+
1)
CH,O
2)
HOCH,OH
3)
NaHSO,
Table I. Formaldehyde Recovery vs. Microsomal Incubation Time
H,O
=+
+
NaHSO,
HOCH,OH~
-1 HOCH,SO,Na
t
H20
Recovery, % Microsomal incubation time, min
0 5 15 30
Range
Averagea
88-103 92-106 78-94 71-77 62-68 92-1 04
95
86 74 64 98b
4) CH,O
B 62 A 31 B 31 A 15 B 15
Recovery, %C
4-
l2
-+
NaHSO,
f
2 HI
+ NH,
2 CH2(COCH,),
+
Figure 3. Chemical events in the Nash determination of formaldehyde in the presence of bisulfitea Altered chemical equilibria peculiar to the protein precipitation-acidification step have been excluded for diagrammatic simplicity. Dilute aqueous CHzO solutions largely contain methylene glycol in equilibrium with small amounts of unhydrated CHzO (4)
a
Table 11. Formaldehyde Recovery at Various Concentrations
A 123 B 123 A 62
H20
lOOb
a Using 246 ~ .IM standard formaldehyde solution as described in the experimental. Each value represents the mean of 3 determinations. b Solutions containing 0.05% NaHSO,.
Formaldehyde concn, wjWa
+
Std Dev
CONCLUSIONS
68
A method for employing the Nash determination for formaldehyde in the presence of bisulfite has been developed. The devised procedure should permit application of this determination to metabolic studies involving readily air-oxidizable compounds where bisulfite is employed as an antioxidant. Furthermore, bisulfite can serve as an alternative to semicarbazide as a formaldehyde-trapping agent in biological studies.
102 64 98 63 100 60
100
A without bisulfite, B with bisulfite. b Solutions containing 0.05% NaHSO,. = Microsomal incubations proceeded for 3 0 min as described in the experimental. Each value represents the mean of 6 determinations. a
LITERATURE CITED
___________
(1) P. Mazei, in "Fundamentals of Drug Metabolism and Drug Disposition", B. N. LaDu. H. G. Mandel, and E. L. Way, Ed., Williams and Wilkins, Baltimore, Md., 1971. (2) T. Nash. Biochern. J., 55, 416 (1953). (3) T. Nash, Nature (London), 170 976 (1952). (4) J. F. Walker, "Formaldehyde", 3rd ed., Reinhold, New York, N.Y.. 1964. (5) L. H. Donnaliy, lnd. Eng. Chern., Anal. Ed., 5 , 91 (1933). (6) J. H. Block, E. B. Roche, T. 0. Soine, and C. 0. Wilson. "Inorganic Medicinal and Pharmaceutical Chemistry", Lea and Febiger, Philadelphia, Pa.. 1974. (7) W. Ledbury and E. W. Blair, J. Chern. SOC.,127, 2832 (1925). (8) R. V. Smith, P. W. Erhardt, and S. M. Leslie, Res. Cornrnun. Chern. Path. Pharrnacol., in press (1975). (9) R. E. Stitzel, F. E. Greene, R. Furner, and H. Conaway. Biochern. Pharrnacol., 15, 1001 (1966). (10) B. N. LaDu, L. Gaudette, N Trousof, and B. B. Brodie, J. Biol. Chern., 214. 741 (19.55) ---, (11) W.k. Frisell and C. H. Mackenzie, in "Methods of Biochemical Analysis", Vol. VI, D. Glick, Ed., Interscience. New York, N.Y., 1958.
work. The use of an inorganic rather than organic substance for this purpose would seem to have merit. Furthermore, certain workers have reported that excess reaction times are required for Nash determinations when semicarbazide is employed as a formaldehyde-trapping agent (9). To test the utility of bisulfite as a formaldehyde-trapping agent, experiments were performed under typical drugmicrosomal incubation conditions. The results of these investigations are indicated in Tables I and 11. I t is clear that formaldehyde losses associated with volatility and/or further metabolic oxidation to formate and carbon dioxide (10, 1 1 ) are completely prevented by bisulfite. The mechanism of this effect is probably due to formation of the bisulfite addition product with formaldehyde. The series of chemical events which occur in the Nash determination of formaldehyde, as described in this report, are depicted in Figure 3.
I
RECEIVEDfor review June 6, 1975. Accepted September 2, 1975. This work was supported by grant NS-12259, National Institute of Neurological Diseases and Stroke, National Institutes of Health.
Mercury Loss from Culture Media Rodger W. Baier, Leonard Wojnowich, and Lloyd Petrie Duke University Marine Laboratory, Beaufort, N.C. 285 16
Loss of mercury from stored aqueous solutions has been reported by a number of investigators. Early explanations for losses followed along the lines of adsorptive processes that had been observed for many metals with respect t o glass ( 1 ) and to other frequently used storage containers. Materials best suited for minimizing adsorption and pro2464
viding satisfactory conditions for storing mercury a t low concentrations were found to be Pyrex glass, polycarbonate, and Teflon ( 2 ) . Newton and Ellis ( 3 )have shown that the adsorptive capacity of containers varies with both solution composition and container material. Precipitates that form during storage, e.g., MnOn from the reduction of
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
KMn04, now a commonly used preservative for Hg solutions, can be efficient scavengers for metal ions ( 4 ) . Volatilization is a second mechanism by which mercury is lost from solution. Loss of Hg(I1) from dilute solutions in uncapped vials was reported by Toribara et al. ( 5 ) .Using isotopic mercury, Shimomura et al. (6, 7) indicated that mercury loss was directly proportional to pH. The loss of Hg(I1) was attributed to volatilization of reduced mercury, which could be avoided by adding strong oxidants such as K M n 0 4 or NaOCl. Unpreserved natural water lost as much as 82% of added mercury during one week (8). Coyne and Collins (9) alluded to the problems that might be encountered with mercury loss under routine conditions of sample collection, storage, and measurement by atomic absorption. Their work showed a 90% loss of mercury added to unpreserved creek water within three days. Mechanisms of mercury volatilization have been the subject of much controversy. Toribara et al. (5) proposed that the reduction of Hg(I1) to Hg(1) is followed by the spontaneous disproportionation of Hg(1) to Hg(I1) and metallic Hg. This loss mechanism can be justified thermodynamically, particularly when Hg(I1) concentration is not too large. These authors along with Shimomura et al. (7) suggest that solutions commonly found in the laboratory are usually contaminated with enough reductants to carry out this process. Conversely, other workers ( 1 0 ) have invoked bacterial action as a means for converting Hg(I1) to the more volatile organic forms. At the concentrations used by these investigators (100 ppb), physical adsorption could not account for more than 1%of the mercury lost from solution. Furthermore, samples enriched with nutrients lost considerably more mercury than did non-enriched samples. Tonomura et al. (11) have isolated microorganisms which perform the conversion of Hg(I1) to elemental Hg and to organomercury compounds. Among these were the aerobic, mercury-resistant Pseudomonas spp. and the anaerobic C l o s t r i d i u m c o c h l e a r i u m . Their work supports quite elegantly the mechanism of bacterial conversion and subsequent volatilization. To determine the relative importance of physical adsorption and bacterial uptake with subsequent volatilization in culture media initially containing 5-20 ppb Hg, two sets of experiments were conducted. The first experiments determined the rate of mercury loss from culture media in plastic and glass vessels, of various surface-to-volume ratios. The second set of experiments also alluded t o the rate of mercury loss from culture media, but more importantly established the actual mercury concentration that crab larvae experience in various stages of development and evaluated loss rates for nearly sterile systems.
EXPERIMENTAL Methods. Mercury analyses were performed with a Varian AA6 atomic absorption spectrophotometer. Source radiation at 253.7 nm passed through an 18-cm tube filled with a gas mixture swept from the SnClpreduced sample. The detectable limit based on 10 cm3 of sample was 0.1 ppb Hg. Aliquots from a Varian-certified HgC12 solution were diluted with deionized water for daily preparation of standards. Oxidation of organomercury compounds in solution was performed by adding 1 ml concd HNOs and 2 ml concd H2.504 to a 10-ml sample, and subsequently adding 1 ml of 10% KMn04 (repeated until the pink color persisted) and 2 ml of 10% K2SaOs. After 10 minutes, the solution was discolored with NHZOH-HCl and, after waiting an additional 10 minutes, was analyzed by the cold vapor technique described by Parker (12). Reagent blanks usually corresponded to twice the noise level and were subtracted from all analyses. Seawater, initially spiked with 20 ppb Hg and stored until the Hg concentration dropped to less than 0.2 ppb, measured 5 ppb after seing treated by this oxidative procedure. Reagent blanks usually measured 0.1 ppb Hg.
3 11
!
1
C
i
2
3
4
'ire
5 ea15
6
'
Figure 1. Mercury loss from culture media initially contsining 20 ppb Hg in vessels of various surface to volume ratios at 21 O C Polyethylene had been exposed to solutions containing 20 ppb Hg prior to its use in this exDeriment without an acid treatment
Radiomercury analyses were performed using soft-Beta liquid scintillation (Beckman LS-100 with a 0-1.72 MeV energy window and PPO/Triton X-lOO/toluene scintillation solution). Container Surfaces. Pyrex glass and polyethylene containers with and without nitric acid washing were used in the studies. The wetted surface-to-volume ratios were 0.56 cm-' (Erlenmeyer) and 1.56 cm-I (culture dish), and all experiments were initiated with water adjusted to 30% salinity and 20 ppb Hg. Several identical containers held at 20-21 "C were each sampled in triplicate on a daily basis over a period of one week. Analyses were then performed on oxidized samples of solution and acid washings from the containers. In one case, a nutrient mixture of mannitol, ferric citrate, and ammonium hydroxide was added to 800 cm3 of solution stored in an Erlenmeyer flask. T h e solution was monitored for mercury over a 1-week period. When the mercury content of the aqueous phase had dropped below the detectable level, acid washings from the walls of the flask were analyzed. Mercury in the liquid phase of all stored samples decreased logarithmically with time (Figure 11, showing higher rates of disappearance when the glass was unwashed and when the container surface-to-volume ratio was higher. Polyethylene used without prior acid washing had a smaller effect on mercury loss, even though the surface to volume ratio was comparable to that of Pyrex. Washings of culture dish walls after the 6-day exposures accounted for only 3% of the mercury initially present. The same oxidative treatment of walls of the Erlenmeyer flask, after pouring out the solution with added nutrients, accounted for 3.7%. Confirmation of a vapor phase transport mechanism was obtained by determining the partitioning of radioactive 203Hg in a closed system. Deionized water containing 20 ppb stable HgC12 and 17,500 cpm *03Hgwas placed in an acid-washed but non-sterilized 60-ml Pyrex beaker. The beaker was placed in a tightly sealed soft glass jar containing 65 ml 10% KMn04. After 10 days, the beaker was carefully removed, an aliquot of KMn04 solution taken, and the jar washed in 25% "03. The *03Hgactivities of the beaker solution, KMn04 sample, and HNOi wash were determined (Table I). It is seen that 67% of the Hg in the beaker was transported as a vapor into the KMn04 solution and onto the walls of ~
~
~
Table I. iMercury Partitioning in Closed Svstem after 10 Days S?rnple
I n i t i a l beaker s o l u t i o n Final beaker s o l u t i o n KMnO, s o l u t i o n HNO, w a s h
Total accounted
Hrj a c t l i
I@,
,
cpm
01I n i t i a l
17,500
100
5,700
33 37
6,500 4,200 16,400
24
94
-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2465
Table 11. Mercury Partitioning in Open System after 3 Days Deionned
I
I
35%. seawater
water,
Sample
M of initial
of initial
Initial solution 3-day solution Dish HNO, w a s h Larvae accumulation Volatilization (by difference)
100 27 3 7 63
100
36 3
... 61
Table 111. Effect of Sterilization on Mercury Loss, ppb Hg Time ( h o u r s )
Sterile seailater
Mercury loss from culture media in the containers used in exposure experiments at 5, 10, and 20 ppb Hg
Sterile d c -
Sterile
plus 20 rnl 1 . o t non-
Dabs
lonired w a t e i
seanater
sterile seawater
0 1 2 3
16.4 15.5 17.9 16.0 13.2
16.4 13.9 16.2 12.9 13.8
... ...
...
14.7 13.9 11.8 10.4 10.3
...
...
11.3
8.9
7.8
Figure 2.
the capped jar. This loss rate compares favorably with an open container experiment in which crab larvae were present. Seawater containing Rhithropanopeus harrisii was placed in an acid-cleaned but non-sterilized 1-1. culture dish. Sufficient *03Hg was added to give an initial Hg(I1) concentration of 3 X 10-13M. After three days, the larvae were filtered from solution and the dish was washed with 25% " 0 3 . Partitioning of *03Hgwas determined as before, and a control was included using deionized water with the same initial *03Hg concentration. Results revealed a 6163% Hg volatilization loss (by difference) during a 3-day period (Table 11). Concentration Effect. Experiments were performed to ascertain what levels of mercury the crab larvae might encounter in their culture media as a function of time. Solutions of 5, 10, and 20 ppb Hg were prepared in filtered seawater having a salinity of 30% in 1-1. volumetric flasks. After approximately 24 hours, 50 ml of each of these solutions was added to acid washed culture dishes, and the mercury loss was monitored by atomic absorption. Figure 2 shows mercury concentration in culture media with time as it was transferred and stored in two containers. The loss was calculated from the time mercury was placed in the flasks even though a solution transfer had occurred, thus providing an estimate of actual mercury concentrations during the exposure period. Mercury loss was not solely dependent on the presence or absence of bacteria but on the concentration of mercury as well. Analysis of a 1000-ppm aqueous HgN03 solution prepared one year before without preservatives showed no loss of mercury. A 10-ppm solution was not as stable, losing 14% of the mercury in a 50-hour period, Losses from the ppb solutions were seen to range from 68-83% as determined from both the stable mercury and radiochemical experiments mentioned earlier, and the similar results obtained by Chau et al. (8) and Shimomura et al. (6, 7). Sterilized Media. To determine if bacteria play a definitive role in the loss of mercury from solution, two 1-1. flasks containing 30% seawater and a third flask containing deionized water were autoclaved and spiked with unsterilized Hg standard to form 20 ppb Hg solutions. In addition, one of the seawater solutions was spiked with 20 ml of non-sterilized seawater. All solutions were monitored over an 8-day period (Table 111).During the first 4 days, the relative rates of mercury loss from the various solutions were: seawater + bacteria > seawater > deionized water. The rate of loss becomes nearly equal for each of the test solutions after this 4-day period. Furthermore, after a 3-day period, the rate of loss from a nonsterilized solution was about 30 times greater than the autoclaved, deionized water solution mentioned above.
DISCUSSION Mercury loss rates determined from these studies at the 0-20 ppb level show the need for before and after monitoring of culture media during biological exposure experiments. Mercury loss from plastic and glass containers demonstrates the need to choose wisely the container material for the culture media. Although this study indicates that polyethylene can be the material best suited for storing mercury solutions, the literature dictates just the opposite 2466
-
4 5
6 7
...
(2, 8). Pretreatment of the surfaces is apparently critical and many factors governing the nature of active sites are not clearly understood. The bactericidal effect of mercury absorbed in polyethylene may be such a factor. A polyethylene container originally containing a 1000-ppm mercury standard was washed several times with deionized water, and then filled with deionized water. Mercury concentration in the water increased rapidly initially, and then more slowly, as an equilibrium was established between mercury adsorbed on the container surface and the solution. This explains the comparatively low rate of mercury disappearance determined in the initial experiment, since the container used had previously stored mercury solutions. A subsequent test using a polyethylene container in which mercury had not been stored yielded results which agreed with the literature as to the poor suitability of polyethylene containers for storing mercury. Another phenomenon involving diffusion of mercury through polyethylene has recently been described by Bothner and Robertson (13), and verified in this laboratory. These authors discovered that diffusion of airborne mercury vapor through polyethylene container walls can create a serious contamination problem. Loss of mercury is shown to be much greater in seawater, which has the necessary nutrients, than in deionized water, where nutrients are lacking. That mercury was lost from sterile solutions at all can possibly be explained by the combined phenomena of wall adsorption, conversion to organic forms by bacteria introduced during initial mercury addition and subsequent sampling, and volatilization. It remains to be shown that volatilization of mercury is completely eliminated in sterile solutions. At higher mercury concentrations (10-1000 ppm range), bacterial growth is probably limited because of a poisoning effect thereby decreasing the rate of mercury loss. At lower concentrations (ppb), bacteria are able to survive and accelerate mercury loss. Survival of bacteria is dependent not only on the concentration of mercury in the media but on available nutrients as well. It appears that the prevalent mechanism of mercury loss in non-acidified media is bacterial conversion to the organ-
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
ic and/or elemental form and subsequent volatilization. The extent of Hg(I1) loss by volatilization is similar to that determined by Corner and Rigler (IO) at the 100 ppb level. A large fraction of the initial inorganic mercury is apparently released near the walls as the organo and/or metallic form. Tonomura et al. (11) demonstrated the release of methylmercury by Clostridium cochlearium and alluded to the formation of other organomercury compounds in different bacterial species as well. Methylmercury is known to be an order of magnitude more volatile than inorganic mercury. Since organic forms of mercury are more toxic, later stages of mercury poisoning and uptake may be quite different than initial stages in batch cultures. Release of mercury in the metallic form was demonstrated with Pseudomonas spp. in an elegantly proposed biochemical pathway by Tonomura et al. These workers elucidated the reductive decomposition of organic and inorganic mercury compounds to the metallic form and the complementary organic ligands. During the initial stages of culturing in glass dishes, the rate of inorganic mercury conversion to organic forms by bacteria can be given by
where R = rate of Hg loss, mol/l./second, k l = rate constant for second-order reaction, l./cell-second N B = number of bacteria, cell/l. C, = inorganic Hg concentration at the wall, mol/l. If mercury transport is not diffusion controlled, then C, = C, where C is the mercury concentration in the bulk solution. The number of bacteria will increase exponentially with time, so that the rate R takes the form
where NBO= finite number of bacteria at time t = 0, and k2 = rate constant for bacterial growth, sec-'. Integrating and solving for In C gives
(4) where COis the concentration of Hg in the bulk solution at time t = 0 showing log C to decrease exponentially with time. This model was applied to two sets of data taken a year apart using an 8.7-hour doubling time for bacteria starting a t an initial concentration of lo9 cells per liter (Figure 3). The second-order rate constant k l , derived from a least
.IO
.20
.30
3
Time ( mega sec )
Flgure 3. Comparison of mercury loss from glass Erlenmeyer flasks with a simple second-order model describing bacterial uptake Circled points were measured one year earlier
squares fit using these values is 3.1 X l./cell-second compared to 7.4 x l./cell-second for measured bacterial uptake of extracellular carbon from Skeletonemu costu t u m (14). Although definitive studies are needed to describe the bacteria taking part in the conversion and their distribution, the proposed mechanism is consistent with measured losses from the culture media. When bacteria cover the walls, the rate expression for mercury conversion would become pseudo-first-order to the extent that the product of k l and N B is constant.
LITERATURE CITED (1) A. E. Baiiard and C. D. W. Thornton, hd. Eng. Chem., 13, 893 (1941). (2) M. R. Greenwood and T. W. Clarkson, J. Am. lnd. Hyg. Assoc., 31, 251 (1970). (3) D. W. Newton and R. Ellis. Jr., J. Environ. Quality, 3, 20 (1974). (4) C. Feidman, Anal. Chem., 46, 99 (1974). (5) T. Y. Toribara. C. P. Shields, and L. Kovai, Talanta, 17, 1025 (1970). (6) S. Shimomura, Y. Nishihara and Y. Tanase, Jpn Anal., 17, 1148 (1968). (7) Ref. 6, 16, 1072 (1969). (8) Y. K. Chau and H. Saitoh. Environ. Sci. Techno/.,4, 839 (1970). (9) R. V. Coyne and J. A. Collins, Anal. Chem., 44, 1093 (1972). (10) E. D. S. Corner and F. H. Rigier, J. Mar. Biol. Assoc. U.K.,36, 449 (1957). (11) K. Tonomura, K. Furukawa, and M. Yamada, "Environmental Toxicology of Pesticides", F. Matsumura, Ed., Academic Press, New York, N.Y., 1972, pp 115-127. (12) C. R. Parker, "Water Analysis by AA Spectroscopy". Varian Techtron Pub., Springvaie, Australia, 1972. (13) M. H. Bothner and D. E. Robertson, Anal. Chem.. 47, 592 (1975). (14) W. H. Bell, J. M. Lang, and R. Mitchell, Limnol. Oceanogr., 19, 833 (1974).
RECEIVEDfor review May 19, 1975. Accepted July 31, 1975. This research was supported by The Environmental Protection Agency under Grant No. R801305.
ANALYTICAL CHEMISTRY, VOL. 47, NO. 14, DECEMBER 1975
2467
