Wastewater Characteristics...

Typical data on the individual constituents found in domestic wastewater are shown below :

"Wastewater Characteristics"...

pH, Acidity, and Alkalinity...

pH...

pH is a measure of how acidic or alkaline a solution is. In pure water at room temperature, a small fraction (about two out of every billion) of the water molecules (H₂O, or really, H-O-H) splits, or dissociates, spontaneously, into one positively charged hydrogen ion (H⁺) and one negatively charged hydroxide ion (OH^-) each. There is an equal number of each ion, so the water is said to be "neutral". Some materials, when dissolved in water, will produce an excess of (H⁺), either because they contain these ions and release them when they dissolve, or because they react with the water and cause it to produce the extra hydrogen ions. Substances which do this are called acids. Likewise, some chemicals, called bases or alkalis, produce an excess of hydroxide ions. The scale which is used to describe the concentration of acid or base is known as pH, for power or potential of the Hydrogen ion. A pH of 7 is neutral. pH's above 7 are alkaline (basic); below 7, acidic. The scale runs from about zero, which is very acidic, to fourteen, which is highly alkaline. The scale is logarithmic, meaning that each change of one unit of pH represents a factor of 10 change in concentration of hydrogen ion. So a solution which has a pH of 3 contains 10 times as many (H⁺) ions as the same volume of a solution with a pH of 4, 100 times as many as one with a pH of 5, a thousand times as many as one of pH 6, and so on. Some common materials and their approximate pH's are ; acids : carbonated beverages, 2 to 4; lemon juice, about 2.3; vinegar, about 3 and bases : baking soda, 8.4; milk of magnesia 10.5; ammonia, 11.7; lye, 14 to 15.

While the pH measures the concentration of hydrogen or hydroxide ions, it may not measure the total amount of acid or base in the solution. This is because most acids and bases do not dissociate completely in water. That is, they only release a portion of their hydrogen or hydroxide ions. A strong acid, like hydrochloric acid, HCl, releases essentially all of its H⁺ in water. The concentration of H⁺ is the same as the total concentration of the acid. A weak acid, like acetic acid (the acid in vinegar), may release only a few percent of the hydrogen that it has available. If you are trying to neutralize an acid by adding a base, like sodium hydroxide, the amount you would need to neutralize a strong acid could be calculated directly from the pH of the acid solution. But for a weak acid, the pH does not tell the whole story; the total amount of base needed would be a lot more. This is because as the OH^- from the base reacts with the H⁺ in solution to form water, more H⁺ will break loose from the undissociated portion of the acid to take its place. The neutralization will not be complete until all of the weak acid has dissociated.

Acidity...

To measure the total acidity, also called base-neutralizing capacity (BNC) of a water sample, it has to be titrated with base. That is, a solution of a base whose concentration is known must be added to the water sample slowly until the neutralization is complete. By measuring the volume of the base added, you can figure out the original concentration of acid.

Alkalinity...

In a similar way, the acid-neutralizing capacity (ANC), or alkalinity of a water sample has to be determined by titrating it with a solution of a strong acid of known concentration.

Significance...

Although there are some microorganisms which can function at extreme pH's, most living things require pH's close to neutrality. Many enzymes and other proteins are denatured by pH's which differ much from pH 7, which disrupts the functioning of the organism and may kill it. Besides the harm to aquatic life in natural waters, pH imbalances can inhibit - or completely wipe out - biological processes in wastewater treatment plants, resulting in incomplete treatment and pollution of the receiving waters. Low (acidic) pH's also cause corrosion in sewers systems and increase the release of toxic and foul-smelling hydrogen sulfide gas. (This gas has been responsible for the deaths of numerous sewer workers.) Low pH's also increase the release of metals, some toxic, from soils and sediments. Alkalinity is an important parameter because it measures the water's ability to resist acidification, for instance, to acid rain. In wastewater treatment, some processes produce acidity. If there is not enough alkalinity to neutralize it, the pH of the process can drop and cause it to become inhibited. Alkalinity can be supplemented by chemical addition to avoid this.

Measurement...

There are indicator solutions which change color in different pH ranges, and these can be used for approximate estimation of pH in solutions which contain high enough concentrations of pH-determining ions. "pH paper", impregnated with such indicators, are commonplace in testing laboratories. For accurate measurements and use in dilute solutions, electrochemical measurement (a "pH meter") is required. Alkalinity and acidity are determined by titration with strong base or acid, respectively, using either indicators or a pH meter to mark the endpoint.

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Dissolved Oxygen ( DO )...

Like solids and liquids, gases can dissolve in water. And, like solids and liquids, different gases vary greatly in their solubilities, i.e, how much can dissolve in water. A solution containing the maximum concentration that the water can hold is said to be saturated. Oxygen gas, the element which exists in the form of O₂ molecules, is not very water soluble. A saturated solution at room temperature and normal pressure contains only about 9 parts per million of DO by weight (9 mg / L). Lower temperatures or higher pressures increase the solubility, and visa versa.

Significance...

Dissolved oxygen is essential for fish to breathe. Many microbial forms require it, as well. The oxygen bound in the water molecule (H₂O) is not available for this purpose, and is in the wrong "oxidation state", anyway. The low solubility of oxygen in water means that it does not take much oxygen-consuming material to deplete the DO. As mentioned before, the biodegradation products of bacteria which do not require oxygen are foul-smelling, toxic, and/or flammable. Sufficient DO is essential for the proper operation of many wastewater treatment processes. Activated sludge tanks often have their DO monitored continuously. Low DO's may be set to trigger an alarm or activate a control loop which will increase the supply of air to the tank.

Measurement...

DO can be measured by a fairly tricky wet chemical procedure known as the Winkler titration. The DO is first trapped, or "fixed", as an orange-colored oxide of manganese. This is then dissolved with sulfuric acid in the presence of iodide ion, which is converted to iodine by the oxidized manganese. The iodine is titrated using standard sodium thiosulfate. The original dissolved oxygen concentration is calculated from the volume of thiosulfate solution needed. Measurements of DO can be made more conveniently with electrochemical instrumentation. "DO - meters" are subject to fewer interferences than the Winkler titration. They are portable and can be calibrated directly by using the oxygen in the air.

Biochemical Oxygen Demand...

General...

Biochemical Oxygen Demand is a common, environmental procedure for determining the extent to which oxygen within a sample can support microbial life. The following tutorial explores the theory and basics of performing this test when one has little or no prior experience. This method is popular in many environmental laboratories analyzing waste water, compost, sludge, and soil samples. Although methods for each matrix are similar, this tutorial focuses on the method associated with only waste water effluents. The main details of this method are taken specifically from Standard Methods for the Examination of Water and Wastewater (Method 507: 1985, and Method 218B: 1971) and the US Environmental Protection Agency of 1979 (Method 405.1). Slight variations and additional insight are added from my experience as an analyst, and modifications will be noted. Other methods may exist amongst laboratories performing this test, so it must be stressed that this method, although approved, is not all inclusive. In addition, this procedure is only suitable for samples void of serious matrix interferences. To gain a broader appreciation of oxygen demand, additional avenues of interest may be explored including CBOD (carbonaceous oxygen demand), COD (chemical oxygen demand), and TOC (total organic carbon).

"Hourly Variations in BOD₅ Concentrations"...

The test for Biochemical Oxygen Demand is especially important in waste water treatment, food manufacturing, and filtration facilities where the concentration of oxygen is crucial to the overall process and end products. High concentrations of dissolved oxygen (DO) predict that oxygen uptake by microorganisms is low along with the required break down of nutrient sources in the medium (sample). On the other hand, low DO readings signify high oxygen demand from microorganisms, and can lead to possible sources of contamination depending on the process. Performing the test for BOD requires a significant time commitment for preparation and analysis. The entire process requires five days, and it is not until the last day where data is collected and evaluated. During this time, samples are initially seeded with microorganisms and supplied with a carbon nutrient source of glucose-glutamic acid. The sample is then introduced to an environment suitable for bacterial growth at reproducible temperatures, nutrient sources, and light within a 20^oC incubator such that oxygen will be consumed. Quality controls, standards and dilutions are also run to test for accuracy and precision. Determination of the dissolved oxygen within the sample can be determined through Winkler titration methods. The difference in initial DO readings (prior to incubation) and final DO readings (after 5 days of incubation) predicts the BOD of the sample. A suitable detection limit as per environmental QC is 1 mg / L.

"BOD and NOD Curves"...

Why 5 Days ?...

It can take as long as 25 days before no further changes can be detected in a bottle in which a BOD test is being conducted. Depending on the nature of the sample, the test may be near completion in a few days. A reasonable compromise between waiting too long to get results and getting unreliable answers is 5 days. As long as the samples are pretty much the same from one sampling period to another, the 5-day test works fairly well. For example, samples from a process in a waste treatment plant will have essentially the same nature over long periods. The 5-day BOD will be highly meaningful in showing variations in plant performance. The following figure shows simulation of the BOD test with different rate coefficients. Note the vertical line for 5 days. If the samples are quite different in their composition, the error in comparing them at 5 days will be great, and a longer time for the test would be better. This must be balanced by a long wait before having results, and delay in making adjustments based on these results may be costly.

Required Equipment...

The following is a list of necessary materials and equipment for starting the procedure : (1) Series of 250-300 ml BOD bottles with ground glass stoppers, and caps, (2) Incubator set at 20^oC, (3) Two large carboys (20 liter capacity depending on sample amount), (4) Series of class A pipets (0.2 mL - 10 mL), (5) Aeration device, (6) pH - meter, (7) Six 500 ml ehrlenmeyer flasks, (8) Millipore water, (9) Phosphate buffer pillows for every 6 liter carboy volume, (10) DO - meter with membrane electrode, (11) Seed, (12) 50 ml class A buret, (13) Stir plates and stir bars, (14) Series of volumetric flasks, (15) Series of beakers and (16) Deionized water.

"Simulation of the BOD Test"...

Required Reagents...

The following is a list of reagents used in this method that are commercially prepared : (1) Concentrated sulfuric acid, (2) 0.0375 N Sodium thiosulfate, (3) Starch indicator, (4) Crystalline D-glucose and glutamic acid, (5) Bleach (5.25 %), (6) Acetic acid, (7) Potassium iodide, (8) Sodium hydroxide pellets, (9) Manganese sulfate and (10) Sodium sulfite. The remaining reagents are prepared within the laboratory. Caution must be taken since the shelf life of these reagents should not exceed 6 months unless otherwise noted. (1) 0.1 N Sulfuric acid : Add 2.8 mls concentrated acid to 1 liter distilled water. (2) 0.1 N Sodium hydroxide : Add 4 grams sodium hydroxide pellets to 1 liter distilled water. (3) Sodium sulfite solution : Prepare daily. Dissolve 1.575 g sodium sulfite up to one liter deionized water. (4) Manganese sulfate solution : Dissolve 364 grams manganese sulfate up to one liter deionized water. Slight heating and filtration may be necessary. (5) Glucose - Glutamic solution : Dissolve 75 mg glucose and 75 mg glutamic acid up to 500 ml deionized water. Sufficient stirring is required. (6) Alkali azide solution : In a 1 liter volumetric, dissolve 500 gram sodium hrdroxide pellets with 150 grams potassium iodide. When dissoliution is complete, add an additional 40 mls distilled water with 10 grams sodium azide. Caution must be taken when handling this solution. (7) 1 + 1 Acetic acid : 500 ml pure grade acetic acid is added to 500 ml deionized water with stirring. Caution must be taken since the temperature of the solution will increase rapidly. (8) Potassium iodide solution : Dissolve 10 grams potassium iodide in 100 ml volumetric flask with distilled water.

"Microbiology of the BOD Reaction"...

Sample Preparation...

Handling of the sample is critical to this procedure. The sample must be incubated within 48 hours of its original sampling time. Analysis after this point will have significant effects on the oxygen concentration within the sample and may often lead to less than accurate results. Usually, the DO of the sample will tend to decrease. When the sample is first brought in for analysis, it must be maintained at a temperature of approximatley 4 degrees Celsius. This is to ensure the fact that the oxygen concentration will remain constant and will also inhibit the further growth of organisms. Once in the lab, the pH of the sample must be adjusted for analysis. The desired pH for this procedure is between 6.5 and 7.5 where bacterial growth is possible. A 100 ml sample is usually adjusted with 0.1 N sulfuric acid or 0.1 N sodium hydroxide depending on the original pH of the sample. Once obtained, the sample content is checked to deterimine chlorine content. Chlorine must be removed from the sample because it introduces an interference with the dissolved oxygen.

Chlorine Content...

The chlorine content on a 100 ml neutralized sample can be determined by adding 10 ml potassium iodide solution and 10 ml 1 + 1 acetic acid as mentioned in the reagents section. When fully mixed, the addition of a starch indicator will denote the presence of chlorine if the sample turns a greyish - black. At this point, dropwise addition of freshly prepared sodium sulfite will diminsh the chlorine within the 100 ml sample. Once the chlorine has been dissipated, a new 100 ml sample must be neutralized and the same number of sodium sulfite drops must be added. At this point, the sample is ready for analysis. If no chlorine is detected in the sample after the 20 ml reagent addition, the addition of sodium sulfite is not necessary, and the sample must be neutralized again from a fresh sample.

"Oxygen Uptake vs Organisms"...

Procedure...

Once reagents have been properly prepared, the sample is ready for analysis. The BOD procedure including the DO analysis is actually quite lengthy, so time maintenance is important. This portion can be carried out in four separate steps including carboy setup, adjustment of DO, preparation of seed inoculum, and BOD sample preparation. It is best to set up the carboy as soon as possible to ensure proper results. In addition, the BOD sample preparation is the last step of the procedure.

Carboy Set Up : The BOD procedure calls for two carboys to be used each time the procedure is carried out. The carboys will contain the water necessary for the procedure, and two are used instead of one to serve as a source of comparison amongst carboys. Also, it is a good idea to have two sources of water in case something goes terribly wrong with one of the carboys, there will be some sort of back up. To ensure that the two carboys involved in the procedure are free of contaminants, the carboys must first be rinsed with acid and water. Sufficient water should be rinsed to eliminate all traces of acid. Once the acid is flushed from the carboys, a small amount of bleach is added to eliminate excess organisms that may serve as sources of interference. The carboys are again flushed with adequate deionized water until the presence of bleach is eliminated. The carboys are then filled with pure water that has been finely filtered. Millipore water seems to work best in this procedure. The carboys should be filled up to supply atleast six liters for the first sample, and three liters for each successive sample. The six liters for the first sample are necessary because quality controls will also be included as part of the run. Once filled, one phosphate buffer pillow is added to the carboy per six liters water. These pillows are commercially available, and save the analyst time from preparing the reagents. After addition, the water solutions must be aerated until the point of saturation. Commonly, many laboratories will prepare this water the day before the procedure, but five hours prior to analysis appears to be sufficient. After aeration, the water is ready to be used to fill the BOD bottles, and must be capped until then.

"Temperature Dependency"...

Seed Preparation : The seed solution must be set up some time before the BOD bottle are filled with water. This is done by adding 0.045 grams of a polyseed inoculum (or sometimes a BOD sample can be used as the inoculum) to 250 mls distilled water. The seed will not dissolve but it is important that the seed is stirred continously at moderate speed for about two hours. At approxiamtley 30 minutes prior to use, the seed solution is allowed to settle undisturbed. Caution must be taken not to disturb the seed particles because the liquid portion of the solution is used in the procedure. Remnants of actual seed within the prepared BOD bottles could greatly effect the results.

DO Preparation : The DO (dissolved oxygen) of the prepared carboy water must be determined to serve as a reference to all other sample and standard readings. This is most often accomplished by performing a Winkler titration on the carboy water, and adjusting the DO meter to this reading. This method is not contained in the BOD procedure, but rather comes from Standard Methods for the Examination of Water and Wastewater (Method 218B: Azide Modification: 1971). In my experience, we often used a pH meter with an electrode that could also determine the DO of the solution as our DO meter. The Winkler titration is carried out by first withdrawing three samples of water from each carboy. Caution must be taken that no air bubbles become trapped in the bottle. As a result, it is best to withdraw the water slowly while the bottles are tilted slightly. Once filled, one bottle from each carboy is set aside until later. The remaining four bottles are used in the actual titration. 2 mls manganese sulfate is added into each bottle under the surface of the water, followed by 2 mls alkali azide solution. The bottles are stoppered and shaken until a brown floc appears. The bottles are allowed to settle until the floc is halfway settled. The bottles are then shaken again and allowed to settle. Once settled, 2 mls concentrated sulfuric acid is added down the neck of each bottle and the bottles are shaken again. At this point, the floc will disappear and the solutions should be amber in color. Now, the solutions are ready for titration. The solutions are first transfered to 500 ml ehrlenmeyer flasks. Each solution is titrated with 0.0375 N sodium thiosulfate until the solution turns a pale yellow. (Standard Methods suggests using 0.025 N sodium thiosulfate for a different volume of sample.) At this point, a few drops of starch indicator are added to turn the solution a dark blue. Titration continues until the solution turns clear. The volume of titrant used in mls directly corresponds to the DO of the sample. The average DO reading from each carboy is calculated and recorded. If the DO readings from each carboy set are not relatively close to each other (within 0.1 mls) the process must be repeated until consistent DO readings are obtained. The DO readings should normally fall between 6.0 and 9.0 mg/L. If values are greater than 9.0 mg/L, the carboy water must be aerated again to reduce the DO. Once the DO readings are calculated, the two bottles that were withdrawn at the beginning are now used. The carboy bottle from which the samples will be made up from is used to adjust the DO meter (in our case, the pH meter). Once adjusted, the DO of the other bottle is measured to determine whether the meter is reading correctly. If the meter reading comes within 0.1 of the Winkler reading, the calibration is complete. If not, the entire Winkler procedure must be completed for both carboys.

BOD Sample Preparation : Finally, the last step in the BOD procedure involves inoculating the sample with various dilutions along with standards and blanks for quality control. The following quality control must always accompany each BOD run : (1) 2 carboy water blanks, (2) 4 standards, (3) 2 seeded blanks and (4) optional control sample. Usually, many laboratories will include one set of QC for every ten samples. Additional QC is necessary for more than 10 samples. Preparation : (1) Water blanks - carboy water is withdrawn to the rim of the bottles. (2) Standards - 4, 6, 6, and 8 mls of standard solution are added to separate bottles. An additional 2 mls of seed solution is added to each bottle and the bottles are then filled to the rim with carboy water. (3) Seeded blanks - 2 mls of seed solution are added to each of 2 BOD bottles. The bottles are filled to the rim with carboy water. (4) Samples- 4-5 bottles are usually necessary for each sample. Some samples may have to be diluted in order for the DO range to be detected by the meter. Observation of the sample will usually give an indication to its dilution. Clean samples usually require small dilutions whereas wastewater samples will need high dilutions due to their high BOD values. Once reasonable dilutions have been determined, the specific volume of sample is added to each bottle along with 2 mls polyseed. The bottles are filled to the rim with carboy water. Once all the bottles have been filled, the initial DO's of each solution is determined on the meter and recorded. Once recorded, the bottles are capped with ground glass stoppers to avoid excess bubbles and capped. The bottles are placed in an incubator at approxiamtely 20^oC where they will remian for five days.

Analysis : After five days of incubation, the samples are ready to be analysed. The samples are removed from the incubator and allowed to equilibrate to room temperature. In the meantime, the analyst should calibrate the meter again with the carboy water as in the DO sample preparation section. It may be desired to use fresh carboy water for the calibration as in the carboy set up section. Once the meter is calibrated, the samples are read starting with the blanks and ending with the actual samples. The final DO of each solution is recorded and the initial and final readings will be used to calculate the BOD. The best results come about when the initial and final DO values for the blanks are similar indicating the absence of organisms and reliable equipment. The blank DO should normally be less than 0.2 mg / L.

"BOD Curve & Equations"...

BOD Calculations...

The calculations for BOD take into account the unseeded blanks and the seeded solutions. These values must be subtracted out in order to obtain reasonable BOD results. The following calculations are taken from Standard Methods (Method 507: 1985, p. 531). (1) The BOD of the blanks are calculated by subtracting the final DO from the initial DO :

BOD_blank = DO₁ - DO₂

(2) The BOD of the seeds are calculated by subtracting the final DO from the initial DO and multiplying this number by the dilution factor :

BOD_seed = ( DO₁ - DO₂ ) x ( Dilution factor per 300 ml )

(3) The BOD of sample and standards are calculated by subtracting the final DO from the initial DO and multiplying this number by the dilution factor. The final value is determined by substracting out the BOD blank and the seed blank for each delta DO. If the testing procedure was carried out correctly and the dilutions of the sample were made appropriately, the analyst should have obtained BOD values that are within a reasonable percent error and relative percent difference. Generally, values are discarded for a specific sample dilution if the final DO of the sample is < 1.0 mg/L of if delta DO is < 1.0 mg/L. This also stresses the importance of using different dilutions for each sample to key in on the appropraite BOD when little is known about how the sample will react and how high its BOD levels are.

Chemical Oxygen Demand...

The COD test is done by heating a portion of sample in an acidic chromate solution, which oxidizes organic matter chemically. The amount of chromate remaining (measured by a titration), or the amount of reduced chromium produced (measured spectrophotometrically), is translated into an oxygen demand value. Biodegradability, toxins, and bacteria are not important, and the test is complete in about two hours. The figure will be higher than the BOD.

Total Organic Carbon...

The TOC is done instrumentally. The organic carbon is oxidized to carbon dioxide by burning or by chemical oxidation in solution. The carbon dioxide gas is swept out and measured by infrared spectrometry or by redissolving it in water and measuring the pH change (the gas is acidic.) Both COD and TOC can often be correlated with BOD for a specific wastewater sample, but each wastewater is different. As a rough guide, the COD of a raw domestic wastewater is about 2.5 times the 5-day BOD.

Solids...

Water, a liquid, can contain quite a bit of solid material, both in dissolved and suspended forms. The term "dissolved" implies that the individual molecules of a substance are mixed in among the water molecules. In practice, solids are classified as "dissolved" if they pass through a standard glass-fiber filter with about one micrometer pore size. Solids captured on the filter are, by definition, "suspended" solids. Solids which settle out of a water sample on standing for a period of an hour are defined as "settlable". Solids are also further classified as "fixed" or "volatile". Fixed solids are basically the ash left over after burning the dried solids; volatile solids are those that are lost in this procedure. The sum of the two is referred to as "total". (This can be confusing, as the word "total" is also used in describing the sum of suspended and dissolved solids). Volatile solids are often used as an estimate of the organic matter present.

Significance...

Solids in wastewater contribute to sediment formation; volatile solids may be associated with oxygen demand.

Measurement...

Total solids (TS) are determined by drying a known amount of a sample at a temperature of 103 to 105 C in a tared (pre-weighed) vessel, such as a porcelain dish, cooling in a dry atmosphere (in a container known as a desiccator), weighing on an analytical balance, subtracting the tare weight, and dividing by the original amount of sample. Results can be expressed in mg/L if the sample was originally measured out by volume; or percent by weight, if the sample was originally weighed. If the sample is then burned in a furnace at about 500 C, cooled, and weighed, the fixed (FS) or volatile solids (VS) can be determined. If the original sample is filtered through a tared glass-fiber filter, which is then dried, the weight of the material captured on the filter is used to figure the total suspended solids (TSS). Burning the filter in the furnace allows measurement of volatile suspended solids (VSS) or fixed suspended solids (FSS). The dissolved solids (DS) can be estimated from the difference between the total solids and the total suspended solids, but the official method calls for drying the filtrate (the liquid which passes through the filter) in a dish at 180 C (and, of course, there are TDS, FDS and VDS). An estimate of total suspended solids can be obtained by an optical/instrumental measurement known as turbidity. The sample is placed in a glass tube; a beam of light is shined through it, and the light scattered at right angles to the beam is measured photometrically. In the same way that COD can be correlated with BOD, turbidity can be correlated with TSS; but the correlation will hold only for the particular sample from which it was derived. Similarly, an estimate of dissolved solids is often made by measuring the water's electrical conductivity. Pure water does not conduct electricity. If substances which dissociate into electrically charged ions are dissolved in the water, they will conduct a current, roughly proportional to the amount of dissolved substances. Conductivity can be used to track sewage pollution. Note, however, that many organic materials dissolve in water without producing ions. So, while a salt solution may have a high electrical conductivity, a concentrated solution of sugar would go undetected by this method.

Nutrients...

Nutrients are usually thought of as compounds of nitrogen or phosphorus, although certainly other elements, such as iron, magnesium, and potassium are also necessary for bacterial and plant growth. Nitrogen occurs primarily in the oxidized forms of nitrates (NO₃^-) and nitrites (NO₂^-) or the reduced forms of ammonia (NH₃) or "organic nitrogen" where the nitrogen is part of an organic compound such as an amino acid, a protein, a nucleic acid, or one of many other compounds. All of these can be used as nutrients, although the organic nitrogen first needs to decompose to a simpler form. Phosphorus is biologically important in the form of phosphate, the most highly oxidized state of the element. The most biologically available form is dissolved orthophosphate, (PO₄^-3). (In solution, there are up to three hydrogens attached to the molecule, each one decreasing the negative charge of the ion by one. How many hydrogens are attached depends on the pH. There are also condensed forms of phosphate, with more than one phosphorus atom per ion, such as pyrophosphate and polyphosphates. There are also organic phosphates, and all of these forms can be either dissolved or particulate (i.e., insoluble). The sum of all the forms is known as total phosphorus.

Significance...

These nutrients are important in natural waters because, in excess, they can cause nuisance growth of algae or aquatic weeds. In wastewater treatment, a deficiency of nutrients can limit the effectiveness of biological treatment processes. In some plants treating industrial wastewaters, ammonia or phosphoric acid must be added as a supplement.

Measurement...

Ammonia can be measured colorimetrically, by the Nessler or phenate methods, after distillation from an alkaline solution to separate it from interferences. It can also be determined by an electrode method, sometimes without distillation, since there are fewer interferences. Organically-bound, reduced nitrogen can be determined by the same methods after a digestion (the Kjeldahl digestion) which converts the nitrogen in those compounds to ammonia. The combination of ammonia and organic nitrogen is known as "Total Kjeldahl Nitrogen," or TKN. (TKN analysis is used for measuring protein content of animal feeds, as well.) Nitrite is determined colorimetrically. Nitrate can also be determined this way; the most popular way is by first reducing nitrate to nitrite chemically using cadmium, then analyzing the nitrite. There is an electrode method for nitrate, but it is not considered too accurate. Finally, ammonia (as the positively charged ammonium ion, NH₄⁺), nitrate, and nitrite can be measured by ion chromatography, as well. Phosphate can be measured by ion chromatography, also. Greater sensitivity, at lower cost, is obtained by colorimetric methods which measure dissolved orthophosphate. Some insoluble phosphates and condensed phosphates - so called "acid-hydrolyzable phosphate" - can be included by heating the sample with acid to convert these forms to orthophosphate. If the organic phosphate is to be included, to measure "total phosphate", then the sample must be digested with acid and an oxidizing agent, to convert everything to the orthophosphate form.

Chlorine...

The pure element exists as the molecule, Cl₂, which is a gas or a liquid at normal temperatures, depending on the pressure. When dissolved in water, most of it reacts to form hypochlorous acid (HOCl) and hydrochloric acid (HCl) which make the water more acidic. The HOCl dissociates, to some extent, to form H⁺ and OCl^-, called hypochlorite ion. (The HCl dissociates completely.) If there is enough alkalinity to react with the hydrogen ions produced and keep the pH around neutral, most of the chlorine will be in the form of hypochlorous acid and hypochlorite ion. Disinfection can be done using solutions of sodium hypochlorite, which produce the same substances in solution. Hypochlorite ion is not considered as strong a disinfectant as HOCl, so the pH can affect the disinfectant efficiency. Dissolved chlorine, hypochlorous acid, and hypochlorite ion, taken together, are all known as "free chlorine". Free chlorine can react with ammonia in solution to form compounds called chloramines, which are weaker disinfectants than free chlorine, but have the advantage of not being used up by side reactions to the extent that free chlorine is. Free chlorine (and chloramines) also react with organic nitrogen compounds to form organic chloramines, which are even weaker disinfectants. The chloramines are termed "combined chlorine," and the sum of the free and combined forms are called "total chlorine". (Note that a large enough amount of chlorine can oxidize ammonia to nitrogen gas; this can be used as a chemical means of destroying ammonia)

Significance...

Chlorine is the most commonly used disinfecting agent for drinking water and wastewater. It is coming into some disfavor because of toxic and carcinogenic byproducts, such as chloroform, which are formed when it reacts with organic matter present in the water. Unless reduced to chloride, chlorine itself is toxic to aquatic life in receiving waters. Pure chlorine liquid or gas is also a storage and transportation hazard because of the possibility of accidental releases to the atmosphere. Some treatment plants are switching to hypochlorite solution because it is safer to handle. Others are eliminating it entirely and using UV light or ozone for disinfection.

Measurement...

There are several choices for chlorine measurement, some of which can distinguish between free chlorine and the various chloramines. There are titrations involving visual, color-indicator endpoints, as well as electrochemically measured endpoints. Some of them can be used to differentiate among the various forms of chlorine depending on whether iodide ion is added to the testing mixture. The indicator known as DPD (full name, N,N-diethylparaphenylenediamine) can be used to measure free or total chlorine both colorimetrically or as a titration indicator. "Amperometric titration" is a sensitive electrochemical method.

Oil and Grease...

Is the name given to a class of materials which can be extracted from water using certain organic solvents. They can be of biological origin (animal fat, vegetable oil); they can be "mineral" (petroleum hydrocarbons); or they can be synthetic organic compounds. Fats and greases from restaurants and food processing industries can clog sewers, causing blockages and backups. Petroleum products can be toxic and flammable, and can coat surfaces and interfere with biodegradation by microorganisms in wastewater treatment plants. They are mostly biodegradable, especially biological oils and greases, but are a problem due to forming a separate phase from the water.

Measurement...

The major method of analysis is liquid-liquid extraction. Currently, the chlorofluorocarbon known as CFC-113 is used, but is due to be phased out in favor of the hydrocarbon, hexane, because of the damage done by CFC's to the stratospheric ozone layer. In the procedure, the sample is acidified, and then shaken several times with the solvent. The solvent portions are combined and evaporated, and the residue is measured by weight. In a CFC solution, the concentration of the oil/grease can also be measured by infrared spectrophotometry without having to evaporate the solvent. To determine petroleum hydrocarbons alone, the extract solution can be treated with the material, silica gel, which absorbs the more polar biological compounds. A newer method, solid phase extraction, passes the water sample through a small column or filter containing solid sorbent material which absorbs the oil and grease. It is then desorbed from the sorbent using a solvent and analyzed as above.

Metals...

Chemically, metals are classified as elements which tend to lose electrons in a chemical reaction. As solids, they have easily movable electrons, which makes them good conductors of electricity and reflectors of light. In compounds, they tend to be positively charged, because they have lost electrons (which carry a negative charge), and they tend to bind with non-metals. This tendency makes some of them, such as iron and magnesium, biologically useful as part of biochemically active compounds like enzymes. Others, such as lead, cadmium, and mercury are highly toxic because they interfere with the normal operation of these biological compounds. The US EPA lists nine metals used in industry (arsenic, cadmium, chromium, copper, lead, mercury, nickel, silver, and zinc) as toxic "priority pollutant" metals.

Measurement...

There are numerous colorimetric methods for metals. Most of them are more useful in a purer medium, such as drinking water, than they are in wastewater, because of the presence of interfering substances. The most popular methods in use today involve one form or another of atomic spectroscopy, as described previously. Another technique, X-ray spectroscopy, is useful primarily for solid samples. There are also electrochemical methods, like polarography and "anodic stripping voltametry" which are quite sensitive; but due to their complexity, they are confined mostly to research purposes.

Cyanide...

Cyanide is the name of an ion composed of carbon and nitrogen, CN^-. It is used in the mining and metal finishing and plating industries - usually as the sodium or potassium salts, NaCN or KCN - because of its ability to bind very strongly to metals to form water-soluble complex ions. This same property makes it highly toxic to living things because it prevents the normal activity of biologically important, metal-containing molecules. It is, however, biodegradable by some bacteria in low concentrations; and they can become acclimated to higher concentrations if given enough time. For unacclimated microorganisms in a wastewater treatment plant, however, a cyanide "dump" by an industry can lead to inhibition or even death, which can cause a severe "plant upset".

Measurement...

Cyanides are usually measured by a sensitive colorimetric / spectrophotometric procedure which can detect levels down to about 5 parts per billion in water. Since much of the cyanide in a sample is likely to be bound to metal ions, a digestion / distillation procedure is necessary to measure "total" cyanide. Cyanide can also be measured by ion chromatography or an electrode method, though the latter is not considered too accurate.

Toxic Organic Compounds...

An organic compound is any compound which contains carbon, with the exception of carbon monoxide and carbon dioxide, carbonates, or cyanides. Organic compounds contain chains and/or rings of connected carbon atoms, often with other elements attached. There are millions of possible compounds, with many useful properties. Many are biologically active, since all living things are made up of organic molecules. Industries use and produce thousands of organic compounds in manufacturing such items as plastics, synthetic fibers, rubber, pharmaceuticals, pesticides, and petroleum products. Some of the compounds are starting materials; some are solvents; some are byproducts. The US EPA lists 116 of them as toxic "priority pollutants"; many states have longer lists. One of the major groupings is volatile organic compounds (VOC's), many of which are chlorine-containing solvents. There are also petroleum hydrocarbons and starting materials for plastics, dyes, and pharmaceuticals. The "semi-volatile" group include solvents, PAH's (polycyclic aromatic hydrocarbons, like naphthalene and anthracene which are coal tar constituents), as well as pesticides (especially chlorinated pesticides) and PCB's (polychlorinated biphenyls, which were formerly used in electrical transformers and other products).

Measurement...

Most of these are analyzed routinely by gas chromatography (GC), often followed by mass spectrometry (MS) for identification. HPLC is also used for some analytes. A technique which is becoming available for field measurements for some of these compounds is immunoassay, sometimes called ELISA, for "enzyme-linked immunosorbent assay". This method, which produces a color reaction related to the concentration of the target compound, or family of compounds, is portable, relatively inexpensive and does not require a great deal of training. It is in use more for surveying hazardous waste sites, however, than for water analysis.

Pathogenic Microorganisms...

Sewage contains large numbers of microbes which can cause illness in humans, including viruses, bacteria, fungi, protozoa and worms (and their eggs or ova). They originate from people who are either infected or are carriers. While many of these can be measured directly by microscopic techniques (some after concentration), the analyses most commonly performed are for so-called "indicator organisms". These organisms, while not too harmful themselves, are fairly easy to test for and are chosen because they indicate that more serious pathogens are likely to be present. For instance, wastewater treatment plants are often required to test their effluents for the group known as "fecal coliforms", which include the species E. coli, indicative of contamination by material from the intestines of warm-blooded animals. Water supplies test for a more inclusive group called "total coliforms", and in some cases, for general bacterial contamination (heterotrophic plate count, or HTP).

Measurement...

The two most commonly used methods of analysis for indicator organisms are the multiple tube fermentation technique and the membrane filter procedure. In the first method, a number of tubes containing specific growth media are innoculated with different amounts of the sample and incubated for a particular time at a prescribed temperature. The appearance of colors, fluorescence, or gas formation indicates the presence of bacteria belonging to the target group. The number of organisms per 100 mL in the original sample is estimated from most probable number (MPN) tables, which list the values of MPN for different combinations of positive and negative results in tubes which contained different initial volumes of the sample. Often, positive results must be confirmed by further innoculation of small amounts of material from the positive tubes into tubes containing a different media, which can extend the test to several days. The second technique involves filtering a known volume of sample through a membrane filter (made of a material such as cellulose acetate) which has a small enough pore size to retain the bacteria. The filter is then placed in a dish of sterile nutrient media, either soaked into an absorbent pad or in a gel such as agar, and sealed. The dish is incubated for the prescribed time and temperature. The media contain a colored indicator which will identify the target bacteria. Each bacterium in the original sample will result in a colony after incubation, which is large enough to see without a great deal of magnification. The concentration in the sample can be determined by direct count of the colonies, knowing the volume of sample used. In some cases, these colonies require further confirmation. Detection and enumeration of HTP or of specific pathogenic bacteria, such as Salmonella, E. coli or Enterococcus can be done by similar methods, but utilizing specific growth media for each type. Viruses are usually measured by concentration, followed by addition to cultures of cells which they infect and counting the number of plaques formed due to cell destruction. Pathogenic protozoa and ova of multicelled organisms are determined by concentration and direct counting under the microscope, often with the aid of fluorescent staining compounds. Besides, direct observation, identification of pathogenic microorganisms can be done by standard techniques used in clinical laboratories involving observing reactions in a battery of different indicating media. Some newer methods use chromatography to identify patterns of compounds which serve as "fingerprints" for certain bacteria; DNA analysis is another recent innovation. Most wastewater treatment plants, however, confine their testing to simply counting the numbers indicator bacteria.