Determination of Pesticide Residues in Fresh Fruits in the Republic of Serbia by LC-MS/MS

1. Introduction

Pesticides play a significant role in food production. They protect or increase yields and may increase the number of times each year a crop can be grown on the same land. Pesticides are used to protect crops against insects, weeds, fungi and other pests. Since they are designed to be biologically active, pesticides are potentially toxic to humans and can have both acute and chronic health effects, depending on the quantity and ways in which a person is exposed [1]. For this reason, pesticide residue levels in food are regulated by national and European legislation. These comprehensive legislative frameworks define rules for the approval of active substances, their uses in plant protection products and their permissible residues in food. To ensure a high level of consumer protection, legal limits, or so-called ‘maximum residue levels’ (MRLs) are established in Regulation (EC) No 396/2005 [2]. European Union harmonized MRLs are set for more than 1,300 pesticides covering 378 food products/food groups [3]. This regulation is being continuously amended by several Regulations and Commission Regulations to update the commodities included and their residue levels, based on the most recent knowledge from the Member States, EFSA and the Commission. In Serbia, Regulation on the ’’maximum residue levels of pesticides in food’’ have changed few times over the past 10 years [4,5,6], with the aim of aligning the national legislation with the EU regulation. The latest Serbian Regulation [7] on the ’’maximum residue levels of pesticides in food’’ is fully harmonized with the European Union Regulations.

There have been many surveys of pesticide residues in fruits/food recently [3,8,9,10,11,12,13,14,15,16,17,18]. Summary of the studies published in available literature dealing with pesticide residues in fruit (from 2010 onwards) are presented in Supplementary material 1 Table S1. The overall conclusion of these studies is that there is a widespread pesticide presence currently in fruits/food in general. The pesticides are frequently detected above the LOQ, however MRL is seldom exceeded. And some of the pesticide residues were detected with concentrations above their MRL. Also, pesticides that are found in these studies are banned and/or unauthorized in many countries because of their high toxicity. Therefore, pesticide residues control is an important activity intended to prevent, reduce or eliminate the chemical hazard in food.

The objective of this study was to investigate the concentrations of pesticide residues in fruits (Table 1), which are collected as a part of the national monitoring program for pesticide residues in Serbia and to compare these levels with maximum residue levels established by the Serbian Regulation [4,5].

2. Materials and Methods

Over 4-year period, concentrations of pesticide residues were determined in 2,164 samples of fresh fruits (Table 1). The analyses were conducted by an accredited state laboratory (A BIO TECH LAB). The method for sample preparation and analysis of the concentrations of pesticide residues in the collected fresh fruits was conducted as described in detail in Kecojević et al. [19]. A total of 173 (Table 2) pesticide residues were detected in these 2,164 samples. The limit of quantitation (LOQ) for all pesticide residues was 0.005 mg/kg (LOQ for cabbage, Kecojević et al. [19]), while reporting limit (RL) was 0.01 mg/kg. Generally, Serbian as well as EU MRLs for pesticide residues in fruits are in the range of 0.01–10 mg/kg, depending on the compound. Only for a few pesticides MRLs are up to 15 and 20 mg/kg. The results were evaluated according to the RL and MRLs that have been established by Serbian regulation.

3. Results and discussion

The individual concentrations of the analyzed pesticide residues in all samples of fruits (N = 2,164) are shown in the Supplementary material 2 (individual results). All pesticide residues at or above the reporting limit (RL ≥ 0.01 mg/kg) are reported.

In this study, a total of 2,164 samples of fresh fruits were analyzed for pesticide residue. A yearly total of 136 (6.28%), 651 (30.08%), 687 (31.75%) and 690 (31.89%) of these samples were analyzed in 2016 (Serbian fruits: 57 samples; imported fruits: 79 samples), 2017 (Serbian fruits: 38 samples; imported fruits: 613 samples), 2018 (Serbian fruits: 53 samples; imported fruits: 634 samples) and 2019 (Serbian fruits: 51 samples; imported fruits: 639 samples), respectively. Detailed characteristics like common name, country of origin and number of samples (without and with pesticide residues) of the analyzed samples are shown in Table 1. The evaluation of the obtained results of 2,164 different fruit samples has shown (Table 1) that 62.57% (1,354 out of the 2,164 samples) of all samples contained pesticide residues (RL ≥ 0.01 mg/kg) and 37.43% (810 out of the 2,164 samples) of the samples contained no pesticide residues (RL < 0.01 mg/kg). All samples of aronia (N = 1), Brazil nut (N = 2), carambola (N = 2), cashew (N = 1), chestnut (N = 2), fig (N = 1), lychee (N = 1), passion fruit (N = 1), pistachio (N = 1), pitaya (N = 2) and quince (N = 1) were pesticide-free. The detection rates (when the sample size ’’N’’ is greater than 30) of pesticide residues in peach (nectarine) (N = 36), pineapple (N = 41), strawberry (N = 46), kiwi (N = 50), peach (N = 56), pomegranate (N = 66), pear (N = 95), grapefruit (N = 115), grape (N = 152), banana (N = 168), mandarin (N = 194) lemon (N = 217), orange (N = 324) and apple (N = 351) samples were 36.11%, 73.17%, 76.09%, 34.00%, 42.86%, 63.64%, 71.58%, 88.70%, 34.87%, 58.33%, 69.07%, 80.65%, 69.75% and 64.10%, respectively. All pesticide residues detected in almond (N = 6), apricot (N = 15), aronia (N = 1), banana (N = 168), blackberry (N = 2), blueberry (N = 5), Brazil nut (N = 2), carambola (N = 2), cashew (N = 1), chestnut (N = 2), clementine (N = 16), coconut (N = 1), currants (N = 2), date palm (N = 8), fig (N = 1), hazelnut (N = 5), Japanese apple (N = 9), kiwi (N = 50), kumquat (N = 4), lychee (N = 1), mango (N = 24), passion fruit (N = 1), peach (N = 56), peach (nectarine) (N = 36), peanut (N = 8), pistachio (N = 1), pitaya (N = 2), plum (N = 26), pomelo (N = 12), quince (N = 1), raspberry (N = 8), strawberry (N = 46), sweet cherry (N = 20) and walnut (N = 6) were below or at the MRLs. The MRLs for pesticide residues were exceeded in 101 out of the 2,164 (4.67%) samples: apple (12 out of the 351 samples, 3.42%; Bosnia and Herzegovina: N = 2, North Macedonia: N = 2, Poland: N = 3, Serbia: N = 5), avocado (1 out of the 19 samples, 5.26%; Peru: N = 1), grapefruit (21 out of the 115 samples, 18.26%; South Africa: N = 4; Turkey: N = 17), grapes (2 out of the 152 samples, 1.32%; North Macedonia: N = 2), lemon (4 out of the 217 samples, 1.84%; Argentina: N = 1, Turkey: N = 2), lime (4 out of the 28 samples, 14.29%; Guatemala: N = 1, Mexico: N = 2, The Netherlands: N = 1), mandarin (14 out of the 194 samples, 7.22%; Spain: N = 2, Swaziland: N = 1, Turkey: N = 11), orange (7 out of the 324 samples, 2.16%; Spain: N = 1, Turkey: N = 6), pear (6 out of the 95 samples, 6.32%; Poland: N = 2, Serbia: N = 4), pineapple (3 out of the 41 samples, 7.32%; Colombia: N = 1, Costa Rica: N = 2), pomegranate (26 out of the 66 samples, 39.39%; Turkey: N = 26) and sour cherry (1 out of the 14 samples, 7.14%; Serbia: N = 1).

The frequency of the detected pesticide residues in fruit samples are shown in Table 2. A total of 173 pesticide residues (distributed as: 49.13% insecticides, 35.84% fungicides and 15.03% herbicides) were detected in all the fruit samples (Table 2). Imazalil, thiabendazole, pyrimethanil, imidacloprid, carbendazim, acetamiprid, boscalid, fludioxonil, chlorpyrifos, prochloraz, pyriproxyfen, tebuconazole, propiconazole, prothioconazole, azoxystrobrin, methoxyfenozide and pyraclostrobin were the pesticide residues most frequently found (occurrence in more than 90 analyzed samples, > 4%) and were detected in 624 (28.84%), 337 (15.57%), 245 (11.32%), 213 (9.84%), 185 (8.55%), 171 (7.90%), 154 (7.12%), 154 (7.12%), 145 (6.70%), 143 (6.61%), 138 (6.38%), 116 (5.36%), 113 (5.22%), 97 (4.48%), 96 (4.44%), 94 (4.34%) and 93 (4.30%) samples, respectively. Of the 173 pesticide residues, 64 (36.99%) of them were detected at least once in fruit samples in level higher than MRLs. A total of 309 pesticide residues (133 in pomegranate, 43.04%; 95 in grapefruit, 30.74%; 33 in mandarin, 10.68%; 13 in apples, 4.21%; 11 in orange, 3.56%; 7 in pear, 2.27%; 5 in lime, 1.62%; 4 in lemon, 1.29%; 3 in pineapple, 0.97%; 2 in grape, 0.65%; 2 in sour cherry, 0.65; 1 in avocado, 0.32%) were found in the 101 fruit samples containing residues above MRLs. The other 109 (63.01%) pesticide residues did not exceed their MRL values. The most frequent pesticide residues found to exceed the MRL were butoxycarboxim (100%, 5 out of 5 samples), carbofuran (100%, 1 out of 1 sample), deltamethrin (60.00%, 12 out of 20 samples), fenvalerate (60.78%, 31 out of 51 samples), formothion (58.82%, 10 out of 17 samples), iprodione (66.67%, 2 out of 3 samples), mepanipyrim (61.54%, 8 out of 13 samples), mepronil (100%, 1 out of 1 sample), nuariomol (66.67%, 2 out of 3 samples), oxamyl (100%, 10 out of 10 samples), piperonyl-butoxide (50.00%, 1 out of 2 samples), pirimiphos-methyl (83.33%, 10 out of 12 samples), promecarb (71.43%, 5 out of 7 samples), prometryn (75.00%, 3 out of 4 samples), sulfentrazone (50.00%, 2 out of 4 samples), tebuthiuron (63.64%, 7 out of 11 samples), terbutryn (50.00%, 3 out of 6 samples) and tricyclazole (100%, 3 out of 3 samples). Among the mostly detected pesticide residues, imazalil was found at the highest concentration (93.349 mg/kg, 18.67 times higher than MRL) in grapefruit.

An overview of the number of residue residues per sample are shown in Table 3a and Table 3b. Many samples contained several pesticide residues. A total of 5,078 individual pesticide residues were found in the 1,354 fruit samples containing residues. Of the 2,164 samples analyzed, a single pesticide residue was detected in 252 (11.65%) samples and two, three, four, five and six pesticide residues in 278 (12.85%), 227 (10.49%), 203 (9.38%), 145 (6.70%) and 103 (4.76%) samples, respectively. Seven or more pesticide residues were detected in 6.75% of the samples. The samples with highest number of pesticide residues were two samples of grapefruit both with 44 pesticide residues.

Table 3. a. Number of pesticide residues in an individual sample.

No. of pesticide residues	0	1	2	3	4	5	6	7	8	9	10	11
No. of samples	810	252	278	227	203	145	103	65	34	19	7	2
%	37.43	11.65	12.85	10.49	9.38	6.70	4.76	3.00	1.57	0.88	0.32	0.09
No. of samples with residues above the MRL	0	7 (7 with 1)	17 (13 with 1; 4 with 2)	10 (7 with 1; 3 with 2)	12 (4 with 1; 6 with 2; 1 with 3; 1 with 4)	8 (3 with 1; 2 with 2; 2 with 3; 1 with 5)	15 (7 with 1; 6 with 2; 1 with 3; 1 with 4)	7 (3 with 1; 1 with 2; 2 with 3; 1 with 5)	8 (3 with 1; 3 with 2; 1 with 3; 1 with 4)	5 (3 with 1; 1 with 2; 1 with 4)	1 (1 with 1)	0

Table 3. a. Number of pesticide residues in an individual sample.

No. of pesticide residues	0	1	2	3	4	5	6	7	8	9	10	11
No. of samples	810	252	278	227	203	145	103	65	34	19	7	2
%	37.43	11.65	12.85	10.49	9.38	6.70	4.76	3.00	1.57	0.88	0.32	0.09
No. of samples with residues above the MRL	0	7 (7 with 1)	17 (13 with 1; 4 with 2)	10 (7 with 1; 3 with 2)	12 (4 with 1; 6 with 2; 1 with 3; 1 with 4)	8 (3 with 1; 2 with 2; 2 with 3; 1 with 5)	15 (7 with 1; 6 with 2; 1 with 3; 1 with 4)	7 (3 with 1; 1 with 2; 2 with 3; 1 with 5)	8 (3 with 1; 3 with 2; 1 with 3; 1 with 4)	5 (3 with 1; 1 with 2; 1 with 4)	1 (1 with 1)	0

Table 3. b. Number of pesticide residues in an individual sample.

No. of pesticide residues	12	13	15	17	26	29	30	31	33	36	39	44
No. of samples	4	4	1	1	1	1	1	1	1	1	1	2
%	0.18	0.18	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.09
No. of samples with residues above the MRL	2 (1 with 1; 1 with 2)	0	0	0	1 (1 with 16)	1 (1 with 17)	1 (1 with 11)	1 (1 with 17)	1 (1 with 18)	1 (1 with 16)	1 (1 with 20)	1 (1 with 19; 1 with 22)

Table 3. b. Number of pesticide residues in an individual sample.

No. of pesticide residues	12	13	15	17	26	29	30	31	33	36	39	44
No. of samples	4	4	1	1	1	1	1	1	1	1	1	2
%	0.18	0.18	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.05	0.09
No. of samples with residues above the MRL	2 (1 with 1; 1 with 2)	0	0	0	1 (1 with 16)	1 (1 with 17)	1 (1 with 11)	1 (1 with 17)	1 (1 with 18)	1 (1 with 16)	1 (1 with 20)	1 (1 with 19; 1 with 22)

4. Conclusions

In this study, pesticide residues were determined in 2,164 samples of the most popularly consumed fruits in Serbia (during 2016–2019). There were 1,354 (62.57%) samples contaminated with pesticide residues, of which 101 (4.67%) samples were higher than the MRLs. Among the 101 fruit samples with MRL exceedances, pomegranate was the fruit with the highest number of MRL exceedances (26 samples, 25.74%), followed by grapefruit with 21 samples (20.79%), mandarin with 14 samples (13.86%), apple with 12 samples (11.88%), orange with 7 samples (6.93%), pear with 6 samples (5.94%), lemon and lime with 4 samples each (3.96%), pineapple with 3 samples (2.97%), grapes with 2 samples (1.98%) and avocado and sour cherry with 1 sample each (0.99%). Pomegranate, grapefruit and mandarin showed the highest number of samples with multiple pesticide residues higher than MRLs. These results highlight the need to continuously monitor pesticide residues in fruits in order to fully protect public health.