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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) arcabrita@icbas.up.pt
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Abstract
2
Detailed mineral profile of a selection of commercially available complete dry dog foods was
3
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
12
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
18
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
22
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
32
hemoglobin-Fe, 7% is myoglobin-Fe and free Fe exists in minute quantities.3 Copper is
33
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
42
selenoproteins such as glutathione peroxidase, thioredoxin and iodothyronine 5’-deiodinase,8
43
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
52
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
54
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.
57
Few studies presented data on essential and NEE elements in commercially available dry and
58
wet dog foods.12-16 However, to the best of our knowledge, a detailed characterization of the
59
mineral profile of dog complete foods was not yet published. This study aimed to determine
60
the mineral profile of a selection of complete dry dog foods obtained in Portugal, which is
61
also commercially available in other European countries and USA. Compliance with
62
nutritional requirements of both adults and puppies with legal limits and the presence of
63
potentially toxic elements were evaluated.
64 65
Material and Methods 4 ACS Paragon Plus Environment
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Dry dog food samples
67
Twenty-six complete dry dog food samples (20 for adults and 6 for puppies) from popular
68
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
71
players in the global dog food market. All samples were labeled as complete for adult dogs
72
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-
75
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.
81
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
88
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
93
microwave heating program of 250 W for 1 min, 0 W for 1 min, 250 W for 5 min, 400 W for
94
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
97
ultrapure water was included in each digestion run. Each sample was digested in duplicate.
98
For accuracy check, the certified reference materials DOLT-4 (dogfish liver) and DORM-3
99
(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)
101
were supplied by the Institute for Reference Materials and Measurements (IRMM, Geel,
102
Belgium). z-Score values for determination of Mg, Ca, Na, K, Fe, Mn, Cu, Zn, Se, Cr, Ni, As,
103
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
107
Ground samples were dried for 6 h at 105 °C to express their mineral content in a dry matter
108
(DM) basis using the method 930.15.19 Total ash content was determined gravimetrically
109
according to the method 942.05.19
110
Trace elements were determined by inductively coupled plasma mass spectrometry (ICP-MS)
111
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
115
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
117
solutions were prepared by appropriate dilution of the corresponding AccuTrace Reference
118
Standard (AccuStandard®, New Haven, USA) solutions: ICP-MS-200.8-IS-1 (100 mg L−1 of
119
Sc, Y, In, Tb, and Bi) and ICP-MS-200.8-TUN-1 (100 mg L−1 of Be, Mg, Co, In and Pb).
120
Calibration standards were prepared from 100 mg L−1 multi-element standard solutions: ICP-
121
MS-200.8-CAL1-1 (Isostandards Material, Madrid, Spain), ICP-MS 200.8-CAL2-1
122
(AccuTrace Reference Standard from AccuStandard®), and Plasma CAL Q.C.N.3 (SCP
123
Science, Quebec, Canada).
124
Responses were corrected using
125
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
130
999.10 (Fe) and 975.03 (Mg and Ca).20 Cathode lamps (Perkin Elmer, Überlingen, Germany;
131
SCP Science) were used as a radiation source. The multi-element calibration standards were
132
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.
143
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).
148
Absorbance was measured at 430 nm. Samples were analyzed in duplicate and readings were
149
performed in triplicate.
150 151
Calculations and statistical analysis
152
Whenever the food metabolizable energy (ME) content was not stated in the label, this
153
parameter was calculated based on the modified Atwater factors considering the labeled
154
protein, fat and carbohydrates contents.1 The daily intake of each essential element was
155
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.
158
Statistical analysis was carried out using IBM SPSS Statistics 24 (IBM Corporation, Armonk,
159
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
161
classification in adult (n=20) and puppy (n=6) foods. Samples of adult foods were divided
162
into quartiles considering the market segment: low (n=6), medium (n=3), premium (n=3) and
163
super-premium (n=6). This segmentation was based on the market price per kg of each food,
164
≤ 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),
168
according to the use of meat or meat by-products without specifying the absence of bone-rich
169
meals (e.g. deboned chicken, chicken liver) as a first or second ingredient. The effect of
170
bone-rich meals on ash, Ca, Mg and P content was evaluated by one-way variance analysis
171
considering the dietary inclusion of bone-rich meals as main factor (fixed effect). Individual
172
means were compared using Tukey's post hoc test. Statistical significance was assumed for P
173
< 0.05.
174 175
Results
176
Moisture, energy, ash and mineral contents
177
The descriptive statistics concerning moisture, ME, total ash, macro and trace element
178
contents of the studied foods are presented in Table 1. For adult and puppy foods, moisture
179
content averaged 80 g kg-1, ranging from 69 to 102 g kg-1. Metabolizable energy content
180
averaged 3915 ± 219 and 4136 ± 235 kcal kg-1 DM, respectively for adult and puppy foods.
181
Total ash and quantified minerals were, respectively, 79.8 ± 21.9 g kg-1 DM and 48.0 ± 10.4 g
182
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.
183
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.
207
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
210
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.
211
Thallium, Be and Ag average concentration was < 10 µg kg-1 DM. Similar results were found
212
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
214
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
217
essential mineral elements contents
218
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,
221
Ca, P, and Mg contents were significantly higher in foods with the inclusion of bone-rich
222
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
230
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
238
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
242
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
246
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,
252
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
259
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
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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
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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
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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
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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
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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
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Journal of Agricultural and Food Chemistry
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.
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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)
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Journal of Agricultural and Food Chemistry
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
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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
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0.192 0.537 selenium
0.311
0.860 analysis.
Page 25 of 32
Journal of Agricultural and Food Chemistry
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
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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