United States
Environmental Protection
Agency
Environmental Research
Laboratory
Corvallis OR 97330
EPA-600 3-79-089
August 1979
Research and Development
xvEPA
The Soil Core
Microcosm—
A Potential
Screening Tool
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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-
-
niTnnTn? "f^"^- Elir™ation of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6, Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This reporthas been assigned to the ECOLOGICAL RESEARCH series Thisseries
describes research on the effects of pollution on humans, plant and animal spe-
cies, and materials. Problems are assessed for their long- and short-term influ-
ences. Investigations include formation, transport, and pathway studies to deter-
mine the fate of pollutants and their effects. This work provides the technical basis
for setting standards to minimize undesirable changes in living organisms in the
aquatic, terrestrial, and atmospheric environments.
the Nationai
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EPA-600/3-79-089
August 1979
THE SOIL CORE MICROCOSM—
A POTENTIAL SCREENING TOOL
Jay D. Gile
James C. Collins
James W. Gillett
Terrestrial Division
Con/all is Environmental Research Laboratory
Corvallis, Oregon 97330
CORVALLIS ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CORVALLIS, OREGON 97330
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DISCLAIMER
Laboratory 5s Environmental Zt^'^ C°rVall1s Env1ron«.ntal Research
tion. Mention of trite names oTrnlT *9?ncy' and aPProved for Publica~
endorsement or recommendation for use '' Pr°dUCtS d°6S n0t Const1tute
11
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FOREWORD
Effective regulatory and enforcement actions by the Environmental Protec-
tion Agency would be virtually impossible without sound scientific data on
pollutants and their impact on environmental stability and human health.
Responsibility for building this data base has been assigned to EPA's Office
of Research and Development and its 15 major installations, one of which is
the Corvallis Environmental 'Research Lboratory (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 and stream
systems; and the development of predictive models on the movement of pollu-
tants in the biosphere.
This report is a product of the Environmental Protection Agency's Alter-
native Chemicals Program, which in part seeks alternative methods for evaluat-
ing environmental impacts of both new and old pesticides. Screening tech-
niques are essential in the registration and re-registration process required
under FIFRA and for chemical testing under TSCA. Both regulatory agencies and
chemical manufacturers will benefit by the use of test methodologies that
provide the necessary data for safe and effective use of agricultural chemi-
cals and other toxic substances in a timely and cost effective manner.
To this end we have examined a microcosm system which may meet those
needs of-both industry and government in avoiding unreasonable impact on man
and his environment.
James C. McCarty
Acting Director, CERL
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ABSTRACT
Two experiments have been performed to determine the suitability of a
soil core microcosm (SCM) as a screening tool under FIFRA and TSCA. The SCM
consisted of a 5- x 10-cm soil core removed intact from a field site and en-
cased in PVC. Experiment I examined 0.25 Ib/a applications of 14C-labeled
dieldrin, methyl parathion and 2,4,5-T. In Experiment II 0.25, 0.50 and 1.0
Ib/a applications of HCB were studied. Weekly leachates were analyzed for
nitrate (NOs-1), phosphate (P04-3), ammonia (NH3), calcium (Ca+2) and dis-
solved organic carbon (DOC) as well as 14C. Transport through the soil column
and subsequent metabolism were followied via 14C. The majority of the chemi-
cals from both experiments found in the soil were in the top 2 cm. Extract-
able metabolites were detected for all but HCB. 2,4,5-T had no effect on any
nutrient losses. Nitrate loss was increased by dieldrin and decreased by
methyl parathion. Phosphate export was decreased by methyl parathion 0 5
Ib/a HCB and 1.0 Ib/a HCB. Ammonia loss was decreased by the two highest
levels of HCB. Calcium export was decreased by methyl parathion and increased
by dieldrin. DOC was significantly decreased by methyl parathion and 1 0 Ib/a
HCB. Treatont levels were below norma1 aPPlication rates for all chemicals
and did not really challenge the system.
These experiments demonstrate that it is possible to assess some effects
of a chemical on a soil ecosystem and its fate simultaneously with the soil
rnra mirr>ni~ncm «-««.* ijr niuu uiie bU I I
core microcosm.
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CONTENTS
FOREWORD 1V
ABSTRACT • -iv
LIST OF FIGURES vii
ACKNOWLEDGEMENTS . . . ill
SECTIONS
1. CONCLUSIONS AND RECOMMENDATIONS 1
2. INTRODUCTION 3
3. METHODS AND MATERIALS 6
4. RESULTS AND DISCUSSION 10
5. OVERVIEW 23
REFERENCES 26
APPENDIX A
1. Soil Core Microcosm Screening Protocol 28
2. Soil Core Experiment I Protocol 31
3. Soil Core Experiment II Protocol 33
APPENDIX B
Standard Rainwater Formula 35
APPENDIX C
Quality Assurance Program .• • • -36
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FIGURES
No.
~~ Page
1 Soil core microcosm _
2 Mean 14C activity in leachate 12
3 Mean nutrient levels in leachate for Experiment 1 15
4 Mean nutrient levels in leachate for Experiment II 20
5 Mean C02 evolution for Experiment II 21
vi
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ACKNOWLEDGEMENTS
The authors gratefully acknowledge the contributions of Anne Burton,
William Davis, Elizabeth Frolander and Kathy VanKirk for their assistance in
the maintenance of the soil core microcosms and sample analysis. This work
was supported by program element 1EA714 of the Office of Research and Develop-
ment.
vli
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SECTION 1
CONCLUSIONS AND RECOMMENDATIONS
CONCLUSIONS
1. Extraction of cores from field sites can be considered an "art", there-
fore, it can be expected that different technicians could produce differ-
ent configurations of cores.
2. Inherent variation in natural ecosystems necessitates the use of at least
25 cores per-treatment, assuming that.some cores will be discarded during
the equilibration period due to aberrant leaching rates and that three
cores per treatment per week should be destructively analyzed for chemi-
cal movement. This will leave 9-12 cores for leaching and terminal 14C
analysis.
3. C02 measurements may be questionable due to high degree of intrinsic
variation plus competition between plants and the chemical trapping agent
for the C02 during photosynthesis.
4. The soil core permits the examination of effects on cycling of selected
nutrients (phosphate, ammonia, calcium). Nitrate and DOC do not appear
to be suitable parameters; nitrate may not be leached because of low
availability in soil and whereas DOC measurements can be obscured by
variations in plant and animal biomass between cores.
5. Fate, including transport and metabolism of a chemical may be followed
throughout the soil system, although not with respect to air loss without
considerable sampling apparatus.
In summary, it is possible to gain some assessment of the effect of a
chemical on the soil ecosystem, its fate, transport and metabolism at a rela-
tively low cost (approximately $40K/chemical).
RECOMMENDATIONS
Future work with soil core microcosms should include:
1. Continued evaluation of the nutrient parameters with emphasis on direct
links to key ecosystem components or processes.
2. Refinement of the C02 sampling system to reduce variation and account for
the competition of plants during the photosynthetic period.
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3. Round-robin testing of the system with three or more laboratories examin-
ing the same chemicals.
4. Comparison of results with larger microcosms (e.g. CERL TMC).
5. Development of mathematical models with emphasis on nutrient cycling and
microbial respiration.
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SECTION 2
INTRODUCTION
TESTING REQUIREMENTS
Under the current guidelines of the Federal Insecticide, Fungicide,
Rodenticide Act (FIFRA) as amended and the Toxic Substances Control Act
(TSCA), chemical manufacturers are required to perform a variety of tests on
the metabolism and mobility of a chemical in soil. For example, under FIFRA
Section 163.62-8 of the "Proposed Guidelines for Registering Pesticides in the
United States" requires data on aerobic and anaerobic soil metabolism reflect-
ing the rate, type and degree of metabolism in at least three soils. In
addition to transformation, information on the effects of the pesticide on
microorganisms is required for such microbial processes as oxygen consumption,
carbon dioxide evolution, nitrogen cycle reactions and measurement of enzyme
activity for dehydrogenase or phosphatase. With respect to mobility (Section
163.62-9) data on the extent and rate of leaching, volatility, and adsorption/
desorption are required (Johnson, 1978).
Similarity, under TSCA the guidelines for premanufacture testing tenta-
tively recommend testing of adsorption of the chemical in soil and sediment as
a means of assigning the chemical to one of four mobility classes (Section
A-3.5) and of aerobic and anaerobic biodegradation to assess persistence in
soil (Section A-4.5). Effects of microorganisms on metabolism of the chemical
and effects of the chemical on the microorganisms as indicated by C02 evolu-
tion are also recommended (Sections A-4.54, A-4.55). Section C-3 further
examines microbial effects using tests on organic matter decomposition and
nitrogen and sulfur transformations (Muir, 1979). Generally these tests
incorporate a variety of different testing apparatus and methods, necessitat-
ing different facilities and handling techniques. A single system capable of
providing the needed data on metabolism, mobility, and microbial effects could
expedite the testing and evaluation processes. Because such a system would
combine the requirements of several tests, it would not necessarily^.the
optimum tool for all tests. Any single system attempting to provide data from
several distinct tests would have to be viewed as a compromise providing
reasonable although not necessarily the best data within its physical^limita-
tions Industry and government must recognize its limitations and determine
the criteria by which such a system could be appropriate and applicable /to any
testing scheme.
THE SOIL CORE MICROCOSM
Such ;a svstem may be a soil core microcosm (SCM) originally developecTat
ona? Soratbry (Draggon, 1976, 1979; O'Neill et a].., 1977;
1977; EPA, 1977). The SCM concept developed around the theory
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that an intact ecosystem or portion thereof could better represent a "real
world" situation than a synthetic system so long as key processes could be
adequately examined in the light of the inherent variability of a natural
system (Ausmus et al_. , In Witt and Gillett, 1979). Early efforts centered
around the loss of certain key nutrients (nitrate, phosphate, ammonia, cal-
cium, DOC) via leachate as an indicator of community integrity and C02 produc-
tion as an indicator of microbial respiration. More recent studies (Van Voris
et al_. , 1978) have demonstrated within a controlled laboratory setting how
these microcosm measurements can be used to evaluate complex relationships,
such as that between ecosystem, diversity and resistance/resilience of the
ecosystem to pollutant impact.
A wide variety of terrestrial microcosm systems have been developed (Witt
and Gillett, 1979), mostly as synthetic or constructed systems for evaluating
the fate and effects of organic chemicals. The recent "Workshop on Terres-
trial Microcosms" (Gillett and Witt, 1979) reviewed the state-of-the-art of
terrestrial microcosm technology and found that current systems had a number
of deficiencies which greatly limited their use as screening tools. The SCM
appeared to be the most satisfactory for effects studies, but had not been
tested with organics. The soil-litter ecosystem respirator (Lighthart and
Bond, 1976) addressed more limited effects (decomposition) and also had been
tested only with inorganics. The plant-soil system of Liechtenstein and co-
workers (1974) had been tested with organic pesticides, but could not be
expected to provide realistic effects for intact ecosystems. Larger systems
(Cole et aj. , 1976; Gillett and Gile, 1976; Nash et ah, 1977) appeared to
have similar limitations, in addition to being less practical and more costly
when used in a screening mode. General criticisms for all systems include-
lack of inter- laboratory validation, lack of field verification of accuracy of
impact predictions, deficiencies in simulating environmental conditions (air
flows, temperature regimes), and deficiencies in criteria for acceptability
(scaling criteria, degree of complexity, etc). Yet the conclusion was that
terrestrial microcosm systems offered very real advantages and showed consid-
9 t0 ^'^ *" inte9rated view of the fate and
Not all of the deficiencies in these systems can be explored Because of
loss through the lelchatV »ST hY aS eyidenced bV changes in micronutrient
by altered CO, Droductfnn MY changes J/1 comniunity respiration as evidenced
synthetTc : organl? chem cais usedT' nth! - *S*"*** in these Stud1es 1s on
materials in the environment Pesticides or appearing as hazardous
achieving9 athcerPtariPnOSTev°ei ^fW?™? T the need to "^ costs of
environment, to compart ^ the /CM tprhn ? ab°-^a chemic^'s behavior in the
gain experience wHh the sv,tpm h 9V Wlt,h that of other systems, and to
H with the system by personnel othe
, ,
personnel other than the originators.
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Indeed work was needed to improve the technology p_er se and determine the
extent to which the SCM could be standardized. The basic operating scheme is
that suggested by Ausmus et a].., 1977 as reported in Gillett and Witt
(Appendix B) 1979. This was modified by conversations with Ausmus, Draggon,
Van Voris, Jackson and others regarding their experience with the system at
Oak Ridge and during the lERL-Environmental Assessment testing of energy-
related pollutants (EPA, 1977). The SCM protocol was then merged with the
procedures of soil sampling, extraction and chromatographic analysis applied
to other terrestrial systems (Cole et al_., 1976; Gile and Gillett 1979a,b)
for chemical fate. Comparisons can thus be made between these studies and
those performed with other model ecosystems or in the field.
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SECTION 3
METHODS AND MATERIALS
SOIL CORE MICROCOSM
The soil core microcosm consisted of a 5- x 10-cm soil core extracted
intact from the field sampling site (a ryegrass pasture adjacent to CERL for
Experiment I; Schmidt Farm for Experiment II). The cores were first trimmed
of any extraneous material and then placed on a perforated polyethylene disk
and encased in heat-shrinkable polyvinyl chloride (PVC), 1.5-mil thick, with a
?Ja;£icizer content of 5"10%' The pvc was molded to the soil surface with
ibU C air from a heat gun for approximately 20 seconds. Prior to shrinkage
the cores were chilled by incubation at 5°C for 12 hr to minimize the heat
f" c microorganisms. Above ground vegetation was clipped at the
'" tK cores in
*nH m? H"^ T6 ^a! thei? cemented on a 9lass funnel with silicon rubber
and fitted with a leachate collection flask (Fig. 1). The assembled S CM was
^
t, .t
permitted control of environmental temperature liqht and
j
Th
ion of the air systems in the growth chamber •
tamed under a 16-hr daily liqht cycle with * tomna V -S were main"
(day) to 16°C (night). The schedule of Teachl no ™u1valent to 0.25 Ib/a
10,10a-hexaChloro-P4-6 "^M ^5 W^' ,DihelHdrin (HEOD> ^^^
— H ^ ',^,^aD67-octahydro-l ,4-6110^6X0-5,8-
,,
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Vegetation
PVC Casing
Soil Core
(intact)
Glass
Funnel
Rubber Stopper
(two hole)
Removable
Glass Cover
C02Trap
Silicon
Rubber
Seal
Perforated
Polyethylene
Disc
Leachate
Collection
Flask
Figure 1. Soil core microcosm.
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TABLE 1. EXPERIMENTAL PROTOCOL
Dayb
0 7 14 21 35 42 49 56 58
Add std. ref. rai
Remove 30 ml of 1
Remove 1 core for
C02 analysis9
Termination
nwater
eachate
14C analysis
X X X X X
X X X X
X
XXX
X
X
X
X
X
X
X
X
X
X
X
X
X
Experiment II only.
Cores were treated with pesticide on Day 28.
dimethanonaphthalene) was obtained from Shell Chemical Company as an analyt-
HFoi/tn^nv.-H9^ PT° ^ mi>d wUh "^eled dieldrin (Amersham 85%
SV pr*°KV.lde ,« nyLe-ne solutlon Wlth 55 ug/ml at 85 mci/mmole. Similarly
methyl parathion (0 0-dimethyl-0-rnitrophenyl phosphorothioate), octyl 245-
tnchlorophenoxyacetate, and hexachlorobenzene (HCB) solutions were oreoared
from analytical standards (Polyscience Corp.) and U-rinq 14C-labelPd rhSlfc
New England Nuclear; 95% purity). In Experiment II ? "C-l££$ ^1$?*
tions were applied at a rate of 0.25, 0.50, and 1.0 Ibs/a equivalent in ?»i
of xylene. Each SCM thus received 55, 110 or 220 ua of "C-HPR ™H n R 1
"C respectively. In addition to the positive control fxvlpnl r : ^^ °f
set of SCM's received no treatment (negative control) carrier), one
--water and the
analyzed for nutrients and total i*C At the end of P16S Wfre taken and
next water application, one core of each «? !T ^ch week, before the
"C-labeled chemical and the ?eSainin cores l r??s Frem°Yed fo^ Analysis of
checked for C02 evolution. Both experliBents wiiS ,Experiment " only) were
which point all cores were analyzed for nnt, * terminated on Day 58, at
•or nutrient content and 14C distribution.
ANALYSES
late method (cadmium reducti'omethod^
(Standard Methods, Rand et al 197?, ™°d Tas"fblc acid reduction method
was determined via atoBiraBsorptlon swrt^J6 1C?cn AutoAnalyzer. Calcium
DOC was determined as total carbon bv rif^t °P-y (?tanda^ Methods, 1975).
International Carbonaceous Analyzer TstanH^ 12J?Stlon 1nto an Oceanography
yw standard Methods, Rand et aj.., 1975).
8
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C02 evolution: A 5-ml beaker containing 4 ml of 0.2 N KOH solution was
carefully placed on the surface of the SCM at 0800 hrs, removed at 1600 hrs
and back titrated with 0.1 N HC1 solution. During the C02-trapping period,
each SCM was covered with an inverted 150-ml beaker which was removed for all
other operations.
Radiochemistry: Liquid scintillation spectrometry was used to determine
14C levels in each sample. A volume of 1 ml of each leachate was checked
immediately by direct addition to the scintillation cocktail (both for anal-
ysis and for worker protection regarding subsequent handling during chemical
analyses). Soil extracts were checked for total activity then chromatographed
on 250-u silica gel G plates using a hexane:ether (1:1) solvent system.
Materials were located by radioautography, extracted from the TLC plates, and
analyzed. Metabolites were tentatively identified by comparative Rf values.
Residual radioactivity after extraction of soil segments with KC1, hexane:
isopropanol (3:2), or acetone was determined by combustion of the dried soil
sample in a Packard Oxidizer and collection of the 14C02.
Termination (harvest) procedures: When a SCM was terminated, the core
was stripped of the PVC coat (which was extracted with ethyl acetate) and
sectioned into three segments approximately 2-, 3-, and 5-cm deep. The sec-
tions were extracted with 200 ml of 0.1 M KC1 solution followed by aspiration
of residual liquid. Each segment was freed of plant material then extracted
in a Waring blender with one of the series of organic solvents and water to
determine 14C distribution of the parent chemical and any metabolites. The
plant material was similarly extracted. Only extracts containing 103 dpm or
greater were subjected to TLC, permitting ready detection of any extractable
metabolites at the 1% level. Air filters were extracted with ethyl acetate
and the extract counted.
QUALITY ASSURANCE
See Appendix C for Quality Assurance procedure.
DATA HANDLING
The results of each analysis were stored in;a Control Data Corporation
3300 computer and accessed by a program which yielded the following computed
values: total 14C mass balance per SCM; fraction of radioactivity as equiv.
of parent in each SCM section as parent, metabolites (including origin mater-
ial and aqueous extracts which were not chromatographed), and bound residue;
total nutrient content, weekly elution rate, and terminal nutrient residue for
nitrate, ammonia, phosphate, cadmium, and DOC; and COg evolution^ raters a
weekly 8-hr value. Leachate data were considered to be log-normally distrib-
uted/Statistical significance was determined by the methods of Snedecor and
Cochran (1969), using Student's t test.
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SECTION 4
RESULTS AND DISCUSSION
EXPERIMENT I [dieldrin (HEOD), methyl parathion (HP), 2,4,5-T (T)].
Mass Balance and Distribution
from the soil core was QeneraVlv &r th™*0™1* °f C fr°m each chemical
iiiiK1-^
EH^nlSH1^
m^jo^^r^t^ ^ S"^4»"-«^'^'tS
**' '^- uncn-^*uuilLcU IlldSS D9 I 3 ni"*P Tnic r*r»ii1*-l <-J 4 -Pf. i_ •
the differing "rainfall" rea' "aiauce. mis couia differ by virtue of:
to be lost by disolacempnt nhanrtmor,, Vc .mi_9 . fa.use more of tne
rt
plant cover (as compared to other terrestrial m^ Ulath« 1969)j the lack of
the "still air layer" above the fsoll and ?h mi?rocosin. systems), decreasing
flow; the different phy*^ exchange with air
SCM as compared to the TMC; or the lowered S ^ .incr ^ as bound
"$1Sis -
HEOD in mobility and T in staMit ^ m°b11e; HP ""» closely
10
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TABLE 2. 14C ACTIVITY AT TERMINATION (% OF APPLIED)'
Component Chemical
Methyl Parathion 2,4,5,-T
Soil 30.0 [0.29d] 41.1 [0.24]
Plant 8.4 [0.51] 6.1 [0.59]
Leachate . 0.5 [1.02] 2.5 [0.89]
Soil Caseb 2.6 [0.79] 1.3 [0.79]
Air Filter0 0.6 0.4
Total 43.1 — 51.4
a Mean of 8 cores for methyl parathion; 9 for 2,4,5-T; and 12 for
b PVC heat-shrinkable tubing and masking tape.
c Composite from a single filter apparatus/treatment.
Coefficient of variation.
TABLE 3. PROFILE OF 14C ACTIVITY IN SOIL AT TERMINATION (% OF
APPLIED)*
Level Pb Mc Bd
HEOD
60.9 [0.15]
14.3 [0.49]
0.2 [0.21]
2.5 [0.55]
i n
1 . U
la. £.
HEOD.
TOTAL 14C
Total
• Methyl Parathion
31- f\ r nc— ————•• n 7^ rn TQ i
-5 cm 0.6 u.b u./o LU.33J
e t n n r n C - , (\ fi4 fO 421
6-10 cm 0.5 u.o u.ot L"-t*J
26.9 [0.36]
2.0 [0.24]
1.6 [0.16]
2,4,5-T ;
Top 2 cm 2.1 [0.28] 3.4 [0.25] 29.0 [0.32]
3-5 cm 1.2 [0.45] 1-0 0.63] 2.9 0.71]
6-10 cm 0.5 [0.47] 1.3 [0.03] 1.5 [0.54]
34.5 [0.26]
5.1 [0.52]
3.3 [0.38]
HEOD ;
Top 2 cm 43.6[0.21] 4.4 [0.48] 3.8 [0.30]
3-5 cm 6.5 [0.50] 0.4 [1.2] 0.5 [0-46]
6-10 cm 1.7 [0.1 9[ ,O...P." °-5 CO. 12]
a Mean of 8 cores for methyl parathion; 9 for 2,4,5-T; and 12 for
Extractable parent.
C • ..•••-•.••• .-.•:•';-'••••"•" ' ' . " • •
Extractable metabolites.
51.8 [0.19]
7.4 [0.52]
1.9 [0.16]
HEOD.
e Coefficient of variation.
11:
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I4C activity in leochate
10000
E
ex
c
a
0)
1000
Methyl Parathion
100
2 3
Weeks
Figure 2. Mean 14C activity in leachate for Experiment I.
12
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Movement of the chemicals through the SCM is controlled by the inter-
action of several properties: volatility and fugacity; solubility; biodegra-
dation rates; and adsorption/desorption rates and equilibria Difference be-
tween SCM's might be due to organic matter content bulk density (or its
inverse, the degree of aeration of the soil by capillary channels), and the
nature of biota present (since the small portion of rhizosphere present woud
be associated with only limited plant species). In spite of great intrinsic
variability between SCM's regarding flow rate, for example, the results indi-
cate that considerable consistency can be achieved.. Prediction of the outcome
should eventually be possible, based upon the P^slcal/b\oc.he"^"l P^P%?;!!
of the test chemical. Sorption phenomena have been related to octanol/water
partition coefficient and organic matter content of the soil <£. Lasisiter
1979); volatility (from water), solubility and partition coefficient a so
have been related (Chiou et ah , In Witt and Gillett, 1979; Metcalf |t al_
1979) Given the properties of these three test chemicals shown in Table 4,
the performance ofPthe SCM reveals the interactions fairly well in intercom-
parisons.
TABLE 4. PHYSICAL PROPERTIES OF TEST MATERIALS
•^-^™' —
Property Methyl Parathion 2,4,5-T HEOD
Mpa 36oc 158°C . 175°C
M a Q -r v in-6 f9n°n 1 x 10-2 (20°C) 7.78 x 10-7 (25°C)
Vapor pressure 9.7 x 10-° UU u; i x iu v.^u ^j
Solubility3 55-60 <25°C) 278 ppm (25°C) 0.05 ppm
0/W partition" 2.8 x 10 ' 9.2x10*. 3.9x10=
Adsorption Kb 5.6x10* 2.2x10' 3.4-x 10*
a
Spencer, 1973.
b Chiou, 1979.
0/W partition coefficient estimated from solubility.
Adsorption K calculations based only on organic matter weight in soil, ignor-
ing the contribution of sorption by other constituents.
reflect only 14C activity.
13
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Two metabolites of methyl parathion were detected in the soil When
their Rf s were compared to those of known metabolites of methyl parathion it
appeared as though ami no-methyl parathion and p-nitrophenol were the maior
metabolites. Two metabolites of 2,4,5-T with Rf's similar to those of un-
identified metabolites detected by Metcalf et al . (1979) were also detected
but no attempt was made to identify them. The ¥etabolites of HEOD, although
unidentified, appear to have Rf's similar to those detected in the CERL TMC.
EFFECTS ON NUTRIENT LOSSES
fNO -IhepO°?3f1S1Hentr *2 Vnnr
cwcL ' h4 ' NH?' Ca ' P°C)
systems; however irrespective of
ent^rel
c/lcu1ated for the five nutrients examined
1 fal1 within the range common to biological
treatment, P04-3, NH, and Ca+2 aooear to be
TABLE 5. COEFFICIENT OF VARIATION (CV). EXPERIMENT I<
Experiment I: methyl parathion, 2,4,5-T and HEOD.
"Tth\*e£r1^ nUttn'ents via the lea'hate over the
:ted N03-i loss relative to th treatments mQtW parathion and dieldrin
• loss the dieldrin treatment h contro1- Despite an initial increase in
'ol within two weeks. Methvl nJSfJ?^ significantly different from the
i/capne an initial increase in
: significantly different from the
initially increased loss of NOa-1,
lw reduced N03-* losses at the 90%
s appear to have reduced P04-3
nmeant effect at the 90% level. NH3
:antly influenced by any of the chemi-
~n significantly (p £ o.l) altered Ca+2
ng export while dieldrin slightly ih-
ichate were only significantly impacted
previous studies an increase "in rnoffVA- *.* Deduced export. Contrary to
consistently associated with a signiSt °Jfva!;iation does'not appear to be
C.V.'s remain relatively constant VnH- V fect on nutnent loss (Table 6).
treatment measurements. C0nstant indicating the reliability of the post-
course
affected N0a-i loss rel
N08-i loss the dieldrin
control within two wek".
but quickly recovered to produce
confidence level. While all t
export, only methyl parathion had
export in the leachate was not
cals. Both dieldrin and methjl
export with methyl parathion
creased export. DOC levels in
(at the 90% level) b>^methyl
prev ous studies an increase in
-------
1000
cr>
i i i i
Control
Methyl Parathion
— HEOD
— 2,4,5-T
100 r
o>
o
Q.
X
0)
ro
X
o
0)
10
I I i I
0.1 r
treatment
J L
_L
4 5
Weeks
C3»
E
o
a.
X
a>
O
O
O
,c
o
a>
0.1 r
o
a.
x
c
O
o
O
c
o
a>
100 k
10 r
i r
i r
treatment
i* i i
8
2 3 45
Weeks
8
I0p—r—r
i r
treatment
_L_ -L
JJ
3 4 5;
Weeks
Figure 3. Mean nutrient levels in leachate for Experiment I.
15
-------
TABLE 6. CUMULATIVE NUTRIENT LOSS IN EXPERIMENT I
Nutrient
a,b
Treatment
Control Methyl Parathion 2,4,5-T
HEOD
NOa-1 (ug)
Initially available
Pre-treatment loss
Post-treatment loss
Total loss
P04-3 (ug)
Initially available
Pre-treatment loss
Post-treatment loss
Total loss
NH3 (ug)
Initially available
Pre-treatment loss
Post- treatment loss
Total loss
Ca+2 (ug)
Initially available
Pre-treatment loss
Post- treatment loss
Total loss
903 [0.89]c
105 [0.89]
167 [0.65]
272 [0.73]
98.5 [0.12]
25.0 [0.44]
11.4 [0.61]
36.5 [0.44]
1125 [0.32]
6.5 [0.34]
8.0 [0.29]
14.5 [0.27]
1862 C0.21]
425 [0.60]
236 [0.49]
662 [0.55]
1344 [0.66]
105 [0.81]
397 [0.84]
503 [0.80]
113.6 [0.23]
42.4 [0.43]
15 [0.61]
57.8 [0.45]
1749 [0.37]
8.4 [0.42]
9.1 [0.16]
17.5 [0.24]
1354 [0.46]
449 [0.63]
514 [0.71]
963 [0.65]
1290 [0.26]
105 [0.69]
342 [0.60]
448 [0.55]
96.4 [0.17]
25.8 [0.35]
11.1 [0.58]
37.1 [0.40]
1504 [0.38]
5.1 [0.19]
8.9 [0.28]
14.0 [0.20]
1231 [0.30]
363 [0.46]
449 [0.46]
812 [0.43]
1464 [0.54]
209 [0.76]
678 [0.75]
887 [0.66]
98.5 [0.16]
33 [0.35]
13.2 [0.39]
46.1 [0.29]
1400 [0.45]
6.5 [0.52]
8.1 [0.12]
14.5 [0.27]
1612 [0.33]
497 [0.47]
666 [0.50]
1162 [0.46]
The use of a KC1 extraction on the core at termination did not permit DOC
analysis.
Mean total nutrient; 8 cores for control and methyl parathion; 9 cores for
2,4,5-T; and 12 cores for HEOD.
c Coefficient of variation.
Of the three pesticide treatments, methyl parathion had the most signifi-
cant impact, which would be judged to be largely beneficial since it resulted
in greater retention- of soil nutrients. Dieldrin had a slightly negative
SlrfeS«fHlffif+ h?fl -in T6 ^ased -losses of Ca+2 and N03-i, whereas 2,4,5-T
h*l ™ ¥ + ^t- Half-life for the chemical and the effect appear to
have no direct relationship, since methyl parathion with the shortest half-
( bTS8? t0,hucnn the m0st s19ni'f i«nt effect on nutrient
' V~J an? HEOD Were 30 and 67 days, respectively. Each
.^st be viewed in the light of possible impact of the
?e?*'. nin-Ce this would have dominated the comparisons. Thus,
ininlaJ ^^l 1n "™*"1 6Xp°rt "sociated withThe HEOD treat-
rnntmu inct ^ % ,K% • hl9he^ cumulati vne ^^ In comparison to controls,
controls lost 30% of their available N03-i in the leachate. Furthermore, the
(%,!f
th«p
16
-------
methyl parathion treatment resulted in an increase in NOs-1 export even though
that loss was rapidly reversed and the rate of loss became lower than the
control rate.
None of the treatments led to severe or irreversible displacements of any
of the nutrients. The concentrations applied were at or below that recom-
mended for actual use of these pesticides and therefore did not really chal-
lenge the system.
EXPERIMENT II [Hexachlorobenzene (HCB)]
Mass Balance and Distribution
Recovery of 14C ranged from 33.6% at the 1 Ib/a treatment to 43.3% at the
0.25 Ib/a treatment (Table 7). The inverse relationship between the amount
applied and that recovered probably reflects a saturation ^4^*^111^
of soil with HCB and a resultant volatilization of the 14C material. In
contrast to the first experiment, plants contain the majority o "C recovered
even though the HCB was applied to the soil. One probable exp anation is the
greater abundance of plant biomass in Experiment II, since above ground vege
tation was removed in Experiment I. No 14C ™*tetBctod™wrftoe]*ac\i
ate analyzed, which is not unexpected for HCB (< 0.02 ug/1 solubility in
water).
TABLE 7. 14C ACTIVITY OF TERMINATION (% OF APPLIED)'
Component
HCB
Soil
Plant
Leachate .
Soil CaseD
Total
Mean of 15 cores
r\\jrt i » i • | i
. . • —
. — —
0.25 Ib/a HCB
20.0 [0.32]C
22.1 [0.42]
0
1.2 [0.37]
43.3
_— —— -
— — . — . —
for al 1 treatments of
-1_ 4-nk-inn anrl mA^kinCI
Chemical
0.50 Ib/a HCB
17.3 [0.30]
22.0 [0.39]
0
1.3 [1.07]
40.6
. — — •
_,
HCB.
taoe.
1.0 Ib/a
12.9 [0.30]
19.8 [0.30]
0
0.9 [0.27]
33.6
Coefficient of variation.
TU • -4. * i4r
The majority of * 4C
recovered from the soil was in the top 2-cm
re between the treatment levels
used, extractable parent predominated in tne Tir&t
17
-------
TABLE 8. PROFILE OF 14C ACTIVITY IN SOIL AT TERMINATION (% OF TOTAL 14C
APPLIED)3
Level
Top 2 cm '
3-5 cm
6-10 cm
Pb
9.7 [0.45]e
0.6 [0.75]
0.2 [0.41]
Mc
0.25 Ib/a HCB
0
0
0
Bd
8.4 [0.35]
0.7 [0.52]
0.4 [0.14]
Total
18.1 [0.34]
1.3 [0.61]
0.6 [0.18]
0.50 Ib/a HCB
Top 2 cm
3-5 cm
6-10 cm
7.0 [0.45]
0.4 [0.79]
0.2 [0.41]
0
0
0
8.3 [0.39]
0.7 [0.49]
0.6 [0.34]
15.3 [0.32]
1.1 [0.59]
0.8 [0.32]
1.0 Ib/a HCB
Top 2 cm
3-5 cm
6-10 cm
a .. _ _
5.9 [0.64]
0.4 [0.51]
0.1 [0.32]
0
0
0
5.5 [0.25]
0.6 [0.52]
0.4 [0.15]
11.4 [0.42]
1.0 [0.46]
0.5 [0.14]
..__.. „ , ,« x,vi v,j |U| aii i/reauilelil/5.
Extractable parent.
c Extractable metabolites.
e Coefficient of Variation.
depth HCB was present exclusively as extractable metabolites and bound resi-
As shown in Tables 9 and 10, most of the material remainina in the olants
was unchanged parent HCB, with no detectable metabolites and an amount of
bound residue proportional to the dose applied to the soil An Tdentical
Gi^U T9S79b ^Plant^t T "" ?llo"d in a *«Vr ^ (G11« Sd
™llll\y}t^ --easing leA propor-
EFFECTS ON NUTRIENT LOSSES AND C02
<«•
of theentsanclung n f^ T l^ <«• J*
the four nutrients examined (Po!-3 NH rJ^ nnr(flClent^°f variatlon for
improvement, in comparison to Exptri^en?3! (Table in ^"n^11^ exhibited, som,e
-^s
18
-------
TABLE 9. PROFILE OF 14C ACTIVITY IN PLANTS AT TERMINATION (% OF TOTAL 14C
APPLIED)3
Pb
MH
Bd
0.25 Ib/a
20.0
0
2.1
0.50 Ib/a
19.8
0
2.2
1.0 Ib/a
12.9
0
1.9
a Mean of 15 cores for all HCB treatments.
Extractable parent.
Extractable metabolites.
Bound residues.
TABLE 10. PPM OF HCB AND BOUND RESIDUES IN PLANTS
0.25 Ib/a
P* 0.55
BD 0.06
0.50 Ib/a
1.08
0.12
1.0 Ib/a
1.38
0.20
TABLE 11. COEFFICIENTS OF VARIATION (CV). EXPERIMENT IIa
Parameter
P04-3
NH3
Ca+2
DOC
C.V.
0.10
;o.i5
0.07;
; .0.24
Experiment II: 0.25, 0.50 and 1.0 Ib/a HCB.
NOg-1 levels in leachate below detection limits.
levels was observed for the xylene carrier alone or with the^O.25 Ib/a equiv-
alent HCB A significant effect at the 95% level for the 0.5 Ib/a and at the
90% level 'for the 1.0 Ib/a treatment was observed, with both treatments reduc-
ing P04-3 export. NH3 loss was reduced by the xylene treatment at the 90%
level a'nd b/the 0.503and 1.0 Ib/a HCB treatment , at the 95% confidence level
Except for a reduction of Ca+* loss by the xylene
treatments had a significant effect at the 90% ^^^^H^f
treatment of HCB reduced the loss of DOC via the leachate at the 90* level.
19
-------
ro
o
100
o
Q.
X
<°
ro
X
c
o
•Control
l/4# HCB
-1/2* HCB
-I* HCB
• Xylene
10
treatment
100
o
o.
X
»
3
Q_
c
o
a>
10
treatment
4 5
Weeks
lOOOr r
o
CL
o
o
O
c
o
a)
100
o
a.
x
a>
O
O
O
o
a>
10
10
treatment
4 5
Weeks
6
8
O.I
treatment
4 5
Weeks
Figure 4. Mean nutrient levels in leachate for Experiment II.
-------
There appears to be a reduction in the amount of C02 evolved with all
levels of HCB and xylene alone followed by a temporary increase in C02 evolu-
tion and then another decline for the HCB treatments (Fig. 5).
As with Experiment I none of the treatment levels severely affected the
soil core. Normally HCB has been applied as a fungicide at much higher levels
and therefore should be tested at 1, 10 and 100 Ib/a equivalents to determine
the effects.
^
1
"c
o
o
<
e
CM
O
0
1.00
0.75
0.50
0.25
-0.25
-0.50
-0.75
-1.00
«
— 1 1 —
I/A& Hm
l/2# HCB
\ I#HCB
-\ Xylene
i\
• ^^
l.%\,
\$>^
/
V*'
-
treotment
j 4 5
Weeks
1
-
-
^^0
S^ **^^^.^
-
-------
SECTION 5
OVERVIEW
In these two experiments, which varied slightly from the suggested proto-
col (Gillett and Witt, 1979), an attempt was made to determine if fate and
effects of a chemical could be evaluated simultaneously in a single test and,
if so, could data be obtained more cost-effectively.
In terms of chemical fate, there were few surprises. More HEOD metabo-
lites were recovered than in any previous tests, but the small fraction °f"JB
metabolites found in other experiments (Gile and Gillett, 1979b; Metcalf,
1979) was absent. Soil degradation rates and volatility losses were con-
founded, at least partially, by the interim sampling techniques and inadequate
air monitoring. The analytical techniques employed radiometry (requiring
l4C-labeled chemicals and liquid scintillation apparatus), but could be
adapted to. standard analytical techniques to the extent that "^Jf ™°°* ^fTf
sensitive and available. This is less a limitation for pesticide registra
tion, where requirements are such that radio-labeled chemicals may be involved
in a variety of necessary studies, than for screening of toxic substances.
However, only radiometry can reveal the extent of bound residues in soil or
Plant and animal tissues. Otherwise, radiometry compares _favorably for the
determination of parent compound in relation to gas-liquid ^hro^°^aP^ith
regard to sensitivity, specificity, and cost Radiometry has decided advant
ages over other methods regarding metabolite detection, particularly as a
screen.
The main difficulty is the volume of the sample load (and associated
qualit? assurance) Experiment I involved the ^tenBination of parent and
metabolites by TLC and of bound residues by combust!on of •over 100 samples
and Experiment II involved over 150 samples Intermediate sampling Oj single
support. This level of effort seems necessary *o overcome the inherent varia
bility of the soil cores, even when they were collected from a single m area.
Performing soil studies on three different types of^oll.at Jour levels
with 12 cores at termination and three cores at each^weekly date (pre treat
ment, 1st, 2nd weeks after treatment) P^P02!1^^
treatment) controls would require analysis of approximately 400 cores
samples) and over 2000 samples of -leachate. ^-If an adequate «
system, such as a polyurethane plug, were added• "f sa"P,^ « w
vals-after treatment, an additional 225 analyses would be requ^ red
elites were identified or bound residues were ^herju^
level of effort would be required, but that wouia oe ueyui.u
23
-------
level envisioned for this test. As set forth above, such a test would cost
approximately $40,000/chemical , to yield the following information:
* Estimate of overall half-life of chemical applied to three different
soil types.
* Estimate of Teachability (qualitative) in three soil types.
* Quantitative estimate of soil volatility under this moisture regime.
* Qualitative picture of metabolites and bound residue and estimate of
mass balance by all known loss routes, with information on rates of
transformation and movement in soil.
* nutrientVss 1>revers1ble chan9e in te™s of soil respiration or
* nutHent P°SSlble reversible 1mPacts on soil respiration and
As more experience is gained with this testing procedure the "estimates"
given above can be associated with more speclffc^rUerll' of performance
These would be vital to efficient use of the SCM as a screening tEol Recom-
(Gtand ' that ^^on of m^crolosms be ^£-
predictive modeis'
communitv°Lsthe,n??H t0 eS\iBI?ie the impact of a chem1cal on a simple soil
1979 O^Neill et 1? T?7°7^ ^"^ ^ ^ ' rep°rted 1n G111ett and W1tt'
and irreversible SP 'ct 1-vV , mTCr°" and mac^nutrient losses are severe
of D?oceSSM (on a geological timescale) changes. Because of the
f
24
-------
at a lesser cost should be valuable. The purpose of the SCM test is not to
label a chemical "bad", but rather to provide a means of discriminating be-
tween levels of concern about problems of fate and effect. Depending on the
structure of a hazard evaluation system in which the SCM test results are
used, these results could justify further testing or permit by-passing such
testing.
25
-------
REFERENCES
Bostick, W. D. and B. S. Ausmus. (In Press). Methodologies for determination
of adenosine phosphates. Anal. Biochem.
Chiou, C. 1979. Oregon State University, personal communication.
Cole, L. K R L. Metcalf, and J. R. Sanborn. 1976. Environmental fate of
S?udiesC10*7-14n terrestrial mode1 ecosystems. Intern. J. Environ.
Draggon, S. 1976 The microcosm as a tool for estimation of environmental
transport of toxic materials. Intern. J. Environ. Studies. 10:76-70.
1979. The effects of substrate type and arsenic dosage level on
"A« t^n!naTr J" 9r?sslan? microcosms, Part I: Preliminary results on
n M wm rf ^v7^eStrJa\Mlcrocosms and Environmental Chemistry
(J. M. Witt and J. Gillett, eds.). Corvallis, Oregon. National Science
Foundation NSF/RA 79-0026, Washington, D.C. pp 102-110. 16nCe
EPA. 1977. Soil core microcosm test. IERL-RIP Procedures Manual- Level I
Environmental Assessment Biological Tests for Pilot Studies. EPA-600/
Gil6't^triad eco^s^^ch^Von^nL^^r^^T3 '™««
G11e'{™^ J^W'£^ sJKt^^fJSS1 FoS^Sem5 Jun^
Gil1creeJninWa Protocol T Vt }p ^ ^^X *'' So11 Core M1crocosm
Microcosms June VR i¥ iqS Proceedings of the Workshop on Terrestrial
Oregon- Nat1onal Sc-nce F—
Glllett, J. W., and J. D. Gile. 1976. Pesticide fate in a terrestrial labor-
atory ecosystem. Intern. J. Environ. Studies 10:15-22.
Jackson, D. R. , C. D. Washburne and B. S. Ausmus. 1977. Loss of Ca and
°f S011 '
Jackson, D. R. and J. M. Hall. 1978. Extraction of nutrients from intact
°* Che"1cal ''
26
-------
Johnson, E. L. 1978. Proposed guidelines for registering pesticides in the
United States. Federal Register 43:(132)29696-29741.
Lassiter, R. 1979. E.P.A. Environmental Research Laboratory, Athens, Ga,
personal communication.
Lee, J. J. and D. E. Weber. 1976. A study of the effects of acid rain on
model forest ecosystems: In: Proceedings of the Air Pollution Control
Association Annual Meeting.
Lichtenstein, E. P., T. W. Fuhremann and K. R. Schulz. 1974. Translocation
and metabolism of 14C-phorate as affected by percolating water in a model
soil-plant ecosystem. J. Agric. Food Chem. 22:99-96.
Lighthart, B., and H. Bond. 1976. Design and preliminary results from soil/
litter microcosms. Intern. J. Environ. Studies JJD: 51-58.
Metcalf, R. L , L. K. Cole, S. G. Wood, D. J. Mandel and M. L. Milbrath.
1979. Design and evaluation of a terrestrial model ecosystem for evalu-
ation of substitute pesticide chemicals. EPA-600/3-79-004. U.S. En-
vironmental Protection Agency, Corvallis, OR. 299 pp.
Muir, W. 1979. Toxic Substances Control Act. Premanufacturing Testing of
New Chemical Substances. Federal Register. 49:(53)16240-16292.
Nash, R. G. , M. L. BeaU, Jr., and W. G. Harris. 1977. Toxaphene and 1.1.1-
Trichloro-2,2-bis [p-chlorophenyl] ethane (DDT) losses from cotton in an
agroecosystem chamber. J. Agric. Food Chem. 25:336-341.
O'Neill, R. V., B. S. Ausmus, D. R. Jackson, R. I. Van Hooks, P. Van Voris, C.
D. Washburne and A. P. Watson. 1977. Monitoring terrestrial ecosystems
by analysis of nutrient export. Water, Air Soil Pollut. 8:271-277.
Rand, M. C., A. E. Greenberry and M. J. Taras (ed.). 1975. Standard Methods
for the Examination of Waste and Wastewater. 14th ed.
Snedecor, G. W. and W. G. Cochran. 1969. Statistical Methods (6th ed.). The
Iowa State University Press.
Spencer, E. Y. - 1973. Guide to Chemicals Used in Crop Protection (6th-ed.).
University of Western Ontario. Publication 1093. 542 p.
Spencer, W. F. and M. M. Cliath. 1969. Vapor density of dieldrin. Environ.
Sci. Techno!. 3:670.
Van Voris> P., R. v. O'Neill, H. H. Shugart, W. R. Emanuel. 1978. Functional
Complexity and Ecosystem Stability: An Experimental Approach. ORNL/
TM-6199. 120 p.
Wl>tt» J. M. and J. W. Gillett (ed.). 1979. "Terrestrial Microcosms and
Environmental Chemistry," The Proceedings of a Symposium on June 13-14,
1977, Corvallis, Oregon. National Science Foundation, NSF/RA 79-0026.
Washington, D.C. 147 pp.
27
-------
APPENDIX A
SOIL CORE MICROCOSM SCREENING PROTOCOL
The soil microcosm test should yield a reasonably rapid yes/no answer to
questions of short-term contaminant effects. Since terrestrial accumulation
sites and remineralization processes are predominantly within soil, intact
soil microcosms excised from representative target systems are used as test
units. The description which follows is taken from the work of Ausmus et aj.
(1977, as reported in Gillett and Witt, 1979).
Obtain soil cores 5- cm diameter x 10-cm depth from representative
terrestrial ecosystems. Encase the cores in a 1- to 3-mil-thick teflon
with 1- to 3-mil-thick shrinkable polyvinvyl chloride and gently heat
shrink until a tight bond with the core (minimum boundary flow) is
achieved. Leave enough lining above the soil surface to use gaseous
export traps if necessary. Mount on glass funnels in test tubes. Cover
sides with opaque wrappings to negate abnormal algal growth. Place in
environmental chamber under as near the field conditions as possible.
Equilibrate 3 weeks, if possible. Leach with rainwater or reconsti-
tuted water (known water chemistry) 2 to 3 times (enough to obtain 20 to
30 ml/date during equilibration). Determine Ca and dissolved organic
carbon (DOC) concentrations in these samples. If possible, use alkali
traps to determine daily C02, flux 3 to 10 days during equilibration.
Use these data to discard dissimilar replicate soil cores and establish
behavior of individual replicates.
Experimental design is preferably a randomized complete block and,
if possible, factorial treatment arrangement of dosages with a minimum of
three cores per dose per terrestrial ecosystem tested. "'Randomized incom-
plete block designs can be used to test a large number of contaminants
simultaneously. Dosages of a wide range should be used for this phase to
maximize the clarity of dose-dependent observations.
Add the test contaminant to the surface of the cores in a carrier,
such as soil (taken from replicate cores to those used as experimental
units). Dosages might be 0, 10, 100, 1000 ppm, for example, based on
core weights. Total amendment to all cores should be equivalent so that,
for ;example, a control replicate would receive carrier (such as soil)
equi valent to the carrier pi us contami nant received by a treatment dose.
Contaminant and carrier should be well mixed prior to uniform deposition
onto the core surface.
Set gas traps to monitor C02 recovery. Collect traps and titrate
with O.I MHC1 at 24-hour intervals, if'possible. The more frequent the
28
-------
measurement, the more complete data analysis can be, however, diurnal
rhythms make daily observation the minimum useful time period. Weekly
measures are practically useless.
After a week, add sufficient rainwater or reconstituted water (known
water chemistry), to collect approximately 20 ml of leachate per core.
Analyze Ca and DOC concentration. Determine contaminant concentration
using standard chemical techniques. On days 14 and 21 repeat leaching.
Perform intact extraction techniques for pools of nutrients left
within the core to estimate mass balances for microcosm units (Jackson
and Hall, 1978). This technique is the addition of 200 ml of 1.0 M KC1
or NaHC03 to cores and measurement of Ca, DOC, and the contaminant,
respectively, in the leachate.
Biotic analyses could also be conducted. Before extraction, core
samples 1 cm in diameter should be removed from the soil microcosms using
a cork borer. The hole should be filled by a glass rod of the approxi-
mate size When extraction for contaminant and nutrients is to be per-
formed.
Biotic analysis could use the ATP assay or adenylate energy charge
(Bostick and Ausmus, in press) which would allow relative microbial pools
to be compared across treatment levels. The procedure for ATP analysis
is to add 1 g of soil (wet weight) to 6 ml of pH 7.4 TRIS buffer with
0.06 g ethylenediaminetetraaecetic acid (EDTA). Vortex briefly. Add 3
ml of chloroform. Vortex again. Sonify in ice water 2 to 5 minutes.
Centrifuge (preferably at low temperature) at 100 x G for 2 to 10 min-
utes. Transfer buffer to new tube. Add 3 ml of CCL4. Recentrifuge
briefly. Sample buffer phase. Assay at 340 nm using standard hexokinase
reaction or a fluorescence spectophotometer (if sensitivity greater than
0.5 ppm is required).
Divide cores into 1-cm depths. Within each depth measure the amount
of contaminant by radiosotopic or standard chemical techniques. This is
an optional step, useful if the distribution of the contaminant is to be
estimated.
Both monitoring data and harvest data will be available on nutrient
processes and microbial activity.
Monitoring Results
Calculate the total export of Ca, DOC, and contaminant for each microcosm
by date using concentrations detected and volumes of leachate collected.
Calculate mean export (with standard error) by treatment dose for each contam-
inant. These data may be expressed as cumulative export and plotted as a
function of time. C02 efflux or other gaseous export data can be similarly
summarized and presented. Statistical comparison can be made by covariance to
determine the effect of treatment on export of nutrients and contaminant.
Previous studies show that C02 efflux and nutrient export often increase as a
function of dose. However, Ca and C02 release may be inhibited by some toxic
29
-------
otoxican nt sh™ biPhas]c behavior, depending on the concentration
dose Transport of the contarmnant is usually greater with increasing
Harvest Results
Calculate the extractable Ca, DOC, or contaminant based on concentrations
measured multiplied by extracted volumes. Calculate mean ^ (wTth standard
errors) across replicates for each treatment dose. Use standard ana vs" of
variance of Duncan's Range Test to determine differences du * to treatmen?
Biotic data may be summarized as ATP per gram of soil by 1-cm depth intervals
for each dosage. ATP concentrations may be increased I or decreased bv thl
contaminant, depending on the specific microbial population impacted
30
-------
Soil Core Microcosm Experiment I
Purpose
The purpose of this experiment is to yield data reflecting the fate and
effects of 14C dieldrin, parathion and 2,4,5-T on a soil ecosystem and to
determine the suitability of the soil core microcosm as a screening tool.
Experimental Approach
Select a 1 sq meter area in the field behind the CERL trailers and clip
grass down to surface: this will serve as the source for the soil cores.
There will be 15 cores per treatment for a total of 60 including control
treatment.
Obtain soil cores 5 cm dia x 10 cm depth and place each in a sealed
plastic bag for transport to lab. Once in the laboratory remove cores and
clip above ground vegetation down to soil surface. Fit each core with poly-
ethylene base and encase in Teflon and shrinkable PVC and gently heat shrink
until a tight bond with the core is achieved. Leave approximately 5 cm of
tubing above soil surface to use with gaseous export traps if necessary.
Mount on glass funnel (seal interface of core and funnel with silicon rubber)
in 250 ml flask. Place in incubator adjusted for field conditions or in
greenhouse.
Equilibrate cores for 28 days. Leach with std ref water (i.e. standard
ref. rainwater) on days 7, 14, 21 and 28 (enough to obtain 20 to 30 ml/date).
NH3; NOs-1 and P04-3 will be determined via Technicon Auto Analyzer by
Northrop personnel. Northrop personnel will prepare and deliver approx. 20 ml
of leachate to LASS for Ca+2 and DOC analysis. Certified standards and blanks
will be run with all samples. The certified standards and methods for prep-
aration are available from W. Griffis (LASS). Standards and blanks should be
verified routinely Exceptional care must be exercised in the preparation of
all samples due to the high probability of contamination from both the human
body and local environment. Also use KOH traps to determine C02 efflux for 24
hr on Monday and Thursday.
On day 28 (after leaching) apply pesticides at the rate of 1 Ib/acre (15
cores/treatment). Use standard carriers for dieldrin, parathion and 2,4,5-T;
treat control with dieldrin carrier. Use a pipette to apply material evenly
to surface of core; rinse pipette and apply rinse to core.
Use 0.2 N KOH traps for C02 efflux, collect traps and titrate with 0.1 M
HC1 for 24 hrs. on every Monday and Thursday.
31
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On day 35 add sufficient std. ref. water to collect 30 ml of leachate/
core. Analyze for same parameters as during equilibration period plus oesti-
cide content, repeat process on days 42 and 49 for all of the above oara-
meters. H
On days 28, 35, 42 and 49 remove 1 core from each treatment at random,
subdivide into three layers, 0-2 cm, 2-5 cm and 5-10 cm. From top layer
remove all biotic material (plant root and shoot and visible animal species)-
weigh and analyze for pesticide. Perform intact extraction of all 3 levels
for pools of nutrients left within the core. Use either 1 M KC1 or NaHC03 for
extraction. After nutrient extraction treat soil samples by prescribed meth-
ods for 14C analysis. Nutrient extraction should also be analyzed for 14C.
On day 56, terminate cores and perform intact extraction process and 14C
analysis.
iFor 3 treatments + control there will be approximately 380 leachate
samples, 960 C02 samples and 180 (60 x 3 subsamples) destructive samples.
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Soil Core Microcosm Experiment II
Purpose
The purpose of this experiment is to yield data reflecting the fate and
effects of 14C labeled HCB on a soil ecosystem, as well as continuing evalua-
tion of the soil core system as a potential screen for TSCA.
Experimental Approach
Select a 5-sq meter area at Schmidt Farm, free of agricultural chemicals.
This will serve as the source for the soil cores. There will be 21 cores per
treatment level (3 levels: 0.25, 0.5, and 1 Ib/acre) for a total of 84 in-
cluding control treatment.
Obtain soil cores 5-cm dia x 8-cm minimum depth and place in a sealed
plastic bag for transport to lab. Once in the laboratory remove cores and fit
each with a polyethylene base and encase in shrinkable PVC. Gently heat
shrink until a tight bond with the core is achieved. Leave approximately 5 cm
pf tubing above soil surface to use with gaseous export traps if necessary.
Mount on glass funnel (seal interface of core and funnel with silicon rubber)
in 250 ml flask. Place in growth chamber adjusted for field conditions.
Equilibrate cores for 28 days. Leach with std. ref. water (i.e. standard
£ef. rainwater) on days 7, 14, 21 and 28 (enough to obtain 30 ml/date).
Determine NOa-1, NH3 and P04-3 concentrations of leachate via Techicon Auto
Analyzer by Northrop personnel. Northrop personnel will prepare and deliver
approx. 20 ml of leachate to LASS for Ca+2 and DOC analysis.
Use 0.2 N KOH traps for C02 efflux, collect traps and titrate from 5
cores/treatment with 0.1 M HC1 for 24 hrs on every Monday. Certified stan-
dards and blanks will be "run with all samples. The certified standards and
methods for preparation are available from W. Griffis (LASS). Standards and
blanks should be verified routinely. Exceptional care must be exercised in
tne preparation of all samples due to the high probability of contamination
from both the human body and local environment.
On day 28 (after leaching) apply HCB at the prescribed rate (15-18 cores/
treatment). Use standard carrier for HCB, treat 1/2 control with carrier and
balance with distilled H20. Use a pipette to apply material evenly to surface
°f core, rinse pipette and apply rinse to core.
°n day 35 add sufficient std. ref. water to collect 30 ml of leachate/
Analyze for same parameters as during equilibration period plus pesti-
cide content, repeat process on days 42 and 49 for all of the above para-
meters.
.0" days 28, 35, 42 and 49 remove 1 core from each treatment at random,
ivide into three layers, 0-2 cm, 2-5 cm and 5-8 cm. From top layer remove
33
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~
56 tpJin ,analyS1S' Nutrl6nt extract1on sh°"ld be analyzed for "C. On day
56, terminate cores and perform intact extraction process and 14C analysis.
F°r ?/,ntr™tments + contro1 tnere Will be approximately 480 leachate
es, 140 C02 samples and 160 (54 x 3 subsamples) destructive samples.
34
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APPENDIX B
Standard Reference Rainwater Formula (Lee and Weber, 1976).
__ Tne standard "rain" solution was made from deionized distilled water
CuS04, MgCl2, KC1, NaCl and NH4N03 to give the following compositon.
Ca+2 0.22 mg/1
NH4+ 0.22 mg/1
Na+ 0.11 mg/1
K+ 0.06 mg/1
Mg+2 0.08 mg/1
S04-2 0.48 mg/1
N03- 0.74 mg/1
Cl- 0.53 mg/1
35
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Treatment
APPENDIX C
QUALITY CONTROL IN PROCEDURES FOR SOIL CORE MICROCOSMS
=
the AA II system.
Setup:
1. Are the manifolds set up
sizes and transmission li
ties.)
2. Are the reagents freshly made and of the correct composition?
3. Is the heating bath on? (If required.)
36
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4. Are the correct filter and cell installed in the colorimeter?
5. Is the colorimeter on?
Operation:
1. Is the proportioning pump on?
2. Are the air bubbles evenly spaced?
3. Is the sampling rate correct?
4. Is the sampler on?
5. Is the recorder on?
6. Is the baseline smooth and stable?
7. Is the digital printer on?
Results
1. Do the standards give peak heights in the proper range and are the
results linear in that range?
2. Do the QC samples give proper values?
Maintenance
1. The pump tubes should be changed each week.
2. The proportioning pump should be lubricated as per the Technicon
manual. ; .>
Pesticide Residue Analysis.
The following fractions of the SCM's are analyzed separately for pesti-
cide residues: leachates, soil, plant material, polyvinylchloride and masking
tape. Liquid scintillation spectrometry is used to trace the residual mater-
lal in the samples. Each type of sample is analyzed using a specific proced-
ure. The activity of the resulting fractions is then determined. Those that
are found to have sufficient activity are analyzed further by thin-layer
cnromatography and autoradiography. The residues found in this manner can be
classified in one of the following groups: intact pesticide, non-polar metab-
olite, polar metabolite and nonextractable (bound) residue. From the data
acquired the biodegradability index and ecological magnification for each type
or sample can be determined.
. ^ne following brief list of precautions in the analysis for pesticide
residues will assist in the maintenance of quality control.
37
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Analysis: (intact pesticide, polar and non-polar metabolites)
1. Is it the proper procedure for the type of sample?
2. Is there complete transfer of material?
3. Are the measurements of masses and volumes accurate?
4. Is all data written down promptly and correctly?
Sample Oxidation: (non-extractable bound residue)
1. Start-up of Packard 306 Sample Oxidizer:
a. Has the waste jug been emptied?
b. Is the methanol gas trap full?
c. Are the reagent reservoirs full?
d. Do the N2 and 02 cylinders have sufficient pressure?
e. Is the distilled water reservoir full?
f. Gas cylinders on?
g. Power on?
h. Is the distilled water switch on PRESSURE?
2. Operation:
a. Do the reagent dispensers have the proper settings? Are thev
working properly?
b. Does the timer have the proper setting? Is it working proo-
erly? -•••
c. Is the ignition basket in good condition? (Clean? Coils
properly spaced?)
d. Are the samples burning properly? (Sufficient oxygen and/or
burn time? High voltage switch stuck? Are there leaks in the
system? Leaking 4-way valve? Pneumatic mechanisms operating
properly?) r s
3. Shutdown:
a. Is the power off?
b. Is the distilled water switch on VENT?
38
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c. Gas cylinders off?
d. Is the instrument cleaned up?
4. Results:
a. Are the AES ratios consistent and in the proper range?
b. Are the values for samples and spikes reasonable?
c. Is the recovery good?
d. Is there low carryover?
Sample Counting:
1. Set-up of the Packard 3385 Liquid Scintillation Spectrometer:
a. Are the samples set up properly?
b. Is there a blank of the same cocktail in each group of samples?
c. Is the channel set for the proper isotope?
d. Are the preset count and preset time of their correct values
for the results desired?
e. Is the desired information printed out?
2. Results:
a. Are the AES values consistent with the type of cocktail being
used and the expected amount of quenching? .
b. Are the activity values reasonable?
c. Is the background value reasonable?
Thin-Layer Choromtography and Autoradiography: The following is a brief
outline of the procedures used to analyze extracts by thin-layer chromatog-
raphy and auto-radiography and precautions taken to assure good results.
1. Thin-Layer Chromatography
Precautions Sample Procedure
1. The volume is measured out which will give 104
dpm or the entire sample if the total activity
is between 10s and 104 dpm.
Correct
volume?
39
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2.
100%
transfer of
material?
3.
4.
Material
left on
column?
100%
transfer of
material?
Proper
solvent
system used?
The volume is reduced (if necessary) to 20 ml
on the rotary evaporator.
The volume is reduced further (if necessary) to
2 ml with a stream of nitrogen.
If necessary, the sample is filtered through a
small column of anhydrous Na2S04.
5. The sample is spotted.
6.
7.
104 dpm of the reference standard is spotted.
The plate is developed.
2. Autoradiography
Precautions Developed Plates
Correct 1.
orientation
in box?
Chemicals in
good condition?
Proper
temperature used?
Correct film-
paper
orientation?
Correct paper-
plate
orientation?
Accurate spot
removal?
2.
3.
4.
5.
Procedure
The plates are placed in box with x-ray
film (stacked alternately) and allowed to
sit 4 weeks.
After 4 weeks the film is developed.
A trace is made of all darkened spots on
the film. The spots are numbered and their
distances from the origin measured.
All identified spots are removed from the
plates and placed in scintillation vials.
Cocktail is added to the vials and the
samples are counted.
°f
1s Spent °n some asPect of
40
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DEPORT NCX " ~
EPA-600/3-79-089
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
4. TITLE ANDSUBTITLE
2.
The Soil Core Microcosm - A Potential Screening Tool
3. RECIPIENT'S ACCESSION NO.
5. REPORT DATE
August 1979 issuing date
6. PERFORMING ORGANIZATION CODE
AUTHOR(S)
Jay D. Gile
James W. Gillett
James C. Collins,
Northrop Services
8. PERFORMING ORGANIZATION REPORT NO.
PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Corvallis, OR 97330
10. P
Wft
ELEMENT NO.
11. CONTRACT/GRANT NO.
-. SPONSORING AGENCY NAME AND ADDRESS
same as #9
13. TYPE OF REPORT AND PERIOD COVERED
Final 6/78 - 7/79
14. SPONSORING AGENCY CODE
EPA/600/2
ELEMENTARY NOTES
This report presents the results of two experiments performed at CERL in an effort to
uetermine the suitability of a soil core microcosm as a screening tool under FIFRA.
ine soil core microcosm consisted of a 5 x 10 cm soil core removed intact from a field
site and encased in PVC. In Experiment I, 0.25 Ib/ac applications of ^C-labeled
aieidrin, methyl parathion and 2,4,5-T were examined, whereas 0.25, 0.50 and 1.0 Ib/a
Mn icaJn°ns of HCB weCe studied in Experiment II. Weekly leachates were analyzed for
NQs- , POi,-1, NH3, Ca+2 and DOC as well as '"C. The majority of the chemicals from
both experiments found in the soil were in the top 2 cm. Extractable metabolites were
oetected for all but HCB. 2,4,5-T had practically no effect on any nutrient losses.
INU3- loss was impacted by dieldrin and methyl parathion. PC*-3 was impacted by
methyl parathion, 0.5 Ib/a HCB and 1.0 Ib/a HCB. NHa was Impacted by the two upper
levels of HCB. Calcium export was altered by methyl parathion and dieldrin. DOC was
significantly impacted by methyl parathion and 1.0 Ib/a HCB. Treatment levels for all
cnemicals were below normal application rates and did not really challenge the system.
It is possible to gain some assessment of chemical effect on a soil ecosystem, its
ate and metabolism with the soil core microcosms.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
Pesticides
Toxics
Soil core
Screening tool
Nutrient cycling
i-nermcal movement and transformation
18~EL I Ml BUI ION STATEMENT "
Release to Public
EpA Form 2220-1 (Rer. 4_
b. IDENTIFIERS/OPEN ENDED TERMS
Alternative chemicals
program
Laboratory microcosms
19. SECURITY CLASS (ThisReport)
unclassified
20.
c. COSATI Field/Group
06/F
21. NO. OF PAGES
50
22. PRICE
77)
PREVIOUS EDITION IS OBSOLETE
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