The method was applied to 10 different product samples (dill, arugula, bell pepper, beetroot, daikon radish, apple, cucumber, zucchini, tomato). The proposed method successfully showed compliance with nitrate levels corresponding to established safety limits. Nitrates (NO₃⁻) are naturally present in soil and are taken up by plants as a nitrogen source in nitrogen life cycles. However, overuse of nitrogen fertilizers in agriculture leads to nitrate accumulation in plant tissues. After ingestion, nitrates can be reduced to nitrites (NO₂⁻), which may interfere with oxygen transport and, under certain conditions, form carcinogenic N‑nitroso compounds. Therefore, maximum residue limits (MRLs) for nitrates have been established for various vegetables — e.g., 2000–4500 mg/kg for leafy greens, 250–1000 mg/kg for root vegetables, depending on the country and season. Existing analytical methods for nitrate quantification (ion chromatography, spectrophotometry with cadmium reduction, mass spectrometry) are precise but require laboratory infrastructure and are expensive. This creates a significant barrier for consumers.

Method

1. Chemicals and Reagent

Potassium nitrate (KNO₃, ≥99 %, Sigma‑Aldrich)- used for standard solutions; zinc powder– reducing agent; Griess reagent: sulfanilamide (5 g) dissolved in 200 mL 5 % phosphoric acid; N‑(1‑naphthyl)ethylenediamine dihydrochloride (0.5 g) dissolved in 200 mL distilled water ((Both solutions were mixed 1:1 immediately before use)); distilled water- for all dilutions.

2. Standard Solutions Preparation

A 10,000 mg/L NO₃⁻ stock solution was prepared by dissolving 16.30 g KNO₃ in 1 L distilled water. Working standards of 0.1, 0.5, 1, 5, 10, 25, 50, 100, 250, 500, and 1000 mg/L NO₃⁻ were prepared by serial dilution to produce color scale. All solutions were used within 24 hours (nitrate standards are stable, but the reduction step is sensitive).

3. Zinc reduction; Griess assay procedure

The method is based on reduction of nitrate to nitrite by zinc powder in acidic medium, followed by reaction with Griess reagent to form a pink‑to‑red color.

1. Sample preparation: fresh vegetables/fruits (50 g) were homogenized with 50 mL distilled water using a blender. The homogenate was filtered through a paper coffee filter.

Fig. 1. Homogenization of dill

2. Dilution: because preliminary tests showed that undiluted extracts caused excessive colour (brown precipitate), samples were diluted 10‑ to 100‑fold with distilled water (depending on expected nitrate content).

Fig. 2. Undiluted extracts of dill caused excessive colour (brown precipitate)

3. In a 10 mL test tube, 1 mL of diluted sample (or standard solution) was mixed with 0.5 mL of 1 % zinc powder suspension in distilled water. The mixture was shaken for 3 minutes at room temperature.
4. Griess reaction: After zinc particles settled (30 sec), 1 mL of the supernatant was transferred to a clean tube, mixed with 0.5 mL of freshly mixed Griess reagent, and allowed to stand for 10 minutes.

Colour reading: The resulting pink‑to‑red colour was visually compared against a colour scale prepared from known nitrate standards (Figure 3).

*When available, absorbance was measured at 540 nm using a portable colorimeter (for validation only; visual reading is the intended method).

Fig. 3. Colour scale from 0.1 to 50 mg/L NO₃⁻

Sample Collection

Ten types of fresh products were purchased from a local market in Moscow in February–May 2023: dill (Anethum graveolens), arugula (Eruca vesicaria), bell pepper (Capsicum annuum), beetroot (Beta vulgaris), daikon radish (Raphanus sativus), apple (Malus domestica), cucumber (Cucumis sativus), zucchini (Cucurbita pepo), tomato (Solanum lycopersicum). All samples were processed within 2 hours of purchase.

User Feedback

After finalizing the method, five members of the target audience were recruited (home vegetable gardeners and students) to test the method. Three success criteria were defined: (1) ability to distinguish samples with nitrate levels above/below the local safety limit (objective, via colour comparison); (2) reproducibility of results between two independent users (objective); (3) overall satisfaction score (subjective, 1–5 scale).

Results

Using the optimized zinc–Griess protocol, standard nitrate solutions produced a clear pink‑to‑red colour progression (Figure 3). Concentrations above 50 mg/L (e.g., 100 mg/L) produced a red colour but required dilution for accurate visual matching. The visual detection limit was approximately 2–3 mg/L. For fresh produce, after applying the sample dilution factor (typically 10‑ to 100‑fold), the working range corresponds to 50–5000 mg/kg fresh weight, which covers all relevant safety limits. Table 1 presents the nitrate concentrations determined by visual matching of the diluted sample colours to the standard scale. For each sample, three independent replicates were analysed; the reported value is the median concentration (in mg NO₃⁻ per kg fresh weight).

Table 1

Nitrate concentrations in selected vegetables and fruits

*MRL = Maximum residue limit based on European Union regulation (EC) No 1881/2006 for summer/autumn harvest, converted to fresh weight basis.

The highest nitrate levels were found in leafy greens (dill, arugula) and beetroot — all within expected ranges. Root vegetables (daikon) showed intermediate levels. Fruits (apple, tomato) and cucurbits (zucchini, cucumber) had low nitrate content.

Discussion

1. Method Performance and Limitations:

The proposed zinc–Griess method provides a semi‑quantitative nitrate estimate with a detection limit of ~10 mg/kg in fresh produce it allows to distinguish compliance vs. non‑compliance with most safety standards (typically 200–3000 mg/kg). Still the main limitation is the need for a fresh Griess reagent (stable for about one week when refrigerated) and careful control of the zinc powder amount.

2. Comparison with Other Low‑Cost Methods:

Commercially available nitrate test strips (e.g., Merckoquant®) cost $1–2 per strip and are not widely accessible in all regions; moreover they are less precise as mostly they are adapted towards aquarium tests. The classical cadmium reduction method is highly sensitive but uses toxic cadmium powder, making it unsuitable for home use. This zinc‑based method is safer (zinc is an essential micronutrient) and cheaper ($0.05 per test for zinc and Griess reagents). Similar zinc reduction approaches have been reported for water analysis but rarely applied to plants due to colour interferences, however dilution and filtration steps effectively removed most chlorophyll and anthocyanin pigments.

Commercially available nitrate test strips tested on known concentrations of nitrates showed no difference, confirming that those tests are useless for vegetables/fruits testing.(image #)

Fig. 4. Commercially available nitrate test strips tested on known concentrations of nitrates

Conclusion

A simple, low‑cost colorimetric method for nitrate detection in vegetables was developed using zinc powder reduction and Griess reagent. The method reliably distinguishes nitrate levels relevant to food safety regulations, with a visual detection limit of 10 mg/kg. User testing confirmed that the method is practical for home gardeners and students and other individuals.

References:

1. EFSA balances the consumer risks from nitrate in vegetables with the benefits of a balanced diet high in vegetables and fruit. — Текст: электронный//efsa.europa.eu: [сайт].—URL:https://www.efsa.europa.eu/en/news/efsa-balances-consumer-risks-nitrate-vegetables-benefits-balanced-diet-high.
2. Zolotov_praktikum.pdf. — Текст: электронный // chembaby.ru: [сайт]. — URL:https://chembaby.ru/wp-content/uploads/2015/09/Zolotov_praktikum.pdf.
3. nitrate-nitrite-background-document.pdf. — Текст: электронный // who.int: [сайт].—URL:https://www.who.int/docs/default-source/wash-documents/wash-chemicals/nitrate-nitrite-background-document.pdf.
4. Bioaccumulation of trace elements in vegetables grown in various anthropogenic conditions. — Текст: электронный // ResearchGate: [сайт]. —URL: https://www.researchgate.net/publication/365510538_Bioaccumulation_of_trace_elements_in_vegetables_grown_in_various_anthropogenic_conditions .
5. Development and Validation of a Method for Determination of Residual Nitrite/Nitrate in Foodstuffs and Water After Zinc Reduction. — Текст: электронный//ResearchGate: [сайт].—URL:https://www.researchgate.net/publication/225808840_Development_and_Validation_of_a_Method_for_Determination_of_Residual_NitriteNitrate_in_Foodstuffs_and_Water_After_Zinc_Reduction.