mercuric ion. Methylmercury, when found in natural
sediments, is always present in small amounts, usually <0.1%
of the total mercury22» 23

The effects of changing from anaerobic to aerobic
conditions on the fate of mercury in water-sediment systems
were examined in a system incubated anaerobically for 14
days, and then aerobically for 14 days (Appendix A). The
system contained mercuric chloride (50 mg/l) and calcium
acetate (10 g/l), which together were shown to result in
production of elemental mercury and methylmercury in
anaerobic systems.

Aeration of the system initially changed the
concentrations of the methylmercury that had been produced
during the 14-day anaerobic incubation (Figure 2). The
decrease in concentration may be a result of microbial
demethylating action22, since methylmercury is not readily
degraded abiotically in water34.

By the end of the aerobic incubation, the methylmercury
concentration in the sediment had increased to 60 na/aram of
sediment. The average rate of methylmercury formation over
both the 14-day anaerobic and the 14-day aerobic incubation
periods was approximately 5 na/q sediment/fay. However
methane, indicating the action of methane bacteria, was
produced during the anaerobic incubation but was not
detected during the aerobic incubation (Tahle 2). Prohably
two physiologically different bacterial populations (one
anaerobic, the other aerobic) were involved in the
methylation of mercury during the 28-day incubation.

Surprisingly, aeration stimulated the release of
elemental mercury from the water-sediment system (Figure 3).
About 3 mq (1.2%) of the added mercuric ion was released
during the 28-day incubation at a rate of 60 ng/gram
sediment/day during the anaerobic period, and at a rate of
160 ng/qram sediment/day during the aerobic period.
Microbial activity, which greatly increased upon aeration,
probably mediated the reduction of mercuric ion to elemental
mercury. The rate of release of elemental mercury was not
constant during the aerobic incubation; the rate decreased
significantly from day 14 to day 28, probably reflecting a
decreasing rate of microbial metabolism.

The concentration of soluble mercury, which was not
methylmercury, increased in the water-sediment system during
aeration (Table 3). Although mercuric chloride was added
initially at a concentration of 50 ma/l, during anaerobic
incubation it was detected in the sediment-free water at

-12-


34,

35.

36.

37.

Baughman, G. L., J. A. Gordon, N. L. Wolfe, and

R. G. Zepp. Chemistry of Organomercurials in Aquatic
Systems. U. S. Environmental Protection Agency,
Washington, DC. EPA-660/3-73-012. September 1973.
97p.

Jernelov, A. Factors in the Transformation of
Mercury to Methylmercury. In: Environmental Mercury
Contamination, Hartung, R., and B. D. Dinman (ed.).
Ann Arbor, Ann Arbor Science Publishers, Inc., 1972.
p. 167-172.

Summers, A., and S. Silver. Mercury Resistance in
a Plasmid-Bearing Strain of Escherichia coli. J.
Bact. 112:1228-1236, 1972.

Rodina, A. G. Decomposition of Salts of Orqanic

Acids. In: Methods in Aquatic Microbiology, Colwell,
Pp. Ro, and N.S. Zombruski (eA.). Raltirore, Maryland,
University Park Press, 1972. p. 235-240.

Waksman, S. A. Species Concept Among the
Actinonycetes. Pacteriol. Pev. 21:1-29, 1957.

-33-


-62-

Table 12. MERCURY IN AQUATIC BIOTA BEFORE AND AFTER DISCONTINUED INPUT

. ug/Mercury/g?
Biota Total Methyl % Methyl

Before> After® Before> After¢ BeforeP Aftere

Libellulidae

(Dragonfly Nymphs) 14.30 0.29 0.06 0.04 0.42 13.79

Corixidae

(Water Boatmen) 0.20 0.09 0.01 0.03 5.00 33.33

Dytiscidae

(Predaceous Diving 1.00 0.42 0.17 0.10 17.00 23.81

Beetles)

Hydrophilidae

(Water Scavenger 2.95 0.77 0.01 0.02 249 2.60

Beetles)

aug Mercury per gram of wet tissue homogenate
baverage of total mercury, methylmercury, percent methyl entries in Table 1.

°Results of one analysis.


APPENDIX C
MERCURY DISTRIBUTION IN AQUATIC BIOTA

Aquatic biota were collected from a drainage area
containing the combined effluent from six artificial stream
channels located near Aiken, South Carolina. The stream in
this drainage area had a continuous flow of about 600 1/min.
and included two distinct habitats. In the first area the
water flowed rapidly over a rocky bottom (Rocky Creek Area).
The second area was a ditch containing the backwater from
the first area. Here, the water flowed slowly over a bottom
with typical pond community emergent vegetation such as
cattails (Cattail Ditch Area).

Low level mercuric ion concentrations of 0.01, 1.0 and 5
/1 were maintained continuously in the artificial streams
for eighteen months and then were discontinued. During
mercuric ion addition the total mercury concentration in the
water from both the Rocky Creek Area and the Cattail Ditch
Area was approximately 0.8 ug/l. Oraanisms were collected
from these areas while mercuric ion was being added and also
two weeks after the mercury input was discontinued.

Control hiota were collected from a ditch containing
slow moving backwater from a constantly flowing artesian
well from the same aquifer as that supplying the artificial
stream channels.

The organisms were drained and homogenized in a chilled
tissue grinder. Depending on size, the number of
individuals incorporated into the homogenate ranged from
three to thirty. Aliquots were removed and weighed wet for
total and methylmercury analyses.

-38-

ug


For total mercury, each sample was digested with aqua
regia and oxidized with permanganate26. Aliquots of the
digest were pipetted into a flask and reacted with a
reducing agent, stannous chloride, for 30 seconds. The
mercury, all in the form of elemental mercury, was
determined using a cold vapor technique27.

Elemental mercury was quantitated by using the cold
vapor technique on the sample directly (without digestion
and without the reducing agent).

An aliquot of undigested sample was reacted for 30
seconds with the reducing agent and analyzed with the cold
vapor technique, yielding a value for the combined
concentrations of mercury present as elemental mercury and
mercuric ion. The difference between the amount of mercury
recovered with and without the reducing agent was considered
to be mercuric ion.

Reference curves from digested and undigested standards
having an average coefficient of variation of 5% were used
to quantitate duplicate mercury analyses.

In this laboratory, for sediments spiked with mercuric
ion, recovery was 95%.

Methylmercury was extracted from fish, sediments, media,
and aquatic biota by the method of Longbottom et al.28 and
quantitated with a Barber-Coleman gas chromatograph equipped
with a Radium-226 electron capture detector. A 1m x 6 mm
Pyrex column packed with 5% DEGS on 89-100 mesh Chromosorb W
coated with 5% KBr was employed for separation. The
nitrogen carrier gas flow was 60 ml/min.; the operating
temperatures for the column, detector, and inlet were 140°,
210°, and 180°C, respectively.

Recoveries of methylmercury hydroxide added to sediments
averaged 67%. A linear calibration curve was ohtained using
methylmercuric bromide (0.01 to 1 ng mercury).

Release of elemental mercury from growth systems was
determined by bubbling effluent gases through two traps
(Figure 1)29. The first trap, containing phosphate-
carbonate, removes volatile organomercury compounds such as
ethyl and methylmercuric chloride; the second trap,
containing acid permanganate, removes elemental mercury from
the effluent gas.


Table 3. TOTAL DISSOLVED MERCURY IN ANAEROBIC
AND AEROBIC INCUBATION PERIODS?

Total Mercury, ug/l
Days Anaerobic Aerobic
2 3
13 15
15 80
27 46

amMercury analyses were completed on water samples in which
the bacteria were removed by centrifugation (18,000 xG
for 15 minutes).

-16-


SFCTION VII
PUBLICATIONS

Holm, Harvey W., and Marilyn F. Cox. Simple ‘lethod
for Introducing Flemental Mercury into Biological
Growth Systems. Applied Microbiology. 27:622-423,
1974,

Holm, Harvey W., and Marilyn F. Cox. “Mercury
Transformations in Aquatic Sediments. Racteriological
Proceedings. p. 25, 1974 (Abstract).

Cox, Marilyn F., Harvey W. Holm, Henry 7. Kania, and
Robert L. Knight. MNethylmercury and Total Mercury
Concentrations in Selected Stream Riota. Submitted
to the Journal of the Fisheries Research Roard of
Canada, 1974.

Holm, Harvey W., and Marilyn F. Cox. Transformations

of Flemental Mercury by Bacteria. To he submitted
to Applied Microbiology. (In Preparation).

-34-


CONTENTS

Abstract
List of Figures

List of Tables

Acknowledgments

Sections

I. Conclusions

It. Recommendations

III. Introduction

Iv. Materials and Methods
v. Results and Discussion
VI. References

VII. Publications

VIII. Appendices

iii

vi

aw ne

10
30
34
35


2. except for the mosquito fish, all groups of the
biota had similar concentrations of methylmercury.

The low percentage of methylmercury in the mosquito fish
compared to those for bass and crappie muscle tissue may be
a result of (1) high inorganic mercury content of fish gut
contents, (2) physiological differences among fish species,
or (3) the age of the fish. It is unlikely that the low
percentages were caused by inadequate extraction techniques,
since analyses in our laboratory with muscle tissue from
large-mouth bass and black crappie yielded high values.

The data do not indicate the source of the methylmercury
acquired by the mosquito fish. If methylmercury were taken
up from the water, then other gilled forms, especially the
tadpoles, could be expected to have high levels. If
methylmercury were taken up in the food, then the predaceous
forms such as damselfly and dragonfly nymphs and mosquito
fish could be expected to have similar concentrations of
methylmercury. Neither pattern was evident in these
samples.

No methylmercury was detected in a bottom community of
algae, fungi, and bacteria exposed continuously to 1 ug/l
mercuric ion, even though their total mercury content was 42
g/g (wet weight).

Concentrations of total mercury in organisms collected
from the contaminated streams were 10 to 100 times the total
mercury concentrations of biota from the stream receiving no
mercuric ion (Table 11); except for trace amounts in the
mosquito fish, no methylmercury was detected in biota from
the control stream.

Biota collected two weeks after the mercury input was
stopped (Table 12) showed significantly lower total mercury
concentrations than biota found in the stream while mercuric
ion was being added continuously; but there was no
significant difference in the methylmercury concentration in
the same species collected two weeks after mercury input was

stopped.

-27-


3

> FENITROGEN (ANAEROBIC) -»=-I—— AIR (AEROBIC
3 le
£ |
— XP |
a |
= |
8 60 F eo e
n |
z |
z 407 ° |
a F
[a4
tu
= 20+ | S
= |
|

2 9 1 j im ! n

0 10 20 30

TIME (days)

Figure 2. Production of methylmercury in water-sediment systems.

-13-


-61-

Table 5,

COMPARISON OF THE STABILITY OF FLEMENTAL MERCURY IN TWO GROWTH MEDIA.

Time (h)?

Basal Salts Medium

Basal Salts Medium + Yeast Extract

Elemental Mercury

Total Mercury

Elemental Mercury

Total Mercury

(ug/l) (ug/1) (ug/l) (ug/1)

0 57.0 57.5 56.5 104.9
(S=6.1, N=10) (S=10.6, N=10) (S=5.6, N=9) (S=19.9, N=9)

48 56.3 52.5 35.0 102.1
(S=4,3, N#10) (S=4.3, N=9) (S=4.9, N=9) (S=22.9, N=9)

a
Hours represent time elapsed after removal of Hg? globule.


300

200

Hg° RELEASED (ug)

100

Figure 3.

1
— NITROGEN (ANAEROBIC)—t| AIR (AEROBIC) ———>}
a I

iN
a

LIN \ Ls
0 10 20 30
TIME (days)

Production of elemental mercury in water-sediment
systems. The value reported is the total amount

of elemental mercury released from 1,900 grams of
sediment each day.

-15-


=8Z-

Table 11.

MERCURY IN AQUATIC BIOTA FROM INPUT AND CONTROL AREA

ug Mercury/g@

Biota Total Methyl
Input Area> Control Area® Input Area Control Areac

Libellulidae
(Dragonfly Nymphs) 14.30 9.21 9.06 0.00
Corixidae
(Water Boatmen) +20 0.02 0.01 9.00
Rana sp.
(Tadpoles) 4.28 0.10 0.01 0.00
Gambusta affints 5.02 0.06 0.32 traced

(Mosquito fish)

aug Mercury per gram of wet tissue homogenate.

baverage of total mercury and methylmercury entries in Table 1.

°Results of one analysis.

4Relow detectability of extraction procedure (0.01 t1g/g).


SECTION V
RESULTS AND DISCUSSION
PHASE I -- TRANSFORMATIONS OF MERCURY IN WATER-SFDIMENT SYSTFMS

Transformations of mercury in various systems (Appendix
A) were monitored as functions of (1) acetate content, (2)
mercuric ion concentration, (3) incubation conditions
(aerobic or anaerobic), and (4) the form of mercury added to
the system.

Methane bacteria produce large amounts of
methylcobalamine, a compound that has been implicated'3,» 31,
in the methylation of mercury. These strictly anaerobic
organisms are probably therefore involved in this reaction
in sediments. Water-sediment systems, containing calcium
acetate (Appendix A) to optimize the growth of these
anaerobes, were used to study the transformations of
mercury.

32

In 25-day anaerobically incubated systems, production of
methylmercury was stimulated by high concentrations of
mercuric chloride and calcium acetate (Tahle 1). In systems
receiving only mercuric chloride, the concentration of
methylmercury was no higher than that of the control. This
suggests that the methane bacteria, which require short
chain fatty acids such as acetate for growth, could he
involved in the methylation of mercury in sediments.
However, investigators have been unable to demonstrate that
pure cultures of methane bacteria methylate mercury'!.

Autoclaved systems containing high acetate and mercuric
chloride concentrations did not form methylmercury, again
suggesting that viable bacteria are responsible for the
production of methylmercury in sediments.

Elemental mercury was the predominate product formed in
these 25-day anaerobically incubated systems (Table 1).
More elemental mercury than methylmercury was produced. As
with methylmercury, highest amounts of elemental mercury
were released from systems containing high concentrations of
acetate and mercuric chloride. Elemental mercury _is
probably formed both biotically?° and abiotically24 in these
systems, although acetate definitely increased the output of
elemental mercury.

The production of elemental mercury is expected since

the Eh of anaerobic environments that is optimum for growth
of methane bacteria is low and permits the reduction of

-10-


SECTION II
RECOMMENDATIONS

Elemental mercury, shown here to be a siqnificant
intermediate product of water-sediment systems containing
mercury, should be intensively studied to determine its role
in the fate of mercury in aquatic systems, Factors to
consider are (1) the transport of elemental mercury to
important food chain organisms, such as fish, and its
possible methylation within the animal; (2) the rate of loss
of elemental mercury from polluted systems so that accurate
predictions can be made concerning the recovery of polluted
waters, and (3) the transport of elemental mercury from the
atmosphere to terrestrial and aquatic environments.

Methylmercury accumulates in fish although it cannot be
detected in natural waters with the analytical methodology
now available. Therefore techniques to measure
environmental levels of methylmercury must be developed to
allow insight into the mechanisms of mercury transport.

The phenomenon of methylation of mercury should he
explored in detail. Because this work showed that bacteria
form only small amounts of methylmercury, if any, other
organisms such as fungi, aquatic insects, and fish should he
evaluated for their abilities to methylate various forms of

mercury.


the test populations between the 0 and 48-hour sampling
periods, shows that elemental mercury killed P. aeruginosa
and decreased the growth rate of P. fluorescens. This
phenomenon demonstrates that the Impact of elemental mercury
on a complex aquatic system cannot be accurately predicted
with a study of only a few microbial cultures.

PHASE IIT -- MERCURY DISTRIBUTION IN AQUATIC BIOTA

Dragonfly and damselfly nymphs, beetles, water bugs,
snails, tadpoles, and mosquito fish, Gambusia affinis, were
analyzed for total and methylmercury (Appendix C).

The data (Table 10) permitted the following conclusions
about the levels of total mercury in biota from a stream
receiving a continuous input of mercuric ion:

1. The levels of total mercury in dragonfly nymphs and
damselfly nymphs were much higher than levels found in the
other biota.

2. The levels of total mercury ranged greatly between
the groups of biota.

3. When several samples of a single species were
analyzed for total mercury, the results ranged widely.

If all the data for total mercury are considered, the
bottom dwelling organisms (damselfly nymphs, dragonfly
nymphs, and tadpoles) appear to have an average mercury
level (x = 12.41 ywg/g) higher than the average calculated
for those forms living in the water column (corixids,
dytiscids, hydrophilids, notonectids, and mosquito fish, xX =
2.47 wo/g). If the biota are grouped according to feeding
habits instead of habitat, then the carnivores (dragonflys,
damselflys, notonectids, dytiscids, and_mosquito fish)
contained on the average more mercury (x = 10.15 ug/g) than
the herbivores and detritivores (corixids, hydrophilids,
snails, and tadpoles, x = 2.98 ug/q). Although small sample
sizes do not permit extensive statistical treatment of these
data, these calculations suggest that both the habitat and
food habits could affect the mercury concentrations in these
aquatic biota.

Quantitation of methylmercury in aquatic biota in the
same stream showed that

l. the mosquito fish contained a greater portion of the

total mercury as methylmercury than did most of
the other biota;

-25-


-2Z-

Table 7. MERCURY ACCUMULATION BY BACTERIA DURING A 48-HOUR GROWTH PERIOD.
Cell-Associated
Organism Total Mercury, yg/l Mercury, ug/l % Cell-Associated

P. aeruginosa

P. fluorescens

Citrobacter sp.

jet

» coli

ihe]

subtilis

Ir

megaterium

47.4
56.4
45.9
57.6
100.8
116.4

(S=3.87, N=5)
(S=10.4, N=6)
(s=10.7, N=4)
(S=2.8, N=2)

($=14.5, N=4)
(S=22.2, N=10)

No Growth
10.5 (S=10.6, N=4)
12.3 (S=4.0, N=4)
13.5 (S=2.1, N=2)
43.1 (S=26.4, N=3)
50.3 (S=19.6, N=7)

18.6
26.8
23.4
42.7
43.2



10

11
12

TABLES

Anaerobic mercury transformations in
water-sediment systems (25 day incubation)

Relative concentrations of methane and carbon
dioxide in anaerobic and aerobic incubation periods

Total dissolved mercury in anaerobic and aerobic
incubation periods

Fate of elemental mercury in water-sediment
systems

Comparison of the stability of elemental mercury
in two growth media

Concentrations of elemental mercury and mercuric
ion at the 0 and 48 hour sampling times

Mercury accumulation by bacteria during a
48-hour growth period

Mercury concentration factors of bacteria grown
in a basal salts medium containing elemental
mercury

Population changes of bacteria in media with and
without elemental mercury

Mercury in aquatic biota exposed to a continuous
input of mercuric ion

Mercury in aquatic biota from input and control area

Mercury in aquatic biota before and after discontinued
input

16

18

19

20

22

23

24

26

28
29


FIGURES

Growth system with mercury traps

Production of methylmercury in water~sediment
systems

Production of elemental mercury in
water-sediment systems

iv

15


Table 1. ANAEROBIC MERCURY TRANSFORMATION IN WATER-SEDIMENT

SYSTEMS

(25-Day Incubation)

Elemental Mercury

Methylmercury

System (g/g sediment) (ug/g sediment)
Control 0.0 0.003

50 mg/l HgCh, 0.012 0.002

50 mg/1 Bach, 0.540 0.028

+ 10 g/l acetate

10 mg/1 HgCl, 0.052 0.009

+ 10 g/l acetate

-ll-


SECTION I
CONCLUSIONS

In water-sediment systems receiving mercuric ion or
elemental mercury, the methylmercury content was always less
than 1.0% of the total mercury; in pure culture studies no
methylmercury was formed.

Elemental mercury was produced in much larger quantities
than methylmercury in water-sediment systems receiving
mercuric ion. The rate of release of elemental mercury to
the atmosphere during aerobic incubation was nearly three
times the rate observed during anaerobic incubation.

Dimethylmercury was not produced in anaerobic or aerohic
water-sediment systems.

Flemental mercury was oxidized in pure cultures of
Escherichia coli, Pseudomonas fluorescens, Pseudomonas
aeruginosa, Citrobacter, Racillus megaterium, and Racillus
subtilis, and the oxidized mercury accumulated on the
bacterial cells.

Of the six cultures tested, only the two Pseudomonas
cultures were inhibited by elemental mercury.

Mosquito fish contained significantly more methylmercury
than did tadpoles, snails, and aquatic insects collected
from the same water. Algal masses did not contain
methylmercury.

-1-


only 3-15 yg/l. During aerobic incubation, however, it
increased to 45-80 ug/l. The sediment obviously acted as a
sink for mercury.

Our experiments show that elemental mercury is released
from water-sediment systems receiving mercuric ion.
However, when mercury in the elemental form is added to
water-sediment systems from an atmospheric source (Appendix
A), it oxidizes in the system and accumulates in sediments
(Table 4). A 4-day exposure of the system to elemental
mercury resulted in a 10-fold increase of mercury in the
sediments; a 33-day exposure resulted in a mercury
concentration in the sediments of 100 times that of the
control.

The atmospheric additions of elemental mercury resulted
in production of small amounts of methylmercury in the
sediments (Table 4). However the amount of methylmercury
produced was not greater than the amount obtained from the
additions of mercuric chloride to sediments discussed in
previous experiments.

PHASE II ~~ TRANSFORMATIONS OF ELEMENTAL MFRCURY IN
MICROBIAL GROWTH SYSTEMS

The objective of Phase II was to determine the fate and
impact of elemental mercury in systems (Anpendiy R)
containing pure cultures of bacteria. Phenomena examined
included (1) the oxidation and methylation of elemental
mercury by bacteria; (2) the accumulation of mercury hy
bacteria; and (3) the toxicity of elemental mercury to
bacteria.

The stahility of elemental mercury in sterile, aerohic
systems is affected by the nature of the oraanic carhon in
the medium (Table 5). Elemental mercury is stable in a
sterile, basal salts medium30 containing alucose, hut is
slowly oxidized in the hasal salts medium supplemented with
0.1% yeast extract. Jernelév35 has also concluded that
elemental mercury should be oxidized in natural waters hy
organic materials, but no reaction rates were provided.

In this investigation, B. subtilis, and B. megaterium
were studied in the basal salts medium supplemented with
yeast extract; F. coli, P. fluorescens, P. aernginosa, andé
Citrobacter were studied in the basal salts mecium

supplemented with glucose.

All of the bacteria growing in the test media stimulated
the oxidation of elemental mercury (Tahle 6), as cetermined

-17-


Ecological Research Series

Mercury In Aquatic Systems:
Methylation, Oxidation-Reduction,

And Bioaccumulation

xtED S74>
> &

2
: SZ
z

B,

%, x
AL prot®

% ‘
N aGeNct

4

National Environmental Research Center
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330


An alternative to the production of methylmercury in the
environment by bacteria, and subsequent uptake by fish, is
the uptake of inorganic mereury followed by methylation
within the fish. Jernelov suggests that organisms in fish
slime may methylate mercury, but no data are presented in
the report. Others ~ have demonstrated that fish liver
homogenates can methylate mercury. To date, however, no one
has reported that live fish can methylate inorganic mercury.

If inorganic mercury is to be taken up and methylated by
fish, in many aquatic environments it must first be released
from the sediment sinks into the water column. Elemental
Mercury, a product of microbial action on mercurials, could
be the transport form. Pure cultures of bacteria can
produce elemental mercury from mercuric chloride 0
phenylmercuric acetate*!, ethylmercuric phosphate?',
methylmercuric chloride2!, and methylmercuric bromide2?.
Over 70% of mercury added as mercuric ion can be released by
mixed bacterial cultures’ as elemental mercury; smaller
amounts can be released from streams23 and sediments?4,

Information is lacking on several points:

@ the relative importance of each mercury transformation
product (methylmercury, dimethylmercury, and elemental mercury);

e@ the microbial fate of elemental mercury; and

@ the contribution of methylmercury in food chain
organisms to fish.

This study was desioned to define environments that
produce mercury transformations; to identify mercury
products and the rates of their formation; and to complete a
food web study to determine total mercury and methylmercury
levels and distribution in organisms other than fish.


SECTION III
INTRODUCTION

Mercury in the environment has been the focus of
intensive research, primarily because of the well-known
presence of methylmercury in fish from mercury-contaminated
waters!» 2, 3, 4, 5. Since the methylated form of mercury
has been shown to be toxic to animal life in small amounts,
its formation, degradation, and concentration in organisms
are of great interest.

Methylmercury can be formed in complex laboratory growth
systems®» 7, 8, Therefore, bacteria are thought to he the
organisms responsible for the methylation of mercury in the
environment. Methylmercury is assumed to be formed in the
sediments, released to the water, and accumulated by the
fish either through direct uptake or a food chain or hoth3, 9. T0
However no data are available to clearly support either
mechanism.

Evidence is needed to define the role of bacteria in the
methylating phenomenon. To date only two laboratories!1, 12
have found pure bacterial cultures that methylate mercury,
and these only in small amounts. However, cell-free
extracts of methane bacteria have been reported to methylate
mercury13. To complicate matters, Spangler’4 reported that
methylmercury is deqradable by bacteria, suggesting that it
may not accumulate in the environment.

The mechanism of transport of methylmercury to fish is
also difficult to determine, in part hecause the compound
has not been detected in natural waters or sediments in
concentrations great enough to cause problems to the fish'5.
In his study using a stream into which mercury had been
discharged, Uthe 19 showed that rainbow trout held in cages
in the stream, after discharge was stopped, accumulated
methylmercury in their bodies. These researchers ahandoned
total mercury measurements for water samples because the
levels of mercury were helow the detection limits of the

methodology used.

As an alternative to direct uptake of methylmercury,
food chain transport to fish has been proposed?» 16, Again,
few data are available to support this hypothesis in natural
waters. Usually the data on food chain transport of
methylmercury to fish have been obtained from short term
experiments in which reed is added to the systems
and uptake rates are studied!


APPENDIX A
TRANSFORMATIONS OF MERCURY IM WATFER-SEDIMENT SYSTFMS

A variety of mixed-culture environments were used to
study the impact of organic carbon, oxygen, and the form and
concentration of mercury on microhial transformations of
mercury in water-sediment systems.

To determine the effects of carbon and mercuric ion
concentration on mercury transformations in anaerobic
environments, different concentrations of carbon (0 and 10
g/l calcium acetate) and mercuric chloride (0, 10, and 50 mg
/\) were incubated in flask systems (Figure 1) containing
590 ml of medium25 and 100 grams of homogenized sediments.
The flasks were bubbled with nitrogen gas at a rate of 20
ml/min at 25°C for the duration of the 25-day incubation,
with the effluent gases trapped by methods of Kimura and
Miller?9, Elemental mercury was quantitated in the effluent
gas; total and methylmercury were quantitated in the
sediments.

The impact of oxygen on the fate of mercury was
determined hy studying mercury transformations in a sediment
incubated anaerobically for 14 days, and then aerobically
for 14 days. One thousand grams of a farm pond sediment, 5
liters of Rarker's Medium37 , 250 mg of mercury as mercuric
chloride, 50 g calcium acetate, and 5 grams of qlucose were
incubated at 32°C with nitrogen gas flowing at a rate of 20
ml/min for days 1-14, and with air flowing at a rate of 20
ml/min for days 15-28. During the aeration, the water-
sediment was continuously mixed at 100 rpm; the system was
thoroughly mixed for 10 minutes (890 rpm) before collectina
water and sediment samples. The effluent gas was passed
through traps to catch dimethylmercury, elemental mercury,
and any other forms of mercury released; the water and
sediments were periodically analyzed for methylmercury and
total mercury. The effluent air was also analyzed for
methane and carbon dioxide.

The fate of elemental mercury in water-sediment systems
was determined in an apparatus designed by Holm and Cox?9,
To each system containing 159 grams of sediment, (sterile or
non-sterile, depending on the test) , were added 590 ml of a
25% soil extract?® and 0.25 g K,HPO,, and the pH was
adjusted to 7.5. Flemental mercury was placed in the closed
system and allowed to equilibrate with the medium for 0, 4,
or 33 days, the total incubation period in all cases lasting
33 days at 25°C. Total mercury, methylmercury, and :
elemental mercury were quantitated in the water and sediments
of the test systems.

-36-


Table 9. POPULATION CHANGES* OF BACTERIA IN MEDIA
WITH AND WITHOUT ELEMENTAL“MERCURY

log,, Population Changes
Organism Control Elemental Mercury
P. aeruginosa +4 - 2
P. fluorescens + 4 + 3
Citrobacter sp. +3 + 3
E. coli +3 +3

4Expressed as log, 4 changes between the 0 and 48-hour
sampling times,

—24—-


-€7-

Table 8.

MERCURY CONCENTRATION FACTORS OF BACTERIA GROWN

IN A BASAL SALTS MEDIUM CONTAINING ELEMENTAL MERCURY

Elemental Mercury in Medium Mercury in Cells? Concentration
Organism ug/l ng/gram Factorb
E. coli 57.4 11,250 196
P. fluorescens 54.6 65,630 1,202
Citrobacter 57.6 12,810 222

4mMercury content of cells was estimated by measuring the mercury concentration in a
centrifuqed 48-hour culture of bacteria and using the assumption that 106 bacteria
weigh 1 yg (wet weight).

brhe concentration factor was obtained by dividing the mercury concentration of the

cells by the mercury concentration of the medium.


ACKNOWLEDGMENTS

Technical assistance from Mr. Heinz P. Kollia and Ms.
Mary Marie Faucher of the Freshwater Ecosystems Branch is
gratefully acknowledged.


Table 2. RELATIVE CONCENTRATIONS? OF METHANE AND CARBON DIOXIDE
IN ANAEROBIC AND AEROBIC INCUBATION PERIODS

Time Methane Carbon Dioxide

Anaerobic

(Day 13) 35 45

Aerobic

(Day 15) 0 244

4Relative concentrations were determined by measuring peak

areas after gas chromatography analysis.

-14-


ll.

12.

13.

14,

is.

16.

17.

18.

20.

21.

22.

Yamada, M., and K. Tonomura. Formation of Methyl-
mercury Compounds from Inorganic Mercury by
Clostridium cochlearium. J. Ferment. Technol.
50:159-166, 1972.

Vonk, J. W., and A. K. Sijpesteijn. Studies on

the Methylation of Mercuric Chloride by Pure Cultures
of Bacteria and Funqi. Antoie Van Leeuwenhoek.
39:505-513, 1973.

Wood, J. M., F. S. Kennedy, and C. G. Rosen. Synthesis
of Methyl-mercury Compounds by Extracts of a Methanocenic
Bacterium. Nature. 220:173-174, 1968.

Spangler, W. J., J. L. Spigarelli, J. M. Rose, and
H. M. Miller. Methylmercury: Bacterial Degradation
in Lake Sediments. Science. 180:192-193, 1973.

Andren, A. W. Personal Communication. Oak Ridge
National Laboratory, Environmental Sciences Division,
1974,

Fagerstrom, T., and B. Asell. Methylmercury
Accumulation in an Aquatic Food Chain. A Model and
Some Implications for Research Planning. Ambio.
2:164-171, 1973.

Hannerz, L. Experimental Investigations on the
Accumulation of Mercury in Water Organisms, Rep.
Inst. Freshwater Res. (Drottingholm). 48:120-176,
1968.

Jernelév, A. Methylation by Microorganisms in Fish
Slime. In: Environmental Mercury Contamination,
Hartung, R., and B. D. Dinman (ed.). Ann Arhor,

Ann Arbor Science Publishers, Inc., 1972. p. 176-177.

Imura, N., S. K. Pan, and T. Ukita. Methylation of
Inorganic Mercury with Liver Homogenate of Tuna
Fish. Chemosphere. 5:197-201, 1972.

Magos, L., A. A. Tuffery, and T. W. Clarkson.
Volatilization of Mercury by Bacteria. Brit. J.
Industr. Med, 21:294-298, 1964.

Furukawa, K., T. Suzuki, and K. Tonomura. Decomposition
of Organic Mercurial Compounds by Mercury-resistant
Bacteria. Agr. Biol. Chem. 33, 128-130, 1969.

Spangler, W. J., J. L. Spigarelli, J. M. Rose, R. S.

-31-


23.

24,

25.

26.

27.

28.

29,

30.

31.

32.

33.

Flippin, and H. H. Miller. Degradation of Methyl-
mercury by Bacteria Isolated from Environmental
Samples. Appl. Microbiol. 25:488-493, 1973.

Kania, H. J. Personal Communication. Savannah
River Ecology Laboratory. Aiken, South Carolina.
1974.

Bongers, L. H., and M. N. Khattak. Sand and Gravel
Overlay for Control of Mercury in Sediments. Martin
Marietta Corp. Washington, DC. Water Pollution
Control Research Series 16080HVA. U. S. Environ-
mental Protection Agency. January 1972. 45p.

Payne, W., and V. Feisal. Bacterial Utilization
of Dodecyl Sulfate and Dodecyl Benzene Sulfonate.
Appl. Microbiol. 11:339-344, 1963.

U. S. Environmental Protection Agency. Methods
Development and Quality Assurance Research
Laboratory. Cincinnati, Ohio. 1972.

Hatch, W., and W. Ott. Determination of Sub-
Microgram Quantities of Mercury by Atomic Absorption
Spectrophotometry. Anal, Chem. 40:2085-2087, 1968.

Longbottom, J., R. Dressman, and J. Lichtenberg.
Gas Chromatographic Determination of Methyl Mercury
in Fish, Sediment, and Water. J. A. 0. A. C.
56:1297-1303, 1973.

Kimura, Y., and V. L. Miller. Vapor Phase Separation
of Methyl- and Ethylmercury Compounds and Metallic
Mercury. Anal. Chem, 32:420-424, 1960.

Holm, H., and M. Cox. Simple Method for Introducing
Elemental Mercury into Biological Growth Systems.
Appl. Microbiol. 27:622-623, 1974.

Imura, N., E. Sukegawa, S. K. Pan, K. Nagao, J. Y.
Kim, T. Kwan, and T. Ukita. Chemical Methylation of
Inorganic Mercury with Methylcobalamin, A Vitamin
B,2 Anlog. Science. 172:1248-1249, 1971.

Bertilsson, L., and H. Y. Neujahr. Methylation of
Mercury Compounds by Methylcobalmin. Biochemistry.
10:2805-2808, 1971.

Andren, A., and R. Hariss. Methylmercury in Estuarine
Sediments. Nature. 245:256-257, 1973.

-32-


-62-

Table 6.

CONCENTRATIONS OF ELEMENTAL MERCURY AND MERCURIC ION
AT THE 0? AND 48-HOUR SAMPLING TIMES

— ee = Ce en ene ed
Elemental Mercury, yg/l Merauric Ion, ug/l

Organism 0-Hour 48-Hour 0-Hour 48-Hour

P. aeruginosa 58.6 54.0 0.6 3.8
(S=7.1, N=6) (S=5.5, N=6) (S=1.3, N=6) (S=4.4, N=6)

P. fluorescens 54.6 38.4 0.3 6.7
(S=6.4, N=8) (S=9.4, N=7) (S=0.7, N=7) (S=3.7, N=7)

Citrobacter sp. 57.6 36.8 1.6 1.5
(S=8.6, N=4) (s=4.5, N=4) (S=1.5, N=4) (S=1.0, N=4)

E. cobi 59.2 34.0 1.1 2.1

~ ~ (S=2.8, N=4) (S#9,0, N=4) (S=1.5, N=4) (S=2.8, N=4)

B. subtilis 49.8 7.2 11.9 8.2

~ (S=8.7, N=4) (S=6.1, N=4) (S=8.5, N=4) (S=10.3, N=4)

B. megaterium 57.9 0.1 9.6 1.8
(S=7.1, N=10) (S=0.2, N=10) (S=6.7, N=10) (S=2.3, N=10)

@Mercury analyses were completed on samples removed prior to adding the inoculum.


a

ABSTRACT

The role of organisms in the fate of mercury in aquatic
environments was evaluated. Objectives were (1) to
quantitate transformations of mercury in water-sediment
systems, (2) to investigate the fate of elemental mercury in
microbial growth systems, and (3) to measure the
concentration of total and methylmercury in food chain
organisms.

In anaerobic water-sediment systems spiked with calcium
acetate and mercuric chloride, elemental mercury was
produced in larger quantities than methylmercury. The rate
of methylation of mercury in aerobic environments was
comparable to that in anaerobic environments; however, the
rate of release of elemental mercury to the atmosphere
during aerobic incubation was nearly three times that
observed during anaerobic incubation. No dimethylmercury
was produced in these systems.

In water-sediment systems, added elemental mercury was
oxidized and deposited in the sediments where small amounts
of methylmercury were formed. Six pure cultures of bacteria
oxidized elemental mercury, but none formed methylmercury.
Two Pseudomonas species did not grow in the presence of
elemental mercury.

In a stream receiving mercuric ion, mosquito fish
contained more methylmercury than did tadpoles, snails, and
aquatic insects. Algae did not contain methylmercury, even

. though their total mercury levels were high.

This report was prepared in fulfillment of ROAP 21AIM,
Task 11, by the Freshwater Ecosystems Branch, Southeast
Environmental Research Laboratory, National Environmental
Research Center-Corvallis, U. S. Environmental Protection
Agency. Work was completed as of June 30, 1974.

ii


SELECTED WATER 1, Rept No.) 2. 3. Accession No,
RESOURCES ABSTRACTS

INPUT TRANSACTION FORM Ww
4. Title 5. Report Dace
Mercury in Aquatic Systems: Methylation, Oxidation- 6.

Reduction, and Bioaccumulation & Performing Organleation
No.

Report

7, Author(s)

Holm, Harvey W., and Marilyn F. Cox 10. Project No.

anization ROAP_21AIM, Task 11

9,
Southeast Environmental Research Laboratory 11, Contract/Grant No.
U. S. Environmental Protection Agency

113. Type cf Report and
Period Covered

12. Sponsoring Organize’‘on U. §. Environmental Protection Agency
15. Supplementary Notes

Environmental Protection Agency report number, EPA-660/3-74-021, August 1974

16. Abstract .

The role of organisms on the fate of mercury in aquatic environments was evaluated.
Objectives were (1) to quantitate transformations of mercury in water-sediment systems,
(2) to investigate the fate of elemental mercury in microbial growth systems, and (3)
to measure the concentration of total and methylmercury in food chain organisms.

In ‘anaerobic water-sediment systems spiked with calcium acetate and mercuric chloride,
elemental mercury was produced in larger quantities than methylmercury. The rate of
methylation of mercury in aerobic environments was comparable to that. in anaerobic
environments; however, the rate of release of elemental mercury to the atmosphere
during aerobic incubation was nearly three times that observed during anaerobic
incubation. No dimethylmercury was produced in these systems.

In water-sediment systems, added elemental mercury was oxidized and doposited in the
sediments where small amounts of methylmercury were formed, Six pure cultures of
bacteria oxidized elemental mercury, but none formed methylmercury. Two Pseudomonas
species did not grow in the presence of elemental mercury.

In a stream receiving mercuric ion, mosquito fish contained more methylmercury than did
tadpoles, snails, and aquatic insects. Algae did not contain methylmercury, even
though their total mercury levels were high.

172, Descriptors

*Heavy metals, *Aquatic bacteria, *Food chain, Aquatic Microbiology, Fish food
organisms, Growth rates

17b. Identifiers
‘Mercury, *Transformation, *Elemental mercury, *Methylmercury, Bioaccumulation

I7¢. COWRR Field & Group 05B

18. Availabilit; 19. S-~urity Class, | 21. Mo. of To:
i Cheport) Pages | Send
‘ i WATER RESOURCES SCIENTIF!
20 Seeursy Class, | 2, Price | MATERAESSUREEEecERTUTGIagonmaTion cenran
Cpe) WASHINGTON, D. C.
Abstractor ] Institution

WRSIC 102 (REV. JUNE 1971)


APPFNDI¥ B
THE FATE OF ELEMFNTAL MERCURY IN MICROBIAL GROWTH SYSTEMS,

Six pure cultures of hacteria (Ff. coli, P. fluorescens,
Pp. aeruginosa, Citrohacter, RB. subtilis, and “Be megaterium)
Were used to study the fate of elemental mercury in
microbial growth systems.

Each of the mercury flasks3° received 500 ml of either a
basal salts medium25 or a basal salts medinm containing 0.1%
yeast extract, and was sterilized at 121°c for 15 minutes.
After the flasks cooled, elemental mercury globules were
added to the mercury holder of selected flasks; and the
systems were equilibrated. After 48 hours, the mercury
globule was removed from the test flasks and sterile qlucose
(0.25%) was added to the flasks not containing yeast
extract. Inocula of the appropriate test hacterium were
added (usually to a concentration of 106/ml), and flasks
were incubated at 25°C at 125 rpm for 48 hours.

B. subtilis and R. megaterium were studie? in medium

supplemented with yeast extract; the other cultures were
studied in medium supplemented with qlucose.

Each experiment contained duplicate sterile controls
containing elemental mercury; duplicate inoculated controls,
with no mercury; and duplicate test systems receiving
elemental mercury and bacteria.

At zero time (just prior to inoculation), duplicate
samples were removed for mercury analyses (total, elemental,
and mercuric ion), after which bacteria were added, the
flask contents were mixed, and samples were removed for
plate counts.

After 48 hours duplicate mercury analyses (elemental
mercury, mercuric ion, total mercury, methylmercury, and
bacterial-associated mercury) and bacterial counts (TGE pour
plates) were completed.

-37-


—_— >) a >)
NITROGEN (
° | ° °
GROWTH FLASK °
SNS ZENS
PHOSPHATE— PERMANGANATE
CARBONATE SOLUTION
SOLUTION

Figure 1. Growth system with mercury traps29


Elemental mercury was equilibrated?° over a 48-hour
period with a sterile basal salts medium25 containing either
0.25% glucose or 0.1% yeast extract.

At zero time (just prior to inoculation), duplicate
liquid samples were removed for mercury analyses (total
mercury, mercuric ion, elemental mercury), after which
bacteria were added, the flask contents were mixed, and
samples were removed for plate counts.

After a 48-hour incubation (25°C, 125 rpm) mercury
analyses (elemental mercury, mercuric ion, total mercury,
methylmercury, and cell-associated mercury) and bacterial
counts (TGE agar pour plates) were completed.

Phase III -- Mercury Distribution in Aquatic Biota
(See Appendix C)

The objective of this study was to determine the
concentration of methyl- and total mercury in a variety of
aquatic organisms exposed to a low level of mercuric ion
over an extendec time period. The mercury levels of these
biota were compared to mercury levels in similar biota taken
from a control area receiving no known mercury input.
Organisms that were analyzed included dragonfly and
damselfly nymphs, beetles, water bugs, snails, tadpoles, and
mosquito fish, Gambusia affinis. Analyses were performed on
homogenized whole organisms.



EPA-660/3-74-021
August 1974

MERCURY IN AQUATIC SYSTEMS:

METHYLATION, OXIDATION-REDUCTION, AND BIOACCUMULATION

by

Harvey W. Holm
Marilyn F. Cox

Southeast Environmental Research Laboratory
Athens, Georgia

ROAP 21 AIM, Task 11
Program Element 1BA023

NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330

"Ror sale by the Superintendent of Documents, U.8. Government Printing Office, Washington, D.C. 20402 - Price $1.05


10.

SECTION VI
REFERENCES

Rivers, J., J. Pearson, and C. Shultz. Total and
Organic Mercury in Marine Fish. Bull. of Envir.
Con. and Tox. 8:257-266, 1972.

Westod, G. Methylmercury as Percentage of Total
Mercury in Flesh and Viscera of Salmon and Sea
Trout of Various Ages. Science. 181:567-568, 1973.

Langley, D. G. Mercury Methylation in an Aquatic
Environment. Jour. Water Poll. Control Fed.
45:44-51, 1973.

Lockhart, W., J. Uthe, A. Kenney, and P. Mehrle.
Methylmercury in Northern Pike (Esox lucius):
Distribution, Elimination, and Some Biochemical
Characteristics of Contaminated Fish. J. Fish.
Res. Bd. Can, 29:1519-1523, 1972.

Zitko, V., B. Finlayson, D. Wildish, J. Anderson,

and A. Kohler. Methylmercury in Freshwater and
Marine Fishes in New Brunswick, in the Bay of Fundy,
a at on the Nova Scotia Banks. J. Fish. Res. Rd. Can.
28:1285-1291, 1971.

Jensen, S., and A, Jernelédv. Biological Methylation
of Mercury in Aquatic Organisms. Nature. 223:753-754,
1969.

Bisogni, J., and A. Lawrence. Kinetics of Microbially
Mediated Methylation of Mercury in Aerobic and
Anaerobic Aquatic Environments. Cornell University
Water Resources and Marine Sciences Center. Ithaca,
New York. Technical Report 63. May 1973. 180p.

Bishop, P. L., and E. J. Kirsch. Biological Generation
of Methylmercury in Anaerobic Pond Sediment. In:
Proceedings of the 27th Industrial Waste Conference.
Purdue University, Lafayette, Indiana. 1972.

Jernelév, A., and H. Lann. Mercury Accumulation in
Food Chains. Oikos (Copenhagen). 22:403-406, 1971.

Uthe, J. F., F. M. Atton, and L. M. Royer. Uptake
of Mercury by Caged Rainbow Trout (Salmo gairdneri)
in the South Saskatchewan River. J. Fish. Res. Bd.
Can. 30:643-650, 1973.

-30-


-st-

Table 4. FATE

OF ELEMENTAL MERCURY IN WATER-SEDIMENT SYSTEMS.

Total Incubation

Mercury in Sediments, ug/g

System Period (Days) Total Mercury Methylmercury
Control 33 0.12 0.0

Hg° (4-day exposure) 33 1.12 0.006

Hg° (33-day exposure) 33 15.6 0.017



RESEARCH REPORTING SERIES

Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:

1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research

4. Environmental Monitoring

5. Socioeconomic Environmental Studies

This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formaticn,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.

EPA REVIEW NOTICE

The Office of Research and Development has reviewed this report
and approved its publication. Mention of trade names or
camercial products does not constitute endorsement or recam-
mendation for use.


-97-

Table 10. MERCURY IN AQUATIC BIOTA EXPOSED TO A CONTINUOUS INPUT OF MERCURIC TON

Number ya Mercury/a?

Biota of Analyses? Total Methy1 @Methyl
Libellulidae-Neurocordulina alabamensis 1 14.40 9,96 0.41
(Dragonfly Nymphs) 1 14.20 0.06 9.42
Coenagrionidae-Arogia sp. 1 22.20 0.10 0.45
(Damselfly Nymphs) 1 23.20 0.06 0.25
Notonectidae-Votonecta tndtea 1 1.39 0209 6.92

(Back Swimmers)

Corixidae-Hesperocoriaza sp. 1 0.06 9.01 16.67
(Water Boatmen) 1 9.33 -- --¢
Dytiscidae
(Predaceous Diving Beetles) + 2.00 9.17 17.90
Hydrophilidae-Troptsternus sp. 9
(Water Scaverger Beetles) 1 2.05 0-01 0.4
Lymnaeidae-Lymnaea sp. 3 2.14 9.01 0.47
(Pond Snails) 2 1.89 -- --
Physidae-Physa sp. z 2.40 0.01 0.42
(Pond Snails) 3 8.20 9.94 0.49
Ranidae-Rana sp. 3 2.08 6.0L 0.48
(Tadpoles) 3 4.36 9.03 0.69

1 6.41 9.00 9.00
Poeciliidae-Gambusia affinis 3 2.42 0.28 11.57
(Mosquito fish) 2 3.35 -- --

1 9.28 0.37 3.99

aug Mercury per gram of wet tissue homogenate.

bNumbers in this column refer to single, duplicate, or triplicate total mercury analyses
performed on one pooled sample.

CAnalysis not done.


c.

SECTION VIII

APPENDICES
Page

Experimental Procedure for Phase I -- 36
Transformations of Mercury in Water-Sediment
Systems
Experimental Procedure for Phase II -- 37
The Fate of Flemental Mercury in Microbial
Growth Systems
Experimental Procedure for Phase III -- 38

Mercury Distribution in Aquatic Biota

-35-


by paired "t" tests (a = 0.05). The amount of mercury
oxidized by the bacteria ranged from small amounts (P.
aeruginosa) to nearly 100% (B. megaterium). The small
amount of oxidation by P. aeruginosa, P. fluorescens, and E.
coli is expected since these species have been shown

to reduce mercuric ion to elemental mercury?»

The elemental mercury oxidized by the bacteria was not
quantitatively recovered as mercuric ion (Table 6).
Apparently most of the oxidized mercury in the growth
systems is complexed in such a manner that it is not
released during reduction by stannous chloride.

Consequently, only two of the populations, P.
fluorescens and R. megaterium sianificantly changed the
concentrations of mercuric ion in the medium within the 48-
hour incubation period. P. fluorescens increased the
concentration of mercuric ion, B. megaterium decreased it.
The differences between these two cultures may be due either
to the different growth kinetics exhibited by the organisms
(Pp. fluorescens grew poorly in the presence of elemental
mercury, therefore there were less cells to bind the
mercuric ion), or to different growth media in which the
cultures were studied.

The elemental mercury that was oxidized in these systems
was not transformed to methylmercury. After an incubation
of 48 hours, all cultures were analyzed for methylmercury,
but none was detected (detection limit, 9.6 ug/l). This
differs from results reported by Vonk and Sijpesteijn'!? who
reported that some of these genera can produce small amounts
of methylmercury from mercuric chloride. The different
results may be explained by the fact that different media,
mercury sources, incubation periods, and analytical
procedures were used in the two studies.

Mercury added to these systems accumulated in the
bacterial cells (Tahle 7). In these six cultures, the
percentage of the total mercury in the system associated
with the bacterial biomass ranged from 18.6 to 43.2%.
Generally those organisms growing in the hasal salts medium
with glucose contained less mercury than those growing with
the yeast extract. The concentration factors for
accumulation of mercury were 222, 196, and 1202 for
Citrobacter, E. coli, and P. fluorescens, respectively
(Table 8).

Growth of bacterial populations may be affected by
elemental mercury. Table 9, a summary of log, 9 changes in

-21-


SECTION IV
MATERIALS AND METHODS

MATERIALS AND ANALYTICAL PROCEDURES

Organisms

Mixed bacterial populations present in local pond
sediments were used as inocula for studying the fate of
mercury in water-sediment systems. No attempt was made to
isolate and identify the bacterial flora present in the
sediments.

Pure cultures of Escherichia coli, Pseudomonas
fluorescens, Pseudomonas aeruginosa, Citrobacter, Bacillus
megaterium, and Bacillus subtilis were used for studying the
transformations of elemental mercury. Inocula of the
Bacillus species were 24-hour cultures (25°C, 125 rpm) grown
In a basal salts medium25 containing 0.1% yeast extract; the
other four cultures were grown on the basal salts medium
containing 0.25% glucose.

Ten different families of aquatic organisms were
collected by personnel at the Savannah River Ecology
Laboratory, Aiken, South Carolina, from a small stream
receiving low levels of mercuric chloride. Organisms from a
control stream and from the test stream two weeks after the
mercury input was discontinued were also collected. Water
was drained from the organisms and the organisms were
nemogenized in a chilled tissue grinder prior to analysis.

Reag ents

Reagent grade chemicals were used for the preparation of
the growth media. Extractions of methylmercury were
completed with spectroanalyzed grade solvents.

Growth Measurements

During the course of experiments, bacteria were counted
by plating serial dilutions, in duplicate, in Tryptone
Glucose Extract Agar (TGE) pour plates, incubating at 25°C
for 48 hours, and counting the colonies.

Analytical Procedures

A Laboratory Data Control (LDC) UV Monitor, Model 1235
was used for quantitating total mercury, mercuric ion, and

elemental mercury.

-5-


phosphate~carbonate trap replaced by an acidified mercuric
chloride trap. Since dimethylmercury is cleaved to form
monomethylmercury in the presence of excess mercuric ion,
this trap should quantitatively trap dimethylmercury as
monomethylmercury. The methylmercury “in the trap was
quantitated by Longbottom's method

A Varian Aerograph Model 90P-3 chromatograph, equipped
with a thermal conductivity detector operated at room
temperature was used to measure production of methane and
carhon dioxide. Separation was obtained using a 1.2 m x 6
mm glass column packed with Porapak Q.

EXPERIMENTAL DESIGN

Each phase of this research required a distinctly
different approach. Only an outline of the experimental
design will be documented here; comprehensive descriptions
of the experiments may be found in Appendices A, B, and C.

Phase I -- Transformations of Mercury in Water-Sediment
Systems (See Appendix A)

The fate of mercury (mercuric ion and elemental mercury)
was determined in laboratory water-sediment systems bubbled
with either nitrogen gas or air (flow rate, 20 ml/min).

Mercury forms quantitated in the effluent air, water, or
sediments, as appropriate, included total mercury, mercuric
ion, elemental mercury, methylmercury, and dimethylmercury.

Transformations of mercury in the systems were monitored
as functions of

(1) calcium acetate concentration;

(2) mercuric ion concentration;

(3) form of mercury added to the systems; and

(4) incubation conditions (anaerobic or aerobic).

Phase II -- Transformations of Elemental Mercury in
Microbial Growth Systems (See Appendix B)

Six pure cultures of bacteria were used to study the
fate of elemental mercury in microbial growth systems.