Ecological Research Series
TRACE ELEMENT RESEARCH USING CONIFEROUS
FOREST SOIL/LITTER MICROCOSMS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, Oregon 97330
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
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The nine series are:
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This report has been assigned to the ECOLOGICAL RESEARCH series. This series
describes research on the effects of pollution on humans, plant and animal spe-
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mine the fate of pollutants and their effects. This work provides the technical basis
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This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-77-091
August 1977
TRACE ELEMENT RESEARCH
USING CONIFEROUS FOREST
SOIL/LITTER MICROCOSMS
B. Lighthart, H. Bond, and M. Ricard
Terrestrial Ecology Branch
Corvallis Environmental Research Laboratory
Corvallis, Oregon
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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Disclaimer
This report has been reviewed by the Corvallis Environmental Research Labora-
tory, U.S. Environmental Protection Agency, and approved for publication.
Mention of trade names or commercial products or sources does not constitute
endorsement or recommendation for use.
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Protection
Agency would be virtually impossible without sound scientific data on pollu-
tants and their impact on environmental stability and human health. Responsi-
bility for building this data base has been assigned to EPA's Office of
Research and Development and its 15 major field installations, one of which
is the Corvallis Environmental Research Laboratory (CERL).
The primary mission of the Corvallis Laboratory is research on the effects of
environmental pollutants on terrestrial, freshwater, and marine ecosystems;
the behavior, effects and control of pollutants in lake systems; and the
development of predictive models on the movement of pollutants in the bio-
sphere.
This report encompasses research to detect and understand the effects of
trace element pollutants on soil/litter components and on decomposition
processes of coniferous forests. It also includes design and development of
a microcosm system useful in measuring pollutant perturbations. Data gener-
ated in this study will, subsequently, be used in predictive models to deter-
mine pollutant impact within the terrestrial ecosystem.
A. F. Bartsch
Director, CERL
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ABSTRACT
Respirometers have been designed, constructed and, to a limited extent,
tested to maintain and measure production and/or consumption of biogenic heat
and carbon dioxide production and oxygen consumption for extended periods of
time in approximately 0.5 1 soil and/or litter microcosms.
Using coniferous soil/litter microcosms, the mean coefficient of varia-
tion within sets of similar microcosms was 10.7% for the oxygen consumption
rate and 3.9% for carbon dioxide production rate.
Microcosm respiratory response, population responses to moisture level
(where measured), succession, and salt effects were similar to those observed
in the natural world.
Respiration of the decomposer communities in coniferous forest soil/
litter microcosms was inhibited by treatment with "real world" salt concentra-
tions of Cd, Se, Zn, Mn, Ni, Cu, Hg, Co, Cr, V , Li, La, Ag, and Pb. These
findings support the thesis that the consequence of these ecosystem disrup-
tions might be to reduce primary and secondary production of the dependent
populations.
Scale drawings of the microcosm "life-support" system and an outline of
procedural details of system maintenance and microcosm preparation are pre-
sented.
This report was submitted as partial fulfillment of in-house research
under Program Element 1AA006, ROAP 21 ALU, Task 3. It covers the period
March, 1974, to November, 1976, and work was completed as of March, 1977.
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CONTENTS
PAGE
Foreword iii
Abstract iv
List of Figures vii
List of Tables xi
Acknowledgments xii
I. Introduction 1
A. Decomposer Organisms in the Organic Matter Cycle -- 1
B. Tyler's Thesis of Soil Pollution Effects 1
1. Laboratory Evidence 1
2. Community Evidence 3
3. Field Evidence 3
C. Purpose 3
II. Methods 5
A. Brief Description of Microcosm System Design 5
B. Experimental 13
1. Natural Variables 13
2. Pollutant Stress 14
III. Results and Discussion 15
A. Natural Variables 15
1. Succession 15
2. Microcosm Variation 15
3. Soil Moisture 20
4. Salt Effects 20
5. Biogenic Heat Production 29
B. Pollutant Stress 29
1. Microcosm Biological Age 29
2. Trace Element Quality 34
3. Trace Element Quantity 34
4. Trace Element Combinations 34
C. Interpretation 45
IV. Future Research 47
V. References 48
VI. Publications 51
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CONTENTS (CON'T)
PAGE
VII. Appendices
A. Microcosm Preparation 53
B. Inter-Experiment Maintenance Schedule 59
C. Intra-Experiment Maintenance Schedule 64
D. Scale Drawings of "Life-Support" System 66
E. Electronic Control Systems 76
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LIST OF FIGURES
FIGURE PAGE
1 Simplified Systems Diagram of the Organic Matter Cycle 2
2 Systems Diagram of the Microcosm in its "Life-Support"
System ----------- -------- - --------------- - ---------- - ---- 6
3 Photograph Showing "Life-Support" Systems -------- -------- 7
4 Close-Up Photograph of a Single Reactor Module --- ------ -- 8
5 Close-Up Photograph of the Reactor Lid Arrangement -- ----- 9
6 Diagram of Hydrogen Generator Portion of Oxygen Generator
Showing Salt Bridge ---------- - --------------------------- ^
7 Diagram of Electrolytic Oxygen Generator
8 Graphs of Heterotrophic Bacteria and Fungal Propogules
(CFU = Colony Forming Units) in Soil and Litter in Five
"Identically" Processed Microcosms (a&b); Nine "Ident-
ically" Processed Microcosms Incubated at 10, 30 and 60%
of Field Water Holding Capacity, Respectively (c&d); and
Three "Indentically" Processed Microcosms Injected with
Distilled Water (0.0), 0.01 and 10.0 ppm Final Mean CdCl2
Concentration, Respectively (e&f) ------------------------ 16
9 Accumulated Oxygen Consumed Through Time in Three
"Identically" Prepared and Incubated Coniferous Forest
Soil/Litter Microcosms, Including the Initial 10 Day Flush
Period After Preparation --------------------------------- 17
10 Accumulated Carbon Dioxide Produced Through Time in Three
"Identically" Prepared and Incubated Coniferous Forest
Soil/Litter Microcosms, Including the Initial 10 Day Flush
Period After Preparation ---------------------- ; ------ ----- 18
11 Accumulated Oxygen Consumed Through Time in 8 "Identically"
Prepared and Incubated Coniferous Forest Soil /Litter
Microcosms ----------------------------------------------- 19
12 Accumulated Carbon Dioxide Produced Through Time in 8
"Identically" Prepared and Incubated Coniferous Forest
Soil/Litter Microcosms ----------------------------------- 19
vii
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LIST OF FIGURES (CON'T)
FIGURE PAGE
13 Accumulated Oxygen Consumed 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 Water Holding
Capacity 25
14 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 Water Holding
Capacity 26
15 Accumulated Oxygen Consumed Through Time in "Identically"
Processed Coniferous Forest Soil/Litter Microcosms Treated
to a Final Mean Indicated Concentration of Sodium Chloride 27
16 Accumulated Carbon Dioxide Produced Through Time in
"Identically" Processed Coniferous Forest Soil/Litter
Microcosms Treated to a Final Mean Indicated Concentration
of Sodium Chloride 27
17 Accumulated Oxygen Produced Through Time From "Identically"
Processed Coniferous Forest Soil/Litter Microcosms Treated
to a Final Mean Indicated Concentration of Sodium Sulfate 28
18 Accumulated Carbon Dioxide Produced Through Time in
"Identically" Processed Coniferous Forest Soil/Litter
Microcosms Treated to a Final Mean Indicated Concentration
of SoHium Sulfate. " 28
19 Salts (NaCl, NaSOj effects (Compared to Initial Control
Rate) on Respiration in "Identically" Prepared Coniferous
Forest Soil/Litter Microcosms Treated to the Indicated
Final Mean Salt Concentration 30
20 Accumulated Carbon Dioxide Produced Through Time From
"Identically" Prepared and Incubated Coniferous Forest *
Soil/Litter Microcosms Treated to 1500 ppm CdCl2 (at
arrows) at Either 0, 5 or 20 Days of Biological Age 33
21 Survival of 0, 5 and 15 Day Old Collembola Populations in
Identically Prepared Microcosms Treated to 1500 ppm CdCl2 36
22 Survival of Nematodes in 0, 5 and 15 Day Old "Identically"
Prepared Coniferous Forest Soil/Litter Microcosms Treated
to 1500 ppm CdCl2 6/
vm
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LIST OF FIGURES (CON'T)
FIGURE PAGE
23 Accumulated Oxygen Consumed Through Time in "Identically"
Prepared and Incubated Coniferous Forest Soil/Litter
Microcosms Treated to a Mean Final Concentration of the
Indicated Heavy Metals (as Salts) ------------------------ 38
24 Accumulated Carbon Dioxide Produced Through Time in
"Identically" Prepared and Incubated Coniferous Forest
Soil/Litter Microcosms Treated to a Mean Final
Concentration of the Indicated Heavy Metals (as Salts) --- 38
25 Accumulated Oxygen Consumed Through Time in "Identically"
Prepared and Incubated Microcosms Treated With the
Indicated Trace Elements --------------------------------- 39
26 Accumulated C02 Produced Through Time in "Identically"
Prepared and Incubated Microcosms Treated with the
Indicated Trace Elements --------------------------------- 40
27 Accumulated Oxygen Consumed Through Time in Three
"Identically" Prepared and Incubated Coniferous Forest
Soil /Litter Microcosms Treated to a Final Mean Concentra-
tion of (a) 0.0, (b) 0.01, and (c) 10.0 ppm Cadmium
Chloride ------------------------------------------------- 41
28 Accumulated Carbon Dioxide Produced Through Time in Three
"Identically" Prepared and Incubated Coniferous Forest
Soil /Litter Microcosms Treated to a Final Mean Concentra-
tion of (a) 0.0, (b) 0.01, and (c) 10.0 ppm Cadmium
Chloride ------------------------------------------------- 42
29 Graph of Relative Oxygen Consumption and Carbon Dioxide
Generation Rate Values for the Initial 200, and Subsequent
Hours After Cadmium Addition (Mean Microcosm Concentra-
tion) to Douglas Fir Soil/Litter Microcosms -------------- 43
30 Accumulated Oxygen Consumed Through Time in Four
"Identically" Prepared and Incubated Coniferous Forest
Microcosms Treated to a Mean Final Concentration of 0.0
ppm CdCl2 or Se02, 25 ppm CdCl2, 10 ppm Se02, and Both --- 44
31 Accumulated Carbon Dioxide Produced Through Time in Four
"Identically" Prepared and Incubated Coniferous Forest
Microcosms, Treated to a Mean Final Concentration of 0.0
ppm CdCl2 or Se02, 25 ppm CdCl2, 10 ppm Se02, and Both --- 44
32 Drawing of Microcosm "Life Support" System Including
Reactor, Pressure Monitoring, 02 Generating and H2
Generating Modules ---------- ----------------------------- 67
ix
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LIST OF FIGURES (CON'T)
FIGURE PAGE
33 Cut-Away View of Respirator Reactor Module 68
34 Schematic Diagram of Reactor Module Components Showing a)
Lateral View of Top Assembly, b) Top View of Top Plate, c)
Lateral View of C0? Trap Assembly and d) Top and Cut-Away
Side View of Bottom Plate 69
35 "U" Tube Pressure Monitoring Module 70
36 Oxygen Generation Module Showing a) Cut-Away View of Base
With Evacuated/Silvered External Shell, b) Top View of
Generator Module, c) Oxygen Probe Component with Electrode
and d) Top View of Oxygen Probe 71
37 Lateral View of Hydrogen Generation Module 72
38 Microcosm Electronic Circuit Diagram 77
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LIST OF TABLES
TABLE PAGE
1 Means, Standard Deviations and Coefficients of Variation
for 02 Consumption, C02 Production and Respiratory
Quotient for all or Non treatment Portions Within and
Among "Identically" Prepared and Incubated Sets of
Coniferous Forest Soil/Litter Microcosms -.-- 21
2 Tabulation of Respiratory Rates for "Identically" Pre-
pared and Incubated Coniferous Forest Soil/Litter Micro-
cosms Within Each Experiment and Subjected to the
Indicated Treatment 22
3 Table Showing Comparison of C02 Production Rate and
Biogenic Heating Rate (as Indicated by Microcosm Heater
On-Time Rate) Changes Due to Microcosm Treatment With
Various Salt Concentrations 31
4 Respiratory Measurements of "Identically" Prepared
Coniferous Forest Soil/Litter Microcosms Treated to 1500
ppm CdCl2 Stress at the Indicated Microcosm Biological
Ages 32
5 Tabulation of the Survival of Nematodes (Cephalobus
persignis) and Collembola (Isotoma sp.) Added to
Coniferous Forest Soil/Litter at the Indicated Microcosm
Age and Stressed with CdCl^ as Shown 35
6 Trouble Shooting 61
7 Tabulation of Soil Ecosystem Respirometer (SER) Components,
Sources and Approximate Costs 73
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ACKNOWLEDGMENTS
To Messrs. R. Shimabuku for microbiological and nematological assistance,
L. Russell for soil entomological assistance, and J. Jacobson for electronic
assistance, we thank you.
For all of the excellent administrative help, we would like to thank
Drs. N. R. Glass, A. S. Lefohn and L. C. Raniere.
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I. INTRODUCTION
A. Decomposer Organisms in the Organic Matter. Cycle
Decomposition is an essential process in all food webs on this planet and
must not be disrupted if biological systems are to remain in temporal equil-
ibrium. The decomposition/primary production system is a cyclic process
(Figure 1) 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 et al., 1974; Ausmus and Witkamp,
1974; Gist, 1972). The extent of this process is shown by the estimates that
80 to 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 decom-
posers is below ground from the roots of the primary producers (McBrayer et
al., 1974).
B. Organic Matter Cycle and Tyler's Thesis.
It can be imagined that any interruption in the decomposition portion of
this cycle might lead to a decrease in formation of readily utilizable plant
nutrients that will subsequently limit plant growth rates (Ausmus and
Witkamp, 1974).
It is increasingly apparent that man is disrupting soil decomposition
processes, particularly through the addition of trace element pollutants.
Examples with anticipated disruptive consequences include fly ash from metal
smelters that settles to the ground producing high heavy metal soil concentra-
tions (Ratsch, 1974; Linzon, et^ a_l_. , 1975). Cadmium contamination of phos-
phate fertilizers (Williams and David, 1973) and miscellaneous trace elements
contained in sewage sludge added to agricultural fields (Page, 1974), combus-
tion emission fallout from coal-fired power plants (Klein and Russell, 1973),
and roadside accumulations of lead from automobile combustion of tetraethyl
lead in gasoline are other examples.
1. Laboratory Evidence.
It is anticipated that many organisms, if not the entire community, in
heavy metal contaminated soils are affected by these pollutants. It has been
documented that heavy metals as well as certain non metals inhibit bacterial
growth (deJong and denDoren, 1971; Salle, 1973; Lamanna and Mallette, 1965),
1
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DECOMPOSERS
(microorganisms)
CONSUMERS
(animals)
*
Figure 1. Simplified systems diagram of the organic matter cycle.
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including nitrogen fixing bacteria (Wilson and Reisenauer, 1970); they affect
the soil fungal flora (Hartman, 1974) and soil protozoa (Bojsova, 1963).
Although little is known about heavy metal and nonmetal effects on the
soil fauna, particularly the arthropods, high concentations of As and Cu in
orchard and+grassland soil have eliminated earthworms (van Rhee, 1973) while
Cu and Ag were toxic to soil nematodes (Pitcher and McNamara, 1972;
Hafkenscheild, 1971).
2. Community Evidence.
Pollutants may alter the natural rate of decomposition in soil systems
with unknown, but crudely predictable, consequences. At low pollutant concen-
trations, a stimulation of the decomposition rate could result in more rapid
remineralization. At higher concentrations, lethal effects on decomposer
organisms result in cessation of decomposition and resultant plant growth in
the affected area. Thus, plant growth rates and standing stock of animals
might be expected to vary as a function of soil decomposition rates.
3. Field Evidence
Jackson and Watson (1976), and Watson et. al. (1976) found that litter
accumulated and there were mineral nutrient abnormalities surrounding a lead
smelter. They attribute these phenomena to disruption of the soil decomposi-
tion process. Dr. J. Wolak (Director, Forest Res. Inst., Warsaw, Poland,
personal communication) has seen marked litter build-up in Polish forest soil
near long standing smelters. Although the litter decomposer processes are
largely inoperative, tree growth occurs to a limited extent. Apparently some
essential nutrients for tree growth are in the air pollutants and/or these
leach through the accumulated litter and carrv soluable nutrient portions of
the litter downward. Tyler has also shown a reduction in the decomposition
In Swedish forests due to heavy metals emitted by local smelters (Tyler,
1972, 1974, 1975; Ruhling and Tyler, 1973).
C. Purpose.
The study of pollutant stressants on decomposition in extramural labora-
tories such as field plots suffers from several shortcomings that make the
use of laboratory microcosms attractive. Variation over short distances- in
natural or prepared soils confounds treatment effects analysis, often negating
definitive results (e.g., Bond, Russell, and Shimabuku, unpublished). To
solve this problem, large and numerous field plots must be used, creating the
problem of site decontamination after use of toxic and persistent pollutants.
Even if an acceptable extramural site were found practical, this site would
have only one of an almost infinite variety of soil types that would need to
be studied.
Soil/litter microcosms can and have been designed small enough to allow
many treatments with replication to be performed in a single experiment and
the contaminated soil/litter is relatively easy to discard. Further,
microcosms simulating a soil and/or litter system may be used to study nutri-
ent or pollutant webs, pore sizes and transfer rates. Those data may be
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useful in construction of predictive computer simulation models of the system
This document summarizes the soil/litter microcosm research conducted through
March, 1977 at the Corvallis Environmental Research Laboratory.
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II. METHODS
The microcosm system was designed and constructed (see Section II. A)
with the following criteria in mind, and experimentally used to understand
(see Section II. B.I), and evaluate pollutant effects (see Section II. B.2)
on coniferous forest soil/litter systems. The criteria were: (a) simplicity
of design and operation, (b) minimization of cost, (c) minimization of instru-
mentation size, (d) operation for extended time periods, (e) ability to
measure biological "integrator-indicators,"!/ (f) ability to measure "differ-
entiated-indicators,"2/ and (g) maximize studies of soil/litter microbiology.
Stotzky (1965), MacFadyen (1970), McGarity, Gilmore, and Bollen (1958),
and Pramer (1965) discuss various aspects of soil microcosm design.
A. Description of_ Microcosm System Design.
The microcosm system consists of 10 "Life Supporting" modules (Figure 2).
The "Life Support" system (Figure 3) for each microcosm consists of (a) an
insulated microcosm or reactor chamber (Figure 4 and 33); (b) heat monitoring
and maintenance module (Figure 5 and 34a); (c) carbon dioxide production
monitoring module (Figure 5 and 34c); (d) internal pressure monitoring module
(Figure 4 and 35); and (e) oxygen monitoring and generating module (Figures 6,
7, 36 and 37). 3/
The reactor chamber module consists of a one liter Dewar flask large
enough to accept the glass microcosm insert (600 ml Berzelius glass beaker).
The flasks were sealed with a foam stopper equipped lid with a small bore
glass tube connected to the oxygen generator module, a thermal regulator 4_/
and wires to a heating resistor connected to the mercury bulb of the thermal
regulator. The thermal regulator and heating resistor were electrically
inter-connected to maintain the temperature of the reactor chamber at the
"set point" of the thermal regulator. Reactor heat loss and resistor heating
constants were measured and used to evaluate heat production in each reactor.
]_/ Integrator-indicators were defined as the community heat, oxygen, and
carbon dioxide measurements.
2/ Differentiated-indicators were defined as the organism groups responsible
~~ for the changes in the integrator-indicators.
3/ The original prototype model of the device shown in Figure 7 was supplied
by Dr. W. W. Anderson, Dept. of Microbiology, Oregon State Univ., Cor-
vallis, OR.
4/ VWR, P.O. Box 3200, San Francisco, Calif., Catalog No. 61845-009.
5
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(Dashed line components are for future development)
OXYGEN
MONITOR &
GENERATOR
PRESSURE
MONITOR
"1
INSULATED
MICROCOSM
CHAMBER
MICROCOSM
INSERT
HEAT
MONITOR a
CONTROL
I
CARBON
DIOXIDE
ADSORBANT
"1
__[__
Ti
Li_.
n_-^J
LIGHT
QUAL- I
I QUANTITY I
L I
. I
-r
f
Figure 2. Systems diaqram of the microcosm in its "life-support" system.
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Figure 3. "Life support" systems,
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Finure A. Sinole Reactor module,
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Figure 5. Close-up of reactor lid arrangement,
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GAS BURET
LEVELING
TUBE
SODIUM SULFATE
ELECTROLYTE
LEVEL
PL AT IN UM ELECTRODE
Figure 6. Diagram of hydronen Generator
electrolytic oxygen generator
bridge modification.
component of
showina salt
10
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GAS BURET
PLATINUM
ELECTRODE (+)
THREE-WAY
STOP-COCK
TO RESPIRATOR
Hg CONTACT
ELECTROLYTE
LEVEL
PLATINUM
ELECTRODE (-)
LEVELING TUBE
Figure 7. Diaqram of electrolytic oxygen generator.
11
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Biologically generated heat (in calories) was calculated and used in Equation
1:
Biologically Generated Heat AQ, K^ Atc Aty
From Test Microcosm = ^- = £- HC ^ H^ ^- (Eq. 1)
(in Calories) c
where: K, H.tT
l^ = rpp- and is evaluated during a calibration run, i.e.,
c c c using a non living microcosm,
H - v2
R Heating rate constant for resistor,
t = Time heating resistance is "on" in minutes,
T = Test reactor,
c = Control reactor,
Q = Heat output by microcosm in calories,
T = Experiment time in minutes,
v = Voltage at heating resistor in volts,
R = Resistance of heating resistor in ohms.
Carbon dioxide within the reactor was trapped in 10.0 ml of 0.60 N_ NaOH.
Every 24 or 48 hours during an experiment the NaOH in the vial was
withdrawn through a permanently placed needle and valve in the reactor vessel
top and titrated (Coleman, 1973). Data are presented as milliliters carbon
dioxide generated during the trapping period.
In an earlier version of the system, oxygen level maintenance and moni-
toring was performed with a hydrolytic oxygen generator similar to that
designed by McGarity, et. al., 1958 (Figure 7). Oxygen consumed during
microcosm respiration in the closed reactor system resulted in reduced intern-
al pressure activating an electrolytic switch that causes oxygen to be deliv-
ered to the reactor. Hydrogen is stoichiometrically generated at the cathode.
One-half of the hydrogen volume generated is equal to the oxygen produced at
the anode.
The system described here uses a sodium sulfate/agar salt bridge embedded
in fritted glass between the hydrogen and oxygen generating electrode,
eliminating fluctuations in the electrolytic switch due to barometric pressure
changes (Figure 6). A further modification in which oxygen will be generated
at the anode and copper plated out at the cathode is now being tested. Ozone
or other gases generated during the electrolytic reaction (Woodland, 1973) is
trapped in activated charcoal and nickel dibutyldithiocarbamate impregnated
glasswool located in a U-tube between the oxygen electrode and reactor
chamber. Any gas leaks in the slightly positive pressure maintained system
"as monitored with a closed manometer system (Figure 4 and 35).
12
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A detailed documentation of the "Life Support" system with scale drawings
is presented in Appendix D.
B. Experimental.
The experiments described are not intended to be comprehensive but
rather to illustrate principles of effects of natural and pollutant stress
variables on soil/litter microcosm systems.
Initially four "Life Support" Systems were constructed and experiments
were designed to observe (1) organismal populations succession between "ident-
ically" prepared and incubated microcosms, (2) variation in respiration, (3)
detection of the initial flush phenomenon, (4) effects of microcosm soil
moisture at 10, 30, and 60 percent of field water holding capacity on "indica-
tor" microcosms. Another experiment (5) was designed to study the effects on
soil respiration to treatment with three levels of cadmium (0.0, 0.01 and 10
mg CdCl2/kg dry soil). Subsequently, eight microcosm systems were prepared to
study the effects of (6) salt quality, i.e., the anions of chloride and
sulfate of sodium, and the cations of lithium and sodium (chlorides), (7) the
quantity at 0.132, 1.32, and 13.2 mM sodium chloride and 0.1761, 1.761 and
17.61 mM sodium sulfate, and three experiments to observe the respiratory
effects of the following trace elements at high naturally found concentra-
tions: (8) Cd, Cu, As, Se, Hg, Zn, Pb; (9) Ni, Cr, Co, Mn, Cd, Mo; and (10)
Ca, V , Li, Sn, La, Sb, and Ag.
1. Natural Variables.
Microcosm vessels were prepared to contain 150 grams dry weight (DW) of
homogenized and sifted soil, overlain with 15 grams (DW) of sifted litter.
Replicate or "identical" microcosms were prepared by simultaneously sifting
moisture-adjusted soil and litter components into a battery of microcosms
until a predetermined weight of material was added. Soil and litter was com-
pacted by setting a weighted cylinder on top of the material for one minute
after each addition of soil or litter. Microcosms were then monitored in the
soil ecosystem respirometer (SER) system to determine variability in respira-
tion rates. Reactors were maintained at 20° C in a constant temperature
room held at 17 to 18°C.
Surface spread plate counts of total heterotrophic bacteria and micro-
fungi were used to determine succession potential by counting in triplicate
on plates of 10-fold sterile phosphate buffer dilutions of agitated soil and
litter samples on the following media: Acidified Potato Dextrose Agar (Difco,
1958) for fungi, and Bunt and Rovira's medium (Bunt and Rovira, 1955) for
bacteria. Incubation was at 20°C for two weeks.
Analyses for soil and litter moisture were determined gravimetrically on
105°C oven-dried''samples. In the soil moisture experiment, soil was moistened
to the desired fraction of field water holding capacity prior to microcosm
loading.
13
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For the trace element and salt experiments, the test materials were
introduced into the microcosm by injecting an aqueous solution of the sub-
stance into the soil and litter at 45 points with a syringe. See Table 2 for
the chemical species and mean microcosm levels used. Also, see Appendix A
for further preparation details.
2. Pollutant Stress
To determine if the biological age of microcosms was an influential
factor on pollutant stress effects or on the establishment of controlled
populations of Collembola or mites, identical microcosms were prepared and
held at -4°C. After attaining the frozen state, groups were removed and
incubated and/or monitored for respiratory activity at 0, 5, and 15 or 20
days of age, respectively; then all groups except the controls were spiked
with CdCl2 at 1500 ppm. Effects on populations planted at 0, 5, and 15 days
were determined by intermittent and final destructive harvests.
Trace element quality, quantity and combination effects were assessed by
spiking the prepared microcosms with aqueous solutions of the candidate
pollutants at controlled concentrations either singly or in combination.
Spiking (injection) techniques are described in Appendix A.
14
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III. RESULTS AND DISCUSSION
A. Natural Variables.
In most instances the data will be presented and discussed in the integ-
rated form, that is, as the slope of the summed measurements over time, e.g.,
the slope of the summation of oxygen consumed or carbon dioxide produced,
over time in a microcosm. These slopes indicate production rates. When
presented, populations of organisms will be referred to in differentiated
terms or numbers of organisms.
Due to the inadequacy of the biogenic heat measuring system at this
writing it will be discussed only briefly.
1. Succession.
Population densities of microorganisms, particularly the bacteria in the
microcosms, change dramatically immediately after microcosm preparation
(Figure 8a). There is a rapid increase in the first 10 days -- from 107 to
108 bacteria/gm (DW) of soil or litter. The population remains almost con-
stant at the higher level for at least 20 days longer.
This rapid microbial growth is also reflected in an initial rapid flush
of C02 and uptake of 02 in the first 10 days in the life support systems
(Figures 9 and 10).
Jenkinson and Poulson (1976) found a similar initial flush and attributed
it to disruption of previously inaccessible microbial nutrients released from
destroyed organisms as a result of soil manipulation. In any event, it
appears that relatively stable populations within the microcosms prevail 10
days after their preparation.
Therefore, all experiments were subsequently initiated only after a 10
day pre-incubation of the prepared microcosms. It was reasoned that the 10
day "flush" was an adjustment period and unless the interest was studying the
effects of stressants on adjustment in the microcosms, the adjustment should
be allowed to proceed to "completion" before stressing the systems. This
would more closely test the stress effect which occurs in nature most of the
time.
2. Microcosm Variation.
Specific experiments to test inter-microcosm variation and those portions
of stress testing experiments with like treatments were used to evaluate the
variation that occurs within and among "identically" prepared and incubated
microcosms (e.g., Figures 11 and 12).
15
-------
10'
10
0 0 ppm Cd Litter
0 01 ppm Cd
10 0 ppm Cd
Soil
(e)
200 ""400 "600
10'
10
10'
O
10'
I01
10
Litter
Soil
Litter
Soil
(b)
10%
(d) :
0 00 ppm Cd
° 0-01 ppm Cd
' 10-0 ppm Cd
Litter
Soil
(f)
EXPERIMENT TIME (hr)
0 200 400 600
Figure 8. 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 microcosws incu-
bated 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 CdCl2 concen-
tration,respectively (e & f).
16
-------
u
X
o
_J
o
100
80
60
40
20
0
O A,
i A D
,D
§
0
200 400
EXPERIMENT TIME (hr)
600
Figure 9. Accumulated oxygen consumed through time in thre-e
"identically" prepared and incubated coniferous
forest soil/litter microcosms.
17
-------
*
E
:
h-
O
1-
OJ
O
O
160
140
120
100
80
60
40
20
n
o u
o
o
O . A
O A Q
0 ° A A °
n ° A D D
O AD
0 A 0
O A ^
° A
0 A
o
n
° 6
O A
OA
CD A
_ O
1 1 1 1 1
0
200 400
EXPERIMENT TIME (hr)
600
Figure 10. Accumulated carbon dioxide produced through tine
in three "identically" prepared and incubated
coniferous forest soil/litter microcosms.
-------
600
£400
x
o
200
o
°
0
CO
200 400
EXPERIMENT TIME (hr)
600
Figure 11. Accumulated oxygen consumed through time in the 8 indicated
and "identically" prepared and incubated coniferous forest
soil/litter microcosms.
e
u
Q
X
O
Q
2
O
CD
CC
O
_l
1-
o
t-
^uu
300
200
100
Q
£f
fa. jf^ 'fff
A &--
A 9 V /-
A 5 \|
A ni i
A G
A 9
A 9
6
9
1 I 1 l I I
0 200 400 600
EXPERIMENT TIME ( hr)
Figure 12. Accumulated carbon dioxide produced through time in the 8
indicated, and "identically" prepared and incubated coni-
ferous forest soil/litter microcosms.
19
-------
The results of 29 experiments (Table 1) show that the mean rates of
oxygen consumption and carbon dioxide production are 0.54 and 0.43 ml/hr,
respectively, for 150 gm (DW) soil plus 15 gms (DW) litter. The variances of
0.05 ml/hr for 02 consumption and 0.03 ml/hr for C02 production indicate that
seasonal changes of metabolism in natural soil are undetectable in these
systems after preparation and pre-incubation in the microcosms for 10 to 14
days.
The large variance for the 02 consumption rate is due to the relatively
less sensitive method of measuring 02 than C02. It is anticipated that
system improvement will reduce this variance.
The respiratory quotient (RQ) of 0.80 (Table 1) indicates the mean
metabolism of compounds is at the protein to lipid oxidation state (White,
Handler and Smith, 1973, p. 283).
The coefficients of variation (CV) for the 02 consumption and C02
production rates provides a means to estimate a significant difference between
the control rates and the test rates in any single set of microcosms, i.e.,
the CV's from these data can be used to determine what may be called a 95
percent confidence level about a test set control rate (Eq. 4).
P{ (-2*CV.) * microcosm < Microcosm < (2*CV.) * microcosm} = 0.95 (Eq. 4)
i I
where i is the CV as calculated from the data found in Table 1.
3. Soil Moisture.
The effect of soil moisture on microbial populations is shown in Figure
8c and 8d where the tendency is for the bacterial populations to prevail
under moist conditions and fungi under drier conditions.
The integrated biological activity as deduced from oxygen consumption
and carbon dioxide production (Table 2, Figures 13 and 14) shows a marked
increase with increased soil moisture, i.e., respiration was two to three
times faster at 60 percent than 10 percent soil moisture.
From the RQ's (Table 2) it is seen that at 30 and 60 percent soil moist-
ure, respiration indicates a "usual" oxidation process compared to other
results in Table 2, but that at 10 percent a more oxidizable substrate may be
metabolized. These observations may be correlated with the relatively high
fungal activity at low soil moisture and their sacdiarophilic reputation.
4. Salt Effects.
As seen in Figures 15, 16, 17 and 18 and tabulated in Table 2, the
quality and quantities of salts, or rather anions, may markedly inhibit
community respiration in the coniferous forest soil/litter microcosms. The
respiratory relationship of C02 production and 02 consumption is not affected
by salt treatment as indicated by the small differences in RQ's between
microcosm treatments. However, there is a substantial anion effect that is
20
-------
TABLE 1. MEAN (BAR); STANDARD DEVIATIONS (S) AND COEFFICIENTS OF VARIATION (CV) FOR OXYGEN CONSUMPTION
(Xi), CARBON DIOXIDE PRODUCTION (X2), AND RESPIRATORY QUOTIENT (Xj/X2) FOR ALL OF NON TREATMENT
PORTIONS WITHIN AND AMONG "IDENTICALLY PREPARED AND INCUBATED SETS OF CONIFEROUS FOREST SOIL/
LITTER MICROCOSMS
Experiment Number
Number in Set
13 7
14 8
18 2
18 4
22 8
Total Number
Mean of Means
S of Means
c
v v
Ai Ai
0.55 y 0.07
0.48 0.06
0.46 0.00
0.55 0.08
0.59 0.05
29
0.54
0.05
CVy
C%fl
12.9
12.0
0.9
14.8
7.8
29
10.7
3.7
h
0.41 -1
0.40
0.39
0.45
0.47
29
0.43
0.03
SX2 C$2
^0.01 1.7
0.01 4.0
0.01 3.4
0.03 6.5
0.02 4.5
29
3.9
1.5
-------
TABLE 2. TABULATION OF RESPIRATORY RATES FOR "IDENTICALLY" PREPARED AND INCUBATED CONIFEROUS FOREST
SOIL/LITTER MICROCOSMS WITHIN EACH EXPERIMENT, AND SUBJECTED TO THE INDICATED TREATMENTS.
Microcosm
Treatment
Experiemnt 14
Control
CdCl2
L»UL» 1 r\
Na9HAsOA
SeO? 4
HgCT?
ZnCK
PbCl2
Experiment 16
Control
NiCl9
CrCi;
CuCl9
MnCl
CdCl
MOO/
Experiment 22
Control
CaCl2
VoOc
Ly
SnCl?
LaCK
Mean
Microcosm
Concentration
(ppm1)
0
1500
3000
1000
680
30
7500
200
0
700
100
50
850
1500
10
0
.6131
60
100
10
100
Oxygen
Consumption Rates
(ml/hr)
Pre-2 Post-2
0.508
0.499
0.537
0.550
0.440
0.376
0.432
0.518
0.585
0.591
0.611
0.581
0.573
0.545
0.449
0.196***
0.416
0.526
0.250***
0.345
0.148***
0.414
0.484
0.250***
0.385
0.380
0.252***
0.217***
0.540
0.510
0.235***
0.481
0.331**
0.503
0.425
Carbon Dioxide
Production Rate
(ml/hr)
Pre- Post-
0.385
0.397
0.402
0.403
0.368
0.391
0.404
0.413
0.498
0.472
0.469
0.472
0.438
0.468
0.361
0.200***
0.184***
0.364
0.213***
0.276***
0.109***
0.348
0.356
0.174***
0.255***
0.292***
0.206***
0.165***
0.414
0.397
0.164***
0.379***
0.264***
0.385
0.355***
Respiratory
Quotient
(C0?/09)
Pre- Post-
0.76
0.80
0.75
0.73
0.84
1.04
0.94
0.78
._._
0.77
0.80
0.77
0.81
0.77
0.86
0.80
1.02
0.44
0.69
0.85
0.80
0.74
0.84
0.74
0.70
0.66
0.77
0.82
0.76
0.77
0.78
0.70
0.79
0.80
0.77
0.83
-------
TABLE 2 (CON'T)
OJ
Microcosm
Treatment
SbCl,
AgNO^
Experiment 133
fnntrnl 1
fnntvnl 7
rnrrf-ynl A
rnrrhvnl ^
fnntrnl fi
Control 1
fnntrnl R
Experiment 3
Mn i c "f~ 1 1 PP»
Mn i c i" r i v£»
Experiment 18a3
Experiment 18b
rl 1 Li UuUolM 0
ivi i crucuoMi D
rl 1 CrULUoin /
M-i f*v*r\r*rtc-m Q.
Mean
Microcosm
Concentration
(ppm1)
10
10
i n°/
Qn°/
fin0/
Long term variation
Short term variation
Oxygen
Consumption Rates
(ml/hr)
Pre-2 Post-2
0.532 0.457
0.683 0.606
n Rfifi
n t^RQ
U . JO;?
n fifi?
n c;i 7
U. D 1 /
n AA7
n d7^
n ^7 - - -
n md -
n n^n
n i?2
(900 hrs)
n ASR
n dfin --
n RDR
n ARQ
n fiifi -- --
n fi?7
Carbon Dioxide
Production Rate
(ml/hr)
Pre- Post-
0.459 0.400
0.506 0.442***
n A07
n /n 7
U . H- 1 /
n AIR __ _
n ADA
u . tut -
n An? - - -
n Aifi -
n An7
n n?s --
n n^fi
n ncn -
n -577
n TQC;
n A?R
n API
n AAR
n ARQ
Respiratory
Quotient
(C0?/0?)
Pre- POst-
0.86 0.88
0.74 0.73
n 71 7
n 7Afi
n F>II
n 7Ri
n Qnn
n R7Q
n 7^R
i in
n 7fi
n 75
n R? __ _
n Rfi
n Rfi
n Q?
n 7^
n 7R
-------
TABLE 2 (CON'T)
Microcosm
Treatment
Experiment
Na SO
Na£sO?
Na^SoJ
NaCI
NaCI
NaCI
LiCl
Mean
Microcosm
Concentration
(ppm1)
24
0.
1.
17.
0.
1.
13.
552.
Mean
Variance
Coe. of
1761 mM
761 mM
61 mM
132 mM
32 mM
2 mM
0 ppm
Variation (%)
Oxygen
Consumption Rates
(ml/hr)
Pre-2 Post-2
0.430
0.459
0.534
0.516
0.404
0.430
0.481
0.540
0.474
0.052
11.0
0
0
0
0
0
0
0
0
0
0
29
.417
.361
.308
.444
.229
.185
.319
.473
.342
.102
.8
Carbon Dioxide
Production Rate
(ml/hr)
Pre- Post-
0.408
0.414
0.446
0.401
0.386
0.382
0.470
0.422
0.416
0.030
7.2
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
27.
338
282
235
347
199
145
293
357
275
076
7
Respiratory
Quotient
(C0p/0p)
Pre- POst-
0.
0.
0.
0.
0.
0.
0.
0.
95
90
84
78
96
89
98
78
0.81
0.78
0.76
0.78
0.87
0.78
0.92
0.76
1 As metal or non metal ** >_ 2 Coefficients of variation from experiment control.
2 Pre- and Post-pollutant treatment *** ^3 Coefficients of variation from experiment control.
3 Rate calculated from entire experimental time.
-------
LU
X
O
o
0
200 400
EXPERIMENT TIME
600
(hr)
Figure 13. Accumulated oxygen consumed through time in three
"identically" prepared and incubated coniferous
forest soil/litter microcosms treated to an initial
soil moisture of (a) 10%, (b) 30%, (c) 60% of field
holding capacity.
25
-------
E
N"~ **
<-
h-
O
1
CM
O
O
IDU
140
120
100
80
60
40
20
n
C) _
n
0
QDa
a D
0°
w
_ D A A A
D A A A
°D AAAA o
Q A AA O
" g^'tto0000000000
So ° i i i i i i
0
EXPERIMENT TIME (hr)
Figure 14. Accumulated carbon dioxide produced throuqh time
in three "identically" prepared and incubated
coniferous forest soil/litter microcosms treated
to an initial soil moisture of (a) 10%, (b) 30%,
(c) 60% of field holding capacity.
-------
500
3 400
LJ
X
O
h-
o
300
200
ofcA-
o
Salt
added
200 400 600
EXPERIMENT TIME (hr)
CONTROL
O.I32mM
l.32mM
!3.2mM
800
Figure 15. Accumulated oxygen consumed through time in "identically" proc-
essed coniferous forest microcosms treated to a final mean indi-
cated concentration of sodium chloride.
1 400
LU
O
x 300
O
O
CO
a:
o
100
0
CONTROL
g O.I32mM
n
A
200 400 600
EXPERIMENT TIME (hr)
800
Figure 16.
Accumulated carbon dioxide produced through time in "identically"
processed coniferous forest microcosms treated to a final mean
indicated concentration of sodium chloride.
27
-------
500
J 400
z
UJ
& 300
X
_, 200
h-
o
t- 100
-A CONTROL
+r
^j* ^O.I76lmM
^^T <2JSRL76lmM
added ^J^'^ QS^^^
i ^^j[ wO^h
l**Sfo ^^
t£JP^
dtffi
\4 1 1 1 1 1 1 1 J
0 200 400 600 800
EXPERIMENT TIME (hr)
Figure 17. Accumulated oxygen produced through time in "identically" proc-
essed coniferous forest microcosms treated to a final mean indi
cated concentration of sodium sulfate.
~ 400
£
UJ
2 300
X
o
o
z 200
O
CD
(T
<
0 100
_l
<
1-
o
I- n
1-
CONTROL
OO./76/mM
O ._, O/.76/ mM
. 8 2 5 A A 17.61 mM
o -» i * 9 U t\
c>0 IT A Q n 2S
added « B E
1^ 3) Ot
ffl
.a8
- *6*
- A4
l_l
A
-E 1 1___ 1 I 1 i I i
200 400 600
EXPERIMENT TIME (hr)
800
Figure 18. Accumulated carbon dioxide produced through time in "identically"
processed coniferous forest microcosms treated to a final mean
indicated concentration of sodium sulfate.
28
-------
seen as a more pronounced decrease in respiration in the Nad as opposed to
Na2S04 treated microcosms. On a molar basis it might be a logarithmic func-
tion of salt concentration (Figure 19). From this experiment one could
conclude that a large portion of the effects of toxic trace substances added
to soils may not be due to the toxicity of the trace substance but rather an
effect of a high salt concentration. Of course, a combination of high salt
and presence of specific toxic substances may synergize their individual
effects resulting in more than additive respiratory inhibition.
Further comparison of Na2S04 treatment salt effects (Figure 19) with a
6.7 mM MgSOi^ treatment gives a 25 and 33 percent reduction in respiration,
respectively, whereas there is an 8 percent difference based on molarity. In
further calculations based on ionic strength, there is only a 3 percent
difference. It appears that at least at high toxicant levels a salt effect
may be the dominating process causing respiratory loss in the soil community.
5. Biogenic Heat Production.
Biogenic heat produced by the microcosm community in the "salts effects"
experiments was detected (Table 3) where positive values are shown for all
changes in heat production rates. However, the trends in the changes in the
heat and C02 production rates do not correlate for both Nad and Na2SOt+
series as one might expect. Further, the coefficient of variations for the
pre- and post-treatment of biogenic heating rate are the same, indicating
only random variation causing rate changes. It is reasoned that the
amount of biogenic heat produced by the microcosms is small compared to the
total heat input to the system as seen from equation (1). The calculation
of biogenic heat is markedly subject to small errors in measurement of heating
resistor on-time heat input. Thus, it is shown that the quantification of
biogenic heat is still uncertain. Efforts are underway to solve this problem.
B. Pollutant Stress.
The desired microcosm biological age at which to add pollutant stresses
(Section III. B.I) was determined and the effects of the quality (Section III.
B.2), quantity (Section III. B.3), and combinations (Section III. B.4) of
pollutants are illustrated.
1. Microcosm Biological Age.
When microcosms are treated with 1500 ppm Cd at 0, 5 and 20 days incu-
bation after preparation 02 consumption and C02 production is reduced 22 - 30-
30% and 38 -43-35%-, respectively (Table 4, Figure 20). Based on the RQ data
which remains relatively constant at 0.71 - 0.75 there is no obvious relation-
ship between biological age and respiration. Because no regular pattern in
the microbial population stress effect with microcosm age has been detected,
we assume that the time of stress on microcosm respiratory activity is rela-
tively small.
For 25 adult Collembola (Isotoma sp.), and approximately 3000 free
living nematodes added to these same microcosms, the time after pollutant
addition was critical. That is, pollutant added similtaneously with the
29
-------
100.0
10.0-
5
E
-10 0 10 20 30 40 50 60
RELATIVE DECREASE IN 02 (- -) C02() RATE (%)
Figure 19. Salts (NaCl, Na2S04) effects (compared to initial
control rate) on respiration in "identically"
prepared coniferous soil/litter microcosms treated
to the indicated final mean salt concentration.
30
-------
TABLE 3. TABLE SHOWING COMPARISON OF CARBON DIOXIDE PRODUCTION RATE AND BIO-
GENIC HEATING RATE (AS INDICATED BY MICROCOSM HEATER ON-TIME RATE)
CHANGES DUE TO MICROCOSM TREATMENT WITH VARIOUS SALT CONCENTRATIONS.
Biogenic Heat
Microcosm
On-Time
Pre-
Treatment Treatment
No Microcosm
H20
0.1761 mM Na2S04
1 .761 mM Na2S04
17.61 mM Na2S04
0.132 mM NaCl
1.32 mM NaCl
13.2 mM NaCl
552.0 ppm LiCl
Mean
Variance
Coe. of Variation (%)
0.700
0.287
0.414
0.414
0.315
0.337
0.404
0.466
0.369
0.375
0.060
16
Heater
Rate (hr/hr)
Post-
Treatment
0.700
0.296
0.450
0.477
0.394
0.427
0.477
0.513
0.404
0.432
0.071
16
Change
0
3
9
15
25
27
18
14
9
CO^
Microcosm
Production
Pre-
Treatment
0
0.422
0.408
0.414
0.446
0.401
0.386
0.382
0.470
0.416
0.030
7
Production
C0? Rate
(hr/hr)
Post-
Treatment
0
0.355
0.338
0.282
0.235
0.347
0.199
0.145
0.293
0.274
0.076
28
Change
0
15.8
17.0
32.0
47.3
12.9
48.5
62.0
37.6
31
-------
TABLE 4. RESPIRATORY MEASUREMENTS OF "IDENTICALLY" PREPARED CONIFEROUS FOREST SOIL/LITTER MICROCOSMS
TREATED AT THE INDICATED BIOLOGICAL AGES TO 1500 PPM CdCL2>
00
ro
Oxygen
Microcosm Biological
Age at Treatment
(Days)
0
5
20
Consumption
(ml/hr;
Control
0.55
0.46
0.43
Rate
)
Test
0.43
0.32
0.30
Difference
From
Control
(*)
-22
-30
-30
Carbon Dioxide
Production Rate
(ml/hr)
Control Test
0.45 0.28
0.40 0.23
0.34 0.22
Difference
From
Control
(*)
-38
-43
-35
Respiratory
Quotient
(co?/o?)
Control Test
0.81 0.75
0.86 0.71
0.80 0.74
-------
^"*
E
LU
Q
X
O
Q
Z
O
CD
a:
o
o
H
400
350
300
250
200
150
100
50
o.
Control
0 *
*
2O * A A ^"O Pays
1 -*A
^
4
4 Q O -5 Pays
A o 8
O A Q, O O Days
ff 9 Q. "
\ + e i 8
JA 9
fl°
1 1 1 1 1
0 200 400 600 800 1000
EXPERIMENT TIME (hr)
Figure 20. Accumulated carbon dioxide produced through time
from "identically" prepared and incubated conif-
erous forest soil/litter microcosms treated to
1500 ppm CdCl2 at either 0, 5 or 20 days of bio-
logical age.
33
-------
organisms (0 day experiment) or within 1 week (5 day experiment) showed
little or no growth of Collembola and a 98 percent decrease in nematodes
after 20-25 days incubation (Table 5, Figures 21 and 22). Later pollutant
additions resulted in the same marked population reduction effects. It
appears that this pollutant stress has the same general characteristics for
both animals regardless of the biological age of the system: cessation of
reproduction and death of adults.
2. Trace Element Quality.
Statistically significant (Table 2) effects of particular trace elements
added as single salts to the microcosm system show only an inhibition of C02
production by Cu, Hg, Cr, Co, V, and La while As showed a stimulation, whereas
both C02 production and 02 consumption were inhibited by Cd, Se, Zn, Ni, Mn,
and Li. It is possible that oxygen consumption is also inhibited for the
former group but the analytic methods cannot detect the change. It does
appear that the statistical method used to limit significance in this case
causes this result because the RQ's appear to be logical, i.e., about 0.7 to
0.8. Additionally, none of the RQ's except Experiment 1 Cd and Co are outside
two coefficients of variation (see Table 1) from experiment controls.
Finally, graphical interpretation of Figures 23, 24, 25, and 26 show
possible additional inhibitory effects of Cr, Hg, and Co on oxygen consumption
and stimulation of oxygen consumption and carbon dioxide production by
As and Mo. Copper causes an initial stimulation after pollutant addition
and subsequently an inhibition of oxygen consumption and C02 production.
In conclusion it-appears that trace elements found in real world
levels can and probably do inhibit soil decomposition processes as seen by
reduced respiratory activity.
3. Trace Element Quantity.
Respiratory response (Table 2, Figures 27 and 28) but not detectable
microbial response (Figure 8e, f) varied with amount of cadmium added to
microcosms and with time of observation. At low cadmium concentrations (0.0]
ppm CdCl2), a transient stimulatory effect (increased respiratory rate) was
noted for the initial 200 hours after amendment, followed by recovery to
control rates. Respiration at high cadmium concentrations (10 ppm CdCl2)
equaled that of the control for approximately 300 hours after amendment;
thereafter, respiration decreased by 40 percent compared to the control,
(Bond, et^ al_, 1975, 1976). The mechanism of the dramatic loss of activity at
high cadmium levels is unknown. There was no decrease in numbers of cultur-
able soil or litter fungi or bacteria, although there was a relative increase
in oxygen consumed over carbon dioxide produced (Figure 29) suggesting un-
coupling of oxidative phosphorylation; a common heavy metal effect (White, e_t
al., 1973). Stimulation at low levels and inhibition at higher levels by
heavy metals is a common microbial response to heavy metals, and is referred
to as oligodynamic action (Salle, 1973) or the Arndt-Schulz Law (Lamanna and
Mallette, 1965, p. 897).
34
-------
TABLE 5. TABULATION OF THE SURVIVAL OF NEMATODES (CEPHALOBUS PERSEGNIS) AND COLLEMBOLA (ISOTOMA SP.)
ADDED TO CONIFEROUS FOREST SOIL/LITTER MICROCOSMS AT THE INDICATED AGES, AND TREATED TO 1500
OJ
en
PPM CdCL2 AT THE INDICATED
MICROCOSM AGES.
Kind of Organisms
Added to Microcosms Number
Arthropods Nematodes Added
+ - 25
+ - 25
+ - 25
+ - 25
+ - 25
+ 3400
+ 3500
+ 3000
+ 3000
+ 3000
Microcosm Age
at Stress
% Viable After
Indicated Microcosm Age
(Days)
Animals
5
0
0
0
0
5
0
0
0
0
CdCl?
-5
0
5
15
NA
-5
0
5
15
NA
0
100
100
100
100
100
100
100
100
100
100
5
60
60
96
66
66
19
34
31
37
37
[Days)
15
28
332
132
132
0.9
18
60
60
20
12
--
--
_ _
0.5
--
--
25
--
116
1100
1780
_ _
3
12
43
35
--
--
400
2140
--
16
44
-------
100
5 10 15 20 25 30
EXPERIMENT TIME (DAYS)
35
Figure 21. Survival of 0, 5 and 15 day old collembola populations in
"identically" prepared coniferous forest soil/litter micro-
cosms treated to 1500 ppm CdCl2.
36
-------
0
5 10 15 20 25 30
EXPERIMENT TIME (DAYS)
35
Figure 22. Survival of nematodes in 0, 5, and 15 day old
"identically" prepared coniferous forest
soil/litter microcosms treated to 1500 ppm CdCl2.
37
-------
DUU
f 400
0 300
x
0
_i 200
0
h 100
°c
X"' ' (IOOO ppm)
^*Cu
g *
8
*
* l 1 1 1 1 I 1 1 1 1
D 200 400 600 800 IOOO
EXPERIMENT TIME (hr)
Figure 24. Accumulated carbon dioxide produced through time in "identically"
prepared and incubated coniferous forest soil/litter microcosms
treated to a mean final concentration of the indicated heavy
metals (as salts).
38
-------
500r
CJ
200 400
EXPERIMENT TIME
600
(hr)
Mo (IOppm)
« Control (Oppm)
CD Cr (IOO ppm)
Co (5Oppm)
Ni (TOO ppm)
CD Mn (85O ppm)
800
Figure 25. Accumulated oxygen consumed through time in "identically" prepared
and incubated microcosms treated with the indicated trace elements.
-------
- 300
E
Q
X
0 200
Q
"ZL
O
CD
o:
S 100
h-
o
i
i
r\
V
v
Pollutant *
Addition v a
1 v - 8 °
1 : s o o °
v JJ o
- i **
m
i i i i
w"*
v
W A
v A a
/» a
w ffi
A °
^ A^ 'D
ID (5O ppm)
(IOO ppm)
o Mn (85Oppm)
O Ni (7OO ppm)
0
200 400 600
EXPERIMENT TIME (hr)
800
Figure 26. Accumulated CP2 produced through time in "identically" prepared and
incubated microcosms treated with the indicated trace elements.
-------
- 100
E
LU
e?
o
80
x 60
O
40
20
0
0
200 400 600
EXPERIMENT TIME (hr)
Figure 27,. Accumulated oxygen consumed through time in three
"identically" prepared and incubated coniferous
forest microcosms treated to a final mean concen-
tration of (a) 0.0 ppm, (b) 0.01 ppm, (c) 10.0 ppm
cadmium chloride.
-------
O
160
140
120
100
80
cvj 60
O
0 40
20
0
6
0 200 400 600
EXPERIMENT TIME (hr)
Figure 28. Accumulated carbon dioxide produced through time
in three "identically" prepared and incubated
coniferous forest microcosms treated to a final
mean concentration of (a) 0.0 ppm, (b) 0.01 ppm,
and (c) 10.0 ppm cadmium chloride.
42
-------
LJ
UJ
>
_J
LJ
cr
l-40r-
1-20 -
1-00
0-80-
Relative to
control
0-60
0-00 0-01 0-10 1-00 10-0
MEAN MICROCOSM CONCENTRATION
(/zg/gm) OF CADMIUM (ADDED AS CHLORIDE)
Figure 29. Graph of the relative oxygen consumption (*),
and carbon dioxide generation (0) rate values
for the initial 200 (solid lines), and subse-
quent (dashed lines) hours after cadmium addi-
tion (mean microcosm concentration) to Douglas
fir soil/litter microcosms.
43
-------
£
LJ
X
0
1
f
0
600
500
400
300
200
100
n
-
55 CADMIUM
Q& ^SELENIUM
c^d0 & CONTROL
ccsP^gB CADMIUM a
^P sffr ^ ^ SELENIUM
A\^~ *~~ ~
d^8
& 3ri
. ^ POLLUTANT
aft*^ ADDITION
oP^ i 1 i i 1
200 400 600 800
EXPERIMENT TIME (hr)
1000
Figure 30. Accumulated oxygen consumed throuqh time in four "identically"
prepared and incubated coniferous forest microcosms treated to
a mean final concentration of 0.0 ppm cadmium chloride or sele-
nium oxide, 25 ppm cadmium chloride, 10 ppm selenium oxide, and
both
E
1
_J
h-
O
h-
CVJ
0
O
600
500
400
300
200
100
r>
<-
-
-
Q
O
oD**
8»
L Be» t
8 POLLUTANT
« ADDITION
9
i i
o °0SELENIUM
o° a ^CONTROL
o ngSl * * CA SELENIUM
no°n i»
o2oS4
D 4^
O £ *
e8
i i i
0 200 400 600 800
EXPERIMENT TIME (hr)
1000
Figure 31. Accumulated carbon dioxide consumed through time in four "identi-
cally" prepared and incubated coniferous forest microcosms treated
to a mean final concentration of 0.0 ppm cadmium chloride or sele-
nium oxide, 25 ppm cadmium chloride, 10 ppm selenium oxide, and
both.
44
-------
4. Trace Element Combinations.
In the combined selenium/cadmium experiment, as with the high cadmium at
three levels experiment (Section III. B.3), there was a delay of several
hundred hours before an inhibitory effect on oxygen consumption was observed
(Table 2, Figures 30 and 31). In the selenium-only treatment, effects were
seen almost immediately after pollutant amendment. The interaction effect of
selenium and cadmium on oxygen consumption rate was 55 percent greater than
when either of the metals where added singly. The reduced respiration in the
control reactor is presumably due to the inadvertent deletion of distilled
water at the time of pollutant addition. Previous results (e.g., see Figures
27 and 28) indicate respiration remains relatively constant with water addi-
tion to the control microcosm.
The point to be drawn from this experiment is that combinations of trace
elements may well have unexpectedly severe effects on soil decomposer pro-
cesses!
C. Interpretation
In lieu of contamination of natural study sites, pollutant materials
research in microcosms appears to be an acceptable alternative. From a
statistical point of view, the so-called "integrator-indicators" of respira-
tion, i.e., oxygen consumption and particularly carbon dioxide reduction,
allow detection of significant treatment effects in microcosms with reasonable
precision, e.g., 10.7 and 3.9 percent coefficient of variation, respectively.
Even the "differentiated-indicators," i.e., organism populations, show reason-
able similarity in density through time for microcosms up to six weeks old.
The similarity between microcosms is dependent upon careful attention
paid to detail in microcosm preparation. A battery of inserts are prepared
simultaneously. All manipulations that can be carried out with the whole
homogenous substrate(s) are performed before microcosm vessel preparation!
Subsequent treatments are as alike as possible!
Microcosms treated to natural variables respond respirometrically and
organismally similar to what is thought to occur in nature. The effect of
moisture reduction from 60 to 10 percent of field water holding capacity both
reduces respiration dramatically and shifts the microcosm population from the
less xerophilic bacteria to the more xerophilic fungi. The drier conditions
also selected for a more saccharolytic-like microflora.
As also might be expected, increased salt treatment of_the microcosm
alters respiration.- Both quality of salts, e.g., Cl" or S04, and quantity
affect respiration. From our experiments with coniferous forest soil and
litter, the chloride ion is significantly more toxic than sulfate ion; this
toxicity increases from 18 to 2400 ppm. The relationship of toxicity to
concentration appears to be logarithmic.
Finally, the successional quality of the microcosm immediately after
preparation also approximates what might be thought to occur after disturbance
in nature by a natural phenomenon. Thus, there is a marked flush of gases
45
-------
(02 and C02) immediately after microcosm preparation which is due to the
rapid growth of the populations, particularly microorganisms, utilizing
nutrients liberated from previously inaccessible microsites in the soil, and
those liberated from organisms damaged during the soil manipulation process.
The respective effects of trace element pollution on the forest soil/
litter microcosms is a function of at least their quality, quantity, and
combinations. What the salt effects of the added pollutants are on respira-
tion is problematical but is likely to be significant as indicated by our
measurements.
Most of our trace element studies showed respiratory effects at concen-
trations found in nature, e.g., Cd, Cu, Se, Hg, Zn, Ni, Cr, Co, Mn, V, Li,
La, and Ag. Cadmium, the only element tested so far, showed an Arndt-Schulz
Law effect in that a stimulatory effect on respiration was seen at low concen-
trations and inhibition at high concentration.
Combinations, such as Cd and Se show a greater than added inhibitory
effect when compared to the individual elements. The mechanism of this
effect is unknown.
46
-------
IV. Future Research
The survey of soil pollution using the microcosms will continue. Levels
and combinations of materials will be tested in the near future. In order to
identify specific soil ecosystem mechanisms an artificial soil system with
known biological and chemical constituents is to be prepared and eventually
used in the microcosm test system. The test system will also act as the
prototype for a predictive computer simulation model that will be used to
test for differences between predicted and observed microcosm treatment
results.
47
-------
V. References
Ausmus, B. S. and M. Witkamp. 1974. Litter and Soil Microbial Dynamics in a
Deciduous Forest Stand. EPFB-IBP-73-10, UC-48-Biol. and Med. Oak Ridge
Natl. Lab., Oak Ridge, Tenn. p. 183.
Bojsova, D. 1963. The Effects of Arsenic on the Bacteriologic and Biologic
Life in the Soil and on its Self-Purifying Faculty. Cesk. Hyg. Prague
8:6:377-382.
Bond, H., B. Lighthart, R. Shimabuku, and L. Russell. 1976. Some Effects of
Cadmium on Coniferous Forest Soil and Litter Microcosms. Soil Sci.
121(5):278-287.
Bond, H., B. Lighthart, R. Shimabuku, and L. Russell. 1975. Some Effects of
Cadmium on Coniferous Forest Soil/Litter Microcosms. EPA-660/3-75-036.
Northwest Environmental Research Laboratory, U.S. Environmental Protec-
tion Agency, Corvallis, Oregon.
Bunt, J. S. and A. D. Rovira. 1955. Microbiological Studies of Some Subant-
arctic Soils. J. Soil Sci. 6:119-128.
Coleman, D. C. 1973. Soil Carbon Balance in a Successional Grassland. OIKOS
24:195-199
deJong, L. and E. denDoren. 1971. Tolerance of Azotobacter for Metallic and
Non Metallic Ions. Antonie Van Leeuwenhoek. J. Microbiol. Serol.
37:119-124.
DIFCO, DIFCO Manual (DIFCO Laboratory, Detroit, Michigan, 1958).
Gist, C. S. 1972. Analysis of Mineral Pathways in a Cryptozoan Foodweb.
Eastern Deciduous Forest Biome, Memo Rep. 72-23. Coneeta Research Site,
Inst. Ecology, Univ. Georgia, Athens, pp.151.
Hafkenschield, H. H. M. 1971. Influence of Cu ions on Trichodorus pachy-
dermus and an Extraction Method to Obtain Active Specimens. Nematologica
17:4:535-541.
Hartman, L. M. 1974. Fungal Flora of the Soil as Conditioned by Varying
Concentrations of Heavy Metals. Amer. J. Bot. 6:5:23.
Jackson, D. R. and A. P. Watson. 1976. Disruption of Macronutrient pools in
Forest-floor Litter Near a Lead Smelter, (personal communication).
48
-------
Jenkinson, D. S. and P. S. Poulson. 1976. The Effects of Biocidal Treatments
on Metabolism in Soil - I. Soil Biol. Biochem. 8:167-177.
Klein, D. H. and P. Russell. 1973. Heavy Metals: Fallout Around a Power
Plant. Environ. Sci. Tech. 7:4:357-358.
Lamanna, C. and M. F. Mallette. 1965. Basic Bacteriology. 3rd Ed., The
Williams and Wilkins Co., Baltimore, pp. 1001.
Linzon, S. N., P. J. Temple, R. G. Pearson, M. L. Smith, and B. H. Chai.
1975. Lead Contamination of Urban Soils and Vegetation by Emission from
Secondary Lead Industries. Proc. 68th Pollution Control Assoc. Paper
7518.2
McBrayer, J. F., D. E. Reichle, and M. Witkamp. 1974. Energy Flow and
Nutrient Cycling in a Cryptozoan Food-Web. EDFB-IBP-73-8, UC-48-Biol.
and Med., Oak Ridge Natl. Lab, Oak Ridge, Tenn. pp.78.
MacFadyen, A. 1970. Simple Methods for Measuring and Maintaining the Propor-
tion of Carbon Dioxide in Air, for Use in Ecological Studies of Soil
Respiration. Soil. Biol. Biochem. 2:9-18.
McGarity, J. W., C. M. Gilmore, and W. B. Bollen. 1958. Use of an Electro-
lyte Respirometer to Study Denitrification. Can. J. Microbiol. 4:303-
316.
Odum, E. P. 1971. Fundamentals of Ecology. 3rd ed., W. B. Saunders Co.,
Philadelphia, PA.
Page, A. L. 1974. Fate and Effects of Trace Elements in Sewage Sludge When
Applied to Agricultural Lands. A Literature Review Study. EPA-670/2-
74-005. National Ecological Research Center, U.S. Environmental Protec-
tion Agency, Cincinnati, Ohio.
Pitcher, R. S. and D. G. McNamara. 1972. The Toxicity of Low Concentration
of Silver and Cupric Ions to Three Species of Plant-Parasite Nematodes.
Nematologica. 18:385-390.
Pramer, D. 1965. Features of a Flask and Method for Measuring the Persist-
ence and Biological Effects of Pesticides in Soil. Soil Sci. 100:1:68-
70.
Ratsch, H. C. 1974. Heavy-Metal Accumulation in Soil and Vegetation From
Smelter Emissions. EPA-660/3-74-012. National Ecological Research
Center, U.St Environmental Protection Agency, Corvallis, OR 97330.
Rtlhling, A. and G- Tyler. 1973. Heavy Metal Pollution and Decomposition of
Spruce Needle Litter. Oikos. 24:402-416.
Salle, A. J. 1973. Fundamental Principles of Bacteriology. Seventh Ed.,
McGraw-Hill, Inc., N.Y.
49
-------
Stotzky, G. 1965. Microbial Respiration. In: Methods of Soil Analysis II.
Chemical and Microbial Properties. C. A. Black, ed., Amer. Soc. Agron-
omy, pp. 1550-1570.
Tyler, G. 1972. Heavy Metals Pollute Nature, May Reduce Productivity, Ambio.
1:52-59.
Tyler, G. 1974. Heavy Metal Pollution and Soil Enzymatic Activity. Plant
and Soil . 41:303-311.
Tyler, G. 1975. Heavy Metal Pollution and Mineralization of Nitrogen in
Forest Soil. Nature (Lond.) 255:701-702.
van Rhee, J. A. 1973. Copper Contamination Effects on Earthworms by Disposal
of Pig Waste in Pastures. Progress in Soil. Zoo. Proceed, of 5th Inatl.
Colloq. on Soil Zoo. Academia, Prague, pp. 451-457.
Watson, H. P., R. I. Van Hook, D. R. Jackson, and D. E. Reichle. 1976.
Impact of a Lead Mining-Smelting Complex on the Forest Floor Litter
Arthropod Fuana in the New Lead Belt of Southeast Missouri. ORNL/NSF/
EATC-30, ESD Publ. Mo. 881. Oak Ridge Nat'l. Lab., Oak Ridge, Tenn.
167 pp.
White, A., P. Handler, and E. L. Smith. 1973. Principles of Biochemistry.
5th Ed. McGraw-Hill Book Co., N.Y. p. 1296.
Williams, C. H. and D. J. David. 1973. The Effect of Superphosphate on the
Cadmium Content of Soils and Plants. Aust. J. Soil Res. 11:43-56.
Wilson, D. 0., and H. M. Reisenauer. 1970. Effects of Some Heavy Metals on
the Cobalt Nutrition of Rhizobium melkoti. Plant Soil. 32:1:81-89.
Witkamp, M. 1971. Soils as Components in Ecosystems. Ann. Rev. Ecol. Syst.
2:85-110.
Whittaker, R. H. 1970. Communities and Ecosystems. Macmillan Co., London.
Woodland, D. J. 1973. The Ozone Problem in Electrolyte Respiration and its
Solution. J. Appl. Ecology. 10:661-662.
50
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VI. PUBLICATIONS
1975. Bond, H., B. Lighthart, R. Shimabuku, and L. Russell. Some Effects
of Cadmium on Coniferous Forest Soil/Litter Microcosms. EPA-660/3-
75-036. National Ecological Research Laboratory, U. S. Environmen-
tal Protection Agency, Corvallis, OR.
1976. Bond, H., B. Lighthart, R. Shimabuku, and L. Russell. Some Effects
of Cadmium on Coniferous Forest Soil and Litter Microcosms. Soil
Sci. 121(5):278-287.
1976. Lighthart, B. and H. Bond. Design and Preliminary Results From
Soil/Litter Microcosms. Internatl. J. Environmental Studies. 9:1-
8.
Lighthart, B. and H. Bond. Trace Element Pollution Affects Decom-
poser Respiration in Microcosms. (Pending).
51
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VII. Appendices
A. Microcosm Preparation
B. Inter-Experiment Maintenance Schedule
C. Intra-Experiment Maintenance Schedule
D. Scale Drawings of "Life-Support" System and Parts List
E. Electronic Control Systems
52
-------
Appendix A
Microcosm Preparation
Soil and litter preparations for use in pollution effect studies have
been standardized to eliminate as many variables as possible within and
between experiments. This includes both handling and processing methodologies
as well as microcosm insert preparation. The descriptive phases set forth
below include collection and pre-preparation, soil and litter processing,
insert preparation and examples concerning soil/litter moisture adjustment
and pollutant spiking information. Simplified flow-diagrams indicate the
various operations.
Collection and Pre-preparation--
Fresh soil and litter samples are placed in separate plastic bags and
transported into the laboratory where they are spread on paper and air-dried
for 24-48 hours. During this period they are stirred several times and
twigs, stones, roots, and other undesirable materials removed.
Soil and Litter Processing--
After drying the soil is homogenized in batches in a Waring Blender set
at low speed with intermittent removal of the blender cannister which is
shaken to dislodge large soil clods and insure good mixing. Each batch is
homogenized - 3 minutes. Homogenized soil is collected in a large pan, and
is sieved through a 1/10" mesh hand held sieve to remove larger lumps and/or
other debris. The sieved soil is then hand-mixed by stirring to achieve the
final homogenate.
Thin layered subsamples of this soil homogenate (- 1 g) are oven-dried
at 110°C for 1 hour to determine the moisture content. The moisture content
is then adjusted upward to attain = 60% field water holding capacity (FWHC)
by addition of distilled water, delivered in a fine mist (chromatograph
sprayer or other suitable apparatus) while stirring. The wetted soil is
allowed to equilibrate 2 hours and moisture determinations are repeated to
assure proper moisture content. Subsamples may also be taken for pH determin-
ations or other" analyses if desired.
Enough sojl is prepared, as described above, to make up a predetermined
number of microcosm inserts within 24 hours after the initial homogenizing
operation. Microcosms are assembled on the same day as the water addition.
After drying,the litter (coniferous) is sieved in a rotating drum sifter
with 16 mesh screening (modified rock polishing apparatus) and collected in
a common container and homogenized with hand stirring. Thin layer subsamples
53
-------
(= 0.5 g) are oven-dried at 110°C for 1 hour to determine the moisture con-
tent. The moisture content is then adjusted upward in the same manner as
soils are wetted to attain - 78% moisture content by weight. After settling
2 hours moisture determinations are again made to confirm that the desired
level has been achieved. At this point subsamples may be taken for pH deter-
minations and/or other analyses.
Enough litter is prepared in this manner to make up the desired number
of microcosm inserts on the same day on which it was wetted.
Insert Preparation --
Berzelius beaker inserts, pretared and numbered, are placed in a circular
configuration on paper or on a large tray. The prepared soil is then poured
onto a 3' x 3' wooden framed screen (1/4" mesh screen overlaying a 1/8" mesh
screen) and sieved into the inserts by shaking the screen in a reciprocating
manner until each insert contains approximately 150 g of soil. The inserts
are then individually weighed and the amount of soil is adjusted to the exact
amount, 150 g (wet weight) per insert. To standardize the interstitial pore
space the soil is then compressed by placing a snug fitting wooden cylinder
into the beaker and carefully placing a 12 kg weight (lead brick) on top of
the cylinder for one minute. The compression apparatus is then removed and
the prepared litter (15 g, wet weight) is added to the top of the soil,
spread to attain even distribution and the compression operation repeated.
Immediately after removing the compression apparatus a 4" x 4" piece of
Teflon film is placed over the top of the insert followed by a 4V x 4V
piece of Saran wrap with a 0.5 cm diameter central perforation. These two
top layers are secured with a rubber band. Studies indicate that this cover
arrangement allows for adequate aeration and cuts moisture loss from the
system to <0.2 ml/day under incubation conditions.
The microcosm inserts are then placed into an incubator at the desired
temperature (20°C) for a 10-14 day equilibrating period prior to use in the
SER system.
Water loss from the inserts is replaced each week by adding distilled
water drop-wise over the litter surface until the original weight is attained.
Soil/litter Moisture Adjustment--
The optimal moisture content for an experimental microcosm is approxi-
mately 60-78% of the field water holding capacity (FWHC) of the soil and
litter. This was found to be 18-23% moisture for soil and 48-63% moisture
for litter, as measured by drying wet samples 20 hours at 110°C.
Determining final weight of soil:
ex. 1: If 100 g final weight of remoistened soil is needed, the amount
of water present would be 23 g per 77 g dry soil.
54
-------
o O f*
-j-, * = 0.299 which is the proportion of water to dry soil at
y 78% FWHC.
ex. 2: The same applies to the litter: therefore in 100 g final weight
sample there are 63 g water and 37 g dry litter.
- 1.703 which is the proportion of water to dry litter
at 78% FWHC.
Microcosms are analyzed on a dry weight basis, and dry weight is the basis
for microcosm preparation.
ex. 3: If 15 g dry weight soil is needed, the amount of water to be
added is:
yg-g - 0.299; x = 4.485 g water.
4.485 g water + 15 g dry soil = 19.485 g final weight.
ex. 4: If 45 g dry weight litter is needed, the amount of water to be
added is:
- 1 .703; x = 76.635 g water.
76.635 g water + 45 g dry litter = 121.635 g final weight.
Pollutant Addition to Microcosms--
As previously described in this report an assessment of the effects of
certain trace elements (e.g., heavy metals) on decomposition was accomplished.
In this regard certain methods and techniques were devised and used to stand-
ardize the addition to or spiking of microcosm inserts with aqueous solutions
of the desired pollutant.
The following information describes the preparation of spiking solutions,
the time of spiking the inserts and the pollutant application technique.
Spiking solutions were prepared by accurately weighing out and solubil-
izing the selected pollutant (e.g., CdCl2) in distilled deionized water. An
example of the calculations to attain a given concentration per a given dry
weight of soil follows:
Basic Formula:
1. Formula Wt. » ug Element/g of Dry Wt. of x Wt. of *
of Compound Soil or Litter Needed dry soil
(Atomic Weight Element)
55
-------
Diagram 1
SOIL/LITTER COLLECTION, PREPARATION AND PROCESSING
Collect soil and litter into
separate plastic bags
Subsample for
moisture determination
Air dry 24-48 hrs/stirring and
remove undesirable materials
I
Disintegrate soil
in Waring Blender
Sieve litter through
drum sieve
Hand sieve soil
Batch homogenization
with hand stirring
Batch homogenization
with hand stirring
I I
Subsample for
moisture determination
if desired
Add distilled water in
mist to wet sample;
Hand stir while wetting
Add distilled water in
mist to wet sample;
Hand stir while wetting
Hold 2 hours for
moisture equilibration
Hold 2 hours for
moisture equilibration
I I
Subsample for
moisture determination
or other analysis
Hold in covered container
for microcosm insert
preparation
Hold in covered container
for microcosm insert
preparation
56
-------
Diagram 2
MICROCOSM INSERT ASSEMBLY
Sieve prepared soil through 3' x 3' screen
into prepared inserts arranged in
circular pattern on a large paper or tray
Weigh insert and soil;
adjust soil weight to 150 g
Compress soil with wooden cylinder
plus weight for 1 minute
Add weighed amount (15 g) of prepared
litter and spread to attain even
distribution over soil surface
Repeat compression process
as above
Cover immediately with Teflon film-
Saran film secured with rubber band
Record weight of total unit
Incubate - equilibrate at 20°C
for 10-14 days
Weigh and adjust moisture loss
each week
57
-------
= number of grams of the compound needed per horizon.
* Soil or litter, a separate calculation for each.
2. (Gram compound per horizon) x (Vol. solution prepared)
(total vol. delivered per horizon)
= the number of grams compound needed to prepare the solution.
ex. 1: Wanted: 1000 ppm of Cd as CdCl2 in 100 g dry soil, will spike
the horizon with 4 ml of the 20 ml of spike solution prepared.
Using Formula 1:
(183.4 g CdC1?)(10"3 parts)(100 g) = 0 163 q
(112.4 g Cd)
Using Formula 2:
(°-16349m1(20m1) - 0.815 g.
Solution concentrations were such that when 4 ml of solution was added
to the soil horizon and 2 ml added to the litter horizon of each insert the
final mean concentration desired was attained.
After the inserts have been monitored in the reactor module for 10 days
to determine background values of oxygen consumed, C02 produced and heat
energy flux they are removed one at a time from the module and the pollutant
is added.
The pollutant, in aqueous form, is delivered via 1 ml, discardable
tuberculin syringes with 2"-2V', 22 gauge luer-loc needles. A small portion
of neoprene rubber is pierced by the needle and serves as a depth marker when
injecting. A disc (3 mm thick) of transparent plastic with 45 holes at 1 cm
square grid points is placed on top of the litter horizon. The needle is
inserted through these grid holes and the pollutant injected by delivering an
equal portion through each hole until 2 ml are delivered at each level. In
this manner 2 ml is placed approximately 3/4" above the bottom of the*soil
layer, 2 ml just beneath the surface of the soil layer (1 cm) and 2 ml of the
appropriate concentration into the center or midway into the litter horizon.
As soon as spiking is completed the insert is returned to the reactor
module.
58
-------
Appendix B
Inter-Experiment Maintenance Schedule
SER Calibration--
The SER units are calibrated to determine the heat demand and loss of
each unit. A heat control is run with each experiment to ascertain heating
fluctuations which occur in a non respiring SER. A beaker containing 500 g
of dry compressed soil is placed in the SER heat control during the actual
SER experiments since the microcosms in the test reactors act as insulation
and alter the heat demand from that of empty dewar flasks.
Calibration Check List--
1. SERs are readied for operation.
a. Reactor moducles are dried.
b. The manometer fluid is adjusted to a medial position.
c. Agar bridge integrity is checked.
d. H2 and 02 electrodes are inspected for deposits, breakage or
electrolyte leaks.
e. NaOH sample port (valve) is lubricated and rubber septums
replaced.
f. Stopcocks and ground glass joints are lubricated.
g. Air filters are checked for excessive liquid absorption.
2. Starting SERs.
a. Main voltage for gas generation is set between 12.5 and 15.5
volts DC, depending on total unit load on the power supply.
Power on.
b. Individual panel voltages are set at approximately 13.0 volts
AC, depending on heating element resistance. Power on.
c. Heat time meters are set to zero. Power on.
d. SER lids are lowered and tightened shut.
59
-------
e. Electrolyte level in 02 probe is set even with 02 generator
electrolyte level.
f. All thermometers (thermoregulators) are set for 20°C.
3. Data collection.
a. Computer data sheets are used for collecting and recording all
SER data.
b. Readings are made three times daily, at approximately 08:30,
12:00, and 16:00. One reading is made on weekends.
c. Readings consist of recording barometric pressure, room temper-
ature, manometer level, time, date, calibration number,
electrolyte level in 02 probe, heating times, and panel volt-
ages for the individual units are recorded, then reset at each
reading to the original setting of 13.00 ± 0.08 volts AC.
d. Amends are made in the apparatus, if any unusual readings or
activity is observed i.e., air leaks causing gas generation.
Duration of Calibration--
Calibration runs were originally made prior to and after each experiment.
Currently, calibration runs are not being made this frequently since heat
demands appear to be linear for each unit. A calibration run extends from
three to seven days depending on the needs of the system.
Trouble Shooting the System and Agar Bridge Preparation--
Certain symptoms have been found to be indicative of the cause of minor
malfunctions within the SER system. These have been tabulated (Table 6) to
aid in correcting these problems. Instructions are provided if it is neces-
sary to find small leaks by submersion of the reactor module and/or to prepare
agar bridges.
1. Submerging a SER: This is done when the SER is empty (i.e.,
without a microcosm).
a. The manometer is replaced by a stopcock and securely ti'fed,
with the stopcock open to allow air flow.
b. The lid is tightened down evenly with the wing nuts.
c. The 3-way stopcock is closed to the atmosphere.
d. The 02 probe is lifted out of the 02 generator. A meter
length of tubing is placed over the 3-way stopcock. The
stopcock is opened and air expelled into the unit, then the
tubing is clamped shut.
60
-------
TABLE 6. TROUBLE SHOOTING
Symptom(s)
Possible Cause(s)
Remedy(s)
1. spontaneous or
over generation
of H and 0,
2. under generation
of H,, and 0?> or a
delayed response
between pressure
changes in the
flask end gas
production
3. not generating
gases (H-, Oj
or the unit not
heating properly
4. discoloration of
agar bridge and/ *
or electrolyte
a. lid not sealed tiditly
b. inadequate tension at
joints
c. stopcock(s) open
d. ground glass joints not
adequately lubricated
e. C09 valve leak
f. hole in buret bulb
g. tygon tubing/glass
connectors loose
2. a. blocked air line
b. all items listed in 1
(a-g).
3. Electrical circuit or
electronics panels
malfunctioning
a. alligator clip off of
electode
b. panel switch off
<_. panel malfunction
d; wire broken or corroded
e. electrodes coated or loose
f. electrolyte contaminated
4. a. microbial growth
b. impurity in Na-SO^
reagent, egar or
distilled water
c. chemical reaction with
the contents of the SCR
1. a. tiahten all three wing nuts on lid
evenly
b, put springs or rubber bands snuggly
around the joint
L. check positions of all stopcocks
d. lubricate stiff joints
e. regrease valve. If necessary,
submerge the unit in a bath of
vater. Lock for air bubbles
around the CO, valve. If air is
leaking at the valve/lid contact,
re-epoxy into place.
f. pressurize buret and submerge in a
water bath! If air bubbles form on
bulb, replace.
g. look for air bubbles between qlass
and tubing. If loose, replace
tubing or add a hose clamp, tighten
snuggly around the joint.
2. a. clean stopcock openings of excess
lubricant.
b. see 1 (a-g).
3. a. check position of alligator clips
and tighten or replace if corroded
b. check all switches on panel and
indicator light
<-. tighten panel in position, clean
contact strips; try replacement
panel
d. check for loose wires, check
terminals
e. if probes are blackened, drain
electrolyte, rir.se with distilled
water. Clean electodes v.'itii 50%
nitric acid (aq.) then let set
15-30 min. Drain and rinse 6-12
tines with distilled water to
remove all traces of acid. Refill
with fresh electrolyte. If elec-
trodes are loose, epoxy in place.
f. replace electrolyte
4. a. if the discoloration is gold-qreen-
brown; remove bridge and remove
aqar. Rinse with 50!' nitric acid
(aq). Reform bridge using an anti-
bacterial agent.
b. prepare fresh electrolyte or aqar
from new reagent stock, use dis-
tilled water direct from source.
c. replace air filters in U-tube.
-------
lABtC 6. (CON'T.)
Symptom(s)
5. manometer fluid
loosing color
6. "water" in air
1 i ne
Possible Causc(s)
Ranedy(s)
5. pH change
6. a. evaporation or condensa-
tion from generators,
manometer fluid, micro-
cosms, or HaOH.
b. inadequate gas produc-
tion i.e. gas produc-
tion is not meeting the
demands of the microcosm
Electrolyte is backing
up the Op electrode and
into the air line.
replace air filters in U-tube. Acidify
nanometer fluid below pH 4.
a. replace air filters, dry the lines
with an air jet. Make certain that
chamber and SER temperatures do not
fluctuate.
b.
electrolyte level should be read-
justed. Filters changed if wet. Hay
need to remove the microcosm if gas
generating capacity is exceeded.
Gas generation can be increased by
increasing power supply voltage out-
put; do not exceed 100 ma total cap-
acity with all units generating with
a nine unit load, maximum voltage
setting is theoretically 31.0 V DC.
62
-------
e. The SER is submerged in a water bath, first allowing trapped
air to escape from under the stand and around the lid edge and
valves.
f. The unit is carefully observed for several minutes, to detect
air bubble formation.
g. The source of any air leak is noted and sealed.
2. Forming agar bridges.
a. Five percent Noble agar is prepared by placing 5 g Noble agar
in 95 ml distilled water. The solution is heated and stirred
until dissolved.
b. The clean glass fritted tubing is heated, then placed in the
agar for 5 minutes to allow the agar to permeate the frit.
Gentle vacuum is applied to draw the agar about 3 cm into the
tubing. Then gentle air pressure is applied to force the agar
back into the pores, until the agar is 2 cm above the frit.
c. The bridge is removed from the agar and dried 10 minutes, then
dipped into the agar to coat the outer surface. The bridge is
allowed to set 10 minutes then placed in the electrolyte (8%
Na2S04 aq. with 0.05% NaN3), until it is replaced in the SER.
63
-------
Appendix C
Intra-Experiment Maintenance Schedule
Experiment Initiation--
At the start of a SER experiment the microcosms are placed inside the
SER units and data are collected from the respiration of these microcosms.
The experiment itself is affixed to the end of the calibration run or unit
warm-up period. All units must be showing proper heating, maintenance of
voltage settings, the ability to produce oxygen gas and no contamination of
the electrolyte or agar bridge, or air leakage in any part of the sealed
system.
Microcosm inserts are placed in the reactor modules after the morning
set of readings for th£ calibration run. This ends the calibration period.
The collection of experimental data begins at the noon set of readings. This
allows the microcosms and SERs to equilibrate for 2-3 hours.
SER Experiment Initiation Procedures--
1. The usual morning calibration readings are taken.
2. Microcosms are brought two at a time from the incubator to the SER
incubation chamber.
3. The unit is opened and the microcosm placed inside with the Teflon
and Saran covering removed.
4. The lid is placed on the reactor module, but not tightened down.
Ten ml of 0.6 N^ NaOH* (aq.) is added to the NaOH cup via the port
on the SER cover. The SER lid is then secured. Two NaOH blanks
are taken at the beginning of the experiment and at the time when
samples are withdrawn.
5. The manometer fluid is set to a medial position, the electrolyte
level in the 02 electrode is set even with the electrolyte level in
the 02 generator.
6. At the time of the noon readings the following adjustments are
made.
The standardized 0.6 N NaOH solution must be made up with C02-free dis-
tilled deionized water and maintained in a sealed container to obtain the
true C02 value of C02 produced and to obtain accurate titration vaTues.
64
-------
a. The tension on the lid thumbscrewsare balanced and they are
retightened.
b. Barometric pressure, room and unit temperatures, manometric
pressure, time, date, experiment number, and electrolyte level
in the 02 electrode are all recorded.
c. The clock meters are all shut off and set to zero, then power
is turned on again.
d. Panel voltages are set to their individual settings.
e. Electrolyte level is set to zero in the buret or at maximum
capacity in the buret bulb of the hydrogen module.
7. 02 and other readings indicated in 6b above are made at noon each
day. Electrolyte level is reset in the hydrogen module.
8. NaOH samples (C02 trapping agent) are collected 24 hours after the
beginning of the experiment. After this, all other samples are
collected at 48 hour intervals. The 02 and C02 readings are made
during the noon data collection.
9. The readings are made three times daily and once on weekends. The
standard readings consist of those items listed in 6b along with
listing clock meter readings, panel voltage readings and reset,
plus 7 and 8 when applicable.
10. SERs are constantly observed at the time of data collection for any
malfunction. These are repaired immediately.
Duration of an Experiment--
SER experiments are run for approximately 31-35 days. The first 10-14
days after placement of the microcosms in the SERs is the internal control
period in which the microcosms equilibrate to a relatively constant level of
respiratory activity. The microcosms can then be spiked with pollutants.
Post spike period run time is 21 days.
65
-------
Appendix D
Scale Drawings of "Life Support" System and Parts List
Figures 32-37 show the various modules and component parts of the "life
support" unit including the hook-up of the total system. Also included is a
tabulation of the parts with identifying information, procurement sources and
approximate costs (Table 7).
66
-------
DRAWING OF MICROCOSM AND "LIFE SUPPORT" MODULES
REACTOR MODULE
SHOWING
MICROCOSM COMPARTMENT
Figure 32. Drawing of microcosm "life support" system including reactor,
pressure monitoring, 02 generatina and H2 generating module.
67
-------
46
COi TRAPPING AGENT
REMOVAL-REPLENISHMENT
PORT WITH RUBBER
SERUM CAP
Figure 33. Cut-away view of respirator reactor module.
68
-------
REACTOR TOP
C02 TRAP ASSEMBLY
c)
SCALE:
[DRAWING c) ONLY]
o
BOTTOM PLATE
SCALE;
DRAWINGS ol, b), AND dll
Figure 34. Schematic diagram of reactor module components showina (a) lateral
view of top assembly, (b) top view of top plate, (c) lateral view
of C02 trap assembly, and (d) top and cut-away side view of bottom
plate.
69
-------
27
31
32
1
{f
(!>!
0
I-
SCALE:
cm
Figure 35. "U" tube pressure monitoring module.
70
-------
OXYGEN GENERATION
MODULE
EQUI LIBRA TING
-OUTLET FOH
HESEHVOIR
EVACUATED
INTERNALLY
SIL VERED
SHELL
o)
c)
b)
d)
SCALE:
Figure 36. Oxygen generation module showing (a) cut-away
view of base with evacuated/silvered external
shell, (b) too view of generator module, (c)
oxygen probe component with electrode and (d)
top view of oxygen probe.
71
-------
41
-40
BASE - TOP VIEW
SHOWING f JOINTS
SCALE:
0 5
1 ' cm ' '
Figure 37. Lateral view of hydrogen generation module.
72
-------
TABLE 7. TABULATION OF SOIL ECOSYSTEM RESPIROMETER (SER) COMPONENTS, SOURCES AND APPROXIMATE COSTS
Diagram Catalog
Number Number
1 61845-009
2 NY-600-1-OR
3 NY-400-1-OR
4
5 B-D #3112
6 86570
7
8
9
10
11
12
13
14
15 46188
16
17
18
19
20
21
22 K-611420
23
24 14020
25
26
Part Description
I. REACTOR MODULE
Thermometer, Bromwil 0-50° C
Swaglok fittings, 9/16-18
Swaglok fittings, 7/16-20
Glass capillary tubing ID 1/16" OD 1/4"
BD luer lock fitting w/rubber septum
Stainless steel and nylon valve; IFLI
Aluminum tubing ID 1/2" OD 3/4", rings 1/4" high
Turned aluminum disk, 1/2" thick, 6-5/8" diameter
Neoprene sheeting for gasket, 1/8" thick
Styrofoam inner lid (insulation)
Threaded 1/2" diameter nylon tubing
1" nylon washer
1/2" brass nut
10 kn resistors (2)
Thistletubes; to custom make NaOH cups
18 gaug^ x 2" mod. hypodermic needle
Microtubing; size 050 x 090, 100 ft. roll
Turned aluminum disk, 1/2" thick with 1/8" lathed
depression, 6-5/8" diameter
Standard wing nut for 3/16" rod
Threaded rod 3/16" diameter
Lock nuts for 3/16" rod
Dewar flask, 95mm I.D., 125mm O.D., 1200ml cap.
Aluminum Dewar base
Berzellus beakers 600 ml capacity; no spout
Tar sealing compound, Matrix Binder
Polyurethane foam
Purchase
Source*
VWR
Swag 1 o k
Swaglok
VWR
B-D
Hamilton
Custom made rings
Custom made
Power Transmission
Albany, OR
Hobby Shop
Hardware store
Custom made
Hardware store
Kierulff Elect.
Seattle, WA
VWR
VWR
Norton Plastics
Akron, OH
Custom made
Hardware store
Hardware store
Hardware store
Kontes-Martin,
Evanston, IL
Ellco Glass Co.
Hardware
Unit
Price
34.00
3.20
3.10
.40
.71
6.00
.10
4.00
.40
1.00
.50
.25
.20
2.00
1.00
1.50
8.00
4.00
.07
45
.05
33.00
2.30
.25
73
-------
TABLE 7. (CON'T.)
Diagram
Number
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
Not
Pict
Not
Pict
Not
Pict
Catalog
Number Part Description
II. MANOMETER MODULE
25 ml boiling flask, for manometer
Snail springs
Modified glass capillary for manometer fluid
Kreb's manometer fluid, acidified
Activated charcoal granules
Fine textured glass wool
Ozone trapping filter
Kimble U-tube; modified 100 mm drying tube
46050
III. OXYGEN GENERATOR MODULE
Oxygen generator
13 cm long platinum wire epoxyed in glass cap.
tubing 3/4" long
NY-810-6-4 Swaglok fittings, nylon reducing union
02 probe glass support
IV. HYDROGEN GENERATOR MODULE
Modified condensation flask; hydrogen reservoir
w/stopcock
Pressure equilization standpipe
Inverted 25 ml pyrex buret
4-8 m pore size fritted glass dispersion tubes
(modify) O.D. 10 mm, length 150, porosity code E-10
Noble agar; use 5% in H,0-> electrolyte support
Hydrogen generator base (cost includes modification
of all glassware)
Platinum anode, secured in epoxy
IV. ELECTRICAL SYSTEM
Single strand electical wire; 22 gauge PVC coating
Snap connectors, U connectors for wiring
Alligator :lips 3/4" length
382 line Minature indicator lamps 14V
2032
Heath kit electronics converter for D.C. current
to generate gases
5C-240634 Clock meters 120V 60 Hz 2.5w
AAAE1
Purchase
Source*
Custom modified
VWR
Custom made
Manometric Techniques
VWR
VWR
Kimax (VWR)
Custom made
Custom made
Swaglok
Custom made
Custom made
Custom made
Custom made
ACE Glass, Inc.
Louisville, KY
Oifco
Custom made
Custom made
Kierulff Elect.
Seattle, WA
Hardware store
Hardware store
GE
Kierulff Elect.
Seattle, WA
GE
.Unit
Price
10.00
.05
10.00
14.58
100.00
5.00
3.70
5.35
140.00
.55
.30
.10
.65
150.00
42.50
1
Manometric Techniques, Umbeit, Stauffer and Burn's, Burgess Publ. Co., Minn., MN, 1964, 4th ed.
Ozone Problem in Electrolyte Respirometry and its Solution, D. J. Hoodland, J. Applied Ecology, 10:661-2, 1973.
74
-------
TABLE 7. (CON'T.)
Diagram Catalog
Number Number
Part Description
Purchase Unit
Source* Price
VI. MISCELLANEOUS
48
49
Not 6403-20
Pict
Not
Pict
Not 8040-00-
Pict 159-4846
Not size 11
Pict 1/2" x
260"
Not 21639-045
Pict
Not 21641-047
Pict
Not 21677-000
Pict
Not
Pict
Not
Pict
Not
Pict
Not 970 V
Pict
Tygon tubing ID 1/4" OD 1/4"
Electrolyte overflow bottle, 200 ml, milk dilution
Hose clamps with thumb screws 1/2" to 3/4"
Silastic sealing compound, GE RTV 108 silicone
rubber adhesive sealant
Epoxy kit (for cementing metal, glass) Devcon
Teflon sealing tape, around swaglok fittings
Clamps for supporting units, 3 prong vinylized
Clamps for supporting units, 3 prong vinylized
Clamp holder, Fisher Castaloy
Nidibutylidithio carbamate, active ingredient in
ozone trap
0.6 N NaOH (aq.) C02 absorbent
8% Na2S04 Caq.) electrolyte
Low/high vacuum/pressure Dow Corning silicone grease
VWR
VWR
Horizon
GSA
Hardware
Lake City Industrial
Products, Lake City,
PA
VWR
VWR
VWR
ICN Pharamcuticals, Inc
VWR
VWR
VWR
.60
.92
1.25
.90
3.60
4.50
1.75
* Where appropriate
75
-------
Appendix E
Electronic Control System
The following schematic (Fig. 38) depicts the electronics control system
for the "life support" unit. Electronic components, procurement sources and
approximate costs are incorporated in Table 7, Appendix D.
76
-------
(VERSION FEBRUARY 1977)
UREACTOR CHAMBER SECTION-*^ ELECTRONICS SECTION-
POWER SUPPLY SECTION-
THERMO
REGULATOR
0
) HEATER (2)
IOK, 5W
| (WIRE WOUND)
READ-OUT SECTION
TIME ELAPSE
METER
NOTES:
I RY-DPST 12 VOLT O.C. COIL RELAY.
Z. ALL RESISTORS 1/4 WATT AT 5%.
}. ADDITIONAL ELECTRONICS SECTION CAN
BE WIRED IN PARALLEL. CONNECT AT
() LOCATION.
Figure 38. Microcosm electronic circuit diagram.
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1 . REPORT NO.
EPA-600/3-77-091
3. RECIPIENT'S ACCESSI Of* NO.
4. TITLE AND SUBTITLE
TRACE ELEMENT RESEARCH USING CONIFEROUS FOREST
SOIL/LITTER MICROCOSMS
5. REPORT DATE
August 1977
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
8. PERFORMING ORGANIZATION REPORT NO.
B. Lighthart, H. Bond and M. Ricard
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Terrestrial Ecology Branch
Environmental Research Laboratory-Corvallis
Corvallis, Oregon 97330
10. PROGRAM ELEMENT NO.
1AA006
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Environmental Research Laboratory-Corvallis
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis., Oregon 97330
13. TYPE OF REPORT AND PERIOD COVERED
In House _,
14. SPONSORING AGENCY CODE
EPA/600/02
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Respirometers have been designed, constructed and to a limited extent, tested to
maintain and measure production and/or consumption of biogenic heat and carbon diox-
ide production and oxygen consumption for extended periods of time in approximately
0.5 1 soil and/or litter microcosms. Using coniferous soil/litter microcosms, the
mean coefficient of variation within sets of similar microcosms was 10.7% for the
oxygen consumption rate and 3.9% for carbon dioxide production rate.
Microcosm respiratory response, population responses to moisture level where mea-
sured, succession, and salt effects were similar to those observed in the natural
world.
Respiration of the decomposer communities in coniferous forest soil/litter micro-
cosms was inhibited by treatment with "real world" salt concentrations of Cd, Se, Zn,
Mn, Ni, Cu, Hg, Co, Cr, Va, Li, La, Ag, and Pb. These findings support the thesis
that the consequence of these ecosystem disruptions might be to reduce primary and
secondary production of the dependent populations. Scale drawings of the microcosm
"life-support" system and an outline of procedural details of system maintenance and
microcosm preparation are presented.
This report was submitted as partial fulfillment of inhouse research under Program
Element 1AA006, ROAP 21 ALU, Task 3. It covers the period March, 1974, to November,
1976. and work was completed as of March, 1977.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COS AT I Field/Group
Microcosms
Trace Elements
Heavy Metal s
Decomposition
Forest Soil/Litter
06/F
3. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)'
Unclassified
21.
OF PAGES
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
78
U. S GOVERNMENT PRINTING OFFICE 1977798-680/9 REGION 10
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