Aflatoxins (AFTs) are a class of secondary metabolites produced by Aspergillus parasiticus and Aspergillus flavus (Piekkola et al., 2012). Aflatoxins are readily produced by Aspergillus flavus under relatively high air humidity and temperature conditions (Yu et al., 2004). Aflatoxins can be classified into several types, such as B₁, B₂, G₁, and G₂. Among them, aflatoxin B₁ (AFB₁) is the most toxic and has been classified as a class I carcinogen by the World Health Organization Cancer Research Unit (Yogendrarajah et al., 2015).

Currently, numerous studies have indicated that there is a risk of aflatoxins in dark tea. Liu et al. (2016) used high performance liquid chromatography–tandem mass spectrometry (HPLC–MS) for testing 10 samples of Pu-erh tea, among which AFB₁ was detected in two samples at 3.1 μg/kg and 7.5 μg/kg, respectively. Wu J. (2013) used HPLC to detect AFB₁ in seven samples at concentrations greater than 5 μg/kg in 60 samples of Pu-erh tea. Cui et al. (2020) examined 158 dark tea samples by liquid chromatography-mass spectrometry, two of which tested positive for aflatoxins. One Kangzhuan tea contained 2.07 μg/kg of AFB₁; the other Fuzhuan tea contained four aflatoxins at concentrations of AFB₁: 1.24 μg/kg, AFB₂: 0.78 μg/kg, AFG₁: 0.81 μg/kg, AFG₂: 1.04 μg/kg. The previous reports have shown that dark tea has the possibility of being contaminated with aflatoxins, but levels and detection rates of aflatoxins are very low, so the risk exposure level of aflatoxin in dark tea is very low. Aflatoxin, as a highly toxic carcinogen, has always been a great concern for tea consumers. Therefore, the most effectively way to prevent dark tea from being contaminated by exogenous aflatoxin during processing and storage is an issue worthy of attention and consideration.

Ochratoxin (OTs) is a group of secondary metabolites produced by fungi Aspergillus and Penicillium (Wang et al., 2018). Ochratoxins were first isolated by Vander Merwe from the metabolites of Aspergillus ochraceus (Van der Merwe et al., 1965). Ochratoxin is classified into several types based on its structure, including A, B, C, and, with ochratoxin A (OTA) ranking first. Available studies have demonstrated that OTA is nephrotoxic, hepatotoxic, immunotoxic, genotoxic, neurotoxic, and teratogenic (Chen et al., 2018). It is classified by the International Agency for Research on Cancer (IARC) as a renal carcinogen in animals and a probable carcinogen in humans. OTA contaminates a variety of crops and teas (Zhang, 2017; Deng et al., 2020; He et al., 2020; Zhao et al., 2022), with the main OTA-producing fungi differing depending on environmental conditions. The main fungi that produce OTA are shown in Table 1 (Sánchez-Hervás et al., 2008; Iqbal et al., 2014; Wang et al., 2016; Deng et al., 2020). Only parts of some strains of Aspergillus niger have the ability to produce OTA. There are differences in the ability to synthesize OTA on different substrates and under different environmental conditions (Deng et al., 2020).

Many researchers have tested OTA in dark tea using various testing techniques, and the results are detailed in Table 2. Combined with the above results, it can be observed that OTA in dark tea has a certain exposure risk. Many Aspergillus spp. fungi are dominant in the processing of dark tea, which is closely related to the formation of dark tea sensory quality (Wu J., 2013; Xiong, 2017). On the other hand, Fungi of the genus Penicillium have been detected during the storage of dark tea (Haas et al., 2013). OTA-producing fungi have similar strains to the dominant fungi in dark tea solid-state fermentation and storage As a result, there is a risk of OTA exposure during both the processing and storage of dark tea. The dominant strains in dark tea during the solid-state fermentation process are similar to many OTA-producing strains, but OTA is not only present in dark tea samples. OTA has been found in black and green tea samples, according to some studies (Santos et al., 2009; Malir et al., 2014). This indicates that the source of OTA in dark tea has two possibilities, therefore we should pay much more attention to fungal production of toxicity and exogenous contamination. And Zhao Z. et al. (2021) and Zhou et al. (2015a,b) isolated OTA-producing strains from dark tea samples.

Citrinin (CIT) was originally isolated from cultures of Penicillium citrinum by Hetherington and Raistrick (1931). CIT is a well-known hepatorenal toxin that causes functional and structural kidney damage as well as debilitation of liver metabolism (Flajs and Peraica, 2009; Vidal et al., 2018). In recent years, CIT has received much attention due to its toxic effects on mammals and its widespread presence in foods, where it is usually found together with OTAs (Silva et al., 2021). The main CIT-producing fungi are similar to the OTA-producing ones, mainly Penicillium and Aspergillus fungi (Sengling Cebin Coppa et al., 2019).

The kidney is the main target organ for the toxic effects of CIT, and there are also reports of CIT effects on the liver and bone marrow (Magan and Olsen, 2004). CIT is a fungal toxin widely present in food, and there is a possibility that dark tea is contaminated with CIT. Li and He (2020) examined 113 tea samples of Liupao tea from different years by HPLC, 37 positive CIT samples with a CIT content of 7.8–206.1 μg/kg were detected. Zhou et al. (2015a,b) isolated an OTA-producing Penicillium chrysogenum dominant strain from Pu-erh tea by inoculating it on raw dark tea with 35% moisture content at 30°C for 25 days, and the CIT content was 26.30 μg/kg.

Since the majority of OTA-producing fungi can produce CIT (Silva et al., 2021), OTA was also detected in many tea samples, which forced us to consider whether OTA-producing fungi in dark tea will also produce CIT. Bugno et al. (2006) isolated 10 genera of molds from herbs, 89.9% of the fungi were Aspergillus and Penicillium spp., and 21.97% of both Aspergillus and Penicillium spp. were able to produce CIT. Two hundred and sixty strains of Penicillium spp. were isolated from cereals and fruits by Andersen et al. (2004), and 85% of Penicillium spp. were able to produce CIT. In recent years, many other researchers have isolated and identified Penicillium citrinum from dark tea. Haas et al. (2013) isolated and identified two strains of Penicillium citrinum from traditional Pu-erh tea loose tea, three strains in organic Pu-erh tea loose tea, and three strains in tightly pressed Pu-erh tea, respectively. Su (2018) isolated and identified 34 fungal strains from four commercially available dark tea samples, including four strains of Penicillium oryzae. Zhao (2012) and Hu et al. (2020) isolated and identified Penicillium citrinum from ‘flowering’ of Fuzhuan tea and finished dark tea, respectively. Aspergillus spp. and Penicillium spp. fungi are very common in the production and processing of dark tea. Penicillium citrinum is also more common in dark tea. Therefore, there is a certain risk exposure level for CIT in dark tea, which poses potential health hazards to consumers. There are relatively few studies on CIT in dark tea samples, and there is almost no standard limit on the amount of CIT in dark tea.

Deoxynivalenol (DON), also known as vomitoxin, is named for its ability to cause vomiting in pigs. This is a secondary metabolite produced by toxigenic Fusarium species and others, combined with the high rate of contamination in both cereals and their products (Chen et al., 2017; Csikós et al., 2020; Wang and Wang, 2021; Yang et al., 2021). DON is potentially harmful to both humans and animals when it enters the food chain, such as causing loss of appetite, immunosuppression, nausea, and vomiting, and has been classified as a Class III carcinogen by the European Union (Pereira et al., 2019).

Although DON is mostly detected in cereals and their products, the researchers have not paid sufficient attention to it. In recent years, it has been reported that tea also has the possibility of being contaminated by DON. Yan (2019) examined 61 fermented dark tea samples, and DON was detected in three samples, including one Pu-erh tea with 8.6 μg/kg and two Hunan dark tea with 70.1 and 299.5 μg/kg, respectively. Hu et al. (2019) examined 174 Pu-erh tea samples, DON was detected in 30.63% of the samples. Although the content of DON in the detected dark tea samples is far below the limit, we should pay much more attention to DON, which can protect the health of consumers and promote the sustainable development of dark tea.

Masked mycotoxins, usually conjugated mycotoxins, are mycotoxin derivatives formed by microbial transformation, degradation, hydrolysis, reduction, glycosylation, formylation, etc. Most masked mycotoxins are less toxic or non-toxic than their original mycotoxins. However, some masked mycotoxins are not completely transformed and degraded, and under some specific conditions, these masked mycotoxins can be reconverted to mycotoxins under certain conditions (Berthiller et al., 2013; Tan et al., 2016).

The detection of masked mycotoxins is relatively cumbersome. In the initial studies, the “indirect method” was usually used as a quantitative method to detect the amount of masked mycotoxins. With the constant development of analytical techniques, the direct detection of masked mycotoxins has become more common. Currently, commonly used direct detection methods include LC–MS/MS and ELISA. LC–MS/MS method has been widely used for the detection of mycotoxin residues in cereals, grains, oils, and other foods for its high selectivity and sensitivity (Lozowicka et al., 2022). LC–MS/MS can simultaneously detect different components in mixed samples, so that masked mycotoxins, their prototypes, metabolites, and even different types of mycotoxins can be quantified simultaneously. Vendl et al. (2009) used LC–MS/MS to simultaneously quantify DON and ZON prototypes and their eight masked mycotoxins in cereal foods. Due to the shortage of commercial standards, studies on the quantitative analysis of masked mycotoxins are significantly limited. However, ELISA may respond to masked mycotoxins (Berthiller et al., 2013). The mycotoxins detection studies in tea, where results of the test by ELISA were mostly positive for mycotoxins (Santos et al., 2009; Liu, 2011), also forced us to think, whether there are also masked mycotoxins in tea.

Also, the detection of mycotoxins exposure levels in dark tea is mainly for their prototypes and little attention has been paid to the risk of masked mycotoxins. There are only a few reports on the preparation of standards, anabolic pathways of masked mycotoxins (Tan et al., 2016), which is the reason that masked mycotoxins deserve further attention and research. The common masked mycotoxins are masked trichothecenes, masked ZEN, masked fumonisins, masked OTA etc. Table 3 shows the common masked mycotoxins in naturally contaminated foods.

In addition to these masked mycotoxins detected in naturally contaminated food, there are also masked mycotoxins that have been identified but not yet observed in naturally contaminated food. All of the prototypes of mycotoxins mentioned above have been found in dark tea. Whether it forms masked mycotoxins in the same condition. Whether the masked mycotoxins are harmful to consumers. These questions force us to investigate more in the future.

Many Aspergillus spp. fungi are the dominant species in the production and processing of dark tea and have a strong relationship with the formation of dark tea quality (Jiang, 2012a; Wu J., 2013; Xiong, 2017). At present, only Eurotium cristatum has been specified by the Chinese national standard as the physicochemical index of Fuzhuan tea.

There are parts of fungi in the genus Aspergillus that produce mycotoxins, such as Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus versicolor.

During the fermentation of dark tea, the number of Aspergillus spp. is always in a dominant position. The fungi Aspergillus spp. have an essential contribution to the quality formation of dark tea. For example, Aspergillus niger metabolism can produce organic acids and enzymes that have a variety of hydrolytic enzymes, including oxidases, glycosidases, and proteases, which can hydrolyze polysaccharides, fats, proteins, and cellulose into monosaccharides, amino acids, and some soluble carbohydrates, so that the biochemical components in the tea can be easily leached out and enhance the thickness of the tea broth. Aspergillus niger is a comparatively safe industrial fungus that was recognized worldwide in the 20th century and is a commonly used industrial fermentation strain. However, in recent years, there have been plenty of studies finding that some strains of Aspergillus niger could produce mycotoxins. Although only a few Aspergillus niger fungi are able to produce mycotoxins, we absolutely need to pay more attention to them.

Fungi of the genus Penicillium are also frequently detected during dark tea processing and in finished dark tea (Haas et al., 2013). It is widely used in the processing and storage of dark tea production.

There are more than 600 species of fungi in the genus Penicillium, and their morphological characteristics are generally similar to each other. Many researchers have also isolated and identified Penicillium spp. fungi from dark tea, as shown in Table 5, and Penicillium spp. fungi that may produce fungal toxins are shown in Table 6.

Li (2019) isolated fungi of the genus Rhizoctonia from Ya’an Tibetan Tea. Zhao (2012) isolated one strain of Fusarium spp., one strain of Trichoderma and three strains of Mucor spp. from the ‘flowering’ processing of Fuzhuan tea. Wu et al. (2021) isolated Fusarium equiseti, Alternaria, and Cladosporium cladosporioides from Fuzhuan tea. Wen et al. (2012) isolated Mucor spp. and Rhizoctonia spp. from Liupao tea fermentation samples. All of the above fungi have the ability to produce mycotoxins, but few researchers have paid attention to them previously. These are the questions and directions that we need to pay close attention to in the future, whether these fungi can produce mycotoxins on tea substrates or not.

These fungal genera that have been isolated and identified as having the potential to cause adverse health effects to consumers are Fusarium, Xylaria, Streptomyces, and, Trichoderma spp. The fungi of the genus Fusarium mainly include Fusarium graminearum, Fusarium verticillioides, Fusarium nivale, Fusarium tricinctum, and Fusarium equiseti etc. (Zu et al., 2021). These fungi may produce toxic secondary metabolites such as trichothecenes, zearalenone, moniliformin, and butenolide (Huang et al., 2017). Some strains of Trichoderma such as T. reesei and T. viride are capable of producing gliotoxin, which belongs to the Tricothecenes (Mao and Zhang, 2018). Some strains of Alternaria can produce a variety of mycotoxins such as alternariol (AOH), alternariolmethylether (AME), and tenuazonicacid (TeA) (López et al., 2016). Mucor spp. is also a pathogenic fungus, often causing mold in food, Mucor can enter the body through the respiratory tract, digestive tract, or skin, causing blood clots and tissue necrosis (Yang and Liu, 2021).

In summary, many researchers have isolated and identified the microorganisms from dark tea. Therefore, a plentiful toxin-producing source has been discovered. The isolation and identification of toxin-producing fungi in dark tea require us to think highly of the safety of dominant fungi in the processing of dark tea and avoid the infestation of toxicity-producing fungi. We need to pay further attention to whether these fungi produce mycotoxins and whether they can cause health effects in consumers. Only a small number of researchers have paid attention to the toxicity-producing properties and isolating conditions of dark tea. However, in order to ensure the safety of dark tea production, research on the infestation of harmful fungi in dark tea is unquestionably required.

The pile fermenting process is crucial in defining the quality of dark tea. The essence of fermentation is the enzymatic reaction of extracellular enzymes produced by the secretion of dominant microorganisms and the action of moist heat (heat of microbial respiration and metabolism) combined.

A series of complex transformations occur to form the unique flavor quality of dark tea (Wang et al., 1991a). During processing, high temperature and high humidity conditions are favorable for microbial growth and the metabolic transformation of black tea, but if the dark tea is contaminated with harmful fungi during the pile fermentation, then there is a risk that the dark tea will be contaminated with fungal toxins.

The microorganisms in the fermentation process are the more abundant stage in the whole dark tea production process (Zhang et al., 2017; Xu Z. et al., 2019). Wen et al. (2012) isolated Aspergillus niger, Aspergillus oryzae, and Aspergillus glaucus from Liupao tea fermentation samples. Wang Q. et al. (2020) and Dong et al. (2009) isolated Aspergillus niger, Aspergillus tamarii, Aspergillus fumigatus, Aspergillus clavatus, Aspergillus oryzae, Aspergillus glaucus, Aspergillus terreus, Aspergillus candidus, Aspergillus wentii var. fumeus, Aspergillus penicillioides, Aspergillus aureolatus, Aspergillus egyptiacus, Aspergillus foetidus, Aspergillus japonicus Saito var. japonicus, Aspergillus restrictus Smith, etc., from the fermentation process of Pu-erh tea.

Dark tea is a kind of post-fermented tea with the characteristic that “flavor improves with ages,” and its flavor is unique. The change of the inner material component of dark tea is intimately related to temperature, humidity, and age (Chen Y. et al., 2016). Therefore, during the storage process, dark tea is always changing silently, and the degree is accountable for the storage conditions. Mildew and deterioration of the dark tea are brought on by improper storage, thus leading to the contamination of dark tea by fungal toxins.

Dark tea during storage is probably infested with harmful fungi due to high humidity conditions and thus. Xu et al. (2016) investigated mycotoxin residues under natural moldy and exogenous inoculation with toxin-producing Aspergillus flavus conditions by artificial high humidity mold-promoting culture, and all three mycotoxins were not detected in naturally moldy dark tea samples, while AFB₁ was detected in tea samples with exogenous inoculation with Aspergillus flavus strains. Zhong et al. (2010) isolated and identified a total of one Aspergillus strain, three Penicillium strains, and one Rhizopus strain for the fungal populations present in the storage of Ya’an Tibetan tea. Zhou et al. (2015a,b) isolated and identified a strain of Aspergillus niger from aged Pu-erh tea for solid-state fermentation of Pu-erh tea, and OTA was detected in the fermented Pu-erh tea samples, which proved that this strain of Aspergillus niger has the ability to produce OTA. In Li’s finding, total of 218 fungi was isolated from Liupao tea, and some of these strains, such as Aspergillus ochraceus, Aspergillus oryzae, Penicillium citrinum, and Penicillium chrysogenum can produce CIT (Li et al., 2020). Zhao et al. (2021) isolated 560 fungal strains from dark tea samples, including seven species of Penicillium spp. and 13 species of Aspergillus spp., and 20 species of other genera. Six different species of isolated fungi were identified had the ability to produce OTA. Nearly all strains belonging to species of Aspergillus carbonarius, Aspergillus ochraceus, Aspergillus nidulans and Penicillium verrucosum were capable of producing OTA, but only part of Aspergillus niger and Aspergillus tubingensis can produce OTA. Zhao et al. (2022) isolated 18 strains of Aspergillus spp. and Penicillium spp. fungi from dark tea samples of different years, and one Aspergillus niger was found to have the ability to produce OTA. Although dark tea has the characteristic of “flavor improves with ages,” if it is not stored properly, it may becomeinfested with harmful fungi and thus become moldy or produce fungal toxins that are harmful to human health.

The possibility of dark tea being contaminated by fungi, which warns that dark tea enterprises need to concentrate on the contamination of harmful microorganisms during the production process. It’s necessary to constantly pay attention to the changes in environmental conditions during the storage of dark tea to prevent it from being contaminated by fungi during the storage process. The standardized of the production of dark tea is urgently needed to ensure the safety of the dark tea production, processing, and storage environments, which is the guarantee for the prosperous development of the dark tea industry.