- Email: [email protected]
Microbial transformations of arsenic in lake ohakuri, New Zealand
Wat. Res. Vol. 20, No. 3, pp. 283-294, 1986 Printed in Great Britain. All rights reserved
0043-1354/86 $3.00+0.00 Copyright © 1986 Pergamon Press Ltd
MICROBIAL TRANSFORMATIONS OF ARSENIC IN LAKE OHAKURI, NEW Z E A L A N D MICHAEL C. FREEMANl'*, JOHN AGGETT2 and GLENNYS O'BRIEN 2 'Biological Sciences Department, University of Waikato, Private Bag, Hamilton and 2Department of Chemistry, University of Auckland, Private Bag, Auckland, New Zealand (Received October 1984)
Abstract--The concentration and chemical speciation of arsenic in the waters and sediments of Lake Ohakuri, New Zealand were examined. Mixed microbial populations from the sediments were tested in vitro for their ability to mediate redox transformations of inorganic arsenic. Under aerobic conditions the mixed microbial cultures were found to be able to reduce arsenic(V) to arsenic(Ill) and also to oxidize arsenic(Ill) to arsenic(V). Under anaerobic conditions only reduction of arsenic(V) to arsenic(III) was observed. Four species of sediment fungi were isolated, grown aerobically and all were found capable of reducing arsenic(V) to arsenic(Ill). The role of microbial heterotrophs in determining the observed mobility and speciation of arsenic in Lake Ohakuri is discussed. Key words--arsenic, arsenic(III), arsenic(V), sediments, bacteria, fungi, biogeochemistry, Lake Ohakuri,
Walkato River
INTRODUCTION The water quality of the eutrophic Waikato River in the North Island of New Zealand is influenced by arsenic released in geothermal discharges from natural sources south of Lake Ohakuri and by the discharges from the Wairakei geothermal power station (Aggett and Aspell, 1980; Coulter, 1977; Strachan, 1979) (Fig. I). The dissolved arsenic concentration is usually between 30-60/~gI -~ and is normally found as the thermodynamically stable arsenic(V) (Ferguson and Gavis, 1972), however, during early spring and summer the thermodynamically less favoured form arsenic(llI) has on occasion been observed as the predominant form (Aggett and Aspell, 1980). A significant proportion of the arsenic becomes adsorbed in the sediments of the Waikato River hydrolakes (Aggett and Aspell, 1980). The mean arsenic concentration of Lake Ohakuri sediments has been reported as 3 3 5 # g g -I dry wt (Aggett, 1981). These sediments are loose black oozes consisting of about 85% water which usually contains 1000-4000 p g I- ~ arsenic with approximately equal amounts of arsenic(Ill) and arsenic(V) (Aggett, 198 I; Aggett and Kadwani, 1983). It has been shown that the arsenic in these sediments has considerable mobility. Studies have shown that in chemically reducing regions of sediment, arsenic is released to the interstitial water and diffuses upwards to the sediment-surface interface where it is either readsorbed or released to the overlying water (Aggett and O'Brien, 1984). *Present address: North Canterbury Catchment Board and Regional Water Board, P.O. Box 788, Christchurch, New Zealand. 283
The latter may be attributable to the growth of phytoplankton and/or submerged macrophytes and evidence has been presented to corroborate this theory (Freeman, 1985, 1986a, b). However, during early spring, surface water temperatures of 8-14°C and phytoplankton densities of 5-10% of the summer maximum (Coulter et al., 1983), would tend to reduce the likelihood of phytoplankton and macrophyte mediated arsenic speciation changes. This paper is an attempt to identify potential microbial transformations of arsenic in the system. METHODS Dissolved oxygen and temperature profiles for the lake were carried out using a Yellow Springs Instrument Model 54 ARC dissolved oxygen meter, calibrated in the field against oxygen-saturated air. Water samples were collected using a 2-I. PVC Van Dorn water sampler. An aliquot was filtered through a 0.45/~m membrane filter and immediately analysed for iron(ll). A second aliquot was filtered through a 0.45 #m membrane filter and acidified to pH 2 for later analysis of total iron, total arsenic and arsenie(IIl). All sample containers were acid-cleaned and rinsed with MiNi Q water. Sediment samples were taken with a Jenkin's sediment sampler. One centimetre sections were transferred to 50 ml centrifuge tubes. The headspaces of the tubes were purged with oxygen-free nitrogen and the tubes sealed and stored in a "chilly-bin". Interstitial water was separated by centrifugation, usually carried out within 4 h of sampling, at 15,000 rpm (4100g) for 15 min at 4°C. Following centrifugation the interstitial water was filtered through a 0.45/~m membrane filter. A small volume was immediately analysed for iron(II) and the remainder was preserved with hydrochloric acid (pH 2) for subsequent analysis of total iron, total arsenic(III). The analytical methods used were those described in Aggett and O'Brien (1984). Sediment samples for the laboratory experiments were collected from a depth of 30 m in Lake Ohakuri on 31 March 1984, with an Eckman grab sampler. The sediment was transported on ice and stored in the laboratory at 4°C.
284
MlCHAEL C. FREEMANet al.
km
Fig. 1. Location of the Waikato River and its hydrolakes in the North Island, New Zealand.
pH and Eh values for the mixed sediment sample were determined at 20°C using an Extech p H / E h meter, model 67 (Golterman et al., 1975). Sediment samples were diluted with sterile Milli Q water, lightly sonicated, (Kontes sonicator, power setting No 5, peak frequency, 30 s for a I0 ml sample), and subsamples were prepared for bacterial counts using dilution plating with nutrient agar made up with MBL medium, as described in Nichols (1973), with vitamins omitted, orthophosphate phosphorus concentration reduced to 120 #g 1-i, arsenic(V) added to approx. 50/~g 1- ~and boron added to l mg 1- ~, the latter two being added to simulate the concentrations found in the Waikato River (Strachan, 1979). Anaerobic plates were incubated in a standard anaerobic jar using a "gas pak" (BBL). Yeast were counted using the following medium, made up with the modified MBL medium (MMBL): 5 g l -I, Bacto-peptone or Caesin Hydrolysate, 10gl -~ glucose, 4 g l -~ yeast extract, 4 g l - t malt extract, 3.5 ml 1-I of 1% (w/v) rose bengal solution and 20g 1-t agar (APHA, 1981; Booth, 1971). The bacterial experiments were carried out in 1 1. conical flasks using MMBL medium with arsenic added as arsenate or arsenite and 10mg 1-I o f each o f the following added:
glucose, sodium glycollate, caesin hydrolysate and yeast extract. Aerobic bacterial cultures were loosely stoppered with cotton wool and foil, and shaken at 120 rpm. Anaerobic bacterial cultures were stoppered with rubber bungs and shaken three times daily. Anaerobic conditions were obtained by purging the flask and medium with a sterile mix of 80% H 2 and 20% CO 2. After each sampling, the anaerobic flask and medium were purged with the gas mix. Incubations were carried out at 20°C. The initial pH of the medium, measured with a Radiometer pH meter 28 was 7.2. The cultures were inoculated with subsamples taken from a lightly sonicated ten-fold dilution of fresh sediment. Controls in the arsenite sediment test were spiked to give a final concentration of 0.2% HgCI2 (w/v) in addition to the sediment inoculation. Fungal cultures were carried out using isolates obtained from dilution plating. Identification of the fungi was carried out with the aid o f keys and illustrations in Ainsworth et al." (1973) and Carmichael et aL (1980). The isolates were grown in magnetically-stirred, continuously aerated 11. conical flasks, initially containing 1 1. of a fungal medium similar to that detailed above with the rose bengal and agar omitted. The possibility that these fungi could produce volatile
Microbial transformation of arsenic 0
20
40
60
80
100 A s ( ~ g 1-1)
4;
4;
1~
1;
4;
2'0
I
I
I
I
I
I
2
4
6
8
'
'
o
A,(m) 10 E
As(~z)
7 I ,
Fe( mg 1-1 )
Temp. (*C)
10 DO(mg I -I )
0.5 I
1.0
1.5
I
I
!/
10
PC /I
,#
J
Fe (111")
.
'
(a}
,o
(b)
20 30'
.~ 3 0
40
40
0
0
' TempTeD0
7
-.
285
1
2
l
I
0.6
3
4
I
1.2
5
I
1.8
6
I
2.4
7
Solid
As(mg g -1)
Interstitial Fe(mg 1-1)
I
3.0
.6 Interstitial
0
1.0
2.0
3.0
£2 ~o g 6 ._E 8 tn 10
"~p(O'-
Solid As
Fe (TIT)
/.o-
Fe (TTE) a."7-~ g At'"
(c)
10 LA
(d)
Fig. 2. The variation of arsenic(V), arsenie(III), iron(III) and iron(II) in the water column and interstitial water of Lake Ohakuri, during stratification.
alkylarsines was investigated by running the sterileair outlets through 50 ml of 2 M HNO 3 placed in an ice bath (Cox and Alexander, 1973; Vidal and Vidal, 1980). Bacterial numbers were counted using acridine orange epifluores~nt microscopy (Meyer-Reil, 1978; Zimmerman et al., 1978). The volume of the sample used for cell counts was varied during the experiment and this resulted in a counting uncertainty (standard error) of 10-15%. Fungal growth was followed using a haemocytometer with the yeast species and medium absorbance at 750 nm with the mould (Calam, 1969; Mailette, 1969). These methods usually had uncertainties of 15-20 and 10% respectively. The epifluorescent technique was used, together with plating onto nutrient agar, as a check for contaminants in the fungal cultures. Samples from the mixed microbial cultures, approx. 70ml, were filtered using successively smaller pore size membrane filters, normally 0.8 #m followed by 0.45 pm. (Sediment material quickly clogged the 0.45/~m filter without a prefilter.) The fungi were separated from the sample by centrifugation at 3000 rpm (164g) for 15 min. The 70 ml of supernatant or filtrate was stored in 50% HNO3-cleaned polyethylene bottles and immediately frozen at - 10°C until analysis. Samples were periodically analysed for the presence of the four environmentally common arsenic species, arsenic(V), arsenie(III), monomethylarsonate and dimethylarsinate using an anion-exchange method (Aggett and Kadwani, 1983). After cheeks with this method revealed the absence of any methylated arsenic a simpler pH selective method for the two inorganic species was used, based on Aggett and Aspell (1976). Arsenic(III) was selectively determined by using a 10 ml sample buffered to pH 4.2 with 5 rul of a 2 M citrate buffer. Total dissolved arsenic was determined using a 10 ml sample acidified with 2 ml concentrated HC1. Arsenic(V) was calculated as the difference. Total microbial arsenic was determined using a nitric acid, nitric-sulphuric-perchloric acid two step digestion according to Maher (1983). Other methods and equipment used for the arsenic analyses were as detailed in Freeman (1985). The reproducibility and detection limit, based on the analysis of five 50pgl -I samples and blanks, were 4.2% and 2 #g 1- ~ respectively.
Dissolved reactive phosphate phosphorus (DRP) was determined by the method of Downes (1978). RESULTS AND DISCUSSION
The typical summer profiles of arsenic(V), arsenic(III), dissolved oxygen (DO), temperature, iron(III) and iron(II) in the body of Lake Ohakuri are illustrated in Fig. 2(a) and (b). The sediment profiles of arsenic(V), arsenic(III), iron(Ill) and iron(II) are shown in Fig. 2(c) and (d). These data illustrate some of the factors affecting arsenic speciation in both the lake water and sediments. As the lake stratified, the concentration of dissolved arsenic in the hypolimnion increased (Fig. 3). The proportion of arsenic(III) also rose, to a maximum at the sediment surface [Fig. 2(a)] (O'Brien, 1983). The diffusion of arsenic from the sediments to the overlying waters is believed to be a consequence of iron(III) reduction and resolubilization of b o u n d arsenic(V) and arsenic(Ill) in a mechanism similar to that of phosphate release from deoxygenated sediments (Wetzel, 1975). The preponderance of arsenic(III) in the hypolimnion was probably due to both the physico/chemical conditions and microbially-mediated speciation changes of desorbed arsenic(V). The preponderance of arsenic(V) in the sediment interstitial waters was probably a reflection of the original arsenic oxidation state during deposition. The potential contribution of the hypolimnetic arsenic to the total lake load after destratification is not great due to the relative volumes of the epilimnion and hypolimnion at this time of year, 1.09 x 10s and 0.05 x 10Sm 3 respectively (O'Brien, 1983). Studies carried out before and after
286
MICHAELC. FREEMANet al. 10
15
20
E ~ 121
3o 35
27/1/E
I
I
I
I
20
40
60
80
Dissolved
I
I
I
I
I
I
100
120
140
160
180
200
orsenic
(/~g
1-1 )
Fig. 3. The dissolved inorganic arsenic profiles of Lake Ohakuri during stratification. destratification in autumn 1982 revealed a small, but significant rise in the total dissolved arsenic of 3 ~ l m g m -3, measured downstream of the Lake Ohakuri dam tailrace. The variation of dissolved arsenic concentration in Lake Ohakuri over a 21 month period appears to have some cyclical tendency, however, the peaks occur at different times in different years (Fig. 4). These studies did not reveal any high concentrations of arsenic(Ill) in the surface waters as were observed in previous years (Aggett and Aspell, 1980). The reason(s) for the irregular occurrence of arsenic(Ill) have not yet been satisfactorily answered. In order to establish whether or not heterotrophic microorganisms had the potential to become involved in arsenic transformations and pathways, bacteria and fungi were isolated from the lake sediments. Preliminary studies showed the sediment microflora to be more diverse and numerous than the surface planktonic populations. Thus, a sediment sample was collected to serve as a diverse source for a mixed heterotrophic culture. The sediment sample used for the experiments was a black ooze, indicating the presence of iron sulphide. The pH and redox potential of the mixed sample were 6.6 and - 5 0 m V respectively. Although sediment oxygen concentration was not determined at the time of collection, studies carried out in situ in April 1981 --
40
o
30
2o >
10
c5 o
I L I I I I NDJ FMAMJ
I I
t I I I I I J A S 0 NDJ 1981 Time
( months
l
I I I ~ I I FMAMJ d 1982
)
Fig. 4. The variation of dissolved inorganic arsenic, at 10 m depth, over 21 months in Lake Ohakuri.
and 1982 during stratification showed that only trace amounts of dissolved oxygen were available at the sediment surface and the temperature in this region was 16.0-16.5°C. The numbers of heterotrophs found in the sample were as follows: aerobic bacteria: l.l x 106 colony forming units (CFU) g-~ dry wt, anaerobic bacteria: 2.7 × 10 6 C F U g - 1 and aerobic fungi: 1.9 x 104CFUg -~. The first arsenic(V) sediment test, carried out under aerobic conditions, exhibited exponential growth of a mixed microbial community with one large (approx. 3 x 5#m) unidentified "yeast sized" species dominating until after 60 h of incubation [Fig. 5(a)]. During the exponential growth phase the DRP concentration dropped from 1741tgl -~ (5.61#M) to 32 g 1-~ (1.03/~M). During this period the arsenic(V) was quantitatively reduced to arsenic(Ill). The total cell density did not increase after 40 h, however, there was a decline of the yeast species density, which was replaced during the 30-60h period, by a mixed bacterial community, consisting of various rods and cocci. This community change indicated that the initial yeast population was most likely responsible for the arsenic(V) reduction to arsenic(Ill). The community change was possibly due to competition for the limited amount of DRP available, the bacterial population was apparently more able to thrive on a lower DRP concentration [Fig. 5(c)]. The increase in aerobic bacterial numbers was concurrent with the rapid oxidation of arsenic(Ill) to arsenic(V). A further drop in the DRP concentration to 20 #g 1-~ (0.64#M) and the presence of the potential competitive analogue arsenic (V) at 54#g As 1 (0.72 #M) probably combined to produce the crash in microbial numbers and the ensuing rise in DRP. In the second arsenic(V) test, carried out under anaerobic (80% H> 20% CO2) conditions, the increase in bacterial numbers, mainly small rods (approx. 0.5 x 1.5 #m), accompanied the reduction of arsenic(V) to arsenic(III) [Fig. 5(b)]. In both experiments it is difficult to determine the
287
Microbial transformation of arsenic
with HgCI:, at 0.2% (w/v). The first test [Fig. 6(a)] carried out under aerobic conditions enabled a microbial community dominated by a "yeast-sized" cell (approx. 3 x 5 #~m) to initially peak at nearly 107 cells ml-L Thereafter, the yeast numbers declined and were eventually replaced by small cocci and rods (approx. 0.5 #xm dia and 0.5 x 1.5 #xm respectively). The disappearance of arsenic(III) and appearance of arsenic(V) was quite slow until the microbial commu-
effect of phosphate limitation on arsenic(V) reduction as in each case, exponential microbial growth, DRP depletion and arsenic(V) reduction all occurred concurrently [Fig. 5(a), (b) and (c)]. The controls in this first set of experiments were run without any sediment additions. The second set of sediment tests were carried out using arsenic(III) as the initial arsenic source and the controls, with sediment/bacteria inoculations spiked Aerobic
8
(o)
/
-
60
6
4
50
i
_=
~
As(V)
L ,.,,7;,, /
/
\
/
'\
'¢~
/J
20
8
6 10
/
/'\, / \\
As(m')/ \\ II ..... l'-~t" /
test
4
..1
"''''m
I
20
i
\¢i
i/\
'\ \
\/
F- - = - - " 60
4O
--
8
--
6
--
~
60 .
/ ~
I80
As 1~) ~
.
.
i-~
b-i '160
(b)
Anaerobic
I . 180
(h )
Time
107
/
i
\
As ( m ) control
•
iI ,
1
106
test
b-" . . . . . . .
-~'"
u
Aerobic
/
control
40 2
microorganisms
.
.
.
.
.
.
control . .
test
" ~
~
4 50
i
2
4o
\\
e (J
.
'~. 30 106 8
20
6
-~
/
~ /
lO 4
~
.
/ \\\\ .....
.....-m
i "
-----"-.~----X7 I
20
AS ira" ) control
~------
40
--i-
T-
60
Time
Fig. 5 ( a - b )
80 ( days
)
k.
'~i
I~
.
.
.
•
i
180
MICHAEL C. FREEMAN et al.
288
(c)
DRP
concentration
200 --
.t trol
/
150
i... 100 --
"~~- " ~ ~.
Anaerobic I"est
~/e
v n C~ E3 50 --
Aerobic l"est
0
I
[
l
I
20
40
60
80
Time
I
FI
I
160
180
(h)
Fig. 5. Arsenic speciation and dissolved reactive phosphate phosphorus (DRP) changes in arsenic(V) heterotrophic culture experiments. nity composition began to change, after which arsenic(Ill) disappeared from the medium being completely oxidized to arsenic(V). The proportion of arsenic(V) and arsenic(Ill) also changed in the control. The initial appearance of arsenic(V) was unexpectedly rapid and may have been due to the sonication procedure. Sonication may have caused the release of inorganic or organic oxidizing agents which, in the absence of a rapidly growing microbial community, caused rapid oxidation of some of the arsenic(llI) at the beginning of the experiment. However, it appears that the system ultimately approached equilibrium with arsenic(Ill) concentration at about 35 # g l -~ and arsenic(V) concentration at about 17#gl J,
The concentration of DRP during the experiment was never observed to drop to the minimum concentrations recorded in the previous experiments [cf. Figs 6(c) and 5(c)]. However, in the period between hours 22 and 46 the initial slope of the graph indicated that the DRP concentration may have reached a minimum during that time [Fig. 6(c)]. If this was the case a very low DRP concentration may have also contributed to the microbial community changes. During the anaerobic experiment, an initial bacterial population composed mainly of clumps of small cocci (approx. 0.4/~m dia) 'crashed' and was replaced by a population dominated by large rods (approx, 0.7 × 2.5 #m) [Fig. 6(b)]. The arsenic(III) was rapidly oxidized to arsenic(V) concurrent with the initial (o)
Aerobic
test
107 60 ~
"~.~
~:~
Aerobic microorgenisms
8 6
4 p---o 30
- "-x3"~ - ~
-~
- "--°'--
I
F--~
lo6 -
iO
/{
J
"/A-~(~')
test
~
....
8 6
"/ 0
,
I
,I
I
20
40
60
8x104 ,
Time Fig.
6(a)
'~=
I 80
( h )
.
.
.
.
.
w--
l lO0
.I
,
1"20 r
j
i
140
Microbial transformation of arsenic
289 (b)
Anaerobic
test
~ Anoerobic booterio
107
I--,
8
6
/
40
~l
~/
~
/
/
AS(TIT)test
E __=
(..) 20
//"
,
////
Io" 1o
8
6
\
/ / /~.~ . . . . F i- "/-
- 7 ,'/ ~"
/
"
\'-
'~_ __~__ ./__ __ - - - - - . - ~ \ /
•
~__~--
I---I---'-'I
~ ~A ~"
~--~
~,
As (~) control As (~r)lC~
0
s (V1 test
I
20
""'~'-"---.~4 \/
40
exlo"i
I
I
I
60
80
100
I I 120
Time ( h )
(c) DRP concetration
600
500
\
400 1
:l 300 O. O~ Q
\
200
\~ ~ o /
100
/.e.~_~ /
N
\
I
,--- - / e " ~ l
I 40
j /
t~.-.e
.._t--"
./
/
\
2O
Aerobic test ~
/-
Anaerobic test I
I
I
60
80
100
I I
120
J--L--
140
Time ( h )
Fig. 6. Arsenic speciation and dissolved reactive phosphate phosphorus (DRP) changes in arsenic(III) heterotrophic culture experiments. exponential growth of bacteria. The initial oxidation of arsenic(III) to arsenic(V) in the anaerobic sediment test was most likely due to the growth of aerobic heterotrophs utilizing the molecular oxygen remaining in the medium [Fig. 6(b)]. After all the molecular oxygen was consumed, the aerobic coccus population diminished and was replaced by an anaerobic community dominated by large rods that probably assisted the attainment of chemical equilibrium by reducing arsenic(V) to arsenic(Ill). The anaerobic control experiment reflected the W.R. 2 0 / ~ C
thermodynamic stability of arsenic(III) under such conditions (Ferguson and Gavis, 1972). The higher initial DRP concentration in the arsenic(III) sediment tests compared to the arsenic(V) sediments tests, was apparently due to a change in the medium composition, caesin hydrolysate (BDH) being replaced by bactopeptone (Difco) [of. Figs 5(c) and 6(c)]. Although the individual microbial species thought responsible for the redox transformations of inorganic arsenic have not been identified in these mixed culture experiments, it has been shown that there are
290
MICHAELC. FREEMANet al. concentrations, 10-50 mg P 1 - ] ( 0 . 3 1 - 1 . 6 mM phosphate), the arsenic(V) reduction pathways were operating. No volatile arsines were found in the traps. The arsenic content of the fungi during the experiments were low compared to the arsenic concentration of Lake Ohakuri phytoplankton cultured in a medium with the same arsenic concentration (Table 1). The ecological logic of microbiological arsenic(V) reduction has been explained in terms of detoxification strategy aimed at avoiding the con-
microorganisms in the sediment capable of effecting the transformations at rates comparable with those observed in the lake. Four fungal species were isolated from sediment samples and tentatively identified as Candida sp., Rhodoturula sp., Trichoderma viride and a species from the phialidic Eurotiaceae. Each of these species reduced arsenic(V) to arsenic(Ill) aerobic conditions of phosphorus surplus [Fig. 7(a)-(d)]. Thus, even at very high external DRP (a)
i v fv
Cells
60
/
~
r
-'~
/
/
/
/
/
v~
/ )-'-- - ----o... 50
•
-
As ( ~ )
/
~
•
------o
•
108
c. E
c~
/
401
rr 30
/
~ 2o
DRP
/
1o 107
\ ./
2O
0
i
i
40
60 Time
6
"--,o
t
r-~
-8_
80
100
120
( h )
-
(b)
4 /
/
60 __. - - - - - - - - @ .
.
.
.
.
•
50 1 I
E
40
-
30
-
20
-
10
-
n
tY o i
lo - / 0
\
/*/ •
f
0
I 20
40
~ ~
~.. I
I
i~
60
80
1oo
Time
Fig. 7(a-b)
(h )
-~ 12o
Microbial transformation of arsenic
291
(c)
60 I-
~o\
2.0
50~
'~, ,,o iE 30
N-:--~ \
1.5
-
I.0
--
.j~ ",,
'\ ~,
•
.~/u
,..j.---
~
Medium O.D.
, ~
-
10
-
0
-
/
'~
0.5
////\ ///
0.0
20
"\
/
/../"
DRP
",%,
../..-
40
60
Cells
x.. A
, J
-'08
40
tic 63
3O
cn
20
40
.,/\
. 6/ J
-
JJ
, \ ~
~,
a..
J
-%t-. ,.
_~ j
E
I 120
(d)
-
5O
I 100
--
-
60
I 80 (h )
Time 6
/
Id
" ,,\ '\As(~)
/
-
~L 2 0
:L
2
A s (I'TT)
/'~.~"\
4
t
-
.
~.~
// ..~-'-
,./As(m, DRP
--- - - o .
~.
/J/ I107
/
~e~
--
7 I [
0
20
~--.
",o._ "~
I 40 Time
"~
~"
I
I
60
80
"'"
"L 0
I
100
120
(h }
Fig. 7. Arsenic speciation and dissolved reactive phosphate phosphorus (DRP) changes in arsenic(V) fungal culture experiments. sequences of incorporating arsenic(V) instead of phosphate into cellular components and has been documented in both, mixed heterotrophic communities (Johnson, 1972; Myers et al., 1973) and pure cultures (Green, 1918; Jones et al., 1984; Myers et al., 1973; Vidal and Vidal, 1980; Woolfolk and Whitely, 1962). Similarly, there are many accounts of heterotrophic microorganisms being able to oxidize arsenic(III) to arsenic(V) (Green, 1918; Johnson and Pilson, 1975; Myers et al., 1973; Osborne and Ehrlich, 1976; Peterson and Carpenter, 1983; Philips and Taylor, 1976; Quastel and Scholfield, 1953; Scudlark
and Johnson, 1982; Turner, 1954). The most likely biochemical explanation of arsenic(III) oxidation to arsenic(V) has been attributed to a co-oxidation mechanism incidental to normal cellular oxidation of reduced carbon compounds (Scudlark and Johnson, 1982). Presumably, these microorganisms are less susceptible to the potential toxic effects of arsenic(V) than those of arsenic(III). The available data on Lake Ohakuri sediment arsenic biogeochemistry can be combined with the results of this study to produce a simple model incorporating both physico/cbemical and micro-
292
MICHAEL C. FREEMAN et al.
Table 1. The average arsenic content of the fungi during the test period Total arsenic (#g g-.i) Number of Fungi )7 a samples . . . . . . . Candida sp. 1.2 1.0 8 Rhodotorula sp. 2.0 1.3 8 Trichoderma viride 7.7 0.4 3 Phialidic Eurotiaceae 3.8 4.0 3 L. Ohakuri Phytoplankton (Freeman, 1985, 1986b) 10-600
Table 2. A summary of the arsenic speciation changes observed in mixed heterotrophic cultures derived from the sediments of Lake Ohakuri Sediment culture conditions . . . . . Initial arsenic Anaerobic speciation (80% H~, 20% CO 2) Aerobic . . . . . . . . . . . . As(V) Reduced to As(IIl) Reduced to As(Ill) then oxidized to As(V) As(Ill) Oxidized to As(V) Oxidized to As(V) then reduced to As(Ill)
biological features of the system (Fig. 8) (Aggett and Aspell, 1980; Aggett and Kadwani, 1983; Aggett and O'Brien, 1984). Under stratified conditions the sediment Eh decreases and adsorbed arsenic(V) is desorbed and a portion is reduced to arsenic(Ill). At the sediment surface similar concentrations of arsenic(V) and arsenic(Ill) are diffusing upwards and the physico/chemical conditions, together with microbial uptake and reduction cause arsenic(Ill) to predominate• In the oxygenated epilimnion, arsenic(V) is favoured and this situation is only occasionally disrupted by highly active growth of arsenic(V) reducing organisms (Freeman 1985,
(DMA) was found in intersitital waters "with 200mgl -] arsenic" from the sediments of Lake Washington (Crecelius, 1975). The Menominee River, grossly polluted by waste from a factory producing methylated arsenic pesticides, has been reported to have up to 6 g 1-t arsenic in interstitial waters, of which 90% was monomethylarsenic acid (MMA) and 10% inorganic arsenic (Iverson et al., 1979). However, compared to the large number of published papers on sediment arsenic very few report the presence of either MMA or DMA, although in some cases they were probably not tested for. The scarcity of reports of methylated arsenicals in sediments is probably due to the tendency of anaerobic sediment microorganisms to demethylate these compounds (Holm et al., 1980). Such demethylation has also been reported for soil microorganisms (Von Endt et al., 1968). While caution should always be exercised when attempting to explain field observations with labora-
1986a, b). Methylated arsenicals were not found in any of these microbial cultures. This result is in agreement with the findings of O'Brien (1983) and Aggett and Kadwani (1983). There have been a limited number of reports of methylated arsenicals found in sediment interstitial waters. One gg 1-l of dimethylarsinic acid
As(~) 1-t-2 AS (1Tr)oq ~
DO
As(~Z) i
0
As(3Zloq 10 20
As (~) AS(I)! AS (]Z)
-.~..A,
I 30
(~
As(ST)oq
As (lTr)aq As 1'~11 'As(V)
IDecreosir
As (Trr)oq
E~
(m)
I+2
As
~
As ( V ) a q
li
I
Dissolved As (/~g I-t)
\]~,._
(cm)
10
As (~Z) (IE) '~"~ Fe(Tn')oq~ Fe(Tr)aq+S = FeS
Usual direction of conditions due to :
reaction
(11 Thermodynamics i,e. low
[
ppt
ppt
( 2 ) Microbial
\\"~
under stratified Eh +
DO, near neutral pH
activity
Fig. 8. A simplified m o d e l describing the p h y s i c o / c h e m i c a l and m i c r o b i o l o g i c a l features of arsenic speciation in stratified L a k e O h a k u r i .
I
Microbial transformation of arsenic tory results, these experiments provide a number of possible microbiological answers to the occasional unexplained early spring observations of arsenic(III) in the eutrophic Lake Ohakuri (and Waikato River) surface waters (Aggett and Aspell, 1980). One plausible explanation is that heavy rainfall, during late winter/spring entrains a large number of actively metabolizing soil heterotrophs and available nutrients (Collins, 1960; Woodbridge and Garret, 1969). These additional bacteria and fungi will have adjusted to live at elevated arsenic concentrations due to the relatively high levels of 28-31 #gg-1 total arsenic reported for soils from the Lake Ohakuri region (Aggett and Aspell, 1980). However, the ability to mediate arsenic speciation changes is not limited to those microorganisms from environments with elevated concentrations of arsenic (for example, see Johnson, 1972; Myers, 1973; Osborne and Ehrlich, 1976; Phillips and Taylor, 1976; Vidal and Vidal, 1980). While no intensive studies have been carried out on heterotrophic numbers and activity in Lake Ohakuri a study carried out in late summer 1977 reported bacterial numbers (acridine orange direct counts) in Lake Ohakuri surface waters to be 1 x 106 cells ml-1, the maximum glucose uptake rate to be 0.05/~g1-1 and the natural substrate turnover time to be 80 h (Spencer and Ramsay, 1978). The relatively low concentrations of organic substrates in the test medium used in this study and the maximum cell densities of approx. 107 cells ml -~ suggest that the nutrient status and microbial activity were similar to those found in Lake Ohakuri. Studies carried out on Lake Ohakuri during 1980-1982 reported arsenic(III) concentrations always below 4 #g 1-1 (O'Brien, 1983). One disadvantage of these latter studies was that samples were usually only taken once every month, and phenomena that only last for a couple of weeks may have been missed. Further intensive studies, involving more frequent sampling, may provide the conclusive reasons for occasional arsenic(III) predominance in Lake Ohakuri surface waters. Acknowledgements--We thank staff of the Water Quality Centre, Hamilton; especially Mr K. J. Costley for carrying out the DRP analyses and Dr J. B. Macaskill for the use of the epifluorescent microscope. M. C. Freeman gratefully acknowledges the Hilary V. Jolly Post-doctoral Fellowship from the University of Waikato. The work was also supported by a grant from the Waikato Valley Authority.
REFERENCES
Aggett J. (1981) Mobility of arsenic in the sediments of the Waikato hydrolakes. In The Waters of the Waikato, pp. 301-311. Seminar Proceedings, University of Waikato, Hamilton, New Zealand. Aggett J. and Aspell A. (1976) The determination of arsenic(liD and total arsenic by atomic absorption spectroscopy. Analyst 101, 341-347. Aggett J. and Aspell A. (1980) Arsenic from geothermal sources in the Waikato catchment. N. Z. 7l Sci. 23, 7%82.
293
Aggett J. and Kadwani R. (1983) Anion exchange method for speciation of arsenic and its application to some environmental analyses. Analyst 108, 1495-1499. Aggett J. and O'Brien G. (1984) Factors affecting the mobility of arsenic in the sediments of Lake Ohakuri, New Zealand. Envir. Sci. Technol. 19, 231-238. Ainsworth G. C., Sparrow F. K. and Sassman A. S. (1973) The Fungi. An Advanced Treatise, Vol. IV A. Academic Press, New York. APHA (1981) Standard Methods for the Examination of Water and Wastewater, 15th edition. American Public Health Association, Washington, D.C. Booth C. (1971) Fungal culture media. In Methods in Microbiology (Edited by Booth C.), Vol. 4, pp. 49-94. Academic Press, London. Calam C. T. (1969) The evaluation of mycelial growth. In Methods in Microbiology (Edited by Norris J. R. and Ribbons D. W.), Vol. 1, pp. 567-592. Academic Press, London. Carmichael J. W., Kendwick B., Conners I. L. and Sigier L. (1980) Genera of Hyphomycetes. University of Alberta Press, Alberta. Collins V. G. (1960) The distribution and ecology of gram-negative organisms other than enterobacteriaceae in lakes. J. appl. Bact. 23, 510-514. Coulter G. W. (1977) The ecological impact on the Waikato River of untreated effluentfrom the proposed Broadlands geothermal power station. New Zealand Energy Research and Development Commission Report No. 26. Coulter G. W., Davies J. and Pickmer¢ S. (1982) Seasonal limnological change and phytoplankton production in Ohakuri, a hydro-electric lake on the Waikato River. N.Z. Jl Mar. Freshwat. Res. 17, 169-183. Cox D. P. and Alexander M. (1973) Effect of phosphate and other anions on trimethylarsine formation by Candida humicola. Appl. Microbiol. 25, 405-413. Crecelius E. A. (1975) The geochemical cycle of arsenic in Lake Washington and its relation to other elements. Limnol. Oceanogr. 20, 441-451. Downes M. T. (1978) An automated determination of low reactive phosphorus concentrations in natural waters in the presence of arsenic, silicon and mecuric chloride. Water Res. 12, 743-746. Ferguson J. F. and Gavis J. (1972) A review of the arsenic cycle in natural waters. Water Res. 6, 1259-1274. Freeman M. C. (1985) The reduction of arsenate to arsenite by an Anabaena assemblage isolated from the Waikato River. N.Z. Jl Mar. Freshwat. Res. 19, 277-282. Freeman M. C. (1986a) The role of growth and senescence of a Ceratophyllum demersum--epiphyte assemblage in arsenic biogeochemistry in the Waikato River, New Zealand. Submitted to Aquat. Bot. Freeman M. C. (1986b) The uptake and reduction of arsenate to arsenite by phytoplankton isolated from the arsenic-rich Waikato River, New Zealand. Submitted to J. Plank. Res. Golterman H. L., Clymo R. S. and Ohnstad M. A. M. (1978) Methods for Physical and Chemical Analysis of Freshwaters, International Biology Program Handbook No. 8, 2nd edition. Blackwell Scientific, London. Green H. H. (1918) Description of a bacterium which oxidizes arsenite to arsenate and of one which reduces arsenate to arsenite isolated from a cattle-dipping tank. S. Afr. J. Sci. 14, 465-467. Holm T. R., Anderson M. A., Stanforth R. R. and Iverson D. G. (1980) The influence of adsorption on the rates of microbial degradation of arsenic species in sediments. Limnol. Oceanogr. 25, 23-30. Iverson D. G., Anderson M. A., Holm T. R. and Stanforth R. R. (1979) An evaluation of column chromatography and fiameless atomic absorption spectrophotometry for arsenic speciation as applied to aquatic systems. Envir. Sci. Technol. 13, 1491-1494.
294
MICHAEL C. FREEMAN et al.
Johnson D. L. (1972) Bacterial reduction of arsenate in sea water. Nature 240, 44 45. Johnson D. L. and Pilson M. E. Q. (1975) The oxidation of arsenite in seawater. Envir. Lett. 8, 157-171. Jones J. G., Davison W. and Gardener S. (1984) Iron reduction by bacteria: range of organisms involved and metals reduced. FEMS Microbiol. Lett. 21, 133-136. Magazda C. H. (1979) Physical and chemical limnology of six hydroelectric lakes on the Waikato River, 1970-1972. N.Z. Jl Mar. Freshwat. Res. 13, 561-572. Maher W. A. (1983) Spectrophotometric determination of arsenic in biological tissue and sediments after digestion with nitric, sulphuric and perchloric acids and preconcentration by zinc column generation and trapping. Analyst 108, 939-943. Malette M. F. (1969) Evaluation of growth by physical and chemical means. In Methods in Microbiology (Edited by Norris J. R. and Ribbons D. W.), Vol. I, pp. 521-566. Academic Press, London. Meyer-Reil L. A. (1978) Autoradiography and epifluorescence microscopy combined for the determination of numbers and spectrum of actively metabolizing bacteria in natural waters. AppL envir. Microbiol. 36, 506-512. Myers D. J., Heimbrook M. E., Osteryoung J. and Morrison S. M. (1973). Arsenic oxidation state in the presence of microorganisms by differential pulse polarography. Envir. Lett. 5, 53-61. Nichols H. W. (1973) Growth media--freshwater. In Phycological Methods (Edited by Stein J. R.), Vol. I. Cambridge University Press, Cambridge. O'Brien G. (1983) Observations on the mobility of arsenic in the sediments in Lake Ohakuri. Ph.D. thesis, University of Auckland, New Zealand. Osborne F. H. and Ehrlich H. L. (1976). Oxidation of arsenite by a soil isolate of Alcaligenes. J. appl. Bact. 41, 295-305. Peterson M. L. and Carpenter R. (1983) Biogeochemical processes affecting total arsenic and arsenic species distri-
butions in an intermittently anoxic t~ord. Mar. Chem. 12, 295-321. Phillips S. E. and Taylor M. L. (1976) Oxidation of arsenite to arsenate by Alcaligenes faecalis. Appl. envir. Microbiol. 32, 392-399. Quastel J. H. and Scholefield P. G. (1953) Arsenite oxidation in soil. Soil Sci. 75, 279-285. Scudlark J. R. and Johnson D. L. (1982) Biological oxidation ofarsenite in seawater. Est. Coast. ShelfSci. 14, 693-706. Spencer M. J. and Ramsay A. J. (1978) Bacterial populations, heterotrophic potentials, and water quality in three New Zealand rivers. N.Z. JI Mar. Freshwat. Res. 12, 415-427. Strachan C. (Ed.) (1979) The Waikato River: A Water Resources Study. Water and Soil Technical Publication No. I 1, Wellington. Turner A. W. (1954) Bacterial oxidation of arsenite--1. Description of bacteria isolated from arsenical cattledipping fluids. Aust. J. Biol. Sci. 7, 452-478. Vldal F. V. and Vidal V. M. V. (1980) Arsenic metabolism in marine bacteria and yeast. Mar. Biol. 50, 1-7. Von Endt D. W., Kearney P. C. and Kaufman D. D. (1968). Degradation of monosodium methanearsonic acid by soil microorganisms. J. agric. Fd Chem. 16, 17-20. Wetzel R. G. (1975) Limnology. Saunders, Philadelphia. Woodbridge D. D. and Garrett W. R. (1969) Relationship between bacteria, nutrients and rainfall in selected bodies of freshwater. Bull. envir, contain. Toxic. 4, 311-319. Woolfolk C. A. and Whiteley H. R. (1962) Reduction of inorganic compounds with molecular hydrogen by Micrococcus lactilyticus--l. Stoichiometry with compounds of arsenic, selenium, Tellerium, transition and other elements. J. Bact. 84, 647-658. Zimmerman R., Iturriaga R. and Becker-Birk J. (1978) Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. envir. Microbiol. 36, 926-935.
0043-1354/86 $3.00+0.00 Copyright © 1986 Pergamon Press Ltd
MICROBIAL TRANSFORMATIONS OF ARSENIC IN LAKE OHAKURI, NEW Z E A L A N D MICHAEL C. FREEMANl'*, JOHN AGGETT2 and GLENNYS O'BRIEN 2 'Biological Sciences Department, University of Waikato, Private Bag, Hamilton and 2Department of Chemistry, University of Auckland, Private Bag, Auckland, New Zealand (Received October 1984)
Abstract--The concentration and chemical speciation of arsenic in the waters and sediments of Lake Ohakuri, New Zealand were examined. Mixed microbial populations from the sediments were tested in vitro for their ability to mediate redox transformations of inorganic arsenic. Under aerobic conditions the mixed microbial cultures were found to be able to reduce arsenic(V) to arsenic(Ill) and also to oxidize arsenic(Ill) to arsenic(V). Under anaerobic conditions only reduction of arsenic(V) to arsenic(III) was observed. Four species of sediment fungi were isolated, grown aerobically and all were found capable of reducing arsenic(V) to arsenic(Ill). The role of microbial heterotrophs in determining the observed mobility and speciation of arsenic in Lake Ohakuri is discussed. Key words--arsenic, arsenic(III), arsenic(V), sediments, bacteria, fungi, biogeochemistry, Lake Ohakuri,
Walkato River
INTRODUCTION The water quality of the eutrophic Waikato River in the North Island of New Zealand is influenced by arsenic released in geothermal discharges from natural sources south of Lake Ohakuri and by the discharges from the Wairakei geothermal power station (Aggett and Aspell, 1980; Coulter, 1977; Strachan, 1979) (Fig. I). The dissolved arsenic concentration is usually between 30-60/~gI -~ and is normally found as the thermodynamically stable arsenic(V) (Ferguson and Gavis, 1972), however, during early spring and summer the thermodynamically less favoured form arsenic(llI) has on occasion been observed as the predominant form (Aggett and Aspell, 1980). A significant proportion of the arsenic becomes adsorbed in the sediments of the Waikato River hydrolakes (Aggett and Aspell, 1980). The mean arsenic concentration of Lake Ohakuri sediments has been reported as 3 3 5 # g g -I dry wt (Aggett, 1981). These sediments are loose black oozes consisting of about 85% water which usually contains 1000-4000 p g I- ~ arsenic with approximately equal amounts of arsenic(Ill) and arsenic(V) (Aggett, 198 I; Aggett and Kadwani, 1983). It has been shown that the arsenic in these sediments has considerable mobility. Studies have shown that in chemically reducing regions of sediment, arsenic is released to the interstitial water and diffuses upwards to the sediment-surface interface where it is either readsorbed or released to the overlying water (Aggett and O'Brien, 1984). *Present address: North Canterbury Catchment Board and Regional Water Board, P.O. Box 788, Christchurch, New Zealand. 283
The latter may be attributable to the growth of phytoplankton and/or submerged macrophytes and evidence has been presented to corroborate this theory (Freeman, 1985, 1986a, b). However, during early spring, surface water temperatures of 8-14°C and phytoplankton densities of 5-10% of the summer maximum (Coulter et al., 1983), would tend to reduce the likelihood of phytoplankton and macrophyte mediated arsenic speciation changes. This paper is an attempt to identify potential microbial transformations of arsenic in the system. METHODS Dissolved oxygen and temperature profiles for the lake were carried out using a Yellow Springs Instrument Model 54 ARC dissolved oxygen meter, calibrated in the field against oxygen-saturated air. Water samples were collected using a 2-I. PVC Van Dorn water sampler. An aliquot was filtered through a 0.45/~m membrane filter and immediately analysed for iron(ll). A second aliquot was filtered through a 0.45 #m membrane filter and acidified to pH 2 for later analysis of total iron, total arsenic and arsenie(IIl). All sample containers were acid-cleaned and rinsed with MiNi Q water. Sediment samples were taken with a Jenkin's sediment sampler. One centimetre sections were transferred to 50 ml centrifuge tubes. The headspaces of the tubes were purged with oxygen-free nitrogen and the tubes sealed and stored in a "chilly-bin". Interstitial water was separated by centrifugation, usually carried out within 4 h of sampling, at 15,000 rpm (4100g) for 15 min at 4°C. Following centrifugation the interstitial water was filtered through a 0.45/~m membrane filter. A small volume was immediately analysed for iron(II) and the remainder was preserved with hydrochloric acid (pH 2) for subsequent analysis of total iron, total arsenic(III). The analytical methods used were those described in Aggett and O'Brien (1984). Sediment samples for the laboratory experiments were collected from a depth of 30 m in Lake Ohakuri on 31 March 1984, with an Eckman grab sampler. The sediment was transported on ice and stored in the laboratory at 4°C.
284
MlCHAEL C. FREEMANet al.
km
Fig. 1. Location of the Waikato River and its hydrolakes in the North Island, New Zealand.
pH and Eh values for the mixed sediment sample were determined at 20°C using an Extech p H / E h meter, model 67 (Golterman et al., 1975). Sediment samples were diluted with sterile Milli Q water, lightly sonicated, (Kontes sonicator, power setting No 5, peak frequency, 30 s for a I0 ml sample), and subsamples were prepared for bacterial counts using dilution plating with nutrient agar made up with MBL medium, as described in Nichols (1973), with vitamins omitted, orthophosphate phosphorus concentration reduced to 120 #g 1-i, arsenic(V) added to approx. 50/~g 1- ~and boron added to l mg 1- ~, the latter two being added to simulate the concentrations found in the Waikato River (Strachan, 1979). Anaerobic plates were incubated in a standard anaerobic jar using a "gas pak" (BBL). Yeast were counted using the following medium, made up with the modified MBL medium (MMBL): 5 g l -I, Bacto-peptone or Caesin Hydrolysate, 10gl -~ glucose, 4 g l -~ yeast extract, 4 g l - t malt extract, 3.5 ml 1-I of 1% (w/v) rose bengal solution and 20g 1-t agar (APHA, 1981; Booth, 1971). The bacterial experiments were carried out in 1 1. conical flasks using MMBL medium with arsenic added as arsenate or arsenite and 10mg 1-I o f each o f the following added:
glucose, sodium glycollate, caesin hydrolysate and yeast extract. Aerobic bacterial cultures were loosely stoppered with cotton wool and foil, and shaken at 120 rpm. Anaerobic bacterial cultures were stoppered with rubber bungs and shaken three times daily. Anaerobic conditions were obtained by purging the flask and medium with a sterile mix of 80% H 2 and 20% CO 2. After each sampling, the anaerobic flask and medium were purged with the gas mix. Incubations were carried out at 20°C. The initial pH of the medium, measured with a Radiometer pH meter 28 was 7.2. The cultures were inoculated with subsamples taken from a lightly sonicated ten-fold dilution of fresh sediment. Controls in the arsenite sediment test were spiked to give a final concentration of 0.2% HgCI2 (w/v) in addition to the sediment inoculation. Fungal cultures were carried out using isolates obtained from dilution plating. Identification of the fungi was carried out with the aid o f keys and illustrations in Ainsworth et al." (1973) and Carmichael et aL (1980). The isolates were grown in magnetically-stirred, continuously aerated 11. conical flasks, initially containing 1 1. of a fungal medium similar to that detailed above with the rose bengal and agar omitted. The possibility that these fungi could produce volatile
Microbial transformation of arsenic 0
20
40
60
80
100 A s ( ~ g 1-1)
4;
4;
1~
1;
4;
2'0
I
I
I
I
I
I
2
4
6
8
'
'
o
A,(m) 10 E
As(~z)
7 I ,
Fe( mg 1-1 )
Temp. (*C)
10 DO(mg I -I )
0.5 I
1.0
1.5
I
I
!/
10
PC /I
,#
J
Fe (111")
.
'
(a}
,o
(b)
20 30'
.~ 3 0
40
40
0
0
' TempTeD0
7
-.
285
1
2
l
I
0.6
3
4
I
1.2
5
I
1.8
6
I
2.4
7
Solid
As(mg g -1)
Interstitial Fe(mg 1-1)
I
3.0
.6 Interstitial
0
1.0
2.0
3.0
£2 ~o g 6 ._E 8 tn 10
"~p(O'-
Solid As
Fe (TIT)
/.o-
Fe (TTE) a."7-~ g At'"
(c)
10 LA
(d)
Fig. 2. The variation of arsenic(V), arsenie(III), iron(III) and iron(II) in the water column and interstitial water of Lake Ohakuri, during stratification.
alkylarsines was investigated by running the sterileair outlets through 50 ml of 2 M HNO 3 placed in an ice bath (Cox and Alexander, 1973; Vidal and Vidal, 1980). Bacterial numbers were counted using acridine orange epifluores~nt microscopy (Meyer-Reil, 1978; Zimmerman et al., 1978). The volume of the sample used for cell counts was varied during the experiment and this resulted in a counting uncertainty (standard error) of 10-15%. Fungal growth was followed using a haemocytometer with the yeast species and medium absorbance at 750 nm with the mould (Calam, 1969; Mailette, 1969). These methods usually had uncertainties of 15-20 and 10% respectively. The epifluorescent technique was used, together with plating onto nutrient agar, as a check for contaminants in the fungal cultures. Samples from the mixed microbial cultures, approx. 70ml, were filtered using successively smaller pore size membrane filters, normally 0.8 #m followed by 0.45 pm. (Sediment material quickly clogged the 0.45/~m filter without a prefilter.) The fungi were separated from the sample by centrifugation at 3000 rpm (164g) for 15 min. The 70 ml of supernatant or filtrate was stored in 50% HNO3-cleaned polyethylene bottles and immediately frozen at - 10°C until analysis. Samples were periodically analysed for the presence of the four environmentally common arsenic species, arsenic(V), arsenie(III), monomethylarsonate and dimethylarsinate using an anion-exchange method (Aggett and Kadwani, 1983). After cheeks with this method revealed the absence of any methylated arsenic a simpler pH selective method for the two inorganic species was used, based on Aggett and Aspell (1976). Arsenic(III) was selectively determined by using a 10 ml sample buffered to pH 4.2 with 5 rul of a 2 M citrate buffer. Total dissolved arsenic was determined using a 10 ml sample acidified with 2 ml concentrated HC1. Arsenic(V) was calculated as the difference. Total microbial arsenic was determined using a nitric acid, nitric-sulphuric-perchloric acid two step digestion according to Maher (1983). Other methods and equipment used for the arsenic analyses were as detailed in Freeman (1985). The reproducibility and detection limit, based on the analysis of five 50pgl -I samples and blanks, were 4.2% and 2 #g 1- ~ respectively.
Dissolved reactive phosphate phosphorus (DRP) was determined by the method of Downes (1978). RESULTS AND DISCUSSION
The typical summer profiles of arsenic(V), arsenic(III), dissolved oxygen (DO), temperature, iron(III) and iron(II) in the body of Lake Ohakuri are illustrated in Fig. 2(a) and (b). The sediment profiles of arsenic(V), arsenic(III), iron(Ill) and iron(II) are shown in Fig. 2(c) and (d). These data illustrate some of the factors affecting arsenic speciation in both the lake water and sediments. As the lake stratified, the concentration of dissolved arsenic in the hypolimnion increased (Fig. 3). The proportion of arsenic(III) also rose, to a maximum at the sediment surface [Fig. 2(a)] (O'Brien, 1983). The diffusion of arsenic from the sediments to the overlying waters is believed to be a consequence of iron(III) reduction and resolubilization of b o u n d arsenic(V) and arsenic(Ill) in a mechanism similar to that of phosphate release from deoxygenated sediments (Wetzel, 1975). The preponderance of arsenic(III) in the hypolimnion was probably due to both the physico/chemical conditions and microbially-mediated speciation changes of desorbed arsenic(V). The preponderance of arsenic(V) in the sediment interstitial waters was probably a reflection of the original arsenic oxidation state during deposition. The potential contribution of the hypolimnetic arsenic to the total lake load after destratification is not great due to the relative volumes of the epilimnion and hypolimnion at this time of year, 1.09 x 10s and 0.05 x 10Sm 3 respectively (O'Brien, 1983). Studies carried out before and after
286
MICHAELC. FREEMANet al. 10
15
20
E ~ 121
3o 35
27/1/E
I
I
I
I
20
40
60
80
Dissolved
I
I
I
I
I
I
100
120
140
160
180
200
orsenic
(/~g
1-1 )
Fig. 3. The dissolved inorganic arsenic profiles of Lake Ohakuri during stratification. destratification in autumn 1982 revealed a small, but significant rise in the total dissolved arsenic of 3 ~ l m g m -3, measured downstream of the Lake Ohakuri dam tailrace. The variation of dissolved arsenic concentration in Lake Ohakuri over a 21 month period appears to have some cyclical tendency, however, the peaks occur at different times in different years (Fig. 4). These studies did not reveal any high concentrations of arsenic(Ill) in the surface waters as were observed in previous years (Aggett and Aspell, 1980). The reason(s) for the irregular occurrence of arsenic(Ill) have not yet been satisfactorily answered. In order to establish whether or not heterotrophic microorganisms had the potential to become involved in arsenic transformations and pathways, bacteria and fungi were isolated from the lake sediments. Preliminary studies showed the sediment microflora to be more diverse and numerous than the surface planktonic populations. Thus, a sediment sample was collected to serve as a diverse source for a mixed heterotrophic culture. The sediment sample used for the experiments was a black ooze, indicating the presence of iron sulphide. The pH and redox potential of the mixed sample were 6.6 and - 5 0 m V respectively. Although sediment oxygen concentration was not determined at the time of collection, studies carried out in situ in April 1981 --
40
o
30
2o >
10
c5 o
I L I I I I NDJ FMAMJ
I I
t I I I I I J A S 0 NDJ 1981 Time
( months
l
I I I ~ I I FMAMJ d 1982
)
Fig. 4. The variation of dissolved inorganic arsenic, at 10 m depth, over 21 months in Lake Ohakuri.
and 1982 during stratification showed that only trace amounts of dissolved oxygen were available at the sediment surface and the temperature in this region was 16.0-16.5°C. The numbers of heterotrophs found in the sample were as follows: aerobic bacteria: l.l x 106 colony forming units (CFU) g-~ dry wt, anaerobic bacteria: 2.7 × 10 6 C F U g - 1 and aerobic fungi: 1.9 x 104CFUg -~. The first arsenic(V) sediment test, carried out under aerobic conditions, exhibited exponential growth of a mixed microbial community with one large (approx. 3 x 5#m) unidentified "yeast sized" species dominating until after 60 h of incubation [Fig. 5(a)]. During the exponential growth phase the DRP concentration dropped from 1741tgl -~ (5.61#M) to 32 g 1-~ (1.03/~M). During this period the arsenic(V) was quantitatively reduced to arsenic(Ill). The total cell density did not increase after 40 h, however, there was a decline of the yeast species density, which was replaced during the 30-60h period, by a mixed bacterial community, consisting of various rods and cocci. This community change indicated that the initial yeast population was most likely responsible for the arsenic(V) reduction to arsenic(Ill). The community change was possibly due to competition for the limited amount of DRP available, the bacterial population was apparently more able to thrive on a lower DRP concentration [Fig. 5(c)]. The increase in aerobic bacterial numbers was concurrent with the rapid oxidation of arsenic(Ill) to arsenic(V). A further drop in the DRP concentration to 20 #g 1-~ (0.64#M) and the presence of the potential competitive analogue arsenic (V) at 54#g As 1 (0.72 #M) probably combined to produce the crash in microbial numbers and the ensuing rise in DRP. In the second arsenic(V) test, carried out under anaerobic (80% H> 20% CO2) conditions, the increase in bacterial numbers, mainly small rods (approx. 0.5 x 1.5 #m), accompanied the reduction of arsenic(V) to arsenic(III) [Fig. 5(b)]. In both experiments it is difficult to determine the
287
Microbial transformation of arsenic
with HgCI:, at 0.2% (w/v). The first test [Fig. 6(a)] carried out under aerobic conditions enabled a microbial community dominated by a "yeast-sized" cell (approx. 3 x 5 #~m) to initially peak at nearly 107 cells ml-L Thereafter, the yeast numbers declined and were eventually replaced by small cocci and rods (approx. 0.5 #xm dia and 0.5 x 1.5 #xm respectively). The disappearance of arsenic(III) and appearance of arsenic(V) was quite slow until the microbial commu-
effect of phosphate limitation on arsenic(V) reduction as in each case, exponential microbial growth, DRP depletion and arsenic(V) reduction all occurred concurrently [Fig. 5(a), (b) and (c)]. The controls in this first set of experiments were run without any sediment additions. The second set of sediment tests were carried out using arsenic(III) as the initial arsenic source and the controls, with sediment/bacteria inoculations spiked Aerobic
8
(o)
/
-
60
6
4
50
i
_=
~
As(V)
L ,.,,7;,, /
/
\
/
'\
'¢~
/J
20
8
6 10
/
/'\, / \\
As(m')/ \\ II ..... l'-~t" /
test
4
..1
"''''m
I
20
i
\¢i
i/\
'\ \
\/
F- - = - - " 60
4O
--
8
--
6
--
~
60 .
/ ~
I80
As 1~) ~
.
.
i-~
b-i '160
(b)
Anaerobic
I . 180
(h )
Time
107
/
i
\
As ( m ) control
•
iI ,
1
106
test
b-" . . . . . . .
-~'"
u
Aerobic
/
control
40 2
microorganisms
.
.
.
.
.
.
control . .
test
" ~
~
4 50
i
2
4o
\\
e (J
.
'~. 30 106 8
20
6
-~
/
~ /
lO 4
~
.
/ \\\\ .....
.....-m
i "
-----"-.~----X7 I
20
AS ira" ) control
~------
40
--i-
T-
60
Time
Fig. 5 ( a - b )
80 ( days
)
k.
'~i
I~
.
.
.
•
i
180
MICHAEL C. FREEMAN et al.
288
(c)
DRP
concentration
200 --
.t trol
/
150
i... 100 --
"~~- " ~ ~.
Anaerobic I"est
~/e
v n C~ E3 50 --
Aerobic l"est
0
I
[
l
I
20
40
60
80
Time
I
FI
I
160
180
(h)
Fig. 5. Arsenic speciation and dissolved reactive phosphate phosphorus (DRP) changes in arsenic(V) heterotrophic culture experiments. nity composition began to change, after which arsenic(Ill) disappeared from the medium being completely oxidized to arsenic(V). The proportion of arsenic(V) and arsenic(Ill) also changed in the control. The initial appearance of arsenic(V) was unexpectedly rapid and may have been due to the sonication procedure. Sonication may have caused the release of inorganic or organic oxidizing agents which, in the absence of a rapidly growing microbial community, caused rapid oxidation of some of the arsenic(llI) at the beginning of the experiment. However, it appears that the system ultimately approached equilibrium with arsenic(Ill) concentration at about 35 # g l -~ and arsenic(V) concentration at about 17#gl J,
The concentration of DRP during the experiment was never observed to drop to the minimum concentrations recorded in the previous experiments [cf. Figs 6(c) and 5(c)]. However, in the period between hours 22 and 46 the initial slope of the graph indicated that the DRP concentration may have reached a minimum during that time [Fig. 6(c)]. If this was the case a very low DRP concentration may have also contributed to the microbial community changes. During the anaerobic experiment, an initial bacterial population composed mainly of clumps of small cocci (approx. 0.4/~m dia) 'crashed' and was replaced by a population dominated by large rods (approx, 0.7 × 2.5 #m) [Fig. 6(b)]. The arsenic(III) was rapidly oxidized to arsenic(V) concurrent with the initial (o)
Aerobic
test
107 60 ~
"~.~
~:~
Aerobic microorgenisms
8 6
4 p---o 30
- "-x3"~ - ~
-~
- "--°'--
I
F--~
lo6 -
iO
/{
J
"/A-~(~')
test
~
....
8 6
"/ 0
,
I
,I
I
20
40
60
8x104 ,
Time Fig.
6(a)
'~=
I 80
( h )
.
.
.
.
.
w--
l lO0
.I
,
1"20 r
j
i
140
Microbial transformation of arsenic
289 (b)
Anaerobic
test
~ Anoerobic booterio
107
I--,
8
6
/
40
~l
~/
~
/
/
AS(TIT)test
E __=
(..) 20
//"
,
////
Io" 1o
8
6
\
/ / /~.~ . . . . F i- "/-
- 7 ,'/ ~"
/
"
\'-
'~_ __~__ ./__ __ - - - - - . - ~ \ /
•
~__~--
I---I---'-'I
~ ~A ~"
~--~
~,
As (~) control As (~r)lC~
0
s (V1 test
I
20
""'~'-"---.~4 \/
40
exlo"i
I
I
I
60
80
100
I I 120
Time ( h )
(c) DRP concetration
600
500
\
400 1
:l 300 O. O~ Q
\
200
\~ ~ o /
100
/.e.~_~ /
N
\
I
,--- - / e " ~ l
I 40
j /
t~.-.e
.._t--"
./
/
\
2O
Aerobic test ~
/-
Anaerobic test I
I
I
60
80
100
I I
120
J--L--
140
Time ( h )
Fig. 6. Arsenic speciation and dissolved reactive phosphate phosphorus (DRP) changes in arsenic(III) heterotrophic culture experiments. exponential growth of bacteria. The initial oxidation of arsenic(III) to arsenic(V) in the anaerobic sediment test was most likely due to the growth of aerobic heterotrophs utilizing the molecular oxygen remaining in the medium [Fig. 6(b)]. After all the molecular oxygen was consumed, the aerobic coccus population diminished and was replaced by an anaerobic community dominated by large rods that probably assisted the attainment of chemical equilibrium by reducing arsenic(V) to arsenic(Ill). The anaerobic control experiment reflected the W.R. 2 0 / ~ C
thermodynamic stability of arsenic(III) under such conditions (Ferguson and Gavis, 1972). The higher initial DRP concentration in the arsenic(III) sediment tests compared to the arsenic(V) sediments tests, was apparently due to a change in the medium composition, caesin hydrolysate (BDH) being replaced by bactopeptone (Difco) [of. Figs 5(c) and 6(c)]. Although the individual microbial species thought responsible for the redox transformations of inorganic arsenic have not been identified in these mixed culture experiments, it has been shown that there are
290
MICHAELC. FREEMANet al. concentrations, 10-50 mg P 1 - ] ( 0 . 3 1 - 1 . 6 mM phosphate), the arsenic(V) reduction pathways were operating. No volatile arsines were found in the traps. The arsenic content of the fungi during the experiments were low compared to the arsenic concentration of Lake Ohakuri phytoplankton cultured in a medium with the same arsenic concentration (Table 1). The ecological logic of microbiological arsenic(V) reduction has been explained in terms of detoxification strategy aimed at avoiding the con-
microorganisms in the sediment capable of effecting the transformations at rates comparable with those observed in the lake. Four fungal species were isolated from sediment samples and tentatively identified as Candida sp., Rhodoturula sp., Trichoderma viride and a species from the phialidic Eurotiaceae. Each of these species reduced arsenic(V) to arsenic(Ill) aerobic conditions of phosphorus surplus [Fig. 7(a)-(d)]. Thus, even at very high external DRP (a)
i v fv
Cells
60
/
~
r
-'~
/
/
/
/
/
v~
/ )-'-- - ----o... 50
•
-
As ( ~ )
/
~
•
------o
•
108
c. E
c~
/
401
rr 30
/
~ 2o
DRP
/
1o 107
\ ./
2O
0
i
i
40
60 Time
6
"--,o
t
r-~
-8_
80
100
120
( h )
-
(b)
4 /
/
60 __. - - - - - - - - @ .
.
.
.
.
•
50 1 I
E
40
-
30
-
20
-
10
-
n
tY o i
lo - / 0
\
/*/ •
f
0
I 20
40
~ ~
~.. I
I
i~
60
80
1oo
Time
Fig. 7(a-b)
(h )
-~ 12o
Microbial transformation of arsenic
291
(c)
60 I-
~o\
2.0
50~
'~, ,,o iE 30
N-:--~ \
1.5
-
I.0
--
.j~ ",,
'\ ~,
•
.~/u
,..j.---
~
Medium O.D.
, ~
-
10
-
0
-
/
'~
0.5
////\ ///
0.0
20
"\
/
/../"
DRP
",%,
../..-
40
60
Cells
x.. A
, J
-'08
40
tic 63
3O
cn
20
40
.,/\
. 6/ J
-
JJ
, \ ~
~,
a..
J
-%t-. ,.
_~ j
E
I 120
(d)
-
5O
I 100
--
-
60
I 80 (h )
Time 6
/
Id
" ,,\ '\As(~)
/
-
~L 2 0
:L
2
A s (I'TT)
/'~.~"\
4
t
-
.
~.~
// ..~-'-
,./As(m, DRP
--- - - o .
~.
/J/ I107
/
~e~
--
7 I [
0
20
~--.
",o._ "~
I 40 Time
"~
~"
I
I
60
80
"'"
"L 0
I
100
120
(h }
Fig. 7. Arsenic speciation and dissolved reactive phosphate phosphorus (DRP) changes in arsenic(V) fungal culture experiments. sequences of incorporating arsenic(V) instead of phosphate into cellular components and has been documented in both, mixed heterotrophic communities (Johnson, 1972; Myers et al., 1973) and pure cultures (Green, 1918; Jones et al., 1984; Myers et al., 1973; Vidal and Vidal, 1980; Woolfolk and Whitely, 1962). Similarly, there are many accounts of heterotrophic microorganisms being able to oxidize arsenic(III) to arsenic(V) (Green, 1918; Johnson and Pilson, 1975; Myers et al., 1973; Osborne and Ehrlich, 1976; Peterson and Carpenter, 1983; Philips and Taylor, 1976; Quastel and Scholfield, 1953; Scudlark
and Johnson, 1982; Turner, 1954). The most likely biochemical explanation of arsenic(III) oxidation to arsenic(V) has been attributed to a co-oxidation mechanism incidental to normal cellular oxidation of reduced carbon compounds (Scudlark and Johnson, 1982). Presumably, these microorganisms are less susceptible to the potential toxic effects of arsenic(V) than those of arsenic(III). The available data on Lake Ohakuri sediment arsenic biogeochemistry can be combined with the results of this study to produce a simple model incorporating both physico/cbemical and micro-
292
MICHAEL C. FREEMAN et al.
Table 1. The average arsenic content of the fungi during the test period Total arsenic (#g g-.i) Number of Fungi )7 a samples . . . . . . . Candida sp. 1.2 1.0 8 Rhodotorula sp. 2.0 1.3 8 Trichoderma viride 7.7 0.4 3 Phialidic Eurotiaceae 3.8 4.0 3 L. Ohakuri Phytoplankton (Freeman, 1985, 1986b) 10-600
Table 2. A summary of the arsenic speciation changes observed in mixed heterotrophic cultures derived from the sediments of Lake Ohakuri Sediment culture conditions . . . . . Initial arsenic Anaerobic speciation (80% H~, 20% CO 2) Aerobic . . . . . . . . . . . . As(V) Reduced to As(IIl) Reduced to As(Ill) then oxidized to As(V) As(Ill) Oxidized to As(V) Oxidized to As(V) then reduced to As(Ill)
biological features of the system (Fig. 8) (Aggett and Aspell, 1980; Aggett and Kadwani, 1983; Aggett and O'Brien, 1984). Under stratified conditions the sediment Eh decreases and adsorbed arsenic(V) is desorbed and a portion is reduced to arsenic(Ill). At the sediment surface similar concentrations of arsenic(V) and arsenic(Ill) are diffusing upwards and the physico/chemical conditions, together with microbial uptake and reduction cause arsenic(Ill) to predominate• In the oxygenated epilimnion, arsenic(V) is favoured and this situation is only occasionally disrupted by highly active growth of arsenic(V) reducing organisms (Freeman 1985,
(DMA) was found in intersitital waters "with 200mgl -] arsenic" from the sediments of Lake Washington (Crecelius, 1975). The Menominee River, grossly polluted by waste from a factory producing methylated arsenic pesticides, has been reported to have up to 6 g 1-t arsenic in interstitial waters, of which 90% was monomethylarsenic acid (MMA) and 10% inorganic arsenic (Iverson et al., 1979). However, compared to the large number of published papers on sediment arsenic very few report the presence of either MMA or DMA, although in some cases they were probably not tested for. The scarcity of reports of methylated arsenicals in sediments is probably due to the tendency of anaerobic sediment microorganisms to demethylate these compounds (Holm et al., 1980). Such demethylation has also been reported for soil microorganisms (Von Endt et al., 1968). While caution should always be exercised when attempting to explain field observations with labora-
1986a, b). Methylated arsenicals were not found in any of these microbial cultures. This result is in agreement with the findings of O'Brien (1983) and Aggett and Kadwani (1983). There have been a limited number of reports of methylated arsenicals found in sediment interstitial waters. One gg 1-l of dimethylarsinic acid
As(~) 1-t-2 AS (1Tr)oq ~
DO
As(~Z) i
0
As(3Zloq 10 20
As (~) AS(I)! AS (]Z)
-.~..A,
I 30
(~
As(ST)oq
As (lTr)aq As 1'~11 'As(V)
IDecreosir
As (Trr)oq
E~
(m)
I+2
As
~
As ( V ) a q
li
I
Dissolved As (/~g I-t)
\]~,._
(cm)
10
As (~Z) (IE) '~"~ Fe(Tn')oq~ Fe(Tr)aq+S = FeS
Usual direction of conditions due to :
reaction
(11 Thermodynamics i,e. low
[
ppt
ppt
( 2 ) Microbial
\\"~
under stratified Eh +
DO, near neutral pH
activity
Fig. 8. A simplified m o d e l describing the p h y s i c o / c h e m i c a l and m i c r o b i o l o g i c a l features of arsenic speciation in stratified L a k e O h a k u r i .
I
Microbial transformation of arsenic tory results, these experiments provide a number of possible microbiological answers to the occasional unexplained early spring observations of arsenic(III) in the eutrophic Lake Ohakuri (and Waikato River) surface waters (Aggett and Aspell, 1980). One plausible explanation is that heavy rainfall, during late winter/spring entrains a large number of actively metabolizing soil heterotrophs and available nutrients (Collins, 1960; Woodbridge and Garret, 1969). These additional bacteria and fungi will have adjusted to live at elevated arsenic concentrations due to the relatively high levels of 28-31 #gg-1 total arsenic reported for soils from the Lake Ohakuri region (Aggett and Aspell, 1980). However, the ability to mediate arsenic speciation changes is not limited to those microorganisms from environments with elevated concentrations of arsenic (for example, see Johnson, 1972; Myers, 1973; Osborne and Ehrlich, 1976; Phillips and Taylor, 1976; Vidal and Vidal, 1980). While no intensive studies have been carried out on heterotrophic numbers and activity in Lake Ohakuri a study carried out in late summer 1977 reported bacterial numbers (acridine orange direct counts) in Lake Ohakuri surface waters to be 1 x 106 cells ml-1, the maximum glucose uptake rate to be 0.05/~g1-1 and the natural substrate turnover time to be 80 h (Spencer and Ramsay, 1978). The relatively low concentrations of organic substrates in the test medium used in this study and the maximum cell densities of approx. 107 cells ml -~ suggest that the nutrient status and microbial activity were similar to those found in Lake Ohakuri. Studies carried out on Lake Ohakuri during 1980-1982 reported arsenic(III) concentrations always below 4 #g 1-1 (O'Brien, 1983). One disadvantage of these latter studies was that samples were usually only taken once every month, and phenomena that only last for a couple of weeks may have been missed. Further intensive studies, involving more frequent sampling, may provide the conclusive reasons for occasional arsenic(III) predominance in Lake Ohakuri surface waters. Acknowledgements--We thank staff of the Water Quality Centre, Hamilton; especially Mr K. J. Costley for carrying out the DRP analyses and Dr J. B. Macaskill for the use of the epifluorescent microscope. M. C. Freeman gratefully acknowledges the Hilary V. Jolly Post-doctoral Fellowship from the University of Waikato. The work was also supported by a grant from the Waikato Valley Authority.
REFERENCES
Aggett J. (1981) Mobility of arsenic in the sediments of the Waikato hydrolakes. In The Waters of the Waikato, pp. 301-311. Seminar Proceedings, University of Waikato, Hamilton, New Zealand. Aggett J. and Aspell A. (1976) The determination of arsenic(liD and total arsenic by atomic absorption spectroscopy. Analyst 101, 341-347. Aggett J. and Aspell A. (1980) Arsenic from geothermal sources in the Waikato catchment. N. Z. 7l Sci. 23, 7%82.
293
Aggett J. and Kadwani R. (1983) Anion exchange method for speciation of arsenic and its application to some environmental analyses. Analyst 108, 1495-1499. Aggett J. and O'Brien G. (1984) Factors affecting the mobility of arsenic in the sediments of Lake Ohakuri, New Zealand. Envir. Sci. Technol. 19, 231-238. Ainsworth G. C., Sparrow F. K. and Sassman A. S. (1973) The Fungi. An Advanced Treatise, Vol. IV A. Academic Press, New York. APHA (1981) Standard Methods for the Examination of Water and Wastewater, 15th edition. American Public Health Association, Washington, D.C. Booth C. (1971) Fungal culture media. In Methods in Microbiology (Edited by Booth C.), Vol. 4, pp. 49-94. Academic Press, London. Calam C. T. (1969) The evaluation of mycelial growth. In Methods in Microbiology (Edited by Norris J. R. and Ribbons D. W.), Vol. 1, pp. 567-592. Academic Press, London. Carmichael J. W., Kendwick B., Conners I. L. and Sigier L. (1980) Genera of Hyphomycetes. University of Alberta Press, Alberta. Collins V. G. (1960) The distribution and ecology of gram-negative organisms other than enterobacteriaceae in lakes. J. appl. Bact. 23, 510-514. Coulter G. W. (1977) The ecological impact on the Waikato River of untreated effluentfrom the proposed Broadlands geothermal power station. New Zealand Energy Research and Development Commission Report No. 26. Coulter G. W., Davies J. and Pickmer¢ S. (1982) Seasonal limnological change and phytoplankton production in Ohakuri, a hydro-electric lake on the Waikato River. N.Z. Jl Mar. Freshwat. Res. 17, 169-183. Cox D. P. and Alexander M. (1973) Effect of phosphate and other anions on trimethylarsine formation by Candida humicola. Appl. Microbiol. 25, 405-413. Crecelius E. A. (1975) The geochemical cycle of arsenic in Lake Washington and its relation to other elements. Limnol. Oceanogr. 20, 441-451. Downes M. T. (1978) An automated determination of low reactive phosphorus concentrations in natural waters in the presence of arsenic, silicon and mecuric chloride. Water Res. 12, 743-746. Ferguson J. F. and Gavis J. (1972) A review of the arsenic cycle in natural waters. Water Res. 6, 1259-1274. Freeman M. C. (1985) The reduction of arsenate to arsenite by an Anabaena assemblage isolated from the Waikato River. N.Z. Jl Mar. Freshwat. Res. 19, 277-282. Freeman M. C. (1986a) The role of growth and senescence of a Ceratophyllum demersum--epiphyte assemblage in arsenic biogeochemistry in the Waikato River, New Zealand. Submitted to Aquat. Bot. Freeman M. C. (1986b) The uptake and reduction of arsenate to arsenite by phytoplankton isolated from the arsenic-rich Waikato River, New Zealand. Submitted to J. Plank. Res. Golterman H. L., Clymo R. S. and Ohnstad M. A. M. (1978) Methods for Physical and Chemical Analysis of Freshwaters, International Biology Program Handbook No. 8, 2nd edition. Blackwell Scientific, London. Green H. H. (1918) Description of a bacterium which oxidizes arsenite to arsenate and of one which reduces arsenate to arsenite isolated from a cattle-dipping tank. S. Afr. J. Sci. 14, 465-467. Holm T. R., Anderson M. A., Stanforth R. R. and Iverson D. G. (1980) The influence of adsorption on the rates of microbial degradation of arsenic species in sediments. Limnol. Oceanogr. 25, 23-30. Iverson D. G., Anderson M. A., Holm T. R. and Stanforth R. R. (1979) An evaluation of column chromatography and fiameless atomic absorption spectrophotometry for arsenic speciation as applied to aquatic systems. Envir. Sci. Technol. 13, 1491-1494.
294
MICHAEL C. FREEMAN et al.
Johnson D. L. (1972) Bacterial reduction of arsenate in sea water. Nature 240, 44 45. Johnson D. L. and Pilson M. E. Q. (1975) The oxidation of arsenite in seawater. Envir. Lett. 8, 157-171. Jones J. G., Davison W. and Gardener S. (1984) Iron reduction by bacteria: range of organisms involved and metals reduced. FEMS Microbiol. Lett. 21, 133-136. Magazda C. H. (1979) Physical and chemical limnology of six hydroelectric lakes on the Waikato River, 1970-1972. N.Z. Jl Mar. Freshwat. Res. 13, 561-572. Maher W. A. (1983) Spectrophotometric determination of arsenic in biological tissue and sediments after digestion with nitric, sulphuric and perchloric acids and preconcentration by zinc column generation and trapping. Analyst 108, 939-943. Malette M. F. (1969) Evaluation of growth by physical and chemical means. In Methods in Microbiology (Edited by Norris J. R. and Ribbons D. W.), Vol. I, pp. 521-566. Academic Press, London. Meyer-Reil L. A. (1978) Autoradiography and epifluorescence microscopy combined for the determination of numbers and spectrum of actively metabolizing bacteria in natural waters. AppL envir. Microbiol. 36, 506-512. Myers D. J., Heimbrook M. E., Osteryoung J. and Morrison S. M. (1973). Arsenic oxidation state in the presence of microorganisms by differential pulse polarography. Envir. Lett. 5, 53-61. Nichols H. W. (1973) Growth media--freshwater. In Phycological Methods (Edited by Stein J. R.), Vol. I. Cambridge University Press, Cambridge. O'Brien G. (1983) Observations on the mobility of arsenic in the sediments in Lake Ohakuri. Ph.D. thesis, University of Auckland, New Zealand. Osborne F. H. and Ehrlich H. L. (1976). Oxidation of arsenite by a soil isolate of Alcaligenes. J. appl. Bact. 41, 295-305. Peterson M. L. and Carpenter R. (1983) Biogeochemical processes affecting total arsenic and arsenic species distri-
butions in an intermittently anoxic t~ord. Mar. Chem. 12, 295-321. Phillips S. E. and Taylor M. L. (1976) Oxidation of arsenite to arsenate by Alcaligenes faecalis. Appl. envir. Microbiol. 32, 392-399. Quastel J. H. and Scholefield P. G. (1953) Arsenite oxidation in soil. Soil Sci. 75, 279-285. Scudlark J. R. and Johnson D. L. (1982) Biological oxidation ofarsenite in seawater. Est. Coast. ShelfSci. 14, 693-706. Spencer M. J. and Ramsay A. J. (1978) Bacterial populations, heterotrophic potentials, and water quality in three New Zealand rivers. N.Z. JI Mar. Freshwat. Res. 12, 415-427. Strachan C. (Ed.) (1979) The Waikato River: A Water Resources Study. Water and Soil Technical Publication No. I 1, Wellington. Turner A. W. (1954) Bacterial oxidation of arsenite--1. Description of bacteria isolated from arsenical cattledipping fluids. Aust. J. Biol. Sci. 7, 452-478. Vldal F. V. and Vidal V. M. V. (1980) Arsenic metabolism in marine bacteria and yeast. Mar. Biol. 50, 1-7. Von Endt D. W., Kearney P. C. and Kaufman D. D. (1968). Degradation of monosodium methanearsonic acid by soil microorganisms. J. agric. Fd Chem. 16, 17-20. Wetzel R. G. (1975) Limnology. Saunders, Philadelphia. Woodbridge D. D. and Garrett W. R. (1969) Relationship between bacteria, nutrients and rainfall in selected bodies of freshwater. Bull. envir, contain. Toxic. 4, 311-319. Woolfolk C. A. and Whiteley H. R. (1962) Reduction of inorganic compounds with molecular hydrogen by Micrococcus lactilyticus--l. Stoichiometry with compounds of arsenic, selenium, Tellerium, transition and other elements. J. Bact. 84, 647-658. Zimmerman R., Iturriaga R. and Becker-Birk J. (1978) Simultaneous determination of the total number of aquatic bacteria and the number thereof involved in respiration. Appl. envir. Microbiol. 36, 926-935.