- Email: [email protected]
The chemical composition of two species of Daphnia, their algal food and their environment
The Science of the Total Environment, 6 (1976) 79-102 © Elsevier Scientific Publishing Company, Amsterdam - - Printed in Belgium
THE CHEMICAL COMPOSITIObl OF TWO SPECIES OF Daphnia, THEIR ALGAL FOOD AND THEIR ENVIRONMENT
U. M. COWGILL
Deparlment o~ BioloKv, UniversiO' t?/ Pittsburgh, PittsbulTzh, Pa. 15260 (U.S.A.) (Received September 28th, 1975)
ABSTRACT
Since the elements of which plankton are composed enter the food chain, it was thought interesting to devote a study to their chemical composition. Daphnia magna and D. pulex were reared in a laboratory for three months maintained on a monoculture of E. gracilis supplemented occasionally with a mixed algal culture. These plankton were raised in spring water. A complete chemical analysis revealed the plankton and the rock from which the spring water flowed to contain 54 detectable elements, while the concentrated water had only 53. Major differences were noted between the two species of Daphnia in their ability to accumulate K, Na, Ca, Sr, P, S, C1, and Co. The Daphnia and algae contained different amounts of Na, K, Mg, Ca, Zn, Al, Sc, La, Nd, Sn, Ti, P, S, Cl, Br, I, Fe, Mn, Ni, Zr, Cu, Ag, Be, Si, Pb and Hg. The composition of the Daphnia is governed by the mixed algal culture and the spring water. The Daphnia reject E. gracilis; the latter does not contribute to their chemical composition. Aside from supplementary additions of N, P, K and C1 to the algal cultures their composition is governed by that of the spring water.
INTRODUCTION
There is a large body of scientific literature concerning the nutrient requirements of the freshwater plankton 1. However, there are few studies of the chemical composition of the plankton themselves. The usual approach to the problem of estimating the nutrient requirements of a specific species has been to add a small quantity of some element to its medium and observe the response. This approach, though old and established, is not without difficulty. Additions of a monovalent or divalent cation to a growth medium may increase productivity merely by altering the ratio of monovalent to divalent ions, even though the added cations themselves are not limiting growth 2. The addition of a particular com79
pound may produce a positive response because the presence of some trace substance satisfies a deficiency while the principal elements involved in the compound in question are present in ample quantity. It, therefore, would appear useful to know something of the chemical composition of some plankton themselves. This paper concerns the elemental composition of two species of Daphnia, the algae they were fed, the spring water in which they were all reared, and the trap rock from which the spring water flows. Unfortunately, there are no available comparative data for these plankton. Since the elements of which the plankton are composed eventually find their way into the food chain, it was thought to be of interest to devote an investigation to their chemical composition. MATERIALS AND METHODS
Descriptions of the site, field and laboratory methods have been previously given 3 so that a brief summary should suffice.
Study site and fieid methods The plankton were cultured in the laboratory in water from West Spring. This spring is located on Springside Ave. which is on the east side of West Rock Ridge in New Haven, Conn. The ridge consists of Triassic Trap Rock with almost continuous exposure of rock, but is partly covered by till and talus 4. Three 10-kg samples of rock were collected from the vicinity from which the spring flowed. Water (174.7 1) was collected weekly for a period of two months.
Laboratory culturing methods Populations of Daphnia magna Straus, 1820 and Daphnia pulex Leydig, 1860 were cultured in autoclaved water from West Spring in glass aquaria, maintained at a constant temperature of 20°C under a 12-h light, 12-h dark cycle. The Daphnia populations were fed daily or once every two days, with a xenic monoculture of Euglena gracilis Klebs supplemented occasionally with mixed cultures containing E. gracilis and varying proportions of Chlamydomonas, Oocystis, Sphaerocystis, Ankistrodesmus, Scenedesmus, Closterium, Closteriopsis, a small filamentous green alga and a small filamentous blue-green alga. Daphnia were collected weekly for a period of three months. Algae used for analyses were cultured in autoclaved water from West Spring in 4-1 Pyrex Erlenmeyer flasks. These flasks were maintained at room temperature in constant light, fluorescent and natural daylight. Cells were kept in suspension by continuous aeration. Rubber tubing and sterile cotton filters were used to connect the aeration system to the flasks in series. Rubber stoppers filled with glass tubing and sterile cotton filters were used to seal the flasks. Both E. gracilis as well as the mixed algal culture were harvested weekly for a period of three months. E. gracilis was obtained from the Culture Collection of Algae, Indiana University, and stocks were maintained on nutrient agar 5. This organism was 80
TABLE 1 PERTINENT RESULTS OF THE CHEMICAL ANALYSES
Substance
D. pul~:x D. magna 15. gracilis Mixed culture Spring H20 (L.) Trap rock (%)
Oxide constituents (dry weight basis)
Ignition
14et H't.
Dry H't.
Loss
Major*
Minor
Total
(g)
(ca
(%)
(%)
(~ )
( ~o)
96.00 66.71 65.61 91.60 174.74
0.30 1.11 1.67 2.97 9.25 1.64
88.82 81.18 87.39 86.54 10.41 2.77
11.028 19.060 13.101 14.074 -95.810
0.085 0.081 0.124 0.111 -0.191
99.93 100.32 100.61 100.73 -100.41
* Na, K, Mg, Ca, AI, Si, Ti, P, S, CI, Mn, Fe.
used for inoculation of the monoculture. Algae for inoculation of the mixed culture came from a fish tank. In order to promote algal growth, occasionally 0.5 ml of a liquid fertilizer was added to 2 1 of spring water prior to autoclaving. A complete analysis of 100 ml of this liquid fertilizer (Hy-trous Corp., Boston, Mass.) showed it to contain 4 % total N, 8 % P205, 4 °/6 K20, 0.8 % CI and no detectable trace elements with the exception of less than 1 ppm Br. This analysis was carried out recently and was not performed on the same solution that was used to encourage the growth of the algae. The total amount used in culturing did not exceed 3 ml, and it is presumed that no detectable elements other than those encountered in the recent analysis of the fertilizer were added to the culturing media. Algae were harvested by centrifugation and Daphnia were collected by filtration through nylon netting which was fine enough to retain all instars but not the algae. C h e m i c a l techniques
Although all precautions were taken to avoid contamination 3 from alien sources, some unavoidable sources should be noted. West Spring, which was condemned several years ago, was fitted with a gravity pipe of unknown composition that had soldered joints. The spring, according to local residents, had been in use since 1919. The solder used during that period consisted largely of Sn and Pb 6. It is, however, quite possible that Cd, Hg and Bi may have been included 7. All cultures were maintained either in pyrex glass or commercial window glass, thus providing a possible source for the inclusion of extraneous B in the media. The effects of these possible sources of contamination will be considered later in this paper. Immediately on arrival at the laboratory the water was evaporated to dryness at 80°C; its total solids weighed 9.25 g. 81
The three samples of rock were weighed, dried, crushed and ground in the usual manner. The samples were manually mixed for 6 h each and two aliquots were taken from each sample. The chemical data presented here for the trap rock is a mean of these six aliquots. All plankton samples were rinsed in three changes of doubly distilled deionized water. The plankton were separated from the rinse water by centrifugation (8000 rpm, 30 rain); microscope examination did not reveal cell breakage while assay for potassium indicated no loss of cell contents. The supernatant liquid was poured off. The centrifuge tubes were weighed on an analytical balance, dried in an oven at 60°C for one week and reweighed. The results are shown in Table 1. All chemical analyses are presented on a dry weight basis. It is clear that the wet weights are anomalously high. Daphnia retain variable amounts of water between the two valves of the carapace, making its removal difficult. Thus, it is thought that the wet weights are in error, and all chemical data are expressed on a dry weight basis. The general technique involved in the preparation of samples for analysis by X-ray emission and optical emission have been previously described s.
Instrumental precision Despite the fact that methods used to gather these data have been previously discussed, the precision with which the concentration of elements here described has been determined should be mentioned. The quantities of the elements detected by X-ray emission spectroscopy were all determined in a helium
TABLE 2 HIGHEST AND LOWEST CONCENTRATIONS OF ELEMENTS FOUND IN THE PLANKTON BY OPTICAL EMISSION SPECTROSCOPY AND THE RELATIVE STANDARD DEVIATION FOR COUNTS FROM 20 DUPLICATES
Element
Low Be Hg Bi Sn Co B Mo V Ag Li Na Cd
82
Relative standard deviation (%)
Conc. (ppnO
0.017 2.68 1.0 1.2 0.7 12.0 0.2 1.3 0.46 0.28 .200 1.24
H~h
Low
High
0.088 4.25 1.8 23.5 3.8 17.8 6.45 6.4 4.7 0.60 1180 1.38
9.8 7.2 8.8 4.7 3.2 2.5 4.6 3.6 8.3 6.2 1.7 6.6
4.9 3.3 9.2 3.8 4.4 2.4 4.4 6.8 3.0 5.8 2.1 6.8
tunnel. All X-ray tubes (Cr, Mo, Pt) were used at maximum rating. Whenever possible, counts were accumulated to 10,000. Thus, .K, Ca, P, S, CI, Fe, Cu, As, Pb, Ga and Sr were determined with a standard counting error or a precision of less than 1%; Y, F and Ce with a precision of less than 2 %; AI, Si, Mn, Zn, Rb and Br with a precision of less than 3 96 ; Nb, Pr, Yb, Se, Ge, Cr and Zr with a precision of less than 4 %; Ni and Mg with a precision of less than 5 °/6; Ba, Nd and Ti with a precision of less than 6 %; La, Sm and Eu with a precision of less than 10 9;, ; Hf, Sc and Er with a precision of less than 12 % ; and finally, I, Cs, Dy and Gd with a precision of less than 15 %. Each of these elements was counted on three separate and non-consecutive occasions, and the results were considered to be reproducible only when the net counts agreed with previous occasions within less than 1%. Table 2 shows the highest and lowest concentrations of elements found in the plankton by optical emission spectroscopy and the percent relative standard deviation for counts from 20 duplicates.
Mineralogical techniques A mineralogical analysis was carried out on the trap rock, the concentrated dried water sample, and the four samples of plankton. Techniques involving the use of an X-ray diffractometer have been previously described 9. All results were confirmed with microscopic techniques. RESULTS
Before discussing the results of this study in detail, it should be noted that the trap rock from which West Spring flowed was collected after the spring had been condemned and closed. In order to ascertain whether it was valid to compare the composition of the spring with that of the rock, a linear correlation coefficient was determined between the concentration of the 54 elements in the spring water (Eu=0) and that in the rocks from which it used to flow. The coefficient obtained (r=0.8421) was statistically significant beyond the 0.1% level.
The alkafi metals Table 3 shows the distribution bf Na, K, Rb, Cs and Li in the four plankton samples, the spring water and the trap rock. The Daphnia clearly accumulate more Na and Rb on the average than do the algae, while the algae concentrate higher quantities of K, Cs and Li than do the Daphnia. Relative to the composition of D. magna, D. pulex contains notably less Na, K and Cs but more Rb, with the Li content being about the same. The Rb:K ratio (Table 4) in the spring water is higher than that of either the earth's crust j° or the trap rock, suggesting that there is an enrichment of Rb relative to K in the water. It would appear, on the basis of the ratios, that both groups of plankton tend to exclude Rb relative to K to some extent. The Cs:K ratio is the same in the trap rock and the spring, both being 83
TABLE 3 ALKALI METALS: Na, K, Rb, Cs, Li Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Conc. (ppnO Na
K
Rb
Cs
Li
861 1 180 252 200 0.103 15 753
8 003 13 839 17 898 13 955 0.258 5 562
35.3 22.9 26.4 20.7 1.9×10 -3 18.9
0.82 1.38 1.77 1.12 5.6x10 -5 1.26
0.38 0.28 0.60 0.42 2x10 -4 6.23
TABLE 4 COMPARISON OF THE Rb:K AND Cs:K RATIOS IN PLANKTON, SPRING WATER, TRAP ROCK AND IN THE EARTH'S CRUST Substance
Rb :K
~s :K
Earth's crust 10 Trap rock Spring H20 Daphnia, m Algae, ffi
4.3× 10-3 3.4x 10-3 7.2x10 -3 2.7x10 -3 1.5x10 -3
1.4× 10-4 2.3 × 10- 4 2.2×10 -4 1.0x10 -4 0.9x10 -4
somewhat higher than that of the earth's crust j°. Relative to K, Cs is excluded to some extent by both groups of organisms. The Rb:K ratio of the two groups of plankton are similar, though that of the Daphnia is more reminiscent of the trap rock than is that of the algae.
Copper and silver Table 5 shows the distribution of Cu and Ag in the two groups of plankton, the water and the trap rock. The algae concentrate both elements to a far greater extent than do the two species of Daphnia. The Ag:Cu ratio of the earth's crust (1.3×10 -3) and that of the spring water are quite similar (1.8x10-3); however, this ratio in the algae (21x10 -3) as well as in the Daphnia (8x10 3) suggest that there is a notable enrichment of Ag relative to Cu in these organisms. When this ratio in the spring water (1.Sx10 -3) is compared to that in the trap rock (0.1xl0 -3) through which it flows, the enrichment of Ag relative to Cu is sizeable. Relative to the earth's crustal composition ~o the trap rock is enriched in Cu and impoverished in Ag. It is noteworthy to mention that this entire study was carried out during the winter. Silver is more concentrated in the winter than in the summer in many lakes 11 in the general region around West Spring. It is believed that this obser84
TABLE 5 Cu AND Ag CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
(one. (ppnO Cu
Ag
70.0 77.5 283.2 156.5 3.8×10 -3 89.9
0.78 0.46 4.7 4.6 6.8×10 -6 0.009
vation results from the seeding of clouds with AgI to encourage snow fall. The possibility of atmospheric contribution to the Ag content of the plankton should, therefore, not be ignored. Despite this possibility, it is clear that the two groups of organisms take up Ag to a different extent.
Beryllium Beryllium exhibits a seasonal distribution in lakes H in the vicinity of West Spring, being more concentrated in the winter than in the summer. It is believed that the source of the contribution is the combustion of fossil fuels. The possibility of atmospheric contribution to the Be content of the plankton should, therefore, not be ignored. The algae accumulate more Be than do the Daphnia (Table 6). The Be:Li ratio for the Daphnia is 0.07, for the algae is 0.17, and for the spring water is 0.075. This would suggest that Be moves more easily from the water to the algae than does Li. Similarly Be moves easily from the trap rock (Be:Li 0.02) to the water than does Li. It will be observed later that the same situation prevails in the case of Mg, where Be is enriched relative to this alkaline earth.
TABLE 6
Be CONCENTRATIONS Substance
Be (ppnO
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
0.017 0.028 0.088 0.084 1.5×10 -5 0.123
85
The alkaline earths Table 7 shows the distribution of Mg, Ca, Sr and Ba in the plankton, the spring water and the trap rock. The Mg content of the algae is 10.6 times that of the Daphnia while the latter accumulate 6.9 times more Ca than the former. As would be expected, Sr follows Ca. D. pulex contains slightly less Mg than does D. magna and considerably less Ca. It is curious that tolerance experiments using D. pulex in continuous culture have shown that 120 ppm of Mg is the upper limit 12. D. pule× accumulates more Ba than any of the other organisms. Relative to their composition in the continental crustl° Sr, Ba and Be are impoverished and Ca and Mg enriched in the trap rock (Table 8). Considering the Sr:Ca ratio in the spring water, Sr is excluded by both groups of organisms relative to Ca. There clearly is an enrichment of Sr relative to Ca in the spring water. Relative to Ca in the spring water, Ba is excluded by the Daphnia and accumulated by the algae. The Be:Mg ratios indicate that Be moves more easily to the Daphnia from the water than does Mg, and to a lesser extent this is also true in the case of the algae. However, in reference to Ca in the spring water, Be moves more easily to the algae while it is almost excluded by the Daphnia. ,The explanation for this peculiar situation lies in the large differences in accumulation of Mg and Ca in the two groups of organisms.
TABLE 7 ALKALINE EARTHS: Mg, Ca, Sr, Ba
Substance
Com'. (ppm)
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Mg
Ca
Sr
Ba
549 633 5 996 6 520 1.88 40 058
36 878 76 643 7 311 9 112 4.49 57 151
45.8 81.9 40.0 35.3 0.033 129.3
84.3 67.1 48.5 44.5 0.014 126
TABLE 8 COMPARISON OF THE Sr:Ca, Ba:Ca, Be:Mg, Be:Ca RATIOS IN PLANKTON, SPRING WATER, TRAP ROCK AND IN THE EARTH'S CRUST
Substance
Sr :Ca
Ba :Ca
Be :Mg
Be:Ca
Eanh's crust Io Trap rock Spring H20 Daphnia Algae
9
10.2x10 -3 2.2× 10-3 3.1×10 -3 1.3× 10-3 5.7× 10-3
1.2 xlO -4 0.03× 10-4 0.08)<10 -4 0.38× 10-4 0.14× 10-4
6.7 ×10 -s 0.2 × 10-5 0.3 ×10- 5 0.03x 10-5 1.04x 10-5
86
x10 -3
2.3× 10-3 7.3)<10-3 1.1 x 10-3 4.6x 10-3
Zinc, cadmium and mercuiy Table 9 shows the distribution of Zn, Cd and Hg in the plankton, the spring water and the trap rock. The mean Zn and Hg content of the Daphnia is lower than that in the algae while the Cd concentration is about the same for the two groups.
TABLE 9 Zn, Cd AND Hg CONCENTRATIONS
Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Conc. (ppnO Zn
Cd
Hg
134.6 102.0 304.0 338.7 0.012 92.4
1.34 1.24 1.38 1.34 1.6×10-5 0.38
2.68 3.30 4"20 4.25 1.3×10 -4 0.025
Goldschmidt 13 has suggested that the geochemistry of Hg is in some ways analogous to that of Cd. The geochemical ratio of Hg to Cd in the continental crust I° is 0.4. That in the trap rock is 0.07, in the spring water is 8.13 while in the Daphnia it is 2.32 and in the algae 3.11. Compared to the ratio in the spring water, Cd moves more easily to both groups of organisms than does Hg. It should be noted that both Cd and Hg could have been contributed to the spring water through its contact with the soldered joints 7 of the gravity pipe through which the spring flowed. A second possible contributory source could have been the Hg contamination in'the culture rooms. A third source may have been the combustion of fossil fuels in the winter 8. There is no question that the Hg content of the plankton is unexpectedly high. Both cultures of algae contain more Hg than the Daphnia. The Daphnia may have obtained their Hg content by consumption of algae and possibly some from the water in which they were reared. However, the Daphnia were cultured in open aquaria in constant temperature chambers, while the algae were raised in sealed flasks and aerated with piped-in building air filtered through cotton wool. It therefore remains unclear what mechanism was proceeding to permit the entrance of Hg into the sealed flasks. Though it seems highly unlikely, the algae may have accumulated Hg to an enormous extent from the water in which they were reared. Only further investigation will elucidate this point. In reference to the above discussion, it is interesting to note that the threshold concentration of Hg for the immobilization of D. magna is 0.0044 ppm when Hg is added as HgClz to Lake Erie water j4. 87
Aluminum, gallium and boron Table 10 shows the distribution of AI, Ga and B in the plankton, the spring water and the trap rock. The AI concentration in the Daphnia is lower than that in the algae but the Ga concentration is higher. The ratio of Ga:AI" in the spring water would suggest that Ga moves more easily to the Daphnia than does AI (Table 11). In reference to AI, however, Ga is vastly enriched in the spring water when the Ga:AI ratio in the trap rock is considered. It is interesting to note that the Ga:AI ratio in the spring water is essentially the same as that in the algae.
TABLE 10 AI, Ga AND B CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppt~O AI
Ga
B
127.0 121.7 190.5 232.9 0.207 53 179
0.94 0.68 0.52 0.59 4.9x10 -4 15.8
14.0 14.0 12.0 17.8 0.058 130
The B concentration of the spring water is only slightly higher (0.047) than the mean proposed by Boyd and Walley 15, and that of the algae is within the range found by B o y d 16. This would suggest that the possible contribution to the B concentration in the algae and Daphnia from the glassware in which they were reared is probably minimal. The mean concentration of B in the two groups of plankton is essentially the same.
Scandium, yttrium and the rare earths Table 12 shows the distribution of Sc, Y and the rare earths in the plankton, the spring water and the trap rock. A comparison between the concentration of Sc, Y and the ten detectable rare earths (Eu=0) in the water and that in the trap rock revealed a linear correlation coefficient of 0.841 which is significant well beyond the 0.1 .% level. All the rare eai'ths of even atomic number were detected; of the odd-numbered members of the group only La, Pr and Eu were found and the latter element was not at a detectable concentration in the concentrated spring water. The others were presumably present in amounts below detection. If the ratios of AI, Sc, Y and the rare earth elements (2; Ln) are compared in the Daphnia and the trap rock, and in the algae and the trap rock (Table 13), it becomes apparent that AI and Sc are absorbed to a moderate extent but Y and the rare earths are accumulated in quantity. 88
~
b'~
',o
x
~
i
x
X
X
X
X
r'~ t'-,,I '~1- o
X ~
×
z
¢~ ×
©
¢.,i ¢.~ e,i e,~ I I I I !
X
X
X
X
X
Z
Z
N~
X
bZ Z
N
X
X
X
X
m z ,<
,,.t XX
X
×
Z 0
~
x~
m
XX
×
1
I
)'(X
X
I
I
X
X
7
g -d z e~
d "~.~ ..1 •.--
~. ~
~
~.~
89
TABLE 13 RATIOS OF AI, Sc, Y AND THE TOTAL LANTHANIDE RARE EARTHS IN THE PLANKTON, THE SPRING WATER AND TRAP ROCK
Com. (ppm)
Earth's crust Io Trap rock Spring H20 Daphnia if5
D. nlagna D. pulex Algae ~
E. gracilis Mixed culture
AI
Sc
)'
ZLn *
82 300 53 179 0.207 124.4 121.7 127.0 211.7 190.5 232.9
22 39.7 6.4x10 -4 18.4 16.8 20.0 6.95 6.8 7.1
33 19.7 1.6×10-3 41.5 50.6 32.4 57.4 53.3 61.5
146.4 112.1 5.04×10 -3 132.9 124.6 141.1 95.9 101.7 90.0
Ratios Daphnia: Trap rock Daphnia: Spring H20 Daphnia: Earth's crust lo
2.3× 10-3 601 1.5x10 -3
46x 10-2 28 750 84x10 -2
2.11 25 938 1.26
1.19 26 369 0.91
Algae: Trap rock Algae: Spring H20 Algae: Earth's crust l°
4.0×10 -3 1023 2.6×10 3
17.5x10 -2 10 859 32x10-2
2.91 35 875 1.74
0.86 19 028 0.66
* Total lanthanide rare earths.
TABLE 14 THE GEOCHEMICAL RATIOS OF SOME ELEMENTS IN THE PLANKTON, SPRING WATER, TRAP ROCK AND THE EARTH'S CRUST lo
Ratio
Earth's crust I0
Trap rock
Spring H 20
Daphnia
AIgae
Sc:Ti Y:Ti La:Ti Ce:Ti Pr:Ti NO:Ti Sm:Ti Eu:Ti Gd:Ti Dy:Ti Er:Ti Yb:Ti AI:Ti Ca:Ti P:Ti
3.9 x10 -3 5.8 ×10 -3 5.3 ×10 -3 10.5 ×10 -3 1.4 x l 0 -3 4.9 ×10 -3 1.1 x l 0 -3 0.21x10 -3 0.94×10 -3 0.53x10 -3 0.49×10 -3 0.53x10 -3 14.39 7.37 184×10 3
5.8 ×10 -3 2.9 ×10 -3 1.7 ~,,10 3 10.5 ×10 3 0.50× 10--3 1.4 ~I0 3 0.46×10 -3 0.16x10 -3 0.40×10 -3 0.11×10 3 0.71×10 3 0.48×10 -3 7.78 8.36 60×10 3
160 x l 0 -3 400 x l 0 -3 225 ×10 -3 600 ×10 -3 57.5x10 -3 237.5x10-3 11 x l 0 -3 -24.5×10 -3 40 x10 -3 15 x l 0 -3 50 x l 0 -3 51.75 1122.5 7250x10 -3
7.83 17.66 21.74 18.23 2.26 7.62 2.06 0.49 1.77 1.36 0.30 0.70 52.91 24 153 6 186 170x10 -3
0.49 3.99 1.59 3.21 0.24 0.60 0.29 0.09 0.27 0.15 0.09 0.13 14.70 570 1 876 910x10-3
90
The Sc concentration in the Daphnia is 2.6 times that in the algae. In the case of Y, the algae contain slightly more than the Daphnia. It may be that both higher and lower aquatic plants exclude Sc from their tissues ~7. On the average the algae absorb less La, Pr and Nd than do the Daphnia. The concentration of Ce, Sm, Eu, Gd, Dy, Er and Yb is essentially the same in both groups of plankton. It is curious that the algae also contain less Ca than the zooplankton. Since La can substitute for Ca, it may be that the Ca concentration in the algae is controlled by the simultaneous uptake of La. Titanium is probably the least soluble and least mobile of the common elements of the earth's crust. This element, therefore, forms a convenient reference point to which other elements may be compared. Table 14 shows the ratios of the rare-earth elements to Ti. Relative to Ti in the earth's crust, Sc and Er are the only elements of this group enriched in the trap rock. As one would expect, relative to the Ti in the trap rock, the whole group is vastly enriched in the spring water. Relative to Ti, this whole group of elements is enriched in the plankton but vastly more so in the Daphnia than in the algae. It is interesting to note that relative to Ti, Ca is enriched in the trap rock, the spring water and the plankton. Phosphorus is impoverished relative to Ti in the trap rock when the P:Ti ratio in the continental crust 1° is considered. The enrichment of P relative to Ti is enormous in the spring water and the plankton when the P:Ti ratio in the trap rock is considered. It is curious that Eu was not detected in the concentrated water. Though the concentration of this rare earth was too low in the spring water to be estimated, it could be found in the plankton, thereby confir~ning its presence in the water.
Silicon, germanium, tin and lead Table 15 shows the distribution of Si, Ge, Sn and Pb in the plankton, the spring water and the trap rock. Bowen 17 suggests as a mean value for freshwater 4×10 -5 for Sn and 5 × 1 0 -3 for Pb. The Pb content of the spring water is below the suggested mean while Sn is much above it. As mentioned earlier, the source
TABLE 15 Si, Ge, Sn AND Pb CONCENTRATIONS
Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppm) Si
Ge
Sn
Pb
892.9 771.3 724.6 1 136.0 6.8 244 252
0.29 0.20 0.25 0.18 1.8× 10-5 1.9
4.2 1.2 23.5 20.0 2× 10-3 0.62
9.8 7.2 12.6 10.0 8.5x 10-4 39.4
91
of the Sn could well be the solder 6 on the joints of the gravity pipe through which the spring water flowed. If this were true, one would expect the Pb also to be high. However, due to the Ca concentration of the water, it may be that the Pb which was extractable had been removed long before sampling. Since the composition of the gravity pipe is unknown, it is also possible that some Sn may have been in the pipe itself. In any case, it is quite clear that algae accumulate far more Sn than do the zooplankton; this is also true of Pb, but to a lesser extent. The threshold concentration for the immobilization of D. magna by Sn is 66.5 ppm (SnC14) and by Pb is 0.93 ppm (PbCI2) when added to Lake Erie water J4. It would certainly seem reasonable to suggest that D. magna excludes Sn from its tissues, especially in view of the fact that D. pulex contains 3.5 times as much. The tolerance level proposed for Pb is rather curious since D. magna accumulates far more Pb than the tolerance level would lead one to suppose. The Ge content is essentially the same in both groups of organisms. The mixed culture contains more Si than any of the other organisms. Of the monocultures, D. pulex contains the most while E. gracilis contains the least. It may be that some of the species in the mixed culture accumulate Si to a large extent.
Zirconium, haJhium and titanium Table 16 shows the distribution of Zr, Hf and Ti in the plankton, spring water and the trap rock. The Daphnia contain more Zr and Hf and considerably less Ti than do the algae. As would be expected, Hf follows Zr. Relative to Zr in the spring water, both groups of organisms tend to exclude Hf from their tissues (Table 11). TABLE 16
Zr, Hf AND Ti CONCENTRATIONS Sttbstance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppnO Zr
HJ
Ti
14.9 15.9 7.6 9.8 6.7x10 -3 82.1
0.012 0.014 0.006 0.009 1.2×10 -4 2.54
2.4 2.3 11.4 17.4 4 x I 0 -3 6834
Phosphorus The distribution of P in the two groups of plankton (Table 17) shows that the Daphnia accumulated about half as much as do the algae. The P in the trap rock is impoverished relative to the earth's crust (1050 ppm) 1°. Reported concentrations of P in water are highly variable 17 but Bowen j8 proposes a mean value of 0.005 ppm for freshwater. The amount in the spring water is far higher. AI92
TABLE 17 P CONCENTRATIONS Substance
P (ppnO
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
13 141 15 934 23 095 30 960 0.029 407
though it has not been possible to ascertain the reason for the closure of the spring, it may be presumed that it was due to contamination from human sources. Kopp and Kroner j9 proposed a mean P concentration for waters of the United States of 0.12ppm with a range of 0.002-5 ppm. West Spring certainly falls within their measured range.
Arsenic and bismuth Table 18 shows the distribution of As and Bi in the plankton, the spring water and the trap rock. The As concentration is slightly higher in the algae than in the Daphnia, while the mean concentration of Bi is about the same in both groups. As more As data are gathered it may turn out that aquatic plants genTABLE 18 As AND Bi CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Conc. (ppnO As
Bi
2.3 2.2 3.6 3.4 2.4×10 -4 2.7
1.8 1.5 1.8 1.0 6.3×10 -5 0.07
erally contain more of this element than animals. Bismuth has no known biological function. Small quantities seem to be present in most living things. The amount in the plankton is higher than would be expected and in any case is accumulated to a great extent over that of the trap rock.
Vanadium and niobium D. pulex accumulates larger quantities of V and Nb than does D. magna (Table 19). E. gracilis contains more V than does the mixed algal culture and slightly more Nb. 93
TABLE 19 V AND Nb CONCENTRATIONS Substance
D. pulex D. magna £. gracilis
Mixed culture Spring H20 Trap rock
(ore. (ppm) 1
Nb
4.4 1.3 6.4 2.8 9×10 -6 48.7
16.1 11.9 14.1 13.3 5.8x10 -4 5.5
Vanadium is far more concentrated in lake waters in the vicinity of West Spring during me winter than the summerJL Again, it is possible that there is some contribution to the atmosphere resulting from the burning of fossil fuels. The amount of V in the spring water is extremely low compared to that suggested as a mean by Bowen ~8 Both V and Nb are impoverished in the trap rock when their quantity is compared with the crustal average ~°. The plankton accumulate Nb in excess of that found in the trap rock.
Sulfur and selenium The mean quantity of Se in the two groups of plankton is the same (Table 20). The mean S content of the algae is slightly higher than that of the Daphnia. Relative to S in the trap rock (Table 11) there is a considerable enrichment of Se in both groups of organisms. Both S and Se are accumulated by the organisms in excess of that found in the trap rock. Relative to the amount in the earth's crust 1° S is enriched and Se impoverished in the trap rock.
TABLE 20 S AND Se CONCENTRATIONS Substance
D. pulex D. magna E. gracilis
Mixed culture Spring H20 Trap rock
94
Com. (ppm) S
Se
3965 5647 5943 4201 0.626 512
2.1 1.6 2.0 1.9 3x 10-5 0.85
Chromium and molybdenum The Mo concentration in the algae is far greater than that in the Daphnia and both groups contain Mo in excess of that found in the trap rock (Table 21). The mean concentration of Cr is about the same for the two groups of organisms. D. magna contains less Cr and Mo than does D. pulex. Both elements are believed to be required for life 18. TABLE 21
Cr AND Mo CONCENTRATIONS Substance
D. pulex D. magna E. gracilis
Mixed culture Spring H20 Trap rock
Conc. (ppnO Cr
Mo
1.3 0.6 1.4 1.2 2.2×10 -5 62.4
0.7 0.2 6.5 5.7 1.3x10 -6 0.102
The halogens The differences in accumulation of CI between D. magna and D. pulex are striking (Table 22). The algae contain far less of the element than either of the Daphnia species. Bromine follows C1 in the four groups of organisms. Iodine is far more enriched in the two groups of algae than in the two species of Daphnia. The mean concentration of F in the two groups of organisms is the same. In reference to the CI in the spring water, the algae are enriched in F and the Daphnia exclude it (Table 11). In reference to Br in the spring water, the Daphnia accumulate C1 while the algae exclude it. Relative to C1 in the spring water, Daphnia excludes I while the algae accumulate it.
TABLE 22 HALOGENS: F, C1, Br, I
Substance
D. pulex D. magna E. gracilis
Mixed culture Spring H20 Trap rock
£ bnc. (ppnO F
CI
Br
I
12.4 9.4 13.1 9.9 0.019 275
3 198 10 130 360 400 1.45 128
27.1 37.1 18.0 14.2 0.02 3.8
0.54 0.69 1.75 1.40 6.7×10 -4 0.035
95
Iron, cobalt, nickel and manganese Table 23 shows the distribution of Fe, Mn, Co and Ni in the plankton, the spring water and the trap rock. The mean Fe, Mn and Co content is impoverished and the Ni enriched in the Daphnia relative to that in the algae. D. magna contains notably less Co than D. pulex. Relative to Mn in the spring water, Fe is accumulated by the algae and excluded by the Daphnia. Relative to Co, Fe is to some extent excluded by both groups of organisms, while compared to Ni, it is accumulated by the plankton (Table 11).
TABLE 23 Fe, Mn, Co AND Ni CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppnO Fe
Mn
Co
Ni
1 014 916 12 961 10 534 0.164 99 624
132 94 279 240 4.4x10 -3 3588
3.3 0.7 3.8 2.8 1.1×10 -5 9.6
4.2 3.6 1.8 2.7 8.9x10 -4 44.7
Statistical analyses Table 24 shows the results of statistical analyses involving elemental comparison in the four plankton samples. The degrees of freedom are only 2 and therefore it is not possible to verify statistically the validity of these apparent relationships by multiple correlation techniques. Although the levels of significance encountered are weak, and the number of samples small, the results may be tentatively considered as indicative of rates of uptake of elements by very different plankton species. The relationships shown by Li, Ag, Be, Sn, Hg, As and Nb would suggest that this group has a biological function. They are all related at varying degrees of significance to elements known to be required for life ~8. Table 25 shows the linear correlation coefficients obtained by comparing the concentration of all elements in the organisms with those in the spring water. There is no statistically significant relationship between either algal culture and the water in which they were reared. Since the water in which the mixed culture and E. gracilis were grown had a liquid fertilizer supplement added prior to autoclaving, the concentrations of P, K and C1 were removed from the calculations and the linear correlation coefficients were again determined. The concentration of the elements in the water compared to that in E. gracilis gave a linear correlation of 0.337 which is well beyond the 1% level of significance, while the linear 96
TABLE 24 LINEAR CORRELATION COEFFICIENTS AMONG THE ELEMENTS FOUND IN THE PLANKTON (N=4) Elements
Na, Zr Na, Hf Na, CI Na, Br K, Cs Cs, S Li, V Cu, 1 Cu, Fe Cu, Mn Ag, Mg Ag, Ti Ag, Mo Ag, Fe Ag, Mn Be, Mg Be, Hg Be, Ti Be, 1 Be, Fe Be, Mn Mg, Ti Mg, Mo Mg, I Ca, Sr Ca, CI Ba, 2Ln* Zn, Sn * 2Ln--total
P=O.I
P=O.05
P=O.02
O. 90(0
O. 9500
O. 9800
0.9491 0.9228 0.9254 0.9850 0.9420 0.9337 0.9054 0.9537 0.9234 0.9137 0.9944 0.9360 0.9970 0.9890 0.9800 0.9880 0.9741 0.9210 0.9800 0.9860 0.9460 0.9630 0.9840 0.9460 0.9633 0.9904 0.9889 0.9694
Elements
Zn, Ti Zn, As Zn, Mo Zn, Fe Zn, Mn Hg, P AI, Ti AI, As Sc, La Sc, Nd Ce, V Sn, Mo Sn, Fe Sn, Mn Zr, Hf Ti, Mo As, Fe As, Mn V, F Nb, Se Se, Cr Se, Co Cr, Co Mo, I Mo, Fe Mo, Mn CI, Br Fe, Mn
P=O.I
P=O.05
P=O.02
P=O.OI
O. 9000
0.9500
O. 9800
O. 9990
0.971 0.9732 0.9748 0.956 0.9523 0.9104 0.9994 0.9070 0.9984 0.9774 0.9551 0.9960 0.9942 0.9963 0.9810 0.9041 0.9977 0.9945 0.9574 0.9381 0.9565 0.9432 0.9973 0.9750 0.9968 0.9896 0.9580
6~9850
L a n t h a n i d e rare earths.
correlation involving the mixed culture provided a coefficient of 0.449 which is well beyond the 0.1 °/6 level of significance. A multiple correlation coefficient employing these linear ones reveals R to be well beyond the 0.5 % level of significance; however, a t test of significance applied to the partial correlation coefficients showed that the composition of the mixed culture reflected that of the spring water, but this was not the case with E. gracilis. The question was posed as to which of the following substances was most important in determining the elemental composition of D. pulex:E, gracilis, the mixed algal culture, or the spring water? A multiple correlation was employed to answer this question (Table 26). The relationships between D. pulex and E. gracilis ~ere totally insignificant. That between D. pulex and the mixed culture was highly significant and that between D. pulex and the spring water was significant well beyond the 0.1 °/6 level.
97
TABLE 25 L I N E A R C O R R E L A T I O N C O E F F I C I E N T S B E T W E E N THE E L E M E N T A L COMPOSITION OF T HE P L A N K T O N A N D THE SPRING W A T E R (?~=54)
Linear correlation coe[/ictents
D. pulex D. magna E. gracilis Mixed culture
D. puh'x
D. magna
E. gracilis
Mixed culture
Spring water
--
0.9887* -0.4154"* 0.4403*
0.5226* 0.4154"* -0.9547*
0.5581 * 0.4403* 0.9547* --
0.5013" 0.5188" 0.1430"** 0.1583"**
0.9887* 0.5226* 0.5581"
* 0.1% level of significance. ** 0.5 % level of significance.
*** Not significant.
Posing the same question in reference to D. magna produced somewhat different results. E. gracilis did not in any way contribute to the composition of D. magna but both the mixed culture and the spring water did. However, the latter is far more significant than the former. It is noteworthy to point out that linear correlation coefficients should be examined by multiple correlation techniques to ascertain their validity. The relationship between D. pulex and E. gracilis is significant well beyond the 0.1% level and that with D. magna beyond the 0.5 % level. These relationships are spurious because they reflect the effect of the mixed algal culture and the spring water. When the effects of these two substances are subtracted from the relationship which either Daphnia species had with E. gracilis, the lack of significance is clearly exposed. The answer to our original question, therefore, is that the elemental composition of the Daphnia is controlled by the mixed algal culture and the spring water. E. gracilis clearly did not contribute.
T ABLE 26 MULTIPLE CORRELATIONS X denotes the dependent variable. N = 5 4 , d f = 3 , 50 for F test; df=50 for t test.
t value oJ partial correlation co~[licients D. magna
D. pulex
X X
* Not significant. ** 0.2 % level of significance. *** 0.1% level of significance,
98
E. gracilis
0.125" 0.365*
F value o f Mixed culture
Spring water
multiple correlation coefficients
3.48"* 5.15"**
4.21 *** 4.18"**
11.15"** 15.69"**
D. pulex behaves essentially like D. magna when feeding patterns are considered J. D. magna is able to obtain larger fragments from the water than D. pulex. However, at 20°C, the difference in mean filtering rate between D. magna and D. pulex was not significant 2°. Richman 21 has shown that the filtration rate per unit dry weight declines with the size of the animal and with per unit animal increases slowly reaching 5 ml per day for the largest specimens. The filtration rate was independent of the quantity of algae used for food. Both species of Daphnia clearly rejected E. gracilis. The reason for this situation is not clear. Pacaud 22 has shown that Euglena can be easily digested by Daphnia, though it is clear from Lef'evre's 23 work that the mixed culture also would be quite successful. It is not possible now to discover the cause of that rejection. A number of possible explanations, however, may be advanced. The cause may be the spatial arrangement between the two species of Daphnia and the Euglena in the glass aquaria. Another possibility may lie in the elemental content of Euglena as compared to the mixed culture. The Euglena contained more K, Cs, Li, Cu, Pb, V, S, Mo, F, Fe and Mn than either species of Daphnia or the mixed algal culture. It may be that E. gracilis was rejected because of its elemental composition. In any case, whatever the mechanism, on the basis of the elemental analysis it would appear that the two species of Daphnia were selective in their food intake. This is especially interesting in view of the fact that their diet was supplemented only occasionally with the mixed algal culture 3. Mineralogy of the plankton, the spring water and the trap rock A mineralogical study of the trap rock revealed the presence of quartz, albite, illite, montmorillonite and chlorite. The concentrated spring water contained an array of various hydrated forms of gypsum, the predominant mineral being the hemihydrate or bassanite. All the plankton showed a prominent band at 4.46/t~ which is characteristic of organic materials. Only D. pulex exhibited the characteristic second major reflection of bassanite at 6.02 A, which has a net intensity of 95 based on 100. The major line at 3.01/~ was absent, presumably due to an orientation effect incurred in the preparation of the sample. DISCUSSION
It is interesting to observe the differences in elemental concentration of the organisms and the trap rock from which the spring water flowed. All the plankton here studied are enriched in K, Rb, Zn, Cd, Hg, Y, La, Ce, Sm, Gd, Dy, Sn, P, Bi, Nb, S, Se, Mo, CI, Br and I relative to the quantity in the trap rock. The algae contain more Cu, Ag, As than does the trap rock. The Daphnia accumulate Ca, Nd and total rare earth elements above that in the trap rock. Cesium is accumulated by D. magna and E. gracilis in excess of that in the rock; Pr is enriched in all specimens except the mixed algal culture; and Eu is accumulated by all plankton save D. magna. 99
All concentrations of elements detected in the organisms are in excess of that found in the spring water. A comparison of these organisms in relation to other living organisms 24, all based on the composition of the continental crust 1°, shows, on the basis of available data, that all are impoverished in Na, Mg, Mn, Fe, AI, Si, Ni and Cr. All are enriched in P, S, Cd, Zn and probably Mo. Such a pattern found for different groups of living organisms supports the suggestion that life began in a water-rich environment in contact with the primitive atmosphere of the earth. The reasoning behind this suggestion lies in the assumption that the basic chemical composition of an organism should reflect its primordial chemical environment. It is further assumed that an organism would not become dependent on a rare element for its existence. This implies that an organism which now requires a rare element for its survival is merely reflecting the fact that the now rare element was more abundant in its ancestral environment. It is frequently assumed that if an organism takes up a given element that this element is used in some fashion. However, it is perfectly possible and even plausible that a given element may be taken up and used to control or limit the uptake of another element by substitution processes. The composition of Daphnia clearly reflects the composition of the water in which they live and the mixed algal culture which they consume. Were there an element present that was toxic to the Daphnia, one solution to the problem, of course, might be death. Another solution might be to precipitate a mineral that has sorbed water on or in which the toxic element could be sorbed. Another solution might to be take up another less toxic or harmless element, similar in charge and size to the toxic one, and engineer a substitution mechanism, thus bringing about an adjustment to a less than perfect environment. All of these suggestions are possible and some have occasionally been observed to occur in other organisms 25. Further research along such lines may clarify these suggestions. Toxicity experiments show that a given amount of an element provides an intolerable environment for the organism, resulting in its death. The results become enigmatic when, for example, less than 1 ppm Pb causes death, but the organism already contains considerably more Pb in its tissues. Anderson 14 examined toxicity levels of 21 elements using D. magna as a test organism and Lake Erie water as a medium. A comparison of the amount required to immobilize D. magna and the amount that D. magna contains in the present study shows that of the 21 elements only Na, Sn, Li, Co and Cr are less concentrated in the organism than the amount required for immobilization. Although it is more than likely that the toxicity level of a given element will vary with the environment to which it is added, and the reaction of the particular organism will vary with the environment and its composition will reflect the environment, the comparison of the Anderson 14 data and the composition of D. magna, may serve as a guideline. It is believed by some 26 that cations may be toxic due to their ability to combine with an essential sulfhydryl group on a key enzyme. It is further believed that this ability is directly proportional to the insolubility of the correspond100
ing metal sulfide. Thus, the most toxic metal ions are those which form the most insoluble sulfides. Of the elements which are less concentrated in the D. magna than in the solution required to immobilize it, only Sn forms a highly insoluble sulfide. Of the ones that are more concentrated, is it possible that some of these elements are accumulated to near lethal amounts and that the small additional amount results in death? It might be considered a protective device to accumulate an element to near toxic quantities. Such a concept, however, is based on the assumption of a stable environment, which almost never is the case. This enigmatic situation might be resolved by examining the elemental composition of an organism before and after toxicity experiments since these experiments inform us only that X amount of a compound makes life intolerable for a specific organism. It does not follow that the elements composing that compound are taken up by the organism. Such an approach, therefore, might also further our understanding of the problem of elemental accumulation in organisms generally.
ACKNOWLEDGEMENTS
This study was supported by the National Science Foundation. The plankton were cultured by Dr. C. W. Burns during her post-doctoral stay at the Biology Department at Yale University. She was ably assisted by Mrs. Helen Gray of the same department. Mrs. Nancy Baratz of the Kline Science Library at Yale University searched the scientific literature for comparative data on the organisms discussed in this paper. I am extremely grateful for the assistance offered by the organizations and persons mentioned above.
REFERENCES 1 G. E. Hutchinson, A Treatise on Limnology, John Wiley, New York, 1967, Vol. II, p. 307. 2 J. D. A. Miller and G. E. Fogg, Arch. MikrobioL, 28 (1957) 1. 3 U. M. Cowgill and C. W. Bums, Limnol. Oceanogr., 20 (1975) 1005. 4 J. S. Brown, U.S. Geol. Surv. Water-Supply Pap., 540 (1928). 5 R. C. Starr, Am. J. Bot., 47 (1960) 67. 6 J. G. Thompson, J. Res. Nat. Bur. Stand., 5 (1930) 1085. 7 U. S. Department of Commerce, Bur. Stand. Circ., 388 (1930) 1. 8 U. M. Cowgill, Sci. Total Environ., 2 (1973) 259. 9 U. M. Cowgill, Arch. Hydrobiol., 71 (1973)421. 10 S. R. Taylor, Geochern. Cosmochim. Acta, 28 (1964) 1273. 11 U. M. Cowgill, Dev. AppL Spectrosc., 10 (1972) 331. 12 G. E. Hutchinson, Int. Rev. Gesamten Hydrobiol. Hydrogr., 28 (1932) 90. 13 V. M. Goldschmidt, Geochemistry, Clarendon Press, Oxford, 1954. 14 B. G. Anderson, Trans. Ant. Fish. Soc., 78 (1950) 96. 15 C. E. Boyd and W. W. Wailey, Am. Midl. Nat., 88 (1972) 1. 16 C. E. Boyd, Ant. Midl. Nat., 84 (1970) 565. 17 U. M. Cowgill, Arch. Hydrobiol. Suppl., 45 (1974) i. 18 H. J. J. Bowen, Trace Elements in Biochemistry, Academic Press, London, 1966. 19 J. F. Kopp and R. C. Kroner, Trace Elements in Waters of the United States, Federal Water Pollution Control Administration, Cincinnati, Ohio, 1970.
lO1
20 21 22 23 24 25 26
C. W. Burns, LimnoL Oceanogr., 14 (1969) 697. S. Richman, Ecol. Monogr., 28 (1958) 273. A. Pacaud, Bull. BioL Ft. Belg. Suppl., 25 (1939) 1. M. Lef'evre, Bull. Biol. Fr. Belg., 76 (1942) 250. A. Banin and J. Navrot, Science, 189 (1975) 550. U. M. Cowgill, Arch. Hydrobiol., 74 (1974) 350. W. H. R. Shaw and B. Grushkin, Arch. Biochem. Biophys., 67 (1957) 447.
102
THE CHEMICAL COMPOSITIObl OF TWO SPECIES OF Daphnia, THEIR ALGAL FOOD AND THEIR ENVIRONMENT
U. M. COWGILL
Deparlment o~ BioloKv, UniversiO' t?/ Pittsburgh, PittsbulTzh, Pa. 15260 (U.S.A.) (Received September 28th, 1975)
ABSTRACT
Since the elements of which plankton are composed enter the food chain, it was thought interesting to devote a study to their chemical composition. Daphnia magna and D. pulex were reared in a laboratory for three months maintained on a monoculture of E. gracilis supplemented occasionally with a mixed algal culture. These plankton were raised in spring water. A complete chemical analysis revealed the plankton and the rock from which the spring water flowed to contain 54 detectable elements, while the concentrated water had only 53. Major differences were noted between the two species of Daphnia in their ability to accumulate K, Na, Ca, Sr, P, S, C1, and Co. The Daphnia and algae contained different amounts of Na, K, Mg, Ca, Zn, Al, Sc, La, Nd, Sn, Ti, P, S, Cl, Br, I, Fe, Mn, Ni, Zr, Cu, Ag, Be, Si, Pb and Hg. The composition of the Daphnia is governed by the mixed algal culture and the spring water. The Daphnia reject E. gracilis; the latter does not contribute to their chemical composition. Aside from supplementary additions of N, P, K and C1 to the algal cultures their composition is governed by that of the spring water.
INTRODUCTION
There is a large body of scientific literature concerning the nutrient requirements of the freshwater plankton 1. However, there are few studies of the chemical composition of the plankton themselves. The usual approach to the problem of estimating the nutrient requirements of a specific species has been to add a small quantity of some element to its medium and observe the response. This approach, though old and established, is not without difficulty. Additions of a monovalent or divalent cation to a growth medium may increase productivity merely by altering the ratio of monovalent to divalent ions, even though the added cations themselves are not limiting growth 2. The addition of a particular com79
pound may produce a positive response because the presence of some trace substance satisfies a deficiency while the principal elements involved in the compound in question are present in ample quantity. It, therefore, would appear useful to know something of the chemical composition of some plankton themselves. This paper concerns the elemental composition of two species of Daphnia, the algae they were fed, the spring water in which they were all reared, and the trap rock from which the spring water flows. Unfortunately, there are no available comparative data for these plankton. Since the elements of which the plankton are composed eventually find their way into the food chain, it was thought to be of interest to devote an investigation to their chemical composition. MATERIALS AND METHODS
Descriptions of the site, field and laboratory methods have been previously given 3 so that a brief summary should suffice.
Study site and fieid methods The plankton were cultured in the laboratory in water from West Spring. This spring is located on Springside Ave. which is on the east side of West Rock Ridge in New Haven, Conn. The ridge consists of Triassic Trap Rock with almost continuous exposure of rock, but is partly covered by till and talus 4. Three 10-kg samples of rock were collected from the vicinity from which the spring flowed. Water (174.7 1) was collected weekly for a period of two months.
Laboratory culturing methods Populations of Daphnia magna Straus, 1820 and Daphnia pulex Leydig, 1860 were cultured in autoclaved water from West Spring in glass aquaria, maintained at a constant temperature of 20°C under a 12-h light, 12-h dark cycle. The Daphnia populations were fed daily or once every two days, with a xenic monoculture of Euglena gracilis Klebs supplemented occasionally with mixed cultures containing E. gracilis and varying proportions of Chlamydomonas, Oocystis, Sphaerocystis, Ankistrodesmus, Scenedesmus, Closterium, Closteriopsis, a small filamentous green alga and a small filamentous blue-green alga. Daphnia were collected weekly for a period of three months. Algae used for analyses were cultured in autoclaved water from West Spring in 4-1 Pyrex Erlenmeyer flasks. These flasks were maintained at room temperature in constant light, fluorescent and natural daylight. Cells were kept in suspension by continuous aeration. Rubber tubing and sterile cotton filters were used to connect the aeration system to the flasks in series. Rubber stoppers filled with glass tubing and sterile cotton filters were used to seal the flasks. Both E. gracilis as well as the mixed algal culture were harvested weekly for a period of three months. E. gracilis was obtained from the Culture Collection of Algae, Indiana University, and stocks were maintained on nutrient agar 5. This organism was 80
TABLE 1 PERTINENT RESULTS OF THE CHEMICAL ANALYSES
Substance
D. pul~:x D. magna 15. gracilis Mixed culture Spring H20 (L.) Trap rock (%)
Oxide constituents (dry weight basis)
Ignition
14et H't.
Dry H't.
Loss
Major*
Minor
Total
(g)
(ca
(%)
(%)
(~ )
( ~o)
96.00 66.71 65.61 91.60 174.74
0.30 1.11 1.67 2.97 9.25 1.64
88.82 81.18 87.39 86.54 10.41 2.77
11.028 19.060 13.101 14.074 -95.810
0.085 0.081 0.124 0.111 -0.191
99.93 100.32 100.61 100.73 -100.41
* Na, K, Mg, Ca, AI, Si, Ti, P, S, CI, Mn, Fe.
used for inoculation of the monoculture. Algae for inoculation of the mixed culture came from a fish tank. In order to promote algal growth, occasionally 0.5 ml of a liquid fertilizer was added to 2 1 of spring water prior to autoclaving. A complete analysis of 100 ml of this liquid fertilizer (Hy-trous Corp., Boston, Mass.) showed it to contain 4 % total N, 8 % P205, 4 °/6 K20, 0.8 % CI and no detectable trace elements with the exception of less than 1 ppm Br. This analysis was carried out recently and was not performed on the same solution that was used to encourage the growth of the algae. The total amount used in culturing did not exceed 3 ml, and it is presumed that no detectable elements other than those encountered in the recent analysis of the fertilizer were added to the culturing media. Algae were harvested by centrifugation and Daphnia were collected by filtration through nylon netting which was fine enough to retain all instars but not the algae. C h e m i c a l techniques
Although all precautions were taken to avoid contamination 3 from alien sources, some unavoidable sources should be noted. West Spring, which was condemned several years ago, was fitted with a gravity pipe of unknown composition that had soldered joints. The spring, according to local residents, had been in use since 1919. The solder used during that period consisted largely of Sn and Pb 6. It is, however, quite possible that Cd, Hg and Bi may have been included 7. All cultures were maintained either in pyrex glass or commercial window glass, thus providing a possible source for the inclusion of extraneous B in the media. The effects of these possible sources of contamination will be considered later in this paper. Immediately on arrival at the laboratory the water was evaporated to dryness at 80°C; its total solids weighed 9.25 g. 81
The three samples of rock were weighed, dried, crushed and ground in the usual manner. The samples were manually mixed for 6 h each and two aliquots were taken from each sample. The chemical data presented here for the trap rock is a mean of these six aliquots. All plankton samples were rinsed in three changes of doubly distilled deionized water. The plankton were separated from the rinse water by centrifugation (8000 rpm, 30 rain); microscope examination did not reveal cell breakage while assay for potassium indicated no loss of cell contents. The supernatant liquid was poured off. The centrifuge tubes were weighed on an analytical balance, dried in an oven at 60°C for one week and reweighed. The results are shown in Table 1. All chemical analyses are presented on a dry weight basis. It is clear that the wet weights are anomalously high. Daphnia retain variable amounts of water between the two valves of the carapace, making its removal difficult. Thus, it is thought that the wet weights are in error, and all chemical data are expressed on a dry weight basis. The general technique involved in the preparation of samples for analysis by X-ray emission and optical emission have been previously described s.
Instrumental precision Despite the fact that methods used to gather these data have been previously discussed, the precision with which the concentration of elements here described has been determined should be mentioned. The quantities of the elements detected by X-ray emission spectroscopy were all determined in a helium
TABLE 2 HIGHEST AND LOWEST CONCENTRATIONS OF ELEMENTS FOUND IN THE PLANKTON BY OPTICAL EMISSION SPECTROSCOPY AND THE RELATIVE STANDARD DEVIATION FOR COUNTS FROM 20 DUPLICATES
Element
Low Be Hg Bi Sn Co B Mo V Ag Li Na Cd
82
Relative standard deviation (%)
Conc. (ppnO
0.017 2.68 1.0 1.2 0.7 12.0 0.2 1.3 0.46 0.28 .200 1.24
H~h
Low
High
0.088 4.25 1.8 23.5 3.8 17.8 6.45 6.4 4.7 0.60 1180 1.38
9.8 7.2 8.8 4.7 3.2 2.5 4.6 3.6 8.3 6.2 1.7 6.6
4.9 3.3 9.2 3.8 4.4 2.4 4.4 6.8 3.0 5.8 2.1 6.8
tunnel. All X-ray tubes (Cr, Mo, Pt) were used at maximum rating. Whenever possible, counts were accumulated to 10,000. Thus, .K, Ca, P, S, CI, Fe, Cu, As, Pb, Ga and Sr were determined with a standard counting error or a precision of less than 1%; Y, F and Ce with a precision of less than 2 %; AI, Si, Mn, Zn, Rb and Br with a precision of less than 3 96 ; Nb, Pr, Yb, Se, Ge, Cr and Zr with a precision of less than 4 %; Ni and Mg with a precision of less than 5 °/6; Ba, Nd and Ti with a precision of less than 6 %; La, Sm and Eu with a precision of less than 10 9;, ; Hf, Sc and Er with a precision of less than 12 % ; and finally, I, Cs, Dy and Gd with a precision of less than 15 %. Each of these elements was counted on three separate and non-consecutive occasions, and the results were considered to be reproducible only when the net counts agreed with previous occasions within less than 1%. Table 2 shows the highest and lowest concentrations of elements found in the plankton by optical emission spectroscopy and the percent relative standard deviation for counts from 20 duplicates.
Mineralogical techniques A mineralogical analysis was carried out on the trap rock, the concentrated dried water sample, and the four samples of plankton. Techniques involving the use of an X-ray diffractometer have been previously described 9. All results were confirmed with microscopic techniques. RESULTS
Before discussing the results of this study in detail, it should be noted that the trap rock from which West Spring flowed was collected after the spring had been condemned and closed. In order to ascertain whether it was valid to compare the composition of the spring with that of the rock, a linear correlation coefficient was determined between the concentration of the 54 elements in the spring water (Eu=0) and that in the rocks from which it used to flow. The coefficient obtained (r=0.8421) was statistically significant beyond the 0.1% level.
The alkafi metals Table 3 shows the distribution bf Na, K, Rb, Cs and Li in the four plankton samples, the spring water and the trap rock. The Daphnia clearly accumulate more Na and Rb on the average than do the algae, while the algae concentrate higher quantities of K, Cs and Li than do the Daphnia. Relative to the composition of D. magna, D. pulex contains notably less Na, K and Cs but more Rb, with the Li content being about the same. The Rb:K ratio (Table 4) in the spring water is higher than that of either the earth's crust j° or the trap rock, suggesting that there is an enrichment of Rb relative to K in the water. It would appear, on the basis of the ratios, that both groups of plankton tend to exclude Rb relative to K to some extent. The Cs:K ratio is the same in the trap rock and the spring, both being 83
TABLE 3 ALKALI METALS: Na, K, Rb, Cs, Li Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Conc. (ppnO Na
K
Rb
Cs
Li
861 1 180 252 200 0.103 15 753
8 003 13 839 17 898 13 955 0.258 5 562
35.3 22.9 26.4 20.7 1.9×10 -3 18.9
0.82 1.38 1.77 1.12 5.6x10 -5 1.26
0.38 0.28 0.60 0.42 2x10 -4 6.23
TABLE 4 COMPARISON OF THE Rb:K AND Cs:K RATIOS IN PLANKTON, SPRING WATER, TRAP ROCK AND IN THE EARTH'S CRUST Substance
Rb :K
~s :K
Earth's crust 10 Trap rock Spring H20 Daphnia, m Algae, ffi
4.3× 10-3 3.4x 10-3 7.2x10 -3 2.7x10 -3 1.5x10 -3
1.4× 10-4 2.3 × 10- 4 2.2×10 -4 1.0x10 -4 0.9x10 -4
somewhat higher than that of the earth's crust j°. Relative to K, Cs is excluded to some extent by both groups of organisms. The Rb:K ratio of the two groups of plankton are similar, though that of the Daphnia is more reminiscent of the trap rock than is that of the algae.
Copper and silver Table 5 shows the distribution of Cu and Ag in the two groups of plankton, the water and the trap rock. The algae concentrate both elements to a far greater extent than do the two species of Daphnia. The Ag:Cu ratio of the earth's crust (1.3×10 -3) and that of the spring water are quite similar (1.8x10-3); however, this ratio in the algae (21x10 -3) as well as in the Daphnia (8x10 3) suggest that there is a notable enrichment of Ag relative to Cu in these organisms. When this ratio in the spring water (1.Sx10 -3) is compared to that in the trap rock (0.1xl0 -3) through which it flows, the enrichment of Ag relative to Cu is sizeable. Relative to the earth's crustal composition ~o the trap rock is enriched in Cu and impoverished in Ag. It is noteworthy to mention that this entire study was carried out during the winter. Silver is more concentrated in the winter than in the summer in many lakes 11 in the general region around West Spring. It is believed that this obser84
TABLE 5 Cu AND Ag CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
(one. (ppnO Cu
Ag
70.0 77.5 283.2 156.5 3.8×10 -3 89.9
0.78 0.46 4.7 4.6 6.8×10 -6 0.009
vation results from the seeding of clouds with AgI to encourage snow fall. The possibility of atmospheric contribution to the Ag content of the plankton should, therefore, not be ignored. Despite this possibility, it is clear that the two groups of organisms take up Ag to a different extent.
Beryllium Beryllium exhibits a seasonal distribution in lakes H in the vicinity of West Spring, being more concentrated in the winter than in the summer. It is believed that the source of the contribution is the combustion of fossil fuels. The possibility of atmospheric contribution to the Be content of the plankton should, therefore, not be ignored. The algae accumulate more Be than do the Daphnia (Table 6). The Be:Li ratio for the Daphnia is 0.07, for the algae is 0.17, and for the spring water is 0.075. This would suggest that Be moves more easily from the water to the algae than does Li. Similarly Be moves easily from the trap rock (Be:Li 0.02) to the water than does Li. It will be observed later that the same situation prevails in the case of Mg, where Be is enriched relative to this alkaline earth.
TABLE 6
Be CONCENTRATIONS Substance
Be (ppnO
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
0.017 0.028 0.088 0.084 1.5×10 -5 0.123
85
The alkaline earths Table 7 shows the distribution of Mg, Ca, Sr and Ba in the plankton, the spring water and the trap rock. The Mg content of the algae is 10.6 times that of the Daphnia while the latter accumulate 6.9 times more Ca than the former. As would be expected, Sr follows Ca. D. pulex contains slightly less Mg than does D. magna and considerably less Ca. It is curious that tolerance experiments using D. pulex in continuous culture have shown that 120 ppm of Mg is the upper limit 12. D. pule× accumulates more Ba than any of the other organisms. Relative to their composition in the continental crustl° Sr, Ba and Be are impoverished and Ca and Mg enriched in the trap rock (Table 8). Considering the Sr:Ca ratio in the spring water, Sr is excluded by both groups of organisms relative to Ca. There clearly is an enrichment of Sr relative to Ca in the spring water. Relative to Ca in the spring water, Ba is excluded by the Daphnia and accumulated by the algae. The Be:Mg ratios indicate that Be moves more easily to the Daphnia from the water than does Mg, and to a lesser extent this is also true in the case of the algae. However, in reference to Ca in the spring water, Be moves more easily to the algae while it is almost excluded by the Daphnia. ,The explanation for this peculiar situation lies in the large differences in accumulation of Mg and Ca in the two groups of organisms.
TABLE 7 ALKALINE EARTHS: Mg, Ca, Sr, Ba
Substance
Com'. (ppm)
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Mg
Ca
Sr
Ba
549 633 5 996 6 520 1.88 40 058
36 878 76 643 7 311 9 112 4.49 57 151
45.8 81.9 40.0 35.3 0.033 129.3
84.3 67.1 48.5 44.5 0.014 126
TABLE 8 COMPARISON OF THE Sr:Ca, Ba:Ca, Be:Mg, Be:Ca RATIOS IN PLANKTON, SPRING WATER, TRAP ROCK AND IN THE EARTH'S CRUST
Substance
Sr :Ca
Ba :Ca
Be :Mg
Be:Ca
Eanh's crust Io Trap rock Spring H20 Daphnia Algae
9
10.2x10 -3 2.2× 10-3 3.1×10 -3 1.3× 10-3 5.7× 10-3
1.2 xlO -4 0.03× 10-4 0.08)<10 -4 0.38× 10-4 0.14× 10-4
6.7 ×10 -s 0.2 × 10-5 0.3 ×10- 5 0.03x 10-5 1.04x 10-5
86
x10 -3
2.3× 10-3 7.3)<10-3 1.1 x 10-3 4.6x 10-3
Zinc, cadmium and mercuiy Table 9 shows the distribution of Zn, Cd and Hg in the plankton, the spring water and the trap rock. The mean Zn and Hg content of the Daphnia is lower than that in the algae while the Cd concentration is about the same for the two groups.
TABLE 9 Zn, Cd AND Hg CONCENTRATIONS
Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Conc. (ppnO Zn
Cd
Hg
134.6 102.0 304.0 338.7 0.012 92.4
1.34 1.24 1.38 1.34 1.6×10-5 0.38
2.68 3.30 4"20 4.25 1.3×10 -4 0.025
Goldschmidt 13 has suggested that the geochemistry of Hg is in some ways analogous to that of Cd. The geochemical ratio of Hg to Cd in the continental crust I° is 0.4. That in the trap rock is 0.07, in the spring water is 8.13 while in the Daphnia it is 2.32 and in the algae 3.11. Compared to the ratio in the spring water, Cd moves more easily to both groups of organisms than does Hg. It should be noted that both Cd and Hg could have been contributed to the spring water through its contact with the soldered joints 7 of the gravity pipe through which the spring flowed. A second possible contributory source could have been the Hg contamination in'the culture rooms. A third source may have been the combustion of fossil fuels in the winter 8. There is no question that the Hg content of the plankton is unexpectedly high. Both cultures of algae contain more Hg than the Daphnia. The Daphnia may have obtained their Hg content by consumption of algae and possibly some from the water in which they were reared. However, the Daphnia were cultured in open aquaria in constant temperature chambers, while the algae were raised in sealed flasks and aerated with piped-in building air filtered through cotton wool. It therefore remains unclear what mechanism was proceeding to permit the entrance of Hg into the sealed flasks. Though it seems highly unlikely, the algae may have accumulated Hg to an enormous extent from the water in which they were reared. Only further investigation will elucidate this point. In reference to the above discussion, it is interesting to note that the threshold concentration of Hg for the immobilization of D. magna is 0.0044 ppm when Hg is added as HgClz to Lake Erie water j4. 87
Aluminum, gallium and boron Table 10 shows the distribution of AI, Ga and B in the plankton, the spring water and the trap rock. The AI concentration in the Daphnia is lower than that in the algae but the Ga concentration is higher. The ratio of Ga:AI" in the spring water would suggest that Ga moves more easily to the Daphnia than does AI (Table 11). In reference to AI, however, Ga is vastly enriched in the spring water when the Ga:AI ratio in the trap rock is considered. It is interesting to note that the Ga:AI ratio in the spring water is essentially the same as that in the algae.
TABLE 10 AI, Ga AND B CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppt~O AI
Ga
B
127.0 121.7 190.5 232.9 0.207 53 179
0.94 0.68 0.52 0.59 4.9x10 -4 15.8
14.0 14.0 12.0 17.8 0.058 130
The B concentration of the spring water is only slightly higher (0.047) than the mean proposed by Boyd and Walley 15, and that of the algae is within the range found by B o y d 16. This would suggest that the possible contribution to the B concentration in the algae and Daphnia from the glassware in which they were reared is probably minimal. The mean concentration of B in the two groups of plankton is essentially the same.
Scandium, yttrium and the rare earths Table 12 shows the distribution of Sc, Y and the rare earths in the plankton, the spring water and the trap rock. A comparison between the concentration of Sc, Y and the ten detectable rare earths (Eu=0) in the water and that in the trap rock revealed a linear correlation coefficient of 0.841 which is significant well beyond the 0.1 .% level. All the rare eai'ths of even atomic number were detected; of the odd-numbered members of the group only La, Pr and Eu were found and the latter element was not at a detectable concentration in the concentrated spring water. The others were presumably present in amounts below detection. If the ratios of AI, Sc, Y and the rare earth elements (2; Ln) are compared in the Daphnia and the trap rock, and in the algae and the trap rock (Table 13), it becomes apparent that AI and Sc are absorbed to a moderate extent but Y and the rare earths are accumulated in quantity. 88
~
b'~
',o
x
~
i
x
X
X
X
X
r'~ t'-,,I '~1- o
X ~
×
z
¢~ ×
©
¢.,i ¢.~ e,i e,~ I I I I !
X
X
X
X
X
Z
Z
N~
X
bZ Z
N
X
X
X
X
m z ,<
,,.t XX
X
×
Z 0
~
x~
m
XX
×
1
I
)'(X
X
I
I
X
X
7
g -d z e~
d "~.~ ..1 •.--
~. ~
~
~.~
89
TABLE 13 RATIOS OF AI, Sc, Y AND THE TOTAL LANTHANIDE RARE EARTHS IN THE PLANKTON, THE SPRING WATER AND TRAP ROCK
Com. (ppm)
Earth's crust Io Trap rock Spring H20 Daphnia if5
D. nlagna D. pulex Algae ~
E. gracilis Mixed culture
AI
Sc
)'
ZLn *
82 300 53 179 0.207 124.4 121.7 127.0 211.7 190.5 232.9
22 39.7 6.4x10 -4 18.4 16.8 20.0 6.95 6.8 7.1
33 19.7 1.6×10-3 41.5 50.6 32.4 57.4 53.3 61.5
146.4 112.1 5.04×10 -3 132.9 124.6 141.1 95.9 101.7 90.0
Ratios Daphnia: Trap rock Daphnia: Spring H20 Daphnia: Earth's crust lo
2.3× 10-3 601 1.5x10 -3
46x 10-2 28 750 84x10 -2
2.11 25 938 1.26
1.19 26 369 0.91
Algae: Trap rock Algae: Spring H20 Algae: Earth's crust l°
4.0×10 -3 1023 2.6×10 3
17.5x10 -2 10 859 32x10-2
2.91 35 875 1.74
0.86 19 028 0.66
* Total lanthanide rare earths.
TABLE 14 THE GEOCHEMICAL RATIOS OF SOME ELEMENTS IN THE PLANKTON, SPRING WATER, TRAP ROCK AND THE EARTH'S CRUST lo
Ratio
Earth's crust I0
Trap rock
Spring H 20
Daphnia
AIgae
Sc:Ti Y:Ti La:Ti Ce:Ti Pr:Ti NO:Ti Sm:Ti Eu:Ti Gd:Ti Dy:Ti Er:Ti Yb:Ti AI:Ti Ca:Ti P:Ti
3.9 x10 -3 5.8 ×10 -3 5.3 ×10 -3 10.5 ×10 -3 1.4 x l 0 -3 4.9 ×10 -3 1.1 x l 0 -3 0.21x10 -3 0.94×10 -3 0.53x10 -3 0.49×10 -3 0.53x10 -3 14.39 7.37 184×10 3
5.8 ×10 -3 2.9 ×10 -3 1.7 ~,,10 3 10.5 ×10 3 0.50× 10--3 1.4 ~I0 3 0.46×10 -3 0.16x10 -3 0.40×10 -3 0.11×10 3 0.71×10 3 0.48×10 -3 7.78 8.36 60×10 3
160 x l 0 -3 400 x l 0 -3 225 ×10 -3 600 ×10 -3 57.5x10 -3 237.5x10-3 11 x l 0 -3 -24.5×10 -3 40 x10 -3 15 x l 0 -3 50 x l 0 -3 51.75 1122.5 7250x10 -3
7.83 17.66 21.74 18.23 2.26 7.62 2.06 0.49 1.77 1.36 0.30 0.70 52.91 24 153 6 186 170x10 -3
0.49 3.99 1.59 3.21 0.24 0.60 0.29 0.09 0.27 0.15 0.09 0.13 14.70 570 1 876 910x10-3
90
The Sc concentration in the Daphnia is 2.6 times that in the algae. In the case of Y, the algae contain slightly more than the Daphnia. It may be that both higher and lower aquatic plants exclude Sc from their tissues ~7. On the average the algae absorb less La, Pr and Nd than do the Daphnia. The concentration of Ce, Sm, Eu, Gd, Dy, Er and Yb is essentially the same in both groups of plankton. It is curious that the algae also contain less Ca than the zooplankton. Since La can substitute for Ca, it may be that the Ca concentration in the algae is controlled by the simultaneous uptake of La. Titanium is probably the least soluble and least mobile of the common elements of the earth's crust. This element, therefore, forms a convenient reference point to which other elements may be compared. Table 14 shows the ratios of the rare-earth elements to Ti. Relative to Ti in the earth's crust, Sc and Er are the only elements of this group enriched in the trap rock. As one would expect, relative to the Ti in the trap rock, the whole group is vastly enriched in the spring water. Relative to Ti, this whole group of elements is enriched in the plankton but vastly more so in the Daphnia than in the algae. It is interesting to note that relative to Ti, Ca is enriched in the trap rock, the spring water and the plankton. Phosphorus is impoverished relative to Ti in the trap rock when the P:Ti ratio in the continental crust 1° is considered. The enrichment of P relative to Ti is enormous in the spring water and the plankton when the P:Ti ratio in the trap rock is considered. It is curious that Eu was not detected in the concentrated water. Though the concentration of this rare earth was too low in the spring water to be estimated, it could be found in the plankton, thereby confir~ning its presence in the water.
Silicon, germanium, tin and lead Table 15 shows the distribution of Si, Ge, Sn and Pb in the plankton, the spring water and the trap rock. Bowen 17 suggests as a mean value for freshwater 4×10 -5 for Sn and 5 × 1 0 -3 for Pb. The Pb content of the spring water is below the suggested mean while Sn is much above it. As mentioned earlier, the source
TABLE 15 Si, Ge, Sn AND Pb CONCENTRATIONS
Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppm) Si
Ge
Sn
Pb
892.9 771.3 724.6 1 136.0 6.8 244 252
0.29 0.20 0.25 0.18 1.8× 10-5 1.9
4.2 1.2 23.5 20.0 2× 10-3 0.62
9.8 7.2 12.6 10.0 8.5x 10-4 39.4
91
of the Sn could well be the solder 6 on the joints of the gravity pipe through which the spring water flowed. If this were true, one would expect the Pb also to be high. However, due to the Ca concentration of the water, it may be that the Pb which was extractable had been removed long before sampling. Since the composition of the gravity pipe is unknown, it is also possible that some Sn may have been in the pipe itself. In any case, it is quite clear that algae accumulate far more Sn than do the zooplankton; this is also true of Pb, but to a lesser extent. The threshold concentration for the immobilization of D. magna by Sn is 66.5 ppm (SnC14) and by Pb is 0.93 ppm (PbCI2) when added to Lake Erie water J4. It would certainly seem reasonable to suggest that D. magna excludes Sn from its tissues, especially in view of the fact that D. pulex contains 3.5 times as much. The tolerance level proposed for Pb is rather curious since D. magna accumulates far more Pb than the tolerance level would lead one to suppose. The Ge content is essentially the same in both groups of organisms. The mixed culture contains more Si than any of the other organisms. Of the monocultures, D. pulex contains the most while E. gracilis contains the least. It may be that some of the species in the mixed culture accumulate Si to a large extent.
Zirconium, haJhium and titanium Table 16 shows the distribution of Zr, Hf and Ti in the plankton, spring water and the trap rock. The Daphnia contain more Zr and Hf and considerably less Ti than do the algae. As would be expected, Hf follows Zr. Relative to Zr in the spring water, both groups of organisms tend to exclude Hf from their tissues (Table 11). TABLE 16
Zr, Hf AND Ti CONCENTRATIONS Sttbstance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppnO Zr
HJ
Ti
14.9 15.9 7.6 9.8 6.7x10 -3 82.1
0.012 0.014 0.006 0.009 1.2×10 -4 2.54
2.4 2.3 11.4 17.4 4 x I 0 -3 6834
Phosphorus The distribution of P in the two groups of plankton (Table 17) shows that the Daphnia accumulated about half as much as do the algae. The P in the trap rock is impoverished relative to the earth's crust (1050 ppm) 1°. Reported concentrations of P in water are highly variable 17 but Bowen j8 proposes a mean value of 0.005 ppm for freshwater. The amount in the spring water is far higher. AI92
TABLE 17 P CONCENTRATIONS Substance
P (ppnO
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
13 141 15 934 23 095 30 960 0.029 407
though it has not been possible to ascertain the reason for the closure of the spring, it may be presumed that it was due to contamination from human sources. Kopp and Kroner j9 proposed a mean P concentration for waters of the United States of 0.12ppm with a range of 0.002-5 ppm. West Spring certainly falls within their measured range.
Arsenic and bismuth Table 18 shows the distribution of As and Bi in the plankton, the spring water and the trap rock. The As concentration is slightly higher in the algae than in the Daphnia, while the mean concentration of Bi is about the same in both groups. As more As data are gathered it may turn out that aquatic plants genTABLE 18 As AND Bi CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Conc. (ppnO As
Bi
2.3 2.2 3.6 3.4 2.4×10 -4 2.7
1.8 1.5 1.8 1.0 6.3×10 -5 0.07
erally contain more of this element than animals. Bismuth has no known biological function. Small quantities seem to be present in most living things. The amount in the plankton is higher than would be expected and in any case is accumulated to a great extent over that of the trap rock.
Vanadium and niobium D. pulex accumulates larger quantities of V and Nb than does D. magna (Table 19). E. gracilis contains more V than does the mixed algal culture and slightly more Nb. 93
TABLE 19 V AND Nb CONCENTRATIONS Substance
D. pulex D. magna £. gracilis
Mixed culture Spring H20 Trap rock
(ore. (ppm) 1
Nb
4.4 1.3 6.4 2.8 9×10 -6 48.7
16.1 11.9 14.1 13.3 5.8x10 -4 5.5
Vanadium is far more concentrated in lake waters in the vicinity of West Spring during me winter than the summerJL Again, it is possible that there is some contribution to the atmosphere resulting from the burning of fossil fuels. The amount of V in the spring water is extremely low compared to that suggested as a mean by Bowen ~8 Both V and Nb are impoverished in the trap rock when their quantity is compared with the crustal average ~°. The plankton accumulate Nb in excess of that found in the trap rock.
Sulfur and selenium The mean quantity of Se in the two groups of plankton is the same (Table 20). The mean S content of the algae is slightly higher than that of the Daphnia. Relative to S in the trap rock (Table 11) there is a considerable enrichment of Se in both groups of organisms. Both S and Se are accumulated by the organisms in excess of that found in the trap rock. Relative to the amount in the earth's crust 1° S is enriched and Se impoverished in the trap rock.
TABLE 20 S AND Se CONCENTRATIONS Substance
D. pulex D. magna E. gracilis
Mixed culture Spring H20 Trap rock
94
Com. (ppm) S
Se
3965 5647 5943 4201 0.626 512
2.1 1.6 2.0 1.9 3x 10-5 0.85
Chromium and molybdenum The Mo concentration in the algae is far greater than that in the Daphnia and both groups contain Mo in excess of that found in the trap rock (Table 21). The mean concentration of Cr is about the same for the two groups of organisms. D. magna contains less Cr and Mo than does D. pulex. Both elements are believed to be required for life 18. TABLE 21
Cr AND Mo CONCENTRATIONS Substance
D. pulex D. magna E. gracilis
Mixed culture Spring H20 Trap rock
Conc. (ppnO Cr
Mo
1.3 0.6 1.4 1.2 2.2×10 -5 62.4
0.7 0.2 6.5 5.7 1.3x10 -6 0.102
The halogens The differences in accumulation of CI between D. magna and D. pulex are striking (Table 22). The algae contain far less of the element than either of the Daphnia species. Bromine follows C1 in the four groups of organisms. Iodine is far more enriched in the two groups of algae than in the two species of Daphnia. The mean concentration of F in the two groups of organisms is the same. In reference to the CI in the spring water, the algae are enriched in F and the Daphnia exclude it (Table 11). In reference to Br in the spring water, the Daphnia accumulate C1 while the algae exclude it. Relative to C1 in the spring water, Daphnia excludes I while the algae accumulate it.
TABLE 22 HALOGENS: F, C1, Br, I
Substance
D. pulex D. magna E. gracilis
Mixed culture Spring H20 Trap rock
£ bnc. (ppnO F
CI
Br
I
12.4 9.4 13.1 9.9 0.019 275
3 198 10 130 360 400 1.45 128
27.1 37.1 18.0 14.2 0.02 3.8
0.54 0.69 1.75 1.40 6.7×10 -4 0.035
95
Iron, cobalt, nickel and manganese Table 23 shows the distribution of Fe, Mn, Co and Ni in the plankton, the spring water and the trap rock. The mean Fe, Mn and Co content is impoverished and the Ni enriched in the Daphnia relative to that in the algae. D. magna contains notably less Co than D. pulex. Relative to Mn in the spring water, Fe is accumulated by the algae and excluded by the Daphnia. Relative to Co, Fe is to some extent excluded by both groups of organisms, while compared to Ni, it is accumulated by the plankton (Table 11).
TABLE 23 Fe, Mn, Co AND Ni CONCENTRATIONS Substance
D. pulex D. magna E. gracilis Mixed culture Spring H20 Trap rock
Cone. (ppnO Fe
Mn
Co
Ni
1 014 916 12 961 10 534 0.164 99 624
132 94 279 240 4.4x10 -3 3588
3.3 0.7 3.8 2.8 1.1×10 -5 9.6
4.2 3.6 1.8 2.7 8.9x10 -4 44.7
Statistical analyses Table 24 shows the results of statistical analyses involving elemental comparison in the four plankton samples. The degrees of freedom are only 2 and therefore it is not possible to verify statistically the validity of these apparent relationships by multiple correlation techniques. Although the levels of significance encountered are weak, and the number of samples small, the results may be tentatively considered as indicative of rates of uptake of elements by very different plankton species. The relationships shown by Li, Ag, Be, Sn, Hg, As and Nb would suggest that this group has a biological function. They are all related at varying degrees of significance to elements known to be required for life ~8. Table 25 shows the linear correlation coefficients obtained by comparing the concentration of all elements in the organisms with those in the spring water. There is no statistically significant relationship between either algal culture and the water in which they were reared. Since the water in which the mixed culture and E. gracilis were grown had a liquid fertilizer supplement added prior to autoclaving, the concentrations of P, K and C1 were removed from the calculations and the linear correlation coefficients were again determined. The concentration of the elements in the water compared to that in E. gracilis gave a linear correlation of 0.337 which is well beyond the 1% level of significance, while the linear 96
TABLE 24 LINEAR CORRELATION COEFFICIENTS AMONG THE ELEMENTS FOUND IN THE PLANKTON (N=4) Elements
Na, Zr Na, Hf Na, CI Na, Br K, Cs Cs, S Li, V Cu, 1 Cu, Fe Cu, Mn Ag, Mg Ag, Ti Ag, Mo Ag, Fe Ag, Mn Be, Mg Be, Hg Be, Ti Be, 1 Be, Fe Be, Mn Mg, Ti Mg, Mo Mg, I Ca, Sr Ca, CI Ba, 2Ln* Zn, Sn * 2Ln--total
P=O.I
P=O.05
P=O.02
O. 90(0
O. 9500
O. 9800
0.9491 0.9228 0.9254 0.9850 0.9420 0.9337 0.9054 0.9537 0.9234 0.9137 0.9944 0.9360 0.9970 0.9890 0.9800 0.9880 0.9741 0.9210 0.9800 0.9860 0.9460 0.9630 0.9840 0.9460 0.9633 0.9904 0.9889 0.9694
Elements
Zn, Ti Zn, As Zn, Mo Zn, Fe Zn, Mn Hg, P AI, Ti AI, As Sc, La Sc, Nd Ce, V Sn, Mo Sn, Fe Sn, Mn Zr, Hf Ti, Mo As, Fe As, Mn V, F Nb, Se Se, Cr Se, Co Cr, Co Mo, I Mo, Fe Mo, Mn CI, Br Fe, Mn
P=O.I
P=O.05
P=O.02
P=O.OI
O. 9000
0.9500
O. 9800
O. 9990
0.971 0.9732 0.9748 0.956 0.9523 0.9104 0.9994 0.9070 0.9984 0.9774 0.9551 0.9960 0.9942 0.9963 0.9810 0.9041 0.9977 0.9945 0.9574 0.9381 0.9565 0.9432 0.9973 0.9750 0.9968 0.9896 0.9580
6~9850
L a n t h a n i d e rare earths.
correlation involving the mixed culture provided a coefficient of 0.449 which is well beyond the 0.1 °/6 level of significance. A multiple correlation coefficient employing these linear ones reveals R to be well beyond the 0.5 % level of significance; however, a t test of significance applied to the partial correlation coefficients showed that the composition of the mixed culture reflected that of the spring water, but this was not the case with E. gracilis. The question was posed as to which of the following substances was most important in determining the elemental composition of D. pulex:E, gracilis, the mixed algal culture, or the spring water? A multiple correlation was employed to answer this question (Table 26). The relationships between D. pulex and E. gracilis ~ere totally insignificant. That between D. pulex and the mixed culture was highly significant and that between D. pulex and the spring water was significant well beyond the 0.1 °/6 level.
97
TABLE 25 L I N E A R C O R R E L A T I O N C O E F F I C I E N T S B E T W E E N THE E L E M E N T A L COMPOSITION OF T HE P L A N K T O N A N D THE SPRING W A T E R (?~=54)
Linear correlation coe[/ictents
D. pulex D. magna E. gracilis Mixed culture
D. puh'x
D. magna
E. gracilis
Mixed culture
Spring water
--
0.9887* -0.4154"* 0.4403*
0.5226* 0.4154"* -0.9547*
0.5581 * 0.4403* 0.9547* --
0.5013" 0.5188" 0.1430"** 0.1583"**
0.9887* 0.5226* 0.5581"
* 0.1% level of significance. ** 0.5 % level of significance.
*** Not significant.
Posing the same question in reference to D. magna produced somewhat different results. E. gracilis did not in any way contribute to the composition of D. magna but both the mixed culture and the spring water did. However, the latter is far more significant than the former. It is noteworthy to point out that linear correlation coefficients should be examined by multiple correlation techniques to ascertain their validity. The relationship between D. pulex and E. gracilis is significant well beyond the 0.1% level and that with D. magna beyond the 0.5 % level. These relationships are spurious because they reflect the effect of the mixed algal culture and the spring water. When the effects of these two substances are subtracted from the relationship which either Daphnia species had with E. gracilis, the lack of significance is clearly exposed. The answer to our original question, therefore, is that the elemental composition of the Daphnia is controlled by the mixed algal culture and the spring water. E. gracilis clearly did not contribute.
T ABLE 26 MULTIPLE CORRELATIONS X denotes the dependent variable. N = 5 4 , d f = 3 , 50 for F test; df=50 for t test.
t value oJ partial correlation co~[licients D. magna
D. pulex
X X
* Not significant. ** 0.2 % level of significance. *** 0.1% level of significance,
98
E. gracilis
0.125" 0.365*
F value o f Mixed culture
Spring water
multiple correlation coefficients
3.48"* 5.15"**
4.21 *** 4.18"**
11.15"** 15.69"**
D. pulex behaves essentially like D. magna when feeding patterns are considered J. D. magna is able to obtain larger fragments from the water than D. pulex. However, at 20°C, the difference in mean filtering rate between D. magna and D. pulex was not significant 2°. Richman 21 has shown that the filtration rate per unit dry weight declines with the size of the animal and with per unit animal increases slowly reaching 5 ml per day for the largest specimens. The filtration rate was independent of the quantity of algae used for food. Both species of Daphnia clearly rejected E. gracilis. The reason for this situation is not clear. Pacaud 22 has shown that Euglena can be easily digested by Daphnia, though it is clear from Lef'evre's 23 work that the mixed culture also would be quite successful. It is not possible now to discover the cause of that rejection. A number of possible explanations, however, may be advanced. The cause may be the spatial arrangement between the two species of Daphnia and the Euglena in the glass aquaria. Another possibility may lie in the elemental content of Euglena as compared to the mixed culture. The Euglena contained more K, Cs, Li, Cu, Pb, V, S, Mo, F, Fe and Mn than either species of Daphnia or the mixed algal culture. It may be that E. gracilis was rejected because of its elemental composition. In any case, whatever the mechanism, on the basis of the elemental analysis it would appear that the two species of Daphnia were selective in their food intake. This is especially interesting in view of the fact that their diet was supplemented only occasionally with the mixed algal culture 3. Mineralogy of the plankton, the spring water and the trap rock A mineralogical study of the trap rock revealed the presence of quartz, albite, illite, montmorillonite and chlorite. The concentrated spring water contained an array of various hydrated forms of gypsum, the predominant mineral being the hemihydrate or bassanite. All the plankton showed a prominent band at 4.46/t~ which is characteristic of organic materials. Only D. pulex exhibited the characteristic second major reflection of bassanite at 6.02 A, which has a net intensity of 95 based on 100. The major line at 3.01/~ was absent, presumably due to an orientation effect incurred in the preparation of the sample. DISCUSSION
It is interesting to observe the differences in elemental concentration of the organisms and the trap rock from which the spring water flowed. All the plankton here studied are enriched in K, Rb, Zn, Cd, Hg, Y, La, Ce, Sm, Gd, Dy, Sn, P, Bi, Nb, S, Se, Mo, CI, Br and I relative to the quantity in the trap rock. The algae contain more Cu, Ag, As than does the trap rock. The Daphnia accumulate Ca, Nd and total rare earth elements above that in the trap rock. Cesium is accumulated by D. magna and E. gracilis in excess of that in the rock; Pr is enriched in all specimens except the mixed algal culture; and Eu is accumulated by all plankton save D. magna. 99
All concentrations of elements detected in the organisms are in excess of that found in the spring water. A comparison of these organisms in relation to other living organisms 24, all based on the composition of the continental crust 1°, shows, on the basis of available data, that all are impoverished in Na, Mg, Mn, Fe, AI, Si, Ni and Cr. All are enriched in P, S, Cd, Zn and probably Mo. Such a pattern found for different groups of living organisms supports the suggestion that life began in a water-rich environment in contact with the primitive atmosphere of the earth. The reasoning behind this suggestion lies in the assumption that the basic chemical composition of an organism should reflect its primordial chemical environment. It is further assumed that an organism would not become dependent on a rare element for its existence. This implies that an organism which now requires a rare element for its survival is merely reflecting the fact that the now rare element was more abundant in its ancestral environment. It is frequently assumed that if an organism takes up a given element that this element is used in some fashion. However, it is perfectly possible and even plausible that a given element may be taken up and used to control or limit the uptake of another element by substitution processes. The composition of Daphnia clearly reflects the composition of the water in which they live and the mixed algal culture which they consume. Were there an element present that was toxic to the Daphnia, one solution to the problem, of course, might be death. Another solution might be to precipitate a mineral that has sorbed water on or in which the toxic element could be sorbed. Another solution might to be take up another less toxic or harmless element, similar in charge and size to the toxic one, and engineer a substitution mechanism, thus bringing about an adjustment to a less than perfect environment. All of these suggestions are possible and some have occasionally been observed to occur in other organisms 25. Further research along such lines may clarify these suggestions. Toxicity experiments show that a given amount of an element provides an intolerable environment for the organism, resulting in its death. The results become enigmatic when, for example, less than 1 ppm Pb causes death, but the organism already contains considerably more Pb in its tissues. Anderson 14 examined toxicity levels of 21 elements using D. magna as a test organism and Lake Erie water as a medium. A comparison of the amount required to immobilize D. magna and the amount that D. magna contains in the present study shows that of the 21 elements only Na, Sn, Li, Co and Cr are less concentrated in the organism than the amount required for immobilization. Although it is more than likely that the toxicity level of a given element will vary with the environment to which it is added, and the reaction of the particular organism will vary with the environment and its composition will reflect the environment, the comparison of the Anderson 14 data and the composition of D. magna, may serve as a guideline. It is believed by some 26 that cations may be toxic due to their ability to combine with an essential sulfhydryl group on a key enzyme. It is further believed that this ability is directly proportional to the insolubility of the correspond100
ing metal sulfide. Thus, the most toxic metal ions are those which form the most insoluble sulfides. Of the elements which are less concentrated in the D. magna than in the solution required to immobilize it, only Sn forms a highly insoluble sulfide. Of the ones that are more concentrated, is it possible that some of these elements are accumulated to near lethal amounts and that the small additional amount results in death? It might be considered a protective device to accumulate an element to near toxic quantities. Such a concept, however, is based on the assumption of a stable environment, which almost never is the case. This enigmatic situation might be resolved by examining the elemental composition of an organism before and after toxicity experiments since these experiments inform us only that X amount of a compound makes life intolerable for a specific organism. It does not follow that the elements composing that compound are taken up by the organism. Such an approach, therefore, might also further our understanding of the problem of elemental accumulation in organisms generally.
ACKNOWLEDGEMENTS
This study was supported by the National Science Foundation. The plankton were cultured by Dr. C. W. Burns during her post-doctoral stay at the Biology Department at Yale University. She was ably assisted by Mrs. Helen Gray of the same department. Mrs. Nancy Baratz of the Kline Science Library at Yale University searched the scientific literature for comparative data on the organisms discussed in this paper. I am extremely grateful for the assistance offered by the organizations and persons mentioned above.
REFERENCES 1 G. E. Hutchinson, A Treatise on Limnology, John Wiley, New York, 1967, Vol. II, p. 307. 2 J. D. A. Miller and G. E. Fogg, Arch. MikrobioL, 28 (1957) 1. 3 U. M. Cowgill and C. W. Bums, Limnol. Oceanogr., 20 (1975) 1005. 4 J. S. Brown, U.S. Geol. Surv. Water-Supply Pap., 540 (1928). 5 R. C. Starr, Am. J. Bot., 47 (1960) 67. 6 J. G. Thompson, J. Res. Nat. Bur. Stand., 5 (1930) 1085. 7 U. S. Department of Commerce, Bur. Stand. Circ., 388 (1930) 1. 8 U. M. Cowgill, Sci. Total Environ., 2 (1973) 259. 9 U. M. Cowgill, Arch. Hydrobiol., 71 (1973)421. 10 S. R. Taylor, Geochern. Cosmochim. Acta, 28 (1964) 1273. 11 U. M. Cowgill, Dev. AppL Spectrosc., 10 (1972) 331. 12 G. E. Hutchinson, Int. Rev. Gesamten Hydrobiol. Hydrogr., 28 (1932) 90. 13 V. M. Goldschmidt, Geochemistry, Clarendon Press, Oxford, 1954. 14 B. G. Anderson, Trans. Ant. Fish. Soc., 78 (1950) 96. 15 C. E. Boyd and W. W. Wailey, Am. Midl. Nat., 88 (1972) 1. 16 C. E. Boyd, Ant. Midl. Nat., 84 (1970) 565. 17 U. M. Cowgill, Arch. Hydrobiol. Suppl., 45 (1974) i. 18 H. J. J. Bowen, Trace Elements in Biochemistry, Academic Press, London, 1966. 19 J. F. Kopp and R. C. Kroner, Trace Elements in Waters of the United States, Federal Water Pollution Control Administration, Cincinnati, Ohio, 1970.
lO1
20 21 22 23 24 25 26
C. W. Burns, LimnoL Oceanogr., 14 (1969) 697. S. Richman, Ecol. Monogr., 28 (1958) 273. A. Pacaud, Bull. BioL Ft. Belg. Suppl., 25 (1939) 1. M. Lef'evre, Bull. Biol. Fr. Belg., 76 (1942) 250. A. Banin and J. Navrot, Science, 189 (1975) 550. U. M. Cowgill, Arch. Hydrobiol., 74 (1974) 350. W. H. R. Shaw and B. Grushkin, Arch. Biochem. Biophys., 67 (1957) 447.
102