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

2. Sampling and data collection

2.1. Initial statement of the experimental site

Before planting out the lettuce plants in the soil, the samples of soil, compost, urine and greywater have been made to known the initial concentration of the microbiological parameters.The parameters or indicators which are analyzed in the different matrix are contained in the table 1.And then, samples of soils are taken for each treatment per week to analyze these parameters in table 1 below.Therefore, samplings were carried out from 10 April to 26 May 2014.

Table 2: Different parameters which are analyzed in the matrix

Matrix	Indicators/pathogens
Soil	E. coli/Faecal coliform, Salmonella, Helminthes eggs, Faecal enterococci
Compost	Helminthes eggs, E.coli/Faecalcoliform, Salmonella
Urine	Faecal coliform, Faecal enterococci, Salmonella
Greywater	E. coli/ Faecal coliform, Salmonella, Faecal enterococci

For all these parameters the microbiological analysis will be used.

2.2. Microbiological analysis of matrix (soil, compost, urine, and greywater)

2.2.1. Enumeration of bacteria in soil and compost

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). Fecal coliforms and E. coli and Enterococci were cultured following a method 9215 A in Standard Methods for the Examination of Water and Wastewater (APHA, 1998). Relevant dilutions were spread on plates in duplicate on the following selective media; chromo cult coliform agar ES (Difco, France) incubated at 44,5°C and for 24 h for Fecal Coliforms,E. coli, and Salmonella, Slanetz Bartley agar at 37°C for 48 h for Enterococci. The bacteria load is expressed in (log₁₀ UFC/g-DW soil or compost) through the equation 1:

(Equation 1)

Where:

N = Bacteria load in compost or soil (Log₁₀UFC/g- DW- soil or compost);

n = Number of colonies in box of Petri;

P = Weight of compost or soil samples (25g);

V_l = Volume of Buffer phosphate used to homogenization of compost or soil samples;

V = Volume of test (1 mL);

d = factor of dilution.

DW= Dry weight is expressed by this equation below:

(Equation 2)

Where:

M₁= 10g fresh weight + empty weight oftube,

M₂= 10g-dry weigth+ empty weight of tube,

M₀= empty weight of tube.

2.2.2. Enumeration of bacteria in urine

The description of E.coli and Faecal Coliform(FC) or Enterococci was done by the method of culture of spreading out in depth.The samples were diluted with sterile ringer. After dilution, 1 mL of the diluted sample was spread out over media (Chromocult 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 coliwere identified by blue colorantpurple 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 expressed by equation (2):

(Equation 3)

Where:

N = Concentration of bacteria in urine (UFC/100 mL);

n = Number of colonies in box of Petri;

Vs = Reference volume (100 mL);

V = Volume of test (1 mL);

d = dilution factor.