The impact of the volume of the respiratory droplets and their evaporation kinetics on the environmental stability of respiratory viruses

In a recent study posted to the bioRxiv* preprint server, researchers measured the impact of initial respiratory droplet volume and relative humidity (RH) on the environmental stability of respiratory viruses, including influenza A and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

Research: The environmental stability of enveloped viruses is influenced by the initial volume and evaporation kinetics of droplets. Image Credit: Corona Borealis Studio/Shutterstock

In addition, they examined a bacteriophage, Phi6, a common surrogate for enveloped viruses. Other determinants of the environmental stability of these viruses are virion structure, droplet composition, fomite surface material and temperature.


During the 2019 coronavirus disease (COVID-19) pandemic, studies are overestimating the risk of SARS-CoV-2 transmission on contaminated surfaces. They demonstrated the importance of fomite transmission based on SARS-CoV-2 stability estimates in droplets up to 50 L. However, they barely explained virus decay in smaller, more physiologically relevant droplet volumes.

The droplet size typically determines the distance traveled by respiratory discharges and the host infection site. Smaller droplets or aerosols travel further, and those less than 10 m in diameter are more likely to settle deep in the airways. Previous studies measuring virus stability in the environment created droplet volumes ranging from five to 50 L, while airway droplets were less than 0.5 µL. Therefore, these studies failed to properly mimic a droplet physiological volume created by a respiratory expansion.

About the study

In the present study, researchers measured the environmental stability of the H1N1 strain of influenza A virus and Phi6 in droplets of 50, five and one µL at 40%, 65% and 85% RH in a controlled humidity chamber. In addition, the researchers examined droplet evaporation rates for which they used a microbalance to measure droplet mass every 10 minutes for up to 24 hours and performed all evaporation experiments in duplicate.

For experiments estimating the stability of SARS-CoV-2, the team used an airtight desiccator at room temperature and 55% RH. For Phi6 and H1N1 virus, they first pipetted droplets onto polystyrene tissue culture-coated six-well plates. They then resuspended droplets at seven time points — zero minutes, 20 minutes, 40 minutes, one hour, four hours, eight hours, and 24 hours. Finally, the team examined the effect of droplet morphology and drying pattern after 24 hours on different droplet volumes.

Study findings

The authors noted that the droplet drying pattern after 24 hours depended on the RH but not on the initial droplet volume, so any differences in viral decay by initial droplet size were not due to final physicochemical differences. At all RVs (40%, 65%, 85%), the droplets linearly lost their mass over time before reaching a plateau, which is called a quasi-equilibrium stage. The researchers defined the period before and after the quasi-equilibrium stage as the wet and dry phase, respectively. The decay of enveloped viruses likely depended on complex interactions of media components with the viral glycoprotein and its changes during and after drying.

The evaporation was faster for smaller droplets and at a lower RH. The time to reach quasi-equilibrium at 40% and 85% RH ranged from 0.5 to 11 hours for one L and 50 µL droplets, respectively. The study data indicated that the initial droplet volume altered the drying kinetics, affecting virus stability. SARS-CoV-2 and H1N1 virus similarly decayed at 65% RH (mean RH), and the differences were only apparent in larger droplets.

Furthermore, for all drop sizes tested, while a droplet was wet and evaporation was still occurring, the viruses were subject to a faster decay rate than after reaching quasi-equilibrium. An earlier preprint showed that biphasic virus decay probably also occurs in aerosols. Therefore, the first phase of viral decay was significant for short-range transmission, while both phases appeared important for longer-distance viral transmission. The first viral decay stage occurred within seconds and further decay occurred at the quasi-equilibrium stage.


The study emphasized the importance of using physiologically relevant media and careful use of surrogates for an accurate assessment of the transmission risk of future emerging pathogens. The study results showed that RH had a greater impact on viral decay in 50 µL droplets than in one µL droplets. Furthermore, the viral decay rates during the wet phase were greater than or comparable to the decay rates in the dry phase, regardless of droplet size and RH. The differences in virus decay were more common in 50 L droplets than in one µL droplets and at low RH.

The study results challenged previous studies estimating viral stability with large droplet volumes. According to the authors, the results of those studies would have been different if they had used smaller droplet volumes, especially over shorter time periods. Over 24 hours, viral decay was similar in all three droplet volumes. Physical and chemical properties of the droplets, initial volume and humidity of the environment probably caused their evaporation at different rates and resulted in these differences.

The study results also warned against extrapolating survival times from surrogates to other viruses and strain selection. In the current study, Phi6 decayed faster than the H1N1 virus and SARS-CoV-2 under experimental conditions; thus relying solely on Phi6 data can lead to potentially erroneous conclusions about pathogenic viruses. In fact, H1N1 matured more like SARS-CoV-2 and could serve as its surrogate while extrapolating its persistence in more physiologically relevant conditions.

Future studies should focus on creating real-world conditions for respiratory droplet volume (ranging from submicrons to hundreds of microns in diameter) and respiratory fluid chemical composition to improve public policy for optimal SARS-CoV-2 transmission control strategies.

*Important announcement

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and therefore should not be considered conclusive, should guide clinical practice/health-related behavior, or be treated as established information.

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