the inactivation of viruses in pig slurries: a review

ELSEVIER Pll:SO960-8524(96)0005

THE INACTIVATION OF VIRUSES REVIEW

Biorrsvurce Technology 61 (1997) 9-20 0 1997 Elsevier Science Limited

All rights reserved. Printed in Great Britain 0960-8524197 $17.00

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IN PIG SLURRIES: A

Claire Turner* & Colin H. Burton

Silsoe Research Institute, Wrest Park, Silsoe, Bedford, MK45 4HS, United Kingdom

(Received 12 January 1997

Abstract This review discusses a number of possible treatments

for pathogen disinfection and evaluates them for their applicability in inactivating viruses in either dilute or concentrated pig slurry, particularly on a large scale. Methods are discussed under the headings of chemical, physical and other treatments. From those methods considered, it appears that the most suitable treatments are the use of heat at about 60°C for up to 30 min, or the application of an appropriate concentration of chemical, such as sodium or calcium hydroxides, or formalin. Aerobic and anaerobic treatments are also known to have virucidal effects, and could be used to assist decontamination on farms where such technolo- gies are already used in routine slurry treatment. 0 1997 Elsevier Science Ltd.

Key words: Viruses, decontamination, inactivation, pathogen disinfection, pig slurry, review.

NOMENCLATURE

AD(V) ASF(V) BOD COD CSTR DNA DS ECBO FMD HAV HCC IU PAA PPm RH RNA SDS SVD(V) Y

Aujeszky’s Disease Virus African Swine Fever Virus Biological Oxygen Demand Chemical Oxygen Demand Continuous Stirred Tank Reactor Deoxyribonucleic Acid Dry Solids Enteric Cytopathogen Bovine Orphan Foot and Mouth Disease Hepatitis A Virus Hepatitis Contagiosa Canis Infectious Unit Peracetic Acid Parts Per Million Relative Humidity Ribonucleic Acid Sodium dodecyl sulphate Swine Vesicular Disease Virus Gamma (irradiation)

*Author to whom correspondence should be addressed.

accepted 1 April 1997)

INTRODUCTION

Faeces, urine, uneaten food and bedding from inten- sively farmed pigs are usually collected as a slurry and stored in lagoons, pits or above-ground tanks until field conditions are suitable for application to agricultural land. In the UK, for example, current recommendations indicate that the storage volume should be sufficient to hold the slurry produced during a period of at least four months, unless an environmentally safe year-round disposal system is available (MAFF, 1991). As a result of this, large slurry stores are common, particularly on large farms. If an outbreak of a particular notifiable viral disease occurs, not only do all the animals have to be slaughtered and the bodies safely disposed of, but the slurry that accumulates needs to be treated to inactivate any residual virus contamination. This review examines ways in which this might be achieved, and evaluates microbial disinfection techniques from both the agricultural sector and the wastewater-treatment industry. It reports some of the studies done in the field of virus inactivation in animal wastes, and also considers information from the related field of wastewater treatment, concerning human and animal virus inactivation, since much of this may be relevant to disinfecting animal effluents. In some cases, bacterial disinfection studies are quoted for comparison. In practice, the contaminated liquids may include both slurry and wash-water since these are usually collected in a single handling system in most intensive pig farms. Consequently, depending on the amount of wash water used and the amount of rain collected in the same system, the primary effect is one of diluting the slurry. This dilution effect is likely to influence the effectiveness of various deactivation processes and therefore the approaches reviewed here will be considered with reference to low-strength and high- strength slurries. For simplicity, these will be referred to as dirty water and slurry, respectively, although the difference is solely that of dilution with water.

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10 C. Turner; C. H. Burton

The conclusions reached in this review are not only based on the efficacy of a particular technique in inactivating the viruses of interest, but also depend on the feasibility of decontaminating up to 5000 tonnes of effluent (or more in some cases), over a reasonable time period. This represents a likely slurry store size on a large pig farm, for instance. The time period should be at least an order of magnitude shorter than the time taken for inactivation to occur naturally during the storage of slurry.

GENERAL INACTIVATION/PERSISTENCE OF VIRUSES

Although it has been reported that there are a number of virucidal agents in sludge or slurry that have varying activities (see later), viruses can persist in liquid animal manure for a long time, depending on the storage conditions. Pesaro et al. (1995) looked at the in situ inactivation of picorna, rota, parvo, adeno and herpes viruses in non-aerated liquid and semi-liquid animal waste. Depending on the ambient temperature, pH and type of waste, the time required for a lo-fold reduction of the titre of infectious virus ranged from one week for the herpes virus to more than six months for rotavirus. Berg et al. (1988) found that enteroviruses survived for up to 38 days without loss of infectious virus titre in extended aeration of sludges at 5°C and Lund and Nissen (1983) found that a lo-fold reduction in the concentration of enterovirus seeded into liquid animal manure took two-four days at 20°C under aerated conditions, or 300 days at 5°C under non- aerated conditions. Scheuerman et al. (1991) studied three enteroviruses and a rotavirus, and found that virus inactivation was more rapid under aerobic than anaerobic conditions, and at higher temperatures. These results imply that seeded slurry may require a great deal of time (probably months) for natural inactivation of viruses in stored sludge, so a more ‘active’ and rapid inactivation technique will be required.

CHEMICAL TREATMENTS

Ozonation Ozone is produced by discharging an electric spark through oxygen, and the resulting ozone is bubbled through the effluent to be disinfected. This is widely used in the wastewater industry, either on its own, or in combination with another technique. It has been found by some researchers (Farooq and Akhlaque, 1983; Hartemann et al., 1983) that in general, viruses are more resistant to ozonation than bacteria such as Escherichia coli. Warriner et al. (1985) claimed that poliovirus was more susceptible to ozone than coliforms. It seems that reductions in the titres of infectious virus can be achieved by the application of

ozone in wastewater, provided a sufficiently high residual concentration is present. A 2 lo4 fold reduction in infectious units, IU, is chosen as the criterion here for virus inactivation, as this is the reduction level required for certifying a disinfectant’s efficacy against a viral agent. The required ozonation is not known for slurries, and it has been noted by a number of researchers that the presence of organic and solid material in wastewater can protect viruses against inactivation by ozone (Foster et al., 1980; Finch and Smith, 1989; Hartemann et al., 1983; Emerson et al., 1982; Farooq and Akhlaque, 1983) due to the reaction of ozone with the organic material present. Greene and Strenstrom (1994) show that total organic carbon levels predict the demand for ozone when trying to reduce coliform numbers, therefore, it seems very likely that higher levels of ozone will be required when inactivating viruses in slurries with higher solids and chemical oxygen demand (COD) levels than wastewater.

There is also some dispute in the literature as to the optimal temperature at which virus inactivation by ozone occurs. Botzenhart et al. (1993) and Her- bold et al. (1989) claim that ozone is more effective at lower temperatures (10°C rather than 20°C) whereas Roy et al. (1980) say that virus inactivation by ozone increases with increasing temperature. They agree that inactivation is more effective at a higher pH; in contrast, Harakeh and Butler (1985) claim that disinfection of viruses is better at a lower pH (best at pH 4), and much better in the presence of 0.5 M sodium bicarbonate. This, they believe, is because the OH. radical formed from ozonation is inhibited by carbonate or bicarbonate ions, implying that molecular ozone is more active against viruses than the OH. radical.

Other researchers have used ozonation in con- junction with other techniques. Venosa et al. (1984) found that if UV irradiation either preceeded or followed ozone treatment (but not when they were applied simultaneously), then the inactivation of Mycobactetium fortuitum was increased synergistic- ally. Burleson et al. (1975) used simultaneous treatments of ozonation and sonication (using the output from a 40 kHz ultrasonic generator to ener- gize a piezoelectric crystal transducer at the bottom of a treatment column) and found that sonication reduced the required contact time of ozone for the complete inactivation of the organisms (viruses and bacteria), although sonication on its own was ineffective.

The advantages of ozonation are that it is a widely used technique in the water and wastewater industries, so equipment and expertise are readily available with many suppliers; it is also known to have a strong virucidal effect in water. The disadvantages are that it is likely to require high ozone levels where organic carbon levels are high, so preclarification will be required. There is also a risk of explosion with high ozone levels. In addition, there

Viruses in pig slurries: A review 11

is the potential risk of toxicity to operators: the Short Term Exposure Limit is 0.3 ppm after 10 min and the Occupational Exposure Standard is 0.1 ppm for an 8 h weighted time average.

Chlorination This has been used for many years to treat potable water and wastewater; particularly to destroy con- taminating bacteria. It has much lower activity against viruses (Tyrrell et al., 1995; Warriner et al., 1985), although some researchers have found good reductions in phage or viruses in wastewater with the application of a chlorine compound. Lothrop and Sproul (1969) demonstrated 104-fold reductions in IU of T2 phage and poliovirus type 1 in various wastewater samples after chlorination, and Bosch et al. (1993) generated Cu2+ and Ag2+ ions electro- lytically in wastewater with low levels of chlorine and found a 104-fold reduction in poliovirus titre. Engel- brecht et al. (1980) and Bosch et al. (1993) both reported that there is a wide range of susceptibilities of different viruses. As with ozone, viruses may also be protected by the presence of organic material during chlorination; Hejkal et al. (1979) found that viruses occluded within particulate matter in faecal homogenates were protected from inactivation by chlorine compounds. It has been noted, however, that chlorination is generally less effective than ozonation in virus inactivation (den Blanken, 1985).

The main advantage of chlorination is that the technique is used widely in the wastewater industry, therefore equipment and expertise are readily available. However, toxic compounds are used and the virucidal activity is not particularly high.

Ethylene oxide treatment Jordy et al. (1975) looked at the virucidal properties of gases containing ethylene oxide mixed with carbon dioxide or with methyl formate. A number of viruses, such as enteroviruses, parvovirus, poxvirus and paramyxovirus known to be resistant to chemical or physical treatments were studied under a number of conditions, including in a dried state in animal manure and in liquid suspension. It was found that in general, ethylene oxide gases were able to inactivate the viruses even after they had been dried in the presence of organic matter, although dried virus seemed more resistant to ethylene oxide than virus in suspension. A disadvantage is that ethylene oxide is highly toxic, making its large-scale outdoor use impractical.

Peracetic acid (PAA) treatment PAA treatment has been examined by the wastewater industry as an alternative disinfection treatment to chlorine, which poses both toxicity and environmental problems. Both Morris (1993) and Baldry et al. (1991) compared PAA with a chlorine- based treatment, and found that chlorine performed better in reducing virus titres than PAA. With the

latter, an extended time period was required for virucidal action. Bacterial inactivation using the two disinfectants was similar, however. Harakeh (1984) found that quite high levels of PAA (at least 140 ppm) were needed for 104-fold reductions of certain viruses in municipal sewage effluent. It is also known that PAA treatment of slurry causes a very large amount of foam to be produced. Herni- man et al. (1973) compared PAA to a number of other chemical treatments and found it to produce the best reduction (104.9-fold reduction in IU) at a concentration of 1%.

An advantage is that it is less toxic than chlorine, however, high levels are required for virucidal activity as it performs worse against viruses than chlorine; in addition, foam production in slurry is likely.

Ammonia It has been shown that slurries themselves have certain virucidal properties, and in many cases this has been shown to be due to bacterial or proteolytic activity. However, other components have also been found to have virucidal properties; one of these is ammonia. Wekerle and Albrecht (1983) found that heating slurry caused rapid inactivation of viruses, and that this effect was not due to heat alone. They found that an increased temperature enhanced ammonia production, and this was responsible for inactivating the viruses. Burge et al. (1983) also found that the virucidal activity of ammonia increased with higher temperatures. They found that picornaviruses, which are single stranded RNA viruses, were susceptible to inactivation by these means, whereas reoviruses, which are double stranded RNA viruses, were resistant. They speculated that double-stranded DNA viruses would also be resistant.

The virucidal effects of ammonia are influenced by pH. Ammonia only has virucidal activity in its uncharged state, so that in order to have an effect, the pH needs to be greater than 8 (Ward and Ash- ley, 1977a; Burge et al., 1983). Hence, ammonia release could be induced in slurry by raising the pH although the amount released is unlikely to be sufficient for full disinfection of the slurry. The alternative approach of sparging gaseous ammonia into slurry would be more effective, although it is more costly, and the ammonia would need to be removed from the system using scrubbers, and recycled.

Ionic detergents Other wastewater-sludge components found to have an effect on virus levels are detergents. Ward and Ashley (1980) noted that ionic detergents in wastewater sludge reduced the heat required to inactivate a rotavirus, and reported similar results with reovirus. Non-ionic detergents, on the other hand, appeared to stabilize the virus in the presence of

C. Turner; C. H. Burton 12

SDS, an anionic detergent. In another paper, Ward and Ashley (1978) commented that ionic detergents seemed to actually protect some viruses such as poliovirus and enteroviruses, hence detergents appear not to have a universal virucidal activity.

Other chemical disinfectants Chemical disinfectants were used initially in the treatment of slurries, but because of toxicity and high volumes/concentrations needed, interest in this method has waned. However, Derbyshire and Arkell (1971) looked at time and temperature effects for the activity of 10 different disinfectants against Tal- fan virus (a porcine enterovirus) and a strain of porcine adenovirus type 2 in cell culture. Many were inactive, or had little activity against both viruses. The most successful disinfectants were ethanol at a concentration of 70% and sodium hypochlorite at a final concentration of 1%. Potassium permanganate and sodium hydroxide were found to have reasonable activity, but again at relatively high concentrations. Formaldehyde (as formalin) activity was shown to be slow and poor, especially at 4°C. Haas et al. (1995) reviewed virus inactivation, including the use of formaldehyde. Little activity was found below 10°C and optimal activity occurred when the temperature was above 20°C. Even then, quite high amounts were needed (20-40 1 me3 of a 35-37% solution), and at least four days were required for inactivation. Herniman et al. (1973) looked at a number of similar disinfectants against swine vesicular disease (SVDV), and found compar- able results with 70% ethanol. Here, good virus inactivation was achieved with formalin, but at a final concentration of 10% (equivalent to a formaldehyde concentration of around 4%). Bohm (1994) noted that slurry treated with formalin could be safely dispersed on land as a fertilizer, with no ill effects to the crop grown on the land, although the use of large quantities of such a chemical may cause some health and safety concerns. Sodium hydroxide can also be used for the virus inactivation of slurry (Haas et al., 1995) when 16-30 1 m-’ of a 50% solution is used, bringing the pH to at least 12 with the exposure time of at least four days. It too can be safely spread on land (Bohm, 1994).

Although relatively high chemical concentrations are required to achieve inactivation, the means of inactivation is simple, and little equipment would be required beyond a large tank and a means of mixing thoroughly. However, many of the chemicals are toxic or corrosive which can introduce potential hazards to the decontamination procedure.

Lime treatment Lime (calcium hydroxide) is sometimes used to stabilize animal wastes and reduce odour emissions before disposal. It is also widely used in agriculture to counteract soil acidification. It has been noted that this treatment reduces pathogen levels in slur-

ries, including levels of viruses, and this has led to some researchers examining the inactivation of viruses using calcium hydroxide. Derbyshire and Brown (1979) seeded porcine enteroviruses type 2 and 3, and bovine enterovirus into pig slurry to a level of about lo4 IU and added calcium hydroxide to bring the pH up to 11.5. After mixing, the slurry was allowed to settle, and it was found that no bovine enterovirus was detected after 1 h, no porcine enterovirus type 2 after 3 h, and no porcine enterovirus type 3 after 24 h. Koch and Euler (1984) looked at lime treatment for inactivating Aujeszky’s disease virus (ADV) in pig slurry. They found that about 30 kg lime was needed per cubic metre of slurry for complete inactivation, and that the pH needed to be at least 11.5 to have an effect. Increased total solids in the slurry meant that more lime was required. Again, the virucidal effect was related to temperature with better virus inactivation at increased temperature. They also believed that high pH itself was not the cause of the inactivation, but that ammonia release due to increased pH provided the virucidal mechanism. Grabow et al. (1978) studied the role of high pH-lime treatment in reducing the number of pathogens in a wastewater treatment process, and found that this treatment extensively reduced the numbers of bacteria and viruses. Because the levels of ammonia are likely to be much lower in wastewater, and lime was still effective at similar concentrations in this medium, it is likely that ammonia released by lime addition is not the only mechanism of action. In contrast to Koch and Euler (1984), Grabow et al. (1978) believed the biocidal action was due mainly to the high pH. Haas et al. (1995) reported that a concentration of about 40-60 1 m-’ of a 40% solution of calcium hydroxide was effective if mixed with slurry with an exposure time of at least four days. The procedure was still effective at sub-zero air temperatures. Very good mixing was required to ensure even distribution of the chemical; for this reason, lime milk is preferable to solid calcium hydroxide.

To sum up, the advantages of lime treatment are that it is inexpensive; treated slurry can be easily disposed of, and it is already in use as a slurry stabi- lization treatment, so reducing the requirements for developing a new treatment plant. However, very good mixing is required, and several days may be needed for treatment.

Iodine treatment Iodine disinfection has been studied as an alternative to chlorine in wastewater treatment. Almost all E. coli were killed in water samples containing stream sediments to which a dose of 3 mg I-’ had been added within a 30 min contact time (Ellis et al., 1993). For samples containing digested sludge, 8 mg I-’ iodine was required to achieve similar disinfection levels, and for samples containing raw sludge (at the lowest turbidities), a minimum of


10 mg 1~’ was required. The disinfectant power of iodine was found to decrease with increasing temperature, which is in contrast with most other treatments examined. Disinfection also decreased with increasing pH and turbidity. Sobsey et al. (1991) looked at the ability of iodine to inactivate hepatitis A virus (HAV) and poliovirus in waters of varying qualities. A 104-fold reduction in HAV was found with 16 mg I-’ iodine in clean water; the other viruses were more resistant.

The main disadvantage of this technique is that disinfection power is reduced with increasing turbidity, so the treatment of concentrated slurry would be a problem. It could possibly be used with dilute slurry or dirty water, although high iodine doses would be required.

PHYSICAL TREATMENTS

UV irradiation This is another widely used technique in the water industry for viral and bacterial decontamination. In general, viruses are more resistant to UV irradiation than bacteria (Harris et al., 1987) and in some cases need three-four times greater UV doses than bacteria (Chang et al., 1985). Viruses, however, unlike bacteria, are not able to photo-reactivate (after nucleic acid damage by UV, bacteria have the cell mechanisms to repair some of that damage when subjected to light of certain wavelengths) and therefore suffer irreversible changes to their nucleic acids.

104-fold reduction in IU in the titres of a range of organisms (including coliforms and coliphage) using a y dose of 5 kGy; a similar dose of electron beam radiation only induced a 103-fold reduction of numbers in secondary effluent and raw sewage. Yeager and O’Brien (1983) found that 10 kGy was sufficient to eliminate bacteria and parasites in sludge, however, viruses were more resistant. They found that the mechanism of inactivation of poho- virus was mostly due to damage to the RNA genome, but also partly due to alterations in the viral protein coat. Vasl et al. (1983) also studied the inactivation of poliovirus, as well as echovirus, and found that 6.5 kGy only reduced the titres by 10’ to 102-fold, which is clearly insufficient. Some researchers have examined the inactivation of SVDV by gamma irradiation, and found it to be one of the most resistant viruses to this kind of treatment. Under the worst case conditions, SVDV in liquid animal faeces required a dose of 40 kGy for it to be reduced to below detectable levels (Thomas et al., 1982). Simon et al. (1983) also noted that SVDV was particularly resistant; at a dose of 30 kGy, all viruses tested except SVDV were completely destroyed in both cell culture and animal manure. An advantage of gamma irradiation is that dose levels can be altered readily by adjusting either the amount of radioactive cobalt in the source or changing the residence time in the reactor. Disadvantages are that viruses are fairly resistant, plus the fact that there are serious safety considerations in the use of radioactive cobalt.

UV is much better for decontamination when the % transmittance of radiation is high. This means that a much greater radiation effect is likely to be observed in liquids containing low concentrations of solids than in animal slurries with higher solid content. Thus, the required radiation dose will need to be much higher in slurry. Immersed lamps also tend to get coated with protein or other organic material cross-linked by inorganic ions such as calcium, iron and phosphate (Gross and Davis, 1991) hence the quartz sleeves need to be cleaned regularly. More frequent cleaning will be needed with higher organic loads in the material. Temperature seems to have little effect on UV disinfection (Severin et al., 1983).

Heat treatment Many techniques discussed in this review are temperature dependent, and virus inactivation is usually enhanced at higher temperatures by one of a number of different mechanisms. This section concerns the specific effects of heat on virus infectivity.

It therefore seems that UV is not viable for inactivating viruses in slurry unless it is preclarified. However, it may be suitable for dirty water although high doses would be required. Equipment and expertise are readily available as the technology is often used in water purification.

Gamma and electron beam irradiation

Monteith et al. (1986) found that bovine enterovirus was inactivated to below detectable levels in digested liquid manure heated to 70°C however, some bovine parvovirus was detected after this treatment. This indicates that some viruses are more heat resistant than others. Botner (1991) looked at the temperature dependent inactivation of ADV during anaerobic storage at temperatures ranging from 5 to 55°C and found that at 5°C the virus was only inactivated after 15 weeks, whereas at 55°C no virus was detected after 10 min. Herniman et al. (1973) looked at SVDV and studied its thermal inactivation in both milk and pig slurry. In milk, the virus was inactivated by heating to 60°C for 2 min, whereas virus in slurry required a slightly higher temperature: 64°C for 2 min for inactivation.

Gamma irradiation has been reported for the pur- Heat may be applied in a number of ways, and pose of reducing virus concentrations in sludges or two are considered here. Firstly, it can be applied by slurries. Most researchers in the field report the use steam injection under pressure, which has the poten- of a 6”Co y source, although the doses used vary tial problem of producing aerosols unless steam is widely. Farooq et al. (1993) observed at least a injected in an enclosed pipe, or into an enclosed


tank with filters installed on air outlets. Heat could be recovered using a heat exchanger, although foul- ing of the heat exchanger may be a problem. Steam could be generated by using an oil fired steam generator, which could be operated at a farm site.

The second possible method for heat production is by using microwave radiation. Microwave heating has rarely been studied for the decontamination of animal slurries; however, it has been noted (Bohm et al., 1984) that microwaves themselves seem to have no virucidal properties and any observed inactivation is caused by temperature effects alone. Bohm et al. (1984) heated small volumes of liquid manure (flowing within tubing at rates of between 100 and 530 ml min-’ from a 20 litre reservoir) in a microwave field of 1 kW at 2450 MHz and observed the effect on suspensions of the DNA virus HCC (hepatitis contagiosa canis) and the RNA viruses reovirus type 1 and ECBO (enteric cytopathogen bovine orphan) strain LCR4 virus. The HCC virus was inactivated to below detectable levels (approximately a reduction in IU of 104-fold) when a temperature of 63-70°C had been reached; ECBO LCR-4 required 58-62”C, and the reovirus 55-58°C. The average holding time in the UHF field was less than 1 s in all cases. Vasl et al. (1983) compared gamma and microwave irradiation, and found microwaves to be more efficient for inactivating viruses such as poliovirus 1 when used under the conditions and doses tested (6.5 kGy for y irradiation and 10 s in a microwave field at 2450 MHz). In contrast to most other techniques, they found that the greater the solids content of the medium, the greater the inactivation efficiency, possibly because the solids in the slurry could preserve heat for longer.

Microwave radiation has the advantage of allowing rapid heating of the effluent, thus causing rapid virus inactivation. However, the electrical power needed for a microwave field capable of heating large volumes of slurry may not be available on many farm sites, although heat recovery could be used to reduce the energy requirement. Another problem is that the capital cost of large-scale microwave equipment is high.

The main advantage of using heat for inactivation of viral pathogens is that, unlike many other methods, the solids and protein present seem to afford the virus particles no protection, and heat retention may be improved by the presence of solids.

Photocatalytic inactivation This technique has been used to reduce the levels of bacteria and/or viruses in water or wastewater. Tita- nium dioxide is suspended in the effluent, which is then irradiated with fluorescent light or sunlight. Watts et al. (1995) reported that with secondary wastewater effluent containing 250 mg 1-l titanium dioxide, a lo’-fold reduction in coliform bacteria was achieved in 150 min under laboratory lights, whereas poliovirus 1 was reduced by a similar

amount in about 30 min. Matsunaga et al. (1988) immobilized titanium dioxide on acetylcellulose membranes in a continuous reactor and used a mer- cury lamp to inactivate E. coli in water, and achieved better than a ,lO*-fold reduction in titre when the cell suspension flowed in the system with a mean residence time of 16 min. This method is relatively inexpensive, but is unlikely to be effective in any- thing other than relatively clean water due to the poor penetration of light in turbid liquids.

Sand column/soil filtration The filtration of effluents through sand or soil, usually in columns, has been used in the water industry for purification of wastes and the removal of pathogens. Powelson and Gerba (1994) looked at the removal of two types of phage and of poliovirus type 1 after percolation through coarse-sand columns under saturated and unsaturated conditions; the latter gave better removal. Ho et al. (1991) also looked at poliovirus removal from wastewater run through a sand column amended with red mud, a bauxite refining residue. They found that the starting virus level of lo4 viruses per ml was reduced to below detectable levels after 2.3 exclusion volumes over 6.7 days, although unamended sand columns showed poor virus removal. Pig slurry has also been run through soil columns. Lam et al. (1993) showed that 100% of E. coli were removed from pig slurry when passed through a volcanic soil column; less was removed by granitic soil. Not all systems reported produce a complete removal of pathogens, however. Gerba et al. (1991) looked at soil aquifer treatment for the removal of viruses in wastewater, and found that only a 10’ to lo*-fold reduction was observed after the movement of sewage effluent through 15 feet of soil at a rate of 50 feet per day. They attained better virus removal with a slower rate of 3 feet per day.

In summary, the method is probably relatively inexpensive, however, it removes viruses rather than inactivates them. The soil type is crucial, and the process is relatively slow as flow rates need to be low for maximal removal of viruses. Soil columns need to be cleaned regularly when they become clogged; and this will be more frequent when using liquids containing high levels of solids, such as slurry.

Drying/evaporation Batty et al. (1979) studied the resistance of SVDV to drying and they found that the virus did not survive drying in environments of high relative humidities (RH), but there was little virus loss at low RH. Inactivation was also influenced by the suspending medium; water afforded the virus no protection, but drying in tissue culture medium allowed the survival of some virus. Ward and Ashley (1977b) looked at water evaporation from raw sludge seeded with poliovirus and found that the recovery of infectious poliovirus decreased gradually until the solids con-

Viruses in pig slurries: A review 1.5

tent reached about 65%, and then a more rapid decrease of virus titre of more than lo”-fold was observed. Coxsackie virus and reovirus were found to behave similarly to poliovirus. This is unlikely to be a very suitable system for animal slurry as viruses are protected by organic material and high water losses are required before the technique becomes effective. Therefore, the technique will be expensive, particularly considering that slurries can contain more than 96% water. In generated by this method.

OTHER TREATMENTS

addition, aerosols may be

Bacterial/proteolytic inactivation Many researchers (Eisenhardt et al., 1977; Deng and Cliver, 1992; Deng and Cliver, 1995a,b; Knowlton and Ward, 1987; Ward, 1982) have noted that viral inactivation in sludges or mixed wastes is caused by microbial activity. This has been demonstrated by showing that viral inactivation occurs more rapidly in mixed wastes than in clean buffer and that auto- claving wastes prior to virus seeding reduces viral inactivation. In many cases, the virus levels have been reduced by the addition of proteases, demon- strating that proteolytic activity is at least partly responsible for this effect, although in some cases, the bacterial activity does not seem to be enzymatic (Deng and Cliver, 1995b). Inactivation is more rapid at higher mesophilic temperatures, but still occurs at 4°C (Knowlton and Ward, 1987). Inactivation is relatively slow; the fastest rate of inactivation reported was about 18 days for a lo-fold IU reduction under aerobic conditions (Deng and Cliver, 1992) or a reduction in IU of lo2 fold in 24 h at 35°C under anaerobic conditions (Eisenhardt et al., 1977). The advantage of this method is that natural virucidal activity is present in the slurry, however, even if slurry is seeded with proteases or microorganisms which secrete proteases, it is likely to be too slow and too inconsistent to be of great use.

Aerobic treatment Many researchers have observed that pathogen numbers are reduced during aerobic treatment of animal slurries, which is usually performed to reduce COD, BOD, ammonia and odour before spreading on agricultural land. Munch et al. (1987) looked at the reduction in a number of different bacteria seeded into batches of aerated and non-aerated slurry, both batches being stored at both 6-9°C and 18-20°C. They found that the reduction always pro- ceeded faster in aerated than in non-aerated slurry, and at the higher temperature. Ginnivan et al. (1981) also looked at the inactivation of bacteria, as well as that of pig parasites, in aerobically treated pig slurry, and found that at thermophilic temperatures, the pathogen levels declined from lo6 to below detectable levels (i.e. close to zero) within 4 h.

Other researchers have looked at virus inactivation. Bohm (1984) studied the inactivation from lo7 to 10’ IU to below detectable levels of several animal viruses in a rotating aeration system. Foot and mouth disease (FMD) virus was undetectable after aeration at a pH of 8 at 50°C for 48 h; ADV needed 5 h aeration at 40°C for inactivation, and swine vesicular disease virus, 48 h aeration at the same temperature. Wekerle and Albrecht (1983) investi- gated the inactivation of vaccinia virus and a bovine enterovirus in a circulating aerator, and they found that heat-dependent inactivation of vaccinia virus occurred at 50°C and of the enterovirus at 56°C when the viruses were in plastic tubes within the slurry, but when the virus was added directly to the slurry, inactivation occurred at the lower temperature of 47°C. Inactivation in this case was considered to be due to the virucidal effect of ammonia released during heat treatment of the slurry. Spill- man et al. (1987) found that rotavirus and coxsackievirus B5 were rapidly inactivated under aerobic thermophilic fermentation at 61°C; parvovirus was more resistant, however.

The advantage of this method is that air supply to a tank is relatively cheap, which could allow resources for supplementing aerobic treatment. In practice, the temperature needs to be above a certain level to be effective, but since aerobic treatment is exothermic, a temperature increase may be achieved by the insulation of a reactor in a batch process. The main disadvantage is that the process is relatively slow, although it is more rapid than anaerobic treatment. Foam production is a potential problem, especially in a batch process. The reactor would need to be enclosed, and the air emissions filtered to prevent the release of aerosols.

Anaerobic digestion Anaerobic digestion is a commonly used technique for the breakdown of organic matter in sludges and slurries. It is also used on farms for the production of biogas and a residue which can be used as a fertilizer or possibly re-fed to livestock (Derbyshire et al., 1986). For the latter reason, some studies have been done to examine the inactivation of viruses during digestion, and this information can be used to evaluate the usefulness of the technique for possible inactivation of viral pathogens. Berg and Berman (1980) found that bacteria were seven-10 times more sensitive than viruses to mesophilic and thermophilic anaerobic digestion. Interestingly, they also found that viruses that occur naturally in raw sludges appear to be inactivated by the digestion process much more slowly than laboratory strains seeded into the sludges. This may occur because naturally present viruses are closely associated with the organic material in the sludges and are thus more difficult to inactivate (Goddard et al., 1981). It also indicates that there may be a general problem in trying to extrapolate results obtained using seeded

16 C. Turnel; C. H. Burton

sludges to those likely to be obtained with in situ organisms. Ward and Ashley (1976) looked at the effect of anaerobically digested sludge on polioviru- ses at 4 and 28°C and found that virus inactivation increased with time and temperature. The inactivation rate ranged from a reduction of more than lo-fold per day at 28°C to about lo-fold every five days at 4°C and seemed to be due to RNA damage. The virucidal activity was not found in raw sludge and therefore seems to be a result of the digestion process. Monteith et al. (1986) seeded a bovine enterovirus and parvovirus into liquid cattle manure, and noted that both were rapidly inactivated by the anaerobic digestion process under thermophilic (55°C) conditions, no virus having been detected after 30 min. They survived for 13 and eight days, respectively, under mesophilic conditions, however. Spillman et al. (1987) also found that animal viruses such as rotavirus and coxsackievirus B5 were inactivated very rapidly at thermophilic temperatures.

These results indicate that virus inactivation does occur during anaerobic digestion, but the rate of inactivation is dependent on the virus, and the temperature and duration of digestion.

An advantage of this method is that gas produced during the digestion process can supply heat needed to maintain thermophilic temperatures. The reactor is, of necessity, closed, so problems of particulate or aerosol emissions would not arise. Capital costs are high, however, and the process would only be worth- while with thermophilic temperatures. This contrasts with a much more rapid (and hence smaller and cheaper) reactor that could be used to provide aerobic treatment.

SLURRY CLARIFICATION

A number of treatments described thus far will be suitable only for very dilute liquid streams, so if they are to be considered at all, some pre-clarification of the slurry will be required. Treatments described earlier that would be enhanced by preclarification are as follows: ozonation, iodine treatment, UV irradiation, photocatalytic inactivation and sand/soil column filtration. Before clarification, the slurry may be conditioned with chemicals to aid flocculation. Then the solids removal step is performed, using some mechanical unit operation. There are a number of options for dewatering slurry, ranging from gravity sedimentation to centrifugation to com- paction. Sedimentation techniques involve allowing the slurry to settle, and then drawing off the dilute liquid layer at the top, or the more concentrated solids layer at the bottom. This technique is relatively inexpensive, but does not produce high levels of dewatering. Better sedimentation may be achieved by the use of a centrifuge; much better dewatering is possible, but capital costs and running costs are both high (Miner et al., 1983). An alterna-

tive technique is to use belt or roller presses where a combination of gravity and a pressing stage squeezes out the liquid through a screen (Jeffrey, 1982). Mounting such devices high above the ground allows the solids to pile up beneath the separator. This technique is probably more common in slurry dewatering than centrifugation.

DURATION OF TREATMENT AND REACTOR TYPE

An important issue that needs to be decided is: what is the maximum time duration over which the treatment should operate, and which is the most suitable mode of operation, i.e. batch or continuous? A batch treatment could involve treating the entire volume of slurry at one time, for example by tipping and mixing the required amount of chemical into a lagoon. It could also involve pumping a smaller volume of slurry into a tank, treating it for a period, and then pumping the treated slurry into a storage tank, and repeating the process for all the slurry. In the case of a continuous process, the treatment could take place in a continuous-stirred tank reactor with constant input and output. Alternatively, it may be possible to operate in a plug-flow manner so that contamination of treated effluent with untreated effluent is avoided.

In most of the treatments described, time is a consideration. In the case of chemical treatment (formaldehyde, sodium hydroxide and lime), the slurry is likely to require contact with the chemical for a minimum time period, believed to be four days (Haas et al., 1995). This means that any continuous stirred-tank reactor process would need to operate over a much greater time period than if a plug flow system was used, to ensure that all the slurry had this required contact time with the chemical. In the case of batch treatment (for instance, chemicals being pumped into a slurry lagoon), the slurry containing chemicals would need to be continually mixed for at least that minimum time period. How- ever, the addition of chemicals to large open surfaces such as in slurry lagoons will present particular hazards of gaseous emissions, which would be more easily solved in a continuous system. In the case of heat treatment, a continuous system is likely to be the most suitable option, although its throughput will depend on the availability of sufficient electrical and thermal power. Thus, although the slurry would need to be heated to a particular temperature only for a short time (say, 30 min), treatment of the total volume of slurry could take weeks. Electrical power could be used for heating (e.g. by microwaves), but would also be needed in the case of oil-burning steam generators, for driving feed water pumps and atomizing the fuel. In general, a lower throughput will reduce the power requirements.


GENERAL COMMENTS

In many of the methods used for virus inactivation, the viruses are protected from damage by organic material present, such as protein, or by adsorption on particulate matter. It is therefore likely that any treatment designed for water disinfection would need to be increased in dose or duration when decontaminating fluids such as pig slurry containing high levels of organic material. An exception to this appears to be heat treatment, in which the high solid content of the slurry appears to retain the heat, and promotes virus inactivation.

In general, viruses seem to be more resistant to inactivation than bacteria, therefore any treatment

designed to destroy bacteria will probably need to be used at higher doses or for a longer duration to deal with viruses. Seeded viruses are less resistant to inactivation than viruses naturally present in the sludge and this needs to be taken into account when designing an inactivation process (Berg and Berman, 1980).

Another point worth raising is that temperature has an effect on most treatment techniques. In most cases, an elevated temperature enhances virus inactivation, but in a very few (sometimes disputed) cases, a lower temperature gives better results. Tempera- ture clearly needs to be monitored and regulated in whatever treatment process is examined. Table 1 provides a summary of the suitability of all the treat-

Table 1. Summary of treatments for virus inactivation in dirty water and slurry

Treatment Advantages Disadvantages Overall suitability

Ozonation

Chlorination

Ethylene oxide Peracetic acid

Widely used Equipment available Strong virucidal effect

Widely used for water

Virucidal activity Less toxic than chlorine

Ammonia Ammonia already present in slurry

Ionic detergents Chemicals

Naturally occurring in slurry Little equipment required;

Mostly cheap

Lime Inexpensive. Used to stabilize slurry

Iodine

UV Irradiation

Gamma and electron beam radiation

Heat treatment

Used in water industry Works at lower temperatures

Widely used. Equipment available. Virucidal

Dose easy to alter

Successful at virus inactivation Solids present will not interfere

Photocatalytic inactivation

Soil/sand column

Drying/evaporation

Low cost

Relatively inexpensive

Many viruses can not withstand drying

BacteriaVproteolytic inactivation

Anaerobic digestion

Natural virucidal activity in slurry

Gas can be used to maintain heat

Aerobic treatment Air supply low cost

Pre-clarification needed High ozone levels needed Explosion risk

Toxic compounds. Weak virucidal activity

Toxic. Little published data Less virucidal activity than

chlorine. Foam production

ASF may be resistant. If added, recycling needed. pH control needed

Virucidal activity unproven Many are toxic. High levels

needed. Temp. control needed

Ammonia released and ASF may be resistant. Good mixing needed. Long duration

High doses needed. Lower disinfection with high solids

Pre-clarification needed. High doses needed

Safety problems. Some viruses resistant

High energy use. High capital costs. Need to look at means of application

Likely not to be effective in slurry

Virus removal, not virus inactivation. Slow

Aerosol production. Need high water losses. Virus protected

Slow Inconsistent

High cost. Non-mobile. Long time needed

Heat required. Slow process

Possible use with dirty water, or slurry if clarified

Not very suitable

Unsuitable Unsuitable

Could have some applications

Generally unsuitable Some chemicals very suitable

Likely to be suitable

Unsuitable for slurry, not very suitable for dirty water

Possible use with dirty water or clarified slurry

Not very suitable

Likely to be most suitable technique

Unsuitable for all except low turbidity dirty water

Unsuitable for all except dirty water

Too expensive and not universally suitable

Only suitable if slow inactivation during storage is acceptable

Suitable if thermophilic anaerobic digester already in place

Possibly suitable, particularly in combination with another technique


ments discussed for inactivating viruses in dirty water or slurry.

CONCLUSIONS

This review has examined a number of different methods for the inactivation of viruses in dirty water and slurry. Dirty water and slurry can, for the most part, be grouped together as most of the technolo- gies will be applicable to both. There are a few exceptions to this. Both ozonation and UV irradiation may be applied to dirty water, but not to slurry, due, in the former case, to the high carbon loading in slurry which will ‘soak up’ the ozone, and in the case of UV, to the turbidity and suspended solids present. These may be applicable to clarified slurry. The use of sand columns, iodine treatment and photocatalytic inactivation may have some limited use in treating dirty water containing low levels of solids, in certain circumstances, but would not be universally acceptable.

Many of the techniques found in the literature have been applied to the water industry, but have been shown to be unsuitable for use in liquid animal effluents. The most suitable techniques seem to be heat treatment (where it is thought that a temperature of about 60°C maintained for up to 30 min should be sufficient to inactivate most viruses), and the addition of a chemical (such as formalin, sodium hydroxide or calcium hydroxide) at the required concentration. In addition, aerobic or anaerobic treatment could be used to assist decontamination, particularly if routinely used on a particular farm for slurry treatment prior to land spreading.

ACKNOWLEDGEMENTS

The authors would like to thank Dr Trevor Cumby and Dr Philip Wilkinson for a critical review of the manuscript, and the Ministry of Agriculture, Fish- eries and Food for funding this work.

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the inactivation of viruses in pig slurries: a review

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