Mycotoxins as human carcinogens—the <Emphasis Type="Italic">IARC Monographs</Emphasis> classification

Humans are constantly exposed to mycotoxins (e.g. aflatoxins, ochratoxins), mainly via food intake of plant and animal origin. The health risks stemming from mycotoxins may result from their toxicity, in particular their carcinogenicity. In order to prevent these risks, the International Agency for Research on Cancer (IARC) in Lyon (France)—through its IARC Monographs programme—has performed the carcinogenic hazard assessment of some mycotoxins in humans, on the basis of epidemiological data, studies of cancer in experimental animals and mechanistic studies. The present article summarizes the carcinogenic hazard assessments of those mycotoxins, especially aflatoxins (aflatoxin B₁, B₂, G₁, G₂ and M₁), fumonisins (fumonisin B₁ and B₂) and ochratoxin A (OTA). New information regarding the genotoxicity of OTA (formation of OTA-DNA adducts), the role of OTA in oxidative stress and the identification of epigenetic factors involved in OTA carcinogenesis–should they indeed provide strong evidence that OTA carcinogenicity is mediated by a mechanism that also operates in humans–could lead to the reclassification of OTA.     

Development of an in vitro method for the prediction of mycotoxin binding on yeast-based products: case of aflatoxin B1, zearalenone and ochratoxin A

To date, no official method is available to accurately define the binding capacity of binders. The goal is to define general in vitro parameters (equilibrium time, pH, mycotoxin/binder ratio) for the determination of binding efficacy, which can be used to calculate the relevant equilibrium adsorption constants. For this purpose, aflatoxin B₁ (AFB₁), zearalenone (ZEA) or ochratoxin A (OTA) were incubated with one yeast cell wall in pH 3, pH 5 or pH 7 buffers. The percentage of adsorption was recorded by quantitation of remaining mycotoxins in the supernatant and amount of mycotoxin adsorbed on the residue. The incubation of yeast cell wall in the presence of mycotoxins solved in buffer, lead to unexpected high adsorption percentage when the analysis was based only on remaining mycotoxins in the supernatant. The decrease of mycotoxins in the supernatant was not correlated to the amount of mycotoxins found in the residue. For this reason we modified the conditions of incubation. Yeast cell wall (5 mg) was pre-incubated in buffer (990 μl) at 37 °C during 5 min and then 10 μl of an alcoholic solution of mycotoxin (concentration 100 times higher than the final concentration required in the test tube) were added. After incubation, the solution was centrifuged, and the amount of mycotoxins were analysed both in the supernatant and in the residue. A plateau of binding was reached after 15 min of incubation whatever the mycotoxins and the concentrations tested. The adsorption of ZEA was better at pH 5 (75 %), versus 60 % at pH 3 and 7. OTA was only significantly adsorbed at pH 3 (50 %). Depending on the pH, the adsorptions of OTA or ZEA were increased or decreased when they were together, indicative of a cooperative effect.     

The production of aflatoxin B1 or G1 by Aspergillus parasiticus at various combinations of temperature and water activity is related to the ratio of aflS to aflR expression

The influence of varying combinations of water activity (a_w) and temperature on growth, aflatoxin biosynthesis and aflR/aflS expression of Aspergillus parasiticus was analysed in the ranges 17–42°C and 0.90–0.99 a_w. Optimum growth was at 35°C. At each temperature studied, growth increased from 0.90 to 0.99 a_w. Temperatures of 17 and 42°C only supported marginal growth. The external conditions had a differential effect on aflatoxin B₁ or G₁ biosynthesis. The temperature optima of aflatoxin B₁ and G₁ were not at the temperature which supported optimal growth (35°C) but either below (aflatoxin G₁, 20–30°C) or above (aflatoxin B₁, 37°C). Interestingly, the expression of the two regulatory genes aflR and aflS showed an expression profile which corresponded to the biosynthesis profile of either B₁ (aflR) or G₁ (aflS). The ratios of the expression data between aflS:aflR were calculated. High ratios at a range between 17 and 30°C corresponded with the production profile of aflatoxin G₁ biosynthesis. A low ratio was observed at >30°C, which was related to aflatoxin B₁ biosynthesis. The results revealed that the temperature was the key parameter for aflatoxin B₁, whereas it was water activity for G₁ biosynthesis. These differences in regulation may be attributed to variable conditions of the ecological niche in which these species occur.     

Einsatz einer kombinierten Immunoaffinitätssäule zum Nachweis von Aflatoxinen (B<Subscript>1</Subscript>, G<Subscript>1</Subscript>, B<Subscript>2</Subscript>, G<Subscript>2</Subscript>), Ochratoxin und Zearalenon

A method for the combined determination of the mycotoxins aflatoxin B₁, G₁, B₂, G₂, ochratoxin A and zearalenone in cereals and feed is described. After extraction with acetonitrile/water or methanol/water the cleaning takes place with new combined immunoaffinity clean-up column “AflaOchraZea” by VICAM. When the mycotoxins are determined in different cereals with this new type of clean-up column low detection limits and high recovery rates can be reached similar to those obtained by using separate immunoaffinity clean-up colums for the said mycotoxins.     

Mycobiota in poultry feeds and natural occurrence of aflatoxins, fumonisins and zearalenone in the Rio de Janeiro State, Brazil

The intake of mycotoxin-contaminated feeds can lead to nutrient losses and may have adverse effects on animal health and on productivity. The aims of this study were (1) to determine the mycobiota present in poultry feed samples, and (2) to evaluate the natural occurrence of aflatoxin B₁, fumonisin B₁ and zearalenone. Fungal counts were similar between all culture media tested (10³ CFU g⁻¹). The most frequent genus isolated was Penicillium spp. (41.26%) followed by Aspergillus spp. (33.33%) and Fusarium spp. (20.63%). High precision liquid chromatography was applied to quantify aflatoxin B₁ and fumonisin B₁. Thin layer chromatography was used to determine zearalenone levels. Aflatoxin B₁ values ranged between 1.2 and 17.5 μg kg⁻¹. Fumonisin B₁ levels ranged between 1.5 and 5.5 μg g⁻¹. Zearalenone levels ranged between 0.1 and 7 μg g⁻¹. The present study shows the simultaneous occurrence of two carcinogenic mycotoxins, aflatoxin B₁ and fumonisin B₁, together with another Fusarium mycotoxin (zearalenone) in␣feed intended for poultry consumption. Many samples contained AFB₁ levels near the permissible maximum and it could affect young animals. A synergistic toxic response is possible in animals under simultaneous exposure.     

Isolation of Bacillus spp. from Thai fermented soybean (Thua-nao): screening for aflatoxin B1 and ochratoxin A detoxification

Aims: To study the interaction between Bacillus spp. and contaminating Aspergillus flavus isolated strains from Thai fermented soybean in order to limit aflatoxin production. To study the detoxification of aflatoxin B₁ (AFB₁) and ochratoxin A (OTA) by Bacillus spp. in order to find an efficient strain to remove these toxins. Methods and Results: One A. flavus aflatoxin-producing strain and 23 isolates of Bacillus spp. were isolated from soybean and fresh Thua-nao collected from the north of Thailand. Inhibition studies of A. flavus and A. westerdijkiae NRRL 3174 (reference strain) growth by all isolates of Bacillus spp. were conducted by dual culture technique on agar plates. These isolates were also tested for AFB₁ and OTA detoxification ability on both solid and liquid media. Most of the strains were able to detoxify aflatoxin but only some of them could detoxify OTA. Conclusions: One Bacillus strain was able to inhibit growth of both Aspergillus strains and to remove both mycotoxins (decrease of 74% of AFB₁ and 92·5% of OTA). It was identified by ITS sequencing as Bacillus licheniformis. The OTA decrease was due to degradation in OTα. Another Bacillus strain inhibiting both Aspergillus growth and detoxifying 85% of AFB₁ was identified as B. subtilis. AFB₁ decrease has not been correlated to appearance of a degradation product. Significance and Impact of the Study: The possibility to reduce AFB₁ level by a strain from the natural flora is of great interest for the control of the quality of fermented soybean. Moreover, the same strain could be a source of efficient enzyme for OTA degradation in other food or feeds.     

Enniatin B and ochratoxin A in the blood serum of workers from the waste management setting

The waste management occupational environment is recognized by the simultaneous presence of several substances and biologic agents. Therefore, workers are exposed simultaneously to multiple contaminants. Occupational exposure to aflatoxin B₁ in one Portuguese waste sorting plant was already reported. However, besides this mycotoxin, data regarding fungal contamination showed that exposure to other mycotoxins could be expected. A study was developed to analyze if exposure to other mycotoxins besides aflatoxin B₁ was occurring in the workers from the waste sorting plant previously assessed and to discuss how these findings need to be considered in the risk assessment process. In addition to aflatoxin B₁ detected previously by ELISA, two additional mycotoxins and one mycotoxin degradation product were detected and quantified by a multi-mycotoxin HPLC-MS/MS approach: Enniatin B and ochratoxin A as well as 2’R-ochratoxin A. Besides the confirmation of co-exposure to several mycotoxins, results probably indicate different exposure routes for the mycotoxins reported.     

New examinations of mycotoxin carryover to cocoa beans

Mycotoxins are secondary metabolites which can form on various foodstuffs through the growth of certain fungi. Ochratoxin A (OTA) and the aflatoxins B₁ B₂, G₁ and G₂ have been detected in low concentrations in cocoa and cocoa products. As regards the question of in what stages of the cocoa production process a contamination with the mycotoxin-producing moulds and the formation of mycotoxins takes place, it is assumed that in the case of cocoa the contamination is not concerning the individual beans but the fermentation units. A model test was carried out to provide information on the process by which a possible carryover of the above-mentioned mycotoxins to cocoa beans occurs during the fermentation process. For this purpose fresh cocoa beans were left to soak in an artificial mycotoxin-containing fermentation solution. The mycotoxin levels in the cocoa beans were regularly determined over a period of 12 days. New findings were made as regards the migration of mycotoxins during the fermentation process. We interpret the divergent uptake behaviour of the mycotoxins to indicate that the transport of OTA and that of aflatoxins does not take place in the same manner. This is possibly caused by chemico-physical effects, such as the different polarities of the mycotoxins. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006     

Synthesis and toxicity evaluation of aflatoxin P

Conversion of aflatoxin B₁ to its demethylated derivative, aflatoxin P₁, has been achieved by treatment of the parent substance with lithium $\text{t}$-butylmercaptide in hexamethyl phosphoramide for 2 hours at 75°C. A preliminary toxicologic evaluation in newborn mice showed aflatoxin P₁ to cause some mortality at 150 mg/kg, whereas aflatoxin B₁ had an LD_{5 0} of 9.5 mg/kg under comparable conditions.     

Seasonal variation in exposure frequency and concentration levels of aflatoxins and ochratoxins in urine samples of boys and girls

Urine samples from children in Sierra Leone (134 boys and 110 girls), were collected during the dry season. During the rainy season samples were collected from 97 boys and 93 girls. Analysis of the dry season samples, revealed that, with the exception of one boy, all children had detectable amounts of aflatoxins and/or ochratoxins in their urine. Similarly, with the exception of four children (two from each sex), rainy season urine samples also contained these two mycotoxins. There were significant differences in the frequency of exposure to some mycotoxins: ochratoxin A (OTA), p < 0.01;4-hydroxyochratoxin A (4R-OTA), p < 0.002; aflatoxin M1 (AFM₁), p < 0.04;aflatoxicol (AFL), p < 0.03; aflatoxin B₂ (AFB₂), p < 0.04 . There were also significant differences in the levels of aflatoxin B₁ (AFB₁), (p < 0.05) and AFB₂, (p < 0.02) detected in dry season samples. Stratification of these results according to season and sex, has indicated significant differences with respect to 4R-OTA (p < 0.04) and AFB₁ (p < 0.02). The results of this study show that in Sierra Leone, children are frequently and constantly exposed to both aflatoxins and ochratoxins. This revised version was published online in August 2006 with corrections to the Cover Date.     

Carbon dioxide production as an indicator of Aspergillus flavus colonisation and aflatoxins/cyclopiazonic acid contamination in shelled peanuts stored under different interacting abiotic factors

Aspergillus flavus is the main xerophylic species colonising stored peanuts resulting in contamination with aflatoxins (AFs) and cyclopiazonic acid (CPA). This study evaluated the relationship between storage of shelled peanuts under interacting abiotic conditions on (a) temporal respiration (R) and cumulative CO₂ production, (b) dry matter losses (DMLs) and (c) aflatoxin B₁ (AFB₁) and CPA accumulation. Both naturally contaminated peanuts and those inoculated with A. flavus were stored for 7-days under different water activities (a_w; 0.77–0.95) and temperatures (20–35°C). There was an increase in the temporal CO₂ production rates in wetter and warmer conditions, with the highest respiration at 0.95 a_w + A. flavus inoculum at 30°C (2474 mg CO₂kg⁻¹h⁻¹). The DMLs were modelled to produce contour maps of the environmental conditions resulting in maximum/minimum losses. Maximum mycotoxin contamination was always at 0.95 a_w although optimal temperatures were 25-30°C for AFs and 30-35°C for CPA. These results showed a correlation between CO₂ production and mycotoxin accumulation. They also provide valuable information for the creation of a database focused on the development of a post-harvest decision support system to determine the relative risks of contamination with these mycotoxins in stored shelled peanuts.     

Preparation, purification and characteristics of an aflatoxin degradation enzyme from Myxococcus fulvus ANSM068

Aims: To prepare, purify and characterize an extracellular enzyme from Myxococcus fulvus ANSM068, designated as myxobacteria aflatoxin degradation enzyme (MADE), which possesses degradation activity against aflatoxin B₁ (AFB₁), G₁ (AFG₁) and M₁ (AFM₁) in solution. Methods and Results: The culture supernatant of strain M. fulvus demonstrated high degradation ability against AFB₁ (71·89%), AFG₁ (68·13%) and AFM₁ (63·82%) after 48 h of incubation. An enzyme was purified from the supernatant of M. fulvus using ethanol precipitation and chromatography on DEAE‐Sepharose and Superdex 75. An overall 166‐fold purification of the enzyme with a recovery of 57% and a final specific activity of 569·44 × 10³ U mg^?1 was obtained using the present purification protocol. The apparent molecular mass of MADE was estimated to be 32 kDa by SDS‐PAGE. AFG₁ and AFM₁ were significantly degraded, by 96·96 and 95·80%, respectively, when treated with pure MADE (100 U ml^?1) produced by strain ANSM068. MADE exhibited the largest amount of activity at 35°C and pH 6·0, with Mg²⁺ ions greatly promoting and Zn²⁺ strongly inhibiting MADE activity. Conclusions: An aflatoxin degradation enzyme from bacterial isolates can effectively remove aflatoxin B₁, G₁ and M₁ in solution. Significance and Impact of the Study: The high activity and wide temperature and pH range of MADE for the degradation of aflatoxin have promising applications in control of mycotoxins during food and feed processing.     

Mycotoxins in several Polish food products in 2004–2005

In 2004–2005, samples of several selected Polish foods such as cereal products, nuts, dried fruits, coffee and culinary spices collected from Warsaw market and taken from food producers were analyzed on presence of aflatoxin B₁, B₂, G₁, G₂ (AF), ochratoxin A (OTA), zearalenone (ZEA) and deoxynivalenol (DON). After extraction and clean-up of extracts on immunoaffinity columns (IAC), mycotoxin analyses were carried out by HPLC using fluorescence and UV detectors. The concentrations of aflatoxins and ochratoxin A depending on the kind of sample ranged from 0.02 to 7.8 (one sample, of peanuts) and 0.02–11.9 μg/kg (one coffee sample), respectively. The levels of ZEA and DON were found to be below 50 °g/kg.     

Simultaneous occurrence of mycotoxins in human food commodities from Cameroon

Eighty-two samples of dried food commodities from Cameroon were screened and quantified for different mycotoxins, including fumonisin B₁ (FB₁), zearalenone (ZEA), deoxynivalenol (DON), aflatoxin (AF) and ochratoxin A (OTA), by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. The percentage of positive samples was as follows: FB₁ 41%, AF 51%, ZEA 57%, DON 65% and OTA 3%. High FB₁ contents were found in maize, averaging 3,684 μg/kg (range: 37-24,225 μg/kg), whereas the highest average ZEA level was found in peanuts (70 μg/kg), followed by maize (69 μg/kg), rice (67 μg/kg) and beans (48 μg/kg) with no ZEA was detected in soybeans. DON contents were low, ranging from 13 to 273 μg/kg, and for AF the average content was 2.6 μg/kg with peanuts and maize as principal substrates. The incidence of OTA was low, with a mean level of 6.4 μg/kg recorded. The majority (79%) of samples contained more than one mycotoxin and the most frequent co-occurrence found was FB₁ + ZEA + DON, detected in 21% of samples (mainly maize) analysed. Co-contamination with FB₁ + ZEA + DON + AF was found in 11% of the samples. Although a large proportion of samples had fairly low levels of individual mycotoxins, this should be of concern as the co-occurrence of mycotoxins may generate additive or synergistic effect in humans, especially if the respective commodities are consumed almost on a daily basis.     

In Vitro Detoxification of Aflatoxin B1, Deoxynivalenol,Fumonisins, T-2 Toxin and Zearalenone by Probiotic Bacteria from Genus Lactobacillus and Saccharomyces cerevisiae Yeast

The aim of the following research was to determine the detoxification properties of probiotic Lactobacillus sp. bacteria (12 strains) and S. cerevisiae yeast (6 strains) towards mycotoxins, such as aflatoxin B₁, deoxynivalenol, fumonisins, T-2 toxin and zearalenone, which pose as frequent feed contamination. The experiment involved analysing changes in concentration of mycotoxins in PBS solutions, after 6, 12 and 24 h of incubation with monocultures of tested microorganisms, measured by high-performance liquid chromatography (HPLC). We found that all strains detoxified the mycotoxins, with the highest reduction in concentration observed for the fumonisin B₁ and B₂ mixture, ranging between 62 and 77% for bacterial strains and 67–74% for yeast. By contrast, deoxynivalenol was the most resistant mycotoxin: its concentration was reduced by 19–39% by Lactobacillus sp. strains and 22–43% by yeast after 24 h of incubation. High detoxification rates for aflatoxin B₁, T-2 toxin and zearalenone were also observed, with concentration reduced on average by 60%, 61% and 57% by Lactobacillus, respectively, and 65%, 69% and 52% by yeast, respectively. The greatest extent of reduction in the concentration for all mycotoxins was observed after 6 h of incubation; however, a decrease in concentration was noted even after 24 h of incubation. Thus, the tested microorganisms can potentially be used as additives to decrease the concentrations of toxins in animal feed.

     

Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries

Alternaria alternata, A. tenuissima, Fusarium graminearum, F. semitectum, F. verticillioides, Aspergillus flavus, and Aspergillus section Nigri strains obtained from blueberries during the 2009 and 2010 harvest season from Entre Ríos, Argentina were analyzed to determine their mycotoxigenic potential. Taxonomy status at the specific level was determined both on morphological and molecular grounds. Alternariol (AOH), alternariol monomethyl ether (AME), aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs), and ochratoxin A (OTA) were analyzed by HPLC and the trichotecenes deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), fusarenone X (FUS-X), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) by GC. Twenty-five out of forty two strains were able to produce some of the mycotoxins analyzed. Fifteen strains of Aspergillus section Nigri were capable of producing Fumonisin B₁ (FB₁); two of them also produced Fumonisin B₂ (FB₂) and one Fumonisin B₃ (FB₃). One of the F. graminearum isolated produced ZEA, HT-2, and T-2 and the other one was capable of producing ZEA and DON. Two A. alternata isolates produced AOH and AME. Four A. tenuissima were capable of producing AOH and three of them produced AME as well. One Aspergillu flavus strain produced aflatoxin B₁ (AFB₁), aflatoxin B₂ (AFB₂), and aflatoxin G₁ (AFG₁). To our knowledge, this is the first report showing mycotoxigenic capacity of fungal species isolated from blueberries that include other fungi than Alternaria spp.     

Mycotoxins in organic and conventional cereals and cereal products grown and marketed in Croatia

In this study, the levels of aflatoxin B₁ (AFB₁), ochratoxin A (OTA), zearalenone (ZEN), deoxynivalenol (DON) and fumonisins (FUM) in unprocessed cereals (n = 189) and cereal-based products (n = 61) were determined using validated ELISA methods. All samples originated from either conventional or organic production corresponded to the 2015 harvest in Croatia. Based on the mean mycotoxin concentrations, the risk for the consumer to exceed the tolerable daily intake (TDI) for these toxins by the consumption of both types of cereals and cereal-based products was assessed. Mycotoxin contamination of organic cereals and organic cereal-based products was not significantly different (p > 0.05). Given that the exposure assessment resulted in a small fraction of the TDI (maximum: DON, 12% of TDI), the levels of the investigated mycotoxins in both types of cereals and cereal-based products from the 2015 harvest did not pose a human health hazard.     

Reduction of aflatoxins by Rhizopus oryzae and Trichoderma reesei

This study evaluated the ability of the microorganisms Rhizopus oryzae (CCT7560) and Trichoderma reesei (QM9414), producers of generally recognized as safe (GRAS) enzymes, to reduce the level of aflatoxins B₁, B₂, G₁, G₂, and M₁. The variables considered to the screening were the initial number of spores in the inoculum and the culture time. The culture was conducted in contaminated 4 % potato dextrose agar (PDA) medium, and the residual mycotoxins were determined every 24 h by HPLC-FL. The fungus R. oryzae has reduced aflatoxins B₁, B₂, and G₁ in the 96 h and aflatoxins M₁ and G₂ in the range of 120 h of culture by approximately 100 %. The fungus T. reesei has reduced aflatoxins B₁, B₂, and M₁ in the 96 h and aflatoxin G₁ in the range of 120 h of culture by approximately 100 %. The highest reduction occurred in the middle of R. oryzae culture.     

In situ absorption of aflatoxins in rat small intestine

To evaluate the rate at which the four main aflatoxins (aflatoxins B₁, B₂, G₁ and G₂) are able to cross the luminal membrane of the rat small intestine, a study about intestinal absorption kinetics of these mycotoxins has been made. In situ results obtained showed that the absorption of aflatoxins in rat small intestine is a very fast process that follows first-order kinetics, with an absorption rate constant (k _a ) of 5.84±0.05 (aflatoxin B₁), 4.06±0.09 (aflatoxin B₂), 2.09±0.03 (aflatoxin G₁) and 1.58±0.04 (aflatoxin G₂) h^–1, respectively.     

Analysis of cocoa products for ochratoxin A and aflatoxins

Eighty-five samples of cocoa products sampled in Canada were analysed for ochratoxin A (OTA) and aflatoxins in 2011–2012. Inclusion of the aflatoxins in this survey required additional method development. Chocolate was extracted with methanol–water plus NaCl, while for cocoa two successive extractions with methanol and methanol–water were made. Extracts were cleaned on an AflaOchra immunoaffinity column (IAC). Determination was by reversed phase high performance liquid chromatography (HPLC). Detection of the aflatoxins was with a post-column photochemical reactor and of OTA by fluorescence detection. Mean limits of quantification (LOQ) of chocolate and cocoa powders were 0.16 ng/g (OTA) and 0.07 ng/g (aflatoxin B₁), respectively. Survey results showed that the incidences of OTA above the LOQ in natural cocoa were 15/15 (mean 1.17 ng/g), 20/21 for alkalized cocoa (mean 1.06 ng/g), 9/9 for baking chocolate (mean 0.49 ng/g), 20/20 for dark chocolate (mean 0.39 ng/g), 7/10 for milk chocolate (mean 0.19 ng/g), 5/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. These results confirm our previous work with OTA. In the same samples, incidences of aflatoxin B₁ above the LOQ were 14/15 for natural cocoa (mean 0.86 ng/g), 20/21 for alkalized cocoa (mean 0.37 ng/g), 7/9 for baking chocolate (mean 0.22 ng/g), 16/20 for dark chocolate (mean 0.19 ng/g), 7/10 for milk chocolate (mean 0.09 ng/g), 4/5 for cocoa liquor (mean 0.43 ng/g), and 0/5 for cocoa butter. Both aflatoxins and OTA were confirmed by HPLC-MS/MS when OTA or aflatoxin levels found were above 2 ng/g in cocoa.