1392 TEX ET AL.: JOURNAL OF AOAC INTERNATIONAL Vor. 95, No. 5,2012 AGRICULTURAL MATERIALS Determination of Ash 942.05 Revisited Naxey Tumex and Lawrexcr Novorsy Animal Feed: AOAC Official Method South Dakota State University, Olsoa Biochemistry Labs, Brookings, SD $7007 ANby CRAWFORD 15737 #5 La Subida Dr, Hacienda Heights, CA 91745 AOAC Official Method 942.05, Ash in Animal Feed, hhas been applied in feed laboratories since its publication in the Official Methods of Analysis in 1942. itis a routine test with renewed interest due to the incorporation of “ash values” into modern equations for the estimation of energy content of dairy feed, beef feed, and pet food. As with other empirical methods, results obtained are a function of the test conditions. For this method, the critical conditions are the ignition time, ignition temperature, and any other furnace or weighing conditions. ‘Complete ignition can be observed by the absence of black color (due to residual carbonaceous material) in the ash residue. To investigate performance of AOAC 942.05, 15 samples were chosen to be representative of a wide range of feed materials. ‘These materials were tested at the conditions of AOAC 942.05 (ignition at 600°C for 2 h) and similar or more rigorous conditions. The additional conditions i ted included: 600°C for 4 h; 600°C for 2h, ool, and ignite 2 additional h; 600°C for 2 h, cool, wet, dry, and ignite 2 additional h; 550°C for 6 h 550°C for 3 h, cool, and ignite 3 additional h; and 550°C for 3h, cool, wet, dry, ignite 3 additional h. Results for all other conditions investigated were found to be significantly different from the current ‘AOAC Method 942.05. All ignition conditions were significantly different from each other except two: 550°C for 3h, cool, ignite 3 additional h; and 550°C for 3h, cool, wet, dry, and ignite 3 additional h. Recommendations for modification to AOAC Official ‘Method 942.08 are suggested based on statistical analysis of the data and a review of the literature. ‘Received Mavch 25,2012, Acoepied by SG Apa Conesponding autor's email: nancy thiex @sdstate edu DOL 10.8740 jaan. 12-129 Iisa “defining” or “empirical” method, meaning the analytical results obtained are defined by the conditions specified forthe method. Any change tothe conditions of the method (ignition time, ignition temperature, and any other furnace of weighing conditions) will bis results, Historically, ash results have posed problems for feed laboratories and their clients, in part due to the empirial nature of the method. Variability in ash results are well documented in Association of American Feed Control Officials (AAFCO) Check Sample Program method performance reports (3) Renewed interest in tightening the variability of results for AOAC Official Method 942.08 (4) ae due tothe incorporation of “ash values” into modem equations for the estimation of energy content of dairy feed (5), beef fed (6), and pet food (7), In these computations to estimate the energy component of feed, a higher ash value computes to a lower energy value Since eneray is an expensive component of feeds, variability in ash esults ean affeet economic value for a given feed produet or ingredient: an incomplete ash will negatively impact economics ‘A perfect residue after ignition of a feed material would be ‘white, with no hint of residual carbon. visual examination of the residue by the analyst ean quickly establish if carbonaceous 1ateral has been oxidized and the ignition is complete. Some methods eal for examination of the residue, while others do not The current AOAC Official Method 942.08 does not Today, there are at least four “official” methods for determination of ash in animal feed: AOAC Official Method 942.05 (1); ISO 5948-2002 (8). Com Refiners Association (CRA)-G4 (9); and American Associaton of Cereal Chemists (AACC) 08-03 (10). Table 1 compares the requirements for each of the methods Historical Information for AOAC Official Method 942.08 Associate Referee IL. St John reported in 1942 (1) thatthree different materials were submitted to each of four laboratories resulting in 12 total materials, Collaborators determined loss (on ignition (ash) atthe temperature ofthe then official method (650°C) and also at $00, $50, 600, and 700°C. They were asked to report results to two decimal places and make observations fn the appearance of the ash. The materials tested were mash barley, alfalfa, oats, mash + 28% shell flour, soybean, wheat tankage, peas, bran and shorts, com, cottonseed. poultry feed, herring, and linseed. St. John reported the averages, average differences between duplicates, maximum range, and percent weight loss due to increase of temperature. Based on these calculations, St Tohn concluded that (J) collaborating analysts were able to obtain approximate agreement between their own duplicates; (2) agreement among analysts’ duplicates was good‘TEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, NO. 5.2012 1393 Table 1. Comparison of current official methods for crude ash ined Year Seo Teatweight| g Teh Prechar Temp, © Surface area, en? ‘OAC 84205 1982 Fe 2 2 Ne 600 = 130 5048-2002 1076; revision 2002 Fees 5 Siottouhte sen? Yes 550 » craw 2008 Feesetute 5 4 Yes 525-800 — AACCORGO1 1861;rav 1976 rev 1978 Feedstue-ropid meted 2 2 Neo 00 - 5 Wroriginal an color back or dark ra, inseatng presence of carbon, a repeated ath cyl is epeifes Jn many cases, although not “so good” with the mash, mash plus added shell flour, and alfalfa and soybean meals: (3) in a ‘majority of instances, the incremental loss on ignition is greatest betoreen $00 and $50°C compared to the 50°C steps above 550°C, (4) with @ majority of feed materials, the incremental Jos asthe temperature is increased by 50°C steps is “within the same general range” between $50 and 700°C (exceptions are ‘mash, mash plus added shel flour, and alfalfa); (5) consistency of results is essentially the same at temperatures of $50 and 600°C, as at 650°C and above (a “till Lower” at 550°C than at higher temperatures: (6) recovery of mineral elements is generally better at the lower temperatures than at the higher Temperatures, with better recovery of potassium at $50 and 600°C than at higher temperatures; and (7) the appearance of the ash was considered of litle or no value in evaluating the loss of carbon since no consistent variation in color was observed at any temperature, with some carbon remaining a temperatures as highs 700°C. Based on the conclusions, St, Jobn recommended (J) the temperature of ashing in the oficial method be reduced to 600 or 350°C (from 650°C): (2) careful adherence to the details ofthe procedure is necessary: and (3) results should be reported to only one decimal place. ‘In 1943, St John reported on additional work (12) to compare results at 550, 600, and 650°C. Four different feed materials \vere submitted to each of four different laboratories, resulting in 16 total feed materials, Feed materials selected were laying mash, dog fish, alfalfa, wheat, starting mash, batley, soybean, sta, developing mash, silage, mill rn, dairy feed, turkey concentrate, com, linseed, and peas. Averages, average differences between duplicates, maximum range, and percent weight loss due to increase of temperature were reported St John concluded that (1) the results confirmed the 1942 work: (2) precision was generally the same at all three temperatures ‘with average difference between duplicates well below 0.1%, with a light favor of 600°C; (3) maximum ranges were similar among temperatures, with a slight favor for $50 or 600°C: (4) smal progressive decrease in ash residue was observed as the temperature increases: (3) collaborators noted the importance of not overloading the mule furnace; (6) the precision ofthe ‘method did not justify utilizing asecond decimal place; (7) most collaborators preferred using 600°C: and (8) there was a critical ‘need fora standardized method ‘Asa result of the work of St. John, AOAC Official Method 942.05 (4), Ash in Animal Feed, was adopted as First Action in 1942, The method simply read “Weigh 2 g test portion into porcelain erucible and place in temperatre-contolled furnace preheated to 600°C, Hold at this temperature 2 b, Transfer crucible directly wo desiccator, cool, and weigh immediately. Report percent ash o first decimal plac Experimental Materials Fiftcen samples for study were selected to represent a wide range of feed materials and to cover a concentration range from 2 to 90% crude ash. These reflect the wide range of materials in animal feeds and ate representative of the great diversity cof combinations/concentrations of mineral content in feed ‘and feed ingredients. The materials studied include fish meal (FM), dehydrated alfalfa (DA), mineral vitamin premix (MVP), beef cattle conditioner (BC). pork finisher medicated (PFM), chick starter grower medicated (SGM), swine feed medicated (SFND, com silage (CS), grass hay (GHD, alfalfa hay (AH), horse feed with molasses (HF), barley (B), adult cat food (ACE) lite cat food (LCF), and com gluten meal (CGM). MVP. BC, PEM, CSGM, SFM, and HF were AAFCO check samples used as supplied. with no additional preparation, for the proficiency-testing program. All remaining materials were prepared in accordance with the National Forage Testing Association (13), AAFCO (4), and AOAC (15) guidelines. CS, GH, and AH were ground to pass a 1 mm screen in a Tecator CCyelotec eyelone mill Foss North America, Eden Prairie, MN) Other feed materials were ground to pass a 0.75 mm sereen in a Retsch ZM 100 centrifugal mill (Retsch GmbH, Haan, Germany, wwwRetsch com). Apparatus (a) Mufie furnace —With programmable controller (Thermolyne F6058, Bamstead Intemational, Dubuque, 14). (b) Analsrical holonce—Readable to 0.1 mg, electonic (Sartorius, Elk Grove, IL) (©) Crucibles Porcelain, low wide form, 30 ml, numbered with furace-proof ink (CoorTek, Golden, CO), (@) Desiccator.—With stoppered lid (Corning, Acton, MA). (©) Drying oven—Set to 100°C (Weiss-Gallenkamp, Loughborough, UK), Methods Fificen feed materials were teste atthe conditions of AOAC {942.08 (jzition at 600°C for 2 h) and similar or more rigorous conditions, Two temperatures were chosen, 600 and 550°C, because these temperatures had been studied fairly thoroughly {nthe past and they had been adopted by AOAC (600°C), AACC (600°C), and 1SO (550°C), and the CRA method allowed for either temperature (525-600°C), Additional heating. times ‘were added to those specified in the AOAC and ISO methods to investigate if a longer time would provide more thorough i seeoe|ioo'dno-ouopene)di Sp1394 THEX ET AL.: JOURNAL OF AOAC INTERNATIONAL Vor. 95, No. 5, 2012 Table 2. Average residue on ignition (crude ash), % Tera ‘Oan_SaaH _GWOANweITH —GOOTNDH 60H _SSOEH _—GBVDHNaRTH _—SGODHIH fw mee 2048 2008 7987 mo aan 2000 Ey Da ar 262 eae as eer aes er eer MvP e070 e942 er7e e578 erste e108 ous: ac wo1s 1018 1010 10.10 reas 1042 1042 1040 Pe aa 407 406 a4 sor 497 502 495 csom 606 670 668 eat mm 788 758 770 srw 149402 208 raat 1508154 1552 1508 cs a7 374 an an ae 381 375 376 oH a3 a8 a2 40 es 8a 830 a2 aH 764 1s 751 155 758758 743 rst 8 2a 238 20 aa 24s 2 220 229 ACF ace 260 ast eet a2 ant er 260 Ler 202 200 aot 00 om ee oat on cow 24s 128 18 123 2210 120 120 Ls Mean 71389 raze r318 081397 rar) ra90" Least square means with same superscript eter are not signcanty dteret (2 = 0.05), ignition. The thoroughness of the ignition was determined by the color of the ash residue and a corresponding reduetion in residue weight, The base conditions investigated were ignition st 600°C for 2 h and ignition at SS0°C for 3 h. The additional conditions investigated inchuded 600°C for 4 b; 600°C for 2b. cool, ignite 2 additional b; 600°C for 2h, cool, wet, dey, and Jgnite 2 additional h; 580°C for 6 h; $50°C for 3 h, cool, and ‘ignite 3 additional h: and S50°C for 3 h, cool, wet, dry” and jgnite 3 additional h. The eight sets of ignition concitions are referred to later inthis manuscript as eight “ignition conditions Test Procedure (a) Remove crucibles, which have been dried for atleast 2 at 100°C, from oven to desiccator. Cool and weigh, recording the tare weight to the nearest 0.1 mg (T). Note: New erucibles reed to be ignited at the specified temperature'time of the method prior to frst use (b) Program mufile furnace to reach desired temperature (see Methods above) in 60 min (@) Weigh 1.5-2.0 g test portion into the crucible, recording the weight of crucible and test portion tothe nenrest 0.1 mg (W), (@) Ignite in furnace at time and temperature (see Methods above) after the furnace reaches temperature, (@) Allow fumace to cool below 200°C, then transfer crucibles to a desiccator with stoppered lid (H Cool to room temperature and weigh within 1b, recording Weight o the nearest 0.1 mg (R). When opening the Aesiceator, very slowly loosen the stopper to allow air to enter slowly, preventing the ash residue from being blown out ofthe crucibles (g) Proceed as follows, depending upon experimental conditions: (D Skip to (k) ifno additional ignition, Q) Skip to() if igniting {G) Proceed t0(h and i if wetting prior to reigiting. (h) Careflly add a few mi of distilled H.0 to the residue to break it up. Dry crucible and contents in a 105°C oven (mechanical convection speeds up the process) fora few hours ‘until all water is evaporated. The concepts that a crust forms on the surface of some materials as they ash. Water is used to break up this crust and allow for a more complete ignition () Reignite crucible and contents in furnace for the specified temperature and time. Repeat steps (e) andl (f) before proceeding woth). G) Place crucibles in desiccator with stopper top. Cool and weigh, recording weight to the nearest 0.1 mg (R), When ‘opening the desiccator, very slowly loosen the stopper to allow air to enter slowly, preventing the ash residue from being blown ‘out ofthe crucibles, (k) Photograph crucibles containing residue for a visual record of residual carbon, () Cateulation: ‘Residue on ignition (crude ash), %= (R-T) x 100/(W— 7) where T = tare (empty) weight of crucible, R = weight of crucible + residue, and W= weight of crucible + test portion Statistical Analysis Statistical analysis was performed using two-way analysis of variance (ANOVA) for the eight sets of ignition conditions ‘and 15 materials (16). The full factorial experimental design was 8 15 treatments with replication in each cell, Significance vas determined with «= 0.05. Having determined a significant difference in the methods, a post hoc Tukey HSD test (16) was used to dstingvish which ignition conditions were significantly different fiom others, i seeoe|ioo'dno-ouopene)di Sp“TimEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95, No. 5, 2012 aes Figure 1. Markers represent the mean % ash estimated fr triplicate analysis 1395 Material x ignition Condition: Ls Means tom aed OH ‘each material for each set of ignition conditions. The frst four makers in each material group represent 600°C conditions and the second {our represent 550°C, so from the let markers represent 600°C/2, 600°Cid h, 600°C/2 hiwet2 h, 600°C/2 2 '550°C/3 h, 550°CI6 h, 550°C/3 hiwet’ h, and S50°C/3 hi h. The height ofthe markers exceeds the 96% confidence interval derived from the ANOVA error term (0.1), Results ‘Means for 15 materials, expressed as percent crude ash forthe eight experiments, are provided in Table 2, Results were found to be significantly different ftom the current AOAC Method 942.05 for all additional ignition conditions investigated. All jgnition conditions were also significantly different from each other exeept two: $50°C for 3 h, cool, and ash 3 additional and 550°C for 3, cool, wet, dry, and ignite 3 additional h Figure 1 illustrates the mean percentage of ash estimated from replicate analysis of eacl material foreach set of ignition conditions. The eight conditions in each material group are Iabeled in Figure 1. The height ofthe markers exceeds the 95% confidence interval derived from the ANOVA ertor ter (20.1), ‘The presence of carbon was noted in the residues for CGM, ‘SFM, GH, and B aftr the initial ignition eyele. The “whiteness” of the ash improved with addtional ignition, as reflected by lower residue weights. CGM was nearly carbon-fee after the second heating eyele; however, the ash was still not entirely white Discussion ‘The results ofthis study emphasize the empirical nature of the method, and that statistically significant bias will result from any change in ignition temperature or time for most feed materials, There is no “perfect” time'temperature combination for all feed materials. Some, such as CGM, are resistant to complete ignition, while other high mineral content feed ‘remixes or ingredients may be susceptible to loss of volatile elements, Figure 1 is an especially reveating visual record of ignition temperature and time effects. “Gaps” between the 600 and 530°C results for a single material are conspicuous, as are “Slopes” with increasing ignition times within a temperature For example, CSGM, HE. and BC have clearly separated gaps beween temperature markers, Sloping lines that continue and do not flatten raise a concem for loss of volatile elements ‘This is clearly the case for MVP and SFM. CGM does not have a temperature eflet, hut does have a clear slope related ‘0 ignition time, fing a either temperature, This observation is confirmed by the black color of the ash residue afer the initial ignition indicating the presence of significant amounts of carbon, Additional sgntion areatly hghtened the color ofthe ash residue, corroborating the additonal release of carbon. For FM, B, and CS, there is itl impact on results for changes to temperature or time. Examination ofthe data indicates that there is no single time and temperature combination suitable forall materials. The ISO 5948-2002 and AOAC 942.05 methods produce statistically different results for mos feed materials, Examination slso raises concer about the 600°C temperature for some materials, such as MVP and SFM, that indicate continual los of weight with time, suggesting loss of more volatile elements. For these materials, 550°C appears to be a preferred ignition tempeatire for a “constant weight” ignition esidve, While a second ash cycle greatly improved results, there was no apparent advantage to wetting the ash reside between igitions Concems about ignition temperature Iead to a review of crude ash methods forall food/fed materials. Most of the rethods were adopted in the early 20th century. Thirty-seven of 4 AOAC Official Methods of analysis methods for ere ash in botanical matrixes (food or products that could be considered animal feed ingredients) specs temperatures below 600°C (17), Similarly. cight of 12 ACC methods for similar products specify’ ignition temperate below 600°C (18). Dry ‘shin is @ common mechanism for removal of organic material ‘fom food and feed material price to elemental analysis. Since fall ecovery ofthe elements is critical and loss of inorganics ‘would have been observed, dry ash procedures used in AOAC Official Methods of alysis metbods to remove organic matter fiom botanical matrixes prior to elemental analysis were reviewed. Of the 27 methods, 21 call for as temperatures of 580°C or below (17) and 10 spevfy the use of “ashing ai, such as Ma(NOs): and #50, 10 speed the ashing process and convert clements into less volatile forms. St.John in 1941, i seeoe|ioo'dno-ouopene)di Sp1396 THHEX ET AL.: JOURNAL OF AOAC INTERNATIONAL Vor. 95, No. 5, 2012 remarked that the effect of ashing temperature on the loss of elements, particularly potassium, indicates a need for a further ‘study of methods for the determination of potassium in plant material. .."(19), The loss of sodium, potassium, and manganese a ignition temperatures over 500-S80°C has been reported in the years since 1942, when AOAC Official Method 942.08 was adopted (20-22), Since the decision onan ignition temperature between 50nd 500°C for AOAC Official Method! 942.08 was long debated and ‘a somewhat subjective decision by St. John (I, 12), the authors ‘suggest that the temperature be reconsidered, or thatthe method conulitions be modified to reduce the seope of application for the 600°C ignition. It has been recently suggested that a fresh air supply to the muffe furnace will provide a more thorough ash in a single 2 or 3 h ignition, but this modification was not tested in this sua AOAC Official Method 942.08 specifies adding crucibles containing test portions to a hot furnace, a practice that is susceptible to problems that may lead to loss of material fom the crucible due to rapid ignition. Placing crucibles into ‘cold fumace and ramping to the final ignition temperature (cither manually or with a programmable controller) resolves issues related to potential loss of residue from crucibles duc to sputtering or flaming as a result of rapid ignition. AOAC Official Method 942.08 does not mention prior ignition of new crucibles nor drying of crucibles prior to use. These practices ‘ze commonly known to be necessary to avoid weighing ervors associated with residual moisture or loss of crucible weight during ignition, Recommendations for Modification of AOAC Official Method 942.05 Based on the results of this study. a review of methods ‘nd literature, and the omissions mentioned in the preceding paragraphs, the authors recommend several modifications to AOAC Official Method 942.08. This method should be modified toallow the ramping of furnaces to the desired temperature tobe consistent with the equipment in current use i laboratories, 10 recommend weighing to the nearest 0.1 mg, and to specify the use of preignited, predried crucibles. The authors recommend Towering the ignition temperature to SS0°C; however, they also recommend! two 3h ignition eyeles to ensure release of carbon from the feed material. To emphasize the empirical nature of the method, the authors suggest revising the title to include the ‘ignition temperature and labeling the method as “Residue on Ignition (Crude Ash).” This title change is consistent with the titling of moisture methods as “Loss on Dry The authors suggest that the AOAC Official Methods Board consider the following asa minor revision to AOAC Official Method 942.08. () The title be changed to: Residue on Ignition (Crude Ash) in Animal Feed at $50°C, (2) The text to read as follows: “Weigh approximately 2g test portion into. preignite. predried, preweighed porcelain crucible, recording all weights tonearest 0.1 mg. Place into temperanure-coatolled furnace and ‘over I h bring (tamp) furnace to $50°C; hold at 550+ 10°C for 3h. (Note: Furnaces with programmable temperature control may he programmed. Manual temperature-controlled furnaces should be manually monitored and adjusted to reach S50°C in | b). Allow famace to cool to below 200°C. Open furnace door to ensure fresh aie supply and repeat the 3h mufile furnace heating cyele to carfon-free residue. Final residue shoul be carbon-free (nearly white), not black or gray. Transfer crucible directly to desiccator, cool, and weigh within 1 h, reporting results tothe appropriate number of significant digits (generally two decimal places). (Note 1: Open desiccator cautiously to allow ais to enter slowly, preventing the ash residue from being blown out ofthe crucible, Nore 2: Care should be taken to not ‘overload the fimace).” (3) Caleulation to appear as follows: Residue on ignition (crude ash) at 850°C, 9 (residue mass, *100)/(test portion mass, g) Acknowledgments. We wish to acknowledge Renata Wink and Christelle Guillaume for the ash analyses and many contributions 0 the project. References (1) Thiex,N. 2012) in Distr: Grats: Production, Properties, ad lization, K. Liv & K.A, Rosetraer (Bas), CRC Pres, Taylor tad Francis Group, Boca Rate, FL, pp 193-217 (2) Codes limentarivs Proedral Mata, 2010, p/w ano. og DitetoryCommineePagesCheekSampleProgam asp (accessed Jy 30, 2010) (3) AAFCO MeshodPerfomace Reports, AAFCO Colabative ‘Chock: Sample Program. p/w ano. (accessed July 30, 2012) (4) Oficiot Methods of ass (2005) 18th Ed, AAC INTERNATIONAL, Gaithersburg, MD, Method 942.08 (6). NimionReqnroments of Datry Cane (2001) National Aendeny Press, Washington, DC. pp 13-18. (6) Tedeschi, L.0., Fox, DG. & Doane, PH. 2005) Prof nim Set 21, 403-415 (Regulation PFS, Statements of Calorie Content (2011) Offi Prbicnon ofthe Americ Fed Control Oca Ine, Oxf Isp (8) 180 $948-2002, anima Feeding Stat Determination of Cre 4 Intentional Organization fr Standardization, Geneva, ‘Switzerland (9) CRAMA, Ash (Residue on Jnition} np e-com xs p- outa plonds’2009/127Mo pf (accessed Janay 24, 2012) (10). A4CC fterartonal Approved Methods of nobis (1978) ith a, Method 08-0301, AACC International, St. Pl, MN DOK 10.1094AACCuMethod-08-03.01, (1) St-Jolun, JL, 1942) AssoviateRefeee Rept, J Assoe Off Agric Chom. 28, 857-864 (02) ‘St John, J (1943) Associate Referee Report J Ass00. Off Agric. Chem. 26,220-226 (13) Thiex,N, Tora, Wolf M4 Collin, M. (1999) Quai Assurance Quay Conro! Guselines for Forage Laboratories, National Forage Testing Associaton, Omaha, NE (14) Thies, N..Novouy.L.. Ramsey. C. Latimer G, Tonna,L. & eine, R-(2000) Guidlines fr Preparing Laboratory Samples, Assocation of American Feed Couto Official, le, Oxford IN (15) Offciat Methods of Anas (2005) Sth Ea AOAC INTERNATIONAL Gnithersburz, MD, Method 968.16 (16) StatSot Ie. 2008) STATISTICA (data analysis software system), version 6, wawstaso com (accessed July 30,2012) i seeoe|ioo'dno-ouopene)di Spo as) 19) en) ‘TEX ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 95,NO. 5, 2012 1397 Officio! Mets af analysis (2008) 18th Ed, AOAC INTERNATIONAL. Gaithersburg, MD. Approved Meds ofthe American Assocation of Creal ‘Chemists (1983) 8h Ed, American Association of Cereal (Chemists, St Paul, MN St Jon, IL, 1941) J Assoc. Off gre. Chem 24, 848-854 Henbook of Reference Methods for Plant Anais (1998) Kai, en @ ‘YP (Ea), Soi and Plant Anaiysis Council, Ine, CRC Press, Boca Raton, FL, p83 Isaac, RA. & Jones, JB, (1972) Comme Soi Se. Plant Ana 3, 261-269 pds dior 10 1081 0010362720936675 Rowan, C.A, Zajeek, O.T, & Calabrese, EJ. (1982) dna (Che, $4, 149-151, bpd or!10.1021/ac002882047 1o'do-atwopede)eday wow PepeoIUNoG 202 1990120 £1 vo 1san6 fa zezssosizéeL/SIS6eRNUE/DeOE!