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Chemical & Bioassay Analyses of Chemical & Bioassay Analyses of Emissions from Biodiesel Fuel Emissions from Biodiesel Fuel

CombustionCombustionR. Kobayashi, X. Liu, C. Vogel, F. Matsumura, N. Ka doR. Kobayashi, X. Liu, C. Vogel, F. Matsumura, N. Ka do

Dept. Environmental ToxicologyDept. Environmental Toxicology

University of California, DavisUniversity of California, Davis

T. Cahill T. Cahill –– Arizona State UniversityArizona State University

California Air Resources BoardCalifornia Air Resources Board

Sacramento, CA.Sacramento, CA.

Research ObjectivesResearch Objectives

•• Study biodiesel and renewable Study biodiesel and renewable diesel emissions:diesel emissions:

•• Chemical characterization of Chemical characterization of toxicstoxics

•• Toxicity studies of emissionsToxicity studies of emissions

Unregulated Toxic EmissionsUnregulated Toxic Emissions

•• PAHsPAHs•• Alkyl Alkyl PAHsPAHs•• NitroNitro--PAHsPAHs•• Selective reactive Selective reactive aldehydesaldehydes

2

Toxicity Studies of EmissionsToxicity Studies of Emissions

•• Tests for markers of Tests for markers of inflammation in human cellsinflammation in human cells

•• Tests for Tests for genotoxicitygenotoxicity•• MutagenicityMutagenicity

•• Chromosomal DamageChromosomal Damage

Test VehiclesTest Vehicles

CARB Diesel, Soy, CARB Diesel, Soy, Animal, and Animal, and Renewable @ 20%, Renewable @ 20%, 50% and 100%50% and 100%

UDDSUDDSDOC,DOC,DPF,DPF,EGREGR

12.812.82008 Freightliner2008 FreightlinerMercedes Benz Mercedes Benz

MBE 4000MBE 4000

CARB Diesel, Soy, CARB Diesel, Soy, Animal, and Animal, and Renewable @ 20%, Renewable @ 20%, 50% and 100%50% and 100%

UDDSUDDS--15152000 Freightliner 2000 Freightliner C15 CaterpillarC15 Caterpillar

Fuels TestedFuels TestedTest Test CycleCycle

Control Control DevicesDevices

Engine Engine Displacement Displacement

(L)(L)Vehicle/EngineVehicle/Engine

MethodsMethods

Sampling System

Hi Volume Sampler

Medium Volume Sampler

3

PM Filter Preparation for PM Filter Preparation for AnalysesAnalyses

Pressurized Fluid Extraction

Hi Vol Filter 8” x 10 “ Med Vol Filter 90 mm

Pressurized Fluid Extraction

Internal std

Chemical Analyses

Bioassay Analyses

Cleanup

Bioassay Chemistry

PUF XAD Preparation for PUF XAD Preparation for AnalysesAnalyses

Pressurized Fluid Extraction

PUF for Bioassay

XAD (Chem only)

Liquid Extraction with Shaking

Internal std

Chemical Analyses

Bioassay Analyses

Pressurized Fluid Extraction

PUF for Chem

Chemical Analyses

Internal std

Cleanup Cleanup

Bioassay Chemistry

MethodsMethods

Chemical Analyses

PAHs, Alkyl PAHs Nitro-PAHs

GC/MS - EIGC/MS -NCI

4

MethodsMethods

Human Cell Markers of Inflammation

Genotoxicity

Bioassay Analyses

Chemical AnalysesChemical Analyses

• PAHs

• Alkyl PAHs

• Nitro-PAHs

Compounds AnalyzedCompounds Analyzed

Dibenzo(a,l)pyrene

Benzo(g,h,i)perylene

Dibenz(a,h)anthracene

7,12-Dimethylbenz(a)anthraceneIndeno(1,2,3-cd)pyrene

1-MethylpyrenePerylene

4-MethylpyreneBenzo(a)pyrene

6N-BaP+1N-BeP1-Methyl & 3-MethylfluoranthenesBenzo(e)pyrene

6N-chrysene2-MethylfluoranthenesBenzo(b+j+k)fluoranthenes coelute

7N-BaA2-MethylanthraceneChrysene+triphenylene

1N-pyrene1-MethylphenanthreneBenz(a)anthracene

3N-fluoranthene9-MethylphenanthrenePyrene

2N-phenanthrene2-MethylphenanthreneFluoranthene

3N-phenanthrene3-MethylphenanthreneAnthracene

9N-anthracene2,3,5-Trimethylnaphthalene coelutePhenanthrene

2N-fluorene1,6-DimethylnaphthaleneFluorene

5N-acenaphthene2,6-Dimethylnaphthalene coeluteAcenaphthene

2N-naphthalene1-MethylnaphthaleneAcenaphthylene

1N-naphthalene2-MethylnaphthaleneNaphthalene

Nitro-PAHsAlkyl PAHsPAHs

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PAH Emissions PAH Emissions PM Associated PM Associated PAHsPAHs

C15 VehicleC15 Vehicle

Draft Benzo(a)pyrene Emission

- C15 Vehicle -

0

100

200

300

400

500

600

CARB-S S20 S50S10

0

CARB-A A20 A50A10

0

CARB-R R20 R50R100

CARB A100

A100

(ng/mile)

PAH Emissions PAH Emissions PM Associated PAHsPM Associated PAHs

Draft Indeno(1,2,3-cd)pyrene Emission

- C15 Vehicle -

0

50

100

150

200

250

300

CARB-SS20 S50

S100

CARB-A A20A50

A100

CARB-R R20 R50R100

CARB A10

0A100

(ng/mile)

PAH Emissions PAH Emissions VaporVapor--Phase PAHsPhase PAHs

Draft Naphthalene Emission

- C15 Vehicle -

0

200000

400000

600000

800000

1000000

CARB-S S20 S50S10

0

CARB-A A20 A50A10

0

CARB-R R20R50

R100

CARB A100

A100

(ng/mile)

6

PAH Emissions PAH Emissions VaporVapor--Phase PAHsPhase PAHs

Draft Fluoranthene Emission

- C15 Vehicle -

0

2000

4000

6000

8000

10000

12000

14000

CARB-S S20 S50S10

0

CARB-A A20 A50A10

0

CARB-R R20R50

R100

CARB A100

A100

(ng/mile)

PAH Emissions PAH Emissions NitroNitro--PAHsPAHs

PM AssociatedPM Associated

Draft 1-Nitropyrene Emission

- C15 Vehicle -

0

200

400

600

800

1000

CARB-SS20 S50

S100

CARB-A A20 A50A100

CARB-RR20

R50R100

(ng/mile)

PAH Emissions PAH Emissions NitroNitro--PAHsPAHs

VaporVapor--PhasePhase

Draft 1-Nitronaphthalene Emission

- C15 Vehicle -

0

200

400

600

800

1000

1200

CARB-SS20 S50

S100

CARB-A A20 A50A100

CARB-RR20

R50R100

(ng/mile)

7

Summary Summary PAHsPAHsC15 VehicleC15 Vehicle

�� PM and SemiPM and Semi--Volatile Volatile PAHsPAHs and Nitroand Nitro--PAHsPAHsdecreased with increasing blend level of decreased with increasing blend level of biodiesels.biodiesels.

PAH Emissions PAH Emissions PM Associated PM Associated PAHsPAHs

MBE 4000MBE 4000

Draft Pyrene Emission

- MBE4000 Vehicle -

0

100

200

300

400

CARB S20 S50 S100

(ng/mile)

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PAH Emissions PAH Emissions VaporVapor--Phase Phase PAHsPAHs

MBE 4000MBE 4000

Draft Naphthalene Emission

- MBE4000 Vehicle -

0

5000

10000

15000

20000

25000

CARB S20 S50 S100

(ng/mile)

PAH Emissions PAH Emissions VaporVapor--Phase Phase PAHsPAHs

MBE 4000MBE 4000

Draft 2-Methylnaphthalene Emission

- MBE4000 Vehicle -

0

1000

2000

3000

4000

5000

CARB S20 S50 S100

(ng/mile)

Draft PAH Emissions Draft PAH Emissions NitroNitro--PAHsPAHsMBE 4000MBE 4000

Draft 1-Nitronaphthalene Emission

- MBE4000 Vehicle -

0

20

40

60

80

100

120

CARB S20 S50 S100

(ng/mile)

9

Summary Summary PAHsPAHsMBE 4000 VehicleMBE 4000 Vehicle

• Low Levels of PM associated PAHs and Nitro-PAHs in CARB and Biodiesel Fuel Emissions

• Lower levels of Vapor-phase PAHs and Nitro-PAHs emissions

Reactive CarbonylsReactive Carbonyls

Reactive Carbonyl Reactive Carbonyl Sampling Sampling

• Sampling from Dilution Tunnel to Mist Chamber

• Samples in parallel to Filtered/Charcoal dilution air

• Samples for single UDDS test cycle

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Reactive Carbonyl Reactive Carbonyl AnalysesAnalyses

• Stable carbonyls formed through reaction with bisulfite

• Carbonyls liberated from bisulfite

• Free carbonyls derivatized by o-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine (PFBHA*)

• Derivatives detected & quantitated by GC/MS - NCI

Carbonyl EmissionsCarbonyl Emissions

C15 VehicleC15 Vehicle

Draft Acrolein EmissionsC15 Vehicle

0

100

200

300

400

500

CARB-SS50

S100

CARB-AA50

A100

CARB-RR50

R100

Fuel

Em

issi

ons

( µµ µµg/

mile

)

Carbonyl EmissionsCarbonyl Emissions

C15 VehicleC15 Vehicle

Draft o,m-Tolualdehyde EmissionsC15 Vehicle

0

10

20

30

40

50

60

CARB-SS50

S100

CARB-AA50

A100

CARB-RR50

R100

Fuel

Em

issi

ons

(µµ µµ

g/m

ile)

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Carbonyl EmissionsCarbonyl Emissions

MBE 4000 VehicleMBE 4000 Vehicle

Draft Acrolein Emissions MBE 4000

0

20

40

60

80

100

120

140

CARB S50 S100

Fuel

Em

issi

on (

ug/m

ile)

SummarySummaryReactive CarbonylsReactive Carbonyls

• C15 Vehicle S50, S100 and A50, A100 were higher in certain carbonyls such as acrolein

• C15 Vehicle Renewable diesel no change over Carb

• MBE4000 Vehicle – carbonyls lower

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GenotoxicityGenotoxicity TestsTests

•• MicrobialMicrobialegeg. Ames Salmonella test. Ames Salmonella test

•• Mammalian cellMammalian cellegeg. Chinese hamster ovary (CHO). Chinese hamster ovary (CHO)

•• In vivoIn vivoegeg. Big Blue transgenic rodent. Big Blue transgenic rodent

GenotoxicityGenotoxicity TestsTestsTwo QuestionsTwo Questions

•• How consistent is it to hypothesized How consistent is it to hypothesized mechanisms of action for mechanisms of action for carcinogens?carcinogens?

•• How does it compare to animal or How does it compare to animal or human carcinogenicity tests?human carcinogenicity tests?

Salmonella Tester StrainsSalmonella Tester Strains

•• TA 98 TA 98

•• FrameshiftFrameshift mutation in the mutation in the HisDHisDgene coding for gene coding for histidinolhistidinoldehydrogenasedehydrogenase

•• Target site: series of 8 Target site: series of 8 GCGCGCGCGCGCGCGC’’ss

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SalmonellaSalmonella TesterTester StrainsStrains

•• TA 100 TA 100

•• BaseBase--pair mutation in the His G pair mutation in the His G gene coding for His biosynthesisgene coding for His biosynthesis

•• Target site: Target site: GGGGGG ((prolineproline) His ) His dependentdependent

Salmonella/Salmonella/microsomemicrosome TestTest

•• A feature of the Test:A feature of the Test:

•• Metabolic enzymes can be Metabolic enzymes can be added to detect activationadded to detect activation

Salmonella/Salmonella/microsomemicrosome TestTest

•• Metabolic enzymes needed for Metabolic enzymes needed for activation of certain compounds activation of certain compounds ––egeg. PAHs. PAHs

•• Enzymes from various tissues can Enzymes from various tissues can be used be used –– e.g. Lung, livere.g. Lung, liver

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MicrosuspensionMicrosuspension

DoseDose--ResponseResponse

0

500

1000

1500

2000

2500

0 0.2 0.4 0.6 0.8 1 1.2

Dose (µg/plate)

Net

Rev

erta

nts/

plat

e

Std Plate

Microsuspension

Urine Urine MutagenicityMutagenicity

Mut Res 157:227, 1985

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Draft C15 PM Mutagen Emissions TA98 +S9

0

500

1000

1500

2000

2500

3000

S-CARB

S20 S50S10

0

A-CARB

A20 A50A10

0

R-CARB

R20 R50R10

0

Fuel

Mut

agen

Em

issi

ons

(Rev

/mi x

10

3 )

Mutagen Emissions TA98 (+S9)Mutagen Emissions TA98 (+S9)C15 Vehicle PMC15 Vehicle PM

Draft C15 PM Mutagen Emissions TA98 -S9

0

500

1000

1500

2000

2500

3000

S-CARB

S20 S50S10

0

A-CARB

A20 A50A10

0

R-CARB

R20 R50R10

0

Fuel

Mut

agen

Em

issi

ons

(Rev

/mi x

10

3 )

Mutagen Emissions TA98 (Mutagen Emissions TA98 (––S9)S9)

C15 Vehicle PMC15 Vehicle PM

Draft C15 Vapor Phase Mutagen Emissions TA98+S9

0

25

50

75

100

125

150

CARB S20 S50 S100 CARB A20 A50 A100 CARB R20 R50 R100

Fuel

Mut

agen

Em

isis

on (R

ev/m

ile x

10

3 )

Mutagen Emissions TA98 (+S9)Mutagen Emissions TA98 (+S9)C15 Vehicle VPC15 Vehicle VP

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Draft C15 Vapor Phase Mutagen Emissions TA100+S9

0

50

100

150

200

250

300

CARB S20 S50 S100 CARB A20 A50 A100 CARB R20 R50 R100

Fuel

Mut

agen

Em

issi

ons

(Rev

/mile

x 1

03

)

Mutagen Emissions TA100 (+S9)Mutagen Emissions TA100 (+S9)C15 Vehicle VPC15 Vehicle VP

Draft Mutagen Emissions TA 98+S9MBE 4000 PM

0.0

2.0

4.0

6.0

8.0

10.0

CARB S20 S50 S100Fuel

Mut

agen

Em

issi

ons

(Rev

/mile

x 1

0 3)

Mutagen EmissionsMutagen Emissions

MBE 4000 Vehicle PMMBE 4000 Vehicle PM

Draft Mutagen Emissions TA 98+S9MBE 4000 Vapor Phase

0.0

2.0

4.0

6.0

8.0

10.0

CARB S20 S50 S100

Fuel

Mut

agen

Em

issi

ons

(Rev

/mile

x 1

0 3)

Mutagen EmissionsMutagen Emissions

MBE 4000 Vehicle VPMBE 4000 Vehicle VP

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Bus EmissionsBus EmissionsCompressed Natural GasCompressed Natural Gas

Mutagen Em issions TA98CBD

0.0

4.0

8.0

12.0

16.0

(+S9) (-S9) (+S9) (-S9) (+S9) (-S9) (+S9) (-S9)

CNG.1 E CD.OC E CD.CR CNG. 2

V ehicle C onf ig urat io n

Mu

tag

en E

mis

sio

n

(Rev

/mil

e) x

10

5

PM

VP

ES&T 39:7638, 2005

SummarySummary

• For C15 Vehicle: Generally decrease in Mutagen emissions with blend level

• For C15 PM Samples TA98 (+ or – S9) more sensitive than TA100 for all fuels

• Vapor Phase samples lower mutagen emissions than PM TA100 slightly more sensitive

• MBE Mutagen Emissions considerably lower than C15

SummarySummary

• Chemical and Biological tests were overall very consistent with each other regarding emission results for the fuels tested

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AcknowledgmentsAcknowledgments

California Air Resources Board – SSD, MTA/MSOD, MSCD, MLD, RD

UCR - CCERT

UCD – Etox

NESTE, SCAQMD, ARB

AcknowledgmentsAcknowledgments

California Air Resources Board – Bob Okamoto, Dean Simeroth, Bart Croes, Linda Smith, Alvaro Alvarado

CE-CERT – Tom Durbin

MTA/MSOD Staff – Keshav Sahay, Bill Robertson, Kwangsam Na, George Gatt, Thomas Ladzinski, Ralph Rodas

MSCD – Don Chernich, Tullie Flower, Wayne Sobieralski, RoelofRoelof RiemersmaRiemersma , , Mark Burnitzki

UCD – Xiaoxue Liu, Reiko Kobayashi, UkaEnkhbayar, Rupinder Kaur, Connie Chao, Brian Do, Christina La

NESTE, SCAQMD, ARB