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Mycotoxins presence in pre- and post-fermented silage from Tunisia
⁎Corresponding author at: Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, University of Valencia, Av. Vicent Andrés Estellés, s/n 46100 Burjassot-Valencia, Spain. ana.juan@uv.es (A. Juan-García)
-
Received: ,
Accepted: ,
This article was originally published by Elsevier and was migrated to Scientific Scholar after the change of Publisher.
Peer review under responsibility of King Saud University.
Abstract
Silage represents a major part of the feed ration for livestock, being a potential cause of mycotoxicosis as it can be contaminated with toxigenic fungi capable of producing mycotoxins in suitable environmental conditions.
In the present work, the presence of natural mycotoxins in samples collected of silage from the main producing areas in Tunisia (Ariana, Bizerte, Béjà and Jendouba) was assessed based on different plant material (oat, barley, triticale, artichoke, sulla or raygrass). Mycotoxins were evaluated during three periods in green forage (P1) and subsequently in ensiled products after 60 days (P2) and 100 days (P3) of fermentation. Samples were extracted by a QuEChERS procedure and analyzed by mass spectrometry for the determination of 23 mycotoxins. The results showed the presence of Fusarium mycotoxins, deoxynivalenol (DON), HT-2 toxin, zearalenone (ZEA), enniatins (ENA1, ENB, ENB1) and beauvericine (BEA); as well as one Alternaria mycotoxin tentoxin (TENT). The highest values detected were for DON in the three periods. DON maximum value were 381, 2053 and 916 µg/kg for P1, P2 and P3, respectively. This study demonstrated the presence of fungi and DON, ZEA and ENs contamination in Tunisian silage without a recognized risk on the ruminants or further in humans. However, a continuous enhancement of the silage quality, and management and control of mycotoxins, should be implemented to ensure safe ensilable plant material.
Keywords
Silage
Post-fermented
Pre-fermented
Mycotoxin
LC-MS/MS
QuEChERS
Tunisia
1 Introduction
The animal feed system in Tunisia is experiencing numerous disturbances related to climatic, economic, social and environmental factors (Ben Salem et al., 2011). This could be explained by irregular and seasonal distribution of feed production as well as the extensive characteristics of livestock farming.
Nowadays, the main issues encountred in livestock farming in Tunisia are related to a significant decrease in rangelands, a considerable drop of the consumable biomass yield as well as the lack of fodder which remains of poor quality (Jemaa et al., 2016; Ben Salem et al., 2011; Mohamed-Brahmi, Khaldi & Khaldi, 2010).
The availability of stored food (wet or dry) during periods of shortages is a crucial solution to overcome the animal weight losses and avoid a potential mortality (Olivier et al., 2015). Silage is a technique for preserving green fodder through lactic fermentation under anaerobic conditions. This fermentation allows the stabilization of the plant material by minimizing losses of dry matter and nutritional value as well as the inhibition of the growth of undesirable microorganisms due to pH value drops (4–4.5) (del Palacio et al., 2016) and the effect of lactic acid towards other microorganisms (Ma et al., 2017). However, under several climatic conditions, presence of water, relative humidity, temperature or inadequate storage practices, allows molds growing and production of harmeful secondary metabolites as mycotoxins, may affect the silage safety (Boudra, 2009).
Furthermore, the silage production is not standerized, so that the silage quality and consequently the mycotoxins levels may vary widely due to various factors such as an inadequate rate of dry matter during harvest, a slow filling of the silo, a bad settlement, losses during anaerobic storage, imperfect hermeticity of the silo and aerobic deterioration after opening the silo (Gallo et al, 2015).
Mycotoxins are secondary metabolites produced by toxicogenic fungi (Aspergillus, Penicillium, Alternaria and Fusarium) in order to increase its competitiveness front other microorganisms and to increase substrates available for its growth (Duarte et al., 2011; Yiannikouris & Jouany, 2002). Mycotoxins can be developed on the plant in the field during its growth, in pasture or during storage (hay, in silage) during poor storage or handling conditions. Fusarium fungal genera can lead not only to mycotoxins contamination but to alter organoleptic and nutritive qualities of stored foods leading to a decrease in zootechnical performance (milk production, body weight gain, reproduction …) (Akande et al., 2006). Consumer can be exposed to mycotoxins throught meat or other animal derived products such as eggs or milk (Duarte, Lino & Pena, 2011). There is currently an important data on mycotoxins in animal nutrition. However, little work has been undertaken to study the adverse effects of mycotoxins on the quality of livestock feed.
In Tunisia, there is not available data on the effects of toxigenic fungi and mycotoxins on the quality of feed and the ruminants performances. To the better of our knowledge, this is the first report ever on silage feedstuffs performed in Tunisian samples. The present work aim to evaluate the natural muti-mycotoxins presence in green forage and subsequent ensiling products after 60 and 100 days fermentation within the optic of a better understanding of the mycotoxins dynamics and the conditions leading to follow up their toxicity problems in ruminants.
2 Materials and methods
2.1 Chemicals and reagents
Solvents (acetonitrile, hexane and methanol) were purchased from Merck KGaA (Darmstadt, Germany). Anhydrous magnesium sulphate was obtained from Alfa Aesar GmbH & Co. (Karlsruhe, Germany); sodium chloride was purchased from Merck and C18 was purchased from Phenomenex (Torrance, USA). The derivatization reagent composed of BSA (N,O-bis(trimethylsilyl) acetamide), TMCS (trimethylchlorosilane) and TMSI (N-trimethylsilylimidazole) were purchased from Supelco (Bellefonte, PA). Sodium dihydrogen phosphate and disodium hydrogen phosphate, used to prepare phosphate buffer, were acquired from Panreac Quimica S.L.U. (Barcelona, Spain). The standards of aflatoxins (AFB1, AFB2, AFG1, AFG2), ochratoxin A (OTA), sterigmatocystine (STG), alternariol monomethylether (AME), alternariol (AOH), tentoxin (TENT), zearalenone (ZEA), nivalenol (NIV), deoxynivalenol (DON), 3-acetyl-deoxynivalenol (3AcDON), 15-acetyl-deoxynivalenol (15AcDON), diacetoxyscirpenol (DAS), neosolaniol (NEO), T-2, HT-2 toxin, beauvericine (BEA), enniatins (ENA, ENA1, ENB and ENB1), were purchased from Sigma Aldrich (Madrid, Spain).
2.2 Sampling
A set of feed samples (n = 84) were collected for the present survey. Fresh grass and consequent silages were taken from different silos where intensive breeding and silage production are practiced, and located in four regions: in Ariana and Bizerte located in Tunisian Northen area and Béjà and Jendouba in the Tunisian North West area. Samples were mainly composed of cereals (oat, barley, tritical) as well as other adjuvant plant material such as artichoke, sulla or ryegrass. Sampling was carried out from April to September 2018. According to the period of fermentation, three different set of samples were collected from each studied silo: i) before compaction (P1), ii) 60 days after (P2) fermentation starts but the material is not used in the feeding process yet; and iii) after 100 days of compaction (P3) when the fermentation is stopped and silage can be used. The sampling procedure is detailed as follows: At P1 stage, fresh material was taken randomly during silo filling; while, at P2 and P3 stages, plant material was collected from three different layer of the silos (upper, middle and lower) at 50 cm depth. Plant material was homogenized and mixed thoroughly to obtain a representative sample of 1 kg placed in sealed plastic bags. Samples were dried at 50 °C for 48 h, milled into fine powder and stored at −20 °C in PTFE tubes prior to the analysis for mycotoxins presence.
2.3 Mycotoxin analysis
2.3.1 QuEChER extraction
Tweenty three mycotoxins were extracted according to the methodology developed by Juan et al., (2017) with few modifications. The parameters of instrumental linearity, matrix effect, sensitivity, accuracy and precision were studied for validation method, according to the EU Commission Decision, 2002/657/EC (EC, 2002). These validation was done previously to apply on studied sample.
Briefly, 5 g of each homogenized sample were extracted with 30 mL of acetonitrile/water /acetic acid (79:20:1, v/v/v) homogenized for 15 min in an horizantal shaker and centrifuged for 5 min at 1792g (G-force) and 5 °C using an Eppendorf Centrifuge 5810R (Eppendorf, Hamburg, Germany).
The upper layer was filtered through Whatman filter paper and transfered into 50 mL centrifuge tube. Then, 4 g of MgSO4 and 1 g of NaCl were added. The mixture was shaken for 2 min and centrifuged (10 min, 1792g, 5 °C). Afterwards, 2 mL of the upper-layer was transferred into 15 mL centrifuge tube and added 100 mg of C18 and 600 mg of MgSO4. The tube was shaken for 1 min and centrifuged for 10 min at 1792g and 5 °C. Finally, the upper layer was filtered through a syringe nylon filter (13 mm/0.22 μm) purchased from Anàlisis Vínicos S.L (Tomelloso, Spain). The final volume was transfered into conical vials, 250 µL for derivatization and GC–MS/MS analysis and the rest for LC-MS/MS analysis.
2.3.2 Derivatization
Before GC–MS/MS analysis, 250 µL of the filtred were dried under a gentle stream of nitrogen using a Zymark TurboVap LV Evaporator. The dry extracts were re-dissolved in 50 µL of BSA + TMCS + TMSL (3:2:3) and left for 30 min to react at room temperature. The derivatized sample was diluted to 250 µL with hexane and vortexed for 30 s. The derivatized was purified with a liquid–liquid extraction adding 1 mL of phosphate buffer (60 mM, pH 7). Finally, 100 µL of the upper layer was transferred to an autosampler vial for its GC analysis.
2.3.3 GC–MS/MS analysis
The final extract was injected in splitless mode at 250 °C in programmable temperature vaporization (PTV) using an Agilent 7890A GC system coupled with an Agilent 7000A triple quadrupole mass spectrometer with inert electron-impact ion source and an Agilent 7693 autosampler (Agilent Technologies, Palo Alto, CA). The mass spectrometer was operating in electron impact ionization (EI, 70 eV). The source and transfer line temperatures were 230 °C and 280 °C, respectively. The collision gas for MS/MS experiments was nitrogen, and the helium was used as carrier gas at fixed pressure of 20.3 psi, both at 99.999% purity supplied by Carburos Metálicos S.L. (Barcelona, Spain). Data have been acquired and processed using the Agilent MassHunter version B.04.00 software. The quantification and confirmation transition were performed as described previously by Oueslati et al., (2018). Eigh mycotoxins were analyzed by GC–MS/MS. NIV, DON, 3-ADON, 15-ADON, DAS, NEO, T-2 and HT-2 and have been separated on a HP-5MS (30 m × 0.25 mm × 0.25 µm) capillary column (Sup. Figure 1). The oven temperature program was initially 80 °C, and the temperature increased to 245 °C progressively at 60 °C/min. After a 3 min hold time, the temperature was increased to 260 °C progressively at 3 °C/min and finally to 270 °C at 10 °C/min and then held for 10 min.
2.3.4 LC–MS/MS analysis
From filtered final volum, as indicate in Section 2.3.1., 0.5 mL was evaporated to dryness at 38 °C under a gentle stream of nitrogen using a multi-sample Turbo-vap LV Evaporator (Zymark, Hoptkinton, USA). Before LC-MS/MS analysis the dry residue was reconstituted to a final volume of 0.5 mL with methanol/water (70:30, v/v) and filtered through a 13 mm/0.22 μm nylon filter purchased from Anàlisis Vínicos S.L (Tomelloso, Spain).
The analysis was performed using a LC Agilent 1200 using a binary pump with automatic injector, and coupled to a 3200 QTRAP® AB SCIEX (Applied Biosystems, Foster City, CA) equipped with a Turbo-VTM source (ESI) interface. The chromatographic separation of the analytes was conducted at 25 °C with a reverse phase analytical column Gemini®NX-C18 (3 μm, 150 × 2 mm ID) and a guard column C18 (4 × 2 mm, ID; 3 μm) from Phenomenex (Madrid, Spain). Mobile phase was a time programmed gradient using methanol as phase A (0.1% formic acid and 5 mM ammonium formate), and water as phase B (0.1% formic acid and 5 mM ammonium formate). The following gradient was used: equilibration for 2 min at 90% B, decrease linearly to 20% of phase B in 3 min, maintain 20% of phase B for 1 min, decrease linearly from 20 to 10% of phase B in 2 min, maintain 10% of phase B for 6 min, decrease to 0% B in 3 min, maintain 100% A for 1 min, finally increase linearly from 0 to 50% B in 3 min, return to initial conditions (90% B) in 2 min and maintain during 2 min. The flow rate was 0.25 mL/min in all steps. Total run time was 21 min. The injection volume was 20 µL.
With regard to mycotoxin analysis, the QTRAP System was used as triple quadrupole mass spectrometry detector (MS/MS) (Sup Fig. 2 and Fig. 3). The Turbo-V™ source was used in positive mode to analyze the 15 mycotoxins (AFs, OTA, STG, AME, AOH, TENT, ZON, ENs and BEA) with the following settings for Source/Gas Parameters: Vacuum Gauge (10e−5 Torr) 3.1, curtain gas (CUR) 20, ionspray voltage (IS) 5500, source temperature (TEM) 450 °C, ion source gas 1 (GS1) and ion source gas 2 (GS2) 50. The precursor ions (Q1), product ions (Q3), collision energies (CE), collision cell exit potential (CXP), declustering potential (DP) and collision cell entrance potential (CEP) (Stanciu et al., 2017). The entrance potential (EP) was the same for all analytes, 10 V. Acquisition and processing data were performed using Analyst® software, version 1.5.2.
3 Results and discussion
Many regions in Tunisia, especially in the northern area, ensiled forages are highly valuable as animal feed used in major dairy farms. In order to assess the quality of silage from Tunisia, the present work evaluated the natural presence of 23 different mycotoxins during all the production process. The analytical method used in this investigation was based on both LC-MS/MS and GC–MS/MS techniques, which LOD and limit of quantification (LOQ) were estimated using an extract of the blank for a signal-to-noise ratio (S/N) ≥3 and ≥10, respectively, from chromatograms of samples spiked at the lowest level validated. Sensibility of which was below the European recommended levels, the limit of detection (LOD) values ranged from 0.5 to 25 µg/kg (Table 1). The validation results indicates that linear regression coefficients of all calibration curves demonstrated a good linearity, with corresponding correlation coefficients (r2) higher than 0.9989. LDs and LQs of the mycotoxins analyzed presented a high variability and were between 0.1 and 500 µg/kg. The accuracy was evaluated for each compound and recovery (n = 6) values were between (67 ± 7) % (ZEA in Triticale) and (108 ± 9) % (BEA in Rayegrass). Regarding precision values evaluated from the relative standard deviation (RSDR) of intraday precision (n = 6) and interday precision (n = 9), these were lower than 9% and 15%, respectively. LOD:Limit of detection; LOQ: limit of quantification.
Mycotoxin
LOD (µg/Kg)
LOQ (µg/Kg)
Recommendation (µg/Kg)
DON
1.31
4.38
900–12000
Rec. 2006/576/EC
15-ADON
1.12
3.73
3-ADON
1.86
6.21
NIV
1.55
5.15
.
NEO
2.17
4.63
.
DAS
1.70
5.66
.
T-2
4.36
8.53
250–2000
Rec. 2013/165/EU
HT-2
1.97
6.55
ZEA
1.31
4.38
900–12000
Rec. 2006/576/EC
ENA
0.51
1.70
–
ENA1
0.51
1.68
–
ENB
0.29
0.98
–
ENB1
0.48
1.60
–
BEA
0.38
1.27
–
AFB1
1.68
5.60
5–20*
Directive 2003/100/EC
AFB2
1.84
6.13
AFG1
1.59
5.29
AFG2
2.09
7.64
STG
2.6
5.2
–
AME
16.43
54.78
–
AOH
24.94
83.14
–
TENT
1.84
4.47
–
OTA
0.84
2.81
50–250
Rec. 2006/576/EC
The analysis revealed the presence of eight mycotoxins amoung the 23 tested being mainly Fusarium mycotoxins (DON, HT-2 toxin, ZEA, ENA1, ENB, ENB1 and BEA) and one Alternaria mycotoxin, TENT (Table 2). These results are in compliance with those reported from various studies Gonzales Pereyra et al., 2008; Richard et al., 2009. McElhinney et al. (2015) reported no AFs, OTA, fumonisins and T-2 toxin in silage samples from Ireland. Another study in Uruguay indicated that freshly harvested samples were not contaminated with AFs (del Palacio et al., 2016). Further, no AFs were reported in silage samples from Poland (Kosicki et al., 2016; Panasiuk et al., 2019). Such observation may be due to the Aspergillus and Penicillium sp. intolerance to anerobic conditions and an acid environment. These previous results suggested the stability of these substances during fermentation (Boudra & Morgavi, 2008), however, it is important to notice that these mycotoxins did not appeared in post-fermentation phase of our study coinciding with Pereyra et al. (2008).
P1 (N = 26)
Mycotoxin
Ariana (N = 4)
Bizerte (N = 7)
Béjà (N = 10)
Jendouba (N = 5)
TOTAL
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I(F, %)
Max(µg/Kg)
M ± SD (µg/Kg)
DON
-
-
1 (14)
54.4 ± 144
3(30)
63.3 ± 105
-
-
4 (15)
381
39 ± 242
ENB
1 (25)
0.01 ± 0.02
3 (43)
0.3 ± 0.51
2 (20)
0.028 ± 0.07
1 (20)
0.036 ± 0.08
7 (27)
0.4
0.035 ± 0.26
BEA
1(25)
0.05 ± 0.1
-
-
-
-
-
-
1 (4)
0.2
0.008 ± 0.14
P2 (N = 26)
Mycotoxin
Ariana (N = 4)
Bizerte (N = 8)
Béjà (N = 9)
Jendouba (N = 5)
TOTAL
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I(F, %)
Max(µg/Kg)
M ± SD (µg/Kg)
DON
–
–
2 (25)
380 ± 599
1 (11)
129 ± 411.2
1 (20)
90 ± 201
4 (15)
2053
179 ± 1325
ENA1
–
–
1 (13)
0.04 ± 0.11
–
–
–
–
1 (4)
0.32
0.01 ± 0.22
ENB
1 (25)
0.075 ± 0.15
4 (50)
0.6 ± 0.82
2 (22)
0.096 ± 0.21
2 (40)
0.048 ± 0.08
9 (27)
2.6
0.24 ± 1.7
ENB1
–
–
1 (25)
0.175 ± 0.5
–
–
–
–
1 (4)
1.4
0.054 ± 0.9
BEA
1 (25)
0.035 ± 0.07
1 (25)
0.0125 ± 0.035
–
–
–
–
2 (8)
0.14
0.0092 ± 0.09
ZEA
1 (25)
4.27 ± 8.5
–
–
–
–
–
–
1 (4)
17.1
0.66 ± 1.2
TENT
1 (25)
7.4 ± 14.8
–
–
–
–
–
–
1 (4)
29.6
1.14 ± 20
P3 (N = 32)
Mycotoxin
Ariana (N = 6)
Bizerte (N = 11)
Béjà (N = 9)
Jendouba (N = 6)
TOTAL
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I (F,%)
M ± SD (µg/Kg)
I (F, %)
M ± SD (µg/Kg)
I(F, %)
Max(µg/Kg)
M ± SD (µg/Kg)
DON
3 (50)
593 ± 131
4 (36)
807.3 ± 48
–
–
2 (33)
537 ± 33
9 (28)
915.9
153.9 ± 538
HT-2
–
–
3 (18)
12.55 ± 42
–
–
1 (17)
5.17 ± 10
3 (9)
116
5.3 ± 78
ENA1
2 (17)
0.083 ± 0.12
–
–
–
–
–
–
2 (3)
0.5
0.016 ± 0.34
ENB
2 (33)
0.167 ± 0.20
5 (45)
0.41 ± 0.65
2 (22)
0.067 ± 0.14
2 (33)
0.07 ± 0.11
11 (34)
3
0.20 ± 1.98
ENB1
1 (17)
0.143 ± 0.35
2 (18)
0.13 ± 0.32
–
–
–
–
3 (9)
0.86
0.071 ± 0.56
The highest mycotoxin values detected in our samples were for DON at all three stages. DOŃs maximum value were 381, 2053 and 916 µg/kg for P1, P2 and P3, respectively. However, the most detected mycotoxin was ENB that was found in the 27% of samples at both P1 and P2, and in the 34% of samples at P3.
3.1 Occurrence of mycotoxins in the studied periods
During the first stage (P1), the sampled green forage intended for silage production was contaminated with three Fusarium mycotoxins: DON, ENB and BEA. ENB was the most frequently detected (27%) followed by DON (15%) and BEA (4%). DON was present in four samples with a maximum amount reaching 381 µg/kg, although it did not exceed the EU maximum guidance levels (EC, 2006). The maxium amouts of DON (198 µg/kg), and ENB (0.4 µg/kg) were found in samples from the region of Bizerte; while BEA (0.2 µg/kg) maximum level was found in Ariana region (Table 2). Both region are located in the Northern side of Tunisia characterized by having a humid climate, high levels of precipitations and average temperature of 26 °C.
During the second stage (P2), DON, ZEA, TENT, ENA1, ENB, ENB1 and BEA contaminated the silage samples being under a fermentation process for 60 days. ENB was the most frequently mycotoxin, detected in 7 samples (27%) with a mean value of 0.7 µg/kg. The highest concentration detected was for DON reaching 2053 µg/kg, which did not either exceed the EU guidance values (EC, 2006). Whereas, ZEA, TENT and BEA were detected at lower levels of 17, 30 and 0.14 µg/kg in 4%, 8% and 4% of analyzed samples, respectively. It is important to notice that the maximum amounts were found in samples from the Bizerte region where both annual relative humdity (73%) and mean temperature (18 °C) are suitable for the growth of Fusarium spp.
Furthermore, in the third stage (P3), where samples were under 100 days of fermentation showed to be contaminated with DON (28%), HT-2 (9%), ENA1 (3%), ENB (34%) and ENB1 (9%). It was observed that DON levels increased compared with the other stages, to reach a mean value of 1539 µg/kg; nonetheless, not even at this stage exceeded the EU guidance values (EC, 2006). In addition, ZEA and BEA decreased to traces levels. However, ENA1, ENB and ENB1 manteined their trend with mean values of 0.02, 0.2 and 0.07 µg/kg, repectively. The high incidence and maximum amounts of mycotoxins, even if the levels decreased, were also found in samples from the Bizerte region. In fact, this observation corroborate the effect of both warm temperature and high humidity for the fungal growth and subsequent mycotoxins production. In our case, it was subsequently supposed that both Fusarium graminearum and Fusarium avenaceum co-existed all over the silage production process even if they are quite affected by the fermentation procedure and anaerobiosis conditions.
DON has been previously described as one of the most common mycotoxin found in silage (Storm et al., 2008; Gallo et al., 2015; Cogan et al., 2016; Kosicki et al, 2016) present at different concentrations in pre- and post-fermentation samples as reported in the literature: in Lithuania in grass mixture silage at 1100 µg/kg; in The Netherlands, in maize silage at 651 µg/kg (Driehuis et al., 2008); in Uruguay, in wheat silage at 6007 µg/kg and in Germany, it was higher than 300 µg/kg (Driehuis et al., 2008). However, other authors have set that the presence of DON in silage is sporadic and if any, at low concentrations (Gallo et al., 2015; Wzmbacq et al., 2016) or on its modified forms (masked) as during the fermentation process they are able to bind to microbial metabolites or their cell walls (Berthiller et al., 2013; Kovač et al., 2018). Here it was revealed that levels and contamination frequency of DON were higher on pre-fermented samples. This suggest that DON contamination decrease during storage. Ensiling process affect fungal growth as pH decrease lead to unsuitable acidified environment and oxygen is consumed reaching anaerobiosis (Manisfield et al., 2005; Boudra & Morgavi, 2008). In a recent study it was indicated that during the ensiling, the presence of lactic acid bacteria increases which contributes to detoxification effect (Ma et al., 2017). Also, del Palacio et al. (2016) have stated that a reduction of the incidence and levels of 43% wheat silage in DON contamination was detected after a 4-months storage period.
This is the first report on emerging mycotoxins performed in silage samples from Tunisia. In compliance with our results, it has been confirmed the presence of ENs and BEA in silage (Sorensen et al., 2008; Shimshoni et al, 2013; Mcelhinney et al, 2015) with sporadic occurrence and/or low frequencies (Sulyok et al, 2010; Shimshoni et al, 2013). For HT-2, which was mainly found in the region of Bizerte, is known to be produced in hot and humid climate (Fels-Klerx, 2010; Xu et al., 2014). Regarding ZEA, higher mean levels have been detected in comparison with the present study as reported by Schmidt et al. (2015) at (181 ± 278) µg/kg and Driehuis et al. (2008) at 146 µg/kg. In addition, ZEA has been frequently reported in silage fodder (Schmidt et al., 2015; Storm et al., 2014).
3.2 Occurrence of mycotoxins according the type of silage
The available data in the litterature about the incidence of mycotoxins in silage are mainly focused on maize, grass and their mixture. Very few information on other types of silage have been published. In the present study, it has been included eight different silage products, four with unique silage variety (oat, tritical, rayegrass and artichoke) and four with combination of two silage variety (oat + triticale, oat + barley, sulla + oat and sulla + rayegrass). According to the studied matrix and the different sampling stages, it was observed that triticale and oat presented the highest incidence of mycotoxins (Table 3). Triticale had four different mycotoxins in both P1 (DON, and ENB) and P2 (ZEA and BEA); while, seven toxins were observed in oat at both P2 (DON, ENA1, ENB and ENB1) and P3 (DON, ENB1, ENB, BEA and HT-2) stages and in artichoke (ENB, BEA and TENT) in the three sampled stages. N: number of analyzed samples
Stage
Oat
Tritical
Rayegrass
Artichoke
N
Mycotoxin (I)
Mean (µg/Kg)
N
Mycotoxin (I)
Mean (µg/Kg)
N
Mycotoxin (I)
Mean (µg/Kg)
N
Mycotoxin (I)
Mean (µg/Kg)
P1
10
–
–
5
DON (2)
558 ± 1517
2
ENB (1)
0.12 ± 0.11
3
DON (1)
832 ± 1266
–
–
EN B (2)
0.8 ± 1.9
–
–
ENB (1)
0.067 ± 0.1
–
–
BEA (1)
0.05 ± 0.1
–
–
–
–
–
–
–
–
–
–
–
–
P2
15
DON (4)
310 ± 666
2
BEA (1)
0.05 ± 0.013
2
–
–
2
ENB (1)
0.15 ± 0.15
EN A1 (1)
0.02 ± 0.4
ZEA (1)
8.55 ± 9.9
–
–
BEA (1)
0.07 ± 0.049
ENB (7)
0.37 ± 0.8
–
–
–
–
TENT (1)
14.8 ± 18.9
ENB1 (1)
0.093 ± 0.026
–
–
–
–
–
–
P3
15
DON (5)
429 ± 148
3
EN A1 (2)
0.1 ± 0.2
2
–
–
3
–
–
ENB (4)
0.09 ± 0.89
EN B1 (2)
0.28 ± 0.072
–
–
–
–
HT-2 (3)
7.71 ± 1.91
ENB (3)
0.05 ± 0.69
–
–
–
–
ENB1 (1)
0.2 ± 0.052
DON (1)
245 ± 63
–
–
–
–
BEA (1)
0.095 ± 0.067
–
–
–
–
–
–
Stage
Oat + Triticale
Oat + Barley
Sulla + Oat
Sulla + Rayegrass
N
Mycotoxin (I)
Mean (µg/Kg)
N
Mycotoxin (I)
Mean (µg/Kg)
N
Mycotoxin (I)
Mean (µg/Kg)
N
Mycotoinx (I)
Mean (µg/Kg)
P1
2
DON (2)
2309 ± 1632
2
ENB (1)
0.1 ± 0.1
2
ENB (2)
0.7 ± 0.18
1
–
–
P2
1
–
–
2
–
–
2
ENB (1)
0.32 ± 0.33
1
–
–
P3
2
DON (2)
412 ± 291.3
2
ENB (2)
0.04 ± 0.028
2
ENB (2)
0.5 ± 0.7
1
HT2 (1)
22.4
–
–
–
–
DON (1)
925 ± 382.4
–
–
Regarding to silage samples containing mixtures of two varieties of forage, the most contaminated was sulla + oat combination, which presented more than one mycotoxin, and oat + triticale which presented only DON at the highest value (2309 µg /kg at P1). Sulla + oat samples presented DON (906 µg/kg) at P3 and ENB in slightly decresing amounts from P1 to P3 (from 0.7 to 0.5 µg/kg). The other two mixtures presented a trace contamination in oat + barley at 0.2 µg ENB/kg as well as HT-2 present in one sample at 22.4 µg/kg. Our results confirmed that the occurrence of mycotoxins is higher in cereals (oat and triticale) than in the other analyzed plant materials or the mixtured silage samples, since mycotoxins mainly fusariotoxins are frequently found contaminating cereals, which may represent a higher source of nutrients (Oueslati et al., 2018).
3.3 Co-occurrence of mycotoxins
In the present investigation, 11 samples were contaminated with more than one mycotoxin. Table 4 shows the combination of Fusarium mycotoxins co-occurring in each sample and its main composition. ENB was the major mycotoxin present in these samples. While, oat based samples were the most co-contaminated matrix with up to four mycotoxins (HT-2 + ENB1 + ENB + BEA) found at P3 in one sample. Moreover, other triticale samples at P3 were contaminated with four mycotoxins simultaneously (DON + ENA1 + ENB + ENB1): with three combined mycotoxins ENA1 + ENB + ENB1 and HT-2 + DON + ENB in triticale and oat, respectively at P3 and ENA1 + ENB + ENB1 in oat at P2. Lastly, with two combined mycotoxins DON + ENB in oat at P2 and P3; ENB + BEA in artichoke sample at P2; and DON + ENB in triticale at P1 were observed. In summary, mycotoxins combination were found mainly in oat based samples at the three stages, in seven samples and only two samples of triticale.
Number of Mycotoxins
P1
P2
P3
Combination
Type of silage
Combination
Type of silage
Combination
Type of silage
2 Mycotoxin
DON + ENB
Oat (1)
DON + ENB
Oat (2)
DON + HT-2
Triticale (1) Triticale + Oat (1)
HT-2 + ENB
Oat (1)
DON + ENB
Oat (2) Sulla + Oat (2)
ENB + ENB1
Oat (1)
ENB + ENB1
Oat (2) Triticale (1)
ENB + BEA
Oat (1)
ENB + 15ADON
Cc Maize (2)
Total
3
2
12
3 Mycotoxin
HT-2 + ENB + ENB1
Oat (1)
ENA1 + ENB + ENB1
Oat (1)
ENA1 + ENB + BEA
Sulla + Oat (1)
ENA1 + ENB + ENB1
Triticale (1)
HT-2 + DON + ENB
Triticale (1)
Total
3
1
1
4 Mycotoxin
15ADON + ENA1 + ENB + ENB1
Oat (1)
HT-2 + ENA1 + ENB + ENB1
Oat (1)
HT-2 + ENA1 + ENB + BEA
Oat (1)
HT-2 + FB2 + ENA1 + ENB
Sulla + Oat (1)
Total
4
5 Mycotoxin
HT-2 + ENA1 + ENB + ENB1 + BEA
Oat (1)
Total
1
Overall, the diversity of the mycotoxins combination was observed mainly at P3 with four different combinations of mycotoxins. In fact, the ready-to-use silage was brought back to the aerobic conditions which is a critical point that ensures the good quality of the silage as the optimal conditions may induce the growth of the existing fungi and the mycotoxins production again. Furthermore, at this stage, farmers also manage their silage products by adding concentrate supplements with regards to the targeted performances of the livestock. This may include other sources of mycotoxins by adding cereal grains (barley, maize, wheat…), plants by-products (tomatos, beets, olives, dates…) and also oilseeds (pea, soy, lupine…). It is a common practice to meet the specific needs of each farmers that have to be investigated further to ensure a safe silage quality at every scale. Although the frequency of co-occurrence of several mycotoxin in samples analyzed are low, observing the co-occurrence of mycotoxins with different chemical properties and modes of action in silage could imply a real issue if considering additive and/or synergistic effects; especially in dairy cows, which are economically and industrially of high importance for their derived products.
4 Conclusion
In the present study, results contribute to a better knowledge of natural mycotoxins contamination of green forage intended to the silage production as no data were published previously in Tunisia.
It is revealed that mycotoxins in silage from Tunisia depends on its composition, especially if they are oat based. Its presence starts in the pre-harvest season without exceeding EU levels along the silage production. Mycotoxin incidence may decrease during the ensiling process due to the fermentation, modification or masking of mycotoxins. Although the incidence of mycotoxin is reduced, amounts increase with the storage period, which decreases the transfer of mycotoxins to the animal by-products and safety in food consumers. The obtained data did not recognize any hazard on the ruminants or further derived products for consumers health in Tunisia. Still, it is important to promote the constant silage monitoring to collect information and build data on the toxigenic fungi dynamics and their mycotoxins under different environmental conditions. Further, a continuous enhancement of the silage quality and mycotoxins management and control should be implemented by developping ensilable plant material with a good resistant to fungal attack and environmental factors.
Acknowledgement
This research has been supported by the Spanish Ministry of Economy and Competitiveness [AGL2016-77610-R]. Special thanks to all members of the Ministry of Agriculture of Tunisia for their participation in the sampling procedure. Finally, thanks to the PhD student A. Mannai and Professor H. Ben Salem for their efforts to supply the samples and their availability.
Declaration of Competing Interest
None.
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Appendix A
Supplementary material
Supplementary data to this article can be found online at https://doi.org/10.1016/j.arabjc.2020.06.029.
Appendix A
Supplementary material
The following are the Supplementary data to this article:Supplementary data 1
Supplementary data 1
