United States
 Environmental Protection
 Agency	
 Environmental Research
 Laboratory
 Narragansett Rl 02882
 Research and Development
 EPA-600/S3-83-055  Sept 1983
Project Summary
Experimental  Marine  Microcosm
Test  Protocol  and Support
Document
K. T. Perez
  The purpose of the  experimental
ecosystem (microcosm) test protocol
is to determine the fates and ecological
effects of various "photostable" chem-
icals within a site-specific marine or
estuarine ecosystem. The protocol,
microcosm facility and associated ex-
perimental procedures correct some of
the limitations imposed by single spe-
cies and single variable or process test
systems by simulating most of the
relevant dimensions of the natural sys-
tem in both the water column and the
benthos. The data resulting from com-
parisons of experimental controls with
the field are far more  applicable to
predictive environmental assessments
than those derived from simpler tests.
Furthermore, since the information
generated via microcosm experiments
is integrated at the system level of
organization, quantitative relationships
can be determined for factors such as
chemical exposure concentrations and
transport rates to measured body bur-
dens, ecological effects, input concen-
trations and loadings.
  There are, however, two constraints
upon the use of the system: 1) Because
of the size of the microcosm  tank,
macrofauna cannot be  tested;  thus,
data on some economically important
species cannot be derived. In addition,
the exclusion of macrofauna could af-
fect significant variables in the system,
but whether or not this would be the
case,  particularly  within the experi-
mental time frame of 30 days, has not
been established; 2) The microcosm
test system was developed from a tem-
perate Northeast (U.S.) estuarine eco-
system where intertidal and reef sub-
systems are either small or lacking.
The applicability of this protocol to
other ecosystems where either shallow
conditions or major reef subsystems
exist has yet to be determined.
  A support document, incorporated
as Part (c) of the full report provides a
rationale for the use of the test system.
  This Project Summary was developed
by EPA's Environmental Research Lab-
oratory. Narragansett. Rl, to announce
key findings of the research project
that is fully documented in a separate
report of the same title (see Project
Report ordering information at back).

Introduction
  Chemical companies are required, under
the Toxic Substances Control Act (TSCA).
to submit information about new and
existing chemicals. This information is
then used by the U.S. Environmental Pro-
tection Agency (EPA) to develop an en-
vironmental or risk assessment for each
chemical. In the marine environment the
assessment of risk from a specific chem-
ical includes the effects of that chemical
on the receiving body or ecosystem and
the potential for indirect human exposure
through the consumption of marine or-
ganisms. Such  information  is usually
derived from  simple test systems. For
example, components of the ecosystem
(single species) or processes (e.g., water
solubility or biological  breakdown) are
isolated and either exposed to or measured
in the presence of the chemical of interest
These data are then used to develop the
environmental assessment However, such
assessments have been subject to ques-

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tion on three major grounds. First the data
base is usually confined to the measure-
ment of only a few variables. Second, the
validity of these data is tenuous, since the
physical, chemical, and biological  com-
plexities of the natural ecosystem are not
simulated. Third, the use of these data to
generate a meaningful and accurate pre-
diction of the fate, transport, and eco-
logical effects of the chemical  in a natural
ecosystem is highly questionable.

Test Protocol
  The Microcosm Test Protocol described
in the full report is designed to overcome
many of the concerns associated with
environmental assessments based upon
existing simple (single species and proc-
esses) test  systems. Specifically,  undis-
turbed, natural pelagic, and benthic com-
munities are coupled within a single sys-
tem, the physical and chemical conditions
of which are  equated  to those  in the
natural system being simulated.
  For purposes of the protocol, the natural
system undergoing assessment is defined
in terms  of its physical  and  temporal
boundaries, its  light and temperature
regime,  its water composition, turbulence
and  turnover rate, the  ratio  of benthic
surface  area to  sea  water volume, the
sediment characteristics, and the water
flow rate over the sediment surface.
  The biota of the water column are char-
acterized by the number and species com-
position  of  phytoplankton, zooplankton,
and  transient larval forms found  at the
designated collection site at mid-tide. The
benthic subsystem is characterized by the
sediment  type  and the structural  com-
position of the benthic community.  If the
natural system has more than one distinct
benthic community, then those organisms
directly linked to human consumption or
important to  the economics  and the
aesthetics of the  area  should  be con-
sidered for  experimental  use.  If  some
benthic communities  contain  species
which are known to be more sensitive to
environmental contaminants than others,
these communities should also be con-
sidered.

Microcosm  Facility
  The  microcosm facility (Figure  1)  is
composed of  two  basic parts:  (1) the
support equipment (room, waterbath, light
and turbulence fixtures, test compartments,
and an air evacuation system), and (2) the
experimental microcosm (tanks, paddles,
benthic cylinders,  pumps and pump air
supply).
  The microcosm test system is  placed in
operation as follows: (1) The test water is
collected from the natural system at mid-
tide by hand bucketing or non-destructive
pumping,  (with a diaphragm pump) and
transported  to  the test facility  in  glass
tanks. The test water is then distributed
equally among the microcosm tanks until
the prescribed volume is reached. (3)
Benthic cylinders are used by divers to
collect undisturbed sediment cores from
the prescribed portion of the natural sys-
tem. The bottoms of the cylinders (cores)
are sealed,  prior to  installation  in  the
microcosm  tanks, by placing  them in
slightly larger diameter  crystallization
dishes. The benthic cylinders and crystal-
lization  dishes containing the sediment
cores are then placed in the tanks, and any
disturbed sediment is allowed to settle (for
approximately 30 minutes). (4) The ben-
thic pumps are placed adjacent  and con-
nected  to the benthic cores. Air lines
supplying low pressure air are attached to
create the desired water flow rates over
the sediment surfaces. (5) Paddles are
installed, and the speed of  rotation is
established to generate a water column
turbulence level equivalent to that in the
natural system. (6) Light intensity to the
water in the tanks is controlled  by ad-
justing the shading of fluorescent lights to
appropriate levels, and the photoperiod is
set equal to ambient conditions. (7) Water
flow in the  bath  is adjusted to provide
temperature  tracking  within  1°C of the
natural system. Flow  rates must also be
sufficient to  maintain  all experimental
tanks within  1°C  of each other.  (8) After
the disturbed sediment in the benthic
cylinders settles,  the  low  pressure air to
the benthic pumps is turned on, and water
flow to the benthic cylinders is begun at an
average rate equal to the average tidal flow
over the benthic  substrate in the natural
system. (9)  Any  resuspended sediment
that settles on the bottom of the micro-
cosm tanks at any time during the experi-
ment should be collected with  a tubing
                            Fluorescent
                            Lamps
                                           Exhaust
                                           Fan
                 Charcoal
                 Filter
      Benthic Pump
      Air Supply and
      Exhaust
      Manifold
Benthic
Pump Air _
Line
  Sampling
  Port
                                                                                                               Paddle
                                                                                                               Drive
                                                                                                               Motor
                                                            Benthic
                                                            Pump
                                                            Air
                                                            Controller
                                                                                           Benthic Pump

                                                                                           Paddle Shaft

                                                                                          Microcosm
                                                                                          Tank
     Waterbath
     Trough
Transverse
Plexiglass
Cover
Figure  1.    Experimental microtosm facility.

                                    2

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pump and replaced into the bentnic cylin-
ders.  (10)  Water turnover volume  and
frequency are established to match the
exchange rate of the natural  system.
Water exchange in the microcosm tanks
should be scheduled at least three times a
week and should coincide with the  bio-
logical and chemical sample collections.
(11) If a surface film containing the  test
substance is formed, a portion of that film
must be removed during each water ex-
change to simulate natural transport  The
area of surface film to be removed by
wicking with filter pads or other suitable
absorbent material is defined by:

             Area (cm2) =
 water turnover
volume/turnover
      day
total microcosm
 water volume.
                 X benthic surface area
Entry of Test Substance
  If the mode (i.e., atmospheric and/or
aqueous) and form (i.e., gas,  liquid, or
solid) of entry of the test substance into
the marine environment is known, as with
existing chemicals,  or predictable, as with
"new" chemicals under TSCA, then similar
mechanisms of entry will be simulated in
the experimental microcosms. If the only
mode of entry is the atmosphere, then this
protocol  is not applicable at this  time.
However, if the mode of entry is partially or
completely aqueous, then the  test sub-
stance, in its realistic form, will be added to
achieve nominal water concentrations (i.e,
those based upon projected or measured
field exposures and varying by at least two
orders of magnitude).  If the mode and
form of entry of the test substance into the
marine environment is unknown, then the
mode of entry into the microcosms will be
aqueous. The form of entry  will  depend
upon the solubility of the test substance in
sea water. If the test substance is insoluble
in sea water, then  an appropriate carrier
will have to be used. An appropriate carrier
would completely dissolve the test sub-
stance and result  in a uniform water
column distribution of the test substance
at the start of the experiment.


Ecological and Chemical
Effects Sampling
  Simultaneous measurements of the test
substance and ecological effects are pre-
scribed for a thirty-day period. This sam-
pling  period enables the investigator to
establish either the time for the test sub-
stance to reach quasi-equilibrium or the
degree to which equilibrium is reached
within the various physical artd^bibtic com-
partments of the experimental microcosm.
  Those measurements required to de-
scribe ecological effects (i.e., phytoplank-
ton,  zooplankton, and benthic organism
abundance, and ammonia levels and flux
rates) are dependent on the characteris-
tics  of  both  the contained biotic com-
munities and the test substance. Ideally, a
structural and functional measure should
be made from each trophic  level. It is
recommended that  additional ecological
measures be  made in  those compart-
ments exhibiting significant accumulation
of or exposure to the test substance.
  The test substance is measured within
the microcosms to establish (1) material
budget  (2) total transport, and (3)  bio-
accumulation and potential trophic transfer.
  It  is necessary to develop  a material
budget of the test substance and resultant
breakdown products throughout the course
of the experiment This indicates the ac-
curacy of measured concentrations  and
rates of removal within selected micro-
cosm compartments. The compartments
identified as essential to a material budget
are the overlying gas phase, surface film (if
produced), water column, sediment glass
surfaces and  benthic macrofauna not in-
cluded in the sediment sub-cores. Water
analysis should include determination of
test substance concentrations  in terms of
the total, dissolved,  and paniculate frac-
tions.
  Bioaccumulation of the test substance
can result in subsequent ecological effects
and  may also be a  potential  source for
human exposure via direct consumption
or upward trophic transfer. It is essential to
be able to define quantitatively the rela-
tionship between  the amount of test
material added (i.e., the input function) and
subsequent bioaccumulation  levels,  tro-
phic  transfer,  and ecological effects.
  Analysis of sediment sub-cores is re-
quired to determine  the vertical profile of
the test substance  and, thus, exposure
concentrations for some  of the benthic
organisms.
  The amount of test substance found in
the surface film and  subsequently  lost
through gas transport or decomposition
can,  depending on the characteristics of
the substance, form a significant fraction
of the total budget As a result  the design
and  performance of any  chemical sam-
pling program must  take the contribution
of the surface film into consideration.
  The temporal dynamics and quantities
of the test substance and breakdown
products within selected microcosm com-
partments enable the investigator to de-
termine:  (a) the exposure concentrations
and the relationships to the body burdens
of associated biota, (b) the concentration
of the test substance at which ecological
effects are observed, and  (c)  the total
transport of the test substance and break-
down products. All of these measures can
then be  related to the three different
quantities of the test substance added to
the microcosms (season and the inter-
action  of season  and test substance,  if
included as a variable in the microcosm).

Cleaning
  The combination of benthic biological
activity and water flow through the ben-
thic chamber will result in significant but
realistic quantities of suspended sediment
in the  microcosm tanks. Under natural
conditions,  this  resuspended sediment
settles back  to  the sediment  surface.
However, in the microcosms, the resus-
pended sediment settles to the bottom of
the larger microcosm tanks. All such settled
sediment is collected at the time of water
turnover and returned to the benthic box in
order to compensate for and minimize the
effects of this artifact

Data Analysis
  One of the major features of the recom-
mended experimental design and statis-
tical analysis  (see Section 6.1 of the full
report) is its ability to establish indepen-
dently  the quantitative effect of (1) the
solvent carrier (if used) and, (2) the test
substance for all the variables measured.
  Furthermore, the  degree of divergent
biotic behavior between the control micro-
cosms  and the natural system provides a
measure of the validity of the microcosm
responses to the test substance.

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     The EPA  author  and Project Officer (see  below)  is K.  T. Perez with the
      Environmental Research Laboratory, Narragansett, Rl 02882.
     The complete report, entitled "Experimental Marine Microcosm Test Protocol and
      Support Document: Measurement of the Ecological Effects, Fate and Transport
      of Chemicals in a Site-Specific Marine Ecosystem," (Order No. PB 83-230 854;
      Cost: $10.00, subject to change) will be available only from:
            National Technical Information Service
            5285 Port Royal Road
            Springfield, VA 22161
            Telephone: 703-487-4650
     The EPA Project Officer can be contacted at:
            Environmental Research Laboratory
            U.S. Environmental Protection Agency
            Narragansett, Rl 02882
                                                ft US GOVERNMENT PRINTING OFFICE- 1983-659-*17/7194
United States
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Information
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