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Deliverable D-JRP-TOXOSOURCES-WP2.2 Report on quantitative exposure data from survey of WP2

Hepatitis E virus (HEV) is endemic worldwide and is the most common cause of viral hepatitis. Infection with HEV genotype 1 and 2 (HEV1 and 2) can give acute (but transient) hepatitis (jaundice). However; fulminant liver failure with high mortality in pregnant women occurs. HEV1 and 2 are transmitted fecally-orally and often causes waterborne outbreaks in tropical and sub-tropical regions. Infection with HEV3 and 4 is usually asymptomatic but can result in acute infection, especially in patients with underlying liver disease or people with a high alcohol intake. HEV3 can also cause chronic disease - cirrhosis and problems in other parts of the body - in people with an impaired immune system. In Europe HEV3 predominantly occurs and is foodborne. Over the last ten years, there has been an increase in the number of reported hepatitis E cases within the EU, including Sweden. Note: this report is published by the Swedish Food Agency (Livsmedelsverket) as part of their Report Series (www.livsmedelsverket.se/bestall-ladda-ner-material/sok-publikationer/artiklar/2019/l-2019-nr-09-del-2-hepatit-e-riskvarderingsrapport), SE; PDF; Efsa.focalpoint@slv.se

Disease-causing microorganisms, pathogens, can be transmitted by drinking water causing acute as well as chronic disease. Waterborne pathogens include a wide range of viruses, bacteria and parasitic protozoa. Since these waterborne pathogens mainly derive from faecal contamination, microbiological drinking water quality is assessed by analysing indicator bacteria such as E. coli, normally occurring in the gut of warm blooded animals, including humans. A quantitative microbiological risk assessment was performed to estimate acceptable water quality. It showed that the probability of stomach illness is greatly increased if the contamination comes from wastewater. If the contamination comes from manure, there is a slightly increased risk of enterohaemorrhagic E. coli. This means that E. coli should not be detected in 100 ml samples to achieve water quality comparable to municipal drinking water. Note: this report is published by the Swedish Food Agency (Livsmedelsverket) as part of their Report Series (www.livsmedelsverket.se/bestall-ladda-ner-material/sok-publikationer/artiklar/2021/l-2021-nr-03-e.-coli-och-koliforma-bakterier-i-brunnsvatten), SE; PDF; Efsa.focalpoint@slv.se

This is a risk assessment on risks with consuming wild boar mead contaminated with Salmonella Choleraesuis prepared by the Swedish Food Agency Note: this report is published by the Swedish Food Agency (Livsmedelsverket) as part of their Report Series (www.livsmedelsverket.se/bestall-ladda-ner-material/sok-publikationer/pm/2021/pm-2021-risker-med-att-ata-kott-fran-vildsvin-som-ar-smittade-med-salmonella-choleraesuis), SE; PDF; Efsa.focalpoint@slv.se

On-farm measures can be used in multi-barrier schemes to manage microbial risks from consumption of wastewater-irrigated vegetables, especially where informality of the practice determines minimal external support for farmers. Evidence indicates that cessation of irrigation greatly reduces microbial contamination on leafy vegetables, but at the expense of produce quality. Replacing wastewater with higher-quality irrigation water during the last days of cultivation is an alternative to cessation of irrigation that does not compromise produce quality. This study evaluated the effect of wastewater substitution under on-farm conditions on different indicators of microbial contamination of lettuce. Lettuce was cultivated in experimental plots and irrigated with three water sources: spring water, water from a wastewater-polluted river and effluent from a primary wastewater treatment plant, but with the river water replaced by spring water in half the plots about two weeks before harvest. The experiment was repeated four times in different seasons. Irrigation water samples collected during cultivation and lettuce samples collected at harvest were analysed for helminth eggs, Escherichia coli and coliphages. Variables characterizing the irrigation practices and environmental conditions were recorded. There were no significant differences in helminth egg or E. coli concentrations on lettuce (medians ranged from -0.7 to -0.1 log(10) eggs g(-1) and 0.6-1.4 log(10) cfu g(-1), respectively) between any of the treatments involving wastewater irrigation; no statistical analysis was possible for coliphages because concentrations on lettuce were mostly at or below the detection limit (94% of samples). Variables associated with temperature and soil explained helminth egg and E. coli concentrations on lettuce, while number of days of irrigation with spring water (representing wastewater substitution) was significant only for E. coli. It was concluded that the experimental conditions were suboptimal for successful implementation of wastewater substitution for on-farm microbial risk management, but key variables for successful implementation were identified.

A risk assessment on the risks of eating meat from CWD-infected deer prepared by the Swedish Food Agency. Note: this report is published by the Swedish Food Agency (Livsmedelsverket) as part of their Report Series (www.livsmedelsverket.se/bestall-ladda-ner-material/sok-publikationer/artiklar/2020/l-2020-nr-19-risker-med-att-ata-kott-fran-cwd-infekterade-hjortdjur), SE; PDF; Efsa.focalpoint@slv.se

A scientific opinion on SARS Coronavirus 2 in food and drinking water prepared by the Swedish Food Agency. Note: this report is published by the Swedish Food Agency (Livsmedelsverket) as part of their Report Series (www.livsmedelsverket.se/bestall-ladda-ner-material/sok-publikationer/pm/2020/pm-2020-coronavirus-i-livsmedel-och-dricksvatten-vetenskapligt-underlag-livsmedelsverket.pdf), SE; PDF; Efsa.focalpoint@slv.se

The request from NFSA and NEA: Antimicrobial agents and microorganisms are introduced to sewage systems by different human activities, from private homes, institutions such as schools and hospitals, office buildings, industrial and commercial activities, i.e., from everywhere where people work and live. The Norwegian Food Safety Authority (NFSA) and Norwegian Environment Agency (NEA) asked the Norwegian Scientific Committee for Food and Environment (Vitenskapskomiteen for mat og miljø, VKM) for an extension of the 2009 VKM report “Risk assessment of contaminants in sewage sludge applied on Norwegian soils” regarding the impact of wastewater (WW)- and sewage sludge treatment methods used in Norway, on the fate and survival of antimicrobial resistant bacteria, fate of antimicrobial resistance genes, and main drivers for resistance (e.g. antibiotics, antifungal agents, heavy metals, disinfectants). The request addressed by VKM: VKM appointed a working group, consisting of three members of the Panel on Microbial Ecology, four external members and VKM staff to prepare a draft Opinion document. The Panel on Microbial Ecology has reviewed and revised the draft prepared by the working group and approved the Opinion document “Assessment of the impact of wastewater and sewage sludge treatment methods on antimicrobial resistance”. The antimicrobial resistance cycle: Exposure to antimicrobial agents is regarded as the most important driver for development and dissemination of AMR in microorganisms. Consequently, an important location for the development of AMR is the gut of humans or animals receiving antimicrobial drug therapy. As ARB, ARG, resistance genes and antimicrobial agents will end up in the WW system, this system could be regarded as a potential hot spot for interactions between different microorganisms, between different antimicrobial agents, and between microorganisms and antimicrobial agents. Hospitals and pharmaceutical companies are regarded as being an important source for antimicrobial drug residues released in WW. At the wastewater treatment plant (WWTP), bacteria and genes end up either in the effluent wastewater fraction or in the sludge fraction. When ARB and ARG are distributed with the WW sludge, they may reach arable land when the sludge is used as soil improver and fertilising product, and thus be recycled into the food-production chain. When following the effluent WW fraction, ARB and ARB will be released into WW recipients, such as lakes, rivers or fjords, and may, from these environments, also be recycled into food production. In each step of these cycles, ARB and ARG will be introduced into new environmental compartments to which they must adapt, and to microbial communities with which they must compete for survival and growth. Depending on the bacterial species, these new environmental compartments will be more or less hostile, but they will also provide opportunities for microbial interactions, like dissemination of ARG due to horizontal gene transfer (HGT) within and between bacterial species. Findings: It is challenging to deliver a general assessment of the nature of as well as the probability for direct discharge of ARB and ARG into effluent WW and applied sludge. This is due to the combined complexity of resistance carriers, traits, various sources of variation, and the WW systems. Moreover, there is currently a lack of harmonized methods and protocols to compare studies from different systems. However, there are no strong indications that there is a significant enrichment of ARB in WWTP operated under European conditions, which, on a general level, also applies to the Norwegian situation. Although some studies indicate a slight increase in the fraction of ARB, the absolute reduction in bacterial load during WW treatment (WWT) is significant; removal of between 99 % to 99.9 % of faecal indicator bacteria is generally achieved by secondary treatment, including biological and physico chemical treatment steps. Effluent WW is often released into water recipients, and there are many mechanisms (physical, mechanical, and chemical) that will limit the extent that ARB of faecal origin are transferred to the food-production chain. However, there are different views on the significance of this release for the development of AMR. Results from single studies indicate that WWTP effluents contribute little to the total AMR exposure of micro – and macro organisms in aquatic and marine environments. On the other hand, freshwater environments in general are regarded as an important reservoir of novel antibiotic resistance determinants, and in some areas, relative abundance of resistance determinants in effluents has been observed to be considerably higher than in pristine natural water sources. Some imprint of AMR in recipient waters, compared to pristine waters, is unavoidable. During WWT, bacteria largely adhere to particles that are aggregated and precipitated to form a solid sludge. The mandatory hygienisation of sludge kills a large proportion of these bacteria, notably all thermosensitive faecal bacteria. However, the resulting hygienised sludge is still rich in bacteria, some of which are carriers of ARGs. The current Norwegian regulations on use of sludge on soil contribute to prevent contamination of food with antimicrobial resistant bacteria and antimicrobial resistance genes from sludge. Yet, soils do contain a pool of both natural and sludge-derived antimicrobial resistance. The contribution of sludge to this antimicrobial resistance pool is probably temporally limited to a period after soil amendment with sludge. A recent, comprehensive study from Sweden showed that long-term application of sewage sludge on farmland only resulted in minor changes of soil bacterial community composition. No evidence could be found for enrichment of antimicrobial resistant bacteria or antimicrobial resistance genes in soil amended with digested and stored sewage. Hospital WW contains more ARB, ARG, and antibiotic residues than municipal sewage, but the difference is not large for ARB and the impact may be minimal in large WW systems. In smaller WW infrastructures, a hospital or similar institution may have a higher impact on the effluent water from the WWTP, and this might suggest that local treatment of the WW at the hospital could be advantageous. A recent Norwegian study monitored bacterial diversity in different WW in the Oslo area, and found the highest concentration of AMR (ARB and/or ARG) in hospital WW. But surprisingly, high concentrations were also found in the studied community wastewater. The relative contribution of hospital effluents seemed low in terms of dissemination of antimicrobial resistant bacteria to the wastewater treatment plant. All measures that can be taken at source to avoid dissemination of antimicrobial agents, ARB, and ARG should be evaluated for their contribution towards combatting AMR emergence. Concentrations of antimicrobial agents, ARB, and ARG are highest in the sewage system and at the inlet to WWTPs. Separation of the different fractions of antimicrobials, ARB, and ARG for individual treatment may therefore reduce the total load reaching the WWTPs. Due to the high concentrations of ARB and ARG in the sewage system, risks from sewage pipe leakages are of concern. Intrusion of contaminated water into the drinking water distribution system should also raise concern. Rehabilitation of the sewage and drinking water networks will considerably mitigate risks. The level of sewage treatment in Norway is rather low, and upgrading will decrease the concentration of bacteria discharged. However, WWTPs are generally not designed for removal of AMR. Membrane processes seem to be the most promising option for increasing such removal rates. Future perspectives: The opinion discusses how the “concept of sensitive recipients” for requirements of the level of WWT could be revisited. This concept is currently based on controlling nutrient loads to the environment, rather than on trace contaminants or contaminants such as ARB and ARG that develop in the stressed environment. In the future, it might be of value to define requirements for WWT based on the relative increase caused by the discharge to the pollution level. Using such a paradigm, a small load with contaminants to a rather unpolluted environment would be rated as being highly critical and the discharge would require further treatment. In addition to the amount of ARB, the type of resistance and their level of horizontal mobility are also important in this aspect. This opinion also proposes the establishment of a new monitoring programme, parallel to the existing NORM and NORM-VET monitoring programmes; “NORM-ECO”. There is relatively little knowledge on AMR in non-clinical compartments, compared with hospital and other clinical settings, and parameters that would trigger immediate responses from NFAS or NEA are not yet identified. However, establishment of a “NORM-ECO”-system requires clarification of that needs further definition.

Toxoplasmosis is a foodborne zoonosis caused by Toxoplasma gondii, a single-cell parasite that is globally prevalent in warm-blooded animals. Eating insufficiently cooked meat is the main cause of infection in humans. Other transmission routes are exposure to contaminated water, vegetables or soil. A pregnant woman infected for the first time can transfer the parasite to her foetus, which in rare cases can lead to miscarriage. People with impaired immune systems such as cancer and transplant patients during treatment may contract encephalitis from an infection. However, most infected people are symptomless. Note: this report is published by the Swedish Food Agency (Livsmedelsverket) as part of their Report Series (www.livsmedelsverket.se/bestall-ladda-ner-material/sok-publikationer/artiklar/2019/l-2019-nr-14---livsmedelsburen-toxoplasmos), SE; PDF; Efsa.focalpoint@slv.se

Klebsiella pneumoniae is a gram-negative, opportunistic pathogen, and a common cause of healthcare-associated infections such as pneumonia, septicemia, and urinary tract infection. The purpose of this study was to survey the occurrence of and characterize K. pneumoniae in different environmental sources in a rural area of Shandong province, China. Two hundred and thirty-one samples from different environmental sources in 12 villages were screened for extended-spectrum β-lactamase-(ESBL)-producing K. pneumoniae, and 14 (6%) samples were positive. All isolates were multidrug-resistant and a few of them belonged to clinically relevant strains which are known to cause hospital outbreaks worldwide. Serotypes, virulence genes, serum survival, and phagocytosis survival were analyzed and the results showed the presence of virulence factors associated with highly virulent clones and a high degree of phagocytosis survivability, indicating the potential virulence of these isolates. These results emphasize the need for further studies designed to elucidate the role of the environment in transmission and dissemination of ESBL-producing K. pneumoniae and the potential risk posed to human and environmental health.

Klebsiella pneumoniae is a gram-negative, opportunistic pathogen, and a common cause of healthcare-associated infections such as pneumonia, septicemia, and urinary tract infection. The purpose of this study was to survey the occurrence of and characterize K. pneumoniae in different environmental sources in a rural area of Shandong province, China. Two hundred and thirty-one samples from different environmental sources in 12 villages were screened for extended-spectrum beta-lactamase-(ESBL)-producing K. pneumoniae, and 14 (6%) samples were positive. All isolates were multidrug-resistant and a few of them belonged to clinically relevant strains which are known to cause hospital outbreaks worldwide. Serotypes, virulence genes, serum survival, and phagocytosis survival were analyzed and the results showed the presence of virulence factors associated with highly virulent clones and a high degree of phagocytosis survivability, indicating the potential virulence of these isolates. These results emphasize the need for further studies designed to elucidate the role of the environment in transmission and dissemination of ESBL-producing K. pneumoniae and the potential risk posed to human and environmental health. Funding Agencies|National Natural Science Foundation of China [41771499, 81361138021]; Fundamental Research Funds of Shandong University [2018JC102]; Swedish Research Council [D0879801]; Swedish Research Council Formas [2016-00640]

Nutrient load rates to the Baltic Sea need to be reduced. Agricultural land is regarded as the most significant contributor to the loads, and measures to reduce losses of nitrogen (N) and phosphorus (P) loads have been proposed, both for the near and far future. Agricultural production was to a large extent considered in these scenarios, whereas effects on other ecosystem services were not evaluated. The question to be answered by this report is whether the measures adopted to reduce N and P losses improve or impair multiple benefits of agriculture. The question is answered for a specific catchment (Svärtaån) located in Sweden, but the method is thoroughly described to provide a potential method to also evaluate other catchments. This work was performed as a part of the Baltic Compass project (2013). The answer to the main question of whether the measures adopted to reduce N and P losses from agricultural fields improved or impaired multiple benefits of agriculture, seems to be that they improved. Most of the BAP measures had a positive influence on most of the MB-categories, the clearest exception being liming in tile drains which only improved the cost effectiveness. Except for water protection, the biosecurity MB-category was positively influenced by the most measures (6 out of 9) and with soil quality the next highest (5 out of 9). It is less clear how the absolute values evaluated for the MB index, can be compared among MB-categories. Among the BAP-measures, structural liming was the most positive measure (summing up the indices of all MB-categories), followed by buffer-zones and spring cultivation the next, although the cost effectiveness of these latter measures was evaluated to decrease. In the “unavoidable” future (Future scenarios) GHG emissions strongly increased. The only measure that mitigated that effect was reduced N fertilisation, providing more arguments for applying reduced fertilisation than only to reduce leaching.

Dricksvattenburna utbrott orsakas av fekalt förorenat (avlopps- eller gödselförorenat) vatten och kan leda till stora kostnader för samhället. Utav de runt hundra potentiella agens som kan spridas fekalt-oralt via vatten är det i huvudsak åtta som är zoonoser, d.v.s. kan ha lantbrukens djur som reservoar. Syftet med denna rapport är att ge en överblick över hälsoriskerna med avrinning från lantbruket, d.v.s. att risken för att vattenburna utbrott ska orsakas av fekal förorening från gödselspriding och/eller strandbetande djur och hur denna risk vid behov kan minimeras. Rapporten innehåller genomgångar av: • vattenburna utbrott som rapporterats i Sverige • barriärer som finns mellan djuren (gris och nötkreatur) och dricksvattenkonsumenten • infektionsdosen hos de mikroorganismer som kan spridas från djur till människa • förekomsten av de mikroorganismer som kan spridas från nötkreatur och grisar till människa via vatten i svenska besättningar • en inbördes riskrankning mellan dessa organismer. Rapporten avslutas med en diskussion utifrån några allmänna principer för utformning av riskkriterier och riskreducerande åtgärder i tre nivåer: på vattenverket, i miljön samt hos lantbrukaren. En infekterad individ (människa, djur) kan dagligen utsöndra tiotals miljarder infektiösa celler. Utanför värden, i miljön, sker en inaktivering av dessa organismer. En barriär kan definieras som en hantering eller process som minskar sannolikheten att exponeras, infekteras eller bli sjuk av en specifik patogen. Det kan t.ex. handla om teknikval, utspädning eller hantering som reducerar halten av patogener i vatten. Den viktigaste barriären i detta sammanhang är dricksvattenreningen där en kontrollerad avskiljning kan ske och inaktiveringen av mikroorganismer kan bedömas. För att nå kvalitetsmålet som ställs på dricksvatten bör halterna i slutändan vara en infektiös cell per 100 000 – 1 000 000 liter. Att nå detta mål underlättas om man har ett råvatten av god kvalitet. Årligen sker mellan fem och tio vattenburna utbrott i Sverige. De orsakas i regel av patogener med låg infektionsdos såsom norovirus, Campylobacter, Giardia och Cryptosporidium. Vissa av dessa kan utsöndras av lantbrukens djur, men registrerade utbrott har i regel orsakats av avloppsförorenat vatten. Utav de vattenburna zoonoserna är det fr.a. Campylobacter och, regionalt, Verotoxinproducerande Escherichia coli (VTEC) som kan tänkas påverka kvaliteten på råvattnet. Dessa bakterier är dock känsliga för desinfektion och fungerar reningen som den ska är risken för vattenburen smitta minimal. Cryptosporidium parvum, som är en klorresistent parasit, kan utsöndras av kalvar upp till ca sex veckors ålder. I de fall dricksvattensäkerheten är baserad på inaktivering med klor och inte har någon effektiv avskiljning med filter, kan djurens påverkan på vattentäkten behöva ses över. Vidare bör vattenreningen byggas ut för att innefatta minst en effektiv barriär mot parasitära protozoer. Utifrån de studier som gjorts i Sverige verkar risken för dricksvattenburna utbrott orsakade av gödselhantering och strandbete liten. Avloppsutsläpp i ett avrinningsområde utgör den största risken då dessa till skillnad från gödsel kan innehålla humanspecifika stammar av Campylobacter och Cryptosporidium samt tarmvirus med låg infektionsdos. Vidare sker utsläpp av tusentals organismer per liter direkt i vattenmiljön. En stor del av den gödsel som sprids har varit lagrat under en längre tid och en viss avdödning har skett. Trots det kan infektiösa organismer finnas kvar. I och med att stora kvantiteter sprids finns risk för ytavrinning och i närhet av råvattenintag rekommenderas det att gödsel hanteras med förnuft. En tumregel är att om man klarar miljömålen med gödselspridning och att råvattnet inte överskrider Svenskt vattens riktvärden för fekala indikatorer (se tabell 4) är risken för smittspridning acceptabel. Om det visar sig att den fekala påverkan på råvattnet ligger högre än riktlinjerna och härrör från djurhållning kan Jordbruksverkets regler för gödselspridning i nitratkänsliga områden rekommenderas: att gödsel appliceras med en säkerhetsmarginal till vattnet och helst brukas ner i marken inom fyra timmar. För strandbete under samma förhållanden kan man tänka sig en begränsning i antalet djur, samt åldern på dessa djur, som har kontakt med vattnet för att minimera risken för smittspridning.

Ecosystem Health and Sustainable Agriculture

Cow manure may contain substantial amounts of pathogenic microorganisms such as Salmonella, Campylobacter, enterohaemorragic E. coll (EHEC), parasites and viruses. Insufficiently disinfected manure has been shown to be a significant factor for the occurrence of pathogens in animal herds. Further, transmission of disease from manure to man has been verified, for example the outbreak of EHEC in Halland this summer (2005). Treatment of the manure before application to land is suggested, to decrease the risk of disease transmission to man or other animals, since microorganisms may survive for extended periods of time in soil. Storage, composting, anaerobic digestion and chemical disinfection are common practises. In outbreak situations, the cormnon practise is lime treatment of the manure. Lime raises pH to around 12 for a period of time, leading to a rapid pathogen die-off The problem is that the handling is complicated with salt precipitation making the incorporation difficult. Further, lime treatment leads to nitrogen losses with a lower agronomic value of the manure. In this project the possibilities of pathogen disinfection with urea or ammonia was evaluated. Urea is the globally most used nitrogen fertiliser. When applied to soil it is converted to ammonia and carbon dioxide by the enzyme urease that is naturally excreted by soil and intestinal bacteria. The same thing will happen if urea is added to manure. With a higher pH, more of the ammonia will be present in the uncharged form, which is disinfecting. At a lower pH, the ammonia will be present in the plant assimilable ion form. lf the ammonia is added as ammonia (aq), which is a stronger base than urea, pH will be higher. Thus, less ammonia needs to be added for the same disinfecting effect providing an economically interesting option. The addition of urea or ammonia raises the agronomic value of manure, however, it is important that the manure well is covered to avoid nitrogen losses. Earlier die-off studies in human faeces have reported a rapid die-off of especially E. coli, but also Salmonella after addition of 6% (w/w) urea. In this study we have added high numbers of Salmonella, enterococci and a bacteriophage to liquid manure (TS 12%) that was collected at Kungsängens gård in Uppsala. Enterococci are, after coliforms, the most common group of faecal indicator microorgnanisms. They are used as a complement to the coliforms since they are more tolerant to many types of environmental stress. Bacteriophages are viruses that infect bacteria, but not eucaryotic cells, and are therefore easy and harmless to handle. They have earlier been used as a model for viral die-off in several reported studies. The microorganisms were added in numbers between 106 and 108 colony- or plackforming units (CFU/PFU) per gram manure. After the organisms had the chance to adapt to the material over night, 2% urea or 0.5% ammonia was added to the material. There was also a control study with no addition of ammonia. The experiments were carried out in triplicates at 4 and 14 °C. Salmonella were severely affected by the treatment and could not be detected after 2 days at 14 °C and 6 days at 4 °C after the addition of ammonia (~pH 9.7). Also urea treatment (~pH 9.2) gave a significant die-off of Salmonella at 14 as well as 4 °C. The time for l log reduction (90%), decimal reduction, was reduced between 76% and 97% compared to the controls (Table, at the end of the summary). Enterococci was not affected in the same way as Salmonella even if the die-off was significantly quicker in ammonia as well as urea treated manure than in the control, in the latter case, however, only at 14 °C (see Table, at the end of the summary). Further, it took some time for the ammonia treatment to be effective. Urea addition seemed to be growth promoting, at least initially. This phenomenon has earlier been documented in studies of enterococci in urine. The phages were not affected at all by ammonia treatment but the reduction took place log- linearly (exponentially) for other reasons, probably by the presence of DNAse (enzyme breaking down DNA) in the material. The die-off was significantly quicker at 14 than 4 °C. Another Salmonella experiment was carried out, where an outbreak situation at a farm was simulated. Ammonia or urea was added to liquid manure in jars that were kept over the weekend. During the following week, 1% salmonella infected manure (107 CFU/ g) was added on top of the disinfected manure. This is what would happen at a farm if disinfection was performed during the time of an asymptomatic outbreak Infected manure was added during five consecutive days without any incorporation into the disinfected manure. After the following weekend, the contents in the jars were mixed and the material sampled. The Salmonella densities were 1 000 times lower in the ammonia treated, and 100 times lower in the urea treated, manure than in the control. After another two days, the Salmonella level was below detection limit in the ammonia treated manure, after four in the urea treated, while it was still possible to detect Salmonella in the control after 35 days when the experiment was ended. Ammonia, added as a water-soluble or in the form of urea, proved to effectively disinfect Salmonella in cow manure (liquid manure with a dry substance, DS, of 12 %). If a six log reduction (99,999%) is desired, less than a week treatment with 0.5% ammonia is needed in 14 as well as 4 °C. Treatment with urea for 10 days at 14 °C or 24 days at 4 °C will also lead to the same hygiene effect. This time can be shortened by raising the pH (and thereby the amount of free ammonia) in the material by the addition of a stronger base such as lime, ashes, sodium- or potassium hydroxide. The time for five log reduction without additives was 170 days at 4 and 42 days at 14 °C. The long-term ammonia disinfection effect in covered compartments leads to further treatment during transports in tanks. Otherwise, reinfection of hygienised material has caused problems during transports. Further, disinfection of manure is a safety barrier in case of an asymptomatic outbreak at a farm. Ruminants are an important reservoir for EHEC and only few strains induces infection in animals that still can shed a lot of bacteria of clinical importance for humans. The use of enterococci as an indicator of Salmonella occurrence after ammonia treatment would lead to an overestimation of the risk due to their tolerance and different behaviour in the material. We suggest that other indicators are used, for example E. coli or faecal coliforms, as indicators of Salmonella occurrence in ammonia treated manure. To be able to account for the uncertainty of the analysis the time to achieve five log reduction was simulated and results presented in the table below. The recommended treatment is based on the upper 95% value and simplified to; at least two weeks treatment with 2% urea at temperatures above 10 °C, one month at lower temperatures. For ammonia one week after the addition of 0.5% is sufficient while storage should include at least one summer season.

This project was initiated to fill knowledge gaps on the occurrence of pathogens in different streams of wastewater, e.g. greywater and domestic wastewater. The aims were also to measure the removal of pathogens in different treatment processes, conventional and innovative, and correlate the removal to that of common microbial process indicators, such as faecal coliforms, enterococci, Cl. perfringens spores and bacteriophages. One study also assessed the correlation between the removal of microorganisms and some commonly measured physico-chemical process indicators. The results can be applied in microbial risk assessments (MRAs) of urban wastewater systems. Indicators and parasitic (oo)cysts were enumerated with standard methods and viruses with rtPCR. High levels of Giardia cysts and enteroviruses were found in untreated wastewater (103.2 and 104.2 L-1 respectively) indicating high incidences in the society. Noroviruses were also often found in high numbers (103.3 L-1) during winter, but less frequent and in lower numbers (102.3 L-1) during the rest of the year. This temporal variation correlated to the clinical laboratory reporting of noroviruses. A temporal variation was also shown for Giardia with significantly lower cyst counts in untreated wastewater during spring. Cryptosporidium oocysts were not as numerous in untreated wastewater (5 L-1) reflecting a lower incidence in the society than for the other pathogens during the time of the study. Since temporal variation had a larger impact than spatial, site-specific measurements may not be necessary to perform screening level MRAs of wastewater discharge and reuse. Good data can be found in the literature and corrected for by recovery of the detection method, flow and incidence in the society. Removal of microorganisms in wastewater treatments varied from 0 to >5.8 log due to process combination and organism in question. Treatment in integrated hydroponics removed microorganism more efficiently than did secondary conventional treatment, though having longer hydraulic retention time. Tertiary treatment and treatment in a membrane bioreactor (MBR) showed better removal potential than treatment in upflow anaerobic sludge blankets (UASB) in a pilot plant. Human virus genomes were less removed and Giardia cysts more removed than all of the studied indicators. Enumeration with PCR, however, may underestimate infectious virion removal. Spores of sulphite-reducing anaerobes and somatic coliphages were significantly less removed than E. coli and enterococci in all the studied processes. Bacterial indicator and spore removals correlated to enterovirus genome removal (p