Mineral Composition of Dry Dog Foods: Impact on Nutrition and

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Food Safety and Toxicology

Mineral composition of dry dog foods: impact on nutrition and potential toxicity Ana Margarida Pereira, Edgar Pinto, Elisabete Matos, Francisco Castanheira, Agostinho Almiro Almeida, Cláudia Baptista, Marcela A. Segundo, António Mira da Fonseca, and Ana Rita Cabrita J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02552 • Publication Date (Web): 28 Jun 2018 Downloaded from http://pubs.acs.org on June 29, 2018

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Journal of Agricultural and Food Chemistry

Mineral composition of dry dog foods: impact on nutrition and potential toxicity

Ana Margarida Pereira1, Edgar Pinto2, Elisabete Matos3, Francisco Castanheira4, Agostinho A. Almeida2, Claudia S. Baptista5, Marcela A. Segundo2, António J. M. Fonseca1, Ana Rita J. Cabrita1

1

LAQV, REQUIMTE, Instituto de Ciências Biomédicas de Abel Salazar (ICBAS),

Universidade do Porto, Rua de Jorge Viterbo Ferreira nº 228, 4050-313 Porto, Portugal 2

LAQV, REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia,

Universidade do Porto, Rua Jorge Viterbo Ferreira nº 228, 4050-313 Porto, Portugal 3

SORGAL, Sociedade de Óleos e Rações S.A., Estrada Nacional 109 Lugar da Pardala, 3880-

728 S. João Ovar, Portugal 4

Alltechaditivos – Alimentação Animal Lda., Parque de Monserrate – Av. Dr. Luis Sá nº 9 –

Arm. A, 2710-089 Abrunheira, Portugal 5

CECA-ICETA, Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas de

Abel Salazar (ICBAS), Universidade do Porto, Rua Jorge Viterbo Ferreira nº 228, 4050-313 Porto, Portugal

Corresponding author: Ana Rita J. Cabrita Instituto de Ciências Biomédicas de Abel Salazar (ICBAS), Universidade do Porto Rua de Jorge Viterbo Ferreira nº 228, 4050-313 Porto, Portugal +351 220 428 000 (ext. 5366) [email protected]

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Abstract

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Detailed mineral profile of a selection of commercially available complete dry dog foods was

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determined using ICP-MS (Se, Cu, Mn and non-essential trace elements) flame photometry

4

(Na and K), atomic and molecular spectrophotometry (Ca, P, Mg, Zn and Fe). The

5

contribution of ingredients to the mineral composition was correlated to the food market

6

segment. Results showed an oversupply of essential elements due to the energy density effect

7

on feed intake. Additives contributed from 40.8 to 55.1% to the total trace elements contents.

8

With the exception of Se, all trace elements were supplied above the nutritional requirements

9

of adult dogs. Legal limits of Cu, Se and Zn were surpassed. The content of non-essential

10

trace elements included values in the range of nanograms to micrograms per kg, without

11

surpassing safe upper limits. This work brings awareness to the need to find supplementation

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strategies that ensure nutritional adequacy and avoid waste.

13 14

Key-words: Dog food; Essential elements; Legal limits; Non-essential elements; Nutritional

15

requirements.

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Introduction

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Minerals can be classified as essential or non-essential elements (NEE). Essential elements

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are micronutrients that can be found in higher (macroelements) or lower (trace elements)

19

amounts in the body. They are supplied by the raw materials and by additives (food

20

fortification), in order to meet the requirements of animals of a certain age and physiological

21

state.1-2 For dogs, essential macroelements comprise Ca, P, Cl, Mg, K, and Na, and essential

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trace elements include Zn, I, Se, Cu, Mn and Fe. Macroelements have functions on several

23

systems and most often their actions are interlinked. Calcium and P play a structural role,

24

being essential components of the skeleton and involved in the synthesis of structural

25

proteins. Sodium, K and Cl are involved in the maintenance of the acid-base balance,

26

membrane permeability and osmotic control of water distribution within the body.

27

Magnesium has catalytical, electrochemical and structural functions, being, to a lesser extent,

28

also a constituent of the bone tissue, along with Ca and P.

29

Although required in small amounts, trace elements strongly affect animal health, well-being,

30

and performance. Iron is mostly complexed to proteins, either heme (e.g., hemoglobin) or

31

non-heme compounds (e.g., transferrin) and particularly in dogs, 57% of total body Fe is

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hemoglobin-Fe, 7% is myoglobin-Fe and free Fe exists in minute quantities.3 Copper is

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involved in the synthesis of hemoglobin through its interaction with Fe, facilitating its

34

intestinal absorption, release and cellular utilization.4 It also participates in connective tissue

35

formation, free radical removal, as well as hair production and pigmentation.5 Manganese is

36

required for the synthesis of mucopolysaccharides through the polymerase and galacto-

37

transferase enzymes and it is a component of arginase, pyruvate carboxylase and

38

mitochondrial superoxide dismutase.6 Zinc is a constituent of hundreds of metalloenzymes,

39

being involved in several functions, including protein and carbohydrate metabolism, nucleic

40

acid synthesis, cell replication and differentiation stabilization of DNA, RNA and ribosomes,



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immune response, skin function and wound healing.7 Selenium is present in several

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selenoproteins such as glutathione peroxidase, thioredoxin and iodothyronine 5’-deiodinase,8

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being involved in free radical removal and thyroid hormone synthesis. Selenium is also

44

needed for neutrophils, macrophages, NK cells, and T lymphocytes functioning, affecting

45

immune responses.9

46

In the European Union (EU), the trace elements added for fortification must be claimed in the

47

label.10 In addition, due to their toxicity for both animals and the environment, a maximum

48

level in feed is legally established and it applies to all life stages. Mineral supplementation

49

should take into account the elements supplied by the raw materials to ensure animal

50

requirements without surpassing the legal limits, which can be found in the FEDIAF

51

publication.2 Authorized feed additives including trace elements and specific EU directives

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that set their maximum content in feed are listed in Annex I of the European Union Register

53

of Feed Additives pursuant to Regulation (EC) No 1831/2003.11 Non-essential elements were

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not proven to have a metabolic role in the body, not necessarily due to the absence of

55

functions, but probably due to the lack of knowledge of their action in some species. If

56

supplied above safe levels, NEE may constitute a variable risk for animal health.

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Few studies presented data on essential and NEE elements in commercially available dry and

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wet dog foods.12-16 However, to the best of our knowledge, a detailed characterization of the

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mineral profile of dog complete foods was not yet published. This study aimed to determine

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the mineral profile of a selection of complete dry dog foods obtained in Portugal, which is

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also commercially available in other European countries and USA. Compliance with

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nutritional requirements of both adults and puppies with legal limits and the presence of

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potentially toxic elements were evaluated.

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Material and Methods 4 ACS Paragon Plus Environment

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Dry dog food samples

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Twenty-six complete dry dog food samples (20 for adults and 6 for puppies) from popular

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international brands and different market segments (low, medium, premium and super

69

premium) were selected. The samples were acquired in supermarkets (n=12), assuming a high

70

volume of sales and at veterinary clinics and specialized stores (n=14), representing leader

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players in the global dog food market. All samples were labeled as complete for adult dogs

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and included a range of main flavors (e.g., cereals, chicken, fish). All samples were packaged

73

in sealed bags. After opening, the samples were ground to pass through a 1 mm sieve and

74

stored in plastic containers. Although not exhaustive, the selected samples provide a snap-

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shot of European dry dog foods from different market segments.

76 77

Reagents and labware

78

Ultrapure water (18.2 MΩ cm) used in all experiments was obtained from a Sartorius Arium®

79

water purification system (Goettingen, Germany). All chemicals were of analytical reagent

80

grade and purchased from Sigma Aldrich (St. Louis MO, USA) unless otherwise stated.

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Sample digestions were performed using high-purity HNO3 (≥ 69% (w/w), TraceSELECT®

82

(Fluka, Seelze, Germany) and H2O2 (30% (v/v), TraceSELECT® Fluka) of p.a. grade. All

83

plastic ware used in sample digestion and elemental analysis were immersed for, at least, 24 h

84

in a 10% (v/v) HNO3 solution to ensure decontamination and then rinsed with ultrapure

85

water.

86 87

Digestion and analytical quality control

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Ground samples (c.a. 500 mg) and certified reference materials were solubilized by

89

microwave-assisted acid digestion using an MLS 1200 Mega high-performance microwave

90

digestion unit (Milestone, Sorisole, Italy) equipped with an HPR-1000/10 S rotor. After



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weighing the sample using a plastic spatula, 3 mL of HNO3 and 1 mL of H2O2 were added to

92

each polytetrafluoroethylene digestion vessel. The sample was subsequently submitted to a

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microwave heating program of 250 W for 1 min, 0 W for 1 min, 250 W for 5 min, 400 W for

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5 min and, finally, 650 W for 5 min.17 Vessels were then allowed to cool to room

95

temperature. Thereafter, the content was transferred to 25 mL polypropylene volumetric

96

flasks and water was added to bring up to total volume. A blank constituted by 500 µL of

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ultrapure water was included in each digestion run. Each sample was digested in duplicate.

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For accuracy check, the certified reference materials DOLT-4 (dogfish liver) and DORM-3

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(fish protein) were supplied by the National Research Council of Canada (CNRC, Ottawa,

100

Canada) while ERM®-BD151 (skimmed milk powder) and ERM®-BB422 (fish muscle)

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were supplied by the Institute for Reference Materials and Measurements (IRMM, Geel,

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Belgium). z-Score values for determination of Mg, Ca, Na, K, Fe, Mn, Cu, Zn, Se, Cr, Ni, As,

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Ag, Cd, Sn, and Pb were < 2 (Supplementary Material, Tables S1, and S2), showing that the

104

applied methods performed satisfactorily.18

105 106

Ash and elemental analysis

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Ground samples were dried for 6 h at 105 °C to express their mineral content in a dry matter

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(DM) basis using the method 930.15.19 Total ash content was determined gravimetrically

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according to the method 942.05.19

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Trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS)

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using an iCAP Q™ (Thermo Fisher Scientific, Schwerte, Germany) instrument, equipped

112

with a MicroMist™ nebulizer, a Peltier cooled cyclonic spray chamber, a standard quartz

113

torch and nickel skimmer and sampling cones. High purity (99.9997%) Ar (Gasin II, Leça da

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Palmeira, Portugal) was used as the nebulizer and plasma gas. The ICP-MS operated under

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the following conditions: RF power 1550 W; auxiliary Ar flow rate 0.80 L min-1; nebulizer 6 ACS Paragon Plus Environment

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flow rate 1.08 L min-1 and plasma flow rate 14 L min-1. Internal standards and tuning

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solutions were prepared by appropriate dilution of the corresponding AccuTrace Reference

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Standard (AccuStandard®, New Haven, USA) solutions: ICP-MS-200.8-IS-1 (100 mg L−1 of

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Sc, Y, In, Tb, and Bi) and ICP-MS-200.8-TUN-1 (100 mg L−1 of Be, Mg, Co, In and Pb).

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Calibration standards were prepared from 100 mg L−1 multi-element standard solutions: ICP-

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MS-200.8-CAL1-1 (Isostandards Material, Madrid, Spain), ICP-MS 200.8-CAL2-1

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(AccuTrace Reference Standard from AccuStandard®), and Plasma CAL Q.C.N.3 (SCP

123

Science, Quebec, Canada).

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Responses were corrected using

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isotopes of each element were used in the analysis: 7Li, 9Be, 27Al, 51V, 52Cr, 55Mn, 59Co, 60Ni,

126

65

127

238

128

Iron, Mg and Ca were determined using an AAnalyst 200 flame (air-acetylene) atomic

129

absorption spectrometer (Perkin Elmer, Überlingen, Germany) according to the method

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999.10 (Fe) and 975.03 (Mg and Ca).20 Cathode lamps (Perkin Elmer, Überlingen, Germany;

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SCP Science) were used as a radiation source. The multi-element calibration standards were

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prepared from 1000 mg L-1 standard solutions from each target element. Lanthanum chloride

133

solution at 0.1% (w/v) was used in the determination of Ca and Mg to eliminate chemical

134

interferences.

135

Sodium and K in food are conventionally determined by flame (butane) atomic emission

136

photometry, method 963.2321. In these work, Na and K were determined in the samples

137

solutions using a Jenway model PFP-7 (Buck Scientific, Norwalk, USA) flame photometer

138

operated under the manufacturer recommended operating conditions. The intensity of the

139

atomic emission was recorded at 589 nm and 766 nm, respectively. Cesium chloride solution

140

at 0.1% (w/v) was used to eliminate chemical interferences. External calibration with Na and

Cu,

66

Zn,

75

As,

82

Se,

85

Rb,

88

45

Sc,

Sr,

95

89

Y,

Mo,

129

107

Tb and

Ag,

111

115

Cd,

In internal standards. The following

118

Sn,

121

Sb,

137

Ba,

205

Tl,

208

Pb and

U.17, 20



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K standards solutions prepared from Sodium and Potassium Standards for AAS,

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TraceCERT®, 1000 mg L-1.

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Phosphorus was determined by spectrophotometry using the molybdovanadate reagent under

144

a microplate format, adapted from method 965.17.20 The sample solutions were allowed to

145

react with the molybdovanadate for 45 min at 25 °C. The spectrophotometric determination

146

was performed in a monochromator-based microplate reader (Cytation™ 3, Bio-Tek

147

Instruments, Vermont, USA) controlled by Gen 5™ software (Bio-Tek Instruments).

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Absorbance was measured at 430 nm. Samples were analyzed in duplicate and readings were

149

performed in triplicate.

150 151

Calculations and statistical analysis

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Whenever the food metabolizable energy (ME) content was not stated in the label, this

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parameter was calculated based on the modified Atwater factors considering the labeled

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protein, fat and carbohydrates contents.1 The daily intake of each essential element was

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calculated based on the recommended food daily allowance and the content of the analyzed

156

element found in the product label. The proportion of total Fe, Cu, Mn, Zn, and Se from

157

additives was calculated using the information provided in the label whenever available.

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Statistical analysis was carried out using IBM SPSS Statistics 24 (IBM Corporation, Armonk,

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NY, USA). Simple regression analysis was performed between ash and total identified

160

mineral content. For descriptive statistics, samples were divided according to the life stage

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classification in adult (n=20) and puppy (n=6) foods. Samples of adult foods were divided

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into quartiles considering the market segment: low (n=6), medium (n=3), premium (n=3) and

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super-premium (n=6). This segmentation was based on the market price per kg of each food,

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≤ 1.20 €, ≤, 4.07 €, ≤ 5.30 € and > 5.30 €, respectively, for the first, second, third and fourth

165

quartile. The effect of the market segment on ME and mineral contents was evaluated by one-


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way variance analysis considering the quartile as main factor (fixed effect). Additionally,

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foods were classified as ‘high bone content’ (n=12) or ‘no/low bone content’ (n=14),

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according to the use of meat or meat by-products without specifying the absence of bone-rich

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meals (e.g. deboned chicken, chicken liver) as a first or second ingredient. The effect of

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bone-rich meals on ash, Ca, Mg and P content was evaluated by one-way variance analysis

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considering the dietary inclusion of bone-rich meals as main factor (fixed effect). Individual

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means were compared using Tukey's post hoc test. Statistical significance was assumed for P

173

< 0.05.

174 175

Results

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Moisture, energy, ash and mineral contents

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The descriptive statistics concerning moisture, ME, total ash, macro and trace element

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contents of the studied foods are presented in Table 1. For adult and puppy foods, moisture

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content averaged 80 g kg-1, ranging from 69 to 102 g kg-1. Metabolizable energy content

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averaged 3915 ± 219 and 4136 ± 235 kcal kg-1 DM, respectively for adult and puppy foods.

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Total ash and quantified minerals were, respectively, 79.8 ± 21.9 g kg-1 DM and 48.0 ± 10.4 g

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kg-1 DM in adult foods and 80.2 ± 2.0 g kg-1 DM and 42.1 ± 11.5 g kg-1 DM in puppy foods.

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Total ash content was significantly correlated with total quantified minerals (r = 0.931, P
50% of

202

analyzed samples, respectively (Figure 1). Selenium was above the legal limit in about 50%

203

of foods, and the maximum value found was 5 times the higher permitted (Figure 1).

204

The trace elements labeled as additives contributed to 40.8 to 55.1% of the total herein

205

determined, the remaining being supplied by the raw materials (Figure 2). However, a large

206

amplitude was observed for all elements.

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The descriptive statistics concerning NEE contents are presented in Table 2. Strontium was

208

the NEE found in the highest concentration, followed by Ba and Rb in both adult and puppy

209

foods. In adult foods, the average content of As, V, Mo, Cr, Li, Pb, Cd, Co and Ni was within

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0.1 to 1 mg kg-1 DM, while U, Sb and Sn had an average content of 0.01 to 0.1 mg kg-1 DM.

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Thallium, Be and Ag average concentration was < 10 µg kg-1 DM. Similar results were found

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in puppy foods with the exception of average Be and Cr contents that were lower (0.023 mg

213

kg-1 DM) and higher (1.50 mg kg-1 DM), respectively. Among NEE, Li, Sn, Sb and Pb

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contents showed the largest variation (RSD > 75%) both in adult and puppy foods.

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Effect of the market segment and dietary inclusion of animal by-products on energy and on

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essential mineral elements contents

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Results in Table 3 show that market segment significantly affected ME, Ca, P, Mg and K but

219

not Na and the trace element contents. Calcium, P and Mg were higher in low segment

220

markets, while ME and K are higher in premium and super premium foods, respectively. Ash,

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Ca, P, and Mg contents were significantly higher in foods with the inclusion of bone-rich

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meals (Table 4).

223 224

Daily intake of essential elements

225

The daily intake of macro and trace elements relative to NRC recommendations

226

presented in Figure 3. All adult foods supplied macroelements above the nutritional

227

requirements and 50% of them supplied > 400% of Ca, > 350% of P, > 750% of Na, > 200%

228

of Mg and > 150% of K. Regarding trace elements, with the exception of Se (that was

229

supplied below the nutritional requirements in 25% of the foods), all trace elements were

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oversupplied to adult dogs (50% of analyzed foods supplied > 600% of Fe, > 3000% of Cu, >

231

1500% of Mn and > 450% of Zn).

1

are

232 233

Discussion

234

Energy content

235

The ME content of puppy foods is higher than that of adult’s in order to meet puppies’ higher

236

energy requirements for growth and development.1 If puppies are fed with low energy and

237

low digestibility diets, a large amount of food is required to meet their nutritional needs. The

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energy content of foods from the low market segment is significantly lower compared to

239

premium segments. This implies a larger daily food allowance to ensure the energy

240

requirements of the animals, most probably accompanied by a larger intake of nutrients,



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including essential elements. To avoid this oversupply of essential elements, mineral

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supplementation should be performed according to nutrient requirements expressed in ME

243

basis instead of DM, therefore eliminating the effect of energy density on mineral allowance.1

244 245

Essential macroelements

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In the analyzed foods, Ca and P were the elements found at higher concentrations followed

247

by K, Na, and Mg, agreeing with earlier results of Alomar et al.22 In the studied adult foods,

248

the daily intake of Ca was always above recommendations, ranging from 199 to 1206% of the

249

NRC recommended values. High-Ca intake is correlated with high-Ca serum, due to Ca

250

intestinal passive absorption.23 Excess Ca lowers the activity of the parathyroid gland and

251

causes osseous lesions (e.g., decreased bone length, osteoporosis of the long bones,

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metaphyseal flaring),24 with greater severity in large breed juveniles.25 Although the effects

253

of an excessive intake of Ca in growing dogs are well known, the long-term effect on adult

254

dogs is weakly established. Recently, Stockman et al.26 suggested that adult dogs seem to be

255

capable of tolerating high-dietary Ca levels of 7.1 g per 1000 kcal up to 40 weeks. Applying

256

the conversion factors proposed by FEDIAF,2 it equals to 28.4 g kg-1, which is below the

257

content of three of the herein analyzed foods.

258

Among the studied macroelements, Na registered the highest daily allowance. There is a

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paucity of information concerning Na excess and deficiency in dogs, but high-salt intake

260

(0.14 mg kg-1 body weight, BW) in adult dogs has been related with increased mesenteric

261

venoconstriction, which can contribute to hypertension.27 An excess of Na may also impair

262

the Ca-homeostasis by increasing its renal clearance.28 The Na levels of the analyzed foods

263

were below the amount suggested to affect Ca homeostasis (< 12 g kg-1),28 and below the safe

264

upper limit established by NRC (1.5 mg kg-1).1


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The daily allowance of Mg and K was above the nutritional requirements in adult foods. The

266

scarce studies on the effects of increased serum Mg levels (hypermagnesemia) suggest a low

267

medical risk of an excessive Mg intake.29 However, as Mg is mainly excreted in urine, it is

268

recommended to avoid Mg over-supplementation in dogs with renal failure. The average

269

content of Mg in the analyzed foods was below the considered safe dose for dogs (1.7 mg kg-

270

1 1

271

elevation of blood K (hyperkalemia) might be life-threatening due to the risk of cardiac

272

arrhythmias.30 Therefore, over-supplementation of K should be avoided, particularly in dogs

273

with cardiac diseases, chronic kidney disease or other medical conditions prone to develop

274

hyperkalemia such as hypoadrenocorticism.

275

Ash, Ca, P and Mg contents were related to the market segment in a significant way, with low

276

market segment foods having the highest amounts, probably reflecting the use of low-grade

277

animal by-product meals. Indeed, the use of bone-rich ingredients as the main or second

278

ingredient significantly increased Ca, P and Mg contents. Conversely, super premium foods

279

presented the lowest contents of Ca, P and Mg and the highest K levels. This may be due to

280

the use of fresh meats, fruits and vegetables such as sweet potato, carrot, spinach, and apple,

281

that have a low content of Ca, Mg and P, and high content of K.31 The use of low-grade

282

ingredients may have negative impact over and beyond the ones highlighted in this work.

283

One example is the presence of antibiotics residues in chicken bones, incorporated as bone

284

meals in pet formulas, that can exert toxic, pro-inflammatory, and pro-apoptotic effects in the

285

animal.32

). Unlike Mg, a safe upper limit for K content in dog foods is not established. However, the

286 287

Essential trace elements

288

Trace element contents of dog foods reported in the literature show a wide range. Median and

289

maximum contents of Fe and Zn in the analyzed foods were above the values reported for 13 ACS Paragon Plus Environment


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commercial dog foods available in the USA (Fe: 89 and 220 mg kg-1 DM; Zn: 140 and 330

291

mg kg-1 DM) 14, but within the range of commercial dog foods for puppy, adult and seniors

292

available in Brazil (Fe: 188 – 646 mg kg-1 DM; Zn: 44 – 633 mg kg-1 DM).13 Maximum

293

contents of Cu and Mn were above those reported for commercial dog foods available in the

294

USA, (8.34 and 70 mg kg-1 DM, respectively)

295

respectively).12,

296

reported values in New Zealand (< 0.4 mg kg-1 DM),15 and in the USA (0.44 mg kg-1 DM).14

297

The Fe daily allowance was above the NRC1 recommendations in the analyzed adult foods.

298

Due to the absence of a physiologic pathway for Fe-excess excretion, the regulation of its

299

absorption is crucial to respond to high dietary content.33 However, if mechanisms of

300

regulation of Fe absorption fail, toxicosis might appear. Mild clinical signs occur when Fe

301

intake is between 20 to 60 mg kg BW-1 and include vomiting, diarrhea, and gastrointestinal

302

bleeding.34 Based on this data, a 10 kg-adult dog would need to consume from 270 to 810 g

303

of the studied food with the highest Fe content (741 mg kg-1) to present signs of toxicosis,

304

which is above the recommended food allowance for this sample (140 g). It suggests that, in

305

this particular case, the risk of toxicosis increases as consequence of overfeeding.

306

The daily allowance of Cu exceeded the NRC recommendations in adult foods.1 A

307

continuous excessive intake of Cu may contribute to the hepatic accumulation of Cu over the

308

years, which increases the risk of development of chronic hepatitis and cirrhosis in middle to

309

old-aged dogs.35

310

In all the analyzed foods, Mn was supplied above the NRC recommendations.1 There are few

311

studies regarding Mn toxicity for dogs,1 thus conclusions cannot be drawn over these results.

312

In all adult foods, Zn was oversupplied relative to NRC (60 mg kg-1 DM)1 and AAFCO (80

313

mg kg-1 DM)36 recommendations. The AAFCO recommendation takes into account the

14

14

and in Turkey (18 and 12.5 mg kg-1 DM,

Selenium content in the analyzed foods was around 20% above the


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interactions of Zn with other dietary constituents, such as Ca, Fe, Cu, Cd and phytate, that

315

reduce Zn intestinal uptake by different mechanisms.37

316

Selenium was below the NRC recommendations1 in 5 adult foods. The remaining were

317

above the nutritional requirements, with 6 adult foods close to or even doubling the NRC

318

recommendations.1 Adult dogs seem to tolerate an intake of Se 22 to 28 times above the

319

nutritional requirements, showing no other clinical signs than weight loss.38 In turn, low

320

dietary Se induces abnormalities in adult hair growth,39 reduces the serum levels of puppies’

321

and thyroid hormones.40

322

Overall, a large variability in trace element allowance was found in the analyzed foods. This

323

dispersity is partially due to the energy density of the foods. Since the feeding plan is

324

determined by ME requirements, the optimum allowance of certain micronutrients may not

325

be ensured.

326

Contrarily to macroelements, trace elements were not affected by market segment. This is

327

probably due to the narrow window imposed by the legal maximum, which limits the total

328

trace element content. Additives contributed from 40 to 55% to the total element content in

329

foods, being the remaining supplied by the raw materials. This suggests that additives of the

330

elements found above the legal limits (Cu, Zn and Se) should be lowered. However, that

331

might be a foregone conclusion. On one hand, the content of trace elements in the ingredients

332

is affected by several factors such as soils and regions. On the other hand, the determinations

333

performed in this study only provided information on the total content and not of the form of

334

the element. So, in order to adjust the additives in the formula, it would be important to run

335

regular food analyses including speciation as a way to estimate the bioavailability of the

336

elements of the raw materials.41 Additionally, the use of more bioavailable forms (e.g.,

337

organic trace elements, such as metal amino acid complexes or proteinates) would constitute



Page 16 of 32

338

a step ahead by assuring animal requirements with a lower amount of additives, complying

339

with the maximum legal limits.

340 341

Non-essential elements

342

Despite the elements described throughout this section being classified as NEE, it is not

343

implicit that they are always toxic neither is dismissed the possibility of positive effects on

344

body function. The harm of an element may be related to the exceeding maximum tolerable

345

levels (MTL), which should, ideally, be species specific. The content of the analyzed dry dog

346

food samples is in accordance with previous reports12-14 with a few exceptions as discussed

347

below.

348

Strontium was the NEE present in the highest amount in both adult and puppy foods. It is

349

deposited mainly in bone and teeth.42 Despite not being considered essential, Sr may have

350

positive effects on bone mineralization, but it was also related to toxic effects, which are

351

dependent on the dose.43 Strontium is mainly sourced by ingredients derived from plants44 but

352

may also be related with the use of bone meals.45 Generally, Sr toxicity is low,46 and no MTL

353

has been reported for dogs. According to NRC,46 Ba and Rb are NEE for dogs, and their

354

concern for animal health is low.

355

Nickel content in animal foods was recently reviewed by EFSA47 and 18 mg kg BW–1 was

356

established as the no observed adverse effect level (NOAEL) while 45 mg kg BW–1 was the

357

level associated with adverse effects (vomiting, polyuria, lung lesions and granulocytic

358

hyperplasia of the bone marrow). For dog foods containing hydrogenated vegetable oils (Ni

359

catalysis used to increase oil stability), an upper Ni dietary concentration of 2.65 mg kg-1 was

360

estimated,47 which is 2-fold the maximum found in the analyzed foods. However, results of

361

Duran et al.

362

times higher the values herein obtained and above the EFSA estimates.

12

reported levels of Ni ranging from 8.10 to 19.7 mg kg-1 DM, more than 10


Page 17 of 32


363

There is no clear evidence of Cr essentiality for animals, since deficiency symptoms were not

364

demonstrated under experimental conditions.48 A MTL for dietary Cr is not established,

365

though NRC46 refers a value of 100 mg kg-1 for mammalian species concerning the more

366

soluble Cr(III), which is far above the maximum found in the analyzed foods.

367

Molybdenum is a cofactor of several enzymes (e.g., sulfite oxidase, xanthine dehydrogenase,

368

aldehyde oxidase) and participates in sulfite excess detoxification, purine catabolism, reactive

369

oxygen production, and aldehyde oxidation.49 Molybdenum serum levels are most probably

370

determined by dietary intake, but neither dietary deficiency nor MTLs was yet reported in

371

dogs.1

372

Data on V in dog nutrition is lacking and an MTL for dogs has not been established.

373

According to NRC,46 V may have a role in the body and its concern for animal health is high.

374

Arsenic has a variable toxicity according to its chemical form. The presence of Ar species in

375

dog food is likely when it includes animal tissues, such as the liver.50 In the study of Byron et

376

al.,51 6-month-dogs fed with less than 50 mg As kg

377

and 17.5 mg kg-1 of sodium arsenite and sodium arsenate, respectively, have survived without

378

clinical or post-mortem effects.51 In another study, 7 to 8-month beagles were initially fed

379

with 0, 1, 2 and 4 mg kg-1 BW of sodium arsenite (containing 0.57, 1.14 and 2.28 mg kg-1 BW

380

of As) for 58 days, the doses being doubled after that period for another 125 days.52 Effects

381

on food consumption were dose-dependent, with higher doses depressing food consumption,

382

thus promoting weight body loss and changes in liver function.52 According to these studies,

383

the As content found in the analyzed foods seems to be of no concern, since the maximum

384

measured was 0.69 mg kg-1, slightly above the minimum dose tested by Neiger and

385

colleagues52. In addition, the As level is below the one reported in pet food available in the

386

Italian market from 2007 to 2012 (min: 0.86 mg kg-1; max: 12.5 mg kg-1).53

-1

of diet, corresponding to 28.5 mg kg-1



Page 18 of 32

387

Lithium is considered of low concern and is, sometimes, added to animal feed or water as

388

therapeutics.46 Besides its use as a mood-stabilizer drug in humans, it is considered a

389

hematopoietic stimulant in dogs.54 Lithium carbonate was orally administered at

390

concentrations ranging from 14 – 16 mg kg-1, corresponding to approximately 1.4 to 1.6 mg

391

kg-1 of Li,54 and at 12.24 mg kg-1, corresponding to approximately 1.2 mg kg-1 of Li 55 and no

392

side effects were reported. In the analyzed foods, Li was not intentionally added, being

393

supplied by the raw materials in concentrations that, according to previous studies, do not

394

constitute a risk for the animals.

395

Lead is an environmental contaminant mainly sourced from anthropogenic activities. Lead

396

nephrotoxicity after oral exposure was proved in humans and several animals, including

397

dogs.56 Levels of Pb in the analyzed foods were 10 times below those reported by Duran

398

(5.04 to 15.5 mg kg-1 DM),12 but above the ones reported by Kelly (0.320 mg kg-1 DM).14 A

399

recent review on the risk assessment of lead intoxication in dogs suggests that 1 mg kg BW-1

400

of lead acetate (corresponding to approximately 0.63 mg kg BW-1 of Pb) is the lowest

401

observed effect level (LOEL), while a single dose of 300 mg kg BW-1 (approximately 191 mg

402

kg BW-1 Pb) might be lethal.57 Considering an adult dog weighing 15 kg fed with the studied

403

food with the highest Pb content (0.84 mg kg-1), the Pb intake would be 0.17 ± 0.01 mg, 55

404

times below the LOEL (9.45 mg).

405

Cadmium is not an essential element for animals, and it is found as a contaminant from both

406

natural and anthropogenic activities. Grains contribute a significant proportion of dietary Cd,

407

the highest amount being found in the endosperm.

408

organs and impairs the homeostasis of other elements such as Zn, Fe and Ca.59 Average Cd

409

levels were found above the values reported by Kelly (0.67 mg kg-1 DM).14 Data on Cd

410

toxicity in dogs is scarce, but the maximum found in the analyzed foods is lower than the

411

MTL of 10 mg kg-1 established for other species. 46

58

Excessive Cd intake affects several


Page 19 of 32


412

Cobalt(III) is the central atom of vitamin B12, being essential for animals that can synthesize

413

this vitamin, such as horses and ruminants.60 Dogs lack the ability to produce vitamin B12 in

414

the gastrointestinal tract, making it indispensable to meet nutritional requirements of this

415

vitamin rather than Co. There is not an MTL for Co in dog foods but the content found in the

416

studied foods was below the legal limit of 1.12 mg kg-1 DM.

417

Uranium is toxic to the kidney, reproductive system and respiratory tract. The range of U

418

content in the analyzed samples was lower than that reported by Elis et al. (0.46 – 3.99 mg

419

kg-1 DM).13 NRC suggested an MTL for U in diets of 100 for rodents and < 100 mg kg-1 for

420

fish, with no information concerning dogs.46 Thus the U content in the analyzed samples

421

appears not to constitute a risk for dogs since the maximum obtained (1.72 mg kg-1 DM) is

422

almost 100 times below the lower MTL for other species.

423

The average contents of Sb, Sn, Be, Tl and Ag were lower than 0.05 mg kg-1 DM both for

424

adult and puppy foods. Data on these elements in dog nutrition and toxicology is lacking and

425

MTLs have not been established for dogs. According to NRC, none of these elements are

426

required for animals and their concern for animal health is low.46

427

This study presented a detailed characterization of the mineral profile of a selection of

428

complete dry dog foods commercially available in Europe. The results highlight the

429

importance to know the content of essential elements (run regular feed analysis) of raw

430

materials before planning the supplementation, bringing awareness to the need to find

431

fortification strategies, that simultaneously ensure animal requirements and avoid waste.

432



Page 20 of 32

Abbreviations BW – Body Weight DM – Dry Matter EU - European Union ICP-MS – Inductively Coupled Plasma Mass Spectroscopy LOEL - Lowest Observed Effect Level ME - Metabolizable Energy MTL - Maximum Tolerable Level NEE - Non-Essential Elements NOAEL - No Observed Adverse Effect Level RSD - Relative Standard Deviation


Page 21 of 32


Funding This work was financed by Project MinDog, funded by Portugal 2020, financed by the European Regional Development Fund (FEDER) through the Operational Competitiveness Program (COMPETE) - reference number 017616. Financial support from FEDER funds POCI/01/0145/FEDER/007265 and National Funds (FCT/MEC) under the Partnership Agreement PT2020 UID/QUI/50006/2013 is also acknowledged. AM Pereira also thanks FCT, SANFEED Doctoral Programme, Soja de Portugal and Alltech for her Ph.D. grant PD/BDE/114427/2016.



Page 22 of 32

Figure 1. Distribution of trace element content as a percentage of the EU legal maximum (dashed line) from directives listed in Annex I of EC No 1831/200311 and transcribed to FEDIAF recommendations2 (median, minimum, maximum, 25 and 75 % percentiles)

Figure 2. Percentage of total trace element content sourced by the labeled additives (grey area): A – minimum; B – median and C – maximum in the analyzed foods. Iron, Cu and Mn were not added to 4, 1 and 2 samples, respectively (content was not declared in the label)


Page 23 of 32


Figure 3. Daily intake of macroelements (A) and trace elements (B) relative to NRC nutritional recommendations for adult dogs 1 (dashed line). Median, minimum, maximum and percentiles (25 and 75%) are represented for all elements except for Cu which were 3209, 570, 6119, 2707 and 3834%, respectively



Page 24 of 32

Table 1. Descriptive statistics concerning moisture (g kg-1), metabolizable energy (ME, kcal kg-1 dry matter, DM), total ash (g kg-1 DM), total quantified minerals (g kg-1 DM), and essential macro (g kg-1 DM) and trace elements (mg kg-1 DM) contents of the studied commercial dry complete dog foods Adults (n=20)*

Mean

s.d.

Median

Minimum

Maximum

Moisture

79.9

9.1

76.2

70.1

ME

3915

219

3928

Total ash

79.8

22.0

Total minerals

48.0

10.4

Puppies (n=6)

Mean

s.d.

Median

Minimum

Maximum

102

Moisture

77.4

5.3

78.7

69.4

82.4

3624

4278

ME

4136

235

4065

3878

4536

71.3

50.8

116

Total ash

80.2

20.1

74.6

57.2

116

46.7

33.0

71.6

Total minerals

42.1

11.5

37.5

29.1

59.5

Macroelements

Macroelements

Ca P

19.4 11.7

7.5 3.1

17.9 11.8

10.1 6.76

34.8 18.5

Ca P

16.6 11.0

7.4 2.8

13.7 9.81

9.12 8.14

27.8 14.6

Ca:P ratio

1.64

0.35

1.51

0.938

2.42

Ca:P ratio

1.45

0.29

1.42

1.12

1.90

Na

6.90

2.03

6.51

3.02

11.0

Na

5.50

2.07

5.81

2.45

8.24

Mg

1.53

0.45

1.39

0.93

2.38

Mg

1.21

0.41

1.13

0.683

1.91

K

7.65

2.31

6.97

5.17

17.7

K

7.00

1.36

7.19

5.02

8.71

Trace elements

Trace elements

Fe

270

160

228

51.8

741

Fe

313

140

308

80.4

478

Cu

22.8

8.6

21.1

11.0

47.0

Cu

22.1

5.1

20.2

15.0

30.2

Mn

79.8

32.2

81.1

37.6

180

Mn

70.4

31.1

77.1

29.9

103

Zn

325

93.5

310

182

566

Zn

276

27.4

268

248

317

Se * n

0.586

0.235

0.519

0.317 19

1.19

Se

0.539

=

for

24


0.192 0.537 selenium

0.311

0.860 analysis.

Page 25 of 32


Table 2. Descriptive statistics concerning non-essential trace elements (mg kg-1 DM) contents of the studied commercial dry complete foods Adult (n=20) Mean

s.d.

Median

Minimum Maximum

(Puppy n=6) Mean

s.d.

Median

Minimum Maximum

Li

0.252 0.205

0.183

0.036

0.909

Li

0.193

0.137 0.159

0.079

0.433

Be

0.009 0.007

0.008

0.001

0.025

Be

0.023

0.027 0.008

0.002

0.059

V

0.358 0.248

0.301

0.121

1.021

V

0.828

0.753 0.648

0.135

2.09

Cr

0.768 0.323

0.680

0.413

1.594

Cr

1.50

1.24

0.830

0.506

3.36

Co

0.147 0.078

0.134

0.050

0.321

Co

0.128

0.047 0.126

0.066

0.199

Ni

0.816 0.322

0.739

0.338

1.325

Ni

0.703

0.296 0.685

0.351

1.14

As

0.318 0.473

0.088

0.009

1.624

As

0.199

0.242 0.114

0.055

0.689

Rb

4.41

1.90

4.15

1.34

10.4

Rb

4.06

1.61

4.19

1.27

6.02

Sr

29.3 21.62

18.0

9.30

78.0

Sr

23.5

19.1

18.1

7.28

59.7

Mo

0.492 0.158

0.446

0.345

1.057

Mo

0.409

0.067 0.398

0.327

0.507

Ag

0.007 0.010

0.003

0.002

0.041

Ag

0.005

0.003 0.004

0.001

0.008

Cd

0.151 0.284

0.070

0.039

1.324

Cd

0.099

0.059 0.073

0.043

0.187

Sn

0.037 0.034

0.027

0.010

0.161

Sn

0.042

0.023 0.034

0.020

0.085

Sb

0.049 0.042

0.0312

0.080

0.17

Sb

0.048

0.066 0.023

0.010

0.181

3.42

6.28

2.69

14.0

Ba

7.26

4.41

2.17

11.5

Tl

0.009 0.010

0.004

0.001

0.031

Tl

0.008

0.008 0.007

0.001

0.023

Pb

0.185 0.179

0.124

0.041

0.840

Pb

0.122

0.053 0.119

0.053

0.201

U

0.062 0.107

0.031

0.005

0.495

U

0.468

0.624 0.197

0.020

1.60

Ba

7.13

7.75

25



Page 26 of 32

Table 3. Effect of market segment of adult commercial dry complete foods on metabolizable energy (ME, kcal kg-1 DM), essential macro (g kg-1 DM) and trace elements (mg kg-1 DM) contents. Means with different superscripts are significantly different (P < 0.05) Low n ME

Medium Premium Super Premium

6

5

3

6

3694a

3966a,b

4070b

4019a,b

a

b

b

SEM

P

76.5

0.013

1.92

Mineral Composition of Dry Dog Foods: Impact on Nutrition and

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