Relation of d–Gossypol to the Toxicity of Some Cottonseed Products1

Ind. Eng. Chem. , 1928, 20 (1), pp 59–63. DOI: 10.1021/ie50217a026. Publication Date: January 1928. ACS Legacy ... a meeting... SCIENCE CONCENTRATES...
0 downloads 0 Views 745KB Size
January, 1928

INDUSTRIAL A N D ENGINEERING CHEMISTRY

bidities, while the Hyattsville raw water ranges from 6.8 on low to 6.4 on high turbidities. The higher p H value of the raw water on low turbidities a t Burnt Mills permits good coagulation with a lower alum dose than a t Hyattsville. Apparently little advantage was gained with the use of sodium aluminate, and its use in regular operation has been discontinued pending the completion of additional laboratory experiments. With higher turbidities in the raw water and a p H value of 6.7, the use of sodium aluminate will probably prove of decided advantage, and it is now being used in regular plant operation for turbidities over 300 p. p. m. With the low doses of alum, using the straight alum treatment on low turbidities, we have had little trouble a t this plant with alumina passing through the filters, and consequently little after-coagulation has occurred. With the very high doses of alum when treating high turbidities without sodium aluminate, however, alumina did pass the filters and caused considerable trouble from after-coagulation. It was impossible to add enough lime to the filtered water to maintain a p H of 7.8 or 8.0 without imparting a milky appearance to the water. During these periods water was allowed to leave the plant with a pH of 7.2 to 7.4 and tests on the distribution system revealed that the p H value had been reduced to 6.8 or 6.7. Using sodium aluminate on high turbidities permits the water to be filtered with only a slight reduction in p H value and it

59

is possible to maintain a passive tap water. Colloidal interference, formerly troublesome a t times a t this plant, has been overcome by the combined treatment. Conclusions

The experience gained a t these plants during the past few months has indicated clearly that the combined treatment of small quantities of sodium aluminate with alum has specific value in the treatment of the water, especially during periods when the raw water contains much colloidal clay. Summarizing briefly the value of the combined treatment, the following outstanding advantages have been noted: (1) Rapid coagulation with complete agglomeration of colloidal clay with lower doses of alum. (2) More effective removal of suspended matter in the coagulation and subsidence basins, resulting in improved filter operation. (3) Less lime to maintain the desired hydrogen-ion concentration in the filtered water. (4) Slightly reduced cost of chemicals and greater over-all efficiency in operation of the plant.

It is probable that the treatment is not adapted t o all classes of waters, and the results obtained elsewhere may not in all cases be comparable with the conclusions stated above. K i t h waters similar to those a t Hyattsville the treatment will be of specific value.

Relation of &Gossypol to the Toxicity of Some Cottonseed Products’ Willis D. Gallup DEPARTMENT OF AORICULTURAL CHEMICAL RESEARCH, OKLAHOMA EXPERIMENT STATION, STILLWATBR, OKLA.

TTEMPTS to determine the toxicity of cottonseed meal by means of a chemical determination of its gossypol content have met with little success. A lack of knowledge of the substances concerned and to which the toxicity is due, together with much misleading information as to the relative toxicity of different cottonseed products, has hindered progress along this line. The results of more recent investigations which have been published from this department2J and elsewhere, as well as those obtained in this study, point to a new interpretation of some of the former results obtained with the use of cottonseed meal as a food. While attempting to devise a rapid and accurate chemical method for the determination of gossypol in cottonseed meal, the fact was revealed that the gossypol content as determined was not indicative of the toxicity of the meal. This might a t first appear contradictory to the results of Schwartze and .41sbergJ4 in which it was demonstrated that the toxicity of cotton seeds is due entirely to gossypol and is directly related to the amount of this substance present. That these investigators were cognizant of considerable variation between the gossypol content of cottonseed meal and the seeds from which it is produced is shown in their use of cottonseed kernels in their studies in preference to the meal “in order to eliminate any effect upon the toxicity of the seed resulting from the heat and pressure em-

A



Received July 27, 1927. Published with the permission of the Director of the Oklahoma Experiment Station, who originated a study of the factors influencing the gossypol content of cottonseed meal in this laboratory. 2 Gallup, J . Dairy Sci., 9, 359 (1926). * Gallup, I n d . En# Chem., 19, 726 (1927). J . Ag7. Research, 26, 173 (1924).

ployed in the process of expressing oil.” Biological tests were also found not to be entirely reliable, owing to individual variations among the animals, and especially between animals of different species. Instances of this are shown in the earlier studies on the feeding value of cottonseed meal, in which some investigators carried out their work with guinea pigs and rabbits, which are very susceptible to the toxic action of gossypol, while others drew their conclusions from the results obtained by feeding the meal to albino rats, which are capable of consuming relatively large quantities before showing any toxic symptoms. The determination of gossypol in cotton seeds as first suggested by Carruth‘ involves the extraction of the ground seeds with ether, which removes the oil and gossypol and is followred by the precipitation of the gossypol with aniline. Schwartze and Alsbergs were able to make the method more nearly quantitative by precipitating the gossypol from a petrolic ether-oil solution. In both cases the gossypol is first removed from the seeds by ether, in which it must be completely soluble if the method is to give quantitative results. Previous experimentation has shown that if the ground seeds are heated previous to the extraction a portion of the gossypol becomes insoluble and cannot be removed by the usual organic solvents. Since cottonseed meal is produced from the seeds after thorough heating, this condition seemed to be responsible for the unsuccessful attempts to correlate the toxicity of the meal with its soluble gossypol content. The extent of this change of gossypol from a soluble to an insoluble form will depend upon the length of time of heating, the temperature attained, and the amount of moisture 6

6

J . B i d . Chem., 32, 87 (1917). J . Agr. Research. 26, 285 (1923).

INDUSTRIAL AND ENGINEERING CHEMISTRY

60

Vol. 20, No. 1

present. As the gossypol is decreased by this heating there is a EXPERIMENTS USING COTTON SEEDS-TWO varieties decrease in the toxicity of the seeds, although not in the same of cotton seeds were used, one containing small amounts proportion, and the seeds remain toxic long after the sol- and t.he other relatively large amounts of gossypol, but uble gossypol has entirely disappeared, as shown by chem- neither showing the presence of d-gossypol. The cottonical determinations for this substance. Continued heating, seed meal used was of good quality, bright yellow meal especially in the presence of excess moisture, destroys the showing traces of gossypol and an average amount of dinsoluble gossypol and ultimately yields a product which gossypol quite comparable with the amounts found in other is free of both forms of gossypol and almost, if not entirely, meals examined in this laboratory. In order to convert deprived of any toxic properties. Proof of this is given the gossypol in the seeds to d-gossypol and note the dein a previous article.3 crease in the toxicity of the product, the seeds were ground The insoluble form of gossypol, which has been given the and heated in thin layers in an electric oven at 110” C. for name “d-gossypol,” may be a combination of gossypol, different lengths of time which varied from 10 minutes to with some other constituent in the seeds or a single com- 16 hours. Gossypol and d-gossypol determinations were pound formed by a rearrangement of the gossypol mole- made on the seeds before and after such treatment and cule through the agency of heat, or perhaps an oxidation some of the products so formed were fed in adequate diets to albino rats. (The change in the gossypol content of Note-The term “d-gossypol” is employed throughout this paper t o denote that form of gossypol which is practically insoluble in ether and these seeds under this treatment is given in a previous paper.3) formed at the expense of gossypol when the seeds are heated. There is some controversy a s t o t h e identity of d-gossypol and its existence as a compound separate from gossypol but the different solubility of the two substances has made it necessary to distinguish one from another. Carruthlo has suggested the name “d-gossypol” for the less soluble form.

Gossy p ol Determinations. These determinations were made in a manner similar to that’ suggested by Schwartze and Alsberg,e in which 50 grams of the ground seeds are e x t r a c t e d with anhydrous ether in a Soxhlet extraction apparatus for 24 hours. The extract is evaporated a t a low temperature and the residue taken up with about ten times or hydrolytic product. its volume of petrolic ether. S c h w a r t z e’ has indicated After standing overnight, the that the formation of this solution is filtered and 1 cc. substance may be due to a of aniline added, the flask being shaken until the aniline physico-chemical property gossypol which could n o t be removed f r o m t h e h e a t e d is completely dissolved. This of g o s s y p o l , “such as its is set aside for precipitation seeds by extraction w i t h ether. T h i s gave evidence of tendency to become bound to take place, which requires the toxicity of d-gossypol. or adherent to the meal.” about 7 days, and a t the end T h e investigation was continued w i t h t h e u s e of cotof this time the precipitate of I n the form in which it tonseed meal, which c o n t a i n s only traces of etheraniline and gossypol is colhas been isolated from the lected in a weighed Gooch soluble gossypol b u t relatively large a m o u n t s of dh e a t e d seeds it resembles crucible. The precipitate is gossypol. The m e a l proved to be toxic when fed in gossypol, and some investiw a s h e d several times with large amounts to a n i m a l s but, in s p i t e of its higher petrolic ether and the crucible gators& believe it to be a c o n t e n t of d-gossypol, was much less toxic than the and contents are dried for 4 single compound similar to, hours a t 105” C. and weighed. heated seeds. It is pointed o u t that the present chemibut not i d e n t i c a l w i t h , d-Gossypol Determinations. cal m e t h o d s f o r the d e t e r m i n a t i o n of gossypol a n d its T h e m e t h o d suggested by gossypol. That this form related c o m p o u n d s d o n o t suffice as a m e a s u r e of the Shenvoodg was followed, in of gossypol which occurs in toxicity of h e a t e d cottonseed products. w h i c h 50 grams of ground relatively large amounts in seeds, after thorough extraccottonseed meal is not toxic tion with ether, are digested with 100 cc. of hot aniline has been suggested by Sherwood,’ althoigh Carruthlo in an earlier investigation suggested for 5 minutes. The temperature is kept constant a t 110’ C. the mixture stirred constantly. The material is filtered that it might be the substance responsible for the toxicity of and through a Biichner funnel while still hot, using suction to recottonseed meal, rather than the soluble form which is found move the excess aniline, and when cool is thoroughly washed in the seeds. The results reported in this investigation are with ether. The ether is recovered from these washings by in harmony with the latter suggestion. Since a rapid chemi- distillation and the residue added to the main filtrate with the of a little ether. The aniline is then distilled from the cal method for determining the toxic substances in cotton- aid filtrate until about 10 cc. remain, which are washed into a small seed meal was desirable, it appeared necessary first to beaker with ether and set aside for precipitation of the aniline confirm previous indications of the toxicity of d-gossypol. d-gossypol compound. This precipitation also requires about 7 days and the precipitate is handled in the same way as described for gossypol. Calculation of the gossypol and d-gossypol Experimental Methods in these precipitates is made by multiplying the weight of the precipitate by the factor 0.74. ~

J . Oil b Fat Ind., 8, 173 (1926).

North Carolina Expt. Sta., 42nd Annual Report. J . Agr. Research, 82, 793 (1926). 10 J . A m . Chcm. Soc., 40, 647 (1918). 8 @

~~~~~~

~

The d a t a obtained in this investigation broadly indicate the extent to which gossypol a n d its related compounds, in particular d-gossypol, a r e responsible f o r the toxicity of certain cottonseed products. Gossypol was converted to a less soluble f o r m called d-gossypol by h e a t i n g c o t t o n seeds in an electric oven f o r several hours. T h e extent of this change was observed by chemical d e t e r m i n a t i o n s of the two comp o u n d s a n d the decrease in the toxicity of the seeds determined by feeding experiments. It was f o u n d that the toxicity decreased as the t i m e of h e a t i n g was increased a n d that the toxicity was d u e t o that f o r m of

Albino rats were used in this investigation, since they are less susceptible to “cottonseed injury” and can be fed cottonseed products for a greater length of time without fatal results than most experimental animals. In fact, their resistance to the effects of gossypol, and especially d-gossypol, is such that some investigators have stated that cottonseed meal is not toxic for them. 7

~~~

The growths made by rats on two of these products, together with the percentage of d-gossypol in the rations, are given in Group 1 of Table 111. These results give strong evidence of the toxicity of d-gossypol, since the heating process when continued for 1 hour completely converted the gossypol to d-gossypol, although the seeds remained toxic even after heating for 16 hours. Such may not always be true, as is shown later, and the length of time of heating necessary to bring this change to completion appears to depend somewhat upon the amount of gossy-

INDU8TRIA.L A N D ENGINEERING CHEMISTRY

January, 1928

61

pol originally present. Furthermore, the methods for deter- heated seeds. A direct comparison was made possible, mining these two forms of gossypol are subject to some error. therefore, between the extract and the residue of seeds Another lot of cotton seeds containing 0.423 per cent heated for 2 and 4 hours, the former containing soluble gossypol, which is considerably more than was originally gossypol and the latter the insoluble or d-gossypol. EXPERIMENTS USING COTTONSEED MEAL-FO~the purpresent in the first group of seeds, was ground and divided into three portions. One portion was completely extracted pose of extending the investigation with the use of a cotwith ether, which removed the gossypol and oil. The tonseed product more widely used as a feed than are the second portion was first heated as described above for 2 seeds, a sample of cottonseed meal containing 0.92 per cent hours and then extracted. The third portion was similarly d-gossypol was obtained. One port,ion of the meal was heated for 4 hours and only half of it extracted with ether. dampened and autoclaved as described in the experiment Another lot of the same seeds was autoclaved a t 20 pounds with the seeds and another portion was thoroughly extracted with ether and the oil so removed replaced with refined (1.4 kg.) steam pressure, as previously d e ~ c r i b e d ,which ~ process is effective in destroying the toxic properties of cottonseed oil. These products, after their gossypol and the seeds. The products were examined for gossypol and d-gossypol contents were determined, were incorporated in d-gossypol and fed to albino rats in the rations shown in the rations shown in Table 11. Table I. Table 11-Cottonseed Meal R a t i o n s Heating the seeds for 2 or even 4 hours did not com(Figures in per cent) pletely convert the gossypol to d-gossypol as was expected, Ration number 25 26 27 52 53 meal (traces of gossypol, which no doubt was due to the relatively large amount origi- Cottonseed 0.92% d-gossypol) 45 nally present. Although longer heating would accom- Extracted meal + refined cottonseed oil (0.92% d-gossypol) 45 plish this, it would also tend to destroy the d-gossypol, Autoclaved meal (0.547, d-gossypol) 45 Basic ration 90 90 which was undesirable, and 4 hours of heating was selected Ether extract of meal (traces of gossypol) 10 as the optimum length of time for producing the best yields Cottonseed oil refined 10 of d-gossypol in these particular seeds. Ether extraction Wheat 50 50 50 CaCOa 1 1 1 of the heated seeds was accomplished for the purpose of N aCi1 1 1 3 3 3 removing any soluble gossypol and thereby leaving a prod- Cod-liver oil uct containing only the d-gossypol. Since this meal showed only a trace of gossypol, it appeared desirable to determine if an ether extract of the meal which T a b l e I-Cottonseed R a t i o n s (Fiaures in per cent) would contain this small amount of gossypol could produce Ration number 33 35 36 38 39 40 41 42 the usual toxic symptoms in animals if incorporated in Cotton seeds (0.42% gossypol) 35 Cotton seeds, extracted 26.2 26.3 26.2 their diet in large quantities. As a control, a ration containCotton seeds, autoclaved for 2 ing the same amount of refined oil was to be used. The hours (0.218% d-gossypol) 35 Cotton seeds, heated 2 hours, ether extract was added to a growing ration in amounts extracted (0.341 yo d-gossypol) 26.2 Cotton seeds, heated 4 hours, equal to 10 per cent of the ration and in which amounts extracted (0.40% d-gossypol) 26.2 it provided over twice as much gossypol as could be present Cotton seeds heated 4 hours (0.09% gossypol, 0.30% d if added as cottonseed meal with the meal composing 45 35 gossYP01) Extract of 2-hour heated seeds per cent of the ration. If the toxicity of cottonseed meal (0.878% gossypol) 8.8 is due t o the ether-soluble gossypol alone, then such a ration Extract of 4-hour heated seeds 8.8 (0.37% gossypol) as this should be extremely toxic, provided this particular Refined cottonseed oil 8.8 8.8 8.8 sample of meal showed toxic properties. More elaborate Wheat 6 0 60 60 60 60 60 60 60 work of this kind dealing with the production of a nonh-aC1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 CaCO. toxic meal will be given in a subsequent paper. The above Cod-liver oil 3 3 3 3 3 3 3 3 rations are presented in Table I1 and the growth made by The results of this feeding work, together with the amounts of the two the animals is included in Group 3 of Table 111. forms of gossypol in the various rations, are tabulated in Group 2 of Table 111.

Discussion

The heated, unextracted seeds represented products containing both forms of gossypol, while the extracts of the heated seeds which were used in combination with unheated seeds freed of gossypol by extraction, represented gossypol in amounts much less than were found in the extract of unRATION GOSSYPOL NUMBER IN RATION Per cent 34 29 33 35 36 38 39 40 41 42 25 26 27 52 53 a

Table 111-Average G r o w t h of R a t s on Cottonseed P r o d u c t D i e t s d-GossuPoL NUMBER OF DURATION INITIAL FINAL IN RATION ANIMALSOB EIPERIMEXT WEIGHT WEIGHT GAIN LOSS Per cent Days Grams Grams Grams Grams GROUP 1-COTTON

... ...

O.OS3 0.040

0.148

...

... 0:077 0 : 032 0.032

Trace

I

... ...

0.076 0.090 0 : io4

o:io4 0.414 0.414 0.243

4 4

20 90

69 45 GROUP 2-COTTON

4 4 4 4 4 4 4 4

30 60 60 45 45 30 30 35

4 150 4 160 4 150 ... 4 40 T&eo ... 4 40 Two and one-half times as much gossypol as in ration

... ...

A study of the data presented in Table I11 reveals a rather astonishing situation as regards the growth of the animals on the rations containing different amounts of the two forms of gossypol. Group 1 shows the usual decrease of d-gossypol and de-

61 71 59 76 74 121 110 55 GROUP I-COTTONSEED 64 72 68 48 49 25.

SEEDS

60 85 SEEDS

48 205 177 66 89 104 172 51 MEAL 201 211 231 111 110

... 40

..9

...

13

134 118

...

15

...

62 ... 137 139 163 63 61

..

.. 10 .. 17 ..

DAILY GAIN Grams

...

0.4

...

2.2 1.9

4

... ... 2.0 ...

.. .. .. ..

0.9 0.9 1.1 1.6 1.5

..

0.3

LIVED

i 2 4 4 3 4 4

4 4

DIED

4 2

i

62

I N D U S T R I A L A N D ENGINEERING CHEMISTRY

struction of gossypol which takes place when ground cotton seeds low in gossypol are heated for a sufficient length of time. Along with this change there is a decrease in the toxicity of the seeds as shown by the growth of the animals. The seeds in ration 29 were heated sixteen times as long as those in ration 34. I n Group 2 the decline of the animals receiving ration 33 gives evidence of the toxicity of the gossypol in the raw seeds, while the excellent growth made by animals receiving the unheated, extracted seeds points to the complete removal of gossypol by ether extraction of the unheated seeds. These results are representative of a large number obtained in this laboratory with different varieties of cotton seeds. The destruction of this toxic substance by steam heat, as reported in previous papers, is again demonstrated in results obtained with ration 36, which was composed of autoclaved seeds, while its incomplete destruction and the formation of d-gossypol under conditions of dry heating for the same and longer periods of time are shown in rations 38 to 42 in Table I and supported by the growth of animals receiving these rations as presented in Group 2 of Table 111. That the insoluble gossypol which is formed by heating the ground seeds for 2 and 4 hours, and which cannot be removed by ether extraction, is toxic, is shown by the decline of the animals receiving these products in rations 38 and 40, the former containing 0,090 per cent and the latter 0.104 per cent d-gossypol. The amount of unchanged gossypol obtained from seeds by ether extraction after heating for 4 hours was much less than was present after 2 hours of heating, being so low in the former case that the animals receiving ration 41, in which the extract was incorporated, made good gains over a short feeding period. The growths made by animals in Group 3 receiving rations 25, 26, and 27, which were made up of cottonseed meal and contained the largest amount of d-gossypol of all the rations reported here, are in striking contrast to those reported immediately previous. The meal contained 0.92 per cent d-gossypol, which could not be removed by ether extraction or completely destroyed by autoclaving for 1 hour. There was present, therefore, in rations 25 and 26, about four times as much of this compound as in any of the cottonseed rations, and yet the animals whose diets were restricted to these cottonseed meal rations made the best growth. That their growth was still below normal is made apparent by comparison with the results obtained with the use of ration 27, containing the autoclaved meal and less d-gossypol. Furthermore, the general condition of these animals, the number of breeding failures, and high death rate among the young born, which are not included in these data, are indicative of a toxic factor which can be greatly reduced by autoclaving the meal. This statement is supported not only by the one feeding trial reported here, but by several others to be reported in their proper order. The point to be emphasized is that the insoluble gossypol found in cottonseed meal is much less toxic than the insoluble gossypol produced by simply heating the seeds, and the chemical determination for this compound does not, therefore, suffice as a measure of its toxicity. I n order to account for these differences in toxicity, reference was made to the studies reported by Carruth,l0 in which several forms of gossypol are given consideration. TWO of these are reported as being formed from gossypol by heatone called b-gossypol and produced along with considerable black resinous material on heating the isolated gossypol to 186-190" C.; and the other, d-gossypol, formed in the seeds during the cooking process which precedes the expression of the oil a t the oil mill. It would hardly seem that b-gossypol could be formed under the dry heating conditions reported here, since the

Vol. 20, No. 1

temperature was maintained around 110' C. a t all times and there was no indication of the formation of dark resinous material. The seeds did develop a yellow color characteristic of b-gossypol, which is more intensely yellow than gossypol but also characteristic of ground seeds which have been stored for some time and is the usual color of the commerical meal. Furthermore, almost quantitative yields of the insoluble gossypol have been obtained by heating the seeds as described which could not result if any large amount of resinous material was being formed by the conversion of gossypol to b-gossypol during the process. The final solution of the problem may be found in the fact that gossypol breaks down in the seeds a t a temperature of 100' C., but when isolated and subjected to test-tube reactions it withstands a temperature up to 186" C. It is quite possible that this compound as it exists in its original state in the seeds is quite different from that which has been isolated. That the isolated gossypol is toxic to the same degree as seeds containing similar amounts of this substance has been demonstrated.' With little evidence to support the view that b-gossypol is a factor determining the toxicity of the heated cotton seeds, attention was given to the amount of d-gossypol remaining in seeds which had been autoclaved for 2 hours and which had no harmful effects when fed to animals, as in ration 36. Ration 34, containing only slightly more d-gossypol produced by dry-heating the seeds for one hour, was extremely toxic and ration 29, containing little more than half as much, was also quite toxic. Evidently some factor other than the d-gossypol as determined by the present chemical methods must be taken into consideration in order to approximate the toxicity of these products. The method of preparation of the meal becomes more important in this respect than the determination of its d-gossypol content, and as yet sufficient data of this nature in an attempt to find a correlation between the toxicity of a cottonseed meal and its d-gossypol content as affected by oil-mill processes have not been obtained. That the presence of moisture is necessary for the destruction of the toxic properties of cotton seeds during the cooking process a t the oil mill seems to be an established fact. It is also well known that the condition of the seeds as regards their moisture content during this process will determine to some extent the amount of oil which can be later expressed from the seeds. For this reason the addition of water is often practiced when cooking very dry seeds, but the amount is small and apparently much less than is necessary for the destruction of the toxic principle. Very few samples of meal examined in this laboratory have contained unchanged gossypol in sufficient amounts to be of physiological importance. The d-gossypol has always been found in relatively large quantities, although its toxic properties have been decreased to a degree much less than can be accomplished by simply heating the seeds in a dry condition. I n view of these findings the manner of cooking the seeds assumes importance, not only in the production of oil, but in improving the quality of the meal. Conclusion The toxicity of cottonseed meal may be due not only to the presence of ether-soluble gossypol as it is found in cotton seeds, but also to the presence of what appears to be a decomposition product formed during the heating of the seeds previous to expressing the oil, and given the name d-gossypol. The toxicity of the meal studied was not reduced by removal of the small amount of ether-soluble gossypol which it contained, nor was this form of gossypol present in sufficient amounts to produce toxic symptoms in animals when the extract was fed in excessive quantities. When the meal is heated in the presence of moisture, as in autoclaving, it

January, 1928

INDUSTRIAL AND ENGINEERING CHEMISTRY

loses its toxic properties although it may still contain a small amount of d-gossypol. Although cotton seeds are extremely toxic, they also may be rendered non-toxic by autoclaving in a wet condition. By heating the seeds for a short time in a dry condition the gossypol becomes partially converted to the insoluble form and a separation made of the two by extraction with ether. The insoluble gossypol so produced is much more toxic than the insoluble form found in cottonseed meal or in

63

seeds which have been subjected to steam heat. The determination of d-gossypol by the present chemical methods is not a safe criterion for estimating the toxicity of cottonseed products. Acknowledgment The writer's thanks are due to V. G. Heller and N. B. Guerrant for their constant interest, and advice during the progress of this work.

Germicidal Efficiency of Sodium Hydroxide and Sodium Hydroxide-Carbonate Mixtures at the Same H-Ion Concentration'j2 Max Levine, E. E. Peterson, and J. H. Buchanan DEPARTMENTS OF CHEMISTRY A N D BACTERIOLOGY, Iowa STATECOLLEGE, AMES, IA.

I

T HAS been suggested that the H-ion concentration might be employed as an index of the germicidal efficiency of alkaline washing solutions. Observations with sodium hydroxide, sodium carbonate, and trisodium phosphate indicated very clearly that the H-ion concentration was not a dependable measure of the relative germicidal efficiencies of theqe alkalies.3 Thus a t p H 12.15 and 70" C:. the killing times for 99.9 per cent of about a million bacterial spores were approximately 5 and 36 minutes for the trisodium phosphate and sodium hydroxide, respectively. Many of the washing compounds consist of sodium hydroxide or mixtures of sodium hydroxide and carbonate. It was the purpose of the following observations t o ascertain whether sodium hydroxide and mixtures of the hydroxide and carbonate a t the same H-ion concentration were equally effective as germicides. The procedure employed was t o determine the surviving bacteria after various periods of exposure to the alkali solution and ascertain the time for effecting a reduction of 99.9 per cent. The organism employed was a spore-former of the Subtilis group. The details as to preparation of the test culture and technic of disinfection were identical with those previously described in studies on the effect of concentration of sodium hydroxide and temperature on disinfection.4 Experimental

At pH 13.20 a 0.48 N sodium hydroxide was only slightly more efficient than a normal alkali mixture consisting of 1.33 per cent sodium hydroxide plus 3.5 per cent sodium carbonate. The hydroxide effected a reduction of 99.9 per cent of the exposed bacteria in 40.9 minutes, as compared with 43.7 minutes for the alkali mixture. At p H 13.32 the sodium hydroxide solution (0.725 N ) was more effective as a germicide than the normal alkali mixture consisting of 2.0 per cent sodium hydroxide and 2.7 per cent sodium carbonate. The killing time for 99.9 per cent of the exposed bacteria a t 50" C. was 22.8 minutes for the hydroxide and 31.1 minutes for the mixture. At p H 13.40 and 50" C. the hydroxide (0.8425 N or 3.4 per cent) effected a reduction of 99.9 per cent in 15 minutes as compared with 18 minutes for the carbonate-hydroxide mixture (2.7 per cent NaOH 1.8 per cent NaEOa).

+

The H-ion concentration does not seem to be an accurate index of the relative germicidal efficiencies of solutions of sodium hydroxide as compared with hydroxide carbonate mixtures. SODIG11 HYDROXIDE AKD A COMMERCIAL -4LK.4Ll \vASHIIUG ComrPouxD-A widely used commercial washing compound consisting of a mixture of sodium hydroxide and carbonate JTas employed, the concentration being adjusted t o give a pH of 13.18 for the 50" and 60" C. series. This correqmnded to a 0.5 sodium hydroxide which gave pH 13.20. The concentration of the commercial mashing compound on titration using methyl orange indicator was equivalent to a normal alkali, and contained 1.99 per cent NaOH and 2.66 per cent SODIUM HYDROXIDE AXD SYXTHETIC HYDROXTDE-CARBOSATE MIXTURES-In this series of experiments normal alkali -Ua?C03on a weight basis. solutions were prepared by mixing various quantities of norFigures 3 and 4 show the results obtained a t 50" and mal sodium hydroxide and normal sodium carbonate. The BO" C., respectively. It is apparent that the commercial germicidal efficiencies of these mixtures a t 50" C. were then washing compound a t p H 13.18 was a better sterilizing agent compared with those of sodium hydroxide solutions of the than sodium hydroxide a t approximately the same reaction same H-ion concentration. (pH 13.20). Thus a t 50" C. the killing time of 99.9 per cent of the exposed bacteria was 34 minutes for the commercial T a b l e I-Composition of Alkali S o l u t i o n s SODIUMHYDROXIDE A N D CARBONATE compound as compared with 40.8 minutes for the hydroxide, SODIUMHYDROXIDE MIXTURES while a t 60" C. the killing times were 8.5 and 11.75 minutes, PH SOLUTION h'aOH NazCOa NaOH liaL!Os respectively. A: J '. _b-v z t . Parts Pavts c7, b..v w t . c7, b-v z t- 13.20 0.48 1.92 1 2 1.3 3.5 An analysis of the test materials shows that the commercial 13.32 0.725 2.90 1 1 2.0 2.7 compound in the concentration used contained practically as 13.40 0,843 3.40 2 1 2.7 1.8 much caustic alkali as the pure sodium hydroxide. The reThe composition of the various solutions is indicated in sults therefore indicate that the presence of the carbonate in Table I. The results are shown graphically j.n Figures 1 the commercial product served to increase the germicidal and 2. They may be summed up as follows: effect of the sodium hydroxide present, although the p H was 1 Received June 29, 1927. not appreciably affected. 2 These studies were made possible through a fellowship maintained by Another series of observations on the relative germicidal the American Bottlers of Carbonated Beverages at Iowa State College. efficiency of sodium hydroxide and the commercial washing a Levine, Peterson, and Buchanan, I n d . Eng. Chem., 19, 1338 (1927). 6 Iowa Sfate College J . Science, 1, No. 4, 379-394 (1927). compound described above was carried out a t 70" C. with I-

,I