Two types of soil (soil-1, low pH, and soil-2, neutral pH) were sampled at a depth of 0–15 cm from uncontaminated rice growing area for this pot study. Soil-1 was collected from the Leeton Field Station, while soil-2 was collected from Yanco Agricultural Institute, New South Wales (NSW), Australia. Approximately 1 ton of soils was collected from each site. Soils were collected from a single sampling point at 4 m × 4 m grid area. Collected soils samples were air-dried under the sun and unwanted materials, e.g., plant debris, stones, pebbles, etc., were cleaned. Each soil was homogenized and crushed to pass through a 4 mm mesh-sieve before being used to fill pots. A subsample (approx. 500 g soil) was further crushed and screened by a sieve (<2 mm mesh) and used for characterization of basic soil properties. Basic physico-chemical characteristics of the soil are summarized in Table 1. The pots used in this experiment were 9 L rectangular plastic pots (length 31 cm, width 22 cm, and height 18 cm) loaded with 6 kg of the test soil. The pot trial was set up in a greenhouse at the University of Newcastle, Australia. The day and night temperatures were adjusted to 28°C and 22°C with 85% RH (relative humidity) under ambient photoperiod (240–350 μmol m⁻²s⁻¹). The entire experimental procedure is illustrated in Figure 1.

Two commonly grown Australian rice cultivars, the Langi rice cultivar—long grain and the Quest rice cultivar—medium grain, were secured from the Department of Primary Industries, NSW, Australia, and used as plant materials in this study. Sodium hypochlorite (NaOCl) solution (1% v/v) and Decon (0.5 ml) were used to sterilize the rice seeds for 20 min followed by 3–4 times washing with ultra-pure water (Lee et al., 2012). Sterilized rice seeds were allowed to germinate on a plastic tray with a bottom portion covered with the moistened paper towel in the a constant room temperature (30°C) of the laboratory. After 72 h, all sprouted rice seeds were then relocated to a seedling growing media and nurtured for 22 days. Healthy uniform size seedlings were chosen and pulled out with care and then transplanted into plastic pots. Each plastic pot was received two hills and two rice seedlings were placed in each hill. The rice plants are cultivated under flooded conditions of about 2–3 cm standing water layer above the soil surface during the whole growth period. Randomized complete block design (RCBD) was utilized to organize each pot.

The pot soils were spiked with three levels of Cd at the rates of 0 (Cd0), 1 (Cd1.0), and 3 (Cd3.0) mg Cd kg⁻¹ soil (as cadmium nitrate: Cd(NO₃)₂.4H₂O) and Fe at the rates of 0 (Fe0), 1 (Fe1.0) and 2 (Fe2.0) g Fe kg⁻¹ soil (as ferrous sulfate: FeSO₄.7H₂O). The levels of Cd were selected based on the concentration usually found in the paddy field soil (Jinadasa, 1999; Machiwa, 2010; Satpathy et al., 2014; Honma et al., 2016; Liu et al., 2016; Duan et al., 2017). Ultra-pure water was used to dissolve Cd(NO₃)₂.4H₂O and FeSO₄.7H₂O separately and properly mixed with the pot soils. The pot with spiked soils was equilibrated for 1 month, maintaining 80% water-holding capacity beforehand transferring rice seedlings. Therefore, the Cd and Fe combination has given a total of nine treatments which designated as Fe₀Cd₀, Fe₀Cd_1.0, Fe₀Cd_3.0, Fe_1.0Cd₀, Fe_1.0Cd_1.0, Fe_1.0Cd_3.0, Fe_2.0Cd₀, Fe_2.0Cd_1.0, and Fe_2.0Cd_3.0. The study consisted of three replications per treatment resulting in a total of 108 pots. Chemical fertilizer used in each pot soil and application rates per kg soil were N:200 mg, P:65 mg, K:160 mg, and S:75 mg in the form of urea (CO(NH₂)₂), triple superphosphate (Ca(H₂PO₄), muriate of potash (KCl), and gypsum (CaSO₄.2H₂O), respectively. All fertilizers except N were applied to the pot soil as basal dose before transplanting of rice seedlings. The N fertilizer addition was done at 7, 30, and 60 days after transplanting rice seedlings in 3 equal splits. Intercultural operations were performed as and when necessary throughout the entire growth period. The pot experiment started in April and continued up to August in 2018.

Soil pore water was collected four times at 10, 30, 60, and 110 days after the transplanting of rice seedlings into a plastic pot during the entire plant growth. Soil pore water was extracted using the Rhizon soil pore water sampler (Rhizosphere Research Products, Netherlands). The Rhizon sampler was vertically inserted into pot soil up to a depth of 10 cm from the soil surface near the center after puddling of pot soil but before transplanting of rice seedlings. 10 ml of soil pore water was collected using a syringe and filtered through a 0.45 um syringe filter into a prewashed plastic tube. The filtered soil pore water was acidified immediately with high purity nitric acid (7 M) to measure dissolved metals using ICP-MS, Agilent 7900, Japan.

At the mature stage, rice plants from each pot were harvested on August 22, 2018, using sharp stainless-steel scissors. After harvesting, whole rice plants were rinsed at least 3-4 times with deionized water followed by ultra-pure water and placed on a plastic sheet for sun drying. Yield-related components were categorized as the number of tillers per hill, plant height, panicle length, and filled grains per panicle from 5 individual rice plants which were randomly selected from each pot. Plant height and panicle length were recorded manually using measuring tape. The number of tillers per pot was recorded before harvesting rice plants. Filled grains were separated from each panicle and counted manually. The straw and grains weights were recorded with the help of electric balance. After oven drying, whole paddy rice was separated into rice grains and husk. Rice grains were finely ground and digested following the procedure described by Rahman et al. (2009). The concentration of Cd in rice grains was measured by ICP-MS (Agilent 7900, Japan). Rice flour-SRM1568b (NIST, United States) was used as standard reference materials for quality control. Blanks, duplicates, and continuing calibration verifications (CCVs) were also employed in each batch of Cd analysis. The observed value of Cd in rice flour was 20.43 ± 0.84 µg kg⁻¹, which indicated 91% recovery of the certified value (22.4 ± 0.1.3 µg kg⁻¹). The translocation factors (TFs) of Cd from root to straw (TFroot-straw), root to grain (TFroot-grain), straw to husk (TFstraw-husk), and straw to grain (TFstraw-grain) was measured by estimating Cd concentration in one part with respect to the other parts as described by Khaliq et al. (2019).

In this study, the potential health risks from Cd contamination through consumption of rice was estimated by calculating the average daily intake (ADI), hazard quotient (HQ), and cancer risk (CR) using the following equations: ADI  = C C d × I n g R × E D × E F B M × A T , (1) HQ = ADI R f D    , (2) CR = ADI × CSF , (3) where

ADI = estimated daily intake of Cd from rice consumption (mg d⁻¹) per B_M (kg);

C_Cd = concentration of Cd in rice (mg kg⁻¹);

IngR = ingestion rate of rice (0.432 kg d⁻¹) for adults obtained from Islam et al. (2017);

B_M = body mass (70 kg) (Pirsaheb et al., 2021);

E_F = exposure frequency (365 days yr⁻¹);

E_D = exposure duration (70 years);

A_T = average exposure time (365 days× E_D = 25,550 days);

R_fD = oral reference dose (1 × 10⁻³ mg kg⁻¹ daily for Cd) as suggested by USEPA (IARC, 2012);

CSF = cancer slope factor (6.3 mg kg⁻¹ per day) (Kusin et al., 2018; Pirsaheb et al., 2021).

In terms of non-carcinogenic risk, HQ > 1 denotes significant adverse human health effects due to the presence of a particular element in the diet, while HQ < 1 indicates no significant risk is anticipated (Zhuang et al., 2016; Abtahi et al., 2017; Antoniadis et al., 2019; Halder et al., 2020). In case of carcinogenic risk, if CR value surpassing 1.0 × 10⁻⁴ is considered as unacceptable whereas CR value below 1.0 × 10⁻⁶ is not considered to develop significant health effect, CR values from 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ are usually supposed to be an acceptable range (IARC, 2012).

JMP Pro software version 14.2.0 software was utilized to perform all statistical analyses. Three-way full factorial analysis of variance (ANOVA) was applied to test the significance of the different treatments. To determine the comparison between variance, a student’s t-test was utilized and a significance test was done at the 0.05 level. GraphPad Prism Software (Version: 9.0.0) was used to generate all figures. Data from the statistical analysis were expressed as means ± standard error (SE) (n = 3).

Yield and yield components of two rice cultivars were determined after harvesting of whole rice plant and results are demonstrated in Tables 2, 3. There was a wide variation in yield components and yield of two rice cultivars. In terms of performance, the Quest cultivar produced the highest number of tillers per hill, plant height, panicle length, number of filled grains per panicle, straw yield, and grain yield than the Langi cultivar (Table 2). From Table 2, it was revealed that the number of tiller per hill, filled grains per panicle, straw yield, and grain yield were decreased by 37,11, 30, and 32%, respectively, in soil 1 (pH 4.6) compared to soil 2 (pH 6.6). However, no changes were observed for plant height and panicle between soils 1 and 2. The results showed that soil pH significantly influenced the grain yield. Results indicated that variation in yield components and the yield of the rice cultivar were dose dependent. The difference between Cd1.0 and Cd3.0 was statistically identical except plant height and filled grains per panicle for Langi and filled grains per panicle for the Quest cultivar (Table 3). The values of yield components and the yield of the rice cultivar were decreased with the increasing Cd level in the soil (Table 3). Compared to control, the number of tillers per hill was reduced by 16.5 and 24.1% for the Langi cultivar while 12.3 and 19.9% for the Quest cultivar at 1 and 3 mg kg⁻¹ Cd treatments. Similarly, plant height, panicle length, the number of filled grains per panicle, straw yield, and grain yield of the Langi cultivar was reduced by 3.9, 5.6, 8.7, 18.3, and 20.3%, respectively, at Cd1.0 while 9.5, 9.3, 17.2, 28.4, and 33.3%, respectively, at Cd3.0 treatment compared to the Cd control. On the other hand, the plant height, panicle length, number of filled grains per panicle, straw yield, and grain yield of the Quest cultivar were decreased by 3.5, 2.7, 6.2, 12.2, and 16.5%, respectively, at Cd1.0, while 5.5, 4.8, 14.8, 17.3, and 29.4%, respectively, at Cd3.0 treatment, compared to the Cd control. The difference in yield components and yield may be related to the inherent tolerance capacity of the rice cultivar to Cd stress and the toxic effect of external Cd was less in the Quest cultivar compared to the Langi cultivar. A decrease in yield and yield components of the rice cultivar through the application of exogenous Cd has been observed by Kanu et al. (2017), and the negative consequences were greatly varied depending on the rice cultivar and levels of Cd toxicity in the soil. Reduction of yield and yield-related components due to Cd toxicity was also reported by Herath et al. (2014). The application of Fe in the soil increased the number of tillers per hill, plant height, panicle length, number of filled grains per panicle, straw yield, and grain yield of the rice cultivar, and this was more pronounced in Fe1.0 than Fe2.0 compared to the control (Table 4). Also, in previous studies, it has been observed that biomass of straw and grain of rice plant was enhanced through the moderate Fe (1 g kg⁻¹) application compared to high Fe exposure (2 g kg⁻¹) to the soil (Liu et al., 2020). This is likely because moderate Fe supplementation can enhance rice growth and promote photosynthesis and a reverse situation is observed when a high Fe dose is applied (Pinto et al., 2016). Yield components and yields of rice cultivar were significantly (p <0 .0001) influenced by combined Fe and Cd supplementation into the soil (Table 5). The addition of 1 and 2 g kg⁻¹ Fe significantly increased rice cultivar's yield components and yield compared to Fe0 with no Cd supply, but the increment was less in Fe2.0 treatment than the Fe1.0. Rice cultivar subjected to moderate Fe supply (1 g kg⁻¹) combined with Cd still produced higher yield components and yield than higher Fe application (2 g kg⁻¹). For example, the grain yield of the Langi cultivar was increased by 67.3 and 33.2% at Fe1.0 and Fe2.0, whereas the grain yield of Quest was increased by 60.1 and 25.9% at Fe1.0 and Fe2.0 treatment under the Cd1.0 addition. At Cd3.0, grain yield increased by 55.8 and 21.6% for the Langi cultivar and 54.7 and 23.4% for the Quest cultivar at 1 and 2 g kg⁻¹ Fe application. Yield components and yield of both rice cultivars were slightly reduced with Cd3.0 application as compared with Cd1.0. Higher values of yield components and yield were observed at moderate Fe supply because moderate Fe supply reduced Cd toxicity in rice plants through enhancing growth, photosynthetic activity, stomatal conductance with no visual symptoms of Fe toxicity (Liu et al., 2010). On the other hand, high Fe supply combined with Cd stress reduced photosynthetic rate and deactivated antioxidative systems resulting in lower biomass and grain yield (Liu et al., 2020). So it is recommended to use suitable doses of Fe in the rice field to promote rice production and combat Cd toxicity in rice plants when cultivated in the Cd-contaminated soil.

There was a significant difference (p < 0.001) in soil pore water Cd concentration over time (Figure 2). The Cd concentration in soil pore water reached 70.75 μg L⁻¹ by 10 days after transplanting (DAT) of rice seedlings and then gradually decreased to 24.48 μg L⁻¹ before harvesting of rice plant (110 DAT) (Figure 2). Soil pore water sampled at the flowering stage (after 60 DAT) contained less Cd than initial samples. Our results are consistent with an earlier study conducted in Canada by Gao et al. (2011), showing that Cd concentration in soil pore water ranged from 20 to 80 μg L⁻¹. Similar results of Cd in soil pore water were reported in previous studies (Meers et al., 2007; Beesley and Dickinson, 2009). The Cd concentration in soil pore water was significantly affected by Cd and Fe addition in soils (Figure 3). Compared to control, application of 1 and 3 mg kg⁻¹ Cd in the soil increased Cd concentration in pore water by 48.3 and 76.1%, respectively, under Fe0 treatment. A recent study by Wang et al. (2021) showed that Cd contents in pore water of soil increased with the increasing level of Cd in the soil and ranged from 0.11 to 1.7 mg L⁻¹ in the red soil and 0.06–1.3 mg L⁻¹ in the yellow brown soil, respectively, when soil spiked with five levels of Cd (0, 1, 2, 2.5, 5.0, 7.5, and 10 mg kg⁻¹ soil). At Cd1.0 treatment, the pore water Cd concentrations were decreased by 31.5 and 58.4% with the addition of 1 and 2 g kg⁻¹ Fe, respectively, as compared to corresponding Fe control. Addition of 1 and 2 g kg⁻¹ soil reduced pore water Cd concentrations by 23.7 and 43.6% under 3 mg kg⁻¹ Cd supplementation, respectively, when compared with Fe control. The results of this study demonstrated that pore water Cd concentration decreased due to the addition of Fe to the soil. It was well accepted that application of Fe increased the formation of Fe oxides in soil pore water having large surface area along with more functional groups with abundant reactive sites to adsorb Cd, thereby reducing Cd concentration in pore water (Jiang et al., 2017; Zhu et al., 2018; Li et al., 2020). Besides, Fe plaque is deposited around the root surfaces with Fe addition that impede Cd uptake and accumulation through adsorption or co-precipitation, thereby decreasing pore water Cd, which reduced potential Cd transfer from root to rice grain (Wan et al., 2018; Deng et al., 2020).

Translocation factors (TFs) of Cd from root to straw, root to grain, straw to husk, and straw to grain were determined and demonstrated in Figures 4, 5. TFs between the different parts of root-rice systems varied greatly and depending on the external Cd and Fe application and rice cultivar. TF of Cd from root to straw (TF_root-straw) was varied between 0.20–0.33, while from root to grain (TF_root-grain), the value lies between 0.01–0.02. TF from root to straw (TF_root-straw) was an order of magnitude higher than root to grain (TF_root-grain) (Figure 4), showing stronger accumulation ability of Cd by straw as compared to the translocation of Cd in rice grain. A similar finding was reported by Deng et al. (2020), who showed that the mean TF of Cd occurred in the order of TF_root-straw > TF_root-grain ranging between 0.38–0.46 for TF_root-straw and 0.07–0.08 for TF_root-grain, respectively. On the other hand, TF of Cd from straw to husk (TF_straw-husk) was varied between 0.12–0.26, while from straw to grain (TF_straw-grain), it ranged between 0.06–0.11. TF from straw to grain (TF_straw-grain) was lower than straw to husk (TF_straw-husk) (Figure 5), showing the inherent ability of rice plants to accumulate more Cd in the husk and less transfer to rice grain. These results were consistent with the outcomes from Deng et al. (2020), who observed that Cd translocation in terms of TF_straw-husk was higher than TF_straw-grain ranging between 0.20–0.24 for TF_straw-husk and 0.18–0.20 for TF_straw-grain, respectively. Similarly, Khaliq et al. (2019) also reported that the TF of Cd in rice grain was much lower as compared to the different parts of the rice plant. The translocation of Cd between different parts of the rice cultivar trended to increase with the increasing Cd level and decrease with increasing Fe concentration. With regard to the cultivar performance, the Quest cultivar had lower Cd translocation factors than Langi cultivars. Similar results were found by Kanu et al. (2017), who studied five rice cultivars and showed that the translocation factor for Xiangyxiangzhan and Guixiangzhan rice cultivars was minimal compared to Meixiangzhan, Basmati, and Nongxiang18 rice cultivars. The translocation factors of Cd into different parts of the rice plant for the Fe2.0 group were significantly lower than those for control and Fe1.0 under Cd1.0 and Cd3.0 treatments. The TF values were increased with the increasing Cd level which is supported by Kanu et al. (2017), who observed the highest TF values when rice plants are subjected to Cd treatment.

The Cd concentration in rice grain was demonstrated in Table 6. The grain Cd concentration ranged from 0.01 mg kg⁻¹ to 1.92 mg kg⁻¹ for all treatment combinations. The rice cultivar cultivated in soil-1 (low pH) had higher grain Cd concentration than soil-2 (high pH). The Cd concentration in two rice cultivars differed significantly (p < 0.001); 0.67 mg k g⁻¹ for the Langi and 0.51 mg kg⁻¹ for the Quest rice cultivar. The rice cultivar treated with Cd1.0 led to the lower grain Cd concentration while Cd3.0 resulted in higher Cd accumulation in rice grain. Fe supplementation significantly reduced the grain Cd accumulation (Table 6). Average daily intake (ADI) was calculated using Cd dataset of two rice cultivars grown in two dissimilar soils and presented in Table 6. In soil-1, the ADI of Cd from the rice grain of the Langi cultivar ranged from 0.84 × 10⁻³ to 11.83 × 10⁻³ (mg ⁻¹kg⁻¹ bw) (weekly 5.88 × 10⁻³ to 82.8 × 10⁻³ (mg⁻¹ kg⁻¹ bw) while for the Quest cultivar, it ranged from 0.56 × 10⁻³ to 9.42 × 10⁻³ (mg⁻¹ kg⁻¹ bw) (weekly 3.92 × 10⁻³ to 65.94 × 10⁻³ (mg⁻¹ kg⁻¹ bw). For soil-2, the ADI value of the Langi cultivar lies between 0.20 × 10⁻³ to 3.85 × 10⁻³ (mg⁻¹ kg⁻¹ bw) (weekly 1.4 × 10⁻³ to 37.3 × 10⁻³ (mg⁻¹ kg⁻¹ bw) while for the Quest cultivar, it ranged from 0.08 × 10⁻³ to 3.85 × 10⁻³ (mg⁻¹ kg⁻¹ bw) (weekly 0.56 × 10⁻³ to 26.95 × 10⁻³ (mg⁻¹ kg⁻¹ bw). The ADI of Cd from rice grain of both rice cultivars was increased with the increasing Cd levels in the soil and the highest value was observed when no Fe was supplied to the soil. Our results also revealed that ADI value was higher for the Langi cultivar compared to the Quest cultivar. Toxicological Profile for Cadmium, Agency for Toxic Substances and Disease Registry (ATSDR) under US Department of Health and Human Services (ATSDR, 2012) and the European Food Safety Authority (EFSA, 2011) set weekly tolerable Cd intake limits of 0.7 and 2.5 × 10⁻³ mg⁻¹ kg⁻¹ bw, respectively. The joint FAO/WHO Expert Committee on Food Additives (JECFA) recommended the provisional tolerable weekly intake (PTWI) value of Cd as 5.8 × 10⁻³ mg⁻¹ kg⁻¹ bw, which is equivalent to 1 × 10⁻³ mg⁻¹ kg⁻¹ bw per day (PTDI) (JECFA, 2010). Table 6 showed that the daily intake of Cd from rice grain was below the provisional tolerable weekly intake (PTDI) when no exogenous Cd was supplied to the soil but much higher than PTDI when additional Cd was supplied to the soil. A recent investigation demonstrated that the average weekly intake of Cd from rice grain was 0.66 × 10⁻³ to 4.03 × 10⁻³ mg⁻¹ kg⁻¹bw, which was lower than this study (Shahriar et al., 2020). Similarly, Naseri et al. (2015) found an average weekly intake of Cd in different types of rice were greatly varied and ranged from 3.7 × 10⁻³ to 5.7 × 10⁻³ mg⁻¹ kg⁻¹ bw based on 110 g rice consumption per day. The higher value of ADI of Cd in this study possibly indicates the influence of exogenous Cd addition, dissimilar soil type, and variation in Cd uptake between the rice cultivars. In the present investigation, the bioaccessibility of Cd was not considered, which might be another reason for the higher ADI of Cd. The bioaccessibility of Cd in different rice brands varied from 17.4 to 79.7% (Sharafi et al., 2019). Another study showed that the bioaccessibility fraction of Cd ranged from 2.57 to 26.9% from rice consumption (Tefera et al., 2021). So, the average daily intake of Cd will be less if we consider the bioaccessibility fraction of Cd in the rice grain of two rice cultivars. Further investigation on the bioaccessibility fraction of Cd in rice grain should be conducted in order to ensure a more accurate Cd risk estimation.

In this study, the non-carcinogenic risk (HQ) of Cd ranged from 0.84 to 11.83 for the Langi cultivar and 0.56 to 9.42 for the Quest cultivar in soil-1 while 0.20 to 5.33 for the Langi cultivar and 0.08 to 3.85 for the Quest cultivar in soil-2. The value of HQ was increased with increasing Cd contamination in both soils and decreased with the addition of Fe (Figures 6A,B). For example, the HQ value was 4.57 and 5.81 with Cd1.0 and Cd3.0 treatment, respectively, when 1 g kg⁻¹ Fe was added to the soil while at 2.0 g kg⁻¹ Fe supplementation, the value was 3.08 and 4.10 at Cd1.0 and Cd3.0 treatment, respectively. For both Cd treatments, the HQ values of two rice cultivars were exceeded the safe level (HQ > 1), indicating a risk that existed due to non-carcinogenic effects, which is supported by published reports (Rai et al., 2019; Pirsaheb et al., 2021). The results of this study are in good agreement with Chen et al. (2018a), who reported HQ value from rice consumption was higher than the threshold level and ranged between 2.35 and 4.17. Shahriar et al. (2020) also demonstrated that the HQ values were varied from 0.09 to 0.58 from rice intake in Bangladesh, which were much lower than this present study. According to USEPA, the permissible value of CR is 1.0 × 10⁻⁴ while the tolerable limit of CR for regulatory purposes ranged from 1.0 × 10⁻⁶ to 1.0 × 10⁻⁴ (IARC, 2012). The CR value of Cd exposure was shown in Figure 6 (C and D). It was observed that the CR value at all treatment combinations was higher than the permissible limit of 1.0 × 10^−4, indicating a potential carcinogenic significant risk of Cd through consumption of rice. The CR value at Cd treatment without Fe application had the highest risk than with Fe application. The Langi cultivar had a higher potential health risk than the Quest cultivar. The results of this present study are in good agreement with the findings by Shahriar et al. (2020), who showed that the mean CR value of different rice brands was ranged from 2.10 × 10⁻³ to 8.70 × 10⁻³. In this study, the results of health risk estimation might be overestimated because the only exposure route of rice consumption was taken into account. The contribution of rice consumption to dietary intake of Cd differed remarkedly depending on gender, age, ingestion rate, and geographical area even within the same study area (Shahriar et al., 2020).