1.1 Study framework and sampling stations
This study took place from June 2018 to June 2019 and was carried out in two phases. The first phase, which lasted 3 months (June to August) consisted of a series of screening tests to determine the ranges of minimal observable effect concentrations on eggs. At the end of this screening, the active Carbon and 6 ranges of mass concentrations were obtained for the analysis, namely (0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L and 0.6 g/L).
1.2 Description of sampling station Discharge of Nomayos (No) With geographical coordinates 04°09'33.7'' N and 11°22'08.9'' E, and an altitude of 629 m, the Nomayos sewage sludge discharge station is located on the periphery of Yaounde. For the time being, it is the sole discharge point for sewage sludge removed from the septic tanks of households in this area (Fig. 1a). Trucks unload their contents on a surface assigned to them by the local authorities, these dumps are generally found in water course located at the downstream of the dump, the population living in the vicinity of this site practice food and market gardening and use the sludge from these excreta as fertilizer (Fig. 1b).
1.3. Preparation of disinfectant solution
We used charcoal particles of plant origin (Marcia et al. 2004; Zhang et al. 2005) which were ground into fine particles and then washed with distilled water, dried and sieved. Activation was carried out chemically using acid and base (Fig. 1c). First the coal crystals were soaked in 98% sulphuric acid (H2SO4) solution (desiccant, oxidant and mineral removal agent) for 24 h and then washed with distilled water until a pH of 6 was reached in the residual liquid. In a second step (25 g) of activated carbon was immersed in 100 ml KOH, the whole was brought under stirring to 85°C for 2 h. The resulting liquid was then filtered off and the fine active charcoal crystals obtained were dried at 120°C for 24 h (Mahmoud et al., 2018). The concentrations used for disinfection were weighed using a balance (0.1 g/L, 0.2 g/L, 0.3 g/L, 0.4 g/L, 0.5 g/L and 0.6 g/L).
1.4 Sampling
Sampling of wastewater and sewage sludge was carried out following the recommendations of Rodier et al. (2009) for physicochemical analysis and following the approach proposed by Keffala et al. (2012) for biological analysis. Samples were collected using sterile 5L bottles and returned to the laboratory. For wastewater, sampling was done directly on the effluent, for sewage sludge the sample was taken directly after discharge by trucks.
1.5. Physico-chemical analyses and determination of helminth eggs
1.5.1. Physico-chemical analyses
The physico-chemical parameters evaluated in this study were measured using conventional techniques described by Rodier et al. (2009) with appropriate reagents. The pH was measured using a multiparameter HANA HI 9829. Turbidity (FTU), suspended solids and colour were measured in the laboratory by colorimetry with a spectrophotometer HACH DR/3900. Measurements of ammoniacal nitrogen contents in mg/L of water were determined by colorimetry, using the spectrophotometer HACH DR/2900. The reagents used Nessler and Rochelle salt. The oxydability contents in mg/L of KMnO4 were measured by volumetry.
1.5.2. Determination of helminth eggs
1.5.2.1 Sample pre-treatment
The sample brought back to the laboratory was sieved for the removal of large particles, then the viable eggs in the sample were previously quantified, non-viable eggs were removed by flotation with n-butanol. Approximately 1ml of n-butanol was added to 500ml of sample to allow the non-viable eggs which are less dense to float, and then these non-viable eggs were removed by suction of the supernatant.
1.5.2.2. Disinfection test
For the treatment and disinfection of the samples, we made a device comprising 1 series of 6 Erlenmeyer flasks, and then 500 mL of previously homogenized sample was introduced into each Erlenmeyer flask. Active carbon was introduced in Erlenmeyer flasks at the different concentrations (C1, C2, C3, C4, C5, C6). The samples were then homogenized using a magnetic device and a baro-magnet to ensure perfect contact between the Active charcoal and the sample. To allow the disinfectant to act, a contact time of 24 hours was observed for each sample (Akam et al., 2005). Then 5ml of pellet was taken from each sample and introduced into a test tube, this pellet was then washed with sterile water twice (Amoah et al., 2017 a). To these 5mL of pellet were added 5mL of distilled water, shaken and centrifuged at 500 rpm for 7 min (Ibañez-Cervantes et al. 2013). The supernatant from this centrifugation was removed by aspiration. The resulting pellet was washed a second time with sodium thiosulphate solution to neutralize adsorption. The resulting pellet was subjected to viability testing by staining and incubation, after concentration of the eggs by sedimentation and McMaster technique. The test was repeated for all sampling campaigns conducted during this study, with observations repeated twice (Khallaayoune and Fatiha 1995).
1.5.3. Viability Analysis
1.5.3.1 Staining Viability Test
For the staining viability test we used the neutral red which is a vital dye (Merward et al. 2011; Karkashan et al. 2015). Neutral red is a dye that has the ability to bind to the structure of viable eggs and stain them red. After concentration of the parasitic elements 1 ml of neutral red was added to each sample and a contact time of 10 minutes was observed to allow the stain to penetrate the viable eggs. These samples were then placed on the McMaster slides for observation, the eggs stained red by the stain were considered viable, and the unstained eggs were considered potentially non-viable (Sarvel et al. 2006). The number of eggs per litre was calculated using the formula proposed by Sengupta et al. (2011).
N = AX/PV
Where N = number of eggs per litre of sample, X = volume of final product (ml), A = number of eggs counted on the McMaster slide or average of the numbers found in two or three slides, P = capacity of the McMaster slide (0,3 ml), V = volume of the initial sample (litres).
1.5.3.2 Incubation viability test
For this technique, 5ml of pellet were incubated on Petri dishes in an oven at 30°C for 30 days (Pecson et al. 2007), during these days the process of reduction of egg viability was demonstrated, starting from the destruction of the egg membrane to the inactivation of the egg. Then the eggs were examined under an optical microscope, so that non-viable eggs segmentation stopped while viable eggs continued their segmentation and development. Eggs containing at least eight blastomers and eggs containing mobile larvae were considered viable (Stien 1989; Keffala et al. 2012 Amoah et al. 2017 b). Identification was done through morphological analysis of egg size, shape and content (Řežábkováa et al. 2019). Egg content and wall changes were examined by light microscopy at 40X and 100X objectives. Measurements were made using an eyepiece micrometer and photos were taken using an Xploview model photographic device attached to one of the microscope's eyepieces. The number of eggs per litre was calculated using the formula proposed by Ajeagah et al. (2014) :
With Vx = volume of pellet in 1 L of sample, Vy = volume of pellet used for observation, y = number of eggs observed in Vy.
1.5.4. Molecular analyse by Polymerase Chain Reaction (PCR)
1.5.4.1. DNA isolation
Each wastewater sample (100 mL) was filtered through a sterile 0.2 µm Sterivex filter (Millipore, USA) and the genomic DNA (gDNA) was extracted from the filters using a PowerWater Sterivex DNA Isolation Kit (MOBIO Laboratories, California, USA). Extraction reagent blank controls (ExCs, n = 6) were included alongside each batch of gDNA extractions. Purified DNA was stored at − 20°C prior to molecular analyses.
1.5.4.2. Next-generation sequencing library preparation
For NGS library preparation and sequencing, the 16S metagenomic sequencing library preparation protocol from Illumina (Part # 15044223 Rev. B; Illumina, USA) was followed, with only minor modifications to the first stage PCRs. The hypervariable 9 (V9) regions of the eukaryotic 18S were amplified, using 2 µl of template DNA (out of a total of 50 µl); these primers were modified to include Illumina MiSeq adapter sequences (Part # 15044223 Rev. B; Illumina, USA). Amplification of 18S V9 with the Euk1391F/EukBr primers was carried out using conventional PCR as per the 18S amplification protocol available from the Earth Microbiome Project. A mammalian blocking primer (Mammal_block_I-short_1391f) was used at a final concentration of 1.6 µM to reduce amplification of mammalian DNA. The libraries were sequenced on the Illumina Miseq platform (San Diego, CA, USA), with v2 sequencing chemistry for the eukaryotic 18S NGS.
1.6. Statistical analysis
The normality of the data was assessed using the Kolmogorov-Smirnov test, while data comparisons were made using the ANOVA test and Students T test. The correlations were made using the Pearson test. All these analyses were carried out using the SPSS version 17.0 software.