The following documents were submitted by the Republic of Ecuador to the international arbitration hearing the case between Chevron/Texaco and the Republic. These documents further show the impact of Chevron/Texaco’s decades’ long oil pollution on the people of Ecuador.

1. EXPERT OPINION OF BLANCA LAFFON, PhD
In the Matter of an Arbitration under the Rules of the
United Nations Commission on International Trade Law
Chevron Corporation and Texaco Petroleum Company vs.
The Republic of Ecuador, PCA Case No. 2009-23
November 7, 2014
Prepared for
Winston & Strawn LLP
1700 K Street N.W.
Washington DC 20006-3817
Prepared by
Prof. Blanca Laffon, PhD
Location A Coruña, Spain

2. EXPERT OPINION OF BLANCA LAFFON, PhD
CONTENTS
1. Executive Summary ............................................................................................................... 1
1.1. Personal qualifications and experience ............................................................................ 1
1. 2. Summary of scope of retention ....................................................................................... 2
1.3. Summary of opinions ....................................................................................................... 2
2. Bases of opinions ................................................................................................................... 3
2.1. Background information regarding the presence of contamination in El Oriente region 3
2.2. Similarity between exposure in the Concession Area and exposure to oil spills ............ 4
2.3. Acute health effects reported in populations exposed to oil spills .................................. 5
2.4. Alterations in the genetic material are in the origin of cancer development ................... 7
2.5. Genotoxicity tests and cancer risk ................................................................................... 9
2.6. Genotoxicity studies in people exposed to oil spills ...................................................... 13
2.7. Immune and endocrine toxicity studies in people exposed to oil spills ........................ 16
2.8. Miscellanea: IARC classification of crude oil ............................................................... 17
3. References ............................................................................................................................ 19
Appendix A – Epidemiological studies on acute toxic effects related to exposure to oil spills
Appendix B – Epidemiological studies on genotoxicity, immunotoxicity and endocrine
toxicity, and studies on potential toxicological assessment, related to exposure to oil spills
Appendix C – Curriculum vitae
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3. 1. Executive Summary
1.1. Personal qualifications and experience
I am an Associate Professor of Psychobiology at the University of A Coruña (Spain),
and I have been accredited by ANECA (Spanish National Agency for Evaluation of Quality
and Accreditation) as full Professor (meaning that I have earned enough merits for that
category) since February 2014.
I obtained my B.S. in Pharmacy from the University of Santiago de Compostela,
Spain, with honors and extraordinary award, in 1996, and my Ph.D. in Pharmacy from the
same University, also with honors and extraordinary award, in 2001. Following several
postgraduate university fellowships, including 23 months at the Portugal National Institute of
Health (Department of Environmental Health), I became an Associate Professor at the
University of A Coruña in December 2008. After completing my doctorate, I conducted
additional postgraduate studies in the fields of genomics, proteomics and bioinformatics
(2002), and genetic and molecular epidemiology (2006).
My research interest is focused on the effects of pollutants on organisms, especially at
the molecular and cytogenetic levels, by conducting in vitro, in vivo and human
epidemiological studies aimed to evaluate the genotoxicity and cytotoxicity associated with
exposure to environmental or occupational contaminants. Genotoxicity studies adverse effects
on genetic material. Cytotoxicity studies adverse effects at the cellular level, specifically on
the cell cycle and viability.
In this context, I have conducted (with my research group) a complete biomonitoring
study of people exposed to the Prestige oil tanker spill, which occurred off the coast of
Galicia (Northwest of Spain) in November 2002. The primary objective was to evaluate the
possible damage to the genetic material (genotoxicity) in people exposed to this oil as a
consequence of participating in the cleanup operations. We also determined other markers of
endocrinologic toxicity (hormones indicating psychophysiological stress), and of
immunologic toxicity (several parameters indicating alterations in the immune system, which
is closely connected to the endocrine and nervous systems). In these studies we identified
significant alterations that my team and I detailed in our published articles.
We completed a follow-up assessment of the alterations observed seven years after the
workers were initially exposed. As a result of these studies, and the related papers published
in international journals, the Institute of Medicine of the National Academies invited me to
participate as an advisor in the Workshop assessing the human health effects of the Gulf of
Mexico oil spill, held in New Orleans (LA-USA) in June 2010 (2 months after the Deepwater
Horizon BP platform accident).
I have led or participated in over a dozen research projects, supported by grants from
the Galician and Spanish Ministries of Science and the European Commission. The results of
these investigations were published in over 90 scientific articles and book chapters I authored
or co-authored (more than 1,000 citations received, h-index = 20 according to Scopus
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4. database), and a great part of them focused on genotoxic effects associated with exposures to
potentially toxic agents (in vitro and human population studies). Six of my research works
were awarded scientific prizes from different private and public entities. Additionally, I am
Associate Editor of several indexed peer-reviewed scientific journals, and I serve as a journal
peer-reviewer for over thirty journals and as a research project peer-reviewer for public
institutions from different countries.
My academic activities include teaching Genetic Toxicology, Environmental
Toxicology and Public Health, Psychopharmacology, and Congenital Alterations of Language
in different Degrees and Masters, and supervising research works. In the past 12 years, I have
supervised 8 Ph.D. theses, 3 Master theses and 7 Honors Degrees. I have provided a detailed
CV in Appendix C.
1.2. Summary of scope of retention
I was retained in August 2014 by Winston & Strawn LLP to provide my expert
opinion regarding the health impacts of petroleum in conjunction with the Bilateral
Investment Treaty (BIT) arbitration between Chevron Corp. and the Republic of Ecuador.
1.3. Summary of opinions
1. The closest exposure situations to the one present in the Concession Area that have been
the subject of genotoxicity research are those experienced as a consequence of major
marine spills of crude oils or fuel oils by residents and workers who participated in the
cleanup tasks.
2. Most investigations carried out after oil spill accidents are cross-sectional epidemiological
studies that analyze acute physical effects or psychological consequences in the exposed
population: cleanup workers or residents. Data obtained in these studies indicate that
people exposed to oil spills experience acute physical consequences, including upper
respiratory tract illnesses, headaches, nausea, vomiting, and more.
3. Although classical epidemiological studies are very useful for establishing causal
relationships between an exposure and an adverse health outcome, molecular
epidemiology studies using genotoxicity biomarkers (indicative of damages in the genetic
material) are an important tool by which to assess cancer risk in people exposed to
occupational or environmental carcinogens. Genotoxicity biomarkers provide early and
reliable warning signals of cancer risk.
4. My molecular epidemiology studies of the genotoxic effects in people exposed to the
Prestige oil spill as a consequence of their participation in the cleanup operations indicate
that this exposure induced DNA damage. That damage became fixed as chromosome
alterations, thus increasing the risk of cancer development, after only several months of
exposure.
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5. Additional studies carried out two years after the exposure to Prestige oil detected that a
higher proportion of exposed participants had structural chromosomal alterations, which
seemed to increase with intensity of exposure.
The most recent Prestige study to be carried out examined individuals seven years after
they were exposed to the oil for a mean of 9 months (range 2-10 months). While this study
suggests that a prolonged period of non-exposure to oil might lead to the removal of DNA
damage induced by the exposure, the plasma cortisol levels and percentage of natural
killer cells continued to be significantly altered in the population that was previously
exposed, notwithstanding that this population had been free from exposure for seven
years. These alterations to the exposed population’s immunological and endocrine
systems lead to an increased risk for developing cancer and/or other diseases.
Accordingly, the study recommended periodic health monitoring for those people who
were exposed to the Prestige oil spill.
5. Taken together these studies show that the exposed population in El Oriente is at risk for
developing health problems, including in particular, cancer. Unlike the populations who
were exposed to marine oil spills for mere months, the Ecuadorians living in the
Concession Area have been exposed to oil for decades and continue to be exposed even
today. Additionally, the people who engaged in the cleanup of the oil spills usually wore
protective gear, meaning their exposure pathways were limited mostly to inhalation. In
contrast, the people living in El Oriente have been exposed to oil through various
pathways and they do not wear protective clothing.
These opinions are given to a reasonable degree of scientific probability. They are
based on my education, training, experience, information and data available in the scientific
literature, and information and data about this lawsuit made available to me at the time these
opinions were formulated. If additional information becomes available, I may supplement my
opinion to reflect such additional information.
The bases for these opinions are provided in this report. The documents I relied upon
to reach these opinions are cited in the document and listed in the references section at the end
of the report.
2. Bases of opinions
2.1. Background information regarding the presence of contamination in El
Oriente region
Oil extraction and production operations in the Ecuadorian Concession Area involved
petroleum exploration surveys, drilling exploration and production wells, processing crude oil
at the wellhead or production facility, maintenance activities, and transporting oil via
pipelines. According to the Louis Berger Group (LBG) report, “these operations resulted in
the uncontrolled release of waste materials and byproducts into the air, surface water, stream
sediment groundwater, and soil. Materials released included crude oil, drilling mud, formation
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6. (produced) water, cleaning solvents, diesel fuel, sanitary wastes, burned and unburned flare
gases, and diesel exhaust”, containing hazardous and toxic chemicals (December 2013 LBG
Expert Report at 47). Many of these chemicals are persistent in the environment.
Extensive data collected by Dr. Harlee Strauss in her opinions show that, as a
consequence of oil extraction and production operations, adults and children residing in the
Concession Area have been exposed to toxic and hazardous contaminants via multiple
exposure pathways (ingestion and dermal exposures) through diverse activities. (December
2013 Strauss Report at 5). These exposures to contaminated environmental media have been
nearly continuous during the time that individuals lived in the vicinity of the facilities, often
counted in many years. Unlike occupational exposures, there have been no recovery periods
from the exposures (nights/weekends/vacations), and vulnerable groups such as the very
young, fetuses, elderly, and the infirm are also part of the exposed population. (December
2013 Strauss Report at 5). In this regard, as pointed out by Goldstein in regards to the Gulf of
Mexico oil spill, “children are at particular risk for effects from environmental exposures”
since, “[a]s compared with adults, they breathe in more air per unit of body mass, their bodies
detoxify many chemicals less effectively, and they explore more adventurously” (Goldstein,
2011 at 1339). In addition, “[t]here is inadequate information about the potential reproductive
and developmental effects of crude-oil components”, thus “[p]regnant women should
particularly avoid dermal contact with oil and should avoid areas with visible oil
contamination or odors” (Goldstein, 2011 at 1339).
As demonstrated in the LBG rejoinder report, such contamination in the Concession
Area is still present and widespread; even some of the sites included in Texaco Petroleum’s
remediation plan continue to be a persistent source of environmental contamination.
Therefore, some exposure is on-going.
2.2. Similarity between exposure in the Concession Area and exposure to oil
spills
As already stated by Dr. Grandjean, perhaps the closest exposure situations to the one
present in the Concession Area are those experienced as a consequence of major marine oil
spills by residents and workers who participated in the cleanup tasks (Grandjean Report (Nov.
22, 2013) at 5). For some of these accidents (9 out of 40 major oil spills), studies on effects of
exposure to diverse aspects of human health have been performed. In 6 of these accidents
(Exxon Valdez, MV Braer, Sea Empress, Tasman Spirit and Hebei Spirit tankers, and
Deepwater Horizon platform), the spill consisted of crude oil; in the 3 other cases, the spill
was caused by fuel oil No. 6 (also named bunker C) (Nakhodka, Erika and Prestige). I
conducted several studies following the Prestige spill, detailed below in this report.
Crude oil is a complex combination of hydrocarbons consisting predominantly of
paraffinic (straight and branched-chain alkanes), naphthenic (cycloalkanes or cycloparaffins),
and aromatic hydrocarbons (API, 2011 at 5). Sulfur, oxygen and nitrogen compounds,
organometallic complexes notably of nickel and vanadium, and dissolved gases, such as
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7. hydrogen sulfide, are also found in crude oil. Similar hydrocarbons, heterocyclics, metals and
other constituents, e.g., hydrogen sulfide, are present in all crude oils but their proportions
vary depending on the crude source (API, 2011 at 5). Fuel oils are produced from crude
petroleum by different refining processes, depending on their intended use, and are composed
of complex and variable mixtures of aliphatic (alkanes, alkenes, cycloalkanes) and aromatic
hydrocarbons, containing low percentages of sulfur, nitrogen, and oxygen compounds
(Laffon, 2014 at 667). The exact chemical composition of each of the fuel oils may vary
somewhat, depending on the source, the refinery involved, the presence of additives or
modifiers, and other factors (Laffon, 2014 at 667).
Dr. Jeffrey Short has compared the crude oil produced in the Oriente to the Prestige
fuel oil and has concluded that the two share similar suites of toxic compounds. (Short Expert
Report (Nov. 7, 2014) at Section 4.6).
2.3. Acute health effects reported in populations exposed to oil spills
Most investigations carried out in human populations after oil spill accidents are cross-sectional
epidemiological studies that analyze acute physical effects or psychological
consequences in the exposed people: cleanup workers or residents. Data obtained in these
studies, reviewed in Aguilera et al. (2010) and discussed in Dr. Strauss’ first report (Strauss
Report (Feb. 18, 2013) at 28-31), indicate that people exposed to oil spills experience acute
physical consequences, including upper respiratory tract illnesses, throat and eye irritation,
headaches, dizziness, nausea, and vomiting. These studies concluded that, although
respiratory symptoms are long-lasting, these consequences generally diminish with time once
exposure has ceased (Aguilera, 2010 at 297-98).
Additional studies published recently, and therefore not included in the Strauss and
Aguilera reviews, also support these general conclusions, the results of which are described
briefly in the following paragraphs. Summarized data of all studies published so far on acute
toxic effects in people exposed to oil spills are presented in Appendix A.
New studies regarding Prestige oil-exposed populations showed persistent respiratory
symptoms in fishermen two years after the exposure (Rodríguez-Trigo et al., 2010 at 489-90),
and a higher prevalence of lower respiratory tract symptoms five years after cleanup in the
exposed fishermen than in the controls (Zock et al., 2012 at 508). Six years after exposure, the
Zock data indicated the persistence of objectively measured indices of respiratory health
impairment in cleanup workers1 (Zock et al., 2014).
After the Tasman Spirit disaster, Meo et al. (2009a) noted higher rates of health
complaints like eye irritations, respiratory problems, headaches, nauseas, and general illness
in oil-exposed individuals. A significant reduction in lung function parameters was observed
in those subjects exposed for more than 15 days (Meo et al., 2009b).
1 The authors recognized that they could not formally demonstrate that this persistence was due to
exposure because of limitations in the study design (mainly related to the selection of the control population).
(Zock et al., 2014).
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8. Regarding the Hebei Spirit spill, significantly increased risks of several physical
symptoms like headache, nausea, dizziness, fatigue, tingling of limb, hot flushing, sore throat,
cough, runny nose, shortness of breath, itchy skin, rash, and sore eyes were observed in
residents from the heavy and moderately oil soaked areas as compared with residents from
light soaked areas (Lee et al., 2010 at 170). Children who lived closest to the oil spill area
showed higher respiratory effects (Jung et al., 2013 at 367-68). In a questionnaire study, the
scientists observed that more frequent and greater exposure in people engaged in cleanup was
strongly associated with a higher occurrence of acute symptoms (Sim et al, 2010 at 51).
Another study reported a similar result, showing associations in residents between physical
symptoms and exposure levels by evaluating urinary metabolites of volatile organic
compounds (“VOC”), polycyclic aromatic hydrocarbons (“PAH”) and heavy metals (Cheong
et al., 2011 at 3-5). Furthermore, longer cleanup work in volunteers was also associated with
an increase in symptoms such as visual disturbance, nasal and bronchus irritation, headaches,
heart palpitations, fatigue and fever, memory and cognitive disturbance, and abdominal pain
(Ha et al., 2012 at 169). The first study to quantify the burden of disease (BOD) due to an oil
spill, which is “necessary to assess the scale of health damage at the population level as well
as the associated compensation costs,” (Kim et al., 2013 at 2) found that the BOD for 1 year
for the residents living near contaminated coastal areas was significant and related to
proximity to the spill (Kim et al., 2013 at 2). The Kim study also found that for persons who
participated in cleanup efforts, asthma and post-traumatic stress disorder comprised the most
prominent disease burden in the contaminated areas. One year after the Hebei Spirit accident,
eye symptoms, headaches, skin symptoms, and neurovestibular symptoms had a longer
duration in people involved with the cleanup efforts than did back pain or respiratory
symptoms (Na et al., 2012 at 1251).
Many studies focused on the health effects of oil on human populations following the
Deepwater Horizon platform disaster in the Gulf of Mexico. Over one-third of children
experienced either physical symptoms or mental health distress, as reported by their parents
(Abramson, 2010 at 4). Additionally, significant alterations were observed in several clinical
parameters such as platelet counts, hemoglobin levels, hematocrit, and a number of liver
enzymes in subjects participating in the cleanup activity when compared to the controls
(D’Andrea and Reddy, 2013 at 967). Data obtained in the same exposed population indicated
that they are at risk of developing alterations in their hematological profile and liver function
(D’Andrea and Reddy, 2014 at 866e.12).
Dr. Moolgavkar in his May 2013 expert report for Chevron (pages 17-18) manifested
his concern that “many of the oil spill studies that Dr. Strauss cites (page 29 of her report)
lack appropriate comparison populations to determine whether the observed health symptoms
are in excess of expectation. Nearly all of these studies evaluated nonspecific self-reported
health symptoms, with a high probability of recall bias.” Nevertheless, the newly published
studies on health effects related to exposure to oil spills often include a comparison group:
either control groups (Rodríguez-Trigo et al., 2010; Zock et al., 2012 and 2014; Meo et al.,
2009a and b; Cheong et al., 2011; D’Andrea and Reddy, 2013), lightly exposed individuals
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9. (Lee et al., 2010), or the same individuals before the exposure started (Ha et al., 2012).
Moreover, although some of these studies evaluated self-reported health symptoms, others
were based on analysis of objective and specific clinical parameters, such as respiratory
parameters (e.g., forced spirometry, methacoline challenge, markers of oxidative stress,
airway inflammation and growth factor activity in exhaled breath condensate, and the skin
prick test for common inhalant allergens) (Rodríguez-Trigo et al., 2010; Zock et al., 2014;
Meo et al., 2009b; Jung et al., 2013), or hematological parameters (e.g., white blood cell and
platelets counts, hemoglobin, hematocrit, blood urea nitrogen, creatinine, liver enzymes)
(D’Andrea and Reddy, 2013 and 2014).
Thus, previous and recently published studies provide evidence sufficient to establish
the relationship between exposure to oil spills and the development of acute physical effects
in the exposed individuals. As pointed out by Levy and Nassetta in their review article, “these
studies found that cleanup workers and community residents who were exposed more
intensively and/or for longer periods of time tended to have a higher frequency of acute
symptoms” (Levy and Nasetta, 2011 at 162).
The health effects reported in all of the studies discussed above in populations exposed
to oil spills are similar to the ones reported in the Concession Area communities, extensively
reviewed in Dr. Strauss’ report. They also provide further support to her opinion that
symptoms and pathologies described in individuals exposed to crude oil and residues from El
Oriente extraction and production activities are consistent with effects reported for exposure
to oil spills.
2.4. Alterations in the genetic material are in the origin of cancer development
There are several epidemiological studies referenced in the expert reports filed in this
litigation that discuss the likelihood of a causal connection between exposure to oil and cancer
(Grandjean, Strauss, Moolgavkar). Cancer is one of the most complex diseases affecting
humans; it remains a major chronic health problem associated with toxicological substances
(Barret, 1993). The cause-effect relationship that represents the basis of the pathological
investigation is not easy to apply to the process of human carcinogenesis. Most of the
population is exposed to a variety of human carcinogens in their daily life, yet only a small
fraction of exposed individuals actually develop cancer (Carbone and Pass, 2004 at 400).
Since fewer than 10% of all cancers are hereditary, and cancers caused by infection are
thought to constitute some 15% of the non-hereditary cancers, the 70% to 80% remaining are
called “sporadic,” because they are essentially of unknown etiology (Brucher and Jamall,
2014 at 2). They are probably related to exposure to chemical and physical agents with
carcinogenic potential. Agents present in food, tobacco smoke, occupational environments,
alcohol, urban pollution, medicine and medical procedures, and industrial products have been
under investigation for at least three decades (Doll and Peto, 1981), and evidence of their
carcinogenesis has now been obtained for many of these agents (reviewed in Clapp et al.,
2008; Irigay et al., 2007).
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10. Cancer is today recognized as a highly heterogeneous disease: more than 100 distinct
types of human cancer have been described, and various tumor subtypes can be found within
specific organs (Grizzi and Chiriva-Internati, 2004). Because all cancers share the properties
of uncontrolled growth, invasion, and metastasis, a common mechanism for their origin has
often been suggested (Couch, 1996 at 136).
The association between genetic alterations and human cancer was first observed
decades ago and explained in Theodor Boveri’s somatic mutation theory of cancer (Balmain,
2001 at 77), which states that a tumor can arise by self-proliferation from a cell that has been
transformed by acquired modification of its genetic material. A causal association between
genetic alterations and cancer is supported by extensive experimental and epidemiological
data (Dixon and Kopras, 2004 at 441), proving that Boveri’s theory is as sound and correct as
any scientific theory ever can be (Heim, 2014 at 138). Thus, somatic gene mutations are
widely accepted as the basic event in the conversion of a normal cell into a cancer cell:
carcinogens interact with DNA resulting in irreversible changes, which predispose the cells to
malignant transformation.
It is generally accepted that chemical carcinogenesis is a multistep process, each step
corresponding to a genetic event in a cell which provides the cell with a selective advantage in
terms of survival and/or proliferation (Monier, 2000 at 603-604). The final risk of cancer
development is a function of the combined probabilities of relatively rare events occurring in
each stage (Franco et al., 2004 at 415). Extensive experimental observations in chemical
carcinogenesis have demonstrated this process can be separated operationally into three
general stages, i.e., initiation, promotion, and progression, through which a normal cell
evolves into a cancer cell as the result of heritable changes in multiple, independent genes
(Vincent and Gatenby, 2008 at 729).
Initiation follows exposure to mutagens and involves the induction of a permanent and
irreversible change in a cell’s genome, which provides it with a growth advantage over its
neighbors, although little or no observable changes in the cellular or tissue morphology can be
observed. Promotion is the experimentally defined process by which the initiated cell expands
by self-proliferation into a visible tumor, often a benign lesion. During progression benign
tumors are transformed into malignant cancers, involving the acquisition of one or more
qualitative changes in the precursor cells. When chronic exposure is involved, few chemicals,
if any, will affect only one stage in the multistep carcinogenic process (Barret and Wiseman,
1987 at 65). In fact, most chemical carcinogens operate via a combination of mechanisms
(they are not mutually exclusive; rather, they probably work in conjunction to result in
neoplastic development), and even their primary mechanism of action may vary depending on
the target tissue/cells (Barret, 1993 at 9).
The vast majority of chemical carcinogens are ‘genotoxic’ in their carcinogenic mode
of action, which means that they (or their metabolites) are capable of interacting with the
genetic material, thereby inducing DNA damage. There is, however, a smaller group of
carcinogens that induce cancer via ‘non-genotoxic’ mechanisms. Hernández et al. (2009)
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11. reviewed these possible mechanisms including endocrine modification, tumor promotion,
tissue-specific toxicity and inflammation, cytotoxicity and immune suppression, and
inhibition of gap-junction intercellular communications, among others.
The correlation between the ability to induce changes in DNA and tumorigenesis is
well established for most chemical initiating agents (Couch, 1996 at 136). Indeed, most
initiating agents are genotoxic. For instance, PAHs are mutagenic agents that act as tumor
initiators. A single exposure to these agents does not typically give rise to a tumor, but may
produce latent damage that can result in tumor formation following a subsequent insult
(Couch, 1996 at 136).
A very important aspect of the chemical carcinogens dose-effect relationship is the
eventual determination of a threshold. As indicated by Monier, “[f]or a non-genotoxic
carcinogen, a threshold [in the dose–effect relationship] can be safely assumed. For genotoxic
drugs [chemicals], however, it is usually difficult to prove or disprove that a threshold does
exist, and the tendency is to accept linear no-threshold relationships in determining
permissible levels of exposures” (Monier, 2000 at 604). Thus, Goldstein et al. (2011) suggest
that “[r]egulatory prudence has led to the use of ‘one-hit models’ for mutagenic end points,
particularly cancer, in which every molecule of a carcinogen is presumed to pose a risk”. In
other words, the safety threshold for genotoxic carcinogens is effectively zero, with the
presumption that any exposure increases risk, or there is no dose free of risk.
Since a malignant cell needs to acquire multiple, heritable alterations at independent
genetic locations, chemical carcinogenesis development involves a long delay (long latency
period) between the causal event and the clinical manifestation of disease (Couch, 1996 at
134). In the case of solid tumors there is a 20 to 40-year interval from the time of exposure of
an individual to a chemical or viral carcinogen until the clinical detection of a tumor (Wogan
et al., 2004 at 482).
2.5. Genotoxicity tests and cancer risk
The traditional epidemiological technique has always been the hallmark approach to
demonstrate associations between exposure to hazardous substances and the development of
disease such as cancer (Bonassi and Au, 2002 at 73). As expressed in his expert opinion of
May 2014 (page 3), Dr. Moolkgavkar contends that epidemiological studies, in contrast to
risk assessment, “are necessary to reach a conclusion that an exposure resulted in adverse
health outcomes”, since they “evaluate what actually did happen.” I agree that
epidemiological studies are very useful when the conditions are appropriate to carry them out.
However, epidemiological methods – the study of the factors that control the patterns of
incidence of disease – normally require large numbers of subjects and/or long periods of time,
because what is measured (the occurrence of disease) is a rare event (Collins, 1998
at 360).
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12. Not surprisingly, therefore, few epidemiologic studies on cancer incidence or mortality
related to exposure to oil have been performed to date. Nonetheless, data useful for assessing
causal associations between oil and cancer risk may be obtained by other scientifically
reliable methodologies. Specifically, molecular epidemiology has developed to attempt to
integrate traditional epidemiological investigation of cancer risk factors with the substantial
expansion of knowledge of the molecular mechanisms of cellular processes (Shields and
Harris, 1991). This approach has a great potential in monitoring cancer risk in people exposed
to occupational or environmental carcinogens, especially when waiting for large scale studies
conducted over decades of time will not sufficiently protect the health of those exposed.
The essential feature of molecular epidemiology is the use of biomarkers, with clear
advantages of economy, speed and precision, to measure in individuals such things as
exposure to agents implicated in the etiology of a particular disease, pre-clinical
manifestations of disease, or features of the disease itself. Biomarkers are measurable
biological parameters (something that can be measured in human subjects) that reflect, in
some way, an individual's risk of disease, because they indicate exposure to a causative agent,
or because they represent an early stage in the development of the disease. Therefore, the
ultimate goal of using biomarkers in molecular epidemiological studies is to provide valuable
information to be able to predict health risks (Collins, 1998 at 360). Thus, biomarkers are
used as meaningful and indispensable tools for investigation into environmental mutagenesis
and cancer risk assessment, since they provide early and reliable warning signals of cancer
risk (Au, 2007 at 241).
In the context of carcinogenicity, biomarkers can mean proof of exposure to a
carcinogen, detection of a reaction product or an indication that a preliminary genotoxic event
or actual DNA damage has occurred (Committee on Carcinogenicity, 2013 at 2). The
following describes biomarkers used in studies on genotoxic effects in oil-exposed
populations, which are frequently employed in cancer molecular epidemiology, and their
association with risk of cancer estimation:
 Chromosomal alterations: In normal circumstances, when a DNA insult is produced, most
of the damage is repaired within hours if not minutes. Importantly, however, some of the
DNA damage may not be repaired. The amount of unrepaired damage depends on the
extent of the damage not only to the DNA, itself, but also to the system that functions to
repair DNA2. Some of the unrepaired damage can result in microscopically visible
changes in chromosomes, which are cytogenetically detected as micronuclei or
chromosome aberrations.
 Micronuclei (MN): MN represent whole chromosomes or chromosome fragments that
are excluded from the re-forming nucleus at the end of nuclear division and remain in
2 Physical and chemical agents that are able to react with DNA and proteins (e.g., DNA repair enzymes)
might at low doses interfere with cellular DNA repair processes. Furthermore, the individual DNA repair
capacity is influenced by the possible presence of numerous polymorphisms in DNA repair genes which may
modify the activity of the encoded proteins.
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13. the cytoplasm forming a small nuclear body (a micronucleus). The use of MN as a
measure of early genotoxic effects has become a standard assay in human
biomonitoring studies (Mateuca et al., 2012 at 317). Regarding populations
occupationally exposed to PAHs, a recent meta-analysis showed that frequencies of
MN in lymphocytes may be indicators of early genetic change in these individuals
(Wang et al., 2012 at 22). MN assessment is a relevant biomarker because MN
represent irreversible biological alterations that can lead to the development of cancer
(Au, 2007 at 241). Thus, MN are considered to be biomarkers of early carcinogenic
effects (through genotoxic mechanisms). Indeed, an analysis performed within the
framework of the HUMN project (HUman MicroNucleus international collaborative
project, http://humn.org) indicates that an increased frequency of MN in peripheral
blood lymphocytes predicts cancer risk in humans (Bonassi et al., 2007 at 625). The
existing evidence linking MN frequencies with cancer risk was also substantiated by a
recent meta-analysis of 37 publications, which clearly showed a 45% increase (28%-
64%, 95% confidence interval) in the baseline MN level of untreated cancer patients
compared to cancer-free referents (Iarmarcovai et al., 2008 at 274). A recent review on
this topic (Bonassi et al., 2011 at 94) concluded that “the presence of association
between MN formation in the leukocytes of healthy individuals and subsequent risk of
cancer is supported not only by theoretical considerations but also by a large range of
experimental findings”.
 Chromosome aberrations (CAs): CAs include breaks, deletions, duplications,
circularisation, dicentrics (i.e., two centromeres on one chromosome) and
translocations. Lymphocytes, when stimulated to proliferate in vitro, may reveal the
effects of accumulated, unrepaired damage as chromosome aberrations at the first cell
division. Many aberrations lead to loss of chromosomal material in one of the
daughter cells, or may even disrupt division itself resulting in a high probability of
cellular dysfunction or death (Collins, 1998 at 372). However, translocation of a
segment of one chromosome to a site on another chromosome tends not to involve
significant loss of genetic material, and translocations tend to be stably transmitted
through generations of cells. They have a potential clinical importance; although genes
are not lost, the regulation of their expression may be altered in the new chromosomal
context (Collins, 1998 at 372). Some CAs are typically found in particular types of
cancer. For instance, the characteristic ‘Philadelphia chromosome’ is present in the
leukemic cells of almost all patients with chronic myelocytic leukemia. It typically
results from a balanced reciprocal translocation, which transposes the abl proto-oncogene
(found on chromosome 9) to a region on chromosome 22. As a result, an
abnormal fusion protein with oncogenic properties is produced (Jabbour and
Kanterjian, 2014 at 548). Other B and T cell lymphomas and leukemias are also
accompanied by specific translocations. CAs have been demonstrated to be an early
predictor of cancer risk. The extensive use of this assay has resulted in the
accumulation of valuable data in many laboratories. This has enabled the examination
of the potential association between previously measured CA frequency and
subsequent cancer outcome. An association between high CA frequency and increased
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14. cancer incidence was originally detected in a collaborative project of 10 Nordic
cytogenetic laboratories (Hagmar et al., 1994 at 2921). An independent study among
10 laboratories in Italy, based on cancer mortality data, arrived at the same conclusion
(Bonassi et al., 1995 at 133). The two cohorts were afterwards updated and examined
together; the results supported the findings that CAs are predictive of cancer risk
(Hagmar et al., 1998 at 2921). Furthermore, a case-control study nested within the
two cohorts indicated that this association is not merely a reflection of smoking or
occupational exposure to carcinogens, but is similarly seen in apparently unexposed
subjects (Bonassi et al., 2000 at 1619).
 Sister chromatid exchanges (SCE): SCEs are reciprocal DNA exchanges occurring during
replication of the genetic material, just before cell division, between the two sister
chromatids of a duplicated chromosome (Mateuca et al., 2012 at 306). It is thought that
SCEs reflect a disruption of the normal replication process by the presence of DNA
lesions (Collins, 1998 at 374). Since SCEs are the manifestation of damage to DNA, i.e.,
they may involve errors and therefore possible mutations, they are direct indicators of the
adverse effects of exposure to DNA damaging agents (Tsongas, 1984 at 988).
 Measurements of primary DNA damage: This includes DNA breaks, altered bases or
adducts. Two of the most common methods to determine this kind of DNA damage are
the evaluation of DNA adducts and the comet assay. DNA adducts are formed by the
chemical reaction of DNA with a variety of classes of DNA-damaging agents. The comet
assay measures breaks in the DNA strands or lesions which give rise to breaks; it is
commonly used in investigations evaluating populations potentially exposed to
genotoxicants. Although exposures to non-genotoxic carcinogens will not be detected
using these assays, they are considered to be valuable methods for detection of genotoxic
exposure in humans. However, the DNA damage measured by the comet assay (and also
by evaluation of DNA adducts) identifies hazard rather than risk, and its value for
predicting cancer is not yet known because it has not been investigated in prospective
cohort studies (Albertini et al., 2000 at 129).
 Mutations in marker genes: Mutations are exceedingly rare events. The mutagenic
potential associated with a given exposure is evaluated by determining mutations induced
in several well-established marker genes. One of them, and probably the most frequently
used in biomonitoring studies, is the hprt gene, commonly studied in lymphocytes.
Although the implications of elevated frequency of hprt mutations for cancer risk have not
been assessed in prospective human studies, molecular analyses of in vivo derived hprt
mutations have shown types of mutations similar to mutagenic changes seen in cancer-related
genes or genomic regions associated with cancer (Albertini and Hayes, 1997; Cole
Page - 12
and Skopek, 1994).
Regarding the relationship between genotoxicity biomarkers and risk of cancer, it is
noteworthy that, in evaluating the carcinogenic potential of chemicals, the International
Agency for Research on Cancer (IARC) reviews data from genotoxicity studies (including

15. DNA damage, gene mutation, SCEs, MN formation, CAs and aneuploidy) in view of the
relevance of these processes to carcinogenesis (IARC, 2006 at 10-12).
The described biomarkers are usually assessed in peripheral blood leukocytes. In
human trials, only a limited range of biological material can be obtained without ethically
unacceptable intrusion. For this reason, to estimate events occurring at the target organs and
to provide early warning signals for health risk, assessment of genotoxicity is normally
carried out in readily available surrogate cells (Mateuca et al., 2012 at 306). The most
frequently used surrogate cells in human studies are the peripheral blood leukocytes
(reviewed in Salama et al., 1999 at 99). The major motive for using leukocytes is that these
cells circulate throughout the body and that they have reasonably long life-span if a suitable
cell type is considered (e.g., T-lymphocytes); therefore, they can be damaged in any
tissue/organ-specific toxic environment (Au, 2007 at 241).
2.6. Genotoxicity studies in people exposed to oil spills
Given the relationship between genotoxicity parameters and cancer risk, several
studies have aimed to evaluate genotoxic effects in people exposed to oil spills (MV Braer
and Prestige). The details on the design and results of these studies are presented in Appendix
B.
The two studies corresponding to the Prestige oil spill were carried out by a research
group of which I was a part. The first study included people involved in autopsies and cleanup
of oil-contaminated birds (Laffon et al., 2006), and the second one, partially published in
several different papers (Pérez-Cadahía et al., 2006, 2007, 2008a, 2008b, 2008c) analyzed
volunteers and workers who participated in the cleanup of beaches and rocks. Results
obtained in the group of volunteers handling oil-contaminated birds showed significant
increase, when compared to the control group, in DNA damage (evaluated by means of the
comet assay), related to the duration of exposure, and also in the chromosomal damage (MN
test), although in this last case significance was not reached.
Exposed individuals included in the second study were divided into three groups:
volunteers who cleaned up oil on the beaches for 5 days; workers who collected oil manually
on the beaches for 3 months (MW), and workers who used high-pressure water jets to clean
rocks on or near the beach for 4 months (HPW). Significant increases in DNA damage over
the control individuals were observed in all exposed groups. Significant increases were also
detected for the MN test in MW, and for SCE test in HPW. It is generally considered that, for
chronic exposures, cytogenetic techniques (such as MN and SCE tests) express cumulative
events, while the comet assay provides information about recent repairable exposure levels
(Maluf and Erdtmann, 2000 at 26). Hence, the results obtained indicate that exposure to
Prestige oil induced DNA damage, and this damage became fixed as chromosome alterations,
thus increasing the risk of cancer development, after only several months of exposure.
Additionally, a cell proliferation index, indicative of toxicity to the cell cycle, was also
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16. evaluated, and again significant effects for this index were observed only in those subjects
exposed for months.
Afterwards, as further confirmation of the results obtained in these two epidemiologic
studies, an in vivo study using a rat model of subchronic exposure to a fuel oil with similar
characteristics to that spilled by the Prestige tanker was carried out by our research group
(Valdiglesias et al., 2012 at 756), in order to determine potential genotoxic effects under
strictly controlled exposure conditions. Results obtained showed that inhalation oil exposure
induced DNA damage in the rats, and also alterations in the DNA repair response, although
the sensitivity to oil substances varied depending on the rat strain. These data supported the
previously described genotoxic effects in humans exposed to Prestige oil during cleanup
tasks.
Regarding the design of the abovementioned epidemiologic studies, Dr. Moolgavkar
in his report (page 5) expressed concerns about epidemiologic studies which are ecologic in
design (not including individual-level data on exposure), and about the lack of control for
potential confounders in these studies, in order to use their results to establish causal
associations. Unlike the studies criticized by Dr. Moolgavkar, the Prestige studies were not
ecologic in design. The oil-exposed subjects in fact reported individual-level data on exposure
— excepting the one Prestige study wherein an automated sampler was used to analyze
environmental levels of VOC in the working room for individuals handling oil-contaminated
birds — and all of the studies reported data on several potential confounders (age, gender and
smoking habits, etc.) known to influence genotoxicity assays results. Moreover, since these
studies were based on experimental laboratory analyses, they were free of recall bias (in
contrast with studies that rely on self-reported health symptoms). Finally, the analyses were
made and the results were analyzed ‘blindly’, i.e., the persons responsible for these tasks did
not have information on the exposure status of the subjects.
Another study, carried out two years after the exposure to Prestige oil in highly
exposed fishermen, detected that a higher proportion of exposed participants had structural
chromosomal alterations, in comparison with the control group, and the risk seemed to
increase with intensity of exposure (Rodríguez-Trigo et al., 2010 at 489). A more thorough
analysis of the chromosomal locations revealed three chromosomal bands commonly involved
in hematological cancer as the most affected by acute oil exposure, and significantly higher
dysfunction in DNA repair mechanisms, expressed as chromosomal damage, in oil-exposed
participants than in those not exposed (Monyarch et al., 2013 at e81726).
The only study that found no relationship between oil exposure and genotoxic damage
was a simple longitudinal study conducted after the MVBraer oil spill. That study was carried
out to assess the primary damage in the DNA (DNA adducts) and the frequency of mutations
in the hprt gene in the peripheral leukocytes of residents in the Shetland Islands polluted area
and controls (who lived about 40 miles [72 km] north of Sumborough Head) at 3 sampling
times (10 days, 10 weeks and 1 year after the accident) (Cole et al., 1997 at 98). These authors
did not obtain any evidence of genotoxicity in DNA adducts or the hprt gene. However, the
Page - 14

17. size of the two groups analyzed was extremely small, especially the control group, thus
precluding the possibility of producing any statistically reliable conclusions.3 Additionally,
participation of the exposed individuals in the cleanup tasks was not specified by the authors
of the study (so it can be assumed to be null). Indeed, for the study participants, only their
status as residents in the polluted area was mentioned but no indication of the absolute or
relative level of their exposure was provided. It may well be that those who participated in the
cleanup work were more exposed to the oil compounds than that those who lived near the
spill but who did not carry out cleanup tasks.
There is only one molecular epidemiology study that analyzed genotoxicity parameters
in people from the parish of San Carlos located in Sachas, Orellana province (Paz y Miño et
al., 2008). That study focused on individuals who were exposed to oil whilst working at the
Sacha South production station. DNA primary damage was evaluated by the comet assay and
chromosomal alterations by the CA test. Results obtained showed a greater percentage of
DNA damage and CA in the exposed individuals than in the controls. These results are in line
with the ones reported for Prestige oil exposed individuals, further supporting the increase in
genotoxic risk (and consequently cancer risk) associated with exposure to oil.
To determine the persistence of the genotoxic alterations observed beyond a two year
period, as was studied in one of the Prestige oil studies (Rodríguez-Trigo et al., 2010), a
follow-up study was carried out seven years later in individuals exposed to Prestige oil for a
mean of 9 months (range 2-10 months). This study reported no significant differences
between the exposed population and the controls in the genotoxicity parameters (Laffon et al.,
2014 at 10). These results suggest that bone marrow hematopoietic stem cells, which produce
leukocytes (the surrogate cells in which genotoxicity was evaluated), do not necessarily have
permanent damage in their DNA, so long as the subjects remain exposure free for a prolonged
period of time.
A potential toxicological risk assessment was also carried out after decontamination of
beaches polluted by the Erika oil spill (Dor et al., 2003). Seven different scenarios of
exposure for people using the beaches were contemplated, selecting the most conservative
available toxicological values for computing risks. Like the 2014 Prestige oil study, the
results obtained in this study indicated that risks were low, both in the long-term and short-term,
for people on holiday and for people working at these cleaned beaches during the
summer period following the oil spill. Cancer risks in decontaminated beaches did not differ
3 For determination of DNA adducts only 20 exposed and 7 non-exposed individuals were analyzed
immediately after the accident, and the number of controls was further reduced to 4 in the samples taken 1 year
later (no samples of 10 weeks were analyzed for DNA adducts). Moreover, the authors described methodological
problems during the analyses, e.g., poor quality of the thin layer chromatography plates. For the hprt mutation
assay, the number of exposed and control samples evaluated was 21 vs. 7, 24 vs. 9, and 20 vs. 5, respectively in
the 3 sampling times. In the experimental design discussion, the authors cite the Robinson et al. (1994) study, as
recommending “that, given the variability in mutant hprt frequency, a minimum of 30-50 individuals per donor
group would be necessary to have a 90% chance of detecting a 1.5-fold increase in mutant frequency over the
control level.” (Cole, 1997 at 106). Thus, sample size of Cole et al. study was clearly too small to detect any
significant effect (Robinson et al., 1994 at 109 (recognizing the limitations of their own study)).
Page - 15

18. substantially from those estimated for control beaches, except when decontamination work
was not completed, as observed in some rocky areas. Consequently, the authors hypothesized
that risks of cancer at beaches not cleaned yet, or recently spoiled by fuel deposits, would be
of concern and would justify temporarily closing the beaches.
The results obtained in the abovementioned studies suggest that carrying out a
properly executed remediation in the Concession Area would be beneficial for the residents,
since according to the current data the risk of long-term adverse effects for their health (at
least the risk of cancer) would decrease and likely reach unexposed levels. It is nevertheless
important to note that we have no data to date that show how long the reversal process takes.
In other words, we do not know how long it would it take for people who have been exposed
for much longer than several months (individuals analyzed were at most 10 months exposed
to Prestige oil) to return to the control level. Here, the residents in El Oriente region have
been substantially exposed through mutually reinforcing media for decades.
In addition, the main exposure pathway for individuals exposed to Prestige oil was by
inhalation, and to a lesser extent through dermal contact. Most people involved in the cleanup
tasks wore boots, protective clothing, gloves, and often but not always face masks. And
ingestion of oil, if it occurred at all, was only accidental. These exposure conditions are
drastically less pronounced than those present in the Concession Area, where no personal
protective devices are used and, as set forth by Dr. Strauss in her report, multiple pathways
are involved.
2.7. Immune and endocrine toxicity studies in people exposed to oil spills
The studies our group carried out in Prestige oil-exposed individuals also included
other parameters reflecting longer-term physiological changes. At the moment of exposure,
decreases in the hormones prolactin and cortisol, both markers of psychophsysiological stress,
were observed in the exposed individuals as compared to the controls, indicating alterations in
the normal endocrine function in the individuals (Pérez-Cadahía et al., 2007, 2008a). Another
study, performed by a group we collaborated with in the study of the same groups of exposed
individuals, analyzed several immunological parameters. This study showed that individuals
exposed for several months to oil had significant modifications in some lymphocyte
subpopulations (increases in %T lymphocytes and %T-helper lymphocytes, and decrease in
%T-cytotoxic lymphocytes), as well as in concentrations of plasma cytokines (increases in
interleukin-2, interleukin-4, interleukin-10 and interferon gamma), but no effects were
detected in the group of short-term exposed volunteers (Gestal et al., 2004). All these effects
indicate that exposure to the oil induced significant changes in the endocrine and immune
systems.
In the follow-up study carried out seven years later (Laffon et al., 2013), significant
endocrine and immunological alterations were observed in the exposed subjects, namely
increase in cortisol concentration and decrease in the percentage of natural killer (NK) cells.
The increase in cortisol in the exposed subjects, contrasting with the decrease initially
detected, suggests an alteration in the endocrine system. Significantly higher levels of plasma
Page - 16

19. cortisol were also reported in outdoor workers chronically exposed to urban pollution, which
shares several compounds with oil (Rosati et al., 2011; Tomei et al., 2003), and it has been
established that a chronic increase in cortisol, subsequent to the increase in hypothalamic
pituitary-adrenal axis activity, is associated with negative health outcomes (Rosati et al.,
2011). Additionally, the NK cells are effector lymphocytes of the innate immune system that
control several types of tumors and microbial infections by limiting their spread and
subsequent tissue damage (reviewed in Vivier et al., 2008). Since cortisol suppresses the
immune response, it may be that the overall decrease of NK cells observed in the exposed
group was an indirect consequence of the increase in cortisol in those individuals. Because
NK cells are the major cell type involved in immune surveillance against cancer cells, the
decrease in NK cells is presumed to increase cancer risk.
The persistence of immunological alterations seven years after the exposure supports
Dr. Strauss’ opinion (page 54 of her initial report) that “the risk of delayed impacts [in
Concession Area inhabitants] is on-going even if there is no additional exposure”, and “the
risk of delayed impacts continues to increase if the exposure remains”. Again, it is necessary
to consider that these immediate and delayed immunological alterations were observed in
people exposed to oil for only several months; the effects on subjects exposed for years, or
even decades (such as El Oriente residents) is for now unknown.
The immunological alterations described in oil-exposed individuals, both at the time
of exposure and also seven years later, provide further support for Dr. Strauss’ opinion (page
48 of her rejoinder report) that exposure to crude oil has immunosuppressive effect, causing a
reduction in the body’s defense against infection and in the immunosurveillance against
cancer cells.
In summary, genotoxic, immunotoxic and endocrine toxicity results obtained in
Prestige oil-exposed individuals refute Dr. Moolgavkar’s conclusion (page 23 of his report)
that “the available epidemiologic evidence does not support a causal effect of environmental
exposure to petroleum from oil exploration and production activities on cancer or other health
outcomes in residents of surrounding communities, either in general or specifically in the
Texpet Concession Area”. The increased risk for developing cancer and/or other diseases
related to dysfunction of the immunological and endocrine systems lead to the
recommendation of periodic health monitoring for those people who were exposed to the
Prestige oil spill. To facilitate the early detection of possible health problems, the
recommendation focused on the determination of cancer biomarkers, clinical immunological
parameters and cortisol levels.
2.8. Miscellanea: IARC classification of crude oil
IARC evaluated the carcinogenic risk of crude oil and included it in group 3 as “not
classifiable as to its carcinogenicity in humans” (IARC, 1989), on the basis of “inadequate
evidence for the carcinogenicity in humans” and “limited evidence for the carcinogenicity in
experimental animals.” As IARC sets forth in the Preamble document for the IARC
Monographs on the Evaluation of Carcinogenic Risks to Humans (IARC, 2006), “agents that
Page - 17

20. do not fall into any other group are also placed in this category” (group 3), and “an evaluation
in group 3 is not a determination of non-carcinogenicity or overall safety”. Instead, “it often
means that further research is needed, especially when exposures are widespread” (as is the
case with crude oils) “or the cancer data are consistent with differing interpretations”. We
now have an additional 25 years of research since the 1989 IARC classification of crude oil,
which was based on only a few studies. In light of the accumulating evidence over the past
two decades showing the genotoxicity of crude oil and its relationship to cancer in humans, a
new review is necessary to include evidence obtained from more recently published studies.
Page - 18

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 Expert Opinion of Kenneth J. Goldstein, M.A., CGWP and Jeffrey W. Short, PhD,
regarding the Environmental Contamination From Texpet’s E&P Activities in the Former
Napo Concession Area Oriente Region, Ecuador. February 2013.
 Expert Opinion of Harlee S. Strauss, PhD, regarding human health‐related aspects of the
environmental contamination from Texpet’s E&P activities in the former Napo concession
area Oriente region, Ecuador. February, 2013.
 Expert Report of Suresh H. Moolgavkar, MD, PhD. May 31, 2013.
 Rejoinder Opinion of Harlee Strauss, PhD, regarding human health risks, health impacts,
and drinking water contamination caused by crude oil contamination in the former
Petroecuador‐Texaco concession, Oriente Region, Ecuador. December, 2013.
 Opinion of Philippe Grandjean, MD. November, 2013.
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28. Page - 26

29. 1
APPENDIX A
Epidemiological studies on acute toxic effects related to exposure to oil spills (ordered by the chronology
of the spills). Black letter: from Aguilera et al. (2010); blue letter: studies not reviewed before by any
expert in this arbitration process.
Accident –
Study characteristics Methods Results
Reference
MV Braer –
Campbell et al.
(1993)
Cross-sectional.
Initial acute effects in
residents (N=420) and
controls (N=92)
Questionnaires of acute
symptoms, peak expiratory
flow, hematology, liver and
renal function tests, blood
and urine toxicology
Principal health effects arose on days 1
and 2 (headaches, itchy eyes, and throat
irritation).
No significant differences between
exposed and controls were found for any
of the biological markers.
Toxicological studies did not show any
exposure that are known to affect human
health
MV Braer –
Campbell et al.
(1994)
Cross-sectional.
Follow up after 6
months of acute effects
in residents (N=344)
and controls (N=77)
General health
questionnaire.
Peak expiratory flow, urine
analysis, hematology, and
liver and renal function
tests
The mean general health questionnaire
score of exposed was significantly
greater than that of controls.
Exposed had greater overall scores for
somatic symptoms, anxiety and
insomnia, but not for personal
dysfunction and severe depression
MV Braer –
Crum (1993)
Cross-sectional.
Affectation of
respiratory tract in
children living close to
Braer shipwreck (N=
44 at 3 days and 56 at
9-12 days after oil spill)
Peak expiratory flow rate Peak expiratory flow rates were within
the normal range in both parts of the
study, and no deterioration was seen over
the study period
Sea Empress –
Lyons et al.
(1999)
Cross-sectional.
Acute health and
psychological effects
in exposed (N=539)
and controls (N=550)
Questionnaires of acute
symptoms.
HAD and SF-36 scores
Exposed showed significantly higher
anxiety and depression scores, worse
mental health, and self-reported
headache and sore eyes and throat
Sea Empress –
Gallacher et al.
(2007)
Cross-sectional.
Acute
symptomathology
attributable to
psychological exposure
in exposed (N=794) and
controls (N=791)
Questionnaires of acute
toxic and non-toxic
symptoms and Hospital
Anxiety and Depression
Scale
Perceived risk was associated with raised
anxiety and non-toxicologically related
symptom reporting.
Toxic symptom reporting was associated
with oil exposure and with raised
perceived risk
Nakhodka –
Morita et al.
(1999)
Cross-sectional.
Acute health problems
in exposed (N=282)
Questionnaires of acute and
toxic symptoms.
Personal air samplers to
assess carcinogenic
benzene, toluene and
xylene.
Metabolites of benzene,
toluene and xylene in urine
Levels of hydrocarbons in air were far
below the occupational acceptable limit.
The principal complaints of symptoms
were low back pain, headache, and
symptoms of eyes and throat
Erika –
Schvoerer et al.
(2000)
Cross-sectional.
Acute health effects in
volunteers and workers
who participated in the
cleanup (N=1,465)
Self-questionnaires sent by
postal mail
The more recurrent health disorders were
lower back pains, headaches and skin
irritations.
Duration of the cleanup activity was
identified as risk factor for the health
problems that occurred

30. 2
Prestige –
Suarez et al.
(2005)
Cross-sectional.
Acute health problems
among subjects
involved in the cleanup
operation after the spill
(N=800)
Questionnaire on exposure
conditions, acute health
problems, and use of
protective material
Bird cleaners accounted for the highest
prevalence of injuries.
Working more than 20 days in highly
polluted areas was associated with
increased risk of injury in all workers.
Toxic effects were higher among
seamen.
No severe disorders were identified.
Prestige –
Carrasco et al.
(2006)
Cross-sectional.
Association between
health information, use
of protective devices
and occurrence of acute
health problems in
exposed (N=799)
Questionnaire on exposure
conditions, acute health
problems, use of protective
material and health-protection
information
received
Health-protection briefing was
associated with use of protective devices
and clothing.
Uninformed subjects registered a
significant excess risk of itchy eyes,
nausea/vomiting/dizziness, headaches
and throat and respiratory problems.
Seamen, the most exposed group, were
the worst informed and registered the
highest frequency of toxicological
problems
Prestige –
Zock et al.
(2007)
Longitudinal 12-24
months after the spill.
Association between
participation in cleanup
work and respiratory
symptoms in exposed
(N= 6,780)
Questionnaires with
qualitative and quantitative
information on cleanup
activities and respiratory
symptoms
The risk of LRTS increased with the
number of exposed days, exposed hours
per day, and number of activities.
The excess risk of LRTS decreased when
more time had elapsed since last
exposure
Prestige –
Rodríguez-
Trigo et al.
(2010)
Cross-sectional, two
years after the
exposure.
Respiratory effects in
fishermen highly
exposed (N=501) and
not exposed (N=177)
Respiratory symptoms,
forced spirometry,
methacholine challenge,
markers of oxidative stress,
airway inflammation, and
growth factor activity in
exhaled breath condensate
Participation in clean-up was associated
with persistent respiratory symptoms and
elevated markers of airway injury in
breath condensate.
The risk for elevated levels of exhaled 8-
isoprostane, vascular endothelial growth
factor, and basic fibroblast growth factor
seemed to increase with intensity of
exposure to clean-up work
Prestige –
Zock et al.
(2012)
Cross-sectional, five
years after cleanup.
Persistence of
respiratory symptoms
in exposed fishermen
(N=466) and non-exposed
individuals
(N=156)
Questionnaire on upper and
lower respiratory tract
symptoms, allergic
conditions, anxiety and
beliefs about the effects of
the oil spill on the
participant’s own health
The prevalence of lower respiratory tract
symptoms had slightly decreased in both
groups, but remained higher among the
exposed. The risk of having persistent
respiratory symptoms increased with the
degree of exposure for moderately and
highly exposed, when compared with
those without any symptoms. Findings
for nasal symptoms and for respiratory
medication usage were similar
Prestige –
Zock et al.
(2014)
Cross-sectional.
Four-year follow-up,
six years after cleanup
work, and comparison
with previous
evaluation (Rodríguez-
Trigo et al., 2010).
Persistence of
functional and
biological respiratory
health effects in never-smoking
fishermen
exposed (N=158) and
non-exposed (N=57) to
the oil
Respiratory symptoms,
forced spirometry,
methacholine challenge,
markers of oxidative stress,
airway inflammation and
growth factor activity in
exhaled breath condensate
During the four-year follow-up period
lung function, bronchial
hyperresponsiveness and the levels of
respiratory biomarkers of oxidative stress
and growth factors had deteriorated
notably more among non-exposed than
among exposed. At follow-up,
respiratory health indices were similar or
better in cleanup workers than in non-exposed.
No clear differences between
highly exposed and moderately exposed
cleanup workers were found

31. 3
Tasman Spirit –
Janjua et al.
(2006)
Cross-sectional.
Acute health effects in
exposed residents
(N=216) and controls
living 2 Km (N=83) and
20 Km (N=101) far
from the coastline
Questionnaires on acute
health symptoms and on
perception about the role of
oil spill in producing ill
health, and anxiousness
about the effect of oil spill
on health
Data showed moderate-to-strong
associations between the exposed group
and the symptoms.
There was a trend of decreasing
symptom-specific prevalence odds ratios
with increase in distance from the spill
site
Tasman Spirit –
Khurshid et al.
(2008)
Cross sectional.
Health parameters of
people working/living
in the vicinity of an oil-polluted
beach
(N=100)
Hydrocarbon/organic
content in seawater and
sand samples.
Hematological and
biochemical parameters.
Liver and renal function
tests
Seawater had no traces of hydrocarbon
content.
Lymphocyte and eosinophil levels were
slightly increased.
About 11 people had raised SGPT, but
this was not significant
Tasman Spirit –
Meo et al.
(2008)
Cross sectional.
Lung function in
exposed (N=20) and
controls (N= 31)
Spirometry Significant reduction in FVC, FEV1,
FEF25%-75% and MVV in exposed.
Lung function parameters were improved
when the subjects were withdrawn from
polluted air environment
Tasman Spirit –
Meo et al.
(2009a)
Cross sectional.
Health complaints
among males involved
in cleanup operations
(N=50) and controls
(N=50)
Standardized questionnaire
on respiratory and general
health complaints
The subjects involved in oil cleanup
operations had significantly higher rates
of health complaints including cough,
runny nose, eye irritation/redness, sore
throat, headache, nausea and general
illness, compared to their matched
controls
Tasman Spirit –
Meo et al.
(2009b)
Cross sectional.
Lung function in
subjects exposed to
crude oil spill into sea
water (N=31) and
controls (N=31)
Spirometry Subjects exposed to polluted air for
periods longer than 15 days showed a
significant reduction in FVC, FEV1,
FEF25–75% and MVV
Hebei Spirit –
Lee et al.
(2009)
Cross-sectional.
Protective effects of
wearing protective
devices on exposure
and symptoms among
the residents (N=288)
and volunteers (N=724)
who participated in the
cleanup
Questionnaires about
symptoms, use of protective
devices and potential
confounding variables.
Analysis of VOCs, PAHs
and heavy metals in urine
Levels of fatigue and fever were higher
among residents not wearing masks than
among those who did wear masks.
Urinary mercury levels were found to be
significantly higher among residents not
wearing work clothes or boots
Hebei Spirit –
Lee et al.
(2010)
Cross-sectional.
Acute health effects in
residents from seashore
villages of a heavy and
moderately oil soaked
area and a lightly oil
soaked area (10 villages
from each area, 10 male
and female adults from
each village)
Questionnaire on the
characteristics of residents,
the cleanup activities, the
perception of oil hazard,
depression and anxiety, and
the physical symptoms
The more highly contaminated the area,
the more likely it was for residents to be
engaged in cleanup activities and have a
greater chance of exposure to oil. The
indexes of anxiety and depression were
higher in the heavy and moderately oil
soaked areas. Significantly increased
risks of several physical symptoms was
obtained
Hebei Spirit –
Sim et al.
(2010)
Acute health problems
in people engaged in
the cleanup (N=846)
Questionnaire on
demographic information,
operation and exposure to
oil, and health status
Residents and volunteers experienced
acute health problems. More frequent
and greater exposure (including lack of
protective suit and mask) was strongly
associated with a higher occurrence of
symptoms

32. 4
Hebei Spirit –
Cheong et al.
(2011)
Cross-sectional.
Physical symptoms in
residents participating
in cleanup work
(N=288) and controls
(N=39)
Questionnaire regarding
subjective physical
symptoms,
sociodemographic
characteristics and cleanup
activities.
Urinary metabolites of
VOCs, PAHs and heavy
metals
Exposed residents showed associations
between physical symptoms and the
exposure levels
Hebei Spirit –
Ha et al. (2012)
Cross-sectional.
Exposure status and
acute health effects on
volunteers that
participated in the
cleanup (N=565)
Questionnaire regarding
physical symptoms.
Urinary metabolites of
VOCs and PAHs before
and after exposure
Volunteers that participated for longer
cleanup work reported an increase in
physical symptoms (visual disturbance,
nasal and bronchus irritation, headaches,
heart palpitations, fatigue and fever,
memory and cognitive disturbance, and
abdominal pain). The levels of t,t-muconic
acid, mandelic acid, and 1-
hydroxypyrene were significantly higher
in samples after cleanup than those
measured before participation
Hebei Spirit –
Na et al. (2012)
Cross-sectional, one
year after the accident.
Health problems of
people involved with
cleanup efforts (N=442)
Questionnaire on
demographic information,
risk factors and the
continuation and duration
of any health symptoms
Eye symptoms, headaches, skin
symptoms, and neurovestibular
symptoms had a longer duration than did
back pain or respiratory symptoms
Hebei Spirit –
Jung et al.
(2013)
Cross-sectional.
Respiratory effects on
children who lived
along the Yellow Coast
(N=436)
Modified International
Study of Asthma and
Allergies in Childhood
questionnaire.
Health examination (skin
prick test, pulmonary
function test, and MBPT),
The children who lived close to the oil
spill area showed a significantly lower
FEV1, an increased prevalence of
‘asthma ever’ (based on a questionnaire),
and ‘airway hyperresponsiveness’ (based
on the MBPT) than those who lived far
from the oil spill area. Male sex, family
history of asthma, and residence near the
oil spill area were significant risk factors
for asthma
Hebei Spirit –
Kim et al.
(2013)
Cross-sectional, 1.5
years after the spill.
Burden of
disease (BOD),
including physical and
mental diseases, of the
residents living in
contaminated
coastal area (N=10,171)
Questionnaires on exposure
and medical problems, and
to assess psychological
health and asthma, and
physical and laboratory
examinations of respiratory,
cardiovascular,
neurological and
psychological systems
The YLD of mental diseases including
PTSD and depression for men were
higher than that for women. The YLD
for women was higher in asthma and
allergies (rhinitis, dermatitis,
conjunctivitis) than that for men. The
effects of asthma and allergies were the
greatest for people in their 40s, with the
burden of mental illness being the
greatest for those in their 20s. Proximity
to the spill site was associated with
increased BOD.
Deepwater
Horizon-
Abramson
(2010)
Cross-sectional.
Short and potential
long-term impact of the
Deepwater Horizon
disaster on coastal
residents (children and
families) (N=1,203)
Telephone interviews on
exposure, physical and
mental health, and
decisions related to oil spill
on a daily basis
Over one-third of parents reported that
their children had experienced either
physical symptoms or mental health
distress as a consequence of the oil spill.
One in five households has seen their
income decrease as a result of the oil
spill and 8% have lost jobs.
Over 25% of coastal residents think they
may have to move from the area because
of the oil spill

33. 5
Deepwater
Horizon –
D’Andrea and
Reddy (2013)
Cross-sectional.
Adverse health effects
in subjects participating
in the cleanup activity
(N=117) and controls
(N=130)
Clinical data (white blood
cell and platelets counts,
hemoglobin, hematocrit,
blood urea nitrogen,
creatinine, ALP, AST,
ALT) and somatic symptom
complaints
Platelet counts were significantly
decreased, and hemoglobin and
hematocrit levels were significantly
increased, among oil spill-exposed
subjects. Similarly, oil spill-exposed
subjects had significantly higher levels
of ALP, AST, and ALT compared with
the unexposed subjects
Deepwater
Horizon –
D’Andrea and
Reddy (2014)
Cross-sectional.
Hematological and liver
function indices in
subjects who
participated in the
cleanup operations
(N=117)
White blood cell and
platelets counts,
hemoglobin, hematocrit,
blood urea nitrogen,
creatinine, ALP, AST,
ALT), and urinary phenol.
Values were compared with
the standardized normal
range reference values
Data obtained indicate that people
exposed are at risk of developing
alterations in hematological profile and
liver function. Results support the earlier
study (D’Andrea and Reddy, 2013)
findings
ALT, alanine amino transferase; ALP, alkaline phosphatase; AST, aspartate amino transferase; BOD, burden of
disease; FEF25%-75%, forced expiratory flow; FEV1, forced expiratory volume in first second; FVC, forced vital
capacity; LRTS, low respiratory tract symptomathology; MBPT, methacholine bronchial provocation test; MVV,
maximum voluntary ventilation; PAH, polycyclic aromatic hydrocarbons; PTSD, post-traumatic stress disorder; SF-
36, short form-36; SGPT, serum glutamic pyruvic transaminase; VOC, volatile organic compounds; YLD, years lived
with disability.

34. 1
APPENDIX B
Epidemiological studies on genotoxicity, immunotoxicity and endocrine toxicity, and studies on
potential toxicological risk assessment, related to exposure to oil spills (ordered by the chronology of the
spills). Black letter: from Aguilera et al. (2010); blue letter: not included in Aguilera et al. (2010).
Accident –
Study characteristics Methods Results
Reference
Braer –
Cole et al.
(1997)
Longitudinal.
Genotoxicity in
residents (N=26) and
controls (N=9) at 3
sampling times (10
days, 10 weeks and 1
year after the accident)
DNA adducts in the
mononuclear cell fraction and
frequency of hprt mutations
in T lymphocytes
No evidence of genotoxicity was
obtained for either end point
Erika –
Baars (2002)
Potential toxicological
risk assessment for
people involved in
cleaning activities and
for tourists
Risk characterizations on the
basis of suppositions of the
potential exposure during
cleaning and tourist activities
The risk for the general people was
limited.
Increased risk for developing skin
irritation and dermatitis, and very
limited risk for developing skin
tumors, were described for people
who had been in bare-handed contact
with the oil
Erika –
Dor et al.
(2003)
Potential toxicological
risk assessment after
decontamination of 36
beaches polluted by
the Erika oil spill and
7 control beaches
Determination of the 16
PAHs selected by the
U.S.EPA in sand, water and
surface of rocks.
Seven scenarios of exposure
for people using the beaches
were contemplated, and the
most conservative available
toxicological values were
selected for computing risks
The sand and water were slightly
polluted, with values similar to those
found in the control beaches. The
rocky areas were still highly polluted.
No lethal risk was found for a young
child who had accidentally ingested
small ball of fuel.
The life-long excess risks for skin
cancer and for all other cancers were
about 10-5 in scenarios including
contact with the polluted rocks.
The hazard quotient for teratogenic
effects was very small, except in
scenarios where pregnant women
would walk among rocks containing
high pollution levels.
Prestige –
Laffon et al.
(2006)
Cross-sectional.
Genotoxicity in
individuals performing
autopsies and cleaning
of oil-contaminated
birds (N=34) and
controls (N=35)
Environmental VOCs.
Comet assay and MN test.
DNA repair genetic
polymorphisms (XRCC1,
XRCC3, APE1)
Significant increase in the comet
assay, but not in the MN test, related
to the time of exposure.
Exposed individuals carrying
XRCC1-399Gln or APE1-148Glu
alleles showed increased DNA
damage.
Prestige –
Pérez-Cadahía
et al. (2006)
Cross-sectional.
Genotoxicity in
volunteers and hired
workers participating
in the cleanup (N=68)
and controls (N=42).
Environmental VOCs.
Comet assay, SCE, MN test
Highest VOC levels in the
volunteer’s environment.
Significant increase in the comet
assay in exposed individuals.
Influence of sex, age and tobacco
smoking on the genotoxicity
variables.
No effect of using protective mask
during cleanup labors