EFFECTS OF REAGENTS AND CHEMICALS USED IN EXISTING SANITATION HELMINTH TEST METHODS ON THE VIABILITY OF ASCARIS SUUM EGGS

Sustainable development goal (SDG) 6 includes all countries worldwide and is aimed at halving the number of people living without access to sustainable sanitation and potable water, improving water quality by reducing water pollution, improving water use efficiency, ecosystem protection, community involvement, and water and sanitation management. SDG 6 also focuses on the development of reuse technologies (Hoekstra et al., 2017), and reuse depends on the complete hygienization of waste material by pathogen removal or destruction (Verbyla et al., 2013). Use of potable water for flushing latrines is wasteful; consequently, there has been a move toward use of nonsewered sanitation systems that have a backend treatment facility and can treat and reuse the flushed water. To ensure that these toilets are safe for users, testing for pathogen inactivation is important.

Ascaris spp. are the most resilient of all helminths in fecal waste and are therefore used as an indicator organism when testing for pathogen inactivation by these sanitation systems. Because of ethical and logistical issues, it is often difficult to source Ascaris lumbricoides (human roundworm) eggs; thus, eggs of the pig roundworm, Ascaris suum, are used as a surrogate. Both roundworm species are morphologically identical in all developmental stages (Daugschies et al., 2013).

New sludge treatments and toilet technologies are constantly being developed, and helminth eggs must be spiked into the system to test inactivation efficacy. The processing and recovery of helminth eggs in fecal sludge should be consistent so that data are comparable. Various laboratories and groups have used variations of the standard U.S. Environmental Protection Agency (USEPA; 2003) method, the Mexican Standard method for wastewater analysis (Secretaría de Economía, 2012), the Bailenger method (Ayres and Mara, 1996), and the Pollution Research Group (PRG) helminth method for helminth testing (Ayres and Mara, 1996; U.S. Environmental Protection Agency, 2003; Secretaría de Economía, 2012; Velkushanova et al., 2021). According to the WHO (World Health Organization, 2006), the recommended limit for helminth eggs should be 1 egg/L of wastewater and 1 egg/g of total dried solids (sludge); however, counts also differ across countries (Maya et al., 2012). This variation in egg load and the fact that egg viability is not taken into consideration calls for these limits to be re-evaluated. There is also no single “gold standard” in existence for helminth egg recovery and analysis from sanitation and environmental samples. A highly sensitive, standard isolation and enumeration method is therefore required for application in laboratories globally.

The basic principles of sample processing and egg recovery are standard across the various methods currently in existence: washing and sedimentation (using a surfactant or wash solution), flotation (using density gradients to allow eggs to separate from particulate matter), extraction (phase separation using hydrophilic and lipophilic solutions to remove protein and lipid contaminants from the remaining sample containing helminth eggs), incubation (to determine egg viability), and microscopic analysis (before and after incubation using a light microscope) (Rocha et al., 2016; Amoah et al., 2017b). Table I summarizes all reagents and chemicals used in existing methods.

To develop such a standard method, the first step would be to evaluate the effects of the listed chemicals and reagents on helminth egg viability, because chemicals used in a method should have no impact on the outcome of the test. The choice of wash solution plays a critical role in the number of eggs recovered from a given sample type (Rocha et al., 2016); however, the effects of wash solutions alone on egg viability have not been widely explored. Some studies have revealed that long-term exposure to some of these chemicals compromises the viability of eggs. Exposure to zinc sulfate (Gaspard et al., 1996) and magnesium sulfate (Smith, 1991) as flotation solutions can be toxic to eggs. Nelson and Darby (2001) found that extraction solutions, such as ethyl acetate and diethyl ether, reduce egg viability. The present study was conducted to determine the effects of various solutions on egg viability and which chemicals and reagents perform best and should thus be used in a standard international processing and enumeration method.

Testing was conducted with 5 experiments, and the viability of A. suum eggs was quantified. Other chemicals and reagents in addition to those listed in Table I were included to ensure a well-rounded dataset. Exposure times were chosen based on existing methods and the logical time frames needed to complete the intended steps when performing the method (including centrifugation times), and all combinations were tested in 5 replicates. Ascaris suum eggs were isolated from the feces of research pigs using the PRG helminth method (Velkushanova et al., 2021).

Experiment 1 involved the exposure of eggs to various wash solutions for different periods, including overnight soaking that is necessary for dry sample types. Solutions tested were ammonium bicarbonate (at 119 g/L), 0.1% Tween® 20, 0.1% Tween® 80, 0.1% 7X®, 1% Triton® X-100, 0.1% Sunlight® Liquid (a commonly used South African brand of dishwashing liquid), 1% bentonite suspension, and tap water as a control. Exposure times were 10 min, 30 min, 2 hr, 6 hr, and 24 hr.

Experiment 2 involved the exposure of eggs to commonly used flotation solutions: zinc sulfate (specific gravity [sp. gr.] 1.3), magnesium sulfate (the solution saturated at sp. gr. 1.25 and formed crystals at the bottom of the bottle when stored), sodium nitrate (sp. gr. 1.3), sodium chloride (saturated at sp. gr. 1.18 even when the solution was heated), and sucrose (saturated at sp. gr. 1.2). Eggs were exposed for 30 min, 1 hr, and 2 hr.

Extractions usually are done in combinations of a hydrophilic solution plus a lipophilic solvent. Experiment 3 involved the exposure of eggs to all extraction solutions separately to determine the individual effect of each solution on egg viability. Then Experiment 4 explored these solutions in combinations to simulate the solutions used in actual extraction methods. Solutions were 10% formalin, acetoacetic buffer, acid-alcohol, ethyl acetate, and diethyl ether individually and combinations of 10% formalin and acetoacetic buffer each with ethyl acetate and diethyl ether, and acid-alcohol with ethyl acetate. Eggs were exposed for 15 min, 30 min, and 1 hr.

Experiment 5 involved the exposure of eggs to various incubation solutions: tap water, physiological saline, 0.1 N sulfuric acid, and 0.5%, 2%, and 5% formalin for 28 days.

Egg stock solutions were made up in deionized water, with an egg count of ca. 300 eggs/ml of suspension. Approximately 13 ml of each test chemical was pipetted into 15-ml conical plastic test tubes (Falcon tubes), and 1 ml of well-mixed egg stock was then spiked into each sample. Exposure times were monitored.

Test tubes containing samples and the respective test chemicals were then poured over a 20-µm-pore-size sieve (100 mm in diameter) after each sample’s exposure times elapsed. The test tube was rinsed several times and poured over the sieve. The sample retained on the sieve was then thoroughly washed with tap water to ensure that all chemical residuewas removed, and the retentate was collected back into the same (well-rinsed) test tube and centrifuging at 1,512 g for 5 min. The supernatant was subsequently discarded, the pellet was analyzed by light microscopy, and the sample was washed back into the test tube, incubated for 28 days at 25–27 C in water, then re-examined microscopically. Eggs were categorized as potentially viable when undeveloped (1 cell), developing (2 cells, multiple cells, or gastrula), motile (plump developed larva that moved), or immotile (plump developed larva that did not move) and as nonviable when necrotic (thin shriveled larva), dead (globular, ruptured, or with irregular contents), and infertile (unfertilized).

For the incubation solution samples, an initial check of the egg stock was done by light microscopy (as detailed above) in triplicate to obtain an average initial egg count and reference point for postincubation samples (i.e., initial viability as a percentage of the total egg count). Eggs (1-ml aliquots of the stock solution) were pipetted into 15-ml Falcon tubes. The tubes were centrifuged at 1,512 g for 5 min, the supernatant was discarded, and 2 ml of each incubation solution was added to the respective tubes. The pellets were dislodged to allow the eggs to be fully exposed to the incubation solution. The tubes were then incubated for 28 days at 25–27 C and subsequently analyzed to determine egg development and viability according to the categories described above.

Potential viability refers to the percentage of viable eggs before incubation and thus includes undeveloped and developing eggs, whereas actual viability refers to the percentage of eggs that reached the larval stage after incubation. The criterion set for successful inactivation for this study was <10% viable eggs recovered after treatment (Naidoo et al., 2016).

Biological development, referred to below as contamination, was noted and differentiated from debris based on what the sample looked like before and after incubation. Debris would have appeared in both instances, but contamination growth would appear only after incubation. After incubation, eggs from some of the exposure times appeared “webby,” “matted,” or clumped. Egg development was halted in these cases, and eggs looked damaged. Inconsistent fluctuations in actual viability after incubation can be seen across exposure times for some of the experiments below, which were attributed to contamination and resultant egg damage.

The wash solution alone had no significant effect on potential egg viability (P = 0.790), but a significant effect was observed on actual viability (P < 0.001). Exposure time alone had a significant effect on both potential and actual viability (P = 0.04 and P < 0.001, respectively). The interactions of wash solutions and exposure times were also significant for both potential and actual viability (P = 0.027 and P < 0.001, respectively). Eggs looked healthy (mostly at the 1-cell undeveloped stage) when analyzed directly after reagent exposure, accounting for the high potential viability values for all reagents and exposure times; however, not all of these eggs developed to the larval stage after incubation. Across exposure times, ammonium bicarbonate and 7X® allowed for the highest larval development of eggs (Table II). The samples looked the healthiest, with minimal contamination and excellent development, and larvae were plump and motile. The water samples had weblike contamination after incubation, with severe clumping of eggs. The same webiness was seen in Tween® 20 and Tween® 80, and eggs appeared dark and unhealthy. Sunlight® Liquid and Triton® X-100 performed second best; samples were clear and easy to analyze with good egg development. Bentonite samples were very particulate and therefore messy to analyze, with severe contamination (fungal hyphae visible), and the eggs were dark.

Flotation solution alone and exposure time alone both had significant effects on the potential viability and actual viability of eggs (P < 0.001 for both). The interactions of flotation solutions and exposure times were also significant for both potential and actual viability (P < 0.001). Eggs that appeared healthy directly after reagent exposure accounted for the high potential viability values; however, larval development was not as high after incubation. At 15 min, zinc sulfate, magnesium sulfate, and sodium nitrate resulted in the highest larval development (Table III). Exposure times >15 min resulted in poor larval development, and sucrose and sodium chloride resulted in severe contamination of the samples postincubation and very poor larval development (Table III). Sodium chloride halted development of eggs, whereas sucrose caused samples to appear murky and slimy.

Extraction solution alone and exposure time alone had a significant effect on potential egg viability (P = 0.044 and P = 0.001, respectively) and actual viability (P < 0.001 for both). The interactions of extraction solutions and exposure times had no significant effect on potential viability (P = 0.095) but had a significant effect on actual viability (P = 0.007). Potential viability values across all extraction solutions and exposure times were ±80%; however, actual viability decreased for samples exposed to the solvents for >15 min (Table IV). The 10% formalin samples were clear with minimal development of contamination. Acetoacetic buffer caused blackening of the eggs, which appeared damaged with some fungal contamination, and samples were not as clear as those exposed to formalin. Ethyl acetate also caused blackening of the eggs, with extensive contamination, but samples were clear of debris and easy to analyze. Diethyl ether caused some eggs to rupture, and samples had extensive contamination, with eggs that appeared most damaged of all 5 flotation solution exposures. Acid-alcohol allowed the hatching of eggs after incubation.

The effect of the extraction combination alone was significant for potential egg viability but not for actual viability (P < 0.001 and P = 0.279, respectively). Exposure time alone had a significant effect on both potential and actual viability (P < 0.001 and P = 0.002, respectively). The interaction of extraction combination and exposure times was also significant for both potential and actual viability (P = 0.002 and P = 0.021). As for the individual exposures, 15 min resulted in the best development of eggs. Actual viability then fluctuated for longer exposures, and although values may appear similar (Table V), the condition of the eggs deteriorated. The formalin and ethyl acetate samples looked clear and easy to analyze, and the developed eggs looked healthy. In the formalin and diethyl ether samples, eggs were blackened, and some contamination was evident. Acetoacetic buffer and ethyl acetate samples were clear, but some contamination was evident. Acetoacetic buffer and diethyl ether samples also were easy to analyze, but eggs again appeared blackened and damaged postincubation. Acid-alcohol and ethyl acetate allowed extensive hatching of eggs.

The incubation solution had a significant effect on both potential and actual viability (P < 0.001). Samples incubated in water and physiological saline were contaminated, and eggs were caught in webby clumps that made microscopic analysis difficult. Sulfuric acid samples allowed the best egg development (Table VI); eggs were well developed with plump, motile larvae. These samples were also very clean and easy to analyze. The formalin samples had some contamination, with the highest at a concentration of 0.5% and the least at 5%, but eggs looked increasingly damaged as the formalin concentration increased.

A variety of methods exist for the isolation and enumeration of helminth eggs from environmental and sanitation samples, but some were designed for wastewater samples and are time-consuming and require consumables (USEPA method and Mexican method for wastewater analysis), and some are not robust enough to isolate all helminth eggs, e.g., the Bailenger method (Ayres and Mara, 1996; USEPA, 2003; Secretaría de Economía, 2012). The chemicals and reagents required for these methods may have inhibitory or detrimental effects on helminth eggs. A single, cost-efficient, and inclusive method is therefore needed.

Ammonium bicarbonate and 7X® performed the best across all exposure times based on the effects of the 8 wash solutions on egg viability, indicating that even prolonged soaking of samples in a wash solution is possible. These solutions resulted in clean deposits postincubation with minimal biological contamination, indicating that these solutions could have sufficient antimicrobial properties to thoroughly clean samples before incubation for viability assessment. Tween®80 and Tween®20 both affected egg viability, and samples developed contamination that could have further impacted viability. Although many studies have been focused on the effects of wash solutions on recovery of helminth eggs from various sample types, few have focused on the isolated effects of the solutions on egg viability. Ravindran et al. (2019) stated that 7X® does not form a precipitate when reacting with a flotation solution, thus making it a good dissociation solution. Amoah et al. (2017a) also stated that ammonium bicarbonate and Tween®80 did not affect egg viability (71.1% and 87.9%) and were adequate for egg recovery, but prolonged exposure to Tween®80 damaged eggs, in agreement with the findings of the present study. Data also suggest that Triton®X-100 and Sunlight® Liquid may be used as alternatives to ammonium bicarbonate and 7X® but for shorter exposure times not exceeding 6 hr. Naidoo et al. (2016) reported that sodium hypochlorite–based detergents (surfactants) damaged eggs, reduced egg viability, thinned the eggshell, and initiated hatching of larval eggs and should thus never be used.

Egg viability was similar for the flotation solutions zinc sulfate, magnesium sulfate, and sodium nitrate when exposed for 15 min, with ±67% of eggs producing viable larvae. The difference between potential and actual viability could be attributed to the contamination that formed after incubation because samples were not processed with a wash solution, which would generally reduce microbial activity. After 15 min of exposure, actual viability dropped rapidly, indicating that exposure time played a role in egg development and should be kept to a minimum. Gaspard et al. (1996) stated that prolonged exposure to flotation solutions may damage the eggs because of the inhibitory nature of some salts; thus, reduced exposure times were advised.

Nelson and Darby (2001) and Amoah et al. (2017a) reported similar findings; zinc sulfate, magnesium sulfate, and sodium chloride did not affect egg viability at sp. gr. 1.2 (viability of 81.2%, 85.0%, and 83.9%) and sp. gr. 1.3 (viability of 88.5%, 88.5%, and not applicable), respectively. We favored zinc sulfate over magnesium sulfate and sodium nitrate. Magnesium sulfate precipitated into salt crystals over time, but reaching a sp. gr. of 1.3 was difficult, and the solution precipitated at sp. gr. 1.25. Smith (1991) reported that magnesium sulfate was toxic to eggs, and Amoah et al. (2017a) further elaborated that increased exposure time exacerbated the toxic effects. Sodium nitrate samples were more particulate, indicating a reduced ability to separate eggs from finer debris and resulted in extensive contamination postincubation. Gaspard et al. (1996) recommended a double flotation with sodium chloride; however, we found that the solution precipitated before reaching sp. gr. 1.3, even when heated.

The extraction experiments indicated that whereas the hydrophilic solutions had little to no effect on egg viability (both potential and actual), with acid-alcohol affecting egg development the least, viability was reduced when eggs were exposed to ethyl acetate and diethyl ether (lipophilic solvents). In combination, acid-alcohol and ethyl acetate performed the best after 15 min of exposure, with a potential viability of 89.6% and actual viability of 79.4%. Acetoacetic buffer and ethyl acetate also produced promising results, but no combinations yielded an actual viability figure >80%, possibly due to the synergistic effects of the hydrophilic and lipophilic solutions on the eggshell. Nelson and Darby (2001) reported that acid-alcohol alone resulted in the inactivation of eggs (52.2% viability), but diethyl ether alone did not have any effect on the viability of eggs (85.3%), contradicting the findings of our study. Amoah et al. (2017a) reported 74.4% and 3% egg viability for ethyl acetate and acetoacetic acid alone, respectively, and 13.2% and 59.1% for acetoacetic acid plus ethyl acetate and acetoacetic acid plus formalin combinations, respectively. Although the acetoacetic acid plus formalin is not a combination of a hydrophilic and a lipophilic reagent but rather 2 hydrophilic reagents, these authors explored the effects of the same chemicals that we had tested, and individual findings aligned with the findings of our study; acetoacetic buffer combinations were less toxic to eggs than were combinations with formalin.

Nelson and Darby (2001) recommended that exposure to the extraction combination (acetoacetic buffer and diethyl ether in this case) should be limited to 30 min, and samples should be rinsed before incubation. After 15 min of exposure, the viability values fluctuated across the extraction combinations in this study. Although potential viability was >80% and actual viability was >65% after 1 hr of exposure to the combinations, the prolonged effects on the eggshell must be considered. Nelson and Darby (2001) reported that acid-alcohol, both alone and in combination with diethyl ether, reduced egg development as a result of increased eggshell permeability. It is possible that solvents also increase permeability by interfering with the lipid layer of the eggshell (Nelson and Darby, 2001). We therefore recommend a maximum exposure time of 15 min, and samples should be rinsed before incubating. Nelson and Darby (2001) also reported 30% egg viability after extraction of sludge samples with acid-alcohol and diethyl ether, but acetoacetic buffer and diethyl ether extractions did not reduce egg viability. We found that acid-alcohol was the least toxic to egg development, both alone and in combination with ethyl acetate, even up to 1 hr of exposure. Therefore, acid-alcohol is the preferred hydrophilic solution for extractions, with acetoacetic buffer as the second-best option.

Diethyl ether is hazardous to human health and more flammable than ethyl acetate and is thus recommended as a suitable alternative with its lower flash point and more effective egg recovery (Rude et al., 1987). Data from the present study indicated that diethyl ether was more toxic to eggs than ethyl acetate and should therefore be replaced by the ethyl acetate for extractions. Nelson and Darby (2001) stated that egg recovery was 48% lower in extracted sludge samples and therefore recommended that the extraction step be removed from the sample processing where possible. The present study was focused solely on the effects of reagents on egg viability; thus, eggs were not spiked into sludge samples for recovery. Based on our data, extractions performed with acid-alcohol and ethyl acetate at the minimum exposure time caused the least amount of damage to Ascaris eggs.

Studies have indicated that fungal development can be toxic to eggs, and some fungal strains are ovicidal and can halt egg development (Ferreira et al., 2011; Blaszkowska et al., 2014). Microbial growth results in oxygen competition that may further damage eggs (Gaspard et al., 1996). An ideal incubation solution should thus have antimicrobial properties thereby inhibiting bacterial and fungal growth that could interfere with egg development (Cruz et al., 2012). We found that sulfuric acid resulted in the best egg development (81.5% potential and 79.6% actual viability) and is therefore recommended for use; eggs looked healthy, larvae within were motile, minimal contamination developed, and samples were easy to analyze. Oksanen et al. (1990) found 90–97% viability for eggs incubated in tap water and sulfuric acid and 88% viability in 1% formalin, with slower egg development. Nelson and Darby (2001) reported 72.8%, 82.4%, and 82.7% viability in water, sulfuric acid, and 0.5% formalin, respectively, and Pecson and Nelson (2005) reported 95% viability in sulfuric acid. Amoah et al. (2017a) reported 89.7% viability in 0.5% formalin. All of these findings support the findings of our study. The highest actual viability seen in the present study was in samples incubated in tap water (85% potential and 82.6% actual viability); however, the eggs clumped together and made sample analysis very difficult. The same situation occurred in the 0.5% formalin.

Although modern enumeration techniques exist, such as real-time PCR for the molecular detection of the DNA of helminth eggs in sludge (Gyawali et al., 2015) and the BacLight staining technique, which requires a specialized confocal microscope for helminth egg quantification (Gyawali et al., 2015), these methods are not suitable for small laboratories in developing countries. PCR results indicate only the presence or absence of eggs and not their viability status (an indicator of risk to human health) (Amoah et al., 2017b). A simple cost- and labor-effective international standard method is therefore needed.

The present study provided the foundation for the development of such a helminth recovery method, and the following recommendations were developed for each step. Ammonium bicarbonate and 7X® were the best wash solutions, with Triton® X-100 and Sunlight® Liquid as acceptable alternatives. Zinc sulfate is recommended for flotation, and magnesium sulfate and sodium nitrate may be used as replacements. For extractions, hydrophilic solutions have a less toxic effect than do solvents in the phase extraction step. Acid-alcohol or acetoacetic buffer are ideal hydrophilic solutions, and ethyl acetate is recommended as the solvent. Acid-alcohol plus ethyl acetate was the best combination, followed by acetoacetic buffer plus ethyl acetate as an alternative. Sulfuric acid produced the clearest samples after incubation, with healthy eggs containing plump, motile larvae. Water may be used as a replacement incubation solution.

The next steps in development of a standardized helminth method are focused on physical aspects, such as washing samples, centrifugation speeds and times, various possible densities of flotation solutions, egg recovery experiments after washing, flotation and extractions, and how to handle all sample types or matrices. These experiments were needed so modifications could be made at each step to optimize and develop a final method adaptable to different sample types while optimizing recovery and maintaining egg viability. This work will be presented in subsequent articles.

We thank the Water Research Commission of South Africa and the National Research Foundation for funding this project and Merissa Naidoo for her invaluable technical assistance and input. Ethical approval for the use of Ascaris suum eggs from our research pigs was granted by the Animal Research Ethics Committee of the University of KwaZulu-Natal (AREC/071/018).