Mycotoxins in feed – An underestimated challenge for the aquafeed industry

MYCOTOXINS IN FEED An underestimated challenge for the aquafeed industry

Ram C. Bhujel, PhD Research Associate Professor Director, Aqua-Centre Asian Institute of Technology (AIT), Bangkok, Thailand Email: director@aqua-centre.org

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More fish will be needed to feed the rapidly growing world population that is expected to double i.e., 15 billion by 2100.

Aquatic food is the largest source of high-quality animal protein1.

An estimated 156.4 million mt aquatic food was consumed in 20182. More than half (82 mt million) came from aquaculture.

The required increase in supply for additional people needs to come from aquaculture (Figure 1). Annual aquaculture production has grown by over 500% from less than 15 million mt during 1986-95.

A rapidly growing aquaculture industry needs more feed which is the major input that accounts for up to 70-80% of the production cost.

Figure 1. Cage culture in rivers and lakes is a growing feedlot Aquaculture in Asia.

High cost and low feed quality are the major problems.

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Mycotoxins – A hidden problem Feed quality depends mainly on the ingredients used and the level of mycotoxin contamination.

Mycotoxins are quite common in food and feed (Figures 2 and 3).

Figure 2. Grains used as ingredients for fish/animal feed.

Figure 3. Training on feed formulation using linear

programming and manufacturing organized at Aqua-Centre, AIT, Thailand (for more information: info@aqua-centre.org).

Potential economic losses and human health hazards due to mycotoxins have not been completely interiorized.

There is a need to create awareness and make people understand the gravity of the problem.

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The purpose of this paper is to highlight the importance of mycotoxins, their origins, and the methods of prevention and mitigation so that their occurrence and impacts can Figure 4. Feed with molds due to problem during storage.

be avoided or minimized.

What are mycotoxins?

Mycotoxins are secondary mold by-products or metabolites with harmful effects.

They are relatively small molecules but, as they are chemically stable, they are not destroyed by normal cooking or processing.

Mycotoxins are produced by diverse groups of fungi from the genera Aspergillus, Fusarium and Penicilium3,4.

Fungal reproduction occurs through the production of spores that are easily dispersed afar by winds. They can also attach on the bodies of insects, birds and rodents and be transported long distances.

Fungal spores can infect plants at any stage.

In fact, a recent study showed that mycotoxins were detected in 60–80% of the samples of agricultural products contaminated annually, which is about three times higher than the figure (25%) earlier reported5.

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The earliest history description of mycotoxins dates back to 1920 in the US: a yellow mold, Aspergillus flavus, was for the first time identified as a pathogen of maize6.

1942-1947

1920

The history of mycotoxins

Real concerns about mycotoxins began in Russia due to massive outbreaks of Alimentary Toxic Aleukia (ATA) in humans from 1942 to 1947, affecting up to 10% of the population in some districts7.

This occurred when food was in short supply due to wars and people consumed the grains left in the field which were colonized by Fusarium sporotrichioides8.

19 60s

During the 1960s, over 500 cases of outbreaks called as “X-disease” in Turkey were reported linked to mycotoxins.

Later on, a mycotoxin present in imported Brazilian peanut-meal9,10 was found to be linked with the similar disease in poultry in Brazil. The same “X-disease” seen in Turkey also appeared in the UK.

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1920 Aspergillus flavus Maize

1960s X-disease Peanut-meal

1960s X-disease Peanut-meal

1942-1947 Fusarium sporotrichoides (ATA) Grains

1960s X-disease Peanut-meal

Mycotoxins in aquaculture The earliest case of mycotoxicosis detected in fish may be the hatchery-reared rainbow trout (Oncorynchus

mykiss ) in the USA with cottonseed meal found to be responsible11.

Feed is the major input for intensive farming, representing 50-70% of production cost. Fishmeal is considered to be the best ingredient for aquafeed but, due to sustainability issues, its use has been discouraged and attempts have been made to replace it with plant-based ingredients.

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As a result, the risk of exposure to mycotoxins is higher because plant-based products have high chances of being contaminated by mycotoxins.

Many types of mycotoxins have been detected in fish feed12,13.

In most cases, more than one mycotoxin is simultaneously present in each batch of feed at unpredictable levels and combination patterns.

Even if mycotoxins are present at a low level, long-term ingestion of such feed may be a cause of unexplained mortalities that occur on aquafarms.

It is very difficult to identify the disease, even when mycotoxins are detected in fish’s body.

The major concern is that the mycotoxins may remain in the fish’s body e.g., liver, kidney and muscles, as residues and finally be ingested by humans14. Therefore, mycotoxins are emerging and an underestimated problem for the Aquaculture industry.

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Types and sources of mycotoxins Over 300 mycotoxins have been identified. but only about 20 are known and produced naturally by fungi from the genera Aspergillus, Fusarium and Penicilium3,4.

The most worrisome mycotoxins are aflatoxins, ochratoxin A and toxins produced by Fusarium molds.

Aflatoxin was thought to be the most common in fish feed15-19. However, recent extensive survey of feeds and feed ingredients covering 79 countries conducted

by Biomin in 2020 showed that Fusarium mycotoxins are the most prevalent ones.

Deoxynivalenol (DON) was found in 65% of the samples, Fumonisin (FUM) in 64% and Zearalenone (ZEN) in 48% of the samples20.

Table 1 shows the major types of mycotoxins and their sources.

Optimal temperature & water activity (aw)

Mycotoxins

Fungi source

Aflatoxins (B1, B2, G1, G2)

Aspergillus flavus Aspergillus parasiticus

T=33ºC aw=0,99

Peanuts, rice, corn, wheat, sorghum, cottonseed, copra, nuts, milk and milk products

Ocratoxin A

Aspergillus ochraceus Penicillium verrucosum Aspergillus carbonarius

T=15-30ºC aw=0,85-0,98

Cereal grain (wheat, barley, oats, corn), dry beans, moldy peanuts, cheese, coffee, resins, grapes, dried fruit, wine, cocoa

Zearalenone

Fusarium graminearum Fusarium culmorum Fusarium crookwellense

T=15-30ºC aw=0,98

Corn, wheat, moldy hay, pelleted commercial feed, waste systems

Fumonisin B1, B2, B3

Fusarium moniliforme Fusarium verticillioides Fusarium proliferatum

T=10-30ºC aw=0,93

Corn, Sorghum, asparagus

Deoxynivalenol

Fusarium graminearum Fusarium crookwellense Fusarium culmorum

T=15-25ºC aw=0,97-0,99

Wheat, Corn, barley

Patulin

Penicillium expansum

T=24ºC aw=0,99

Moldy feed, rotten apples, wheat straw residue

Citrinin

Penicillium expansum

T=24ºC aw=0,99

Cereal grain (wheat, barley, corn, rice)

Penicillic acid

Aspergillus ochraceus

T=15-30ºC aw=0,85-0,98

Stored corn, cereal grains, dried beans

Trichothecenes

Fusarium graminearum Fusarium sporotrichiodes; Fusarium poae

T=10-30ºC aw=>0,93

Corn, wheat, commercial cattle feed, mixed feeds, barley oats

Commodities

Table 1. shows the major types of mycotoxins and their sources.

Sources 21,22,23,24

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Based on the sources, fungi can be divided into four major categories:

SOIL-BORNE FUNGI Soil is the main source of fungi. Contamination of agricultural products or feed ingredients occurs during the production process in the field or during the handling, transportation and storage.

Soil-borne “filamentous fungi” are the main source of mycotoxins25,26.

AIR-BORNE FUNGI Most fungi reproduce through spores that are very light and can be dispersed long distances by air or winds in the field, infecting plants at any stage.

Spores can enter together with grains into storage room and stay in vessel or equipment used for storing the grains, feeds and feed ingredients, growing when suitable conditions prevail.

SEED-BORNE FUNGI Many crops may get fungal infections from the seed if they are stored in bad conditions where fungi can remain and grow over long periods27.

ANIMAL-BORNE FUNGI Fungi spores can also attach themselves to the bodies of insects, birds and rodents, which allows them to be transported long distances.

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Factors affecting mycotoxin occurrence The two most important factors (Table 1) that affect the fungal life cycle are: TEMPERATURE

WATER AVAILABILITY.

Occurrence of different species of fungi may vary with geographic regions, seasons and weather conditions, and the production of mycotoxins depends on several factors during the whole process of production, harvesting, handling, transportation and storage:

Physical factors

Biological factors Plant variety, stress, insects and fungi spore load

Temperature, moisture and water activity, relative humidity, and mechanical damage of grains.

Chemical factors Oxygen, carbon dioxide, composition of substrate, pesticide and fungicides used.

Optimal conditions for each type of mycotoxin producing fungi are available23.

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Impacts of mycotoxins on fish Mycotoxins are dangerous food or feed contaminants with a high absorption rate. For example, bioavailability assays show that 85% of AFB1 may be absorbed by fish28.

Presence of mycotoxins in feed critically reduces aquaculture productivity and profitability by reducing feed intake, causing abnormality and cancer, damaging gills and liver, reducing growth, increasing feed conversion ratio, suppressing immunity and increasing disease occurrence, causing toxicity and high mortality and reducing spawning frequency and fecundity.

The impact of mycotoxins depends on various factors3,29:

Type and quantity of mycotoxins in the feed Feeding level Duration of exposure Fish species Sex, age, and health condition of the fish Nutritional status of the exposed species

Channel catfish and tilapia appear to be able to detoxify dietary aflatoxins more efficiently than other species11.

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In fact, up to 275 ppb and 250 ppb of aflatoxins had no effects on catfish and tilapia, respectively. However, higher doses i.e. >1,000 ppb (>1ppm) can cause damages.

Similarly, for ochratoxin A (OTA) the dose of 2-4 ppm is likely to have negative effects in catfish, while DON has to be above 15 ppm in the diet to causes harm. T-2 toxin, at a dose above 0.625 ppm was associated with reduction in weight gain.

In addition to catfish and tilapia, numerous studies have been carried out in trout and salmon, but studies are limited in other species. Therefore, further research is needed.

Effects of mycotoxins in humans The ultimate and most concerning consequences of mycotoxin contamination are their effects on human health.

Various reports have demonstrated the presence of different amounts of mycotoxins and their residues in fish muscles, liver and kidney30-34, with the liver containing the highest amounts19,35,36.

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Mycotoxins enter the human body through the ingestion of fish, causing public health problems due to their genotoxic, carcinogenic, immunosuppressive and endocrine disrupting effects26.

Aflatoxin B1 targets the liver and it has genotoxic and carcinogenic effects.

Fumonisin B1 disturbs the metabolism of sphingolipids.

Deoxynivalenol and T-2 toxin inhibit protein synthesis in eukaryotic cells.

Zearalenone is associated with reproductive problems.

Ochratoxin A is known for its nephrotoxic properties.

Monitoring mycotoxins Approximately 100 countries covering 85% of the global population have specific regulations or detailed guidelines for mycotoxins in human food. However, for animal feed and feed ingredients regulations may not exist and, if they exist, they may not be strict.

Despite having policies and regulations, most countries lack monitoring of mycotoxins in food and agricultural products. Many people and animals get sick or die from unknown illnesses that may be associated to the presence of mycotoxins in food or feed.

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Soybean, rice, corn, wheat and their byproducts are heavily used in fish feed, and mycotoxins may develop during their production, processing and storage.

Aquatic animals are at high risk for exposure to mycotoxins if low-quality plant-based feed ingredients are used.

Therefore, monitoring and assessment of the products is needed at different stages of the production chain. However, it is difficult to continuously monitor in many spots.

A portable assay for detecting the presence of mycotoxins has proven to be a useful tool that allows for testing anywhere37,38.

Ultimately, frequent testing is necessary to monitor production animals, especially when the probability of exposure to fungi and mycotoxins is high.

A combination of immunohistochemistry with genotoxicity assays has been suggested as an attractive biomonitoring tool in aquaculture16.

Other lines of research are focusing on a method consisting of the application of nanomaterial-based electrochemical biosensors which may lead to the development of a highly sensitive, reliable, sophisticated, rapid, and cost-effective sensing technique to monitor products in real-time39.

Similarly, a dual DNA tweezers nanomachine has been developed for one-step simultaneous detection of AFB1 and OTA in food samples40.

14

Any tool to assess the risk of mycotoxin exposure will surely be useful, as there are plenty of residues due to the inclusion of land-based feed ingredients. The risk is calculated through Bayesian models that determine the critical concentrations 5% (CC5) for different mycotoxins.

Based on the analysis of 97 commercial fish feeds, the most predominant mycotoxins in fish feed are deoxynivalenol, zearalenone, fumonisins and enniatins27.

In the US, the Food and Drug Administration (FDA) has established advisory levels for other mycotoxins (e.g., DON and fumonisins) for the feed industry (Table 2) with the levels that are considered adequate to protect human and animal health.

Each agricultural product has its own maximum limit, and the United States Department of Agriculture Agricultural Marketing Service (USDA-MS) and the Federal Grain Inspection Service (FGIS) have provided verified test kit names.

Their manufacturers, detection methods, range and the list of commodities for aflatoxins, fumonisins, deoxynivalenol, zearalenone and ochratoxins are available at FGIS42.

Maximum level of aflatoxins

Table 2. Information pertaining to the Food and Drug Administration (FDA) limits for aflatoxin levels applicable to human food and animal feed products41.

Intended use

20ppb

All foodstuffs for human consumption except milk (milk should be less than 0.5 ppb)

20 ppb

Corn, peanut products, cottonseed meal and other animal feed and feed ingredients intended for dairy animals.

For animal species or uses not specified below, or when the intended use is not known

20 ppb

Corn, peanut products and other animal feeds and feed ingredients, excluding cottonseed meal, intended for immature animals

100 ppb

Corn and peanut products intended for breeding beef cattle, breeding swine or mature poultry

200 ppb

Corn or peanut products intended for finishing swine (100 lbs (45,35 kg) or more)

300 ppb

Corn and peanut products intended for finishing (i.e., feedlot) beef cattle

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HOW CAN WE ESTIMATE THE LEVEL OF MYCOTOXINS IN FEED? When feed manufacturers purchase ingredients, they should also ask for

If the information for each ingredient is available, the total amount of mycotoxins contributed to the total diet can be estimated by multiplying the amount of mycotoxins in the feedstuff by the ratio of feedstuff (%) in the total diet: Total diet Deoxynivalenol (DON) or Vomitoxin Level

=

Level of DON in feedstuff (%) x (Feedstuff (lbs. DM) Total Diet (lbs. DM))

INFORMATION NOT AVAILABLE

INFORMATION AVAILABLE

the test results.

Toxins

If information about mycotoxin levels in the ingredients is not available from suppliers, then feed manufacturers should arrange for testing so that they can test ingredients as well as feed.

Table 3 provides the maximum level of mycotoxins in feed for different animals.

Dairy

Feedlot

Swine

Poultry

Trout

Catfish

Tilapia

Shrimp

20

20

20

20

20

2,000

2,000

20

0.5-1.0

10

1

2

-

1

-

0.2

Fumonisin (ppm)

2

7

10

20

-

20

100

-

T-2 Toxin (ppb)

100

500

100

100

2,500

600

-

-

Zearalenone (ppb)

400

5,000

300

10

-

-

-

5,000

5.000

700

700

4,000

2,000

-

1,000

500

500

500

750

-

-

-

-

Aflatoxins (ppb) Deoxynivalenol (ppm)

Ochratoxin A (ppb) Ergot Toxins (combined) - ppb

Table 3. Potentially harmful dietary limits for mycotoxins in total diet on a dry matter basis11 41,43.

Note – empty cells mean data are not available

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The European Commission legislation also stipulates maximum admissible levels for mycotoxins.

These levels are defined in the valid version of Commission Regulation (EC) No. 1881/2006 of 19 December 200644 setting maximum levels for certain contaminants in foodstuffs and in Directive 2002/32/EC of the European Parliament and of the Council of 7 May 2002 on undesirable substances in animal feed.

Additionally, maximum levels have been established for citrinin in food supplements based on rice fermented with red yeast, Monascus purpureaus45.

The European Commission also has the Recommendation 2012/154/EU of 15 March 2012 for the monitoring of the presence of ergot alkaloids in feed and food.

In the feed sector, maximum levels are defined only for aflatoxin B1 and Ergot alkaloids indirectly via ergot, which indicates that more is needed.

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Sampling For mycotoxin analysis, sampling and preparing the samples are the key.

Samples taken from a large volume of grains/feed ingredients or feed samples should represent the whole stock/lot, and multi-stage sampling is usually necessary.

Sample size, minimum volume and the steps for sampling are described in the Mycotoxin Handbook41.

Once samples are taken, they need to be handled correctly so that moisture never exceeds 20%.

At the same time, the EU also has its own methods for sampling and analysis for the official control of the levels of mycotoxins in foodstuffs under the Commission Regulation (EC) No 401/2006 of 23 February 200644.

Sampling protocols are also available from the Codex Alimentarius standard CODEX STAN 193-199546.

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Preventing mycotoxin occurrence Prevention of mycotoxins occurrence requires careful application of protocols throughout the entire value chain, including crop production, harvest, transport, distribution processing and storage as summarized in Table 4.

Major categories

Factors and descriptions

1. Crop production and harvesting

Select resistant varieties Rotate crops Control insect pests, birds, and rodents Use biofertilizers Harvest soonest possible Don’t leave in the field Clean properly Dry properly

2. Storage, transportation and distribution

Control temperature Control moisture/humidity Avoid mechanical injury Clean packaging Clean and dry room Control insects, birds, and rodents

3. Storage, transportation and distribution

Discard broken/damaged Control temperature Control moisture Test and monitor regularly Detect and discard infected ingredients Select and avoid inadequate ingredients

4. Quality monitoring system (testing)

System in place to test regularly Select right methods: Visual inspection Testing with equipment Standardize sampling size/method Standardize for selection/discarding

Table 4. Method of exclusion of mycotoxin occurrence in the food/feed value chain (Modified from47 and mycotoxinsite.com).

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Some plant varieties are naturally resistant to fungi. Identifying and selecting these varieties may be a good option to avoid mycotoxin occurrence.

For example, improved corn varieties that are resistant to Aspergillus

flavus are available in the USA. Identifying those varieties and hybridizing may work.

In addition, selecting the genes or DNA loci responsible for resistance against certain fungus and inserting them into high yielding varieties should be future lines of research48.

Bt (Bacillus thuringiensis) technology has been applied in some crops to control insect pests so that chances of fungi production and spreading reduces considerably e.g., Bt corn or cotton11.

As shown in Table 4, good agricultural practices such as crop rotation, soil tillage, and chemical and biological control of plant diseases are essential to minimize fungal growth.

Harvesting the products as quickly as possible, avoiding drying in the field, moving to better facilities as soon as possible, and drying on platforms raised above the ground can avoid mycotoxin occurrence.

Raw material suppliers should implement methods for quick testing to detect the presence of mycotoxins in cereal products in order to be able to accept or reject a batch.

Unfortunately, these quick tests can only detect a few mycotoxins and the regulatory limits vary among different countries. Thus, the responsibility of mycotoxin management in aquafeeds comes to the feed producers.

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Potential treatments A number of ways and substances have been tested and used to reduce fungal growth or bind the mycotoxins to make them unavailable for absorption through the intestinal tract.

There are some commercially available detoxification products and enzymes that appear to have a high selectivity for transforming mycotoxins into less toxic forms. However, this strategy is only effective when aquafeeds contain a single type of mycotoxin but, as mentioned previously, a cocktail of mycotoxins is likely to be present in the feeds, making it unfeasible to supplement with an enzyme for each potential toxin.

As a result, attention is shifting to more practical and universal solutions, such as mycotoxin binders.

In general, organic adsorbents bind to a larger spectrum of mycotoxins than inorganic adsorbents.

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CONCLUSIONS AND RECOMMENDATIONS

Among the more than 300 known mycotoxins, only approximately 20 have been demonstrated to be harmful to animals and humans. However, more research on mycotoxins is needed.

The negative consequences associated with mycotoxins have not been fully internalized by crop producers, aquaculture practitioners, feed manufacturers, fish farmers, researchers and policy makers.

There is a need to create more awareness and make people understand the magnitude of the problem.

More training, workshops and policy dialogues among stakeholders should be organized to transfer the knowledge and safe techniques of crop production practices, transportation, processing, storage and handling.

Furthermore, clear policies on mycotoxins, more laboratory facilities for testing, more trained human resources and clear guidelines for regular monitoring are required in each country or state.

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