Presence of 31 mycotoxins in pig feed in Spain

PRESENCE OF 31 MYCOTOXINS IN PIG FEED IN SPAIN

L. Gámiz-Gracia1, N. Arroyo-Manzanares2, V. Rodríguez-Estévez3 and A.M. García-Campaña1 Department of Analytical Chemistry, Faculty of Sciences, University of Granada.

1

Department of Analytical Chemistry, Faculty of Chemistry, University of Murcia, Spain.

2

Department of Animal Production, Faculty of Veterinary Medicine, University of Cordoba.

3

1

Mycotoxins are fungal toxins produced

According to the Food and Agriculture

by several hundred species of molds that

Organization of the United Nations

can grow on crops or food under certain

(FAO), it is estimated that 25% of cereal

conditions. The most important are

production and 20% of plant production

those produced by molds of the genera

worldwide are affected by mycotoxins,

Aspergillus, Fusarium and Penicillium.

although recent studies consider that this figure may be underestimated2.

In addition, the effects of climate change are expected to increase the production of toxigenic Mycotoxins have become one of the

fungi in the coming years3.

most reported contaminants worldwide, a fact that is reflected in the latest report from the EU Rapid Alert System for Food and Feed (RASFF)1.

It should be noted that, despite the current state of knowledge and improvements in production and storage practices, it has not been possible to eradicate the development of fungi and molds and, therefore, the presence of mycotoxins in a large number of foods.

2

Despite the wide variety of known mycotoxins with various toxicological effects, the EU has set maximum permitted or recommended levels for only 14 of them (aflatoxins B1,

Among farm animals, pigs are

B2, G1, G2 and M1, ochratoxin A, patulin,

considered to be one of the

deoxynivalenol, zearalenone, fumonisins

most vulnerable species to

B1 and B2, T-2 and HT-2 toxins, citrinin) as

the effects of mycotoxins.

well as for ergot sclerotia, in various food intended for human consumption4,5.

Such effects may include changes In the case of animal feed, the number of mycotoxins with maximum permitted or recommended levels is

in immune response (resulting in reduced vaccination efficacy), loss of appetite, abortions, agalactia, etc.

even lower, being limited to aflatoxin B1, ochratoxin A, deoxynivalenol, zearalenone, fumonisins B1 and B2, T-2 and HT-2 toxins, and ergot sclerotia6,7.

Mycotoxins with established maximum permitted levels or recommendations in food intended for human consumption in the EU Aflatoxins B1, B2, G1, G2 and M1 Ochratoxin A Patulin Deoxynivalenol Zearalenone Fumonisins B1 and B2 T-2 and HT-2 toxins Citrinin

Mycotoxins with fixed maximum permitted levels or recommendations in animal feedstuffs in the EU

Aflatoxin B1 Ochratoxin A Deoxynivalenol Zearalenone Fumonisins B1 and B2 T-2 and HT-2 toxins

3

Table 1 shows a summary of the mycotoxins regulated in the European legislation for pig feed, their permitted or recommended limits, as well as their most common health effects.

Table 1. Maximum permitted or recommended values in pig feed.

Mycotoxin

AFB1 a

Aspergillus flavus

Fusarium graminearum Fusarium culmorum

DON b

ZEN b

OTA

Main producing fungi

Fusarium graminearum

Penicillium verrucosum Aspergillus ochraceus Aspergillus carbonarius

b

FB1 + FB2

b

T-2 + HT-2 b

Ergot sclerotia a

Fusarium verticillioides Fusarium proliferatum

Product Raw materials for animal feed

0.02

Compound feed for pigs (except piglets)

0.02

Compound feed for piglets

0.005

Raw materials for animal feed: Cereals and cereal-based products, with the exception of corn by-products

Common effects described in pigs8,9,10 Low performance, liver damage, immunosuppression, neoplasia

8

Corn by-products

12

Compound feed for pigs

0.9

Raw materials for animal feed: Cereals and cereal-based products, with the exception of corn by-products

2

Corn by-products

3

Compound feed for piglets and gilts

0.1

Compound feed for sows and fattening pigs

0.25

Raw materials for animal feed: cereals and cereal-based products

0.25

Compound feed for pigs

0.05

Raw materials for animal feed: corn and corn-based products

60

Compound feed for pigs

5

Oat milling products (hulls)

2

Fusarium sporotrichioides Fusarium langsethiae Other cereal-based products Claviceps purpurea

Maximum or recommended content (mg/kg) c

Loss of appetite, vomiting, poor performance

Low performance, infertility, prolapse, abortions

Low performance, liver and kidney damage

0.5

Compound feed

0.25

Feed materials and compound feed containing unground cereals

1,000

Pulmonary edema, immunosuppression, intestinal disorders, weight loss Anorexia, weight loss, lesions in the mouth and esophagus, leukopenia, reproductive problems

Mycotoxins with maximum permissible contents 6 Mycotoxins with guideline values 7 c Maximum content in mg/kg in animal feed calculated on the basis of a moisture content of 12%

a

b

4

Ergot alkaloids, known since ancient times for their toxic effects, are not included in legislation (there is only a maximum content for ergot sclerotia, listed in Table 1), although they also pose a risk to livestock and it is expected that maximum contents for these compounds will be established in the near future.

Focusing on emerging mycotoxins In addition to the mycotoxins listed in Table 1, there are other mycotoxins that are currently under study as potentially toxic and for which maximum permitted levels have not yet been established.

These are the so-called “emerging mycotoxins”, including some produced by fungi of the Fusarium genus, such as enniatins and beauvericin, which colonize cereals in particular and can accumulate in the grain11.

These groups of mycotoxins are discussed in more detail in the following section.

5

ERGOT ALKALOIDS Ergot alkaloids, or rye ergot,

This disease can cause different symptoms depending

characterized by the spur or “horn”

on the species in question. Thus, in pigs, ergot alkaloids

that the fungus produces on the grain

can cause poor performance, loss of appetite,

as it grows, are produced by different

agalactia, reproductive problems, neonatal mortality,

fungi from the orders Hypocreales

liver damage and gangrene13-16. This wide variety of

and Eurotiales, Claviceps purpurea

symptoms makes it difficult to identify the problem.

being the most widespread in Europe.

This fungus infects grains of various cereals such as rye, wheat, barley, millet and oats, frequently used in animal feed.

The toxicity of ergot alkaloids has been known since the Middle Ages, since it caused the epidemics known as “St. Anthony’s fire” or ergotism and, although it is

Ergot alkaloids

Performance Loss of appetite Reproductive problems Neonatal mortality Liver damage Gangrene

considered an eradicated disease in humans, sporadic outbreaks of ergotism in cattle have been described in recent decades12,13.

6

To date, more than 50 different ergot alkaloids with the ergoline ring as a common structure have been described (Figure 1).

Figure 1. Ergoline ring from the basic structure of ergot alkaloids.

Monitoring of six most common ergot alkaloids (ergometrine, ergotamine, ergosine, ergocristine, ergokryptine, ergocorninine) and their corresponding epimers (ergometrinine, ergotaminine, ergosinine, ergocristinine, ergokryptinine, ergocorninine) is recommended.

Main monitored ergot alkaloids Ergometrine Ergotamine Ergosine Ergocristine Ergokryptine Ergocornine

Epimers

Ergometrinine Ergotaminine Ergosinine Ergocristinine Ergokryptinine Ergocorninine

While the C8-(R) isomers are biologically active, the C8(S) epimers are considered to have little or no activity.

However, as the conversion between isomers is rapid, contaminated samples often present both forms, so, when determining the concentration of ergot alkaloids in a sample, they must be considered together17,18,19.

7

The maximum permitted levels for ergot alkaloids are currently under study, although the industry recommends maximum levels in swine feed between 0.2-0.5 mg/kg20.

However, some studies suggest that lower values consumed over prolonged periods of time could cause intestinal and liver damage21.

In the meantime, and based on the available data and to avoid vasoconstrictive effects of ergot alkaloids in cattle, an acute reference dose of 1 µg/kg body weight and a tolerable daily intake (TDI) of 0.6 µg/kg body weight per day have been estimated22.

In addition, as a precautionary measure, the EU has established a maximum level for ergot sclerotia in cereals of 1,000 mg/kg6.

The European Food Safety Authority (EFSA) has recently pointed out the need to collect more information on the presence of these alkaloids and to develop analytical methods for their control.

Acute reference dose:

1 μg/kg body weight Tolerable daily intake (TDI):

Ergot alkaloids in pig feed:

0.6 μg/kg body weight per day

0.2-0.5 mg/kg

Rye ergot sclerotia in cereals:

1,000 mg/kg

8

ENNIATINS

ENNIATINS AND BEAUVERICIN

Enniatins are produced by fungi of the

The structure of the main enniatins and

genus Fusarium, such as F. avenaceum,

beauvericin is shown in Figure 2.

F. oxysporum, F. poae or F. tricinctum, and present cyclic hexadepsipeptide structure alternating D-α-hydroxyisovaleric acids and N- methyl-L-amino acids.

a)

b)

The amino acid residues of type A and B enniatins are aliphatic N-methyl-valine or N-methylisoleucine, or mixtures of these amino acids.

Up to 29 different enniatins are known, enniatins A, A1, B and B1 being the most studied,

BEAUVERICIN

especially in cereals and derived products.

Beauvericin is produced by Fusarium proliferatum,

F. subglutinans, F. verticillioides and F. oxysporum, and has a structure related to that of the enniatins, although unlike the latter, the three amino acid residues are aromatic N-methyl-phenylalanines.

R1

R2

R3

ENNA

Sec-but

Sec-but

Sec-but

ENNA1

i-pr

Sec-but

Sec-but

ENNB

i-pr

i-pr

i-pr

ENNB1

i-pr

i-pr

Sec-but

Figure 2. Chemical structure of the main enniatins (ENN) (a) and beauvericin (b).

9

Thus, beauvericin has insecticidal

Enniatins inhibit the acyl-CoA and cholesterol acyl .

11,23

transferase enzymes

properties capable of inducing apoptosis in mammalian cells. It also has antiviral, cytotoxic and immunosuppressive activity23-25.

Like the enniatins, beauvericin facilitates the transport of mono or divalent cations across the cell membrane, altering the normal physiological concentrations of these ions.

Enniatins Inhibition of acyl-CoA and cholesterol acyl transferase

Beauvericin Insecticidal, antiviral, cytotoxic and immunosuppressive activity

Although in vitro studies have demonstrated the toxicity of these mycotoxins (showing possible genotoxic and reproductive effects), there is still insufficient evidence of their toxicity in vivo26.

Therefore, EFSA issued a scientific opinion on the need to collect more toxicological data produced by chronic exposure to these compounds, concluding that it is necessary to have more data on the simultaneous presence of enniatins and beauvericin with other Fusarium toxins, as well as to study their possible combined effects27.

Some recent studies have demonstrated the cytotoxicity of these compounds in in vitro studies, using the porcine intestinal epithelial cell line IPEC-J2, with beauvericin being the mycotoxin with the highest bioavailability28.

The presence of enniatins and beauvericin in cereals has been widely documented through various studies that have revealed the high incidence of these compounds, sometimes at concentrations of the order of mg/kg29-32.

10

Case Study Presence of 31 mycotoxins in pig feed Our research group carried out the analysis of 228 samples of swine feed for the presence of 31 mycotoxins:

The samples (2 grain corn samples and 226 feed samples, of which 183 were

Mycotoxins with permitted

meal and 43 pellets) were obtained from

or recommended limits:

Spanish farms and feedmills between

aflatoxins B1, B2, G1 and G2,

February and August 2017 and included:

ochratoxin A, deoxynivalenol, zearalenone, fumonisins B1 and B2, and T-2 and HT-2 toxins.

Enniatins (B, B1, A and A1)

71 feed and 2 corn samples destined to fattening pigs 42 feeds for sows 111 feeds for piglets

Beauvericin 12 most common ergot alkaloids

The mycotoxins were extracted from the matrix by solid-liquid extraction, while

Other mycotoxins considered

liquid chromatography with a fluorescence

of interest: citrinin, fusarenon

detector (LC-FLD) was used for the analysis

X and sterigmatocystin

of aflatoxins, and liquid chromatography coupled to tandem mass spectrometry

The results were published in

(LC-MS/MS) for the rest of the mycotoxins.

two research articles33,34.

It is worth mentioning that LC-MS/ MS allows the measurement of a large number of analytes with good sensitivity, selectivity and confirmatory power that have made it an essential tool for multi-mycotoxin analysis35.

11

A summary of the data obtained is presented in Figure 3 and Table 2.

The most frequent mycotoxins were: Enniatin B (100% of the samples).

Enniatin B1 (83.3% of the samples) Enniatin A1 (73.2% of the samples)

All samples met the requirements for maximum permitted or recommended levels for aflatoxin B1, ochratoxin A, fumonisins B1+B2, Toxins T-2 and HT-2, deoxynivalenol and sum of the twelve ergot alkaloids

Beauvericin (98.2% of samples) Fumonisin B1 (66.2% of samples) However, some samples (3.1%) On the other hand, aflatoxin G2 and the ergot alkaloids ergotaminine, ergocorninine, ergocristine, ergokryptine and ergokryptinine were not detected in any sample.

As for the levels found, the highest

showed zearalenone concentrations above the recommended level: 1 sample of corn (7,681 µg/kg) 5 samples of piglet feed (concentrations between 125 and 956 µg/kg)

concentrations were recorded for zearalenone (7,681 µg/kg), fumonisin B1 (3,959 µg/kg) and

1 feed for fattening pigs (290 µg/kg)

enniatin B (1,222 µg/kg).

100% 90% 80% 70% 60% 50% 40% 30% 20% 20% 0% AFB1 AFB2 AFG1 AFG2 FB1 FB2 ZEN OTA CIT T-2 HT-2 DON F-X STE ENNA ENNA1 ENNB ENNB1 BEA Em Emn Et Etn Es Esn Eco Econ Ecr Ecrn Ekr Ekrn

Percentage of positive samples *

Figure 3. Incidence of mycotoxins found in 228 samples of pig feed.

*considering samples with concentrations above the detection limit of the method used AFB1: aflatoxin B1; AFB2: aflatoxin B2; AFG1: aflatoxin G1; AFG2: aflatoxin G2; FB1: fumonisin B1; FB2: fumonisin B2; ZEN: zearalenone; OTA: ochratoxin A; CIT: citrinin; T-2: T-2 toxin; HT-2: HT-2 toxin; DON: deoxynivalenol; F-X: fusarenon X; STE: sterigmatocystin; ENNA: enniatin A; ENNA1: enniatin A1; ENNB: enniatin B; ENNB1: enniatin B1; BEA: beauvericin; Em: ergometrine; Emn: ergometrinine; Et: ergotamine; Etn: ergotaminine; Es: ergosine; Esn: ergosinine; Eco: ergocornine; Econ: ergocorninine; Ecr: ergocristine; Ecrn: ergocristinine; Ekr: ergokriptine; Ekrn: ergokryptinine.

12

Table 2. Summary of the results obtained in the determination of 31 mycotoxins in 228 samples of pig feed. %CV *

Concentration range (µg/kg) *

Quartile 0.25 (µg/kg) *

Quartile 0.50 (µg/kg) *

Quartile 0.75 (µg/kg) *

0.94

89.9

0.29-2.91

0.46

0.57

0.95

1.3

0.60

55.0

0.28-1.06

0.38

0.47

0.77

2

0.9

0.33

33.3

0.22-0.44

0.28

0.33

0.39

AFG2

0

0.0

---

---

---

---

---

---

FB1

151

66.2

403

132.6

4.09-3,959

106

209

536

FB2

96

42.1

184

101.0

3.63-961

63,0

133

219

ZEN

31

13.6

741

244.0

101-7,681

127

137

463

OTA

2

0.9

---

---

---

---

---

---

CIT

43

18.9

147

80.3

10.7-512

73.0

109

196

T-2

17

7.5

31.9

12.4

28.0-35.9

29.9

31.9

33.9

HT-2

6

2.6

117

4.8

112-123

115

117

120

DON

35

15.4

237

50.6

153-555

165

182

229

F-X

21

9.2

291

82.5

65.1-821

111

212

343

STE

7

3.1

104

115.4

11.2-308

11.4

12,8

179

ENNA

87

38.2

9.82

170.3

3.29-65.0

3.61

3.93

6.46

ENNA1

167

73.2

19.0

127.5

4.54-140

6.69

10.5

20.4

No positive samples

Incidence (%)

AFB1

7

3.1

AFB2

3

AFG1

Average concentration (µg/kg) *

ENNB

228

100

118

137.2

2.01-1,222

14.9

54.3

164

ENNB1

190

83.3

34.3

141.3

2.58-247

6.55

15.1

37.2

BEA

224

98.2

20.7

270.2

1.71-747

4.94

8.73

19.2

Em

18

7.9

59.1

73.8

18.6-145

31.2

37.3

65.8

Emn

5

2.2

---

---

---

---

---

---

Et

8

3.5

9.47

37.5

6.32-16.0

6.58

7.42

12.8

Etn

0

0.0

---

---

---

---

---

---

Es

9

3.9

5.72

50.9

3.41-10.1

3.63

3.85

8.14

Esn

11

4.8

10.7

30.7

5.87-17.5

8.16

10.84

13.16

Eco

1

0.4

---

---

---

---

---

---

Econ

0

0.0

---

---

---

---

---

---

Ecr

5

2.2

12.2

14.0

10.2-14.9

10.5

12.5

12.6

Ecrn

0

0.0

---

---

---

---

---

---

Ekr

0

0.0

---

---

---

---

---

---

Ekrn

0

0.0

---

---

---

---

---

---

*considering only samples with concentrations above the limit of quantification of the method employed AFB1: aflatoxin B1; AFB2: aflatoxin B2; AFG1: aflatoxin G1; AFG2: aflatoxin G2; FB1: fumonisin B1; FB2: fumonisin B2; ZEN: zearalenone; OTA: ochratoxin A; CIT: citrinin; T-2: T-2 toxin; HT-2: HT-2 toxin; DON: deoxynivalenol; F-X: fusarenon X; STE: sterigmatocystin; ENNA: enniatin A; ENNA1: enniatin A1; ENNB: enniatin B; ENNB1: enniatin B1; BEA: beauvericin; Em: ergometrine; Emn: ergometrinine; Et: ergotamine; Etn: ergotaminine; Es: ergosine; Esn: ergosinine; Eco: ergocornine; Econ: ergocorninine; Ecr: ergocristine; Ecrn: ergocristinine; Ekr: ergokryptine; Ekrn: ergokryptinine.

13

CO-OCCURRENCE OF MYCOTOXINS As for the presence of several mycotoxins in the same sample (considering only those

Figure 4. Presence of several mycotoxins in the same sample of pig feed.

mycotoxins present at concentrations above the detection limit of the method),

12 mycotoxins 0.44%

it can be summarized as follows:

11 mycotoxins 0.44%

2-3 mycotoxins: 9.2% 4 mycotoxins: 16.7%

10 mycotoxins 3.51%

7 mycotoxins: 18.9%

2 mycotoxins 1.75%

9 mycotoxins 4.39% 3 mycotoxins 7.46%

5 mycotoxins: 16.7% 6 mycotoxins: 16.2%

13 mycotoxins 0.44%

8 mycotoxins 13.16%

4 mycotoxins 16.67%

8 mycotoxins: 13.2% 9-13 mycotoxins: 9.2%

Figure 4 shows the results obtained.

7 mycotoxins 18.86% 6 mycotoxins 16.23%

5 mycotoxins 16.67%

14

CONCLUSIONS Of the 228 samples analyzed, only 7 showed levels above the permitted levels for zearalenone.

The low incidence of ergot alkaloids should also be noted, which could be explained by the fact that many of the feeds were made from corn, a cereal that is not very susceptible to contamination by Claviceps purpurea.

On the other hand, the high incidence of emerging mycotoxins, particularly enniatin B (100% of the samples), enniatin B1 (83.3%), enniatin A1 (73.2%) and beauvericin (98.2%), not currently included in the legislation, but with evidence of toxicity to pigs, should also be highlighted.

An important aspect to take into account is the simultaneous presence of different mycotoxins in the same sample (up to 13 mycotoxins), given the possible interrelationship between the different mycotoxins consumed together, which may present additive, synergistic, potentiated or antagonistic effects on health.

These effects cannot be predicted from the individual mycotoxin effects.

Moreover, there is dependence on the dose, species and toxin, and even on the factors linked to the experimental methodology used in their study.

However, given the importance of this issue and taking into account that, in risk analysis, the maximum permitted or recommended levels and TDI are set according to the individual effects of each mycotoxin, these values should be reviewed taking into account these possible effects and the potential risk of chronic exposure to multiple mycotoxins, even if they are at lower levels than those permitted36,37,38.

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