4th ISPTS, Beijing, Nov 19, 2007
Exposure Assessment of
Environmental Samples and Contaminants:
TIE Approach
Jong Seong Khim and John P. Giesy
Toxicology Centre, University of Saskatchewan, CANADA
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4th ISPTS, Beijing, Nov 19, 2007
CONTENTS
Background
Objectives
Approach
Results and Major Findings
Conclusion
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4th ISPTS, Beijing, Nov 19, 2007
BACKGROUND
Before Our Study: mid 1990s
z Only a few studies reported for classical POPs for sediments
of the Korean coast, and no reports on some EDCs
z Moreover, above studies focused sorely on analytical concentrations
z The current risk assessment paradigm of the U.S. and Canada, which
considers biological effects in addition to chemical concentrations,
has never been introduced for Korean coastal sediment
thus, the concept of biological effects together with
measurement of chemical concentrations was
first employed in Korea during our study
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4th ISPTS, Beijing, Nov 19, 2007
BACKGROUND
lots of industrial complexes
along the Korean coastal areas
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4th ISPTS, Beijing, Nov 19, 2007
BACKGROUND
some sediment samples collected from
Korean coastal areas
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4th ISPTS, Beijing, Nov 19, 2007
APPROACH
sediment
sediment
property
property
metal
metal
analysis
analysis
Scheme
Korean
Sediments
(>700 stns)
Organic
Organic
analysis
analysis
Chemical
Concentration
benthic
benthic
community
community
in
invivo
vivo
bioassay
bioassay
In
InVitro
Vitro
bioassay
bioassay
COMPARISON
6
Biological
Response
4th ISPTS, Beijing, Nov 19, 2007
ACTIVITIES
Study began in Dec 1995
(long-term research plan)
96
SEDIMENT STUDY
99
98
97
DATA obtained in Dec 2001
(~700 stations)
00
01
SEDIMENT TIE STUDY
SURVEYS in Korean coast
122
Namyang (5)
Incheon (7)
Shihwa (20)
Gyeonggi (60)
238
Gwangyang (75)
Mokpo (91)
Incheon (9)
Ulsan (63)
121
Shihwa (20)
Masan (31)
Jinhae (70)
ANALYSES of
Sed. Property
Metal
Community
Organics
In vitro bioassay
63
Ulsan (30)
Onsan (23)
133
Pohang (34)
Masan (24)
Ulsan (34)
Onsan (33)
Shihwa (8)
11 locations
Gwangyang (11)
Amphipod bioassay
Microtox bioassay
Sea-urchin bioassay
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4th ISPTS, Beijing, Nov 19, 2007
SEDIMENT TIE STUDY
SEDIMENT TIE
Seoul
1
East Sea
KOREA
West Sea
6
AREA
n
from
year
1 Shihwa
11
8
11
lake
inland river
bay
1998
2000
2001
28
8
13
9
16
16
26
8
bay
inland river
bay
inland river
bay
inland river
bay
inland river
1998
2000
1999
1999
1999
1999
2000
2000
2 Gwangyang
5
3
- applied since 1998
(analyzed for samples >150
among total of >700)
3 Masan
4
4 Onsan
2
5 Ulsan
South Sea
6 Pohang
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4th ISPTS, Beijing, Nov 19, 2007
OBJECTIVES
Assessment
Assessmentof
ofSediment
SedimentContamination
Contamination
Sediment TIE
z Bioassay-directed Fractionation
z Mass Balance Analysis
Concentration
(Level)
Distribution
(Sources)
Dioxin-like
Activity
Specific Objectives
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Estrogenic
Activity
4th ISPTS, Beijing, Nov 19, 2007
KEY CONCEPT (1)
Pollutants
(Mixture)
Bioassay-directed
Fractionation
sig.
TIE
RE
RE
fractionation
TIE
start
*
fractionation
*
*
F1
F2
bioassay
F3
F1
F2
F3
chemical stop! further
treatment
fractionation
*
F4
F5
F6
F7
F4 F5 F6 F7
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TIE
end
4th ISPTS, Beijing, Nov 19, 2007
KEY CONCEPT (2)
Instrumentally
derived
Mass Balance
Analysis
calculated by multiplying
the analytical concentrations
of compounds identified
by their REPs and summing
Σ (Conc.ⅹREPs)
e.g.
TEQs or EEQs
=
>
<
11
Bioassay
derived
estimated directly from
dose-response curves
from bioassay analysis
of a sample and standard
EC50std/EC50sample
e.g.
TCDD-EQs or E2-EQs
4th ISPTS, Beijing, Nov 19, 2007
TARGET COMPOUNDS
Compounds
(used for / found in)
1 PCBsa: Polychlorinated biphenyls
2 OC pesticides: Organochlorine pesticides
(structure)
(coolants, lubricants, capacitors)
a
(insecticides, herbicides)
Cl(m)
Cl(n)
m+n=1-10
- HCB: Hexachlorobenzene
- CHLs: Chlordanes
b
- HCHs: Hexachlorocyclohexanes
- DDTsb: Dichloro diphenyl trichloroethanes
3 PAHsc: Polycyclic aromatic hydrocarbons
(coal tar, crude oil, dyes, plastics) c
4 APs: Alkylphenols
(detergents, paints, agents)
d
- NP : Nonlyphenol
d CH
- OP: Octylphenol
- BP: Butylphenol
5 BPAe: Bisphenol A
(plastics, resins, containers, dental sealants)
CC l3
Cl
Cl
9
e
Standards for Bioassays
OH
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CH3
HO
OH
CH 3
Cl
O
Cl
Cl
O
Cl
f
2,3,7,8-TCDDf: 2,3,7,8-tetrachlorodibenzo-p-dioxin (most toxic dioxin)
6
7 E2g: 17-β-estradiol (natural estrogen)
CH OH
3
g
HO
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4th ISPTS, Beijing, Nov 19, 2007
METHODS
Sediment
Samples
organic extraction
initial bioassay screening
bioassay-directed fractionation
second bioassay determining potencies
instrumental quantification
1
1
Raw Extract (RE)
2
H4IIE/MVLN
5
3
(RE)
(RE)
(FEs)
(FEs)
(FEs)
Florisil Column Fractionation: FEs (F1, F2, & F3)
(1 ml DCM)
(1 ml each for 3 FEs)
4
5
F1
F2
F3
(1 ml Hex)
(1 ml Hex)
(1 ml ACN)
H4IIE/MVLN
H4IIE/MVLN
H4IIE/MVLN
(0.5 ml)
(0.5 ml)
(0.5 ml)
GC
GC/HPLC
HPLC
(0.5 ml)
(0.5 ml)
(0.5 ml)
PCBs
HCB, p,p’-DDE
PAHs
HCHs, CHLs, DDTs
APs (NP, OP, BP)
Bisphenol A
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4th ISPTS, Beijing, Nov 19, 2007
IN VITRO BIOSSAYS
H4IIE-luc or MVLN Cells
Standard for
H4IIE-luc assay
2,3,7,8-TCDD
Cl
O
Cl
Cl
O
Cl
72 hr of
Exposure
(dosing)
ARNT
AhR-luc.
Luc.-mRNA
Sediment
Extracts
(RE or FEs)
AhR
ER-luc.
ER
Luciferase
Standard for
MVLN assay
17-b-estradiol
CH3 OH
Light
HO
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Luciferin
4th ISPTS, Beijing, Nov 19, 2007
RESULT (1): CHEMICAL CONCENTRATIONS
I Inland areas
POPs in Korean Sediments
I
1) Concentration
z inland > bay
SH
2) Distribution
z APs: much greater in inland (ppm levels)
z PAHs: widespread distribution (ppm levels)
z PCBs & OCPs: relatively uniform (ppb levels)
I
APs
PAHs
I
PH
I
I
MS
US
OS
3) Sources
z generally agree between inland and bay
z hot spot and multiple sources
GY
4) Overall,
z low to moderate, compared with other studies
z mostly lower than SQGs
APs PCBs
PAHs pesticides
2000 ppb
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4th ISPTS, Beijing, Nov 19, 2007
RESULT (2): BIOLOGICAL EFFECTS
I Inland areas
SH
Dioxin-like Activity (H4IIE-luc Cells)
1) Screening Response
z >90% of REs showed significant responses
I
2) Fraction Response
z F2 ≥ RE > F3 > F1; PAHs responsible
z ΣFEs > RE; interaction between compounds
I
PH
I
I
US
I
MS
OS
3) Distribution
z inland > bay
z very high in river sediment; point sources
4) Mass Balance
z TEQs < TCDD-EQs
z Sometimes good agreement (TEQs = TCDD-EQs)
GY
RE F1 F2 F3
20%-TCDD-max.
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4th ISPTS, Beijing, Nov 19, 2007
RESULT: MVLN BIOASSAY
I Inland areas
SH
Estrogenic Activity (MVLN Cells)
1) Screening Response
z ~50% of REs showed significant responses
I
I
GY
PH
US
I
I
I
2) Fraction Response
z RE > F2 > F3 > F1; PAHs and APs responsible
z ΣFEs > RE; interaction between compounds
z Cytotoxic in many F3
3) Distribution
z inland > bay
z very high in river sediment; point sources
OS
MS
4) Mass Balance
z EEQs << E2-EQs (average 20%)
RE F1 F2 F3
20%-E2-max.
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4th ISPTS, Beijing, Nov 19, 2007
SUMMARY: COMPARISON
POPs & EDCs levels
low to moderate
RE < FEs responses
interaction (non-additive)
Localized distribution
point sources from inland
F2 (H4IIE-luc) & F2-3 (MVLN)
greatest responses
Major contaminants
PAHs and APs
Korean
Sediments
n=154
F3 (MVLN)
sometimes, cytotoxic
Chemical
Concentration
TEQs < TCDD-EQs
EEQs << E2-EQs
Biological
Response
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4th ISPTS, Beijing, Nov 19, 2007
ENDING REMARK
OVERALL
z TIE and MBA were useful to determine chemical composition
and concentrations that responsible for the biological responses
z however, known composition and concentrations of target chemicals
could not fully account for biological responses
more elaborate characterization of compounds
associated with some fractions should improve
further understanding
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4th ISPTS, Beijing, Nov 19, 2007
FUTURE TIE WORKS
Chemicals
Media
Tools
existing POPs
emerging EDCs
environmental
samples
bioanalytical
methods
for
with
by use of
PCDD/DFs, PAHs,
APs, PFCs, PBDEs
Sediment, Water,
Biological Samples
in vitro, in vivo,
in situ
MORE INTEGRATED APPROACH &
UNDERSTANDING NEEDED
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4th ISPTS, Beijing, Nov 19, 2007
Acknowledgement to:
Research Teams of Profs. Giesy & Koh
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