7.2 Biochemical Oxygen Demand (BOD5/CBOD5) & Total Suspended Solids (TSS) Analysis

7.2 Biochemical Oxygen Demand (BOD5/CBOD5) & Total Suspended Solids (TSS) Analysis

Exam Focus: Biochemical Oxygen Demand (BOD5) and Total Suspended Solids (TSS) represent the core regulatory metrics under the Clean Water Act National Secondary Treatment Standards. Class I operators must demonstrate thorough competency in sample dilution chemistry, nutrient water preparation, incubation thermodynamics, quality assurance validity criteria (including dilution blank depletion limits and glucose-glutamic acid standards), the enzymatic inhibition of nitrification using TCMP, and gravimetric filtration and muffle furnace ignition calculations.

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1. Biochemical Oxygen Demand (BOD5) Principles & Biochemical Kinetics

Biochemical Oxygen Demand (BOD) is an empirical bioassay measuring the mass of dissolved oxygen consumed by aerobic microorganisms as they biologically oxidize organic matter in water over a specified time and temperature. Rather than measuring a specific chemical compound, BOD evaluates the potential of a wastewater stream to deplete dissolved oxygen in receiving rivers, streams, and lakes.

Carbonaceous vs. Nitrogenous Oxygen Demand

Oxygen uptake in untreated wastewater proceeds in two distinct biochemical phases:

1. Carbonaceous Stage (CBOD): Heterotrophic bacteria metabolize carbon-based organic molecules (carbohydrates, proteins, fats) into carbon dioxide and water: $\text{Organic Matter } (C_xH_yO_z) + O_2 \xrightarrow{\text{Heterotrophs}} CO_2 + H_2O + \text{New Bacterial Cells}$ In raw domestic wastewater, carbonaceous oxidation begins immediately upon incubation, with approximately 60% to 70% of total carbonaceous demand satisfied within the first 5 days at 20°C.
2. Nitrogenous Stage (NBOD): Autotrophic nitrifying bacteria (Nitrosomonas and Nitrobacter) oxidize reduced nitrogen compounds (ammonia and organic nitrogen) into nitrite and nitrate: $\text{Step 1: } 2NH_4^+ + 3O_2 \xrightarrow{\textit{Nitrosomonas}} 2NO_2^- + 4H^+ + 2H_2O$ $\text{Step 2: } 2NO_2^- + O_2 \xrightarrow{\textit{Nitrobacter}} 2NO_3^-$ $\text{Overall: } NH_4^+ + 2O_2 \rightarrow NO_3^- + 2H^+ + H_2O$ Stoichiometrically, oxidizing 1.0 mg of ammonia nitrogen ($NH_4^+$-N) consumes 4.57 mg of dissolved oxygen ($O_2$)! In raw sewage, nitrifiers have a lag phase of 6 to 10 days, meaning standard 5-day tests primarily reflect carbonaceous demand. However, in biologically treated secondary effluents, nitrifiers are already abundant and active, which can cause significant nitrogenous oxygen demand to interfere during the 5-day test.

+---------------------------------------------------------------------------------------------------------+
|                                 BIOCHEMICAL OXYGEN UPTAKE CURVE                                         |
|                                                                                                         |
|   Dissolved Oxygen Consumed (mg/L)                                                                      |
|      ^                                                                                                  |
|      |                                                /====== Total Oxygen Demand (BOD)                 |
|      |                                               /                                                  |
|      |                                 +------------+========= Nitrogenous Demand (NBOD)                |
|      |                                /             |          (Begins Day 6-10 in raw sewage;          |
|      |                  /============+              |           active immediately in secondary)        |
|      |                 /                            |                                                   |
|      |    /===========+ Carbonaceous Demand (CBOD)  |                                                   |
|      |   /                                          |                                                   |
|      +----------------------------------------------+---------------------------------------> Time (Days)|
|          0    1    2    3    4    *5 DAYS*     6    7    8    9   10   12   14   20                      |
|                                   (Standard Test)                                                       |
+---------------------------------------------------------------------------------------------------------+

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2. BOD5 Laboratory Setup, Dilution Water & Nutrient Buffers

Standard laboratory testing uses 300 mL glass BOD bottles fitted with ground-glass flared stoppers. The flared neck is filled with deionized water to form a liquid seal that prevents atmospheric air from entering, capped with a plastic cup to prevent evaporation.

Incubation Specifications

• Temperature: Maintained strictly at 20.0 ± 1.0°C in a calibrated, dark environmental incubator.
• Incubation Duration: Exactly 5 days ± 6 hours (120 ± 6 hours).
• Darkness Requirement: Incubation must occur in complete darkness to prevent photosynthetic algae present in the sample from generating oxygen, which would artificially lower the measured DO depletion.

Dilution Water Preparation

Because raw wastewater BOD (200 to 250 mg/L) far exceeds the solubility of dissolved oxygen in water (~9.1 mg/L at 20°C), samples must be diluted with nutrient-fortified dilution water. Dilution water is prepared from high-purity deionized water aerated with oil-free filtered air until saturated with DO. For each liter of water, 1.0 mL of four essential reagent solutions is added:

1. Phosphate Buffer Solution: Formulated with $KH_2PO_4$, $K_2HPO_4$, $Na_2HPO_4\cdot 7H_2O$, and $NH_4Cl$. Buffers the solution at pH 7.2 and supplies essential phosphorus and nitrogen.
2. Magnesium Sulfate Solution ($MgSO_4\cdot 7H_2O$): Supplies magnesium ions essential for bacterial enzymatic function.
3. Calcium Chloride Solution ($CaCl_2$): Supplies calcium for cell wall structural stability.
4. Ferric Chloride Solution ($FeCl_3\cdot 6H_2O$): Supplies trace iron for bacterial electron transport chains.

Seed Microorganisms

Samples that do not contain an abundant, viable biological population—such as chlorinated effluents, industrial wastes, or heat-treated sludges—must be inoculated with an active bacterial "seed" (typically settled raw domestic sewage, unchlorinated primary effluent, or a commercially freeze-dried microbial consortium). Fresh raw sewage contains abundant native bacteria and does not require external seed.

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3. Rigorous Test Validity Criteria & Quality Assurance

Under Standard Methods 5210 B, a BOD5 sample dilution is considered legally and technically valid only if it satisfies all of the following criteria:

1. Minimum DO Depletion: The sample dilution must consume at least 2.0 mg/L of dissolved oxygen between Day 0 and Day 5 ($\text{Initial DO} - \text{Final DO} \ge 2.0\text{ mg/L}$). Depletions $< 2.0\text{ mg/L}$ exhibit high relative analytical error.
2. Minimum Residual DO: The sample dilution must retain at least 1.0 mg/L of dissolved oxygen at the end of the 5-day incubation period ($\text{Final DO} \ge 1.0\text{ mg/L}$). If DO drops below 1.0 mg/L (or reaches 0.0 mg/L), aerobic bacteria become oxygen-starved, slowing decomposition and invalidating the test.
3. Dilution Water Blank Limit: The unseeded dilution water blank must not show a DO depletion exceeding 0.20 mg/L over 5 days ($\text{Blank Initial DO} - \text{Blank Final DO} \le 0.20\text{ mg/L}$). A depletion $> 0.20\text{ mg/L}$ indicates organic contamination of dilution water, dirty glassware, or failing water purification systems.
4. Glucose-Glutamic Acid (GGA) Standard Check: A primary QA standard prepared with 150 mg/L reagent-grade glucose and 150 mg/L glutamic acid. A 2% dilution (6.0 mL of GGA in a 300 mL bottle) must yield a 5-day BOD of $198 \pm 30.5\text{ mg/L}$ (acceptable range: $167.5\text{ to } 228.5\text{ mg/L}$). Results outside this range indicate seed toxicity, inactive microorganisms, or incubator temperature malfunction.

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4. Carbonaceous BOD (CBOD5) & Nitrification Inhibition

Publicly Owned Treatment Works (POTWs) utilizing secondary biological nitrification often encounter significant nitrogenous oxygen uptake during final effluent testing. To isolate carbonaceous organic loading from ammonia oxidation, NPDES permits often specify Carbonaceous Biochemical Oxygen Demand (CBOD5) limits (typically 25 mg/L monthly average instead of 30 mg/L for BOD5).

The Chemical Inhibitor: TCMP

To perform CBOD5, a chemical nitrification inhibitor is dosed into each 300 mL bottle prior to filling with dilution water:

• Inhibitor Chemical: TCMP (2-chloro-6-(trichloromethyl)pyridine) or alternatively allylthiourea (ATU).
• Dosage: Typically 10 mg of TCMP per 300 mL BOD bottle (or approximately 3 mg per bottle depending on formulation).
• Biochemical Mode of Action: TCMP selectively halts the metabolic enzymes of autotrophic nitrifiers (Nitrosomonas and Nitrobacter), completely suppressing the oxidation of ammonia ($NH_4^+$) to nitrite and nitrate. It does not inhibit heterotrophic bacteria, allowing unrestricted measurement of true carbonaceous oxygen demand.

+---------------------------------------------------------------------------------------------------------+
|                                   CBOD5 VS. TOTAL BOD5 MEASUREMENT                                      |
|                                                                                                         |
|   Sample Stream              Bottle Chemistry               Microbial Pathway        Measured Parameter |
|   ---------------------------------------------------------------------------------------------------   |
|   Secondary Effluent  -----> Standard Dilution Water -----> Heterotrophs +         = TOTAL BOD5         |
|   [Organics + NH3]           (No Inhibitor)                  Nitrifiers Active       (Permit Limit 30)  |
|                                                                                                         |
|   Secondary Effluent  -----> Dilution Water + TCMP  -----> Heterotrophs Active;    = CBOD5              |
|   [Organics + NH3]           (Nitrification Inhibitor)       Nitrifiers Blocked      (Permit Limit 25)  |
+---------------------------------------------------------------------------------------------------------+

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5. BOD5 Mathematical Calculations

Unseeded BOD5 Calculation

When testing raw sewage or unchlorinated primary effluent with native biological populations, the unseeded formula is used:

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

Where:

• $DO_{\text{initial}} = $ initial dissolved oxygen concentration immediately after preparation (mg/L),
• $DO_{\text{final}} = $ dissolved oxygen concentration after 5 days of incubation at 20°C (mg/L),
• $P = $ decimal dilution fraction $= \frac{\text{Sample Volume (mL)}}{\text{Total Bottle Volume (300 mL)}}$.

Seeded BOD5 Calculation

When an external biological seed is added, the oxygen depletion caused by the seed itself must be subtracted:

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

Where:

• $B_1 = $ initial DO of the seeded dilution water control blank (mg/L),
• $B_2 = $ final DO of the seeded dilution water control blank after 5 days (mg/L),
• $f = $ ratio of seed volume in sample bottle to seed volume in seed control bottle $= \frac{% \text{ seed in sample bottle}}{% \text{ seed in control bottle}}$.

Worked Calculation Example:

An analyst sets up an unseeded BOD5 test using 6.0 mL of primary effluent in a 300 mL BOD bottle. The initial DO is measured at 8.40 mg/L. After 5 days of incubation at 20°C, the final DO is 3.60 mg/L. The dilution water blank initial DO was 8.50 mg/L and final DO was 8.40 mg/L (depletion = 0.10 mg/L, valid).

1. Check validity: $\text{Depletion} = 8.40 - 3.60 = 4.80\text{ mg/L}$ (meets $\ge 2.0\text{ mg/L}$). Final $\text{DO} = 3.60\text{ mg/L}$ (meets $\ge 1.0\text{ mg/L}$).
2. Calculate dilution fraction: $P = 6.0\text{ mL} / 300\text{ mL} = 0.02$.
3. Calculate BOD5: $\text{BOD}_5 = \frac{8.40 - 3.60}{0.02} = \frac{4.80}{0.02} = 240\text{ mg/L}$.

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6. Total Suspended Solids (TSS) Gravimetric Methodology

Total Suspended Solids (TSS) measures the non-filterable particulate matter in a wastewater sample. The standardized gravimetric protocol (Standard Methods 2540 D / EPA Method 160.2) requires filtering a measured volume of sample through a pre-weighed glass fiber filter disk, followed by thermal drying.

Laboratory Apparatus & Preparation

• Filter Disk: Glass fiber filter without organic binder, nominal pore size of 1.5 µm (Whatman 934-AH, Gelman A/E, or Millipore AP40).
• Filtration Apparatus: Gooch crucible or vacuum filtration funnel mounted on a side-arm vacuum flask.
• Analytical Balance: Precision balance sensitive to 0.1 mg (0.0001 g).
• Drying Oven: Gravity convection or forced-air oven maintained strictly between 103°C and 105°C.
• Desiccator: Airtight chamber charged with active indicating silica gel desiccant to prevent ambient moisture absorption during cooling.

Step-by-Step Analytical Workflow

1. Filter Pre-Conditioning: Place filter disk on vacuum base. Wash with three successive 20 mL portions of reagent-grade deionized water under vacuum. Dry in oven at 103°C to 105°C for 1 hour, cool in desiccator to room temperature (20–30 minutes), and weigh on analytical balance to nearest 0.1 mg. Record as Tare Weight ($W_1$).
2. Sample Filtration: Vigorously shake sample container to thoroughly resuspend solids. Immediately measure a representative volume using a wide-bore pipet or graduated cylinder while swirling. Pour through filter under vacuum. Filter volume is selected to yield 2.5 to 200 mg of dried solids (typically 25 to 50 mL for raw influent; 200 to 500 mL for final effluent).
3. Deionized Water Rinsing: Wash filter with three successive 10 mL portions of deionized water to rinse all dissolved mineral salts through the filter. Failure to rinse leaves dissolved salts that crystallize upon drying, falsely elevating TSS.
4. Drying and Desiccation: Dry filter in oven at 103°C to 105°C for at least 1 hour. Cool in desiccator to room temperature and weigh ($W_2$). Repeat the drying cycle (30 minutes) until constant weight is attained (weight loss $< 0.5\text{ mg}$ or $< 4%$ of previous weight).

TSS Calculation Formula

$\text{TSS (mg/L)} = \frac{(W_{\text{final}} [\text{g}] - W_{\text{tare}} [\text{g}]) \times 1{,}000{,}000}{\text{Sample Volume (mL)}}$

Where multiplying by $1{,}000{,}000$ converts the gram weight difference to milligrams ($1\text{ g} = 1{,}000\text{ mg}$) and milliliters to liters ($1\text{ L} = 1{,}000\text{ mL}$).

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7. Volatile Suspended Solids (VSS) Ignition Protocol

Volatile Suspended Solids (VSS) represents the organic, combustible fraction of TSS, serving as a primary indicator of biological biomass in activated sludge mixed liquor (MLVSS) and digester sludges.

Muffle Furnace Ignition Workflow

1. Take the dried, weighed filter disk from the TSS test ($W_{\text{final}}$).
2. Transfer filter into a muffle furnace preheated to $550 \pm 50^\circ\text{C}$ and ignite for 15 to 20 minutes (up to 30 minutes for heavy solids loadings).
3. Allow filter to cool partially in air, transfer to a desiccator, cool to room temperature, and weigh on the analytical balance to the nearest 0.1 mg. Record as Ignited Weight ($W_{\text{ignited}}$).
4. Interpretation: Organic matter volatilizes into carbon dioxide, water vapor, and nitrogen gases. The remaining residue is inorganic ash (Fixed Suspended Solids - FSS). The mass lost during ignition equals VSS.

$\text{VSS (mg/L)} = \frac{(W_{\text{final}} [\text{g}] - W_{\text{ignited}} [\text{g}]) \times 1{,}000{,}000}{\text{Sample Volume (mL)}}$

$\% \text{ Volatile Solids} = \frac{W_{\text{final}} - W_{\text{ignited}}}{W_{\text{final}} - W_{\text{tare}}} \times 100\%$

In raw domestic wastewater, VSS typically constitutes 70% to 80% of TSS; in activated sludge mixed liquor, MLVSS typically comprises 75% to 85% of MLSS.