Quick insights
- The WHO cone test is used to assess the efficacy of insecticide-treated materials
- WHO cone test measures two endpoints – mosquito knockdown after 60 minutes (KD60) and mortality after 24 hours (M24)
- Knockdown indicates the immediate effect of insecticide exposure, whereas mortality indicates the delayed lethal effect
- Both KD60 and M24 are both important to interpret WHO Cone test results and assess product performance
- High knockdown does not always mean high mortality because some mosquitoes are capable of recovering after initial paralysis
- WHO Cone testing is applicable to insecticide-treated nets, textiles, residual sprays, coatings and other vector control products
Summary
The WHO Cone test measures both immediate mosquito lockdown (KD60) and delayed mortality (M24) to evaluate the efficacy of insecticide-treated materials. These measured parameters provide reliable WHO Cone test results that help manufacturers, researchers and public health organizers in evaluating product performance, detect potential insecticide resistance and support quality assurance.
WHO Cone test: Knockdown vs mortality – What do the results really mean?
The WHO Cone test, developed by World Health Organization, is a laboratory bioassay for vector control guidelines to determine the biological activity of insecticide applied on treated surfaces. The WHO Cone test is one of most widely referenced tests to evaluate insecticide-treated materials used for mosquito control.
During the test, mosquitoes are exposed to a treated surface using a transparent plastic cone attached to the material. Mosquitoes are kept inside the cone for a defined contact time and then transferred to clean holding containers and observed over time.
It is applicable for treated mosquito net, textile, wall coating or residual surface treatment and provides valuable information about how mosquitoes respond after contacting the treated surface.
The two primary parameters measured are –
Knockdown at 60 minutes (KD60) which measures the immediate incapacitating effect of insecticide after exposure
Mortality at 24 hours (M24) which measures whether the mosquitoes ultimately die following a 24-hour recovery period.
Understanding the difference between these parameters is important to interpret WHO Cone test results. This combined assessment provides a complete assessment of insecticidal efficacy and long-term performance
WHO Cone test is commonly applicable to:
- Long-lasting insecticidal nets (LLINs)
- Insecticide-treated textiles
- Spray-treated surfaces
- Vector control coatings
- Other insecticide-treated materials used to reduce mosquito contact and disease transmission
Test mosquito species used in WHO Cone testing:
- Aedes
- Culex
How does WHO Cone testing generate KD60 and M24 results?
The WHO Cone test follows a standard procedure to generate results:
- WHO Cones are attached to the treated surfaces
- A known number of female mosquitoes are introduced into the cone for fixed exposure time
- After exposure, Knockdown is observed at 60 minutes
- Knocked down mosquitos are then transferred into a clean chamber and kept under controlled conditions with 10 % sucrose solution for 24 hours observation period Following the exposure period, mortality rates are assessed
Understanding Knockdown (KD60)
KD60 is the percentage of mosquitoes knocked down within 60 minutes after being exposed to the insecticide treated surfaces. Knocked down mosquitoes are the ones that are immobilized or unable to fly normally due to the insecticide. The knocked down mosquitoes are observed by looking at these behaviour patterns and recorded. This parameter helps in understanding the instantaneous effect of the product on mosquitoes.
Understanding mortality (M24)
24-hour mortality measures the percentage of mosquitoes that die within 24 hours after the exposure period and holding time under controlled conditions.
Evaluating mortality rate is useful while testing long-term insecticidal properties of products. Some products may show immediate knockdown effects, but this effect can be temporary, and resistant mosquitoes can recover and continue to transmit disease
For this reason, 24-hour mortality is an important parameter in mosquito control product testing. It allows researchers to assess true product efficacy and long-term vector control performance.
Knockdown vs Mortality: Key differences
| Parameter | Knockdown (KD60) | Mortality (M24) |
| Observation time | 60 minutes after exposure | 24 hours after exposure |
| What it measures | Immediate incapacitation | Confirmed death of mosquitoes |
| What it indicates | Speed of insecticide action | Overall lethal efficacy |
| Primary use | Early evaluation of activity | Final efficacy evaluation |
Why does high knockdown not always mean high mortality?
All mosquitoes that are knocked down cannot be dead. Some insecticides work by interfering with mosquito’s nervous system, leading to temporary paralysis quickly after exposure. But, if the dose of insecticide is insufficient to cause irreversible damage, some mosquitoes can recover normal movement during the 24-hour holding period. Hence, high KD60 values cannot indicate high mortality without evaluating M24 results
Can mortality be high even when knockdown is moderate?
Yes, during evaluating WHO cone test results, some insecticides cause slower toxic effects in mosquitoes that are not immediately visible during 60-minutes observation time. In these cases, mosquitoes are active for a while after exposure but undergo progressive physiological damage which causes death in the next 24 hours. Hence, in some cases, high mortality can be observed even if the knockdown values are moderate
Why do some mosquitoes recover after knockdown?
Several factors can contribute to this behaviour in mosquitoes in WHO cone test results, such as:
- Knock-down resistance (KDR) mutations: This genetic mutation is found in insects that build resistance to pyrethroid insecticides and DDT.
- Metabolic detoxing: Resistant mosquitoes can also produce excess detoxifying enzymes that break down or sequester insecticides before the chemicals cause permanent damage
- Low levels of insecticide uptake during exposure – Some mosquitoes may absorb only a small amount of insecticide during the exposure period
What do KD60 and M24 reveal about insecticide resistance?
Behaviour of mosquitoes when evaluating KD60 and M24 can provide useful insights to understand potential insecticide resistance. If the KD60 or M24 values are lower than expected, it may indicate that mosquitoes possess resistance mechanisms that reduce/tolerate efficacy of insecticide
However, WHO Cone test cannot alone confirm resistance. Unusual WHO Cone test results can mean that there is a need for additional resistance investigation should be done using specialized susceptibility assays.
Also read
- WHO Cone Test vs Tunnel Test: A Practical Comparison for Insecticide Treated Product Testing
- Understanding the WHO Cone Test: Evaluating Insecticide-Treated Surfaces and Materials
What is considered a good WHO Cone test result?
There is no universally accepted “good” result for this test because passing criteria depends on product type, insecticide used and applicable regulatory requirements.
Generally, strong WHO Cone test results are :
- High KD60 values / high M24 values
- Low mortality in untreated control groups
- Consistent results across replicate tests
Need help with WHO Cone testing?
At Microbe Investigations Switzerland (MIS), we provide WHO Cone Test services to assess the effectiveness of insecticide-treated materials through standardized mosquito knockdown and mortality evaluations.
Our expert entomology team performs tests in accordance with internationally recognized protocols, generating reliable and reproducible results that support product development and regulatory submissions.
To further discuss your testing needs, contact our experts today
FAQs
1. Can mosquitoes recover after knockdown?
Yes, some mosquitoes may recover after being knocked down if the insecticide exposure is not sufficient to cause irreversible physiological damage
2. Is mortality more important than knockdown?
Importance of parameters depends on the product type and action of insecticide on mosquitoes. Generally both mortality and knockdown are considered equally important
3. What mosquitoes species are used in WHO Cone tests?
The test uses Anopheles, Aedes and Culex species for the procedure.
4. Why are both KD60 and M24 needed?
Evaluating both parameters allows a comparison between temporary incapacitation and true lethal efficacy, providing a complete assessment of product performance
5. Can WHO Cone test be used for insecticide treated textiles?
Yes, WHO Cone test is applicable for insecticide-treated textiles
6. Can high knockdown occur without high mortality?
Yes, some insecticides can cause rapid paralysis in mosquitoes but do not deliver lethal dose to kill the mosquitoes. In such cases, Knockdown can be high without high mortality
7. Does WHO prioritize knockdown or mortality?
WHO considers both measurements equally important as they evaluate different biological responses
8. Can insecticide resistance affect KD60 and M24?
Yes, insecticide resistance can lead to lesser-than-usual KD60 and M24 values
9. What products commonly undergo WHO Cone testing?
WHO Cone test is commonly applicable to:
- Long-lasting insecticidal nets (LLINs)
- Insecticide-treated textiles
- Spray-treated surfaces
- Vector control coatings
- Other insecticide-treated materials used to reduce mosquito contact and disease transmission
