Rare earth element labeling as a tool for assuring the origin of eggs

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Food and Beverage Chemistry/Biochemistry

Rare earth element labeling as a tool for assuring the origin of eggs and poultry products Donata Bandoniene, Christoph Walkner, Daniela Zettl, and Thomas Meisel J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b03828 • Publication Date (Web): 12 Oct 2018 Downloaded from http://pubs.acs.org on October 14, 2018

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Journal of Agricultural and Food Chemistry

Rare Earth Element Labeling as a Tool for Assuring the Origin of Eggs and Poultry Products Donata Bandoniene1*, Christoph Walkner1, Daniela Zettl1, Thomas Meisel1 1Montanuniversität

Leoben, General and Analytical Chemistry, Franz-Josef-Straße 18, Leoben,

Austria, 8700 *Corresponding

Author: Donata Bandoniene, Montanuniversität Leoben, General and Analytical

Chemistry, Franz-Josef-Straße 18, Leoben, 8700, Austria [email protected] Fax: + 43 3842 402 1202; Phone: +43 3842 402 1207

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Abstract

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Laying hens were fed terbium and thulium supplemented feed in order to introduce a distinctive

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rare earth element pattern that allows discrimination of labeled from unlabeled poultry

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products. Samples of egg yolk, egg shells, meat, bones, liver, blood and feces were analyzed

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using either conventional or laser ablation ICP-MS. Already after a short time of administering

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supplemented feed, terbium and thulium enrichment could be unambiguously detected in the

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products, while absolute terbium and thulium contents remained low enough to ensure safety

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for the customer. This method could potentially be applied to specifically label foodstuffs

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produced in certain regions or under certain conditions, in order to ensure food authenticity.

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Keywords

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food authentication, rare earth elements, chemical labeling, ICP-MS, laser ablation ICP-MS,

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eggs, poultry products


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Introduction

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The regionality, the origin of foodstuff, has taken a high priority in today's society as a result

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of globalization and the free trade in goods. Recently, growing concern about food authenticity

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has spurred research into analytical techniques and methods capable of verifying the

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geographic origin of foodstuff. Amongst others, studies on the determination of the geographic

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origin of food items such as meat,1-3 milk,4 rice,5 potatoes,6 tomatoes,7 onions,8-10 artichokes,11

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chili peppers,12 fruits,13 pistachios,14 oils,15 including pumpkin seed oil,16-18 wine,19,20 coffee21

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and tea22 were undertaken.

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Food products with a direct relationship to the local soil reflect the regional distribution of

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trace elements, through absorption from the soil into the plant or by feed intake into the

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animal. Thus, the use of trace element fingerprinting, combined with statistical methods, is

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suitable for classification of such food products according to their origin. In contrast, for food

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products from conventional agriculture, in many cases this close connection between soil, plant

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and animal is not given. Animals are usually fed commercial complete feed and vegetables are

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grown in glasshouses on artificial substrates of diverse origin. In these cases it is not possible to

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verify the origin of such products based on a region-specific trace element fingerprint.

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The objective of the present study is to develop a method for labeling poultry products by

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selective enrichment of two rare earth elements (REE), namely terbium and thulium, in the feed

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for laying hens. Through this process a distinctive REE pattern is artificially introduced, which

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can be detected by suitable measurement methods in poultry products such as eggs and meat.

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This method could be applied to specifically label foodstuffs produced in a certain region or 3 ACS Paragon Plus Environment


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under certain conditions, such as organic products or free-range eggs, and distinguish them

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from (unlabeled) products of other origin. In a recent study conducted in our laboratory, the

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feasibility of REE labeling of glasshouse tomatoes has been shown,23 and a similar method has

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been proposed for labeling farmed salmon in order to distinguish escaped animals from wild

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salmon.24

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In our definition, the REE comprise 16 elements, specifically the 15 lanthanides plus yttrium.

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The REE can be classified into two groups according to their atomic number: The light rare earth

45

elements (LREE), including elements with atomic numbers 57 to 63 (lanthanum to europium),

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and the heavy rare earth elements (HREE), including elements with atomic numbers 64 to 71

47

(gadolinium to lutetium) plus yttrium.25 REE appear to be ideally suited for chemical labeling

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since their natural background levels in plants and animals are very low, allowing introduction of

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labels using very small quantities of REE, provided that sensitive measurement techniques are

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available for detection.

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REE are generally considered to be of low toxicity, especially when administered orally, with

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LD50 values in the single digit g/kg body mass range, comparable to table salt.26,27 This is thought

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to be mainly due to the poor absorption of REE from the gastrointestinal tract, with the bulk of

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ingested REE being quickly excreted via feces.28 In fact, low concentration dietary

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supplementation of REE has been noticed to improve body weight gain and feed conversion in

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various farming animals, including pigs, cattle, sheep and chickens, and has been practiced in

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China for decades.28-30 The use of a feed additive containing REE citrates (LancerTM, Treibacher

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Industrie AG, Althofen, Austria) for weaned piglets has been permitted in Switzerland, and

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authorization in the European Union is being pursued.31 4 ACS Paragon Plus Environment

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Due to the generally low REE levels in animal tissues, their quantitation can be quite

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challenging. A combination of acid digestion and solution nebulization ICP-MS (SN-ICP-MS) is

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most commonly applied. However, the large amounts of matrix elements such as calcium

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contained in samples such as egg shells or bones may impair ICP-MS measurement due to

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spectral and non-spectral interferences.

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Laser ablation ICP-MS (LA-ICP-MS) has only recently been introduced to the field of food

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analysis.32-35 Compared to SN-ICP-MS, LA-ICP-MS is less prone to some matrix effects, and

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measurements can be performed with limited or even without sample preparation. On the

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downside, limits of detection achieved using LA-ICP-MS are typically several orders of

69

magnitude higher compared to SN-ICP-MS, and accurate quantitation of the results obtained is

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often problematic, especially if matrix-matched calibration standards and reference materials

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are unavailable.36 However, higher limits of detection are at least partly compensated by the

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absence of dilution factors in solid state analysis. Moreover, the present study aims at

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determination of relative REE enrichment rather than absolute mass fractions, avoiding some of

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the issues related to calibration. Therefore, LA-ICP-MS was chosen as a complementary

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technique for REE analysis.

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Within the present study, a novel approach to assuring the geographic origin of poultry

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products by chemical labelling using REE is presented. The goal was to develop a simple, safe,

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fast and universally applicable food labelling and detection procedure.

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Materials and Methods

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Chemicals The REE chlorides supplemented to the complete feed were TbCl3 x 6 H2O and TmCl3 x 6 H2O,

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provided by Treibacher Industrie AG (Althofen, Austria). Nitric acid 65% m/m p.a. (Roth,

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Karlsruhe, Germany) was additionally purified by subboiling distillation. Hydrogen peroxide

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solution (30 % m/m, suprapur, Merck KGaA, Darmstadt, Germany), ammonia solution (25 %

87

m/m, ROTIPURAN, Carl Roth GmbH, Karlsruhe, Germany) and acetic acid (96 % m/m,

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ROTIPURAN, Carl Roth GmbH, Karlsruhe, Germany) were used as received. High purity water

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was prepared using the Siemens Ultra Clear system (18.2 MΩ cm resistivity, Siemens Water

90

Technologies, Barsbüttel, Germany). 1% m/v HNO3 was used for dilution of samples and

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calibration standard, and as carrier and rinsing solutions for SN-ICP-MS. Because REE concentrations in foodstuff are not equally abundant and occur in patterns

92 93

similar to Earth crust (lighter REE are more abundant than heavier REE, in addition to the Oddo-

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Harkins rule17,25), calibration solutions containing REE in equal concentrations might impede

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accurate measurements due to memory effects. Therefore, a custom made REE multi-element

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standard (AHF-CAL-7, Inorganic Ventures, New Jersey, USA) was used, with a distribution

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pattern similar to continental crust. The REE concentrations are: 1000 µg mL-1 cerium, 500 µg

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mL-1 lanthanum, neodymium and yttrium, 100 µg mL-1 praseodymium, 150 µg mL-1 thorium,

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50 µg mL-1 dysprosium, gadolinium, samarium and uranium, 20 µg mL-1 erbium, europium and

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ytterbium, 10 µg mL-1 holmium and terbium, and 5 µg µg mL-1 lutetium and thulium in 7% m/v

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HNO3. A 50 ng mL-1 stock solution (calculated for lanthanum, neodymium and yttrium) was

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prepared and diluted accordingly with 1% m/v HNO3 for a calibration range from 0 to 10 ng mL-

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1.

Internal standard solutions (100 ng mL-1 indium and rhenium for SN-ICP-MS, 10 µg mL-1 6 ACS Paragon Plus Environment

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indium for LA-ICP-MS) were prepared from 1000 mg L-1 single-element standard solutions

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(Merck KGaA, Darmstadt, Germany). Reference materials MACS-337 (microanalytical calcium

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carbonate standard, synthetic calcium carbonate pressed pellet in 19 mm ring) and MAPS-438

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(microanalytical calcium phosphate standard, synthetic calcium phosphate pressed pellet in 19

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mm ring) for LA-ICP-MS analysis were obtained from the United States Geological Survey,

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Reston, VA, USA.

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Preparation of REE Marker Feed

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Terbium and thulium were chosen as ideal labeling elements for this study because of their

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monoisotopic nature, the lower abundance of these HREE compared to LREE in nature and their

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relatively low price. The basis for the hens´ diet was the commercially available complete feed

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OGT G-61, manufactured by Uitz Mühle GmbH (Knittelfeld, Austria). A REE marker solution was

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prepared by dissolving the REE chlorides TbCl3 x 6H2O (2 g L-1) and TmCl3 x 6 H2O (1 g L-1) in

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deionized water. The obtained solution was then added to two batches of the complete feed

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and mixed in a horizontal mixer, in order to achieve an approximate 500-fold enrichment.

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Marking Experiment

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All animal experiments were reported to and approved by the Austrian Federal Office for

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Food Safety according to applicable Austrian law. The experiments were conducted at the

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Agricultural Research and Education Centre Raumberg-Gumpenstein in July – September 2013.

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Fifty-two laying hens of the breed Lohmann Brown-Classic BIO at the age of 140 days were kept

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in a closed stable equipped in accordance with animal welfare standards. At the age of 150 –

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160 days, the laying hens reach approximately 50 % of their full egg production capacity. The

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stable was 5.85 x 2.22 m and equipped with two water tanks (15 L each) and two feed tanks (15 7 ACS Paragon Plus Environment


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kg each), providing feed and water for ad libitum consumption; the dietary intake of one layer is

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125 – 130 g per day. Five perches were arranged above a feces dump at the back side of the

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stable. 15 layer moulds were mounted in a slightly elevated position. Aeration was provided by

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three windows positioned on the shady side of the stable in order to avoid overheating. The

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stable was illuminated for 10 h per day at the beginning of the experiment, gradually increasing

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up to a maximum of 14 h. In order to allow the animals to acclimatize to the stable and feed,

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they were kept for three weeks under the same conditions as during the marking experiment,

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but without REE enrichment in feed. In the following text, this acclimatization phase will be

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referred to as days -20 to 0, or weeks -3 to 0. From day 1 onwards, REE spiked feed was

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administered to all hens for a five weeks labeling phase, until day 35 (weeks 1 – 5).

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Subsequently, unspiked feed was fed again for a four weeks dilution phase from day 36 to day

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65 (weeks 6 – 9).

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Sampling

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The complete feed with and without REE spiking were sampled for chemical analysis. Four

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subsamples from different places in the horizontal mixer were taken with a sample scoop and

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pooled. From that pooled sample, a portion of 500 g was taken as lab-sample.

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Samples of four hens each were randomly taken in the first week of the acclimatization

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phase, at the end of the acclimatization phase and then every week until the end of the

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experiment. Hens were slaughtered and from each hen a chicken drumstick, a piece of breast

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meat, blood and liver were sampled and stored at -20 °C. Feces samples were taken directly

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from the intestine of the hens in order to study the digestibility of the tested elemental markers


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and to avoid contamination through dust in the stable. These samples were also stored at -20

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°C. An individual body weight was recorded for every sampled hen.

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Initially, two eggs were randomly sampled every day; starting at day 6, when the layers had

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reached their full egg production performance, the sample size was increased to ten eggs per

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day.

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Sample Preparation

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Eggs were rinsed using deionized water and egg shell, yolk and egg white were separated.

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From all eggs sampled during a day, yolk and egg white were pooled separately yielding one

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daily composite sample each. From egg yolk composite sample ca. 20 g were subsampled and

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dried at 60-70 °C in an oven to complete dryness which took about 2-3 days and subsequently

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stored in a refrigerator. Meat and liver samples were cleaned with deionized water after

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defrosting.

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Feed, egg white, yolk, liver, meat, blood and feces samples were digested using a high

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pressure asher (HPA-S, Anton Paar, Graz, Austria) for SN-ICP-MS analysis. Subsamples of

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approximately 1 g of compound feed, 2 g of egg white, 4 – 5 g of liver or meat, 1 g of blood, 1.5

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g of feces (each wet weight) and 1 g of oven-dried yolk were weighed in 90 mL quartz glass

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digestion vials, and 10 mL concentrated sub-boiled HNO3 were added. Digestion was carried out

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at a temperature of 280 °C and a pressure of approximately 125 bar for 2.5 h. The resulting

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solutions were transferred into 15 mL round bottomed PFA vials and dried on a hot plate at 50-

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70 °C hot plate surface temperature. The residues were redissolved in 5 mL 1 % m/v HNO3, and

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100 µL portions of the indium and rhenium internal standard solution were added

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subsequently. Two replicate subsamples were digested and analyzed for each sample. 9 ACS Paragon Plus Environment


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For LA-ICP-MS analysis, egg shell fragments of 30 - 50 mm2 size, without membrane, were

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mounted onto glass microscope slides by means of double-sided adhesive tape, the inner side of

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the shell facing upwards. Meat and bone samples were prepared for LA-ICP-MS by means of dry

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ashing, following a well-established procedure.39 Samples of meat (20 – 30 g wet weight) and

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bones (5 – 10 g wet weight) were dried at 120 °C over night and ashed in ceramic crucibles over

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a Bunsen burner until formation of smoke ceased. After cooling to room temperature, the

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residues were coarsely crushed using a glass rod, and 500 µL of a 10 µg mL-1 indium solution

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were added as an internal standard. The crucibles were then placed in a muffle furnace at a

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temperature of 550 °C for 5 h. Residues that appeared to contain residual carbon (i. e. dark grey

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to black in color) after this treatment were heated for another 5 h with addition of a few drops

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of 30 % w/w H2O2 solution to facilitate complete ashing. This step was repeated if necessary.

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The remaining ash was homogenized in an agate mortar, and 200 – 250 mg of each sample were

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pressed into a 13 mm diameter pellet using a hydraulic press, applying 105 N for 1 min. For LA-

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ICP-MS measurement the pellets were mounted onto glass microscope slides by means of

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double-sided adhesive tape.

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ICP-MS Measurement

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The REE mass fractions of digested samples were determined by SN-ICP-MS using an Agilent

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7500 ce (Agilent Technologies, Tokyo, Japan) equipped with a 100 µL PFA nebulizer, a Peltier-

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cooled Scott-type spray chamber and nickel sampler and skimmer cones. Prior to each

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experiment, the instrument was tuned to maximum sensitivity while keeping oxide formation

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ratio (CeO+/Ce+) below 1 % using a solution containing 1 µg/L lithium, cobalt, yttrium, cerium

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and thallium. Samples were taken up by an autosampler (SC-2 DX, Elemental Scientific, Omaha, 10 ACS Paragon Plus Environment

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NE, USA), loaded into a 2 mL sample loop via a six-port valve and conveyed to the nebulizer by

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means of a carrier solution (1 % m/v HNO3) by a peristaltic pump. Limits of quantitation (LOQ)

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were calculated as 10 times standard deviation of the calibration blank.

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For the analysis of a part of the meat, liver and blood samples, the ICP-MS was equipped with

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an automated on-line solid phase extraction system (seaFAST, Elemental Scientific, Omaha,

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Nebraska, USA). The seaFAST system is supposed to improve limits of detection for a range of

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elements through preconcentration and matrix removal. The system loads approximately 4 ml

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sample onto a chelation column (SeaFAST concentrator column CF-N-0200, Elemental Scientific,

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Omaha, Nebraska, USA), where transition metal ions (including REE) are retained. Matrix

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components such as alkali and alkaline earth metal ions are washed from the column using an

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ammonium acetate buffer solution at pH 6, and pass through to waste. The chelated REE are

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then eluted with a small volume of 10 % (v/v) HNO3 directly into the nebulizer for ICP-MS

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detection.

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LA-ICP-MS analyses were carried out using a NWR 213 laser ablation system (Electro

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Scientific Industries, Portland, OR, USA) equipped with a TV 2 two-volume cell and coupled to an

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Agilent 8800 ICP-MS/MS (ICP-QQQ, Agilent Technologies, Tokyo, Japan). Sample aerosol was

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transported into the plasma by a He flow of 0.8 L/min, which was mixed with the carrier gas in a

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Liebig gas mixer. In order to increase the sensitivity, the ICP-MS/MS was operated in single

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quadrupole mode since no polyatomic interferences were expected for the m/z used, and the

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instrument was equipped with an additional foreline pump.40 For LA-ICP-MS analysis of egg

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shells, an s-lens set of ion lenses (Agilent Technologies, Tokyo, Japan) was used instead of the

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standard lenses in order to further increase sensitivity. Five lines of 3 mm length were ablated 11 ACS Paragon Plus Environment


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on the inner surface of each egg shell sample, with 250 µm spot diameter, 20 Hz repetition rate,

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70 µm/s scanning speed and 75 % laser energy (approx. 2.5 J/cm2 fluence). Reference material

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MACS-3 was used for element bias and drift correction. Sets of 5 samples (i. e. 25 line scans)

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were bracketed by sets of 2 spot ablations of MACS-3, with 80 µm spot diameter, 10 Hz

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repetition rate, 45 s dwell time and approx. 2 J/cm2 fluence. For meat and bone samples, five

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lines of 2 mm length per sample were performed, with 110 µm spot diameter, 20 Hz repetition

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rate, 50 µm/s scanning speed and approx. 2.5 J/cm2 fluence. Sample-standard-bracketing was

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carried out as described above, using reference material MAPS-4.

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Time-resolved profiles for m/z = 43 (43Ca+), m/z = 89 (89Y+), m/z =159 (159Tb+), m/z = 169

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(169Tm+) and, for meat samples, m/z = 115 (115In+) were recorded and exported to a spreadsheet

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for data reduction. Calcium was used as an internal standard for quantitative analyses of egg

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shell and bone samples, while indium was used for meat samples. Analyte/internal standard

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count ratios as well as 159Tb+/89Y+ and 169Tm+/89Y +count ratios were calculated for each data

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sweep, and from all data sweeps acquired during ablation after a 5 s stabilization period, 10 %

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trimmed means were calculated in order to reduce spikes caused by incomplete atomization of

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large particles in the plasma. Results were then corrected for mass bias and drift by sample-

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standard-bracketing, assuming linear time-dependent drift between each 2 sets of standards.

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Element mass fractions were calculated by means of one point calibration using the above-

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mentioned reference materials, i. e. response factors (analyte/internal standard count ratio per

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mass fraction in ng/g) for the reference materials were calculated for each measurement

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session, and count ratios acquired for unknown samples were divided by the respective


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response factor. Limits of quantitation (LOQ) were calculated as 10 times standard deviation of

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25 consecutive gas blank measurements.

236 237

Results and Discussion

238 239 240

Terbium and Thulium Contents in Chicken Feed The REE contents of the feed without terbium and thulium spiking (baseline feed) and with

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terbium and thulium spiking (marker feed) were determined using SN-ICP-MS after acid

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digestion. Terbium and thulium mass fractions for baseline feed were 0.017 ± 0.003 mg/kg and

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0.011 ± 0.002 mg/kg (mean ± standard deviation, n = 12), for marker feed 7.7 ± 1.0 mg/kg and

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4.1 ± 0.6 mg/kg (mean ± standard deviation, n = 6), respectively. This corresponds to

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approximately 450-fold and 400-fold enrichment of terbium and thulium in the marker feed,

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respectively. Although the variance of the results is relatively high, it was considered fit for the

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purpose of the experiment.

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Calculation of Terbium and Thulium Anomaly

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The natural distribution of lanthanides in animals and animal products is in accordance with

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the Oddo-Harkins rule, with the even-numbered elements being more abundant than the odd-

251

numbered ones.25 In order to identify anomalies in the REE profiles, it is customary to normalize

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REE mass fractions by division by a suitable reference data set. In the present work, unlabeled

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samples were used as references (day -7 egg yolk sample for yolk, day 0 feces sample for feces,

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day 0 breast meat sample for other sample types). As an example, Supplementary Figure 1 13 ACS Paragon Plus Environment


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shows normalized REE mass fractions determined using SN-ICP-MS in egg yolk samples from

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days -10, 4 and 15. Ideally, for an unlabeled sample (day -10), normalized values for all elements

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are approximately 1, while terbium and thulium values for labeled samples (days 4 and 15)

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clearly stand out. The deviation of these values from the baseline, or REE anomaly, can be

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calculated as REEn/REEn*, where REEn is the normalized mass fraction of the respective element

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and REEn* is the expected normalized value interpolated from the two respective “neighboring”

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elements, i. e. gadolinium and dysprosium for terbium and erbium and ytterbium for thulium

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(equations 1 and 2):41 Tbn Tbn* =

2 × Tbn Gdn + Dyn

Tmn Tmn* =

2 × Tmn Ern + Ybn

(1)

(2)

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REEn/REEn* values provide a demonstrative measure of REE enrichment in food samples,

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which is also relatively robust towards variations in the absolute REE levels that may arise due

265

to factors such as tissue heterogeneity, water content or, potentially, contamination or sample

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loss during sample preparation. Median REEn/REEn* values determined using SN-ICP-MS in food

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samples from acclimatization, labeling and dilution phase are summarized in Table 1.

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Due to the low natural levels of HREE naturally present in food samples, it was not possible

269

to acquire complete REE profiles by LA-ICP-MS. Therefore, yttrium was chosen as a reference

270

element with ionic radius and chemical behavior similar to the HREE, but much higher

271

abundance, and normalized REE/Y ratios (REEn/Yn) were calculated from REE/Y intensity ratios

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acquired by LA-ICP-MS as a measure of REE enrichment, following equations 3 and 4:


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Tbn Yn =

Tb/Y Tbref Yref

(3)

Tmn Yn =

Tm/Y Tmref Yref

(4)

Reference values REEref/Yref are calculated as the mean values for all measured REE/Y ratios

274

for unlabeled samples of the same type. As long as the ratio between yttrium and the HREE

275

(gadolinium, dysprosium, terbium, erbium) is approximately equal in sample and reference

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(which can be assumed for samples of the same type), REEn/REEn* and REEn/Yn can be expected

277

to give comparable values, and therefore for the rest of the discussion they will be regarded as

278

equivalent measures of REE anomaly. Median REEn/Yn values determined using LA-ICP-MS in

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food samples from acclimatization, labeling and dilution phase are summarized in Table 2.

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Terbium and thulium levels in unlabeled egg shell samples and thulium levels in unlabeled

281

bone samples were below the respective limits of quantitation (Table 2), and calculating

282

REEref/Yref and, consequently, REEn/Yn ratios based on these data would result in high

283

uncertainty or even erroneous values. Therefore, Tb/Y and Tm/Y ratios of calcium carbonate

284

reference material NIM-GBW07129,42 0.014 and 0.015, respectively, were used as REEref/Yref

285

instead. Similarly, Tmref/Yref for bone samples was extrapolated from the respective Tbref/Yref

286

ratio using the Tb/Tm ratio of calcium phosphate reference material Durango apatite,43 2.8.

287

Since the main constituents of these reference materials match the samples and the differences

288

in ionic radii and hence chemical behavior between yttrium, terbium and thulium are small, the

289

extrapolated values can be assumed to approximate the true values. It should also be noted

290

that the purpose of using these reference values for REEn/Yn calculation is merely to give an

291

estimation of the magnitude of REE enrichment in labeled samples, and that the ability of the 15 ACS Paragon Plus Environment


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present method to discriminate between labeled and unlabeled samples does not depend on

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their trueness. All egg shell, meat and bone samples from the labeling phase analyzed using LA-

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ICP-MS showed Tb/Y and Tm/Y ratios significantly higher than the respective acclimatization

295

phase samples, confirmed by one-tailed t-tests (P = 0.01, data not shown).

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Terbium and Thulium Enrichment in Eggs

297

While no REE enrichment in egg white could be detected (data not shown), the accumulation

298

of terbium and thulium takes place in the egg yolk. Supplementary Figure 1 shows normalized

299

REE mass fractions in samples from days -10, 4 and 15, while Figure 1 shows the development of

300

terbium anomaly in egg yolk recorded during the complete experiment. During the

301

acclimatization (up to day 0) and dilution phase (days 36 to 65), hens were fed baseline feed,

302

while during the labeling phase marker feed was dispensed. Terbium contents remain on a low

303

(natural) level during the acclimatization phase. After 4 days of feeding with marker feed, a

304

distinct increase in terbium contents is already visible. After approximately 7 days, terbium

305

levels remain relatively constant for the rest of the labeling phase, at approximately 50-fold

306

enrichment. During the dilution phase terbium levels slowly decrease, but even after 30 days

307

without marker feed an approximately 15-fold enrichment can be detected. In general, terbium

308

and thulium anomalies developed very similarly throughout the experiment; therefore, for the

309

rest of the discussion only terbium will be considered. Median values for terbium and thulium

310

anomalies and REE mass fractions for all sample types investigated are summarized in Table 1

311

(results acquired using SN-ICP-MS) and Table 2 (LA-ICP-MS).

312 313

The fact that REE enrichment occurs in egg yolk but not in egg white is not surprising since mineral content, including calcium, of egg white is generally very low.44 It is assumed that in 16 ACS Paragon Plus Environment

Page 17 of 38


314

biological systems, REE ions can replace Ca2+ ions to a certain extent, due to their similar ionic

315

radii,26,45 resulting in extremely low REE levels in egg white.

316

Among the sample types analyzed in this study, egg shells can be sampled most easily, and

317

are most suitable for LA-ICP-MS analysis due to their mainly inorganic matrix. However, egg

318

shells naturally are in close contact with REE spiked feed and feces, and therefore their external

319

surface can be assumed to be contaminated. Consequently, also REE contents determined in

320

homogenized egg shell samples can be expected to be influenced by surface contamination.

321

Therefore the internal surface of egg shell samples was analyzed using LA-ICP-MS in order to

322

determine the amount of REE actually incorporated into the shell; results are shown in Table 2

323

and Figure 2. Unfortunately, egg shells were not sampled during the entire experimental period.

324

One sample per day was investigated for days -11 to -6 (acclimatization phase), 5 samples every

325

3 days for days 16 to 35 (labeling phase) and 5 samples every 5 days for days 36 to 65 (dilution

326

phase). Terbium and thulium contents in all samples from the acclimatization phase were below

327

the limits of quantitation. Although the scatter amongst individual samples (i. e. eggs laid by

328

different hens) for one day is relatively large, terbium enrichment is clearly visible for all

329

samples from the labeling period. During the dilution phase, terbium levels decrease again, but

330

are still elevated at the end of the experiment. Again, thulium anomaly follows the same general

331

trend. Compared with egg yolk, terbium anomalies in egg shells are lower (approximately 20-

332

fold enrichment during the labeling phase), and appear to decrease faster, which can be

333

assumed to be due to the sequence of egg generation: The generation of egg yolk begins

334

approximately 10 days before egg deposition, while egg shell is established only during the last

335

24 h,44 when the overall levels of marker elements in the organism are already lower due to 17 ACS Paragon Plus Environment


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336

natural excretion. In view of potential practical application, both egg yolk and shell samples

337

appear suitable for authentication of eggs. While egg yolk samples have the advantage of higher

338

REE contents, LA-ICP-MS analysis of egg shells is possible with very little sample preparation, in

339

principle even without breaking the eggs.

340

Terbium and Thulium Enrichment in Meat

341

Meat samples typically contain very low amounts of REE but substantial amounts of matrix

342

elements such as potassium, calcium or magnesium, complicating their analysis by ICP-MS.

343

Therefore, two different approaches were tested: SN-ICP-MS analysis after wet digestion and

344

LA-ICP-MS analysis of pressed pellets after dry ashing.

345

For the analysis of a part of the meat samples, as well as some liver and blood samples, the

346

ICP-MS was equipped with the seaFAST system for preconcentration and matrix removal.

347

However, the application of the SeaFAST system, originally intended for direct analysis of sea

348

water samples, for food samples has several drawbacks: Analysis time per sample is

349

considerably extended due to the flow resistance of the chelation column, which is also prone

350

to clogging by particles contained in incompletely digested samples. In addition, no significant

351

improvement in detection capacity could be achieved. Therefore, after a few attempts using the

352

SeaFAST system, conventional SN-ICP-MS was applied again.

353

Figure 3 shows the terbium anomaly determined using both SN-ICP-MS and LA-ICP-MS for

354

breast (a) and drumstick meat samples (b). Terbium enrichment in meat is even more

355

pronounced than in eggs, with anomalies between 50 and 100 in the labeling phase, slowly

356

decreasing during the dilution phase. Although the values measured for individual specimens (4

357

hens were sampled per week) differ considerably, the results obtained by both methods are 18 ACS Paragon Plus Environment

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358

comparable. The dry ashing/LA-ICP-MS method can be expected to be more robust towards

359

sample heterogeneity and superficial contamination due to larger sample size (20 – 30 g vs. 4 –

360

5 g for SN-ICP-MS), and in fact, LA-ICP-MS analysis yielded fewer extremely high values. On the

361

downside, quantitative LA-ICP-MS results (Table 2) have to rely on one-point calibration using

362

not strictly matrix-matched reference material MAPS-4, and should therefore be regarded with

363

some caution. Both breast and drumstick meat could be distinctively labeled with terbium and

364

thulium, while total REE contents remain low enough that consumer safety should be

365

maintained (see below).

366

Terbium and Thulium Enrichment in Bones

367

Similar to egg shells, bones appear to be a sample type very suitable for LA-ICP-MS analysis,

368

due to their relatively high REE content and mostly inorganic constituents. In experiments with

369

rats, REE have been found mainly in liver (LREE) and bones (HREE) after intravenous

370

injection.46,47 Retention of REE in skeleton is reported to be longer in comparison to soft

371

tissues.45 Therefore, bone samples show promise for potential practical application.

372

For the present study, leg bone samples were ashed, homogenized and pressed into pellets

373

in order to facilitate quantitative LA-ICP-MS analysis. However, for applications where

374

determination of REE anomalies is sufficient, direct analysis of bone samples is certainly

375

conceivable.

376

Figure 4 shows terbium anomaly detected in 4 bone samples per week throughout the

377

labeling experiment. Although the hens were fully-grown at the beginning of the labeling phase,

378

an approximately 10-fold terbium enrichment was established after one week and remained

379

more or less constant until the end of the experiment. 19 ACS Paragon Plus Environment


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Page 20 of 38

Terbium and Thulium Enrichment in Liver, Blood and Feces

381

In order to further elucidate the behavior of REE in the organism, also samples of liver, blood

382

and feces were analyzed. For liver and blood, samples from 4 individual animals per week were

383

analyzed throughout the experiment. Terbium anomalies determined by SN-ICP-MS in liver and

384

blood samples are shown in Figure 5 (a) and (b), respectively. Terbium anomaly in liver increases

385

during the labeling phase, and remains relatively constant throughout the dilution phase. In

386

contrast, terbium anomaly in blood quickly stabilizes at approximately 20-fold enrichment and

387

remains at this level until the end of the experiment. Neither terbium anomalies nor terbium

388

mass fractions (Table 1) determined in liver samples are distinctly larger than in meat samples.

389

However, it is worth noting that thulium mass fractions in labeled liver samples are almost equal

390

to terbium mass fractions and consequently thulium anomalies are distinctly higher than

391

terbium anomalies. Supplementary Figure 2 shows median REE profiles for liver samples from

392

acclimatization, labeling and dilution period. Normalized mass fractions for thulium in labeling

393

and dilution period are significantly higher than for terbium. In addition, the other HREE also

394

appear to be slightly enriched relative to the LREE. In all the other sample types that were

395

analyzed, including feces (see below), no systematic differences between terbium and thulium

396

enrichment could be detected. Based on the data available, it is not clear whether this

397

fractionation is a result of preferential thulium uptake or of preferential terbium excretion.

398

Altogether, terbium anomalies in the sample types analyzed follow the trend meat ≈ liver >

399

egg yolk > egg shell > blood > bone, whereas terbium mass fractions descend in the order bone

400

> meat ≈ liver > egg yolk > egg shell > blood; similar results were achieved for thulium.


Page 21 of 38


401

As expected, REE uptake from feed was very low, and the bulk of the marker elements were

402

excreted with feces. Feces samples were analyzed from each 4 hens sampled at the end of the

403

acclimatization, labeling and dilution phase (Figure 5 (c)). At the end of the labeling phase, a

404

median terbium anomaly of 500 was determined, which corresponds to the enrichment in the

405

marker feed. Even at the end of the dilution phase, a terbium anomaly of approximately 300

406

persisted.

407

REE labeling of all sample types (eggs, meat, bones, blood, liver, feces) through spiked

408

chicken feed was found to be successful. This method makes it possible to assure the origin of

409

any poultry products. For practical application, the choice of labeling REE can of course be

410

varied in proportion and element combination. At the same time, all terbium and thulium mass

411

fractions determined in poultry products were in the ng/kg or single digit µg/kg range, low

412

enough to virtually exclude negative effects on potential consumers. Acceptable daily intake

413

values proposed for REE range between 0.1 – 2 mg/kg body weight,48,49 which would

414

correspond to nearly 2000 kg of labeled chicken breast meat (total REE content 3.6 µg/kg) per

415

day for a person of 70 kg. It should also be noted that, since terbium and thulium are among the

416

least abundant REE, total REE content in labeled chicken meat is only about twice as high as in

417

unlabeled meat.

418 419

Funding Source

420 421

The authors gratefully acknowledge financial support from the Office of the government of

422

Styria (program Zukunftsfonds Steiermark, Exciting Science, PN: 6016). 21 ACS Paragon Plus Environment


Page 22 of 38

423 424

Acknowledgements

425 426

The authors would like to thank Treibacher Industrie AG for providing terbium and thulium

427

chlorides used in the labeling experiments and Agilent Technologies, Inc. for providing an s-lens

428

assembly free of charge. The authors would also like to thank the project partner Agricultural

429

Research and Education Centre Raumberg-Gumpenstein for collaboration in carrying out the

430

animal experiments, and especially Renate Mayer for the support through project management

431

and Eduard Zentner for organizing and supervising the experiments. Special thanks go to

432

student Brigitte Maier, who participated in the study in the context of her final year project, and

433

did a great job in attending to the chickens with great care and diligence, sampling and sample

434

preparation.

435 436

Abbreviations

437

HREE, heavy rare earth element; ICP-MS/MS, inductively coupled plasma tandem mass

438

spectrometer; LA-ICP-MS, laser ablation inductively coupled plasma mass spectrometry; LOQ,

439

limit of quantitation; LREE, light rare earth element; REE, rare earth element; SN-ICP-MS,

440

solution nebulization inductively coupled plasma mass spectrometry

441 442

Supporting Information


Page 23 of 38


443

Supplementary Figure 1 – 2: Two graphs showing selected REE profiles for egg yolk and liver

444

samples.



445

Page 24 of 38

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Figure Captions

Figure 1: Terbium Anomaly (Tbn/Tbn*) detected in egg yolk using SN-ICP-MS (daily pooled samples of 2 egg yolks in acclimatization phase, 10 egg yolks in labeling/dilution phase) during the experiment.

Figure 2: Terbium anomaly (Tbn/Yn) determined using LA-ICP-MS in egg shell samples taken during the labeling experiment. Days 16 – 33: labeling phase (hatched symbols); days 36 – 61: dilution phase. Terbium contents in samples from acclimatization phase were below the LOQ.

Figure 3: Terbium anomaly determined by means of SN-ICP-MS (Tbn/Tbn*) and LA-ICP-MS (Tbn/Yn) in breast meat (a) and drumstick (b) samples taken during the labeling experiment (4 samples per week). Weeks -3 – 0: acclimatization phase; weeks 1 – 5: labeling phase (hatched symbols); weeks 6 – 9: dilution phase.

Figure 4: Terbium anomaly (Tbn/Yn) determined using LA-ICP-MS in bone samples taken during the labeling experiment. Weeks -3 – 0: acclimatization phase; weeks 1 – 5: labeling phase (hatched symbols); weeks 6 – 9: dilution phase.

Figure 5: Terbium anomaly (Tbn/Tbn*) determined using SN-ICP-MS in liver (a), blood (b) and feces (c) samples taken during the labeling experiment. Weeks -3 – 0: acclimatization phase; weeks 1 – 5: labeling phase (hatched symbols); weeks 6 – 9: dilution phase.


Page 31 of 38


Table 1: Median values for REE mass fractions (dry weight for egg yolk, wet weight for other sample types) and terbium and thulium anomalies (Tbn/Tbn*, Tmn/Tmn*) determined using SNICP-MS in egg yolk, meat, liver, blood and feces samples taken during acclimatization (Acc.), labeling (Lab.) and dilution phase (Dil.). Limits of quantitation (LOQ) are the average of 10 calibrations and incorporate the average dilution factors due to sample preparation of 5 for yolk and blood and 150 for feces samples. Y Acc. Lab. Dil. Acc. Breast Lab. meat Dil. Acc. DrumLab. stick Dil. Acc. Liver Lab. Dil. Acc. Blood Lab. Dil. Acc. Feces Lab. Dil. meat, liver LOQ yolk, blood feces Egg Yolk

0.14 0.15 0.15 0.20 0.12 0.07 0.13 0.10 0.10 0.08 0.15 0.15 0.08 0.05 0.10 228 107 146

La 0.21 0.21 0.14 0.26 0.16 0.14 0.17 0.15 0.16 0.36 0.16 0.12 0.13 0.10 0.11 106 62 88

Ce 0.45 0.40 0.33 0.71 0.44 0.46 0.53 0.34 0.47 0.67 0.27 0.24 0.47 0.21 0.28 177 60 126

Pr 0.037 0.032 0.038 0.058 0.073 0.033 0.038 0.039 0.032 0.085 0.060 0.041 0.024 0.015 0.021 24 15 20

Nd

Sm

0.19 0.15 0.12 0.23 0.14 0.11 0.12 0.12 0.11 0.26 0.17 0.10 0.22 0.28 0.16 99 58 80

0.004 0.003 0.006 0.002 0.01 0.02

0.02

0.03

0.6

0.5

0.9

0.01 0.06 0.3

2

Eu

0.069 0.051 0.057 0.071 0.046 0.034 0.047 0.039 0.051 0.053 0.038 0.039 0.026 0.020 0.025 23 14 18

Gd µg/kg 0.057 0.038 0.071 0.039 0.081 0.036 0.017 0.051 0.013 0.039 0.012 0.026 0.015 0.035 0.015 0.032 0.012 0.033 0.016 0.056 0.020 0.034 0.016 0.027 0.009 0.028 0.012 0.027 0.010 0.025 6.3 26 3.6 13 4.4 18

Tb 0.021 0.70 0.50 0.029 1.62 0.77 0.024 0.82 1.01 0.018 0.89 1.19 0.079 0.22 0.26 6.1 1420 1041

Dy 0.048 0.045 0.044 0.044 0.034 0.023 0.031 0.028 0.028 0.049 0.029 0.024 0.024 0.023 0.021 30 14 19

Ho 0.010 0.008 0.007 0.006 0.019 0.021 0.013 0.017 0.017 0.012 0.015 0.012 0.021 0.12 0.005 6.6 2.8 4.0

Er 0.033 0.027 0.020 0.037 0.026 0.018 0.023 0.021 0.022 0.037 0.023 0.018 0.018 0.018 0.016 20 8 12

Tm 0.010 0.41 0.22 0.011 0.80 0.35 0.007 0.39 0.49 0.007 0.76 1.02 0.005 0.06 0.06 3.4 707 509

Yb 0.033 0.032 0.027 0.030 0.023 0.016 0.021 0.019 0.019 0.033 0.020 0.016 0.016 0.016 0.015 17 7.6 11

Lu 0.007 0.016 0.016 0.004 0.017 0.008 0.003 0.008 0.009 0.003 0.011 0.011 0.006 0.005 0.004 2.5 14 14

Tbn Tmn Tbn* 1.0 45 26 1.3 93 72 1.5 59 66 0.7 56 104 8.1 18 25 1.3 500 276

Tmn* 0.8 40 24 1.0 89 70 1.0 61 65 0.5 81 178 0.7 11 7.5 1.2 523 270

0.01 0.003 0.002 0.004 0.004 0.001 0.003 0.001 0.003 0.001 0.06

0.01

0.01

2

0.4

0.3

0.02 0.02 0.005 0.7

0.7

0.2

0.01 0.005 0.4

0.2

0.02 0.004 0.5

0.1



Page 32 of 38

Table 2: Median values for yttrium, terbium and thulium mass fractions and normalized REE/Y ratios (Tbn/Yn, Tmn/Yn) determined by means of LA-ICP-MS in egg shell, meat and bone samples from acclimatization (Acc.), labeling (Lab.) and dilution phase (Dil.). Values for egg shells were acquired in s-lens configuration; Limits of quantitation for meat and bone samples incorporate the average dilution (or actually preconcentration) factors due to sample preparation of 0.25 and 0.01 for bones and meat, respectively. T mn Tb Tm Tbn µg/kg Yn Yn Acc. 1.0 < LOQ < LOQ n/a n/a Egg Lab. 1.0 0.5 0.2 21a 8.4 a shells Dil. 1.1 0.2 0.1 9.5 a 3.9 a Acc. 0.12 0.01 0.005 0.9 0.8 Breast Lab. 0.19 1.00 0.48 53 59 meat Dil. 0.08 0.44 0.21 42 44 Acc. 0.09 0.01 0.004 1.0 1.0 DrumLab. 0.18 0.98 0.43 65 79 stick Dil. 0.10 0.54 0.24 46 51 Acc. 1.5 0.3 < LOQ 1.0 n/a Bone Lab. 1.2 1.8 1.0 7.1 12 b Dil. 1.3 2.0 1.1 6.0 9.6 b shells 0.6 0.1 0.1 LOQ meat 0.008 0.003 0.002 bone 0.2 0.07 0.05 a Ratios calculated based on reference values published for calcium carbonate reference material NIM-GBW07129. b Ratios calculated based on reference values published for calcium phosphate reference material Durango apatite. Y


Page 33 of 38


Figure 1 egg yolk

100

labeling

acclimatization

dilution

Tb Anomaly

75

50

25

0 -20

-10

0

10

20

days

30

40

50

60

70



Page 34 of 38

Figure 2 120

egg shells

Tb Anomaly

100 80 60 40 20 0 16 18 21 24 27 30 33 36 41 46 51 56 61

days


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Figure 3 350

(a)

breast meat

300

SN-ICP-MS LA-ICP-MS

250 Tb Anomaly

drumstick

(b)

SN-ICP-MS LA-ICP-MS

200 150 100 50 0 -3

0

1

2

3

4

5

6

7

8

9

weeks

-3

0

1

2

3

4

5

6

7

8

9



Page 36 of 38

Figure 4 30

bones

Tb Anomaly

25 20 15 10 5 0 -3 0 1 2 3 4 5 6 7 8 9 weeks


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Figure 5

Tb Anomaly

250

(a)

liver

(b)

blood

1000

200

800

150

600

100

400

50

200

0

0 -3 0 1 2 3 4 5 6 7 8 9

-3 0 1 2 3 4 5 6 7 8 9 weeks

(c) feces

0

5

9



Page 38 of 38

Table of contents graphic

Tb

REE normalized

Tm


Rare earth element labeling as a tool for assuring the origin of eggs

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