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Bioaccessibility Testing 18/06/14

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Bioaccessibility Testing


Paul Gribble

Technical Director

XXXXXXX

Dr Alexander Lee

Technical Director

0131 344 2309

Bioaccessibility Testing


A Sticky Idea to Selling

• A sticky idea is understood, it’s remembered, and it changes something. Sticky ideas of all kinds—ranging from the “man flu does not exist” urban legend to JFK’s “Man on the Moon” speech—have several common traits

• The more traits the stickier.


Principle 1 Simple Message

• Likely to overestimate the risk;

• Result in unnecessary determinations;

• Result in unnecessary remediation and costs;

• Result in fear and concern (fright factors).

• But resolving is not simple (Principle 1), Concrete (Principle 2), Credible (Principle 4)

• But albeit incomplete science invoking its use CAREFULLY can assist in clarifying risks

Under Part IIA and planning reliance on total contaminant soil concentrations in the assessment of “significant harm ….or.. The significant possibility of significant harm” arising from unacceptable intake is


Principle 1 Simple Message

• Do you think bioaccessibility is a useful tool ?

– 151 LA England and Wales 40.3% returns

– 70.2% yes

• Do you think that use of bioaccessibility data leads to more cost effective site management

• How many of you have accepted bioaccessibility data? (half!)

• Do you make any distinction between bioavailability and bioaccessibility?

Taken after “Survey on Bioaccessibility implementation” 2009”


Lets start with Simple

• Bioaccessibility is the fraction of a substance that is released from the soils during such processes as digestion into solution making it available for absorption (measured in vitro)

• Bioavailability relates the fraction that reaches the blood system via the gastrointestinal tract (measured in vivo – almost impossible see Pb TOX report)

• In vitro approaches to establishing the bioaccessible fraction are taken as a surrogate of oral bioavailability

• Simple message it can help

• Recognised as a decision support tool – but not statutorily defined in regards to how (not concrete!)

Bioaccessibility based estimates of bioavailability Less Conservative Assessments maybe


Fundamentals

• Fundamental assumption behind MOST the SGV is that 100% of the contaminant in soil is in a form that is bioavailable and therefore contributes to uptake and dose - Precautionary

• Widely believed that most contaminants are likely to be less bioaccessible than in the materials used to derive tolerable daily intakes.

• Most discussions focus on oral bioavailability

• Correction of the oral exposure pathway in CLEA will give more realistic estimate of uptake and risk.


The Truth (Credible – Principle 4)

Misuses Commentary 1. Insufficient samples A minimum of 10 samples per averaging zone is typical in order to gain an

adequate appreciation of the variation in bioaccessibility.

2. Use of peer review literature rather than site specific values

There is not necessarily a relationship between literature values and site specific bioaccessibility.

3. Application to non ingestion pathways The PBET seeks only to simulate the direct oral ingestion pathway.

4. Application to other substances Inappropriate appreciation of substance specific bioaccessibility.

5. Lack of lines of evidence Bioaccessibility test results may not be compatible with geological history, geochemistry.

6. Mixing samples from different soil/ ground types

Bioaccessibility varies with medium.

7. Poorly documented test procedure Bioaccessibility tests are empirical and interpretation should be based on the specific method applied.

8. Analysis of samples not representative of concentrations of concern

Bioaccessibility varies with total concentration but the relationship is not necessarily either linear or positive.

9. Inappropriate use of statistics Statistical summaries of bioaccessibility tests may

result in discordant matching of bioavailability estimate and total concentration

10. Application of summary (average) or single values to a dataset

The relationship between total and bioaccessible concentrations is not necessarily linear.

11. Use of wrong test Results are not relevant to the risk estimation.

12. Lack of details in reports Reviewer cannot evaluate the robustness of the risk estimate and the compliance of the risk evaluation with the specific legal context.

13. NOT using bioavailability at all Over conservative risk assessments and unnecessary remediation

(taken after CIEH Professional Practice Note)


Common Myths

Myth Consequence Truth 1. The regulator completely rejects

the use of bioavailability Over conservative risk assessments and unnecessary remediation

The regulator cautions that bioavailability may only be useful in limited circumstances

2. Bioavailability is positively correlated with total

concentration

Use of low bioaccessibility results underestimates risks posed by high total

concentrations

The relationship between total and bioavailable fractions is much more complex.

3. No other country uses bioaccessibility test results

Reluctance to inform risk evaluation with estimates of bioavailability

Several countries accept various in vivo and in vitro based approaches to incorporating bioavailability in risk assessments.

4. PBET is the only show in town Less than optimal test conditions for specific circumstances.

The PBET is only one of several physiologically based tests available worldwide.

5. All laboratories carry

out the “PBET‟ in the

same way

Lack of comparability between results Different laboratories may well have made minor modifications to improve efficiency or reduce costs.

6. All laboratories carry out “total‟ analysis in the same way

Lack of comparability between results Laboratories may differ in the way extraction and analysis is carried out.

7. The regulator does not allow the use of bioaccessibility in human health risk

assessment

Over conservative risk assessments and unnecessary remediation

The regulator cautions that its use may only be justified in limited cases.

8. We understand why a given soil has the bioavailability it has

Over reliance on lines of evidence and literature values rather than site specific measurements

We are only just beginning to understand the biogeochemical controls on bioavailability.


Can we deliver Concrete (3) and Credible (4)

• Yes but only

• If considered with other lines of evidence e.g. history geochemical data etc.

• We recognise current tests not suitable for all substances of concern

• We recognise bioavailability varies with soil composition

• Anchored to the sites CSM

• Appropriate no of samples (LQM >10)

• Recognise pathway specific

• Applied carefully BUT ……


Things have moved on …….

• EA survey (R&D Technical Report P5-062/TR/01 – 2002) • 3 soils As, Pb, Ni to 11 labs (2 abroad)

• Large variation in results observed • Application of standard method to different substances a concern irrespective of

analyte, concentration and matrix • Challenges validity

• In-vivo vs in-vitro correlation (See also EA Science update 2)

• Strong correlation between in-vitro bioaccessibility and in-vivo bioavailability is key condition for acceptance i.e. credibility principle 4

• UK 2 main tests modified PBET and buffered acid solution (SBET) • PBET simulates digestion in the stomach and small intestine and seeks to replicate

chemical not microbiological conditions • Neither designed specifically for Arsenic – applicability found limited • The environment Agency 2007 noted that in vitro tests should be used cautiously

with respect to arsenic as the bioavailability relationship remains uncertain! • See R&D Technical Report P5-062/TR02 2002


Other Tests – to confusing!

• PBET – Physiologically Based Extraction Test modified by BGS

(As Cd, Pb validated! others lower levels of validation)

• (SBRC) SBET – Simplified Bioaccessibility Test (originally developed for Pb – validation data are available)

• CePBET – Colon Enhanced Physiologically Based Extraction Test (CePBET) for PAH

• IVG - Invitro Gastrointestinal Method

• FOREhST – Fed Organic Extraction Human Simulation (incl. PAH)

• US P – US Pharmacopoeia Method

• TIM – TNO Gastrointestinal

• SHIME – University of Ghent

• RIVM in vitro Digestion model

• DIN 00 19738 – German DIN

• UBM


• BARGE (the Bioaccessibility Research Group of Europe)

• But BARGE – looking at standardisation, cross validation

• BARGE Unified Bioaccessibility Method (UBM) • Aim: to harmonise the use of bioaccessibility

in human health risk assessments for contaminated soils in Europe (Based on RIVM (Dutch))

• Modified RIVM methodology to ensure adequate conservatism, applicable to the local geological conditions in a range of different countries,

• These studies have been published in the peer reviewed literature and are related to Cd, Pb and Zn contamination of urban and agricultural soils) and As, Cr & Pb contamination in urban UK soils In Vivo Validation of the Unified BARGE Method to Assess the Bioaccessibility of Arsenic, Antimony, Cadmium, and Lead in Soils, Journal of Environmental Science and technology


An Example

Stane Gardens Shotts: Site investigation and remedial discussions with multiple stakeholders. Groundwater and human health risks identified. £2.75M remediation on-going including soils treatment, enhanced capping and river restoration and isolation. Largest SEPA funded river restoration Project to date.


• PBET-determined RBA results applied to the “soil” bioavailability parameter in the CLEA model, as this test approximates stomach and intestinal absorption contaminants in soil (which does not directly equate to the bioaccessibility of dust within lungs).

• British Geological Society, no standard laboratory methods presently exist for deriving airborne dust bioaccessibility

Analyte Location

Total Concentration Reported by Alcontrol

mg/kg

Total Concentration, Reported by ESG

mg/kg

Relative Bioaccessibility (RBA) %

Arsenic GS9 0.5m 35.4 28.24 6

Arsenic GS10 0.4m 42.4 22.34 11

Arsenic GS12 0.5m 44 40.22 5

Chromium GS11 0.3m 4.9 4.7 9

Copper GS10 0.4m 16500 731.9 36

Lead GS10 0.4m 550 482.1 14

Nickel GS4 0.3m 207 420.5 21

Nickel GS5 0.4m 375 174.9 24

Nickel GS6 0.2m 157 349.4 25

Nickel GS8 0.3m 138 502.2 25

Nickel GS9 0.5m 371 256.9 12

Nickel GS10 0.4m 550 222.6 14

Nickel GS12 0.5m 473 398.6 17


Terminology Reminder

Bioavailability

In-vivo: tested on whole, living organisms In-vitro: in a tube

Bioaccessibility


Any Volunteers?

Image courtesy of BGS


Reminder on Terminology

Bioaccessibility | Bioavailability


Basis of Bioaccessibility testing

• Designed to be representative of Gastrointestinal tract

• Need to be robust and repeatable

• Includes synthetic human

digestive fluids:

– saliva

– gastric fluid

– duodenal fluid

– and bile


Outline laboratory procedure

Extraction done in two consecutive stages

Stage 1 – Gastric

– Replicates conditions stomach conditions

– Uses saliva and gastric fluid

– Extracted using end-over-end mixers at 37°C

Designed with conservatism


Outline laboratory procedure

Extraction done in two consecutive stages

Stage 2 - Gastro-Intestinal

– Replicates conditions in small intestine following on from the stomach stage ie sample goes through stage 1 and stage 2

– Duodenal fluid and Bile added to samples

– Extracted further using end-over-end mixers at 37°C


BARGE

Bioaccessibility Research Group of Europe

Referred to as Unified BARGE Method (UBM)

- Simulates the unfed state

- Used for bioaccessibility of metals

- Validated for robustness and repeatability

- Arsenic, Cadmium and Lead validated to in-vivo

by the BARGE group collaborators

- Other metals have limited data at present

- Stage 1 extraction 1 hour – 1 set of samples analysed

- Stage 2 extraction an additional 4 hours – 2nd set of samples analysed


FOREhST

Fed ORganic Estimation human Simulation Test

- Simulates the fed state

- Uses porridge and sunflower oil as well as the synthetic human solutions

- Used for PAH’s

- Validated for robustness and repeatability

- Stage 1 extraction 2 hours

- Stage 2 extraction an additional 2 hours


Where do we go from here?

Lab data should be back just in time for BARGE on lead. Hope to tie back to C4SL for lead.

Message is • Simple (Principle 1) • Increasingly Credible and Concrete (Principles 3 and 4) • Adds to the Consultants Narrative (Principle 6 the

Story and “Lines Of Evidence approach”.


Questions?

Please type any relevant questions in ‘Q&A box’.


Post webinar questions and discussion

For more information please contact – [email protected]

or

[email protected]

Group Discussion – at ‘Soil Testing and Analysis’ on Linkedin

Certificate of Participation available

mailto:[email protected]

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