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Formation of methylmercury in the marine sediment underin vitro conditions
ENVIRONMENTAL
RESEARCH
Formation
20, 325-334 (1979)
of Methylmercury in the Marine under in Vitro Conditions
ISRAELABERDICEVSKY,* HAYA~HOYERMAN,~ *Department Engineering and
of Microbiology. Biotechnology,
Sediment
AND SHMUELYANNAI~.'
Faculty of Medicine and I’Department Technion-Israel Institute of Technology.
of Food Haifa. Israel
Received July 27. 1978 The purpose of this study was to find out whether there is an appreciable conversion of inorganic mercury into methylmercury in the marine bottom sediment. Samples were taken from the Eastern Mediterranean coast, in the Haifa Bay area. H&I, in various concentrations was added to flasks containing samples of sterile or nonsterile bottom sediment and growth medium. and the flasks were incubated under aerobic or anaerobic conditions, for different time intervals. We found that indeed methylmercury was formed, probably by microbial action (since no methylmercury was detected in any of the flasks containing sterile sediment samples). Several types of bacteria which could tolerate considerable concentrations of mercury were isolated, and each type was innoculated into flasks containing medium and a sterile sediment sample. Methylmercury was later found in the latter flasks also.
INTRODUCTION
When evaluating the potential hazard of mercury in foods it is now generally recognized that the most important factor to be considered is their methylmercury content, rather than merely the amount of total mercury. Methylmercury is probably the most toxic of all forms of the metal commonly found as a food contaminant, and it is also readily absorbed and slowly excreted by living organisms (Nelson et al., 1971; Wood, 1972). It has been found that the major food source for methylmercury in the human diet is fish and other seafoods, which derive their mercury from the aquatic habitat (Nelson et al., 1971). In the past it was believed that since most of the mercury compounds are water insoluble, no environmental contamination should result from industrial discharge of this metal, as it would remain inert in the bottom sediment (Jones, 1971). More recently, however, it became evident that this view was incorrect. Several reports indicated that the supposedly inert mercurials are in fact transformed, spontaneously or microbiologically, into the water-soluble methylmercury (Jensen and Jernelov, 1969). The conversion of inorganic ‘mercury into methylmercury was shown to take place in fresh water (Jensen and Jernelov. 1969), and this process may account for the appearance of methylmercury in the aquatic food chain. However, to the best of our knowledge very little information has been published to date on the possibility that a similar transformation of inorganic mercury into methylmercury may also take place in the marine ecosystem. The only reports that we know of concerning such a possibility are those of Olsen and Cooper (1974), Nelson et al. (1972), and Blair et al. (1974), which show ’ All correspondence should be addressed to: Dr. Shmuel Yannai. Dept. of Food Eng. and Biotechnology. Technion-I.I.T.. Haifa, Israel. 325 0013-9351/79/060325-10$02.00/0 Copyright All rights
@I 1979 by Academic Press. Inc of repmductlon in any form reserved.
326
BERDICEVSKY,
SHOYERMAN,
AND
YANNAI
that such a process can take place as a result of the activity of microorganisms in estuaries and bay areas. The goal of the study reported here was to investigate whether methylation of inorganic mercury into methylmercury can actually occur in sediments taken from the Mediterranean, and whether microorganisms isolated from the sediment may be responsible for it. The sampling area was shown previously to contain a relatively high level of inorganic mercury (Levitan er al., 1974). MATERIALS AND METHODS Growth Medium Nelson medium containing per liter of distilled water (w/v): 2.0 g glucose, 5.0 g Casamino acids (Difco), 1.0 g yeast extract (Difco), 10.0 g NaCl, 2.3 g MgC12.6Hz0, 3.0 g KCI. The pH was adjusted to 7.3, and the medium was autoclaved at 121°C for 15 min. A solid medium for stock culture maintenance, and for the isolation of microorganisms, was prepared by adding 25.0 g of agar (Difco) per liter of broth (Nelson et al., 1972). Growth Conditions a. Selection for relative mercury tolerance. To screw-capped test tubes containing 10.0 ml of Nelson medium and 1.0 g of marine sediment, the following concentrations of inorganic mercury were added: 0, 1, 5, 10, 50, 100, 1000, 1500, 2000, and 3000 pg/ml. Microbial growth was monitored turbidimetrically after 48 and 96 hr. b. Main experimental system. To 21 Erlenmeyer flasks containing 1.5 liter of Nelson medium and 100 g of freshly collected marine sediment (which was mostly coarse sand) 0, 3, 10, or 30 pg of HgCl, were added per ml of medium. (in duplicates). The flasks were incubated at room temperature under aerobic and anaerobic conditions (the latter in stoppered flasks). Sterilized sediments (autoclaved for 1 hr at 121°C) were used as controls. Samples were collected after 0, 1,2, 5, 12, and 19 days of incubation and assayed for the contents of total mercury and methylmercury, and the different microorganisms that could be isolated on solid Nelson medium were incubated aerobically and anaerobically. Laboratory Setup a. Anaerobic system. The flasks containing the sediment and growth medium were stoppered with rubber stoppers. b. Aerobic system. Filtered (sterile) air was continuously passed through a trap containing a mixture of sulfuric acid and potassium permanganate solution (to eliminate all mercury vapor from the air) and then through the culture flask. From the latter the air was driven through another trap containing H,SO, and KMnO, solution, to absorb all the mercury liberated from the culture flasks. The air was pumped from the headspace of the second trap by a vacuum pump, which in turn facilitated air circulation through the entire system. One half of the flasks used in both systems were autoclaved at 121°C for 1 hr, in order to make it possible to distinguish between microbially induced and spontaneous transformations of mercury compounds, that may take place during the incubation period. The assay for total mercury was described previously (Levitan et al., 1974), except that here there was no need to concentrate the mercury from large water
METHYLMERCURY
IN MARINE
327
SEDIMENT
samples, as its levels in the growth medium were always high enough to permit direct determination. Methylmercury was determined following the method described by Longbottom et al. (1973). RESULTS
AND DISCUSSION
Tolerance of Various Microorganisms
to Mercuq
This was a range-finding experiment, designed to select the practical mercury concentrations in the medium in which the microorganisms can still grow on one hand, but which will also show definite growth inhibition for even tolerant microbes. The data obtained were used in further experiments. The results presented in Table 1 indicate that the concentrations of 1 to 30 &ml used in our experiments are included in this effective range. The conditions under which each of the types of microorganisms was isolated from the different media are described in Table 2. Microorganisms 2 and 5 appeared in all media, under both aerobic and anaerobic conditions, where there was any growth at all. This proved their relatively high tolerance for mercury. Microbe 3 appeared only on the 5th day, in the media into which 3 &ml of mercury were added, under both aerobic and anaerobic conditions. As regards microbe 1. it was detected in all aerobic media, whereas in media in which the supply of oxygen was limited it did not appear until the 12th day, when apparently much of the added mercury left the media by binding to the sediment, to the glass walls of the flasks, or by evaporation (see Table 3). In the case of the anaerobic medium containing 30 &ml, not until the 19th day of incubation could it be detected. As for microorganism 4, its presence, in any of the media, was not revealed before the 12th day, and even then-only in those with 3 and 10 pg of added mercury/ml. Yet, on the 19th day it was detected in all of the growth media without exception. It should be noted, though, that the bacteria growing in the unaerated flasks were not strictly anaerobic: rather, chances are that these were facultative anaerobes. TABLE GROWTH
Concentration of added HgCl, (expressed as Hg) ( ,ugiml) 0 5 10 SO 100 500 loo0 2ooo 3000
1
OF MICROORGANISMS EXPOSED TO VARIOUS CONCENTRATIONS IN THE GROWTH MEDIUM AS A FUNCTION OF TIME”
Growth on Nelson medium under anaerobic conditions 48 hr
OF MERCURY
Growth on Nelson medium under aerobic conditions
96 hr
48 hr
96 hr
++++ ++ ++ + +
++++ f-t +++ + +
++++ ++++ ++++ +++ -
++++ ++++ ++++ ++++ -
-
-
-
-
u The number of plus signs denotes the relative turbidity of the growth medium, and a minus sign, a clear medium (no microbial growth).
328
BERDICEVSKY,
TYPES OF BACTERIA
Conditions of system Anaerobic
Aerobic
SHOYERMAN,
AND
YANNAI
TABLE 2 FOUND AFTER EXPOSURE TO DIFFERENT CONCENTRATIONS DURING THE ~%DAY EXPERIMENTAL PERIOD
Concentration of HgCl, added (I*dml)
Day 1
Day 2
0 3 10 30
1, 2, 5 2, 5 -
1, 2, 5 2, 5 2, 5 -
0 3 10 30
1, 2, 5 1, 2, 5 -
1, 2, 5 1, 2, 5 1, 2, 5 -
OF
HgClz
Type of bacteria” Day 5
Day 12
Day 19
1, 2, 5 2, 3, 5 2, 5 -
1, 2, 4, 5 1, 2, 4, 5 1, 2, 4, 5 2, 5
1, 2, 4, 5 1, 2, 4, 5 1, 2, 4, 5
1, 2, 5 1, 2, 3, 5 1, 2, 5 -
1, 2, 1, 2, 1, 2, 1, 2,
1, 2, 1, 2, 1, 2, 1, 2,
4, 5 4, 5 4. 5 5
1,2,4,5
4, 4, 4, 4,
5 5 5 5
a Five different unidentified microorganisms were isolated in this experiment. No attempt was made to identify them; however, they did differ morphologically from one another. The type of microorganism is characterized as follows: (1) Gray, spreading, and smooth colonies of large gram-negative bacilli. (2) Transparent small colonies, opaque in the middle, of gram-negative bacilli. (3) Small white colonies of gram-positive cocci. (4) Small orange colonies of pleomorphic gram-negative baccilli. (5) Creamy-white colonies of short gram-negative bacilli.
These are qualitative data, and no attempt was made to evaluate the microbial growth quantitatively; however, it was obvious that there was an inverse proportion between the mercury level and the total microbial growth, as judged by the turbidity of the different media. Distribution of Mercury in the Different System Components The different components of the system were analyzed for mercury content after 19 days of incubation. The results, presented in Table 3, show that the higher the mercury concentration in the medium the higher the percentage of mercury lost by evaporation. This effect was probably due to the formation of volatile forms of the metal (alkyl and elemental mercury), induced by microorganisms which possess the capacity to metabolize mercury (Brinckman et al., 1976). It is noteworthy that this increase in microbial mercury transformation capability was not a direct function of the amount of mercury added to the medium; rather, it was enhanced by increasing concentrations of the metal in the medium. This fact can possibly be attributed to the growth-suppressive effect of mercury on most microorganisms, except those that are capable of metabolizing the metal, and which can, therefore, tolerate more of it than the microbes which cannot metabolize mercury (Fagerstrom and Jernelov, 1972). In the absence of their natural competitors, the latter microorganisms dominated the medium and, consequently, much more mercury could be transformed into its volatile derivatives. The presence of bacteria relatively resistant to mercury in the growth medium was discussed above. The quantity of mercury remaining in the sterile aerobic and anaerobic media after different time intervals is presented in Fig. 1. There was a sharp drop during the first 5 to 7 days of incubation, and later the mercury concentration reached a
Nonsterile aerobic system
6.0 5.2 Il.6
5.5 1.4 3.8
Evaporated
IN THE
Sterile aerobic system
DISTRIBUTION
Hg”
0.5 3.8 7.8
W’
Corrected evaporated
SYSI EM COMPONENTS,
” Percentage of the amount of Hg added to the system. ’ The difference between the sterile and nonsterile aerobic action.
0 3 10 30
Concentration of added mercury (abnl)
MERCURY
system
5.0 10.0 16.3
Sterile aerobic system
in
3
is supposed
6.7 11.5 21.3
Sterile anaerobic system
Hg remaining solution”
AFTER
TABLE 19 DAYS
OF DIFFERENT
to constitute
51.7 55.0 33.4
37.8 33.6 46.5
Sterile aerobic system
CONCENTRA
induced
29.4 32.5 39.0
Sterile anaerobic system
by microbial
29.8 37.3 38.7
Nonsterile aerobic system
to flask”
OF ‘THE METAL
Hg adsorbed
UONS
the loss of Hg due to volatilization.
55.0 47.5 46.5
Sterile anaerobic system
Hg bound to the sediment” Sterile aerobic system
ADDITION
3
K 2 z
$ 5 n
2 3
s *
F5 0”
5
:
330
BERDICEVSKY,
SHOYERMAN,
n
11 02
I
2
I
I
4
III
Aerobic,
III
6
Time
AND YANNAI
I
with
III
I
I
I
II
(days)
FIG. 1. Rate of decrease in total Hg content of the sterile media as a function of time and the amount of mercury added.
constant level. The rate of decrease was slower, and the amount eventually remaining was higher, in the media into which the greater amounts of mercury were added. The level of mercury in the anaerobic media was checked only at the termination of the incubation period (after 19 days), and was found to be in all cases somewhat higher than in aerobic media with the same initial amount of the metal. Similar findings with regard to the rate of mercury evaporation from sterile culture media under anaerobic conditions were reported by Baier et al. (1975). The change in pH in the various growth media is given in Table 4. The initial pH was, in all instances, 7.2-7.4. After 5 days there was still no change in the sterile system, at any mercury level, but in the nonsterile media the pH dropped appreciably in all cases, except for those into which 30 &ml of mercury were added. The decrease in pH is indicative of the extent of microbial growth (i.e., acid production from sugars). Production of Methylmercury As can be seen in Table 5, methylmercury was produced in all of the systems maintaining anaerobic conditions. However, the percentage of methylmercury in the medium was in inverse proportion to its total mercury content; and this fact suggests that the highest efftciency of conversion of the inorganic into the methyl TABLE pH VALUEOFTHE
DIFFERENTMEDIA
4
5 DAYSAFTERTHE
STARTOF~NCUBATION
Level of Hg added to the medium (pg/ml) Nature of system
0
3
10
30
Sterile Nonsterile, aerobic Nonsterile, anaerobic
7.3 6.2 6.4
7.3 6.3 6.4
7.2 6.6 6.5
7.3 7.2 7.4
0.10 3.1 10.1 30.1
Aerobic
n.d. n.d. n.d. n.d.
11.3 n.d.l n.d. n.d.
I-Q
FORM~I)
n.d. n.d. n.d. n.d. in the medium
Hg
time.
of total
n.d. n.d. n.d. n.d.
98.0 3.15 0.23 0.068
5th day Hg
as Hg)
AND OF ‘rtrt
(expressed
Percentage
of CH,Hg
at zero
n.d. n.d. n.d. n.d.
98.0 97.9 23.3 20.4
/a
Level
TABLE 5 or- DURATION OF INCUBA-rlON, PRESENT IN THE SYSTF.M
of total 71.3 n.d. n.d. n.d.
present
Percentage
2nd day
AS A FUNCTION
” In terms of percentage of the amount of mercury ” Concentration of mercury in the medium itself. ( Not detected.
0.1” 3.1 10.1 30.1
Amount of Hg in medium b.4ml)
0~ M~VHI'LM~RCUR~
Anaerobic
Nature of system
LEVEL"
4.25 n.d. 11.6 n.d.
5.2 2.6 2.4 2.4
E-Lg
AMOUNT
Percentage
4.25 n.d. 0.115 n.d.
5.2 0.084 0.024 0.008
of total
MERCURY
12th day
OF TO-I-AL
Hg
i i-z
3
$
2 r
2 <
;s z z
2
3
332
BERDICEVSKY,
SHOYERMAN,
AND
YANNAI
derivative of the metal was attained by the system into which no mercury had been added. The media themselves contained 100 pg total mercury and this level is hereafter referred to as “background mercury level.” Only two samples of aerobic system were analyzed for their methylmercury content, since we assumed that little methylmercury would be produced under these conditions, as reported in some papers. It turned out, however, that at least as far as the media into which 10 pg of mercury were added per milliliter-the level of methylmercury after 12 days under aerobic conditions was higher than in the medium with the same initial mercury concentration kept under anaerobic conditions. The reason for the fact that not much methylmercury was found in the media after 12 days, and that its level decreased between the 5th and the 12th day, is apparently that much of it disappeared from the medium by evaporation, binding to the sediment, and adsorption to the glass surface of the flasks. Another phenomenon is the almost complete absence in the medium of methylmercury at the end of the incubation period. It seems likely that when the cultures grew older and/or became overcrowded they stopped producing methylmercury. This was also the case with the system into which no mercury was added. In the latter case a considerable amount of methylmercury was detected, which is ascribable to the fact that the medium itself contains 0.1 pg mercury/ml of medium, originating mainly from the materials constituting the growth medium. As can be seen in Table 5, the higher the mercury level in the growth medium the lower the percentage of methylmercury produced. Even in absolute amounts the culture containing the lowest total concentration showed the highest methylmercury content. This observation can be accounted for by the fact that the media with the high levels of the metal supported poor growth of microorganisms, as discussed above. It is also possible that at the range of mercury levels present in the media employed in this study there is no correlation between the total mercury content and the amount of the methyl derivative produced. The methylmercury level present in the control medium merely reflects the fact that the microbial growth in that medium was the highest. A similar amount of methylmercury was also produced in the medium into which 3 pg/ml of inorganic mercury were added. This latter culture showed a microbial growth similar to that of the control culture, as estimated visually. As indicated above, the production of methylmercury from inorganic mercury may have resulted from biotransformation involving microorganisms. Yet, the possibility of spontaneous methylation of mercury cannot be ruled out. The existence of such processes had, in fact, been reported by several investigators (Imura et al., 1971; Bertilson and Neujahr, 1971; Wood, 1974). Therefore, a further experiment was designed with a view to finding out whether the latter possibility could have accounted for a considerable part of the methylmercury produced, or whether it was even the major pathway involved. The possible spontaneous methylation of inorganic mercury was checked with a series of flasks containing sterile culture media into which 0 or 3.75 pg of mercuric chloride was added per milliliter of medium. In addition, four out of the five types of the microorganisms, isolated and characterized as described above, were cultured in several Erlenmeyer flasks. The setup used for this purpose was similar to that described under
IN
METHYLMERCURY
TABLE TOTAL
MERCURY IN THE
CONTENT PRESENCE
OF THE DIFFERENT OF THE BACTERIA
MARINE 6
MEDIA ISOLATED
AS A FUNCTION OF INCUBATION FROM A MARINE SEDIMENT
Type
(sterile Zero
time:
pg Hgiml
4 Days: 6 Days: 8 Days:
None medium)
5 3.75 100
2. IO 56
1.83 48.8
2.00 53.3
1.65 44.0
I. 15 30.7
pg Hgiml
7%
2.10 56
1.70 48.5
1.85 49.3
1.58 42.1
1.05 28.0
pg Hgiml cr,
2.02 53.9
1.43 38.1
1.80 48.0
1.00 26.6
0.90 24.0
pg Hgiml
2.02 53.9
1.35 36.0
1.80 48.0
0.90 24.0
0.80 21.3
2.00 53.3
1.35 36.0
1.73 46. I
0.72 19.2
0.62 16.5
2.00 53.3
1.25 33.3
1.70 45.3
0.62 16.5
0.55 14.7
pg Hg/ml pg Hgiml
of Hg content
OF METHYLMERCURY IN THE
at zero
time.
PRESENCE
IN THE OF THE
DIFFERENT
BACTERIA
7 MEDIA
ISOLATED
AS A FUNCTION FROM
Type 1 2 Days:
4
wg Hgiml 7%
TABLE PRODUCTION
2
3.75 100
% ” Percentage
1
3.75 100
9% 12 Days:
of bacteria
3.75 100
7% 10 Days:
TIME,
3.75 100
75” 2 Days:
333
SEDIMENT
pg CHR Hgiml
5%”
A MARINE
OF INCUBATION
TIME
SEDIMENT”
of bacteria 4
2
5
0.00198 0.053
n.d.’
0.0004 0.01
n.d.
4 Days:
&g CH, 95
Hgiml
0.00198 0.053
0.00119 0.032
0.04JO8 0.021
n.d.
6 Days:
wg CH, 5%
Hgiml
0.00119 0.032
0.0004 0.01
0.0016 0.042
n.d.
0.0004 0.01
0.0004 0.01
0.0008 0.021
n.d.
0.0004 0.01
n.d.
0.0004 0.01
n.d.
n.d.
n.d.
0.0004 0.01
n.d.
8 Days:
wg CH:, Hgiml
% IO Days:
pg CHR Hg/ml
% 12 Days:
pg CHR Hg/ml
5% rr Values for methylmercury are expressed in terms media. * Percentage of mercury content at zero time. ” Not detectable.
of Hg.
No CH,Hg
was detected
in the sterile
334
BERDICEVSKY,
SHOYERMAN,
AND YANNAI
Materials and Methods, except that in this case the volume of the flasks was 250 ml, and 10 g of bottom sediment and 200 ml of growth medium were placed in each flask. The total mercury and methylmercury contents of the cultures were determined at zero time, and after 2, 4, 6, 8, 10, and 12 days of incubation. The results for total mercury are presented in Table 6, and those for methylmercury, in Table 7. AS can be seen in Table 6, there was a gradual decrease in the concentration of total mercury in all media. There was, however, a different rate of disappearance of the metal from each, probably indicating that the type of microorganism had some influence on the process. We offer no explanation for this observation. As regards the amount of methylmercury produced in the media, it is obvious that no spontaneous methylation could account for it, since no methylmercury was detected in any of the sterile media. The methylmercury reached its peak level after 4 to 6 days of incubation, and then started to drop, probably by evaporation and/or adsorption to the sediment and the glass surfaces. REFERENCES Baier, R. W., Wojnowich, L., and Petrie, L. (1975). Mercury loss from culture media. Anal. Chem. 47, 2464-2467. Bertilsson, L., and Neujahr, H. Y. (1971). Methylation of mercury compounds by methylcobalamin. Biochemistry 10, 2805-2808. Blair, W., Iverson, W. P., and Brinckman, F. E. (1974). Application of a gas chromatograph-Atomic absorption detection system to a survey of mercury transformations by Chesapeake Bay microorganisms. Chemosphere 3, 167-174. Brinckman, F. E., Iverson, W. P., and Blair, W. (1976). “Approaches to the Study of Microbial Transformations of Metals.” Proc. 3rd Internat. Biodegradation Symp., pp. 919-936. The University of Long Island, Kingston, R.I. Fagerstrom, T., and Jernelov, A. (1972). Some aspects of the quantitative ecology ofmercury. Wafer Res. 6, 1193-1202. Imura, N., Sukegawa, E., Pan, S. K., Nagao, K., Kim, J. Y., Kwan, T., and Ukita, T. (1971). Chemical methylation of inorganic mercury with methylcobalamin, a vitamin Bi2 analog. Science 172, 1248- 1249. Jensen, S., and Jernelbv, A. (1969). Biological methylation of mercury in aquatic organisms. Nature (London) 223, 753-754. Jones, H. R. (1971). “Mercury Pollution Control.” Noyes Data Corp., Park Ridge, N.I. Levitan, S., Rosner, L., and Yannai, S. (1974). Mercury levels in some carnivorous and herbivorous Israeli fishes, and in their habitats. Israel J. Zoo/. 23, 135- 142. Longbottom, J. E., Dressman, R., and Lichtenberg, J. (1973). Gas chromatographic determination of methylmercury in fish, sediment and water. J.A.O.A.C. 56, 1297-1303. Nelson, N., Byerly, T. C., Kolbye, A. C., Kurland, L. T., Shapiro, R. E., Shibko, S. I., Stickel, W. H., Thompson, J. E., Van Den Berg, L. A., and Weissler, A. (1971). Hazards of mercury. Environ. Res. 4, i-69. Nelson, J. D. Jr., McClam, H. L., and Colwell, R. R. (1972). “The Ecology of Mercury Resistant Bacteria in Chesapeake Bay.” Proc. Marine Technology Society, 8th Annual Conference, Washington, D.C., pp. 303-312. Olson, H. R., and Cooper, R. C. (1974). In situ methylation of mercury in estuarine sediment. Nature (London) 252, 682-683. Wood, J. M. (1972). A progress report on mercury. Environmen? 14, 33-39. Wood, J. M. (1974). Biological cycles for toxic elements in the environment. Science 183, 1049-1052.
RESEARCH
Formation
20, 325-334 (1979)
of Methylmercury in the Marine under in Vitro Conditions
ISRAELABERDICEVSKY,* HAYA~HOYERMAN,~ *Department Engineering and
of Microbiology. Biotechnology,
Sediment
AND SHMUELYANNAI~.'
Faculty of Medicine and I’Department Technion-Israel Institute of Technology.
of Food Haifa. Israel
Received July 27. 1978 The purpose of this study was to find out whether there is an appreciable conversion of inorganic mercury into methylmercury in the marine bottom sediment. Samples were taken from the Eastern Mediterranean coast, in the Haifa Bay area. H&I, in various concentrations was added to flasks containing samples of sterile or nonsterile bottom sediment and growth medium. and the flasks were incubated under aerobic or anaerobic conditions, for different time intervals. We found that indeed methylmercury was formed, probably by microbial action (since no methylmercury was detected in any of the flasks containing sterile sediment samples). Several types of bacteria which could tolerate considerable concentrations of mercury were isolated, and each type was innoculated into flasks containing medium and a sterile sediment sample. Methylmercury was later found in the latter flasks also.
INTRODUCTION
When evaluating the potential hazard of mercury in foods it is now generally recognized that the most important factor to be considered is their methylmercury content, rather than merely the amount of total mercury. Methylmercury is probably the most toxic of all forms of the metal commonly found as a food contaminant, and it is also readily absorbed and slowly excreted by living organisms (Nelson et al., 1971; Wood, 1972). It has been found that the major food source for methylmercury in the human diet is fish and other seafoods, which derive their mercury from the aquatic habitat (Nelson et al., 1971). In the past it was believed that since most of the mercury compounds are water insoluble, no environmental contamination should result from industrial discharge of this metal, as it would remain inert in the bottom sediment (Jones, 1971). More recently, however, it became evident that this view was incorrect. Several reports indicated that the supposedly inert mercurials are in fact transformed, spontaneously or microbiologically, into the water-soluble methylmercury (Jensen and Jernelov, 1969). The conversion of inorganic ‘mercury into methylmercury was shown to take place in fresh water (Jensen and Jernelov. 1969), and this process may account for the appearance of methylmercury in the aquatic food chain. However, to the best of our knowledge very little information has been published to date on the possibility that a similar transformation of inorganic mercury into methylmercury may also take place in the marine ecosystem. The only reports that we know of concerning such a possibility are those of Olsen and Cooper (1974), Nelson et al. (1972), and Blair et al. (1974), which show ’ All correspondence should be addressed to: Dr. Shmuel Yannai. Dept. of Food Eng. and Biotechnology. Technion-I.I.T.. Haifa, Israel. 325 0013-9351/79/060325-10$02.00/0 Copyright All rights
@I 1979 by Academic Press. Inc of repmductlon in any form reserved.
326
BERDICEVSKY,
SHOYERMAN,
AND
YANNAI
that such a process can take place as a result of the activity of microorganisms in estuaries and bay areas. The goal of the study reported here was to investigate whether methylation of inorganic mercury into methylmercury can actually occur in sediments taken from the Mediterranean, and whether microorganisms isolated from the sediment may be responsible for it. The sampling area was shown previously to contain a relatively high level of inorganic mercury (Levitan er al., 1974). MATERIALS AND METHODS Growth Medium Nelson medium containing per liter of distilled water (w/v): 2.0 g glucose, 5.0 g Casamino acids (Difco), 1.0 g yeast extract (Difco), 10.0 g NaCl, 2.3 g MgC12.6Hz0, 3.0 g KCI. The pH was adjusted to 7.3, and the medium was autoclaved at 121°C for 15 min. A solid medium for stock culture maintenance, and for the isolation of microorganisms, was prepared by adding 25.0 g of agar (Difco) per liter of broth (Nelson et al., 1972). Growth Conditions a. Selection for relative mercury tolerance. To screw-capped test tubes containing 10.0 ml of Nelson medium and 1.0 g of marine sediment, the following concentrations of inorganic mercury were added: 0, 1, 5, 10, 50, 100, 1000, 1500, 2000, and 3000 pg/ml. Microbial growth was monitored turbidimetrically after 48 and 96 hr. b. Main experimental system. To 21 Erlenmeyer flasks containing 1.5 liter of Nelson medium and 100 g of freshly collected marine sediment (which was mostly coarse sand) 0, 3, 10, or 30 pg of HgCl, were added per ml of medium. (in duplicates). The flasks were incubated at room temperature under aerobic and anaerobic conditions (the latter in stoppered flasks). Sterilized sediments (autoclaved for 1 hr at 121°C) were used as controls. Samples were collected after 0, 1,2, 5, 12, and 19 days of incubation and assayed for the contents of total mercury and methylmercury, and the different microorganisms that could be isolated on solid Nelson medium were incubated aerobically and anaerobically. Laboratory Setup a. Anaerobic system. The flasks containing the sediment and growth medium were stoppered with rubber stoppers. b. Aerobic system. Filtered (sterile) air was continuously passed through a trap containing a mixture of sulfuric acid and potassium permanganate solution (to eliminate all mercury vapor from the air) and then through the culture flask. From the latter the air was driven through another trap containing H,SO, and KMnO, solution, to absorb all the mercury liberated from the culture flasks. The air was pumped from the headspace of the second trap by a vacuum pump, which in turn facilitated air circulation through the entire system. One half of the flasks used in both systems were autoclaved at 121°C for 1 hr, in order to make it possible to distinguish between microbially induced and spontaneous transformations of mercury compounds, that may take place during the incubation period. The assay for total mercury was described previously (Levitan et al., 1974), except that here there was no need to concentrate the mercury from large water
METHYLMERCURY
IN MARINE
327
SEDIMENT
samples, as its levels in the growth medium were always high enough to permit direct determination. Methylmercury was determined following the method described by Longbottom et al. (1973). RESULTS
AND DISCUSSION
Tolerance of Various Microorganisms
to Mercuq
This was a range-finding experiment, designed to select the practical mercury concentrations in the medium in which the microorganisms can still grow on one hand, but which will also show definite growth inhibition for even tolerant microbes. The data obtained were used in further experiments. The results presented in Table 1 indicate that the concentrations of 1 to 30 &ml used in our experiments are included in this effective range. The conditions under which each of the types of microorganisms was isolated from the different media are described in Table 2. Microorganisms 2 and 5 appeared in all media, under both aerobic and anaerobic conditions, where there was any growth at all. This proved their relatively high tolerance for mercury. Microbe 3 appeared only on the 5th day, in the media into which 3 &ml of mercury were added, under both aerobic and anaerobic conditions. As regards microbe 1. it was detected in all aerobic media, whereas in media in which the supply of oxygen was limited it did not appear until the 12th day, when apparently much of the added mercury left the media by binding to the sediment, to the glass walls of the flasks, or by evaporation (see Table 3). In the case of the anaerobic medium containing 30 &ml, not until the 19th day of incubation could it be detected. As for microorganism 4, its presence, in any of the media, was not revealed before the 12th day, and even then-only in those with 3 and 10 pg of added mercury/ml. Yet, on the 19th day it was detected in all of the growth media without exception. It should be noted, though, that the bacteria growing in the unaerated flasks were not strictly anaerobic: rather, chances are that these were facultative anaerobes. TABLE GROWTH
Concentration of added HgCl, (expressed as Hg) ( ,ugiml) 0 5 10 SO 100 500 loo0 2ooo 3000
1
OF MICROORGANISMS EXPOSED TO VARIOUS CONCENTRATIONS IN THE GROWTH MEDIUM AS A FUNCTION OF TIME”
Growth on Nelson medium under anaerobic conditions 48 hr
OF MERCURY
Growth on Nelson medium under aerobic conditions
96 hr
48 hr
96 hr
++++ ++ ++ + +
++++ f-t +++ + +
++++ ++++ ++++ +++ -
++++ ++++ ++++ ++++ -
-
-
-
-
u The number of plus signs denotes the relative turbidity of the growth medium, and a minus sign, a clear medium (no microbial growth).
328
BERDICEVSKY,
TYPES OF BACTERIA
Conditions of system Anaerobic
Aerobic
SHOYERMAN,
AND
YANNAI
TABLE 2 FOUND AFTER EXPOSURE TO DIFFERENT CONCENTRATIONS DURING THE ~%DAY EXPERIMENTAL PERIOD
Concentration of HgCl, added (I*dml)
Day 1
Day 2
0 3 10 30
1, 2, 5 2, 5 -
1, 2, 5 2, 5 2, 5 -
0 3 10 30
1, 2, 5 1, 2, 5 -
1, 2, 5 1, 2, 5 1, 2, 5 -
OF
HgClz
Type of bacteria” Day 5
Day 12
Day 19
1, 2, 5 2, 3, 5 2, 5 -
1, 2, 4, 5 1, 2, 4, 5 1, 2, 4, 5 2, 5
1, 2, 4, 5 1, 2, 4, 5 1, 2, 4, 5
1, 2, 5 1, 2, 3, 5 1, 2, 5 -
1, 2, 1, 2, 1, 2, 1, 2,
1, 2, 1, 2, 1, 2, 1, 2,
4, 5 4, 5 4. 5 5
1,2,4,5
4, 4, 4, 4,
5 5 5 5
a Five different unidentified microorganisms were isolated in this experiment. No attempt was made to identify them; however, they did differ morphologically from one another. The type of microorganism is characterized as follows: (1) Gray, spreading, and smooth colonies of large gram-negative bacilli. (2) Transparent small colonies, opaque in the middle, of gram-negative bacilli. (3) Small white colonies of gram-positive cocci. (4) Small orange colonies of pleomorphic gram-negative baccilli. (5) Creamy-white colonies of short gram-negative bacilli.
These are qualitative data, and no attempt was made to evaluate the microbial growth quantitatively; however, it was obvious that there was an inverse proportion between the mercury level and the total microbial growth, as judged by the turbidity of the different media. Distribution of Mercury in the Different System Components The different components of the system were analyzed for mercury content after 19 days of incubation. The results, presented in Table 3, show that the higher the mercury concentration in the medium the higher the percentage of mercury lost by evaporation. This effect was probably due to the formation of volatile forms of the metal (alkyl and elemental mercury), induced by microorganisms which possess the capacity to metabolize mercury (Brinckman et al., 1976). It is noteworthy that this increase in microbial mercury transformation capability was not a direct function of the amount of mercury added to the medium; rather, it was enhanced by increasing concentrations of the metal in the medium. This fact can possibly be attributed to the growth-suppressive effect of mercury on most microorganisms, except those that are capable of metabolizing the metal, and which can, therefore, tolerate more of it than the microbes which cannot metabolize mercury (Fagerstrom and Jernelov, 1972). In the absence of their natural competitors, the latter microorganisms dominated the medium and, consequently, much more mercury could be transformed into its volatile derivatives. The presence of bacteria relatively resistant to mercury in the growth medium was discussed above. The quantity of mercury remaining in the sterile aerobic and anaerobic media after different time intervals is presented in Fig. 1. There was a sharp drop during the first 5 to 7 days of incubation, and later the mercury concentration reached a
Nonsterile aerobic system
6.0 5.2 Il.6
5.5 1.4 3.8
Evaporated
IN THE
Sterile aerobic system
DISTRIBUTION
Hg”
0.5 3.8 7.8
W’
Corrected evaporated
SYSI EM COMPONENTS,
” Percentage of the amount of Hg added to the system. ’ The difference between the sterile and nonsterile aerobic action.
0 3 10 30
Concentration of added mercury (abnl)
MERCURY
system
5.0 10.0 16.3
Sterile aerobic system
in
3
is supposed
6.7 11.5 21.3
Sterile anaerobic system
Hg remaining solution”
AFTER
TABLE 19 DAYS
OF DIFFERENT
to constitute
51.7 55.0 33.4
37.8 33.6 46.5
Sterile aerobic system
CONCENTRA
induced
29.4 32.5 39.0
Sterile anaerobic system
by microbial
29.8 37.3 38.7
Nonsterile aerobic system
to flask”
OF ‘THE METAL
Hg adsorbed
UONS
the loss of Hg due to volatilization.
55.0 47.5 46.5
Sterile anaerobic system
Hg bound to the sediment” Sterile aerobic system
ADDITION
3
K 2 z
$ 5 n
2 3
s *
F5 0”
5
:
330
BERDICEVSKY,
SHOYERMAN,
n
11 02
I
2
I
I
4
III
Aerobic,
III
6
Time
AND YANNAI
I
with
III
I
I
I
II
(days)
FIG. 1. Rate of decrease in total Hg content of the sterile media as a function of time and the amount of mercury added.
constant level. The rate of decrease was slower, and the amount eventually remaining was higher, in the media into which the greater amounts of mercury were added. The level of mercury in the anaerobic media was checked only at the termination of the incubation period (after 19 days), and was found to be in all cases somewhat higher than in aerobic media with the same initial amount of the metal. Similar findings with regard to the rate of mercury evaporation from sterile culture media under anaerobic conditions were reported by Baier et al. (1975). The change in pH in the various growth media is given in Table 4. The initial pH was, in all instances, 7.2-7.4. After 5 days there was still no change in the sterile system, at any mercury level, but in the nonsterile media the pH dropped appreciably in all cases, except for those into which 30 &ml of mercury were added. The decrease in pH is indicative of the extent of microbial growth (i.e., acid production from sugars). Production of Methylmercury As can be seen in Table 5, methylmercury was produced in all of the systems maintaining anaerobic conditions. However, the percentage of methylmercury in the medium was in inverse proportion to its total mercury content; and this fact suggests that the highest efftciency of conversion of the inorganic into the methyl TABLE pH VALUEOFTHE
DIFFERENTMEDIA
4
5 DAYSAFTERTHE
STARTOF~NCUBATION
Level of Hg added to the medium (pg/ml) Nature of system
0
3
10
30
Sterile Nonsterile, aerobic Nonsterile, anaerobic
7.3 6.2 6.4
7.3 6.3 6.4
7.2 6.6 6.5
7.3 7.2 7.4
0.10 3.1 10.1 30.1
Aerobic
n.d. n.d. n.d. n.d.
11.3 n.d.l n.d. n.d.
I-Q
FORM~I)
n.d. n.d. n.d. n.d. in the medium
Hg
time.
of total
n.d. n.d. n.d. n.d.
98.0 3.15 0.23 0.068
5th day Hg
as Hg)
AND OF ‘rtrt
(expressed
Percentage
of CH,Hg
at zero
n.d. n.d. n.d. n.d.
98.0 97.9 23.3 20.4
/a
Level
TABLE 5 or- DURATION OF INCUBA-rlON, PRESENT IN THE SYSTF.M
of total 71.3 n.d. n.d. n.d.
present
Percentage
2nd day
AS A FUNCTION
” In terms of percentage of the amount of mercury ” Concentration of mercury in the medium itself. ( Not detected.
0.1” 3.1 10.1 30.1
Amount of Hg in medium b.4ml)
0~ M~VHI'LM~RCUR~
Anaerobic
Nature of system
LEVEL"
4.25 n.d. 11.6 n.d.
5.2 2.6 2.4 2.4
E-Lg
AMOUNT
Percentage
4.25 n.d. 0.115 n.d.
5.2 0.084 0.024 0.008
of total
MERCURY
12th day
OF TO-I-AL
Hg
i i-z
3
$
2 r
2 <
;s z z
2
3
332
BERDICEVSKY,
SHOYERMAN,
AND
YANNAI
derivative of the metal was attained by the system into which no mercury had been added. The media themselves contained 100 pg total mercury and this level is hereafter referred to as “background mercury level.” Only two samples of aerobic system were analyzed for their methylmercury content, since we assumed that little methylmercury would be produced under these conditions, as reported in some papers. It turned out, however, that at least as far as the media into which 10 pg of mercury were added per milliliter-the level of methylmercury after 12 days under aerobic conditions was higher than in the medium with the same initial mercury concentration kept under anaerobic conditions. The reason for the fact that not much methylmercury was found in the media after 12 days, and that its level decreased between the 5th and the 12th day, is apparently that much of it disappeared from the medium by evaporation, binding to the sediment, and adsorption to the glass surface of the flasks. Another phenomenon is the almost complete absence in the medium of methylmercury at the end of the incubation period. It seems likely that when the cultures grew older and/or became overcrowded they stopped producing methylmercury. This was also the case with the system into which no mercury was added. In the latter case a considerable amount of methylmercury was detected, which is ascribable to the fact that the medium itself contains 0.1 pg mercury/ml of medium, originating mainly from the materials constituting the growth medium. As can be seen in Table 5, the higher the mercury level in the growth medium the lower the percentage of methylmercury produced. Even in absolute amounts the culture containing the lowest total concentration showed the highest methylmercury content. This observation can be accounted for by the fact that the media with the high levels of the metal supported poor growth of microorganisms, as discussed above. It is also possible that at the range of mercury levels present in the media employed in this study there is no correlation between the total mercury content and the amount of the methyl derivative produced. The methylmercury level present in the control medium merely reflects the fact that the microbial growth in that medium was the highest. A similar amount of methylmercury was also produced in the medium into which 3 pg/ml of inorganic mercury were added. This latter culture showed a microbial growth similar to that of the control culture, as estimated visually. As indicated above, the production of methylmercury from inorganic mercury may have resulted from biotransformation involving microorganisms. Yet, the possibility of spontaneous methylation of mercury cannot be ruled out. The existence of such processes had, in fact, been reported by several investigators (Imura et al., 1971; Bertilson and Neujahr, 1971; Wood, 1974). Therefore, a further experiment was designed with a view to finding out whether the latter possibility could have accounted for a considerable part of the methylmercury produced, or whether it was even the major pathway involved. The possible spontaneous methylation of inorganic mercury was checked with a series of flasks containing sterile culture media into which 0 or 3.75 pg of mercuric chloride was added per milliliter of medium. In addition, four out of the five types of the microorganisms, isolated and characterized as described above, were cultured in several Erlenmeyer flasks. The setup used for this purpose was similar to that described under
IN
METHYLMERCURY
TABLE TOTAL
MERCURY IN THE
CONTENT PRESENCE
OF THE DIFFERENT OF THE BACTERIA
MARINE 6
MEDIA ISOLATED
AS A FUNCTION OF INCUBATION FROM A MARINE SEDIMENT
Type
(sterile Zero
time:
pg Hgiml
4 Days: 6 Days: 8 Days:
None medium)
5 3.75 100
2. IO 56
1.83 48.8
2.00 53.3
1.65 44.0
I. 15 30.7
pg Hgiml
7%
2.10 56
1.70 48.5
1.85 49.3
1.58 42.1
1.05 28.0
pg Hgiml cr,
2.02 53.9
1.43 38.1
1.80 48.0
1.00 26.6
0.90 24.0
pg Hgiml
2.02 53.9
1.35 36.0
1.80 48.0
0.90 24.0
0.80 21.3
2.00 53.3
1.35 36.0
1.73 46. I
0.72 19.2
0.62 16.5
2.00 53.3
1.25 33.3
1.70 45.3
0.62 16.5
0.55 14.7
pg Hg/ml pg Hgiml
of Hg content
OF METHYLMERCURY IN THE
at zero
time.
PRESENCE
IN THE OF THE
DIFFERENT
BACTERIA
7 MEDIA
ISOLATED
AS A FUNCTION FROM
Type 1 2 Days:
4
wg Hgiml 7%
TABLE PRODUCTION
2
3.75 100
% ” Percentage
1
3.75 100
9% 12 Days:
of bacteria
3.75 100
7% 10 Days:
TIME,
3.75 100
75” 2 Days:
333
SEDIMENT
pg CHR Hgiml
5%”
A MARINE
OF INCUBATION
TIME
SEDIMENT”
of bacteria 4
2
5
0.00198 0.053
n.d.’
0.0004 0.01
n.d.
4 Days:
&g CH, 95
Hgiml
0.00198 0.053
0.00119 0.032
0.04JO8 0.021
n.d.
6 Days:
wg CH, 5%
Hgiml
0.00119 0.032
0.0004 0.01
0.0016 0.042
n.d.
0.0004 0.01
0.0004 0.01
0.0008 0.021
n.d.
0.0004 0.01
n.d.
0.0004 0.01
n.d.
n.d.
n.d.
0.0004 0.01
n.d.
8 Days:
wg CH:, Hgiml
% IO Days:
pg CHR Hg/ml
% 12 Days:
pg CHR Hg/ml
5% rr Values for methylmercury are expressed in terms media. * Percentage of mercury content at zero time. ” Not detectable.
of Hg.
No CH,Hg
was detected
in the sterile
334
BERDICEVSKY,
SHOYERMAN,
AND YANNAI
Materials and Methods, except that in this case the volume of the flasks was 250 ml, and 10 g of bottom sediment and 200 ml of growth medium were placed in each flask. The total mercury and methylmercury contents of the cultures were determined at zero time, and after 2, 4, 6, 8, 10, and 12 days of incubation. The results for total mercury are presented in Table 6, and those for methylmercury, in Table 7. AS can be seen in Table 6, there was a gradual decrease in the concentration of total mercury in all media. There was, however, a different rate of disappearance of the metal from each, probably indicating that the type of microorganism had some influence on the process. We offer no explanation for this observation. As regards the amount of methylmercury produced in the media, it is obvious that no spontaneous methylation could account for it, since no methylmercury was detected in any of the sterile media. The methylmercury reached its peak level after 4 to 6 days of incubation, and then started to drop, probably by evaporation and/or adsorption to the sediment and the glass surfaces. REFERENCES Baier, R. W., Wojnowich, L., and Petrie, L. (1975). Mercury loss from culture media. Anal. Chem. 47, 2464-2467. Bertilsson, L., and Neujahr, H. Y. (1971). Methylation of mercury compounds by methylcobalamin. Biochemistry 10, 2805-2808. Blair, W., Iverson, W. P., and Brinckman, F. E. (1974). Application of a gas chromatograph-Atomic absorption detection system to a survey of mercury transformations by Chesapeake Bay microorganisms. Chemosphere 3, 167-174. Brinckman, F. E., Iverson, W. P., and Blair, W. (1976). “Approaches to the Study of Microbial Transformations of Metals.” Proc. 3rd Internat. Biodegradation Symp., pp. 919-936. The University of Long Island, Kingston, R.I. Fagerstrom, T., and Jernelov, A. (1972). Some aspects of the quantitative ecology ofmercury. Wafer Res. 6, 1193-1202. Imura, N., Sukegawa, E., Pan, S. K., Nagao, K., Kim, J. Y., Kwan, T., and Ukita, T. (1971). Chemical methylation of inorganic mercury with methylcobalamin, a vitamin Bi2 analog. Science 172, 1248- 1249. Jensen, S., and Jernelbv, A. (1969). Biological methylation of mercury in aquatic organisms. Nature (London) 223, 753-754. Jones, H. R. (1971). “Mercury Pollution Control.” Noyes Data Corp., Park Ridge, N.I. Levitan, S., Rosner, L., and Yannai, S. (1974). Mercury levels in some carnivorous and herbivorous Israeli fishes, and in their habitats. Israel J. Zoo/. 23, 135- 142. Longbottom, J. E., Dressman, R., and Lichtenberg, J. (1973). Gas chromatographic determination of methylmercury in fish, sediment and water. J.A.O.A.C. 56, 1297-1303. Nelson, N., Byerly, T. C., Kolbye, A. C., Kurland, L. T., Shapiro, R. E., Shibko, S. I., Stickel, W. H., Thompson, J. E., Van Den Berg, L. A., and Weissler, A. (1971). Hazards of mercury. Environ. Res. 4, i-69. Nelson, J. D. Jr., McClam, H. L., and Colwell, R. R. (1972). “The Ecology of Mercury Resistant Bacteria in Chesapeake Bay.” Proc. Marine Technology Society, 8th Annual Conference, Washington, D.C., pp. 303-312. Olson, H. R., and Cooper, R. C. (1974). In situ methylation of mercury in estuarine sediment. Nature (London) 252, 682-683. Wood, J. M. (1972). A progress report on mercury. Environmen? 14, 33-39. Wood, J. M. (1974). Biological cycles for toxic elements in the environment. Science 183, 1049-1052.