Pesticide residues analysis involves sensitive instrumental techniques and proper skills in handling the whole experimental procedure. A small negligence can give rise to gross errors in the results. Therefore, it should be demonstrated that the method being used is fit for the intended purpose.
In the present study, EU-citrate buffered QuEChERS method has been validated for the extraction of selected organochlorine, organophosphate, and synthetic pyrethroids in brinjal and cauliflower. The method showed linearity (R2 > 0.99) in the working range (1×MRL, 2×MRL, and 4×MRL) and the residual concentration was within the limit of ± 20 percent as per SANTE Guidelines (2019). The method accuracy was within the acceptable limits. The sensitivity of the method was sufficient enough to be used for routine pesticide residue monitoring studies. The matrix effect was in the range of -11.4 to 54.7 percent indicating sufficient cleaning efficiency of the procedure. Quality control (QC) checks at the limit of quantification (LOQ) were run with real samples in each sequence.
A total of 25 brinjal samples and 26 cauliflower samples were analyzed among which 22 and 18 samples (88, 69 percent) were contaminated and 20 percent were exceeding MRLs set by EU. Chlorpyrifos was the most frequently detected pesticide found in both brinjal and cauliflower. It was detected in brinjal samples from Dera Ismail Khan, Mansehra, and Mandi-bahauddin and cauliflower samples originated from Swabi, Mansehra, Huzro, Sawat, Haripur, and Multan. Chlorpyrifos is an organophosphorus insecticide classified as WHO category II (Turner, 2018) that is widely used for the treatment of pests (leaf hopper, fruit borer) affecting brinjal and cauliflower production (Omprakash, 2014). The results were consistent with other studies reporting chlorpyrifos in brinjal samples from district Gujranwala (Iqbal et al., 2009; Latif, et al., 2011). It has been recognized as endocrine disruptor chemicals (UNEP, 2016). Due to the perilous effect of chlorpyrifos on human health and the environment, the European Union legislated to ban the use of chlorpyrifos in 2020 (EFSA, 2020).
Another interesting finding of the study is that endosulfan has not been detected in any sample but the presence of one of its isomer endosulfan sulfate in 24 percent brinjal and 3.8% of cauliflower samples indicates its translocation/uptake from drift or previous applications from the soil. These samples were supplied in the Islamabad market from Mandi bahauddin, Bhakkar, Gorakhpur, Faisalabad, Sargodha, Khushab, and Narowal. Endosulfan is an organochlorine compound and its use has recently been prohibited in Pakistan as it has been enlisted as Persistent Organic Pollutants (POPs) under Stockholm Convention on POPs in 2017. Since most of the organochlorine pesticides (OCPs) are banned and organophosphate (OPs) are highly toxic and hazardous compounds hence pyrethroids are among the most extensively used pesticides in the world for vegetables. Among the four pyrethroids viz. bifenthrin, cypermethrin, ʎ-cyhalothrin, and deltamethrin were investigated, only the residues of ʎ-cyhalothrin were detected in both brinjal and cauliflower samples. ʎ-cyhalothrin is recommended and widely used for the treatment of bugs and fruit borer which affects brinjal production severely in Pakistan (Ali, 2018). The contaminated samples were originated from Multan, Faisalabad, Mandi bahauddin, Layyah, Bhakkar, Mardan, Sahiwal, Attock, Sargodha, DI khan, and Mansehra. The residues of different PYs in vegetables have also been reported in Pakistan and other countries like Bangladesh (Amjad et al., 2019; Hossain et al., 2013, Ali, et al., 2019). In the present study although the concentration of λ-cyhalothrin in brinjal samples was below EU-MRL. Its higher detection frequency may be of great concern as dietary intake from other sources might pose serious accumulative implications on human health. Long-term exposure to a low dose of pyrethroid residues could lead to chronic diseases e.g nervous and immune system impairment, cardiovascular, and genetic disorders (Zhao et al., 2014). This compound is more toxic than the majority of the active ingredients approved in the European Union (EU 2017).
The causes of the occurrence of multiple residues in brinjal and cauliflower might be because different compounds are used individually or as a mixture. Another cause might also be spray drift from neighboring plots (Lozowicka, 2015). Endosulfan is a highly persistent pesticide and may travel long distances in the air. The researcher has reported residues of endosulfan in crops approximately 121 miles from the sprayed agricultural site (Bradford et al., 2010). Moreover, this pesticide may persist in soil for several years before degradation. The concentration of pesticide mixture applied to the crop before harvesting above the recommended level may be the reason for the high level of chlorpyrifos in cauliflower samples.
From a consumer health perspective, it is important to compare exposure estimates to established toxicological criteria such as EDI, ESTI, which are the realistic estimation of pesticide residues exposure (Lehmann et al., 2017; Li et al., 2018). All the aHIs for selected pesticides were less than unity except chlorpyrifos, which meant there was a negligible short-term or acute risk except chlorpyrifos. Chlorpyrifos presented an HQ close/exceeding the unity. Chlorpyrifos may induce hematological malignancies, behavioral and developmental anomalies, oxidative stress, genotoxicity, and immunotoxicity (ur Rahman et al., 2021). Ventura et al., (2016) reported chlorpyrifos is one of risk factor for breast cancer. It proliferate the human breast cancer cells by enhancing progesterone receptor (PgR), decreased serum the progesterone, estradiol, and luteinizing hormone levels (Ventura et al., 2016). In study conducted on Thai pesticide sprayer, Kongtip, et al., 2021 concluded its acute exposures adversely affect the hypothalamic–pituitary–thyroid (HPT) axis, which result in disturbance of human thyroid hormone levels (Kongtip, et al., (2021). Other pesticides (λ-cyhalothrin, endosulfan sulfate) were found in concentrations yielding smaller HQ values. From the public health point of view, the observed levels of pesticide residues do not pose a serious health risk to the consumers, but the presence of pesticide residues in food items even at the lowest possible level should be a matter of concern as the low level does not remain low forever.
The difference in potential health risks in gender-based two sub-population may be to the fact that everyone does not necessarily consume both vegetables each day and in similar proportions. For this reason, a deterministic approach or average portions might underestimate or overestimate an individual's dietary intake. To overcome this shortage, the assumption was made that only 68 percent of the Pakistan population consuming these vegetables. This assumption is supported by the fact that the global diet is generally poor and monotonous (Savy et al., 2003). In absence of extreme intake values, these average estimates yielded no acute or chronic risk. A food frequency questionnaire (FFQ) could be used in further studies to validate the estimation of propensity-to-consume items and refined the presented assessment in the future.
Exposure to pesticide mixtures in cumulative risk assessment (HI) was associated with a larger intake of pesticides with the same mode of action thus resulting in a higher hazard for the consumer. As chlorpyrifos and λ-cyhalothrin do not share the same mode of action thus they are not considered in the same cumulative assessment group (CAG). Under these conditions, ∑HIs which correspond to the sum of HIs from the same CAG will not yield a value significantly different from the HIs of these pesticides. Joint use of several formulation/trademarks alone or in combination with a single vegetable is the probable reason for multiple pesticide detections. Moreover, illiteracy, lack of awareness, and poor labeling quality (foreign language, etc.) and counterfeiting could have led to misinterpretations.