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ORGANIC MATTER It is not possible to determine the amount of organic matter pres ent in a sample of water by any direct method. As all protein matter contains nitrogen, methods have been devised to determine the total amount of nitrogen and also the amount of nitrogen in various com binations. From such data valuable information concerning the sani tary history and sanitary quality of the water may be inferred. The nitrogen is determined as (1) total nitrogen ; (2) nitrogen as free am monia; (3) nitrogen as albuminoid ammonia; (4) nitrogen as nitrites; (5) nitrogen as nitrates.

The organic matter in water is of animal and vegetable origin and exists both in solution and in suspension. Some of it is in the body of living beings; some of it is in their dead bodies; and some of it is in various stages of decomposition until the final stable compounds, such as ammonia and nitrates, are reached. The total amount of or ganic matter present in a sample of water is represented by the amoun) of nitrogen as free ammonia and albuminoid ammonia. The presence of nitrogen as nitrites and nitrates indicates the amount of self-purifica tion which the water has undergone. Their significance will be dis cussed separately.

Free Ammonia. If there is much "free" ammonia in the water the sample may be Nesslerized directly. If the water contains comparatively little, as is usually the case, the ammonia must first be concentrated by distillation and condensation.

Place 500 c. c. of the sample of water in a metal or glass still con nected to a tin or aluminium condenser in such a way that the dis tillate may be conveniently delivered directly into Nessler tubes. The entire apparatus must first be freed from ammonia by blowing steam through it until the distillate shows no trace of free ammonia. When this has been done the distilling flask is emptied and 500 c. c. of the sample water measured into it. The distillation should be carried on at a rate so that not more than 10 c. c. nor less than 6 c. c. condense per minute; that is, it should take from 5 to 10 minutes to distill 50 c. c., which is the quantity Nessler tubes are ordinarily graduated to contain. Four Nessler tubes of the distillate containing 50 c. e. each are collected from the first portion that comes over; these contain the free ammonia.

If the sample is acid, or if the presence of urea is suspected, about one-half gram of sodium carbonate should be added previous to dis tillation, otherwise the ammonia will not come off. Sodium carbonate is omitted, when possible, as it tends to increase "bumping." The amount of ammonia is determined by adding 2 c. c. of Nessler reagent to each tube and comparing the depth of color with a set of standard tubes prepared with a known quantity of ammonium chlorid solution, plus an equal quantity of Nessler reagent.

Nessler's reagent is prepared by dissolving 50 grams of potassium iodid in a minimum quantity of cold water. To this add a saturated solution of mercuric chlorid until a slight permanent precipitate per sists. Then add 400 c. c. of 50 per cent. potassium hydroxid solution which has been allowed to clarify by sedimentation before using; dilute to one liter, allow to stand, and The solution should give the required color with ammonia within 5 minutes after addition, and should not precipitate with small amounts of ammonia within 2 hours. The reaction between Nessler's reagent and ammonia is an empiric one. The which constitutes the Nessler's reagent in the presence of ammonia, forms a brownish compound which is known as mercurammonium iodid, and has the formula NHg2IH2O.

Ammonia-free water may readily be obtained by distilling water containing about 0.2 per cent. dilute sulphuric acid.

Standard Solution. The standards for comparison con sist of ammonium chlorid dissolved in ammonia-free water. Dissolve 3.82 grams of ammonium chlorid in 1 liter of water; dilute 10 c. c. of this to 1 liter with the ammonia-free water. One c. c. will then equal 0.00001 gram of nitrogen as ammonia.

A gram molecule of NII,C1 weighs 53.5 grams—that is: N 14 H 4 + Cl 35.5 = 53.5 The equation would then be : 14 : 53.5 : : 1 : x x = 3.82 That is, if there are 14 grams of nitrogen in 53.5 grams of b,M monium chlorid, then 1 gram of nitrogen is contained in 3.82 grams of ammonium chlorid. It is to be noted that, while the method deter mines the amount of ammonia, the results are expressed in terms of nitrogen. In the same way the nitrites and nitrates are also expressed in terms of nitrogen.

Prepare a series of 16 Nessler tubes, which contain the following number of cubic centimeters of the standard ammonium chlorid solu tion, namely: 0.0, 0.1, 0.3, 0.5, 0.7, 1.0, 1.4, 1.7, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, and 6.0; dilute each one to the 50 c. c. mark with ammonia-free water. These will contain 0.00001 gram of nitrogen for each cubic centi meter of the standard solution used. Add about 1 c. c. of the Nessler reagent to each tube; do not stir the contents of the tubes.

The color produced in the distillate from the sample under exam ination is now compared with standards by looking vertically down ward through them at a white surface placed at an angle in front of a window so as to reflect the light upward. The tubes should be allowed to stand at least 10 minutes after Nesslerizing before making the com parison.

The last 50 cubic centimeters of the distillate examined should con tain no ammonia, or at most a trace, otherwise it may be inferred that all has not been collected, or some error has crept into the work. It is not uncommon for the last tube to contain a little ammonia when the organic matter is of plant origin. Ammonia determinations should be carried out in a special room, where at least volatile ammonia re agents are not exposed. Special care must be exercised not to contam inate the Nessler tubes with soiled fingers, rags, etc. Care must be exer cised thoroughly to wash the tubes free from alkaline soaps, and to rinse with ammonia-free water. The Nessler tubes containing the standard Significance of Free Ammonia. The free ammonia which comes off with the first part of the distillate usually exists in the water as chlorids or carbonates. It is called "free ammonia" because these salts are readily decomposed and the ammonia is expelled by boiling.

Rain water washes down some free ammonia which is found in the atmosphere. Angus Smith and Boussingault place the average amount of ammonia in the rain of temperate climates as 0.5 part per million.

The amount of ammonia in rain water was studied by Filhol. He found that in the city of Toulouse the rain water contained 6.60 parts per million, while the rain water collected near the city contained only from 0.44 to 0.77 part per million. These figures show the marked dif ference between city and country rain.

In a surface or ground water free ammonia represents one of the latter stages of putrefaction of organic matter; thus, the bacterial de composition of sewage yields ammonia in abundance.

The ammonia itself ordinarily found in drinking water is harmless; its significance lies in the fact that it indicates the presence of putre fying organic matter.

The presence of free ammonia in clean and properly stored rain water has much less significance than in a surface or ground water.

Free ammonia in water results not only from the decomposition of nitrogenous organic matter, but is also formed during the process of denitrification, by which nitrates are again reduced to nitrites and nitrites to ammonia. This action only takes place near the surface of the soil, and to a limited extent. Deep well waters of exceptional purity upon chemical analysis and practically sterile upon bacteriologi cal examination, may contain a relatively high percentage of free am monia. This is supposed to come from a chemical reduction under high pressure and perhaps temperature of the geological nitrogenous matter in coal and alluvial deposits.

A definite permissible limit for the amount of free ammonia which good water should contain cannot be fixed. Its significance must be judged from the other constituents of the water and a sanitary survey of its source. As a rule, safe water may contain from 0.015 to 0.03 or even 0.055 part per million. In general, free ammonia is less of a danger signal than the fixed or albuminoid ammonia.

Albuminoid Ammonia. Nitrogen as albuminoid ammonia is always determined in conjunction with and as a continuation of the method for determining nitrogen as free ammonia. After obtaining 200 c. c. (that is, 4 Nessler tubes of 50 c. c. each) from the first portion of the distillate, for the purpose of determining nitrogen as free ammonia, withdraw the flame, disconnect the flask and add 40 c. c. or more of hot alkaline potassium permanganate, and continue the distillation until at least 4 portions of 50 c. c. each, or, preferably, 5 portions, of the distillate have been collected in separate Nessler tubes.

The calculation is as follows : The alkaline potassium permanganate solution is made by pouring 1,200 c. c. of distilled water into a porcelain dish holding 2,500 c. c.; boil 10 minutes and turn off the gas. Add 16 grams of C. P. potas sium permanganate and stir until dissolved. Then add 800 c. c. of 50 per cent. clarified solution of potassium or sodium hydrate and enough distilled water to fill the dish. Boil down to 2,000 c. c. Test each batch of this solution for albuminoid ammonia by making a blank determi nation. Correction should be made accordingly.

After the readily decomposed ammonia salts have been broken up and the ammonia driven off in the steam which condenses to form the first 200 c. c., the remainder of the sample of water in the still con tains nitrogenous organic matter that requires a strong oxidizing agent to disintegrate it. This is accomplished by the alkaline potassium per manganate. The nitrogen in the complex protein molecule finally forms ammonia, and hence this is called albuminoid ammonia ; the amount of it is determined by Nesslerization, precisely as for free ammonia. In ground waters and surface waters containing but little pollution the nitrogen as albuminoid ammonia usually approximates about one-half of the total organic nitrogen. In sewage and other liquids containing considerable nitrogenous organic matter the percentage of ammonia forming organic matter is variable. For this reason the amount of albuminoid ammonia obtained by the alkaline permanganate method is less valuable than the total organic nitrogen determined by the Bjel dahl method.

If it is desired to determine how much of the organic matter is in solution and how much in suspension, the sample of water should be passed through a Berkefeld filter. The albuminoid ammonia in the filtrate represents the dissolved organic matter, and the difference be tween the albuminoid ammonia in the total sample and the filtered sample gives the suspended nitrogen as albuminoid ammonia.

The albuminoid ammonia is a fairly correct index of the amount of organic pollution in the water. It comes from minute organisms, both living and dead, that are in the sample, also from particles of animal and vegetable matter in suspension, and finally from the nitrog enous substances in solution and in various stages of decomposition. The organic matter in itself is not dangerous to health, but is unde sirable because it putrefies and thus gives a water disagreeable tastes and odors; further, it offers food for bacterial growth. The amount of albuminoid ammonia is therefore an index of pollution, but if of vegetable origin it has much less sanitary significance than if of animal origin. Organic matter of animal origin yields a much larger amount of albuminoid ammonia than a similar amount of vegetable matter. Whether the organic matter comes from sewage, from a dead carcass, of from the swamps, cannot be stated with certainty from this test, but if the albuminoid ammonia comes over quickly, that is, if most of it appears in the first Nessler tube, it is presumably of animal origin; whereas, if the ammonia comes over more slowly and the second and third Nessler tubes contain appreciable amounts, the organic matter is presumably of vegetable origin.

No arbitrary standard can be set as to the maximum amount of albuminoid ammonia a good water may contain. Waters considered "pure" often contain as much as 0.079 to 0.34 part of nitrogen as al burninoid ammonia per million.

Nitrites. Nitrites in water are regarded as a special danger signal. The reason for this is that nitrites indicate that active putrefaction of nitrogenous organic matter is going on as the result of bacterial activ ity. The presence of nitrites, therefore, at once suggests organic pollu tion. The presence of nitrites in water represents the transitional stage in the oxidation of organic matter between ammonia and nitrates, and therefore indicates incomplete oxidation of the protein and the active growth of bacteria.

Nitrites are never present except in small amounts, for they are soon oxidized to the higher and more stable nitrates, but a minute amount, according to some authorities, is sufficient to condemn a water. As a rule, pure water contains no nitrites, or traces only; on the other hand, nitrites may be absent from an impure water, owing to the fact that the oxidation has not reached this stage, or perhaps has entirely passed it. The absence of nitrites, therefore, does not mean that the water is necessarily safe, while their presence in any but the smallest measurable amounts shows pollution. We must not give to the nitrites an exag gerated importance: they are a danger signal in the same sense that the colon bacillus is a danger signal, indicating pollution but not neces sarily infection, for they do not tell the source or nature of the organic matter. The presence of nitrites in spring and deep well water may be without sanitary significance, for in these cases they may be gen erated by the deoxidation of the nitrates which is brought about either by the action of reducing substances, such as ferrous oxid, or by organic matter. It should be remembered that the colorimetric test for nitrites with sulphanilic acid and a-amidonaphthylamin is one of the most deli cate tests in chemistry. With this method we are able to detect quan tities as small as one part in a hundred million. When, therefore, a water analyst reports a trace of nitrites it means an exceedingly minute quantity.

Nitrites are not only formed by the nitrifying bacteria in the soil from ammonia, but are also formed from the denitrification of nitrates by a variety of microorganisms. The typhoid bacillus, the colon bacillus, and many other bacteria have the power of producing nitrites in culture media.

Nitrates are poisonous, but the minute amounts found in water can scarcely have a pharmacological effect.

Method for Estimating Nitrogen as (1) Sul phanilic acid solution. Dissolve eight grams of the purest sulphanilic acid in 1,000 c. c. of 5 N. acetic acid (sp. gr. 1.041). This is prac tically a saturated solution.

(2) Naphthylamin acetate or chlorid solution. Dissolve 5.0 grams solid a-naphthylamin in 1,000 c. c. of 5 N. acetic acid; filter the solution through washed absorbent cotton, or an alundum filter.

(3) Sodium nitrite, stock solution. Dissolve 1.1 grams silver ni trite in nitrite-free water; precipitate the silver with sodium chlorid solution and dilute the whole to one liter.

(4) Standard sodium nitrite solution. Dilute 100 c. c. of solution (3) to one liter; then dilute 5 c. c. of this solution to one liter with sterilized nitrite-free water; add one c. c. of chloroform and preserve in a sterilized bottle. One c. c. = 0.0005 mg. nitrogen.

Procedure.Measure out 50 c. c. of the decolorized sample (de colorized by adding aluminium hydrate free of nitrite—sce under Chlo rin), or a smaller portion diluted to 50 c. c., into a Nessler tube. At the same time make a set of standards by diluting various volumes of the standard nitrite solution in Nessler tubes to 50 c. c. with nitrite free water, for example, 0.0, 0.1, 0.2, 0.4, 0.7, 1.0, 1.4, 1.7, 2.0, and 2.5 c. c. Add 1 c. c. each of reagents Nos. 1 and 2 (above) to each 100 c. c. of the sample and to each standard. Mix ; allow to stand 10 min utes. Compare the samples with the standards. Do not allow the sam ples to stand over one-half hour before being compared, on account of ab sorption of nitrites from the air. Make a blank determination in all cases to correct for the presence of nitrites in the air, the water and other reagents. Dilute all samples which develop more color than the 2.5 c. c. standard before comparing. Mixing is important.

When 50 c. c. of the sample are used, then 0.01 times the number of c. c. of the standard gives the parts per million of nitrogen as nitrite.

Calculation.One c. c. of the standard equals 0.0005 mg. N. as nitrites. 50 c. c. of the sample is used and is found to equal 0.5 c. c. of the standard.

Then 50 c. c. sample contains 0.00025 mg. N. as nitrites and one liter will contain 20 X 0.00025 or 0.005 mg. of N. or 0.005 part per million.

When 50 e. c. of the sample is used 20 X 0.0005 X the number c. c. of the standard will give the number mg. N. per liter or parts per million. This can be shortened to 0.01 X the number c. c. standard used equal parts per million of N. as nitrites.

Nitrates. Nitrates are the end products of the mineralization of organic matter. Their presence, therefore, signifies past or distant pollution. While the absence of nitrates does not necessarily mean purity, their presence, on the other hand, does not necessarily indicate immediate danger. If a water contains an appreciable quantity of ni trates and no nitrites, it shows that the source of pollution has been distant and that the organic matter has been completely oxidized. In waters considered pure the nitrates are rarely less than 0.3 part, or they may run as high as 1.6 parts, per million. Polluted waters usually contain very much more, as 17 to 20, or more parts per million. Nitrates usually exist in water as salts of alkaline bases.

Young ° has shown that the ground waters of Kansas sometimes contain large amounts of nitrates—as much as 500 parts per million. The medicinal dose of potassium nitrate is 0.3 gram. Less than a liter of water would therefore contain sufficient nitrates to produce therapeutic effects duch as irritation of the mucous membrane of the stomach, resulting in gastritis, and also diuresis, with irritation of the mucous membrane of the bladder.

The test for nitrates depends upon the fact that they react with phenoldisulphonic acid to form a compound resembling picric acid, which is yellow in the presence of an alkali. The amount of nitrates is deter mined colorimetrically by comparison with standard solutions.

Phenoldisulphonic Acid Method. Reagents: (1) Phenoldisulphonic acid. Dissolve 25 grams of pure iirhite phenol in 150 c. c. of pure con centrated sulphuric acid. Add 75 c. c. of fuming sulphuric acid (15 per cent. stir well, and heat for 2 hours at about 100° C.

(2) Potassium. hydroxid solution. Prepare an approximately 12 N. solution, 10 c. c. of which will neutralize about 4 c. c. of the phenol disulphonic acid.

(3) Standard nitrate solution. Dissolve 0.72 gram of pure re crystallized potassium nitrate in 1 liter of distilled water. Evaporate cautiously to dryness 10 c. c. of the solution on the water bath. Moisten residue quickly and thoroughly with 2 c. c. of phenoldisulphonic acid and dilute to 1 liter. This is the standard solution, 1 c. c. of which equals 0.001 mg. of nitrate nitrogen.

(4) Standard silver sulphate solution. Dissolve 4.4 grams of silver sulphate free from nitrate in 1 liter of water. One c. c. of this solution is equal to 1 mg. of chlorid.

Procedure.The alkalinity, chlorid and nitrite content, and color of the sample must first be determined. If the sample is highly colored decolorize it with freshly precipitated aluminium hydroxid. Measure into an evaporating dish 100 c. c. of the sample, or if nitrate is very high such volume as will contain about 0.01 mg. of nitrate nitrogen. Add sufficient sa sulphuric acid nearly to neutralize the alkalinity. Then add sufficient standard silver sulphate to precipitate all but about 0.1 mg. of chlorid. The removal of chlorid may be omitted if the sample contains less than 30 parts per million of chlorid. Heat the mixture to boiling, add a little aluminium hydroxid, stir, filter, and wash with small amounts of hot water. Evaporate the filtrate to dryness, and add 2 c. c. of the phenoldisulphonic acid, rubbing with a glass rod to insure intimate contact. If the residue becomes packed or appears vitreous be cause of the presence of much iron, heat the dish on the water bath for •Young, C. C.; Jour. d. M. A., June 24, 1911, LVI, p. 1881.

a few minutes. Dilute the mixture with distilled water, and add slowly a strong solution of potassium hydroxid or ammonium hydroxid until the maximum color is developed. Transfer the solution to a Nessler tube, filtering if necessary. If nitrate is present a yellow color will be formed. Compare the color with that of standards made by adding 2 c. c. of strong potassium hydroxid or ammonium hydroxid to various amounts of standard nitrate solution and diluting them to 50 c. c. in Nessler tubes. The following amounts of standard nitrate solution are suggested : 0, 0.5, 1.0, 1.5; 2.0, 4.0, 6.0, 8.0, 10.0, 15.0, 20.0, and 40.0 c. c. These standards may be kept several weeks without deterioration. If 100 c. c. of water is used the number of cubic centimeters of the stand ard multiplied by 0.01 is equal to parts per million of nitrate ni trogen.

Standards that will remain permanent for several years if stored in the dark may be prepared from tripotassium nitrophenoldisulphonate.

If nitrate nitrogen is present in excess of 1 part per million it should be oxidized by heating the samples a few minutes with a few drops of hydrogen peroxid, free from nitrate, repeatedly added, or by adding dilute potassium permanganate in the cold until a faint pink coloration appears; the nitrogen equivalent of the nitrite thus oxidized to nitrate is then subtracted from the final nitrate nitrogen reading.

ammonia, water, nitrogen, solution, nitrites, free and sample