Chapter 3 – Methodology

3.1 Research Design / Model Specification / Architectural

Design

This study employed a field-based observational design, leveraging my firsthand experience

traversing the Ido Local Government Area of Ibadan. The investigation was centered on

cassava processing sites scattered across this semi-rural region, which is characterized by

extensive cassava farming and small-scale garri production. I travelled to multiple villages

(such as Apata, Omi-Adio, Akufo, and Apete) over unpaved roads and footpaths, observing

how cassava tubers are transformed into food products and noting the environmental context.

At each local cassava mill, farmers grated and pressed fermented cassava mash to produce

starch or garri, discharging the residual cassava mill effluent (a cyanide-rich, acidic

wastewater) directly onto nearby land or into shallow pits. The pervasive sour odour and pale

coloration of the effluent-soaked soils were evident, underscoring anecdotal concerns that

such waste could be altering soil chemistry and posing risks to crops and water quality 1.

These field insights shaped the study’s design, prompting a systematic assessment of

contaminants in the soils, plants, and wastewater around these processing centres.

Population and Sample: The population of interest included all cassava processing sites and

the surrounding agricultural lands in Ido LGA. For practicality, a purposive sampling

approach was used to select representative processing centres known for high production

volumes and continuous effluent discharge. A total of eight (8) cassava processing sites were

selected, encompassing different communities within the LGA to capture geographic

variability. At each site, the target sample types were soil (at two depths), cassava plant
leaves, and cassava wastewater (effluent). By focusing on these matrices, the study aimed to

elucidate the contamination pathway from effluent to soil and crops.

Sampling Technique and Layout: Within each selected processing site, samples were

collected along a transect radiating outwards from the effluent source to represent a gradient

of exposure. Four distances were established for sampling: 0 m, 50 m, 200 m, and 1 km from

the effluent discharge point. The 0 m location (often at the point where effluent is released

onto soil) captures maximum impact, while 50 m and 200 m sites represent intermediate

exposure as one move away from the cassava mill. The 1 km site served as a control, located

in the same general area but sufficiently far (≥1000 m) from any cassava effluent influence.

This control site was typically upwind/upstream and outside the drainage path of the

processing waste, chosen to reflect baseline environmental conditions 2. At each distance,

sampling was done in replicates (generally in triplicate) to ensure that the data are

representative. In practice, multiple subsamples were taken and later composited to form a

representative sample per distance. The sampling layout is thus a nested design: at each of 8

sites, samples were taken at 4 distances, with each sample type collected in triplicate. This

design enables both a between-site comparison (to account for site-specific differences) and a

within-site analysis of contamination attenuation with distance.

Humanized Field Reflections: Implementing this design required considerable field

coordination. I set out at dawn, moving from the city centre to remote villages by motorcycle

or four-wheel drive, guided by local informants who knew the location of each cassava mill.

Travel between sites revealed a patchwork of farmlands and fallows; one could see women

peeling cassava under trees, youth operating grating machines, and shallow greyish pools

where effluent collected near processing areas. These on-the-ground observations reinforced

the rationale for the study: in many locations, cassava effluent was simply left to seep into the
soil, and cassava plants were sometimes growing in or around these discharge zones. The

personal interactions with villagers also built trust, allowing me to sample on private

farmlands. Overall, the study’s architectural design was both scientific and pragmatic –

scientific in its structured approach to sampling distances and media, and pragmatic in

adapting to field realities (such as impassable roads or the need to sample when processing

was actively occurring to obtain fresh effluent). This design provided a framework to

systematically assess how far and in what ways the pollutants (PAHs and heavy metals) from

cassava industrial activity might spread in the environment.

3.2 Sample Collection

Soil Samples: At each of the four designated distances (0 m, 50 m, 200 m, 1 km) from a

cassava processing point, soil samples were collected from two depth intervals –

surface/topsoil and subsoil. The topsoil samples were taken from 0–15 cm depth, while

subsoil samples were taken from 15–30 cm depth, using a clean stainless steel hand auger or

soil corer3. In each case, the soil surface debris was cleared, and the auger was driven to the

required depth to extract the soil. Three subsamples from the vicinity (within a 5 m radius of

the distance marker) were combined to form a composite sample for that depth and distance,

reducing spatial heterogeneity2. The composite was placed in a pre-cleaned plastic sample

bag. Large stones, roots, and debris were manually removed from the sample 2. The soil

samples were air-dried in the shade for 72 hours to reduce moisture, then gently crushed with

a sterile mortar and pestle. The dried soil was sieved through a 2 mm mesh (for

homogenization) and then through a 0.5 mm sieve for the portion intended for chemical

analysis2. Each processed soil sample was stored in an acid-washed polypropylene container,

labelled with the site, distance, and depth, and kept in a cool, dry place prior to analysis.
Cassava Plant (Leaf) Samples: Cassava leaves were sampled from cassava plants present at

or near the same 0 m, 50 m, 200 m, and 1 km points. At each location, several cassava plants

(3–5 plants) were selected randomly (or at 0 m, the plants visibly growing on effluent-

exposed soil were chosen). From each plant, a few mature green leaves (third or fourth fully-

expanded leaves from the top of the cassava stem) were cut using a stainless-steel knife and

pruning shears. The leaves (petioles removed) from the multiple plants were combined into

one composite leaf sample per location to account for plant-to-plant variability. The

composite fresh leaves were placed in clean paper bags. In the field, the leaves were gently

rinsed with distilled water to remove surface dust or soil particles, then air-dried. In the lab,

the leaves were further processed: they were oven-dried at 60 °C to constant weight (to

prevent mould and to stop enzymatic activity)2. The dried leaves became brittle and were then

ground into fine powder using a clean ceramic mortar or a laboratory mill. The powdered leaf

samples were sieved (<0.5 mm) to homogenize the particle size. Each sample was stored in a

sealed polyethylene bag for subsequent analysis of contaminants. By compositing multiple

leaves and plants, the sample captures an average contaminant level in cassava foliage at each

distance, which is relevant for assessing potential food-chain transfer (though cassava leaves

can be used as vegetables, here they serve as a bioindicator of soil/effluent contamination

uptake).

Cassava Effluent (Wastewater) Samples: Fresh cassava mill effluent was collected directly

from each processing site (typically at the point of discharge from the press or collection pit).

Using sterile polypropylene bottles (1 litre capacity), samples of the liquid effluent were

carefully taken. If the effluent was flowing, the bottle was held mid-stream (to avoid

sediment) and allowed to fill; if it was pooled, the sample was scooped from just below the

surface. About 2–3 litre of effluent was collected per site to ensure sufficient volume for all

laboratory analyses. Immediately upon collection, the effluent samples were sealed and
placed in a cooler with ice packs at ~4 °C to preserve their integrity4. The opaque sample

kegs protected the samples from sunlight, which is important because parameters like

biochemical oxygen demand (BOD) or certain metal species could change with biological

activity or photolysis. All effluent samples were collected during active processing periods to

ensure they were fresh and representative (cassava effluent characteristics can vary daily, so

timing was coordinated with processing schedules).

After collection, sample handling followed strict protocols to avoid cross-contamination.

New disposable gloves were worn for each sample type and site. Soil sampling tools were

cleaned with distilled water and wiped with isopropanol between sites. Plant samples were

handled with washed tools and clean surfaces. Effluent bottles were pre-rinsed with the

effluent before final filling to condition the container. All samples – soil, leaves, and effluent

– were clearly labeled with an anonymized code (no personal identifiers, only site/distance

info) and logged in a field notebook with GPS coordinates of each sampling point. At the end

of each sampling day, the cooler with effluent and plant samples (which required cooler

temperatures) was transported back to base. Chain-of-custody procedures were observed:

samples were accompanied by documentation from field to laboratory, and no unauthorized

persons handled them.

Finally, all collected samples were transported under sterile, controlled conditions to the

analytical laboratory. The soil and plant samples, being dry, were kept in clean dry boxes,

while the effluent samples remained in the iced cooler. The destination was the

Environmental Investigation and Support Laboratory (EISL) in Port Harcourt – a certified

analytical lab. Samples arrived at EISL within 24–48 hours of collection. There, they were

stored appropriately (effluents refrigerated at 4 °C, soils and plant powders at room

temperature in a desiccated environment) until analysis commenced. This careful collection

thereby yielding reliable analytical results.

3.3 Sample Analyses

The analysis phase was subdivided based on sample type, with standardized procedures

applied to each category of sample. All methods were selected to quantify either polycyclic

aromatic hydrocarbons (PAHs) or heavy metals (the target contaminants), as well as general

physicochemical parameters of environmental significance. Wherever possible, analyses

followed internationally recognized standard methods (such as APHA Standard Methods or

ASTM protocols) to ensure accuracy and comparability. Each subsection below details the

approach for crops (cassava leaves), soils, and effluents respectively.

3.3.1 Crop Analysis (Cassava Leaves)

Preparation of Samples: The powdered cassava leaf samples obtained from Section 3.2

were used for all analyses in this section. Prior to chemical analysis, the leaf powders were

further dried in an oven at 105 °C for 2 hours to remove any residual moisture 2. This step

prevents moisture from interfering in weight-based concentrations and ensures consistent

results. The dry weight of each sample was recorded for use in concentration calculations

(results for heavy metals and PAHs in plants are typically reported on a dry-weight basis).

Heavy Metal Analysis in Leaves: The concentrations of heavy metals in cassava leaves

(specifically, metals of concern such as lead (Pb), cadmium (Cd), chromium (Cr), zinc (Zn),

nickel (Ni), and possibly iron (Fe) and copper (Cu)) were determined by atomic absorption

spectrophotometry (AAS). Prior to AAS measurement, the leaf samples underwent acid

digestion to extract the metals into solution. Approximately 1.0 g of the dried leaf powder
was weighed into a borosilicate glass digestion flask. A mixture of concentrated acids –

typically nitric acid (HNO₃) and perchloric acid (HClO₄) in a ratio of 4:1 – was added to the

sample2. (This nitric-perchloric acid digestion is a standard method for plant tissues,

effectively destroying organic matter and releasing metals into the liquid phase). The flask

was heated gently on a hotplate under a fume hood until dense white fumes cleared,

indicating the completion of digestion. After cooling, the digest was filtered (Whatman

No. 42 filter paper) into a 50 mL volumetric flask and diluted to volume with deionized

water. This clear solution – containing the dissolved metals from the plant – was then

analysed by AAS. The AAS instrument was calibrated with certified standard solutions for

each target metal (for example, Pb at 283.3 nm, Cd at 228.8 nm, etc.)5. Calibration curves

with multiple points were run to ensure linearity, and a blank digest (with no plant material)

was also prepared to check for contamination. Each metal’s concentration in the leaf digest

was read at the appropriate wavelength and optimized lamp conditions on the AAS, following

the instrument’s standard conditions. The method used corresponds to APHA and AOAC

guidelines for metals in plant/biological samples; the atomic absorption readings at specific

wavelengths provide quantitative results5. Quality control measures included analysing a

standard reference plant material and spiking a sample with a known metal amount to check

percent recovery. Heavy metal concentrations were reported in milligrams of metal per

kilogram of dry leaf (mg/kg dry weight).

PAH Analysis in Leaves: To assess polycyclic aromatic hydrocarbon contamination in

cassava leaves, a solvent extraction followed by chromatographic analysis was performed.

About 10 g of dried cassava leaf powder was placed in a Soxhlet extractor thimble. PAHs

were extracted using an organic solvent mixture (commonly n-hexane: acetone or

dichloromethane) over a period of several hours (e.g., 6–8 h)6. The warm solvent

continuously cycled through the sample, dissolving PAH compounds out of the plant matrix.
The resulting extract was concentrated (evaporated) to a small volume (1–2 mL) using a

rotary evaporator at low temperature. This concentrate was then subjected to gas

chromatography–mass spectrometry (GC–MS) analysis, following the U.S. EPA Method

8270 protocol for semi-volatile organics6. Before GC–MS injection, an internal standard mix

of deuterated PAHs was added to each sample extract to aid quantification and recovery

tracking. The GC–MS was operated in selected ion monitoring mode targeting the PAH

compounds of interest (such as naphthalene, phenanthrene, fluoranthene, chrysene,

benzo[a]pyrene, etc.). The separation occurred on a fused-silica capillary column, and the

mass spectrometer scanned diagnostic fragment ions for each PAH. Identification of each

PAH in the cassava leaf extract was based on matching retention times and mass spectra with

those of authentic standards. Quantification used the internal standard method, comparing the

peak areas of target PAHs to those of calibration standards run under identical conditions 6.

Standard quality checks (including calibration verification, method blanks, and replicate

analyses) were implemented according to EPA 8270 guidelines. The results were expressed

in micrograms of PAH per kilogram of leaf (µg/kg dry weight). This method aligns with

widely accepted procedures for PAH determination in environmental plant samples – solvent

extraction followed by GC–MS provides both sensitivity and specificity for detecting the

suite of priority PAHs6. Detecting PAHs in cassava leaves can indicate atmospheric

deposition of these pollutants (e.g. from burning biomass or vehicular emissions) or uptake

from contaminated soils; however, cassava leaves are not typically a significant sink for

PAHs, so detection (if any) was expected to be low.

The soil samples (topsoil and subsoil composites from each distance) were subjected to a

comprehensive set of analyses to characterize both their general physicochemical properties

and the levels of contaminants (heavy metals and PAHs).

Physicochemical Parameters of Soil: Several key soil properties were measured using

standard soil science methods to understand the baseline soil conditions and possible impact

of cassava effluent:

 pH: Soil pH was determined in a 1:2.5 soil-to-water suspension using a calibrated

digital pH meter7. For each sample, 10 g of air-dried soil was mixed with 25 mL of

distilled water and shaken for 30 minutes. After allowing the suspension to settle, the

pH electrode (glass electrode) was inserted into the supernatant and the reading

recorded once stabilized8. The pH meter was standardized with pH 4.0, 7.0 (and 10.0

if needed) buffer solutions before measurements. This method corresponds to AOAC

and APHA standard procedures for soil pH8. Soil pH is important as cassava effluent

can be acidic; changes in soil pH at effluent-exposed sites could indicate buffering or

acidification due to the waste.

 Total Organic Carbon (TOC): The organic carbon content of the soil was measured

by the Walkley-Black wet oxidation method 9. In this classic procedure, ~0.5 g of

finely ground soil was treated with a known excess of 1 N potassium dichromate

(K₂Cr₂O₇) solution and concentrated sulfuric acid, which oxidizes organic carbon to

CO₂. After digestion (and cooling), the remaining dichromate was titrated with

ferrous ammonium sulphate using ferroin indicator to determine how much

dichromate was consumed by oxidizing the soil organic matter 8. The TOC (%) was

calculated from the titration results, applying a correction factor for incomplete
 oxidation (Walkley-Black typically assumes ~77% efficiency unless a heat catalyst is

used). This method is a standard in soil chemistry. It provides insight into soil organic

matter, which can influence contaminant binding; for instance, soils high in organic

carbon might retain more PAHs (since PAHs are hydrophobic and bind to organic

matter).

 Available Phosphate (PO₄³⁻): Available phosphorus in soil was determined by the

Bray P-1 method (suitable for acidic soils common in Southern Nigeria) 9. A known

weight of soil (5 g) was extracted with Bray P-1 extractant (0.03 N NH₄F + 0.025 N

HCl) by shaking for 5 minutes. The extract was filtered, and the phosphate in the

extract was then measured calorimetrically by the ascorbic acid molybdenum blue

method8. In this colorimetric test (APHA 4500-P E), ammonium molybdate and

antimony potassium tartrate react with orthophosphate in the sample to form a

phosphomolybdate complex, which is reduced by ascorbic acid to yield a blue-

coloured solution8. The intensity of the blue colour, measured at 882 nm on a

spectrophotometer, is proportional to the phosphate concentration8. A series of

phosphate standard solutions was run to prepare a calibration curve. Results were

reported as mg of available P per kg of soil. This method (Bray-1 with ascorbic acid

finish) is widely used and referenced in agronomic studies for available soil

phosphorus. It helps to determine if cassava effluent (which may contain some

phosphorus from cell material) has altered soil fertility or not.

 Exchangeable Cations (Ca²⁺, Mg²⁺, K⁺, Na⁺): The major exchangeable bases in

soil – calcium, magnesium, potassium, and sodium – were extracted using neutral 1 M

ammonium acetate solution (at pH 7) according to standard soil testing protocols 2. In

this procedure, 10 g of soil was leached with ammonium acetate, which displaces Ca,

Mg, K, and Na from the soil’s cation exchange sites. The filtrate extract was
 collected, and the concentrations of Ca²⁺ and Mg²⁺ were determined using the

Atomic Absorption Spectrophotometer (AAS), while K⁺ and Na⁺ were determined by

flame photometry or AAS10. The AAS for Ca and Mg was calibrated with known

standards and operated at the appropriate wavelengths (e.g., Ca at 422.7 nm, Mg at

285.2 nm), and emission readings for K and Na were taken (or AAS in emission

mode)10. This method follows standard procedures in soil analysis (Jackson, 1962; or

current ASTM D** methods) and has been validated for accuracy in numerous

studies10. Exchangeable cation results (in cmol(+)/kg or in mg/kg) indicate soil

fertility and reflect if effluent inputs (which might add sodium or other salts) have

affected soil chemical balance. For instance, a high Na level could indicate sodicity

from the effluent, whereas Ca and Mg could be diluted if effluent leaching occurs.

 Other Soil Parameters: Additional soil tests were performed such as moisture

content (by gravimetric oven-drying at 105 °C), particle size distribution (via

hydrometer or sieve analysis to classify soil texture), and possibly total nitrogen (by

Kjeldahl method) if relevant to understanding fertility. These are ancillary to the main

aims but help describe the soil matrix. Moisture content ensures comparison on a dry-

weight basis, and texture can influence contaminant retention (clay vs sand). For

brevity, detailed methods for these are not expanded here, but standard protocols

(ASTM D2216 for moisture, hydrometer method for particle size, etc.) were followed.

Heavy Metal Analysis in Soil: The determination of heavy metals in soil samples was

executed using acid digestion followed by Atomic Absorption Spectrophotometry, analogous

in concept to the plant analysis but with a stronger digestion to account for the soil’s mineral

matrix. Approximately 1.0 g of the dried, sieved soil was placed in a Teflon digestion vessel.

A two-step acid digestion was performed: first, the sample was treated with 10 mL of

concentrated nitric acid (HNO₃) and heated gently to oxidize organic matter and leach
readily soluble metals; after cooling, 5 mL of concentrated hydrochloric acid (HCl) was

added (a procedure similar to EPA Method 3050B or the aqua regia digestion approach) 2. The

mixture was heated to near boiling for about 2 hours. Optionally, a few drops of perchloric

acid or hydrogen peroxide could be added to ensure complete breakdown of organics. (In

some cases, for total metals including those in silicate lattices, a hydrofluoric acid step is

used, but given safety concerns, the aqua regia digest suffices for most heavy metals of

interest, though it may not capture all silicate-bound fractions.) After digestion, the sample

was filtered and diluted to a known volume with deionized water. The resulting solution was

then analysed by AAS for the target metals Pb, Cd, Cr, Zn, Ni, Cu, and Fe. Each metal was

quantified at its specific analytical wavelength with calibration standards run in the same acid

matrix. Blanks and duplicate digestions were also carried through to monitor any

contamination or analytical precision. The AAS method is consistent with APHA (3120 B for

ICP or 3111 B for AAS) and ASTM standards for metals in environmental samples. For

quality assurance, standard reference soil materials (with known metal contents) were also

digested and analyzed, and the recovery was checked to fall within acceptable ranges

(generally 85–115%). Heavy metal concentrations in soil were reported in mg/kg (dry

weight). These data reveal the degree of soil contamination at varying distances; for instance,

significantly elevated heavy metals at 0 m relative to 1 km would indicate contamination

from the cassava effluent, especially if the metals follow a decreasing trend with distance.

PAH Analysis in Soil: The analysis of PAHs in soil involved extraction of soil samples

followed by GC–MS, similar to the procedure for plant samples but with a more rigorous

extraction method due to the complex soil matrix. About 20 g of dried soil from each sample

was thoroughly mixed with anhydrous sodium sulfate (to ensure dryness) and loaded into a

Soxhlet extractor or an automated extractor system. PAHs were extracted using an organic

solvent such as dichloromethane or hexane/acetone (EPA Method 3540C – Soxhlet

typically ran for 16–24 h in Soxhlet (or a shorter cycle in pressurized extraction) until the

solvent in the thimble was clear, indicating exhaustive extraction. The extract was then

concentrated and subjected to a clean-up step: it was passed through a silica gel or alumina

column to remove polar interferents, yielding a cleaner hydrocarbon fraction. The purified

extract was concentrated to 1 mL and spiked with surrogate/internal standards. PAH analysis

was carried out on a GC–MS system as described for leaves. The instrument was calibrated

for 16 EPA priority PAHs (from naphthalene to benzo[g,h,i] perylene) across a suitable range

(e.g., 0.1 to 10 mg/L standards). The GC–MS method (comparable to EPA 8270D for solids)

was tuned to detect PAHs with appropriate ions; for example, the molecular ion and key

fragments for each PAH were monitored. Identification was confirmed by matching the

retention time and ion ratios to those of calibration standards. To ensure method fidelity, a

method blank (with clean Ottawa sand) and a spiked matrix sample were run with each batch

of soil samples. The surrogate recovery (often deuterated PAHs added at the start of

extraction) was checked to be within control limits (typically 70–130%). PAH concentrations

in soil were reported in µg/kg. This analysis allows us to see if cassava processing sites

(which might involve wood fires for roasting garri, or fuel use, etc.) have PAH residues in

soil. However, it’s more expected that heavy metals might be present from machinery (wear

of grater teeth, etc.)1 or from contaminated water, whereas PAHs might not be a primary

contaminant from cassava processing itself unless there is significant smoke deposition. The

results would nonetheless show the environmental baseline for PAHs in these agricultural

soils.

3.3.3 Effluent (Wastewater) Analysis

parameters to characterize the wastewater and identify potential pollutants. The methods

largely followed Standard Methods for the Examination of Water and Wastewater (APHA)

protocols and are itemized below:

 pH: The pH of the effluent was measured in the lab using a calibrated pH meter with

a glass electrode, as per APHA 4500-H⁺ method. Before measurement, each effluent

sample was well mixed. The electrode was standardized with pH 4.0, 7.0, and 10.0

buffers. Then it was immersed in a beaker containing the effluent sample and the pH

was recorded once stable. Cassava effluent is typically acidic (due to organic acids

from fermentation), so pH is a critical parameter indicating its corrosivity and

potential impact on soils.

 Electrical Conductivity (EC): EC was measured using a conductivity meter (APHA

2510 B). The meter (calibrated with KCl standard solution for 1413 µS/cm) had its

probe dipped into the effluent sample at room temperature, and the reading in

microSiemens per centimeter (µS/cm) was taken when stabilized. Conductivity

reflects the total ionic content (salts) of the wastewater, which could include cyanide,

chloride, sulfate, etc. A high EC in effluent can lead to soil salinization.

 Total Dissolved Solids (TDS): TDS was determined gravimetrically following

APHA 2540 C. A known volume of filtered effluent (e.g., 100 mL) was passed

through a pre-weighed glass-fiber filter paper. The filtrate (containing dissolved

solids) was collected in a clean evaporating dish. Separately, the increase in weight of

the filter paper represents total suspended solids, and the residue from filtrate upon

evaporation represents TDS. In practice, APHA suggests evaporating the filtered

sample in a drying oven at 180 °C until dry, then cooling and weighing. However, the

procedure in this study followed a simplified approach where the filter paper was
 dried at 105 °C for a short time and re-weighed to get TSS, and by difference from a

total solids measurement the TDS was inferred. For clarity: we measured Total Solids

(TS) by evaporating 100 mL of well-mixed effluent in a pre-weighed flask at 105 °C

to constant weight. We also measured Total Suspended Solids (TSS) by capturing

solids on a filter and drying it. Then TDS was calculated as TS – TSS. Results were

expressed in mg/L. These parameters indicate the load of dissolved and particulate

matter in the effluent, which affect water quality and soil pore clogging.

 Total Alkalinity and Bicarbonate: The alkalinity of the effluent (mainly due to

bicarbonate since cassava effluent contains fermentative CO₂ which can form

HCO₃⁻) was measured by titration (APHA 2320 B). 50 mL of effluent was titrated

with 0.1 N sulfuric acid using phenolphthalein and methyl orange indicators in a two-

step titration. Phenolphthalein alkalinity was first determined (dropwise addition of

acid until pH ~8.3, phenolphthalein endpoint faint pink to clear), then methyl orange

was added and titration continued to pH ~4.5 (methyl orange endpoint from yellow to

orange). The volumes of acid used allowed calculation of bicarbonate (HCO₃ ⁻) and

total alkalinity (expressed as mg/L CaCO₃). Bicarbonate concentration was derived

from phenolphthalein alkalinity if any (for carbonate) and total for bicarbonate. Given

cassava effluent is often acidic, alkalinity might be low; nonetheless this test indicates

buffering capacity.

 Chloride (Cl⁻): Chloride content in the effluent was determined by the

Argentometric method (Mohr’s titration), consistent with APHA 4500-Cl⁻ B. 100 mL

of effluent sample was placed in a flask, and 1 mL of potassium chromate (K₂CrO₄)

indicator was added. This solution was titrated with standard 0.014 N silver nitrate

(AgNO₃) solution. Silver nitrate reacts with chloride to form silver chloride

precipitate; once all chloride is consumed, the first excess drop of AgNO₃ reacts with
 the chromate indicator to form a reddish-brown silver chromate precipitate, marking

the endpoint (color change from yellow to light reddish brown). The volume of

AgNO₃ used was plugged into the formula to compute chloride concentration in

mg/L. This method is a classic argentometric titration and was performed carefully to

avoid overshooting the endpoint. Chloride in cassava effluent could originate from the

cassava itself or from process water; it’s important for assessing potential

groundwater contamination and corrosion potential.

 Sulphate (SO₄²⁻): Sulphate was measured using a turbidimetric method (APHA

4500-SO₄²⁻ E). A measured volume of effluent (typically 50 mL) was treated with

conditioning reagents including glycerol and NaCl (to control solubility and ionic

strength) and then barium chloride crystals were added. Sulphate reacts with Ba² ⁺ to

form a barium sulphate suspension. After a set time of gentle stirring, the turbidity of

the suspension was measured at 420 nm with a spectrophotometer, comparing against

a calibration curve prepared with known sulphate standards. The result gives sulphate

concentration in mg/L. (Alternatively, if available, an ion chromatography technique

could be used; however, the turbidimetric method is straightforward and commonly

used in water analysis.) Sulphate in cassava effluent might be low unless sulphate-rich

water or additives were used; measuring it helps in understanding if any significant

anionic load besides organic acids is present.

 Nitrate (NO₃⁻): Nitrate was determined by a colorimetric method (such as the

brucine sulphate method or cadmium reduction method) per APHA 4500-NO₃ ⁻. In

this study, a simple UV-spectrophotometric screening was done: the effluent sample

was filtered, and its absorbance at 220 nm (where nitrate has a strong absorbance) and

275 nm (for interference correction) were measured. Using APHA guidelines, the

difference in absorbance (corrected for organic interference at 275 nm) was used to
 estimate nitrate concentration, with calibration from KNO₃ standards. Because

cassava effluent is rich in organic nitrogen (from proteins in cassava tubers) and not

initially in nitrate, this step was more to see if any nitrification had occurred in

standing effluent or if background water contributed nitrates. Results were expressed

in mg/L NO₃⁻-N.

 Dissolved Oxygen (DO): DO in freshly collected effluent was measured on-site

where possible using a portable DO meter (or by Winkler titration in the lab if the

field measurement was not feasible). The Winkler method (APHA 4500-O) involves

fixing the oxygen with manganese sulphate and alkaline iodide reagents in a BOD

bottle at the time of collection, then titrating the liberated iodine with sodium

thiosulfate in the lab. However, cassava effluent typically has very low DO (high

BOD load depletes oxygen). Field DO meter readings (after calibration in air) were

taken by immersing the probe in the effluent pool; values were recorded in mg/L.

Low or zero DO was observed, as expected, indicating a strong oxygen demand.

 Chemical Oxygen Demand (COD): COD was determined by the dichromate reflux

titration method (APHA 5220 B). In this method, 2.5 mL of effluent was refluxed

with 1.5 mL of standard potassium dichromate solution in the presence of 3.5 mL

concentrated sulfuric acid and silver sulphate catalyst (mercury sulphate can be added

to mask chloride if chloride is high). The mixture was heated for 2 hours at 150 °C.

After digestion, the remaining dichromate was titrated with standardized ferrous

ammonium sulphate (FAS) using ferroin indicator. The colour changes from bluish-

green to reddish-brown signifies the endpoint. A blank (distilled water) and a standard

glucose or potassium hydrogen phthalate solution were run in parallel for quality

control. COD results were calculated in mg/L O₂. COD represents the amount of

organic (and oxidizable inorganic) matter in the effluent and is a key pollution
 parameter. Cassava effluent is known to have extremely high COD due to starches,

sugars, and cyanogenic compounds; this test quantifies that load.

 Phosphate (PO₄-P): Soluble orthophosphate in the effluent was measured by the

same ascorbic acid colorimetric method described for soil extracts, but here applied

directly to the water sample (after appropriate dilution if needed). 5 mL of filtered

effluent was treated with ammonium molybdate-antimony reagent and ascorbic acid,

and the blue colour measured at 882 nm. The APHA 4500-P E method was followed,

and results were reported as mg/L PO₄³⁻ (or as P). Cassava effluent might contain

some phosphate from cell contents, though not typically very high. This parameter,

along with nitrate, indicates nutrient pollution potential (eutrophication risk).

 Other Parameters: In addition to the above, the study measured Biochemical

Oxygen Demand (BOD₅) on the effluent. A 5-day BOD test was set up by diluting

the effluent with oxygen-saturated water, seeding with microbial inoculum, and

incubating in BOD bottles at 20 °C in the dark for 5 days. DO was measured initially

and after 5 days; the drop in DO gave the BOD₅ (with corrections for dilution and any

seed). This standard test (APHA 5210 B) indicates the biodegradable organic load.

Given the high COD, BOD was expected to be high as well; values were expressed in

mg/L O₂ consumed. Furthermore, cyanide content was an important parameter

(cassava effluent contains linamarin which can hydrolyse to cyanide). Although not

explicitly listed in the prompt, literature suggests cyanide is a critical effluent

pollutant. If measured, it would be by a titrimetric or colorimetric method (e.g.,

APHA 4500-CN with pyridine-pyrazalone or an ion-selective electrode). For

completeness, we note that total cyanide was analysed by first distilling the sample in

an acid setup to release HCN, which was captured in NaOH and then quantified

calorimetrically. Results (if measured) would be in mg/L CN⁻.

standards) to ensure data quality and acceptance. For example, pH, alkalinity, chloride,

sulphate, etc., followed APHA 23rd edition guidelines; heavy metals followed APHA or

ASTM D methods for AAS; and organics (PAHs) followed U.S. EPA methods integrated

into standard lab practices. Each test included proper blanks, calibration standards,

duplicates, and spikes where applicable. The laboratory (EISL) that conducted the analyses is

ISO-certified, and all instruments were calibrated and maintained as per manufacturers’

recommendations.

In summary, the effluent analysis provided a profile of the cassava wastewater: low pH, high

organic load (COD/BOD), significant cyanide, and possibly elevated suspended solids and

nutrients, all of which help explain the potential for soil and crop impact. These data

complement the soil and crop analyses by linking the source characteristics to the observed

environmental concentrations.

3.4 Data Analysis

After laboratory analyses, the raw data were compiled and subjected to statistical analysis to

address the research questions. The approach combined descriptive statistics (to summarize

concentrations and properties) with inferential statistics (to test hypotheses about differences

between groups, such as between distances or between sample types).

Initially, all concentration values for heavy metals, PAHs, and other parameters were

tabulated for each site, distance, and sample type. Using Microsoft Excel and SPSS

(Statistical Package for the Social Sciences, Version 25), summary statistics were computed:

mean, standard deviation (SD), minimum, maximum, and coefficient of variation for each

variable. These descriptive stats provided an overview of central tendencies and variability.
For example, the mean concentration of Pb in topsoil at 0 m versus at 1 km could be

compared, and the SD indicated how much variation existed among the replicate samples

To infer patterns and significance:

 Analysis of Variance (ANOVA): A one-way ANOVA was performed for key

response variables (e.g., heavy metal levels in soil) with distance as the factor. For

each metal in soil, we tested whether the mean concentrations at 0 m, 50 m, 200 m,

and 1 km were significantly different (at α = 0.05). The assumption of ANOVA

(approximate normality and equal variances) was checked; data were log-transformed

if needed to stabilize variances. If the ANOVA F-test indicated a significant effect of

distance (p < 0.05), it implied that contamination levels differed depending on

proximity to the effluent source. In such cases, a post-hoc test (Tukey’s HSD) was

applied to determine which pairwise distances differed. Similarly, ANOVAs were

conducted for heavy metal concentrations in cassava leaves across distances, and for

comparing topsoil vs subsoil (two-way ANOVA if treating depth and distance as two

factors). For effluent parameters, which were measured per site (not by distance),

ANOVA was not applicable; instead, simple comparisons with regulatory standards

or literature values were made.

 Descriptive Comparisons: The means of parameters at effluent-exposed soils (0 m)

were compared with means at control soils (1 km). A t-test (independent two-sample

t-test) was used for each metal to see if the 0 m vs 1 km difference was statistically

significant. This serves as an alternative test directly addressing contamination (since

control sites ideally reflect background levels). Likewise, cassava leaves from

polluted sites were compared to control-site leaves. These tests complement ANOVA
 and can be easier to interpret (effectively testing the hypothesis that effluent has no

effect, by comparing impacted vs control).

 Correlation Analysis: Pearson correlation coefficients were computed to explore

relationships between variables, for instance between metal levels in soil and in

cassava leaves. A strong positive correlation (r close to 1) between, say, soil lead and

leaf lead across sites would suggest uptake of lead by cassava plants. Also,

correlations among different heavy metals in soil were examined to see if they

originate from a common source (effluent) or have different behaviors.

 Regression Modeling: In a few cases, simple linear regression was used to model the

decay of contaminant concentration with distance. Distance (in log scale) was the

independent variable and concentration the dependent. The fit of these models (R²

values) provided insight into how predictably contamination decreases away from the

source. For example, a high R² in a regression of soil Zn vs log(distance) would imply

a clear gradient attributable to the effluent source.

The statistical analyses were carried out using SPSS, ensuring that each dataset was coded

correctly (factor levels for distances, etc.). Results were considered significant at the 95%

confidence level (p < 0.05). Findings were typically reported with the mean ± SD for each

group. Where ANOVA indicated differences, the text specifies which groups were higher or

lower (e.g., “significantly higher Pb in 0 m soils than in 200 m and control soils, p < 0.01”).

Data were presented in tables and graphs for clarity – for instance, bar charts with error bars

(±SD) showing concentration vs distance for various parameters.

Through this statistical evaluation, the study distilled the laboratory results into meaningful

evidence regarding the impact of industrial cassava activities. The combination of descriptive

and inferential statistics helped confirm whether the hypothesized patterns (e.g., highest
pollution at the source, diminishing with distance, and elevated levels in soils and crops near

the source) were supported by data. This rigorous analysis adds credibility to the conclusions

drawn and helps quantify the uncertainties in measurement.

3.9 Ethical Approval

No formal ethical approval was required for this study. The research did not involve human

subjects or personal data, focusing instead on environmental samples (soil, plants, and water)

and standard laboratory analyses. However, the study was conducted with a strong sense of

ethical and professional responsibility. Permission was obtained from local landowners and

cassava processors before collecting samples on their premises, and the purpose of the study

was explained to all stakeholders in understandable terms. All sampling and disposal of

chemical wastes in the lab were done in an environmentally responsible manner, following

the guidelines of the University’s research ethics and safety committees (where applicable).

Since only environmental materials were involved, institutional ethical review was not

mandatory; nevertheless, the researcher adhered to all relevant regulations and best practices

to ensure safety and integrity in the research process.

Endnotes (Turabian References):

2023. “Removal of Toxic Substances from Cassava Effluent Using Alum and

Moringa oleifera Seeds as Coagulants: A Comparative Study towards Improved

Agricultural Practice.” Ife Journal of Science 25(1) (May): 75–[Link]

2. Harrison, U. E., S. R. Osu, and J. O. Ekanem. 2018. “Heavy Metals Accumulation in

Leaves and Tubers of Cassava (Manihot esculenta Crantz) Grown in Crude Oil

Contaminated Soil at Ikot Ada Udo, Nigeria.” Journal of Applied Sciences and

Environmental Management 22(6): 845–[Link]

3. Adejoh, I. P. 2016. “Assessment of Heavy Metal Contamination of Soil and Cassava

Plants within the Vicinity of a Cement Factory in North Central, Nigeria.” Advances

in Applied Science Research 7(3): 20–[Link]

4. Okoye, Rosemary, Samuel Onuorah, and Oluchi Abba. 2023. “Evaluation of the

Impact of Untreated Cassava Mill Effluent on the Physicochemical Properties of Soil

in Aba, Abia State, Nigeria.” Malaysian Journal of Science and Advanced

Technology 3(2): 101–[Link]

5. American Public Health Association (APHA). 2017. Standard Methods for the

Examination of Water and Wastewater. 23rd ed. Washington, DC: APHA-AWWA-

[Link]

6. Zhang, Yu-ge, Min Xiao, Yi-hua Dong, and Yong Jiang. 2012. “Determination of Soil

Exchangeable Base Cations by Using Atomic Absorption Spectrophotometer and

Extraction with Ammonium Acetate.” Guang Pu Xue Yu Guang Pu Fen Xi

(Spectroscopy and Spectral Analysis) 32(8): 2242–[Link]

7. Teklab, Inc. 2023. “EPA Method 8270 Analysis – Semivolatile Organic Compounds

(SVOCs) by GC/MS.” Teklab Analytical Testing Services. Accessed May 26, 2025.

[Link]
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