Alleviating cobalt and lead toxicity in rice using zero valent iron (Fe°) amendments

Abstract

Simultaneous impact of zero valent iron (Fe°) and rice cultivar on uptake, translocation, and bioaccumulation of cobalt (Co) and lead (Pb) in rice (Oryza sativa L.) was investigated to alleviate Co and Pb toxicity in rice. Kilombero and Faya rice cultivars, amended with Fe° dosages of 0, 6.20, and 12.40 g Fe° kg⁻¹ soil, were cultivated under continuous flooding in pots in a greenhouse. Shoot and grain-Co and Pb concentrations were determined using inductively coupled plasma mass spectrometry (ICP-MS). For Co, amending Faya rice with at least 6.20 g Fe° kg⁻¹ reduced grain-Co accumulation by 33% or more compared to control plants (F = 17.5; p < 0.001) while inconsistent results were obtained for Kilombero. For Pb, Faya also accumulated more than 39% less grain-Pb than control plants (272 μg kg⁻¹) while Kilombero accumulated more than 55% less grain-Pb than control plants under the same conditions. Despite reducing grain-Pb accumulation in both cultivar, Fe amendments of at least 6.20 g Fe° kg⁻¹ reduced grain-Pb accumulation with greater magnitude in Kilombero (55%) than in Faya (39%). Nonetheless, Fe amendments inhibited greater shoots-Co and Pb translocation (≥32%) to grains in Faya compared to Kilombero (≤20%). The work provides a novel promising agronomical practice of reducing Co and Pb bioaccumulation in rice.

Highlights

• •Impact of rice cultivar x Fe° amendments on Co and Pb accumulation in rice grains was studied.
• •Fe° dosages markedly inhibited available Co and Pb in porewater.
• •Fe° dosages marked inhibited translocation of Co and Pb from shoots to grain.
• •Uptake of Co from soil was higher in Kilombero than in Faya while uptake of Pb was vice versa.
• •Co and Pb accumulation in rice grains varied with cultivar and Fe° dosages.

Cobalt (Co); Faya, Kilombero, Lead (Pb); Rice; Zero valent iron (Fe°); Cultivar.

1. Introduction

Contamination of food crops such as rice, maize and wheat with toxic metal(loid)s such arsenic (As) [1, 2], cadmium (Cd) [3], cobalt (Co) [4] and lead (Pb) [5] is worsening globally due to inappropriate anthropogenic activities such as mining, indiscriminate use of toxicant-containing agrochemicals such as pesticides and fertilizers; and agronomical practices that stimulate uptake of toxic metal(loid)s [6]. It is becoming apparent that rice could be a major source of ingesting Co and Pb among the communities that solely depend on rice diets for subsistence living. Contamination of rice with elevated Co and Pb concentrations threatens food safety which calls for global collective efforts of adopting agricultural practices for mitigating the contamination [7].

Pb, as a toxic pollutant, is of public health concern and has no known benefits even at low non-phytotoxic concentrations [6]. Pb is ranked second after arsenic amongst the most hazardous metals [4]. In humans, elevated Pb concentrations can damage the nervous system and gastrointestinal and renal organs [8], and cause brain disorders and convulsions and/or death [9, 10]. Background concentrations of Pb ranges from 30000 - 100000 μg kg⁻¹ in uncontaminated soils [11] and 100–500 μg kg⁻¹ in less contaminated soils [11]. Rice plants readily uptake Pb through roots. Rice plants also efficiently transport Pb to edible parts such as grains which ultimately allows Pb enter the food chain. Rice cultivated in Pb-contaminated paddy soils bioaccumulate more Pb as compared to rice cultivated in uncontaminated soils [6]. The World Health Organisation (WHO) [12] and the European commission (EC) [13] set the maximum permissible limit for Pb in rice grains at 200 ug kg⁻¹ to protect trade and human health.

For Co, it is not considered as an essential element to humans in its inorganic form. Excessive consumption of Co has genotoxic, hepatotoxic, nephrotoxic, neurotoxic, and immunotoxin effects to human and animal health [14, 15]. Co consumption has been linked to certain diseases such as Alzheimer's, Parkinson's and autism [14, 15, 16]. There are as yet no maximum permissible levels set for Co concentrations in rice and other food commodities by either WHO or other organisation [17].

Several agronomical practices, such as use of Fe° and silicon amendments, use of alternate wetting and drying irrigation, and selecting low metal(loids) accumulating cultivars and their interactions have been studied and reported to effectively regulate metal(loids), predominantly arsenic, accumulation in rice [18, 19, 20, 21]. Agronomical practices of interest in this investigation were the use of Fe° and the selection of low metal(loids) accumulating cultivars and their interactions on Co and Pb. For Fe° amendments, Fe° has been reported to effectively remove pollutants including heavy metals from groundwater, wastewater and soil [22, 23]. In this study, Fe° was chosen because due to its low cost, simplicity of application, being a non-toxic material and relatively environmental friendly [23]. Nevertheless, presence of certain nanoscale zero-valent iron at elevated levels reduce soil microbial biomass and activity [22, 23]. Nevertheless, the presence of certain nanoscale zero-valent iron at elevated levels can reduce soil microbial biomass and activity [22, 23].

Previous studies showed that Fe° and iron materials markedly regulate As and Cd bioaccumulation in rice [18, 19, 20, 21] by immobilizing As and Cd in soil or reducing their availability in soil porewater through sorption of As or Cd particulates or ions onto ironoxides or iron hydroxdes groups [18, 24, 25, 26]. Subsequently, As and Cd sorbed onto oxidised Fe° may co-precipitate together with the iron materials which immobilise them [18, 24, 25, 26]. Considering that the chemistry of Co and Pb is similar to that of As and Cd, we expect Co and Pb to show similar trends of adsorption and co-precipitation processes which will eventually reduce availability and accessibility of Co and Pb to rice plants [18, 24, 25, 26]. Thus, the sorption of Co and Pb onto Fe° can be utilised in paddy fields to regulate Co and Pb bnioaccumulation in rice which has high potential of reducing health hazards associated with these metalloids.

There are many studies that have reported a positive impact of Fe° amendments on the reduction of As and Cd accumulation in rice grains. For instance, amending rice with Fe° has been reported to markedly reduce As and Cd accumulation in rice grains under certain soil conditions and in certain rice cultivars [11, 18, 21, 26, 27]. Mlangeni et al. [18, 21]showed that Fe° amendments reduced grain-Cd and As concentrations in rice by more than 51% and 61%, respectively, in rice cultivated under low water (LW) and alternate wetting and drying irrigation compared to respective control treatments. Farrow et al. [26] also reported that amending soils with ferrous ion immobilized soil-As and reduced grain-As accumulation in rice grains.

However, most of these studies on impact of iron materials or Fe° on metal(loids) transfer from soil to plants in rice fields refers to As and/or Cd amended with iron materials or Fe°. Few studies, if not none, have investigated impact of iron materials or Fe° on Co and Pb transfer to rice grains. Considering that Co and Pb are emerging contaminant that require attention, it is thus necessary to study impact of Fe° on alleviating Co and Pb bioaccumulation in rice using similar experiments as have been carried out with As or Cd.

Therefore, uptake and bio-accumulation of Co and Pb in two most popular Malawian rice cultivars (Faya and Kilombero) [28] were investigated through greenhouse pot experiments. Both cultivars are aromatic and pure line selections from the Faya genotype pool. However, Faya rice flowers earlier than Kilombero rice cultivar [28]. The main aim of the study was to evaluate and understand the extent to which simultaneous use of Fe° amendment and cultivar selection alleviate bioaccumulation of Co and Pb in rice. Specifically, the study evaluated simultaneous impact of various Fe° amendment dosages and rice cultivar on uptake and bioaccumulation of Co and Pb in rice grains in order to alleviate Co and Pb toxicity in rice.

2. Methods and experiments

2.1. Greenhouse experiments

The greenhouse pot experiments used agricultural sand-loam soils collected at soil depth of 0–12 cm in Aberdeenshire, Scotland. Experimental soils, dried to a constant mass in a drying chamber [18, 21], were sieved using 4 mm sieve. The purpose for sieving was to remove plant materials, large soil aggregates and stones from the soils. One part of the sieved soils was mixed with one part of sand to make a homogeneous soil-sand mixture with 1:1 soil to sand ratio (v/v). The soil-sand mixture was used because it allows easy drainage of water into soil. Besides, it also enable easy extraction of roots from soil when determining root biomass [18, 21]. Overall metal(loids) concentrations plus physical and chemical properties of the soil-sand mixture were predetermined (Table SM1).

1.0 kg of soil-sand mixture was spiked with one of the three levels of Fe° dosages (0, 6.20 and 12.40 g Fe° kg⁻¹) before being placed in PVC pots (height: 25 cm and diameter: 22 cm) lined with plastic-liners [18, 21]. The plastic liners were used to reduce the loss of solubilised Fe, Co and Pb and other soil nutrients. The Fe° used in this experiment had an average diameter of 3.9 ± 1.01 mm (Beijing North Yongbang Science and Technology, China). Rice plants were grown in the greenhouse with natural lighting, temperature range from 22 to 35 °C, and humidity ranging from 45 - 55%. Initial soil sample pH (6.1) was measured in deionized water (with soil to water ratio of 1:2.5) after shaking for one (1) hour [18, 29].

All treatment pots, planted with one seeding per pot of either Faya 14M69 or Kilombero rice, were continuously flooded with no less than 3 cm water level above soil surface to replicate continuous flooding (CF) irrigation practiced in smallholder rice farms in Malawi [30]. Four replicates were arranged for each treatment and all other conditions were as specified in our previous publication [21].

2.2. Collection of porewater and determination of Co and Pb concentrations in porewater

At predetermined time points (25^th, 40^th, 90^th, 110^th and 130^th date after transplanting (DAT), porewater was sampled from each pot experiment using Rhizon samplers inserted into the soil at a 45⁰ angle [18]. The porewater was collected through syringes attached to Rhizon samplers. The pH of porewater was determined on raw porewater (Table SM2) whereas Co and Pb concentrations were determined on 2 mL of porewater. Porewater reserved for determination of the studied metal(loids) was acidified with 1% HNO₃ immediately after collection. Each porewater sample was diluted to 20 mL with 18.2 MΩ cm Milli-Q water prior to metal(loids) measurements using inductively coupled plasma mass spectrometry (ICP-MS/MS 8800, Agilent Technologies, Santa Clara, CA, USA) in inorganic mode [31, 32, 33]. The instrument (ICP-MS/MS) was optimized using continuous introduction of a tuning solution having 1 μg L⁻¹ Pb, Co, yttrium (Y) and rhodium (Rh). The ICP-MS/MS operating conditions, including solution flow rate, used in this study are summarized in Table SM3.

2.3. Shoots and grain sample preparation and Co and Pb determination

Total Co and Pb concentrations in shoots and grains and certified reference material (CRM) were determined on 0.20 g (±0.01 g) of ball-milled samples in triplicate, digested in 2.00 mL of 70% nitric acid overnight assisted by open vessel digestion using Mars 5 digestion system (CEM, US). Analytes (Co and Pb) concentrations in shoots, grains and CRM were determined using ICP-MS/MS (8800 model: Agilent Technologies, USA) in inorganic mode. Similarly, the instrument operating conditions were optimized as stated in section 2.3) by continuous introduction of a tuning solution having 1 μg/L Pb, Co, Y and Rh (Table 2).

Table 2.

Interaction impact of cultivar x Fe° dosages on Co and Pb accumulation in rice shoots and grains and on Shoot-to-grain Co and Pb-TF showing F-values and level of significance (asterisk). TF is the translocation factor of elements from shoot-to-grain.

Parameter	Cultivar	Fe°	Cultivar x Fe°
	F-value	F-value	F-value
Shoot-Co	5.3∗∗	1.3∗	13.2∗∗∗
Shoot-Pb	3.5∗∗∗	3.7∗∗	21.4∗∗
Grain-Co	11.6∗∗	15.2∗∗∗	17.5∗∗
Grain-Pb	7.4∗	4.4∗	Ns
Shoot-to-grain Co-TF	Ns	112.6∗∗∗	Ns
Shoot-to-grain Pb-TF	62.1∗∗∗	8.3∗∗	Ns

Note: ∗ ∗, ∗∗ and ∗∗∗ indicate level of significance at 0.05, 0.01 and 0.001, respectively whereas ns indicate non-significant outcome.

2.4. Translocation factor

The soil-to-shoots cobalt translocation factors (soil-to-shoots Co-TF), soil-to-shoots lead translocation factors (soil-to-shoots Pb-TF), shoots-to-grains Co-TF and shoots-to-grains Pb-TF were calculated as ratios of Co and Pb concentrations in shoots or grains to that in corresponding soil or shoots [34] (Equation1).

$${\mathrm{T}\mathrm{r}\mathrm{a}\mathrm{n}\mathrm{s}\mathrm{l}\mathrm{o}\mathrm{c}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n}\mathrm{f}\mathrm{a}\mathrm{c}\mathrm{t}\mathrm{o}\mathrm{r},\mathrm{T}\mathrm{F}=[\mathrm{C}]}_{\mathrm{a}}/{[\mathrm{C}]}_{\mathrm{b}}$$ Equation 1

where [C]_a is Co or Pb concentration in shoots or grains and [C]_b is Co or Pb concentration in soil or shoots. Considering that TF values for metals vary with plant species, soil pH, soil chemistry and soil amendments [35], TF were compared across soil metal content, cultivar and Fe° dosages and their interactions. Higher TF indicates either greater efficiency of plants in translocating the metal(loids) from soil to shoots and grains or poor metal(loids) retention in soil. In contrast, lower soil-to-shoot TF indicates either lower efficiency of plants in translocating the metal(loids) from soil to shoots and grains or stronger sorption of metal(loids) to soil particles or both [35, 36].

2.5. Quality assurance and control

Ultra-pure Milli-Q water (18.2 MΩ cm) was used in sample solutions preparation. Working standards and blanks were prepared in a 2% (v/v) nitric acid solution. Nitric acid solution (10%) was used to wash all glassware and equipment as well as to rinse sample introduction systems of the ICP-MS/MS in order to remove any possible contaminant [37]. Accuracy of measuring of shoots and grain-Co and Pb was checked with NIST1568a - rice flour (Table 1) while that of porewater-Co and Pb was checked with CRM - DC73319 (Table 1). Percentage recoveries of Co and Pb in the CRM ranged from 84% to 106% (mean: 95.0%) and 76% to 100% (mean: 87.5%), respectively, across all samples (Table 1) and these were satisfactory.

Table 1.

Accuracy checks of shoot-Co and Pb and grain-Co and Pb measurements using CRMs DC73319 and NIST1568a showing certified and mean measured values, and recoveries (%). Values are given as means ± SD in μg kg⁻¹ (n = 6).

Metal(loid)	Certified value	Mean measured value	Recovery, %
DC73319
Co	14.2 ± 1.0	12.9 ± 4	92%
Pb	98.0 ± 6.0	101 ± 6	102%
NIST1568a
Co	17.7 ± 0.5	16.9 ± 2.0	95 ± 11%
Pb	4.1 ± 0.3	3.5 ± 0.5	85 ± 3%

Note: SD, standard deviations.

2.6. Statistical analyses

Impact of Fe° amendments, rice cultivar and their interactions on shoot-Co and Pb and grain-Co and Pb accumulation was analysed using Minitab 20.4 Statistical Software. Analysis of variance (ANOVA) was used to evaluate level of significance for multiple comparisons between treatment means. Pearson correlation relationships amongst the tested variables were computed using Microsoft Excel.

3. Results

3.1. Impact of Fe° amendments on porewater-pH and Co and Pb concentration

3.1.1. Soil-pH

Porewater was sampled for determination of porewater-pH at 25^th, 40^th, 90^th and 110^th date after transplanting (DAT) (Data not shown). No significant porewater pH differences were observed at all sampling points. However, mean porewater-pH under Kilombero (pH = 6.1) was lower than that under Faya (pH = 6.3) which indicated that Kilombero induced more acidic conditions in the porewater. Furthermore, mean porewater-pH in soils amended with 6.20 g kg⁻¹ Fe° (pH = 6.2) and 12.40 g kg⁻¹ Fe° (pH = 6.3) were higher than that under control (pH = 5.8) showing that Fe° amendments enhanced alkalinity of porewater (Data not shown).

3.1.2. Porewater-Co

Results showed that Fe° dosages (p = 0.005; F = 8.09) and interactions of rice cultivar x Fe° dosages (p = 0.035; F = 4.41; Figure 1a) markedly affected porewater-Co mobilization whereas cultivar alone had no impact (p = 0.758; F = 0.10; Figure 1a). For impact of Fe° dosages, mean Co concentrations in porewater amended with 6.20 g Fe° kg⁻¹ (9400 μg kg⁻¹) and 12.40 g Fe° kg⁻¹ (7500 μg kg⁻¹) were 38% and 53%, respectively, lower compared to the control (15900 μg kg⁻¹; Figure 1a) showing that Fe° reduced available Co concentrations in porewater. For impact of interaction of cultivar x Fe° amendment, Fe° markedly reduced Co concentrations from 18740 μg kg⁻¹ in control treatment to 7110 μg kg⁻¹ (62%) and 9140 μg kg⁻¹ (51%; Figure 1a) in porewater (under Kilombero cultivation) amended with 6.20 g Fe° kg⁻¹ and 12.40 g Fe° kg⁻¹, respectively. Conversely, while Fe° dosages of 6.20 g Fe° kg⁻¹ had no significant impact on Co concentrations, Fe° dosage of 12.40 g Fe° kg⁻¹ reduced Co concentrations from 13080 μg kg⁻¹ in control treatment to 5080 μg kg⁻¹ (62%) in porewater under Faya cultivation.

Figure 1.

Interaction impact of cultivar x Fe° dosages (a) Co and (b) Pb concentrations (μg kg⁻¹) in porewater under Faya and Kilombero rice cultivars. Error bars are standard deviation (n = 4). Bars with different letters are significantly different (P < 0.05).

3.1.3. Porewater-Pb

Two-way ANOVA revealed that porewater-Pb was markedly affected by Fe° amendment (p < 0.001; F = 81.28) only but not by Fe° amendment (P = 0.499; F = 0.48) and interaction of cultivar x Fe° amendment (p < 0.481; F = 0.77; Figure 1b). For impact of Fe° dosages, Pb concentrations in treatments amended 6.20 g kg⁻¹ Fe° (6900 μg kg⁻¹) and 12.40 g kg⁻¹ Fe° (6800 μg kg⁻¹) were 5% and 6%, respectively, higher than that under control (7100 μg kg⁻¹; Figure 1b) showing that porewater Pb mobilisation was markedly impacted by Fe°.

3.2. Interaction impact of cultivar x Fe° dosages on shoots-Co and Pb concentrations

3.2.1. Shoot-Co

Statistical description of measured parameters showing independent impact of cultivars type and Fe° dosages and interaction impact of cultivars type and Fe° dosages on Co and Pb accumulation in shoots and grains are summarized in Figure 2 and Table 2. Simple linear correlations and general linear regression analysis were also performed to evaluate impact of various Fe° dosages (Figure 2; Table 3).

Figure 2.

Interaction impact of cultivar x Fe° dosages on Co (a & b) and Pb (c & d) accumulation in rice shoots (a & c) and grains (b & d) in Faya and Kilombero rice cultivars. Error bars are standard deviations (n = 4). Bars with different letters are significantly different (P < 0.05).

Table 3.

Linear regression between Fe° dosages and shoots and grains-Co and Pb, concentrations in Faya and Kilombero showing linear equations and regression coefficients.

Plant type		Shoots			Grains
Cultivar	Metal(loid)	Equation	r2	Equation	r2
All cultivars	Co	y = –4.6x + 320	0.61	y = –2.3x + 81	0.92
All cultivars	Pb	y = –3.5x + 106	0.89	y = –17x + 283	0.96
Faya	Co	y = –13x + 435	0.93	y = –4x + 90	0.95
Kilombero	Co	y = 4x + 231	0.44	y = –0.08x + 54	0.002
Faya	Pb	y = –1x + 69	0.57	y = –18x + 273	1.00
Kilombero	Pb	y = –8x + 168	0.88	y = –19x + 308	0.90

Note: x is Fe° dosages (g kg⁻¹); y is either Co or Pb concentration in shoots or grain; r² is the linear regression coefficient; ∗, ∗∗ and ∗∗∗ indicate level of significance at 0.05, 0.01 and 0.001; ns indicate non-significant outcome.

Accumulation of Cobalt in shoots was markedly affected by cultivar (P < 0.01; F = 5.3), Fe° amendment (p < 0.05; F = 1.3) and interaction of cultivar x Fe° dosages (p < 0.001; F = 13.2; Figure 2a, b, c, and d; Table 2). Regardless of Fe° dosages, the mean shoot-Co was measured to be 338 ± 86 μg kg⁻¹and 264 ± 59 μg kg⁻¹ in Faya and Kilombero rice, respectively (Figure 2a). Thus, Faya shoots accumulated 22% more Co than Kilombero rice (Figure 2a). For Fe°, rice amended with 6.20 and 12.40 g kg⁻¹ of Fe° dosages accumulated mean shoot-Co concentrations of 335 ± 32 and 265 ± 29 μg kg⁻¹, respectively, while shoot-Co concentrations in control plants averaged 319 ± 18 μg kg⁻¹ (Figure 2a). While Fe° amendment of 6.20 g Fe° kg⁻¹ soil induced no significant differences, Fe° amendment of 12.40 g Fe° kg⁻¹ soil reduced shoot-Co accumulation by 17% (Figure 2a). Linear regression equation (r² < 0.61; p < 0.01) and linear correlation (r = -0.78; p < 0.01) between Fe° dosages and shoot-Co concentrations were significant (Figure 3a; Table 3) and suggested an inverse linear and negative correlation relationship.

Figure 3.

Linear regression between Fe° dosages and Co and Pb concentrations in (a) shoots and (b) grains showing linear correlation (r), p-values, linear regression lines for Co (solid lines) and Pb (dashed lines) against Fe° dosages (g kg⁻¹) in shoots (filled triangles and squares) and grain (un-filled triangles and squares) in Kilombero (triangles) and Faya (squares); linear regression (equations) have removed from the graphs to reduce crowding and are presented in Table 3.

For interaction impact of cultivar x Fe° dosages, mean shoots-Co concentrations in rice amended with 0, 6.20 and 12.40 g Fe° kg⁻¹ soil averaged 423 ± 51, 382 ± 36 and 284 ± 14 μg kg⁻¹, respectively, in Faya (Figure 2a) and 275 ± 34, 289 ± 56 and 265 ± 37 μg kg⁻¹, respectively, in Kilombero (Figure 2a). While Fe° dosages induced inconsistent and non-significant shoots-Co variations in Kilombero rice, corresponding Fe° dosages of 6.20 and 12.40 g Fe° kg⁻¹ reduced shoot-Co concentration by 10% and 33%, respectively, in Faya rice (Figure 2a). The observation shows that Fe° amendments are more effective in Faya compared to Kilombero. Furthermore, Fe° dosages strongly and negatively correlated with shoot-Co concentrations in Faya rice (r = -0.97; r² = 0.93, p < 0.001; Figure 3a; Table 3) whereas weaker and positive correlations were observed in Kilombero (r = 0.66; r² = 0.42, p < 0.05; Figure 3a; Table 3).

3.2.2. Shoot-Pb

Shoot-Pb concentration was markedly affected by cultivar (P < 0.001; F = 3.5), Fe° dosages (p < 0.01; F = 3.7) and interactions of cultivar x Fe° dosages (F = 21.4; p < 0.01; Figure 2c; Table 2). For cultivar, mean shoot-Pb concentrations averaged 67 ± 19 in Faya and 150 ± 22 μg kg⁻¹ in Kilombero (Figure 2c). The observation showed that Faya accumulated 55% less Pb in shoots compared to Kilombero had (Figure 2c). For Fe°, mean Pb concentrations in shoots amended with 0, 6.20 and 12.40 g Fe° kg⁻¹ soil averaged 141 ± 38, 76 ± 25 and 68 ± 16 μg kg⁻¹, respectively. The observation revealed that Fe° dosages of 6.20 and 12.40 g Fe° kg⁻¹ soil reduced Pb concentrations by 46% and 52%, respectively (Figure 2c). Furthermore, linear regression (r² < 0.88; p < 0.001) and linear correlation (r = -0.79; p < 0.001) analyses of Fe° dosages versus shoot-Pb concentrations produced significant and negative relationships (Figure 3a) which suggested existence of inverse relationships between the studied parameters.

For interaction impact of cultivar x Fe°, Fe° dosages of 6.20 and 12.40 g Fe° kg⁻¹ soil reduced Pb accumulation from 239 ± 43 μg kg⁻¹ in control treatments to 125 ± 25 μg kg⁻¹ (30%) and 77 ± 17 μg kg⁻¹ (57%), respectively, in Kilombero (Figure 2c) whereas corresponding Fe° dosages stimulated inconsistent and non-significant variations in Faya (Table 2; Figure 2c). Linear regression and correlations analyses revealed strong negative linear correlation between shoot-Pb concentrations and Fe° dosages with higher coefficients of determination (r² = 0.88; r = –0.76; p < 0.001; Figure 3c; Table 2) in Faya compared to that in Kilombero (r² = -0.44; r = 0.66; p < 0.05; Figure 3c; Table 3). These observations showed that impact of Fe° amendment on shoot-Pb bioaccumulation depends on both Fe° dosages and rice cultivars.

3.3. Impact of cultivar, Fe° and their interactions on Co and Pb accumulation in rice grains

3.3.1. Grain-Co

Grain-Co concentration was markedly impacted by cultivar (P < 0.01; F = 11.6), Fe° (p < 0.001; F = 15.2) and interactions of cultivar x Fe° (p < 0.01; F = 17.5) (Table 2; Figure 2b). For cultivar, Faya accumulated 53% greater grain-Co (78 ± 28 μg kg⁻¹) than Kilombero had (51 ± 19 μg kg⁻¹; Figure 2b). For Fe°, Fe° markedly reduced grain-Co accumulation from 83 ± 12 μg kg⁻¹ in control treatments to 61 ± 10 μg kg⁻¹ (27%) and 54 ± 13 μg kg⁻¹ (35%) in grains amended with 6.20 and 12.40 g Fe° kg⁻¹ soil dosages, respectively (Figure 2b). Furthermore, linear regression analyses revealed significant negative linear correlation (r² < 0.918; r = -100; p < 0.001) between Fe° dosages and grain-Co concentrations (Figure 3b; Table 3).

For interaction of cultivar x Fe°, Fe° markedly reduced grain-Co accumulation from 93 ± 26 μg kg⁻¹ in control treatment to 62 ± 34 μg kg⁻¹ (33%) and 48 ± 11 μg kg⁻¹ (48%) rice grains amended with 6.20 and 12.40 g Fe kg⁻¹ dosages, respectively, in Faya (Figure 2b); whereas corresponding Fe° dosages stimulated inconsistent and/or non-significant variations in Kilombero (Figure 2b). While Fe° dosages had weak and/or non-significant linear and correlation relationships with grain-Co concentration in Kilombero, Fe° had a strong linear (r² = 0.96; p < 0.001) and negative correlation (r = –0.98; p < 0.05) relationships with grain-Co concentration in Faya (Figure 3b; Table 3).

3.3.2. Grain-Pb

Grain-Pb accumulation in rice was markedly affected by cultivar (P < 0.05; F = 7.4) and Fe° amendment (P < 0.01; F = 7.4) while interaction of cultivar x Fe° amendment had no significant impact (p > 0.05; Figure 2d; Table 2). For cultivar, Faya accumulated 70% less Pb (128 ± 25 μg kg⁻¹) in grains than Kilombero had (423 ± 22 μg kg⁻¹; Figure 2d). For Fe°, Fe° dosages of reduced grain-Pb accumulation from 279 ± 12 μg kg⁻¹ in control treatment to 152 ± 90 μg kg⁻¹ (49%) and 86 ± 65 μg kg⁻¹ (71%) in rice grains amended with 6.20 and 12.40 g Fe° kg⁻¹, respectively (Figure 2d). Similarly, linear regression and correlation analysis revealed that Fe° dosages had a significant negative correlation (r > -0.95; p < 0.001) and strong linear (r² > 0.90; p < 0.01) relationships with grain-Pb concentrations (Figure 3c & 2d; Table 3) in both cultivars. However, both correlation (r = -1.00) and linear regression coefficients (r² = 1.00) were higher in Faya rice compared to Kilombero rice (r = -0.95; r² = 0.90; p < 0.01) though not significantly different.

3.4. Impact of interaction of rice cultivar and Fe° on soil-to-shoot Co-TF and Pb-TF

3.4.1. Soil-to-shoot Co-TF

Soil-to-shoot Co-TF was markedly affected by Fe° dosages (p < 0.001; F = 112.6), cultivar (p < 0.001; F = 10.1) and interaction of cultivar x Fe° (p < 0.01; F = 7.3; Figure 4a; Table 2). For cultivar, soil-to-shoot Co-TF in Faya rice (1.44) was 28% greater than that in Kilombero rice (1.12) showing that Faya translocates greater Co to grains than Kilombero had (Figure 4a). For Fe°, soil-to-shoot Co-TF in rice amended with 6.20 g Fe° kg⁻¹ (1.12) and 12.40 g Fe° kg⁻¹ (1.17) were more than 27% less than that in control (1.41) (Figure 4a) showing that Fe° dosages of 6.20 and 12.40 g Fe° kg⁻¹ markedly reduced Co translocation. For interaction of cultivar x Fe°, Fe° dosages of 6.20 and 12.40 g Fe° kg⁻¹ reduced soil-to-shoot Co-TF by 10% (1.61) and 40% (1.12), respectively, in Faya rice while no significant changes were observed in Kilombero rice (Figure 4a).

Figure 4.

Interaction impact of Cultivar x Fe° dosages on soil-to-shoot Co-TF (a) and soil-to-shoot Pb-TF (b) in Faya (blue bars) and Kilombero (red bars) rice cultivars. Error bars are standard deviation (n = 4); Bars with different letters are significantly different; Co-TF, cobalt translocation factor; Pb-TF, lead translocation factor; TF, translocation factors.

3.4.2. Soil-to-shoot Pb-TF

While soil-to-shoot Pb-TF was not markedly affected by interactions of cultivar x Fe° amendment (p > 0.05), soil-to-shoot Pb-TF was markedly impacted by cultivar (P < 0.05; F = 62.1) and Fe° amendments (p < 0.001; F = 8.3) (Table 2; Figure 4b). For cultivar, soil-to-shoot Pb-TF in Faya rice (1.9) was 2.3 folds less than that in Kilombero rice (6.3) (Figure 4b) showing that Faya rice translocated Pb from soil to grains compared to Kilombero rice (Figure 4b). For Fe°, Fe° dosages of 6.20 and 12.40 g kg⁻¹ reduced soil-to-shoot Pb-TF from 4.4 in control treatment to 2.3 (49%) and 2.7 (71%), respectively (Figure 4b), showing that Fe° dosages markedly regulated Co translocation.

3.5. Impact of cultivar, Fe° and their interaction on shoot-to-grain Co-TF and shoot-to-grain Pb-TF

3.5.1. Shoot-to-grain Co-TF

Shoot-to-grain Co-TF were markedly affected by Fe° dosages (p < 0.001; F = 112.6) and interactions of cultivar x Fe° (p < 0.01; F = 7.3) but not by cultivar (P > .0.05; Table 2; Figure 5a). Foe cultivar, shoot-to-grain Co TF in Faya and Kilombero rice averaged 0.23 and 0.19, respectively. Thus, Faya rice had translocated 16% less shoot-Co to grains than Kilombero rice had, though not markedly different (Figure 5a).

Figure 5.

Interaction impact of cultivar x Fe° dosages on shoot-to-grain Co- TF (a) and Pb-TF (b) in Faya and Kilombero; Error bars are standard deviation (n = 4); Bars with different letters are significantly different; TF, translocation factors.

For Fe°, shoot-to-grain Co-TF in rice amended with 0, 6.20 and 12.40 g Fe° kg⁻¹ soil were 0.25, 0.23 and 0.20, respectively, indicating that plants amended with Fe° amendment dosage of 12.40 g Fe° kg⁻¹ soil had translocated 21% less shoot-Co to grains than the control plants had (Figure 5a) while shoot-Co translocated by plants amended with Fe° amendment dosage of 6.20 g Fe° kg⁻¹ was not markedly different from that the control plants had. For interaction of cultivar x Fe° amendment, the shoot-to-grain Co-TF stimulated by Fe° dosages of 0, 6.20 and 12.40 g Fe° kg⁻¹ soil averaged 0.22, 0.16 and 0.18, respectively, in Faya rice and 0.17, 0.23 and 0.17, respectively, in Kilombero rice. The observation showed that Faya rice amended with 6.20 g Fe° kg⁻¹ soil had translocated 26% less shoot-Co to grains but translocated 34% more shoot-Co to grain in Kilombero rice (Figure 5a).

3.5.2. Shoots-to-grain-Pb translocation factor (TF)

Shoots-to-grain-Pb TF were markedly affected by cultivar (P < 0.05; F = 62.1), Fe° amendment (p < 0.001; F = 8.3) and interactions of cultivar x Fe° amendment (p < 0.01; F = 4.1; Table 2; Figure 5b). Regardless of amending soils with Fe°, shoot-to-grain-Pb TF in Faya and Kilombero rice averaged 1.9 and 4.2, respectively, with Faya rice translocating 2-folds less shoot-Pb to grains than Kilombero rice had (Figure 5b). For Fe°, rice amended with 6.20 (2.0) and 12.40 g kg⁻¹ of Fe° dosages (1.3) translocated more than 26% less shoot-Pb to grains than control treatment had (2.7; Figure 5b) showing that Fe° amendments markedly inhibited translocation of shoot-Pb to grains.

4. Discussion

The presence of contaminants in soil reduce soil microbial biomass as well as microbial activities [22]. It is reported that Fe° negatively affect bacterial abundance and microbial quality of contaminated soils but not of non-contaminated soil [22]. Considering that soils used in this study was not contaminated and was uniform in all treatments, impact of various Fe° dosages on rhizosphere microorganisms was not evaluated in this study. Nevertheless, impact of cultivar, Fe° dosages and their interaction were evaluated on porewater-pH and porewater-Co and Pb concentrations (Figure 1; Table SM3).

Porewater-pH is an important parameter because it can decide solubility and bioavailability of metal(loid)s such as Co and Pb. In this study, lower pH was observed in porewater planted with Faya (Table SM3). The observation suggested that Faya excreted exudates that induced more acidic conditions compared to that of Kilombero. Lower pH might be responsible for increased Co and Pb uptake in Faya under control treatments considering that bioavailability of metal(loids) increases with soil pH decrease, resulting in increased uptake of Co and Pb [38]. Moreover, most micronutrients, including Co and Pb, are less available in alkaline soils (soil pH > 7.5) and are optimally available in slightly acidic soils and/porewater (6.5 < soil-pH < 6.8) [38, 39].

In this study, Faya rice cultivar accumulated higher Co and lower Pb in shoots and grains compared to Kilombero rice. In addition to accumulating lower grain-Pb, Faya cultivar accumulated lower As concentrations (412 ± 116 μg/kg) in grains compared to Kilombero (700 ± 29 μg kg⁻¹) [21]. These observations confirm that selecting Faya cultivar compared to Kilombero has added benefits of regulating a wide spectrum of carcinogen bioaccumulation in rice grains. Furthermore, selecting Faya cultivar has also the observation confirms previous observations that rice cultivars have different ability of rice in up-taking of and bio-accumulating Co and Pb in shoots and grains varies considerably with rice plant species [40]. Consistent to our results, Rahman et al [17] reported that different rice cultivars show different Co accumulation rates in rice shoots and grain. For instance, Rahman et al [17] identified Japanese sushi rice as a low Co accumulating cultivars (mean: 9–14 μg kg⁻¹; range: 9–15 μg kg⁻¹) compared to Thai rice (mean: 21,700 μg kg⁻¹; range: 11,700–35,400 μg kg⁻¹). For Pb, Rahman et al [17] reported significant differences in grain-Pb accumulation ranging from 2,700 μg kg⁻¹ to 4,800 μg kg⁻¹. Furthermore, Ashraf et al. [41] and Fangmin et al [42] reported that uptake, translocation, and accumulation of Pb markedly across plant genotypes. A study conducted by Liu e al [11]. in China confirmed genotypic variation of Pb translocation from roots to shoot in which higher translocation factors (TF) was reported in hybrid rice cultivars followed by indica and japonica in that order. The higher Co content in Faya rice could be attributable to the observed higher soil-to-shoot Co-TF and shoot-to-grain Co-TF which is consistent with the observed data. Lower Pb content seen in Faya rice (2-folds) compared to Kilombero rice (4-folds) infers that Faya rice translocated lower soil and shoot-Pb to grain compared Faya rice which suggests soil-to-shoot Pb-TF and shoot-to-grain Pb-TF are prime contributors to grain-Pb bioaccumulation. The observation suggest that selecting Faya rice over Kilombero rice would markedly regulate quantity of Pb entering the food chain.

Considering that, soil-Co and soil-Pb interacts with all metals that are geochemically associated with iron [40], soil amendments with Fe°, which generates enormous amounts of iron oxides and iron hydroxides upon oxidation of Fe° interferes with bioavailability, mobility, and uptake of metal(loids) including Co and Pb in rice systems. In this study, iron oxides and iron hydroxides might have sorbed Co and Pb on its surface thereby immobilising Co and Pb which made soil-Co and Pb less available for rice plants uptake which consequently reduced Co and Pb burden in rice grain. Furthermore, greater shoot-Co, grain-Co, shoot-Pb and grain-Pb reduction observed in plants amended with greater Fe° dosages suggested that higher dosages of Fe° generated greater magnitude of iron oxides and iron hydroxides in the rhizosphere (considering that higher soil-Fe° is linked to elevated formation of iron hydroxides which has greater surface area with greater capacity to sorb Co and Pb [43]. Thus, elevated sorption of Co and Pb onto iron hydroxides substantially limited uptake and translocation of Co and Pb from soil to shoots and from shoots to grains which is consistent with data obtained in this study for soil-to-shoot Co-TF and Pb-TF as well as with previous reports for As and Cd accumulation in rice [18, 21]. Thus, Fe° regulated Co and Pb bioaccumulation in rice by immobilizing soil-Co and Pb through adsorption of Co or Pb ions onto Fe oxides - similar to reaction reported for As and Cd [18, 24, 25, 26]. For instances, rice amended with 12.40 g Fe° kg⁻¹ soil was reported to accumulate more than 17% less As in shoots [21] and more than 67% less As in grains compared to the control treatments [21, 26]. The inverse relationship between Fe° dosages and Co and Pb concentrations could also be attributed to competition for uptake of Fe, Co and Pb considering that these metal(loids) are up-taken through same sites in crystalline structures and form similar metallo-organic compounds [40, 44].

Furthermore, higher soil-Fe can result in formation of greater iron plaque on roots of rice which can retain greater soil-Co and Pb in the same way As is retained [40, 44]. Thus, reduced grain-Co and Pb accumulation in rice grains amended with greater Fe° dosages could also be linked to elevated iron plaque on rice roots which might have reduced soil-to-shoot Co-TF and soil-to-shoot Pb-TF which limited a pool of shoot-Co and Pb translocated to grains. The observation is in agreement with previous findings [18, 21, 45, 46]. For instance, Siddique et al. [46] reported that iron plaque formation increased with extra addition of iron to soi and that iron plaque has high affinity for metal(loids) which impacts plant uptake of metal(loids). Furthermore, reduced soil-to-grain Co-TF and soil-to-grain Pb-TF in plants amended with higher Fe° dosages might be linked to elevated chelation of Co and Pb in shoots which might have been stimulated by higher concentration of iron oxides and iron hydroxides resulting from Fe° dosages.

Our earlier study indicated that amending Kilombero with at least 6.20 g/kg Fe° markedly reduced accumulation of both As and Cd in rice grains to values lower than that legislated by the European Commission's (200 μg/kg) without impairing rice grain yields benefit. In this study, our results indicate that Fe° amendments stimulated lower inhibitory effect on shoot and grain-Co and Pb accumulation in Kilombero but greater inhibitory impact on shoot and grain-Co accumulation in Faya which suggest that impact of Fe° on shoot and grain-Co and Pb accumulation is dependent on both rice cultivar and Fe° dosages which is supported by stronger and more negative correlations observed in Faya than Kilombero for both shoots and grain-Co concentrations. Thus, use of Fe° amendment does not produce benefits of reducing As and Cd concentration but also limit bioaccumulation of Co and Pb concentration. Considering that greater TF may imply poor Co and Pb retention in soil or greater efficiency of the plant in translocating Co and Pb from soil to shoot or from shoots to grains; while lower transfer factors may indicate strong sorption of metal(loids) to the soil colloid/particles [35, 36], greater inhibitory effect obtained in Faya amended with Fe° dosage of at least 6.20 g kg⁻¹ suggests that amended Faya rice has lower efficiency to absorb both Co and Pb while amended soils had higher sorption of Co and Pb which indicates that Fe° dosages influenced similar uptake and accumulation of grain-Pb and Co concentrations in Faya. For Kilombero, corresponding Fe° dosages had no effect on grain-Co accumulation but had a decreasing effect on Pb uptake and accumulation. The finding shows that impact of Fe° amendment in regulating grain-Co accumulation is dependent on both cultivar and Fe° dosages with Faya rice having greater grain-Co and Pb reduction potential. The reduction could be linked to formation of greater magnitude of iron plaque on Faya rice roots which has greater potential of sequestering greater magnitude of soil-Co and Pb. Thus, the observation suggests genotypic differences between Faya and Kilombero in formation of iron plagues which is consistent with a previous report [47]. Wu et al. [48] reported significant positive correlation between Fe concentrations and As for hybrid cultivars and significant negative correlation for indica cultivars and attributed the differences to cultivar variation in formation of iron plaque.

Comparisons between grain-Pb concentrations with maximum contaminant limit (MCL) for Pb concentrations in rice (200 μg kg⁻¹) [12, 49], indicated that grain-Pb concentrations obtained in this study for control treatments of both Faya and Kilombero exceeded the MCL. Conversely, grain-Pb concentrations obtained in rice amended with Fe° were lower than MCL. The observation suggests that amending rice with Fe° of at least 6.20 g kg⁻¹ soil markedly reduce Pb bioaccumulation to safe levels for human consumption.

5. Conclusions

Our present study has revealed positive impacts of interaction of Fe° x cultivar on quality of rice by reducing bio-accumulation of cobalt and lead in rice grains cultivated in soils under practical environmental conditions. The study has also increased our understanding of interaction impact of Cultivar x Fe° dosages in rice systems. For instance, variable cultivar response and dosage dependent effects of zero valent iron amendments on both shoots and grain Cobalt and lead content and translocation seen in both cultivars suggested that Fe° can effectively alleviate excessive accumulation of lead in rice and/or diminished Co supply in diets simultaneously. For Malawi, there is need to jealously guard contamination of rice paddies for Malawi to continue producing high quality rice with low grain-cobalt and lead content.

Declarations

Author contribution statement

Angstone Thembachako Mlangeni, PhD: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.

Andrea Raab: Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Joerg Feldmann: Conceived and designed the experiments; Analyzed and interpreted the data; Contributed reagents, materials, analysis tools or data; Wrote the paper.

Funding statement

Dr Angstone Thembachako Mlangeni was supported by Commonwealth Scholarship Commission [MWCS-2015-334].

Data availability statement

Data will be made available on request.

Declaration of interest’s statement

The authors declare no conflict of interest.

Additional information

No additional information is available for this paper.

Appendix A. Supplementary data

The following is the supplementary data related to this article: