BANAT’S UNIVERSITY OF AGRICULTURAL SCIENCES
AND VETERINARY MEDICINE
TIMIŞOARA
FACULTATY OF VETERINARY MEDICINE
DOCTORAL THESIS
EVALUATION OF CONTAMINATION LEVELS WITH OCHRATOXIN A
AND AFLATOXINS OF FEEDSTUFFS AND FOODSTUFFS IN THE WEST
PART OF ROMANIA
Ing. chim. Oprea Lăcrămioara Aurora Ruxanda
(căs. Damiescu)
SCIENTIFIC COORDINATION,
PROF. Dr. dr. h.c. ALEXANDRA TRIF
TIMIŞOARA 2010
ABSTRACT
Key words: ochratoxin A, aflatoxin B1, aflatoxin M1, feedstuffs, foodstuffs, imunoenzymatic test,
liquid cromatography coupled with mass spectrometry
The doctoral thesis contains 178 pages, 113 tables, 101 graphics, 36 figures, 2 anexes. It is
structered in two principal parts: Bibliographic study and Own research.
PART I
BIBLIOGRAPHICAL BACKGROUND
Bibliographic synthesis is composed of five chapters and it is extended on 42 pages, 205
bibliographical titles.
Chapter 1, INTRODUCTION, contains a presentation of the historical knowledge about
mycotoxinic contamination and the risk for human consumer regarding the presence of mycotoxins in
foodstuffs.
Chapter 2, TOXIGENIC FUNGI, contains three subchapters :
Subchapter 2.1. General considerations contains general considerations about fungi, are presented
the characteristics of fungi (framing, structure, reproduction and types of fungi with the classification in four
major divisions :Zigomicetes, Ascomicetes, Basidiomicetes and Deuteromicetes, structural specifications
and reproduction, main representatives).
Subchapter 2.2 Factors that influence the development of fungi and the elaboration of mycotoxins
contains the presentations of factors that influence the development of fungi and the elaboration of
mycotoxins (substrate / chemical composition of the substrate, pH value, water activity factor, relative
umidity of air, temperature, aeration, luminosity, biological agents, competitor micro-organisms, genetic
capability, period of time from contamination, agrotechnical achievments, agrotechnical and plant protection
works), with specification of their contribution to fungi development and the elaboration of mycotoxins.
Subchapter 2.3. The main fungi producers of aflatoxins and ochratoxins contains the
presentation of the main fungi producers of aflatoxins and ochratoxins, Aspergillus and Penicillium spp
(taxonomic clasification, preferential substrates, general cultural characteristics, mycroscopical general
features) and the species that produce aflatoxins and ochratoxins, A. flavus, A. parasiticus, A. fumigatus,
A.niger, A. nidulans, A. ochraceus, A. versicolor, P. verrucosum sin. viridicatum, (cultural characteristics,
mycroscopic characteristics).
Chapter 3, AFLATOXINS, contains six subchapters:
Subchapter 3.1. : description of the fungi producers of aflatoxins;
Subchapter 3.2.: presentation of favorite substrates for aflatoxin’s elaboration;
Subchapter 3.3.: physical-chemical properties of aflatoxins;
Subchapter 3.4.: biosynthesis of aflatoxins;
Subchapter 3.5.: toxicity contains two parts:
Subchapter 3.5.1. : metabolism of aflatoxins, with specification of the importance to acknowledge
and to understand it in order to understand the toxicity and the carcinogenic effects of aflatoxins, with
explanations of the metabolic pathways, of correlation between metabolites and toxicity, metabolism –
mutagenic effects, carcinogenic;
Subchapter 3.5.2 :lethal doses for different species;
Subchapter 3.6 : effects regarding health and productivity starting from biochemical lesions,
immunotoxic effects: suppression of median cellular immunity, and humoral immunity, haematotoxic
effects, hepatotoxic – liver is the main target, carcinogenic - hepatic, renal, intestinal, pulmonary,
reproductive effects
Chapter 4, OCHRATOXINELE contains six subchapters:
Subchapter 4.1. description of the fungi producers of ochratoxins;
Subchapter 4.2. : presentation of favorite substrates for ochratoxin’s elaboration;
Subchapter 4.3.: physical-chemical properties of ochratoxin A;
Subchapter 4.4 - biosynthesis of ochratoxins;
Subchapter 4.5 Toxicity contains two parts:
Subchapter 4.5.1. - metabolism of ochratoxins, presentation of pathways and premises for
detoxification - duodenum, ileum, pancreas ability for superior hydrolysis over liver and kidneys, increased
ability for detoxification through hydrolysis of ruminants;
Subchapter 4.5.2 - lethal doses for different species;
Subchapter 4.6.: effects regarding health and productivity starting from biochemical lesions,
immunotoxic effects: immunosuppression - affectation of immunocompetent organs, neurotoxic effects
with hippocampus and cerebellum affectation, sever nephrotoxic effects, hepatotoxic effects less important
as the nephrotoxic effects, carcinogenic effects renal, hepatic, reproductive effects: affectation of
spermatogenesis, levels of testosterone, embryo toxic and teratogenic effects –bones and visceral anomalies,
other effects: weight loss, reduction of eggs production, milk production
Chapter 5, MYCOTOXINS IN FOODSTUFFS,
Subchapter 5.1. : presence of aflatoxins and ochratoxin A in foodstuffs in different parts of the
world and the risk of different nutritive products to the human consumer:
Subchapter 5.2.: influence of mycotoxins concerning the quality of foodstuffs (modification of
nutritive value through reduction of glucide levels, lipolysis, oxidation of lipids, proteins, sulphur depletion,
phosphorus, magnezium and oligoelements depletion
Subchapter 5.3 : maximum permitted levels in Romanian legislation accordingly with European
legislation
PART TWO
PERSONAL RESEARCH
It is extended on 138 pages and contains five chapters, plus the bibliography.
Chapter 6. MOTIVATION, PURPOSE AND RESEARCH OBJECTIVES
6.1. MOTIVATION
 The precariousness of research regarding the prevalence and the aflatoxins and ochratoxins levels in
fodders and food products from the west part of Romania
 The necessity, imposed by high toxicity, to assess the feeds and foodstuffs contamination, with the
purpose of protecting human and animal health
 EFSA’s recomandation of studying the potential increase of contamination with aflatoxin B1 and
ochratoxin A growth on cereals in the EU, as a result of climatic changes, with the purpose to create
predictiv models based on the data analisys, to define scenarios and to create maps, which will
locate the areas in which a potential contamination of cereals is possible.
6.2. PURPOSE
Researches had the purpose of assessing the mycotoxic contamination level of different fodder types
and foodstuffs of animal and non-animal origins in the west part of Romania during the 2004-2009 period.
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6.3. OBJECTIVES:
following the annual aflatoxin B1 (AFB1) and ochratoxin A (OTA) prevalence dynamics in : volume
fodders, silage fodders, raw materials for combined fodders, combined fodders, mineral-vitamin
supplements and protein-mineral-vitamin concentrates ;
estimation of aflatoxin B1 and/or ochratoxin A contamination levels of different fodder types;
prevalence of AFB1 and/or OTA in meat from different species, in pork sausages and in spices used
for pork sausages products;
contamination levels of meat samples from different species with AFB1 and/or OTA, of pork
sausages and of spices used for pork sausages;
AFM1 frequency in raw milk, milk for direct human consumption, powder milk and milk products yoghurt, cheese, butter;
levels of contamination with aflatoxin M1, aflatoxin’s B1 metabolite
AFB1 and/or OTA frequency in foodstuffs of non-animal origin (hazelnuts, pistachio, nuts, dryed
fruits, bier, wine, coffee)
levels of contamination with AFB1 and/or OTA for products of non-animal origin ;
optimization of the method of analysis Varian 394, using the liquid chromatography technique
coupled with mass spectrometry;
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comparative study of the common analitical method of identifying and dosing of the aflatoxin B1 and
ochratoxin A (imunoenzimatic test ELISA) with ultraperformant method - liquid-chromatography
coupled with mass-spectometry (LC-MS/MS)
Chapter 7 MATERIALS AND METHODS
Researches were made in the west part of Romania, in the Arad, Bihor, Caraş-Severin, Mehedinţi,
Satu-Mare, Timiş counties, during the 2004-2009 period, on a significant number of samples: 2174 fodder
samples, 5796 food products of animal origin: 1284 meat samples from different species and pork sausages,
4234 samples of imported meat, 278 samples of poultry meat for export, 371 samples of spices used for pork
sausages, 1106 samples of products of non-animal origin (hazelnuts, pistachio, nuts, dryed fruits, roasted
coffee, ground coffee, bier, wine).
The methods used for analysing the samples are validated and accredited.
Chapter 8 RESULTS, DISCUSIONS AND CONCLUSIONS contains four subchapters:
Subchapter 8.1. :researches regarding the prevalence and levels of aflatoxin B1 and/or ochratoxin A
in different types of fodders (volum fodders - Subcapitolul 8.1.1., silage fodders - Subcapitolul 8.1.2, raw
materials for combined fodders - Subcapitolul 8.1.3, combined fodders - Subcapitolul 8.1.4 , vitamin-mineral
supplements and protein-vitamin-mineral supplements - Subcapitolul 8.1.5.)
Subchapter 8.2. : research regarding the contamination of animal foodstuffs (meat from different
species - Subchapter 8.2.1), pork sausages (Subchapter 8.2.2), spices used for pork sausages (Subchapter
8.2.3)
Subchapter 8.3. : subchapter includes prevalence and aflatoxin M1 level in milk and diary products
(raw milk - Subcapitolul 8.3.1, milk for direct human consumption - Subcapitolul 8.3.2., powder milk Subcapitolul 8.3.3., diary products - Subcapitolul 8.3.4.)
Subchapter 8.4. : subchapter includes researches regarding the contamination of non-animal
foodstuffs (nuts - Subcapitolul 8.4.1, roasted and ground coffee - Subcapitolul 8.4.2)
Subcapitolul 8.5. contains researches concerning the optimisation of the LC-MS/MS method for
determination of mycotoxins and the comparison between this method and the immunoenzimatic method
(optimisation of method - Subcapitolul 8.5.1., ELISA mrthod - Subcapitolul 8.5.2., comparison of methods Subcapitolul 8.5.3.)
Chapter 9. GENERAL CONCLUSIONS
Researches made during the 2004-2009 period, regarding the fodder’s and food product’s
contamination levels with ochratoxin A and aflatoxins, have revealed:
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Prevalence and the level of aflatoxin B1 and/or ochratoxin A in different types of fodders
from the west part of Romania
 the growing dynamic of the contamination prevalence with aflatoxin B1 and/or ochratoxin A
during the 2004-2007 periond and the important decrease registered during 2008-2009 at all
fodder categories;
 sample frequency variation depending on the type of contaminated feed:
aflatoxin B1: volume fodders (30,2%), silage fodders (23,8%), combined fodders
(22,4%), raw materials for combined fodders (21,6%), vitamino-protein supplements
and concentrates (17%);
ochratoxin A: silage fodders (16,2%), volume fodders (14,9%), vitamino-protein
supplements and CPVM (12,4%), raw materials for combined fodders (12,1%),
combined fodders (12,1%);
aflatoxin B1 and ochratoxin A: silage fodders (12,5%) vitamino-protein supplements
and CPVM (11,2%), volume fodders (9,9%), raw materials for combined fodders
(9%), combined fodders (8.9%);
 differences between counties regarding the studied mycotoxins without being able to
establish a hierarchy depending on the type of fodder and mycotoxin;
 differences of the mean level of contamination between different categories of fodders:
aflatoxin B1: volume fodders (26,50 ppb), silage fodders (22,05 ppb), raw materials
for combined fodders (17,19 ppb), combined fodders (4,43 ppb), vitamino-protein
supplements and CPVM (3,50 ppb);
ochratoxin A: raw materials for combined fodders (4,97 ppb), combined fodders
(4,81 ppb), silage fodders (4,63 ppb), volume fodders (4,61 ppb), vitamino-protein
supplements and CPVM (3,04 ppb);
aflatoxin B1 and ochratoxin A, for aflatoxin B1 : volume fodders (15,40 ppb), silage
fodders (10,50 ppb), raw materials for combined fodders (9,59 ppb), combined
fodders (3,44 ppb), vitamino-protein supplements and concentrates (3,36 ppb);
aflatoxin B1 and ochratoxin A, for ochratoxin A: silage fodders (3,58 ppb), raw
materials for combined fodders (3,36 ppb), volume fodders (3,32 ppb), vitaminoprotein supplements and concentrates (3,20 ppb), combined fodders (3,0 ppb).
 exceeding the maximum permited limit by applicable law (non-compliant samples, the
percentage of positive samples)
aflatoxin B1: volume fodders (54,8%), silage fodders (40,4%), raw materials for
combined fodders (30,6%), combined fodders (20,6%), vitamino-protein
supplements and concentrates (0%);
ochratoxin A: volume fodders (55,5%), silage fodders (41%), raw materials for
combined fodders (38,2%), combined fodders (32,3%), vitamino-protein
supplements and concentrates (0%);
absence of non-compliant samples in mixt contaminated samples, unable to make a
difference on type of fodder;
 exceeding precentage variations of the maximum permited limits:
aflatoxin B1: volume fodders (minimum 23%, maximum 47,4%), silage fodders
(minimum 33,3%, maximum 50%), raw materials for combined fodders (minimum
27,3%, maximum 40%), combined fodders (minimum 15,3%, maximum 36,3%),
vitamino-protein supplements and concentrates (0%);
ochratoxin A: volum fodders (minimum 50%, maximum 57,2%), silage fodders
(minimum 33,3%, maximum 42,8%), raw materials for combined fodders
(minimum 36,3%, maximum 42,8%), combined fodders (minimum 22,6%,
maximum 42,1%), vitamino-protein supplements and concentrates (0%);
 insignificant differences between counties regarding the contamination level of fodders with
aflatoxin B1 and/or ochratoxin A;
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Prevalence and the aflatoxins B1 and/or ochratoxins A level in different food products of
animal origin, in the west part of Romania
 Meat and pork sausages
 the absence of aflatoxin B1 and/or ochratoxin A contamination of domestic meat samples
and from import, from different species (beef, pork, mutton, horses, poultry, fish);
 the contamination of pork sausages with aflatoxin B1 (15,9%), ochratoxin A (7%), and
aflatoxin B1 asocciated with ochratoxin A (3,8%);
 low contamination level, with aflatoxin B1 (under 1,5 ppb) and/or ochratoxin A (under 1,3
ppb) of the pork sausages;
 different prevalence of aflatoxin B1 in spices used for pork sausages: paprika (6,6%),
allspice (3,7%), pepper (3,5%), nutmeg (3,1%), ginger (2,8%), coriander (2,4%), thyme and
garlic powder (1,9%);
 exceeding the maximum permited limits for aflatoxin B1 in all the analised spice samples,
the spices hierarchy depending on the exceeding grade of maximum permitted level: paprika
(+110,8%), pepper (+87,8%), thyme (+42,6), allspice (+21,4%), nutmeg (+16%), coriander
(+15,2%), ginger (+14,6%), garlic powder (+14,2%);
 different prevalence of ochratoxin A in spices used for pork sausages: pepper (2,1%),
coriander (1,8%), thyme (1,3%), ginger (0,9%), allspice (0,7%), paprika (0,3%);
 spice’s hierarhy depending on the contamination level with ochratoxin A: thyme, paprika,
pepper, garlic powder, allspice, nutmeg, coriander and ginger (in the absence of any legislative
specifications refering to the maximum permited limit for ochratoxin A in spices, all the samples
were considered non-compliant);
 the different frequency of aflatoxin B1 and ochratoxin A in spices used for the pork sausages,
the percentage of exceeded maximum permited limit of aflatoxin B1 in: thyme (+109%), paprika
(+83,8%), pepper (+56%) and garlic powder (+11%).
 Raw milk and milk products (aflatoxin M1)
 the presence of aflatoxin M1 in 15,3% of raw milk samples;
 26,1% of the raw milk samples are non-compliant, with exceedings of the maximum
permited limit of minimum 80% and maximum 140%;
 15,8% of the samples of milk for direct human use and 17% of powder milk samples are
contaminated, but without exceeding the maximum permited limit;
 absence of contamination in yoghurt, cheese and butter samples
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Prevalence and aflatoxins B1 and/or ochratoxins A level in food products of non-animal origin
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the absence of contamination in hazelnuts, pistachio, fruit juice, dryed fruits, bier, wine
samples;
contamination of 20,8% of the nut samples with aflatoxin B1, but without exceeding the
maximum permited limit;
contamination with ochratoxin A of 7,4% of roasted coffee beans samples and 4,6% of
ground coffee samples, but without exceeding the maximum permited limit.
The researches conducted with the purpose of optimisation of the LC-MS/MS Varian 394
method and for comparison of the optimized LC-MS/MS method with the ELISA method, have
highlighted:
 the optimised LC-MS/MS method, through changing the working parameters (gradient, flow,
dwell time, mobile phases) is superior to the Varian 394 method, having higher recovering
percentages for all the analysed compounds;
 hierarchy of the methods depending on the recovery percentage is optimised LC-MS/MS,
ELISA and LC-MS/MS Varian 394;
 higher sensitivity of the optimised LC-MS/MS method compared to the LC-MS/MS Varian 394
method (the percentage difference of determined mycotoxin values is +37,39%);
 percentage differences similar between the average recovery percentage (-5,51%) and the
average level (-5,44%) of the analytes determined between the ELISA method and the
optimised LC-MS/MS;
 The advantage of the optimised LC-MS/MS method compared to the ELISA method, based on
the possibility of quick, simultaneous determination of more mycotoxins and easier sample
preparation.