ENCLOSED SYSTEMS FOR
TESTING MICROBIAL PEST
CONTROL AGENTS
Dr. John A. Couch, Coordinator
U.S. Environmental Protection Agency
Environmental Research Laboratory
Gulf Breeze, FL 32561
Prepared For
U.S. Environmental Protection Agency
Office of Pesticide Programs and
Office of Research and Development
This document is a preliminary draft. It has not
been formally released by the U.S. Environmental
Protection Agency and should not at this stage be
construed to represent Agency policy. It is being
circulated for comments on its technical merit and
policy implications.
ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
GULF BREEZE, FLORIDA 32561
-------
LIBRARY COPY
TABLE OF CONTENTS
I. Organization of Workshop ii
II. Introduction 1
III. Descriptions and Documentation of Suggested Enclosed Systems 4
1. Estuanne Systems 7
2. Freshwater Systems 67
3. Terrestrial Systems 134
IV. Recommendations of the Pathology-Microbiology Working Group 153
V. Attendees and Participants, Their Addresses and Areas
of Expertise 157
i WOPERfTYOP
ENVIRONMENTAL PROTECTION AGENCY
-------
WORKING GROUPS
TERRESTRIAL SYSTEMS
Chairman - Ray Seidler
Lowell Etzel
Joe Gorsuch
Pete Van Voris
ORGANIZATION OF WORKSHOP
(WORKING GROUPS AND EFFORTS)
COORDINATOR - JOHN COUCH
Environmental Research Laboratory
Sabine Island, Gulf Breeze, FL 32561
Office of Pesticide Programs - ZIG VAITUZIS
Office of Research and Development - MORRIS LEVINE
FRESHWATER
Chairman - Dick Anderson
Gary L. Phipps
Lyle Shannon
Paul Franco
ESTUARINE
Co-Chairman - Tom Duke
Co-Chairman - Ken Perez
Steve Foss
Fred Genthner
Larry Goodman
John Couch
PATHOLOGY-MICROBIOLOGY
Chairman - Clint Kawanishi
Paul Baumann
Drion G. Boucias
Max Summers
Loy Volkman
-------
INTRODUCTION
by
John A. Couch
Background
This report stems from a workshop held at the EPA, Environmental
Research Laboratory, Gulf Breeze, Florida on February 18 and 19, 1986.
The workshop and report were requested by the Hazard Evaluation Division
of the Office of Pesticide Programs.
The report consists of descriptions and documentation of some
enclosed, multispecies systems that may be used for laboratory testing of
both natural and genetically altered microbial pest control agents (MPCA's-
viruses, bacteria, fungi, and protozoa) for possible effects in nontarget
species, and ecosystems.
Laboratory tests systems relative to estuarine, freshwater, and
terrestrial habitats, related species, and physical-chemical factors were
considered by the members of the four working groups (p. ii) and the workshop
as a whole. Each working group wrote a section of this report relevant to
the needs for risks evaluations appropriate to their respective ecosystem
types (Sections III, 1, 2, and 3).
Goals and Attributes of Test Systems
The purpose of this report is to provide specific recommendations which
would permit properly trained biologists to construct an enclosed, laboratory
system to evaluate the effects of microbial pest control agents (MPCA's) at
several levels of biological complexity. These described systems may provide
the opportunity to examine possible effects of MPCA's in nontarget species
in regard to ecosystem function as it may be altered by such phenomena as
gene transfer, persistence of agent, microbial metabolic perturbation, or
1
-------
changes in population and species compositions due to unexpected competition
or replacement. Other, very important endpoints of great concern, centered
below the ecosystem, species or population levels may also be studied in
the suggested systems. These primary effect endpoints, such as infectivity.
pathogenicity, and toxicity are expressed via the modes of action of most
MPCA's and may be studied in individuals of nontarget species selected to
inhabit the enclosed systems. Therefore it may be feasible to study
possible activity of MPCA's at several biological/ecological levels in
these suggested test systems.
The attributes of the test systems desired include:
1. Containabilityl
2. Reproducibility
3. A variety of trophic levels available in one or more of the systems
4. Reasonable cost and size
5. Capacity for testing viruses, bacteria, fungi and protozoa.
Most of these attributes have been considered in the range of systems
suggested in this report for estuarine, freshwater, and terrestrial
conditions.
Qualifications of and Limitations on Systems Described Herein
The enclosed systems described in this report are presented as
suggested systems in which to attempt to develop methods for examining
possible ecosystem and population level effects and impacts of MPCA's.
These systems have not been validated yet in terms of efficacy with either
^•Containability as used in this report means the reasonable containment
necessary to prevent release of the microorganisms under study to the
work place (laboratory) or larger environment.
-------
natural or genetically engineered MPCA's. They are presented here as a
few of many possible systems that may be adapted for testing MPCA's for
possible environmental impacts.
A final caveat is in order for those who may attempt to use these
suggested, described systems for use with MPCA's without due adaptation:
most of these suggested systems were developed to study the effects of
chemical toxicants; therefore the different nature and modes of actions
of MPCA's and chemicals must be taken into consideration if users of
these systems are going to successfully adapt them to MPCA testing and
study (See Section IV for further discussion on this point).
Each of the system sections were authored by those listed on title
pages, and should be considered the results of the authors' effort and
expertise. Further, each section or system description stands on its own
merits in that it represents the authors' expertise and viewpoints.
-------
ESTUARINE SYSTEMS
AUTHORS:
John A. Couch
Thomas W. Duke
Steven S. Foss
Kenneth T. Perez,
-------
PREAMBLE TO
MARINE AND ESTUARINE SYSTEMS
Three types of marine microcosms were identified to establish the
potential environmental risks of genetically engineered and indigenous
microorganisms. Specifically, simple, gnotobiotic and simulative systems
(see table) vary from simple, two or more species systems to complex,
interacting natural assemblages, respectively. The general underlying
assumption was that the greater the physical and biological complexity
of the experimental system, the greater the likelihood of observing
ecological effects and ultimate fate of the test microorganism. In
addition, the ability to relate ecosystem effects to natural marine
environments was assumed to be directly related to the complexity of
environmental simulation- »The converse is probably valid for determining
specific effects in individual, nontarget organisms, i.e., infectivity,
pathogenicity; and toxicity endpoints are more optimally dealt with in
simpler, single species tests.
In this section, a simple and two simulative systems are described;
to the best of our knowledge, appropriate gnotobiotic systems in the marine
area have not been developed. The simple system does not attempt to
simulate any marine environment, but rather contains selected, trophically
important species to which the test microorganism can be exposed through
the surrounding seawater medium. The two simulative systems, the seagrass
microcosm and the coastal microcosm, relate to shallow intertidal and
deeper water marine environments, respectively.
The level of containment of the test microorganism will be dependent
upon whether the microbe is natural, i.e., derived originally in the
-------
natural environment or genetically engineered, i.e., an organism altered or
manipulated anthropogenically and, as a result, not found in natural
environments. If the former, then the containment procedures will not be
as stringent as those for the latter. Each system has either explicitly
or implicitly addressed the degree of containment relative to the type of
microorganism tested.
-------
General Characteristics and Properties of "Microcosms"
or Multispecies Test Systems Currently in Use
Microcosm"
Type
Components
Configuration of
Components
External
Exchange
Conditions
imple
>_ 2 species; water, food,
or air only as exposure
medium; artificial
medium (e.g. filtered
or nutrient enriched
water).
Direct or indirect
species interaction
via medium (e.g., air or
water, etc.). Simulation
conditions investigator
dependent (e.g., water
turbulence, air flow rates,
etc.).
Usually static;
completely
contained
inotobiotic _>_ 2 trophically re-
lated species chosen
by investigator;
sometimes water and
soils/sediments
artificial media {e.g.
nutrient enriched*water
screened and/or steri-
lized soils, etc.).
All species and media
capable of direct and
indirect interactions;
simulation conditions
investigator dependent.
Static or man-
ipulated flow-
through (e.g.
nutrient addi-
tions, artificial
predation, etc.).
Simulative
» 2 species; natural
intact biotic assem-
blages taken from field;
natural and unmanipu-
lated media.
All species and media
capable of direct and
indirect interactions,
simulation conditions
dependent upon natural
system being simulated.
Unmanipulated
flow-through or
static based upon
turnover time of
natural system
being simulated
as situation
requi res.
-------
A Simple, Enclosed, Multispecies
System for the Evaluation of Infectivity,
Toxicity, and Pathogenesis of MPCAs in
Nontarget Aquatic Species
Office of Pesticides
Programs
ORD
USEPA
by
SteveVs. Foss, and John A. Couch
USEPA
Environmental Research Laboratory
Gulf Breeze, Florida
-------
1. SCOPE.
1.1 The application of pathogenic microorganisms for the control of
pests has brought worldwide attention to the importance of these
agents as microbial pesticides in their natural insect hosts.
With continued efforts of geneticists, microbiologists, and insect
pathologists, improved and more effective microbial pest control
agents (MPCA's) will be available to potential producers of commercial
MPCAs in the future. (Margalit et al., 1985). The possible intensive
application of natural MPCAs and the introduction of genetically
altered organisms into the environment presents a question of
safety with regard to nontarget species. Because of the present
use and the probable increase in registration, production and
application of these agents in the future, the USEPA was given the
responsibility for providing potential producers of MPCAs with
«»
guidelines for tests to determine the safety of MPCAs in nontarget
species. (Couch et al., 1985).
1.2 To provide these methods, we are concerned with the design, develop-
ment, and laboratory testing of a simple, enclosed aquatic system
that can maintain nontarget species from at least three different
phyletic groups for a minimum of 30 days for agent safety tests.
We consider this a system to initiate preliminary studies on the
potential impact of MPCAs on nontarget species. Our system does
not attempt to simulate natural environments but rather to provide
multispecies testing with limited interaction capability for
evaluating endpoints of infectivity, pathogenicity and/or toxicity.
1.3 Other systems presented and discussed at this workshop will provide
a larger opportunity for closer simulation of natural environments
-------
and for studies of more complex interactions among microbial
agents and nontarget species.
1.4 Preliminary studies indicate that three to four different non-
target species can be successfully maintained in the same system
for a 30 day period. The design of this test system enables us to
expose simultaneously a greater number of animals to MPCAs, and to
provide some species interaction, thus expediting evaluation of
the risks of these agents in nontarget aquatic hosts.
2. SELECTION OF NONTARGET SPECIES
2.1 Species from three representative estuarine phyletic groups were
selected for testing:
1.) a fish, Cyprinodon variegatus; 2.) a shrimp, Palemonetes pugio,
and 3.) two molluscs, Rangia cuneata and Crassostrea virginica.
These animals were1 thosen because of their availability, laboratory
adaptability, widespread estuarine and geographical distribution,
and ecological role in the aquatic environment. (Couch, 1983).
All 4 of these species can be readily found and collected in most
typical estuaries along the entire Atlantic and Gulf coasts of the
United States. Inland laboratories wishing to use these estuarine
species as test animals, but unable to make collections themselves,
can often obtain them from commercial sources.
2.1.1 The fish, Cyprinodon variegatus. inhabits shallow brackish areas
of pools (small, sandy-bottomed sites in bays, coves, bayous,
creeks, tidal cuts or channels) that are left isolated during low
tides. They are tolerant of a wide range of salinities (0-142ppt)
and temperatures (2-39°C) (Courtney & Couch, 1984). Collection
using either seine net or bait trap can be easily made during
10
-------
warmer months throughout the range, and anytime during the year in
warmer latitudes. The grass shrimp, Palemonetes pugio, can be
found in beds of submerged vegetation in shallow estuarine waters
(Williams, 1984). They are normally most abundant in a salinity
range of 10-20ppt, however this grass shrimp can be successfully
maintained in lower or higher salinity range under laboratory
conditions. During the warmer months they can easily be collected
using a push net or fine seine net. The most favorable habitat
for the clam, Rangia cuneata, is a combination of low salinity,
high turbidity, and a substrate of sand, mud and vegetation. The
common rangia is usually found in areas where the salinity seldom
exceeds 18ppt and can be successfully maintained at lower or
higher salinities. (LaSalle et al., 1985). This clam can be
collected by hand or with tongs in shallow waters. The oyster,
* '
Crassostrea virginica, usually can be found growing on a variety
of substrates in shallow water, halfway between high and low tide
levels. The range of salinities favorable for £. virginica falls
within two zones, the polyhaline from 18-30ppt and the mesohaline,
from 5-18ppt. This oyster is tolerant of low salinities, but its
survival is dependent on the range of fluctuations and the suddenness
of the changes (Galtsoff, 1964). Oysters can be collected anytime
of the year by hand or by the use of tongs.
2.1.2 Animals we tested were collected from the estuarine system located
in the Pensacola/Gulf Breeze, Florida area. Adult C_. variegatus
and _P_. pugio were collected from Range Point, Santa Rosa Sound, by
seine and push net; R_. cuneata (2.5-4.5cm in size) were collected
by hand in Escambia Bay from the shallow water near the mouth of
11
-------
the Escambia River. Substrate consisted of a sandy-clay bottom
intermittently covered with Thalassia beds. The clams were found
partially or completely buried in the sediment. Small C_. vjrqinica_
(4-6cm in size) were collected from seawater troughs at the EPA
Laboratory on Sabine Island.
3. TEST SYSTEM AND METHODS
3.1 Two 76 liter glass aquaria were filled to a 3 cm depth with clean
crushed coral shell over a recirculating filtration system.
Seawater from Santa Rosa Sound (25 ppt salinity) was added to
these tanks and diluted to 15ppt with deionized water. Two shrimp
were placed in each of 24 holding cups. Each cup consisted of a
fine mesh screen cylinder cemented to a glass Petri dish bottom.
Twelve shrimp holding cups were placed in a suspended cup tray in
each tank with 20 "fish, 16 clams, and 10 oysters, (See fig. 1 )
and were allowed to acclimate for 1 week. Animals that died
before the maintenance observation period (30 days) began were
replaced.
3.2 Two types of food, a commercial flake or live artemia, were used
for feeding. In one tank the shrimp were fed one pressed flake-food
pellet (approximately 0.056g/pellet) per holding cup. Approximately
0.5 g of unprocessed flake food was added to the open tank space
for the fish. Shrimp in the second tank received approximately 10
ml of a suspension of live artemia per holding cup and 300 ml of
live artemia suspension was added to the fish tank. It was noted that
a greater amount of algae grew in the flake food tank, compared to
the live Artemia tank. Animals were fed 3 times a week for 30
days. Oysters and clams probably filter-feed upon bacteria, some
12
-------
algae and/or detritus available in the animals environment.
3.3 This test system can be adapted in several ways. To accommodate
and maintain a greater or lesser number of animals, the size and
shape of the test chamber can be varied. Any desired salinity can
be maintained by either diluting existing seawater or by mixing
commercially available sea salts with fresh water. Temperatures
can be easily controlled by using common aquarium heaters or coolers.
4. PRELIMINARY EFFICACY OF SYSTEM AS MAINTAINED FOR 30 DAYS
4.1 The recirculating, static multispecies system maintained for 30
days proved to be very successful. Apparently, either method of
feeding (flake vs. Artemia) can be used when applicable. The fish
and clams had 100% survival with both feeding methods. A 99%
survival rate was noted for the oysters in both feeding systems.
Eighty percent(80%«)'of the shrimp that were fed live artemia and
84% of the shrimp fed flake food pellets survived. The higher
mortality for the shrimp, compared to the other species, is within
acceptable limits for this system. (Couch et al. 1983). This
mortality can be explained by the shrimps' direct contact with one
another in the holding cups, and their cannibalistic and antagon-
istic tendencies. A sampling of animals for each species group
were examined grossly and appeared to be healthy.
Interaction among species in the system included direct ingestion
of oyster fecal strings by fish, filtering of fecal materials from
fish by oysters and clams, and exposure to fecal material from grass
shrimp for all other species. This provides desirable interaction
among test species because MPCA material may thus be exposed to
potential natural degrading or enhancing factors.
13
-------
5. RECOMMENDATIONS FOR USE OF SYSTEM WITH MPCA's
5.1 Two methods of exposing animals to MPCAs using this system are
possible. Dosing thru feeding appears to be an efficient, natural,
and certain method for ensuring internal exposure to certain MPCAs
such as viruses, bacteria, and protozoans. (Couch, 1983). Fungi,
such as Lagenidium giganteum, which attach externally to the
cuticle of their-hosts, could be introduced directly as zoospores
into the water in test system in known concentrations.
5.2 Tissues from the exposed test animals can be examined, using
specific histopathological, ultrastructual, serological, and
genetic methods, to evaluate possible effects associated with
infectivity, pathogencity, and toxicity.
14
-------
Palemonetes pjjgig
(Grass Shrimp)
Cypjjnodon
vaneggtus
(Sheepshead Minnow)
3,
Underground
Filter
System
fri6
Crassostrea yirginica
(Oyster)
-------
TABLE I COMPONENTS OF A SIMPLE MULTISPECIES (3-4 SPECIES) LABORATORY TEST SYSTEM FOR MPCAs
(COMPARE COMPONENTS IN THIS TABLE TO ITEMS IN FIGURE 1)
COMPONENTS
TEST TANK
AQUARIUM
FI6*
REF.
1
DESCRIPTION
ANY SIZE GLASS AQUARIUM
DEPENDING ON DESIRED SYSTEM
SOURCE
COMMERCIALLY AVAILABLE FROM AQUARIUM
SUPPLY OR, CAN BE CONSTRUCTED
SEAWATER
CRUSHED CORAL
GRAVEL
RECIRCULATING UNDER-
GRAVEL FILTER SYSTEM
0 - 33% SALINITY
COARSE GRAVEL SUBSTRATE COVERING
THE UNDERGRAVEL FILTER SYSTEM
TRAPS DIRT AND DEBRIS IN SUBSTRATE
ALSO CREATES A WATER-AIR MIXTURE
COMMERCIALLY AVAILABLE SEA SALTS OR
DILUTION OF SEAWATER
COMMERCIALLY AVAILABLE FROM AQUARIUM
SUPPLY
COMMERCIALLY AVAILABLE FROM AQUARIUM
SUPPLY
AIR SUPPLY
GLASS TRAY
SHRIMP HOLDING
CUPS
NONTARGET TEST
ANIMALS
FOOD
MPCAs TO BE
TESTED
CENTRAL AIR COMPRESSOR OR PORTABLE
ELECTRIC AIR PUMPS
A REMOVABLE GLASS TRAY WITH SHORT
SIDES SUSPENDED IN THE TEST AQUARIUM
FINE MESH SCREEN CYLINDER CEMENTED TO
GLASS PETRI DISH BOTTOMS
FISH, SHRIMP, CLAMS AND OYSTERS.
OTHERS MAY BE SUITABLE.
FISH FLAKE FOOD
BRINE SHRIMP (ARTEMIA)
(MICROORGANISMS) VIRUSES, BACTERIA,
PROTOZOA AND FUNGI
COMMERCIALLY AVAILABLE FROM AQUARIUM
SUPPLY
CONSTRUCTED USING COMMERCIALLY
AVAILABLE MATERIALS
CONSTRUCTED USING COMMERCIALLY
AVAILABLE MATERIALS
COLLECTION FROM ESTUARIES OR OBTAINED
FROM COMMERCIAL SOURCES
COMMERCIALLY AVAILABLE FROM AQUARIUM
SUPPLY
*SEE FIGURE OF TESTSYSTEM FOR PICTORIAL DESCRIPTION OF COMPONENT
-------
References
Couch, J.A., S.S. Foss and L. Courtney, 1985. Progress Report on
Evaluation for Risks of an Insect Virus, Bacterium, and Protozoon to a
Nontarget, Estuarine Crustacean. Office of Pesticides Program, ORD,
USEPA, 2 p.
Couch, J.A., S.M. Martin, G. Tompkins, and J. Kinney. 1983. A Single
system for the Preliminary Evaluation of Infectivity and Pathogenesis of
Insect Virus in a Nontarget Estuarine Shrimp. Journal of Invert.
Pathology 43, 351-357.
Courtney, L.A. and J. Couch, 1984. Usefullness of Cyprinodon variegatus
and Fundulus grandis in Carcinogenicity Testing: Advantages and Special
Problems. Natl. Cancer. Inst. Monogram 65:83-96.
Galtsoff, P.S. 1964. The American Oyster. Fishery Bulletin, Vol 64.
United States Dept. of the Interior, Fish and Wildlife Service, Bureau of
Commercial Fisheries, pp 4-5, 405-406.
LaSalle, M.W. and A.A. de la Cruz. 1985 Species Profiles: Life
Histories and Environmental Requirements of Coastal Fishes and Invertebrates
(Gulf of Mexico) Common Rangia. U.S. Fish Wild!. Serv. Biol. Rep 82
(11.31). U.S. Army Corps of Engineers, TR EL-82-4. 16 pp.
Margalit, J. and D. Dean. 1985. The Story of Bacillus thuringiensis
var. israelensis. J. Am. Mosq. Control Assoc. Vol 1, No 1 p5
Williams, A.B. 1984. Shrimps, Lobsters, and Crabs of the Atlantic
Coast of the Eastern United States, Maine to Florida. Smithsonian
Institution Press, pp 76-78.
16
-------
A Microcosm System for Evaluating System-Level Infectivity and
Effects Of Microbial Pest Control Agents on Near-Shore,
Nnn-Tarnot f*r» minimi t.i P^
Non-Target Communities
For Office of Pesticides
By
Thomas W. Duke
U.S. Environmental Protection Agency
Office of Research and Development
Environmental Research Laboratory
Gulf Breeze, FL
17
-------
INTRODUCTION
Several commercial chemical companies are developing microbial pest
control agents for application to agricultural crops to control specific
pests. Under the Federal Insecticide, Fungicide and Rodenticide Act
(FIFRA), they are required to submit information on the potential impact
of these, agents on the environment. Since these agents cannot normally
be "field-tested" to determine their impact on non-target organisms,
laboratory tests can be extremely useful in evaluating potential impact.
The near-shore marine area could receive these agents by direct
application for control of noxious insects or by transport of the agents
from adjoining freshwater and terrestrial environments. A method is
needed to test sensitive near-shore organisms in a holistic manner without
an actual release in the environment. The procedure described herein
captures some of the interactive complexities that characterize the
structure and functions of the near-shore environment.
This test procedure orginally was developed to evaluate the effects
of complex mixtures of chemicals on a rooted aquatic plant (seagrass)
community (Morton, et al., 1986). Microbial pest control agents have
not yet been tested in the system. Also, larger predators were excluded
from the microcosms and this could result in some difficulties when
extrapolating results to field situations. Nevertheless, the proposed
microcosm test systems appears to be a reasonable tool for determining
the potential impact of these agents on the near-shore environment.
18
-------
NEAR-SHORE TEST PROTOCOL
1. SCOPE and PURPOSE
1.1 This test is designed to develop data on the impact of microbial pest
control agents (MPCA) on the near-shore environment using a seagrass
community as a sensitive test system. Effects of the MPCA on the
microcosms can be determined through measurements of productivity;
numbers, species richness and biomass of macroinvertebrates; and
degradation rates.
2. MICROCOSM and SUPPORT FACILITIES
2.1 The microcosm is designed to simulate desirable physical, chemical
and biological conditions existing in the natural environment where
seagrass samples are taken. The support facilities (room, light
fixtures, air system) ensure proper environment for testing.
The experimental microcosms provide a community testing chamber that
reflects conditions in the environment as much as possible.
2.2 The microcosms are plexiglass cylinders, 16.0 cm inside diameter,
50 cm high that are attached to a plate on the bottom. Cores
collected from the seagrass beds are slipped intact from the core
cylinder into the test cylinder.
2.3 Samples of the seagrass community are taken by divers with plexiglass
cylinders 14.0 cm inside diameter and 50 cm high. The cores
penetrate 10 cm deep in the grass beds and upon withdrawal, contain
sediment with attendent macroinvertebrates and grass.
2.4 In the laboratory, microcosms can be operated with continuous flow
of seawater or by replenishing the water in the microcosms daily.
19
-------
(a) Flowing Water System
Unfiltered seawater of known quality is pumped into a settling
reservoir above the test apparatus (Figure 1 (modified from
Morton et al., 1986)). A 1-mm Nitex® filter is fitted over
the delivery standpipe in the settling reservoir to exclude
larger material from reaching and clogging the delivery tubes
to the microcosms. Seawater is flowed from the settling
reservoir to a baffled 20-liter Plexiglas® primary head-box
suspended over the microcosms. Twelve glass standpipes, six
at each end of the head-box, are fitted with silicone stoppers
and calibrated to deliver approximately 200 mL/min. A larger
diameter glass overflow standpipe at each end of the primary
head-box maintains the water at a constant level. From the
twelve standpipes, the water enters glass mixing tubes that
« *
deliver the seawater to twelve secondary headboxes. From
each secondary head-box, seawater is delivered separately to
four microcosms at a rate of about 40 mL/min. A glass cover
is placed over the open end of the microcosm cylinder.
(b) Static (Renewal) System
In this system, 8 liters of seawater are placed in the micro-
cosms. The water is drained (syphoned) from each microcosm
every 24 hours and replaced by fresh seawater of known salinity,
pH, temperature and dissolved oxygen.
2.5 Decomposition
Leaf litter or detritus bags can be added to each microcosm
in order to determine impact of the microbiological pest
control agents on decomposers. (Weight loss of bags in
20
-------
UNFILTERED OLj
SEAWATER
SETTLING
RESERVOIR
PRIMARY HEAD BOX
INJECTION OF
MirRQBIAL PEST CONTROL AGENT
MIXING
TUBE
MIXING
RESERVOIRS
STIR
PLATES
SECONDARY
HEAD BOX
PERISTALTIC
PUMP
FIG. 1
-------
treated microcosm is compared with that in control - weight
loss is equated to degradation). Bags are constructed of 2 mm
mesh Nytex nylon netting and 10 cm square. Glass rods anchor
the bags in the microcosms just above the sediment.
2.6 Lighting
Lighting for the test is provided by four 400-WATT multivapor
lamps and four high-intensity fluorescent bulbs rated at 250 watts,
Suspended 46.0 cm above the microcosms, the bulbs provided an
average light energy of 217 yE/m^/S. Lights are connected to
a 24 hour timer (12h light and 12h dark).
2.7 A summary of components of the microcosms is found in Table 1.
3. DECONTAMINATION
3.1 Tests must be conducted to determine the methods and materials
required to decontaminate microcosm glass tubing, or other
materials that may contain the test organisms. Appropriate
disposal procedures must be employed if decontaminating agent
is classified as a hazardous material. Care must be taken to
collect and decontaminate flow-through or renewal water.
4. Definitive Test
4.1 The flow chart for conducting the test is found in Table 2.
(Modification of Morton et al., 1986). There should be at least
12 replicates of exposed microcosm in each test. The test is
conducted for six weeks with flowing seawater and for two weeks
with replenished water.
4.2 Harvest and Treatment of Samples.
4.2.1 The 48-microcosm test system is harvested randomly at the end of
21
-------
the test period. Fifteen seagrass leaves with associated epiphytes
are taken from each treatment for analysis of biomass and chlorophyll
content. Remaining contents are sieved to separate plant and
sediment portions and placed into 0.5 mm nylon bags. All portions
should be immersed in propylene phenoxetol in seawater to relax
organisms. Plant materials are fixed in five percent formalin
until the animaU are picked from them and grass weights determined.
Sediment portions are placed in ten percent formalin in seawater
with 100 mg/liter rose bengal stain. Sediments are rinsed with
freshwater, then sieved through 1.0 and 0.5-mm sieves. Sieved
material is stored in 60 percent 2-propanol until the macroinvertebrates
can be sorted and identified. All macrofauna are identified to
the lowest taxonomic level possible.
4.2.2 Litter bags can be removed at intervals for measurements of rates
.'
of decomposition or can be removed at the end of the test. Bags
are removed from the microcosms, rinsed in freshwater, dried at
100° C for 24 h, weighed, and the weight change (loss) calculated.
4.2.3 Measurements of the Thalassia leaf samples includes chlorophyll a_
per g tissue or per cm2 blade area. Effects on epiphytic communities
are observed by measuring differences in epiphytic biomass (ash
free dry weight-AFDW) per cm2 of Thalassia leaf as a biomass
indicator, chlorophyll a_ per g AFDW epiphyte tissue as a community
health indicator, and chlorophyll a_ per cm2 leaf area as an
indicator of photosynthetic potential. Details of these analyses
to determine the health and productivity of the epiphytes can be
found in Price, et al. (19).
4.2.4 A summary of criteria for effects is presented in Table 3.
22
-------
Table 1.
Descriptive Table - Seagrass Microcosm
Component
Seagrass
Macrobenthos
ro
CO
Description
Seagrass beds cored to a depth
of approximately 10.0 cm by
Plexiglas cylinder (14.0 cm in-
side diameter, 50.0 cm height)
grass, sediment and attendant
macroinvertebrates are sampled.
Taken as part of seagrass core.
Larger macroinvertebrates eli-
minated from microcosms.
Leaf Litter Bags of 2 mm nylon netting,
Detritus Bags 10 cm square. Either dried
or fresh seagrass are added to
bags that are anchored in the
microcosm by glass rods.
Source
Within euphotic zone of
near-shore environment,
particularly temperate
sub-tropic, and tropical
waters.
Taken in same location
as grasses.
Leaves for bags taken from
same grass beds as sea-
grass cores.
Species
Thalassia testudinum used in
this protocol, however, other
grasses such as Zostera marina
may be employed.
The kinds and numbers of species
vary with season and water
quality at time of sampling.
Examples of macroinvertebrates
collected in the fall in Santa
Rosa Sound, FL, are as follows:
Annelida
Aricidae philbinae
Armandia macu!ata~
Fabriciola spp.
Mediomastus californiensi s
Nereis pelagica
Pista sp. A
Prionospio heterobranchia
Tharyx marioni
ArthropoHa
Elasmopus laevi s
Mollusca
Bittium varium
The leaves in the bags should be
the same as those used in the
microcosms.
-------
Table 1. (Cont'd)
Component Description
Source
Species
Microcosms
Lights
Consist of Plexiglas cylinders
slightly larger than coring
apparatus (16 cm inside dia-
meter; 50 cm high). Seagrass
cores slipped inside microcosm
so each one contained intact
core of seagrass and attendent
sediment, macroinvertebrates,
and epiphytes. Experimental
apparatus shown in Figure 1.
A combination of 4 400-watt
multi vapor lamps (Model Mur
400 U) and 8 215-W cool-white
fluorescent lamps (Model E96
T12/CW/1511). Lights are
connected to 24 hour timer
(12h light and 12h dark). An
average of 217 jjE/cm^/S were
delivered to the surface of
the microcosms.
Plexiglas tubes can be pur-
chased at most local plastic
pipe supply companies or
plumbing supply companies.
Electrical Supply Co.
-------
Table 2.
FLOW CHART
Collect Cores From Field
Establish Microcosm in Laboratory
Begin Daily
Measurements
of pH, Tem-
perature,
Salinity
Acclimate to Laboratory Conditions
Add Microbial Test Agents
Expose for Six Weeks
(Daily Water Renewal)
or
Expose for Two Weeks
(Static Test)
Harvest Microcosms
Analyze
Macroinvertebrates:
Number
Kinds
Species Diversity
Patterns of recognition
Rate of Decomposition (Weight Loss of
Litter Bags)
Chlorophyll Content and Growth of Seagrass
Kinds and Density of selected Microbes
25
-------
Table 3.
Criteria for Effects - Seagrass Microcosm
Component Criteria
Seagrass Chlorophyll a_ per gram
blade area
Growth rate
Macroinvertebrates Number of individuals
per species
Species richness
Total number of
individuals
Species diversity
Cluster or discrimi-
nate "(analysis or
other methods of
pattern recognition
Microbes
Enumeration of desired
functional groups
(Nitrification, Sulfur,
etc.)
Method
Acetone extraction
Spectrophotometer
analysis
Movement of alumi-
num tape during
experiment
Microcosms are
harvested at the
end of desired
time. Macroinver-
tebrates are seived,
(1.0 and 0.5 mm),
identified and
enumerated. 10
numerically dominant
species can be
analyzed.
Plate counts
Reference
Strickland and
Parsons (1972)
Modification of
Zieman (1974)
Morton et al.
(1986)
26
-------
References
Morton, D.R., Duke, T.W. Macauley, J.M., Clark, J.R., Price W.A.,
Hendricks, S.J., Owsley-Montgomery, S.L., and Plaia, G.R. In Press.
"Impact of Drilling Fluids on Seagrasses: An Experimental Community
Approach." Proceedings of a Symposium entitled "Community Toxicity
Testing." 1986. 25 pp.
Price, W.A., Macauley, J.M., and Clark, J.R. In Press
"Effects of Drilling Fluids on Thalassia testudinum and Its Epiphytic
Algae." 1986, 21 pp.
Strickland, J.D.H. and Parsons T.R. (1972). A Practical Handbook of
Seawater Analysis. Bulletin 167 (2nd ed). Fish. Res. Bd. Can. 310 p.
Zieman, J.C. (1974). Methods for the study of the growth and
production of turtle grass, Thalassia testudinum Konig. Aquaculture
4, 139-143.
27
-------
EXPERIMENTAL MARINE MICROCOSM TEST PROTOCOL
AND
SUPPORT DOCUMENT
Measurement of the Ecological Effects, Fate
and Transport of Living Microorganisms in
in a Site-Specific Marine Ecosystem
by
Kenneth Perez
Ecosystems Analysis Branch
Environmental Research Lab
E.P.A.
Narragansett, R.I.
28
-------
TABLE OF CONTENTS
Page
(A) PREFACE 31
(B) TEST PROTOCOL
1. Scope ancLPurpose 34
2. Definition of Natural System 34
3. Test Organism 36
4. Microcosm Facility 37
5. Test Procedures
5.1 Test Purpose 43
5.2 Assumptions 43
5.3 Test of Assumptions 44
5.4 Use of Test Results 44
5.5 Preliminary Testing 44
5.6 -definitive Test 45
5.7 Waste Di sposal 55
6. Data Analysis 55
7. Test Protocol Bibliography 58
8. List of Figures 59
(C) SUPPORT DOCUMENT
Purpose of the Support Document 60
Rationale for the Use of Microcosms 60
Rationale for Microcosm Design 61
Rationale for Test Procedures 62
Test Water Collection and Distribution 62
Test Organism Addition 62
29
-------
Page
Sampling 63
Ecological Effects 63
Ecological Fate 63
Data Analysis 64
Restrictions of Test Protocol 64
Support Document Bibliography 65
30
-------
PREFACE
Commercial chemical companies are required, under the Federal
Insecticide, Fungicide and Rodenticide Act (FIFRA) to submit information
about the agricultural use of indigenous and genetically engineered
organisms. This information is used by the Environmental Protection
Agency to develop an environmental and/or risk assessment for each test
organism prior to the approval of a production permit. This assessment,
in the marine environment, would include effects of the organism on the
receiving body or ecosystem and the potential for indirect human exposures
through the consumption of marine organisms.
The microcosm test protocol described herein possesses many of the
physiochemical and biological complexities of natural coastal marine
systems. This was achieved by coupling natural and undisturbed assemblages
of the water column and benthic communities in a realistic manner. This
test system, therefore, will provide a diversity of microenvironments in
which the fate and effects of the introduced test organism can be measured.
If the test organism survives and proliferates in the microcosm, this
protocol has the potential to estimate the specific impacts at the ecosystem
level of organization. It may also provide estimates of the air and
advective transport of the test organism to adjacent areas. Such information
enables the user to develop a more accurate environmental risk assessment
of the test organism as compared to those assessments founded upon test
systems possesing lower levels of physical and biotic complexities. In
addition, the recommended experimental procedure has some internal checks
designed to establish the validity of the test results. Furthermore, the
knowledge and control of inputs and outputs of variables, perturbations
31
-------
and processes minimize the variance between replicate microcosms. This
dramatically enhances the ability to detect differences between the
exposed and control sets of microcosms utilizing standard statistical
procedures.
ASSUMPTIONS AND CONSTRAINTS
The protocol described below is a modification of one designed to
establish the fate and effects of toxic chemicals (Perez, 1983). Because
of the limited information regarding such test procedures using living
microorganisms, these modifications were made, primarily, on a conceptual
basis. As a result, there are a number of assumptions and constraints
associated with the use of this protocol.
First, we believe that number of replicates per treatment condition
and the error rate found to be adequate for toxic chemicals testing, will
also be adequate for detecting differences between control and exposed
treatments when used for microorganism testing.
Second, we assume that the ecological effects and fate of the test
organism in the laboratory microcosms will be sufficiently similar to
that realized in a field system when exposed to the test organism.
However, until field validation studies are performed, environmental risk
assessments, based on data derived from this protocol, will be constrained
by this assumption.
The last constraint is that natural sunlight is not simulated in
this protocol. If the test organism (1) accumulates in the surface
microlayer (i.e., the interface between the water and the overlying gas
phase) and (2) is sensitive to ultraviolet radiation, this protocol will
potentially overestimate the survivability and proliferation of the test
organism in the marine environment and thus provide a worst case scenario.
32
-------
Finally, some words of caution in the application of this test
protocol are necessary. The data and ensuing assessments derived from
this procedure are constrained in two primary areas. (1) Because of
the size of the microcosm tank, macrofauna such as fishes and large
crustaceans have been excluded. As a result, some economically important
species cannot be tested in these systems. In addition, macrofauna
exclusion could possibly affect the responses of some of the measured
ecological effects. Whether macrofauna significantly influence such
variables (phytoplankton, zooplankton, nutrients, chemical distribution,
etc.) for different ecological systems, particularly within the experi-
mental time frame of 30 days, remains to be established. (2) The micro-
cosm test system was developed from a temperate Northeast (U.S.) estuarine
ecosystem where intertidal and reef (e.g., oyster, coral, etc.) subsytems
are either small in area or missing. The applicability of this protocol
to other site-specific marine ecosystems where either shallow conditions
and/or major reef subsystems exist has yet to be determined.
CONTAINMENT
This protocol is written and the system designed to minimize environ-
mental release and human exposure to any Genetically Engineered Material
(GEM). The level of containment designed, in our view, exceeds, or at
least equals, the National Institutes of Health, Class III biological
safety level (Anon. ,1984). If the test organism is an indigenous micro-
organism, that is, one naturally found in field environments, containment
conditions can be relaxed to the degree that the primary concern will be
to prevent the contamination of the experimental control tanks.
33
-------
TEST PROTOCOL
1. SCOPE AND PURPOSE
1.1 The standards of this subsection are designed to develop data
on the fate and ecological effects of living microorganisms on
marine ecosystems. The United States Environmental Protection
Agency (EPA) will use these data to assess the hazards these
organisms may present to the marine environment and potentially,
human health, through uptake and accumulation in human food species.
2. DEFINITION OF A NATURAL SYSTEM
2.1 The natural marine system (designated as "natural system") at a
particular geographic location consists of a coupled water column
and benthic subsystem. Within both the water column and benthic
subsystems, certain.physical and chemical conditions, and
biological properties exist. These conditions and properties are
simulated in laboratory microcosms as described in Section 4.2
and 5.6.2.
2.1.1 The physical and chemical conditions of the natural system are
defined in terms of its:
(1) Boundaries - The boundaries of the natural system are
delineated by the maximum excursion of water above and below a
selected point during one complete tidal cycle.
(2) Light - The light regime of the natural system is
characterized by the photoperiod, the best estimate of average
monthly incident radiation (total incident radiation per day
divided by photoperiod), the average seasonal water extinction
coefficient, the average daytime vertical light intensity for the
34
-------
entire water column, and the depth at which the average light
intensity occurs.
(3) Sea water - That fluid existing within the boundaries of the
natural system.
(4) Sea water turbulence - The average water motion realized for
the entire water column of the natural system during the period
of experimentation.
(5) Sea water turnover rate - The time required for one complete
water turnover or exchange within the defined boundaries of the
natural system.
(6) Ratio of benthic surface area to sea water volume - The
ratio obtained by dividing the calculated benthic surface area of
the natural system by the best estimate of water volume of the
system.
(7) Sediment - The bottom substrate existing, at the mean water
depth, within the boundaries of the natural system.
(8) Sea water flow rate over sediment surfaces - The average
tidal velocity over the bottom as measured in the natural system.
2.2 Biological Conditions and Descriptions
2.2.1 The biota of the natural system is characterized by the organisms
in the water column and benthic subsystem.
(1) The water column is characterized by the numbers and species
composition of phytoplankton, zooplankton, and transient larval
forms found in the sea water at the designated collection site at
a mid-tide. The water column, with its assemblages of biota, is
obtained from the natural system by the methods described for
filling the microcosms (Section 5.6.2(1)).
35
-------
(2) The benthic subsystem is characterized by the sediment type
and the structural composition of the benthic community, i.e.,
numbers, species composition, and feeding types of organisms
collected from the benthic boxes (Section 4.2.1 (2,3)) and re-
tained on a 0.5 mm screen.
(3) If the natural system has more than one distinct benthic
community, then those having direct routes to human consumption
or which are of significant economic and aesthetic importance
should be considered for experimental use.
3. TEST ORGANISM
3.1 Information Required
3.1.1 A detailed description of the test organism including levels
at which human health may be affected, and any known methods for
identifying and measuring the organism must be supplied. All
known information regarding the decontamination of air, water,
and surfaces contaminated with the test organism should be
included.
3.1.2 The degree of purity and the identification and quantification
of any contaminating organisms, if known, should be determined.
3.1.3 It is extremely important to have a knowledge of, or a best
estimate of the test organism's mode and form of entry into the
marine environment. Specifically, does the test organism enter
by atmospheric or aqueous sources? Is the test organism in
association with other materials such as a dispersing agent?
As will be discussed in Section 5.5.1, the extent of such
information will determine the method by which the test organism
is added to the experimental microcosms.
36
-------
3.2 Methods must be described for collecting and measuring the test
organism in and/or on the air, sea water, surface microlayer,
sediment, biota (both whole animal and specific tissues if
necessary), and microcosm surfaces.
4. MICROCOSM FACILITY
4.1 The microcosm facility (Figure 1) is designed to simulate
relevant physical, chemical, and biological conditions existing
in the natural system described in Section 2. It 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 microcosms (tanks,
paddles, benthic cylinders, pumps and pump air supply). Two
physically separate units (one each for exposed and control
microcosms) must be'used when biological substances are being tested.
4.1.1 Support Equipment (See Figure 1)
(1) All testing must be performed in a room that eliminates any
light source other than that provided by the microcosm facility.
(2) The microcosm tanks must be held in a waterbath of sufficient
dimensions, with sufficient heating/cooling capacities, as to
provide uniform test temperatures within all treatment conditions
and maintain those temperatures within 1 C of the natural system
at the time of the experiment.
(3) A canopy above the waterbath should hold sufficient fluorescent
lighting to provide an even light distribution at the desired light
intensity over all the experimental microcosms. Light is provided
by "Cool White" fluorescent bulbs, and the photoperiod controlled
by a 24 hour timer. A plexiglass cover, the same length and width
37
-------
of the canopy, is mounted directly below the fluorescent lamps.
(4) The open area between the canopy and waterbath is enclosed
and sealed with plexiglass. The front plexiglass covers are re-
movable to facilitate setting up and filling the microcosm tanks.
These covers are equipped with rubber gloves and air locks (Figure
6) to facilitate the safe collection and removal of samples. The
air locks will be designed to allow the application of an appro-
priate decontamination agent (5.5.5) to the exterior of sampling
vials, pipets, etc. prior to removal from the chamber. The air
locks are vented to the air exhaust system (Section 411.1[6]).
Plexiglass panels are also mounted transversly within the waterbath/
canopy module. These panels are joined and sealed to the horizontal
plexiglass sheet mounted below the fluorescent lights and extend
approximately ten cm. below the water surface, to provide airtight
test compartments (Figure 6). The first three compartments will
each hold two microcosm tanks. The fourth compartment will hold
three tanks. All nine tanks within a given waterbath/canopy unit
(Figure 1) will be reserved for one specific treatment condition
(i.e., exposed or control (Section 5.6.1 (1)). The individual test
units (exposed and control) must be physically separated from each
other to reduce the chance of contamination of the control tanks.
Access to the microcosm tanks during a test is via the rubber gloves
and air locks mounted in the front of each test unit.
(5) Turbulence is provided by a system that consists of an electric
motor, motor controller, chain drive, mounts and drive shafts.
The motor should have the capability of maintaining speeds of 1 to
40 RPM. This system is mounted on the canopy with the drive shafts
38
-------
extending through, directly above each microcosm tank (Figure 1).
The paddles are bolted to the drive shafts.
(6) A slightly negative air pressure is maintained within each
test compartment. An exhaust fan draws air from the test compart-
ments through a PVC pipe manifold. The exhaust air is passed
through HEPA particle filters capable of removing the test organism
(Figure 1). The^filtered air is vented through a stack outside of
the laboratory building. The exhast stack should be as far removed
as possible from the laboratory air intake.
4.2 Experimental Microcosms
4.2.1 The experimental microcosms are designed to provide a multi-trophic
level experimental chamber that has coupled pelagic (water column)
and benthic communities similar to those existing in the natural
system.
(1) The microcosm tanks are 140 liter, cylindrical fiberglass
containers (Figure 3). The tanks are held in position by a plywood
"collar" mounted in the water bath trough and encircling each tank
approximately 15 cm below the rim.
(2) The benthic boxes are thick walled PVC cylinders, open at both
ends, and are approximately 30 cm long (Figure 5). The inner
diameter of the benthic cylinder must be such that the microcosms
sediment surface area-to-water volume ratio equals that of the
natural system (Section 2.1.1(6)).
(3) The benthic cylinders are used by divers to collect undisturbed
sediment cores from the prescribed portion of the natural system
2.1.1 (7) and 2.2.1 (3). The depths of the core should be equal
to or greater than the depth of biological habitation or activity
39
-------
but not exceed 25 cm. The bottoms of the cylinders (cores) are
sealed, prior to installation in the microcosm tanks, with PVC
plugs secured by PVC bolts (Figure 5). The cores, at this point,
should be watertight i.e., the water overlying the sediment core
will not flow downward between the sediment periphery and the
cylinder. If shells and/or other debris, biologically derived
tunnels, etc. preclude this condition, then such cores should be
rejected. The length of time between collection of the cores and
initiation of the test should not exceed two weeks. During the
interim period the cores can be held in the laboratory, submerged
in flowing, well aerated sea water.
(4) Waterflow over the sediment core in the microcosm is provided
by an air displacement pump (Figures 3,4,5). The dimensions of the
pump should be sufficient to provide a flow comparable to the
average bottom water flow rate of the natural system. The pump
is positioned in the tank adjacent to the benthic core (Figures 1,
3). The benthic core is mounted in the microcosm tank so that the
cylinder overflow port is approximately 1 cm. above the tank water
level. The cylinder and pump are supported from fiberglass angle
brackets bolted to the inside of the tank (Figures 3,5). Water
from the microcosm tank enters the pump through a small (2 cm)
hole in bottom. When air pressure is applied to the pump, a
silicon rubber check valve inside the pump is forced downward and
seals the bottom hole. This forces water in the pump through a
connecting tube and into the benthic cylinder. Water entering the
cylinder is directed into several horizontal streams by a diffuser
(Figure 5) in order to minimize sediment disturbance and re-
40
-------
suspension. Air pressure to the pumps is controlled by an
electronic timer operating a solenoid valve. The air continues
to force water out of the pump for a preselected period of time.
The pumping time must be less than that required to force all the
water out of the pump. The timer then activates the solenoid
valve which stops the air flow, allowing water to re-enter the
pump from the microcosm tank. Again, after a pre-defined refill
period (at least long enough to allow the pump to completely
refill), the air resumes flowing and the cycle is repeated. Water
overflows from the benthic cylinder back into the microcosm tank
through a small hole located approximately 5 cm. above the sediment
level. The rate of waterflow should approximate the mean tidal
velocity measured at the sediment surface of the natural system
(Section 2.1.1(8))
(5) The light intensity over the sediment surface should approxi-
mate that incident in the bottom meter of the natural system. This
is accomplished by placing a light filter or cover over the top
and sides of the benthic cylinder. See Section 5.5.4 for establish-
ment of water column light intensity.
(6) Turbulence is generated by paddles that are connected to the
drive shafts described in Section 4.1.1(5) and Figures (1,2). The
paddle consists of a vertical shaft with two rows of smaller
diameter shafts fused to it radiating horizontally outward (Figure
2). The length and number of arms are dependent on the microcosm
tank, benthic cylinder and pump configurations, and the level of
turbulence desired. The turbulence level in the microcosms is
controlled by paddle configuration as well as the speed of rotation,
41
-------
Once the paddle configuration is established, the speed of rotation
is adjusted so that the dissolution rate (described as weight
loss/time) of pure gypsum (CaS04) blocks (1.5cm x 2.5 cm x 1.0
cm), distributed throughout a microcosm tank, is statistically
equivalent to the dissolution rates of gypsum blocks measured in
the water column (top to bottom) of the natural system during the
season of the experiment. Dissolution rates should be measured and
water turbulence adjusted prior to each experiment.
(7) Several small fiberglass disks, approximately 1 cm2, are sus-
pended below the water surface. These disks should be constructed
from the same material as the tank. They will be used to provide
estimates of the amount of test organism adhering to the test
tank.
(8) The tank is covered with a plexiglass cover. The cover serves
to prevent the release of the test organism to the laboratory
atmosphere and allows for the monitoring of the atmosphere above
the water surface for aerosolization of the test organism. The
cover is made of 0.25 inch thick plexiglass and is mounted, with
an air tight seal, to the tank (Figure 3). The cover has a 1.5
inch hole in the center over which is mounted a ball bearing
pillow block (flanged unit). A fiberglass rod, approximately 18
inches long, is inserted in the pillow block. The pillow block
unit is seated on a silicon gasket and makes an air tight seal
with the plexiglass cover. The fiberglass rod is attached, with
couplings, to the paddle (lower end) and the drive shaft (upper
end). Another hole is drilled near the edge of the cover and
fitted with an air line coupler for the benthic pump (Figure 7).
42
-------
Air line fittings (Figure 7) are also provided to allow air to be
drawn across the water surface. The rate of air flow must be
sufficient to maintain the concentrations dissolved gases eqivalent
to field levels. Other openings can be made in the cover to
accommodate additional sampling if necessary. Any openings in
this cover must be equiped with plugs or covers which allow them
to be sealed.
(9) All test equipment and sampling devices that come in contact
with the sea water and test organisms must have been decontaminated
by appropriate means (5.5.5), washed with detergent and thoroughly
rinsed with deionized water prior to use.
5. TEST PROCEDURES
5.1 Test Purpose
5.1.1 The purpose of the-experimental ecosystem (microcosm) test is to
determine the fates and ecological effects of introduced micro-
organisms within a site specific marine or estuarine ecosystem
within a 30 to 60 day period.
5.2 Assumptions
5.2.1 The ability to extrapolate the results derived from the experi-
mental microcosms to the natural system is based on the correspon-
dence of the biological events expressed in the experimental
control tanks and those realized in the natural system at the time
of the experiment. For those biological events that are statist-
ically equivalent (a = 0.05), it is assumed that the results
observed in the experimental tanks receiving the test organism
would also be realized in the natural system, if similarly stressed,
5.2.2 It is assumed that the arbitrarily chosen mode and form of entry
43
-------
of the test organisms into the microcosms will simulate the known
or predicted entry into the natural system.
5.3 Test of Assumption 5.2.1
5.3.1 The fate and ecological effects of the test organism can be
ascertained in the experimental microcosms and related to the
expected perturbation behavior in a natural system provided the
levels of all relevant independent variables, either simulated
and/or realized in the microcosms, are equal to those in the
natural system at the time of the test.
5.4 Use of Test Results
5.4.1 The data generated from these microcosms will be used to better
understand the potential fate, transport and ecological effects
of the test organism in marine ecosystems. The assessment should
discuss the potential ecological effects and the possibility of
indirect human exposures associated with the mode and form of
release of the test organism in a specific marine ecosystem.
5.5 Preliminary Testing
5.5.1 Input of Test Organism: Mode, form and exposure levels. The
manufacturers recommended mode (i.e., atmospheric, aqueous) and
form (i.e., liquid, solid, with or without carrier) of application
of the test organism into the natural environment will be simulated
in the experimental microcosms.
5.5.2 Input of Test Organism: Desired exposure concentration. The
nominal exposure concentration of the test organism within the
microcosms should be at the manufacturers recommended application
level.
44
-------
5.5.3 Sampling and measurement. Procedures and techniques must be
obtained or developed for sampling and measuring the test
organism in sea water, air, surface microlayer, sediment,
tissues, and microcosm surfaces prior to the definitive test.
5.5.4 Light Intensity. Preliminary tests should be performed (without
the test organism) to establish the proper light intensity over
the microcosms. -These tests should be done with all the microcosm
equipment and facilities (i.e., waterbath, tank, paddle, benthic
box and pump) in place. The tests should be performed at several
light levels, based upon conditions listed in Section 2.1.1 (2),
with photoperiod equal to ambient conditions, for at least 14
days. Those light levels producing phytoplankton abundances that
are statistically equivalent to the natural system should be used
in the definitive test.
5.5.5 Decontamination: Tests must be performed to determine the methods
and materials required to decontaminate microcosm test compartments,
air locks, and sample containers that may contain the test organism.
Appropriate disposal procedures must be employed if the decontaminat-
ing agent is classified as, or the investigator feels is, a hazardous
material.
5.6 Definitive Test
5.6.1 The definitive test consists basically of (a) the initial setup and
filling, (b) addition of the test organism, (c) daily sampling and
water exchange, and (d) final sample collections.
(1) Experimental design: There should be at least nine replicate
control and nine replicate exposed microcosms established for each
experiment.
45
-------
5.6.2 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, (diaphragm
pump) and transported to the test facility. If the natural
system is vertically stratified in terms of temperature and/or
salinity, the initial filling water should be a mixture (average)
of water collected from various depths. Subsequent collections
for water exchange (Section 5.6.4(4)) and biological sampling
(Section 5.6.6) are performed in the same manner.
(2) During the filling process, the water is distributed equally
among the microcosm tanks until the prescribed volume is reached.
(3) The benthic cylinders containing the sediment cores (Section
4.2.1(2,3)) are carefully placed in the tanks (Section 4.2.1(4))
so as to minimize-sediment disturbance.
(4) The benthic pumps are placed adjacent and connected to the
benthic cores (Figure 3). The plexiglass covers, with paddles,
water sampling and exchange tubes attached (Figure 3), are placed
on the tank. A short flexible tube is used to connect the benthic
pump to the air line fitting on the under side of each cover. The
covers are then bolted or otherwise clamped to the tank rims to
provide an air tight seal. Air lines (Figure 3) supplying low
pressure air to the benthic pumps are attached to the air line
fitting on the top of the covers.
(5) The paddles are connected to the drive shafts. The speed
of rotation is established (see Section 4.2.1(6)) to generate a
turbulence level equivalent to that in the natural system.
(6) Light intensity at the microcosm tank water surface is
46
-------
controlled by adjusting the shading of fluorescent lights with
aluminum foil or similar material to achieve levels as determined
in Section 5.5.4. Photoperiod is set equal to ambient conditions.
(7) Temperature in the water bath is adjusted to the average daily
temperature of the natural system being simulated.
(8) After any 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 begun at an average rate equal to
the average tidal flow over the benthic substrate in the natural
system. (Sect. 4.2.1(4)).
5.6.3 Addition of the test organism
(1) The test organism is added to the water column of the experi-
mental microcosms in the mode, form, and concentration established
in Section 5.5.1 and 5.5.2.
(2) If a carrier other than water is used to disperse the test
organism in the natural system (Section 5.5.1), it should also
be used to apply it in the test system. Analysis of effects will
be based on the integrated effects of both the test organism and
carrier.
(3) If input of the test organism to the natural system is other
than a one dose application (i.e., multiple application, runoff),
the test organism must be added to the microcosm tanks in the
same manner as the initial dose whenever there is a water exchange
(Section 5.6.4(3,4)) but only in quantities sufficient to achieve
the desired test concentrations in the replacement water.
5.6.4 Performance of the test
(1) The experiment is run for 30 days. It is recommended that this
47
-------
test be conducted during the temperature extremes of an annual
cycle. Two tanks (both from the same compartment) from each
treatment condition must be sacrificed and comprehensively sampled
(at days 10, 20, and 30) in order to determine the fate dynamics
of the test organism. If, after thirty days, the test organism in
the water column has not reached steady state, the operation of
the remaining si* tanks (three each of control and exposed) will
be continued for an additional thirty days.
(2) Water turnover volume and frequency is established to match
the exchange rate of the natural system (Section 2.1.1(5)). Water
exchange in the microcosm tanks should be scheduled at least
three times a week and should coincide with the biological and
chemical sample collections. The discreet volume of water
removed during the water exchange equals the calculated amount
minus that volume removed during biological and chemical sampling.
(3) Water removal from the microcosm tanks for water exchange,
biological, and chemical sampling is accomplished by drawing the
desired volume of tank water into the waste water vacuum flask.
Each flask is equiped with a float switch (Figure 6) which will
turn off the vacuum pump should the water volume in the flask
exceed the desired volume. Samples of this water can then be
taken for zooplankton enumeration. The remaining water can be
drained into the waste water tank (Figure 1). The waste water
tank will have had a sufficient volume of the disinfectant added
(5.5.5) to kill the test organisms present in the waste water.
This process is repeated for each experimental tank. The waste
water must be tested to insure that the test organism is not
48
-------
present before being disposed.
(4) On water exchange days, the refill water tank is filled with
newly collected water (5.6.2 [1]). Phytoplankton and zooplankton
samples are collected from the refill water for comparison with
those taken from the control and exposed microcosm tanks. The
refill tank is placed in the refill chamber and the chamber sealed.
The water and vacuum lines are connected via the gloved ports.
Water replacement is accomplished by first drawing the desired
volume of newly bucketed water from the refill water tank into the
refill water vacuum flask. This water is then allowed to drain
into its respective experimental tank. After all the tanks have
been refilled, the refill water tank is disconnected (via the
gloved ports) from the refill line and the chamber and refill tank
disinfected as a precaution in case of accidental or inadvertent
contamination. The refill tank can then be removed, washed and
readied for the next use.
(5) Open Petri dishes containing an appropriate growth medium
can be placed at various locations within the test, replacement
water, and waste water chambers during the test period. Any
indication of the test organism growing in the dishes would be
positive proof of contamination within the chamber. Negative
results (i.e., no growth), however, would not necessarily be proof
of no contamination.
5.6.5 Test Organism Fate Sampling
(1) Samples of the test organism in the air and water column
should be taken on water exchange days prior to the actual
replenishment. Samples from the surface microlayer, sediments,
49
-------
benthic organisms and microcosm surfaces will be taken from those
tanks sacrificed at day 10, 20, and 30 (5.6.4 [1]). All sampling
and other microcosm operations (water exchange, etc.) must be
performed using the gloved ports mounted in the front plexiglass
covers. All samples must be placed into bottles, tightly sealed
and decontaminated in the air lock before removal from the test
chamber. Likewise, all sampling materials (pipettes, forceps, etc.)
must be decontaminated before removal from the test chamber. The
samples must be opened and analyzed at an appropriately equipped
site remote from the test chambers.
(2) Water samples, to determine test organism levels in the water
column, should be taken through the sampling port in the plexiglass
microcosm cover. All such samples must be tightly sealed before
removal from the microcosm test chamber. Water samples should be
taken prior to the water exchange on those days when water exchange
is scheduled. (3) Water samples for test organism analysis must
be collected: (a) While the turbulence (paddle) system is operating.
(b) At a point at least three centimeters from the side of the tank
and 10 centimeters below the air-water interface.
(4) Representative samples of the various zooplankton species
should be collected once a week (see Section 5.6.6 [4]) to analyze
for uptake and accumulation of the test organism . These samples
will be collected with the water exchange flask at a rate to
preclude zooplankton avoidance. The sample is then passed through
the zooplankton collector (Figures 1,7). The water, after passing
through the collector, drains into the waste water tank and is
decontaminated. The zooplankton sample is rinsed with filtered
50
-------
sea water and drained into sample bottles. The bottles are tightly
capped and decontaminated before removal for analysis. As with
all other sampling procedures, access to the zooplankton sampler
is gained through the air lock and gloved ports in the waste water
chamber.
(5) Potential transport of the test organism to the atmosphere by
aerosolization should be tested prior to water exchanges and prior
to sacrificing the tanks (5.6.4 [1]) at days 10, 20, and 30. The
test organism is collected on a membrane filter inserted in the
air vacuum line leaving the plexiglass tank cover (Figure 7). The
duration of sampling must be sufficient to collect a representative
sample while not exceeding the life expectancy of the test organism
on the membrane filter. This will be a conservative estimate of
the aerosolization and subsequent gas phase transport because
there is no microcosm simulation of wave activity. Methods of
analysis to establish the presence of the test organism in aerosols
will be dependent upon the characteristics of the test organism.
(6) All samples for test organism analysis should be processed
in a time frame consistent with the life expectancy of the test
organism in the sample.
5.6.6 Ecological Effects Sampling
(1) During the operation of the definitive test, the plexiglass
covers of both the microcosm tank and the test facility (Figure 1)
are installed and all biological and chemical samples collected
either from the waste water vacuum flask or through the sampling
port in the plexiglass cover.
(2) All samples should be taken from at least 10 cm below the
51
-------
water surface and away from the side of the tank. Sampling must
be performed with the paddle system in operation.
(3) The following ecological information is considered to be
essential:
(a) Identification of all benthic organisms reported in
numbers/m per species.
(b) Zooplankton_(including transient larval forms) abundance in
numbers/liter per life stage per species.
(c) Phytoplankton abundance in numbers/ml per species.
(d) Ammonia levels in micro moles N/liter.
(e) Ammonia flux rates from the sediment in ugat N/m /hr.
(f) Oxygen uptake rates by the sediment in mg 02/1/m /hr.
Additional analyses such as bacteria counts or growth measures
of benthic organism.may be performed depending on conditions in
the natural system being simulated and the analytical resources
available to the investigators.
(4) The type of biological sample, volume of sample, collection
frequency, method of collection and analysis are listed below.
All samples must be placed in appropriate containers and sealed
before being removed from from the microcosm facility.
(a) Two milliliter phytoplankton samples are collected through
the sampling port in the tank cover. The algae are counted and
identified with methods described by Utermohl (1931).
(b) Zooplankton samples are collected twice weekly. Two liters
from the waste water flask are passed through, and the organisms
collected on, a 28 urn mesh nylon screen (Figures 1,7). The
zooplankton is rinsed into a collection bottle and preserved for
52
-------
numerical counting. Numbers and species composition are determined
by examination under a dissection microscope. Wastewater from the
2 liter sample drains into the waste water holding tank for sub-
sequent decontamination and disposal.
(c) Ammonia level in the water column is determined once a week.
Twenty milliliter samples, collected with a syringe through the
sampling ports, are analyzed colorimetrically (Slawyk and Maclsaac
1972).
(d) Ammonia flux rates from the benthic box cores are measured
at the time of tank sacrifice. After the cover is removed water
flow to the benthic cylinders is halted for one to three hours
(length of the incubation period is inversely related to temperature)
The diffuser is removed and a thin plastic cover is floated on the
water surface of the benthic cylinder. Water samples of 20
milliliters are collected at the start and conclusion of the
incubation period. Analysis for ammonia is identical to (c) above.
(e) Samples for oxygen uptake (or release) are taken at the same
time as those for ammonia flux. Fifty milliliter water samples are
collected with a syringe and overflowed (to the tank) into 20 ML BOD
bottles. A modification of the Azide Modification (Standard Methods,
1976) (0.1 ml of KI and MgS04 and 0.2 ml of H2S04) is used to prepare
the sample. The samples are titrated with a microburet. Results
are reported as the flux of oxygen in mg 02/l/m2/hr.
5.6.7 Procedure for Final Microcosm Sampling
(1) The plexiglass tank covers are removed from the appropriate tanks,
after routine sampling has been performed, on days 10, 20, and 30.
Cover removal is accomplished via the gloved ports.
53
-------
(2) Those microcosm tanks that have been selected for sacrifice
and sampling at days 10 and 20 (Section 5.6.4 [1]) and all remaining
tanks following the exposure period must have the following samples
taken, sealed in bottles, decontaminated and removed from the test
chamber.
(a) Triplicate sediment sub-cores (2.5 cm diam. x 10 cm deep) to
determine the vertical distribution of the test organism in the
benthic substrate. These cores include the sediment and associated
biota.
(b) The overlying water from the benthic box is removed with a
syringe. The top one-to-two centimeters of sediment remaining in
the benthic box are removed and sealed in bottles for subsequent
screening.
(c) Estimates of the amount of test organism accumulated on the
microcosm surfaces are made at the time of the tank sacrifice.
Replicate samples of the suspended fiberglass disks (4.2.1 [7])
are sealed in vials and removed for analysis.
(3) After all samples have been collected and removed from the
test chamber, sufficient disinfectant is added to the two tanks
and benthic boxes to kill the test organisms therein. These
decontaminated tanks are recovered and left untouched until the
end of the experimental period.
(4) If a steady state condition of the test organism concentration
in the water column is achieved by day 30 the three remaining
exposed tanks will be sacrificed and sampled as described in Sect.
5.6.7 to determine the fate and mechanism of effect of the test
organism in the affected ecological compartment. Otherwise, the
54
-------
test, with the remaining six tanks, will be extended to 60 days
and sampled as prescribed in Section 5.6.7.
(5) At the conclusion of the experiment, disinfectant is added
to the waterbath, all interior surfaces of all test chambers, air
locks, replacement and waste water chambers. The covers of the
tanks and test chambers can then be removed, water and sediment
discarded after growth detection tests show the absence of test
organism within the system.
5.7 Waste Disposal
5.7.1 Liquid and solid wastes generated through sampling and water
turnover must be collected and held until decontamination treat-
ments have rendered the test organism safe for disposal.
5.7.2 Sufficient volumes of the disinfectant (Section 5.5.5) must be
used to decontaminate the water in the exposed microcosm tanks.
5.7.3 Contaminated solid wastes such as sediment, filters, charcoal,
ion exchange resin, glassware, gloves, etc, must be disinfected
before disposal. If the disinfectant is a toxic material it must
be disposed in an appropriate manner.
6. DATA ANALYSIS
6.1 Ecological Effects Analysis
Analysis of variance procedures (Steel and Torrie 1960) and
regression techniques (Snedecor and Cochran 1980) should be used
to analyze all data. The level of significance is set at the 5%
level (a = 0.05) unless otherwise defined. Documentation of all
analysis used in the study must be provided.
6.1.1 Test Organism Effects: Perturbed Case
The model of repeated measures, analysis of variance or a completely
55
-------
random design will be used to detect differences in the 30 day
(and if necessary, 60 day) responses due to the test organism
relative to the laboratory control set. The specific replicates
for the laboratory control set will depend on the results of
6.1.2.
6.2 Test Organism Fate Analysis
The analysis should consist of two phases. First, determine the
total biomass (density) of the test organism in the various
microcosm compartments (water, surface microlayer, sediment,
aerosol and microcosm surfaces). This includes attempts to document
the presence of the test organism in the control tanks. A standard
regression analysis should be performed on all time series data to
determine if the test organism is increasing or decreasing relative
to the addition rate. Secondly, the investigators should establish
whether the means and associated variances of each compartment are
independent of one another. If dependence is found, then appropriate
transformations (e.g., Log 10) should be applied so that the independ-
ence assumption can be satisfied. Statistical analyses are performed
on the transformed data, if necessary, to test for differences
between control and exposed sets.
6.3 Fate of Test Organism
Determine the concentration of the test organism in the following
microcosm compartments on days 10, 20, and 30.
(a) Surface microlayer
(b) Water column
(c) Sediment core
(d) Selected biota
56
-------
(e) Microcosm surfaces
6.3.1 Fate Analysis
Test for compartmental differences in the concentration of the
test organism from each of the compartments listed in 6.3.
Establish a regression line and test for differences over time.
6.4 Transport of Test Organism
Estimate the total biomass of the test organism exported from the
gas and liquid compartments via water exchange and aerosolization
over the 30 day period. Test for changes in biomass over time.
6.4.1 Transport Analysis
Test for concentration differences of the test organism for each of
the compartments listed in 6.3 at days 10, 20, 30, and if necessary
60. Perform the same analysis as above for the integrated
concentration of the test organism transported during the term
> •
of the experiment. Attempt to develop an expression that will
determine the net growth of the test organism over the term of
the experiment.
GROWTH = (INPUT - (TRANS + SED + WATER + SML + WALL))
INPUT = The total amount of test organism added during experiment.
TRANS = The total removed during water exchange plus aerosol.
SED = The total found in the sediment.
WATER = The amount remaining in the water column at test conclusion,
SML = The total measured in the surface micro!ayer.
WALL = The total adhering to tank wall (liquid and gas phases).
6.5 Bioaccumulation
Compute both (a): the concentration of the test organism in either
the microcosm animals and/or their specific tissues, in units of
numbers/wet weight and (b): the ratio of animal or tissue
concentration to medium concentration (Bioaccumulation Factor).
Choice of species should be based upon commercial importance
57
-------
(e.g., hard shell clam or close analogue) or known or suspected
linkages to man-based food chains. At least three replicates,
(each from separate microcosm systems) of a given species/
concentration combination should be measured if possible.
Indicate feeding type in the case of benthic species.
6.5.1 The choice of medium, for bioaccumulation calculations, will be
dependent upon the species feeding type. If the medium is
sediment, the concentration of the test organism in the top 1 cm
of the sediment core should be used to define the exposure level.
6.5.2 Bioaccumulation Analysis
Test for test organism accumulation differences for selected
(Section 6.5) species.
6.6 Environmental Assessment of Test Organism
Provide an environmental assessment of the potential fate and
effects of the test organism based on the analysis performed
above and literature findings.
7. TEST PROTOCOL BIBLIOGRAPHY
American Public Health Association, American Water Works
Association, Water Pollution Control Federation. 1975 Standard
Methods for the Examination of Water and Waste Water. 14th Edition.
New York.
Anonymous. 1984. Biosafety in microbiological and biomedical
laboratories. Public Health Service, Center for Disease Control
and National Institutes of Health. U.S. Dept. Health and human
Services, HHF Publication no.(CCDC)84-8395, 1st ed.
Perez, K.T. 1983. Experimental marine microcosm test protocol
and support document: Measurements of the ecological effects,
58
-------
fate, and transport of chemicals in site-specific marine ecosystems.
Accession no. PB83-230854. Technical Information Service,
Springfield, VA.
Slawyk, G., Maclssac, I. 1972. Comparison of two automated
methods in a region of coastal upwelling. Deep Sea Research,
19:521-524.
Steele, R.D-G., Torrie, B.H. 1960. Principals and Procedures of
Statistics. McGraw-Hill Book Co., N.Y. 481 pp.
Snedecor, G.W., Cochran, W.G. 1980. Statistical Methods. 7th
Edition. Iowa State University Press, Ames, Iowa.
Utermohl, H. 1931. Neue Wege in der quantitativen Erfassung des
Planktons. Verh. int. Verein. theor. angew. Limnol., 5:567-595.
List of Figures
a) Figure 1 Experimental Microcosm Facility
b) Figure 2 Paddle Drive and Speed Control System
c) Figure 3 Experimental Microcosm
d) Figure 4 Air Control and Supply System for Benthic Pumps
e) Figure 5 Benthic Subsystem
f) Figure 6 Exposure Chamber
g) Figure 7 Microcosm Cover and Zooplankton Sampler
59
-------
SUPPORT DOCUMENT
Purpose of the Support Document
The purpose of the support document is to provide a general rationale
for the specifications and use of the experimental microcosm system to
determine the fate, ecological effects, and transport of selected test
organisms in coastal marine environments.
Rationale for the Use of Microcosms
The fate and ecological effects of an organism introduced into a given
ecosystem are necessarily dependent on both (a) the characteristics of the
organism and (b) the physical, chemical, and biological properties and
interrelationships specific to that ecosystem. Any evaluation of potential
environmental impact, therefore, must be based on data derived with these
complexities in mind. Ideally the microcosm of a specific marine ecosystem
should be as realistic as*Possible and allow for the: (1) prediction of
ecological effects and test organism fate at the system level of organization,
(2) production of verifiable data, (3) standardization and replication
among different laboratories, and (4) application of standard statistical
techniques for data analysis.
The microcosm methodology described in the Test Protocol is used as an
experimental, physical model of a specific portion of a natural ecosystem.
Much of the rationale for the design, conditions, and operation of the
microcosm system is derived from attempts to duplicate the biotic and abiotic
conditions of a natural system (Perez et.al., 1977). This experimental
laboratory system meets the criteria listed above and should provide a pre-
dictive capability for assessing the potential fate and effects associated
with the release of a specific organism into coastal marine environments.
60
-------
Rationale for Microcosm Design
Much of the rationale for the microcosm's materials and methods are
stated in the test protocol or are intuitively obvious. These areas will
not be covered in this support document.
It is specified that the exposure and control microcosm systems be
physically separated. This is to isolate the control tanks and prevent
their contamination with the test organism.
Since the microcosm test protocol is intended to be a site-specific
test simulating a marine system, a number of system components and experi-
mental conditions will be dependent on environmental conditions at the
site (natural system) being simulated. The physical variables are light
(intensity and photoperiod)(Section 5.6.2 [6]), temperature (Section
5.2.6 [7]), water turbulence (Section 5.6.2 [5]), water turnover rate
(Section 5.6.2 [10]), and ratio of benthic surface area to water volume
(Section 2.1.1 [6]). The biotic components are the endemic water column
and benthic communities (Section 5.6.2 [1J and [3]).
In the microcosm, environmental conditions of photoperiod, temperature,
turbulence, water exchange rate, benthic surface area, and benthic pumping
rate are all established at levels that best duplicate conditions in the
natural system. Previous experiments (Perez et al., 1977) have show that
the field-derived conditions are essential in order for the biotic responses
of the microcosms to be equivalent to those in the natural system. Appropriate
light intensity, however, must be empirically determined. Performance of
the test at the temperature extremes of the natural system is specified
to insure that the test organism is exposed to the full range of expected
temperatures. This will allow better estimations of the organism's fate
and environmental effects.
61
-------
The selection of tank volume of 140 liters (Section 4.2.1) is somewhat
arbitrary. However, this tank size, and associated box core, does result
in a representative benthic component when simulating most coastal and/or
estuarine ecosystems. In addition, the inclusion, where applicable, of
a commercially important bivalve is insured when the investigator uses
this protocol. The primary consideration should be that the tanks be as
large as possible while allowing for sufficient numbers of replicates
within personnel, space, and/or monetary constraints.
Rationale for Test Procedures
Test water collection and distribution
The protocol requires the use of a water collection method which is
nondestructive to planktonic organisms (Section 5.6.2). The application
of such a method insures that any divergent behavior of the water column
communities between the experimental microcosm and the natural system is
due to microcosm effects and not the artifacts of the water collection
and distribution methods (e.g., pumping). The simultaneous filling of
all microcosm tanks at the start of an experiment (Section 5.6.2. [2])
satisfies a major assumption of the recommended analysis of variance
procedure (Section 6.1), specifically, that the experimental units
(microcosms) are as identical as possible prior to the application of the
treatment conditions (concentrations of the test organism).
Test organism addition
The manner of preparation and addition of the test organism in a
stock solution is dependent upon the nature of both the test organism and
receiving water. In general, the test organism should be added in a
manner similar to that observed or predicted in the natural system.
62
-------
It is recommended that test organism concentration be established at
known or projected field levels.
Sampling
Simultaneous measurements of the test organism and ecological effects
are prescribed for a thirty day period (Section 5.6.4 and 5.6.5). This
sampling period enables the investigator to establish either the time for
the test organism to re-ach quasi-equilibrium or the degree to which
equilibrium is reached within the various physical and biotic compartments
of the experimental microcosm. An extension of the experimental time period
(Section 5.6.1 [3]) is prescribed when the test organism concentration
in the water column has not reached a steady state. This extension allows
more time for the test organism to reach equilibrium in the experimental
microcosms and, thus, permits more accurate estimates of test organism
fate.
Ecological effects
Those measurements required to describe ecological effects (Section
5.6.5 [4]) are dependent on the characteristics of both the contained
biotic communities and the test organism. Ideally, a structural and/or
functional measure should be made from each trophic level. It is recommended
that additional ecological measures be made in those compartments exhibiting
significant accumulation of and/or exposure to the test organism.
Ecological fate
Test organism compartments: The test organism is measured within the
microcosms to establish whether the test organism is capable of surviving
and being transported to adjacent environments as well as bioaccumulating
in man based food chains. (Section 5.6.4).
Bioaccumulation of the test organism can result in subsequent ecological
63
-------
effects and may also be a potential source for human exposure via direct
consumption or trophic transfer. It is essential, from a management
viewpoint, to be able to quantitatively define the relationship between
the amount of test organism added i.e., the input function, to subsequent
bioaccumulation levels, trophic transfer, and ecological effects. Analysis
of sediment sub-cores is required to determine the vertical profile of
the test organism and, thus, exposure concentrations for some of the
benthic organisms. Field validation studies have shown that the top one
centimeter of the sediment (Section 5.6.1) is where most deposit feeding
and associated bioaccumumlation occurs. It is necessary to develop a
budget of the test organism throughout the course of the experiment
(Section 6.2). This information will indicate survivability of the test
organism within the experimental microcosms. The compartments identified
as essential to an organism budget are the overlying gas phase, water
column, sediment, microcosm surfaces and benthic macrofauna not included
in the sediment sub-cores.
Data Analysis
One of the major features of the recommended experimental design and
statistical analysis (Section 6.1) is the ability to independently establish
the quantitative effect of the test organism for all the ecological
variables measured. In addition, the degree of divergent biotic behavior
between the control microcosms and the natural system provides a measure
of the validity of the microcosm responses to the test organism.
Restrictions of Test Protocol
Scalar dimensions (Section 4.2.1) and simulation complexity (Section
4.1) limit the use and interpretation of results from experimental microcosms,
Specifically, the size of the microcosm results in the exclusion of
64
-------
macrofauna such as fishes and large crustaceans. Since most, if not all,
microcosms are so restricted, such a limitation is characteristic of this
experimental approach. In addition, the microcosm in its present design
does not simulate the effects of ultraviolet light. Because the microcosm
light lacks the U.V. component of the natural system, its possible effects
on survival, growth, and reproduction of the test organsim, especially if
it accumulates at the aJr-water interface, cannot be estimated.
Support Document Bibliography
Perez, K., Morrison, G., Lackie, N., Oviatt, C., Nixon, S., Buckley, B.,
and Heltshe, J. 1977. The importance of physical and biotic scaling to the
experimental simulation of a coastal marine ecosystem. Helgolander wis
Meeresunters. 30:144-162.
65
-------
Summary
Three laboratory marine systems are presented that may be used to
evaluate the impact of genetically engineered and naturally occurring
MPCA's on the near-shore environment. The microcosms, one simple with
fish, shrimp and two molluscs, one a simulative assemblage of shallow,
intertidal organisms including plants and another simulative assemblage
representing deeper water communities. The systems differ in complexity;
the simple system with four selected species and the simulative systems
with populations and numbers of organisms as collected in the environment.
They also differ in other factors such as the manner in which water is
supplied. The simple system is static, the near coastal system can be
static or flowing and the deeper water system is maintained with flowing
seawater.
Of the three proposed microcosm tests, only the simple system has
been operated with microorganisms. The simulative systems were developed
for testing the impact of toxic organic chemicals and the authors believe
that these systems must be tested with microorganisms before they are used
in routine testing with microbial pest control agents.
66
-------
FRESHWATER SYSTEMS
AUTHORS:
Richard L. Anderson
Terry E. FT urn
Paul J. Franco
Jeffrey M. Giddiflgs
Gary W. Hoi come
John W. Leffler
Gary L. Phipps
Lyle Shannon
Carolyn L. Thomas
J. David Yount
67
-------
This section contains the following:
1. Methods for conducting multi-species, vertebrate and invertebrate
freshwater acute tests with Microbial Pest Control Agents (MPCA's).
Gary L. Phipps and Gary W. Holcome, ERL-D, Duluth, MN 55804.
2. A procedure for exposing stream invertebrates to microbial pest control
agents in the laboratroy. Terry E. Flum and Richard L. Anderson, ERL-D,
Duluth, MN 55804.
3. Methods for a microcosm test to determine survival and ecosystem level
effects of Microbiert Pest Control Agents. John W. Leffler, Dept. of
Biology, Ferrum College, Ferrum, VA 24088; Lyle Shannon, Dept. of Biology,
University of Minnesota, Duluth, Duluth, MN 55812; Carolyn L. Thomas,
Dept. of Biology, Ferrum College, Ferrum, VA 24088; J. David Yount,
ERL-D, Duluth, MN 55804.
4. Methods for establishing microcosms of a aquatic littoral ecosystems for
determining safe levels of agent exposure. Paul J. Franco, Oak Ridge
National Laboratory, Oak Ridge, TN 37381; Jeffrey M. Giddings,
Springborne Bionomics, Wareham, MA 02571.
68
-------
INTRODUCTION
Interest in microbial pest control agents (MPCA's) for insect control
is increasing. Concurrently, there is an increasing need to develop an
understanding of the potential for adverse effects from these agents to
populations, communities and ecosystems. The need is particularly great in
freshwater systems because of the variety of habitats that may receive
biological control agents from either direct application or from applications
to land adjacent to water.
Microbial control agents are unique when compared to other pest control
agents, because thay are alive. Life adds questions of infectivity, patho-
genicity and competition to the usual concerns of toxicity, decreases in
growth an reproduction and adverse changes in population, communities and
ecosystems. In a tiered evaluation system, the first step to develop an
understanding of effects is laboratory acute bioassays. The acute tests
can be followed by chronic life cycle exposures which examine reproduction,
growth and mortality. The final laboratory tests are microcosms, which are
designed to simulate outdoor systems. In this report, systems which were
developed for chemical toxicity determination are described. They include
two laboratory systems that can test many species at one time and two
microcosm systems that provide the complexity of interacting populations and
allow measurement of system effects.
The first test described in this section is a multi-species exposure
system that was designed to provide chemical toxcity data on a variety of
species from a single test. In its current design a diluter is used to
provide water to 12 exposure tanks. This system may be adapted for specific
MPCA use by reducing the number of exposure tanks and altering the diluter
or developing other methods to deliver water and maintaining animals in the
69
-------
system. The key item in this test is the mechanism for exposing eight or
more species.
The second test in this section describes an exposure system for stream
invertebrates. The exposure tank is a trough, about 10 cm wide and 13 cm
deep, that is turned in an oblong form to provide a continuous channel.
Physical.factors such as flow and temperature are controlled by a circulating
pump and a water bath. ,Direct exposure of animals can be accomplished by
enclosing animals in screen containers or by simple release into the tank.
The impact of sediments, rocks, or plants on the expression or toxicity of
an agent can be tested with this system.
The remaining protocols in this seciton describe methods to produce
laboratory microcosms. One system was designed to provide data on ecosystem
effects of chemicals but is being applied to questions of survival and
ecosystem effects of MPCAs.in freshwater systems. The microcosm development
is started by collecting biota from natural outdoor sources and establishing
animal stocks in the laboratory. These stocks can be long lived, often
lasting more than a year which allows tests to be started at anytime.
Material from the stock tank is used to prepare the microcosm. The microcosm
containers are one liter beakers which contain a sediment, nutrient
supplimented water and, after an equilibration period, stable communities
of primary producers, consumers and decomposers. The method, shows good
reproducibility and has been field validated with chemical toxicants. The
small size of the microcosms permits many to be used simultaneoulsy in a
small enclosure.
The other protocol in this section describes procedures for establishing
freshwater littoral communities in the laboratory with materials collected
from natural ponds. This system was also developed to assess the effects
70
-------
of chemical toxicants on littoral communities. The procedure utilizes 72
liter aquaria as exposure tanks and establishes microcosms by collecting
sediment and water from natural, outdoor systems and placing the material
with its biota into the aquaria. This system has the advantages of easy
assembly, low maintenance and replicability. Because the microcosms are
established from natural ecosystems, a site-specific evaluation of effects
can be established.
In summary, this section describes four protocols. The first two
systems can provide exposure data on many species at one time. The two
microcoms systems can provide data on effects and expression of an agent in
complex environments. However, a key consideration for all these protocols
is that they may be adaptable to MPCA registration uses.
All the systems were developed to test chemicals using the endpoints
of death, reproduction or ecosystem changes. None have been extensively
tested with MPCAs and all the writers acknowledged that research is necessary
before these systems can be used with the same confidence associated with their
use in chemical hazard assessment.
The writers have identified seven areas that need to be addressed
before high confidence can be associated with MPCA use as follows:
1) Operation of the procedure with a specific MPCA
2) Endpoints
3) Positive Control
4) Quantitation
5) Dosing
6) Containment
7) Decontamination
The primary research goal is to operate these procedures with an MPCA.
Because of the variety of MPCAs, it will be necessary to test the procedures
with an agent from each group of interest. The results of the first
experiments may affect the other research areas which indicate a need for
71
-------
comprehensive studies. Each procedure has specific endpoints such as death,
reproduction changes or system effects. Methods that measure other effects
that may be uniquely associated with MPCAs such as infection, pathogenicity,
environmental expression, persistence and competition which may lead to
species replacement are yet to be developed for use with these freshwater
systems. These methods can be developed through research or evaluation of
adaptable procedures duping the protocol evaluation. The third research
need is the addition of a positive control. In the usual bioassay or
toxicity test, concentrations can be selected that will cause a lethal
effect. Because MPCAs are not general toxicants, many animals may not be
sensitive so it is important that the target animal (if aquatic) be tested
in the system to show that the system allows an effect to occur. In the
multispecies tests, this may be done by adding a container with target
animals to the system. In the microcosm tests, the target animal could be
added to a test unit or a separate positive control test unit could be
established to verify that the agent, at the test concentration, was causing
the observed effect. Areas four through seven point to specific questions
that must be answered before the procedures can be widely used. All risk
assessments require information of the exposure concentration and effect
relationship so quantitation methods for each system are necessary. The
last areas, dosing, containment and decontamination are included to show
their importance and the need to have methods available before the testing
of an MPCA.
72
-------
METHODS FOR CONDUCTING MULTI-SPECIES,
VERTEBRATE AND INVERTEBRATE FRESHWATER ACUTE TESTS WITH MICROBIAL
PEST CONTROL AGENTS (MPCAS)
by
GARY L. PHIPPS and W. HOLCOMBE
U.S. ENVIRONMENTAL PROTECTION AGENCY
ENVIRONMENTAL RESEARCH LABORATORY-DULUTH
6201 CONGDON BOULEVARD
DULUTH, MINNESOTA 55804
73
-------
1. SCOPE.
This multi-species test procedure was designed to simultaneously
test 8-12 freshwater vertebrates and invertebrates against toxicants.
The toxicants include industrial organics, pesticides and metals and
the procedure has been used successfully to produce LC50 data on
selected species (Phipps et al., 1985). Since it has not been used
for testing Microbial Pest Control Agents (MPCAs), several modifications
will be suggested to adapt the procedure to those unique MPCA testing
needs. This procedure presumes a working knowledge of aquatic
toxicity testing so many basic concepts will not be included. This
procedure, used a modified Mount-Brungs diluter (1967) with 5 experi-
mental toxicant concentrations and a control, all duplicated. MPCA
testing, however, is different in that the need is to determine
whether a concentration-independent effect is caused by the MPCA,
therefore any apparatus which will provide an exposure concentration
and a control is satisfactory.
2. APPLICABLE DOCUMENTS.
2.1 1976 Annual Book of ASTM Standards, Part 31;
D8888 Oxygen, dissolved in water
D1067 Acidity and alkalinity
D1125 Conductivity
D1126 Hardness
D1293 pH
D1426 Ammonia
E729-80 Conducting acute tests with fishes, macroinvertebrates
and amphibians.
74
-------
2.2 EPA-660/ Ecological Research Series
3-75-011 Acquisition and culture of research fish:
Rainbow trout, fathead minnows, channel catfish,
and bluegills.
3-75-009 Methods for acute toxicity tests with fish, macro-
invertebrates, and amphibians.
3. SUMMARY.
This procedure differs from standard acute toxicity tests in a few
key areas as follows:
3.1 The MPCA target organism may be tested simultaneously with a diverse
variety of nontarget organisms that might be found in the same body
of water or area.
3.2 Several vertebrate and invertebrate species are tested simultaneously,
yielding considerably more data for the time, effort, and money
.. •
expended.
3.3 All species are exposed in the same test chamber, in the same water
column, assuring uniform exposure conditions for all tested species
thereby decreasing test-to-test variability.
3.4 Warm and cold water species are tested together at 17° C, which is a
recognized standard warm water test temperature (ASTM E729, 1980),
and within the physiological optimum for rainbow trout (Lichtenheld
1966; Lavrosky 1968; Dickson and Kramer 1971; McCauley and Pond
1971; Hokanson et al. 1977).
3.5 A self-starting siphon was used (Benoit and Puglisi 1973) which
varied the height of the test water in the exposure chamber (Fig. 1)
which effects water exchange in the Daphnia and Chironomid exposure
beakers (Fig. 1) making the MPCA concentration in the beakers the
75
-------
same as that in the exposure chamber.
3.6 It was easier to distinguish changes in behavior and to determine
the most sensitive species.
4. SIGNIFICANCE.
4.1 This test was designed to produce an acute aquatic toxicity data
base for a minimum of time, effort and finances. In addition, the
test organisms were selected for their diversity in representing the
freshwater community, their ease of procurement, availability and
ease of culturing.
4.2 Because all of the organisms in each concentration were tested in
the same water columnm, species differences in toxicty became very
apparent, the more sensitive species were easily identified, and
behavioral differences could be observed.
5. QUALITY CONTROL.
.. •
5.1 Organisms.
It is recommended that organisms be brought into the laboratory and
acclimated to the dilution water for at least 2 weeks. During this
time mortalities should be less than 10 percent in 2 weeks. If
mortalities exceed this limit, the organisms should be treated for
disease, destroyed, or new ones procured. After treatment, the
organisms must be reacclimated.
5.2 Control Mortality.
Control mortality should never exceed 10 percent per species per test.
A test must be repeated if mortality is greater than 10 percent.
Practically speaking, however, if any control mortality takes place,
serious scrutiny of the test should take place to isolate the cause
because the quality of the test is jeopardized.
76
-------
5.3 Test Conditions.
Test conditions must be maintained as close to constant as possible.
Strict attention must be given to temperature, dissolved oxygen,
fighting and any other variable which affects the well-being of the
organism. The ideal test has only one variable between the control
and experimental treatments, so all the other variables must be
controlled as closely as possible.
6. FACILITIES.
The test and animal holding systems must be located in a laboratory
with adequate physical characteristics which include:
6.1 Water Supply.
A water supply of satisfactory quality for aquatic testing must be
available for the holding and test systems. The water must be
tested for adequacy.using methods described by APHA (1980).
.. •
6.2. Electrical Supply.
An electrical supply for powering testing equipment, dosing pumps
and fluorescent lights must be protected by ground-fault-interrupt
to protect the researchers from electrocution.
6.3 Air Supply.
An-oil free air supply for aeration of the fish tanks and brine
shrimp cultures must be supplied.
6.4 Temperature Control.
Adequate control (^ 2° C) of room temperature is essential to maintain
uniform test temperatures (+_ 1° C) in static test systems. Water
temperature control of dilution water should be sufficient to maintain
the test water temperature within + 1° C.
77
-------
6.6 Ventilation.
Negative pressure ventilation must be provided to assure the
protection of the researchers from MPCAs or their products.
6.6 Effluent Water Treatment.
Facilities should include a water treatment system to sterilize the
effluent to avoid an accidental environmental release.
6.7 Sterilizer.
A sterilizer of adequate size to accommodate exposure tanks and
dosing equipment is essential.
7. EQUIPMENT.
7.1 Dilution Apparatus.
Since MPCA testing uses only one toxicant concentration, a standard
dilution apparatus is not needed. A variety of apparatuses listed
in EPA-660/3-75-009- section No. 3 (1975) should work for MPCA
testing. The Mount-Brungs diluter (1967) may be easily modified to
give the one experimental concentration and control, by disconnecting
4 of the 5 experimental concentrations.
7.2 Exposure Tanks (Fig. 1).
Glass aquaria measured 60 x 30 cm deep with a 17 cm standpipe and
had a volume of 30.6 L. Aquaria were divided into six 16.6 cm x
15.0 cm compartments and one 10.0 x 30.0 cm compartment with stainless
steel dividers. The standpipe located in the 10 x 30 cm compartment
had a self-starting siphon which varied the water level in the tank
by 2.5 cm to allow water exchange in the Daphnia and Chironomid
chambers.
7.3 Daphnia and Chrionomid Exposure Beakers (Fig. 1).
Beakers were 250 ml in volume. Two holes, 19 mm in diameter, were
78
-------
drilled opposite each other with their centers 2 cm up from the
bottom. These holes were covered by stainless steel screen which
was silicone-glued to the outside of the beaker. The screen-covered
holes effected water exchange in the beakers without the escape of
organisms. A wire was attached (Fig. 1) to the beaker and the
beakers were hung in the exposure aquaria so that the lowest water
level in the aquarium was 3 cm above the bottom of the beaker.
7.4 Snail Exposure Chambers.
The exposure chambers consisted of stainless steel screen cylinders
10 x 3 cm diameter, closed on one end by stainless steel screen and
on the other by a No. 7 neoprene stopper. The chambers prevent the
snails from leaving the water during the testing.
8. PROCEDURES.
8.1 Test Preliminaries.-
. .
Once the experimental concentration of MPCA has been determined,
the MPCA stock concentration must be determined and the stock
prepared. The diluter is then started and the experimental tanks
filled with MPCA-laden water. The test begins when the species that
are tested for 96 hours are counted out and randomly distributed to
the exposure chambers.
8.2 Test Start, Day 1
8.2.1 The test started at hour zero on day one when the species were
introduced into the different screen chambers in the exposure
chambers. Records must be kept of the following: (1) Mortalities
for each species at least once every 24 hours. (2) Agent information
such as purity, source, lot number, stock concentration and nominal
or calculated exposure concentrations. (3) Water temperature.
79
-------
(4) Dissolved oxygen. (5) Test start date and time. (6) Any other
pertinent information.
8.2.2 After the test is started with the species that will be tested for
96 hours, the Daphnia that will be tested for 48 hours must be
cultured. Adult Daphnia magna are isolated and fed, and the time of
isolation is noted. Exactly 24 hours later the adults are removed.
The resulting young Daphnia are then counted into the exposure
beakers and hung in the exposure tanks.
8.2.3 The third to fouth instar Chironomid larvae are placed into exposure
beakers which had about 1 ml acetone-washed quartz sand previously
spread on the bottom. In addition, about 1 ml of midge food is
placed into the beakers. Once the larvae are in the beakers, they
should be set aside for 24 hours to acclimate and to build tubes.
8.3 Day 2 Testing.
— * •
On day 2 or 24-30 hours after the test is started, the Daphnia and
midges, as well as any other organism that are tested for 48 hours,
are placed into the exposure chambers. A mortality count is made on
the other species and the dead are removed. The water temperature
is recorded. Snails are counted at 96 hours. Daphnia and Chrionomids
are counted only at 48 hours.
8.4 Day 3 Testing.
On day 2 or 24-30 hours after the test is started, the Daphnia and
midges, as well as any other organism that are tested for 48 hours,
are placed into the exposure chambers. A mortality count is made on
the other species and the dead are removed. The water temperature
is recorded. Snails are counted at 96 hours. Daphnia and Chrionomids
are counted only at 48 hours.
80
-------
8.4 Day 3 Testing.
Mortalities are counted and the dead are removed. Water chemistry
measurements such as dissolved oxygen, alkalinity, pH and hardness
are determined and recorded. The water temperature is recorded.
Exactly 48 hours after the organisms were introduced into the test,
Daphnia immobilization and Chironomid and Daphnia mortalities are
determined for each test beaker and recorded.
8.5 Day 4 Test Termination.
At exactly 96 hours after the start of the test, the final count is
made on all species tested for 96 hours. The snails are removed
from their exposure chambers and mortalities are counted and recorded.
To determine mortality, snails should be inspected with a binocular
dissecting scope and the foot probed with a blunt instrument about
paper clip wire size to stimulate movement. After mortalities are
counted, 10 control fish of each species should be ice-anesthetized
and weighed to determine fish size for each species. Removal and
disposal of the remaining fish in the test now takes place, and the
test system is cleaned in preparation for the next test.
81
-------
REFERENCES
American Public Health Association, American Water Works Association,
and Water Pollution Control Federation. 1980. Standard methods for the
examination of water and wastewater, 15 edn. Washington, DC.
American Society for Testing and Materials. 1980. Standard practice
for conducting acute toxicity tests with fishes, macroinvertebrates and
amphibians, E279-80. Philadelphia, PA.
Benoit, D.A. and F.A. Puglisi. 1973. A simplified flow-splitting
chamber and siphon for proportional diluters. Water Res. 7:1915-1916.
Biesinger, K.E. and G.M. Christensen. 1972. Effects of various metals
on survival, growth and reproduction of Daphnia magna. J. Fish. Res. Board
Can. 29(12):1691-1700.
Brauhn, J.L. and R.A. Schoettger. 1975. Acquisition and culture of
research fish: Rainbow trout, fathead minnows, channel catfish, and
bluegills. Ecological Research Series No. EPA-660/3-75-011. U.S.
Environmental Protection Agency, Corvallis, OR. 54 pp.
Dickson, I.W. and R.H. Kramer. 1971. Factors influencing scope for
activity and active and standard metabolism of rainbow trout (Salmo gairdneri),
J. Fish. Res. Bd. Can. 28:587-596.
Hokanson, K.E.F., C.F. Kleiner and T.W. Thorslund. 1977. Effects of
constant temperatures and diel temperature fluctuations on specific growth
and mortality rates and yield of juvenile rainbow trout, Salmo gairdneri.
J. Fish. Res. Bd. Can. 34:639-648.
Lavrosky, V.V. 1968. Raising of rainbow trout (Salmo gairdneri)
together with carp (Cyprinus carpio) and other fishes. In: Proc. FAO
World Symp. on Warm-Water Pond Fish Culture, ed. by T.V.R. Pillay, FAO
(FAOUN) Fish Rep. 44(5):213-217.
82
-------
Litchtenheld, R.W. 1966. Effect of light, temperature, and gamma
radiation on the locomotor activity of juvenile steelhead trout, Salmo
gairderni. Ph.D. thesis, University of Washington.
McCauley, R.W. and W.L. Pond. 1971. Temperature selection of rainbow
trout (Salmo gairdneri) fingerlings in vertical and horizontal gradients.
J. Fish. Res. Bd. Can. 28:1801-1804.
Mount, D.I. and W.A. Brungs. 1967. A simplified dosing apparatus for
fish toxicology studies. Water Res. 1:21-29.
Phipps, G.L. and G.W. Holcombe. 1985. A method for aquatic multiple
species toxicant testing: Acute toxicity of 10 chemicals to 5 vertebrates
and 2 invertebrates. Environ. Pollut. (Series A) 38:141-157.
U.S. Environmental Protection Agency: Committee on Methods for Toxicity
Tests with Aquatic Organisms. 1975. Methods for acute toxicity tests with
fish, macroinvertebrates, and amphibians, Ecol. Res. Ser., EPA-660/375-
009, NTIS No. PB242105 (National Technical Information Service, Springfield,
VA 22161).
83
-------
GO
Species
Fathead minnow
Pimpephales promelas
Channel catfish
Ictaluarus punctatus
Bluegill
Lepomis macrochlrus
Goldfish
Carassius auratus
White sucker
Catostomus commersoni
Chinook salmon
Oncorhynchus
tshawytscha
Rainbow trout
Salmo gairdneri
Crayfish*
Onconectes Immunis
Leech
Nephelopsis obscura
Snail
Aplexa hypnorum
Daphnia magna3
Midge3
Canytarsus dissimilis
African Frog
Xenopus laevis
Source
Lab
Culture
Commercial
Hatchery
Commercial
Hatchery
Lab culture or
Commerical
Hatchery
Commercial
Hatchery
Commercial
Hatchery
Commercial
Hatchery
Commercial
Hatchery
Bait
Dealers
Lab
Culture
Lab
Culture
Lab
Culture
Lab
Culture
TABLE 1
SPECIES TESTED
Size or
Lifestage Age
Juv. 6-8 wks.
0.3-0.8g
Juv. l-40g
Juv. 0.2-2g
Juv. l-20g
Juv. Approx.
l-15g
Juv. 3-7g
Juv. 0.5-20g
Juv. 2-4g
Juv. <_ 1 year
Adult 1-2 month
1-2 instar 0-24
3-4 instar 7-8 day
Tadpole 1-4 weeks
Holding
Tank Volume
57L
530L
530L
57L
530L
530L
530L
530L
57L
57L
2L
2L
57L
Holding
Temperature
20°C
17°C
17°C
17°C
17°C
15°C
15°C
17°C
17°C
20°C
17°C
17°C
20 °C
Food
Type
Artemia
nauplii
Dry food2
Frozen Brine
Shrimp
Frozen Brine
Shrimp
Dry Food
Dry Food
Dry Food
Dry Food
Fish & Liver
Artemia
nauplii
Daphnia4
food
Daphnia
food
Artemia
nauplii
Feeding
Frequency
2/daily
2/daily
2/daily
2/daily
2/daily
2/daily
2/daily
2/daily
I/daily
2/daily
I/daily
I/daily
I/daily
Number Tested
Chamber
10
10
10
5-10
5-10
5-10
5-1
10
10
10
10
10
10
1 May be held in same tank as bluegills.
2 Made to U.S. Fish and Wildlife Specifications.
3 Reared in static conditions.
* Biesinger and Christensen 1972.
-------
A PROCEDURE FOR EXPOSING STREAM INVERTEBRATES TO
MICROBIAL PEST CONTROL AGENTS IN THE LABORATORY
by
Terry E. Flum and Richard L. Anderson^
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
^Corresponding Author
85
-------
1. SCOPE.
1.1 This method, developed from experience obtained in toxicity testing
of stream invertebrates, has been used to examine the impact of biotic
and abiotic factors on the distribution of a Microbial Pest Control
Agent (MPCA). The primary unit in the system is a stainless steel
trough and a circulating pump. Each trough is about 2 meters long,
13 cm deep and 11 cm wide. The ends of the trough are connected
and the system is formed into an oval with an overall length of
about 92 cm. The trough is filled to a depth of 10 cm and the pump
circulates the water. The stream may be used as a simple bioassay
chamber by enclosing the test animals in screen containers suspended
in the flow. The stream may also be used for more complex simulations.
The complex studies would be accomplished by the addition of sediment,
rocks or other debris and animals would not be enclosed but allowed
to move freely in the system.
1.2 Successful operation of this procedure assumes a knowledge of toxicity
testing and an understanding of the techniques and procedures described
in the following texts:
1. Standard Methods for the Examination of Water and Wastewater, 1985.
2. 1976 Annual Book of ASTM Standards, Part 31:
D888 Oxygen, dissolved in water
D1067 Acidity and Alkalinity
D1125 Conductivity
D1126 Hardness
D1293 pH
D1426 Ammonia
E729-80 Conducting acute tests with-fishes, macroinvertebrates
and amphibians
86
-------
3. U.S. EPA Ecological Research Series.
660/3-75-009 Methods for acute toxicity tests with fish,
macroinvertebrates, and amphibians,
2. SUMMARY.
Wild-caught or cultured invertebrates are enclosed in screen containers
and exposed in a laboratory stream system. Flow rates and temperature
are controlled and animals can be maintained for at least 30 days,
if food is provided. Effects criteria may be death or animals can
be sampled for analysis by microscopy or other techniques.
3. SIGNIFICANCE.
3.1 Lotic environments range from springs to shallow streams to rivers.
These environments contain a range of habitats that can receive
inputs of MPCAs from planned and accidental events. Unfortunately,
stream invertebrates are not often used as test animals in the
laboratory because of their need for flowing water. This procedure
describes a contained, controlled-flow test system. It can be used
as a multiple species bioassay chamber or as microcosm for more
complex environmental testing. It is small, low cost and all
equipment can be reused.
4. APPARATUS.
4.1 Facilities. A holding and a test area are needed in which the
animals used in the test can be cultured in the laboratory, collected
from natural habitats or obtained from commercial sources. If
animals are cultured, then the systems used in the culturing will
be adequate or can be adapted to maintain the collected or purchased
animals. If the testing laboratory does not culture invertebrates,
then holding facilities must be established. Designs and conditions
87
-------
for rearing and holding are available in reports in section 1. Both
facilities should provide an adequate water source, temperature
control, and electricity for lights, timers and pumps. In
addition, the test area should provide a clean section for test set-
up and an adequate waste treatmeant and clean-up facility.
4.2 Test System Apparatus.
4.2.1 Stream Chamber. -The streams are contained in stainless steel
troughs, each about 2 meters long, 13 cm deep and 11 cm wide. The
ends of the trough are connected and the system is formed into an
oval with an overall length of 92 cm. In this form, each stream
has a 60 cm straight section and curves on each end that have a
radius of 16 cm.
4.2.2 Temperature Control. Stream invertebrates may be exposed at
temperatures that are less than 20° C so systems that can maintain
lower than room temperatures must be developed. Options include
cooling the test area air or using a refrigerated water bath.
4.2.3 Light. Light is provided by wide spectrum fluorescent lamps at an
intensity of 270 lux (25 foot candles). A combination of Gro-Lux®
and Vita-Lite® bulbs has been used successfully.
4.2.4 Circulation. Water circulation in the trough is provided by a
submersible pump. In our system, a water bath was used and the
pump was located in the water bath. This was necessary because
the pump can heat the exposure water. The pump inlet line was
constructed from PVC pipe and positioned just below the surface on
the side opposite the outlet pipe. Water that passed through the
pump passes through an adjustable float gauge that allows control
of flow rates. The water returns to the trough through a dispersing
88
-------
orifice located at the water surface, parallel to the trough bottom.
In our system, the oulet was constructed from a PVC plumbing "T"
that was plugged on the ends of the "T" and had 3, 2 mm holes
drilled to allow the water to be forced out under pressure.
4.2.5 Screen Containers. Stainless steel screen containers are recommended
because of their durability. The containers can be constructed
in either cylinder or cube form with largest mesh size that retains
the test animals. The design should include either a built-in cap
or have another provision for capping. The containers are placed
on the bottom of the trough or suspended by hooks from the side of
the trough. The containers should extend above the water surface
at lest 3 cm.
5. WATER.
Test waters commonly used in bioassays or toxicity testing are
- •
from wells or the surface. The water should be uncontaminated and
of constant quality. Specifications for the test water are provided
in the references in Section 1. A common guide for water quality
is its suitability for rearing Daphnia. If Daphnia can be reared
or if the water is adequate for culturing other invertebrates, it is
assumed be adequate for use in this procedure. Water should be
added to replace evaporative loss. The water can be slowly added
by pouring through a tube directly in front of the outlet.
6. TEST ORGANISMS.
Selection processes for lotic test organisms should include a
prediction of what populations will be exposed if the agent is
directly applied for controlling an aquatic pest or what aquatic
populations will be exposed if the material is applied to a
89
-------
terrestrial system. If the preferred nontarget animals are not
available then substitutions should be closely related to the
selected animals. There are few commercial sources of stream
invertebrates and local collections may be the best source of
animals.
6.1 Collection and Laboratory Acclimation. Collection techniques for
invertebrates include kick-nets, seines, screens and dredges
(Merritt, Cummins and Resh, 1978). After collection, the animals
should be quickly transferred to the laboratory. Special care
should be given to maintaining the dissolved oxygen near saturation
and providing a current through a portable aerator. A record of
the collection and maintenance procedures is essential. Animals
should be kept at the test temperature for at least 1 week before
testing. Dead, diseased or damaged individuals should not be
used.
6.2 Experimental Design. A minimum test would include one exposure
concentration and a control that would receive no additions of the
MPCA. A position control would be accomplished by a container in
the exposure stream that contains the target organism (if aquatic).
This is included to assure that the agent is toxic at the test
concentration. Each test is done in duplicate so 4 troughs would
be required. If additional concentrations were needed, two troughs
would be added for each concentration.
7. PROCEDURE.
7.1 Preparation. Some of the preparation for an exposure must be
completed one or two weeks before the test so the procedure will
be presented chronologically:
90
-------
-14 days - Test animal collections should be completed at least
one week before the test to provide time for acclimation to temp-
perature, food and usually a different water.
-7 days - All collected animals should be acclimated to the
test conditions. Begin daily checks for animal health. Monitor
dissolved oxygen, temperature and pH of the holding system. If
reared animals ape being used, then monitor those systems to assure
similarity to the collected animals holding condition.
-3 days - The troughs should all be filled and operating by this
day. Dissolved oxygen, pH and temperature should be checked each
day. These values must be stable for at least 2 days before the
animals are added. All the screen containers should be cleaned
and sterilized.
-2 days - Distribution of the test animals. Two containers of
10 animals each are necessary for each trough so a total of 4
containers (40 animals) are needed for the minimum test. To
prepare the containers for 1 species, carefully remove the animals
from the holding/rearing tank and transfer to a shallow white
enamel pan with enough water to allow easy movement of the animals.
Transfer small animals with a pipet or large animals with a piece
of screen shaped into a scoop to each of 4 beakers. Continue the
process until each beaker has 10 animals. Place labeled screen
containers into each trough and randomly select a beaker and gently
pour the animals into the screen container. Continue until all
animals in each beaker are transferred and then count the animals
in each screen container. Repeat this process for each species
until all the test animals are in the test troughs. Most species
91
-------
will not leave the water but caps may be required on the screen
enclosures.
-1 day - Check all the screen enclosures for number and condition.
Replace any that do not appear healthy. Select the streams that
will receive an MPCA.
0 day - Check all the animals before adding the MPCA. The
methods for adding the test agent depend on the formulation (liquid,
powder, or granule) and other special considerations that cannot
be forseen. Before the addition, the dissolved oxygen and pH
should be measured. After addition, these same measurements
should be made at intervals that will describe their changes during
the test. Also, a schedule for monitoring the agent concentration
should be prepared before the exposure begins and should be followed
throughout the exposure.
+1 day - On each day, the animals in the screen enclosures
should be counted and recorded along with any observations on
fitness. If a sampling schedule or a decision that any "impaired"
individual would be removed for analysis has been made, those
animals can be removed at this time.
The animals should be checked daily until the test is complete.
Exposures up to 30 days have been successful in this system.
7.2 Temperature. The temperatures should be maintained within +_ 1° C.
Temperature measurements should be continually recorded. Minimum
requirement is a daily measurement.
7.3 Dissolved Oxygen. Aeration should not be necessary because of
the recirculating system. Dissolved oxygen should be measured
before the test and daily during the exposure. At least one set
92
-------
of measurements should be made throughout a 24 hour period to
monitor for sags in oxygen content. A preferred system would use
a probe that measures oxygen content and an automatic recording
system.
7.4 pH. The pH should be monitored on the same schedule as the
dissolved oxygen. A continually recording system is preferred to
spot sampling.
7.5 Feeding. Feeding requirements may be specific for some animals
and those foods should be provided. For many stream invertebrates,
soaked hardwood leaves (maple, aspen, alder, birch) are acceptable.
The leaves are most acceptable if soaked for up to 30 days in
control water. The leaves can be added directly to the screen
enclosures and replaced when only the skeleton remains. If the
containers are suspended, the fecal material and debris will fall
to the bottom of the trough.
7.6 Concentration Monitoring. A critical part of the test is the
concentration of the test agent in the system. Before the test
begins, a prediction of the probable distribution of the agent in
the system must be completed and a sampling schedule must be
devised to monitor these locations. After the test starts, the
schedule may change.
8. QUALITY ASSURANCE.
8.1 Criteria for accepting a test rests on definite statements and
good judgement. Chemical testing criteria include:
8.1.1 At least 80% of the control animals survive.
8.1.2 Temperature deviation does not exceed 1° C.
8.1.3 Dissolved oxygen does not drop below 40% saturation.
93
-------
8.1.4 pH does not deviate more than 1 pH unit.
If a test is awry, good judgment requires that it be terminated
and repeated. It is critical, even in those tests, that the
observations be used to improve the next test.
9. OPTIONS.
The recirculating trough can also be the basis for a stream
microcosm. The basic system has been adapted to examine the fate
of an MPCA in a more complex system and the impact of nontarget
animals on the fate and distribution of an MPCA. The basic system
was altered by ttie addition of a sand substrate and rocks were
simulated by unglazed clay tiles. In the studies, beach sand was
sieved through a 250 urn screen and rinsed in water before it was
added to the troughs. The sand was added to a depth of about 3
cm. The unglazed tiles were used to simulate natural rock. Each
commercial tile was quartered to produce small squares, about 7.5
cm on a side. The tiles were inserted into the sand at about a 45
degree angle, leaning away from the flow. About one-half of each
tile remained above the sand. The tiles were centered to allow
flow on both sides. Twenty-two tiles were placed in each trough,
about 10 cm apart. In the sand-tile system, free stoneflies
(Pteronarcys) have been successfully maintained for 30 days.
Other nonpredaceous invertebrates could also be added to this
system.
10. ADDITIONAL INFORMATION.
Many references are in the reports mentioned in Section 1. Additional
information about testing and maintenance of stream invertebrates
can be obtained from the following sources:
94
-------
Nebeker, A.V. and A.E. Lemke. 1968. Preliminary studies on the
tolerance of aquatic insects to heated waters. J. Kansas Entomological
Society 41(3):413-418.
Nebeker, A.V. 1972. Effect of low oxygen concentration on survival
and emergence of aquatic insects. Trans. Am. Fish. Soc. 101(4) -.675-679.
Anderson, R.L. 1982. Toxicity of fenvalerate and permethrin to several
nontarget aquatic invertebrates. Environ. Entomol. 11(6):1251-1257.
Anderson, R.L. and P. Shubat. 1984. Toxicity of flucythrinate to Gammarus
lacustris (Amphipoda), Pteronarcys dorsata (Plecoptera) and Brachycentrus
americanus (Trichoptera): Importance of exposure duration.
Environ. Pollut. (Series A) 35:353-365.
95
-------
References
Merritt, R.W., Cummins, K.W., and V.H. Resh. 1978. Collecting,
Sampling and Rearing Methods for Aquatic Insects. In: An Introduction
to the Aquatic Insects of North America, Ed. R.W. Merritt and K.W. Cummins,
Kendall/Hunt Publishing Co., Dubuque, Iowa. pp. 13-31.
96
-------
METHODS FOR A MICROCOSM TEST TO DETERMINE SURVIVAL AND ECOSYSTEM
LEVEL EFFECTS OF MICROBIAL PEST CONTROL AGENTS
John W. Leffler, Lyle J. Shannon,
Carolyn L. Thomas, and J. David Yount
U.S. Environmental Protection Agency
Environmental Research Laboratory-Duluth
6201 Congdon Boulevard
Duluth, Minnesota 55804
97
-------
1. SCOPE
1.1 Description of test. This protocol is designed to provide data on
survival and ecosystem-level effects of microbial pest control
agents (MPCAs) introduced into a freshwater environment. Such
microbial agents are those subject to test regulations under the
Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA). The
EPA will use these data to assess the ecological hazards an
introduced microorganism presents to the freshwater environment.
The test systems are generic aquatic microcoms containing stable
communities of primary producers, consumers and decomposers.
Changes are monitored at the ecosystem-level to provide a measure
of effect that is integrated across population and community
boundaries.
1.2. Justification for test. The EPA has recognized for some time that
evaluation of chemical hazard to environmental systems requires testing
at levels of the ecological hierarchy above the single species level
(Environmental Protection Agency, 1979). A recent study by the
National Academy of Science (1981) concluded that tests at the
population and ecosystem levels as well as single species tests
"are needed before sound judgements can be made about the potential
environmental hazard of any chemical." The same conclusions apply
to evaluations of the survival and effects of MPCAs.
This protocol provides data which are complementary to those
obtained from single species tests. The main advantage of this
protocol is that it represents an increase in "ecological realism"
over single species tests (Schneider, 1981). It permits evaluation
of effects on functional properties at the ecosystem level of
98
-------
organization. Effects due to species interactions, trophic
organization, and biogeochemical cycling can be asesessed.
Sensitivity of microcosms to microbial agents may vary greatly,
being either greater or less than sensitivity values obtained
from single species tests.
The microcosms of this protocol do not simulate any natural system,
but do posses certain functional characteristics common to all
freshwater lentic systems. Since many kinds of organisms are absent
from the microcosms, commercially important species of fish and
shellfish should be tested independently. The test outlined in
this protocol should be used as a first approximation in identifying
microbial agents that are persistent and/or potentially harmful to
environmental integrity.
2. SUMMARY OF TEST
The purpose of the microcosm test is to determine fate and
ecosystemlevel effects of agents introduced into aquatic environments.
This test assumes that certain ecosystem-level functions are common
to all ecosystems and are not the result of specific taxonomic
groups present in a particular system.
2.1 Test systems. The test systems are aquatic micro-ecosystems
established in one liter beakers and maintained in an environmental
chamber.
2.2 Durations of test. A complete test requires twelve weeks.
Microcosms are constructed and allowed to develop and mature for
six weeks. The microbial agent is then added and monitored for an
additional six week period.
2.3 Measurement of effects. Mature microcosms are inoculated with
99
-------
varying densities of the agent to be tested. Effects of the
organism are determined through periodic measurements of pH, Eh
(redox potential), dissolved oxygen (DO) and 14C glucose decompositon.
These variables are all correlates to such ecosystem processes as
primary production, community respiration, secondary production,
decompositon and nutrient flux.
2.4 Determination of-survival rates. Survival rates of the organism
are determined through periodic enumeration of the agent.
2.5 Output. This test can provide estimates of minimum effect densities
and minimum survival densitites for the microbial agent tested.
It also provides data on survival rates and the magnitude of
environmental effect.
3. SIGNIFICANCE.
The data generated from this test can be used to develop a first
stage assessment of the ecological hazard an organism would present
to aquatic environments. The results of this test together with
other early assessment data can determine the need for further
environmental testing.
3.1 Importance of ecosystem level testing. Ecosystem level tests
provide an intermediate between single species effects tests and
full scale environmental tests of survival and effects. As
Schneider (1981) and others have noted, they yield a more
"ecologically realistic" estimate of fate and effects than do
single species tests.
3.2 Significance of variables measured. This protocol yields minimum
effect densities for the test microorganism expressed in terms of
the following ecosystem level parameters:- pH, Eh, net daytime community
100
-------
production (oxygen gain), night community respiration (oxygen
loss); and decomposition rate of orgnaic matter. The procedure
also determined the minimum densities necessary for survival of an
introduced microbe.
A deviation in pH and Eh may have significant impact on an
aquatic ecosystem. Changes in the equilibrium of chemical species
may alter normal,nutrient dynamics with resultant impacts on the
biota (Schindler, et aj_., 1980).
A deviation in the autrophic and heterotrophic components of
an ecosystem could alter food chains with resultant impact on
species important to_ human commerce and recreation. Because of
the laws of thermodynamics, energy is lost at each step of a food
chain. A decrease in autotrophy at the base of a grazing food
chain may ultimately lead to a reduction in game fish biomass
several levels removed. Similarly an increase in heterotrophy,
most notably by bacteria, may also decrease the food supply passing
to game fish at the top of detrital food chains. The decrease in
dissolved oxygen resulting from increased heterotrophy may also
adversely affect game fish such as trout.
A deviation decomposition rate could adversely affect the
nutrient cycles of an aquatic system by altering the mineralization
rates of organic matter. An excess of nutrients could lead to
algal blooms and lowered dissolved oxygen concentrations which
stress game fish of commercial or recreational importance. A
deficit in available nutrients could reduce primary productivity.
This effect could be passed through the food chain resulting in
reduced biomass of commercial or recreationally important organisms,
101
-------
4. DEFINITION OF TERMS.
4.1 Microcosm: A small laboratory aquatic ecosystem containing primary
producers, grazers, detritivores, and decomposers and displaying
the biogeochemical cycling found in natural ecosystems.
4.2 Ecosystem: A region of the physical environment and its associated
organisms.
4.3 Community: A unit composed of all the populations of different
species occupying a given ecosystem.
4.4 Population: A unit composed of all the individuals of a single
species within a given ecosystem.
4.5 Primary Producer: An appropriate plant species. An organism that
obtains its energy from sunlight through photosynthesis (i.e., an
autotroph). In these microcosms all the primary producers are algae.
4.6 Grazer: An organism which obtains its energy by feeding on primary
producers.
4.7 Heterotroph: An organism which obtains its energy from organic
compounds manufactured by primary producers.
4.8 Detritivore: An organism which feeds on dead oraganic material
and its associated microbial community.
4.9 Decomposer: An organism, usually a bacterium or fungus, which obtains
energy by breaking down the remains or waste products of other
organisms.
4.10 Benthic: A organism whose activities are primarily confined to
the substrate.
4.11 Planktonic: A small, nonmotile or weakly motile organism which
spends most of its time suspended in the water column.
of microbial test agent.
102
-------
4.12 Treatment: A group of microcosms treated with an identical density
4.13 Oxygen Gain: The daily increase in dissolved oxygen caused by algal
photosynthesis.
4.14 Oxygen Loss: The overnight decrease in dissolved oxygen in response
to plant and animal respiration.
4.15 Heterotrophic Activity: An indicator of the density of the
microbial population determined by the rate at which ^C glucose
is decomposed.
4.16 Minimum Effect Density: The lowest treatment level at which a
microbial agent causes a measureable ecosystem effect.
4.17 Minimum Survival Density: The lowest treatment level at which an
introduced microbial agent can survive in a microcosm system.
4.18 Maximum Hazard Test: A test to determine the effects of an MPCA on
a nontarget species when appled at several times the manufacturers'
recommended application rate.
5. APPARATUS.
5.1 Facilities
5.1.1 Growth Chambers. Microcosms are maintained in an environmental
chamber under controlled conditions in order to enhance replicability
and reproducibility.
5.1.2 Temperature Requirements. Temperature is maintained at 20° C within
a +_ 1° C range.
5.1.3 Light Requirements. The light cycle is set so that there are 12
hr of darkness and 12 hr of light daily.
Lighting is obtained from "cool light" fluorescent lights. The
lamps are arranged throughout the chamber to provide even lighting
at an intensity of about 500 f.c. (5381 lumens/M^).
Microcosms are distributed among all shelves of the chamber and
103
-------
are spaced to receive equal lighting.
One replicate microcosm from each treatment is placed on each
shelf of the chamber. This creates a randomized block design,
reducing variability caused by environmental gradients within the
chamber. All microcosms assigned to a particular shelf are randomly
situated on that shelf.
All microcosms on a particular shelf are moved to another shelf
within the chamber twice weekly. This rotation of microcosm within
the chamber at regular intervals also reduces within-chamber
variability.
Microcosms remain in the environmental chamber at all times except
during brief sampling periods.
5.2 Microecosystems
5.2.1 Vessels. The microcosm container is a one liter pyrex beaker
.'
covered with a large glass petri dish cover. Because of the lip
on the beaker, the system is not completely sealed and gas exchange
with the atmosphere can occur. The cover prevents contamination
and minimizes the chance for acccidental release of the agent
being tested.
Prior to use, the microcosm beakers should be washed according
to the procedure in 10.1.2.
5.3 Monitoring Instruments
5.3.1 pH and Eh. A pH selective ion meter with a multiple electrode
switch is used to measure pH and Eh. The meter should permit pH
readings to 0.01 pH unit and Eh readings to 0.1 millivolt. Glass
combination pH electrodes and glass metallic (Ag-AgCl/Pt) Eh
electrodes are used to determine pH and Eh of the microcosms.
104
-------
5.3.3 Dissolved Oxygen. A dissolved oxygen meter with two polarographic
electrodes is used to measure die! oxygen changes. The instrument
should permit DO readings to 0.1 mg/L.
5.3.4 Heterotrophic Activity. A liquid scintillation counter calibrated
for l^C counting is required for measuring heterotrophic activity.
An instrument with an automatic sample changer and hard copy
printout is recommended
5.3.5 Microscopic Examinations. A good quality microscope is required
to identify organisms in the microcosm inoculum. While species
counts are not a monitored parameter, assurance that specific
functional groups are present is required.
5.3.6 Microbial Agent Enumeration. Required equipment will vary depending
on the agent tested and the methods of enumeration.
6. REAGENTS AND MATERIALS.
6.1 Media
6.1.1 Water Quality. All water used in preparing media and replacing
evaporative loss from microcosm systems should be distilled and
deionized.
6.1.2 Stock solutions. The chemically defined medium is a modification
of Taub #82 (Taub and Read, 1982). Stock solutions are prepared
and stored at 4° C.
105
-------
Stock Solution
A
B
C
D
E
F*
G*
H*
IA
IB
Compound
NaN03
MgS04.7 H2)
KH2P04
NaOH
CaCl2 or
CaCl2.2H20
- NaCl
EDTA
NaOH
FeS04.7H20
EDTA
NaOH
H3B03
ZnS04.7H20
MnCl2.4H20
Na2Mo04.2H20
CuS04.5H20
Co(N)3)2.6H20
AL2(S04)3;18H20
Na2Si03.9H20
g/L Stock ml Stock/L Medium
8.5 5
24.65 1
13.6 1
3.2
11.1 20
14.7
5.84 30
26.1
10.7
24.9
29.0
12.0
1.854
0.287
1.98
0.024
0.049
0.291
3.2 1
4.55 5
* Combined to make stock solution J, consisting of:
25 ml. F stock solution
50 ml. H stock solution
6 ml. G stock solution
19 mL. distilled, deionized water
This stock (J) is added to the final medium at the rate of .05
ml per liter.
900 ml of medium are required for each microcosm beaker.
6.1.3 Media preparation. At the time microcosms are started the appropriate
proportions of stock solutions and distilled water are thoroughly
mixed in a large polypropylene container. When mixing is complete,
the medium will be quite alkaline and must be neutralized to pH
7.0 with 10 percent HC1.
106
-------
Ideally, the mixing container should be large enough to hold the
entire volume of media required. If this is not possible, the
media should be mixed in two separate batches, and 450 ml of each
batch added to each microcosm. This will ensure that the media
composition is identical in all microcosms.
6.2 Sediment
6.2.1 Type. Washed, fine-grained sand is used as a sediment. It
contains no nutient matter, and is used as a substrate for growth
and a refuge for organisms. Beach sand is acceptable if it is
clean and not mixed with organic matter.
6.2.2 Preparation. Each microcosm requires 50 ml of sand. The total amount
of sand required for a test is placed in a container, covered with
10 percent HC1 and mixed. After two hours, the acid is decanted
and the sand flooded with distilled water. The water is mixed with
the sand and poured off. This rinsing process is repeated until
the rinse water reaches a pH of approximately 7.0. After rinsing
the sand is oven dried at 75° C.
6.3 Stock Aquaria
6.3.1 Size. Stock communities of the organisms used in the test are
maintained in 38 liter all glass aquaria. The aquaria may be held
at room temperature and should be illuminated by a bank of
fluorescent lights. A sheet of glass may be used to partially
cover the top of the tank and retard evaporation.
6.3.2 Media. The stock communities are raised in T82, the same medium
as used in the microcosms. The bottom of the stock tanks should
be covered to a depth of approximately 10 cm with acid-washed
sand.
107
-------
6.4 Cleaning solutions. The stock aquaria and all microcosm vessels
are cleaned according to the procedures suggested by Struempler
(1973) and Leffler (1982). These procedures require laboratory
detergent, 200 ,g/L hypochlorite solution and 10 percent HC1 (see
section 10.1.2).
7. SAFETY PRECAUTIONS.
7.1 Use of Cleaning -Solutions. The solutions used in cleaning the
vessels should be handled with caution. Rubber gloves and goggles
should be worn during the cleaning process.
7.2 Avoiding Release of Test Organism. Of major importance in performing
these tests is the use of proper aseptic technique. In addition
to the recognized practices associated with the handling and
transfer of microorganisms the following precautions should be
taken.
7.2.1 Covering of test vessels. Microcosms are covered with petri dish
lids except during sampling. This minimizes the possibility of
airborne transfer of the test microorganisms.
7.2.2 Sanitizing monitoring probes. All monitoring probes should be
sanitized after use. The sanitizing procedure of choice will
depend on the microbe being tested.
7.2.3 Proper disposal of vessels and petri dishes. All used microcosm
vessels, petri dishes, stirring rods and other glassware which
come in contact with the tested agent microorganism should be
autoclaved prior to disposal or cleaning and reuse.
8. MICROBIAL AGENTS.
To determine minimum effect densities and minimum survival densities,
the microorganism tested should be inoculated into test microcosms
108
-------
at a range of cell densitites.
8.1 Determining Inoculation Densities. Determination of the cell
densities will be governed to some extent by the organism being
tested. If initial tests at maximum hazard cell densities indicate
no effects and no survival of the organism, further tests at lower
concentrations are probably not required. If either effects or
survival are sees at maximum concentrations, further tests should
be performed to determine minimum effect and survival densities.
9. TEST ORGANISMS
9.1 Establishing the stock tank community.
9.1.1 Aquaria specifications. Stock cultures for inoculating microcosms
are maintained in 38 L all-glass aquaria. These tanks may be kept
in the laboratory at room temperature. Several banks of "cool
white" fluorescent lights suspended over the tanks are
* •
controlled by a 12 hr light-12 dark time switch.
9.1.2 Preparing the stock culture. To prepare a stock culture, the tank
is first thoroughly cleaned and rinsed (6.4). Washed sand (6.2.2)
should be added to a depth of 1 - 2 cm and the tank filled with
approximately 36 L of microcosm medium (6.1.2). Samples collected
from natural ecosystems (approximately 2 L) should then be added to
the aquarium.
At least one new stock culture should be prepared every six months
to ensure a healthy, growing system. Inoculum from older stock
cultures in addition to new "wild" material should be used to
start a new culture.
A stock culture may be used for several years unless obvious
changes suddenly occur in the the system. Periodic additions of
109
-------
nutrient medium will aid in maintianing a healthy culture. In
preparing inoculum for microcosms samples from several different
aged stock cultures should be mixed together. No stock culture
less than three months old should be used as a source of microcosm
inoculum.
Distilled water is added to the stock cultures as needed to replace
evaporative lossss.
9.1.3 Source of organisms. Samples taken from natural ecosystems are
used to start the stock cultures. A variety of system should be
sampled in order to ensure a diversity of species in the stock
cultures. Suitable sources include small ponds, marshes, vernal
pools or other standing water sources.
9.1.4 Required functional groups. The following minimum criteria must
be met in terms of species composition in the stock cultures. The
diversity will most likely be far greater than this minimum.
(a) two species of single-celled green algae or diatoms.
(b) one species of filamentous green alga.
(c) one species of nitrogen-fixing blue-green alga.
(d) one grazing macroinvertebrate.
(e) one benthic, detritus-feeding macroinvertebrate.
(f) bacteria and protozoa species.
9.2 Evaluation of stock tank cultures. Stock tank cultures should be
examined periodically to verify the presence of the required
functional groups. Any groups that are missing should be added
either by taking samples from other stock tanks or collecting fresh
natural material.
If algae in the stock tanks appear to be-in poor condition (e.g.
110
-------
showing shrunken chloroplasts or reduced chlorophyll content)
additional nutrient medium should be added.
9.3 Minimum time requirement for "co-adaption." The stock communities
are allowed to mature for at least three months prior to use as a
source of inoculum for the microcosms. This development period
allows a relatively stable species assemblage to develop. Those
species which are unable to coexist with other members of the
community disappear, leaving behind a "co-adapted" group of species.
10. PROCEDURE.
10.1 Setup
10.1.1 Size of experiment - Number of microcosms. The number of treatment
groups will depend on the number of cell concentrations to be
tested. A typical chemical test requires 30 microcosms. These
are divided into seven treatment groups (a control and five test
groups) each containing five replicates. To ensure that the group
of 30 is uniform at the time of treatment, a larger group of 35
microcosms is started and culled to 30 (see 10.4).
10.1.2 Vessel preparation. Microcosm vessels are washed five times with
tap water, five times with 200 mg/L hypochlorite solution, five
times with 10 percent HC1, and five times with distilled water.
The cleaned vessels are kept inverted in a dust-free environment
until needed.
10.1.3 Sediment. Each microcosm receives 50 ml of acid-washed, neutralized
and oven dried (see 6.2.2) sand.
10.1.4 Media. Each microcosm recieves 900 ml of neutralized T82 medium
(see 6.1.2).
10.1.5 Inoculation. The stock culture aquaria are stirred gently to bring
111
-------
all particulate matter into suspension. A 1 L beaker is used to
remove aliquots from each stock culture. These aliquots are
combined in a 6 L polypropylene container. Transfers are handled
gently and never poured through the air because of injury to the
organisms.
Prior to inoculation, verify the presence of each of the
required functional groups in this mixed stock culture. Following
this, microscopic counts, using a Palmer counting slide (Wetzel
and Likens, 1984), should be made to determine the algal density
in the mixed stock. The algal density should be approximately
10^ cells per mL. If necessary, adjust the inoculum to this
concentration with either distilled water or aliquots of algae
from more densely populated stock aquaria.
A stir bar is added to the 6 L container which is placed on
a stir plate. Stirring is just sufficient to maintain particulates
in suspension without injuring the organisms.
A 50 ml beaker is used to remove 50 ml aliquots from the 6 L
container. These are carefully transferred to each beaker containing
50 mL of sand and 900 mL of medium. Again care is taken in the
transfer not to injure the organisms.
Each microcosm is assigned a number which is marked on the outer
surface of the beaker.
The microcosms are placed in the environmental chamber such that
no two replicates of the same treatment are located on the same
shelf. This creates a randomized block design.
10.1.6 Cross-seeding. Twice weekly for three weeks following inoculation
all microcosm are cross-seeded.
112
-------
Each microcosm is stirred gently and a 50 mL beaker is used to
remove a 50 ml from the 6 L container and return them gently to
each microcosm.
10.1.7 Reinoculation. Throughout the life of the microcosm, each is
reinoculated weekly using the procedure outlined. 10.1.6.
Following the weekly reinoculation distilled water is added to
each microcosm t« return its volume to 1000 mL.
10.2 Measurements. Ecosystem level effects are measured through periodic
monitoring of the variables listed below. It is important that all
measurements be made at at the same time of day during each sampling
period. The following results must be reported for each microcosm:
(a) pH value to 0.1 pH units
(b) pE value to 1 millivolt
(c) Rate of oxygen gain 0.01 mg02/L/hr.
(d) Rate of oxygen loss 0.01 mg02/L/hr.
(e) Heterotrophic Activity in dpm/15ml_/2 hr.
(f) Population density of the microbial agent.
If the growth chamber lights are set to come on a 8 AM and off at
8 PM, the morning measurements should be started at approximately
6:30 AM, or early enough to finish before the lights come on.
Afternoon measurements can be started at approximately 4:30 PM,
while the lights are still on. The light regime must be a 12 hr-
on 12 hr-off schedule, but the on-off times can be shifted from 8
AM and 8 PM. If such shifts are made, sampling times should be
adjusted accordingly.
10.2.1 Dissolved Oxygen. Oxygen gain and loss are estimated by the three-
point diel dissolved oxygen method (McConnell, 1962; Abbott, 1966;
113
-------
Leffler, 1978; and Hendrix et al., 1981). Measurements are
scheduled such that morning and afternoon readings are taken on
the first day of a sampling period, and a second morning reading
is taken on the following day. The dissolved oxygen electrode
must be standardized against air-saturated water before each sampling
session. The procedures are given by the American Public Health
Association (1975).
To measure oxygen levels in the microcosms, the oxygen electrode
is inserted into the center of the microcosm and gently stirred
until the readings stabilize. A separate electrode should be used
for the control group (10.6.2).
The electrodes should be thoroughly rinsed with distilled water
before being moved from one microcosm to another.
10.2.2 pH and Redox potential (Eh). Before each sampling session, the pH
and Eh electrodes should be standardized. A separate set of
electrodes must be used for the control group.
Phosphate buffer of pH 7 is suitable for calibrating the pH electrode.
The glass metallic combination (Ag-AgCl/Pt) Eh electrode may be
standardized against the solution described by Light (1972). Both
of these standards are of relatively high ionic strength and tend
to slow the response of the electrodes to conditions in the
relatively low ionic strength of the microcosms. Accordingly,
both electrodes should be held in an extra microcosm until readings
stabilize before beginning the actual sampling.
A glass combination pH electrode is inserted into the microcosm
such that its tip is in the center of the beaker halfway between
the bottom and the surface. The microcosm is not stirred. The
114
-------
electrode is allowed to equilibrate, usually 3-5 minutes, before
the reading is taken. Otherwise the procedures follow standard
practice (American Public Health Association, 1975).
The Eh electrode is inserted into the microcosm such that
its tip is in the center of the beaker halfway between the bottom
and the surface. The microcosm is not stirred. Readings usually
stabilize in about 15 seconds.
Since Eh is influenced by pH, Eh values should be corrected to
standard pH 7 (Wetzel, 1983, p. 299) by the relationship E7 (mv) =
Eh (mv) + 58 (pH - 7). The electrodes should be thoroughly rinsed
with distilled water before being moved from one microcosm to
another.
10.2.3 Heterotrophic Activity. Heterotrophic Activity is measured by the
14C method described by Wright and Hobbie (1966) and Thomas (1985).
Sampling is achieved by placing the microcosm on a stir plate.
A stir bar is inserted and rotated at approximately 60 rpm. A
wide mouth pipet is inserted so that its tip is halfway between
the center of the beaker and the wall and halfway between the
bottom and the surface. A 15 mL sample is withdrawn. If sampling
occurs on reinoculation days, it should precede the reinoculation
procedure.
A 0.3 ml aliquot of 14C-glucose (0.15 uCi) is added to a 15 ml
sample from each microcosm. After a 15 minute incubation period,
the sample is acidified and incubated for two hours during which
C02 is trapped on a strip of filter paper saturated with a C02
absorbing agent. The ^C radioactivity of the paper is then
counted by liquid scintillation.
115
-------
The stir bar and pipet should be thoroughly rinsed with distilled
water when they are moved from one microcosm to another.
10.2.4 Population density of MPCA or GEM. Measurement of survival of the
microbial agent is critical. The enumeration system will vary
with the organism being evaluated so specific procedures cannot
be given here.
10.3 Pre-treatment monitoring. Microcosms should be sampled twice
during the six week development phase. All variables (10.2) should
be measured in each microcosm on day 31 and day 38 of the pre-
treatment period.
In addition, well stirred samples should be withdrawn from each
microcosm and examined microscopically to verify the presence of
the required functional groups.
10.4 Culling Procedure. To improve replicability, the initial group of
microcosms must meet a set of quality control criteria. Microcosms
failing to meet these criteria or varying significantly from the
rest of the group are culled before treatment.
10.4.1 Failure to achieve minimum quality control standards. Microcosms
failing to meet the minimum quality control cirteria (Sec. 11) for
successful development are removed and not used in the test.
10.4.2 Calculation of group means. After removal of any microcosm for
failure to meet minimum criteria, any remaining extra systems are
culled by calculating the group mean for each of the ecosystem-
level variables measured. Those microcosms showing the greatest
deviation from these means are culled until the remaining group is
reduced to the desired size (usually 35).
The remaining set of systems is randomly assigned numbers, and
116
-------
arranged into treatment groups of five replicate microcosms.
The remaining set of systems is randomly assigned numbers, and
arranged into treatment groups of five replicate microcosms.
10.5 Treatment
10.5.1 Technique for adding toxic agent. The method of adding the test
organism will vary depending on its formulation. It may be added
in dry form or djluted in a series of sterile blanks. If media
dilution blanks are used, an equivalent amount of sterile media
must be added to each contol microcosm.
10.6 Post-treatment monitoring.
10.6.1 Schedule. Microcosms are normally monitored for six weeks following
the additon of the test organism. For the first two weeks all
ecosystem-level variables (Sec. 10.2) are measured twice a week
(i.e., on days 2, 4, 9, and 11) with the exception of heterotrophic
activity which is measured once each week. Thereafter, all
variables are measured once a week on days 18, 25, 32, and 39.
If no deviations from controls are observed in any treatment being
tested for a two week period the monitoring may be terminated prior
to the end of six weeks.
Determinations of the survival of the microbial agent should be
made once a week on a schedule corresponding to that of the
heterotrophic activity measurements.
10.6.2 Use of separate set of control and treatment electrodes. To avoid
contamination of the controls with the microbial agent, a separate
set of electrodes should be used for all oxygen, pH, and Eh
measurements on controls.
10.6.3 Sterilization of equipment. Waste disposal. Liquid wastes generated
117
-------
through test organism additions and 14C methodology must be
collected and disposed in accordance with EPA, NIH, and NRC
regulations.
Contaminated solid wastes such as biomass, filters, charcoal, ion
exchange resin, vermiculite, glassware, gloves, etc., must be
packaged and disposed in accordance with EPA and NRC regulations.
11. QUALITY ASSURANCE.
To assure that a test has been successful, the following criteria
must be met:
11.1 Pre-treatment criteria. All functional groups should be present
in the systems at the last pre-treatment measurement.
By the end of the development period the morning dissolved oxygen
levels should not fall below 4.0 mg/L and evening dissolved oxygen
levels should reach,at least 11 mg/L.
11.2 Post-treatment criteria. If two control microcosms become
significantly different from the rest of the control group, for any
variable, that control should be discarded and not used for any
calculations.
If the microbial agent appears in two or more of the controls
the experiment should be aborted.
If the microbial agent appears in one of the controls, that system
should be discarded and not used in any calculations.
12. DATA ANALYSIS.
12.1 Determination of minimum effect. If the microbial agent was tested
at several initial cell densities and at least one of the treament
groups showed no significant effect a minimum effect or no effect
concentration should be estimated.
118
-------
12.1.1 Dunnett's procedure. Dunnett's procedure (Steel and Torrie, 1960)
is used to determine which, if any of the treatments are different
from the control (p < = 0.5). This test should be performed on
each variable measured for each sampling day. This permits a
determination of which variables are significantly affected and
the duration of the effect.
These calculations may be summarized by plotting the difference
(or deviation) of each treatment group mean from the control mean.
In these figures the control mean appears as a straight line
centered at zero. An area corresponding to the upper an lower
bounds of Dunnett's significant difference (p < .05) is plotted
around the zero line (control mean) and defines a region where the
various treatments are not significantly different from the control
Points lying outside the shaded area are significantly different
(p < .05) from the control.
From these calculations, the minimum and no-effect concentrations
of the microbial agent are determined.
12.2 Determination of Organism Survival. From the periodic counts of
the density of the microbial agent, determinations of the survival
rate of the organism can be reported.
References
Abbott, W. 1966. Microcosm studies on estuarine waters. I. The
replicability of microcosms. J. Water Poll. Cont. Fed. 39:258-270.
American Public Health Association. 1975. Standard Methods for the
Examination of Water and Wastewater. American Public Health Assoc.,
Washington, DC, 1193 pp.
119
-------
Betz, F., M. Levin, and M. Rogul. 1983. Safety aspects of genetically-
engineered microbial pesticides. Rec. DNA Tech. Bull. 6:4 pp 135-141.
Environmental Protection Agency. 1979. Toxic Substance Control
Act: Premanufacture testing of new chemical substances. Federal Register,
March 16, 19079, pp. 16240-16292.
Environmental Protection Agency. 1985. Freshwater Aquatic Invertebrate
Toxicity and PathogenicJty Testing: Tier 1 ln_ FIFRA Regulations, Subdivision
M, Section B: Microbial Testing. Washington, DC.
Hendrix, P.F., C.L. Langer, E.P.Odum, and C.L. Thomas. 1981.
Microcosms as Test Systems for the Ecological Effects of Toxic Substances:
An Appraisal with Cadmium. Final Report of EPA Grant No. R805860010.
U.S.E.P.A. Environmental Research Laboratory, Athens, Georgia.
Leffler, J.W. 1978. Ecosystem response to stress in aquatic
microcosms. In Energy and.Environmental Stress in Aquatic Systems. J.H.
Thorp and J.W. Gibbons (eds.), DOE Symposium Series, Augusta, GA, Nov. 2-
4, 1977. National Technical Information Service, Springfield, VA.
Leffler, J.W. 1982. Microcosms and Stress Criteria for Assessing
Environmental Impact of Organic Chemicals. Final Report, Battelle
Columbus Laboratories, Columbus, Ohio.
Mallory, Larry M., Chang-Soo Yuk, Li-Nuo Liang, and Martin Alexander.
1983. Alternative prey: A mechanism for elimination of bacterial species
by protozoa. Appl. and Envr. Micro. 46:5 pp. 1073-1079.
McConnell, W.J. 1962. Productivity relations in carboy microcosms.
Limnol. and Oceanogr. 7:336-343.
National Academy of Sciences. 1981. Testing for Effects of
Chemicals on Ecosystems. A Report by the Committee to Review Methods for
Ecotoxicology. National Academy Press, Washington, DC, 98 pp.
120
-------
Schindler, J.E., J.B. Waide, M.C. Waldron, J.J. Mains, S.P. Schreiner,
M.L. Freedman, S.L. Benz, D.R. Pettigrew, L.A. Schissel, and P.J. Clark.
1980. A microcosm approach to the study of biogeochemical systems. 1.
Theoretical rationale. In Microcosms in Ecological Research, J.P. Giesy
(ed.), DOE Symposium Series, Nov. 8-10, 1978. Augusta, GA, National
Technical Information Service, Springfield, VA.
Schneider, R.A. 1981. Classes of ecotoxicological tests: their
advantage and disadvantages for regulation. In Working Papers Prepared
as Background for Testing for Effects of Chemicals on Ecosystems. National
Academy of Sciences, National Academy Press, Washington, DC.
Steel and Torrie. 1960. Principles and Procedures of Statistics
McGraw-Hill New York. 481 pp.
Stotsky, G. and H. Babich. 1984. Fate of genetically-engineered
microbes in natural environments. Recom. DNA Tech. Bull. 7:4 pp 163-188.
Struempler, A.W. 1973. Adsorption characteristics of silver, lead,
cadmium, zinc, and nickel on borosilicate glass, polyethylene, and
polypropylene container surfaces. Anal. Chemistry. 45:2251-2254.
Taub, F.B. and P. Read. 1982. Standardized Aquatic Microcosm Protocol.
Final Report, Vol. II. FDA Contract 223-80-2352.
Thomas, C.L. 1985. Development of a Test System for Screening
Toxic Substances; A Comparison Using Organic Substances. Ph.D. Dissertation.
Virginia Polytechnic Institute and State University, Blacksburg, VA. 183
pp.
Wetzel, R.G. 1983. Limnology. Saunders College Publishing.
Philadelphia, PA. 767 pp.
Wetzel, R.G. and G.E. Likens. 1984. Limnological Analyses. W.B.
Saunders Co. Philadelphia, PA. 357 pp.
121
-------
Wright, R.T. and J.E. Hobble. 1966. The use of glucose and acetate
by bacteria and algae in aquatic ecosystems. Ecology. 47:447-464.
122
-------
METHODS FOR ESTABLISHING MICROCOSMS OF AQUATIC LITTORAL
ECOSYSTEMS FOR DETERMINING SAFE LEVELS OF AGENT EXPOSURE1.2
by
Paul J. Franco
Jeffrey M. Giddings^
Environmental Sciences Division
Oak Ridge National Laboratory
Oak'Ridge, Tennessee, 37831
^-Research sponsored by the Office of Health and Environmental Research,
U.S. Department of Energy, under Contract No. DE-AC05-840R21400 with
Martin Marietta Energy Systems, Inc.
^Publication No. 2750, Environmental Sciences Division, ORNL.
3Present Address: Springborn Bionomics, Wareham, Massachusetts 02571
123
-------
1. SCOPE.
This protocol describes procedures for establishing freshwater littoral
communities in a laboratory with materials collected from natural
ponds. The methods were developed to assess effects of chemical
toxicants on aquatic communities (4), however, the systems may be
adaptable to testing the potential impacts of microbial pesticdes on
freshwater ecosystems.
2. APPLICABLE DOCUMENTS.
2.1 1976 Annual Book of ASTM Standards, Part 31:
D888 Oxygen, dissolved in water
01067 Acidity and Alkalinity
D1125 Conductivity
D1126 Hardness
01293 pH
01426 Ammonia
3. SUMMARY.
Littoral ecosystems are established in 72 L glass aquaria using
sediments, water, and biota from natural ponds. The microcosms are
easily replicated and require little maintenance. These microcosms
are static systems (no inflow or outflow) and retain a functional
similarity to natural ponds for more than 6 months under artificial
lighting. In testing for effects of toxicants, at least five chemical
concentrations, a no-treatment control, and a solvent control (if a
carrier solvent is used) are recommended with replication. Chemical
(pH, hardness, conductivity, and dissolved oxygen) and biological
(e.g., zooplankton and bacterial densities, and chlorophyll
concentrations) are made periodically throughout the experimental
period. No-observable-effect-concentrations (NOEC) are determined
using Dunnett's procedure.
124
-------
4. SIGNIFICANCE.
Single-species laboratory tests are useful for measuring the
relative toxicity of chemical pollutants, but a priori they cannot
predict effects that may occur in natural environments. The
microcosms described here closely resemble, both structurally and
functionally, the ecosystems from which they are derived.
Responses of these systesm to chemical toxicants and microbial
pesticides are likely to mimic those of natural communities.
Such microcosms have been used in parallel experiments with outdoor
ponds to evaluate effects of organic contaminants on an aquatic
community (6). The results from these experiments confirm that
the microcosms may be used as surrogates for ponds in toxicity
tests.
The microcosms are easy to assemble, require minimal maintenance,
are replicable, and are stable for periods of at least 6 months.
Because the microcosms are established from natural ecosystems, the
nature of the microcosm communities will vary with the source. This
allows for site-specific evaluation of toxicant effects.
5. DEFINITION OF TERMS.
5.1 Agent - chemical or microbial toxicant for whose effects on aquatic
ecosystems are being measured.
5.2 Littoral - describing shallow-water habitats that are transitional
between terrestrial and deep-water habitats.
5.3 Net Primary Production - a measure of the net amount of carbon
fixed by the community. In the system described here, the carbon
fixation rate is estimated from the net rate of oxygen production
(oxygen produced - oxygen consumed), allowing for oxygen diffusion
125
-------
into or out of the microcosm.
5.4 Overnight Respiration - the amount of oxygen consumed by the
community during the 12 h dark period, allowing for oxygen diffusion.
In this protocol, the lengths of dark and light periods are equal
and overnight respiration is assumed to be equal to daytime respiration.
5.5 Ecosystem Metabolism - a measure of the energy balance within the
community. It is estimated based on the ratio of net primary
production (P) to overnight respiration (R). If P:R is greater
than 1.0, the system is autotrophic (fixes more energy than it
consumes). If P:R is less than 1.0, the system is heterotrophic
(consumes more energy than it fixes). Ecosystem metabolism exerts
a strong influence upon chemical conditions within the system.
6. APPARATUS.
6.1 Facilities. Microcosms may be set up in either an environmental
chamber or a laboratory area with restricted traffic, to minimize
disturbance. Temperature should be kept relatively constant since
the systems will respond to rapid temperature fluctuations.
6.2 Lighting. Wide-spectrum fluorescent lamps are used to supply 150 to
250 uE m2 s'1 (1050 to 1900 foot-candles) of photosynthetically
active radiation (400 to 700 nm) with a 12-h daily photoperiod.
As is shown in Figure 1, nine lamps (e.g., Westinghouse Colortone
50, high output) will provide sufficient light for ten microcosms,
with minimal hotspotting.
6.3 Aquaria. The microcosms are contained in 72-L glass aquaria
(40 cm deep x 60 cm long x 30 cm wide). (Smaller microcosms are
difficult to replicate; larger ones take up too much space.)
Before use, aquaria and all other glassware are cleaned using a
126
-------
nonphosphate detergent and are rinsed successively with dilute
HC1, saturated N32C03, and distilled water.
6.4 Sampling Tubes. Glass tubes, about 40 cm long and with an
inner diameter of 2 cm, are used to collect water samples.
6.5 Delivery System. Chemical toxicant solutions can be introduced
into the microcosms using an adjustable volumetric pipette.
A delivery system will be developed for each microbial agent.
7. MATERIALS.
7.1 Sediment. Sediment is collected from the upper 10 cm of the bottom
of a natural pond. In shallow ponds, the sediment may be collectd
using a shovel. In deeper ponds, an Ekman dredge can be used. It
is best to collect sediments when ambient water temperatures are
within 10°C of the temperture at which the experiment will be
conducted to avoid exposing biota to temperature shock.
7.2 Water. Water is collected into clean plastic carboys from an
undisturbed area of the pond in the same general location from
which the sediment is taken.
7.3 Plants. Macrophytes and/or filamentous algae are collected from
the same area of the pond from which sediment and water were taken.
Care should be taken to prevent overexposing of the plants to
sunlight.
7.4 Agents. Chemical toxicants should be of reagent grade. Solutions
of the toxicant should be made using distilled water or reagent-
grade solvents. For microbial agents, the strains tested should be
of the same degree of purity and activity as those for whose
release is anticipated.
127
-------
8. PRECAUTIONS.
8.1 Some chemical and microbial agents have the potential to affect
humans adversely if precautions are not taken. Therefore, contact
with all these materials should be minimized. Information on
toxicity to humans and recommended handling procedures should be
studied before tests are begun.
8.2 Care should be taken to ensure that large crustaceans, (e.g.,
crayfish) are excluded from the microcosms. The burrowing activities
of these animals will severely disrupt the microcosms.
9. TEST ORGANISMS.
9.1 The organisms that would comprise a microcosm community depend
upon the source of materials. The dominant organisms in microcosms
described in reference 3 were El odea canadensis (macrophyte); Spi rogyra
sp., Gonium sp., Euglena sp., and Phacus sp. (algae); Alona costata,
Chydorus sphaericus, and Simocephalus vetulus (cladocerans); and Cyclops
spp., Eucyclops agilis, and Macrocyclops albidus (copepods).
10. PROCEDURES.
10.1 Microcosm Construction. Up to twenty microcosms can be assembled
by two persons in about 8 h. Sediment is collected and sieved to
remove large debris and stones. The sediment is distributed
equally among the aquaria until each has at least a 5-cm layer
(~10 L per microcosm). The aquaria are filled with pond water to
within 2 cm of the top. The water should be added so as to prevent
undue disturbance of the sediment. A simple device, such as an
inverted beaker or a plastic bottle with holes punched around the
bottom, can be used to dissipate the force of the water. If
sufficient care is taken, any suspended sediment will settle
128
-------
overnight. Once the water column is clear, about 100 g (drained
weight) of macrophyte or algae is put into each aquarium, and a
portion of each plant is gently pushed into the sediment to anchor
it in place. All other biota enter the microcosm along with the
sediment, water, or plants.
About 7 to 14 d are required for the microcosms to reach an
equilibrium. Cross-inoculation during this pretreatment period
improves replicability among microcosms.
10.2 Temperature. The temperature range selected will depend upon the
communites and conditions being modeled. Caution should be taken
to avoid rapid fluctuations in temperature that might perturb the
microcosms.
10.3 Experimental Design. To measure ecosystem responses to chronic
exposure and determine safe levels of chemical toxicants, it is
necessary to adequately define an exposure-response curve. Standard
practice is to use a geometric series of five exposure levels and
a no-treatment control (if a carrier solvent is used for the
toxicant, a solvent control should also be tested). Each exposure
level is replicated at least once, preferably two or more times.
The microcosms are monitored during all phases of the experiment:
pretreatment, to determine attainment of equilibrium; treatment,
to measure responses and post-treatment, to evaluate recovery.
10.4 Application of the Agent. For chemicals, the toxicant is dissolved
either in water or an organic solvent (usually methanol). Appropriate
volumes of toxicant solution are injected below the water surface,
using an adjustable volumetric pipette, according to a predetermined
schedule. The pipette is moved around the microcosm during the
129
-------
application to promote distribution of the toxicant. Methods of
applying microbial pesticides will be developed for each agent.
10.5 Water Chemistry. Conductivity and water temperature are measured
in situ three times a week, using commercially available portable
instruments. Samples integrating chemical conditions throughout
the water column are collected by vertically immersing a glass
sampling tube touwithin a few centimeters of the sediment. The
top of the tube is sealed with a stopper and the tube and its
contents are lifted out of the microcosm. Samples taken from two or
three locations within the microcosm are sufficient to characterize
the water chemistry. Hardness, ammonia, pH, and other parameters
are measured from such samples twice weekly.
10.6 Dissolved Oxygen. Dissolved oxygen (DO) is measured using a DO
meter twice daily: at its peak concentration (at the end of the light
period) and its lowest concentration (at the end of the dark period).
10.7 Zooplankton samples. Zooplankton are collected by rapidly submerging
a 2-L beaker into a microcosm. The zooplankton are concentrated
in a Wisconsin-type plankton bucket equipped with 80 micron mesh, stained
with methylene blue, narcotized with Neo-Synephrine hydrochloride,
and preserved in sucrose-formaldehyde solution. Zooplankton are
counted and identified to genus or species. Whole samples are counted
unless debris or high zooplankton density make subsampling necessary.
10.8 Planktonic Bacteria. Once a week 0.2 to 0.5 ml aliquots of 100 mL
samples are stained with acridine-orange fluorescent dye and
filtered through 0.4 urn polycarbonate filters. Filters are examined
by epifluorescent microscopy (2). Bacteria in twenty 2500-um2
fields are counted.
130
-------
11. CALCULATIONS.
11.1 Ecosystem metabolism is estimated from diurnal changes in DO (5).
Net primary production (P) and overnight respiration (R) are
calculated by:
P = A - 12 (D) and R = B + 12 (D)
where A is the increase in DO during the day, B is the decrease in
DO at night, and-D = the rate of oxygen diffusion into the microcosms
(units for A, B, P, and R are mg oxygen per L water per hr). The
rate of oxygen diffusion is related to the percentage saturation and
can be estimated by measuring the rate at which DO concentration
increases in an aquarium filled with sterile, oxygen-depleted
water.
11.2 Weekly means of parameters at each toxicant concentration are compared
with control means ysing Dunnett's method (1). Toxicant concentrations
at which responses are not significantly different from control
values are NOECs.
12. QUALITY ASSURANCE.
Because determination of toxicant effects is based on comparison of
treated microcosms and controls, it is necessary to establish stable
control conditions with as little variance as possible among control
replicates. Several factors impact the replicability of microcosms
and care should be taken to consider these during the conduct of the
experiment. Collection, transportation, and distribution of water,
sediment, and biota should be done in such a manner as to minimize
stress on plants and animals. Even distribution of collected
materials during set-up and cross-innoculation during the pretreat-
ment period will help reduce variability among microcosms.
131
-------
Adequate and uniform lighting will promote development of similar
macrophyte communities. Because these microcosms are sensitive to
rapid fluctuations in temperature, ambient air temperature should
be kept to within 3°C of the selected experimental temperature.
13. REPORT
The results reported should include the following:
13.1 Name of the test^. investigator, laboratory, and test date.
13.2 A brief description of the toxicant or agent, including its source
and lot number or other identifying features.
13.3 A description of the source of the sediment, water, and biota;
the methods of collection; and the date materials were collected.
13.4 A detailed description of the communities that develop in the
microcosms, including a list of the dominant species in each and
any variations among microcosms.
13.5 A description of the significant or observable responses at each
treatment level, including the NOEC.
REFERENCES
Chew, V. 1977. Comparisons Among Treatment Means in an Analysis of
Variance. U.S. Department of Agriculture, Agriculture Research Service,
ARS/H/6, Washington, DC.
Daley, R.J. 1977. Direct Epifluorescence Enumeration of Native
Aquatic Bacteria: Uses, Limitations, and Comparative Accuracy. In
Native Aquatic Bacteria: Enumeration. Activity, and Ecology, J.W.
Costerton and R.R. Colwell, Eds., ASTM STP 695, American Society for
Testing and Materials, Philadephia, PA, pp. 29-45.
132
-------
Franco, P.J. et al. 1984. Effects of Chronic Exposure to Coal-derived
Oil on Freshwater Ecosystems: I. Microcosms. Environ. Toxicol. Chem.
3:447-463.
Giddings, J.M. 1985. A Microcosm Procedure For Determining Safe
Levels of Chemical Exposure in Shallow-Water Communities. Presented at
the Symposium on Community Toxicity Testing, Colorado Springs, Colorado,
May 6-7, 1985.
Giddings, J.M. and G.K. Eddlemon. 1978. Photosynthesis/Respiration
Ratios in Aquatic Microcosms Under Arsenic Stress. Water Air Soil Pollut.
9:207-212.
Giddings, J.M. and P.J. Franco. 1985. Calibration of Laboratory
Bioassays with Results from Microcosms and Ponds. In Validation and
Predictability of Laboratory Methods for Assessing the Fate and Effects
of Contaminants in Aquatic-Ecosystems, T.P. Boyle, Ed., ASTM STP 865,
^ .
American Society for Testing and Materials, Philadelphia, PA, pp. 104-119.
133
-------
TERRESTRIAL SYSTEMS
AUTHORS:
Ramon J. Seidler
Lowell Etzell
Joe Gorsuch
Pete Van Von's
134
-------
"ENCLOSED SYSTEMS FOR TESTING
MICROBIAL PEST CONTROL AGENTS"
by
Terrestrial Systems Working Group
Ramon J. Seidler, EPA, Chairman
Lowell Etzel, U.C. Berkeley
Joe Gorsuch, Eastmen Kodak
Pete Van Voris, Battelle, N.W. Lab
135
-------
INTRODUCTION
The United States Environmental Protection Agency's Office of Pesticide
Programs is responsible for providing registrants with protocols regarding
risk assessment issues for microbial pest control agents (MPCAs). This
responsibility includes the new generation of MPCAs, microbes modified
by recombinant DNA or other technologies to produce genetically modified
microbial pesticides (GMMPs).
Enclosed multispecies test systems could offer a convenient means to
conduct exposure assessments in habitat-simulating fashion eliminating
the need for environmental release of GMMPs during the test evaluation
period. Some of the challenges in establishing protocols for the enclosed
multispecies test systems include the very limited database on effects
research involving GMMPs, the lack of appropriate bioassays for measuring
possible effects from GMMPs, and the modest experiences in maintaining
multispecies in enclosed test systems.
Virtually all that is available in the literature concerning multi-
species tests comes from toxicity evaluations involving the effects of
man-made chemicals on nontarget organisms. Information supplied by the
participants of this working group strongly reflects this literature
database. Information is provided on commercially available biota for
possible inclusion into an enclosed system and a considerable amount of
information is provided concerning the design and application characteristics
of one of the many terrestrial microcosms which have been described in
the literature. The greatest challenge of this working group has been to
determine the selection of nontarget species, the number and population
size to include in any enclosed test system, how those species may be
136
-------
maintained and for how long, and a lack of data to determine appropriate
end-points for measuring effects from MPCAs and GMMPs on nontarget species
and ecosystems.
ENCLOSED TEST SYSTEMS
A terrestrial microcosm can be defined as a confined portion of a
terrestrial ecosystem, subject to controls, that can be used to investigate
and evaluate ecologicaLprocesses (Van Voris, et al., 1985). Terrestrial
microcosms provide flexibility to the investigator in permitting the
control of numerous variables relevant to the determination of risk
assessment issues. Microcosms are more likely to reflect natural ecosystem
responses then most single species bioassays (Tolle, Arthur, and Van
Voris, 1983). Single species assays will not reveal potential perturbations
in population densities when certain predators, competitors, or ecosystem
functional activities are altered. For populations of several species to
exist in enclosed terrestrial microcosms, suitable trophic levels and
certain habitat simulating features must be maintained. If predatory
insects are enclosed for example, a suitable and consistent supply of
prey, of course, must be included in the system. This could complicate the
experimental microcosm if unusual nutritional requirements arise in
maintaining one or more predator/prey combinations. Furthermore, one
should expect fluctuations in predator/prey numbers as a function of each
species density, stage of growth, growth rate and conditions under which
the microcosm is maintained. How changes in beneficial prey cell numbers
can be specifically related to harm caused by the GMMPs will be a very
difficult issue to resolve since there are many factors which influence
their number.
Terrestrial microcosms come in numerous shapes and designs each
137
-------
offering the unique creativity of the designer and the novel purpose(s)
for which it is to be used (see Figs 1-9; Gillett, Witt, and Watt, 1979).
Discussions of this working group did not consider in any detail the
diversity of existing terrestrial microcosms or microcosm technologies.
Discussions of the Working Group centered predominantly on the Experimental
Terrestrial Soil-Core microcosm (TSCM) developed at Battelle, NW Laboratory
(Fig. 9). The advantages of this terrestrial microcosm are summarized in
the following items.
1. The TSCM has been adopted as a standard by the American Society
for Testing and Materials and is an EPA/OTS terrestrial microcosm test
protocol for evaluating toxicity of chemicals (Van Voris, 1985; Van
Voris, Tolle, and Arthur, 1985).
2. The TSCM is adaptable in terms of sizes of the intact soil core,
the number of soil core units which simultaneously can be replicated, and
in the enclosure design which uses HEPA filters.
3. The experimental design involves the incorporation of an undisturbed
soil core which maintains the natural biological and physical stratification
of the soil(Van Voris et al., 1985).
4. The TSCM can be used to evaluate a number of parameters relevant
to the risk assessment of GMMPs including: a) an evaluation of fate and
survival characteristics; b) genetic stability; and c) effects on nontarget
species and possible influences on various ecological processes such as
niche displacement and microbial functional activities such as nutrient
cycling.
The containment aspects of the TSCM have been anticipated in the
design which involves a Lexan® wall enclosure and a quartz glass top with
a stainless steel air-tight lower compartment to enclose the soil and
138
-------
microcosm tubes. The entire enclosed system is to be maintained under
slight negative air pressure and all air passing through the system will
be double HEPA filtered prior to exhaust. Two HEPA filters of laminar
flow grades with 99.97% and 99.99% efficiency would be installed in
sequence to insure some 8-log reduction in particles of 0.3 urn and larger
from moving out of the enclosure. Since virus particles are of the order
of 0.002-0.05 urn in size, an additional feature provides for installation
of an incinerator on the exhaust side of the microcosm to insure that
smaller particles would not escape.
Additional features of the enclosed TSCM include the use of glove box
type access ports and an ethylene oxide gas flush sample transfer chamber
(Fig. 9). Since the enclosed TSCM is on wheels it can be readily moved
between various environmental chambers. As designed, the TSCM meets the
BL-3 containment level guideline of the National Institutes of Health for
research with recombinant DNA. The estimated cost for construction of
two complete enclosed TSCM units is about $34,000. The dimensions of
such a unit are approximately 12 ft long, 4 ft high with about 32 cubic
ft of space within the enclosure to accommodate 12 soil core units. The
size of the individual cores is approximately 60cm deep and 20cm in
diameter. The depth is sufficient to allow for the establishment of
deep-rooted plants.
BIOTA FOR MICROCOSMS
A. Insects
Many resource materials were provided to the working group including
a listing of 20 groups of beneficial arthropods which might be included
in a terrestrial line missing details). Information provided includes a
listing of commercial insectaries, source materials, as well as methods
139
-------
for rearing both the beneficial arthropods and, as appropriate, the
corresponding prey or host species of the beneficial arthropod. The
original list of 20 beneficial arthropods provided in Appendix A was
reduced here to those that are most readily available and are easier to
propagate or maintain. A list which contains only a single representative
from each taxonomic group from Appendix A is summarized in Table 2.
Table 2. Systems of beneficial arthropods suggested for inclusion in
risk assessment testing of microbial pesticides.
SYSTEM BENEFICIAL SPECIES
#1 Amblyseius hibisci
#4 Geocoris punctipes
#5 Chrysoperla carneS
#7 01 la abduminalis
#9 Trichogramma sp.
#13 Lysiphlebus testaceipes
#14 Cotesia melanoscelus
#15 Metaphycus helvolus
#16 Aphytis lingnanensis
COMMENTS
a beneficial mite;
11 day life-cycle at 25C
big-eyed bug; a variety
of food will suffice
including cabbage looper;
34 day life-cycle at 26.7C
green lacewing;
24-27 day life-cycle at 26.7C
apparently only available
by field collection;
15 day life-cycle at 26C
parasite of insect eggs,
primarily lepidopterans
8-10 day life-cycle at 26.7C
parasite of the greenbug;
not active below 18.3C;
13 day life-cycle at 21C
parasite of gypsy moth;
15 day life-cycle at 23C
parasite of black scale
13 day life-cycle at
27 ,8C
parasite of California
red scale; 17 day life-cycle
at 26C
140
-------
Table 2. (Cont'd)
#17
Brachymeria intermedia
parasite of lepidopterans
15-30 day life-cycle at 24C
#18 Lixophaga diatraeae
#19 Apis mellifera-
#20 Vanessa cardui
fly parasite on sugarcane
borer; USDA mass produces;
18-20 day life-cycle at 26C
honeybee; freshly emerged
bees from a brood comb
should live the longest
in confinement (perhaps 30
days)
The only butterfly considered for
tests;feeds on over 100 plant
species; life of about 7 weeks
B. Plants and invertebrates.
The following list of plants and other invertebrates represents those
species commonly employed'"n enclosed microcosm test systems. The species
are readily available from various sources such as seed companies and
biological supply houses (See Appendix B).
Table 3. Plants suggested for inclusion in risk assessment testing
of microbial pesticides.
1. alfalfa
2. clover
3. oats
4. ryegrass
5. timothy
6. winter wheat
7. soybean
8. tomato
9. pine and Douglas
fir seedlings
10. corn
11. cotton
12. trifolium
141
-------
Table 4. Other invertebrates and non-beneficial insects suggested
for inclusion in risk assessment testing of microbial
pesticides.
1. cricket 3. earthworm 5. pillbug
2. nematode 4. collembola 6. millipede
Sources of the above biota are given in many of the references
appended and a noninclusive list of supply organizations is provided
in Appendix B.
142
-------
FACTORS TO CONSIDER IN THE DESIGN AND EXECUTION OF MICROCOSM TESTS.
In establishing the risk assessment of a GMMP, the working group
sees the need for duplicate enclosed microcosms. Both microcosms would
receive suitable nontarget species as well as the target (pest) species
of the GMMP. Only one microcosm would receive the GMMP itself. Possible
effects in the negative control (no GMMP) would be compared in severity
and number to those recorded in the experimental microcosm. One can
gauge the relative merits of the test by ascertaining rates of population
decline in the negative control microcosm. One may also use population
declines in the negative control to assess the level of species compatibility
and general"health" of the microcosm.
The materials used in the TSCM should reasonably reflect those at the
site destined to receive the GMMP in the small scale open field test.
Thus, the soil core shoultl be taken from this field site along with a
representative amount of natural vegetation.
The lists of flora and fauna previously listed above are intended to
provide flexibility to the agency program offices regarding selection and
requirements of further nontarget species. The number of different
species included in the microcosm tests should probably not exceed 5 to 7
added test species. The workgroup recommends that similar representatives
from the list of flora and fauna should be suggested to all registrants
for enclosed test trials for purposes of continuity of test evaluations
and consistency in agency requirements. The list of test species added
to the microcosm may include, as examples; a grass, broadleaf and cereal
grain (e.g., ryegrass, alfalfa, wheat), cricket, and earthworm as well as
two or three species from the list of beneficial arthropods. In addition
to the indigenous plant species taken with the soil core, these recommended
143
-------
species should provide a reasonable measure of species diversity for
nontarget tests. Higher species of organisms such as avian and mammals
cannot be satisfactorily maintained in this type of enclosed microcosm.
The possible effects of GMMPs on these species will be evaluated in
single species tests.
Inclusion in the microcosm of the intended target species of the
GMMP is strongly recommended by this working group. The target species
can serve as an internal control to verify the efficacy of the GMMP risk
assessment by making sure sufficient GMMP numbers were added to result in
an effective dose. The presence of infected target species will also
provide localized high concentrations of the GMMP which can therefore be
more "naturally" evaluated for ecological fate and hazards. If a pre-
determined population of target species is not eliminated, then the
enclosed microcosm risk assessment experiment is probably invalid.
The kinds of evaluations and duration of the exposure tests in the
enclosed microcosm will be influenced by the nature and number of the
species introduced into the enclosure. Thus, life-cycles of most beneficial
arthropods are greatly influenced by temperature and will be completed
within 3 to 4 weeks for most species. However, the three to four week
life-cycle should not be considered as a short-coming of the microcosm
evaluation system. The completion of life-cycles with sexual maturity
and egg laying, could become a part of the end-points selected for
assessment of risks.
Choices of stocking densities of target and nontarget species repre-
sent a challenge. Thus, stocking densities of plants may have physical
constraints and may be based upon common agricultural practices. However,
insufficient experiences do not allow more than best "estimates" for
144
-------
stocking densities of other species. In the 20 cm X 60 cm soil core, it
should be possible to maintain 4 to 8 earthworms for several weeks, without
significant disturbance of the ecosystem. On the other hand, densities
of suitable prey/host numbers will largely determine the population
density of beneficial predator/parasite that can be supported. In a
space of-32 cubic ft perhaps 50 specimens of each beneficial arthropod
can be maintained if onJy two or three species are used. Obviously prey
or host specimens must also be provided. This will necessitate adding to
the microcosm suitable nurse plants or other food sources for supporting
and maintaining viable prey or host specimens.
It seems most likely that the major benefit of conducting enclosed
testing will be the information gained on the ecological hazards as
opposed to hazards involving toxicity, virulence, and pathogenicity
effects on non-target species. The microcosm will provide a realistic
.. •
measure for testing the recovery of the GMMP from natural habitat material.
Furthermore, it will also be possible to gain information on the fate,
survival, ecological competitiveness, and genetic stability of the test
agent. The specific procedures for conducting these ecological risk
assessments are not available. However, identification, detection, and
fate analyses can be conducted by most microbial ecologists if appropriate
genetic tags and selective media are used to facilitate the detection of
the GMMP.
Specific recommendations for recovering microbes from plant, insect,
and invertebrate tissues, from soil, and the use of appropriate buffers,
blending conditions, etc. are beyond the scope of this document. Some of
these particulars will necessitate research and all will certainly require
trial and error efforts to optimize recovery. -Nevertheless, flora and
145
-------
fauna will be analyzed qualitatively and quantitatively for the GMMP.
These analyses should be conducted until two successive attempts to
recover the GMMP are negative. If the GMMP is found in association with
nontarget specimens, the biological nature of the association must be
determined. Thus, histopathology and/or electron microscopy coupled with
suitable-means to locate and identify the GMMP must be conducted. Evidence
should be provided so that it is obvious beyond any reasonable doubt that
the association of the GMMP with a nontarget species is transient and is
of no biological significance.
Measurement of effects of GMMPs on other microbes, microbial processes,
and activities is a current topic of research now being supported by this
agency. However, there are certain assays that can be conducted.to
assess ecological risks and EPA should consider these in evaluating risks
of new GMMPs. The TSCM is designed so that fluid leachate from soil
cores can be collected. Thus, GMMP cell densities and environmental fate
and survival including mobility through soil can be quantitatively measured
in these leachates. A basic investigation of the total microbial profile
in leachates collected from the experimental and control microcosms
should be made. A total bacterial plate count should be reported as well
as estimates of numbers of different colony types appearing on a standard
medium. A similar evaluation should be conducted for fungal populations.
These measurements can be used to evaluate impact of the GMMP on microbial
species diversity and population size.
Various mineral analyses can also be conducted on the leachate. Some
comparison of leachate chemical quality and quantity in the experimental
system must be made with.the negative control microcosm which did not
receive the GMMP. Specific analyses of the leachate should involve
146
-------
measurement of the solution pH plus organic carbon, total carbon, ammonia,
nitrate, and inorganic phosphate. Replication of analyses involving leachates
from different soil cores is appropriate.
Bibliography
Ausmus, B. S., D. R. Jackson, and P. Van Voris. 1979. The accuracy
of screening techniques. In: J. M. Witt and J. W. Gillett, eds. Symposium
on Terrestrial Microcosms and Environmental Chemistry, June, 1977, Corvallis,
OR. NSF/RA 79-0026.
Beall, M. L., Jr., R. G. Nash, and P. C. Kearney. 1976. Agroecosystem--a
laboratory model ecosystem to simulate agricultural field conditions for
monitoring pesticides. In: Environmental Modeling and Simulation, W.
Ott, ed. EPA-600/9-76-016. U.S. Environmental Protection Agency,
Washington, D. C. pp. 790-793.
Bezark, L.G., and H. Yee. 1985. Suppliers of beneficial organisms.
Calif. Dept. Food and Ag. Biological Control Services Program. Sacramento,
CA. 95832. pp. 1-6.
Cole, L. K., R. L. Metcalf, and J. R. Sanborn. 1976. Environmental
fate of insecticides in a Jerrestnal model ecosystem. Intern. J. Environ.
Studies. 10:7-14.
Oraggan, S. 1976. The microprobe analysis of 60-Cobalt uptake in sand
microcosms. Xenobiotica 6:557-563.
Gile, J. D., J. C. Collins, and J. W. Gillett. 1982. Fate and impact
of wood preservatives in a terrestrial microcosm. J. Agric. Food Chem.
30:295-301.
Gile, J. D. 1983. 2,4-D--Its distribution and effects in a ryegrass
ecosystem. J. Environ. Qual. 12:406-412.
Gillett, J. W., and J. D. Gile. 1976. Pesticide fate in terrestrial
laboratory ecosystems. Intern. Environ. Studies. 10:15-22.
Gillett, J. W., J. M. Witt, and C. J. Watt (eds). 1979. Symposium
on Terrestrial Microcosms and Environmental Chemistry. June, 1977,
Corvallis, OR. NSF/RA 79-0026.
Lichtenstein, E. P., T. W. Fuhrmann, and K. R. Schulz. 1974. Trans-
location and metabolism of 14C-phorate as affected by percolating in a
model soil-plant ecosystem. J. Agric. Food Chem. 22:991-996.
Lichtenstein, E. P., K. R. Schultz, and R. R. Liang. 1977. Fate of
fresh and aged soil residues of the insecticide 14C-N-2596 in a soil-corn-
water ecosystem. J. Econ. Entomol. 70:169-175.
147
-------
Lighthart, B., H. Bond, and B. G. Volk. 1977. The use of soil/litter
microcosms with and without added pollutants to study certain components
of the decomposer community. In: J. M. Witt and J. W. Gillett, eds.
Symposium on Terrestrial Microcosms and Environmental Chemistry, June,
1977, Corvallis, Or. NSF/RA 79-0026.
Metcalf, R. L., G. K. Sangha, I. P. Kapoor. 1971. Model ecosystem for
the evaluation of pesticide degradability and ecological magnification.
Environ. Sci. Technol. 5:709-713.
Tolle, D. A., M. A. Arthur, and P. Van Voris. 1983. Microcosm/field
comparison of trace element uptake in crops grown in fly ash-amended soil.
Sci. Total Environ. 31^242-261.
Van Voris, P. 1985. Standard guide for terrestrial soil-core microcosm.
ASTM Test Procedure. Draft for E47.08 subcommittee.
Van Voris, P., D. A. Tolle, and M. F. Arthur. 1985. Experimental
terrestrial soil-core microcosm test protocol. PB85-213338, EPA/600/3-85/047.
U.S. Environmental Protection Agency, Corvallis, OR. 97333.
Van Voris, P., D. A. Tolle, M. F. Arthur, and J. Chesson. 1985.
Terrestrial microcosms: applications, validations, and cost-benefit analysis.
In: J. Cairns, Jr., (ed.). Multispecies toxicity testing, Pergamon Press,
New York, pp 117-142.
148
-------
APPENDIX A
Included in Appendix A is a recommended list of beneficial arthropods
for enclosed systems testing. Provided with this material is information
for insect propagation, life-cycle characteristics, a list of suitable
prey/hosts, source materials and relevant literature citations. Also
included is a copy of the pamphlet, "Suppliers of Beneficial Organisms in
North America", published by the State of California, Department of Food
and Agriculture.
In the last section of the Appendix A is a copy of the Standard Guide
for Terrestrial Soil-Core Microcosm, that was submitted to ASTM by Dr.
Van Voris.
149
-------
Proposed Non-Target Terrestrial Organisms for Testing Microbial Pest Control Agents (L«Ka Etzel)
rbhropod-Plant Systems
Predaceous Mites
A* Acarina:Phytoseiidae
(1) Amblyseiua hlbisci
on Tetranychug urtioae
(2-spotted mite)
on lima bean plants
References Related to Rearing
Kennett & Hamai, 1960
(2) Metaseiulus occidentalie Ditto
on T. urticae
on lima bean plants
(3) HiytoseiuluB persimilis Ditto
on T, urticae
on lima bean plants
Sources for
Starting Colonies
CDFA listf
EntSoo list
Ditto
Ditto
Notes
A. hibiaci easily reared
on pollen (oak, magnolia,
& cattail pollens good)
Insect Predators
A. Heteroptera:Lygaeidae
(4) Geocoria punctipes
on noctuid-moth eggs
on corn plants
B. NeuropteratChrysopidae
(5) Chrysoperla earnea
(green laoewing)
on Acyrthosiphon pisxim
(pea aphid)
on fava bean plants
or alfalfa
, Coleoptera:Coccinellidae
(6) Hippodamia, convorgena
on A_. pi gum
on fava bean plants
or alfalfa
Cohen, 1981
Cohen & Debolt, 1983
Morrison, 1985a (for £. earnea)
Forbes et al.,1985 (for aphids)
Hagen & Sluas, 1966 (for
Hippodamia)
Forbes et al., 1985 (for aphida)
IhtSoo list)
Cohen (?)
CDPA listf
EntSoo list
CDFA list;
EntSoo list (for
aphid)
Common in North America,
Can be reared on
artificial diet.
-------
Arthropod~Plant Systems
(7) Olla abdominalis
on A_. pi sum
on fava bean plants
or alfalfa
(8) Cryptolaemus montrouzieri Fisher, 196*3
on Pseudococcidae
(mealybugs) on citrus
trees or oleanders
or potato sprouts
References Related to Rearing
!II. Insect Parasites
A. Hymenopterazlrichogrammatidae
(9) Trichogramma spp.
on Ostrinia nubilalis
(Eur. corn borer) eggs
or Heliothis zea (corn
eorworm) eggs
on corn plants
B. Hymenoptera:!fymaridae
(10) Anaphes flavipes
on Oulema melanopua
(cereal leaf beetle)
eggs on cereal grain
plants
C. gymenoptera:Aphidiidae
(11) Aphidius ervi or smithi
on A_. pi stan
on fava bean plants
or alfalfa
(12) Maretiella rapae
on Myzus persioae
(green peach aphid) or
Brevicorynae brassioae
(cabbage aphidl
on Chinese cabbage
or kale
Burbutis & Goldstein,
Morrison, 1985b
Anderson, 1968
UC-Berkeley (xinpubl.)
Forbes, et al.,1985 (for aphids)
Simpson et al., 1975
Sources Tor
Starting Colonies
Field collection;
EntSoc list (for
aphid)
CDFA list;
EntSoc list
Motes
Common arboreal species
in North America.
Artificial diet soon to
be developed.
CDFA list;
EntSoc list
EntSoc list
EntSoo List
Field collection;
EntSoo list (for
aphids)
Common parasite in
North America*
(V)
-------
Arthropod-Plant Systems
(13) Lysiphlebua testaoeipes
on SchizapMB graminum
(greenbug) on oat plants
D. HymenopterarBraconidae
(14) Coteaia (»Apanteles)
melanoacelus
on Lymantria dispar
(Gypsy Moth)
E, Hymenoptera:Enoyrtidae
(15) Metaphycus helvolus
on Saissetia oleae
(black scale)
on oleander
F. RymenopteratAphelinidae
(16) Aphytis melinus
Cor A. llagnanensia)
on Aspidiotis nerii
(oleander scale)
on butternut pumpkins:
(Cucurbita moschata)
G. Iftnnienoptera:Chal(tididae
(17) Braohymeria intermedia
on Trichoplusia ni
(cabbage loqper)
on cabbage or other
suitable plant
H. DipterazTachinidae
(18) Lixophaga diatraeae
on Diatraea saccharalis
(sugaroane borer)
on plants (?)
References Related to Rearing
Starks & Burton, 1977
Sources for
Starting Colonies
CDFA listf
EntSoc list
Chianese, 1985 (for C. mglan^ftCelua) EntSoo list
ODell et alM 1995 (for '"
PlanderB,
CDPA 'list
DeBach & White, 1960
Papacek & Smith, 1985
v
fecale )
Palmer, 1985 (for B. intermedia)
King & Hariley, 1985a (for Gallaria
mellonella)
Guy et alM 1985 (f^r T. ni)
King & Hartley. 1985>
L. diatraeaej
tog & Hartley, jt£05d
-------
Arthropod-Plant Systems
III. Other Beneficial or Innocuous
Insects
A, HymenopterarApidae
(19) Apis mellifera
(honeybee) In cage
with honey, water,
and plants (alfalfa or
mustard)
B. LepidopterajNymphalidae
(20) Vanessa cardui (painted
lady butterfly) on
host plants
Referenoea Related to Rearing
Sourcee for
Starting Colonies
Notes
Morton, 1979
Dietz, etal., 1976
Apiary;
Carolina Biol. Supply
Co.}
Wards Natural Science
Establishment, Inc.
Carolina Biol. Supply
Co.;
Wards Natural Science
Establishment, Inc.
The most widely distri-
buted butterfly. Hosts
include burdook, thistles,
sunflowers, nettles. The
references listed are for
artificial diets given in
Singh (1985).
-------
-5-
Proposed Non-Target Terrestrial Organisms
for Testing Microbial Pest Control Agents (L.K. Etzel)
I. General References
1. Bezark, L.G. and H. Yee
1985, 1985 Suppliers of Beneficial Organisms in North America. California
Department of Food, and Agriculture, Biological Control Services
Program, 3288 Meadowview Road, Sacramento, CA 95832. 6 pp.
2. Dickerson, V.A., J.B. Hoffman, E.G. King, N.C. Leppla, and T.M. OBell
1979. Arthropod Species in Culture in the United States and Other
Countries. Entomological Society of America, College Park, MD. 93 pp.
3. Singh, P. and H.F. Moore (eds.)
' 1985. Handbook of Insect Rearing. Volumes I and II. Elsevier, New
York. 488 pp. & 514 PP.
II. Specific References
4* Anderson, B.C.
1968. The biology and ecology of Anaphes flavipes. an exotic egg parasite
of the cereal leaf beetle. Ph.D. Thesis. Purdue University, 148 pp,
5. Burbutis, P.P. and L.F. Goldstein
1983. Mass rearing Trichogramma nubilale on European corn borer, its
natural host. Protection Ecology, 5_s269-275«
6. Chianese, R.
1985. Cotesia melanoscelus. In "Handbook of Insect Rearing" (P. Singh
and R.F. Moore, eds.), Vol. I, pp. 395-400, Elsevier, New York,
488 pp.
7. Cohen, A.C.
1981. An artificial diet for Geoooris -punctrpes (Say), Southwest.
Entomol., £:109-113.
8. Cohen, A.C. and J.V. Debolt
1983. Rearing Geocoris punctipes on insect eggs. Southwest. Entomol.,
8_:6l-64.
9. DeBach, P. and E.B. White
1960. Commercial mass culture of the California red scale parasite
Aphytis lingnanensis. Calif. Agric. Exp. Stn. Bull. No. 770, 58 PP.
10. Deitz, L.L., J.W. Van Duyn, J.R. Bradley, Jr., R.L. Rabb, V.M. Brooks,
and R.E. Stizmer
1976. A guide to the identification and biology of soybean arthropods in
North Carolina. Agric. Exp. Sta. Tech. Bull. 238, 264 pp.
11. Fisher, T.V.
1963. Mass culture of Cryptolaemus and Leptomastix. natural enemies of
citrus mealybug. Calif. Agr. Expt. Stn. Bull., 797:39.
-------
-6-
12. Flanders, S.E.
1942. Metaphycna helvolus, an encyrtid parasite of the black scale.
Jour. Econ. Entomol., ^5.: 690-698.
13. Forbes, A.R., B.D. Frazer, and O.K. Chan
1985. Aphids. In "Handbook of Insect Rearing" (p. Singh and R.F. Moore,
eds.), Vol. I, pp. 355-359. ELsevier, New York, 488 pp.
14. Guy, R.H., N.C. Leppla, and J.R. Rye
1985. Trichoplusia pj. in "Handbook of Insect Rearing" (p. qingh and
H.F. Moore, eds.), Vol. II, pp. 487-494. ELsevier, New Tork, 514 pp.
15. Eagen, K.S. and R.R. Sluss
1966. Quantity 'of aphids required for reproduction by HiTypodanii*. spp. in
tHa laboratory. Pp. 47-59. In "Ecology of Aphidophagoua Insects"
(I. Hodek, ed.), Dr. W. Junkr Publ., The Hague, Netherlands, 360. pp.
16. Kennett, C.E. and J. Hamal
1980, Oviposition and development in_predaceous mites fed with artificial
and natural diets (Acari:Phytoseiidae). Ent. Erp. Appl., 28;116-
122.
17* King, E.G. and G.G. Hartley
1985a. Galleria mellonella. In "Handbook of Insect Rearing" (P.Singh
and R.F. Moore, eds.), Vol. II, pp. 301-305, Elsevier, Bew York,
514 PP.
18. King, E.G. and G.G. Hartley
1985b. Libcophaga diatraeae. In "Handbook of Insect Rearing" (P. Singh
and R.F. Moore, eds.), Vol. II, pp. 119-123, Elsevier, New York,
514 PP.
19. King, E.G. and G.G. Hartley
1985c. Diatraea saccharalis. In "Handbook of Insect Rearing" (P. Singh
and R.F. Moore, eds.), Vol. II, pp. 265-270, Elsevier, Bew York,
514 PP.
20. Morrison, R.K.
1985a. Chrysopa carnea. In "Handbook of Insect Rearing" (P. Singh and
R.F. Moore, eds.), Vol. I, pp. 419-426, Elsevier, New York, 488 pp.
21. Morrison, ~R.K.
1985b. !Prichograwia spp. In' "Handbook of Insect Rearing" (P. Singh
and R.F. Moore, eds.), Vol. I, pp. 413-417» Elsevier, New York,
488 pp.
22. Morton, A.C.
1979. Rearing butterflies on artificial diets. J. Res. Lepid., 18_:221-
227.
23. ODell, T.M., C.A. Butt, and A.W. Bridgeforth
1985. Lymantria diapar. In "Handbook of Insect Rearing" (P. Singh and
R.F. Moore, eds.), Vol. II, pp. 355-367, Elsevier, New York, 514 PP.
-------
-7-
24. Palmer, D.J.
1985. Brachymeria intermedia. In "Handbook of Insect Hearing" (p. Singh
and H.P. Moore, eds.), Vol. I, pp. 383-393, ELsevier, New York,
488 pp.
25. Papacek, D.F. and D. Smith
1985. Aphytis lingnanensis. In "Handbook of Insect Hearing" (p. Singh
and R.F. Moore, eds.), Vol. I, pp. 373-381, ELsevier, New York,
488 pp.
26. Simpson, B.A., V.A. Shands, and G.V. Simpson
1973. Mass rearing of the parasites Praon sp. and Diaretiella ra-pae.
Aon. Qrfcomol. Soc. Am., 68:257-260.
27. Singh, P.
19&5. Multiple-species rearing diets. In "Handbook of Insect Hearing"
(P. Singh & H.F. Moore, eds.), Vol. I, pp. 19-44, ELsevier, New
York, 488 pp.
28. Starks, K.J. and R.L. Burton
1977. Greeribugs: determining bib-types. TISDA-ARS Tech. Bull., 1556:12.
-------
State of California
Department of Food and Agriculture
1985
SUPPLIERS OF BENEFICIAL ORGANISMS
IN NORTH AMERICA
Larry G. Bezark
Helen Yee
Biological Control Services Program
Division of Pest Management Environmental Protection and Worker Safety
"A beneficial mite attacking a pest mite"
Artwork by Linda Heith
Insects, mites, snails and diseases, although usually thought of as undesirable, can be very useful for controlling various
pests. Biological control Is the use of beneficial organisms for pest control These beneficial organisms can be predators
such as ladybugs. lacewings and praying mantlds. which feed on other Insects. Others, such as nematodes and wasps
Including Trichogramma, are parasites. Trtchogramma. wasps lay their eggs In the eggs of caterpillar pests, where they
develop by feeding on the pest's egg. Some beneficial Insects attack noxious weeds. Fungi, bacteria and viruses can also
be used to control Insect and weed pests.
"Hie 1982 version of this list has been sent in response to thousands of requests, to Individuals, universities, extension
entomologists, and many others. The list has appeared In part or In Its entirety. In several magazines and has been
walled out separately as part of a larger publication on resources for organic pest control. Since this list was last
published three years ago. we have added 23 new suppliers. In addition, there are 13 organisms now available that were
not for sale In 1982.
-------
Green OU»er
Lace- Lady- Praying Egg Organisms
Wings Bugs Mantids Wasps (see last page)
CGP
CWP.PMP
Supplier
Ag Bio Chem Inc.
3 Fleetwood Ct
Orlnda, CA 94563
(415) 254-O789
Applied Bionomics
P.O. Box 2637
Sidney. B.C
Canada V8L4C1
(6O4) 656-2123
Mail Phone Whole-
Order Order Retail sale No
XX x Free
Brochu
X X X X Free
Catalog
Need
Permit
For US
Import
MD.RSM
Associates Insectary
P.O. Box 969
Santa Paula, CA 93060
(805) 933-1301
FPG. FPS
1*2. MB
FPM. FPN
FPS. GHP
Beneficial Bloaystetn*
1603-P 63rd Street
Emeryville. CA 94608
(415)655-3928
Beneficial Insectary
245 Oak Run Road
Oak Rua CA 96O69
(916)472-3715
XXX Whole-
sale
For
PCA's
X X X X Postage
Paid
Beneficial Insects
Company
P.O. Box 556
Brownsville. CA 95919
(916) 675-2251
X X Free
Brochun
X FP
Beneficial Insects Ltd.
P.O. Box 154
Banta, CA 95304
Ltcewlng-
April-Ju]]
APA, GWP
PMM. PMP
Better Yield Insects
13310 Riverside Drive E.
Tecumseh. Ontario
Canada N8N 1B2
(519h 735-OO02
X X X X Need pen
For US
Import
DS
Bio-Con Systems
P.O. Box 377
Sunnymead. CA 92388
(714) 656-1712
X X X X Free
Brochure
X .X X X FP Bio-Control Company
P.O. Box 247
Cedar Ridge. CA 95924
(916) 272-1997 *
X X X X Free
Brochure
PMA
Biogenesis. Inc.
P.O. Box 36
Mathls. TX 78368
(512) 547-3259
X X X X
XX X .BSP. FPM. FPS
GHP. GWP. LT. MD
NPV. PMC. PMO
PMP. RSM
PMC. PML
PMO. PMP
Bio Insect Control X X X X
710 5. Columbia
Plalnvlew. TX 79072
(806) 293-5861"
Blotactlcs. Inc. X X X X Free
7765 Lakeside Drive Brochure
Riverside. CA 92509
(714) 685-7681 after 6 pm
-------
jiten
|IBC. Lady- Praying Egg Other
«gM Bugs Mantids Wasps Organisms Supplier
PN. PNB B.R. Supply Company
PNC PNC £.O. Box 845
Exeter. CA 93221
(2O9) 732-3422
BP Burgess Seed & Plant Co. .
Department 69
905 Four Seasons Road
Bloomlngton. IL 61701
(309) 663-9551
XXX Burpee Seed Company
3OO Park Avenue
Warmlnster. PA 18974
v X FP.NOW California Green Lacewlngs
NOWP P.O. Box 2495
Merced. CA 95340
(209) 722-4985
X CHP. PNC Colorado Insectary
P.O. Box 3266
Durango. CO 81302 -
(3O3) 247-536O
DS Decollate Snails
P.O. Box 972
. Lake Arrowhead. CA 92352
(714) 337-2282 after 7 pm
BP Fairfax Biological
Laboratories. Inc.
Clinton Comers. NY 12514
(914) 266-3705
• BP. GHP Fanners Seed and Nursery
MB Department 72
22O7 East Oakland Avenue
Bloomlngton. IL 61701
(309) 663-9551
DS Flllmore Decollate
Snail Farm
Mail Phone Whole-
Order Order Retail sale Notes
X X X X
XXX Free
Catalog
X Free
Catalog
X X X X Free
Literature
& Price
List
X X X X Free
Brochure
X X X X Free
Brochure
SendSASE
X X X X Free
Brochure
XXX Free
Catalog
X X X X Free
Brochure
2841 West Young Road
FUlmoreCA 93015
(805) 524-103O
(714) 781-7643
X BSP. DS. FP
MO.RSM
Foothill Ag. Research Inc.
510W. Chase Drive
Corona. CAS 1720
(714)371-0120
X Free
Literature
Fountain's Sierra Bug Co.
P.O.Box 114
Rough & Ready. CA 95975
(916) 273-0513
X Free
Brochure
Cothard Inc.
P.O. Box 7
Mesllla. NM 88046
(505) 522-9031
X Free
Brochure
X X FP. CHP
Curney Seed & Nursery Co.
Yankton. SD 57079
(605)665-4451
Free
Catalog
-------
Green
Lace-
Wings
X
X
X
X
X
X
X
X
X
X
Lady Praying Egg Other
Bugs Mantids Wasps Organisms
X X D&FPMJPP
FPRFPS.GHP
GWP.MRMD.NPV
PMCJ»MUPMO
PMP.RSB.RSM
X X X BP.FP
GHP. MB
X X BSP.CMDJDS
FPM.FPS.FPT
GHP.GWP.MD.PMC
PMO.PMP.RSM
X X X BP.FP
GWP.MD
PM
X X FP.FPS
PMA
XX FP
XXX. BP. FPS.FPZ
GHP. GWP. MCP
MO
BP
DS
X
XXX BSP. FPM. FPN
FPS.GWP. MB
MD. MF. PBP.
PMC. PML. PMO
PMP. RSM
X GWP.MD
. PMC. PML
PMO. PMP
X X BBP. BP. CPP
FP. GHP. GWP. MB
MD. NPV. PMC
PMO. PMP. PNC
Supplier
Harmony Farm Supply
P.O. Box 451
Graton. CA 95444
(707) 823-9123
Henry Field's Seed
and Nursery Company
Shenandoah. IA 51602
(712) 246-1888
Integrated Pest Management
305 Agostino Road
San Gabriel CA 91776
(818)287-1101
King's Natural Pest Control
224 Yost Avenue
Spring City. PA 19475
Kunafin Trichogramma
Insectaries
Route 1. P.O. Box 39
Quemado. TX 78877
(512) 757-1468
(512) 773-0149
Lakeland Nurseries Sales
Inc. 340 Poplar Street
Hanover. PA 17331
(717) 637-5555
Mellinger's Nursery
2310 W. South Range Road
North Lima. OH 44452
(216) 549-9861
Miller Nurseries
5060 West Lake Road
Canandalgua. NY 14424
Chuck Musgrove
Riverside. CA
(714) 78O-8733
Nationwide Seed & Supply
4801 Fengenbush Lane
Louisville. KY 40228
Natural Pest 'Controls
8864 Little Creek Drive
Orangevale. CA 95662
(916) 726-0855
Nature's Control
P.O. Box 35
Medford. OR 97501
(503) 773-5927
Necessary Trading Company
P.O. Box 603
New Castle. VA 24127
(703) 864-5103
Mall Phone Whole
Order Order Retail sale Notes
X XX X Free
Catalog
XXX Free
Catalog
J( JW A Jt £rQ1llf
Literature
X XX Postpaid
Free
Literature
X X X X Free
Information
XXX Free
Catalog
XXX Free
Catalog
X X Free
Catalog
X X Wffl not
Ship
X Postage
Paid
X X X Free
Brochure
X X X X Free
Brochure
X X X X Charge for
Catalog
PN
The Nematode Farm. Inc.
3335 Birch Street
PaJo Alto. CA 94306
(415)494-8630
-------
^*^ lady- Praying Egg Other
jl^l Bugs Mantids Wasps Organisms
X X'
x X X BSP. FP. GWP
MB. MD. PMC.
PML. PMO. PMP
RSM
BP
DS
X X X X BPBSP.DSJPM
FPS.GHP.GWP.MD
MF.NOW.NPV.PMC
PMHJMUPMO.PMP
PN.PNCRSBJISM
BP. GHP. MB
XX X X APA-Canada only
FP-US only
GWP-Canada only
PMP-Canada only
XX X BSP.DS. FP
GWP. MD. PMC
PMO. PMP
BBP
X FPG. FPM. FPS
GWP. MD
x X X X FP.GWP
X X
PMO
Supplier
Organic Control Inc.
5 132 Venice Blvd.
Los Angeles. CA 90019
Organic Pest Control
Naturally
P.O. Box 55267
Seattle. WA 98 1 55
(206) 367-0707
Richard Owen Nursery
Department 74
2300 East Lincoln Street
Bloomlngton. IL 61701
(309) 663-9551
Pacific Tree Farms
4301 Lynwood Drive
Chula Vista. CA 92O1O
(619) 422-2400
Peaceful Valley Farm Supply
1 1 1 73 Peaceful Valley'Road
Nevada City. CA 95959
(916) 265-3276
Reuter Laboratories
P.O. Box 346
Haymarket VA 22069
1-800-368-2244
Rich ten
P.O. Box 26
Goodwood. Ontario
Canada LOC 1AO
(416)640-6677
Rlncon-VUova
Insectartes. Inc.
P.O. Box 95
Oak View. CA 93O22
(805) 643^5407'
• Rocky Mountain Inacctary
P.O. Box 152
Palisade. CO 81526
(303)245-0406^
Spaldlng Laboratories
76O Prtntz Road
Arroyo Grande. CA 93420
(605) 489-5946
Unique Insect Control
P.O. Box 15376
Sacramento. CA 95851
(916) 967-7082
West Coast Ladybug Sales
P.O. Box 903
Grldley. CA 95948
(916) 534-0840
Wllk. Kltayama and Mead
9093 Troxel Road
Chico. CA 95928
Mall Phone Whole
Order Order Retail sale Notes
X X X Free
Catalog
Postpaid
X XX Charge for
Catalog
XXX Free
Catalog
X X X X Charge for
Catalog
X X X X Free
Catalog
X X X X Free
Brochure
XXX Canada
Orders
Only In June
Need permit
For US
Import
X X X X Free
Brochure
Visa and MC
X X X X Free
Brochure
X X X X Free
Brochure
X X X X Free
Literature
X X X X Free
Literature
XXX Sulfur & OP
Resistant
Strain
-------
California Department of Food and Agriculture
Biological Control Services Program
3288 Meadowview Road
Sacramento. CA 95832
. POSTAGE
X>00 AND AGRICULTURE
1220 M STREET
SACRAVEN7O. CA 95314
OREHI lAaCWINGS-Qvyjopo. canxa
LADTBUCA-Hlppodamto canurrgnu
PKATINO MAirnD*-r
BOO WAftPV-rnrnaonimnia mlniuum
TntlKUjrumma platntrt
APA-AphM paruile->lphhio/r«f* aphldtmyza
BBP-Bean beetle p*z*tUr-PtdtobiiaJbvtotatu3
BP-MIIky ipocr-BortUua popiltlae
CCT-Crown gill pmrntlvc-^gnitaetcrfum rodloboct«-
CtCD-Cllrus mcah-bug p*n*lir-L«p(oniasiu daclylopll
CPf-Colondo potato beetle panuile-E^ivum putllert
OS-DrmlUte MUil-ffumliM dcrallala
TMTjr pumte* variola iprcte*
CPH-Crmuhoppcr pathogavNa
GWFM5reenhou>e whltefly pum&if
LT-Cnenbu( puwiie-Ly«(p/i/ebta fntarrtpo
MB-Mo«qulio bacterlum-Bacll/u* l/iurtngtafutt (iractKnK*
MCF-Mormon cricket pathogen
afD-M«Jybug de*trayer-Oyp trutd
PMC-Predaiory mHe-Amblyselia calljornlcut
PMH-Pred*toiy mlle-Ambfyscluj ftHMid
na^Prrdatory mlir-PfiyluetuJu* lonptpe*
PMM-Predxoiy mlir-Amb/ysMuc mcknuKt
PMO-Predalory mlie-M««a«rtu(u» occldcniaJU
rm-Fty pumsllv-Moionia cttrtptnnte
Fm-Tly parMtte-Carrtnaox sp.
FFHTJr pcnsllr-Spalanola «iH«ia
rTT-riy puulir-SpfitiDfoawrr lachfnarpfiagus imlaiulana earpocoptar
PNO-Panslllc ntnuitoac-Neoapleaana ata«rrt
MSB-Red sole panaue-Gomprrtrlla btfdmclata
MSM-Red icmle para>lte-Xpfi|/ll« nw/lnu*
The oinplUar kuval bacterium Baettlu* thuHngtmtU to (valliblr through many reuU
and wholoate concern* under wioua brand names.
Inclusion in this pamphlet does not imply an endorsement of the suppliers or their products by the California Department of
Food and Agriculture, nor does it imply criticism of other suppliers not listed. This pamphlet may be freely reproduced. One
copy per request is available from:
Biological Control Services Program
3288 Meadowview Road
Sacramento. CA 95832
(916) 427-4590
-------
Draft No. 2
May 17, 1985
Peter Van Voris
509/375-2498
Designation: EXXXX-85
AMERICAN SOCIETY FOR TESTING AND MATERIALS
1916 Race St., Philadelphia, PA 19103
Standard Guide for
TERRESTRIAL SOIL-CORE MICROCOSM
This standard 1s Issued under the fixed designation EXXX; the nunber Imedlately following
the designation Indicates the year of original adoption or, In the case of revision, the
year of the last revision. A nunber In parentheses Indicates the year of last approval.
This guide Is under the Jurisdiction of ASTM Committee E*7 on Biological Effects and
Environmental Fate, and Is the direct responsibility .of Subcommittee E47.08 on Biological
Field Tests.
1.0 SCOPE
1.1 This standard guide defines the requirements and procedures for
testing the environmental fate, ecological effects and environmental
transport of chemicals 1n terrestrial ecosystems using the soil -core
microcosm. Additionally, 1t provides a general rationale for the approach
and materials suggested for use in the soil -core microcosm.
1.2 This test procedure is designed to supply site-specific or
possibly regionalized information on the probable chemical fate and
ecological effects resulting fron release or spills of chemicals in the
environment in either liquid or solid form.
1.3 Experience has shown that microcosms are best applied chemical
in the assessment process at stages after aquisltion of preliminary
knowledge about a chemical's properties and biological activity. The data
resulting from performance of this standard can then be used to compare
the potential terrestrial environmental hazards of a chemical.
'M» document l'i In process of development and Is for ASTM committee use only. It sh«ll not
be reproduced or circulated or quoted, In whole or 1n part, outside of ASTM coonittee
activities except with the approval of the chairman of the committee with Jurisdiction or
the President of the Society.
-------
1.4 This standard guide may involve hazardous materials, chemicalst
radiolabeled compounds, hazardous operations and wastes. This standard
guide does not purport to address all of the safety problems associated
with testing the environmental toxicity and environmental fate of a pure
chemical or complex mixture of chemicals. It is the responsibility of
whoever uses this standard to consult and establish the appropriate safety
and . health protocols and determine the applicability of regulatory
limitations for wastes prior to performing the test.
2. APPLICABLE DOCUMENTS
2.1 ASTM Standards:
D18 Test Methods for Soils and Rocks
(D422-63, D2167-84, D2216-80, and D2488-84)
D19 Test Methods for Water Quality Analysis
(0511-84, D515-82, D1254-67, D1426-79,.and D3867-79)
2.2 Environmental Protection Agency 1982. Environmental Effects
Test Guideline. Washington, DC. Office of Pesticides and Toxic
Substances, U. S. EPA EPA 560/6-82-002. Available from NTIS,
Springfield, VA PB82-232992.
2.3 Environmental Protection Agency. 1982. Environmental Effects
Test Guideline. Washington, DC. Office of Pesticides and Toxic
Substances, U.S. EPA EPA 560/6-82-002. Available from NTIS,
Springfield, VA PB82-232992.
2.4 Environmental Protection Agency. 1982. Chemical Fate Test
Guideline. Washington, DC. Office of Pesticides and Toxic
Substances, U.S. EPA EPA 560/6-82-003. Available from NTIS,
Springfield, VA PB82-233008.
3.0 DEFINITION OF TERMS
3.1 Soil-Core Terrestrial Microcosm - The terrestrial microcosm or
micro-ecosystem is defined here as a physical model of an Interacting
,^-^r—
This tfocu»ent it In process of development and is for ASTH committee use only. It shall not
be reproduced or circulated or quoted, in whole or In part, outside of ASTM committee
activities except with the approval of the chairman of the committee with Jurisdiction or
the President of the Society.
-------
cormunity of autotrophs, omm'vores, herbivores, carnivores and decomposers
within an intact soil profile. Specifically, it is an intact soil-core
containing the natural assemblages of biota surrounded by the boundary
material. The system includes all equipment, facilities, and
instrumentation necessary to maintain, monitor, and control the
environment. The forcing functions, e.g., light intensity and duration,
water quality and watering regime, temperature, and toxicant dose for the
test system, are under the investigator's control. This test system is
distinguished from test tube and single species toxicity tests by having a
natural assemblage of organisms present, resulting 1n a higher order of
ecological complexity and, thus, the capacity to evaluate chemical effects
on component interactions and ecological processes. Certain features of
this test system however, set limits oi\. the types of questions that can be
legitimately addressed. Those .limitations are related to scale and
sampling, which in turn constrain both (a) the type of ecosystems and
species assemblages on which one can hope to gain information, and (b) the
longevity of the test system. .,_
3.2 Physical, Chemical and S-iolog€sal Conditions - The physical,
chemical, and biological conditions-jof the test system are determined by
'§&.•:
the type of ecosystem from which th% test system was extracted and by
either the natural vegetation in the^ecosystem or the selection of crops
for planting. Vegetation and cr^s selection are constrained and
determined by the size (width and depth) of the soil core extracted.
Boundaries - The boundaries of the test system are determined by the
size of the core and the growth space needed for the vegetative component.
Light - Light for the test-^iystem can be supplied by artificial
lighting in either a growth chamber^"fir-greenhouse or can be the natural
photoperiod occurring in a greenhouse. Because of the flexability of this
test guide, lighting period is not specified; however, if the test is
performed in a growth chamber, the daily photoperiod should be at least
the average monthly incident radiation (quantity and duration) for the
month in which the test is being performed. During extremely short
-3-
This document irs
-------
natural photoperiods, which might not allow for l=flowering or seed set to
occur, the photoperiod should be artificially lengthened. If the test is
performed in a growth chamber, the daily photoperiod should be at least
the average monthly incident radiation (quantity and duration) for the
month in which the test is being performed.
Water - Water for the test system should either be purified
untreated laboratory water with a known chemical composition or should be
precollected, filtered rainwater from the site being evaluated. ASTM D19
Test Methods for Water Quality Analysis should be followed.
Soil - The soil for the microcosm should be an Intact, undisturbed
(non-homogenized) core extracted from a soil type typical of the region or
site of interest and be of sufficient depth to allow a full growing season
for the natural vegetation or the crops selected, without causing the
plants to become significantly rootbound. Disturbances during extraction
and preparation should be kept to a minimum. It should be noted that soil
characteristics will play an important role in how the microcosm responds
to a test substance. In addition, within-site soil heterogeneity will
also influence the microcosm response* The approach used in this test
system however, is based on comparison of responses among and between
treatments rather than on tte atejolitfte values measured for an individual
microcosm.
Biota - The biota of the microcosm are characterized by the organisms
in the soil at the time of extraction (Parkinson et al. 1971; Phillipson
1971) and by the natural vegetation or crops introduced as the autotrophic
component. The biota includes all heterotrophic and carnivorous
invertebrates in the soi3 antf all soil and plant bacj^J^^ungi, and
viruses.
4.0 SIGNIFICANCE AND USES
4.1 This test method establishes a4procedure to assist industry
in evaluating the potential ecological impacts and environmental transport
-4-
This document 1* in process of development and 1s for ASTM ccwnittee use only. It shall not
be reproduced or circulated or quoted, In whole or 1n part, outside of ASTM conwittee
activities except with the approval of the chairman of the committee with Jurisdiction or
the President of the Society.
-------
of a chemical that may be released or spilled in the environment. The
suggested test procedures are designed to supply site specific information
for a chemical without having to perform field test.
4.2 Specifically, this test is used to determine the effect of a
chemical on; (1) growth and reproduction of either natural vegetation or
crops, and (2) nutrient uptake and cycling within the soil/plant system.
Additionally, the soil-core microcosm will provide Information on; (1)
potential for bioaccumulation (i.e., enrichment) of the chemical into plant
issues, and (2) the potential for and rate of transport of the chemical
through soil to ground water.
4.3 The results of this test should be used 1n conjunction with
information on the chemical and biological activity of the chemical of
interest to compare the relative environmental hazard and the potential for
environmental movement once released.
4.4 The test methods described here are designed specifically
for liquid or solid type materials. Significant modifications of the
exposure system would be necessary to accommodate chemicals that may be
released in a gaseous or aerosol form.
4.5 Correlation of multi-year soil-core microcosm test results
with data derived from a series of multi-year field plot tests has been
determined for a limited number of materials. Information relating to
microcosm to field correlation can be found in Van Voris et aj_. (1984),
Van Voris et al_. (1985) and Tolle et.af. (1983)
t " •'
5.0 CHEMICAL CHARACTERIZATION OF TEST SUBSTANCE AND SOIL
5.1.1 Infcmation Fequired or. lest Substance - Minimum
information required to properly design and conduct an experiment on a
test chemical includes the chemical's source, composition, degree of
purity, nature and quantity of any impurities present, and certain
physiochemical information such as water solubility and vapor pressure at
This document is in process of development and \\ for ASTM comnittee use only. It shall net
6« reproduced or circulated or quoted, in »hole or in part, outside of ASTM cotmitte*
activities except wfth the approval of the chairman of ttie committee with Jurisdiction or
the President of the Society.
-------
25°C (Laskowski £t a_K 1982; Swann et jil_. 1983). Ideally, the structure
of the test chemical should also be known, including functional groups,
nature and position of substituting groups, and degree of saturation.
Octanol-water partition coefficient, dissociation constant, degree of
polarity, and pH of both pure and a series of serial dilutions shall also
be known. Where mixtures are involved or where a significant impurity
(>1%) occurs, data must be available on as many components as practicable.
However, the octanol-water partition coefficient (Kow or P) stands out as
the key value. In'combination with other chemical characteristics, log
can be used to estimate Henry's Law Constant to predict volatility from
soil solutions. Soil sorption can be estimated from log, the organic
matter content, or from those data that suggest a relationship exists.
Water solubility can be predicted with some degree of accuracy from log
if this value is less than 7.
5.1.2 Several tests may be needed to supply information on
environmental mobility and stability. Support information on
phytotoxicity, the physicochemical nature of the chemical, its mammalian
toxicity, invertebrate toxicity or its ecological effects (e.g.,
species-specific LD5Q, biodegradability) not only assist in proper design
of the microcosm experiment, but also are useful in assessing the fate and
effects of the chemical in a terrestrial microcosm. If the chemical is
radioactively labeled, the position and specific element to be labeled
should be specified.
5.1.3 It is imperative to have an estimate of the test
substance's toxicity to mammals as a precaution for worker safety. In
addition, hydrolysis or photolysis rate constants should be known in order
to determine necessary handling precautions. When a radiolabeled material
is used, normal laboratory techniques for radiation safety provide an
ample margin of safety (Wang and Willis 1965), except for chemicals in the
"very highly toxic" category (i.e., rat oral LD5Q <1 mg/kg).
5.1.4 The above information required for a test chemical is con-
sidered to be the minimum that is necessary to handle and apply the
-6-
This document is in process of devel opfl>ent and is for ASTM cotmiittee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM conwittee
activities except with the approval of the chairman of the committee with jurisdiction or
the President of the Society.
-------
chemical safely (Walters 1980), as well as provide a perspective from
which to interpret the test results. Water solubility, soil sorption and
octanol-water partitioning, and vapor pressure largely will control the
physical transport and bioavailability of a test chemical in soil. Water
soluble chemicals are likely to move with soil water into the water films
surrounding soil particles and root surfaces. Most microbially-mediated
biodegradation occurs in the water-containing microsites of soil
particles. Plant uptake and bioaccumulation is largely a function of
water transfer to roots and solubility in fatty tissues. In addition,
water-soluble chemicals and their transformation products may be leached
to ground water. Water solubility of an organic chemical is a function of
the dissociation of ionic compounds and the polarity of non-ionic
compounds.
5.2.1 Information Required on Soil- Soil sorption of an organic
molecule depends on several properties of the chemical (i.e., molecular
size, ionic speciation, acid-base properties, polarity, and nature of
functional groups) and of the soil (e.g., organic matter content, clay
content, clay mineralogy and nature, pH, water content, bulk density).
Highly sorbed chemicals may displace inorganic nutrient ions from exchange
sites in the soil and also may be effectively immobilized, depending on
soil pH. Thus, chemicals attracted more strongly to soil surfaces than to
water may be very immobile in soil. In some cases, this may render the
compound relatively resistant to biodegradation. In other cases, however,
immobilization of the compound on soil particles may render it susceptible
to extracellular enzymatic degradation. Specific information on
descriptive data required for soil can be found in Section 6.2.2.
5.2.2 Compounds with very high vapor pressures (boiling point
<80°C or vapor pressure >25 mm Hg-) are not suitable for testing in the
terrestrial soil core microcosm as described here. Modification of the
test system such as that described in Van Voris et al. (1977) and Van
Voris .et £K (1981) should be useful for handling gaseous or aerosol type
chemicals.
_ r -7-
This document is in process of development and is for ASTM committee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM cowr ttee
activities except with the approval of the chaiman of the cormittee with ju'i sdi ction or
the President of the Society.
-------
6.0 TERRESTRIAL MICROCOSM EXTRACTION AND MAINTENANCE
6.1 Microcosm and Cart Design - The 60-cm-deep by 17-cm-dlameter
terrestrial microcosm is designed to yield pertinent information about a
chemical for either a natural grassland ecosystem (Figure 1) or an
agricultural ecosystem planted with a multiple-species crop (Van Voris et
aj_. 1982, 1984, 1985; Zwick ert aj_. 1984). The agricultural microcosm is a
17-cm-diameter tube of Driscopipe* containing an intact subsoil core (40
cm) covered by homogenized topsoil (20 cm). Driscopipe is an ultra-high
molecular weight, high-density, non-plasticized polyethylene pipe.
Driscopipe^
High Density
Polyethylene
Glass Wool
Bi.'Chner Funnel
Intact
Soil Core
Figure 1. Microcosm Structure and Materials
(After Van Voris et_ al_. 1985)
-8-
This document is in process of development and is for ASTM cw«ittee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM committee
activities except with the approval of the chairman of the co*«ittee with jurisdiction or
the President of ^e Society.
-------
non-plasticized polyethylene pipe. It is impermeable to water,
lightweight, tough, rigid, and highly resistant to acids, bases, and
biological degradation. Additionally, Driscopipe* does not release
plasticizers or other compounds that may interface with test results. The
tube sits on a Buchner funnel covered by a thin layer of glass wool. The
funnel and tube are washed with 0.1 HC1 prior to use and are reuseable for
future tests.
6.1.2 Six microcosms are typically contained in a moveable cart,
which is packed with Styrofoam* beads to reduce drastic changes in
temperature profile (Van Voris e± aK 1982, 1984;) (Figure 2)., Cart
dimensions are based on the environmental chamber size or the
maneuverability required in the greenhouse. Carts are mounted on wheels
so the cart can be moved within the greenhouse in a manner defined by
statistical requirements to make exposure to light and temperature more
uniform.
Soil-Core
.Microcosms
Agricultural
Microcosm
Styrofoam
Insulation's^
Dnscopipe
®
leachate
Figure 2. Arrangement of Microcosms in Styrofoam Filled Cart
(After EPA Test Prctoco1 (In Press))
-9-
This document i'i in process of development and is for A.STH committee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTH committee
activities except with the approval of the chairman of the covittee with jurisdiction or
the President of the Society.
-------
6.2.1 Soil Core Extraction - Soil cores are extracted from either
a natural grassland ecosystem or a typical agricultural soil in the region
of interest. The intact system is extracted with a specially designed,
steel extraction-tube (Van Voris et al_. 1982, 1983, 1984; 1982; Zwick et
a]_. 1984) (Figure 3) and a backhoe. The steel extraction- tube encases
the polyethylene Driscopipe* to prevent the tube from warping and/or
splitting under pressures created during extraction. For the agricultural
microcosm, the plowed topsoil is moved aside and saved. Once the core is
cut by the leading'edge of the driving tube, it is forced up into the
microcosm tube. For the natural grassland ecosystem, the vegetation is
clipped before the core is extracted. The soil core microcosm is later
removed as a single unit (soil and Driscopipe*) from the extraction tube
and taken to the laboratory. For the agricultural microcosm, the topsoil
Driscopipe®
Rolled
Steel
Weld
Stainless Steel
Cutting Edge
Cap
Soil Core
Polyethylene
Microcosm Tube
Handles
Tube Holder
Stainless Steel
Cutting Edge
Figure 3. Diagram of Microcosm Extraction Tube
(After Van Voris et £L 1985)
-1.0-.
This document Is in process of development and is for ASTM committee use only. It shall not
be reproduced or circulated or quoted, in whole or In part, outside of ASTM committee
activities except with the approval of the chairman of the coanittee with Jurisdiction or
the President of the Society.
-------
1s backfilled Into the upper 20 cm of the microcosm tube. It is
recognized that this extraction procedure does disrupt and compress the
soil-core to a certain extent; however, the tests are being performed to
compare different treatment levels and controls within the experiment.
6.2.2 Detailed chemical and physical properties of the soil in
the test systems need to be determined using USDA description
nomenclature. Information such as pedologic identity, according to the
USDA 7 Approximation Soil Classification System, percent organic matter,
field capacity, cation exchange capacity, bulk density, macro- and
micro-nutrient content, organic matter content, mineralogy, exchange
capacity, particle size distribution, hydraulic characteristics, and other
Important characteristics need to be measured before and after the
experiment (Black 1965; 1982; ASTM D422-63, D2167-84, D2216-80, and
D2488-84). The history of the soil, including previous crops grown and
pest control and other management practices used, should be documented in
order to assist in the interpretation of the results.
6.3.1 Microcosm - For the natural ecosystem (undisturbed
grassland) test system, natural plant cover should be sufficiently diverse
to be representative of plant species in the ecosystem of interest. When
the agricultural microcosm is used, a mixture of grasses and broad leaves
(e.g., legumes) should be included. Two or three species of grasses or
legumes that are typically grown together as an agricultural crop in the
region of interest should be chosen. The species chosen must be
compatible regarding growth habit and be able to grow to maturity in the
small surface area (240.5 cm2) of the microcosm. In some cases, it may be
appropriate to select a grain crop in order to evaluate the uptake of the
radiolabeled test substances and their degradation products in grain
normally grown for human consumption (Van Voris et al 1984).
6.3.2 The seed application rate should duplicate standard farming
practice for the region of interest. Seeds should be planted evenly and
covered with an appropriate depth of soil. Similarly, the test substance
application form should approximate a reasonable scenario of how the test
-11-
This document is In process of development and is for ASTM conmittee use only. It shall not
b« reproduced or circulated or quoted, In whole or in part, outside of ASTH eowiittee
activities except with the approval of the chairman of the committee with jurisdiction or
the President of the Society.
-------
substance might arrive at the site in question. If the test substance is
a solid, one option is to mix it with the topsoil prior to planting, thus
mimicking the plowing of an agricultural field prior to sowing seed.
Alternatively, it may be dusted on the surface to simulate dry deposition.
6.3.3 Plant tissue analysis should be consistent with those
practices that may be region specific. Plants from either the natural
grassland ecosystem or the agricultural test unit are harvested from each
microcosm at the end of the test period (Van Voris et al. 1982; Tolle et
al. 1982), and air dried, and then oven-dried. In the range-finding
test (see Section 7.3.1), the crop is harvested four weeks after first
exposure to the test substance. In the definitive test (see Section
7.4.1), plants may be harvested one or two times during the 12-week
growing period or at the end of the test. The definitive test may need to
be extended beyond the 12-week test period to accommodate plant species
which take longer to reach the desired maturity (e.g., seed production).
6.4.1 Microcosm Watering and Leachate Collection - Microcosms are
watered as dictated by a predetermined water regime based on site history
with either purified laboratory water (e.g., distilled, Reverse Osmosis)
or with rainwater that has been collected, filtered and stored in a cooler
at 4°C (Van Voris et a]_. 1980, 1982, 1983). If comparisons are being made
between microcosms and field plots, then parallel watering in both units
should be attempted. Care needs to be taken to maintain sufficient water,
but over-watering can induce fungal disease and stress.
6.4.2 Microcosms are leached at least once before and once every
two or three weeks after dosing. However, if natural rainfall amounts are
higher or lower this should be used to guide selection of a leaching
regime. Caution should be exercised to not over water because this may
drastically alter the rate of degradation, transformation, trans!ocation
and transport within the microcosm.
6.4.3 Leachate is collected in 0.1N HC1 washed flasks after
excess laboratory water or rainwater has been added to each microcosm.
The 500-ml flask is attached to the Buchner funnel with Tygon tubing,
^ -12-
This document is in process of development and ij for ASTM co™ittee use only. It shall not
be reproduced or circulated or quoted, in whole or in pert, outside of ASTM coimittee
activities except with the approval of the chairman of the cooB'ttee with jurisdiction or
the President of the Society.
-------
(see Figure 2) 1n an attempt to Insure that all test microcosms will leach
within a 2-day period, 15% more microcosm soil cores are extracted than
are required for a combination of both the range finding and definitive
tests. When the microcosms are leached before planting, those which do
not leach, leach too quickly, or take longer than two days to produce 100
ml of leachate after the soil has been brought to field capacity are
discarded.
6.5 Greenhouse and Grovth Chamber Environments - Microcosms in
Insulated carts or other such devices .are kept in a greenhouse or
environmental chamber that has temperature and light control. Greenhouse
and environmental chamber temperatures are designed to approximate outdoor
temperatures that occur during a typical growing season 1n the region of
Interest. If the experiment is not conducted in the greenhouse during the
normal agricultural growing season, then lights suitable for plant growth
on timing devices are used to simulate the photoperiod and Intensity
typical for the growing season in the area of interest. If the experiment
1s conducted in the greenhouse during periods when the photoperiod of the
natural light is not long enough to Induce Cowering and seed set, then
supplimental lighting will be required.
7.0 TEST PROCEDURES
7.1 Test Purpose aid Assumptions - The purpose of the
terrestrial soil-core microcosm test is to determine the fate and
ecological effects of a test substance, including transformation products,
withir a site- specific natural grassland or agricultural ecosystem. The
relationship of fate and effects data from treated versus control
microcosms is assumed to be very similar to that from treated versus
control field test plots (Van Voris et al.. 1982, 1983, 1984). This
assumption is supported by the microcosm/field comparisons reported by Van
Voris et aj_. (1982, 1984), Jackson et a]_. (1979) and Tolle et al.. (1983).
The fate and effects from the microcosm test should then be related to
-13-
fhij doci»ert Is in process of development and is for ASTM cowaittee u»e only. It thai! not
b* reproduced or circulated or quoted, In whole or in part, outside of ASTM committee
activities except with the approval of th« chairman of the cwwn'ttee with Jurisdiction or
the Pr«$ide.-t of the Society.
-------
either the natural or agricultural ecosystems which have the same
combination of soil type, vegetation, crop species, and environmental
variables used during the microcosm test.
7.2.1 Evaluation of Teat Substance - Physicochemical information
supplied for the test substance (see Section 6.1.1) is used to tailor the
general range-finding test procedures to the specific substance.
Phytotoxicity and/or bacteriostatic action, if known, should be taken into
account in designing the exposure concentrations of the range-finding
experiment. Only one concentration above that known to cause at least 50?
change in plant growth or 50* change in bacterial growth/respiration needs
to be tested. In addition, the lowest treatment level should not be lower
than a factor of 10 in soil and 100 in water. These factors are the
analytical limits of detectability of the parent compound at the start of
the experiment.
7.2.2 The water solubility and soil sorption capacity can be used
to determine the appropriate frequency of leachate analyses for the
radiolabeled test substance and its transformation products. This same
information will also determine the design of the soil sampling procedures
for the range-finding test. Chemical structure and any degradation
information are used to determine which transformation products to analyze
for in the soil, leachate, and plant tissue collected.
7.2.3 As was stated in Section 6.3.2, exposure should approximate
a reasonable scenario. The water solubility, dissociation constant(s),
and soil pH must be taken into account in determining the formulation of
the chemical. The maximum concentration of a chemical in water solution
should not exceed half of saturation. However, solubility may be markedly
altered by ionization in soil. If the soil pH is such that a more soluble
form is likely, the test substance should be adjusted accordingly with
either sodium hydroxide or hydrochloric acid prior to introduction into
the microcosm. If the pH adjustment to increase solubility is extreme
(49), chemical and photolytic degradation may be enhanced when
-14-
This document is in process of development and is for ASTM committee use only. It shall not
be reproduced or circulated or quoted, in nhole or in part, outside of ASTM conwittee
activities except with the approval of the chairman of the committee with Jurisdiction or
the President of the Society.
-------
preparing the chemical solutions. In all cases, the exposure formulation
should be consistent with the hypothetical scenario forming the basis for
the test.
7.3.1 Range-finding Test Experimental Design- The range-finding
test should last four weeks from first exposure of the test substance to
plant harvest. At the start of the test, the microcosms are dosed (see
Section 7.5) with a minimum of five concentrations of the test substance.
Three replicate microcosms are used for each of the four or five treatment
levels and the controls, resulting in a total of 15 or 18 microcosms.
Concentrations typically used for dosing are 0.1, 1.0, 10, 100 ppm, if a
hypothetical scenario is not known and, if deemed appropriate, 1000 g/g
of topsoil in the upper 20 cm of the microcosm. The logarithmic scale for
dose levels in a range-finding test is suggested by Rand (1980). The bulk
density (g/cm3) of the dry topsoil is used to calculate the exposures.
Depending on whether the potential mode of release of the test chemical is
likely to be a single accidental spill or a repeated effluent release.
Either a single, "acute" dose, or a multiple application, "chronic" dose,
should be considered and selected based on a reasonable exposure
scenario. In either case, the total amount of test chemical applied
should be equal to five of the recommended test concentrations.
7.3.2 Each microcosm cart, holding one replicate of each of the
four or five test concentrations and a control, is moved randomly on a
weekly basis in the greenhouse to avoid location induced effects. If no
discernible greenhouse position effects are recorded during the
range-finding test, then this process need not be followed during the
definitive test.
7.3.3 The range finding tests yield two necessary types of
Information. These include: (1) estimates of the bounds of toxicity
within which the 50* response (e.g. LD50) lies and (2) initial estimates
of variance 1n response. Given the identification of bounds of toxicity
for the initial doses, the dose levels for the definitive tests may be
refined. The variance estimates should be used to determine sample sizes
-15-
This document is 1n process of development and is for ASTM cowr-'tte* use only. It shall not
b« reproduced or circulated or quoted, in whole or in part, outside of ASTH conmittee
activities except with the approval of the chairman of the coa»ittee with jurisdiction or
the President of the Society.
-------
needed in the definitive tests to achieve statistical tests able to detect
specified differences (A) among dose levels with a specified power (1-B).
7.4.1 Definitive Test Experimental Design- The definitive test
lasts for 12 or more weeks from first exposure of the test chemical to
final harvest. Test results may be influenced by extraneous environmental
sources of variation, such as temperature or light gradients within a
greenhouse. These sources of variation may be accounted for by randomly
repositioning the ,cards (see 7.3.2) and/or by employing experimental
designs such as randomized block, latin-square, or other more complex
designs. If such extraneous sources of variability in test results are
not accounted for, results may be biased, thus jeopardizing the outcome of
the experiment. The types of statistical analyses to be performed are
decided at this point, dictated largely by the experimental and treatment
designs. The experimental designs determine the method of randomization
of the treatments to account for extraneous sources of variability in the
experiment environments, while the treatment design determines number of
treatments and the arrangement of treatments with respect to one another.
7.4.2 At the start of the test, the microcosms are dosed with
three concentrations of the test substance. The number of microcosms to
be dosed should be determined by the desired power of the statistical
tests. Power is influenced by the variance of the response (estimated
from range finding tests), the size of the difference to be detected among
the treatments, and the alpha (x) level. The desired power, alpha level
and detectable difference are specified by the researcher, the variance
estimates are obtained from the range finding tests. Based on these four
values, the sample size, or nunber of replicates for each dose level
should be determined (see Neter and Wasserman, 1974 and Kasterbaum avel
Hoel, 197C for discussion of power of a test). The three treatment levels
chosen are estimated from the range-finding test data to produce a 20 to
25% change in productivity for each subsequent concentration of the test
chemical. In order to reduce analytical costs associated with the fate
studies, the replicate microcosms in each treatment level are employed as
~—i——,
This document ij in process of development and it for ASTM cownittee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM cowiittee
activities except with the approval of the chairman of the committee with jurisdicticm or
the President of the Society.
-------
replicate pairs. Thus, leachate and plant tissue analyses are conducted on
the polled samples from paired microcosms. Productivity data, on the
other hand, are analyzed for each individual microcosm. Each cart holds
six to eight microcosms (see Figure 2). The microcosms paired for
analyses are placed in different carts to insure that all microcosms are
housed under the most uniform conditions possible.
7.4.3 Plant Productivity - Depending on the type of natural
vegetation or crop planted, it may be possible to harvest nore than once
(e.g., during the middle and at the end of the test). If growth is
vigorous, grasses may be harvested at a pre-arranged height, e.g., 2 to 6
cm above soil surface during the middle of the 12 week test period.
Multiple harvests permit evaluation of both gross plant yield and plant
uptake of the test substance with respect to time (Elseewi et jil_. 1980;
Van Voris et al_. 1984, 1985).
7.5,1 Exposure Techniques - If the primary mode of exposure of
the test chemical is anticipated to be by addition of pH adjusted
laboratory water or rainwater containing appropriate concentrations of the
test substance, then this procedure should be followed. Again, the
following procedure is used unless information is available that suggests
some other regime should be used or that the chemical is associated with
other chemicals or solvents. In no case shall the total aqueous volume of
exposure be sufficient to cause microcosm leaching (i.e., > field
capacity). Recommended concentration levels are discussed in Sections 7.3
and 7.4. Test- substances which are likely to be released into the
environment as a liquid or powder, and which can be mixed with water, are
applied as a single dose of liquid sufficient in volume to bring the
microcosm soil surface horizon to field capacity. The volume of
laboratory water or rainwater required for exposure can be determined on
an unplanted microcosm of the same soil type on site; the volume selected
should be identical for all microcosms. Carriers ether than water are not
recommended unless they are likely to be released into the environment in
conjunction with the effluent stream or accidental spill of the test
-17-
This document is In process of development and it for A5TM comrnttee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTX cownittee
activities except with the approval of the chair-nan of the committee with jurisdiction or
the President of the Society.
-------
substance. If a carrier 1s necessary, then acetone or ethanol should be
considered; however, the use of carriers should be avoided unless they are
essential to produce a realistic exposure.
7.5.2 Several typical exposure modes are suggested for particular
types of test substances if either a hypothetical or real (actual)
exposure scenario is not available. If the test substance is likely to be
a contaminant of irrigation water, it should be applied every week in
proportionate concentrations, such that the total amount applied equals
the desired treatment level. If the test substance does not mix with
water, it should be applied as evenly as possible to the top of the
unplanted microcosm and mixed into the topsoil prior to planting. If the
test substance is normally sprayed on growing plants (e.g., pesticide),
then the desired amount should be mixed with the volume of water necessary
to wet the soil surface and wet the plants to the point where they begin
to drip. A chromatography sprayer or nebulizer should be used to spray
plants that are past the seedling stage. The recommendations by the test
substance's manufacturer for field spraying should be followed as closely
as possible, but the test should be terminated (last harvest) at least
eight weeks after the plants are sprayed.
7.6.1 Waste Disposal - All liquid (leachate) and solid (soils and
plant tissues) samples must be retained for proper disposal. All sample
collection bottles, collection apparatus, microcosm tubes (Driscopipe )
and sampling tools should be thoroughly cleaned (acid washed) and analyzed
for radioactive contamination before they are stored or used on another
test system. All samples and the remaining, undisturbed portion of the
test system should be disposed of in accordance with EPA and Nuclear
Regulatory Commission (NRC) regulations, if radiolabeled compounds were
used. Soil leachate and all other aqueous-sample wastes should be treated
prior to disposal using one or more of the following techniques:
(a) filtration
(b) activated charcoal filtration
(c) ion exchange.
^-^—T
This document is in process of development and Is for ASTM ccxwrittee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM contm'ttee
activities except with the approval of the chairman of the cowiittee with jurisdiction or
the President of the Society.
-------
7.6.2 Soils contaminated with organic residues and/or
radio!abeled compounds as well as the Driscopipe , sample bottles,
glassware, gloves, masks, filters, activated charcoal from aqueous
cleanup, and any other potentially contaminated equipment must be either
certified as uncontaminated or packaged and disposed of in accordance with
existing EPA and NRC guidelines and regulations.
8.0 FATE AND EFFECTS SAMPLING PROCEDURFS
Sampling procedures have "been divided into tvo basic categories:
ecological effects sampling and test-chemical fate sampling. Ecological
effects sampling may include productivity measurements, physical
appearance of plants, and nutrient less or uptake measurements.
Test-chemical fate sampling may include leachate, soil, and plant
analyses.
8.1.1.1 Ecological Effects Sampling - Productivity Measurements -
Primary productivity is a commonly measured parameter in terrestrial
effects testing. Depending on the plant species, it may be desirable to
report total yield or yield by plant part. For example, in the case of
grain crops such as soybeans, oats, and wheat, the total biomass yield can
be reported in addition to the grain yield. This will allow a relative
comparison of total biomass yield with grain yields typically reported for
local agriculture. In addition, separate grain samples may be useful for
later tissue analyses to determine whether the test chemical was enriched
in potentially edible plant parts. For other systems, such as natural
grassland microcosms, segregation into plant parts may be unnecessary.
8.1.1.2 At a minimum, productivity should be reported as oven-dry
weight. Jones and Steyn (1973) recommend 65°C for 24 hours as adequate
conditions for drying without unnecessary thermal decomposition of plant
material. Information on the chemical's volatility should be evaluated
when selecting a drying temperature. It may be desirable in some
circumstances to report air-dry productivity or to be able to calculate
-19-
This document i~» In process of development and Is for ASTM conw'ttee use on1/. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM committee
activities except with the approval of the chairman of the cotw'tte* with jurisdiction or
the President of the Society.
-------
air-dry yields based on moisture loss after oven-drying. These data could
be useful if agricultural crops are the plants used 1n the microcosm and
if it is desirable to compare productivity with yields reported 1n local
agriculture.
8.1.1.3 The number of harvests will depend on the plants grown. An
agricultural crop, alfalfa/timothy for example, may require two or more
harvests over the course of the testing period (Malanchuk et jil_. 1980;
Van Voris et al_. 1984).
8.1.2 Physical Appearance of Plants - Throughout the test period,
it is desirable to record the physical, appearance of plants 1n the
terrestrial microcosm. Symptoms of nutrient deficiency or toxicity,
pathogenicity, water stress, or test-chemical-induced toxicity should
be monitored. These observations may be useful in interpreting the
specific ecological effects of a test chemical relative to responses
in plants elicited by known environmental toxicants or stresses
(Daubenmire 1959). Careful observation on physical appearance in controls
vs. treated microcosms may also aid in determining whether abnormal
physical appearance is a result of the test chemical or 1s a manifestation
of microcosm management.
8.1.3.1 Nutrient Loss Measurements - An important ecological
effects sampling procedure is to monitor nutrient losses in leachates
(O'Neill et a_l_. 1977; Van Voris et al_. 1980; Jackson et al_. 1978; Jackson
et aj_. 1979). The rationale for such monitoring is explained in detail in
the accompanying support document. One of the desirable attributes of the
terrestrial microcosm approach to testing chemicals 1s the relative ease
with which soil leachates can be collected. This offers the potential to
construct nutrient budgets for the model ecosystem (Schindler £t al_. 1980;
Gile and Gillett 1979).
8.1.3.2 The final suite of nutrients monitored in leachates
probably will depend on the nature of the test chemical Ausmus e£ £l_.
1978; Ausmus et al_. 1979; Jackson et £l_. 1979; Harris et al_. 1980). Those
nutrients which initially should be considered during the range-finding
-20-
This document is in process of development and Is for ASTH cownittee use only. It shall not
be reproduced or circulated or quoted, In whole or 1n part, outside of ASTM cownittee
activities except with the approval of the chairman of the comtiittee with jurisdiction or
the President of the Society.
-------
test include calcium, potassium, nitrate-nitrogen, ortho-phosphate,
ammonium- nitrogen, and dissolved organic carbon (DOC). Depending on the
results of nutrient losses measured during the range-finding test, a set
of nutrients can be selected for monitoring during the definitive test.
8.1.3.3 Various methods exist to analyze for the nutrients.
Standard techniques which have proven useful Include atomic absorption
spectrophotometry for Ca and K and analysis using a Technicon Autoanalyzer
II for nitrate-nitrogen, ortho-phosphate, DOC, and ammonium-nitrogen
(ASTM .0511-84, D515-82, D1254-67, D1426-79, and D3867-79; Association of
Official Analytical Chemists (AOAC) 1975; American Society for Testing and
Materials 1979; EPA 1979). For less rigorous determinations, e.g., during
the range-finding test, Ion-specific electrodes may be useful for nitrate-
and ammonium-nitrogen.
8.1.3.4 A standard procedure, described below, has proven to be
useful in handling leachates. As soon as soil water samples are
collected, the sample volume 1s recorded and the pH determined using a
glass electrode. Samples are centrifuged at low speed (e.g. 5000 rpm) to
remove large particulates and filtered through a 0.45-micron filter. The
sample should be divided into two aliquots prior to storage in the dark at
4°C. Blanks consisting of distilled water and reference standards in
Instrument calibration quantities should be prepared and stored similarly.
8.2.1 Teet-Chertical Pate Sampling - The fate of the test chemical
(see EPA 1982b) will be determined by methods appropriate to the test,
including sensitivity factors adequate to verify exposure and distinguish
between parent material, transformation products, and naturally occurring
materials present in the test system. Usually this will involve use of a
radiolabeled parent compound and subsequent analysis of microcosm
components for radioactivity end chemical identity. Methods appropriate
to the latter may be adequate for quantification of fate, but usually
cannot reveal bound residues in soil or plants and frequently are
Inadequate for cost-effectively tracing movement and transformation. To
the extent that the fate 1n soil and plants is well enough understood from
other experiments and depending on the degree to which the microcosm test
-21-
TMs document la In process of development and 1* for ASTM conmltte* use or.ly. It shall not
be reproduced or circulated or quoted, In ottol* or 1n pert, outside of ASTM coomltte*
activities eicept «1th th* approval of the chairman of the conmlttee with jurisdiction or
the Pre»1dent of the Society.
-------
is being employed to verify fate and exposure hypotheses, analytical
requirements may be reduced (Harvey 1983; Lichtenstein et al_. 1972;
Lichtenstein et aj_. 1973; Metcalf et £l_. 1973; Cole et aK 1976).
8.2.2 Radiolabeling the Parent Compound - The parent compound may
be labeled with 1I+C either in an appropriate aromatic, cyclic carbon
group, or linear chain (Lichtenstein e£ al. 1974). Other labels,
including stable isotopes such as 15N may be more useful and Informative.
In order for the microcosm test to permit an analysis of the fate of the
parent compound and/or its metabolites, attention should be paid to known
or hypothesized metabolic pathways for test substances. Hence the site
and form of label is an integral part of the total test design. The
laboratory conducting the test is not required to have the capability for
radiolabeling, since this is routinely handled by speciality chemical
firms. Sufficient radioactivity must be present in order to detect at
least \% of the initial parent compound in a typical sample of leachate,
soil or plant tissue.
8.2.3.1 Compartment Analysis for Labeled Compomds Several
compartments of the terrestrial microcosm can be analyzed for
radioactivity, which include: samples of soil leachate, plant tissue,
including roots and shoots; and soil from different depths. The different
soil depths used for radiochemical analyses should be selected based on
information on soil sorption of the compound of interest. From previous
experience, these depths should be relatively close to the soil surface
(1-2 cm) for radiolabeled chemicals that are strongly sorbed to soils; if
any isotope appears in the leachate, the depth selection should be lower
in the soil profile. Samples will be homogenized and extracted with
solvents appropriate for the parent compound. Additional extraction steps
may be necessary such as acidification and extraction with non-polar
solvents, sohxlet extractions with polar and/or non-polar solvents,
alkaline or acid hydrolysis with or without heat, detergent extractions,
or protease digestion. The 1UC in the soil or plant samples which cannot
be extracted will be oxidized and analyzed as lt*CQ2 as described by
____ ~22~
This document Is in process of development and is for ASTM committee use only. It shall not
b« reproduced or circulated or cuoted, ir whole or in part, outside of ASTM coomittee
activities except with the approva'. of the chairman of the committee with jurisdiction or
the President of the Society.
-------
Lichtenstein £t a]_. (1972) or dissolved in Protosol and Aquasol as
described by Cole et al. (1976). The extracts and the oxidized or
dissolved samples will be counted by !I*C liquid scintillation (Metcalf et
a]_. 1973; Cole^et al_. 1976).
8.2.3.2 At the termination of the range-finding test, soil samples
will be collected from the top, middle, and bottom of the 60-cm soil
cores. If the labeled compounds or Its metabolites are not detected by
liquid scintillation in the deeper soil samples, then soil samples at the
end of the definitive test should be taken closer to the top of the soil
column.
8.2.4 Identification of Degradation Products Liquid
scintillation will identify the presence of ll4C-labeled compounds in
sample extracts, but the identification and quantification of the parent
compound or its degradation products will require gas-liquid
chromatography (GLC), and thin-layer chromatography (TLC) or high
performance liquid chromatography (HPLC) (I). S. EPA 1982b). TLC
autoradiography using no-screen X-ray film for chromatographed fractions
found to be radioactive by liquid scintillation counting (Cole et al.
1976; Metcalf .et_ a]_. 1973) is most cost-effective. Whenever possible, the
identity of the parent compound and probable degradation products in
fractions found to be radioactive by liquid scintillation counting (Cole
et_ al_. 1976; Metcalf et. ^1_. 1973) will be verified by gas-liquid
chromatography methods. Also, the concentration of the parent compound
and degradation products should be verified by an alternative
chromatographic methods system (e.g., HPLC or GLC) with known standards.
9.0 DATA ANALYSIS
9.1 Ecological Effects Analysis- A combination of statistical
analysis procedures are suggested to evaluate the data collected to
discern ecological effects. Regression, correlation, and covariance
analysis, as well as analysis of variance (ANOVA) procedures described in
-23-
This document fs in process of development »nd is for ASTM committee use only. It shall not
b« reproduced or circulated or quoted, in whole or in part, outside of ASTM conitiittee
activities except with the approval of the chairman of the committee with jurisdiction or
the President of the Society.
-------
the following section may be as appropriate methods for data analysis. Use
of these or other analysis techniques will be determined by the objectives
of the experiments and the original experimental and treatment designs. A
number of statistical references describe the common methods of
statistical analysis (Sokal and Rohlf 1981; Snedecor and Cochran 1980;
Steel and Torrie 1980). The level of significance for all statistical
tests of hypothesis is set at the 5? level (a - 0.05) and power of the
test (1-B) at 0.90 or .095 unless otherwise defined. The results of all
statistical tests performed on all data must be fully documented for
evaluation. In addition, graphs displaying the data and tables containing
all raw numbers should be appended to the submittal.
9.1.1 Test Material Effects- Because the mode of exposing the
test system can be done without the use of carrier chemicals (see Section
6.2.3 and 6.5.1), there is no need to perform separate analyses on carrier
effects as suggested in the marine and freshwater microcosm protocols.
This may be mandatory however, if it becomes advisable to use carrier
chemicals.
9.1.2 Productivity of Natural or Planted Vegetation The sum
total of both air-dry and oven-dry biomass expressed in g/m2 collected
during and at the end of the definitive test should be evaluated initially
by using side-by-side histograms displaying the calculated means,
and 95% confidence intervals for controls and all exposure levels. This
will allow early visual evaluation of the effects of the chemical by
exposure level. Variance estimates may indicate whether logarithmic or
some other transformation of the data may be necessary for graphic display
and analysis. Analysis of variance (ANOVA) calculations (Sokal and Rohlf
1981; or Steel and Torrie 1980) should be carried out first to test for
position effects within the carts and within the environmental area where
the test was performed. Position effects may be accounted for in the
design of the experiment (e.g., by blocking) and any effect of position
can then be accounted for in subsequent analyses. If these tests prove to
be significant at the 5% level (a = 0.05), then this will need to be
__.__ ~24~
This document is in process of development and it for ASTW committee use only. It shall not
be reproduced or circulated or quoted, in whole or ir. part, outside of ASTM conwittee
activities except with the approval of the chairman of the committee with jurisdiction or
the President of the Society.
-------
accounted for in the remainder of the statistical analyses. Pair-wise
comparisons of variables that are measured only once during the 12-week
experiment may be necessary (Snedecor and Cochran 1980).
9.1.3.1 Statistical Methods - In order for any statistical
procedures to be applicable, all experimental microcosms must be assigned
to the experimental treatment level by some random process. This may be
accomplished through use of a completely randomized experimental design,
randomized block, Latin-square or other appropriate experimental designs.
As stated earlier, if an appropriate experimental design is not used, the
results and analysis may be biased, thus jeopardizing the outcome of the
experiment. Analysis of variance (ANOVA) procedures should be performed
on biomass data in order to determine if an ecological effect resulted
from the treatment levels of the parent compound and transformation
products. If the ANOVA is significant, then Orthogonal comparisons or a
multiple-range comparison such as Duncan's should be performed in order to
determine which of the treatment means were different from the others.
The undosed controls are considered to be one of the treatment levels.
Again, the 5i level (o = 0.05) should be considered as the level of
significance for all tests and the power should be 0.90 or 0.95. All
values, whether significant or not, must be reported for each statistical
test being performed. Where more than a single factor or treatment is
incorporated into the original experimental design a factorial ANOVA is
the appropriate test to conduct.
9.1.3.2 Regression analysis should subsequently be performed on the
productivity results. Outlier data, defined as an obvious data recording
or reporting error, should be excluded; however, these data and the fact
that they have been excluded must be reported. If a substantial number of
data points have been declared as outliers, deficiencies in quality
control may necessitate rerunning the test. Once outlying values have
been detected and removed from further statistical evaluations, regression
models or Probit analysis can be used to estimate the concentration where
501 of the productivity realized in the controls occurred for the test
-25-
This document i» In process of development and it for ASTM co»»ittee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM committee
• ct1v1ties except with the approval of the chairman of the cownittee with jurisdiction or
the President of th« Society.
-------
substance (EC5Q). Ordinary linear least squares regression analysis
may be performed to define the response of relevant production parameters
as functions of dose. If it appears that productivity 1s nonlinear with
respect to dose, it may be necessary to transform the data or fit either a
quadratic or cubic least-squares regression model to the data for this
type of response. Utilization of computer software packages such as SAS
(Statistical Analysis System) or BMDP (Biomedical Computer Program) may
prove useful.
9.1.4 Physical Appearance of Plants - Changes in physical
appearance in plants in the terrestrial microcosms should be reported for
all test units. No statistical evaluations of the effects of the chemical
and transformation products on appearance are to be performed unless there
is a clearly identifiable pattern of effects. In this case, clearly
recognizable patterns of injury to plants may be ranked in terms of
severity. A non-parametric test, such as Kruskal-Wallis test, may be
conducted to test for differences in plant injury.
9.1.5.1 nutrient Losses - Based on the nutrient or nutrients
selected for analysis in the soil leachate (see Section 8.3.1) collected
from each microcosm, the total cumulative nutrient loss for each test
system for each nutrient should be calculated by (a) multiplying nutrient
loss concentration from each collection date times the total volume
leached from that microcosm for that collection date, and (b) then adding
it to the previous sum of total loss. Plots of the collection date means
(±SE) of the cumulative nutrient losses for each treatment level should be
developed as a function of days after seeding for the agricultural
microcosm or days after exposure for the natural grassland microcosm.
Zero loss should be the starting point; if there was no leachate for any
microcosm during a particular collection period, the data point should be
recorded as zero so that no data are considered missing.
9.1.5.2 A one-way ANOVA should be performed on the total cumulative
nutrient loss data at the end of the experiment, to evaluate the treatment
level effects. A multiple comparison procedure, such as Duncan's, may be
_ -26-
This document i» in process of development and is for ASTM conmittee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM conwnittee
activities except with the approval of the chairman of the coawittee with jurisdiction or
the President of the Society.
-------
used to determine which specific treatment means were different from each
other.
9.1.5.3 Regression/correlation analysis comparing nutrient losses
versus productivity should be performed to determine the relationship
between these two independently measured variables. In order to perform
this analysis the productivity and cumulative leachate loss measures, must
be matched unit for unit. This test should be performed for each nutrient
analyzed versus productivity.
9.2 Chemical Tate Analysis - At the end of the experiment, the
budget or distribution of the parent compound and transformation products
1s calculated for each exposure (concentration) level. This entails
determining the amount added and the subsequent distribution of the
radioactivity of the parent compound and transformation products through
chemical analysis in each of the primary compartments, soil, H20, plant
tissue and air of the test system as well as in the soil leachate. Fate
analysis should result in distribution values for above-ground plant
tissues, plant roots, each soil depth (see Section 8.2.3), and loss to
soil leachate. Gaseous loss can be estimated. This is followed by
performing statistical analyses for each exposure level on any differences
in distribution of the compound throughout the test system.
Multi-compartmental modeling and multivariable analyses such as
multivariate analysis of variance may also prove useful in assessing the
fate of parent compounds and transformation products.
9.2.1.1 Radioactivity Budgets - The calculation of a complete
budget of all radioactivity is required to be submitted with the results
of this test. The budget must show the percent of the compound that was
tagged and the location of the tag. The label may be 1UC, stable 15N, or
another suitable label and should be located in a portion of the molecule
expected to persist and/or have biological activity.
9.2.1.2 Total Radioactivity Added - The total radioactivity added
per test unit is based on the decay rate of the radioactive tag, the total
amount of radioactive tag added to the compound when initially formulated,
-27-
Ihfs document is in process of development and 1s for ASTM co«»itte* use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outtide of ASTM committee
activities except with the approval of the chairman of the corwitte* irith jurisdiction or
the President of the Society.
-------
the length of time between formulation and test unit exposure (radioactive
decay), and the particular exposure level of the test unit.
9.2.1.3 Total Radioactivity Removed - The total radioactivity
removed from the microcosm is based on the concentration of the radiolabel
in (a) the soil leachate concentration times the volume of soil leachate
lost per collection date, (b) the calculated gas phase losses of the
compound, and (c) the type of radiolabel and the rate of radioactive decay
of that label over the length of the experiment.
9.2.1.4 Total Radioactivity Remaining - The total radioactivity
remaining in the microcosm is based on radiolabel analysis of each of the
primary compartments (a) above-ground plant tissues, (b) plant roots
(i.e., cleaned of soil particles) and (c) the distribution of the .label
through the different soil depths. The soil depths to be analyzed will be
-based on a range-finding evaluation as suggested in section 8.2.3.
9.3.1 Fate of Parent Compound and Transformation Products -
Calculations should be made for each exposure (concentration) level
relative to the percent distribution of the test substance for (a) above-
ground plant tissues, (b) below-ground plant tissues, (c) each depth
through the soil profile, (d) losses to soil leachate, and (e) calculated
total vaporization or gaseous losses. In addition, an analysis of the
time to reach steady-state loss of the chemical compound in the soil
leachate and the time to initiate compound leaching from each exposure
level should be calculated. For those exposure levels where the time to
reach steady state loss in the soil leachate is greater than the length of
the experimental period (12 weeks) then an extrapolation of a regression
model may be used to estimate the time necessary to reach steady state
(Snedecor and Cochran 1980). This should be done cautiously and with the
assumption that the shape of the response beyond the experimental period
is the same as that estimated for the experimental period.
-28-
This document is in process of development and Is for A5TM committee use only. It shall not
be reproduced or circulated or quoted, in whole OP in part, outside of ASTM comnittee
activities except with the approval of the chairman of the cownittee with jurisdiction or
the President of the Society.
-------
9.3.2 The calculations for the final distribution of the test
material and transformation products are based on the measured radio-
activity 1n that compartment on a per gram basis times the total weight or
volume of material 1n that compartment, expressed on a dry weight basis
where appropriate. All calculations, are subsequently corrected for the
radioactive decay occurring since the beginning of the experiment. The
quantities of compound are to be expressed as a percent of the original
compound added to the test system. An ANOVA test should be performed on
the calculated mean percentage remaining for each of the four main
compartments to discern any differences among exposure concentrations. It
may be necessary to transform the percentage data prior to ANOVA 1n order
to satisfy the assumptions of the analysis. In addition, regression
analyses should be performed comparing above-ground productivity versus
concentration of parent compound and transformation products in
above-ground plant tissues, and cumulative nutrient losses for each
exposure concentration versus parent compound and transformation products
in the soil leachate.
9.4.1 B-ioconcentraticn and Enrichment - Calculate the concentra-
tion of the radioactivity in the above-ground plant tissues and in the top
15 cm of soil on a concentration per unit dry weight basis. The ratio of
the plant tissue concentration to soil concentration is then defined as
the Bioconcentration Factor (BF). Side-by-slde histograms displaying the
BF ratios should then be compared for statistical differences.
9.4.2 Enrichment ratios (ER) should be calculated for the higher
exposure levels by dividing the activity expressed on a dry weight basis
in a higher exposure level by the lowest exposure level in which a
significant (i.e., detectable) concentration could be analytically
determined. The enrichment ratio is then regressed against exposure
concentration to determine the linearity of the uptake of the chemical as
measured through the radiolabeled portion of the chemical. If it appears
that an ordinary least squares regression analysis is not the appropriate
function defining the enrichment of the chemical then an attempt to fit a
quadratic or cubic equation should be made. The above guidelines for
-29-
This document 1* in process of development and 1» for ASTM committee u»e only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM committee
activities except with the approval of the chairman of the committee with jurisdiction or
the President of the Society.
-------
calculating BFs and ERs assume that no wet analytical chemistry has been
performed to separate the parent compounds from the transformation
products in each compartment. If this has been done, then individual BFs
and ERs should be calculated as outlined above.
-30-
Tr.is document Is In process of development and is for ASTM committee use only. It shall not
be reproduced or circulated or quoted, in whole or In part, outside of ASTX co»«ittee
activities except with the approval of the chairman of the committee with jurisdiction or
the President of the Society.
-------
10.0 TEST PROTOCOL BIBLIOGRAPHY
Association of Official Analytical Chemists. 1975. Official Methods of
Analysis of the Association of Official Analytical Chemists. 12th
Edition. Washington, D.C., 1094 pp.
American Society for Testing and Materials. 1979. Annual book of ASTM
Standards. Part 31, Water. American Society for Testing and Materials,
Philadelphia, PA. 128 pp.
Ausmus BS, Dodson, GF, & Jackson DR. 1978. Behavior of heavy metals in
forest microcosms: III. Effects on litter-soil carbon metabolism. Water
A1r Soil Pollut. 10:19-29.
Ausmus BS, Kimbrough S, Jackson DR, & Llndberg S. 1979. The behavior of
hexachlorobenzene in pine forest microcosms: transport and effects on
soil processes. Environ. Pollut. 20:103-111.
Black, CA (ed). 1965. Methods of Soil Analysis I. Physical and
Minerological Properties. American Society of Agronomy, Madison, WI.
770 pp.
Black, CA (ed). 1982. Methods of Soil Analysis II. Chemical and
Microbiological Properties. 2n Edition American Society of Agronomy,
Madison, WI. 802 pp.
Cairns J, Jr. 1981. Testing for effects of chemicals on ecosystems.
National Academy of Science, Washington, D.C. 103 pp.
Cairns J, Jr. ed. 1985. Multispecies Toxicity Testing. Pergamon Press,
New York. 253 pp.
-31-
TM» docunent is 1" process of development and 1» for ASTH coewittee use only. It shall not
be reproduced or circulated or quoted, 1n »hole or in part, outside of AST* co«»1ttee
activities except with the approval of the chairman of the co««iitte« with jurisdiction or
the President of the Society.
-------
Cole LK, Sanborn OR, & Metcalf RL. 1976. Inhibition of corn growth by
aldrin and the insecticide's fate in the soil, air, crop, and wildlife of
a terrestrial model ecosystem. Environ. Entomol. 5:583-589.
Daubenmire, R. 1959. A canopy cover method of vegetation analysis. N.W.
Science 33: 43-64.
Elseewi AA, Straughan IR, & Page AL. 1980. Sequential cropping of fly
ash-amended soils: Effects on soil chemical properties and yield and
elemental composition of plants. Sci. Tot. Environ. 15:247-259.
Environmental Protection Agency. 1979. Manual of Analytical Methods for
the Analysis of Pesticide Residues 1n Human and Environmental Samples.
Health Effects Research Laboratory. Research Triangle Park, N.C.
Environmental Protection Agency. 1982a. Environmental Effects Test
Guidelines. Washington, DC. Office of Pesticides and Toxic Substances,
U.S. EPA. EPA 560/6-82-002. Available from NTIS, Springfield, VA
PB82-232992.
Environmental Protection Agency. 1982b. Chemical Fate Test Guidelines.
Washington, DC. Office of Pesticides and Toxic Substances, U.S. EPA. EPA
560/6-82-003. Available from NTIS, Springfield, VA PB82-233008.
Environmental Protection Agency (In Press). Experimental Terrestrial
Soil-Core Microcosm Test Protocol. A Method for Measuring the Potential
Ecological effects, Fate, and Transport of Chemicals in Terrestrial
Ecosystems. Con/all is Environmental Research Laboratory Research Report
71.
Gile JD, & Gillett JW. 1979. Fate of selected fungicides in a terres-
trial laboratory ecosystem. J. Agric. Food Chem. 27:1159-1164.
-32-
TMs document is in process of development and is for ASTM committee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM committee
activities except with the approval of the chairman of the committee with jurisdiction or
the President of the Society.
-------
Harris WF, Ausmus BS, Eddlemon GK, Giddings JM, Jackson DR, Luxmore RJ,
O'Neill EG, O'Neill RV, Ross-Todd M, and Van Voris P. 1980. Microcosms
as potential screening tools for evaluating transport and effects of
toxic substances. ORNL/EPA-4, EPA-600/3-80-042. Environ. Sci. Div.
Pub!-No. 1506, Oak Ridge, TN. 453 pp.
Harvey J, Jr. 1983. A simple method of evaluating soil breakdown of lkC
pesticides under ffeld conditions. Residue Rev. 85:149-158.
Jackson DR, Selvidge WJ, & Ausmus BS. 1978. Behavior of heavy metals in
forest microcosms: II. Effects on nutrient cycling processes. Water Air
Soil Pollut. 10:13-18.
Jackson DR, Ausmus BS, & Levin M. 1979. Effects of arsenic on nutrient
dynamics of grassland microcosms and field plots. Water Air Soil Pollut.
11:3-12.
Jones JB, & Steyn WJA. 1973. Sampling, handling, and analyzing plant
tissue samples, pp. 249-270. In: LM Walsh and JD Beaton eds. Soil
Testing and Plant Analysis. Soil Science Society of America, Inc.,
Madison, WI.
Kastenbaum, M. A. and D. G. Hoel. 1970. Sample size requirements:
one-way analysis of variance. Biometn'ka 57(2):421-430
Laskowski DA, Goring CAI, McCall PJ, & Swann RL. 1982. Terrestrial
environment, pp. 198-240. In: RA Conway ed. Environmental Risk Analysis
for Chemicals. Van Nostrand Reinhold Company,-New York, NY.
Lichtenstein EP, Schulz KR, & Fuhremann TW. 1972. Movement and fate of-
dyfonate in soils under leaching and nonleaching conditions. J. Agr. Food
Chem. 20:831-838.
-33-
TMs document ii 1" process of development and Is for ASTX conittee use only. It shall not
be reproduced or circulated or quoted, In whole or 1n part, outside of ASTM committee
activities except with the approval of the chairman of the co*e;ttee with jurisdiction or
the President of the Society.
-------
Lichtenstein EP, Fuhremann TW, Schulz KR, Hang TT. 1973. Effects of
field application methods on the persistence and metabolism of phorate 1n
soils and its translocation into crops. J. Econ. Entomol. 66:863-866.
Lichtenstein EP, Fuhremann TW, & Schulz KR. 1974. Translocation and
metabolism of (UC) phorate as affected by percolating water 1n a model
soil-plant ecosystem. J. Agr. Food Chem. 22:991-996.
Malanchuk JL, Mueller CA, & Pomerantz SM. 1980. Microcosm evaluation of
the agricultural potential of fly-ash-amended soils, pp. 1034-1049. In:
JP Giesy, Jr. ed. Microcosms 1n Ecological Research, CONF-781101. U.S.
Department of Energy, Technical Information Center.
Metcalf RL, Kapoor IP, Lu P, Schuth CK, & Sherman P. 1973. Model
ecosystem studies of the environmental fate of six organochlorlne
pesticides. Environ. Hlth. Perspect. 4:35-44.
O'Neill RV, Ausmus BS, Jackson DR, Van Hook RI, Van Voris P, Washburne C,
& Watson AP. 1977. Monitoring terrestrial ecosystems by analysis of
nutrient export. Water Air Soil Pollut. 8:271-277.
Neter, J & Wasserman W. 1974. Applied linear statistical models, Irwin.
Homewood, IL.
Parkinson, D, TRG Gray, ST Williams. 1971. Methods for Studying the
Ecology of Soil Micro-organisms. IBP-Handbook No. 19. Blackwell
Scientific Publications, Oxford, England. 115 p.
PhilHpson, J. 1971. Methods of Study in Quantitative Soil Ecology,
Population, Production and Energy Flow. IBP-Handbook No. 18. Blackwell
Scientific Pub-lications, Oxford, England. 297 p.
-34-
TMs document is in process of development and Is for ASTM coowittee use only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM coawittee
activities except with the approval of the chairman of the cownittee with jurisdiction or
the President of the Society.
-------
Rand GM. 1980. Detection:bioassay. Pages 390-403. In: Guthrle FE &
Perry JJ., eds. Introduction to Environmental Toxicology. Elsevier North
Holland, Inc., N.Y. 484 pp.
Schlndler JE, Waide JB, Waldron MC, Mains JJ, Schreiner SP, Freeman ML,
Beng SL, Pettigrew DR, Schissel LA, & Clark PJ. 1980. A microcosm
approach to the study of biogeochemical systems. I Theoretical
rationale, pp. 192-203. In: JP Giesy, Jr. ed. Microcosms in Ecological
Research, CONF-781101. U.S. Department of Energy, Technical Information
Center.
Snedecor GW & Cochran WG. 1980. Statistical Methods. 7th Edition.
Iowa State University Press, Ames, Iowa.
Sokal RR 4 Rohlf FJ. 1981. Biometry: The principles and practice of
statistics in biological research 2nd Edition. W. H. Freeman and
Company, San Francisco, CA.
Steele RDG & Torrie BH. 1980. Principals and Procedures of Statistics
2nd Edition. McGraw-Hill Book Co., N.Y. 481 pp.
Swann RL, Laskowski DA, McCall PJ, Vander Kuy K, & Dishburger HJ. 1983.
A rapid method for the estimation of the environmental parameters
octanol/water partition coefficient, soil sorption constant, water to air
ratio, and water solubility. Residue Reviews. 85:17-28.
Tolle DA, Arthur MF, Van Voris P, Feder PI, Matthews MC, Chesson J,
Larson ME, & Zwick TC. 1982. Evaluation of terrestrial microcosms for
environmental studies of utility wastes. Third Ann. Prog. Rep. on RP
1224-5 to Electric Power Research Institute, Palo Alto, CA.
-35-
Th" $ document is In process of development and Is for ASTM coa»ittee ute only. It shall not
be reproduced or circulated or quoted, in whole or in part, outside of ASTM conwittee
activities except with the approval of the chairman of the coawitte* with jurisdiction or
the President of the Society.
-------
Tolle DA, Arthur MF & Van Vorls P. 1983. Microcosm/field comparison of
trace element uptake in crops grown in fly ash-amended soil. Sci. Tot.
Environ. 31: 243-261.
Van Voris P, O'Neill RV, Emanuel WR, & Shugart Jr, HH. 1980. Functional
complexity and ecosystem stability. Ecology 61(6):1352-1360.
Van Yoris P, Arthur MF, & Tolle DA. 1982. Evaluation of terrestrial
microcosms for assessing ecological effects of utility wastes. EPRI
Publication No. EA-2364. Electric Power Research Institute, Palo Alto,
CA. pp. 178.
Van Voris P, Arthur MF, Tolle DA, Morris JP, & Larson M. 1983. Use of
microcosms for monitoring nutrient cycling processes in agroecosystems. p.
171-182. In: R. Lowrance, R. Todd, L. Asmussen, and R. Leonard (eds.)
Nutrient Cycling in Agricultural Ecosystems. University of Ga. College of
Agric. Exp. Station, Spec. Pub. 23. Tifton, GA.
Van Voris P, Tolle DA, Arthur MF, Chesson J, & Zwick TC. 1984.
Development and validation of terrestrial microcosm test system for
assessing ecological effects of utility wastes. Final Project Report
RP1224-5. EPRI Publication No. EA-3672. Electric Power Research
Institute, Palo Alto, CA. pp. 185.
Van Voris P, Tolle DA, Arthur MF, & Chesson J. 1985. Terrestrial
Microcosms: validation, applications, and cost-benefit analysis.
pp 117-142 In: J Cairns, Jr. ed. Multispecies Toxicity Testing. Pergamon
Press, New York.
Walters. DB. (ed) 1980. Safe Handling of Chemical Carcinogens, Mutagens,
Teratogens and Highly Toxic Substances. Volumes I and II. Ann Arbor
Science Publishers Inc., Ann Arbor Michigan. 652 pp.
-36-
rnis document is in process of development and Is for ASTM comnittee use only. It shall not
be reproduced or circulated or quoted, 1n whole or In part, outside of ASTM cowMttee
activities except with the approval of the chairman of the committee with jurisdiction or
th« President of the Society.
-------
Wang, CH and DL W1THs. 1965. Radlotracer Methodology in Biological
Sciences. Prentice-Hall, Inc. Englewood Cliffs, New Jersey. 382 pp.
Zwick TC, Arthur MF, Tolle DA, & Van Voris P. 1984. A unique laboratory
method for evaluating agro-ecosystem effects of an industrial waste
product. Plant Soil. 77: 395-399.
-37-
shall not
TM» document is in process of development «nd it for ASTH eowittee use only. It sh
be reproduced or circulated or quoted, in whole or in part, outside of AST* committee
activities except irlth the approval of the chairwan of the coanittee with jurisdiction or
the President of the Society.
-------
APPENDIX B
A noninclusive list of sample sources of biological materials for
enclosed species tests
1. Ward's Natural Science Establishment, Inc.
11850 E. Florence Ave.
Santa Fe Springs, CA. 90670-4490
2. Harris Moran Seed Co.
1155 Harkins Rd.
Salinas, CA. 93901
3. Carolina Biological Supply Co.
Burlington, North Carolina 27215
4. California Department Food and Agriculture
Biological Control Services Program
Sacramento, CA. 95832
150
-------
-1-
Life-Cycle Characteristics of Non-Target Terrestrial Organisms
for Testing Microbial Pest-Control Agents
Prepared by L.K. Etzel
System
A. Amblyseius hibisci
1. Length of life cycle:
Egff - Egg: 11 days @ 25.5'C; 6C% R.H.
«
2. Immature stage:
Type of foodr Pollen of oak, magnolia, cattail, Mesembryanthemum spp.;
Panonychus citri (citrus red mite);
Oligonychus punicae (brown almond mite)
Quantity:
5. Adult:
Type of food: Same
Quantity: Ovipositing female eata 3 to 8 adult 0_. punicae per day
Longevity: X ovi positional period = 19.6 days
Fecundity: Ca. 2 eggs/female/day; Total ca. 40 eggs
4. Other notes:
B. Panonychus citri
1. Length of life cycle:
Egg - Adult: 14 days @ 2J.9*C; 65% R.H.; on lemon fruit
33 days @ 15.6"C; 6596 R.H.; on lemon fruit
2. Immature stage:
Type of food: Citrus seedlings or lemon fruit
Quantity:
3w Adult
Type of food: Same
Quantity:
Longevity: 23 days @ 23.9'C; 65% R.H.; on lemon fruit
36 days @ 15.6*C; 65% R.H.; on lemon fruit
Fecundity: Total ca. 36 eggs 0 23.9*0 as above
Total ca. 50 eggs @ 15.6'C as above
4. Other notes:
C. References
1. Davis et al. (1979)
-------
-2.
Life-Cycle Characteristics (con't)
System #1 Ccon't)
2.
3.
4.
5.
6.
7.
8.
9.
10.
Kennett and Hamai (1980)
McMurtry (1970)
McMtortry and Johnson 1966
McMurtry and Scriven 1964
McMurtry and Scriven 1965
McMurtry and Scriven 1966
McMurtry, Huffaker, and van de Vrie (1970)
Monger (1963)
van de .Vrie, McMurtry, and Hnffaker (1972)
-------
-3-
Life-Cycle Characteristics (con't)
System #2
A. Metaaeiulus occidental is
1. Length of life cycle:
Egg - Adult: 6.3 days ® 24* C
2. Immature stage:
Type of food: Tetranychus urticae (two—spotted mite)
T. pacificus (Pacific spider mite)
Various other Tetranychid mites
Quantity: 16-17 mite eggs during development
3. Adult:
Type of food: Same
Quantity: Ovipositing female eats ca. 11 small two-spotted mite
nymphs/day
Longevity: X ovipositional period =17.3 days
Fecundity: Ca. 2 eggs/female/day; Total ca. 35 eggs
4. Other notes:
B. Tetranychus urticae
1. Length of life cycle:
Egg - Egg: 19 days @ 20.3'C; 15.5 hr. light/day
2. Immature stage:
Type of food: Attacks an extremely wide host range of plants
Quantity:
3. Adult:
Type of food: Same
Quantity i
Longevity: 18 days @ 20.3'C
Fecundity: Ca. 2.4 eggs/female/day; Total ca. 38 eggs
4* Other notes:
C. References
Davis et al., (1979)
Kennett and Hamai (1980)
McMurtry and Scriven (19^5)
McMurtry, Huffaker, and van de Vrie (1970)
van de Vrie, McMurtry, and Huffaker (1972)
8
(0.
11. IgliMky and Rainwater (1954)
12. Lfting (1969)
(1970
-------
-4-
Life-Cycle Characteristics (con't)
System #1
A. Phytoseiulus persi'milis
1. Length of life cycle:
Egg - Adult: 3.8 days @ 30'C
4.6 days @ 25'C
7.4 days @ 20'C
2. Immattote stage:
Type of food! Tetranychua -urticae (two-spotted spider mite)
Quantity:
3. Adult:
Type of food: Same
Quantity* Ovipositing female eats ca. 3 adtilfr female two-spotted
mites/day or ca. 34 mite eggs
Longevity: X ovipositional period ca. 14 days @ 26*0
Fecundity: Ca. 4 eggs/female/day; Total ca. 54 eggs
4. Other notes:
B. Tetranychus urticae
See info under System #2.
C. References
Same as for System #2.
-------
-5-
Life-Cycle Characteristics (con't)
System #4
A. Geocorla punctities (big-eyed bug)
1. Length of life cycle:
Egg - Adult: 34 days @ 26.7*C (incl. ca. 9 days in the egg stage)
2. Immature stage:
!Iype «of food: Lygus bugs, spider mi tea, aphids, leafhoppers, and
moth eggs and larvae
Quantity: 20-30 moth eggs/day/nymph
3. Adult:
Type of food: Same
Quantity: 50 - 60 moth eggs/day/adult
Longevity: Female ca. 68 days @ 25»5"C
Male ca. 42 days @ 25.5"C
Fecundity: Ca. 3.6 eggs/female/day; Total ca. 75 eggs @ 23.9*C (20& hatch)
Ca. 7.2 eggs/female/day; Total ca. 126 eggs 0 26.7*C (4956 hatch)
4. Other notes:
B. Trichoplusia ni (cabbage looper)
1. Length of life cycle:
Egg - Adult: 20 - 21 days @ 28*C (incl. 1J - 14 days in the larval stage)
2. Immature stage:
Type of food: A variety of plants, including cabbage, turnip, lettuce,
pea, and cotton; artificial diet.
Quantity:
3. Adult:
Type of food: Sugar water
Quantity:
Longevity: Ca. 8 days
Fecundity: X = ca. 400 eggs
4. Other notes:
C. References
14. Champlain and Sholdt (1967)
15. Cohen (1981)
16. Cohen (1985)
17. Cohen and Debolt (1983)
-------
Life-Cycle Characteristics (con't)
System #4 Ccon't)
18. Dunbar and Bacon
19. Duribar and Bacon (1972b)
20. Guy et al.(l985)
-------
-7-
Life-Cycle Characteristics (con't)
System #5
A. Chrysonerla carnea (green lacewing)
1. Length of life cycle:
Egg - Adult: 24 - 27 days 0 26.7'C; 75% R.H. (incl. 15-16 days as larva)
2. Immature stage:
Type tof food: Principally aphids and moth eggs and young larvae
Quantity: Ca. 30 - 40 mg moth eggs/larva; ca. 80 mg aphids/larva
3. Adult:
Type of
Quantity:
Longevity: 80 - 82 days @ 20*C & 8096 R.H. with proper food
Fecundity: Depends on food; artificial diet - ca. 890 eggs over 75
days; honey - 32 eggs over 46 days
4. Other notes: Rearing must be conducted with at least 14 hr. of light
per day to prevent diapause.
B. Acvrthosiphon pisxim (pea aphid)
1. Length of life cycle:
Kymph - Nymph: 9-10 days 0 21*C
2. Immature stage:
Type of food: Pava bean plants;(alfalfa^ other legumes
Quantity:
3. Adult:
Type of food: Same
Quantity:
Longevity: Estimate 10 days
Fecundity: Ca. 9 nymphs/female/day; ca. 90 nymphs total
4. Other notes:
C. References:
(1) Davis et al..(l979)
21. Canard et al. (1984)
22. Forbes et al. (1985)
23. Morrison (I985a)
-------
-8-
Life—Cycle Characteristics (con't)
System #6
A. Hi-p-podamia convergens (convergent ladybird beetle)
1. Length of life cycle:
Egg - Adult: 17 days @ 26*0 (incl. ca. 9 days as larva)
2. Immature stage:
Type of foodv Aphids
Quantity: Estimate 15 mg/larva/day; or 140 - 170 mg/larval stage
3. Adult:
Type of food: Same
Quantity: Estimate 25 mg/adult/day; or ca. 1350 mg/adnlt stage
Fectmdity: Ca. 1270 eggs; ca. 25 eggs/female/day
Longevity: X = 60 days @ 22.8*0
4. Other notes: Bearing should be conducted with at least 11 hours of light.
B. Acyrthosi-phon. pisum
See info under System #5.
0. References:
(1) Davis et al. (1979)
(22) Porbes et al. (1965)
24. Clausen (1916)
25. Hagen (1962)
26. Hagen and Sluss (1966)
27. Simpson and Burkhardt (i960)
-------
Life-Cycle Characteristics (con't)
System -#7
A. Olla abdominalis (ashy-gray ladybird beetle)
1. Length of life cycle:
Egg - Adult: 15 days 0 26'C (incl. 8 days as larva)
2. Immature stage:
Type ,of. food: Aphids
Quantity: Estimate 15 ing/larva/day; or 125 ug/larval stage
3. Adult:
Type of food: Same
Quantity: Estimate 15 mg/adult/day; or ca. 675 ing/adult stage
Longevity: Estimate 45 days
Fecundity: Ca. 295 eggs; ca. 8 eggs/female/day
4. Other notes:
B. Acyrthosi-ahon pi sum
See info under System #5.
C. References:
(1
22
24
25
27
Bivis et al. (1979)
Forbes et al. (1985)
Clausen
Hagen
Simpson and Burkhardt (i960)
-------
-10-
Life-Cycle Characteristics (con't)
System #8
A. Cryptolaemus montrouaieri (mealybug destroyer)
1. Length of life cycle:
Egg - Adult: 28 - 29 days @ 26.7*C (incl. ca. 15 days as larva)
43 - 47 days 0 21.1*0 (incl. oa. 25 days as larva)
2. Immature stage:
Type of food: Mealybugs
Quantity:
3. Adult:
Type of food: Same
Quantity:
Longevity: X = 51 days @ 25"C
Fecundity: Ca. 440 eggs; ca. 9 eggs/female/day @ 25*C
4. Other Notes:
B.' Pseudocoecus citri (citrus mealybug)
1. Length of life cycle:
Egg - Adult: Estimate 4-5 weeks © ca. 21.1'C
2. Immature stage:
l^pe of food: Vide host range of plants; especially severe on citrus
Quantity:
3. Adult:
Type of food: Same
Quantity:
Longevity: Estimate 4 weeks
Fecundity: Ca. 300 - 500 eggs
4. Other notes:
C. References:
28. Fisher (1963)
-------
-11
Life-Cycle Characteristics (con't)
System #9
A. Trichogramma. spp. (parasites of insect eggs)
1. Length of life cycle:
Egg - Adult: 8-10 days 0 26.?*C; 80# R.H.; continuous light
2. Immature stage:
Type °f food: Primarily attack eggs of lepidopterans
Quantity: 1-2 parasites/egg
3. Adult z
Typ* of food: Honey
Quantity:
Longevity:
Fecundity: Less than 100 eggs
4* Other notes:
B. Qatrinia nubilalis (European corn borer)
1, Length of life cycle:
Egg - Adult: 21 - 28 days @ 27*C; 75 - 8C$ R.H.; contirruoxis light
(incl. 13-19 days as larva)
2. Immature stage:
Type of food: Vide host range, including corn, potatoes, beans,
celery, etc.; artificial diet
Quantity:
3. Adult:
T^pe of food: Water
Quantity:
Longevity: 12 days
Fecundity: X = 300 eggs/female
4* Other notes:
C. References:
29. Burbutis and Goldstein (1983)
30. Guthrie et al. (1985)
31. Morrison (l985b)
-------
-12-
Life-Cycle Characteristics (con't)
System #10
A. Anaphea flavipes (egg parasite of cereal leaf beetle)
1. Length of life cycle:
Egg - Adult: 8-9 days @ 30*C; 80-$ R.H.
2. Immature stage;
Type of food: Eggs of cereal leaf beetle
Quantity: 3+ parasites/egg
3. Adult:
Type of food: Honey
Quantity:
Longevity: 4-5 days @ 21 *C; 8O$ H.H.
Fecundity: Ca. 21 eggs/female
4* Other notes:
B. Oulema melanopua (cereal leaf beetle)
1. Length of life cycle:
Egg - Adult:
2. Immature stage:
Type of food:
Quantity:
3. Adult:
Cfype of food:
Quantity:
Longevity:
Fecundity:
4* Other notes:
C. References:
32. Anderson and Paschke (1968)
-------
Life-Cycle Characteristics (con't)
Sstem
A. Aphidiua ervi (parasite of pea aphid)
1. Length of life cycle:
Egg - Adult: Ca. 14 days © 21.1*0; 55% R.H.
2. Immature stage:
Type ,of food: Pea aphids
Quantity: 1 parasite/aphid
3. Adult:
Type of food: Honeydew or honey
Quantity:
Longevity: 8-10 days @ 21.1*0
Fecundity: Ca. 50 adult progeny/female with large-scale rearing
4. Other notes:
B. Acyrthosiphon pisum
See info under System #5,
C. References:
(22) Forbes et al. (1985)
33. Fox et al. (19*7)
34* Quezada (unpubl.)
-------
-14.
Life-Cycle Characteristics (con't)
System #12.
A. Diaretiella rapae (parasite of green peach aphid and cabbage aphid)
1. Length of life cycle:
Egg - Adult: 10.4 days @ 25*0
14.7 days 0 20'C
20.2 days 0 15'C
2. Immature stage:
type of foodii Green peach aphids or cabbage aphids
Quantity: 1 parasite/aphid
3. Adult:
type of food: Honeydew or honey
Quantity:
Longevity: Maximum of 6 days @ ca. 19*0
Fecundity: Ca. 230 progeny/female
4. Other notes:
B. Brevicorynae brassicae (cabbage aphid)
1. Length of life cycle:
Nynph - IJymph: Estimate 10-14 days @ 21.1*0
2, Immature stage:
type of food: Wide variety of cole plants, including cabbage, collards,
and rape
Quantity:
3. Mult: . •
type of food: Same
Quantity:
Longevity: Ca. 30 days in nature in summer
Fecundity: Ca. 80 - 100 nymphs
4. Other notes:
C. References:
(22) Forbes et al. (1985)
35. Hagen and van den Bosch (1968)
36. Simpson et al. (1975)
-------
-15-
Life—Cycle Characteristics (con't)
System #13
A. Lysiphlebus testaceipes (parasite of greenbug)
1. Length of life cycle:
Egg - Adult: 11.1 days @ 21 - 32*C
10.8 days @ 27'C
13.2 days @ 21*C
2. Immature stage:
*
Type of food: Greenbugs
Quantity: 1 parasite/aphid
3. Adult:
Type of food: Eoneydew or honey
Quantity:
Longevity: 2-3 days @ 23.3*0
Fecundity:
4. Other notes: The parasite is not active at temperatures below 18.3'C,
but the greenbug can reproduce at temperatures as low as
4.4'C.
B. Schizatihis grannnuin (greenbug)
1. Length of life cycle:
Nymph - Nymph: 6 days @ 26.7"C
9 days @ 21.1*0
15 days @ 15.6*C
2. Immature stage:
Type of food: Wheat, barley, oats, rye, and sorghum
Quantity:
3. Adult:
Type of food: Same
Quantity:
Longevity: Ca. 20 days @ 22.2*C
Fecundity: Ca. 100 nymphs/female
4. Other notes:
C. References:
(22) Forbes et al. (1985)
37. Headlee (1914)
38. Hight et al. (1972)
39. Starks and Burton (1977)
-------
-16-
Life-Cycle Characteristics (con't)
System #14
A. Cotesia (sApanteles) melano3celTi3 (parasite of gypsy moth)
1. Length of life cycle:
Egg - Adult: Ca. 15 days @ 23*C; 4<$ R.H.; 18 hr. light daily
2. Immature stage:
Type of food: Larvae of gypsy moths
Quantity: 1 parasite/larva
3. Adult:
Type of food: Honey
Quantity:
Longevity: At least 2-3 weeks
Fecundity: Ca. 100 progeny/female
4. Other notes:
B. Lyaantria dispar (gypsy moth)
1. Length of life cycle:
Egg - Adult: Ca. 40-44 days @ 25*C; 50 - 6096 R.H.; 16 hr. light daily
2. Immature stage;
Type of food: Foliage of over 300 species of trees and shrubs, expecially
oaks; artificial diet
Quantity: Ca. 10 ml diet per larva
3. Adult:
Type of food: None
Quantity: N/A
Longevity: Males =3-5 days; females =2-3 days
Fecundity: Ca. 900 eggs/female
4. Other notes:
C. References:
40. Chianese (1985)
41. ODell et al. (1985)
-------
-17-
Life-Cycle Characteristics (con't)
System #15
A. Hetaphycus helvolua (parasite of black scale)
1. Length of life cycle:
Egg - Adult: 13 days 0 27.8*0
2. Immature stage:
lype of food: Black scale
Quantity: 1 parasite/scale
3. Adult:
TVpe of food: Honey
Quantity:
Longevity: 50 days Q 26.7"C, if fed honey; but can live much longer
with cool temperatures and host feeding
Fecundity: Ca. 400 eggs/female
4. Other-notes:
B. Saisaetia oleae (black scale)
1. Length of life cycle:
Egg - Adult: ?0 - 90 days @ 21"C
2. Immature stage:
*Eype of food: Wide host plant range among the ornamentals; can be
severe pest of citrus and oliv>_
Quantity:
3. Adult:
Type of food: Same
Quantity:
Longevity: Estimate 1 month @ 21' C
Fecundity: 1,000 - 4,000 eggs
4. Other notes:
C. References:
42. Clausen (1978)
43. Flanders (1942)
-------
-18-
Life-Cycle Characteristics (con't)
System #16
A. Aphytia. lingnanensis (parasite of California red scale)
1. Length of life cycle:
Egg - Mult: Ca. 15 days © 27'C; 60# R.H.
17 days @ 26*C; 6C# R.H.
2. Immature stage:
Type'of food: California red scale; oleander scale (in lab)
Quantity: 2'JOT more parasites/scale
3. Mult:
lype of food: Honey
Quantity:
Longevity: Ca. 10 days @ 26*C; 60$ R.H.
Fecundity: Ca. 5-6 eggs/day; ca. 57 eggs/female total on red scale;
more than twice this number on oleander scale; each female
destroys ca. an additional 46 scales by predation
4. Other notes:
B. Aspidiotis nerii (oleander scale)
1. Length of life cycle:
Crawler - Crawler: 60 days © 25*C; 50 - 6C% R.H.
2. Immature stage:
Type of food: Oleander plants; banana squash or butternut pumpkins
Quantity:
3. Adult:
Type of food: Same
Quantity:
Longevity: 10-14 days
Fedundity: X = 94 crawlers/female
4. Other notes:
C. References:
44. DeBach and White (1960)
45. Papacek and Smith (1985)
-------
-15-
Life-Cycle Characteristics (con't)
System #17
A. Brachymeria intermedia (parasite of gypsy moth and many other lepidopterans)
1. Length of life cycle:
Egg - Adult: 15 - 30 days @ 24*C
2. Immature stage:
Type of food: Larvae in several lepidopterous families
Quantity: 1 parasite/larva
3. Adult:
Type of food: Honey
Quantity:
Longevity: 4-6 weeks @ 24* - 27*C under forced lab prod.; 150 days
@ 21* - 24*C with honey and water and no hosts
Fecundity: Ca. 300 progeny/female
4. Other-notes:
B. 'Priehoplusia ni (cabbage looper)
See info under System #4.
C. References:
(20) Guy et al. (1935)
46. King and Hartley (1985a)
47. Palmer (1985)
-------
-20-
Life-Cycle Characteristics (con't)
System #18
A. Lixophaga diatraeaeslfly parasite on sugarcane borer)
1. Length of life cycle:
Egg - Adult: 18.2 - 20.9 days @ 26'C; 80# H.H.
2. immature stage:
Type ^of food: Larvae of the sugarcane borer and of the greater wax
moth (lab only)
Quantity: 1 'parasite/larva
3. Adult:
Type of food; Raw brown sugar
Quantity:
Longevity: Female = ca. 24 days @ 26* C
Male- = ca. JO days
Fecundity: Peak fecundity = 78 maggots in uterus on 12th day
4. Other notes:
B. Diatraea saccharalis (sugarcane borer)
1. Length of life cycle:
Egg - Adult: 36 - J8 days @ 26*0
2. Immature stage:
Type of food: Sugarcane or grain sorghum; artificial diet in lab
Quantity: Artificial diet - ca. 3.3 ml/larva
Grain sorghum stalk - 1.5"/larva
Sugarcane tops - ca. 15 gm/larva
3. Adult:
Type of food: Water
Quantity:
Longevity: Ca. 7 .days @ 26*C
Fecundity: 435 eggs/female @ 26*C
730 eggs/female @ 24*C
4. Other notes:
C. References:
(46) King and Hartley (l985a)
48. King and Hartley (l985b)
49. King and Hartley (1985c)
50. King and Martin (1975)
-------
-21
Life-Cycle Characteristics (con't)
System #18 (con't)
51. King et al. (1975!
52. King et al. (1975'
53. King et al. (1979,
54. Miles and King (1975)
55. Pan and Long (1961)
56. Wongsiri and Randolph (1962)
-------
-22-
Life-Cycle Characteristics (con't)
System #19
A. Apis mellifera (honeybee)
1. Adult:
type of food: Honey, pollen, and water
Quantity:
Longevity: Foragers live ca. 3 weeks in nature; what would happen in
a caged environment is unpredictable. Freshly emerged bees
i£E@0 the brood comb should live the longest (at least a.
month in the proper conditions) but may need to be fed pollen
in addition to honey. There should be at least 50 bees
placed in a microcosm so they can aggregate to maintain
suitable environmental conditions.
-------
-23-
Life-Cycle Characteristics (con't)
System #20
A. Vanessa cardui (Painted lady butterfly)
1. Length of life cycle:
Egg - Adult:
Egg - ca. 1 week in nature in summer
Larva - ca. 1 month in nature in summer
Pupa - ca. 2 weeks in nature in summer
Total - ca. 7 weeks
Immature stage j
Type of food:
Can feed on over 100 spp. of plants (chiefly in the
Malvaceae, Compositae, Leguminosae); plants include
thistles (a favorite), burdock, sunflowers, nettles;
can be a serious pest at times on maize, alfalfa, beans,
sunflowers, and soybeans. Artificial diets available.
Quantity:
Adult:
4.
Type of food:
Quantity:
Longevity: Ho info found, but should be quite long-lived because of
potential for migration.
Fecundity:
Other notes:
B. References:
57. Brooks and Knight (1982)
58. Singh (1985)
59. Williams, C.B. (197°)
-------
-24-
Proposed Non-Target Terrestrial Organisms
for Testing Hierobial Pest Control Agents
(Revised Bibliography)
Prepared by L.Z. Etzel
I. General Beferences
A, Bezark, L.G. and H. Tee
t985. 1985 Suppliers of Beneficial Organisms in North America. California
Department of Pood and Agriculture, Biological Control Services
Program,' ,3288 Meadowview Roady "Sacramento, CA 95852. 6 pp.
B. Dickerson, W.A., J.D. Hoffman, E.G. King, N.C. Leppla, and T.M. ODell
1979. Arthropod Species in Culture in the United States and Other
Countries. Entomological Society of ^SeTica, College Park, HD. 93 PP.
C. Singh, P. and R.P. Moore (eds.)
1985. Handbook of Insect Rearing. Volumes I and II. Elsevier, New
York. 488 pp. 4 514 PP.
II. Specific References
System #1
1. Davis, D.W., S.C. Hoyt, J.A. McMurtry, and M.T. AliNiazee (eds.)
1979. Biological Control and Insect Pest Management. University of
California Press, Berkeley. 102 pp.
2. Kennett, C.E. and J. Hamai
1980. Oviposition and development in predaceous mites fed with artificial
and natural diets (Acari:Phytoseiidae). Ent. Exp. Appl., 28;116-
122.
3. McMurtry, J.A.
1970« Some factors of foliage condition limiting population growth of
Oligonychus -punicae (AcarinatTetranychidae). Ann. Entomol. Soc.
Amer.. 631406-412.
• 4. McMurtry, J.A. and E.G. Johnson
1966. An ecological study of the spider mite Oligonychus punicae (Hirst)
and—its natural enemies. Hilgardia, 37;363-400.
•
5. McMurtry, J.A, and G.T. Scriven
1964. Studies on the feeding, reproduction, and development of Amblvseiua
hibisci (Acarina:Phytoseiidae) on various food substances. Ann.
Entomol. Soc. Amer., 57t649-655.
6. McMurtry, J.A. and G.T. Scriven
1965. Insectary production of phytoseiid mites. Jour. Econ. Entomol.,
5J3:282-284.
7. McMurtry, J.A. and G.T. Scriven
1966. The influence of pollen and prey density on the number of prey
consumed by Amblyseius hibisci (Acarina:Phytoseiidae). Ann.
Entomol. Soc. Aaer., 59:147-149*
-------
-2C
8. McMurtry, J.A., C.B. Huffaker, and M. van de Vrie
1970. Ecology of Tetranychid mites and their natural enemies: A review.
I. Tetranychid enemies: Their biological characters and the inpact
of spray practices. Hilgardia, 40:331-390.
9. Munger, P.
1963. Factors affecting growth and multiplication of the citrus red mite,
Panonychtia citri, Ann. Entomol. Soc. Am., 56:867-874.
10. van de Vrie, M., J.A. McMurtry, and C.B. Euffaker
1972. Ecology of Tetranychid mites and their natural enemies: A review.
HI. Biology, ecology, and pest status, and host-plant relations
of tetranychids. Hilgardia, 4^:343-432.
System #2
References 1, 2, 6, 8, 10
11. Iglinsky, W., Jr., and C.F. Rainwater
1954. Life history and habits of Tetranychus desertorua and bimaculatus
on cotton. Jour. Econ. Entonol., 47t1084-1006.
12. Laing, J.E. :
1969. Life history and life table of Tetranychus •qrticae Koch.
Acarologia, 9j 32-42.
13. Scriven, G.T. and J.A. McMurtry
1971. Quantitative production and processing of Tetranychid mites for
large-scale testing or predator production. Jour. Econ. Entomol.,
64:1255-1257.
System #3
References 1, 2, 6, 8, 10, 11, 12, 13
System #4
14. Champlain, R.A. and L.L. Sholdt
1967. Life history of Geocoris punetipes (Hemiptera:Lygaeidae) in the
laboratory. Annals Entomol. Soc. Amer., 60:881-883.
15. Cohen, A.C.
1981. An artificial diet for Geocoris puncti-pes (Say). Southwest.
Entoaiol., 6_:109-113. '
16. Cohen, A.C.
1985. Simple method for rearing the insect predator Geocoris punctrpes
(Heteroptera:Lygaeidae) on a meat diet. Jour. Econ. Entomol.,
78:1173-1175.
17. Cohen, A.C. and J.W. DeBolt
1983. Rearing Geocoris -punctipes on insect eggs. Southwest. Entomol.,
8.: 61-64.
18. Dunbar, D.M. and O.G. Bacon
1972a. Feeding, development, and reproduction of Geocoris punctipes
(Heteroptera:Lygaeidae) on eight diets. Ann. Entomol. Soc. Aner.,
65:892-895.
-------
-26-
19. Dunbar, D.M. and O.G. Bacon
1972b. Influence of temperature on development and reproduction of
Geocoris atricolor, G. pallens, and G. punetipes (Heteroptera:
Lygaeidae) from California. Envir. Entomol., j.:596-599.
20.. Guy, R.H., N.C. Leppla, and J.R. Rye
1995. Trichoplusia ni. In "Handbook of Insect Rearing" (p. Singh and
R.F. Moore, eds.)f Vol. II, pp. 487-494. ELsevier, New York, 514 pp.
System #5
Reference 1
21. Canard, M. Y. Semeria, and T.R. New (eds.)
1984. Biology of Chrysopidae. Dr W. Junk Publishers, Boston. 294 pp.
22. Forbes, A.R., B.D. Frazer, and O.K. Chan
1985. Aphida. In "Handbook of Insect Rearing" (P. Singh and H.P. Moore,
eds.), Vol. I, pp. 353-359- ELsevier, New York, 488 pp.
23. Morrison, R.K.
1985a. Chrysopa carnea. In "Handbook of Insect Rearing" (P.Singh and
H.F. Moore, eds.), Vol. I, pp. 419-426, ELsevier, New York, 488 pp.
System #6
References 1, 22
24. Clausen, C.P.
1916. Life-history and feeding records of a series of California
Coccinellidae. Calif. Univ. Pub. Entomol., 1.:251-29.9.
25. Hagen, K.S.
1962. Biology and ecology of predaceous Coccinellidae. Annu. Rev.
Entomol., J_:289-326.
26. Hagen, K.S. and R.R. Sluss
1966. Quantity of aphids required for reproduction by HiTipodamia spp. in
the laboratory. Pp. 47-59. In "Ecology of Aphidophagous Insects"
(I. Hodek, ed.), Dr. W. Junk, Publ., The Hague, Netherlands, 360 pp.
27. Simpson, R.G. and C.C. Burkhardt
1960. Biology and evaluation t>f certain predators of Therioa-phis maculata
(Buckton). Jour. Econ. Entomol., 53:89-94.
Svsten #7
References 1, 22, 24, 25, 27
System "#8
28. Fisher, T.W.
1963. Mass culture of Cryptolaemus and Letitomastix. natural enemies of
citrus mealybug. Calif. Agr. Expt. Stn. Bull., 757;39.
-------
-27-
System #9
29. Burbutis, P.P. and L.P. Goldstein
1983. Mass rearing Tricho gramma nubilale on European corn borer, its
natural host. Protection Ecology, £:269-275.
30. Guthrie, W.D., J.C. Bobbins, and J.L. Jarvis
1985. Ostrinia nubilalis. In "Handbook of Insect Rearing" (p. Singh and
R.F. Moore, eds.), Vol. II, pp. 407-413. Elsevier, New York, 514 pp.
31. Morrison, R.K.
1985"b. TJrichograrmtfi spp. In "Handbook of Insect Rearing" (p. Singh
and H.F. Moore, eds.), Vol. I, pp. 413-417, Elsevier, New York.
408 pp.
System
32. Anderson, B.C. and J.D. Paschke
1968. The biology and ecology of Anaphea flavipes (HymenopterarMymaridae),
an exotic egg parasite of the cereal leaf beetle. Ann. Entomol.
Soc. Amer., 61 ;1-5.
System
Reference 22
33. Pox, P.M., B.C. Pass, and R. Thurston
1967- laboratory studies on the rearing of Anhidius smithi (Hymenoptera:
Braconidae) and its parasitism of Acyrthosiphon pisxun (Homoptera:
Aphididae). Ann. Entomol. Soc. Amer., 60; 1083-1 087.
34. Quezada, J.
unpubl. Notes on rearing Acyrtho s i ph on pisxtm and Aphidius ervi.
System #12
Reference 22
35. Hagen, Z.S. and R. van den Bosch
1968. Impact of pathogens, parasites, and predators on aphids. Arum.
Rev. Entomol., 13.: 325-384.
36. Simpson, B.A., W.A. Shands, and G.W. Simpson
1975. Mass rearing of the parasites Praon sp. and Diaretiella rapae.
Ann. Itatomol. Soc. Am., 68; 257-2 60.
System
Reference 22
37. Headlee, T.J.
1914. Some data on the effect of temperature and moisture on the rate
of insect metabolism. Jour. Scon. Entomol., 2.5413-417.
38. Hight, S.C., R.D. Eikenbary, R.J. Miller, and K.J. Starks
1972. The greenbug and Lysiphlebus testacei-pea. Environ. Entomol.,
1:205-209.
-------
-28-
39. Starks, K.J. and R.L. Burton
1977. Greenbugs: determining biotypes. USDA-ARS Tech. Bull., 1536:12.
System #14
40. Chianese, R.
1985. Cotesia melanoscelus. In "Handbook of Insect Rearing" (p. Singh
and R.F. Moore, eds.), Vol. I, pp. 395-400, Elsevier, New York,
488 pp.
41. ODell, T.M., C.A. Butt, and A.W. Bridgeforth
1985. Lymytria dispar. In "Handbook of Insect Rearing" (p. Singh and
H.P. Moore, eds.), Vol. II, pp. 355-367 t Elsevier, New York, 514 pp.
System
42. Clausen, C.P. (ed.)
1978. Introduced parasites and predators of arthropod pests and weeds t
A world review. TTSDA Agric. Handbook No. 480, 545 pp.
43. Planders, S.E.
1942. Metaphyeus helvolus, an encyrtid parasite of the black scale.
Jour. Econ. Entomol., 35:690-698.
System #16
44. DeBach, P. and E.B. White
1960. Commercial mass culture of the California red scale parasite
Aphytis lingnanenais. Calif. Agric. Exp. Stn. Bull. No. 770, 58pp.
45. Papacek, D.F. and D. Smith
1985. Aphytis lingnanensis. In "Handbook of Insect Rearing" (P. Singh
and R.P. Moore, eds.), Vol. I, pp. 373-381, Elsevier, New York,
488 pp.
System
Reference 20
46. King, E.G. and G.G. Hartley
1985a. Galleria mellonella. In "Handbook of Insect Rearing" (p. Singh
and R.P. Moore, eds.), Vol. II, pp. 301-305, Elsevier, New York,
514 PP.
47. Palmer, D.J.
1985. Brachymeria intermedia. In "Handbook of Insect Rearing" (P. Singh
and R.P. Moore, eds.), Vol. I, pp. 383-393 , Elsevier, New York,
488 pp.
System #18
Reference 46
48. King, E.G. and G.G. Hartley
1985b. Lixophaga diatraeae. In "Handbook of Insect Rearing" (p. Singh
and R.P. Moore, eds.), Vol. II, pp. 119-123, Elsevier, New York,
514 PP.
-------
-2?-
49. King, E.G. and G.G. Hartley
1985c. Diatraea saccharalis. In "Handbook of Insect Rearing" (p. Singh
and R.P. Moore, eds.), Vol. II, pp. 265-270, ELsevier, New York,
5H PP.
50. King, E.G. and D.F. Martin
1975. Lixophaga diatraeae; Development at different constant temperatures.
Environ. Entomol., 4_: 329-332.
51. King, E.G., P.D. Brewer, andJJ.F. Martin
1975. Development of Diatraea saccharalia (Lep. :Pyralidae) at constant
temperatures. Entomophaga, 20i 301-306.
52. King, E.G., D.P. Martin, and L.R. Miles
1975. Advances in rearing of Lixophaga diatraeae (Dipt.: Tachinidae).
Entomophaga, 20:307-311.
53. King,. E.G., G.G. Hartley, D.P. Martin, J.W. Smith, T.E. Summers, and
R.D. Jackson
1979. Production of the Tachinid Lixophaga diatraeae on its natural host,
the sugarcane borer, and on an unnatural host, the greater wax moth.
IT.S. Dept. of Agric., SEA, AAT-S-3/April 1979.
54. Miles, L.R. and E.G. King
1975. Development of the Tachinid parasite, Lixophaga diatraeae, on various
developmental stages of the sugarcane borer in the laboratory.
Environ. Entomol., 4_s81 1-814.
55. Pan, Yung-Song and W.H. Long
1961. Diets for rearing the sugarcane borer. Jour. Econ. Entomol.,
: 257-261.
56. Wongsiri, T. and N.M. Randolph
1962. A comparison of the biology of the sugarcane borer on artificial
and natural diets. Jour. Econ. Entomol., 3_5? 472-473*
System #19
No references.
System #20
57. Brooks, M. and C. Knight
1982. A Complete Guide to British Butterflies. Jonathan Cape, London.
159 PP.
58. Singh, P.
1985. Multiple-species rearing diets. In "Handbook of Insect Rearing"
(P. Singh & R.P. Moore, eds.), Vol. I, pp. 19-44, Elsevier, New
York, 488 pp.
59. Williams, C.B.
1970. The migration of the painted lady butterfly, Vanessa cardui
(Nymtjhalidae), with special reference to North America. J. Lepid.
Soc/, 24.: 157-1 75.
-------
PATHOLOGY-MICROBIOLOGY SUBGROUP
AUTHORS:
C.Y. Kawanishi
D.G. Boucias
P. Bauman
L. Volkman
M. Summers
151
-------
Draft Report
Microbiology-Pathology Subgroup
Members
Dr. C.Y. Kawanishi, Chairman
Developmental Biology Division
Health Effects Research Laboratory
U.S. EPA
Research Triangle Park^NC 27711
Dr. Drion G. Boucias
Dept. of Entomology and Nematology
345 Archer Road Labs.
University of Florida
Gainesvill, FL 32601
Dr. Paul Bauman
Department of Bacteriology
University of California-Davis
Davis, GA 95616
Dr. Loy Volkman
Dept. of EntomO'
338 Hilgard Hall
University of California
Berkeley, CA 94720
Dr. Max Summers
Department of Entomology
Texas A&M University
College Station, TX 77843
Dr. Loy Volkman
Dept. of Entomology and Parasitology
338 Hilgard Hall
lln-i uorci +• v nf ralifnrnis
152
-------
Pathology - Microbiology Subgroup Critique and Recommendations
This subgroup was formed to facilitate adaptation of the terrestrial,
freshwater and estuarine enclosed systems to testing microbial pesticides.
This charge was fulfilled through evaluation of the systems and by providing
background information to the other subgroups on the properties, pathology,
taxonomy and ecology of the various groups of agents that are the active
components of microbiaVpesticides (Federal Register, 1984). The objective
was to ensure, as far as was possible, that the enclosed systems resulting
from the workshop were (1) compatible with the biological, chemical and
physical properties of the agents, and (2) the endpoints measured in the
systems were appropriate for infectious and toxic living agents. The
micro-biology-pathology subgroup members participated in the discussions of the
other subgroups concerned with the design, function and composition of the
enclosed systems. ;
The following is a summary of our critique and recommendations. A
concensus developed among members of the subgroup during the course of the
workshop that a common fault, with most of the enclosed systems was that they
were developed for environmental chemical toxicants and microbial agents
have not been tested with them. There is concern that the endpoints and
parameters measured in some of them may possibly be inappropriate for
microbial agents or may be too insensitive for the generally milder and
more specific effects of microbial pesticidal agents. Because of the use
for which they were originally intended, some of the design features and
conditions of the enclosed systems may either be incompatible with or
disadvantageous to the microbial agents because their tolerance limits are
usually narrower than those of chemical toxicants. Consequently, their
full potential effect may not be realized in the enclosed system. As an
153
-------
example, during discussions of the freshwater subgroup it was found that
the diurnal variation in pH in the aquatic littoral microcosm can attain
alkalinities in the range which affect viral occlusion bodies (Edgawa and
Summers, 1972; Kawanishi, et al., 1972) and certain bacterial pesticide
protoxin crystals (Cooksey, 1971). Thus, the effects observed with these
agents in this system may be dependent on when during the diurnal cycle the
pesticidal agent is introduced. Closer examination of other enclosed
systems may reveal analogous situations which must be circumvented to
effectively utilize them for ecological assessments.
The following are the recommendations of the committee: (1) The
compatibility of each enclosed system and the specific properties of the
agent being carefully examined on a case-by-case basis before it is
recommended for testing. The basis for this recommendation are the following
considerations. Most systems were originally developed for toxicity testing
., •
and not designed specifically with microbial pesticides in mind. Additionally,
the subgroup can neither anticipate the diverse types of microbial agents
nor the variety of modes of action to be tested in these systems. Frequently
pesticidal action is accomplished through infectivity and pathogenicity
(Payne, 1982). Even where toxicity is the mode of action, both the nature
of the toxins (Wilcox et al., 1986) as well as the mechanisms of action
(Luthy and Ebersold, 1981) appear to be different from most environmental
chemical toxicants. Additionally, a single microbial pesticide may have
multiple modes of action such as a combination of infectivity and toxicity
and one action may be dependent upon the other (Heimpel and Angus, 1963).
Also, because the systems were designed to test the relatively severe effects
frequently manifested by chemical pesticide toxicity, their utility with
microbial pesticidal agents which may not act through acute or lethal
154
-------
effects but rather through chronic and debilitating action resulting in reduced
vigor and fecundity of the affected species (e.g., Katagiri, 1981) in
question; (2) Where possible positive controls should be included; (3) The
systems should eventually be validated with microorganisms of known effects.
References
Cooksey, K.E. 1971. The protein crystal toxin of Bacillus thuringiensis:
biochemistry and mode of action, p.247-274. In H.D. Surges and H.W. Hussey
(ed.), Microbial Control of Insects and Mites. Academic Presss, Inc., New York.
Egawa, K. and Summers, M.D. 1972. Solubilization of Trichoplusia ni
granulosis virus proteinic crystal I. Kinetics. J. Invertebr. Pathol. 19,
395-404.
Fereral Register. 1984. Data Requirements for Pesticide Registration.
Vol. 49, 42856-42905.
Heimpel, A.M. and Arvgtis, T.A. 1963. Diseases caused by sporeforming
bacteria, p.42. In Steinhaus, E.A. (ed.), Insect Pathology - An Advanced
Treatise. Academic Press, New York and London.
Katagiri, K. 1981. Pest control by cytoplasmic polyhedrosis viruses,
p. 433-440. In Surges, H.D. (ed.), Microbial Control of Pests and Plant
Diseases 1970-1980. Academic Press, London, New York, Toronto, Sidney,
San Francisco.
Kawanishi, C.Y. Egawa, K. and Summers, M.D. 1972. Solubilization of
Trichoplusia ni granulosis virus proteinic crystal II. Ultrastructure. J.
Invertebr. Pathol. 20, 95-100.
Luthy, P. and Ebersold, H.R. 1981. Bacillus thuringiensis delta-endotoxin:
Histopathology and molecular mode of action, p. 235-267. In E.W. Davidson
(ed.), Pathogenesis of Invertebrate Microbial Diseases. Allanheld, Osmun &
Co. Publishers, Inc.
155
-------
Payne, C.C. 1982. Insect viruses as control agents. Parasitol. 84,
35-77.
Wilcox, D.R., Shivakumar, A.G., Mel in, B.E., Miller, M.F., Benson,
T.A., Schopp, C.W., Casuto, D., Gundling, G.J., Boiling, T.J., Spear, B.B.
and Fox, J.L. 1986. Genetic Engineering of Bio-Insecticides. In Protein
Engineering: Applications in Science, Industry and Medicine (In press).
156
-------
WORKSHOP PARTICIPANTS AND THEIR AREAS OF EXPERTISE
NAME
Dick Anderson
Paul Baumann
Drion B. Boucias
John A. Couch
Tom Duke
Lowell Etzel
Steven Foss
Paul Franco
Larry Goodman
Fred Genthner
ADDRESS & AFFILIATION
U.S. Environmental Protection Agency
Environmental Research Laboratory
6201 Congdon Boulevard
Dulyth, Minnesota 55804
Department of Bacteriology
University of California-Davis
Davis, California 95616
Department of Entomology & Nematology
345 Archer Road Laboratory
University of Gainesville
Gainesville, Florida 32601
U.S. Environmental Protection Agency
Environmental Research Laboratory
Sabine Island, Gulf Breeze, FL 32561
U.S. .Environmental Protection Agency
Environmental Research Laboratory
Sabine Island, Gulf Breeze, FL 32561
Division of Biological Control
University of California
1050 San Pablo Avenue
Albany, California 94706
U.S. Environmental Protection Agency
Environmental Research Laboratory
Sabine Island, Gulf Breeze, FL 32561
Oakridge National Laboratory
Environmental Science Division
Building 1504, P.O. Box X
Oakridge, Tennessee 55812
U.S. Environmental Protection Agency
Environmental Research Laboratory
Sabine Island, Gulf Breeze, FL 32561
U.S. Environmental Protection Agency
Environmental Research Laboratory
Sabine Island, Gulf Breeze, FL 32561
AREA OF EXPERTISE
Freshwater Systems,
Entomology
Bacterial Pathogens
Fungal Pathogens of
Insects
Virology, Invertebrate
and Fish Pathology
Aquatic Ecology
Biological Control
Estuarine Biology
Freshwater Biology
Estuarine Toxicology
Microbiology,
Biotechnology
157
-------
Clint Kawanishi
Gary Phipps
Ken Perez
Lyle Shannon
Ray Seidler
Max Summers
Pete Van Voris
Loy Volkman
ADORES & AFFILIATION
Environmental Science Section, HAEL
Eastman Kodak Company
Kodak Park Building 306
Rochester, New York 14650
U.S. Environmental Protection Agency
Health Effects Research Laboratory
Research Triangle Park, NC 27711
U.S_, Environmental Protection Agency
Environmental Research Laboratory
6201 Congdon Boulevard
Duluth, Minnesota 55804
U.S. Environmental Protection Agency
Environmental Research Laboratory
South Ferry Road
Narragansett, Rhode Island 02882
Department of Biology
University of Minnesota
Duluth, Minnesota 55812
U.S. Environmental Protection Agency
Environmental Research Laboratory
200 SW 35th Street
Corvallis, OR 97333
Department of Entomology
Texas A&M University
College Station, TX 77843
Battelle Memorial Laboratories
Pacific Northwest Laboratory
Richland, Washington 99352
EXPERTISE
Environmental Sciences
Microbiology,
Biotechnology
Aquatic Biology
Aquatic Ecology
Biology
Microbiology,
Biotechnology
Virology &
Biotechnology
Terrestrial
Envi ronments
Ecology
and
Department of Entomology & Parasitology Virology and
338 Hilgard Hall Immunoassay
University of California
Berkeley, California 94720
158
------- |