10.3 Wastewater Laboratory Analysis: BOD5, COD, Total Suspended Solids (TSS) & Settleometry

Wastewater Laboratory Analysis: BOD5, COD, Total Suspended Solids (TSS) & Settleometry

Wastewater treatment facilities operate under strict effluent discharge limitations governed by the Clean Water Act (CWA), EPA 40 CFR Part 133 (Secondary Treatment Standards), and CDPHE Regulation 62 (Colorado Discharge Permit System). Regulatory compliance and biological process control depend directly upon precise laboratory quantification of organic loading, solids partitioning, and sludge settleability.

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1. 5-Day Biochemical Oxygen Demand (BOD5 & CBOD5)

Biochemical Oxygen Demand (BOD5) is the empirical bioassay measuring the mass of dissolved oxygen consumed by heterotrophic microorganisms while biochemically oxidizing organic matter in wastewater over a 5-day period.

$\text{Organic Matter} + \text{Microorganisms} + \text{O}_2 \longrightarrow \text{CO}_2 + \text{H}_2\text{O} + \text{New Biomass}$

Standard Analytical Conditions (Standard Methods 5210B)

• Incubation Bottle: $300\text{ mL}$ borosilicate glass BOD bottles with tapered ground-glass stoppers and flared liquid-seal necks. Bottles must be filled with water seals and capped with plastic covers to prevent evaporation.
• Incubation Temperature: Thermostatically controlled incubator maintained at $20.0^\circ\text{C} \pm 1.0^\circ\text{C}$.
• Incubation Duration: Exactly $5\text{ days} \pm 6\text{ hours}$ (120 hours).
• Total Darkness: Incubator interior must be completely dark to prevent photosynthetic oxygen generation by microscopic algae.
• Dilution Water Preparation: High-purity deionized water aerated to saturation with dissolved oxygen (approx. $8.5–9.0\text{ mg/L}$ at sea level; $7.0–7.5\text{ mg/L}$ at Colorado mountain elevations). Dilution water is buffered using analytical nutrient pillows containing phosphate buffer ($\text{pH } 7.2$), magnesium sulfate ($\text{MgSO}_4$), calcium chloride ($\text{CaCl}_2$), and ferric chloride ($\text{FeCl}_3$).

Dilution Water Blank QC Criterion: Dissolved Oxygen Depletion <= 0.20 mg/L over 5 Days

Seeding & Carbonaceous BOD (CBOD5)

• Microbial Seed: Disinfected (chlorinated/UV-treated) effluents, industrial wastes, or high-temperature samples lack viable heterotrophic bacteria. They must be inoculated with an acclimated microbial seed (fresh unchlorinated secondary effluent or commercial bacterial seed capsules such as PolySeed).
• Carbonaceous BOD (CBOD5): Autotrophic nitrifying bacteria (Nitrosomonas and Nitrobacter) oxidize ammonia nitrogen to nitrite and nitrate, exerting Nitrogenous Biochemical Oxygen Demand (NOD): $\text{NH}_4^+ + 2\text{O}_2 \longrightarrow \text{NO}_3^- + 2\text{H}^+ + \text{H}_2\text{O}$ To measure only carbonaceous oxygen demand, add TCMP (2-chloro-6-(trichloromethyl)pyridine), a chemical nitrification inhibitor that selectively halts nitrifying bacteria without inhibiting carbon-oxidizing heterotrophs.

Regulatory Acceptance & Quality Control Criteria

For an analytical BOD dilution bottle to be valid for compliance reporting, it must satisfy two mandatory criteria:

1. Minimum DO Depletion: The sample must consume at least $2.0\text{ mg/L}$ of dissolved oxygen over the 5-day incubation ($DO_{\text{initial}} - DO_{\text{final}} \ge 2.0\text{ mg/L}$).
2. Minimum Residual DO: The sample bottle must retain at least $1.0\text{ mg/L}$ of dissolved oxygen at the conclusion of Day 5 ($DO_{\text{final}} \ge 1.0\text{ mg/L}$).
3. Glucose-Glutamic Acid (GGA) Standard Check: A standard solution containing $150\text{ mg/L}$ reagent-grade glucose and $150\text{ mg/L}$ glutamic acid ($6.0\text{ mL}$ of GGA standard in a $300\text{ mL}$ bottle) must achieve a 5-day BOD recovery of $198 \pm 30.5\text{ mg/L}$ (valid acceptance range: $167.5\text{ to } 228.5\text{ mg/L}$).

Mathematical BOD Formulas & Calculations

1. Unseeded BOD Formula:

$\text{BOD}_5\text{ (mg/L)} = \frac{DO_{\text{initial}} - DO_{\text{final}}}{P}$

Where $P = \text{Dilution Fraction} = \frac{\text{Sample Volume (mL)}}{300\text{ mL}}$.

2. Seeded BOD Formula:

$\text{BOD}_5\text{ (mg/L)} = \frac{(DO_{\text{initial}} - DO_{\text{final}}) - (B_1 - B_2) \times f}{P}$

Where:

• $B_1$ = Initial DO of seed control bottle ($\text{mg/L}$)
• $B_2$ = Day 5 DO of seed control bottle ($\text{mg/L}$)
• $f$ = Ratio of seed volume in sample bottle to seed volume in seed control bottle: $f = \frac{\text{mL seed in sample bottle}}{\text{mL seed in seed control bottle}}$

Worked Example 9.3.1: Seeded BOD5 Calculation

A laboratory technician sets up a seeded BOD5 test on primary effluent. A $300\text{ mL}$ bottle receives $15.0\text{ mL}$ of wastewater sample and $2.0\text{ mL}$ of seed. A seed control bottle containing $10.0\text{ mL}$ of seed in $300\text{ mL}$ yields an initial DO of $8.60\text{ mg/L}$ and a Day 5 DO of $5.10\text{ mg/L}$. The wastewater sample bottle yields an initial DO of $8.40\text{ mg/L}$ and a Day 5 DO of $3.20\text{ mg/L}$. Calculate the wastewater BOD5.

Step 1: Check QC criteria for the sample bottle:

• DO Depletion = $8.40 - 3.20 = 5.20\text{ mg/L}$ (Satisfies $\ge 2.0\text{ mg/L}$ requirement)
• Residual DO = $3.20\text{ mg/L}$ (Satisfies $\ge 1.0\text{ mg/L}$ requirement)

Step 2: Calculate seed correction factor ($f$) and seed DO depletion ($B_1 - B_2$): $f = \frac{2.0\text{ mL}}{10.0\text{ mL}} = 0.20$ $(B_1 - B_2) = 8.60 - 5.10 = 3.50\text{ mg/L}$ $\text{Seed Correction} = 3.50\text{ mg/L} \times 0.20 = 0.70\text{ mg/L}$

Step 3: Calculate sample dilution fraction ($P$): $P = \frac{15.0\text{ mL}}{300.0\text{ mL}} = 0.050$

Step 4: Calculate seeded BOD5: $\text{BOD}_5 = \frac{(8.40 - 3.20) - 0.70}{0.050} = \frac{5.20 - 0.70}{0.050} = \frac{4.50}{0.050} = 90.0\text{ mg/L}$

2. Chemical Oxygen Demand (COD)

Chemical Oxygen Demand (COD) is a rapid chemical test that measures the total oxygen equivalent of all organic matter susceptible to oxidation by a strong chemical oxidant. While BOD5 requires 5 days of incubation, COD results are obtained in 2.5 to 3 hours, making COD an invaluable tool for real-time plant process control.

Closed Reflux Colorimetric Method (Standard Methods 5220D)

• Chemical Reagents: Sample is pipetted into a sealed borosilicate digestion vial containing potassium dichromate ($\text{K}_2\text{Cr}_2\text{O}_7$) as the primary oxidant in concentrated sulfuric acid ($\text{H}_2\text{SO}_4$).
• Catalyst: Silver sulfate ($\text{Ag}_2\text{SO}_4$) is added to catalyze the oxidation of straight-chain aliphatic hydrocarbons and alcohols.
• Chloride Interference Suppressor: Mercuric sulfate ($\text{HgSO}_4$) is added to complex chloride ions ($\text{Cl}^-$) as soluble mercuric chloride complexes, preventing chloride from reacting with dichromate to cause false-positive COD elevations.
• Digestion Conditions: Vials are heated in a pre-calibrated heating block at $150^\circ\text{C} \pm 2^\circ\text{C}$ for exactly 2 hours (120 minutes).
• Photometric Measurement: As organics are oxidized, hexavalent chromium ($\text{Cr}^{6+}$, orange) is reduced to trivalent chromium ($\text{Cr}^{3+}$, green). Spectrophotometric absorbance is measured at $600\text{ nm}$ (for high-range COD $100–1500\text{ mg/L}$) or $420\text{ nm}$ (for low-range COD $0–150\text{ mg/L}$).

COD:BOD Ratio Dynamics in Wastewater

• Typical Raw Domestic Wastewater: $\text{COD}:\text{BOD}_5$ ratio is typically $1.5:1\text{ to } 2.5:1$.
• Final Treated Effluent: Ratio rises to $3.0:1\text{ to } 5.0:1$ because biological treatment removes readily biodegradable organics, leaving refractory (non-biodegradable) organic compounds.
• Industrial Waste Streams: A high $\text{COD}:\text{BOD}_5$ ratio ($> 3.0:1$) in raw influent indicates toxic, refractory, or synthetic organic compounds resistant to biological oxidation.

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3. Gravimetric Solids Analysis: TSS, TDS, and VSS

Wastewater solids are categorized by physical separation (filtration) and thermal behavior (drying and combustion).

+---------------------------------------------------------------------------------------------------------+
|                                 TOTAL SOLIDS (TS) THERMAL PARTITIONING                                  |
+---------------------------------------------------+-----------------------------------------------------+
| TOTAL SUSPENDED SOLIDS (TSS)                      | TOTAL DISSOLVED SOLIDS (TDS)                        |
| - Retained on 1.5 um glass fiber filter           | - Passes through 1.5 um glass fiber filter          |
| - Dried at 103°C to 105°C                         | - Evaporated and dried at 180°C                     |
| +-----------------------+-----------------------+ | +-------------------------+-----------------------+ |
| | Volatile Suspended    | Fixed Suspended       | | | Volatile Dissolved      | Fixed Dissolved       | |
| | Solids (VSS)          | Solids (FSS)          | | | Solids (VDS)            | Solids (FDS)          | |
| | - Burned at 550°C     | - Mineral ash residue | | | - Burned at 550°C       | - Mineral residue     | |
| | - Organic biomass     | - Sand, silt, grit    | | | - Soluble organics      | - Dissolved salts     | |
| +-----------------------+-----------------------+ | +-------------------------+-----------------------+ |
+---------------------------------------------------+-----------------------------------------------------+

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

1. Place a standard Whatman 934-AH or GF/C glass fiber filter disc ($1.5\text{ }\mu\text{m}$ pore size) in a vacuum filtration apparatus. Wash with deionized water, dry in an oven at $103–105^\circ\text{C}$, cool in a desiccator, and record tare weight ($W_{\text{tare}}$) on an analytical balance ($0.1\text{ mg}$ precision).
2. Filter a well-mixed sample volume ($V$, typically $25–100\text{ mL}$ for mixed liquor or $250–1000\text{ mL}$ for final effluent).
3. Dry filter and retained residue in a laboratory drying oven at $103^\circ\text{C}–105^\circ\text{C}$ for a minimum of 1 hour.
4. Cool to room temperature in a desiccator and weigh to constant weight ($< 0.5\text{ mg}$ weight change):

$\text{TSS (mg/L)} = \frac{(W_{\text{dry}} - W_{\text{tare}})\text{ in grams} \times 1{,}000{,}000}{\text{Sample Volume (mL)}} = \frac{(W_{\text{dry}} - W_{\text{tare}})\text{ in mg} \times 1{,}000}{\text{Sample Volume (mL)}}$

2. Total Dissolved Solids (TDS) Procedure (Standard Methods 2540C)

The filtrate passing through the TSS glass fiber filter is transferred to a pre-weighed evaporating dish and evaporated to dryness in a drying oven at $180^\circ\text{C} \pm 2^\circ\text{C}$ for 1 hour. Drying at $180^\circ\text{C}$ removes occluded water of crystallization and decomposes mineral bicarbonates.

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

The dried TSS filter disc is placed in a muffle furnace at $550^\circ\text{C} \pm 50^\circ\text{C}$ for 15 to 20 minutes. Organic volatile carbon volatilizes to $\text{CO}_2$, while inorganic mineral ash remains as Fixed Suspended Solids (FSS). In activated sludge mixed liquor, the $\text{MLVSS}/\text{MLSS}$ ratio is typically $0.70\text{ to } 0.85$ (70–85% active biological biomass).

$\text{VSS (mg/L)} = \frac{(W_{\text{dry at } 105^\circ\text{C}} - W_{\text{ash at } 550^\circ\text{C}})\text{ in mg} \times 1{,}000}{\text{Sample Volume (mL)}}$

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4. Settleometry & Sludge Volume Index (SVI)

Clarifier performance and activated sludge settling kinetics are evaluated using the 30-Minute Settleometer Test.

Settleometer Protocol

• A fresh sample of mixed liquor suspended solids (MLSS) is collected from the aeration basin effluent weir.
• Pour the sample immediately into a standard $2.0\text{ L}$ Mallory settleometer or $1.0\text{ L}$ wide-diameter graduated cylinder. Gently stir with a paddle to halt rotation, then allow the sludge blanket to settle under quiescent conditions for 30 minutes.
• Record the Settled Sludge Volume after 30 minutes ($\text{SSV}_{30}$ in $\text{mL/L}$). Also observe settling characteristics: settleability speed, interface clarity, and pin-point floc presence.

Sludge Volume Index (SVI) Formula

Sludge Volume Index (SVI) is defined as the volume in milliliters occupied by 1 gram of activated sludge solids after 30 minutes of settling:

$\text{SVI (mL/g)} = \frac{\text{SSV}_{30}\text{ (mL/L)} \times 1{,}000\text{ mg/g}}{\text{MLSS (mg/L)}} = \frac{\text{SSV}_{30}\text{ (mL/L)}}{\text{MLSS (mg/L)} / 1{,}000}$

Operational Interpretation of SVI Ranges