Office of Naval Research. Contract N8onr-520/III. Project NR-083-01

Duxbury, A.C.

Yentsch, C.S.

Name not available

Plankton Pigment Nomographs

Nomographs for computing the amounts of plankton pigments aa determined by the spectrophotometric method have been constructed. The methods used for alignment and scaling are discUBSed. The use of the nomographs shortens computation time considerably

Duxbury, A. C.

Yentsch, Charles S.

Yale University

      P.O. BOX 208118 | NEW HAVEN CT 06520-8118 USA | PEABODY.YALE. EDU   JOURNAL OF MARINE RESEARCH The Journal of Marine Research, one of the oldest journals in American marine science, published important peer-reviewed original research on a broad array of topics in physical, biological, and chemical oceanography vital to the academic oceanographic community in the long and rich tradition of the Sears Foundation for Marine Research at Yale University.  An archive of all issues from 1937 to 2021 (Volume 1–79) are available through EliScholar,  a digital platform for scholarly publishing provided by Yale University Library at  https://elischolar.library.yale.edu/.  Requests for permission to clear rights for use of this content should be directed to the authors, their estates, or other representatives. The Journal of Marine Research has no contact information beyond the affiliations listed in the published articles. We ask that you provide attribution to the Journal of Marine Research.  Yale University provides access to these materials for educational and research purposes only. Copyright or other proprietary rights to content contained in this document may be held by individuals or entities other than, or in addition to, Yale University. You are solely responsible for determining the ownership of the copyright, and for obtaining permission for your intended use. Yale University makes no warranty that your distribution, reproduction, or other use of these materials will not infringe the rights of third parties.  This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. https://creativecommons.org/licenses/by-nc-sa/4.0/   PLANKTON PIGMENT NOMOGRAPHS1 BY A. C. DUXBURY AND CHARLES S. YENTSCH2 Department of Oceanography University of Washington Seallle 5, W ashingtcn ABSTRACT Nomographs for computing the amounts of plankton pigments aa determined by the spectrophotometric method have been constructed. The methods used for alignment and scaling are discUBSed. The use of the nomographs shortens computa-tion time considerably. Introduction. The semi-micro method described by Richards with Thompson (1952) for analysis of plankton samples may be used to determine pigment types in plankton populations. In this method the total absorbencies of plankton extracts are measured at specified wave lengths. In brief, the plankton samples are concentrated, the pigments are extracted in 90% acetone, and the optical density of the extract is measured at wave lengths 665, 645 and 630 mµ for chlorophylls a, b, and c and at 480 and 510 mµ for animal and plant carotenoids. Where large numbers of samples are involved, con-siderable time is spent in computing the pigment types because of the numerous steps involved in solving the simultaneous equations of three or four variables. This time may be shortened considerably by converting the Richards with Thompson equations into nomo-graphic form. Development of the N omographs. The nomographs designed for solving these equations comprise a series of alignment diagrams which are readily constructed and which can be used rapidly for determining the amounts of pigment types. The nomographs consist of a series of parallel lines which are spaced and scaled by means of ratios between scale moduli. The scale modulus is a factor which will reduce the coefficient of a component of an equation, such as that of Richards with Thompson, to a unit value. The use of scale moduli allows the different components to be treated with equal scale spacing and 1 This work was supported in part by the Office of Naval Research under Contract No. N8oNRIIII with the University of Washington. 2 Present address: Woods Hole Oceanographic Institution, WO<Yls Hole, Maas. 92 1956] Duxbury and Yentsch: Pigment N omographs 93 facilitates the construction of uniform scales for these components. The method used in preparing these nomographs is taken, in part, from Levens (1948) and Kulmann (1954). The Richards with Thompson equations for determining chloro-phyll are: C,. (mg/L) = 15.6 Dw. - 2.OD,46 - O.8Dsao Cb (mg/L) = 25.4 DM6 - 4.4Dw, - 10.3D830 C. (MSPU/L) = 109 D&ao - 12.5DMli - 28.7D646, where the D terms to the right are optical densities. The equationl'I for determining animal and plant carotenoids, knowing the optical densities and chlorophyll values, are: or D, .. 610 = D610 - o.0026c,. - o.ooa5cb - O.OO21c. D,.,,so = D,so - O.0019C,. - 0.0136Gb - 0.0054C. Nonastacian (MSPU/L) = 7.6 (D,.,480 - 1.49 D,..610) Astacian (MSPU/L) = 2(4.45 D,.,610 - D,..4so) The nomograph of the equation for determining chlorophyll a is set up in the following manner. Since the equation has three com-ponents or variables that are multiplied by constant coefficients, scale moduli that reduce or change these coefficients to equal values must be determined. This can be done by multiplying each coef-ficient by its reciprocal, which reduces all coefficients in the equations to unity. These reciprocals are the scale moduli. The equation now has all components weighted equally. A vertical line is drawn for D646 near the left side of the paper, and then another line at some distance is drawn parallel to it for D630• The distance between these lines is quite arbitrary. The D666 line is then drawn parallel and at an arbitrary distance to the right of the first two lines. On an 8 X 10 piece of paper the distance between D646 and D630 can conveniently be about three inches, the distance between De:io and De66 about four inches. The D646 line should be subdivided from zero to about 0. 7 unit in optical density, zero being placed at the top. The spacing of the subdivisions should be great enough to assure reading accurately to about .002 unit. The Deso line is now graduated similarly with corresponding divisions located on a line perpendicular to the divisions on D646· To allow for both positive and negative signs that occur in the equation, the direction from bottom to top of the page is considered positive. The D,u line is now scaled with zero at the bottom on a common perpendicular with the 0. 7 mark ovthe D646 and D630 lines, the 0. 7 at the top in line with the zero mark of the latter lines. 94 Journal of Marine Research [15, 1 The equation C,. (mg/L) = 15.6D045 - 2.0D"' - 0.8Deao is now divided into two parts. The last two components are set equal to a factor called Q. Thus, - l.0D046 - 0.8D&ao = Q and f5.6Dee1 + Q = C,. (mg/L) The scale moduli for Du6 and D63o are 1/2.0 and 1/0.8 respectively. The Q line is placed between Du6 and D63o at a location determined by the ratio of these scale moduli. In this case the ratio is 1/2.0 divided by 1/0.8 or 0.8/2.0. The position of Q is conveniently de-termined by assuming that the number of units of distance between Du6 and D 630 is 2.8 units and by locating Q 0.8 units from Du, and 2.0 units from D63o. To simplify further calculations, a substitution is made by referring to the scale moduli with letters and subscripts as follows: Mu6 = 1/2.0; Maao = 1/.08; and Mf'M = 1/15.6 . Q is now located on the nomogram, but a scale modulus for it, Mq, must now be determined. M q gives the ratio between the size of the divisions of Q and those of Dw., DM6 and D63o. However, Q need not be graduated, and M q is merely used later for determining the place-ment of the chlorophyll a line. Mq is found in the following manner: M 646 X M &ao 1/2.0 X 1/0.8 1 ---- =Mq =-----=- . M 646 + Me3o 1/2.0 + 1/0.8 2.8 The chlorophyll a line is now positioned. The ratio of Mq/Mw. or 1/2.8/1/15.6 places the C,. line 15.6 units from Q · and 2.8 units from Dr.e6, if the distance between Q and Dee, is assumed to be 18.4 units. The final calculation is the scale modulus for chlorophyll a. It is determined in much the same manner as for M q. Thus, Mc,. = Mq X Mr.e6 = 1/2.8 X 1/15.6 = _1_ M q + Mee6 1/2.8 + 1/15.6 18.4 This means that each unit on the C,. scale is 1/18.4 times as large as the units on the optical density scales. Since the optical density scales are in tenths, the scale modulus, M ca, may be multiplied by ten ~o convei:t, the chlorophyll units from tenths to units. Thus, l(mg/L) 1s equal m length to 1/1.84 times the length devoted to 0.1 optical de~ity. Graduation of the chlorophyll a line is accomplished by sol~mg the nomogram ~o determine the zero point on the chlorophyll a line and then marking off the successive units along the scale. The nomog~am is used in the following manner. A straight edge is emplo~ed to li_ne up t~e values ?f D046 and D1110 as determined by the analysIS; the mtersect1on of this edge with Q is then noted. The 1956] Duxbury and Yentsch: Pigment N om-0graphs 95 straight edge is then moved to join the intersection on Q with the value obtained for Dw,. This line now crosses the chlorophyll a line, and the value of chlorophyll a (in mg/I,) is read directly. All other nomograms in this series are constructed in the same manner, using appropriate scale moduli. It should be noted that the equations for the carotenoids necessitate solving first for the D,..,80 and D,..610- These equations involve four variables instead of the three as in the case of chlorophyll; otherwise, the same procedure is followed. Chlorophyll a and chlorophyll b combine to designate an intersection on the Q line; Q and chlorophyll c combine to designate a W line intersection and W and D,80 or D610 combine to designate the intersecting D,.. lines. The solving of these diagrams consists of the same stepwise procedure described for chlorophyll a. Errors and Range of Values. The amount of error introduced by using the nomographs depends largely upon the accuracies to which D,es, 480 D,H, 410 • D410 - .0019 Ca - 01 36 Cb - .00!54Cc 4~Cb 0 2 3 4 6 7 10 Figure 1 D,n 00 480 480 10 10 • 9 8 8 7 7 6 6 !5 !5 4 4 3 3 2 2 0 0 96 Q Journal of Marine Research Ores, 510 D•10 - .002• Co - .oou - .0021 Co o,.,, a,o • .. [15, 1 the nomographic scales may be read. In all instances the reading obtained from the nomographs are indicative of the number of signi-ficant decimal places to which the pigment. values should be calculated. In some instances the equations have constants that approximate the errors in the optical density readings. This reduction in accuracy will show quite distinctly on the nomographs. However, the signi-ficant accuracy and relative accuracy is still maintained. The range of values shown on the nomographs (Figs. 1-6) have been based largely on pigment values commonly found in the waters of Puget Sound and adjacent areas. These values, in all instances, were obtained by concentrating the plankton in one or two liters of sea water and extracting the pigments with 5.0 cc of 90% acetone (Creitz and Richards 1955). In order to read accurately on these scales, it was necessary to fold one or two of the scales to meet the restrictions of paper size. The range given to chlorophyll b is quite arbitrary, since this pigment was seldom found in large amounts in 1956] OD Q 645M.,II 0 .I .2 .3 .4 .5 .6 .7 Duxbury and Yentsch: Pigment N omographs 97 CHLOROPHYLL a C0 (Mg /L) • 15 .6 0665 - 2.0 D6 45 - 0 .8 0630 C0 OD OD 630M.,u 0 Mg/L 665M,.u .7 .I .6 8 .2 7 .5 6 .4 5 4 .4 . 3 3 2 .5 .2 0 . I 0 Figure 3 98 Journal of Marine Research [15, 1 CHLOROPHYLL b Cb (Mg/ L l • 25.4 0645 - 4.4 0665 - 10.3 Duo a 00 Cb OD 00 645M..U 665M,IJ 630MJI Mg/L 10 .4 0 0 9 ,6. 8 0 1 .I ,I 6 .3 5 4 .4 3 .2 .2 .I 2 .2 .5 0 .3 .6 .3 .I .2 .1 .4 .4 0 .8 .3 Figure 4 marine plankton. The range of values shown for the animal (astacian) carotenoids may too low, since some zooplankters are not caught in standard hydrographic sampling bottles. No optical density values are extended farther than 1.0, since an extract with a concentration giving an optical density reading higher than 0.90 will not follow 1956] OD 665M,.a 0 .I t::i. .4 .2 .3 .4 ·8 Duxbury and Yentsch: Pigment N omographs Cc CHLOROPHYLL C (MSPU/L)• 109 0630 Q 00 645M..P 0 .I .2 . 3 .4 6 0 .I .2 .3 Figure 5 -12.5 0665 -28.7 Cc MSPU/L 40 30 20 10 0 99 0645 00 630M~ .4 .3 .2 . I 0 Beer's Law and therefore the extract must be diluted (Richards with Thompson, 1952). Greater color sensitivity may be obtained by using longer light paths. The plankton pigment nomographs, following the original equations, are based on optical density values using a 1 cm light path. When longer light paths are employed, either the optical density readings or the pigment values may be corrected accordingly. 100 Journal of Marine Research (15, 1 CAROTENOI OS Ase (MSPU/Ll • 2 (4.450,ea, 610 - Ores, 480 l Nae (M SPU/ L l • 7. 6 tD,81, 490 - 1.49 0 re,,s,o > D,es NONASTACIN ASTACIN o, .. TYPES TYPES 480 MSPU/ L MSPU/L 510 06 6. 06 0 .7 5 .7 0 4 .I .6 .6 .I 3 2 . 2 .5 .5 0 .3 .4 .4 .3 0 .6 . , .I .& 7 0 0 .7 Figure 6 1956] Duxbury and Yentsch: Pigment N omographs 101 Directions for the Use of the N omographs. To obtain chlorophyll values from the nomographs, optical density readings on scales adjacent of line Q are aligned and the intersection on the Q line is de-termined. This point is then aligned with the reading for the third optical density scale. The intersection of this line with the chlorophyll scale gives the value of the chlorophyll in question. For the carotenoid nomographs, residual absorbencies 510 and 480 mµ. must be considered together with values for the chlorophyll observed in the same sample. Chlorophyll a and b values are aligned to determine the intersection of the Q510 scale. This intersection is aligned with the chlorophyll c value and the intersection on W610 is determined. The final step consists of aligning the intersection of W61o with the correct value of Duo and reading the value of D,..610 directly at the intersection. The same procedure is followed for obtaining Drea480 values. The amounts of astacian and nonastacian carotenoids are obtained by aligning residual absorbencies 510 and 480 mµ. and reading the amounts of either pigment at the intersection of the respective scales. REFERENCES CREITZ, G. I. and F. A. RICHARDS 1955. The estimation and characterization of plankton populations by pigment analysis. III. A note on the use of "millipore" membrane filters in the estimation of plankton pigments. J . Mar. Res., 14 (3): 211-216. KCJLMANN, A. C. 1954. Nomographic Charts. McGraw-Hill Book Co. Inc., New York. 244 pp. LEVENS, A. s. 1948. Nomography. John Wiley and Sons, New York. 175 pp. RICHARDS, F. A. with T. G. THOMPSON 1952. The estimation and characterization of plankton populations by pigment analysis. II . A spectrophotometric method for the estimation of plankton pigments. J . Mar. Res., 11 (2): 156-172. 

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