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Risk e Learning
Metals – BioavailabilityApril 9, 2003
2:00 – 4:00 pm EDT
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Assessing the Oral Bioavailability of Lead in Soil in Humans
J.H. Graziano1, N.J. LoIacono1,
M. Maddaloni2, S. Chillrud3, C.B. Blum4
1Environmental Health Sciences, Mailman School of Public Health, Columbia University; 2US Environmental Protection Agency, Region 2;
3Lamont-Doherty Earth Observatory, Columbia University, 4College of Physicians & Surgeons, Columbia University
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Assessing the Oral Bioavailability of Lead in Soil in Humans
• Stable isotope dilution
• Previous work on Bunker Hill, ID, soil
• Results from ongoing studies of amended soils (Joplin, MO)
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Lead Isotope Ratio Midpoint
Lead Isotope Ratio Midpoint
- Australian subjectsN
umbe
r of
Sub
ject
sN
umbe
r of
Sub
ject
s
- US subjects
1.095 1.105 1.115 1.125 1.135 1.145 1.155 1.165 1.175 1.185 1.195 1.205 1.2150
2
4
6
8
10
12
14
16
18
20
A
Lead Isotope Ratio Midpoint
Num
ber of
Sub
ject
s
1.095 1.105 1.115 1.125 1.135 1.145 1.155 1.165 1.175 1.185 1.195 1.205 1.2150
1
2
3
4
5
6
7
8
9
10
B
1.095 1.105 1.115 1.125 1.135 1.145 1.155 1.165 1.175 1.185 1.195 1.205 1.2150
1
2
3
4
5
6
7
8
9
10
C
- Scottish subjects
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Lead Isotope Ratios of Select Sites
Location Superfund Site 206Pb/207Pb
Kellogg, ID Bunker Hill 1.057
Butte, MT Silver Bow Creek 1.148
Leadville, CO California Gulch 1.170
Buffalo, NY Bern Metal 1.205
Triumph, ID Triumph Mine 1.253
Tar Creek, OK Tar Creek 1.350
Joplin, MO Oronogo-DuenwegMining Belt
1.378
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Clinical Protocol
• Screening and physical exam
• Obtain informed consent
• Three day admission
• Subject dosed at 250 µg Pb/70 kg body wt
• Collect blood and urine samples
• Standardized meals
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Time (hrs)
Subject # 5
0 5 10 15 20 25 30 35
Lea
d 2
06
/20
7
1.190
1.191
1.192
1.193
1.194
1.195
1.196
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Results of Previous Studies from Bunker Hill Soil
Group(n=6)
Age(yrs)
Weight(kg)
Pb Dose(ug)
Soil Dose(mg)
Bioavailability(%)
Fasted 28 59.7 213 72.9 26.2(18.0-35.6)
Fed 28 67.9 242 82.9 2.52(0.2-5.2)
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Lead Isotope Ratio Distributions
0123456789
101112
Nu
mb
er
of
Su
bje
cts
1.15
1.18
1.21
1.24
1.27 1.3
1.33
1.36 1.4
Lead Isotope Ratio
Joplin SoilSample
Subjects
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Soil Homogeneity
SampleLead(ppm)
Weight(mg) 206Pb/207Pb
Untreated 5,200 75.5 1.3816
“ 69.3 1.3826
“ 62.2 1.3817
Phosphate-Amended*
4,240 66.8 1.3790
“ 65.9 1.3784
“ 66.2 1.3796
* 1% Phosphate, field–treated aged 18 months
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Subject Demographics
Subject Soil Type Age Race/Ethnicity Gender Weight Pb Dose Soil Dose(kg) (ug) (mg)
1 Non-amend 30 African Amer F 60 239.2 462 Amended 37 Caucasian F 53.6 195.5 46.13 Non-amend 23 Caucasian M 66 257.4 49.54 Amended 25 Caucasian M 89.5 324.4 76.55 Amended 40 African Amer M 73.5 262.5 61.96 Non-amend 35 Asian F 60.5 215.8 41.57 Amended 41 African Amer F 81 296.4 69.98 Non-amend 34 Middle Eastern M 79 282.4 54.39 Amended 20 Caucasian M 62.7 224.7 5310 Non-amend 23 Caucasian F 59 210.6 40.5
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Examples of Pb isotope data : for 2 different soil types .
1.180
1.190
1.200
1.210
1.220
1.230
1.240
1.250
1.260
1.270
0 5 10 15 20 25 30 35
Time from ingestion (hr)
Pb
20
6/P
b2
07
Subj 103 :Untreated Soil
Subj 105 :Treated Soil
Lea
d
206
/207
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Subject Change in Blood Lead Subject # Soil Type BPb Start BPb End Change Mean
101 Non-Amended
4.19 5.91 1.72
103 “ 1.30 2.22 0.92
106 “ 1.49 3.32 1.83
108 “ 2.06 2.50 0.44
110 “ 1.11 2.20 1.09 1.20
102 Amended 1.84 2.32 0.48
104 “ 3.62 4.05 0.43
105 “ 4.88 5.78 0.90
107 “ 2.95 3.82 0.87
109 “ 2.27 2.60 0.33 0.60
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Preliminary Results
GroupAge(yrs)
Weight(kg)
PbDose(ug)
SoilDose(mg)
Bioavailability(%, Absolute)
Untreated 29.0 64.9 241.1 46.4 35.8(14.6-54.2)
Amended 32.6 72.1 260.7 61.5 15.3(8.0-26.2)
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Comparative Results: Animal, In vitro & Human
Animal*In vitro**(pH 2.3) Human
% Reduction inBioavailability
38 38 57
* Casteel et. al., ** Ruby et. al.
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Relative Costs:Remedies for Metal-Contaminated Soils
0 0.5 1 1.5 2
$ (Millions)
Phosphate Inactivation
Soil Cap
Asphalt Cap
Phytoextraction
Soil Wash
Excavate & Landfill
* Source: EPA Technical Innovation Office
Net Present Cost for 1 Hectare Site
1.62
0.79
0.25
0.16
0.14
0.06
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Manuscripts in Progress
• Graziano, JH, LoIacono, NJ, Chillrud, SN, Ross, J, Blum, C. Oral bioavailability of lead in phosphate-amended and non-amended soils from a Missouri smelter site.
• Chillrud, SN, Hemming, G, Wallace, S and Ross, J. Determination of lead isotopes in blood with separation by iron hydroxide co-precipitation and analysis by multi-collector ICP-MS, In preparation for Journal of Analytical Atomic Spectroscopy
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Risk e Learning
Metals – BioavailabilityApril 9, 2003
2:00 – 4:00 pm EDT
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Determination of Metal Speciation in Aquatic Ecosystems Using Equilibrium Gel Samplers
Martha Chang Jen Galvin
Sarah Griscom Chris Lewis
Dave Senn Jim Shine
Crista Trapp
Harvard School of Public Health - Dept. Environmental Health
NIEHS Superfund Basic Research Program
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Outline:
1. Importance of Metal Speciation
2. Gel Flux Samplers
3. ‘Gellyfish’ Equilibrium Gel Sampler
- As a metal speciation tool
- As a bio-mimic
4. Conclusions
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Knowledge of the Free Metal Ion Concentration is Important:
- Key determinant describing partitioning amongst different phases:
- DOC bound
- POC bound (settling particles)
- Biological Uptake
-Thus a determinant describing:
- Transport
- Fate
- Effects
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Cd2+
Cd
Transmembrane Protein
Dissolved
Humic Acid
Complex
Water
Cell
Cd
Biotic Ligand Model:
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Zn2+
Zn
Cd2+
Cd
Transmembrane Protein
Dissolved
Humic Acid
Complex
Water
Cell
Need to Understand Competitive Interactions:
-Interactions can be antagonistic, protective, or neutral
-Need simultaneous data for multiple metals
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Current Metal Speciation Techniques:
- Indirect
- titrations to characterize ligands
- labor Intensive
- Methodological Uncertainties
-titration window
- L1 & L2 ligands ??
- One metal per titration
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Acrylic Plate
Gel + Chelex 100
Gel only
(Diffusive Gel Layer)
[dz
Diffusion Gradient (dC)
Gel Flux Samplers: - Diffusion Gradient Thin-film (DGT) Samplers
- Zhang & Davison. 1995. Anal. Chem. 67:3391-3400
Assuming C=0 at Chelex/Gel interface:
a) Determine a flux rate of metals to the Chelex 100
b) F = -D dC/dz solved for C, the external concentration
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Strengths and Weaknesses of Flux Samplers:
(-) Measures a flux, not a concentration
(-) Unclear what metal species is measured
(+) Flux of individual metals independent of the underlying complex mixture
(-) Flux of individual metals independent of the underlying complex mixture
- less suitable as a biotic ligand analog?
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9101112
Actual Free Cu2+ (CLE-ACSV, pCu)
7
8
9
10
11
DG
T 'L
ab
ile' C
u (
pC
u)
Twiss and Moffett (2002)
- “Impacted Sites”
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Equilibrium Sampler (Gellyfish) : Design Criteria
Polyacrylamide gelToyopearl AF Chelate-650M resin
21 mm
3 mm
Toyopearl Resin (Tosohaas, Inc.):
- Mean bead size: 65 µm
- exchange capacity: 35 µeq/ml
Gel Crosslinker: DGT Crosslinker (DGT Research, Ltd.)
Final Resin Concentration Inside Gel:
- 3 x 10-4 eq/L- Designed for less than 5% depletion of metal from surrounding 2 L solution
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Strengths and Weaknesses of Gellyfish Sampler:
(+) Is in equilibrium with a concentration, not a flux
(+) Clearer what metal species is measured (free metal ion)
(-) Accumulation of individual metals related to the underlying complex mixture
- possible correction procedures?
(+) Accumulation of individual metals related to the underlying complex mixture
- more suitable as a biotic ligand analog?
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General Differences
Flux Sampler = Empirical
- What you see is what you get
- Is what you get what you want to see?
Gellyfish = Mechanistic
- What you see is the free metal ion concentration
- BUT… you must be able to account for all possible
confounding factors
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The ‘Gellyfish’:
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Equilibration, Back Extraction, and Analysis:-Experiments conducted with artificial seawater
- AQUIL salts recipe (no nutrients)
- Metal speciation controlled with EDTA
- Equilibration
- Gellyfish suspended in 2L sample with teflon
string for appropriate equilibration period
- After Equilibration:
- Gellyfish removed from test solution,
rinsed in DI water
- Gellyfish placed in 10 ml 10% HNO3
- After 24 - 48 hrs, back extract analyzed for Cu
- ICP-MS (Perkin Elmer ELAN 6100 DRC)
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Equilibration Times:
Range of t90 equilibration times: 15 - 30 hours
Coefficient of Variation for Repeated Measures: ~ 5%
0 10 20 30 40 50 60 70 80
Equilibration Time (hours)
0
1
2
3
Ma
ss
Cu
Co
llec
ted
(µ
g)
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Calibration with Free Metal Ions:
Salinity = 20 ppt (pH = 7.9 - 8.1)
Total Copper:
1.5 x 10-6 M (nominal pCu = 7, 8, 9, 10)
1.5 x 10-7 M (nominal pCu = 11, 12)
r2 = 0.97
7891011
Sample Free Zn 2+ (pZn2+)
1e-10
1e-09
1e-08
5e-08
Gellyfi
sh
Zn
mass c
ollecte
d (
mo
l)
6789101112
Sample Free Cu2+ Concentration (pCu)
1e-12
1e-11
1e-10
1e-09
1e-08
1e-07
Ge
llyfi
sh
Cu
Ma
ss
Co
llec
ted
(m
ol) Cu:
Salinity = 20 ppt (pH = 7.8 - 8.0)
Total Zinc:
5 x 10-7 M (nominal pCu = 7, 8, 9)
5 x 10-8 M (nominal pCu = 10, 11)
Zn:
r2 = 0.94
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Correction Procedure for Complex Mixtures:
-K’Cu-Toso = [CuToso]/[Cu2+]*[Tosofree] independent of other
solution components
-Requirement: Semi-conservative solution component
- Conservative behavior in water (Xtotal = Xfree)
- Reacts with Tosohaas resin
- Candidate: Sr
- Assumption: Srtotal = free Sr2+
- therefore: K’Sr-Toso = [SrToso]/[Srtotal]*[Tosofree]
- solve above equation for [Tosofree]
- substitute into original equation
- Result: Cu2+ = K* * [Cugel] *{[Srtotal]/[Srgel]}
where K* = K’Cu/K’Sr
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Model Test of Sr Correction Procedure:
- 5 free Cu ion levels: pCu range: 10 - 12
- 4 salinity levels : 10, 15, 20, 25 o/oo (= 20 total treatments)
- No Correction: Cugel at fixed pCu varies w/ salinity
- Correction Applied: Data collapse to a single line
1e-13 1e-12 1e-11 1e-10 1e-090.0000
0.0001
0.0002
0.0003
0.0004
0.0005
Gel
lyfi
sh C
u (
mo
l/L)
Before Sr Correction
Free Cu2+ (mol/L)
1e-13 1e-12 1e-11 1e-10 1e-090.0000
0.0001
0.0010
0.0100
[Cu
gel]
*{[S
r tota
l]/[S
r gel]}
After Sr Correction
Free Cu2+ (mol/L)
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Me2+
Alternate Use: Bio-mimic:
Phytoplankton
Zooplankton Planktivorous Fish
Piscivorous Fish
Gellyfish
Concordance?
Direct Uptake
Trophic Transfer
Trophic Transfer Trophic
Transfer
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The ‘Gellyfish’: Field Deployment Apparatus
Gellyfish placed in a piece of LDPE sheet with a hole punched through it, sandwiched by polycarbonate filters, and held togehter with snap-together slide holders.
Slides mounted in a plastic basket for field deployment
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Initial Experiments: Calibrate with Biomonitoring Organisms
A) Water:
B) Sediment:
Gellyfish
Gellyfish
Mytilus edulis
Neries Virens
Field Study
Lab Study
Megel
(mol)
Me
org
an
ism
(m
ol/g
)
m = ??
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Theoretical Basis:
- Competetive Metal Interactions governing metal uptake by Gellyfish = interactions governing uptake by biological organisms
PbCd
CuMn
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Potential Advantages as Monitoring Tool:
1) Gellyfish Don’t Die (can they be eaten?)
2) Gellyfish don’t undergo gametogenesis
3) Gellyfish are all the same (good or bad?)
4) Equilibration time can be controlled
- Surface area/volume ratio
- Can select a desired integration period
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Current/ Near Future Experiments:
- Effect of complex mixtures on Metal uptake
-Proof of Sr correction concept
- Use with other metals (Pb, Cd)
- similar analytical windows for different metals?
-Calibrate with uptake into aquatic organisms
- select desired equilibration time?
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Conclusions:
- Gel Flux (DGT) Samplers
- are the strengths weaknesses?
- ‘Gellyfish’ Sampler
- Equilibrium based
- Are the ‘weaknesses’ strengths?
- Proof of concept for Cu, Zn promising
- Potential Biotic Ligand Analog?
- Supporting tool for speciation-based WQ approaches?
- Allow new types of experiments in Metal Speciation Research?
- ligand specificity studies
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