Analyst, December, 1966, Vol. 91, p p . 783-789 783 The Thermal Analysis of Lichens Growing on Limestone BY B. D. MITCHELL, A. C. BIRNIE (The Macaulay Institute for Soil Research, A berdeen, Scotland) AND J. K. SYERS (Department of Soil Science, Lincoln College, New Zealand) In an investigation of the pcdogenic activities of lichcns growing on limestones it was found that they varied greatly in their calcium oxalate content. As chemical methods of determining calcium oxalate in such material are somewhat tedious, detailed qualitative and quantitativc studies of oxalate in specific lichen species were made by using differential thermal and thermogravimetric analysis. The results show that in a controlled atmosphere of oxygen-frec nitrogen, thermal methods provide a rapid means of identifying and determining calcium oxalate in lichens, and.also enable an assessment to be made of the non-oxalate derived carbonate in the sample. IT has been recognised for some time that there is a correlation between the amounts of calcium and oxalate in many plants, and it has been claimed that calcium oxalate is abundant in all plant species inhabiting limestone. However, in the course of an investigation of the pedogenic activity of lichens, Syersl found that those growing on limestone varied widely in their oxalate content. Chemical methods of determining oxalate in plant materials are tedious, especially for small amounts. Recently, Mitchell and Knight2 demonstrated that oxalate in higher plants, for example, Beta vulgaris and Efiilohizcm Zaizceolatiinz, may be conveniently monitored by thermal methods, and in view of this a detailed study of calcium oxalate in specific lichen species was conducted by using thermal methods.Because the energy changes occurring in a material when heated may vary according to the atmosphere enveloping it, the thermal characteristics of lichen species were determined in oxygen, nitrogen and carbon dioxide to ascertain under which of these atmospheres the calcium oxalate reactions were distinct from other pyrolysis reactions occurring in the lichens. Further, as the area of a peak in a thermal curve is indicative of the amount of reactant, it was necessary to establish which of the thermal peaks associated with the decomposition of calcium oxalate was the most satisfactory for its quantitative determination.METHODS MATERIALS- Seven lichen species were examined, namely, Asfiicelia calcaria, Calofilaca hefipiana, Physcia adscendem, Physcia caesia, Rhizocavpon calcaveii m, Verrzccaria 12 ipescens and Xantlzoria parietina. The samples, which were obtained by scraping the surface of lichen-colonised limestone, were dried at 40" C and ground to pass through a O.5-rnm sieve. PROCEDURES- The differential thermal analysis curves were determined under controlled-atmosphere conditions ; equipment and experimental details have been described elsewhere by Stewart, Birnie and MitchelL3 The tliermogravimetric characteristics of the lichens in a nitrogen atmosphere were also determined by using a TR 01 Stanton thermobalance from which the differential thermogravimetric curves were derived.RESULTS The complete-combustion curve for calcium oxalate monohydrate (Fig, 1 , curve D) exhibits an endothermic peak at 243" C, indicating the loss of water of hydration. The series of exothermic effects between 450 and 500" C traces the oxidation of the oxalate to carbonate, and the decomposition of the latter to calcium oxide is shown by the 900" C endothermic peak.784 MITCHELL, BIRNIE AND SYERS: THERMAL [Analyst, Vol. 91 In an inert atmosphere calcium oxalate decomposes in the following three stages- CaC,O,.H,O = CaC,O, + H,O.. . . . . * * (1) . . - * ( 2 ) . . . - (3) CaC,O, = CaCO, + CO . . . . CaCO, = CaO + CO, . . . . All three reactions are endothermic and the heats of reaction (H" 298" K, kcal. per mole) are 13.3, 15.5 and 42-5, respectively (Simons and Newkirk,).Under the dynamic heating con- ditions of differential thermal analysis, the peaks associated with these reactions occur at about 235O, 500" and 900" C (Fig. 2, curve D). Thus, in the interpretation of the thermal curves of lichens, endothermic effects in the 150" to 250" C region are regarded as indicative of the dehydration of calcium oxalate, effects between 400" and 600" C of oxalate decomposi- tion, and in thc 700" to 900" C range of carbonate decomposition. Reasons for the fluctu- ations between the peak temperatures of calcium oxalate alone and the peaks on the lichen curves due to the presence of calcium oxalate will be dealt with later. The thermal characteristics of the seven lichen species fall into three well defined groups, depending on the amount of calcium oxalate and calcium carbonate present. Thus Physcia adscendens, Physcia caesia and Xanthoria parietina, which contain small amounts of calcium oxalate, have closely similar thermal curves, as do the oxalate-rich species Rhizocarpoiz calcareum, Aspicilia calcaria and Caloplaca heppiana.The Verrucaria nigrescens sample is unique in that it contains an appreciable amount of calcium carbonate. EFFECTS IN AN OXYGEN ATMOSPHERE- The complete-combustion curves for Physcia adscendens and Verrucaria nigrescens (Fig. 1, curves A and B) exhibit exothermic peaks at about 300", 430" and 500" C and the peak areas decrease with increasing temperature. Complete-combustion patterns of this type are common for fresh plant material and are thought to reflect concentrations of carbohydrates of high molecular weight.2 The combustion curve of Rhizocarpon calcareum (curve C) is D .I- v 900 243 I A Temperature, "C - Fig.1. Differential thermal analysis curves in an oxygen atmosphere (10 ml per minutc) for: -4, Physcia adscendens; B, Verrucaria nagrescens ; C, Khizocavfion calcareurn ; and D, calcium oxalate monohydrate, (15-mg samples diluted with 50 mg of calcined kaolin, loosely packed : recording sensitivity, 170 pvolts pcr inch; hcating rate, 10" C per minute)December, 19661 ANALYSIS OF LICHENS GROWING ON LIMESTONE 785 markedly different. The endothermic peak at about 150" C indicates the dehydration of calcium oxalate, and the exothermic system is limited to peaks at 296" and 447" C.The latter peak, from a comparison with that of curve D, is affected by the combustion of calcium oxalate, but the complete-combustion curves are obviously not satisfactory for the un- ambiguous detection of calcium oxalate. EFFECTS IN A NITROGEN ATMOSPHERE- Temperature range 150" to 250" C-The small endothermic peaks at 175" and 177" C on the pyrolysis curves of Physcia adscendens and Verrucaria nigrescens indicate that these materials contain only small amounts of oxalate (Fig. 2, curves A and B). The dehydration of the oxalate in these two lichen species occurs 50" to 60" C lower than that in Rhizocarpon calcareum (curve C ) and that of pure calcium oxalate (curve D). Further, the dehydration of the oxalate in Physcia and Verrucaria occurs in a single stage.The reason for the doubling of the dehydration peak, which is particularly marked in the curve for Rhizocarpon (Fig. 2, I Temperature, "C - Fig. 2. Differential thcrmal analysis curves in a nitrogcn atmosphere (200 ml per minute) for: A, Physcia adscendens ; B, Verrucaria nigrescens ; C, Khizocarpon culcareum ; and D, calcium oxalatc monohydrate, (50-mg samples diluted with 120 mg of calcined kaolin, hard packed; recording sensitivity, 70 pvolts per inch; heating rate, 10" C per minute) curve C), is not absolutely clear. Substantial amounts of magnesium oxalate dihydrate are known to accumulate in certain plant tissue. This magnesium salt dehydrates in the 200" C region. The presence of magnesium oxalate in the lichens would result also in an endothermic peak between 600" and 700" C, marking decomposition to magnesium carbonate, and there is no indication of such a peak.Further, the magnesium contents of these lichens were determined chemically and found to be very low, indeed that of Rhixocarpon was the lowest, being 149 p.p.m. Physcia and Verrucaria contained 500 and 974 p.p.m., respectively. De- hydration of oxalic acid dihydrate occurs at 100" C and the monohydrate decomposes between786 MITCHELL, BIRNIE AND SYERS : THERMAL [Analyst, Vol. 91 180" and 190" C. Free oxalic acid, however, cannot be present in the lichens because the exchangeable calcium is high : Rhizocarpon, 898 milli-equivalents per 100 g ; Physcia, 120 milli- equivalents per 100 g; and Verrucaria, 946 milli-equivalents per 100 g.From evidence of infrared-absorption spectroscopy, not only is calcium oxalate the only oxalate present, but it appears to be exclusively in the monohydrate form. Although the energy changes shown on a differential thermal analysis curve that indicate the loss of sorbed moisture in the sample are not necessarily reflected exactly by the differential thermogravi- metric curve, it is nevertheless significant that the differential thermogravimetric curves of Rhizocarpon calcareum and calcium oxalate (Fig. 3, curves C and D) give no indication of a two-stage dehydration of oxalate. The variation in the dehydration pattern of oxalate, as reflected by the differential thermal analysis curve, may be related to the dilution and hard-packing technique developed for examination of the sample under inert-atmosphere conditions.Hard packing would tend to restrict the egress of water vapour and the effect would be enhanced by increased concentration, particularly in the initial stages. Temperature, "C - Fig. 3. Uiflcrcntial thermogravimetric curvt's in a nitrogen atmosphere (200 1111 per minute) for: 4 , PIzyscia adscrrzdezzs; €3, Verrucavza 9zigvescens; C, Hizizocavpon cnlcaveunz ; and I), calcium oxalatc monohydrate, (sample weight, 100 mg; heating rate, 4' C per minute) Temperature raizge 250" to 400" C-Substantial weight losses between 300" and 400°C are shown on the differential thermogravimetric curves of lichens determined in an inert atmos- phere (Fig. 3, curves A to C), Physcia adscendens losing 52 per cent., and Rhizocarpon calcareum, 21 per cent, Infrared spectroscopy indicated that the Physcia species contained considerably larger amounts of carbohydrate than the Rhizocurpon species.The pyrolysis curves of cellulose and carbo- hydrates of low molecular weight exhibit well defined endothermic peaks about 300" C,5 and attributing, therefore, the weight loss in the 350" C region to carbohydrate decomposition, it is noteworthy that no endotliermic effect is recorded in this region of the differential thermal analysis curves of lichens under inert atmosphere conditions (Fig. 2, curves A to C). There There is no weight loss in this region for calcium oxalate (curve D).December, 19661 AKALYSIS OF LICHENS GROWING ON LIMESTONE 787 is, however, an exothermic reaction, whose intensity decreased with reduction in carbo- hydrate content.As a peak on a differential thermal analysis curve need not necessarily represent a single reaction, but may be the record of the summation of simultaneous reactions, it is conceivable that the endothermic effect associated with the initial decomposition of the carbohydrate component is completely masked by an exothermic reaction involving the products of decomposition. As care was taken to exclude oxygen in these determinations, the exothermic peaks probably resulted from auto-oxidation by organic components of the lichens. Temperature range 400" to 600" C-The absence of an endothermic peak in the region of 500" C on the differential thermal analysis curves of Physcia adscendens and Verrucaria nigrescens (Fig.2, curves A and B) is in accord with a low calcium oxalate content. However, the differential thermogravimetric curve of the former (Fig. 3, curve A) shows a weight loss about this temperature which, by comparision with that for calcium oxalate, corresponds to a content of less than 5 per cent. The corresponding weight loss for Rhixocarpon (curve C) is equivalent to a calcium oxalate content of 60 per cent. Temperature range 700" to 950" C-The pyrolysis curves of Verrucaria izigrescens and Rhixocarpon caZcareum in a nitrogen atmosphere (Fig. 2, curves B and C) exhibit a large endothermic peak at 800" C resulting from the decomposition of calcium carbonate, which in the latter species is derived from calcium oxalate. However, for the Verrucaria sample the calcareous substratum is probably the principal source.The carbonate decomposition peak on the curve of Yhvscia adscendens (curve A) is much smaller and occurs at a considerably lower temperature (705" C), both features being in accord with a lower calcium oxalate content. 3 20 Tern pera t u re, "C -I-- Fig. 4. Differential thermal analysis curves in a carbon dioxide atmosphere (200 ml per minute) for: A, Physcia adscendens ; B, Verrucaria nigrescens ; C , Rhizocarpon cakareum ; and D, calcium oxalate mono- hydrate; E, 1 + 1 mixture of Physcia adscendens and calcium oxalate monohydrate, (50-mg samples diluted with 120 mg of calcined kaolin, hard packed: recording sensi- tivity, 70 pvolts per inch; heating rate, 10" C per minute)788 MITCHELL, BIRNIE AND SYERS: THERMAL [Analyst, Vol.91 EFFECTS IN A CARBON DIOXIDE ATMOSPHERE- The differential thermal analysis curves of lichens determined in a carbon dioxide atmosphere (Fig. 4, curves A to C) are similar to those observed in a nitrogen atmosphere, except in the 700" to 950" C range of curves B and C for Verrucaria and Rhizocarpon, respec- tively, where a doubling of the peak reflecting carbonate decomposition occurs. Although the curves for Physcia and calcium oxalate monohydrate (curves A and D) do not show this doubling phenomenon, that of a mixture (1 + 1) of these materials (curve E) does possess a complex high temperature - peak system, but only when the lichen and oxalate have been intimately mixed (ground for 1 minute in a vibratory ball mill). The lichen samples on reaching 700" C consist essentially of a carbonaceous residue PLUS calcium carbonate and, as the temperature is raised, increasing amounts of carbon dioxide will be produced as decomposition of the latter proceeds.The high temperature - peak pattern could arise from the oxidation of the carbon residue by carbon dioxide, and consequently would represent an endothermic effect with a superimposed exothermic reaction. This effect, however, only occurs under a dynamic carbon dioxide atmosphere when an additional and substantial amount of carbon dioxide is produced in situ, as in the decomposition of samples containing appreciable amounts of calcium carbonate. Under these conditions, the voids in the sample well are completely filled with carbon dioxide. The doubling effect is not observed when nitrogen is the con- trolling atmosphere, but in this instance the carbon dioxide from carbonate decomposition would diffuse more easily from the reaction site.DISCUSSION The results show that calcium oxalate in naturally occurring materials such as lichens may be unambiguously identified on the differential thermal analysis curve, but only when determined in a nitrogen atmosphere. Comparison of the weight loss in the 500" C region of the differential thermogravimetric curves of the lichens with the corresponding weight loss on the differential thermogravimetric curve for a known quantity of calcium oxalate provides a simple and direct estimate of the oxalate in the lichens. The oxalate content of these plants was also determined chemically by a micro method6 and it will be noted (Table I) that while agreement between thermogravimetry and the chemical method is good for high concentrations of oxalate, at low levels the former method is less satisfactory.TABLE I AMOUNTS (PER CENT.) OF CALCIUM OXALATE AND CALCIUM CARBONATE IN LICHENS DETERMINED BY DIFFERENTIAL THERMOGRAVIMETKIC, DIFFERENTIAL THERMAL ANALYSIS AND CHEMICAL METHODS Calcium oxalate, per cent., determined by- differential thermo- gravimetric method Physcia adscendens 5 Rhizocarpon calcareum 60 Verrucaria nigrescens nil Aspicelia calcarea 42 differential* thermal analysis chemical method method 2.7 2.2 0.8 1.4 60 56 48 39 Calcium carbonate, per cent., determined by- - diff erentialt thermo- gravimetric chemical: method method 2.3 ( 5 52 54 7.0 11 21 24 * Determined from 170" C peak area for Physcia and Verrucaria (Fig.2, curves A and B) t Obtained by subtracting the calculated weight loss for calcium oxalate derived carbonate Obtained by treating the dry matter with 0.1 N hydrochloric acid overnight, filtering, and and from 500" C peak area for Rhizocarpon (Fig. 2, curve C). from total weight loss between 700" and 800°C (Fig. 3, curves ,4 to C). then back-titrating the unused acid with alkali. Differential thermal analysis can also provide a quantitative result as the area enclosed by a peak is proportional to the amount of reactant. As the 500" C peak on the differential thermal analysis curve of calcium oxalate resulting from the oxalate - carbonate reaction is, unlike the dehydration peak, invariably free from doubling, it may be used for the quantitative determination of oxalate by simply measuring the area of this peak on the lichen curve and comparing this with the peak area for the known amount of calcium oxalate monohydrate (see, e.g., Rhizocarpon calcareum, Table I). For low levels of oxalate this 500" C peak is eitherDecember, 19661 ANALYSIS OF LICHENS GROWING ON LIMESTONE 789 absent or poorly defined (Physcia adscendens and Verrucaria nigrescens, Fig.2, curves A and B). Here, however, oxalate dehydration is recorded as a single peak (170” C), and integration of this provides a good assessment of the oxalate content of the lichen (Table I). Indeed, the agreement between differential thermal analysis and the chemical determinations of oxalate is remarkably close.Finally, consideration of the weight losses on the differential thermogravimetric curves associated with carbonate decomposition in relation to the effects on these curves resulting from the first and second stages of calcium oxalate decomposition, enables an assessment to be made of the non-oxalate derived carbonate (Table I), which is in good agreement with the chemical determination of this component. Ability to distinguish these two forms of calcium in the sample has shown that the three lichen species containing appreciable amounts of calcium oxalate, namely, A spicelia calcaria, Calofilaca heppiana and Rhizocarpovt calcareum are confined to calcareous rocks (i.e., obligate calicoles) , whereas Physcia adscendens, Physcia caesia and Xaizthoria fiarietina, which contain 5 per cent. or less of calcium oxalate, are ubiquitous. These results, supported by other unpublished evidence, show that thermal techniques provide a rapid and reliable means of determining oxalate in plant materials. Magnesium oxalate commonly occurs with calcium oxalate in plants; however, there is no indication of the presence of magnesium salt in the lichen species examined, all the evidence pointing to the oxalate being present exclusively in the form of the calcium oxalate monohydrate. REFERENCES 1 . 2. 3. 4. Simons, E. L., and Newkirk, A. E., Talanta, 1964, 11, 649. 5. Mitchell, R. D., Sczent. Proc. R. Dubl. Soc., i3, 1960, 1, 105. 6. Syers, J, K., “Study of Soil Formation on Carboniferous Limestone with Particular Reference t o Lichens as Pedogenic Agents,” 1’h.D. Thesis, L’nivcrsity of Durham, 1964. Mitchell, €3. D., and Knight, A. H., J . Exp. Rot., 1965, 16, 1 . Stewart, J. M., Rirnie, A. G., and Mitchell, B. n., Agrochimica, in the press. Joy, K. W., Ann. Bot., 1964, 28, 689. Received February 25th, 1966