ot
May ' 992
Mien
-------
EPA/600/R-92/066
May 1992
WORKSHOP: APPLICATION OF MICROCOSMS FOR ASSESSING THE RISK OF
MICROBIAL BIOTECHNOLOGY PRODUCTS
May 19, 1992
Hunt Valley, Maryland
edited by
C.R. Cripe and P.H. Pritchard
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, Florida 32561
A.M. Stern
U.S. Environmental Protection Agency
Office of Toxic Substances
Health and Environmental Effects Division
Washington, DC 20460
U.S. ENVIRONMENTAL PROTECTION AGENCY
OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL RESEARCH LABORATORY
GULF BREEZE, FLORIDA 32561
-------
Disclaimer
The information in this document has been funded wholly or in part by the United
States Environmental Protection Agency. It has been subject to the Agency's peer and
administrative review, and it has been approved for publication as an EPA document.
Mention of trade names or commercial products does not constitute endorsement or
recommendation for use.
-------
Abstract
The U.S. Environmental Protection Agency (EPA) develops testing methods to
support assessments of the environmental risks associated with the release of microorgan-
isms and microbial pest control agents. Microcosms may be used as one step in the
progression of product development from laboratory to field experimentation. The utility
of microcosms in this process is, in some measure, dependent on the capacity of the test
system to simulate environmental complexity, and consequently, to provide relevant
answers to questions of environmental concern that may be raised by the regulatory
community. The usefulness of current microcosm systems to evaluate and provide relevant
information on a variety of regulatory endpoints pertinent to environmental risk assess-
ment of microbial products was examined by workshop participants who met at Hunt
Valley, MD, on January 23-27, 1989. A total of 14 generic and site-specific microcosms,
portraying terrestrial and aquatic habitats with varying degrees of ecosystem complexity,
was examined. The endpoinis of ecological effects and other performance characteristics
were compared for each microcosm system. Finally, future directions of microcosm
research that appear to be required to fill gaps in the state-of-thc-scicncc were recom-
mended.
HI
-------
Contents
Page
Abstract iii
1. Introduction 1
2. Workshop Background 1
3. Workshop Objectives 1
4. Microcosm Descriptions 2
5. Research Needs , 2
6. Summary 4
Appendices
A, Workshop Participants 5
B. Aquatic Microcosms 7
1. Benthic-Pelagic Microcosm 9
2. Compartmentalized La^r Microcosm 19
3. Mixed Flask Culture Microcosm 29
4. Pond Microcosm 37
5. Sediment Core Microcosm 45
6. Standard Aquatic Microcosm 57
7. Stream Microcosm 67
8. Waste Treatment Microcosm 77
***ซ***ซ*ซ*ซ*ซป**ซซ**ซ********** 8 /
1. Root Svstem Microcosm _.._ .. __ ... ~. .~... 89
J u mm 11 ปซ***ปปป w ป ปซปซซปซปปซ ป****ป>ปป v v
2. Soil Core Microcosm ..-..ซ.................ซ...^..^._ซ_..........^ 99
3. Soil in a Jar Microcosm 109
4. Terrestrial Microcosm Chamber 119
5. Terrestrial Microcosm System ^. 129
6. Versacore Microcosm -.._..................................~.............. 137
-------
Application of Microcosms for Assessing the
Risk of Microbial Biotechnology Products
1. Introduction
The EPA, under the Toxic Substances Control Act (TSCA)
and the Federal Insecticide, Fungicide, and Rodenticide Act
(FTFRA), is charged with the regulation of microbial biotech-
nology products such as genetically engineered microorgan-
isms (OEMs) and microbial pest control agents (MPCAs) that
might be released to the environment. EPA's regulatory pro-
grams developed to evaluate that risk must be able to analyze
data gathered from a variety of experimental approaches
ranging from relatively simple laboratory studies to more
complex field studies.
Field studies, although providing relevant information
concerning a particular site, suffer from many drawbacks: (1)
they are subject to disruption by meteorological events, (2)
they do not allow easy examination of the influence of indi-
vidual variables (e.g., temperature, nutrients, soil composi-
tion, water content) on the interactions of introduced microor-
ganisms with their environment, and (3) introduction of mi-
croorganisms at a field test site for research purposes may, by
itself, pose an unacceptable risk.
Evaluations of chemical fate and effects have utilized
laboratory test systems, such as microcosms, to provide risk
assessment information while avoiding some of the problems
of field testing. Some test systems are simple enough to offer
the advantages of replication and experimental manipulation
while maintaining sufficient complexity to include many im-
portant ecosystem processes.
Tests conducted in microcosms may be diagnostic in
themselves or a surrogate for small-scale field testing, thus
allowing regulatory decisions to be made on laboratory-scale
testing and reducing the time and expense of the first stage of
field testing.
In assessing the risks of microorganisms, the Office of
Toxic Substances (OTS) and the Office of Pesticide Programs
(OPP) currently use separate but similar criteria called "Points
to Consider" (regulatory endpoints) which outline the catego-
ries of information that are useful in risk assessments by the
EPA. Although the OPP and OTS lists differ somewhat,
overall risk assessments address similar issues. Some experi-
mentally-derived information that would satisfactorily ad-
dress the points in the lists may be obtained by testing in
microcosms. It is, therefore, appropriate to evaluate the use-
fulness of the quality and quantity of information that micro-
cosm systems can provide relative to these regulatory end-
points. This document summarizes such an evaluation per-
formed by a group of scientists.
2. Workshop Background
The Microcosm Workshop was a joint effort of EPA's
Office of Research and Development (ORD) and the Office of
Pesticides and Toxic Substances (OPTS). Fourteen micro-
cosm systems judged appropriate for testing the fate and
potential ecological effects of introduced microorganisms were
selected before the workshop for discussion by the partici-
pants. These microcosms were not chosen to represent the
entire field of appropriate test systems but, rather, to be
representative of systems that had provided useful informa-
tion for chemical risk assessment or that were specifically
designed-for testing microorganisms. A brief description of
each microcosm was contributed by its developer before the
workshop.
Participants worked in both plenary sessions and small
subgroup sessions to generate information about the potential
uses and limits of the selected microcosms with respect to the
assessment of the survival and ecological effects of intro-
duced microorganisms as well as their potential for transfer-
ring genetic material to indigenous microorganisms. Work-
shop participants also identified areas in microcosm technol-
ogy where further research was required to expand microcosm
applicability or to increase confidence in data outputs. This
information was supplemented by the results of_ question-
naires distributed to microcosm developers which requested
more details about their test systems after the workshop had
concluded.
3. Workshop Objectives
The overall workshop objectives were to determine the
current state-of-the-art in microcosm design and to ascertain
the extent to which microcosms could be applied to biotech-
nology risk assessment Specific goals were to:
1. Identify the most appropriate of the currently
available microcosms to evaluate the fate and
effect parameters of microorganisms released to
the environment.
2. Provide sufficient information to allow
assessment of advantages and disadvantages of
each microcosm with respect to:
-------
Table 1. Summary of test systems examined by workshop.
Name of Microcosm
Benthic-Pelagic
Compartmentalized Lake
Mixed Flask Culture
Pond
Sediment Core
Standard Aquatic
Stream
Waste Treatment
Root System
Soil Core
Soil in a Jar
Terrestrial Chamber
Terrestrial System
Versacore
Habitat
Marine
Freshwater
Freshwater
Freshwater
Marine
Freshwater
Freshwater
Wastewater
Terrestrial
Terrestrial
Terrestrial
Terrestrial
Terrestrial
Terrestrial
Oeveloper(s)
Perez
Kroer
Shannon
GkJdings
Prtehard/Clark
Taub
Bon
Gealt
Klein
Fredrickson
Stotzky
Gillett
Seidler/Armstrong
Holben/Jansson
Page
9
19
29
37
45
55
65
75
87
97
109
119
129
137
a. Potential for, and confidence in, the ex-
trapolation of laboratory data to field pre-
dictions with regard to critical fate and ef-
fect endpoLnts.
b. Cost and expertise required to construct and
operate the microcosms.
c. Potential for the development of possible
modifications to expand microcosm utility.
3. Identify gaps in current knowledge regarding
microcosm development and application for
biotechnology risk assessment
4. Microcosm Descriptions and
Questionnaire Design
Selection of an appropriate microcosm design to assess
the potential environmental risk of a microorganism requires
knowledge of microcosms that have demonstrated value in
other types of risk assessment activities (e.g., chemical ef-
fects, fate, transport). A questionnaire was completed by
developers of each of the 14 microcosms listed in Table 1 to
provide specific information about their potential use in as-
sessing the risk of microbial biotechnology products. Collat-
ing this information produces a useful, structured, comparison
of these systems relative to risk assessment needs and to each
other.
The queaoanaire examines general characteristics of
each test system: a description of the physical design and size,
lighting, temperature control, purpose for which microcosm
was originally designed, habitat represented as well as trophic
levels and method of establishing communities, sampling of
environmental media, provisions for air or water exchange/
circulation, equilibrium period prior to use, lifespan of test
system, and environmental parameters routinely monitored.
These ancillary details may find important application in
simulation or assessment modeling.
Questions concerning containment focus on whether cur-
rent designs are adequate for working with genetically engi-
neered microorganisms or if specific modifications would
improve containment. A section on protocols details the de-
velopment of standard operating procedures for microcosm
construction, operation or output analysis. Modifications (other
than those related to containment) that would improve a test
system's use for risk assessment are solicited. Sampling strat-
egies (repetitive, destructive, etc.) are examined, along with
information on test system cost
A section on applicability for evaluating ecological pa-
rameters describes techniques that have been used to monitor
five types of ecological effects factors in the test system:
communky structure, trophic interactions, energy flow, bio-
geochemical cycling, or other effects. Results of field calibra-
tion tests (comparison of the responses of ecological param-
eters in microcosms with the field in the absence of stress
agents) for each of these five factors was also solicited, as was
information on problems encountered with making these com-
parisons.
A final questionnaire section addresses field verification
studies; these are tests with genetically engineered organisms
or surrogate organisms to compare survival, colonization, and
microbial/gene mobility observed in microcosms with those
observed in the field.
Microcosm questionnaire responses are grouped accord-
ing to aquatic or terrestrial application (Appendix B and C,
respectively). At the end of each summary is listed additional
information such as the name, address, and telephone number
of the microcosm developer or contact person, pertinent pub-
lications, protocols, CHher documents relating to the micro-
cosm, data that have been derived from its use, and, if avail-
able, a diagram of the test system.
It is acknowledged that any microcosm selected for a risk
assessment application may incorporate specific features (such
as size, containment, or ecological endpoints) from one or
more of the 14 systems examined here or elsewhere, to
ouuicoS QUbOuOilS uIU^UC 1C tปC
-------
microorganisms and microbial biotechnology products, but
they also generated substantial lists of related research efforts
required to maximize the utility of microcosms for this pur-
pose. These suggestions have been incorporated into narra-
tives of microcosm research topics.
5.2 Conducting Comparison Studies
More studies of field calibration (baseline studies of
various ecological parameters that are observed in a micro-
cosm in the absence of a stress agent relative to those ob-
served in the field) and field validation (comparison of stress-
response relationships among ecological endpoints in a mi-
crocosm and in the field in the presence of a specific stressor)
are needed to improve confidence in the ability to extrapolate
microcosm-derived data on microbial fate, effects, and/or
gene transfer to field data. Seasonal information from micro-
cosm studies of site-to-site comparisons between geographi-
cal areas and among habitats is also required to examine the
effects of spatial and temporal variability. Effects of succes-
sional changes in microcosms and extrapolation of those data
to natural systems also should be examined.
Comparisons between different types of microcosms uti-
lizing a variety of endpoints should substantially improve the
selection and design features of test systems used for specific
risk assessments. Finally, interlaboratory comparisons of the
same test systems were suggested to assess lab-to-lab variabil-
ity.
5.3 Evaluating Increased Test System
Diversity and Interactions
It is necessary to expand the scope of microcosm research
to include the study of higher trophic levels, greater species
diversity, and community-level ecological processes and in-
teractions. Although careful consideration must be given to
cost effectiveness, such expansions would appreciably in-
crease the utility of microcosms and the relevance of the data
obtained from their use.
5.4 Developing Mathematical Models and
Appropriate Statistical Methodologies
A greater emphasis on the development of field-validated
mathematical models to enhance the ability to extrapolate fate
and effects data obtained with microcosms to a field site is
required. Development and application of appropriate quanti-
tative methods to measure the effects of potential perturbation
of ecosystems with respect to specific variables as they vary in
both laboratory test systems and in the field is also necessary
to achieve a sufficient level of confidence in the use of
microcosms and models.
The lack of appropriate aquatic transport microcosms
suggested a special need for hydrodynamic modeling, as it
relates to microbial transport; chemical and particle move-
ment models are probably not aHgqijg^. for this purpose.
Effects of factors such as microbe size, shape, and physical
surface characteristics on physical transport should be exam-
ined.
5.5 Developing Test Organisms/Markers
Model test organisms (Le., bacteria, fungi, viruses) with
appropriate markers for assessing fate and effects must be
identified and developed. Methods of detection, must be
improved, and the spectrum expanded and tested for applica-
bility to different types of microcosms and field tests. Markers
should not pose an ecological (or health) risk or affect micro-
cosm structure or function.
There is also a need to develop techniques to measure the
movement and expression of genetic material introduced into
a microcosm.
5.5 Identifying New and Relevant Endpoints
Additional development of structural and functional end-
points, especially those requiring non-destructive sampling
techniques, is needed. The scope of the endpoints should
allow testing for ecological effects that include investigating
the increased susceptibility of a system to secondary distur-
bances (e.g., invasion, chemical stress, physical stress) when
the microbial agent is introduced simultaneously with, or
subsequent to, the introduction of a secondary stress agent
5.7 Basic Microbial Ecology
Limitations in the understanding of microbial ecology
remain one of the most serious hindrances to microbial risk
assessment. For example, a variety of factors that control
microbial production and biomass (e.g., substrate and preda-
tor control) may be known, but the extent of their influence,
and the effects introduced in a system as a consequence of the
interactions taking place among its components, are not known.
5.8 Microcosm Design and Testing
Considerations
The mode and magnitude of introduction of a microbial
agent may affect fate, ecological effects, or transfer of novel
genetic material and, thus, should be considered typical vari-
ables in microcosm testing.
It is not clear which or how many environmental vari-
ables (e.g.. temperature, light, water content) should be mea-
sured and controlled for microcosm tests, although this will
probably depend on the specific application. The degree of
environmental control necessary for field comparisons also
needs to be determined.
The effects of measures to contain microbial biotechnol-
ogy products may reduce the capability of a microcosm to
simulate a real ecosystem. Likewise, containment of a field
test site may alter normal community structure or functions;
this potential should be considered when comparing results
from a microcosm with those in a field test.
5.9 Final Considerations
The successful use of microcosms and models for risk
assessment will depend on definitive articulation of the objec-
tives of a particular application. For example, attention must
be given to study objectives (e.g., screening vs. a more
definitive assessment) and to the degree of detail required
(e.g., the required levels of confidence and ability to extrapo-
late to the field) to meet these objectives. Such decisions
affect the practicality of expanding the scope of microcosm
research and the further development of mathematical models
and microcosms to accommodate this expansion.
-------
6. Summary
Fourteen microcosm designs, using a variety of terrestrial
and aquatic habitats, are described. Most systems were origi-
nally designed to assess the fate/effects of xenobiotic com-
pounds. Only a few were actually developed with microbial
biotechnology risk assessment in mind, but all should provide
some useful information in evaluating microbial products.
Initially, the workshop focused on the suitability of each
microcosm to assess persistence, ecological effects, or ex-
change of novel genetic material. However, it became appar-
ent that confidence in these assessments must be tempered by
gaps in our knowledge of microbial ecology. A variety of
relevant research topics was compiled by each subgroup to
address the information necessary for risk assessment testing
of microbial biotechnology and for interpretation of test re-
sults.
The 14 microcosms described in Appendices B and C
provide a basis for the selection of microcosm designs appro-
priate for specific applications. These systems should be
viewed as tools for the generation of some of the information
necessary for microbial biotechnology risk assessment Vari-
ous aspects of a selected system (e.g.. trophic levels, structural
or functional endpoints, physical habitat) may have to be
modified to answer a specific question. Information provided
by such microcosms will only be as applicable for extrapola-
tion to the natural environment as the ecological processes
included in the test systems. Thus, field calibration and field
verification remain two of the most critical components of
microcosm development and testing.
-------
Appendices
Appendix A
Workshop Participants
Dr. Dick Anderson
U.S. Environmental Protection Agency
Environmental Research Laboratory
6201 Congdon Blvd.
Duluth, MN 55804
(FTS) 780-5616
Dr. Thomas Boa
Stroud Water Research Center
Division of Environmental Research
Academy of Natural Sciences
R.D. #1 Box 512
Avondale, PA 19311
(215) 268-2153
Dr. James Clark
Environmental Toxicology Division
Exxon Biomedical Sciences, Inc.
Mettlers Road, CN 2350
East Millstone, NJ 08875-2350
(908)873-6039
Dr. Rick Coffin
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, FL 32561
(FTS) 228-9255
Mr. Rick Cripe
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, FL 32561
(FTS) 228-9340
Dr. Bob Frederick (RD-682)
U.S. Environmental Protection Agency
401 "M" Street, S.W.
Washington. DC 20460
(FTS) 382-5989
Dr. James Fredrickson
Bauelk Pacific NW Laboratories
Po.O. Box 999
Richland, WA 99352
(509) 375-3908
Dr. Michael Gealt
Depc of Bioscience & Biotechnology
Drexel University
32nd and Chestnut Streets
Philadelphia, PA 19104
(215) 885-5092
Dr. Jeffrey Giddings
Springborn Bionomics. Inc.
790 Main Street
Wareham, MA 02571
(508) 295-2550
Dr. James Gillett
16 Femow Hall
ICET Cornell University
Ithaca, NY 14853-3001
(607) 255-2163
Dr. Mike Heitkamp
Monsanto Company
800 LJndberg Ave.
SL Louis, MO 63167
(314) 694-3296
Dr. William Holben
Michigan State University
East Lansing, MI 48824
(517)355-9282
Dr. Donald Klein
Colorado State University
Depc of Microbiology
Fort Collins, CO 80523
(303) 491-6947
-------
Mr. Niels Kroer
National Environmental Research Institute
Department of Marine Ecology and Microbiology
Frederiksborgvej 399
P.O. Box 358
DK4000 Roskilde, DENMARK
(45 46 30 13 88)
Dr. Wayne Landis
Institute of Environmental Toxicology
and Chemistry
Huxley College
Western Washington University
Bellingham, WA 98225
(206) 647-6109
Dr. Mark Luckenbach
Virginia Institute of Marine Science
College of William and Mary
Gloucester Point, VA 23062
(804) 642-7000
Dr. Robert Miller
Loyola University
Department of Biochemistry and Biophysics
2160 South First Avenue
Maywood, EL 60153
(312) 531-3360
Dr. Vincent J. Nabholz (TS-796)
U.S. Environmental Protection Agency
401 "M" Street, S.W.
Washington, DC 20460
(FTS) 3824271
Dr. P.H. Pritchard
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze. FL 32561
(FTS) 228-9260
Dr. Gary Sayler
University of Tennessee
Microbiology/Ecology Department
10515 Research Drive
Suite 200
Knoxville,TN 37996
(615) 974-5219
Dr. Mark Segal CTS-796)
U.S. Environmental Protection Agency
40i "M* Strees, S.W.
Washington, DC 20460
(FTS) 382-3389
Dr. Ray Seidler
U.S. Environmental Protection Agency
Environmental Research Laboratory
200 S.W. 35th Street
Corvallis, OR 97333
(FTS) 4204661
Dr. Lyle Shannon
University of Minnesota
Biology Department
Duluth, MN 55812
(218) 726-8000
Dr. Francis Sharpies
Oak Ridge NAS Laboratory
P.O. Box Y
Oak Ridge, TN 38731
(615) 576-0524
(615) 691-0452
Dr. Frank Stay
U.S. Environmental Protection agency
Environmental Research Laboratory
6201 Congdon Blvd.
Duluth. MN 55804
(FTS) 780-5542
Dr. Art Stern (TS-796)
U.S. Environmental Protection Agency
401 "M" Street, S.W.
Washington, DC 20460
(FTS) 382-4281
Dr. Guenther Stotzky
New York University
Department of Biology
1009 Main Washington Square
New York, NY 10003
(212) 998-8266
Dr. Glenn W. Suter n
Environmental Sciences Division
Oak Ridge National Lab
Oak Ridge. TN 37831
(615) 574-7306
Dr. Frieda Taub
School of Fisheries, HF-15
University of Washington
Seattle, WA 98195
(206)682-2115
Dr. In-Soon You (TS-796)
U.S. Environmental Protection Agency
401 "M" Street, S.W.
Washington. DC 20460
(FTS) 3824237
-------
Appendix B
Aquatic Microcosms
-------
BENTHIC-PELAGIC MICROCOSM
GENERAL CHARACTERISTICS
I. Briefly describe the physical design, including
microcosm vessel material. If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
4. If environmental media are used, how is the
environment sampled?
DEVELOPER: K. PEREZ
Each microcosm consists of a glass or fiberglass tank
containing a pelagic phase (ISO liters of hand-
bucketed seawater) and a coupled benthic phase
(relatively undisturbed 169 cm3 x 20 cm deep benthic
box cote). The two phases are linked by an air-driven
displacement pump continuously exchanging seawater
from the water column to the benthic box core. The
water turbulence of the pelagic phase is controlled by
a rotating, reversible stirring paddle.
Yes x No neuston, plankton, benthos
Yes x No phytoplankton
Yea x No' amphipods, bivalves, polychaetes &
hydroids
Yes x No intertidal fish larvae GOW frequency)
Whole sampling of environment, i.e., no
reconstruction. Whole assemblages of organisms are
determined by the size of water column (volume)
and benthic core (cross-sectional diameter and vertical
depth); surface microlayer communities develop after
the microcosm is established in the laboratory.
Sediment is cored; water is hand-dipped.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Sediment habitat: benthic organisms, aerobic and
anaerobic sediment zones; water column: pelagic
fauna.
Size limits the incorporation of an intertidal zone and
large top carnivores.
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Dimensions (cm)
Depth =100
Soil/Sediment
Volume (L) Surface Area (cm1)
150
170-500
Ratio of the sediment surface area to water column
volume of the natural system being simulated
Approximately 1 mj
7. For what purpose was the microcosm originally
designed?
To estimate the fate and ecological effects of
chemicals in natural aquatic environments.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
Seawater containing living organisms and other
material is collected from the natural system and
exchanged with the seawater in the microcosm. The
volumes removed and added are equal; the water
turnover time is equal to that of the natural system
being simulated. Seawater is aerated by the physical
motion of the stirring paddle. The rate of stirring is
adjusted so that the dissolution rate of a solid material
is similar to that of the natural system.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
Yes
Typically 30 days, but longer tests are possible.
An adequate cleaning regime to eliminate significant
fouling on the microcosm walls.
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
Fluorescent lamps
Average water column irradiation =ป 38^E nr* s'1
Irradiation is constant during the light period of a
particular season; photoperiod is seasonaiiy-
dependent and controlled by an electric timer.
10
-------
GENERAL CHARACTERISTICS
(CONTINUED)
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
Water column partial I atrs. vertical profile of oxygen
in sediment
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
14. How is water/air circulated/mixed?
Natural water temperatures are reproduced by placing
all microcosms in a water bath which is continuously
and rapidly flushed with seawater derived from the
natural system. Natural temperatures could be
simulated by placing a temperature control in the
water bath.
Water mixing is controlled by a paddle rotating at a
speed such that the dissolution rate of a known solid
material is equivalent to that of the natural system.
CONTAINMENT
1. a. Is containment with current microcosm
design adequate for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
Yes.
. (See Protocol Draft)
Gas phase containment over microcosms. Water bath
containment by using closed circulation. Exit and
entry to microcosms is by a sterilizable compartment.
Yes.
Better filters and sterilization methodologies.
__ a. Considerable resources, drill, or time.
x b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
11
-------
PROTOCOLS
1. Has a detailed protocol (e.g., standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or* Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
2. What levels of difficulty would be involved in
making the modifications in (1) above?
Yes.
Yes.
Yes_
Yes.
Yes.
Yes.
Yes.
Yes.
.No.
.No.
No_
.No_
.No.
No
.No.
No
. a. Considerable resources, skill or
time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
.d. Can81 estimate at this time.
12
-------
SAMPLJNG
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
No limit to water column or surface microlayer
sampling. Benthic sampling is restricted to completion
of study or if additional replicate microcosms are
used to sample before the completion of the study.
2. Is destructive sampling during the course of a
test run required?
Yes x No with regard to benthos
only
3. Would design modifications allow the use of
alternative sampling strategies?
Yea No x (additional microcosms would
allow measurement of benthos
dynamics)
COST FACTORS
1. What is the relative capital cost of a single
complete microcosm unit (i.e.. one vessel, stirrer,
etc., without temperature control, flowing water,
etc.)?
2. How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
a. Less than $100
b. Between S100 and S500
c. Between $500 and $1000
x d. More than S1000
3 - 5 replicates/treatment
x a. Less than $5000 (excluding vessel
cost, for a chemical test)
b. Between $5000 and $20000
c. Over $20000
d. An estimate has not been made
13
-------
APPUCAB1LJTY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisrrts by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-dueti vity in phytoplankton by '*C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIAyPROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
Phytoplankton - direct cell counting
Zooplankion & transient larval forms: direct count
Benthos - sieve (0.5 mm), stain (Rose Bengal), count
Surface microlayer ATP analysis
Zooplankton age structure:juv./adult; naupVjuv.
Relationships developed using above data
same
same
same
same
ENERGY FLOW
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
PRIMARY PRODUCTION Estimated from temporal dynamics
SECONDARY PRODUCTION Same
P/R RATIO
OTHER (SPECIFY)
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Phytoplankton idem, (diatoms, bluegreen, etc.)
Sediment bioturbTresusp.: radioactive microspheres
Size excludes large macrofauna from these micro-
cosms. [However in natural systems macrofauna
are usually transient in time and space.]
Reasons that a parameter cannot be addressed in
your microcosm
14
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the Held in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
- WITHHELD WAS:
H=fflGH; I=INTERMEpIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
ENERGY FLOW
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Sed. resusp.,
bioturb.
If comparability studies have been conducted,
briefly discuss major problems encountered in
making comparison, cite the reference^), and in-
clude a copy, if possible.
(1) Sampling problems - Define the spatial scale
of natural system being simulated,
(2) Ca'iซr-T for observed deviation or divergent
behavior of laboratory system from natural
system.
(3) Ease of measurement in field is sometimes
difficult in laboratory and vice-versa.
15
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
I=Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival,
colonization or mioobial gene mobility potential
that have been field verified in your microcosm?
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes.
.No.
Yes.
.No.
Yes
No
Yes_
No.
FURTHER INFORMATION ON BENTHIC-PELAGIC MICROCOSM
Or. Kenneth Perez
U.S. Environmental Protection Agency
Environmental Research Laboratory
South Ferry Road
Narragansett, RJ 0288
(FTS) 838-3056
Dwyer, R.L., and K.T. Perez. 1983. An experimental
examination of ecosystem linearization. Am. Nat.
121:305-323.
Experimental marine microsom test protocol and
support document: Measurement of the
ecological effects, fate and transport of living
micro-organisms in a site-specific marine
ecosystem. U.S. Environmental Protection
Agency, Environmental Research Laboratory,
Narragansett, RL Preliminary Draft 42 p.
Federal Register. 1987. ง797.3100 Site-specific
aquatic microcosm test 52(187):36352-36360.
Perez, K.T., EW. Davey, N.F. Lactic. G.E. Morrison,
G.G. Murphy, Aฃ. Soper, and D.L. Winslow.
1983. Environmental assessment of a phthalate
ester. di(2-ehtylhexyl) phthalate (DEHP), derived
from a marine microcosm. In: W.E. Bishop,
RD. Cardwell, and BJ3. Heidolph (eds.). Aquatic
Toxicology and Hazard Assessment: Sixth
Symposium, ASTM STP 802. American Society
for Testing and Materials, Philadelphia, pp. 180-
191.
G.E. Soper, R. J. Blasco, D.L. Winslow, Rl.
Johnson, P.G. Murphy, and J.F. Heltshe. 1991.
Influence of size on fate and ecological effects
of Kepone in physical models. Ecological
Applications. (3):237-248.
16
-------
Air Exhaust
Manifold
Fluorescent
Lamps
Benthic Pump
Air Supply and
Exhaust Manifold
Exhaust Fan
Fitter
Sampling
Port
Watarbaih
Trough
Transverse
Plexiglass
Cover
Microcosm Tank
Benthic
Pump
Air
Controller
Paddle Shan
Figure 1. Benthic pelagic microcosm unit
Coupling Sleeve .
Fiberglass Connecting Rod
Pillow Block
Benthic Box
a
nsr
mm
Drive Shaft
Air tor Benlhic Pump
Air Line Fitting
T"T~}"* Plexiglas;
ซ Cover
Benthic Pump
Check Valve
Figure 2. Bentfite pelagic microcosm facility.
17
-------
COMPARTMENTALIZED LAKE MICROCOSM
GENERAL CHARACTERISTICS
I. Briefly describe the physical design including
microcosm vessel material If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
i
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
4. If environmental media are used, how is the
environment sampled?
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
DEVELOPER: N. KROER
Microcosm consists of 3 units: algal and herbivore
(216 and 27-1 glass aquariums), and benthic
community (sediment core(s) in plexiglass tube(s)).
A Peristaltic pump recycles water through silicone
tubing at 2.5 L/h (algal unit -*-*herbivore unit -ป-ป
sediment cores in series ->-ป algal unit). A 150 n
nylon screen prevents escape of zooplankton from
herbivore unit but allows movement of smaller
organisms. Row between units can be adjusted to
control grazing and geochemicaJ cycling. Water
volume to sediment-surface ratio may be adjusted.
Yes.
Yes.
Yes.
Yes.
.No.
.No.
.No.
No
Bact, flagel., diatoms
Phytoplankton
Zooplankton, benthic
Sediment: Intact cores.
Water Water in algal unit flushed through a 150 [un
sieve to remove zooplankton. The water in the
herbivore unit is unfiltered and contains the
zooplankton removed by sieving water for the algal
tank.
Intact sediment cores are collected in clear plexiglass
tubes. Water is collected in plastic carboys. Water
for the algal unit is filtered through a 150 pun sieve to
remove zooplankton. The zooplankton is placed in
the herbivore unit (with unfiltered water). The
microcosms are set up within 4-5 h of sampling.
Benthic and pelagic (water column)
19
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
Dimensions (an) Volume (L)
Algal unit:
60x60x60
Herbivore unic
30x30x30
216L
27 L
SoWSediment
Surface Area (ctrf)
Depends on
number and
size of
sediment cores
The size of the units may be varied, but relatively
large units may be preferable to property scale surface
area to volume.
c. How much space is required per microcosm
unit?
Space for a rack with 3 shelves; overall dimensions:
200 cm (H) x 80 cm (W) x 80 cm (D)
7. For what purpose was the microcosm originally
designed?
For testing OEMs
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
10% of the water in the algal unit is replaced on a
daily basis (workdays). The percentage may either
be increased or decreased to simulate the natural
water residence time. The microcosms are not aerated.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factors in es-
tablishing the lifespss of this microcosm?
Yes_
3-4 weeks
Wall growth on the sides of the microcosm may limit
the lifespan. However, nc effects on the bacterial
community due to wall growth have been observed.
20
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What land of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
The algal unit is illuminated by 12 Phillips TLD
fluorescent tubes. The herbivore unit and the sediment
cores are not illuminated
Max 350 |iฃ nv2 sec'1 measured at water surface.
Light intensity may be regulated by turning off
individual tubes.
Light cycles are controlled by a PC. Every week the
photoperiod is adjusted to the average light/dark
ratios for that week.
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
(for primary production measurements)
pH
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
The microcosms are housed in a cold room at appro*.
S-10ฐC. The algal tank and the water-bath with the
sediment cores are heated with immersed heating
elements.
14. How is water/air circulated/mixed?
Water in algal units is mixed by a Teflon<&-coated
stainless steel paddle adjustable to various speeds.
Paddle is40 x 17cm with42 holes (1.5 cm diameter).
21
-------
CONTAINMENT
1.
a. Is containment with current microcosm
design microcosm design adequate for
working with GEMs?
b. If so, describe containment design.
c. Could containment be unproved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
PROTOCOLS
1. Has a detailed protocol (e.g.. standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
x Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of iiฃr2Srฃ descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
All units are placed in stainless steel pans that drain
into a 300-L container in the event of breakage.
Extra-strength glass is used in aquaria. Seals and a
glass cover prevent escape of aerosols. HEP A filters
are used to filter environmental chamber air. Some
containment problems may arise while cleaning the
zooplankton filter or sampling water.
Yes.
Yes.
Yes.
Yes.
Yes.
Yes_
Yes.
.No.
. a. Considerable resources, skill, or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
.No.
.No.
.No.
.No.
.No.
.No.
Yes.
Yes.
.No.
.No.
. A manuscript is in preparation.
22
-------
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
Replacing the herbivore unit with a large volume of
water above the sediment in the sediment cores
probably will make it easier to conduct microcosm
tests. At the same time (he ratio of surface area to
water volume would be reduced O&ss effect of wall
growth)
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
. a. Considerable resources, skill or
time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
Currently, 10% of the water (Le., 25 L) is removed
for sampling and replaced with new filtered water
daily (workdays). More (or less) water could probably
be replaced. Given the microcosm size, there is
almost no limit to repetitive sampling.
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
I. What is the relative capital cost of a single
complete microcosm unit (i.e., one vessel, stirrer,
etc., without temperature control, flowing water,
etc.)?
Yes
.No_
2. How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
Yes_
.No_
. (e.g., sediment sampling)
a. Less than $100
b. Between S100 and S500
c. Between $500 and $1000
x d. More than S1000 (<$2000)
Three
a. Less than $5000
b. Between $5000 and $20000
c. Over $20000
x d. An estimate has not been made
23
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
ENDPOINT
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving, Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-ductivity in phytoplankton by 14C-carbonate uptake or in macro-
phytes by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia
concentrations or fluxes, etc.)- Also indicate if an endpoint could not be used in your
microcosm, and if not why.
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
PLANTS
ANTMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
chl a extraction from phytoplankton
Bacteria-AODC; flagellates/ciliates-phmulin stain
TROPHIC
INTERACTIONS
Turnover of free amino acids; DOC concentration
Grazing by flagellates/ciliaies: filtration
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERB IVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
ENERGY FLOW PRIMARY PRODUCTION 'XT-carbonate uptake
SECONDARY PRODUCTION 5H-lhytnidine incorp.; bac^flag. production in filtered water
P/R RATJO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Analysis: NH^, NO, concentrations
Analysis of PO4' concentrations
Reasons that a parameter cannot be addressed in
your microcosm
Fish, clams etc. are excluded as they may change
the behavior of the microcosm by eating zoopLank-
ton or filtering the wate (affecting phytoplankton
and microheterotroph populations). A larger vol-
ume of water would be required if these organisms
are to be included
24
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=fflGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
ENERGY FLOW
BIOGEOCHEM
CYCLING
OTHER
EFFECTS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
L-I . (phytoplankton)
If comparability studies have been conducted,
briefly discuss major problems encountered in
making comparison, cite the references), and in-
clude a copy, if possible.
Light limitation seems to reduce the algal biomass
(chl a) and primary production. However, this is
not reflected in the microbial community. Vari-
ability due to organisms filtering the water (zoop-
lankton and benthic invertebrates) tend to be less
relative to a single container with water and sedi-
ment. The reason ia probably that zooplankion and
clam/polychaete grazing is limited by the flow rate
between units. A manuscript is in preparation that
will discuss the problems in more detail
25
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for GEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes.
No.
Yes
No
2. If the answer to la, (above) is "yes," please rate
the degree of comparability (H=High;
I=Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival.
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
Yes.
No
Yes
No
FURTHER INFORMATION ON COMPARTMENTALIZED LAKE MICROCOSM
Mr. Niels Kroer
National Environmental Research Institute
Department of Marine Ecology and Microbiology
Frederiksborgvej 399
P.O. Box 358
DK-4000 Roskilde, DENMARK
(4546301388) ~
Coffin, R., N. Kroer, and N. Jorgensen. 1990. Heterotrophic microbial dynamics in aquatic microcosms: Design
ccnsidsrasens and field v*iid3ticn. In: Review of Progress in use EkjicCunology-cvuCrobiai Fcsi Control Agent
Risk Assessment Program, EPA/600/9-90AJ29, U.S. Environmental Protection Agency, Environmental Research
Laboratory, Cormallis, OR and Environmental Research Laboratory, Gulf Breeze, FL, pp. 137-138
26
-------
*=?
ซ3
^
Algal Unit
Hertivore Unit
Sedmant Cores
Rgur* 3.
Comp*rtmซitallzปd Uka microoocm.
27
-------
MIXED FLASK CULTURE MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material. If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: L SHANNON
Mixed flask culture (MFC) microcosms are relatively
small systems consisting of SO ml of sand sediment,
900 ml of nutrient medium and 50 ml of inoculum
(stock community collected from natural ponds) in
1 L beakers. The beakers are covered with a large
petri dish to prevent contamination. The test typically
consists of 4 treatment groups, each containing 5
replicate microcosms.
Yes.
Yes.
Yes.
Yes_
.No.
.No.
.No.
No
genera unknown
variety of green and blue
green algae and diatoms,
cladocerans, copepods,
rotifers, amphipods
chironomid larvae, snails
Communities are established from a mixed stock
culture derived from samples collected from a variety
of natural ponds.
"Wild" samples are allowed to "co-adapt" in the
laboratory for 3 months before use.
4. If environmental media are used, how is the
environment sampled?
Samples collected in small buckets, mixed in 4Q-L
aquaria. Nutrient medium (T82) is added and systems
equilibrated for 3 months.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Small eutrophic ponds
(1) Size is the main limiting factor. Because of their
small size these systems would be probably be poor
surrogates for large pelagic systems. (2) Since these
are static they could not represent k>tic systems.
29
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Dimensions (cm) Volume (L)
1L beaker
10cm dia.
14.5 cm height
1L
Soil/Sediment
Surface Area (cm?)
78.5 cm1
The upper limit is a function of incubator space.
These microcosms could not be much smaller or they
would be unable to support zooplankton populations.
Approximately 2088 cm3
7. For what purpose was the microcosm originally
designed?
This test system was designed to provide data on the
effects of chemicals or microorganisms introduced
into a freshwater environment It can also be used to
monitor survival of introduced microorganisms.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
There is free exchange with the air. These are static
systems with replacement for evaporative loss.
Three months for the initial stock culture which can
then be maintained for many months (we have
maintained some for 1-1/2 to 2 years). The
microcosms are allowed to equilibrate for 6 weeks
prior to treatment
To allow time for development of algae and
zooplankton populations. Equilibration is determined
on the basis of primary production (oxygen gain) and
zooplankton population density.
10, Microcosm "ufespan :
a. How long are microcosm tests generally
run? - -
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
Usually 42 days; although they have been run over 1
year. . ^ ,._.....
Ability to maintain algae and zooplankton
populations.
30
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
14. How is water/air circulated/mixed?
"cool light" fluorescent tubes.
- 500 foot candles.
12:12,L:D.
. (controlled @ 20ฐC)
. (controlled)
.(pH.Eh)
Microcosms are kept in environmental chamber.
Fans circulate air in the environmental chamber.
Water is not mixed.
31
-------
CONTAINMENT
1. a. Is containment with current microcosm
design aij^iatg for working with GEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
PROTOCOLS
1. .Has a detailed protocol (e.g., standard oper-
ating procedures, publication, etc.) been de-
veloped covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no,* could a competent technician, wim ihe
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
Yes_
No_
Each beaker is covered with a large petri dish cover.
All beakers are contained in a growth chamber.
Yes_
No_
Add appropriate filters to the air intake and exhaust
ports on the growth chamber.
____ a. Considerable resources, skill, or time.
_____ b. Moderate resources, skill or time.
x c. Minimal resources, skill or time.
d. Can't estimate at this time.
Yes x No_
Yes x No.
Yes x No
Yes,
Yes.
Yes_
Yes.
Yes_
No.
No.
No
No.
No.
32
-------
MICROCOSM MODIFICATION POTENTIAL
List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
Sediments are currently being modified to provide
substrate for a richer, more diverse microbial
community.
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
2. Is destructive sampling during (he course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
1. What is the relative capital cost of a single
complete microcosm unit (Le.. one vessel, stirrer.
etc., without temperature control, flowing water.
etc.)?
2. How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
. a. Considerable resources, skill or
time.
. b. Moderate resources, skill or time.
, c. Minimal resources, skill or time.
. d. Can't estimate at this time.
Generally, population sampling is accomplished by
withdrawing subsamples (50 ml for zooplankton, 13
ml for microorganisms, 2 ml for protozoa). The 50
ml zooplankton subsamples are replaced. The others
are not. These systems are generally able to withstand
the removal of 50 mL per week with no ill effects.
The volume removed each week is replaced with
deionized H,O.
Yes.
Yes
(although it might be
used in some tests)
No_
x a. Less than $100
b. Between S100 and $500
c. Between $500 and S1000
d. More than $1000
Five
a. Less than $5000
x b. Between $5000 and $20000
c. Over $20000
d. An estimate has not been made
33
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-duetivity in phytoplankton by '*C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.)- Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
Algae counted (microscope) in Palmer-MaJoney cell
Direct count or microscopic count of subsamples
Direct count
plate count/DAPI staiiVXT-gluc. degradVselec. media
density/activity of bact funct. groups in sediment
protozoan vs bacterial functional group densities
algal taxa vs zooplanloers. snails, insect density
Usually not measured few predators in the system
ENERGY FLOW
PRIMARY PRODUCTION oxygen gain
SECONDARY PRODUCTION zooplankton counts
P/R RATIO oxygen gain/oxygen toss
OTHER (SPECIFY) Total carbon, total dissolved carbon
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
auto analyzer NO". NO', NH,
auto analyzer ortho- and total phosphate
Silica
pH,Eh
Reasons that a parameter cannot be addressed in your microcosm
34
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a Held calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=HIGH; ^INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
H(algae); L(macrophy.)
_H(zooplank); I(insect)
I(snails); L(insects)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted,
briefly discuss major problems encountered in
making comparison, cite the reference^), and in-
clude a copy, if possible.
Studies are currently being conducted. This micro-
cosm and a new "aquatic core" microcosm are
being compared to 9 natural ponds. Parameters
being compared include pH. production, respira-
tion, P/R, nutrients and nutrient cycling rates, and
populations of: (1) microbial functional groups,
(2) algae (3) zooplankton (4) insects, (5) molluscs.
35
-------
HELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
I=Iniermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
Survival/
Colonization
Yes.
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes.
.No.
Yes
No
Yes.
.No
Need improved methods for monitoring the organism;
resolution with current techniques is not as fine as
would be desired.
5. Please discuss any factors other than survival,
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
FURTHER INFORMATION ON MIXED FLASK CULTURE MICROCOSM
Dr. Lyle Shannon
University of Minnesota
Biology Department
Duluth. MN 55812
(213) 726-80CG
Flum, TJr. and LJ. Shannon. 1987. The effects of
three related amides on microecosystem stability.
Ecotoxicol. Enviom. Saf. 13:239-252.
Shannon, LJ.. T.E. Flum, ILL. Anderson, and J.D.
"fount. 1989. Adaptation^mixed uask culture
microcosms for testing the survival and effects
of introduced microorganisms. In: U.M, Cowgill
and L.R. Williams (eds.). Aquatic Toxicology
and Hazard Assessment: 12th Volume, ASTM
STP 1027, American Society for Testing and
Materials, Philadelphia, pp. 224-239.
Shanon, LJ., T.E. Flum, and JJ). Yount. 1989. Draft
Protocol for a Mixed Flask Culture Microcosm
Toxiciry Test
Yount, JD. and LJ. Shannon. 1988. Slate changes in
laboratory microecosysicins in response to
chemicals from three structural groups. In: I.
Cairns, Jr., and J.R. Pratt (:eds.) Functional
Testing of Aquatic Biota for Estimating Hazards
of Chemicals, ASTM STP 988, American Society
for Testing and Materials, Philadelphia, pp. 86-
96.
36
-------
POND MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material. If possible, include a
labeled diagram.
DEVELOPER: J. GIDDINGS
The system consists of glass aquaria (generally 80-L,
although 8-L and 120-L systems have also been
used), containing natural pond water and a 5- to 10-
cm sediment layer. The microcosm contains the
natural macrophytic, pelagic and benthic
communities.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
Yes_j No Pelagic, benthic
Yes_j_ No Algae, macrophytes
Yes x No Zooplankton, benthos
Yes No x (Fish could be included)
3. Describe how communities of organisms are
established in the microcosm.
4. If environmental media are used, how is the
environment sampled?
Pond water and sediment are collected from natural
sources and placed into aquaria. Macrophytes
(community from natural sources) are planted.
Community may be supplemented by zooplankton or
macroinvertebrates from natural sources or from
cultures.
Sediment collected with shovel or dredge. Water
collected with pump, sampling bottle, or depth-
integrated column sampler. Macrophyte communities
collected en masse by hand.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
System normally includes aerobic and anaerobic
sediment, macrophyte, and free-swimming habitats
corresponding to typical littoral freshwater
environments.
Shallow depth, absence of circulation and water
renewal. Lotic or deep pelagic systems cannot be
simulated except in general sense.
37
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Soil/Sediment
Dimensions (cm) Volume (L) Surface Area (cm2)
60 x 30 x 40(D)
or
60 x 30 x 60(D)
80-120
2000
Lab space (controlled light and temperature) is only
limitation. Systems less than 80 L possible but harder
to sample, more variable.
Less than 4 m1 for 12 to 20 replicates.
7. For what purpose was the microcosm originally
designed?
Measuring fate and effects of toxicants on typical
freshwater ecosystems.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
Aeration can be provided but usually isn't;
macrophytes supply plenty of oxygen. Pond water
added to replace water removed in sampling; distilled
water added to replace water lost by evaporation.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
ran?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
Yes_
6-8 weeks
Criteria: Photosynthesis/respiration ratio should be
approximately one (as determined by D.O.
concentrations). The pH usually levels off at - 8-9.
Macrophytes become well-established.
Purpose: Achieve representative productivity; reach
relative stability (conditions relatively constant day-
to-day); replicates become more uniform.
6-12 months.
Eventually, macrophytes senesce (nutrient limitation?)
and replicates diverge.
38
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
Sun-simulating fluorescent lights
150-250 piE m-1 sec1 (about 1/3 full sunlight)
12:12photoperiod
(N.P)
(pH, alkalinity)
conductivity, organic carbon, suspended
solids
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
14. How is water/air circulated/mixed?
Environmental chamber (usually)
Not done.
39
-------
CONTAINMENT
I.
a. Is containment with current microcosm
design ariTiat* for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so. what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain*
ment, what degree of difficulty would be
encountered in making these modifications?
PROTOCOLS
1. Has a detailed protocol (e.g.. standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no." do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If uic susws to o*> of :hs cbov:
is "no," could a com pi* tent
la, Ib, cr!c)
with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
Yes_
Yes_
.No.
Use filters, anteroom in environmental chamber.
a. Considerable resources, skill, or time.
____ b. Moderate resources, skill or time.
x c. Minimal resources, skill or time.
d. Can't estimate at this time.
Yes.
Yes.
Yes_
Yes.
Yes.
Yes_
Yes.
Yes_
.No.
.No.
.No_
.No.
.No.
.No_
.No_
(Standard ANOVA or
regression analysis is
sufficient)
40
-------
MICROCOSM MODIFICATION POTENTIAL
I. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
Develop sampling techniques for sediment
Apply microbiological techniques to benthic and
planktonic communities.
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
. a. Considerable resources, skill or
time.
, b. Moderate resources, skill or time.
. c. Minimal resources, skill or time. (If
microb. tech. exist).
. d. Can't estimate at this time.
Sediment sampling would be limited by quantity of
sediment available (roughly 10-20 L). Repeated
destructive sampling would disturb ecological
conditions. Otherwise, there are few practical limits.
Repeated sampling and monitoring are normal
2. Is destructive sampling during the course of a
test run required?
Yes x No x (Yes, for enumeration/moni-
toring of benthic or pelagic
communities.)
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
1. What is the relative capital cost of a single
complete microcosm unit (i.e., one vessel, surer,
etc., without temperature control, flowing water,
etc.)?
Yes.
No
2. How many replicate vessels are generally used
per treatment?
x a. Less than $100
b. Between $100 and $500
c. Between $500 and $1000
d. Morethan$1000
Three
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
a. Less than $5000
b. Between $5000 and $20000
_j_c. Over $20000
d. An estimate has not been made
(Main cost is labor for monitoring which varies
depending on test objectives.)
41
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-duetivity in phytoplankton by I4C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC SUBSTRATE^ACTERIA
INTERACTIONS BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
pigment analysis; periphytometers; macrophyte observed
zooplank. collect.; macroinven. obs. final harvest
macroinvertebrate obs.; final harvest (sieving)
any applicable ecological techniques
litter bags & glucose uptake have been measured
Not studied; could use enclosures/repeated sampling
ENERGY FLOW
PRIMARY PRODUCTION Diurnal D.O., "C
SECONDARY PRODUCTION Diurnal D.O
P/R RATIO Diurnal D.O
OTHER (SPECIFY)
BIOGEOCHEM.
OTHER
EFFECTS
NITROGEN
CYCLING PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Any applicable ecological techniques; water anal.
Same
Same
Fish survival and growth; on site bioassays
Reasons that a parameter cannot be addressed in your microcosm
42
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=HIGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL(SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted, briefly discuss
major problems encountered in making comparison, cite the
reference(s), and include a copy, if possible.
43
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
Questions
1. Has your microcosm response to this factor been
compared to field data?
FACTOR
Survival/ Environmental Mobility
Colonization (Specify organism or gene)
Yes No x Yes No _x_
2. If the answer to la. (above) is "yes," please'rate
the degree of comparability (H=High;
^Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival.
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
Yes
Possibly
No
Yes_
No
FURTHER INFORMATION ON POND MICROCOSM
Dr. Jeffrey Giddings
Springborn Bionomics, Inc.
790 Main Street
Wareham. MA02571
(508) 295-2550
Franco. PJ. J.M. Giddings. S.E. Herbes, LA. Hook,
J.D. Newbold, W.K. Roy. GJfc. Southworth. and
AJ. Stewart 1984. Effects of chronic exposure
to coal-derived oil on freshwater ecosystems: I.
Microcosms. Environ. Toxicol. Chem. 3:447-
463.
Giddings. J.M. 1986. A microcosm procedure for
dฃtฃ?!siciฃ9 s0fฑ !svc!s of chstnics! exposure in
shallow-water communities. In: J. Cairns, Jr.
(ed,). Community Toxicity Testing, ASTM STP
920, American Society for Testing and Materials,
Philadelphia, pp. 121-134.
Giddings, J.M.. and PJ. Franco. 1985. Calibration of
Laboratory bioassays with results from
microcosms and ponds. In: TP. Boyle (ed.).
Validation and Predictability of Laboratory
Methods for Assessing the Fate and Effects of
Contaminants in Aquatic Ecosystems, ASTM
STP 865, American Society for Testing and
Materials, Philadelphia, pp. 104- 119.
44
-------
SEDIMENT CORE MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material. If possible, include
a labeled diagram.
2. Which of ihe following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: PRITCHARD/CLARK
Three borosilicate glass vessel designs have been
used, each with an intact sediment core and an
overlying water column: Ecocore uses 35 mm (diam.)
x 40cm glass tubes; Ecocore n uses 3 or 4 L reaction
kettles .(Coming 6947) or 27-L Jars (Corning 6942-
27L). or Seagrass Communities of clear acrylic tubes
(16 cm diam. x 50 cm) with flat, acrylic bottoms.
Yes_
Yes.
Yes_
Yes_
,No_
.No_
.No_
No
Bacteria, protozoa
Phytoplankton, seagrasses
Benthic, epibenthic
Natural assemblages of water column plankton are
added to microcosms containing intact sediment cores
with their associated benthic and/or seagrass
communities.
4. If environmental media are used, how is the
environment sampled?
Water is collected in a carboy, and sediment in
acrylic or glass coring devices.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Usually salt marsh or shallow estuarine bay, vegetated
or barren substrates. Freshwater systems (including a
eutrophic lake) have been simulated.
Scaling considerations for deep bodies of water.
45
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
Dimensions (cm) Volume (L)
3.5 (diam) x 40 0.175
13 (diam) x 24/32 3.0/4.0
29 (diam) x 45 27
16 (diam) x 50 10
SoUlSediment
Surface Area (cm?)
9.6
133/133
660
200
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Lower limit: sampling frequency and volumes,
inclusion of larger animals/plants.
Upper limit Decontamination of vessels, effluent
arid containment considerations.
Ecocore: 25 cm2
Ecocore II: 0.3 m1
Seagrass system: 0.2 m3
7. For what purpose was the microcosm originally
designed?
Ecocore and Ecocore n (reaction kettles): to determine
the fate of xenobiotic compounds; 27-L system: GEM
Risk Assessment; seagrass microcosm: ecological
effects of test chemicals.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
Ecocore: static operation, aerated (and mixed) with a
long stainless steel needle; Reaction kettle: both
static and flow-through (40 ml/h) modes; 27-1 system:
daily batch replacement (10%); seagrass community:
flow-through design (7 L/h) with airstone for mixing
and aeration
T
Yes_
No_
At least overnight. ;
Primarily to allow settling of paniculates suspended
as a result of sampling.
10. Microcosm "lifespan":
27C J^J
run?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
Usually, 2 to 6 weeks
Wall growth and food/nutrients limitations if operated
in a static mode.
46
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
Earlier tests: fluorescent (40-W, cool white; 250-W
GE Power Groove).
900 Einsteins nv2 s-1, measured at water surface, with
two 400-W Multi-Vapor lamps.
Timer controls photoperiod, typically 14:10
(Light: Dark).
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Cnher (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
14. How is water/air circulated/mixed?
. (NH, concentration)
. Salinity
Clear acrylic bath, with refrigerated circulator
attached.
Ecocore: Aeration through needle.
Ecocore 0: 300 rpm motor and glass stirrer.
Seagrass Community: Water flow and air stone.
47
-------
CONTAINMENT
1. a. Is containment with current microcosm
design ad^q"3^ for working with OEMs?
b. If so. describe containment design.
c. Could containment be improved by design
modification?
d. If so. what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
PROTOCOLS
1: Has a detailed protocol (e.g., standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib. or Ic)
is "ncv" could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
Yes_
Yes_
No x
No_
An enclosure with HEP A filters would be required,
and the effluent would have to be treated. Sealed tops
may be added to the microcosm vessels.
a. Considerable resources, skill, or time.
x b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
Yes.
Yes.
Yes.
Yes.
Yes.
Yes.
Yes.
Yes.
No.
No.
No
No,
No
48
-------
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
2. What levels of difficulty would be involved in
making the modifications in (1) above?
None
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
2. Is destructive sampling during the course of a
test run required?
3.- Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
I. What is the relative capital cost of a single
complete microcosm unit (Le., one vessel, stirrer,
etc., without temperature control, flowing water,
etc.)?
2. How many replicate vessels are generally used
per treatment?
. a. Considerable resources, skill or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
Static systems (i.e., Ecocore) are limited by the
relatively small volume of water and sediment, while
the larger systems which use periodic water
replacement or flow-through design do not share
these problems. All systems can be replicated (more
easily with smaller systems) and may be destructively
sampled, however.
Yes
No
. (But is desirable for Ecocore)
Yes
No
a. Less than $100 (Ecocore, Seagrass com.)
b. Between $100 and S500 (Reaction kettle,
27-Ljar)
.c. Between$500and $1000
.d. More than S1000
Two for small systems, up to eight for seagrass
systems
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
. a. Less than S5000
. b. Between $5000 and S20000
.c. Over $20000
d. An estimate has not been made
49
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-ductivity in phytoplankton by I4C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
Plant composition for seagrass, epiphytes abundance
Epifauna colonizing seagrass
Sieving. Rose Bengal staining, and sorting
AO Direct Counts; CPU; bact. diversity by morphol.
5-amino acid total pool/turnover
Selective filtration, staining, and counting
Leaf litter loss rate
ENERGY FLOW PRIMARY PRODUCTION Phytoplankton 14C-optake; macrophyte-growth
SECONDARY PRODUCTION Thymidine uptake; leucine uptake
i P/R RATIO 24-hour dissolved oxygen cycle
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
OTHER
hFFECli
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Ammonia concentration
Phosphate concentration
Thalassia-chl a; epiphyte: chl a, dry wt
Gene exchange
Reasons that a parameter cannot be addressed in
your microcosm
Large vertebrates or invertebrates may not be
appropriate due to small vessel size, or flow of
water necessary to provide plankionk food.
50
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
FACTORS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=fflGH; I=1NTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS Diversity-L; ADOC-H; CFU-H
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
PRIMARY PRODUCTION L
SECONDARY PRODUCTION Thiamine uptake-H; glut. assim7min.-H
P/R RATIO
OTHER (SPECIFY) L pH
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
_ Ammonia
.Phosphate
If comparability studies have been conducted,
briefly discuss major problems encountered in
making comparison, cite the references), and in-
clude a copy, if possible.
Statistical problems (i.e., how many samples, what
sampling intervals, choice of statistical tests, etc.
to detect significant differences), selection of sen-
sitive endpoints, and interpretation (what do dif-
ferences mean?).
51
-------
FIELD VERIFICATION OF M1CROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
I. Has your microcosm response to this factor been
compared to field data?
If the answer to la. (above) is "yes." please rate
the degree of comparability (H=High;
I=Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival
colonization or microbial gene mobility potential
that have been field verified in your
microcosm?
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes
No.
Yes
No
Yes x
Possibly
No
Yes
No
52
-------
FURTHER INFORMATION ON SEDIMENT CORE MICROCOSM
Dr. PJi Pritchard
U.S. EPA
Environmental Research
Laboratory
Gulf Breeze, FL 32561
(FTS) 228-9340
Dr. James Clark
Environmental Toxicology Division
Exxon Biomedical Sciences, Inc.
Mettlers Road, CN 2350
East Millstone, NJ 08875-2350
(908) 873-6039
Clark, J.R., and J.M. Macauley. 1990. Comparison
of the seagrass Thalassia testudinum and its
epiphytes in the field and in Laboratory test
systems. In: W. Wang, J.W. Gorsuch, and WJL
Lower, (eds.). Plants for Toxicity Assessment,
ASTM STP 109-1, American Society for Testing
and Materials, Philadelphia, pp. 59-68.
Coffin, R.. N. Kroer, and J. Jorgensen, 1990.
Heterotrophic microbial dynamics in aquatic
microcosms: Design considerations and field
validation. In: Review of Progress in the
Biotechnology-Microbial Pest Control Agent
Risk Assessment Program, EPA/600/9-90/029,
U.S. Environmental Protection Agency,
Environmental Research Laboratory, Corvallis,
OR, and Environmental Research Laboratory,
Gulf Breeze, FL, pp. 137-138.
Cripe, C.R., and P.H. Pritchard. 1990. Aquatic test
systems for studying the fate of xenobiotic
compounds. In: Aquatic Toxicology and Risk
Assessment: Thirteenth Volume, ASTM STP
1096, W.G. Landis and W.H. van der Schalie
(eds.), American Society for Testing and
Materials, Philadelphia, PA, pp. 29-47.
Kroer, N., and R.B. Coffin. Microbial trophic
interactions in aquatic microcosms developed
for testing genetically engineered micro-
organisms: A field comparison. In press.
Microbial Ecology.
Macauley, J.M.. JJ*. Clark, and A.R. Pins. 1990.
Use of Thalassia and its epiphytes for toxicity
assessment: Effects of a drilling fluid and
tributyltin. In: W. Wang. J.W. Gorsuch, and
W.R. Lower, (eds.), Plants for Toxicity
Assessment. ASTM STP 1091, American Society
for Testing and Materials, Philadelphia, pp. 255-
266.
Morton, R.D., T.D. Duke. J.M. Macauley, J.R. Clark,
W.A. Price, SJ. Hendricks, S.L. Owsley-
Montgomery. and G.R. Plaia. 1986. Impact of
drilling fluids on seagrasses: An experimental
community approach. In: J. Cairns, Jr. (ed.),
Community Toxicity Testing, ASTM STP 920,
American Society for Testing and Materials,
Philadelphia, pp. 199-212.
O'Neill, E J.. C .R. Cripe, L .H. Mueller. J J>. Connolly.
P.H. Priichard. 1989. Fate of Fenthion in Salt-
marsh Environments: II. Transport and
Biodegradanon in Microcosms. Environ. Toxicol.
Chem. 8:759-768.
53
-------
Microcosm Chamber
Sediment
o -*
Water
Sillcone Stopper
Figur* 4. Ecocor* microcosm.
54
-------
Air
Outflow
(Volume Leveler)
Gasket
To Constant
Temperature
Circulator
Water Jacket
Sediment
From
Constant
Temperature
Circulator
Figure 5.
ccocofe I nucrocoeJit*
55
-------
Unfiltered -ป
Seawater
Setting ป
fil
y
Reservoir
n
Overflow
-* 1
nv
Primary Head Box
Injection of Drilling
Mud or Clay
Mixing
Reservoirs
Stir
Plates
Peristaltic
Pump
Secondary
Head Box
Rgur* e.
Scagrsaa community.
56
-------
STANDARD AQUATIC MICROCOSM
GENERAL CHARACTERISTICS
DEVELOPER: F. TAUB
1. Briefly describe the physical design including
microcosm vessel material If possible, include a
labeled diagram.
Each microcosm consists of a 4-L glass container,
covered with a petri dish. Substrate is washed sand
plus chitin and cellulose. Medium is distilled water
and reagent grade salts. Algae and invertebrates are
added from laboratory cultures.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
4. If environmental media are used, how is the
environment sampled?
Yes_
Yes_
Yes_
Yes
.No_
.No_
.No_
No
10 species of algae.
5 species
Laboratory cultures are the source for the organisms.
Reinoculation of organisms is done once per week at
numbers below the detection limit (are likely to be
counted only if reproduction occurs). This allows
populations to develop after temporary pehods of
toxicity or random extinction.
N/A
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Early spring through summer of a temperate aquatic
community, e.g., pond.
Size is a limitation; large carnivores cannot be
included. Preliminary work was done on a marine
system.
57
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Dimensions (cm)
SoilJSediment
Volume (L) Surface Area (cm2)
3L
convenience, number of replicates
314.2
A typical SAM microcosm experiment using 24-30
microcosms can be run on a 2.6 x .85 meter table in a
temperature controlled room or reach-in incubator.
7. For what purpose was the microcosm originally
designed?
This microcosm was designed to measure ecological
effects of a test chemical or to explore the potential
of a novel organism to invade and become established,
and its effects such as changes in nutrient cycling or
species displacement.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
The petri dish cover allows some exchange with die
atmosphere, especially when it is removed for
sampling the community. Aeration is avoided because
dawn-night-dawn oxygen measurements are used to
estimate net photosynthesis and respiration.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
No.
7 days
The growth of algae and reproduction of animals are
checked and outlier(s) (if any) or cracked microcosms
are eliminated.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
K What are the most important factors in es-
tablishing the iifespan of foปy niicxocosm?
SOP is 63 days, but some have been maintained for
up to a year.
Volume removed in twice-weekly sampling.
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
Two 8-foot (high intensity, warm white) fluorescent
tubes (GE F96PG17WW).
80 ME nrป sec4 (850-1000 ft-c).
12:12 L:D photoperiod
58
-------
GENERAL CHARACTERISTICS
(CONTINUED)
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
. NC3 , NO2 . NH,, pH, O, (3 point), pH
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
Environmental chamber, or temperature controlled
room.
14. How is water/air circulated/mixed?
Manually, before sampling.
CONTAINMENT
1. a. Is containment with current microcosm
design adequate for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
Yes
Yes
No
No_
Unbreakable containers (e.g., change from glass to
plastic). Sampling procedures would require change.
. a. Considerable resources, skill, or time.
. b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
59
-------
PROTOCOLS
I. Has a detailed protocol (e.g., standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib. or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
Yes.
Yes.
Yes.
Yes.
Yes
No.
No.
No
Yes
Yes
Yes
_ No
_ No
_ No
No.
No
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
Define the microbial community (concurrently,
algaeincluding blue-greensand protozoa, rotifers,
etc. are enumerated), but not (usually) specific
bacterial, fungal species.
2. What levels of difficulty would be involved in
making the modifications in (1) above?
. a. Considerable resources, skill or time.
. b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
. d. Can't estimate at this time.
60
-------
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
Sampling of algae, protozoa and rotifers requires
removing a few ml.
Sampling of pH and O, currently involves electrode
introduction; perhaps these could be chemically
decontaminated after use.
Sampling of zooplankton (remove, pour subsamples,
return) would have to be modified. Photography is a
possibility.
Yes_
Yes.
No
No
COST FACTORS
I. What is the relative capital cost of a single
complete microcosm unit (i.e., one vessel, stirrer,
etc.. without temperature control, flowing water.
etc.)?
2. How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
x a. Less than $100
b. Between $ 100 and S500
c. Between $500 and $1000
d. More than $1000
Five or Six
a. Less than $5000
x b. Between $5000 and $20000
c. Over S20000
d. An estimate has not been made
61
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-ductivity in phytoplankton by 14C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
ENERGY FLOW
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Algae: count (10 sp.); dominance, diversity index
Count 5 species of animals; species dominance,
Ostracod and amphipods are part of system
CPU select, media; Electron Transport System; ATP
CPU and microscopic protozoan counts
Algal counts and herbivore counts
(might use invertebrate predators/small fish)
Analysis NO3 (plant uptake), NO2, NH, (from zooplankton)
Algal uptake and recycling by zooplankton
Changes in algal dominance, species diversity
Changes in animal dominance, species diversity
Antibiotic resistance
Reasons that a parameter cannot be addressed in
vour microcosm
System is too small for fish population. Small fish,
such as juvenile Medaka would be a possibility.
62
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=fflGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY) .
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted,
briefly discuss major problems encountered in
making comparison, cite the references), and in-
clude a copy, if possible.
A dissertation by F. Joan Hardy (1984, "Re-
sponses of naturally-derived aquatic microcosms
to selective chemical stress," doctoral disserta-
tion. University of Washington, Seattle. WA,
276 p.) compared the responses of indoor and
outdoor microcosms derived from Lake Wash-
ington and Green Lake to the "Standardized
Aquatic Microcosm" during two sequential years.
Although the test utilized streptomycin as a stres-
sor, comparison of the controls should provide
information relevant to field calibration of this
system.
63
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for GEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the Geld. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes.
No
Yes.
No
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
I=Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
Yes No
Yes No
Yes x No Yes No
Depends on funding
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
Copper, insecticide, and streptomycin effects.
64
-------
FURTHER INFORMATION ON STANDARDIZED AQUATIC MICROCOSM
Dr. Frieda Taub
School of Fisheries, HF-15
University of Washington
Seaule,WA98195
(206)685-2115
ASTM, 1991. Practice for Standardized Aquatic
Microcosms: Fresh Water. 1991 Annual Book
of ASTM Standards. Vol. 11.04, American
Society for Testing and Materials, Philadelphia.
Conquest, LJ... and F.B. Taub. 1989. Repeatability
and reproducibility of the Standardized Aquatic
Microcosm: Statistical properties, p. 159-177. In
U. Cowgill (ed.) Aquatic Toxicology and Hazard
Assessment, 12th Volume ASTM STP 1027,
American Society for Testing and Materials,
Philadelphia, PA.
Haley, M.V.. E.L. Vickers, T.-C. Cheng. J. DeFrank,
T.A. Justus, and W.G. Landis. 1990.
Biodegradation and reduction in aquatic toxicity
of the persistent riot control material 1,4-Dibenz-
Oxazepine. In: Aquatic Toxicology and Risk
Assessment: Thirteenth Volume, ASTM STP
1096, W.G..Landis and W.H. van der Schalie
(eds.). American Society for Testing and
Materials. Philadelphia, PA, pp. 60-76.
Harrass. M.C.. and F. B. Taub. 1985. Comparison of
laboratory microcosms and field responses to
copper. In: TJ>. Boyle (ed.). Validation and
Predictability of Laboratory Methods for
Assessing the Fate and Effects of Contaminants
in Aquatic Ecosystems, ASTM STP 865,
American Society for Testing and Materials,
Philadelphia, pp. 57-74.
Swartzman, G.L., F.B. Taub, J. Meador, C. Huang,
and A.C. Kendig. 1990. Modeling the effect of
algal biomass on multispecies aquatic
microcosms response to copper toxicity. Aquat.
Toxicol. 17:93-118.
Taub, F.B.. Pi. Read, A.C. Kindig, M.C. Harrass,
HJ. Hartmann, LI. Conquest, FJ. Hardy, and
P.T. Munro. 1983. Demonstration of the
ecological effects of streptomycin and malaihion
on synthetic aquatic microcosms. In: W.E.
Bishop. R.D. Cardwell. B.B. Heidolph (eds.),
Aquatic Toxicology and Hazard Assessment:
Sixth Symposium. ASTM STP 802, American
Society for Testing and Materials, Philadelphia,
pp. 5-25.
Taub, F.B., and Read, P.L. "Standard aquatic
microcosm protocol," Draft final report. U.S.
Food and Drug Administration Contract No.
223-83-7000 with FDA, Washington, DC 20204
(1986). Available from Dr. B J.. Hoffmann, U.S.
FDA, HFF-304, Rm. 511157.200 C Street. SW,
Washington, DC 20204.
65
-------
STREAM MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material. If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: T. BOTT
Each microcosm is constructed of vinyl coated heavy
gauge steel with plexiglass end plates. A drilled,
plexiglass buffer plate is used to establish laminar
flow. Surface sediments (2 cm) from the stream are
placed into 40 plastic trays (0.1 m square x O.OS1 m
deep) with the bottoms removed and replaced with
400-nm mesh nylon screen. This allows for exchange
of water, dissolved nutrients, and biota between the
surface sediments and those under the trays and
reduces the likelihood of generating anaerobic
conditions. Microcosms are housed in a greenhouse.
Yes.
Yes.
Yes.
Yes
No_
No_
.No_
No
Bacteria/fungi/algae/ protozoa
Algae
Insects, snails, meiofauna
Seeding from natural "parent" stream.
4. If environmental media are used, how is the
environment sampled?
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Surface sediments are removed from White Clay
Creek with a shovel and transferred to a pail, brought
to a greenhouse, and placed in trays. Coarser
sediments underneath are collected similarly and
placed in the microcosms. The trays are then placed
on top.
Flowing stream (presently simulates a slow run);
specialized habitats such as leaf packs, rocks or
pools can be added to the system.
Size and slope limit flow to moderate velocity; fast
ripple would be hard to duplicate; size also limits
number of habitats included when sample replication
is factored in.
67
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Soil/Sediment
Surface Area (err?)
Dimensions (cm) Volume (L)
223 cm (L) x 20.3 cm
(W) x 12.7 cm (D)
c. Greenhouse is 3.69 m wide x 4.62 m long.
7. For what purpose was the microcosm originally
designed?
Testing effects of introduced bacteria on benthic
community and stream ecosystem parameters.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
Water from the creek is pumped to a 140 L header
tank from which it is distributed to water jackets and
microcosms. The water from each microcosm is
collected through five 2.54 cm i.d. tubes into 20 L
collection tank (also in a water jacket) from which it
is recycled to the head of each microcosm. Water is
discharged to the parent stream after filtration
(cartridge filters) and treatment by ultraviolet radiation
(Sanitron Sterilizer).
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
Yes x No
4 weeks
Purpose: To let sediments resettle and surface
communities to reestablish. Criteria were not
established or used - but would involve testing for
chlorophyll a concentrations, algal species occurrence,
insect species occurrence.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
1 to 4 months; several years may be possible.
1) Construction material. 2) Sediment build-up from
repeated storms (water coming in carries silt from
parent stream during storms which settles out because
microcosm flow rate is always the same).
68
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
Ambient solar radiation
Water jackets and the use of vinyl coated metal
maintains near-ambient stream water temperatures.
14. How is water/air circulated/mixed?
Water (35L) is recirculated through the systems with
the addition of 0.9 L of new water/min. This can be
varied. Overflow is returned to stream after treatment
(see 8). Teel Pumps (IP677A) are used for
recirculation from collection tanks to the top of the
microcosm stream.
69
-------
CONTAINMENT
1. a. Is containment with current microcosm
design tf^'f*** for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
Yes_
.No_
Partially
PROTOCOLS
1. Has a detailed protocol (e.g.. standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, ib. or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
See 8 (above) for treatment of discharge water.
Yes x No
Increase isolation of each stream. Cement greenhouse
floor (presently gravel). Filter air in greenhouse and
use negative pressure. Need larger collection pool in
event of pump failure.
a. Considerable resources, skill, or time.
_S b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
Yes.
Yes.
Yes
Yes.
Yes_
.No.
.No.
No
Yes
Yes
Yes
_No_x__
_No_x_
_No_x__
.No.
.No.
70
-------
MICROCOSM MODIFICATION POTENTIAL
I. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
Make slightly deeper and enlarge exit ports to allow
for greater water velocity and simulation of faster
flows in riffles.
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
I. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
2. Is destructive sampling during the course of a
test run required?
. a. Considerable resources, skill or time.
. b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
. d. Can't estimate at this time.
Removal of trays from system for measures of
photosynthesis and respiration in respirometers
followed by destructive sampling of sediments for
analyses of ATP, chlorophyll a. total bacterial
densities, densities of added bacterial population,
enzyme activities, protozoa and meiofaunal densities
(if desired), uptake of radio-actively tagged nutrients,
bacterial productivity measurements. Number of trays
limits sampling of the system.
Yes
3. Would design modifications allow the use of
alternative sampling strategies?
Yes
71
-------
COST FACTORS
I. What is the relative capital cost of a single
complete microcosm unit (Le., one vessel, stirrcr,
etc., without temperature control, flowing water,
etc.)?
2.
How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
a. Less than $100
. b. Between $100 and $500 (Includes
^circulation, not water supply)
c. Between $500 and $1000
d. More than $1000
Two
. a. Less than $5000
. b. Between $5000 and S20000
. c. Over $20000
. d. An estimate has not been made
72
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
ENDPOINT
Indicate which of the following parameters nave been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving, Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-dactivity in phytoplankton by 14C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
Algal biomass by chlorophyll a, spp. by microscopy
Sieve, sort, count, weigh, identify, ATP
FA/DAPI/AO counts; biochem. markers-FAME/lipid-P/ATP_
TROPHIC SUBSTRATE/BACTERIA Radio-substrate incorp.; DOC change; POC: wgt., chem.
INTERACTIONS BACTERIA/PROTOZOA Feeding studies; fluores.-labeled bact; bact den.
PLANTS/HERBIVORES
HERBIVORES/PREDATORS x
OTHER (SPECIFY)
ENERGY FLOW PRIMARY PRODUCTION D.O. change; I4C-bicarbonate uptake
SECONDARY PRODUCTION M
P/R RATIO D.O. change in flowing water respirometeni
OTHER (SPECIFY) Leaf litter decomp.: leaf pack wt. change over rime
BIOGEOCHEM. NITROGEN
CYCLING PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER PLANT (SPECIFY)
ht-HiCTS ANIMAL (SPECIFY) ^
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Reasons that a parameter cannot be addressed in
your microcosm
Herbivores, predators: Size and water velocity
might limit the inclusion of some herbivores and/
or predators.
Secondary production; Size and water velocity
limitations for some organisms.
Animals: Size and water velocity will limit the
study of riffle organisms and fish.
73
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS: .
H-fflGH; lalNTERMEDtATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
. Algadchl a)
_(Chl a. bact. dens.)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
Community Respir.
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
. (Litter decomp.)
If comparability studies have been conducted,
briefly discuss major problems encountered in
making comparison, cits she references), and in-
clude a copy, if possible.
Major problem. Differing storm effects in micro-
cosms and the parent stream. In microcosms, sedi-
menaQon of the silt load occurs; In the parent
storm there is scour, and no scour occurs in the
micro-cosm because flow rates are constant.
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes.
No.
Yes
No
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
^Intermediate; L=Low).
3. If the answer to la. (above) is "no." do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival
colonization or mioobial gene mobility potential
that have been field verified in your microcosm?
Yes
Yes
No
FURTHER INFORMATION ON STREAM MICROCOSM
Dr. Thomas Bott
Stroud Water Research Center
Division of Environmental Research
Academy of Natural Sciences
RD. #1 Box 512
Avondale,PA19311
(215) 268-2153
Boo, T.L., and L.A. Kaplan. 1990. Cellulytic bacteria as surrogates for a genetically engineered microorganism:
Microcosm studies of persistence and effects in streambed sediments. In: Review of Progress in the Biotechnology-
Microbial Pest Control Agent Risk Assessment Program, EPA/600/9-90/Q29, U.S. Environmental Protection
Agency, Environmental Research Laboratory, Corvallis, OR and Environmental Research Laboratory, Gulf
Breeze, FL, pp. 139-143.
75
-------
WASTE TREATMENT MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
4. If environmental media are used, how is the
environment sampled?
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
DEVELOPER: M. GEALT
This microcosm simulates a municipal waste facility
with four replicates of each type of holding tank.
Materials are primarily plexiglass, PVC, glass, Tygon,
and epoxy. Medium (see below) is pumped from a
holding tank to the primary settling tanks (ST1) with
a peristaltic pump; liquid hows by gravity to aerator
tanks and then to secondary sealing tanks (ST2).
Sludge from ST2 is pumped back to the aerator
tanks. The final effluent from the ST2's goes to a
100-L tank to which bleach is added.
Yes.
Yes.
Yes.
Yes.
.No.
.No.
.No.
No
Depends on medium used
Depends on medium used
Authentic Wastewater from raw wastewater, settling
tank, etc.. may be used to supply the growth medium
and culture. Artificial Medium consisting of a synthetic
wastewater or 0.03% nutrient broth, can be used with
either a combination of pure cultures from wastewater,
etc. (characterized) or bacteria derived from primary
or raw sewage (uncharacterized).
Urecharacterized bacteria are obtained as a grab sample
from raw wastewater, sealing tank, etc. Authentic
wastewater, when used, is pumped from a municipal
treatment facility into 200- to 300-L holding tanks
(enough for a S - 6 day test) and maintained at room
temperature until used.
Waste treatment system.
No limitations-as long as microcosm is applied to
waste treatment systems.
77
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
SoUlSediment
Surface Area (cm?)
N/A
Dimensions (cm) Volume (L)
Settling tank (each) 7 L
Aerator (each) 5 L
Lines (total) 1 L
Lagoons (if used) 10 L
Dimensions for one complete system: 500 cm (L) x
300 cm (H) x 200 cm.
Room size, getting the common feed, etc. to function,
and engineering so that sampling is physically
possible.
Each replicate system requires 7.5 m3; four replicates
require 30 m'
7. For what purpose was the microcosm originally
designed?
To model GEM survival and gene transfer in a waste
treatment system.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
The microcosm is not directly connected to the
environment. Medium is pumped according to the
desired system retention time. Aerators use "house"
compressed air and aquarium air stones to produce
continuous bubbling (like a boil) in the tank.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
Yes.
1-2 days without test organisms (GEMs) but with
wastewater organisms.
Uncertain. We assume it allows biofilm to form
which aids in gene transfer mechanisms.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
6 days (beyond 2 day acclimation period)
Nutrient level High nutrients lead to high growth
which tend to clog return activated sludge lines.
78
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What land of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
14. How is water/air circulated/mixed?
Room light (Two 100 or 150 watt) overhead.
Generally on during day and off at night.
Optical density (for cell growth)
Air-conditioned room maintained at 20-25ฐ C.
Peristaltic pump (one for medium flow, one for
return activated sludge)
79
-------
CONTAINMENT
I. a. Is containment with current microcosm
design adequate for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
Yes_
No
PROTOCOLS
I. Has a detailed protocol (e.g., standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib. or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm/
b. Operate a microcosm?
Plexiglass covers on tanks contain' aerosols.
Environmental chamber has its own AC and exhaust
system. To facilitate cleaning, floor and walls are
made of ceramic tile, and there is a floor drain.
Yes
No
1. Tight-fitting lids with air exchange filters.
2. Time-controlled chlorine bleach addition to waste
holding tank.
3. Automatic sampling devices not requiring
removal of the tank tops for sampling.
. a. Considerable resources, skill, or time.
. b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
Yes_
Yes_
Yes
Yes_
Yes.
Yes
No.
No.
No_
No_
No_
No
Yes.
No
80
-------
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
1. Automatic sampling and monitoring, e.g.,
temperature, D.O., pH, etc.
2. Restructure activated sludge return lines to
decrease clogging (using larger diameter tubing,
different pump heads, etc.)
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
. a. Considerable resources, skill or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
5 ml samples can be obtained from any or all of the
following:
ST1, ST2: influent, settled solids, and effluent.
Aerator (return sludge container).
Lagoon
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
I. What is the relative capital cost of a single
complete microcosm unit (i.e., one vessel, stirrer,
etc., without temperature control, flowing water,
etc.)?
Yes
No
2. How many replicate vessels are generally used
per treatment
Yes.
No_
a. Less than $100
b. Between S100 and $500
x c. Between S500 and S1000
d. More than S1000
Four
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
.a. Less than SSOOO
. b. Between $5000 and $20000
. c. Over $20000
. d. An estimate has not been made
81
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-duetivity in phytoplankton by 14C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.)- Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
ENERGY FLOW
BIOGEOCHEM
CYCLING
OTHER
EFFECTS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
NITROGEN
PHOSPHORUS
SULFUR
OTHERJSPECEFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Standard methods
Standard methods (BOD, TOC, suspended solids)
Reasons that a parameter cannot be addressed in your microcosm
82
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=HIGH; ^INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
N/A
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY) Wastewater
operation parameters
If comparability studies have been conducted, briefly discuss
major problems encountered in making comparison, cite the
reference(s), and include a copy, if possible.
83
-------
FIELD VERIFICATION OFM1CROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes
.No.
Yes
No
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
I=Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival,
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
Yes
No.
Yes
No.
FURTHER INFORMATION ON WASTE TREATMENT MICROCOSM
Dr. Michael Gealt
Depc of Bioscience & Biotechnology
Drexel University
32nd and Chestnut Streets
Philadelphia. PA 19104
(215) 885-5092
Mancini, P., S. r-eneis, u. Nave, and M.A. oeaii.
1987. Mobilization of plasmid pHSV106 from
Escherictua coli HB101 in a laboratory-scale
waste treatment facility. Appl. Environ.
Mioobiol. 53:665-671.
Sagik, B Jป., and C-A. Sorber. 1979. The survival of
host-vector systems in domestic sewage treatment
plants. Recombinant DNA Bull. 2:55-61.
84
-------
Inculaand
Medium Feed
Primary
Settling
Tanks
Aerators
Return Activated
Sludge Line
\ \
Effluent Uneซ
Side View
Figure?.
Laboratory waste treatment facility.
85
-------
Appendix C
Terrestrial Microcosms
87
-------
ROOT MICROCOSM SYSTEM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material. If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: D. KLEIN
Seeds are sterilized (20% Chlorox) and germinated
on sterile 1/10 strength nutrient agar. Noncon-
taminated plants are transferred to a 1-liter Pyrex jar
containing autoclaved. fritted clay covered with 2 cm
of sand, Hollands's solution (1/4 strength, 400 ml)
buffered to pH 7 with Sorensens phosphate is added,
and the jar is sealed with a lid containing 3 holes: 1
for sterile air input, 1 for sterile nutrient input, and
one for the plant (surrounded by silicone sealant).
For nonsterile treatments, a 10 ml mixture of
rhizosphere organisms can be added.
Soil microbiota
Plant seedlings
Could be included
Sterile seedlings are transplanted. Natural mixed
inocula, or specific single or combined microbial
isolates can be added.
4. If environmental media are used, how is the
environment sampled?
Autoclaved, fritted clay is used.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Grass and forb systems
The plants must be limited in size. Small trees could
possibly be used, but only in scaled-up root
microcosm system.
89
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
7. For what purpose was the microcosm originally
designed?
Soil/Sediment
Dimensions (cm) Volume (L) Surface Area (cm1)
Approx.
12 x 12 cm
1L
Approx. 100 cm1
To measure plant root and microbial respiration, and
to separate the two processes.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
With the tubing connections, air and water in the
Root Microcosm Systems can be exchanged when
desired.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
Yes.
No
Approximately 90 days.
Plant establishment and viability, and lack of system
contamination.
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
Standard greenhouse or growth chamber conditions;
depends on the environmental conditions to be
duplicated.
90
-------
GENERAL CHARACTERISTICS
(CONTINUED)
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
These depend on experimental design
. Dissolved organic matter
Constant temperature room or growth chamber.
14. How is water/air circulated/mixed?
A syringe is used to exchange water in each individual
unit to slowly pass the liquid through the filters.
CONTAINMENT
I. a. Is containment with current microcosm
design ?ซJT'at* for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
Yes.
No
Physical barrier on top of unit.
Membrane filters on gas and water inlet and outlet
Yes No x
. a. Considerable resources, skill, or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
91
-------
PROTOCOLS
I. Has a detailed protocol (e.g.. standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. -Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g.. additional trophic
levels, reduction of analytical time/costs, etc.).
2. What levels of difficulty would be involved in
making the modifications in (1) above?
Yes.
Yes_
Yes
Yes.
Yes.
No.
No.
No.
Yes
Yes
Yes
No
_ No
No
No.
No
Improvement of ability to sample plant growth matrix
before completion of an experiment, and to remove
root sub-samples.
. a. Considerable resources, skill or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
d. Can't estimate at this time.
-------
SAMPLING
I. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
Gas and liquid sampling, and microbial sampling of
liquid medium. Periodic sampling of solid material
can be accomplished by setting up replicate units
which can be taken apart at desired intervals.
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
1. What is the relative capital cost of a single
complete microcosm unit (i.e.. one vessel, stirrer.
etc., without temperature control, flowing water,
etc.)?
Yes
No
2. How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
Yes
.No_
.a. Less than S100 (Appro*. S5/unit)
b. Between SI00 and S500
. c. Between S500 and S1000
d. More than $1000
Three to four
_ a. Less than $5000
. b. Between $5000 and $20000
. c. Over S20000
. d. An estimate has not been made
93
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-duetivity in phytoplankton by 'XT-carbonate uptake or in macrophvtes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
Microscopic and viable populations; lipid analyses
Microscopic and viable pop.; exudate analysis
ENERGY FLOW
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
PRIMARY PRODUCTION Plant growth and respirometry
SECONDARY PRODUCTION Microbial responses in the rhizosphere
P/R RATIO
OTHER (SPECIFY)
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Chemical analysis
Same
Same
Growth responses (dry weight) and respirometry
Community structure and function characteristics
Reasons that a parameter cannot be addressed in
your microcosm
With appropriate construction and sampling modi-
fications, it should be possible to sample a full
range of plant/microbe interactions in smaller plant
systems.
94
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-caJibraied a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=HIGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R.RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted, briefly discuss
major problems encountered in making comparison, cite the
reference(s), and include a copy, if possible.
95
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes
No.
Yes
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
^Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
Yes x No
If funding is available.
Yes
No
The major variables tested to date have been nitrogen
level, plant type and microbial inoculation presence
in the plant root zone.
5. Please discuss any factors other than survival
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
I*.:-, r\ A
-
Trlica. 1988. Rhizosphere microorganism effects
on soluble amino acids, sugars, and organic
acids in the root zone of Agropyron crisuuum,
A. smiitiii and Boutelona gracilis. Plant Soil.
110:19-25.
Dr. Donald Klein
Colorado State University
Depc of Microbiology
Fort Collins, CO 80523
(303)491-6947
0twiiuiiu,
__ .
LS./V. IWCUL, dJIU C.U-. I\CUC1IIC. 1700.
Carbon and nitrogen losses through root
exudation by Agropyron cristaaun, A. smitfui
and Bouulona gracilis. Soil Biol. Biochem.
20:477482.
96
-------
Drying Tube Filled With
Nonabsorfaant Cotton
Millipore Filter **\_3
Silicone Rubber Sea
Nutrient Solution Out
Nutrient Solution In
Flgur* 8. Root microcosm ปyปiซnv
97
-------
SOIL CORE MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material. If possible, include a
labeled diagram.
2. Which of the following trophic levels are
. normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: J. FREDRICKSON
The 60-cm-deep by 17-cm-diameter microcosm
consists of a 17-cm-diameter tube of Driscopipe
(polyethylene pipe) containing an intact soil core (40
cm) covered by homogenized topsoil (20 cm). The
natural grassland microcosm is an intact, totally
undisturbed 17-cm-diameter by 60-cm-deep test
system. This tube sits on a Buchner funnel that is
covered by a thin layer of glass wool. Six to eight
microcosms are typically contained in a moveable
can, which is packed with insulated beads or a
comparable material to reduce drastic changes in
temperature profile.
Yes.
Yes_
Yes.
Yes_
.No.
.No.
.No.
No
indigenous soil microflora
plants w/size, time limits
soil microfauna
possibly small mammals
They are "pre-established" as the microcosm consists
of an intact soil core it harbors indigenous
communities. Plants, microorganisms, microfauna
etc., can be readily introduced.
4. If environmental media are used, how is the
environment sampled?
A steel coring tube is driven into soil and extracted to
obtain an intact core housed in a Driscopipe liner.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Limited to terrestrial environments, mainly soils and
unsaturated sediments.
Physical limitations for saturated sediments and water.
99
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Dimensions (cm)
SoillSediment
Volume (L) Surface Area (cm1)
60 (depth) x 17 (diam)
Physical ability to extract an intact core. They can be
quite large if the proper heavy equipment is available.
- 1 FtJ
7. For what purpose was the microcosm originally
designed?
Toxicological studies of impacts of chemicals on soil
biota and nutrient cycling processes.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
Moisture: Water characterized using ASTM D19,
Test Metjiods for Water Quality Analysis.
Microcosms are leached at least once before dosing
and once every two or three weeks after dosing,
based on natural rainfall amounts. Leachate is
collected in 500-ml flasks attached to the Buchner
funnel.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
Yes_
Soil is saturated and allowed to drain. The length of
time varies with soil texture but can be < 24 h for a
coarse grained soil to 3-4 days for a clay soil.
In general, one pore volume of water is leached
through the core to remove initial concentrations of
nutrients. Following this initial leaching, no additional
time is required for equilibration.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factor in estab-
lishing fo* Jjfesnsi?! f,f ?hiซ ***
Microcosms generally operated over 2-3 week periods
although there is essentially no restraint on lifespan.
Microcosms have been operated for up to 8 months
with/vit ntonr*
ป ป *ปW-^* w ' ^ ~ i
100
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What land of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
Light for the test system can be natural or artificial,
depending on the use of a growth chamber or a
greenhouse.
400 nEinsteins nr1
That which is optimal for plant growth or mimics
specific field photoperiod.
Microcosms in insulated carts or other devices are
kept in a greenhouse or environmental chamber where
temperature and light can be controlled.
14. How is water/air circulated/mixed?
Air is circulated via greenhouse or growth chamber
fans.
101
-------
CONTAINMENT
I. a. Is containment with current microcosm
design microcosm design adequate for
working with GEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
Microcosms are contained in a greenhouse or in a
growth chamber within a laboratory.
Yes
An improved HEPA-filtered containment chamber
for housing the soil-cores. Such a chamber has been
designed and a prototype was constructed. Designs
are available.
a. Considerable resources, skill, or time.
j_ b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
PROTOCOLS
1. Has a detailed protocol (e.g., standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la. Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
3. If the answer to any of the above (la. Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
Yes.
Yes.
Yes
Yes.
Yes_
.No.
.No.
No
Yes
Yes
Yes
_No
_No
_No
.No.
.No.
102
-------
MICROCOSM MODIFICATION POTENTIAL
l: List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
1) Development of a system that would allow
maintenance of soil water potential This could
be done either (a) manually by weighing cores
every day and adding water to a pre-determined
constant weight or (b) automatically by developing
a computer-controlled system that would add
water when the weight of a core dropped below a
certain value.
2) Use in a programmable environmental chamber
that spans the temperature-humidity values in the
field.
a. Considerable resources, skill or
time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
Sampling plants without destruction is difficult but
can be done.
Subsampling soil is accomplished easily but can
destroy the physical integrity of the core for transport
(leaching) studies.
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
Yes
(in general, but is dependent on
nature of the experiment)
103
-------
COSTFACTOflS
1. What is the relative capital cost of a single
complete microcosm unit (Le., one vessel, slirrer,
etc., without temperature control, flowing water,
etc.)?
2. How many replicate vessels are generally used
per treatment?
x a. LesstnanSlOO
b. Between $100 and S500
c. Between S500 and $1000
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving, Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary productivity in phytoplankton by I4C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC SUBSTRATE/BACTERIA
INTERACTIONS BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
introduced indigenous soil microbes (bacteria, fungi)
earthworms, aphids, com borers (GEM vectors)
mineralization of 14C-labeled cellulose
aphids & corn borers on plants
bacterial colonization, nodulation of plant roots
ENERGY FLOW PRIMARY PRODUCTION plant biomass (root & shoot), microbial respiration
SECONDARY PRODUCTION soil microbial biomass
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM. NITROGEN
CYCLING PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER PLANT (SPECIFY)
EFFECTS ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
"N uptake, mineralization, pool partitioning
Plant assimilation, leaching
rhizome popVdivers.; enzymes: dehydVglucosidVperox.
Reasons that a parameter cannot be addressed in
your microcosm
Animals in soil-core microcosms generally cause
out-of-scale problems (e.g., excess grazing of
plants)
105
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=fflGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
M-H
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
M-H
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
M-H
OTHER PLANT (SPECIFY)
EFFECTS ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)*
OTHER (SPECIFY)
* rhizosphere & soil populations: diversity/enzyme activities.
If comparability studies have been conducted, briefly discuss
major problems encountered in making comparison, cite the
reference(s), and include a copy, if possible.
106
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the Held. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
If the answer to la. (above) is "yes." please rate
the degree of comparability (H=High;
^Intermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
FACTOR
Survival/
Colonization
Environmental Mobility
(Specify organism or gene)
Yes
No
Yes
No
Pseudomoruu sp. and
Strepiomyces lividan
An Azospirillum
and a Pseudomonas
I-H
Yes
.No
Yes
No
Growth chamber favored microbial growth & function
over field. Comparability better between plant growth
stages than on actual time basis. Field temperature
and humidity changes were difficult to simulate.
5. Please discuss any factors other than survival,
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
Effects: 14C-Cellulose mineralization; rhizosphere
populations; enzyme activity (dehydVglucosid.) I4N
transformation; microbial biomass; transport by
leaching, root growth, earthworms; nutrient uptake
& leaching.
107
-------
FURTHER INFORMATION ON SOIL CORE MICROCOSM
Dr. James Fredrickson
Battelle Pacific NW Laboratories
P.O. Box 999
Rkhland, WA 99352
(509) 375-3908
Federal Register.
microcosm test
1987. 797.3775 Soil-core
52(187):36363-36371.
El 197 Guide for Conducting a Terrestrial Soil-Core
Microcosm Test In 1991 Annual Book of ASTM
Standards, Vol. 11.04, American Society for
Testing and Materials, Philadelphia, PA.
Bentjen, S.A., J.K. Fredrickson, P. Van Voris, and
S.W. Li. 1989. Intact soil-core microcosms for
evaluating the fate and ecological impact of the
release of genetically engineered
microorganisms. Appl. Environ. Microbiol.
55:198-202.
Fredrickson, J.K., S.A. Bentjen. and H. Bolton, Jr.,
S.W. Li, and P. Van Voris. 1989. Fate of Tn5
mutants of root growth-inhibiting Pseudomonas
sp. in intact soil-core microcosms. Can. J.
Microbiol. 35:867-873.
Fredrickson, J.K., H. Bolton, Jr., S.A. Bentjen, K.M.
McFadden. S.W. Li, and P. Van Voris. 1990.
Evaluation of intact soil-core microcosms for
determining potential impacts on nutrient
dynamics by genetically engineered micro-
organisms. Environ. Toxicol. Chem. 9:551-558.
Bolton, H. Jr., J.K. Fredrickson. JM. Thomas, S.W.
Li, D.W. Workman, S.A. Bentjen, and J.L. Smith.
1991. Field calibration of soil-core micro
microcosms: Ecosystem structural and functional
comparisons. Mkrob. EcoL 21:175-189.
Bolton, H. Jr., J.K. Fredrickson. S.A. Bentjen, D.W.
Workman, S.W. Li, and J.M. Thomas. 1991.
Field calibration of soil-core microcosms: Fate
of a genetically altered rhizobacterium. Microb.
EcoL 21:161-173.
High Density
High Molecular Weight
Polyethylene
Intact
Soil Core
Glass
Wool
Buchner
Funnel
Soi! ccra iic.oc3s.-fi.
108
-------
SOIL IN A JAR MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
. Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: G. STOTZKY
Sieved (1 cm) soil is stored in a greenhouse or
laboratory. Two weeks before use, soil water tension
adjusted to -33 kPa, and soil is mixed with a glucose
solution (1% wt/wt) and ca. 20 mg fresh garden soil
g-i soil SO-g (oven-dry equivalent) of sieved (2 mm)
soil adjusted to -33 kPa water tension is added to 8 -
10 100-ml glass vials, which are placed in a 1-gal
wide-mouth jar. A manifold, attached to a scrubber
system (to saturate air with water and remove oil,
COr nitrogen compounds, and other contaminants),
provides air to the jar. CO2 in exiting air is trapped
and quantified.
Yes x No soil microbiota
Yes. No_s
Yes_s No soil microinvertebrates.
Yes No_x
Sieved soil (1 cm mesh) from the top 5 cm of a field
contains microbiological and microinvertebrate
communities.
4. If environmental medium are used, how is the
environment sampled?
Soil is collected from the surface of a field.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Soil from a tilled or untilled field
109
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Soil/Sediment
Dimensions (cm) Volume (L) Surface Area (en?)
16 x 26 cm - 3.8 L
Convenience
Approximately 26 cm1
7. For what purpose was the microcosm originally
designed?
Used for soil microbiological research, testing the
effects of pollutants (e.g.. heavy metals, acid
precipitation, pesticides) on microbial activity in soil.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so. what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
Soil containers are continuously flushed with water-
saturated air
Yes-
No
Days, weeks, or months
Design and purpose of study; maintenance of soil at
-33 kPa water tension
1L What !sod of ligmns is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod.
means of control, etc.):
Constant darkness or light/dark cycle may be used
110
-------
GENERAL CHARACTERISTICS
(CONTINUED)
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
. (at beginning and end of test)
. (maintained constant)
. (maintained constant)
. (at beginning & perhaps end of test)
(pH, species diversity, enzyme activity, and
survival of OEMs at beginning and end of
test)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
14. How is water/air circulated/mixed?
Constant temperature incubator or room.
Continuous flushing with water-saturated, CO2-free
air.
CONTAINMENT
1. a. Is containment with current microcosm
design adequate for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
Yes x No_
Soil contained in glass vessels is autoe laved before
disposal
Yes No x
. a. Considerable resources, skill, or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
d. Can't estimate at this time.
111
-------
PROTOCOLS
1. Has a detailed protocol (e.g., standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib. or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, ex.).
Yes.
Yes.
Yes
Yes.
Yes
.No.
.No.
.No
Yes
Yes
Yes
_No
_ No
_No
.No.
No
Greater degree of automation for measuring CO,
evolved (e.g., capacitance measurements; automatic
sampling for iteration)
2. What levels of difficulty would be involved in
making the modifications in (1) above?
. a. Considerable resources, skill or time.
. b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
.
-------
SAMPLING
I. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
Repetitive sampling is limited by the number of soil
vials within the master jar.
2. Is destructive sampling during the course of a
test run required?
Yes x No (For analyses in #12 above; no, if
only respiration is measured.)
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
1. What is the relative capital cost of a single
complete microcosm unit (i.e.. one vessel, stirrer,
etc., without temperature control, flowing water,
etc.)?
Yes.
No
2. How many replicate vessels are generally used
per treatment?
x a. Less than SI00 (without titrator. etc.)
b. Between S100 and S500
c. Between S500 and S1000
d. More than $1000
Three to five
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
i. Less than S5000 (without labor)
b. Between S5000 and S20000 (with labor)
. c. Over $20000
1 An estimate has not been made
113
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
ENDPOINT
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-duetivity in phytoplankton by 14C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
Species diversity by selective media; probes
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
Addition of specific substrates
Selective media
ENERGY FLOW
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Can use 'XT-labeled substrates
Soil anal.-perfusion apparatus (EPA/600/3-90/011)
Soil anal
Soil anal
Soil anal
Addition of GEMs
Reasons that a parameter cannot be addressed in
your microcosm
System is limited to soil It could be modified to
include plants, but this would not be practical.
114
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H.fflGH; ^INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted,
briefly discuss major problems encountered in
making comparison, cite the references), and in-
clude a copy, if possible.
Not conducted
115
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
^Intermediate; L=Low).
3. If the answer to la, (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
FURTHER INFORMATION ON SOIL IN A JAR
FACTOK
Survival/
Colonization
Environmental Mobility
(Specify organism or gene)
Yes
.No
Yes.
Yes
Yes
Dr. Guenther Stotzky
New York University
Department of Biology
1009 Main
Washington Square
New York, NY 10003
(212) 998-8266
Stotzky, G. 1965. Microbial Respiration. In Methods
of Soil Analysis, C.A., Black et al., (eds),
American Society of Agronomy, Inc., Madison,
WI.pp. 1550-1570.
Stotzky, G. 1989.-Methods to measure the influence
of genetically engineered bacteria on ecological
processes in soil. U.S. Environmental Protection
Agency, EPA/600/3-90/011. 36 pp.
Stotzky. G. 1991. Evaluation of selected biochemical
and ecological methods to assess effects of
recombinant bacteria in terrestrial ecosystems.
U.S. Environmental Protection Agency. In press.
pp.
116
-------
CO2 -Free Air
(To Atmosphere)
_ซ-8.9cml.D.-M
15 Rubber Stopper
Wire Spring
21
Wire Loop Nฎ**
Bubble
Tower
Glass Beads
(6mm)
3/16 in. I.D.
Latex Rubber Tubing
Quick Disconnect
One-Way Valve
I 3/16 in. 1.0
Latex Rubber Tubing
CO 2- Free Air
(From Manifold)
3/16 in. O.D. Pyrex
Air Outlet Tuba
Individual
Incubation Jars
Soil
(50 g Oven-Dry
Wt. Equivalent)
Gallon Glass
Master Jar
Figure 10.
Soil In Jar.
117
-------
TERRESTRIAL MICROCOSM CHAMBER
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material. If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: J. GILLETT
The Terrestrial Microcosm consists of a chamber (1
x 0.75 x 0.75 m) constructed of gia*$ plate, plexiglass,
a UV-transparent glass top cover, and removable
side panels (with glove openings). This chamber
rests on a polyethylene box (1 x 0.75 x 0.55 m). It
contains soil and a variety of biota, including
seedlings. Soil is mixed and sieved through a coarse
(1 cm) screen to remove rocks, roots and other
debris; then it is sieved through a 2 mm screen after
being tumbled in a portable cement mixer. Each
system requires about 200 to 300 kg of sieved soil
which is added in 5-cm layers saturated with water
and packed by application.
Yes x No Indigenous
Yes_5 No Indig. or agric. plants
Yes_s No Indig. & earthworms
Yes_5 No Voles/quail with 730 cm soil
Through the use of unsterilized soil.
4. If environmental media are used, how is the
environment sampled?
Air polyurethane foam filters and direct air sampling;
soil: coring; water leachate sampling.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
a. Agroecosystems
b. Plant size, temperature means and extremes.
119
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
SoWSedunent
Dimensions (cm) Volume (L) Surface Area (cm2)
Upper chamber
100(L)x75(W)
x5
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What kind of lighting is used?
' a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
14. How is water/air circulated/mixed?
1000- watt sylvania metal halide lamp, positioned 55
cm above the chamber.
18:6 L:D cycle
(Soil and air)
. (In leachate)
. (Water output: leachate & air moisture)
A constant temperature room containing the chambers
is heated and cooled to ฑ 1ฐC.
Negative flow through baffled filters
121
-------
CONTAINMENT
1. a. Is containment with current microcosm
design ad**yia** for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
Yes x No.
Glove box
Yes x No_
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain*
menu what degree of difficulty would be
encountered in making these modifications?
PROTOCOLS
1. Has a detailed protocol (e.g., standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib. or Ic)
is "no." could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a
. a. Considerable resources, skill, or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
(Depends on desired level of
containment)
Yes_j
Yes_j
Yes.,
Yes
Yes
Yes
Yes_a
v*ซ ,
i No (Note, however, that the
purpose of this system is to
, No I have flexibility in protocols)
i No
_No
_No
__No
L_No
vซ
122
-------
MICROCOSM MODIFICATION POTENTIAL
1. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g;, additional trophic
levels, reduction of analytical time/costs, etc.).
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
a. Considerable resources, skill or time.
b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
(1) Soil cores are replaced with "control plugs."
(2) No limits to air and water sampling.
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
I. What is the relative capital cost of a single
! complete microcosm unit (i.e., one vessel, stirrer,
etc., without temperature control, flowing water,
etc.)?
Yes_
No_
Yes_
No
a. Less than S100
, b. Between $100 and $500
, c. Between $500 and $1000
.d. More than$1000
2. How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
. a. Less than $5000
. b. Between $5000 and $20000
.c. Over $20000
. d. An estimate has not been made
123
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-ductivity in phytopiankton by 14C-carbonate uptake or in macro-
phyies by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia
concentrations or fluxes, etc.)- Also indicate if an endpoint could not be used in your
microcosm, and if not why.
ENDPOINT
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
TROPHIC
INTERACTIONS
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
Biomass measurements
Soil sampling and enumeration
Soil sampling and enumeration
NA
Consumption
Predation rate
ENERGY FLOW
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
NITROGEN Leachate analysis
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Same
Same
Reasons that a parameter cannot be addressed in
your microcosm
Destructive sampling for a number of parameters
limits repetitive observations over time.
124
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H-fflGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted, briefly discuss
major problems encountered in making comparison, cite the
reference(s), and include a copy, if possible.
125
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
FACTOR
Survival/
Colonization
Environmental Mobility
(Specify organism or gene)
Yes
.No.
Yes
No
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
I=lntermediate; L=Low).
3. If the answer to la. (above) is "no," do you plan
to conduct field verification studies with microbes
in the next three years.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival,
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
Yes
No
Yes
No
Chemical fate and mass balances are very close in
field results, but no specific studies using GEMs
have been made.
FURTHER INFORMATION ON TERRESTRIAL MICROCOSM CHAMBER
Dr. James Gillea
16 Fernow Hall
ICET Cornell University
Ithaca, NY 14853 3001
(607) 255-2163
Gillett, J.W., and J.O. Gile. 1976. Pesticide fate in
terrestrial laboratory ecosystems. Intern. J.
Environ. Stud. 10:15-22.
Gile, JJX, J.C. Collins, and J.W. Gillett. 1982. Fate
and impact of selected wood preservatives in a
terrestrial model ecosystem. J. Agric. Food Chem.
30:295-301.
126
-------
Rain Inlet
Air Intel
Rain Nozzle
~ซc
^
a^
r^^^m
"X
-*|~ '
to
to
La
'
to
to
to
' ^T^ A
^9.
^
*
ooo
ooo
ooo
ooo
ooo
ooo
nOO
x
^
*ป
G
V
C
ป .
.XX
r*i "
i
^I^H
T
1
. I
1
I
1
i/oun
/atar
lutlet
f
L_jcL/ + $
8
*
Lysimetef Tube
.^ป.*\
^H
^M
1^
*
" " "' *
?X~
X
*
*
^
^^
x*
^**
^^
X
'^^
ซ*i '''fj ',
o-
(*itt
*^^*
^rt*
-^H
^ -
II
II
11
II
II
VS*ฑ
A
.1
9
f
^l
X
x^
~~~^
;v L AirOutJet
^ PolyunBthane Foat
^
-------
TERRESTRIAL MICROCOSM SYSTEM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: SEIDLER/ARMSTRONG
The Terrestrial Microcosm makes use of the Gillett
microcosm (See Terrestrial Microcosm Chamber)
for small scale experiments. Each chamber holds 2
wooden trays (47 x 37 x 7 cm) lined with polyethylene
bags, supported by a metal rack in the chamber. 50
cm above the floor. Trays are planted with beans or
other selected indigenous plants. A humidifier is
located below the trays, and single-pass air is forced
through the chambers and exhausted through HEPA
raters.
Yes.
Yes.
Yes.
Yes.
.No_
.No_
.No.
No.
Indigenous bacteria, fungi
Indigenous; planted seeds
Indigenous; cutworms, etc.
Indigenous species collected with soil sample. Insects
are introduced from lab cultures. Seeds are planted.
4. If environmental media are used, how is the
environment sampled?
Soil is collected with shovel and put in wooden trays
lined with plastic bags.
5. What habitats are represented?
a. Typically:
b. What factors) limit the habitats that could
be represented?
Agricultural field
Size of the trays that hold soil and plants
129
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
Dimensions (cm)
SoUfSediment
Volume (L) Surface Area (cirf)
Upper chamber
100 x 75 (w) x
75 (d)
Lower chamber
100 x 75 x 55 3480
Each microcosm contains two wooden trays (47 x 37
x 7 cm) holding soil and plants.
Size of larger chamber holding the trays and
convenience of lifting trays with soil.
About 3mx3mx4m (overhead) for plastic box
that contains the trays.
7. For what purpose was the microcosm originally
designed?
Effects of toxic chemicals on ecosystem processes.
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
Air passing through chamber is controlled by manual
valves and is exhausted through a HEP A filter. Each
chamber contains an industrial grade humidifier below
the trays, and water accumulating on the chamber
floor is suctioned through a tube, collected in a 5 gal
container and disinfected with bleach.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so. what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
Yes_
May not be required
Three days to allow plants/soil to acclimate to air
temperature, relative humidity, and light/dark cycle.
To acclimate contents of trays to chamber
environment
10. Microcosm "lifespan":
a. How long are microcosm tests generally
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
3-4 weeks
Loss of plants due to destructive sampling
130
-------
GENERAL CHARACTERISTICS
(CONTINUED)
11. What kind of lighting is used?
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod,
means of control, etc.):
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
A 1000-watt GTE Sylvania metal halide lamp is
centered over each system.
Unknown
Light/dark cycle - variable with timer
(on/off)
No controltotally determined by lights and
temperature of room (room is heated/refrigerated to
ฑ2ฐQ
14. How is water/air circulated/mixed?
Air passage through system by fan controlled
manually with valve
131
-------
CONTAINMENT
1. a. Is containment with current microcosm
design adequate for working with OEMs?
b. If so, describe containment design.
c. Could containment be unproved by design
modification?
d. If so. what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
PROTOCOLS
1. Has a detailed protocol (e.g., standard operating
procedures, publication) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
Plastic doors with glove-box type design. HEPA
filter traps particles/ bacteria before exhausting to
outdoors
Yes
No.
Depends on what experiments are to be done (e.g.,
could put whole chamber into negative pressurized
room)
a. Considerable resources, skill, or time.
b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
_j d. Can't estimate at this time.
(Depends on experimental design)
Yes.
Yes.
Yes.
Yes.
Yes.
Yes_
.No.
.No.
.No
.No.
.No_
.No.
b. Operate a microcosm?
Yes.
No.
132
-------
MICROCOSM MODIFICATION POTENTIAL
I. List any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM rislcassessmem use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
Install computerized control of environment so
chambers can mimic variability of outdoor conditions.
Suggest controls for RH, air and soil temperature,
soil moisture, and light intensity.
2. What levels of difficulty would be involved in
making the modifications in (1) above?
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
a. Considerable resources, skill or
time.
b. Moderate resources, skill or time.
c. Minimal resources, skill or time.
d. Can't estimate at this time.
Leaves and soil are currently sampled.
If destructive sampling is used, sampling is limited
by small number of plants.
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
1. What is the relative capital cost of a single
complete microcosm unit (Le., one vessel, stirrer,
etc.. without temperature control, flowing water,
etc.)?
Yes.
No
Yes_
No.
a. Less than $100
,b. Between S100 and S500
c. Between S500 and S1000
d. More than $1000
2. How many replicate vessels are generally used
per treatment?
Two "boxes" are used in an experiment; replicate
experiments are performed with plants and soil in
pairs of boxes
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
. a. Less than $5000
. b. Between $5000 and $20000
. c. Over $20000
d. An estimate has not been made
133
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
ENDPOINT
Indicate whichof the following parameters have been measured in your microcosm by briefly
listing the technique (i.e., benthos by sieving. Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-duetivity in phytoplankton by l4C-carbonaie uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
OTHER
EFFECTS
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Reasons thai a parameter cannot be addressed in your microcosm
134
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in 'the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=fflGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted, briefly
major problems encountered in making comparison, cite the
reference(s), and include a copy, if possible.
135
-------
FIELD VERIFICATION OF M1CROBIAL FATE
Field verification tests with GEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide an indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
compared to field data?
If so, please cite the reference(s), and, if possible,
enclose a copy.
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
I=Iniermediate; L=Low).
3. If the answer to la. (above) is "no." do you plan
to conduct field verification studies with microbes
in the next three yean.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival.
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
FURTHER INFORMATION ON POND MICROCOSM
Survival/
Colonization
FACTOR
Environmental Mobility
(Specify organism or gene)
Yes
.No
Yes
No
Yes
No
Yes_
No
Dr. Ray Seidler
U.S. EPA
200 S.W. 35th Street
Corvallis, OR 97333
(FTS) 4204708
Dr. John L. Armstrong
U.S. EPA
200 S.W. 35th Street
Corvallis, OR 97333
(FTS) 420-4718
Armstrong. Jl. Protocol for application of microcosms to study of fate and survival of recombinant bacteria
associated with plants and herbivorous insects. U.S. Environmental Protection Agency, Environmental Research
Laboratory. Corvallis, OR. Preliminary Draft 16 p.
138
-------
VERSACORE MICROCOSM
GENERAL CHARACTERISTICS
1. Briefly describe the physical design including
microcosm vessel material If possible, include a
labeled diagram.
2. Which of the following trophic levels are
normally represented?
Microorganisms (specify)
Primary producers (specify)
Invertebrates (specify)
Vertebrates (specify)
Other (specify)
3. Describe how communities of organisms are
established in the microcosm.
DEVELOPER: W. HOLBEW J. JANSSON
Constructed of clear
-------
GENERAL CHARACTERISTICS
(CONTINUED)
6. Microcosm size:
a. Typically:
b. What factors) limit these size characteris-
tics?
c. How much space is required per microcosm
unit?
7. For what purpose was the microcosm originally
designed?
SoillSediment
Dimensions (cm) Volume (L) Surface Area (cm1)
9.5 cm (O.D.) tubes
Could be scaled-up if desirable
1 cubic foot
57cm2
To monitor transport, survival, and gene exchange of
bacterial populations, in soil (bulk or rhizosphere).
8. Discuss any provisions for exchanging air and
water in your microcosm with the environment.
For aquatic systems, describe aeration and water
exchange (static, static-replacement, flow-
through); for terrestrial systems, indicate air
exchange and addition of water.
Cores are brought to field capacity (approx. 23%
moisture) by setting them in distilled water overnight.
Cores were drained, both ends covered with
Parafilmฎ. After germination, Parafilm is removed
from the top of the core and moisture is maintained
by daily weighings and additions of water.
9. Equilibrium period:
a. Is laboratory equilibrium required before
testing?
b. If so, what is the equilibration period?
c. If required, what is the purpose of the equi-
librium period and what criteria are used to
determine when it is equilibrated.
10. Microcosm "lifespan":
a. How long are microcosm tests generally
run?
b. What are the most important factors in es-
tablishing the lifespan of this microcosm?
i i. What Jdfld of lighting U
a. Type of lights (wattage, model, source, etc.):
b. Typical light intensity:
c. Lighting control (intensity, photoperiod.
Yes-
1-3 weeks
Fluorescent light
Constant light.
138
-------
GENERAL CHARACTERISTICS
(CONTINUED)
12. Which of the following environmental parameters
are routinely monitored?
a. Soil moisture
b. Relative humidity
c. Temperature
d. Light intensity
e. Inorganic nutrients
f. Carbon dioxide
g. Dissolved oxygen
h. Other (specify)
13. How is temperature controlled (constant
temperature room, water bath, etc.)?
Incubation room maintained at desired temperature
and humidity.
14. How is water/air circulated/mixed?
Environmental chamber provides air exchange.
CONTAINMENT
1 .a. Is containment with current microcosm
design adequate for working with OEMs?
b. If so, describe containment design.
c. Could containment be improved by design
modification?
Yes_
Small scale allows benchtop work in lab so that
regular laboratory containment practices can be
followed.
Yes
d. If so, what is the nature of the modifications
needed to improve containment?
e. If modifications would improve contain-
ment, what degree of difficulty would be
encountered in making these modifications?
. a. Considerable resources, skill, or time.
, b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
. d. Can't estimate at this time.
139
-------
PROTOCOLS
1. Has a detailed protocol (e.g.. standard operating
procedures, publication, etc.) been developed
covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
2. If the answer to any of the above (la, Ib, or Ic)
is "no," do you expect to develop protocols
within the next 2 years covering:
a. Microcosm construction?
b. Microcosm operation?
c. Output analysis?
3. If the answer to any of the above (la, Ib, or Ic)
is "no," could a competent technician, with the
aid of literature descriptions:
a. Construct a microcosm?
b. Operate a microcosm?
MICROCOSM MODIFICATION POTENTIAL
1. Us* any additional modifications (other than
containment) that you would recommend to
improve the effectiveness of this microcosm for
GEM risk assessment use (e.g., additional trophic
levels, reduction of analytical time/costs, etc.).
Yes.
Yes.
Yes.
Yes.
Yes.
Yes.
Yes.
Yes_
.No.
.No.
No
.No.
No
. No.
No
The system is pretty well suited to small scale studies
and large numbers of replicates can be handled
effectively.
2. What levels of difficulty would be involved in
making the modifications in (1) above?
. a. Considerable resources, skill or time.
. b. Moderate resources, skill or time.
. c. Minimal resources, skill or time.
, d. Can't estimate at this time.
140
-------
SAMPLING
1. What sampling strategies are currently possible
without design modification, and what are the
limits for repetitive sampling?
2. Is destructive sampling during the course of a
test run required?
3. Would design modifications allow the use of
alternative sampling strategies?
COST FACTORS
I. What is the relative capital cost of a single
complete microcosm unit (ue., one vessel, stirrer,
etc.. without temperature control, flowing water,
etc.)?
2. How many replicate vessels are generally used
per treatment?
3. What is the estimated minimal cost of a complete
microcosm test, including vessels?
Total destructive sampling
Collect and plate leachate
Take small diameter "minicores" through profile
leaving bulk of microcosm largely untouched
Disassemble in 2.5 cm increments to sample vertically
through the profile
Yes_
Yes
.No.
No
x a. Less than $100
b. Between $100 and $500
c. Between $500 and $1000
d. More than $1000
Three
x a. Less than $5000
b. Between $5000 and $20000
c. Over $20000
d. An estimate has not been made
141
-------
APPLICABILITY FOR EVALUATING ECOLOGICAL PARAMETERS
ENDPOINT
Indicate which of the following parameters have been measured in your microcosm by briefly
listing the technique (i.e.. benthos by sieving, Rose Bengal Staining, and sorting; microor-
ganisms by lipid analysis; bacteria/protozoa interactions by selective filtration, staining, and
counting; primary pro-ductivity inphytoplankton by "C-carbonate uptake or in macrophytes
by measuring plant growth; an aspect of nitrogen cycling by measuring ammonia concentra-
tions or fluxes, etc.). Also indicate if an endpoint could not be used in your microcosm, and
if not why.
PARAMETER
TECHNIQUE
COULD NOT
BE STUDIED
IN THIS
MICROCOSM
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
Usually 1-3 seedlings are used
DNA probes, selective plating, direct counts
TROPHIC SUBSTRATE/BACTERIA
INTERACTIONS BACTERIA/PROTOZOA
PLANTS/HERBIVORES
HERBIVORES/PREDATORS
OTHER (SPECIFY)
Substrate depletion analyses (e.g. HPLQ
Sterile/nonsterile systems; eukaryotic inhibitors
ENERGY FLOW PRIMARY PRODUCTION
SECONDARY PRODUCTION Could study label uptake by microbes from plants
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM- NITROGEN
CYCLING PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY)
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
Gene exch.; transport thru soil, population inputs
Reasons that a parameter cannot be addressed in
your microcosm
Scale is too small for many of these parameters.
Biogeochemical cycling has not been tested but
probably is not appropriate at this scale
142
-------
FIELD CALIBRATION OF ECOLOGICAL PARAMETERS
Field calibration tests compare the responses of ecological parameters in microcosms with
the field in the absence of stress agents, and may provide an indication of extrapolation
potential. If a field calibration test has been performed with your microcosm for any of these
parameters, please signify high, intermediate, or low comparability with the field. If you have
not field-calibrated a parameter but plan to do so in the next 3 years, please indicate this, also.
FACTORS
PARAMETERS
PARAMETER HAS BEEN
STUDIED; COMPARABILITY
WITH FIELD WAS:
H=HIGH; I=INTERMEDIATE;
L=LOW
PARAMETER HAS NOT
BEEN FIELD CALIBRATED
BUT IS EXPECTED
TO BE WITHIN 3 YEARS
COMMUNITY
STRUCTURE
PLANTS
ANIMALS
BENTHOS
MICROORGANISMS
OTHER (SPECIFY)
TROPHIC
INTERACTIONS
SUBSTRATE/BACTERIA
BACTERIA/PROTOZOA
PLANTS/HERBIVORES
OTHER (SPECIFY)
ENERGY FLOW
PRIMARY PRODUCTION
SECONDARY PRODUCTION
P/R RATIO
OTHER (SPECIFY)
BIOGEOCHEM.
CYCLING
NITROGEN
PHOSPHORUS
SULFUR
OTHER (SPECIFY)
OTHER
EFFECTS
PLANT (SPECIFY) _
ANIMAL (SPECIFY)
MICROBIAL (SPECIFY)
OTHER (SPECIFY)
If comparability studies have been conducted, briefly discuss
major problems encountered in making comparison, cite the
reference(s), and include a copy, if possible.
143
-------
FIELD VERIFICATION OF MICROBIAL FATE
Field verification tests with OEMs or microbes used as surrogates for OEMs may be
conducted to compare the survival, colonization, and microbial/gene mobility observed in
microcosms with the field. These tests may provide and indication of extrapolation potential.
Questions
1. Has your microcosm response to this factor been
. compared to field data?
2. If the answer to la. (above) is "yes," please rate
the degree of comparability (H=High;
I=Intermediate; L=Low).
3. If the answer to la. (above) is "no." do you plan
to conduct field verification studies with microbes
in the next three years.
FACTOR
Survival/ ' Environmental Mobility
Colonization (Specify organism or gene)
Yes.
.No_x
Yes
No_x_
Yes No _x Yes No _x_
Depends on funding availability.
4. If field verification studies have been conducted
with microbes, briefly discuss major problems
encountered in making the comparisons.
5. Please discuss any factors other than survival
colonization or microbial gene mobility potential
that have been field verified in your microcosm?
FURTHER INFORMATION ON VERSACORE
Dr. William Holben
Michigan State University
East Lansing, M! 48S24
(517) 355-9282
Jansson, J.K., W.E. Holben. J.M. Tiedje, and BJC Chelm. 1989. The fate of recombinam pseudomonads in modified
soil-core microcosms (Versacores). In JJC Fredrickson and RJ. Seidler (eds.). Evaluation of Terrestrial
Microcosms for Detection, Fate, and Survival Analysis of Genetically Engineered Microorganisms and Their
Recombinam Genetic Material Report (PNL-6828) prepared for U.S. EPA, Environmental Research Laboratory,
Corvallis. by Pacific Northwest Laboratory. Rkhland. WA. Pp. 3-1-3,23.
144
-------
Probe with
Specific Gene
Mini-Core
t
Extract Soil
ONA
t
Sample
Dilute
1
Platoon
Selective
Media
Leochat*
Section 1
Section 2
Section 3
Wire Screen
Flgur*12.
145
------- |