Jul 10, 1972 - TREVOR PLATT. The standard error of the estimate is 0.154. BRIAN. IRWIN cal mg-l ( r2 ... Bedford Institute of Oceanography most other ...
306
NOTES
of the backwaters around Willington Island (Co&in). J. M ar. Biol. Ass. India 5: 170177. SHAH, N. M. 1970. Studies on seasonal variation of phytoplankton pigments in the Laccadive Sea off Cochin. Ph. D. thesis, Univ. Kerala. 92 p.
R. 1960. Observations on the effect of the monsoons in the production of phytoplankton. J. Indian Bot. Sot. 39: 7S89.
SUBRAH~XANYAN,
Submitted: 10 July 1972 Accepted: 1 December 1972
Caloric content of phytoplankton ABSTRACT
There are important theoretical and empirical reasons for expressing calorific values in temls of grams of carbon rather than grams dry weight. For phytoplankton, calorific value may be predicted with fairly high accuracy from carbon content, enabling much smaller samples to be used and resulting in a considerable saving in time. This removes the biggest stumbling block to the routine measurement of the energy efficiency of primary production.
A study of the flux of energy is an important step in the analysis of the dynamics of an ecosystem. Toward this end, much effort has been expended in measuring the caloric content of the tissues of organisms. Generalized food chain calculations, of the type made by Ryther ( 1969) for the potential productivity of the oceans, rely heavily on the use of standard conversion factors for interpretation of biomass in terms of calories. The range of observed values for such conversions is from 3,300 to 9,400 cal g-l of ash-free dry weight ( Cummins and Wuycheck 1971)) a threefold variation. Clearly, for generalized calculations, any method that reduces the spread in these conversions is of some interest. Our purpose here is to point out that, for both theoretical and empirical reasons, the possible range of conversion factors is much reduced if caloric values are expressed in terms of grams of carbon rather than grams dry weight, and in addition that the calorific value of a tissue sample may be predicted with fairly high precision from its carbon content. The latter point can be of considerable significance during field studies on very small organisms, such
as phytoplankton, for which it is difficult to collect samples suitable for caloric analysis. METHODS
Samples were collected during the spring phytoplankton bloom of 1969 in St. Margaret’s Bay, Nova Scotia (44” 35’ N, 64” 02’ W), 1.6 km from the shore in 53 m of water (see Platt and Subba Rao 1970). On 10 occasions during the bloom, phytoplankton was collected by towing horizontally at 5 m a net 0.5 m in diameter with mesh size 76 p. The samples were washed with filtered seawater through a nylon sieve (mesh size 153 p), collected on a 76-p nylon mesh, rinsed briefly in distilled water, transferred to a plastic bag, frozen immediately on Dry Ice, and stored in the dark at -20°C. After the frozen samples had been lyophilized, the dry material was stored in sealed glass vials in a desiccator at -20°C. Between 0.5 and 2.0 g of dry material were recovered from each sample. The abundance of zooplankton in St. Maragaret’s Bay was low during this study. This, together with the fact that fresh samples had been screened through the fine mesh, ensured us a series of dried phytoplankton samples essentially uncontaminated by zooplankton, a point which was checked by suspending some of the freeze-dried material in cedar oil and looking at it under the microscope. The dried material was analyzed as shown in Table 1. RESULTS
AND
DISCUSSION
The chemical characteristics of the phytoplankton did not remain constant throughout the spring bloom ( Table 2) ;
NOTES
Table
1.
Summary
Sample (mg)
Quantity
of technical
Units (dry
wt.)
307
data for the analytical
procedures
followed
No. replicates per sample
Primary standard
Method
Carbohydrate
20-50
%
3
Dextrose
Raymont et al. 1964
Protein
20-50
%
3
Bovine albumen
Itzahaki and Gill 1964
Lipid
20-50
%
2
Stearic acid
Bligh and Dyer 1959; Pande et al.
Ash
20-50
%
2
Calorific value
10-20
Cal/mg
2
Benzoic acid
Phillipson micro bomb calorimeter
'L5
%
2
Cyclohexane 2:4 dinitrophenylhydrazone
F&M model 185 C,H,N analyzer
1963
Carbon and nitrogen
Ashing at 500°C to constant weight
Table 2. Analyses of samples of dried phytoplankton
Date Apr/69
1 3 8 10 14 16 18 22 24 28 anic
Carbohydrates %
Protein
Lipid
Ash
%
%
%
14.2 14.1 19.7 20.8 30.3 27.6 33.3 28.7 22.4 28.7
25.1 25.0 17.8 17.4 14.7 16.3 13.6 16.3 20.1 18.7
16.5 19.0 16.1 12.1 3.4 3.5 8.1 6.5 3.5 11.3
*For the vast majority of plankton (see Curl 1962). carbon”
38.6 33.8 41.8 43.7 50.1 50.2 48.4 46.6 50.5 44.7 samples
Carbon* % 32.2 34.7 30.0 24.9 16.5 19.0 18.6 24.9 18.7 23.8 I’% carbon”
Nitrogen
C:N
Cal/mg dry wt
6.0 5.5 5.8 8.2 12.8 12.2 17.2 10.3 10.2 9.1
3.529 3.746 3.121 2.578 2.062 2.278 2.538 2.515 2.151 2.799
% 5.36 6.33 5.20 3.04 1.29 1.56 1.08 2.41 1.83 2.61
is synonymous with
“:i org-
308
NOTES
Table 3. Comparison of measured and calculated (A-calculated from culated from simple regression; C -calculated from multiple regression)
Date Apr
Measured
A
B
proximate composition; B-calcalorie values for phytoplankton
C
69
1
3.529
3.223
3.411
3 8 10
3.746 3.121 2.578
3.445
3.627 3.221
14
2.062 2.278
3.103 2.750
16 18 22 24
2.538 2.515 2.151
2.218 2.176 2.740 2.502 2.112
28
2.799
3.059
the relative proportions of carbohydrate and ash and the ratio of carbon to nitrogen went through maxima whereas protein, lipid, and calorific value went through minima. From the proximate composition of the phytoplankton we can calculate the expected calorific value using the following conversion factors (Prosser and Brown 1961) : for protein 4.19 cal mg-l, for carbohydrate 4.2, and for fat 9.5 (Table 3). The agreement between these calculated values and the measured values is reasonably good (T = 0.95). The absolute values of caloric content per ullit carbon given in Table 3 are lower than those given in Platt and Subba Rao (1970). Those earlier values were based on carbon determinations by the wet oxidation method, which underestimates the carbon available. However, the relative changes in calories they noted and the conclusions they drew remain valid, The coefficient of variation among the calorific values is reduced from 20% (X = 2.732) when they are expressed in terms of dry weight to 10% (2 = 11.403)
3.422 3.678
Cal/mg C measured
10.969 10.800 10.412
2.701
3.162 2.713
10.360
2.056 2.272
2.010 2.261
12.483 11.974
2.237 2.781 2.246
2.487 2.827 2.119
13.664 10.107 11.495
2.686
2.637
11.770
when they are expressed in terms of weight of carbon (Table 3). The reason for this, as may be checked by simple arithmetic, is that, on the average, the carbon content of lipid is about 1.8 times higher than that of carbohydrate and of protein. Thus, whereas the caloric content of lipid per unit dry weight is about 2.3 times that of carbohydrate and of protein, the caloric content per unit carbon of lipid is only 1.3 times that of carbohydrate and protein. The theoretically possible range of conversion factors for expressing grams of organic carbon in terms of calories is much narrower, therefore, than the corresponding range for grams dry weight. This leads to the hypothesis that the percent carbon in dry tissue should be a good predictor of its calorific value. For the samples of dry phytoplankton described in Table 2 we find the following equation wg. 11, cal mg dry wt-l = 0.632 + 0.086 ( %C ) . The standard error of an estimate from this equation is 0.181 cal mg-l (+ = 0.91).
309
NOTES
ple, taken with a net, and involves washing, freeze-drying, and sensitive weighing. Again, in primary production studies using the 14C technique the experimental results are calculated directly in terms of grams of carbon produced per unit time. The simplest way to convert those to energy units is by means of a factor relating carbon to calories. This is particularly important in calculating, for example, the efficiency index kb (Platt 1969) which relates phytoplankton production (in energy units) to the available radiant energy. Finding the caloric equivalent of the material produced by the phytoplankton is currently the biggest obstacle to the use of the coefficient kb either as a predictive tool or as an index of comparison between difCarbon % by weigh1 ferent waters. Within the range of their Fig. 1. Relationship between percent of carbon validity, the regressions given here permit and calorific value measured on phytoplankton the prediction of caloric equivalents with samples. fairly high accuracy. The precision could be improved by making a much more extensive survey of the relationship between If the nitrogen content is also known, calories, carbon, and nitrogen in phytothis equation can be refined by allowing plankton. for the relative proportions of carbon and A useful general conversion factor for nitrogen, which reflect the proximate comphytoplankton is 1 mg carbon = 11.40 calposition of the tissue. We find ories ( Table 3). cal mg dry wt-l = -0.555 + 0.113 (%C) + TREVOR PLATT 0.054 (C:N). The standard error of the estimate is 0.154 cal mg-l ( r2 = 0.94). There seems no reason why similar relationships should not hold between the carbon and caloric contents of the tissues of most other organisms. We can therefore recognize valid theoretical and empirical reasons for expressing caloric contents in terms of weight of carbon. There are also important operational Carbon content is much considerations. easier to determine than caloric content, especially when a C, H, N analyzer is available, Only 100 pg of sample is required for a carbon analysis compared to about 10 mg for a calorie measurement. Thus, at least in coastal waters, 1 liter of seawater filtered onto a silver filter is sufficient for carbon determination. A caloric determination requires a much bigger sam-
BRIAN
IRWIN
Fisheries Research Board of Canada Marine Ecology Laboratory Bedford Institute of Oceanography Dartmouth, Nova Scotia REFERENCES BLIGH,
E. G., AND W. J. DYER. 1959. A rapid method of total lipid extraction and purifi37: 911cation. Can. J, Biochem. Physiol.
917. K. W., AND J, C. WUYCHECK. 1971. Caloric equivalents for investigations in ecoMitt. Int. Ver. Theor. logical energetics. Angew. Limnol. 18. 158 p. of carbon in marine CURL, H. 1962. iinalyses plankton organisms. J. Mar. Res. 20: 181188. ITZAHAKI, R. F., AND D. M. GILL. 1964. A micro-Biuret method for estimating proteins. Anal. Biochem. 9: 401-404.
CUMMINS,
310
NOTES
PANDE, S. V., R. PARVIN KHAN, AND T. A. VENKITASVBRAMANIAN. 1963. Microdetermination of lipids and serum total fatty acids. Anal. Biochem. 6: 415-423. PLATT, T. 1969. The concept of energy efficiency in primary production. Limnol. Oceanogr.
14: -,
653-659.
AND D. V. SUBBA RAO. 1970. Primary production measurements on a natural plankton bloom. J. Fish. Res. Bd. Can. 27: 887-899.
PROSSER, C. L., AND F. A. BROWN. 1961. Comparative animal physiology, 2nd ed. Saunders. RAYMONT, J. E. G., J. AUSTIN, AND E. LINFORD. 1964. Biochemical studies on marine zooplankton. 1. J. Cons., Cons. Perm. Int. Explor. Mer 28: 354-363. RYTHER, J. H. 1969. Photosynthesis and food production in the sea. Science 166: 72-76.
Submitted: 16 August 1972 Accepted: 28 November 1972
An evaluation of liquid scintillation counting techniques for use in aquatic primary production studies1 ABSTRACT Methods evaluated for the liquid scintillation counting of samples from aquatic primary production studies include the counting of intact filters and cells in a toluene fluor, solubilization and counting of membrane filters and cells in various reagents, and counting of a suspension of cells mixed with the fluor. A 1: 1 Triton X-1OO:toluene fluor was very effective for the suspension of cells and gave high counting efficiencies. Up to 5 ml of sample could be mixed with the fluor, but this was considered inadequate for most primary production studies. The filter standardization method for the counting of intact filters was investigated further and found to be applicable over a wide range of conditions but only accurate when the weight of algae on the filters was small (< 1 mg). Direct solubilization of the filters and cells in a naphthalene-dioxane or 2-methoxyethanoltoluene fluor were the simplest, most accurate, and economical methods for primary production studies; the latter dissolved both wet and dry cellulose nitrate membrane filters and gave excellent replicate counting.
The use of l”C-tracer techniques in aquatic primary production studies has increased as a result of the need for more accurate measurements of the rate of production, both in situ and in the laboratory, The radiocarbon taken up by phytoplankton was originally measured (e.g. Steemann ’ This work was carried out under the terms of Grant NO. GR/3/644 from the Natural Environment Research Council made to Prof. G. Chapman.
Nielsen 1952) with Geiger-Miiller counters, which generally had low counting efficiencies; often the results were difficult to standardize. When attempts were made to increase the counting efficiences of the system, as by using windowless counters and very thin samples, the counting efficiency was higher than predicted by the normal zero extrapolation procedure (Jitts and Scott 1961), and this standardization method was thrown into disrepute (Goldman 1968). Wood ( 1970, 1971) reviewed previous problems with the Geiger-Miiller counting system and concluded that the zero extrapolation procedure is applicable so long as the counting efficiency is kept below 25%, thus excluding the very weak P-activity (O-30 keV) from detection. Wood ( 1971) also pointed out other factors which contribute errors of some magnitude. For these reasons, together with the compulsory low counting efficiency in comparison with liquid scintillation techniques, the latter methods have been investigated in the hope that comparisons between the results of different workers may be made easier and more reliable, In liquid scintillation counting for primary production studies, Schindler ( 1966) adapted a naphthalene-dioxane fluor to solubilize membrane-filtered algae, and Wolfe and Schelske (1967) counted the intact filters and cells in a toluene fluor. Lind and Campbell (1969) proposed some advances to this latter method but, as I
