DOUGLAS CARVALHO AMARAL 
 
 
 
 
 
 
 
 

STRATEGIES TO DECREASE ARSENIC AND 

CADMIUM IN RICE GRAINS BY RICE 

RESIDUE MANAGEMENT AND FLOODING  

 

 

 

 

 

 

LAVRAS – MG 

2017 



 

 

DOUGLAS CARVALHO AMARAL 
 
 
 
 
 
 
 
 

STRATEGIES TO DECREASE ARSENIC AND CADMIUM IN RICE 

GRAINS BY RICE RESIDUE MANAGEMENT AND FLOODING  

 

 

 

 

 

Tese apresentada à Universidade Federal de 
Lavras, como parte das exigências do Programa 
de Pós-Graduação em Ciência do Solo, área de 
concentração em Recursos Ambientais e Uso da 
Terra, para a obtenção do título de Doutor. 

 

 

  

 
 

Orientadores 
Dr Angelia Lyn Seyfferth 

Dr Luiz Roberto Guimarães Guilherme 
 

 

 

LAVRAS-MG 

2017 



 

 

        

Ficha catalográfica elaborada pelo Sistema de Geração de Ficha Catalográfica da Biblioteca 
Universitária da UFLA, com dados informados pelo(a) próprio(a) autor(a). 

 

        

        

  

Amaral, Douglas Carvalho. 
       Strategies to decrease arsenic and cadmium in rice grains by 
rice residue management and flooding / Douglas Carvalho Amaral. 
- 2017. 
       107 p. : il. 
 
       Orientador(a): Luiz Roberto Guimarães Guilherme. 
        
       Tese (doutorado) - Universidade Federal de Lavras, 2017. 
       Bibliografia. 
 
       1. Water management. 2. Si-rich residues. 3. Trace elements. I. 
Guilherme, Luiz Roberto Guimarães. II. Título. 

   

      

        

O conteúdo desta obra é de responsabilidade do(a) autor(a) e de seu orientador(a). 
 



 

 

STRATEGIES TO DECREASE ARSENIC AND CADMIUM IN RICE 

GRAINS BY RICE RESIDUE MANAGEMENT AND FLOODING  

 

 

 

 

Tese apresentada à Universidade Federal de 
Lavras, como parte das exigências do Programa 
de Pós-Graduação em Ciência do Solo, área de 
concentração em Recursos Ambientais e Uso da 
Terra, para a obtenção do título de Doutor. 

 

 

 

 

APROVADA em 27 de março de 2017. 

 

Dr Angelia Lyn Seyfferth UDel - USA 

Dr Antônio Chalfun Junior UFLA 

Dr Carlos Alberto Silva UFLA 

Dr Leônidas Carrijo Azevedo Melo UFLA 

  

 

 
Dr Luiz Roberto Guimarães Guilherme 

Orientador 
 
 
 
 

LAVRAS – MG 
2017 



 

 

ACKNOWLEDGEMENTS 
 

Firstly, I would like to express my sincere gratitude to my advisors Dr 

Luiz Roberto Guimarães Guilherme and Dr Angelia Lyn Seyfferth, and co-

advisor Dr Guilherme Lopes for the continuous support of my Ph.D study and 

related research, for their patience, motivation, and immense knowledge. Their 

guidance helped me in all the time of research and writing of this dissertation. I 

could not have imagined having a better advisors and mentors for my Ph.D 

study.  

I would also like to acknowledge Dr Antonio Chalfun Junior, Dr 

Leonidas Carrijo Azevedo Melo, and Dr Carlos Alberto Silva as the committee 

members of this dissertation, and I am gratefully indebted to them for their very 

valuable comments on this dissertation. 

A very special gratitude goes out to all down at the University of 

Delaware and Federal University of Lavras, as well for FAPEMIG, CAPES, 

CNPq, and NSF for helping and providing the funding for the work. 

I thank my fellow labmates in for the stimulating discussions, for the 

sleepless (synchrotron) nights we were working together before deadlines, and 

for all the fun we have had in the last four years.  

I also take this opportunity to express gratitude to all of the Department 

faculty members and staff for their help and support. 

Last but not the least, I must express my very profound gratitude to my 

parents and to my brother and wife for providing me with unfailing support and 

continuous encouragement throughout my years of study and through the 

process of researching and writing this dissertation. This accomplishment would 

not have been possible without them.  

Finally, my sense of gratitude to one and all, who directly or indirectly, 

have lent their hand in this venture. Thanks for all your encouragement! 



 

 

RESUMO GERAL 
 

O arroz (Oryza sativa L.), alimento básico para a metade da população 
mundial, é uma fonte potencial de arsênio (As) e cádmio (Cd). Elevados teores 
de As e Cd no solo e/ou água de irrigação podem afetar o crescimento e 
desenvolvimento, assim como decrescer a produção e qualidade do arroz. Em 
adição, o arroz contaminado com As e Cd pode afetar diretamente a saúde 
humana. Portanto se faz necessário desenvolver diferentes estratégias para 
minimizar o risco de acúmulo de As e Cd em grãos de arroz. Diferentes técnicas 
e manejos de irrigação tem sido introduzidas para decrescer o acúmulo de As e 
Cd em grãos de arroz. No entanto, As e Cd tem respostas opostas aos diferentes 
manejos de irrigação e podem ainda se acumular nos grãos. Em contraste, 
técnicas que visam o manejo do silício (Si) podem ser altamente efetivas em 
combater o problema de acúmulo excessivo de As e Cd nos grãos de arroz. 
Assim, investigações adicionais são necessárias para desenvolver técnicas 
simples e economicamente viáveis para decrescer o acúmulo de As e Cd em 
grãos de arroz. No presente estudo, dois experimentos foram conduzidos onde 
nós (1) desenvolvemos uma técnica para amostrar e concentrar minerais 
presentes nas placas de ferro formadas nas raízes de arroz para entender o 
impacto da aplicação de Si na quantidade e composição mineralógica destas 
placas e seu efeito na tranferência de As para a parte aérea das plantas de arroz, e 
(2) investigamos os efeitos de diferentes estratégias de irrigação na absorção de 
As e Cd por plantas de arroz em solo altamente intemperizado utilizando como 
amenizantes resíduos de arroz ricos em Si. No primeiro experimento, a técnica 
desenvolvida foi efetiva em concentrar placas de ferro das raízes de arroz. Os 
resultados mostram que estratégias que promovem mais Fe e As nas placas ainda 
podem transferir As para a parte aérea e acumular nos grãos de arroz, mas 
estratégias que promovam mais Si nas placas podem decrescer o acúmulo de As 
tanto na parte aérea quanto em grãos de arroz. No segundo experimento, nossos 
resultados indicam que a utilização de resíduos ricos em Si é uma técnica 
promissora para decreascer o acúmulo de As e Cd em grãos de arroz sob 
diferentes manejos de irrigação sem afetar negativamente a produção e os teores 
de Zn e Fe nos grãos de arroz. Nós sugerimos que a incorporação de casca de 
arroz (FH) e cinzas da casca de arroz (RHA) podem ser estratégias promissoras 
dependendo da escolha do amenizante sendo que os resíduos tiveram diferentes 
efeitos em difentes condições de manejo. Pesquisas adicionais são necessárias 
para avaliar o efeito residual dos amenizantes assim como confirmar ambos os 
riscos e benefícios de diferentes técnicas de manejo na produção de arroz. 
 

Palavras-chave: Oryza sativa L. Manejo de irrigação. Resíduos ricos em Si. 

Elementos-traço. Saúde humana.  



 

 

GENERAL ABSTRACT 
 
Rice (Oryza sativa L.), a staple food for half of the world’s human 

population, is a potential source of arsenic (As) and cadmium (Cd). Elevated As 
and Cd levels in soil and/or irrigation water can affect growth and development 
of plants, decreasing rice yield and quality. Moreover, contaminated rice grain 
may threaten human health worldwide. It is therefore imperative to investigate 
the effects of different strategies to develop new techniques for minimizing the 
risk for As and Cd transfer into rice grains. Water management has been 
introduced to decrease As and Cd accumulation in rice grains. However, As and 
Cd have opposite responses to water management and may still accumulate in 
rice grains. In contrast, Si management techniques may prove to be highly 
effective in combating the problem of excessive accumulation of As and Cd in 
rice grains. Therefore, additional investigations are needed to develop simple 
and cost-effective methods to decrease As and Cd in rice grains. In this study 
two experiments were conducted where we (1) developed a technique to sample 
and concentrate intact Fe plaque minerals to understand the impact of increasing 
solution-phase Si on the quantity and mineral composition of Fe plaque and 
controls on shoot transfer of As, and (2) investigated the effects of different 
water management strategies on As and Cd uptake by rice in well-weathered soil 
amended with Si-rich rice residues compared to non-amended controls. In the 
first study, the technique reported was effective at concentrating As-bearing Fe 
plaque from root systems. Our data indicate that strategies that promote more Fe 
and As in plaque may still transfer As to shoots but strategies that promote more 
Si in plaque decrease both shoot As and grain As. In the second experiment, our 
data indicate that utilizing Si-rich rice amendments is a promising strategy to 
decrease heavy metals contamination in rice under different water managements 
without negatively affecting rice yield, and grain Zn or Fe. We suggest that soil-
incorporation of fresh husk (FH) and rice husk ash (RHA) may be a promising 
strategy depending on the choice of amendment in view of the fact that the 
amendments had different behavior over different conditions. Further research is 
needed to evaluate the residual effects of the amendments as well to confirm 
both risks and benefits for different management strategies. 
 

Keywords: Oryza sativa L. Water management. Si-rich residues. Trace elements. 

Human health.  

 

 

 



 

 

SUMMARY 

 

CHAPTER I  

1 INTRODUCTION ............................................................................. 08 

2 LITERATURE REVIEW ................................................................. 12 

2.1 Human health exposure to trace elements through rice ............. 12 

2.2 Trace elements accumulation in rice ............................................ 14 

2.3 Management practices to control trace elements in rice ............. 17 

REFERENCES ................................................................................. 21 

CHAPTER II: ARTICLES  

A new approach to sampling intact Fe plaque reveals Si-induced 
changes in Fe mineral composition and shoot As in rice. Reprinted 
with permission from Amaral, D. C.; Lopes, G.; Guilherme, L. R. G.; 
Seyfferth, A. L., A New Approach to Sampling Intact Fe Plaque 
Reveals Si-Induced Changes in Fe Mineral Composition and Shoot As 
in Rice. Environmental Science & Technology 2017, 51, (1), 38-45. 
Copyright © 2017, American Chemical Society .................................... 28 

Impacts of Si-rich rice residues on grain As and Cd under flooded 
and non-flooded conditions .................................................................. 66 



8 

 

 

CHAPTER I 

 

1 INTRODUCTION 

 

 Environmental pollution is considered major threat to arable lands, 

water reserves and ultimate to food chain (COMMISSION OF THE 

EUROPEAN COMMUNITIES - CEC, 2002). This contamination is originated 

from various sources including combusting, industries, mining, weathering as 

well as human activities (NAGAJYOTI; LEE; SREEKANTH, 2010). Among 

the toxic substances, trace elements arsenic (As), cadmium (Cd), chromium (Cr), 

mercury (Hg), and lead (Pb), may enter the food chain and pose health risks to 

humans and animals (MCLAUGHLIN; PARKER; CLARKE, 1999). In this 

respect, there is a growing need to manage toxic elements to protect the 

agricultural production and human health. 

Rice (Oryza sativa L.) is the second most extensively cultivated cereal 

crop throughout the world and it provides food for more than half of the world’s 

population (ORGANISATION FOR ECONOMIC CO-OPERATION AND 

DEVELOPMENT; FOOD AND AGRICULTURE ORGANIZATION OF THE 

UNITED NATIONS - OECD; FAO, 2011). Rice is also a major source of As 

and Cd, particularly in China, Japan, Taiwan and many other countries (ARAO 

et al., 2009; CHEN, 1991; WILLIAMS et al., 2009). Elevated As and Cd levels 

in soil and/or irrigation water can affect growth and development of plants, and 

reduce yield and could add considerable amounts of these elements to the diet 

intake through plant-based item for consumption (BRAMMER; 

RAVENSCROFT, 2009). Arsenic and Cd contents in rice grains varies widely, 

with most reported concentrations found in the range 0.03 to 1.83 mg kg−1 for As 

and 0.01 to 0.1 mg kg−1 for Cd (CORGUINHA et al., 2015; MEHARG et al., 

2009, 2013; MEHARG; RAHMAN, 2003). At high concentrations, long-term 



9 

 

 

consumption of As-contaminated rice is involved in various health problems 

including cancers typically involving the bladder, skin and lungs (AGENCY 

FOR TOXIC SUBSTANCES AND DISEASE REGISTRY - ATSDR, 2007); 

and Cd-contaminated grains is involved in kidney diseases, cancer and 

osteoporosis (ATSDR, 2008). 

Changes in soil pH and redox potential (Eh) state affect the solid-phase 

speciation and solubility of As and Cd and their availability for uptake by rice. 

Decrease in Cd solubility or extractability during paddy soil reduction was 

observed in numerous laboratory studies and was attributed to the precipitation 

of Cd-sulfide under sulfate reducing conditions (CORNU et al., 2007; DE 

LIVERA et al., 2011; HUANG et al., 2013). The extent of Cd-sulfide formation 

during paddy soil flooding may also be relevant with respect to Cd 

remobilization during paddy soil drainage and re-oxidation (FULDA; 

VOEGELIN; KRETZSCHMAR, 2013). Additionally, when paddy soil is under 

aerobic conditions, As is strongly sorbed to mineral soil components such as Fe 

and Al (hydr)oxides as As(V), and less mobile (GOLDBERG, 2002). In 

contrast, when anaerobic conditions develop in flooded soils, solubilized As is 

vigorously taken up by rice plants as As(III) and highly accumulates in grains 

(HONMA et al., 2016). It is therefore imperative to investigate the effects of 

different strategies on the dissolved As and Cd concentrations in soil, As and Cd 

uptake by rice, and As speciation in rice grains to develop new techniques for 

minimizing the risk for As and Cd transfer into rice grains. 

Water is an integral component of a rice paddy system. Various water 

management methods have been introduced to decrease As and Cd accumulation 

in rice as well to increase rice production, such as shallow water depth with 

wetting and drying (LIN et al., 2005), alternate wetting and drying throughout 

the crop period (YE et al., 2013), and intermittent irrigation followed by shallow 

water management (UPHOFF; KASSAM; HARWOOD, 2011). However, there are 



10 

 

 

significant obstacles to the adoption of different systems, principally limited 

information on water management at large scales and the risk of yield loss with 

improper management (LAHUE et al., 2016). Furthermore, As and Cd have 

opposite responses to water management. Thus, alternative management 

techniques under natural flooded and non-flooded rice systems are therefore 

needed to reduce the environmental problem associated with rice cultivation 

without affecting rice production and global food security. 

Silicon (Si) is an agronomically important element that can have 

beneficial effects on crop production (IMTIAZ et al., 2016). For example, the 

accumulation of Si in rice results in positive impacts on rice yield and quality by 

protecting against abiotic and biotic stresses including heavy metals toxicity (LI 

et al., 2009; SEYFFERTH; FENDORF, 2012). Rice husk residues, an 

agricultural waste, is abundantly available in rice producing countries like 

China, India, Bangladesh, Brazil, United States, Cambodia, Vietnam, Myanmar, 

and South East Asia (PODE, 2016). However, only a small quantity of rice husk 

is utilized in agriculture for the purpose of soil-incorporation of Si-rich rice husk 

at present. 

Soil-incorporation of Si-rich rice husk residues such as fresh husk (FH) 

and rice husk ash (RHA) has positive impacts increasing dissolved and plant-

available Si (PENIDO et al., 2016). Silicon addition into soil has shown to 

decrease both inorganic As and Cd in rice without negatively affecting grain 

nutrients and improving rice yield and quality (SEYFFERTH et al., 2016). 

Recent studies have shown that soil-incorporation of Si alleviated the toxicity of 

Cd in rice, which partly overcame the reduction in growth due to Cd exposure 

(SHI et al., 2005). In addition, Si is also reported to decrease As in rice plants. 

The main pathway identified to uptake As in rice plants include silicate transport 

pathways since As(III) is an analogue of silicic acid and enter into the root by 

aquaporin channels (MA et al., 2008). Thus, increasing dissolved Si through 



11 

 

 

soil-incorporation of Si-rich rice residues may decrease As and Cd toxicity in 

rice plants. Consequently, the FH and RHA amendments could become a 

potential resource of low cost to improve rice yield and quality decreasing heavy 

metals accumulation in rice grains. 

Here, we (1) developed a technique to sample and concentrate intact Fe 

plaque minerals from aquatic plant root systems to understand the impact of 

increasing solution-phase Si on the quantity and mineral composition of Fe 

plaque and controls on shoot transfer of As, and (2) investigated the effects of 

different water management strategies on As and Cd uptake by rice in well-

weathered soil amended with Si-rich rice residues compared to non-amended 

controls.  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



12 

 

 

2 LITERATURE REVIEW 

 

2.1 Human health exposure to trace elements through rice 

 

Rice (Oryza sativa L.), a staple food for half of the world’s human 

population, is a potential source of arsenic (As) and cadmium (Cd) (HONMA et 

al., 2016; TSUKAHARA et al., 2003). These elements are known to have many 

toxic effects in humans and are ranked first and seventh, respectively, in the 

priority list of hazardous substances compiled by the US Environmental 

Protection Agency (USEPA) (ATSDR, 2011). Arsenic exposed humans may 

develop skin lesions, neuropathy, gastrointestinal diseases, cardiovascular 

diseases, and cancer (ATSDR, 2007). Cadmium contamination also causes 

serious damage to human health, such as stomach irritation, kidney disease, 

cancer and osteoporosis (ATSDR, 2008). 

Rice is the second most extensively cultivated cereal crop throughout 

the world and has multiple uses for humans particularly in most developing 

countries in Latin America, sub-Saharan Africa, South and South-east Asia 

(AZAM; SARKER; NAZ, 2016). According to FAO, rice ingestion contributes 

a large intake of energy in various countries such as Bangladesh 438 g day-1, 

Vietnam 452 g day-1, India, 195 g day-1, Thailand 278 g day-1, China 212 g day-1, 

Japan 165 g day-1, Ivory Coast 153 g day-1, 158 Brazil 108 g day-1, and the 

United States 25 g day-1 (FOOD AND AGRICULTURE ORGANIZATION OF 

THE UNITED NATIONS - FAO, 2010). Therefore, the evaluation of As and Cd 

exposure pathways through rice and rice-based products in the Cd- and As-

contaminated zones with high rice consumption will form a basis for risk 

assessment to develop agro-environmental sustainability of world rice supplies. 

Food crops have highly variable percentages of As compounds. In most 

dietary As models, the food component is dominated by organo-As compounds 



13 

 

 

(MCLAUGHLIN; PARKER; CLARKE, 1999). However, concentration of As 

in rice followed the trend: As(III) > MMA > As(V) > DMA (AZAM; SARKER; 

NAZ, 2016). Moreover, rice has been identified as an important source of 

inorganic As, which may vary from 10% to 90% of total As (WILLIAMS et al., 

2005, 2007). Inorganic As(III) and As(V) are considered the far greater health 

hazards than organic As compounds (CHAPPEL; ABERNATHY; COTHERN, 

1994). Arsenic contents in rice varies widely, with most reported concentrations 

found in the range 0.03 to 1.83 mg kg−1 in different countries such as United 

States, Bangladesh, India, and China (BHATTACHARYA et al., 2010; 

MEHARG et al., 2009; MEHARG; RAHMAN, 2003; NORTON et al., 2012). 

Recently, the Codex Alimentarius Commission adopted a maximum level of 

inorganic As of 0.2 mg kg−1 in polished rice grain (FAO, 2014). However, grain 

samples from supposedly uncontaminated paddy fields have been shown to 

contain over 0.2 mg kg−1 of inorganic As in polished rice grain, exceeding the 

maximum permissible concentration established by the Codex Alimentarius 

Commission (HONMA et al., 2016). Since rice is a potential source and 

exposure pathway of As, further studies understanding how As is taken up by 

plants and subsequently transformed is essential for estimating the risks posed 

by arsenic-contaminated soils to human.  

In addition to As, Cd poisoning has largely occurred and has been 

detected in the majority of food samples analyzed (CLEMENS et al., 2013). 

Cadmium toxicity especially affects humans rather than animals, because of 

their longevity and the accumulation of Cd in their organs by eating Cd-

contaminated food (TUDOREANU; PHILLIPS, 2004). Moderate Cd 

contamination of arable soils can result in considerable Cd accumulation in 

edible parts of crops. Such levels of Cd in plants are not toxic to crops but can 

contribute to substantial Cd dietary intake by humans (WAGNER, 1993). It was 

reported that Cd can be readily taken up by rice and translocated to shoot and 



14 

 

 

then to grains (WANG et al., 2014). Thus, increased consumption of white and 

especially brown rice contributes to the recent increase of the dietary Cd intake. 

In the case of “Itai-itai disease”, Cd-polluted rice was the major source of Cd 

intake in the patients (YAMAGATA; SHIGEMATSU, 1970). This is the early 

case of chronic Cd toxicity in general populations without specific industrial 

exposure (URAGUCHI; FUJIWARA, 2012). In japan, even though Cd 

concentrations in rice grains are usually much lower than the limit established by 

the Codex Alimentarius Commission of FAO/WHO (0.4 mg kg-1), recent 

general populations have shown internal Cd level higher than those of other 

countries (SHIMBO et al., 2001). It is due to daily consumption of rice which 

contains relatively high Cd (TSUKAHARA et al., 2003). Many reports suggest 

importance to consider chronic effects of Cd exposure through foods and suggest 

the importance of reducing grain Cd accumulation in rice and other cereals for 

better human health (GAO et al., 2016; URAGUCHI; FUJIWARA, 2012). In 

short, paddy rice contaminated by Cd in the soil challenges the food safety as a 

whole. 

Contaminated food particularly rice grain may threaten human health 

worldwide, which speaks for the necessity of evaluating food safety with respect 

to the presence of non-essential trace elements such as Cd and As. Thus, 

research on As and Cd bioavailability is necessary for detecting As and Cd 

intake of human from rice for the use in accurate risk assessments to develop a 

mitigation strategy to minimize As and Cd accumulation in rice grains.  

 

2.2 Trace elements accumulation in rice 

 

Trace elements, for example arsenic (As), cadmium (Cd), chromium 

(Cr), mercury (Hg), and lead (Pb), may enter the food chain and pose health 

risks to humans and animals (MCLAUGHLIN; PARKER; CLARKE, 1999). 



15 

 

 

The sources of these elements vary, and the propensity for plants to accumulate 

and translocate them to edible and harvested parts depends on soil and climatic 

factors, plant genotype and agronomic management. Bioavailability of the 

contaminants to humans also depends on the speciation of the contaminant in the 

food, dietary composition and the nutritional status of the individual.  

Of the elements listed above, the elements that have most commonly 

given rise to health concerns about food safety are the heavy metals Cd and As. 

These elements are two of the most challenging environmental problems in food 

crops especially in rice, affecting millions of people worldwide (HONMA et al., 

2016). Understanding how As and Cd are taken up by plants and subsequently 

transformed is essential for estimating the risks posed by As- and Cd-

contaminated soils to human population.  

The principal causes of elevated soil As are the historically widespread 

use of As compounds as insecticides and herbicides for agricultural production 

(MANDAL; SUZUKI, 2002). The toxicity of As compounds depends on a 

number of factors, including the chemical form present: inorganic As forms are 

more toxic than organic, and arsenite (As(III)) is more toxic than arsenate 

(As(V)) (WORLD HEALTH ORGANIZATION - WHO, 1981). The availability 

of As in soil depends on Eh and pH. Arsenic (V) dominates in aerated soils and 

typically has limited mobility at circumneutral pH due to its strong affinity for 

Al and Fe (hydr)oxides (FENDORF; KOCAR, 2009); thus, the concentrations of 

arsenate in soil solutions are usually low. Under anaerobic conditions, the 

propensity for As migration increases due to the reductive dissolution of Fe 

(hydr)oxides and the reduction of As(V) to As(III), which has a limited affinity 

for most solids other than Fe (hydr)oxides (HERBEL; FENDORF, 2006). 

Two main pathways identified to uptake As in rice plants include 

phosphate transport pathways since As(V) is an analogue of phosphate and new 

evidence reveals that As(III) (silicic acid analogue) enter into the root by 



16 

 

 

aquaporin channels, which are used as silicate transport (MA et al., 2008; WU et 

al., 2011). It is therefore established that As(V) and phosphate share the similar 

transport pathway in rice roots. Thus, under aerobic condition, the phosphorus 

availability may strongly affect the As bioaccumulation in plants with the 

transporters having a higher affinity for phosphate than for As(V) (MEHARG; 

NAYLOR; MACNAIR, 1994). In contrast, As(III) uptake shares the highly 

efficient silicon (Si) pathway of entry to root cells and efflux towards the xylem 

(ZHAO et al., 2009). Thus, As(III) uptake is of particular importance for rice 

and other aquatic plants with their roots growing in anaerobic or semi-anaerobic 

environments. Rice is a strong accumulator of Si, with Si concentration in the 

shoots typically varying from 5 to 10% (HODSON et al., 2005); thus, the 

efficient Si uptake pathway in rice allow inadvertent passage of As(III), which 

explain why rice is efficient in accumulation of As (ZHAO et al., 2009). 

Arsenic content of rice plant parts generally follow the pattern: root > 

straw > husk > grain (LEI et al., 2013). Moreover, the concentration of As in all 

plant parts increased with an increase in soil As, and As concentrations in rice 

grains is approximately 10 times higher than those in other cereals such as wheat 

and barley (WILLIAMS et al., 2007). Since the predominant As species in rice 

grain is inorganic As, minimizing the intake of As from rice in the diet is an 

important health issue.  

In addition to As contamination, it has been reported that human 

activities contribute a major share of annual Cd addition to the environment 

(CLEMENS et al., 2013). In agricultural soils, atmospheric deposition is known 

as a major source of Cd input (KELLER; SCHULIN, 2003). In rice fields, 

application of phosphate fertilizers and water irrigation is the major 

anthropogenic Cd polluting sources (DU et al., 2013). Cadmium accumulation in 

plant-derived food is mostly due to the uptake of soil Cd. Although reported 

mean values for topsoils around the world are usually in the range of 0.2–1.0 mg 



17 

 

 

kg-1 dry soil, there is enormous variation, with maximum levels near 50 mg kg-1 

in some agricultural soils (UNITED NATIONS ENVIRONMENT 

PROGRAMME - UNEP, 2008). 

Cadmium solubility and bioavailability in soil mainly depend on soil 

redox potential (Eh) and pH (HONMA et al., 2016). In general, the enrichment 

of Cd in paddy rice grain occurs during soil oxidation, which accompanies pre-

harvest drainage of the flooded paddy fields as well in upland rice cultivation. 

The predominant form of Cd taken up by rice plants is Cd2+, and the uptake is 

suppressed under reduced conditions (URAGUCHI; FUJIWARA, 2012). 

Drainage of soils results in aerobic conditions that enhance the availability of Cd 

for plant uptake through oxidation of CdS to Cd2+ and SO4
2−, which has a much 

higher solubility than CdS formed under flooding conditions (BINGHAM et al., 

1976). After absorption by the roots, Cd is transported to the stele by passing 

through endodermis and Casparian strips and then Cd is translocated to shoot via 

xylem and finally accumulates in grains (SONG et al., 2013). It has been 

extensively reported that Cd stress caused a reduction in rice growth and 

biomass. Furthermore, Cd may affect the uptake and translocation of essential 

nutrients by rice (LI et al., 2012). 

Cadmium and As are highly toxic heavy metals that can readily 

accumulate in rice grains. Moreover, the rice grain contaminated with Cd and As 

represents a major risk to human health. Therefore, more detailed studies are still 

required to understand the mechanisms of different strategies in reducing these 

elements toxicity in rice. 

 

2.3 Management practices to control trace elements in rice 

 

As described above, rice can accumulate Cd and As in grains and could 

transfer them to humans via the food chain. Thus, reduction of Cd and As in rice 



18 

 

 

plants is among the major issues for sustainable agriculture and human health. 

During the past decades, several practices have been employed in the 

management of Cd and As toxicity in rice plants to minimize the health impacts 

of As and Cd from rice consumption.  

Water management is a promising method that affects Cd and As 

bioavailability in soils and their subsequent uptake by rice (ARAO et al., 2009). 

For example, alternate wetting and drying (AWD) of rice paddies, in which 

fields are drained and re-flooded one or more times during the growing season, 

has been promoted as a strategy to decrease irrigation water use and reduce trace 

elements without affecting rice production (LAHUE et al., 2016; RICHARDS; 

SANDER, 2014). However, despite these potential benefits, there are significant 

obstacles to the adoption of AWD across the world; principally limited 

information on AWD management at large scales and the risk of yield loss with 

improper management (LAHUE et al., 2016). In addition, to make it more 

difficult, As and Cd have opposite responses to water management. When a 

paddy field is flooded and the soil has a low redox potential, any Cd present in 

the soil combines with sulfur (S) to form CdS, which has low solubility in water. 

Thus, flooding of paddy fields during the growing season, especially during later 

stages of plant growth, can effectively reduce Cd concentrations in rice grains 

(ARAO et al., 2009). In contrast, anaerobic conditions in paddy soils usually 

lead to the reduction of As(V) to As(III), which enhances the bioavailability of 

As to the rice plants (HONMA et al., 2016). It is therefore necessary to 

investigate the feasibility of simultaneously mitigating both As and Cd 

accumulation in rice grains through appropriate management technique. 

Silicon (Si) is the second most abundant element in the earth’s crust and 

plays a number of important roles in the mineral nutrition of plants (DUAN et 

al., 2013). The scientific documentation on the benefits of Si to crops has helped 

establish Si fertilization as an agronomic practice in many agricultural lands 



19 

 

 

worldwide (TUBANA; BABU; DATNOFF, 2016). For example, Si fertilizers 

have been widely used in Japan to increase rice yield. This can be attributed to 

the fact that Si fertilization leads to better structural support and enhanced 

biomass and, consequently, higher yield bearing capacity, as well as increased 

resistance against various biotic and abiotic stresses that would otherwise cause 

yield decline (LI et al., 2009). It was estimated that off-take of Si by a rice crop 

is 270 kg/ha/year and if this plant-incorporated Si is not recycled back into the 

paddy ecosystem, it is a serious loss with major implications for rice fertility 

(MEHARG; MEHARG, 2015). Studies illustrate that continuous input of Si, 

either through straw recycling or as mineral fertilizer, is essential to ensure long-

term productivity of paddy soils.  

In addition to the role of Si in alleviating various stresses, Si appears to 

be fundamental to regulating grain nutrition by playing an important role with 

respect to assimilation of the problematic toxins such as As and Cd in rice. 

Arsenite (As(III)), the prevalent form of As under anaerobic conditions in paddy 

soils, is a silicic acid analogue and rice is efficient at assimilating As(III). It was 

observed that higher Si in soil solution outcompeted As assimilation by rice. In 

addition, Si fertilization is also known to reduce cadmium uptake and transport 

by rice. However, the mechanisms are still not well understood.  

It is shown that soil incorporation of Si leads to higher rice yields, as 

well improves the quality of rice grain through decreasing Cd and As contents. 

Therefore, finding sustainable approaches to improve Si availability is of 

extreme importance. Si-rich residues are abundantly available in rice producing 

countries like China, India, Bangladesh, Brazil, United States, Cambodia, 

Vietnam, Myanmar, and South East Asia, which are continually available to 

farmers and could be incorporated into paddy soils with minimal change and 

cost to current practices (PODE, 2016). For example, rice husk (e.g. fresh, and 

ash) is an underutilized Si-rich material with high potential agronomic benefit if 



20 

 

 

reincorporated to paddy soil to increase plant-available Si (SEYFFERTH et al., 

2016). Rice husk residues are advantageous over straw because contains less 

labile carbon, an order of magnitude less As, and upon incorporation to plant-

less soil leads to 50% more porewater Si (PENIDO et al., 2016). Thus, rice husk 

may be an economically viable resource to improve rice nutrition and decrease 

rice As and Cd due its trivial increase in soil-dissolved Si. However, the 

biogeochemical impact of incorporating these residues into soils under typical 

flooded and non-flooded water managements is unknown.  

Perhaps a more sustainable strategy with respect to Si fertilization is to 

increase the once common practice of reincorporating rice residues back into 

paddy fields. Furthermore, optimization of biogeochemical cycling of Si in 

paddy environments is, therefore, a good long-term agricultural policy with great 

potential benefit to the global economy and human health. Overall, continued 

research in these areas may contribute to the effort of reducing toxic As and Cd 

in rice, and can greatly help to reduce overall intake in regions prone to As and 

Cd problems.  

 

 

 

 

 

 

 

 

 

 

 

 



21 

 

 

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27 

 

 

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28 

 

 

Chapter II: Articles 
 

 
 
Article 1: A new approach to sampling intact Fe plaque reveals Si-induced 

changes in Fe mineral composition and shoot As in rice 

 

Published: Environmental Science & Technology 
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



29 

 

 

A new approach to sampling intact Fe plaque reveals Si-induced changes in 

Fe mineral composition and shoot As in rice 

 

 

Douglas C. Amaral1,2, Guilherme Lopes2, Luiz R.G. Guilherme2, Angelia L. 

Seyfferth1* 

 

 

1Department of Plant and Soil Sciences, College of Agriculture and Natural 

Resources, University of Delaware, Newark, DE 19716, USA. 

2Soil Science Department, Federal University of Lavras, Lavras MG, Brazil. 

 

 

*Corresponding author: Angelia L. Seyfferth, 531 S. College Avenue, 152 

Townsend Hall, Newark, DE, USA 19716, Phone: (302) 831-4865, Fax: (302) 

831-4865, email: angelias@udel.edu 

 
 

 
 
 
 
 
 
 

 



30 

 

 

ABSTRACT 

The Fe (oxyhydr)oxide rind, or Fe plaque, that forms on aquatic plant roots is an 

important sorbent of metal(loid)s and plays a role in the attenuation of 

metal(loid) uptake into higher plants. However, the mineral composition of Fe 

plaque and thus its potential to sorb metal(loid)s is affected by solution 

chemistry. The predominant strategy to characterize Fe plaque using dithionite-

citrate-bicarbonate (DCB) extraction and elemental analysis reveal total Fe 

quantity but misses the mineral structure of the Fe (oxyhydr)oxide. Here, we 

developed a new technique using gentle sonication to sample intact Fe plaque 

from the root system and concentrate it for subsequent mineralogical 

characterization using synchrotron-based X-ray diffraction and X-ray absorption 

spectroscopy. We then coupled that data with conventional DCB extraction. The 

sample preparation method was effective at concentrating As-bound Fe plaque 

minerals in a uniform coating onto membranes that could easily be analyzed 

with X-ray techniques. Using these methods, we show that the percentage of 

poorly-ordered Fe minerals in Fe plaque increases with increasing pore-water Si 

in flooded rice paddy soils. These findings have implications for understanding 

mineral controls on As cycling in the soil-rice nexus, and the sampling approach 

can be adopted for other aquatic plant systems. 

 

 



31 

 

 

INTRODUCTION 

Iron (Fe) plaque is a heterogeneous mixture of Fe oxides, hydroxides, and 

oxyhydroxides that form on aquatic plant roots and plays a role in the cycling of 

metal(loid)s at the soil-water-plant nexus.1-3 These neoformed Fe plaques are 

precipitated at the oxic/anoxic interface of aquatic rhizospheres where Fe(II) 

from reductive dissolution of Fe (oxyhydr)oxides and O2 from radial oxygen 

loss intercept.3, 4 This process may be biotic or abiotic,5, 6 and the type of Fe 

phase that forms is dependent upon the solution chemistry and rate of oxidation.6 

For example, dissolved organic C and Si are known to slow or inhibit the 

transformation of poorly-crystalline Fe (oxyhydr)oxides to higher ordered 

phases.7 The mineral composition, or crystallinity, of Fe plaque is important for 

understanding the sorption capacity of plaque for metal(loid) retention8, 9 and 

thus rhizospheric cycling.10, 11 However, the characterization of Fe plaque phases 

and how they may change with treatments is currently challenging due to limited 

strategies for collecting and concentrating intact Fe plaque. Several techniques to 

investigate Fe plaque on aquatic plant roots from the natural environment2, 12, 13 

and under experimental conditions14-17 have been employed. The conventional 

characterization of Fe plaque by dithionite-citrate-bicarbonate (DCB) extraction 

and elemental analysis to quantify the total Fe and other elements is certainly 

useful18, but does not provide information about the mineral phase(s) of the Fe 

plaque. Much of what we know about the mineral composition of Fe plaque was 



32 

 

 

gleaned from a relatively small number of studies that utilized micro-X-ray 

fluorescence imaging (µXRF), micro-X-ray absorption spectroscopy (µXAS), 

and micro-X-ray diffraction (µXRD).1, 6, 11, 13, 19-21 These studies revealed that the 

composition of Fe plaque is heterogeneous and consists of a mix of minerals 

including ferrihydrite, lepidocrocite, goethite, and siderite depending on local 

conditions.22-25 However, these techniques focus on a microscopic portion of one 

root, and even with multiple “spots” investigated they do not necessarily capture 

the mineral composition of the root system and how the mineral composition, 

and thus metal(loid) retention, might change due to changes in solution 

chemistry. Bulk XRD coupled to bulk XAS can provide information about the 

type (XRD) and quantity (XAS with linear combination) of Fe mineral phases 

on the root system,6, 26 but the preparation of homogenous Fe plaque samples for 

these techniques have limitations. For example, XAS spectra of freeze-dried and 

powdered rice roots with intact plaque will have spectral interferences between 

Fe (and As) in the plaque and inside of the root. Here, we describe a technique to 

sample and concentrate the Fe plaque associated metal(loid)s from aquatic roots 

using the Si-treated rice rhizosphere as a model system. Seyfferth et al.27 

recently showed that the addition of Si-rich rice husk residues (fresh and ashed 

husk) to flooded paddy soil increased solution-phase Si without affecting pH and 

decreased inorganic grain As by 25-50%. While several possible mechanisms 

were likely at play that attenuated As uptake in rice including differences in the 



33 

 

 

expression of Si transporters in roots,28, 29 microbial activity,30, 31 and competition 

for uptake,10, 28 another possible mechanism is that increased solution-phase Si 

due to husk amendments27 affected the quantity and mineral composition of As-

scavenging Fe (oxyhydr)oxides that comprise Fe plaque.32 Here, we 1) 

developed a technique to sample and concentrate intact Fe plaque minerals from 

aquatic plant root systems for subsequent mineralogical characterization using 

bulk XRD and XAS, and 2) used this technique and conventional techniques 

(DCB extraction and elemental analysis) to understand the impact of increasing 

solution-phase Si on the quantity and mineral composition of Fe plaque and 

controls on shoot transfer of As. We hypothesized that Si addition to rice paddy 

soils leads to Fe plaques on roots comprised of more poorly-ordered minerals. 

We developed a systematic approach to obtain DCB-extractable elements in root 

plaques and intact samples of Fe plaque for XRD and XAS using sonication. 

This technique uses low-powered sound waves where the ultra-sound energy 

emitted by the frequency generator is transmitted through water and removes Fe 

plaques without the use of chemical reagents, which preserves the integrity of 

the Fe plaque samples. The sonicating solution is then filtered through nitro 

cellulosic membranes and Fe plaques on the filter membranes can be directly 

analyzed at the bulk XRD and XAS beamlines. 

 

 



34 

 

 

MATERIALS AND METHODS 

Plant Growth and Root Samples 

A full description of the study from which root and root plaque samples were 

obtained can be found in Seyfferth et al.27 Briefly, three cultivars of rice (Oryza 

sativa L. cv. M206, IR66, and Nipponbare) were grown in well-weathered, 

flooded soil amended with silica-rich fresh husk (FH) or rice husk ash (RHA) at 

1% (w/w) and compared to a non-amended control in a replicated pot study 

utilizing 3.5 kg of soil. Solution-phase Si decreased in the order FH- amended > 

RHA-amended > nonamended control, and pH was not affected by Si 

treatment.27 Total As in the soil was 16 mg kg-1 and plants were kept flooded 

with deionized water until grain filling. At maturity, shoots were separated from 

roots, and the entire root system was carefully removed from soil and thoroughly 

washed by a series of submergence in DDI water and gentle massaging. Washed 

roots were placed onto paper towels and allowed to air dry. Once dry, roots were 

hand shaken to remove any residual soil particles, and washed again. This 

process was repeated until no visible soil particles remained on the root system, 

at least four times. Once clean and air-dried, the root system was bisected 

longitudinally in half creating two mirror images of the root system: one half 

was utilized for DCB extraction and total elemental analysis in sequence,27 and 

the other half was utilized for intact Fe plaque sampling via sonication and 



35 

 

 

characterization with bulk XRD and XAS. Plaque-free roots, straw, husk, and 

unpolished grain were digested and analyzed as reported previously.27 

 

Dithionite-citrate-bicarbonate (DCB) extraction and total elemental 

analysis 

Fe plaque on root surfaces was extracted using a cold DCB solution, modified 

from Taylor and Crowder.33 Briefly, one half of the entire root system, bisected 

longitudinally, was placed into a 50 mL falcon tube to which 40 mL of 0.3 M 

sodium citrate (Na3C6H5O7·2H2O), 5 mL of 1.0 M sodium bicarbonate 

(NaHCO3), and 3 g sodium dithionite (Na2S2O4) was added. After 3 h of 

agitation at room temperature, roots were removed from the solution and rinsed 

thoroughly with 18 MΩ●cm (DDI) water, with the rinse solution added to the 

extract solution. The DCB extract solution was brought to 100 mL volume and 

then analyzed for total Fe, As, Si, P, and other elements using ICP-OES with 

matrix-matched standards after 10- to 100-fold dilution. Plaque-free roots were 

dried at 65°C, ground to a fine powder, digested with hot nitric acid, and the 

digestates were subject to elemental analysis with ICP-OES.27 

 

Intact Fe plaque sampling for mineral and speciation characterization 

The remaining half root system with intact Fe plaque was subject to gentle 

sonication to obtain samples of Fe plaque minerals for characterization using 



36 

 

 

bulk XAS and XRD techniques. Roots with Fe plaque were placed into 400-mL 

glass beakers partially filled with DDI water, and the beakers were placed into a 

sonicating bath. Sonics at 20 kHz were applied for 2 hours at 25°C, utilizing ice 

packs in the water bath to ensure standard temperature. This method physically 

removed the Fe plaques from the roots without the use of chemical reagents. 

After sonication, roots were lifted above the sonication solution and gently 

rinsed with DDI water, carefully ensuring the rinses were collected into the 

sonication solution. The sonication solution containing the dislodged Fe plaques 

was then vacuum filtered through an acid-washed glass filtration device 

equipped with a 0.2 µm nitro cellulosic filter membrane (47 mm disk). The Fe 

plaques were collected onto the nitro cellulosic filter (Fig. 1), which was 

carefully placed into a petri dish and loosely covered. Once dry, dishes were 

capped and transported to Stanford Synchrotron Radiation Lightsource (SSRL) 

for bulk XRD and XAS analyses. 

 

Fe plaque mineral characterization 

The mineral composition of Fe plaque deposited on the filters was analyzed with 

bulk XRD at SSRL on beamline 11-3 utilizing an incident energy of 12724 eV 

(λ = 0.9744 Å) with MAR 345 Image Plate Detector. Lanthanum hexaboride 

was used to calibrate the MAR 345 detector parameters upon integration of the 

diffractograms in Q space between 0 and 6 Å-1 at 0.006 steps and total Q of 1000 



37 

 

 

counts per 500 s with the Area Diffraction Machine (v2 Beta 248, SSRL, 2007). 

An integrated diffractogram of a blank nitro cellulosic filter membrane was 

utilized as a blank and its signal was subtracted from the signal of each sample 

using XRD-BS. The integrated peaks were analyzed using Match! Software (v3, 

Crystal Impact, 2015). These data were used to constrain the fitting parameters 

for X-ray absorption spectroscopy. In order to quantify the mineral composition 

of Fe plaques and to detect short-range ordered Fe plaque minerals, the intact Fe 

plaques were further characterized with extended X-ray absorption fine structure 

(EXAFS) spectroscopy on beam line 11-2 at SSRL. This beam line is equipped 

with a liquid nitrogen-cooled double crystal monochromator (Si (2 2 0) phi 90) 

and Kirkpatrick-Baez mirrors. The incident energy was calibrated to the first Fe 

inflection point at 7112 eV obtained from a standard Fe foil placed in between I1 

and I2. Iron K-edge spectra were collected with a Lytle detector from 150 eV 

below the absorption edge to k values of 12.5 Å-1. For each sample, four spectra 

were obtained and averaged. These averaged spectra were background 

subtracted, normalized, fitted with a spline function (k-weight = 3) and fitted by 

linear combination (k range of 3 – 12 Å-1) with Athena software34 utilizing both 

ferrihydrite and the results of the XRD analyses to constrain the fits, which were 

2-line ferrihydrite (Fe(OH)3·nH2O), lepidocrocite (γ-FeOOH), and goethite (α-

FeOOH); see Seyfferth et al.6 and Hansel et al.1 for a detailed explanation of the 

preparation of these standards. 



38 

 

 

 

Arsenic speciation on Fe plaques 

The oxidation state of As in the Fe plaque samples were determined by X-ray 

absorption near edge structure (XANES) at SSRL on beamline 11-2. The 

incident energy was calibrated using a prepared sodium arsenate standard, which 

was assigned its first inflection point at 11874.0 eV. The As fluorescence was 

monitored using a 100-element Germanium detector. Three spectra per sample 

were averaged, background-subtracted, normalized, and fitted by linear 

combination in the region 11850–11900 eV using Athena software.34 The 

following materials were included as As standards: As(III)i as NaAsO2, and 

As(V)i as Na3AsO4. 

 

RESULTS 

Fe plaque sampling method. The sonication and deposition method was 

effective at removing As-bound Fe plaque minerals from rice roots and evenly 

depositing them onto nitro cellulosic membranes (Fig. 1). The technique 

concentrated Fe and As onto the membranes by two orders of magnitude. The 

method removed 47 (± 9.2)% of the DCB-extractable Fe and 57 (± 18)% of the 

DCB-extractable As from roots with minimal loss of Fe (0.02% of DCB-

extractable) or As (bdl) through the filter. Deposited Fe plaques were clearly 

visible on the filter membrane disks and varied in color depending on the 



39 

 

 

amendment-induced differences in solution-phase Si (Fig. 1). Independently of 

the cultivars, the color of the Fe plaque deposited onto nitro cellulosic 

membranes was similar between treatments showing a light orange color for the 

FH amended (~500 µM Si) soils, a dark orange color for the RHA amended (~ 

200 µM Si) soils, and a medium orange color for the nonamended (~ 70 µM Si) 

soils. These membrane filters were easily placed directly in front of the X-ray 

beam for bulk XRD and XAS analyses, and could be divided for simultaneous 

analyses on multiple beamlines due to the large (47 mm) size of the filters and 

homogenous deposition of the Fe plaques. 

 

XRD and EXAFS characterization of Fe plaque minerals. The integrated 

diffractograms of Fe plaques deposited onto filter membranes reveal differences 

in dominant Fe (oxyhydr)oxides due to Si amendment and cultivar (Fig. 2). 

Peaks of α-FeOOH and γ-FeOOH were observed in all samples, but the peak 

sharpness and magnitude differed among cultivar and amendment (Fig. 2). 

Secondary XRD peaks of scorodite (FeAsO4·nH2O), pyrite (FeS2), and 

magnetite (Fe3O4) were also observed in some samples, but were minor 

components (Fig. 2). Due to weak diffraction intensity relative to other more 

crystalline minerals, Fe(OH)3·nH2O was not detected with XRD. The k3-

weighted EXAFS spectra and linear combination fits (LCF) of the Fe plaque 

samples show that Fe(OH)3·nH2O, α-FeOOH, and γ-FeOOH were the dominant 



40 

 

 

Fe minerals but the magnitude of each varied among cultivars and soil 

amendments (Fig. 3 and Table 1). Soil amendments that led to the highest 

average pore water Si (FH, ~ 500 µM Si) led to a higher percentage of 

Fe(OH)3·nH2O and a lower percentage of γ-FeOOH in plaque (Table 1). While 

the change was subtle for Nipponbare, M206 and IR66 plants grown in soil 

amended with FH had 30 and 19% more Fe(OH)3·nH2O, respectively, in plaque 

compared to nonamended control plants (Table 1). RHA amendment, which 

resulted in a “medium” level of average pore water Si (~ 200 µM Si) also 

increased the percentage of Fe(OH)3·nH2O in plaque relative to nonamended 

control by 12% for IR66 and 2% for M206, but not Nipponbare. Plaque from 

M206 roots was dominated by Fe(OH)3·nH2O, with lesser amounts of α-FeOOH 

and γ-FeOOH. Plaque from Nipponbare roots was dominated by α-FeOOH 

followed closely by Fe(OH)3·nH2O with minor amounts of γ-FeOOH. IR66 

plaques were comprised of equal proportions of Fe(OH)3·nH2O and α-FeOOH as 

dominant phases and minor amounts of γ-FeOOH. The addition of other Fe 

phases including scorodite (FeAsO4·nH2O), pyrite (FeS2), or magnetite (Fe3O4) 

in the LCF that were indicated as possible phases by XRD did not improve fits; 

these phases are thus possibly present only in trace amounts. 

 

Speciation of Arsenic in Fe plaques. LCF fits of the As-XANES spectra (Fig. 

4) show clear differences in oxidation states of As due to amendment and 



41 

 

 

cultivar. For all samples, sorbed As on plaques was mainly in the As(V) 

oxidation state (60-80%) with lesser amounts of As(III) (20-40%), but the ranges 

varied with amendment and cultivar (Table 2). In general, the percent arsenite 

sorbed decreased in the order RHA > FH > non-amended control for all 

cultivars. 

 

Role of Fe plaque mineral composition on transfer of As to rice shoots. In 

general, the amount of As in shoots increased and the mass ratio of shoot/root 

As decreased as the amount of total Fe in plaque from DCB extraction increased 

(Fig. 5A, D); however, this relation differed when considering the role of each 

mineral and among cultivars (Fig. 5). When considering the proportion of DCB-

extractable Fe in plaque as Fe(OH)3·nH2O, α-FeOOH, or γ-FeOOH, differences 

in their impact on shoot As were observed (Fig. 5 B-D). While an increase in 

shoot As was observed as Fe(OH)3·nH2O or α-FeOOH increased, the increase in 

shoot As per unit increase in DCB-extractable Fe was greater for γ-FeOOH (Fig. 

5 B-D). Similarly, the decrease in the mass ratio of shoot/root As per unit 

increase in DCB-extractable Fe was greater for γ-FeOOH than for 

Fe(OH)3·nH2O or α-FeOOH (Fig. 5 F-H). 

 

Relationship between shoot As and plaque Fe, As and Si and P. We observed 

strong and significant relationships between grain and shoot As and DCB-



42 

 

 

extractable Si in plaque, and weak or no relations between shoot As and DCB-

extractable Fe or As (Fig. 5A, Fig. 6). A significant power regression was found 

between inorganic grain As and plaque Si for Nipponbare (r2 = 0.41, p = 0.031), 

and significant linear regressions were found between inorganic grain As and 

plaque Si for M206 (r2 = 0.672, p = 0.007) and IR66 (r2 = 0.608, p = 0.013) (Fig. 

6A). Significant linear regressions between shoot As and DCB-extractable Si 

were observed with negative slopes for M206 (r2 = 0.776, p = 0.002), IR66 (r2 = 

0.57, p = 0.019), and Nipponbare (r2 = 0.648, P = 0.009) (Fig. 6C). In contrast, 

relations between inorganic grain or shoot As and DCB-extractable Fe or As 

were less evident and not significant, but generally trended toward a positive 

relationship when all cultivars were considered together (Fig. 5A and Fig. 6B 

and D). There was also no relation between shoot As or grain As and plaque P 

(data not shown). 

 

DISCUSSION 

The sample preparation technique was effective at concentrating and 

depositing As-bound Fe plaque minerals in a uniform coating onto nitro 

cellulosic membranes that could easily be analyzed with synchrotron X-ray 

spectroscopic methods. This technique has several advantages over existing 

methods aimed to characterize Fe plaque on aquatic plant roots. In contrast to 

other techniques where a microscopic portion of a single root is exposed to the 



43 

 

 

X-ray beam,2, 3, 6, 11 the method reported here extracts Fe plaque from a root 

system, which does not bias the Fe mineral characterization to a few “hot spots”. 

By characterizing the roots as a system, changes in the proportion of different Fe 

minerals that comprise plaque can be attributed to differences in solution 

chemistry, rather than the inherent heterogeneity of the Fe plaque 

microenvironment.1 Moreover, the method concentrates Fe and As by two orders 

of magnitude compared to DCB-extractable totals, resulting in higher signal to 

noise ratios6 and thus less repeat scans needed per sample (Figs. 3 and 4). This 

method can be applied not only to rice, but to any aquatic plant root system on 

which Fe plaques form.  

Limitations to the technique also exist, which represent a trade-off 

between complete plaque removal and undesired As(III) oxidation. The 

technique removes close to 50% of the DCB-extractable Fe and nearly 60% of 

the DCB-extractable As from the plaque-coated roots, and we observed patchy 

pigmented areas remaining on the roots after sonication. While the technique 

does not remove 100% of the DCB-extractable amounts, it is difficult to 

conclude exactly how much of the Fe plaque was removed because DCB 

extraction is also known to strip metal(loid)s from root cell membranes.35 Thus, 

at least 50%, and perhaps more, As-bound Fe plaque was removed with gentle 

sonication (i.e. 20 kHz for 2 hours). This compromise in duration and power of 

sonics was an advantage for minimizing As(III) oxidation, which can occur up to 



44 

 

 

10% when sonicated at over 180 kHz.36 It should be noted that the intent of the 

present approach was not to remove all plaques, but assess whether soil 

treatments (i.e. Si amendments) would cause global changes in Fe plaque 

phases, as current methods to spot-check micron-scale areas on root-bound 

plaque is simply not feasible to answer that question due to the heterogeneity of 

root plaque and low signal to noise. Because the plaque removed represented a 

large fraction of the outer plaque that As-bearing rhizosphere solution would 

encounter during solute delivery to the root, the present technique is an advance 

over existing methods. Furthermore, there were large fractions of Fe(OH)3·nH2O 

and α-FeOOH present, indicating that no selection for poorly-crystalline 

materials had occurred during sampling.  

The dominant method of Fe plaque characterization with DCB 

extraction and elemental analysis can also capture information about the entire 

root system; however, mineral information is lost in the DCB extraction method 

because the Fe minerals reductively dissolve. Our data show that solution 

chemistry impacts the proportion of minerals that comprise plaque, and each 

mineral affects plant concentrations of As differently (Fig. 5, Table 1). These 

observations corroborate previous work that shows the amount of DCB-

extractable Fe differs with solution chemistry due to the formation of different 

minerals that comprise Fe plaque.37 Moreover, the reactive surface area for As 

sorption differs among Fe plaque minerals, which affects availability of As to 



45 

 

 

roots for uptake and translocation to shoots.38, 39 Thus, it is important to not only 

know how much Fe is present in plaque, but also the proportion of different 

minerals that may impact As retention (Fig. 5).  

Our data corroborate previous findings that used µXAS and µXRD on 

whole root mounts and revealed Fe(OH)3·nH2O, γ-FeOOH, and α-FeOOH, as 

dominant Fe phases in plaque.3, 6, 11 In addition, our data also show that the 

proportion of these minerals differs as solution-phase Si changes and also among 

rice cultivar, differences that may not have been revealed with µXAS and µXRD 

on whole root mounts. As hypothesized, the proportion of Fe(OH)3·nH2O in Fe 

plaque increased with increasing solution-phase Si, which was achieved with the 

FH treatment. We also observed a decrease in γ-FeOOH with increasing 

solution-phase Si, and γ-FeOOH seems to have a greater proportional impact on 

the shoot As concentrations than Fe(OH)3·nH2O or α-FeOOH (Fig. 5). Thus, the 

alteration of the proportion of Fe minerals that comprise plaque (Table 1) due to 

amendment-induced changes in solution-phase Si is at least partially responsible 

for the differences in As accumulation in rice shoots and grains reported 

previously.27  

The present study shows that in addition to amendment-induced changes 

in solution-phase Si, the mineral composition of Fe plaque and its relation to 

shoot As levels also differed among rice cultivars (Table 1 and Fig. 5). Whereas 

Fe(OH)3·nH2O was dominant in M206 plaque, α-FeOOH was dominant in 



46 

 

 

Nipponbare plaque and dominant phases in IR66 plaque were roughly equal 

proportions of Fe(OH)3·nH2O and α-FeOOH. The formation of Fe coatings on 

aquatic plant roots is a function of several abiotic and biotic factors,6 including 

solution chemistry (e.g., dissolved Fe2+, Si, pH) as well as biologic factors such 

as the rate of oxygen loss (ROL) from roots.4, 32, 40, 41 Since all cultivars were 

grown in the same soil with the same treatments that did not affect pH, the 

differences observed were likely due to different extents or rates of ROL 

between cultivars, and illustrate the need for assessing multiple cultivars in rice 

studies involving Fe plaque and As cycling.26, 32, 40, 41  

It is commonly observed that inorganic As – both As(III) and As(V) – 

are the predominant forms of As encountered by rice roots in most paddy soils;3, 

6, 11, 42 however, some evidence suggests that Si addition may increase the 

methylated As forms in soil solution. Here, we observed only inorganic As 

associated with Fe plaque (Table 2), but in the previously reported study from 

where the roots were obtained, Seyfferth et al. 27 observed an increase of 

methylated As in pore water and in grain with increasing solution-phase Si.27 A 

mixture of As(III) and As(V) with no detectable methylated As on Fe plaque of 

rice roots has been previously reported.3, 6, 11, 21, 41-43 These findings indicate that 

as As is methylated, it has a lower affinity for sorption onto Fe plaque compared 

to inorganic As species, and is thus more plant available.44 



47 

 

 

Our data show that the type of Fe mineral and the amount of DCB-

extractable Si were important factors that govern shoot As concentrations, 

whereas the role of total DCB-extractable Fe or As was less clear (Figs. 5-6). A 

higher proportion of γ-FeOOH in plaque and lower proportion of Fe(OH)3·nH2O 

in plaque increases accumulation of As in rice shoots. As solution-phase Si 

increases, both the amount of Fe(OH)3·nH2O and the ability of Si to precipitate 

and encrust As-bearing Fe(OH)3·nH2O and decrease inorganic grain As 

increases (Table 1; Figure 6).7 This Si coprecipitation with As-bearing 

Fe(OH)3·nH2O, and more Fe(OH)3·nH2O in plaque, renders As less mobile and 

thus less plant available. These mechanisms thus seem likely for explaining the 

decrease in shoot As as solution-phase Si increases (Fig. 6).27 

 

Implications. The technique reported here was effective at concentrating 

As-bearing Fe plaque from a root systems for subsequent mineral 

characterization synchrotron X-ray characterization with improved signal to 

noise ratios. When coupled to traditional DCB extraction and elemental analysis, 

the proportion of Fe phases that comprise plaque and how they change with 

solution chemistry can be evaluated free from heterogeneity between individual 

"hot spots" of plant roots. This technique can be applied to any aquatic plant that 

forms Fe plaque. Our data indicate that strategies that promote more Fe and As 

in plaque may still transfer As to shoots, thus potentially leading to higher levels 



48 

 

 

of As in shoots and grain. However, strategies that promote more Si in plaque 

decrease both shoot As and grain As. Incorporation of Si-rich rice husk residue 

to flooded soils increases pore-water Si, promotes more Fe(OH)3·nH2O 

formation, likely leads to coprecipitation of opaline Si and As-bearing 

Fe(OH)3·nH2O, more As methylation, and less methylated As retention onto Fe 

plaque, which all play a role in decreasing shoot As and inorganic As in grain. 

However, cultivar differences, especially with respect to ROL should be 

considered when devising management strategies aimed to lower shoot As. 

 

ACKNOWLEDGEMENT 

We thank Caroline Golt and the UD Soil Testing Laboratory for analytical 

assistance, Rattandeep Gill, Andrew Morris, Gang Li, Izzy Sibbers and Kelli 

Kearns, Matt Limmer and Fred Teasley for sampling assistance. ALS 

acknowledges support from the UD Research Foundation grant No. 14A00765, 

and the National Science Foundation (grant Nos. 1338389 and 1350580). DCA 

acknowledges support from CNPq. Use of the Stanford Synchrotron Radiation 

Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. 

Department of Energy, Office of Science, Office of Basic Energy Sciences under 

Contract No. DE-AC02-76SF00515. 

 



49 

 

 

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41.  Syu, C. H.; Lee, C. H.; Jiang, P. Y.; Chen, M. K.; Lee, D. Y., 

Comparison of As sequestration in iron plaque and uptake by different 

genotypes of rice plants grown in As-contaminated paddy soils. Plant Soil 2014, 

374, (1-2), 411-422. 

42.  Voegelin, A.; Weber, F.-A.; Kretzschmar, R., Distribution and 

speciation of arsenic around roots in a contaminated riparian floodplain soil: 

Micro-XRF element mapping and EXAFS spectroscopy. Geochim. 

Cosmochim.Acta 2007, 71, (23), 5804-5820. 



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43.  Voegelin, A.; Kaegi, R.; Frommer, J.; Vantelon, D.; Hug, S. J., Effect of 

phosphate, silicate, and Ca on Fe(III)-precipitates formed in aerated Fe(II)- and 

As(III)-containing water studied by X-ray absorption spectroscopy. Geochim. 

Cosmochim. Acta 2010, 74, (1), 164-186. 

44.  Li, R. Y.; Ago, Y.; Liu, W. J.; Mitani, N.; Feldmann, J.; McGrath, S. P.; 

Ma, J. F.; Zhao, F. J., The rice aquaporin LSi1 mediates uptake of methylated 

arsenic species. Plant Physiol. 2009, 150, (4), 2071-2080. 

 

 

 

 

 

 

 

 

 

 

 

 

 

 



57 

 

 

FIGURES 

 

 

Figure 1. Fe plaque minerals sampled from rice root systems of M206, IR66, or 
Nipponbare (NB) cultivars grown in well-weathered soil and amended with 
either fresh rice husk (FH, ~500 µM Si), rice husk ash (RHA, ~ 200 µM Si) (1% 
w/w) or a nonamended control (C, ~ 70 µM Si).  Fe minerals were deposited 
onto 47 mm nitro cellulosic filter membranes after sonication. 

 



58 

 

 

 

Figure 2. Integrated XRD patterns in Q space at an X-ray photon energy of 
12724 eV (λ = 0.9744 Å) of intact Fe plaque sampled from rice roots of M206, 
IR66, or Nipponbare (NB) cultivars grown in well-weathered soil and amended 
with either fresh rice husk (FH, ~500 µM Si), rice husk ash (RHA, ~ 200 µM Si) 
(1% w/w) or a nonamended control (C, ~ 70 µM Si). Letters above peaks 
indicate Fe minerals: L = lepidocrocite, G = goethite, S = scorodite, M = 
magnetite, P = pyrite.   

 



59 

 

 

 

Figure 3. Linear combination fits of k3-weighted Fe-EXAFS spectra of intact Fe 
plaque sampled from rice roots of M206, IR66, or Nipponbare (NB) cultivars 
grown in well-weathered soil and amended with either fresh rice husk (FH, high 
Si), rice husk ash (RHA, medium Si) (1% w/w) or a nonamended control (C, 
Low Si). 

 

 

 

 

 



60 

 

 

 

Figure 4. Normalized As K-edge XANES spectra of Fe plaques from rice roots 
of M206, Nipponbare (NB), and IR66 cultivars grown in soil amended with 
fresh rice husk (FH) or rice husk ash (RHA) (1% w/w) compared to those grown 
in a nonamended control soil (C).  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



61 

 

 

 

Figure 5.  Relationships between shoot As (A-D) or shoot/root As (E-H) and 
DCB-extractable Fe in plaque as total Fe (A, E), ferrihydrite-Fe (B, F), goethite-
Fe (C, G) or lepidocrocite-Fe (D, H) from M206, IR66, or Nipponbare (NB) rice 
cultivars grown in well-weathered soil and amended with either fresh rice husk 
(FH) or rice husk ash (RHA) compared to a nonamended control soil. Insets in 
E-H are zoomed in versions without Nipponbare to make M206 and IR66 more 
evident. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



62 

 

 

 

Figure 6. Relationships between inorganic grain As and plaque Si (A) or As (B) 
and shoot As and plaque Si (C) or As (D) from M206, IR66, or Nipponbare 
(NB) rice cultivars grown in well-weathered soil and amended with either fresh 
rice husk (FH) or rice husk ash (RHA) compared to a nonamended control soil. 
A significant power regression was found between inorganic grain As and 
plaque Si for Nipponbare (r2 = 0.41, p = 0.031). Significant linear regressions 
were found between inorganic grain As and plaque Si for M206 (r2 = 0.672, p = 
0.007) and IR66 (r2 = 0.608, p = 0.013), and for shoot As and plaque Si for 
M206 (r2 = 0.776, p = 0.002), IR66 (r2 = 0.57, p = 0.019), and Nipponbare (r2 = 
0.648, P = 0.009). 
 
 
 
 



63 

 

 

TABLES 

 

Table 1. Linear combination fitting results of first-shell Fe EXAFS 
spectra of sonicated and deposited Fe plaque from rice roots of M206, 
Nipponbare (NB), and IR66 cultivars grown in soil amended with fresh 
rice husk (FH, ~500 µM Si), rice husk ash (RHA, ~ 200 µM Si) (1% 
w/w) or a nonamended control (C, ~ 70 µM Si). 

Treatments 
Percent of fitted species 

R value 
Fe(OH)3·nH2O γ-FeOOH α-FeOOH 

M206 C 53 20 27 0.024 

M206 FH 69 13 18 0.035 

M206 RHA 54 18 30 0.025 

NB C 40 8 52 0.033 

NB FH 41 7 52 0.034 

NB RHA 39 7 54 0.033 

IR66 C 42 17 41 0.031 

IR66 FH 50 10 40 0.040 

IR66 RHA 47 14 40 0.028 

 

 

 

 

 



64 

 

 

Table 2. Linear combination fitting of normalized As K-edge 
XANES spectra of sonicated and deposited Fe plaque from rice roots 
of M206, Nipponbare (NB), and IR66 cultivars grown in soil 
amended with fresh rice husk (FH, ~500 µM Si), rice husk ash 
(RHA, ~ 200 µM Si) (1% w/w) or a nonamended control (C, ~ 70 
µM Si). 

Treatments 
Percent of fitted species 

R value 
As(V) As(III) 

M206 C 60 40 0.006 

M206 FH 66 34 0.008 

M206 RHA 71 30 0.015 

NB C 69 31 0.016 

NB FH 62 38 0.005 

NB RHA 61 39 0.008 

IR66 C 80 20 0.015 

IR66 FH 73 27 0.014 

IR66 RHA 60 40 0.007 

 

 

 

 

 

 

 



65 

 

 

 

TOC art 

 

 

 

 

 

 

 

 

 

 



66 

 

 

Article 2: Impacts of Si-rich rice residues on grain As and Cd under flooded 

and non-flooded conditions 

 

Submitted: Environmental Science & Technology 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



67 

 

 

Impacts of Si-rich rice residues on grain As and Cd under flooded and non-

flooded conditions 

 

Douglas C. Amaral1,2, Matthew A. Limmer1, Luiz R.G. Guilherme2, Angelia L. 

Seyfferth1* 

 

1Department of Plant and Soil Sciences, College of Agriculture and Natural 

Resources, University of Delaware, Newark, DE 19716, USA. 

2Soil Science Department, Federal University of Lavras, Lavras MG, Brazil. 

 

*Corresponding author: Angelia L. Seyfferth, 531 S. College Avenue, 152 

Townsend Hall, Newark, DE, USA 19716, Phone: (302) 831-4865, Fax: (302) 

831-4865, email: angelias@udel.edu 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 



68 

 

 

ABSTRACT 

Rice is an important staple food and may be negatively impacted by As 

and/or Cd in grain.  While water management aimed to increase the soil Eh may 

decrease grain As, it may increase grain Cd. Strategies such as increasing Si 

through soil amendment of Si-rich rice residue have proven effective at 

decreasing inorganic without affecting Cd levels in grain under flooded soil 

conditions. However, the impacts of these amendments on Cd and As uptake by 

rice under different water managements have not been reported. We investigated 

the effects of different water management strategies on As and Cd uptake by rice 

in well-weathered soil amended with fresh rice husk (FH) and rice husk ash 

(RHA) to assess their effects on As and Cd accumulation in rice grain. Our 

results show that FH and RHA decreased grain As under both flooded and non-

flooded conditions. Under flooded conditions where grain As and yield is 

higher, FH additionally decreased inorganic grain As.  Under non-flooded 

conditions where grain Cd is highest and yield is lower, RHA decreased grain 

Cd.  These data illustrate that a combined approach of manipulating both Eh and 

pore water Si may alleviate Cd and As in grain. 

 

 

 

 



69 

 

 

INTRODUCTION 

 Rice is the most widely consumed staple food for 50% of the world’s 

population1, 2 and is a potential source of arsenic (As) and cadmium (Cd)3 

worldwide.4-6 There is a growing important health issue underlying the transfer 

of trace-elements such as Cd and As into rice grains7-11 in the soil-water-rice 

nexus.12-14 Arsenic may be present in soil in different chemical forms, including 

inorganic As (As(III) and As(V)) and methylated As species 

(monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA). Under 

flooded rice cultivation, inorganic As(III) is the predominant form found in soils 

and is taken up by plants mediated by Si transporters.15, 16 However, the 

availability of As in soil depends on pH and redox potential (Eh).17 Under 

different rice management systems, flooding and drainage cycles have a major 

impact on As dynamics in soil and its bioavailability to plants.3, 18, 19 In paddy 

fields under anaerobic conditions, As is released through reductive dissolution of 

Al and Fe (oxyhydr)oxides into the aqueous phase and the reduction of As(V) to 

the more soil-mobile As(III).20 When oxidized conditions develop such as during 

drainage periods, As(V) is strongly sorbed to Fe and Al (oxyhydr)oxides and is 

less mobile in soil.21 Reductive dissolution of Fe (oxyhydr)oxides and reduction 

of As into soil solution is promoted under decrease of Eh below +100 mV, 

which is considered to be the point where As(V) reduces to As(III) at neutral 

pH.22 Maintaining Eh above this value (e.g., through mid-season drainage) 



70 

 

 

renders As less mobile and thus less plant available, but also increases Cd 

availability.  

Cadmium availability and solubility in soil also depend on soil pH and 

Eh variations.3 When Cd is present in soils under flooded conditions and low Eh, 

plant-available Cd is decreased due to sulfide formation (CdS).23, 24 However, 

under typical non-flooded conditions or after drainage of paddy fields the 

predominant form of Cd taken up by rice plants is available through oxidation of 

CdS to Cd2+ and SO4
2-.25 It has been clearly shown that increasing soil Eh 

decreases As plant-availability but increases Cd availability and accumulation in 

grain.3, 18 Therefore, water management has important impact on soil Cd and As 

speciation, availability, and absorption by rice plants as well their accumulation 

in rice grains. 

 Studies have shown it may be possible to control Cd and As absorption 

by rice only through careful water management.3, 18, 26 For example, alternate 

wetting and drying (AWD) of rice paddies, in which fields are drained and re-

flooded one or more times during the growing season, has been shown to 

decrease total grain As.27  Honma et al.3 show that maintaining soil Eh to -100 

mV will keep As and Cd to minimum values.  However, according to LaHue et 

al.28 there are obstacles to implement this management practice in rice paddies 

due to lack of information on AWD management at large scales and the risk of 

yield loss with improper management. For example, rice yield is highest when 



71 

 

 

soils are flooded, despite higher As availability.  Therefore, it may be difficult to 

implement careful redox control at the field scale, particularly in developing 

countries, due to limited technology and resources, and also due to unpredictable 

field conditions (e.g., water availability or limited drainage conditions). Thus, it 

is imperative to develop practical management practices that can decrease both 

Cd and As in grain without compromising yield.    

One promising strategy to combat As in rice is through Si addition to 

soil, but its simultaneous impacts on Cd has not been thoroughly investigated. Li 

et al.19 showed that Si fertilization not only decreased As accumulation in rice 

shoots and grain but also benefited on rice yield under flooded condition.  

Recently, soil incorporation of   Si-rich rice husk residues (e.g. fresh husk and 

rice husk ash) has been investigated as a means to improve plant-available Si 

without some of the negative consequences associated with straw residues (i.e., 

higher As and CH4).29-31 Si-rich rice husk was shown to decrease inorganic As in 

grain without negatively affecting grain Cd or  rice yield under flooded 

conditions.31 However, the effects of Si-rich rice amendments incorporation to 

soil on Cd and As uptake by rice under non-flooded conditions and how they 

compare to flooded conditions has not been reported.  Here, we investigated the 

effects of fresh rice husk and rice husk ash on As and Cd uptake by rice under 

flooded and non-flooded conditions.  We found that incorporation of fresh rice 

husk (FH) and rice husk ash (RHA) decreased grain As under flooded and non-



72 

 

 

flooded conditions.  Under flooded conditions where grain As and yield was 

higher, fresh husk incorporation additionally decreased inorganic grain As.  

Under non-flooded conditions where grain Cd is highest and yield was lower, 

RHA decreased grain Cd.  These data illustrate that a combined approach of 

manipulating both Eh and pore water Si may alleviate Cd and As in grain 

depending on local condition