91 BACTERIAL LARVICIDE TECHNICAL CONCENTRATES (TK) Note For Preparation

9.1. bacterial larvicide technical concentrates (tk) note for preparation of draft specifications. do not omit clauses or insert additional

9.1. BACTERIAL LARVICIDE TECHNICAL CONCENTRATES (TK)
Note for preparation of draft specifications. Do not omit clauses or
insert additional clauses, nor insert limits that are more lax than
those than given in the guidelines, without providing justification.
From the “Notes” provided at the end of this guideline, incorporate
only those which are applicable to the particular specification.
…… [Genus, species, subspecies and strain of bacterium] TECHNICAL
CONCENTRATE
[CIPAC number]/TK (month & year of publication)
9.1.1 Description (Note 1)
The material shall consist of …… [Genus, species, subspecies and
strain of bacterium] together with related by-products of the route of
manufacture and shall be in the form of [physical description], free
from visible extraneous matter and added modifying agents, except for
stabilizers (preservatives) and free-flow agents (Note 2), if
required.
9.1.2 Active Ingredient (Note 3)
9.1.2.1 Identity
The active ingredient shall comply with an identity test and, where
the identity remains in doubt, shall comply with at least one
additional test.
9.1.2.2 Active ingredient content (biopotency)
The …… [Genus, species, subspecies and strain of bacterium] content
shall be declared in International Toxic Units (ITU/mg product), and
when determined by the method described in Note 4, the average
biopotency shall not be less than 90% of the declared minimum content.
9.1.3 Relevant impurities and contaminants
9.1.3.1 Microbial contaminants and impurities
(Note 5.)
9.1.3.2 Chemical impurities
The material shall be free from beta-exotoxin when tested with the fly
larvae toxicity test (Notes 6 and 7) or an equivalent HPLC method.
9.1.3.3 Water (WHO test method M7R1)
Maximum … g/kg (Note 8).
9.1.4. Physical properties
9.1.4.1 pH range (CIPAC MT 75.3), if required
pH range … to …
9.1.5 Storage stability
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9.1.5.1 Stability at elevated temperature
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(Method to be developed – Note 9.)
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Note 1 The technical concentrate is the axenic (“pure”) single
organism, with all relevant biological components associated with it,
e.g., toxins, cellular parts and spores. The description must include
information on any genetic modifications of the strain used.
Note 2 A free-flow agent may be required to minimise static
electricity and the agglomeration of particles.
Note 3 Information must be provided on the source and identification
characteristics of reference material obtainable from an
internationally recognised institution.
Note 4 Determination of the biopotency (toxicity) of Bacillus
thuringiensis subsp. israelensis and B. sphaericus products.
Principles
Biopotency is tested by comparing mosquito larval mortality produced
by the product under test with the mortality produced by the
corresponding reference standard. Biopotency is measured in
International Toxic Units (ITU) per mg of product.
Presently, there are two internationally recognized reference powders
that allow determination of biopotency using bioassays of bacterial
preparations to mosquito larvae, when used in conjunction with the
methods described below.
The biopotency of products based on Bacillus thuringiensis subsp.
israelensis (Bti) is compared against a lyophilized reference powder
(IPS82, strain 1884) of this bacterial species, using early
fourth-instar larvae of Aedes aegypti (strain Bora Bora). The toxicity
of IPS82 has an arbitrarily assigned toxicity of 15,000 ITU/mg powder
against this insect strain.
The biopotency of products based on Bacillus sphaericus (Bsph) is
determined against a lyophilized reference powder (SPH88, strain 2362)
of this bacterial species using early fourth-instar larvae of Culex
pipiens pipiens (strain Montpellier). The toxicity of SPH88 has an
arbitrarily assigned toxicity of 1,700 ITU/mg of powder against this
insect strain.
The toxicity of all bacterial preparations based on Bti or Bsph can be
determined against the above standard powders. The toxicity (ITU/mg)
of products tested is determined according to the following formula:
titre (ITU/mg) of product tested = titre standard (ITU/mg) x LC50
(mg/l) standard LC50 (mg/l) unknown "X"
The use of alternative bacterial larvicide reference powders and/or
alternative strains of mosquitoes in this test must be approached
cautiously, because it is inevitable that different results will be
obtained with them. Such alternatives must be the subject of careful
cross-calibration against the reference powders and/or strains
identified above. Ideally, such cross-calibration should be conducted
by a group of independent expert laboratories. The alternative
powders/strains, and the cross-calibration data which support them,
should be made available to anyone who wishes to use, or check, the
test with the alternative powders/strains.
Method
Apparatus and reagents
Top-drive homogenizer or stirrer.
Ice bath (container of crushed ice).
Analytical balance (accurate to ± 0.1 mg).
Top-pan balance (accurate to ± 10 mg), preferably with tare facility.
Deionised water.
Wetting agent (e.g. Tween 80).
200 ml borosilicate glass or plastic beakers.
500 ml wide-necked, screw-capped, clear glass bottle.
100 ml screw-capped clear glass bottles.
Micropipette.
10 ml pipette.
12 ml plastic tubes with stoppers or caps.
200 ml plastic or wax-coated paper cups.
(i) Preparation of reference standard suspensions for calibration of
the bioassay
Before preparing the suspension, check that stirring/blending of the
wetting agent/water mixture, described in the following paragraph,
does not lead to foaming. If it does, dilute (e.g. 1:10) the wetting
agent before use.
Accurately weigh about 50 mg (to the nearest 0.1 mg) of the reference
standard powder and transfer it to a 200 ml beaker with 100 ml
deionised water (it can be transferred directly to the 500 ml bottle
if the neck is wide enough to accept the stirrer/blender head). Allow
the mixture to stand for 30 min and add a small drop (about 0.2 mg) of
wetting agent. Place the beaker in the ice bath and either stir or
blend the mixture for 2 min. Check visually for any large particulates
remaining and repeat the stirring/blending if there are any. Weigh or
tare the 500 ml bottle and transfer the suspension/solution to it,
rinsing carefully and thoroughly the beaker and stirrer/blender. Add
further deionised water to make the weight of contents to 500 g (500
ml), cap the bottle and shake vigorously to mix the contents. Confirm,
by microscopic examination of a small aliquot, that no aggregates of
spores and crystals persist. If any are present, the contents must be
subjected to further stirring/blending in the ice bath. This primary
suspension/solution contains 1 mg/10 ml and must be shaken vigorously
immediately before removing aliquots.
Transfer 10 ml aliquots of the primary solution/suspension to clean 12
ml tubes that are stoppered/capped immediately. If transferring a
number of aliquots, cap and shake the primary suspension/solution at
intervals not exceeding 3 min, because the spores and crystals
sediment quickly in water. The aliquots can be stored for a month at
4°C and for 2 years in a freezer at ‑18°C. Each contains 1 mg standard
powder.
To prepare a “stock solution”, weigh or tare a 100 ml bottle. Transfer
one of the 10 ml aliquots into the 100 ml bottle, rinsing carefully at
least twice with deionised water, and fill to a total of 100 g. Shake
the mixture vigorously (or use the blender) to produce a homogeneous
suspension. Frozen aliquots must be homogenised thoroughly before use,
because particles agglomerate during freezing. The “stock solution”
contains 10 mg/l.
From the “stock solution”, subsequent dilutions are prepared directly
in plastic cups filled (by weighing) with 150 ml de-ionized water. To
each cup, 25 early L4 larvae of Aedes aegypti or Culex pipiens
(depending on the bacterial species to be tested: Aedes for Bti and
Culex larvae for B. sphaericus) are added first by means of a Pasteur
pipette, prior to addition of bacterial suspensions. The volume of
water added with the larvae is removed from the cup (by weighing) and
discarded, to avoid changing of the volume in the cup. Using
micropipettes, 600 µl, 450 µl, 300 µl, 150 µl, 120 µl and 75 µl of
“stock solution” are added to separate cups and the solutions mixed to
produce final concentrations of 0.04, 0.03, 0.02, 0.01, 0.008 and
0.005 mg/l, respectively, of the reference standard powder. Four
replicate cups are used for each concentration and one for the
control, which contains only 150 ml de-ionized water.
(ii) Preparation of suspensions of the product to be tested
For bioassay of preparations of dry products (TK, WP, WG, WT) of
unknown toxicity, an initial homogenate is made in the same manner as
described for the reference standard powder, above, except that the
replicate determinations must be made on dilutions prepared by
weighing separate test portions of the product. That is four replicate
primary suspension/solutions must be prepared. For assay of a liquid
formulation (SC), after suitable agitation, 100 mg is weighed instead
of 50 mg (the “stock solution” then corresponding to 20 mg/l). Cups
and larvae are prepared as described above and comparable dilutions
are prepared as for the reference standard.
For products of unknown toxicity, perform range-finding bioassays,
using a wide range of concentrations of the product under test, to
determine its approximate toxicity. The results are then used to
determine a narrower range of concentrations for a more precise
bioassay.
(iii) Determination of toxicity
No food is added for Aedes larvae. For the Culex bioassay, finely
ground yeast extract (1.5 mg) is added to the water and mixed to
produce a concentration of 10 mg/l. All tests should be conducted at
28 + 2°C, with a 12-h light/12-h dark cycle. To avoid the adverse
effects of evaporation of water in low humidity, the relative humidity
should be maintained at 50 ± 15%, if possible.
Each bioassay series should preferably involve 6 concentrations x 4
replicates x 25 larvae for the reference standard and the unknown and
100 larvae for the control. The aim is to identify a range of
concentrations that give mortality between 5 to 95 % (because 100
larvae are used). Data giving 0 or 100 % mortality are ignored for the
calculation of the LC50. To prepare a valid dose-response curve, only
concentrations giving values between 95% and 5% mortality should be
used. A minimum of two dilution points must be above the LC50 and two
below, to ensure the validity of the value. The sensitivity of the
insect colony may require a slightly different 6 dilution series to be
used.
Mortality is determined at 24 and 48 h by counting the live larvae
remaining. If pupation occurs, the pupae should be removed and their
numbers excluded from the calculations. If more than 5% of larvae
pupate, the test is invalidated because larvae do not ingest 24 hours
before pupation and too many larvae may have survived simply because
they were too old. Because of the very rapid killing action of Bti,
usually there is no difference between the 24 and 48 h mortality. In
this case, the 48-h count confirms the 24-h reading and provides a
check on the possible influence of factors other than Bti components.
Mortality is recorded at 48 h for Bsph preparations, due to its slower
rate of action.
If the control mortality exceeds 5%, the mortalities of treated groups
should be corrected according to Abbott's formula [Abbott, W. S.
(1925). A method for computing the effectiveness of an insecticide.
Journal of Economic Entomology, 18, 265-267]:
X – Y
percentage (%) control = ————
X x 100
where X = % survival in untreated control,
Y = % survival in treated sample.
Tests with a control mortality greater than 10%, or any pupation
greater than 5%, should be discarded. Mortality-concentration
regression lines may be drawn on gausso-logarithmic paper but this is
rather subjective. It is preferable to use a statistical program, such
as SAS, which incorporates a Log Probit Analysis. With such a
statistical program, Abbott’s formula is not required because the
correction is automatically carried out by the program. The toxicity
of an unknown preparation is determined by estimation and comparison
of the LC50s of the tested product and reference standard
preparations, using the formula described above. The toxicity of Bti
preparations is defined by the count at 24 h after initiation of the
test, whereas the toxicity of Bsph is defined by the count after 48 h
of larval exposure.
For increased accuracy, bioassays should be repeated on at least three
different days, concurrently with the assay of the reference standard,
and the standard deviation of the means calculated. A test series is
valid if the relative standard deviation (RSD or coefficient of
variation, CV) is less than 25%.
(iv) Production of test larvae
L4 larvae are representative of the total sensitivity of the target
population and convenient to handle. It is very important to use a
homogenous population of early fourth instars, which are obtained
within five day of hatching using standardized rearing methods.
For Aedes aegypti, eggs are laid in a cup lined with filter paper and
filled one third with deionised water. The paper is dried at room
temperature and kept for several months by storing in a sealed plastic
bag at room temperature. When larvae are needed, the paper is immersed
in de-chlorinated water. To synchronise hatching, add larval feed to
the water 24 hours prior to adding the eggs. The bacterial growth will
deoxygenate the water and this triggers egg hatching. This usually
induces the first instars to hatch within 12 h. These larvae are then
transferred to a container (25 x 25 x depth cm) containing 2 litres of
de-chlorinated water, to obtain a population of 500 to 700 larvae per
container. Larval feed may be flakes of protein as used for aquarium
fish, or powdered cat biscuit, and the containers are held at 25 + 2°
C. It is important that the amount of food is kept low to avoid strong
bacterial growth that kills the larvae. Several feedings with one or
two days interval and daily observation of the larvae is optimal. If
the water becomes turbid, replace all water by filtering out the
larvae and transfer to a clean container with clean water and feed.
Five to seven days later a homogenous population of early fourth
instars (5 days old and 4 to 5 mm in length) should be obtained.
For Culex pipiens pipiens larvae, it is more difficult to obtain a
homogenous population of fourth instars. Firstly, a large number of
egg rafts must be laid and collected on the same day. These can be
stored at 15-18°C in order to accumulate more eggs for hatching. The
first instars are fragile and thus should not be handled. Development
to the second instar usually takes 3-4 days at 25 + 2° C after the
eggs are laid. When ready, second instars are grouped in a tray with 3
L dechlorinated water of 4-6 cm depth, 800 – 1000 larvae per tray.
Food (yeast extract and dog or cat biscuits) is provided as needed.
Early fourth instars suitable for testing are usually obtained within
7 days, though sometimes 8 or 9 days are required.
Note 5 The maximum acceptable levels of microbial contaminants have
not yet been determined.
Note 6 Fly larvae toxicity test: Bond R. P. M., et al. The
thermostable exotoxin of Bacillus thuringiensis. In: Burges H. D. and
Hussey N. W., eds. Microbial control of insects and mites. Academic
Press, London, 1971.
Note 7 No test is required if the manufacturer has shown that the
Bacillus thuringiensis strain is not capable of producing beta
exotoxin. No test is required for Bacillus sphaericus, because this
species is not known to produce exotoxins.
Note 8 Generally, the water content should not exceed 5%, to preclude
premature degradation of the product.
Note 9 Microbial larvicides should be stored at cool temperatures but
accelerated storage stability tests would be most useful for rapid
checks on the storage stability of products. At present, no
standardised method is available. In the absence of an accelerated
storage stability test, it is recommended that the following minimum
standards be met:
a) no more than 10% loss in biopotency below the labelled potency
value when stored at 5ºC for 2 years; and
b) no more than 10% loss in biopotency below the labelled potency
value when stored at 20 to 25ºC for 1 year.
These storage stability tests shall be performed using representative
product samples and the biopotency shall be assessed using the test
method described in Note 3.
Results from the biopotency test may vary by up to ± 25% from the
average and this must be taken into account in determining the potency
loss. If one- and two-year test data are not available at the time of
drafting a specification, an estimate of the storage stability may be
acceptable, pending completion of the tests.