Effects of Cleaning Procedures on the Concentration of Pesticide Residues on Crisp Fresh-Cut Lettuce (cv. Vera) ^†

Abstract

Decontamination procedures are needed for ready-to-eat foods, especially vegetables. In this work, we focused on the effects of four cleaning solutions and ultrasound baths on the amount of pesticide residue left on lettuces. Five pesticides were applied to lettuces grown in controlled conditions. The residues were analyzed with an acetate QuEChERS method and a HPLC-MS/MS system. All the tested methods resulted in diminution of residues without significant differences among them. Out of 16 pesticides analyzed on commercial samples, only five were found on seven of the samples, without exceeding the MRLs.

1. Introduction

The consumption of fresh fruits and vegetables are part of a healthy lifestyle as they are a source of vitamins, fiber and many nutrients. Food safety is a major issue for fresh produce as it is usually eaten raw to maintain more of their benefits; however, this may lead to foodborne illnesses if not washed properly [1]. Regarding raw vegetables, ready-to-eat foods have increased in popularity as they present an easy way to get produce that is already washed and cut.

To prepare vegetables for consumption several parameters need to be taken into consideration. This work focuses on pesticide residues, considering the maximum residue limits (MRL), which are set by the Codex Alimentarius in Uruguay [2]. During production, farmers use pesticides to avoid plagues, to improve production and to prevent diseases that could affect their crop [3]. When applied using good agricultural practices, pesticides should leave residues that are below the established MRLs; furthermore, these residues can be reduced by different cleaning procedures.

Ultrasound baths are an environmentally-friendly decontamination method and are used widely to reduce microorganisms and residues in different industries [4]. Cavitation bubbles formed by the ultrasonic waves can detach residues from surfaces and when they burst, they have the ability to break molecules [5]. This latter ability has proven to be effective with pesticides [6]; therefore, the intention was to study whether or not the different frequencies applied and different times of application have different effects on the residues, without damaging the leaves so as to preserve the fresh look of the lettuces.

The main objective of this paper is to compare and contrast some of the methods used on vegetables in the industry nowadays.

2. Materials and Methods

2.1. Materials and Equipment

Work mixes were prepared from pesticide stock solutions that had been made by dissolving high purity standards in the appropriate solvent and were kept at −40°. HPLC grade acetonitrile (MeCN) and methanol (MeOH) as well as deionized water from a MilliQ system were used. Sodium acetate (NaOAc), ammonium formate, formic acid 88%, sodium chloride (NaCl) and glacial acetic acid (HAc) were also employed during this work.

The analysis was performed with an Agilent 1200 series HPLC system (Agilent Technologies, Palo Alto, CA, USA), coupled to a 4000 QTRAP^® mass spectrometer system from Applied Biosystems SCIEX™ (Conrad, Canada). The column employed was a ZORBAX Eclipse XDB-RP-C18 (150 mm × 4.6 mm, 5 µm) from Agilent Technologies. The spectrometer was working in Multi Reaction Monitoring (MRM) mode with nitrogen as a collision gas. The mobile phase solutions were A: deionized water with 2% MeOH, 0.1% formic acid and 5 mM ammonium formate, and B: MeOH with 2% deionized water, 0.1% formic acid and 5 mM ammonium formate. The gradient started with 30% of mobile phase B for a minute, then increased to 100% after 11 min; it was kept constant for two minutes and then reduced back to 30% after two minutes. It remained there until the 21 min of run-time were up. A flow of 0.6 mL/min was kept throughout the analysis. The software employed was the Analyst Software, version 1.8 (SCIEX).

2.2. Lettuce Acquirenment

This study was performed using two groups of lettuces. On one hand, commercial lettuces were purchased from local producers. One the other hand, lettuces were grown in a greenhouse in controlled conditions and obtained at the ripe stage. Both groups were analyzed for pesticide residues with their own associated blanks.

2.2.1. Commercial Samples and Processing

All in all, 22 lettuces were bought from different local producers and shops over a two-month period. Three of these lettuces were obtained from an organic producer, and were used as blank samples for the validation procedure and for calibration curves.

Each head of lettuce was shredded and milled with liquid nitrogen to a fine dust as soon as it got to the laboratory, and was kept frozen until analysis.

2.2.2. Lettuce Production and Pre-Harvest Treatments

In order to obtain lettuces with similar levels of pesticides, an experiment in controlled conditions was carried out. Crisp lettuce (Lactuca sativa, cv Vera) was grown in a greenhouse with daily watering in Estación Experimental Dr. M.A. Cassinoni, near Paysandú, Uruguay. A total of 70 lettuces were cultivated following Good Agricultural Practices during September and October 2020.

From the pool of pesticides available for use on lettuce in Uruguay, acetamiprid, boscalid, carbendazim, chlorpyrifos ethyl and methyl, cyromazine, dimethoate, fluvalinate, imidacloprid, iprodione, methomyl, pyraclostrobin, pyrimethanil, pirimicarb, propamocarb and spynosad were chosen for this study after consulting with lettuce producers from the north-west region. Five of these products were chosen to be used during the controlled-conditions experiment; the selected pesticides were chlorpyrifos ethyl, pirimicarb, imidacloprid, boscalid and pyraclostrobin. The application of the pesticide was performed by following each label recommendations, using a hand spray applicator.

2.2.3. Lettuce Harvest and Post-Harvest Processing

The lettuce heads were harvested at the ripe stage at four to six-weeks-old and immediately processed. Applied (A) and non-applied (NA) heads were shredded manually with a stainless-steel knife to obtain homogenous batches.

NA-samples were processed with liquid nitrogen and kept frozen until analysis. Additionally, an A-sample was taken to generate positive controls, while the rest of the batch was used for different decontamination procedures.

2.2.4. Chemical Treatments

Three disinfectant solutions were tested by simulating domestic conditions, including 100 ppm of sodium hypochlorite, 80 ppm of peracetic acid and 40,000 ppm acetic acid (4%), together with tap water. For this purpose, six liters of each solution was placed in ten-liter tubs with leaves that were the equivalent of half a lettuce from the A-samples. They were kept submerged for ten minutes and stirred mid-way. Once the time was up, the excess liquid was removed with a domestic salad spinner, each sample was milled into a fine dust with liquid nitrogen and kept frozen until analysis.

2.2.5. Ultrasound Treatments

Two stainless steel ultrasound units (Elma, Germany) Transsonic TI-H (25 kHz, 45 kHz) and Elmasonic P (37 kHz, 80 kHz) with operating powers of 100–120 W were used. Ultrasound power dissipation (P) was determined calorimetrically, recording the temperature of the bath throughout the different procedures.

For each of the four frequencies, 70 g of chopped lettuce and three liters of cold distilled water were placed in the ultrasound baths to be treated for two time periods, ten or fifteen minutes. Each combination was tested twice.

After the application of the treatments, the excess water was eliminated using a domestic salad spinner; then, each sample was milled into a fine dust with liquid nitrogen and kept frozen until analysis.

2.3. Development and Validation of Extraction Method

After testing the three traditional QuEChERS methods (original, acetate and citrate), the acetate version without clean-up was validated according to the SANTE guidelines [7] for the 16 pesticides selected. It began with 5 g of lettuce in 50 mL Falcon tubes to which 10 mL of 1% HAc in MeCN were added. The mixture was shaken manually for one minute and then 4 g of MgSO₄ and 1 g of AcONa were added; after three minutes of manual shaking, the tube was centrifuged for 5 min at 3500 rpm. The extract was filtered through a 0.45 µm pore syringe filter into 2 mL glass vials for HPLC-MS/MS analysis.

3. Results

3.1. Method Validation

The acetate-buffered QuEChERS method without clean-up was chosen for this analysis after testing its performance against the original and citrate-buffered methods [8,9,10], and then against itself with a clean-up procedure. Once testing was done, the SANTE guidelines were followed in order to validate the selected methodology [7]. Three levels of concentration with five replicates each were put to the test, achieving limits of quantitation of 10 μg/kg for 12 pesticides, 25 μg/kg for carbendazim, chlorpyrifos methyl and pyraclostrobin, and 50 μg/kg for iprodione. This implies recoveries between 70–120% and standard deviations below 20%. The matrix effect was below 20% for all pesticides except for carbendazim, therefore this method has an advantage as it can be used without a matrix-matched calibration curve. Details of the validation parameters are shown in

Table 1. Recoveries, relative standard deviations (RSD), limits of quantitation (LOQ), linear ranges and matrix effects obtained for each pesticide.

Pesticide	10 μg/kg		25 μg/kg		50 μg/kg		LOQ (μg/kg)	Lineal Range (μg/kg)	Matrix Effect (%)
	Recovery (%)	RSD (%)	Recovery (%)	RSD (%)	Recovery (%)	RSD (%)
Acetamiprid	87	1	96	3	98	2	10	5–100	−14
Boscalid	92	10	90	9	108	9	10	5–50	1
Carbendazim	---	---	82	3	83	4	25	5–100	−29
Chlorpyirifos ethyl	112	5	100	5	100	3	10	5–100	1
Chlorpyrifos methyl	---	---	92	8	117	14	25	5–50	−8
Cyromazine	94	4	88	1	85	1	10	5–100	1
Dimethoate	97	3	97	1	98	1	10	5–100	−7
Fluvalinate	93	20	96	17	101	15	10	5–100	1
Imidacloprid	97	11	94	4	94	8	10	5–100	2
Iprodione	---	---	---	---	91	8	50	25–100	−7
Methomyl	104	4	96	2	96	3	10	5–100	0
Pirimicarb	101	2	96	2	96	1	10	5–100	0
Propamocarb	95	2	91	2	90	2	10	5–100	4
Pyraclostrobin	---	---	117	3	109	4	25	10–100	−3
Pyrimethanil	95	7	96	7	99	4	10	5–100	−4
Spynosad	102	2	100	3	101	3	10	5–100	−11

.

3.2. Commercial Lettuce Samples

Once the method was validated, it was challenged through the analysis of 22 commercial samples. Only seven of these lettuces showed pesticides residues at quantifiable levels for five pesticides (acetamiprid, boscalid, iprodione, propamocarb and pyraclostrobin). These results are presented in

Table 2. Maximum residue limits (MRL) for Codex Alimentarius (CA) and European Union (EU), results of positive samples.

Pesticide	MRL		Sample 12 (mg/kg)	Sample 13 (mg/kg)	Sample 14 (mg/kg)	Sample 15 (mg/kg)	Sample 16 (mg/kg)	Sample 17 (mg/kg)	Sample 18 (mg/kg)
	CA (mg/kg)	EU (mg/kg)
Acetamiprid	---	1.5	ND	ND	ND	ND	0.115	0.414	ND
Boscalid	---	50	ND	ND	ND	ND	0.315	0.985	ND
Carbendazim	5	0.1	ND	ND	ND	ND	<0.010	<0.010	ND
Iprodione	10	0.01	ND	ND	ND	ND	0.053	0.131	ND
Propamocarb	100	40	2.5	4.2	0,033	<0.010	ND	ND	10
Pyraclostrobin	40	2	ND	ND	ND	ND	0.125	0.308	ND
Pyrimethanil	---	20	ND	ND	ND	ND	<0.025	<0.010	ND
Spynosad	---	10	ND	< 0.010	ND	ND	<0.025	<0.010	ND

together with the maximum residue levels (MRL) from the Codex Alimentarius [2] and the European Union [11]. As it stands, all the samples were below the established regulations for Uruguay but iprodione failed EU [11] standards.

3.3. Decontamination Procedures

Regarding the cleaning solutions used, all four resulted in a reduction in the concentration of the applied pesticides when compared with the positive control. Chlorpyrifos proved to be the most resilient with reductions as little as 5% while pyraclostrobin was the easiest to remove with a maximum decrease of 90%. Still, no significant difference was found between the selected solutions according to the ANAVA test Pillai Bartlett with alfa 0.05.

Ultrasound baths were also demonstrated to be an effective method of pesticide-residue reduction. Each combination of time–frequency managed to lower the selected pesticides where, again, chlorpyrifos was a tough contestant with an average diminution of just 0%. On the other hand, pyraclostrobin had an average of 58%, with a maximum of 70%. Once more, despite the differences among the pesticides, the combinations did not show differences when analyzed with the ANAVA test Pillai Bartlett with alfa 0.05.

All in all, though vastly different in their mode of action and cost, both the ultrasound and the cleaning solutions managed to achieve what was expected, and amazingly, there was no significant differences among the 12 methodologies when using the ANAVA test Pillai Bartlett with alfa 0.05, as the p-value was 0.28. All results are shown in Figure 1.

4. Conclusions

A fit-for-purpose methodology based on the QuEChERS approach for the determination of pesticide residues in lettuce was developed and validated for 16 compounds. The method had its applicability tested in the analysis of 22 commercial samples, where seven samples showed pesticide residues, below the corresponding MRL.

Both the ultrasound and the cleaning solutions managed to reduce pesticide residues without significant differences among them. Chlorpyrifos proved to be the most resilient while pyraclostrobin was the easiest to remove.