Aflatoxin B1 (AfB1) is a genotoxic and carcinogenic mycotoxin that is produced by fungi, in particular Aspergillus flavus
and Aspergillus parasiticus.
AfB1 can be metabolized to aflatoxin M1 (AfM1) by cows, sheep, and goats. Like AfB1, AfM1 is also considered to be genotoxic and carcinogenic to animals and humans [1
]. Elevated concentrations of AfB1 in feed result in elevated levels of AfM1 in milk and milk products. Due to their toxic effects on human and animal health, the presence of AfB1 and AfM1 in foodstuffs is strictly regulated within the European Union (EU). Regulation (EC) No 1881/2006 sets the maximum levels for aflatoxin M1 in dairy products, i.e., 0.05 µg/kg for raw milk, heat-treated milk, and milk for the manufacture of milk-based products. In addition, a maximum level for AfB1 in all feed materials was set at 0.02 mg/kg, as well as for compound feed for cattle, sheep, and goats, with the exception of dairy cattle, dairy sheep, and dairy goats at 0.005 mg/kg (Directive 2002/32/EC, Consolidated version 27 February 2015), all relative to a feed with a moisture content of 12%.
Historically, aflatoxins have been mainly found in products originating from countries with tropical weather conditions favourable for the growth of Aspergillus
spp., like India, Brazil, and Colombia. However, during the last decade, serious contaminations of maize with aflatoxins have been reported in southern Europe. These include maize grown in 2003 in Italy [2
] and maize from the 2012 harvest from the Balkan region [3
]. Since climate change is increasingly affecting the formation of mycotoxins in Europe [4
], these recent incidents are most likely not the last cases of aflatoxins in maize for European farmers.
Maize is a commonly used ingredient for feeding dairy cows. Feed manufacturers produce feed by mixing and grinding maize and other feed ingredients. In recent decades, the maize consumption of dairy cows has increased due to the prices of raw materials, compound feed composition, and the increase in the amount of concentrated feeds in diets. For example, in the Netherlands, the inclusion rates of maize in compound feed for dairy cows has increased from approximately 1% to 18% in the period 2010–2013 [5
]. Additionally, the milk production of dairy cows is steadily increasing, which will coincide with an increased intake of feed, particularly compound feed, by cows.
The abovementioned developments have possibly increased the total exposure of dairy cows to aflatoxins, which might in turn lead to a higher probability of dairy cows’ milk to be contaminated by AfM1.
The aim of this study was to estimate the AfM1 contamination in dairy cows’ milk, using transfer modelling under multiple scenarios of compound feed composition, feed contamination with AfB1, feed consumption, and milk yield. The inclusion rate of maize in compound feed and the contamination of maize by AfB1 were modelled by Monte Carlo simulations, and hence the AfM1 concentration in dairy cows’ milk with the intake distribution could be investigated. Monte Carlo simulation is a computerized mathematical technique that is often used for quantitative analyses and decision-making. The strength of this simulation technique is that it provides the decision-maker with a range of possible outcomes together with their probabilities.
This study aimed at investigating the potential exceedance of the maximum limit for AfM1 concentrations in milk, under different scenarios for AfB1 contamination of feed ingredients, inclusion rates of ingredients in compound feed, milk production, and transfer rates. Monte Carlo simulation modelling was used to investigate the AfM1 contamination of milk for the wide range of situations that realistically occur in practice. The results show that milk production of the farm and milk yield appeared to have minimal effects on the outcomes; a higher milk production did not result in increased concentrations of AfM1 in the farm milk, due to a potential dilution effect. While, according to transfer equations, a higher milk yield will result in a higher transfer rate, the concentration in the milk produced on the farm changes only slightly due to an increased milk production.
The Monte Carlo approach was applied to simulate the AfB1 contamination in each individual feed ingredient, using monitored mean and standard deviation AfB1 concentrations in the respective feed ingredient monitored over a 10-year period. Thus, the monitoring results were used to fit a distribution for AfB1 in each feed ingredient, and this distribution was in turn used as an input in the Monte Carlo simulation. The same approach was used to simulate the compound feed composition for Scenario 3, provided a known minimum and maximum percentage inclusion for each ingredient. This approach allowed us to simulate 1000 different contamination/composition pairs for the daily feed intake for AfB1, changing every two weeks. With 10-year monitoring data of AfB1 contamination in feed ingredients and the guidelines available for feed composition, less than 0.6% of all the simulations for individual two-week periods were above the EC limit for AfM1 concentration in milk, with a maximum concentration of 0.08 µg/kg (over all five transfer equations). This resulted from either a high inclusion rate of maize with an AfB1 concentration below the EC limit in compound feed, or from a low inclusion rate of maize above the EC limit for AfB1 in the compound feed. Nevertheless, it can be concluded that given the current practices, the probability of exceeding the EC limit of AfM1 in dairy milk is very low. In our model we also tested the effect of corn silage contaminated with AfB1 on the AfM1 content in milk. The contamination of corn silage was set to the limit of quantification of AfB1. With an inclusion rate of corn silage in the dairy cow diet of 27% and a low AfB1 contamination level, a minimal change in the AfM1 content in milk was observed.
Our model concludes that under current inclusion rates of ingredients in compound feed and current AfB1 levels in feed ingredients, the probability of exceeding this EC limit is very low, and only a highly contaminated batch would be cause for concern. When considering the same lactation scenario and the same compound feed composition scenario, an increase in the milk yield and the daily feed intake by a factor of 1.3 resulted in a maximum increase of 0.2% in the probability of exceeding the EC limit in milk. Hence, using the current guidelines for compound feed composition, and the current limits on AfB1 in feed materials, an increase in milk yield does not appear to increase the probability of exceeding the limit of AfM1 in milk.
In 2013, a contaminated shipment of maize intended for feed materials was imported into the Netherlands [3
]. The batch had a mean AfB1 concentration of 50.2 (±36.1) µg/kg, which is much higher than the EC legal limit for using maize as an ingredient (being 0.02 mg/kg at a moisture content of 12%). This batch was, however, not found to be contaminated during regular monitoring and was used for the production of a compound feed for dairy cattle. Such a batch was included in our transfer model, also using Monte Carlo simulations for the simulation of the AfB1 contamination of the feed ingredients and for the inclusion rate of each ingredient for a two-week period (weeks 25 and 26). Under normal lactation, 5%–28% of the simulations exceeded the EC limit for AfM1 in milk in the respective weeks. However, the probability, when including this contaminated feed in our model, was considerably dependent on the transfer equation used.
The transfer rate used to set the EC legislative limit in feed for dairy cows was 1%–2%. However, several studies found higher transfer rates of aflatoxins in cows with a higher milk yield [6
] and in early/mid-lactating cows [7
]. This was incorporated in our model through a 45-week lactation cycle, with milk yield varying through the cycle. The transfer rate varied, possibly due to differences between the metabolisms of the cows, the milk yield, and the source of contamination. In fact, the source of the contamination, milk yield, and cow breed in the studies available varied. Veldman et al. [7
] used contaminated groundnut meal, Britzi et al. [8
] and Masoero et al. [6
] used contaminated corn meal, and the model by Van Eijkeren et al. [9
] was fitted to data by Frobish et al. [16
] using contaminated cottonseed. Concerning breed, Britzi et al. [8
] carried out their study on Israeli Holstein cows (high-yield cows with an average yield of 11,400 kg milk
/cow), Masoero et al. [6
] carried out their study on Holstein cows, and Veldman et al. [7
] did not specify which cows were used. The maximum transfer rate from Veldman et al. [7
] and Britzi et al. [8
] was about 6%; however, in the model set up by Van Eijkeren et al. [9
], the maximum transfer rate was 3.2%. When the model set up by Van Eijkeren et al. [9
] was applied to the results from Masoero et al. [6
] and Veldman et al. [7
], the model did not agree with the data. The rate of exceedance of the EC threshold varied considerably depending on the equation used. However, it is unclear which of these equations is most suitable within our model, and hence all should be considered.