Health risk assessment associated with the reuse of compost, urine and greywater in agricultural field in sahelian climate.

2.2.3. Enumeration of bacteria in greywater

The description of E. coli and Faecal coliform (FC) or Enterococci was done by the method of culture of spreading out in surface.The samples were diluted with sterile ringer. After dilution, 0.1 mL of the diluted sample was spread out over media (Chromo cult Agar for E. coli/Faecal coliform and Slanetz Bartley for Enterococci), contained in box of Petri which were then carried to the drying oven for incubation with 44 °C during 24h for E. coli/Faecal coliform and with 37 °C during 48 h for Faecal Enterococci. E coli were identified by blue color and purple and Faecal Enterococci by whitish. The colonies obtained were counted thereafter and numbers obtained was allotted to the number of E coli or enterococci present in the sample. This is why the concentration is expressed in unit forming colony (UFC) reported to 100 mL of sample. Bacteria load is determined by equation 2 above in similar conditions.

2.2.4. Enumeration of Salmonella

- Compost and soil

Compost or soil samples 25 g (w/v) were homogenized in 225 mL of buffer phosphate water and a 10-fold dilution series was performed in maximum recovery diluents (ringer solution). 10 mL of Rappaport Vassiliadis media were added in test tubes of different dilutions (10⁰ to 10^-6) where three to five repetitions are made per dilution and 1 mL of sample is added in the test tubes. It is illustrated by figure 3below.Then, test tubes are introduced in incubator during 24h at 37°C for testing process before sowing in ChromAgar media on Petri box and then incubating at 37°C during 24h to confirm the result of first observation. Final result is obtained by the tables of Mac Grady (annex i)where it is expressed in Most Numbers Probable per gramme (MNP/g).

- Urine and greywater

Process is similar as compost and soil analysis (figure 3). However, dilution is made directly without homogenization with buffer phosphate water. Final result is expressed in Most Numbers Probable per liter (MNP/L).

10 mL Rappaport solution + 1 mL sample (soil or compost)

T₃

T₃

T₃

T₃

T₃ 

T₂

T₂

T₂

T₂

T₂

T₁

T₁

T₁

T₁

T₁

Figure 3: Illustration of step of Salmonella analysis

10⁰ 10^-1 10^-2 10^-3 10^-4

2.2.5. Enumeration of helminth eggs in soil and compost

Briefly, analysis was performed on compost or soil and was based on the recognition of forms and structure of helminth eggs in microscope. Sludgewas prepared by adding 225 mL of 0.1% Tween 80 to 25g compost sample. The mixture was homogenized for 1 min using a blender and screened through 4 layers of wet gauze folded. The filtrate was collected in round bottom flasks and allowed to settle for 3 hours and submitted to analysis. Helminth eggs were determined by the US EPA protocol (1999) modified by Schwartzbrod (2003) with a modified density of zinc sulfate (ZnSO₄) saline solution.Quantification of Helminth eggs is made through the equation 3:

(Equation 4)

Where:

N = Number of helminth eggs/L

V= Volume of initial sample compost or soil (225 mL);

k = Constant related to the performance of the method (k = 1.42).

Then, result of equation 3 is reduction of the weight of dry compost or soil diluted (25g) where the final result is expressed in eggs/g.