16.2 Wastewater Lab Analysis: BOD5, TSS, COD & Volatile Solids

8.2 Wastewater Lab Analysis: BOD5, TSS, COD & Volatile Solids

Municipal wastewater treatment plants operate to protect receiving streams and groundwater aquifers from organic over-enrichment, solids sedimentation, and severe dissolved oxygen depletion. To evaluate treatment performance and enforce compliance with National Pollutant Discharge Elimination System (NPDES) permits issued by the Oregon Department of Environmental Quality (DEQ) under OAR Chapter 340, Division 045, wastewater operators must master standard analytical laboratory procedures.

Key compliance parameters include Biochemical Oxygen Demand ($\text{BOD}_5$), Carbonaceous Biochemical Oxygen Demand ($\text{CBOD}_5$), Chemical Oxygen Demand ($\text{COD}$), Total Suspended Solids ($\text{TSS}$), Volatile Suspended Solids ($\text{VSS}$), and Settleable Solids. Understanding the analytical mechanics, chemistry, QA/QC criteria, and mathematical formulations of these tests is fundamental to utility operations.

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Biochemical Oxygen Demand (BOD5 & CBOD5) Testing

Biochemical Oxygen Demand ($\text{BOD}_5$) measures the quantity of dissolved oxygen consumed by living heterotrophic microorganisms while biochemically oxidizing decomposing organic carbon and oxidizable inorganic matter in wastewater. The standard test (Standard Methods 5210B) measures dissolved oxygen depletion over a 5-day incubation period in the dark at $20.0 \pm 1.0^\circ\text{C}$.

A 5-day incubation period was historically selected to reflect the maximum travel time of treated sewage down typical river basins before reaching the ocean. At $20^\circ\text{C}$, a 5-day test captures approximately $65\% \text{ to } 70\%$ of the total ultimate carbonaceous biochemical oxygen demand ($\text{BOD}_u$).

 Dissolved Oxygen (mg/L)
 9.0 ┌───────────
     │\ 
     │ \  Carbonaceous Demand (CBOD)
     │  \   (Heterotrophic bacteria oxidize organic carbon)
 5.0 │   \────────────────┐
     │                    │\  Nitrogenous Demand (NBOD)
     │                    │ \   (Autotrophic nitrifiers oxidize NH3 -> NO3)
 1.0 │                    │  \─────────────
 0.0 └────────────────────┴────────────────────► Time (Days)
     0                    5 (Standard Test)   20 (Ultimate)

1. Test Apparatus & Dilution Water Preparation

Samples are incubated in specialized $300 \text{ mL}$ glass BOD bottles featuring tapered ground-glass stoppers and flared necks. The flared lip must be filled with high-purity deionized water and sealed with a plastic cap to form a continuous water seal, preventing atmospheric oxygen from diffusing into the sample bottle during the 5-day test. Bottles are incubated inside a light-tight incubator to prevent photosynthetic algae from generating dissolved oxygen.

Because raw wastewater exerts far more oxygen demand (typically $150 \text{ to } 400 \text{ mg/L}$) than can be dissolved in water (maximum oxygen saturation is only $\sim 9.1 \text{ mg/L}$ at $20^\circ\text{C}$), samples must be diluted using nutrient-buffered dilution water. High-purity deionized water is aerated with filtered oil-free air to achieve saturation ($8.0 \text{ to } 9.0 \text{ mg/L}$ DO) and dosed with $1.0 \text{ mL/L}$ each of four reagent solutions:

1. Phosphate Buffer Solution (pH 7.2): Provides buffering capacity to neutralize organic metabolic acids produced during decomposition and maintains optimal neutral pH.
2. Magnesium Sulfate ($\text{MgSO}_4$) Solution: Provides essential magnesium ions.
3. Calcium Chloride ($\text{CaCl}_2$) Solution: Provides structural calcium ions.
4. Ferric Chloride ($\text{FeCl}_3$) Solution: Provides trace iron essential for bacterial respiratory enzymes.

2. Microbial Seeding & Inhibitors

Samples that contain an active, healthy microbial population (such as raw, untreated municipal sewage or primary clarifier effluent) do not require supplemental seeding. However, samples that have undergone disinfection (chlorination/UV), industrial discharges, high-temperature processes, or membrane filtration lack viable heterotrophic bacteria. These samples must be seeded with an active biological inoculant—such as settled domestic wastewater, unchlorinated secondary effluent, or commercially stabilized bacterial consortia (e.g., PolySeed®).

3. Carbonaceous BOD (CBOD5) & Nitrification Inhibition

In biologically treated secondary effluents, autotrophic nitrifying bacteria (Nitrosomonas and Nitrobacter) proliferate. These organisms oxidize ammonia ($\text{NH}_4^+$) to nitrite ($\text{NO}_2^-$) and nitrate ($\text{NO}_3^-$), consuming $4.57 \text{ mg of } \text{O}_2$ per mg of ammonia-nitrogen oxidized:

$2\text{NH}_4^+ + 3\text{O}_2 \xrightarrow{\text{Nitrosomonas}} 2\text{NO}_2^- + 4\text{H}^+ + 2\text{H}_2\text{O}$ $2\text{NO}_2^- + \text{O}_2 \xrightarrow{\text{Nitrobacter}} 2\text{NO}_3^-$

This exertion of Nitrogenous Biochemical Oxygen Demand (NBOD) can falsely suggest treatment plant failure when the facility is actually achieving advanced biological nitrification. To isolate carbonaceous organic demand, Oregon DEQ permits often specify $\text{CBOD}_5$. The analyst adds a chemical nitrification inhibitor—TCMP (2-chloro-6-(trichloromethyl) pyridine) or ATU (allylthiourea)—which selectively inactivates Nitrosomonas enzymes without harming heterotrophic carbon-oxidizing bacteria.

4. QA/QC Criteria for Valid BOD5 Determinations

Under Standard Methods 5210B, strict quality control criteria must be satisfied for a test run to be legally defensible:

• Dilution Water Blank: Dilution water with no sample must experience a DO depletion of $\le 0.20 \text{ mg/L}$ after 5 days (ideally $\le 0.10 \text{ mg/L}$). Greater depletion indicates contaminated DI water, dirty glassware, or contaminated reagents.
• Glucose-Glutamic Acid (GGA) Standard: A reference solution containing $150 \text{ mg/L}$ reagent-grade glucose and $150 \text{ mg/L}$ glutamic acid must be analyzed with each batch. A $2.0\%$ dilution ($6.0 \text{ mL}$ of GGA in a $300 \text{ mL}$ bottle) must yield a 5-day BOD of $198 \pm 30.5 \text{ mg/L}$ (acceptable range: $167.5 \text{ to } 228.5 \text{ mg/L}$). This validates seed viability and analytical technique.
• Depletion Criterion: A sample dilution is valid only if the initial-to-final DO depletion is at least $\ge 2.0 \text{ mg/L}$.
• Residual Criterion: A sample dilution is valid only if the final DO remaining on Day 5 is at least $\ge 1.0 \text{ mg/L}$.

5. BOD Calculations

For an unseeded sample: $\text{BOD}_5 \text{ (mg/L)} = \frac{\text{DO}_0 - \text{DO}_5}{P}$ Where $\text{DO}_0$ is initial DO (mg/L), $\text{DO}_5$ is final DO on Day 5 (mg/L), and $P$ is the decimal volumetric fraction of sample in the bottle ($P = \frac{\text{Sample Volume (mL)}}{300 \text{ mL}}$).

For a seeded sample: $\text{BOD}_5 \text{ (mg/L)} = \frac{(\text{DO}_0 - \text{DO}_5) - (B_0 - B_5)f}{P}$ Where $(B_0 - B_5)$ is the DO depletion in the seed control blank, and $f$ is the ratio of seed volume in the sample bottle to seed volume in the seed blank ($f = \frac{\text{Seed in Sample (mL)}}{\text{Seed in Blank (mL)}}$). If multiple dilutions meet the depletion and residual criteria, their calculated values are averaged.

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Chemical Oxygen Demand (COD)

Chemical Oxygen Demand ($\text{COD}$) measures the total oxygen equivalent required to chemically oxidize all organic matter in a wastewater sample using a powerful chemical oxidant under harsh thermal and acidic conditions (Standard Methods 5220).

Chemistry of Digestion

The standard closed reflux micro-method uses potassium dichromate ($\text{K}_2\text{Cr}_2\text{O}_7$) in boiling $50\%$ concentrated sulfuric acid ($\text{H}_2\text{SO}_4$). The sample and reagents are sealed in heavy-walled glass culture tubes and digested in a dry heating block at $150 \pm 2^\circ\text{C}$ for 2 hours:

$\text{Organics} + \text{Cr}_2\text{O}_7^{2-} + 8\text{H}^+ \xrightarrow{\text{Ag}_2\text{SO}_4, 150^\circ\text{C}} 2\text{Cr}^{3+} + 4\text{H}_2\text{O} + \text{Carbon Dioxide}$

• Silver Sulfate ($\text{Ag}_2\text{SO}_4$) Catalyst: Straight-chain aliphatic hydrocarbons are resistant to oxidation; silver sulfate acts as a catalyst to ensure complete destruction.
• Mercuric Sulfate ($\text{HgSO}_4$) Interference Suppressor: Chloride ions ($\text{Cl}^-$) are readily oxidized by dichromate, which would generate enormous false-positive COD values. Mercuric sulfate complexes chloride ions to form soluble, unoxidized mercuric chloride complexes ($[\text{HgCl}_4]^{2-}$).

COD Measurement & Ratios

Following digestion, COD is quantified either by back-titrating residual unreduced hexavalent chromium ($\text{Cr}^{6+}$) with ferrous ammonium sulfate (FAS) using ferroin indicator (sharp color change from blue-green to reddish-brown), or spectrophotometrically at $600 \text{ nm}$ (measuring green trivalent chromium $\text{Cr}^{3+}$ formed).

In typical domestic municipal wastewater, the ratio of $\text{COD} : \text{BOD}_5$ ranges from $1.5 : 1 \text{ to } 2.5 : 1$. A sudden spike in this ratio ($> 3.0 : 1$) indicates the arrival of refractory industrial waste, synthetic surfactants, petroleum hydrocarbons, or toxic chemicals that cannot be broken down biologically.

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Total Suspended Solids (TSS) & Volatile Suspended Solids (VSS)

Solids in wastewater exist in physical fractions categorized by filterability and volatility. Solids retained on a standard filter are suspended, while those passing through are dissolved.

 ┌────────────────────────────────────────────────────────┐
 │ Total Solids (TS) - Dried at 103-105°C                 │
 ├────────────────────────────┬───────────────────────────┤
 │ Total Dissolved Solids     │ Total Suspended Solids    │
 │ (TDS) - Passes 1.5 µm      │ (TSS) - Retained on 1.5 µm│
 └────────────────────────────┼───────────────────────────┤
                              │ Volatile Suspended Solids │
                              │ (VSS) - Burned at 550°C   │
                              │ (Active biological mass)  │
                              ├───────────────────────────┤
                              │ Fixed Suspended Solids    │
                              │ (FSS) - Inert mineral ash │
                              └───────────────────────────┘

1. Total Suspended Solids (TSS) Procedure (Standard Methods 2540D)

TSS represents the non-filterable particulate fraction retained on a glass fiber filter under vacuum:

1. Filter Preparation: A binder-free glass fiber filter disk (Whatman 934-AH, $1.5 \ \mu\text{m}$ nominal pore size) is placed wrinkled-side up in a Buchner funnel or Gooch crucible, washed with three $20 \text{ mL}$ volumes of reagent-grade deionized water under vacuum, dried in a laboratory drying oven at $103 \text{ to } 105^\circ\text{C}$ for 1 hour, cooled in a desiccator, and weighed on an analytical balance sensitive to $0.1 \text{ mg}$ ($0.0001 \text{ g}$). This yields the tare weight ($B$ grams).
2. Filtration: A well-mixed, representative sample aliquot ($V \text{ mL}$) is vacuum-filtered through the pre-weighed disk. The volume selected must yield a dry residue mass between $2.5 \text{ mg}$ and $200 \text{ mg}$. The filter and funnel walls are rinsed with three $10 \text{ mL}$ portions of deionized water to wash away residual dissolved salts.
3. Drying: The filter is dried in the oven at $103 \text{ to } 105^\circ\text{C}$ for a minimum of 1 hour (or until constant weight is achieved). This temperature evaporates all liquid water without volatilizing organic solids or decomposing thermally unstable mineral salts.
4. Cooling & Weighing: The filter is placed in a desiccator containing active blue cobalt-indicating silica gel desiccant. Desiccation cools the filter to ambient balance temperature while preventing hygroscopic absorption of atmospheric humidity. The cooled filter is weighed to obtain gross weight ($A$ grams).

$\text{TSS (mg/L)} = \frac{(A - B) \times 1,000,000}{V \text{ (mL)}}$ Where $A$ is weight of filter + dried residue (g), $B$ is tare weight of clean filter (g), and $V$ is sample volume filtered (mL).

2. Volatile Suspended Solids (VSS) Procedure (Standard Methods 2540E)

VSS quantifies the organic, biologically active fraction of suspended solids:

1. The dry filter and residue from the TSS determination ($A$ grams) are placed in a muffle furnace preheated to $550 \pm 50^\circ\text{C}$ for $15 \text{ to } 20 \text{ minutes}$.
2. At $550^\circ\text{C}$, all combustible organic carbonaceous material ignites and oxidizes to carbon dioxide and water vapor, while inorganic mineral salts remain behind as inert ash.
3. The filter is partially cooled in air, transferred to a desiccator to reach room temperature, and weighed ($C$ grams).

$\text{VSS (mg/L)} = \frac{(A - C) \times 1,000,000}{V \text{ (mL)}}$ $\text{FSS (Fixed Suspended Solids, mg/L)} = \text{TSS} - \text{VSS} = \frac{(C - B) \times 1,000,000}{V \text{ (mL)}}$

Operational Importance of VSS in Activated Sludge

In activated sludge process control, operators measure Mixed Liquor Suspended Solids (MLSS) and Mixed Liquor Volatile Suspended Solids (MLVSS). The ratio of $\frac{\text{MLVSS}}{\text{MLSS}}$ reflects the proportion of active microbial biomass versus inert mineral debris (sand, clay, calcium precipitates) in the aeration basin. A healthy activated sludge plant maintains an MLVSS/MLSS ratio of $0.70 \text{ to } 0.85$ ($70\% \text{ to } 85\%$). A declining ratio indicates mineral accumulation, alerting the operator to increase waste activated sludge (WAS) pumping rates.

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Settleable Solids & Imhoff Cone Analysis

Settleable solids measure the volume of suspended particulate matter that settles to the bottom of a quiescent fluid column under gravity within a 1-hour period (Standard Methods 2540F). This test serves as a rapid, direct physical indicator of primary clarifier performance and solids-liquid separation efficiency.

Standard Procedure

1. A standardized $1.0 \text{ L}$ Imhoff cone (a steep-walled conical glass or transparent polycarbonate vessel with inverted milliliter graduations at the tip) is filled exactly to the $1.0 \text{ L}$ mark with well-mixed raw wastewater, mixed liquor, or treated effluent.
2. The sample settles undisturbed for 45 minutes.
3. At 45 minutes, a glass rod or the cone itself is gently spun or rotated along its vertical axis to dislodge solids adhering to the sloped conical sides.
4. The solids settle for an additional 15 minutes (total settling time: 60 minutes).
5. The volume of settled solids compacted into the conical tip is read directly in $\text{mL/L}$.

Process Control Applications

• Primary Clarifier Performance: Raw domestic municipal influent typically contains $5.0 \text{ to } 20.0 \text{ mL/L}$ of settleable solids. An efficiently operating primary clarifier removes $95\% \text{ to } 99\%$ of settleable solids and $50\% \text{ to } 65\%$ of Total Suspended Solids.
• Secondary Clarifier Troubleshooting: Settleable solids in secondary effluent should be negligible ($< 0.1 \text{ to } 0.5 \text{ mL/L}$). Higher readings indicate secondary clarifier solids washout, hydraulic overloading, or activated sludge bulking.