The apparent persistence of about one tenth of the benzene rings of the 6&R seems somewhat anomalous for a pure compound. At first glance one would expect that if 90% of the molecules were degraded, the other 10%, having exactly the same structure, should also be degradable. Three possible explanations come to mind. First, the resistant 10% may originate in some impurity of the 6 R. This is not likely because the 66S behaved essentially the same as the 64R in this respect, even though the latter had undergone two further recrystallizations. If impurities are responsible they are remarkably tenacious. Second, the B-C medium is not a particularly rich one, and the bacteria may become dormant after a few weeks, without vitality enough to degrade the last of the intermediate. Or, third, the biodegradation of the 6-phenyl isomer may follow two different pathways (for example by initial attack on the No. 1 or No. 12 carbon of the chain), one of which may lead to a more resistant intermediate than the other. Or the true reason may lie elsewhere.

Attempts were made to develop further acclimation toward degradation of these residual UV materials by inoculation from older cultures. The degree of success could not be ascertained because of nitrite interference.

Interference by Nitrite

Attempts to develop greater activity of the cultures toward the more advanced biodegradation products involved reinoculation at 7 and 14 days from the parent 21- and 28-day cultures. Although such acclimation may have been developed thereby, it could not be detected because nitrite formation was promoted also and the intense absorption band of the nitrite ion centered at 210 mμ obscured everything in the region of interest. When the practice of reinoculation was discontinued the nitrite formation gradually abated and caused no more trouble after a few further transfers.

Setzkorn and Huddleston (1965) reported interference in their studies on biodegradation of benzene, toluene, and xylene sulfonates, characterized by development of strong absorption below about 240 mp in their control cultures. This was undoubtedly due to nitrite or nitrate formation. In the present work nitrite developed in the surfactant cultures as well as in the controls.

Cross Acclimation and Biodegradation Pathways

Earlier work has shown that a shake culture acclimated to C1, LAS can accomplish primary degradation of C11 LAS with no lag, but that a C11 LAS culture does require acclimation before it can handle C1 LAS (Swisher, 1966a). Such a nonreciprocal situation also exists with the present cultures, as demonstrated by cross-feeding experiments during the eleventh to fourteenth transfers on B-C medium. Results from the thirteenth are typical (Figure 5).

The curves at the left show the fate of 34S in both the 34S acclimated culture (top) and in the 64R culture (botton) which had never before seen 34S. The 64R culture accomplished the primary biodegradation in 3 days, just as readily as the 34S culture itself. Ring degradation by the 6pR culture was slightly slower, but nevertheless neared completion by 7 days, compared to 5 days for the 34S acclimated culture.

In contrast, the 34S culture was unable to accomplish ring degradation of 64R at all (right, top), and required 8 days for the primary biodegradation compared to 4 days for the 64R acclimated culture (right, bottom).

These results differ somewhat from those found in similar experiments with activated

FIG. 5. Primary biodegradation (MBAS) and benzene ring biodegradation (Aabsorbance) accomplished on 345 (left) and 64R (right) by 345 acclimated culture (top) and 6 R acclimated culture (bottom). Thirteenth transfers of cultures in B-C medium.

sludge (Swisher, 1967). There, neither sludge could degrade the rings of the other isomer upon first exposure, and acclimation required several days in each case.

The relations between primary and ring biodegradation, (i.e., between MBAS and ▲ absorbance) illustrated in Figure 5 are typical of those found throughout this work. The 3-phenyl isomer characteristically shows an interval of at least a day or two between the disappearance of methylene blue activity and the onset of ring degradation, even with a well-acclimated culture. With the 6-phenyl isomer in its acclimated culture, there appears to be no such delay; ring degradation begins at the same time as primary biodegradation, although it does lag considerably in the later stages.

There is an even greater contrast between the two isomers on comparing the total net absorbance instead of the A absorbance. As shown in Figure 4, the 3-phenyl typi cally undergoes a noticeable increase in the net absorbance during the first few days, whereas no such change occurs with the 6-phenyl. Actually the total absorbance of the 34S system does not increase beyond the initial value it had at day zero, measured against 0.01 molar KH,PO, as reference, but only the net absorbance measured against the corresponding control culture which is itself changing in absorbance.

These differences between the 34S and the 64R cultures, and between the two isomers during biodegradation, indicate that the chemical pathways involved may be quite different. Previous work (Swisher, 1963, 1964) has shown that primary biodegradation of LAS begins with the oxidation of a terminal carbon atoin of the side chain to a carboxyl, followed by B-oxidation of the chain, and furthermore that the B-oxidation occurs very quickly, since there is no accumulation of long chain carboxylates in the system. However, certain shorter chain carboxylates, still containing the benzene ring, do accumulate temporarily, for example y-sulfophenyl caproic acid from the 3-phenyl isomer and probably ẞ-sulfophenyl adipic acid from the 6-phenyl. Three

possibilities for the subsequent degradation of the 3-phenyl intermediate can be enumerated: a) continued oxidation on down the chain from the end already oxidized and then through the ring, b) attack from the other end of the chain and then through the ring, or c) direct attack on the ring itself.

If pathway b) were followed, the next step beyond the y-sulfophenyl caproic acid would be B-sulfophenyl adipic acid, the intermediate likely formed in the 6-phenyl degradation. If such is the case, the 34S culture should be able to degrade the 64R rings, which it is not. Thus, these present results suggest that the biodegradation of the 3-phenyl isomer does not go via pathway b). This is only tentative, because at the present stage of this work there is still insufficient basis for drawing any firm conclusions.

Deacclimation

It seems obvious that if acclimation of a culture to the biodegradation of a given substrate can be developed by exposure to that substrate, continued exposure under the same conditions should only improve the acclimation. This is not necessarily the case in actual practice.

Throughout the history of the 34S culture in B-C medium from the first to the 15th transfer the biodegradation pattern was substantially the same as in the 13th (Fig. 5, upper left). Primary biodegradation was complete within 3-4 days, ring degradation within 7. Starting with transfer 16, however, the onset of ring degradation was delayed to 7 days or more, and in some cases was not complete in 14, although primary degradation was still completed within 3-5 days. The parallel 64R culture underwent no such change in behavior, showing a pattern similar to Figure 5, lower right, throughout, except that completion of primary biodegradation did vary somewhat, from 4 days to 8 days..

This change in the capability of the 34S culture was not an actual loss of acclimation for ring degradation, but rather was a change in the speed. Perhaps this reflects some change in the bacterial species-population distribution whereby the ring-degrading species have become outnumbered by invaders who find the medium desirable for other reasons, but which do not or cannot degrade the detergent. Perhaps the use of sterilization and aseptic techniques in the maintenance and propagation of the cultures would have improved the stability; this is by no means certain in the case of a mixed culture, since population distribution will still undoubtedly drift unless all species present have exactly identical responses to the culture conditions.

The bacterial species predominating in the three B-C cultures, determined after 14 days growth of the 19th transfers, were those which might be anticipated under such circumstances: 34S culture, Alcaligenes faecalis, Pseudomonas sp; 6+R culture, Intermediate coliform, Pseudomonas sp; Pseudomonas sp., Bacillus sp. They were each present to the extent of several million/ml. There is, of course, no evidence as to which, if any, of these is responsible for the ring degradation, but there is likewise no evident reason why they could not all develop the ability.

CONCLUSIONS

The benzene rings of 3-phenyl- and 6-phenyldodecane sulfonate are biodegradable under the conditions of the shake culture test, after suitable provision for acclimation of the cultures.

Acclimation to 6-phenyl ring degradation includes acclimation to the 3-phenyl

isomer also, but not vice versa. This suggest that the biodegradation pathways may be significantly different for the two isomers.

ACKNOWLEDGMENTS

The shake cultures were maintained and transferred by F. M. Smith. Methylene blue analyses by F. M. Smith and R. G. Schwartz. Bacterial isolation and identification by Laboratory for Experimental Biology, St. Louis, Mo.

LITERATURE CITED

American Public Health Association. 1965. Standard Methods for the Examination of Water and Wastewater. 12th ed. APHA, New York.

Bunch, R. L., and C. W. Chambers. 1967. A biodegradability test for organic compounds. J. Water Pollution Control Fed. 39: 181-187.

Setzkorn, E. A., and R. L. Huddleston. 1965. Ultraviolet spectroscopic analysis for following the biodegradation of hydrotropes. J. Am. Oil Chemists Soc., 42: 1081-1084.

Soap and Detergent Association Subcommittee on Biodegradation Test Methods. 1965. A procedure and standards for the biodegradability of alkyl benzene sulfonate and linear alkylate sulfonate. J. Am. Oil Chemists Soc., 42: 986-993.

Swisher, R. D. 1963. Transient intermediates in the biodegradation of straight chain ABS. J. Water Pollution Control Fed., 35: 1557-1567.

Swisher, R. D. 1964. LAS: major development in detergents. Chem. Eng. Progr., 60 (12): 41-45.

Swisher, R. D. 1966a. Shake culture biodegradation of surfactants without inoculation. Develop. Ind. Microbiol., 7: 271-278.

Swisher, R. D. 1966b. Biodegradation of surfactant benzene rings. 36th International Congress on Industrial Chemistry, Brussels, September 1966.

Swisher, R. D. 1967. Biodegradation of LAS benzene rings in activated sludge. 22nd Annual Purdue Industrial Waste Conference, Lafayette, May 1967.

Swisher, R. D., J. T. O'Rourke, and H. D. Tomlinson. 1964. Fish bioassays of LAS and intermediate biodegradation products. J. Am. Oil Chemists Soc., 41: 746-752.

Weber, W. J., Jr., J. C. Morris, and W. Stumm. 1962. Determination of alkylbenzenesulfonates by ultraviolet spectroscopy. Anal. Chem., 34: 1844-1845.

AFTERNOON SESSION

Senator SPONG. The hearings will resume, Mr. Lee H. Bloom.

STATEMENT OF LEE H. BLOOM, VICE PRESIDENT, LEVER BROS. CO., NEW YORK

Senator SPONG. Mr. Bloom, we are pleased to have you with us. Mr. BLOOM. Thank you, Mr. Chairman. You have been very patient this morning; I will try to be brief.

Senator SPONG. Thank you.

Mr. BLOOM. You do have my extensive statement for the record? Senator SPONG. We will accept the statement in its entirety and you may be as brief as you wish.

Mr. BLOOM. Mr. Chairman, my name is Lee H. Bloom, administrative vice president and general counsel of Lever Bros. Co.

We certainly agree that the public is confused about detergents. In view of that, we think it particularly fortuitous that this hearing is well covered by TV and the press, because one of the things the public is very confused about is the point that eutrophication is not a problem in many important areas of the country such as New York City, for example.

We hope and feel that the press could do a great service to the public to help clear up confusion by fairly reporting that fact and we hope that they will do so.

Now that fact, in hearings here and in the House and at the Federal Trade Commission in the spring, has been brought out. The EPA has committed itself to identify those areas where eutrophication is a problem to determine what the controlling nutrient is and to take appropriate steps to deal with it.

We welcome that effort and support it. In the meantime, my company is doing these things. We have reduced approximately a year ago the phosphate level of all our powdered detergents to 8.7 percent which is the lowest practical level at which effective cleaning can occur. We find that when we drop below that level for normal detergents in normal use conditions, there is a severe drop at that point in cleaning effectiveness, and that is demonstrated in the graph in our booklet which Mr. Hurlbert showed you this morning.

We also are continuing our extensive research effort which is the largest single effort in our company, and as has been said by the other companies that appeared today, and I think it is our language that has been quoted by others as well, we have pursued every lead which seemed likely to be fruitful and continue to do so without any limitation of money or manpower devoted to that purpose.

We have a few promising compounds in our laboratory which, if they are to be fully tested to insure their safety to people, garments and machines, and the environment, must be quite awhile off before they can actually be adopted. We are hopeful they will be successful.

We support and believe that in the meantime, while EPA is finding its answers, and until substitutes which are safe and effective for people and the environment can be developed, an 8.7 level of phosphorous in detergents is a rational and responsible thing to do and we urge that a national standard be adopted for that purpose and that in so doing,

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