Table of Contents

Basic Laboratory Procedures for Wastewater Examination

1. Introduction to the Laboratory

· Background

· Collection of Samples

· Laboratory Equipment

· Weighing on the Analytical Balance

I. Rules for Weighing

II. Units of Measurement

· Uniformity of Reagents

I. Laboratory-Grade Water

II. Standard Solutions

· Laboratory Safety

· References

2. Laboratory Analysis

· Volumetric Analysis

· Gravimetric Analysis

· Colorimetric Analysis

· Electrometric Analysis

· Accuracy, Precision, and Quality Assurance

I. Accuracy

II. Precision

1. Determinate Errors

2. Indeterminate Errors

III. Quality Assurance

1. Check Standards

2. Duplicate Analysis

3. Blanks

4. Known Additions

5. Externally Supplied Standards

6. Validation of Results and Technician Duplicates

7. Control Charts

· References

3. Physical and Chemical Examination

· Settleable Matter

I. General

II. Apparatus

III. Procedure

· pH

I. General

II. Procedure

· Volatile Acids in Wastewater Sludge

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Total Alkalinity of Wastewater and Sludge

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Suspended Solids (Total Nonfilterable Residue)

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Volatile and Fixed Residue in Wastewater

I. General

II. Reagents

III. Procedure

IV. Calculations

· Total Solids, Volatile Matter, and Fixed Matter in Sludge

I. General

II. Apparatus

III. Procedure

IV. Calculations

· Suspended Matter for Mixed Liquor and Return Sludge

I. General

II. Apparatus

III. Procedure

IV. Calculation

· Settleability

I. General

II. Apparatus

III. Procedure

IV. Calculation

· Sludge Volume Index

· Centrifuge Method for Estimating Suspended Matter

I. Apparatus

II. Procedure

1. Part I—Determination of Centrifuge Factor

2. Part II—Estimation of Suspended Solids

· Oxygen-Consumption Rate

I. Apparatus

II. Procedure

III. Calculations

1. Oxygen Uptake Rate

2. Consumption Rate

· Dissolved Oxygen—Winkler Technique

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Dissolved Oxygen—Membrane Electrode Method

· Biochemical Oxygen Demand

I. General

II. Apparatus

III. Reagents

IV. Pretreatment of Wastewater Samples

V. Procedure for Biochemical Oxygen Demand Determination without Seeding

VI. Procedure for Biochemical Oxygen Demand Determination with Seeding Calculation

· Chemical Oxygen Demand—Closed Dichromate Reflux Method, Colorimetric

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Chloride

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculations

· Chlorine Residual

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Chlorine Requirement

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Ammonia Nitrogen—Ammonia-Selective Electrode Method

I. General

II. Apparatus

III. Reagents

IV. Procedure

· Nitrate Nitrogen—Nitrate Electrode Method

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Total Kjeldahl Nitrogen

I. General

II. Safety Considerations

III. Apparatus

IV. Reagents

V. Procedure

· Total Phosphorus

I. General

II. Apparatus

III. Reagents

IV. Procedure

V. Calculation

· Temperature

· Specific Gravity

I. General

II. Apparatus

III. Procedure

IV. Calculation

· Reference

4. Microbiological Examinations

· General

· Total Coliform and Fecal Coliform Membrane Filter Procedure

I. General

II. Apparatus

1. Nondisposable Equipment

2. Expendable Equipment and Materials

III. Media, Buffer, and Neutralizing Solution Preparation

1. M-Endo Broth (Total Coliform)

2. M-FC Broth (Fecal Coliform)

IV. Phosphate Buffer

V. Sodium Thiosulfate Solution

· Sterilization of Equipment and Materials

I. Phosphate Buffer

1. Autoclave Technique

2. Membrane Filter Technique

II. Sample and Dilution Bottles

III. Filter Holders

1. Steam Autoclaving

2. Ultraviolet Light

3. Dry Heat

IV. Alternate Sterilization Procedures

· Sampling

· Sample Volume Selection/Dilutions

· Test Procedures

I. Preparation of Petri Dishes

1. Filtration

2. Incubation

II. Identification and Counting

III. Counting and Recording

IV. Stressed Organisms

V. Calculations

· Microbiological Examinations

· Reference

5. Data Recording and Reporting

· Data Recording

I. Bench Sheets

II. Log Book

· Expression of Results

I. Significant Figures

II. Rounding Off Numbers

III. Rounding Off Single Arithmetic Operations

1. Addition

2. Subtraction

3. Division and Multiplication

· Reference

Appendix A Recommended Laboratory Supplies

Appendix B Preparation of Common Types of Distilled Water and Desk Reagents

· Common Types of Distilled Water

I. Distilled Water

II. Carbon Dioxide Free Distilled Water

III. Redistilled Water

IV. Deionized Distilled Water

V. Deionized Water

· Acid Solutions

· Alkaline Solutions

I. Stock Sodium Hydroxide

II. Ammonium Hydroxide Solutions

· Indicator Solutions

I. Phenolphthalein Indicator Solutions

II. Methyl Orange Indicator Solution

Index

List of Tables

1. Summary of special sampling or handling requirements.

2. Some SI units.

3. Conversion of SI units to U.S. customary units.

4. Acceptance limits for duplicate samples and known additions to water and wastewater.

5. Estimating sample size for BOD₅ determination.

6. Photometer light paths suitable for various concentration ranges.

7. Conversion of temperature from Fahrenheit to Celsius degrees.

8. Temperature correction factor.

9. Testing guide.

10. Suggested sample volumes required for tests.

11. Sample BOD work sheet.

12. Wastewater laboratory work sheet for total suspended solids (TSS) and total volatile suspended solids.

13. Wastewater laboratory work sheet for total suspended solids and total volatile suspended solids.

14. Wastewater laboratory work sheet for chemical oxygen demand (COD) (from dichromate).

15. Wastewater laboratory work sheet.

16. Wastewater laboratory work sheet—oil and grease.

B.1 Preparation of uniform acid solution.

B.2 Preparation of uniform NaOH solution.

List of Figures

1. Two-hour pH sampling showing a drop in pH at hour 6.

2. Heat-resistant glass or plastic bottles are suitable for sampling.

3. Volumetric flasks are used for making standard solutions.

4. Measurements made with graduated cylinders are sufficiently accurate for most experiments.

5. The buret and stand are used for titrating.

6. Some typical pipets.

7. Safety bulbs or pipet fillers.

8. Typical spectrophotometer.

9. Dessicators are used for cooling such items as crucibles and dishes.

10. The two primary types of balances used in the laboratory.

11. A ventilated hood should be used when using volatile solvents, bases, or acids.

12. Care should be used in making rubber-to-glass connections.

13. After filling the Imhoff cone to the 1-L mark, allow to settle for 45 minutes, then stir gently and allow to settle for 15 minutes.

14. The acidic range on the pH scale is 0 to 7. The basic range is 7 to 14.

15. Typical pH meter.

16. Distill off 150 mL at the rate of approximately 5 mL/min.

17. Titrate the solution with 0.02 N NaOH until the pink color of phenolphthalein first appears.

18. Add measured sample to a 100-mL beaker.

19. Titrate to pH 4.5.

20. Filter 10 to 25 mL of sample through Gooch crucible, using 380-mm Hg vacuum.

21. For volatile solids determination, place the weighed crucible in a muffle furnace set at 550 ºC.

22. Place evaporating dish containing 25- to 50-mL sample on steam bath and evaporate to dryness.

23. Place dish in drying oven set at 103 ºC.

24. After wetting filter paper with distilled water, add 20 to 100 mL of sample.

25. A 1000-mL graduated cylinder or Mallory settleometer is used to determine settleability of mixed liquor.

26. Siphon sample into BOD bottle with great care to avoid air bubbles.

27. Add 1 mL each of manganous sulfate and alkali–iodide–azide solution.

28. Shake BOD bottle well by inverting gently, then allow floc to settle.

29. Carefully add 1 mL H₂SO₄ slowly along neck of bottle.

30. Titrate 201 mL of the solution with sodium thiosulfate.

31. Add 100 mL of well-mixed sample to an Erlenmeyer flask.

32. Carefully add 1 mL of H₂SO₄to flask.

33. Titrate 100 mL of the solution with Na₂SO₃.

34. Add dilution water to BOD bottles carefully to avoid air bubbles.

35. Chemical oxygen demand digester with culture tubes.

36. Titrate the sample with AgNO₃ solution after adding 1 mL of K₂CrO₄ indicator.

37. Kjeldahl digestion and distillation apparatus.

38. Preparation of a 1:10 dilution.

39. Preparation of a 1:100 dilution.

40. Insertion of absorbent pad to petri dish.

41. Dispensing media contents from glass ampoule.

42. Filtration apparatus.

43. Loading a presterilized membrane, grid-side up, evenly centered onto the filter holder support base.

44. Dispensing sample into the funnel.

45. Rinsing of funnel walls with sterile buffer.

46. Transferring filter pad to prepared petri dish.

47. Incubation of petri dishes.

48. Microscopic examination of bacteria colonies.

49. Counting procedure for coliform colonies.

50. Locating borderline colonies.

51. Comparison of compound and stereoscopic binocular microscopes.

52. Common organisms observed or identified in wastewater treatment.

53. Sample size determination for BOD test.

54. Sample size determination for chemical oxygen demand test.

55. A guide to sample size determination.