Dominance of Fumonisins and Aflatoxins in aquafeed samples from 10 countries

DOMINANCE OF FUMONISINS AND AFLATOXINS IN AQUA FEED SAMPLES FROM 10 COUNTRIES

Jog Raj*, Hunor Farkaš, Nemanja Todorovic, Zdenka Jakovčević, Robert Čepela and Marko Vasiljević PATENT CO, DOO., Mišićevo, Serbia *Corresponding author: jog.raj@patent-co.com

1

The most recent data estimates that

Now, in most circumstances,

aquatic animals (capture fisheries and

mycotoxins occur in many, if

aquaculture taken together) currently

not all, feed raw materials of

contribute approximately 40% of the

plant origin if the environmental

global animal protein production, up

conditions are favorable.

from about 25% in the early 2000s.

40% of the global animal protein production

To some extent, the presence of mycotoxin-producing fungi is less frequent in feed ingredients coming from animals (marine

To meet this target the demand for

fishmeal and fish oil), but

aquatic feed is also increasing and

it is not unheard of.

efforts to minimize the use of marine fish-based oils and protein, multiple alternatives are being considered, for example other (even aquaculture) fish offal and slaughter or farming by-products, including animal manure.

With today’s globalization of trade However, the focus is mainly on

and the use of feed raw materials

plant-based sources of protein and

from all over the world, it is crucial to

oil such as corn, soybeans, various

screen for contaminated ingredients

post-processing by-products such

when preparing the commercial

as oil-pressed cakes and meals,

feed for aquaculture species.

bioethanol by-products, silage (from plants or animals), and many more.

2

Surveillance is routinely carried out in larger companies, but it remains a problem for small feed mills and use of contaminated feeds in farms is a risk.

In this survey, more than

+200

samples were 200 samples were analysed analysed for multiple for multiple mycotoxins mycotoxins using LC-MS/MS triple

quadrupole (Agilent 6460 series) based multi-mycotoxin method for quantification of all mycotoxins present.

Therefore, the aim of the present study was to screen aqua feed samples received from different countries between January and November 2021 to determine their level of contamination with mycotoxins:

These included Aflatoxin B1 (AFB1), B2, G1 and G2, Ochratoxin A (OTA), Zearalenone (ZEN), Deoxynivalenol (DON), Fumonisin B1 and B2 (FB1 and FB2), T-2 and HT-2 toxins, all regulated in the EU in

Serbia Spain

feed by EU Directives 2002/32/EC, 2006/576/EC, and 2013/165/EU.

Vietnam Turkey Egypt Senegal

The results of this survey per country are presented as below.

Philippines Uganda

Results of mycotoxin levels for each country

Kenya Thailand

Results of mycotoxins in aqua feed for all 10 countries Effects of mycotoxins on aquaculture species (fish and shrimp) Conclusions

3

Results of mycotoxin levels for each country SERBIA

In 2021, 29% of the aqua feed samples

DON with an average of 128 ppb, FB1 with

from Serbia were not contaminated with

86 ppb and FB2 with an average of 95 ppb

mycotoxins. 14% of the samples were

were detected in aqua feed samples.

contaminated with one mycotoxin and 57% samples were found to contain more than 1 mycotoxin (Figure 1).

Figure 1. Number of mycotoxins per sample in aqua feed from Serbia.

FB1 was detected in 57% of the samples followed by DON in 29 % and then FB2 in 14 % of aqua feed samples from different regions of Serbia (Table 1).

14 Table 1. Mycotoxin contamination levels (ppb) in aqua feed samples from Serbia in 2021.

57

29

1 mycotoxin

DON

FB1

FB2

Average (ppb)

128

86

95

Maximum of positives (ppb)

185

147

95

Minimum (ppb)

72

47

95

% of positives

29

57

14

> 1 mycotoxin < LOQ

4

SPAIN

In 2021, 46 % of the aqua feed samples

DON with an average of 93 ppb, FB1 with

from Spain were not contaminated

103, FB2 with 89 ppb, ZEN with 75 ppb,

with mycotoxins whereas 7% had one

and AFB1 with an average of 0.6 ppb were

mycotoxin and 47% samples were found

detected in aqua feed samples from Spain.

to contain more than 1 mycotoxin (Figure

2). So, in net 54% of aquafeed samples were contaminated with mycotoxins.

FB1 and ZEN were detected in 47% of the samples followed by

Figure 2. Number of mycotoxins per sample in aqua feed from Spain.

AFB1 in 27 % and DON in 13 % of aqua feed samples from different regions of Spain (Table 2).

Table 2. Mycotoxin contamination levels (ppb) in aqua feed samples from Spain in 2021.

7

AFB1

ZEN

DON

FB1

FB2

Average (ppb)

0.6

75

93

103

89

Maximum of positives (ppb)

1.0

134

102

186

127

Minimum (ppb)

0.4

29

84

62

53

% of positives

27

47

13

47

47

46

47

1 mycotoxin > 1 mycotoxin < LOQ

5

THAILAND

In 2021, 63% of the aqua feed samples

DON with an average of 135 ppb, FB1 with

from Thailand were not contaminated

143, FB2 with 245 ppb, and AFB1 with an

with mycotoxins whereas 23% had one

average of 0.7 ppb were detected in aqua

mycotoxin and 14% samples were found

feed samples from Thailand in 2021.

to contain more than 1 mycotoxin (Figure

3). So, in net 37% of aquafeed samples were contaminated with mycotoxins.

FB1 and AFB1 were detected in 11% of the samples whereas DON

Figure 3. Number of mycotoxins per sample in aqua feed from Thailand.

was detected in 17% and FB2 in 6% samples from different regions of Thailand (Table 3).

Table 3. Mycotoxin contamination levels (ppb) in aqua feed samples from Thailand in 2021.

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AFB1

DON

FB1

FB2

Average (ppb)

0.7

135

143

245

Maximum of positives (ppb)

1.1

305

387

247

Minimum (ppb)

0.4

64

44

243

% of positives

11

17

11

6

23

63

1 mycotoxin > 1 mycotoxin < LOQ

6

VIETNAM

In 2021, 36% of the aqua feed samples

DON with an average of 527 ppb, FB1

from Vietnam were not contaminated

with 317, FB2 with 124 ppb, ZEN with

with mycotoxins, whereas 21% had one

58 ppb and AFB1 with an average of

mycotoxin and 43% samples were found

0.6 ppb were detected in aqua feed

to contain more than 1 mycotoxin (Figure

samples from Vietnam in 2021.

4). So, in net 64% of aquafeed samples were contaminated with mycotoxins.

FB1 and DON were detected in 36% of the samples whereas

Figure 4. Number of mycotoxins per sample in aqua feed from Vietnam.

FB1 was detected in 36% and AFB1 was detected in 18% of the samples from Vietnam (Table 4).

43

36

21

Table 4. Mycotoxin contamination levels (ppb) in aqua feed samples from Vietnam in 2021.

AFB1

ZEN

DON

FB1

FB2

Average (ppb)

0.6

58

527

317

124

Maximum of positives (ppb)

1.4

209

1556

2490

673

Minimum (ppb)

0.4

18

73

55

45

% of positives

18

58

36

36

33

1 mycotoxin > 1 mycotoxin < LOQ

7

TURKEY

In 2021, 29% of the aqua feed samples

FB1 with an average of 124 ppb, FB2

from Turkey were not contaminated

with 154 ppb, and AFB1 with an average

with mycotoxins whereas 4% had one

of 1.8 ppb were detected in aqua feed

mycotoxin and 67% samples were found

samples from Turkey in 2021.

to contain more than 1 mycotoxin (Figure

5). So, in net 71% of aquafeed samples were contaminated with mycotoxins.

FB1 was detected in 67% of samples and AFB1 was detected in 63% of

Figure 5. Number of mycotoxins per sample in aqua feed from Turkey.

the samples from Turkey (Table 5).

Table 5. Mycotoxin contamination levels (ppb) in aqua feed samples from Turkey in 2021.

4 29 67

AFB1

FB1

FB2

Average (ppb)

1.8

124

154

Maximum of positives (ppb)

6.9

336

535

Minimum (ppb)

0.4

46

49

% of positives

63

67

63

1 mycotoxin > 1 mycotoxin < LOQ

8

KENYA

UGANDA

In 2021, all the aqua feed samples

In 2021, all the aqua feed samples

from Kenya were contaminated

from Uganda were contaminated

with more than one mycotoxin.

with more than one mycotoxin.

FB1 with an average of 264, FB2 with

FB1 with an average of 370 ppb, FB2

76 ppb and AFB1 with an average of

with 185 ppb and AFB1 with an average

31.7 ppb were detected in aqua feed

of 133 ppb were detected in aqua feed

samples from Kenya in 2021.

samples from Uganda in 2021.

FB1, FB2 and AFB1 were detected in all

FB1, FB2 and AFB1 were detected in

(100%) samples from Kenya (Table 6).

all (100%) the samples (Table 7).

Table 6. Mycotoxin contamination levels (ppb) in aqua feed samples from Kenya in 2021.

Table 7. NMycotoxin contamination levels (ppb) in aqua feed samples from Uganda in 2021.

Average (ppb)

AFB1

FB1

FB2

32.4

265

76

AFB1

FB1

FB2

Average (ppb)

133

370

185

252

391

201

Maximum of positives (ppb)

41.7

421

120

Maximum of positives (ppb)

Minimum (ppb)

31.7

207

69

Minimum (ppb)

14

349

169

% of positives

100

100

100

% of positives

100

100

100

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PHILIPPINES

EGYPT

In 2021, all the aqua feed samples

In 2021, all the aqua feed samples

from Philippines were contaminated

from Egypt were contaminated with

with one mycotoxin (AFB1).

more than one mycotoxin.

AFB1 with average of 0.8 ppb, ZEN with 78 ppb, DON with 159 ppb, FB1 with 78 ppb and

AFB1 with average of 4 ppb was detected in 100 % of aqua feed samples tested

FB2 with an average of 84 ppb were detected in aqua feed samples from Egypt (Tabla 9).

from Philippines in 2021 (Table 8).

FB1 was the most predominant mycotoxin detected in 100% aqua feed samples

Table 8. Mycotoxin contamination levels (ppb) in aqua feed samples from Philippines in 2021.

from Egypt, followed by AFB1 in 69%, DON in 62% and ZEN that was detected in 54% samples (Table 9).

AFB1 Average (ppb)

4

Maximum of positives (ppb)

7

Minimum (ppb)

2

% of positives

100

Table 9. Mycotoxin contamination levels (ppb) in aqua feed samples from Egypt in 2021.

AFB1

ZEN

DON

FB1

FB2

Average (ppb)

0.8

78

159

78

84

Maximum of positives (ppb)

1.7

151

237

162

147

Minimum (ppb)

0.5

25

67

46

48

% of positives

69

54

62

100

54

10

SENEGAL

In 2021, 29% aquafeed samples from Senegal

AFB1 with average of 1.6 ppb, ZEN

were not contaminated with mycotoxins

with 120 ppb, DON with 78 ppb, FB1

whereas 71% samples were contaminated

with 55 ppb and FB2 with an average

with more than one mycotoxin (Figure 6).

of 116 ppb were detected in aqua feed samples from Senegal (Tabla 10).

Figure 6. Number of mycotoxins per sample in aqua feed from Senegal.

FB1 was the most predominant mycotoxin as it was detected in 71% aqua feed samples from

0

Senegal, followed by ZEN and FB1 in 43% samples and then DON in 29% samples (Tabla 10).

29 71

1 mycotoxin > 1 mycotoxin

Table 10. Mycotoxin contamination levels (ppb) in aqua feed samples from Senegal in 2021.

AFB1

ZEN

DON

FB1

FB2

Average (ppb)

1.6

120

78

55

116

Maximum of positives (ppb)

2.3

187

88

74

159

Minimum (ppb)

0.8

52

68

40

83

% of positives

71

43

29

43

43

< LOQ

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Results of mycotoxins in aqua feed for all 10 countries Summary of the mycotoxins in aqua feed survey results from Serbia, Spain, Vietnam, Turkey, Egypt, Senegal, Philippines, Uganda, Kenya and Thailand received between January and November 2021

In 2021, 36% of the aqua feed samples received from Serbia, Spain, Vietnam, Turkey, Egypt, Senegal, Philippines, Uganda, Kenya and Thailand between January and November 2021 were not contaminated

DON with an average of 265 ppb, ZEN with 82 ppb, AFB1 with 6 ppb, FB1 with 181 ppb, FB2 with 134 ppb and OTA with an average of 38 ppb were detected in 2021 in aqua feed received from these 10 countries.

with mycotoxins whereas 64% samples were found to contain more one or more than one mycotoxin (Figure 7).

FB1 was the most predominant This data showed cooccurrence of

mycotoxin as it was detected in 47%

mycotoxins in 50% of aqua feed

of the tested samples followed by

samples from Serbia, Spain, Vietnam,

AFB1 in 36%, ZEN in 29% and DON in

Turkey, Egypt, Senegal, Philippines,

26 % of aqua feed samples (Table 11).

Uganda, Kenya and Thailand (Figure 7).

Figure 7. Number of mycotoxins per sample in aqua feed from Serbia, Spain, Vietnam, Turkey, Egypt, Senegal, Philippines, Uganda, Kenya and Thailand recieved between January and November 2021.

14 1 mycotoxin

36

50

> 1 mycotoxin < LOQ

Table 11. Mycotoxin contamination levels (ppb) in aqua feed samples from Serbia, Spain, Vietnam, Turkey, Egypt, Senegal, Philippines, Uganda, Kenya and Thailand received between January and November 2021.

AFB1

OTA

ZEN

DON

FB1

FB2

Average (ppb)

6

38

82

265

181

134

Maximum of positives (ppb)

252

80

364

1556

2490

673

Minimum (ppb)

0.4

2

18

64

40

45

% of positives

36

4

29

26

47

39

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Effects of mycotoxins on aquaculture species Literature has shown that, while aflatoxins in recent decades have been the “most famous” of all the mycotoxins, it should be kept in mind that there are over a hundred different mycotoxins and many of them are known to cause human health problems for centuries (e.g., ergotism) and still are causing animal health problems.

A summary of different effects of mycotoxins on aquatic animals is shown in Figure 8. The toxic effects of different mycotoxin groups are similar in terrestrial and aquatic animals.

For example, aflatoxins cause liver damage and ochratoxins are toxic for kidneys.

Figure 8. Effects of mycotoxins on aquatic animals.

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Although in aquaculture the phenomenon

Similar discrepancies in OTA

of synergism has been scarcely described,

studies have also been reported,

it has been demonstrated that different

pointing out that there is still quite

mycotoxins can have synergistic

a lot that we don’t know about the

effects in aquaculture species.

metabolism and accumulation of mycotoxins in aquatic animals.

This means that combined negative effects of mycotoxins on productivity and health appear greater than the

Even if mycotoxins are present at a

sum of their individual effects.

low level, long-term ingestion of such feed may be a cause of unexplained mortalities that occur on aquafarms.

The presence of diverse mycotoxins can also cause general immunosuppression and opens the door for further

Clinical signs, which are only tip

health problems, including increased

of the iceberg, are inexistent and

susceptibility to infectious diseases.

easily can be associated with other pathologies. In most cases, mycotoxins affect growth, feed efficiency, reduce survival rate

The risk associated with

causing enormous economic losses.

the consumption of aquatic animals that have been fed mycotoxin contaminated feed is still not well understood.

Some studies suggest that there is a relatively low risk of bioaccumulation of aflatoxins in the muscle tissue of fish and shrimp, while there is

Taking the example of catfish production, a 5% increase in FCR due to mycotoxin contamination can result in $250 million in extra feed costs to produce the same quantity of fish.

also some evidence that AFB1 residues are present in fish muscle in relevant concentrations and cause human health problems.

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FUMONISINS, A RISING CONCERN

Fumonisins are naturally occurring toxins produced by several species of Fusarium fungi (molds), with

Fusarium moniliform recognized as the largest producer of this mycotoxin.

The presence of fumonisins in fish feed is associated with: Reduced growth rate Decreased feed intake

Fumonisins inhibit the sphinganine (sphingosine) N-acyltransferase (ceramide synthase), a key enzyme in lipid metabolism, resulting in disruption of this pathway.

As fumonisins are relatively stable at high temperature and processing conditions, it is expected that they will be found in finished feeds as well.

In recent surveys, including the data in this report, fumonisins has been the most predominant mycotoxins in corn (Raj et al., 2022) and animal/fish feed samples.

Poor feed conversion rate Disturbances in sphingolipid metabolism

In an experiment with one-year old carps (Cyprinus carpio), the toxic effect was accompanied by changes in the exocrine and endocrine pancreas and inter-renal tissue.

In rainbow trout, the presence of FB1 makes AFB1 more carcinogenic than if either were alone.

15

In young channel catfish, the

It appears that fish that are highly

final weight gain of fish exposed

dependent on lipid metabolism can

to moniliformin, AFB1 and

be very sensitive to fumonisins since

FB1 poly contamination was

they disrupt sphingolipid metabolism

42% lower than the control.

and consequently reduce growth and feed utilization, even at levels of (168 µg/kg feed FB1 and 333 µg/kg FB2).

In comparison, feeding feeds with equal contamination levels of either moniliformin or FB alone, resulted in a 16% and 23% lower final weight gain, respectively.

A study conducted by Pérez Acosta (2016) also described the synergistic effect between FB1 and AFB1.

CARRY-OVER OF MYCOTOXINS – HUMAN HEALTH CONCERN

Mycotoxins enter the food chain directly through their residues

The combination of these mycotoxins induced worse

in edible fish meat, posing an immediate threat to human health.

hepatopancreas damage, as compared to each of these mycotoxins alone.,

Gonçalves et. al. (2020) showed that low level of fumonisins

Bioaccumulation of mycotoxins in aquaculture seafood products

can affect sea bass and sea

is not widely reported and

bream growth performances.

consequently not regulated.

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PREVENTION AS A SOLUTION

Given the known problems that mycotoxins can

The ultimate solution may lie in the use

cause, prevention of fungal development in

of feed additives that eliminate or reduce

feed and prevention of mycotoxin uptake in

the uptake of mycotoxins that adversely

aquatic species are imposed as a solution.

affect the growth, development, health, and immunity of aquatic species.

Deactivation strategies include: Prevention includes: Adsorption: clays, yeasts, and algae 1. Implementation of various multi-level strategies to apply feed safety criteria.

Biotransformation: pure enzymes or microorganisms

2. Traceability of the feed production chain from the field to fork.

Organ protection: different plant extracts, monoterpenoids. etc.

Each of these supplements should meet the following parameters: Pre-harvest control, such as the application of “Good Agricultural Practice” (GAP), can only reduce but cannot eliminate mycotoxins.

That is why focus should be on post-harvest control measures, maintaining acceptable storage conditions and producing feed in accordance with “good manufacturing practice “(GAP).

Efficiency Broad spectrum action Stability and speed of action Selectivity Safety Positive effect on health and production parameters Economic profitability

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CONCLUSIONS In 2021, 64% of aqua feed samples received from Serbia, Spain, Vietnam, Turkey, Egypt, Senegal, Philippines, Uganda, Kenya, and Thailand received between January and November 2021, were contaminated with one or more than 1 mycotoxin.

Fumonisins (47%) and aflatoxins (36%) were the most prevalent mycotoxins detected in aquafeed samples using LC-MS/MS.

The literature data have shown that Fumonisins and aflatoxins can have synergistic effects on fish/shrimp production.

Cooccurrence of more than one mycotoxin was also detected in 50 % of samples from these 10 countries.

The co-occurrence of more than one mycotoxin can lead to additive or synergistic effects when fed to sensitive animal species (e.g., shrimps and fish amongst the others) and can result in carry over of mycotoxins for humans via fish/shrimp food.

This study suggests that use of additives to combat mycotoxins and reduce their carry over to humans should be considered while feeding aquatic animals.

References on request.

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