determination of fungicide residues in grape by gc/ms...determination of fungicide residues in grape...

DETERMINATION OF FUNGICIDE RESIDUES IN GRAPE BY GC/MS

SanjaLazić, Vedrana Komlen, Dragana Šunjka, Nada Grahovac, Jadranka Pejčić, Alma Rahimić, Milenko Blesić

Faculty of Agriculture, Novi Sad, SERBIA Agromediterannian faculty, Mostar, BiH

Plant protection products (pesticides) are

used worldwide within agriculture to protect

crops and ensure the quantity and quality of

the harvest.

Fungicides represent one of the most relevant

groups of pesticides applied to vineyards.

These compounds are sprayed directly on

fruit and leaves to prevent the attack of fungi,

which reduce the yield of fruit.

If fungicides are not degraded, some of them will

penetrate plant tissues and appear in the pulp and juice.

Once present in the pulp, pesticides are difficult to

completely be removed.

Residues of fungicides are one of the most important

pollution sources in food production and might pose

potential threat to public health.

The concentration of pesticide residues may increase

during post processing, where in general pesticide

concentrations in processed juices are higher than that in

the natural fruit.

Having in mind the health risk of pesticide residues in fruit and wine, it is

of particular importance to provide precise, accurate and reliable test

result of residues as the scientific basis for ensuring food safety and fair

practice in international trade.

• single component

• multi-residue analysis method (different

components of different types of pesticides)

Fungicide residues

Analysis of fungicide residues

PRESENCE OF BENALAXYL, DIMETHOMORPH AND

TETRACONAZOLE RESIDUES IN GRAPE SAMPLES

Dimethomorph

Tetraconazole

Benalaxyl

Aim of the study

Field experiment

The vineyards choice was done to research on four different

locations in the vineyards of Mostar.

• Žilavka

• Blatina

Grape varieties

• famous Hercegovinian grape varieties

• Special clime conditions

• 2290 sunshine hours per year

Vineyards location

Area of vineyard 2 ha

Age of vineyard 9 years

Variety Žilavka

Blatina

Humčine – plantation vineyard

Area of vineyard 13 ha

Age of vineyard 5 years

Variety Žilavka

Mukoša – plantation vineyard

Area of vineyard 30 ha

Age of vineyard 7 years

Variety Blatina

Žitomislić – plantation vineyard

Area of vineyard 0,4 ha

Age of vineyard 6 years

Variety Žilavka

Blatina

Hodbina – small vineyard

Protection program

Treatments were carried out air assistance sprayers (atomizer) with

300 l with average interval between treatments was of 10 days.

The protection program in the vineyard Mukoša and Žitomislić

Date treatment Product Active substance Quantity

kg or l/ha

May 5th

Delan Dithianon 0.3

Sulfur Sulfur 1.5

May 15th Delan Dithianon 0.4

Thiovit jet Sulfur 2.0

May 25th Pergado F Mandipropamid+ folpet 2.0

Talendo Proquinazid 0.25

June 5th Fantic F Benalaxyl + folpet 2.5

Domark Tetraconazole 0.75

June 18th Forum star Dimethomorph + folpet 2.0

Collis Kresoxim-methyl + boskalid 0.4

June 30th Mikal premium Fosetyl + folpet+ iprovalicarb 3.0

Postalon Quinoxyfen + myclobutanil 0.125

July 12th Cabrio top Pyraclostrobin + methiram 2.0

August 9th Mythos Pirimethanil 2.5

The protection program in the vineyard Hodbina

Date treatment Product Active substance Quantity

kg or l/ha

April 20th Cuprablau Copper 3.0

Chromosul Sulfur 2.5

April 28th Chromosul Sulfur 2.5

May 7th Chromosul Sulfur 2.5

May 20th

Ridomil gold Metalaxyl + mankozeb 2.5

Chromosul Sulfur 2.5

Topas Penconazole 0.25

June 1st Ridomil gold Metalakxyl + mankozeb 2.5

Chromosul Sulfur 2.5

June 11th

Fantic F Benalaxyl + folpet 2.5

Domark Tetraconazole 0.75

Pirus Pirimetanil 2.5

June 24th Acrobat Dimethomorph + mankozeb 2.5

Systane 24 E Myclobutanil 1.0

July 4th Cuprablau Copper 3.0

Thiovit jet Sulfur 4.0

July 20th Nordoks 75W Copper 1.25

Thiovit jet Sulfur 4.0

August 5th Mythos Pirimetanil 2.5

The protection program in the vineyard Humčine

Date treatment Product Active substance Quantity

kg or l/ha

April 21st

Folpan Folpet 2.05

Kossan WG Sulfur 4.1

May 6 th Folpan Folpet 2.5

Kossan WG Sulfur 4.9

May 14 th Manfil 75 WG Mancozeb 3.3

Karathane Gold 350 Metyldinocap 0.9

May 23rd Manfil 75 WG Mancozeb 3.3

Karathane Gold 350 Meptyldinocap 0.9

May 27 th Fantic M Benalaxyl + mancozeb 4.9

Domark Tetraconazole 1.4

June 16 th Fantic F Benalaxyl + folpet 4.9

Crystal Quinoxyfen 0.4

June 29 th Fantikc F Benalaxyl + folpet 4.9

Crystal Quinoxyfen 0.4

July 15th Mythos Pirimetanile 2.5

The field sampling was conducted during the technological maturity of the

grape varieties of "Žilavka" and "Blatina“.

Sampling procedure

Sampling procedure

The average weight of each laboratory sample of grapes was

approximately 2 kg with at least five clusters. The samples were packed

in plastic bags and delivered to the laboratory refrigerator.

Location Varieties Number of

samples

Humčine Žilavka

Blatina

4

4

Mukoša

Žitomislić

Žilavka

Blatina

4

4

Hodbina Žilavka

Blatina

4

4

50 g sample + 100 ml ACN - homogenize 5 min

Extraction procedure

add 10 g NaCl - homogenize 5 min

Clean up - Octadecyl (C18) SPE tubes

Clean up - Carbon SPE tubes / Aminopropyl SPE tubes

Evaporation to drynes

Extract diluted in acetone

GC/MS analisys

Metod by Fillion et al., 2002.

GS/MS analysis

Determinations were performed on Thermo type

Focus DSQ II GC/MS

Operating mode Splitless

Column HP-5MS, 5% phenyl methyl siloxan

(30m x 0.25mm x 0.25µm)

Helium flow rate 0.9 ml/min

Injector temperature 250 C

Temperature detector 285 C

Initial column temperature 70 C

Initial time 2 min

Speed of temperature rise 25 C/min

Final temperature 280 C

Determination conditions

Results

LOD 0.001 mg/kg

LOQ 0.003 mg/kg

Recovery values for

benalaxyl, dimethomorph

and tetraconazole

> 90 %

GS-MS chromatogram of standard mixture

Results

GS-MS chromatogram of grape samples

tetraconazole benalaxyl

Results

Locality Investigated

compound

Determinted

value (mg/kg)

LOD

(mg/kg)

Humčine

Benalaxyl < LOD 0.001

Dimethomorph < LOD 0.001

Tetraconazole 0.003 0.001

Mukoša

Benalaxyl 0.002 0.001

Dimethomorph < LOD 0.001

Tetraconazole 0.003 0.001

Hodbina

Benalaxyl < LOD 0.001

Dimethomorph < LOD 0.001

Tetraconazole 0.002 0.001

Determined value of fungicides in grape variety Žilavka by GC/MS

Locality Investigated

compound

Determinted

value (mg/kg)

LOD

(mg/kg)

Humčine

Benalaxyl 0.014 0.001

Dimethomorph < LOD 0.001

Tetraconazole 0.017 0.001

Žitomislić

Benalaxyl < LOD 0.001

Dimethomorph < LOD 0.001

Tetraconazole 0.001 0.001

Hodbina

Benalaxyl < LOD 0.001

Dimethomorph < LOD 0.001

Tetraconazole < LOD 0.001

Determined value of fungicides in grape variety Blatina by GC/MS

In grape samples variety Zilavka tetraconazole residues

were found in samples from all localities, while

benalaxyl residues were found only in samples from

locality Mukosa.

Conclusions

In grape samples variety Blatina tetraconazole residues

were found in samples from locality Humcine and

Zitomislic, whereas benalaxyl residues were found in

grape samples from locality Humcine.

Residues of dimethomorph in analyzed samples of

grape, variety Žilavka and Blatina were not detected.

The concentrations of benalaxyl in the analyzed grape

samples were 0.002-0.014 mg/kg.

Conclusions

The concentrations of tetraconazole in the analyzed

grape samples were 0.001-0.017 mg/kg.

The highest value of the fungicide residues were found

in grape variety Blatina, locality Humcine. It is can be

explain with intensive application of fungicide on this

locality.

Conclusions

The concentrations of fungicides were below the

permissible levels (MRL) set by EU regulations (The

Annexes of Commission Regulation (EC) No

396/2005) and by Serbian regulations (Official

gazette, No 25/2010 i 28/2011) for tetraconazole (0.5

mg/kg) and benalaxyl (0.3 mg/kg) in grape.

The obtained positive results for tetraconazole and

benalaxyl could be attributed to the high sensitivity of

the developed GC/MS method.