Mycotoxin contamination of sorghum and its contribution to human dietary exposure in four sub-Saharan countries

This research aimed at evaluating the safety, and the type, level and prevalence of mycotoxins in grain sorghum of four sub-Saharan African (SSA) countries (Burkina Faso, Ethiopia, Mali and Sudan). A multi-analyte LC-MS/MS method for quantification of 23 mycotoxins (nivalenol, deoxynivalenol, fusarenon X, neosolaniol, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, diacetoxyscirpenol, roquefortine C, HT-2 toxin, alternariol, T-2 toxin, FB1, FB2, FB3, zearalenone, aflatoxin G₁, aflatoxin G₂, aflatoxin B₁, aflatoxin B₂, sterigmatocystin, OTA, altenuene, alternariol monomethylether) was applied to different sorghum matrices. Of the 1533 analysed samples, 33% were contaminated with at least one of the following mycotoxins: aflatoxins, fumonisins, sterigmatocystin, Alternaria toxins, OTA and zearalenone. Country of origin, colour, source and collection period of sorghum samples significantly influenced the type, level and prevalence of mycotoxins. Sterigmatocystin (15%), fumonisins (17%) and aflatoxins (13%) were the most prevalent. FB1 (274 ± 585 µg/kg) had the highest mean concentration followed by FB2 (214 ± 308 µg/kg) while diacetoxyscirpenol (8.12 ± 19.2 µg/kg) and HT-2 (11.9 ± 0.00 µg/kg) had the lowest concentrations. Neosolaniol, fusarenon-X, 3-acetyl deoxynivalenol, 15-acetyl deoxynivalenol, T-2 toxin, nivalenol and roquefortine C were not detected in any of the samples. Sudan had the lowest prevalence and mean concentration of all mycotoxins. Pink sorghum had the highest concentrations of fumonisins and aflatoxins. Mycotoxins from Aspergillus spp. and Alternaria spp. are the mycotoxins of concern in SSA grain sorghum with regard to prevalence, concentration and possible health risk from exposure. Based on the performed risk characterisation, daily consumption of sorghum containing aflatoxins, alternariol, alternariol monomethyl ether, sterigmatocystin and OTA could result in exceeding the established health-based guidance values for these toxins.     

Aflatoxins and heavy metals in animal feed in Iran

The occurrence of aflatoxin (aflatoxin B₁, aflatoxin B₂, aflatoxin G₁ (AFG₁) and aflatoxin G₂ (AFG₂)) and heavy metal (Pb, Cd, As and Hg) contamination was determined in 40 industrially produced animal feed samples which were collected from the southwest of Iran. The results indicated that 75% of samples were contaminated by four aflatoxins and the level of AFB₁ and sum of aflatoxins were higher than the permissible maximum levels in Iran (5 and 20 µg kg^?1, respectively) in all feed samples. A positive correlation was found between four types of aflatoxins in all the tested samples (p < 0.01) and the positive correlation between AFG₁ and AFG₂ was significant (r² = 0.708). All feed samples had lead concentrations lower than the maximum EU limit, while 5%, 17% and 42.5% of feed samples had As, Cd and Hg concentrations higher than the maximum limits, respectively.     

Mycotoxin contamination of animal feedingstuff: detoxification by gamma-irradiation and reduction of aflatoxins and ochratoxin A concentrations

Mycotoxins are fungal secondary metabolites identified in many agricultural products screened for toxigenic moulds. They have been reported to be carcinogenic, teratogenic, tremorogenic, haemorrhagic and dermatitic to a wide range of organisms. With the increasing stringent regulations for mycotoxins imposed by importing countries such as those of the European Union, many cereals that are not safe for human consumption are used in formulations intended for animal feed. Gamma-rays are reported in the scientific literature to destroy ochratoxin A and aflatoxin in food crops and feed. The present study provides preliminary data for establishing the effect of dose of gamma-irradiation, ranging from 0 to 15 kGy, on aflatoxins and ochratoxin A reduction in commercial animal feed. The mycotoxin levels were determined by means of immunoaffinity clean-up (IAC) and HPLC with fluorescence detection (HPLC-FLD). The maximum reductions found at 15 kGy were 23.9%, 18.2%, 11.0%, 21.1% and 13.6% for ochratoxin A, aflatoxin B₁, aflatoxin B₂, aflatoxin G₁ and aflatoxin G₂, respectively. Results showed that the gamma-rays even at 15 kGy were not effective in the complete destruction of ochratoxin A and aflatoxins in the tested feed.     

Aflatoxins in dairy cow feed,raw milk and milk products from Turkey

This study aims to detect aflatoxins (AFs) in dairy cow feed, milk and milk products using a high-performance liquid chromatography coupled with fluorescence detection (HPLC-FLD) method. All the validation parameters met the method performance criteria of the European Union. The samples comprised 76 dairy cow feeds and 205 milk and milk products (including yoghurt and yoghurt-based beverage, ayran). AFs were present in 26.3% of the feed samples. Two feed samples exceeded the maximum limit (ML) of 5 µg kg^?1 for AFB₁ as established by the EU. Nineteen milk samples (21.1%) contained aflatoxin M₁ (AFM₁) of which three exceeded the EU ML of 0.05 µg l^?1. In addition, only two yoghurt samples and one ayran sample contained AFM₁, but the levels were lower than the EU ML.     

Survey of roasted street-vended nuts in Sierra Leone for toxic metabolites of fungal origin

Consumption of mycotoxin contaminated foodstuffs is common in regions where foods are not adequately controlled and routinely monitored, and this could have adverse effects on the health of consumers. In this study, 100 samples of roasted nuts (50 cashew nuts and 50 peanuts) vended within two cities of Sierra Leone were analysed for mycotoxins and other microbial metabolites by a LC-MS/MS method. The peanut samples contained detectable levels of 17 microbial metabolites, including aflatoxins B₁, B₂, G₁ and G₂ and alternariol, while none of these metabolites were found in the cashew samples. Aflatoxins (max: 5,729 μg/kg; mean: 487.8 μg/kg) and alternariol (3 μg/kg) were found in 24% and 2% of the peanut samples, respectively. One-third of the aflatoxin-contaminated peanut samples contained aflatoxins at levels exceeding the total aflatoxin limit of 4 μg/kg set by the European Union. Aflatoxin contamination of Sierra Leonean peanuts is high and requires urgent intervention to reduce consequent exposure.     

SCREENING AND QUANTIFICATION OF AFLATOXINS AND OCHRATOXIN A IN DIFFERENT CEREALS CULTIVATED IN ROMANIA USING THIN-LAYER CHROMATOGRAPHY-DENSITOMETRY

ABSTRACT

The main focus of our study was to implement a rapid, inexpensive and reliable method that could be utilized to check the cereals for safety (i.e., screening for total aflatoxins, as well as individual B₁, B₂, G₁, G₂ aflatoxins and ochratoxin A). We developed a protocol by which we were able to isolate mycotoxins from cereals collected from different regions of Romania. After extraction in chloroform, the mycotoxins were separated by thin‐layer chromatography (TLC) and quantified using densitometry. Forty‐three samples of different cereals (wheat, maize, rye and Triticale) were analyzed. Twenty‐five of the 43 samples (58.14% of the total number) were found to be contaminated with different mycotoxins in various concentrations: aflatoxin B₁ (1.6–5.7 µg/kg), aflatoxin B₂ (0.89–4 µg/kg), aflatoxin G₁ (1.2–5.76 µg/kg), aflatoxin G₂ (0.96–3.4 µg/kg) and/or 4.3–30 µg/kg ochratoxin A. The concentration of total aflatoxin contamination ranged from 11.2 to 10.8 µg/kg. Among the different cereals, the highest number of contaminated samples was found to be in the wheat samples (62.5%). The method outlined in this study has been adopted already by our laboratory for current analyses of mycotoxins. The results obtained using this method is in compliance with the strict regulatory guidelines developed both in Romania, as well as in the European Union.

PRACTICAL APPLICATIONS

Thin‐layer chromatography (TLC) is a rapid, inexpensive and convenient method that can be used routinely to screen for mycotoxins. This article describes the detailed procedures for the extraction, purification and quantification of aflatoxins and ochratoxin A, using TLC. Using this method one can identify concomitantly five different mycotoxins and by coupling it with densitometry a quantitative determination is also possible. Therefore, this could become a routine technique in regional laboratories responsible for checking agrifood safety.     

AFLATOXIGENIC ASPERGILLI IN FOODS AND FEEDS IN UGANDA

Studies conducted during the sixties and the seventies on food crops in Uganda showed that the populace was exposed to consumption of aflatoxin-contaminated foods. These studies also linked the highest incidence of liver cancer in the world to the presence of high levels of aflatoxins in the food and beverages. After a lapse of a decade, it was of interest to investigate the occurrence of aflatoxins and aflatoxigenic fungi in staple Ugandan food crops and poultry feeds derived from these foodstuffs. A simple, rapid and reproducible procedure was used. The procedure consisted of growing or culturing feed grains on a selective medium, Aspergillus flavus/parasiticus agar (AFPA) followed by screening for aflatoxin producing fungi on a coconut agar medium (CAM) under UV light with a subsequent confirmatory screening method for aflatoxin production by the fungi in pure culture. Fifty-four samples consisting of corn and peanuts, soybean and poultry feed were analyzed for content of aflatoxigenic. A. flavus/parasiticus and 25 of the samples were also screened for aflatoxins B₁ and G₁, zearalenone, sterigmatocystin, ochratoxin A, citrinin, vomitoxin, and diacetoxyscirpenol (DAS). Aflatoxigenic A. flavus/parasiticus was detected from the majority of corn (77%), peanuts (36% human food and 83.3% animal feed) and poultry feed (66.6%). but not from soybean samples. Two samples out of 25 contained detectable levels of aJatosin B, (20 ppb). For the jirst time other mycotoxins, zearalenone (3 samples) and vomitoxin (2 samples) were detected in corn from Uganda.     

Natural co-occurrence of ochratoxin A,ochratoxin B and aflatoxins in Sicilian red wines

The natural occurrence of ochratoxin A, ochratoxin B, aflatoxin B₁, aflatoxin B₂, aflatoxin G₁ and aflatoxin G₂ (OTA, OTB, AFB₁, AFB₂, AFG₁, AFG₂) in red wines was investigated by HPLC/FLD after immunoaffinity column clean-up in 57 market samples produced in Sicily (Italy). The results showed a very low incidence of these mycotoxins in analysed samples, confirming the high degree of quality and safety of Sicilian red wines. The results indicated 71.9% and 64.9% positive samples for OTA and OTB respectively, with an average level of 0.13 μg l^–1, well below the European maximum permitted levels (MLs). The aflatoxin most frequently detected in the samples was AFG₁, present in 57.9% of samples, while the other aflatoxins were rarely present. Recovery experiments were carried out on eight mycotoxin-free red wines spiked with OTA, OTB, AFB₁, AFB₂, AFG₁ and AFG₂ at two different levels. The limits of detection (LODs) in wines were 0.02 µg l^–1 for OTA, 0.04 µg l^–1 for OTB, 0.03 µg l^–1 for AFG₁, AFG₂ and AFB₂, and 0.05 µg l^–1 for AFB₁. A good correlation was found, with good performances in term of precision for the method.     

Aflatoxins in cotton seeds and cotton seed cake from Pakistan

ABSTRACT

A study was conducted to evaluate the natural occurrence of aflatoxins (AFB1, AFB2, AFG1, AFG2) in cotton seeds (n = 110) and cotton seed cake (CSC; n = 110) from Pakistan. All samples were screened by Enzyme-Linked ImmunoSorbent Assay (ELISA). Positive samples were further quantified by IAC-HPLC-FLD. Total contamination frequency and aflatoxins mean levels were 80% and 69 μg/kg in cotton seeds and the corresponding values for cotton seed cake 88% and 89 μg/kg, respectively. Aflatoxin B1was found in all positive samples and co-occurred with AFB2, AFG1, and AFG2. Sixty-four cotton seeds and 71 CSC samples contained aflatoxins levels higher than the ML set for animal feed (20 µg/kg). The results of the present study will help the regulatory authorities to formulate strategies for monitoring aflatoxins in animal feeds.     

Development and validation of an UHPLC-MS/MS method for the determination of mycotoxins in grass silages

An ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) multi-mycotoxin analytical method was developed to simultaneously identify and quantify 20 mycotoxins in grass silages, inclusive of mycotoxins that are currently regulated in European Union feeds. Extraction of mycotoxins from dried grass silages was performed using of a modified QuEChERS extraction employing an acidified aqueous extraction (0.1 N HCl) with no further clean-up. Following chromatographic separation, analytes were detected using a fast polarity-switching MS/MS method that allowed both positive and negative ions to be analysed from a single injection, thus the reducing time and cost of analysis. The limits of detection and quantification ranged between 3 µg kg^–1 DM (aflatoxin B₁, beauvericin and enniatin A and A₁) and 200 µg kg^–1 DM (deoxynivalenol), and between 10 µg kg^–1 DM (aflatoxin B₁, beauvericin and enniatin A₁) and 500 µg kg^–1 DM (deoxynivalenol), respectively. Inter-assay accuracy and precision ranged between 90% and 107% and between 3.9% and 15.0% CV, respectively. The accuracy of the method was assessed through the application to a range of incurred samples in an inter-laboratory study.     

Occurrence of certain mycotoxins in corn and corn‐based products and thermostability of fumonisin B1 during processing

A total of 57 samples of corn and corn‐based products collected from various districts of Egypt were analyzed for Fusarium mycotoxins (T‐2, diacetoxyscripenol (DAS( deoxynivalenol (DON) and fumonisin B₁ (FB₁) and aflatoxins. FB₁ was detected in about 80%, 53.85%, 33.3%, and 28.57% of yellow corn, corn meal, white corn, and popcorn samples, respectively. The levels of FB₁ ranged from 10 to 780 μg/kg. T‐2 and DAS were detected in 5% and 10% of yellow corn samples, respectively, and DON was detected in white corn and popcorn samples at levels of 28.8 and 10.1 μg/kg, respectively. Starch samples were found to be free from Fusarium mycotoxins. Baking balady bread at 450°C/min reduced FB₁ to 72.4% while baking Franco bread at 250°C/20 min reduced FB₁ to 57.4%. Boiling of macaroni and corn in water completely removed FB₁ from contaminated samples. On the other side, corn flakes samples were found to be contaminated with aflatoxins B₁ and G₁ at levels ranging from 6 to 10 ppm, whereas 2.9% of samples were contaminated with aflatoxin B₁ > 35 ppm and G1 > 16 ppm.     

Natural and in vitro coproduction of cyclopiazonic acid and aflatoxins

Eighty samples of animal feeds of different origins were screened for the natural co-occurrence of cyclopiazonic acid (CPA) and aflatoxins in Portugal. Forty-five strains of Aspergillus flavus were collected from those samples and studied for their ability to produce these mycotoxins, in vitro. CPA was detected by thin-layer chromatography using Erhlich's reagent for confirmation. Aflatoxins were determined by high-pressure liquid chromatography with postcolumn iodination. Only 5 of the 80 samples (6.2%) were naturally contaminated with cyclopiazonic acid (0.16 mg/kg) and 36 (45.0%) with aflatoxin B1 (AFB1) (from 0.001 to 0.016 mg/kg). An in vitro study of the 45 strains of A. flavus was performed in cracked corn at 25 degrees C (water activity, a(w) = 0.96), incubated for 21 days to CPA production. For in vitro production of aflatoxins, the same substrate was incubated at 28 degrees C for 14 days. Nineteen of the strains (42.2%) produced CPA (ranging from 0.5 to 1.45 mg of CPA/kg) and 23 of them (51.1%) produced AFB1 (from 0.001 to 0.844 mg/kg). Only 10 isolates (22.2%) produced both CPA and AFB1 (0.05 to 0.10 mg/kg and 0.001 to 0.230 mg/kg, respectively). Thirteen strains did not produce either CPA nor AFB1.     

Total aflatoxins in complementary foods produced at community levels using locally available ingredients in Ethiopia

This study was conducted to determine the occurrence and levels of total aflatoxins in complementary foods (CFs) and their ingredients. A total of 126 samples collected from 20 Districts from Amhara, Tigray, Oromia, and Southern Nations Nationalities and Peoples (SNNP) regions were analysed for levels of total aflatoxins using enzyme linked immunosorbent assay (ELISA). Aflatoxins were detected in 62 out of 66 pre-milling samples with mean range of 0.3–9.9 µg/kg. Aflatoxins were also detected in 19 out of 20 post-production CFs and in all of the one-month stored CFs at households and grain banks, with a mean range of 0.5–8.0, 3.6–11.3, and 0.2–12.4 µg/kg, respectively. Overall, 3 out of 126 samples exceeded the maximum limit (10 µg/kg). Although most aflatoxin levels were below the maximum limit and thus considered to be safe for consumption, more effort should be implemented to reduce contamination, as these CFs are intended for consumption by young children.     

Screening of aflatoxins in duck feedstuffs in West Java,Indonesia

During the period 1979-1985, incoming feed ingredients (some of imported origin) for duck diets were screened for their aflatoxin level. As a result. records of aflatoxin data from 533 corn, 88 rice, 136 rice bran, 47 wheat pollard, 207 soya bean meal. 55 copra meal and 166 fish meal samples were obtained. Aflatoxin B ₁ was the predominant form, the incidence of samples containing it ranging from 69 to 94%; then accompanied by aflatoxin G₁, 37 to 81%: by B ₂, 33 to 70%; by G₂, 13 to 66%. Levels of total aflatoxin > 20 μg kg ^?1 were most frequently encountered in corn (71%) and copra meal (58%), followed by soya bean meal (20%), wheat pollard (15%), rice bran (12%), fish meal (10%) and rice (4%). Most feed ingredients contained aflatoxins < 100 μg kg^?1 except in corn of which 202 samples (38%) had a higher level. Corn is concluded to be the main source of alfatoxin contamination in corn-based mixed feeds.     

Determination of aflatoxin M1 in breast milk as a biomarker of maternal and infant exposure in Colombia

Chronic exposure to aflatoxins, and especially to aflatoxin B₁ (AFB1), causes hepatocellular carcinoma with prevalence 16–32 times higher in developing compared with developed countries. Aflatoxin M₁ (AFM1) is a monohydroxylated metabolite from AFB1 that is secreted in milk and which can be used as a biomarker of AFB1 exposure. This study aimed to determine AFM1 levels in human breast milk using immunoaffinity column clean-up with HPLC and fluorescence detection. Breast milk samples were obtained from 50 nursing mothers. Volunteers filled in a questionnaire giving their consent to analyse their samples as well as details of their socioeconomic, demographic and clinical data. The possible dietary sources of aflatoxins were assessed using a food frequency questionnaire. A total of 90% of the samples tested positive for AFM1, with a mean of 5.2 ng l^?1 and a range of 0.9–18.5 ng l^?1. The study demonstrated a high frequency of exposure of mothers and neonates to AFB1 and AFM1 in Colombia, and it points out the need to regulate and monitor continuously the presence of aflatoxins in human foods. Further research is needed in order to determine the presence of other mycotoxins in foods and in human samples as well as to devise protection strategies in a country where mycotoxins in human foods are commonly found.     

Aspergillus and aflatoxin in groundnut (Arachis hypogaea L.) and groundnut cake in Eastern Ethiopia

This study was conducted to assess major Aspergillus species and aflatoxins associated with groundnut seeds and cake in Eastern Ethiopia and evaluate growers’ management practices. A total of 160 groundnut seed samples from farmers’ stores and 50 groundnut cake samples from cafe and restaurants were collected. Fungal isolation was done from groundnut seed samples. Aspergillus flavus was the dominant species followed by Aspergillus parasiticus. Aflatoxin analyses of groundnut seed samples were performed using ultra performance liquid chromatography; 22.5% and 41.3% of samples were positive, with total aflatoxin concentrations of 786 and 3135 ng g^?1 from 2013/2014 and 2014/2015 samples, respectively. The level of specific aflatoxin concentration varied between 0.1 and 2526 ng g^?1 for B₂ and B₁, respectively. Among contaminated samples of groundnut cake, 68% exhibited aflatoxin concentration below 20 ng g^?1, while as high as 158 ng g^?1 aflatoxin B₁ was recorded. The study confirms high contamination of groundnut products in East Ethiopia.     

Single-Laboratory Validation for the Determination of Aflatoxin B1, B2, G1, and G2 in Foods Based on Immunoaffinity Column and Liquid Chromatography with Postcolumn Derivatization and Fluorescence Detection

This study presents a method validation procedure for the determination of aflatoxin B₁, B₂, G₁, and G₂ in hazelnut, hazelnut paste, walnut, peanut, pistachio, corn, and wheat. The method consisting of clean-up with immunoaffinity column, high performance liquid chromatography with postcolumn derivatization and fluorescence detection was validated in accordance with Commission Regulation 2004/882/EC. The selectivity, linearity, decision limit, detection capability, detection and quantification limits, precision, recovery, ruggedness, and measurement uncertainty of the method were determined. The limit of detection and limit of quantification values (μg/kg) were: aflatoxin B₁, 0.02, 0.07; aflatoxin B₂, 0.01, 0.02; aflatoxin G₁, 0.02, 0.07; and aflatoxin G₂, 0.01, 0.03. The relative standard deviation values for the repeatability and within-laboratory reproducibility were below 4 and 5 %, respectively. The recovery values of the spiked samples ranged from 80 to 105 %. These results complied with minimum performance criteria established by regulation 2006/401/EC. Therefore, the procedure can be implemented for the routine analysis of aflatoxins in the studied matrices.     

Aflatoxin M<Subscript>1</Subscript> in pasteurized and raw milk from organic and conventional systems

Aflatoxin M₁ is one of the most common toxic natural substances found worldwide. It metabolizes from aflatoxin B₁ that is present in the diet of mammals. In this study, 84 milk samples were investigated in total, and 63 (75 %) were contaminated with aflatoxin M₁ above the limit of detection. No difference was observed between the samples from organic and conventional systems (0.021 vs. 0.018 µg/kg; p > 0.05). There was no difference between pasteurized and raw milk samples (0.018 vs. 0.020 µg/kg; p > 0.05). None of the samples contained aflatoxin M1 above the maximum level permitted by Brazilian Legislation (0.5 µg/kg for fluid milk). The estimated daily intake (EDI) of aflatoxin M₁ through organic and conventional milk consumption was also evaluated. In this study, the EDI-values for aflatoxin M₁ did not pose a toxicological risk for the population. To our best knowledge, this is the first report on aflatoxin M₁ levels in organic milk from Brazil.     

Water availability and calcium propionate affect fungal population and aflatoxins production in broiler finisher feed during storage

The aim of this study was to investigate the effects of calcium propionate, water activity (a_w) and incubation time on the total fungal count and aflatoxins B₁ (AFB₁), B₂ (AFB₂), G₁ (AFG₁) and G₂ (AFG₂) production in the broiler finisher feed. The feed was added with calcium propionate (5 g kg^–1), adjusted to 0.85, 0.90 and 0.95 a_w and stored for 28 days at 25°C, analysing for mould growth and aflatoxins production every 7 days. Analysis of variance indicated that all the factors (preservative, a_w and storage time) alone and in combination significantly (p < 0.001) affected the total fungal count and aflatoxins production in the feed. Minimum total fungal counts (1.99 × 10² CFU g^–1) were observed in calcium propionate feed at 0.85 a_w on day 1 and the highest (4.36 × 10⁹ CFUs g^–1) in control sample at 0.95 a_w on day 28 of storage. During the storage period, AFB₁ content in control samples increased from 11.35 to 73.44, from 11.58 to 81.81 and from 11.54 to 102.68 ng g^–1, whereas in preserved feed the content of B₁ increased from 11.47 to 37.83, from 11.54 to 49.07 and from 11.20 to 53.14 ng g^–1 at 0.85, 0.90 and 0.95 a_w, respectively. Similar patterns were noted for AFB₂, AFG₁ and AFG₂ contents. All the aflatoxins readily increased over storage time; however, the increase was much slower in preserved feed that contained a lower amount of available water. This study reveals that calcium propionate addition to poultry litter along with water activity amelioration is an effective tool for controlling mould incidence and aflatoxin production in poultry feed.     

Occurrence of aflatoxins in selected spices in Poland

The aim of this study was to determine the contamination of aflatoxins B₁, B₂, G₁, G₂ in some selected spices (n = 84) in the Kuyavian-Pomeranian province of Poland. Aflatoxins were found in 63.1 % of the analysed samples. The presence of these compounds was confirmed in all the samples of pepper, nutmeg and turmeric. The maximum content for the sum of aflatoxins B₁, B₂, G₁, G₁ exceeding the acceptable level (10 μg/kg) was 16.91 μg/kg in one sample of nutmeg and 12.1 μg/kg in one sample of pepper, whereas in one sample of nutmeg the maximum content was equal to the regulatory limit. The lowest degree of contamination was found in black pepper.