BBAGEN-28507; No.

of pages: 7; 4C: 2
Biochimica et Biophysica Acta xxx (2016) xxxxxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

journal homepage: www.elsevier.com/locate/bbagen

A paler shade of green? The toxicology of biodiesel emissions: Recent

ndings from studies with this alternative fuel,
Michael C. Madden
National Health and Environmental Effects Research Laboratory, Ofce of Research and Development, U.S. EPA, Chapel Hill, NC 27514, United States

a r t i c l e

i n f o

Article history:
Received 29 February 2016
Received in revised form 24 May 2016
Accepted 25 May 2016
Available online xxxx
Keywords:
Biodiesel emissions
Petroleum diesel
Lung toxicity
Cardiovascular toxicity
Mutagenicity

a b s t r a c t
Background: Biodiesel produced primarily from plants and algal feedstocks is believed to have advantages for
production and use compared to petroleum and to some other fuel sources. There is some speculation that exposure to biodiesel combustion emissions may not induce biological responses or health effects or at a minimum
reduce the effects relative to other fuels. In evaluating the overall environmental and health effects of biodiesel
production to end use scenario, empirical data or modeling data based on such data are needed.
Scope of review: This manuscript examines the available toxicology reports examining combustion derived biodiesel emissions since approximately 2007, when our last review of the topic occurred. Toxicity derived from
other end uses of biodiesel - e.g., spills, dermal absorption, etc. - are not examined. Findings from biodiesel emissions are roughly divided into three areas: whole non-human animal model exposures; in vitro exposures of
mammalian and bacterial cells (used for mutation studies primarily); and human exposures in controlled or
other exposure fashions.
Major conclusions: Overall, these more current studies clearly demonstrate that biodiesel combustion emission
exposure- to either 100% biodiesel or a blend in petroleum diesel- can induce biological effects. There are reports
that show biodiesel exposure generally induces more effects or a greater magnitude of effect than petroleum diesel, however there are also a similar number of reports showing the opposite trend. It is unclear whether effects
induced by exposure to a blend are greater than exposure to 100% biodiesel. Taken together, the evidence suggest
biodiesel emissions can have some similar effects as diesel emissions on inammatory, vascular, mutagenic, and
other responses.
General signicance: While acute biodiesel exposures can show toxicity with a variety of endpoints, the potential
effects on human health need further validation. Additionally there are few or no ndings to date on whether biodiesel emissions can induce effects or even a weaker response that petroleum diesel with repeated exposure scenarios such as in an occupational setting.
This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding, Andy Ghio and Weidong Wu.
2016 Published by Elsevier B.V.

1. Introduction
Biodiesel is a liquid fuel produced by reactions of fatty acids with
methanol resulting in fatty acid methyl esters (FAMEs). Typically plant
based oils rich in triglycerides are utilized though animal derived lipids,
and waste oils including phospholipids, can supply the fatty acids but to
Disclaimer: The research described in this article has been reviewed by the National
Health and Environmental Effects Research Laboratory, U.S. Environmental Protection
Agency and approved for publication. Approval does not signify that the contents
necessarily reect the views and the policies of the Agency nor does mention of trade
names or commercial products constitute endorsement or recommendation for use.
This article is part of a Special Issue entitled Air Pollution, edited by Wenjun Ding,
Andy Ghio and Weidong Wu.
U.S. Environmental Protection Agency, 104 Mason Farm Road, Mail Code: 58B, Chapel
Hill, NC 27514, United States.
E-mail address: madden.michael@epa.gov.

a much lesser extent. In the US, soy is the primary feedstock utilized
while in Europe and Canada, rapeseed and canola are the main sources;
canola oil is rapeseed oil that is low (b2%) in erucic acid (docosenoic
acid) [19], though a variety of feedstocks have been utilized in both regions. Blends of biodiesel with petroleum diesel are typically used for
many onroad and offroad applications [44]. In the US, a 20% and 5% biodiesel in petroleum diesel blend (B20 and B5 respectively) are the typical ratios for vehicles, though neat biodiesel (B100) can be utilized in
engines designed for pure petroleum diesel (B0). (Throughout the rest
of this paper, diesel refers to petroleum diesel.)
Though the B100 is intended to be primarily methylated or ethylated
fatty acids, some nonderivatized fatty acids are contained in the fuel. For
soy-based biodiesel, the main fatty acids that were methylated to produce the fuel were C18 fatty acid derived (oleate (C18:1), linoleate
(C18:2), linolenate (C18:3)) [28,50]. Additionally other classes of compounds, e.g., aromatics, carbonyls, branched alkanes and alkenes, can

http://dx.doi.org/10.1016/j.bbagen.2016.05.035
0304-4165/ 2016 Published by Elsevier B.V.

Please cite this article as: M.C. Madden, A paler shade of green? The toxicology of biodiesel emissions: Recent ndings from studies with this
alternative fuel, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.05.035

M.C. Madden / Biochimica et Biophysica Acta xxx (2016) xxxxxx

be detected in the precombustion phase within the engine [23], making

the combustion components mixture more complex, and likely the
emission compounds more complex as well. Processes that better optimize the availability of cellulose-derived fatty acids for methylation
have come under more intense research investigations, due in part to a
projected increase use of cellulosic waste to meet U.S. Congressional targets for renewable fuels; cellulosic material will increase from approximately 0.8 million gallons in 2013 to a projected 206 million gallons in
2016 [14,47]. The methylation step is usually catalyzed with potassium
hydroxide [35] though enzymatic conversion is an alternative catalyst
[8]. Byproducts of the reactions include glycerin and methanol; what to
do with the large volume of these byproducts is a current issue. Biodiesel
production in the US as well as globally has been rapidly expanding in
terms of both production and use (Fig. 1; U.S. Department of Energy).
The expansion in production is dependent on multiple and complex
factors including nancial incentives and government policies and
regulations.
Biodiesel fuel use offers potential advantages over petroleum based
fuels. Production of the fuel could be done locally, which could possibly
minimize concerns of availability and transportation issues. Emissions
of carbon dioxide (CO2), a greenhouse gas, are lower with combustion
of biodiesel compared to petroleum diesel (US EPA, [26]). Particulate
matter (PM), of concern related to premature mortality and increased
morbidity [49], also is emitted at lower concentrations [46]. Lower
PM2.5 concentrations were observed in an occupational setting with
B20 emissions compared to B0 [41]. The total (methylene chloride) soluble organic fraction of biodiesel PM is a greater percentage than diesel;
but what must be factored into the amount of organics emitted per distance or per engine work load is that there is an overall reduction in
total mass of PM [12]. McCormick [26], citing data primarily generated
with heavy duty engines, more comprehensively reviewed the impact
of burning biodiesel on the production of regulated ambient air criteria
pollutants, greenhouse gases, as well as PM size (which affects the lung
deposition distribution and percent deposited within lung sites), and
hydrocarbons which may be active in the induction of human health effects and biological responses. His conclusions were than B20 use made
little difference in NOx emitted and overall airshed NOx levels, that the
greenhouse gas CO2 decreased with B20 and B100 combustion, and that
more data were needed to make rmer conclusions concerning changes
in PM size changes and air toxics concentrations emitted.
In order to better understand the balance of advantages and disadvantages of the use of biodiesel in the nation's energy structure, a comprehensive, up to date, life cycle analyses is needed to be in order to
determine the overall utility of using a liquid fatty acid based fuel. Life
cycle analyses include cradle to grave scenarios, from the production
of the feedstock with effects on the environment (soil quality, water
usage, wildlife composition changes), production issues (energy input,
transportation issues, occupational health protection, byproduct management, etc), storage and transport of the fuel (with potential issues

such as spills and leaks), plus issues related to consumer exposure to

the fuel (dermal, inhalation) and combustion emissions released occupationally and into the ambient environment, such as discussed in an
Oak Ridge National Laboratory Report from a 2009 Cradle to Grave
Workshop [9]. One component of the life cycle analyses lacking in
adequate data and ndings is the endstage use where potential
human toxicity could occur. With regards to human biological and
health responses, potentiating the toxicity would be predisposing sensitivity factors such as disease states, increased exposures levels, and decreased detoxication mechanisms, among some factors.
The purpose of this review is to examine more recent ndings of the
toxicity of specically biodiesel combustion emissions related to human
health. Throughout this report when biodiesel or petroleum diesel is
mentioned, it refers to combusted fuel products and not the liquid fuel
itself. Potential biological responses to biodiesel not combusted, e.g.,
dermal absorption of the fuel, are not examined in this review. The reader is referred to a 2007 review [38] of then reported effects of biodiesel
toxicology with additional literature reported in a book chapter related
to biodiesel emission toxicology [25]. A search with the term biodiesel
in PubMed revealed 66 and 129 hits for the years 2006 and 2007, respectively, but an increase to 1812 and 1620 hits in 2014 and 2015, respectively. Included in this update are reports utilizing a variety of
approaches that assist in determining potential health related outcomes
from exposures to biodiesel or components of biodiesel. Typically surrogates of health effects are measured and reported as an early biomarker
in a pathway believed to be involved in the development of an adverse
response or disease. The reader is referred to the most recent US Environmental Protection Agency (U.S. EPA) Integrated Science Assessment
(ISA) nal reports for PM [49], carbon monoxide [45], and oxides of nitrogen [48] for toxicological information related to those specic emissions found in biodiesel components.
2. In vivo exposures using animal models
Exposure of mice to B100 and B0 (i.e., petroleum diesel) PM via pharyngeal aspiration induced increases in total cells recovered in the bronchoalveolar lavage uid relative to unexposed control mice at 1 and
7 days post exposure, with the outcome resolving at day 28 after exposure. [51]. The increase at day 1 was attributed to mainly neutrophilia,
and at day 7 to an increased macrophage number. B100 induced a greater increase in neutrophils (Day 1) and macrophages (Day 7) than B0 implying a greater inammatory potency. Similarly total protein in lavage
uid as a marker of permeability was increased more in B100 treated
mice compared to B0. In lung tissue homogenate, only B100 induced
an increase in lactate dehydrogenase activity (as a marker of cell viability) on Day 1 post exposure, while both PM types induced increases on
Day 7 and 28. B100 PM induced signicantly more on Day 28 than B0.
There was a persistence of increased tissue oxidation markers (carbonyls and 4-hyroxynonenal) out to 28 days with B100 exposure, but

Fig. 1. U.S. biodiesel gures (production, consumption, exports) up to 2015. Numbers are given in millions of gallons.
Source: U.S. Department of Energy. http://www.afdc.energy.gov/data/10325 Accessed 11 January 2016.

Please cite this article as: M.C. Madden, A paler shade of green? The toxicology of biodiesel emissions: Recent ndings from studies with this
alternative fuel, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.05.035

M.C. Madden / Biochimica et Biophysica Acta xxx (2016) xxxxxx

not observed with B0 exposures. B100 aspiration tended to increase

several inammatory cytokines in lung tissue 1 day post exposure
whereas B0 did not; however as measured in lavage uid both PM induced similar changes. With chemokines and growth factors, there
were similar trends of increased mediators in both lavage uid and tissue for both B0 and B100.
Aspiration of B20 and B0 PM induced similar increases in cell numbers and inammatory cell types in the lung lavage of mice 3 days
post exposure [15]. B20 increased three cytokines (IL-6, G-CSF, and IP10) in both lavage uid and lung tissue homogenate compared to control and B0 PM, demonstrating consistent potency of the biofuel compared to the petroleum product. Greater levels of protein carbonyls
(markers of endogenous tissue oxidation) were found in lung tissue of
B20 exposed mice compare to controls and B0 exposed animals,
which did not induce a change from control.
A comprehensive study examining acute pulmonary and
extrapulmonary effects in mice of whole biodiesel exhaust exposure
(i.e., gases and PM) was reported [7]. It should be noted that the
commercial diesel fuel utilized was high in S (500 ppm) and
contained 3% biodiesel. The biodiesel utilized was soy based but
with ethyl esters of the fatty acids. Exposure to B100, B50, and B0 induced neutrophilia in lung lavage, but decreased macrophage numbers. In tissue parenchyma, total phagocytes (macrophages plus
neutrophils) increased but neutrophilia was observed only with B0
exposure. Extensive evaluation of blood cells showed B0 did not induce alterations, but a few changes induced by B100 and B50, including an increased reticulocyte % and absolute lymphocyte numbers,
respectively. Similarly with bone marrow cell analyses, only the
total cells increased with B0 exposures, but B50 and B100 exposures
also induced the increase in total cell numbers and also several cell
types. Cardiac electrophysiological changes related to heart rate,
heart rate variability, and blood pressure were monitored 30 and
60 min post exposure. With the 7 endpoints reported, B0 and B100
each changed (relative to unexposed control) two of the parameters
at both time points with both altering the low frequency domain of
the heart rate variability (HRV). Additionally B0 altered the HRV
root mean square of successive differences in heart beat interval
(i.e., RMSSD) while B100 changed the heart rate. In stark contrast,
B50 did not alter any cardiac-related endpoint. Macrophages from
both exposures at 1 day post showed the presence of particulate matter
inside the cell (as discerned by TEM); but unlike B0 exposure, at 7 and
28 days post exposure, cytoplasmic inclusions were observed in macrophages exposed to B100, which resembled lipid or foam droplets. Pulmonary inammation, measured as the numbers of neutrophils and
macrophages in lung lavage uid and also lung parenchyma pathology,
appeared greater in lung tissue out to 28 days post exposure to B100
compare to controls and B0 exposures.
BalbcJ mice were exposed to whole exhaust at multiple concentrations for 4 weeks and liver endpoints examined as well as lung endpoints [36]. [The tissues analyzed in this paper were collected from
exposures at the U.S. EPA in North Carolina, and are described in more
detail below.] Generally the B100 exposures were more potent than
the B0 exposures with B100 inducing more of an increase in both lung
and liver of LDH activity, myeloperoxidase ( a marker of inammatory
cells), 4-hydroxynonenal, and total carbonyls, and more of a decrease
in the antioxidants reduced glutathione and total thiols. The alterations
in inammation/allergy-related cytokines was not as clear in terms of
B100 having a greater potency and generally lacked exposure concentration responsiveness. With some cytokines, the responses were different, e.g., MCP-1, a monocyte chemoattractant, increased in the liver of
B100 exposed mice, but decreased in B0 exposed animals.
Recently reports have been published concerning interrelated acute
and repeated exhaust exposure studies with biodiesel and biodiesel
blend examining the potential toxicity of biodiesel. The studies
were performed at the US EPA facilities in RTP, NC using primarily rodent models and also cell in vitro exposures and Ames assays with

appropriate bacterial tester strains. Additionally B0 emissions exposures were performed to allow comparative evaluation for the biological effects observed in this set of studies. Concentrationresponse relationships were examined with exposure concentrations up to approximately 500 g PM/m3 in some studies. The effects
of the biodiesel composition, in terms of the % blend, on the responses was also studied. These reports are all contained within
the journal Inhalation Toxicology volume 27, issue 11, in 2015.
In the EPA studies, the fuel and emissions were characterized. Biodiesel blends were created by splash blending of the neat biofuel and
petroleum fuel. Splash blending of biodiesel can result in creation of
compounds not present in either the parent fuel, resulting in different
fuel composition that to some extent than would be expected [39].
Some components, or fragments of some components, were found in
the engine PM emissions due to the incomplete combustion of the fuel
including FAMEs on the PM [28]. A blend dependent increase in
FAMEs was observed as the % biodiesel increased. As in other reports,
there was a decrease in PM PAHs as the percentage of biodiesel increased; there was a greater percentage of organics (extractable in
DCM) and organic acids present on biodiesel PM in a blend-dependent
fashion. These components of biodiesel emissions (i.e., decreased
PAHs, increased organic acids and organic extractable material in general) make the exhaust components dissimilar to petroleum diesel exhaust. Additional differences in B100 exposures were characterized
including an increasing trend of methylene chloride extractable material compared with B0, B20, and B50. Previous reports [7] have shown
very little in linear changes for chemical components of biodiesel emissions as the biodiesel proportion increased in blends, with the exception
of metals, with most components such as CO, PAHs, BC, being nonlinear
(i.e., concave or convex type of relationships). It is not clear why these
nonlinear emission component relationships exist at present. It should
be noted that the Brito studies were performed with an ethylated conversion of fatty acids and with a higher sulfur content in the petroleum
diesel, which may have affected the linearity of the components with
the biodiesel blend. These observations of linear trends in biomarkers
of exposure (e.g., FAMEs, organic acids) with biodiesel blends in the
EPA studies, as compared to nonlinear relationships in some emissions
components noted in Brito et al. [7], offer a better chance for discerning
an association between an internal exposure marker and a biological
response.
As for the toxicology ndings observed in the EPA studies, several
study designs employing rodent in vivo exposures to whole exhaust
were reported. For lung responses, inammatory changes in two rat
strains (Wistar Kyoto and Spontaneously Hypertensive) demonstrated
few systematic changes with acute (1 day) or repeated (4 weeks) exposures. [5]. Neutrophilia (assessed in lavage uid) was decreased in rats
exposed to the highest B100 acute exposure (compared to clean air),
but was observed with the B20 exposure in the second strain. Changes
in the lavage concentrations of most soluble mediators were not observed except for increases -glutamyl-transferase (as a marker of cell
damage). B0 exhaust exposure as well as the particulate fraction has
been shown to alter the lymphotype type of response (i.e., Th1 vs
Th2) [11,37]. In unsensitized Balbc mice, the mice did not show lung
neutrophilia with acute B100 exposures but in great contrast B0 exhaust
induced approximately an 8-fold increase [16]. Mice exposed to B20 had
an intermediate increase (less than B0, more than B100 exposures) in
PMNs. With house dust mite sensitization involving pretreatment,
mice, after 4 weeks of repeated B100 or B20, had no altered lymph
node proliferation score compared to ltered air exposure responses,
in contrast to an increased score with B100 exposure. The T-cells of
B100 exposed mice were responsive to an agonist with increased production of several cytokines (relative to ltered air responses). The B0
exposures seemed to have induced an inhibitory effect on the ConAinduced cytokine responses; the B0-exposed lymph node tissue no longer had signicant increases relative to ltered air in response to ConA
incubation.

Please cite this article as: M.C. Madden, A paler shade of green? The toxicology of biodiesel emissions: Recent ndings from studies with this
alternative fuel, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.05.035

M.C. Madden / Biochimica et Biophysica Acta xxx (2016) xxxxxx

Exposure to criteria air pollutants can induce extrapulmonary effects

[10,40]. Examining for extrapulmonary effects, spontaneous hypertensive (SH) rats exposed to whole exhaust B100 and B0 for 4 h showed
primarily decreased heart rate variability (HRV) endpoints and heart
rate (HR), though not in a systematic manner; but B20 did not. HRV
changes were demonstrated at some of the lower B100 exposures
therefore the data suggested greater potency for the neat biofuel emission source [13]. Changes induced in the blood (cells and uid) related
to immune function and clotting were examined for pollutant-induced
effects. Angiotensin Converting Enzyme was increased with B100 and
B0 exposures, with B20 exposure again being the exception showing
no change. Fibrinogen was increased only in B0 exposed rats. Creactive protein, a marker of inammation, was the only biomarker to
increase with B100 exposure; interestingly, some of the markers related
to atherosclerosis (HDL, LDL, and triglycerides) along with blood urea
nitrogen (BUN) and total protein decreased after B100 exposure. BUN
levels are related to liver and kidney function and may therefore indicate involvement of these extrapulmonary systems in the response to
combustion emissions. The data support more potency for B100 compared to B0 for these endpoints.
With SH rats arrhythmias were discerned in acutely (1 day) or repeatedly (5 days) exposed animals when challenged with aconitine
(to induce stress) post exposure [21]; all fuels were equipotent in inducing the responses i.e., a decreased dose of aconitine needed to induce
specic types of arrhythmias. Five days of B20 exposure were needed
to observed these induced arrhythmias while the other two blends induced the response with one day of exposure. More adverse effects,
i.e., cardiac arrest, were observed at greater emission exposure concentrations for all 3 fuel types.
3. In vitro exposures with cells and bacteria
With the US EPA studies performed in RTP, three papers reported on
mutagenicity and DNA damage. Using dichloromethane-extracted PM
from the biodiesel (B0, B20, B50, B100) emissions, mutagenicity was determined in more than a dozen combinations of Salmonella strains with
and without S9 fraction activation [29,30]. For B0, B20, B50, and B100
about 60% of the organic extractable material was nonpolar. Revertant
values were normalized for energy generated by the combustion in
order to standardize for fuel and combustion technology variability
among studies. The data can best be summarized overall as showing decreased mutagenicity with an increasing proportion of biodiesel in the
fuel. The authors showed the relative mutagenicity of the extracted
PM in context of PM from other combustion types from reports in the
literature; the PM from the neat biodiesel, petroleum diesel and blends
fall in the middle of these multiple types of combustions ranging from
open burns to better controlled utility oil, coal, and gas combustion. Follow-up work with the PM extractions from the different combusted
fuels used column separation to demonstrate that most of the mutagenicity was found in the polar and weakly polar fractions. B100 had less
mutagenicity likely due to decreased amounts of these polar and weakly
polar fractions relative to B0. B20 had highly polar compounds contribute to the mutagen load seen in the Ames assays. Using P32 postlabeling techniques with DNA from calf thymus and Salmonella TA100
strain to detect markers of damage, the adduct levels of the extracts
after S9 metabolism were examined. The DNA damage were greater
with B100 PM extracts compared to B0, with B20 inducing the lowest
levels of adducts [33].
Using a combination of exposure approaches, the mutagenicity of B7
and B30 (rapeseed methyl ester with 50 ppm S containing petroleum
diesel) was examined [3]. Extracts, whole PM, and whole exhaust exposures of several tester strains, A549 epithelial cells, and rat lung slices
were studied. The effects of emissions passing through an oxidation catalyst and in some cases a particle lter were also studied. Endpoints
were bulky DNA adducts and bacterial mutagenicity in three bacterial
strains. The data showed that PM produced little or no DNA bulky

adducts in A549 cells or rat lung slices. As well documented with B0

PM, B0 PM methylene chloride extracts induced mutagenicity, especially with an S9 metabolism, in TA98 and YG1041, but not TA102, strains.
Mutagenicity was also observed in the same two strains with B7 and
B30 PM. An oxidation catalyst eliminated the mutagenicity of the extracts. When whole PM (in DMSO after methylene chloride extraction)
were added to the cultures, there was a greater mutagenicity in B30
treated TA98 and YG1041 cultures compared to B0. Whole B30 exhaust
and the exhaust with the majority of B30 PM removed still exhibited
mutagenic capability in TA98 strains (but not other strains) suggesting gas phase mutagenic components; the mutagenicity was decreased after passage through the oxidation catalyst. In contrast,
PM traps removed most or all of the B0 mutagenic agents while the
oxidation catalyst did not. The authors noted that some experiments
with the bacteria were characterized by cytotoxicity at the highest
exposure concentrations.
The effect of a PM trap on biological responses of differentiated (via
air-liquid interface incubation) normal human bronchial epithelial
(NHBE) cultures upon exposure to B0 and B99 was investigated. The authors examined mRNA transcript levels as surrogates for heme oxygenase and cytochrome P450 1A1 proteins. Both B0 and B99 whole exhaust
exposures increased the two biomarkers after 60 min of exposure. [20].
Removal of PM via the trap did not eliminate the increases in the two
mRNAs; the data showed greater magnitudes of increases in the
mRNAs with the PM removed suggesting that the PM fraction attenuated the cellular responses. It should be noted that the exposure system
utilized placing an electrostatic charge on the PM emissions to improve
PM deposition on the cells.
The oxidative potential of B0 and B50 (rapeseed methyl ester) PM
extracts was examined in a cell free system examining the disappearance of ascorbic acid or dithiothreitol in separate assays. Under an
urban driving cycle, B0 PM had showed a higher oxidative potential
than B50 PM; the PM collected after passing through a diesel particle lter had less potential for both fuel types but a similar trend of the B0
being more potent. With a rural driving cycle, there was no clear trend
for which fuel type had more potency or if the lter altered the potency
[17]. The authors noted that general ndings could be dependent on the
normalization process, i.e., be it per distance unit driven or per mass
equivalent, as well as the type of driving cycle. For instance, using a
rural driving cycle, B50 PM were more potent for induction of cytotoxicity (EC20; expressed as propidium iodide staining) if expressed on a
per mass basis, but not on a per distance basis. The induction of cytokine
interleukin (IL) 6 production was more potent for B50 PM generated
with an urban driving cycle when the PM lter was in place on a per
mass but not on a per distance basis. PM concentrations used for the
cell assays ranged up to approximately 200 g/ml.
With the lung epithelial cell line (A549), B20 PM extracted with
methylene chloride induced more multinucleated cells (52%) compared
with B0 (12%), while unexposed cells were at 7% at one concentration
(25 g/ml); it is unclear if the values were statistically signicant.
More apoptosis was observed in the B20 exposed cells [1] relative to
control cultures.
Rat alveolar macrophages exposed to B20 particles at as low as10 g/
ml released more of the immune/inammatory lipid mediator prostaglandin E2 [6] relative to untreated macrophages; the release was not altered in cultures incubated with B0. B20 and B0 were noncytotoxic to
macrophages up to 100 g/ml; cytotoxicity was observed with greater
exposure concentrations.
The effects of PM matter from B0 and biodiesel (as B20) at multiple
exposure concentrations using a variety of cell types and endpoints
were studied [22]. It should be noted that two Euro driving cycles (i.e.,
Euro2 and 4) with two engines meeting these standards (i.e., an older
and newer engine, respectively) and biodiesel fuel feedstocks (animal
fat, rapeseed oil) were utilized to produce the PM utilized, and due to
the complexity of the exposure conditions and endpoints, the ndings
summarized in general here. With vascular human umbical vascular

Please cite this article as: M.C. Madden, A paler shade of green? The toxicology of biodiesel emissions: Recent ndings from studies with this
alternative fuel, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.05.035

M.C. Madden / Biochimica et Biophysica Acta xxx (2016) xxxxxx

endothelial cell (HUVEC) cultures, expression of phagocyte adhesion

molecules was affected only by B0 exposure. With HUVECS, there was
overall concentration-related suppression, relative to a cell free system,
in reactive oxygen species production (dichlorouorescein formation)
with both B20 exposures but only one B0 exposure. Using airway epithelial cells (the cell line A549), DNA damage endpoints strand breaks
and formamidopyrimidine glycosylase sensitive sites showed primarily
concentration-dependent increases in both DNA damage markers compared to control exposures with all PM with small differences between
the B20 and B0 particles. There was no induction of inammatory gene
(interleukin 8 (IL8) and monocyte chemotactic protein 1 (CCL2))
changes with the B0 and B20 exposures using a monocyte cell line
(THP-1). The authors summarized the various ndings and compared
which driving cycle and fuel produced more toxicity overall with 10
endpoints and 3 cell types. The authors concluded that the B0 emissions
PM was generally more toxic and that the Euro 4 driving cycle induced a
greater magnitude of effect.
In vitro assay of B20 in THP-1 cells differentiated with phorbol
ester, used to make the cell line more macrophage-like in terms of
e.g., increased phagocytosis, oxygen radical production, demonstrated
an increase in unspecied general oxidative species compared to control 12 h post incubation, however the levels of the ROS were lower
compared to B0 values [15]. [The probe used in these assays detected
total reactive oxygen species, not specifying which ones.] No changes
in the ROS levels were observed in BEAS-2B, an airway epithelial cell
line. Responses of two human airway epithelial cell lines (10 KT and
NuLi-1) to exposures to combusted B100 (made with canola oil), B20,
B0, and also canola oil not converted too FAMEs after 1 h were studied
[52]. Exposures All exposures induced increased apoptosis (with B0
the most potent), and decreased viability (with pure canola oil the
most potent), relative to clean air in both cell types post exposure. All
exposures, with the exception of canola oil emissions), increased the
production in the 24 h post exposure of the inammatory cytokines
Interleukin 6 and 8 and the chemokine RANTES (Regulated on
Activation, Normal T Cell Expressed and Secreted) relative to clean air
levels; canola oil emissions induced a decreased level compared to airexposed 10 KT control cells. PM concentrations were approximately
510 ug/m3 for B0, B20, and B100, and 35 ug/m3 for combusted canola
oil.

for B20 exposure, that amounted to 20 children always exposed to B20

after the switchover, and 72 exposed sometimes to B20 during year 3.
There was no alteration after the switchover to B20 in exhaled NO,
FEV1, FVC, and school absences, which was in contrast to the improvements observed with other technologies.
Human volunteers were exposed to B0 and B75 for 200 min. in an
underground mine. Subjects were exposed during a 110 min mucking
operation and 80 min vehicle operation with a 10 min break between
the two operations. Forced expiratory volume in 1 s (FEV1) was
decreased both post B0 and B75 exposure (relative to the baseline
preexposure value), with the B0 being a greater decrement compared
to the B75 [27]. No change in FVC was observed for B75 exposures,
but only for the B0 exposure. With exhaled breath markers, while
both B0 and B75 increased NO and CO, there was no difference
between the two exposures. Use of sputum induction revealed increased neutrophil, macrophage, and total white blood cell numbers
post both exposures; sputum soluble mediator concentrations related to inammation sound increases in several (including IL-8 and -6)
post exposure; and plasma soluble mediator production also increased for ET-1, and P-selectin. The increases post exposures (compared to pre-exposure) in sputum cells as well as soluble mediators
showed no statistically signicant magnitude differences between
the B0 and B75. Urinary markers of oxidative DNA damage were
not changed post exposure.
An assessment of biodiesel use (primarily as B20) from heavy duty
on road engines on Canadian health outcomes was performed using A
Unied Regional Air Quality Modeling System (AURAMS) air quality
modeling and MOBILE6.2C emissions model [34]. In Canada, canola is
the fuel feedstock, and B5 can be used year round but B20 only in
warm months (MaySeptember). [B0 was assumed to replace B20 in
the cold months.] Effects were analyzed for the 2006 year as well as
projected for 2020. It was estimated that the use of biodiesel resulted
in seven fewer premature deaths under 2006 model parameters (primarily driven by ozone and PM2.5 reductions) with net economic benet of a total of $48M and up to approximately 21,000 less days of acute
respiratory symptoms for all pollutants combined. Estimates for the
year 2020 reported increases of two premature mortalities and economic costs of approximately $15M.
5. Reports addressing other concerns with biodiesel emissions

4. Human studies
5.1. Source of the biodiesel stock
Human vascular responses to 1 h exposures with B0, B30, and B100
(rapeseed methyl ester) whole emissions were examined in two studies
[43]. In the rst study, healthy volunteer exercised on a bike with
15 min alternating rest/exercise period with B30 or B0 exposures, and
in the second study B100 or B0 exposures. The ndings from both studies were similar, showing that the B30 and B100 exposures inhibited vasodilation responses (measured as forearm blood ow) as much as
B100 exposures (all relative to clean air). [Previous studies demonstrated B0 exposure induces inhibition of vasodilation to endothelial dependent agonists for up to 6 h. [24]] B30 and B0 exposures induced similar
magnitudes of effects related to blood pressure, central arterial stiffness,
heart rate variability, respiratory function, and exhaled NO; there was
only a smaller pulse wave velocity with B30 exposure. Platelet clotting
ex vivo was similar with B0 and B30 exposures. There was a transient
increase in blood neutrophils and total leukocytes which resolved over
24 h. With B100 exposures, platelet clotting ex vivo, and soluble clotting
factor levels in vivo were similar to B0 exposure, with both exposures
again demonstrating a short transient increase in blood neutrophils.
A panel study of 275 participating US schoolchildren riding B0
powered school buses that would be switched to lower PM emission
technologies over 4 years examined exhaled nitric oxide (NO), specic
lung function parameters, and school absenteeism [2]. The emissions
lowering technologies included use of oxidation catalysts and low S
fuel, but also ~7% buses changed to B20 fuel. In terms of children studied

Because biodiesel fuel can be derived from different starting materials, and therefore different fatty acids, the potential exists for different
physicochemical proles of compounds to be emitted after combustion.
One report concluded that the chemical composition of the biodiesel
fuel was more important than physical properties (e.g., density, viscosity) in controlling PM emissions [31]. The PM emitted could therefore
modulate toxic responses due to size (affecting lung site of deposition
and the % deposited, and interactions with lung cell types including
phagocytes). It is unclear whether the different fuel components and/
or combustion endproducts induce different biological responses or a
different magnitude of response. Hemmingsen et al. [22] concluded
that for ROS production there was little different in B20 derived from
animal fat compared to B20 derived from rapeseed. However there are
many types of plants used for biodiesel production, including jatropha
in arid lands, and algae, which are intensively research due to high
levels on fatty acids in these feedstocks.
5.2. Blend effects
As previously discussed, some reports [7] have shown very little in
linear changes for chemical components of biodiesel emissions as the
biodiesel proportion increased in blends. The lack of linear blend dependent effects extends to observed biological outcomes, e.g., with

Please cite this article as: M.C. Madden, A paler shade of green? The toxicology of biodiesel emissions: Recent ndings from studies with this
alternative fuel, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.05.035

M.C. Madden / Biochimica et Biophysica Acta xxx (2016) xxxxxx

B20 exposures having more or less of a biological effect than both B0

and B100 when one would expect the magnitude to be between neat
fuels, and could be considered almost a common theme. This lack of a
linear blend dependent effect was observed, for instance, with rodent
arrhythmias in the Hazari et al. [21] paper, the general toxicity endpoints in the Bass et al. [5] paper, and the DNA adducts reported by
Ross et al. [33]. Further studies examining the reason for these observed
(nonlinear) trends in emissions characteristics and biological endpoints
needs further evaluation. Potentially the blends may be the intended
homogeneous solution if not mixed adequately and stratication of
the fuels exist in a tank, as one possibility or changes in composition
due to oxidative instability over time [39].
5.3. Factors affecting emissions
Beside fuel stock and blending, engine factors such as the size, type,
load, and lubricant may play a role in inducing effects due to different
emissions [28]. Due to the cost and time involved in with assay of multiple engine run conditions and biological endpoints, high through
screening would potentially be one approach to initially examine factors
that appear to inuence a biological response. Another approach would
be hypothesis driven studies where a particular chemical emissions
class would be examined for a role in eliciting a response.
5.4. Human heterogeneity
It is unclear at present with biodiesel exposure scenarios (blends or
neat biodiesel) if any sensitive groups exist. From research with controlled exposures to diesel PM [18] or ozone [4], certain genotypes, in
these reports glutathione-S-transferases present in ~40% of the US and
European population, can infer sensitivity relative to the whole population. Other factors that have been demonstrated to alter the effects of air
pollutants include age, medications, obesity, and diet. What these determinants that confer resistance or sensitivity might be with biodiesel exhaust remain to be elucidated, and the mechanisms examined.
6. Summary
These data from the studies performed by Mutlu et al. [29,30] are important due to the often conicting information in the literature about
the relative mutagenicity of biodiesel combustions. Incorporation of
normalization factors as performed in the analyses by Mutlu, in addition
to a per mass basis for comparative toxicity evaluations, will be needed
as part of the risk assessment process. The ndings from Gerlofs-Nijland
et al. [17] additionally emphasize the need for incorporation of metrics
related to distance traveled or engine work performed as well as per
mass basis when assessing the pollutants emitted from environmental
exposures.
Emissions from biodiesel have generally been thought of as less
toxic; however a more critical evaluation has concluded that based solely on some of the emitted components, that the biofuel is not completely green [42]. This article concentrates on health effects and comes to a
similar nding that biodiesel emissions can induce biological responses
that are considered surrogate markers of possible adverse effects should
exposures continue. As such, a view of biodiesel emissions being without impacts in end stage use is not plausible. What remains to be determined is how toxic exposures can be, especially related to repeated
exposures such as in an occupational setting, and how to predict the
risk to the population especially in the context of variable economic
and legislative inuences upon biodiesel production. Further efforts in
unraveling key health outcomes seen with petroleum diesel exposures,
such as increased lung cancer, may be the most important way to
proceed for the immediate future. However longer term research efforts
should likely incorporate identication of potentially susceptible
human populations that may be affected by unique or increased
amounts of components found in biodiesel emissions, such as fatty

acids or fatty acid fragments (from incomplete conversion to methylated species and subsequent incomplete combustion) [28,32].
Transparency Document
The Transparency document associated with this article can be
found, in online version.
Acknowledgements
The author wishes to thank Kimberly Swanson (Charlottesville VA)
for her input, usually on a daily basis, on comprehensive and global
topics related to biodiesel production and use issues. Funding for the authors was derived from internal U.S. Environmental Protection Agency
funds.
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Please cite this article as: M.C. Madden, A paler shade of green? The toxicology of biodiesel emissions: Recent ndings from studies with this
alternative fuel, Biochim. Biophys. Acta (2016), http://dx.doi.org/10.1016/j.bbagen.2016.05.035