Analysis of E.U. Rapid Alert System (RASFF) Notifications for Aflatoxins in Exported U.S. Food and Feed Products for 2010–2019

The most common, toxic, and carcinogenic mycotoxins found in human food and animal feed are the aflatoxins (AFs). The United States is a leading exporter of various nuts, with a marketing value of $9.1 billion in 2019; the European Union countries are the major importers of U.S. nuts. In the past few years, border rejections and notifications for U.S. tree nuts and peanuts exported to the E.U. countries have increased due to AF contamination. In this work, we analyzed notifications from the “Rapid Alert System for Food and Feed (RASFF)” on U.S. food and feed products contaminated with mycotoxins, primarily AFs, for the 10-year period 2010–2019. Almost 95% of U.S. mycotoxin RASFF notifications were reported for foods and only 5% for feeds. We found that 98.9% of the U.S. food notifications on mycotoxins were due to the AF contamination in almond, peanut, and pistachio nuts. Over half of these notifications (57.9%) were due to total AF levels greater than the FDA action level in food of 20 ng g−1. The Netherlands issued 27% of the AF notifications for U.S. nuts. Border rejection was reported for more than 78% of AF notifications in U.S. nuts. All U.S. feed notifications on mycotoxins occurred due to the AF contamination. Our research contributes to better understanding the main reasons behind RASFF mycotoxins notifications of U.S. food and feed products destined to E.U. countries. Furthermore, we speculate possible causes of this problem and provide a potential solution that could minimize the number of notifications for U.S. agricultural export market.


Introduction
Mycotoxins are unavoidable and unpredictable toxic fungal secondary metabolites produced by three major genera of soil-borne molds: Aspergilli, Fusarium, and Penicillium [1,2]. The most common, toxic, and carcinogenic mycotoxin found in human food and animal feed are the aflatoxins (AFs), especially aflatoxin B1 (AFB1) [3][4][5]. AFs have been reported to be present in a wide variety of crops, including corn, wheat, soy, rice, cottonseed, tree nuts, oilseeds, herbs, and spices. Animal byproducts such as milk, meat, and egg can also be at risk of AF contamination [1,[6][7][8][9]. Moreover, AF contamination is one of the key foodborne risks that is greatly influenced by climate conditions. High temperatures, humid weather, and drought stress are favorable conditions for dissemination of and infestation by the primary AF-producing fungus Aspergillus flavus [10,11]. Therefore, the ongoing global warming is expected to elevate the levels of AF contamination, especially in fields at the pre-harvest stage [11][12][13][14][15]. The United States is continuing to be a major producer and exporter of tree nuts such as almonds, pistachios, walnuts, pecans, and hazelnuts, and is one of the leading exporters of groundnuts (peanuts) worldwide Tables 1 and 2 [16]. In 2019, the market value of the U.S. tree nut and peanut exports to the world was $9.1 billion and $675 million, respectively. The European Union countries are the largest market for U.S. tree nuts, importing more than a third of all exported U.S. tree nuts, and they represent the third greatest market for exported U.S. peanuts [17][18][19][20]. Unfortunately, all these edible nuts are prone to fungal infestation and contamination with mycotoxins, especially AFs, that hamper the flow of the nuts across borders. Worryingly, increasing numbers of incidents where U.S. tree nuts exported to the E.U. countries have been rejected at the border because of AF contamination have occurred in the past decade.   Union  2977  2585  2707  2769  3114  2  India  606  521  738  663  823  3  Canada  686  598  643  696  696  4  Hong Kong  846  1156  1251  1052  692  5  China  208  182  243  328  606  6  United Arab Emirates  430  310  301  304  439  7  Japan  480  374  398  433  416  8  Mexico  269  253  256  371  344  9  Turkey  300  365  308  279  341  10  South Korea  354  296  305  290  290  All Others  1285  1261  1329  1331  1314  Total Exported  8441  7901  8479  8516  9075 Values in millions of dollars. Source: United States Agricultural Export Yearbook, USDA, 2019.
Mycotoxin contamination results in more notifications than any other hazard in the Rapid Alert System for Food and Feed (RASFF), and the foremost toxin that has been associated with the notifications is AFs, especially in the nuts and nut products [1,[21][22][23][24]. RASFF was established in 1979 by the E.U. countries to allow swift exchange of information on hazards in food and feed (chemical, biological, physical, and allergens) among the E.U. countries. All food and feedstuff imported into E.U. are checked by the competent authorities of the Member States, and when risks to public health are detected during these checks, information is disseminated through the RASFF to all E.U. Member States and to the exporting countries [24][25][26]. In recent years, certain audits have been conducted in U.S. by the European Commission/Directorate-General for Health and Food Safety in response to continuing high number of RASFF notifications for AF contamination in tree nuts and peanuts imported from the U.S [1,27,28].
Despite tremendous efforts to control fungal toxin contamination of food and feed products, U.S. tree nut and peanut consignments destined to the E.U. countries are often being rejected due to AF levels exceeding not only the E.U. maximum limit but even the FDA action level. This serious matter prompted us to analyze RASFF notifications for the occurrence of mycotoxins in food and feed products of U.S. origin exported to the E.U. countries during the period of 2010 to 2019. We mostly focused on food samples because they accounted for 95.7% of all food and feed notifications, and we considered AF contamination as the target for this study as it represents 98.9% of mycotoxin notifications. In this report, we examined the levels of AF contamination in food products of U.S. origin, notification years, the types of rejection, the countries issuing the most notifications, and the product category that is frequently contaminated with AFs. We aim to provide readyto-access data on mycotoxin contamination of food and feed products originating from the U.S. and exported to the E.U. countries. Moreover, the numbers of RASSS mycotoxin notifications issued for food products from other countries exported to E.U. countries were compared to those issued for U.S. food products. Along with other tracing tools, this study will help to explore the root problems contributing to AF contamination in U.S. tree nuts and may help to assess risk factors associated with the problem.

Levels of AF Contamination in the U.S. Nuts as Specified in RASFF Notifications
The percentage of U.S. nuts containing AFs from 2010 to 2019 notified by RASFF is illustrated in Figure 2. We allocated the RASFF notifications by the levels of AF contamination in U.S. nuts into three groups: >4 to ≤10 ng g −1 as group one, >10 to <20 ng g −1 as group two, and ≥20 ng g −1 (FDA action level) as group three. Based on this classification, we found that 19% of notifications fell into group one (n = 81), 23% into group two (n = 98), and 57.9% of the notifications into group three (n = 247).

Levels of AF Contamination in the U.S. Nuts as Specified in RASFF Notifications
The percentage of U.S. nuts containing AFs from 2010 to 2019 notified by RASFF is illustrated in Figure 2. We allocated the RASFF notifications by the levels of AF contamination in U.S. nuts into three groups: >4 to ≤10 ng g −1 as group one, >10 to <20 ng g −1 as group two, and ≥20 ng g −1 (FDA action level) as group three. Based on this classification, we found that 19% of notifications fell into group one (n = 81), 23% into group two (n = 98), and 57.9% of the notifications into group three (n = 247).  Figure 3A shows the numbers of the RASFF AF notifications based on the type of notifications for 2010 to 2019. The RASFF notifications for AF in the U.S. originated nuts were classified as border rejection (78%), alert (5.8%), and information for attention and follow-up (15.8%). The top five major notifying countries were the Netherlands with 27% of notifications (n = 126), Spain with 12.33% of notifications (n = 57), Italy and the United Kingdom with 11.4% of notifications each (n = 53 each), and Germany with 10.3% of notifications (n = 48). Other E.U. countries reported 27% of RASFF notifications ( Figure 3B). In 2019, the top five major notifying countries were the Netherlands with 39% of notifications, the United Kingdom with 16% of notifications, Italy with 13% of notifications, Spain with 11% of notifications and Germany with 7.0% of notifications. Other E.U. countries reported 14% of RASFF notifications ( Figure 3C).  Figure 3A shows the numbers of the RASFF AF notifications based on the type of notifications for 2010 to 2019. The RASFF notifications for AF in the U.S. originated nuts were classified as border rejection (78%), alert (5.8%), and information for attention and follow-up (15.8%). The top five major notifying countries were the Netherlands with 27% of notifications (n = 126), Spain with 12.33% of notifications (n = 57), Italy and the United Kingdom with 11.4% of notifications each (n = 53 each), and Germany with 10.3% of notifications (n = 48). Other E.U. countries reported 27% of RASFF notifications ( Figure 3B). In 2019, the top five major notifying countries were the Netherlands with 39% of notifications, the United Kingdom with 16% of notifications, Italy with 13% of notifications, Spain with 11% of notifications and Germany with 7.0% of notifications. Other E.U. countries reported 14% of RASFF notifications ( Figure 3C).

RASFF Mycotoxin Notifications on the U.S. Originated Feed Products and the Affected Feed Category
Twenty mycotoxin notifications for feed products of U.S. origin were reported by RASFF during 2010 to 2019. All of these notifications were reported for AF contamination A B C

RASFF Mycotoxin Notifications on the U.S. Originated Feed Products and the Affected Feed Category
Twenty mycotoxin notifications for feed products of U.S. origin were reported by RASFF during 2010 to 2019. All of these notifications were reported for AF contamination of groundnuts for birdfeed and wildlife. Levels of total AF contamination ranged from 28 ng g −1 to 220 ng g −1 . Ten notifications reported total AF levels in the range of >20 and ≤50 ng g −1 , seven notifications fell in the range of >50 and ≤100 ng g −1 , and three notifications reported AFs levels of more than 100 ng g −1 . About 80% of these notifications were classified as border rejections and the other 20% were classified as information for attention. of groundnuts for birdfeed and wildlife. Levels of total AF contamination ranged from 28 ng g −1 to 220 ng g −1 . Ten notifications reported total AF levels in the range of >20 and ≤50 ng g −1 , seven notifications fell in the range of >50 and ≤100 ng g −1 , and three notifications reported AFs levels of more than 100 ng g −1 . About 80% of these notifications were classified as border rejections and the other 20% were classified as information for attention.

Summary of the Analysis of the RASFF Notifications
To the best of our knowledge, there are no publications or available reports which assess RASFF notifications for AF contamination of the U.S. nuts. In this work, we primarily analyzed RASFF AF notifications for U.S. food products exported to E.U. countries during the last ten years (2010 to 2019). We found that 98.9% of the notifications were reported for AF contamination of nuts (almonds, peanuts, and pistachios). The most frequent notifications were reported for pistachios (42%) followed by peanuts (34%) and almonds (22%). All kinds of nuts such as shelled nuts, nuts in the shell, kernels nut, or roasted and salted nuts were reported to have AFs in the RASFF notifications, with different prevalences and levels of contamination. More than half of these notifications (57.9%) reported total AF levels greater than the U.S. FDA action level for food (20 ng g −1 ). About 19% of notifications reported AF levels in the range of >4 to ≤10 ng g −1 , and 23% reported AF levels between >10 and ≤20 ng g −1 . The Netherlands issued more of these notifications than any other E.U. country, with issuing more than 27% of AF notifications for U.S. nuts. Border rejections constituted more than 78% of notifications for AF contamination in nuts that originated in the U.S. Only 1.13% of U.S. nuts were reported to be contaminated with other mycotoxins. Ochratoxin A contamination was reported in two notifications in ground corn and spaghetti. Deoxynivalenol was reported in three notifications associated with wheat and maize. No notifications were reported for patulin, fumonisins, zearalenone, and patulin.
We analyzed the RASFF notifications for mycotoxins in feed products of U.S. origin. We identified 20 relevant notifications. Interestingly, all these notifications reported AF contamination in groundnuts for birdfeed and wildlife. The levels of AF contaminations were 28 ng g −1 to 220 ng g −1 . About 80% of these notifications were classified as border rejections, with the other 20% classified as information for attention. The country issuing the most notifications for mycotoxins in feed products of U.S. origin was the United Kingdom followed by the Netherlands.
For the years 2010 to 2019, RASFF reported 5045 and 439 notifications on mycotoxin contaminations in food and feed products, respectively, exported to E.U. countries from all countries around the world. The U.S. is the fourth top country linked to the notifications, behind Turkey, China, and India. Among the notifications for food products from all countries, 89% (n = 4487) of the reported notifications attributed to AF contamination. The second most reported mycotoxin in food products was ochratoxin A, which was responsible for 10% (n = 507) of the RASFF notifications. Deoxynivalenol, fumonisins, zearalenone, and patulin were reported in less than 2% of notifications.
Regarding the feed products from all countries, AF contamination was reported in 98.4% of the RASFF notifications. Of these, 77.9% of the contaminated feed products were groundnuts and 11.6% were maize. Sunflower seeds, cottonseeds, rice bran, sorghum, and compound feed were also reported to be contaminated with AFs.

Problem Characterization
Due to frequent RASFF notifications for U.S. nuts destined to E.U. countries in recent years, some assessments have been conducted by the European Commission/Directorate-General for Health and Food Safety in the U.S. for pistachios and peanuts [27,28]. Overall, highly limited official U.S. control measures to tackle AF contaminations were noted, according to the findings of the most recent audit on peanuts (7-15 October 2019). In addition, sampling for AFs and analysis and validation of data for peanuts intended for export to the E.U. has not been performed to meet the 100% of the requirements of Regulation (EC) No. 401/2006 [27]. Another audit on U.S. pistachios that was conducted 5-12 September 2017, found that there were no official controls or requirements applicable to pistachios intended for export to E.U. In addition, there were no legal requirements applicable to these exports to ensure those sampling methods, analyses or reporting procedures fulfilled Regulation (EC) No 401/2006. Furthermore, samples rejected by RASFF due to AF contamination were not adequately followed up in the U.S. system to investigate possible root causes or to implement preventive measures [28].
While the USDA has implemented an instrument called AF certification for peanuts and tree nuts and has also adopted pre-export controls and check on AFs in U.S. nuts, most of these programs are voluntary. Most companies exporting to E.U. have signed memorandum of understanding to comply with these programs. No official controls are performed to verify compliance with this program, and there is no official supervision of consignments destined for the E.U. relating to AF contamination [27,28].
Another possible explanation for the large number of RASFF notifications on U.S. nuts is the gap between the FDA action level and the E.U. maximum level of contamination. The U.S. FDA has established an action level of 20 ng g −1 for total AFs (B1, B2, G1 and G2) for foods, peanut, and peanut products, and pistachio nuts [1,29]. European maximum levels for AF contamination of groundnuts, tree nuts, and processed nut products for direct human consumption are 2.0 ng g −1 for AFB1 and 4.0 ng g −1 for total AFs. If the nuts are subject to sorting or other physical treatment before consumption or will be used as an ingredient in foodstuffs, the limits for AFB1 and total AFs are 8.0 ng g −1 and 15 ng g −1 for groundnuts and 5 ng g −1 and 10 ng g −1 for tree nuts, respectively [30].
The lack of a surveillance program and a regular monitoring system to detect AF contamination and levels of contamination represent significant drawbacks that the U.S. is facing in the fight against AFs. No studies for the occurrence and levels of AFs in the U.S. nuts for 2010~2019 have been published. Regular monitoring and testing are insufficiently employed at the level of individual states. The results of the states' monitoring and surveillance packages could serve as a useful tool if the levels of AFs are elevated in a geographical area, alerting growers to the need for more attention and testing.

Conclusions and Recommendations
Almost 95% of U.S. mycotoxin RASFF notifications were reported for foods and only 5% for feeds. The number of E.U. RASFF notifications and border rejections of U.S. nuts, mainly pistachios, almonds, and peanuts, due to contamination with AFs have increased over last ten years (2010 to 2019). More than 50% of notifications were due to AF levels not only exceeding the E.U. maximum limits but also the U.S. FDA's action level. Pistachios were the nut type responsible for the most notifications over the last ten years. Border rejections constituted more than 78% of RASFF notifications for AF contamination in U.S. nuts destined to E.U countries.
RASFF reported 5045 and 439 notifications for mycotoxin contamination in food and feed products, respectively, exported to E.U. countries from around the world during the years 2010 to 2019. About 89% of food and 98.6% of feed notifications for mycotoxin contamination were attributed to AF contamination.
The growing numbers of reports on the health benefits of eating nuts will likely lead to increased consumption of these products. In addition, the food industry using large numbers of tree nuts to manufacture pastries, sweets, ice cream, and confectionary products. As U.S. continues to be the largest supplier of tree nuts to the globe, especially to E.U. countries, it is therefore of great importance to keep U.S. nuts sheltered from AF contamination by implementing a mandatory and enforceable legal framework for official export control procedures concerning AFs in nuts.
To avoid escalating numbers of E.U. RASSF notifications, U.S. sampling, method validation, and results and reporting should comply with the E.U. requirements of Regulation (EC) No 401/2006. Importantly, comprehensive surveillance data on the occurrence and levels of AFs in almonds, pistachios, and peanuts are urgently needed to assess the current and ongoing conditions of the problem. The rejected shipments of the nuts exported from U.S. to E.U. countries due to AF contaminations should be adequately followed up on in order to identify possible root causes and/or to implement preventive measures.

Material and Methods
Data were obtained from the RASFF portal (https://webgate.ec.europa.eu/rasffwindow/portal/?event=SearchForm&cleanSearch=1). Search criteria for the RASFF mycotoxins notifications in the U.S food and feed products over ten years (2010 to 2019) were as follows: Type "food and feed", Hazard category "mycotoxins", notified between "01/01/2010" and "31/12/2019", product flagged as "origin", product country "United States (US)". Search criteria for the worldwide RASFF notifications in food products over ten years ((2010 to 2019) were: Type "food", Hazard category "mycotoxins", notified between "01/01/2010" and "31/12/2019", product flagged as "origin", product country "not specified". The extracted data were exported from the RASFF portal directly to Microsoft Excel 2010 (Microsoft 365 MSO) to generate descriptive statistics. Each single notification list contained data in the following order: product category, date, reference, product type, notification type, notification basis, notified by, countries concerned, subject, action taken, distribution status, and risk decision. When more than one originating country was mentioned, or two countries (one for raw product origin and the other for processing and packaging), we considered the country of origin of the raw food.
Levels of AF contamination were extracted from the "subject" category into the Excel column. Usually, the RASFF portal presents the results on AFB1 and total AFs (B1, B2, G1, G2). In this report, we considered the total AFs because the FDA action level is set for total AFs. Therefore, AF concentrations in this report represent the summation of four AFs (B1, B2, G1, G2).
There are three major types of RASFF notifications: alert, information, and border rejection. Alert notifications typically are delivered through RASFF when the hazard is detected in food and feed that are already present in the E.U. market and a rapid action is required to protect the public. Information notifications are used when a hazard is detected in food or feed placed in the market of one E.U. country but has not reached other E.U. members' markets. In this case, the risk does not require rapid actions. Regulation (E.U.) No 16/2011 defines two sub-types of information notifications: 'information notifications for follow-up' and "information notifications for attention". Border rejections are concerned with food and feed consignments that have been rejected at the external borders of the E.U. due to the presence of hazard in food and feed [21,24,31].

Data Availability Statement:
The data presented in this study are openly available in the European Commission RASFF portal database (https://webgate.ec.europa.eu/rasff-window/portal).