For liquid and slurry types of faecal sludge (TS < 15%), sub-samples for dilution are measured out volumetrically. Mix the sample as described in the 'homogenisation' section and measure out an appropriate volume for dilution into a volumetric flask, and then add distilled water to a specific volume. A serial dilution will be necessary depending on the required concentration range, and could be followed by filtration or centrifugation to separate the suspended solids depending on the specific method. For semi-solid and solid sludge (TS > 15%), tare a beaker on an analytical balance and then weigh out the sample into the beaker. Calculate the volume of required dilution water, and weigh it out in a separate tared container. Add a volume of the dilution water to the sample, and mix it well, then transfer to the blender. Reserve some of the volume of dilution water to rinse the beaker that held the sample and also transfer the wash water to the blender. For example, this could mean weighing out between 1.8 g and 2.0 g into a 50 mL beaker, and then rinsing it in in series to obtain the required volume of 200 mL. Blend the diluted sample at the highest setting until homogenised, then pour the diluted sample into a bottle for analysis or storage. It is not necessary to remove all of the solids from the blender as the sample has been thoroughly mixed.

The results should be represented as either a volumetric concentration (mg/L) or as a mass fraction by total dry solids (g/g TS). In general, it is not recommended to report results as a wet basis mass fraction (g/g wet sample), due to the high variability in percentage solids or moisture in faecal sludge samples. This means that density (mass per volume) and TS (mass of TS per volume) should also be measured for each sample for accurate reporting and to allow for comparison between results. The definition of the dilution factor that is used in the methods is:

Dilution factor =

Final adjusted volume of dilution Original volume of sample aliquot to be diluted

For example, if 10 mL is the final dilution volume and 1 mL was the original sample volume, then the dilution factor would be 10.

#### **<IP_ADDRESS> Filtration**

Filtration of samples could include sieving of dried ground samples prior to metals digestion, or filtration of liquid samples to remove suspended solids. For example, as described in Method <IP_ADDRESS> for total suspended solids, acceptable glass fibre filters will range in pore size from 0.45 μm to 2.0 μm depending on the thickness of sludge and clogging of the filters. In addition to pore size, clogging is addressed through the process of sample dilution for semi-solid to solid samples, as explained in the method description. The type of filter material will also depend on the intended method, and is explained in each section; for example, glass fibre filters are specific to methods that use a 550 °C muffle furnace, and paper filters can be used in nutrient methods.

## **<IP_ADDRESS> Centrifugation**

Centrifugation can be used instead of, or in addition to, filtration to separate suspended particles from the sample. For liquid faecal sludge (TS < 5%), mix and blend the sample as described in the 'homogenisation' section and transfer an aliquot of the sample into a clean glass beaker. Withdraw sample aliquots of equal volume into one or more centrifuge tubes. Place an even number of centrifuge tubes diagonally opposite each other to balance the centrifuge. If there are an odd number of centrifuge tubes filled with sample, fill an additional tube with the same volume of water to balance the centrifuge. Centrifuge the tubes at a specific g-force and time. Decant the filtrate (supernatant) into a clean glass beaker and discard the pellet. Centrifuge the samples until the required volume for sample analysis is obtained. Centrifugation can also be used for separation of the suspended solids in slurry, semi-solid, and solid samples (TS > 5%); however, centrifugation will be more effective if the samples are diluted beforehand. In cases where the sludge is very difficult to filter, centrifugation can be used prior to filtration to remove most of the suspended material.

## **8.4.3 Sample and chemical disposal**

Waste minimisation and pollution prevention in the laboratory is the preferred approach to managing laboratory waste. However, when the remaining material is disposed of, it is important to ensure the protection of public and environmental health, and disposal needs to be included in each SOP. For more detailed information on methods of disposal, see National Research Council (2011).

Faecal sludge samples and laboratory equipment should be contained and sterilised to eliminate all pathogens. All contaminated equipment should be disinfected before washing, storage, or disposal. If possible, it is recommended to autoclave materials contaminated with faecal sludge before discarding them. After sterilisation, waste can be handled safely and disposed of in the local waste collection. Incinerated or pasteurised samples can also be contained and disposed of in the general waste. If autoclaving is not possible, small volumes of faecal sludge samples (*e.g.* 1-5 L) should be flushed down a drain that is connected to the sewer, and the whole area then properly sterilised. If this is not possible, store the waste samples in a sealed container in a cold room or refrigerator at 4 °C. The person responsible for the samples (*e.g.* the student, researcher, or laboratory personnel) then needs to arrange for the waste samples to be discarded at a local wastewater or faecal sludge treatment plant. If a faecal sludge treatment plant is not available, the samples may be discarded in the laboratory toilet facilities. This, however, should be done cautiously due to potential blockages and contamination. The toilet facilities should be well washed and disinfected after the sample disposal.

It is important to document in SOPs which acids, bases, salts, and solutions can be poured down the drain with dilution, and which chemicals may not be disposed of in this fashion under absolutely any conditions (*e.g.* heavy metals, pesticides, oils, nitrite). A management plan needs to be in place for disposal of toxic compounds.

Containers need to be labelled for separate collection of waste that is safe to be managed with the local waste collection, *e.g.* biohazard and sharps, and waste chemicals. Material such as broken glassware, sharp objects and fine powders that could harm workers during collection also needs to be separately collected. For more information on developing waste handling procedures, refer to Prudent Practices in the Laboratory: Handling and Management of Chemical Hazards (National Research Council, 2011).

## **8.5 SHIPPING AND RECEIVING OF FAECAL SLUDGE SAMPLES AND EQUIPMENT**

In some cases, faecal sludge samples may be shipped locally or internationally; for example, when the required analysis is not locally available, when samples are collected from remote areas for comparison among multiple laboratories of the same samples, or for research studies. In this case, the sending and receiving parties need to fulfil specific regulatory requirements and documentation based on the local context and requirements of the shipping company. These may include import-export permits for biological samples issued by the relevant authorities (*e.g.* see the Department of Health example in Figure 8.2), and a material transfer agreement (MTA) signed by the sender and receiver organisations. Further information and details on specific local regulations can be obtained by local courier companies. Sufficient time between application for all the documents and the shipping date must be planned, including customs regulations. For example, in some countries this process may take up to 3 months.

The sample packaging method depends on the type of faecal sludge samples to be shipped. For example, oven- dried and/or autoclaved samples are relatively easy to ship due to their reduced risk of leakage and contamination whereas more liquid samples need special care. Liquid samples need to be contained in order to minimise the risk of leakage. Samples should be packed in sealed, airtight containers with headspace, wrapped in double containment (*e.g.* watertight plastic bags) along with absorbent material in case of leakage, and shipped with ice packs (for very short transport times) or dry ice. Below in Figure 8.3 are examples of packaging, but specific guidelines must be obtained from each courier company, and further information on packaging instructions can be found through the International Air Transport Association (IATA). Samples should be shipped as soon as possible after collection to ensure that they are well preserved. In addition, the shipping time should be as short as possible to avoid biodegradation processes in the samples and accumulation of biogas in the shipping containment.

**Figure 8.2** Example of an export permit application in South Africa (source: Department of Health, Pretoria, South Africa).

For primary containment, samples should be shipped in rigid plastic bottles or containers sealed with a watertight, screw-on cap (see Figure 8.3 A). Samples should be packed directly from the refrigerator to maintain temperature. Absorbent material should be packed around the primary containers before sealing the plastic bags. For secondary containment, primary containment containers are packed into watertight, sealed, strong plastic bag/s (see Figure 8.3 B and C). Absorbent material must be packed on the outside of the secondary containment. There must be a sufficient amount of absorbent material inside of the package to ensure absorbance of the sample volume in the event of leakage. Examples of absorbent materials are sponges, vermiculite, paper towels, or wooden chips. Sealed plastic bags with the sample containers must be packed inside a rigid container to ensure that samples are fixed during shipping (see Figure 8.3 E). A cardboard or a rigid outer packing (*e.g.* a hardplastic cooler box) is recommended (see Figure 8.3 F). It may be additionally packed into a larger box if required by shipping regulations. Include paperwork inside the shipping box explaining the contents of the shipment and the regulations it falls under.

Cooling packs, ice or dry ice may be used in the shipping container (see Figure 8.3 D). Samples should be kept at 4 °C for the entire duration of shipping. General cooling rules for samples are:

**Figure 8.3** Examples of packaging in the laboratory (photos: UKZN PRG).

## **8.6 CHEMICAL AND PHYSICO‐CHEMICAL PROPERTIES**

## **8.6.1 Solids and moisture content**

Total solids is a term applied to the material left in a vessel after evaporation of a sample and its subsequent drying in an oven at a defined temperature. Total solids are comprised of total suspended solids (TSS), total dissolved solids (TDS), fixed solids (FS) (ash) and volatile solids (VS). Total solids are determined for all types of faecal sludge – liquid, slurry, semisolid and solid. The same methods are used to determine TS and moisture content; the total mass of a sample before the analysis is the sum of its TS and moisture content. Sand content (measured as silica as an indicator of soil content in faecal sludge) is the concentration of sand in the TS of an unfiltered faecal sludge sample. Sand can influence faecal sludge treatment processes (*e.g.* dewatering), increase abrasion of mechanical equipment, and affect the quality of faecal sludge treatment end products.

## **<IP_ADDRESS> Total solids and moisture content – volumetric and gravimetric methods by oven drying3**

#### <IP_ADDRESS>.1 Introduction

Total solids (and/or moisture content) is one of the most commonly used faecal sludge parameters, and is used for almost every design or management decision. For example, for making decisions on treatment design, settling, or emptying. A known volume (the volumetric method) and/or weight (the gravimetric method) of a thoroughly-mixed sample is evaporated to a constant weight in a crucible (porcelain or silica) or an aluminium weighing boat, in a drying oven at 103-105 °C; the remaining solids are cooled down to room temperature in a desiccator to avoid absorption of air moisture and then re-weighed. The residual material remaining in the crucible are TS, and can consist of organic and inorganic material, and dissolved, suspended or volatile matter.

The gravimetric method is recommended for semi-solid and solid types of sludge, as it is often difficult to measure volumes accurately for sludge with higher TS concentrations. For more liquid types of sludge, either the gravimetric or volumetric method can be used. However, these are general recommendations, and a final decision of which method to use needs to be assessed for each application individually. Conversion between volumetric and gravimetric measurements can be done if the density is known (Chapter 2). Density of faecal sludge can easily be measured by weighing a known volume of sludge (Method <IP_ADDRESS>). When doing such conversions, it is always recommended to measure the actual density of the specific samples, and this becomes even more important with samples at the higher range of % TS.

Solid and semi-solid sludge types can form a water-trapping crust if the initial rate of drying is too high. This can be avoided by placing the samples in the drying oven at a lower temperature, and gradually increasing the temperature of the oven until the prescribed temperature of 103-105 °C is reached.

#### <IP_ADDRESS>.2 Safety precautions

3 The volumetric method is based on Method 2540B of the Standard Methods for the Examination of Water and Wastewater. The gravimetric method is based on ASTM E1756−08 Method A and on Method 2540G of the Standard Methods for the Examination of Water and Wastewater. Both methods should be cited as: Rice *et al.* (2017) as described in Velkushanova *et al*. (2021).

## <IP_ADDRESS>.3 Apparatus and instruments

## <IP_ADDRESS>.4 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

between 2.5 and 200 mg of the dried sample (in general approximately 30 mL for the volumetric method, or 10-20 g for the gravimetric method, but this will depend on the type of sludge).

## <IP_ADDRESS>.5 Sample preservation

Samples should be analysed as soon as possible. If samples cannot be analysed immediately, they should be stored in a refrigerator at 4 °C for no longer than 7 days and, if TSS or VSS analysis is conducted, no longer than 48 hours. Before starting analysis, let the samples return to ambient temperature. Do not freeze the samples.

#### <IP_ADDRESS>.6 Sample preparation

 Uniformly mix all the samples using a stainless steel rod (or other appropriate tool) in order to have thoroughly mixed representative samples. If desired, samples can also be blended (see Section 8.4.2).

 Measure out an appropriate sample volume/mass (indicatively 30 mL for the volumetric method, or 10-20 g for the gravimetric method) which will yield a residue between 2.5 and 200 mg of dried sample, by using a volume measuring cylinder or analytical balance. With very dilute faecal sludge samples, a pipette can be used. For other sludge types, clogging of the pipette will occur, and therefore using a graduated cylinder to measure volume is recommended.

## <IP_ADDRESS>.7 Analysis protocol

## *Preparation of equipment*

## *Procedure*

	- For the gravimetric method (semi-solid to solid sludge):
	- Weigh out 10-20 g mass of the sample to the weighed crucible using a spatula.
	- Record the wet mass + mass of the crucible (W2).

## <IP_ADDRESS>.8 Calculation

*Liquid and slurry samples (volumetric method):*  Total Solids in wet sample (mg/L) =

$$\frac{\left(\text{W}\_{\text{3}}\left(\text{g}\right)\text{-W}\_{\text{l}}\left(\text{g}\right)\right)\times\text{1,000,000}}{\text{V}\_{\text{sample}}\left(\text{mL}\right)}$$

Total Solids in wet sample (g/L) =

$$\frac{\left(\text{W}\_{3}(\text{g})\text{-W}\_{1}(\text{g})\right)\times 1,000}{\text{V}\_{\text{sample}}\left(\text{mL}\right)}$$

*Semi-solid and solid samples (gravimetric method):* Total Solids in wet sample ( g g⁄ ) =

(W3(g)-W1 (g)) (W2ሺgሻ-W1 ሺgሻ)

Moisture content in wet sample (g/g) =

ሺW2 ሺgሻ-W3(g)ሻ (W2 ሺgሻ-W1(g))

Moisture content ሺ%ሻ =

ሺW2 ሺgሻ-W3(g)ሻ (W2 ሺgሻ-W1(g)) × 100(%)

Where:

W3 = Dry sample mass + crucible mass after drying (g)

Vsample = Volume of sample used (mL)

For an explanation of the conversion of these units into %TS, refer to Chapter 2, Section 2.2.

#### <IP_ADDRESS>.9 Data set example

Described in Engund *et al*. (2019) and Strande *et al*. (2018) are the collection of 60 faecal sludge samples in Hanoi, Vietnam, and 180 samples in Kampala, Uganda. Solids analysis for TS, TSS, VS, VSS, and fixed solids were carried out and reported as concentrations. The complete raw data set is available using the link below4 .

A faecal sludge sample was collected from a ventilated improved pit latrine in Durban, South Africa. It was analysed gravimetrically in six replicates using Method <IP_ADDRESS>. The average COD (g/g wet sample) was 0.23. The results for TS and moisture content are presented in Table 8.4 (source: unpublished data UKZN PRG).

## **<IP_ADDRESS> Volatile and fixed solids – ignition method5** <IP_ADDRESS>.1 Introduction

The dry sample residue from Method <IP_ADDRESS> is ignited at 550 °C for 30 min or until constant weight. The remaining ash represents the fixed (inorganic) solids, while the weight lost on ignition represents the volatile solids (organic matter) in faecal sludge. For more details, see Chapter 2.

## <IP_ADDRESS>.2 Safety precautions

 General health and safety (H&S) procedures specific for conducting laboratory analysis of faecal sludge are presented in Section 8.2. Before conducting this method, it is important to be familiar with Section 8.2.3 to ensure safety measures are properly carried out.

4 https://doi.org/10.25678/0000tt.

5 This method follows Method 2540E of the Standard Methods for the Examination of Water and Wastewater, and should be cited as: Rice *et al.*, (2017)

#### <IP_ADDRESS>.3 Apparatus and instruments

#### <IP_ADDRESS>.4 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

weigh. Adjust the balance manually if necessary. Make sure to use a standard weight of a mass similar to the mass of the expected sample + crucible.

#### <IP_ADDRESS>.5 Sample preservation

Samples should be analysed as soon as possible. If samples cannot be analysed immediately, they should be stored in a refrigerator at 4 °C for no longer than 7 days and, if TSS or VSS analysis is conducted, no longer than 48 hours. Before starting the analysis, let the samples return to room temperature (20 °C). Do not freeze the samples.

#### <IP_ADDRESS>.6 Sample preparation

Dry the samples to constant weight to remove moisture content, following Method <IP_ADDRESS>.

## <IP_ADDRESS>.7 Analysis protocol *Preparation of equipment*

## *Procedure*

 Ignite the residue from the TS in a muffle furnace at a temperature of 550 °C for 20 min. Note: for some solid and semi-solid faecal sludge samples, 20 min might not be enough, as they might need more time to combust all the volatile matter. For each type of sludge, check that constant weight is achieved after 20 min. To do this, cool and weigh the sample as described below, place the sample back in the furnace for 10 min and cool and weigh again. Repeat the steps of drying, cooling and weighing until a constant weight is obtained, or until weight change is less than 4% of the previous measurement. The length of combustion time needs to be evaluated for each specific type of sample, and revisited periodically.

#### <IP_ADDRESS>.8 Calculation

*Liquid and slurry samples (volumetric method):*  Volatile solids in wet sample (g/L) =

$$\frac{(\text{W}\_{\text{3}}\text{ (g)}\text{-W}\_{\text{4}}\text{ (g)}) \times 1,000}{\text{V}\_{\text{sample}}\text{(mL)}}$$

Fixed solids in wet sample (g/L) =

$$\frac{\left(\text{W}\_4\text{(g)-W}\_1\text{(g)}\right) \times 1,000}{\text{V}\_{\text{sample}}\text{ (mL)}}$$

Where:

W1 = Crucible mass (g) W2 = Crucible mass + wet sample mass (g) W3 = Crucible mass + sample after drying (g) W4 = Crucible mass + sample after incinerating (g) (W3-W1) = Sample mass after drying (g) (W4-W1) = Sample mass after incinerating (g) Vsample = Sample volume used (mL)

*Slurry, semi-solid and solid samples (gravimetric method):*

Volatile solids in wet sample (g/g) =

 (W3 ሺgሻ-W4(g)) (W2(g)- W1 (g))

Volatile solids in dry sample (g/g) =

(W3 ሺgሻ-W4 (g)) (W3 ሺgሻ-W1(g))

Volatile solids ሺ%TSሻ =

$$\frac{\left(\mathrm{W\_3(g)-W\_4(g)}\right)}{\left(\mathrm{W\_3(g)-W\_1(g)}\right)} = \frac{\mathrm{VS}\left(\frac{\mathrm{g}}{\mathrm{g}}\right)}{\mathrm{TS}\left(\frac{\mathrm{g}}{\mathrm{g}}\right)} \times 100\left(\%\right)$$

Fixed solids in wet sample (g/g) =

$$\frac{\left(\mathbf{W\_{4} (g)-W\_{1} (g))}\right)}{\left(\mathbf{W\_{2} (g)-W\_{1} (g))}\right)}$$

Fixed solids in dry sample (g/g) =

$$\frac{\left(\mathbf{W}\_4(\mathbf{g})\text{-W}\_1(\mathbf{g})\right)}{\left(\mathbf{W}\_3(\mathbf{g})\text{-W}\_1(\mathbf{g})\right)}$$

Fixed solids ሺ%TSሻ =

$$\frac{\left(\text{W}\_4\text{(g)-W}\_1\text{(g)}\right)}{\left(\text{W}\_3\text{(g)-W}\_1\text{(g)}\right)} = \frac{\text{Fixed solids}\left(\frac{\text{g}}{\text{g}}\right)}{\text{TS}\left(\frac{\text{g}}{\text{g}}\right)} \times 100\left(\%\right)$$

Note: for values of W1 to W3 and how to calculate them, see Method <IP_ADDRESS>.

#### <IP_ADDRESS>.9 Data set example

A faecal sludge sample was collected from a ventilated improved pit latrine in Durban, South Africa. It was analysed in six replicates using Method <IP_ADDRESS>. The average initial samples mass was 5 g dry weight - the same dry samples from Section <IP_ADDRESS>.9 were used for ignition. The average VS (g/g dry sample) was 0.56. The results for VS and fixed solids are presented in Table 8.5 (source: UKZN PRG).

**Table 8.5** Mass of samples before and after the analysis for the ignition method.

## **<IP_ADDRESS> Total suspended solids and total dissolved solids – oven drying method6**

#### <IP_ADDRESS>.1 Introduction

The TSS method is used to determine the efficiency of treatment technologies, such as settling tanks and biological filters. The measured volume of a thoroughly-mixed sample is vacuum-filtered through a dried, pre-weighed glass fibre filter. The filters and residue are then dried to a constant weight at 103-105 °C. The increase in weight of the filter represents the total suspended solids. Total dissolved solids are the TS minus the TSS.

For faecal sludge, clogging of the filters is a common problem. For this reason, this method is only suitable for liquid and slurry samples. If clogging occurs, the method can be adapted by dilution of the sample and/or choosing a larger pore size (maximum up to 2.0 μm), but needs to be carefully documented.

#### <IP_ADDRESS>.2 Safety precaution

#### <IP_ADDRESS>.3 Apparatus and instruments

#### <IP_ADDRESS>.4 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

6 This method is adapted from Method 2540D of the Standard Methods for the Examination of Water and Wastewater, and

should be cited as: Rice *et al.* (2017) as adapted in Velkushanova *et al*. (2021).

while cooling down in the desiccator. Routinely dry the desiccant in the oven at 105 °C (or at a different temperature, depending on the manufacturer's instructions), prior to the colour indicating that the desiccant is nearly saturated.

## <IP_ADDRESS>.5 Sample preservation

Samples should be analysed as soon as possible. If samples cannot be analysed immediately, they should be stored in a refrigerator at 4 °C for no longer than 48 hours. Before starting the analysis, let the samples return to ambient temperature. Do not freeze the samples.

## <IP_ADDRESS>.6 Sample preparation

## <IP_ADDRESS>.7 Analysis protocol *Equipment preparation*

potential residual organic material from previous measurements. Only porcelain crucibles should be used (see Method <IP_ADDRESS>).

## *Procedure*

## <IP_ADDRESS>.8 Calculation

Total suspended solids (g/L) =

ሺW2(g)-W1(g)ሻ Vsample(L) (× DF if using dilution factor)

Vsample = Volume of sample used (L)

DF = Dilution factor

Total dissolved solids ሺg L⁄ ሻ ൌ

Total solids ( g L⁄ ) ̶ Total suspended solids ( g L) ⁄

## <IP_ADDRESS>.9 Data set example

A faecal sludge sample was collected from a ventilated improved pit latrine in Durban, South Africa. It was analysed in six replicates using Method <IP_ADDRESS>. The average TSS (g/L) was 0.37. The results for TSS are presented in Table 8.6 (source: unpublished data UKZN PRG).

**Table 8.6** Total suspended solids obtained by oven drying methods.

## **<IP_ADDRESS> Volatile suspended solids – ignition method7** <IP_ADDRESS>.1 Introduction

The dry sample residue from Method <IP_ADDRESS> is ignited at 550 °C to constant weight. The remaining ash represents the fixed (inorganic) suspended solids, while the weight lost on ignition represents the volatile suspended solids (organic matter).

## <IP_ADDRESS>.2 Safety precaution

direct contact with hot surfaces. Always wear heat resistant gloves suitable for withstanding high temperatures.

## <IP_ADDRESS>.3 Apparatus and instruments

## <IP_ADDRESS>.4 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

7 This method follows methods 2540D and E of the Standard Methods for the Examination of Water and Wastewater, and should be cited as: Rice *et al.*, (2017).

magnesium, chloride and sulphate can be hygroscopic and require prolonged drying, proper desiccation, and rapid re-weighing.

#### <IP_ADDRESS>.5 Sample preservation

Samples should be analysed as soon as possible. If samples cannot be analysed immediately, they should be stored in a refrigerator at 4 °C for no longer than 48 hr. Before starting analysis, let the samples return to ambient temperature. Do not freeze the samples.

#### <IP_ADDRESS>.6 Sample preparation

Filter and dry the samples to constant weight, following Method <IP_ADDRESS>.

## <IP_ADDRESS>.7 Analysis protocol

## *Preparation of equipment*

#### *Procedure*

 Weigh the crucible on the analytical balance as soon as it has cooled to ambient temperature and record the weight (W3).

#### <IP_ADDRESS>.8 Calculation

Volatile suspended solids in wet sample (g/L) =

$$\frac{\text{(W}\_2\text{ (g)-W}\_3\text{ (g))}}{\text{V}\_{\text{sample (L)}}} \text{(}\times \text{DF, if used)}$$

#### <IP_ADDRESS>.9 Data set example

A faecal sludge sample was collected from a ventilated improved pit latrine in Durban, South Africa. It was analysed in six replicates using Method <IP_ADDRESS>. The average VSS (g/L) was 0.03. The results for VSS are presented in Table 8.7 (source: unpublished data UKZN PRG).

**Table 8.7** Volatile suspended solids obtained by oven drying and ignition methods.

## **<IP_ADDRESS> Total solids and moisture content – thermal balance (moisture analyser) method8**

#### <IP_ADDRESS>.1 Introduction

A moisture analyser is designed to determine relative moisture content in small samples of various substances, by measuring the change of weight due to water evaporation during convective drying. This method is applicable for all types of sludge – liquid, slurry, semi-solid and solid; however, samples with a higher moisture content will have a longer drying and measurement time. Method <IP_ADDRESS> and Method <IP_ADDRESS> are equally suitable to determine TS and moisture content, and should be selected depending on the availability of equipment.

## <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Apparatus and instruments

#### <IP_ADDRESS>.4 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

#### <IP_ADDRESS>.5 Sample preservation

Samples should be analysed as soon as possible. If samples cannot be analysed immediately, they should be stored in a refrigerator at 4 °C for no longer than 7 days. Before analysis, let the samples return to ambient temperature. Do not freeze the samples.

#### <IP_ADDRESS>.6 Sample preparation

Thoroughly mix all the samples using a stainless steel rod (or other appropriate tool) in order to have representative samples. If desired, samples can also be blended (see Section 8.4.2).

## <IP_ADDRESS>.7 Analysis protocol *Equipment preparation*

#### *Procedure*

8 This method follows Method B of the ASTM Standard Test Method for Determination of Total Solids in Biomass (E1756- 08) and should be cited as: ASTM (2015).

and a flashing icon to indicate that the machine is ready for loading the sample.

## **<IP_ADDRESS> Sand content9**

#### <IP_ADDRESS>.1 Introduction

The residual ash from Method <IP_ADDRESS> is washed with 0.1 M HCl and combusted at 550 ºC. The leftover residual is silica, and is typically reported as the 'sand' content as an indicator of soil content in faecal sludge.

#### <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

#### <IP_ADDRESS>.4 Required apparatus and instruments

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

9 This method is from the German Association for Water Wastewater and Waste guidelines, and should be cited as:

Method DWA-M 383 for the determination of sand content (DWA, 2008).

#### <IP_ADDRESS>.6 Sample preservation

Samples have to be 100 % dry and should be stored in a desiccator after volatile solids analysis (Method <IP_ADDRESS>) until the start of the sand content analysis.

#### <IP_ADDRESS>.7 Sample preparation

Sand content analysis is performed on the 100% dry ash remaining from the volatile solids analysis (Method <IP_ADDRESS>).

### <IP_ADDRESS>.8 Analysis protocol

paper. Wash the residue on the paper filter with the 0.1 M HCl solution. Keep rinsing until the mass of residue stays constant and the filtrate is clear. If the filtrate is clear from the beginning, rinse at least three times with an aliquot of HCl solution.

<IP_ADDRESS>.9 Calculations *Liquid and slurry samples (volumetric method):*  Sand content ሺ g L ሻ =

$$\frac{\text{(W2 (g) - W1 (g))}}{\text{Sample volume from TS analysis (mL)}} \times 1,000 \, (\frac{\text{mL}}{\text{L}})$$

$$\text{Sand content (\\$\circledast\text{TS})} = \text{}$$

$$\frac{\text{Sand content}\left(\frac{\text{g}}{\text{L}}\right)}{\text{TS}\left(\frac{\text{g}}{\text{L}}\right)} \times 100\left(\%\right)$$

Sand content ሺ% ashሻ =

$$\frac{\text{Sand content}\left(\frac{\text{g}}{\text{L}}\right)}{\text{Ash}\left(\frac{\text{g}}{\text{L}}\right)} \times 100\left(\%\right)$$

*Slurry, semi-solid, and solid samples (gravimetric method):*

Sand content of wet weight ൬ g g ൰ =

൫W2 ሺgሻ ̶ W1 ሺgሻ൯ Wet sample weight from TS analysis (g)

Sand content of dry weight ൬ g g ൰ =

$$\frac{\text{(W2 (g) - W1 (g))}}{\text{Weight of dry solids from TS analysis (g)}}$$

Sand content ሺ%TSሻ =

$$\frac{\text{Sand content}\left(\frac{\text{g}}{\text{g}}\right)}{\text{TS}\left(\frac{\text{g}}{\text{g}}\right)} \times 100\left(\%\right)$$

W1 = Weight of dried filter + crucible W2 = Weight of filter + crucible + residue

#### <IP_ADDRESS>.10 Data set example

In Gold *et al.* (2017), 73 faecal sludge samples from septic tanks, lined pit latrines, unlined pit latrines and johkasou10 tanks in Uganda, Vietnam and Japan, and 18 samples from wastewater treatment plants in Switzerland, were analysed for sand content following Method <IP_ADDRESS>. The results were used to determine influence on dewaterability. They report a 75% confidence interval (where 75% of the data is expected to lay) ranging from 7-9% sand of TS for lined johkasou septic tanks from Japan, 9-33% sand of TS for wastewater sludges from Switzerland, to a maximum of 45-69% sand of TS from unlined pit latrines in Uganda. When higher %TS of dewatered sludge are due to high sand content, it can have a negative impact on resource recovery.

## **8.6.2 Chemical oxygen demand (COD)**

Chemical oxygen demand (COD) is defined as the amount of oxygen that is required for the chemical oxidation of organic matter using a strong chemical oxidant. For faecal sludge, it is used as a proxy for the organic matter content of a sample. For example, it is measured to characterise sludge prior to treatment, or during monitoring of treatment processes. COD is one of multiple parameters to measure organic content of a sample. Other parameters include biochemical oxygen demand (BOD), volatile fatty acids (VFA), total organic carbon (TOC), and biomethane potential (BMP), which are not yet included in this book. Common perception in the sector, is that COD is more accurate than BOD for faecal sludge analysis, due to the high variability and concentrations of organic matter. More information on these methods can be found in Rice *et al.* (2017).

## **<IP_ADDRESS> Chemical oxygen demand – closed reflux spectrophotometric method11**

#### <IP_ADDRESS>.1 Introduction

Chemical oxygen demand (COD) is a proxy measurement of the amount of organic matter in water or wastewater. The principle of the COD test is that organic compounds are oxidised to carbon dioxide with a strong oxidising agent such as dichromate under acidic conditions. The amount of oxidising agent required to completely oxidise the organic matter in the sample is compared to the equivalence of oxygen. COD can be determined by an open reflux method, a closed reflux titrimetric method (Method <IP_ADDRESS>) or a closed reflux colorimetric method. The method described here is the closed reflux colorimetric method which uses a spectrophotometer to measure absorbance. The principle is that when a sample is digested, the dichromate ion oxidises COD in the sample and the chromium is reduced from Cr6+ to Cr3+. These chromium species are coloured and absorb in the visible region of the spectrum. Dichromate ion (Cr2O7 2–) is extremely absorbent in the 400-nm region, whereas the chromic ion (Cr3+) is extremely absorbent in the 600-nm region.

Commercial test kits based on standard methods for measuring COD are available, with pre-packaged individual aliquots of the necessary chemical in pillows (dry chemicals) and vials (liquid chemicals). Commonly used COD test kits from manufacturers such as Hach and Merck employ the closed reflux colorimetric method.

The example provided here is the Merck COD spectrophotometric testD (Merck, 2020d) for samples with concentrations of 15-300 mg COD/L, and it is based on the manufacturer's protocol for water and wastewater using the standard method 5220 (Rice *et al.,* 2017). For faecal sludge, samples must be diluted

10 Johkasou is the Japanese word for on-site wastewater treatment.

11 This method should be cited as: Method 5220 D (Rice *et al.,* 2017), and if test kits are used, also as per the manufacturer's directions, including any modifications.

to prevent false high readings associated with turbid solutions.

## <IP_ADDRESS>.2 Safety precautions

## <IP_ADDRESS>.3 Required chemicals

Distilled water

## <IP_ADDRESS>.4 Required apparatus and instruments

## <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

serial dilutions based on the type of sludge for accurate measurements.

## <IP_ADDRESS>.6 Sample preservation

## <IP_ADDRESS>.7 Sample preparation

#### *For liquid, slurry, semi-solid or solid samples:*

 Samples containing concentrations of COD beyond the range of the test kit must be diluted appropriately with distilled water, following serial dilutions.

## *For slurry to solid samples with an estimated range of 15-300 mg COD/L:*

#### <IP_ADDRESS>.8 Analysis protocol

## *Calibration*

Follow the spectrophotometer manufacturer's instructions for calibration, since calibration procedure differs between instruments. It may be necessary to calibrate the instrument before every reading, or it may only be necessary to perform periodic calibration checks to determine when calibration is necessary.

## <IP_ADDRESS>.9 Procedure

## *This method is for a measuring range of 15-300 mg/L COD.*

<IP_ADDRESS>.10 Calculation *Liquid and slurry samples:*  Result of analysis (mg/L)

$$\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right) = \text{A } \left(\frac{\text{mg}}{\text{L}}\right) \ge \text{DF}$$

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

$$\mathbf{A} = \begin{array}{c} \text{Spectrophotometric measurement value } (\text{mg/L}) \end{array}$$

DF = Dilution factor (F/I)

F = Final diluted volume (L)

I = Initial sample volume (L)

*Slurry, semi-solid and solid samples:*

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right) \times \frac{\text{V}}{\text{M}} (\frac{\text{L}}{\text{g}})}{\text{Total solids content} \left(\frac{\text{g}}{\text{g}}\right)}$$

Where:

#### <IP_ADDRESS>.11 Data set example

COD is measured in most characterisation studies for faecal sludge and it is essential for the design of faecal sludge treatment plants. In a study to quantify and characterise faecal sludge from urban areas in Douala, Cameroon, Maffo *et al.,* (2019) used the COD spectrophotometric test kit to measure the COD of seven faecal sludge composite samples collected within a day from trucks at a dumping site in Douala. The COD concentrations measured ranged from of 28,900-73,150 mg/L with an average of 39,925 mg/L and a standard deviation of 15,034 mg/L.

Similarly, Semiyaga *et al*. (2016) used the COD spectrophotometric method in the characterisation of faecal sludge from lined and unlined pit latrines. For 22 faecal sludge samples from lined pit latrines, the average COD was 65,521 ± 43,960. The 10 unlined pit latrines sampled had an average COD of 132,326 ± 43,786 mg/L. Variability in COD concentration was evident in the 3 settlements where samples were taken, with an average COD of 107,137 ± 32,542 mg/L, 75,120 ± 28,778 mg/L, and 20,794 ± 8,456 mg/L for the Bwaise II, Kibuye and Kamwokya settlements, respectively.

Gold *et al*. (2017) also determined the COD concentration of faecal sludge and wastewater samples from different types of containments in Uganda, Vietnam, Japan and Switzerland in a crosscountry analysis of FS dewatering. The results of COD for septic tanks in Uganda, septic tanks in Vietnam, johkasou systems (onsite technologies) in Japan and digested wastewater sludge in Switzerland were in the range of 7.2-21.5 g/L, 15.3-31.5 g/L, 10.1- 16.5 g/L and 17.0-22.1 g/L, respectively. Using percentage TS (% TS) after centrifugation as a metric of dewaterability, it was observed that COD correlated to a decreased dewaterability (decreased final % TS), a relationship that has also been observed in wastewater.

## **<IP_ADDRESS> Chemical oxygen demand – closed reflux titrimetric method12**

#### <IP_ADDRESS>.1 Introduction

A sample is digested at 150 ºC for 2 hours in a concentrated dichromate solution, using silver sulphate as a catalyst and mercuric sulphate as a masking agent to prevent chloride interference. The dichromate will be partially reduced by the oxidisable material present in the sample. The excess dichromate is then titrated with ammonium iron (II) sulphate and the COD value calculated from the amount of dichromate used.

The half reaction for the reduction of dichromate is:

$$\mathrm{CrzOr}^{2-} + 14\,\mathrm{H}^{+} + 6\mathrm{e}^{-} \to 2\,\mathrm{Cr}^{3-} + 7\,\mathrm{HzO}$$

The remaining dichromate is titrated with a standard ammonium iron (II) sulphate solution:

$$\text{CrzO}\text{r}^{2-} + 6\text{ Fe}^{2+} + 14\text{ H}^{\*} \to 6\text{ Fe}^{3+} + 7\text{ HzO} + 2\text{ Cr}^{3+} $$

The equivalence point is indicated by a sharp colour change from blue-green to reddish brown as the ferroin indicator undergoes reduction from iron (III) to the iron (II) complex.

## <IP_ADDRESS>.2 Safety precautions

12 This method is adapted from Method 5220C of the Standard Methods for the Examination of Water and Wastewater and should be cited as: Rice *et al.* (2017), as described in Velkushanova *et al.* (2021).

## <IP_ADDRESS>.3 Required chemicals

## *Standard potassium dichromate (K2Cr6O) - digestion solution: 0.13 M*

## *Sulphuric acid (H2SO4)/silver sulphate reagent (Ag2SO4) - COD reagent*

## *Ferroin indicator*

## *Ferrous ammonium sulphate (FAS) (Fe(NH4)2(SO4)2ꞏ6H2O): 0.10 M*

## *Potassium hydrogen phthalate (KHP) - standard solution*

## <IP_ADDRESS>.4 Required apparatus and instruments

Pestle and mortar

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

and reagent volumes, consult Table 5220:I in Rice *et al*. (2017).

 Rinse the glassware with 20% H2SO4 to remove any organic residues from previous use.

## <IP_ADDRESS>.6 Sample preservation

## <IP_ADDRESS>.7 Sample preparation

*This method is valid within a concentration range of 40-3,600 mgCOD/L*

## *For liquid, slurry, semi-solid or solid samples:*

 Samples containing concentrations of COD beyond the concentration range must be diluted appropriately with distilled water, following serial dilutions. The sample needs to be homogenised thoroughly (with a blender or stirring rod) before diluting.

## *For slurry to solid faecal sludge:*

## <IP_ADDRESS>.8 Analysis protocol

*Preparation of reagents, chemicals and standard solutions*

	- Dry K2Cr6O7 in the oven (150 °C) for 2 hr and let it cool to ambient temperature in a desiccator. Afterwards, weigh 39.93 g of dried K2Cr6O7 and transfer into a 1 L volumetric flask, and add approximately 500 mL distilled water.
	- To this volumetric flask, add 167 mL of 32% concentrated sulphuric acid (H2SO4) and 13.3 g mercuric sulphate (HgSO4). This reaction produces heat!
	- Completely dissolve the mercuric sulphate by swirling the volumetric flask or by using a magnetic stirrer. Allow the flask contents to cool to room temperature and then dilute the mixture with distilled water to the mark of the volumetric flask.
	- Add 26 g silver sulphate crystals or powder to the 2.5 L volumetric flask of concentrated sulphuric acid while mixing using a magnetic stirrer.
	- Leave for 2 days to dissolve and mix well before use.
	- Dissolve 1.485 g 1,10-phenentroline monohydrate and 0.695 g ferrous sulphate (FeSO4ꞏ7H2O) in distilled water and dilute to 100 mL in a 100 mL volumetric flask.
	- Dissolve 39.2 g Fe(NH4)2(SO4)2ꞏ6H2O in distilled water.
	- Add 20 mL of 98% concentrated sulphuric acid and dilute to 1 L in a volumetric flask.

## *Calibration*

#### *Standardisation*

Use a standard K2Cr2O7 digestion solution (daily, or before every analysis set) to correct any variation in the concentration of the FAS, following the COD measurement procedure below. The molarity of the FAS is calculated by the following equation:

Molarity of FAS =

Volume K2Cr2O7 solution titrated (mL) Volume FAS used in titration (mL)× 0.10

## *Procedure*

## *Equipment preparation*

 If using a heating block for digestion, preheat the heating block to 150 °C.

*Sample digestion*

## *Titration procedure*

#### <IP_ADDRESS>.9 Calculations

Molarity of FAS =

Volume K2Cr2O7 solution titrated (mL) Volume FAS used in titration (mL)× 0.10

COD ሺmg/Lሻ =

$$\left(\frac{\text{Volume FAS used in blank titration (mL)}}{\text{Sample volume (mL)}}\right) \times 1$$

(Molarity of FAS × 8,000 × DFሻ ̶

൬ Volume FAS used in titration (mL) Sample volume ሺmLሻ ൰×

ሺMolarity of FAS × 8,000 × DFሻ

COD on dry basis ሺg/gሻ =

COD ሺmg/Lሻ × 0.001

Total solids (g/g)

Where:

#### <IP_ADDRESS>.10 Data set example

In Ward *et al*. (2021), COD was measured using the closed reflux titrimetric method for 465 faecal sludge samples from 421 pit latrines and septic tanks in Lusaka, Zambia. For septic tanks, the median COD was 53.3 g/L and the average COD was 72.1 g/L, with a standard deviation of 56.9 g/L. For pit latrines, the median COD was 121.1 g/L and the average COD was 122.6 g/L, with a standard deviation of 65.5 g/L. The heterogeneity of faecal sludge is reflected in the high standard deviations. As a quality control measure, laboratory triplicates were done for every 10th sample. The relative standard error on the replicates was 8%. The entire raw data set is included with publication13.

Two faecal sludge samples were collected in Durban, South Africa: one from a ventilated improved pit latrine and another one from a urine diversion toilet. These were analysed in four replicates using Method <IP_ADDRESS>. The average COD (g/g dry sample) were 0.77 and 0.64 for VIP and UDDT toilets, respectively. The results for COD are presented in Table 8.8 (source: unpublished data UKZN PRG).

## **8.6.3 Fat and fibre**

Crude fat and crude fibre are components of the organic matter in a sample. Characterisation of crude fat and crude fibre content is useful in assessing the resource recovery potential of faecal sludge. Crude protein is not covered in this section, but may also be of interest for characterising samples for resource recovery applications. Methods for characterisation for crude protein can be found in FAO (2011) and Regulation (EC) No 152/2009 – Determination of the Content of Crude Protein. These methods, however, could require additional method development for faecal sludge samples.

## **<IP_ADDRESS> Crude fat – Soxhlet extraction method14**  <IP_ADDRESS>.1 Introduction

In the proximate system of analysis, crude fat is the fraction of the sample that is soluble in organic solvents, such as ether. The crude fat fraction contains mostly fatty acids, but can also include waxes. Crude fat can be used to characterise resource recovery end products from faecal sludge treatment, for example black soldier fly larvae, to determine their value as animal fodder and/or use as bio-oil or a feedstock for biodiesel. Crude fat can also be measured directly on dewatered or dried faecal sludge to determine its suitability as a feedstock or co-feedstock for larvae rearing or vermiculture. Since several lipid extraction methods may be considered, the source material and lipid type present in the sample play an important role

13 https://doi.org/10.25678/00037X

14 This method is adapted from Crude Fat - Ether Extract Method (FAO, 2011) including recommendations from Regulation (EC) No 152/2009 Determination of Crude Oils and Fats. This

method should be cited as: Crude Fat - Ether Extract Method (FAO, 2011) as adapted in Velkushanova *et al.* (2021).

in determining the appropriate extraction method. The most widely used is the Soxhlet extraction method. This method uses a selected solvent of choice, such as petroleum ether, to extract fat from a dried, ground sample.  The fat is then dried and quantified by weighing, and is reported as a mass fraction (% crude fat).

#### <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

#### <IP_ADDRESS>.4 Required apparatus and instruments

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operation conditions, and interferences that are specific to this method include:

#### <IP_ADDRESS>.6 Sample preservation

To preserve, samples can be freeze-dried, oven-dried at 100 ± 3 °C, or dried in a vacuum oven at 80 ± 2 °C to a constant weight, following Method <IP_ADDRESS>. Freeze-dried or oven-dried samples can be stored in a cool, dry place for an extended period of time.

#### <IP_ADDRESS>.7 Sample preparation

 Determine the amount of faecal sludge required for analysis - *e.g.* 5.0 g of dried, ground sludge is required per crude fat analysis. It is recommended to prepare at least 11 g of dried, ground sludge to allow for duplicate measurements and determination of the moisture content of a dried sample. Ensure that a sufficient amount of faecal sludge is dried to allow for at least 11 g of dried, ground sample, taking into account that some sample may be lost during the grinding and sieving. The amount of wet sludge required will be dependent on the TS of the sample.

#### <IP_ADDRESS>.8 Analysis protocol

#### *Calibration*

Standard curve and error estimation:

## *Hydrolysis pre-treatment (optional, based on sample characteristics)*

conical flask. Cover the beaker with a watch glass or connect a reflux condenser to the conical flask.

## *Extraction*

 Turn off the heating mantle and allow the 250 mL round-bottomed flask to cool down to room temperature.

### *Drying*

#### <IP_ADDRESS>.9 Calculation

$$\text{\% crude fat} = \frac{\text{W}\_3 - \text{W}\_2}{\text{W}\_1} \times 100\%$$

Where:

W1 = Initial weight of sample (g)

#### <IP_ADDRESS>.9 Data set example

Gold *et al*. (2020) quantified lipids in human faeces to assess their potential as a feedstock for black soldier fly larvae rearing. Lipids were extracted from freezedried samples using ether extraction with petroleum ether at 40-60 °C following hydrolysis with 3 M hydrochloric acid following Regulation (EC) No 152/2009 Determination of Crude Oils and Fats. Crude fat content in human faeces was measured at 20.9 % of the dry mass. Nyakeri *et al*. (2019) used ether extraction to quantify crude fat in black soldier fly larvae reared using faecal sludge as a feedstock. Larvae were dried and ground, and the residue was dried at 110 °C after extraction, instead of the 80 °C specified in this method. Analyses were performed in triplicate. The average crude fat content in larvae fed with faecal sludge was reported as 184 ± g/kg, which can be converted to 18.4% of dry mass.

Cooling water for circulation in condenser

Soxhlet with thimble

Condenser Heating mantle with roundbottomed flask

**Figure 8.4** Soxlet setup (photo: UKZN PRG).

## **<IP_ADDRESS> Crude fibre – filtration method15**

<IP_ADDRESS>.1 Introduction Determinations of crude fibre are used for quality

control and specifications of animal feeds by regulatory agencies (Mertens, 2003). This method involves the extraction of fibre from a defatted sample using dilute sulphuric acid followed by potassium hydroxide solution. The mass difference after extraction, filtration, and incineration in a muffle furnace is equal to the mass of crude fibre (reported as % crude fibre). While this method is the standard for regulatory agencies, and is robust and easy to measure for many feedstocks and in many laboratories, crude fibre is not reflective of actual available dietary fibre, and does not fully capture all cellulose, pentosans, and lignins in the sample. Other methods of characterising dietary fibre may be more appropriate, depending on the purpose of characterisation, including acid detergent fibre (ADF), neutral detergent fibre (NDF), and total dietary fibre (TDF). These methods are described in detail in FAO (2011) and Novotny *et al*. (2017).

This method is applicable for determination of samples with a crude fibre content higher than 1%. If the sample contains >10% fat, the fat must first be extracted with petroleum ether prior to beginning the analysis. Fat extraction can be performed as part of the characterisation of crude fat, described in Method <IP_ADDRESS>. The defatted sample from the crude fat analysis can be subsequently analysed for crude fibre following this method.

## <IP_ADDRESS>.2 Safety precautions

15 This method is adapted from the Crude Fibre – Filtration Method described in the FAO Quality Assurance for Animal Feed Analysis Laboratories Manual (FAO, 2011) including

suggestions from Novotny *et al*. (2017). This method should be cited as: Crude Fibre – Filtration Method (FAO, 2011) as adapted in Velkushanova *et al.* (2021).

## <IP_ADDRESS>.3 Required chemicals

	- Potassium hydroxide (KOH) pellets
		- Distilled water
	- Concentrated sulphuric acid (H2SO4) (98%)
	- Distilled water

## <IP_ADDRESS>.4 Required apparatus and instruments

## <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

283

16 https://ehs.berkeley.edu/workplace-safety/glove-selectionguide#comparison

additional trials with specific sludge to be analysed may be necessary to determine the best pore size for a specific sample. The pore size of the Gooch crucibles should be reported with the results. Filtration of a sample should not take more than 10 min. Information about modifications to this method to deal with difficult-to-filter samples can be found in Novotny *et al*. (2017).

## <IP_ADDRESS>.6 Sample preservation

To preserve them, samples can be freeze-dried or oven-dried (at 80 °C if crude fat will also be analysed in the sample, otherwise dry at 103 °C) to constant weight, following Method <IP_ADDRESS>. Freeze-dried or oven-dried samples can be stored for an extended period in a cool, dry place.

## <IP_ADDRESS>.7 Sample preparation

representative samples. If desired, samples may also be blended (see Section 8.4.2).

#### <IP_ADDRESS>.8 Analysis protocol

## *Preparation of reagents, chemicals, and standard solutions*

	- Add 12.9042 g KOH pellets to a 1 L volumetric flask.
	- Fill to the 1 L mark with distilled water and swirl to fully dissolve the KOH pellets.
	- Add some distilled water to a 1 L volumetric flask.
	- Add 8.1582 mL concentrated sulphuric acid (98%) to the 1 L volumetric flask.
	- Top up the 1 L volumetric flask to the mark with distilled water.

#### *Preparation of equipment*

### *Defatting pre-treatment*

Perform defatting pre-treatment if the sample has more than 10% fat, and fat has not already been extracted for the crude fat analysis.

## *Digestion and filtration*

	- Transfer the residue quantitatively to a 500 mL beaker using hot distilled water.
	- Add 150 mL 0.15 M sulphuric acid.
	- Add a glass rod to avoid bumping, see Figure 8.5.
	- Boil on a heating mantle for 30 ± 1 min under a fume hood (maintain the volume with hot distilled water).
	- Leave to cool.
	- Filter through the Gooch crucible using the vacuum pump, see Figure 8.6.
	- Wash the residue 5 times, each time with 10 mL hot distilled water (check with litmus paper for neutrality).
	- Just cover the residue with acetone, and leave for a few minutes.
	- Apply slight suction to remove the acetone.
	- Transfer the residue quantitatively to a 500 mL beaker using hot distilled water.
	- Add 150 mL 0.23 M potassium hydroxide.
	- Add a glass rod to avoid bumping, see Figure 8.5.

## *Oven drying*

## *Muffle furnace*

**Figure 8.5** Acid‐base digestion setup (photo: UKZN PRG).

**Figure 8.6** Vacuum filtration setup (photo: UKZN PRG).

<IP_ADDRESS>.9 Calculation

% crude fibre =

Where:

W1 = Weight of the sample (g)

#### <IP_ADDRESS>.10 Data set example

An example of a different acid digestion fibre method for the determination of fodder quality for plants grown on planted drying beds is presented in Gueye *et al*. (2016). Acid digestion fibre (ADF) was measured by boiling a sample of forage in a detergent under acid conditions (pH = 2) and filtering the boiled sample through filter paper. In addition to fibre, TKN was determined by the Kjeldahl digestion method, crude protein (CP) was calculated as TKN × 6.25, crude cellulose (CC) was determined by first digesting in sulfuric acid (0.26 N), and then potassium hydroxide (0.23 N). TP was extracted by dry ashing in a muffle furnace diluted with an acid mix (HCL/HNO2) and analysed by the molybdate procedure.

## **8.6.4 Nitrogen**

Measurements of the different forms of nitrogen indicate the stabilisation and nutrient availability in treatment and resource recovery processes. Nitrogen is present in water and wastewater in different concentrations and chemical forms, including inorganic forms (ammonia, ammonium, nitrite and nitrate) and organic nitrogen. Organic nitrogen consists of a complex mixture of compounds including amino acids, amino sugars, and proteins. Total nitrogen consists of the sum of organic and inorganic fractions, and Kjeldahl nitrogen consists of organic nitrogen and ammonia/ammonium, which can be determined together analytically. In faecal sludge, nitrogen concentrations can be 10-100 times greater than observed in influent to sewer-based, municipal wastewater treatment plants, so special care needs to be taken with sample preparation and method selection. For more information on the nitrogen cycle, refer to Strande *et al*. (2014).

Ammonia exists either as free ammonia (NH3) or in the ionic form ammonium (NH4 + ). In aqueous solutions, a pH-dependent equilibrium exists between the two forms, and thus ammonia occurs partly in the form of ammonium and partly as free ammonia. In faecal sludge treatment processes, it is important to monitor ammonia measurements because high ammonia concentrations can inhibit biological processes, such as anaerobic digestion and stabilisation ponds. In planted drying beds, high ammonia concentrations can inhibit plant growth, which is important for treatment processes. The leachate from planted and unplanted drying beds typically also still has high concentrations of ammonia. Due to the ability to inhibit biological activity, ammonia concentrations can also be used to slow the growth of pathogens, and during alkaline stabilisation of faecal sludge, the addition of lime increases the pH, which results in greater NH3 concentrations (than NH4 + ) (Englund and Strande, 2019). In this book, methods for determination of ammonia include test strips, and spectrophotometric and titrimetric methods. Other methods not yet included in this book are the ammonia selective electrode method and flow injection analysis.

Nitrite, which is an intermediate of the oxidation of ammonia to nitrate (nitrification), is usually present in low to non-detectable concentrations in wastewater and faecal sludge. However, in the event of an imbalance in the nitrification process, nitrite may accumulate. Nitrite is toxic and effluents with high nitrite concentration can adversely affect aquatic life when dilution in the receiving water is insufficient. In aerobic treatment processes, accumulation of nitrite may signify deficiencies in the treatment process. In this book, methods for determination of nitrite include nitrite colorimetric and spectrophotometric methods.

Nitrate and ammonium are considered to be plantavailable nutrients. However, ammonia/ammonium is rapidly oxidised to nitrate in the environment, and nitrate can leach rapidly through soils. Nitrate becomes toxic and affects public health when it enters the food chain through contamination of surface or groundwater. Eutrophication and algal blooms of surface water are direct environmental impacts of pollution from excess nutrients. Nitrate management in faecal sludge treatment is thus essential to protect public and environmental health. In this book, methods for determination of nitrate concentrations include colorimetric and spectrophotometric methods.

## **<IP_ADDRESS> Total nitrogen – spectrophotometric method17**

Total nitrogen can be determined by several different methods, including in-line UV/persulfate digestion and oxidation with flow injection. The method described here is total nitrogen quantified with spectrophotometry, based on the oxidation of all the nitrogenous compounds with the persulfate digestion to nitrate, followed by a cadmium reduction process to measure the nitrate.

Commercial test kits based on standard methods for measuring total nitrogen are available, with prepackaged individual aliquots of necessary chemical in pillows (dry chemicals) and vials (liquid chemicals). Commonly used total nitrogen test kits from manufacturers such as Hach and Merck employ a variety of methods. The example provided here is the Hach total nitrogen spectrophotometric testD (Hach, 2020) for samples with concentrations of 2-150 mgN/L, which is based on the manufacturer's protocol for water and wastewater using the standard method 4500-N C (Rice *et al.,* 2017). For faecal sludge, samples must be diluted to prevent the false high readings associated with turbid solutions.

### <IP_ADDRESS>.1 Safety precautions

17 This method should be cited as: Method 4500-N C (Rice et al., 2017), and if test kits are used, also as per the manufacturer's directions including any modifications.

## <IP_ADDRESS>.2 Required chemicals

## <IP_ADDRESS>.3 Required apparatus and instruments

## <IP_ADDRESS>.4 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

 Perform quality control with the Hach ammonia nitrogen standard solution with every test batch (or on a daily or weekly basis, depending on the testing load).

Note: quality control for other methods can also include nitrate standard solutions.

concentrations refer to the manufacturer's instructions.

 Suspended solids and turbid solutions interfere with measurement, thus faecal sludge samples should be diluted with an appropriate dilution factor based on the type of sludge for accurate measurements. Always use serial dilutions.

## <IP_ADDRESS>.5 Sample preservation

## <IP_ADDRESS>.6 Sample preparation

## *For liquid, slurry, semi-solid or solid samples:*

## *For slurry to solid samples:*

 Total solids analysis should be performed on the samples so that the results of the total nitrogen measurement can be expressed on a mass per mass basis (gN/gTS).

## <IP_ADDRESS>.7 Analysis protocol *Calibration*

Follow the spectrophotometer manufacturer's instructions for calibration. It may be necessary to calibrate the instrument before every reading, or it may only be necessary to perform periodic calibration checks to determine when calibration is necessary. For this method, calibration can be performed as follows:

## *Procedure*

#### *For measuring the range of 2-150 mgN/L:*

## <IP_ADDRESS>.8 Calculations

## *Liquid and slurry samples:*

Result of analysis (mg/L) Final concentration ቀ mg L ቁ = Aቀ mg L ቁ × DF

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

$$\mathbf{A} = \begin{array}{c} \text{Spectrophotometric measurement value } (\text{mg/L}) \end{array}$$

DF = Dilution factor (F/I)

F = Final diluted volume (L)

I = Initial sample volume (L)

$$Sherry, \text{\textquotedblleft} semi-solid \text{\textquotedblright} and \text{\textquotedblleft} solid \text{\textquotedblright} samples; \text{\textquotedblleft}$$

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right) \times \frac{\text{V}}{\text{M}} (\frac{\text{L}}{\text{g}})}{\text{Total solids content} \left(\frac{\text{g}}{\text{g}}\right)}$$

Where:

$$\mathbf{A} = \begin{array}{c} \text{Spectrophotometric measurement value } (\text{mg/L}) \end{array}$$

V = Volume of dilution (L)

M = Wet weight of sludge used in sample preparation (g)

#### <IP_ADDRESS>.9 Data set example

While developing methods to reliably estimate faecal sludge qualities and quantities, Strande *et al*. (2018) measured the total nitrogen of faecal sludge (in addition to other physicochemical parameters) from pit latrines and septic tanks located in households, non-households, and public toilets. 180 samples were analysed, with an uneven distribution and high variability. Hence, the median rather than the mean of the results were used for the statistical analysis. In spite of the high variability in the results, as illustrated in Figure 8.7 of the TN results for pit latrines and septic tanks, there were differences based on households, non-households and public toilets, and the pit latrines in general had a higher TN concentration. The complete raw data set is available at the link provided below18.

**Figure 8.7** Total nitrogen for septic tanks (blue boxes) and pit latrines (green boxes) from households, non‐households, and public toilets (Strande *et al.,* 2018).

## **<IP_ADDRESS> Ammonium – colorimetric (test strip) method19**

#### <IP_ADDRESS>.1 Introduction

Measurement of ammonium is important for assessing the impact on receiving water bodies, evaluating treatment performance, and to check for inhibitory concentrations in treatment processes. In the test strip method, ammonium ions react with Nessler's reagent to form a yellow-brown compound. The concentration of ammonium is measured semi-quantitatively by visual comparison of the reaction zone of the test strip with the fields of a colour scale. This method is based on the specific manufacturer's protocol for water and wastewater. The example provided here is one of many ammonium colorimetric test methods for samples with concentrations of 10-400 mg/L NH4 + . Ammonium measurement results are expressed either as mg/l NH4 + or NH4-N, and it is important to note how the results are expressed by the selected test method. For faecal sludge, samples must be diluted to prevent masking of the resultant colour change.

#### <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

The required chemicals will be specific to each manufacturer's test kit; specific information on test kits can be found on the manufacturers' websites.

Distilled water

#### <IP_ADDRESS>.4 Apparatus and instruments

18 https://doi.org/10.25678/0000tt

19 This method should be cited as the specific manufacturer's method along with any modifications. The example used here is the Merck MQuant Ammonium Test (Merck, 2020j)D.

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

#### <IP_ADDRESS>.6 Sample preservation

Samples should be analysed as soon as possible after sampling. For analysis within 24 hours of collection, the sample must be refrigerated at 4 °C. Refrigeration reduces biological activity, which can increase NH4 + concentration.

#### <IP_ADDRESS>.7 Sample preparation

### *For liquid, slurry, semi-solid or solid samples:*

## *For slurry to solid samples:*

#### <IP_ADDRESS>.8 Analysis protocol

#### *For measuring range of 10-400 mgNH4 +/L:*

## <IP_ADDRESS>.9 Calculation *Liquid and slurry samples:*

## Result of analysis (mg/L)

Final concentration ቀ mg L ቁ = Aቀ mg L ቁ x DF

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Spectrophotometric measurement value (mg/L)

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids content } (\frac{\text{g}}{\text{g}})}$$

Where:

A = Spectrophotometric measurement value (mg/L)

V = Volume of dilution (L)

M = Wet weight of sludge used in sample preparation (g)

## **<IP_ADDRESS> Ammonium – phenate spectrophotometric method20**

#### <IP_ADDRESS>.1 Introduction

There are various methods to measure ammonia; selection of the appropriate method is based on the concentration and the presence of interferences. In this method, ammonia first reacts with hypochlorite to form monochloramine. Chloramine then reacts with phenate to form 5-aminophenate which is oxidised to indophenol, a reaction catalysed by sodium nitroprusside. Indophenol has an intense blue colour that is measured spectrophotometrically. This method measures both ammonium ions and dissolved ammonia in a concentration range of 2–150 mgNH4-N/L.

It is also possible to conduct this method with commercially available test kits. Commonly used test kits from manufacturers such as Hach, Merck, and Hanna employ different methods in the kits used for ammonia measurement; the Nessler method, the salicylate method and the phenate method are common methods. For example, the Hach test kit uses the salicylate method while Merck kits are based on the phenate method. The test kits are also based on standard methods, with pre-packaged individual aliquots of necessary chemical in pillows (dry chemicals) and vials (liquid).

The example provided here is the Merck ammonium spectrophotometric test kit (Merck, 2020f)D for samples with concentrations of 2-150 mgNH4-N/L, and uses the manufacturer's protocol for water and wastewater based on the standard method 4500 (Rice *et al*., 2017). For faecal sludge, samples must be diluted and filtered to prevent false-high readings associated with turbid solutions.

#### <IP_ADDRESS>.2 Safety precautions

## <IP_ADDRESS>.3 Required chemicals

Distilled water.

	- Reagent NH4-1 (supplied by the manufacturer).
	- Reagent NH4-2 (supplied by the manufacturer).

#### <IP_ADDRESS>.4 Required apparatus and instruments

20 This method should be cited as: Method 4500 (Rice *et al*., 2017), and if test kits are used, also the manufacturer's directions including any modifications.

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

#### <IP_ADDRESS>.6 Sample preservation

#### <IP_ADDRESS>.7 Sample preparation

## *For liquid, slurry, semi-solid or solid samples:*

 Turbidity will falsely increase the spectrophotometric reading, thus samples might have to be filtered to prevent false-high measurements. Report any preparation steps with the results.

 Samples containing more than 150 mgNH4-N/L must be diluted with distilled water.

### *For slurry to solid samples:*

## <IP_ADDRESS>.8 Analysis protocol *Calibration*

Follow the spectrophotometer manufacturer's instructions for calibration. It may be necessary to calibrate the instrument before every reading, or it may only be necessary to perform periodic calibration checks to determine when calibration is necessary. For this method, calibration can be performed as follows:

## *Procedure*

*For measuring range of 2.0–75 mgNH4-N/L (2.6–96.6 mgNH4 +/L):*

#### *For measuring ranges of > 75-150 mgNH4-N/L (> 96.6-193 mgNH4 +/L):*

#### <IP_ADDRESS>.9 Calculations

Result of the analysis (mg/L NH4-N) = measurement value, A (mg/L) × dilution factor

### *Liquid and slurry samples:*

Result of analysis (mg/L) Final concentration ቀ mg L ቁ = Aቀ mg L ቁ x DF

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Spectrophotometric measurement value (mg/L)

DF = Dilution factor (F/I)

F = Final diluted volume (L)

I = Initial sample volume (L)

$$Slurry,\ semi\text{-solid}\ and\ solid\ samples;$$

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids content} \left(\frac{\text{g}}{\text{g}}\right)}$$

Where:

A = Spectrophotometric measurement value (mg/L)

V = Volume of dilution (L)

$$\mathbf{M} = \begin{array}{c} \text{We get weight of sludge used in sample} \\ \text{preparation (g)} \end{array}$$

#### <IP_ADDRESS>.10 Data set example

In evaluating the effect of drying on the physical and chemical characteristics of faecal sludge for its reuse, Septien *et al*. (2020) measured the ammonium concentration of faecal sludge from pit latrines before and after drying at different temperatures using a Merck Spectroquant®,D. Raw faecal sludge samples had ammonium concentration of 24 ± 4 g/g TS. By using the convective drying rig, the ammonia concentration reduced to 4 ± 2 g/g TS when it was dried at 40 ° C.

Hach test kits using the salicylate method and a Hach-Lange DR2800 spectrophotometer, following the manufacturers' directions, were used to evaluate 60 samples in Hanoi, Vietnam, and 180 in Kampala, Uganda, for NH4 + -N concentrations. In Hanoi concentrations ranged from 54 to 1,700 NH4 + -N/L, and in Kampala, Uganda from 24 to 3,000 mgNH4 + -N/L (Englund *et al*., 2020; Strande *et al*., 2018). The complete raw data set is available using the link below21.

21 https://doi.org/10.25678/0000tt

## **<IP_ADDRESS> Ammonia – distillation and titration method22**

#### <IP_ADDRESS>.1 Introduction

Acid-base titrations are useful in determining concentrations of acids and bases in samples. An acid or a base is neutralised with a known volume of corresponding acid or base. The exact concentration of the titrant solution must be known in order to establish the concentration of the titrated solution. The endpoint of this reaction is when the solution reaches neutral pH, where the determinant in the sample is equivalent to the analyte, and is only visible when an indicator is used. This is used to calculate the concentration of other determinants from the known ratio of the reaction.

Ammonia exists either as free ammonia (NH3) or ammonium ions (NH4 + ). In aqueous solutions, a pHdependent equilibrium exists between the two forms and thus ammonia occurs partly in the form of ammonium ions and partly as free ammonia. Measurement of ammonia is important for assessing the impact on receiving water bodies, evaluating treatment performance and nutrient management, and checking for inhibitory concentrations in treatment processes. The sample is adjusted to pH 9.5 using a borate buffer to allow the measurement of organic nitrogen compounds. The sample containing ammonium nitrogen is distilled in boric acid and then titrated using 0.02N H2SO4 as follows:

H2SO4 + 2 NH4OH → 2 H2O+ (NH4)2SO4

This method is based on the Standard Methods for the Examination of Water and Wastewater (Rice *et al*., 2017). Based on local experience, the method might need to be adapted for different types of faecal sludge. For example, the UKZN PRG in Durban adapted the sample volume to 70 mL, based on the characteristics of faecal sludge in Durban and the semi-automated distillation setup used in the laboratory.

#### <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

Ammonia-free distilled water

Ammonium chloride standard

NaOH solutions (1 N and 0.1 N)

Mixed indicator solution

Indicator boric acid solution

22 This method is based on Method 4500-NH3 B and C of the Standard Methods for the Examination of Water and Wastewater and should be cited as: Rice *et al*. (2017) as described in Velkushanova *et al*. (2021).

23 https://ehs.berkeley.edu/workplace-safety/glove-selectionguide#comparison, (Berkeley Environment, Health & Safety).

## <IP_ADDRESS>.4 Required apparatus and instruments

 Titration setup with burette in metal clamp (or automatic titration unit).

## <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

## <IP_ADDRESS>.6 Sample preservation

 Samples should be analysed as soon as possible after sampling. For analysis within 24 hr of collection, samples must be refrigerated at 4 °C. For longer storage durations of up to 28 days, the pH should be reduced to below pH 2 with concentrated sulphuric acid and stored at 4 ºC, or the sample should be frozen at -20 ºC without acidifying. If acid preservation has been used, neutralise the samples to pH 7 with NaOH or KOH immediately before carrying out the analysis.

	- Ammonium-free water is very difficult to store without interference by gaseous ammonia, and storage should therefore be avoided.
	- Prepare a fresh solution of ammonium chloride monthly.
	- Prepare mixed indicator solution monthly.
	- Prepare indicator boric acid solution monthly.
	- Standard sulphuric acid titrant should not be stored longer than one week.

## <IP_ADDRESS>.7 Sample preparation

## *This method is valid within a concentration range of 5-100 mg/L NH3-N:*

This method is valid for all sludge types (liquid, slurry, semi-solid, and solid), as long as they are prediluted using serial dilutions within the specified range. Ideally, dilutions should aim to be within the range of 10-25 mg/L NH3-N. Use ammonium-free distilled water for making dilutions.

## *For liquid faecal sludge:*

Filter turbid samples using 0.45 μm filter.

## *For slurry to solid faecal sludge:*

#### <IP_ADDRESS>.8 Analysis protocol

*Preparation of reagents, chemicals and standard solutions*

## Ammonia-free water

Eliminate traces of ammonia in distilled water by adding 0.1 mL concentrated sulphuric acid to 1 L distilled water and then redistill. Alternatively, treat distilled water with enough bromine or chlorine to produce a free halogen residual of 2-5 mg/L and redistill after standing for 1 hr. Discard the first 100mL of distillate, and check the ammonia concentration in the collected water before use.

	- Prepare ammonium chloride standard by weighing and dissolving 3.819 g of NH4Cl into a 1 L volumetric flask with distilled water and dilute the flask content to the mark with ammonia-free distilled water. This makes an ammonium chloride standard solution where 1.0 mL = 1 mg N = 1.22 mg NH3.
	- Then prepare a working standard solution by transferring 10 mL stock solution into 1 L in a volumetric flask and further dilute with ammonia-free distilled water to give a concentration of 12 mg/L NH3.
	- Weigh 4 g of NaOH and dissolve in 1 L of ammonia-free distilled water.
	- Weigh 40 g NaOH and dissolve in 1 L of ammonia-free distilled water.
	- Add 88 mL of 0.1 N NaOH solution to 500 mL of 0.025 M disodium tetraborate (Na2B4O7) solution (9.5 g Na2B4O7ꞏ10H2O hydrous/L or 5.0 g Na2B4O7 anhydrous/L) into a 1 L volumetric flask and dilute to the mark with ammonia-free distilled water.
	- Weigh and dissolve 200 mg methyl red indicator in 100 mL of 95% organic polar solvent (isopropyl alcohol or ethanol) in a beaker. In a separate beaker, dissolve 100 mg methylene blue in 50 mL of the same solvent. Then combine the solutions.
	- Weigh 20 g H3BO3 and dissolve in ammoniafree distilled water. Add this to a 1 L volumetric flask, add 10 mL mixed indicator solution, and dilute to the mark.
	- First make a 0.1 N sulphuric acid solution by diluting 2.71 mL concentrated sulphuric acid to 1L with distilled water.
	- Dilute 200 mL 0.1 N sulphuric acid solution to 1 L with distilled water to make a 0.02 N solution.
	- Allow it to cool down to ambient temperature.
	- Dry anhydrous sodium carbonate (Na2CO3) at 270 °C, cool down in a desiccator, and weigh 1.325 g. Dissolve in ammonia-free distilled water and make up to 500 mL in a volumetric flask (0.05 N solution). Do not keep longer than 1 week.
	- Titrate the sulphuric acid solution against 25 mL of sodium carbonate solution using 5 drops of methylene blue-methyl red mixed indicator.
	- Calculate the exact normality of the sulphuric acid:

Normality of H2SO4 solution =

25 × 0.05 Volume of H2SO4 used

#### <IP_ADDRESS>.9 Calibration

## *Preparation of the setup*

Add 500 mL ammonia-free water and 20 mL borate buffer to a distillation flask and adjust the pH to 9.5 with 6.0 N NaOH solution. Add a few glass beads and use this mixture to steam-clean the distillation apparatus until the distillate shows no traces of ammonia.

## *Procedure*

 Add the sample to a distillation flask. Use the sample volume as specified here:

	- Note the starting volume of the sulphuric acid in the burette.
	- Titrate until the indicator turns pale purple.
	- Note the final volume of sulphuric acid in the burette.

## <IP_ADDRESS>.10 Calculations

*Liquid and slurry samples:*  NH3-N ሺmg L⁄ ሻ ൌ

ሺA ̶ Bሻ × 280 Sample volume ሺmLሻ × DF

NH3-N on dry basis (g/g) ൌ

NH3-N ( mg L⁄ )

Total solids concentration (mg/L)

#### Where:

*Slurry, semi-solid and solid samples:*

NH3-N on dry basis (g/g) =

(A ̶B) × 280 Sample volume (mL) × V M × 1 1,000 ×

$$\frac{1}{\text{Total solids content} \left(\text{g/g}\right)}$$

#### Where:

V = Volume of dilution (L)

M = Wet weight of sludge used in sample preparation (g)

#### <IP_ADDRESS>.11 Data set example

Spit *et al*. (2014) sampled three pit latrines in Blantyre, Malawi, and determined the ammonia using the titration method. Each sample was analysed in triplicate and the results were reported volumetrically, as shown in Table 8.9.

**Table 8.9** The results from the ammonia and TS analysis from three pit latrines in Blantyre, Malawi.

Two faecal sludge samples were collected from two ventilated improved pit latrines in Durban, South Africa. They were analysed in four replicates each, using Method <IP_ADDRESS>. The average ammonia content (g/g dry sample) was 0.71 and 0.74 for samples one and two respectively. The results for ammonia are presented in Table 8.10 (source: unpublished data UKZN PRG).

**Table 8.10** VIP faecal sludge analysis using the ammonia distillation and titration method.

## **<IP_ADDRESS> Nitrite – colorimetric (test strip) method24**  <IP_ADDRESS>.1 Introduction

Nitrite is a toxic intermediate of nitrification. Wastewater effluents with high nitrite concentration can adversely affect aquatic life when dilution in the receiving water is insufficient. In aerobic faecal sludge treatment processes, accumulation of nitrite signifies deficiencies in the treatment process. This method describes the Merck 110007 MQuant Nitrite TestD as an example of a commercially available nitrite colorimetric test strip method for samples with concentrations of 2-80 mgNO2 - /L. It is based on the manufacturer's protocol for water and wastewater. In the presence of an acidic buffer, nitrite ions react with aromatic amine to form a diazonium salt, which in turn reacts with N-(1-naphthyl)-ethylenediamine to form a red-violet azo dye. The nitrite concentration is measured semi-quantitatively by visual comparison of the reaction zone of the test strip with the fields of a colour scale. Nitrite measurement results are expressed as either mgNO2 – /L or mgNO2-N/L, and it is important to note how the results are expressed by the selected test method. Depending on the expected nitrite concentration in the sample, kits with the appropriate measurement range should be selected. For faecal sludge, samples must be diluted to prevent masking of the resultant colour change.

#### <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

#### <IP_ADDRESS>.4 Required apparatus and instruments

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

24 Test strip methods should be cited as the specific manufacturer's method along with any modifications. The example used here is the Merck MQuant Nitrite Test (Merck, 2020h)D.

## <IP_ADDRESS>.6 Sample preservation

## <IP_ADDRESS>.7 Sample preparation

#### *For liquid, slurry, semi-solid or solid samples:*

## *For slurry to solid samples:*

## <IP_ADDRESS>.8 Analysis protocol

#### *For measuring range of 2-80 mgNO2 - /L*

 Immerse the reaction zone of the test strip in the diluted, filtered sample (15 - 30 °C) for 1 second. Shake off excess liquid from the strip and after 15 sec determine which colour field on the label the colour of the reaction zone coincides with most exactly. Read off the corresponding result in mgNO2/L or mgNO2-N/L.

## <IP_ADDRESS>.9 Calculation

The dilution factor used must be stated.

Result of the analysis (mg/L NO2 – ) = measurement value, A (mg/L) × dilution factor (DF)

## *Liquid and slurry samples:*

$$\stackrel{\circ}{\text{Final concentration}} \text{ (mg/L)} = \text{A } \left(\frac{\text{mg}}{\text{L}}\right) \times \text{DF}$$

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Colorimetric measurement value (mg/L)

DF = Dilution factor (F/I)

*Slurry, semi-solid and solid samples:*

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right) \times \frac{\text{V}}{\text{M}} (\frac{\text{L}}{\text{g}})}{\text{Total solids content } (\frac{\text{g}}{\text{g}})} $$

Where:

## **<IP_ADDRESS> Nitrite – spectrophotometric method25**  <IP_ADDRESS>.1 Introduction

Nitrite can be measured using different methods, such as chromatography or spectrophotometry. This method describes nitrite measurement by spectrophotometry. The principle is that in an acidic solution nitrite ions react with sulfanilic acid to form a diazonium salt. The diazonium salt reacts with N-(1-naphthyl) ethylenediamine dihydrochloride to form a red-violet azo dye at a pH of 2.0-2.5, which is then determined spectrophotometrically. Nitrite measurement results are expressed in different ways (*i.e.* mg/L NO2 – or mg/L NO2-N), and it is important to know how the results are expressed by the selected test method. Spectrophotometric measurements ranging from 0.01-1.0 mg/L is applicable for this method. However, for lower concentrations < 0.01 mg/L, spectrophotometric measurements can be made by adapting this method using a 5 cm light path and a green colour filter. The sample is filtered prior to analysis, as solids interfere with the spectrophotometric method, based on the assumption that NO2 – will be solution, and not with the solids fraction. It is possible to conduct this method with commercially available test kits. The test kits are based on standard methods, with pre-packaged individual aliquots of the necessary chemicals in pillows (dry chemicals) and vials (liquid chemicals). Commonly used test kits from manufacturers such as Hach, Merck, and Hanna vary slightly in the methods they use for nitrite measurement. The ferrosulphate method and diazotization method are most commonly used in test kits.

The example provided here is the Merck Spectroquant Nitrite Test spectrophotometric test kit (Merck, 2020e)D for samples with concentrations of 0.02-1.0 mgNO2-N/L, and it is based on the manufacturer's protocol for water and wastewater using the standard method 4500 (Rice *et al.,* 2017). For faecal sludge, samples must be diluted and filtered to prevent false high readings associated with turbid solutions.

#### <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

#### <IP_ADDRESS>.4 Required apparatus and instruments

25 This method should be cited as: Method 4500-NO2–B (Rice *et al.,* 2017) and, if test kits are used, also as per the manufacturer's directions including any modifications.

## <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

## <IP_ADDRESS>.6 Sample preservation

 Samples should be analysed immediately after sampling to prevent bacterial conversion of NO2 – to NO3 – or NH3. For short-term preservation of 1 to 2 days, samples should be stored at 4 °C or frozen at −20 °C (Rice *et al.,* 2017). Samples should be thawed to room temperature before analysis.

## <IP_ADDRESS>.7 Sample preparation

## *For liquid, slurry, semi-solid or solid samples:*

## *For slurry to solid samples:*

 Weigh out between 1.8 g and 2.0 g of thoroughlymixed faecal sludge sample into a beaker.

## <IP_ADDRESS>.8 Analysis protocol *Calibration*

Follow the spectrophotometer manufacturer's instructions for calibration, since calibration procedure differs between instruments. It may be necessary to calibrate the instrument before every reading, or it may only be necessary to perform periodic calibration checks to determine when calibration is necessary.

## *Procedure*

This method is for a measuring range of 0.02-1.0 mg/L NO2-N

#### <IP_ADDRESS>.9 Calculation

### *Liquid and slurry samples:*

Result of analysis (mg/L) Final concentration ቀ mg L ቁ = Aቀ mg L ቁ × DF

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Spectrophotometric measurement value (mg/L)

DF = Dilution factor (F/I)

F = Final diluted volume (L)

I = Initial sample volume (L)

*Slurry, semi-solid and solid samples:*

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids content} \left(\frac{\text{g}}{\text{g}}\right)}$$

Where:

A = Spectrophotometric measurement value (mg/L)

V = Volume of dilution (L)

M = Wet weight of sludge used in sample preparation (g)

#### <IP_ADDRESS>.10 Data set example

Septien *et al*. (2020) evaluated the effect of drying on the physical and chemical characteristics of faecal sludge for resource recovery. The nitrite concentration of faecal sludge from pit latrines before and after drying at different temperatures were measured using a Merck Spectroquant®,D. The sample was prepared by blending it with water, centrifugation, recovering the liquid fraction and using the commercial test kit. Nitrite in a solid sample dissolves and remains in a soluble state which can be measured. In this example, nitrite was measured in the faecal sludge sample which was further subjected to drying in infra-red and convective drying rigs. For duplicate samples, nitrite concentration of 0 g/kg dry solid was measured for untreated faecal sludge and dried faecal sludge samples after drying in a convective drying rig at 30 °C, 60 °C and 80 ° C. Nitrite is usually oxidised to nitrate and thus concentrations are expected to be very low to zero in faecal sludge samples. However, nitrite concentrations in untreated faecal sludge for infra-red drying were 13 ± 0 g/kg dry solid. After drying, the concentration of nitrite decreased to 2.3 ± 0.1 g/kg dry solid. The decrease in nitrite and other nitrogen forms determined in this work did not translate into a decrease in total nitrogen, and thus drying of faecal sludge was concluded to induce changes in the chemical forms of nitrogen.

## **<IP_ADDRESS> Nitrate – colorimetric (test strip) method26**  <IP_ADDRESS>.1 Introduction

Nitrate is a major source of available nitrogen for plants and microorganisms. However, nitrate becomes toxic and affects public health when it contaminates surface or groundwater used as drinking water. Eutrophication and algal blooms of surface water are also environmental impacts of nitrate pollution. Nitrate management in faecal sludge treatment is thus essential to protect public and environmental health. This method describes the Merck MQuant® Nitrate TestD as one example of a commercially available nitrate colorimetric test strip method, for samples with concentrations of 10-500 mgNO3 – /L. It is based on the manufacturer's protocol for water and wastewater. In this method, nitrate ions are reduced to nitrite ions in the presence of a reducing agent. In the presence of an acidic buffer, nitrite ions react with aromatic amine to form a diazonium salt, which in turn reacts with N-(1 naphthyl)-ethylenediamine to form a red-violet azo dye. Nitrate measurement results are expressed as

26 Test strip methods should be cited as the specific manufacturer's method along with any modifications. The example used here is the Merck MQuant Nitrate Test (Merck, 2020g)D.

either mgNO3 – /L or mgNO3-N/L, and it is important to note how the results are expressed by the selected test method. The nitrate concentration is measured semi-quantitatively by visual comparison of the reaction zone of the test strip with the fields of a colour scale. Depending on the expected nitrate concentration in the sample, kits with the appropriate measurement range should be selected. For faecal sludge, samples must be diluted to prevent masking of the resultant colour change.

## <IP_ADDRESS>.2 Safety precautions

## <IP_ADDRESS>.3 Required chemicals

## <IP_ADDRESS>.4 Required apparatus and instruments

## <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

## <IP_ADDRESS>.6 Sample preservation

## <IP_ADDRESS>.7 Sample preparation

## *For liquid, slurry, semi-solid or solid samples:*

## *For slurry to solid samples:*

#### <IP_ADDRESS>.8 Analysis protocol

## *For measuring range of 10-500 mgNO3/L:*

#### <IP_ADDRESS>.9 Calculation

The dilution factor used must be stated.

Result of analysis (mg/L NO3 – ) = measurement value, A (mg/L) × dilution factor

## *Liquid samples:*

Result of analysis (mg/L)

$$\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right) = \text{A } \left(\frac{\text{mg}}{\text{L}}\right) \times \text{DF}$$

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Colorimetric measurement value (mg/L)

DF = Dilution factor (F/I)

F = Final diluted volume (L)

I = Initial sample volume (L)

*Slurry, semi-solid and solid samples:*

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids content} \left(\frac{\text{g}}{\text{g}}\right)}$$

Where:

$$\mathbf{A} = \begin{array}{c} \text{Coloricmetric measurement value (mg/L)} \end{array}$$

## **<IP_ADDRESS> Nitrate – cadmium reduction spectrophotometric method27**

#### <IP_ADDRESS>.1 Introduction

The principle of this method is that nitrate interacts with cadmium particles in aqueous solutions which converts all the nitrate in the sample into nitrite. The nitrite formed is determined by diazotising with sulfanilamide and coupling with N-(1-naphthyl) ethylenediamine dihydrochloride to form a highly coloured azo dye that is measured spectrophotometrically. Nitrate measurements are easily influenced by interfering components such as dissolved organic matter, surfactants, nitrite and various inorganic compounds in water. The concentration ranges for the different methods used for nitrate measurement are also limited. For instance, the concentration ranges of the electrode method, the cadmium reduction spectrophotometric method and the automated cadmium reduction method are 0.14 to 1,400 mg NO3-N/L, 0.01 to 1.0 mg NO3-N/L and 0.001 to 10 mg NO3-N/L, respectively. This means

27 This method should be cited as: Method 4500-NO3-E (Rice *et al*., 2017), and if test kits are used, also as per the manufacturer's directions including any modifications.

that higher concentrations should be diluted to the range of the selected method. Methods for nitrate determination are based on the oxidizing properties of nitrate. However, other oxidants present in water and wastewater may interfere making nitrate measurement difficult.

It is possible to conduct this method with commercially available test kits. The test kits are based on standard methods, with pre-packaged individual aliquots of necessary chemical in pillows (dry chemicals) and vials (liquid chemicals). Commonly used test kits from manufacturers such as Hach, Merck, and Hanna employ different methods in the kits used for nitrate measurement. The cadmium reduction method is used in Hach and Hanna test kits.

The example provided here is the Hach nitrate spectrophotometric test kit (Hach, 2020)D for samples with concentrations of 0.3-30 mgNO3-N/L, which is based on the manufacturer's protocol for water and wastewater using the standard method 4500-NO3-E (Rice *et al*., 2017). For faecal sludge, samples must be diluted and filtered to prevent false high readings associated with turbid solutions.

## <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

#### <IP_ADDRESS>.4 Required apparatus and instruments

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

## <IP_ADDRESS>.6 Sample preservation

#### <IP_ADDRESS>.7 Sample preparation

## *For liquid, slurry, semi-solid or solid samples:*

## *For slurry to solid samples:*

#### <IP_ADDRESS>.8 Analysis protocol

## *Calibration*

Follow the spectrophotometer manufacturer's instructions for calibration. It may be necessary to calibrate the instrument before every reading, or it may only be necessary to perform periodic calibration checks to determine when calibration is necessary. For this method, calibration can be performed as follows:

## *Procedure*

## *For measuring range of 0.3-30.0 mgNO3-N/L*:

#### <IP_ADDRESS>.9 Calculation

*Liquid and slurry samples:*  Result of analysis (mg/L) Final concentration ቀ mg L ቁ = Aቀ mg L ቁ × DF

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Spectrophotometric measurement value (mg/L)

DF = Dilution factor (F/I)

$$Slurr\text{y, } semi\text{-solid and } \underline{\text{sọlid samples:}}$$

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right) \times \frac{\text{V}}{\text{M}} (\frac{\text{L}}{\text{g}})}{\text{Total solids content } (\frac{\text{g}}{\text{g}})} $$

Where:

#### <IP_ADDRESS>.10 Data set example

Strande *et al*. (2018) determined the nitrate concentrations of faecal sludge from pit latrines and septic tanks located in households, non-households, and public toilets using the Hach nitrate test kits. As shown in Figure 8.8, 180 samples were analysed and the results showed an uneven distribution and a high variability. Hence, the median rather than mean of the results was used for the statistical analysis. Similar to other forms of nitrogen determined, nitrate concentrations in pit latrines from households, nonhouseholds and public toilets were all found to be higher compared to septic tanks. The complete raw data set is available at the link below28.

**Figure 8.8** Nitrate concentrations in septic tanks (blue boxes) and pit latrines (green boxes) from households, non‐ households, and public toilets (Strande *et al.,* 2018).

## **<IP_ADDRESS> Total Kjeldahl nitrogen – distillation and titration method29**

#### <IP_ADDRESS>.1 Introduction

Kjeldahl nitrogen is defined as the total organic nitrogen together with the inorganic compounds ammonia and ammonium (NH3/NH4 + ). In the presence of sulphuric acid, potassium sulphate, and cupric sulphate catalyst, amino nitrogen in the organic matter in the sample and free ammonia are converted into ammonium during digestion. Upon addition of a base, ammonium is converted into ammonia, and the ammonia is then distilled from an alkaline medium and absorbed in boric or sulphuric acid. The ammonia may be determined spectrophotometrically, by ammonia selective electrode or by titration with a standard mineral acid. The titration method is described here. The titrimetric and selective electrode methods of measuring ammonia in the distillate are suitable for determining a wide range of organic nitrogen concentrations.

Degradation:

Organic N in sample + H2SO4 → (NH4)2SO4(aq) + CO2(g) + SO2(g) + H2O(g)

Liberation of ammonia: (NH4)2SO4(aq) + 2NaOH → Na2SO4(aq) + 2H2O(l) + 2NH3(g)

−

Capture of ammonia: B(OH)3 + H2O + NH3 → NH4 + + B(OH)4

Back-titration: B(OH)3 + H2O + Na2CO3 → NaHCO3(aq) + NaB(OH)4(aq) + CO2(g) + H2O

All dilutions and reagents should be made with ammonia-free distilled water.

Various apparatus is available for Kjeldahl analysis, ranging from low-tech heating blocks for digestion to fully automatic Kjeldahl setups that do digestion and/or distillation and titration. This method might need to be adapted based on the type of apparatus used, and for automated systems the

28 https://doi.org/10.25678/0000tt

29 This method is adapted from Method 4500-Norg B and C of the Standard Methods for the Examination of Water and

Wastewater and should be cited as: Rice *et al*. (2017), as described in Velkushanova *et al.* (2021).

manufacturer's instructions should be followed. In addition, the method might need to be adapted for different types of faecal sludge, based on local experience and the specific characteristics of faecal sludge. The total Kjeldahl nitrogen distillation and titration (TKN) method is widely accepted and has been tested for multiple substances; however, some steps in the process are affected by the composition of the sample and need to be adjusted accordingly (*e.g.* amounts of acid used, and digestion temperature and time). A useful guide with further information on considerations for Kjeldahl analysis can be found in 'A Guide to Kjeldahl Nitrogen Determination Methods and Apparatus' by Labconco Corporation (1998), and the Labconco website30. Methods should always be adapted according to rigorous quality control principles. For example, UKZN PRG in Durban uses a semi-automated digestion setup, and for samples with a high organic/fat content they mix samples overnight on the digestion apparatus prior to digestion.

## <IP_ADDRESS>.2 Safety precautions

## <IP_ADDRESS>.3 Required chemicals

	- Distilled water
	- Concentrated sulphuric acid, or bromine or chlorine, depending on the method used; see Section <IP_ADDRESS>.8.
	- K2SO4
	- CuSO4
	- Alternatively, commercial pre-mixed Kjeldahl tablets or powder, free of Hg and Se, are available and can be used instead of mixing digestion reagent manually.
	- Boric acid
	- Ammonia-free distilled water
	- Concentrated sulphuric acid
	- Ammonia-free distilled water
	- NaOH
	- Ammonia-free distilled water
	- Methyl red indicator
	- Bromocresol green indicator
	- Ethanol.

## <IP_ADDRESS>.4 Apparatus and instruments

30 https://www.labconco.com/articles/a-brief-introduction-tokjeldahl-nitrogen-determ

31 https://ehs.berkeley.edu/workplace-safety/glove-selectionguide#comparison

## <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

## <IP_ADDRESS>.6 Sample preservation

 The most reliable results are obtained from fresh samples. If immediate analysis is not possible, preserve the samples for Kjeldahl digestion by acidifying between pH 1.5 and 2.0 with concentrated sulphuric acid and storing in a refrigerator at 4 °C. Let the samples return to room temperature before starting the analysis. If acid preservation was used, neutralise the samples to pH 7 with NaOH or KOH immediately before starting the analysis.

	- Mixed indicator solution should be freshly prepared every month.
	- Sulphuric acid titrant should not be stored longer than one week.

## <IP_ADDRESS>.7 Sample preparation

## *For liquid faecal sludge:*

 Filter turbid samples using a 0.45 μm pore size filter.

## *For slurry to solid faecal sludge:*

## <IP_ADDRESS>.8 Analysis protocol

## *Preparation of reagents, chemicals and standard solutions*

## Ammonia-free water

 Eliminate traces of ammonia in the distilled water by adding 0.1 mL concentrated sulphuric acid to 1 L distilled water and redistill. Alternatively, treat the distilled water with enough bromine or chlorine to produce a free halogen residual of 2-5 mg/L and redistill after standing for 1 hr. Discard the first 100 mL of distillate, and check the ammonia concentration in the collected water before use.

Boric acid - 4%

 Dissolve 40g of boric acid in the distilled water in a 1L volumetric flask, and dilute up to the mark.

Sulphuric acid - 0.1 N

 Dilute 2.71 mL 98% concentrated sulphuric acid in the distilled water in a 1 L volumetric flask, and dilute up to the mark.

Mixed indicator

 Weigh 0.02 g methyl red and 0.10 g bromocresol green indicator in a 100 mL volumetric flask and fill to the mark with ethanol.

## *Calibration*

## *Procedure*

 Include standards and a blank with every analysis batch.

Digestion

Distillation

Distil the sample into the boric acid.

## Titration

## <IP_ADDRESS>.9 Calculation

$$\begin{array}{l} \text{Liquid and slowry samples:}\\ \text{TKN (mg/L)} = \frac{(\text{A} - \text{B}) \times 0.1 \times 14 \times 1,000}{\text{Sample volume (mL)}} \times \text{DF} \end{array}$$

Where:

F = Final diluted volume (L)

I = Initial sample volume (L)

TKN on dry basis (g/g) =

$$\text{TRN} \left(\text{mg/L}\right)$$

Total solids concentration (mg/L)

*Slurry, semi-solid and solid samples:*  TKN on dry basis ሺg/gሻ =

$$\frac{(\text{A} - \text{B}) \times 0.1 \times 14 \times 1,000}{\text{Sample volume (mL)}} \times \frac{\text{V}}{\text{M}} \times \frac{1}{1,000} \times 1$$

$$\text{The first-order coupling between the two-dimensional } \mathcal{N} \text{-matrices is the only possible } \mathcal{N} \text{-matrices with } \mathcal{N} = \{0, 1, 2, \dots, N\} \text{ and } \mathcal{N} = \{0, 1, 2, \dots, N\}.$$

1 Total solids content ( g g⁄ ) Where:

V = Volume of dilution (L)

M = Wet weight of sludge used in sample preparation (g)

#### <IP_ADDRESS>.10 Data set example

Faecal sludge samples were collected from a ventilated improved pit (VIP) latrine and from a urine diversion toilet (UDDT) in Durban, South Africa, and were analysed using the total Kjeldahl nitrogen distillation and titration method. Each sample was analysed in four replicates and reported gravimetrically. The average TKN values (g/g dry sample) were 0.02 and 0.03 for the VIP and UDDT, respectively. The results for TKN are presented in Table 8.11 (source: unpublished data UKZN PRG).

**Table 8.11** TKN values (g/g dry sample) for a VIP and a UDDT toilet analysed by the titrimetric method.

## **8.6.5 Phosphorus**

Phosphorus is important for monitoring the nutrient availability in both faecal sludge treatment and resource recovery processes. It is an essential nutrient for the growth of plants and organisms, and also a potential environmental pollutant (Tchobanoglous *et al.*, 2003). Phosphorus is present in faecal sludge as orthophosphates, polyphosphates, and organically bound phosphates (Strande *et al.,* 2014).

There are three ways of determining phosphorus in wastewater or faecal sludge: orthophosphate, acid hydrolysable phosphate, and total phosphorus, which are used to analyse orthophosphate, polyphosphates (condensed phosphate), and organic phosphate. Orthophosphate is also called reactive or available phosphorus, as it is soluble, readily interacts with other positive elements or compounds, and is bioavailable. It is the only phosphorus form that can be determined directly without preliminary hydrolysis or oxidative digestion of the sample. To measure the concentration of polyphosphates, acid hydrolysis of samples at boiling water temperature is used to convert the dissolved and particulate phosphates into dissolved orthophosphate which is then quantified. Organic phosphates can be determined by an oxidative destruction of the organic matter in the sample, which converts organic phosphates into orthophosphates, which are then quantified. Total phosphorus concentrations will be greater than the orthophosphate concentration. In this book, a spectrophotometric method for determining total phosphorus and orthophosphate concentrations is described.

## **<IP_ADDRESS> Total phosphorus and orthophosphate ‐ spectrophotometric method32**

#### <IP_ADDRESS>.1 Introduction

The determination of total phosphorus and orthophosphate is based on the principle that in sulphuric acid solution, orthophosphate ions react with molybdate ions to form phosphomolybdic acid. This molybdate compound is reduced by ascorbic acid to form an intense phosphomolybdenum blue (PMB) that is then quantified with a spectrophotometer. To measure total phosphorus, other phosphorus forms are initially converted into orthophosphate before measurement.

Commercial test kits based on standard methods for measuring total phosphorus and orthophosphates are available, with pre-packaged individual aliquots of any necessary chemicals in pillows (dry chemicals) and vials (liquid chemicals). Commonly used total phosphorus and orthophosphate test kits from manufacturers such as Hach and Merck employ a variety of methods. For example, the Merck test kit uses the ascorbic acid method. The example provided here is the Merck phosphate spectrophotometric testD

32 This method should be cited as: Method 4500-P E (Rice *et al*., 2017), and if test kits are used, also as per the manufacturer's directions including any modifications.

which is used for the determination of total phosphorus and orthophosphate for samples with concentrations of 0.05-5 mg PO4-P/L, and it is based on the manufacturer's protocol for water and wastewater using the standard method 4500-P E (Rice *et al*., 2017). Phosphate measurement results are expressed as either mg PO4 3–/L or mg PO4-P/L (or gTP/gTS or gPO4-P/gTS with TS analysis) and it is important to note how the results are expressed by the selected test method. For faecal sludge, samples must be diluted (for total phosphorus) and filtered (for orthophosphate) to prevent false high readings associated with turbid solutions.

## <IP_ADDRESS>.2 Safety precautions

## <IP_ADDRESS>.3 Required chemicals

## <IP_ADDRESS>.4 Required apparatus and instruments

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

type of sludge for accurate measurements. Always use serial dilutions.

 The colour remains stable for at least 60 min following the end of the reaction time, which takes 5 min.

## <IP_ADDRESS>.6 Sample preservation

## <IP_ADDRESS>.7 Sample preparation *For liquid, slurry, semi-solid or solid samples:*

## *For slurry, semi-solid or solid samples:*

## <IP_ADDRESS>.8 Analysis protocol *Calibration*

Follow the spectrophotometer manufacturer's instructions for calibration. It may be necessary to calibrate the instrument before every reading, or it may only be necessary to perform periodic calibration checks to determine when calibration is necessary. For this method, calibration can be performed as follows:

## *Procedure*

Orthophosphate measurement

*For measuring range of 0.05-5.0 mg/L PO4-P:*

## Total P measurement

#### *For measuring range of 0.05-5.0 mg/L PO4-P:*

#### <IP_ADDRESS>.9 Calculation

### *Liquid and slurry samples:*

$$\begin{array}{l} \text{Result of analysis (mg/L)}\\ \text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right) = \text{A } \left(\frac{\text{mg}}{\text{L}}\right) \times \text{DF}^2 \end{array}$$

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Spectrophotometric measurement value (mg/L)

DF = Dilution factor (F/I)

F = Final diluted volume (L)

I = Initial sample volume (L)

$$Slarry,\text{\textquotedblleft}semi\text{-solid}\text{\textquotedblright}and\text{\textquotedblright}samples;\text{\textquotedblleft}$$

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right) \times \frac{\text{V}}{\text{M}} (\frac{\text{L}}{\text{g}})}{\text{Total solids content} \left(\frac{\text{g}}{\text{g}}\right)}$$

Where:

#### <IP_ADDRESS>.10 Data set example

Phosphate was measured by Septien *et al*. (2020) while evaluating the effect of drying on the physical and chemical characteristics of faecal sludge for resource recovery. Phosphate concentrations of faecal sludge from pit latrines were measured before and after drying under convective and infra-red drying rigs using a Merck Spectroquant. Different concentrations of phosphate were determined for untreated faecal sludge samples. Before drying in the convective drying rig, average phosphate in untreated faecal sludge was 2.4 ± 0.7 g PO4 3–/g dry solid. The untreated faecal sludge used for infra-red drying was 13 ± 0 gPO4 3–/g dry solid. Drying under the convective drying rig did not affect the phosphate concentrations because 2.3 ± 0.9, 2.5 ± 0.6 and 2.1 ± 0.7 g PO4 3–/g dry solid were measured after drying at 40 ° C, 60 ° C and 80 ° C, respectively. For infra-red drying, phosphate concentrations were slightly reduced to 11 ± 0 g PO4 3–/g dry solid after drying at 80% medium infra-red for 9 min. It was concluded from the study that drying does not affect the phosphorus concentration because most phosphorus forms are bound to the solids in faecal sludge. This further suggests that dried faecal sludge pellets present an attractive nutrient composition for agricultural applications.

Phosphate was measured with the Hach test kit for 60 samples in Englund *et al*. (2020) and phosphate and total phosphorus for 180 samples in Strande *et al*. (2018) and reported as mg/L. The range of observed PO4 in septic tanks in Hanoi, Vietnam was 3.5 to 33 mg PO4-P/L. The range of PO4 was 4-367 mgPO4-P/L and TP was 6-2,040 mg TP/L for a range of household and non-household sources in pit latrines and septic tanks in Kampala, Uganda as shown in Figure 8.9. Both complete raw data sets can be downloaded using the link below33.

**Figure 8.9** Range of total phosphorus (above) and phosphate (bellow) concentrations in faecal sludge samples from Kampala, Uganda. 180 samples consisting of pit latrines (green boxes) and septic tanks (blue boxes) (*Strande et al., 2018*).

## **<IP_ADDRESS> Orthophosphate ‐ colorimetric (test strip) method34**

#### <IP_ADDRESS>.1 Introduction

Similar to nitrates, phosphorus is a major nutrient that contributes to eutrophication and it is present in aqueous solutions mainly as orthophosphate, polyphosphate or organically-bound phosphate. Concentrations of phosphorus in faecal sludge are usually 2-5 times higher than in wastewater and thus orthophosphate determination is crucial in faecal sludge treatment. The colorimetric method described here is one of many orthophosphate colorimetric test methods for samples with concentrations of 10-500 mg/L PO4 3–. It is based on the Merck MQuant Phosphate TestD protocol for water and wastewater. In this method, orthophosphate ions (PO4 3–) react with molybdate ions in the presence of a sulphuric solution to form molybdophosphoric acid, which is reduced to phosphomolybdenum blue (PMB). The phosphate concentration is measured semi-quantitatively by visual comparison of the reaction zone of the test strip with the fields of a colour scale. Phosphate measurement results are expressed as either mgPO4 3–/L or mgPO4-P/L or mgP2O5/L and it is important to note how the results are expressed by the selected test method. Depending on the expected phosphate concentration in the sample, kits with the appropriate measurement range should be selected. For faecal sludge, samples must be diluted and filtered to prevent masking of the resultant colour change.

#### <IP_ADDRESS>.2 Safety precautions

33 https://doi.org/10.25678/0000tt

34 Test strip methods should be cited as the specific manufacturer's method along with any modifications. The

example used here is the Merck MQuant Phosphate Test (Merck, 2020i)D.

## <IP_ADDRESS>.3 Reagents, chemicals and standard solutions

The required chemicals will be specific to each manufacturer's test kit; specific information on test kits can be found on the manufacturers' websites.

## <IP_ADDRESS>.4 Apparatus and instruments

#### <IP_ADDRESS>.1 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

 The orthophosphate colorimetric test method is used for qualitative to semi-quantitative measurement. For a quantitative measurement, refer to Method <IP_ADDRESS> (the total phosphorus and orthophosphate spectrophotometric method).

#### <IP_ADDRESS>.6 Sample preservation

#### <IP_ADDRESS>.7 Sample preparation

#### *For liquid, slurry, semi-solid or solid samples:*

#### *For slurry, semi-solid or solid samples:*

#### <IP_ADDRESS>.8 Analysis protocol

#### *This method is valid for a measuring range of 10-500 mgPO4 3-/L.*

#### <IP_ADDRESS>.9 Calculation

## *Liquid samples:*

Result of analysis (mg/L) Final concentration ቀ mg L ቁ = Aቀ mg L ቁ × DF

$$\text{Dry basis } \left(\frac{\text{g}}{\text{g}}\right) = \frac{\text{Final concentration } \left(\frac{\text{mg}}{\text{L}}\right)}{\text{Total solids concentration } \left(\frac{\text{mg}}{\text{L}}\right)}$$

Where:

A = Colorimetric measurement value (mg/L)

DF = Dilution factor (F/I)

*Slurry, semi-solid and solid samples:*

$$\text{Dry basis } (\frac{\text{g}}{\text{g}}) = \frac{\text{A} \left(\frac{\text{mg}}{\text{L}}\right) \times \frac{\text{V}}{\text{M}} (\frac{\text{L}}{\text{g}})}{\text{Total solids content } (\frac{\text{g}}{\text{g}})} $$

Where:

## **8.6.6 pH and electrical conductivity**

pH, electrical conductivity (EC), and alkalinity are measurements of solution properties in faecal sludge samples. pH is a measure of how acidic or basic an aqueous solution is. The pH scale is logarithmic, and is inversely related to the concentration of hydrogen ions in the solution. A pH of 7 is neutral, an acidic solution has a lower pH (< 7) and a high concentration of hydrogen ions, while a basic solution has a high pH (>7) and a low concentration of hydrogen ions. Biological treatment processes and conditioning for improved dewatering commonly require a pH between 6 and 9, and therefore pH is a standard design and operational parameter for faecal sludge treatment. pH values outside of this range may indicate contamination by other solid or liquid wastes, and can contribute to issues with biological processes or problems with corrosion. EC is a measure of dissolved salts, and can be used as a proxy for salinity. The concentration of dissolved salts influences the flocculation and dewatering properties of faecal sludge, and high ECs may inhibit biological treatment processes. EC is also an important parameter in resource recovery from faecal sludge as irrigation or soil conditioner, as high concentrations of dissolved salt may be harmful to crops and contribute to the accumulation of salinity in soils. Measurements of pH and EC are temperature-dependent, and thus temperature should always be recorded together with pH and EC. Alkalinity is also an important solution property of faecal sludge, and is important for biological reactions as it is a measure of the capacity of the solution to resist acidification. A method for measuring alkalinity in faecal sludge is not included in this section, but a thorough explanation of how to measure alkalinity can be found in Method 2320 of the Standard Methods for the Examination of Water and Wastewater (Rice *et al.,* 2017).

## **<IP_ADDRESS> pH – electrode method35**

#### <IP_ADDRESS>.1 Introduction

This method is an electrometric measurement procedure to determine the pH of faecal sludge samples with different TS contents. This method is preferred over the colorimetric test strip method for faecal sludge due to its increased accuracy and precision, and the fact that pH readings obtained with this method are not influenced by the colour of the sample and can be made at higher TS contents than the pH test strip method. In this method, different sample preparation and measurement steps may be necessary depending on the TS of the sample. If the electrode does not produce a stable reading when inserted into a sample, the sample needs to be diluted before measuring the pH. For most types of faecal sludge (liquid, slurry, semi-solid, and some solid samples), dilution will not be necessary. However, for some solid samples or samples that have been dewatered or dried, dilution will be required.

#### <IP_ADDRESS>.2 Safety precautions

#### <IP_ADDRESS>.3 Required chemicals

#### <IP_ADDRESS>.4 Required apparatus and instruments

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

35 This method is adapted from USEPA Method 9040C (USEPA 2004a), USEPA Method 9045D (USEPA 2004b), Method 4500 from the Standard Methods for the Examination of Water and Wastewater (Rice *et al.,* 2017), and the UK Method for Measuring the pH of Food Products (Vijayakumar and Adedeji, 2017). This method should be cited as described in Velkushanova *et al.* (2021).

	- be cleaned with an ultrasonic bath, or,
	- be washed with detergent, rinsed several times with water, placed in 1:10 HCl so that the lower third of the electrode is submerged, and then thoroughly rinsed with water, or,
	- be cleaned as per the manufacturer's instructions.
	- It should be noted that adding distilled water to the sample may change the pH. If the pH of a sample can be measured without adding water, it should not be diluted.

## <IP_ADDRESS>.6 Sample preservation

Samples should be analysed as soon as possible.

## <IP_ADDRESS>.7 Sample preparation

## *For liquid and slurry samples:*

## *For semi-solid and solid samples:*

## *For dry samples (e.g. very 'thick' or 'dry' solid samples, dried end products such as pellets):*

## <IP_ADDRESS>.8 Analysis protocol

## *Calibration*

## *Measurement*

## *For liquid and slurry samples:*

magnetic stir bar to collide with the electrode (see Figure 8.10).

## *For semi-solid and solid samples:*

#### *For dry samples requiring water addition:*

## <IP_ADDRESS>.9 Calculation

No calculation required - direct reading.

#### <IP_ADDRESS>.10 Data set example

Ward *et al*. (2021) measured the pH of 465 faecal sludge samples collected from pit latrines and septic tanks in Lusaka, Zambia. pH was measured either on the day of collection or on the following day, with samples stored at 4 °C. Samples were homogenised with a blender before measuring the pH. The median pH value for all the samples was 7.7. TS values of the samples in this study ranged from < 0.5% ds to approximately 20% DS. None of the samples required dilution prior to pH measurement. The entire raw data set is included with publication36.

**Figure 8.10** Analysis of pH for liquid faecal sludge sample. Note position of pH electrode: submerged in the well‐mixed sample, but with enough clearance above the magnetic stir bar (photo: Eawag).

#### 36 https://doi.org/10.25678/00037X

## **<IP_ADDRESS> Electrical conductivity – electrode method37** <IP_ADDRESS>.1 Introduction

Electrical conductivity (EC) is a measure of the ability of an aqueous sample to conduct electric current. Samples with higher concentrations of ions have higher EC, and EC can be used as a proxy measurement for dissolved salt concentrations. EC in faecal sludge is reported in units of millisiemens per centimetre or microsiemens per centimetre. The symbol for siemens is written either as 'S' or 'mho' so EC measurements for faecal sludge may be reported as any of the following: mS/cm, mmhoS/cm, µS/cm, µmho/cm.

Different sample preparation and measurement steps may be necessary depending on the TS concentration of the sample. If the sample does not have high enough water content for a reading to be obtained, dilution will be required. For most types of faecal sludge (liquid, slurry, semi-solid, and some solid samples), dilution will not be necessary. However, for some solid samples or samples that have been dewatered or dried, dilution will be required.

## <IP_ADDRESS>.2 Safety precautions

## <IP_ADDRESS>.3 Required chemicals

#### <IP_ADDRESS>.4 Required apparatus and instruments

 Analytical balance capable of weighing 0.1 g (if pre-dilution of the sample is required)

#### <IP_ADDRESS>.5 Quality control

General information on quality assurance and quality control (QA/QC) is provided in Section 8.3. Information on standards, operating conditions and interferences that are specific to this method includes:

#### <IP_ADDRESS>.6 Sample preservation

Samples should be analysed as soon as possible.

## <IP_ADDRESS>.7 Sample preparation

#### *For liquid and slurry samples:*