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Human Bioaccessibility of Heavy Metals and PAH from Soil

5 Quantification of bioavailability

The relative bioavailability factors required for adjusting MCLs for variations in contaminant bioavailability from soils, see chapter 2, can be obtained at different levels:

• characterisation of source and site chemistry
• in vivo tests
• in vitro tests

Each approach has benefits and disadvantages, and each has a separate role in the implementation of bioavailability in risk assessment. It should be noted that bioavailability may be taken into account in the toxicity studies behind the MCLs.

5.1 Characterisation of source and site

A characterisation of the source and the site with respect to the chemistry of the contaminants and the soil is mandatory in advance of deciding in favour of a bioavailability study, see also chapter 2. The main objective for this characterisation is to evaluate, whether bioavailability is likely to be reduced with the current contaminant and soil chemistry. The evaluation should at the least include (originally elaborated for metal contaminated soils):

• species of contaminants at source (e.g.: is the original contamination metallic copper scrap of limited bioavailability or more bioavailable copper sulphate solution?)
• number and concentrations of contaminants (e.g.: do we face many contaminants at high concentrations or a few with concentrations close to nominal MCL?)
• soil geochemistry and its potential for contaminant (im)mobilisation (e.g.: do we deal with a highly organic soil with large potential for reduction of Cr(VI) to the less toxic and less bioaccessible Cr(III) or a sandy soil without this potential?)
• species and vehicle comparison between site and the toxicity studies behind the nominal MCL (e.g.: was the toxicity or epidemiological study made with an aqueous solution of lead nitrate compared to the insoluble lead phosphate in the soil at the site?)
  

Very high contaminant concentrations suggest that even with very low contaminant bioavailabilities, the safe, revised MCLs will not approach actual concentrations. In this phase, geochemical modelling (with e.g.: MINEQL or MINTEQ) can assist in identifying probable metal species in soil water /10;35/.

Access to previous bioavailability data for the same site type (source, soil and age) can assist the evaluation.

Due to the complexity of the soil matrix, the chemical characterisation alone is not considered sufficient to allow for quantitative bioavailability adjustments /9/.

5.2 In vivo tests

The ultimate bioavailability test is measurements in humans, followed by animal experiments and then by in vitro tests.

In vivo tests are generally considered the best bioavailability tests available, as the animal uptake measured in these tests is believed to resemble the conditions applied during toxicity testing. Oral in vivo tests generally include both dissolution (bioaccessibility), absorption and reduction. Absorption of soil contaminants is not covered by the present review, but an overall understanding of the techniques used for in vivo bioavailability studies is useful as a reference for subsequent chapters on bioaccessibility and in particular on the correlation between bioavailability and bioaccessibility, chapters 7 and 8.

Different approaches have been taken for in vivo bioavailability tests /4;14;47/:

• intestinal perfusion
• excretion measurements
• blood kinetics
• target tissue measurements
  

In the intestinal perfusion techniques, a section of the intestine of an experimental animal is separated, the contaminated matrix for testing is introduced in the intestine, and the concentration of contaminant is subsequently measured in the matrix after passing the section. The absorbed fraction is the fraction of contaminant that disappeared during intestinal passage. Strictly speaking, the intestinal perfusion techniques are not real in vivo techniques, as the intestinal section is separated from the animal to varying degrees in different versions of these techniques. Pros et contras are:

+  dissolution and transport close to real conditions
+  removal by absorption close to real conditions
-  transport over the epithelium membrane during absorption is not included
-  reduction in membrane cells and liver not included
-  metabolites formed in the intestine are not considered
-  removal by degradation (in lumen and at membrane surface) measured as available
-  costly
-  only experimental animals available for contaminants

In excretion measurements, experimental animals are fed the contaminated matrix and the excreted (faeces) fraction measured. The non-excreted or retained fraction of contaminant is the bioavailable fraction. Pros et contras are:

+    dissolution and transport close to real conditions
+    removal by absorption close to real conditions  
-     the transport over the epithelium membrane during absorption is not included  
-     reduction in membrane cells and liver not included  
-     metabolites formed in the intestine not considered  
-     removal by degradation (in lumen and at membrane surface) measured as available  
-     excretion with bile is measured as non-available  
-     time consuming and costly  
-     only experimental animals available for contaminants

Distinguishing the initial excretion of unabsorbed contaminant with faeces and the re-excretion of contaminant occurring later may refine the mass balance technique. Further refinements include measurements of urinary excretion and blood concentrations. Also, urinary excretion alone has been used to give a lower boundary for bioavailability of contaminants that are not metabolised /5/.

An experimental approach combining the perfusion and excretion techniques is the in situ test. Here, the full gastrointestinal system of the experimental animal is used for digestion and uptake while the animal is anaesthetised but still alive. This technique exhibits the pro et contras of the perfusion and excretion techniques but is more comprehensive and consistent with true in vivo conditions.

In blood kinetic studies (traditional bioavailability studies), the contaminated matrix is ingested and approximately the same amount is injected intravenously. The blood concentration of contaminant is measured over time and the bioavailability is calculated as the ratio between the area under the concentration curves for oral administration and for intravenous injection. Pros et contras are:

+     dissolution and transport under to real conditions
+     removal by absorption under to real conditions
+     removal by degradation (in lumen and at membrane surface) under real conditions
+     the transport over the epithelium membrane during absorption included
+     reduction in membrane cells and liver included
-     metabolites not considered, unless specifically analysed for
-     demands sensitive analytical methods due to limited amount of blood available
-     demands larger experimental animals than rodents or many experimental animals
-     very costly
-     only experimental animals available for toxic contaminants

In target tissue measurements, the contaminated matrix is ingested and after due delay, the resulting concentration is measured in the target tissue, such as the liver if liver cancer is the effect driving the MCL. Pros et contras are:

+    dissolution and transport close to real conditions
+    removal by absorption close to real conditions
+    removal by degradation (in lumen and at membrane surface) close to real conditions
+    the transport over the epithelium membrane during absorption included
+    reduction in membrane cells and liver included
+    distribution and potential tissue accumulation included  
-     metabolites not considered, unless specifically analysed for  
-     demands identification of target tissue  
-     demands specific target tissue(s) without general effects  
-     very costly  
-     only experimental animals available for contaminant

Interpretation of liver concentrations as estimates of overall bioavailability has been suggested based upon the assumption that the liver reflects the overall systemic level of the contaminant /5/. Use of this method is valid only for contaminants where the liver is the major organ for distribution and metabolisation and this should be verified in advance.

All in vivo methods for bioavailability measurements are subject to large variability, as are all biological systems. Conversely, all the methods address the overall bioavailability including both bioaccessibility and absorption, see chapters 3 and 4, but reduction is included in the blood kinetic and target tissue approaches only.

Epidemiological studies where exposure and health effects are recorded and correlated for large population groups are rarely available for MCL derivation, compare the US TDI for arsenic, see chapter 2.

5.3 In vitro tests

Bioavailability tests in vitro are based upon two different approaches /4;14;47/:

• bioaccessibility or dissolution tests
• absorption tests

Test simulating the dissolution processes of the contaminants from the matrix, i.e.: the bioaccessibility, are addressed in chapter 6. The common in vitro tests for absorption are using:

• membrane chambers
• everted sacs
• cell culture chambers
  

In the membrane chamber technique, a sheet of intestinal epithelium (the mucosa) is set up as a membrane between two chambers. One chamber is filled with a solution of the contaminant, the other with a medium that receives the contaminant transported over the membrane. After incubation, the resulting concentration of contaminant is measured in the receiving medium. The pros et contras are:

+    absorption close to real conditions
+    removal by degradation (at membrane surface) close to real conditions
+    the transport over the epithelium membrane during absorption included
+    fast and simple
+    interspecies comparisons possible  
-     includes only absorption and excludes matrix effects  
-     metabolites not considered  
-     reduction in liver not included  
-     effect of blood supply and lymph drain not included

In the everted sac technique, a small peace of intestinal epithelium is taken out and everted to a small sac “inside out”, i.e.: with the inner part of the epithelium facing the outside of the sac. The sac is filled with a medium that receives the contaminant, closed and situated in a solution of the contaminant. After incubation, the resulting concentration of contaminant is measured in the receiving medium. The pros et contras are:

+    absorption close to real conditions
+    removal by degradation (at membrane surface) close to real conditions
+    the transport over the epithelium membrane during absorption included
+    fast and simple
+    interspecies comparisons possible  
-     includes only absorption and excludes matrix effects  
-     metabolites not considered  
-     reduction in liver not included  
-     demands sensitive analytical methods due to limited amount of receiving medium available  
-     effect of blood supply and lymph drain not included

In cell culture techniques, intestinal epithelium cells (e.g.: Caco-2 cultured from a human colon carcinoma) are cultured to form a cell monolayer on a filter support. The monolayer is polarised, i.e.: exhibits the physiological features of in vivo epithelium cells with an upper and an under side, and it tolerates artificial soil digests after slight dilution. The filter with the cell culture is set up as a membrane between two chambers. One chamber is filled with a solution of the contaminant, the other with a medium that receives the contaminant transported over the membrane. After incubation, the resulting concentration of contaminant is measured in the receiving medium and in the cells. The pros et contras are:

+    absorption close to real conditions
+    removal by degradation (at membrane surface) close to real conditions
+    the transport over the epithelium membrane during absorption included
+    can be used with soil digests
+    cell cultures more reproducible than most biological tests  
+    fast and simple  
-     includes only absorption, unless combined with bioaccessibility pre-test  
-     metabolites not considered  
-     reduction in liver not included  
-     comparability between original intestinal epithelium and the cultured cells can be disputed  
-     currently available only for research purposes and not for routine testing

Preparation of filters coated with monolayers of original intestinal membrane cells has not yet been successful.

The absorption tests are designed to address the absorption step and consequently, most of the techniques cover only one of the two main processes susceptible two matrix and speciation variations, see chapter 3. An exception is the cell culture method that can be used with digests of contaminants from soil and thus may include matrix effects upon the absorption process. The absorption tests can though be useful to elucidate differences in uptake among different species of a contaminant.

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