(Cu, Fe, Mn, and Zn) and Toxic (Pb)

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Bioaccessibility vs. bioavailability of essential (Cu, Fe, Mn, Zn) and toxic (Pb) elements from phyto hyperaccumulator P. stratiotes - potential risk of dietary intake Zuzana #adková, Ji#ina Száková, Daniela Miholová, Barbora Horáková, Old#ich Kopecký, Daniela K#ivská, Iva Langrová, and Pavel Tlustos J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/jf5058099 • Publication Date (Web): 09 Feb 2015 Downloaded from http://pubs.acs.org on February 18, 2015

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Journal of Agricultural and Food Chemistry

Bioaccessibility vs. bioavailability of essential (Cu, Fe, Mn, Zn) and toxic (Pb) elements from phyto hyperaccumulator P. stratiotes - potential risk of dietary intake Zuzana Čadková1, Jiřina Száková2, Daniela Miholová3, Barbora Horáková1, Oldřich Kopecký1, Daniela Křivská1, Iva Langrová1, Pavel Tlustoš2 1

Department of Zoology and Fisheries, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 957, Prague 6, 165 21, Czech Republic 2

Department of Agroenvironmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 957, Prague 6, 165 21, Czech Republic 3

Department of Chemistry, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 957, Prague 6, 165 21, Czech Republic

Corresponding author: Čadková Zuzana Address: Department of Zoology and Fisheries, FAFNR, CULS Prague; Kamycka 957, Prague 6, 165 21, Czech Republic Phone: +420 224 383 451 e-mail: [email protected]

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Abstract

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Aquatic weeds are widely used as animal feed in developing countries. However, information

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concerning element bioavailability from these plants is lacking. A combination of an in vitro

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method (PBET) and an in vivo feeding trial was conducted in the present study in order to

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investigate potential element bioaccessibility and estimated bioavailability from Pistia

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stratiotes (PS). Cu, Fe, Mn, Zn and Pb concentrations in PS biomass, artificial gastrointestinal

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fluids and rat tissues were determined using ET-AAS and ICP-OES. PS exhibited elevated Fe,

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Mn and Pb levels. The PBET revealed high bioaccessibility of all monitored elements from

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PS biomass. The results of the in vivo trial were inconsistent with those of the PBET, because

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animals fed PS exhibited low levels of essential elements in the tissues. The consumption of a

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PS supplemented diet significantly decreased total Fe levels and increased total Pb

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accumulation in exposed animals. Significantly reduced amounts of essential elements in the

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intestinal walls indicated a potential disruption in nutrient gastrointestinal absorption in

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animals fed PS.

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Key words:

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Pistia stratiotes, microelements, risk elements, biokinetics, physiologically based extraction

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test, feeding trial


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Introduction

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Due to a crucial shortage of livestock fodder in developing countries of tropical and

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subtropical regions, utilizing aquatic plants as animal feed seems to be a suitable option.

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Aquatic plant cultivation is cheap and less-laborious than conventional plant production, and

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it is less demanding on agricultural land. It is also very effective since most aquatic plants

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used as animal feed comprise water weeds with exceptional biomass productivity under

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favorable conditions. On the other hand, such plant species are able to grow in poor

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environmental condition, e.g., contaminated sewage canals, eutrophic water reservoirs etc1.

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Biota, living at these polluted sites, can be threatened by a high level of risk elements in the

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aquatic environment2, 3, 4.

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Although FEEDIPEDIA, a compendium of up-to-date information on feed resources, lists just

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8 aquatic plants as animal forage, a broader spectrum is used world-wide, in which the most

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common species are water hyacinth (Eichhornia crassipes) and duckweed (Lemnacae family).

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Based on their nutritional values and digestibility, 12 aquatic plants have been considered

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potential livestock feed5. Apart from conventional aquatic fodder, this list also includes Pistia

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stratiotes (PS).

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Pistia stratiotes (L. 1753), often referred to as water cabbage, water lettuce, Nile cabbage, or

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shellflower, is a floating perennial macrophyte from the family Araceae. It consists of a

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rosette of leaf blades that arise from a central meristem (a very short stem axis) and long

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feathery roots. P. stratiotes is native to South America, most likely Brazil. However, this

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species currently occurs in nearly all tropical and subtropical fresh waterways and water

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reservoirs, and has been included in the Global Invasive Species Database6, 7.

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PS is traditionally used for pig, cattle and duck food in China, Malaysia and Singapore8. A

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comprehensive feeding trial with whole PS plants was also conducted under controlled 3 ACS Paragon Plus Environment


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conditions on adult cattle, sheep, goats, horses, pigs and albino rats9. Water lettuce can

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possibly be used to produce Se-enriched plants for animal nutrition10. Moreover, leaf protein

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extracted form PS leaves significantly improves the nutritional value of protein deficient diet

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in ruminants, monogastric livestock and humans, because its digestibility (in vitro as well as

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in vivo) is even higher than that of other aquatic weeds11,12. Finally, fibrous byproducts from

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protein extraction could be utilized as an additional food for ruminants. Since PS has been the

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subject of miscellaneous studies, the basic nutritional values of this plant from former studies

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as well as from our study are shown in Table 1.

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However, the use of PS as livestock feed has a potential limitation because the plant is

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reported to accumulate considerable quantities of heavy metals, and it even possesses hyper-

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accumulative properties for several risk elements such as Cd, Hg and Pb13. Whereas Pb

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concentration in conventional plants is usually very low, Pb levels in PS growing on polluted

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areas can increase by up to 1.4814 and 15.76 mg.kg-1 15. Moreover, (cultivation in 1 mmol L−1

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Pb solution), an incredibly high Pb concentration of 203 g.kg-1 was observed16 under artificial

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conditions.

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The EU Directive 2002/32/EC17 set the maximum permissible limits for Pb in green fodder at

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30 mg.kg-1. However, even slightly increased levels of this metal in forage crops may

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negatively affect the health of both animal and human consumers because Pb is toxic to the

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organism, even when absorbed in small amounts. Recently, it has been demonstrated that the

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trace element toxicity of food is dependent on more than just their total concentration.

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Regarding the risk of feed intake, the bioaccesssibility and bioavailability of trace elements

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are crucial factors. Bioaccessibility (BAC) is theoretically the amount of substance that is

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soluble in the gastrointestinal environment, i.e., fraction that is released from the matrix into

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the gastrointestinal tract during the digestion process and, thus, becomes available for

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intestinal absorption and can enter the blood stream18. Bioavailability (BAV) is the general 4 ACS Paragon Plus Environment

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term used to describe the absorption of a contaminant into the body of an exposed subject.

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Absolute bioavailability (ABAV) is the proportion of the contaminant in the administered

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dose that is absorbed into the test organism.

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To evaluate bioaccessible fractions of contaminants, several in vitro approaches have been

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developed in attempts to mimic the effects of the digestion process in mammals. Generally,

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these approaches are commonly called simulated in vitro gastro-intestinal extraction

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procedures. The physiologically based extraction test (PBET), an in vitro test system for

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predicting the bioavailability of metals from a solid matrix, is the most widely used to date.

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The PBET was designed primarily to evaluate the absolute risk element bioavailability from

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contaminated soil in the digestive tracts of children19. However, various modifications of this

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original technique have been recently carried out. Concerning risk element bioaccessibility in

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animal and human food, the PBET was previously used to determine bioaccessible portions of

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both essential and toxic elements in contaminated plant samples20,21, seaweed22, medicinal

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plants23, raw vegetables24, uncooked rice25, cooked vegetable soup26, walnuts27 and mussel

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tissue28. However, to the best of our knowledge, both the BAC and BAV of trace elements

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have never been evaluated simultaneously in any phyto hyper-accumulators, including P.

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stratiotes.

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Because data concerning the total concentrations of both essential and risk elements in

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contaminated plant material provide little information about the possible nutritional values

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and toxic effects of this kind of forage, the PBET can be a useful tool in assessing the

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potential risk of its diet intake. Nevertheless, the results of in vitro procedures should always

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be accompanied and confirmed by in vivo trials. Therefore, the aims of this study were as

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follows: i.) to determine potential bioaccessibility and real bioavailability of both essential

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and risk elements from P. stratiotes; and ii.) to determine the effect of P. stratiotes biomass

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feed intake on element accumulation in mammalian organisms. For this purpose, an in vitro 5 ACS Paragon Plus Environment


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PBET using artificial digestive solutions was conducted, followed by an in vivo feeding trial

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on laboratory rats. In the course of this experiment, essential (Cu, Fe, Mn, Zn) and risk (Pb)

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element levels were determined within a simulated gastrointestinal tract and within several rat

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tissues (liver, kidney, muscle, bone, intestinal wall).

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Material and methods

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Chemicals

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Nitric acid 65%, p.a. ISO (Merck)

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H2O2 30%, TraceSelect (Fluka)

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Standard solution ASTASOL - CZ9098MN1 (Analytika, CR)

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Ammonium dihydrogen phosphate GR (Merck)

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Certified reference material BRC 12-02-01 (Bovine liver)

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Apparatus

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MWS-3+ microwave digestion system (Berghof Products+ Instruments, Germany) equipped

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with Teflon digestion vessel DAP-60S

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Atomic absorption spectrometer with electrothermal atomization (ET-AAS, Varian AA 280Z,

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Australia) with a graphite tube atomizer GTA 120 and a PSD 120 programmable sample

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dispenserInductively coupled plasma-atomic emission spectrometry (ICP-OES, Varian

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VistaPro, Australia) equipped with a two channel peristaltic pump, a Struman-Masters spray

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chamber and a V-groove pneumatic nebulizer made of inert material.


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In vitro evaluation of bioaccessibility

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The PBET20 was performed to assess bioaccessible portions of elements in the gastrointestinal

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tract as follows:

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i)

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0.84 mL of lactic acid and 1 mL of acetic acid was mixed with deionized water. The pH of the

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mixture was adjusted with concentrated HCl to 2.5 (± 0.05). Then 0.5 g of sample was mixed

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in an 85 mL polypropylene bottle with 50 mL of the prepared gastric solution. The bottle was

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placed in a shaker bath at 37 °C and shaken for 1 hour at 150 rpm. After centrifugation, a 5

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mL aliquot was taken off and measured for element content.

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ii)

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reaction mixture, and the pH was adjusted to 7 with a saturated NaHCO3 solution. Later, 25

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mg of pancreatin and 87.5 mg of bile salts were added. The sample was again shaken in the

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bath for 2 hours, centrifuged, and the extract was then measured. The element contents in the

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extracts were determined by ICP-OES. Bioaccessible portions of Cu, Fe, Mn, Zn and Pb were

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expressed as a percentage of total element concentration in both the control diet and P.

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stratiotes biomass.

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In vivo evaluation of bioavailability

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Experimental animals and diet

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Adult male Wistar rats (Rattus norvegicus) were used as models for bioavailability

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evaluation. The animals were obtained from a commercial supplier (Institute of Physiology of

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the AS CR, Prague, Czech Republic) and randomly divided into 2 groups based on plant

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material intake: CO – commercial diet only (n=6), PS – P. stratiotes intake (n=9). The rats

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were placed separately into plastic metabolic cages (TecniPlast, Italy) and left to acclimatize

Gastric solution - 1L volumetric flask: 1.25 g pepsin, 0.5 g of citric acid, 0.5 g malate,

Pancreatic solution: 5 mL of the fresh gastric solution was added to the remaining



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for 7 days. They were kept in controlled conditions (temperature of 22±2 °C; 12/12 hours

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light/dark cycle), given commercial feed and allowed to drink tap water ad libitum.

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A complete feed mixture for SPF breeding rats was purchased from a commercial supplier

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(Velaz s.r.o., Czech Republic) and used as a control diet. The nutritional values, as declared

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by the manufacturer, are as follows: Moisture – 12.5 %; Nitrogenous compounds – 24 %;

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Fiber – 4.4 %; Fat – 3.4 %; Ash – 6.8 %; Lysin 14g; Methionin 4.8g; Ca – 11g; P – 7.2g; Na

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– 1.8g; Cu – 20 mg; Se – 0,38 mg DW. The PS supplemented diet was prepared as follows: P.

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stratiotes specimens were cultivated in a Pb-enriched medium under greenhouse conditions29,

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harvested biomass was dried, dry mass was ground to a fine powder and each single dose (25

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mg) was then weighed and thoroughly mixed with 25 g of commercial feed. These batches

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were administered to rats daily for a period of 6 weeks. PS biomass constituted approximately

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0.1 % of the feed ration. Data concerning feed consumption was recorded daily (see Table 3),

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whereas individual animal weight gain was recorded twice a week (Figure 1).

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All experiments with laboratory animals were conducted in compliance with the current laws

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of the Czech Republic (Act No. 246/1992 coll. on Protection Animals against Cruelty) and

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EC Directive 86/609/EEC. The experimental project was approved by the committee of the

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Ministry of Education, Youth and Sports, Czech Republic (approval code MSMT-

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31220/2014-7). Animal care was supervised by authorized personnel: Zuzana Čadková,

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Ph.D., recipient of the Central Commission for Animal Welfare Certificate No. CZ 02201.

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Element analysis of rat tissues

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At the end of feeding trial, experimental animals were euthanized. Individual autopsies were

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carried out with Teflon® instruments in order to obtain appropriate tissue samples (liver,

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kidney, testes, duodenal wall, femoral muscle and bone) for element analyses. All tissues

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were weighed, properly washed in redistilled water, placed into Petri dishes and stored at a


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temperature of -20°C until they underwent chemical analysis. Prior to element analysis, tissue

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samples (except bones) were freeze-dried and microwave digested in an acid-based mixture

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using MWS-3+ microwave digestion system (Berghof Products+ Instruments, Germany). The

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ET-AAS technique was used to determine element concentrations in all digested tissue

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samples of control groups, as well as in the liver, muscle, testes and intestinal wall of PS

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exposed groups30. The calibration curve for the measurement was prepared using the standard

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solution ASTASOL CZ9098MN1 (Analytika, CR). The evaluation of the obtained data was

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carried out using a standard addition method, and ammonium dihydrogen phosphate GR

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(Merck) was used as a matrix modifier. Samples with elevated Pb levels (kidneys and bones),

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along with the total element content in the PS biomass and in the control diet, were analyzed

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using inductively coupled plasma optical emission spectrometry (ICP-OES, Varian VistaPro,

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Australia). Certified reference material BRC 12-02-01 (Bovine liver) was simultaneously

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analyzed under the same conditions to assess analytical data quality (Table 2). Experimental

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data were adjusted using the mean element concentrations in blanks (± 3 SD of blanks), which

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were prepared under the same conditions as were the tissues samples. Tissue samples were

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analyzed in two replicates, which were averaged for subsequent statistical processing. The

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relative element concentration in rat tissues was expressed in mg.kg-1 dry weight

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Total element levels in rat tissues

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Total amounts of particular elements accumulated in rat tissues were determined using the

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element concentrations (mg.kg-1 fresh weight) together with the weight coefficient of

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individual rat tissues31, according to the following formula:

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TEAT (mg) = EC (mg.kg-1 FW) × animal weight (kg) × SOWC × 10-2

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TEAT – total element amount in tissue

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EC – element concentration



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FW – fresh weight

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SOWC – specific organ weight coefficient31

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Additionally, P - portions (%) of the total Pb intake accumulated in a particular animal tissues

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were determined according to the folowing formula:

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P(%) = TEAT (mg) / TI (mg) × 100

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TI – total intake

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Statistical procedure

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Data obtained from the in vitro PBET was assessed in statistical software Statistica, ver. 1232

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using the Student´s T-test for dependent variables. Results of the in vivo feeding trial

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(differences in element tissue accumulations between animals from CO and PS groups) were

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analyzed using R statistical software, ver. 3.1.133. Due to the low number of animals used in

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our experiment (n = 15) and a significant number of potential interactions, we used separate

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tests for each monitored element. The basic lme model tested general influence of fixed effect

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“group” (CO vs. PS) with control for random effect “animal” with nested factor “organ”. The

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influence of individual animal variability was tested using AIC value comparison between a

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model that includes factor “animal” and a simplified model without this factor. In all cases

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(Cu, Fe, Mn, Pb and Zn) AIC differences exceeded 50, favoring simpler model. Finally, after

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factorial ANOVA with factors “group” and “organ”, post-hoc Tukey HSD tests were used to

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determine effect of diet (CO vs. PS) on element concentrations in particular organs. An

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analogical approach was used for evaluation of differences in TAET values between PS and

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CO animals. To fit normality, data were log-transformed before applying the statistical

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models. However, in order to show the patterns more clearly, original values were presented

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in graphs and tables. The threshold for statistical significance was set at α = 0.05.

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Results

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During the course of our experiment, the supplementing of daily feed rations with 0.1 % of PS

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dry mass did not significantly affect animal weight gains (T-test; CO vs. PS group; see Figure

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1). The individual variance was mainly caused by non-uniform initial animal weights at the

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beginning of the experiment. Figure 1 shows that individual variation actually decreased

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during the feeding trial.

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Total and extracted element concentrations

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Artificially cultivated P. stratiotes dry mass contained significantly higher Fe, Mn and Pb

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concentrations than did the control pellet feed. All 3 element concentrations were also

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significantly higher in the medium that simulated the gastric phase of PS digestion (pepsin

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extraction). In the artificial intestinal environment (pancreatin extraction), PS digest showed

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significantly higher concentrations of all of the monitored elements - Cu, Fe, Mn, Zn and Pb

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(Table 4).

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In vitro bioaccessibility

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The PBET revealed that element extraction from PS biomass during the digestive process

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differs from that of conventional feed (Figure 2, 3). In control feed, the BAC (%) of the

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majority of the monitored elements (Cu, Mn, Zn and Pb) was higher (25 – 53 %) in the gastric

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phase than in the intestinal fluid (9 – 20 %). The most significant decrease was observed for

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Zn - more than a five-fold decrease (from 53 % in GP to 9 % in IP). Fe BAC (%) from control

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feed showed a trend opposite to previous elements. It was extremely low in GP (1%) and

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increased to 5 % in a simulated intestinal environment. In PS biomass, the BAC (%) of all

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essential elements decreased significantly during the transition from the gastric phase (5 – 73

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%) to the intestinal phase (3 – 31 %). As with control feed BAC, Fe BAC in PS was very low

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(5 % in GP and 3 % in IP), and Zn BAC dropped considerably (from 73 to 31 %). Unlike to 11 ACS Paragon Plus Environment


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control feed digestion, PS feed digestion exhibited almost twice the Pb BAC levels in the

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gastric phase (5 %) than in the intestinal fluid (9 %).

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In vivo bioavailability

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Element accumulation in rat tissues was expressed in two ways: i.) relative concentration in

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mg.kg-1 DW (Table 5) and ii.) total amount in milligrams - TEAT (Figures 4A-F and 5). PS

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biomass intake affected element bio-kinetics in a rat organism. Supplementing the diet with

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Pb-loaded PS caused a significant increase in total Pb accumulation (lme, F = 38.332, p

(Cu, Fe, Mn, and Zn) and Toxic (Pb)

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