SOW_for_lab_tests.docx

USDA-Agricultural Research Service Statement of Work (SOW) Testing of Pseudomonas fluorescens strain ACK55 for EPA bioherbicide registration

The following analyses are to be conducted on Pseudomonas fluorescens strain ACK55 (P.f. ACK55) for EPA bioherbicide registration. The laboratory conducting these tests is to be AAALAC accredited, GLP compliant, and USDA registered. All tests are listed and explained on the EPA website.

www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-885-microbial-pesticide-test-guidelines and https://www.epa.gov/test-guidelines-pesticides-and-toxic-substances/series-870-health-effects-test-guidelines

Pseudomonas fluorescens strain ACK55 with 50% glycerol in cryovials will be shipped from the Pullman ARS Lab using overnight shipping. The P.f. ACK55 cells in TSB and glycerol 9 (1:1) are to be stored frozen until used. The bacteria in the cryovial will be inoculated into Tryptic Soy Broth (TSB) for growth to mid-log stage (32 hr) to obtain the correct titer for each test. Generally the bacteria shaken in TSB at 22oC for 32 hours attains mid-log phase and yields populations of log 8 cells mL-1. The bacterial concentration stated on the bioherbicide label is 4 X 108 colony forming units per mL or 0.24% (Upper limit 0.264, Lower limit 0.216%). The bacteria are to be enumerated on Sands and Rovira selective medium (1970). Individual tests may have additional tasks to be conducted. Please identify pre-tests and costs of pre-tests in quote. Bacterial colonies of interest will be grown on Sands and Rovira (1970) selective agar medium, frozen, and sent to Pullman ARS Lab for further identification using MIDI-ID (Sherlock™ GC & GC-MS Software, MIDI-Inc., Newark, Delaware).

Nine major categories of analyses are requested (Table 1). The Acute IV Injection Toxicity/Pathogenicity Study (OPPTS 885.3200) is to be established so that the study can be extended to 100 days. Additional cohorts of test animals are to be included. After harvests at approximately day 21, 42, 64, and 100 days the ARS and EPA request results, so that the decision can be made to extend the tests. All analyses are to be conducted during the work week. Costs for sensitivity, variability and analytical verification, recovery rate, and enumeration trials used to support the main pathogenicity studies are to be identified.

Acute Dermal Toxicity (OPPTS 870.1200) will consist of ten replicates for a total of 14 days with additional dose levels if irritation persists. Pre-tests for validation of sample processing and enumeration Method should be included and costs assigned.

Primary Dermal Irritation (OPPTS 870.2500) will be conducted with at least three replications for up to 72 hr. If irritation persists then additional observations will be made 14 days.

Acute Eye Irritation (OPPTS 870.2400) will be conducted with at least three replications for at least 72 hr. If irritation persists then the experiment will be extended and observed at 7 and 21 days.

Freshwater Aquatic Invertebrate Testing (OPPTS 885.4240) will be conducted over 21 days in suspension. If toxicity is evident then LC50 study may be needed.

Freshwater Aquatic Vertebrate Testing, Tier I, (Fish 885.4200) consists of one dose rate with 7 to 10 replicates for 96 hours. If toxicity is evident then LC50 study may be needed.

Avian Oral, Tier I (OPPTS 8850.4050) will be tested at least one dose rate using ten replicates per dose rate given over 5 days and observed over thirty days. If toxicity is evident, then observations will be extended and LD50 study may be needed.

Avian Inhalation (OPPTS 885.4100, Tier 1) will be tested at three dose rates using ten replicates per dose given over 5 days and observed over thirty days. If toxicity is evident, then observations will be extended and LD50 study may be needed.

Acute Toxicity to Honey Bees (OPPTS 885.4380) will consist of a laboratory range finding study using 3 to 4 concentration levels and 2 to 3 replicates. Acute Oral and Contact Toxicity to Honey Bee will also be a laboratory study to determine the LD50. This study is to consist of 5 test rates, control, and reference item with 4 replicates, 10 bees each. Please identify any pre-tests and costs for solubility determination and method establishment.

Report of the treatments and findings is required at the end of each study. Reports should include information on the Microbial Pest Control Agent, dose route and frequency, replications, sample preparation, observations, measurements taken, treatments and controls, replications, statistical analyses, and a thorough reporting of the results of the study. The reports should conform to EPA GLP.

www.regulations.gov/document?D=EPA-HQ-OPPT-2009-0159-0002

Table 1. List of the nine testing groups required for analysis of Pseudomonas fluorescens strain ACK55.

observe for 14 days

observe for 21 days

observe at 7, 21 days

Non-OPPTS References Sands, D.C., and A.D. Rovira. 1970. Isolation of fluorescent pseudomonads with a selective medium. Appl. Microbiol. 20: 513–514.