1. welcome

2. Quality control and registration standards for
biocontrol agents.
Karthik, C. M.
PGS16AGR7259
Jr.MSc (Agri)
presentation on

3. Introduction
• Biological control- it’s a pest control method where large
numbers of natural enemies are periodically introduced to
control the pests.
• It is a popular control method applied by professional and
progressive farmers because of its benefits when compared
with chemicals.
• For a long time, natural enemies were produced without proper
quality control procedures. Poorly performing natural enemies
resulted in a failures of biological control and a low profile of
this pest control methods (P. DeBach, 1976 and Berkel, 1980)

4. • Quality control was touched upon by several biocontrol
workers in the initial part of 20th century, but the first papers
seriously addressing the problem appeared only in the 1980s
(Van Lenteren, 1986).
• Several examples of poor functioning of organisms when
quality control guidelines are not applied and that resulted in
failure of biological control were observed.
Eg: Failure of Trichogramma brassicae in swiss in controlling
the European corn borer (Ostrinia nubilalis) in maize (Bigler,
1994).
• The extremely artificial rearing conditions, compared to the
habitat where they are released, call for the establishment of
sophisticated quality control concepts.

5. • The best combination of a set of laboratory methods which are
quick, simple and reliable must be developed to control the
quality of mass reared Arthropods.
• Initial developments in the area of mass production, quality
control, storage, shipment and release of natural enemies have
decreased production costs and led to better product quality.
• Innovations in long-term storage (e.g., through induction of
diapause), shipment and release methods, may lead to a further
increase in natural enemy quality with a concurrent reduction
in costs, thereby making biological control easier and
economically more attractive to apply.
• Even if the natural enemies leave the insectary in good
condition, shipment and handling by the producers,
distributors and growers may result in deterioration of the
biological control agents before they are released.

6. Quality control
• Quality of an organism can be defined as the ability to function
as intended after release into the field.
The aim of quality control programmes is
• To check whether the overall quality of a species is maintained
(general).
• To determine the characteristics that affect overall quality
(straightforward).
• To determine whether a natural enemy is still in a condition to
properly control the pest to an acceptable level rather than
maximum or optimum level.

7. Total Quality
Management

8. • The type of biological control programme determines the
selection criteria for natural enemies and strongly related to
the quality control aspects.

9. • Before starting a quality control programme, one should
realize there are many basic considerations and obstacles to be
overcome; careful evaluation of these obstacles and
considerations is essential.
Obstacles in mass production
1. Production of qualitatively good natural enemies at
economical costs.
2. Lack of effective mass production technique on artificial
diets.
3. Lack of techniques that prevent the selection pressures
leading to the genetic deterioration of mass produced one.
4. Cannibalism among the predators.
5. Behavioral changes and reduced vigour due to rearing at
unnatural condition and on artificial diet.
6. Rearing can be infected by the pathogen.

10. Development of quality control
The problem of quality control can be
approached from two sides:
• Measure how well the insect functions in its
intended role, if it does not function well enough,
trace the cause and improve the rearing method.
• List the changes we can expect whn the mass
rearing is started, measures these and if the
changes are undsired, improve the rearing
method.

11. The main effects on laboratory cultures during mass
production are
• Genetic variation and revolution – can be overcome by
introduction of wild individuals from the field regularly.
• Inbreeding due to mating with the related ones. It can be
prevented by
1. Precolonization methods: selection and pooling of founder
insects from throughout the range of species.
2. Postcolonization method:
 Varying the laboratory environments
 Gene infusion – the regular rejuvenation of the gene pool
with the wild species

13. • The 5th workshop of the International Organization for
Biological Control (IOBC) global working group "Quality
Control of Mass Reared Arthropods" (Bigler, 1991) in
Wageningen, the Netherlands, formed the starting point for a
heated discussion among producers of natural enemies and
scientists on how to approach quality control in the
commercial setting at that time.
• As a result of the meetings, quality control guidelines were
written for more than 20 species of natural enemies.
• Elements of the IOBC guidelines are used for quality control
of natural enemies by several companies in most of the
countries.

14. General quality control criteria for mass reared
natural enemies
Criteria alrealy in use:
• Quantity: number of live natural enemy organisms in container
• Sex ratio: minimum percentage females (male biased ratio may indicate
poor rearing conditions)
• Emergence: emergence rate to be specified for all organisms sold as eggs
or pupae
• Fecundity: number of offspring produced during a certain period (for
parasitoids fecundity is also an indication of the host kill rate)
• Longevity: minimum longevity in days
• Parasitism: number of hosts parasitized during a certain period
• Predation: number of prey eaten during a certain period
• Adult size: hind tibia length of adults, sometimes pupal size (size is often
a good indication for longevity, fecundity and parasitization/predation
capacity)
Criteria to be added in near future:
• Flight: short- or long-range flight capacity
• Field performance: capacity to locate and consume prey or parasitize
hosts in crop under field conditions

15. Comments:
- Quality control is done under standardised test
conditions of temperature (usually 22 ± 2o C or
25 ± 2o C), relative humidity (usually 75 ± 10 %)
and light regime (usually 16 L : 8 D), that are
specified for each test
- All numbers / ratios / sizes should be mentioned
on the container or packaging material
- Fecundity, longevity and predation capacity tests
can often be combined
- Expiration date for each shipment should be given
on packaging material
- Guidelines should be usable for all product
formulations

16. • Flight tests are supposed to be essential to determine quality if
the natural enemy has been reared under conditions where
flight was not needed to find hosts or prey, which is often the
case under crowded mass rearing conditions.
• Flight tests are also needed when the natural enemy is
seriously manipulated during mass rearing and preparation for
shipment and when storage periods are long.
• Correlation between values obtained at laboratory testing and
field performance is important to be able to select a limited set
of laboratory criteria that give meaningful information about
performance after release.
Eg: A short-range flight test has been developed for Encarsia
formosa, i.e. a test where the parasitoid has to fly a distance of
4 - 20 cm (van Lenteren et al., 2003)

17. Amblyseius degenerans Berlese (Acarina: Phytoseiidae)
• Test conditions Temperature: 22+1o C
• RH: 70+5%
• Light regime: 16L:8D
Quality control criteria
• Quantity: The number of mites as specified on the label, excluding
eggs
• Sex-ratio: ≥ 50% females; n=100; an annual test
• Fecundity: ≥ 7 eggs/female over a period of 7 days; n= 30; an
annual test
Description of testing methods
• Quantity: take 4 random samples of each 3 g. Spread each sample
on a white paper and count the mites by gently stirring the
material (vermiculite). First, count the live predators running
away; the counted mites will be killed. Next, sieve the material (90
µm) so that the mites will be collected in it. Count the remaining
mites.

18. • Sex-ratio: Identification of sex is done by mounting individuals
on microscopic slides (100 X). Distinguish between females and
males by checking the ventral and genital shields.
• Fecundity: The average number of eggs per female should be ≥
7. Do not include the eggs laid during the first day of the
experiment.

19. Aphidius colemani Viereck (Hymenoptera: Braconidae)
• Test conditions Temperature: 25 ± 2°C RH: 75 ± 5%
• Light regime: 16L:8D
Quality control criteria for mummies
• Quantity and emergence: ≥ The number of live adults that
should emerge from the package as specified by the manufacturer. A minimum
of three containers should be counted.
• Emergence rate: ≥ 45 % (n=500). A weekly or batch-wise test.
• Sex ratio: ≥ 45% females, a seasonal test, n=150
• Fecundity: ≥ 60 mummies/female in the first day when tested on Aphis
gossypii; n=30, an annual test.
≥ 35 mummies/female in the first day when tested on Myzus persicae; n=30, an
annual test.
Description of testing methods
Quantity and emergence: Put the mummies with the carrier material in a
container with a cork in the bottom and ventilation. Put some droplets of honey
on the outer side of the gauze. Transfer the contents to a new container every
day. The container with emerged adults can be frozen and subsequently
counted. Continue until no more wasps emerge. Run the test for a maximum of
7 days.
calculate the emergence rate by using formula:
(no. of adult wasps/ no. of mummies) x 100.

20. Sex ratio: Take a sample of 100 adults and count the number of female wasps.
Females are distinguished from males by their pointed abdomen (ovipositor).
The length of the female abdomen is almost equal to wing length. The male
abdomen is more rounded at the end and is always shorter than the wings. The
females should amount to more than 45% of the total.
Fecundity: This test can be done with leaf discs on whole plants.
• Day 1: Use potted cucumber plants (30x) of two/three leaves. Place a plastic
ventilated cylinder over the plant. Put 30 adult A. gossypii or 30 Myzus persicae
onto each plant using a fine brush.
• Day 2: Remove the adult aphids from the dishes with a moist brush. Check the
number of offspring (>100 / tray). Place the container with emerged wasps in a
cold room (8-12 °C) for 5 minutes. Select 30 females by checking them under a
stereo microscope. Release individual females onto the Petri dishes in the cold
room. Place the dishes upside down at 25°C for 24 hours.
• Day 3: Remove the wasps from the dishes after 24 hours.
• Day 4-10: Check the quality of the leaves. If the quality is poor remove the
aphids to a new petri-dish with a fresh leaf.
• Day 11: Count the number of mummies per dish.

21. Aphidius ervi (Haliday) (Hymenoptera: Braconidae)
Test conditions
• Temperature: 22 ±2°C
• RH: 75 ± 5%
• Light regime: 16L:8D
Quality control criteria for adults
• Quantity: ≥ The number of live adults as specified on the container.
• Adult mortality: < 8 % of the number of adults present in the container,
based on 3 containers sampled and n = 500 or more; a weekly or batch-
wise test.
Quality control criteria for mummies
• Quantity and Emergence: ≥ The number of live adults that should
emerge from the package as specified by the manufacturer.
• Emergence rate: ≥ 75 % (n=250 ). A weekly or batch-wise test.
• Sex ratio: ≥ 45% females, a seasonal test, n=150
• Fecundity: ≥ 35 mummies/female in 2 hrs when tested on Macrosiphum
euphorbiae, an annual test.

22. Encarsia formosa Gahan (Hymenoptera: Aphelinidae)
Test conditions
• Temperature: 22°C±2°C
• RH: 60-90%
• Light regime: 16L:8D
Quality control criteria
• Emergence rate: ≥ the number of adults specified on the label which will
emerge over a two-week period; n=1000; a weekly or batch-wise test
• Sex-ratio: ≥ 98% females; n=500; an annual test
• Fecundity: ≥ 7 eggs/female/day for days 2, 3 and 4 after emergence of the
adult; n=30 females; an annual test
Description of testing methods
• Emergence: Take 3 sub samples which make up 1,000 or more full black
pupae in total in a closed container for two weeks and then determine the
number of emerged adults.
• Sex-ratio: Take a sample of 500 of the adults from the emergence test and
count the number of male wasps. These are completely black and easily
distinguished from the females which have a yellow abdomen. The number of
females should be ≥ 98%.
• Fecundity: test will carried out on whitefly larvae

23. • Day 1: take black pupae which are close to emergence in a container.
• Day 2: Collect 30 freshly emerged females, put each into a small
container with a droplet of honey until the following day. This is to feed
them and to get them through the pre-oviposition period.
• Day 3: The test is conducted on individual females in small round
plastic "petri dish type" trays which can be closed very tightly. A nylon
mesh is incorporated into the lid to facilitate air exchange. Trays are
filled with agar solution (1%) to a depth of 10 mm. Just before the agar
solidifies a tobacco leaf disc is placed with its upper surface in contact
with the agar. The leaf disc should contain at least 25 whitefly larvae
(Trialeurodes vaporariorum) in the 3rd and 4th instar. Prepare 30 trays
in total and release 1 female per tray.
• Day 4: Provide the female with a new supply of whitefly larvae by
placing her in a new tray. Do this around 10 0'clock in the morning,
again.
• Day 5: Repeat day 4.
• Day 6: Remove the parasites from the whitefly larvae. Keep all whitefly
that were exposed to E. formosa in closed containers to prevent
unwanted parasitism after the test. Count all black pupae after 14 days.
• The average number of black pupae per female per day should be ≥7.
This test should be performed in the period August to October.

24. Leptomastix dactylopii Howard (Hymenoptera: Encyrtidae)
Test conditions
• Temperature: 25°C±2°C
• RH: 70±5%
• Light regime: 16L:8D
Quality control criteria
• Quantity: ≥ the number of live adults as specified on the label; a weekly or
batch-wise test
• Adult mortality: < 10%, based on 3 containers sampled and n=500 or more; a
weekly or batch-wise test
• Sex-ratio: ≥ 45% of the number specified on the label should be females; the
sex ratio does not necessarily have to be 45% as long as there are enough
females in the container; a four-weekly test.
• Fecundity: ≥ 40 offspring/female/14 days; n=30 females; an annual test
Description of testing methods
• Fecundity: Place a single potato tuber with short sprouts and infested by an
ample amount of L3 females of citrus mealybug, Planococcus citri, in a
ventilated container. Introduce a single pair of Leptomastix dactylopii into the
container. Leave the system as it is for 14 days. By the end of the 14 days, take
out the pair of wasps from each container. Collect emerging adults of L.
dactylopii from the 21st day up to the 31st day from the beginning of the
experiment. Calculate the cumulative number of adults emerging during this
period.

25. Orius spp. (O. laevigatus, O. insidiosus, O. majusculus,
O. aldibipennis) (Hemiptera: Anthocoridae)
Test conditions
• Temperature: 22-25o C
• RH: 70+5%
• Light regime: 16L:8D
Quality control criteria
• Quantity: The number of live adults/nymphs as specified on the container.
Species name(s) to be indicated on the label. A weekly test.
• Sex-ratio: ≥ 45% females; n=100 (picked at random; to distinguish between
male and female, see figure); a seasonal test
• Fecundity: ≥ 30 eggs/female/14 days; n=30 pairs; an annual test

26. Trichogramma brassicae Bezd. (=T. maidis)
(Hymenoptera: Trichogrammatidae)
Test conditions
• Temperature: 23+2o C
• RH: 75+10%
• Light regime: 16L:8D
• Rearing hosts: Ephestia kuehniella, Sitotroga cerealella
Species identification: The species is specified on the label and verified
by the producer
Quality control criteria
• Sex-ratio: ≥ 50% females; 100 adults assessed on 10 release units each
or 5 x 100 adults of bulk material; at least weekly or batch-wise test if
batches were exposed to special treatments (e.g. storage)
• Number of females: As indicated on label; determined as for sex-
ratio.
• Fecundity and longevity: ≥ 40 offspring / 7 days / female; 80% of
females should live at least 7 days; monthly or batch-wise test; n=30.
• Natural host parasitism: ≥ 10 parasitized hosts / 4 hours / female

27. Description of testing methods
• Fecundity and longevity: 30 females (age 24 hrs) are confined
individually in glass tubes; at least 200 factitious host eggs (< 24
hrs) are glued with water on a small cardboard strip. Eggs of E.
kuehniella (< 24 hrs old) are UV irradiated and provided at day 1
and removed after day 7; fresh eggs of S. cerealella are provided
at day 1, 3 and 5. The number of living adults is recorded after
day 7. Egg-cards are incubated and the number of black eggs is
counted not earlier than at day 10
• Natural host parasitism: 30 females (age 24 hrs) are confined
individually in tubes; two fresh egg-masses of at least 20
eggs/egg-mass of Ostrinia nubilalis (< 24 hrs old) are added for
4 hrs; after separation of the egg-masses from the females they
are incubated for 3 days; the number of black eggs is counted;
the mean number of black eggs is ≥ 10 per female. The host
cluster acceptance rate (= females parasitizing at least one host
egg) should be ≥ 80%.

28. Registration standards
Bio-pesticides Registration
At present, microbial pesticides like fungi, NPV etc., are
included in the schedule of Insecticides Act, 1968 and
Insecticides rule, 1971. This ensures the quality of bio-
pesticides at farmers level.
Directorate of Plant Protection Quarantine and Storage,
Department of Agriculture and Cooperation, Ministry of
Agriculture, GOI have issued guidelines/data requirements for
registration of bio-pesticides in the country. As per this, all the
units have to meet the Indian standards and technical
specifications to be eligible for registration under the
Insecticides Act, 1968.

29. Registration of Insecticides Under Insecticides Rules, 1971
1.a. An application for registration of an insecticide under the Act shall be made in Form I and the said
Form including the verification portion, shall be signed in case of an individual by the individual
himself or a person duly authorized by him in case of partnership firm by the managing partner ; in
case of a company, by any person duly authorized in that behalf by the Board of Directors ; and in
any other case by the person in-charge or responsible for the conduct of the business.
b. The Registration Committee may, if necessary, direct inspection of the 'testing facility' for
establishing the authenticity of the data.
2. An application form duly filled together with a bank draft of Rs. 100 only, drawn in favour of the
Accounts Officer, DPPQ&S, payable at Faridabad towards registration fee shall be sent to the
Secretary, Registration Committee, DPPQ&S,, NH-IV, Faridabad-121001, Haryana. One Self
addressed stamped envelope and one stamped envelope must be enclosed along with the
application.
3. The registration fee payable shall be paid by a demand draft drawn on the State Bank of India,
Faridabad, in favour of the Accounts Officer, Directorate of Plant Protection, Quarantine and
Storage, Faridabad, Haryana.
4. The certificate of registration shall be in Form II or Form II-A, as the case may be and shall be
subject to such conditions as specified therein.
Issue of duplicate certificate of registration
A fee of rupees five only shall be paid for a duplicate copy of the Certificate of Registration if the
original is defaced, damaged or lost.

31. Sl.
No.
Parameters 9(3B) 9(3)
1.
1.1
Systematic name (Genus and species)
Strain name
R
R
R
R
2 Common name, if any R R
3. Source of origin as Annexure-1.1 R R
4. Specification of the product as per Annexure-I. R R
5.
5.1
5.2
5.3
5.4
Composition of the product
Viral Unit: POB/Capsule count pr ml/g of the product
Percent content of the bio-control organism in the formulation and nature of biomass
Percent of carrier/filler, wetting/dispersing agent, stabilizers/ emulsifiers, containments/ impurities etc.
Moisture content
R
R
R
R
R
R
R
R
R
R
6. Manufacturing process R R
7. Test procedure and criteria used for identification by DNA test (Restriction enzymes analysis test). R R
8.
8.1
8.2
Method of analysis
Viral unit:
NPVs 1x109 POB/ml or g. minimum
GVs: 5x10 9 capsules /ml or g. minimum
(For NPV/GV, POB/Capsule Count will be taken with Haemocyto meter as detailed in Appendix-I)
Biological assays for determining the LC50 / LD50 of the formulation
Bioassay for NPV by the Diet Surface Contamination Method as detailed in Appendix-II
Bioassay for GV against Chilo infuscatellus as detailed in Appendix-III
Bioassay for GV against Plutella xylostella as detailed in Appendix-IV
Bioassay for GV against Acheae janta as detailed in Appendix-V
R
R
R
R
9.
9.1
9.2
9.3
Contaminants:
Pathogenic contaminants (Salmonella, Shigella, Vibrio etc.)
Other microbial contaminants
Chemicals and botanical pesticide contaminants
R
R
R
R
R
R
R
R
10.
10.1
Shelf life claim: Not less than 6 months
Data on storage stability as detailed in Note 2.
R
R
R
R
11. A sample for verification (100 ml or g) R R
GUIDELINES / DATA REQUIREMENTS FOR REGISTRATION OF BACULOVIRUSES - NUCLEAR POLYHEDROSIS
VIRUS (NPV) & GRANULOSIS VIRUS (GV) UNDER SECTION 9(3B) AND 9(3) OF THE INSECTICIDE ACT, 1968
R = Required
NR = Not
Required
R** = Two
seasons/years data
on
bioeffectiveness
from minimum
two agro climatic
conditions
R*** = Two
seasons/years data
on
bioeffectiveness
from minimum
three agro climatic
conditions
A. BIOLOGICAL CHARACTERISTICS AND CHEMISTRY

32. 12. Field studies :data from SAU’s/ICAR
Institute certified by Director Research
of SAU or Head of the ICAR Institute
R** R***
13. Laboratory studies data on LC50 values
for each target insect species should be
generated at a laboratory under ICAR/
SAU/ CSIR/ICMR.
R R
14.
14.1
14.2
14.3
14.4
14.5
For mother culture
Single Dose Oral (rat and mouse)
Single dose pulmonary
Single dose intravenous
Cell culture
Human safety records.
R
R
R
R
R
R
R
R
R
R
15
15.1
15.2
15.3
15.4
15.5
15.6
For formulation
Data on mother culture as in 14 above
Single Dose Oral (Rat & Mouse)
Single dose pulmonary
Primary skin irritation
Primary eye irritation
Human safety records
R
R
R
R
R
R
R
R
R
R
R
R
16
16.1
16.2
16.3
16.4
16.5
16.6
For formulated product to be directly manufactured:
(Mammalian toxicity testing of formulations)
Single Dose Oral (Rat & Mouse)
Toxicity/Infectivity/Pathogenicity
Single dose pulmonary
Toxicity/Infectivity/Pathogenicity
(Intra-tracheal preferred)
Single dose intravenous
Toxicity/Infectivity/Pathogenicity
Human safety records (Effect/Lack of effects)
Primary skin irritation
Cell culture
R
R
R
R
R
R
R
R
R
R
R
R
17.
17.1
17.1.1
17.1.2
17.1.3
17.2
17.2.1
17.2.2
Environmental safety testing: Core Information
requirements
(For formulation only)
Non-target Vertebrates
Mammalsa
Birds(two species)b
Fresh water fishc
Non-target invertebrates
Terrestrial Invertebratesd
Soil invertebratese
NR
NR
NR
NR
NR
R
R
R
R
R
18.
18.1
18.2
18.3
18.4
Manufacturing process/process of formulation
Raw material
Plant and Machinery
Unit Process operation/Unit process
Out-put (Finished product and generation of
waste)
R
R
R
R
R
R
R
R
19
19.1
19.2
19.3
19.4
Packaging:
Classification-solid, liquid or other types of
product.
Unit pack size – In metric system
Specification – Details of primary, secondary and
transport pack
Compatibility of primary pack with the product
R
R
R
NR
R
R
R
R
20. Labels and leaflets
As per Insecticides Rules, 1971 indicating
the common name, composition, antidote,
storage, statements etc
R R
D. Processing, Packaging & Labelling
B. Bioefficacy: C. Toxicity:

33. INDIAN STANDARD BACULORIVUS SPECIFICATIONS
1. Form and composition of the product
1.1 Viral Unit: POB/Capsule count pr ml/g of the product
1.2 Percent content of the bio-control organism in the
formulation and nature of biomass
1.3 Percent of carrier/filler, wetting/dispersing agent,
stabilizers/ emulsifiers, containments/ impurities etc.
1.4 Moisture content
2. pH
4. Viral Unit:
NPVs (Helicoverpa & Spodeptera) - 1x109 POB/ml or gm (minimum ) (POB –Polyhedral
Occlusion Body)
GV (Chilo, Plutella & Acheae) - 5x109 Capsules/ml or g (minimum).
5. Contaminants:
5.1 Biological contaminants:
5.1.1 Pathogenic contaminants: Pathogenic contaminants such as gram negative bacteria
Salmonella, Shigella, Vibrio etc. should be absent.
5.1.2. Other microbial contaminants: Other microbial contaminants should not exceed
1x104 /ml or g
5.2 Chemical/botanical pesticides contaminants should be absent.

34. 6. Identification of Baculovirus by DNA test (Restriction enzyme analysis test).
7. An undertaking should be submitted that the strain is indigenous, naturally
occurring and not exotic and not genetically modified as per Annexure-1.1
8. Method of analysis:
Viral Unit:
NPVs (Helicoverpa and Spodeptera) =1x109 POB/ml or gm. minimum
GVs = 5x109 Capsules/ml or gm. minimum.
8.1. In case of NPVs/, POB/Capsule count should be taken with
Haemocytometer using shallow depth counting chamber as detailed in
Appendix – I
8.2. Biological assay for determining the LC50 or LD50 of the formulation:
8.2.1 Bioassay for NPV by the Diet Surface Contamination Method as detailed
in Appendix-II OR
8.2.2 Bioassay for GV against Chilo infuscatellus as detailed in Appendix-III
OR
8.2.3 Bioassay for GV against Plutella xylostella as detailed in Appendix-IV.
8.2.4 Bioassay for GV against Acheae janta as detailed in Appendix-V.
8.3 Plating for contaminants on specified media.