Wastewater Laboratory Analyses

Learn common wastewater laboratory analyses, including BOD, COD, TSS, VSS, pH, alkalinity, dissolved oxygen, ammonia, nitrate, phosphorus, settleability, solids tests, and operator interpretation.

Wastewater operators rely on laboratory and field analyses to understand influent loading, biological activity, solids separation, nutrient removal, treatment efficiency, and final effluent quality. Individual results are useful, but the strongest operational decisions come from interpreting several related measurements together.

This article focuses on the meaning and practical use of common wastewater analyses rather than detailed permit limits or analytical procedures, which should always be checked against the applicable method and facility requirements.

Why Wastewater Laboratory Analyses Matter

Laboratory and field data help operators answer questions such as:

How much organic load is entering the plant?
Is biological treatment removing that load effectively?
Are solids settling and separating properly?
Is nitrification occurring?
Is phosphorus being removed?
Is aeration meeting process demand?
Are final effluent conditions changing?

Field Measurements Versus Laboratory Analyses

Some parameters are commonly measured directly in the process because they can change rapidly.

Examples include:

temperature;
pH;
dissolved oxygen;
chlorine residual;
ORP.

Other tests generally require laboratory preparation, incubation, filtration, digestion, weighing, or colorimetric analysis.

Biochemical Oxygen Demand

Biochemical Oxygen Demand, or BOD, is a measure of the oxygen used by microorganisms while degrading biodegradable organic material under defined test conditions.

BOD is one of the most important wastewater measurements because it represents the biodegradable organic load placed on the treatment process.

BOD5

A common form is BOD5, in which oxygen depletion is measured over a five-day incubation period under specified conditions.

The result is commonly reported in:

mg/L

BOD Is Not the Same as DO

Dissolved oxygen measures oxygen currently present in water.

BOD represents the amount of oxygen microorganisms consume while degrading biodegradable material.

High Influent BOD

High influent BOD can increase:

biological loading;
oxygen demand;
aeration requirements;
sludge production.

Effluent BOD

Elevated effluent BOD can indicate:

incomplete biological treatment;
solids carryover;
process upset;
poor clarification;
sample or analytical problems.

BOD Removal Efficiency

A common process calculation is:

Removal Efficiency, % = (Influent BOD - Effluent BOD) ÷ Influent BOD × 100

BOD Removal Example

If influent BOD is 220 mg/L and effluent BOD is 22 mg/L:

Removal = (220 - 22) ÷ 220 × 100

Removal = 90%

BOD Test Limitations

BOD is useful but has limitations.

It:

requires several days;
depends on biological activity;
can be affected by toxic substances;
can be affected by nitrification unless the method addresses it appropriately.

Chemical Oxygen Demand

Chemical Oxygen Demand, or COD, measures the oxygen equivalent of material oxidized by a strong chemical oxidant under defined test conditions.

COD generally provides results much faster than BOD.

COD Versus BOD

COD and BOD are related but different.

COD may include material that is:

biodegradable;
slowly biodegradable;
not readily biodegradable in the BOD test.

COD is therefore often higher than BOD for the same wastewater.

Using COD as a Process Indicator

Facilities may use COD trends to obtain faster information about changing organic loading.

A plant-specific historical relationship between COD and BOD can be useful, but it should not be assumed to be identical at every facility.

Total Suspended Solids

Total Suspended Solids, or TSS, measures suspended material captured according to the analytical method.

TSS is commonly reported in:

mg/L

What TSS Represents

TSS may include:

biological solids;
inorganic particles;
precipitated chemicals;
other suspended material.

Influent TSS

Influent TSS represents suspended solids entering the plant.

Higher influent TSS can increase:

primary sludge production;
solids loading;
downstream treatment demand.

Effluent TSS

Elevated effluent TSS may indicate:

poor clarifier settling;
solids washout;
rising sludge;
filamentous bulking;
hydraulic overloading.

TSS Removal Efficiency

A common calculation is:

Removal Efficiency, % = (Influent TSS - Effluent TSS) ÷ Influent TSS × 100

Volatile Suspended Solids

Volatile Suspended Solids, or VSS, are the portion of suspended solids lost during ignition under the analytical method.

VSS is often used as an approximate indicator of the organic or biological fraction of suspended solids.

Fixed Suspended Solids

The portion remaining after ignition is often described as fixed or inorganic suspended solids.

A simplified relationship is:

Fixed Suspended Solids = TSS - VSS

VSS Percentage

A useful calculation is:

VSS, % of TSS = VSS ÷ TSS × 100

VSS Example

If TSS is 3,000 mg/L and VSS is 2,250 mg/L:

2,250 ÷ 3,000 × 100 = 75%

Approximately 75% of the suspended solids are volatile by this analytical definition.

Mixed Liquor Suspended Solids

Mixed Liquor Suspended Solids, or MLSS, is the suspended-solids concentration in an activated-sludge aeration basin.

MLSS includes:

biological solids;
inert organic solids;
inorganic suspended solids.

Mixed Liquor Volatile Suspended Solids

Mixed Liquor Volatile Suspended Solids, or MLVSS, represents the volatile portion of MLSS.

It is commonly used as an approximate indicator of biological and organic solids in mixed liquor.

MLVSS Is Not Pure Active Biomass

MLVSS includes more than living microorganisms.

It may also include:

dead biomass;
nonliving organic material;
other volatile solids.

Settleability Test

A settleability test evaluates how activated-sludge solids settle in a graduated container over a specified time.

A common observation is the settled sludge volume after 30 minutes.

30-Minute Settled Sludge Volume

The result may be reported as:

mL/L after 30 minutes

This provides information about sludge settling behavior.

Sludge Volume Index

Sludge Volume Index, or SVI, relates the 30-minute settled sludge volume to MLSS concentration.

A common formula is:

SVI, mL/g = Settled Sludge Volume, mL/L × 1,000 ÷ MLSS, mg/L

SVI Example

Suppose:

30-minute settled sludge volume = 300 mL/L;
MLSS = 3,000 mg/L.

SVI = 300 × 1,000 ÷ 3,000

SVI = 100 mL/g

Interpreting SVI

SVI is useful for comparing sludge settleability over time.

An increasing SVI can indicate poorer settling or more bulky sludge.

However, interpretation should also include:

sludge blanket;
microscopic condition;
effluent TSS;
process loading;
DO.

Do Not Use SVI Alone

A single SVI value does not identify the cause of poor settling.

Possible causes of abnormal settleability include:

filamentous growth;
young sludge;
old sludge;
denitrification;
toxicity;
hydraulic effects.

Dissolved Oxygen

Dissolved oxygen, or DO, is a key activated-sludge process measurement.

It helps operators evaluate the balance between:

oxygen supply;
biological oxygen demand.

Low DO

Low DO may result from:

high organic loading;
high ammonia loading;
insufficient airflow;
diffuser fouling;
poor mixing;
instrument error.

High DO

High DO can indicate excessive aeration relative to process demand.

This can increase energy use without improving treatment.

pH

Wastewater pH affects:

biological activity;
nitrification;
chemical precipitation;
disinfection;
toxicity of some compounds.

Sudden pH Change

A sudden pH change may indicate:

industrial discharge;
chemical spill;
chemical-feed problem;
loss of alkalinity;
instrument error.

Alkalinity

Alkalinity provides acid-neutralizing and buffering capacity.

It is particularly important in nitrifying wastewater processes.

Nitrification and Alkalinity

A commonly used operator relationship is:

Approximately 7.14 mg/L alkalinity as CaCO3 is consumed for each 1 mg/L ammonia-nitrogen nitrified.

Residual Alkalinity

Low residual alkalinity can indicate risk of:

pH decline;
reduced nitrification;
process instability.

Ammonia-Nitrogen

Ammonia-nitrogen is one of the most useful measurements for evaluating nitrification.

High effluent ammonia may indicate:

low DO;
low pH;
insufficient alkalinity;
low temperature;
insufficient solids retention time;
toxicity.

Nitrite

Nitrite is an intermediate product of nitrification.

Elevated nitrite may indicate incomplete oxidation from ammonia to nitrate.

Nitrate

Nitrate is the more oxidized nitrogen form produced by nitrification.

Nitrate can be reduced through denitrification under anoxic conditions.

Interpret Ammonia, Nitrite, and Nitrate Together

These three measurements can provide a useful picture of nitrogen conversion.

For example:

high ammonia and low nitrate may indicate poor nitrification;
low ammonia and high nitrate may indicate good nitrification but limited denitrification;
low ammonia and lower nitrate may indicate effective nitrification and denitrification when total nitrogen also supports that conclusion.

Total Kjeldahl Nitrogen

Total Kjeldahl Nitrogen, or TKN, includes:

organic nitrogen;
ammonia and ammonium nitrogen.

It does not include nitrate or nitrite.

Total Nitrogen

A simplified relationship is:

Total Nitrogen = TKN + Nitrite-N + Nitrate-N

Phosphorus

Common wastewater phosphorus measurements include:

total phosphorus;
orthophosphate.

Orthophosphate

Orthophosphate is a dissolved inorganic phosphorus form that is readily available for biological uptake and chemical reaction.

Total Phosphorus

Total phosphorus includes dissolved and particulate phosphorus measured by the analytical method.

Total phosphorus and orthophosphate should not be treated as the same measurement.

Phosphorus Removal Troubleshooting

Poor phosphorus removal may be associated with:

poor biological uptake;
insufficient chemical dose;
poor mixing;
poor solids separation;
inadequate sludge wasting;
high phosphorus recycle loads.

Chlorine Residual

Wastewater effluent disinfection may use chlorine, depending on facility design.

Residual measurements may include:

free chlorine;
total chlorine.

Residual should be measured promptly because chlorine continues reacting after collection.

Dechlorination Monitoring

Where dechlorination is used, operators may monitor residual after the dechlorination process to evaluate chemical-feed performance.

Fecal Indicator Organisms

Microbiological analyses may be used to evaluate effluent disinfection performance.

Sampling and analytical technique are critical because contamination can produce misleading results.

Temperature

Temperature affects:

biological reaction rates;
oxygen solubility;
nitrification;
settling behavior;
instrument response.

Conductivity

Conductivity can be useful for detecting changes in:

dissolved salts;
industrial wastewater;
sidestreams;
chemical addition.

ORP

Oxidation-Reduction Potential, or ORP, can help evaluate whether biological zones are more oxidizing or reducing.

It may support interpretation of:

aerobic conditions;
anoxic conditions;
anaerobic conditions;
nutrient-removal processes.

ORP Is Not a Direct DO Measurement

ORP reflects combined redox chemistry and should not be interpreted as if it were dissolved oxygen.

Solids Tests

Wastewater laboratories may measure several solids fractions, including:

total solids;
total suspended solids;
total dissolved solids;
volatile solids;
fixed solids.

Total Solids

Total Solids represent the combined dissolved and suspended material measured by the analytical method.

Total Dissolved Solids

Total Dissolved Solids, or TDS, represent dissolved material remaining after suspended material is removed according to the analytical procedure.

Suspended and Dissolved Relationship

A simplified conceptual relationship is:

Total Solids = Suspended Solids + Dissolved Solids

Actual analytical methods and reporting conventions should be followed.

Sludge Percent Solids

Sludge concentration is often expressed as percent solids.

A simplified relationship is:

Percent Solids = Dry Solids Mass ÷ Wet Sample Mass × 100

Percent Solids Example

If a 100 g sludge sample contains 4 g dry solids:

Percent Solids = 4 ÷ 100 × 100 = 4%

Why Percent Solids Matters

Percent solids affects:

sludge pumping;
thickening;
digestion;
dewatering;
haul volume.

Volatile Solids in Sludge

Volatile-solids testing can help evaluate the organic fraction of sludge.

It is commonly used in solids-handling and digestion process evaluation.

Mass Loading

Wastewater laboratory concentrations become more useful when combined with flow.

A common relationship is:

Load, lb/day = Flow, MGD × Concentration, mg/L × 8.34

BOD Loading Example

If influent flow is 3.0 MGD and BOD is 200 mg/L:

Load = 3.0 × 200 × 8.34

Load = 5,004 lb/day BOD

Why Loading Matters

Two days can have the same concentration but different total loading if flow changes.

Operators should distinguish:

concentration;
mass load.

Process Samples

Useful wastewater sampling points may include:

influent;
primary effluent;
aeration basin;
return activated sludge;
waste activated sludge;
secondary effluent;
final effluent;
sidestream returns.

Sample Location Changes Interpretation

An ammonia concentration in influent answers a different question from ammonia measured in final effluent.

Always connect the result to the correct process location.

Influent Data

Influent tests can help evaluate:

organic loading;
solids loading;
nutrient loading;
industrial contributions;
diurnal variation.

Aeration Basin Data

Useful aeration-basin measurements include:

DO;
MLSS;
MLVSS;
pH;
temperature;
settleability;
ammonia;
nitrate.

Secondary Clarifier Data

Useful information includes:

sludge blanket depth;
effluent TSS;
settleability;
return-sludge concentration;
visual observations.

Sidestream Samples

Recycle streams from solids handling can contain high concentrations of:

ammonia;
phosphorus;
solids;
organic matter.

A low-flow sidestream can still contribute a significant mass load.

Use Paired Data

Useful pairs include:

BOD and COD;
TSS and turbidity;
DO and ammonia;
ammonia and nitrate;
pH and alkalinity;
MLSS and settled sludge volume;
total phosphorus and orthophosphate.

Example: Rising Effluent TSS

Review:

settleability;
SVI;
sludge blanket;
hydraulic loading;
return-sludge operation;
microscopic condition where available.

Example: High Effluent BOD and High TSS

If both BOD and TSS rise, solids carryover may be contributing to the BOD result.

Review clarification performance before concluding that only biological oxidation has failed.

Example: High Effluent BOD but Normal TSS

This pattern may point more strongly toward incomplete soluble organic treatment or another analytical/process issue.

Example: High Ammonia and Low DO

This pattern is consistent with possible oxygen limitation of nitrification.

Also review:

pH;
alkalinity;
temperature;
solids retention time.

Example: High Ammonia with Adequate DO

If DO is adequate, investigate other nitrification limitations such as:

low temperature;
low pH;
insufficient alkalinity;
toxicity;
insufficient solids retention time.

Example: High Nitrate in Final Effluent

If nitrification is complete but final nitrate remains high, denitrification may be limited.

Review:

anoxic-zone DO;
carbon availability;
internal recycle;
anoxic detention time.

Example: Poor Settleability

Review:

SVI;
DO;
F/M relationship where used;
sludge age;
microscopic observations;
filamentous growth;
toxicity;
nutrient conditions.

Laboratory QA/QC Still Applies

An unusual wastewater result should be reviewed for:

sample location;
sample handling;
holding time;
calibration;
blanks;
duplicates;
calculations;
units.

Wastewater Samples Can Change Quickly

Biological activity continues after sample collection.

Improper handling can change:

DO;
pH;
nitrogen species;
organic concentrations;
solids distribution.

Composite Samples

Composite sampling is commonly useful for parameters intended to represent average wastewater conditions over time.

The required sample type should always follow the applicable procedure.

Grab Samples

Grab samples are useful when the parameter:

changes rapidly;
must be measured immediately;
represents a specific process condition.

Units Matter

Common reporting mistakes include confusing:

mg/L with lb/day;
ammonia as N with ammonia as NH3;
nitrate as N with nitrate as NO3;
phosphorus as P with phosphate as PO4;
percent solids with mg/L.

Concentration Versus Loading

A concentration may remain unchanged while total load increases because flow increases.

This is why plant loading calculations require both:

flow;
concentration.

Trend Laboratory Data

Trend review can reveal gradual changes in:

organic loading;
solids separation;
nitrification;
phosphorus removal;
sludge settleability;
sidestream loading.

Common Wastewater Laboratory Mistakes

Confusing BOD with DO.
Assuming COD and BOD are interchangeable.
Interpreting concentration without considering flow and mass loading.
Confusing TSS with VSS.
Treating MLVSS as pure living biomass.
Using SVI alone to diagnose a settling problem.
Ignoring alkalinity when troubleshooting nitrification.
Looking only at ammonia without reviewing nitrite and nitrate.
Confusing total phosphorus with orthophosphate.
Ignoring sample location and sample type.
Changing process operation based on one questionable result.
Ignoring laboratory QA/QC and units.

A Practical Wastewater Result Review

Confirm sample location and sample type.
Confirm parameter and units.
Review laboratory QA/QC.
Compare with historical trend.
Compare with related process measurements.
Review flow and calculate loading where relevant.
Review current equipment and process conditions.
Verify unusual results before major process changes.
Document the operational interpretation and response.

A Practical Biological Process Review

Review influent BOD or COD.
Review flow and organic loading.
Review aeration-basin DO.
Review MLSS and MLVSS.
Review settleability and SVI.
Review pH and alkalinity.
Review ammonia, nitrite, and nitrate.
Review effluent BOD and TSS.
Review sludge wasting and return-sludge operation.
Compare all values with historical plant performance.

What to Remember for the Exam

BOD measures oxygen used by microorganisms to degrade biodegradable material under defined test conditions.
COD measures the oxygen equivalent of chemically oxidizable material and generally produces results faster than BOD.
BOD and DO are different measurements.
TSS measures suspended solids, while VSS represents the volatile portion of suspended solids.
Fixed suspended solids can be estimated as TSS minus VSS.
MLSS is the suspended-solids concentration in activated-sludge mixed liquor.
MLVSS is the volatile fraction of MLSS and is not identical to active living biomass.
A 30-minute settleability test measures settled sludge volume, commonly in mL/L.
SVI relates settled sludge volume to MLSS concentration and is commonly reported in mL/g.
SVI should be interpreted with other process and settling information.
DO is a key indicator of the balance between aeration supply and biological oxygen demand.
Alkalinity supports pH stability and is consumed during nitrification.
Ammonia, nitrite, and nitrate should be interpreted together when evaluating nitrogen conversion.
TKN includes organic nitrogen plus ammonia/ammonium but excludes nitrate and nitrite.
Total phosphorus and orthophosphate are different measurements.
Wastewater mass loading depends on both flow and concentration.
A common loading formula is lb/day = MGD × mg/L × 8.34.
Elevated effluent BOD together with elevated TSS can indicate solids carryover.
Wastewater laboratory results should be interpreted with sampling information, QA/QC, process observations, and historical trends.
Operators should always confirm units and reporting basis before making process decisions.