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Environmental Science
Cite this: Energy Environ. Sci., 2012, 5, 7381
www.rsc.org/ees REVIEW
Toxicological and ecotoxicological potencies of biofuels used for the transport
sector—a literature review†
Kerstin Bluhm,a Sebastian Heger,a Thomas-Benjamin Seiler,a Arnold V. Hallare,ac Andreas Sch€afferb
and Henner Hollert*a
Published on 20 March 2012. Downloaded by Virginia Tech on 19/07/2013 00:25:12.

Received 31st October 2011, Accepted 16th March 2012

DOI: 10.1039/c2ee03033k

In the past few years, the development and use of biofuels for the transport sector have attracted growing
attention worldwide due to their promising benefits including a reduced dependence on fossil fuels and
a potential to slow down the effect of global climate change. Nevertheless, concerns have also started to
emerge regarding their potentially adverse environmental impacts and possible effects on human health.
In this context, literature research was carried out to obtain an overview of the current research activities
on the (eco)toxicological relevance of biofuels. The literature review revealed an increase in research
activities on biofuels, in general, especially within the last four years. In contrast, comparatively few
research activities were focused on the (eco)toxicological effectiveness of biofuels or their emissions even
though this topic will be of great relevance as soon as a biofuel becomes commercially marketed in the
future. Furthermore, the results of the available studies vary widely. Several findings on acute and
mechanism-specific toxicity indicate less or comparable effects induced by biofuels in comparison to
fossil diesel fuels. However, indications for negative impacts that are inducible both by the biofuels
themselves and their emissions were found. Based on the data available, an (eco)toxicological relevance
or human health risks associated with spills or the use of biofuels currently cannot be ruled out.
Therefore, additional experimental studies are necessary to provide a more comprehensive dataset for the
identification of future alternative fuels with low environmental impact.

a
1. Introduction
RWTH Aachen University, Institute for Environmental Research,
Department of Ecosystem Analysis, Worringerweg 1, 52074 Aachen, ‘Biofuel’ pertains to all renewable energy sources derived from
Germany. E-mail: Henner.Hollert@bio5.rwth-aachen.de; Fax: a wide variety of organic materials such as starch, vegetable oils,
+4924180-22182; Tel: +4924180-26669
b
RWTH Aachen University, Institute for Environmental Research, Chair of
animal fats, or cellulose. The term encompasses solid biomass,
Environmental Biology and Chemodynamics, Aachen, Germany liquid fuels, and various biogases. In very recent years, biofuels
c
University of the Philippines Manila, College of Arts and Sciences, have attracted growing attention worldwide, especially in the
Department of Biology, Ermita, Manila, Philippines 1000 transport sector, where the issues of oil price stability and energy
† Electronic supplementary information (ESI) available: Table S1, security are of paramount concern. As a consequence, the
overview of reviewed publications arranged according to endpoints
investigated. See DOI: 10.1039/c2ee03033k number of scientific publications on biofuels has grown

Broader context
The use of biofuels for the transport sector has drawn increasing interest in many countries during the past few years. Beside their
promising benefits, especially regarding the reduced dependence on fossil fuels, concerns arose regarding potential negative envi-
ronmental and social impacts and the sustainability of biofuels. The concerns regarding the environment and sustainability are
mainly based on potentially negative GHG emissions and energy balances within the context of life cycle assessments in comparison
to the fossil fuels they are supposed to replace. Beside the various efforts to investigate and verify the advantages of biofuels in terms
of sustainability and reduction of emissions, further potential impacts on the environment are deemed relevant. Here, the (eco)
toxicological evaluation of biofuels is a particular topic scarcely taken into account. Some studies with a focus on ecotoxicological
investigations revealed significant hazard potentials. However, the currently available data do not allow an unambiguous conclusion
of the potential effects on the environment and human health. Therefore, further research on (eco)toxicological potencies of biofuels
is required to allow the identification of future fuels with a low (eco)toxicological potential compared to conventional fossil fuels.

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Fig. 1 Estimated number of published articles on biofuels between 1949
and 2010. Searched in Thompson ISI – Web of Science, topic ‘‘biofuel*’’.
exponentially during the past few years. This increase in publi-
cations is illustrated in Fig. 1.
So far, up to three generations of biofuels have been described
in the literature and are referred to as biodiesel, bio-oils, bio-
ethanol, biomethane, biobutanol, biomass-to-liquid (BTL)-
diesel and hydrogen. First generation biofuels are mainly derived
from traditional domestic production processes and traditional
feedstocks such as plant seeds and grains that yield starch or oil,
which are then converted into fuels via conventional technology.
On the other hand, second (and third) generation biofuels could
be produced using lignocellulosic biomass,1,2 oils or carbohy-
drates, respectively, from non-food plant parts (e.g., stems, twigs,
and leaves),3 non-food crops (e.g., switch grass, jatropha,
microalgae, and meadowfoam seed oil)4–9 or industrial waste
products (e.g., wood chips and fruit peels)10–12 and, thus,
perceived to have the advantage of being more sustainable, and
of having better environmental performance.13,14 Improvements
in environmental performance of a biofuel technology can be
gauged from the following indicators: (1) lower energy inputs
through optimization in feedstock processing, (2) better energy
balance and (3) lower greenhouse gas (GHG) emissions. In
addition, (4) specific fuel design and (5) extended engineering
efforts to ensure optimal combustion of fuel components and
enhanced engine performances are necessary and under investi-
gation to achieve improvements with respect to reducing
hazardous exhaust gas components.15
Being a fast-growing technology, biofuel production and
utilization offer many advantages when compared to the tradi-
tional use of fossil fuels (gasoline/diesel). In contrast to fossil
fuels, which are depletable resources, biofuels are naturally
renewable alternatives that can be easily replenished, e.g. by crop
productivity. The shift from fossil fuels to biofuels is also of high
relevance in terms of environmental protection. Biofuels have the
potential to cause less GHG emissions compared to conventional
types of transport fuels. Consequently, biofuels are frequently
perceived to have a significant role in curbing effects of global
climate change.16,17 A supposed positive potential of biofuels to
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reduce greenhouse gas emissions and pollution served as an
impetus for the creation of legal policies to promote renewable
energy sources in the EU (COM (2006) 848)18 and the US
(Energy Security and Independence Act of 2007).19 Accordingly,
the US government supports the increase in biofuel production
from 6.5 billion gallons in 2007 to 36 billion gallons by 2022.19
Correspondingly, the European Commission has set a 10% target
for biofuels of the overall consumption of petrol and diesel in the
transport sector in the EU for 2020.18 Aside from environmental
benefits, the use of biofuels is also expected to benefit the
domestic or local economy by creating jobs for local farmers and
by reducing too much dependence on imported foreign oils.
Despite these benefits afforded by biofuel technology, many
concerned sectors, especially the environmentalists, are wary
about the potential short term and long term consequences that
this technology will have for the environment and human society.
For instance, land use changes may result in habitat loss,
a decline in biodiversity, a release of GHG and an excessive use
of fertilizers and pesticides.17,20,21 Concerns likewise exist about
competition with agriculture for arable land used for food
production22,23 and an enhanced competition for freshwater
resources.24 The potential impacts of biofuel production depend
on several parameters which were already discussed for various
scenarios.25–27 Furthermore, the possible future role of bioenergy
was presented and suggestions for a sustainable biomass
production were published.28 However, (eco)toxicological
potencies were not addressed. The same applies for life cycle
assessment (LCA) approaches. In LCAs, GHG emissions play
a major part in assessment of the environmental impact.29 Beside
this, impacts on the environment in terms of ecotoxicological
effects and impacts on human health were already included in
several studies.16,30–33 However, the majority of such investiga-
tions based the assessment of environmental impacts solely on
the release of known hazardous substances in the production
process and on emission rate data of regulated or ‘a priori’
pollutants. No definite bioassay results have been shown
regarding the toxic potential of the fuels themselves or their
emissions. In the case of formation or release of exhaust emis-
sions as an eligible factor to quantify impacts on human health
and the environment, a draft technical report (U.S.-EPA 2002)34
on a comprehensive analysis of emission impacts of biodiesel was
published. For biodiesel it revealed decreased total emissions of
hydrocarbons but also indicated a shift in the composition
towards an increase of the mass ratio of unregulated hazardous
air pollutants to total hydrocarbons. In this particular case the
results of chemical analysis reflect the problem of considering
only a particular part of total emissions such as regulated
pollutants. In addition, bioassay results can differ from toxicity
assessments based on chemical analysis. Therefore, a combina-
tion of bioassays and chemical analysis is recommended for
evaluation of the toxicity of samples contaminated with complex
chemical mixtures found in sediments, water, air or exhaust
emissions.35–38
Fossil fuels are a complex mixture of hydrocarbons. Potential
negative effects of the use of fossil fuels on the environment and
human health have been intensely examined39–43 but the use of
fossil fuels was introduced without the awareness of any future
related environmental or human health problems. Today the
production and use of alternative fuels are much more
This journal is ª The Royal Society of Chemistry 2012

questioned and discussions regarding the potential impact on the
environment and human health of such alternatives started
already several years ago. However, beside the efforts in inves-
tigating and verifying the advantages of biofuels in terms of
sustainability and reduction of emissions, further potential
impacts like (eco)toxicological potencies should be increasingly
considered. Any potential problems that might occur with the
use of biofuels should be investigated before a fuel is commer-
cially available to avoid or minimize any unintended environ-
mental problems that might occur in the future. However, as
biofuels will be used to replace fossil fuels, an understanding of
the toxic potential of biofuels compared to fossil fuels is impor-
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tant and crucial for many parts of further policy decisions and
might also influence trade agreements. The (eco)toxicological
potencies of a product, for example, are important regarding
regulations or restrictions of their production or transportation.
However, comparatively few publications are available about
biofuels and their impact, in particular with respect to (eco)
toxicological effects. Regarding this, there is an urgent need for
comprehensive risk assessments of biofuels as suggested by Davis
and Thomas.44 It is in this light that our review has been
undertaken on the current state of biofuel research. The paper
will provide a comprehensive survey of the literature that high-
lights the impacts of biofuels on the environment and human
health, with a focus on (eco)toxicological and mechanism-
specific toxicological investigations. Hopefully, this review will
encourage more studies that will aim at comprehensive risk
assessments of biofuels and help to provide a clear and sound
basis for decision-making on this emerging issue.
2. Data sources
The data used for this literature review were obtained by using
a broad search strategy including the electronic databases
Thomson Reuters-ISI-Web of Science, PubMed, Toxnet, and
Google Scholar. Several search terms regarding the topics fossil
fuel and biofuels, respectively, as well as terms related to acute
(aquatic) toxicity and mechanism specific toxicity were used to
find and select publications that address (eco)toxicological
investigations on biofuels for the transport sector.
3. Publications on (eco)toxicological investigations
of biofuels
The endpoints investigated in the studies considered for this
review as well as the organisms used for the tests are summarized
in Fig. 2. As indicated in the Venn diagrams, mainly biofuel
exhaust was investigated using mutagenic endpoints or a combi-
nation of several endpoints. In contrast tests to reveal the acute
toxic and/or the acute aquatic toxic potential were used for
investigations of biofuels themselves.
3.1 Mutagenicity/genotoxicity studies on biofuel emissions
The occurrence of mutagenic and genotoxic pollutants continues
to pose significant public health concern. These pollutants are
usually associated with carcinogenicity as well as induction of
some genotypic and phenotypic alterations in organisms,
including humans. Such changes can also precede the occurrence
This journal is ª The Royal Society of Chemistry 2012
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Fig. 2 Overview of endpoints and organisms used for (eco)toxicological
investigations of biofuels. Overlapping circles illustrate sets of different
endpoints investigated in a study. Numbers in the circles/overlapping
areas represent the number of publications reported on the same set of
investigations.
of teratogenic malformations, reproductive damage, and
immune deficiency. Therefore, in this section (eco)toxicological
investigations are reviewed that are related to mutagenic and
genotoxic hazards of exhaust emissions from biofuels and biofuel
blends in comparison to fossil fuels.
Mutagenic activity of biofuel emissions has been reported
already in several studies.45-56 In most cases, mutagenic screening
of particulate matter (PM) extracts was carried out using the
Ames or Salmonella/microsome bioassay. Data from above
studies were conflicting. Several publications reported that bio-
fuel emissions induced lower mutagenic potency when compared
to the exhaust of fossil fuels.46–48 Other studies revealed either no
differences or an increase in mutagenic activity of biofuel emis-
sion extracts.45,53,54
In most studies and depending on the test strain, lower to no
mutagenicity at all was found for particulate emissions of rape-
seed oil methyl ester (RME) compared to fossil diesel fuel
(DF).46–48 This is the case even if there was a parallel increase in
the amount of total emitted mass and extractable fraction
derived after combustion of RME fuel.46 Moreover, with the
exception of one loading mode and metabolic activation in the
Ames tester strain TA98, soybean oil methyl ester (SME) PM
extract also revealed lower mutagenicity compared to DF.
Nevertheless, it is worth mentioning that the soluble organic
fraction (SOF) of PM derived from the combustion of low sulfur
diesel fuel (LS-DF) induced similar numbers of revertants as the
emissions of RME and SME in a later study by B€ unger et al.47
Another study evaluated the mutagenicity of RME exhaust
particle extracts and condensates by using the Ames plate
incorporation assay but also rat hepatocytes.49 They observed
a significant mutagenic potential of RME and diesel exhausts.
Depending on defined measuring points (motor load and
Energy Environ. Sci., 2012, 5, 7381–7392 | 7383

revolutions per minute) the mutagenic potential was similar for
RME and diesel emissions or higher for the diesel exhaust. The
results were referred to a specified sample volume.
An idea of the compounds that might induce mutagenic effects
in the exhaust of biofuels is given by Finch et al.57 They report on
investigations of a soybean derived biofuel in the Ames muta-
genicity assay. By using, among others, the tester strains TA98
and TA98/1,8-DNP6 with and without metabolic activation, they
compared a relative mutagenicity calculated as revertants per mg
per plate of particulate SOFs and semi-volatile extracted frac-
tion, respectively. These tester strains respond differently with an
elevated relative mutagenicity calculated for the tester strain
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TA98. Based on their results, they concluded that for particulate
SOF one-third to one-half of the mutagenicity observed in strain
TA98 could be attributed to nitro-aromatics. For the semi-
volatile extracted fraction the results indicated that the effect
observed in TA98 with metabolic activation and most of the
mutagenicity observed in TA98 without mutagenic activation
could be attributed to nitro-aromatics. Kooter et al.52 reached
a similar conclusion in their study. Based on their results for
biodiesel and RSO emissions they assumed a potential interre-
lation between the increased mutagenicity and nitro-polycyclic
aromatic hydrocarbons (nitro-PAHs). The results of a study by
Turrio-Baldassarri et al.58 also indicated the presence of nitro-
aromatics in emission extracts. However, they investigated
a biodiesel blend instead of a pure biofuel. Beside vegetable oil
derived biofuels, other potential biofuels, namely, pork lard
methyl ester, beef tallow methyl ester and yellow grease methyl
ester, were also examined for their mutagenic potentials by Kado
and Kuzmicky.51 The results varied depending on the test
conditions. However, they draw the conclusion that the use of
biofuels can reduce emissions of particle-bound toxic mutagenic
compounds.
In contrast, several studies reported higher mutagenic effects
of biofuels. For instance, rapeseed oil (RSO) was found to
significantly increase mutagenicity of particle emission extracts
derived from combustion in a heavy truck diesel engine
compared to DF or even with other biofuels.45,54 B€ unger et al.45
investigated the particle emission extracts and condensates of
biofuels derived from the same feedstock but differently pro-
cessed (RSO and RME). Results showed that the mutagenic
potential of RME particle extract was mostly comparable to the
mutagenic potential of the petro-diesel particle extract. An
increase of mutagenic effects for RME particle emission extracts
was significant only for the tester strains TA98 (with metabolic
activation) and TA100 (without metabolic activation). On the
other hand, the RSO particle extract induced stronger mutagenic
potentials with all tester strains. Even the condensates of RSO
showed a greater mutagenic response in comparison to conden-
sates of other fuels like RME and DF.45,54 Biofuel emissions,
therefore, differ considerably in their toxic potentials even when
derived from the same feedstock. Krahl et al.54 added that the
significant increase in mutagenic effect of RSO emissions could
not be explained by the regulated exhaust emissions measured.
Kooter et al.52 also reported higher mutagenic effects of biofuel
emissions compared to the emissions of DF. They investigated
RSO, biodiesel (EN14214) and DF emission extracts of a Euro
III truck engine in the Ames assay. The results indicate a dose-
dependent response of the test strains (TA98 and YG1024)
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without metabolic activation after exposure to biodiesel blend
emission extracts, the RSO fuel emission extract and the emission
extract of a pure biodiesel. Calculated fold inductions also
indicate an increased mutagenic potential of the emissions of
RSO and biodiesel in the test strain YG1024 without metabolic
activation. In contrast, no dose-dependent response or increased
induction factors were found for the DF emission extract.
A further study on diesel–biodiesel blends reported an overall
similar mutagenic potency and genotoxic profile for both emis-
sions coming from diesel and a diesel blend (containing 20%
RME).58 Thus, the addition of 20% biodiesel RME to fossil diesel
fuel did not have a significant increasing or lowering effect on
total mutagenicity of the resulting emissions. On the contrary,
Gagnon and White50 reported on a reduction of mutagenic
activity for the polar fraction of fuel emissions with increasing
biodiesel content in the fuel compared to ultra-low sulfur diesel
(ULSD).
Differences in mutagenic responses were not only reported for
biodiesels or diesel–biodiesel blend emissions. An example is
given by Krahl et al.53 They compared mutagenic potencies of
emission extracts of RME, gas-to-liquids (GTLs) and a DF with
a biofuel–DF mixture containing 60% DF, 20% RME, 20%
GTL, and an additive. The blend showed a significant reduction
of some emissions (e.g., CO and particulate matter) but slightly
higher hydrocarbon emissions compared to the expected emis-
sion values of its individual components. The data also showed
a higher mutagenic potential for the exhaust of the blend than the
exhaust of each of its constituents. These results illustrate that it
is both impossible and impractical to draw conclusions regarding
health effects on the basis of the regularly monitored exhaust
emissions alone.
Jalava et al.59 investigated exhaust particles but used the comet
assay instead of the Ames assay. The PM extracts of three fuels
(diesel, RME and a hydrotreated fresh vegetable oil) combusted
in an industrial diesel engine increased the DNA damage
significantly at least at the two highest concentrations tested
(300 mg mL 1 and 150 mg mL 1). DNA damage was also induced
by all fuel PM extracts at a concentration of 50 mg mL 1 when the
engine was running without a catalysator. Taking into account
the emission factor (mg MJ 1) a slightly higher genotoxicity of
the DF exhaust particles was indicated.
In addition to biodiesel or oil methyl esters other biofuels like
ethanol were tested for their mutagenic or genotoxic potential
after combustion. Song et al.56 found out that PM extracts of
pure diesel and ethanol–diesel blends contain direct-acting as
well as indirect-acting mutagens. To express the mutagenicity of
PM extracts, they introduced the concept of ‘brake specific
revertants’ to refer to mutagenic activity in revertants per milli-
gram and the term ‘brake specific emission’ of SOF given in gram
per kilowatt hour. By comparing the highest concentrations of
the SOF of PM applied in this study they observed the highest
number of brake specific revertants in the emission extract of
ethanol–diesel blend containing 20% ethanol (E20). In addition
to the Ames test, genotoxicity of the SOF of PM was also
examined using the comet assay. For all blends investigated, the
emissions of both the DF and E20 induce similar effects with
higher comet tail length than the emissions of other ethanol–
diesel blends (E5, E10 and E15). Although increasing sample
concentrations caused a corresponding increase in DNA
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damage, there was no statistical difference among the increasing 3.2 In vitro studies on cytotoxicity and inflammatory effects
ethanol fractions of the diesel fuel blends applied with the same
Few studies could be found comparing cytotoxic effects of bio-
quantity of corresponding SOF. At any rate, Song et al.56 were
fuel emissions to petroleum diesel fuel emissions. A pilot study by
able to conclude that the lowest toxicity can be achieved by E5
Swanson et al.61 examined the inflammatory potential of bio-
PM extracts. This was in agreement with their PAH emission
diesel extracts (SME) and soybean oil ethyl ester (SEE). There-
analysis. Zhang et al.60 investigated the engine emission exhaust
fore, they used cytokines IL-8 and IL-6 as markers of a pro-
of pure methanol instead of ethanol blends. They compared the
inflammatory response. For the dose range applied (with highest
genotoxic potential of organic extracts of condensate, PM as well
concentrations of 40 mg PM equivalent per mL) no cytotoxicity
as semi-volatile organic compounds when a bus (engine without
measured as a lactate dehydrogenase (LDH) activity increase
exhaust catalytic converter) was running on methanol or gaso-
(CytoTox 96 cyotoxicity assay) or a reduction in cell viability (3-
line. Therefore, they used the micronucleus assay, comet assay
(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide
and Ames test. The results indicate stronger effects for gasoline
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(MTT) proliferation assay) for biodiesel emission particulate

exhaust in comparison to methanol exhaust as the tests revealed
extracts was observed. Despite the low concentrations applied,
increased micronucleus formation, induced DNA damage in
a dose-dependent increase in IL-8 and IL-6 was found in
human lung carcinoma A549 cells and a higher number of TA98
a transformed human bronchial epithelial cell line (BEAS-2B).
revertants in the presence of metabolic activating enzymes only
Furthermore, they reported that the SOF of biodiesel emissions
for gasoline engine exhaust.
appeared to be a more potent inflammatory stimulant compared
To conclude the results of mutagenicity studies with emission
to diesel fuel PM extracts. The data provided in that paper were
extracts, the use of biofuels and blending with biofuels do not
only preliminary and not appropriate to rate one type of soy ester
necessarily lead to a reduced mutagenic potential of emissions
as more potent than the other. Furthermore, the concentration
and even increased mutagenic effects might be possible.
units for the comparison of inflammatory stimulation (mg PM
However, a direct comparison of the results obtained with the
equivalent per mL) were discussed as not useful for providing
Ames assay from the studies presented in this section is quite
a clear idea of actual relative potencies of the organic extracts
complicated since the methodological approaches applied (e.g.,
and there is no relation to the rate at which the engine worked. A
reference fuel) were different in each case. The purchase of fossil
study associated with the same subject compared the cardio-
fuels from different providers allowed the composition (e.g.,
vascular and inflammatory toxicity potential of diesel and bio-
aromatics and sulfur) and, consequently, the properties of fossil
diesel exhaust particles in mice.62 Although they used another
fuels used in the investigations to vary. Furthermore, various
kind of engine, their results partly corroborate the findings of
test setups were chosen, including vehicle or engine types not
Swanson et al.61 Biodiesel exhaust exposure induced an increase
optimized for combustion of biofuels, different operating
in bronchoalveolar lavage inflammatory cells, bone marrow
conditions and test cycles as well as differences in sample
activation, increased blood platelets and increased heart rate
collection and extraction (e.g., Soxhlet extraction vs. sonication
variability. They concluded from their results that biodiesel PM
bath or the use of dichloromethane vs. methanol). These
causes equal or even more toxic effects than PM of diesel,
scenarios influence the composition of the exhaust as well as the
especially due to promoted cardiovascular alterations as well as
composition of the extracts obtained and thus, affect the
pulmonary and systemic inflammation. The effects of the PM of
mutagenic potential of the tested extracts. Consequently, results
DF, RME and a hydrotreated fresh vegetable oil seem to be
of several different mutagenic studies can hardly be compared.
different when mouse macrophages (RAW264.7) were exposed
Additionally, the variability of presenting results (e.g., number
and inflammatory mediators measured. The work of Jalava
of revertants per plate, number of revertants per weight unit of
et al.59 revealed an increase of inflammatory mediator (TNF-
PM, revertants per hour of engine running time, and revertants
a and MIP-2) responses but relative responses were more
per defined litre of exhaust or related to kW h) can hamper
pronounced after exposure to hydrotreated fresh vegetable oil
a direct comparison. Thus, to provide results that will allow
and DF PM extracts compared to RME PM extracts. Accord-
comparison of mutagenic potentials of biofuel emissions, the
ingly, additional investigations are recommended to allow
use of a defined reference fossil fuel, defined test setups,
a better understanding of the inflammatory responses associated
extraction methods and a calculation procedure would be
with biofuels as well as a better characterization of the inflam-
necessary. However, it is remarkable to note that not only fossil
matory potencies of fuel emissions depending on the type of fuel
fuel emissions but also emission particles of some biofuels have
and the engine used.
been shown to contain direct- and indirect-acting polar
Besides inflammatory potencies, Jalava et al.59 also investi-
aromatic mutagens. This is quite interesting with respect to new
gated the cytotoxicity of RME, DF and hydrotreated fresh
developments in the field of biofuels. Results revealed that it is
vegetable oil emission extracts. The PM extracts of all fuels
both impossible and impractical to draw conclusions regarding
significantly reduced cell viability (MTT test) and increased
health effects on the basis of the regularly monitored exhaust
apoptosis at least at the highest concentrations tested (cytotox-
emissions alone. Therefore, emissions of new developed biofuels
icity: 300, 150 and 50 mg mL 1; apoptosis: 300 and 150 mg mL 1).
should not only be chemically investigated but also biologically
In addition, measuring the apoptotic responses the hydrotreated
for a better assessment of their mutagenic potential. Further-
fresh vegetable oil induced an apoptotic response at a concen-
more, in order to take better account of the indications of DNA
tration of 50 mg PM per mL. No significant or only small
damage found for biofuel or blend emission extracts, greater
differences were found between the fuel samples when tests were
focus on the application of tests like the comet assay should be
performed with or without a catalytic converter. Calculations of
considered.

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the so-called relative responses (adjustment with the emission
factor) revealed stronger relative cytotoxic and apoptotic
responses for the DF and the vegetable oil compared to RME.
The lower responses of RME might be associated with a reduced
particulate emission. However, engines operating on hydro-
treated fresh vegetable oil also revealed less PM emissions
compared to DF but slightly stronger relative apoptotic
responses were found. The effect inducing compounds could not
be clarified.59 In contrast, Kooter et al.52 revealed a significant
higher relative cytotoxicity of the biodiesel compared to DF.
They also investigated PM extracts using the mouse macrophage
cell line RAW264.7 but instead of the MTT assay they applied
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the LDH cytotoxicity assay.
B€unger et al.46 evaluated and compared cytotoxic effects of
diesel engine exhaust from a light duty diesel engine running on
biodiesel (RME) and DF, respectively. Using the test cycle FTP-
75, investigated particulate extracts revealed slightly lower cell
viability for RME exhaust compared to DF but the observed
difference was not significant. Likewise, particulate extracts
derived from DF and biodiesel combustion in two other test
cycles did not induce significant differences. These results indi-
cate a similar cytotoxic effect for fossil diesel and biodiesel
emissions. However, total PM was increased when the engine
was running on biodiesel. This was ascribed to an incomplete
combustion as the engine was not optimized for the use of RME.
Although a more efficient combustion may alter the composition
of PM resulting in a different test result, cytotoxic effects of
biofuel emissions cannot be ruled out. B€ unger et al.48 compared
the cytotoxicity in the L929 mouse fibroblast cell line of particle
extracts from the exhaust of a farm tractor fueled with RME or
petroleum diesel and found that the biodiesel extract reduced the
cell viability to 50% at a 4 times lower exhaust concentration than
the DF. Therefore, RME was more toxic when the tractor was at
idle. In contrast, there was little difference between the extracts
when the tractor was operated at its rated load.
Different findings were reported by Liu et al.63 They examined
particulate exhaust emissions and, additionally, semi-volatile
emissions to determine the relationship between the biodiesel
blends and potential adverse effects on the environment and
human health. Therefore, they used the Microtox test and the
MTT assay and investigated DF, blends with palm fatty acid
methyl ester (10%, 30%, 50% and 75%) as well as the pure bio-
fuel. For this study a generator was used with a constant output
power of 13 kW. Results of the Microtox tests will be discussed in
more detail in the next section. The MTT assay was used for
evaluation of the impact of the samples on the growth and
metabolism of lung epithelial cells, BEAS-2B. For all particulate
extracts tested the cell viabilities exceeded 80%. Consequently,
particulate engine exhaust extracts of both biodiesel-blends and
DF were considered to be non-cytotoxic. In contrast, semi-
volatile extracts (containing e.g., hydrocarbons and PAHs) from
B50 and B75 strongly inhibited cell growth with the strongest
cytotoxic effect revealed for B50. Furthermore, B100 did not
show inhibitory effects on cell growth. Thus, stronger cytotoxic
effects were not directly related to a higher content of biodiesel in
diesel fuels in this study. However, the results indicate that
blending with biodiesel does not necessarily have beneficial
effects on emissions emitted into the environment. Blending may
even have adverse health effects when the biofuel concentration
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is low. By again using the MTT assay to evaluate actual toxicity
of gaseous compounds in the exhaust, at a specific engine load
Liu et al.64 reported stronger acute toxicity and cytotoxic effects
for B10 than DF, indicating that the gaseous emissions from the
biodiesel blend had more adverse health effects than those from
diesel.
Similar to the results on 100% biodiesel, results of another
potential biofuel and fuel additive (methanol) showed no inhib-
itory effects on cell growth. By using the MTT assay and human
lung carcinoma cells Zhang et al.60 compared the cytotoxicity of
methanol engine exhaust to the gasoline engine exhaust.
Concentrations of 0.05–0.8 L engine exhaust per mL after 2 and
24 h of application were examined. They found stronger cyto-
toxic effects for gasoline compared to methanol engine exhaust.
3.3 Studies on dioxin-like activity
Few in vivo and in vitro studies were available that investigated
dioxin-like biological effects of biofuels (cf. Fig. 2). Gagnon and
White50 assessed the dioxin-like activity of biodiesel emissions by
using the DR-CALUX assay. They revealed an increased
activity of more than 100% for a biodiesel fuel blend (B20, not
described in more detail) compared to ULSD. This increase was
found for both fractions investigated (polar and non-polar). In
another study by Poon et al.65 both LS-DF and SME signifi-
cantly increased the activity of ethoxyresorufin-O-deethylase
(EROD), benzyloxyresorufin-O-dealkylase (BROD), pentoxyr-
esorufin-O-dealkylase (PROD) and glutathione S-transferase
(GST). These results were found after repeated oral treatment of
male rats for 4 weeks. The increased liver enzyme activities of
EROD, BROD, PROD and GST found by Poon et al.65 after
treatment with SME could not be confirmed by Poon et al.66 The
only biofuel inducing GST activity in this study is the reclaimed
frying oil of animal origin methyl ester (FraME) at the highest
dose of 500 mg kg 1. They also report a significant increase in
enzymatic activity of acyl-CoA oxidase in groups treated with
SME, RME, FraME and ULSD. In the earlier study only LS-DF
treatment caused an increased activity of acyl-CoA oxidase. In
this case, inconsistent results do not allow a reasonable inter-
pretation. Other biofuels such as ethanol did not cause dioxin-
like biological effects in a study by Chu et al.67 After inhalative
exposure of rats to unleaded gasoline, ethanol or a mixture of
85% ethanol and 15% gasoline a significant increase in liver
microsomal EROD activity was only observed in male rats
exposed to gasoline.
3.4 Ecotoxicological studies on biofuels—acute aquatic
toxicity
In this section studies were taken into account that investigated
the effects of fuel oil in water dispersions (OWDs), water
accommodated fractions (WAFs) and emission extracts to
aquatic organisms.63,64,68–71
Khan et al.70 evaluated the acute toxicity of biodiesels derived
from recycled cooking oils and fats and diesel–biodiesel blends
on daphnids (Daphnia magna) and rainbow trout (Oncorhynchus
mykiss). Oil in water dispersions (OWDs) were used in the
experiments. Their results demonstrated that biodiesel as well as
diesel–biodiesel blends were considerably less toxic to 6 week old
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rainbow trouts compared to conventional DF. Nevertheless, it
was shown in the same study that, just like the common DF,
biodiesel and blends could also pose a quite substantial hazard to
aquatic organisms as the concentrations that indicate 50%
mortality in a given time (LC50-values) were quite low (e.g.,
LC50: 4.65 ppm for daphnids exposed to 100% biodiesel from
recycled cooking oil compared to LC50: 1.78 ppm for diesel
exposure). This, therefore, implies that the release of biofuels to
the environment due to an accidental spill or inadvertent
discharge during transportation, storage, or use represents
a potential danger to aquatic ecosystems.
Hollebone et al.69 conducted a more comprehensive work and
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investigated three different biodiesels (two based on vegetable
oils of canola and soy crops, respectively, and one animal-source
waste fry oil) for their acute toxicity against Daphnia magna,
Oncorhynchus mykiss and the bacterium Vibrio fischeri (Micro-
tox assay). For the tests, they also used OWD but additionally,
for Daphnia magna assay, a water accommodated fraction
(WAF) was applied. The Microtox assay results revealed that
LS-DF and ULSD were significantly more toxic than biodiesel.
In the same study, soy-biodiesel was found to be the most toxic of
the biodiesels. These results were consistent with their results
obtained for fish toxicity and with data of Khan et al.,70 who also
reported petro-diesels (LS-DF and ULSD) to be significantly
more toxic in the fish test than the biodiesels. In contrast, no
coherent data could be established for Daphnia magna toxicity in
the studies. For biodiesel derived from rapeseed vegetable oil and
applied as OWD, Hollebone et al.69 reported a more than fifteen-
fold higher LC50 value and thus, a lower toxicity in the Daphnia
magna test compared to petro-diesel. The biodiesel derived from
soybean vegetable oil showed, by contrast, a similar toxic
potential to petro-diesel. By varying the test approach, this time
using WAF for exposure, a different result was observed. In this
case, biodiesel derived from both animal oil and soy oil yielded
lower LC50 values compared to biodiesel derived from rapeseed
oil and LS-DF. However, all LC50 values were much higher
compared to the application of OWD and, therefore, imply
a lower toxic potential. An explanation for the higher toxic
potential of OWD is given and might be the formation of oil
layers observed in both studies. These oil layers might induce
either physical smothering or trapping of D. magna resulting in
a higher mortality rate of the OWD. This explanation implies
a much lower toxicity of the dissolvable fraction compared to the
physical danger posed by the oily fuel. Such a scenario raises
concerns for small species of arthropods and other invertebrates
in aquatic ecosystems in the case of a biofuel spill.69,70
Other results on aquatic toxicity due to biodiesels derived from
vegetable oil have been published.68,71 Reece et al.71 compared the
acute aquatic toxicity of three vegetable oil esters (RME, rape-
seed oil ethyl ester (REE) and SME) and a LS-DF. The results
indicate lower acute toxicity in daphnids for all biofuels
compared to the DF (<1.43 ppm) with RME (LC50: 23 ppm)
being more toxic than REE (LC50: 99 ppm) and SME (LC50:
332 ppm). Therefore, biodiesels seem to be less toxic than petro-
diesel fuel. In addition to the comparison of biofuels to DF, the
studies by Reece et al.71 and Hollebone et al.69 allow a compar-
ison of different rapeseed oil based biodiesels. The SME used by
Reece et al.71 appears to be less toxic to daphnids than the bio-
diesel derived from soy oil used in the study by Hollebone et al.69
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On the contrary, the LC50 value of the rapeseed oil based bio-
diesel used by Hollebone et al.69 exceeded the LC50 values of
REE and RME indicating a lower acute toxicity. However,
a comparison of the two studies appears to be questionable when
comparing the results of the reference DF. Even though both
Reece et al.71 and Hollebone et al.69 investigated LS-DF as
a reference fuel, their results for this substance differ by a factor
of at least 12 (LC50: 17.9 mg L 1, Hollebone et al.;69 LC50:
<1.43 ppm, Reece et al.71). Hollebone et al.69 used a homogenizer
to prepare the dispersions; Reece et al.71 only stirred the fuel into
water and observed a sheen of fuel on top of each chamber
comparable to observations by Khan et al.70 Therefore, physical
smothering or trapping of D. magna resulting in a higher
mortality rate might explain the difference in the LC50-values in
particular with regard to similar LC50-values calculated by Khan
et al.70 and Reece et al.71 Although these studies are more suitable
for comparison in terms of dispersion preparation, different
feedstocks were used for the biodiesel tested and thus,
a comparison does not appear to be appropriate.
Gateau et al.68 provided information on acute aquatic toxicity of
four vegetable oil methyl esters (RME, sunflower oil methyl ester
(SuME), erucic rapeseed oil methyl ester (eRME) and high oleic
sunflower oil methyl esters (HoSuME)). WAFs or water soluble
fractions (WSFs), respectively, of all these vegetable oil methyl
esters revealed half maximal effective concentrations (EC50) >
1000 mg L 1 for daphnid toxicity, EC50 > 10 000 mg L 1 for algae
toxicity and even LC50 > 100 000 mg L 1 for fish toxicity, i.e., at
least 10-fold higher than those found for the DF. Precise LC50/
EC50-values were also not calculated for DF but specified as
<100 mg L 1 for daphnids and algae toxicity as well as <150 mg L 1
for fish toxicity. For fish toxicity a direct comparison to the studies
by Hollebone et al.,69 Khan et al.70 or Reece et al.71 was not
possible. In this particular study, Gateau et al.68 used WAF instead
of OWD, exposed the fish only for 48 h instead of 96 h and tested
a different species (zebrafish instead of rainbow trout). Beside 96 h,
Khan et al.70 also calculated an LC50-value after 48 h of exposure
to OWD of petro-diesel fuel. The LC50 of 350 ppm after 48 h of
incubation indicates a lower toxicity for OWD compared to the
WAF tested by Gateau et al.68 However, due to the use of different
test species and the fact that the diesel tested is unspecified in terms
of the sulfur content a direct comparison between the LC50-values
for diesel is not possible. The latter is also a problem for the
comparison of the daphnid toxicity results. In this case, the results
of DF appear to be inconsistent. Gateau et al.68 present an EC50-
range much lower than the LC50-value calculated for a WAF by
Hollebone et al.69 but the differences might be due to different
diesel fuels examined. Hollebone et al.69 and Gateau et al.68
investigated WSF/WAF of rapeseed oil derived biodiesel. The
results exceeded both 1000 mg L 1 and, therefore, indicate a higher
toxicity for OWD (280 mg L 1, Hollebone et al.69 and 23 ppm,
Reece et al.71) compared to WSF/WAF. Based on their results,
Gateau et al.68 concluded that vegetable oil methyl esters like RME
or SuME are not toxic to aquatic organisms like fish, daphnids and
algae according to the German classification of substances
hazardous to water. In a further study investigating aquatic
toxicity of biofuel emissions, Womac et al.72 compared the lethality
to fish of water laden with different concentrations of exhaust from
engines burning either SME or petroleum diesel fuels. They
reported that both fuels induced low mortalities.
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Two further studies that investigated the acute toxicity of
biodiesel in aquatic organisms (Vibrio fischeri; Microtox assay)
have been published recently.63,64 Whereas Hollebone et al.69
investigated the toxic potential of neat biodiesels, Liu et al.64
examined the emissions of a biodiesel blend and reported quite
different findings. They compared extracts of gaseous emissions
from DF and the biodiesel blend (B10, 10% palm fatty acid
methyl ester) collected at different loading modes (idling, 10%,
33%, and 55%). The addition of biodiesel increased the toxicity
of emission extracts compared to DF for all operation modes.
These results are consistent with an earlier report that evaluated
not only gaseous (semi-volatile) emissions but also PM in the
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exhaust.63 In addition, they investigated several biodiesel blends
(B10, B30, B50, B75, and B100) derived from palm fatty acid
methyl ester but only one loading mode (13 kW equal to 33%).
This study revealed a higher toxicity of semi-volatile pollutants
generated from biodiesel combustion compared to particles
emitted from the unmodified engine. Consequently, higher values
for the toxicity of semi-volatile pollutants indicate the generation
of more toxic gas products than toxic particles by the use of
biodiesel. In this case toxicity was expressed in TUV (toxic unit
per litre of exhaust; for calculation of the toxic unit see Liu
et al.63). Additionally, the calculated TUV for semi-volatile
emissions indicates higher toxicity for biodiesel and biodiesel
blend combustion products compared to diesel emission. In
contrast, the TUV calculated for the particulate emission toxicity
of each fuel differed rather slightly especially regarding the values
for B50, B75 and DF. Furthermore, by calculating and
comparing the TUW (toxicity unit per mg particle SOF) diesel
particulates indicated to be more acute toxic than particles
generated by biodiesel combustion. However, a definite differ-
entiation between fuels is difficult since a statistical analysis to
determine differences between the TUVs of the fuels was not
performed. Nevertheless, a lower TUW for biodiesel compared
to DF could be explained by a higher amount of PM emitted
when the engine is running on biofuel and blends. Thus, even
though combustion of biodiesel produces fewer compounds that
are toxic or compounds that are less toxic to the bacterium Vibrio
fischeri as indicated by the TUW an overall higher amount of
emitted particles per litre exhaust results in a TUV comparable to
that of DF. A potential explanation for the difference in the toxic
potential of PM extracts is given by Liu et al.63 They attributed
the higher toxicity of biofuel blends to the inefficient combustion
in unmodified engines which apparently required a larger
amount of biodiesel to maintain their functionality. In the future,
a reduction in emissions from biofuel combustion might be
achievable through engine modification. Modifications, for
example in the injection timing, injection pressure or exhaust gas
recirculation rates, can improve the fuel combustion perfor-
mance and lower exhaust emissions.73–75 Therefore, the use of an
engine not optimized for the combustion of the biofuel could
have resulted in an overestimation of the toxic potential of the
biofuel. Nonetheless, a hazard of biofuel emission to the envi-
ronment cannot be ruled out with regard to the studies available.
Overall, the results regarding WAFs and OWDs have been
shown to vary from being virtually as toxic as petro-diesel to
1000 times less toxic, but biofuels predominantly induced less or
comparable toxic effects on aquatic organisms than the DF used
for comparison. However, the toxic potentials depend on the
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type of application (OWD, WAF), fuel batch and feedstock
source and substantial hazards to aquatic organisms might be
still possible. Additionally, a substantial hazard of biofuel
emission to the environment could not be ruled out with regard
to the studies by Liu et al.63,64 even though several biodiesels were
reported to be less toxic to soil microorganisms than DF.68,76
3.5 Further ecotoxicological studies on biofuels
The toxicities of a biofuel (without details regarding the feed-
stock) and of DF on soil microorganisms were determined by
measuring the respiration and enzyme activity of soil dehydro-
genases.76 Their data analysis indicated that DF was toxic when
concentrations in the soil exceed 3% w/w. At higher concentra-
tions the respiration as well as the activity of dehydrogenases
constantly decreased with increasing concentrations. Contrary to
the results for DF, application of biodiesel revealed a significant
increase of dehydrogenase activity with increasing biofuel
concentrations up to the highest concentration tested (12% w/w).
Furthermore, their data revealed a steady increase of respiration
with higher concentrations of biofuel in the soil. Thus, they
concluded that the biodiesel fuel is not toxic at concentrations of
up to 12% w/w and that the DF exhibits toxic properties at
concentrations higher than 3% w/w.
In accordance to these results, toxicity data on the bacterium
Pseudomonas putida exposed to WAFs of several vegetable oil
methyl esters (RME, eRME, SME and HoSuME) and a petro-
diesel also demonstrated a higher toxic potential for the petro-
diesel.68 EC0-values (representing the extrapolated concentration
giving 0% inhibition) presented were >1000 mg L 1 for bacteria
(Pseudomonas putida) exposed to biofuels. The EC0-value
obtained for bacterial toxicity after exposure to petro-diesel, on
the contrary, was <10 mg L 1. Thus, petro-diesel appears to have
a more than 100-fold higher toxic potential towards Pseudo-
monas putida.
An advantage of biodiesels over petro-diesel fuels lies in their
biodegradability. They are readily biodegradable76,77 and
enhance the biodegradability of fossil fuels.78–80 Thus, the expo-
sure time of organisms might be reduced. However, beside the
direct effects of biodiesels an increase of the mobility of poten-
tially hazard substances should be considered. Indications for an
enhanced dissolution81 or dispersion69 of DF components were
reported which might result in an increased mobility and wider
spreading of these components. This effect might also occur if
a biodiesel enters a polluted site and remobilizes contaminants.
Therefore, even though a biodiesel is readily biodegradable and
has the potential to enhance the biodegradability of diesel, the
spreading of potential hazard substances over a wide area should
be taken into consideration. Consequently, this topic should be
addressed in further studies to assess the distribution and fate of
fuels or fuel components after entering the environmental
compartments.
3.6 In vivo toxicity and health effects
3.6.1 Acute oral and dermal toxicity. Few studies on acute
oral and dermal toxicity of biofuels in comparison to petrol
based fuel could be found. According to the studies, there are no
differences between biofuels and DF in terms of an EC50 for
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acute oral and dermal toxicity. An acute oral toxicity was not
found for DF and its WAF, respectively,68,71 REE71,82 RME and
its WAF, respectively,68,71 the WAFs of SuME, eRME and
HoSuME68 as well as several fuel biodiesel–DF mixtures (20%
RME or REE and 50% RME or REE71) at the highest admin-
istered dose of 5000 mg kg 1. However, Reece et al.71 found that
the occurrence of clinical observations increased with increasing
content of DF.
Results of acute dermal toxicity tests indicated an LD50 (dose
at which 50% of the tested organisms will die) greater than 2000
mg kg 1 when LS-DF, RME and REE were administered on
rabbit skin.71 Vegetable oil methyl esters like RME and SuME
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were also investigated according to the OECD guidelines for
testing acute dermal irritation/corrosion and acute eye irritation/
corrosion.68 According to this study, both biofuels are neither
irritating for the skin of rabbits (0.5 mL) nor for their eyes
(0.1 mL) after 72 h of observation.
3.6.2 Clinical, histopathological observations and alterations
of biochemical parameters after oral treatments. Poon et al.65
investigated three biodiesels derived from soy oil, rapeseed oil
and fish oil and a LS-DF in a 4 week oral toxicity study. The
doses were administered 5 days per week for four consecutive
weeks. The investigated endpoints were effects on urinary
parameters, hematological analysis, selected serum biochemical
values, body and organ weights, histopathological evaluation
and effects on liver enzyme activities. Effects were mainly found
for LS-DF and SME and to a lesser extent for RME. Histo-
pathological changes were observed for all biodiesels and
LS-DF. Most changes are referred to as minor and adaptive in
nature with the exception of effects on the kidney. In the SME
and LS-DF treatments histopathological changes and albumin-
uria occurred and were associated with a kidney dysfunction. In
a study by Poon et al.66 albuminuria occurred only in the RME
and a fossil fuel (ULSD) treatment and similar histopathological
changes were reported. However, histological changes in the
outer cortex of the kidney tubules observed in both studies were
described as typical of hyaline-droplet nephropathy. This effect
occurred in rats after treatment with fossil fuel but also with
biofuels. They discussed hyaline-droplet nephropathy to be
specific to male rodents and unlikely to occur in humans and
therefore the significance in risk assessments as minimal.
Additionally, hepatomegaly was reported by Poon et al.65 after
oral administration of SME, RME and LS-DF and also changes
in the activity of phase I and phase II xenobiotic metabolism
enzymes in the S9 fraction were found after oral administration
of SME and LS-DF (cf. section ‘Studies on dioxin-like activity’).
After LS-DF and SME treatments they found no indication of
histological changes indicative of liver damage but an increase of
urinary ascorbic acid, interpreted as a biomarker of hepatic
response to xenobiotics. They concluded that RME and fish oil
methyl ester are less likely to be hepatotoxic than SME and
LS-DF. Overall, they considered SME to potentially be more of
a concern for human health than RME or fish oil methyl ester
after oral exposure but call for future work encompassing both
toxicological and analytical work to elucidate the potential
health effects of individual biodiesels.
Poon et al.66 investigated RME, SME, FraME and ULSD.
They could not confirm all results of the study by Poon et al.65
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Although the liver, thyroid and kidneys were again identified as
target organs after oral intake of biodiesel and ULSD, hepato-
megaly was only observed for ULSD and not reported for RME
or SME as in the earlier study. Furthermore, in the study by
Poon et al.65 the strongest but still moderate histological effects
on the thyroid were an increased epithelium height after LS-DF
and SME treatments and the vesiculation of nuclei in the thyroid
epithelium for the LS-DF treatment. In this context, the authors
referred to a potential connection with the elevated UDP-glu-
curonosyl-transferase activity in the liver. The increased enzyme
activity found after treatment with LS-DF might have had an
effect on the thyroid through increased thyroxin glucuronidation
but there are only few studies to confirm such an interaction.65 In
the study by Poon et al.66 ULSD was used instead of LS-DF and
no significant changes in the UDP-glucuronosyl-transferase
activity were measured. Additionally, the results of the groups
treated with SME and RME differed between the studies. A
statement on a sound scientific basis can, therefore, not be made.
In the study by Poon et al.65 only the LS-DF treatment
produced significant changes in the lipid profile with increased
serum free fatty acids and decreased liver triglycerides. In
contrast, Poon et al.66 report a decrease in serum free fatty acids
for ULSD. Liver triglycerides were not measured and no
significant changes occurred in the concentration of triglycerides
in the serum. Furthermore, treatment with both biodiesels
FraME and RME also induced significant changes in the lipid
profile that were not found by an earlier study.65 Therefore, these
results again are inconsistent. Taken together, however, in both
studies the fossil fuels caused greater histopathological and
biochemical effects and the effects caused by the biodiesel
depended on the feedstock. Nevertheless, results of these studies
indicate oxidative stress after both, treatment with fossil fuels
and biofuels.
3.6.3 Inhalation toxicity. A study investigating inhalation
toxicity of ethanol–gasoline vapor in rats revealed increased
kidney weight and elevated liver microsomal EROD activity.67
Combined exposure to ethanol and gasoline appeared to exert an
additive effect on growth suppression. Inflammation of the upper
respiratory tract was only observed in ethanol–gasoline mixture
groups. They concluded that their treatments with ethanol and
gasoline produced mild, reversible, biochemical, hematological
and histological effects, with some indications of interactions
when they were co-administered.
4. Further publications on (eco)toxicological
investigations of additives and potential biofuels
The gasoline additives ethyl tertiary-butyl ether (ETBE) and
methyl tertiary-butyl ether (MTBE) are fuel oxygenates used to
improve the combustion and reduce the emission of pollutants.
For both substances reviews and comprehensive studies on (eco)
toxicological effects have already been published. Data on e.g.
acute toxicity, eye- and skin-irritation, developmental and
reproductive toxicity and genotoxicity were compiled for the
substance ETBE after oral and inhalation exposure.83,84 For
MTBE, studies on acute toxicity, neurotoxicity, endocrine
changes, reproductive toxicity, genotoxicity and carcinogenicity
after exposure to pure MTBE or MTBE containing gasoline with
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Table 1 Overview of main data gaps and recommendations for further investigations
Main data gaps
A direct comparison of studies is not possible due to
variations in the test procedure:
- no standardized reference fuel
- no standardization regarding sample sampling
(emissions) and preparation
- different procedures for the calculation of the
toxic potentials of biofuel emissions
A sound data basis is missing that would allow
a more accurate evaluation of potential
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(eco)toxicological or health effects of biofuels
Engine optimization was not accounted for or
was not mentioned in most studies
a focus on human health were reviewed.40,85,86 In addition,
overviews on the toxicity of MTBE to marine species87 and
freshwater organisms88 were published. Beside the aggregated
information that is already available, ETBE and MTBE are not
necessarily derived from renewable sources and, therefore, were
not further discussed in this review.
Further publications reviewed toxicological data not only of
ETBE and MTBE but also of further gasoline additives, i.e.
ethanol and methanol.40,86 These substances can be received from
biomass feedstock and used as biofuels. However, for methanol
and ethanol there are many areas of application. Due to their
relevance, several studies have been conducted and also reviews
published regarding their potential toxic effects (e.g. ref. 89–96).
Therefore, effects of methanol or ethanol themselves were not
addressed in this review. However, some recently published
studies on the effects of engine exhaust when methanol or
ethanol is used as a fuel and of a gasoline–ethanol mixture in an
inhalation study had been considered (see Sections 3.1, 3.2, 3.3
and 3.6.3).
A table listing the reviewed publications arranged on the basis
of the endpoints investigated and highlighting the methods and
main findings of each study is available in the ESI†. Further-
more, Table 1 provides an overview of the remaining data gaps
and recommendations for future research activities.
5. Conclusions
The literature reviewed proved toxicological and ecotoxico-
logical investigations as suitable for investigations of biofuels
and revealed quite contrasting and inconclusive findings con-
cerning the toxic effects of biofuels. In some studies a hazard
potential of biofuels was reported. However, most studies are not
directly comparable as different biofuels and reference fossil fuels
were investigated. In addition to this, different engines, operating
cycles and extraction procedures were used for the investigation
of emissions. General variations in toxicities induced by bio-
diesels derived from different feedstocks or by biofuels derived
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Key recommendations
It is crucial to find an agreement on a reference fuel and sample sampling/
preparation to allow the comparison of results among various studies.
Furthermore, to calculate the toxic potential of biofuel emissions,
a reference related to the differences in the energy content of the fuels is
more advisable than a reference to the weight unit, as a fuel might produce
emissions that contain fewer compounds that are toxic or compounds that
are less toxic to an organism but an overall higher amount of emitted
emissions to produce the same amount of energy
Further (eco)toxicological investigations of biofuels are essential, e.g.
regarding
- dioxin-like activity
- inflammatory potencies
- genotoxicity/mutagenicity
- histopathological and biochemical effects
- aquatic toxicity
Further research on engine optimization is advisable to reduce the toxic
potential of engine emissions. In this context, the toxic potential should not
only be determined based on the chemical analyses of regulated emissions
but also by (eco)toxicological investigations
from the same feedstock but processed in a different way (plain
oil, oil methyl or ethyl esters) were noted and identified. RME
and SME, for example, can produce lower or similar emissions of
mutagenic compounds compared to DF, whereas RSO may also
have strong opposite effects. In general, lower mutagenic activity
was observed for the diesel engine exhaust generated from neat
biodiesel in comparison to petro-diesel fuels. However when
mixed with fossil diesel fuels (blends), biodiesel did not neces-
sarily induce a lowering effect on the total mutagenicity of
emissions. Findings concerning the cytotoxicity, the inflamma-
tory potential, histopathological observations, biochemical
effects and the general toxicity to aquatic organisms also varied
widely among the published studies. Depending on the types of
application, species investigated, fuel batches, feedstock sources,
and further processing, the toxicity of biofuels and their emis-
sions can range from being less toxic to having toxicity equal to
petro-diesel. Moreover, there are first indications for biodiesel
emissions being more potent in terms of toxicity than DF emis-
sion extracts. Based on the currently published studies revealing
the toxic potential of biofuels and their exhaust, adverse impacts
on the environment and human health cannot be ruled out.
However, to date, only comparatively few (eco)toxicological
investigations on biofuels have been conducted and the data do
not allow a definite conclusion on the potential effects on the
environment and human health. Engine optimization, e.g., for
the run on biofuels, was not accounted for or was not mentioned
in most studies. Optimization of engines, however, might have
the potential to reduce the toxicity of biofuel exhausts and,
therefore, should be considered to avoid an overestimation of the
toxic potential.
Overall, for a better understanding of the risks associated with
the use of biofuels, blends, and additives including their envi-
ronmental behaviours and whole environmental performance,
additional experimental studies are advisable to avoid unin-
tended adverse effects. Besides chemical analyses, bioassays for
the screening of potential adverse effects should be performed. In
this context, the generation of scientifically sound results is
This journal is ª The Royal Society of Chemistry 2012
 View Article Online

absolutely essential to allow the comparability of data for
a better assessment of toxicological potentials of biofuels while
avoiding redundant testing. To allow a better comparability of
data, it would be helpful to unify the collection of emission
material and its further processing. The same applies for the
method used for an application of fuels in aquatic toxicity tests.
Furthermore, there is still a need to fill data gaps regarding
subchronic and chronic toxicity of biofuels to help characterize
and compare their toxicity. These investigations should be done
optimally at the time of product development to examine their
(eco)toxicological effects as early as possible. All of these
recommendations are necessary to avoid extensive use of biofuels
Published on 20 March 2012. Downloaded by Virginia Tech on 19/07/2013 00:25:12.
Excellence Initiative of the German federal and state govern-
ments to promote science and research at German universities.
The authors would like to thank the anonymous reviewers for
improving this manuscript with their constructive comments.
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