Method validation and analytical quality assurance
A single-laboratory validation was carried out following the standard protocol adopted in the laboratory. The following parameters were evaluated: linearity, working range, detection and quantification limits, precision, recovery, and accuracy. Calibration curve was plotted, and linearity was evaluated by the values of determination and variation coefficients of the method after the application of several statistical tests. The calibration curves were obtained using a series of nitrate and nitrite standard solutions. All calibration curves were linear with correlation coefficients from the linear regression ranging from 0.992 to 0.999. Method performance data for nitrate and nitrite determination in baby-leaf salads are shown in Table 1. SPSS for windows version 17.0 and JMP windows version 7.01 (JMP SAS Institute, Cary, NC) were used to calculate all statistical parameters (means, standard deviations, coefficient of variation, minimum and maximum, correlation coefficient), and a t-test was used for determination of significant differences between the mean values.
Table 1. Method performance data
|Analyte||Matrix||LOD (mg/kg)||LOQ (mg/kg)||Recovery range (%)||RSDr (n = 6) (%)||Measurement uncertainty (%)||Accreditation (Yes/No)|
|NO3||Baby-leaf salad (lettuce)||1.2||1.4||73–105||3.30||7.7||Noa|
|NO2||Baby-leaf salad (lettuce)||0.1||0.1||70–110||14.2||22||Noa|
Nitrate contents of baby-leaf salads
To our knowledge, this is the first report that compares the nitrates/nitrites levels of fresh and baby-leaf salads in two types of agriculture. Individual results obtained for nitrate and nitrite levels are shown in Tables 2 and 3.
Table 2. Nitrate and nitrite concentrations in baby-leaf salads from organic and conventional farming in Northern Portugal.a,b
|Plant material||Agriculture production system||[ ] Nitrites (mg/kg FW)||[ ] Nitrates (mg/kg FW)|
|Green lettuce||Conventional||0.25 ± 0.05a||26.05 ± 2.09b|
|Organic||0.32 ± 0.13a||6.40 ± 1.48a|
|Red lettuce||Conventional||1.41 ± 0.11a||45.19 ± 4.54b|
|Organic||1.89 ± 0.02b||5.16 ± 2.26a|
|Watercress||Conventional||0.81 ± 0.19a||42.76 ± 7.19b|
|Organic||0.93 ± 0.01a||1.45 ± 0.30a|
Table 3. Nitrate and nitrite concentrations in baby-leaf salads from conventional farming in Northern Portugal.a,b
|Plant material||[ ] Nitrites (mg/kg FW)||[ ] Nitrates (mg/kg FW)|
|Rucola (Rocket)||0.18 ± 0.02a||17.82 ± 3.84b|
|Chard||3.48 ± 0.30b||23.13 ± 6.77b|
|Corn salad||0.14 ± 0.02a||10.57 ± 1.189a|
The results showed a considerable significant variation in the average levels of nitrate contents between the two production systems. The average levels of nitrates were higher (P < 0.05) in conventional produce, and as expected, the averages levels of nitrites were lower when compared with nitrates. The average levels of nitrites, except red lettuce, were very similar (P > 0.05) in both organic and conventional agriculture system (Tables 2 and 3). It seems that only the nitrate levels were significantly affected by the type of production. Similar tendency was found by Pussemier et al. (2006), who reported significant differences in the average levels of nitrate contents from organic and conventional produce. They reported lower levels of nitrates in organic (1703 mg/kg) and higher in conventional (2637 mg/kg) produces. Also, our findings showed a nitrate variation with plant family being the Asteraceae (lettuce) and Brassicaceae (watercress) – the families with the highest average levels. This result is in agreement with Santamaria (2006), who stated that families like Brassicaceae (rocket, radish, mustard and cress), Chenopodiaceae (beetroot, Swiss card, spinach), Asteraceae (lettuce), and Apiaceae (celery, parsley) are usually, among the vegetables, the plant families with highest nitrate contents. This tendency was confirmed in the present study.
The limits detected for nitrate in our samples are within the legal limits (<3000 mg/kg FW for lettuce and similar samples) recommended by European Union regulations (Regulation [EC] No. 1258/2011); thus, from the point of view of nitrates, this type of vegetables are safe. Moreover, the average content of nitrates is very far from those presented by Mor et al. (2010), which makes them very interesting from nutritional perspective. These plant materials can be used safely in adults, but also in infant meals, which oblige very low levels of nitrates and nitrites (Greer and Shannon 2005; Chan 2011).
One important aspect particularly to human health is related to contamination with nitrites. It is well accepted that when nitrates is reduced to nitrites, nitrite may react with amines or amides to form carcinogenic compounds (Savino et al. 2006). With regard to nitrite content of baby-leaf salads studied, our results showed a variation between 0.14 mg/kg FW for corn salad and 1.89 mg/kg FW for red lettuce. Moreover, only in the red lettuce, significant differences in nitrite contamination between organic and conventional produce (P < 0.05) were observed. For green lettuce and watercress, there were no significant differences. Nevertheless, the average nitrite content was higher in organic produce. Compared with literature, our average values are very similar to those reported in fresh vegetables (González et al. 2010; Mor et al. 2010). It is commonly assumed that the nitrite levels in fresh leafy vegetables are usually less than 2 mg/kg FW (Santamaria 2006). In this study, nitrite levels, except in chard, were lower than 2 mg/kg FW and very lower than the limits considered toxic (EU 1995).
Chung et al. (2004) and Prasad and Chetty (2008) have demonstrated that well-storage conditions are necessary to maintain the nitrites in low concentrations, due to minor activity of the enzyme reductase, responsible for reduction of nitrates in nitrites, and/or microbiological reduction of nitrates into nitrites. Under refrigerated storage, the nitrite accumulation tends to be reduced or even totally inhibited (Prasad and Chetty 2008). It seems to be the case of the samples studied in the current study. In fact, a poor storage must be avoided; otherwise, it results in bacterial growth, which can contribute to the increasing accumulation of high nitrite levels. Nitrate or even nitrite accumulation is dependent not only of agriculture system and respective practices but also of soil properties, fertilizer usage, cultivation, weather conditions, harvesting time, and size of vegetables and storage conditions (Tamme et al. 2006), which are unknown and whose effects are impossible to account in this study.
To conclude, based on our results, it seems that baby-leaf salads produced in organic and conventional systems in Northern Portugal have low levels of nitrates and nitrites. Therefore, they are toxicologically safe, and their consumption might be incremented without risk for health. The differences between organically and conventionally cultivated plants are less but exist. Nevertheless, it does not represent any type of risk for human health. These results are important not only for adults but particularly for children, in which the toxicological aspects related with nitrates and nitrites accumulation assume more importance.