Analysis of Cadmium Accumulation in Linseed (Linum Usitatissimum L.) Depending on Soil pH and Fertilizer Level

ABSTRACT Flax (Linum usitatissimum L.) has great potential for the production of oil with functional properties. There are several factors drastically reducing the quality of linseeds. One of them is the cadmium uptake and accumulation in seeds. The aim of this study was to assess the control capability of Cd uptake level by fertilization and soil pH. The experiment was carried with four Polish varieties of linseed: Bukoz, Szafir, Olivin, and Jantarol. The soil with two different pH was used in the experiment: acidic (pH 4.5) and alkaline (pH 7.5). Two concentrations of aqueous solution of cadmium nitrate (Cd(NO3)2) were added to the soil to obtain the final concentration of Cd: 2 mg/kg and 4 mg/kg of soil, respectively. The phosphorous fertilizer was supplied in the amounts of: 50 kg/ha, 100 kg/ha, 150 kg/ha. The results revealed that the pH level is important factors in the cultivation of linseed. The cadmium uptake by linseed can be significantly weakened by increasing the soil pH. Among the four flax varieties examined, the highest seed yield with the lowest Cd content at the same time was obtained for “Bukoz” during cultivation with 100 kg/ha phosphorous fertilization and pH 7.5.


Introduction
Flax (Linum usitatissimum L.) is an annual, self-pollinating crop from the Linaceae family, widely distributed in the temperate climate zone.Flax grows best in regions with as moderate warmth, high moisture, and well drained, medium to heavy soils (Worku, Heslop-Harrison, and Adugna 2015).
During cultivation, flax requires a wide crop rotation of about 7 years.Cereals and maize are good preceding crops.Mineral fertilization is recommended for flax cultivation (Berti et al. 2009;Zhao et al. 2020).The standard requirement of flax for macronutrients, depending on the abundance of soil, is 40-60 kg N, 30-50 kg P 2 O 5 , and 60-80 K 2 O per hectare.Micronutrients such as boron and zinc improve plant health.
The area under annual cultivation of oilseed flax is more than 4 million ha, accounting for 1% of the total world production of plant oil.The five major producers of oilseed flax are Canada, China, the United States, and the European Union (FAOSTAT 2021).
L. usitatissimum is grown either for stem fiber (fiber flax) or for seed oil (oilseed flax, linseed) (Liu et al. 2011;Singh et al. 2011).These two morphotypes differ in terms of morphology and agronomic traits.Fiber type is taller, has fewer branches, greater straw weight and seed protein content.Oilseed flax is shorter, has more branches, more seeds, and higher seed oil content (Diederichsen and Ulrich 2009;You et al. 2017).Therefore, many breeding programs now focus on producing dual purpose varieties that combine high seed yield with high fiber yield (Fila et al. 2018).
In recent years, the demand for unconventional oils has been increasing as a result of their health benefits.Flax has great potential for the production of oil with functional properties.
Its daily consumption aids in the prevention of atherosclerotic cardiovascular disease due to the presence of LIN, phytoestrogen, lignan, and soluble fiber, which makes it suitable as a source of drugs and therapeutics.Beneficial effects, related to the consumption of linseed oil, are mainly associated with its high levels of omega-3 fatty acids.It was demonstrated that linseed oil consumption results in a reduction in the incidence of renal injury and some cancers and reduction in blood pressure (Harper and Jacob 2005;Ogborn et al. 2002).The beneficial effect of lignans on human health was also described.It was confirmed that lignans can inhibit the division of cancer cells of lung tumors (Adlercreutz 2008).
There are several factors drastically reducing the quality of linseeds.One of them is the presence of toxic chemical elements.Cadmium (Cd) is one of the toxic heavy metals naturally occurring as a pollutant in the agricultural and industrial aeries.It is present only in inorganic form and in one oxidation state (2+).Cd can damage different cellular structures and a variety of tissues causing oxidative stress (Gratao et al. 2009).
Cadmium is not an indispensable element for plant growth and reproduction, however, it accumulates in organisms of plants and consequently animals with a long half-life of about 25-30 years (Genchi et al. 2020).Exposure to cadmium primarily occurs through the ingestion of contaminated food.In humans, it can result in a variety of adverse effects, such as renal and hepatic dysfunction, pulmonary edema, osteomalacia, and damage to the adrenals and hemopoietic system (Tinkov et al. 2018).In addition to its cytotoxic effect, cadmium is a proven human carcinogen (group I of International Agency for Research on Cancer classification) (Clemens and Ma 2016;Sharma, Rawal, and Mathew 2015).Cadmium exposure has been related to breast, lung, prostate, pancreas, and urinary bladder cancers (Mezynska and Brzóska 2018).
Flax is one of the species of high ability to accumulate cadmium from soil (Bjelkova, Gencurova, and Griga 2011;Praczyk et al. 2015).There are currently no recommendations for acceptable level of Cd in flaxseed; however, the European Food Safety Authority recommends weekly maximum dietary intake at the level of 2.5 µg of Cd per kg of body weight (EFSA 2011).The concentration of Cd in flaxseed may influence food processor and consumer choices, particularly in the health food sector.To improve marketability and considering human health, the decrease of cadmium uptake by seeds of flax is crucial.
The previous studies on Cd accumulation in other plant species confirm that the pH of the soil and fertilization level are important factors for controlling the uptake of heavy metals (Baldantoni et al. 2016;Wielgusz et al. 2022).It was shown that a low pH promotes Cd accumulation, and phosphate reduces Cd uptake by some plant species (Baldantoni et al. 2016) while in other plant species Cd accumulation was increased under higher pH (Linger, Ostwald, and Haensler 2005).
Therefore, the purpose of this study was to assess the control capability of Cd uptake level by fertilization and soil pH.The effect of fertilizer supply, pH and cadmium concentration in the soil on seed yield and fatty acid concentration were also assessed.Genetic factor was also one of the factors studied -four typically oily flax varieties were selected for the study.

Plant material
The experiment was carried with four Polish varieties of linseed, bred in Poland: Bukoz, Szafir, Olivin, and Jantarol.All oilseed flax varieties are characterized by a lower height compared to the fibrous varieties.In addition, they have more branches in the panicles, on which there are more seed pods.Bukoz variety, bred at the Institute of Natural Fibres and Medicinal Plants registered in 2009, is resistant to lodging and very resistant to fungal diseases (Andruszewska, Byczyńska, and Silska 2009).The "Bukoz" is characterized by a high seed yield (1.8 t/ha).The seeds of this variety are brown in color and have a relatively high fat content.The length of the vegetation period is 112 days from the day of sowing.
The varieties Szafir, Jantarol, and Olivin were bred in Company Plant Breeding Strzelce.The Szafir variety (registered in 1991) has the shortest vegetation period − 109 days.Due to the lowest average height, it is very resistant to lodging but susceptible to fungal diseases.The variety yields 1.2 t of seeds per hectare.Seeds of the Szafir variety are brown in color.The fat content and composition of fatty acids is not as high as in the case of other varieties, which, however, affects the high taste of the oil obtained from the seeds of this variety.Varieties Jantarol (registered in 2000) and Olivin (registered in 2005) are characterized by yellow seeds.The seeds have a high content of fat and fatty acids, but a much lower seed yield compared to brown-seed varieties.In addition, these varieties are much more susceptible to infection by fungal pathogens.The Jantarol variety is the most susceptible to lodging compared to the other described varieties.
Soil samples were analyzed at the beginning of the experiment.The granulometric composition was examined by determining the percentage of sand, silt, and clay according to the Casagrande aerometric method with Pruszyński's Modification (Mocek 2015).The content of P and K in the soil was assessed by the Egner-Riehm method (extraction with a calcium lactate solution) (Mercik 2004).The Mg content in the soil was assessed using the Schachtschabel method (extraction with calcium chloride solution).The determination of the percentage of organic C was carried out using the Tiurin method (Mocek 2015).The total N content in the soil was determined using the Kjeldahl method.Soil pH was measured using the potentiometric method (Ostrowska, Gawliński, and Szczubiałka 1991).Table 1 represents average value content of clay, silt, sand fractions (%), C organic (%), Cd (mg/kg), and N (%), P, K (mg/100 g of soil) for all soil samples from the experiment.

Flame atomic absorption spectrophotometry
The initial cadmium content in the soil and the final post-harvest cadmium content were assessed.The cadmium content in the soil was determined by the method of flame atomic absorption spectrophotometry (Van Lonn 1996).During the first stage, the mineralization of the samples was carried out with the use of concentrated nitric acid.The samples were mineralized in a microwave oven under the following conditions: mineralization temperature: 210°C, digestion time: 5 min, digestion pressure: 13 788 951,458 Pa, power used (for 24 trials): 500 W.After the completion of mineralization and cooling of the samples, the mineralization products were quantitatively transferred to 10 cm 3 calibrated tubes and the cadmium content was assessed.The absorption of the cathode ray tube radiation by the free atoms of the element in the sample solution was measured and introduced into the flame in a sprayed form.
For the determination of the calibration curves, multi-element standard solutions (0, 0.05, 0.25, 0.50, 0.75, 1, 1. 25, 2, 5, 5.50, 6, 6.50, 7, 7.50, 8, 8.50, 9, 9.50, 10 mg/kg) were prepared from a concentrated standard solution (100 mg/kg).The standard solutions were acidified with nitric acid, and then the standard solution was added to the prepared sample solutions and the cadmium content was determined.Table 1 represents average content of initial cadmium content in the soil from the experiment.

Fertilization
Three levels of fertilizer with P element were supplied: 50 kg/ha, 100 kg/ha, and 150 kg/ha.Properly calculated amounts of phosphate fertilizer Polifoska 6 (manufactured by Grupa Azoty, Poland) per area were put into the soil in the pots.This fertilizer in the form of granules contains 6% nitrogen (N) in the ammonium form, 20% phosphorus (P 2 O 5 ) soluble in neutral ammonium citrate and water, i.e. available in the form of mono-and diammonium phosphate, and 18% soluble in water.The fertilizer also contained 30% water-soluble potassium (K 2 O) as a potassium chloride and 7% water-soluble sulfur trioxide (SO 3 ) as a sulfate.
No other fertilizers with micro and macro elements were added to the soil.Before setting up the experiment, the abundance of the soil in nitrogen, potassium, phosphorus, and magnesium was examined (Table 1).The amounts of these elements were not changed, as they were not the factors tested in the experiment.

Determination of Cd concentration in the soil
Two concentrations of cadmium as an aqueous solution of cadmium nitrate (Cd(NO 3 ) 2 ) were added to the soil to obtain: 2 mg/kg and 4 mg/kg of soil.The crystalline form of Cd(NO 3 ) 2 was dissolved in water to obtain a solution with a concentration of 35%.Based on the atomic mass of Cd (112,411), proper volume of solution was added to each pot.
Appropriate amounts of the solution were added to the pots 14 days before sowing the seeds to ensure equilibrium time.
The experiment was arranged as follows: all measurements were conducted in four replicates (each combination in four pots, each pot comprised 25 plants).The pot of capacity of 150 dm 3 (120 cm high and 40 cm in diameter), was filled with the soil prepared as described in subsections 2.2-2.4.
The temperature and humidity were controlled during the entire period of the experiment.From the sowing to the end of the emergence, the temperature was maintained at about 10°C (±2°C).In following phases of hemp development, the temperature in the greenhouse was maintained in the range of 15-22°C, in both years of the experiment.

Plasma optical emission spectrometry
The Cd content in seeds of flax was determined with a plasma optical emission spectrometry (ICP-OES).The plant material samples were mineralized in a closed system using a mixture of concentrated acids (HNO 3 , HClO 4 , H 2 SO 4 , HCl, HF, H 2 O 2 ).For the determination of the calibration curves, multielement standard solutions (0, 0.05, 0.25, 0.50, 0.75, 1, 1.25, 2 mg/kg) were prepared from a concentrated standard solution (100 mg/kg).The standard solutions were acidified with nitric acid, and then the standard solution was added to the prepared sample solutions and the Cd content was determined (Ferreira et al. 2007).

The yield measurements
Mature flax seeds have been harvested separately from each combination and have been weighed.The seed yield results are the mean of the four replications (pots) for each experimental combination.The cadmium content in the flax seed was determined after the harvest from each combination.

Seed oil extraction and fat content
The Soxhlet extraction apparatus was used to extract oil from seed flax, the extraction solvent was n-hexane, the liquid-to-material ratio was 1:30, and the extraction time was 8 h.Hexane was removed with a rotary vacuum evaporator (RE-52AA, Shanghai Instruments Ltd.) in a water bath at 40°C and then dried in oven (DGG-9240A, Shanghai Senxin Instruments Ltd.) at 105°C.

Statistical analysis
The statistical analyses and models were prepared based on discriminant analysis.It was checked which variables influence seed yield.The Cd concentration in flax seeds was measured.Canonicalcorrelation analysis (CCA) was used to construct the model.
The following parameters were included in the analysis: four flax varieties ("Bukoz," "Szafir," "Olivin," and "Jantarol"), soil pH (pH 4.5, 7.5), phosphorous fertilization level (P 50, P 100, P 150), cadmium concentration (Cd 4, Cd 8).A stepwise progression analysis was used to find which variables have the most influence on seed yield of flax varieties and also on Cd concentration in the soil and seeds.All variables were assessed.The model included the variables that contributed the most to the discrimination of groups based on p and F values for the analyzed variable.The process was repeated until the P value was more significant than 0.05 for the variable under investigation.To determine the significance limit level, a Monte Carlo permutation test was done (individually for each variable and then for the whole model).All comparisons, calculations, and graphic elements were created using Canoco for Windows and Microsoft Excel spreadsheets.The following tools of Canoco for Windows were used: Canoco for Windows 4.5, CanoDraw for Windows, and WCanoIMP.
The data were also analyzed by two-way ANOVA.Comparison of relevant means was conducted using by a post hoc T-test.
Bioconcentration factor (BCF) was calculated for the seeds according to the formula: BCF = metal content (plant part)/metal content (soil)

Results
In all combinations of the experiment, significant uptake of Cd from the soil by plants and accumulation in seeds was observed.As the cadmium concentration in the soil increased, the flax varieties took up and accumulated more of this metal in the seeds.
The seeds of the oilseed flax varieties Jantarol and Szafir had the highest cadmium concentrations (above 3 mg•kg −1 ).This was observed with phosphorus fertilization at doses of 50 and 150 kg•ha −1 .The uptake and accumulation of cadmium in seeds for these varieties was shown at a dose of 100 kg•ha −1 of phosphorus in fertilizer.Bukoz and Olivin varieties accumulated less cadmium in seeds (less than 3 mg•kg −1 ) (Figure 1).For all flax varieties, cadmium accumulation in seeds was higher on soil with pH 4.5.On soil with a slightly alkaline pH, the Jantarol and Bukoz varieties showed the lowest cadmium accumulation in seeds (0.8 and 1.0 mg•kg −1 respectively).No significant effect of phosphorus fertilization level was observed in these combinations.
The highest seed yield (0.5 kg from the combination) was recorded in the Bukoz variety when the seeds were sown in soil with lower Cd levels, with a slightly alkaline pH, after the application of phosphorus at a rate of 100 kg•ha −1 .This yield was twice as high compared to other varieties.
For all varieties, seed yield was the highest on alkaline soil with a lower Cd concentration and phosphorus fertilization at a dose of 100 kg•ha −1 (Figure 2).
The highest level of fat content was observed in Olivine cultivar (more than 42%).The soil pH and fertilization level did not affect the fat content in cultivar tested (Figure 3).
The two-way analysis of variance (ANOVA) shows the significant differences between cultivars regarding an average seed yield (Table 2) and an average straw yield (Table 3) with variable output Cd level, fertilization level, and soil pH.The statistical analysis confirmed that the highest seed yields were achieved at lover Cd concentration in soil and pH 7.5.Both in the case of straw and seed yields, differences in yield within varieties were observed for all tested variables.
It was confirmed that Bukoz cultivar displayed the highest average yield of seeds and straw, while the highest fat content was found in the case of Olivin cultivar (44%) (Table 4) The Bukoz variety was also characterized by the lowest bioconcentration index in seeds (average 5.81).In the conducted experiment, the mutual influence of many factors was analyzed.The following CCA model shows the complex relationships between all statistically significant parameters (Figure 4, Table 5).Analysis confirmed the strongest influence of soil pH on all variables in model.Low pH influences Cd content in seeds, especially under conditions of lower fertilization (P 50) and at higher Cd levels (Cd 4).

Discussion
Concerning the dual purpose of L. usitatissimum use, the quality of the raw material seems to be crucial.Recent years brought a renewal of interest in linseed utilization as a food crop.Linseed oil offers important nutritional benefits but only if it does not contain a significant amount of heavy metals.Cadmium is one of the major industrial soil pollutants, which is toxic to nearly all living organisms (Sterckeman and Thomine 2020;Wielgusz et al. 2022).The process of cadmium uptake by the plant is irreversible and the plants cannot return it to the environment during vegetation.The level of heavy metal uptake depends not only on the total content of this metal in the soil, but above all on the amount of their active forms available to the plant (Sterckeman et al. 2018).The physical and chemical properties of the soil have a major impact on Cd uptake -in adverse conditions, even at sites  with natural heavy metal content, plants can contain elevated amounts of heavy metals, especially cadmium (Tye et al. 2003).
In the undertaken research, we tested what factors actually affecting the accumulation of cadmium in oilseed flax, from which oil is obtained for food and pharmaceutical purposes.It was also checked whether farmers can reduce the Cd level in seeds, thanks to agrotechnical treatments, phosphorous fertilization, and proper selection of cultivation site.In our experiment, controlled concentrations of Cd were introduced into the soil of two pH levels and phosphorous fertilizer was supplied in three different amounts.
As mentioned above, flax is a crop used in cultivation either to obtain seeds for food or fiber.Due to its high activity of accumulating heavy metals, flax is used for phytoremediation of contaminated soils too (Kozłowski and Mackiewicz-Talarczyk 2020;Mańkowski et al. 2020).The conducted experiment allowed to identify agrotechnical factors that affect the reduction of cadmium accumulation activity from the soil by flax plants.
Obtained results revealed that all flax varieties accumulated Cd in seeds to varying extents.The highest concentration of Cd was found in seeds of cv.Szafir and Jantarol, taking into account all factors of experiment (above 3.5 mg•kg −1 of cadmium).In contrast, the lower level of Cd in seeds was shown in cv.Bukoz and Olivin.Previous studies (Bjelkova, Gencurova, and Griga 2011;Sauvé et al.   have shown that plants accumulate heavy metals mainly in the roots, much less in the seeds but some species, such as hyperaccumulating plants, accumulate a higher concentration of the metal in their above ground parts than in their roots (Sterckeman and Thomine 2020).Conducted research proved that the amount of cadmium in the seeds of the tested flax varieties was high, exceeding the standards for food raw material (1.0 mg•kg −1 (European Commission Regulation)) and even more so for pharmaceutical (0.5 mg•kg −1 (European Pharmacopoeia 2023)).Moreover, our study revealed a significant influence of soil pH on the intensity of cadmium uptake by all tested flax varieties.The capacity for absorbing heavy metals for plant species can differ significantly, in some species increases at a higher pH (Linger, Ostwald, and Haensler 2005) but in the other plants low pH promotes Cd uptake (Baldantoni et al. 2016).In the case of flax, the conducted studies showed higher accumulation of Cd in seeds in an acidic environment (pH 4.5).On soil with a slightly alkaline pH, the cv.Jantarol and Bukoz showed the lowest cadmium accumulation in seeds, what should be taken into account when growing flax for food purposes (0.8 and 1.0 mg•kg −1 respectively).Seeds were found to contain the lowest levels of cadmium at very high concentrations of this metal in the soil.In addition, it was revealed that acidic pH and higher Cd content in soil significantly decreased seed yield.As it was shown earlier, Cd uptake may negatively influence plant growth and yield, even at relatively low concentrations (Aslam, Okal, and Waseem 2023).
In the experiment, very high concentrations of cadmium in the soil were artificially introduced, so in all combinations of the experiment the content of accumulated cadmium in the seeds was high.The purpose of such an experiment was to check the relationship and amount of cadmium accumulation, with the factors studied (pH, variety, phosphorus fertilization).Cadmium contents in soil under natural conditions are not that high.With the results obtained, it is possible to indicate to producers of seeds for the food or pharmaceutical industry what agrotechnical treatments they should use and what sites they should choose (e.g., in case of unfavorable acidic soils, it is advised to raise the pH level).
Differences between varieties confirmed that the genetic factor is also important in the ability of a plant to accumulate cadmium (Bjelkova, Gencurova, and Griga 2011).It depends on their genetic, anatomical, and physiological structure (Farid et al. 2014).Similarly, the genetic background was closely related to fat content in seeds and among the examined varieties of flax, "Olivin" had the highest content of fats.
No conclusive effect of phosphorus fertilization level was observed in this experiment.In general, the presence of phosphorus in the soil is an important factor limiting the uptake of heavy metals by plants, because at a higher content of easily soluble forms of this element, sparingly soluble phosphates -including zinc, cadmium, lead, and copper -can be precipitated (Weng et al. 2002).According to previous study, phosphate reduces Cd uptake by some plant species (Baldantoni et al. 2016) but we did not find this for flax cultivars.
Intermediate dose of phosphorous fertilization was associated with the highest seed yield.Probably extreme doses of fertilizer limit seed setting -too low fertilization causes undernutrition of plants, too high fertilization stimulates growth of vegetative parts.
Among the four flax varieties examined, the highest seed yield was obtained for "Bukoz" (two times more than others) during cultivation with 100 kg/ha phosphorous fertilization and pH 7.5.Under these conditions, seeds contain the lowest amount of cadmium (the lowest bioconcentration index).Relatively high seed yield was also specified for cv."Szafir" but in the case of this cultivar the control of pH level is essential to avoid high concentration of Cd in seeds.In conclusion, it was shown that the soil pH is an important factor for the intensity of uptake of heavy metals as well as for seed yield.The fertilization level and pH should be adjusted to reflect production goals.In the case of flax varieties intended for the food or pharmaceutical industries, the problem with high concentration of Cd in seeds can be addressed to some extent by regulating pH levels.The cadmium uptake by oilseed flax can be significantly lowered by increasing the soil pH.On the other hand, fiber flax can be used for phytoremediation, the efficiency of which, in the case of cadmium absorption, can be also adjustable by soil pH.Extended research is crucial in order to assess the precise dose of fertilizer to maintain a high seed yield with low cadmium levels at the same time.

Highlights
• Factors affecting cadmium accumulation in linseed, intended for the food and pharmaceutical industries, were assessed.
• Acidic pH significantly increased cadmium uptake by flax and decreased seed yield.
• Flax seeds accumulated cadmium in excess of the permitted standards for food and pharmaceutical goals.
• The highest seed yield and the lowest cadmium accumulation were obtained for "Bukoz" cultivated with 100kg/ha phosphorous fertilizer and pH equal 7.5.• Intermediate level of phosphorous fertilization resulted in higher seed yield for all tested cultivars.

Figure 1 .
Figure 1.The values of the cadmium content in the seeds of linseed varieties with acidic and alkaline soil, two cadmium levels in the soil and three different doses of fertilizer introduced (two-year average).Cd 2, Cd 4 -cadmium levels introduced to soil [mg/kg of soil].

Figure 2 .
Figure 2. Seed yield of linseed varieties with acidic and alkaline soil, two cadmium levels in the soil and three different doses of fertilizer introduced (two-year average).Cd 2, Cd 4 -cadmium levels introduced to soil [mg/kg of soil].

Figure 3 .
Figure 3. Fat content in the seed of linseed varieties with acidic and alkaline soil, two cadmium levels in the soil and three different doses of fertilizer introduced (two-year average).Cd 2, Cd 4 -cadmium levels introduced to soil [mg/kg of soil].

Figure 4 .
Figure 4. CCA Model (n = 96).Relationships between the examined features (initial Cd content in soil, Cd content in seeds, yield of straw and seeds, fertilizer level).Cd initial -initial content of Cd in the soil.Cd 2, Cd 4 -levels of cadmium improved in the soil (mg/kg).P150 -fertilization dose (150 kg/ha).

Table 2 .
ANOVA analysis for seed yield [10 −3 kg] for four varieties of flax for different levels of fertilization, two Cd concentrations in the soil and two pH levels.

Table 3 .
ANOVA analysis for straw yield [10-3 kg] for four varieties of flax for different levels of fertilization, two Cd concentrations in the soil and two pH levels.

Table 4 .
The values of minimum, maximum, mean, and standard deviation for the parameters tested and bioconcentration factor for seeds (for Bukoz, Szafir, Oliwin, and Jantarol cultivars).

Table 5 .
Correction coefficients for CCA model.