changeable hydrogen atoms, such as is achieved in the present study, would lead to an even greater loss of tritium, perhaps as much as 40%. Nearly one half of all the hydrogen atoms in carbohydrates and protein would be exchangeable under the conditions of the present study. It is concluded that the overall isotopic discrimination against tritium, by photosynthesizing C. pyrenoidosa, is about 30%, tritium being incorporated to 70 % of the extent of ‘H. Within biochemical pathways, leading to specific end products, there are large differences in the discrimination against tritium, with incorporation and retention of tritium varying from 0.41 to 1.05. The probable maximum error in these figures is i0.05. In contrast to the incorporation of carbon, which is taken up mostly by a single carboxylation in the reductive pentose phosphate cycle in C. pyrenoidosa (Bassham and Kirk, 1964), hydrogen is incorporated at many points along the biosynthetic pathways. The variation in tritium content of different compounds can be expected as a consequence of a variety of biosynthetic mechanisms. The higher levels of tritium found in the intermediate compounds of the tricarboxylic acid cycle and derivative compounds of that cycle may be an indication of greater retention of tritium than of ‘H in oxidative reactions. Citric acid is formed by condensation of oxalacetate with acetyl CoA which is formed by oxidative decarboxylation of pyruvate. While it is true that the fatty acid moieties of the lipids are also formed from acetyl CoA and the lipids retain less tritium, some of the hydrogen atoms in fatty acids come from reductive reactions. Perhaps more important, the oxalacetate which condenses with acetyl CoA is formed by a series of four oxidative reactions from citrate. The reductive amination of a-ketoglutarate to give glutamate should discriminate against tritium. In fact, the R value for glutamate is significantly lower than the R value of citrate. Similarly, aspartate formed by transamination from oxalacetate has significantly lower R values than malate. It is interesting that in expt 5, which ended with a dark period, the sugar phosphates, which in the dark would be formed by oxidative reactions, have higher R values than
in expt 4 (continuous light). Especially noteworthy is the difference in R values for 3-phosphoglycerate, 0.58 in expt 4 and 0.88 in expt 5. The increases in tritium content during the transformations of sugar phosphates and 3-phosphoglycerate to malate and citrate (expt 4) are significant, the R values rising from 0.78 and 0.58 to 1.04 and 0.97, or more. Thus, it appears that discrimination against tritium in oxidative reactions leads to retention of tritium in metabolic compounds. This conclusion may have some implications for other organisms which are not autotrophic, and, therefore, depend on oxidative metabolism of food. Once the amino acids are formed, there seems to be no significant further change in tritium content as the amino acids are incorporated into proteins. In the case of starch formation from sugar phosphates there may be some discrimination, since the R values for sugar phosphates and for sucrose range from 0.74 to 0.88, whereas the R values for glucose released from starch by hydrolysis are 0.69 and 0.75 in the two experiments. Literature Cited Bassham, J. A . , Calvin, M., “The Path of Carbon in Photosynthesis,” p 31, Prentice-Hall, Englewood Cliffs, N.J. (1957). Bassham, J. A., Kirk, M., Biochim. Biophys. Acta, 90, 553 (1964). Benson, A . A., Bassham, J. A . , Calvin, M., Goodale, T. C., Haas, V. A., Stepka, W., J . Amer. Chem. SOC., 72, 1710 (1950). Kanazawa, T., Kanazawa, K., Kirk, M. R., Bassham, J. A,, Plant Cell Physiol., 11, 445 (1970). Markley, J. L., Putter, I., Jardetzky, O., Science, 161, 1249 (1968). Pedersen, T. A,, Kirk, M., Bassham, J. A . , Physiol. Plant., 19, 219 (1966). Y Weinberger, D., Porter, J. W., Science, 117, 636 (1953). Wyatt, G. R., Biochem. J., 48, 584 (1951). Receiued for reciew October 12, 1971. Accepted January 27, 1972. Work sponsored by US.Atomic Energy Commission.
Spectrophotometric Determination of Oxidized Manganese with Leuco Crystal Violet M ; A ; Kessick, Jasenka Vuceta, and J . J . Morgan’ W. M. Keck Laboratory of Environmental Health Engineering, California Institute of Technology, Pasadena, CA 91109
F
ew methods for the specific determination of low concentrations of naturally occurring oxidized manganese are to be found in the literature. The most sensitive are necessarily colorimetric, since other techniques of high sensitivity, such as amperometry, atomic absorption spectrophotometry, and neutron activation analysis, are not generally capable of distinguishing between manganous manganese and the tri- or tetravalent states of the element commonly found in nature in the form of hydrous oxides. The colorimetric reagents hitherto considered most promising for the deter642 Environmental Science & Technology
mination of manganese in this form have been p-aminophenyls such as benzidine, o-tolidine, and leuco malachite green, 4,4‘tetramethyldiaminotriphenylmethane (Morgan and Stumm, 1965; Ormerod, 1966). 4,4’,4’’-methylidynetris (N,N-dimethylaniline), constitutes another member of this family. Since the higher manganese oxides are the only naturally occurring substances of importance that can oxidize these
To whom correspondence should be addressed.
Oxidized manganese may be determined by reaction in aqueous solution at p H 4.0 with 4,4',4"-methylidynetris (N,N-dimethylaniline), to give the dye crystal violet, followed by measurement of the color produced at 591 mp. For samples of low ionic strength, Beer's Law is obeyed and the molar absorptivity for crystal violet under the conditions of the
analysis was measured as 8.5 X l o 4l./mol-cm. The sensitivity of the method is estimated at 0.13 pg of tetravalent manganese, and may be increased approximately fourfold by an extraction procedure using isobutanol/benzene as solvent. For use with samples of high ionic strength, such as seawater, special calibration is required.
bases to their intensely colored dye forms, the bases may be considered specific reagents for these oxides within the context of the natural water environment.
measurable 5-cm absorbance of 0.01 the method should be sensitive to 0.13 pg of tetravalent manganese, which represents a concentration of 4.8 X lO-*Min a 50-ml sample. To investigate the possibility of interference in the analysis by excess manganous manganese, two samples were made up by adding 0.5- and 1.0-ml quantities of 1.00 X 10-4M stock suspension to 50-ml aliquots of a solution 10-3M in bicarbonate and in manganous perchlorate. The samples were then analyzed for manganese dioxide using the above procedure and calibration graph (Figure 1). The results, which indicate no significant interference, appear in Table I.
Analysis Without Extraction A dilute colloidal suspension of hydrous manganese dioxide was prepared at room temperature by slowly adding slight excess of stock manganous perchlorate solution to a dilute, neutral solution of standardized potassium permanganate, with vigorous stirring. After standing for several days, the manganese dioxide concentration was determined by addition of potassium iodide to aliquots of the suspension, previously deaerated by flushing with nitrogen, followed by acidification, and titration of the liberated iodine under nitrogen with sodium thiosulfate solution. The thiosulfate solution had been previously standardized against potassium iodate. Suitable aliquots of the stock manganese dioxide suspension were then added to approximately 50-1111 quantities of 10-3M sodium biocarbonate in 100 ml volumetric flasks. T o each sample, 2 ml of a 0.1 % solution of 4,4'4"-methylidynetris (N,N-dimethylaniline) in 0.1N perchloric acid was then added, followed immediately by 5 ml of a 6 M acetic acid-acetate buffer to provide a final pH of 4.0, and the resulting violetcolored solutions made up to the mark with distilled water. The 5-cm absorbance was measured at 591 mp against a distilled water blank without delay. A reagent blank was carried through the same procedure and its absorbance subtracted from those of the samples containing manganese. Although the violet coloration was stable at pH 4.0 for an hour or more, prompt spectrophotometric measurement minimized error caused by a slow increase in the absorbance of the reagent blank, probably as a result of reaction of the base with dissolved oxygen or by oxidation with uv light. Figure 1 is a plot of absorbance vs. pmoles of oxidized manganese determined. The molar absorptivity of crystal violet is calculated as 8.5 X l o 4 l./mol-cm. [cf., that for o-tolidine-6.6 X l o 4 l./mol (Morgan and Stumm, 1965)l. Based on a lowest
Analysis with Extraction To increase the sensitivity of the method, the oxidized dye may be extracted from the aqueous phase by a mixture of equal volumes of isobutanol and benzene. Standard manganese dioxide suspensions were prepared by diluting suitable quantities of the stock suspension to 100 ml with 10-3M sodium bicarbonate solution. Each sample was then transferred to a separatory funnel, and 2 ml of a 0.1 % solution of 4,4',4"-methylidynetris (N,N-dimethylaniline) in 0.1N perchloric acid added, followed immediately by 5 ml of the 6M acetic acid-acetate buffer and 25 ml of the isobutanol-benzene solvent. The mixture was shaken for 1 min and allowed to separate for 2 min. The lower aqueous phase was drawn off, and 15 ml of the remaining organic layer pipetted into a 25ml volumetric flask, and made up to the mark with methanol. The 5-cm absorbance of the resulting solution was measured promptly at 591 mp against a solvent blank. A reagent blank was carried through the same procedure, and its absorbance subtracted from those of the standard solution samples. Although 4,4',4"-methylidynetris (N,N-dimethylaniline) was found to decompose to its oxidized form rather rapidly under uv light when dissolved in isobutanol-benzene solvent, this did not provide a significant source of error in these particular determinations. However, low-actinic glassware was used wherever possible. Figure 2 shows a plot of absorbance of the final solvent solution vs. pmoles of oxidized manganese in the original 100-ml samples. The results indicate that the extraction procedure has further increased the sensitivity of the method by a factor of approximately four. This method was also tested for possible interference by manganous manganese by adding 0.1- and 0.3-ml quantities of 1.00 X 10-4M stock
Table I. Analysis in the Presence of Excess Manganous Manganese
Figure 1. Absorbance of crystal violet solution vs. pmoles of manganese dioxide
Amount of MnOn in sample, pmoles x 102
Amount of MnOl determined by analysis, Mmoles x lo2
5 .'OO 10.00
5.09 10.04
Final solution volume 100 ml Volume 6, Number 7, July 1972 643
1
G.6,
L V O _ E S MANC.AUESE D I 3 X DE x 0’
Figure 2. Absorbance of crystal violet in final solvent solution vs. Fmoles of manganese dioxide in original 100-ml samples
manganese dioxide suspension to 100-ml aliquots of a solution 10-3M in bicarbonate and lO-5M in manganous perchlorate. The amounts of added manganese dioxide were then estimated using the extraction procedure and calibration graph (Figure 2) as 1.00 x 1W2and 3.01 X pmoles, respectively, again indicating no appreciable interference in the method from manganous manganese. Samples of High Ionic Strength Since the coloration produced by the oxidation of 4,4’4”methylidynetris (N,N-dimethylaniline) in solution can be attributed to the formation of the resonant cation of crystal violet, the methods with and without extraction were examined for use with samples of high ionic strength. One half- and 1.O-ml quantities of 1.OO x 10-4M stock manganese dioxide suspension were added t o 50-ml aliquots of a solution 10-3M in sodium bicarbonate and 0.65M in sodium chloride, and the procedure followed as described under “Analysis Without Extraction.” Similarly 0.1- and 0.3-mi quantities of the stock suspension were added to 100-ml aliquots of the sodium bicarbonate-sodium chloride solution, and the pro-
Table 11. Comparison of Absorbances for Samples of Low and High Ionic Strength Absorbance Measured absorbance with low MnOz in sample, with high ionic ionic strength #moles strength samples samples 1 . 0 x 10-2 0 . 090a 0 . 160b 3 . 0 x 10-2 0 . 334a 0 . 478b 5.0 x 0. loxc 0 . 208d 1 0 . 0 x 10-2 0,259‘ 0.417d d
a With extraction. From Figure 2.
From Figure 1 .
c
Without extraction.
cedure detailed under “Analysis with Extraction” carried out. The measured absorbances (after subtraction of blank values) are compared in Table I1 with values estimated from Figures 1 and 2. The results indicate that in cases where the sample contributes considerable ionic strength to the final 100-ml solution in the analysis without extraction, or to the aqueous phase in the analysis with extraction, when compared with the ionic strength contribution of the acetic acid-acetate buffer (approximately 0.04 for 5 ml of 6 M buffer a t p H 4.0 in 100 ml), calibration curves need to be constructed for the particular conditions encountered. Such would be the case, for instance, with seawater samples. Literature Cited Morgan, J. J., Stumm, W., J . Amer. Water Works Ass., 57, 107 (1965). Ormerod, J. G., Limnol. Oceanogr., 11, 635 (1966). Receiwd for reciew September 13,1971. Accepted December 31, 1971.
Mercury: Vertical Distribution at Two Locations in the Eastern Tropical Pacific Ocean Herbert V. Weiss’ and Sachio Yamamoto Naval Undersea Research and Development Center, San Diego, C A 92132 Thomas E. Crozier and James H. Mathewson Department of Chemistry, San Diego State College, San Diego, CA 92115
rn Mercury concentrations a t two locations off the west coast of Mexico have been determined in the water column. At a station 60 km from the shore, pronounced fluctuations and greater mercury concentrations, 22-173 nglkg, were measured compared with samples taken 150 km from the coast in which the concentration range was 12-27 ng/kg.
C
urrent interest in environmental contamination by mercury has led t o extensive investigations of mercury levels in a variety of organisms and environments (Ackefors et al., 1970). However, little information has been gathered about the distribution of mercury in the oceans. Mercury concentration was measured in the sea a t two stations 60 and 150 km off the west coast of Mexico to determine 644 Environmental Science & Technology
the mercury levels in areas remote from direct input by man. Samples of seawater were collected in July 1970, over the western edge of the Middle America Trench in 3450 meters of water (16”00’N, 101“40’W, Station V) and directly over the Trench in 4800 meters of water (17”52’N, 103”50’W, Station IX). Vertical profiles of mercury were constructed (Figure 1). Sample Collection
Samples of seawater were collected at depths between the surface and near the bottom in 8-1. “Niskin” bottles attached t o a standard hydrowire. One-liter, unfiltered samples were drawn off within 2 hr after collection into polyethylene bottles which were capped, frozen immediately, and kept
To whom correspondence should be addressed.
