EPA Methods List with Links

US EPA Method 304a: Determination Of Biodegradation Rates Of Organic Compounds (Vent Option)

1.0 Scope and Application.

1.1 Applicability. This method is applicable for the determination of biodegradation rates of organic compounds in an activated sludge process. The test method is designed to evaluate the ability of an aerobic biological reaction system to degrade or destroy specific components in waste streams. The method may also be used to determine the effects of changes in wastewater composition on operation. The biodegradation rates determined by utilizing this method are not representative of a full-scale system. The rates measured by this method shall be used in conjunction with the procedures listed in appendix C of this part to calculate the fraction emitted to the air versus the fraction biodegraded. 2.0 Summary of Method.

2.1 A self-contained benchtop bioreactor system is assembled in the laboratory. A sample of mixed liquor is added and the waste stream is then fed continuously. The benchtop bioreactor is operated under conditions nearly identical to the target full-scale activated sludge process. Bioreactor temperature, dissolved oxygen concentration, average residence time in the reactor, waste composition, biomass concentration, and biomass composition of the full-

2005 scale process are the parameters which are duplicated in the

benchtop bioreactor. Biomass shall be removed from the target full-scale activated sludge unit and held for no more than 4 hours prior to use in the benchtop bioreactor. If antifoaming agents are used in the full-scale system, they shall also be used in the benchtop bioreactor. The feed flowing into and the effluent exiting the benchtop bioreactor are analyzed to determine the biodegradation rates of the target compounds. The flow rate of the exit vent is used to calculate the concentration of target compounds (utilizing Henry's law) in the exit gas stream. If Henry's law constants for the compounds of interest are not known, this method cannot be used in the determination of the biodegradation rate and Method 304B is the suggested method. The choice of analytical methodology for measuring the compounds of interest at the inlet and outlet to the benchtop bioreactor are left to the discretion of the source, except where validated methods are available. 3.0 Definitions. [Reserved] 4.0 Interferences. [Reserved] 5.0 Safety.

5.1 If explosive gases are produced as a byproduct of biodegradation and could realistically pose a hazard, closely monitor headspace concentration of these gases to

2006 ensure laboratory safety. Placement of the benchtop

bioreactor system inside a laboratory hood is recommended regardless of byproducts produced. 6.0. equipment and supplies.

NOTE: Figure 304A-1 illustrates a typical laboratory apparatus used to measure biodegradation rates. While the following description refers to Figure 304A-1, the EPA recognizes that alternative reactor configurations, such as alternative reactor shapes and locations of Probes and the feed inlet, will also meet the intent of this method. Ensure that the benchtop bioreactor system is self-contained and isolated from the atmosphere (except for the exit vent stream) by leak-checking fittings, tubing, etc.

6.1 Benchtop Bioreactor. The biological reaction is conducted in a biological oxidation reactor of at least 6 liters capacity. The benchtop bioreactor is sealed and equipped with internal Probes for controlling and monitoring dissolved oxygen and internal temperature. The top of the reactor is equipped for aerators, gas flow ports, and instrumentation (while ensuring that no leaks to the atmosphere exist around the fittings).

6.2 Aeration gas. Aeration gas is added to the benchtop bioreactor through three diffusers, which are glass tubes that extend to the bottom fifth of the reactor depth.

2007 A pure oxygen pressurized cylinder is recommended in order

to maintain the specified oxygen concentration. Install a blower (e.g., Diaphragm Type, 15 SCFH capacity) to blow the aeration gas into the reactor diffusers. Measure the aeration gas flow rate with a rotameter (e.g., 0-15 SCFH recommended). The aeration gas will rise through the benchtop bioreactor, dissolving oxygen into the mixture in the process. The aeration gas must provide sufficient agitation to keep the solids in suspension. Provide an exit for the aeration gas from the top flange of the benchtop bioreactor through a water-cooled (e.g., Allihn-type) vertical condenser. Install the condenser through a gas- tight fitting in the benchtop bioreactor closure. Install a splitter which directs a portion of the gas to an exit vent and the rest of the gas through an air recycle pump back to the benchtop bioreactor. Monitor and record the flow rate through the exit vent at least 3 times per day throughout the day.

6.3 Wastewater Feed. Supply the wastewater feed to the benchtop bioreactor in a collapsible low-density polyethylene container or collapsible liner in a container (e.g., 20 L) equipped with a spigot cap (collapsible containers or liners of other material may be required due to the permeability of some volatile compounds through

2008 polyethylene). Obtain the wastewater feed by sampling the

wastewater feed in the target process. A representative sample of wastewater shall be obtained from the piping leading to the aeration tank. This sample may be obtained from existing sampling valves at the discharge of the wastewater feed pump, or collected from a pipe discharging to the aeration tank, or by pumping from a well-mixed equalization tank upstream from the aeration tank. Alternatively, wastewater can be pumped continuously to the laboratory apparatus from a bleed stream taken from the equalization tank of the full-scale treatment system.

6.3.1 Refrigeration System. Keep the wastewater feed cool by ice or by refrigeration to 4EC. If using a bleed stream from the equalization tank, refrigeration is not required if the residence time in the bleed stream is less than five minutes.

6.3.2 Wastewater Feed pump. The wastewater is pumped from the refrigerated container using a variable-speed peristaltic pump drive equipped with a peristaltic pump head. Add the feed solution to the benchtop bioreactor through a fitting on the top flange. Determine the rate of feed addition to provide a retention time in the benchtop bioreactor that is numerically equivalent to the retention time in the full-scale system. The wastewater shall be fed

2009 at a rate sufficient to achieve 90 to 100 percent of the

full-scale system residence time. 6.3.3 Treated wastewater feed. The benchtop

bioreactor effluent exits at the bottom of the reactor through a tube and proceeds to the clarifier.

6.4 Clarifier. The effluent flows to a separate closed clarifier that allows separation of biomass and effluent (e.g., 2-liter pear-shaped glass separatory funnel, modified by removing the stopcock and adding a 25-mm OD glass tube at the bottom). Benchtop bioreactor effluent enters the clarifier through a tube inserted to a depth of 0.08 m (3 in.) through a stopper at the top of the clarifier. System effluent flows from a tube inserted through the stopper at the top of the clarifier to a drain (or sample bottle when sampling). The underflow from the clarifier leaves from the glass tube at the bottom of the clarifier. Flexible tubing connects this fitting to the sludge recycle pump. This pump is coupled to a variable speed pump drive. The discharge from this pump is returned through a tube inserted in a port on the side of the benchtop bioreactor. An additional port is provided near the bottom of the benchtop bioreactor for sampling the reactor contents. The mixed liquor from the benchtop bioreactor flows into the center of the clarifier. The

2010 clarified system effluent separates from the biomass and

flows through an exit near the top of the clarifier. There shall be no headspace in the clarifier.

6.5 temperature Control Apparatus. Capable of maintaining the system at a temperature equal to the temperature of the full-scale system. The average temperature should be maintained within ±2 EC of the set point.

6.5.1 temperature Monitoring Device. A resistance type temperature Probe or a thermocouple connected to a temperature readout with a resolution of 0.1 EC or better.

6.5.2 Benchtop Bioreactor Heater. The heater is connected to the temperature control device.

6.6 Oxygen Control System. Maintain the dissolved oxygen concentration at the levels present in the full-scale system. Target full-scale activated sludge systems with dissolved oxygen concentration below 2 mg/L are required to maintain the dissolved oxygen concentration in the benchtop bioreactor within 0.5 mg/L of the target dissolved oxygen level. Target full-scale activated sludge systems with dissolved oxygen concentration above 2 mg/L are required to maintain the dissolved oxygen concentration in the benchtop bioreactor within 1.5 mg/L of the target dissolved oxygen concentration; however, for target full-scale activated sludge systems with dissolved oxygen concentrations above 2

2011 mg/L, the dissolved oxygen concentration in the benchtop

bioreactor may not drop below 1.5 mg/L. If the benchtop bioreactor is outside the control range, the dissolved oxygen is noted and the reactor operation is adjusted.

6.6.1 Dissolved Oxygen Monitor. Dissolved oxygen is monitored with a polarographic Probe (gas permeable membrane) connected to a dissolved oxygen meter (e.g., 0 to 15 mg/L, 0 to 50 EC).

6.6.2 Benchtop bioreactor Pressure Monitor. The benchtop bioreactor pressure is monitored through a port in the top flange of the reactor. This is connected to a gauge control with a span of 13-cm water vacuum to 13-cm water pressure or better. A relay is activated when the vacuum exceeds an adjustable setpoint which opens a solenoid valve (normally closed), admitting oxygen to the system. The vacuum setpoint controlling oxygen addition to the system shall be set at approximately 2.5 ± 0.5 cm water and maintained at this setting except during brief periods when the dissolved oxygen concentration is adjusted.

6.7 Connecting tubing. All connecting tubing shall be Teflon or equivalent in impermeability. The only exception to this specification is the tubing directly inside the pump head of the wastewater feed pump, which may be Viton, Silicone or another type of flexible tubing.

2012 Note: Mention of trade names or products does not

constitute endorsement by the U.S. Environmental Protection Agency. 7.0 Reagents and Standards.

7.1 Wastewater. Obtain a representative sample of wastewater at the inlet to the full-scale treatment plant if there is an existing full-scale treatment plant (see section 6.3). If there is no existing full-scale treatment plant, obtain the wastewater sample as close to the point of determination as possible. Collect the sample by pumping the wastewater into the 20-L collapsible container. The loss of volatiles shall be minimized from the wastewater by collapsing the container before filling, by minimizing the time of filling, and by avoiding a headspace in the container after filling. If the wastewater requires the addition of nutrients to support the biomass growth and maintain biomass characteristics, those nutrients are added and mixed with the container contents after the container is filled.

7.2 Biomass. Obtain the biomass or activated sludge used for rate constant determination in the bench-scale process from the existing full-scale process or from a representative biomass culture (e.g., biomass that has been developed for a future full-scale process). This biomass is

2013 preferentially obtained from a thickened acclimated mixed

liquor sample. Collect the sample either by bailing from the mixed liquor in the aeration tank with a weighted container, or by collecting aeration tank effluent at the effluent overflow weir. Transport the sample to the laboratory within no more than 4 hours of collection. Maintain the biomass concentration in the benchtop bioreactor at the level of the full-scale system +10 percent throughout the sampling period of the test method.

8.0 Sample Collection, Preservation, Storage, and Transport.

8.1 Benchtop Bioreactor Operation. Charge the mixed liquor to the benchtop bioreactor, minimizing headspace over the liquid surface to minimize entrainment of mixed liquor in the circulating gas. Fasten the benchtop bioreactor headplate to the reactor over the liquid surface. Maintain the temperature of the contents of the benchtop bioreactor system at the temperature of the target full-scale system, ±2 EC, throughout the testing period. Monitor and record the temperature of the benchtop bioreactor contents at least to the nearest 0.1 EC.

8.1.1 Wastewater Storage. Collect the wastewater sample in the 20-L collapsible container. Store the container at 4 EC throughout the testing period. Connect the container to the benchtop bioreactor feed pump.

8.1.2 Wastewater flow Rate. The hydraulic residence time of the aeration tank is calculated as the ratio of the volume of the tank (L) to the flow rate (L/min). At the beginning of a test, the container shall be connected to the feed pump and solution shall be pumped to the benchtop bioreactor at the required flow rate to achieve the calculated hydraulic residence time of wastewater in the aeration tank.


Qtest = Qfs =

wastewater flow rate (L/min) average flow rate of full-scale process (L/min) volume of full-scale aeration tank (L)


Eq. 304A-1


fs Vfs

Vfs = The target flow rate in the test apparatus is

the same as the flow rate in the target full-scale process multiplied by the ratio of benchtop bioreactor volume (e.g., 6 L) to the volume of the full-scale aeration tank. The hydraulic residence time shall be maintained at 90 to 100 percent of the residence time maintained in the full-scale unit. A nominal flow rate is set on the pump based on a pump calibration. Changes in the elasticity of the tubing in the pump head and the accumulation of material in the tubing affect this calibration. The nominal pumping rate shall be changed as necessary based on volumetric flow measurements. Discharge the benchtop bioreactor effluent to a wastewater storage, treatment, or disposal facility, except during sampling or flow measurement periods.

8.1.3 Sludge Recycle Rate. Set the sludge recycle rate at a rate sufficient to prevent accumulation in the

2016 bottom of the clarifier. Set the air circulation rate

sufficient to maintain the biomass in suspension. 8.1.4 Benchtop Bioreactor Operation and Maintenance.

temperature, dissolved oxygen concentration, exit vent flow rate, benchtop bioreactor effluent flow rate, and air circulation rate shall be measured and recorded three times throughout each day of benchtop bioreactor operation. If other parameters (such as pH) are measured and maintained in the target full-scale unit, these parameters, where appropriate, shall be monitored and maintained to target full-scale specifications in the benchtop bioreactor. At the beginning of each sampling period (Section 8.2), sample the benchtop bioreactor contents for suspended solids analysis. Take this sample by loosening a clamp on a length of tubing attached to the lower side port. Determine the suspended solids gravimetrically by the Gooch crucible/glass fiber filter method for total suspended solids, in accordance with Standard Methods3 or equivalent. When necessary, sludge shall be wasted from the lower side port of the benchtop bioreactor, and the volume that is wasted shall be replaced with an equal volume of the reactor effluent. Add thickened activated sludge mixed liquor as necessary to the benchtop bioreactor to increase the suspended solids concentration to the desired level. pump this mixed liquor to the benchtop bioreactor through the

2017 upper side port (Item 24 in Figure 304A-1). Change the

membrane on the dissolved oxygen Probe before starting the test. Calibrate the oxygen Probe immediately before the start of the test and each time the membrane is changed.

8.1.5 Inspection and Correction Procedures. If the feed line tubing becomes clogged, replace with new tubing. If the feed flow rate is not within 5 percent of target flow any time the flow rate is measured, reset pump or check the flow measuring device and measure flow rate again until target flow rate is achieved.

8.2 Test Sampling. At least two and one half hydraulic residence times after the system has reached the targeted specifications shall be permitted to elapse before the first sample is taken. Effluent samples of the clarifier discharge (Item 20 in Figure 304A-1) and the influent wastewater feed are collected in 40-mL septum vials to which two drops of 1:10 hydrochloric acid (HCl) in water have been added. Sample the clarifier discharge directly from the drain line. These samples will be composed of the entire flow from the system for a period of several minutes. Feed samples shall be taken from the feed pump suction line after temporarily stopping the benchtop bioreactor feed, removing a connector, and squeezing the collapsible feed container. Store both influent and effluent samples at 4 EC

2018 immediately after collection and analyze within 8 hours of

collection. 8.2.1 Frequency of Sampling. During the test, sample

and analyze the wastewater feed and the clarifier effluent at least six times. The sampling intervals shall be separated by at least 8 hours. During any individual sampling interval, sample the wastewater feed simultaneously with or immediately after the effluent sample. Calculate the relative standard deviation (RSD) of the amount removed (i.e., effluent concentration - wastewater feed concentration). The RSD values shall be < 15 percent. If an RSD value is > 15 percent, continue sampling and analyzing influent and effluent sets of samples until the RSD values are within specifications.

8.2.2 Sampling After Exposure of System to Atmosphere. If, after starting sampling procedures, the benchtop bioreactor system is exposed to the atmosphere (due to leaks, maintenance, etc.), allow at least one hydraulic residence time to elapse before resuming sampling. 9.0 Quality Control.

9.1 Dissolved Oxygen. Fluctuation in dissolved oxygen concentration may occur for numerous reasons, including undetected gas leaks, increases and decreases in mixed liquor suspended solids resulting from cell growth and

2019 solids loss in the effluent stream, changes in diffuser

performance, cycling of effluent flow rate, and overcorrection due to faulty or sluggish dissolved oxygen Probe response. Control the dissolved oxygen concentration in the benchtop bioreactor by changing the proportion of oxygen in the circulating aeration gas. Should the dissolved oxygen concentration drift below the designated experimental condition, bleed a small amount of aeration gas from the system on the pressure side (i. e., immediately upstream of one of the diffusers). This will create a vacuum in the system, triggering the pressure sensitive relay to open the solenoid valve and admit oxygen to the system. Should the dissolved oxygen concentration drift above the designated experimental condition, slow or stop the oxygen input to the system until the dissolved oxygen concentration approaches the correct level.

9.2 Sludge Wasting.

9.2.1 Determine the suspended solids concentration (section 8.1.4) at the beginning of a test, and once per day thereafter during the test. If the test is completed within a two day period, determine the suspended solids concentration after the final sample set is taken. If the suspended solids concentration exceeds the specified concentration, remove a fraction of the sludge from the

benchtop bioreactor. The required volume of mixed liquor to remove is determined as follows:


Vw ' Vr

Vw is the Vr is the (Liters), Sm is the Ss is the

Sm&Ss Sm

Eq. 304A-2

wasted volume (Liters), volume of the benchtop bioreactor

measured solids (g/L), and

specified solids (g/L). 9.2.2 Remove the mixed liquor from the benchtop

bioreactor by loosening a clamp on the mixed liquor sampling tube and allowing the required volume to drain to a graduated flask. Clamp the tube when the correct volume has been wasted. Replace the volume of the liquid wasted by pouring the same volume of effluent back into the benchtop bioreactor. Dispose of the waste sludge properly.

9.3 Sludge Makeup. In the event that the suspended solids concentration is lower than the specifications, add makeup sludge back into the benchtop bioreactor. Determine the amount of sludge added by the following equation:


Vw ' Vr

Vw is the Vr is the (Liters), Sw is the Sm is the Ss is the

Ss&Sm Sw

Eq. 304A-3

volume of sludge to add (Liters), volume of the benchtop bioreactor

solids in the makeup sludge (g/L), measured solids (g/L), and specified solids (g/L).

10.0 Calibration and Standardization.

10.1 Wastewater pump calibration. Determine the wastewater flow rate by collecting the system effluent for a time period of at least one hour, and measuring the volume with a graduated cylinder. Record the collection time period and volume collected. Determine flow rate. Adjust the pump speed to deliver the specified flow rate.

10.2 calibration Standards. Prepare calibration standards from pure certified standards in an aqueous medium. Prepare and analyze three concentrations of calibration standards for each target component (or for a mixture of components) in triplicate daily throughout the analyses of the test samples. At each concentration level,

2022 a single calibration shall be within 5 percent of the

average of the three calibration results. The low and medium calibration standards shall bracket the expected concentration of the effluent (treated) wastewater. The medium and high standards shall bracket the expected influent concentration.

11.0 Analytical Procedures.

11.1 Analysis. If the identity of the compounds of interest in the wastewater is not known , a representative sample of the wastewater shall be analyzed in order to identify all of the compounds of interest present. A gas chromatography/mass spectrometry screening method is recommended.

11.1.1 After identifying the compounds of interest in the wastewater, develop and/or use one or more analytical techniques capable of measuring each of those compounds (more than one analytical technique may be required, depending on the characteristics of the wastewater). Test Method 18, found in appendix A of 40 CFR 60, may be used as a guideline in developing the analytical technique. Purge and trap techniques may be used for analysis providing the target components are sufficiently volatile to make this technique appropriate. The limit of quantitation for each compound shall be determined1. If the effluent

2023 concentration of any target compound is below the limit of

quantitation determined for that compound, the operation of the Method 304 unit may be altered to attempt to increase the effluent concentration above the limit of quantitation. Modifications to the method shall be approved prior to the test. The request should be addressed to Method 304 contact, Emissions Measurement Center, Mail Drop 19, U.S. Environmental Protection Agency, Research Triangle Park, NC 27711.

12.0 Data Analysis and Calculations.

12.1 Nomenclature. The following symbols are used in the calculations.

Ci = Co = X = t =

V = Q =

Average inlet feed concentration for a compound of interest, as analyzed (mg/L) Average outlet (effluent) concentration for a compound of interest, as analyzed (mg/L) Biomass concentration, mixed liquor suspended solids (g/L)

Hydraulic residence time in the benchtop bioreactor (hours) Volume of the benchtop bioreactor (L) flow rate of wastewater into the benchtop bioreactor, average (L/hour)

12.2 Residence Time. The hydraulic residence time of the benchtop bioreactor is equal to the ratio of the volume of the benchtop bioreactor (L) to the flow rate (L/h)

t ' VQ

Eq. 304A-4

12.3 Rate of Biodegradation. Calculate the rate of biodegradation for each component with the following equation:

Rate mg ' Ci&Co L&h t

Eq. 304A-5

12.4 First-Order Biorate Constant. Calculate the first-order biorate constant (K1) for each component with the following equation:

K1L 'Ci&Co g&h tCoX

Eq. 304A-6

12.5 Relative Standard Deviation (RSD). Determine the standard deviation of both the influent and effluent sample concentrations (S) using the following equation:

RSD ' 100 jn (Si&S)2 1/2 Eq. 304A-7 S i'1 (n&1)

12.6 Determination of Percent Air Emissions and Percent Biodegraded. Use the results from this test method

2025 and follow the applicable procedures in appendix C of 40 CFR

part 63, entitled, "Determination of the Fraction Biodegraded (Fbio) in a Biological Treatment Unit" to determine Fbio.

13.0 14.0 15.0 16.0

Method Performance. [Reserved] Pollution Prevention. [Reserved] Waste Management. [Reserved] References.

1. "Guidelines for data acquisition and data quality evaluation in Environmental Chemistry", Daniel MacDoughal, Analytical Chemistry, Volume 52, p. 2242, 1980.

2. Test Method 18, 40 CFR 60, appendix A.

3. Standard Methods for the Examination of Water and Wastewater, 16th Edition, Method 209C, Total Suspended Solids Dried at 103-105 EC, APHA, 1985.

4. Water7, Hazardous Waste Treatment, Storage, and Disposal Facilities (TSDF) - Air Emission Models, U.S. Environmental Protection Agency, EPA-450/3-87-026, Review Draft, November 1989.

5. Chemdat7, Hazardous Waste Treatment, Storage, and Disposal Facilities (TSDF) - Air Emission Models, U.S. Environmental Protection Agency, EPA-450/3-87-026, Review Draft, November 1989.

17.0 Tables, Diagrams, flowcharts, and Validation Data.








D.O. meter












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