A total of 77 dairy feeds from smallholder establishments of Limpopo and Free State were tested for multi-mycotoxin contamination using UPLC-MS/MS technology and were found to be contaminated by a range of 2–16 mycotoxins across feed type classifications. Summary statistics across these feed types for means, ranges, numbers and percentages of positive samples are given in Table 2
. Total mixed rations (TMR) were found to be the most contaminated feed type with up to 16 mycotoxins found, possibly due to the nature of TMR, with several ingredients (compound and forage) mixed, of-ten to the farmers’ discretion. Additionally, commercially bought compound feed such as the dairy concentrates, meals and pellets were also found to contain 8–9 different mycotoxins. Overall, 20 of the 23 mycotoxins assessed were detected across all feed samples with 86% (66 of 77) of the samples evidenced to have at least one mycotoxin above respective decision limits. FUS-X, HT-2 and NEO were absent across all samples with trace amounts (<CCα) of FB3
detected in one sample. Overall incidence rates inclusive of trace amount detections across all mycotoxins are depicted in Figure 1
, and associated contamination data is given in Supplementary Materials Table S1
Findings demonstrate that DON, STERIG, AOH, and ENN B were the most com-monly found mycotoxins with detection rates of 63.6, 45.5, 42.8 and 32.5%, respectively. Indicative of less prevalent patterns were 3-ADON, 15-ADONs, FB1
and ZEN (at 23.4–9.1%), while the rest (NIV, AFB1
, DAS, OTA, AME and ROQ-C) appear to occur marginally across feed samples. In view of mycotoxin detections with concentrations above respective decision limits, additional descriptive statistical analysis of the data on the natural occurrences per farm and by specific feed type presented in Supplementary Materials Tables S3 and S2
2.2.1. Regulated Mycotoxins: A South African and European Commission Perspective
Concerning the presence of regulated mycotoxins; these were in this study found in line with the findings of Gruber-Dorninger et al. [20
], who reported that DON, FUM, and ZEN were the most prevalent regulated mycotoxins in SA feeds. Where SA regulations [21
] for mycotoxins in dairy feedstuff stipulate maximum permissible limits of 5 µg/kg for AFB1
, 3000 µg/kg for DON, 500 µg/kg for ZEN, 50,000 µg/kg for fumonisins (FUMs), and no specifications for OTA, the EC regulatory limits and guidelines [22
] specify a higher 5000 µg/kg for DON, with similar to SA limits for AFB1
, FUMs, ZEN and an additional 250 µg/kg for OTA; thus, few instances of concentrations in excess of regulatory limits were in our case identified.
As demonstrated in Table 2
, even though the Fusarium
toxin DON showed the highest prevalence of 63.6% at mean and maximum levels of 477.7 and 2385 µg/kg, ZEN, the common DON co-contaminant in animal feed [23
], at its much lower prevalence rate of 8.9%, demonstrated a higher mean of 666.7 µg/kg with an upper range of 1793 µg/kg. Despite only two samples (dairy meal and soya bean stover) exceeding the 500 µg/kg SA and EC regulatory limits, general DON and ZEN occurrence patterns appear comparable with [24
], whose studies on global feeds across various feed types report the common simultaneous occurrence of DON and ZEN due to similar fungal production lines, but with DON occurring at a higher degree.
Despite differences in intrinsic toxicities [25
], the derivatives 3-ADON and 15-ADON, which are also contemplated to be of equivalent toxicity to DON in the animal via their de-acetylation at absorption [26
], were found in this study to be less prevalent in overall feed samples occurring more in compound feeds than forages. 3-ADON was found present in 13/77 (16.9%) of overall samples at concentrations ranging from <CCα to 300 µg/kg (mean level: 55.5 µg/kg). Similarly, 15-ADON was detected in 16/77 (20.8%) of all samples at levels ranging 16.0–858.8 µg/kg (mean level 169.6 µg/kg). The highest 3-ADON (300 µg/kg) and 15-ADON (858.8 µg/kg) concentrations detected were from the same maize stover sample that also showed the highest concentration of DON (2385 µg/kg). Of the 49 times DON was detected, the detection ratio for co-occurrence of DON + 3-ADON + 15-ADON: DON + 15-ADON: DON + 3-ADON was 2: 2: 1.25 with DON + 3-ADON + 15-ADON detected in 10% of overall samples.
Regarding ZEN, the previously mentioned low incidence rates appear common-place for South African feed and raw materials with the perception that ZEN maybe a minor yet insistent contaminant in these matrices [10
]. The absence of ZEN in grasses, lucerne and silages (Supplementary Materials Table S2
) may be attributed to the presence of a less predominantly ZEN producing fungi in these matrices given the specific micro-climates relative to the study. Additionally, as noted by Driehuis [29
], proliferation and growth of ZEN producing fungi happen in the field, as the plant grows, thus in the case of grazed grasses and ensilaging forage, which tend to be cut or consumed before full maturity of the plant, it may be rational to assume much lower level contamination in these feeds.
Contrary to the normally reported high rates of fumonisin (FUM) contamination in South African feedstuff [12
], this study demonstrated lower prevalence rates for FB1
at 23.4, 19.5 and 1.3%, respectively, with accompanying contamination means and maximum values well within the legislated limits (FB1
—mean level 189.8 µg/kg: maximum level 485.2 µg/kg; FB2
—mean level 132.4 µg/kg: maximum level 416.9 µg/kg; FB3
single detection: <CCα) (data shown in Table 2
). The single detection of FB3
in trace amounts was from a sample of dairy pellets (Supplementary Materials Table S1
). It is clear, though, in comparison to forage material, that compound feeds demonstrated on average much higher FB levels, which appears common for this category of feeds. Additionally, this study reports on a total absence of FUMs in lucerne and molasses meals (Supplementary Materials Table S2
), which Knusten et al. [30
] assert may be due to the presence of sugar-rich ingredients that may favour the formation of differently structured modified fumonisins from Maillard-type reactions between reducing sugars. Although measurement of these modified fumonisins can ideally be done using indirect approaches, this was not covered in the framework of this work.
Aflatoxins (AFs) and OTA were predominantly absent in most of the samples with marginal prevalence rates of 3.9% each for both total AFs and OTA. Considering the potential risk that AFs pose on human and animal health, this group of mycotoxins is the most commonly monitored and regulated to ensure outbreaks of associated mycotoxicosis are not in question. As infrequent or marginal as these detections may be, they none-the-less contribute to an increased AF intake among lactating dairy cows, which in turn may have carry-over effects of direct proportion in animal tissues and by-products [31
]. However, contradictory to studies reporting high AF prevalence in SA feeds [9
], this study observed much lower detection rates for AFB1
(3.9%) and AFG2
(1.3%) (Table 2
). The totality of AF contamination in our study appears to have been from one farm, where the major source might have been a contaminated dairy concentrate used in the formulation of other mixed rations tested. In instances of quantifiable detection, AFB1
(mean level: 26.1 µg/kg; maximum level 30.2 µg/kg) and AFG1
(mean level: 20.2 µg/kg; maximum level 23.1 µg/kg) independently showed mean levels of contamination exceeding both the SA 10 µg/kg and EU 20 µg/kg regulatory limits for total aflatoxins and the 5 µg/kg SA and EU limit for AFB1
in dairy feeds.
Correspondingly, low AF detection rates in SA feeds and feed ingredients have been reported for AFB1
in varying ratios of B/G analogues [10
]. In the current study, total AFs in two of the three positive samples in question were at least 2.5 times above the 20 µg/kg EU regulated limit for total aflatoxins (dairy concentrate: 51.7 µg/kg and total mixed ration 2: 62.9 µg/kg). In the same two samples, the highly toxic AFB1
was found at levels up to four times the 5 µg/kg regulatory limit for AFB1
alone. Research on AFB1
contamination in relation to storage time has demonstrated increases in AFB1
levels in compound feeds stored for an excess of one month [36
]. Additionally, as with this study, mixed feed rations, when found AF positive, have been reported to be either more frequently or more severely contaminated, this possibly due to their multi-ingredient nature [37
]. As with studies such as those of [38
], an absence of AFs in forage material is also reported.
Further ascertaining the assertion that low OTA detections may be the norm in South African dairy feeds over the past years [17
], the current study found (Table 2
) a 3.9% incidence rate for OTA in the 77 tested samples (mean level: 85.6 µg/kg; maximum level 187.9 µg/kg) with all positives having levels below the 250 µg/kg EC guidance limits specified for cereal-based feeds. While these results are lower than the guidance values, long-term persistent exposure may lead to losses in yield alongside other chronic toxicities in animals [39
]. This low prevalence could be accounted for by the complete absence of this mycotoxin in forages as corroborated by other studies [37
], possibly due to most of the OTA producers’ inability to tolerate high acetic acid concentrations characteristic of ensilaging or grass/hay bailing preservative processes [43
]. Furthermore, the fact that OTA production largely occurs in species-specific temperatures ranges of 25 to 37 °C and associated lower water activity (below ≈ 0.84) [44
], can also account for the comparative absence of this mycotoxin in the Limpopo samples vs. Free State samples. OTA occurrence patterns seem to follow that of AFs, possibly due to the common Aspergillus
species production lines.
Due to the complexities of matrix effect in both commercial feeds and forages, calibration data were inadequate for the correct quantification of T-2 and HT-2 according to confirmatory criteria specified by the method employed. However, a clear outlier for T-2 contamination at the estimated but unconfirmed concentration of 11 002.6 µg/kg was recorded in a single sample of lucerne, this result was nonetheless disregarded as a confirmed value in the overall results.
2.2.2. Non-Regulated Mycotoxins
The other non-regulated mycotoxins STERIG, AOH, and ENN B were found most frequently at prevalence rates of 45.5, 42.8 and 32.5% at mean concentrations of 25.8 μg/kg (range: <CCα–139.1 μg/kg) 279.2 μg/kg (range: 15.5–3088.2 μg/kg), and 1195.1 μg/kg (range: <CCα–14,230.4 μg/kg), respectively. Enniatins only became an issue of high concern as emerging mycotoxins in recent years, thus information on their occurrence in sub-Saharan Africa (SSA) feedstuff is scarce [28
]. However, in compliance with our results for the confirmed presence of the most bioactive of the group (ENN B), literature on global feed occurrences is representative of moderate to high detections in compound cereal-based feeds and low to sporadic detections in forages [46
]. To note however would be the high levels found in this study with up to 9 of the 22 detections comparatively in excess of results from Rasmussen and Storm [46
] on visibly moldy hotspot silages.
Although data on the adverse health effects of STERIG on dairy cattle are scarce, the AFB1
-structurally related toxic precursor of AF production is occasionally reported as a contaminant of feeds [40
]. Owing to the shared structural similarities between STERIG and AFs, commonalities in prominent toxicities (hepatotoxicity, genotoxicity and carcinogenicity) remain apparent, with the potency of AFB1
considered up to 10 times that of STERIG [43
], maximum limits, however, remain unestablished. This study documents the moderate to high-level STERIG contamination with the highest levels found in samples of grass (89.7 μg/kg) and lucerne (139.1 μg/kg) and all three AFB1
occurrences were accompanied by STERIG (6.3–30.9 μg/kg).
With much less known about Alternaria
mycotoxin occurrences in Southern African feeds, this study documents somewhat comparable prevalence rates of AOH with [12
], though at much higher levels (maximum level: 3088.2 μg/kg). Maximum concentrations of AOH were herein found in forage material with highest levels detected in grasses. Monitoring of these possibly mutagenic, genotoxic and precancerous [50
] representatives of field spoilage may increasingly be a noteworthy endeavor in SSA given climate change and the insistent need to preserve the farm to fork food chain. Overall, the highest concentrations of AOH, STERIG and ENN B were found in respective samples of grass, lucerne and molasses meal. The remaining mycotoxins, i.e., NIV, DAS, AME and ROQ-C were only found marginally in feed samples.