SECTION 10

LABORATORY

Table of Contents 10-01

General Information 10-02

Laboratory Tests 10-07

Activated Sludge Process Control Tests 10-18

Biochemical Oxygen Demand Overview 10-22

Solids Overview 10-31

Nitrate-Nitrogen 10-41

Phosphorus (Ortho and Total), EPA Method 365.1 10-42

Copper 10-42

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GENERAL INFORMATION

Weight Measurement

Weight provides the foundation on which exact chemical knowledge rests. The precision
with which analytical measurements can be made depends ultimately on proper
operation of the correct kind of balance. The quality of work, therefore, depends on the
balance and how it is used.

The electronic balance. Electronic balances should be calibrated according to the

manufacturer’s instructions and should be turned off when not in use. Re-zero or tare
as often as needed and be sure to close the doors and wait for the instrument to
stabilize before recording the weight.

Volumetric Measurements

Volume flasks are designed to contain a specified volume of liquid. Since accuracy is not
highly dependent on technique, calibration is often unnecessary for work at the level of
a part per thousand. The principal source of error is variation in temperature, which
causes enough expansion or contraction of aqueous solutions to give errors on the
order of 0.1 percent per 5 degrees centigrade.

Before using a volumetric flask, it should be cleaned thoroughly. The solution to be

diluted or the solid to be dissolved is then transferred to the flask with the aid of a
funnel. It is often more convenient for a solid to be dissolved in a beaker or a conical
flask first, then resulting solution transferred quantitatively. In the most accurate work
before final volume adjustment, the flask is filled to just below the mark and immersed
in a water bath at the calibration temperature. Alternatively, the temperature of the
solution may be taken and a correction made. In careful work, after the meniscus has
been adjusted but before mixing, droplets of solvent above the meniscus may be
removed with a lint-less towel. Finally, the solution is mixed thoroughly by inverting,
shaking, and turning upright at least 10 times.

Pipets. Graduated pipets are used for delivering small volumes of liquid with an
accuracy of about one percent. Volumetric pipets are used to precisely deliver a single
volume of liquid.

The tip must meet stringent requirements because drainage time is controlled by the
diameter of the tip. (According to “Circular 602” of the National Bureau of Standards,
Washington, D.C.,” the tip should be made with a gradual taper from 2 to 3 cm; the
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taper at the extreme end of the tip must be ground perpendicular to the axis of the tube.
The outside edge should bevel slightly…”) The amount of liquid delivered depends on
how a pipet is used. Accuracies of one part per 1,000 can be attained readily, provided
the pipets are used in reproducible manner.

Keep in mind the following points concerning the proper use of a pipet. First, clean the
pipet so that water drains smoothly from the interior surface. Rinse the interior with the
aid of a suction bulb by drawing a portion of the liquid to be pipetted into the pipet.
Then tilt and turn the pipet until all the inner surface has been wetted. Discard this
portion of solution and repeat operation twice.

Next, draw the solution above the mark, carefully wipe the tip and stem of the pipet to
remove external droplets, and allow the solution to drain until the bottom of the
meniscus is even with the calibration mark. Touch the tip momentarily against a beaker
wall to remove excess solution. Move the pipet to the receiving vessel and allow the
solution to drain freely. During drainage, the pipet should be held vertically. After free
solution flow has stopped, the remaining liquid in the tip is left there; do not blow out
this portion.

A rubber suction bulb is required for all pipetting. DO NOT PIPET BY MOUTH. Thoroughly
rinse the pipet after use. Avoid drawing liquid into the bulb. If the bulb is accidently
contaminated, thoroughly rinse and dry it before reusing.

Pipets are marked to deliver a known volume of water at a defined temperature, usually
20 or 25 degrees centigrade when used in a specified manner. When a pipet is used
correctly, a manufacturer’s markings are seldom in error by more than a part or two per
thousand. Since the volume of liquid delivered depends on how the pipet is used, the
technique needs to be carefully specified.

Burets. Burets are designed to accurately deliver measurable volumes of liquid,

particularly for titrations. A 50-mL buret, the most common size, has 0.1-mL graduations
along its length and can be read to the nearest 0.01 mL by interpolation.

In reading, parallax errors are minimized by extending every tenth graduation around
the tube. For reproducible delivery, proper design of the tip is important. Changes
affecting the orifice will affect reproducibility; a buret with a chipped or fire-polished tip
should not be employed for accurate work. Drainage errors are usually minimized if the
tip is constricted so that the meniscus falls at a rate not exceeding 0.5 cm/sec.

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The accuracy of graduation marks on volumetric burets depends on the uniformity of
the bore’s diameter. The most convenient way to determine actual volumes is to weigh
the amount of water delivered. Burets can be purchased already calibrated and
accompanied by a calibration certificate. Nevertheless, the calibration operation is best
done by the user; it is smile and is an excellent means of learning to correctly use
burets.

Because graduations on a 50-mL buret are 0.1 mL apart, volumes between the marks
must be estimated. In this estimation, the width of the lines must be taken into account.
The thickness of a line on a 50-mL buret is usually equivalent to about 0.02 mL and so
takes up one-fifth of the distance from one mark to the next.

Generally, it is preferable to read the bottom of a meniscus and to take, as the value for
a given line, the point where the meniscus bottom just touches the top of the line. For
most people, this “edge-to-edge” technique gives consistent results.

Parallax, another source of error in reading a buret, occurs if the eye is above or below
the level of the meniscus. This error can be minimized by the use of encircling markings
on the buret as guides to keep the eye level with the meniscus.

The apparent position of the meniscus is significantly affected by the way it is

illuminated. Lighting errors are minimized by use of a reading card placed behind and
against the buret.

Before using a buret, clean it thoroughly with soap and a buret brush, taking care not to
scratch the interior surface. Rinse it well with distilled water; the buret is clean when
water drains from the inside surface uniformly without the formation of droplets. Store
a buret filled with distilled water and with the Teflon stopcock nut slightly loosened.
Before use, rinse the buret at least twice with titrant solution and tighten the stopcock.
Note: Do not store anything other than distilled or deionized water in a buret. The
characteristics of chemicals and reagents are likely to change and/or may be absorbed
into the glass.

Drying and Storing Materials

Some materials pick up varying amounts of water from the atmosphere and need to be
dried before being weighed for analysis. Such treatment is especially necessary for filter
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papers and finely powdered chemicals which have large surface areas. Drying is usually
carried out in an oven set at atmospheric pressure; although some substances which
tend to decompose on heating can be effectively dried at lower temperatures under
reduced pressure.

Once dried, samples must be kept dry until weighing is completed. Uptake of
atmospheric moisture or carbon dioxide is reduced if samples are placed in a well-
stoppered bottle. For added protection, dry samples are often kept in a larger container
called a desiccator. Desiccators, generally of glass or aluminum, are designed to contain
a drying agent such as magnesium perchlorate, phosphorus (V) oxide, calcium sulfate, or
calcium chloride. A desiccator maintains a dry atmosphere, provided that the lid is not
removed frequently or the door left open ant he desiccant is kept fresh. Desiccant can
be restored by drying overnight in a drying oven.

Normally, a desiccator is not used to dry materials, but it is convenient for storage and
transport of objects within the laboratory, such as dried samples or ignited crucibles.

Chemicals and reagents should not be transferred from the original containers to other
containers until needed for analysis. The characteristics of various substances may
change by degradation or by absorption of constituents in the atmosphere. Under no
circumstances should chemicals or reagents be left in open containers or unmarked
containers. This could easily lead to accidents resulting for utilization of “unknown”
substances.

This policy should also apply to samples; The characteristics of a sample stored in an
open container are likely to change as a result of biological activity that is continuing to
court in the sample or changes in physical characteristics such as temperature.

Glassware and Cleaning

For general laboratory use, the most suitable material for containers is resistant
borosilicate glass (Pyrex, Kimax, or equivalent). Choose stoppers, caps, plugs that resist
the attack of material contained in the vessel. Metal screw caps are a poor choice for
samples which will cause them to readily corrode. Glass stoppers are unsatisfactory for
strong alkaline liquids because of their tendency to stick fast. Rubber stoppers are
excellent for alkaline liquids, but unacceptable for organic solvents in which they sell or
disintegrate.

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Collect and store samples in bottles made of borosilicate glass, hard rubber, plastic, or
other inert material. There are specific requirements for the containers used in sample
collection and storage that be found in 40 CFR, Part 136.

Lab Ware Cleaning

After each use all lab ware is washed with phosphate-free Laboratory grade Detergent
(Liqunox Critical Cleaning Liquid Detergent) and rinse several times with tap water. All
glass ware is then autoclaved at 15 PSI for 15 minutes and stored until ready to use.
BOD Bottles are soaked for a minimum of 2 hours with chlorine solution then thoroughly
rinsed with tap water and autoclaved at 15 PSI for 15 minutes after each use. BOD
Dilution Water Carboy is rinsed and soaked overnight with chlorine solution then
thoroughly rinsed with tap water then with distilled water after every use.

Always check glassware and sample collection bottles for residue. Water should easily
“sheet” from the glassware if it is clean, and there should be no rough places on the
inside of the glassware that would prevent the appropriate volume from being
transferred.

Always check glassware and sample collection bottles for damage. A pipet or buret with
a broken tip will not dispense the volume that it has been designed and calibrated to
dispense. As well, a sample bottle with a broken lid or “no lid” will leave the sample
exposed to characteristic changes, which will result in a sample that doesn’t truly reflect
the characteristics of the sample stream from which it was collected.

Significant Figures

To indicate the uncertainty in a quantity, the correct number of significant figures must
be selected. For instance, if a graduated cylinder is used to measure a certain volume of
water, the reported result might be 41 mL – two significant figures.

If a buret is used, the result might be 42.21 mL – four significant figures. If the water is
weighed on a top-loading balance, the result might be 41.206 mL – five significant
figures are appropriate. In each case, no fewer digits should normally be used and no
more are justified.

Confusion may arise in the selection of significant figures where zeros are involved.

To avoid ambiguity, remember that any zero needed to locate the decimal point is not a
significant figure. Thus, 0.005 has one significant figure, gut 2.0400 has five. If confusion
is likely, numbers should be written in exponential form. For instance, 6.00 x 10E ¹ºis not
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ambiguous; it has 3 significant figures. The number 40,000 has but one significant figure
because the four zeros are necessary to locate the decimal point that is understood to
be present after the last zero; to prevent ambiguity, it can be written 4 x 104.

When rounding off numbers, if the insignificant number to be rounded off is less than 5,
drop it; if it is greater than 5, increase the last significant figure by 1; if it is 5, round to
the nearest even number. Rounding to the nearest even number ensures that results of
a series of calculations will not be biased up (if all 5s are rounded up) or down (if all 5s
are dropped). For example, 4.25, 6.35, and 1.05 are rounded off to 4.2, 6.4, and 1.0.
During calculations, at least on digit more than the allowed significant figure should be
carried along for rounding off purposes. Avoid rounding off in calculations until the final
answer is obtained. The calculation procedure for each analysis should specify how
many significant figures are to be reported. This will depend on the detection limit of
the instrument being utilized.

The results of analyses that are compiled for the Discharge Monitoring Reports should
be transferred to those reports according to the significant figures of limitation. Each
parameter will have a limitation provided on DMR. The results should be reported on
the DMR with the same number of significant figures as there are on limitations. Care
must be taken in reporting because the limitations are on pounds/day and others are on
concentration.

LABORATORY TESTS

General

Once the necessary samples have been property taken and stored, it is important that
they be analyzed in a precise manner which yields reliable and reproducible results. To
help in this regard, consult the following publications:

1. U.S. Environmental Protection Agency, Methods for Chemical Analysis of Water

and Waste, EPA-600/4-79-020, 1979

2. APHA-AWWA-WPCF, Standard Methods

3. Analytical Quality Control Handbook, Environmental Protection Agency Analytical

Quality Control Laboratory, 1014 Broadway, Cincinnati, Ohio 45202

4. Water Pollution Control Federation (WPCF) Manual of Practice No. 18, Simplified
Laboratory Procedures for Wastewater Examination, 1970
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Interpretation of Laboratory Tests

The WPCF Simplified Laboratory Procedures of Wastewater (Reference 4) discusses the

significance of some of the laboratory tests for specific treatment processes. A brief
discussion of the laboratory test used in the treatment plant is given below.

Temperature

Generally, all biological processes proceed more rapidly at higher temperatures. This is
true in secondary biological treatment such as activated sludge. Temperature records
should be maintained on the raw sewage and the oxidation ditch.

Industrial wastes may increase the temperature of raw sewage. Excessive infiltration
can be detected at times by raw sewage temperature records. When the dissolved
oxygen test is run, it is desirable to know how near saturation it is at a certain
temperature since dissolved oxygen saturation varies with temperature. Temperature
should be measured on a grab sample immediately after collection.

Temperatures are checked on all lab equipment Daily and on all samples using
Standardized Laboratory Thermometers all Thermometers are checked against a
Thermco Precision Digital Thermometer that is certified by Quality Control Services
annually

pH

pH is the negative logarithm of the hydrogen ion concentration and is a measure of the
acidity of a sample; a pH of 7 is neutral, a pH of less than 7 is acidic, and a pH greater
than 7 is basic. At pH values below 6.0 or above 9.0, organisms that stabilize wastes are
inhibited. When a plant is treating normal sewage, the pH of the raw sewage should be
in the range of 6.8 to 7.6. A wide prolonged variation from this range should be
investigated. An abnormal pH probably is caused by:

 Industries discharging materials of high or low pH.

 Slow flow velocity and hence septic sludge deposits in the sewers.
 Abnormal pH in the water supply.
 Septic sewage from any cause.

Determination of pH, Electrometric Method:

Reference: Standard Methods 20th Edition, Procedure 4500-H+B
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Apparatus
1. Thermo Dual Star Meter
2. Orion ATC Probe
3. Stirring Apparatus

Reagents
1. Buffers 4.00, 7.00, 10.00 and pH Check Standard 6.86

Sample Collection
1. Samples are collected in clean Polypropylene Containers and brought to Lab. And
Analyzed immediately

Calibration of pH Meter
1. Check the Probe’s electrolyte level, fill to weep hole
2. Rinse Probe and place in pH 4 Buffer, turn on stir bar. Let Stabilized
3. When Stable, press F2 (Calibrate), Press F2 again (Accept) then press F3 (Start)
4. When stable type in 4.00 then press F2 (Accept)
5. Rinse Probe and place in pH 7 Buffer, pressF2 (Next), then press F3 (Start)
6. When stable type in 7.00 then press F2 (Accept)
7. Rinse Probe and place in pH 10 Buffer, press F2 (Next), then press F3 (Start)
8. When stable type in 10.00 then press F2 (Accept) then press F3 (Cal Done)
9. Record Slope percent on bench sheet then press F2 (Log/Print)

Calibration Check
1. Rinse Probe and place in pH buffer 4 and let stabilize once stable for a few
minutes record value on bench sheet. Repeat procedure for Buffers 7, 10, and
Check Standard 6.86 and record values on Bench Sheet.

Test Procedure
1. Calibrate Meter as described above
2. Pour sample into glass beaker with stir bar
3. Place sample on stir plate and turn on to mix sample
4. Immerse Probe into sample
5. Let stabilize when stable for at least a couple minutes record value and
temperature
6. When finished rinse Probe and store in electrode Storage Solution

Biochemical Oxygen Demand (BOD)

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The BOD test determines how much oxygen is required for the biological decomposition
of organic matter under aerobic conditions and at a standardized time and temperature.
It is the principal test to determine the pollutant strength, in terms of oxygen required,
of domestic wastewater. BOD is used widely to evaluate the efficiency of various
treatment processes and to estimate the effects of pollution on receiving waters.

BOD tests results are usually reported in mg/L of oxygen consumed at the end of the 5-
day test period. These results are referred to as the 5-day BOD (BOD5) and should not
be confused with the ultimate BOD (BODu) of the sample, which includes nitrification.
The BOD can be used for soluble BOD or total BOD. Soluble BOD tests use the filtrate
remaining from the sample used during the suspended solids test, while total BOD tests
use an unfiltered sample.

Determination of Biochemical Oxygen Demand:

Reference: Standard Methods 20th Edition, Procedure 5210B

Apparatus
1. 300 ml BOD Bottles
2. Carboy that holds at least 6 liters
3. BOD Incubator (20 degrees)
4. Pipets
5. Graduated cylinders
6. Plastic sample Containers
7. Stir Plate
8. Stir Bars
9. YSI DO Meter & Probe
10. pH Meter and Probe

BOD Dilution Water

Rinse carboy with distilled water then add 3 liters of distilled water to carboy, add 1 –
Hach Nutrient buffer for 6 liters Pillow or (2- for 3 liters)add drops of diluted acid as
needed to try to maintain a pH of 7.0 typically 4 drops (pH should always be between
6.5 to 7.5). Add 3 more liters of distilled water. Shake Carboy to mix contents
thoroughly. Place in BOD Incubator and loosen cap and allow to sit for at least 3 hours
prior to test.

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BOD Test Procedure
1. Collect the 24 hour Effluent and Influent Composite Samples when collecting
morning grab samples. Be sure to also collect seed for BOD.

2. Bring the BOD Dilution Water, Influent and Effluent Samples to 20 c prior to
starting test. Use freezer or hot water bath if needed to adjust. Make sure seed
sample remains settled so that the supernatant can be used to seed samples.

3. To begin test, fill out BOD Bench Sheet. Volumes of Sample used are determined
by past data, experience, and current organic strength, the total Suspended Solids
test should help in determining this. Set out 13 BOD Bottles, 3 will be Blanks, 1 for
the seeded blank, 3 for the Glucose samples, 3 for Effluent Samples, and 3 for the
Influent Samples.

4. Add small amount of Dilution Water to all Bottles (never add seed or sample to
dry bottle). Add Seed to each Seeded Bottle. Mix Influent Sample and add amount
Determined to each of the Influent Bottles using sterilized Pipet. Add 6 MLS
Glucose/Glutamic Acid to each of the Check Standard Bottles. Mix Effluent Sample
and add amount determined to each of the Effluent Bottles. Fill all Bottles with
BOD Dilution Water starting with Blanks and working from cleanest to dirtiest.
Calibrate DO Meter let Bottles set for 10 Minutes then record the D.O on first
Blank Record the result for all blanks then using the Probed Bottle check pH and
Temperature and record. The Probed Blank Bottle can discarded. Rinse the Probe
thoroughly and check the D.O on the first Glucose. And record the results for all
Glucose Bottles the Probed bottle can then be discarded. Rinse Probe Thoroughly
and check D.O, pH, and Temperature on Middle Bottle of Effluent and Record
Results for all Effluent Bottles. Rinse Probe Thoroughly and Check D.O, pH, and
Temperature on Middle Bottle of Influent Samples and Record Results for all
Influent Samples. Rinse Probe Thoroughly and check D.O on Seeded Blank.

5. Insert Sterilized glass stoppers and caps on all Bottles and put Bottles in BOD
Incubator and let incubate for 5 Days at 20 c. After 5 Days check D.O on All Bottles
and Record.

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Dissolved Oxygen

Determination of Dissolved Oxygen, Membrane Electrode Method

Reference: Standard Method 20th Edition, Procedure 4500-0

Apparatus
1. YSI Model 50 B Dissolved Oxygen Meter
2. YSI BOD Probe
3. Sterilized BOD Bottles

Sample Collection
1. Samples are Collected in Clean Polypropylene Container, brought to the Lab and
transferred into BOD Bottle and tested immediately.

Air Calibration in mg/L

1. Set function switch on D.O Meter to mg/L
2. Press the Cal. Key once
3. Turn the Function Switch to mg/L , the display will show CAL then change to the
appropriate Calibration Value in mg/L
4. Turn the function key to Degrees c and record temperature, Calibrate Value, and
percent on Bench Sheet

Calibration Check
1. Make sure the reading on the Meter is the same as the Solubility of Oxygen
Chart’s pressure vs. Temperature Value.

Total Solids

The total solids content of domestic wastewater or sludge is composed of floating

matter, matter in suspension, colloidal matter, and matter in solution. Analytically, the
total solids content of a sample is all the matter that remains as a residue after
evaporation at 103 to 105 C.

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Suspended Solids

Suspended solids are the undissolved component of the wastewater total solids and can
be measured by passing the sample through a filter and then drying the captured
residue. Suspended solids test results are reported as milligrams per liter. The test for
suspended solids is used extensively in determining wastewater strength and process
efficiencies. For example, primary clarifiers usually remove from 50 to 65 percent of the
suspended solids, and secondary treatment usually brings the reduction to 85 to 90
percent.

Determination of Total Suspended Solids

Reference: Standard Method 18th Edition, Procedure 2540D

Apparatus
1. Whatman 47mm Glass Fiber Filters
2. Aluminum Weighing Dishes
3. Filtration Apparatus
4. Drying Oven
5. Indication Desiccant
6. Desiccator
7. Balance

Preparation of Filters
1. Using Forceps, place Filter Wrinkled side up on Filtration Apparatus
2. Apply Vacuum and wash 3 times using at 20 MLS of Distilled Water each time
3. Place Filters on Numbered Aluminum Weighing Dishes
4. Dry in Oven at 103-105 degrees c for 2 hours
5. Cool Filters for ½ hour in Desiccator to Balance Temperature

Sample Collection
1. Set Composite Samplers up for 24 hour Collection

Procedure
1. Weigh 6 Filters and Record the Weights on Bench Sheet.
2. Using Forceps place the Blank Filter on Filtration Apparatus (Funnel).
3. Pour 100 MLS of Distilled Water into Funnel and Vacuum for 3 Minutes remove
with forceps and place back in Aluminum Dish
4. Place 30 mg/L check Standard filter on the funnel. Mix check Standard by shaking
and pour 100 MLS in the funnel. Rinse Graduated Cylinder and funnel at least 3
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 times. Remove Filter and place in Aluminum Dish. Repeat this procedure on
Influent and Effluent Samples doing two Samples of each. In order to vacuum
Influent Samples you may need to use smaller samples (25MLS)
5. Place Aluminum Dishes with Filters in Drying Oven for 2 hours. Remove Dishes
and Filters and place in desiccator for ½ hour. Removed and Weigh and Record on
Bench Sheet.

Settable Solids

Settable solids are the fraction of the suspended solids that will settle to the bottom of
an Imhoff cone in a 60-minute period. This test is widely used to determine the
efficiency of sedimentation units. If the settable solids that are in the flow leaving a
clarifier or the plant are high, the operator should look for:

 Zero dissolved oxygen

 Too much sludge buildup
 Short circuiting in clarifiers
 Hydraulically overloaded clarifiers
 Warm sewage

Settable solids are also an approximate measure of the quantity of sludge that will be
removed by sedimentation.

Depth of Blanket

The depth blanket (DOB) test tells the operator the amount of sludge in a secondary
clarifier. This knowledge, along with the information gained from the centrifuge spins,
allows accurate control of return and waste flows. The DOB results should be shown
routinely on a worksheet.

Chlorine Residual

Chlorine residual is the chlorine remaining in wastewater at the end of a specified

contact period. Test results are reported in terms of mg/L. This test determines whether
too little or too much chlorine is being added. The Pacific Beach W.W.T.F doesn’t
normally use Chlorine but has it to control filamentous Bacteria, Odors, or Back up
Disinfection if needed.

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Determination of Total Residual Chlorine

Reference: Standard Methods 4500-CI G 20th Edition

Apparatus
1. HACH Colorimeter II Chlorine (cl 2)

Procedure
1. All Samples must be analyzed immediately and cannot be preserved for later
analysis
2. Using HACH Pocket Colorimeter fill a 10ml cell with sample and cap (blank)
3. Press the power key and turn on colorimeter arrow should indicate low range.
4. Remove the meter cap place blank in the cell holder with diamond mark facing
the keypad. Fit the meter cap over the cell compartment to cover cell (be sure to
wipe excess liquid and finger prints off of sample cells)
5. Press zero/scroll key the display will show ---- then 0.00 remove blank cell from
holder
6. Fill a second cell to the 10 ml with Sample
7. Add the contents of one DPD Free Chlorine powder Pillow or one DPD Total
Chlorine Powder Pillow (Total should be used for Wastewater) to the Sample Cell.
8. Cap and shake for 20 Seconds
9. For Free Chlorine wipe excess liquid and finger prints from cell and put prepared
cell in the holder then cover Press Read/Enter the instrument should show ----
follower by results
10. For Total wait 3 to 6 Minutes after adding The DPD Total Pillow. After reaction
time wipe excess liquid and finger prints from cell. Put the prepared sample in the
cell holder and cover the cell with cap. Press Read/Enter the instrument will show
----- then the results in mg/l

Checking Colorimeter
Color meter is checked monthly using Chlorine Standard 1000ppm as CI for DPD
Colorimetric tests

Preparation of check solution

Pipet 10 ml of solution into a 1000 ml volumetric flask and dilute to the mark with
distilled water. This is your 10 ppm working solution. Each ml of the working solution
diluted to 100 ml will give you 0.1 ppm as chlorine. For example: 2 ml of working

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solution diluted to 100ml gives 0.2 ppm as Chlorine , 10 ml of working Solution diluted
to 100ml gives 1.0 ppm as Chlorine

Note: DPD Indicator must be added to your standards

Ammonia

Determination of Total Ammonia as N

Reference: Standard Methods 18th Edition, Procedure 4500-NH3-F

Apparatus
1. Orion Duel Star Meter
2. Orion Ammonia Probe
3. Magnetic Stirrer
4. Stir Bars
5. Glass Beakers
6. Pipets

Sampling
1. Collect 24 hour Composite Sample from U.V. Effluent
2. Most Reliable Results are from fresh Samples. If prompt Analysis can’t be done,
lower pH to around 2 with Concentrated Sulfuric Acid and refrigerate at 4 degrees
c. Samples must be Analyzed within 24 hours
3. Samples and Standards must be at the same Temperature (20-25 degrees c)
4. pH adjusting ISA must be added to all Samples and Standards before
Measurement

Checking Electrode Operation

1. Remove Ammonia Probe from storage solution and Rinse with Distilled Water.
Unscrew top and place enough drops of Ammonia Filing Solution inside so that
when top is screwed back in a small amount of Solution comes out weep hole.
Rinse with distilled water and gently pat dry
2. Using a Sterilized 150 ML Glass Beaker, place 100 MLS of distilled water and clean
stir bar in beaker. Immerse Ammonia Probe at 20 degree angle ensure that there
are no air bubbles trap on Probe Membrane. Add 2 MLS of pH Adjusting ISA. After
reading stabilizes (give at least 10 minutes to stabilize between readings) Record
Reading on Bench Sheet. Add 1 ML of Ammonia Standard to Beaker when reading
Stabilizes Record Reading on Bench Sheet as 1 ml. Add 9 MLS of Ammonia
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 Standard to Beaker when Stabilizes Record Reading as 10 MLS. The Difference
between 1ML AND 10 MLS Reading should be between 54 mV to 60mV Rinse
Probe. Place a Second Beaker on stir plate with stir bar and 100 MLS of Distilled
Water. Immerse Probe at 20 degree angle and check for any trapped air bubbles
on membrane. Add 2MLS of pH adjusting ISA. Add 0.1 ML of Ammonia Standard.
After reading Stabilizes (give at least 10 minutes) record value. Add 0.4 MLS of
Ammonia Standard and let stabilize and record reading as the 0.5 reading this will
be used as the Check Standard.

Procedure

Using a sterilized 150 ML glass Beaker place 100 MLS of mixed U.V. Effluent and stir
bar into beaker then set on stir plate and stir at moderate rate. Immerse Ammonia
Probe at 20 degree angle and check to make sure no air bubbles are trapped on
Membrane. Add 2 MLS of pH adjusting ISA. After reading stabilizes record reading on
bench sheet.

On the graph record the 0.1ml reading on bottom left side of graph and record the
10ml value on the bottom right side of graph. The difference between these two is
divided by 14 (since there are 14 Y axis points). Starting at the 0.1 value subtract the
value you obtained by dividing by 14 from the 0.1 value and record on the next Y
axis. Repeat by subtracting the value you obtained from dividing by 14 from the
value on the Y axis continue this sequence until all Y axis points have a value.

Use the 1ml value from bench sheet to find corresponding point on the Y axis. Mark
this point. Using ruler draw a straight line from the upper Right corner of graph to
the 1ml point and then draw a straight line from the 1ml point to the lower left
corner of graph. Next find the point that the value for the 0.5ml Standard crosses the
line on the graph and the corresponding mg/L value on the Y axis Record this value as
the 0.5 standard. Multiple value by 10 and record as value mg/L on bench sheet.
Next find the point that sample value crosses line on graph and the corresponding
mg/L value on the Y axis. Multiple value by 10 and record as sample mg/L value if
sample value is lower then the 0.1ml and off the chart record result value as 0.03
mg/L

(All results need to be multiple by 10) i.e 0.52 x 10 = 5.2 mg/L

17
Fecal Coliform

Coliform bacteria thrive in the intestines of warm blooded animals and are relatively
harmless themselves. They are always present in water that contains human waste.
Because they are easy to isolate and normally survive longer than disease producing
organisms, coliforms are a useful indicator that pathogenic bacteria and viruses can be
present. Usually, water that is free of total coliform or fecal coliform bacteria is
considered free of disease producing organisms. The membrane filter technique is an
excellent means of determining fecal contamination. Coliform is reported as a monthly
geometric mean.

Determination of Fecal Coliform

Reference: Standard Methods 18th Edition , procedure 9222D

Apparatus
1. Water Bath
2. Sterilized funnel for filtering assembly
3. Sterilized Pipets
4. Sterilized 100 ml graduated Cylinder
5. Sterilized Forceps
6. Petri Dishes
7. Absorbent pads
8. MFC Broth
9. Sterile Filter Membrane
10. Whirl Pak. Bags

Sampling
Collect a grab sample from U.V. Effluent in a sterilized 300 ml BOD Bottle

Procedure
Sterilize Counter and work area. Fill out the Fecal Bench Sheet. Set out 4 Petri Dishes
and label first one Pre- Blank the next two U.V and the amount filtered (usually 100 ml)
and last one Post Blank. Dispense absorbent pads into each dish. Pour 1 ample of MFC
Broth onto each pad. Pour off excess broth after pad has absorbed broth. Replace
covers

Place 1 Sterile Filter Membrane using sterilize Forceps (dip in alcohol and burn off) onto
Funnel. Add Fecal Dilution Water and 100 ml of distilled water through Filter. Using
Sterilized Forceps remove filter and place on Pre-Blank Pad. Next place a filter on the
18
funnel (always using sterilized Forceps for each step). Add about 50 ml Fecal Dilution
Water. Add 100ml of vigorously shaken (min. 25 times) U.V. Sample. Vacuum through
filter. Thoroughly rinse both graduated cylinder and funnel. Remove filter and place on
pad for U.V Sample Repeat procedure for second sample filter. Repeat procedure for
pre-blank for post-Blank Filter.

Place all 4 dishes in Whirl Pak Bags and place in Water Bath so that dishes are upside
down for 24 hours (+/- 2 hours) at 44.5 degrees c

ACTIVATED SLUDGE PROCESS CONTROL TESTS

These process control tests will assist operators in controlling the activated sludge
process:

F/M : Food to Microorganism Ratio

The F/M ratio is a process control number that helps to determine the proper numbers
of microorganisms for your system. To do this calculation you will need the following
information:

1. Influent flow into your activated sludge system (Flow MGD)

2. Influent BOD (mg/L) into your aeration tank(s).
3. Mixed Liquor Volatile Suspended Solids Concentration (mg/L).
4. Volume (MG) or your aeriation system.

The term Food to Microorganism Ration (F/M) is actually a measurement of the amount
of incoming food (Lbs. of influent BOD) divided by the pounds of Microorganisms in your
system. Some calculations also included the volume of activated sludge in the clarifiers;
the one demonstrated here does not. If you have an activated sludge system you should
determine the F/M ratio on a regular basis.

Calculate the F/M as follows:

F = Influent flow, MGD X influent BOD concentration, mg/L X 8.34 lbs./gal.

M = Volume of aeration basin(s), MG X MLVSS, mg/L X 8.34 lbs./gal.
F/M = F:M Ratio

19
Settleability

Settleability is important in determining the ability of the solids to separate from the
liquid in the final clarifier. The activated sludge solids should be returned to the
aeriation tank(s) and the quality of the effluent is dependent on the absence of the
solids flowing over the weir.

The mixed liquor suspended solids test, which is explained in a following section, should
be run on the same sample that is used for the Settleability test. This will allow for a
calculation of the Sludge Volume Index (SVI) and Sludge Residence Time (SRT), both
explained in following sections.

The 2,000 mL graduate that is filled with mixed liquor in the Settleability test is
supposed to indicate what will happen to the mixed liquor in the final clarifier - the rate
of sludge settling, turbidity, color and volume of sludge at the end of 30 minutes. Record
the 30 minute reading on the daily bench sheet.

Sludge Volume Index (SVI)

The Sludge Volume Index (SVI) is used to indicate the condition of sludge (aeration
solids or suspended solids) for Settleability in a secondary or final clarifier. The SVI is the
volume in mL occupied by one gram of mixed liquor suspended solids after 30 minutes
of settling. It is a useful test to indicate changes in sludge characteristics. The proper SVI
range for your plant is determined at the time your final effluent is in the best condition
regarding solids and BOD removals and clarity.

To calculate the SVI:

30 minute Settleability reading X 1000 / Mixed Liquor Suspended Solids, mg/L.

Sludge Residence Time (SRT)

Sludge Residence Time (SRT) is a control guide used to determine the amount of time
that the sludge has remained in the oxidation ditch. To determine the SRT you must
calculate the amount of Total Suspended Solids in the oxidation ditch:

#1. Mixed Liquor Suspended Solids (MLSS), mg/L X Ditch Volume, MG X 8.34
lbs./gal.

20
The MLSS is obtained by doing a Suspended Solids test when the aeration brushes are at
the end of their cycle but still running.

The amount of Effluent Suspended Solids (ESS) is also calculated from the effluent that is
leaving the plant.

#2. Effluent Suspended Solids (ESS), mg/L X Influent Flow, MGD X 8.34 lbs./gal.

The Wasted Aerated Sludge (WAS) is determined by calculating the lbs. of sludge wasted
from the system for a 24 hour period.

#3. Return Aeriated Sludge (RAS) mg/L X Gal. Wasted (MG), for 24 hr. X 8.34
lbs./gal.

The formula is:

SRT = #1, lbs./day divided by #2, lbs./day + #3, lbs./day.

Mixed Liquor Suspended Solids (MLSS)

The Mixed Liquor Suspended Solids (MLSS) test is a very important control test as the
results are used in other control tests, as stated in pervious sections. The Mixed Liquor
Suspended Solids is the concentration of suspended solids in an aeration tank during the
activated sludge process, which occurs during the treatment of wastewater. Mixed
Liquor is a combination of raw or unsettled wastewater and activated sludge, mostly
microorganisms and non-biodegradable suspended matter, within an aeration basin.
The MLSS is an important part of the activated sludge process to ensure that there is
sufficient amount of biomass available to consume applied quantity of organic pollutant
at any time, known as the food to microorganism ratio.

The Mixed Liquor Suspended solids test is performed as follows:

Collect sample from the oxidation ditch while both brushes are running. It is
recommended to collect the sample at the same time each day. Filter 5 mils of mixed
sample through a pre-washed, dried, and weighed filter. Duplicate samples should be
run. Dry the filters at 103 to 105 degrees Celsius for a least one hour. Re-weigh filters.
Record the difference as the Mixed Liquor Suspended Solids in mg/L.

Example:

21
Filter 5 mils of MLSS.

Initial weight of filter only is 0.1200g. Final weight of filter is 0.1297g. Difference is
0.0097g.

0.0097 X 1000 (1000mg/gram) = 9.7 mg. 9.7 mg X 1000 mg/L (multiply by 1000 to get
mg/L) = 9,700 mg/L.

9,700/5 mils filtered = 1940 mg/I MLSS.

BIOCHEMICAL OXYGEN DEMAND OVERVIEW

Biochemical oxygen demand (BOD) is the amount of oxygen that bacteria require for the
consumption of biodegradable matter. The test measures the oxygen required for the
biochemical degradation of organic material (carbonaceous demand), the oxygen used
to oxidize reduced forms of nitrogen (nitrogenous demand, unless their oxidation is
prevented by an inhibitor).

Most wastewater contains more oxygen-demanding materials than the amount of

dissolved oxygen (DO) available in air-saturated water. Therefore, to bring the oxygen
demand and supply into balance, it is necessary to dilute the sample before incubating
it.

For bacteria to metabolize and consume organic material, certain minerals must be
present. These are provided by the four nutrients or buffering solution added to the
dilution water. Also, temperature must be rigidly maintained at 20 degrees C +/- 1
degree.

The environmental provided for the bacteria must be favorable; therefore, conditions
which are deadly must be eliminated. For example, samples with an extreme pH must
be neutralized (with 1N H2SO4 or 1 N NaOH), chlorinated samples must be Dechlorinated
(with sodium sulfite solution), and hot or cold samples must be temperature adjusted to
20 degrees C +/- 1 degree.

Although these factors are eliminated by the analyst, an assumption must be made that,
due to the initial presence of those factors, the bacteria population in the sample is
dead or dying. Therefore, a new population of bacteria or seed must be supplied.
Domestic wastewater and unchlorinated or un-disinfected effluents from waste
treatment plants are usually satisfactory for seeding purposes. If domestic wastewater is
used, let a sufficient volume settle at 20 degrees C +/- 1 degree for at least one hour but

22
no longer than 36 hours. Pour off the supernatant for use as the seeding material and
discard the settled solids.

As the definition of BOD states, BOD is a measure of the amount of oxygen the bacteria
requires to consume organic matter. This oxygen is dissolved in water. The maximum
amount the water can dissolve at 20 degrees C +/- 1 degree is 9.0 mg/L. Higher
concentrations represents unstable conditions in which the excess oxygen may be lost
physically, not biological consumption. A dissolved oxygen (DO) concentration that is
too low will cause the bacteria to stop metabolizing. Either problem can be corrected by
aerating the dilution water prior to its use.

Once the bacteria is supplied with a known initial quantity of oxygen, several
precautions must be taken to prevent physical loss or entry of oxygen.

A siphon must be used to dispense dilution water and to avoid further aeration. Water
seals must be maintained during incubation so that oxygen cannot enter the bottles.
When prepared, the dilution water must be at 20 degrees C +/- 1 degree to avoid
contraction of water in BOD bottles as it cools to 20 degrees C and draws in air.

DO values must be determined carefully by either the Winkler method or a calibrated

DO probe.

The source and quantity of organic matter must be carefully controlled. The ONLY
sources must be the sample and the seed. Organic matter from other sources (i.e.,
siphon tubes, dirty bottles, dirty probes) will introduce error and result in falsely high
BOD concentrations.

The rule is that at least 2.0 mg/L of oxygen is consumed (depletion) and at least 1.0
mg/L of oxygen remains (residual). In order to obtain DO uptake in this range, several
dilutions of the sample must be made. Experience with a particular sample gave the
following dilutions:

Strong industrial wastes 0.0 to 1.0 percent

Raw and settled wastewater 1 to 5 percent
Biologically treated effluent 5 to 25 percent
Polluted river waters 25 to 85 percent

23
This gives the expected BOD range from several percent concentrations of sample
added to a 1 liter or a 300 mL BOD bottle.

In order to demonstrate that the analyses performed were properly executed, a sample
should be analyzed for which the BOD value is known. This quality control test is
performed regularly by analyzing a glucose-glutamic acid (GGA) solution. The GGA
control test will check the dilution water quality, seed effectiveness, and analytical
technique. Low results are sometimes obtained when distilled water is contaminated
(with metals) or when the seed is inactive. Use a mixture of 150 mg/L glucose and 150
mg/L glutamic acid as a standard solution. Determine the 5-day, 20 degree C BOD of a 2
percent concentration of the GGA standard solution. Depending on the see you use, the
BOD will be approximately 200 mg/L. Standard Methods states that the average 5-day
BOD for the 300 mg/L mixed GGA standard solution will be 198 mg/L, with a standard
deviation of 30.5 mg/L. If the value is outside the range, seek the cause of the problem.
Refer to Tables 10-3, 10-4, and 10-5 (BOD Troubleshooting Charts I, II, and III).

Table 10-1. BOD Range/Sample Dilution for Liter Method

Sample (ml) Dilution Factor BOD Range (ppm)

1 1,000 2000-6000
2 500 1000-3000
4 250 500-1500
5 200 400-1200
6 167 334-1016
8 125 250-750
10 100 200-600
15 67 134-416
20 50 100-300
25 40 80-240
33 30 60-180
40 25 50-150
50 20 40-120
67 15 30-90
100 10 20-60
111 9 18-54
125 8 16-48
167 6 12-36
24
 250 4 8-24
500 2 4-12
1,000 1 2-6

Table 10-2. BOD Range/Sample Dilution for 300-ml Bottle Method

Sample (ml) Dilution Factor BOD Range (ppm)

3 100 200-600
4 75 150-450
5 60 120-360
6 50 100-300
7 43 86-258
8 38 76-228
9 33 66-198
10 30 60-180
12 25 50-150
15 20 40-120
20 15 30-90
25 12 24-72
30 10 20-60
40 7.5 15-45
50 6 12-36
60 5 10-30
75 4 8-24
100 3 6-18
150 2 4-12
200 1.5 3-9
300 1 2-6

Notes:

25
 1. Raw sewage is usually between 200 and 300 mg/L BOD.

2. Plant effluent is usually between 3 and 50 mg/L BOD5.

Table 10-3. BOD Troubleshooting Chart I

Symptom Possible Causes Remedy

Symptom: Unseeded dilution water depleted by more than 0.2.mg/L in 5 days in both
bottles.

Contaminated Clean distilled water storage tank.

distilled water
Reduce still’s cooling water
supply.

If deionizer is used, install

micro-filter.

Contaminated Buffers should appear crystal

buffers clear and be less than one year
old; replace phosphate buffer
first.

Thio wrong Re-standardized thio.

normality

Dirty glassware Review and revise procedure for

washing glassware.

Initial DO > 9.0 Reduce DO to saturation at 20

mg/L degrees C by slaking prior to
incubation.
26
Symptom: Unseeded dilution water depleted by more than 0.2 mg/L in 5 days in only
one bottle.
Dirty glassware Recheck procedure for washing
glassware, especially pH of final
rinse water.

Table 10-4. BOD Troubleshooting Chart II

Symptom Possible Causes Remedy

Symptom: 10% - Seed controls have very small depletion (<1.0 mg/L).

Insufficient seed Increase amount of seed used.

Weak seed Collect seed from a different

source or when strength
of wastewater is greater.

Seed was Change seed source.

chlorinated or
contained toxic
industrial waste

Toxics in distilled Try redistilled or distilled-

water deionized water.

Incubator Check and adjust temperature.

temperature too
low

Symptom: 10% - Seed Controls run out

27
 Too much seed Reduce see, use depletion of
1% seed controls for calculations.

Incubator Check and adjust temperature.

temperature too
high

Thio wrong Re-standardize thio.

Normality

Table 10-4. BOD Troubleshooting Chart II (continued)

Symptom Possible Causes Remedy

Symptom: One-tenth of 10% seed depletion not equal to 1% seed depletion.

Erratic consumption This is normal and is the reason

of DO using 10% seed.

BOD standard controls too high (more than 15%)

Contaminated Drain distilled water storage

tank.
distilled water Reduce still’s cooling water
supply.
Contaminated Where deionizer is used ahead
of distilled water still, micro-
filter.
Contaminated Buffers should appear crystal
buffers clear and be less than one year
old; replace phosphate buffer
first.
Dirty glassware Review and revise procedure
for washing glassware.
Improperly Prepare new standard.
prepared standard Purchase new standard.

28
 Math error Check calculation.
Incubator Check and adjust temperature.
temperature
too high

Table 10-5. BOD Troubleshooting Chart III

Symptom Possible Causes Remedy

Symptom: BOD standard control too low, more than 15%.

Weak or no seed Increase seeding.

used

Toxic in distilled Try redistilled or

distilled-water deionized water.

Toxic residue on Review and revise

glassware procedures for
washing glassware.
Check pH of final rinse.
Rinse with distilled or
deionized water.

Used “old” standard Prepare fresh standards:

store standards
only in
concentrated form in
sealed sterile ampules.

Improperly prepared Prepare new standard.

standard Purchase prepared
standard.

.
29
 normality

Math error Check calculations.

Incubator Check and adjust

temperature temperature.
too low

Table 10-5. BOD Troubleshooting Chart III (continued)

Symptom Possible Causes Remedy

Symptom: Two or more proper dilutions (depletion more than 2.0 mg/L, residual DO
more than 1.0 mg/L ) yield widely differing results.

Influent samples Toxics present Check and correct for

residual chlorine,
pH extremes, and
heated samples.
Seed the dilution water.
Check industrial
discharges for other
toxics present.

Effluent samples Toxics present Check for & remove the

residual chlorine;
seed & dilution water.

Chemical demand Use titration technique

when removing
residual chlorine
with sodium
sulfite.

30
SOLIDS TESTING

General Discussion

The terms “solids” refers to matter suspended or dissolved in water or wastewater.

These solids may adversely affect water quality; therefore, the primary goal of a
wastewater treatment plant is to remove as many solids as possible. The solids tests
that are most applicable for use in a wastewater treatment plant include suspended
solids, volatile suspended solids, total dissolved solids, total solids, and total volatile
solids. The results of these tests have the following uses in process control:

1. Evaluating the organic strength of the wastewater

2. Evaluating clarifier solids loading
3. Determining the sludge recycle rate by calculation
4. Calculating clarifier solids capture
5. Estimating the solids inventory
6. Measuring the organic matter proportional to the microorganism concentration in
the mixed liquor

Suspended solids are the solids retained on the glass fiber filter after filtration and
drying to a constant weight at 103 – 105 degrees C. The volatile suspended solids are
then determined by ignition at 550 degrees C. Suspended and volatile suspended solids
are tests normally performed on all samples in a wastewater treatment plant, except on
highly concentrated sludge where filtration is impractical.

31
PHOSPHORUS (ORTHO AND TOTAL), EPA METHOD 365.1

Phosphorus samples are sent out to Water Management Laboratories for analysis.

COPPER

Copper samples are sent out to Water Management Laboratories for analysis.

32