10.2 Wastewater Laboratory Testing: BOD5, COD, Total Suspended Solids & Settleability

Wastewater Laboratory Testing: BOD5, COD, Total Suspended Solids & Settleability

Core Principle: Wastewater treatment facilities operate biological and physical separation barriers to remove oxygen-depleting organics, nutrients, and suspended solids. Rigorous laboratory testing provides plant operators with real-time operational feedback to adjust aeration, sludge wasting, and chemical feeds, while producing legally defensible compliance data for National Pollutant Discharge Elimination System (NPDES) permits administered by ADEM.

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1. Biochemical Oxygen Demand (BOD5) & Carbonaceous BOD (CBOD5)

Biochemical Oxygen Demand ($\text{BOD}$) measures the mass of dissolved oxygen consumed by aerobic microorganisms as they decompose biodegradable organic matter in water over a standardized incubation period. When organic-rich wastewater is discharged into receiving streams, bacterial decomposition consumes dissolved oxygen, suffocating fish and degrading aquatic ecosystems. The standard laboratory test quantifies oxygen consumption over 5 days at $20.0 \pm 1.0^\circ\text{C}$ in the dark ($\text{BOD}_5$).

The Nitrification Problem & CBOD5

In standard aerobic biological oxidation, two distinct oxygen demands occur:

1. Carbonaceous Oxygen Demand (CBOD): Heterotrophic bacteria oxidize organic carbon compounds to carbon dioxide and water: $\text{Organic Carbon } (C_a H_b O_c) + O_2 \xrightarrow{\text{Heterotrophs}} CO_2 + H_2O + \text{New Cells}$
2. Nitrogenous Oxygen Demand (NBOD): Autotrophic nitrifying bacteria (Nitrosomonas and Nitrobacter) oxidize reduced nitrogen compounds (ammonia and organic nitrogen) to nitrite and nitrate: $NH_4^+ + 1.5 O_2 \xrightarrow{\text{Nitrosomonas}} NO_2^- + H_2O + 2H^+$ $NO_2^- + 0.5 O_2 \xrightarrow{\text{Nitrobacter}} NO_3^-$

In raw municipal wastewater, nitrifiers are present in low concentrations, and NBOD typically does not exert significant oxygen demand until day 6 to 10 of incubation. However, in modern secondary effluents, especially facilities practicing biological nitrification or extended aeration, large, acclimated populations of nitrifying bacteria are discharged into the sample bottle. These nitrifiers immediately consume large amounts of oxygen ($4.57\text{ mg } O_2$ per $1.0\text{ mg } NH_3\text{-N}$ oxidized), creating a falsely elevated apparent organic load.

To isolate organic carbon removal performance, standard methods add a nitrification inhibitor—2-chloro-6-(trichloromethyl) pyridine (TCMP)—to the sample bottle prior to incubation. TCMP selectively suppresses nitrifier metabolism without inhibiting heterotrophic carbon oxidizers. The result is reported as Carbonaceous Biochemical Oxygen Demand (CBOD5), which many NPDES permits, including many ADEM municipal permits, use as the oxygen-demand limit.

Dilution Water Preparation (Standard Methods 5210B)

Because raw wastewater ($100-300\text{ mg/L BOD}$) and even secondary effluent ($10-30\text{ mg/L BOD}$) exceed the maximum dissolved oxygen solubility in water ($\sim 9.0\text{ mg/L}$ at 20°C), samples must be diluted with nutrient-fortified, oxygen-saturated water.

• Water Source: High-purity deionized or distilled water free of toxic heavy metals, chlorine, and organic compounds. The water must be aerated with clean, oil-free compressed air until dissolved oxygen reaches near-saturation ($8.0-9.0\text{ mg/L}$).
• Nutrient Buffers: Add $1.0\text{ mL}$ per liter of each of four essential stock solutions:
  1. Phosphate Buffer Solution: ($KH_2PO_4$, $K_2HPO_4$, $Na_2HPO_4 \cdot 7H_2O$, $NH_4Cl$) buffers the water at pH 7.2 and supplies essential phosphorus and nitrogen.
  2. Magnesium Sulfate Solution: ($MgSO_4 \cdot 7H_2O$) supplies magnesium ions for bacterial enzymatic transfer.
  3. Calcium Chloride Solution: ($CaCl_2$) provides essential calcium for cell wall structure.
  4. Ferric Chloride Solution: ($FeCl_3 \cdot 6H_2O$) provides trace iron required for microbial respiratory cytochromes.
• Dilution Water Quality Blank: An unseeded dilution water blank must be incubated alongside every batch. The DO depletion of the blank must not exceed $0.20\text{ mg/L}$ after 5 days. Depletion greater than $0.20\text{ mg/L}$ indicates organic contamination in the reagents, deionized water system, or glassware, invalidating all associated test runs.

Microbial Seed & Seed Correction

Samples that lack viable, acclimated microorganisms—such as disinfected (chlorinated/UV) secondary effluents, extreme pH industrial discharges, or boiled wastes—must be inoculated with an active biological seed (typically settled domestic primary effluent or commercial bacterial seed cultures):

• Seed Control: Prepare a series of dilution water bottles containing varying seed volumes (e.g., 2, 4, and 6 mL) without sample to determine the DO depletion attributable strictly to the seed.
• Seed Depletion Ratio ($S$): The seed correction factor is the DO depletion per mL of seed added: $S = \frac{DO_{\text{seed, initial}} - DO_{\text{seed, final}}}{\text{mL of seed in control bottle}}$

Test Validity Gates & GGA Standards

Under Standard Methods 5210B, a BOD determination is legally valid only if it satisfies three strict criteria:

1. Minimum DO Depletion Gate: The sample bottle must consume at least $2.0\text{ mg/L}$ of DO over the 5-day incubation period ($DO_i - DO_f \ge 2.0\text{ mg/L}$). Depletions $< 2.0\text{ mg/L}$ exhibit high analytical noise and cannot be used.
2. Minimum Residual DO Gate: The sample bottle must contain at least $1.0\text{ mg/L}$ of DO remaining after 5 days ($DO_f \ge 1.0\text{ mg/L}$). If residual DO drops below $1.0\text{ mg/L}$, bacterial respiration becomes oxygen-limited or anoxic, under-measuring true organic demand.
3. Glucose-Glutamic Acid (GGA) Standard: An analytical check standard consisting of $150\text{ mg/L}$ reagent-grade glucose and $150\text{ mg/L}$ reagent-grade glutamic acid ($300\text{ mg/L}$ total solids). A $2.0\%$ dilution ($6.0\text{ mL}$ of GGA in a $300\text{ mL}$ bottle) must yield an average 5-day BOD of $198 \pm 30.5\text{ mg/L}$ (acceptable window: $167.5\text{ to } 228.5\text{ mg/L}$). Values outside this window indicate toxic inhibitory substances or weak, inactive microbial seed.

Mathematical Calculations

Unseeded BOD5 Formula

$\text{BOD}_5\text{ (mg/L)} = \frac{DO_i - DO_f}{P}$

Where $DO_i$ is initial dissolved oxygen (mg/L), $DO_f$ is final dissolved oxygen on day 5 (mg/L), and $P$ is the decimal sample dilution fraction ($P = \text{sample volume (mL)} / 300\text{ mL}$). For example, for a $15\text{ mL}$ sample in a standard $300\text{ mL}$ BOD bottle, $P = 15 / 300 = 0.05$.

Seeded BOD5 Formula

$\text{BOD}_5\text{ (mg/L)} = \frac{(DO_i - DO_f) - (S \times V_s)}{P}$

Where $S$ is seed DO depletion per mL of seed added, and $V_s$ is the volume of seed (mL) added directly into the sample bottle.

Dissolved Oxygen Measurement Methods

• Azide Modification of the Winkler Titration (Standard Methods 4500-O C):
  • Standard wet chemistry titration based on the oxidation of manganous ($Mn^{2+}$) to manganic ($Mn^{3+}$ or $Mn^{4+}$) hydroxide precipitate by dissolved oxygen under alkaline conditions.
  • Reagents: Manganous sulfate ($MnSO_4$), alkaline-iodide-azide ($NaOH + KI + NaN_3$), and concentrated sulfuric acid ($H_2SO_4$).
  • Role of Sodium Azide ($NaN_3$): Destroys nitrite ($NO_2^-$) interference, which is prevalent in wastewater effluents. Without azide, nitrite reacts catalytically with iodide to continuously generate excess iodine, producing false high DO readings: $2NaN_3 + 2HNO_2 \rightarrow 3N_2 \uparrow + N_2O \uparrow + 2NaOH$
  • Acidification dissolves the floc and oxidizes iodide ($I^-$) to free elemental iodine ($I_2$) in direct proportion to original dissolved oxygen. The solution is titrated with $0.0250\text{ N}$ sodium thiosulfate ($Na_2S_2O_3$) using starch indicator (blue to colorless endpoint). For a $200\text{ mL}$ sample, $1.0\text{ mL}$ of $0.0250\text{ N } Na_2S_2O_3 = 1.0\text{ mg/L DO}$.
• Luminescent Dissolved Oxygen (LDO) / Optical Membrane Probe:
  • Utilizes a luminescent sensor cap containing an organometallic platinum or ruthenium dye excited by a blue LED.
  • Oxygen molecules quench the luminescence lifetime. The phase shift of the emitted red light is inversely proportional to dissolved oxygen partial pressure.
  • Operational Advantages: LDO sensors consume zero oxygen during measurement (no stirring or flow velocity required), require no electrolyte solutions or membrane replacements, and are completely unaffected by chemical poisoning from hydrogen sulfide ($H_2S$), ammonia, or heavy metals.

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2. Chemical Oxygen Demand (COD)

Chemical Oxygen Demand measures the total quantity of oxygen required to chemically oxidize organic and oxidizable inorganic matter in water using a powerful chemical oxidant under severe thermal and acidic conditions (Standard Methods 5220D Closed Reflux).

Digestion Chemistry & Reagent Roles

[Sample + Digestion Reagents]
  • Oxidant: Potassium Dichromate (K2Cr2O7 in H2SO4)
  • Catalyst: Silver Sulfate (Ag2SO4)
  • Complexing Agent: Mercuric Sulfate (HgSO4)
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[Closed Reflux Digestion]
Heated in sealed culture tubes at 150°C for 2.0 hours
Cr⁶⁺ (hexavalent, orange) is reduced to Cr³⁺ (trivalent, green)
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[Quantification]
Spectrophotometric absorption at 600 nm (Cr³⁺) or 420 nm (Cr⁶⁺)
Direct concentration output in mg/L COD within 2.5 hours total time

• Potassium Dichromate ($K_2Cr_2O_7$): Strong oxidant in boiling concentrated sulfuric acid ($H_2SO_4$). Dichromate oxidizes nearly all organic carbon compounds to $CO_2$ and $H_2O$, while hexavalent chromium ($Cr^{6+}$, bright orange) is reduced to trivalent chromium ($Cr^{3+}$, deep green): $C_n H_a O_b + c\,Cr_2O_7^{2-} + 8c\,H^+ \rightarrow n\,CO_2 + \frac{a + 8c}{2}\,H_2O + 2c\,Cr^{3+}$
• Silver Sulfate ($Ag_2SO_4$): Functions as an indispensable catalyst. Straight-chain aliphatic hydrocarbons, volatile fatty acids, and alcohols resist dichromate attack; silver ions catalyze the rapid electron transfer required to oxidize these refractory chains.
• Mercuric Sulfate ($HgSO_4$): Eliminates chloride interference. Municipal and industrial wastewaters contain chloride ions ($Cl^-$), which dichromate oxidizes to free chlorine gas ($Cl_2$), consuming oxidant and generating massive false high COD readings. Mercuric sulfate binds chloride at a strict 10:1 ratio ($HgSO_4 : Cl^-$) to form stable, soluble mercuric chloride complexes ($HgCl_4^{2-}$), suppressing chloride interference up to $2,000\text{ mg/L}$.
• Digestion Conditions: Samples are incubated in tightly capped borosilicate culture vials inside a preheated digestion block at $150 \pm 2^\circ\text{C}$ for precisely 2 hours.

COD vs. BOD Comparison & Operational Correlation

• The Municipal COD:BOD Ratio: In raw municipal domestic wastewater, the typical $COD:\text{BOD}_5$ ratio ranges between $1.5:1\text{ and } 2.5:1$. In well-oxidized, treated secondary effluent, the ratio climbs to $3:1\text{ to } 5:1$ because bacteria have consumed the biodegradable fraction, leaving biologically refractory organics behind.
• Troubleshooting Significance: If an operator observes the influent $COD:\text{BOD}_5$ ratio suddenly spike from $2:1$ to $> 3.5:1$, it signals an illegal industrial discharge containing toxic compounds that inhibit biological BOD uptake, or refractory industrial wastes (e.g., plastics, petrochemicals, textile dyes) requiring industrial pretreatment enforcement.

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

Solids testing provides the primary quantitative basis for assessing clarifier efficiency, calculating sludge inventory, establishing Mean Cell Residence Time (MCRT), and ensuring NPDES permit compliance.

Gravimetric TSS Procedure (Standard Methods 2540D)

Total Suspended Solids represents the non-filterable particulate residue retained on a standardized glass fiber filter and dried to constant weight at $103^\circ\text{C to } 105^\circ\text{C}$.

1. Filter Preparation:
   • Place a binderless borosilicate glass fiber filter disk (Whatman 934-AH, nominal pore size $1.5\ \mu\text{m}$) on a vacuum filtration apparatus.
   • Apply vacuum and wash the filter with three successive $20\text{ mL}$ volumes of reagent-grade water to remove loose glass fibers.
   • Transfer the filter to an aluminum weighing dish and dry in an oven at $103-105^\circ\text{C}$ for at least 1 hour.
   • Cool in a desiccator containing active indicating silica gel to room temperature. Weigh on a calibrated analytical balance to the nearest $0.1\text{ mg}$ ($0.0001\text{ g}$) to establish the tare weight ($W_{\text{tare}}$).
2. Sample Filtration:
   • Thoroughly mix the sample container by vigorous inversion.
   • Measure a known volume of sample ($V$, in mL)—typically 25 to 50 mL for raw wastewater, 50 to 100 mL for primary effluent, and 200 to 1,000 mL for final secondary effluent—aiming to yield between $2.5\text{ and } 200\text{ mg}$ of dry dried residue.
   • Filter the sample under vacuum. Rinse the graduated cylinder and filter funnel walls with three successive $10\text{ mL}$ portions of reagent water, allowing complete drainage between rinses to remove dissolved salts.
3. Drying & Weighing:
   • Dry the filter in a drying oven at $103-105^\circ\text{C}$ for at least 1 hour.
   • Transfer to a desiccator, cool to room temperature, and weigh ($W_{\text{dry}}$). Repeat the cycle of drying, desiccating, and weighing until the weight change is less than $0.5\text{ mg}$ (constant weight).

TSS Mathematical Calculation

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

Where $(W_{\text{dry}} - W_{\text{tare}})$ is the net dry residue mass in grams, and $1,000,000$ converts grams to milligrams and milliliters to liters ($1,000\text{ mg/g} \times 1,000\text{ mL/L}$). For example, if a $100\text{ mL}$ sample produces a tare weight of $1.5320\text{ g}$ and a dried weight of $1.5356\text{ g}$:

$\text{TSS} = \frac{(1.5356 - 1.5320) \times 1,000,000}{100} = \frac{0.0036 \times 1,000,000}{100} = 36.0\text{ mg/L}$

Volatile Suspended Solids (VSS) (Standard Methods 2540E)

Volatile Suspended Solids measures the organic (biological) fraction of the suspended solids. After recording the dry TSS weight, the filter disk is transferred into a muffle furnace at $550 \pm 50^\circ\text{C}$ for 15 to 20 minutes:

• Combustion: At 550°C, all organic biological carbon burns off as $CO_2$ and water vapor, leaving behind inert mineral ash (Fixed Suspended Solids, FSS).
• Weighing: Partially cool the filter in air, place in a desiccator to reach room temperature, and weigh on the analytical balance ($W_{\text{ash}}$).

VSS and FSS Formulas

$\text{VSS (mg/L)} = \frac{(W_{\text{dry, g}} - W_{\text{ash, g}}) \times 1,000,000}{\text{Sample Volume (mL)}}$

$\text{FSS (mg/L)} = \frac{(W_{\text{ash, g}} - W_{\text{tare, g}}) \times 1,000,000}{\text{Sample Volume (mL)}}$

• Process Control Application: In an activated sludge basin, Mixed Liquor Suspended Solids (MLSS) includes both living biomass and inert mineral grit. Mixed Liquor Volatile Suspended Solids (MLVSS) represents the active biological mass responsible for treating wastewater. The $\text{MLVSS} / \text{MLSS}$ ratio in healthy municipal activated sludge systems ranges between $0.70\text{ and } 0.85$ (70% to 85%). A decline below 70% indicates severe accumulation of inorganic silt, clay, or chemical precipitants, signaling insufficient solids wasting.

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4. Settleable Solids & Imhoff Cone Test

Settleable solids quantifies the volume of suspended solids that settle out of a liquid sample under still, gravity conditions over a 1-hour period. It is reported in $\text{mL/L}$ (Standard Methods 2540F).

Procedure

1. Thoroughly mix the wastewater sample.
2. Fill a graduated 1.0-liter transparent Imhoff cone exactly to the 1.0-liter mark.
3. Allow the liquid to settle undisturbed for 45 minutes.
4. Gently run a glass stirring rod along the inside conical walls, or gently rotate the cone between hands through a 45-degree arc, to dislodge solids clinging to the sloped walls. Crucial rule: Do not disturb or stir the settled sludge blanket at the cone's apex.
5. Allow the sample to settle for an additional 15 minutes (total settling time: 60 minutes).
6. Read the volume of settled solids directly from the graduated markings at the cone tip, reporting as $\text{mL/L}$.

Operational Applications

• Raw Wastewater: Typical municipal domestic raw wastewater contains $5\text{ to } 15\text{ mL/L}$ settleable solids.
• Primary Clarifier Efficiency: Primary clarifiers are designed to remove settleable solids before secondary biological treatment. Primary effluent should contain $< 0.5\text{ to } 1.0\text{ mL/L}$. Primary clarifier settleable solids removal efficiency must exceed 90% to 95%; lower removal indicates short-circuiting, excessive hydraulic overflow rates, or damaged sludge collector flights.