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Calorific values of the soft parts of the tellinid bivalve Macoma balthica (L.) as determined by two methods
J. e.~p. mar. Biol. Ecol., 1979, Vol. 37. pp. 19-30 0 Elsevier’North-Holland Biomedical Press
CALORIFIC VALUES OF THE SOFT PARTS OF THE TELLINID BIVALVE MACOMA
(L.) AS DETERMINED BY TWO METHODS
BALTHZCA
J. J. Netherlands
BEUKEMA
and W.
DE BRUIN
Institute for Sea Research, Trxel. The Netherlands
Abstract: Calorific values of the ash-free dried soft parts of Macoma halrhica (L.), sampled monthly during 1 year at a tidal flat station in the Dutch Wadden Sea, have been estimated by two methods. The indirect method started from an analysis of the biochemical composition and used conversion factors for the various components to calculate the calorific value of the total tissue. The direct method employed a micro-bomb calorimeter and a furnace for separate ash determinations. The indirect method yielded estimates that were consistently higher than those from the direct one. Two sources, explaining most of the difference. could be traced, viz. incomplete drying of the samples used in the calorimeter and a too high conversion factor for weight to calories applied conventionally for lipids. This conversion factor was found to depend on the method of extraction of the lipids. but was always significantly below the commonly used 9.3 to 9.7 cal.mg-‘. The best estimates for the annual mean of the calorific value at complete combustion of the ash-free and well-dried soft parts of Macoma were 5.47 for the direct and 5.59 cal.mg-’ ash-free dry wt for the indirect method. Both values are higher than estimates for Macoma published earlier by various authors. Calorific values at physiological oxidation amount to only about 4.6 cal.mg-’ or less. due to incomplete protein oxidation. For this reason. it is concluded that biologically significant calorimetry cannot dispense with biochemical analysis. On the other hand, calculation of calorific values from biochemical composition should be accompanied by some calorimetry. as the conversion factor for the lipid fraction. at least. may deviate significantly from commonly applied values.
INTRODUCTION
As a part of the analysis of the functioning of an ecosystem, a study of the transfer of energy between populations is an important step. Accurate values for the energy content of the tissues of organisms are essential for such studies. Calorific values are commonly estimated in either of two ways: 1) by direct bomb calorimetry, or 2) by computation from results of a study of biochemical composition, using appropriate conversion factors for the various components. The first method is direct and exact in well-dried material that is relatively poor in ash (Paine. 1971). The values found need only minor corrections which may be omitted in many cases (Paine, 1971; Schroeder, 1977). The method does, however, yield energy values for complete combustion. For biologically more realistic heat values, based on protein metabolism, the nitrogen content of the material should be determined in addition (Kersting, 1972). The second method calls for a cumbersome complete biochemical analysis of the material. Moreover, there is reason to doubt whether the commonly used conversion 19
20
J.J.BEUKEMAANDW.DEBRUIN
factors. based on Brody (1945), viz. 4.1 kcal .g-’ for carbohydrate, 5.65 for protein, and 9.45 for lipid, are applicable to the specific material in question. All of these values are in fact averages of varying values found for pure compounds. Morowitz (1968) assumes the following mean values: 4.1,5.5, and 9.3 kcal-g-’ for carbohydrate, protein, and lipid, respectively. It cannot be taken for granted beforehand that any biological material will yield values close to these averages. The second method is, therefore, incomplete without some direct calorimetry and the first method needs some biochemical analysis. Although desirable, such complete studies appear to be rare. In fact, there appear to be only few sets of data on calorific values of biological material that allow a comparison of the results of different methods applied to the same material. In the great majority of papers on ecological energetics, results of only one method are accepted without any discussion of the probable bias. The aim of the following study, therefore, is to compare results obtained by the two methods, applied to the dried soft parts of Mucoma balthica (L.), to get some insight into the accuracy of the calorific values obtained. A Mucomu population has been sampled throughout the year in order to have access to material with varying contributions of the basic compounds (Beukema & De Bruin, 1977).
MATERIALANDMETHODS
At an intertidal station in the westernmost part of the Dutch Wadden Sea, about 100 adult Mucomu were sampled 15 times at approximately monthly intervals during the period June 1974 to July 1975. The sampling station is close to Station 4 in Fig. 1 in Beukema et al. (1977). At this station growth rate and condition of Mucomu are close to the average for the whole intertidal Balgzand area (Beukema et al., 1977). The approximately monthly sampled Mucomu were used for determinations on growth rate, condition, and chemical composition, as described in Beukema & De Bruin (1977). In short, the animals were stored alive in running sea water for one day and then killed by briefly immersing in boiling water. The soft parts were dried for 3-5 days at 60°C. Part of the dry material was used for determinations of ash content, i.e., the weight left after 2 h in a furnace at 500-600 “C (the amounts of ash recovered from the calorimeter were found to be only 60-80x of the amounts found by these separate furnace ashings). Most of the material was used for chemical analysis after pulverizing and homogenizing and short (generally less than one month) storage at -20°C. Lipid content was measured by Soxhlet refluxing extractions with various solvents (either pentane, chloroform or chloroform-methanol). Chloroform yielded consistently higher (by 30% in summer to 80% in winter and early spring) amounts of lipids than pentane, and even slightly higher amounts than the chloroform-methanol mixture recommended by Bligh & Dyer (1959).
CALORIFICVALUESOFMACOMA
21
The lipid-free fraction was dried again and used for determinations of glycogen (by the calorimetric anthrone method according to Handel, 1965) and nitrogen (by a standard micro-Kjeldahl procedure, with 6.25 as a multiplication factor to obtain protein figures). Caloritic values were obtained by burning dried 2&35 mg pelleted samples of the lipid and lean fractions in a Gentry-Wiegert micro-bomb calorimeter (described in Phillipson, 1964) using benzoic acid as a standard. The values obtained were corrected for ash content (as determined separately) and are thus expressed in cal .mg -’ ash-freedryweight (ADW). Foreachofthe 15 sampling dates, the material was divided into l-3 lots, each delivering 4-5 pellets. Depending on the amount of material available, 416 pellets from each sampling date were burned. Corrections for acid production and endothermic reactions within the bomb were omitted, because these were expected to be insignificant at the sample size used and the low-ash contents observed (Paine, 1971; Schroeder, 1977).
RESULTS BIOCHEMICAL COMPOSITION
The composition of the soft parts of Macoma undergoes an annual cycle (Beukema & De Bruin, 1977). Glycogen rapidly increases during the short growing season (late March to mid June) and declines during the remainder of the year, especially during the July-December period. Lipids do roughly the same, but build up less rapidly during the second half of the growing season, probably as a consequence of simultaneous losses as a result of spawning. Proteins, though increasing in absolute amounts during the growing season, decrease as a percentage during this period, due to the more rapid increase of glycogen and lipids. During the remainder of the year, proteins are used up at a lower rate than glycogen, resulting in an increase of the relative share of proteins during the second half of the year. Fig. 1 shows the seasonal changes in the proportions of protein, glycogen, and lipids during the 19741975 sampling period. Comparison with Fig. 4 in Beukema & De Bruin (1977) shows that the cycles observed during this year hardly differed from those found during the preceding year. As lipids and glycogen differ greatly in caloritic value, such cycles will affect the stability of the calorific value of the entire tissues. The fluctuations in the shares of these two components, however, run more or less parallel during most of the year (Fig. 1). Consequently, the seasonal changes in the calorific values of Macoma tissues will be limited. Summation of the three main components yielded totals varying from X8-101% (mean 94% of the ADW). The mean deficit of 6% would have consisted almost completely of free sugars and tightly bound water. The material dried at only 60°C would have contained 2 or 3% of water (Beukema & De Bruin, 1977).
J. J. BEUKEMA
22
AND W. DE BRUIN
,I ,a
,.nn:
e IO
n
d
/
f
m
on
I
,~ Dther
~dCflCI1~
I
,975
1974
Fig. I, Annual cycle of the main components of the ash-free dried soft parts of Macomu, expressed as percentages of ADW: protein from 6.25 x N-content; glycogen from calorimetric anthrone estimates; lipids from weighing chloroform extractions; figures for ‘other’ material obtained by difference: note the change of vertical scale between 35 and 450,,.
CALORIFIC
VALUES
OF THE MAIN
COMPONENTS
Lipids
Samples of the lipids extracted by various solvents were burned in the microbomb calorimeter and yielded rather different results (Table I). Pentane extracted the lipids incompletely, but the lipids obtained in this way were of a relatively high calorific content (9.1 cal.mg-‘). Apparently these lipids consisted of a high proportion of long-chain fatty acids that have a calorific value of about 9.3 calsmg- (Morowitz, 1968). Other lipids, not soluble in pentane, but
CALORIFIC
VALUES
23
OF MACOMA
extracted subsequently by chloroform, yielded only 7.4 calmmg -I. This fraction would have contained a higher proportion of short-chain fatty acids and impurities. As expected, the calorific value of the total chloroform extract as calculated from the two fractions (0.25 x 7.38 + 0.75 x 9.10 = 8.67) fits the value found directly (8.62. see third line in Table I). Wet extraction according to Bligh & Dyer (1959) yielded estimates only slightly different from the chloroform extraction of dried material, in accordance with the almost equal amounts of material extracted.
of lipid fractions of soft parts of M~comu, as extracted by various solvents: Calorific values in cal.mg-’ mean values of samples obtained during the May-October period; chloroform-methanol extraction according to Bligh & Dyer (1959) from wet material. other extractions from dried and pulverized material. Mean cal.mg-’
Solvent Pentane Chloroform Chloroform Chloroform
after pentane + methanol
9.10 7.38 8.62 8.42
95” conf. Ii&its 8.98-9.23 7.32-7.44 8.5G8.74 8.22-8.62
No. of samples 7 5 12 IO
Proportion extracted 0.75 0.25 1.00 0.90
In the following, the chloroform-extraction values will be used, as chloroform was found to extract the lipids most completely. As a matter of course. the appropriate calorilic value of 8.62 will be applied. Lean material
Caloritic values of the dried material remaining after lipid extraction and corrected for ash content, were found to vary between 4.74 and 5.17 calvmg-’ ADW (Fig. 3: open squares). Highest values were found at the start and lowest values at the end of the growing season. During this season the share of glycogen increases at the cost of that of protein (Fig. 1). As glycogen has a lower calorific value (about 4.1 calemg -‘) than protein (about 5.65 cal.mgm’ at complete combustion), such seasonal change is to be expected. Calorific values of the lean material were indeed positively correlated with the protein content (Spearman’s Y= +0.69; P < 0.01) and negatively with that of glycogen (Y= -0.75, P < 0.001). All calorific values found for the lean material are well below the values expected if this material consisted only of protein and glycogen with calorific values of 5.65 and 4.10 calemg-‘, respectively (Fig. 2). On an average, the calorific values observed are 0.3 to 0.4 calamg- or about 67% lower than the expected ones. Several reasons for this discrepancy may be suggested. In the first place, incomplete combustion and incomplete drying should be considered. It is clear that incomplete combustion could explain any negative difference between observed
24
J. J. BEUKEMA AND W. DE BRUIN
and expected values. Burning off the remaining ash for 1 or 2 h at 600°C resulted in a mean weight loss of only 0.33% of the dry weight of the original pellet (957; confidence limits 0.27 and 0.39, n = 10). Such weight loss would only add the negligible amount of about 0.02 cal .mg-’ to the calorific value. There is no reason to correct for this error, as it compensates for the acid correction which was not applied and which would have been about -0.02 calemg -I at the sample size used (Schroeder, 1977).
Fig. 2. Calorific values (Cal.mg -’ ADW), with 95% confidence limits (n = 4 or 5), for 26 lots of lean material, sampled on 15 dates throughout the year, and plotted against glycogen content of the same material: solid line, the expected values if all the lean and ash-free non-glycogen material were protein with a calorific value of 5.65 caiemg-’ and if the cabrific value of the glycogen is 4.10 calqmg -’ ; broken line shows best fit by geometric mean regression (n = 26).
Incomplete drying may have been a more serious source of error. Further drying of lean pellets (already routinely dried for three days at 60°C) at various higher temperatures for three days resulted in weight losses of 3% at 100°C and 3.2% at 710°C percentages of ash-free dry weight of lean material). A water content of 3% moved the points in Fig. 2 by 0.15 to 0.17 Cal-mg-’ downwards and by about 1% glycogen to the left as compared with data (not available) for more completely dried material. Thus, about half of the observed discrepancy can be explained by the
CALORIFIC
VALUES
OF MACOMA
25
remaining water content of the dried material (the reason why we dried at only 60 “C was for a possibly unfounded fear of unnoticed disappearance of volatile substances). The still unexplained difference of, on average, x3% between the expected and observed calorific values shown in Fig. 2 may be due to wrong assumptions used in calculating the expected values. The sum of the protein and glycogen accounted for a mean of 94% of the ash-free dried lean material. Half of the remaining 6% will have been water. The other half may have had a calorilic value lower than the 5.65 calemg-’ used in the calculations of the expected values. If all of the other 3% were free sugars with a caloritic value of about 4 calsmg-‘, only about 0.05 cal mg-’ or one third of the still unexplained part of the difference would be explained. There is thus some room left for other errors, e.g., overestimation of the protein content. An unknown part of the nitrogen found will have been non-protein material with a probably lower calorific value than the supposed 5.65 cal .mg-‘.
ESTIMATES
OF THE CALORIFIC
VALUE
OF MACOMA
TISSUES
At complete combustion
A naive calculation, starting from the biochemical composition (Fig. l), and using the commonly applied conversion factors for calamg-’ of 9.45 for lipids, 5.65 for proteins and 4.10 for carbohydrates, and considering all other substances like free sugars as carbohydrates with a calorific value of 4.10, yields figures between 5.5 and 6.0 calwmg-’ (solid circles in Fig. 3). An annual mean calculated from these figures amounts to 5.68 calamg-’ ADW. The highest value is just before the onset of spawning, reflecting the high lipid/glycogen ratio at that time (Fig. 1). The subsequent decrease till the end of the growing season reflects the rapid rise of the share of glycogen at a constant or falling percentage of lipids. The above figures will be maximal ones and can easily be challenged: the conversion factor for at least one of the components (viz. the lipids, see Table I) is too high and the assumption that the ‘other substances’ consist of carbohydrates only is unrealistic. Some corrections can be applied to make these figures more realistic. Assuming that the ‘other substances’ consist of water for a percentage up to 3% of the ADW, the annual mean of the calorific values decreases to 5.57 calsmg-’ ADW. When the conversion factor for lipids is lowered from 9.45 to 8.62 cal.mg-‘, the calorific value of the material decreases to 5.59 calvmg- ADW. Note that the term ADW has been used here with two different meanings, viz. without any water (the 5.68 and 5.59 figures) and with the water remaining after drying at 60°C (the 5.57 figure). Application of both corrections yields an estimate of 5.48 calemg-’ for the ash-free soft parts of Macoma, dried at 60°C (Table II). Starting from the bomb calorimetry data (Table I and Fig. 2) and using the data included in Fig. 1 for the share of the lipids, figures between 5.26 and 5.42 calamg-’ were found for the calorific value of the ash-free, 60°C dried, soft parts of Macoma
26
J. J. BEUKEMA
AND W. DE BRUIN
Fig. 3. Annual cycle of the calorific values (cal.mg -’ ADW) of the ash-free and dried soft parts of Mrrcotna. as obtained by different methods (see text): growing season (from Beukema & De Bruin, 1977) and spawning season (from De Wilde & Berghuis. 1978. Fig. 6. for 1975) indicated at the top of figure.
TABLE Estimates
pressed
Annual Method
II
of the caloritic value of the dried soft parts of Macomcr halrlzica by various methods. exin cal.mg-’ ADW. with separate data for ‘complete’ drying and drying at only 60 C; annual means from n sampling dates.
(\ee also text)
n
dried at 60 C
Bvxh.
camp..
convent.
I5
Bioch.
camp..
O-3”” water
I5
Bioch.
camp..
lipid
corr.
I5
Bioch.
camp..
both con.
I5
5.4X
Bomb
calonmetry
I5
5.33
Bomb
cal.. water-free
15
mean dried completely 5.68
5.57 5.59
5.47
95” 0 conf.
limits
Refcrencc
paper
5.61-5.75
This
5.49-5.65
This paper
5.51-5.67
This paper
5.41-5.56
This paper
5.30. 5.36
Thk
5.43-5.50
This paper
4.65-4.90
Chambers
paper
Bomb
calonmetry
9
4.77
Bomb
calorunetry
8
4.05
Gilbert
(1973)
Bomb
calonmetry
2
5.08
Thayer
~‘f 111. 1973)
& M~lne (1975)
I
CALORIFIC
VALUES
OF MACOMA
27
(open circles in Fig. 3). The annual mean amounts to 5.33 calsmg-‘. To obtain the corresponding value for thoroughly dried material, the calorilic values observed for the lean material should be increased by about 3:/,. The resulting annual mean for the total material amounts to 5.47 calsmg-‘. All of the above values are higher than those found by other authors for the calorific value of the ash-free and dried soft parts of Macoma balthica (Table 11). Our mean values are ~0.35 calemg-’ or w6:/, of the mean. and when compared those obtained from bomb calorimetry are consistently lower than the corresponding values calculated from the biochemical composition (Table II). The largest difference (5.68-5.33) may be explained by incomplete drying, a conventional conversion factor which is too high for lipids, and unknown factors (discussed on p. 25) each of which contributes about equally to the difference. For thoroughly dried soft parts of Macoma. the true calorilic value will be within the limited range of 5.47 to 5.59 calsmg-’ ADW, because both the drying correction for the samples used in bomb calorimetry and the corrected conversion factor for lipids are justified. At phJ>siologicaloxidation The value of biological material as a source of energy for living organisms should not be estimated from heat production at complete combustion. because living organisms cannot oxidize proteins completely (Krebs, 1964; Kersting, 1972). Part of the carbon skeleton of amino acids is not burned. Moreover, end products of nitrogen oxidation are not N2, but such products as uric acid (e.g., in birds). urea (e.g., in mammals) or ammonia (e.g.. in many marine invertebrates). The caloritic value of proteins (and consequently of most biologicdl materials) at physiological oxidation is, therefore, lower than that found by bomb calorimetry (usually ~5.7 calsmg-‘). Kersting (1972) calculates minimal nitrogen corrections, taking into account only incomplete N-oxidation. These corrections amount to z -0.9 calsmg-’ protein in the case of NH, as an end product and to - 1.4 calsmg-’ protein in the case of urea (the latter correction should be 8.5 cal mgm’ N instead of the stated 7.2, K. Kersting, pers. comm.). Physiologically realistic calorific values for animal protein are thus not higher than 4.3 to 4.8 calmmg-’ protein, and probably still lower by ~0.5 calemgdue to incomplete oxidation of C and H (Krebs, 1964). Such corrections may be applied to the data obtained by bomb calorimetry because the protein content of the dried soft parts of Macoma have been estimated routinely (Fig. 2). A correction of - 1.4 calsmg- protein will be applied to the calorimetry values corrected for incomplete drying. A protein correction of this magnitude may be thought to represent either an oxidation of N to urea (with complete oxidation of the C and H) or an oxidation of N to NH, with incomplete oxidation of the C and H.
28
J. J. BEUKEMA
AND W. DE BRUIN
The resulting ‘physiological’ caloritic values for the ash-free and thoroughly dried soft parts of Mucoma range from 4.3 to 4.8 calsmg -’ ADW (lower line in Fig. 3) with a yearly average of 4.57 calsmg-’ ADW. This is 0.90 calemg-’ or 16% less than the corresponding value at complete combustion by bomb calorimetry (Table II). This difference is both larger and of more fundamental importance than the differences shown by the distances between the two top lines in Fig. 3, representing extreme outcomes of various ways of estimating caloritic values at complete combustion. Still lower calorific values should be used for the soft parts of Macoma if the end product of N-oxidation is urea or uric acid and also the oxidation of C and H is incomplete. In fact, no invariably applicable physiological heat value for protein containing material can be’stated, as it will depend on the metabolic capacities of the oxidizing organism. DISCUSSION
Calorific values for biological material that are calculated from the biochemical composition, using commonly applied conversion factors for calsmg-’ (viz. 9.45 for lipids, 5.65 for proteins and 4.10 for carbohydrates), may be overestimates in many cases. The conversion factor for lipids, especially, should often be lower than 9.45, as the lipid fractions extracted by the usual methods contain a lot of impurities. The less lipid we extracted (by choosing more selective solvents), the higher was its caloritic value (Table I). Craig (1977) also observed calorific values lower than 9.45, viz. 8.49 calsmg-’ for lipids extracted by chloroform-methanol from tissues of perch. This tigure agrees well with our value of 8.42 obtained by the same methods (Table I). By using a conversion factor of 9.45 instead of 8.62 for the lipid fraction, the calorific value of Mucoma tissue is overestimated by 1 or 2%, depending on the season (Fig. 1). The generally low share of lipids in the tissues of bivalves will keep this error relatively small in calculated calorific values for the tissues of these animals. Actually, the difference between the calculated and calorimetrically estimated heat values in Mucoma were greater, viz. on an average 6% (Table II: 5.68 compared with 5.33). About one third of this difference could be explained by the incomplete drying of the pellets used for the calorimetry. Thus about one third of the difference or only x2% of the calorific values remains to be explained, e.g., by too high a conversion factor for protein. In the literature published on caloritic values, we found only a few comparions between the results of calculation from biochemical composition and direct calorimetry. Craig (1977) observed a significant difference of 7.2% in favour of the former method. He considers that the calculated value is an overestimate due to the too high conversion factor for lipids discussed above.
CALORIFIC
VALUES
OF MACOMA
29
Pandian (1967) measured calorific values by bomb calorimetry in various stages of developing eggs of Cvangon and also determined percentages of ash. protein and non-protein nitrogen in the same material. He assumed the remainder to be fat with a caloric value of 9.4 calsmg- and adopted 5.65 calmmg- as a conversion factor for protein to calories. With these conversion factors, we calculated caloritic values for the various stages of the eggs (omitting the small fractions of non-protein nitrogen) and found figures which were 7-18:; higher than the ones Pandian had observed by direct bomb calorimetry. Platt & Irwin (1973) measured calorific values by bomb calorimetry in samples of phytoplankton and analyzed the same material for carbohydrate. protein. lipid, and ash. With conversion factors of 4.2 for both proteins and carbohydrates and 9.5 for lipids. they obtained good agreement between measured and calculated values. With a more realistic conversion factor of 5.65 instead of 4.2 for complete combustion of proteins (and, therefore, for comparisons with results from bomb calorimetry). we recalculated their figures and found values which were 2-20”{, higher (on average 11%) than the measured ones. As in the case of Pandian (1967), such difference could be reduced significantly by the use of lower conversion factors for lipids. A separate estimate of the calorific value of at least the lipids appears to be a prerequisite for a valid use of the calculation method. A high proportion of the published calorific values for bivalve tissues obtained by bomb calorimetry are unexpectedly low. Though calorific values of bivalve tissues will be rather low due to the frequently low lipid and high carbohydrate content, values below 5.0 calemgg ADW would be improbable. With a lipid content rarely below 55’6and a carbohydrate content rarely exceeding 50”/1, a lower limit to the calorilic value for the soft parts of bivalves may be estimated at about 0.05 x 8.5 + 0.45 x 5.6 + 0.50 x 4.1 = 5.0cal.mg-’ ADW. Observations significantly below this value are suspect. They might result from such errors as incomplete drying, incomplete combustion, or incorrect determination of ash content. Paine (1971) gives a more complete discussion of sources of error in calorimetry. The direct method appears to yield under-estimates more commonly than over-estimates, just the reverse of the calculation method. A biochemical analysis of the material in question may suggest such an error in calorimetry. Calorific values of biological materials are used mainly in bioenergetical studies. especially in ecological energetics (Phillipson, 1966). to express all parameters in comparable units (calories or Joules). Which of the two methods, direct bombcalorimetry or indirect computation from biochemical composition, should be preferred in such studies? The direct method appears to be simpler, but its results may generally be slightly biased by incomplete drying or combustion and may be not infrequently erroneous. Moreover, as reaction products at complete combustion differ from those at physiological heat production, a nitrogen correction (Kersting. 1972) should generally be applied, necessitating, at least. a partial chemical analysis. The indirect method is more cumbersome, but has as a clear advantage that in-
30
J. J. BEUKEMA
AND W. DE BRUIN
sight is gained into which materials are involved in processes like growth and starvation. Conversion factors, however, are not universal and preferably should be estimated separately, requiring some bomb-calorimetry. In conclusion : direct calorimetry for biological use is not complete without some chemical analysis and calculation of calorific values from biochemical composition cannot be used without some bomb-calorimetry.
REFERENCES BrclKINA.
J.J. & W. DF BRIJIN. 1977. Seasonal changes in dry weight and chemical composition of the soft parts of the tellinid bivalve Mucoma halrhica in the Dutch Wadden Sea. Ncvh. J. .‘+a Rer.. Vol. I 1. pp. 42-55. BFC’KEM,~. J. J.. G.C. CADGE & J. J. M. JANSFN. 1977. Variability of growth rate of Mucomtr /~/l/h& (L.) in the Wadden Sea in relation to availability of food. In. Biology of’hrnthic organisms. edited by B. F. Keegan, P. 0. Ceidigh & P. J. S. Boaden, Pergamon Press. Oxford. pp. 69-77. BLIGH. E.G. & W. J. DYFR. 1959. A rapid method of total lipid extraction and purification. CNII. J. Biochem. Physiol.. Vol. 37. pp. 91 I-9 17. BRODY. S.. 1945. Bioenwgckx andgrowth. Reinhold, New York. 1023 pp. CtiAMaFas. M. R. & H. MILNE. 1975. The production of Mncomrr haltl?icn (L.) in the Ythan estuary. Estuar. cstlmar. Sri.. Vol. 3. pp. 443-455. CRAIG. J. F.. 1977. The body composition of adult perch, Perca /luvirrtilis in Windermere. with reference to seasonal changes and reproduction. J. Anim. Ecol.. Vol. 46. pp. 617-632. GILBERT. M. A.. 1973. Growth rate. longevity and maximum size of Mac,oma halthicu (L.). Biol. Bull. war. hi&. Lab.. Woods Hole. Vol. 145. pp. 119-126. HANDEL. E. VAN. 1965. Estimation of glycogen in small amounts of tissue. Ana1J.r. Biochcm.. Vol. I I. pp. 256-~265. KrRsriNc;, K.. 1972. A nitrogen correction for caloric values. Limnol. Oceanogr.. Vol. 17. pp. 643-644. KRr as. H. A.. 1964. The metabolic fate of amino acids. In. Mammalian prorein metoholism. li)l. 3. edited by H. N. Munro & J. B. Allison. Academic Press. New York. pp. 125-176. MOROWITL. H. J.. 1968. Energ~,,flolc in hiolo~qy. Academic Press. New York. 179 pp. PAINL. R.T.. 1971. The measurement and application of the calorie to ecological problems. il. Rev. Ecol. ,Sw.. Vol. 2, pp. 145.-164. P~LDIAN. T. J.. 1967. Changes in chemical composition and caloric content of developing eggs of the shrimp Crangon crangon. Hc(qoliindw wi.s.v. Mwr~sunter.s.. Vol. 16. pp. 316-224. PHILLIPSO\. J.. 1964. A miniature bomb calorimeter for small biological samples. 0iko.y. Vol. 15. pp. 13&139. PHILLIPSOX. J.. 1966. Ecological ener~c/ic.c. E. Arnold. London. 57 pp. PLr-r-1. T.&B. IRWIN. 1973. Caloric content of phytoplankton. Limnol. Oceanogr.. Vol. IX. pp. 306 -310. SC HKOI-DI K. L.A.. 1977. Caloric equivalents of some plant and animal material. Orcokqia (Brrl.). Vol. 28. pp. 261-267. THZ\.TR, G. W.. W. E. S~HAAF. J. W. AhGr L.O~IC & M. W. LACROIX. 1973. Caloric measurements of some estuarine organisms. Fishrr~~ Bull. ntrm. mar. Fi.vh. Serv.. U.S.. Vol. 71. pp. 289 296. Wit DF, P.A. W. J. DE & E. M. BERGHUIS. 1978. Laboratory experiments on the spawning of !Mrrcomu hrritkicrr : its implication for production research. In. P/?~~sio&>~ L& hchaviour of’ marirw or~ani.sm.v. Proc. 12th Europ. mar. hiol. .S,~wq., edited by D. S. McLusky & A. J. Berry. Pergamon Press. Oxford. pp. 375 384.
CALORIFIC VALUES OF THE SOFT PARTS OF THE TELLINID BIVALVE MACOMA
(L.) AS DETERMINED BY TWO METHODS
BALTHZCA
J. J. Netherlands
BEUKEMA
and W.
DE BRUIN
Institute for Sea Research, Trxel. The Netherlands
Abstract: Calorific values of the ash-free dried soft parts of Macoma halrhica (L.), sampled monthly during 1 year at a tidal flat station in the Dutch Wadden Sea, have been estimated by two methods. The indirect method started from an analysis of the biochemical composition and used conversion factors for the various components to calculate the calorific value of the total tissue. The direct method employed a micro-bomb calorimeter and a furnace for separate ash determinations. The indirect method yielded estimates that were consistently higher than those from the direct one. Two sources, explaining most of the difference. could be traced, viz. incomplete drying of the samples used in the calorimeter and a too high conversion factor for weight to calories applied conventionally for lipids. This conversion factor was found to depend on the method of extraction of the lipids. but was always significantly below the commonly used 9.3 to 9.7 cal.mg-‘. The best estimates for the annual mean of the calorific value at complete combustion of the ash-free and well-dried soft parts of Macoma were 5.47 for the direct and 5.59 cal.mg-’ ash-free dry wt for the indirect method. Both values are higher than estimates for Macoma published earlier by various authors. Calorific values at physiological oxidation amount to only about 4.6 cal.mg-’ or less. due to incomplete protein oxidation. For this reason. it is concluded that biologically significant calorimetry cannot dispense with biochemical analysis. On the other hand, calculation of calorific values from biochemical composition should be accompanied by some calorimetry. as the conversion factor for the lipid fraction. at least. may deviate significantly from commonly applied values.
INTRODUCTION
As a part of the analysis of the functioning of an ecosystem, a study of the transfer of energy between populations is an important step. Accurate values for the energy content of the tissues of organisms are essential for such studies. Calorific values are commonly estimated in either of two ways: 1) by direct bomb calorimetry, or 2) by computation from results of a study of biochemical composition, using appropriate conversion factors for the various components. The first method is direct and exact in well-dried material that is relatively poor in ash (Paine. 1971). The values found need only minor corrections which may be omitted in many cases (Paine, 1971; Schroeder, 1977). The method does, however, yield energy values for complete combustion. For biologically more realistic heat values, based on protein metabolism, the nitrogen content of the material should be determined in addition (Kersting, 1972). The second method calls for a cumbersome complete biochemical analysis of the material. Moreover, there is reason to doubt whether the commonly used conversion 19
20
J.J.BEUKEMAANDW.DEBRUIN
factors. based on Brody (1945), viz. 4.1 kcal .g-’ for carbohydrate, 5.65 for protein, and 9.45 for lipid, are applicable to the specific material in question. All of these values are in fact averages of varying values found for pure compounds. Morowitz (1968) assumes the following mean values: 4.1,5.5, and 9.3 kcal-g-’ for carbohydrate, protein, and lipid, respectively. It cannot be taken for granted beforehand that any biological material will yield values close to these averages. The second method is, therefore, incomplete without some direct calorimetry and the first method needs some biochemical analysis. Although desirable, such complete studies appear to be rare. In fact, there appear to be only few sets of data on calorific values of biological material that allow a comparison of the results of different methods applied to the same material. In the great majority of papers on ecological energetics, results of only one method are accepted without any discussion of the probable bias. The aim of the following study, therefore, is to compare results obtained by the two methods, applied to the dried soft parts of Mucoma balthica (L.), to get some insight into the accuracy of the calorific values obtained. A Mucomu population has been sampled throughout the year in order to have access to material with varying contributions of the basic compounds (Beukema & De Bruin, 1977).
MATERIALANDMETHODS
At an intertidal station in the westernmost part of the Dutch Wadden Sea, about 100 adult Mucomu were sampled 15 times at approximately monthly intervals during the period June 1974 to July 1975. The sampling station is close to Station 4 in Fig. 1 in Beukema et al. (1977). At this station growth rate and condition of Mucomu are close to the average for the whole intertidal Balgzand area (Beukema et al., 1977). The approximately monthly sampled Mucomu were used for determinations on growth rate, condition, and chemical composition, as described in Beukema & De Bruin (1977). In short, the animals were stored alive in running sea water for one day and then killed by briefly immersing in boiling water. The soft parts were dried for 3-5 days at 60°C. Part of the dry material was used for determinations of ash content, i.e., the weight left after 2 h in a furnace at 500-600 “C (the amounts of ash recovered from the calorimeter were found to be only 60-80x of the amounts found by these separate furnace ashings). Most of the material was used for chemical analysis after pulverizing and homogenizing and short (generally less than one month) storage at -20°C. Lipid content was measured by Soxhlet refluxing extractions with various solvents (either pentane, chloroform or chloroform-methanol). Chloroform yielded consistently higher (by 30% in summer to 80% in winter and early spring) amounts of lipids than pentane, and even slightly higher amounts than the chloroform-methanol mixture recommended by Bligh & Dyer (1959).
CALORIFICVALUESOFMACOMA
21
The lipid-free fraction was dried again and used for determinations of glycogen (by the calorimetric anthrone method according to Handel, 1965) and nitrogen (by a standard micro-Kjeldahl procedure, with 6.25 as a multiplication factor to obtain protein figures). Caloritic values were obtained by burning dried 2&35 mg pelleted samples of the lipid and lean fractions in a Gentry-Wiegert micro-bomb calorimeter (described in Phillipson, 1964) using benzoic acid as a standard. The values obtained were corrected for ash content (as determined separately) and are thus expressed in cal .mg -’ ash-freedryweight (ADW). Foreachofthe 15 sampling dates, the material was divided into l-3 lots, each delivering 4-5 pellets. Depending on the amount of material available, 416 pellets from each sampling date were burned. Corrections for acid production and endothermic reactions within the bomb were omitted, because these were expected to be insignificant at the sample size used and the low-ash contents observed (Paine, 1971; Schroeder, 1977).
RESULTS BIOCHEMICAL COMPOSITION
The composition of the soft parts of Macoma undergoes an annual cycle (Beukema & De Bruin, 1977). Glycogen rapidly increases during the short growing season (late March to mid June) and declines during the remainder of the year, especially during the July-December period. Lipids do roughly the same, but build up less rapidly during the second half of the growing season, probably as a consequence of simultaneous losses as a result of spawning. Proteins, though increasing in absolute amounts during the growing season, decrease as a percentage during this period, due to the more rapid increase of glycogen and lipids. During the remainder of the year, proteins are used up at a lower rate than glycogen, resulting in an increase of the relative share of proteins during the second half of the year. Fig. 1 shows the seasonal changes in the proportions of protein, glycogen, and lipids during the 19741975 sampling period. Comparison with Fig. 4 in Beukema & De Bruin (1977) shows that the cycles observed during this year hardly differed from those found during the preceding year. As lipids and glycogen differ greatly in caloritic value, such cycles will affect the stability of the calorific value of the entire tissues. The fluctuations in the shares of these two components, however, run more or less parallel during most of the year (Fig. 1). Consequently, the seasonal changes in the calorific values of Macoma tissues will be limited. Summation of the three main components yielded totals varying from X8-101% (mean 94% of the ADW). The mean deficit of 6% would have consisted almost completely of free sugars and tightly bound water. The material dried at only 60°C would have contained 2 or 3% of water (Beukema & De Bruin, 1977).
J. J. BEUKEMA
22
AND W. DE BRUIN
,I ,a
,.nn:
e IO
n
d
/
f
m
on
I
,~ Dther
~dCflCI1~
I
,975
1974
Fig. I, Annual cycle of the main components of the ash-free dried soft parts of Macomu, expressed as percentages of ADW: protein from 6.25 x N-content; glycogen from calorimetric anthrone estimates; lipids from weighing chloroform extractions; figures for ‘other’ material obtained by difference: note the change of vertical scale between 35 and 450,,.
CALORIFIC
VALUES
OF THE MAIN
COMPONENTS
Lipids
Samples of the lipids extracted by various solvents were burned in the microbomb calorimeter and yielded rather different results (Table I). Pentane extracted the lipids incompletely, but the lipids obtained in this way were of a relatively high calorific content (9.1 cal.mg-‘). Apparently these lipids consisted of a high proportion of long-chain fatty acids that have a calorific value of about 9.3 calsmg- (Morowitz, 1968). Other lipids, not soluble in pentane, but
CALORIFIC
VALUES
23
OF MACOMA
extracted subsequently by chloroform, yielded only 7.4 calmmg -I. This fraction would have contained a higher proportion of short-chain fatty acids and impurities. As expected, the calorific value of the total chloroform extract as calculated from the two fractions (0.25 x 7.38 + 0.75 x 9.10 = 8.67) fits the value found directly (8.62. see third line in Table I). Wet extraction according to Bligh & Dyer (1959) yielded estimates only slightly different from the chloroform extraction of dried material, in accordance with the almost equal amounts of material extracted.
of lipid fractions of soft parts of M~comu, as extracted by various solvents: Calorific values in cal.mg-’ mean values of samples obtained during the May-October period; chloroform-methanol extraction according to Bligh & Dyer (1959) from wet material. other extractions from dried and pulverized material. Mean cal.mg-’
Solvent Pentane Chloroform Chloroform Chloroform
after pentane + methanol
9.10 7.38 8.62 8.42
95” conf. Ii&its 8.98-9.23 7.32-7.44 8.5G8.74 8.22-8.62
No. of samples 7 5 12 IO
Proportion extracted 0.75 0.25 1.00 0.90
In the following, the chloroform-extraction values will be used, as chloroform was found to extract the lipids most completely. As a matter of course. the appropriate calorilic value of 8.62 will be applied. Lean material
Caloritic values of the dried material remaining after lipid extraction and corrected for ash content, were found to vary between 4.74 and 5.17 calvmg-’ ADW (Fig. 3: open squares). Highest values were found at the start and lowest values at the end of the growing season. During this season the share of glycogen increases at the cost of that of protein (Fig. 1). As glycogen has a lower calorific value (about 4.1 calemg -‘) than protein (about 5.65 cal.mgm’ at complete combustion), such seasonal change is to be expected. Calorific values of the lean material were indeed positively correlated with the protein content (Spearman’s Y= +0.69; P < 0.01) and negatively with that of glycogen (Y= -0.75, P < 0.001). All calorific values found for the lean material are well below the values expected if this material consisted only of protein and glycogen with calorific values of 5.65 and 4.10 calemg-‘, respectively (Fig. 2). On an average, the calorific values observed are 0.3 to 0.4 calamg- or about 67% lower than the expected ones. Several reasons for this discrepancy may be suggested. In the first place, incomplete combustion and incomplete drying should be considered. It is clear that incomplete combustion could explain any negative difference between observed
24
J. J. BEUKEMA AND W. DE BRUIN
and expected values. Burning off the remaining ash for 1 or 2 h at 600°C resulted in a mean weight loss of only 0.33% of the dry weight of the original pellet (957; confidence limits 0.27 and 0.39, n = 10). Such weight loss would only add the negligible amount of about 0.02 cal .mg-’ to the calorific value. There is no reason to correct for this error, as it compensates for the acid correction which was not applied and which would have been about -0.02 calemg -I at the sample size used (Schroeder, 1977).
Fig. 2. Calorific values (Cal.mg -’ ADW), with 95% confidence limits (n = 4 or 5), for 26 lots of lean material, sampled on 15 dates throughout the year, and plotted against glycogen content of the same material: solid line, the expected values if all the lean and ash-free non-glycogen material were protein with a calorific value of 5.65 caiemg-’ and if the cabrific value of the glycogen is 4.10 calqmg -’ ; broken line shows best fit by geometric mean regression (n = 26).
Incomplete drying may have been a more serious source of error. Further drying of lean pellets (already routinely dried for three days at 60°C) at various higher temperatures for three days resulted in weight losses of 3% at 100°C and 3.2% at 710°C percentages of ash-free dry weight of lean material). A water content of 3% moved the points in Fig. 2 by 0.15 to 0.17 Cal-mg-’ downwards and by about 1% glycogen to the left as compared with data (not available) for more completely dried material. Thus, about half of the observed discrepancy can be explained by the
CALORIFIC
VALUES
OF MACOMA
25
remaining water content of the dried material (the reason why we dried at only 60 “C was for a possibly unfounded fear of unnoticed disappearance of volatile substances). The still unexplained difference of, on average, x3% between the expected and observed calorific values shown in Fig. 2 may be due to wrong assumptions used in calculating the expected values. The sum of the protein and glycogen accounted for a mean of 94% of the ash-free dried lean material. Half of the remaining 6% will have been water. The other half may have had a calorilic value lower than the 5.65 calemg-’ used in the calculations of the expected values. If all of the other 3% were free sugars with a caloritic value of about 4 calsmg-‘, only about 0.05 cal mg-’ or one third of the still unexplained part of the difference would be explained. There is thus some room left for other errors, e.g., overestimation of the protein content. An unknown part of the nitrogen found will have been non-protein material with a probably lower calorific value than the supposed 5.65 cal .mg-‘.
ESTIMATES
OF THE CALORIFIC
VALUE
OF MACOMA
TISSUES
At complete combustion
A naive calculation, starting from the biochemical composition (Fig. l), and using the commonly applied conversion factors for calamg-’ of 9.45 for lipids, 5.65 for proteins and 4.10 for carbohydrates, and considering all other substances like free sugars as carbohydrates with a calorific value of 4.10, yields figures between 5.5 and 6.0 calwmg-’ (solid circles in Fig. 3). An annual mean calculated from these figures amounts to 5.68 calamg-’ ADW. The highest value is just before the onset of spawning, reflecting the high lipid/glycogen ratio at that time (Fig. 1). The subsequent decrease till the end of the growing season reflects the rapid rise of the share of glycogen at a constant or falling percentage of lipids. The above figures will be maximal ones and can easily be challenged: the conversion factor for at least one of the components (viz. the lipids, see Table I) is too high and the assumption that the ‘other substances’ consist of carbohydrates only is unrealistic. Some corrections can be applied to make these figures more realistic. Assuming that the ‘other substances’ consist of water for a percentage up to 3% of the ADW, the annual mean of the calorific values decreases to 5.57 calsmg-’ ADW. When the conversion factor for lipids is lowered from 9.45 to 8.62 cal.mg-‘, the calorific value of the material decreases to 5.59 calvmg- ADW. Note that the term ADW has been used here with two different meanings, viz. without any water (the 5.68 and 5.59 figures) and with the water remaining after drying at 60°C (the 5.57 figure). Application of both corrections yields an estimate of 5.48 calemg-’ for the ash-free soft parts of Macoma, dried at 60°C (Table II). Starting from the bomb calorimetry data (Table I and Fig. 2) and using the data included in Fig. 1 for the share of the lipids, figures between 5.26 and 5.42 calamg-’ were found for the calorific value of the ash-free, 60°C dried, soft parts of Macoma
26
J. J. BEUKEMA
AND W. DE BRUIN
Fig. 3. Annual cycle of the calorific values (cal.mg -’ ADW) of the ash-free and dried soft parts of Mrrcotna. as obtained by different methods (see text): growing season (from Beukema & De Bruin, 1977) and spawning season (from De Wilde & Berghuis. 1978. Fig. 6. for 1975) indicated at the top of figure.
TABLE Estimates
pressed
Annual Method
II
of the caloritic value of the dried soft parts of Macomcr halrlzica by various methods. exin cal.mg-’ ADW. with separate data for ‘complete’ drying and drying at only 60 C; annual means from n sampling dates.
(\ee also text)
n
dried at 60 C
Bvxh.
camp..
convent.
I5
Bioch.
camp..
O-3”” water
I5
Bioch.
camp..
lipid
corr.
I5
Bioch.
camp..
both con.
I5
5.4X
Bomb
calonmetry
I5
5.33
Bomb
cal.. water-free
15
mean dried completely 5.68
5.57 5.59
5.47
95” 0 conf.
limits
Refcrencc
paper
5.61-5.75
This
5.49-5.65
This paper
5.51-5.67
This paper
5.41-5.56
This paper
5.30. 5.36
Thk
5.43-5.50
This paper
4.65-4.90
Chambers
paper
Bomb
calonmetry
9
4.77
Bomb
calorunetry
8
4.05
Gilbert
(1973)
Bomb
calonmetry
2
5.08
Thayer
~‘f 111. 1973)
& M~lne (1975)
I
CALORIFIC
VALUES
OF MACOMA
27
(open circles in Fig. 3). The annual mean amounts to 5.33 calsmg-‘. To obtain the corresponding value for thoroughly dried material, the calorilic values observed for the lean material should be increased by about 3:/,. The resulting annual mean for the total material amounts to 5.47 calsmg-‘. All of the above values are higher than those found by other authors for the calorific value of the ash-free and dried soft parts of Macoma balthica (Table 11). Our mean values are ~0.35 calemg-’ or w6:/, of the mean. and when compared those obtained from bomb calorimetry are consistently lower than the corresponding values calculated from the biochemical composition (Table II). The largest difference (5.68-5.33) may be explained by incomplete drying, a conventional conversion factor which is too high for lipids, and unknown factors (discussed on p. 25) each of which contributes about equally to the difference. For thoroughly dried soft parts of Macoma. the true calorilic value will be within the limited range of 5.47 to 5.59 calsmg-’ ADW, because both the drying correction for the samples used in bomb calorimetry and the corrected conversion factor for lipids are justified. At phJ>siologicaloxidation The value of biological material as a source of energy for living organisms should not be estimated from heat production at complete combustion. because living organisms cannot oxidize proteins completely (Krebs, 1964; Kersting, 1972). Part of the carbon skeleton of amino acids is not burned. Moreover, end products of nitrogen oxidation are not N2, but such products as uric acid (e.g., in birds). urea (e.g., in mammals) or ammonia (e.g.. in many marine invertebrates). The caloritic value of proteins (and consequently of most biologicdl materials) at physiological oxidation is, therefore, lower than that found by bomb calorimetry (usually ~5.7 calsmg-‘). Kersting (1972) calculates minimal nitrogen corrections, taking into account only incomplete N-oxidation. These corrections amount to z -0.9 calsmg-’ protein in the case of NH, as an end product and to - 1.4 calsmg-’ protein in the case of urea (the latter correction should be 8.5 cal mgm’ N instead of the stated 7.2, K. Kersting, pers. comm.). Physiologically realistic calorific values for animal protein are thus not higher than 4.3 to 4.8 calmmg-’ protein, and probably still lower by ~0.5 calemgdue to incomplete oxidation of C and H (Krebs, 1964). Such corrections may be applied to the data obtained by bomb calorimetry because the protein content of the dried soft parts of Macoma have been estimated routinely (Fig. 2). A correction of - 1.4 calsmg- protein will be applied to the calorimetry values corrected for incomplete drying. A protein correction of this magnitude may be thought to represent either an oxidation of N to urea (with complete oxidation of the C and H) or an oxidation of N to NH, with incomplete oxidation of the C and H.
28
J. J. BEUKEMA
AND W. DE BRUIN
The resulting ‘physiological’ caloritic values for the ash-free and thoroughly dried soft parts of Mucoma range from 4.3 to 4.8 calsmg -’ ADW (lower line in Fig. 3) with a yearly average of 4.57 calsmg-’ ADW. This is 0.90 calemg-’ or 16% less than the corresponding value at complete combustion by bomb calorimetry (Table II). This difference is both larger and of more fundamental importance than the differences shown by the distances between the two top lines in Fig. 3, representing extreme outcomes of various ways of estimating caloritic values at complete combustion. Still lower calorific values should be used for the soft parts of Macoma if the end product of N-oxidation is urea or uric acid and also the oxidation of C and H is incomplete. In fact, no invariably applicable physiological heat value for protein containing material can be’stated, as it will depend on the metabolic capacities of the oxidizing organism. DISCUSSION
Calorific values for biological material that are calculated from the biochemical composition, using commonly applied conversion factors for calsmg-’ (viz. 9.45 for lipids, 5.65 for proteins and 4.10 for carbohydrates), may be overestimates in many cases. The conversion factor for lipids, especially, should often be lower than 9.45, as the lipid fractions extracted by the usual methods contain a lot of impurities. The less lipid we extracted (by choosing more selective solvents), the higher was its caloritic value (Table I). Craig (1977) also observed calorific values lower than 9.45, viz. 8.49 calsmg-’ for lipids extracted by chloroform-methanol from tissues of perch. This tigure agrees well with our value of 8.42 obtained by the same methods (Table I). By using a conversion factor of 9.45 instead of 8.62 for the lipid fraction, the calorific value of Mucoma tissue is overestimated by 1 or 2%, depending on the season (Fig. 1). The generally low share of lipids in the tissues of bivalves will keep this error relatively small in calculated calorific values for the tissues of these animals. Actually, the difference between the calculated and calorimetrically estimated heat values in Mucoma were greater, viz. on an average 6% (Table II: 5.68 compared with 5.33). About one third of this difference could be explained by the incomplete drying of the pellets used for the calorimetry. Thus about one third of the difference or only x2% of the calorific values remains to be explained, e.g., by too high a conversion factor for protein. In the literature published on caloritic values, we found only a few comparions between the results of calculation from biochemical composition and direct calorimetry. Craig (1977) observed a significant difference of 7.2% in favour of the former method. He considers that the calculated value is an overestimate due to the too high conversion factor for lipids discussed above.
CALORIFIC
VALUES
OF MACOMA
29
Pandian (1967) measured calorific values by bomb calorimetry in various stages of developing eggs of Cvangon and also determined percentages of ash. protein and non-protein nitrogen in the same material. He assumed the remainder to be fat with a caloric value of 9.4 calsmg- and adopted 5.65 calmmg- as a conversion factor for protein to calories. With these conversion factors, we calculated caloritic values for the various stages of the eggs (omitting the small fractions of non-protein nitrogen) and found figures which were 7-18:; higher than the ones Pandian had observed by direct bomb calorimetry. Platt & Irwin (1973) measured calorific values by bomb calorimetry in samples of phytoplankton and analyzed the same material for carbohydrate. protein. lipid, and ash. With conversion factors of 4.2 for both proteins and carbohydrates and 9.5 for lipids. they obtained good agreement between measured and calculated values. With a more realistic conversion factor of 5.65 instead of 4.2 for complete combustion of proteins (and, therefore, for comparisons with results from bomb calorimetry). we recalculated their figures and found values which were 2-20”{, higher (on average 11%) than the measured ones. As in the case of Pandian (1967), such difference could be reduced significantly by the use of lower conversion factors for lipids. A separate estimate of the calorific value of at least the lipids appears to be a prerequisite for a valid use of the calculation method. A high proportion of the published calorific values for bivalve tissues obtained by bomb calorimetry are unexpectedly low. Though calorific values of bivalve tissues will be rather low due to the frequently low lipid and high carbohydrate content, values below 5.0 calemgg ADW would be improbable. With a lipid content rarely below 55’6and a carbohydrate content rarely exceeding 50”/1, a lower limit to the calorilic value for the soft parts of bivalves may be estimated at about 0.05 x 8.5 + 0.45 x 5.6 + 0.50 x 4.1 = 5.0cal.mg-’ ADW. Observations significantly below this value are suspect. They might result from such errors as incomplete drying, incomplete combustion, or incorrect determination of ash content. Paine (1971) gives a more complete discussion of sources of error in calorimetry. The direct method appears to yield under-estimates more commonly than over-estimates, just the reverse of the calculation method. A biochemical analysis of the material in question may suggest such an error in calorimetry. Calorific values of biological materials are used mainly in bioenergetical studies. especially in ecological energetics (Phillipson, 1966). to express all parameters in comparable units (calories or Joules). Which of the two methods, direct bombcalorimetry or indirect computation from biochemical composition, should be preferred in such studies? The direct method appears to be simpler, but its results may generally be slightly biased by incomplete drying or combustion and may be not infrequently erroneous. Moreover, as reaction products at complete combustion differ from those at physiological heat production, a nitrogen correction (Kersting. 1972) should generally be applied, necessitating, at least. a partial chemical analysis. The indirect method is more cumbersome, but has as a clear advantage that in-
30
J. J. BEUKEMA
AND W. DE BRUIN
sight is gained into which materials are involved in processes like growth and starvation. Conversion factors, however, are not universal and preferably should be estimated separately, requiring some bomb-calorimetry. In conclusion : direct calorimetry for biological use is not complete without some chemical analysis and calculation of calorific values from biochemical composition cannot be used without some bomb-calorimetry.
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J.J. & W. DF BRIJIN. 1977. Seasonal changes in dry weight and chemical composition of the soft parts of the tellinid bivalve Mucoma halrhica in the Dutch Wadden Sea. Ncvh. J. .‘+a Rer.. Vol. I 1. pp. 42-55. BFC’KEM,~. J. J.. G.C. CADGE & J. J. M. JANSFN. 1977. Variability of growth rate of Mucomtr /~/l/h& (L.) in the Wadden Sea in relation to availability of food. In. Biology of’hrnthic organisms. edited by B. F. Keegan, P. 0. Ceidigh & P. J. S. Boaden, Pergamon Press. Oxford. pp. 69-77. BLIGH. E.G. & W. J. DYFR. 1959. A rapid method of total lipid extraction and purification. CNII. J. Biochem. Physiol.. Vol. 37. pp. 91 I-9 17. BRODY. S.. 1945. Bioenwgckx andgrowth. Reinhold, New York. 1023 pp. CtiAMaFas. M. R. & H. MILNE. 1975. The production of Mncomrr haltl?icn (L.) in the Ythan estuary. Estuar. cstlmar. Sri.. Vol. 3. pp. 443-455. CRAIG. J. F.. 1977. The body composition of adult perch, Perca /luvirrtilis in Windermere. with reference to seasonal changes and reproduction. J. Anim. Ecol.. Vol. 46. pp. 617-632. GILBERT. M. A.. 1973. Growth rate. longevity and maximum size of Mac,oma halthicu (L.). Biol. Bull. war. hi&. Lab.. Woods Hole. Vol. 145. pp. 119-126. HANDEL. E. VAN. 1965. Estimation of glycogen in small amounts of tissue. Ana1J.r. Biochcm.. Vol. I I. pp. 256-~265. KrRsriNc;, K.. 1972. A nitrogen correction for caloric values. Limnol. Oceanogr.. Vol. 17. pp. 643-644. KRr as. H. A.. 1964. The metabolic fate of amino acids. In. Mammalian prorein metoholism. li)l. 3. edited by H. N. Munro & J. B. Allison. Academic Press. New York. pp. 125-176. MOROWITL. H. J.. 1968. Energ~,,flolc in hiolo~qy. Academic Press. New York. 179 pp. PAINL. R.T.. 1971. The measurement and application of the calorie to ecological problems. il. Rev. Ecol. ,Sw.. Vol. 2, pp. 145.-164. P~LDIAN. T. J.. 1967. Changes in chemical composition and caloric content of developing eggs of the shrimp Crangon crangon. Hc(qoliindw wi.s.v. Mwr~sunter.s.. Vol. 16. pp. 316-224. PHILLIPSO\. J.. 1964. A miniature bomb calorimeter for small biological samples. 0iko.y. Vol. 15. pp. 13&139. PHILLIPSOX. J.. 1966. Ecological ener~c/ic.c. E. Arnold. London. 57 pp. PLr-r-1. T.&B. IRWIN. 1973. Caloric content of phytoplankton. Limnol. Oceanogr.. Vol. IX. pp. 306 -310. SC HKOI-DI K. L.A.. 1977. Caloric equivalents of some plant and animal material. Orcokqia (Brrl.). Vol. 28. pp. 261-267. THZ\.TR, G. W.. W. E. S~HAAF. J. W. AhGr L.O~IC & M. W. LACROIX. 1973. Caloric measurements of some estuarine organisms. Fishrr~~ Bull. ntrm. mar. Fi.vh. Serv.. U.S.. Vol. 71. pp. 289 296. Wit DF, P.A. W. J. DE & E. M. BERGHUIS. 1978. Laboratory experiments on the spawning of !Mrrcomu hrritkicrr : its implication for production research. In. P/?~~sio&>~ L& hchaviour of’ marirw or~ani.sm.v. Proc. 12th Europ. mar. hiol. .S,~wq., edited by D. S. McLusky & A. J. Berry. Pergamon Press. Oxford. pp. 375 384.