Comparisons of Mercury( 11) Chloride and Sulfuric Acid as Preservatives for Nitrogen Forms in Water Samples Lyman H. Howe, 111, and C. Wayne Holley Water Quality Laboratory, Division of Health and Safety, TVA, Chattanooga, Tenn. 37401
Ammonia, organic nitrite, and nitrate nitrogen forms in surface water samples underwent biological changes which were more effectively prevented by addition of 42 mg. of mercury(I1) chloride per liter of sample than by 0.8 ml. of concentrated sulfuric acid. Sulfuric acid was particularly unsuitable for preserving samples containing nitrite or a mixture of nitrite and nitrate because it caused the chemical conversion of nitrite to nitrate. Addition of 42 mg. per liter of mercury(I1) chloride did not interfere with analytical procedures employed. However, 80 mg. per liter of mercury(I1) chloride lowered distillation recoveries of free ammonia from distilled water.
A
lthough mercury(I1) chloride and sulfuric acid have been examined as preservatives for nitrogen forms in water samples (American Public Health Assoc., 1965 ; Brezonik and Lee, 1966; Hellwig, 1964, 1967; Jenkins, 1967), detailed evaluations and comparisons of their biological effectiveness are scant. Very few studies have appeared concerning chemical reactions and interferences of these preservatives with nitrogen forms. Brezonik and Lee (1966) found that sulfuric acid preservation makes nitrite disappear and attributed its disappearance to the acid-catalyzed chemical reaction of nitrite with amino acids or ammonia to produce nitrogen gas. The nitrite losses they observed and attributed to sulfuric acid could occur by a different mechanism, such as the chemical conversion of nitrite to nitrate. This conversion can be deduced from chemistry of nitrite and nitrous acid (Cotton and Wilkinson, 1966). Nitrite is known to be a weak base; therefore, nitrous acid forms in the presence of sulfuric acid, a strong proton donor. Nitrous acid, known to be unstable, might convert to nitric acid according to the following equations :
+ NOa- -+ H20 + 2 N 0 2N0 + e 2NO2 2NOs + H10 e H + + Nos- + HNOz
3HNOZ
H+
0 2
+
0 2
2Hf
+ 2N03-
(2) (3)
(4)
Hellwig (1964, 1967) observed that high concentrations of mercury(I1) chloride lower distillation efficiencies of free ammonia from water samples, while low concentrations d o not. Although Hellwig (1967) avoided low recoveries of free ammonia by treatment with hydrogen sulfide or thiosulfate prior to distillation, he has not defined precisely what concentrations of mercury(I1) chloride interfere, what ones d o not, and the chemical conditions for interference. His hydrogen sulfide trzatment is lengthy and time-consuming for routine analyses. If alkaline thiosulfate were used, 478
Environmental Science & Technology
HgCI?
+ 2NH3 S HgNHzC1 +
t CI-
(5)
In light of foregoing discussions, purposes of this study were to examine the sulfuric acid conversion of nitrite to nitrate. to find a concentration of mercury(I1) chloride which would not interfere in distillation of free ammonia from distilled water? and to make detailed comparisons of the biological effectiveness of surfuric acid and mercury(I1) chloride as preservatives for nitrogen forms.
(1)
Providing sufficient sulfuric acid and oxygen are present to encourage complete reactions, the disappearance of nitrite could be explained by the sum of Equations 1 to 3 which is : 2HN02
as he implies, to break up complexes formed between mercury (11) chloride and free ammonia (American Public Health Assoc., 1965), then certain forms of organic nitrogen might be hydrolyzed by alkali to ammonia (Nichols and Foote, 1931). O n the other hand, complexation and hydrolytic difficulties might be avoided simply by using a sufficiently low concentration of mercury(I1) chloride and no alkaline thiosulfate. Moreover. Hellwig (1967) found that mercury(I1) chloride reacts with suspended solids in settled sewage to make dissolved mercury(I1) chloride concentrations equal to as little as 20 of added concentrations. Therefore, the concentration-dependent interference of mercury(I1) chloride he observed in distillation of free ammonia also depends on concentrations of suspended solids. So, to avoid interference, lower mercury(I1) chloride concentrations would be required for those samples without suspended solids than those with suspended solids. Thus, to be sure that the mercury(I1) chloride concentration is low enough not to affect distillation recoveries of free ammonia from very clean surface water samples as well as other samples, its concentration should be low enough so that no interference would occur in distilled water. Besides mercury(I1) chloride concentration in distilled water, other factors in examining this interference would be ammonia or ammonium ion concentration and pH. since these could encourage formation of reaction products which might prevent liberation of free ammonia during distillation. A typical product is given by the following equation (Cotton and Wilkinson, 1966) :
Experimental Procedures Preservative Studies. Samples studied included distilled water, surface water, a 4-to-1 mixture of surface water and settled sewage, and settled sewage and raw sewage. Each sample was thoroughly mixed in a 13-gallon carboy or borosilicate jug. Some samples were enriched with nitrogen forms. Subsequently, each sample was divided into three 2.5-gallon portions. One portion was left unpreserved ; concentrated sulfuric acid, 0.8 ml. per liter, was added to the second portion; and the third portion was treated with the desired amount of mercury(I1) chloride solution. Mercury(I1) chloride was introduced by preparing a nearly saturated standard solution containing 60 grams per liter. Usually the standard solution was diluted to a known volume because such a dilution allowed accurate measurement of volumes with macropipets and produced solutions less sensitive to normal temperature fluctuations. Samples were stored at room temperature (22 i 2" C.) in contact with air and light and were thoroughly shaken before
withdrawing an aliquot for analysis. Concentration of each nitrogen form was determined initially and at time intervals thereafter. Plots were made of each of these concentrations cs. time. Analytical Procedures. Ammonia and organic nitogen forms were determined by distillation and nesslerization procedures described by the American Public Health Assoc. (1965), except that ammonia was trapped in distilled water, and absorbance was measured at 450 mp. using 5-cm. cells. Nitrate and nitrite were measured on a Technicon AutoAnalyzer using a procedure similar to that described by Kainphake, Hannah, et ul. (1967). Decomposition of nitrite and subsequent appearance of nitrate in distilled water samples containing nitrite and preserved with sulfuric acid were studied by different procedures. Nitrite and nitrate nitrogen in these samples were determined at various times from standard curves obtained by. respectively, diazotization and ultraviolet spectrophotometry (American Public Health Assoc., 1965). Results and Discussion Chemical Reactions. Figure 1 clearly shows effect of sulfuric acid preservation on a distilled water sample initially spiked with 10.00 mg. per liter of nitrite nitrogen. As represented by Figure 1, nitrite nitrogen is quantitatively converted to nitrate nitrogen, so that each mole of nitrite lost appears as a mole of nitrate. Under normal laboratory exposures to air and light, nitrite oxidizes according to Equation 4, and perhaps by the mechanism given by Equations 1 to 3. Oxidation did not occur in the absence of sulfuric acid o r in the presence of 42 mg. per liter of mercury(I1) chloride. Thus, sulfuric acid preservation of a sample containing nitrite results in low nitrite values and correspondingly high nitrate values. To establish that the nitrite conversion represented by Figure 1 was not affected by other nitrogen forms, a distilled water mixture was frequently analyzed for all its nitrogen forms over a period of a week. This mixture contained 0.50 mg. per liter of ammonia nitrogen introduced as avmonium chloride, 0.50 mg. per liter of organic nitrogzn introduced as L-glutamic acid, 0.50 mg. per liter of nitrate nitrogen introduced as potassium nitrate, 10.00 mg. per liter of nitrite nitrogen introduced as sodium nitrite, and a preservative or none at all. The nitrogen forms in the unpreserved sample and the sample preserved with 42 mg. per liter of mercury(I1) chloride did not change appreciably for a week. However, in the sulfuric acid preserved sample, the nitrite converted according to Equation 4. The concentrations of organic and ammonia nitrogen remained constant. These data indicate that oxidation of ammonia nitrogen and organic nitrogen to nitrogen gas by nitrite or nitrous acid in the sulfuric acid-preserved mixture to be of no import under normal laboratory exposures to air and light, although Brezonik and Lee (1966) have suggested that this oxidation might account for nitrite losses. A mixture similar to that previously cited but without nitrite was prepared to assure that no changes take place in the absence of nitrite. As expected, the nitrogen forms in this sample did not change with or without addition of sulfuric acid or 42 nig. per liter of mercury(I1) chloride. Chemical Interferences. Distillation of free ammonia (See “Analytical Procedures” for method.) resulted in 98% 3% recoveries from synthetic distilled water samples containing a mixture of 0.50 to 10.00 mg. per liter of ammonia nitrogen introduced as ammonium chloride and 42 mg. per liter of mercury(I1) chloride: however, 14%3 z lower recoveries were obtained from samples containing 80 mg. per liter of mercury (11) chloride. Recoveries were lowered 22 =k 3 a t 90 mg. per
CONC lO8rnl/li
H,SO,
4
K
2
z 0
I
2
3
4
TIME
IN
5
6
7
DAYS
Figure 1. Effect of sulfuric acid on nitrite nitrogen in distilled water
liter of mercury(I1) chloride. Lower recoveries might be caused by irreversible formation of nonvolatile salts, such as the one given by Equation 5 . Apparently these salts or complexes are not easily decomposed into free ammonia. Several samples were synthesized to examine effects of p H o n irreversible formation of mercury salts? for example, formation of HgNHyCl given by Equation 5 . These samples were prepared in distilled water by first adjusting p H values of 1.00 mg. per liter of ammonia nitrogen solutions to 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0 with dilute sodium hydroxide o r sulfuric acid, and then spiking each sample to make it contain 42 mg. per liter of mercury(I1) chloride. Free ammonia recoveries were unaffected. However, addition of mercury(I1) chloride to the solution with p H of 9.0 changed the p H to 6.1. Nevertheless, readjustment of the p H to 9.0 did not affect recovery of free ammonia. Because mercury(I1) salts can be used under certain conditions as catalysts for converting organic nitrogen forms to ammonia (American Public Health Assoc., 1965), free ammonia recovery of 0.50 mg. per liter of ammonia nitrogen from distilled water was examined in the presence of 42 mg. per liter of mercury(I1) chloride and 0.50 mg. per liter of L-glutamic acid nitrogen. However, mercury(I1) chloride did not convert the L-glutamic acid to ammonia, since free ammonia recoveries were unaffected. The above studies show that 42 mg. per liter of mercury(I1) chloride will not affect free ammonia recoveries. However, concentrations of 80 and 90 mg. per liter of mercury(I1) chloride which lowered recoveries of ammonia from distilled water did not lower recoveries of ammonia from a 440-1 mixture of surface water and settled sewage. T o be certain that ammonia recoveries from very clean surface waters would not be lowered, the recommended concentration of mercury(I1) chloride is 42 mg. per liter, even though Hellwig (1964) used 60 to 80 mg. per liter. Concentrations of 21 to 198 mg. per liter of mercury(I1) chloride were examined for interference with the organic nitrogen, nitrite, and nitrate procedures. However, no interference was observed in either distilled o r surface water samples. Biological Changes. Little is known about the qualitative effectiveness of mercury(I1) chloride and sulfuric acid for preventing bacteriological growth. Therefore, their relative preserving power was rated as follows. Nutrient broth was prepared according t o Difco Laboratories (1963). Ten-milliliter portions of broth were put into seven different tubes and sterilized according to the foregoing manual. A 4-to-1 mixture of Volume 3, Number 5, May 1969 479
surface water and settled sewage was prepared. Part of this was preserved with sulfuric acid, another portion was preserved with 42 mg. of mercury(I1) chloride per liter of sample, and the final portion was unpreserved. One milliliter of the sulfuric acid-preserved portion was added to a tube of broth ; 1 ml. of the mercury(I1) chloride-preserved portion to the second tube of broth ; and finally, 1 ml. of the unpreserved sample added to the third tube. A duplicate experiment with a different 4-to-1
? $
0-UNPRESERVE
I
X-HzS04 ( 0 8 rnl/l)
1
3 c
A--HqCIZ ( 2 2 o r 6 6 ~ 9 / 1 1
I
0-UNPRESERVED
[L
A-HgCIz
w
mixture of surface water and settled sewage utilized three more tubes; and 1 ml. of distilled water was added to the seventh tube as a control. Within a day, the tubes containing the unpreserved samples became turbid, indicating bacteriological activity. The sulfuric acid-preserved tubes did not appear active until the tenth day, and the mercury(I1) chloride tubes showed no activity until the twentieth day. The distilled water control remained clear for a month. These observations qualitatively indicate that mercury(I1) chloride was more effective than sulfuric acid in delaying certain forms of bacteriological growth. Biological effectiveness of these preservatives for preventing quantitative changes in the nitrogen forms was evaluated by examining plots of their concentrations cs. time. These studies were made to evaluate quantitatively preservation techniques used by others (American Public Health Assoc.. 1965; Brezonik and Lee, 1966; Hellwig, 1964, 1967; Jenkins. 1967). Figure 2 represents observations made o n a 4-to-1 mixture of surface water and settled sewage enriched with ammonium chloride and L-glutamic acid. Inspection of this plot clearly shows the superiority of 22 o r 66 mg. of mercury(I1) chloride per liter of sample as compared with sulfuric acid for preserving ammonia nitrogen. Similarly, Figure 3 shows mercury(I1) chloride to be the better preservative for organic nitrogen in a 440-1 mixture of surface water and settled sewage enriched with L-glutamic acid. Figure 4 clearly shows that sulfuric acid preservation of nitrite nitrogen in surface water does more harm than good in agreement with the distilled water study given by Figure 1. As illustrated by Figure 5, a concentration of 66 mg. of mercury(I1) chloride per liter of sample will prevent gross changes of nitrite in a 440-1 mixture of surface
I 2 2 or 6 6 r n q / l )
c
1
I
0
l-
OL
1 i k d 1' 0 ,I TIME
lk
Ik
;1
IN DAYS
20
'
Figure 3. Preservation of organic nitrogen in a 440-1 mixture of surface water and settled sewage
z o
0
X - H ~ S O ~ I O ~ ~ I / I I
4
-A
8
TIME
IN
10 DAYS
A
I 12
A ,
16
14
Figure 5. Preservation of nitrite nitrogen in a 4-to-1 mixture of surface water and settled sewage
1
4
A- H g C I , I 6 6 r n g / l )
0
0
2
0-U N P R E S E R V E b
8x 5 C
01
!
6
A
0
I
I
I
I 3
2 TIME
IN
DAYS
Figure 4. Preservation of nitrite nitrogen in surface water 480 Environmental Science & Technology
I 4
1
1 0
2
4
6
8 10 12 14 TIME IN D A Y S
16
18
20
Figure 6. Preservation of nitrate nitrogen in a 440-1 mixture of surface water and settled sewage
water and settled sewage. Other studies of similar mixtures showed that a concentration of 22 mg. per liter was as effective a preservative as the higher concentration. Equal effectiveness of sulfuric acid or 22 or 66 mg. of mercury(I1) chloride per liter of sample as a preservative for preventing nitrate changes in a 440-1 mixture of surface water and settled sewage which did not initially contain nitrite is shown by Figure 6. Even though a concentration of 42 nig. of mercury(I1) chloride per liter of sample is the optimum concentration for preserving nitrogen forms in surface water containing about 20 settled sewage. further studies showed that this concentration did not effectively preserve settled or raw sewage.
Cotton, F. A., Wilkinson, G . , “Advanced Inorganic Chemistry,” pp. 343, 346, 349,617, Interscience, New York, 1966. Difco Laboratories, “Difco Manual of Dehydrated Culture Media and Reagents,” 9th ed., pp. 29-30, Detroit, Mich., 1963. Hellwig, D. H. R., Intern. J. Air Water Pollution 8, 215-28 (1964). Hellwig, D. H. R., WaterRes. 1,79-91 (1967). Jenkins, D., J . Water Pollution Control Federation 39, 159-80 (1967). Kamahake. L. J.. Hannah. S. A.. Cohen. J. M.. Water Res. 1. 205-16 (1967). Nichols, M. S., Foote, M. E., Ind. Eng. Chern., Anal. Ed. 3, 311-13 (1931).
Lirerature Cited American Public Health Assoc., “Standard Methods for the Examination of Water and Wastewater,” 12th ed., pp. 187-93,200-2.205-11,366,401, New York, 1965. Brezonik, P . L., Lee. G. F., Intern. J . Air Water Pollution 10, 549-53 (196h).
Receired for reciew December 6, 1967. Accepted February 3, 1969. Dirision of Wafer, Air, and Waste Chemistry, 154th Meeting, A C S , Chicago, Ill., September 1967. Mention of specijic nianufacturers and models is illustratice and does not imply endorsement by the Tennessee Valley Authority.
COMMUNICATIONS
MBAS and LAS Surfactants in the Illinois River, 1968 William T. Sullivan Water Quaiit) Section, Illinois State Water Survey, Peoria, Ill. 61601
K. D. Swisher Inorganic Chemicals Division, Monsanto Co., St. Louis, Mo. 63166
~~
~
~~
The MBAS content of the Illinois River has dropped to about 0.05 mg. per liter 2 years after the nationwide changeover to the readily biodegradable LAS surfactants; this is a further decline from the level existing 1 years ago. LAS content of the water was less than 0.01 mg per liter, determined by a specific, unequivocal gas chromatographic method.
T
he U. S. industrywide changeover from “hard” tetrapropylene alkyl benzene sulfonate (ABS) surfactants to the more readily biodegradable “soft” linear alkylate sulfonate (LAS) compounds was 21/a years old in January 1968. The methylene blue method is the most widely used technique for the quantitative measurement of anionic surfactants, but it does not differentiate between ABS, LAS, and other methylene blue-chloroform-extractable substances, man-made o r natural. For this reason, the results of monitoring studies have been expressed only in terms of methylene blue active substances (MBAS). We have now applied a method which is specific for LAS.
Comparisons of MBAS levels in the Illinois River before and after the change have been reported by Sullivan and Evans (1968). These data showed a marked decline in anionic surfactants by the end of the first full year following the changeover. Similar results were found by Lawton (1967) for 12 surface water sampling stations in Wisconsin, and in other studies in the United States (Brenner, 1968), England (Waldmeyer, 1968), and Germany (Husmann, 1968). Following the completion, in 1966, of a seven-year monitoring program, no further MBAS analyses were performed on Illinois River water a t Peoria by the Illinois State Water Survey until January and April, 1968. The MBAS concentrations found in the samples at that time, as well as the results of determinations by the Federal Water Pollution Control Administration (FWPCA) from samples collected from the Illinois River in the vicinity of Peoria are shown in Table I. These data indicate that the trend of decreasing concentrations found in 1965-1966 has continued, nearing the minimum limit of applicability for the analytical method in the form employed. T o define more closely the nature of the MBAS still being detected in the Illinois River and to assess the biodegradation of LAS originating from the abundant upriver sources, Volume 3, Number 5, May 1969 481
