Case Studies in Food Safety and Environmental Health

Preface
Case Studies in Food Safety and Environmental health is intended as a reference

for incorporating case studies into a traditional food safety lecture course. It will also
be useful in developing case studies for problem-based learning. The book is divided
into three main sections which contain case studies that can be used, individually or
together as a series, to discuss a range of food safety issues covering microbial food
safety, chemical residues and contaminants, risk assessment and food legislation.
Chapters begin with a summary of the issues that will be examined and the objectives
and learning outcomes for each case study are outlined. Discussion questions are
included to focus the reader on the major issues but also to consider how other rele-
vant factors affect the outcome or decisions made by the persons involved. A list of
references is included at the end of each chapter that will allow the reader to examine
the case study in greater detail.
Part I contains case studies that examine, in a more general framework, a num-
ber of specific issues. Chapter 1 deals with the potential hazards associated with
acrylamide originating from processed foods, while Chapter 2 considers nitrate con-
tamination of the environment in Romania and efforts made to address this problem.
Residual pesticides in olive oil that are endocrine disrupting compounds are the con-
cern of Chapter 3, with microbial food safety issues tackled in the final two chapters.
Chapter 4 examines Clostridium botulinum and Botulism while Chapter 5 looks at
Listeria monocytogenes and Listeriosis.
In part II, food safety issues are examined from an historical perspective. Chapter
6 looks at the Toxic Oil Syndrome case that occurred in Spain in the 1980s, while
Chapter 7 covers a case on aflatoxins in Hungarian paprika in 2004. Botulism and
listeriosis are again the topic of microbial food safety. Chapter 8 looks at an out-
break of Botulism related to mascarpone cheese that occurred in Italy in 1996, while
Chapter 9 deals with a case on listeriosis from butter in Finland between 1998 and
1999 that affected hospital patients.
Finally part III takes on food safety from the perspective of the researcher. Cases
are based around experimental data and examine the importance of experimental
planning, design and analysis. The reader is required to not only consider the food
safety issues at hand but also how to approach an investigation. The possibility of
VI Preface
replacing chemical fertilizers and soil additives with alternative treatment methods
for plant growth is examined in Chapter 10, with Chapter 11 looking at heavy
metals in organic and non-organic milk. Chapter 12 deals with aflatoxins in fish
feed affecting rainbow trout in Estonian fish farms in the 1980s, while Chapter 13
examines mycotoxins in cereal products.
We hope that this book will eventually be part of an arsenal of tools that will
be available for teaching food safety concepts and issues, in a practical and applied
framework, to an ever demanding community of learners.
Portugal, Peter Ho
March 2006 Maria Margarida Cortez Vieira
Contents
Part I Specific Issues
1 Acrylamide and Human Health

Semih Otles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
2 Nitrates in Food, Health and the Environment
Carmen Socaciu, Andreea Stănilă . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
3 Endocrine disrupting compounds in olive oil
Antonis Skilourakis, Elefteria Psillakis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
4 Clostridium botulinum and Botulism
Paul Gibbs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
5 Listeria monocytogenes and Listeriosis
Panagiotis Skandamis, Antonia Gounadaki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Part II Historical perspectives
6 The Toxic Oil Syndrome in Spain

Victoria Ferragut . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
7 A ban on Paprika in Hungary
Cecilia Hodúr, Zsuzsanna Lászlķ, Zsuzsa Hovorka Horváth . . . . . . . . . . . . . . . 53
8 An Outbreak of Botulism in Italy
Laura Bigliardi, Giuliano Sansebastiano . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
9 Listeriosis from butter in Finland
Riitta Maijala . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
VIII Contents
Part III Research-based
10 Alternative solutions for the treatment of food produce

Anna Aladjadjiyan . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
11 Heavy metals in Organic milk
Jelena Zagorska, Inga Ciprovic̆a, Daina Kārklina . . . . . . . . . . . . . . . . . . . . . . . 75
12 Aflatoxins in farmed fish in Estonia
Risto Tanner, Erge Tedersoo . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
13 Mycotoxins in Cereal Products from Romania
Mihaela Avram, Mona Elena Popa, Petru Nicultita, Nastasia Belc . . . . . . . . . . 85
Part I
Specific Issues
1
Acrylamide and Human Health
Semih Otles1,2
1
Department of Food Engineering, Ege University, TR-35100, Izmir, Turkey.
2
[email protected]
Summary
Acrylamide is a versatile organic compound that finds its way into many products
in our everyday life. This compound, identified previously as a potential industrial
hazard, has now been found in many cooked foods. Reports of the presence of acry-
lamide in a range of fried and oven-cooked foods have caused world-wide concern
because of its probable carcinogenicity in humans.
Key words: acrylamide; fried and oven-cooked foods; carcinogenicity; risk commu-
nication
1.1 Objectives and learning outcomes
(1) Explain and apply key concepts regarding acrylamide in foods and identify factors
that effects its formation.
(2) Identify and describe the differences between bioavailability, potential toxicity
and carcinogenicity of acrylamide.
(3) Recognize the requirements of food legislation in relation to acrylamide.
(4) Understand current knowledge of minimizing existing food risks.
(5) Define risks associated with the consumption of acrylamide formed by high tem-
perature heating of certain carbohydrates in the presence of some amino acids
and amino compounds.
(6) Interpret exposure and toxicity data regarding acrylamide in food matrices.
(7) Propose the need for a risk communication policy.
(8) Propose and defend the need for a multifaceted approach of improving safety of
food supplies, with respect to a foodborne toxins of recent or emerging concern,
based on scientific evidence and ethical considerations.
4 Semih Otles
1.2 Introduction
Acrylamide, also known as 2-propenamide, and its analogues have been widely used
since the last century for various chemical and environmental applications. Some
of the common uses of acrylamide are in the paper, dyes, cosmetics and toiletry
industries. It is produced commercially as an intermediate from the production and
synthesis of polyacrylamides [Otles and Otles, 2004b, Rudén, 2004].
Effects on human health resulting from exposure to acrylamide hinges on its
carcinogenic and genotoxic impact. It is known to cause cancer in animals and there
is no scientific reason to doubt that similar effects occur in humans. However, the
degree of risk in humans from contracting cancer through the intake of acrylamide-
containing foods cannot be reliably estimated at present [U.S. EPA, 1985, Posnick,
2002, FDA/CFSAN, 2003, Rice, 2005].
Acrylamide is a versatile organic compound that finds its way into many prod-
ucts in our everyday life. Acrylamide exists as a monomer or as polyacrylamide, its
polymeric form. The single unit acrylamide is toxic to the nervous system. It is a car-
cinogen in laboratory animals and a suspected carcinogen in humans. The multiple
unit or polymeric form is not known to be toxic [Friedman, 2003, Otles and Otles,
2004a].
Human exposure to acrylamide is mostly attributed to the consumption of home
cooked potato chips and other comparable products. i.e. approximately 20% of total
acrylamide exposure. The amounts of acrylamide found in unfried frozen chips are
relatively low compared to the deep-fried product, whereby the amount present will
depend on both frying temperature and frying time. Thus, cooking conditions probably
influences the formation of acrylamide [Rice, 2005].
Most acrylamide in food is formed when asparagine, a natural amino acid, reacts
with certain naturally occurring sugars like glucose. However, the mechanism of
acrylamide formation in foods is not well understood. Acrylamide appears to be
formed as a by product of the Maillard reaction. The Maillard reaction contributes to
the production of a tasty crust and golden color in fried and baked foods. This only
happens when the temperature during cooking is sufficiently high, which may vary
according to the properties of the product and the method of cooking [Rudén, 2004].
The use of acrylamide as a synthetic chemical and the surprising discovery that it
occurs naturally in foods that are treated at high temperatures raises a complex issue.
Although, it is known that acrylamide is formed by baking certain types of breads
and frying potato chips, other chemicals like benzo[a]pyrene that are formed during
grilling or frying, which have been recognized as potential cancer causing agents of
similar potency, may also pose a problem. Although, reported levels of acrylamide
are higher than those for other contaminants, it is difficult to ascertain to what extent
acrylamide exposure effects human health [Claeys et al., 2005, Konings et al., 2003].
1.3 Human health

Preliminary analyzes from existing limited data have indicated that potato and potato
products, such as crisps, chips and other high-temperature cooked potatoes (e.g.,
1 Acrylamide and Human Health 5
roasted, baked), are the main contributors to total mean acrylamide intake, particu-
larly when considered together. This has been observed in data from studies in Nordic,
central European and Mediterranean countries (e.g., Spain, France) and in other re-
gions of the world (e.g., Australia, United States). However, other food groups with
lower concentration of acrylamide that might have been consumed on a daily (or
more regular) basis (e.g., bread, crisp bread), as well as other foods whereby levels
of acrylamide are currently unknown, may also have contributed substantially to the
total intake, with magnitudes varying across countries or study populations [WHO,
2005, Rosen, 2002].
It is known that a high absorption of acrylamide occurs from exposure by in-
halation and its bioavailability following oral administration in drinking water is
also high. i.e. approximately 50-75%. However, the bioavailability of acrylamide in
food matrices is not known. It must be assumed that acrylamide in foods is at least
partially absorbed, since data based on non-smokers that are not occupationally ex-
posed to acrylamide had adducts of haemoglobin with acrylamide and its metabolite
glycidamide (which are sensitive biomarkers for acrylamide exposure). It has been
observed that the most sensitive effect of repeated administration of acrylamide in ex-
perimental animals had been damage to peripheral nerves (peripheric neuropathy1). At
higher dosages, muscular and testicular atrophy also occurs and decreases in erythro-
cyte parameters2 was also observed. Peripheral neuropathy and haemoglobin adduct
formation can also be seen in occupationally exposed humans. In 1985, the WHO de-
rived a TDI3 of 12 mg/kg bw/day that was based on neurotoxicity in sub-chronically
exposed rats. The US-EPA defined a RfD4 of 0.2 mg/kg bw/day, using the same set
of data. In a chronic toxicity and carcinogenicity study, peripheric neuropathy was
1
Peripheric neuropathy is a medical term for damage to nerves of the peripheral nervous
system, which may be caused either by diseases of the nerves or from the side effects of
systemic illness. Peripheral neurophaties vary in their presentation and origin, and may
affect the nerves or the neuromuscular junction.
2
The hematological values for the quantification of size and cell hemoglobin content or
concentration of the erythrocyte, usually derived from erythrocyte count, blood hemoglobin
concentration, and hematocrit.
3
Tolerable daily intake (TDI) is an estimate of the amount of a substance in air, food or
drinking water that can be taken in daily over a lifetime without appreciable health risk.
TDIs are calculated on the basis of laboratory toxicity data to which uncertainty factors
are applied. TDIs are used for substances that do not have a reason to be found in food,as
opposed to substances that do, such as additives, pesticide residues or veterinary drugs in
foods.
4
Reference dose (RfD) is an estimate of the daily exposure to a substance for humans that
is assumed to be without appreciable risk. It is calculated using the number of observed
adverse effect and is more conservative than the older margin of safety.
6 Semih Otles
observed in rats with a LOAEL1 of 2 and a NOAEL2 of 0.5 mg/kg bw/day. These
values of LOAEL and NOAEL should be used for risk assessment [Girma et al., 2005,
Otles and Otles, 2004a, Richmond and Borrow, 2003].
According to the WHO, the additional carcinogenic risk from a lifelong daily
intake of 1 mg per person, amounts to 1 case per 100,000 exposed people. This is
equivalent to a unit life time cancer risk at 1 mg/kg bw/day in 0.7 per 1000, or an
additional carcinogenic risk of 1 per 10,000 exposed people at a lifelong intake of 0.14
mg/kg bw/day). The US-EPA has conservatively estimated the carcinogenic risk for
a unit lifetime cancer risk at 1 mg/kg bw/day of 4.5 per 1000, which is equivalent to
an additional carcinogenic risk of 1 per 10,000 exposed people with a lifelong intake
of 0.02 mg/kg bw/day. Whereas, the Scientific Committee of the Norwegian Food
Control Authority has recently given an estimate of a unit lifetime cancer risk at 1
mg/kg bw/day of 1.3 per 1000, after considering all available data. This is equivalent
with an additional carcinogenic risk of 1 per 10,000 exposed people at a lifelong
intake of 0.08 mg/kg bw/day. For the purpose of risk assessment, the estimate from
the WHO is normally used. However, it must be noted that although recent evaluations
by international bodies have all agreed upon acrylamide being probably carcinogenic
to humans, they also conclude that estimations made from theoretical models are
insufficiently reliable to quantify the actual risk to humans. One of the important
reasons why theoretical models cannot give more reliable estimates, is the lack of
knowledge about the bioavailability of acrylamide in foods. Cancer risk estimates are
determined after administering acrylamide via drinking water to laboratory animals
and, as such, they should be utilized with extreme care [Claeys et al., 2005, Johnson
et al., 1996, Vattem and Shetty, 2003].
1.4 Risk Communication

A joint committee has been set up by FAO/WHO to encourage transparent and open
risk assessment and risk management processes. It recognizes the importance of in-
volving interested parties (e.g., consumer, industry, retail) in this process at certain
stages. An adequate risk communication policy could facilitate the crucial commu-
nication process between risk assessor and risk manager and among all other parties
involved [WHO, 2005, Richmond and Borrow, 2003, Simonne and Archer, 2002, Tyl
and Crump, 2003].
1
Lowest-observed-adverse-effect-level (LOAEL) is the lowest concentration or amount of
a substance, found by experiment or observation, which causes an adverse alteration of
morphology, functional capacity, growth, development, or life span of a target organism
distinguishable from normal (control) organism of the same species and strain under defined
conditions of exposure.
2
No-observed-adverse-effect-level (NOAEL) is the highest concentration or amount of a sub-
stance, found by experiment or observation, which causes no detectable adverse alteration
of morphology, functional capacity, growth, development, or life span of the target organism
under defined conditions of exposure.
Many potentially harmful chemicals are present at extremely low levels in the en-
vironment and in foods [Anon, 2003, Otles and Otles, 2004b]. In many cases, levels
of these chemicals are far below the amounts that are expected to have an adverse
effect on human health. In recent years, analytical methods and instrumentation have
advanced considerably, allowing the detection of very small levels of chemicals that
may or may not have adverse effects on human health. Although the information on
acrylamide in foods and its implications for human health is not yet complete, the
FAO and WHO have issued the following interim advice, based on current knowledge
to minimize existing risks:
(1) Foods should not be cooked excessively (i.e., for too long or at too high a tem-
perature), but they should be cooked thoroughly enough to destroy foodborne
pathogens;
(2) People should eat a balanced and varied diet that includes plenty of fruits and
vegetables, and should moderate their consumption of fried foods.
1.5 Conclusion
The joint FAO/WHO expert committee has so far concluded that further research is
necessary to determine how acrylamide is formed during the cooking process and
whether acrylamide is present in foods other than those already tested. They have
also recommended population based studies of cancers that could potentially develop
from exposure to acrylamide. Despite the uncertainties of extrapolating incidences
of tumours in humams from experimental data for animals, they do provide some
insight into the maximum risk for humans, resulting from the exposure to acrylamide
in foods. From exposure estimations, it appears that the additional risk of cancer for
the average human population (aged 1-97 years), for children (aged 1-6 years), and
for youngsters (aged 7-18 years), might not be negligible.
1.6 Discussion Questions

1. Do foods represent the largest source of acrylamide?
2. How does acrylamide form when food is cooked at high temperatures?
3. Is any level of acrylamide in foods acceptable? Discuss the differences in food
legislation at a national, European and international level.
4. What can be done to avoid the presence of acrylamide in foods?
5. What is the risk of contracting cancer from acrylamide exposure?
6. What conclusions have so far been made from previous risk assessments on
acrylamide?
7. Does a risk communication analysis indicate any adjustment to public commu-
nication needs?
8 Semih Otles
8. How would you design a multifaceted approach for improving safety of food
supplies, with respect to a foodborne toxins of recent or emerging concern?
Prepare a proposal, based on scientific evidence and ethical considerations.
References
Anon. European Commision. Acrylamide Workshop, 20-21.10.2003., 2003.
W. L. Claeys, K. D. Vleeschouwer, and M. E. Hendrixs. Quantifying the forma-
tion carcinogens during food processing: acrylamide. Trends in Food Science &
Technology, 16(5):181–193, 2005.
FDA/CFSAN. Detection and quantitation of acrylamide in foods, 2003. URL http:
//www.cfsan.fda.gov/~dms/acrylami.html. http://www.cfsan.fda.gov/~dms/
acrylami.html.
M. Friedman. Chemistry, biochemistry, and safety of acrylamide. Journal of Agri-
cultural and Food Chemistry, 51(16):4504–4526, 2003.
K. B. Girma, V. Lorenz, S. Blaurock, and F. T. Edelman. Coordination chemistry of
acrylamide. Coordination Chemistry Reviews, 249(11-12):1283–1293, 2005.
K. Johnson, S. Gorzinski, K. Bodner, R. Campbell, C. Wolf, M Friedman, and R. Mast.
Chronic toxicity and oncogenicity study on aa incorporated in the drinking water
of fischer 344 rats. Toxicology and Applied Pharmacology, 85(2):154–168, 1996.
E. Konings, A. Baars, D. van Klaveren, M. Spanjer, P. Rensen, M. Hiemstra, J. van
Kooij, and P. Peters. Acrylamide exposure from foods of the dutch population and
an assessment of the consequent risks. Food and Chemical Toxicology, 41(11):
1569–1579, 2003.
S. Otles and S. Otles. Acrylamide in food: chemical structure of acrylamide. The
Electronic Journal of Environmental, Agricultural and Food Chemistry, 3(5):1–
8, 2004a. URL http://ejeafche.uvigo.es/3(5)2004/001352004.pdf. http://
ejeafche.uvigo.es/3(5)2004/001352004.pdf.
S. Otles and S. Otles. Acrylamide in food: formation of acrylamide and its damages
to health. Electronic Journal of Polish Agricultural Universities, Food Science and
Technology, 7(2):1–12, 2004b. http://www.ejpau.media.pl/series/volume7/
issue2/food/art-02.html.
L. M. Posnick. Acrylamide testing at cfsan exploratory survey results, 2002. URL
http://www.cfsan.fda.gov/~dms/acryposn/sld001.html. http://www.cfsan.
fda.gov/~dms/acryposn/sld001.html.
J. M. Rice. The carcinogenicity of acrylamide. Mutation Research, 580:3–20, 2005.
P. Richmond and R. Borrow. Acrylamide in food. The Lancet, 361(9355):
361–362, 2003.
J.D. Rosen. Acrylamide in Food: Is It a Real Treat to Public Health? American
Council on Science and Health, 2002. http://www.acsh.org/docLib/20040330_
acrylamide2002.pdf.
C. Rudén. Acrylamide and cancer risk- expert risk assessments and the public debate.
Food and Chemical Toxicology, 42(3):335–349, 2004.
H. Simonne and L. Archer. Acrylamide in Foods: A review and Update. University

of Florida. Institute of Food and Agricultural Sciences., 2002. http://edis.ifas.
ufl.edu/pdffiles/fy/fy57800.pdf.
R. Tyl and K. Crump. Acrylamide in food. Food Standards Agency, 5:215–222, 2003.
U.S. EPA. Health and environmental effects profile for acrylamide. U.S. Environmen-
tal Protection Agency, Washington, D.C., EPA/600/X-85/270 (NTIS PB88170824),
1985. URL http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=52015.
http://cfpub.epa.gov/ncea/cfm/recordisplay.cfm?deid=52015.
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its reduction. Innovative Food Science and Emerging Technologies, 4(3):331–338,
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http://who.int/ipcs/food/jecfa. http://who.int/ipcs/food/jecf.
2
Nitrates in Food, Health and the Environment
Carmen Socaciu1,2 and Andreea Stănilă1

1
Department of Chemistry and Biochemistry, University of Agricultural Sciences and
Veterinary Medicine Cluj-Napoca, str. Mănăştur 3-5, 400372 Cluj-Napoca, Romania.
2
[email protected]
Summary
Over the last 25 years, many national and international agencies have been concerned
with pollution of the Romanian environment, particularly in soil and water, food (such
as animal and plant products), and its impact on human health. Contamination with
nitrates due to an overloading of soil with fertilizers, especially before 1989, com-
bined with an inappropriate management of animal waste (rich in organic nitrogen),
resulted in a cascade of problems with food safety and health. An examination of such
events and conflicts associated with nitrate contamination in Romania over the last 20
years and solutions to this problem, from different national and international studies,
are presented here. The European Union’s Council Directive 91/676/EEC [Official
Journal, 1991], known as the Nitrates Directive, was formulated to prevent health and
ecological problems resulting from farm practices. This EU Directive, which also in-
cludes the Code for Good Agricultural Practices, is being implemented at a national
level as part of a national strategy for reducing Romania’s impact on environmental
pollution.
Key words: nitrates; methemoglobinemia; water; environment; Romania
(1) Define the recycling of nitrogen-based compounds in the environment, in relation

to food and health.
(2) Interpret the risks and impact of nitrate/nitrites on human health.
(3) Analyze the factors which have influenced in the past, and still are influencing,
the exposure risks to nitrates and nitrites in Romania.
(4) Formulate the main control points necessary to avoid environmental contamina-
tion, according to initiatives taken at a regional and national level.
12 Carmen Socaciu and Andreea Stănilă
2.2 Introduction
Nitrates (NO3– ) and nitrites (NO2– ) are naturally occurring forms of inorganic ni-
trogen, which are a part of the nitrogen cycle in the environment. Organic nitrogen
decomposes into ammonia, by the action of microorganisms in the soil or waste water,
which is in turn oxidized to nitrites and nitrates.
Norganic −−→ NH3 −−→ NO− −→ NO−

2 − 3 (2.1)
Levels of these compounds that are found in plant tissues are affected by growing
conditions, the use of nitrogen fertilizers and the genetic characteristics of the plant.
Plants take up nitrogen as nitrates and convert it into proteins via photosynthesis-
mediated mechanisms. Poor exposure to light can result in a lower rate of photosyn-
thesis causing an accumulation of nitrate in tissues. Green leafy vegetables (lettuce
and spinach, etc.) are the main source of nitrates in the diet which represent 70-90%
of total nitrate intake. Soil contamination from high nitrogen-containing fertilizers,
including ammonia as well as natural organic wastes from animals or humans, can
raise the concentration of nitrates found in water and vegetables. Nitrate-containing
compounds in the soil are generally soluble and readily migrate into groundwater,
which most likely result in nitrate contamination of rural domestic wells. This is es-
pecially so in areas where there are widespread use of nitrogen-based fertilizers or
where septic sewer systems are in use; that is, in wells less than 30 metres deep. In
1998, almost 36% of wells in Romania were found to contain levels of nitrate over 45
mg/L. It is generally accepted that potentially adverse health effects are more likely to
occur (i.e., the risk increases), from drinking water with nitrate levels greater than 10
mg/L [Hoering and Chapman, 2004]. The United Nations World Health Organization
recommends a maximum level of nitrate no higher than 5 mg/L in water.
2.2.1 Nitrates in Food
Nitrates in food may have both beneficial and potentially detrimental health effects.
In 1995, the European Commission Scientific Committee on Food (SCF) agreed to
retain its earlier Acceptable Daily Intake (ADI) for the nitrate ion of 3.7 mg/kg body
weight [European Comission, 1997]. The maximum limits of nitrates and nitrites in
vegetables and fruits, as established by the Romanian Ministry of Health are given in
table 2.1.
Beside spinach and lettuce, green vegetables like cabbage, broccoli, cucumber
and root vegetables have naturally a greater nitrate content than other plant foods.
Nitrates in a typical diet comes from drinking water (about 21%), vegetables and
fruits (more than 70%), but may also originate from meat products (about 6%) in
which sodium nitrate is used as a preservative and color-enhancing agent.
Physiologically, nitrates are reduced to nitrites in the stomach. Nitrite enters the
bloodstream and binds to hemoglobin, changing it to methemoglobin, which inter-
feres with the blood’s ability to carry oxygen to tissues in the body. An excess of
nitrates in food, water or the use of topical silver nitrate, induces symptomatic methe-
moglobinemia in children. Methemoglobinemia, commonly known as “blue baby
2 Nitrates in Food, Health and the Environment 13
Table 2.1. Maximum levels of nitrates and nitrites permitted in vegetables and fruits in Romania
(mg/kg)
Product Level Product Level

Apple 60 Lettuce 2000 (3000)
Cabbage 900 Pepper 150 (400)
Carrot 400 Potato 300
Cauliflower 400 Spinach 2000
Cucumber 200 (400) Tomato 150
Eggplant 300 Vegetable marrow 500
Grapes 60 Water melon 100
a
Numbers show permitted levels in vegetables and fruits grown in fields and in greenhouses
(Numbers in parenthesis).
b
Data from Anon [1998].
syndrome”, can occur in infants under six months if nitrate levels are greater than 10
mg/L. A baby with methemoglobinemia literally turns blue due to oxygen starvation
of tissues, causing suffocation. Nitrates can damage insulin-producing cells in the
pancreas through the generation of free radicals and from the etiology of diabetes
mellitus [Virtanen et al., 1991, Parslow et al., 1997]. Nitrate exposure may also play
a role in the development of thyroid disease, causing thyroid hypertrophy at nitrate
concentrations greater than 50 mg/L and decreasing the levels of the serum thyroid
stimulating hormone. Other studies suggest a potential link between high nitrates in
the drinking water and an increased incidence of gastrointestinal cancer via nitrite by-
products that combine with amines to form N-nitroso compounds, which are known
to be cancer-causing agents.
2.3 Retrospectives
During the communist era in the 70’s, Romanian agriculture developed intensively in
parallel with the chemical industry that was strongly directed towards a high pro-
duction of nitrogen fertilizers. Before 1989, the government demanded intensive
fertilization of lands in order to produce larger and larger amounts of agricultural
products, consequently ignoring the environmental risks and the harmful impact on
human health. During this period a strong accumulation of nitrates in soil and water
was noticed by the authorities, but official reports failed to recognize the real concerns
to the population.
After 1989, there was a general concern among many national and international
agencies regarding the general pollution of the Romanian environment, including soil,
water, animal and plant products. The Romanian Environmental Protection Agency
set a maximum contaminant level of 10 mg/L for nitrate and nitrite in public water
supplies. It was estimated that 1.5 million people were potentially exposed to nitrates
from rural domestic wells. Reported levels of nitrate contamination reached 13%
in 1990. Between 1992 and 1994, the mean incidence of nitrate contamination in
Transylvania (North-West of Romania) varied between 0.12 and 0.37%, which was
significantly lower than in 1990. After December 1989, the main reason for the de-
crease in nitrates, was the reduction of nitrogenous fertilization that allowed the soil
to regain its the self-purifying properties [Prejbeanu and Badulescu, 2005].
A very high incidence of methemoglobinemia and a significant mortality rate
among children (i.e., 2346 registered cases for children under 1 year and 80 deaths),
were reported in Romania between 1984 and 1996. In the Transylvania region, methe-
moglobinemia incidence rates was found to range between 24 to 363 cases per 100,000
live births between 1990 and 1994 [Ayebo et al., 1997]. In 2000, the infant exposure
to nitrate-contaminated water became a common public health problem in Eastern
Europe [Knobeloch et al., 2000].
Between 1996 and 1999, 45 persons were diagnosed with acute nitrate poisoning
in Calarasi, South of Romania. Many children under one year old were hospitalized
for acute intoxication with nitrites and/or viral hepatitis type A, epidemic in the area,
with 90% of all cases affecting school and pre-school children. Three years later,
in 2002, many more cases of methaemoglobinemia were discovered in a village of
3,500 inhabitants in Garla Mare. Water quality tests showed three prominent types of
pollution in the village: fecal bacteria, nitrates and the endocrine-disrupting pesticide,
atrazine, which is now banned in a number of countries. None of the 78 wells tested
had safe water.
In 2003, nitrate pollution levels were reevaluated. Statistically significant dif-
ferences (p < .001) were found between the nitrate hydrogen level in 33 villages,
compared to results from 1970 [Prejbeanu and Badulescu, 2005]. Nitrate levels also
significantly decreased in vegetables, fruits and water.
2.4 Addressing the problem

Over 7 million people in rural Romania are still drinking water drawn from wells,
which are often polluted with nitrates, bacteria and pesticides, originating from la-
trines, waste dumps and agriculture [Gabizon et al., 2004]. Problems associated with
background pollution of the Romanian environment and health-related consequences
of nitrate exposure stem from:
(1) Excessive use of fertilizers;

(2) Poor hygienic conditions and contamination with organic nitrogen from animal
and septic wastes;
(3) Use of wells, where there is non-controlled waste, in a large number of villages;
(4) Lack of interest from national agencies and the shortage of laboratories with
screening methods to supervise the quality of water and food;
(5) Exposure of children to contaminated water from individual wells, especially in
villages where no water treatment is available.
2.5 Exposure assessment studies and pollution control projects
Since 2000, regional, national and international initiatives, governmental programs

and actions sponsored by private companies have addressed the protection of health
and environmental pollution. This has been in response to debates, both at a govern-
mental level as well as in the media, on further contamination incidents. Among those
worth mentioning are:(1) an exposure assessment study [Zeman et al., 2002], (2) the
Garla Mare Project [Gabizon et al., 2004], (3) the Romania Agricultural Pollution
Control GEF Project [The World Bank, 2001], (4) an environmental pollution study
in the Dolji county [Prejbeanu and Badulescu, 2005].
2.5.1 Exposure assessment studies
Two epidemiological studies, a cohort study and a nested case/control study, were
conducted with children in Transylvania, which formed the basis for an exposure
assessment study, aimed at “developing an exposure model and determining a nu-
merical point estimate of the amount of biological relevant nitrate/nitrite exposure
that occurred for each child” [Zeman et al., 2002]. The cohort study and the nested
case/control study were conducted respectively to examine the relationship between
high nitrate/nitrite exposure and neuropsychological development, and the relation-
ship between methemoglobinemia and various risk factors of the disease. Their anal-
ysis indicated that children with methemoglobinemia, were children at a 2-month-
of-age point estimate that received the highest exposure to nitrate/nitrite in their diet
[Zeman et al., 2002].
In 2003, 564 well water samples (taken in the spring and autumn from three
representative wells), from the Dolj County, and 1521 fruit and vegetables samples
(from 52 villages) were analyzed for levels of nitrates and nitrites. 60.6% of water
samples were found to have levels of nitrates below MAL1 (with the highest sample
containing 285 mg/L). These villages could be classified into three groups, based
on their level of nitrate found in well water: Group 1 with nitrate levels of less than
45 mg/L (43.6% of villages), Group 2 with nitrate levels between 45 and 100 mg/L
(43.6% of villages), and Group 3 with nitrate levels between 100 and 200 mg/L
(12.8% of villages). 34.1% of samples tested negative for nitrites, with the remaining
372 samples containing up to 0.75 mg/L. These results showed a significant decrease
from levels recorded in 1979. Significant decreases in the levels of nitrates were also
found in fruits and vegetables compared to those analyzed in the 1970s [Prejbeanu
and Badulescu, 2005].
2.5.2 The Garla Mare Project
The Garla Mare Project was conducted between 2002 and 2003, involving Medium &
Sanitas, a Romanian Non-Governmental Organization (NGO), and Women in Europe
1
Maximum Admitted Level
for a Common Future (WECF), “to develop replicable, low-cost, short-term solutions
to Romania’s water-related health hazards” [Gabizon et al., 2004].
During this period, a 12 member project committee was set up to consider ways
of reducing water pollution. In conjunction with the major of the village of Gare Mare
and Medium & Sanitas, they undertook the following initiatives:
(1) The committee opened a project information office to allow villagers to check
nitrate levels of waters samples brought from wells in the area, in order to convince
them of its importance;
(2) A survey was conducted by Medium & Sanitas to determine the level of knowl-
edge and awareness of health effects of water pollution among the inhabitants;
(3) An in-depth social economic and gender analysis was also conducted.
The survey and analysis revealed, among other things, that villagers were unaware
of how polluted water affected health, there was a high unemployment rate, and that
they recognized that they would have to pay for improving the supply of water in the
village, eventhough most were unable to do so, as they had difficulties in covering food
and electricity expenses. These and other results, which included a gender analysis
of villagers’ roles and activities, were discussed and solutions to these problems
were presented to villagers at a town hall meeting. The lack of funding, at a local
government level, made it impossible to implement any of the solutions presented
[Gabizon et al., 2004].
The report also outlined a range of short, medium and long term preventative
actions that were undertaken by the village community to tackle the problems that
had been identified: the installation of a water filter in one of the village schools (short-
term), hygienic dry compost toilets in a school and in two private homes (medium-
term), a cooperation initiative between farmers in the Garla Mare areas with organic
farmers from Constanta, Sibiu and the Netherlands (long-term). From 2002 to 2003,
no new cases of blue-baby syndrome were reported [Gabizon et al., 2004].
2.5.3 The Global Environment Facility (GEF) Project
The Global Environment Facility (GEF) is an independent multilateral financial mech-

anism that assists developing countries to protect the global environment in four areas:
biodiversity, climate change, international waters, and ozone layer depletion. The Ro-
mania Agricultural Pollution Control GEF Project, which was implemented in 2003
in partnership with the United Nations Development Program, the United Nations
Environment Program, and the World Bank, is an environmental project involving
21 villages (25,700 residents) in Calarasi, a predominantly agricultural county in
Southeastern Romania. The project aims to protect human life and livelihood while
ensuring a healthy environment. Details of this project has been described elsewhere
[Reynolds and Volovik, 2004].
The Danube River receives much of the excess manure, chemical fertilizers, and
pesticides that are washed from neighbouring farmlands, which in turn, feeds this
excess to the Black Sea. The Black Sea has suffered a severe assault upon its ecological
stability during the last three or four decades, largely through eutrophication (an excess
of nutrients that affect the balance of oxygen). Romania contributes about 27% of
the nutrients entering the Black Sea, through wastes from farms, which is by far the
largest of the 17 contributors to Black Sea eutrophication.
2.6 Conclusion
Official and public awareness of nitrate contamination levels has encouraged the de-
velopment of new initiatives in different regions of Romania. Some specific aims and
objectives need to be fulfilled:
(1) Environmentally-friendly local and regional practices are needed for crop rota-
tion, conservation tillage systems, crop cover, and a better livestock management
system. The efficient application of fertilizer, based on soil tests, will mean farm-
lands will get only the necessary amount of fertilizer. This will reduce nutri-
ent run-off into surface and ground water, protect long-term fertility of soils by
maintaining organic matter levels, foster soil biological activity through the use
of vegetables in crop rotation schemes, as well as effective recycling of organic
materials (includes crop residues and livestock wastes);
(2) Training and awareness of “good practices” in waste management (e.g., compost-
ing, testing, and field application), and water and soil quality monitoring (i.e., the
development and application of a rapid screening tests);
(3) Strengthening the involvement of national institutions and organizations in the
regulatory framework for environmental protection in agriculture, with the sup-
port of the Ministry of Agriculture and Rural development and the Ministry of
Environmental Protection. It also includes the harmonization of relevant national
laws with legal requirements of the European Union, of which Romania hopes
to become a member state.These requirements include the EU’s Nitrates Direc-
tive, formulated to prevent health and ecological problems resulting from farm
practices;
(4) The GEF project will help develop a national strategy for reducing Romania’s
contribution of nutrients to the Danube and the Black Sea. It will support also the
crafting of a Code for Good Agricultural Practices for Romania as a whole;
(5) Organization of regional workshops, field trips, training activities, and the pub-
lication (e.g., articles and press notes) in international agricultural and environ-
mental journals.
The hope is that, with the help of these and other activities, Romania may serve
also as a model for similar initiatives in neighboring countries in Eastern Europe.

1. What are the main factors which may influence the contamination of soil, water
and vegetables with nitrates?
2. Identify food sources that may contain nitrates in a typical diet. What are the
maximum levels permitted in these foods in Romania?
3. Identify at least four main social-economic problems associated with the exposure
and pollution with nitrates.
4. Describe one of the recent projects running in Romania that aims to find solutions
against water-related health diseases?
5. What are the main initiatives and factors that can contribute to environmental
protection?
References
Anon. Health Ministry Order nr.975 regarding the hygiene-sanitary norms for food.
Official monitor, Romanian Government, December 16 1998.
A. Ayebo, B. Kross, M. Vlad, and A. Sinca. Infant methemoglobinemia in the Tran-
sylvania region of Romania. International Journal of Occupational and Environ-
mental Health, 3:20–29, 1997.
European Comission. Opinion on Nitrate and Nitrite (expressed on 22 September
1995). In Food Science and Techniques. Report of the Scientific Committee for
Food, Thirty Eight Series, pages 1–33, 1997. http://europa.eu.int/comm/food/
fs/sc/scf/reports/scf_reports_38.pdf.
S. Gabizon, M. Samwel, and Bentvelsen. Case E: Romania. A village improves
drinking water and women’s participation. In Women and the Environment, Policy
Series, pages 72–75. United Nations Environment Programme, 2004. http://www.
unep.org/PDF/Women/ChapterFive.pdf.
H. Hoering and D. Chapman, editors. Nitrate and Nitrite in Drinking Water. WHO
Drinking Water Series. IWA Publishing, London, 2004.
L. Knobeloch, B. Salna, A. Hogan, J. Postle, and H. Anderson. Blue babies and nitrate-
contaminated well water. Environmental Health Perspectives, 108:675–678, 2000.
Official Journal. Council Directive 91/676/EEC of 12 December 1991 concerning the
protection of waters against pollution caused by nitrates from agricultural sources.
Official Journal of the European Union, (L375), 1991. http://europa.eu.int/
eur-lex/lex/LexUriServ/LexUriServ.do?uri=CELEX:31991L0676:EN:HTML.
R. C. Parslow, P. A. McKinney, G. R. Law, A. Staines, R. Williams, and H. J. Bo-
dansky. Incidence of childhood diabetes mellitus in yorkshire, northern england,
is associated with nitrate in drinking water: an ecological analysis. Diabetologia,
40(5):550–556, 1997.
A. Prejbeanu and N. Badulescu. Aspects of Environmental Pollution in Dolj
county-Romania. In Nachhaltigkeit für Mensch und Umwelt, pages 675–
678, 2005. http://www.mec.utt.ro/~tmtar/lucrari_avh05/vol%20I%20Pdf/
5Ecology/5210%%20Prejbeanu%20Ileana-%20Fl.%20Badulescu.pdf.
P. Reynolds and Y. Volovik. Report to the Danube / Black Sea Strategic
Partnership. Technical report, UNDP /GEF Black Sea Ecosystem Recov-
ery Project, November 2004. http://www.blacksea-environment.org/
Text/eLibrary/.%5CResources%5C47_BS%ERP%20RER_01_G33_A_1G_31%20Ex%
20Summary%20Final7.pdf.
The World Bank. Romania-Agricultural Pollution Control GEF Project. Technical re-
port, 2001. http://www-wds.worldbank.org/servlet/WDSContentServer/WDSP/
IB/2000/01/2%0/000094946_00011905350872/Rendered/PDF/multi0page.pdf.
S. M. Virtanen, L. Rasanen, A. Aro, J. Lindstrom, H. Sippola, R. Lounamaa, L. Toiva-
nen, J . Tuomilehto, and H. K. Akerblom. Infant feeding in finnish children less
than 7 yr of age with newly diagnosed iddm. childhood diabetes in finland study
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etary nitrate exposures in children of Transylvania, Romania. Journal of Exposure
Analysis and Environmental Epidemiology, 12:54–63, 2002.
3
Endocrine disrupting compounds in olive oil
Antonis Skilourakis1 and Elefteria Psillakis1,2

1
Department of Environmental Engineering, Technical University of Crete,
Polytechnioupolis Chania 73100, Greece.
2
[email protected]
Summary
Olive oil is an important component of the Mediterranean diet and its consumption
is believed to be beneficial to human health. However, there has been concerns about
the presence of pesticides in olive oil, from Greece, Italy and Spain, having endocrine
disrupting properties. The toxicity of these pesticides and their ability to interact with
the endocrine system is discussed.
Key words: pesticides; olive oil; endocrine disrupting compounds

(1) Discuss the toxicity of pesticides used in olive tree cultivation.
(2) Define endocrine disrupting compounds (EDCs).
(3) Examine pesticide levels found in olive oil from several Mediterranean countries.
(4) Evaluate the safety of olive oil consumption.
(5) Discuss and evaluate the adequacy of European Maximum Residue Limits
(MRLs), in meeting food safety requirements, for the use of pesticides.
3.2 Introduction
The olive tree is the oldest known cultivated tree in history and has played a crucial
role in civilizations, economies as well as the diet of Mediterranean countries. Olive
tree cultivation began in Africa before the Phoenicians brought it to Morocco, Algeria,
and Tunisia. Olea europaea was first cultivated in Crete and Syria over 5000 years ago
and subsequently spread to Greece, Italy and other Mediterranean countries around
600 BC. Today, the three major olive-producing countries are Spain, Italy and Greece.
Other major producers are Tunisia and Turkey.
22 Antonis Skilourakis and Elefteria Psillakis
Greece has the highest per capita level of olive oil consumption of approximately
220,000 tons per year, which corresponds to an average consumption of 20 kg per
person. In general, 90 % of the world’s olive oil production is consumed by the
producer countries themselves. Nonetheless, there has been a steady increase in olive
oil consumption in non-producing countries on account of its beneficial health effects
[Boskou, 1996].
The concept of the Mediterranean diet originated from the Seven Countries Study
initiated by Ancel Keys1 . The study showed that the population of the island of Crete
had a very low rate of coronary heart disease and certain types of cancer and life
expectancy was high, despite a high fat intake [Hu, 2003]. The typical traditional
dietary patterns in Crete, much of the rest of Greece, and southern Italy in the early
1960s, were considered to be largely responsible for good health observed in these
countries. It is generally believed that the beneficial health effects of olive oil can
be attributed to both its high content of monounsaturated fatty acids and its high
content of antioxidants [Roche et al., 2000]. No other naturally produced oil has
as large an amount of monounsaturated fatty acids (i.e., mainly oleic acid). Studies
have shown that olive oil offers protection against heart disease by controlling LDL2
(“bad”) cholesterol levels while raising HDL3 (“good”) cholesterol levels. Olive oil
is well tolerated by the stomach and its protective function has a beneficial effect on
ulcers and gastritis, it activates the secretion of bile and pancreatic hormones much
more naturally than prescribed drugs, thereby leading to less incidents of gallstone
formation4.
3.3 Pesticides
Olive tree cultivation is mainly conventional, although organic cultivation is becoming
more popular. A range of pesticides5 , which are mainly organophosphorus, are used
in order to protect the tree and crop against pests, such as the olive fly (i.e., Bactrocera
(Dacus) oleae), the olive moth (i.e., Prays oleae) and the black scale (i.e., Saissetia
oleae) [Vreuls et al., 1996].
Cumulative research has shown that pesticides are toxic not only to pests, but also
to humans, animals and the environment in general. Levels of residue pesticides should
be controlled in Food. Therefore, the Food and Agriculture Organization (FAO) has
produced a manual concerning the estimation of maximum residue levels for pesti-
cide residues in Food and Feed, based on Good Agricultural Practice (GAP), which
considers the equilibrium between welfare production and the protection of the envi-
ronment [FAO, 1997]. However, it should be mentioned that the persistence of some
1
US scientist that studied the influence of diet on health in the 1950s [Anon, 1999]
2
Low Density Lipoprotein.
3
High Density Lipoprotein.
4
Also reported in National Geographic News [Anon, 2005c].
5
In general, pesticides can be classified based on whether their action is against insects (i.e.,
insecticides), fungus (i.e., fungicides), and weeds (i.e., herbicides) [Hughes, 1996].
3 Endocrine disrupting compounds in olive oil 23
pesticides once they enter the environment, as well as their ability for bioaccumula-
tion1 , makes them extremely dangerous even at very low concentrations [Oudejans,
1991].
The toxicity of pesticides are due to their interaction with different systems (e.g.,
nervous, endocrine, respiratory, cardiovascular) of the human body. In general, pes-
ticides, as well as other chemical groups, belong to a class of chemicals, known as
Endocrine Disrupting Compounds (EDCs), that are capable of mimicking, blocking
and destroying natural hormones even at low concentrations. The effects of EDCs
on an organism depends on which hormone system is targeted [Birkett and Lester,
2003]. For example a pesticide that can mimic the adrenaline hormone is expected to
cause behavior problems. DTT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane) was
the first pesticide correlated with endocrine disrupting action that was capable of
interacting with estrogen2 [Birkett and Lester, 2003]. Several other pesticides have
since also been found to interact with masculine hormones and glands, causing disor-
ders such as testicular cancer, cryptorchidism, hypospadias [Potrolli and Mantovani,
2002, Eertmans et al., 2003]. Fenthion, fenitrothion and endosulfan, which are widely
used and commonly found in olives and olive oil, were found to interact with andro-
gens [Kitamura et al., 2003] and estrogens [Fang et al., 2001]. Table 3.1 shows other
pesticides that are known to have endocrine disrupting properties.
Table 3.1. Some pesticides with endocrine disrupting properties and their effect on health
Pesticide Description
Cypermethrin A pyrethroid pesticide similar to deltamethrin that can cause testicular
degeneration. However, it is not considered an EDC.
Diazinon An organophosphate herbicide that is believed to affect pheromone
recognition in male fish.
Dimethoate An organophosphate insecticide that is classified as a compound that
may cause thyroid dysfunction and testicular degeneration.
Parathion Classified an adrenal disrupter.
3.4 Pesticides in Olive Oil
According to the MRC Institute for Environment and the Commission of the Eu-
ropean Communities, the list of pesticides used in olive tree cultivation, that have
known endocrine disrupting action are: Amitrole, Carbaryl, Deltamethrin, Diazi-
non, Dimethoate, Diuron, Endosulfan, Fenthion, Glyphosate, Malathion, Mancozeb,
Maneb, Manganese, Methomyl, Paraquat, Simazine, Urea, Zinc, Ziram.
1
Accumulation of substances in an organism
2
Female hormone responsible for reproduction
Some typical pesticides and their residue values in olive oil from major producing
countries (i.e., Italy, Greece and Spain) have been reported and are described below:
(1) Organophosphorus pesticide residues in Italian virgin olive oil [Rastrelli et al.,
2002]
Nine organophosphorus pesticides were found in 31 out of 65 samples of vir-
gin olive oil, obtained from olive oil production areas in the Cilento National
Park in Campania during 1999-2000. Average levels of aziphos-ethyl found in
2 samples were 0.090 mg/kg, while 4 samples had an average of 0.080 mg/kg
of cloropyriphos methyl. Diazinon was detected in 3 samples and dimethoate in
29 samples, averaging 0.083 and 0.061 mg/kg, respectively. Eighteen samples
contained on average 0.073 mg/kg of fenthion, whereas formothion was found
only in a single sample (i.e., 0.082mg/kg). Finally, there were 3 samples with an
average of 0.063 mg/kg of methidathion, 2 with an average of 0.080 mg/kg of
parathion, and a single sample with 0.056 mg/kg of parathion methyl.
(2) Fenthion residues in Sicilian and Apulian olive oil [Dugo et al., 2005]
A total of 79 Italian olive oils were sampled from individual growers in Sicily
(i.e., 51 samples) and Apulia (i.e., 28 samples) during the period between 2002-
03. Fenthion residues were detected in only 7 Sicilian samples and 6 Apulian
samples, ranging from 0.09 to 0.42 mg/kg and 0.18 to 0.73 mg/kg, respectively.
(3) Endosulfan and pyrethroid insecticides in Greek virgin olive oil [Lentza-Rizos
et al., 2001b]
From a total of 338 samples that were analyzed, 22% were contaminated with
endosulfan, ranging from 0.02 to 0.57 mg/kg. Two samples contained residues
of pyrethroid pesticides, λ-cyhalothrin at 0.02 mg/kg and cypermethrin at 0.04
mg/kg.
(4) Fenthion and dimethoate pesticides in Greek olive oil [Tsatsakis et al., 2003]
Olive oil samples from Crete were examined for residues of fenthion and
dimethoate. Average levels of dimethoate found in organic olive oil, sampled
between 1997 and 1999, were 0.0098, 0.0038 and 0.0010 mg/kg, whereas sam-
ples of conventional olive oil contained 0.0226, 0.0264 and 0.0271 mg/kg, re-
spectively. On the other hand, average levels of fenthion, found in samples of
organic olive oil for the same period were 0.0215, 0.099 and 0.035 mg/kg, with
samples of conventional olive oil containing 0.1222, 0.1457 and 0.1702 mg/kg,
respectively.
(5) Organophosphorus and organochlorine pesticides in Spanish virgin olive oil
[Yagüe et al., 2005]
Nineteen samples of virgin olive oil from Arágon were analyzed for organophos-
phorus (OP) and organochlorine (OC) pesticides. Acephate was found in a single
sample at 0.01 mg/kg. Low levels of OC pesticides (i.e., α-hexachlorocyclohexane,
β-hexachlorocyclohexane, lindane, endosulfan sulfate, p,p’-DDE, p,p’-DDT)
were found, ranging from 0.0015 to 0.005 mg/kg. For example, endosulfan sul-
fate was present in 2 samples with an average of 0.003 mg/kg.
3.5 Conclusion
Levels of pesticide found in organic olive oil samples in Greece suggest that organic
olive oil is safer, although not free from pesticides. Pesticide residues levels detected
in olive oil usually do not surpass MRLs set by the European Union. However, MRLs
for pesticides, that are known to endocrine disrupting compounds, should be recon-
sidered, since endocrine disrupting action of pesticides is activated even at very low
concentrations, and their use restricted. In the case of Fenthion, the most commonly
found pesticide residue in olive oil, concentrations as low as 10 and 1 ppm are sufficient
to interact with male hormones [Kitamura et al., 2003]. Their ability to bioaccumulate
in living organisms together with the daily consumption in several countries may be
of concern for consumers [Lentza-Rizos et al., 2001a]. Lypophilic pesticides may
accumulate in the body fat at levels sufficient to cause endocrine disrupting action.
For example, bioaccumulated pesticides in mammals may be transferred from the
mother to the infant during breast-feeding.
Given the known heath risks associated with exposure to pesticides, steps should
be taken to minimize their presence in food. This can be achieved by adopting organic
agricultural methods and using the codes of “Good Agricultural Practices”. Nonethe-
less, continuous monitoring of pesticides residues in olive oil is essential, in order to
prevent and control bioaccumulation in living organisms.

1. What levels of pesticides are sufficient to activate their endocrine disrupting
action? How does these levels compare to that of MRLs set by the European
Union?
2. How does bioaccumulation of pesticides correlate with endocrine disrupting ac-
tion?
3. What other endocrine disrupting compounds can be found in olive oil?
4. Do you think that consumers of organic olive oil are less likely to be exposed to
endocrine disrupting pesticides? Why?
5. What additional measures, if any, should be taken by governments of olive oil
producing countries to protect the quality of olive oil?
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oil from organic and conventional cultivation. Food Additives and Contaminants,
20(6):553–559, 2003.
J. J. Vreuls, R. J. J. Swen, V. P. Goudriaan, M. A. T. Kerkhoff, G. A. Jongenotter,
and U. A. T. Brinkman. Automated on-line gel permeation chromatography-gas
chromatography for the determination of organophosphorus pesticides in olive oil.
Journal of Chromatography A, 750:275–286, 1996.
C. Yagüe, S. Bayarri, P. Conchello, R. Lázaro, C. Pérez-Arquillué, A. Herrera, and
A. Ariņo. Determination of pesticides and PCBs in virgin olive oil by multicolumn
solid-phase extraction cleanup followed by GC-NPD/ECD and confirmation by
ion-trap GC-MS. Journal of Agricultural and Food Chemistry, 53(13):5105–5109,
2005.
4
Clostridium botulinum and Botulism
Paul Gibbs1,2
1
Leatherhead Food International, Randalls Road, Leatherhead, Surrey KT22 7RY, UK.
[email protected]
2
Escola Superior de Biotecnologia, Universidade Católica Portuguesa, Rua Dr. António
Bernardino de Almeida, 4200-072 Porto, Portugal. [email protected]
Summary
Botulism is caused by neurotoxins produced by Clostridium botulinum during growth

in foods. Many different types of foods have been implicated in cases/outbreaks, gen-
erally from under processing, lack or change of preserving agents, improper storage
conditions, etc. In all cases a proper HACCP or risk analysis by appropriately quali-
fied personnel would have identified a botulinal risk. Two examples of botulism will
be considered: Hazelnut yoghurt from the UK and Garlic in Oil from Canada and the
USA.
Key words: Clostridium botulinum; botulism; hazelnut yoghurt; garlic in oil

(1) Define the physiological characteristics of Clostridium botulinum in relation to
food preservation by water activity, pH value and thermal processing conditions.
(2) Identify and describe the toxicological properties of Clostridium botulinum in
relation to human disease.
(3) Interpret the physiological characteristics of Clostridium botulinum in relation to
methods of food preservation, in order to prevent botulism.
(4) Identify and evaluate the control parameters preventing growth and toxin produc-
tion by Clostridium botulinum in foods.
(5) Analyze the effects of changes in food preservation conditions with respect to
botulism.
(6) Propose the need for a HACCP or risk assessment on changes in food character-
istics.
(7) Recognize the requirements of food regulations in relation to botulism.
30 Paul Gibbs
4.2 Introduction
Botulism is a neuroparalytic disease caused by toxins secreted by a small group

of clostridia when growing in foods. Seven neurotoxins (A-G) are recognized by
immunological methods, all of which cause paralysis of the voluntary muscles by
blockade of the release of acetylcholine at the nerve-muscle junctions. Clostridium
botulinum is a strictly anaerobic, spore-forming bacterium, capable of growth over
a wide range of conditions of temperature, pH and water activity. As a species, it
consists of 4 groups of organisms distinguished on the basis of toxins produced, pro-
teolytic or saccharolytic reactions in media, and ability to grow at 4 C (psychrotrophic
strains). The organism must be regarded as ubiquitous, particularly in soil and ma-
rine sediments, usually occurring as spores, and appropriate measures must taken in
food formulation, processing or storage to inhibit growth in the food, or to destroy
the spores and prevent re-contamination, as in canned foods of pH >4.5. For canned
foods of pH >4.5, a “Botulinum Cook” is mandatory (coldest part of the can contents
must reach 121 C held for 3 minutes or the equivalent using a z-value of 10 C).
Only strains in Groups I (proteolytic and mesophilic: toxins types A, B, and F)
and II (non-proteolytic and psychrotrophic: toxins types B, E, and F) are causative
agents of human botulism.
The proteolytic strains are able to grow at lower aw (ca. 10% w/v salt on water)
than the non-proteolytic strains (inhibited by ca. 3.5% w/v salt on water). A few
strains of C. butyricum and of C. barati are also capable of producing botulinal toxins
(types E and F respectively).
4.3 Hazelnut yoghurt (UK)

Yoghurt is produced from pasteurized milk, milk powder, and thickeners, heated to
about 82 C, cooled and inoculated with starter cultures. This mixture is then fermented
for 2-3 hours to produce the desired acidity, flavors are added together with any
sweeteners, and the product packed in plastic cartons. These products generally have
a chilled shelf-life of 21-30 days.
Hazelnut flavoring was made by a company known for its excellent fruit flavorings
and toppings for cakes, etc. These sweetened (ca. 60% w/w sugar) acidic products
were hot-filled into A10 cans (ca. 2.5 Kg), sealed and pasteurized in boiling water
for about 30 min, cooled and dispatched as an ambient temperature stable product.
Hazelnut conserve was produced in a similar manner with no apparent problems.
However, a “low calorie” product was requested and the sugar was replaced with a
high intensity sweetener (i.e., aspartame) in products manufactured in 1988. Some
companies receiving the aspartame-sweetened hazelnut conserve, reported “blowing”
of cans and the producer added sorbate to control what was thought to be yeast
spoilage.
In 1989, 27 persons in the UK developed symptoms of botulism following con-
sumption of hazelnut-flavored yoghurt. The age of those affected was from 14 months
4 Clostridium botulinum and Botulism 31
to 74 years, and all but one were hospitalized; the 74-year old patient died due to as-
piration pneumonia. The yoghurt and the hazelnut conserve were shown to contain
proteolytic C. botulinum type B and Type B toxin of about 600-1800 mouse lethal
doses/ml in the conserve and 14-30 mouse lethal doses/ml in the yoghurt.
4.3.1 Discussion Questions
1. What are the factors controlling the growth and toxin production by C. botulinum
in fruit flavorings and similar products?
2. Could C. botulinum grow and produce toxin in yoghurt?
3. What were the differences in the aspartame-sweetened hazelnut flavoring that
lead to growth and toxin production?
4. Why would the producer have thought that spoilage was due to yeast contamina-
tion?
5. Should a HACCP analysis or risk assessment have been carried out on the new
formulation of hazelnut conserve?
6. What other controls could have been used to minimize the occurrence and growth
of C. botulinum in hazelnut conserve?
7. Should the producer have investigated the causes of the “blowing” of cans of
hazelnut conserve?
8. What food regulations were contravened in producing aspartame-sweetened
hazelnut conserve?
4.4 Garlic in Oil (Canada & USA)

Chopped garlic in oil is made using either fresh garlic or, commercially, with dried
garlic, rehydrated and added to oil. No further processing is carried out, i.e. heat
processing, addition of salt, acidulants or other preservatives. Instructions are usually
given for refrigerated storage, but in general, consumers often only refrigerate after
opening the jars, and the product is generally stored for extended periods at ambient
temperatures. Such products are frequently incorporated into spreads, e.g. for garlic
bread.
In 1985, 36 diners at a Canadian restaurant, suffered botulism as a result of con-
suming garlic butter made from chopped garlic in oil (soybean or olive oil). Initially,
in many cases the patients were mis-diagnosed with other illnesses due to the variety
of early symptoms, dispersal of those affected and unfamiliarity of physicians with
botulism. All those affected were hospitalized, but although 7 persons required me-
chanical assistance in breathing, there were no deaths. Type B toxin was found in the
serum of three of the patients, and C. botulinum type B was recovered from the faeces
of one of the patients.
In 1989, a similar outbreak of three cases was recorded in the USA; the causative
agent in this case was Type A and the product had been stored for 18 months at
ambient conditions. Again, although all three patients were hospitalized, there were
no deaths.
32 Paul Gibbs
4.4.1 Discussion Questions
1. Were there any factors present in this product that should have controlled the
growth of C. botulinum?
2. Would a HACCP assessment have identified this type of product as a botulinal
risk?
3. What was the likely source of the organism?
4. What factors could be incorporated into the product to prevent botulinal growth
and toxin productions?
5. Would the recommendation to keep refrigerated, control the growth of all strains
of C. botulinum?
6. Were any food regulations contravened in the manufacture of this type of product?
7. To your knowledge, are there any similar, flavored oil products, e.g. containing
a herb or spice, in the marketplace? If so, could these present a botulism risk?
What recommendations could you make to a manufacturer of such products?
References
A. H. W. Hauschild. Clostridium botulinum. In M. P. Doyle, editor, Foodborne
Bacterial Pathogens, pages 111–189, New York, 1989. Marcel Dekker.
M. O’Mahoney, E. Mitchell, R. J. Gilbert, D. N. Hutchinson, N. T. Begg, J. C. Rod-
house, and J. E. Morris. An outbreak of foodborne botulism associated with con-
taminated hazelnut yoghurt. Epidemiology and Infection, 104:389–395, 1990.
S. H. St Louis, M. E. Peck, D. Bowering, G. B. Morgan, J. Blatherwick, S. Banerjee,
G. D. Kettyls, W. A. Black, M. E. Milling, A. H. Hauschild, and et al. Botulism
from chopped garlic: delayed recognition of a major outbreak. Annals of Internal
Medicine, 108(3):363–368, 1988.
5
Listeria monocytogenes and Listeriosis
Panagiotis Skandamis1 and Antonia Gounadaki2

1
Laboratory of Quality Control and Hygiene of Foods and Beverages, Department of Food
Science and Technology, Iera Odos 75, 118 55, Athens, Greece. [email protected]
2
Laboratory of Microbiology and Biotechnology of Foods, Department of Food Science and
Technology, Iera Odos 75, 118 55, Athens, Greece.
Summary
In 2003, the Hellenic Food Authority initiated a monitoring program for biological
and chemical hazards in Ready-to-Eat (RTE) food products in Greece. In total, 605
products from diverse catering enterprises and retailers in Athens, Patra, Thessaloniki,
and Crete were examined for the presence of pathogenic and hygiene indicator bacteria
(e.g. coliforms). Detailed inspections of premises were also performed to evaluate the
hygiene level of enterprises offering RTE foods, as well as their compliance to Good
Hygiene Practices. Only 0.7% of analyzed samples were found positive for listeria.
Different RTE meat products have been recalled for possible listeria contamination
since that initial study, showing a sharp increase in the number of contaminated
products (i.e., 8.5 tons) in 2005. Five deaths were also been recorded, resulting from
the consumption of bacon that was presumed contaminated with listeria.
Key words: Listeria monocytogenes; listeriosis, Ready-to-Eat foods

(1) Evaluate the potential impact of underestimating the incidence of listeriosis due
to under reporting.
(2) Assess the contribution of obligatory reporting on the prevention and/or reduction
of listeriosis.
(3) Examine post-processing contamination points and methods to control L. mono-
cytogenes in Ready-To-Eat products.
(4) Understand the survival/growth responses of L. monocytogenes and identify the
most significant environmental factors that favor or inhibit growth.
(5) Address the necessity for continuous training in Food Safety Management Sys-
tems of persons involved in one or more stages of the food chain from “farm-to-
fork”.
34 Panagiotis Skandamis and Antonia Gounadaki
(6) Defend the position that food safety involves everybody along the food chain, in-
cluding primary producers (i.e., food, feed, crop), secondary food manufacturers,
wholesalers, retailers, and consumers.
(7) Convince food producers that compliance with food legislation is not only a typi-
cal demand by the authorities, but a crucial ethical aspect for safe food production
and protection of public health.
5.2 Introduction
Listeria monocytogenes is a pathogen that can cause serious illness in humans. It is
Gram-positive, psychrotrophic (i.e., capable of growing at -0.4 C), facultative anaer-
obic, a non-sporeforming rod, with minimum reported pH and aw for growth of 4.4
and 0.92, respectively. The pathogen is ubiquitous in nature, and normally found in
soil, decaying vegetation, animal and human feces, sewage, silage and water.
Infections that can be caused by L. monocytogenes have been classified into
five categories [Bell and Kyriakides, 1998]: (1) zoonotic infection of skin lesions;
(2) Neonatal infection (i.e., usually by infected mothers or cross-infection by other
neonates); (3) infection during pregnancy due to consumption of contaminated food;
(4) infection of non-pregnant adults, as in (3); (5) food poisoning, which is caused by
consumption of food with exceptionally high levels of L. monocytogenes (i.e., >107
CFU/g). Symptoms that are associated with listeriosis include abortion, stillbirths,
meningitis with or without septicaemia, vomiting or diarrhoea. Despite the low inci-
dence of human listeriosis of between 2 to 15 per million inhabitants, the high concern
that has been expressed for this pathogens is associated with the high fatality rate (i.e.,
approximately 20% for healthy individuals and up to 70% for immunocompromised
individuals).
It is difficult to control L. monocytogenes, as it is able to persist in agricultural
(e.g., soil, water, plants) and food processing environments (e.g., surfaces of industrial
equipment), as well as in distribution, retail and home environments [Tompkin, 2002].
Meat processing facilities are often the source of contaminated carcasses, and/or boxed
beef, poultry, or other meats [Farber and Peterkin, 1991, Tompkin, 2002, Scanga
et al., 2000, Gande and Muriana, 2003]. Epidemiological studies have shown that the
organism can be transferred through cross-contamination from employees, drains,
standing water, residues, floors, and food contact surfaces [Nesbakken et al., 1996,
Samelis and Metaxopoulos, 1999, Gande and Muriana, 2003]. It grows in many foods
during refrigerated storage and can also tolerate and grow in relatively acidic foods,
in foods with relatively low moisture content and in foods with a high salt content
(i.e., Ready-To-Eat foods).
5.3 Ready-To-Eat foods

Ready-To-Eat (RTE) foods are intended for consumption without further bacterici-
dal or viricidal heat treatment or processing with an equivalent effect. Such foods
5 Listeria monocytogenes and Listeriosis 35
are regarded as potentially high risk, because they do not receive any treatment that
would destroy pathogens before consumption. These products may receive a lethality
treatment to eliminate the pathogen but they are likely re-contaminated by exposure to
the pathogen after the lethality treatment (e.g., during peeling, slicing, repackaging)
This is due to the ability of the microorganism to form persistent biofilms on indus-
trial equipment, which can survive common sanitation procedures. In addition, the
number of foods that are already prepared from retail establishments, grocery stores
and delicatessens are increasing and it is likely that adequate food safety measures
may not be applied to control or prevent contamination with L. monocytogenes, thus
increasing the risk for consumer exposure to this pathogen. Certain RTE products,
such as non-reheated frankfurters and sliced meat products, soft cheeses, and veg-
etable salads, pose a greater threat to public health. This was outlined in a report on
the L. monocytogenes risk assessment for selected RTE foods [FDA/CFSAN, 2003]
RTE foods are frequently implicated in listeriosis outbreaks, due to the aforemen-
tioned reasons, and have therefore forced authorities to establish control measures
to reduce the risk of consumption of highly contaminated products. In Greece, the
institute that deals with surveillance and control of foodborne infections and intoxi-
cations is the Center for Surveillance and Intervention (CSI) of the Hellenic Center
for Infectious Diseases Control (HCIDC). The Hellenic Food Authority has initiated
a survey for the presence and level of certain foodborne hazards, with an emphasis on
listeria in various enterprises that are involved in the production, distribution, retail
display and serving of RTE products.
5.4 Listeriosis in Greece

According to the 7th (i.e., between 1993 and 1998) and 8th (i.e., between 1999
and 2000) reports of World Health Organization [BgVV-FAO/WHO, 2000, BfR-
FAO/WHO, 2003], the only known cases of listeriosis in Greece were one in 1998,
seven in 1999 and six in 2000, whereas the HCIDC reported zero cases of listeriosis
for the period 1998-2002 and only one case in 2003. However, it should be noted
that data from the HCIDC was obtained from the system of obligatory reporting
of illnesses, and thus publicly available reports may have underestimated the actual
incidence rates. Despite the existence of the WHO Food Surveillance Programme
for the Control of Foodborne Diseases in Europe that was initiated 20 years ago,
collection of national epidemiological data is difficult, as official agencies access
only 1 to 10% of cases and significant under-reporting is likely [Tirado and Schmidt,
2001]. Another reason for under-reporting is the delay between the occurrence of the
outbreak and the reporting day.
5.4.1 RTE Products

In July 2003, the Hellenic Food Authority initiated the monitoring of chemical and
biological hazards in RTE products. Microbiological analysis was performed by ac-
credited microbiological laboratories in Greece, using ISO methods (i.e., ISO 11290-
1:1996 and ISO 11290-2:1996 for detection and enumeration, respectively). Of the
605 samples tested, four samples (i.e., 0.7%) were found to be contaminated with
listeria. The positive samples consisted of composite plates with minced meat or
seafood, cold sandwiches and lettuce salad.
5.4.2 Meat products

Between March 2003 and April 2005, food authorities disposed of approximately
3,500 kg of meat products. An outbreak of gastroenteritides occurred in May 25,
2005, at a National Health Institute in Greece, involving 37 employees and 55 patients.
Inspectors from the Hellenic Food Authority subsequently destroyed approximately
8.5 tonnes of meat products that were characterized unsafe in Thessaloniki on June
1, 2005. Five deaths were reported during the following nine days. Initially, deaths
were attributed to pathological causes, although the possibility of listeriosis was also
considered. The Central Laboratory of Public Health, reported the detection of listeria
in bacon samples that were used in the National Health Institute to prepare food, on
June 13, 2005. However, it remains unclear as to whether the presence of listeria
was associated with the manufacturing process or with the storage conditions of the
products within the Institute. The processing plant immediately recalled the products
of this specific batch and isolated them until disposal.
5.5 Prevention and Control

The results of the national survey for pathogens and hygiene indicators (i.e., coliforms)
in 605 RTE products indicated promising results with regards to pathogens prevalence
and concentration, as opposed to the concerns expressed about the hygienic level (i.e.,
due to high coliforms loads) of establishments that prepare cold-composite foods, such
as sandwiches with cheese and deli meats (e.g., bacon, ham, frankfurters). The low
incidence rates due to under-reporting and the limited surveillance data available so
far, showing low frequencies of contamination with pathogens, do not imply zero
risk, especially if hygiene conditions in manufacturing, distribution and retail display
are not ensured.
Tolerable levels of L. monocytogenes in foods and especially in RTE foods, is
a matter of great concern at an international level. However, European Community
legislation does not provide microbiological standards for L. monocytogenes in all cat-
egories of RTE products. Current national legislation requires absence of the pathogen
in 25 g of foods, even for fresh meat. However, despite the current “zero tolerance”
policy for L. monocytogenes, the complete absence of this pathogen for certain RTE
foods is not feasible. Risk assessments that have been conducted by the United States
Department of Agriculture and the World Health Organization over the last 5 years
have prompted food safety authorities to discuss the establishment of tolerable levels
of L. monocytogenes (i.e., 100 CFU/g) in those RTE foods that do not allow growth of
the pathogen until consumption, and in foods that will be heated before consumption.
In addition, risk assessment data are under consideration for setting Appropriate Lev-
els of Protection (ALOP) and Food Safety Objectives (FSO) that will ensure tolerable
risk until consumption [van Schothorst, 1998].
5.6 Conclusion
In order to establish microbiological standards, it is essential to categorize foods
based on criteria related to: (1) application of lethality treatment (i.e., mainly heat)
during manufacturing; (2) application of post-lethality treatment, that may consists of
either the immersion of product into antimicrobial solutions or treatment (e.g., heat,
irradiation) of the whole packaging; (3) the possibility of recontamination during dis-
tribution or retail display; (4) the ability of food to support the growth of the pathogen
(i.e., dependent on the intrinsic properties of the food) during the shelf life, in re-
sponse to storage temperature. These considerations may significantly improve the
effectiveness of inspections and the safety level of foods. Greek inspection authorities
will not be forced to withdraw suspect products without having a scientifically-based
criterion, and food manufacturers should target specific performance objectives to
ensure safety of final products, while consumers should be aware of proper handling
of foods. Other considerations include:
(1) Rapid and proper reporting of foodborne illnesses to official agencies in order to
increase the availability of epidemiological data, and hence enable the effective
monitoring of hazards from “farm-to-fork”.
(2) Increase training of food manufacturers and those that are involved in RTE food
preparation on Good Hygiene Practices.
(3) Consideration of research and official surveillance data by the national inspection
authorities and the development of risk assessment for different RTE products.
(4) Increase number of inspections by the Hellenic Food Authority would enhance
the national surveillance programs for foodborne pathogens, provide better mon-
itoring of Hygiene in the food industry and finally assist both manufacturers and
consumers in better handling of foods.

1. To what extent has listeriosis been reported in the last 12 years?
2. What is the balance between obligatory reporting and the use of voluntary surveil-
lance programs1?
3. How variable are diagnostic methods between different Laboratories, such as
hospital microbiological laboratories, national reference centers and laboratories
of district health authorities, which are significant contributors of surveillance
data to the HCIDC?
4. Is “zero tolerance” for L. monocytogenes feasible on/in RTE meat and poultry
products?
5. What measures can be taken to control the potential post-processing contamina-
tion of RTE products with L. monocytogenes?
6. Which environmental factors affect the survival and growth of L. monocytogenes
during distribution and retail display of RTE products?
1
Refer to Tirado and Schmidt [2001]
7. Are most people aware of the significance of training in Food Safety principles
and especially the current food safety strategy from the “farm-to-fork”1?
8. Is compliance with food law a matter of awareness of food safety principles, or is
it simply dependent on the frequency of inspection and the severity of punishment
in the case of non-compliance? Explain why this is the case.
References
C. Bell and A. Kyriakides. Listeria - a practical approach to the organism and its
control in foods. Blackie Academic Professional, London, 1998.
BfR-FAO/WHO. WHO Surveillance Programme for Control of Foodborne Infections
and Intoxications in Europe. Eighth Report 1999-2000. FAO/WHO Collaborating
Centre for Training and Research in Food Hygiene and Zoonoses, 2003. http:
//www.bfr.bund.de/internet/8threport/8threp_fr.htm.
BgVV-FAO/WHO. WHO Surveillance Programme for Control of Foodborne Infec-
tions and Intoxications in Europe. Seventh Report 1993-1998. FAO/WHO Collab-
orating Centre for Training and Research in Food Hygiene and Zoonoses, 2000.
http://www.bfr.bund.de/internet/7threport/7threp_fr.htm.
Commision of the European Communities. White paper on food safety. brussels,
12/1/2000, com (1999) 719., 2000. http://europa.eu.int/eur-lex/en/com/
wpr/1999/com1999_0719en01.pdf.
J. M. Farber and P. I. Peterkin. Listeria monocytogenes, a food-borne pathogen.
Microbiological Reviews, 55:476–511, 1991.
FDA/CFSAN. Quantitative assessment of the relative risk to public health from
foodborne Listeria monocytogenes among selected food categories of ready-to-
eat foods. Food and Drug Administration/Center for Food Safety and Applied
Nutrition, 2003. http://www.foodsafety.gov/~dms/lmr2-toc.html.
N. Gande and P. Muriana. Prepackage surface pasteurization of ready-to-eat meats
with a radiant heat oven for reduction of listeria monocytogenes. Journal of Food
Protection, 66:1623–1630, 2003.
T. Nesbakken, G. Kapperud, and D. A. Caugant. Pathways of Listeria monocytogenes
contamination in the meat processing industry. International Journal of Food Mi-
crobiology, 31:161–171, 1996.
J. Samelis and J. Metaxopoulos. Incidence and principle sources of Listeria spp. and
Listeria monocytogenes contamination in processed meats and a meat processing
plant. Food Microbiology, 16:465–477, 1999.
J. A. Scanga, A. D. Grona, K. E. Belk, J. N. Sofos, G. R. Bellinger, and G.C. Smith.
Microbiological contamination of raw beef trimmings and ground beef. Meat
Science, 56:145–152, 2000.
C. Tirado and K. Schmidt. Who surveillance programme for control of foodborne
infections and intoxications: preliminary results and trends across greater europe.
Journal of Infection, 43:80–84., 2001.
1
see white paper on Food Safety [Commision of the European Communities, 2000]
R. B. Tompkin. Control of listeria monocytogenes in the food-processing environ-

ment. Journal of Protection, 65:709–725, 2002.
M. van Schothorst. Principles for the establishment of microbiological food safety
objectives and related control measures. Food Control, 9:379–384, 1998.
Part II
Historical perspectives
6
The Toxic Oil Syndrome in Spain
Victoria Ferragut1,2
1
Centre Especial de Recerca Planta de Tecnologia dels Aliments (CERPTA), Departament
de Cičncia Animal i dels Aliments, Facultat de Veterināria, Universitat Autōnoma de
Barcelona, 08193 Bellaterra. Spain.
2
[email protected]
Summary
In the early 1980s, an estimated 20,000 people from central Spain were reported
ill from Toxic Oil Syndrome. This intoxication illness caused the death of over 600
people. By the end of 1997, the death toll reached 1,800 cases. The officially held
theory was that adulterated industrial oil was illegally imported from France in 1981,
rerefined, and subsequently sold for human consumption in Spain. However, other
studies pointed to the use of a plaguicide1 which contains organophosphates.
Key words: Toxic Oil Syndrome; intoxication; anilides; fatty acid esters of propane-
diol

(1) Recognize the importance of food labelling to the consumer.
(2) Relate the public health implications of fraudulent practices on food products.
(3) Identify when a toxicological problem could be considered an epidemic.
(4) Examine the importance of process variables, the transformation of compounds
during processing and storage on final product characteristics.
(5) Evaluate the incorrect procedures that were taken in the process of collecting the
toxic oils.
6.2 Introduction
Early in May 1981, six members of a family in central Spain were hospitalized
with an unknown illness and later diagnosed with lung infection. One member of
that family subsequently died while being transferred to another hospital. A few
1
A pesticide
44 Victoria Ferragut
days later, two brothers from the same neighborhood were urgently hospitalized with
similar symptoms. Fifteen days later, more than one thousand patients were reported
to have the same illness. The rapid increase in the number of reported cases and the
geographical location of the illness, in and around Madrid, lead the Spanish sanitary
authorities to raise the alarm of an impending infectious outbreak.
6.3 Sequence of events
In the early stages of the outbreak, the authorities suspected the origin of the ill-
ness to be bacterial, most likely caused by Legionella1 and/or Mycoplasma2 , as the
clinical symptoms of the patients were similar to acute infectious pneumonia. Unsuc-
cessful attempts to cure the patients were made by administrating antibiotics (e.g.,
erytromicin, tetracycline).
A number of reports and rumours began to emerge around this time as to the cause
of this illness, eventhough the precise nature of the causal agent was still unclear. Con-
sumption of onions, strawberries and asparagus were all suggested. Many domestic
animals, mainly cats, dogs, and birds, were put down as they were also implicated as
possible vehicles of transmission. An in-depth study of the clinical histories of the
patients affected by the “atypical pneumonia”3, and an analysis of the geographical
distribution and the socioeconomic status (i.e., lower social classes) of the patients,
suggested food intoxication as the more probable cause of the illness.
An epidemiological study on children affected by the illness was carried out
on June 10, 1981, alongside analysis conducted by the customs central laboratory.
Results suggested that the cause of the epidemic was the consumption of rapeseed oil,
containing aniline and acetaniline, that was fraudulently sold by itinerant salesmen to
consumers [Hernández, 1982]. Further analysis of oil samples showed the presence
of high quantities (i.e., several hundreds of ppm) of fatty acid anilides. By observing
the clinical evolution of affected patients, the illness was considered a form of “toxic
syndrome”4. This was later designated by the World Health Organization (WHO), as
“toxic oil syndrome” (TOS).
Most experts had concluded that the suspected oil was the likely vehicle of the
TOS, as evidence for other alternative theories were not credible. However, one hy-
pothesis did receive a lot of press coverage and was outlined in the Cambio 16 mag-
azine [Anon, 1984]. The article suggested that the epidemic occurred as the result
of the consumption of tomatoes, grown in Andalucia, that were contaminated with
1
Legionella pneumophila causes an acute respiratory infection known as Legionnaire’s dis-
ease. See the MedlinePlus Medical Encyclopedia [Levy, 2003a] for more details
2
Mycoplasma pneumoniae causes an infection of the lungs and is known as Mycoplasma
pneumonia. See the MedlinePlus Medical Encyclopedia [Levy, 2003b] for more details
3
Atypical pneumonia is caused by bacteria, such as Legionella pneumophila, Mycoplasma
pneumoniae and Chlamydophila pneumoniae. See the MedlinePlus Medical Encyclopedia
[Hart and Wener, 2004]
4
A range of signs and symptoms that a person exhibits from exposure to a toxic substance
6 The Toxic Oil Syndrome in Spain 45
organophosphates, namely, Fenamiphos1. Fenamiphosis produced by Bayer and sold

under the commercial name of Nemacur .
6.4 Analysis of the Problem
6.4.1 Probable hypotheses
Two studies have appeared that reviewed the events and discussed the probable cause
of the illness [Guitart, 1984, Gelpı́ et al., 2002]. The hypothesis that a pesticide was
the probable causal agent of the epidemic was first formulated by Dr. Muro, the deputy
director of the Hospital del Rey in Madrid, who was fired on the spot after making this
declaration. This hypothesis was later substantiated, two years later, by Dr. Frontela,
a professor of legal medicine and director at the Institute of Forensic Sciences of the
University of Sevilla. He conducted laboratory assays after administering the plagui-
cide to laboratory animals, which provoked similar symptoms to those observed in
patients affected by the toxic syndrome. Two other scientists from the Epidemiolog-
ical Commission of the Toxic Syndrome, Dr. Martinez Ruiz and Dr. Clavera, made
similar claims and were also dismissed.
The alternative hypothesis was also analyzed by Dr. Muro, by mapping the illness
from sociological studies. Some of the patients affected by the toxic syndrome were
found not to have consumed the toxic oil. However, all of the patients had instead
consumed a tomato variety that was only grown in a few known areas in Spain. Due
to a bad harvest that year, tomatoes grown in Roquetas de Mar (Almeria) were dis-
tributed to local markets around the outskirts of Madrid and sold below the commercial
value. Laboratory studies on rats led Dr. Muro to conclude that the toxic agent from
Nemacur was 10 times more potent in tomatoes, than when it was administered
directly to animals. However, these studies were mainly part of unpublished reports
and were not continued due to the lack of financial support offered by the Spanish
government at the time. This alternative hypothesis was later dropped in favor for the
hypothesis of the toxic oil.
6.4.2 Studies on the toxic oil
Chemical analysis of suspect oils revealed the presence of a number of foreign sub-
stances (i.e., anilines and their derived products), rendering the oils unfit for human
consumption. However, toxicological tests were inconclusive as to the identification
of the toxic substances or the biochemical mechanism of action. Researchers were
1
Fhenamiphos is a systemic pesticide which kills parasites when they eat the foliage of a
plant. An explanation of how pesticides work can be found on the webpage of the New
South Wales Environmental Protection Authority [EPA, 2004]. An International Chemical
Safety Card (ICSC) on essential health and safety information for the use of Fenamiphos
is also available online [IPCS, 1998]. A period of three weeks after the application of the
pesticide is suggested in order to guarantee its elimination. Incorrect use of the pesticide
can be fatal.
also baffled as to the variation in the response within a family to the illness (i.e., some
were not affected at all, while others died), or why no cases were reported from the
“Catalonian circuit”1 .
6.4.3 Epidemiological studies
The highest number of cases (approximately 1,800 known cases) were reported at the
beginning of June 1981, which subsequently declined thereafter until no further cases
were reported in late 1982 [Catalá and Mata, 1982, Toxic Epidemic Syndrome Study
Group, 1982]. TOS affected mainly central regions of Spain: Madrid (14,702 cases),
Valladolid (1,469 cases), Leķn (1,043 cases), Palencia (626 cases) and Segovia (571
cases). Only seven cases were found within the whole of Catalonia, all of which were
believed to have been contracted outside of that region.
Over 20,000 cases of TOS were reported. 97% of all recorded cases were found to
have consumed the toxic rapeseed oil. Most patients were from lower social classes,
of which 60.4% were women and 20.9% less than 15 years old. A study had also
estimated that 50,000 people could have consumed the toxic oil [Picot, 1982]. In
the first 20 months after the outbreak, 262 further deaths occurred (75 men and 187
women) among individuals who had been affected by TOS [Grandjean and Tarkowski,
1984]. This study also suggested that the mortality rate among women was higher,
in particular among younger women, thus substantiating similar conclusions made
from previous studies in 1981.
The rate of reported deaths declined rapidly after the end of 1982. A total of 823
men and 858 women died from the illness, between 1983 and 1997. Only women
under the age of 40 continued to record high rates of death. Of the 62 women who
died before 1994, clinical records showed that 31 cases showed persistant signs of
severe conditions, which was typical of the intermediate or early chronic phases of
TOS [Posada de la Paz et al., 1999].
6.4.4 Origin and processing
Six hundred and thirty-five tons of denatured2 rapeseed oil, containing 2% of aniline,
was legally bought in France by the industrial group RAPSA early in 1981. One
hundred and ten tons of the denatured oil was sold to RAELCA, a company based
in Alcorcķn (Madrid). RAELCA commissioned two refineries to reprocess the dena-
tured oil, by removing the aniline. Sixty tons was sent to the Industria Trianera de
Hidrogenaciķn (ITH) refinery in Sevilla and the remaining 50 tons to the DANESA
BAU refinery in Madrid.
The resulting “edible” reprocessed rapeseed oil was mixed with other edible oils
of dubious quality, in order to change the physical properties (notably color and
odor). Finally, it was distributed by RAELCA and JAP, a packaging company from
1
See section 6.4.4 for an explanation
2
Spanish laws in the 1980s prohibited the importation of rapeseed oil, which could be used
for soap manufacturing and in the iron and steel industries, unless it was made inedible.
Extremadura. The oil was packed, without a commercial label, into 5-liter plastic
bottles and subsequently sold, without any sanitary control, door-to-door as olive oil
[Tabuerca et al., 1983, Koch, 1981].
However, questions still remain as to other possible sources of the epidemic,
namely, two companies from Catalonia (known as the “Catalonian circuit”). RAPSA
supplied 330 tons to Industrias Quı́micas Salomķ from Reus and 49 tons to JORPI
from Prat de Llobregat. JORPI also received an additional 68 tons directly from the
supplier of RAPSA in France. Surprisingly, no cases of TOS were reported to have
resulted from the consumption of oil sold by the “Catalonian circuit” [Rodriguez-
Farré, 1982]. This was apparently due to the fact that the oil was rerefined from
dissimilar batches.
6.4.5 Identifying the causal agent
In June 1981, consumers began to return the suspect toxic oil in exchange for safe
olive oil [Pestaņa et al., 1983]. However, there was no systematic control over this
process. The exact source of only a few samples were known (e.g., families with TOS
cases, number and degree of infection), with only a handful of samples taken from
family abodes [Koch, 1981].
Rapeseed oils, refined using a process simulating those leading to the production
of the toxic agent in 1981, failed to produce relevant toxic effects in laboratory animals
[WHO, 1992]. Toxic agents in the rapeseed oil could not identified. However, aniline
could be ruled out, as its toxic effects are totally unrelated to changes typical of
TOS. In the early 1990s, further investigations focused on the natural history of TOS,
the composition of case-related oils and their effects on laboratory animals, and the
simulation of the refining process [Aldridge, 1992]. A toxico-epidemiologic (“toxi-
epi”) case-controlled control study, which provided new insights into the real etiology,
was conducted. Oil from one particular container, produced at the ITH in Sevilla and
distributed by RAELCA, was identified as the source of the epidemic.
During the 1990s, stored case-related oils and other suspected oils were analyzed
with new, more sophisticated techniques. A group of chemicals associated with the
risk of disease were identified [Hill et al., 1995]. These compounds were fatty acid
esters of 3-(N-phenylamino)-1,2-propanediol (PAP) and its 1-oleyl-ester (O PAP) and
1,2-di-oleyl ester (OO PAP). In fact, their presence in case-related oils was reported
as early as 1984 [Hill et al., 1995]. A reanalysis of the oils used in the “toxi-epi”
study, showed OO PAP to be a more specific marker of case-related oils than OA. OO
PAP was also found in a sample of oil retrieved from the ITH refinery, but not in an
oil derived from aniline-denatured rapeseed oil, illegally produced at other refineries,
nor in unrefined aniline-denatured samples of rapeseed sent to the ITH [Aldridge,
1992].
6.4.6 An accidental incident?
It has been established, from experimental testing of denatured oils, that the production
of the toxic oil may have resulted from faults arising from one or more processes of the
refining process [Gelpı́ et al., 2002]. In a typical process, phospholipids and free fatty
acids are removed by the degumming and neutralization steps, color from a bleaching
process, and the elimination of odoriferous compounds is achieved through distillation
at high temperature under vacuum, using steam as the stripping gas [Vazquez Roncero
et al., 1984].
This hypothesis was supported by evidence that showed no presence of PAP esters
in oils refined by DANESA BAU, and also by the lower amounts of OA present, as
compared to case-related oils [Hill et al., 1995]. A number of possibly toxic oils were
produced from experiments in the laboratory. Assays on toxicity with laboratory
animals showed generally negative results for these oils, although chemical analyzes
showed interesting results [Gelpı́ et al., 2002].
A number of studies have shown that PAP esters can be formed at a temperature
of 300 C. Initial studies in 1995 had identified PAP esters in two samples of rapeseed
oil, after 4 h at 300 C [Hill et al., 1995]. Further research conducted provided more
insights [Ruı́z Méndez et al., 2000, 2001]. These studies showed that the formation of
PAP esters occurred when oil samples were heated at 300 C, eventhough they were
subsequently degraded while at that temperature. No esters were formed at 250 C.
The study also revealed differences in the level of residual PAP esters for samples that
were stored for different durations before processing at 300 C. Denatured oil samples
that had been stored for two weeks prior to refining had higher amounts of PAP esters
than samples stored only for one week. Contrary to these findings, anilides can also
form spontaneously, independent of any step of the refining process, from the aniline
originally added to denature the oil, if in contact with oil constituent fatty acids [Ruı́z
Méndez et al., 2000]. The duration of the distillation during refining of the oil does
not result in significant decreases in these preformed anilides [Ruı́z Méndez et al.,
2000]. The use of different temperature combinations, the rate at which the required
temperature is reached, and pressure in the deodorization step, have provided new
insights as to the formation route and in the identification of more specific toxicity
markers (PAP esters) [Ruı́z Méndez et al., 2001].
These studies have provided a number of conflicting conclusions as to the actual
route for the formation of PAP esters. Findings suggest that these toxic compounds
might have been formed accidently and may only occur under exceptional circum-
stances, which could explain why toxicity seemed to be linked to only a single set of
oil produced in one refinery [Gelpı́ et al., 2002].
6.5 Conclusion
After nearly 25 years of research in searching for the sources of the toxic oil syn-
drome, attempts at identifying the causal agents and the search for the biological
mechanisms of TOS, many questions still remain unanswered. Further research has
been suggested in a review examining 20 years of research into the TOS [Gelpı́ et al.,
2002]. They noted that:
(1) long term effects of TOS still needs to be determined;

(2) studies have shown the causal agents may have been fatty acids esters of PAP.
These compounds were synthesized in experiments simulating industrial condi-
tions of refining. In addition, samples of oil, which were consumed by families
with TOS cases, had higher levels of these compounds than control samples;
(3) the only acceptable etiologic theory at present, based on scientific findings, is the
adulteration of oil;
(4) the unavailability of an animal model, which have shown the “natural” occurrence
of TOS, has hindered attempts at understanding the biological mechanisms of
TOS. Unsuccessful attempts have been made in reproducing symptoms of TOS
in laboratory animals, using synthesized oils containing high levels of aniline
derivatives;
(5) numerous questions still remain with regard to the metabolism of chemicals,
which hinders the evaluation of the significance of TOS.
Finally, it is worth nothing the legal implications of this case. The Spanish judiciary
has been evaluating some 17,000 cases of TOS. In 1997, the courts identified 101 cases
of people, considered to be suffering from “great incapacity” (i.e., those who require
the help of others to carry out any type of work) and 209 cases of “absolute permanent
incapacity” (i.e., inability to carry out any type of work) [Audiencia Nacional de la
Sala de lo Penal, 1989]. In addition, the Spanish National Institute of Social Security
has identified 3,477 cases of people that are considered to have “total permanent
disability” (corresponding to an inability to carry out common activity), two thirds of
whom are women with an average age of 37 years [Martin Arribas et al., 2001].

1. What evidence led to the conclusion that the epidemic resulted from the com-
sumption of toxic oil?
2. Why were there no toxic oil produced by the Catalonianian circuit”?
3. Why was aniline present in the rapeseed oil originating from France?
4. Which compounds were found to be most likely the cause of TOS?
5. Why were further investigations into alternative hypotheses not supported?
6. Why was it difficult to synthesize the compounds, believed to be responsible for
TOS, in laboratory experiments that were aimed at simulating known industrial
conditions?
7. A collection of oil samples was established in June 1981, which were used in the
toxi-epi studies mentioned [Gelpı́ et al., 2002]. Discuss what information would
be needed to adequately classify these samples.
8. What procedures and policies should be in place to prevent the sale of fraudulent
products?
References
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Anon. Según nuevas investigaciones cientı́ficas. un producto bayer envenenķ espaņa.
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F. J. Catalá and J. M. S. Mata. In Simposium nacional sobre el sı́mdrome tķxico, pages
544–551. Ministerio de Sanidad y consumo, 1982. Madrid.
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pesthwwrk.htm. Environmental Protection Authority. Department of Environ-
ment and Conservation. NSW, Australia. http://www.epa.nsw.gov.au/envirom/
pesthwwrk.htm.
E. Gelpı́, M. Posada de la Paz, B. Terracini, I. Abaitua, A. Gķmez de la Cámara, E. M.
Kilbourne, C. Lahoz, B. Nemery, Philen R. M., L. Soldevilla, and E. Tarkowski.
The spanish toxic oil syndrome 20 years after its onset; a multidisciplinary review
of scientific knowledge. Environmental Health Perspectives, 110:457–464, 2002.
P. Grandjean and S. Tarkowski. Toxic oil syndrome: mass food poisoning in Spain–
report of a WHO meeting, Madrid 21-25 March 1983. Copenhagen. Technical
report, World Health Organization Regional Office for Europe, 1984.
R. Guitart. Estudio de aceites implicados en el syndrome tķxico y de los efectos que
las anilidas tienen sobre el metabolismo lipoxigenasico del ácido araquidķnico.
Memoria para optar al grado de licenciado en Ciencias. Technical report, Univer-
sitat Autōnoma de Barcelona, 1984.
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June 2004. URL http://www.nlm.nih.gov/medlineplus/ency/article/000079.
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M. Hernández. In Simposium nacional sobre el sı́mdrome tķxico, pages 544–551.
Ministerio de Sanidad y consume, 1982. Madrid.
R. H. JR. Hill, H. Schurz, M. Posada, I. Abaitua, R. M. Philen, E. M. Kilbourne, S. L.
Head, S. Bailey, W. J. Driskell, J. R. Barr, and et al. Possible etiologic agent for toxic
oil syndrome: fatty acid ester of 3-(N-phenylamino)-1,2-propanediol. Archives of
Environmental Contamination and Toxicology, 28:259–264, 1995.
IPCS. Fenamiphos. International programme on Chemical Safety, Novem-
ber 1998. URL http://www.ilo.org/public/english/protection/
safework/cis/products/icsc%/dtasht/\_icsc04/icsc0483.pdf. http:
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7
A ban on Paprika in Hungary
Cecilia Hodúr1,2, Zsuzsanna Lászlķ1 , and Zsuzsa Hovorka Horváth1

1
College Faculty of Food Engineering, University of Szeged, Mars sq. 7, H-6724 Szeged,
Hungary.
2
[email protected]
Summary
Paprika, a staple ingredient in Hungarian cuisine and also considered a cultural sym-
bol, was taken off the shelves of food retailers and withdrawn from restaurants by the
Hungarian Ministry of Health on the 27th of October 2004, after aflatoxin was discov-
ered in Hungarian paprika by authorities in Slovenia. This potentially carcinogenic
toxin can only be found in paprika originating from the tropics. Suspect products
contained paprika that apparently originated from South America. Some Hungarian
producers were illegally mixing imported and Hungarian paprika before selling them
to the public. Hungary exports approximately 5,500 tons of the spice every year.
Key words: paprika; aflatoxin

(1) Identify and describe factors that prevent aflatoxins in paprika.
(2) Review and analyze food safety requirements and legislation regarding aflatoxins
in foods.
(3) Achieve the familiarity with EU food regulation.
(4) Evaluate the role and responsibility of national, European and international food
authorities in food safety.
(5) Examine the role of HACCP, risk assessment and traceability systems in food
safety control.
7.2 Introduction
The paprika plant has been cultivated for several hundred years in Hungary. First
introduced by the Turks in the 17th century, it is traditionally cultivated in two regions
54 Cecilia Hodúr, Zsuzsanna Lászlķ, and Zsuzsa Hovorka Horváth
of the country, Szeged and Kalocsa. Paprika is the national spice of Hungary and the
vast majority of Hungarian dishes contain paprika. It can be the main ingredient in
some dishes and is used with meat, fish, and in sauces and salad dressings. Ground
paprika, from the dried fruit of Capiscum annum L, contributes flavor and color to
food. Manuel harvesting of the ripened paprika fruit takes place when 60 % humidity
is achieved, by which time it becomes a bright red color. The fruit is harvested with
care to avoid bruises and injuries to the peel and placed in baskets, before they are
transported to the processing plant to the classified, whereby deteriorated and infested
fruit are discarded. This reduces the possibility of finding aflatoxins in the fruit.
Aflatoxins are naturally occurring contaminants found a wide range of plant prod-
ucts that are known to be carcinogenic. Aflatoxins are produced by certain members of
the aspergillus family. There are four main aflatoxins, B1 , B2 , G1 and G2 , commonly
found in various foods. Fungal contamination can occur during plant growth or after
harvest, as the result of drought, insect damage or under poor storage conditions (i.e.,
temperature and humidity).

In August 2004, Hungarian authorities seized 63 metric tonnes of spice in Bács-
Kiskun after aflatoxin was discovered. A month later, Slovenian authorities found a
consignment of paprika powder containing levels of aflatoxins that were higher than
legally permitted in the EU. The consignment that arrived from Austria, originated
from Hungary. Hungarian authorities were notified through the Rapid Alert System
for Food and Feed (RASFF)1 . The Hungarian Ministry of Health was officially in-
formed on October 25, about the contaminated consignment of paprika, and on the
following day they requested from the Public Health Authority and Food Safety Of-
fice the appropriate documentation regarding the incident. An analysis conducted on
October, 27, showed samples from certain brands of paprika containing fifteen times
above acceptable levels of aflatoxin. The investigation led the authorities to suspect
that paprika containing aflatoxins had been imported from Brazil and mixed with Hun-
garian paprika, since normal climatic conditions in Hungary prevented the growth of
molds. A drought occurred the previous summer that had caused a decrease in the
production of paprika in Hungaria. Hungarian producers mixed the cheaper imported
paprika into their products and packed then without any declaration of the amount of
imported paprika.
On October 28, the Hungarian government banned the sale of paprika and pro-
hibited its use. The population was informed to refrain from using paprika until it was
certified to be safe by the national health inspection authorities. The state hygiene
service identified 62 paprika products to be safe the next day and on November, 1,
the Hungarian Public Health Authority (ANTSZ) gave the all-clear for the sale of an-
1
The purpose of the RASFF is to provide control authorities with an effective tool for exchange
of information on measures taken to ensure food safety. See Anon [2006a] for more details.
7 A ban on Paprika in Hungary 55
other 20 paprika products. On November, 15, the European Food Safety Authority1
conducted a five day investigation examining the incident.
7.4 Conclusion
In the beginning of the incident, the Hungarian food quality and safety authorities
reacted indifferently, followed later by a state of panic. Sampling was also inconsis-
tent and included fresh whole paprika. Authorities never established a safe level of
aflatoxin in paprika products. Concrete steps should be taken by both producers and
governmental agencies in improving safety in these products, by setting up HACCP
and traceability systems in order to prevent such an incident from occuring in the
future.

1. What do you know the paprika plant, its cultivation and the production of ground
paprika? What factors should be taken into consideration in order to prevent mold
contamination and subsequent production of aflatoxins?
2. What levels of aflatoxins are considered acceptable in paprika powder? Are there
Maximum Residue Limits (MRLs) for aflatoxins in spices?
3. What analytical methods can be used to determine aflatoxins in paprika? Should
testing be restricted to certified laboratories? Why?
4. Why did producers mix Hungarian and imported paprika and not indicate the
correct composition of the products? What are the legal requirements for labelling
of this and other similar products?
5. Why was there no control over the quality and safety of imported paprika? Who
should have been responsible for ensuring the safety of these products? Why?
6. Was the sampling procedure adopted by the Hungarian food safety authorities at
the time of the incident correct? What would you have done differently? WHy?
7. What was the reaction of the Hungarian food safety authorities when they were
notified of the contaminated paprika products? What procedures should be fol-
lowed in the event of another occurrence?
8. What European legislation is in place for controlling levels of aflatoxins in spices?
What monitoring procedures are present at a national and European level? How
does this compare to control procedures found in other non-European countries?
9. How would HACCP, risk assessment and traceability systems improve the safety
of spices? What recommendations would you make to a producer on how to
control the safety of their products?
1
The European Food Safety Authority (EFSA) provides objective scientific advice on all
matters with a direct or indirect impact on food and feed safety [European Food Safety
Authority, 2004].
56 Cecilia Hodúr, Zsuzsanna Lászlķ, and Zsuzsa Hovorka Horváth
References
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http://www.eum.hu/eum/eum.news.page?pid=DA_39424.
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//www.eduport.hu/cikk.php?id=10940.
Anon. Bővült a forgalmazhatķ és fogyaszthatķ paprikatermékek listája. Szeged Portal,
2004c. http://www.szegedportal.hu/index.php?pg=hir\&id=228.
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Anon. Rendkı́vüli ellenőrzést indı́t az EU a szennyezett paprika mi-
att. Magyarország.hu, 2004f. http://www.magyarorszag.hu/hirek/egeszseg/
paprika20041102.html.
Anon. EUROPA - Food Safety - Rapid Alert System for Food and Feed (RASFF) - In-
troduction, 2006a. http://europa.eu.int/comm/food/food/rapidalert/index_
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Anon. Paprika. Wikimedia Foundation, 2006b. http://en.wikipedia.org/wiki/
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8
An Outbreak of Botulism in Italy
Laura Bigliardi1 and Giuliano Sansebastiano1,2

1
Dipartimento di Sanitā Pubblica, Sez. di Igiene - Universitā, Via Volturno, 39, 43100
Parma, Italy.
2
[email protected]
Summary
In August 1996, an outbreak of botulism in southern Italy affected eight people,

aged between 6 and 23, which resulted in the death of a single hospitalized case.
Microbiological analysis performed on blood and stool samples of people affected,
confirmed the presence of Clostridium botulinum type A spores and botulinum toxin.
The epidemiological investigation showed that a commercial cream cheese known as
mascarpone, produced by a major food company in northern Italy, was the source of
the botulinum toxin. All mascarpone cheese that was produced by this food company
were immediately removed from the market in Italy and abroad. A national alert
was initiated to improve public awareness and health services were informed to be
aware of patients showing symptoms similar to those of botulism. Several hypotheses
suggested that a dairy factory was the source of incident, although, none of them have
so far been proven.
Key words: mascarpone; Clostridium botulinum; botulism

(1) Discuss the importance of reporting infectious diseases in the control and pre-
vention of outbreaks.
(2) Discuss the importance of collecting information on affected subjects.
(3) Describe routes of food contamination and explain how different factors affect
microbial growth.
(4) Interpret epidemiological data and formulate probable hypotheses of the source
of food intoxications.
(5) Propose measures for preventing microbial contamination and food intoxication.
58 Laura Bigliardi and Giuliano Sansebastiano
8.2 Introduction
There are three main issues in the prevention of infectious diseases: (1) measures un-
dertaken relating to the source of infections, (2) the mode of transmission (e.g., water,
food), (3) measures aimed directly at people (e.g., vaccination, serum prophylaxis).
The reporting of infectious diseases is an important part of controlling and preventing
outbreaks. The Communicable Diseases Unit of the Italian Ministry of Health uses
a reporting system, based on the Corpus of Sanitary Laws (Testo Unico delle Leggi
Sanitarie) and the Ministerial Decree of 15 December 1990 [Squarcione et al., 1999].
The collection of information relating to the characteristics of affected subjects (e.g.,
age, sex, residence, personal habits) and all other relevant information, that might be
available for the diagnosis of the pathology and onset of a disease, plays an important
role in the implementation of preventive measures.
Botulism in humans result from the ingestion of toxin produced by Clostridium
botulinum. Foodborne botulism can be a serious public health problem, if inadequate
procedures and conditions are used for the processing of preserved foods. This case
study examines an outbreak of foodborne botulism that occurred in two regions of
southern Italy, in which eight of the confirmed cases were associated with the con-
sumption of fresh cheese.

Only a summary of reported cases will be given in this section, as details of cases
have been described in more detail elsewhere [Aureli et al., 2000]. There were four
reported incidents:
(1) August 12, 1996: A twenty-four-year-old man and a nine-year-old girl from
Campania (a region in southern Italy) showed symptoms belonging to botulism.
This included a range of gastroenteric disorders (i.e., repeated vomiting, diar-
rhoea, asthenia) and neurological symptoms (i.e., diplopia, dyspnea, dysphagia,
lipothymia, cranial and facial nerve dysfunction, tetra hypostenia, drooping eye-
lids, difficulty in speaking, ptosis, lingual paresis, dysphagia and respiratory fail-
ure).
(2) August 30, 1996: Two brothers, aged 15 and 12, and their fourteen-year-old
neighbour, also from Campania, showed the same symptoms as those observed
during the first incident. They were all hospitalized. The fifteen-year-old boy died
37 days after being hospitalized.
(3) September 3, 1996: Two brothers, aged 18 and 15, this time from the town of
Vibo Valentia in Calabria, suffered from nausea, vomiting and diplopia, before
they were taken to hospital. Botulism was again suspected.
(4) August 21, 1996: A six-year-old boy was hospitalized at the Catanzaro Hospital
in Calabria, showing symptoms of botulism (i.e., vomiting, pharyngodynia, dys-
phagia, ptosis, astenia, lipothymia).
8 An Outbreak of Botulism in Italy 59
8.4 Epidemiological and Clinical Studies

The epidemiological investigation was a cohort study that consisted of interviewing
hospitalized patients and other individuals (i.e., parents) present at the meals im-
plicated in the intoxication. Individuals working in the production factories, local
warehouse managers and food venders were also interviewed, in order to examine the
production, distribution, storage and purchase information [Aureli et al., 2000].
Thirty serum and 37 stool samples, were taken from patients before they were
treated with trivalent botulinum antiserum, 29 additional samples from individuals
unrelated to the incident, 39 mascarpone samples (i.e., taken from retail stores and
distributors warehouses) originating from the same production plant, as well as en-
vironmental samples from the suspected production plant, were taken to test for the
presence of toxins and bacterial spores [Aureli et al., 2000].
Both the epidemiological and clinical investigations pointed to the likelihood that
the intoxication resulted from the ingestion of mascarpone cheese used in the cooking
of a homemade dessert called tiramisù. Confirmation of the presence of C. botulinum
type A and botulinum toxin type A, in biological samples from patients and samples
of mascapone, were made by the Istituto Superiore di Sanitā on September 5. As
the result of these findings and a Ministry of Health alert on September 3, three
commercial brands of mascapone cheese from the same producer were subsequently
removed from markets in Italy and other countries [WHO, 1996].
8.5 Routes of Contamination

One likely origin of the microbial contamination could have been the use of infected
raw milk in the production process for mascarpone cheese (i.e., primary contamina-
tion). If the raw milk was contaminated by Cl. botulinum spores, then pasteurization
would have been an insufficient thermal process for the inactivation of spores. Contam-
ination might have also occurred from contact with infected surfaces and unhygienic
process equipment (i.e., secondary contamination).
A further possibility could have been an interruption of the cold chain during trans-
port or storage. Extended periods of time at higher than normal storage temperatures
would have created conditions of growth for aerobic bacteria within the packaging,
resulting in the exhaustion of available oxygen, thus permitting conditions that would
have been ideal for the survival of Cl. botulinum spores. This would have resulted
in a larger amount of positive samples of individually packed mascarpone cheese.
However, this did not occur in this case.
8.6 Preventive measures

Adequate measures should be in place to prevent the occurrence of foodborne dis-
eases, by evaluating all critical control points of a food production process (e.g.,
raw materials, every stage of production, hygiene of personnel and the environment),
60 Laura Bigliardi and Giuliano Sansebastiano
storage and distribution, and until it reaches the consumer. In this particular case, the
following points should be considered:
(1) Improving the quality control of raw materials and reliability of suppliers
(2) Improving the effectiveness of sterilization processes for the inactivation of spores
from pathogenic microorganisms.
(3) Providing proper training of personnel on adequate hygienic practices (i.e., per-
sonal, working environment).
(4) More accurate information, proper measures and procedures for food preservation
during transport and storage.
(5) More attention from the consumer when buying food (e.g., checking package
integrity, the expiry date, instructions for storage), especially if products are be
eaten raw.
1. How does reporting cases of infectious diseases aid in the control and prevention
of food poisoning outbreaks?
2. Why is it necessary to collect information on affected subjects?
3. Explain how you would identify the source of the botulism outbreak?
4. Describe the possible routes of food contamination and explain how different
factors affect microbial growth. What do you think was the most likely cause of
the food intoxication?
5. What preventive measures can be taken to reduce the probability of Botulism
outbreaks?
References
P. Aureli, M. Di Cunto, A. Maffei, G. De Chiara, G. Franciosa, L. Accorinti, A. M.
Gambardella, and D. Greco. An outbreak in italy of botulism associated with a
dessert made with mascarpone cream cheese. European Journal of Epidemiology,
16:913–918, 2000.
S. Squarcione, A. Prete, and L. Vellucci. Botulism surveillance in Italy: 1992-1996.
European Journal of Epidemiology, 15:917–922, 1999.
WHO. Food safety - Outbreak of botulism, Italy. Weekly Epidemiological Record,
71(49):374–375, 1996.
9
Listeriosis from butter in Finland
Riitta Maijala1,2
1
National Veterinary and Food Research Institute, P.O.Box 45, FIN-00581, Helsinki,
Finland.
2
[email protected]
Summary
Twenty-five cases of listeriosis were reported between June 1998 and April 1999 in
Finland, caused by Listeria monocytogenes serotype 3a. Six deaths were reported.
Epidemiological and microbiological investigations identified the source of infection
as butter. On February 19, 1999, the dairy plant involved voluntarily recalled the small
packages of butter from the market, when the connection between the brand of butter
and the human cases was established. The investigation found that the contaminated
packages had been sold to 11 hospitals and to the retail market for many months.
It is worth considering if this outbreak would have been detected had the butter not
been sold to hospitals or, in the case of one hospital, given abundantly to patients
in certain wards. Adequate resources and proper risk communication should be a
priority in situations where many different authorities and laboratories, both at a local
and national level, are involved together with industry and the media.
Key words: butter; Listeria monocytogenes; listeriosis; risk groups

(1) Explain why certain people belong to risk groups of listeriosis and describe these
groups in your country.
(2) Define the legislation aiming to control Listeria monocytogenes in foods.
(3) Identify and evaluate the control parameters preventing contamination and growth
of Listeria monocytogenes in foods.
(4) Categorize different types of foods in low, medium and high risk for causing a
Listeria monocytogenes outbreak.
(5) Design a HACCP plan which will be able to detect and control Listeria monocy-
togenes.
62 Riitta Maijala
9.2 Introduction
Listeria monocytogenes is ubiquitous in various environments, especially in soil, ani-

mal and human faeces, sewage and water. It causes illness mainly in certain risk groups
(e.g., pregnant women, newborns, elderly people, immunocompromised adults), such
as invasive listeriosis, noninvasive gastrointestinal disease, as well as local skin and
eye symptoms. The invasive form is often associated with high death rates (i.e., 20-
40%), whereas the noninvasive form is usually self-resolving. Therefore, even low
number of listeriosis cases are regarded to be a significant public health problem in
many countries, as compared to, for example, salmonellosis. Reported levels of L.
monocytogenes in foods that have caused listeriosis in healthy adults have varied be-
tween 105 and 109 CFU/g. For risk groups, reported levels in foods have been around
104 CFU/g, but has also been reported at less than 10 CFU/g [Maijala et al., 2001].
L. monocytogenes can grow at low temperatures (e.g., in freezers), is a facultative
anaerobe and has also the ability to attach itself to steel surfaces, making it often
difficult to control in food processing. In fact, there have been reports of the same
clone of L. monocytogenes isolated from dairy, ice cream and other food processing
plants for many years. Outbreaks caused by L. monocytogenes have been traced back
to foods such as milk and other dairy products, vegetables, salads, meat products and
fishery products [Ryser, 1999]. However, most of these cases are sporadic and cannot
be linked to any specific source of infection.
The events in this case relate to an outbreak in hospitals caused by butter, involving
immunocompromised patients, (i.e., persons belonging to risk groups), between June
1998 and April 1999. At the end of the outbreak investigation, twenty-five case pa-
tients had been identified with different symptoms (i.e., twenty with sepsis, four with
meningitis, one with abscess). Six of these patients died.
Early in 1999, several L. monocytogenes cases, belonging to a rare serotype 3a,
were reported in Finland. This finding resulted in the initiation of investigations at a
national level on February 4, 1999. Based on epidemiological investigations, most but
not all of the 3a human cases reported in 1998-1999 had been immunocompromised
patients treated at a tertiary care hospital (TCH). Furthermore, it was also noticed that
only one strain of serotype 3a was in the culture collection of the central laboratory.
It had been isolated in 1997 from butter produced by dairy plant A.
Food control authorities visited dairy plant A, as well as the TCH kitchen, and took
several samples on February 17, 1999. They also noticed that according to records,
dairy plant A had begun to deliver 7 g butter packages to the TCH in June 1998.
Furthermore, a local food control laboratory had recently isolated L. monocytogenes
from a 7 g package of butter produced by dairy plant A. The dairy company immedi-
ately stopped production of 7 g, 10 g and 500 g butter packages and started intensive
cleaning of the plant.
9 Listeriosis from butter in Finland 63
The strain isolated by the local food control laboratory was sent to the central
laboratory for typing. It was found to be of the same serotype (i.e., 3a) that had been
isolated from the patients. In addition, preliminary results showed on February 19,
1999, that the 7 g butter packages sampled at the TCH kitchen, as well as samples
(i.e., 7 g and 10 g packages) taken at a wholesale store of dairy plant A, also tested
positive for L. monocytogenes. Additional samples were subsequently taken.
The dairy plant issued a press release and decided voluntarily to recall all their 7
g and 10 g butter products on February 19, 1999. The public health authorities also
issued a statement and requested all hospitals to refrain from serving butter, produced
by the implicated dairy. An official ban on butter from the dairy plant was issued by
the food control authorities on February 22, 1999.
The national food control authorities reported results from quantitative and envi-
ronmental laboratory analyses, as well as deaths caused in this outbreak, to the local
authorities and the press on February 23, 1999. In quantitative analyses, L. monocy-
togenes had been detected, at the level of over 100 CFU/g, only in one butter sample
(i.e., 11,000 CFU/g). At that time, the European Community Directive on milk and
milk-based products [OJEC, 1992], as well as the Finnish milk hygiene legislation,
specified the absence of L. monocytogenes in 1 g of butter. L. monocytogenes was not
detected in the 500 g and 25 kg packages examined. Environmental samples, taken
from the screw conveyor of the butter wagon and two floor drains, gave positive re-
sults for L. monocytogenes. Additional samples were subsequently taken for further
analysis. During next two days, a university laboratory reported quantitative results
(i.e., 7-79 MPN/g) from small butter packages sampled from the TCH kitchen. The
genotype was the same isolated from the patients previously. Furthermore, L. mono-
cytogenes was isolated from 500 g packages.
The national food control authorities and the central laboratory issued a joint
press release, on February 26, 1999, stating that L. monocytogenes had also been
detected in 500 g packages. In addition, the national public health authorities gave
a press release stating that no more cases had been reported after February 2, 1999,
but a fourth death had occurred. Local food control authorities were also informed by
national authorities to recall 500 g packages from the market and advised on control
procedures for listeria in dairy plants.
The dairy plant stopped production of the 25 kg packages on March 4, 1999. No
human cases of listeriosis caused by the outbreak strain were detected since April
1999 and the dairy restarted the production of 25 kg and 500 g butter.
9.4 Conclusion
Without continuous surveillance and typing of L. monocytogenes strains isolated from
humans and the food industry, this outbreak would probably not have been detected
at all. Several parties were involved in this outbreak:
(1) Industry: the dairy plant and the parent company, retail, the hospital kitchens.
64 Riitta Maijala
(2) Authorities: national food control authorities, national public health authorities,
local food control authorities, local public health authorities, customs.
(3) Laboratories: central food laboratory, university laboratory, national public health
laboratory, local food control laboratory, hospital laboratories.
(4) Media: national and local media, also internationally.
Investigations into any outbreak should involve adequate competencies and good
co-operation between food control and public health authorities, as well as with lab-
oratories. A common communication strategy must be agreed on, especially when
deaths are involved, which should be updated regularly and enough resources should
be in place.

1. In spite of detecting of L. monocytogenes several times in the production plant
before the outbreak investigation was initiated, the dairy plant had been unable
to solve the problem. Why?
2. Should the dairy plant have stopped all production of butter, when the connection
between the human cases and the butter was established, instead of on the 4th of
March? What would have been the consequences of that action?
3. Did the central laboratory have the right to reveal the fact that the rare serotype 3a
had been previously been detected only in a dairy plant, eventough this probably
resulted in saving lives?
4. Were the measures taken by the authorities too stringent for the industry, as
compared to the public health risk?
5. Are there any different criteria in selling foods to risk groups (e.g., hospital
kitchens) than to the normal population? Who is responsible for the safety level
of food served for risk groups?
6. What other foods belong to the same category as butter, when the risk from
listeriosis is considered?
7. Why does microbiological criteria for L. monocytogenes differ between conti-
nents? What are the consequences of these differences for local producers and
on international trade?
8. What factors are important in a food processing plant in order to control L.
monocytogenes? How can they be incorporated into the HACCP plan?
References
O. Lyytikainen, T. Autio, R. Maijala, P. Ruutu, T. Honkanen-Buzalski, M. Mietti-
nen, M. Hatakka, J. Mikkola, V. J. Anttila, T. Johansson, L. Rantala, T. Aalto,
H. Korkeala, and A Siitonen. An outbreak of Listeria monocytogenes serotype
3a infections from butter in finland. The Journal of Infectious Diseases, 181(5):
1838–1841, 2000.
9 Listeriosis from butter in Finland 65
R. Maijala, O. Lyytikainen, T. Autio, T. Aalto, L. Haavisto, and T. Honkanen-Buzalski.

Exposure of Listeria monocytogenes within an epidemic caused by butter in finland.
International Journal of Food Microbiology, 70(1-2):97–109, 2001.
OJEC. Council directive 92/46/EC laying down the health rules for the production
and placing on the market of raw milk, heat-treated milk and milk-based products.
Official Journal of the European Communities, L268, 1992.
E. T. Ryser. Listeria, listeriosis, and food safety., chapter Foodborne listeriosis, pages
299–358. Marcel Dekker„ 1999.
Part III
Research-based
10
Alternative solutions for the treatment of food produce
Anna Aladjadjiyan1,2
1
Agricultural University, 4000 Plovdiv, 12 Mendeleev Str, Bulgaria.
2
[email protected]
Summary
A private laboratory in Bulgaria recently found levels of nitrates in vegetables, sold in

markets in Sofia, to be more than 10 times the legal nutritional limit for infants. It is
well known that high levels of nitrates can cause a form of toxic poisoning, known as
methemoglobinemia, in infants. Chemical additives are used for improving the pro-
duction yield of food produce and their application often causes the contamination of
raw materials (i.e., soil, fertilizers for food production, which can be dangerous for
the health of consumers. Alternative methods need to be developed and implemented
to improve and ensure the safety of on-farm production methods. The substitution of
chemical fertilizers and soil additives with alternative treatment methods, such as irra-
diation, ultrasound and the use of electromagnetic energy in the form of microwaves,
are discussed.
Key words: food produce; amelioration; magnetic fields; irradiation; ultrasound;

microwaves

(1) Define production methods for food produce and identify methods used by farm-
ers for increasing yield.
(2) Describe and identify food safety hazards and their source.
(3) Examine the role of chemical amelioration (i.e., fertilizers, soil additives, fumi-
gants, herbicides) as sources of toxic components in food.
(4) List the composition of substances used in chemical amelioration.
(5) Recognize problems associated with nitrate and nitrite intoxication and formulate
procedures to prevent their presence in food.
(6) Compare and question the adequacy of governmental legislation concerning max-
imum residue levels (MRLs).
70 Anna Aladjadjiyan
(7) Review and propose alternative treatment methods for stimulating plant and
growth.
(8) Describe the effect of alternative treatment methods and levels of treatment factors
on seed vitality indices.
(9) Recognize the need for adequate experimental planning and formulation of hy-
potheses.
(10) Design an appropriate experiment and analyze data to test a hypothesis.
10.2 Introduction
The risk of food safety hazards1 affecting on-farm production2 of food produce3 is
closely related to the use of herbicides, fertilizers, and other chemicals. Soil and water
contamination generate toxic compounds that deteriorate the quality of food produce.
In order to improve food safety, the concentration of harmful substances should be
reduced and controlled.
Chemical fertilizers and soil additives, traditionally used by farmers to increase
production yield, may be substituted by alternative treatment methods, such as mag-
netic fields, microwaves, laser irradiation, and ultrasound, to reduce food safety haz-
ards. The substitution of chemical amelioration with alternative methods can result
in an accelerated initial development in plants and reduce levels of chemical toxins
(i.e., nitrates, nitrites), thus improving the safety of food produce.
High levels of toxins, exceeding maximum residue levels (MRLs), may be found
in food produce, from the use of soil or foliar fertilizers, soil additives, fumigants,
herbicides, that are applied for crop nutrition and the management of pests and disease.
Soil contamination of growing sites can lead to the contamination of food produce.
However, the amount of the chemical present in the food produce is more important
than that found in the soil. MRLs permitted for these persistent chemicals in food
produce have been set by governmental agencies [Anon, 2004a,b].
Soil nutrients and fertilizers are composed of one or more plant nutrients (or
fertilizing elements) and also include different chemical compounds. Nitrates and
nitrites are major components of soil nutrients and fertilizers and have been used for
many years in field treatments. Without the addition of these compounds, crops would
deplete nitrogen from the soil. Unfortunately, the use of nitrogen fertilizers can result
in contamination of wells and groundwaters, causing health risks especially among
young people [Lutynski et al., 1996].
Water pollution from nitrates is causing problems in all EU Member States. The
source of nitrate pollution is often difficult to locate. The main polluters are farms,
1
Biological, chemical or physical substances or properties that can be present in food produce,
thus making them an unacceptable health risk to the consumer. Chemical toxins are included
in this definition.
2
Production methods cover growing, harvesting, packing, storage, and dispatch of produce
to consumers.
3
This includes fruit, vegetables, herbs and nuts
10 Alternative solutions for the treatment of food produce 71
eventhough farmers are strongly sensitive to anything which affects the economic via-
bility of their activity [Anon, 2005]. According to EU statistical data from the 1980s,
a progressive worsening of the situation has been detected (i.e., nitrate concentra-
tions in water rose by an average of 1 mg/l per year). As a result, EU directives and
regulations have been published urging Member States to consider establishing and
implementing action programmes in respect of vulnerable zones [OJEC, 1991, 2003].
They must include measures prescribed in the codes of good agricultural practice and
measures to limit the spreading on land of any fertilizer containing nitrogen.
This case study examines the influence of different alternative treatments on
seed vitality indices (i.e.,germinating energy, germination, germ length, and fresh
weight) [Svetleva and Aladjadjian, 1996, Aladjadjian and Svetleva, 1997, Aladjad-
jiyan, 2002b, 2003].
10.3 Experimental
Experiments were conducted on vegetable production (i.e., tomatoes, carrots, pep-
pers) as well as in grain production (i.e., maize, soybean, beans). Alternative treatment
methods used in these studies, as replacements for chemical additives included mag-
netic field, laser irradiation, microwave irradiation, and ultrasound. The use of these
methods led to changes in seed vitality indices (i.e., germinating energy, germination,
uniformity of germination). Germination (%), germ length (mm) and fresh weight
(g), showed maximum values at a specific level of treatment for a given plant.
Table 10.1. Effect of magnetic field on soybean seeds
Exposure time (min) Germination (%) Germ length (mm) Fresh weight (g)
0 52 23.0 5.8
10 88 32.0 15.8
15 96 30.0 11.7
20 88 22.0 8.1
30 72 25.55 9.6
a
Data from Aladjadjiyan [2003].
Table 10.1 shows the results of treating soybean seeds with a magnetic field
[Aladjadjiyan, 2003]. The highest value of germination was achieved with an exposure
time of 15 min. However, an exposure time of 10 min. to a magnetic field resulted
in the highest values for germ length and fresh weight. Similar results have been
observed for Zea mais seeds [Aladjadjiyan, 2002b]. The effect of treatment with
magnetic fields in both cases increased germ length and fresh weight by about 25%.
Table 10.2 examines the use of irradiation on the growth of dry bean seeds by
comparing the fresh weight of seed roots for dry bean seed cv. Plovdiv 564 and dry
bean seed cv. Dobrudjanski. After 21 days, fresh weight for dry bean seed cv. Plovdiv
564 increased to about 30%, when exposed to He-Ne laser irradiation for 5 min. A
Table 10.2. Effect of two irradiation methods on the growtha of bean seeds
Without Water With Water
Treatment time (min) Day 14 Day 21 Day 14 Day 21

He-Ne-laser irradiationb
Control 3.8 4.4 — —

5 3.8 5.8 3.90 4.6
10 4.6 6.2 2.60 4.5
15 5.1 5.9 5.05 5.1
Microwave irradiationc
Control 0.097 0.106 — —

10 0.129 0.136 0.147 0.159
20 0.157 0.161 0.172 0.192
35 0.176 0.197 0.194 0.213
a
Fresh weight (g) of roots
b
Bean seed cv. Plovdiv 564 [Svetleva and Aladjadjian, 1996]
c
Bean seed cv. Dobrudjanski [Aladjadjian and Svetleva, 1997]
more pronounced effect was noted using microwave irradiation on dry bean seed cv.
Dobrudjanski. An increase of up to 80% was achieved with a wavelength of 12 cm
on dry bean seed cv. Dobrudjanski. Similar increases in germ length were also found
in both studies [Svetleva and Aladjadjian, 1996, Aladjadjian and Svetleva, 1997].
Soaking of bean seeds in water prior to treatment seemed to also enhance growth.
However, exact comparison of the two types of treatments is not possible because
they had been carried out on different dry bean varieties.
Finally, the effect of ultrasound treatment on carrot seed vitality indices are pre-
sented in table 10.3. Vitality indices for carrot seed cv. Nantes were highest for samples
exposed for 5 min [Aladjadjiyan, 2002a]. Fresh weight showed a 22% increase.
Table 10.3. Effect of ultrasound treatment on carrot seeds, cv. Nantes
Exposure time (min) Germination (%) Germinative energy (%) Fresh weight (g)
0 76.3 65.3 67.6
1 75.3 65.6 68.0
5 89.3 79.6 82.6
10 83.0 68.6 73.6
a
Data from Aladjadjiyan [2002a].
10 Alternative solutions for the treatment of food produce 73
10.4 Conclusion
The use of chemical additives, through the take of chemical compounds by the plant,
improve plant growth. When alternative methods are use, improved growth of plants
can be explained using an energetic basis. Different forms of energy may be trans-
formed and absorbed by different molecules and then used for accelerating seed
metabolism. The risk of food safety hazards in food produce can be reduced by
the replacement of chemical soil additives and fertilizers with alternative treatment
methods described. They can improve the quality of food produce, achieve higher
productivity and at the same time reduce the risk of contamination from soil and
water.

1. How does food become contaminated with nitrates and nitrites?
2. What are the health risks associated from nitrate and nitrite intoxication?
3. What are the dangers of using chemical amelioration methods for production of
food produce? Is there adequate information available to the general public on
its use?
4. What role does national and European legislation play in controlling chemical
toxins in food produce? Discuss their relevance and how they are applied in
practice?
5. Discuss the different alternative treatment methods for stimulating plant growth.
What are the advantages and disadvantages of using each approach? What other
ways and methods can be applied for stimulating plant growth?
6. Do you think that sufficient and adequate measures have been taken by gov-
ernmental agencies (i.e, from both a national and European perspective) in the
prevention of food safety hazards?
7. What was the objective of the experiments? Discuss the importance of the pa-
rameters analyzed and the experimental factors examined. Were the experiments
adequate (i.e., experimental design and selection of experimental factors and lev-
els) in answering the main purpose of the study? What was the purpose of the
“control”?
8. Discuss and present alternative methods to represent the data. Explain why you
chose these methods. Which methods give an adequate representation of the data?
9. Design and propose an experimental design and statistical analysis for comparing
all the treatment methods in a single experiment. What important factors should
you consider that will affect your analysis of the results?
10. What hypothesis was proposed to explain the effect of alternative treatment meth-
ods for stimulating plant growth? Can you propose another hypothesis?
References
A. Aladjadjian and D. Svetleva. Influence of magnetron irradiation on common bean

(Phaseolus vulgaris L.) seeds. Bulgarian Journal of Agricultural Science, 3:741–
747, 1997.
A. Aladjadjiyan. Increasing carrot seeds (Daucus carota l.), cv. Nantes, viability
through ultrasound treatment. Bulgarian Journal of Agricultural Science, 8:469–
472, 2002a.
A. Aladjadjiyan. Study of the influence of magnetic field on some biological charac-
teristics of Zea mais. Journal of Central European Agriculture, 3(2):89–94, 2002b.
A Aladjadjiyan. Use of physical factors as an alternative to chemical amelioration.
Journal of Environmental Protection and Ecology (JEPE), 4(1):662–667, 2003.
Anon. Guidelines for on-farm food safety for fresh production. Australian Gov-
ernment. Department of Agriculture, Fisheries and Forestry, 2004a. URL http:
//www.affa.gov.au. http://www.daff.gov.au/corporate_docs/publications/
pdf/food/nfis/guidel%ines_onfarm_food_safety_fresh_produce_2004.pdf.
Anon. Regulation(BG) No 31/ 2003 relating to maximum admissable level of pes-
ticides in food. Bulgarian Government. State Gazette No. 14, 2004b. URL
http://www.mzgar.government.bg. http://www.mzgar.government.bg.
Anon. Pollution caused by nitrates from agricultural sources. http://europa.eu.
int/scadplus/leg/en/lvb/l28013.htm, 2005.
R Lutynski, M. Steczek-Wojdyla, Z. Wojdyla, and S. Kroch. The concentrations of
nitrates and nitrites in food products and environment and the occurrence of acute
toxic methaemoglobinemia. Przegl Lek, 53(4):351–355, 1996.
OJEC. Council Directive 91/676/EEC of 12 December 1991 concern-
ing the protection of waters against pollution caused by nitrates from
agricultural sources. Official Journal of the European Union, (L375),
1991. URL http://europa.eu.int/eur-lex/lex/LexUriServ/LexUriServ.
do?uri=CELEX:319%91L0676:EN:HTML. http://europa.eu.int/eur-lex/lex/
LexUriServ/LexUriServ.do?uri=CELEX:319%91L0676:EN:HTML.
OJEC. Regulation (EC) No 2003/2003 of the European Parliament and of the Council
of 13 October 2003 relating to fertilisers. Official Journal of the European Union, 46
(L304):1–194, 2003. URL http://europa.eu.int/eur-lex/en/archive/2003/
l\_30420031121en.html. http://europa.eu.int/eur-lex/en/archive/2003/
l_30420031121en.html.
D. Svetleva and A. Aladjadjian. Effect of helium - neon laser irradiation of dry bean
seeds. Bulgarian Journal of Agricultural Science, 2(5):587–593, 1996.
11
Heavy metals in Organic milk
Jelena Zagorska1, Inga Ciprovic̆a1,2, and Daina Kārklina1

1
Department of Food Technology, Latvia University of Agriculture, 2 Lielā street, Jelgava,
LV3001, Latvia.
2
[email protected].
Summary
Organic agriculture addresses the public demand to reduce environmental pollution

of agricultural production. These methods are used to minimize pollution of air, soil
and water, although they cannot ensure that products are completely free of residues,
because of general environmental pollution [FAO, 2000]. Most heavy metals do not
undergo biological or chemical degradation and may remain in the soil for a long
time after their introduction. The content of heavy and trace metals in milk obtained
by conventional and organic agricultural methods are examined
Key words: organic milk; heavy metals; trace elements; chemical pollution

(1) Define organic agriculture as a sustainable agricultural system and discuss its
importance for human nutrition.
(2) Analyze and contrast the differences in levels of heavy metals and trace elements
found in organic and non-organic milk.
(3) Identify and assess the importance of setting maximum permitted levels in food.
(4) Evaluate the data and examine the need for statistical methods to support the
analysis of the problem.
(5) Propose and argue the necessity of reducing heavy metals in milk.
11.2 Introduction
Organic agriculture is a production management system that aims to promote and
enhance the ecosystem, including biological cycles and soil biological activity, and
is seem as an alternative production method for reducing chemical pollution of the
76 Jelena Zagorska, Inga Ciprovic̆a, and Daina Kārklina
environment. It is based on minimizing the use of external inputs (e.g., pesticides) and
represents a deliberate attempt to make the best use of local natural resources. Meth-
ods are used to minimize the pollution of air, soil and water, however they cannot
ensure that agricultural products are completely free of residues, because of gen-
eral environmental pollution [FAO, 2000]. Apart from pesticide residues, there are
several other chemical hazards associated with foods that originate from general envi-
ronmental pollution. Contaminants can include agricultural and industrial chemicals,
heavy metals and radioactive nuclides. EC regulations (i.e., OJEC [1999]) require
livestock to be fed on organically produced feedstuffs, if they are to be considered
to be produced organically. Even though contamination with pesticide residues and
other agricultural chemical is greatly reduced by organic farming methods, heavy
metals may still pose a problem.
11.3 Heavy metals and trace elements

"Heavy Metals" is a quasi-scientific term, used to describe a group of toxic metallic
elements and their compounds, of which a few are known to travel long distances
through the atmosphere via the grasshopper effect [Arora et al., 2003]. Lead, mercury
and cadmium are heavy metal pollutants that can accumulate, through different human
activities, to levels that are considered toxic to our health and the environment. Under
certain circumstances, exposure to high levels of these metals in the environment has
been linked to adverse effects on human health or wildlife (e.g.subtle neurobehavioral
effects from lead, chronic kidney damage from cadmium, sensory or neurological
impairments from mercury).
Cadmium is regarded as the most serious contaminant of the modern age. It is
absorbed by many plants and, because of its toxicity, is a major problem in foodstuffs.
Contamination through fertilizers is becoming and increasing problem. Cadmium is
similar to lead, as it is a cumulative poison and the danger lies in regular consumption
of foodstuffs that contain cadmium at low levels of contamination . However, in
contrast to lead, the definition of an exact toxicity limit in not possible for cadmium.
The decisive point is whether absorption of the existing cadmium actually takes place.
This is, firstly, dependent upon the composition of the diet as a whole and, secondly,
on the bio-availability of the cadmium compound present [Palmer and Moy, 1991].
Some heavy metals, such as copper, zinc, iron, are essential at very low concen-
trations for the survival of all forms of life. They are considered to be essential trace
elements, but can be toxic when present in large quantities. Daily intake levels of
essential elements are normally significantly lower that the recommended desirable
levels of 3-5 mg/kg and 0.5-1 mg/kg for zinc and copper respectively. A copper in-
take of 0.1-0.2 mg/kg body weight has been found to cause digestive disturbances in
sensitive consumers. Iron is another essential trace element found in milk, of which
the essential content of iron in milk should not exceed 0.5 mg/kg [Harding, 1995].
11 Heavy metals in Organic milk 77
11.4 Experimental
Milk from conventional and organic production systems were obtained to compare the
environmental impact of these systems on the safety of milk. A total of nine organic
bulk milk and nine conventional bulk milk samples were collected from different
regions of Latvia to determine the content of heavy and trace metals. Lead, cadmium,
iron, copper and zinc were determined by atomic absorption spectrophotometry. The
content of heavy metals in organic and conventional milk samples from Latvia is
shown in Table 11.1. The mean for each heavy metal was calculated and compared
with the acceptable or regulated maximum levels. All heavy metals were below legally
accepted upper limits for both organic and non-organic milk, except for lead.
Table 11.1. Levels of heavy metals found in organic and non-organic milk in Latvia (mg/kg
w.w)
Organic Non-organic
Metals Limits Max Min Meana ±SD Max Min Meana ±SD
Lead 0.02b 0.038 0 0.024±0.014 0.035 0.025 0.031±0.004
Cadmium 0.03c 0.013 0 0.006±0.002 0.012 0.005 0.007±0.002
Copper 1.00c 0.258 0.194 0.230±0.020 0.356 0.240 0.290±0.050
Iron — 2.690 1.220 1.590±0.620 1.180 1.149 1.330±0.120
Zinc — 4.850 3.560 4.020±0.540 4.660 3.690 3.940±0.410
a
Mean values with a ±standard deviations for 5 replicates.
b
Maximum permitted levels permitted based on EC Regulation 466/2001 [OJEC, 2002].
c
Maximum permitted levels [Anon, 1999].
Mean levels for lead in organic and conventional samples ranged between 0.024
and 0.031 mg/kg wet weight, which exceeded permissible levels. Lead contamination
in organic and conventional milk samples could have been resulted from feeding cows
with fodder collected from along the sides of roads. It suggests that farmers should
consider better ways to organize the growing of grass and grains for feed, so as to avoid
chemical pollution from roads. The content of cadmium in organic and conventional
milk samples was very low and fairly constant for all types of milk, which was
probably due to the absence of industrial processes (e.g., metal melting and refining,
coal and oil-fired power stations) in Latvia.
11.5 Conclusion
Milk and milk products are an important part of the diet in Latvia. Consumers demand
high quality and safe milk products that are produced with minimal environmental
pollution, under optimal conditions for animal welfare and health. Food free from
residues (i.e., heavy metals) are regarded by the consumer as the norm and not the
exception. However, environmental contaminants are not the primary risk factors.
78 Jelena Zagorska, Inga Ciprovic̆a, and Daina Kārklina
Unfavorable habits, insufficient hygiene and natural toxic substances may be more
important.
1. Why are organic agricultural products important for human nutrition?

2. Why is it important to determine heavy metals in food?
3. What are the differences between heavy metals and trace elements?
4. Were there any differences in the levels of heavy metals and trace elements be-
tween organic and conventional milk? Discuss the statistical methods you would
use to determine if differences exist. Select an appropriate method to test this
hypothesis. What can you conclude from the analysis you have conducted?
5. What is the significance of the term “maximum permitted level”? How is this
determined? Why is it important that these levels are not exceeded?
6. Are the levels for each heavy metal and trace element analyzed in the samples of
organic and non-organic milk higher than maximum permitted levels? How can
you test your conclusion?
7. Why were maximum permitted levels for Iron and zinc not reported? Are there
any legislation regarding maximum permitted levels of these compounds in milk
and milk products?
8. From your analysis of the data presented, what suggestions can you make to
consumers from Latvia regarding the consumption of organic and non-organic
milk?
9. Suggest steps and procedures that farmers can undertake to minimize high levels
of heavy metals in raw milk.
References
Anon. The rules of Cabinet Ministers No 292/1999 of 20 August 1999 on the re-
quirements for food contamination. http://www.likumi.lv/doc.php?id=18618,
1999.
B. Arora, N. Chan, D. Choy, J. Eng, M. Ghods, P. Gutierrez, G. Kemp, A. Reyes,
H. Schneider, and C. Villamayor. Amount and leaching potential of heavy metals
in bark mulch and compost used on the university of british columbia grounds.
http://www.sustain.ubc.ca/pdfs/seedreport04/barkmulch.pdf, 2003.
FAO. Food safety and quality as affected by organic farming. Twenty Second FAO re-
gional conference for Europe. Porto, Portugal, 24-28 July 2000. Food and Agricul-
ture Organization of the United Nations, 2000. URL http://www.fao.org/docrep/
meeting/X4983e.htm. http://www.fao.org/docrep/meeting/X4983e.htm.
F. Harding, editor. Milk Quality. Blackie Academic & Professional, London, 1995.
OJEC. Council Regulation (EC) No 1804/1999 of 19 July 1999 supplementing
Regulation (EEC) No 2092/91 on organic production of agricultural products
11 Heavy metals in Organic milk 79
and indications referring thereto on agricultural products and foodstuffs to in-

clude livestock production. Official Journal of the European Union, 42(L222):
1–28, 1999. http://europa.eu.int/eur-lex/pri/en/oj/dat/1999/l_222/l_
22219990824en00%010028.pdf.
OJEC. Commission Regulation (EC) No 257/2002 of 12 February 2002 amend-
ing Regulation (EC) No 194/97 setting maximum levels for certain contami-
nants in foodstuffs and Regulation (EC) No 466/2001 setting maximum lev-
els for certain contaminants in foodstuffs. Official Journal of the Euro-
pean Union, 45(L041):12–15, 2002. URL http://europa.eu.int/eur-lex/en/
archive/2003/l\_30420031121en.html. http://europa.eu.int/eur-lex/lex/
JOHtml.do?uri=OJ:L:2002:041:SOM:EN:HTM%L.
S. Palmer and G. Moy. Environmental pollution, food contamination and public
health. European Journal of Clinical Nutrition, 45:144–146, 1991.
12
Aflatoxins in farmed fish in Estonia
Risto Tanner1 and Erge Tedersoo2,3

1
National Institute of Chemical Physics and Biophysics, Estonia.
2
Department of Food Processing, Tallinn University of Technology, Estonia.
3
[email protected]
Summary
Early in the 1980s, researchers from the Baltic Fishery Research Institute and Institute
of Experimental and Clinical Medicine investigated liver disorders in fish farmed in
Estonia. This case study examines some of their findings.
Key words: fish feed; rainbow trout; molds; aflatoxins

(1) Define and describe toxicological properties of Aspergillus flavus in relation to
animals and human disease.
(2) Identify and choose valuable information needed for solving problems.
(3) Discuss and manage aspects of scientific investigation.
(4) Identify and propose control parameters that prevent growth and toxin production
by Aspergillus flavus in foods and feeds.
12.2 Introduction
There are an estimated 800 species of fungi (i.e., yeasts and molds), some of which
produce metabolites known as mycotoxins and are known carcinogens (i.e., capable
of causing malignant tumours). Among the mycotoxins, aflatoxins are produced by
molds, such as Aspergillus flavus and Aspergillus parasiticus. Four main aflatoxins,
B1 , B2 , G1 and G2 , have been identified whose main sources are contaminated feed
and food. Aflatoxin B1 is considered one of the most toxic. Alfatoxins are known
to cause acute liver damage and cancer. It is important to note that mycotoxins can
remain in food long after the organism that produced them has died, thus they can
present in food that is not visibly moldy. Moreover, most mycotoxins are relatively
stable substances that survive the usual methods of processing and cooking.
82 Risto Tanner and Erge Tedersoo
The best way to control aflatoxins is to control their production, by ensuring

that adequate controls of food and feed quality are in place, during growth, harvest,
transportation, processing and storage. The most important means of controlling mold
growth and subsequently the production of aflatoxin is preventing damage to crops
during harvest and reducing post harvest moisture levels below those required for
fungal growth (i.e., moisture levels below 18.5% in cereal grains and below 9% in
oilseeds) Aflatoxins can be inactivated using organic acids and ultraviolet irradiation
can be used to reduce their toxicity
12.3 Events
An unknown disease swept Estonian fish breeding farms early in the 1980s. Farmed
fish, mainly rainbow trout, were discovered with various forms of liver disorders,
cancer and malformation. The case was investigated by the scientists of the Tallinn
department of the Baltic Fishery Research Institute in collaboration with researchers
from the Institute of Experimental and Clinical Medicine. The frequency of the oc-
currence of cancer in different fish breeding farms was very different, ranging from
only a few percent to 50-60%. In some cases, fish were found with livers weighing
approximately 3 times more than the rest of fish. Histological investigations indi-
cated even higher frequency of liver disorders (i.e., up to 80% of fishes that had been
investigated in some fish farms).
Fish feed was considered as a possible source of the aflatoxins, however no signif-
icant quantities of these carcinogenic compounds were found. In most farms, paste-
feed that contained ground small fresh fish with added feed yeast, wheat flour and a
vitamin premix was mainly used. There were no known differences in the composition
of fish feed used in fish farms. Extensive comparative research of cancer frequency
in different fish farms were conducted (table 12.1 ), which indicated a possible cor-
relation with fish incubators where young fish were bought. In Estonia, rainbow trout
used to be sold when they reach 3 years old. Most farms did not incubate fish roe
themselves, but bought 1-year-old fish for growing in fish farms.
Table 12.1. Frequency of of liver cancer found in fish from different fish farms in Estonia
Fish farm Number Frequency (%)

Carnikavaa 10 50
Carnikavaa 19 47
Roosna-Alliku 20 25
Roosna-Alliku 44 41
Põlula 27 4
Põlula 32 6
Põlula 17 6
Pidula 10 22
Väike-Maarja 10 60
a
Farms in Latvia
12 Aflatoxins in farmed fish in Estonia 83
A closer examination of the situation indicated that baby fish from incubator farms
which had recently been started on a new feed, originating from Latvia, had the highest
frequency of cancer. The analysis of this starter feed showed aflatoxins in several
batches of the feed (table 12.2 ). Separate analyses of feed components indicated that
the aflatoxins originated from feed, containing silkworm cocoons bought from silk
factories in the Fergana Valley of Uzbekistan. Silkworm cocoons are used as a feed
component due to their high nutritive value, particularly for trout, but in this case
the raw feed was contaminated and contained 492 µg/kg of aflatoxin B1 in the most
contaminated batch.
The dry desert climate of Middle Asia is not normally favorable for the growth of
molds such as Aspergillus flavus. However, in autumn a small amount of molds can
be observed on cotton seeds. Silkworm cocoons are normally killed with hot steam
in the silk factories, silk is removed and the mass of wet cocoons are stored under
open air conditions on factory premises, normally permitting rapid drying in a low
humidity climate. However, mold can quickly multiply under a warm artificial humid
environment before the completion of the drying process. Prior to being used as a
feed component for trout, the cocoons had been used to enrich pig feed due to its
high value proteins and lipids. Its percentage never exceeding 5%. No complaints
regarding toxicity were ever recorded. There were probably three main reasons why
trout, and not pigs, were affected:
(1) percentage of cocoon meal present in the feed (i.e., up to 50%);

(2) age of the trout (i.e., 3-year-old fish in comparison to nine- to twelve-month-old
pigs);
(3) rainbow trout as a species are more sensitive to the influence of aflatoxins in
comparison to the majority of other warm-blooded animals.
Table 12.2. Content of aflatoxin B1 in trout feed (µg/kg)
Fish feed Total Contenta

Trout feed A 6 15
Trout feed B 9 6
Silkworm cocoons A 2 492
Silkworm cocoons B 20 50
a
Determined by HPLC.
12.4 Conclusion
There was shortage of raw material with a high protein content for feed at the time in
the USSR. The silk industry was only concerned with the sale of cocoons and factories
were not interested in additional investment to ensure effective drying of cocoons.
However, trout farmers had to give up one of their primary sources of fish feed. It
84 Risto Tanner and Erge Tedersoo
is worth noting that at the time, disclosure of data about food and environmental
contamination was prohibited in the Soviet Union and publishing of research results
was under strict control of the Communist Party. The researcher who had discovered
the source of aflatoxins was dismissed from the position of head of the laboratory. The
editor of a professional journal on fish farming in the Soviet Union, who had accepted
the article for publication, died of a heart attack. The article was never published and
the case was subsequently closed.

1. What are the most common sources of aflatoxin?
2. What factors control toxin production produced by Aspergillus flavus in food and
feed?
3. What are the effects of exposure to different types of aflatoxins in humans and
animals?
4. If you were the researcher called to examine this incident, describe how you
confirmed the cause of the liver disorders in the trout, using the information
available. Would you have done anything different? Justify your decision. What
would your hypothesis have been? How you would have confirmed the presence
of aflatoxins?
5. Is there sufficient evidence presented in table 1 and 2 to suggest the cause of liver
disorders in the trout? What additional information, if any, would you need to
confirm your conclusions? How would you go about doing this?
6. What other analytical methods and procedures can be used for the detection of
aflatoxins? Discuss the advantages and disadvantages of each method. Which of
these methods and procedures, if any, would you have used instead or in addition
to what was used? Justify your decision.
7. What kind of control measures could have been used to minimize the presence
and growth of Aspergillus flavus in the fish feed? Why would you use them and
how would you implement them?
8. What recommendations would you suggest to the producers of fish feed? Why?
References
S. P. Bogovski and B. L. Sergejev. Determination of aflatoxins in feeds of rain-
bow trout. In Fish cancers, pages 48–51. Institute of Experimental and Clinical
Medicine, Tallinn, 1983.
R. H. Tanner, M. M. Iling, V. V. Kadakas, and B. L. Sergejev. HLPC analysis of
aflatoxins in connection with growing frequency of appearance of trout hepatomas
in fish farms of Estonia. In Fish cancers, pages 52–57. Institute of Experimental
and Clinical Medicine, Tallinn, 1983.
13
Mycotoxins in Cereal Products from Romania
Mihaela Avram1,3, Mona Elena Popa1,4 , Petru Nicultita1 , and Nastasia Belc2
1
University of Agronomic Sciences and Veterinary Medicine, Marasti, no. 59, Bucharest,
Romania.
2
Institute of Food Bioresources, Dinu Vintila, no. 6, Bucharest, Romania.
3
[email protected]
4
[email protected]
Summary
Mycotoxins are important compounds of secondary metabolism from molds, which

can cause detrimental effects on animal and vegetal life. This case study is based on
research in Romania on the assessment of different mycotoxin found in cereal based
products. Concentrations of aflatoxins, ochratoxin A and deoxynivalenol, in cereal
based products are examined. Samples of wheat, mostly from regions in southern
Romania, contained levels of mycotoxins ranging between 0.1 and 4.4 ppb for afla-
toxins. Ochratoxin A was present at between 0.7 and 4.1 ppm and deoxynivalenol
between 0.1 and 0.5 ppm. Levels for deoxynivalenol in corn were between 0.1 and
0.6 ppm. The study examined the correlation between mycotoxins levels and mold
count.
Key words: ochratoxin A; deoxynivalenol; molds; cereal based products

(1) Define mycotoxins and indicate the relationship between mold growth and storage
condition of cereals.
(2) Describe the main effects on human and animal health and identify methods for
the determination of mycotoxin.
(3) Relate regulations regarding maximum permitted levels for mycotoxins.
(4) Assess mycotoxin levels from wheat, corn and derivate products by using ELISA
method.
(5) Examine the correlation between mycotoxin level and mold count.
86 Mihaela Avram, Mona Elena Popa, Petru Nicultita, and Nastasia Belc
13.2 Introduction
Mycotoxins in food are produced by molds, such as Penicillium, Aspergillus and

Fusarium, during preharvest, harvest and storage. The growth of molds in food dur-
ing storage can be influence by a number of factors such as water activity, substrate
aeration and temperature, microbial interactions, mechanical damage and insect infes-
tation. A number of analytical methods (e.g., ELISA, HPLC, GC) has been developed
for the determination of different mycotoxins found in agricultural food products.
Mycotoxins are a potential risk to human and animal health and, as such, their
control should be a major priority for governments and international organizations
that are involved in food safety. Mycotoxins can affect the digestive, respiratory,
circulatory, urinary and reproductive systems. They can cause a range of symptoms,
namely, vascular fragility, hemorrhages, diarrhoea, hepatotoxicity, hepatic necrosis,
pulmonary edema, nephrosis and infertility.
Mycotoxins levels in foods are controlled in many countries by regulatory limits
and laws permitting their presence in certain foods and feed. Table 13.1 shows levels
of mycotoxins in cereal and cereal products permitted in Romania. Romania has
adopted European regulations regarding mycotoxin levels permitted in cereal and
cereal products. Aflatoxins are permitted in cereal and cereal products at levels of 4
ppb, while ochratoxin A is permitted in cereals ar levels of 5 ppb.
Table 13.1. Maximum levels of mycotoxins permitted in cereal and cereal based products in
Romania (ppb)
Product Level
Deoxynivalenol (DON)
Unprocessed cereals, other then durum wheat and barley 1250
Durum wheat, barley 1750
Unprocessed corn —
Cereal flour 750
Bread, bakery product, biscuits, breakfast cereals 500
Dry pasta 750
Baby Food 200
Aflatoxin
Cereal and cereal products 4
Ochratoxin A (OTA)
Cereal 5
Cereal products 3
a
Data from Order of Ministry of Agriculture, Forestry and Rural Development, National Au-
thority for Veterinary and Food Safety, Ministry of Health, National Authority for Consumer
Protection no 1050/97/1145/505/2005.
13 Mycotoxins in Cereal Products from Romania 87
Table 13.2. Mycotoxin level - wheat samples 2002
Variety Aflatoxins (ppb) Ochratoxin A (ppb) DON (ppm) Molds (x104 )

Dropia
Calarasi 0 0 0.1 0.10
Calarasi 1.5 0 0.1 0.10
Ialomita 1.0 0 0.1 0.08
Ialomita 2.6 0 0.1 3.00
Flamura 85
Calarasi 1.2 0.7 0 2.00
Calarasi 0.1 0 0 0.10
Calarasi 2.8 0 0.1 0.10
Ialomita 2.4 4.1 0.1 0.09
Ialomita 1.4 0 0.2 2.00
Fundelea 4
Vaslui 4.4 0 0.1 0.03
a
Data from [Avram et al., 2004b,a]
Variety DON (ppm) Molds (x104 )

Alex
Timis-Lovrin 0.4 0.70
Apullum
Covasna-Catalina 0 0.20
Ariesan
Bistrita 0.3 0.40
Dropia
Ialomita-Fetesti 0.3 23.00
Olt-Bals 0.2 10.00
Fundelea 4
Arges-Stefanesti 0.5 1.00
Lovrin 34
Arad-Vladimirescu 0.2 0.70
Novisad 57
Bihor-Batar 0.1 0.20
Transilvania
Covasna 0 1.00
Covasna-Ilieni 0.1 0.10
Bihor-Cefa 0.3 1.00
a
Data from [Avram et al., 2004a]
13.3 Experimental
Thirty two wheat samples were collected different regions in Romania for the period
of three years, in 2002, 2003 and 2004. Corn and wheat samples and other derived
products were collected from different mills and were used for monitoring the pro-
cessing chain and the effect of technology on mycotoxin content reduction. Corn and
wheat samples were cleaned to remove foreign seeds by sieving and subsequently
milled with a laboratory mill.
13.3.1 Mycotoxin analysis
The mycotoxins, aflatoxins, ochratoxin A and deoxynivalenol (DON), were deter-

mined using a range of ELISA test kits. Test kits from R-Biopharm used for mycotoxin
analysis were Ridascreen Fast Aflatoxins Total, Ridascreen Fast Ochratoxin A,
Ridascreen Fast DON. Five g of ground sample was extracted with 25 ml of 70
% methanol for aflatoxins, 12.5 ml of 70 % methanol for ochratoxin A, or 100 ml
distilled of water for deoxynivalenol Samples were shaken for 3 min before being
filtered through Whatman no. 1 filter. All filtered extracts were diluted, except for
deoxynivalenol, and 50 µl was used per well for each test. Enzyme conjugate and
anti-mycotoxins antibodies were added and allowed to react for 3 to 5 min, before
washing to remove any unbound enzyme conjugate. A solution is subsequently added
to stop the reaction, resulting in a color change that is measured at 450 nm with a
microstrip reader.
Variety DON (ppm) Molds (x104 )

Alex
Teleorman - Bogdan, Balaci, Poecu 0 0.01
Ialomita - Tandarei 0 0.04
Dropia
Calarasi-Independenta 0 0.02
Teleorman - Piatra, Furculesti, Crangu 0.1 —
Calarasi - Dorobantu - Dropia 0 —
Flamura 85
Teleorman - Traian, Bogdan, Plosca 0 0.06
Calarasi - Sarulesti 0.1 0.01
Ialomita - Urziceni 0.2 0.20
Ialomita - Fetesti 0 0.14
Fundelea 4
Dambovita - Petrescu 0 —
Romulus
Ialomita - Slobozia 0 0.50
a
13.3.2 Microbiological tests
Wheat, corn and their derivate products were tested for number of molds placing 1 ml
of inoculum, obtained from diluted sample, into petri dishes and adding sterile agar
13 Mycotoxins in Cereal Products from Romania 89
Table 13.5. Mycotoxin level in wheat and derivate products
Bran Flour 550 Flour 650 Semolina Wheat
DON Moulds DON Moulds DON Moulds DON Moulds DON Moulds
0.2 0.26 0 0.40 0 1.20 0.2 1.60 0 0
0 150.00 0.1 0.27 0 0.43 0.1 0.06 0 1.00
a
Table 13.6. Mycotoxin level in corn and derivate products
Corn Corn flour Embrio
DON Molds DON Molds DON Molds

0 45 0.6 12 0 —
0.4 40 0 — 0 —
0.2 0.1 — — — —
0 0.6 — — — —
0 0.4 — — — —
0.5 — — — — —
0.1 — — — — —
0.1 — — — — —
a
media and then incubate at 25 C for 5 days. After incubation colonies of molds were
counted, taking into account the characteristics of colonies.
1. What are mycotoxins? Why are they considered to be food contaminants?

2. Why it is important to evaluate mycotoxins in cereals?
3. What factors control the growth of molds in cereals and other products, such as
peanuts, almonds, spices, and cotton seeds?
4. What can be done to reduce or avoid mold growth and toxin production in foods?
5. What other methods can be used to determine mycotoxins. How do these methods
compare to the ELISA method in terms of accuracy and repeatability?
6. What should be done with cereals and cereal products that have quantities of
mycotoxins above permitted levels?
7. What was the objective of the study? Was the experimental methods and proce-
dures (e.g. sampling, experimental plan) appropriate?
8. Examine the results presented in the tables. Is there a correlation between my-
cotoxin level and number of moulds? Justify your conclusions. What other rela-
tionships can be found in the results?
9. Taking into account the variability of results for the same lot, what follow up
experiments would be needed to confirm the results obtained? What would be
the hypothesis and experimental procedures and methods for these experiments?
References
M. Avram, N. Belc, and V. Gagiu. Incidence of mycotoxin don in wheat, corn and
derivated products. In Symposium works-scientific progress in food industry, pages
45–49, 2004a.
M. Avram, V. Gagiu, N. Belc, M. Popa, and P. Niculita. Romanian mycotoxins
assessment as quality crop indicators. In Food Micro 2004 New tools for improving
microbial food safety and quality, page 402, 2004b. Book of abstracts.
CAST. Mycotoxins: Risk in plant, animal, and human system. Number 139 in Task
Force Report. Council for agricultural science and Technology, 2003.
FAO/WHO. Safety Evaluation of Certain Mycotoxins in Food. Fifty-sixth meeting of
the Joint FAO/ WHO Expert Committee on Food Additives (JECFA). Number 47
in WHO Food Additives Series. World Health Organisation, 2001.
J. C. Larsen, J. Hunt, I. Perrin, and P. Ruckenbauer. Workshop on trichotecenes with
a focus on DON: summary report. Toxicology Letters, 153:1–22, 2004.

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Preface

Case Studies in Food Safety and Environmental health is intended as a reference

Part I Speciﬁc Issues

1 Acrylamide and Human Health

Part II Historical perspectives

6 The Toxic Oil Syndrome in Spain

Part III Research-based

10 Alternative solutions for the treatment of food produce

1.1 Objectives and learning outcomes

1.3 Human health

1.4 Risk Communication

1.6 Discussion Questions

H. Simonne and L. Archer. Acrylamide in Foods: A review and Update. University

Carmen Socaciu1,2 and Andreea Stănilă1

Key words: nitrates; methemoglobinemia; water; environment; Romania

2.1 Objectives and learning outcomes

(1) Deﬁne the recycling of nitrogen-based compounds in the environment, in relation

Norganic −−→ NH3 −−→ NO− −→ NO−

2.2.1 Nitrates in Food

Product Level Product Level

2.4 Addressing the problem

(1) Excessive use of fertilizers;

2.5 Exposure assessment studies and pollution control projects

Since 2000, regional, national and international initiatives, governmental programs

2.5.1 Exposure assessment studies

2.5.2 The Garla Mare Project

2.5.3 The Global Environment Facility (GEF) Project

The Global Environment Facility (GEF) is an independent multilateral ﬁnancial mech-

2.7 Discussion Questions

Antonis Skilourakis1 and Elefteria Psillakis1,2

Key words: pesticides; olive oil; endocrine disrupting compounds

3.1 Objectives and learning outcomes

3.4 Pesticides in Olive Oil

3.6 Discussion Questions

a range of substances suspected of interfering with the hormone systems of hu-

H. M. Roche, M. J. Gibney, A. Kafatos, A. Zampelas, and C. M. Williams. Beneﬁcial

Botulism is caused by neurotoxins produced by Clostridium botulinum during growth

Key words: Clostridium botulinum; botulism; hazelnut yoghurt; garlic in oil

4.1 Objectives and learning outcomes

Botulism is a neuroparalytic disease caused by toxins secreted by a small group

4.3 Hazelnut yoghurt (UK)

4.3.1 Discussion Questions

4.4 Garlic in Oil (Canada & USA)

4.4.1 Discussion Questions

Panagiotis Skandamis1 and Antonia Gounadaki2

Key words: Listeria monocytogenes; listeriosis, Ready-to-Eat foods

5.1 Objectives and learning outcomes

5.3 Ready-To-Eat foods

5.4 Listeriosis in Greece

5.4.1 RTE Products

5.4.2 Meat products

5.5 Prevention and Control

5.7 Discussion Questions

R. B. Tompkin. Control of listeria monocytogenes in the food-processing environ-

6.1 Objectives and learning outcomes

6.3 Sequence of events

organophosphates, namely, Fenamiphos1. Fenamiphosis produced by Bayer and sold

6.4 Analysis of the Problem

6.4.1 Probable hypotheses

6.4.2 Studies on the toxic oil