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1MICROBIOLOGICAL ASSESSMENTS OF COMPOST TOILETS: IN-SITU

Archived at http://orgprints.org/5861

2MEASUREMENTS AND LABORATORY STUDIES OF THE SURVIVAL OF 3FECAL MICROBIAL INDICATORS USING SENTINEL CHAMBERS 4 5 6L. Tnner-Klanka*, J. Mllerb, A. Forslunda, A. Dalsgaarda 7aDepartment of Veterinary Pathobiology, The Royal Veterinary and Agricultural 8University, Grnnegrdsvej 15, DK-1870 Frederiksberg C., Denmark 9bDepartment of Agricultural Sciences, The Royal Veterinary and Agricultural 10University, Thorvaldsensvej 40, DK-1871 Frederiksberg C., Denmark 11 12 13 14 15 16 17 18 19 20*Corresponding author 21email: ltj[a]kvl.dk 22Phone nr. 0045 3528 2722 23Fax nr. 0045 3528 2755

1Abstract (100-200 words) 2 3Compost toilet systems were assessed for their ability to reduce microbial indicators and 4pathogens. Bacterial pathogens were not detected in any samples indicating a low 5survival rate in composting feces and/or an initial low occurrance. Indicator bacteria 6showed large variations with no clear trend of lower bacterial numbers after longer 7storage. 8 9In controlled composting experiments , thermophilic conditions were only reached 10when amendments were made (grass and a sugar solution). Even then it was impossible 11to ensure a homogenous temperature in the composting faecal material and therefore 12difficult to achieve a uniform reduction and killing of indicator organisms. 13 14Thermotolerant coliforms, Salmonella Typhimurium Phage 28B and eggs of Ascaridia 15galli proved useful as indicators. However, regrowth was detected for enterococci and 16total numbers of bacteria grown at 36 C. These indicator parameters may therefore 17overestimate the level of other (pathogenic) bacteria present in the material and can 18based on our studies not be recommended for use as indicator organisms in composting 19toilet systems. 20 21Addition of indicator bacteria to fecal material contained in semi-permeable capsules 22proved to be a useful technique. Thus, microorganisms, e.g. bacteria, virus and parasites 23can be added in known concentrations to materials to test their survival over time. 24

1 2Keywords: 4-6 3Composting, composting toilet, Bacterial indicator, Salmonella Phage, Ascaridia galli, 4sentinel chamber 5

11. Introduction 2In Denmark and elsewhere in Europe, there has been an increased interest in using 3compost toilet systems with urine diversion, typically in eco-villages and in summer 4cottages with several people wishing to use both fecal matter and urine as fertilizers in 5local horticulture and agriculture productions. If such human wastes are to be used as 6fertilizers, it is necessary to ensure a safe handling and disposal of the material from 7such toilets. However, pathogen die-off and associated health risks from use of stored 8feces have been inadequately assessed despite the fact that compost toilets have been 9used for decades worldwide, particularly in developing countries (Jansen and Boisen 102000). 11 12The Danish legislation stipulates that for fecal waste to be used in private gardens it 13must have undergone hyginization, e.g. been exposed to 70C for at least one hour or 1455C for two weeks which would inactivate all pathogens (The Danish Ministry of 15Environmental Affairs, 2003; Feachem et al., 1983). However, such temperatures are 16not normally reached during storage of feces in single household compost toilets 17(Carlander and Westrell 1999). When composting in a small-scale compost toilet 18system and thermophilic temperatures are to be achieved throughout the year, 19optimization in the form of amendments and insulation of the composting containers is 20necessary as shown in a recent Swedish study (Vinners et al., 2003). Another challenge 21in small-scale composting system is to achieve a homogenous temperature throughout 22the fecal material. 23

1Diverted urine to be used as fertilizers presents a relatively smaller hygienic problem 2compared with fecal matter as it contains low concentrations of pathogens, mostly 3originating from fecal contamination and as pathogen numbers in urine can be 4effectively reduced by prolonged storage for 3-6 months (Hglund et al. 1998, 5Jrgensen et al., 2003). In contrast, the faecal matter contains high numbers of naturally 6occurring enteric bacteria, and occasionally disease-causing pathogens like Salmonella, 7Campylobacter, enteric viruses, and parasites of which the latter survives well 8prolonged external environmental exposures. 9 10The hygienic quality of treated organic waste may be assessed using naturally occurring 11and/or added indicator organisms to model the survival of bacteria, viruses and 12parasites. Salmonella is a relatively common excreted pathogen, and test systems in 13different countries therefore often demand absence of Salmonella after treatment of 14fecally polluted material. Salmonella Senftenberg 775W has been used as a 15representative for the Salmonella genus and as a test hygiene indicator for assessing 16thermophilic composting processes as it survives temperatures up to 60C (Henry et al., 171969; Hsel et al. 1995). Enterococci and thermotolerant coliforms are other examples 18of commonly used fecal bacterial indicator organisms (Redlinger et al. 2001). 19Salmonella Typhimurium phage 28B belongs to the group of F-specific phages and 20does not occur naturally in the environment. Phage 28B has previously been used in 21evaluating thermophilic composting processes as a model of virus survival (Sahlstrm, 222003) and for tracking contamination of groundwater (Carlander et al., 2000). 23

1Ascaris lumbricoides is an important helminth infection in humans. Ascaris spp, show 2high survival rates and resistance to environmental stresses and have therefore for many 3years been used as important hygiene indicators in assessing the quality of composted 4feces. Most published work has used Ascaris suum as an indicator of helminth egg 5survival, but eggs of Ascaridia galli, which is commonly found in poultry, are 6considered to be equally resistant to environmental impacts and may be preferred used 7as they are not pathogenic to humans and large numbers of eggs can be made available 8at low costs (Stott et al., 1994). Furthermore, A. galli has not a migratory phase as part 9of its life cycle, which is in contrast to A. suum. This makes it easier to assess infectivity 10of A. galli. 11 12Sentinel chambers, i.e. small cylinders with different sized pore membranes at either 13end, offer an opportunity to measure the survival of added test microorganisms in 14known concentrations without spreading them to the entire study environment. Sentinel 15chambers have previously been used to study the survival of Cryptosporidium parvum 16oocysts in soil, animal waste, sludge and human urine (Jenkins et al., 1999, Udeh et al., 172003, Jrgensen et al., 2003) and are commercially available in various dimensions and 18membrane pore sizes (Excelsior Sentinel, Inc.). 19 20The main purpose of our studies was to assess the ability of compost toilet systems 21commonly used in Denmark to reduce microbiological indicators and pathogens. This 22was done by analyses of ubiquitous fecal indicator microorganisms and added test 23organisms and by measurements of the process temperatures obtained in the toilet 24systems. In the first study (study A), existing toilet systems in Denmark and southern

1Sweden were analyzed for a number of microorganisms and pathogens. This was 2followed up by two experimental studies (studies B and C) of survival of added 3indicator organisms where feces were composted under different controlled temperature 4conditions. Finally, the use of sentinel chambers for studying pathogen survival in 5small-scale composting systems was assessed. 6 7

12. Methodology 2This study consisted of three parts, A, B and C that are described in the following text. 32.1 Experimental set-up 4Study A - Compost toilet systems studied in Denmark and Sweden 5Batch type and continuous type toilet compost systems were studied. In the batch 6systems, plastic containers from 140 to 280 l were connected to a urine-diverting toilet 7unit through a plastic tube. Containers filled with fecal matter were replaced with empty 8containers, and the filled containers were then stored for up to six months for 9degradation of the fecal material. The continuous systems typically consisted of one 10large chamber with a volume of 1 to 2.8 m3, with a flat or sloped bottom where fresh 11feces was added continuously at one end and old material removed at approximately 12yearly intervals from the opposite end. The toilet systems were used at two eco-villages 13(five batch and two continuous systems) in Denmark, and at individual households in 14the Southern and Eastern parts of Sweden (six continuous systems). 15 16Study B Compost toilet units under controlled conditions 17Two 220-L plastic containers were filled with approximately six months old fecal 18material from toilets at one of the eco-villages included in study A. The collected fecal 19material was mixed and homogenised manually and then divided into two equal volume 20portions that were introduced into the two containers. One container was placed outdoor 21along a wall facing south, whereas the other container was placed indoor in an unheated 22room. 23

1Study C Optimization of the composting process in compost toilet units under 2controlled conditions. 3Two 220-L plastic containers were filled with fecal material from toilets located in the 4same eco-village providing fecal matter for study B. The fecal material was up 12 5months old at the start of the study. The collected fecal material was homogenised 6manually, amendments added, mixed and then divided equally into two portions of 31.5 7kg which were put back into the two containers. Each of the containers were added 25.5 8kg fresh-cut ryegrass and a nutrient- and energy-rich solution consisting of 1.8 kg 9sucrose and 200 g commercial fertiliser (18 % nitrogen content) dissolved in 3 litres of 10tap water. Both containers were placed indoor in an unheated room and were insulated 11with glass fiber mats (4 cm thick) wrapped around the sides and the top of the 12containers which were then placed on a 5-cm thick polystyrene plate. Adequate aeration 13of the composting process was ensured through two plastic drainage tubes (80 mm in 14diameter) placed vertically near the container center with an individual distance of 20 15cm from each other. 16 172.2 Temperature measurements 18In study A, the temperature was measured and logged during a 8 month period in four 19of the batch systems every three hours with temperature data loggers (TinyTalk II, 20Gemini Dataloggers, UK). At two occasions, the temperature was measured in selected 21composting containers only, using a 1-m long metal rod with a temperature probe fixed 22at the end. The metal rod was positioned in the centre of the composting material. In 23studies B and C, 5-channel data loggers (Ninasoft, Kolding, DK) were used to log 24temperatures every 10 minutes. In study B, temperature loggers were placed at two

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1depts in the containers, approximately 7 cm and 22 cm from the top. In study C, 2temperature loggers were placed at three levels, approximately 7 cm from the top 3(position: TOP), 30 cm from the top (position: MIDDLE) and at 50 cm from the top 4(position: BOTTOM). At each dept level, one probe was placed near the outer wall of 5the container and the other probe in the centre of the container (position: CENTRE). In 6both studies B and C, the ambient room temperature was measured and in study B the 7outdoor temperature as well. Due to technical problems with the temperature data 8loggers in study B, temperature data were only obtained for three shorter periods (days 90-9, days 84-101 and days 146-155 after study start). 10 112.3 Sentinel chambers 12Sentinel chambers with a semi-permeable membrane (diameter 0.45m) at either end 13were used (Excelsior Sentinel, Inc., NY, USA) in studies B and C to avoid the spread of 14and contamination with pathogens in the experimental environment. The sentinel 15chambers were cylindrical with a volume of about 2.5 ml. In study B, nine sentinel 16chambers were positioned in the composting containers at each of the two dept levels 17described above, never closer than 10 cm to the outer sidewalls of the container and 18with a distance of approximately 10 cm between individual sentinel chambers. In study 19C, two sentinel chambers were positioned in the centre of one of the composting 20containers at the three dept levels specified above. In addition, a nylon bag containing 21eggs of the parasitic indicator A. galli was positioned in both containers at the same 22three dept levels as described above. 23 242.4 Sampling intervals

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1Study A. At each location and for each individual system, fecal samples were taken at 2either two or three points in time, i.e. in March and December 2001 from the batch type 3toilet systems and in March, October and December from the continuous toilet systems. 4It was, however, not possible to collect samples from the same container on more than 5one occasion and differences over time in a particular toilet system could therefore not 6be assessed. The age of the fecal material ranged from one month up to several years. 7All microbiological analyses, except that for parasite eggs, were performed by an 8accredited laboratory in Denmark. Details of microbiological methods are described in 9Table 1 and in the reference list. 10 11Study B. At monthly intervals, one sentinel chamber was removed from the composting 12container placed outdoor and one chamber was removed from the compost container 13placed indoor. The sentinel chambers were transported to the laboratory for analysis 14which was undertaken within six hours after sample collection. 15 16Study C. After 21 days, all sentinel chambers (one container) and nylon bags 17containing parasite eggs were collected from both compost containers. The sentinel 18chambers and nylon bags were transported to the laboratory for further analysis which 19was undertaken within the day of arrival to the lab. 20 212.5 Indicator microorganisms preparation of test solutions and analysis 22The fecal material was taken out of the chambers for microbiological analyses and 23weighed under sterile conditions. Peptone water (1%, Difco) was added to obtain a 24concentration of 1:10. The mixture was homogenized in a stomacher for 30 seconds at

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1XXX rpm, and subsequently serially diluted before enumeration of Salmonella 2Senftenberg 775W, thermotolerant coliforms, Enterococcus spp., total amount of 3bacteria grown at 36C and Salmonella Typhimurium phage 28B. 4 5Total numbers of bacteria grown at 36C, suspected thermotolerant coliforms and 6enterococci. Suspected thermotolerant coliforms, Enterococci spp. and total numbers of 7bacteria grown at 36C were enumerated as ubiquitous bacterial indicators. For analysis 8of total numbers of bacteria grown at 36C, 1 ml of sample dilutions was mixed with 20 9ml of Water Plate Count Agar (WPCA, Oxoid, Hampshire, UK). Plates were then 10incubated at 36C for 44 4 h after which visible colonies were enumerated (DS/EN 11ISO 6222, 2000). The detection limit was 10 CFU g-1. For analysis of suspected 12thermotolerant coliforms and Enterococcus spp., 0.1 ml sample dilutions were spread 13onto agar plates. Suspected thermotolerant coliforms was counted as typical yellow 14colonies on membrane lauryl sulphate agar (Difco) after incubation at 44C for 21 3 h 15(ISO/DIS 9308-1 mod. 1998). Numbers of Enterococcus spp. were determined as 16typical red-red brown colonies on Slanetz & Bartley agar (Difco) following incubation 17at 44C for 48 4 h (DS 2401 1999). The detection limit for both suspected 18thermotolerant coliforms and Enterococcus spp was 10 CFU g-1. 19 20Salmonella Senftenberg 775W. The heat-resistant Salmonella Senftenberg 775W was 21cultured overnight in Luria broth (Lennox, Difco, Sparks, Maryland, USA) to a 22concentration of 3 x 109 bacteria per ml. The semi-quantitative analysis for S. 23Senftenberg 775W was done by transferring one ml from respective sample dilutions to 24nine ml buffered peptone water (Oxoid) which were then pre-enriched at 37C for 16 h.

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1From this resuscitation step, 0.1 ml was transferred to nine ml tubes of Rappaport2Vassiliadis soy peptone broth (Oxoid) and incubated at 42.0C for 24 h. A sterile loop 3was used to streak enriched samples onto Brilliant green Lactose Saccharose Phenol red 4agar (BLSF; Oxoid) and incubated at 37C for 24 h. Representative suspected colonies 5were confirmed as Salmonella by agglutination with polyvalent Salmonella-O6antiserum (DS 266 1988). The detection limit for Salmonella Senftenberg 775W was 10 7CFU g-1. 8 9Salmonella Typhimurium phage 28B. Salmonella Typhimurium phage 28B was used 10as an virus indicator and grown in Nutrient Broth (Oxoid) with its host strain 11Salmonella Typhimurium type 5 (Lilleengen 1948) until a concentration of 1010 PFU 12was reached. The host strain was then killed by adding chloroform, (10ml/L) and the 13solution containing Salmonella Typhimurium phage 28B was centrifuged and filtered 14through a 0.45m filter to remove any cellular debris. Phage 28B was enumerated by a 15double-agar layer method (Adams, 1959). The host strain Salmonella Typhimurium 16type 5 was grown in nutrient broth at 37C for four hrs. From the 10-fold diluted fecal 17samples, one ml was taken and mixed with one ml broth culture of the host strain and 18three ml of soft agar (a mixture of 70% Blood agar base (Oxoid) and 30% nutrient 19broth). The mixture was spread on a well-dried Blood agar base plate which was 20incubated at 37C for 18 h. Clear zones (plaques) were counted as PFU. When a high 21bacterial background flora was expected (mainly at the lower sample dilutions), the 22samples were filtered through 0.45 m pore size filters before mixed with the soft agar. 23The detection limit for phage 28B was 102 PFU g-1. 24

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1Preparation of sentinel chambers A total of 250 g fecal material obtained from the 2container was mixed with 4 ml of the Salmonella Typhimurium phage 28B solution. A 3volume of 1.5 g fecal material which contained the Salmonella Typhimurium phage 428B was mixed with 0.1 ml Salmonella Senftenberg solution. This fecal material was 5then added to the sentinel chambers which were sealed with acrylate glue. All fecal 6material used in the experiments were initially tested negative for Salmonella 7Typhimurium phage 28B and Salmonella spp.. 8 9Ascaridia galli eggs the parasite indicator 10Preparation of parasite eggs and nylon bags containing eggs. Female worms of A. galli 11were removed from the small intestine of a hen from an organic farm in Denmark. The 12uterus of each female worm was located, removed and the eggs taken out. The eggs 13were stored in 0.1 N H2SO4 at 10C until used. A stock solution of 5000 eggs/ml was 14introduced into the nylon bags. Nylon mesh with a pore size of 20 m was used for the 15nylon mesh bags. A piece of nylon mesh measuring 6 x 12 cm was folded and glued 16together along the sides with melted glue. On the day 0 of the study, 2 ml stock 17solution, containing a total of approximately 10,000 A. galli eggs, were added to the 18nylon mesh bags which were then sealed at the top with more glue. Until placed in the 19compost containers, the nylon bags were kept in distilled water. Also at day 0, a control 20assay, using 2 ml of stock solution in a total volume of 28 ml 0.1 N H2SO4, was set up 21to assess the initial viability of parasite eggs used in the nylon bags. Eggs were 22incubated at 20C for four weeks and examined for their ability to embryonate as 23described in the following. 24

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1Ascaridia galli embryonation assay At day 21 of study C, the nylon bags were removed 2from the compost containers and transported to the laboratory. The bags were rinsed 3with distilled water before being opened and the contents washed directly into a large 4Petri dish (diameter 18 cm) using 0.1 N H2SO4 to a total volume of 28 ml. The Petri 5dishes were incubated in the dark at 20C for four weeks. To allow for adequate 6aeration during the incubation period, lids were taken off the Petri dishes twice a week 7for a time period of 10-15 minutes. After incubation, the content of a Petri dish were 8washed into a 50 ml centrifuge with distilled water and left for overnight sedimentation 9at 4C. The supernatant was removed to a volume of 5 ml and a sub-sample was taken 10from the bottom of the tube and examined under a microscope at 100X magnification to 11identify the developmental stages of the parasite eggs. This allowed for a calculation of 12the percentage of embryonated eggs (eggs containing an infective larva).

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13. Results 23.1 Study A 3Temperature. The temperatures measured in-situ in the existing composting systems 4generally reflected the ambient temperature. The maximum temperature measured in all 5systems was 37C and as such, thermophilic conditions were not present at any of the 6sampling times. 7 8Microbiological indicators. There were no obvious differences in microbiological 9measurements between the toilet systems tested and the results were therefore grouped 10according to sampling time (March and December) and listed as ranges (CFU/g faecal 11material) (Table 2). Numbers of suspected thermotolerant coliforms and enterococci 12correlated to some degree although there was a large variation within both parameters. 13This variation could not be explained by a difference in age of the faecal material; 14however, there was some indication of re-growth in faecal material that was more than 2 15years old. Spores of Clostridium perfringens were found in most samples and due to 16their high resistance to environmental impacts, their numbers did not correlate well with 17the numbers of indicator bacteria. The pathogenic bacteria, Salmonella, Listeria and 18Campylobacter were not detected in any of the 25 g sized samples analyzed. A small 19number of cestode and nematode eggs were found in some samples and might have 20been of human origin although this was not confirmed. 21 223.2 Study B 23Temperature. In the composting container placed outside, a maximum temperature of 2449C was measured during the first days of the study, with temperatures of above 40C

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1maintained for approximately 4 days. The study started in the middle of July and it was 2only during the first nine days that the temperature in the composting container was 3found to be higher than the ambient temperature. In the container placed inside, a 4similar picture was seen, except that the maximum temperature reached in the beginning 5of the study was 43C and that temperatures above 40C were only maintained for 1 6day. 7 8Microbiological indicators. The microbiological findings in fecal material placed in 9the middle of the composting containers are presented in figures 1 (container placed 10indoor) and 2 (container placed outdoor) whereas the measurements from the top of the 11containers are only described in the following text. Suspected thermotolerant coliforms 12and Salmonella Senftenberg were reduced to the detection level (10 CFU/g) after 2-3 13months of storage and stayed at this level throughout the study except in January 2003 14where numbers of suspected thermotolerant coliforms rose to 1,000 CFU/g in the 15container placed indoor. In the same container, enterococci numbers decreased slowly 16over several months from a level of 107 CFU/g to 1,000 CFU and remained above the 17detection level throughout the study period. In the container placed outdoor, numbers of 18enterococci were reduced to the detection level (10 CFU/g) after 2 months but 19increased to a steady level of 104 CFU/g for the remaining study period. Numbers of 20Salmonella Typhymurium phage 28B were reduced slowly in both containers and this 21parameter remained above the detection level (100 PFU/g) throughout the study period 22in the container placed indoor whereas it was reduced to the detection level in the 23container placed outdoor. There was a small reduction to 107 CFU/g in the total viable 24counts at 36C in both containers during the entire study period.

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13.3 Study C 2Temperature. The temperature development was very similar in the two composting 3containers and measurements are therefore only presented for the container which had 4nylon bags with parasite eggs and microbiological indicators placed in the sentinel 5chambers (Figure 3). A maximum temperature of 56.8C was reached after 8-9 days 6(57C in the other container) and a temperature above 55C was maintained for 52.3 7hours (30.2 hours in other container). Temperatures at all dept levels in the container 8were below 40C after 13-14 days and were below 30C at day 21 when the sentinel 9chambers and nylon bags were removed. A similar temperature development was 10observed in the other container. 11 12Measurements of pH were made on day 21 of the study. The pH value was 7.9 in 13material from both containers. 14 15Microbiological indicators. The results are presented as the mean of measurements 16from two sentinel chambers (Figure 4). Suspected thermotolerant coliforms were 17reduced from 105 CFU/g to the detection level (10 CFU/g) in the top and the middle of 18the container whereas approxomately 100 CFU was measured in the bottom of the 19container. Numbers of enterococci were reduced by one log unit in the top and the 20middle of the container. However, there was a 2 log increase in enterococci in the 21bottom of the container, suggesting that regrowth had taken place. Salmonella 22Senftenberg was reduced to below the detection level (10 CFU/g) in the bottom of the 23container whereas there was a 1-2 log reduction in the middle and top of the container. 24Numbers of Salmonella Typhimurium phage 28B was reduced to the detection level

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1(100 PFU/g) in the top of the container, to 104 in the middle and maintained its day 0 2level in the bottom of the container. There was a small reduction 1-log in the total viable 3counts at 36C in the top of the container but unchanged levels were found in the 4middle and in the bottom of the container. 5 6A total of 41% of A. galli eggs were able to embryonate in the control assays, i.e. 7develop a live larvae inside the egg and therefore be potentially infective (Table 3). No 8eggs were able to embryonate in any samples collected at any of the three dept levels. In 9this container slightly higher temperatures were achieved and sustained longer than in 10the other container, where a number of eggs placed at bottom level were able to 11embryonate (23.6%), where as very few eggs (0.9%) placed in the middle and none 12eggs placed in the top were able to embryonate. 13

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14. Discussion 2A maximum temperature of 37C was measures in the small-scale compost toilet 3systems at private households. Thus, little temperature developments were found and 4numbers of microbiological parameters were only slightly reduced . In the experimental 5studies B and C where faeces were composted under controlled conditions and various 6amendments were made to optimize the composting process, temperature developments 7were obtained and some reduction in microbiological parameters took place, although 8not sufficient to provide a complete safe hygienisation of the faecal material. Sentinel 9chambers, however, proved useful in studies of pathogen survival in small-scale 10composting systems. 11 12Even though some faecal material used for the experiments was several years old, 13numbers of indicator bacteria were found well above the detection limit along with 14some parasite eggs. The limited reduction of bacterial indicators and parasite eggs was 15most likely associated with the absence of thermophilic conditions during storage. 16Temperature is considered to be the most important factors for a successful 17hygienisation of faecal material (Sahlstrm et al., 2003, Vinners et al., 2003). 18Normally, pathogens are expected to be killed after two weeks at 55C (Feachem et al. 191983), but other factors such as a desiccation and pH may also play a significant role in 20reducing pathogen levels (Redlinger et al., 2001, Eriksen et al., 1995). 21 22Although important pathogenic bacteria of faecal origin such as Salmonella, 23Campylobacter, and Listeria were not detected in any of the samples from existing 24toilet systems in study A, the survival of the bacterial phage and possible re-growth of

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1indicator bacteria during a study period of several months in study B, suggests that 2some pathogens like viruses may survive for prolonged time periods. Virus survival was 3measured in studies B and C using Salmonella Typhimurium phage 28B which has 4previously successfully been used as an indicator (Holmquist and Stenstrm, 2001). 5Overall, results of the controlled composting studies B and C showed a good survival of 6this indicator except in the top of the composting container in study C. Desiccation is 7the most likely explanation for the poor survival in the top location of the container as 8this is known to have a detrimental effect on virus survival (Guardabassi et al., 2003). 9Our results indicate that viruses may survive prolonged storage in a small-scale 10composting systems and as such may pose a health threat when the faecal material is 11handled and used as fertilizer. Bendixen (1995) found that at a constant temperature of 1230C, 800-900 hours would be required to achieve a 4-log reduction in the numbers of 13viruses. As temperatures above 30C were only reached for short time periods initially 14in study B, this may explain the lack of reduction in numbers of the viral indicator. Still, 15Holmquist and Stenstrm (2001) have found some reduction in phage survival under 16mesophilic conditions (25-30C). 17 18Holmquist and Stenstrm (2001) found that enterococci and E. coli were reduced to the 19detection limit after five days. Our study B showed that enterococci maintained their 20level for several months and it took up to two months to get a significant reduction in 21numbers of thermotolerant coliforms. The difference may have been due to the use of 22straw amendments in the study by Holmquist and Stenstrm (2001) which ensured a 23homogenous increased temperature in the faecal material. Vinners et al. (2003) 24certainly found that using amendments was essential to achieving thermophilic

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1composting conditions. This was also the case in our study C where amendments made 2in the form of fresh grass clipping and a sugar solution caused a 3thermophilictemperature increase for up to two days in the centre of the composting 4container. 5 6Redlinger et al. (2001) found that in only 35.8 % of dry-composting toilets (continuous 7type) were thermotolerant coliforms reduced to levels below 1 x 103 CFU after six 8months storage. Drying out of the material was found to be the primary factor 9associated with bacterial die-off. In our study, composting containers were not open at 10the top and we expect drying-out to have had a smaller effect than in Redlingers study 11(2001). The failure to reduce enterococci numbers in both studies B and C questions the 12use of enterococci as indicators of the hygienic quality of the compost. However, when 13thermophilic conditions are present during a composting process, thermotolerant 14coliforms or E. coli appear to be more appropriate indicators of the survival of 15pathogenic bacteria. Thus, in study C, thermotolerant coliforms were reduced to the 16detection level in the middle of the composting container where thermophilic conditions 17were present for the longest period of time. In the top, the added effect of drying out of 18the faecal material is likely to have compensated for the somewhat lower temperature 19achieved at this position. In the bottom of the container, the temperature was 20comparable to that in the top, but there would have been no effect of drying out, hence 21thermotolerant coliforms remained above the detection level at this position. 22 23Salmonella Senftenberg was used as an indicator of Salmonella spp survival in 24composting faecal material. This indicator organism survived up to 2 months when

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1sentinel capsules were placed in the middle of the composting containers and were not 2exposed to thermophilic conditions (study B). When exposed to thermophilic conditions 3for a time period of up to two days, Salmonella Senftenberg was reduced by a 2-log 4factor (study C). Surprisingly, Salmonella Senftenberg was reduced to the detection 5level in the top of the composting container where a maximum temperature of approx. 637C was achieved. This reduction may have been caused by low moisture content as 7has been described for thermotolerant coliforms (Redlinger et al., 2001). 8 9Eggs of the parasitic indicator, A. galli, were not able to embryonate and were therefore 10not infective when eggs were placed in faecal matter in the top of the composting 11container. In the middle of the containers where thermophilic conditions were reached, 12no or very few eggs could embryonate. This difference could be due to the fact that 13temperature probes and parasite eggs were not placed in exactly identical positions. 14Varying temperatures at the position of the eggs may have facilitated the embryonation 15of some eggs. Following exposure in the bottom of the container, no eggs from one 16container could embryonate, whereas 23.6 % of eggs were able to develop after storage 17in the other container (41 % in the control assay). The maximum temperatures achieved 18in the bottom of the two containers were 46.0 and 37.3 C, respectively. The difference 19in temperature may explain why there was a difference in the percentages of eggs that 20could embryonate. A pH of 8 was measured at day 21 of the study. Since a pH of 12 or 21more for a prolonged time period (three months) is needed to have a killing effect on 22Ascaris suum eggs (Eriksen et al., 1995), pH is not likely to have had any effect on the 23ability of parasite eggs to embryonate in this study. Overall, A. galli eggs proved to be a

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1sensible and useful indicator to study the effect of thermophilic conditions for 2composting of faecal material. 3 4The use of sentinel chambers in composting systems was an excellent way of placing 5known concentrations of indicator organisms in larger volumes of composting material. 6Using sentinel chamber also prevented the spread of indicator organisms and pathogens 7to the entire volume of compost material. Sentinel chambers have previously been used 8to measure Cryptosporidium parvum survival in soil and animal waste with similarly 9satisfactory results (Jenkins et al., 1999; Udeh et al, 2003) and may as such be 10recommended for experimental set-up where a small sampling volume is desired and 11where it is important to avoid the spread of micro organisms to the entire experimental 12environment. 13

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15. Conclusions 2A major finding of our studies was that we did not measure any temperature 3developments in existing composting toilet systems. Only when amendments were 4made and when composting containers were insulated was it possible to obtain 5thermophilic conditions. Even then it was impossible to ensure a homogenous 6temperature in the composting faecal material and therefore difficult to achieve a 7uniform reduction and killing of indicator organisms. 8 9Our studies also showed the usefulness of some micro-organisms as indicator organisms 10and the failure of others. Thermotolerant coliforms, Salmonella Typhimurium Phage 1128B and eggs of Ascaridia galli proved useful as indicators to measure the effect of 12temperature developments during composting processes. However, re-growth were 13detected for enterococci and total viable counts at 36 and 22 C. Analyzed numbers of 14these indicators may therefore overestimate the level of other pathogenic bacteria 15present in the material and the indicators can based on our studies not be recommended 16for use as indicator organisms in composting toilet systems. We propose that 17thermotolerant coliforms be used instead as these are better indicators of the survival of 18pathogenic bacteria. 19 20The usefulness of sentinel chambers for testing the ability of a composting process to 21reduce or kill indicator organisms was demonstrated by our study and sentinel chambers 22may be recommended for the type of experimental set-up used in our studies.

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1Acknowledgements 2Danish Environmental Protection Agency for their financial support 3Sentinel Inc. 4We would like to thank Mrs Margrethe Pearman and Mr.Assoumane Idi at the 5Department of Veterinary Pathobiology, KVL for carrying out the parasitological 6analyses in study A and for providing the Ascaridia galli eggs for study C.

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16. References 2 3Adams, M.H., 1959. Bacteriophages. Interscience Publishers Inc., New York 4 5Bendixen, H.J. (Ed.), O. Bennetzen, F. Boisen, B. Espensen, M. Holfort, J.C. Jrgensen, 6U.S. Mikkelsen, E. Tribler, M. Thisgaard, M. Eskildsen, P. Have, V.F. Jensen, B. 7Ahring, B. Lund, and G. Jungersen, 1995. Smitstofreduktion i biomasse. In Danish. 8Technical report concerning the veterinary programme in biogas plants. Volume 1 and 92. The Veterinary Directorate, The Ministry of Fisheries and Agriculture, Copenhagen, 10Denmark. 11 12Carlander, A., and T. Westrell. 1999. A Microbial and Sociological Evaluation of 13Urine-diverting Double-vault Latrines in Cam Duc, Vietnam. Minor Field Studies No. 1491, ISSN 1402-3237. International Office, Swedish University of Agricultural Sciences, 15Uppsala, Sweden. 16 17Carlander, A., P. Aronsson, G. Allestam, T.A. Stenstrm, and K. Perttu, 2000. 18Transport and Retension of Bacteriophages in Two Types of Willow-cropped 19Lysimeters. Journal of Environmental Science and Health 35, 1477-1492 20 21Danish Ministry of Environmental Affairs (2003) Bekendtgrelse nr. 623 af 30. juni 222003 om anvendelse af affald til jordbrugsforml Slambekendtgrelsen (in Danish). 23Available at: http://mst.dk/ 24

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