Presentation - Association of Public Analysts

Authenticity of Rice 

W.Nader, R.Schubbert, T.Brendel, D.Arthur 

Eurofins 

Presentation during APA conference, 

Newcastle, October 30th, 2009 

October 2009 

www.eurofins.com

Joint effort of the Eurofins network 

Eurofins Public Analyst Service, UK 

Eurofins Global Control, Hamburg 

Eurofins Medigenomix, Munich 

RiceLab, Pakistan 

Eurofins India 

October 2009 Association of Public Analysts Conference 2009 2

Current issues with rice 

Nr. Topic Status Future development 

1 

Issue probably resolved for the USA, 

GMO 

persistent in China 

Many new GMOs in agriculture expected soon 

2 

3 

4 

5 

6 

7 

8 

Mycotoxins 

Pirimiphos-methyl 

Other pesticides 

Rice authenticity 

Arsenic 

Packaging materials 

Cadmium 

Issue seems resolved with new 

Commission Regulation 669/2009, 5 ppb 

for afla B1 and 10 ppb for sum in cargo rice 

Reduction of MRL from 5 to 0.05 ppm 

possible. 

So far pesticides no big issue for rice on 

EU/RASFF level 

Issue of quinclorac has been resolved with 

new EU pesticide regulation 

Code of Practice for Basmati rice brought 

clarity for UK 

FSA recommendation against rice milk for 

toddlers 

Migration of phthalates observed from 

recycled paper package 

Review of current limits in food 

Ochratoxin A and Fusarium toxins not considered 

to be a risk in rice 

Commission requires data to support further use. 

Further reduction of MRLs probable for certain 

pesticides. 

Water soluble pesticides like acetamiprid might 

become an issue 

Interpretation of import duty exemption rules are 

not clear yet ((EC) No 972/2006). Thai fragrent 

rice authenticity under evaluation in France 

New scientific opinion expected from EFSA and 

new limits for rice 

Comprehensive declaration of compliance required 

for packages made out of plastic since May 

Rice not considered to be a risk, but will be 

monitored 


Basmati rice – some facts 

• Export India (2006/2007): 1.1 million tons / 700 million $ 

• and Pakistan (same FY): 900.000 tons/ 570 million $ 

EU Import (2002/2003): > 270.000 tons, 70 % to UK 

• Price: 600 to 700 €/ton, dehusked Pusa Basmati rice 

• In comparison non-Basmati long grain rice: 300 to 350 €/ton, dehusked 

US long grain 

Yields: 

• traditional Basmati rice (Type III): 3 – 3.5 tons / ha 

• inbred Basmati rice (PUSA): 4 – 7 tons / ha 

• long grain non-Basmati rice: > 8 tons /ha 

•EU rice import tariffs: 65 € / ton for dehusked and 175 € / ton for milled 

rice 

• Import tariff exemption: 9 basmati varieties (dehusked) 


Basmati Varieties 

Taraori 

Basmati 370 

Haryana 

Paddy 

Dehusked 

Milled 


Developments on the European level 


Approved Basmati varieties 

Basmati rice varieties eligible for a zero EU import duty 

Basmati 217 (I) 

Ranbir basmati (IET 11348) (I) 

Basmati 370 (I, P) 

Super basmati (P) 

Basmati 386 (I) 

Taraori basmati (HBC-19, Karnal Local) (I) 

Kernel basmati (Basmati Pakistan) (P) 

Type –3 (Dehradun) (I) 

Pusa basmati (IET 10364) (I) 

Other Basmati rice varieties approved by India and Pakistan 

Basmati 198 (P) 

Kasturi (IET 8580) (I) 

Basmati 385 (P)Mahi Suganda (I) 

Haryana Basmati (HKR 228/IET 10367) (I) 

Punjab Basmati (Bauni Basmati) (I) 


Survey by Eurofins Public Analyst Service 

Survey 2008, Eurofins Public Analysts 

21,6% 

3,7% 

42,5% 

32,1% 

0 % (PURE) 

0.1 %- 7 % (PASS) 

7 % - 13 % (BORDERLINE) 

> 13 % (FAIL) 

FSA 2002/2003 Eurofins 2008 

% non-Basmati 363 samples 132 samples 

0 54,0% 3,7% 

< LOQ 10,0% 6,7% 

LOQ- 10 14,0% 44,8% 

10 - 20 6,0% 34,3% 

20 - 30 4,0% 3,7% 

30 - 40 2,0% 3,7% 

40 - 50 1,0% 0,7% 

50 - 60 1,0% 0,0% 

> 60 9,0% 2,2% 


Results from ÖKOTEST Study, 2006 


Satellite DNA 

Different alleles arising from 

variation of tandem repeat length 

during crossing over 


Alleles of microsatellite markers for rice varieties 

RM1 RM223 M16 RM202 RM44 RM201 RM229 RM241 RM171 RM55 

Basmati Varieties 

Taraori W W Z Y Y X Y Y Y X 

Basmati 386 W W Z Y Y X Y Y Y X 

Kernal W W Z Y Y X Y Y Y X 

Basmati 370 W W Z Y X X Y Z Y Z 

Dehradun W W Z Y X X Y Z Y Z 

Basmati 198 Y W Z X (Y) X X Y Z Y Z 

Kasturi W V Y Y W X Y Z Z Y 

Basmati 385 Y W Z X Y X Y Z Y Z 

Super Basmati W W Z Y X X Y Z Z X 

Pusa Basmati W W Y Y Y X X Y Z Y 

Ranbir Basmati W (-1) Y Z Y X (+1) X Y Z(+2) Y Z 

Mahi Sugandha Y X Z X W Z X Z Z Y 

Punjab Basmati Y(+ 2) Y Z X X (+1) X Y X (-4) Y Z (+3) 

Punjab Basmati Z (+5) Y Z X X (+1) X Y Z Y Z 

Haryana Basmati Z Y Y X W X X Y Z Y 

Haryana Basmati Z Y Z Y W X X Y Z Y 

typical Basmati adulterations 

Sherbati W (Z) Z (Y) Y X (Y) W Z X X (Y) W (Z,Y) Y 

Pak 386 Y X Y Y W Z Y Y Z Y 

Thai scented rice 

Hom Mali Z (+5) V Y Y W (+2) W X Y (-2) Z Z (+2) 

Pathumthani Z (+5) V Y Y (+4) W W X Y Z (+4) Y 

Additional markers in use: RM212, RM263, 

RM222, RM282, RM339 

Source: Mark Woolfe (FSA), John Gorham (Bangor 

University), Rainer.Schubbert, Eurofins Medigenomix 


DNA fingerprinting of Basmati 

A: mixture of Pusa and Taraori Basmati and non-Basmati 

B: Pusa Basmati 

C: Super Basmati 

Markers: RM201 (CT) 17 RM171 (GATG) 5 RM229 (TC) 11 (CT) 5 C 3 (CT) 5 

Method: amplification of marker loci with fluorescently labelled primers and analysis of 

amplified DNA on an ABI Prism 3100 sequencer (capillary electrophoresis). 

Green channel: JOE (6-Carboxy-4,5-Dichloro-2,7-Dimethoxy-Fluorescein) 

Blue channel: FAM (6-Carboxylfluorescein) 


Quantitative evaluation of DNA fingerprint 

Pusa 

Taraori 

Sherbati 

RM1 RM223 M16 RM202 RM44 RM201 RM229 RM241 

W W Y Y Y X X Y 

W W Z Y Y X Y Y 

W (Z) Z (Y) Y Y W Z X X (Y) 

RM171 

Z 

Y 

W (Z,Y) 

RM55 

Y 

X 

Y 

Main W 72% W 52% Y 91% X 60% W 48% X 51% X 43% Y 98% W 43% Y 87% 

Minor Z 24% Y 39% Z 9% Y 40% Y 52% W 32% X+2 31% Z 2% Z 41% X 12% 

Y 4% Z 9% _ 0% _ 0% _ 0% Z 18% Y+2 16% _ 0% Y 10% Z+2 1% 

_ 0% _ 0% _ 0% _ 0% _ 0% _ 0% Y 10% _ 0% X 5% _ 0% 


Minor Z 24% Y 39% Z 9% Y Y 11% W 32% X+2 31% Z 2% Z X 12% 

Y 4% Z 9% _ 0% _ 0% _ 0% Z 18% Y+2 16% _ 0% Y 10% Z+2 1% 

_ 0% _ 0% _ 0% _ 0% _ 0% _ 0% Y 10% _ 0% X 5% _ 0% 


Minor Z 24% Y 39% Z Y Y 2% W 32% X+2 31% Z 2% Z X 3% 

Y 4% Z 9% _ 0% _ 0% _ 0% Z 18% Y+2 16% _ 0% Y 1% Z+2 1% 

_ 0% _ 0% _ 0% _ 0% _ 0% _ 0% Y 1% _ 0% X 5% _ 0% 

Result: Content (%) 

Pusa 41% 

Taraori 9% 

Sherbati (non Basmati) 2% 

Other non Basmati 

not identified 48% 

Total Basmati 50% 

Total non Basmati 50% 


You are half-way through! 


Validation at Eurofins for ISO 17025 accreditation 

1. Precision 

a) Repeatability (multiple measurements), s r : 3.2 % (absolute) 

b) Reproducibility (intra-lab variation), S R : 3 % (absolute) 

2. Accuracy dR: 2.4 % absolute (95 % confidence level, 5 

degrees of freedoms) 

3. Uncertainty U: +/- 6 % (derived from S R with an expansion 

factor of 2), or +/- 6.4 %, as derived from U = √(dR) 2 + (2 *s R ) 2 

4. Limit of detection: 0.1 – 1 % for non-Basmati rice 

5. Limit of quantification: 1 % for non-Basmati rice 

6. Linear range of analysis: between 1 to 99 % in a mixture of 

a Basmati variety with non-Basmati rice 


% Pusa Basmati rice by DNA analysis 

Linearity of test 

Linear range of non-Basmati rice quantification 

100 

90 

80 

70 

y = 0,991x 

R 2 = 0,9976 

60 

50 

y = 0,986x 

R 2 = 0,9988 

Pusa/Jasmin 

Pusa/Jasmin (w/oRM44/55) 

40 

30 

20 

y = 0,9563x 

R 2 = 0,9927 

y = 0,8877x 

R 2 = 0,9613 

Pusa/long grain 1 

Pusa/long grain 2 

10 

0 

0 20 40 60 80 100 

% pure Pusa rice (weight/weight) 


Results from the FSA quantitative ring trial 

Uncertainty: Standard deviation 

of reproducibility s R x expansion 

factor 2 = 6 % 

Source: FSA report 


Mandi in India 


Quarantine area in Indian rice mill 


Sampling in Pakistan 


Stuffing and discharge of container 


non-Basmati (%) 

Analysis of 553 rice samples over 2 years, one 

customer, one rice mill 

Results from Basmati Testing 

35 

30 

25 

20 

15 

10 

5 

0 

20.03.2006 28.06.2006 06.10.2006 14.01.2007 24.04.2007 02.08.2007 10.11.2007 


Structure of the fragrance gene (fgr) 

2-acetyl-1-pyrroline 

Source: Bradbury et al.: The gene for fragrance in rice. Plant Biotechnology Journal (2005) 


Fragrancy gene analysis 

Results from single kernel analysis 

Sample RM1 RM223 M16 RM202 RM44 RM201 RM229 RM241 RM171 RM55 RM263 fgr genotype 

Italian fragrant rice W+8 X Z X-3 X X Y+10 Y+6 W Y 159 fgr + 



Italian fragrant rice W+10 V Z Y-8 X X+2 Y+10 Y W Y 159 fgr - 

Italian fragrant rice W+10 V Z Y-8 X X+2 Y+10 Y-5 W+4 Y 159 fgr - 

Italian fragrant rice W+10 V Z X-2 X X+2 Y+10 Y+6 W Y 159 fgr - 

Italian fragrant rice W+10 V Z Y-8 X X+2 Y+10 Y W+4 Y 159 fgr - 

Italian fragrant rice - - Z X-3 - X Y+10 Y+6 W Y 159 fgr - 

Thai Pathumthani Z+5 V Y Y+4 W X Y Z+4 Y 185 fgr + 





Thai Pathumthani Z V Y Y W Y+2 Y W Y 185 fgr - 

Thai Pathumthani Z+5 Y Y Y+4 W X Y Z+4 Y 185 fgr + 


Comparision fgr - testing with DNA fingerprint 

Results from DNA 

fingerprint 

Results from fgr - 

analysis 

non Basmati (%) fgr - content (%) 

Pakistan Super Basmati 11% 5% 

Basmati rice 100% 25% 


Further applications of DNA fingerprinting 

Thai rice 

• Hom Mali, Pathumthani, Vietnamese, Cambodian 

Risotto 

• Arborio, Carnaroli, Roma 

Angus Beef 

• Test has been developed in cooperation with the 

Aberdeen-Angus Cattle Society 

• Currently being re-evaluated with further additional reference materials 

provided by the society 

• Test can be used to differentiate pure bred, sired, non-Angus 


HAVE A RICE DAY! 

Eurofins Laboratories Ltd Public Analysts 

Duncan Arthur, Public Analyst 

28-31 Brunel Road 

Acton, W37XR, England 

DuncanArthur@eurofins.co.uk 

Eurofins Global Control GmbH 

Silke Heimbecher, Sascha Schmidt, 

Dr. Werner Nader 

Am Neuländer Gewerbepark 1 

21079 Hamburg, Germany 

+49 (0) 40 49294-731; global@eurofins.de 

Eurofins Medigenomix GmbH 

Forensic & Applied Genetics 

Dr. Rainer Schubbert, Dr. Torsten Brendel 

Ebersberg / Munich 

schubbert@medigenomix.de 


Content Pusa, measured (%) 

Accuracy 

Accuracy 

Pusa content (w/w) 100,0% 95,0% 50,0% 5,0% 0,0% 

1 100,0% 95,0% 51,0% 8,0% 0,0% 

2 100,0% 95,0% 45,0% 10,0% 0,0% 

3 100,0% 96,0% 50,0% 8,0% 0,0% 

4 100,0% 93,0% 57,0% 10,0% 0,0% 

5 100,0% 93,0% 55,0% 5,0% 0,0% 

6 100,0% 94,0% 51,0% 4,0% 0,0% 

Average 100,0% 94,3% 51,5% 7,5% 0,0% 

standard deviation 0,0% 1,2% 4,2% 2,5% 0,0% 

t-Test 0,0 2,0 1,2 2,9 0,0 

100,0% 

90,0% 

80,0% 

70,0% 

60,0% 

50,0% 

40,0% 

30,0% 

20,0% 

10,0% 

0,0% 

0,0% 10,0% 20,0% 30,0% 40,0% 50,0% 60,0% 70,0% 80,0% 90,0% 100,0% 

Content Pusa expected, w/w (%) 

t < 2.57 (95 % confidence level, 5 degrees of freedom)

Presentation - Association of Public Analysts

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