Advances in PCB Analysis

Presented by Andy Beliveau Region 1 USEPA Office of Environmental Measurement and Evaluation

November 2002

1 Historical Analysis By Aroclor Pattern Recognition GC/ECD detector with packed and capillary column. Relies on matching the standard pattern to the sample pattern. Quantitated only if the pattern matches (otherwise determined to be a non- detect). 3 to 5 peaks used for quantitation.

2 Limitations to Aroclor Analysis

Mixtures of Aroclor patterns overlap . Which peaks represent which Aroclor? Weathered PCB patterns do not match the standards. Interpretation is left to the analyst. Resulting quantitation can be compromised by all of the above.

3 Why use congener analysis

Eliminates interpretation. Weathering is not a problem. It is possible to quantitate all peaks . Sum of the congeners = Total PCB. Congeners can be quantitated individually. Dioxin-like congeners can be quantitated. Non Aroclor PCBs can be identified. Environmentally modified PCBs can be identifed.

4 Types of Congener Analyses

High resolution GC/ ECD (HRGC) – Long column 60 meters. – Long run time - 60 to 90 minutes . – Requires clean rigorous clean-up of sample. HRGC/ Low resolution MS (bench top MSD) – Medium column 30 meters. – Medium run time 30-60 minutes. – Eliminates interferences. – Allows for level of chlorination(homologue) analysis.

5 Type of Congener analysis (cont)

HRGC/ High Resolution MS – Requires a expensive instrument and highly qualified operator. – Requires a 10,000 resolution. – Allows for wide concentration range. – Medium length column- 60 meters. – Run time - 30 to 60 minutes. – Requires rigorious clean-up of sample extract for the best results.

6 Congener Analysis by HRGC/ECD No standard or promulgated EPA method. Can quantitate 125+ congeners (99%). Quantitates and identifies by retention time. Some congeners co-elute depending on column packing, column length, run time, and GC conditions. May require multiple columns to resolve all congeners. Requires individual standards or known retention time mixed standards.

7 Congeners by HR/GC/LRMS

No Standardized EPA method. Method #680 not promulgated. Can quantitate 125+ congeners(99%). Quantitates by retention time and molecular ions for each chlorination level. Requires individual congener standards. Can achieve HRGC/ECD detection limits. Eliminates non PCB interferences. Can identify non PCB compounds or environmentally modified PCBs. 8 Congeners by HRGC/HRMS

Method 1668a is not yet promugated but is used by a handful of laboratories. Can quantitate all 209 congeners in water, soil, or biota. Has a very wide concentration range ( ppm to ppt in soil and ppb to ppqt in water). Can quantitate WHO dioxin-like congeners. Can quant total PCB by sum of congeners. Requires rigorous clean-up and sometimes silica carbon column clean-up. 9 Conclusion: Major uses of congener analysis Identify congeners in biota and sediments where weathering or metabolizm changes the PCB pattern and Aroclor analysis is unusable. Confirms the actual concentration of total PCB for cancer risk assessment. Quantitates WHO doixin-like PCBs for dioxin cancer risks. Possible to Identify environmentally modified PCBs.

10 Sensing Superfund Chemicals with Recombinant Systems

X. Guan,1 W. Luo,2 J. Feliciano,1 R. S. Shetty,1 A. Rothert,1 L. Millner,1 S. K. Deo,1 E. D'Angelo,2 L. G. Bachas,1 and S. Daunert1*

1Department of Chemistry and 2Department of Agriculture University of Kentucky Lexington, KY

11 Whole Cell Sensing System Employing Reporter Protein

Signal

Whole Optical Analytes Cell Transducer Reporter Protein Sugars GFP Metal ions Luciferases Organic pollutants: Alkaline phosphatase PCB metabolites β-Galactosidase 12 Whole Cell Sensing System Employing Reporter Protein

REPORTER PLASMID • Reporter Gene • Regulatory protein controlling the expression of the reporter gene • Specificity of the sensing element towards the analyte

13 •Whole Cell-Based Sensing Systems

Analyte Signal

Reporter protein

14 Arsenic Poisoning

Applications of Arsenic • Agriculture • Treatment for diseases • Industrial uses

New Bangladesh Disaster:Wells that Pump Poison...New York Times, November 10, 1998

Long Exposure to Low Doses of Arsenic • Skin Hyperpigmentation and Cancer C&EN November 9, 1998 • Other Cancers • Inhibition of Cellular Enzymes 15 Arsenic contamination in the USA

U. S. Geological Survey, Fact Sheet FS 063-00, May 2000

16 Schematic Representation of the Antimonite/Arsenite Pump

- - AsO2 SbO2 Cytoplasm ADP ATP AsO - ATP 3- 2 AsO4 ADP ArsA ArsA ArsC

Periplasm Membrane ArsB

- - AsO2 SbO2

17 Sensory Process in Bacteria-Based Sensing Systems

reporter gene Regulatory protein bound to promoter inducer (toxin)

reporter gene + Regulatory protein Protein Expression bound to inducer

Reporter Enzyme

Substrate Product + Signal Signal

(analyte, M)

18 Reporter Genes

Bacterial Luciferase bacterial luciferase ν(λ Decanal + FMNH2 + O2 Decanoic Acid + FMN + H2O + h max = 490 nm)

β−Galactosidase ELECTROCHEMICAL: β−galactosidase p-aminophenyl-β−galactopyranoside p-aminophenol + galactopyranoside

+ - HO NH2 O NH + 2H + 2e

CHEMILUMINESCENCE: β−galactosidase ν(λ AMPGD h max = 463 nm)

19 Dose-Response Curve for Arsenite (β-galactosidase reporter)

60000 E. coli strain AW10 arsR 50000 Induction Time: 10 min ampr

pBGD23 40000 lacZ'

30000

20000

10000 Light Intensity (counts) Light Intensity

0 -9.5 -8.5 -7.5 -6.5 -5.5 -4.5 -3.5 log (Arsenite, M) 20 Sensitivity of the Bioluminescence Sensing System

1 amol of Antimonite = 6 x 105 molecules

Bacterial density = 8 x 104 bacteria / 100 µL

Therefore, on the average each bacterium detects: 6 x 105 ≈ 7 molecules of 8 x 104 Antimonite

21 Comparison of Sensing Systems

β-Galactosidase Chemiluminescence hν Electrochemistry

Detection limit for antimonite: 10-15 M Detection limit for antimonite: 10-7 M

Bacterial Luciferase Bioluminescence hν

Detection limit of antimonite : 10-15 M 1011 - fold selective over phosphate, sulfate, arsenate and nitrate

22 4000 Sensing System for Copper Using pYEX-GFPuv 3000

2000

1000

0

Fluorescence Intensity ,(a.u.)u. -6.0 -5.0 -4.0 -3.0 -2.0 250

log (Copper sulfate, M) 200

150

Pcup1

gfpuv 100

50

pYEX-GFPuv (%)(5) Intensity Fluorescence 0 ampR leu2-d Zn2+ Co2+ Fe2+ Ni2+ Cu2+ Cd2+ Ag+ URA-3

23 In vivo Luminescence Emission of Aequorea victoria

24 ampR arsR Calibration Plot for Antimonite pSD10

gfpuv

2.5 × 105 Induction time = 30 min 2.0 × 105

1.5 × 105

1.0 × 105 (counts) 5.0 × 104

0 Fluorescence Intensity -6.5 -6.0 -5.5 -5.0 -4.5 -4.0 -3.5 -3.0 log (Potassium antimonyl tartrate, M)

25 Selectivity Studies

80000 10-5M 60000 10-6M

40000

20000

0 Fluorescence Intensity (cps) Fluorescence Intensity

- 3- 2- 3- 2- SbO2 As04 Cr2O7 Fe(CN)6 SO4

26 Fluorescent Reporter Proteins in Array Detection

Protein Excitation Emission λ λ max max

GFP 395 (470) 509 EGFP 488 509 BFP 380 440 GFPuv 395 509 YFP 513 527 CFP 433 475 CobA 357 605 RFP 558 583

27 Production of Fluorescent Porphyrinoid Compounds P H C 3 A A P

H3C N HN

oxidation NH P A P H C A N 3 A A A A P P NH HN P P UMT H3C N HN

NH HN SAM P H C A NH HN 3 A A A A A UMT P P H3C N HN P P Urogen III P SAM Dihydrosirohydrochlorin (Precorrin-2) NH N A -CH2COOH A CH3

P -CH2CH2COOH A

SAM - S-adenosyl-L-methionine P P Trimethylpyrrocorphin UMT- methyltransferase III 28 δ- in Urogen Pathway

P A P A

COOH COOH A P A P COOH NH HN NH HN ALA PBG Urogen III HO O dehydratase deaminase synthase N NH HN NH HN H P A A A H N H N 2 2

ALA PBG A P P P HMB Urogen III ALA - δ-aminolevulinic acid PBG - HMB- Hydroxymethylbilane Urogen- Uroporphyrinogen

29 Effect of δ-Aminolevulinic Acid (ALA)

10000 NO ALA NO ALA 8000 1mM ALA

6000

4000

2000 Fluorescence (cps) 0

-2000 024681012 Time (h) 30 Calibration Plot (In the Presence of δ-Aminolevulinic Acid)

45000 O/P 40000 arsR 35000 30000 pSD601 cobA R 25000 amp 20000 15000 Selectivity Study

Fluorescence (cps) 10000 40 5000 35 Induction time = 1 h Analyte concentration = 5 x 10-6 M 0 30 -8 -7 -6 -5 -4 -3 25 log (sodium-m-arsenite) 20 15 10 5 Fluorescence (cps) 0 - - - 3- 2- - - Br Cl NO3 PO4 SO4 AsO2 SbO2

31 Chlorinated Organic Compounds

OH Cl Cl Cl0-5 Cl0-5 0-5 0-4 OH-PCBs Toxic Effects Polychlorinated biphenyls (PCBs) • Carcinogenic • Neurological OH Cl0-4 OH OH • Estrogenic Cl Cl 0-5 0-3 OH PCB-diols Chlorocatechols

32 Biphenyl and 3-Chlorocatechol Catabolic Pathways

Chlorinated biphenyl 3-Chlorocatechol BphA ClcA Dihydrodiol 2-Chloromuconate BphB ClcB 2,3 diOH-ClBP 4-Carboxymethylene- 2-en-4-olide BphC Meta-cleavage compound ClcD Maleylacetic acid BphD Chlorobenzoate Chloro- dihydroxy-benzoate

33

This slide illustrates how the bph and clc pathways can relate to each other. If 3-chlorobenzoate is one of the products of the bph pathway, it can be further degraded to Kreb cycle intermediates via the clc pathway. Not all chlorobenzoates produced by the bph pathway are degraded by the clc pathway, only 3-chlorobenzoates. Other chlorinated benzoates go through other pathways (for example there is a separate pathway for 2-chlorobenzoate). I’m not sure what happens to benzoates that are highly chlorinated. I suspect they must be dechlorinated before they can be degraded, but I’m not sure. P. putida clc Operon

ClcA ClcB ClcD O HOOC COOH OH O COOH COOH O COOH Cl OH Cl - + P/O clc R clc A clc B clc D Clc R

COOH

COOH

Cl

34

Regulation of the clc operon The enzymes responsible for 3-chlorocatechol degradation are found on a plasmid isolated from Pseudomonas. The clcABD genes are transcribed as a single message, while clcR is divergently transcribed from a promoter next to the clcABD promoter (P/O). clcR codes for the regulatory protein that controls expression of both the clc ABD and clcR genes. ClcR is bound to the promoter in the presence and absence of the inducer, 2-chloromuconate. When the inducer binds to ClcR ,there is a conformational change in the progtein resulting in induction of the clcABD genes (+ above right red arrow) and inhibition of clcR gene expression (- above left red arrow). Red arrows indicate the direction of transcription. clcA gene - catechol oxygenase II (chlorocatechol dioxygenase) clcB gene - muconate cycloisomerase II clcD gene - dienelactone hydrolase Selectivity Studies 1.5

) clcR 6 clcA 10

× clcB′ pSMM50R-B′

1.0 3-chlorocatechol ampr lacZ

0.5

4-chlorocatechol

Light Intensity (counts Catechol 4-chlorobiphenyl Biphenyl 0 2-chlorophenol 4-chlorophenol -10 -9 -8 -7 -6 -5 -4 -3 log (concentration, M) plasmid courtesy of S.M. McFall & A.M. Chakrabarty 35 Response to Catechol and Chlorocatechols

5 )

5 Time of induction: 5 min 10

× 4

3-chlorocatechol 3

2 4-chlorocatechol

1

catechol Light Intensity (counts 0

-10 -9 -8 -7 -6 -5 -4 -3 -2 log (concentration, M) 36 Analysis of Chlorocatechols in Simulated Fresh Water (SFW) and Simulated Seawater (SSW)

Sample Theoretical Experimental value±SD(mg•L-1) value (mg•L-1 ) HPLC Bacterial Sensor

0.5 0.50 ± 0.01 0.51 ± 0.05 SFW1.0 0.96 ± 0.06 10 9.70 ± 0.35

0.5 0.49 ± 0.02 0.48 ± 0.03 SSW 1.0 1.03 ± 0.04 10 9.60 ± 0.52 and: Not determined 37 Analysis of Chlorocatechols in Soils HPLC Bacterial Sensor

Sample Theoretical Experimental Theoretical Experimental Value(mg•kg-1) ±SD(mg•kg-1) Value(mg•kg-1) ±SD(mg•kg-1) 0.5 0.49 ± 0.02 0.5 0.51 ± 0.02 Sand 2.0 2.03 ± 0.04 2.0 1.95 ± 0.08 10 9.75 ± 0.55 50 52.0 ± 2.6 Not detectable ± Woolper 0.5 0.5 0.52 0.04 2.0 Not detectable 2.0 1.90 ± 0.18 10 9.55 ± 0.75 50 54.0 ± 4.5 Maury 0.5 Not detectable 0.5 0.43 ± 0.08 2.0 Not detectable 2.0 2.24 ± 0.30 Organic 0.5 Not detectable 0.5 0.43 ± 0.09 Potting Soil 2.0 Not detectable 2.0 1.73 ± 0.28

38 Light Intensity (counts) 2 4 6 8 × × × × 10 10 10 10 0 5 5 5 5 Selectivity Study Selectivity 2-chlorophenol or 4-chlorophenol or 2-chlorophenol 3-chloro-benzoic acid 4-chlorobiphenyl biphenyl 4 3,4-PCB-diol 4-chlorocatechol ′ -chloro-PCB-diol 39 Field Challenges

Conventional Analytical Techniques

Field-kits

Background Signal

Viability of the cells

! Freeze Drying ! Liquid Drying

Strips (β-galactosidase) Vials (luciferase) 40 Response to Arsenite Using β-galactosidase

Test Strips

0 0.1 0.2 0.5 1 Arsenite (µM)

41 Fiber Optic Sensor

Tip of Optical Fiber Bioluminescence Dialysis Membrane Collected by Fiber

Bacteria with Induced Bioluminescence Genetically Engineered Cells Analyte

42 Microfluidic Platform

Prototype CD for Six Simultaneous Analyses

43 Calibration Plot for Antimonite in the Microfluidic Set-up

2.5 ampR arsR 2.0

pSD10 gfp 1.5 uv

1.0

0.5

0.0 Fluorescence (A.U.) -0.5 -5.5 -5.0 -4.5 -4.0 log (Potassium antimonyl tartrate, M)

44 Whole Cell Sensing Systems

• Feasible to sense Superfund chemicals with recombinant systems • Employ these whole cell systems in detection of water and soil samples • Design methods that are amenable to field studies: • Handeling of liquid and freeze dried reagents • Incorporate whole cells in a microfluidics platform • Remote sensing • Incoporation of whole cell sensing systems in fiber optic set-ups

45 Collaborators

Dr. Marc J. Madou Dr. Barry Rosen Dr. Jan Roelof van der Meer

46 Thank You

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