Thesis for master degree (MD)

2010-09-13

Impact of vehicle exhaust emitted by the combustion of

biofuels on human health

by

Luiza Panosyan

A thesis submitted in partial fulfilment of the requirements for the degree

of

Master Sciences

in

Environmental Science

under the supervision of

Professor Lars-Gunnar Franzén

Final Version

Halmstad University

Halmstad 2010

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Abstract of the Thesis

INTRODUCTION: Significant changes in the global ecosystem, together with a potential shortfall in

oil resources, have stimulated intense interest in the development of other sources of energy, and

most particularly biofuels since these are basically considered to be less harmful to human health

than petroleum-based fuels. However, information about the impact of biofuel-derived vehicle

emissions on human health is limited and incomplete.

AIM OF THE STUDY: To identify those biofuels that are less detrimental to human health on the basis

of published results from toxicological and chemical studies of vehicle emission products.

TASKS OF THE STUDY: To review systematically all conventional and alternative fuels used in

internal combustion engines, to identify all known toxic emission products formed by such fuels, to

review their toxic effects on human health, and to analyse the data collected in order to develop

conclusions concerning the possible health benefits deriving from the use of alternative fuels.

MATERIALS AND METHODS: In order to fulfil the requirements of a complete, comprehensive and up-

to-date review of the toxic effects of automotive exhaust, an extensive search of official scientific

data sources has been performed. Relevant publications were retrieved from public domain

databases with a toxicological focus such as Toxcenter and CAplus, as well as from the websites of

the US Environmental Protection Agency and the US Agency for Toxic Substances and Disease

Registry. Keywords employed in the literature search were: petrol, gasoline, diesel exhaust,

emission, biofuel, biogas, biodiesel, bioethanol, bioalcohol, toxicity, methanol and ethanol. A total

of 295 references were initially selected relating to the period 1962 to 2008, and 142 of these

presented titles and abstracts that met the main inclusion criteria, i.e. describing toxicological and

epidemiological studies in humans. In cases where eligible studies relating to the goals and tasks of

the review were limited or not available, some in vitro or in vivo toxicological studies involving

animal models were included.

RESULTS: In comparison with petroleum diesel, the emissions derived from biodiesel contain less

particulate matter, carbon monoxide, total hydrocarbons and other toxic compounds including

vapour-phase C1-C12 hydrocarbons, aldehydes and ketones (up to C8), selected semi-volatile and

particle-phase polycyclic aromatic hydrocarbons (PAHs). Whilst sulphur-containing compounds

appear to be undetectable in biodiesel, nitrogen oxide and a soluble organic fraction comprising

unregulated pollutants including the ―aggregated toxics‖ (i.e., formaldehyde, acetaldehyde, acrolein,

benzene, 1,3-butadiene, ethylbenzene, n-hexane, naphthalene, styrene, toluene and xylene) are

present at elevated levels. Toxicological studies have shown that the mutagenicity of exhaust

particles from biodiesel is normally lower than those obtained from petroleum diesel, however,

rapeseed oil-derived biodiesel exhibits toxic effects that are 4-fold greater than petroleum diesel.

Such enhanced toxicity is probably caused by the presence of carbonyl compounds and unburnt

fuel. The toxicity of highly volatile components of biofuel exhaust has not yet been evaluated

accurately. A substantial portion of these compounds was apparently lost in the process of preparing

the test samples used for the assays (during the evaporation). The overall recoveries of these

compounds have not been evaluated and the accuracy of the sample preparation method has not

been validated. Hence, it could be that the cytotoxic effect of biodiesel exhaust is higher than that

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reported. Moreover, compared with fossil diesel, fuel derived from rapeseed oil emits particulate

matter with increased mutagenic effects. Epidemiological investigations of the effects of biofuels on

humans are very sparse but have revealed dose-dependent respiratory symptoms following exposure

to rapeseed oil biodiesel, although the observed differences between this fuel and petroleum diesel

are not significant. Such data, however, give rise to serious concerns about the future usage of this

plant material as a replacement for established diesel fuels. Combustion of alcohol-based fuels leads

to a reduced formation of photochemical smog in comparison with gasoline or diesel, however, the

emission of aldehydes (officially classified as carcinogenic or potentially carcinogenic) is several

times higher. The toxicity of the exhaust emissions of gasoline-fuelled engines is generally

significantly greater than that of alcohol-burning engines. However, some harmful effects from

ethanol blends might be expected, such as enhanced emissions of carcinogenic PAHs and increased

ozone-related toxicity associated with the high level of aldehydes emitted. The use of ethanol–diesel

fuel blends gives rise to increases in regulated exhaust emissions and, possibly, to greater emissions

of aldehydes and unburnt hydrocarbons. The most promising fuels, in terms of reduced toxicity and

genotoxicity of exhaust emissions, are methanol-containing blends. However, the emission from

these fuels still contains formaldehyde, which is a carcinogen. The use of biogas can significantly

reduce emissions of total PAHs and formaldehyde and, consequently, the risk of lung toxicity. On

the other hand, the emissions of particulate matter by compressed natural gas, and the mutagenic

potencies of the exhaust, are similar to those associated with gasoline and diesel fuels.

CONCLUSIONS: The use of biofuel is currently viewed very favourably and there are suggestions that

the exhaust emissions from such fuel are less likely to present risks to human health in comparison

with gasoline and diesel emissions. However, the expectation of a reduction in health effects based

on the chemical composition of biodiesel exhaust is far from reality. Thus, although toxicological

evidence relating to the effects of biofuels on humans is sparse, it is already apparent that emissions

from the combustion of biofuel and blends thereof with petroleum-based fuels are toxic. In addition

to the regulated toxic compounds, such as total hydrocarbons, carbon monoxide, nitrogen oxides,

particulate matter and polycyclic aromatic hydrocarbons, biofuel emissions contain significant

amounts of various other harmful substances that are not regulated, e.g. carbonyls (including

formaldehyde, acetaldehyde, benzene, 3-butadiene, acrolein, etc.). Whilst biofuels may be

potentially less damaging to human health than petroleum fuels, considerable harmful effects must

still be expected. Substitution of conventional fuel by biofuel decreases the concentration of

regulated toxic pollutants in vehicle exhaust, but increases the concentration of some unregulated

toxic pollutants emitted from on-road engines. Generally, the toxicity of biofuels decreases in the

order biodiesel>biogas>ethanol>=methanol. In this respect, methanol produced by the oxidation of

biogas appears to represent an alternative fuel that exhibits the least potential for damage to human

health, however, this alcohol represents a source of formaldehyde pollution and is carcinogenic. .

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Table of Contents

Abstract of the thesis 2

Table of contents 4

1. Abbreviations 6

2. Introduction 7

2.1. Background 7

2.1.1. Definition of the problem 7

2.1.2. Research questions of the study 7

2.2. Automotive fuels 7

2.2.1. Conventional fuels 7

2.2.2. Alternative fuels 8

2.2.2.1. Vegetable oils 9

2.2.2.2. Biodiesel 10

2.2.2.3. Bioalcohols 10

2.2.2.4. Biogas 11

3. Aim of the study 12

4. Tasks of the study 12

5. Materials 12

5.1. Research strategy 12

5.2. Literature sources 13

6. Methods 14

6.1. Inclusion and exclusion criteria 14 6.2. Study selection 14

6.3. Data abstraction 15

6.4 Data synthesis and analysis 15

7. Results 15

7.1. Toxic compounds emitted from the exhaust systems of motor vehicles 16

7.1.1. Regulated pollutants 17

7.1.1.1. Particulates 19

Total hydrocarbons 20

Polyaromatic hydrocarbons 21

7.1.1.2. Carbon monoxide 22

7.1.1.3. Nitrogen oxides 22

7.1.1.4. Sulphur oxides 23

7.1.1.5. Heavy metals 23

7.1.1.6. Ozone 24

7.1.2. Unregulated pollutants 24

7.1.2.1. Carbonyl compounds 24

Formaldehyde 24

Acetaldehyde 25

Acrolein 25

7.1.2.2. Other oxygenated hydrocarbons in exhaust emissions 26

Methanol 26

Ethanol 26

Formic acid 26

7.1.2.3. Hydrocarbons in exhaust emissions 27

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Benzene. 27

1,3-Butadiene. 27

7.2. Acute and chronic adverse health effects of vehicle exhaust produced by

the combustion of petrofuels, gasoline and diesel 27

7.2.1. Acute and chronic adverse health effects 31

7.2.2. Carcinogenic effects 31

7.2.3. Mutagenic effects 32

7.3. Chemical composition and toxic effects of vehicle exhaust produced by the

combustion of biofuels 32

7.3.1. Biodiesel and vegetable oils 32

7.3.1.1. Chemical composition of the emissions from biodiesel engines 32

7.3.1.2. Biodiesel exhaust emissions and human health 37

7.3.2. Bioalcohol 41

7.3.2.1. Composition of bioalcohol exhaust. 41

7.3.2.2. Toxicological studies of exhausts from bioethanol and alcohol-

blended fuels. 45

Ethanol-based blends 45

Methanol-based blends 47

7.3.3. Biogas 48

7.3.4. Other biofuels 49

8. Discussion and overall conclusions 50

9. Acknowledgements 52

10. References 52

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1. Abbreviations

ALA-D – 9-aminolevulinic acid dehydratase

ANOVA – analysis of variance

ATSDR - US Agency for Toxic Substances and Disease Registry

BaPeq – benzo(a)pyrene equivalents

BFC – brown-fat-cell

CNG - compressed natural gas

CNS – central nervous system

DEP - diesel exhaust particles

DPM - diesel particulate matter

GTL- gas-to-liquid

GWP - global warming potential

HSDB – Hazardous Substances Data Bank

IARC – International Agency for Research on Cancer

LFG - lead-free gasoline

LPG - liquefied petroleum gas

LS-PDD – low sulphur content petroleum-derived diesel

MCA – methanol-containing additive

NOx – nitrogen oxides

PAC - polynuclear aromatic compounds

PAH - polyaromatic (polycyclic) hydrocarbons

PAN - peroxyacyl nitrates

PDD - petroleum-derived diesel

PLG - premium leaded gasoline

PM - particulate matter

RFG - reformulated gasoline

RME - rapeseed oil methyl esters

RSO - rapeseed oil

SME - soybean oil methyl esters

SOx – sulphur oxides

TCDD – 2,3,7,8-tetrachlorodibenzo-p-doxin

US EPA - US Environmental Protection Agency

WHO – World Health Organization

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2. Introduction

2.1. Background

2.1.1. Definition of the problem

There are several significant problems associated with the current interest in biofuels:

Problem 1. Significant changes in the global ecosystem first became apparent in the 20th

century

and are mainly related to the enormous impact of the petrochemical industry on the ecological

balance.

Problem 2. Oil resources are limited and the depletion mid-point (the date at which roughly

half of anticipated global oil resources have been exploited) will be reached within the next 10

to 20 years). Possible solutions to the future potential energy shortfall involve the greater use of

biofuels together with wind, solar, tidal, geothermal and hydro-power, and other renewable

energy sources. If alternative energy sources are not exploited effectively, it is likely that the

price of crude oil will increase exponentially with a doubling time of 11 years (Munack and

Krahl, 2007).

Problem 3. Permanent dependence on supplies of oil and gas from countries such as Iran, Iraq,

Russia, Venezuela, Libya etc could generate shifts in the balance of world power that may be

unacceptable to the main energy consumers.

The benefits that could derive from the use of biofuels include:

reduction in greenhouse gas emissions,

reduction in the amounts of fossil fuel consumed,

increased rural development,

decreased dependence on politically sensitive regions for supplies of energy,

a sustainable fuel supply for the future.

Although such ideas currently dominate public thinking, the hard information available concerning

the impact of biofuel-derived vehicle emissions on human health is limited and incomplete.

2.1.2. Research questions of the study

What is the impact of biofuel-derived vehicle emissions on human health?

It is generally believed that biologically-derived products are less harmful to the human organism

than industrially-generated chemicals. Similarly, biofuels are believed to be less toxic than gasoline

or diesel and, therefore, biofuels derived vehicle emissions should considered to be less damaging

than those derived from petroleum. The validity of this generally-held belief still remains open to

question, however, and a systematic assessment of the comparative safety/toxicity of biofuel-

derived emission is urgently required.

2.2. Automotive fuels

2.2.1. Conventional fuels

Currently, the main source of fuel for road vehicles is petroleum oil. Oil is a naturally occurring,

fossil liquid comprising a complex mixture of hydrocarbons of various molecular weights together

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with various other organic compounds. These components are separated by fractional distillation in

an oil refinery in order to produce gasoline, diesel fuel, kerosene and other products, namely:

hydrocarbons containing from 5 to13 carbon atoms are refined into gasoline (petrol),

hydrocarbons with 9 to 16 carbons are processed to yield diesel fuel and kerosene (the

primary component of many types of jet fuel),

hydrocarbons with 17 to 25 carbons form the basis of fuel oil and lubricating oil,

higher molecular weight hydrocarbons (typically with 35 or more carbons) can either be

used to produce asphalt or may be cracked in modern refineries to provide more valuable

products,

hydrocarbons with less than 5 carbon atoms are normally gaseous and are processed as

natural gas (Speight, 2006).

Gasoline, or petroleum spirit (petrol), is a complex mixture consisting of mainly aliphatic

hydrocarbons [C5-C13 n-alkanes (17.7%) and C4-C13 branched alkanes (32%), C6-C8 cycloalkanes

(5%), C6 olefins (1.8%), aromatics (benzene, toluene, xylene, ethylbenzene, C3-benzenes, C4-

benzenes (30.5%)] and other possible components such as octane enhancers (MTBE, TBA, ethanol,

methanol), antioxidants, metal deactivators, ignition controllers, icing inhibitors, detergents and

corrosion inhibitors (Harper and Liccione, 1995).

Diesel, or diesel fuel, refers in general to any fuel that can be used in a diesel engine. Petroleum-

derived diesel (PDD) is composed of ca. 64-75% saturated hydrocarbons (primarily paraffins

including n-, iso-, and cyclo-paraffins), l-2% olefinic hydrocarbons and 25-35% aromatic

hydrocarbons (including naphthalenes and alkylbenzenes) (Agency for Toxic Substances and

Disease Registry, 1995; Briker et al., 2001). Although the average chemical formula for common

diesel fuel is C12H23, components range from approximately C10H20 to C15H28.

2.2.2. Alternative fuels

Biofuels are defined as solid, liquid, or gaseous fuels derived mainly from recently dead biological

materials. This distinguishes biofuel from fossil fuel, which is derived from long dead biological

material. Biofuel can theoretically be produced from any biological carbon source, however, the

most common sources of biofuels are photosynthetic plants and plant-derived materials. There are

three main strategies to obtain biofuels (Olsson and Ahring, 2007; Worldwatch Institute, 2007;

Mousdale, 2008):

to grow plants that naturally produce oils, such as corn, switchgrass and soya (in the United

States), rapeseed, wheat and sugar beet (in Europe), sugar cane (in Brazil), palm oil and

Miscanthus (in South-East Asia), sorghum and cassava (in China), and Jatropha (in India).

Since the viscosities of these oils diminish with heating they can be burnt directly in a diesel

engine, although the oils can be treated chemically in order to produce biodiesel,

to grow crops containing high levels of sugar (sugar cane, sugar beet and sweet sorghum) or

starch (corn/maize) and subsequently ferment the biomass with yeast in order to produce

bioalcohols (mainly ethanol),

to harvest biomass and submit it to anaerobic digestion in order to produce biogas (mainly

methane). Such biomass can, in fact, derive from almost any form of waste organic material.

The world leaders in the development and use of biofuels are, at the time of writing, Brazil, USA,

France, Sweden and Germany.

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First generation biofuels refers to biofuels made from:

seeds that are pressed to yield vegetable oil for use as biodiesel,

grains that yield starch for fermentation into bioethanol,

natural waste materials for anaerobic degradation to biogas.

The most common first generation biofuels are listed below.

2.2.2.1. Vegetable oils. The use of a plant oil as a fuel for a compression ignition (diesel) engine

dates back to the very early years of the invention of the devise itself. Indeed the first engine of this

type presented by Diesel at the world exhibition in Paris in the year 1900 was fuelled by peanut oil.

Vegetable oil alone is, however, too viscous for continuous use in the diesel engine (Knothe, 2005),

although it can be used in many older diesel engines (equipped with indirect injection systems) but

only in warm climates.

The composition of vegetable oil is quite different from that of petrol-derived fuels, the major

constituents being lipid triglycerides (triacylglycerols). The triglycerides in native fats or oils

comprise a glycerol backbone, the –OH groups of which are esterified with palmitic acid, oleic acid,

α-linolenic acid, etc (Kates, 1972). The triglycerides of sunflower oil comprise predominantly

linoleic acid (68%) together with palmitic, stearic and oleic acids (Table 1) (Somerville, 1993;

Krahl, 2009). It should be noted, however, that there can be significant variation, strongly

influenced by both genetics and climate, in the unsaturated fatty acid profile of the oil. The

vegetable oil also contains minor amounts of lecithin, tocopherols, waxes (long chain alkanes,

alcohols and their esters), carotenoids, phytosterols (e.g. stigmasterol, β-sitosterol, campesterol etc),

acyl-phytosterols, diacylglycerols, monoacylglycerols, glycolipids, glyceroglycolipids, free fatty

acids and other minor non-polar lipid fractions (Kates, 1972).

Table 1. Main fatty acids present in the triglycerides of vegetable oils used as biofuels.

(Table from Krahl, 2009).

a Very small amounts of 20:2 (0.08%), 22:0 (0.34%) and 22:1 (0.29%) are also present (Krahl,

2009)

Vegetable

oil

Fatty acid composition (number of carbon atoms; number of double bonds)

6:0 8:0 10:0 12:0 14:0 16:0 18:0 20:0 16:1 18:1 20:1 18:2 18:3

Rape seed a 0.06 4.62 1.7 0.6 0.25 61 1.3 20.4 9.2

Soybean 11 4 34 54 7

Corn 11 2 28 58 1

Cotton seed 1 22 3 1 19 54 1

Palm 1 45 4 40 10

Peanut 11 2 1 1 48 2 32

Olive 13 3 1 1 71 10 1

Canola 4 2 62 22 10

Sunflower 7 5 19 68 1

Coconut 1 8 6 47 18 9 3 6 2

Palm kernel 3 4 48 16 8 3 15 2

Cocoa 26 34 1 34 3

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For all of the vegetable oils mentioned, each lipid fraction comprises several homologues

containing different numbers of C=C bonds (usually from 0 to 3) separated by methylene (CH2)

groups. In the presence of high temperatures or light, these moieties readily oxidize to yield

hydroperoxides which, in turn, are transformed into a plethora of autoxidation products that are

potentially toxic to living organisms (Kates, 1972).

2.2.2.2. Biodiesel. Combustion of crude vegetable oils causes severe damage to normal diesel

engines, but such oils can be used to manufacture biodiesel that is compatible with most diesel

engines when blended with conventional diesel fuel. Currently biodiesel and PDD blends are used

in unmodified diesel-engined vehicles. Typically, biodiesel comprises fatty acid methyl or ethyl

esters produced by trans-esterification of vegetable oil (Figure 1).

Figure 1. Transformation of triglycerides of vegetable oil into biodiesel and glycerol.

In this process the plant oil is reacted with an alcohol, generally methanol, in the presence of a

catalyst such as potassium hydroxide to produce the corresponding ester and glycerol (Schuchardt

et al., 1998). Methyl esters are less dense than glycerol and can be readily separated from the latter

on the basis of differential density. The glycerol by-product is removed from the bottom of the

reaction vessel and can be further refined for use in the pharmaceutical or cosmetic industries. Feed

stocks for the production of biodiesel include animal fats, algae and various vegetable oils such as

soy, mustard, flax, sunflower, palm, hemp, field pennycress, Jatropha and Madhuca.

2.2.2.3. Bioalcohols. Alcohol fuels can be obtained from petroleum-based or biological sources,

although when they are derived entirely from the latter they are referred to as bioalcohols (e.g.

bioethanol). Methanol, ethanol, propanol and butanol (alcohols with the general formula CnH2n+1OH

where n = 1, 2, 3 or 4) are of interest as fuels because they can be synthesised biologically and they

have characteristics that allow them to be used in unmodified engines. Bioalcohols are produced by

the fermentation of sugars or starch present in wheat, corn, sugar beet, sugar cane, molasses or, in

fact, any carbohydrate-containing material (such as potato and fruit waste, etc) from which

alcoholic beverages can be formed. Typically, the production of ethanol employs enzymatic

digestion in order to release sugars from stored starches, followed by fermentation of the sugars,

distillation and drying (Worldwatch Institute, 2007; Mousdale, 2008). The presence of trace

amounts of many other fermentation products including alcohols with 3 to 12 carbon atoms as well

as numerous fusel oils (ethyl esters of short chain fatty acids; Table 2) varies appreciably depending

on the feedstock employed as well as different processing factors.

BIODIESEL

VEGETABLE OIL COOCH3

COOCH3

COOCH3CH2OH

CH2OH

CHOH+

CH3OH KOH

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glucose pyruvate acetaldehyde ethanol

Table 2. Composition of volatile compounds found in the drinking ethanol. (Utsunomiya et al.,

2010)

Component Component

Methanol Diethyl Succinate

1-Propanol Ethyl-9-decenoate

Isobutanol Ethyl Undecanoate (C11Ethyl)

Isoamylalcohol 1-Decanol

Ethyl acetate Phenylethyl acetate

Isoamyl acetate Ethyl Dodecanoate (C12Ethyl)

Ethyl Hexanoate (C6Ethyl) 2-Phenylethanol

Ethyl Octanoate (C8Ethyl), 1-Dodecanol

Isoamyl Hexanoate (C6Isoamyl) Ethyl Myristate (C14Ethyl)

Furfural Octanoic acid (C8)

Ethyl Nonanoate (C9Ethyl) 1-Tetradecanol

1-Octanol Ethyl Palmitate (C16 Ethyl)

Ethyl Decanoate (C10Ethyl) Ehtanol

The combustion of ethanol follows the following equation: C2H5OH + 3O2 → 2CO2 + 3H2O + heat.

Ethanol can be used in petrol engines as a replacement for gasoline, and can also be mixed with

gasoline in a range of percentages. Most existing automotive petrol engines can run on blends, E85

(15% gasoline, 85% ethanol) is the highest ethanol/gasoline blend that existing flex-fuel vehicles in

the United States can use today [Ginnebaugh et al., 2010].

Methanol is currently produced from natural gas, but it can also be produced from biomass as

biomethanol. Methanol has been proposed as a future biofuel since, compared with ethanol, it has a

primary advantage in that it provides very high ―well-to-wheel‖ efficiency when produced from

synthetic gas. The combustion of methanol follows the equation: 2CH3OH + 3O2 → 2CO2 + 4H2O

+ heat. However, both propanol and butanol are considerably less toxic and less volatile than

methanol. Butanol, for example, has a high flashpoint of 35°C, which is a benefit for fire safety but

may present a difficulty for starting engines in cold weather.

2.2.2.4. Biogas. Biogas is the gas resulting from the breakdown of organic matter by

microorganisms in the absence of oxygen. Biogas produced by anaerobic fermentation of manure,

sewage, municipal waste or energy crops normally contains methane (50-75%) and CO2 (25-50%),

with nitrogen (0.1-10%) as the principal minor constituent. Varying amounts of hydrogen, hydrogen

sulphide, oxygen, ethane, propane, iso-butane, n-butane, and other hydrocarbons can be detected as

is shown in the chromatogram displayed in Figure 2 (Schumacher and Thompson, 2005).

Polysaccharide

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Figure 2. Gas chromatogram of natural gas. Each of the peaks represents one (or possibly more)

compound, and the intensity of the peak provides a rough indication of the relative amount. (Figure

taken from Schumacher and Thompson, 2005)

3. Aim of the study

The aim of the present study was to determine which biofuels are less harmful to humans by

comparing the published results of chemical studies and toxicological investigations of the emission

products of road vehicles.

4. Tasks of the study

Task 1 was to review all conventional and alternative fuels presently in use in internal

combustion-engined vehicles together with those likely to be used in the future (see section

2.2),

Task 2 was to identify all known toxic compounds present in fuel emission,

Task 3 was to review the toxic effects of such compounds on human health,

Task 4 was to analyse the collected data in order to draw conclusions concerning the

possible health benefits of using alternative fuels.

5. Materials

5.1. Research strategy

The following steps were undertaken in order to perform the thorough search of the scientific

literature required to meet the stated objective:

Step 1. An initial literature survey was conducted in order to understand:

the background of the problem – why conventional fuels need to be replaced by alternative

fuels,

the chemical nature of fossil-derived oils and alternative biofuels,

the chemical nature of compounds generated and emitted into the atmosphere as a result of

the combustion of fuels in the automobile engine.

Dete

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resp

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se

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Step 2. A literature search was performed in order to define the nature of the harmful effects

generated by the principal toxic substances identified in vehicle exhausts.

Step 3. A review of the findings of toxicological and epidemiological studies on the effects of

vehicle exhausts on human health was conducted in order to establish possible differences in

harmful effects between petroleum-derived fuels and biofuels, and among the different types of

biofuels.

The bibliographies of the articles highlighted by the initial computer-assisted search were studied in

detail and any relevant studies cited were considered for inclusion in the data base assembled for the

present review.

5.2. Literature sources

In the introductory step 1, Google and the Google book engine, e.g.

http://books.google.com/books?cd=1&q=&btnG=Search+Books, websites were surveyed in order

to gain a rapid understanding of the current classification of fuels, their chemical compositions, and

the recent trends in the development of new energy sources. The keywords used were: petrol,

gasoline, biofuel, biogas, biodiesel, bioethanol and bioalcohol. As a result, a number of books on

chemistry, analysis and technology of fuels, oils and biofuels were used (Speight, 2006; Kates,

1972; Yinon, 2004, 2008; Olsson and Ahring, 2007; Worldwatch Institute, 2007) together with

various articles concerning the chemical composition of fuels that were located on the internet using

the keywords: fuel oil, chemical composition, GC analysis, diesel and gasoline (Agency for Toxic

Substances and Disease Registry – ATSDR, 1995a; Schumacher and Thompson, 2005).

In order to fulfil the requirements of a complete and comprehensive up-to-date review of the toxic

effects of automotive exhaust, an extensive search of official scientific data sources was performed.

Relevant publications were retrieved from public domain data bases with a toxicological focus,

namely, Medline, Toxcenter (http://toxnet.nlm.nih.gov/) and CAplus

(http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed; http://stneasy.fiz-

karlsruhe.de/html/english/login1.html). The keywords used in this stage of the literature search

were: petrol, gasoline, diesel exhaust, emission, biofuel, biogas, biodiesel, bioethanol,

bioalcohol, toxicity, methanol and ethanol.

The website of the US Environmental Protection Agency (US EPA)

(http://www.epa.gov/epahome/quickfinder.htm) provided facile access to relevant information and

official summaries on toxicity and regulatory issues associated with toxic compounds. Thus the

health effects of formaldehyde (one of the major toxins of biogas and biomethanol) can be found at

this URL, whilst complete information on its health assessment and a comprehensive review of

chronic toxicity data can be viewed at http://www.epa.gov/iris/subst/0419.htm. A further sources of

information concerning the toxicity of auto-exhaust compounds and their carcinogenic activity are

the websites of the US Agency for Toxic Substances and Disease Registry (ATSDR;

http://www.atsdr.cdc.gov/toxprofiles/index.asp; http://www.atsdr.cdc.gov/substances/index.html)

and International Agency for Research on Cancer (IARC) - http://monographs.iarc.fr/index.php

where all monographs released by WHO expert committees summarising the results of evaluation

carcinogenic activity of substances are described in details. The US Department of Health and

Human Service Report on the Toxicological Profile of Polyaromatic Hydrocarbons published in

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1995 was accessed at http://www.atsdr.cdc.gov/toxprofiles/tp69.pdf (Agency for Toxic Substances

and Disease Registry, 1995b).

The key words employed in these searches were: methanol, ethanol, formaldehyde, carbon

monoxide, acetaldehyde, acrolein and polyaromatic hydrocarbons.

6. Methods

6.1. Inclusion and exclusion criteria

The only articles included in the present review were those describing: (i) the results of

toxicological studies of biofuel and petrofuel emissions, (ii) health effects of toxic compounds

found in biofuel and petrofuel emissions, (iii) the content of potentially toxic compounds in

petrofuel and biofuel emissions. Numerous publications covering many other issues related to

petrofulel emission and air pollution were excluded. Articles in Chinese, Russian and any other

languages except of English were excluded.

6.2. Study selection

A total of 218 articles published between 1962 and 2009 were initially selected from the Medline

database, although a number of these were duplicated in other databases. A more specific search in

the Toxcenter database using the keywords: biofuel, exhaust emission, biogas and bioethanol

produced 25 references published between 1984 and 2008. A search of the CAPlus database using

the keywords: biofuel, exhaust emission, biogas and bioethanol yielded 42 references published

between 1975 and 2008, whilst a similar search in the Hazardous Substances Data Bank (HSDB)

database (http://toxnet.nlm.nih.gov/cgi-bin/sis/search) gave 12 references dated between 2002 and

2008.

Flow chart of the study selection process

Total number of citations identified through database searching: n=297

Additional references selected for the reviewing: n=16

Total number of references included

in the review, n=146

Number of articles assessed for eligibility: n=113, including:

articles selected for the review of health effects of individual toxic compounds emitted

from petrofuels (sections 7.1 and 7.2) : n = 46, including full text articles - 27

articles selected for the systematic review of biofuels (section 7.3) : n=67, including full

text articles - 27

Citations excluded after screening of titles and abstracts as

well as after removal duplicates: n = 167

- 15 -

In order to ensure the validity of the information provided in the articles selected, only official

reports released by governmental regulatory organisations and papers published in peer-reviewed

journals were considered. Of the references selected, 142 presented titles and abstracts that met the

main inclusion criteria, i.e. toxicity and epidemiological studies in humans, etc. In cases where

eligible studies relating to the goals and tasks of the review were limited or not available, some in

vitro or in vivo toxicological studies involving animal models were also included. All selected

articles were incorporated into the next step of the selection process.

6.3. Data abstraction

Data, including the details of the chemical composition, the content of potentially toxic compounds

from petrofuel and biofuel emissions, and results of toxicological studies were extracted using

predefined criteria. For each eligible article, information related to the task and aim of the study was

abstracted, with particular attention being given to any possible benefits of a biofuel in terms of

reduced toxicity to human health. For this comparison, comprehensive reviews entitled ―Health

Assessment Document for Diesel Engine Exhaust‖ (US EPA, 2002a), ―A Comprehensive Analysis of

Biodiesel Impacts on Exhaust Emissions‖ (US EPA., 2002b), ―Health Assessment Document for

Diesel Engine Exhaust‖ (US EPA, 2002a), and ―Recent Advances in Investigations of Toxicity of

Automotive Exhaust” (Stupfel, 1976), in which the toxic effects of gasoline and diesel engine

exhausts on human health were systematically evaluated, were considered to be of particular value.

A number of references cited in these articles are included in the present treatise by virtue of their

importance in the assessment of comparative toxicity of alternative and conventional biofuels.

At an early stage in the review process it became clear that, regardless of the origin of the fuel (i.e.

fossil or biological), combustion produced essentially the same toxic compounds but in different

ratios and amounts, particularly when the fuels were blends of diesel and biodiesel or gasoline and

ethanol. This finding influenced further data abstraction and analysis of publications, and led to a

focus on the toxicity of regulated and unregulated pollutants. Thus, several of the most important

studies concerning the mutagenic effect of biofuels are considered in some detail in the foregoing

discussion.

6.4. Data synthesis and analysis

The most relevant parts of selected articles containing specific information comprising the complete

body of evidence described in Section 7 (Results), were analysed and summarized in tables as well

as in the form of conclusions at the end of every sub-section, and finally at the end of Section 8

(Discussion and overall conclusions). The final conclusions are unexpectedly alerting, and certainly

not in accord with current public opinion regarding the harmless nature of biofuels.

7. Results

The main fuels currently used in road vehicles are petrol, diesel and fossil gas. The strategy of

substituting these fuels by alternative fuels from renewable sources such as biodiesel, bioalcohols

and biogas in order to reduce traffic emissions is very attractive, but needs to be supported by

rigorous scientific evidence verifying the reduced effect of such emissions on human health.

- 16 -

Since biofuel is chemically quite different from petrol it is possible that the chemical compositions

of the emissions derived from these two fuels vary and, consequently, present diverse impacts on

human health. This was the basic hypothesis of the present study, the objective of which was to

identify fuels that exhibit the least harmful effects on human health. However, in the course of the

review process it became obvious that the combustion of petrofuel and biofuel produced essentially

the same toxic compounds but in different ratios and amounts (sections 7.2.-7.4), particularly when

the fuels were blends of diesel and biodiesel or gasoline and ethanol.

There follows a short description of the major pollutants emitted from the engine and exhaust

system of internal combustion engines.

7.1. Toxic compounds emitted from the exhaust systems of motor vehicles

Primary emissions are a complex mixture containing hundreds of organic and inorganic constituents

Table 3. Chemical composition of diesel exhaust. (Table modified from US EPA, 2002a).

Particulate phase Gas phase

Heterocyclics, hydrocarbons (C14-C35), and Heterocyclics, hydrocarbons (C1-C10), and

PAHs and derivatives: derivatives:

Acids Cycloalkanes Acids Cycloalkanes, Cycloalkenes

Alcohols Esters Aldehydes Dicarbonyls

Alkanoic acids Halogenated cmpds. Alkanoic acids Ethyne

n-Alkanes Ketones n-Alkanes Halogenated cmpds.

Anhydrides Nitrated cmpds. n-Alkenes Ketones

Aromatic acids Sulphonates Anhydrides Nitrated cmpds.

Quinones Aromatic acids Sulphonates

Quinones

Elemental carbon Acrolein

Inorganic sulphates and nitrates Ammonia

Metals Carbon dioxide, carbon monoxide

Benzene

1,3-Butadiene

Formaldehyde, formic acid

Hydrogen cyanide, hydrogen sulphide, methane, methanol

Nitric and nitrous acids

Nitrogen oxides, nitrous oxide, sulphur dioxide

Toluene

- 17 -

Table 4. Major components of gas phase diesel engine emissions, their known atmospheric

products and the biological impact of such products. (Table modified from US EPA., 2002a).

Gas phase Atmospheric reaction product Biological impact

CO2 - Global warming

CO - Highly toxic, blocks oxygen

uptake

NOx Nitric acid Irritates respiratory tract

SO2 Sulphuric acid Irritates respiratory tract

C1-C17 Alkanes Aldehydes, ketones, alkyl nitrates Irritates respiratory tract

C1-C3 Alkenes Aldehydes, ketones Carcinogenic and mutagenic

Formaldehyde Carcinogenic

Higher aldehydes (e.g.

acetaldehyde, acrolein)

CO, hydroperoxyl radical Irritates respiratory tract

Monocyclic aromatic

compounds (benzene,

toluene, xylene)

Hydroxylated and

hydroxylated nitro derivatives

Carcinogenic and mutagenic

PAH Nitro-PAH Carcinogenic and mutagenic

Nitro-PAH Quinones and hydroxylated nitro

derivatives

Mutagenic

Some compounds that are initially emitted in the exhaust gases may be wholly or partially

transformed in the atmosphere into other toxic substances, as outlined in Tables 4 and 5 (US EPA,

2002a).

Table 5. Major components of particulate phase diesel engine emissions, their known atmospheric

products and the biological impact of such products. (Table modified from US EPA., 2002a).

Particle phase Atmospheric reaction product Biological impact

Elemental carbon - Absobs deep into the lung

Sulfate and Nitrate - Irritation of respiratory tract

C14-C35 Hydrocarbons Aldehydes, ketones, alkyl nitrates ?

PAH Carcinogenic and mutagenic

Nitro PAH Carcinogenic and mutagenic

7.1.1. Regulated pollutants

In the USA, the Clean Air Act (US EPA, 2006). requires the US EPA to set National Ambient Air

Quality Standards for the six principal pollutants defined in Table 6. These so-called "criteria"

pollutants are considered to be harmful to public health and to the environment, and their levels are

restricted according to the following criteria:

Primary standards set limits to protect public health, especially the health of "sensitive"

populations such as asthmatics, children, and the elderly.

Secondary standards set limits to protect public welfare, including protection against

decreased visibility, damage to animals, crops, vegetation, and buildings (US EPA, 2006).

- 18 -

Table 6. The National Ambient Air Quality Primary Standards (Table modified from National

Ambient Air Quality Standards (NAAQS, 1990)

Pollutant Level Averaging Time

Carbon monoxide 9 ppm (10 mg/m3) 8 h

35 ppm (40 mg/m3) 1 h

Lead 1.5 µg/m3 Quarterly average

Nitrogen dioxide 0.053 ppm (100 µg/m3) Annual (arithmetic mean)

0.100 ppm 1 h

Particulate matter (PM10) 150 µg/m3 24 h

Particulate matter (PM2.5) 15.0 µg/m3 Annual (arithmetic mean)

35 µg/m3 24 h

Ozone 0.075 ppm (2008 std) 8 h

0.08 ppm (1997 std) 8 h

0.12 ppm 1 h

Sulphur dioxide 0.03 ppm Annual (arithmetic mean)

0.14 ppm 24 h

Figure 3. Typical diameters of particulate matter in relation to the size of grains of fine beach sand

and the diameter of a human hair. (Figure taken from US EPA, 2006).

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7.1.1.1. Particulates. Both Otto and Diesel engines emit large quantities of solid or liquid

material suspended in particulates. Such particulates, which are often referred to as particulate

matter (PM), are of micron size and can coagulate rapidly. As shown in Figure 3, PM typically

comprises ―fine particles" with diameters ≤ 2.5 µm (PM2.5), and ―inhalable coarse particles" with

diameters in the range 2.5 – 10 µm (PM10). The larger particles are less harmful than their smaller

counterparts since they present a reduced surface area for the absorption of toxic compounds.

Additionally, in the less likely event that larger particles were to enter the human body, they would

be eliminated more rapidly than particles with a smaller diameter. US EPA regulations refer only to

exhalable particles with diameters < 10 µm.

Core PMs (i.e. soot and black smoke) are formed by nucleation and coagulation of primary

spherical particles comprising solid carbonaceous material (mainly elemental carbon) and ash (trace

metals and other elements). Organic and sulphur-containing compounds (i.e. sulphate) may add to

these particles through coagulation, adsorption and condensation, and ultimately combine with

other condensed materials (Figures 4 and 5). In this manner, black carbon cores may adsorb known

or suspected mutagens and carcinogens, e.g. polycyclic aromatic compounds (PAC) as organic

phase (US EPA 2002a). Indeed, organic solvent extracts of particulate matter have been shown to

exhibit mutagenic activity in various in vitro bacterial and mammalian assays including the

Salmonella typhimurium mammalian microsome assay. The direct mutagenic effect of extracts of

particulate matter has been reported in many studies), and is mainly associated with substituted

polyaromatic hydrocarbons (PAH) and particularly with nitrobenzanthrones (US EPA 2002a).

Figure 4. Schematic diagram of typical PMs derived from diesel engine exhaust. (Figure taken

from US EPA, 2002a).

- 20 -

Figure 5. Chemical composition of 2.5 μm diameter PMs derived from diesel engine exhaust.

(Figure taken from US EPA, 2002a).

The region of the respiratory tract where the inhaled particles rest depends on the diameter of the

PMs. The smallest particles (< 100 nm) may be extremely harmful to the cardiovascular system

Moreover, it has been shown that PM0.1 (and smaller) can pass through cell membranes and migrate

into other organs including the arteries, where deposition of plaque can induce vascular

inflammation and atherosclerosis (Pope et al., 2002), and the brain, where damage similar to that

found in Alzheimer patients has been detected. PM2.5 and PM10 materials also tend to penetrate into

the gas-exchange regions of the lungs, while smaller particles may be absorbed by the bronchi and

the lungs, enter the bloodstream, and hence cause serious disorders, including:

increased respiratory symptoms, such as irritation of the airways, coughing, or difficulty

breathing,

decreased lung function,

aggravated asthma,

development of chronic bronchitis,

irregular heartbeat,

non-fatal heart attacks,

premature death in people with heart or lung disease.

Typically, children and older adults are more sensitive to exposure to particle pollution than the

general population (US EPA, 2006).

Total hydrocarbons. These toxic substances are derived from unburnt or partially burnt fuel and are

considered to be a major contributor to the toxicity of urban smog, which can cause liver damage

and cancer. Many of these hydrocarbons are regulated in the USA by the Occupational Safety and

Health Administration. Thus, the typical composition of gasoline hydrocarbons (% volume) is as

follows: 4-8% alkanes; 2-5% alkenes; 25-40% isoalkanes; 3-7% cycloalkanes; l-4% cycloalkenes;

and 20-50% total aromatics (0.5-2.5% benzene) (IARC 1989).) (Harper and Liccione, 1995). It is

accepted that concentrations of alicyclic hydrocarbons in excess of 500 ppm are toxic, although

levels detected in the ambient atmosphere are generally 100 – 1000 fold lower than this (Stupfel,

- 21 -

1976). Concentrations greater than 1% have been found to produce narcosis and convulsions,

supposedly due to a hypoxic effect, whilst eye irritation caused by these hydrocarbons has been

attributed to their chemical structure (Stupfel, 1976). The potential danger of hydrocarbons found in

car exhaust is not limited, however, to their nature per se. As outlined in Tables 4 and 5, alicyclic

hydrocarbons interact in the presence of UV irradiation with nitrogen oxides (NOx) and oxygen-

containing free radicals giving rise to other toxic substances, including aldehydes and ketones that

are characteristic of photochemical smog. One group of such products, the peroxyacyl nitrates

(PANs), are formed from a hydrocarbon-derived peroxyacyl radical and nitrogen dioxide (NO2) in

the presence of UV light according to the equation (US EPA 2002a)

Hydrocarbons + O2 + NO2 + light

Peroxyacetyl nitrate (CH3COOONO2) is known to cause skin cancer (Jacobson, 2007).

Polyaromatic hydrocarbons. Polynuclear aromatic compounds (PACs), or PAHs (syn.), are by-

products of the incomplete combustion of fuel (fossil or biomass). More than 100 different PAHs

have been characterised, and the chemical structures of some of these are shown in Table 7. PAHs

are mainly solids, and occur as complex mixtures that are carried in the air on soot and other

particles onto which they have been absorbed. For this reason it is technically very difficult to

measure PAH concentrations in urban air. As pollutants, PAHs are of great concern because many

have been found to possess carcinogenic, mutagenic and teratogenic activities (ATSDR, 1995; EPA

2002a; IARC, 1983).

Table 7. Polycyclic aromatic hydrocarbons identified in PM from diesel engine exhaust (US EPA,

2002a).

Compound Concentration

ng/mg extract

Concentration

ng/mg extract

Acenaphthylene 30 Pyrene 3,532–8,002

Trimethylnaphthalene 140–200 Ethylmethyl anthracene 590–717

Fluorene 100–168 Methyl(fluoranthene/pyrene) 1,548–2,412

Dimethylbiphenyl 30–91 Benzo[a]fluorene/benzo[b]fluorene 541–990

C4 -Naphthalene 285–351 Benzo[b]naphtho[2,1-d]thiophene 30–53

Trimethylbiphenyl 50 Cyclopentapyrene 869–1,671

Dibenzothiophene 129–246 Benzo[ghi]fluoranthene 217–418

Phenanthrene 2,186–4,883 Benzonaphthothiophene 30–126

Anthracene 155–356 Benz[a]anthracene 463–1,076

Methyldibenzothiophene 520–772 Chrysene or triphenylene 657–1,529

Methylphenanthrene 2,028–2,768 1,2-Binapthyl 30–50

Methylanthracene 517–1,522 Methylbenz[a]anthracene 30–50

Ethylphenanthrene 388–464 3-Methylchrysene 50–192

4H-Cyclopenta[def]phenanthrene 517–1,033 Phenyl(phenanthrene/anthracene) 210–559

Ethyldibenzothiophene 151–179 Benzo[j]fluoranthene 492–1,367

2-Phenylnaphthalene 650–1,336 Benzo[b]fluoranthene 421–1,090

Dimethyl(phenanthrene/anthracene) 1,298–2,354 Benzo[k]fluoranthene 91–289

Fluoranthene 3,399–7,321 Benzo[e]pyrene 487–946

Benzo[def]dibenzothiophene 254–333 Benzo[a]pyrene 208–558

Benzacenaphthylene 791–1,643 Benzo[ah]anthracene 50–96

- 22 -

Seventeen PAHs have been identified as most harmful to human health, namely, acenaphthene,

acenaphthylene, anthracene, benzo[ah]anthracene, benzo[a]pyrene, benzo[e]pyrene,

benzo[b]fluoranthene, benzo[ghi]perylene, benzo[j]fluoranthene, benzo[k]fluoranthene, chrysene,

dibenzo[a,h]anthracene, fluoranthene, fluorene, indeno[1,2,3-cd]pyrene, phenanthrene and pyrene

(ASTDR, 1995).

7.1.1.2. Carbon monoxide. CO is a product of the incomplete combustion of fuel in an internal-

combustion engine and is formed according to the equation O2 + 2 C → 2 CO. Even very small

amounts of CO can induce hypoxic injury, neurological damage, and possibly death. When CO is

inhaled, it strongly binds to hemoglobin and blocks oxygen binding, resulting in oxygen starvation

throughout the body. Prolonged exposure to fresh air (or pure oxygen) is required for the CO-bound

hemoglobin to clear.

The specific effects of CO at various concentrations are listed below (Goldstein, 2008):

35 ppm (0.0035%) headache and dizziness within 6 to 8 h of constant exposure,

100 ppm (0.01%) slight headache in 2 to 3 h,

200 ppm (0.02%) slight headache within 2 to 3 h,

400 ppm (0.04%) frontal headache within 1 to 2 h,

800 ppm (0.08%) dizziness, nausea and convulsions within 45 min: unconscious within 2 h,

1,600 ppm (0.16%) headache, dizziness and nausea within 20 min: death in less than 2 h,

3,200 ppm (0.32%) headache, dizziness and nausea in 5 to 10 min: death within 30 min,

6,400 ppm (0.64%) headache and dizziness in 1 to 2 min: death in less than 20 min,

12,800 ppm (1.28%) unconsciousness after 2-3 breaths: death in less than 3 min.

The earliest symptoms of CO poisoning, particularly at low level exposure, are often non-specific

including depression, chronic fatigue syndrome, chest pains, and migraine or other headaches (Ilano

and Raffin, 1990). CO poisoning can also promote myocardial ischemia, arterial fibrillation,

pneumonia, pulmonary oedema, hyperglycemia, muscle necrosis, acute renal failure, skin lesions,

visual and auditory problems and respiratory arrest (Choi, 2001). Chronic exposure to CO may

increase the severity of cardiovascular symptoms, persistent headaches, light-headedness,

depression, confusion and nausea (Fawcett et al., 1992). Severe neurological manifestations may

occur days or even weeks after acute CO poisoning and are associated with cognitive functions,

such as impaired short-term memory, dementia, irritability, gait disturbance, speech disturbances,

Parkinson-like syndromes, cortical blindness and depression (Roohi et al., 2001). A recent report

concluded that exposure to CO can lead to a significant reduction in lifespan owing to damage to

the heart muscle (Henry et al., 2006). Additionally, teratogenic effects of CO have been

demonstrated in chick embryos (Baker and Tumasonis, 1972).

7.1.1.3. Nitrogen oxides. NOx [typically nitric oxide (NO) together with nitrogen dioxide (NO2)]

are generated when nitrogen in the air reacts with oxygen under the high temperature and pressure

conditions present inside the engine. During combustion, nitrogen bound in the fuel is released as a

free radical and forms free nitrogen or NO, the latter being potentially further oxidised to NO2.

Acute exposure to NOx for 0.5 - 24 h induces inflammatory symptoms in the respiratory system of

healthy subjects and aggravates existing symptoms in asthmatic patients. Atmospheres containing 5

ppm or more of mixtures of NO and NO2, as encountered in the occupational exposure of welders,

- 23 -

cause a decrease in maximum breathing capacity and an increase in expiratory resistance

accompanied by methemoglobinemia (Stupfel, 1976). Exposure to NOx at levels between 25 to 75

ppm induces bronchitis and bronchopneumonia, while concentrations in the region of 500 ppm

provoke pulmonary oedema and, ultimately, death. Moreover, a positive association has been

detected between short-term exposure to elevated NO2 concentrations and increased visits to

emergency departments and hospital admissions (US EPA, 2006). In this context, children, elderly

people and asthmatic patients are more sensitive to NO2 exposure than the general population.

Additionally, it should be noted that concentrations of NO2 within about 50 meters of a roadway are

typically between 30 and 100% greater than levels measured in the same area but away from the

roadway.

Interaction of NOx with ammonia in the presence of water vapour and other compounds can

generate fine particles that may be absorbed in the lungs inducing respiratory disorders, such as

bronchitis, and the exacerbation of existing heart disease, leading to increased hospital admissions

and early death. (US EPA 2006). Recent studies have raised concern over the use of oxidative

catalytic converters with diesel engines. It is suggested that such converters can boost the formation

of direct-acting mutagens under certain conditions by reaction of NOx with PAH resulting in the

formation of toxic nitrated-PAHs (Bünger et al., 2006).

7.1.1.4. Sulphur oxides. Sulphur dioxide (SO2) is emitted at concentrations in the range 0.2 to 1.66

ppm by coal-burning heaters and power sources, and by those using sulphur-contaminated fuel.

Intoxication with SO2 mainly affects the upper airways, and a strong association between SO2 levels

and the incidence of lung cancer has been demonstrated (Abbey et al., 1999). Fortunately, man can

detect the odour of SO2 at concentrations between 1 - 3 ppm (Stupfel, 1976). Short-term exposure

(from 5 min to 24 h) to SO2 induces bronchoconstriction and asthmatic symptoms.

Sulphur oxides (SOx) can react with other compounds in the atmosphere resulting in the formation

of aggressive particles that readily penetrate into the lungs and stimulate respiratory diseases such

bronchitis and emphysema (US EPA, 2006).

7.1.1.5. Heavy metals. Small amounts of various heavy metals, including platinum, palladium, lead,

cadmium, zinc and nickel, are emitted following the combustion of gasoline in automobile engines.

Roadside soils and vegetation are often found to be contaminated by deposits of the last four of the

metals listed. Lead, in particular, is easily absorbed into the blood and subsequently distributed

throughout the whole body. The metal interacts with the oxygen transportation system of the

blood. (US EPA, 2006), and affects the nervous, immune, cardiovascular and reproductive systems

as well as the kidneys. The diagnostic criteria of lead intoxication in man are anaemia,

coproporphyrinuria, urinary excretion of 9-aminolevulinic acid dehydratase (ALA-D), and

decreased levels of ALA-D in red blood cells (Stupfel, 1976). Around 90% of absorbed lead is

accumulated in the bones, but long-term experiments involving both man and animals continuously

inhaling high concentrations of lead (3.5 to 20 g/m3) revealed increased blood lead and decreased

blood ALA-D, but an absence of apparent modifications to health (Stupfel, 1976). Infants and

children are especially sensitive to very low levels of lead, which can induce mental disorders

affecting behaviour , learning capacity and cognitive functions. (US EPA, 2006).

- 24 -

Currently, the gasoline sold in most of developed countries is free of lead. That is beneficial for

human health not only because of elimination of lead toxicity. The absence of the lead in gasoline

is associated with reduced emission of PAH, which are highly carcinogenic. Thus, it has been

shown that if premium leaded gasoline is replaced by 92 octane lead-free gasoline, the emissions of

total PAHs and total benzo(a)pyrene equivalents (BaPeq) would be decreased (Mi et al., 2001). The

lead containing gasoline still is widely used in developing countries. That is the problem not only

for people living in these countries, but also for people living in all other countries, where the lead

containing gasoline is forbidden for sale. The reason is that the lead emitted from cars can be

absorbed in plants and disseminated all over the world via the food imported to other countries.

Meat of animals feed with lead contaminated grass will also contain the lead which is easily

accumulated in animal tissue.

It has been found that cadmium accumulates in the kidneys causing functional disturbances, whilst

nickel may increase the risk of bronchial cancer (Stupfel, 1976).

7.1.1.6. Ozone. Ozone is formed when NOx and volatile organic compounds interact at high

temperature in the presence of sunlight. High levels of ozone in the atmosphere induces

coughs, airway irritation, breathing difficulties, lung impairment and pain,

inflammation, similar to sunburn on the skin,

aggravation of asthma and increased susceptibility to respiratory diseases (pneumonia,

bronchitis etc.).

Patients with respiratory tract disorders, children and older adults are especially sensitive to ozone

exposure (US EPA, 2006).

7.1.2. Unregulated pollutants

So called ―aggregated toxics‖, namely, acetaldehyde, acrolein, benzene, 1,3-butadiene,

ethylbenzene, n-hexane, naphthalene, styrene, toluene, and xylene are currently unregulated.

7.1.2.1. Carbonyl compounds. These represent an important class of pollutants emitted from

vehicles fuelled by oxygenated fuels (Sharp et al., 2000; Grosjean et al., 2001; Poulopoulos et al.,

2001; Cardone et al., 2002; Magnusson et al., 2002). Included in this group of contaminants are:

formaldehyde, acetaldehyde, acrolein, acetone, propionaldehyde, crotonaldehyde, methylacrolein,

butyraldehyde, o-tolualdehyde, m-tolualdehyde, valeraldehyde, hexaldehyde and

dimethylbenzaldehyde (Pang et al., 2008). These compounds are precursors of free radicals, PANs

and ozone in the atmosphere (Carter, 1995; Gaffney et al., 1997), while some of them are toxic,

mutagenic and/or carcinogenic (Carlier et al., 1986). Various aldehydes are potent eye irritants:

thus, the eye irritation threshold of formaldehyde is estimated to be 0.01-1.0 ppm, the olfaction and

eye irritation threshold of acrolein is 0.25 ppm, whilst the irritant threshold of acetaldehyde is 50

ppm. Many of the unpleasant odour and irritant properties of diesel and gasoline exhausts are

caused by the presence of aldehydes. Moreover, the upper airways are very sensitive to these

products, and concentrations as low as 0.20 - 1.20 ppm of total aldehydes, or 0.05 - 4.12 ppm of

formaldehyde, are physiologically active in the air (Stupfel, 1976). The ciliary activity of rat and

sheep trachea is inhibited in vitro by very small amounts of aldehydes, and this could inhibit

resistance to air particles penetrating into the lungs (Stupfel, 1976)..

- 25 -

Formaldehyde

Acute effects - the main toxic effects caused by acute exposure to formaldehyde via

inhalation are irritation to the eye, nose and throat, and inflammation in the nasal cavity. In

humans, exposure to high levels of formaldehyde may result in spasms of coughing and

wheezing, and to chest pains and bronchitis. (US EPA, 1988; WHO, 1989).

Chronic effects (non-cancer) - chronic inhalation exposure of humans to formaldehyde is

associated with various respiratory symptoms and irritation of the eye, nose and throat.

Animal studies have revealed chronic effects of formaldehyde exposure on the nasal

respiratory epithelium and lesions in the respiratory system (US EPA, 1988; WHO, 1989;

Calabrese and Kenyon, 1991).

Reproductive/developmental effects - an increased incidence of menstrual disorders and

problems in pregnancy have been reported in women workers using urea-formaldehyde

resins, although possible confounding factors were not evaluated in the study (US EPA,

1988; WHO, 1989).

Cancer risk – WHO and INTERNATIONAL AGENCY FOR RESEARCH ON CANCER

(IARC) consider formaldehyde to be a human carcinogen (cancer-causing agent) and it has

been ranked in EPA group B1 (IARC, 2006; US EPA, 1993). There is sufficient evidence in

humans for the carcinogenicity of formaldehyde.There is sufficient evidence in experimental

animals for the carcinogenicity of formaldehyde. (IARC, 2006).

Acetaldehyde

Acetaldehyde is the major metabolite of ethanol and is also released into the environment by

combustion and photo-oxidation of hydrocarbons (US EPA, 1994a). In air, acetaldehyde reacts with

singlet oxygen, OH radicals, NO3 and NO2 to form, amongst other products, the highly toxic

peroxyacetyl nitrate together with methyl nitrate, methyl nitrite and nitric acid (US EPA, 1994a).

The acute effects of acetaldehyde in humans include irritation of the eyes and respiratory tract and

altered respiratory function. According to the International Agency for Research on Cancer (IARC,

1994a), sub-chronic and chronic dermal exposure to acetaldehyde can cause erythema and burns in

humans, whilst repeat contact may give rise to dermatitis resulting from irritation or sensitisation

(IARC, 1985). In animals, repeat doses of acetaldehyde, administered in high concentrations by

inhalation, caused adverse respiratory tract effects. Additionally, direct administration of

acetaldehyde to rats established alcohol dependency, and it is claimed that many of the adverse

effects of ethanol itself may be attributable to acetaldehyde (US EPA, 1994a).

Cancer risk – There is inadequate evidence in humans for the carcinogenicity of acetaldehyde.

There is sufficient evidence in experimental animals for the carcinogenicity of acetaldehyde. (IARC,

1987). Overall evaluation: acetaldehyde is possibly possibly carcinogenic to humans (Group B2)

(IARC, 1987; US EPA, 1994a). Additionally, acetaldehyde has been shown to possess neurotoxic

activity since it is able to penetrate the human blood-cerebrospinal fluid barrier and can cause

depression of the central nervous system (IARC, 1985; 1987).

Acrolein

Acrolein is a strong dermal irritant and causes skin burns in humans. Exposure to acrolein in the

range 0.17 - 0.43 ppm induces irritation of the upper respiratory tract and congestion, whilst at the

level of 10 ppm it can cause death. The provisional reference dose calculated by US EPA for

- 26 -

acrolein is 0.02 mg/kg body weight/day (ATSDR, 2007). No information is available concerning

the embryotoxic or carcinogenic effects of acrolein in humans. However, a toxic effect on the

reproductive system was observed when the aldehyde was injected directly into embryonic tissue of

rats, and a tumour promotion effect on the respiratory tract and adrenocortical tissue could also be

demonstrated in the murine model.

7.1.2.2. Other oxygenated hydrocarbons in exhaust emissions

Methanol

Acute methanol intoxication is manifested by symptoms of narcosis followed by accumulation of

formic acid in the body causing metabolic acidosis (US EPA, 1994b). Symptoms of lesions of the

central nervous system (CNS) include headache, dizziness, severe abdominal, back and leg pain,

delirium (that can lead to nausea and coma) and visual degeneration that can bring about blindness.

Chronic exposure to methanol vapour (time of exposure and dose not stated) caused conjunctivitis,

headache, giddiness, insomnia, gastric disturbances and bilateral blindness, whilst vision loss

reportedly occurred after exposure to 1200 to 8000 ppm vapour for 4 years (US EPA, 1994b). The

lethal dose of methanol when administrated orally to humans is in the range of 80 - 150 mL, whilst

in the vapour phase a dose of 4000 -13,000 ppm (equivalent to 1140 - 3705 mg/kg) over 12 h is

fatal (US EPA, 1994b).

A genotoxic effect of methanol, in the form of increased sister chromatid exchange, was observed

in Syrian hamster embryo cells in vitro, whilst aneuploidy and chromosome aberrations were

detected in Neurospora crassa (US EPA, 1994b). Methanol also exhibits a neurotoxic effect in

humans and animals, causing depression of the central nervous system as well as degenerative

changes in the brain and visual system.

In polluted air, methanol reacts with NO2 to yield methyl nitrite, and with photochemically-formed

hydroxyl radicals to produce formaldehyde. The half-life of methanol in the atmosphere is 17.8

days, whilst levels of methanol in the air around Stockholm were reported to be in the range 3.83 -

26.7 ppb in 1994 (US EPA, 1994b).

Ethanol

Ethanol exhibits the lowest degree of acute toxicity (category IV) for all effects tested including

acute oral and inhalation toxicity, and primary eye and skin irritations (US EPA, 1995). Ethanol is

generally recognized as a human developmental neurotoxicant causing foetal alcohol syndrome. In

an industrial environment, however, the risk from ethanol appears to be minimal (US EPA, 1995).

Formic acid

Formic acid presents very low toxicity when ingested via the oral route, but in the vapour phase it

causes severe eye and skin irritation. Inhalation of the vapour at 100 ppm is immediately dangerous

to life and health in humans causing respiratory irritation, lachrymation, coughing and headache,

followed within 6 - 8 h by pulmonary oedema, dizziness, frothy expectoration and cyanosis (bluish

skin discoloration caused by lack of oxygen in the blood). Breathing lower concentrations over time

can lead to erosion of the teeth, local tissue death in the jaw, bronchial irritation with chronic cough,

frequent attacks of bronchial pneumonia and gastrointestinal disturbances (Federal Register, 1997).

- 27 -

7.1.2.3. Hydrocarbons in exhaust emissions

Benzene.

Exhaust benzene is produced either from unburned benzene or benzene formed during the use of

other aromatic and nonaromatic compounds found in gasoline. Brief exposure (5–10 minutes) to

very high levels of benzene in air (10,000–20,000 ppm) can result in death. Lower levels (700–

3,000 ppm) can cause drowsiness, dizziness, rapid heart rate, headaches, tremors, confusion, and

unconsciousness. Long-term exposure to benzene cam can lead to anemia, suppress immune

system, cause .acute myeloid leukemia (AML). The International Agency for Cancer Research and

the EPA have determined that benzene is carcinogenic to humans (Group A). Exposure to benzene

may be harmful to the reproductive organs and induce irregular menstrual periods. (Wilbur et al.,

2007)

1,3-Butadiene.

Brief exposure to 1,3-butadiene exposure may cause nausea, dry mouth and nose, headache, and

decreased blood pressure and pulse rate in humans. In laboratory animals, 1,3-butadiene induces

inflammation of nasal tissues, changes to lung, heart, and reproductive tissues, neurological effects,

blood changes, reduced fetal body weight, and birth defects. Long term exposure to 1,3-butadiene in

humans may have an increased risk for cancers of the stomach, blood, and lymphatic system. The

International Agency for Research on Cancer (IARC), National Toxicology Program (NTP), and

EPA all classify 1,3-butadiene as a human carcinogen. EPA has set a reference concentration in

breathing air of 0.9 ppb for 1,3-butadiene. (Ashizawa et al., 2009).

7.2. Acute and chronic adverse health effects of vehicle exhaust produced by the

combustion of petrofuels, gasoline and diesel

More than three decades ago, Stupfel published a comprehensive review of numerous in vitro and in

vivo studies on animals, and epidemiological and experimental studies on humans, in which the

toxic effects of automotive exhaust, and individual components thereof, had been reported (Stupfel,

1976). Table 8 presents a summary of the levels of the major components of the exhausts from

gasoline and diesel-fuelled engines and reveals some differences in their chemical compositions.

Overall, diesel cars emit less hydrocarbons, CO and lead than petrol cars, but produce more noxious

gases and significantly more particulates. (Stupfel, 1976). Stupfel (1976) has pointed out that the

acute toxicity of gasoline exhaust emission is mostly due to CO, and describes one of the first

experiments carried out in 1921-1922 by Henderson and his colleagues to confirm this assumption.

These researchers exposed themselves, together with several other volunteers, to the exhaust gas of

a Ford car containing levels of CO in the range 200-300 ppm, as a result of which the participants

suffered dizziness, headaches, fainting and vomiting. These symptoms were then compared with

those observed following exposure to similar levels of CO, from which it was concluded that CO

was the main toxic constituent of exhaust gas derived from the combustion of gasoline.

- 28 -

Table 8. Concentrations of the main components in automotive exhausts. (Table modified from

Stupfel, 1976).

Gases and particles Gasoline exhaust Diesel exhaust Biological activity

threshold

Air quality

standards

Nitrogen (%) 76-90

Hydrogen (%) 2-6 0.05-8

Carbon dioxide (%) 5-15 1-14 5 0.5

Carbon monoxide 2-6% 0-0.1% 200-300 ppm 30-120 ppm

NOx (ppm) 30-4000 30-2000 3-10

NO2 (ppm) 0.5-40 0.5-5 0.2-0.5

SO3 (ppm) 0-80 100-300 1-5 0.03-0.1

Total aldehydes (ppm) 40-300 10-120 0.06-0.1

Formaldehyde (ppm) 10-300 5-30 0.5-16

Total hydrocarbons 0.03-1.5% 0.01-0.10% 0.2-5 ppm 0.24 ppm

Methane 200-800 ppm 1-20% 1000 ppm

Benzopyrene 1-10 μg/m3 0.01-100 μg/m

3

Lead 79-80% of the lead

of gasoline

0 15.5 μg/m3

Oils (mg/m3) 200-900

Particles 0.2-3 mg/g of

burned gasoline

150-450 mg/m3 60-75 μg/m

3

Data shown in table 8 demonstrate the main differences in gasoline and diesel exhaust from old cars

used more than 40 years ago. In my review, the results obtained in the last two decades will be used

for the comparisons of the effects of biofuel and petrofuel exhausts on human health.

Nowadays, the amounts of many toxic compounds in gasoline and diesel exhaust is significantly

lower, due to invention and implementation of catalytic converters (table 9), particle filters used in

cars working on diesel fuel (Schifter et al., 2001; Franklin et al., 2001; US EPA, 2002a) and many

other innovations in engine technology resulting both in decreased emission of regulated toxics

(table10) and significant changes in a smoke standards for on-road diesel engines (US EPA, 2002a),

figures 6 and 7. For example, table 10 shows US EPA emission standards from 1970 to 2007 for

heavy duty highway diesel engines, wherefrom is obvious that the upper limit for particulates (PM)

and NOx is reduced in 50-60 (!!!) times. Figure 6 shows changes in engine certification data for

NOx, PM, aldehydes, soluble organic fraction emissions reported in the many studies that have

employed the transient test over the 25 years (US EPA 2002a). Figure 7 shows real-world, in-use

emissions measurements of PM, total hydrocarbons, CO and NOx and therefore more accurately

reflects emission factors than engine test data during this period (US EPA, 2002a).

In the past, organic lead compounds were usually used as anti-knock/isooctane booster agents in

gasoline, however, they have been almost completely replaced by methyl-tertiary-butyl ether as an

anti-knock agent in the production of unleaded gasoline. Leaded gasoline has been produced in

much smaller quantities for use in engines not equipped with catalytic converters (Harper and

Liccione J, 1995). For old engines not equipped with catalytic converters other additives in some

extent fulfil the same function as lead.

Comparison of tailpipe and exhaust emission studies showed that reactions in the catalytic converter

are quite effective in destroying most hydrocarbons, methyl tertiary-butyl ether by-product species,

including CO, table 9 (Schifter et al., 2001; Franklin et al., 2001).

- 29 -

Figure 6. Engine certification data for NOx, PM, aldehydes, soluble organic fraction emissions

reported in the many studies that have employed the transient test over the 25 years (Modified from

US EPA 2002a).

Figure 7. Model year trends in PM, NOx, THC, and CO emissions from HD diesel vehicles (Taken

from US EPA 2002a).

- 30 -

Table 9. Emissions of particle-bound PAHs (µg/ml), carbone monooxyde (g/km), nitrogen oxides

(g/km), total hydrocarbons (g/km) and ozone (g of ozone produced by g of nonmethane organic

gases emitted) in gasoline exhaust from vehicles with oxidative catalyst, tree-way catalytic

converter and without catalyst. Effect of ethanol (10% in gasoline-ethanol blend) is shown as well

(Table from data in Schifter et al., 2001 and from US EPA, 2002a).

Gases No catalyst Oxydative catalyst Tree-way catalytic

converter

Gasoline Gasoline-

ethanol

Gasoline Gasoline-

ethanol

Gasoline Gasoline-

ethanol

Carbon monoxide 18 13 13 8 3 3

NOx (ppm) 1.73 1.75 1.08 1.23 0.41 0.48

Total hydrocarbons 1.56 1.45 0.7 0.56 0.22 0.22

Ozone 4.19 4.27 3.95 3.66 3.12 3.08

Pyrene 45 4

Fluoranthene 32 3.6

Benzo[a]pyrene 3.2 3.0

Benzo[e]pyrene 4.8 3.6

Table 10. US EPA emission standards for HD highway diesel engine, (modified from US EPA

2002a).

It is noteworthy that filtered diesel exhaust or the exhaust from the gasoline-catalyst engines are less

harmful than diesel engine emissions (Brightwell et al., 1989). There was no significant changes in

the incidence of lung tumours in the group of rats exposed to filtered diesel exhaust or to the

exhaust from the gasoline or gasoline-catalyst engines for two years (16 hours per day, 5 days per

week), while a statistically significant increase in the incidence of lung tumours was observed in the

group of rats exposed to diesel engine emissions (Brightwell et al., 1989).

Multivariate statistical methods have been applied in order to identify factors that affect the

biological potency of car exhausts. The physical and chemical characteristics of 10 diesel fuels were

determined, and extracts of the particulate exhaust emissions derived from the combustion of these

fuels were assayed chemically and for mutagenic activity using the Ames test and a test involving

Model year Pollutant (g/b hp-hr THC CO NOx Particulate

(PM), 1970 - - - - 1974 - 40 - - 1979 1.5 25 - - 1985 1.3 15.5 10.7 - 1988 1.3 15.5 10.7 0.6 1990 1.3 15.5 6 0.6 1991 1.3 15.5 5 0.25 1993 1.3 15.5 5 0.1-0.25 1994 1.3 15.5 5 0.07-0.1 1996 1.3 15.5 5 0.05-0.1 1998 1.3 15.5 4 0.05-0.1 2004 1.3 15.5 - 0.05-0.01 2007 15.5 0.2 0.01

- 31 -

the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) to the Ah receptor (Sjögren et al.,

1996). The strongest associations between the biological effects of the extracts and the chemical

characteristics of the fuels related to the levels of sulphur, PACs and naphthalenes. Fuel density and

flash point were positively correlated with genotoxic potency, whilst octane number and upper

distillation curve points were associated negatively with mutagenicity and Ah receptor affinity. The

mass of emitted particles and the levels of 1-nitropyrene, particle bound nitrate, and indeno[1,2,3-

cd]pyrene were highly correlated with both mutagenicity and Ah receptor affinity. Exogenous

metabolic activation (S9)-dependent mutagenicity was highly associated with certain PACs, while

S9-independent mutagenicity was more related to the concentrations of nitrates and 1-nitropyrene

present. The results of the study revealed that fuels presenting low levels of biological hazard were

those that contained the least PAC and sulphur (Sjögren et al., 1996).

7.2.1. Acute and chronic adverse health effects

Increases in hospital admissions and daily mortality have been observed in patients with chronic

obstructive pulmonary disease, chronic bronchitis, asthma and cardiovascular disease during short-

term exposure to high levels of PM in the ambient air (Dockery and Pope 1994; Samet et al., 1995;

Katsouyanni et al., 1997). It has been reported that exposure to diesel exhaust particles (DEP)

increases many symptoms of systemic and pulmonary inflammation in healthy humans (Salvi et al.,

1999). In several observational studies, a correlation between mortality and chronic exposure to air

pollution from PM10 has been reported (Dockery et al., 1993; Schwartz 1993; Pope et al., 1995;

Abbey et al., 1999).

7.2.2. Carcinogenic effects

Lung tumours were reproducibly observed in rats, but not in other experimental rodents, following

chronic inhalation of diesel engine exhausts at high concentrations (US 2002a; Mauderly et al.,

1987). In a more detailed study of the potential carcinogenic effects of inhaled automobile exhaust,

rats and hamsters were subjected to the emissions from (i) a gasoline engine, (ii) a gasoline engine

fitted with a three-way catalytic converter, (iii) a diesel engine and (iv) a diesel engine fitted with

particle filtration (US EPA 2002a). Following exposure over a 2 year period (5 days per week and

16 h per day), a statistically significant increase in the incidence of lung tumours in rats exposed to

diesel engine emissions was observed compared with controls. There were, however, no increases

in the incidence of lung tumours in rats exposed to filtered diesel exhaust or to the exhausts from

gasoline engines with or without catalytic converters.

Diesel engine emissions generate strong carcinogenic effects in animals and potential effects in

humans according to a number of extensive reports from scientific and governmental institutions in

USA and Europe (IARC, 1989; US EPA, 2002a).

A prospective environmental cohort study demonstrated a strong correlation between incidence of

lung cancer in non-smokers and the concentration of PM10 in the air (Abbey et al., 1999). The

increased risk of lung cancer following long-term occupational exposure to high concentrations of

diesel emissions has been revealed in several epidemiological studies (Mauderly,; 1994; EPA,

2002a). Similar results were obtained in other studies involving workers in the potash mining

industry and operators of heavy equipment who had been exposed to diesel engine emissions

(Bruiske-Hohlfeld et al., 1999; EPA, 2002a).

- 32 -

7.2.3. Mutagenic effects

Strong mutagenic activities of DEP extracts in the Salmonella typhimurium/mammalian microsome

assay is associated with the presence of substituted PACs, in particular nitro-PAC, while

unsubstituted PACs exhibited a mutagenic effect in the experimental model with metabolic

activation by S9 (ATSDR, 1995; EPA, 2002a; IARC, 1983).

7.3. Chemical composition and toxic effects of vehicle exhaust produced by

combustion of biofuels

In Sweden, there is intense interest in the use of biofuels, particularly for city buses, delivery trucks

and light duty vehicles, with the intent of reducing emissions of the main toxic pollutants. In 1991

the Swedish Government launched an extensive research program, administrated by the Swedish

Transport and Communications Research Board, in order to demonstrate the feasibility of using

ethanol in Flexible Fuel Vehicles and biogas in both light and heavy-duty buses and delivery trucks.

Several Swedish Universities and Institutes of Technology have conducted projects in support of

this program (Egeback and Foxbrook, 1998).

7.3.1. Biodiesel and vegetable oils

7.3.1.1. Chemical composition of the emissions from biodiesel engines. Assuming that the

chemical compositions of petrofuel and biofuel are very different [section 2.2., e.g. RME contains

almost no aromatic compounds whereas fossil diesel fuels comprise 3–40 % aromatics including

PACs at levels up to 340 mg/l (Sjögren et al., 1995)], it might, perhaps, be expected that the

emissions from oil and biodiesel-fuelled engines would be quite dissimilar and, in the case of the

latter, relatively harmless. Despite the compositional differences, however, the combustion products

and exhaust emissions of both types of diesel still contain the same irritant gases, including NOx,

aldehydes and a wide range of organic compounds, many of which can induce oxidative stress in an

organism (Krahl et al., 1996a,b; Mauderly, 1997). An initial comparative study of biodiesel and

rapeseed oil exhaust demonstrated that emissions from biodiesel-fuelled engines were qualitatively

similar to those running on PDD (Krahl et al., 1996b) in spite of significant quantitative differences

in the content of various groups of compounds, such as PAHs, aldehydes, ketones, hydrocarbons

and particulates (Figure 8).

Thus biodiesel emission contains amounts of particulates that are 2-3 times higher compared with

PDD, but 3-4 times less PAHs, while rapeseed oil emission is extremely rich in carbonyl

compounds (with ≤ 4.5 fold higher levels of aldehydes/ketones) and hydrocarbons compared with

PDD and biodiesel. This is most probably associated with the glycerol moiety in the triglycerols,

which can be transformed into acrolein and other aldehydes at high temperatures during the

incomplete burning of rapeseed oil (Graboski and McCormick, 1998), and the residual levels of

methanol or ethanol that could suffer partial combustion to form formaldehyde or acetaldehyde,

which are considered to possess actual or potential carcinogenic properties, respectively (IARC,

1985). Thus, the quality of biodiesel depends substantially on the content of free glycerol in the fuel

after refining, and on the amount of free alcohol (employed in the transesterification procedure)

remaining in the fuel following down-stream processing.

- 33 -

RSO RME0

100

200

300

400

500Smoke Nr

Particulate

Hydrocarbons

CO

NOx

PAH

Aldehydes

Emission from RSO and RME

Rela

tive v

alu

es i

nco

mp

ari

sio

n t

o d

iesel

(10

0%

)

Figure 8. Emissions from engines fuelled with rapeseed oil (RSO) and rapeseed diesel (RME) in

comparison with levels emitted by a diesel-fuelled engine (taken as 100%). Figure modified from

Krahl et al., 1996b.

Further studies have revealed that, in comparison with petroleum diesel, the combustion of

biodiesel produces less CO, solid material (in the form of total particulate matter), total

hydrocarbons and other toxic compounds, including vapour-phase C1-C12 hydrocarbons, aldehydes

and ketones (up to C8), and certain semi-volatile and particle-phase PAHs (Krahl et al., 1998;

Mauderly, 1997; Sharp et al., 2000; Graboski et al., 2003; McCormick, 2007). Thus, the amounts of

the toxic aromatic hydrocarbons benzofluoranthene and benzopyrenes in biodiesel exhaust are,

respectively, 56 and 71% less than in petroleum diesel exhaust, whilst sulphur-containing

compounds appear to be undetectable in biodiesel emissions.

Typically, a significant inverse correlation between the emissions of PM, CO and total

hydrocarbons with increasing content of biodiesel in blends with PDD has been observed in a

number of studies. The US EPA has released details of a comprehensive study of the emission

impact of biodiesel in which statistical regression analysis was employed to correlate the

concentration of biodiesel in admixture with conventional diesel fuel with changes in regulated and

unregulated pollutants emitted from on-road heavy-duty diesel engines (Durbin and Norbeck, 2002;

US EPA 2002a; Zou and Atkinson, 2003). It was concluded that a significant reduction in total

hydrocarbons, CO and PM can be achieved using biodiesel instead of PDD (Figure 9).

- 34 -

Figure 9. Average emission impacts of biodiesel on emission of regulated pollutants [PM, CO,

hydrocarbons (HC) and NOx] compared with those of diesel exhaust from heavy-duty highway

engines. (Figure taken from US EPA. 2002b).

In another study, biodiesel manufactured from canola oil was blended, prior to combustion, with

petroleum diesel in various proportions ranging from 0 to 100%, and the DPMs and gaseous

emissions were determined with particular attention to the levels of PAHs (Zou and Atkinson,

2003). It was found that the concentration of particulate matter was reduced by up to 33% when the

engine burnt 100% biodiesel compared with 100% PDD, and that the diminution in particulate

emissions was related to the increased percentages of biodiesel in the fuel blends. Among 16

targeted PAHs, 9 were detected in the particulate phase of biodiesel exhaust at much lower levels

than in the gaseous phase, but the particulate PAH emissions, which have greater importance with

regard to adverse health effects, were virtually unchanged and did not show a statistically

significant reduction with increased percentage of biodiesel. Some marked reductions were

observed in the levels of the less toxic gaseous PAHs, such as naphthalene, with 100% biodiesel

fuel.

In contrast, the combustion of biodiesel in a diesel engine typically leads to an increase in the

release of NOx if the fuel is burnt without additives (Figure 8; US EPA 2002a; Hansen et al., 2006;

McCormick 2007). For example, the tail-pipe emission of NOx from pure biodiesel (B100) is about

10-12% higher than that of PDD but, since the former has a very low sulphur content, it has been

suggested that NOx could be readily reduced through the use of a catalytic converter (Beer et al.,

2004; McMillen et al., 2005; Hansen et al., 2006).

- 35 -

Figure 10. Typical changes in the levels of NOx emissions according to percentage of biodiesel in

admixture with PDD (A) together with examples of variations according to the source of the

biodiesel (B). (Figure taken from US EPA, 2002b).

The release of NOx depends significantly on the source of the biodiesel (see Figure 10b).

Detailed analyses have shown that molecular the structure of the fatty acids in biodiesel has a

substantial impact on the emission of NOx, which increases with increasing numbers of C=C

double bounds (measured in terms of iodine number, figure 11a), decreases with hydrocarbon chain

length (data obtained using 18, 16, and 12 carbon chain molecules), and increases with cetane

number, which is correlated with un-branched open chain alkanes, figure 11b; (McCormick et al.,

2001).

Figure 11. Effects of iodine number (a) and cetane number (b) on emissions of NOx from biofuels.

(Figures taken from McCormick et al., 2001 with permission of the author).

A B

a b

- 36 -

The direct tailpipe-emission of particulates by biodiesel engines fitted with particulate filters is

reduced by as much as 20% compared with low-sulphur (< 50 ppm; LS) diesel and by ca. 50%

compared with fossil-sourced diesel (Beer et al., 2004), whilst the ranges of size of particles emitted

by PDD and biodiesel engines are roughly the same. However, in comparison with fossil fuel,

biodiesel emits 40-49% less of the sub-micron particles that are believed to deposit deeply in the

respiratory tract and cause respiratory diseases such as lung cancer (Chen and Wu, 2002). In this

respect, it might be considered that the emissions from biodiesel are potentially less detrimental to

humans than those of PDD.

In contrast to the above, it was found that the particles emitted from biodiesel engines typically

contain in the order of 40% more soluble organic material than petroleum diesel soot (Sharp, 1998;

Durbin et al., 1999). This increase in soluble organic fraction most probably results from the

incomplete combustion of fatty acid methyl esters in the plant oil, a situation that is exacerbated by

the fact that the tested engines were optimised for the combustion of PDD fuel (Syassen, 1998). A

lower production of particles with a higher concentration of soluble organic mater may impact on

the biologic effect and toxicity of biodiesel exhaust emissions.

In addition to impacts of regulated pollutants in biodiesel emissions, those of unregulated hazardous

air pollutants, such as the ―aggregated toxics‖ (i.e. acetaldehyde, acrolein, benzene, 1,3-butadiene,

ethylbenzene, n-hexane, naphthalene, styrene, toluene and xylene), have also been investigated. The

results of one study revealed that the mass ratio of total toxics to hydrocarbons actually increased

with the addition of biodiesel, thus, although the total hydrocarbon content decreased, the

concentration of unregulated pollutants in biodiesel emission increased (US EPA, 2002a). Quite

different results were reported in another study, however, where the content of these unregulated

aldehydes was found to be significantly lower in biodiesel exhaust compared with that of PDD

(Figure 12; Krahl et al., 2007a).

Figure 12. Specific emission rates of (a) aromatic hydrocarbons and (b) aldehydes and ketones in

particle extracts of, Swedish low sulphur diesel fuel (MK1), German biodiesel (RME), fossil diesel

fuel (DF), low sulphur diesel fuel with a high aromatic compound content and flatter boiling

characteristics (DF05). (Figures taken from Krahl, et al., 2007a).

a b

- 37 -

However, it should be mentioned that such a big discrepancy in the results is due to different

combustion conditions (as suggested by Krahl el al. 2007a), and because of systematic

methodological errors. The present reviewer (LP) further suggests that there are problems with the

methods of analysis used in these studies since they were apparently not validated for accuracy and

the recoveries of the rather volatile compounds were not estimated. In fact, no data relating to the

validation of the analytical methods employed can be found in the published articles (Krahl et al.,

2007, 2009; Buenger et al., 2000, 2006, 2007; Song et al., 2007). In these studies, all gases emitted

during combustion were collected in dichloromethane and the solution was concentrated by vacuum

evaporation prior to analysis using the Ames test. However, the boiling points of some of the

analytes (e.g. acrolein, 53°C) are very close to the boiling point of the organic solvent

(dichloromethane, 40°C,) whilst others [e.g. formaldehyde (-15°C), acetaldehyde (20°C)] are

actually lower. Obviously they will all evaporate to some extent together with the solvent and their

residual content in the final test sample will be very different from that of the original sample prior

to evaporation. This can lead to large deviations and discrepancies in the results. Consequently the

final results related to toxicity of emitted gases might be distorted and final conclusions might

underestimate the real mutagenic and toxic potential of the constituents of the original sample,

section 7.3.1.2 below.

7.3.1.2. Biodiesel exhaust emissions and human health. Overviews of recent studies of the impact

of biodiesel on air pollutant emissions and health effects are available (McCormick, 2007; Swanson

et al., 2007). However, more than two decades ago it was shown that most of the mutagenic activity

of biodiesel exhaust may be attributed to minor components of the soluble organic fraction,

particularly the PAHs (Mauderly 1987). Sub-chronic exposure of rats to emissions from a diesel

engine burning biodiesel derived from soybean oil induced a dose-related increase in particle-

containing alveolar macrophages (Mauderly, 1994; Finch et al., 2002). However, no, or little, lung

neutrophilia or centriacinar fibrosis were observed in the majority of the exposed rats, and no

significant differences between this group of animals and an air-exposed control group were

detected in respect of most of the health parameters evaluated (Finch et al., 2002).

In an earlier in vitro study of the mutagenicities of the soluble organic fractions of diesel fuel

exhausts, it was observed that the mutagenic effect, as determined by the traditional Ames assay,

was lower for RME biodiesel than for fossil diesel (Eckl et al., 1997). Ames genotoxicity test is

normally used to detect whether a substance is mutagenic and eventually carcinogenic. The

bacteria Salmonella used in this test can not normally grow in the absence of histidine, but after

exposure to a mutagenic substance inducing DNA changes , no longer need histidine in order to

grow. Usually two Salmonela strains are used (TA98 or TA100) with or without rat liver-derived

metabolic activation system (S9). Additionally, the mutagenic (Ames genotoxicity test) and

cytotoxic (assayed against mouse lung fibroblast line L929) effects of diesel engine exhausts from a

modern passenger car burning RME biodiesel or PDD fuel have been compared directly (Bünger et

al., 1998). In comparison with the control, particle extracts of both fuels induced significant

increases in the number of mutations, although for PDD fuel the revertants were significantly higher

compared with RME. The results of the study suggested a higher mutagenic potency of PDD

compared with RME, which was probably due to the lower content of PAC in RME exhaust,

although the emitted masses of RME were higher in most of the test procedures applied in this

study.

- 38 -

In a further detailed comparative study of the toxic potentials of biodiesel and PDD, Bünger et al.

(2000) assayed the particulate matter of diesel engine exhausts from four different fuels (RME,

SME, PDD and LS-PDD for their PAC content, and mutagenic and cytotoxic effects (Table 11).

Parameter PDD LS-PDD SME RME

Mutagenic effect 100 <100 <100 <100

Cytotoxic effect 100 400

PAH 100 <100 <100 <100

Particulate matter 100 ~50 100 ~50

Insoluble matter 100 <100 <100 <100

Sulphur 100

Table 11. Comparison of the main characteristics of PDD and LS-PDD fuels with those of plant-

derived biodiesels (SME and RME). (Table modified from Bünger et al., 2000).

The results indicated that diesel exhaust particles from RME, SME and LS-PDD contained less

carbon black and total PACs, indicating that they should be significantly less mutagenic in

comparison with PDD. Indeed, when the soluble organic fractions were assayed for mutagenic

effect using the Ames assay, the extract from PDD showed an activity that was 4-fold higher with

tester strain TA98 (and 2-fold with TA100) than that of the RME extract. It may be deduced,

therefore, that the lower mutagenic potency of biodiesel exhaust particles compared with fossil

diesel exhaust is likely due to lower emissions of PAC. High sulphur content of the fuel and high

engine speeds (rated power) and loads are also believed to be related to an increase in mutagenicity

of the diesel exhaust particles (Bagley et al., 1998; Bünger at al., 2000b). Despite higher total

particle emission, the solid particulate matter (or soot) in the emission from RME was less than in

those from PDD. While the size distributions and the numbers of emitted particles at "rated power"

were nearly identical for the two fuels, at "idling" speed PDD fuel emitted substantially higher

numbers of smaller particles than RME. However, Bünger et al. (2000a) reported that RME extracts

exhibit a 4-fold stronger toxic effect on mouse fibroblasts at "idling" speed, but not at "rated

power", than PDD extracts. The higher toxicity observed is probably caused by increased levels of

carbonyl compounds and unburned fuel. Results such as these illustrate the benefits, as well as the

disadvantages, for humans and the environment arising from the use of RME as a fuel in, for

example, light and heavy duty trucks and tractors.

Although combustion of the biodiesel blend gave rise to small reductions in the emission of most of

the aromatic and polyaromatic compounds in comparison with PDD, such differences were not

statistically significant at the 95% confidence level. On the other hand, the increase of 18% in the

amount of formaldehyde produced by the biodiesel was statistically significant. However, in vitro

toxicological assays showed that the overall mutagenic potencies and genotoxic profiles of the two

fuels were similar (Turrio-Baldassarri et al., 2004).

A number of economic incentives have recently led to an increase in the use of crude unmodified

vegetable oil as a fuel in Germany. In response to this growing tendency, a systematic comparison

of the mutagenic activity of cold-pressed crude rapeseed oil (RSO) with RME, natural gas derived

synthetic fuel (gas-to-liquid; GTL) and reference PDD has been performed on the basis of the

emissions from a heavy-duty diesel truck engine running the European Stationary Cycle, (Bünger et

- 39 -

al., 2007; Krahl et al. 2007c, 2009). PMs from the exhausts were sampled onto polytetrafluoro-

ethylene-coated glass fibre filters and extracted with dichloromethane in a Soxhlet apparatus, while

the gas phase constituents were sampled as condensates. The mutagenicities of both extracts and

condensates were assayed using Ames assay with tester strains TA98 and TA100. Compared with

PDD, the mutagenic effects of the RSO fuel extracts were significantly larger, showing a 9.7-fold

increase with strain TA98, and a 5.4-fold increase with strain TA100 (Figure 13; Bünger et al.,

2007; Krahl et al. 2007c, 2009).

Figure 13. Numbers of revertants in tester strains TA98 (upper panel) and TA100 (lower panel)

with or without rat liver-derived metabolic activation system (S9) (mean ± standard deviations of

quadruple tests) induced by particle extracts and the corresponding exhaust condensates per L of

exhaust following the combustion of diesel fuel (DF), natural gas derived fuel (GTL), rapeseed

methyl ester (RME), rapeseed oil (RSO) and preheated rapeseed oil (mRSO). TA98-S9" and

"TA98+S9" - tester strain TA98 with or without rat liver-derived metabolic activation system (S9)

Significance of the differences between mean values for DF compared with the other fuels were

analysed using two-tailed Student’s t-test for independent variables (* P < 0.01, ** P < 0.001, *** P

< 0.0001). (data taken from the table in Bünger et al., 2007).

Additionally, the condensate obtained from RSO fuel exhibited a 13.5-fold higher mutagenic

activity compared with the reference fuel. The mutagenicities of RME extracts were also

significantly higher than those of PDD in both TA98 assays with metabolic activation and in TA100

assays without metabolic activation, but the increases were more moderate than those observed with

RSO fuel. No significant differences in mutagenicities were observed between GTL samples and

those derived from PDD fuel. As was mentioned above, there was a big difference in

aldehydes/ketones emitted from rape seed oil compared with biodiesel, and this is apparently due to

the glycerol moiety that can be transformed into acrolein and other carbonyl-containing volatile

compounds. It might be speculated that some of these volatile compounds can be lost during rotary

TA98-S9

DF GTL RME RSO mRSO0

500

1000

1500Extracts

Condensates

*

*****

***

Fuel

Revera

nts

, m

S

D

TA98+S9

DF GTL RME RSO mRSO0

500

1000

1500Extracts

Condensates

**

***

***

***

**

Fuel

Revera

nts

, m

S

D

TA100+S9

DF GTL RME RSO mRSO0

500

1000

1500Extracts

Condensates

**

***

***

Fuel

Revera

nts

, m

S

D

TA100+S9

DF GTL RME RSO mRSO0

500

1000

1500Extracts

Condensates

**

***

***

Fuel

Revera

nts

, m

S

D

- 40 -

evaporation of the dichloromethane solutions prior to bioassay, since their recovery has not been

estimated in the course of preparation of analytical samples and the method was not validated for

the accuracy.

It should be mentioned that some unregulated volatile pollutants, such as formaldehyde,

acetaldehyde [lethal concentration 50 (LC50) 37 g/m3 in rats, lowest-observed-adverse-effect-level

(LOAEL) 728 mg/m3 in rats), and acrolein (LC50 3750 mg/m

3 in rats, LOAEL 4.6 mg/m

3 in rats),

although present in small amounts, are known to exhibit mutagenic and/or carcinogenic activities

(US EPA 2002b). Acetaldehyde forms peroxyacetyl nitrate (via formation of peroxyl radicals and

reaction with NO2), which has been shown to be a direct-acting mutagen toward S. typhimurium

strain TA100 (Kleindienst et al., 1985; Shepson et al., 1986).

To date, only one epidemiological study is available concerning the acute effects on health resulting

from exposure to biodiesel exhaust fumes (Hasford et al., 1997). In this investigation, a

questionnaire was applied to groups of workers who might be constantly exposed to diesel fumes,

i.e. delivery truck drivers, road-maintenance workers and industrial fork lift truck drivers. The

results revealed a range of dose-dependent respiratory symptoms following exposure to RME and

PDD fumes, but no significant differences between the two fuels could be established.

It conclusion it should be emphasised that:

the substitution of PDD by biodiesel fuel requires greater consideration than has been given

to date in respect of a number of important factors relating to human health,

biodiesel is usually employed in the form of a blend (containing between 2 – 20%) with

petroleum diesel, and this is somewhat unlikely to affect significantly the quantity and

composition of emissions and the potential biologic effects of the exhaust, since a negative

effect of blends has been observed with respect to the level of exhaust gas emission, whilst

an increase of mutagenic potency was been reported for such blends showing the maximal

mutagenic effect using B20 (20% of biodiesel and 80% of PDD), which must be considered

as a critical blend (Krahl et al., 2008),

numerous additives are present in biodiesel fuel and these may have significant effects on

human health,

low-quality biodiesels (for example, those that have received inadequate post-

transesterification refining) emit larger quantities of aldehydes and their exhaust emissions

may be associated with varying impacts on the indices of human health,

the use of pesticides on the crop plants with subsequent contamination of feedstocks could

possibly affect human health and this requires further investigation (Van Gerpen et al.,

2004).

Compared with emissions from PDD, those from biodiesel have been shown to contain less PM,

CO, total hydrocarbons and other toxic compounds including vapour-phase C1-C12 hydrocarbons,

aldehydes and ketones (up to C8), certain semi-volatile and particle-phase PAHs. Whilst sulphur-

containing compounds appear to be undetectable in biodiesel exhausts, NOx and soluble organic

fraction comprising unregulated pollutants including formaldehyde, acetaldehyde, acrolein,

benzene, 1,3-butadiene, ethylbenzene, n-hexane, naphthalene, styrene, toluene, and xylene, are

elevated. The mutagenic potential of these unregulated pollutants may have been underestimated

and should be carefully and extensively evaluated.

- 41 -

Toxicological studies have demonstrated the lower mutagenicity of exhaust particles from biodiesel

in general, although fuel derived from rapeseed oil presented toxic activity that was 4-fold higher

than that of PDD. Such increased toxicity is probably caused by the presence of carbonyl

compounds and unburnt fuel. The single epidemiological investigation carried out in humans

demonstrated dose-dependent respiratory symptoms after exposure to RSO or to PDD, but no

significant differences between the two fuels could be detected. The available data represent a cause

for deep concern regarding the future application of rapeseed oil as a replacement for fossil fuels.

7.3.2. Bioalcohol

7.3.2.1. Composition of bioalcohol exhaust.

Initial studies regarding the use of bioalcohol as an alternative fuel were rather encouraging and

provided evidence that the addition of alcohol to gasoline or diesel could reduce the formation of

regulated toxic compounds in the exhaust emissions (Schifter et al., 2001). Indeed, Hansen et al.

(2006) found that NOx emission was suppressed by the addition of 5% ethanol to biodiesel-PDD

blends. It was concluded that ethanol could act as an effective additive to reduce NOx emissions

from biodiesel (Hansen et al., 2006).

Arapatsakos et al. (2003) measured gaseous emissions (CO and hydrocarbons) and fuel

consumption when different ethanol-gasoline mixtures (containing 10, 20, 30 or 40% alcohol) were

burnt in an engine functioning at idle speed and under full load (1 kW). Significant decreases in

gaseous emissions were obtained with blended fuels, and these were roughly proportional to the

percentage of ethanol in the fuel mixtures. It has been shown that blending gasoline with ethanol

leads to a reduction in tailpipe emissions of CO by as much as 30%, volatile organic compounds by

up to 12%, and particulate matter by more than 25%. Since volatile organic compounds readily

form ozone in the atmosphere, the use of ethanol could play an important role in smog reduction.

Ethanol 85 (E- 85) is a commercially available alternative fuel that contains 85 percent ethanol and

15 percent unleaded gasoline. Sheehan et al. (2004) demonstrated that burning E-85 instead of

gasoline reduced the emissions of CO, NOx and total hydrocarbons. However, whilst some authors

support the view that ethanol reduces total hydrocarbon emissions (typically by between 2 and

17%) (Hsieh et al., 2002; Al-Hasan, 2003), others have reported increases in total hydrocarbon

emissions by using ethanol (Poulopoulos et al., 2001; Magnusson et al., 2002).

As for the unregulated pollutants, the results of recent studies are rather frustrating. Thus, the

tailpipe emissions of aldehydes (including formaldehyde, acetaldehyde, acrolein , acetone,

propionaldehyde, crotonaldehyde, methylacrolein, mutyraldehyde, benzaldehyde , o-tolualdehyde,

m-tolualdehyde, valeradehyde, hexaldehyde, dimethylbenzaldehyde) from E10 ethanol-blended

gasoline (containing 10% of ethanol) and ethanol-biodiesel were compared with those from

gasoline and diesel (Pang et al., 2008). Total aldehydes from E-10 varied in the range of 9.2-

20.7 mg/kW.h, values that were 3.0 – 61.7% greater than those from gasoline emissions. The total

aldehyde emissions from ethanol-biodiesel were 1–22% higher than those from PDD under

different engine conditions. Compared with fossil fuels, E-10 presented a slight reduction in CO

emission whilst ethanol-biodiesel showed a substantial decrease in PM emission. However, both

alternative fuels exhibited slight increases in NOx and acetaldehyde emissions.

- 42 -

The problem is that the incomplete combustion of alcohols (oxidative transformations below) in an

internal-combustion engine generates a number of intermediate toxic compounds, e.g. acetaldehyde

and formaldehyde, which have been found in the exhaust emissions [Pang et al., 2008].

[O] [O] [O] [O]

CH3OH HCHO HCOOH CO CO2

Methanol formaldehyde formic acid carbon monoxide carbon dioxide

[O] [O] [O] [O]

C2H5OH H3CCHO H3CCOOH CO CO2

Ethanol acetaldehyde acetic acid carbon monoxide carbon dioxide

Ethanol and methanol are aldehyde precursors and could be partially combusted to form

formaldehyde or acetaldehyde, which are considered to possess actual or potential carcinogenic

properties, respectively (International Agency for Research on Cancer IARC, 1985). Moreover, in

the atmosphere, acetaldehyde increases formation of highly toxic ozone, which can significantly

increase mortality, hospitalization, emergency room visits (Jacobson 2007) and peroxyacetyl nitrate

(PAN), which is known as a potent carcinogen.

Winebrake et al. (2001) estimated toxic emissions of vehicles operating on a variety of fuels,

including conventional gasoline (CG), reformulated gasoline (FRFG), conventional diesel (CD),

conventional natural gas (CNG), ethanol (E85) , methanol (M85), liquefied petroleum gas (LPG)

and electricity (EV). A version of Argonne National Laboratory’s Greenhouse Gas, Regulated

Emissions, and Energy Use in Transportation(GREET) model was used for evaluation of a total

fuel-cycle analysis that calculates emissions of the four most common unregulated mobile air toxics

(acetaldehyde, benzene, 1,3-butadiene, and formaldehyde) from both downstream(e.g., operation of

the vehicle) and upstream (e.g., fuel production and distribution) stages of the fuel cycle. For almost

all of the fuels studied (except of ethanol) , emissions of volatile organic compounds (VOC) were

reduced in comparison with conventional gasoline (Figure 14 c). On the other hand, the addition of

ethanol to gasoline (as in E85) or to RFG was found to lead to a significant increase in acetaldehyde

and formaldehyde emissions (Figure 14 a, b), and the use of methanol or compressed natural gas

(CNG) may result in increased formaldehyde emissions. Considering the emission levels of the four

air toxics (i.e. benzene, 1,3-butadiene, acetaldehyde, and formaldehyde), together with their cancer

risk factors, the authors concluded that all of the alternative fuels and vehicle technologies

presented emission reduction benefits (Winebrake et al., 2001).

In a similar study Jacobson (2007) used a combination of US inventory field data and modeling to

conclude that the high acetaldehyde, formaldehyde, ozone and PAN can be significantly higher in

USA due to the use of ethanol-gasoline blend (E85) instead if conventional gasoline (figure 15 )

- 43 -

Figure 14. Changes in fuel-cycle of: (a) - VOC, (b) - formaldehyde, and (c) - acetaldehyde

emissions for varieties of mobile fuels {reformulated gasoline (FRFG), conventional diesel (CD),

conventional natural gas (CNG), ethanol (E85) , methanol (M85), liquefied petroleum gas (LPG)

and electricity (EV)} relative to conventional gasoline (CG) vehicles. Figures taken from

Winebrake et al., 2001with permission of the author.

.

a b

c

- 44 -

Figure 15. Modeled differences in the August 24-hour average near-surace mixing ratios of (b)

acetaldehyde, (c) formaldehyde, (f) ozone, and (g) PAN in Los Angeles and the United States when

all gasoline vehicles (in2020) were converted to E85 vehicles. Figures taken from Jacobson 2007

with permission of the author.

In conclusion, whilst substantial reductions in PM from ethanol-biodiesel and in CO from E-

gasoline could be achieved, the significant augmentation of carbonyl emissions from the alternative

fuels should arouse public concern because of their potential effects on human health and the

environment

- 45 -

7.3.2.2. Toxicological studies of exhausts from bioethanol and other alcohol-blended fuels.

In fact, very few comparative investigations have focused on the toxic emissions from vehicles

running on alternative fuels.

Comparative acute and chronic toxicity studies have been carried out in which rodents were

maintained for periods of 5 weeks in chambers into which exhaust fumes from gasoline and ethanol

engines were introduced (Massad et al., 1985, 1986). The results showed that the acute toxicity (in

terms of LC50) and the chronic toxicity (assessed in terms of various biological parameters

including pulmonary function and mutagenicity along with haematological, biochemical and

morphological examinations) of gasoline exhaust was significantly higher than that of ethanol.

Ethanol-based blends

The first attempt to evaluate the toxicities of the particulate phases of exhaust emissions from

vehicles fuelled with gasoline and alcohol-mixed fuels was based on the cellular test system known

as the brown-fat-cell (or BFC) test (Bernson, 1983). The results were evaluated by analysis of

variance (ANOVA) with multiple comparisons, and revealed that the exhaust extracts from cars

running on commercially available gasoline were significantly more toxic to cellular oxygen

consumption than equivalent extracts derived from alcohol-mixed fuels. The lowest toxicities were

found in extracts obtained from exhaust gases that had been submitted to catalytic processing.

However the results of two latest studies are somewhat confusing (Song et al., 2007; Jacobson,

2007). One of them is an experimental study (Song et al., 2007), while the second one is based on

field data, simulation of mathematical/ computerized models and cancer risk factors values for the

variety of pollutants (Jacobson, 2007). U.S. Environmental Protection Agency (EPA) estimated

Cancer Unit Risk Estimate (CURE) values are for acetaldehyde 2.7 x 10–6 (µg/m3)–1; benzene 2.9

x 10–5 (µg/m3)–1; 1,3-butadiene1.7 x 10–4 (µg/m3)–1; and formaldehyde 6.0 x 10–6 (µg/m3)–

1;Diesel PM - 3.0 x 10–4 (Winebrake et al., 2001).

In has been shown that the gradual addition of ethanol to diesel (from 0 to 20% ethanol) increases

mutagenic activity of the exhaust emissions (Song et al., 2007). The experiments were conducted

using a heavy-duty diesel engine, and the five fuels tested were E0 (pure diesel fuel), E5 (diesel +

5% ethanol), E10 (+ 10% ethanol), E15 (+ 15% ethanol) and E20 (+ 20% ethanol). Regulated

emissions (total hydrocarbons, CO, NOx and PM) as well as PAH emissions were monitored, and

the Ames and comet assays were used to investigate the mutagenicity and genotoxicity of these

particulate extracts, respectively. The comet assay is a method of detection of DNA fragments

(visible like comets when analyzed by gel electrophoresis) indication on DNA damages in isolated

cells. It was shown that total hydrocarbons and CO emissions significantly increased in parallel

with the ethanol content in the blends up to 53.1% and 70.0% respectively (E20 vs E0). Similarly,

a significant increase of PAH (Figure 16) in the exhaust emitted from E20 was detected by GC-MS

in comparison to E0. Additionally, E20 showed the highest mutagenic effect with both TA98 and

TA100 tester strains (with or without metabolic activation) while E5 was the least mutagenic

(Figure 17). Data relating to DNA damage revealed the lower genotoxic potencies of E10 and E15

overall (Song et al., 2007).

- 46 -

Figure 16. The content (µg/kWh) of various PAH and their total amount in exhaust emissions from

ethanol-gasoline blends. Data from table 3 published in the article Song et al., 2007.

Figure 17. Numbers of brake specific revertants (1000 reverants/kWh) in tester strains TA98 and

TA100 with rat liver-derived metabolic activation system (S9) (mean ± standard deviations of

quadruple tests) induced by extracts (0.1 mg/plate) of exhaust emissions from ethanol-diesel blends

(E0 (pure diesel fuel), E5 (diesel + 5% ethanol), E10 (+ 10% ethanol), E15 (+ 15% ethanol) and

E20 (+ 20% ethanol). Significance of the differences between mean values for DF compared with

the other fuels were analysed using two-tailed Student’s t-test for independent variables (* P < 0.01,

** P < 0.001, *** P < 0.0001). Figure modified from Song et al., 2007.

It should be mentioned, however, that such an erratic concentration-effect curves might be due to

different losses of volatile mutagenic compounds (presumably aldehydes) during the evaporation of

dichlormethane solutions in the course of preparation of analytical samples for Ames test.

In a model stimulation study, assuming that E85 will partially replace gasoline in 2020, Jacobson

(2007) estimated that this replacement may be associated with an increase in asthma and O3-related

E0 E5 E10 E15 E200

50

100

150E0

E5

E10

E15

E20

Ethanol, %

To

tal

PA

H,

g/(

kW

h)

0 5 10 15 20 250

5

10

15Naphthalene

Acenaphthylene

Acenaphthene

Fluorene

Anthracene

Phenanthrene

Pyrene

Fluoranthene

Benzo(a)anthracene

Chrysene

Benzo(a)pyrene

Benzo(b)fluoranthene

Benzo(k)fluoranthene

Benzo(g,h,i)perylene

Dibenz(a,h)anthracene

Indeno(1,2,3-cd)pyrene

Ethanol, %

PA

H,

g/(

kW

h)

0 5 10 15 200

100

200

300

400 TA98 + S9 TA100 + S9

Ethanol, %

BS

R,

revera

nts

10

3/k

W h

- 47 -

hospitalisation and mortality. In Los Angeles, E85 was apparently responsible for a 9% growth in

O3-related effects, but the percentage increase for the US as a whole was only 4% since the rises in

the northeast were partially offset by decreases in the southeast. Moreover, it has been shown that

no benefits can be expected in reductions of population-weighted number of cancer cases relative to

gasoline, suggesting that E85 may result in cancer rates similar to those of gasoline

("40/yrinLosAngelesand"430/yr in the U.S. E85 also led to an increase in the emissions of

peroxyacetyl nitrate, but it was estimated that this would cause little change in cancer risk. Because

of its ozone effect, the use of E85 may present a greater overall public health risk than gasoline.

Additionally, the emission of unburnt ethanol from E85 may result in a global-scale source of

acetaldehyde that is greater than that derived from direct emissions. However, since future emission

regulations relating to E85 are currently uncertain, it could only be concluded with confidence that

E85 is unlikely to improve air quality over future gasoline vehicles.

In conclusion, it would seem that harmful effects as well as distinct benefits must be expected to

accrue from the use of ethanol fuel blends.

Methanol-based blends

The exhaust from gasoline-fuelled engines induced DNA and chromosomal damage, thus indicating

a distinct genotoxicity, while emissions from vehicles burning methanol did not exhibit such

genotoxic potential (Zhang et al., 2005b). Gasoline exhaust showed mutagenicity in the Ames assay

(with strain TA98) with or without metabolic activation (S9), although when S9 was used

mutagenicity increased and a good dose-response relationship could be established. In contrast,

methanol exhaust presented no mutagenic potential in Ames assay (with strains TA98 or TA100)

with or without S9. With respect to in vitro A549 cell micronucleus mutagenicity tests, no

significant differences were detected between exhausts from methanol-fuelled vehicles and the

control, whilst the test was positive for gasoline exhausts. The results strongly suggested that

gasoline-fuelled vehicle exhaust was potential mutagenicity whilst that from methanol-fuelled

engines was not (Zhang et al., 2005a).

Detailed study of the genotoxicity and cytotoxicity of the organic extracts of condensate, PM and

semi-volatile organic compounds emitted by gasoline and absolute methanol-fuelled engines were

examined using Ames test and methyl-thiazol-tetrazolium, micronucleus and Comet assays (Zhang

et al., 2007). The results revealed that gasoline engine exhaust increased micronucleus formation in

human lung carcinoma A549 cells, induced DNA damage in A549 cell lines and increased TA98

revertants in a concentration-dependent manner in the presence of metabolic activating enzymes. In

contrast, methanol engine exhaust did not to exhibit these adverse effects suggesting that methanol

may represent a cleaner automobile fuel.

The effects of diesel fuel blended with methanol-containing-additive (MCA) on the toxicities and

genotoxicities of the exhaust emissions have been investigated by Lin et al. (2002). Five blends of

diesel fuel containing 0, 5, 8, 10 or 15% by volume of MCA were tested, and the results of

Microtox assays indicated that fuels with additive presented moderately lower toxicities of particle-

associated samples, but generally higher vapour-phase associated toxicities. Fuels containing 5 or

8% of additive showed relatively lower genotoxicities according to the Mutatox test in comparison

with those of base diesel.

- 48 -

A recently conducted health assessment on rodents of the combustion emissions of a diesel fuel

emulsion comprising diesel-water-methanol was consistent with results obtained with PDD exhaust

(Reed et al., 2006). Animals were exposed to exhaust atmospheres for 61 to 73 days at the rate of 6

h/day for 5 days/week for the first 11 weeks and for 7 days/week thereafter. Indicators of general

toxicity (body and organ weight, and clinical, pathological and histopathological examinations),

neurotoxicity (glial fibrillary acidic protein assay), genotoxicity (Ames assay, micronucleus assay

and sister chromatid exchange) and reproduction and development were monitored. The outcomes

of the combustion emissions of the emulsion were mild, and no significant reproductive or develop-

mental toxicity, neurotoxicity or in vivo genotoxicity effects were observed. However, some

emission sub-fractions did exhibit positive mutagenic responses in several strains of S.typhimurium.

The presence of methanol in a fuel blend has no significant effect on the rates of emission of

regulated exhaust components (organic material, CO or NOx) or on the composition of total

hydrocarbons, with the exception of methane which is increased significantly (Gabele, 1990).

However, as the percentage of methanol in the blend rises, formaldehyde and methanol account for

increasingly larger proportions of the organic material while hydrocarbons comprise less.

In conclusion, the combustion of alcohol-containing fuels leads to reduced formation of

photochemical smog in comparison with gasoline or diesel, although the emission of potentially

carcinogenic aldehydes may be several-fold higher. The toxicity of the emission from a gasoline-

fuelled engine is significantly higher than that from an engine fuelled by alcohol, however, some

harmful effects from alcohol blends must be expected including increased emissions of

carcinogenic PAHs and an increase in O3-related toxicity associated with the emission of high

levels of aldehydes. Moreover, ethanol-diesel fuel blends could increase the emission of unburnt

hydrocarbons and aldehydes.

The most preferable fuels in terms of reduced toxicity and genotoxicity in comparison with PDD

exhaust emissions are those containing methanol, however, this alcohol represents a source of

formaldehyde pollution and is potential carcinogenic.

7.3.3. Biogas

The methane component of biogas would normally suffer incomplete combustion in an internal-

combustion engine and, hence, the following toxic compounds could be present in the exhaust:

[O] [O] [O] [O] [O]

CH4 CH3OH HCHO HCOOH CO CO2

Methane methanol formaldehyde formic acid carbon monoxide carbon dioxide

In addition, nitrogen can be converted into nitric oxides and hydrogen sulphide into sulphur oxides:

[O] [O]

N2 NO NO2

[O] [O] [O]

H2S SO SO2 SO3

- 49 -

Very few publications are available concerning the toxic effects of biogas-derived emissions.

Although CNG is often regarded as a clean-burning fuel, characterisation of the emissions from

vehicles fuelled by CNG has revealed that the exhaust contains potentially toxic materials (Lapin et

al., 2002; Ristovski et al., 2004; Seagrave et al., 2005). In comparison with diesel-fuelled vehicles,

those burning CNG appear to produce less particulate matter but more total hydrocarbons

(McCormick et al., 2000). In contrast, Ristovski et al. (2004) demonstrated that conversion of an

engine from gasoline fuel to CNG led to a significant reduction in the emissions of total PAH and

formaldehyde but had no effect on the emission of particles.

The potential health risks of particulate exhaust emissions from natural gas fuelled vehicles have

been investigated by Lapin et al. (2002). Organic solvent extracts of PMs from natural gas fuelled

heavy-duty engine tested positive against Salmonella strains TA98 and TA100. The mutagenic

activity of natural gas-derived particulate matter was similar to that of the PM from a light-duty

diesel vehicle.

Seagrave et al. (2005) compared the composition, mutagenic potential and acute lung toxicity of the

collected emissions from heavy-duty vehicles fuelled by CNG and PDD. One important outcome of

the study was that it confirmed and extended previous data (Lapin et al., 2002) relating to the

organic extracts of the PM fraction of a CNG-powered heavy-duty truck and showed that such

emissions contained substantial amounts of direct-acting mutagens. Vapour-phase semi-volatile

organic compounds and PMs were collected from three CNG-fuelled buses, powered by three

different engines including a new technology engine with an oxidative catalytic converter. The

emissions were analysed chemically and their mutagenicities were assessed using the Salmonella

reverse mutation assay. The emission samples were also compared with those collected from

normal and ―high-emitter‖ gasoline and diesel engines. While the range of mutagenic potencies of

the CNG emission samples was similar to those determined for the gasoline and diesel emission

samples, lung toxicity potency factors were generally lower than those for gasoline and diesel

samples (Seagrave et al., 2005). Among the engines tested, those with oxidative catalytic converters

showed lower mutagenic effects.

It may be concluded that the use of biogas, and particularly CNG, can significantly reduce the

emissions of total PACs and also diminish lung toxicity potency. However, the emission of PM and

the mutagenic potencies of the exhaust gases are similar to those found in gasoline and diesel

emission samples. These findings require follow-up studies in order to develop a database relating

to natural gas fuelled vehicles that would allow informed decisions to be made on the use of

alternative fuels to reduce air pollution health risks.

7.3.4. Other biofuels.

Handling cellulosic (second generation) biofuels such as straw, wood chips etc may release dust

particles that contain high concentrations of hazardous micro-organisms, thus representing a

significant potential occupational health problem. It has been found that the storage of such biofuels

outdoors over summer months increases the microbiological contamination (or dustiness) and

should therefore be avoided. The bacterial dustiness of straw is much greater than of wood chips,

whereas the fungal dustiness does not differ much (Sebastian et al., 2006). Moreover, ecological

straw generally contains less microbe-laden dust than conventional straw and is hence the preferred

- 50 -

feedstock. Samples taken from the inner part of biofuel materials are typically dustier than samples

taken from the surface, except for fungal and bacterial biomass in wood chips and total fungi and

fungal biomass in ecological straw.

8. Discussion and overall conclusions

It is very good idea to use a renewable energy source, but it does not necessarily mean that such

fuels would necessarily be "healthy for humans‖. At present, the use of biofuels is favourably

viewed and there are suggestions that its emissions are less hazardous to human health compared

with petroleum gasoline and diesel. As shown by an advertisement in Arlanda airport in Stockholm

(Figure 18), biofuel is currently believed to be ―clean enough to drink‖.

However, the less harmful effects of biodiesel on human health is speculative and far from reality.

Indeed, the combustion of biofuel alone or blended with petroleum fuel produces toxic emissions

that are, in many ways, similar to those present in the exhaust of petrofuels. Thus, alongside the

regulated toxic emissions, such as total hydrocarbons, CO, NOx, PM and PAH, other non-regulated

emissions, especially carbonyls (i.e. formaldehyde, acetaldehyde and acrolein), are also present in

the exhaust from burnt biofuel and these compounds are potentially detrimental to human health.

Figure 18. An advertisement in Arlanda airport in Stockholm

It might be reasonable to classify biofuels into different categories depending on their toxicity:

A. crude or refined vegetable oil, which should not be recommended for use as fuel due to the

extremely high mutagenicity of the exhaust.

B. biodiesel, which has substantially lower amounts of carcinogenic and mutagenic PAH than

PDD,

C. biogas, which can significantly reduce the emissions of PAH, but not the emission of PM

and the mutagenic potencies of the exhaust gases which is comparable to those found in

gasoline and diesel emission.

D. ―pure‖ bioalcohol fuels, which do not emit highly toxic PAH and particulates, but still

contain mutagenic and carcinogenic non-regulated pollutants

- 51 -

A serious problem of biodiesel is associated with NOx levels, which are normally enhanced (up to

15%), and especially with carbonyls (e.g. acetaldehyde), which can rise substantially.

In general, the safety/toxicity of highly volatile components of biofuel exhaust has not yet been

evaluated accurately. A substantial portion of these compounds can be lost during the treatment of

filters and condensates for the non regulated pollutants assay and the Ames test. Moreover, the

overall recoveries of these compounds have not been evaluated and the accuracy of the sample

preparation method has not been validated. Hence, it could be that the cytotoxic effect of biodiesel

exhaust is higher than that reported in several in vitro and animal studies (Buenger et al., 2000,

2006, 2007; Krahl et al., 2009; Song et al., 2007).

There is a great deal of additional research required in relation to these components, as well as on

the fuel blends (i.e. mixtures of PDD with up to 20% biofuels) that are currently promoted by

policies in Europe and the USA.

Additionally, toxicological studies in humans are lacking. According to the knowledge of the

present reviewer only one epidemiological study, involving a very small cohort of road workers, is

available and this has reported irritation of mucosal membranes and an unpleasant odour (similar to

old cooking oil) arising from the emissions of vehicles fuelled with biodiesel (Hasford et al., 1997).

Unfortunately, much less information is available concerning the other biofuels.

Finally, it can be concluded that:

Biofuels are potentially less harmful to human health than petroleum fuels, but may be

considerably more toxic than expected (Figure 19),

substitution of conventional fuels for biofuels in on-road engines decreases the

concentration of regulated toxic pollutants in car exhaust emissions but increases the

concentration of some non-regulated pollutants especially with carbonyls (e.g.

acetaldehyde), which can rise substantially (Figure 19),

the safety/toxicity of highly volatile components of biofuel exhaust has not been yet

accurately evaluated, and detailed toxicological studies of biofuels in humans are lacking,

from the information that is available, it appears that the toxicity of biofuels decreases in the

order biodiesel>biogas>ethanol=methanol,

on this basis, methanol (produced by the oxidation of biogas) is the alternative fuel that

appears to present the least potential for damage to human health, however, this alcohol

represents a source of formaldehyde pollution and is potential carcinogenic.

With respect to the future availability of resources for the production of biofuels (e.g. biodiesel), it

should be mentioned that these are rather limited as well. Thus, an area of about 20 000 square km

(i.e. two million ha), if cropped with the plant sources currently available, would provide a

contribution of just 25% of the fuel actually used in Germany: in this context, the total territory of

Germany comprises 357 021 square km) (Munack and Krahl, 2007).

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Figure 19. A simplified and presumably more realistic advertisement of the effect of petrofuel and

biofuel emission on human health, partially reflecting my conclusions above.

This opinion/conclusion is in line with the conclusion of Swedish scientists from Chalmers

University in Gothenburg, where the authors stated that "Hitherto, the disadvantages of renewable

products have been neglected in research and development. The advantages of renewable products

are advocated strongly by their proponents urging for a quick and subsidized market introduction."

(Pedersen et al., 1999).

9. Acknowledgements

The author wishes to thank Halmstad University, and especially Professor Lars-Gunnar Franzén for

helpful critical comments and Professor Stefan Weisner for the opportunity to carry out further

study and to research the subject area of this thesis.

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