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                                                  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

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                            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.              	

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                               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

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                                          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
                           •*•*•*•••••«•••*•••••*•*•«••••*•«•*••••«*•«»••*••••*••«••••••«*•*•«•••**•**••••••••••••*•••••••*••*••••••••••••••••*•••••** 8 /
     1.   Root Svstem Microcosm _.._        ..        __  ...   ~.  .~...	89
          — ——  J u mm   • 	  • 11 • • •  •»«**•••••*•»••»••»•••••• •••••••••w •••••••» »•«»«•«»«»»«•• •••••••••»•*••••••*•*•*••»•••••>»•••••» v v
     2.   Soil Core Microcosm ..-™..«.......™..........«...^..^._«_..........^	99
     3.   Soil in a Jar Microcosm		109
     4.   Terrestrial Microcosm Chamber	 119
     5.   Terrestrial Microcosm System	^.	129
     6.   Versacore Microcosm -.._..................................~..............	137

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             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:

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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 t»C 
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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.

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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.

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                                     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

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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

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    Appendix B
Aquatic Microcosms

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                            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.

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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 =» 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

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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

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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'i«r-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

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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. §797.3100 Site-specific
        aquatic microcosm test 52(187):36352-36360.
 Perez, K.T., EW. Davey, N.F. Lactic. G.E. Morrison,
     G.G. Murphy, A£. 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;
                                                       « 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 -»-»
    sediment cores in series ->-»  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  |i£ 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-10°C. 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 ii£r2Sr£ 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

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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

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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

-------
                                                                                     *=?
                                                                                          «3
                                                                                                    ^
                     Algal Unit
                                   Hertivore Unit
Sedmant Cores
Rgur* 3.
Comp*rtm«itallz»d Uka microoocm.
                                                         27

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                       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 @ 20°C)

      . (controlled)
      .(pH.Eh)
Microcosms are kept in environmental chamber.
Fans circulate air in the  environmental chamber.
Water is not mixed.
                                                     31

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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

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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

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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

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                                      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

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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

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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

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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
        d£t£?!sici£9 s0f± !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

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                             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

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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

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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

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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 -•»• —
Seawater
Setting »
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

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                         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

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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 fo»y 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 nr» 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

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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,
algae—including blue-greens—and 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

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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

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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

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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

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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

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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

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                                   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

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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

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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

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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

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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

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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

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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 20-25° 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 J»., 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 Une«
                                                                          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 ?«JT'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

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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

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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 »y»i«nv
                                                   97

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                                  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

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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 ?hi« •**•*
                                                             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*
                                                             » » *»W-^* 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

	
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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

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                               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

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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)
                                                            _«-8.9cml.D.-M
                                                                             •15 Rubber Stopper
                                                                                   Wire Spring
                                                                                            21
                                                                                  Wire Loop N®**
 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 ± 1°C.
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*« ,
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 	
v«
                                                      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

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                       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 control—totally determined  by lights  and
temperature of room (room is heated/refrigerated to
±2°Q
    14.  How is water/air circulated/mixed?
Air passage  through  system by  fan  controlled
manually with valve
                                                     131

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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

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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

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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
Parafilm®. 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

-------