Introduction

Saffron (Crocus sativus) belongs to the Iridaceae family and possesses high nutritional and medicinal value due to its biologically active ingredients1,2. The synthesize of apocarotenoids, such as picrocrocin and crocin, is a unique ability of a few species, including Crocus. This distinctive feature, coupled with numerous medicinal, food and health applications, makes saffron as an attractive candidate for organic production3. Organic crop production, particularly of medicinal plants, is crucial for public health, as the consumption of contaminated food and medicinal products poses serious risks to humans4.

Historically, saffron has been primarily produced in Iran, India, Italy, Greece, Spain, Morocco, and Turkey. However, its cultivation has recently expanded as a new crop in various parts of the world, including the USA, Afghanistan, and South Africa2,5,6. Given that approximately 90% (439 t y−1 dried stigma) of the world’s saffron originates from Iran1,7, the development of organic and healthy production methods of this plant in Iran, can significantly ensure the quality of saffron available in global markets.

In Iran, saffron production has traditionally relied on local knowledge. Many saffron producers use limited off-farm and chemical inputs, primarily depending on on-farm and local resources5,8. Agronomic practices aligned with organic farming principles, such as the use of organic fertilizers, non-chemical pests and weeds control, family labor and participatory production, are common among Iranian saffron farmers9. The use of chemical inputs is not widespread in the majority of small fields, which constitute the most common type of saffron fields in Iran. Therefore, regardless of the high economic value, saffron is a suitable candidate for organic systems in terms of sustainability, strengthening local communities and meeting the needs of consumers and related pharmaceutical and food industries5,10.

Although the historical and current use of chemical inputs in Iran’s saffron fields has been limited8, their application has relatively begun in recent years, mainly in some large fields within the primary saffron-producing countries8,11,12. Despite the slow global shift towards chemical management in saffron fields, the emergence of this trend should be a cause for concern and vigilance8. Research has indicated that the continuous use of chemical inputs can increase the concentration of heavy metals in saffron field soil over time13. Therefore, scientific investigations are necessary to assess the potential for increased levels of pollutants, such as heavy metals, and reduced saffron stigma quality due to the long-term application of chemicals, allowing for timely preventative measures.

A one-year study comparing organic and conventional production systems found no significant differences in the quality parameters of the resulting stigmas. However, when these management systems were continued for two years, the levels of crocin and picrocrocin in stigmata from the organic system were 4% and 5% higher, respectively, compared to the conventional system14. The results of a similar experiment showed that the content of crocin and safranal in the stigmata obtained from fields fertilized organically was higher than in those receiving chemical fertilizers15. Another experiment demonstrated a relative increase in crocin and safranal content in stigmata through ecological nutrition methods, including humic acid and mycorrhiza16. Behdani and Hoshyar17 also reported that the contents of crocin, picrocrocin and safranal in the stigmas from organic fields were 18%, 17% and 29% higher, respectively, than in conventional fields.

The medicinal properties of saffron are also positively affected by the organic production systems. Askary et al.18 found that the phytochemical, antioxidant, and anti-cancer properties of stamen and tepal extracts obtained from organically cultivated saffron were more than those from conventionally produced saffron. Results of another study revealed that crop management strategy is crucial for increasing the qualitative and medicinal properties of saffron, where healthier products, higher qualitative yields and increased expression of pro-apoptosis genes were obtained by organic management compared to conventional farming19.

The heavy metal content in saffron stigma is also significantly affected by field management practices. Feizy et al.20 in a study of 21 stigma samples from different parts of Khorasan province, Iran, reported that harmful elements, Cd and Pb, were not detected. Mehraj et al.21 found higher heavy metal contamination in saffron fields located near cement factories. Chichiricco et al.22 also showed that saffron pollen and anthers, which can be used as food supplements, exhibit a high tendency to absorb toxic metals from the soil. Ebrahimi et al.23 found that by organic fertilization of saffron fields, the concentrations of arsenic, lead and mercury were much lower than the permissible limits. A study showed that irrigating saffron with domestic wastewater significantly increased the heavy metal content in the stigma, with the highest accumulation in corms, followed by petals and stigmas24.

Given the impact of field management on the quality and medicinal properties of saffron stigma, it is essential to document the risks associated with the use of chemical inputs in saffron fields. Accordingly, this research compared the content of apocarotenoids and heavy metals in stigma samples obtained from organic and conventional fields, ranging in age from one to 10 years, across different regions of Razavi Khorasan and South Khorasan provinces, the two main saffron cultivation areas in the world.

Materials and methods

Study area

This research was conducted across 14 different fields (seven organic and seven conventional) located in seven distinct areas within five cities in the South Khorasan and Razavi Khorasan provinces of Iran. Two cities (Birjand and Darmian: Gazik) were situated in South Khorasan province, and three cities (Torbat-Heydariyeh, Gonabad and Fariman) were in Razavi Khorasan province. These cities represent the primary regions for saffron production in Iran and globally. Six of the experiment fields (three organic and three conventional) were located in Birjand, while in each of the other cities, two fields (one organic and one conventional) were selected. Within each region, the organic and conventional fields were situated approximately 30–40 m apart. This proximity aimed to ensure maximum similarity in climatic and edaphic characteristics between the two production systems while preventing the transfer of inputs, particularly chemical fertilizers and pesticides, from conventional to organic fields. In addition to establishing a buffer zone, attention was paid to the distance of the selected fields from potential pollution sources and the absence of runoff from adjacent lands8.

Treatments and sample collection

In this research, the effects of organic (OPS) and conventional (CPS) production systems of saffron was investigated on stigma apocarotenoids content and accumulation of heavy metals in stigma. To achieve this, seven organic and seven conventional saffron fields were selected across the five aforementioned cities in Razavi Khorasan and South Khorasan provinces. The key inputs used in the organic and conventional fields within each region are detailed in Table 1. From each experimental area, two stigma samples, one from an organic field and one from a conventional field, were collected during the autumn of 2022. The selected fields were under the supervision of the researcher in the year leading up to sampling, with the coordination and consent of the landowner. Obtaining land history information, monitoring, and collecting samples from the fields were done with the permission and informed consent of the landowner farmer. Collection, harvesting, transportation and storage of plant material, were comply with national guidelines provided by the Iranian National Standardization Organization25.

Table 1 The main characteristics of selected fields for preparing organic and conventional saffron samples.
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The organic saffron fields included in this study were managed for at least one year under the supervision of the investigator. They were managed in accordance with the Iranian National Standard for Organic Products (No. 11000)26, the Iranian National Standard for High-Quality Saffron Production (No. 5230)25, and the recommended guidelines for organic saffron production in Iran9. The fields in Birjand were established and managed directly by the researcher and according to the mentioned standards. Thereby, it was ensured that the organic production principles were followed throughout the entire management period. Field visits and interviews with field’s operators were conducted to independently confirm organic practices, including no use of chemical fertilizers or pesticides, soil health maintenance, maintaining a safe distance from polluting sources, etc. Some of these fields were also directly supervised by a certification body (EU Organic Reg, Bio-Inspecta, Switzerland) or two important saffron production companies (Saharkhiz & Arnika) in Iran.

Within each experimental region, the two selected fields were similar in terms of agronomic management practices, including the field age, soil characteristics, irrigation water properties, the characteristics of the planted corms, planting date, planting method and density, irrigation intervals, etc., while the primary distinction between the paired fields was the type of inputs used (chemical or organic), as detailed in Table 1. The selected fields ranged in age from 1 to 10 years. This variation in field age allowed for the investigation of both the short-term and long-term effects of chemical versus organic input application on stigma quality and heavy metal accumulation.

To control the effects of potential confounding variables such as field age, location, and management history, organic and conventional fields were selected in matched pairs within each region. In each region, the two fields had similar conditions in terms of age, soil type, climate, topography, water source, etc., and the only major difference between them was the management system (organic vs. conventional). This matched-pair design helped to control environmental and age-related variations at the sampling stage, allowing a more accurate comparison between the two management systems. However, due to the limited number of paired fields (seven regions, 14 stigma samples in total), including variables such as field age or region as covariates in the statistical model was not statistically reliable and could lead to model over-fitting. In studies with small sample sizes, it is generally recommended to use a matched-pair design instead of adding covariates, an approach that was applied in this study. Nevertheless, minor differences among regions (e.g., in microclimate or age) may not have been completely controlled in the statistical analysis and were therefore acknowledged as a limitation of this study.

Measurement of stigma apocarotenoids content

Crocin and picrocrocin by HPLC

To assess the effect of production system on stigma quality, the levels of crocin and picrocrocin in the stigma samples were measured using High-Performance Liquid Chromatography (HPLC), based on the method describer by García-Rodríguez et al.27. The HPLC device (Waters HPLC 1525 binary pump) was equipped with a GL Sciences Inc C18 column (150 mm × 4.6 mm; 5.0 μm particle size) and a Waters 2489 UV/visible detector. The detector’s absorbance wavelength was set at 440 nm for crocin and 254 nm for picrocrocin. The mobile phase consisted of water (phase A) and acetonitrile (ACN) (phase B), which were injected according to the program outlined in Table 2.

Table 2 The procedure used for mobile phase injection.
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For sample preparation, 10 mg of stigma powder was dissolved in 5 ml of methanol: water (80:20, v/v). The resulting solution (unknown sample) was shaken in the dark for one hour. Subsequently, it was centrifuged at 4000 rpm for 15 min. Finally, a 10 µL of the solution was injected into the HPLC system. The column temperature was maintained at 30 °C, the run time was 25 min, and the injection loop volume was 20 µL.

The concentrations used from the standard known sample (linear range of standards) to create the standard curve were 5, 10, 15, 20 and 50 ppm (5000 to 50,000 ppb). In instances where the concentration of an unknown sample exceeded the highest standard concentration (50 ppm), the unknown sample was diluted with the mobile phase (methanol: water, 80:20, v/v) until its concentration fell within the 5–50 ppm range of the standard curve. Figure 1 and Fig. 2 show the chromatograms of the standard samples for crocin and picrocrocin, respectively. Crocins consist of crocetin linked to sugar units via ester bonds; Crocin-I (C44H64O24) has one more glucose unit than Crocin-II (C38H54O19), and the two isomers have close but distinct retention times in HPLC28,29, as shown in Fig. 1. The measurement method for crocin-I and crocin-II is the same, and the two compounds are distinguished based on their retention times in HPLC30. Figure 1 displays two peaks, which correspond to crocin-I and crocin-II. The first peak, with a retention time of 15.68 min, represents crocin-I, and the second peak, with a retention time of 16.45 min, represents crocin-II. Crocin-I and Crocin-II have very similar chemical structures, which naturally results in close retention times in HPLC29. Therefore, the proximity of the peaks in the chromatogram is expected.

Fig. 1
figure 1

The chromatogram of standard sample for crocin (Note: In the inset table of the figure, the numbers 1 and 2 indicate crocin-I and crocin-II, respectively, and RT represents the retention time for each of these two compounds).

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Fig. 2
figure 2

The chromatogram of standard sample for picrocrocin.

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Safranal by GC/MS

Gas chromatography/mass spectrometry (GC/MS) analysis was performed by using an Agilent 7890B gas chromatograph coupled with a 5977A mass detector (Agilent Technologies, USA). The gas chromatograph was equipped with a HP-5MS capillary column (phenyl methyl siloxane, 30 m × 0.25 mm ID, 0.25 μm, Agilent Technologies). The injector temperature was set at 230 °C, and the oven temperature was programmed as follows: initial temperature of 80 °C (held for 1 min), followed by an increase to 110 °C at a rate of 10 °C/min, and then held constant at 110 °C for 5 min. Helium was used as the carrier gas with a flow rate of 1 mL/min, and the injection volume was 1 μL. Also, the mass spectrometer was set in EI mode at 70 eV. The interface temperature was adjusted at 270 °C and the mass scan range was set from 50 to 450 m/z. Figure 3 shows the chromatogram for the standard sample of safranal.

Fig. 3
figure 3

A standard chromatogram for safranal (Area: 40,584,373.99, RT: 8.622).

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Measurement of stigma heavy metals content

The content of key heavy metals, including lead (Pb), cobalt (Co), arsenic (As), chromium (Cr), copper (Cu), and cadmium (Cd), in the saffron stigma was determined using the method described by Nóbrega and Donati31, employing an atomic absorption device (AA-6300, GFA-EX7i; Shimadzu). For this procedure, 1 g of the dried stigma sample was carefully weighed and placed in a Teflon vessel suitable for microwave digestion. Subsequently, 50 ml of a TISAB mixture (prepared with 3 parts hydrochloric acid and 1 part nitric acid) was added to the vessel containing the sample. The Teflon vessel was then tightly sealed to prevent vapor loss. The vessel was placed in a microwave digestion system, and a digestion program with a temperature setting of 180 °C and a duration of 20 min was started. After the digestion, the vessel was allowed to cool to ambient temperature. Then, the lid of the vessel was carefully opened to release any remaining vapors. The resulting solution was filtered through a filter paper and diluted to a final volume of 50 ml with deionized water. This final solution was then introduced into the atomic absorption device for the measurement of the target elements. Finally, the determined heavy metal concentrations in the stigma samples were compared with the safe limits established by FAO/WHO and USDA as reported in Abou Fayssal et al.24, which are 0.5, 0.1, 1, 20 and 0.3 mg kg−1 (500, 100, 1000, 20,000 and 300 ppb) for As, Cd, Cr, Cu and Pb, respectively.

Calibration curves were prepared for each heavy metal using standard solutions at different concentrations, and the corresponding absorbance values were recorded. The calibration equations, correlation coefficients (R2), and limits of detection (LOD) and quantification (LOQ) are summarized in Table 3. The measurements of heavy metals and the preparation of the calibration curves were conducted by the Central Laboratory of University of Birjand.

Table 3 Calibration parameters for the determination of six heavy metals.
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Statistics

The apocarotenoid and heavy metal content in stigma samples from the two production systems (OPS and CPS) were compared using an independent samples t-test, with each field within each production system serving as a replicate. For three heavy metals (cobalt, lead and arsenic) where a substantial portion of the measurements were below the limit of quantification (LOQ), or limit of detection (LOD), no inferential statistical tests (e.g., t-test or Mann–Whitney U test) were applied. To avoid biased or unreliable results, these parameters were analyzed descriptively. This approach follows standard analytical practice for censored data and ensures that interpretation remains scientifically sound. While detection frequency analysis or non-parametric methods are generally recommended for censored data, in this study a high proportion of three mentioned heavy metal measurements were below the limit of quantification (LOQ) (Table 9), and the sample size was small (n = 7 per group). Under these conditions, applying such statistical methods would be unreliable. Therefore, a descriptive approach was used, reporting observed concentration, ensuring scientifically sound interpretation without introducing bias.

To evaluate the statistical power of the independent t-tests used to compare organic and conventional saffron samples, a post-hoc power analysis was conducted for each trait, using SAS 9.4. Power was calculated based on t-value and degrees of freedom (df). Results indicated that the power was low for most traits (typically below 30%), particularly for traits like picrocrocin and Cu (Table 4), suggesting limited ability to detect real differences. The low power is likely due to the small sample size, with only seven fields included in each group, which may have limited the ability to detect significant effects. The limited sample size (n = 7 per group) was due to strict selection criteria. Only fields that had both organic and conventional management in close proximity, and with highly similar climatic conditions, soil characteristics, topography, and irrigation water properties, were included. Furthermore, the selected organic fields had to comply as closely as possible with the Iranian national organic standard26 and proposed guidelines for organic saffron production8,9. Due to these constraints, it was not possible to include more than seven paired fields in the study. Considering that saffron is not as widely cultivated as common field crops, and given the above-mentioned strict criteria applied for field selection, it was not possible to identify a large number of suitable fields. Therefore, despite the limited sample size, the results of this study can be considered as a pilot study and provide valuable preliminary insights.

Table 4 Post-hoc power analysis, multiple comparison-adjusted p-values (Bonferroni and FDR) and effect size (Cohen’s d) for independent t-tests comparing organic and conventional saffron stigma samples.
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To account for the increased risk of type-I error due to multiple statistical comparisons, p-values were adjusted using Bonferroni and False Discovery Rate (FDR) corrections, using SAS 9.4. Both raw and adjusted p-values are reported in Table 4. In addition to the significance testing, the effect size for each comparison between organic and conventional saffron stigma samples was calculated using Cohen’s d for independent t-tests, by SAS 9.4. Effect sizes are often categorized as small (d = 0.2), medium (d = 0.5), and large (d = 0.8) according to the benchmarks proposed by Cohen32. However, these thresholds are not fixed and should be interpreted with flexibility33. Therefore, here the magnitude of the effect was interpreted as following: d < 0.2 (negligible), 0.2 ≤ d < 0.5 (small), 0.5 ≤ d < 0.8 (medium), and d ≥ 0.8 (large) (Table 4). Effect size values were reported to provide a complementary measure of practical significance in addition to p-value.

The correlation between apocarotenoids concentrations and heavy metal content in the stigma was evaluated using SAS 9.4 software. The effect of Cr, Cu, Cd and total heavy metals on the apocarotenoids content was modeled using three types of polynomial regression models (linear, quadratic, and cubic) in Sigmaplot 15. The selection of the best-fitting model was initially based on the p-value (significance level) and then by the adjusted R-squared (R2) value.

Results and discussion

Apocarotenoids content

Although the mean contents of crocin-I, crocin-II, and total crocin were numerically higher in most organic production system (OPS) samples compared to the conventional production system (CPS), these differences were not statistically significant (P = 0.55–0.57). Specifically, crocin-I was higher in 85.7% of OPS samples, crocin-II in 57.1%, and total crocin in 85.7% of the OPS samples. The mean total crocin values were 15.0 g per 100 g dry stigma in OPS and 13.8 g per 100 g in CPS, corresponding to an 8.8% numerical increase that did not reach statistical significance (Table 5). The differences between organic and conventional saffron for Crocin-I, Crocin-II, and total Crocin were also associated with negligible effect sizes (Cohen’s d = 0.16–0.17) (Table 4), suggesting minimal practical impact despite the observed variation.

Table 5 The content of crocin in saffron stigma samples produced under organic or conventional production systems, in different regions of Iran.
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In line with the results of the present study, which showed a tendency for relatively higher, but non-significant, crocin content under OPS compared to CPS, Askary et al.34 also reported an increase in crocin content with both OPS and CPS compared to a control (no-fertilizer), with a more increase under OPS. Similarly, Behdani and Hoshyar17 and Fallahi and Mahmoodi15 found OPS to be more effective in enhancing the stigma crocin content. The higher apocarotenoid content in stigma under OPS is likely attributed to the multifaceted role of organic inputs compared to chemicals. The comprehensive and balanced supply of nutrients from organic inputs can stimulate the production of phytohormones, organic acids, and vitamins, which act as indirect factors in improving plant quality14.

Contrary to expectations, crocin-II content was numerically higher in CPS than in OPS in 42.9% of the stigma samples (Table 5). This observation aligns with findings from a study indicating that the application of OPS does not guarantee an improvement in all quality parameters of saffron. It emphasizes the necessity for all inputs used in organic fields to adhere strictly to organic farming standards, particularly ensuring that organic fertilizers are free from pollutants and heavy metals35. Accordingly, in the current study, it seems that some inputs used in OPS might have lacked the required quality standards, leading an increase in the content of some heavy metals in the stigma. The accumulation of these metals may have caused a reduction in certain qualitative parameters. Supporting this hypothesis, the regression analysis results revealed a significant relationship between the cadmium content and the Crocin-II content in the stigma (Table 6). As the amount of cadmium in the stigma increased, the content of crocin-II in this organ showed a sharp decline (Fig. 4). Exposure to heavy metals in medicinal plants can induce oxidative stress, thereby triggering the synthesis of antioxidants. Although antioxidant production serves as a defense mechanism against oxidative stress, it may divert metabolic resources away from the synthesis of other bioactive compounds36.

Table 6 Mean of square for the effect of cadmium content is stigma on the concentration of Crocin-II using quadratic regression model.
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Fig. 4
figure 4

Quadratic regression between cadmium and the picrocrocin content in stigma.

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For picrocrocin, the uncorrected P-value was 0.041 (Fig. 5), which initially suggests a statistically significant difference between the organic and conventional saffron groups. However, after applying multiple comparison corrections using Bonferroni (P-value = 0.741) and False Discovery Rate (FDR) (P-value = 0.741), the difference is no longer statistically significant (Table 4). This indicates that while the initial result was significant, the correction for multiple comparisons shows that the observed difference was likely due to random variation. Across all studied regions, the content of picrocrocin in samples from OPS was higher than those from CPS. Based on the mean picrocrocin values across all regions, OPS resulted in a 53.4% increase in the amount of this compound compared to CPS, which was statistically significant (P = 0.04), but based on uncorrected P-value (Fig. 5). Picrocrocin content also showed a medium effect size (Cohen’s d = 0.66) (Table 4), indicating a substantial difference between organic and conventional samples, which aligns with the significant p-value, and may have meaningful implications for flavor and quality. In another study, the positive effect of OPS on increasing the picrocrocin content of stigma has been confirmed17. The higher quality of stigma under OPS may be linked to increased carbohydrate production resulting from a more appropriate and balanced availability of nutrients, consequently leading to improved plant photosynthesis14.

Fig. 5
figure 5

The effect of production system on the stigma picrocrocin content in different regions. O = organic production system, C = conventional production system, B = Birjand, D = Darmian, T = Torbat, F = Fariman, G = Gonabad, Numbers show the field age (year).

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In the majority of the studied regions (six out of seven), safranal content was numerically higher in OPS than in CPS. Based on the mean values across all regions, safranal content showed an 11.5% increase under OPS, although this difference was not statistically significant (P = 0.3) (Fig. 6). Furthermore, safranal content exhibited a small effect size (d = 0.30) (Table 4), indicating partially low differences between production systems; however, such an effect may still be biologically or commercially relevant. The results of similar studies also corroborate the findings of the current study14,17. The application of organic inputs, particularly manure, in OPS stimulates the production of hormones and primary compounds, which are beneficial in glucosides biosynthesis and their subsequent decomposition into secondary compounds37.

Fig. 6
figure 6

The effect of production system on the stigma safranal content in different regions. O = organic production system, C = conventional production system, B = Birjand, D = Darmian, T = Torbat, F = Fariman, G = Gonabad, Numbers show the field age (year).

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In one of the studied regions, however, the safranal content was numerically higher in the CPS than in OPS (Fig. 6). Results from a similar study showed that, with the exception of crocin percentage, the OPS did not improve all quality characteristics of the stigma, probably due to the adverse effects of heavy metal accumulation in the soil resulting from the application of contaminated organic fertilizers35. Safranal is a volatile oil, and its synthesis relies on a continuous supply of photosynthetic carbon. Disruption of carbon supply by heavy elements can reduce the production of essential oils38.

Supporting the hypothesis of a decrease in the content of active compounds in the stigma due to heavy metal accumulation, Pearson correlation results showed a predominantly negative relationship between the levels of cadmium, chromium, and total heavy metal content with the active constituents of the stigma, although these correlations were not statistically significant (Table 7). Similarly, the linear regression analysis showed a negative and significant effect of total heavy metal content on the amount of picrocrocin in the stigma (Table 8; Fig. 7). Mushtaq et al.11 also reported that increased heavy metal concentration in the soil of saffron fields might be associated with a decline in saffron quantity and quality. The presence of heavy metals in medicinal plants can alter metabolic pathways by affecting the activity of enzymes involved in the biosynthesis of bioactive compounds and/or by the disrupting various biosynthetic pathways within plants, leading to changes in the production of secondary metabolites36.

Table 7 Pearson correlation coefficients matrix of the content of apocarotenoids and heavy metals in saffron stigma samples (N = 14).
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Table 8 Mean of square for the effect of total content of heavy metals is stigma on the concentration of picrocrocin using linear regression model.
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Fig. 7
figure 7

Linear regression between total content of heavy metals and the picrocrocin content in stigma.

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Heavy metals

Results showed that the cobalt content in all organic stigma samples was below the limit of quantification (LOQ). However, only 43% of conventional stigma samples showed cobalt levels below the LOQ, while the remaining samples ranged from 0.66 to 12.08 ppb (Table 9). The lead content in 100% of OPS samples and 85.7% of CPS samples was below the LOQ. In addition, the content of arsenic in all 14 samples, regardless of the production system, was below the limit of detection (LOD) (Table 9). In contrast, a study on 48 stigma samples from four regions of Morocco reported arsenic levels between 1.1 and 5.6 µg kg1 and lead levels ranging from 0.4 to 2.8 µg kg−14. Similarly, Abou Fayssal et al.24 found higher arsenic (120–240 ppb) and lead (110–240 ppb) concentrations in stigma produced with irrigation water of varying quality in India compared to the present study (Table 9). Results of a meta-analysis by Amir Sadeghi et al.39, exhibited that the mean concentration of lead in saffron stigma was 100 ppb, which is considerably higher than the levels observed in both OPS and CPS samples in this research (Table 9).

Table 9 The content of some heavy metals in saffron stigma samples produced under organic or conventional production systems, in different regions of Iran.
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Although the concentrations of the three heavy metals presented in Table 9, were below the established safe limits, planting saffron under OPS was numerically more effective in reducing the concentrations of these elements. Supporting this, a study reported that fertilizing saffron fields with various organic fertilizers caused the concentration of arsenic, lead and, mercury in the stigma be much lower than the permitted levels23. Similarly, it has been confirmed that the increased application of chemical inputs in saffron fields is a primary factor contributing to the contamination of the produced stigma with heavy metals5. Behdani et al.13 also reported that the continuous use of chemicals, particularly inorganic fertilizers, in saffron fields leads to increased concentrations of heavy metals such as Co, Cr, and Cd, in the soil of older fields. Taghavi et al.40 similarly confirmed that the excessive application of chemical inputs (chemical fertilizers, pesticides, herbicides and polluted manure) has resulted in the accumulation of both toxic and non-toxic metals in many field soils.

Most values of heavy metals (Co, Pb, As) were below LOQ or LOD. Therefore, no statistical tests were performed, and the data were presented descriptively.

In four of the studied regions, chromium content in the stigma was numerically higher in CPS, while in the remaining three regions, it was numerically higher in OPS. On average, chromium concentration in CPS showed a 6.5% numerical increase compared to OPS, although this difference was not statistically significant (Fig. 8). Moreover, chromium differences had a negligible effect size (d = 0.1) (Table 4), suggesting that variation between organic and conventional samples is minor and likely biologically irrelevant. Notably, the highest and lowest chromium levels across all 14 stigma samples were 29.6 and 4.93 ppb, respectively, which are well below the safe limit of 1000 ppb (Fig. 8).

Fig. 8
figure 8

The effect of production system on the stigma chromium content in different regions. O = organic production system, C = conventional production system, B = Birjand, D = Darmian, T = Torbat, F = Fariman, G = Gonabad, Numbers show the field age (year). No statistically significant differences were observed (P >0.05).

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The higher numerical chromium concentrations observed in some organic samples could be attributed to factors such as the possible use of contaminated organic fertilizers and the transfer of pollutants via runoff and air from adjacent conventional fields. A similar study highlighted that the amount of heavy metals in organic fertilizers is the main and primary factor affecting the amount of these elements in the soil and plants41. Saffron cultivation under OPS when organic fertilizers contaminated with heavy metals are used or when organic field is situated near polluting sources, can lead to the accumulation of heavy metals in the stigma8,9. Heavy metals in organic fertilizers exhibit high bioavailability. Furthermore, organic acids released during the decomposition of organic fertilizers may mobilize heavy metals bound to soil particles. However, humus from organic fertilizers can mitigate the availability and enhance the fixation of heavy metals in the soil through complexation or chelation reactions41.

In four of the studied regions, cadmium content in the stigma was numerically higher in CPS, while in the remaining three regions, it was numerically higher in OPS. Based on the mean values across all seven regions, cadmium concentration in CPS showed a 71.5% numerical increase compared to OPS, although this difference was not statistically significant (P = 0.3) (Fig. 9). Furthermore, cadmium levels showed a small effect size (d = 0.43), indicating slight but potentially meaningful differences between the two production systems, despite the non-significant p-value (Table 4). Among all stigma samples, the highest cadmium level (40.08 ppb) was observed in a CPS sample; however, this value remained well below the safe limit of 100 ppb (Fig. 9).

Fig. 9
figure 9

The effect of production system on the stigma cadmium content in different regions. O = organic production system, C = conventional production system, B = Birjand, D = Darmian, T = Torbat, F = Fariman, G = Gonabad, Numbers show the field age (year). No statistically significant differences were observed (P >0.05).

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The application of phosphate chemical fertilize is a primary factor contributing to increased cadmium concentrations in agricultural products20. However, the widespread use of feed additives has also elevated the risk of heavy metal accumulation in animal manure, and this can even cause the accumulation of these elements in products from OPS42. Supporting this, results of a long-term study revealed that continuous application of organic manure had no significant effect on soil total Cu and Pb, but caused the accumulation of Zn, Cd, and Cr in the soil, particularly during the late growing seasons, which exerted a negative impact on food safety and quality41. Feizy et al.20 found that reducing the application of chemical fertilizers and increasing the use of high-quality organic fertilizers is an effective strategy to decline the content of heavy metals such as Pb, Cr and Cd in saffron. Accordingly, in organic saffron fields, careful attention should be paid to the quality of the organic fertilizer35 and the absence of contamination in the water used for irrigation24, as contamination of these sources with heavy metals can increase the concentration of these elements in the produced stigma. The results of another study also demonstrated that industrial activities have a concerning effect on the concentration of heavy metals in saffron fields located near these centers43. Amir Sadeghi et al.39, in a meta-analysis study on saffron reported that the mean value of Cd was 9 ppb, with a range of 8–10 ppb, which is near to the mean data obtained in both OPS and CPS, in the present study (Fig. 9).

Based on the mean values across all studied regions, copper content in stigma samples was numerically higher in OPS (8.34 ppb) than in CPS (6.18 ppb), representing a 34.9% numerical increase, although this difference was not statistically significant (P = 0.12) (Fig. 10). In addition, based on Table 4, copper content differences displayed a small effect size (d = 0.47), implying slight but possibly meaningful variation between organic and conventional saffron. Copper is an essential micronutrient for plants at low concentrations, while it’s excessive amounts causes negative effects44. Considering the positive, albeit insignificant, correlation of copper with safranal, crocin, and picrocrocin, and its negative and significant correlation with the content of chromium and cadmium in stigma (Table 7), it appears that the amounts of copper present in both OPS and CPS in the current experiment were within the range required by the plant. Accordingly, the content of copper in all stigma samples (ranging from 1.79 to 12.75 ppb), was considerably lower than safe limit of 20,000 ppb (Fig. 10). Likewise, copper concentration in both production systems was lower than the mean value of 22 ppb reported in a meta-analysis by Amir Sadeghi et al.39.

Fig. 10
figure 10

The effect of production system on the stigma copper content in different regions. O = organic production system, C = conventional production system, B = Birjand, D = Darmian, T = Torbat, F = Fariman, G = Gonabad, Numbers show the field age (year). No statistically significant differences were observed (P >0.05).

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In a previous study, due to the contamination of used manure with some heavy metals, including copper, the content of this element in the stigma samples from OPS was higher than in CPS35. This highlights that the application of any type of organic input in OPS does not guarantee product quality. Before using inputs in organic agro-ecosystems, it must be ensured that they are not contaminated with compounds such as heavy metals8. Results from a similar study on saffron fields in three selected sites of Kashmir revealed that the concentration of copper in soil was 14,100–17,800 ppb, which reduced the stamen weight11.

With the exception of one studied region, total heavy metal content in the stigma was numerically higher in CPS than in OPS. Across all seven regions, mean concentrations were 48.22 ppb in CPS and 38.05 ppb in OPS, representing a 26.7% numerical increase, although this difference was not statistically significant (P = 0.2) (Fig. 11). Moreover, the effect size for total heavy metals was small (Cohen’s d = 0.48) (Table 4), suggesting potential differences between the two production systems that, while not statistically significant, could still be relevant from a quality perspective.

Fig. 11
figure 11

The effect of production system on the total content of heavy metals in stigma in different regions. O = organic production system, C = conventional production system, B = Birjand, D = Darmian, T = Torbat, F = Fariman, G = Gonabad, Numbers show the field age (year). No statistically significant differences were observed (P >0.05).

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Conventional agricultural products may contain higher levels of heavy metals due to the intensive use of chemicals such as pesticides, herbicides, and fertilizers in their production45. In a study, Ayoub et al.43 reported that accumulation of heavy metals in saffron fields is associated with the substantial application of chemical fertilizers. Taghavi et al.40 in a study on saffron fields in Gonabad, Iran, reported that soils were safe for planting in terms of trace elements, based on FAO/WHO and Iranian soil standards. They found that the mean concentrations of the trace elements decreased in the order of Cu >Cr >Pb >Co >As >Cd. However, in the current experiment, the order of decreasing concentration of heavy metals in the stigma was Cr >Cd >Cu >Co & Pb >As (Table 9, Figs. 8, 9, 10). Taghizadeh Tousi46, in a study in Torbat Heydarieh, on the levels of some toxic trace elements (aluminum, bromine, chlorine, mercury, arsenic, and thorium) in saffron, found that the edible part of the plant was somewhat contaminated by Hg, but only in the urban area. Moreover, it was found that saffron is a very poor absorber of the studied elements. Overall, it can be stated that although there is a risk of heavy metal accumulation in soil and plants in OPS, mainly due to the potential use of contaminated fertilizers42, the risk of contamination in CPS is considerably higher9.

Conclusion

The findings of this study suggest trends indicating that the use of organic production systems (OPS) may be associated with slightly higher levels of crocin, picrocrocin, and safranal in saffron stigmas compared to conventional production systems (CPS) in some regions. However, these differences were not statistically significant for most traits. The study suggests that issues such as the use of organic fertilizers contaminated with heavy metals in certain OPS likely led to heavy metal accumulation and a subsequent reduction in stigma quality characteristics. This is supported by the observed negative correlations between the content of some heavy metals and certain quality parameters of the stigma. While some heavy metals such as cobalt, chromium, and cadmium tended to be numerically higher in CPS than OPS, copper showed the opposite trend. Considering the negative relationship between copper content with the content of chromium and cadmium and its positive relationship with quality characteristics of the stigma, the copper levels in both production systems appeared to be within the plant’s requirement and below toxic thresholds. Importantly, the concentrations of all measured heavy metals in both OPS and CPS across the studied areas were below the safe limits established by the FAO/WHO and USDA.

This study has several limitations that should be considered when interpreting the results. First, the small sample size (7 paired fields per group) limits the statistical power and prevents full use of covariates to control for environmental and field-age variables. Second, although differences in environmental conditions and fields management history were largely controlled using a matched-pair design, minor residual effects cannot be completely ruled out. Third, some heavy metal measurements were below the limit of detection or quantification, limiting statistical analyses for these traits to descriptive reporting. Finally, despite post-hoc power analyses and multiple testing corrections, results should be interpreted cautiously, and future studies with larger sample sizes are recommended to increase statistical power.