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Natural occurrence of the naphthoquinone mycotoxins, xanthomegnin, viomellein and vioxanthin in cereals and animal feldstuffs
J. srored Prod. Res. Vol. 22, No. 2, pp. 81-84, 1986 Printedin Great Britain
0022-474X/86 $3.00+ 0.00 PergamonPressLtd
NATURAL OCCURRENCE OF THE NAPHTHOQUINONE MYCOTOXINS, XANTHOMEGNIN, VIOMELLEIN AND VIOXANTHIN IN CEREALS AND ANIMAL FEEDSTUFFS KEITH A.
SCUDAMORE*,
PAULINE
M.
ATKIN*
and
ANTHONY
E. BUCKLE?
*Ministry of Agriculture, Fisheries and Food, Slough Laboratory, London Road, Slough, Berkshire, SL3 7HJ and tMinistry of Agriculture, Fisheries and Food, Agricultural Development and Advisory Department, Shardlow Hall, Shardlow, Derby DE7 2GN, England (Received 19 November 1985)
occurrence of xanthomegnin, viomellein and vioxanthin in commercial samples of wheat and oil seed rape, and of xanthomegnin and vioxanthin in barley, is reported for the first time. Analytical methodology for detection and confirmation of these compounds is described.
Abstract-The
INTRODUCTION
In the United Kingdom samples of stored grain and animal feedstuffs are commonly invaded by a range of moulds of Peniciflium, AspergiZk and other species of fungi (Buckle, 1983). Although the overall contamination of animal feedstuffs in the United Kingdom by mycotoxins is probably very low, ochratoxin A and citrinin, often occurring together, are the toxins found most frequently in samples received at laboratories of the Ministry of Agriculture, Fisheries and Food for mould and mycotoxin examination (Buckle, 1983). Ochratoxin A has been well authenticated as being at least a contributory factor in the occurrence of nephropathy in poultry (Hamilton et al., 1982) and pigs (Krogh, 1978). Reports of surveys carried out in Denmark, Sweden and Hungary (Krogh, 1977; Rutqvist et al., 1977; Sandor et al., 1982) showed that 25 to 39% of swine kidneys with nephrotoxic lesions contained residues of ochratoxin A. However, since ochratoxin A was only found in a fraction of the affected kidneys, it was suggested that other nephrotoxins such as the fungal naphthoquinones, e.g. xanthomegnin and viomellein might be involved. The isolation and identification of xanthomegnin, viomellein and related compounds in cultures of isolates of PeniciNium viridicatitm, Penicillium cyclopium and Aspergilius ochraceus has been described (Stack et al., 1977; Stack and Mislivec, 1978; Robbers et al., 1978; Ciegler et al., 1981; Stack et al., 1983). The detection of viomellein in 1 batch of barley associated with field cases of mycotoxic porcine nephropathy has been reported (Hald et al., 1983). This work describes the finding of xanthomegnin, viomellein and vioxanthin in a number of samples of wheat, barley and in 1 sample of oil seed rape. These samples were also found to be positive for other toxins such as ochratoxin A, citrinin or sterigmatocystin.
MATERIALS
AND
METHODS
For extraction of naphthoquinones, a well mixed sample of milled feedstuff (25 g) was placed in a 250 ml Erlenmeyer flask with 10 ml of 0.1 M orthophosphoric acid and 100 ml of chloroform. The flask was stoppered and shaken for 30 min before the contents were filtered through a fluted filter paper containing anhydrous sodium sulphate. The solvent was collected and evaporated to near dryness before adding to a silica gel Sep Pak cartridge (Carmen et al., 1983) for initial clean up. The naphthoquinones were eluted with chloroform-acetic acid (98:2, v/v) and the solvent evaporated to dryness under nitrogen. The residue was re-dissolved in a small volume of chloroform, either for direct examination using thin-layer chromatography (TLC) or reverse phase high performance liquid chromatography (HPLC) or (and this was usually necessary) for 81
a2
KEITH A. SCUDAMORE et al.
additional clean up using semi-preparative normal phase HPLC. This last procedure was carried out by injecting several successive portions of the chloroform extract into a 10 or 25 cm HPLC column packed with 20 /J or 5 ,u silica respectively and eluting with chloroform-acetic acid (98:2, v/v). Fractions eluting between 3 min and 7 min from the lower efficiency semi-preparative 10 cm column or between 3 min and 5 min from the analytical 25 cm column were collected and bulked. This solution was then split into 2 portions and each evaporated to dryness under nitrogen at 25°C. One portion was re-dissolved in chloroform for TLC and the second portion was made up in methanol-0.2% orthophosphoric acid (70:30, v/v) for additional confirmation of naphthoquinones using reverse phase HPLC on a 25 cm ODS column. Detection of naphthoquinones was by U.V. at 390 nm or with electrochemical detection (Carmen et al., 1984) using both oxidative and reductive modes. Xanthomegnin eluted as 2 unresolved peaks between 7 and 8 min, viomellein as two peaks at 13 and 14 min and vioxanthin at 22 min. Limits of detection following HPLC clean up were approx 10 pg/kg using U.V. detection at 390 nm and significantly lower using electrochemical detection. Two-dimensional TLC was carried out on silica gel 60 plates treated by dipping in 10% tartaric acid in methanol and dried at 75°C for 15 min. The solvents used to develop the plates were (1) benzene-methanol-glacial acetic acid (18: 1: 1, by vol) and (2) toluene-ethyl acetate-90% formic acid (6:3: 1, by vol) [Robbers et al., 19781. The position of each spot after 2-dimensional TLC was designated by the R, value in the first direction, Rn and the Rr value in the second direction, RfL. Plates were allowed to dry in a fume hood and examined under daylight when xanthomegnin appeared as a yellow spot RF, 0.39 Rn 0.33, viomellein as a yellow spot R, 0.50 Rn 0.49 and vioxanthin could not be detected. Under long wave U.V.light, vioxanthin gave an orange spot Rr, 0.74, Rn 0.71 and viomellein and xanthomegnin appeared dark brown. After exposure to ammonia for 5 min, xanthomegnin and viomellein appeared under long wave U.V. light as brown-mauve spots and vioxanthin as a yellow spot. Samples of wheat, barley and oil seed rape grown in the United Kingdom together with 2 compound pig feeds were chosen for examination since previous analysis using established analytical methods (Marti et al., 1978 and Stack et al., 1976) had shown them to be positive for either ochratoxin A, citrinin or sterigmatocystin. Samples were also examined for moulds using a pour-plate method on OAES medium (Kaufman et al., 1963). A 40 g sub-sample was added to 360 ml of peptone saline (0.1% peptone, 0.85% sodium chloride, pH 7.2) in a plastic bag, then homogenised using a Coloworth Stomacher for 5 min. From this suspension a series of lo-fold dilutions was prepared in peptone saline and 1 ml aliquots were dispensed into Petri dishes, to which the OAES medium was added. Two sets of plates were prepared for each sample for incubation at 25°C for 7 days and 37°C for 4 days. The number of mould colonies developing after incubation was counted and the moulds identified.
RESULTS
AND DISCUSSION
Nineteen cereal and compound feed samples were examined for the presence of naphthoquinones and the results are given in Table 1. Eleven of the samples proved positive for all 3 naphthoquinones. These samples also contained citrinin and, except for wheat G and the sample of oilseed rape, ochratoxin A. The mycological results are shown in Table 2. In most of the samples the predominant moulds isolated at 25°C were Penicillium species including P. viridicatum which is consistent with the occurrence of naphthoquinones. The mould counts at 37°C were invariably lower than at 25°C by approx lOO-fold in most samples. The predominant moulds isolated at 37°C were thermophilic Aspergilli, notably A. candidus, A. flavus and A. fumigatus. The detection of aflatoxin Bl in wheat A, in which Aspergillusflavus formed the predominant isolate at 37°C is noteworthy though not unique. The occurrence of A. Jlavus in home-grown cereals has been previously reported when it was found in conjunction with aflatoxin Bl on mouldy barley stored on a farm in Wales (Hacking and Biggs, 1979). Several other cases of aflatoxin contamination of mouldy cereals on farms in England and Wales have subsequently been encountered (Buckle, 1983). Sterigmatocystin was also detected in 9 cereal samples and in 1 oilseed rape sample. Some of these samples were also found to contain Aspergillus versicolor and A. nidulans which have been reported to produce sterigmatocystin.
Natural occurrences in animal feedstuffs Table 1. Occurrence of naphthoquiaoncs
83
and other mycotoxins in cereals and animal feedstuffs Mycotoxin @g/kg)
Commodity
Xanthomegnin
Viomellein
Vioxanthin
Ochratoxin A
Citrinin
200 120 450 200 1100 450 600
1800 300 1800 300 1500 1200 800
400 350 800 200 1200 350 250
<50 1,800 3,500 80
270 470 3000 160 4800 400 + 20 400 20 30 1600 + 800
Wheat Wheat Wheat Wheat
A B c D WheatE Wheat F Wheat G Wheat H Wheat I Wheat J Wheat K Barley A Barley B Barley C Barley D Compound pig feed Compound pig feed Maize Oil seed raDe
-
-
-
600 + + -
90 20 10 -
-
-
100
2,500
150 14 210 11,ooo
75
1,000
Other ST+, B,+ ST 180 ST400 ST13 ’
ST 27 ST8 ST+ ST+ ST+ -
1
80
-
8600
-
450 4100
+
B,+B,+ B,+B,+G,+G,+ ST40
ST = Sterigmatocystin, B,, B, = Aflatoxin B,, 8, G,, G, = Aflatoxin G,, G,. + = present but not quantified. - = not detected.
Evidence for the presence of the naphthoquinones is considered extremely strong on the grounds that (1) Rf values obtained using TLC corresponded exactly with those for standards confirmed by co-chromatography both before and after the additional normal phase HPLC clean up stage. (2) Colours of TLC spots were very characteristic for these compounds both before and after treatment with ammonia. (3) Retention times on reverse phase HPLC corresponded with those for toxin standards and exhibited characteristic double peaks (Wall and Lillehoj, 1983). (4) The U.V. Table 2. Mycological results of samples found positive for the presence of xanthomegnin, Log number of propagules/g Sample identity
25°C
37°C
Wheat A
8.0
6.1
Wheat B
7.2
5.8
Wheat C
7.9
7.4
Wheat D Wheat E
7.1
4.1
Wheat F
8.0
6.0
25°C Penicillium spp. Scopulariopsis sp. Penicillium spp. Aspergih candidus Penicillium spp. Aspergih candidus
No data
Wheat G
3.7
1.7
Wheat I
4.8
2.8
Barley A
8.0
6.8
Barley B
5.1
3.8
Penicillium spp. Gliocladiwn sp. Scopulariopsis sp. Aspergilh versicolor Penicillium spp.
No data on other species Penicillium spp. Fwarium sp. Aspergillw glaucus Scopulariopsis sp. Aspergilh uersicolor Penicillium sp. A. candidus Penicillium spp. Aspergih candidus A. J%VUS Penicillium spp.
Aspergillur ca&idus A. nidulans A. terretu Barley C Oil seed rape
‘Listed in order of predominance.
37°C Aspergilh Jlaous A. jiimigatus Aspergilh candidus A. jhI.9 Aspergillus candidus A. flaws No data Aspergih A. Jauus
Moisture content (%1 30.7 16.4 20.8
fumigates
20.5
Absidia sp. Aspergillus candidus
12.2
Aspergillw Jaws Absidia sp.
12.5
Mucor sp.
Aspergillw candidus A. J%lVuS Aspergilh flaw A. nidalans A. fwnigahts Mucor sp. Paecilomyces sp.
12.6
No data
No data 7.6
viomellein and vioxanthin
Fungal species isolated*
7.5
Aspergilh candidus Penicilhm spp. A. versicolor
Aspergilh
candidus
A.pnuUS Absidia ~1).
22.8
84
KE3tH
A.
SCUDAMORE
et al.
wavelength, 390 nm, used for detection in HPLC was relatively selective in that many compounds that absorb U.V.light at 250 nm do not absorb at this wavelength and the sample extracts produced spectra which were relatively clean in this region. (5) The extracted compounds were electrochemically active at the same reduction or oxidation potentials as the standards. (6) In all positive samples Penicillium sp’p. were present in relatively large numbers. Members of this genus of fungi are known to be capable of producing naphthoquinones in culture (Stack et al., 1977; Stack and Mislivec, 1978; Ciegler et al., 1981). Detection and confirmation of these naphthoquinones in a range of cereals indicates that they can occur at high levels in relatively mouldy cereals and animal feedstuffs in the United Kingdom along with several other mycotoxins. However the samples examined were selected on the basis of being positive for other toxins and as such are not representative of the overall situation. Samples negative for both ochratoxin A and citrinin have yet to be examined in detail. The development of suitable analytical methods coupled with improvements in clean up procedure and sensitive methods of detection such as electrochemical detection may ultimately show that naphthoquinones occur much more frequently in this type of commodity than has previously been suspected. Acknowledgements-We thank Dr M. Stack of the U.S. F.D.A., Washington for the supply of naphthoquinone standards, EDT Research and Severn Analytical for the loan of electrochemical detectors, and Dr J. H. Clarke for helpful advice and discussion. REFERENCES Buckle A. E. (1983) The occurrence of mycotoxins in cereals and animal feedstuffs. Vet. I&s. Commun. 7, 171-186. Carman A. S., Kuan S. S., Francis 0. J., Ware G. M., Gaul J. A. and Thorpe C. W. (1983) High pressure liquid chromatographic determination of xanthomegnin in grains and animal feeds. J. Ass. ofl analyt. Chem. 66, 587-591. Carman A. S., Kuan S. S., Francis 0. J., Ware G. M. and Luedtke A. E. (1984) Determination of xanthomegnin in grains and animal feeds by liquid chromatography with electrochemical detection. J. Ass. oj? analyt. Chem. 67, 10951098. Ciegler A., Lee L. S. and Dunn J. J. (1981) Production of naphthoquinone mycotoxins and taxonomy of Penicillium viridicatum. Appl. Envir. Microbial. 42, 446449.
Hacking A. and Biggs N. R. (1979) Aflatoxin B, in barley in the U.K. Nature 282, 128. Hald B., Christensen D. H. and Krogh P. (1983) Natural occurrence of the mycotoxin viomellein in barley and the associated quinone-producing Penicillia. Appl. Envir. Microbial. 46, 13 1l-l 3 17. Hamilton P. B., Huff W. E., Harris J. R. and Wyatt R. D. (1982) Natural occurrences of ochratoxicosis in poultry. Pouit. Sci. 61, 1832-1841. Kaufman D. D., Williams L. E. and Sumner C. B. (1963) Effect of plating medium and incubation temperature on growth of fungi in soil dilution plates. Can. J. Microbial. 9, 741-751. Krogh P. (1977) Ochratoxin A residues in tissues of slaughter pigs with nephropathy. Nood. Ye&fed. 29, 402405. Krogh P. (1978) Causal associations of mycotoxic nephropathy. Acta path. microbial. stand. SectA 269, l-28. Marti L. R., Wilson D. M. and Evans B. D. (1978) Determinations of citrinin in corn and barley. J. Ass. ofl analyt. Chem. 61, 1353-1358. Robbers J. E.. Hong S., Tuite J. and Carlton W. M. (1978) Production of xanthomegnin and viomellein by species of Aspergillus correlated with mycotoxicosis produced in mice. Appl. Mcrobiol. Envir. 36, 819-823. Rutqvist L., Bjijrklund N.-E., Hult K. and Gatenbeck S. (1977) Spontaneous occurrence of ochratoxin residues in kidneys of fattening pigs. Zentbi. VetMed. 24, 402-408. Sandor G., Glavits R., Vajda L., Vanyi A. and Krogh P. (1982) Epidemiologic study of ochratoxin A associated porcine nephropathy in Hungary. In Proceedings of the Vth International IUPAC Symposium on Mycotoxins and Phycotoxins, Vienna, pp. 349-352. Stack M. E., Nesheim S., Brown N. L. and Pohland A. E. (1976) Determination of sterigmatocystin in corn and oats by gel permeation and high pressure liquid chromatography. J. Assoc. 08 analyt. Chem. 59, 966970. Stack M. E., Eppley R. M., Dreifuss P. A. and Pohland A. E. (1977) Isolation and identification of xanthomegnin, viomellein, rubrosulphin and viopurpurin as metabolites of Penicillium viridicatum. Appl. Envir. Microbial. 33,351-355. Stack M. E. and Mislivec P. B. (1978) Production of xanthomegnin and viomellein by isolates of Aspergillus ochraceus, Penicillium cyclopium and Penicillium viridicatum. Appl. Envir. Microbial. 36, 552-554. Stack M. E., Mislivec P. B., Denizel T., Gibson R. and Pohland A. E. (1983) Ochratoxins A and B, xanthomegnin, viomellein and vioxanthin production by isolates of Aspergillus ochraceus from green coffee beans. J. Fd Pro?. 46, 965-968.
Wall J. H. and Lillehoj E. B. (1983) High performance liquid chromatographic J. Chromat. 268, 463468.
separation of xanthomegnin and viomellein.
0022-474X/86 $3.00+ 0.00 PergamonPressLtd
NATURAL OCCURRENCE OF THE NAPHTHOQUINONE MYCOTOXINS, XANTHOMEGNIN, VIOMELLEIN AND VIOXANTHIN IN CEREALS AND ANIMAL FEEDSTUFFS KEITH A.
SCUDAMORE*,
PAULINE
M.
ATKIN*
and
ANTHONY
E. BUCKLE?
*Ministry of Agriculture, Fisheries and Food, Slough Laboratory, London Road, Slough, Berkshire, SL3 7HJ and tMinistry of Agriculture, Fisheries and Food, Agricultural Development and Advisory Department, Shardlow Hall, Shardlow, Derby DE7 2GN, England (Received 19 November 1985)
occurrence of xanthomegnin, viomellein and vioxanthin in commercial samples of wheat and oil seed rape, and of xanthomegnin and vioxanthin in barley, is reported for the first time. Analytical methodology for detection and confirmation of these compounds is described.
Abstract-The
INTRODUCTION
In the United Kingdom samples of stored grain and animal feedstuffs are commonly invaded by a range of moulds of Peniciflium, AspergiZk and other species of fungi (Buckle, 1983). Although the overall contamination of animal feedstuffs in the United Kingdom by mycotoxins is probably very low, ochratoxin A and citrinin, often occurring together, are the toxins found most frequently in samples received at laboratories of the Ministry of Agriculture, Fisheries and Food for mould and mycotoxin examination (Buckle, 1983). Ochratoxin A has been well authenticated as being at least a contributory factor in the occurrence of nephropathy in poultry (Hamilton et al., 1982) and pigs (Krogh, 1978). Reports of surveys carried out in Denmark, Sweden and Hungary (Krogh, 1977; Rutqvist et al., 1977; Sandor et al., 1982) showed that 25 to 39% of swine kidneys with nephrotoxic lesions contained residues of ochratoxin A. However, since ochratoxin A was only found in a fraction of the affected kidneys, it was suggested that other nephrotoxins such as the fungal naphthoquinones, e.g. xanthomegnin and viomellein might be involved. The isolation and identification of xanthomegnin, viomellein and related compounds in cultures of isolates of PeniciNium viridicatitm, Penicillium cyclopium and Aspergilius ochraceus has been described (Stack et al., 1977; Stack and Mislivec, 1978; Robbers et al., 1978; Ciegler et al., 1981; Stack et al., 1983). The detection of viomellein in 1 batch of barley associated with field cases of mycotoxic porcine nephropathy has been reported (Hald et al., 1983). This work describes the finding of xanthomegnin, viomellein and vioxanthin in a number of samples of wheat, barley and in 1 sample of oil seed rape. These samples were also found to be positive for other toxins such as ochratoxin A, citrinin or sterigmatocystin.
MATERIALS
AND
METHODS
For extraction of naphthoquinones, a well mixed sample of milled feedstuff (25 g) was placed in a 250 ml Erlenmeyer flask with 10 ml of 0.1 M orthophosphoric acid and 100 ml of chloroform. The flask was stoppered and shaken for 30 min before the contents were filtered through a fluted filter paper containing anhydrous sodium sulphate. The solvent was collected and evaporated to near dryness before adding to a silica gel Sep Pak cartridge (Carmen et al., 1983) for initial clean up. The naphthoquinones were eluted with chloroform-acetic acid (98:2, v/v) and the solvent evaporated to dryness under nitrogen. The residue was re-dissolved in a small volume of chloroform, either for direct examination using thin-layer chromatography (TLC) or reverse phase high performance liquid chromatography (HPLC) or (and this was usually necessary) for 81
a2
KEITH A. SCUDAMORE et al.
additional clean up using semi-preparative normal phase HPLC. This last procedure was carried out by injecting several successive portions of the chloroform extract into a 10 or 25 cm HPLC column packed with 20 /J or 5 ,u silica respectively and eluting with chloroform-acetic acid (98:2, v/v). Fractions eluting between 3 min and 7 min from the lower efficiency semi-preparative 10 cm column or between 3 min and 5 min from the analytical 25 cm column were collected and bulked. This solution was then split into 2 portions and each evaporated to dryness under nitrogen at 25°C. One portion was re-dissolved in chloroform for TLC and the second portion was made up in methanol-0.2% orthophosphoric acid (70:30, v/v) for additional confirmation of naphthoquinones using reverse phase HPLC on a 25 cm ODS column. Detection of naphthoquinones was by U.V. at 390 nm or with electrochemical detection (Carmen et al., 1984) using both oxidative and reductive modes. Xanthomegnin eluted as 2 unresolved peaks between 7 and 8 min, viomellein as two peaks at 13 and 14 min and vioxanthin at 22 min. Limits of detection following HPLC clean up were approx 10 pg/kg using U.V. detection at 390 nm and significantly lower using electrochemical detection. Two-dimensional TLC was carried out on silica gel 60 plates treated by dipping in 10% tartaric acid in methanol and dried at 75°C for 15 min. The solvents used to develop the plates were (1) benzene-methanol-glacial acetic acid (18: 1: 1, by vol) and (2) toluene-ethyl acetate-90% formic acid (6:3: 1, by vol) [Robbers et al., 19781. The position of each spot after 2-dimensional TLC was designated by the R, value in the first direction, Rn and the Rr value in the second direction, RfL. Plates were allowed to dry in a fume hood and examined under daylight when xanthomegnin appeared as a yellow spot RF, 0.39 Rn 0.33, viomellein as a yellow spot R, 0.50 Rn 0.49 and vioxanthin could not be detected. Under long wave U.V.light, vioxanthin gave an orange spot Rr, 0.74, Rn 0.71 and viomellein and xanthomegnin appeared dark brown. After exposure to ammonia for 5 min, xanthomegnin and viomellein appeared under long wave U.V. light as brown-mauve spots and vioxanthin as a yellow spot. Samples of wheat, barley and oil seed rape grown in the United Kingdom together with 2 compound pig feeds were chosen for examination since previous analysis using established analytical methods (Marti et al., 1978 and Stack et al., 1976) had shown them to be positive for either ochratoxin A, citrinin or sterigmatocystin. Samples were also examined for moulds using a pour-plate method on OAES medium (Kaufman et al., 1963). A 40 g sub-sample was added to 360 ml of peptone saline (0.1% peptone, 0.85% sodium chloride, pH 7.2) in a plastic bag, then homogenised using a Coloworth Stomacher for 5 min. From this suspension a series of lo-fold dilutions was prepared in peptone saline and 1 ml aliquots were dispensed into Petri dishes, to which the OAES medium was added. Two sets of plates were prepared for each sample for incubation at 25°C for 7 days and 37°C for 4 days. The number of mould colonies developing after incubation was counted and the moulds identified.
RESULTS
AND DISCUSSION
Nineteen cereal and compound feed samples were examined for the presence of naphthoquinones and the results are given in Table 1. Eleven of the samples proved positive for all 3 naphthoquinones. These samples also contained citrinin and, except for wheat G and the sample of oilseed rape, ochratoxin A. The mycological results are shown in Table 2. In most of the samples the predominant moulds isolated at 25°C were Penicillium species including P. viridicatum which is consistent with the occurrence of naphthoquinones. The mould counts at 37°C were invariably lower than at 25°C by approx lOO-fold in most samples. The predominant moulds isolated at 37°C were thermophilic Aspergilli, notably A. candidus, A. flavus and A. fumigatus. The detection of aflatoxin Bl in wheat A, in which Aspergillusflavus formed the predominant isolate at 37°C is noteworthy though not unique. The occurrence of A. Jlavus in home-grown cereals has been previously reported when it was found in conjunction with aflatoxin Bl on mouldy barley stored on a farm in Wales (Hacking and Biggs, 1979). Several other cases of aflatoxin contamination of mouldy cereals on farms in England and Wales have subsequently been encountered (Buckle, 1983). Sterigmatocystin was also detected in 9 cereal samples and in 1 oilseed rape sample. Some of these samples were also found to contain Aspergillus versicolor and A. nidulans which have been reported to produce sterigmatocystin.
Natural occurrences in animal feedstuffs Table 1. Occurrence of naphthoquiaoncs
83
and other mycotoxins in cereals and animal feedstuffs Mycotoxin @g/kg)
Commodity
Xanthomegnin
Viomellein
Vioxanthin
Ochratoxin A
Citrinin
200 120 450 200 1100 450 600
1800 300 1800 300 1500 1200 800
400 350 800 200 1200 350 250
<50 1,800 3,500 80
270 470 3000 160 4800 400 + 20 400 20 30 1600 + 800
Wheat Wheat Wheat Wheat
A B c D WheatE Wheat F Wheat G Wheat H Wheat I Wheat J Wheat K Barley A Barley B Barley C Barley D Compound pig feed Compound pig feed Maize Oil seed raDe
-
-
-
600 + + -
90 20 10 -
-
-
100
2,500
150 14 210 11,ooo
75
1,000
Other ST+, B,+ ST 180 ST400 ST13 ’
ST 27 ST8 ST+ ST+ ST+ -
1
80
-
8600
-
450 4100
+
B,+B,+ B,+B,+G,+G,+ ST40
ST = Sterigmatocystin, B,, B, = Aflatoxin B,, 8, G,, G, = Aflatoxin G,, G,. + = present but not quantified. - = not detected.
Evidence for the presence of the naphthoquinones is considered extremely strong on the grounds that (1) Rf values obtained using TLC corresponded exactly with those for standards confirmed by co-chromatography both before and after the additional normal phase HPLC clean up stage. (2) Colours of TLC spots were very characteristic for these compounds both before and after treatment with ammonia. (3) Retention times on reverse phase HPLC corresponded with those for toxin standards and exhibited characteristic double peaks (Wall and Lillehoj, 1983). (4) The U.V. Table 2. Mycological results of samples found positive for the presence of xanthomegnin, Log number of propagules/g Sample identity
25°C
37°C
Wheat A
8.0
6.1
Wheat B
7.2
5.8
Wheat C
7.9
7.4
Wheat D Wheat E
7.1
4.1
Wheat F
8.0
6.0
25°C Penicillium spp. Scopulariopsis sp. Penicillium spp. Aspergih candidus Penicillium spp. Aspergih candidus
No data
Wheat G
3.7
1.7
Wheat I
4.8
2.8
Barley A
8.0
6.8
Barley B
5.1
3.8
Penicillium spp. Gliocladiwn sp. Scopulariopsis sp. Aspergilh versicolor Penicillium spp.
No data on other species Penicillium spp. Fwarium sp. Aspergillw glaucus Scopulariopsis sp. Aspergilh uersicolor Penicillium sp. A. candidus Penicillium spp. Aspergih candidus A. J%VUS Penicillium spp.
Aspergillur ca&idus A. nidulans A. terretu Barley C Oil seed rape
‘Listed in order of predominance.
37°C Aspergilh Jlaous A. jiimigatus Aspergilh candidus A. jhI.9 Aspergillus candidus A. flaws No data Aspergih A. Jauus
Moisture content (%1 30.7 16.4 20.8
fumigates
20.5
Absidia sp. Aspergillus candidus
12.2
Aspergillw Jaws Absidia sp.
12.5
Mucor sp.
Aspergillw candidus A. J%lVuS Aspergilh flaw A. nidalans A. fwnigahts Mucor sp. Paecilomyces sp.
12.6
No data
No data 7.6
viomellein and vioxanthin
Fungal species isolated*
7.5
Aspergilh candidus Penicilhm spp. A. versicolor
Aspergilh
candidus
A.pnuUS Absidia ~1).
22.8
84
KE3tH
A.
SCUDAMORE
et al.
wavelength, 390 nm, used for detection in HPLC was relatively selective in that many compounds that absorb U.V.light at 250 nm do not absorb at this wavelength and the sample extracts produced spectra which were relatively clean in this region. (5) The extracted compounds were electrochemically active at the same reduction or oxidation potentials as the standards. (6) In all positive samples Penicillium sp’p. were present in relatively large numbers. Members of this genus of fungi are known to be capable of producing naphthoquinones in culture (Stack et al., 1977; Stack and Mislivec, 1978; Ciegler et al., 1981). Detection and confirmation of these naphthoquinones in a range of cereals indicates that they can occur at high levels in relatively mouldy cereals and animal feedstuffs in the United Kingdom along with several other mycotoxins. However the samples examined were selected on the basis of being positive for other toxins and as such are not representative of the overall situation. Samples negative for both ochratoxin A and citrinin have yet to be examined in detail. The development of suitable analytical methods coupled with improvements in clean up procedure and sensitive methods of detection such as electrochemical detection may ultimately show that naphthoquinones occur much more frequently in this type of commodity than has previously been suspected. Acknowledgements-We thank Dr M. Stack of the U.S. F.D.A., Washington for the supply of naphthoquinone standards, EDT Research and Severn Analytical for the loan of electrochemical detectors, and Dr J. H. Clarke for helpful advice and discussion. REFERENCES Buckle A. E. (1983) The occurrence of mycotoxins in cereals and animal feedstuffs. Vet. I&s. Commun. 7, 171-186. Carman A. S., Kuan S. S., Francis 0. J., Ware G. M., Gaul J. A. and Thorpe C. W. (1983) High pressure liquid chromatographic determination of xanthomegnin in grains and animal feeds. J. Ass. ofl analyt. Chem. 66, 587-591. Carman A. S., Kuan S. S., Francis 0. J., Ware G. M. and Luedtke A. E. (1984) Determination of xanthomegnin in grains and animal feeds by liquid chromatography with electrochemical detection. J. Ass. oj? analyt. Chem. 67, 10951098. Ciegler A., Lee L. S. and Dunn J. J. (1981) Production of naphthoquinone mycotoxins and taxonomy of Penicillium viridicatum. Appl. Envir. Microbial. 42, 446449.
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separation of xanthomegnin and viomellein.