Fact Sheet Removal and inactivation of in the environment

Introduction

Cryptosporidium oocysts commonly occur in surface and exposure to 0.06 M ammonia causing 5 log10 inactivation recreational waters due to faecal contamination from wild after 8.2 days at 24°C or 55 days at 4°C (Jenkins et al. 1998). animals or human activity. Oocysts have many character- Considering this sensitivity, it is likely that some level of istics that allow them to persist in aquatic environments inactivation by ammonia will occur in and withstand water treatment processes. Direct exposure animal faeces even at low temperatures, to contaminated recreational or can cause particularly if animal waste is stored prior outbreaks. However, as outlined below, the environment to disposal (Hutchison et al. 2005) can also be hostile to oocysts. This fact sheet provides an overview of the processes that can remove or inactivate As mentioned, oocysts are extremely Cryptosporidium oocysts in catchments and surface waters. sensitive to desiccation, which can occur More information about Cryptosporidium, including the within faeces because of processing by identification and detection of infectious oocysts, is available dung beetles (Ryan et al. 2011) or once in the Cryptosporidium toolbox factsheet. oocysts have been washed into soil. Inactivation by desiccation can be rapid, The terrestrial environment with oocysts air-dried onto glass slides inactivated by 2.5 log10 within 2 hours The two main ways in which oocysts can enter surface and completely inactivated after 4 hours waters are by direct deposition (eg, animal excretion or (Robertson et al. 1992). In addition to sewage discharge) or by rainfall runoff from land. The desiccation, the physical, chemical and terrestrial environment can expose oocysts to temperature biological properties of soil may reduce extremes and desiccation, which are particularly effective oocyst survival (Ferguson et al. 2003). at inactivating oocysts. Cow faeces exposed to sunlight can Soils with low water potential (osmolality), reach internal temperature peaks between 40°C and 70°C in combination with temperature and once the air temperature exceeds 25°C (Li et al. 2005). freeze-thawing, resulted in enhanced Oocysts suspended in water are highly sensitive to these oocyst degradation, with degradation more rapid in dry soil compared with calf faeces or in temperatures, with 6 log10 inactivation after exposure to 60°C for 15 seconds or 55°C for 30 seconds. These rates water at low temperatures (Walker et al. 2001). Oocysts in may be slower in faeces due to the presence of solids and saturated loamy soil had comparable inactivation to oocysts in distilled water, with 0.93 log inactivation after 10 days fats, but an inactivation rate of 3.3 log10 /day in cow faeces 10 has been reported for daily temperature cycles typical of at 30°C (Nasser et al. 2007). In comparison, oocysts in dry spring/autumn in a Mediterranean climate (Li et al. 2005). In loamy soil had 2.5 log10 inactivation after 10 days at 32°C (Nasser et al. 2007). winter the inactivation rates are much slower, with 0.2 log10/ day when the internal faecal matrix temperature was 30°C Aside from inactivation while on land, oocysts can also be and 0.03 log10 / day when the matrix was at 20°C (Li et al. 2010). Although sensitive to heat, oocysts can survive short removed prior to or during transport into receiving waters. periods of freezing (Fayer and Nerad 1996). However, cycles The transport of oocysts through soils is highly dependent of freeze/thawing rapidly inactivate oocysts, particularly in on soil type and conditions. Under some conditions oocysts soil where mechanical contact with particles causes abrasion do not appear to attach to soil particles in water (Brookeset and fragmentation of oocysts (Jenkins et al. 1999, Kato et al. 2006, Dai and Boll 2003, Kaucner et al. 2005). However, al. 2002). Chemicals within faeces also cause significant oocysts will attach to clay loam and sandy loam in the inactivation. Oocysts are highly sensitive to ammonia, with presence of manure (Kuczynska et al. 2005). Irrespective

Water Research Australia Research solutions though collaboration April 2016 Page 1 of binding, soils can act as a filter to limit oocyst mobility of 10. In contrast, these values were 10.8 hours and 6.5 (Tufenkji et al. 2004), with the majority of oocysts retained in hours respectively for water with high (12.3 mg/L) DOC and the top 2 cm of soil (Mawdsley et al. 1996). Oocysts can pass high (77 HU) colour (King et al. 2008). The presence of DOC through saturated macroporous soils, with oocyst removals reduces penetration of UV in water, protecting oocysts that are at sufficient depth. However, solar inactivation may still of 1 – 3 log10 under such conditions (Darnault et al. 2003). Removal of oocysts through soil appears to be variable, occur from UV-A, which has a longer wavelength than UV-B possibly due to variation in the health of oocysts within the and may be able to better penetrate surface waters, and from oocyst population, with the observed transport not consistent resuspension events such as warm water inflows. with colloid theory (Santamaria et al. 2011). Vegetation can also be effective at removing Cryptosporidium (Tate et al. Oocysts in water are not exposed to the same temperature 2004), with vegetation on sandy loam removing 1 – 2 log extremes as those on land. Inactivation still occurs, but the 10 rate is relatively slow, requiring 16 – 24 weeks at 15°C for 1 oocysts/meter compared with a reduction of 2 – 3 log10 oocysts/meter on silty clay or loam (Atwill et al. 2002). log10 oocyst inactivation (Keegan et al. 2008). Inactivation rates increase for higher water temperatures, with more than

Surface waters 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 In addition to providing a medium for oocyst transmission the same in sterile reservoir water and . to a host, aquatic environments can support oocyst survival by providing a thermal buffer against temperature extremes. Oocysts surviving thermal or solar inactivation can be removed However, many factors can remove or inactivate oocysts in by biological interactions, especially at interfaces such as water, including particle interactions, temperature, sunlight sediments and biofilms. Despite the potential importance of exposure and predation by protozoans or zooplankton. biological interactions for the removal of oocysts from water, this field has been little studied. A wide range of organisms Oocyst transport in reservoirs is predominantly driven isolated from reservoir water can ingest oocysts, including by inflows, particularly from rainfall (Brookeset al. rotifers, , amoebae, gastrotrichs and platyhelminths 2004). Inflow temperature, velocity, insertion depth and (King et al. 2007). The kinetics of removal has not been entrainment rate all determine the distribution of oocysts determined using environmentally relevant oocyst numbers. in lakes and reservoirs (Brookes et al. 2004). The position of Predation may lead to removal of oocysts through degradation oocysts can influence survival, with oocysts at the surface of ingested oocysts or by causing clumping of excreted exposed to sunlight, whereas oocysts at depth can escape oocysts that are not digested (King et al. 2007). The infectivity sunlight inactivation but be removed by predation. The of oocysts excreted by freshwater predators has not been sedimentation of individual oocysts is relatively slow (0.27 – determined. However, predation of oocysts by zooplankton

0.35 µm/s) under laboratory conditions (Dai and Boll 2006, isolated from a waste stabilisation pond caused 0.7 to >2 Log10 Medema et al. 1998). Such a sedimentation rate makes it inactivation depending on the types of zooplankton present in unlikely that single oocysts will settle in large water bodies. the microcosm experiment (Salas Iglesias 2014). In addition to However, attachment to particles or organic aggregates can providing an environment for interaction between predators greatly increase sedimentation rates by 100-fold (Medema and oocysts, biofilms might also directly interact with oocysts. et al. 1998, Searcy et al. 2005). Oocyst sedimentation rates The attachment to and detachment of oocysts from biofilms measured in Lake Burragorang were 57.9 – 115.7 µm/s, likely can be variable and appears to be influenced by season due to oocyst aggregation with other oocysts or particles (Wolyniak et al. 2010). Of significance, oocysts embedded (Hawkins et al. 2000). Oocysts that enter sediment can at the bottom of a biofilm retain infectivity up to two times potentially be remobilised by re-suspension events such as better than oocysts at the top of a biofilm or oocysts with no turbulence from underflows (Michallet and Ivey 1999). biofilm following sunlight exposure (DiCesare et al. 2012). Another potential source of oocyst removal in freshwaters is Sunlight, in particular ultraviolet (UV) light, can be harmful by filter feeders such as bivalve molluscs, which concentrate to a wide variety of organisms and is a potent environmental oocysts from contaminated waters (Graczyk et al. 2001, Izumi stress for Cryptosporidium inactivation. Different et al. 2006). Only 1-3% of oocysts are retained following wavelengths of UV light cause different types of damage, ingestion, with the excreted oocysts retaining infectivity (Izumi with UV-B (280nm to 320nm) causing DNA damage and UV-A et al. 2006). Based on the high clearance rate, it is possible (320nm-400nm) causing damage to cellular components that detection of low numbers of oocysts in mollusc tissue such as lipids and proteins (Caldwell 1971, Friedberg et al. represents previous contamination events, while high numbers 1995, Malloy et al. 1997, Ravanat et al. 2001). Solar radiation of oocysts represents a recent event. 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 log 10 An amoeba (Mayorella sp.) containing approximately thirteen ingested oocyst inactivation was 3.2 hours on a winter day with a UV oocysts of . (from King et al, CRCWQT Report 47) index of 3 and 0.9 hours on a summer day with a UV index

Water Research Australia Research solutions though collaboration April 2016 Page 2 Unlike oocysts in animal faeces, oocysts in water are unlikely Dai X and Boll J (2003) Evaluation of attachment of to be exposed to pH extremes or high concentrations of Cryptosporidium parvum and Giardia lamblia to soil particles. chemicals that might cause inactivation, such as ammonia. Journal of Environmental Quality 32(1), 296-304. However, anthropogenic activity could introduce other Cryptosporidium stressors. For example, stabilised hydrogen peroxide is a Dai X and Boll J (2006) Settling velocity of parvum Giardia lamblia candidate being evaluated for the control of cyanobacterial and . Water Research 40(6), 1321-1325. blooms. Hydrogen peroxide is effective at inactivating oocysts Darnault CJ, Garnier P, Kim YJ, Oveson KL, Steenhuis TS, (Weir et al. 2002), but it is not known if the dosing strategy Parlange JY, Jenkins M, Ghiorse WC and Baveye P (2003) used to control cyanobacteria will provide a high enough Preferential transport of Cryptosporidium parvum oocysts concentration to be effective against Cryptosporidium oocysts. in variably saturated subsurface environments. Water Environment Research 75(2), 113-120. Conclusions DiCesare EA, Hargreaves BR and Jellison KL (2012) Biofilms There are many opportunities for removal or inactivation reduce solar disinfection of Cryptosporidium parvum oocysts. of oocysts in the environment, starting with excretion of Applied Environmental Microbiology 78(12), 4522-4525. contaminated faeces onto land, followed by transport into surface waters and transport through a water storage to Fayer R and Nerad T (1996) Effects of low temperatures a water treatment . Considering the effectiveness of on viability of Cryptosporidium parvum oocysts. Applied many of these stressors for inactivating Cryptosporidium, Environmental Microbiology 62(4), 1431-1433. accurate risk assessment of Cryptosporidium does not just require determination of the number of oocysts in a water Ferguson C, Husman AMD, Altavilla N, Deere D and Ashbolt sample but also if the detected oocysts are infectious. For N (2003) Fate and transport of surface water pathogens in some sources of inactivation, such as temperature and solar watersheds. Critical Reviews in Environmental Science and radiation, there are sufficient data to allow inclusion in fate Technology 33(3), 299-361. and transport models. However, there are still knowledge Friedberg E, Walker G and Siede W (1995) DNA and gaps in terms of the contribution of predation to oocyst mutagenesis., ASM Press, Washington, DC. removal and inactivation, as as gaps in understanding thermal inactivation in the terrestrial environment, Graczyk TK, Marcogliese DJ, de Lafontaine Y, Da Silva AJ, especially for particular Australian climate zones. Mhangami-Ruwende B and Pieniazek NJ (2001) Cryptosporidium parvum oocysts in zebra mussels (Dreissena polymorpha): evidence from the St Lawrence River. Parasitology Research 87(3), 231-234.

Hawkins PR, Swanson P, Warnecke M, Shanker SR and Nicholson C (2000) Understanding the fate of Cryptosporidium and Giardia in storage reservoirs: a legacy of Sydney‘s water contamination incident. Journal of Water Supply Research and Technology-Aqua 49(6), 289-306.

Hutchison ML, Walters LD, Moore T, Thomas DJ and Avery SM (2005) Fate of pathogens present in livestock wastes spread onto fescue plots. Applied Environmental Microbiology71(2), 691-696.

References Izumi T, Yagita K, Endo T and Ohyama T (2006) Detection system of Cryptosporidium parvum oocysts by brackish water Atwill ER, Hou L, Karle BM, Harter T, Tate KW and Dahlgren RA benthic shellfish (Corbicula japonica) as a biological indicator (2002) Transport of Cryptosporidium parvum oocysts through in river water. Archives of Environmental Contamination and vegetated buffer strips and estimated filtration efficiency. Toxicology 51(4), 559-566. Applied Environmental Microbiology 68(11), 5517-5527. Jenkins MB, Bowman DD and Ghiorse WC (1998) Inactivation Brookes JD, Antenucci J, Hipsey M, Burch MD, Ashbolt NJ and of Cryptosporidium parvum oocysts by ammonia. Applied Ferguson C (2004) Fate and transport of pathogens in lakes Environmental Microbiology 64(2), 784-788. and reservoirs. Environment International 30(5), 741-759. Jenkins MB, Walker MJ, Bowman DD, Anthony LC and Ghiorse Brookes JD, Davies CM, Hipsey MR and Antenucci JP (2006) WC (1999) Use of a sentinel system for field measurements Association of Cryptosporidium with bovine faecal particles of Cryptosporidium parvum oocyst inactivation in soil and and implications for risk reduction by settling within water animal waste. Applied Environmental Microbiology 65(5), supply reservoirs. Journal of Water and Health 4(1), 87-98. 1998-2005. Caldwell MM (1971) Photophysiology. Giese AC (ed), pp. Kato S, Jenkins MB, Fogarty EA and Bowman DD (2002) Effects 131-177, Academic Press, NewYork. of freeze-thaw events on the viability of Cryptosporidium parvum oocysts in soil. Journal of Parasitology 88(4), 718-722.

Water Research Australia Research solutions though collaboration April 2016 Page 3 Monis PT, King BJ and Keegan A (2014). Removal Kaucner C, Davies CM, Ferguson CM and Ashbolt NJ (2005) and inactivation of Cryptosporidium from water. In Evidence for the existence of Cryptosporidium oocysts as Cryptosporidium: parasite and disease. (eds. Caccio, S.M., single entities in surface runoff. Water Science and Widmer, G.) pp 515-552. Springer-Verlag, Vienna, Austria. Technology 52(8), 199-204. Nasser AM, Tweto E and Nitzan Y (2007) Die-off of Keegan A, Daminato D, Saint CP and Monis PT (2008) Effect Cryptosporidium parvum in soil and wastewater effluents. of water treatment processes on Cryptosporidium infectivity. Journal of Applied Microbiology 102(1), 169-176. Water Research 42(6-7), 1805-1811. Ravanat JL, Douki T and Cadet J (2001) Direct and indirect King BJ and Monis PT (2007) Critical processes affecting effects of UV radiation on DNA and its components. Journal Cryptosporidium oocyst survival in the environment. of Photochemistry and Photobiology B 63(1-3), 88-102. Parasitology 134(Pt 3), 309-323. Robertson LJ, Campbell AT and Smith HV (1992) Survival King BJ, Hoefel D, Daminato DP, Fanok S and Monis PT (2008) of Cryptosporidium parvum oocysts under various Solar UV reduces Cryptosporidium parvum oocyst infectivity environmental pressures. Applied Environmental Microbiology in environmental waters. Journal of Applied Microbiology 58(11), 3494-3500. 104(5), 1311-1323. Ryan U, Yang R, Gordon C and Doube B (2011) Effect of King BJ, Keegan AR, Monis PT and Saint CP (2005) dung burial by the dung beetle Bubas bison on numbers Environmental temperature controls Cryptosporidium and viability of Cryptosporidium oocysts in cattle dung. oocyst metabolic rate and associated retention of infectivity. Experimental Parasitology 129(1), 1-4. Applied Environmental Microbiology 71(7), 3848-3857. Salas Iglesias S (2014) The Fate of Cryptosporidium oocysts in King BJ, Monis PT, Keegan AR, Harvey K and Saint C Bolivar waste stabilisation ponds. Masters, Flinders University (2007) Investigation of the survival of Cryptosporidium in of South Australia. environmental waters, Cooperative Reserarch Centre for and Treatment, Australia. Santamaria J, Quinonez-Diaz Mde J, Lemond L, Arnold RG, Quanrud D, Gerba C and Brusseau ML (2011) Transport of Kuczynska E, Shelton DR and Pachepsky Y (2005) Effect Cryptosporidium parvum oocysts in sandy soil: impact of length of bovine manure on Cryptosporidium parvum oocyst scale. Journal of Environmental Monitoring 13(12), 3481-3484. attachment to soil. Applied Environmental Microbiology 71(10), 6394-6397. Searcy KE, Packman AI, Atwill ER and Harter T (2005) Association of Cryptosporidium parvum with suspended Li X, Atwill ER, Dunbar LA, Jones T, Hook J and Tate KW particles: impact on oocyst sedimentation. Applied (2005) Seasonal temperature fluctuations induces rapid Environmental Microbiology 71(2), 1072-1078. inactivation of Cryptosporidium parvum. Environmental Science and Technology 39(12), 4484-4489. Tate KW, Pereira MD and Atwill ER (2004) Efficacy of vegetated buffer strips for retaining Cryptosporidium parvum. Li X, Atwill ER, Dunbar LA and Tate KW (2010) Effect of Journal of Environmental Quality 33(6), 2243-2251. daily temperature fluctuation during the cool season on the infectivity ofCryptosporidium parvum. Applied Tufenkji N, Miller GF, Ryan JN, Harvey RW and Elimelech M Environmental Microbiology 76(4), 989-993. (2004) Transport of Cryptosporidium oocysts in porous media: role of straining and physicochemical filtration. Environmental Malloy KD, Holman MA, Mitchell D and Detrich HW, 3rd Science and Technology 38(22), 5932-5938. (1997) Solar UVB-induced DNA damage and photoenzymatic DNA repair in antarctic zooplankton. Proceedings of the Walker M, Leddy K and Hager E (2001) Effects of combined National Academy of Science USA 94(4), 1258-1263. water potential and temperature stresses on Cryptosporidium parvum oocysts. Applied Environmental Microbiology 67(12), Mawdsley JL, Brooks AE and Merry RJ (1996) Movement of 5526-5529. the protozoan pathogen Cryptosporidium parvum through three contrasting soil types. Biology and Fertility of Soils Weir SC, Pokorny NJ, Carreno RA, Trevors JT and Lee H (2002) 21(1-2), 30-36. Efficacy of common laboratory on the infectivity of Cryptosporidium parvum oocysts in cell culture. Applied Medema GJ, Schets FM, Teunis PF and Havelaar AH (1998) Environmental Microbiology 68(5), 2576-2579. Sedimentation of free and attached Cryptosporidium oocysts and Giardia cysts in water. Applied Environmental Wolyniak EA, Hargreaves BR and Jellison KL (2010) Seasonal Microbiology 64(11), 4460-4466. retention and release ofCryptosporidium parvum oocysts by environmental biofilms in the laboratory. Applied Michallet H and Ivey GN (1999) Experiments on mixing due to Environmental Microbiology 76(4), 1021-1027. internal solitary waves breaking on uniform slopes. Journal of Geophysical Research-Oceans 104(C6), 13467-13477.

Water Research Australia Research solutions though collaboration April 2016 Page 4