The inactivation of Ascaris eggs was assessed in three fractions of urine/Ca(OH)₂ solution (Supernatant, Mixed liquor and Precipitates—Figure 1) and in three solution treatments: 1) drying at 20°C with storage at 20 and 35°C in sealed containers; 2) storage (no drying) in sealed containers at 20 and 35°C; and 3) thermal treatment in sealed containers at 42 and 50°C (Table 1 and 2).

The collected urine (>10 L) was pooled and calcium hydroxide (Ca(OH)₂; VWR Chemicals, Pennsylvania, United States) was added at a ratio of 10 g Ca(OH)₂ per liter urine to achieve pH 12.5. The mixture was agitated for 10 min by magnetic stirrer and then left to stand undisturbed in closed containers for 3 h at 20°C to form what is referred to as stabilized urine.

Urine Fractions

From the stabilized urine/Ca(OH)₂ solution, three fractions were formed (Figure 1). The fraction referred to as Mixed liquor was the whole agitated mixture treated and dried together. The Supernate and Precipitate fractions were made by decanting the solution after standing for 3 h. The Supernate represented the situation where after Ca(OH)₂ stabilization the top liquid would be pumped out of the reactor for drying, while the precipitates would be removed and dried separately. All three fractions were stored in sealed containers overnight.

The inactivation of Ascaris eggs in the three fractions was assessed by three solution treatments, as described in Table 1 and 2. The volumes of the urine fractions for each treatment are described in Table 3. All treatments had an addition of 0.1 g feces with Ascaris eggs (100 000 eggs per gram epg).

Solution Treatments

Drying-storage: The effect of drying prior to storage was investigated by drying the three urine/Ca(OH)₂ fractions (Mixed liquor, Supernate, and Precipitate) at 20°C to either a Partially wet condition (73–82% moisture content (MC)) or a Dried condition (13–33% MC) followed by storage in sealed containers at 20 or 35°C.

Stored: Urine/Ca(OH)₂ fractions were stored directly in sealed containers at 20 and 35°C (>90% MC).

Thermal Treatment: The third aspect investigated was effect of elevated temperature (42 and 50°C) during storage of the Supernate fraction to assess the thermal pre-treatment time required to achieve a 3 log₁₀ reduction in Ascaris egg viability.

Wet Conditions: For setting up the studies of the three urine/Ca(OH)₂ fractions under wet conditions (>90% MC), 6 ml from each the three different fractions were aliquoted into 7 ml polypropylene tubes with O-ring lined screw caps (Sarstedt AG & Co., Sweden) already containing 0.1 g of Ascaris eggs in feces. After sealing, the tubes were vortexed for 5 s. The capped tubes were then placed at the target temperatures (in incubators or in an insulated box at room temperature) (Table 2).

Partially Wet Conditions: urine/Ca(OH)₂ solution of the three different fractions were divided into 45 ml aliquots, which were transferred to 50 ml centrifuge tubes already containing 0.1 g fecal material and vortexed for 3 s (or to <1 mm lumps). The tubes were then centrifuged for 5 min at 3000 rpm to bring particles, including Ascaris eggs, down from the walls. The tubes were placed without lids on a table and dried with a fan at 20°C until no liquid was visible (73–82% MC) and then the tubes were sealed and stored at target temperature.

Dried conditions: 150 ml of the urine/Ca(OH)₂ solution from the Supernate and Mixed liquor fractions and 45 ml of the precipitates fraction were added to 0.1 g feces with Ascaris eggs (the addition of fecal material was made according to the total solids content). After vortexing for 3 s, the fractions were transferred to open foil trays lined with waxed paper, which were kept on a table and dried with a fan at room temperature to the target moisture content of <33%. The dried material was transferred to 7 ml tubes, which were sealed and stored at the target temperature.

The duration of the drying period varied from 2 to 8 days (Table 1) and the time was included in the duration of the treatment. The variation in drying period was due to the tubes from the Partially wet treatment being dried in smaller containers than the open trays in the Dried treatment. The study was conducted as independent singlets using destructive sampling.

Extraction of Eggs

The extraction procedure for the A. suum eggs was a modification of the USEPA (2003) method. To extract the eggs from crystals formed in the three fractions, diluted 0.1 N sulphuric acid was added to the vials and they were vortexed for 30 s and then centrifuged at 4000 rpm for 3 min. The Supernate was decanted over Tyler sieves (⌀ 38 μm, Cat. No. L3-400, and ⌀ 300 μm, Cat. No. L3-50). This was repeated 5–10 times, until the crystals formed by the urine and the Ca(OH)₂ were nearly dissolved. Any remaining crystals were decanted over Tyler sieves and further washed with 0.1 N sulphuric acid. Sampling frequency depended on the intensity of the treatment and ranged from every 2 weeks at 20°C to every 8 h at 50 °C.

Experiments were conducted in the presence and absence of feces to assess if fecal matter exerts a protective effect of viruses during alkaline conditions (pH 9, 11.5 and 12.3). Additional control experiment with virus in feces exposed to virus dilution buffer (VDB; 5 mmol L⁻¹ NaH₂PO₄, 10 mmol L⁻¹ NaCl, pH 7.5) were also performed to examine the effect of feces itself on virus survival. Each experimental condition was tested in independent singlet experiments.

Experiments at pH 11.5 and 12.3 were conducted in phosphate buffer. Experiments at pH were conducted in ammonium carbonate buffer. The specific composition of the buffers to attain a given pH was determined with PHREEQC (version 2.18.00) (Parkhurst and Appelo 2013) and a database using the Pitzer approach for calculating the ion activity. Solutions were made according to SI, Supplamentary Table S1. by mixing salt with sterilized milliQ water, stored at targeted temperature for at least half day, and the solution pH was then measured and adjusted with NaOH/HCl. The pH and electrical conductivity (EC) were measured at experimental temperature with 780 pH Meter with primatrode with NTC no. 6.0228.010 (Metrohm, Herisau, Switzerland) and a Cond315i conductivity meter and a TetraCon 325 probe (WTW, Weilheim, Germany) respectively.

ΦX174 was used as the test organism at pH 11.5 and 12.3, because MS2 inactivation was too rapid to be measured. Conversely, MS2 were used as the test organism at pH 9.0, because ΦX174 did not exhibit measurable inactivation over the timescale considered.

At the start of the experiment, a sample of the A. suum eggs (n = 100) was observed under a microscope to confirm that the eggs were undeveloped. After extraction from the treatments, 100 eggs were directly examined to assess any development or damage occurring during the treatment period. The viability of the eggs was determined by incubating them at 28°C in 0.1 N H₂SO₄ for 28 days to allow larval development (Arene, 1986). Viability counts were performed under × 10 and × 20 magnification and unfertilized eggs, which constituted a minor proportion of all eggs, were excluded from further counting. Eggs developing to the larval stage were considered viable. Initial viability of the A. suum eggs was 55% in the treatment applying storage temperatures of 20 and 35°C, and 77% in the treatment applying thermal treatment at 42 and 50°C.

Infectivity was assessed using the double agar layer method and was expressed as plaque forming units per mL (PFU ml⁻¹) (Standard Methods 2018). The quantification limit for all phages was 100 PFU ml⁻¹. Number of phages in feces (PFU mg⁻¹) was calculated from the phages in solution PFU mL⁻¹ extracted from feces, multiplied by the total amount of feces in mg mL⁻¹. Natural presence of MS2 and ΦX174 used in this study in feces was assessed to be under the fore-mentioned detection limit.

Confidence intervals for proportions of viable Ascaris eggs were derived using the Wilson score interval (Minitab Inc., US). The logarithmized egg viability data, which showed a two-phase inactivation pattern, were fitted against a non-linear inactivation model (Eq. 1) from (Harm 1980). For the treatments that were repeated, the viability data were modelled as a combined dataset: l o g 10 N t A s c a r i s =   l o g 10 N 0 A s c a r i s [ 1 − ( 1 − 10 k A s c a r i s ∗ t ) 10 n ] (1) where log₁₀N_tAscaris is the log(base 10) proportion of viable eggs at time t (in days), log₁₀N_0Ascaris is the log proportion of viable eggs at start, k _Ascaris is the inactivation rate constant, which describes the change in viability over time during the exponential decay phase (log₁₀ proportion viable eggs day⁻¹), and n is a dimensionless parameter determining lag phase duration. The values of k _Ascaris and n were determined by non-linear regression using the Gauss-Newton algorithm (Minitab 17, Minitab Inc., US). This enabled calculation of the lag phase, i.e., the initial period where there is no significant inactivation (Eq. 2): L a g   p h a s e = l =   n | k | (2)

Many samples had no lag phase according to this model. For these datasets, the model gave n = 1, which reduced the model to a linear regression with the y-intercept set to zero, i.e., exponential decay.

Virus inactivation was modelled as a log-linear decay process according to Eq. 3: l o g 10 ( N v i r u s N 0 ,   v i r u s )   =   − k v i r u s     t   (3) Where N _virus and N _0,virus are the concentration of infectious viruses at times 0 and t, and k _virus is the first-order inactivation rate constant (day⁻¹). The linear regressions were performed on log₁₀ transformed data with Excel software. The 95% confidence interval (95% CI) of k _virus was calculated from the standard error of the slope from one experiment.

Ascaris eggs were inactivated at 20°C, with times ranging from 53 to 111 days for a 3 log₁₀ reduction in egg viability with shorter time required for inactivation in the dry compared to the wet treatment (Table 4). In a related study high pH (12.5) and drying had no effect on the inactivation of Ascaris eggs kept for 80 days at 20°C under similar conditions in terms of pH, moisture, and temperature, but without urine or NH₃ (Senecal et al., 2020). Pecson et al., 2007 confirmed that pH alone will not affect Ascaris egg viability at this temperature whereas Naidoo et al., 2016 showed an effect of moisture content alone, with a 1 log₁₀ reduction in Ascaris egg viability after 6 weeks at MC 20%. It is thus likely that presence of NH₃ have contributed to the inactivation in the current study, while any carbonate formation would have been negligible (Fidjeland et al., 2016). At 35°C, the elevated pH of 12.3 and temperature were the driving factors for the inactivation of Ascaris eggs, which took between 12 and 79 days for a 3 log₁₀ reduction in egg viability, depending on the fraction and moisture conditions (Senecal et al., 2020). At 35°C, a higher degree of drying had no clear effect on the inactivation rate, an effect that was observed at 20°C. In the related study with similar conditions pH, moisture, and temperature levels, but without urine or NH₃, there was a 3 log₁₀ reduction in egg viability within 23 days (Senecal et al., 2020).

The Ascaris inactivation studies could have been strengthened with controls studying the drying media without urine, to assess whether the medium itself had an effect. Formation and accumulation of ammonia could have been monitored in the different fractions and at different moisture conditions to gain a better understanding of the results.

Ascaris eggs require O₂ during development (Pawlowski 1982), but are able to remain viable in anaerobic conditions for >80 days (Gaasenbeek and Borgsteede 1998). In the present study, lack of oxygen should not have affected the egg inactivation rates at 20°C, as demonstrated by no reduction in egg viability after 126 days at pH 12.5 in a sealed container (Senecal et al., 2020). However, the lack of O₂ could have been a compounding stress factor for the Ascaris eggs at higher temperatures. The increase in osmotic pressure caused by drying the urine was not likely to have causes stress for the Ascaris eggs (Decrey et al., 2011), as Ascaris eggs have been shown to survive in concentrated urine mixed with ash at 20°C for >100 days (Senecal et al., 2018).

The lag phases were longest in the partially wet condition at 20°C, for two probable reasons: 1) this condition had less NH₃ than the Wet and Dried condition (took longer to dry, see Table 1 and 2 this condition had no moisture stress compared with the dried condition. This difference in the lag phase duration between the conditions was not observed at 35°C in this study. However, in another study with similar moisture content (60% MC) and temperature, but no NH₃ present, inactivation time was longer compared with wet (>90% MC) and dried (<20% MC) conditions (Senecal et al., 2020). This indicates that partially wet conditions may be ideal for the survival of the Ascaris eggs.

The precipitates at 35°C seemed to have a protective effect on the Ascaris eggs, as the time for a 3 log₁₀ reduction was longer for this fraction than for the other fractions at 35°C (Table 4). For the dried precipitate at 35°C, the MC was higher than in other dried conditions (33%, compared with 18% for the dried Supernate and 24% for the dried mixed liquor fraction), which could have caused the smaller k value. Feces has been shown to have a protective effect on Ascaris eggs (Ghiglietti et al., 1997; Capizzi-Banas et al., 2004), and the precipitates fraction may have provided some similar protection.