Fact Sheet
Removal and inactivation of Cryptosporidium
in the environment
Introduction
Cryptosporidium oocysts commonly occur in surface and
recreational waters due to faecal contamination from wild
animals or human activity. Oocysts have many characteristics that allow them to persist in aquatic environments
and withstand water treatment processes. Direct exposure
to contaminated recreational or drinking water can cause
outbreaks. However, as outlined below, the environment
can also be hostile to oocysts. This fact sheet provides an
overview of the processes that can remove or inactivate
Cryptosporidium oocysts in catchments and surface waters.
More information about Cryptosporidium, including the
identification and detection of infectious oocysts, is available
in the Cryptosporidium toolbox factsheet.
The terrestrial environment
The two main ways in which oocysts can enter surface
waters are by direct deposition (eg, animal excretion or
sewage discharge) or by rainfall runoff from land. The
terrestrial environment can expose oocysts to temperature
extremes and desiccation, which are particularly effective
at inactivating oocysts. Cow faeces exposed to sunlight can
reach internal temperature peaks between 40°C and 70°C
once the air temperature exceeds 25°C (Li et al. 2005).
Oocysts suspended in water are highly sensitive to these
temperatures, with 6 log10 inactivation after exposure to
60°C for 15 seconds or 55°C for 30 seconds. These rates
may be slower in faeces due to the presence of solids and
fats, but an inactivation rate of 3.3 log10 /day in cow faeces
has been reported for daily temperature cycles typical of
spring/autumn in a Mediterranean climate (Li et al. 2005). In
winter the inactivation rates are much slower, with 0.2 log10/
day when the internal faecal matrix temperature was 30°C
and 0.03 log10 / day when the matrix was at 20°C (Li et al.
2010). Although sensitive to heat, oocysts can survive short
periods of freezing (Fayer and Nerad 1996). However, cycles
of freeze/thawing rapidly inactivate oocysts, particularly in
soil where mechanical contact with particles causes abrasion
and fragmentation of oocysts (Jenkins et al. 1999, Kato et
al. 2002). Chemicals within faeces also cause significant
inactivation. Oocysts are highly sensitive to ammonia, with
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exposure to 0.06 M ammonia causing 5 log10 inactivation
after 8.2 days at 24°C or 55 days at 4°C (Jenkins et al. 1998).
Considering this sensitivity, it is likely that some level of
inactivation by ammonia will occur in
animal faeces even at low temperatures,
particularly if animal waste is stored prior
to disposal (Hutchison et al. 2005)
As mentioned, oocysts are extremely
sensitive to desiccation, which can occur
within faeces because of processing by
dung beetles (Ryan et al. 2011) or once
oocysts have been washed into soil.
Inactivation by desiccation can be rapid,
with oocysts air-dried onto glass slides
inactivated by 2.5 log10 within 2 hours
and completely inactivated after 4 hours
(Robertson et al. 1992). In addition to
desiccation, the physical, chemical and
biological properties of soil may reduce
oocyst survival (Ferguson et al. 2003).
Soils with low water potential (osmolality),
in combination with temperature and
freeze-thawing, resulted in enhanced
oocyst degradation, with degradation
more rapid in dry soil compared with calf faeces or in
water at low temperatures (Walker et al. 2001). Oocysts in
saturated loamy soil had comparable inactivation to oocysts
in distilled water, with 0.93 log10 inactivation after 10 days
at 30°C (Nasser et al. 2007). In comparison, oocysts in dry
loamy soil had 2.5 log10 inactivation after 10 days at 32°C
(Nasser et al. 2007).
Aside from inactivation while on land, oocysts can also be
removed prior to or during transport into receiving waters.
The transport of oocysts through soils is highly dependent
on soil type and conditions. Under some conditions oocysts
do not appear to attach to soil particles in water (Brookes et
al. 2006, Dai and Boll 2003, Kaucner et al. 2005). However,
oocysts will attach to clay loam and sandy loam in the
presence of manure (Kuczynska et al. 2005). Irrespective
April 2016 Page 1
of binding, soils can act as a filter to limit oocyst mobility
(Tufenkji et al. 2004), with the majority of oocysts retained in
the top 2 cm of soil (Mawdsley et al. 1996). Oocysts can pass
through saturated macroporous soils, with oocyst removals
of 1 – 3 log10 under such conditions (Darnault et al. 2003).
Removal of oocysts through soil appears to be variable,
possibly due to variation in the health of oocysts within the
oocyst population, with the observed transport not consistent
with colloid theory (Santamaria et al. 2011). Vegetation can
also be effective at removing Cryptosporidium (Tate et al.
2004), with vegetation on sandy loam removing 1 – 2 log10
oocysts/meter compared with a reduction of 2 – 3 log10
oocysts/meter on silty clay or loam (Atwill et al. 2002).
Surface waters
In addition to providing a medium for oocyst transmission
to a host, aquatic environments can support oocyst survival
by providing a thermal buffer against temperature extremes.
However, many factors can remove or inactivate oocysts in
water, including particle interactions, temperature, sunlight
exposure and predation by protozoans or zooplankton.
Oocyst transport in reservoirs is predominantly driven
by inflows, particularly from rainfall (Brookes et al.
2004). Inflow temperature, velocity, insertion depth and
entrainment rate all determine the distribution of oocysts
in lakes and reservoirs (Brookes et al. 2004). The position of
oocysts can influence survival, with oocysts at the surface
exposed to sunlight, whereas oocysts at depth can escape
sunlight inactivation but be removed by predation. The
sedimentation of individual oocysts is relatively slow (0.27 –
0.35 µm/s) under laboratory conditions (Dai and Boll 2006,
Medema et al. 1998). Such a sedimentation rate makes it
unlikely that single oocysts will settle in large water bodies.
However, attachment to particles or organic aggregates can
greatly increase sedimentation rates by 100-fold (Medema
et al. 1998, Searcy et al. 2005). Oocyst sedimentation rates
measured in Lake Burragorang were 57.9 – 115.7 µm/s, likely
due to oocyst aggregation with other oocysts or particles
(Hawkins et al. 2000). Oocysts that enter sediment can
potentially be remobilised by re-suspension events such as
turbulence from underflows (Michallet and Ivey 1999).
Sunlight, in particular ultraviolet (UV) light, can be harmful
to a wide variety of organisms and is a potent environmental
stress for Cryptosporidium inactivation. Different
wavelengths of UV light cause different types of damage,
with UV-B (280nm to 320nm) causing DNA damage and UV-A
(320nm-400nm) causing damage to cellular components
such as lipids and proteins (Caldwell 1971, Friedberg et al.
1995, Malloy et al. 1997, Ravanat et al. 2001). Solar radiation
can inactivate oocysts suspended in tap water and
reservoir waters, with UV-B causing approximately two
thirds of the observed inactivation (King et al. 2008).
The rate of solar inactivation is affected by the level of
dissolved organic carbon (DOC) in the water and the
level of solar radiation (predicted by the UV index). In
the case of water with low (2.8 mg/L) DOC and low (2
Hazen units (HU)) colour, the time required for 1 log10
oocyst inactivation was 3.2 hours on a winter day with a UV
index of 3 and 0.9 hours on a summer day with a UV index
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of 10. In contrast, these values were 10.8 hours and 6.5
hours respectively for water with high (12.3 mg/L) DOC and
high (77 HU) colour (King et al. 2008). The presence of DOC
reduces penetration of UV in water, protecting oocysts that
are at sufficient depth. However, solar inactivation may still
occur from UV-A, which has a longer wavelength than UV-B
and may be able to better penetrate surface waters, and from
resuspension events such as warm water inflows.
Oocysts in water are not exposed to the same temperature
extremes as those on land. Inactivation still occurs, but the
rate is relatively slow, requiring 16 – 24 weeks at 15°C for 1
log10 oocyst inactivation (Keegan et al. 2008). Inactivation
rates increase for higher water temperatures, with more than
3 log10 inactivation after 12 weeks at 20°C, 8 weeks at 25°C and
3 weeks at 30°C (King et al. 2005). The rate of inactivation was
the same in sterile reservoir water and tap water.
Oocysts surviving thermal or solar inactivation can be removed
by biological interactions, especially at interfaces such as
sediments and biofilms. Despite the potential importance of
biological interactions for the removal of oocysts from water,
this field has been little studied. A wide range of organisms
isolated from reservoir water can ingest oocysts, including
rotifers, ciliates, amoebae, gastrotrichs and platyhelminths
(King et al. 2007). The kinetics of removal has not been
determined using environmentally relevant oocyst numbers.
Predation may lead to removal of oocysts through degradation
of ingested oocysts or by causing clumping of excreted
oocysts that are not digested (King et al. 2007). The infectivity
of oocysts excreted by freshwater predators has not been
determined. However, predation of oocysts by zooplankton
isolated from a waste stabilisation pond caused 0.7 to >2 Log10
inactivation depending on the types of zooplankton present in
the microcosm experiment (Salas Iglesias 2014). In addition to
providing an environment for interaction between predators
and oocysts, biofilms might also directly interact with oocysts.
The attachment to and detachment of oocysts from biofilms
can be variable and appears to be influenced by season
(Wolyniak et al. 2010). Of significance, oocysts embedded
at the bottom of a biofilm retain infectivity up to two times
better than oocysts at the top of a biofilm or oocysts with no
biofilm following sunlight exposure (DiCesare et al. 2012).
Another potential source of oocyst removal in freshwaters is
by filter feeders such as bivalve molluscs, which concentrate
oocysts from contaminated waters (Graczyk et al. 2001, Izumi
et al. 2006). Only 1-3% of oocysts are retained following
ingestion, with the excreted oocysts retaining infectivity (Izumi
et al. 2006). Based on the high clearance rate, it is possible
that detection of low numbers of oocysts in mollusc tissue
represents previous contamination events, while high numbers
of oocysts represents a recent event.
An amoeba (Mayorella sp.) containing approximately thirteen ingested
oocysts of Cryptosporidium parvum. (from King et al, CRCWQT Report 47)
April 2016 Page 2
Unlike oocysts in animal faeces, oocysts in water are unlikely
to be exposed to pH extremes or high concentrations of
chemicals that might cause inactivation, such as ammonia.
However, anthropogenic activity could introduce other
stressors. For example, stabilised hydrogen peroxide is a
candidate being evaluated for the control of cyanobacterial
blooms. Hydrogen peroxide is effective at inactivating oocysts
(Weir et al. 2002), but it is not known if the dosing strategy
used to control cyanobacteria will provide a high enough
concentration to be effective against Cryptosporidium oocysts.
Conclusions
There are many opportunities for removal or inactivation
of oocysts in the environment, starting with excretion of
contaminated faeces onto land, followed by transport into
surface waters and transport through a water storage to
a water treatment plant. Considering the effectiveness of
many of these stressors for inactivating Cryptosporidium,
accurate risk assessment of Cryptosporidium does not just
require determination of the number of oocysts in a water
sample but also if the detected oocysts are infectious. For
some sources of inactivation, such as temperature and solar
radiation, there are sufficient data to allow inclusion in fate
and transport models. However, there are still knowledge
gaps in terms of the contribution of predation to oocyst
removal and inactivation, as well as gaps in understanding
thermal inactivation in the terrestrial environment,
especially for particular Australian climate zones.
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