United States       Prevention, Pesticides     EPA712-C-96-304
          Environmental Protection    and Toxic Substances     February 1996
          Agency         (7101)
&EPA    Microbial Pesticide
          Test Guidelines
          OPPTS 885.2350
          Analytical Methods-

     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

     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
(7U.S.C. I36,etseq.).

     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,    internet:     http://
fedbbs.access.gpo.gov, or call 202-512-0132 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."

OPPTS 885.2350 Analytical methods—animals.
     (a) Scope—(1) Applicability. This guideline is intended to meet test-
ing requirements of the  Federal Insecticide,  Fungicide, and  Rodenticide
Act (FIFRA) (7 U.S.C. 136, et seq.}.

     (2) Background. The source material used in developing this har-
monized OPPTS test guideline is the OPP guideline  153A-8a.

     (b) Analytical  methods. Analytical methods  are required both  for
data collection to support proposed tolerances and  for the  enforcement of
such regulations (40  CFR 180).  Note that a monitoring method is required
for the  determination of all microbial pest control agents (MPCAs) that
are exempt  from the requirements of tolerance. The Agency must have
the monitoring  method available in times of need and cannot afford  the
potentially long  method  development period  in the event  adverse effects
are observed subsequent  to registration. The methods must not be subject
to interference due to substrate, reagents, or residues (cells, virions, toxin,
etc.) of related  or unrelated microbial agents whether naturally occurring
and unregistered or whether an MPCA. A confirmatory procedure is also
required for  each residue of concern for both data collection and tolerance

     (1) Description  of  method. Each method  must be  fully described,
or a reprint must be provided  as well, as any  necessary modifications.
Each method must be validated by submitting recovery data and analyses
of untreated control samples of representative animal commodities. The
estimated  sensitivity/detection limit must be provided for each tested com-
modity. Attempts must be made to determine if residues in or on treated
commodities (aged  or weathered) are extracted with the  same efficiency
as those from spiked samples used for recovery experiments.

     (2) Choice of analytical method. Widely  differing analytical meth-
ods may be  required to identify and  quantify all residues of toxicological
concern derived from  a  given MPCA. If a  biologically active microbial
product is of concern, the  more conventional analytical procedures such
as gas chromatography, mass spectrometry,  or high-pressure  liquid  chro-
matography  are typically used. If the MPCA per se,  a mutant, or a viable
recipient of  MPCA  genetic material is a residue  of toxicological concern,
various  immunological methods (such as enzyme-linked  immunosorbent
assay, dot-immunoassay) or molecular probe methods (such as dot hybrid-
ization  procedure,   Southern  hybridization procedure,  or restriction
endonuclease mapping) may be used for identification and/or quantifica-
tion. Since  the  above procedures do not necessarily determine  viable
MPCAs, culturing of tissues (maceration followed  by dilution plating) or
infectivity assays will frequently be necessary. Culturing or bioassays  are
also important as means of detecting  MPCAs at levels below the detection
limits of the above  methods and, theoretically,  as  few as  one viable mi-

crobe can be detected using enrichment techniques, if necessary. In some
cases, microscopy may be useful.

     (3) Purity  of MPCAs. It may be  possible to purify some viral
MPCAs to the point of crystallization whereas other  MPCAs may  not be
isolatable  from host cells  or host  membranes in a viable form.  Some
MPCA residues may be bound (actively or passively) to cellular structures/
components and their release must be attempted using procedures such
as sonication, use  of detergents, or hydrolytic steps  (enzymatic, acid, or
alkaline).  Care  must be taken to determine background levels of cross-
reacting MPCAs since antigenic similarities and/or nucleic acid homology
may exist in indigenous microbes to  a greater or lesser extent. In some
cases, care must also  be  taken to detect different viable forms  of the
MPCA in question (such as spores vs. vegetative cells, encapsulated vs.
nonencapsulated, or yeast vs. mycelial forms) since antigenic determinants
may be different or may  be masked.  In  the case of genetically altered
MPCAs,  the  methods must be  specific enough to determine the MPCA
in the presence of the parent, unmodified, indigenous strain of the same
microbe which,  generally,  will differ only in a relatively small portion
of nucleic acid (chromosomal or extrachromosomal).

     (4) Limitations on methods. The regulatory methods must be rel-
atively simple, rapid, specific, and sensitive and should  not require blank
samples,  exotic equipment or reagents, or use of internal or procedural
standards. If the Agency finds the regulatory methods adequate, they will
be published or referenced in the FDA Pesticide Analytical Manual after
an exemption from tolerance or a  permanent tolerance has been  estab-