Mycotoxins in aquaculture – Underestimated but unavoidable problem

MYCOTOXINS IN AQUACULTURE Underestimated but unavoidable problem

Nemanja Todorović, Jog Raj and Marko Vasiljević PATENT CO, DOO., Vlade Ćetkovića 1A, Mišićevo 24211, Serbia

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World fish production has experienced incredible, growth in just a few decades.

In almost five decades, the worldwide per capita consumption of seafood (fish, crustaceans, mollusks, and other aquatic animals except mammals) has more than doubled, from 9.0 kg in 1961 to 20.2 kg in 2015.

9.0 kg in 1961

20.2 kg in 2015

Aquaculture has, in fact, been growing and continues to grow faster than any other major food production sector, at an average annual rate of 5.8% (2000–2016)1.

To keep fish industry sustainable, it is necessary to find an alternative to the basic ingredients to produce fish food, fish oil and fishmeal.

The aim of the fish feed production is the replacement of fishmeal with less expensive sources of protein, usually of plant origin.

5.8%

A low level of fibers, carbohydrates and indigestible antinutrients, high protein level, good amino acid profile, high digestibility, and good palatability are appropriate characteristics of good plant ingredients in fish feed2,3.

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The main problem caused by the higher inclusion of plant-based ingredients in aquaculture feed is the appearance of mycotoxins that can adversely affect fish health and raise production costs.

Mycotoxins are secondary metabolites produced by various types of fungi that can be found in a variety of plant feedstuffs such as corn, soybean meal, peas, rice bran, wheat, barley, and other commercially available crop by-products (DDGS).

The production of these metabolites can take place before and after harvest, during transport, as well as during storage of raw material and fish feed.

In general, research shows that contamination of fish feed with mycotoxins is a significant and widespread problem in aquaculture around the world, both economically and health-wise.

Mycotoxins-contaminated fish feed is a widespread problem, especially in tropical regions and developing countries where fish feeds are often made by the farmers themselves under inappropriate conditions with improper milling and/or storage.

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Aquatic species have different levels of sensitivity to mycotoxins

Aquatic species show different levels of sensitivity to mycotoxins depending on: Type and quantity of mycotoxins Duration of exposure Age, species and sex

Feed formulation and production region is key to understand if mycotoxin exposure is a risk!

Diet Possible presence of other feed contaminants

Most of the mycotoxins that have the potential to adversely affect production parameters and cause health disorders in fish are the product of three genera of fungi: Aspergillus, Penicillium and Fusarium spp.

Different molds produce around 400 mycotoxin types but the most important ones regarding fish and shrimp health are aflatoxins (AFs) (AFB1, B2, G1, and G2), fumonisins (FBs) (FB1, FB2, and FB3), zearalenone (ZEN), ochratoxin A (OTA), trichothecenes, T-2 and deoxynivalenol (DON), enniatin (ENs) and beauvericin (BEA), as emerging mycotoxins4.

The mycotoxins and their toxic metabolites are known to be either carcinogenic (e.g. aflatoxin B1, ochratoxin A, fumonisin B1), estrogenic (zearalenone), neurotoxic (fumonisin B1), nephrotoxic (ochratoxin), dermatoxic (trichothecenes) or immunosuppressive (aflatoxin B1, ochratoxin A and T-2 toxin).

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All mycotoxins

Aflatoxin B1

1. Immunosuppression

1. Small cell carcinoma

2. Increased mortality

2. Oxidative damage

3. Poor productive performance

3. Pale gills

Zearalenone 1. Affects reproductive parameters in different species 2. Reducing spawning frequency 3. Change of relative fecundity

4. Reduction of growth, feed conversiรณn rate , weight gain

Liver necrosis

Ochratoxin A 1. Mutagenic and toxic effect 2. Degeneration of kidneys and liver, followed by increased mortality 3. Poor feed conversiรณn ratio

4. Acceleration of sexual maturation

5. Increasing mortality

5. Reduced growth in shrimp

6. Abnormalities in shrimp hepatopรกncreas

6. Leaves residues in meat

Pale kidneys and renal necrosis

Fumonisins 1. Lesions in exocrine and endocrine pancreas 2. Lesions in inter-renal tissue

Reduced spawning frequency

Reduced growth, consumption and poor feed conversion

Trichotecenes 1. Decreased average weight gain 2. Decreased production of bacterial cell wall breaking enzymes 3. Immunosuppression at low dosaje 4. Decreased resistance to oxidative damage 5. Inhomogeneous growth and physiological disorders in shrimps

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Although studies on fish represent just 3% of in vivo animal model studies of mycotoxins, to date, the effects of various mycotoxins have been investigated in many fish species such as African catfish (Clarias gariepinus), salmon (Salmo salar), cod (Huso huso), catfish (Ictalurus punctatus), common carp (Cyprinus carpio), prussian carp (Carassius auratus gibelio), rainbow trout (Oncorhynchus mykiss), rohu carp (Labeo rohita), sea bass (Dicentrarchus labrax), various species of tilapia and crustaceans such as whiteleg shrimp (Litopenaeus vannamei) and tiger shrimp (Penaeus monodon).

Clinical signs, which are only top of the iceberg, are inexistent and easily can be associated with other pathologies.

In most cases, mycotoxins affect growth, feed efficiency, reduce survival rate causing enormous economic losses.

Taking the example of catfish production and a 5% increase in FCR, mycotoxins contaminations can cause $250 million in extra feed costs to produce the same quantity of fish5.

250

millon USD

Carry – over of mycotoxins – Human health concern Mycotoxins enter the food chain directly through their residues in edible fish meat, posing an immediate danger to human health.

Bioaccumulation of mycotoxins in aquaculture seafood products is not widely reported and consequently not regulated.

growing crops

humans

harvesting

human food

storage

processing distribution

fish and shrimps

food

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ENVIRONMENTAL HAZARD As previously indicated, many factors pay a role in species susceptibility to mycotoxins, but also, we need to think in the evolutionary path of aquaculture species.

Most of the freshwater herbivorous species are exposed naturally to mycotoxins in a similar way that occurs with livestock, while for carnivorous species, mycotoxins are absolutely alien.

Water soluble mycotoxins, such as fumonisins and DON, can contaminate the water in closed or semi-closed aquaculture systems.

This means that mycotoxins that contaminate the water can consequently get into the soil, posing a threat to the surrounding ecosystem6.

In practice, this means that the low concentrations of mycotoxins commonly observed are more hazardous to carnivorous fish, especially salmonids, while herbivorous freshwater are more resistant species.

Synergism – current knowledge is not enough

Bearing in mind that fish feed contains more than one plant feedstuff and mycotoxin occurrence is common, aquatic organisms are in constant risk of developing mycotoxicosis.

When mycotoxins are present together in feed mixtures, their interactive effects can be classified as additive, antagonistic or synergistic7.

Although in aquaculture the phenomenon of synergism has been scarcely described, it has been demonstrated that different mycotoxins can have synergistic effects in aquaculture species.

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

IIn rainbow trout, the presence of FB1 makes AFB1 more carcinogenic than if either were alone8.

In young channel catfish, the final weight gain of fish exposed to moniliformin and FB1 polycontamination was 42% lower than the control.

In comparison, feeding feeds with equal contamination levels of either monilformin or FB1 alone, resulted in a 16% and 23% lower final weight gain, respectively9.

A study conducted by Perez Acosta, 2016 also describes the synergistic effect between FUM and AFB1.

The combination of these mycotoxins induced worse hepatopancreas damage, comparing to each of these mycotoxins alone10.

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Mycotoxin co-occurrence in feed The large number of aquaculture species as well as the different plant meals that are selected in different parts of the world, primarily on the basis of availability and price, make it difficult to form a general picture mycotoxins contamination in aquaculture feed.

However, the results of plant raw material surveys, such as corn, wheat or soybean can be used to theoretically assess the risk of mycotoxins occurrence in aquaculture feed.

Every new survey performed worldwide finds more mycotoxins in the analysis, including those commonly neglected, also called

The PATENT CO. World corn Mycotoxin Survey 2019 data in Figure 1 reveals that 5 out of 6 samples tested contained 2 or more mycotoxins.

6% of samples were contaminated by one mycotoxin and only 8% of the samples did not contain detectable levels of any of mycotoxins that are regulated in the EU.

Figure 1. Number (%) of mycotoxins per sample in 2019.

emerging mycotoxins.

<LOQ Knowing that multiple contamination may impair the performance of highly productive animals, feed polycontamination by mycotoxins has become an important issue.

1 Mycotoxin >1 Mycotoxins

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Fumonisins, rising concern

The occurrence, level of prevalence and implications that may arise from the presence of fumonisins have been gaining increasing attention in recent years.

The reason for this lies in the fact that the results of all raw material mycotoxin surveys show that fumonisins are the most abundant contaminant.

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

If you compare results of two consecutive PATENT CO. corn surveys (Figure 2), you can see that corn harvested in 2019 had higher occurrence of fumonisins, with higher contamination levels than year before.

% of samples

Figure 2. Percentage of mycotoxins detected in corn samples in 2018 vs 2019.

AFB1

OTA

ZEN

DON

FB1

FB2

HT-2

T-2

Mycotoxins detected % 2018 positives

% 2019 positives

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Fumonisins are naturally occurring toxins produced by several species of Fusarium fungi (molds), with Fusarium moniliforme recognized as the largest producer of this mycotoxin.

Other species such as F. proliferatum, F. nygamai, F. anthophilum, F. dlamini and F. napiforme, are also producers.

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

This enzyme inhibition by fumonisins produces a disruption of sphingolipid metabolism resulting in increased sphinganine and sphingosine along with a decrease in complex sphingolipids in the serum and tissues of animals, which is commonly accepted as the mechanism of action for fumonisins toxicity in most species11.

The presence of fumonisins in fish feed is associated with: Reduced growth rate Decreased feed intake and poor feed conversion rate Disturbances in sphingolipid metabolism

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The available literature mainly describes the influence of fumonisins on freshwater species, primarily on the channel catfish (Ictalurus punctatus).

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 tissue13.

It is known that fumonisins damage liver tissue of rainbow trout (Oncorhynchus mykiss), causing sphingolipid metabolism disturbance consequently inducing cancer in one-month-old trout14.

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Prevention as a solution

Given the known problems that mycotoxins can cause, prevention of fungal development in feed and prevention of mycotoxin uptake in aquatic species are imposed as a solution.

Prevention includes: 1. Implementation of various multi-level strategies to apply feed safety criteria.

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

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).

The ultimate solution may lie in the use of feed additives that eliminate or reduce the uptake of mycotoxins that adversely affect the growth, development, health, and immunity of aquatic species.

Deactivation strategies include: Adsorption: clays, yeasts and algae Biotransformation: pure enzymes or microorganisms Organ protection: different plant extracts, monoterpenoids. etc.

Each of these supplements should meet the following parameters: Efficiency Broad spectrum action Stability and speed of action Selectivity Safety Positive effect on health and production parameters Economic profitability

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Novel approaches in mycotoxin elimination include 360° TOTAL MYCOTOXINS PROTECTION. PATENT CO. has developed a special patented technological process of clinoptilolite modification by firmly attaching long-chain organic cations on the surface of the mineral, forming a new active center.

As a result of this process, a unique product, MINAZEL PLUS® was created.

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Figure 3. Effects of MINAZEL PLUS additive (2kg/MT of MINAZEL PLUS) on weight in common carp.

13 Average body weight (g)

Average body weight (g)

45 40 35 30 25 20

11 9 7 5 3 1

Start

7

14

21

28

35

Trial duration (days) MINAZEL PLUS

42

50

Start

7

14

21

28

33

40

47

61

75

90

Trial duration (days) Control

MINAZEL PLUS

Control

Figure 4. Effects of MINAZEL PLUS additive on weight in Tilapia.

This product can ameliorate the overall adverse effects of mycotoxins and protect fish and shrimp health during exposure to mycotoxin contamination by fast, strong, broad spectrum and high-level bonding.

In combination of strengthening primary antioxidant systems and enhancing immune response, MINAZEL PLUS® offers the most complete coverage against the negative effects of mycotoxins, ensuring optimal growth and performance.

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REFERENCES 1. FAO. The State of World Fisheries and Aquaculture 2018 - Meeting the Sustainable Development Goals; FAO: Rome, Italy, 2018; doi:10.1093/japr/3.1.101 2. Marković, Z., Poleksić, V., Dulić, Z., Stanković, M. (2009): Carp production intensification in traditional semiintensive culture systems by application of extruded feed and selected fish fry. Aquaculture Europe, August 14-17, Trondheim, Norway, Book of Abstracts, pp. 498-499.

References 1-45 are available on request.

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