Future of environmental_forensics_inef2016

© 2016
Chemistry Ma2ers Inc. 1 INEF 2016 – Örebro, Sweden
Making chemistry data meaningful
Future of Environmental Forensics
Maybe it should be more complicated?
June 30, 2016
6th Interna8onal Network of Environmental Forensics Conference,
Örebro, Sweden

© 2016
Chemistry Ma2ers Inc.
Summary of INEF 2016
2 INEF 2016 – Örebro, Sweden
•  I recently gave the ﬁnal plenary talk at the Interna8onal
Network of Environmental Forensics (INEF) in Örebro,
Sweden.
•  The conference was held at Örebro Castle from June
27-30, 2016.
•  It was a pleasure to provide the audience my thoughts
on the future of environmental forensics.

© 2016
Summary of INEF 2016

© 2016
Acknoweldgments
•  Phil Richards, MChem, MRes, PhD, P.Chem

© 2016
Quote for the day
A people without the knowledge of their past
history, origin and culture is like a tree without
roots. – Marcus Garvey

© 2016
Selected History of POPs
•  PCB manufacturing – 1930s
•  DDT manufacturing – 1940s
•  245-T manufacturing begins – 1940s
•  Hexachlorophene produc8on – 1948
•  DDT measured in biota – 1950s
•  Vietnam War – Agent Orange use 1961 - 1972
•  PCBs discovered in biota - 1966
•  Times Beach - 1971
•  Seveso Italy - 1976
https://www.yahoo.com/news/bp/december-23-road-contamination-prompts-evacuation-times-beach-145517324.html
http://www.toxipedia.org/download/attachments/9353/Seveso.chem.jpg?version=1&modificationDate=1238498605000&api=v2
https://upload.wikimedia.org/wikipedia/commons/3/3e/Agent_Orange_at_Johnston_Atoll_1976.jpg

© 2016
PCBs

© 2016
DDT, PCBs & Environmental Research
•  1950-60s – DDT measured in environment
•  1962 – Rachel Carson publishes “Silent Spring”
•  DDT/DDE research in Sweden in White Tailed
Eagles by Dr. Soren Jensen in 1964
Jensen , Ambio 1972 Vol 1, No. 4

© 2016
AnalyPcal Chemistry of the Time (1960s)
•  GC-ECD (only seeing electronega8ve
compounds) and GC-FID very common
•  Mass spectrometry s8ll quite specialized
(but needed for iden8ﬁca8on)
•  Most analy8cal standards not made yet,
need to be synthesized
•  Packed GC columns provided very limited
resolving power

•  No personal computers or google searches
•  Literature search began in library, with
paper journals and took days/weeks

© 2016
Current Day PCB Analysis
•  Isotopically labeled standards available
•  Large propor8on of the 209 congeners
can be chromatographically resolved
(in 1D-GC
•  Aroclor analysis s8ll quite common
•  If congener analysis conducted, only
for a reduced number of congeners

•  Why?
1242
1254
1260
Dataphobia?

© 2016
Aroclors
Herring Gull
Human
Polar Bear
CB153
CB180
CB138/
CB163
CB170/
CB190
CB99
• up to 120 congeners
• CDC monitors top 38
How Many PCBs to Measure?

© 2016
PCB Metabolites Discovered
•  A decade later (1976), Dr. Soren Jensen s8ll
analyzing PCBs in biota
•  Leg the laboratory in a hurry one evening
•  Data was collec8ng on a Bal8c Seal sample
•  A group of peaks elu8ng late from the GC-
ECD
•  Chemically synthesized standards to confirm
iden8fica8on
•  Iden8fied new compound group -
metabolites of PCBs
–  methylsulfonyl-PCBs
–  Opened new research into the field of POP
metabolites
•  Comparing PCBs to metabolites, need as
many PCBs as possible measured in the
samples
Jensen , Ambio 1976 Vol. 5, No. 5/6, 257-260
Swedish Environmental Protection Agency, Persistent
Organic Pollutants, 1998, pp. 152.

© 2016
Was the discovery of PCBs and PCB
metabolites Environmental Forensics?
•  Not for li8ga8on
•  Not determining source
•  Not determining 8ming of the release
•  It was iden8fying the contaminants for the ﬁrst 8me. That
seems to be forensics

•  Making scien8ﬁc sense of the data…

© 2016
Environmental forensics is NOT only for
li8ga8on
Point 1

© 2016
Forensics
•  Ancient roman 8mes
•  Forensics is derived from
la8n “forens”, meaning of,
belonging to the forum,
public
http://www.crystalinks.com/romelaw.html
•  Environmental forensics – determining idenBty, source,
or Bming of contaminant release for the purpose of
public communicaBon and/or liBgaBon.

© 2016
PCDD/Fs

© 2016
Selected History of POPs
•  PCB manufacturing – 1930
•  DDT manufacturing – 1940
•  245-T manufacturing begins - 1940
•  Hexachlorophene produc8on – 1948
•  DDT measured in biota – 1950s
•  Vietnam War – Agent Orange use 1961 - 1972
•  PCBs discovered in biota - 1966
•  Times Beach - 1971
•  Seveso Italy - 1976
Clement R et al., Analytical Chemistry, 1991, 63, 1130A–1139A.
1971
1988

© 2016
Early InvesPgaPons in Dioxin Contaminated
Agent Orange and Chlorinated Phenols
•  Chromatogram of early dioxin
analysis of Agent Orange
•  Inves8gators began by
iden8fying all congeners for
chlorinated dioxins and furans in
the chromatograms
–  210 congeners exist
•  Using mass spectrometry and
glass packed columns
•  Synthesized compounds for
posi8ve iden8ﬁca8on
Rappe C et al., Chemosphere, 1978, 5, 431–438.

© 2016
Rappe C, Gara A, Buser H.R, Chemosphere, 1978, 12, 981 – 991.
A study of PCDD/Fs in Chlorophenol
Formulations

© 2016
Toxicology Takes Over
•  In 1973, AhR iden8fied as
mediator of dioxin toxicity
•  Extensively studied to iden8fy
structural requirements for
binding to AhR
•  TCDD and 2378 subs8tuted
PCDDs/PCDFs fit nicely in
binding site
–  mid-1980s – binding site
defined
•  Toxic equivalents and toxicity
equivalency factors born
Okey A, Toxicological Sciences, 2007, 98, 5–38.
Pandini A. et al, Biochemistry,
2009, 48, 5972–5983

© 2016
Toxicity Equivalency Factors
•  EPA proposed TEFs 1989
•  Based on AhR binding aﬃnity
•  Need chlorines in the 2,3,7,8 posi8ons
for eﬃcient binding
•  Only 17 congeners PCDD/Fs have the
required 2378 subs8tu8on
•  Have been measured ever since
1
2
3
4
9
6
8
7
5
PnCDDs

© 2016
Fast Forward to Present Day
•  Li8ga8on regarding
contaminants at Superfund sites
across the US
•  Source of dioxin at these
loca8ons in ques8on
•  With only 2378 subs8tuted
congeners for forensics, looks
like it is all 2378TCDD
INEF 2016 – Örebro, Sweden
Photograph by A. L. Young
Or is it…
22

© 2016
Company
Diamond Alkali
Syntex (NEPACCO)
Monsanto (Nitro)
Occidental (Hooker)
Dow (Midland)
Hercules and
Transvaal/Vertac
Order of Addition
caustic to TeClBz
caustic to TeClBz
TeClBz to caustic
All in at start
caustic to TeClBz
All in at start
Expect slight differences in PCDD/F
fingerprint due to the manner of
manufacture (and temperature).
Different 245-TCP production
processes
•  245-T produced from 245-
trichlorophenol
•  In 1960s, US Military (under Defense
Produc8on Act of 1950) required all
capacity of 245-TCP to produce
tac8cal military herbicides
•  245-TCP produced from
tetrachlorobenzene

1245-Tetrachlorobenzene 245-Trichloroanisole 245-Trichlorophenate
Base
Heat

© 2016
+
+
-HCl
-Cl2
-2HCl
245-TCP
245-TCP
245-TCP
2345-TeCBz 2378-TCDF
2378-TCDD
A. Hay, 1982, Plenum Press.
Formation of TCDD and TCDF During
245-TCP Production

© 2016
Production of 245-TCP – Process 1
0
20
40
60
80
100
0 50 100 150 200 250 300
CompositionofReactionMass:
PercentofmolesofComponentsIndicated
Reaction Time:
Minutes
Caustic added to Tetrachlorobenzene

© 2016
Reaction Vessel - 0 minutes
Methanol and caustic
Tetrachlorobenzene
and PCE
0
20
40
60
80
100
0 50 100 150 200 250 300
+
+
= 2378-TCDF
= 2378-TCDD
HEAT

© 2016
Mostly Tetrachlorobenzene
and few molecules of
245TCP
0
20
40
60
80
100
0 50 100 150 200 250 300
+
+
= 2378-TCDF
= 2378-TCDD
245-TCP forms in reactor
HEAT

© 2016
50% Tetrachlorobenzene
50% 245TCP
0
20
40
60
80
100
0 50 100 150 200 250 300
+
+
= 2378-TCDF
= 2378-TCDD
HEAT

© 2016
Trace Tetrachlorobenzene
Mostly 245TCP
0
20
40
60
80
100
0 50 100 150 200 250 300
+
+
= 2378-TCDF
= 2378-TCDD
HEAT

© 2016
Chemistry Ma2ers Inc. 30
Formation of Other PCDD/Fs During
245-TCP Production
Any chlorinated
benzene
Any chlorinated
phenol formed
Multiple PCDD/F
Congeners
Add Base
•  Chlorinated benzenes not 100% pure
•  Any other chlorinated benzenes present would form
uninten8onal chlorinated phenols which would form mul8ple
PCDDs/PCDFs (other than 2347-TCDD)


© 2016
ProducPon of 245-T from 245-TCP
245-T contains predominantly 2378-
TCDD as a contaminant BUT…

Hypothesis
•  The different produc8on processes
for 245-TCP (thus, 245-T) would
result in different PCDD/F
fingerprints
•  Fingerprints of PCDD/Fs other than
the 2378 subs8tuted congeners
would poten8ally dis8nguish
between manufacturers

Rappe C et al., Chemosphere, 1978, 5, 431–438.

© 2016
Dataphobia
The fear of having too much data…
And the Cl1 to Cl3 homologues
TEQ in Soil
61800 ng/g TEQ
210 (136 standards) 17 1

© 2016
Environmental forensics inves8ga8ons
should not be limited to regulatory
compounds.
Point 2

© 2016
PAHs

© 2016
Chemistry Ma2ers Inc. www.chemistry-matters.com 35
US EPA 16 Priority Pollutants
PAH CompoundsWhen were the 16 PAHs selected?
acenaphthene
1,2-dihydroacenaphthylene
1,8-ethylnaphthalene
Naphthyleneethylene
peri-ethylenenaphthalene
acenaphthylenenaphthalene anthracene
paranaphthalene
phenanthrene
3-Helicene
ravatite
9H-fluorene
Fluorene
2,2’-methylenebiphenyl
o-biphenylenemethane
diphenylenemethane
chrysene
1,2-benzophenanthrene
benzo(a)phenanthrene fluoranthene
Pyrene
benzo(def)phenanthrene
tetraphene
benz(a)anthracene
benzo(a)anthracene
1,2-benzanthracene
2,3-benzophenanthrene
benzo(b)phenanthrene
benzo(e)acephenanthrylene
benzo(b)fluoranthene
2,3-benzfluoranthene
3,4-benz(e)acephenanthrylene
benzo[k]fluoranthene
11,12-benzofluoranthene
2,3,1’,8’binaphthylene
dibenzo(b,jk)fluorene
8,9-benzofluoranthene
benzo(k)tetraphene
1,2:5,6-dibenzanthracene
dibenzo(a,h)anthracene
benzo(pqr)tetraphene
benzo(a)pyrene
3,4-benzpyrene
1,2-benzpyrene
benzo(def)chrysene
benzo(ghi)perylene
1,12-benzoperylene
indeno[1,2,3-cd]pyrene
1,1-(1,2-phenylene)pyrene
1,10-(o-phenylene)pyrene
2,3-o-phenylenepyrene
35INEF 2016 – Örebro, Sweden
*Established in 1976

© 2016
Double RaPo Plots to DisPnguish Source
36
0.0#
0.2#
0.4#
0.6#
0.8#
1.0#
1.2#
1.4#
0.0# 0.2# 0.4# 0.6# 0.8# 1.0# 1.2# 1.4# 1.6#
IcdP#/#BghiP#
FLRN#/#PYR#
Field&Data& MGP&Source&1& MGP&Source&2& Petroleum&Source&3&
Petroleum)
Liquid)Fuel)
Coal)pyrolysis/combus7on)
Pyrolysis/Combus7on)
Kinetically
Favoured
Thermodynamically
Favoured

© 2016
Know the fundamental principles behind the
graphs and diagnos8c ra8os.
Point 3

© 2016
Expanding the List of PAHs
Alkylated-PAHs and Other PAHs
C1-Phenanthrenes
1
2
3456
7
8
9 10
CH3
C2-Phenanthrenes
Phenanthrene
CH3
CH3
CH3
H3C
CH2 CH3
C3-Phenanthrenes
C4-Phenanthrenes… (?)
CH3
CH3
CH3
CH2
H2C
CH3
(5)
(30) (?)

© 2016
Chromatography Gets Busy
C1-DBT
C2-DBT
C3-DBT
C4-DBT

© 2016
Expanding the List of PAHs
•  Heteroatom PAHs need to be
included (collec8vely called NSO-
heterocycles)
•  Proposed 40 PAHs and 23 NSO-
heterocycles
Anderson JT et al., PACs, 2015, 35, 330.
Carbazole Benzothiophene Quinolinone

© 2016
Petroleum Hydrocarbons

© 2016
TPH
nC32
nC8
nC9
nC10
nC12
nC11
nC13
nC14
nC15
nC16
nC17
nC18
nC19
nC20
nC21
nC22
nC23
nC24
nC25
nC26
nC27
nC29
nC28
nC30
nC31
nC33
nC34
nC35
nC36
nC37
nC38
Gasoline
Diesel
Lubricating Oils
C6
C10
F1 C16
F2
C34
C50
F4F3
PAHs
Biomarkers

© 2016
Muskeg is a very rich organic environment > 50% TOC
PHC Fractions analysis cannot differentiate natural
hydrocarbons (phytogenic) from petroleum hydrocarbons
(petrogenic).

© 2016
Origin of PHCs – Recent Case Study
PHCs Present?

© 2016
PHC FracPons Results
45
PHC F3 – above guidelines in background areas.

© 2016
Natural Peat
46
F2 F3 F4
290 2300 800
Prominent peaks
Typical paoern for peat

© 2016
Mixture of Source Material and Natural Peat
47
F2 F3 F4
320 3100 1300
Clear mixture of
petroleum source and
peat chemicals

© 2016
Biomarker Analysis
48
Chemicals found in petrogenic hydrocarbons and not found in
phytogenic hydrocarbons.
o  Terpanes – prokaryotes (archaea and bacteria)
o  Steranes – eukaryotes (plants and animals)

© 2016
Diagenesis
Sterols to Steranes
49
Cholestadienol Cholestane
Diagenesis
Plant structural
membrane chemical
Oil biomarker
Diagenesis – low pressure and low temperature change in
deposited sediment as it undergoes solidiﬁca8on.

© 2016
Source Sample
Steranes and Hopanes
50
C23, C24, C25, C26
Tricyclic Terpanes
Hopanes

© 2016
Biomarkers
in Site Samples
Petroleum
Biomarker
Standard
Site
Sample

© 2016
Peat Sample
Fa2y Acids and Alcohols
52
1. Hexadecanoic acid (C16)
2. Octadecanoic acid (C18)
3. Eicosanoic acid (C20)
4. Docosanoic acid (C22)
5. Tetracosanoic acid (C24)

6. Hexacosanol (C26)
7. Octacosanol (C28)
8. Decacosanol (C30)
1 2
3 4
5
6
7 8

© 2016
The science needs to be communicated so
non-scien8sts can understand and
communicated with scien8ﬁc certainty (as
much as possible).
Point 4
Science is not ﬁnished unBl it is communicated. - Mark Walport

© 2016
State of Knowledge – AnalyPcal Chemistry
•  We can:
– extract more things and analyze more frac8ons
easier
– separate more compounds for iden8ﬁca8on and
quan8ﬁca8on
– measure compounds to extremely low
concentra8ons (ppq)

© 2016
12C-2378-
TCDD
in Serum
Using cryo-focusing and HRMS, can achieve
540 ag 2378-TCDD, assuming 70 % recovery
S/N 474 (4 Sigma)
m/z 319.8965 [M]
How Low Can We Go?
Jef Focant, Donald G. Patterson Jr, Wayman Turner
Zoex - loop
modulator

© 2016
Extreme Sensitivity
(2,3,7,8-TCDD)
Weight Moles Molecules
Microgram (10-6) 3 Nanomoles (10-9) 2,000,000,000,000,000
Nanogram (10-9) 3 Picomoles (10-12) 2,000,000,000,000
Picogram (10-12) 3 Femtomoles (10-15) 2,000,000,000
Femtogram (10-15) 3 A2omoles (10-18) 2,000,000
A2ogram (10-18) 3 Zeptomoles (10-21) 2,000
Zeptogram (10-21) 3 Yaktomoles (10-24) 2
Yaktogram (10-24) 3 Fantomoles (10-27) 0.002
Currently at the attogram level with cryofocusing coupled
with high resolution mass spectrometry
Dr. Donald G. Patterson, Jr.

© 2016
Complexity?
Conventional GC Analysis
Unresolved Complex Matter
Nelson et al. Environmental Forensics, 7:33–44, 2006

© 2016
Two-Dimensional
Gas Chromatography
Nelson et al. Environmental Forensics, 7:33–44, 2006
A few more
than 16 PAHs!
(0.2 - 7% of oil)

© 2016
Environmental Forensics in the
Gulf Oil Spill
•  Reddy et al. sampled oil directly above well to conduct
comprehensive characterization
–  Gas isotopes, GOR, fluid characteristics, API etc.
–  Fingerprinted with 2D-GC-TOF
–  Could distinguish between crudes
Reddy et al. 2011, PNAS Early Edition, p.1-6

© 2016
Environmental Forensics in the
Gulf Oil Spill
•  Hall et al. took 41 samples (slicks, scrapings off rocks,
grasses and debris, sand patties etc)
•  Oxidized hydrocarbons comprised of more than 50% of
extractable hydrocarbons
•  Used 2D-GC-TOF of samples compared to oil to assess
weathering of oil spill using chemometric techniques
Hall et al. 2013, Marine
Pollution Bulletin, 75 (1–2), 140

© 2016
MulP-Variate StaPsPcal Analysis
•  Even a PCA requires data input, cannot just
dump in 2D-GC raw data ﬁles

•  More eﬃcient data handling techniques are
required

© 2016
2D-GC Chromatogram
•  Benzene
•  Toluene
•  C2-benzenes
•  C3-benzenes
•  C4-benzenes
•  C5-benzenes
•  C1-Naphthalenes
•  Indanes (throughout)
6th Multidimensional Chromatography Workshop
Jan 6 & 7, 2015

© 2016
PCA Data Interpretation
-8
-6
-4
-2
0
2
4
6
8
10
-10 -8 -6 -4 -2 0 2 4 6 8 10 12
PC2(13.31%)
PC1 (61.03%)
PC1 describes process of fire-alteration
6th Multidimensional Chromatography Workshop
Jan 6 & 7, 2015
Fresh gasolinesBurnt gasolines

© 2016
Big Data is Coming – Are you Ready?
http://itknowledgeexchange.techtarget.com/IT-watch-blog/files/2013/05/shutterstock_134968730.jpg

© 2016
Concluding Statements
•  Environmental forensics should be founded on
fundamental science
–  Know the background science so it can be explained
•  Communica8on of EFIs is more important than the
inves8ga8on itself
–  There is some mistrust of scien8sts saying “trust me”
•  Don’t be afraid of more data
–  Expand chemicals for inves8ga8ons beyond regula8on
requirements
•  Need to prepare for data

© 2016
Thank you for your attention
Contact Info:
Chemistry Matters Inc.
Court Sandau
Cell: 403.669.8566
Email: csandau@chemistry-matters.com
URL: chemistry-matters.com
Visit my website and sign up for email for automatic
receipt of my blogs!
Twitter: @Chem_Matters
Slideshare: www.slideshare.net/csandau
74 www.chemistry-maoers.com

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