944 Analyst, October, 1980, Vol. 105, #$. 944-949 A New Type of Biological Reference Material for M u I t i - e I e m e n t An a I ysi s-t h e F u n g us PenicNium ochro-ch/oron ATCC 36741 Miwako Suzuki, Yukiko Dokiya, * Sunao Yamazaki and Shozo Toda Department of Agricultural Chemistry, Faculty of Agriculture, Universaty of Tokyo, Bunkyo-ku, Tokyo, Japan 113 Several series of standard disk samples were prepared using dried mycelia of Penicillaum ochvo-chloron, a fungus extremely tolerant to heavy metals. Be- cause of the ready availability of the homogeneous dry material with arbitrary metal concentrations, the fungus is very suitable for calibration in metal analysis by X-ray fluorescence spectrometry, especially for biological samples. Based on the calibration graph for the reference material, the metal contents of edible wild plants were determined. The reliability of the data was checked by using biological reference materials and by atomic-absorption spectrometry.Keywords: Biological reference material; Penicillium ochro-chloron fungus; heavy metals; biological materials; X-ray fluorescence spectrometvy Many standard reference materials or certified reference materials have been reported for use in calibration and checking the reliability of biological samp1es.l-5 Kale powderl, NBS orchard leaves and bovine liver5 are the best known and widely utilised examples. As the requirements for reference materials are still increasing, the authors have participated in studies to prepare new biological reference materials in Japan, such as tea leaves,6 shark paste7 and shark powder.8 In this paper, the preparation of a fungal-pellet reference material is described and pro- posed for use as a calibration material in the X-ray fluorescence determination of metals in biological samples.As a non-destructive multi-element method of determination, X-ray fluorescence spectrometry is widely applied to biological samples, but interference by matrix elements may limit the accuracy obtainable. Hence there is a need for reliable calibration utilising the reference material. For the analysis of alloy and ore samples by X-ray fluorescence spectrometry, NBS SRM 625-629 zinc-base alloys, SRM 461-464 low-alloy steels, SRM 1636-1638 lead in fuel, SRM 330-333 copper in ores and so on are well established.However, no trials have been carried out with biological samples, partly because of the difficulty in obtaining appropriate concen- tration ranges using biological reference materials. Penicilliuwa ochro-chloron ATCC 36741 is a fungus tolerant to heavy metals that has been studied for several years in the authors’ laboratory. This fungus can grow well even in media containing 105 pg ml-l (saturated solution) of copper, zinc and mangane~e.~ It also has a high tolerance against lead, cadmium and iron (2 x lo5 pg ml-l).lo The fungus has the important specific characteristic that the cellular contents of rnetals can be controlled arbi- trarily over a wide range for the metals added to the media. The ranges of cellular metal contents are much wider than for other biological samples.Investigations were carried out with this fungus in order to prepare a suitable calibration material for X-ray fluorescence spectrometry. Incubation conditions of the fungus were studied, and determination of metals was performed by X-ray fluorescence spectrometry and atomic-absorption spectrometry. Experimental Preparation of Penicillium ochro-chloron Reference Material After pre-incubation for 7 d on Czapek’s agar slant, the spores, of P. ochro-chloron were inoculated into 150 ml of sterilised culture media containirig various concentrations of metals. The compositions of the basal medium and the metals and concentrations * Present address: Geochemical Laboratory, Meteorological Research Institute, Nagamine, Yatabe, Tsukuba-gun, Ibaragi, Japan 305.SUZUKI, DOKIYA, YAMAZAKI AND TODA 945 After incubation for 96 h on a rotary shaker a t 30 "C, The added are shown in Tables I and 11.the fungal mycelia were harvested, lyophilised and dried a t 105 "C to constant mass. dried mycelia were pulverised and homogenised in an agate mortar. TABLE I COMPOSITION OF BASAL MEDIUM Component Content/g 1-1 Component Content (as metal)/mg 1-l Glucose . . .. .. 40 FeSO, .. .. .. 5 (XH,) $0,. . . . .. 3.3 ZnSO, ,. .. .. 5 KH,PO, . . .. . . 2.5 MnSO, .. .. .. 1 MgS0,.7H,O . . .. 1 Na,MoO, . . .. . . 0.5 Ca(N0,),.4Ha0 . . . . 0.5 c u s o , .. .. .. 0.1 coso, .. .. . * 0.1 hTaVO, . . .. . . 0.01 Determination of Metals X-ray j%uorescence spectrometry To make standard disk samples for X-ray fluorescence calibration, 100 mg of the dried fungal powder were weighed, mixed with 400 mg of "binder" [made with poly(viny1 alcohol); Somar Co.] and pressed into disks (1500 kg cm-2 for 10 min) using an oil hydraulic press (Lumis Products Co.).Copper, zinc and manganese were determined by X-ray fluorescence spectrometry using an Ortec TEFA 6110 energy-dispersive X-ray fluorescence spectrometer. The intensity of the K, line of each metal was determined using a molybdenum target and molybdenum filter (40 kV, 50 A). A silicon (lithium) counter was used as the detector, requiring 200-1000 s per sample. c u Zn TABLE I1 COMBINATIOKS AND CONCENTRATIONS OF METALS ADDED TO THE BASAL MEDIA First metal Concentration/pg ml-1 Second metal Concentration/pg ml- 1 . . . . 0.1 (control) Ni .. . . . . 100 10 200 100 c o .. . . . . 100 500 500 1000 1000 10000 2 000 50 000 1000 100000 5 (control) 100 1000 10000 50000 100 000 Cd . . . . Zn . . .. . . Mn . . . . . . Ni . . . . . . c o . . . . . . . . . . . . Cd 5000 10000 1000 10000 1000 10 000 100 200 100 1000 2000 1000 5 000 10000 Atomic-absorption spectrometry A 500-mg amount of dried powder sample was transferred into Kjeldahl flasks and digested with a mixture of concentrated nitric and sulphuric acids. The digest was diluted to 50 ml with de-ionised water and used for determinations. Copper, zinc and manganese were determined by atomic-absorption spectrometry using a Seiko SAS 721 atomic-absorption spectrophotometer. The analytical lines for copper, zinc and manganese were a t 324.8, 213.8 and 279.5 nm, respectively, with a spectral band width of 1.0 nm.An air - acetylene flame was used under regular burning conditions.946 SUZUKI et al. : NEW BIOLOGICAL REFEREKCE MATERIAL FOR Analyst, Vol. 105 Results and Discussion Metal Characteristics in Penicillium ochro-chloron The copper, zinc, manganese, cobalt, nickel, iron, potassium, calcium and magnesium contents in P. ochzro-chloron are compared with those in NBS Biological Standard Reference Materials in Table 111. The metal contents of the fungus were determined by atomic- absorption spectrometry. The most typical characteristic of this fu.ngus is the high and wide concentrations of metals that can be controlled by appropriate addition of metals to the basal media. The ranges of the metal contents of the fungus are generally consistent for plants species.TABLE I11 ELEMENTAL COMPOSITIONS OF NBS BIOLOGICAL STANDARD REFERENCE MATERIALS AND PENICILLIUM OCHRO-CHLORON Metalcontent/wg g-1 (dry mass) -- > Material cu Zn Mn co Ni Fe IK Ca Mg Spinach . . Orchard leaves Tomato leaves Pine needles Oyster tissue Bovine liver Ranges , . .. 1212 5012 16516 (1.5) , . 12+1 2513 9114 (0.2) , . 1151 6216 2381.7 . . 3.010.3 61-74 675115 /ili] . . 63.0 13.5 852 114 17.5 11.2 , , 193 110 130i10 10.3 +LO (0.18) .. 3.0-193 25-852 10-675 0.1-1.5 (6) 1.3 10.2 1.03 i0.19 I:::\ - 0.2-6 550 f 20 300 120 690 +25 200 1 10 195 134 270 +20 195-690 36 600 1300 14 700 i.300 $4 600 1300 3 700 = 200 9 690 +50 9 700 160 3 700-44 600 13 500 i300 20 900 *300 30 000 h300 4 100 = 200 1 500 = 200 123-30 000 (123) - 6200 1200 ( 7 0 ~ 0 ) - 1280 i90 (605) 605-7 000 P .ochro-c/2~07on. . 5.0-4 700 5.0-5 500 0-2 000 0-940 10-490 0-1300 140-40 000 0-900 10-6 000 The metal contents of ten samples of P. ochro-chloron (each 500 mg of powder of dried The homogeneities with respect to No apparent change mycelia) selected at random are given in Table IV. copper, zinc and manganese were found to be within 6.5% (relative standard deviation). in the colour or the state was observed. Dried mycelia were preserved in a silica gel desiccator for 14 months. The change in mass was also negligible. TABLE IV ELEMENTAL COMPOSITION OF PENICILLIUM OCHRO-CHLORON Bottle a . . . . . . . . b . . . . .. . . d . . . . . . . . f . . . . .. .. g ” h . . . . . . . . i . . . . . . . . c . . . . . . . . e .. . . I . . . . . . . . . j . . . . .. . . Average . . . . . . deviation, % . . . . Relative standard Metal contentlpg g-1 (dry mass) r---L-‘p- 7 c u Zn Mn 1800 115 1500 1850 112 1500 1800 120 1500 1800 100 1500 1800 105 1700 1800 115 1500 1800 110 1500 1800 112 1500 1800 112 1700 1900 108 1400 1820 110 1530 1.8 4. I 6.2 Preparation of Calibration Samples for X-ray Fluorescence Spectrometry In order to establish the appropriate mixing ratio of dried mycelia and binder [poly(vinyl alcohol) powder], X-ray fluorescence intensities were measured for amounts of mycelia from 100 to 400 mg veysus 400 mg of binder. As linearity between the mass of mycelia and the X-ray fluorescence intensities was obtained, 100 mg of dried mycelia in 400 mg binder were adopted in further work.No visual changes and no changes in composition and metal contents occurred after 14 months in a desiccator. The mass of the disks increased slightly, but this might be improved by changing the binder material. In addition, samples in a disk shape are suitable for storage and repeated utilisation. The results are summarised in Fig. 1. The preservation of such “disk” samples was also tested.October, 1980 MULTI-ELEMENT ANALYSIS-FUNGUS Penzcillizlm ochro-chloron m m o o / - 0 3 2 0 947 0 I I 100 200 300 400 100 200 300 400 Dried mycelia in 400 mg of binderimg Fig. 1. Relationship between sample mass in disk binder and X-ray fluorescence intensities. Concentration of copper in culture media: (a) 4500 pg ml-l; (b) 225 p g ml-l; and (c) 5 p g ml-I. Concentration of zinc in culture media: ( d ) 900 p g ml-l; and (e) 115 p g ml-1.The relationships between the X-ray fluorescence intensities and the metal concentrations The correla- Hence the calibration for copper, determined by atomic-absorption spectrometry were linear, as shown in Fig. 2. tion coefficients were 0.973, 0.987 and 0.989, respectively. zinc and manganese in biological samples is reliable. 50 7 40 v) m 4- C 2 30 >: 4- m .- 5 20 +- C U .- 10 80 ... m +- Y) 60 C 3 8 .- 5 40 C C 4- ._ & 20 X 6 ... - 5 YI 4- C ; 4 >: ‘I 3 5 2 a 4- C c U x 1 0 2 000 4000 0 1000 2000 3000 4000 5000 0 500 1000 1500 2000 cu content in mycelia/pg g-’ Zn content in mycelialpg g-’ (dry mass) (dry mass) (dry mass) M n content in mycelialpg g-’ Fig. 2. Relationship between X-ray fluorescence intensities and metal contents determined by atomic- absorption spectrometry: (a) copper; (b) zinc; and (c) manganese.Application of Penicillium ochro-chloron Calibration Graph to Other Biological Samples Some NBS Standard Reference Materials and other biological materials whose metal contents are known were used to check the method. Table V shows a comparison between values obtained for most plant and animal samples, except for copper and zinc in orchard leaves, copper in pine needles and manganese in oyster tissue. It can be concluded that calibration for other biological materials is also reliable, with few exceptions.948 SUZUKI et al. : NEW BIOLOGICAL REFERENCE MATERIAL FOR Analyst, VoZ. 105 TABLE V RESULTS OF APPLICATION OF THE PROPOSED METHOD Metalcontentlwg g-1 (dry mass) cu Zn Mn Values reference values Values reference values Values reference values determined r-'-, determined determined ,--*-, Type Material by XRF' Source? Value by XRF' Source? Value by XRF' Source? Value ,-- -----, I 7 Certified or Certified or Certified or Plants .. Orchard leaves Pine needles Tea leaves B Tea leaves D Alfalfa . . Timothy . . .. .. .. .. .. 30 53 15 18 6 20 A 12A1 3.0 ~ 0 . 3 12-15 27-31 9 13 45 67 56 56 23 39 25 i3 61-74 56-66 65-95 27 50 98 727 532 760 69 21 165 * 6 675 215 530-620 890-1010 48 25 Animals . . Bovine liver . , 167 A 1 9 3 ~ 1 0 112 A 130 4.10 19 A 10.311.0 Oyster tissue . . 61 A 63.013.5 750 A 852+14 3 A 17.5+1.2 Shark powder 2 . , 10 F 1.1 14 F 14 10-:?2 19 F < 1 - F - Shark powder 1 , .11 F 1.4 12 F ' X-ray fluorescence spectrometry. t A Certified values for NBS Biological Standard Reference Materialss; B, H. L. Rook personal #communication; C, certified values for JapaAese tea leavesB; D, samples provided by Dr. Jones, Jr., of the Ohio Agricultural Research and Development Centre, U'ooster, Ohio; E, values determined by atomic-absorption spectrometry; F, T. Uchida, personal communication. Next, the metal contents of wild plants were determined by using the proposed method. Some edible wild plants that have traditionally been used in daily dishes in Japan have recently aroused interest from the point of view of sources of trac,e metals for human nutrition. Fifty kinds of edible wild plants were collected in Fukushima Prefecture, dried at 105 "C to constant mass and homogenised in an agate mortar.A 100-mg amount of each was mixed with 400 mg of binder and pressed in the same way as for P. ochro-ckloron. Their metal contents were then measured by X-ray fluorescence and atomic-absorption spectrometry. Some results obtained by X-ray fluorescence analysis utilising the calibration graplis for P. ochyo-chloron reference materials are shown in Table VI, and the values are compared with those obtained by atomic-absorption spectrometry. TABLE VI DETERMINATION OF METALS IN EDIBLE WILD PLANTS Botanical name Sambucus sieboldiana (bud) . . . . Houttuynia cordata . . . . . . Petasites japonicus (bud) . . . . Laportea macrostachya (leaf) . . . . Vicia unijuga . , . . . . . . Plantago asiatica . . . . .. Matteuccia struthiopteris .. . . Cacalia farfaraefolia . . . . . . Acarthopamax spinosum (bud) . . Elatostema umbellatum . . . . * X-ray fluorescence spectrometry. t Atomic-absorption spectrometry. Japanese name Niwatoko-no-me Dokudami Fuki-no-me Miyama-irakusa-no-ha Nanten-hagi Ohbako Tara-no-me Kogomi Uwabami-so Tamabuki Metal. contentlpg g-l (dry mass) -------A----- Cll Zn Mn r - y - v - 7 XRF* AASt XRF* AASt XRF* AASt 43 38 59 67 184 190 14 11 81 67 151 140 22 11 22 25 112 110 20 14 88 85 103 70 25 13 49 53 83 85 19 11 31 33 112 110 20 14 74 75 268 240 35 35 63 90 41 40 11 10 24 29 89 82 15 19 65 65 78 100 7 From these results, it is concluded that the dried mycelia of P. ocho-chloyon serve as a good calibration material for the determination of metals in biological samples by X-ray fluorescence spectrometry. The next step in this study might be to find better binder ingredients and to determine the optimum diameter and thickness of the disks.Further studies on other metals such as cobalt and nickel are under investigation. The authors thank Dr. Yoko Saito of Koriyama Women's College for her help in collecting the Japanese wild plants, and Mr. Toshihiro Aota and Mr. Nobuo Matsumori of Daini Seiko-sha for their assistance in measuring the elements in fungal mycelia and wild plants by X-ray fluorescence spectrometry.October, 1980 MULTI-ELEMENT ANALYSIS-FUNGUS Penicillium ochro-chloron 949 References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Bowen, H. J . M., A~zalyst, 1967, 92, 124. Jones, J . B., Jr., “Abstracts of the Pittsburg Conference on Analytical Chemistry and Applied Yamagata, N., Bunseki Kagaku, 1971, 20, 515. Fukai, R., “Intercalibration of Analytical Methods on Marine Environmental Samples,” Progress “Catalog of NBS Standard Reference Materials,” NBS Special Publication 260, 1978-79 Edition, Fuwa, K., Notsu, K., Tsunoda, K., Kato, H., Yamamoto, Y . , Okamoto, K., Dokiya, Y., and Toda, S., Dokiya, Y . , Taguchi, M., Toda, S., and Fuaw, K., 4 n a l . C h e w , 1978, 50, 533. Dokiya, Y , , Kurosawa, S., Toda, S., and Fuwa, K., Bull. Chem. SOC. Jpn., 1978, 51, 3649. Okamoto, K., and Fuwa, K., Agvic. Bid. Chern., 1974, 38, 1405. Okamoto, K., Suzuki, &I., Fukami, >I., Toda, S., and Fuwa, K., Agric. Bid. Chem., 1977, 41, 17. Spectroscopy, Pittsburg, Pa., 1967,” paper no. 200. Report KO. 13, International Laboratory of Marine Radioactivity, Monaco, 1976. US Department of Commerce, Washington, D.C., 1978. Bull. Chern. Soc. J p ~ z . , 1978, 51, 1078. Received March 20th. 1980 Accepted d p r i l 24th, 1980