Fate of Ascaris at various pH, temperature and moisture levels.

Soil-transmitted helminths (STH) are intestinal worms that infect 24% of the world's population. Stopping the spread of STH is difficult, as the eggs are resilient (can withstand high pH) and persistent (can remain viable in soils for several years). To ensure that new sanitation systems can inactivate STH, a better understanding of their resilience is required. This study assessed the inactivation of Ascaris eggs under various conditions, in terms of moisture content (MC) (<20 to >90%), temperature (20-50 °C) and pH (7-12.5). The results highlight that the exposure of Ascaris eggs to elevated pH (10.5-12.5) at temperatures ≤27.5 °C for >70 days had no effect on egg viability. Compounding effects of alkaline pH (≥10.5) or decreasing MC (<20%) was observed at 35 °C, with pH having more of an effect than decreasing MC. To accelerate the inactivation of STH, an increase in the treatment temperature is more effective than pH increase. Alkaline pH alone did not inactivate the eggs but can enhance the effect of ammonia, which is likely to be present in organic wastes.

Ascaris eggs are most persistent during dormancy (preembryo development) when the eggshell is only permeable to organic solvents, lipid-permeable vapours (such as ammonia (Weiner & Hamm )) and respiratory gases (Barrett ; Clarke & Perry ). The protective eggshell needs to be permeable to respiratory gases to access oxygen (Wharton ). As a consequence of this, the eggshell is also permeable to water vapour (kinetic diameter 265 pm), which is a smaller molecule than oxygen gas (kinetic diameter 346 pm) (Hinton ). The shell restricts water loss while still allowing adequate oxygen gas diffusion (Hinton ; Wharton ). However, the ability of the shell to restrict water loss decreases exponentially with increasing temperature (Wharton ).

Material collection
Ascaris suum eggs were retrieved by sieving swine faeces (Excelsior Sentinel, Inc., USA) and were then stored at 4 C in a diluted formalin solution during transportation and storage prior to use. Human faeces (in total 300 g) were collected fresh from two individuals and stored in a freezer until use. At start-up, the faecal material was thawed overnight and 40 g was inoculated with Ascaris egg solution to a level of 100,000 eggs g À1 faeces. The Mineral soil was used as an inert medium for to achieve the different moisture levels. The soil, collected from Uppsala, Sweden, was rinsed with distilled water, autoclaved wet at 120 C for 20 min and dried at 70 C.
After this preparation, the soil was analysed. The MC and the soil organic matter (SOM) were analysed by drying ∼20 g soil at 105 C for 24 h followed by ignition at 550 C for 4 h. The water moisture was determined to the weight loss on drying and the SOM was determined to the weight loss on ignition. To measure the pH and EC, 50 mL of deionised water was added to 20 g soil in a small beaker with lid and places on a shaker table for 2 h (15 rpm). The pH and EC were measured in the soil/water solution (soil:water ration 1:2.5). The analysis of total nitrogen (N Tot ) in the soil carried out using the Dumas combustion method. The soil had the following characteristics: pH 7.8;

Extraction of eggs from material
The extraction procedure for the Ascaris suum eggs was a modification of the USEPA (2003)

Analyses
At the start of the experiment, a sample of the Ascaris suum eggs (n ¼ 100) was observed under a microscope to confirm that the eggs were undeveloped. The initial viability was The logarithmised egg viability data which showed a two-phase inactivation pattern were fitted against a nonlinear inactivation model (Equation (1)) from Harm ().
For some of the treatments that were repeated, the viability data were modelled as a combined data set. log 10 N t ¼ log 10 N 0 [1 À (1 À 10 kÃt ) where log 10 N t is the logged (base 10) proportion of viable eggs at time t (in days), and log 10 N 0 is the logged proportion of viable eggs at start. The reduction rate constant (k) described the change in viability over time during the exponential decay phase (log 10 proportion of viable eggs day À1 ).
The k and the parameter determining lag phase duration (n; dimensionless) were determined by non-linear regression using the Gauss-Newton algorithm (Minitab 17, Minitab Inc., US). This enabled the calculation of the lag phase (Equation (2)).
The lag phase is the initial period where there is no significant inactivation. Many samples had no lag period according to this model. For these datasets, the model gave n ¼ 1, which reduced the model to a linear regression with the y-intercept set to zero, i.e. exponential decay.

RESULTS
At 20 and 27 C, the different pH and moisture contents studied had little or no inactivation during !70 days (  Ascaris eggs were subjected various conditions: five temperatures (20-50 C); three moisture contents (wet >90% MC; partially wet ¼ 60% MC; dry <20% MC) and three pH levels (7.2, 10.5, and 12.5). When no inactivation was observed, the time of the viability study is given.
na ¼ not applicable due to fast inactivation in relation to sampling frequency. a Combined data from repeated study. *Significant difference compared with initial viability but, due to low inactivation, not suitable for model fitting.
within the samples, since no decrease in egg viability was detected at pH 12.5 during the same period.
Compounding effects of temperature and pH were observed at 35 C (Figure 1). The reduction in egg viability followed the biphasic model and inactivation parameters were derived from the data. The increase in pH from 7.2 to 12.5 decreased the time for a 3 log 10 reduction of egg viability by four folds, from 85 days to less than 22 days ( Table 2). The MC had no linear effect on the time required for a 3 log 10 reduction of egg viability. For the Partially Wet (60% MC) treatment, the time required for a 3 log 10 reduction in egg viability increased by 35 days to that of the Wet (>90% MC) treatment. The difference between Wet and the Dry (<20% MC) treatment decreased by 28 days.
Due to fast inactivation in relation to sampling frequency at 42 and 50 C, the inactivation could only be described by a log-linear relationship and the derived k and the time for 3 log 10 reduction of egg viability are conservative values ( Table 2). The sampling frequency was not sufficient to detect any compounding effect of pH and temperature due to the rapid inactivation. At 42 C, no viable eggs were observed by day 8, with the exception of the pH 10.5 treatment, which had one larva in 1,011 counted eggs (a 2.75 log 10 reduction). At 50 C, all eggs were inactivated below the 3 log 10 detection limit within 8 h. Our results highlight that a pH increase alone is not enough to inactivate STH during 70 days of treatment.

DISCUSSION
Ascaris spp. eggs have also been observed to persist in low pH treatments (ranging from 2-5), with the viability of Ascaris eggs being affected by the uncharged carboxylic acids rather than the pH (Harroff et al. ).
Elevating the pH and the temperature to 12.5 and 35 C, respectively, had a compounding effect on the inactivation rates. Our study indicated that the inacti-   (1)). Error bars show the 95% confidence interval, however too small to be visible for some data points. Square symbols denote detection limit with no viable eggs detected.

Moisture content and temperature
The effect on the inactivation rate of Ascaris eggs different  (Table 2). In the derivation of inactivation parameters k and n (Equation (1)), the detection limit data were used when in line with the inactivation trend or showing faster inactivation than the earlier trend, so the time for inactivation could potentially be shorter.
Temperature was the driving factor for inactivation. This observed effect is likely due to egg shell's ability to restrict water loss that decreases exponentially with increased temperature (Wharton ). At the elevated temperatures, pH had a compounding effect, which could be due to temperature affecting the shell's structure. The temperature at which the lipid layer starts to change in permeability and/or deteriorate depends on the amount of time that the eggs are exposed to an elevated temperature, as the effect on the lipid layer may not be permanent (Wharton ). Dye is reported to be able to penetrate the shell of A. lumbricoides eggs exposed to 44 C, whereas eggs that were allowed to cool before being exposed to a dye retained their shell function after exposure to temperatures as high as 65 C for 12 h (Barrett ; Wharton ). A study testing the effectiveness of chemical disinfectants on Ascaris spp. eggs observed that sludge gave higher protection to the eggs than wastewater (Amoah et al. ).

Ascaris persistence
Ascaris eggs are able to withstand the elevated temperatures better than the bacteria and phages (Senecal et al. ). This may be due to different stress responses and production of protective peptides and proteins. In order to be able to per- Agi ). Pathogenic organisms that pass through various hosts and environments, such as Ascaris, can be exposed to sudden changes in their surroundings, such as temperature (Pérez-Morales & Espinoza ) and salinity (Abaza ). An increase in temperature is not the only stress to induce the expression of heat shock proteins. Other stresses, such as anoxia and ethanol (and NH 3 ), can also induce the expression to help protect the egg (Lindquist & Craig ).
Based on the minimal effect that the different moisture contents had on the Ascaris eggs, when treating organic material, an increase in the pH in combination with increasing temperature may be a more efficient way to accomplish faster Ascaris inactivation than drying the material. As excreta will contain some amount of NH 3 , most treatments of excreta would benefit from being covered to retain the NH 3 . The effectiveness of NH 3 treatment also increases with even small temperature increases (Nordin & Vinnerås ).

CONCLUSIONS
Exposure of Ascaris suum eggs to elevated pH (10.5-12.5) at temperatures 27.5 C for >70 days was shown to have no effect on egg viability. Compounding effect of alkaline pH or moisture <20% MC was observed at 35 C, with pH having more effect than decreasing MC. To accelerate the inactivation of STH, an increase in the treatment temperature is more effective than pH increase. However, using alkali to hygienise organic wastes can enhance the sanitising effect of ammonia if performed in closed containers.