United States
Environmental Protection
Agency
Prevention, Pesticides
and Toxic Substances
(7101)
EPA 712-C-96-135
April 1996
&EPA Ecological Effects Test
Guidelines
OPPTS 850.1950
Field Testing for Aquatic
Organisms
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Introduction
This guideline is one of a series of test guidelines that have been
developed by the Office of Prevention, Pesticides and Toxic Substances,
United States Environmental Protection Agency for use in the testing of
pesticides and toxic substances, and the development of test data that must
be submitted to the Agency for review under Federal regulations.
The Office of Prevention, Pesticides and Toxic Substances (OPPTS)
has developed this guideline through a process of harmonization that
blended the testing guidance and requirements that existed in the Office
of Pollution Prevention and Toxics (OPPT) and appeared in Title 40,
Chapter I, Subchapter R of the Code of Federal Regulations (CFR), the
Office of Pesticide Programs (OPP) which appeared in publications of the
National Technical Information Service (NTIS) and the guidelines pub-
lished by the Organization for Economic Cooperation and Development
(OECD).
The purpose of harmonizing these guidelines into a single set of
OPPTS guidelines is to minimize variations among the testing procedures
that must be performed to meet the data requirements of the U. S. Environ-
mental Protection Agency under the Toxic Substances Control Act (15
U.S.C. 2601) and the Federal Insecticide, Fungicide and Rodenticide Act
(7 U.S.C. 136, etseq.).
Public Draft Access Information: This draft guideline is part of a
series of related harmonized guidelines that need to be considered as a
unit. For copies: These guidelines are available electronically from the
EPA Public Access Gopher (gopher.epa.gov) under the heading "Environ-
mental Test Methods and Guidelines" or in paper by contacting the OPP
Public Docket at (703) 305-5805 or by e-mail:
guidelines@epamail.epa.gov.
To Submit Comments: Interested persons are invited to submit com-
ments. By mail: Public Docket and Freedom of Information Section, Office
of Pesticide Programs, Field Operations Division (7506C), Environmental
Protection Agency, 401 M St. SW., Washington, DC 20460. In person:
bring to: Rm. 1132, Crystal Mall #2, 1921 Jefferson Davis Highway, Ar-
lington, VA. Comments may also be submitted electronically by sending
electronic mail (e-mail) to: guidelines@epamail.epa.gov.
Final Guideline Release: This guideline is available from the U.S.
Government Printing Office, Washington, DC 20402 on The Federal Bul-
letin Board. By modem dial 202-512-1387, telnet and ftp:
fedbbs.access.gpo.gov (IP 162.140.64.19), or call 202-512-0135 for disks
or paper copies. This guideline is also available electronically in ASCII
and PDF (portable document format) from the EPA Public Access Gopher
(gopher.epa.gov) under the heading "Environmental Test Methods and
Guidelines."
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OPPTS 850.1950 Field testing for aquatic organisms.
(a)	Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of both the Federal Insecticide, Fungicide, and
Rodenticide Act (FIFRA) (7 U.S.C. 136, et seq.) and the Toxic Substances
Control Act (TSCA) (15 U.S.C. 2601).
(2) Background. The source material used in developing this har-
monized OPPTS test guideline are OPP 72-7 Simulated or Actual Field
Testing for Aquatic Organisms (Pesticide Assessment Guidelines, Subdivi-
sion E—Hazard Evaluation; Wildlife and Aquatic Organisms) EPA report
540/09-82-024, 1982 and subsequent guidance on aquatic mesocosm tests
under paragraph (e)(3) of this guideline.
(b)	Test standards—(1) Test substance. Unless specified otherwise,
data should be derived from testing conducted with an end-use product.
An end-use product may be the applicant's own product or a typical end-
use product.
(2)	Concentration analysis. The concentration of the test substance
in the water should be determined at the start of the study and samples
should be collected periodically for analysis to verify concentrations.
(3)	Test conditions. The test conditions for conducting field tests
should resemble the conditions likely to be encountered under actual use.
Specifically, the pesticide should be applied according to the rate, fre-
quency, and method specified on the label.
(4)	Endangered species. Studies should not be conducted in critical
habitats or areas containing, or suspected to contain, endangered or threat-
ened plants or animals which may be threatened by the tests to be con-
ducted.
(5)	Residue levels. When the test substance is applied under simu-
lated or actual field condition testing, residues should be determined in
appropriate vegetation, soil, water, sediments, and other environmental
components, and in selected tissues of test organisms.
(6)	Other standards. Any additional standards for conducting these
tests will be provided by the Agency in writing following consultation
between the applicant and the Agency, and will take into account the
mechanisms by which a pesticide may enter the environment, and the food
sources and habitats that may be affected.
(c)	Simulated field studies (mesocosm)—(1) Physical description—
(i) Experimental design. (A) One acceptable design is a minimum of four
experimental treatments consisting of a control which receives no test
compound, an X treatment level representing expected exposures, an X+
treatment level representing an upper bound, and an X- treatment level
representing a lower bound. At least three replicates per treatment level
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are needed to provide the requisite resolution of effects and probability
of their occurrence. However, it is recommended that the number of rep-
licates be dictated as a function of the parameters of interest and the sen-
sitivity of their analysis.
(B) Alternative designs which emphasize regression analysis and uti-
lize more treatment levels with fewer or no replicates may also be appro-
priate. Regression designs are most useful for determining maximum expo-
sure conditions which provide no significant impacts or a specified level
of effect in test systems.
(ii)	Mesocosm number. A minimum of 12 mesocosms is required,
with additional mesocosms added as replicates or treatments when needed
to increase the sensitivity of analysis for specific parameters.
(iii)	Mesocosm size. Dimensions of a mesocosm must be large
enough to accommodate a viable finfish population. Depth should be suffi-
cient to provide a representative open water area, and sloped sides should
provide a littoral area for macrophyte growth and finfish reproduction. An
acceptable design would occupy approximately 0.1 acre surface area with
a volume of at least 300 m3 and a maximum depth of 2 m. Sides of the
mesocosm should be sloped approximately 1 unit of drop for every 2-
3 units of linear distance.
(iv)	Mesocosm features. (A) Mesocosms can be constructed as dug-
out ponds or enclosures of existing impoundments. The mesocosms should
be lined with an impervious material of known adsorption for the test
compound. The sediment used should be well-defined and representative
in composition (percent clay, silt and sand, organic carbon, and organic
nitrogen and ion exchange capacity) to pond sediments in the intended
use area of the pesticide. The sediment depth at the bottom of the systems
should be a minimum of 15 cm. Sediments may consist of natural pond
sediment or top soil. If top soil is used, the complete mesocosm should
be seasoned for 1 year prior to experimental use. This time is necessary
to develop benthic biota. If pond sediments are used, a shorter seasoning
period (e.g. 6 mon) is adequate. Organic content of the top soil should
be at least 2 percent.
(B) A means of interchange (circulation, fill-drain-refill, etc.) of the
water between the systems during initial establishment is desirable to en-
sure even distribution of biota among the mesocosms. Once the systems
have become established or at initiation of a test the circulation should
be stopped and each system kept separate from all other systems. The
required precautions to ensure no cross contamination from pond overflow
during rainstorms, leakage in the circulation system, etc., should be taken
from the outset.
(v)	Mesocosm biota. (A) The mesocosms must contain a representa-
tive pond biota. It is recommended that an established pond with diverse
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biota act as a parent pond. The water in the mesocosm should be equiva-
lent to the water of the parent pond and biota collected from the parent
pond should be evenly distributed to each mesocosm to act as a starter
base. Biota from other sources may be used to augment a natural assem-
blage to ensure adequate representation of important taxa.
(B) Phytoplankton are expected to reach a concentration consistent
with the nutrient levels of the system prior to introduction of
macroinvertebrates. Nutrient levels should be within a mesotrophic classi-
fication. The macroinvertebrate fauna should include representatives of the
rotifers, annelids, copepods, cladocerans, amphipods, aquatic insects, and
gastropods. Introduced macroinvertebrates, if necessary to augment natu-
rally colonized populations, should not exceed 10 g wet-weight/m3 and
finfish should not be introduced at more than 2 g wet-weight/m3. Fish
species used in the test must be of known sensitivity to the test compound
(determined from acute toxicity tests) and appropriate to small pond enclo-
sures. Finfish species used must be native North American species
(bluegill sunfish alone or in combination with largemouth bass are rec-
ommended).
(vi) Mesocosm treatment. Treatment levels of the mesocosms should
be based on exposure models and residue monitoring data if available.
In a three-replicate by four-treatment design, the three experimental treat-
ments should be separated into a low, intermediate, and high treatment
(dosed) and a control treatment (undosed). The intermediate treatment
should approximate the estimated environmental concentration determined
through modeling and experiential data for the intended pesticide use. It
is recommended that the low treatment be 1/10 and the high treatment
lOx the intermediate concentration. Regression designs should bracket ex-
pected exposures and expected response concentrations. Loading of pes-
ticide into the mesocosms is to be by direct overspray to simulate drift
and aerial deposition and with a sediment/water slurry channeled into the
system at predetermined points to simulate runoff. Model predictions with
available monitoring data will dictate the timing, frequency, and mode of
introduction of the test material.
(2) Measured parameters—(i) Chemical/physical properties. (A)
Mesocosm water should be monitored for pH, temperature, transparency
(turbidity), dissolved oxygen, alkalinity, total nitrogen, total phosphorus,
conductivity (total hardness), and particulate and dissolved organic carbon
at appropriate intervals (e.g., biweekly). Observations are to be made at
several locations throughout the mesocosm (which will be dictated by the
physical design of the mesocosm) and at appropriate depths to allow quan-
tification of vertical and horizontal variations. A complete water analysis
should be conducted at test initiation and termination, and at significant
periods during the test (i.e., pesticide inputs, substantial changes in other
observed parameters, etc.). Temperature, pH, and dissolved oxygen should
be monitored on a continuous basis for 24 h on a biweekly schedule and
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at significant periods during the test to provide an estimate of gross pro-
duction and community respiration.
(B) Mesocosm sediment must be analyzed for pesticide content, par-
ticle size, cation exchange capacity, organic content, and pH at the initi-
ation of the test.
(ii)	Biological structure. (A) Biota will be identified to species or
lowest taxonomic unit practical. The schedule for sampling and collection
of biological samples will depend on the design and composition of the
mesocosm and must be determined prior to the initiation of the test. Col-
lections should not be so frequent as to disrupt the system.
(B)	Phytoplankton are to be collected from the water column, domi-
nant species identified, and biomass determined by measuring chlorophyll
a and phaeophytin. All samples should be preserved for archival reference.
Periphyton are to be collected from glass slide substrates placed in the
mesocosm and exposed for a minimum of 2 weeks. Periphyton should
be analyzed for chlorophyll and ash-free weight. Macrophytes are to be
identified to species, biomass determined by dry weight, and percent cover
of the mesocosm determined.
(C)	Zooplankton will be collected weekly with tube cores of the water
column and vertical net tows. All samples are to be archived for future
reference. Zooplankton samples will be analyzed biweekly by enumerating
and identifying dominant species. Cladocerans should be identified to
genus and differentiated by size (e.g., measured for length of muon).
Macroinvertebrates, at a minimum, should be collected from emergent in-
sect traps and artificial substrates. Sampling of sediment directly (e.g.,
Ekman dredge), should be employed cautiously, if necessary for tracking
benthic community parameters, to minimize disruption to the benthic com-
munity. Samples should be enumerated, identified to lowest practical
taxon, and archived.
(D)	Finfish will be identified to species, enumerated, sexed (when
possible) and measured in length and weight (wet) at introduction into
the mesocosms and at test termination. Also at test termination, females
will be assessed for fecundity and all collected fish will be examined for
gross pathology. Spawning substrates will be placed in the systems and
periodically surveyed for number of deposited eggs.
(E)	Toxicity testing and bioassays with indigenous fauna on-site and
in the laboratory may be used to assist in confirming cause and effect
relationships.
(iii)	Residue analysis. Residues of the test material and major
degradates/metabolites will be analyzed at appropriate intervals to the envi-
ronmental properties of the compound in the water, sediments, and biota
at a sensitivity consistent with concentrations of concern.
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(iv) Meteorological conditions. Continuous monitoring of air tem-
perature, wind velocity, precipitation, evaporation, and solar radiation are
required within 1 mile of the mesocosm test facility.
(d)	Actual field studies. Data from an actual field study are required
on a case-by-case basis to support registration of an end-use product in-
tended for outdoor application. Consultation with the Agency is advised
before undertaking these tests. Whenever data are required, the determina-
tion will be made in writing by the Agency and will state which properties
and use patterns of the product were used in the determination.
(e)	References. The following references can provide useful back-
ground information for conducting a simulated or actual field study for
aquatic organisms.
(1)	Graney, R.L. et al. (Eds.). Aquatic Mesocosm Studies in Ecologi-
cal Risk Assessment, Lewis, Boca Raton, FL (1994).
(2)	Hill, I.R. et al. (Eds). Freshwater Field Tests for Hazard Assess-
ment of Chemicals, Lewis, Boca Raton, FL (1994).
(3)	Touart, L.W. Aquatic Mesocosm Tests to Support Pesticide Reg-
istrations. U.S. Environmental Protection Agency, Hazard Evaluation Divi-
sion; Technical Guidance Document. National Technical Information Serv-
ice, Springfield, VA) (1988).
(4)	Voshell, Jr., J.R. (Ed.). Using Mesocosms to Assess the Aquatic
Ecological Risk of Pesticides: Theory and Practice. MPPEAL 75 (1989).
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