LABORATORY TESTING: SAFETY PARAMETERS...- Directive 2002/32/EC - EU Recommendation 2006/576/EC - EU...

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LABORATORY TESTING: SAFETY PARAMETERS

Technical base

Resources

Competence

DECEMBER 2020




Laboratory organization (example)

MAIN DIRECTIONS / Types of analysis

CONTAMINANTS

MOLECULAR GENETICS

QUALITYSYSTEMS

Pesticides, Mycotoxins,

Dioxins/ PCBs,

Heavy metals, etc.

GMO, Allergens, Identification

of type (plant / animal origin)

PROXIMATE CHEMISTRY

Moisture, Protein,

Wet gluten, Oil content, Fiber,

Ash, etc.

MICROBIOLOGY

Food & feed microbiology

PHYTOSANITARY/QUARANTINE

Ento-, Herbo-, Myco-, Bacterio-,

Phytohelmintho- & Virological

expertise's

ANALYTICAL RESEARCH

Research and Development

Department

ISO17025/ILAC accreditation example

NB. Check sphere of accreditation




Laboratory assurance program (example)

* Limit of detection/quantification

QUALITY SYSTEMS

LEVEL 2

ANALYTICAL METHOD VALIDATION

LEVEL 1 LEVEL 4

INTER-LABORATORY

CONTROL

LEVEL 3

INTERNAL QUALITY CONTROL

Unique laboratory number

of the sample on all steps

• Sample registration

• Sample preparation

• Analytical process

• Reporting

Validation parameters

• Linearity

• Selectivity

• LOD/Q*

• Precision

• Accuracy

• Uncertainty

Additional parameters

• System suitability

• Robustness

• Others (on request)

Multiplex control

• Spiked Sample• Calibration curve• Quality Control sample• “Blind samples”• Analysis in doubling• Reagent blank• Solvent calibration vs

Matrix calibration

International and local

proficiency tests

• GAFTA• FOSFA• FAPAS• Bipea• Local providers

TRACKING SYSTEM




Food safety parameters in terms of priority*

# TESTFOOD FEED

ANALYTMETHOD

Testing importance CHROMATOGRAPHY OTHERS

1 Pesticides + +Organochlorine, Organophosphorus,

Pyrethroids, Carbamates, Glyphosate /

Glufosinate, Diquat, Mepiquat etc.

GC/MS, GC/MS/MS,

HPLC/MS/MS-

2 Mycotoxins

+ +

Aflatoxins (B1,B2,G1,G2 and summ) HPLC/RF, LC/MS/MS ELISA

Ochratoxin А HPLC/RF, LC/MS/MS ELISA

Zearalenone HPLC/RF, LC/MS/MS ELISA

DON (+ 3-Ас-DON, 15-Ac-DON) LC/MS/MS ELISA

Т2/НТ2 toxins and summ LC/MS/MS ELISA

Optional Optional DAS LC/MS/MS -

Maize only Fumonisins LC/MS/MS ELISA

3Toxic elements

(“Heavy metals")

+ + Lead (Pb) - ААС / ICP

+ + Cadmium (Cd) - ААС / ICP

Optional + Mercury (Hg) - ААС / ICP

Optional + Arsenic (As) - ААС / ICP

4Sanitary

microbiologyOptional +

Bacteria (Salmonella, E.coli etc.) - Culture medium

Yeast & Molds - Culture medium

5 Dioxins and PCBs Optional Optional PCDD/F & PCBs APGC/MS/MS -

6 PAHs Optional Optional Benzo[a]pyrene and PAH4 HPLC/RF -

Regulations: CODEX, EU Reg 32 (feed); 1881 (food), 396; other* As per IFIA Bulletins 11-01 and 14-01




Chromatography detectors

1. Gas Chromatography (GC):

№ Detectors Tests

1 FID (flame ionization)FAC, Solvent residues, Waxes,

Mineral oils (MOSH/MOAH), Sterols, Phthalates

2 MS or MS/MS (mass-spectrometric) Thermostable pesticides, Dioxins

2. High Performance Liquid Chromatography (HPLC):

№ Detectors Tests

1 SPD (spectrophotometric, UV) Glucosinolates, Antioxidants

2 RF (fluorescent) Mycotoxins (Afla, Ochra, Zeara), BaP, PAHs, tocopheroles

3 MS or MS/MS (mass-spectrometric) Thermo-sensitive and polar pesticides, Mycotoxins




Mycotoxins




MycotoxinsMycotoxins are fungal metabolic products.

- EC Regulation 1881/2006

- Directive 2002/32/EC

- EU Recommendation 2006/576/EC

- EU Recommendation 2013/165/EU

MycotoxinsType of

producer

Cargoes, prone to

affection

Method

(+ LOQ, ppb)

Max allowed

for FOOD by ЕС Reg., ppb

Max allowed

for FEED by ЕС Reg., ppbMax allowed

as per

GMP+, ppbCereals Maize Cereals Maize

Aflatoxin B1

Aspergillus Cereals (esp. Corn)

HPLC/RF (0,8 ppb);

ELISA (1 ppb);

HPLC/MSMS (0,5 ppb)

2 5 20 5

Aflatoxin total

(B1, B2, G1, G2)

HPLC/RF (2 ppb);

ELISA (2 ppb);

HPLC/MSMS (1 ppb)

4 10 NL NL

OchratoxinAspergillus &

PenicilliumCereals (esp.Oat)

HPLC/RF (0,4 ppb);

ELISA (2 ppb);

HPLC/MSMS (1,6 ppb)

5 250 600

Zearalenone Fusarium Cereals (esp. Corn)

HPLC/RF (5 ppb);

ELISA (25 ppb);

HPLC/MSMS (5 ppb)

100 350 2000 100

DON (Vomitoxin) FusariumCereals (esp. Wheat,

Corn)

ELISA (250 ppb);

HPLC/MSMS (50 ppb)1250 1750 8000 1000

Т2/НТ2 Fusarium Cereals (esp. Corn)ELISA (25 ppb);

HPLC/MSMS (10 ppb)50 100 100

200

Maize and

Barley

200

Fumonisin Fusarium Cereals (esp. Corn)ELISA (250 ppb);

HPLC/MSMS (60 ppb)NL 4000 NL 60000 60000




percentage of tested samples with the presence of a pest percentage of tested samples with mass defeatin green In blue

Region

Pest

Reg

ion

1

Reg

ion

2

Reg

ion

3

Reg

ion

4

Reg

ion

5

Reg

ion

6

Reg

ion

7

Reg

ion

8

Reg

ion

9

Reg

ion

10

Reg

ion

11

Reg

ion

12

Reg

ion

13

Reg

ion

14

Reg

ion

15

Reg

ion

16

Reg

ion

17

Percentage of samples with the presence of a pest

Fusarium moniliforme 6 13 4 94 2 52 36 40 23 29 46 5 74 52 49 17 22

Fusarium graminearum 11 12 17 12 16

Fusarium culmorum 5 6 13 20 2

Sorosporium reilianum 12 21 30 13 21 7 7 14

Cladosporium herbarum 26 12 8 22 13 25 25 36 26 12 23 16 42 50 16 36 13

Alternaria alternata 44 76 80 64 87 90 65 25 66 98 99 92 81 70 87 60 60

Helminthosporium maydis 11 2 8 13 2 21 14

Ustilago zeae 12 17 6 11 16 29 33 50

Epicoccum neglectum 11 62 12 40 16 42 10 24

Puccinia maydis 5 3 1 3

Nigrospora oryzae 20 9 3 12 2 5 7

Cephalosporium maydis 1 1

Cercospora zeae-maydis 2 3

Ascochyta maydis 2 5 4 3 3

Curvularia lunata 1

Aspergillum spp. 15 13 7 8 10 7

Mycological monitoring before harvesting (example)

not detected in the tested samples




Mycotoxins in Corn (2019; 2020) (example)

crop 2019

crop 2020 Aflatoxin B1 Aflatoxin Sum(B1, B2, G1, G2)

Ochratoxin A Zearalenone DON T2/HT2 Fumonisins B1+B2




Aflatoxins

Aflatoxins form one of the main groups of mycotoxins.

Aflatoxin is produced by two kinds of fungi: Aspergillus flavus produces B1 and B2, andAspergillus parasiticus produces all 4 types of aflatoxin B1, B2, G1 and G2. They are verytoxic even at low concentrations. In addition, they are among the strongest naturalcarcinogens.

• Cereals

• Corn

• Sunflower seeds

• Soybeans

• Peanut

These compounds are slightly soluble in water (10 - 20 μg /l), insoluble in nonpolar

solvents. They are not subject to destruction under the influence of temperatures.

Complete destruction of aflatoxins can be achieved by treating them with ammonia or

sodium hypochlorite.




Afla G2

Afla G1

Afla B2

Afla B1

DIRECTIVE 2002/32/EC OF THE EUROPEAN PARLIAMENT AND OF THE COUNCIL of 7 May 2002 on undesirable substances in animal

feed

Chromatogram of the sample containing Aflatoxins




Ochratoxins are a group of mycotoxins that are formed as secondary metabolites of moldfungi of the Penicillium verrucosum and Aspergillus ochraceus genus, as well as some otheraspergillus species, including A.carbonarius and A. niger.

Ochratoxin is usually formed when the grain is not properly stored in moist and warmconditions (> 20 °C), therefore they are called mycotoxins of storage.

It is readily soluble in polar organic solvents and aqueous solution of sodiumhydrogencarbonate, slightly soluble in water. In its pure form it is unstable, sensitive to lightand oxygen, it is stable in solutions.

Ochratoxin A

• Corn

• Barley

• Wheat

• Rice

• Coffee

• Grape

Ochratoxin A




Deoxynivalenol (DON, vomitoxin) belongs to the toxic mycotoxins of thetrichothecene group formed as a result of the activity of mold fungi of the Fusariumgraminearum and Fusarium culmorum genus, affecting plants on the field.

DON is less toxic than other trichothecenes (e.g., than T-2 toxin)

DON, like related metabolites, is readily soluble in polar organic solvents –acetonitrile, acetone, chloroform.

• Wheat

• Corn

• Barley

Deoxynivalenol (DON, vomitoxin)

Vomitoxin

Fusarium graminearum




Zearalenone

• Corn

• Barley

• Wheat

• Oats

Zearalenone (ZON) is produced by some species of Fusarium graminearum and F. roseum.

Zearalenone itself is not toxic, but as a result of metabolism, its end products show estrogenic

activity.

Attack by microscopic fungi of the Fusarium genus occurs both in the field, on the root, and

during storage.

Heat treatment in neutral or acidic medium does not destroy zearalenone, but in alkaline

medium at 100 °C for 60 minutes about 50% of toxin is destroyed. Treatment of contaminated

corn with 0.03% ammonium persulphate solution or 0.01% hydrogen peroxide solution also

leads to the destruction of zearalenone.

zearalenone




Trichothecene T-2 is formed as a result of the activity of various Fusarium fungi,

including F. Sporo trichioides, F. poae and F. Acuinatum, which can grow on

different crops during their storage, especially in regions with a cold climate and

high humidity.

HT-2 toxin is a derivative of T-2 toxin, and its effect on the animal body is largely

similar to that of T-2.

T-2 toxin - non-volatile, resistant to the action of various factors, chemically stable

in air.

Diacetoxyscirpenol (DAS) - the greatest amount of DAS on average is formed byF. langsethiae strains.

Out of all the trichothecenes, T-2 toxin and DAS attract special attention becauseof their high toxicity.

• Wheat

• Corn

• Barley

• Oat

• Soybeans

T-2/HT-2

DiacetoxyscirpenolFusarium poae




Fumonisin is a mycotoxin, which is a product of the activity of the fungusFusarium moniliforme, which affects plants on the field.The most common and dangerous is mycotoxin fumonisin B1.

Fumonisin is readily soluble in water and polar solvents. At a temperature of +125 °C, only 25-30% of these toxins are destroyed, and more than 90% isdestroyed when heated above + 175 °C.

Fusarium moniliformae

Fusarium contaminated wheat grains

• Wheat

• Corn

• Barley

Fumonisin




Pesticides




№ Name of pesticide Type of biological effect Method Units

Max allowed for

Wheat

по ЕС. Reg.

396/2005

LOQ Result

Organochlorine pesticides

… ….

Lindane (HCH, gamma) Insecticide, Acaricide BS EN 15662:2018 (GC/MSMS) ppm 0,01 0,01 ND, (< 0,01)

Endrin Insecticide, Rodenticide BS EN 15662:2018 (GC/MSMS) ppm 0,01 0,01 ND, (< 0,01)

Organophosphorus pesticides

…

Bromophos-ethyl Insecticide BS EN 15662:2018 (GC/MSMS) ppm 0,05 0,01 ND, (< 0,01)

Butamifos Herbicide BS EN 15662:2018 (GC/MSMS) ppm 0,01 0,01 ND, (< 0,01)

Cadusafos Insecticide, Nematocides BS EN 15662:2018 (GC/MSMS) ppm 0,01 0,01 ND, (< 0,01)

Pyrethroids

…

Cypermethrin Insecticide BS EN 15662:2018 (GC/MSMS) ppm 2,00 0,01 0,09

Carbamates

…

Methomyl Insecticide, Acaricide BS EN 15662:2018 (GC/MSMS) ppm 0,02 0,01 ND, (< 0,01)

Termostable pesticides (Laboratory report example)

Chemical classes, Biological effect, Limits




№ Name of pesticide Type of biological effect Method Units

Max allowed for

Wheat

по ЕС. Reg.

396/2005

LOQ Result

Triazoles/ Triazines

…

Triasulfuron HerbicideBS EN 15662:2018

(HPLC/MS/MS)ppm 0,05 0,01 ND, (< 0,01)

Others

…

Benomyl FungicideBS EN 15662:2018

(HPLC/MS/MS)ppm 0,10 0,01 ND, (< 0,01)

Polar pesticides

Glufosinate Herbicide (HPLC/MS/MS) ppm 0,03 0,01 ND, (< 0,01)

Glyphosate Herbicide (HPLC/MS/MS) ppm 10,00 0,01 ND, (< 0,01)

Diquat Herbicide (HPLC/MS/MS) ppm 0,02 0,01 ND, (< 0,01)

Mepiquat Growth regulator (HPLC/MS/MS) ppm 3,00 0,01 ND, (< 0,01)

Termo-sensitive pesticides (Laboratory report execution)

Chemical classes, Biological effect, Limits




Max allowed limits for Pesticides as per EU Regulationshttps://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/products/?event=details&p=240

Oil soluble

* means MRL = LOQ

https://ec.europa.eu/food/plant/pesticides/eu-pesticides-database/products/?event=details&p=240




Pesticides in Wheat 2020 (example)

0.0

1

0.0

89

0.0

1

0.0

16

0.0

1

0.0

82

0,5

72

0.0

36

2

0,0

1

0,67

EU Regulation limits, ppm

2 10 5 0,3

12 2

23

2 0,5

0,0

1

3

USA limits, ppm

0,2 30

0,0

1

0,1

5

2 0,5

0,0

1

2

CODEX limits, ppm

2

0,0

1

0,0

1

0,1

5




Heavy metals




Toxicity of heavy metals

• Heavy metals are toxic to human health

• Most common heavy metals are lead (Pb), mercury (Hg),cadmium (Cd) and arsenic (As)

• They are mainly produced by industrial activities, and depositslowly in the surrounding water and soil

• Mercury is highly toxic in vapor form but lead, cadmium andarsenic are more toxic in their cationic form

• Toxicity arises from strong affinity of the heavy metal cationsfor sulfur




Toxicity of trace heavy metals

Metal Toxicity Effect

ArsenicIrritation of respiratory system, liver and kidney damage, loss of appetite, nausea and

vomiting etc

Cadmium Lung, liver and kidney damage; irritation of respiratory system

Chromium Lung damage and irritation or respiratory system

Mercury Irritation of respiratory system; lung, liver and kidney damage

Lead Lung and liver damage; loss of appetite, nausea etc

Nickel Lung, liver and kidney damage




Methods for measurement of trace metals

Most common method of collecting particulate matter

is through filters Identification and concentration of

individual trace metals like lead, cadmium, arsenic,

mercury and chromium is determined by:

• Atomic absorption spectrophotometry (AAS);

AAS detects elements through the application of

characteristic wavelengths of electromagnetic radiation

from a light source. AAS takes advantage of the different

radiation wavelengths that are absorbed by different

atoms.

• Inductively coupled plasma (ICP);

ICP is an analytical technique, used to identify and quantify

chemical elements present within a certain sample. It uses

ionized Argon gas, which is called plasma. An Argon

plasma can reach a temperature of 10000K that allows it to

ionize all the elements present in the material, quantifying

themAtomic absorption spectrophotometer ContrAA800 (Analytik Jena)




Heavy metals limits by EU requirements

Metals

Max allowed

for FOOD

by EC Regulation 1881/2006

and

CODEX STAN 193-1995

Max allowed

for FEED

by Directive 2002/32/EC, ppm

Wheat Corn Feed materials

Lead, ppm 0,2 10

Cadmium, ppm 0,2 0,1 1

Arsenic, ppm No limits 2

Mercury, ppm No limits 0,1




crop 2019

crop 2020

Statistics for Heavy metals in Wheat (2019; 2020) (example)

Arsenic (As) Mercury (Hg) Cadmium (Cd) Lead (Pb)

Ave

rag

e, p

pm

LOQ, ppm

2,0 0,1 1,0 10,0EU Reg, ppm

Feed (32)

NL NL 0,1 0,2EU Reg, ppm

Food (1881)




Microbiology Testing




Express-testing Classic Microbiology ELISA PCR-analysis

Methods of microbiology testing

Total bacterial count, Mold and Yeast, E. coli and Coliform, Salmonella, Listeria and etc.

https://faculty.weber.edu/coberg/class/3853/3853%20Microbial%20Testing%20Procedures.htm




Maximum allowable microbiological limits for grain and feed materials by country

Country / Document CargoMolds,

CFU/g

Salmonella,

in 25 gr

E. coli,

CFU/g

Coliforms,

CFU/g

S. aureus,

CFU/g

APC*,

CFU/g

Europe / Customs Union

No. 299

Cereal grains

Oilseeds- - - - - -

Europe / GMP+BA1 Feeding stuffs 1x106 Absent - - - -

Spain / Info list Raw materials for feeds - Absent Free - < 10 -

USA, Philippines / FDA Cereal grains 1x104 Absent 1x104 1x104 - 1x106

Egypt / ESS:1683 Corn - - Free - - -

China / GB 13078Corn, wheat bran

Feeding stuffs

4x104

4,5x104Absent

Absent

-

-

-

-

-

-

-

-

Jordanian /Tech Reg 1200 Wheat 1x105 - - - - -

Iran / ISIRI 1445-2018 Corn 1x103 Absent ˂ 10 - - -

Sudan / SDS 2013 Cereal grains 1x104 - - - - -

Lebanon / NL 242 Wheat 1X105 Absent - - - -

“-” no limits

* CFU/g – a colony-forming units per gram

** Aerobic Plate Count




Dioxins and PCBsand

Polycyclic aromatic

hydrocarbons

(PAHs)




PAHs vs Dioxins/PCBs: main sources PAHsAnthropogenic human activity: - Heating - combustion products of wood, coal or other biomasses- Operation of mechanisms - exhaust from motor vehicles, products of

incomplete combustion of diesel and solid fuel engines- Chemical and oil refining industry- Road surfaces - asphalt, bitumen

Human activity:- Some food products - smoked meats, fried meats, coffee/chocolate - as

a result of surface charring- Tobacco smokeNatural sources and natural disasters- Volcanoes- Forest fires

Benz[a]pyrene Chrysene

NO CHLORINE

Dioxins/PCBs

Anthropogenic human activity:

- Organochlorine industrial synthesis plants

- Pulp and paper industry using chlorine for bleaching

- Incineration of household waste containing obsolete polyvinyl chloride

products

- Production of chlorphenol herbicides, especially, 2,4-D and 2,4,5-T;

Human activity:

--

Natural sources and natural disasters- Forest fires after forest treatment with organochlorine pesticides

Dioxin (PCDD)

YES CHLORINE

PAHs

Drying of grain

Dioxins/PCBs




Sample preparation consists of three steps 1 – 3 (for solid matrices - grain and oilseeds, cakes/meals):

Dioxins: APGC/MSMS DetectionDioxins: sample preparation

2. Purification of the extract

Cleaning system DEXTech

3. Concentration

Extraction system Velp Concentration system FMS APGC/MSMS system Waters

1. Extraction

and two steps 2 – 3 (for liquid matrices - vegetable oils):

Dioxins/PCBs testing




PAH: HPLC/RF DetectionPAH: sample preparation

Sample preparation consists of two steps for vegetable oils:1. Purification 2. Concentration

PAHs testing




[email protected]

+38 067 361 88 88

Please contact us for any further questions:

Arina KorchmaryovaMBA, BSc Chemistry and Biotechnology

DECEMBER, 2020

LABORATORY TESTING: SAFETY PARAMETERS...- Directive 2002/32/EC - EU Recommendation 2006/576/EC - EU...

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Transcript of LABORATORY TESTING: SAFETY PARAMETERS...- Directive 2002/32/EC - EU Recommendation 2006/576/EC - EU...