

nmen

t P

roce

du

re

SB

RC

R

IVM

D

IN

Co

mm

ents

D

ryin

g <

40°C

A

ir dr

ying

for

one

wee

k A

ccor

ding

to IS

O50

114

64 o

g IS

O 1

4507

Rem

oval

of l

arge

pa

rtic

les

- R

emov

al o

f peb

bles

and

twig

s <

1 m

m e

nsur

ed b

y re

mov

al o

f pa

rtic

les

or b

y si

evin

g

Grin

ding

/mill

ing

Not

spe

cifie

d I R

etsc

h m

ill

Not

spe

cifie

d

Sie

ving

<

250

µm

<1

mm

<

1 m

m e

nsur

ed b

y re

mov

al o

f pa

rtic

les

or b

y si

evin

g

Hom

ogen

izat

ion

Sam

ple

divi

der

Mix

ing

with

a s

hove

l and

sub

-sa

mpl

ing

with

teflo

n co

ated

st

ainl

ess

stee

l spo

on

Not

spe

cifie

d

50

Inte

rnat

iona

l Sta

ndar

diza

tion

Org

anis

atio

n


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Test for bioaccessibility of metals and PAH from soil report · 2006-07-20 · Test for...

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