Contaminants: a dark side of food supplements?

Abstract

Food supplements (FS) are often consumed as one of the strategies to fight ageing-associated pathologies, especially in the case of oxidative stress-related diseases. Despite the popularity of FS, some concerns about their quality and safety have been raised, especially regarding the presence of contaminants. This paper reviews and discusses the occurrence of contaminants in marketed samples of FS in the last two decades, considering both scientific literature and notifications registered on RASFF portal. The most relevant classes of contaminants were included namely metals, toxins, pesticides, dioxins and PCBs, as well as pharmacologically active ingredients. Variable amounts of contaminants were reported in a significant number of commercially available FS. Although the presence of contaminants does not necessarily mean that their levels exceed the regulatory limits or that the FS intake constitutes a risk to human health, it alerts for the need to further monitor FS safety. The evaluation of the risk associated to the consumption of FS, especially in the elderly population, is particularly challenging due to the frequent exposure to multiple toxicants and to different exposure sources, as well as due to possible pre-existing diseases and respective therapeutics. Therefore, improved quality control procedures and monitoring programs should be pursued in order to avoid undesirable products and assure the safety of FS.

Keywords:

• Chemical contaminants
• food supplements
• oxidative-stress-related diseases

Introduction

The demographic changes across Europe, characterised by increasingly ageing population, lead to a rise in age-related diseases such as cardiovascular, diabetes, or neurodegenerative conditions, which includes an oxidative stress component in the underlying pathological processes [1]. Therefore, redox food bioactive nutrients and antioxidants have gained increasing importance as a strategy to promote healthy ageing [1–3]. Along with dietary habits, food supplements (FS) have emerged as one of the possible intervention strategies against pathologies associated with ageing and oxidative stress [4]. In fact, many FS including those constituted by plant extracts, phytochemicals, and multivitamin/mineral (MVM) claim antioxidant properties [3,5]. The COST action NutRedOx is a network focused on the redox control of major age-related diseases [1]. Therefore, in the scope of this action, FS are highly relevant and must be addressed.

Although FS are not intended to substitute a varied and balanced diet [6], they might provide additional nutritional support for an organism during inadequate intakes and/or increased nutrient requirements over the whole life cycle [7]. The use of these products is particularly prevalent among older adults [8–12]. This population is especially vulnerable to micronutrient deficiency due to physiological changes in aging, to the use of chronic medication therapy, or both [7]. In addition to nutritive support, common motivations for food supplements use include improvement of health and wellbeing, as well as prevention and control of diseases [11,13,14].

Vitamins and minerals, alone or in combination, present the most commonly used categories of foods supplements [9,15,16]. Nevertheless, there is a growing trend in fish oils, probiotics and other non-nutritive compounds consumption [17,18]. Mistrust in conventional medicine and the perception that what is natural is healthy [19] are some factors that contribute to the popularity of plant-based FS [20]. Additionally, supplementation with isolated phytochemicals such as curcumin, epigallocatechin gallate, quercetin or resveratrol aims to influence different molecular pathways to delay or prevent age-associated declines in mental and physical functioning [21].

Based on recent years’ surveys results, it was estimated that more than half of the adults in the USA [17] and European countries [20] use FS. Besides elderly people [8–11], the use of FS is more common in women [8–11,16,18,22,23], highly educated people [9,16,22] and those who are following a favourable lifestyle [8–11,16,22].

As a result of the misleading impression that the usage of products of natural origin has fewer side effects compared to that of synthetic drugs, consumers estimate the risk associated with food supplements usage as low [24,25]. Furthermore, self-administration of food supplements without health professional’s advice is widespread not only in healthy people [10,26,27] but also among different patients’ groups [28,29].

From a regulatory point of view, availability and popularity of food supplements raise concerns about their quality, efficacy, and safety. Since they represent a concentrated source of nutrients and other substances with physiological effects, overdose, long-term consumption, as well as the presence of contaminants increase the risk of harmful effects [30]. Mycotoxins, heavy metals, pesticide residues, and other environmental contaminants like polychlorinated biphenyls and dioxins, have been often detected and quantified in FS. In addition, FS may also contain intentionally added unapproved substances. Paradoxically to the antioxidant claims of many FS, contaminants commonly found in these products often have pro-oxidant potential. In fact, oxidative stress has been shown to be involved in the mechanisms of toxicity of heavy metals [31], mycotoxins [32–34], pesticides residues [35], and other environmental contaminants [36].

Generally, the use of FS does not contribute significantly to the total exposure to harmful contaminants [37]. However, prolonged and/or combined exposures to contaminants from FS may lead to deleterious effects on human health. It is important to note that this issue has been addressed by different authorities in the past years [38–45]. Nevertheless, considering the increasing consumption of FS in our aging society, it is essential to give more attention to the quality and safety of such products. Therefore, this paper reviews and discusses the occurrence of the most relevant types of contaminants in marketed samples of FS reported in the last two decades.

Materials and methods

A comprehensive literature search in English was performed to identify relevant articles (original research and reviews). Different types of FS and diverse classes of contaminants were systematically searched in PubMed, within the timeframe of the last two decades. The keywords used were “food supplement” or “dietary supplement,” plus “contaminant,” plus a specific contaminant category (e.g. metals, toxins, pesticides). Electronic copies of the articles were obtained and were then further reviewed to identify other relevant publications addressing FS contamination. Furthermore, the notifications available at the Rapid Alert System for Food and Feed (RASFF) portal were examined. The timeframe was set from 1998 to 2018, and different hazard categories covered. The selected product category was “dietetic foods, food supplements, fortified foods” and, among the notifications obtained, those specifically relative to FS were selected. The European Food Safety Authority (EFSA) scientific opinions accessed through the Wiley online library were also considered.

Contaminants in food supplements

Metals

Metal contamination has been reported in different types of FS, thus, being a relevant topic in the framework of the present review. This kind of contamination might occur as a consequence of a single factor or as a combination of sources that may vary according to the type of supplement. For instance, for plant-based supplements, the chemical composition of the soil, the characteristics of plant, and its growing conditions, as well as other aspects related with the lack of purity, extraction techniques, formulation/manufacturing, transport, and storage conditions can be responsible for the contamination [46]. Most of these factors can also contribute to metal contamination observed in other types of supplements.

As above mentioned, many of the FS are intended to aid elderly patients to cope with age-related illnesses. In addition, some of these products also claim to confer vitality, longevity, and health in general. Among the most common commercially available products that have been suspected of metal contamination, we consider of particular interest the (a) Plant-based supplements (e.g. containing herbs and other botanicals; (b) supplements containing (micro)algae; (c) single- or multivitamin/mineral supplements (MVM), and (d) shark cartilage and other FS of animal origin. Particularly over the last two decades, several original research articles have been devoted to the analytical determination of metals/metalloids specifically in FS. Optimised methodologies have been developed and employed for this purpose. Various analytical methods, especially atomic spectrometric and plasma-based techniques [46], have been valuable for exposure assessment, in order to estimate the toxicological risks associated with metal contamination in FS. Some techniques were also implemented to determine the speciation of certain elements (e.g. arsenic), which is an important aspect to take into account in terms of health effects.

A brief overview of unintentional metal contamination present in FS available worldwide is addressed herewith. Special attention is given to metals that are particularly toxic, and for which no physiological function is actually recognised in humans. However, it should be noted that even essential metals, e.g. iron, copper, cobalt or chromium, can be regarded as contaminants in some types of FS (e.g. mineral supplements) and have therefore been subjected to regular chemical analyses. Dietary mineral supplements containing essential metals, e.g. iron [47], calcium [48–50], or magnesium [51], might also incorporate toxic heavy metals, metalloids, and even radionuclides. In a different perspective, the intake of single/multimineral dietary supplements might display toxic features due to potential metal overload, an issue that should also be considered, especially upon long-term exposure. This aspect is, however, beyond the scope of the present review. The safety of MVM supplements has been recently discussed in the literature [52,53].

Some research articles provide analytical data concerning single metals/metalloids while others report the concentrations of multiple metals, sometimes including not only toxic but also essential elements [54–57]. In this latter case, the information is more representative of the global contamination burden present in the supplements. Among the most common toxic elements, lead, mercury, arsenic, and cadmium are particularly worrying in view of their regular presence in FS and of the inherent toxicological concern that they raise. In this sense, Table 1 summarises some available information on these four relevant toxicants. Examples of representative FS that have been evaluated and for which the authors found quantifiable levels are provided. In some of these reports, in addition to the four contaminants mentioned, other metals were also analysed and quantified, although this information is not specified in Table 1. Importantly, in several reports, the authors compare the estimated exposures, according to the metal concentrations found, with tolerable limit values (e.g. MRL, RDA, PTWI) and conclude on the potential risk associated. As a consequence, in some of these studies, although metal contamination was confirmed, the concentrations obtained were considered as acceptable at that time by the authors. An example of such is the report from Raman et al. [54] that analysed plant-based supplements. This type of conclusion towards the safety of the FS analysed is also stated in some reports mentioned in Table 1 (e.g. [57]). In other cases, only a particular toxic metal present in a given FS was considered as a potential health concern for the general population. For instance, in the report by Dolan et al. [62], the concentrations of arsenic and cadmium measured were considered below the tolerable limits (defined as 15 and 7 µg/kg (b.w.)/week, respectively) and only one FS yielded an unacceptable exposure to mercury of 13.6 µg/kg (b.w.)/week. Nevertheless, the authors argued that this concern could be somehow reduced if part of the total mercury found corresponded to an inorganic mercury form rather than methylmercury. Importantly, the same study also found 11 products that provide estimated exposures of lead exceeding the provisional tolerable intake of lead defined for sensitive populations (e.g. children and women of childbearing age, especially if pregnant; 6 and 25 µg/kg (b.w.)/week, respectively). These segments of consumers should obviously be considered with particular attention. In this context, Schwalfenberg et al. [67] recently reported that diverse prenatal vitamin supplements were contaminated with different toxic metals. While the levels of mercury (present in 14/50 samples) and cadmium (present in all samples) were within the tolerable limits, the levels of lead and arsenic (both present in all samples) surpass the accepted standards in some FS (0.5 and 0.1 µg/day for Pb and As, respectively), with one FS yielding 4 µg/day of lead.

Table 1. Metal contaminants in food supplements. Examples of studies reporting quantifiable levels of arsenic (As), cadmium (Cd), lead (Pb), and mercury (Hg)

Type of food supplement	Contaminants^a	Nr^b	Country of sale	Year	Reference
Calcium
Bone, milk, oyster/clam shell, others	As, Cd, Hg	55	Korea	2004	[50]
Natural – oyster shells/Refined	Pb	23	USA	2000	[48]
Organic/inorganic compounds, dolomite, shells	^210Pb	17	Poland	2016	[49]
Diverse^c
Calcium supplements, vitamins	Pb	45^d	USA	2007	[58]
Macro/microelements, vitamins	Hg	33	Poland	2015	[59]
Plant based, MVM, algae	Cd, Pb, Hg	24	Mexico	2007	[60]
Vitamins, minerals, MVM, herb-based	As, Cd, Pb, Hg	30	Croatia	2010	[61]
Mainly plant-based	As, Cd, Pb, Hg	95	USA	2003	[62]
Mainly plant-based, minerals	Hg	40	USA	2005	[63]
Vitamins, MVM, minerals, herb-based	As, Cd, Pb, Hg	33	Lebanon	2013	[55]
Plant-based and algae	As	16	Denmark	2013	[38]
Health clays products	As, Cd, Pb, Hg	27	Netherlands	2013	[64]
Iron supplements	As	15	Brazil/Spain	2017	[47]
Magnesium (organic/inorganic compounds; dolomite)	^210Pb	14	Poland	2017	[51]
Multimineral and MVM supplements	Pb	168	Poland	2018	[57]
Plant-based
Garlic, equinacea, gingko, ginseng, others	As, Cd, Pb	67	USA	2004	[54]
Herbal	Hg	24	Poland	2018	[65]
Prenatal and children supplements	As	10	USA	2014	[66]
Prenatal vitamin supplements	As, Cd, Pb, Hg	51	Canada	2018	[67]
Shark cartilage powder	Hg	16	USA	2014	[68]

MVM: multi-vitamins/minerals.

^a In these studies the metal contaminant was quantified at least in one of the supplements analysed.

^b The term Nr, refers to the number of FS analysed.

^c The designation diverse means that at least three different types of FS were analysed.

^d Pharmaceutical products included in this number.

Another topic that should not be disregarded is the information present in multidisciplinary reports describing clinical toxicity features associated with the intake of FS. Some examples include the report by Rzymski et al. [56] that addresses two cases of human poisoning in Poland after the simultaneous consumption of FS obtained from online orders containing the microalgae Spirulina and Chlorella. Samples of these FS were analysed, revealing the presence of high levels of various metals including cadmium, lead, and mercury. In addition, some authors advocate that inquiring about the use of FS is an important aspect to include in the clinical context of a patient’s medical history. An example is the potential inadvertently arsenic toxicosis described in a case report on a 54-year-old woman that consumed herbal kelp supplements at a daily basis [69]. However, it should be mentioned that further commentaries on this report were also published, providing clear divergent viewpoints [70–72].

In summary, the diversity of FS and the plethora of potential metal contaminants, each one with diverse toxic effects, undoubtedly contributes to the complexity of this issue, which should be pursued in a holistic manner considering also additional deleterious chemicals from other classes and their possible toxicological interaction with toxic metals.

Toxins

Toxins are defined as toxic substances produced by biological systems such as plants, animals, fungi, or bacteria [73]. This review will focus on mycotoxins and cyanotoxins, since these toxins are the ones that most commonly contaminate FS.

Mycotoxins are structurally diverse secondary metabolites produced by filamentous fungi [37,73,74], such as Aspergillus, Penicillium, and Fusarium species [75]. These fungi can grow on plants both, in the field, and during harvesting, transportation, processing, and storage [74–76]. The presence of toxicogenic fungi does not necessarily imply the production of mycotoxins. This production is influenced by many aspects, including genetic factors, growth conditions, weather, geographical area, humidity, CO₂:O₂ ratio, and the presence of fungicides or other competitive microbial species [37,77,78]. Nevertheless, the presence of toxicogenic moulds constitutes a risk of contamination with mycotoxins [37]. The contamination of raw materials may result in the occurrence of mycotoxins in plant-based FS [74,75]. Since toxigenic fungi are widespread in the environment, the presence of mycotoxins cannot be entirely avoided [74].

Although over 400 mycotoxins are known, some classes are particularly relevant in foodstuff and FS. Table 2 summarises relevant studies reporting the analysis of mycotoxins in FS carried out in the last two decades. Aflatoxins, ochratoxin A (OTA), zearalenone (ZEN), trichothecenes, and fumonisins are especially important due to their frequent occurrence and to the well-established adverse effects on human health [75,77]. EFSA delivered a scientific report on human and animal dietary exposure to T-2 and HT-2 toxins, trichothecenes, which form part of the group of Fusarium mycotoxins. Very high levels were reported in a small number of data on specific plant- and herb-based FS. In the elderly and very elderly, FS may represent an important contribution to the dietary exposure to these xenobiotics [39].

Table 2. Examples of studies reporting quantifiable levels of mycotoxins in food supplements

Type of food supplement	Contaminants^a	Nr^b	Country of sale	Year	Reference
Plant-based
Green tea	Aflatoxin B1	10	Spain	2016	[79]
Grape	Ochratoxin A	24	Italy^c	2015	[80]
Ginkgo	Aflatoxin B1, aflatoxin B2, T-2	9	Spain, Poland, USA	2015	[81]
Green coffee	Ochratoxin A, ochratoxin B, fumonisin B1, mycophenolic acid	50	USA^c	2013	[74]
Milk thistle	Aflatoxins	79	USA	2012	[78]
Different plants	Deoxynivalenol, trichothecenes, zearalenone, enniatins, alternariol, tentoxin, mycophenolic acid	69	Czech Republic, USA	2015	[76]
	Fumonisins, ochratoxin A	62	Belgium^c	2009	[75]
Diverse (mostly plant-based and Red yeast rice)	Citrinin	7	Croatia	2013	[82]
Red yeast rice	Citrinin	4	Taiwan	2005	[83]
Brewer’s yeast	Ochratoxin A	46	Germany^c	2016	[84]
	Ochratoxin A	51	Germany	2002	[85]

^a In these studies at least one toxin was quantified at least in one of the supplements analysed.

^b The term Nr refers to the number of food supplements analysed.

^c Includes samples obtained online.

Of concern are also findings on the occurrence of the so-called “emerging mycotoxins,” potential contaminants for which the amount of data available is not yet sufficient to allow a comprehensive risk assessment and therefore are not included in the standard monitoring and regulation of mycotoxins. According to recent reports, the occurrence [76] and toxicological properties [86], especially of Alternaria toxins, i.e. secondary metabolites formed by black moulds, might be of relevance in the context of FS consumption [76]. Although the toxic effects depend on the type of mycotoxin and exposure conditions, hepatotoxic, immunotoxic, nephrotoxic, carcinogenic, teratogenic, and endocrine effects, among others, have been ascribed to mycotoxins [32,73,77]. Besides plant-based FS, ochratoxin A has also been found in brewer’s yeast supplements [84,85]. In addition, contamination with mycotoxins, especially citrinin, has been reported in supplements based on fermentation products of Monascus purpureus (also known as red yeast rice) [82,83]. Citrinin is a fermentation by-product of Monascus strains with recognised nephrotoxicity and hepatotoxicity [82,83]. The European Commission defined the maximum levels of citrinin in FS based on rice fermented with red yeast Monascus purpureus (2 mg/kg) [87], as well as the methods of sampling, performance criteria for T-2, HT-2 toxin, and citrinin, and screening methods of analysis [88]. The sampling procedure is defined based on the supposition that the FS based on rice fermented with red yeast Monascus purpureus are marketed in retail packages containing usually 30–120 capsules per retail package.

It is important to mention that the FS mentioned in Table 2 are commonly consumed with the purpose of fighting the effects of aging. Some examples are plant-based FS intended for general health support, to reduce menopause effects, to improve liver function [76], or to reduce memory loss, Alzheimer sickness, and vascular diseases [81]. FS of red yeast rice are used as a cholesterol-lowering strategy [82]. Although both the efficacy and the safety should be supported on human studies, that is not always the case and the benefit/risk ratio may be questioned for some mixtures in the market.

The consumption of FS of marine origin has also increased over the past decades due to their perceived health benefits [68,89]. Previous studies have detected cyanotoxins (i.e. toxins produced by certain species of cyanobacteria), arising from the contamination of marine raw materials used in these FS [89,90].

Although FS of marine sources frequently contain nontoxic cyanobacteria, the methods of cultivation in natural waters may allow the contamination by toxin-producing species naturally present in the environment [90]. The contamination may result from cyanobacterial blooms originated by eutrophic conditions in the presence of heat, light, shallow waters, and nutrients [90]. For example, Aphanizomenon flos-aquae is a cyanobacterium commonly used in blue-green algae (BGA) supplements. One relevant source of this BGA is the Upper Klamath Lake (Oregon, USA), where the toxinogenic cyanobacterium Microcystis aeruginosa occurs regularly, allowing the contamination of BGA products during harvesting [89]. The culture conditions commonly used to produce the BGA Spirulina and the green algae Chlorella make these raw materials less likely to be contaminated with unwanted cyanobacterial species [89]. In accordance, in the study of Vichi et al. [91], products containing Spirulina-only were free of contamination, while A. flosaquae-based supplements presented significant levels of total microcystins (up to 5.2 µg/g, with around 40% of samples exceeding 1 µg/g).

Different cyanotoxins have been detected in FS of marine origin, as summarised in Table 3. Among those, microcystins (MC) are especially relevant because of their widespread occurrence [92] and their toxic effects on humans that include hepatotoxic and potential tumour-promoting effects [89]. In addition, beta-methylamino-l-alanine (BMAA), a cyanotoxin with neurotoxic effects, has also been detected in different types of FS [68,90]. In a study performed by Mondo et al [68], BMAA was detected in fifteen out of 16 products with concentrations ranging from 86 to 265 µg/g (dry weight).

Table 3. Examples of studies reporting quantifiable levels of cyanotoxins in food supplements of marine origin

Type of food supplement	Contaminants^a	Nr^b	Country of sale	Year	Reference
Blue green algae	Microcystins, Anatoxin-a, dihydroanatoxin-a, epoxyanatoxin-a, Beta-methylamino-L-alanine	18	Canada^c	2017	[90]
	Microcystins	17	Italy	2012	[91]
Blue green algae and Chlorella	Microcystins	18	Germany	2012	[89]
Shark cartilage	Beta-methylamino-l-alanine	16	USA	2014	[68]

^a In these studies at least one toxin was quantified at least in one of the supplements analysed.

^b The term Nr refers to the number of food supplements analysed.

^c Includes samples obtained online.

As mentioned above for the case of mycotoxin-containing FS, also FS of marine origin are consumed with respect to health claims with apparent interest in aging. FS based on BGA are proposed as health-promoting products with potential benefits for weight loss, to increase energy, as mood improvers, or providing anti-inflammatory, anti-infectious, anticancer, anti-dyslipidaemic and immune-stimulating properties [89,91]. Cartilage-based FS claim potential benefits in diseases like cancer, arthritis, and osteoarthritis [68].

Several analytical techniques have been used to analyse toxins in FS. Most of the reports have used methodologies employing liquid chromatography (LC) coupled to mass spectrometry (MS), including LC–MS/MS [89,91], ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) [68,74,76], or UHPLC coupled to high resolution mass spectrometry (HRMS) [90]. These techniques are rapid, broadly applicable, highly specific, and allow the simultaneous quantitation of multiple toxins present at low concentrations [74,75]. In addition, enzyme-linked immunosorbent assays (ELISA) have also been used [89,91].

Although many studies found quantifiable levels of toxins in FS of different types, as shown in Tables 2 and 3, it must be mentioned that many reports and analyses led to negative results [56,79]. In some reports, toxin contamination was found only in a small part of the samples analysed [79,82]. In the plant-based FS analysed by Diana Di Mavungu et al. [75], none of the 23 mycotoxins investigated was detected in 56 out of 62 samples. In addition, in some analyses, some toxins were investigated and not found. For example, Heussner et al. [89] analysed nodularins, saxitoxins, anatoxin-a, and cylindrospermopsin and obtained negative results. Cylindrospermopsin and saxitoxin were also absent in the samples analysed by Roy-Lachapelle et al. [90].

On the contrary, different studies reported the quantification of toxins in a significant percentage of the samples analysed. Solfrizzo et al. [80] found OTA in 75% of the plant-based FS samples analysed. Contamination with OTA was also found in 63 and 87% of the brewer’s yeast FS analysed by Gareis et al. [85] and by Gottschalk et al [84], respectively. Martinez-Dominguez [81] measured aflatoxins and T-2 toxin in 6 out of nine samples of Ginkgo products. In green coffee bean supplements, ochratoxin A, ochratoxin B, fumonisin B1, and mycophenolic acid were found in 36%, 32%, 10%, and 16% of tested samples, respectively [74]. Regarding microcystins, their presence was detected in 85 of 87 samples tested by Gilroy et al [93].

Besides mycotoxins and cyanotoxins, pyrrolizidine alkaloids (PA) are plant toxins also worth of note. Previous reports have detected PA in FS based on beehive products [94] and plant materials [95,96]. EFSA included FS together with tea and herbal infusions in a risk assessment task. The EFSA Panel on Contaminants (CONTAM Panel) stated that exposure to these plant toxins in food, in particular for frequent and high consumers of tea and herbal infusions, is a possible long-term concern due to their potential carcinogenicity. From the analysis of the available occurrence data, the experts identified 17  PA in food and feed that should continue to be monitored and recommended further toxicity studies on those most commonly found in food, including the development of more sensitive and specific analytical methods. The CONTAM Panel established a new Reference Point of 237 µg/kg body weight per day to assess the carcinogenic risks of PAs and concluded that there is a possible concern for human health related to the exposure to PAs. The Panel noted that consumption of FS based on PA-producing plants could result in exposure levels causing acute/short-term toxicity [42].

Although some studies reported high levels of toxins in FS [89–91], posing potential concerns for consumers, this is frequently not the case. The detection of toxins in FS does not necessarily mean that their levels are above the regulatory limits or that the intake of such FS constitutes a significant risk for human health. In fact, in different studies reporting the presence of toxins in FS, the authors concluded that the contribution of FS to the total weekly intake of toxins is low [75,84]. However, the presence of toxins in some FS samples indicates the need to reinforce the quality control of these products [37,75].

Pesticides residues

Pesticides are xenobiotics intended to kill other forms of life, including insects (insecticides), small rodents (rodenticides), or vegetation (herbicides) [73]. The occurrence of pesticide residues in FS is due to the presence of such compounds in the raw materials. As can be seen in Table 4, the majority of the contaminations of FS with pesticides have been reported in plant-based products, including supplements containing green tea [79,97], ginseng [98], soya [99], Ginkgo biloba [81], or other plants [101]. As many popular FS are based on plants that are grown using conventional agricultural practices, including the application of pesticides during cultivation and/or storage [99], pesticides residues may be present in the final FS product. The pesticides detected in plant-based FS include insecticides, herbicides, and fungicides which could have been applied in the plants for pest control and consequently appear in the final FS [79,81,101]. Moreover, if the FS is prepared by concentration of extracts of the raw material, pesticides, and other contaminants might be concentrated in the final product [99]. This may happen with very polar compounds that have been widely used in conventional farming. If the extraction of plant products is carried out using polar solvents like ethanol, methanol, acetone, or water, polar pesticides can be present in the final FS. This happens, for example, with chlorate, that although forbidden in the European Union, was detected in soya-based supplements at concentrations up to 1642 µg/kg [99]. Besides the detection of pesticides in plant-based FS, the use of pesticides residues in plants presumably justifies the occurrence of pesticides in royal jelly supplements, possibly due to its presence in pollen [79].

Table 4. Examples of studies reporting quantifiable levels of pesticides in food supplements

Type of food supplement	Class of pesticides^a	Nr^b	Country of sale	Year	Reference
Plant-based
Green tea	Insecticides (OPs, CBs, PIs, others) Fungicides Herbicides	10	Spain	2016	[79]
	Acaricides	10	Spain	2014	[97]
Ginseng	Insecticides (OCs, OPs, PIs) Fungicides	23	USA	2016	[98]
Soya	Herbicides	14	Spain	2016	[99]
	Insecticides (OPs, others)	11	Spain	2015	[100]
Ginkgo	Insecticides (OPs, CBs, PIs, others) Fungicides Herbicides	9	Spain, Poland, USA	2015	[81]
Different plants	Insecticides (PIs) Fungicides Herbicides	32	Spain	2015	[101]
Royal jelly	Herbicides	8	Spain	2016	[79]
Oil-based
Fish, seal and vegetable	Insecticides (OCs)	30	Canada^c	2009	[102]
Cod liver	Insecticides (OCs)	15	Italy	2004	[103]
Fish and vegetable	Insecticides (OCs)	21	UK	2004	[104]

OCs: organochlorine insecticides; OPs: organophosphate insecticides; CBs: carbamate insecticides; PIs: pyrethroid insecticides.

^a In these studies, at least one pesticide of each class was quantified at least in one of the supplements analysed.

^b The term Nr refers to the number of FS analysed.

^c Includes samples obtained online.

As can be seen in Table 4, supplements based on fish, mammals (seal), and vegetable oils may also contain pesticides, especially those of lipophilic nature. These oils constitute a source of long-chain n-3 polyunsaturated fatty acids (n-3 PUFA) with health benefits and are thus commonly used in FS. However, oils can, in turn, be contaminated with lipophilic organic chemicals, such as organochlorine pesticides (OCPs) [103,104]. Organochlorine pesticides are ubiquitous contaminants of marine ecosystems [104] with well-established adverse effects on human health [103]. The use of most OCPs was restricted or banned in the European Union since the early 1970s and therefore the levels of these contaminants in marine environments have been decreasing [103]. However, as these pesticides are long-lived compounds that bind strongly to sediments and soils, they are very persistent and can still be found in the environment [73,103,105]. Elevated concentrations of OCPs have been reported in fish products around the world, particularly in farmed fish [102]. As a consequence, FS based on fish oil can also be contaminated with OCPs. The levels may vary with the type of fish used, the location from which the fish was collected [102], and with the oil processing procedures [103]. In addition to fish, oils from other sources, namely from seal, may also contain OCPs. For example, Rawn et al have found OCPs in shark, salmon and seal oils in levels higher than those measured in mixed fish oils or fish oils mixed with vegetable oils [102]. Importantly, recent FS samples show less OCPs contamination compared with past reports [104].

Some previous studies screened a very high number of pesticide compounds [97,98,100,101]. Although many negative results were found, in some cases, pesticides contamination was detected in a relevant number of samples [79,81,98,102]. It is also important to mention that the detection of pesticides does not necessarily imply that it is present at nonauthorised concentrations or constitutes a risk to consumers. In some reports, the levels found were below the Maximum Residue Levels (MRLs) established for raw materials [79,101]. Another relevant aspect is that some FS samples presented more than one pesticide [79,97,101]. The simultaneous exposure to different toxicants, even at low concentrations, may represent additional risks as will be discussed ahead in this paper. Pesticides may undergo transformation in different degradation products, which can be more toxic to humans and to the environment than the parent compounds. Therefore, a proper screening of transformation products should be included in the quality control of FS [101].

The analyses of FS have many technical challenges. Food supplements constitute very complex matrices for pesticide residue analysis [100,106]. Therefore, a proper sample preparation methodology is needed to isolate and concentrate the target compounds [100]. The Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method has been applied for the multiresidue pesticide analysis of plant-based FS [79,97,98,100,101]. The use of selective determination techniques, namely gas and liquid chromatography coupled to tandem mass spectrometry (GC–MS/MS and LC–MS/MS), is very helpful in separating analyte signals from coeluting matrix interferences [100,106]. In fact, while most pesticide determinations are based on GC-MS [97,98,100,103], new pesticides that are more polar, thermally labile or not easy vaporised are better analysed using LC-MS [101]. Nowadays, high-resolution mass spectrometry (HRMS) methods have also been used, with advantages in terms of sensitivity, number of compounds determined and precision [79,101].

As mentioned before, the consumption of FS to fight age-related conditions is very popular [107] and the FS mentioned in Table 4, where pesticides were detected, are not an exception. Green tea is rich in polyphenols and has therefore antioxidant properties [79]. Moreover, it can contribute to weight loss, lowering cholesterol levels, mental alertness, and skin protection [97]. Ginseng supplements are commonly consumed by elderly people [107] due to its potential benefits for the management of aging-related and frailty symptoms [108], including improvements in fatigue, hypertension and cardiovascular diseases, lung function, renal function, and cognitive performance [108]. Soya-based FS are a source of phytoestrogens frequently used to treat menopausal symptoms, especially vasomotor effects, as well as for lowering cholesterol [99]. Ginkgo biloba has been associated with a spectrum of medicinal properties particularly relevant in elderly people [107], including the prevention of memory loss, cognitive dysfunction, vascular pathologies, vertigo, and glaucoma [81,107]. Garlic supplements included in the pesticide analyses performed by Palenikova et al [101] are commonly consumed by elderly people due to its antilipidemic, antihypertensive, and antiatherosclerotic effects. Moreover, antidiabetic, antithrombotic, anticancer, immunostimulant, antioxidant, antiparasitic, and antibacterial properties have also been reported [107]. Royal jelly is considered an antioxidant, anti-inflammatory, antiviral, antiulcerous, and antibacterial product [79]. The consumption of omega-3 fatty acids through fish oil is associated with cardiovascular benefits, namely with improved vascular health and reduced risk of coronary heart disease in humans [102]. Other potential therapeutic advantages of fish oils include the reduction in diabetes mellitus incidence, the alleviation of rheumatoid arthritis symptoms [102,103], as well as immunological benefits [104].

Considering the widespread use of FS, especially in older people, and the not unusual presence of pesticides in these supplements, better quality control procedures and monitoring programs should be established in order to avoid undesirable products and assure the safety of FS.

Dioxins and PCBs

Dioxins (polychlorinated dibenzo-p-dioxins [PCDDs] and polychlorinated dibenzofurans [PCDFs]) and PCBs (polychlorinated biphenyls) are a wide group of ubiquitary and persistent chemicals [40,41,109]. The term “dioxins” include more than 200 substances, which are not produced intentionally and are formed mainly during combustion processes and as industrial subproducts [109,110]. Regarding PCBs, more than two hundred compounds are known, with great similarities to dioxins. PCBs are stable aromatic chlorinated compounds with one to ten chlorine atoms per molecule and with coplanar or mono-Ortho chemical structures [110,111]. These compounds were intentionally produced and used with different applications in the past, such as lubricants, coatings, plasticizers, or inks [109,110]. Nowadays, PCB manufacture is prohibited, but their release to the environment still occurs from the disposal of waste or electrical equipment [109].

Dioxins and dioxin-like substances (e.g. PCBs) are frequently classified as persistent organic pollutants (POPs). The polybrominated diphenyl ethers (PBDEs) are another class of POPs that share similarities with dioxins and PCBs. PBDEs have the element bromine rather than the chlorine element found in the PCBs [110,112]. Therefore, POPs comprise a large group of compounds with related chemical structures, toxicity profiles, and mechanisms of action. They typically bind to the aryl hydrocarbon receptor (AhR) [41,109], an intracellular ligand-activated transcription factor [73].

Long-exposure to dioxins and PCBs can result in a wide range of adverse health effects, including immunotoxicity, nervous system pathologies, dermal toxicity (e.g. chloracne and hyperpigmentation), gastrointestinal disorders, developmental and neurodevelopmental impairments, endocrine alterations (e.g. diabetes and thyroid disorders), reproductive toxicity, liver damage, and carcinogenicity [41,73,109,111,113,114]. The most toxic dioxin, 2,3,7,8-tetrachlorodibenzodioxin (TCDD) and some PCBs, such as 3,4,5,3′,4′-pentachlorobiphenyl (PCB-126) are classified by the International Agency for Research on Cancer (IARC) in Group 1 as carcinogenic to humans [115]. In recent years, we have observed a growing concern about the potential adverse effects of some PBDEs on human health. Some toxicological studies indicated that PBDEs could induce neurotoxicity, cognitive and behaviour problems, immunotoxicity, endocrine, and reproductive system alterations [110,116–119].

Dioxins and PCBs have high chemical resistance, which leads to their accumulation in the environment, and subsequent entry into the food chain [41,104,114]. Most of the human exposure to these toxic compounds results from the consumption of food of animal origin with high-fat content, due to the high accumulation of these compounds in fatty tissues [41,120]. The half-life of elimination presents wide individual variation and depends on the chemical structure of each compound, varying between few weeks to several years [111,121]. FS obtained from animal or vegetal sources with a high lipophilic content have more risk of containing residues of dioxins and PCBs. On the market, there are several antiaging FS obtained from fish (mostly cod liver, but also salmon, and shark oils), marine mammal oils, vegetable oils, and mixtures. They are available at local shops and markets, by mail or online orders and they are commonly sold either as soft capsules or liquid solutions. These FS are known to be a source of lipophilic vitamins, such as vitamin D frequently used to prevent and treat osteoporosis. Additionally, most of those FS contain also polyunsaturated fatty acids (PUFAs), which are often associated with cardiovascular protection. In the last two decades, the popularity of these FS has increased together with data about their benefits in several health conditions, such as osteoporosis, rheumatoid arthritis, autoimmune disorders, cardiovascular diseases, stroke, high blood pressure, and renal injury [122]. However, scientific research on PUFAs’ efficacy is sometimes insufficient and contradictory. In the production of these types of FS, natural oils are often refined and purified by processes such as distillation. Nevertheless, these techniques do not completely remove contaminants, including dioxins and PCBs [123].

The presence of dioxins and PCBs, mainly in fish oil-based FS, has been investigated worldwide, namely in Spain [113,120,124], UK [104,122,125], USA [112,126,127], Canada [102,128,129], or Japan [123,130]. Some of the available and published information regarding the contamination of FS with dioxins and PCBs is summarised in Table 5.

Table 5. Dioxins, PCBs, and PBDEs residues in food supplements.

Type of food supplement	Type of contaminants^a	Nr^b	Country of sale	Year	Reference
Oil-based
Fish	PCDDs, PCDFs, PCBs	25	Japan	2018	[123]
Vegetable and animal mix		12
Marine mammal		4
Egg yolk		3
Mixtures		2
Fish	PCDDs, PCDFs, PCBs, PBDEs	9	Spain	2011	[124]
Vegetable and fish mix		3
Vegetable		3
Fish	PCBs	11	Canada	2010	[128]
Vegetable		6
Fish	PCBs	5	UK	2002	[125]
Vegetable		1
Fish, vegetable and mix	PCBs	30	Canada^c	2009	[102]
Fish	PCBs, PBDEs	14	UK	2004	[104]
Vegetable and animal mix		3
Vegetable		4
Fish	PCBs	13	USA^c	2013	[127]
	PCBs, PBDEs	1	Norway	2011	[114]
	PCBs, PBDEs	10	USA^c	2010	[126]
	PCBs	3	Spain	2010	[131]
	PCDDs, PCDFs, PCBs, PBDEs	30	Canada^c	2009	[129]
	PCBs, PBDEs	1	USA^c	2009	[112]
	PCBs	16	Czech Republic	2009	[132]
	PCDDs, PCDFs, PCBs, PBDEs	6	Switzerland	2006	[133]
	PCBs, PBDEs	11	Japan	2006	[130]
	PCDDs, PCDFs, PCBs	33	UK	2006	[122]
	PCBs	15	Italy	2004	[103]
	PCBs	38	Australia, Austria, Belgium, Brazil, Canada, France, Japan, NL, NZ, Norway, Spain, UK, USA	1998	[134]
Omega-3	PCDDs, PCDFs, PCBs	9	Spain	2017	[113]
Diverse
Fish oil	PCDDs, PCDFs,	7	Spain	2010	[120]
Vegetable oils	PCBs, PBDEs	5
Vegetable and fish oils mix		2
Mineral mix		1
Fish oil	PCDDs, PCDFs,	15	Ireland	2005	[110]
Fish and vegetable mix oil	PCBs	5
Fish oil and vitamins mix		7
Vitamins		2
Vegetable oils		5
Omega-3 and/or Omega-6		8

PCDDs: polychlorinated dibenzo-p-dioxins; PCDFs: polychlorinated dibenzofurans; PCBs, polychlorinated biphenyls; PBDEs: polybrominated diphenyl ethers.

^a In these studies, dioxins (PCDDs and PCDFs), PCBs, and PBDEs contaminants were quantified at least in one of the food supplements analysed.

^b The term Nr, refers to the number of dietary supplements analysed.

^c Includes samples obtained online.

The concentrations of dioxins and PCBs varied widely in FS. In some studies, other sources relevant to human exposure (e.g. food) were analysed and/or other classes of toxics (e.g. pesticides or metals) were detected, although these data were not included in Table 5. In some reports, the authors did not detect any level of dioxins or PCBs in the FS analysed. For example, Melanson et al. [135] analysed five commercial fish oil supplements and the levels of PCBs were below the detection limit. In the Smutna et al.’s [132] study, PCBs were not detected in half of the FS analysed (sixteen), while in the remaining samples different PCBs were found. Nevertheless, in most of the studies performed, POPs were detected in FS and, specifically, in some reports all the FS analysed contained dioxins and/or PCBs [120,126,127,131]. In some studies, the level of POPs detected in FS exceeded the maximum legislative levels. For example, Marti et al. (2010) detected levels up to 12.1 pg TEQ g⁻¹ of dioxin-like PCBs in FS [120], which is markedly higher than the maximum level established for “marine oil intended for human consumption” set at 6.0 pg TEQ g⁻¹ by Regulation EC 1259/2011 [136]. Although some authors considered the consumption of FS as safe and healthy, others emphasised the high levels of POPs detected in some FS and pointed out the potential risks, essentially focused on dioxins and PCBs. Table 5 also comprises studies reporting contaminations of FS with PBDEs.

In the last decades, there have been deep concerns about the presence of different toxicants on FS, including dioxins and PCBs. In the 1990s, the attention of the general public and food safety agencies was drawn to some adverse contamination incidents [40]. Recently, there has been scientific discussion worldwide and regulatory concern, particularly from food agencies about the presence of toxic residues in food and FS [40,41]. Even small quantities of those compounds in long-term exposure could have a significant toxicological impact. In this regard, EU strategies are focused on the reduction and control of dioxins and PCBs in food and most recently in FS. Nevertheless, there is a general lack of information and of legislation addressing the potential toxicological risks associated with the consumption of FS. It is important to emphasise that some people take FS on a daily basis, which might induce toxicity per se by the presence of different toxicants or by the combination with other exposure sources (e.g. food). In several reports the presence of POPs and organochlorine pesticides [102–104,114,125,131,134], metals [132], and other toxics (e.g. hexabromocyclododecane) [114,124] were detected in the same FS. In such cases, potential toxicological interactions and the possibility of a higher toxicological impact should not be underestimated.

Generally, the exposure to POPs through FS appears to be relatively low and below the relevant legislative limits. Nevertheless, in some situations, FS may contain significantly high concentrations of such compounds, particularly dioxins and PCBs. It is also important to mention that some FS are consumed daily and for long periods of time. Therefore, it is crucial to monitor the presence of POPs in FS.

Pharmacologically active ingredients

Over the last two decades, adulterated food supplements have become a rising public health threat [137]. One of the common economically motivated adulteration strategies is the addition of some undeclared drugs, their synthetic analogues or other unauthorised compounds to FS in order to intensify its claimed effects [138]. Recent data show that in the USA, between 2007 and 2016, more than 750 undeclared pharmaceutical agents have been identified in FS, mainly in the plant-based category [139]. The structural complexity of plants makes them suitable matrices for intentional addition of unauthorised or prohibited compounds [140]. In recent years, the trend of FS adulteration with pharmacologically active ingredients has also been raising in EU countries [137]. Among the common adulterated categories [139], the potential presence of pharmaceutical ingredients in FS marketed for weight loss and sexual performance enhancement is of particular relevance for this review.

Anorexics (including sibutramine and rimonabant), and other adulterants like stimulants (e.g. amphetamine, ephedrine and its analogues, yohimbine and synephrine), antidepressants (e.g. fluoxetine, sertraline), anxiolytics (mainly benzodiazepines such as diazepam), diuretics (e.g. furosemide and hydrochlorothiazide) as well as stimulant laxatives (including phenolphthalein and bisacodyl) are illegally added to weight-loss FS [140,141]. Sexual enhancement supplements are most frequently adulterated with phosphodiesterase type-5 enzyme (PDE-5) inhibitors (sildenafil, tadalafil, and vardenafil) and more often with their analogues [140,142]. Some literature data related to adulteration FS with these pharmaceuticals is listed in Table 6.

Table 6. Examples of studies reporting adulteration of food supplements with pharmacological active ingredients

Type of food supplement	Pharmaceutical adulterants	C^a/Nr^b	Country of sale	Year	Reference
Diverse^c (e.g. plant-based, vitamins)	Sibutramine, phenolphthalein	14/20^e	France^d	2010	[143]
	Sibutramine and its analogues, fluoxetine, bisacodyl	62/188^f	South Korea	2014	[144]
	Sibutramine and its analogues, rimonabant, fluoxetine, bisacodyl, phenolphthalein	55/193^f	South Korea	2016	[145]
	Sibutramine, fluoxetine, phenolphthalein	76/370^f	South Korea^d	2018	[146]
	Sibutramine and its analogues, furosemide, bisacodyl, phenolphthalein, thyroid agents	119/447	Singapore	2016	[147]
Plant-based	Sibutramine and its analogues, phenolphthalein	11/22	China	2008	[148]
	Sibutramine and its analogues	23/25	Poland	2011	[149]
	Sibutramine and its analogues, rimonabant, phenolphthalein	24/50	Netherland	2014	[150]
	Sibutramine	26/52	Switzerland^d	2014	[151]
	PDE-5 inhibitors and their analogues	10/64	Czech	2019	[152]
	PDE-5 inhibitors and their analogues	3/11	Italy	2014	[153]
	PDE-5 inhibitors and their analogues	3/26^e	Italy	2015	[154]
	PDE-5 inhibitors, flibanserin, phentolamine, dehydroepiandrosterone, testosterone	92/150	Switzerland^d	2015	[155]
	PDE-5 inhibitors and their analogues, phenolphthalein	11/50	Romania	2017	[156]
	PDE-5 inhibitors and their analogues	74/91	USA	2013	[157]
	PDE-5 inhibitors and their analogues	23/71	Netherland	2013	[158]

^a The term C, refers to the number of food supplements contaminated with pharmacological active ingredients.

^b The term Nr, refers to the number of food supplements analysed.

^c The designation diverse means that at least different three types of food supplements were analysed.

^d Includes samples obtained online.

^eHerbal medicine included in this number.

^fSlimming food products included in this number.

Some of these adulterants are prescriptions or over-the-counter drugs, while others have been banned after concerns about their adverse effects [141]. For instance, sibutramine, an appetite suppressant used in the treatment of obesity, was withdrawn in 2010 due to associated cardiovascular risk [159]. Despite this, sibutramine and/or its derivates is one of the most popular adulterants found in weight loss FS, often in combination with other drugs (Table 6). In some FS, sibutramine reached high concentrations of up to 35 mg per dose [143,151], exceeding the maximum daily dose previously accepted for this drug. Due to genotoxic and carcinogenic risks, phenolphthalein, once used as a laxative, was also withdrawn many years ago [160]. Since constipation is one of the adverse effects of sibutramine, its combination with phenolphthalein has been found in plant FS [161]. Also, Mateescu et al. have alerted that adding phenolphthalein can avoid detection of some primary adulterants by spectroscopic analysis [156].

Other common adulterants of weight-loss FS include approved medicines such as bisacodyl [144,145,147], fluoxetine [145,146], thyroid hormones [147], and furosemide [147]. Although the concentrations of most pharmacologically active ingredients ranged widely, several samples presented very high levels of bisacodyl (up to 199.70 mg/g) and fluoxetine (up to 201.10 mg/g) [144,145].

In the past, dinitrophenol (2,4-DNP) was also used in the treatment of obesity before withdrawn due to its severe toxicity. Considering the interindividual variations in sensitivity, even low 2,4-DNP ingestion may result in serious health effects [162]. However, there is raising evidence of contamination with this toxic substance, especially in online purchased fat burners. Taking all this into account, some authors used the term “Russian roulette” for the consumption of this type of FS [163].

Apart from synthetic compounds, the presence of some natural substances like ephedrine alkaloids (from Ephedra species) has also been detected in weight loss products and is linked to cardiovascular and central nervous system severe effects [164]. Since ephedra and its preparations were prohibited for use in FS [44], the use of the alternative compound p-synephrine is rising. This phenylethylamine derivative with sympathomimetic action is present in weight loss and sports performance enhancement supplements prepared from bitter orange (Citrus aurantium) [165]. However, consumption of high amounts of synephrine also presents adverse cardiovascular effects concerns, especially in the common association with caffeine [166]. Therefore, the combined effects of different naturally occurring substances, illegal pharmaceutical ingredients or both, in weight loss supplements may have risks that should not be underestimated.

As above mentioned, sexual enhancement plant-based supplements are often intentionally contaminated with analogues of PDE-5 inhibitors. Up to now, more than 50 structurally modified analogues of PDE-5 inhibitors were reported as adulterants [167], alone or in combinations [142,155,158]. For those synthetic analogues, especially those never used in clinical practice before, the safety profiles are unknown, and the side effects are not predictable [141]. PDE-5 inhibitors have been used in FS in the same doses [154] or even over 110% of the highest approved drug product strength [157]. Concomitant use of these products with nitrates or α-blockers may cause life-threatening hypotension [168]. Moreover, the presence of different classes of adulterants in the same product, like PDE-5 inhibitors and glyburide, might result in synergistic effects and lead to severe hypoglycaemia, including fatal outcomes [169].

Muscle building/sport performance supplements are another category of FS in which adulterations have been reported. These products may contain anabolic-androgenic steroids (AAS), also called prohormones or other steroid-like substances [137,139,140]. Ingestion of AAS may have numerous side effects, from hypogonadism and infertility in reproductive age men to acute hepatotoxicity [170]. Additionally, it was reported that about 15% of vitamin, mineral, or amino acids based FS may contain undeclared AAS, especially dehydroepiandrosterone (DHEA). Besides intentional adulteration, there is also the possibility of cross-contamination of FS with AAS during the different production stages [171]. Although in some non-European countries DHEA is widely promoted as antiaging supplements, the long-term benefits, as compared with potential adverse health effects, are still unknown [172].

Other pharmaceutical adulterants that can be found in antiaging FS include hypoglycaemic drugs, like metformin and glibenclamide [173,174] and nonopioid analgesics [144]. In addition, a recent study has reported that more than one-quarter of melatonin supplements, commonly used as sleep aids and additionally claimed with many antiaging benefits, are contaminated with unlabelled 5-hydroxytryptamine (5-HTM). Since 5-HTM is a precursor of serotonin, its concurrent ingestion with medicines that modulate synaptic serotonin levels might lead to exacerbation of a potentially lethal serotonin syndrome [175].

In addition to the toxicological and clinical concerns, a rise in adulteration FS with pharmaceutical ingredients presents analytical challenges [141]. Besides conventional spectroscopic and chromatographic methods, many new advanced hyphenated methods have been successfully applied for the detection and structural identification of a large number of different illegal adulterants [176–178]. However, long-term sample processing and available equipment are recognised as the main limiting factors for their application in routine quality/safety controls [179]. Therefore, the improvement of current techniques and the development of new analytical methods for the fast screening and quantification of adulterants in complex mixtures are of great importance.

Notifications in RASFF portal

In addition to a systematic analysis of the information available from academic publications, the data reported by the European Union Rapid Alert System for Food and Feed (RASFF) Safety Alerts portal (https://ec.europa.eu/food/safety/rasff/portal_en) are also highly relevant. RASFF was created in 1979 and has worked since then as a tool to facilitate the flow of information between its members (EU-28 national food safety authorities, Commission, EFSA, ESA, Norway, Liechtenstein, Iceland, and Switzerland), enabling a quick response when a risk to public health is detected in the food chain. This includes an alert system to notify food and feed contaminants that is made public through a portal at the RASFF website. During the period comprised between 1979 and 1990 the number of RASFF alert reports concerning “Dietetic foods, food supplements and fortified foods” rose only 0.5%. In contrast, in the much shorter period from 2011 to 2014 the observed increase was 7.5% [180]. The observed trend in report case number increase highlights the importance of the topic.

Table 7 shows the number, nature, and seriousness of RASFF reports where a specific contaminant was found in FS. Regarding FS adulterations, the number and seriousness degree of RASFF reports indicating the presence of several relevant pharmaceutically active ingredients that were previously found in FS [137] are shown in Figure 1. The number and relevance of metal contamination are clearly demonstrated by the number of “serious” cases reported (Figure 2(A)) from a great number of countries of origin (Figure 2(B)).

Figure 1. Number of cases reported by RASFF concerning adulteration of food supplements with the most common unauthorised pharmaceutical active substances from 1998 to 2018. Risk seriousness is expressed according to the RASFF classification.

Figure 2. Number of cases reported by RASFF concerning contamination of food supplements with metals from 1998 to 2018. The proportion of reports where the contamination was considered serious is shown in (A) and main country of origin of raw material is shown in (B). Data presented in these graphs was obtained from the RASFF report database. Risk seriousness is expressed according to the RASFF classification.

Table 7. RASFF reports regarding food supplements from 1998 to 2018.

Hazard type	Number of reports	Serious risk^a (%)	Contaminants	Main countries of origin (raw material)
Adulteration^b	251	53(134/251)	See Figure 1
Metals	117	45 (53/117)	See Figure 2(A)	See Figure 2(B)
Mycotoxins	1	100 (1/1)	Citrinin	Slovenia
Natural toxins	7	29 (2/7)	Toxic herbal extracts and Solanum nigrum	India, USA, Netherlands, China, Sweden
Pesticide residues	4	25 (1/4)	Triazophos, germanium, lithium, sulphur, etofenprox, chlorate	Germany, Slovakia, Spain, India
Industrial contaminants	2	0 (0/2)	Melamine, semicarbazide	China, India

Data presented in this table was obtained from the RASFF report database.

^a Risk seriousness according to the RASFF classification.

^b The types of adulteration considered in this table are further specified in Figure 1.

Risk assessment considerations

As for food contamination in general, also for FS in particular, the topic of multiple contaminations represents an important issue. In fact, if the impact of a single contaminant is more easily foreseeable, the interaction of different toxicants opens multiple possibilities in the corresponding biological effects. In this respect, the complexity associated to the intake of FS has been touched several times over the last two decades: several problematic factors are arising in this specific manner, both at the analytical and toxicological level. Comprehensive and detailed characterisation of all the ingredients and potential interference of the food/extracts matrices represent one of the major “analytical” challenges associated to the exposure assessment of the contaminants in FS [181]. Similarly, the characterisation and the collection of the information about botanical source, origin of the raw material, processing and formulation can provide precious information for the adequate comparison of the results [182,183]. In addition, correct identification of the material is an essential prerequisite for the formulation of the appropriate conclusions to be translated in the exposure assessment [184].

From the regulatory perspective, the challenge begins with the “definition” of the products per se. In fact, as recently pointed out by Dwyer and colleagues [30], the definition of “Dietary/Food Supplements” and respective regulation, as well as the requirements mandatory for the commercialisation, varies tremendously across the countries especially, when comparing Europe with other markets, such as American and Asiatic. Accordingly, any subsequent toxicological evaluation is hampered by this initial bias and becomes exponentially more difficult.

As emerging in the present work, but also outlined in the “Guidance on safety assessment of botanicals and botanical preparations intended for use as ingredients in food supplements” published by EFSA [43], the possibility of contaminations can extend from biological to chemical contaminants and comprises the most diverse chemical entities and biological pathways. For this reason, a systematic workflow was proposed by the Scientific Committee in 2009, suggesting a tiered-approach based on the previous knowledge available for the supplement of interest [43,185].

Food contaminants risk is a permanent issue for EFSA in Europe. Calls for continuous collection of chemical contaminants occurrence data in food and feed, including FS, are in place for national food authorities, research institutions, academia, food business operators, and other stakeholders. For a call launched in 2019 (https://www.efsa.europa.eu/en/consultations/call/190410), EFSA focus is on the process contaminants, organic and inorganic contaminants, mycotoxins, and plant toxins, thus, covering major critical hazards.

In the case of FS or fortified food, a crucial point for the toxicological evaluation is represented by the exposure to multiple chemicals or “mixtures.” Unspecific or partial description of the ingredients is often accompanied by the possibility of multicontamination scenarios. This includes the possibility of contamination related to the same class of contaminants, as described for instance the contamination with multiple hormones in “antiageing” FS described by He and colleagues [186]. Similarly, in the field of mycotoxin research comparable scenarios might occur. These might originate from the competition of different moulds for the same food item or from subsequent infestations in the pre-, post-harvesting, or storage stages, leading to the co-occurrence of different toxins and related metabolites [187]. In the case of FS, the use of extracts/preparations from different origin contributes to increase the likelihood of multiple contaminations. Especially for not regulated compounds, as in case of the so-called “emerging mycotoxins” [188], there is even no need for the screening from a regulatory perspective. However, the potential for biological effects remains and, in this scenario, the toxicological potential of the single compounds might be substantially enhanced. Multiple contaminations can occur also in case of naturally occurring substances. By definition, the preparation of the FS aims to provide the active constituents in a more concentrated fashion. This process might indeed result in the accumulation of naturally occurring compounds, that simply due their presence in the raw material and are carried over and accumulate in the final formulation of the supplements. As an example, the co-occurrence of structurally-related genotoxic alkenylbenzenes (i.e. elemicin, methyl eugenol, myristicin, safrol) in nutmeg-based plant FS might be mentioned [189,190]. Within this context, it is worthy to note the effort to develop a structured assessment scheme that provides a practical method for assessing botanicals and botanical preparations through a so-called Quality Presumption of Safety (QPS) approach [191]. However, either the preassessment or testing should be subsequently legally mandatory, and, thus, the Threshold of Toxicological Concern (TTC) approach should apply accordingly as a screening and prioritisation tool for the safety assessment when hazard data are incomplete and human exposure can be estimated [192]. The proposed TTC for genotoxic compounds of 0.0025 µg/kg bw/d, based on linear extrapolation for known genotoxic carcinogens, is considered sufficiently protective. Exceptions are high potency carcinogens, i.e. aflatoxin-like, azoxy- or N-nitroso-compounds and benzidines, which are excluded from the current TTC approach. Carcinogens that are not DNA reactive are adequately covered by the other TTC tiers. For those categories of substances as inorganic chemicals, metals, organometallics, proteins, steroids, organo-silicon compounds, TTC is not applicable [192]. Nevertheless, even the QPS approach does not seem to have been adopted for regulatory measures since FS may reach the market without adequate chemistry composition information and toxicological data available, as well as investigated reported adverse effects.

Defining quality for FS is challenging and could be even more difficult than for pharmaceuticals, since FS can be very complex, often containing dozens to hundreds of different chemical substances, many derived from botanicals or other biological sources. Nevertheless, quality control of FS should follow the same standards as pharmaceutical products as both have concentrated ingredients for consumer use. Quality control measures should include the setting of specifications, sampling, testing and analytical clearance, to ensure that raw materials, intermediates, packaging materials and finished products conform to established specifications for identity, strength, purity, and other characteristics. In this regard, the application of hazard analysis and critical control point (HACCP) methodology to FS should be mandatory. Furthermore, Good Manufacturing Practices (GMP) standards should be adapted to FS manufacturing facilities as part of quality assurance. In the case of the Food & Drug Administration, Good Manufacturing Practice in Manufacturing, Packing, Labelling, or Holding Operations for Dietary Supplements were already established [193]. Applying GMPs to FS would be a further step to ensure products consistently produced and controlled to the quality standards appropriate to their intended use.

Complex scenarios can be foreseen by the possibility of the combination of different classes of contaminants [194], for instance, heavy metals and natural toxins [195] or in case of active ingredients and contaminants [196]. In this respect, systematic combinatory studies only started in recent years and risk assessment is still predominantly based on single compound studies. The extension of that classical concept to the evaluation of chemical mixtures is currently a hot topic in toxicology. In addition to interactions among different contaminants, interesting modulatory effects have been already described for their interaction with the so-called “bioactive” food constituents, e.g. polyphenols and mycotoxins [197–199]. In line with the increasing importance of mixtures in food risk assessment, efforts have also been invested by EFSA in the definition of guidelines for the risk assessment of combined exposure to multiple chemicals [200]. To add complexity to the potential evaluation of the risk associated to the consumption of FS, especially in elderly people, it has to be considered that intake of these supplements is often combined with pre-existing diseases and respective therapeutic regiments. In this case, risk assessment may vary a lot on a personal basis and tailored approaches probably represent the most adequate option. Furthermore, it has to be pointed out that regular intake of a contaminated FS might individually enhance the exposure to contaminants, thus potentially exceeding the estimated exposure of the general population based on overall nutrition consumption pattern, which are currently used by food safety authorities for risk assessment as a basis for the definition of regulatory limits.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This article is based upon work from COST Action NutRedOx-CA16112 supported by COST (European Cooperation in Science and Technology). B. V. also acknowledges the COST Action NutRedOx, CA16112 for her STSM Grant. The authors acknowledge the support from Fundação para a Ciência e a Tecnologia (FCT, Portugal), through funding UID/DTP/04567/2019 to CBIOS and UID/DTP/04138/2019 to iMed.ULisboa, as well as from the Ministarstvo Prosvete, Nauke i Tehnolo_skog Razvoja of the Republic of Serbia, through the project III46009.