Survey and risk assessment of the mycotoxins deoxynivalenol,zearalenone, fumonisins,ochratoxin A,and aflatoxins in commercial dry dog food

The aim of the present study was to investigate the occurrence of mycotoxins in commercial dog food, as a basis to estimate the risk of adverse effects. Seventy-six dry dog food samples from 27 producers were purchased from retail shops, supermarkets, and specialized pet food shops in Vienna, Austria. The frequency and levels of deoxynivalenol (DON), zearalenone (ZEA), fumonisins (FUM), ochratoxin A (OTA). and aflatoxins (AF) in dry dog food were determined. Mycotoxin analysis were performed by commercial enzyme-linked immunosorbent assay (ELISA) test kits. Confirmatory analyses were done for DON, ZEA, and FUM by high performance liquid chromatography (HPLC) after extract clean-up with immunoaffinity columns. The correlations between ELISA and HPLC results for DON and ZEA were acceptable and indicated that ELISA could be a simple, low cost, and sensitive screening tool for mycotoxins detection, contributing to quality and safety of pet food. DON was the mycotoxin most frequently found (83% positives; median 308 μg/kg, maximum 1,390 μg/kg). ZEA (47% positives, median 51 μg/kg and maximum 298 μg/kg) and FUM (42% positives, median 122 μg/kg and maximum 568 μg/kg) were also frequently detected in dog food. OTA was less frequently found (5%, median 3.6 μg/kg, maximum 4.7 μg/kg. AF were not detected (<0.5 μg/kg) in any sample. The results show that dry dog food marketed in Vienna are frequently contaminated with mycotoxins (DON > ZEA > FUM > OTA) in low concentrations, but do not contain AF. The high frequency of Fusarium toxins DON, ZEA, and FUM indicates the need for intensive control measures to prevent mycotoxins in dog foods. The mycotoxin levels found in dry dog food are considered as safe in aspects of acute mycotoxicoses. However, repeated and long-time exposure of dogs to low levels of mycotoxins may pose a health risk.     

Untersuchungen zu DON-Gehalten in Getreideerzeugnissen aus dem deutschen Einzelhandel im Jahr 2006

Samples of soft wheat flour (n=78), durum wheat semolina (n=6), and pasta (made from durum wheat, n=49) were purchased in January-April 2006 from retail outlets in Hesse, Germany. Samples were analysed for deoxynivalenol (DON) by enzyme immunoassay. The detection limit of the method was 10 μg/kg, with recoveries of 81–85% (RSD_r: 12–17%). DON was detected in 84% of all samples, but the contamination level was low. Median/maximum values for DON in wheat flour, wheat semolina, and pasta were 28μg/kg/217 μg/kg, 38μg/kg/203 μg/kg, and 24μg/kg/119 μg/kg, respectively. Compared with results obtained from previous years, significantly lower DON levels were observed in these commodities.     

Deoxynivalenol in Mehlproben des Jahres 1999 aus dem Einzelhandel

To study the levels of deoxynivalenol (DON) in retail cereal products, wheat and rye samples were purchased in 1999 from supermarkets and “organic food” shops in Munich, Germany. DON was analysed by an enzyme immunoassay (EIA), 78 of these samples were additionally analysed by HPLC. The following contamination rates (%) and mean DON levels were found: wheat flour type 405 (n=42): 71%, 200 µg/kg; flour type 550 (n=9): 33%, 410 µg/kg; flour type 1050 (n=11): 91%, 370 µg/kg; bread-baking wheat premixes (n=14): 79%, 210 µg/kg; whole grain flour (n=20): 65%, 300 µg/kg; whole grain wheat (n=8): 75%, 280 µg/kg, wheat bran (n=20): 85%, 830 µg/kg; rye flour and grits (n=7): 29%, 120 µg/kg. HPLC confirmed the results obtained by EIA. Further analysis of 16 wheat flour (405) samples in May 2000 showed a similar frequency (69%) and mean DON level (270 µg/kg) as for samples from 1999. It is concluded that with DON levels in wheat for human consumption ranging from 200–400 µg/kg, the intake of DON has to be taken seriously in the light of the temporary tolerable daily intake of 1 µg DON per kg body weight as proposed within the European Union.     

Deoxynivalenol (DON) in hartweizen

Deoxynivalenol (DON) in Durum Wheat High contamination levels of deoxynivalenol (DON) were found during an investigation of noodles in 2001 and initiated an additional survey of Durum wheat used as raw material for these products. Analyses of 53 samples of Durum wheat by HPLC revealed 89% positives and a median concentration of 790 ug/kg, which clearly exceeded the EU action level of 500 μg/kg. Based on these findings, producers increased quality controls on Durum wheat. In 2002 a total of 60 samples were analysed and 85% positives found. The median, however, lay at 215 μg/kg, below a probable maximum level of 500 μg/kg and demonstrated the positive efforts of food producers to decrease the contamination levels of DON in pasta products.     

Further survey of the occurrence of fusarium toxins in wheat grown in southwest Germany

A total of 53, 54, 57, 52 and 60 wheat samples for feed use were collected randomly after the 1989, 1990, 1991, 1992 and 1993 crops, respectively, from farms in an area of southwest Germany. Deoxynivalenol (DON), 3‐ and 15‐acetyldeoxynivalenol (3‐, 15‐ADON), nivalenol (NIV), HT‐2 toxin (HT‐2), T‐2 toxin (T‐2), diacetoxyscirpenol (DAS), and fusarenon‐X (FUS‐X) were determined by gas chromatography, combined with mass selective detection (GC‐MS), zearalenone (ZEA), α‐ and β‐zearalenol (α‐ß‐ZOL) were determined by HPLC. DON was the major toxin, with incidences at 77 to 93% and mean contents at 167 to 735 μg/kg. In contrast, incidences of ZEA, 3‐ADON, NIV, HT‐2, and T‐2 were at 13 to 37%, 10 to 44%, 15 to 67%, 0 to 11%, and 0 to 12%, respectively, with mean contents in positive samples between 2 and 73 μg/kg, except for 948 μg/kg 3‐ADON in samples from 1993. 15‐ADON and FUS‐X were assayed in samples from 1991, 1992 and 1993. 15‐ADON was found in 0 to 11% of samples at mean levels ≤ 17 μ/kg, DAS, α‐ and β‐ZOL, and FUS‐X were not detected in any sample. Over the years, incidences and levels of toxins remained constant, decreased or increased, with most differences between years being slight and insignificant. The risk for livestock due to DON, HT‐2 and ZEA was estimated based on maximum tolerated levels recommended for these toxins in some countries.     

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.     

Metabolisierung von Deoxynivalenol beim Schwein: Bestimmung von DON und DOM-1 im Urin vom Schwein

This paper describes a feeding study with 7 pigs, which were fed with deoxynivalenol contaminated oats at a level of 0.23 mg/kg body mass/day over 16 experiment days. The contamination level of consumed feed was 14.4 mg DON/kg in the ration. The parallel control group of 7 pigs were fed with DON free oats. Urine samples were taken each two days. The content of DON and DOM-1 (de-epoxy deoxynivalenol) in urine was determined. The mean concentration of DON in urine of animals in trial group was 580 μg/l, whereas DOM-1 32 μg/l.     

Fusarium toxins in cereals — Results from eight German Federals States in 2000

A total of 353 cereal samples from 8 German Federal States were analysed for Fusarium toxins in the 2000' survey. The level of mycotoxin contamination of the samples was higher than in the previous year due to higher moisture during flowering in some regions. Concerning deoxynivalenol (DON), the prevailing mycotoxin in all the samples, we have to consider, that 24% of the samples as well as the average concentration exceed the advisory level of 500 μg/kg. The application of Fusarium active fungicides reduced the DON level from 963 μg/kg (samples without treatment) to 630 μg/kg (all samples from treated and untreated fields). It has to be emphasised that the number of nivalenol (NIV) positive samples increased in comparison to the previous year from 9% to 17%. In addition, an increase in the level of NIV was detected, the maximum concentration in winter wheat was 3480 μg/kg. Cereals were rather moderately contaminated with zearalenone (ZEA), more than 80% of the samples contained less than 50 μg/kg.     

Occurrence of deoxynivalenol and zearalenone in brewing barley grains from Brazil

Barley (Hordeum vulgare L.) is an important cereal crop for food and represents one of the main ingredients in beer production. Considering the importance of barley and its derived products, the knowledge about the mycotoxin contamination in the barley production is essential in order to assess its safety. In this study, the levels of deoxynivalenol (DON) and zearalenone (ZEN) in brewing barley were determined using a LC-MS/MS method. A survey was conducted in 2015 to estimate the mycotoxin levels in these products (n?=?76) from four crop regions in Brazil. The results showed high levels of DON and ZEN in the analyzed samples, with contamination levels of 94 and 73.6%, respectively. The mean levels of DON and ZEN ranged from 1700 to 7500 μg/kg and from 300 to 630 μg/kg, respectively. Barley samples from regions 1 and 2 presented higher levels of ZEN and DON, respectively, and those from region 4 presented lower levels of both. Co-occurrence of DON and ZEN was seen in the majority of the barley grain samples, and the mycotoxin content was above the maximum levels established by the Brazilian and European regulations.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination.

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

Immunochemical assessment of mycotoxins in 1989 grain foods: evidence for deoxynivalenol (vomitoxin) contamination

To assess the potential for mycotoxin contamination of the human food supply following the 1988 U.S. drought, 92 grain food samples were purchased from retail outlets in the summer of 1989 and surveyed for aflatoxin B1, zearalenone, and deoxynivalenol (DON [vomitoxin]) by monoclonal antibody-based competitive enzyme-linked immunosorbent assay (ELISA). Only one sample (buckwheat flour) was found to contain aflatoxin B1 (12 ng/g), whereas zearalenone was found in 26% of the samples at a mean concentration of 19 ng/g. In contrast, the DON ELISA was positive in 50% of the samples at a detection level of 1.0 micrograms/g. Between 63 and 88% of corn cereals, wheat flour/muffin mixes, rice cereals, and corn meal/muffin mixes yielded positive results for DON, whereas 25 to 50% of oat cereals, wheat- and oat-based cookies/crackers, corn chips, popcorn, and mixed-grain cereals were positive for DON. The mean DON content of the positive samples was 4.0 micrograms/g, and the minimum and maximum levels were 1.2 and 19 micrograms/g, respectively. When positive ELISA samples were also analyzed by high-performance liquid chromatography, a strong correlation between the two methods was found. The presence of DON in the two highest samples, corn meal and mixed-grain cereal, which contained 19 and 16 micrograms/g, respectively, was quantitatively confirmed by gas chromatography-mass spectrometry. The results indicated that DON was present in 1989 retail food products at concentrations that exceeded those found in previous market surveys and that have been experimentally associated with impaired animal health.     

Preliminary exposure assessment of deoxynivalenol and patulin in South Africa

Deoxynivalenol (DON) and patulin (PAT) are mycotoxins widely regulated internationally. DON is frequently found in cereals, whereas PAT is commonly found in apple juices. A survey of South African commercial products was conducted on DON levels in maize meal and wheat flours, and on PAT levels in apple juices. DON levels in 23 wheat flour samples (mean of 16 positives, 29 μg/kg) were equal to or below 100 μg/kg and in wheat consumers contributed 6–13% of the provisional maximum tolerable daily intake (PMTDI; 1 μg/kg body weight per day) for DON set by the Joint FAO/WHO Expert Committee on Food Additives (JECFA). Sixteen of 18 maize meal samples were contaminated, with a mean for positive samples of 294 μg/kg, and the probable daily intakes ranged from 3.67 μg/kg body weight per day in rural infants to 1.39 μg/kg body weight per day in urban adults. PAT levels in 20 of 30 apple-juice samples were below the detection level of 10 μg/l. Mean of positive samples was 210 μg/l, with three samples exceeding the South African legal limit of 50 μg/l and the highest level (1,650 μg/l) showing the possibility of a brief but high exposure of 37 μg/kg body weight per day (or 9,250% of the JECFA PMTDI of 0.4 μg/kg body weight per day) in young children.     

Need to determine the relative developmental risks of Fusarium mycotoxin dexoynivalenol (DON) and Benomyl (BEN) in wheat

The wheat supply is known to have levels of deoxynivalenol (DON) from fungal infections. The fungal infections can be suppressed with benomyl (BEN). Under certain exposure laboratory conditions DON and BEN are developmental toxins. Since use of BEN may result in concurrent dietary exposure to both compounds, evaluation of the relative developmental risks is needed. The dietary NOAELs for pup decreased body weight for DON and BEN are 0.375 mg/kg/day in mice and 5 mg/kg/day in rat, respectively. Human exposure to DON can be estimated from surveys of wheat grain and finished products which gave a mean DON concentration of 0.86 ppm. The tolerance level for BEN in grain is 0.2 ppm. Presence of DON and BEN in wheat at these concentrations would not impact wheat consumption. A modified margin of exposure (modMOE) was developed using “a”; kg (food) /kg (body weight) as the constant for wheat consumption. The modMOE for DON is 28/a and that for BEN is 1667/a. Comparative relative risks can be expressed as the ratio of BEN risks to DON risks (60). Treatment with BEN at the most efficacious time decreased DON levels to 34% of controls, and decreased the comparative risk ratio from 60 to 18. The potential for fungicides to decrease mycotoxin health risks is important in the safety assessment for foodstuffs. This approach improves on current practices for food safety assessment by including the expected reductions in risks from naturally occurring mycotoxins in the regulatory analysis.     

Occurrence of type A,B and D trichothecenes in barley and barley products from the Bavarian market

Fifty-nine samples of barley and barley products were analysed for 18 trichothecene mycotoxins by a sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method (detection limits 0.062-0.70 μg/kg) after sample extract clean-up on MycoSep®-226 columns. The samples were collected in 2009 from barley processing facilities (mills and malt houses) and at wholesale and retail stage from the Bavarian market. The predominant toxins were T-2 toxin (T-2), HT-2 toxin (HT-2) and deoxynivalenol (DON). For all samples, the mean levels of T-2 and HT-2 were 3.0 μg/kg and 6.8 μg/kg with rates of contamination of 63% and 71%, respectively. The maximum values were 40 μg/kg for T-2 and 47 μg/kg for HT-2. The rate of contamination with DON was high (95%) with a low mean level of 23 μg/kg. The DON levels ranged between 3.4 to 420 μg/kg. For T-2 tetraol, a mean level of 9.2 μg/kg and a maximum level of 51 μg/kg with a rate of contamination of 71% were determined. NIV was detected in 69% of the samples with a mean level of 11 μg/kg and a maximum level of 72 μg/kg. Other type A and B trichothecenes were detected only in traces. Type D trichothecenes, fusarenon-X, verrucarol and 4,15-diacetylverrucarol were not detected in any sample. Winter barley and malting barley were the most contaminated groups of all samples in this study. In malting barley, the highest levels of contamination with type A trichothecenes were found. In contrast, winter barley showed the highest contamination with type B trichothecenes. The lowest mycotoxin concentrations were found in de-hulled and naked barley and in pearl barley.     

Bestimmung von Deoxynivalenol und Deepoxy-Deoxynivalenol in Milch

A HPLC method with UV/diode array detection for the determination of deoxynivalenol (DON) and deepoxy-deoxynivalenol (DOM-1) in milk was developed. Milk was incubated with β-glucuronidase and then defatted. After purification by immunoaffinity chromatography, DON and DOM-1 were separated on a C18 reversed phase column with acetonitril/water (10/90) as the mobile phase and detected at 218 nm. Limits of quantification were 1 μg/l for both toxins, with mean recoveries (1–10 μg/l) of 97% (DON) and 84% (DOM-1), respectively. Milk samples (pasteurized, UHT; n=32) from German retail shops were analysed by this method. Neither DON/DOM-1 nor their glucuronides were found in any sample. These results are consistent with published studies indicating that in lactating cows, DON and DOM-1 are mostly eliminated through urine, and that the carry-over into milk is negligible.     

Mycotoxins in horse feed

A total of 62 samples of commercial horse feed preparations (complementary feeds) containing cereal mixtures (“muesli” or mash, n = 39; pelleted feeds, n = 12), and plain horse feed grains (maize, n = 5; oats, n = 4; barley, n = 2) were purchased from 21 different producers/distributors from the German market. All samples were analysed by competitive enzyme immunoassays (EIA) for six different mycotoxins (mycotoxin groups). Analytes (detection limit, mean recovery) were: deoxynivalenol (DON, 10 μg/kg, 84%), zearalenone (ZEA, 5 μg/kg, 93%), fumonisin B₁ (FB₁, 2 μg/kg, 113%), T-2 toxin (T-2, 0.1 μg/kg, 71%), sum of T-2 + HT-2 toxin (T-2/HT2, 0.2 μg/kg, 97%), ochratoxin A (OTA, 0.2 μg/kg, 67%), and total ergot alkaloids (Generic Ergot Alkaloids “GEA”, 30 μg/kg, 132%). All samples contained DON (16–4,900 μg/kg, median 220 μg/kg), T-2/HT-2 (0.8–230 μg/kg, median 24 μg/kg), and T-2 (0.3–91 μg/kg, median 7 μg/kg). ZEA was detected in 98% of the samples (7–310 μg/kg, median 61 μg/kg). Most samples (94%) were positive for FB₁ (2–2,200 μg/kg, median 27 μg/kg). Ergot alkaloids were detected in 61% of samples (28–1,200 μg/kg, median 97 μg/kg), OTA was found in 42% of samples (0.2–4 μg/kg, median 0.35 μg/kg). The results demonstrate that a co-contamination with several mycotoxins is very common in commercial horse feed from the German market. The toxin concentrations were in most cases well below the levels which are usually considered as critical or even toxic. The highest mycotoxin concentrations were mostly found in single-grain cereal feed: the maximum values for DON and FB₁ were found in maize, the highest T-2/HT-2 toxin concentrations were found in oats, and the highest concentration of ergot alkaloids was found in barley. In composed feeds, no correlation between cereal composition and mycotoxin levels could be found.     

Zearalenone, deoxynivalenol and fumonisins in mixed-feed for laying hens

Fusarium toxins are secondary metabolites produced byfungi of these genera in many commodities under certain conditions. A study was carried out to investigate the co-occurrence of zearalenone (ZEN), deoxynivalenol (DON) and fumonisins (FB₁ and FB₂) in 52 samples of mixed-feed for poultry contaminated withFusarium verticillioides. The zearalenone and deoxynivalenol were checked using immunoaffinity column and the extraction of fumonisin was performed by strong anion exchange (SAX) solid phase column. Detection and quantification were determined by high performance liquid chromatography (HPLC). The limit of detection was 5 μg/kg for ZEN, 100 μg/kg for DON and 50 and 100 μg/kg for FB₁ and FB₂ respectively.Fusarium toxins were detected in 20 samples. Sixteen samples were positive for ZEN (30.7%) presenting levels that ranged from 7.4 μg/kg to 61.4 μg/kg (mean=27.0 μg/kg). 13.5% of the samples presented contaminations of DON, with levels ranging from 100.0 μg/kg to 253 μg/kg (mean=l18.07 μg/kg). FB₁ was detected in 19.2% of samples, with levels ranging from 50.0 μg/kg to 110.0 μg/kg (mean=73.6 μg/kg). FB2 was not detected in any sample. In positive samples simultaneously contamination with two or three mycotoxins were detected in 9 of them (17.3%).     

<Emphasis Type="Italic">Fusarium</Emphasis> toxins in cereals of integrated and organic cultivation from the Federal State of Brandenburg (Germany) harvested in the years 2000–2007

Over a period of 8 years (2000–2007), wheat (n = 407) and rye (n = 510) samples of integrated and organic cultivation in the Federal State of Brandenburg were analyzed by HPLC for the Fusarium toxins deoxynivalenol (DON) and zearalenone (ZEA). In the years 2002 and 2007, the overall contamination level was higher than in the other years. The percentage of DON-positives (>50 μg/kg) varied from 5 to 86%, the median and maximum levels varied from 50 to 380 μg/kg and from 50  to 10,400 μg/kg, respectively. The percentage of ZEA-positives (>3 μg/kg) varied from 2 to 41%, the median and maximum levels varied from 8 to 84 μg/kg and from 10 to 451 μg/kg. In the 8 years of testing, frequency and levels of DON and ZEA were significantly lower in cereals of organic cultivation compared with cereals of integrated cultivation.     

Deoxynivalenol in Lebensmitteln

Within a joint research project entitled “Analysis and occurrence of importantFusarium toxins (deoxynivalenol and zearalenone) and dietary intake of these toxins by the German consumer”, supported by the German Federal Ministry of Consumer Protection, Food and Agriculture (BMVEL), representative analytical data are generated on the contamination level of foods withFusarium mycotoxins. This paper gives a comprehensive summary concerning the contamination of foods from the German market with deoxynivalenol (DON) in the period from August 2001 to April 2004. More than 4700 food samples (mostly cereals and cereal-containing foods) were purchased from food shops in Germany and analysed for DON by enzyme immunoassay, HPLC, and LC-MS/MS, respectively. All analytical methods were validated through intra- and interlaboratory studies and gave mean recoveries of >80% for each matrix. Although DON was detected with high frequency in all cerealcontaining samples, the mean and median levels were in most products well below the recently established maximum permitted limits in Germany.     

Occurrence of deoxynivalenol (DON) and ochratoxin A (OTA) in dog foods

The commercially available dog food samples (29 dry foods and 11 wet foods) were analysed for deoxynivalenol (DON) and ochratoxin A (OTA) using ELISA. All (100%) dry foods were contaminated with DON with various amount of the toxin (22-1837 μg/kg). In wet food 3 samples were found to be positive for DON in the range of 95-170 μg/kg. There were a few samples contaminated with OTA: 3 samples in dry foods (7-40 μg/kg) and 2 samples in wet foods (45 and 115 μg/kg).