Ecological Research Series
Some Effects of Cadmium on
Coniferous Soil/Litter Microcosms
National Environmental Research Center
Office of Research and Development
U S Environmental Protection Agency
Corvaliis, Oregon 37330
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EPA-660/3-75-036
JUNE 1975
SOME EFFECTS OF CADMIUM ON CONIFEROUS FOREST SOIL/LITTER
MICROCOSMS
by
Harold Bond
Bruce Lighthart
Raymond Shimabuku
Loren Russell
National Ecological Research Laboratory
National Environmental Research Center
Corvallis, Oregon
Program Element 1AA006
ROAP 21ALU, Task 3
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
For sale by the Superintendent of Documents, U.S. Government
Printing Office, Washington, D.C. 20402
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ABSTRACT
Description and criticism is given of a preliminary design and use of a
soil/litter microcosm in which oxygen, temperature and humidity are kept
constant and oxygen generation and carbon dioxide and heat evolution
rates are monitored. Using four microcosms, one acting as a dead
control, experiments were performed giving the following results: for
"identically" prepared and incubated microcosms, the coefficient of
variation was as small as 3.8 percent for carbon dioxide evolution rate
and as large as 9.9 percent for oxygen consumption rates. It was also
found that an adjustment period of seven to ten days after microcosm
preparation was necessary to approach relatively constant production
rates. For microcosms adjusted to 10, 30, and 60 percent of field water
holding capacity, oxygen and carbon dioxide rates, and bacterial densities
vary directly whereas the fungi and actinomycetes varied inversely;
while for cadmium amended microcosms, 0.01 ppm and initial stages in the
10 ppm CdClp unit, oxygen consumption was stimulated suggesting respir-
atory enzyme uncoupling while in the later stages the 10 ppm cadmium
amended soils reduced both 0^ and C02 respiration by 40 percent. No
organismal density changes due to cadmium were detected indicating the
cadmium initially affects respiration, possibly by uncoupling respiratory
phosphorylation, and that longer experiments might be necessary to
detect population density changes.
This report was submitted in fulfillment of ROAP 21ALU, Task 3, by the
National Ecological Research Center. Work was completed as of February,
1975.
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CONTENTS
Page
Abstract ii
List of Figures iv & v
List of Tables vi
Acknowledgements vii
Sections
I. Conclusions 1
II. Introduction 4
III. Methods 7
Microcosm system design & preparation 7
Experimental 11
Sample analysis 12
Data analysis 13
V. Results 14
"Identical" microcosm experiment 14
Soil moisture experiment 19
Cadmium treatment experiment 22
VI. References 26
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FIGURES
No. Page
1 Wiring diagram for reactor module heat generating/
regulation/monitoring system. SSCO stands for
Scientific Supplies Company Catalog Stock number. 9
2 Diagram of electrolytic oxygen generator. 11
3 Accumulated oxygen consumed through time in
three "identically" prepared and incubated
coniferous forest soil/litter microcosms. 16
4 Accumulated carbon dioxide produced through
time in three identically prepared and incubated
coniferous forest soil/litter microcosms. 17
5 Graphs of heterotrophic bacterial and fungal
propogules (CPU = colony forming units) in
soil and litter in three "identically"
processed microcosms (a & b); three "identically"
processed microcosms incubated at 10, 30 and 60%
of field water holding capacity respectively
(c & d); and three "identically" processed
microcosms injected with distilled water (0.0),
0.01 and 10.0 ppm final mean CdCl? concentration
respectively (e & f). 19
6 Accumulated oxygen consumed through time in
three "identically" prepared and incubated
coniferous forest soil/litter microcosms
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FIGURES (Cont.)
No. Page
treated to an initial soil moisture of (a)
10%, (b) 30%, and (c) 60% of field holding
capacity. 21
7 Accumulated carbon dioxide produced through
time in three "identically" prepared and
incubated coniferous forest soil/litter
microcosms treated to an initial soil moisture
of (a) 10%, (b) 30%, and (c) 60% of field
holding capacity. 22
8 Accumulated oxygen consumed through time in
three "identically" prepared and incubated
coniferous forest soil/litter microcosms
treated to a final mean concentration of (a)
0.0, (b) 0.01, and (c) 10.0 ppm cadmium as
chloride. 25
9 Accumulated carbon dioxide produced through
time in three "identically" prepared and
incubated coniferous forest soil/litter
microcosms treated to a final mean concentration
of (a) 0.0, (b) 0.01, and (c) 10.0 ppm cadmium
as chloride. 26
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TABLES
No. Page
1 Tabulation of the oxygen consumption and carbon
dioxide generation rates for the two phases (ages)
of each of the 3 indicated experiments. 18
VI
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ACKNOWLEGEMENT
Gratitude is expressed for the statistical assistance from Dr. Larry
Male and Mr. John Jacobson for his technical assistance. To Professor
A. W. Anderson, Department of Microbiology, Oregon State University, we
express our appreciation for the use of a prototype to our oxygen generator.
vn
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SECTION I
CONCLUSIONS
Overall microcosm systems design was satisfactory for detecting treatment
effect differences in carbon dioxide generation and oxygen consumption
over time. Albeit, the oxygen generation system was effected by baro-
metric pressure shifts, particularly as waves of winter fronts passed
through the area. They were presumed to average out over time.
Several aspects of the thermal system proved unsatisfactory and are the
reasons for deletion of thermal data from this report. First, the
thermister occasionally "burned out"; whether this was due to their
moisture permeability is problematical. In any event, two parallel
ceramic thermisters are to be used in future systems. It is imperative
that each thermister as well as each reactor module be carefully calibrated
for heat production and loss rate constant respectively! Finally, it is
necessary to produce or account for any heat losses due to water vapor
escapement from the microcosm/Dewar system, and temperature differential
of the replacement carbon dioxide adsorbing solution in order to measure
the biological heat production.
A future communication is in preparation and will detail the design
improvements used to solve the problems just mentioned.
Similarly prepared and incubated soil/litter microcosms had an initial
adjustment period after preparation that lasted approximately a week to
ten days. The adjustment period is seen at both "indicator" and organismal
levels as a relatively rapid respiration rate, i.e., oxygen utilization,
carbon dioxide generation rate and microbial and nematode numerical
increases. After the adjustment period, respiration decreases without a
concomitant decrease in organisms indicating a slowdown of organismal
activity presumably to a level more conducive to long-term survival at
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low nutrient levels. For the microbes, this would be expressed by
Winogradski as zymogenous growth of the fraction of the autochothonous
population feeding on the altered nutrient supply following microcosm
preparation from undisturbed soil. It is speculated that the zymogenous
population in these experiments would be those autochothonous organisms
that have relatively high growth rates (y ) and substrate saturation
(K ) constants (Jannasch, 1968, 1974) allowing their numerical increase
in relatively high nutrient levels over those adjusted for lower nutrient
levels. Upon depletion of the readily utilized substrate, the zymogenous
population would slowly starve as reserves were exhausted, thus the
populations would revert to the original autochothonous phenotypes
equipped to operate at low nutrient concentrations, i.e., low K and y .
Decline in the zymogenous population in these experiments appears to
occur after approximately two to three weeks incubation depending upon
at least soil moisture conditions.
Organism numerical variation among similarly prepared and incubated
microcosms was dependent upon taxonomic groups which in general may be
ranked: fungi > nematodes > bacteria > actinomycetes, while the variation
among the "integrator" variables,carbon dioxide production and oxygen
consumption, was much less than the organism groups. It is anticipated
that better control of soil/litter compaction, particle size, nutrients,
and initial organism numbers and species would further greatly reduce
variation among microcosms.
On the basis of this first experiment it appears that cadmium chloride
amended soil markedly alters soil respiration. At low cadmium concen-
trations there was community stimulation of oxygen consumption as compared
to carbon dioxide degeneration suggesting uncoupling of respiratory
enzymes whereas high cadmium levels produced a general stoichiometrically
equal reduction (40 percent) in both gas rates. The mechanism for the
high level inhibition is unknown but information about excess heat
-------
evolution from cadmium treated units would have been useful in confirming
possible uncoupling.
The effect of cadmium treatment on organsism densities over the four-
week duration of experiment indicated no numerical changes beyond those
expected from inter-microcosm variations. It would be likely that upon
further incubation, population densities shifts would be seen. Further,
it would also be expected that successional events would be altered
because of differential sensitivities of different organisms to the
toxic effects of cadmium.
The reduction in respiratory activity brought on in cadmium amended soil
would eventually be felt at the primary producer level when plant growth
would be limited by the reduction of essential nutrient delivery rates
as the result of inhibition of organisms performing organic reminerali-
zation in the soil.
It is concluded, that natural soil and litter effects,as well as "man-
produced" effects such as cadmium amendment, may be successfully detected
in soil microcosms using oxygen consumption and carbon dioxide generation
The use of heat generation remains problematical, but is a realistic
goal.
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SECTION II
INTRODUCTION
Decomposition is an essential process in all food webs if they are to
remain in temporal equilibrium. The nature of the forest decomposition/
primary production system is as a cyclic process in which primary
produced organic matter in the form of litter fall, leachates, root
exudates and sloughage is remineralized by soil macro-and micro-organisms
(the decomposers) to plant nutrients in a series of "shredding" and
solubilizing steps (McBrayer, Reichle, and Witkamp, 1974; Ausmus and
Witkamp, 1974; Gist, 1972). The extent of this process is shown by the
estimates that 80-90 percent of net primary production in terrestrial
ecosystems is ultimately acted on by decomposer organisms (Odum, 1971;
Witkamp, 1971; Wittaker, 1970), which along with the primary producers
may make up to 95 percent of the total biomass in deciduous forests
(Odum, 1971). It has been estimated that up to 70 percent of the
caloric or biomass input to the decomposers is below ground from the
roots of the primary producers (McBrayer, et a_l_., 1974). It can be
imagined that any interruption in the decomposition portion of this
cycle might lead to a decrease in formation of readily utilizable
essential plant nutrients that will subsequently limit plant growth
rates.
Both natural and man modified environmental conditions in the soil and
litter may markedly affect rates of decomposer activities. It is known
that temperature, soil moisture, oxygen concentration, particle size,
and quality and quantity of organic matter among others affect soil
decomposition processes (in Gray and Parkinson, 1968). Relatively
little is known about many man modified conditions, e.g., what are the
effects of addition of the heavy metal cadmium as it accumulates as a
contaminant of phosphate fertilization of agricultural lands (Williams
and David, 1973). Further, many materials are known to stimulate organisms
-------
at very low concentration and inhibit at higher concentrations (Loomis,
1971).
Cadmium is known to stimulate and/or inhibit plants, animals and micro-
organisms. In eucaryotic organisms, the action of cadmium is known to
involve (1) blockage of the electron transfer from the TCA cycle to
electron transport chain, (2) blockage of enzymes necessary for the
synthesis of ATP by respiratory chain enzymes and (3) binding to the
enzyme ATPase which is required to hydrolyze the reaction of ATP to ADP
(Berry, Osgood, and St. John, 1974; White, Handler and Smith, 1973). It
is thought that cadmium reacts with the sulfahydral groups in the
affected enzymes. The mechanism of reaction appears to be the entrance
?+
of the cadmium ion (Cd ), the most active form of cadmium, into the
cell where it binds to the membrane of the mitochondria. In certain
yeasts, cadmium effects result in respiratory deficiencies related to
the loss of "cristate" mitochondria producing petite colonies (Lindegren
and Lindegren, 1973). In this case, cadmium might be thought of as a
mutagenic agent. In procaryotic cells, both (deJong, 1971) neutral and
inhibitory effects, depending upon cadmium concentration (Zwarum, 1973)
were demonstrated on Azotobacter sp., Escherichia coli and other bacteria.
For a review of pollutant stress effects on soil litter decomposers see
Wiley (In preparation by U.S. E.P.A.).
In the study of effects of stressers on soil decomposition, particularly
toxic substances such as cadmium and other airborne pollutants, it is
difficult to "sort out" the treatment effect from the natural variation
within and among soil horizons. Further difficulties often arise when
it is necessary to clean up large land areas used for toxic substance
studies upon completion of an experiment. The former problem was encoun-
tered by Bond, et_ aj_. , (1974) who were unsuccessful in "sorting out" the
effects of natural variation from the air pollutant treatments on different
soil plots obtained from adjacent locations. To obviate the natural
-------
variation confounding soil treatment experiments it was reasoned that if
"identical" forest soil/litter simulating microcosms could be prepared
and incubated that one might then be able to more readily detect the
effects of stressor treatment on soil processes. This communication
presents a description of the use of a coniferous forest soil/litter
microcosm system, and how the microcosms responded to (a) similar prepar-
ation, (b) three levels of soil moisture, and (c) three levels of cadmium
stressor.
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SECTION III
METHODS
Three experiments with four soil/litter microcosms, one acting as a
thermobarometer, were performed to obtain a preliminary evaluation of
the soil microcosm technique under soil stressing conditions. The first
experiment was performed to crudely observe variation between three
"identical" microcosms, the second the effects of soil moisture on
microcosm performance, and the third to test the effects of three levels
of cadmium on the system performance. Microcosm performance was measured
by so-called "integration" variables such as oxygen consumed, carbon
dioxide produced and heat generated, and changes in "differentiated"
variables such as microbial and invertebrate populations, and chemical
changes in the soil/litter substrate.
Microcosm System Design and Preparation
Four soil microcosm systems similar to those used by McGarity, Gilmour
and Boll en (1958) were constructed in four modular components: the
soil/litter microcosm or reactor "insert" module, the reactor chamber
module, the electrolytic oxygen generator module, and carbon dioxide
trap module.
The soil/litter "insert" module was the microcosm proper and consisted
of a plastic lined number 300 can layered with (starting from the bottom
of the can), 0.5 cm glass wool, 150.00 gm of soil, 1/16 in mesh plastic
screen, and 15.00 gm of litter sifted through l/16th inch mesh screen.
The soil and its overlying litter were obtained from a visually uniform
area in a Douglas Fir forest stand in the Oregon Coast Mountain Range
during the period June through November. Variation between up to 50
replicate microcosm "inserts", was minimized by sifting large amounts of
homogenized (Waring Blended) and specifically prepared soil into all
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similarly treated "inserts" simultaneously. Compaction of the soil was
crudely achieved by tapping the bottom of the microcosm to a predetermined
soil depth. The plastic screen was used to later facilitate soil/litter
separation for destructive analysis.
The reactor chamber module consisted of a one quart Dewar flask large
enough to accept the microcosm "insert." The flasks were closed off at
the top with a number 17 neoprene stopper through which were inserted a
small bore glass tube connected to the oxygen generator module, a
thermoregulator (Scientific Supplies Company Catalog No. 61845-009), and
wires to a heating resistor taped to the mercury bulb of the thermo-
regulator. The thermoregulator and heating resistor were electrically
connected (Figure 1) so that a mercury switch would be thrown to turn on
both an electric clock and the heating resistor when the temperature in
the reactor was below the "set-point" of the thermoregulator. All of
the reactors were maintained at 20°C in a constant temperature room set
at 17 to 18°C. A heat loss and resistor heat constant must be measured
and used to evaluate the heat production in each reactor. Biologically
generated heat was calculated in calories using equation 1:
Biologically test/control
Generated reactor heat
heat from = loss constant
test microcosm ratio
(calories)
or
heat generated
x in control
microcosm
Heat generated
in test (Eq. 1)
microcosm
^Test
T
— x (RC x tc) - (RT x tT)
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NORMALLY
CLOSED
MERCURY
RELAY
SWITCH
(SSCO 4*61841-020)
PLUG
-o
ON/OFF
SWITCH
V
-------
where
KT = Heat loss rate constant (cal/hr) for the test reactor.
KC = Heat loss rate constant (cal/hr) for the control reactor.
RC = Heat production rate (cal/hr) constant for the control
resistor.
RT = Heat production rate (cal/hr) constant for the test
resistor.
t« = The time (hr.) the control resister was heating.
tT = The time (hr.) the test resistor was heating.
QT t May be converted to calories produced per unit surface
es area and time by multiplication with appropriate factors.
Carbon dioxide within the reactor was trapped in 10.00 mis of 0.6 N NaOH
held in a polyethelene vial hanging just above the "insert". Every 24
or 48 hours during an experiment the remaining alkali in the vial was
titrated using the Coleman, et al., (1972) and Colman (1973) method and
these data presented as milliliters carbon dioxide generated during the
trapping period.
The oxygen consumed and carbon dioxide trapped during soil respiration
in the closed reactor resulted in a reduced pressure that was transmitted
to the electrolytic oxygen generator module (See Figure 2). Oxygen was
generated when an electrolyte "switch" was closed at the positive elec-
trode. Upon relief of the pressure by the generated oxygen the switch
turned off. Hydrogen gas was generated at the negative electrode and
trapped in an inverted and closed burette. One-half of the hydrogen gas
volume is equal to the oxygen gas produced. A charcoal filter in the
Tygon conducting tube between the oxygen generator and the reactor
chamber absorbed toxic gases such as ozone and chlorine produced during
the electrolytic process (Woodland, 1973). The electrolyte in the
generator was an aqueous solution of 8 percent Na9SO,,.
10
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GAS BURETTE
PLATINUM
ELECTRODE (+)
THREE-WAY
STOP-COCK
TO RESPIRATOR
Hg CONTACT
ELECTROLYTE
LEVEL
PLATINUM
ELECTRODE (-)
LEVELING TUBE
Figure 2. Diagram of electrolytic oxygen generator.
11
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EXPERIMENTAL
The first experiment was designed to observe the variation between three
"identically" prepared reactor inserts and incubation conditions. These
inserts were prepared as above with the soil and litter adjusted to 21
and 44 percent soil moisture, respectively (Table 1). Incubation of the
inserts in the reactors began immediately after their preparation, i.e.,
without any preincubation. The second experiment considered effects of
soil moisture [measured as field water holding capacity (FWHC)J. Each
of three sets of 16 inserts were simultaneously prepared. One set was
prepared with 10 percent, the second set with 30 percent, and the third
set with 60 percent of field water holding capacity of soil and litter.
These inserts were preincubated for two weeks prior to random selection
of one from each treatment and introduced into one of the reactors. The
third experiment tested the effects of three levels of cadmium i.e.,
mean microcosm concentrations of 0.0, 0.01, and 10.0 milligrams cadmium
as CdClp/kg of dry weight soil or litter. This experiment was commenced
after two weeks preincubation at 20°C of "identically" prepared inserts.
The CdCl- solution was injected into the soil and litter at 30 evenly
spaced sites with a tuberculin syringe. The solution was made to a
concentration that would just replace the 2-5 milliliters of evaporative
water loss during the previous 2 days of the preincubation period.
Enough microcosm inserts were prepared at any one time to use in the
reactors and for periodic destructive analysis during an experiment
which were performed at circa 0, 3, 6, 12, and 24 days incubation after
the inserts were introduced into the reactors. On the 24th day the
reactor inserts themselves were also destructively analyzed.
12
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SAMPLE ANALYSIS
Soil and litter moisture were determined gravimetrically on 105°C oven-
dried samples. Surface spread plate counts of total heterotrophic
bacteria, actinomycetes, and micro-fungi were counted on triplicate
plates of 10-fold sterile phosphate buffer (APHA, 1970) dilutions of
agitated soil and litter samples on the following three media: Acidified
Potato Dextrose Agar (DIFCO 1953) for fungi, sodium albuminate agar
(Pramer and Schmidt, 1964) for actinomycetes, and Bunt and Rovira's
medium (1955) for bacteria. Incubation was at room temperature for two
weeks.
Nematodes, rotifers and tardigrades, and other small metazoans were
extracted from soil and litter samples with a modified Baermann funnel
technique. Fifteen gm. soil and 1.5 gm. litter samples were wrapped in
one-ply tissue paper and placed on a brass screen in a 13 cm diameter
polypropylene funnel. The samples were flooded with tap water, and the
nematodes were harvested after 6 days at 20°C. Nematode samples were
counted immediately or stored at 6°C. Although the Baermann funnel is
often inefficient, the small, thin samples used should result in effective
extractions (Oostenbrinck 1971, Southey, 1970).
Microarthropods were recovered from 8-10 gm of litter and 100-120 gm of
soil using a Tullgren technique with an apparatus similar to the high-
gradient canister extraction of MacFadyen (1961). The "funnels" were
heated with incandescent bulbs with the heat being progressively increased
by use of a dimmer switch. For the present samples, the cover portion
of the canisters were not air-conditioned as is usually the case for
this type of apparatus. Specimens were collected into 70 percent ethanol
(see also Bond, et al., 1974).
13
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DATA ANALYSIS
Graphical and numerical methods were used to reduce the data. Reactor
oxygen consumption, carbon dioxide and heat generation were summed for
each reactor over time and the linear portions of computer drawn curves
fitted by the least squares method to evaluate their rates of formation.
Microbial data was plotted and a sample mean calculated for each analysis
time.
14
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SECTION IV
RESULTS
"IDENTICAL" MICROCOSM EXPERIMENT
In three "identically" prepared and incubated microcosms,the "integrator"
indicators, rates of oxygen consumption and carbon dioxide production,
decreased from a relatively more rapid near constant rate during the
initial 200 hours after microcosm preparation to a slower near constant
rate after 200 hours (Figure 3 and 4 and Table 1). Oxygen consumption
for the three microcosms ranged from 0.239 to 0.215 ml hours" with a
coefficient of variation of 6.65 percent in the initial 200 hours and
i
from 0.160 to 0.131 ml hours with a coefficient of variation of 9.93
percent in the final 350 hours. Also, during the first 200 hours incu-
bation, carbon dioxide production ranged from 0.363 to 0.396 ml hours"
with a coefficient of variation of 5.03 percent while after this period
the range was 0.190 to 0.176 ml hour with a coefficient of variation of
3.83 percent.
Bacteria, actinomycetes, and nematodes in the initial 150 hours of
Q
incubation increased in the litter 3, 30 and 2.5 times to 1.3 x 10 , 2.5
X 10 , and 7.5 X 10 /gm (DW), respectively, and in the soil 4, 37 and
1.3 times to 6 x 101, 2.5 x 107 and 2.4 x loVgm (DW) respectively
(Figure 5), while the fungi decreased by 1/3 from 3 x 10 /gm (DW) in the
first 100 hours in the soil and litter, then returned to their initial
value by 150 hours. The reason for this initial decrease and return in
the fungi is unknown. For the remainder of the experiment, the bacteria
and actinomycetes in the soil and litter, and fungi in the soil remained
at the higher densities, while the nematodes in the soil and litter and
fungi in the litter slowly decreased. Arthropods remained constant or
increased slightly during the experiment.
15
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LU
100
80
X 60
o
< 40
I—
O
K 20
0
0
OD
o A
<3D A D
200 400
EXPERIMENT TIME (hr)
600
Figure 3. Accumulated oxygen consumed through time in three "iden-
tically" prepared and incubated coniferous forest soil/litter
microcosms.
16
-------
—
E
_j
<
^**»
O
1 —
r^
CO
O
O
1 DU
140
120
100
80
60
40
20
O
0"
o
o
O A A
0 A AD
0 A n D
0 AD
0 AD
~ O A D
° AD
0 AD
0 A °
0° 6*
o A n
0 A
a
0 £
°,&
a
°h
°£
°^
CD^
_ O ™
A
1 1 I i I
'0
200 400 600
EXPERIMENT TIME (hr)
Figure 4. Accumulated carbon dioxide produced through time in three
identically prepared and incubated coniferous forest
soil/litter microcosms.
17
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TABLE 1
TABULATION OF THE OXYGEN CONSUMPTION AND CARBON DIOXIDE
GENERATION RATES FOR THE TWO PHASES (AGES) OF EACH OF THE 3 INDICATED EXPERIMENTS
Phase
Experiment Treatment (Duration of
Number Measurement in Days ^ Water
of Microcosm Age) (Soil/L1tter)
1
1
1
1
1
1
2
2
2
2
2
2
3
3
3
3
3
3
None
None
None
None
None
None
Soil Moisture 10%
of Field Capacity
Soil Moisture 3(1%
of Field Capacity
Soil Moisture 60%
of Field Capacity
Soil Moisture 10%
of Field Capacity
Soil Moisture 30%
of Field Capacity
Soil Moisture 60%
of Field Capacity
None
Cadmium (0.01 ppm)*
Cadmium (10.0 ppm)*
None
Cadmium (0.01 ppm)*
Cadmium (10.0 ppm)*
0-14
0-14
0-14
14-24
14-24
14-24
14-26
14-26
14-26
26-38
26-38
26-38
14-20
14-20
14-20
20-38
20-38
20-38
21.0/44.5
21.0/44.5
21.0/44.5
20.5/37.2
21.6/36.5
20.8/35.9
15.6/20.8
20.5/47-2
26.9/58.3
14.7/43.5
19.3/34.9
26.2/49.5
16.8/38.0
16.8/38.0
16.8/38.0
18.8/35.4
18.6/33.0
17.4/32.6
Oxygen
Uptake
Rate
(ml/hr)
0.249
0.239
0.215
0.160
0.147
0.131
0.028
0.076
0.123
0.039
0.046
0.133
0.140
0.178
0.131
0.137
0.140
0.081
Carbon
Dioxide Coefficient
Generation of
Rate Variation
(ml/hr) (%)
0.396
0.364
0.363
0.190
0.183
0.176
0.054
0.123
0.195
0.104
0.077
0.147
0.251
0.256
0.210
0.157
0.137
0.101
CVCQ 6.65
CVn 5.03
U2
Cvco2 9-93
CVn 3.83
U2
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
N/A
*Theoretical final mean environmental concentrations.
18
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HETEROTROPHIC BACTERIA (cfu/gm (D.W.))
o
o
o
o
m
FUNGI (cfu/gm (D.W.))
o
o
o
o
o
o
o
o
m
-
o
o
A
\ 5oo ^
\ °2° V
CX3 TD ~O "O CO I
- --*,
H
I
, ooo
Q.Q-CL
O D CD D
i ill
O
O
'81° a
Figure 5 Graphs of heterotrophic bacterial and fungal propogules (CFU = colony forming units)
in soil and litter in three "identically" processed microcosms (a & b); three
"identically" processed microcosms incubated at 10, 30 and 60% of field water
holding capacity respectively (c & d); and three "identically" processed microcosms
injected with distilled water (0.0), 0.01 and 10.0 ppm final mean CdCU concentration
respectively (e & f).
-------
The dispersion of values about the analysis times for counts from the
indicated number of microcosms had a maximum coefficient of variation
for either soil or litter at any given harvest time of 17.6 percent for
bacteria (for 7 microcosms), 45.7 percent for actinomycetes (5), 52.4
for fungi (7), 39.2 percent for nematodes (9), and 75.2 percent for
arthropods (5). In all cases for all three experiments in reactor
incubated units terminal analysis measurements were scattered among the
externally-incubated microcosms indicating close agreement between
external and reactor incubated "insert" microcosms.
SOIL MOISTURE EXPERIMENT
The experiment concerned with soil moisture showed marked effects at
both the "integrator" and organismal levels. As soil moisture increased,
so did both oxygen consumption and carbon dioxide generation (Figures 6
and 7, Table 1). The rate of oxygen consumption over the duration of
the experiment changed little for the 60 percent FWHC, but diminished by
about 50 percent in the 30 percent FWHC microcosm and increased by 1/3
in the 10 percent FMHC microcosm. For C02 the 60 and 30 percent FWHC
microcosms decreased 75 and 50 percent respectively and the 10 percent
unit doubled its production.
The microbial population densities in replicate microcosms showed such a
marked dispersion of counts that only general trends could be observed.
(Figure 5). In both the litter and soil the numerically predominantly
organisms were the bacteria and actinomycetes (in that order) in the 30
and 60 percent field holding capacity microcosms whereas the fungi were
numerically dominant in the 10 percent units. The actinomycetes in both
soil and litter in the 10 percent moisture microcosm and fungi in the 10
percent microcosm litter were the only microorganisms thought to be
increasing in number over time.
20
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UJ
X
o
o
0
200 400
EXPERIMENT TIME
600
(hr)
Figure 6. Accumulated oxygen consumed through time in three "iden-
tically" prepared and incubated coniferous forest soil/
litter microcosms treated to an initial soil moisture of
(a) 10%, (b) 30%, and (c) 60% of field holding capacity.
21
-------
x .
E
^
i
i
t-
o
[—
OJ
O
O
160
140
120
100
80
60
40
20
n
c) a
DD
"
u
n
n
n
na
i^
b{-
n A A A A
D A A A
n AA AA ,,0
° AAA ^0°
D A n O
n A O
- g^Aooo0ooooO°
Bo ° i i i i i i
0 200 400 600
EXPERIMENT TIME (hr)
Figure 7, Accumulated carbon dioxide produced through time in three
"identically" prepared and incubated coniferous forest
soil/litter microcosms treated to an initial soil moisture
of (a) 10%, (b) 30%, and (c) 60% of field holding capacity.
22
-------
Nematode densities in the litter increased linearly with time at a rate
of 0.0 nematodes/gm (DW)/day in the 10 percent FWHC microcosm, 0.17
nematodes/gm (DW)/day in the 30 percent and 10.8 nematodes/gm (DW)/day
in the 60 percent FWHC microcosms. In the soil of these same microcosms,
nematode densities increased from undetectable in the 10 percent unit to
a plateau of 25 nematodes/gram (DW) after 300 hours incubation, after an
initial 200-hour delay. Nematodes in the 60 percent soil moisture unit
increased from 15 to 300 worms/gm (DW), and 30 percent showed no change
from 40 worms/gm (DW).
Due to large variation between microcosms, arthropod populations were
judged to showed no differences between treatments.
CADMIUM TREATMENT EXPERIMENT
Cadmium introduced into the microcosms produced detectable effects at
the integrated oxygen consumption and carbon dioxide generation levels
with no obvious effect at the organismal level (Figures 5, 8 and 9,
Table 1). The 0.0 ppm cadmium control produced a constant oxygen uptake
of between 0.140 and 0.137 ml 02 hr ) during the initial and final 200
hours of the experiment respectively, while compared to the control, the
0.01 ppm cadmium microcosm showed a possible 27 percent stimulatory
effect (0.178 ml 02 hr"1) during the initial 200-hour interval of the
experiment, and the 10 ppm cadmium microcosm showed a 41 percent inhibitory
effect (0.081 ml 02 hr"1) after the 200-hour time period.
Carbon dioxide production in all cases decreased throughout the duration
of the experiment (Table 1). The 0.01 ppm cadmium microcosm treatment
was very similar to the control rate, whereas the treatment 10 ppm
23
-------
cadmium microcosm had not only a large CL consumption rate decrease but
also a 36 percent reduction in C02 production compared to the control
after the initial 200 hrs.
At the organismal level, frank cadmium treatment effects were not
detectable in any group.
24
-------
LU
- 100
80
x 60
O
40
20
O
I-
0
0
6
200 400 600
EXPERIMENT TIME (hr)
Figure 8. Accumulated oxygen consumed through time in three "iden-
tically" prepared and incubated coniferous forest soil/litter
microcosms treated to a final mean concentration of (a)
0.0, (b) 0.01, and (c) 10.0 ppm cadmium as chloride.
25
-------
^ s
—
E
h-
0
1—
OvJ
O
O
IOU
140
120
100
80
60
40
20
n
a)
2)\
i~]
nflfi
O
X---5'
^l"n°°
8 D D
^ D D
o n
- $
6
0 i I i i ' i i
0
200 400 600
EXPERIMENT TIME (hr)
Figure 9. Accumulated carbon dioxide produced through time in three
"identically" prepared and incubated coniferous forest
soil/litter microcosms treated to a final mean concentration
of (a) 0.0, (b) 0.01, and (c) 10.0 ppm cadmium as chloride.
26
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SECTION V
REFERENCES
American Public Health Association. 1971. Standard Methods For the
Experimentation of Water and Wastewater. 13th Ed. APHA, Washington,
D.C. 874 p.
Difco Laboratories. 1953. Difco Manual, 9th Ed. Detroit, Michigan.
350 p.
Ausmus, B. S., and M. Witkamp. 1974. Litter and Soil Microbial Dynamics
in a Deciduous Forest Stand. Oak Ridge Nat!. Lab., Oak Ridge, TN.
EPFB-IBP-73-10, UC-48-Biol. and Med. 183 p.
Berry, J. W., D. W. Osgood, and P. A. St. John. 1974. Chemical Villains:
A Biology of Pollution. The C. V. Mosby Co., St. Louis. 189 p.
Bond, H., L. Russell, and R. Shimabuku (in manuscript). Effect of
Sulfur Dioxide and Ozone Fumigation on a Forest Soil/Litter Ecosystem,
National Ecological Research Laboratory, U.S.E.P.A., Corvallis,
Oregon. 143 p.
Bunt, J. S. and A. D. Rovira. 1955. Microbiological Studies of Some
Subantarctic Soils. J. Soil Sci. 6:119-128.
Coleman, D. C., J. E. Ellis, J. K. Marshall, and F. M. Smith. Basic
Field Data Collection Procedures for the Grassland Biome 1972
Season. USIBP Grassland Tech. Rept. #145. 75 p.
Coleman, D.C. 1973. Soil Carbon Balance in a Successional Grassland.
Oikos 24:195-199.
27
-------
deJong, L. E. Den Dooren. 1971. Tolerance of Azotobacter for Metallic
and Non-metallic Ions. Ant. von Leeuwenhoek. 37:119-124.
Gist, C. S. 1972. Analysis of Mineral Pathways in a Cryptozoan Foodweb
Eastern Deciduous Forest Biome. Coneeta Research Site, Inst.
Ecology, Urn'. Georgia, Athens, GA. Mono. Rpt. 72-23. p. 151.
Gray T. R. C. and D. Parkinson, (eds.). 1968. International Symposium
on the Ecology of Soil Bacteria. U. Toronto Press, Toronto, Canada,
pp. 681.
Jannasch, H. W. 1968. Competitive Elimination of Enterobacteriaceae
from Seawater. Appl. Microbiol. 16(1):1616-1618.
. 1974. Steady State and the Chemostat in
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Lindegren, C. C., and G. Lindegren. 1973. Mitochondrian Modification
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Loomis, T. A. 1964. Essentials of Toxicology (2nd Ed.). Lea and
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MacFadyen, A. 1961. Improved Funnel-type Extractors for Soil Arthro-
pods. J. Anim. Ecol. 30:171-184.
McBrayer, J. F., D. E. Reichle, and M. Witkamp. 1974. Energy Flow and
Nutrient Cycling in a Cryptozoan Food-Web. Oak Ridge Natl. Lab.,
Oak Ridge, TN. EDFB-IBP-73-8, UC-48-Biol. and Med. 78 p.
28
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McGarity, J. W., C. M. Gilmore and W. B. Bollen. 1958. Use of an
Electrolytic Respirometer to Study Dentrification in Soil. Can. J.
Microbiol. 4:303-316.
Odum, E. P. 1971. Fundamentals of Ecology, 3rd Ed. W. B. Saunders
Co., Philadelphia, PA. 574 pp.
Oostenbrink, M. 1971. Comparison of Techniques for Population Estima-
tion of Soil and Plant Nematodes. pp. 72-82. In: Methods of
Study in Quantitative Soil Ecology: Population Production and
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Scientific Publications, Oxford, England.
Pramer, D and E. L. Schmidt. 1964. Experimental Soil Microbiology.
Burgess Publishing Co., Minneapolis, Minnesota, p. 53.
Ruhling, A. and G. Tyler. 1973. Heavy Metal Pollution and Decomposition
of Spruce Needle Litter. Oikos. 24:402-416.
Southey, J. F. (editor). 1970. Laboratory Methods for Work With Plant
and Soil Nematodes. His Majesty's Stationery Office, London.
Technical Bulletin 2. 148 pp.
White, A., P. Handler, and E. L. Smith. 1973. Principles of Biochemistry
(5th Ed.). McGraw-Hill Book Co., N.Y. p. 1296.
Wiley, W. T. Unpublished Manuscript. Pollutant Stress Effects on Soil-
Litter Decomposition. National Ecological Research Laboratory,
U.S. EPA, Corvallis, OR.
29
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Williams, C. H. and D. J. David. 1973. The Effect of Superphosphate on
the Cadmium of Soils and Plants. Australian J. Soil Sci. 11(1):43-
56.
Witkamp, M. 1971. Soils as Components in Ecosystems, pp. 85-110. In:
Ann. Rev. Ecol. and Systematics, Vol. 2. (R. F- Johnston, P. W.
Frank, and C. D. Michener Ed.). Ann. Rev., Inc., Palo Alto, CA.
Whittaker, R. H. 1970. Communities and Ecosystems. MacMillan Co.,
London. England. 161 pp.
Woodland, D. J. 1973. The Ozone Problem in Electrolytic Respirometry
and Its Solution. J. Appl. Ecology 10:661-662.
Zwarum, A. H. 1973. Tolerances of Escherichia coli to Cadmium. J.
Environ. Quality 2(3):353-355.
30
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TECHNICAL REPORT DATA
(/'lease read Instructions on the reverse before completing}
2.
1. REPORT NO.
EEA.-&6 Q/3.-LZ 5^036
4. TITLE AND SUBTITLE
Some Effects of Cadmium on Coniferous Torest Soil
and Litter 1 icrocosms
3. RECIPIENT'S ACCESSION-NO.
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
. AUTHOR(S)
H. Bond, B. Lighthart, R. Shimabuku, L. Russell
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORG "VNIZATION NAME AND ADDRESS
National Ecological Research Laboratory
200 SW 35th St.
Corvallis, Oregon 97330
10. PROGRAM ELEMENT NO.
1AA006
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
13. TYPE OF REPORT AND PERIOD COVERED
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Description and criticism is given of a preliminary design and use of a soil/litter
microcosm in which oxygen, temperature and humidity are kept constant and oxygen
generation and carbon dioxide and heat evolution rates are monitored. Using four
microcosms, one acting as a dead control, experiments were performed giving the follow-
ing results; for "identically" prepared and incubated microcosms, the coefficient of
variation was as small as 3.8 percent for carbon dioxide evolution rate and as large
as 9.9 percent for oxygen consumption rates. It was also found that an adjustment
period of seven to ten days after microcosm preparation was necessary to approach
relatively constant production rates. For microcosms adjusted to 10, 30, and 60 per-
cent of field water holding capacity, oxygen and carbon dioxide rates, and bacterial
densities vary directly whereas the fungi and actinomycetes varied inversely; while
for cadmium amended microcosms, 0.01 ppm and initial stages in the 10 ppm CdCl unit,
oxygen consumption was stimulated suggesting respiratory enzyme uncoupling while in the
later stages the 10 ppm cadmium amended soils reduced both 0 and CO respiration by 40
percent. No organismal density changes due to cadmium were detected indicating the
cadmium initially affects respiration, possibly by uncoupling respiratory phosphoryla-
tion, and that longer experiments might be necessary to detect population density
changes.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Cadmium
microcosm
decomposition
b.lDENTIFIERS/OPEN ENDED TERMS
COSATI Field/Group
13. DISTRIBUTION STATEMENT
19. SECURITY CLASS (ThisReport)
21. NO. OF PAGES
20. SECURITY CLASS (Thispage)
22. PRICE
EPA Form 2220-1 (9-73)
ft U.S. GOVERNMENT PRINTING OFFICE: 1975-699-074 (16 REGION 10
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