Humates and Other Natural Organic Substances in the Aquatic Environment
Cornelius Steelink
University of Arizona Tucson, 8 5 7 2 1
...
the bulk of the organic matter in surface waters is derived from natural sources, not from man-made byproducts ...Long before the current interest in trace pollutants, people were concerned with the color o f drinking water and with methods o f color removal. The presence of organic compounds in surface waters has received ct,nsidernble uuhlirity in the popular pew. In recent of water in rivers, years, public concernwith the streams, and lakes has stimulated renewed scientific interest in [he aquatic envinmnent. The citizenry has been exposed to numenus news accounts of toxic materials lurking in surface wnrerr. It is not surprising that the term .'dissolved organic marter" [DOMI wokei images of chiorinnred hydrocxhons, pulp mill effluent, steel mill phenolics, and other noxious substances. In reality, the bulk of the organic matter in surface waters is derived from natural sources, not from man-made byproducts. One can travel to remote Alaskan lakes or to pristine Alpine streams and still find dark-colored organic-laden water. Lone before the current interest in trace pollutants, people were concerned with the color of drinking water and 4 t h methods of color removal. The food and beverage industries have also wrestled with this natural phenomenon, since the quality of their products was dependent on the clarity of water. Natural leaching of soil and leaf litter in drainage basins accounts for color and dissolved organic material. What types of substances are these organics? Where do they come from? How do they interact with minerals, with aquatic organisms and plants, with municipal water treatment facilities? People who manage surface waters would like to know the answer to these questions. The engineer whose desalting membranes are c l o r r ~ dwith relatinous material would like to know. The chief of i i e muniGpal water works who finds chloroform in his drinking water would like to know. The fish and game warden who discovers massive fish kills in non-polluted waters would like to know. The purpose of this article is to provide some answers to these questions and to illustrate the dominant role of humic substances in the aquatic environment. In introducing the topic of the origins and properties of the
organic materials in surface waters, one is compelled to impose certain limits on definitions. Surface waters include rivers, streams, and lakes uhich are free of immediate upstream human disposal enterprises. Oceans are excluded. Natural sources are restricted to run-off from drainage basins of soil, grassland, bogs or forest litter, and lake sediments. certain secondarv sources of organic materials such as algae and aquatic plants will be mentioned, as well as a noteon mechanical pulping effluents. Agricultural fertilizers will not he discussed, although these compounds are leached into surface waters. A limit on concentrations will also be imposed; trace amounts (less than 10 ppb) of organics will be excluded, although the subiect of trace chemicals in surface waters is a fascinating one. Of course, the number of possible organic compounds is limitless; this paper will be confined to humic acids, fulvic acids, and organic acids, with a few remarks on some esoteric organics. Humic Substances
The humates are dark-colored materials which make up the major part of soil organic matter. These substances are subdivided into two general classes: humic acids and fulvic acids. The humic acid fraction is defined as that part of soil organic matter which is soluble in dilute base and insoluble in alcohol and acid. Fulvic acid is the water-soluble portion which remains in solution after neutralization and after all the humic acid has precipitated out. Both substances are operationally defined. A completely defined humate would include soil type, method of extraction, (1). and method of nreci~itation . . Humates are extracted from water by a number of methods. The most current method includes the followine .. stem: . (1) passing the water solution through a basic ion-exchange resin to remove 311 acids: 121 eluting the resin column with NaCl to put the acids into solutionl(3) passing the acid solution through a XAD-2 column to adsorb all phenolics, after acidifying to pH, 1; (4) eluting the XAD-2 column with water or weak base to remove fulvic acid and then with strong base to
Table 1. Elementary Composltlon of Humlc Aclds and Fulvic Acids ( 1) C (%)
H(%)
N(%)
S(%)
O(%)
5.5 5.8 5.2
4.1 3.2 2.3 1.6 1.9 3.6
1.1 0.4 0.4 0.6 0.4 ...
32.9 36.8 35.4 35.6 33.6 31.9
Table 2. Oxygen-Containlng Functional Groups In Humlc and Fulvic Aclds (meqlg) ( 1 )
Soil HA's
56.4 53.8 56.7 56.4 60.4 60.2
5.8
3.7 4.3
Soil FA'S
42.5 47.6 50.9
5.9 4.1 3.3
2.8 09 0.7
1.7 0.1 0.3
47.1 47.3
5.9
2.6
...
45.3
Water FA 46.2 a
Not determined.
44.8
Total Acidity
Carboxyl
Phenolic OH
Alcoholic OH
4.5 3.0 1.5 4.7 4.7
2.1 5.7 4.2 5.5 3.6
2.8 3.5
8.5 9.1 9.1
5.7 3.3 2.7
Carbonyl
Methaxyl
0.3 ...
0.2 ...
4.4 1.8 09 5.2 3.1
3.4 3.6 4.9
1.7 3.1 1.1
...
Soil HA'S
6.6 8.7 5.7 10.2 8.2
2.8
Soil FA'S
14.2 12.4 11.8
... ...
0.3
0.5 0.3
Not determined.
Volume 54, Number 10, October 1977 1 599
Humic and fulvic acids are dark-colored materials found in soil andsurface waters. They may nourish aquatic organisms or they may bephytotoxic, performingfunctions such as transport, concentration, and scavenging of metal ions, the regulation of growth and the formation of sedimen ts. remove humic acid. Fulvic acids can be precipitated from aaueous solutions bv the addition of Ba ion at DH. 4-5. Hoth soil materials are polymeric, polyelectrolytes of illdefined comoosition. The chief differences amear to exist in the carhoxyi content and the base exchange capacity (I). Other measurements indicate that fulvic acids have more aliphatic carhon than humic acids and fewer aromatic carbon atums than humic acids ('l'ahles I and 2). Molecular weight measuremenu are not precise and vary widely between sources, investigators, and methods. By gel filtration, the most common method, humic acids range from 5000-100,000; fulvic acids range from 300-2000. How do these properties for soil humates compare to surface water humates? Apparently, the two environments contain the same chemical s~ecies.Christman (2)r e ~ o r t e dthe same from soil and water humates. Analogous degradation ~ r u ~ e r t i e s aalso r e reoorted bv Weher and Wilson (3).Blounr i4)'demonstrated that most (90%) of the humic acids in Florida rivers are in the 1000-5000 molecular weight range; the remainder was in the range above 5000. When f;esh.waier solutions of humic acid meet ocean waters, the high molecular weight fraction of humic acid precipitates out first. From all this information, we know that humic substances are polycarhoxylic acids, with phenolic and alcoholic groups, carbonyl groups and aromatic rings. How does all this fit into a chemical structure? That is not so easv to formulate. Information on chemical structure comes m"ainly from degradative studies. In the last decade, Schnitzer and coworkers ( I ) have carried out extensive investigations in this field. They have used two methods: (1) a non-degradative fractionation of humic and fulvic acids, followed by analysis by glc-ms, and (2) Dermanganate oxidation followed by glc-ms. Their results hive led them to propose structures fo;h;mic acid and fulvic acids as shown in Figures 1and 2. These structures are consistent with all of the known properties of humic materials, such as: (1) metal chelation, (2) base exchange, (3) affinity for proteins, (4) adsorption of numerous materials, and (5) free radical content. The "openness" of the structure allows for the inclusion of organic materials, such as alkanes and substituted hydrocarbons. Some of these included compounds are liberated upon methylation of fulvic acid: i.e., after the strong hydrogen bonds between carhoxyl groups are removed. The inclusion of water-insoluble substances suggests that fulvic acid may contain hydrophobic cavities. This unusual property is also shown by certain proteins, crown ethers, and cyclodextrins. Very recently, it has been demonstrated for a water-soluble synthetic material (5). The "open" structure of fulvic acid proposed by Schnitzer (I)would accommodate such a cavity. Humic acids are stable organic free radicals whose spin content increases with increasing pH. This has been ascribed to quinhydrone structures 16)which could arise from air oxidation of para- or ortho-diphenols (Fig. 3). The number of such species in the humic acid macromolecule is low; however, a low concentration of quinhydrone can impart a large spin content to a molecular species. This, in turn, can impart a number of properties to the macromolecule, including catalysis of polymerization, inhibition of electron-transport metabolism, and the scavenging of halogen containing pesti-..... .
Once they are leached into surface waters, what properties nf these humates become significant? Color is one of the major 600 1 Journal of Chemical Education
Carbohydrates
Peptides
i
,'
I' k... Metals 8
...-..- - - -- - - - - - - - - - - - -. ->: ,
,a '
Phenolic Acids
Figure 1. Structure ot humlc acid.
,........,..,
C-OH
-.......
OH'.,
.... , , , , ,, ,
O 'H C -OH
0
OH
Figure 2. Structure of fulvic acid after Schnitzer (L).
Figure 3.Ouinhydrone-radical equillbrlum. contributions of humates to surface waters. This suhject was well-reviewed by the Committee on Water Problems (7)in 1966 (see ref. ( 2 )also). Absorbance in the visible spectrum (465 nm) is a standard spectroscopic parameter for quantitative analysis of humates. Humates also fluoresce ( I ) under visible light irradiation. Fluorescence is associated with lower molecular weight fractions ( 8 ) .Christman ( 2 )reported that the color-producing humates are in the 10,000-50,000 molecular weight range: fulvic acid contributes 15-25% of the color and humic acid contrihutes 7545%. There is evidence that the iron-humate complexes are the real color producers. The suhject is complex. Since a large amount of ferric oxide is
+ Hf + 0 , + Humic Acid AulHumatel'+ + Humic Acidr Au
-
(reducedl
+
~ u l H u m a t e ) ~ +H,O +
A""-colloid t Humic Acidr loxidizedl
+ H+ Au' + Humater-H+ transport of gold.
A""-colloid Figure 4. Mechanism for
-
hound into a colloid by organic mitter, particle size is critical to color. Lamar (9) claims that 50% of the color is due to iron-humates of paiticle size less than 0.01 p, and that most of the total iron in solution does not contribute to the dark color. This leads us to one of the most fascinating aspects of humic acid chemistry: the ability to chelate or coordinate metal ions. Humates can absorb 1-17% of their own weight of metal ion (10). In fact, my own curiosity in this subject was aroused by a personal experience. Some years ago, I received a handscrawled letter from an old Arizona placer miner. He had read about my work with humic acid in a local journal and wrote that he had heard renorts of secondarv enrichment of sedimentary gold depos;ts. These depoiits could be due to chemical solution of cold and subseauent denosition of the mrtal ~lownstreamfrom the original source. 'l'here were some earl\. references 1 1 l a . h . con ~ thissubierr. Recenrlv,One . - and ~wa"nson(12) confirmed part of t i i s hypothesis. Organic compounds in the 3-30 ppm concentration range in natural waters have the capacity to reduce AuCla to negatively charged colloidal gold, which is stable for up to eight months. The gold is precipitated from the colloids by contact with acidic or brackish waters (Fig. 4). Of course, there are less esoteric metal-humate compounds which commonly exist in waters. Schnitzer found the stability constants for metal-humates to decrease in the following order (Table 3). Fulvic acids formed tighter metal bonds. Interestindv. u - , metal-fulvic acid-nhosnhate . . comnlexes are orevalent: in fact, most of the phosphate in water may be in this form. Geochemical enrichment of trace heavy metals occurs with humic acids. Peat humic acids have enrichment factors over supernatant waters of 104:l. (Table 4). There are a number of reported physiological properties of these metal-humate comulexes. The most often mentioned are the root stimulating properties (13) of economic plants, Table 3. Order ot Stability of Metal-Humates ( 1 ) Pbzi
Most Stable
cu2+ NiZf
which mav be due to the transport of chelated iron. Humic materials also exhibir pron~~unred effect. on the senziti\,ity of . vhvtonlankron akoe 114, towards trare m e t a l as w t 4 a i . . promoting algal growth (7). One of the most fascinating studies of the effects of chelated iron on phytoplankton was recently reported by D. F. Martin and coworkers (15a,b).Catastrophic fish kills were observed as a result of an outbreak of Florida red tide organism (Gymnodiurn breve, Dauis). Chelated iron is known to promote the erowth of G. breve. This event occurred after a heaw rainfall in which humic acids were washed into the streams feedine into Florida waters. Positive correlations were obtained for the high concentration of humates, tannins, and iron. Another internretation was offered: the organic materials could have w s v e n ~ e dphyrotouic ('u" ~whirhizmarc firmly bound than iron) which would also v r u m o t ~the growth ol C; breve. Another property common to both tannins and humates is a pronounced tendency to adsorb proteins. Other materials which are adsorbed are hydrous metal oxides, certain insecticides, herbicides, Nylon, and alkanes. Both hydrogen bonds and ionic bonds seem to be involved in the bonding. For example, Schnitzer ( I ) has shown that atrazine and other triazines are adsorbed to the extent of 1 pmolelg by humic acids. but not bv fulvic acids. (Fie. 5) Even more impressive is the ability bf fulvic acids to>dsorh, or include, nlasticizers. At DH2.35.950 e of fnlvic acid will comulex with (16). Thus, fuivic acids 1560 g of hi~-(2:eth~lhe~~l)&thalate could onerate as concentrators of these trace toxic materials in sediments, or as transporters of them to other environments precipitate them. Sodium in which DH or ionic strength would . . humatesact as surface active agents, increasing the solubility of DDT twenty-fold. Like most polycarboxylic acids, humic and fulvic acid precipitate in the presence of calcium or magnesium ions. Ion-exchange capacities are in the range 400-600 meqlg. Surface waters which are high in these metallic ions will undergo removal of the organic acids: much of the humate which is transported into high calcium regions will end up as sediment (17). Humic and fulvic acids will also nrecinitate . . out as iron or al'uminum complexes, if the ratio of metal:fulvic acid is hieher than unitv (18). Lower ratios than unitv result in soluble complexes."lt'should be noted that one OF the most common methods of removine color from water is to treat the water with iron or aluminumsalts, especially in the presence of calcium (19). A recent renort claims that humic acids are photosensitizers for the degradation of phenolics in water (20). ~~~~~~
~
Organic Acids
C"2+
The normal range of humic plus fulvic acid concentrations in surface waters is 1-5 ppm, although some reports find more than 10 ppm. Organic acids, on the other hand, constitute
Mnzt MgZ+
Least Stable
Table 4. Approximate G.E.F. (Geochemical Enrichment Factor) Values tor Some~M~alllc Elemenls ( 1 ) in Peat10"
Form
uo:' Fe3+ Fez+
G.E.F.
pH
I X lo4 2.65 X lo" 9.1 X 10'
5.0 4-4.5 4-4.5
HUMlIC ACID Figure 5.
Structure of atrazine-humic acid complex. Volume 54, Number 10, October 1977 I 601
Figure 6. Structure of galtotannins and condensed tannins,
much less than these amounts (21 ).The most common acids in rivers and lakes are butyric, valeric, pyruvic, lactic, succinic, adipic. oxal~c,malonic, citric, tartaric, and malic; these constitllte less than 2 nom . .I. . . in lakes 1'. 221. and 0.5 DDm .. in rivers (21 hlany of these acids are nwmal m~taholitpsor metabolic intermedintri of suil, plant, and ttacterial species. .Most of then1 are xmd chelatnrs of transition metal ious. L'ronic acids constitute 1-3bofsoil oraanic - matter and can h c e x ~ e c t e dto he in surface waters (6). Tannins Tannins sometimes are found in hieh concentration in lakes A S ~result I of leaching of hark and i d i i t t e r . Martin (21)found ;1.I . DDm . In a Florida lakv. The tannins add color Lo waters and possess adsorption properties akin to humates. Leaf extracts contain a laree auantitv of tannins and tannin Drecursors. When leaf litter is abundant, one can expect to f h d tannins in lakes (8).Tannic acids chelate ferrous ion so well. that the reduced iron-tannin complex is stable to oxidation (1'0). It has been shown ( 1 0 ) that not onlv will tannicacid stabilize ferrous ion, but that it will reduce firric ion a t pH values below 3. At higher p H values, tannic acid must compete with OHfor Fez+. Thus, a t p H above 3, one would expect to find soluble ferrous-tannin compounds plus insoluble hydrous iron oxide. The structure of hydrolyzable tannins (the most abundant in hardwoods) reveals its reducing characters (Fig. 6). In the presence of oxygen in aqueous solution it is oxidized to quinones and polymers. Bacteria may also attack the catechol portions of the molecule, yielding dicarboxycylic acids. However. tannins are normallvstable to microbioloeical attack because of their ability to inactivate enzymes a d proteins. They can effectively inhibit the metabolism of microorganisms of such choice substrates as carbohydrates (24). I t would be interesting to see if tannin-rich lakes and streams do have different levels of microfloral activity than tannin-deficient natural waters. Other Orsanics There has been considerable interest in the excretions of aauatic plants and algae. There is some evidence that aleae excrete 'high molecu'lar weight (>50,000) mateiials (25); however, most of the metabolites are monomeric compounds. The detection of these low molecular weight c o m p o k d s in natural waters mav be difficult, since their half-lives in solution may be short d u e to their rapid metabolism by bacteria or other aleae. Laboratory studies of isolated aauatic plants and phytoplankton revealan active excretion p&ss. ~ e t z e l 602 1 Journal of Chemical Education
(26) has shown that aauatic aneios~ermsexcrete oreanics which arc utilized hy aliae and h&e;in and also preci&ated by calcium and maenesium iuns. Staha (271has anahzed the constituents of numerous aquatic plants and found tannins, flavonoids, alkaloids, saponins, and phytosterols. The amounts excreted into thk aquatic environment were not determined. Murphy et al. (28) have shown that hlue-green algae in a eutrophic bay are able to dominate other algal species by excreting a phytotoxic material. This substance, a siderochrome (that is, an iron-complexiug compound synthesized by an organism to overcome an iron deficiency) is a hvdroxamic acid of mol wt = 1000. which chelates iron. Jacob (i9)has isolated a dihydroxyanthraquinone excretion product from Nostoc muscorum. a fresh-water aha. which inhibits growth of several algal species. This material is a powerful metal chelator. A host oi trace urganic compounds (in the pph rangel exist in natural waters. \ f a t i \ . ~ fthem are the natural metabolirf:~ of bacterial processes, such as dicarboxylic acids, aminoacids, fatty acids, sugars, etc. Sophisticated instrumentation (30) has been applied to their identification, as a result of increased interest in trace pollutants. However, reports of these compounds are too scattered and varied to allow a coherent patt e r n to be presented. A number of resin acids common to softwood species have been found to be toxic to juvenile trout in Canadian rivers (31a). However, these substances were found in rivers downstream from a mechanical )non-chemical) pulping mill (316). Is this a "natural source"" How much resin acid would one expect from "natural" leaching processes? No one knows. Very Recent Studles Within the past year, reports that chloroform exists in chlorinated drinking waters have received wide publicity. Epidemiological studies have correlated the presence of chloroform with high incidence of cancer of the bladder (320). Rook (326) and others claim that chloroform and other haloforms arise from the chlorination of humic acids. Confirmation by direct experimental evidence will be required to substantiate these claims. Other possible substrates for chloroform-formers could be tannins and certain carbohydrates. Another fascinatine asnect of water-borne humates has been reported. ~umic-acidappears to he photosensitizer for the degradation of phenolic compounds (20). If such a phenomenon exists in surface waters, it would constitute a natural process for the detoxification of phenolic effluents. Finally, humic materials appear to survive municipal sewace treatment (33) and to exist in effluents. If this claim is sibstantiated, then some of the proposed industrial uses of reclaimed sewage could be seriously affected. Summary We have shown that natural surface waters contain a variety of organic compounds. Most abundant are the humic and fulvic acids, which come from the soil. In turn, the soil generates these compounds by microbiological degradation of plant litter and plant extracts. Other organic compounds which are ubiquitous metabolites of microorganisms and plants also find their way into natural waters. Being lowmolecular weight substances and less resistant to microbioloeical attack. thev do not maintain their inteeritv .. . or conrentration as much ah the humbfes, fulvates, and tannins. Despite the f x t that they are auite resistant 10 chemical and biological destruction,-humates and fulvates perform many functions. These include metal transport. . . metal concentmtion, metal scavenging, growth regulation, and sediment formation. They may nourish aquatic organisms or may be phytotoxic. They are very sensitive to p H or ionic strength. For man's purposes, they may not be so welcome. They do add color to drinking water, foul ion-exchange resins and mem-
hranes, peptize salts, transport and concentrate inorganic and organic pollutants, and produce chloroform. Other than the macromolecular s~ecies( h u m a t e s . fulvates. tannins), which of the organic compounds are natural, rather than man-made? Given the high population density around waterways, maybe that question is impossible to answer. On the other hand, if one assumes that the hulk of man's synthetic organic compounds come from petroleum products, then a technique does exist, to answer the auestion of oriain. The C-14 le;el in fossil fuels is very low compared to recent or actively metabolizing com~ounds.By determinina the C-14 I e d s of i n r i i v i d ~ l n lorganic nmpounrli in uaters, une may identih a suhitance as 'man-made" or "nnturnl" I:~?I.
Future Studies
There remain many unanswered questions about the structures, role, and function of humates in the aquatic environment. fruitful fields of investigation await the i n t r e ~ i dscientist. No systematic structural elucidation has heen-carried out on water-borne humates. Are they indeed identical to soil humates? Do they contain prosthetic groups such as carbohydrates, proteins, and phenolic acids? Do their structures depend only on their ultimate source in the soil, or do the" 1 1 n d r & nlteiation in the w a t t r environment? W h a t chemical moiety w i t h i n thr itrurture of humates gives rise t o chloroform? Do humates contain hvdroohohic cavities: if so. - . what is the size of these cavities? Certainlv. the advent of s ~ e c i aresins l will make the collection andi$olation of waterIhorne humates much easier and much less destructive. The availability of modern degradative and analytical methods should accelerate the speed and certainty of structural studies. Once we have a better idea of the chemical structures of these humates, we can make predictions on their role and function, especially on their response to man-made chemical insult to the aquatic environment.
any
~
~~
Literature Cited 111 Schnitrer. M., and Khan. S. U.. "Humir Substances in the Environment." Marcel Dekkor. Inc, NevYurk, 1972. 121 Chrirtman, R..Symposium Org. Matter af Natural Watels, 1968. Published 1970. p.
, "Sail Biahemiatry:' (Editors: M ~ LA. D., ~ and ~ Pe, 161 S t d i n k , C , and ~ ~ l l i . G.. lerron,G. H.1, MarcelDekker, Inc.,NewYork, 1967.Chap.6. .!. .Amsr 69 . .. ... . WnrrrWnrbn .. . ... .. .. ....Ae. ... ., . ., + R. , ln77(19f71 ... .,....,. I81 Hall, K. J., and h e , G. F., Water Re,worch. 8,239 119741. 191 Lamar. W.. U S Geological Survey Professional Papers #6WD. 1024 119681. (LO) Theu.T. L.,and Singer, P.C.."Tme Metalsand M e ~ - o r g a n i e I n ~ m e l i o " ~Natural in Waters." [Editor Sineer.P. C.!. Ann A,hm Science Publishers. Ann Arbor. Mich171 ,. ,
119691. 113) Schnilzer, M.,sndPoapst,P.A.,Nolure. 213,598 119671. 1141 B d a t e . R. J.. "PsthwsvsofTrseeElemenfs in Arctic Emswtems." Promesa . . - Rooort . t o ~ E d . 2 0 - 3 6119721.. 1151 la1 Kim, Y.S..andMartin.D. F.. WaWRssmrch, 8,607 11974):1b)Manin,D.F.,Doig, M. T., 111, and Pieme. R. H., Jr.. ACS Division of Water, Air, Waste Chemirtry. General Paper. 9. 124 119691. 1161 Omer.C.,and Schnitzer.M..Sciencp. 170.317 119701. 1171 W e t 4 R.G.,Curners.H., and OUuki,A..Arehiu. Hydmbiolo~iea,73.31 119741. 1181 Schnitmr, M., "Organic Compounds in Lhe Aquatic Environment..l (Editors: Faust and Hunler1,MsrcelDekker. New York, 1971.Chap. 13. (19) Chrirlman, R. F..and Minoar.R. A.,Raf (181. Chap. 6. 1201 Maboy, W.. Stanford R e m r c h Institute, private communication. I211 Lamar, W.,snd G w r h h , D. F.,ACS Division of Water. Waste Chemistry, Preprinfs # 4 , I41 11964). 1221 Khomenku. A. N., and Goneharova, I. A,. Cidrokhimich~diyeMol~rioly.55, 32 11971). 123) Martin,D. F.,Virtor,D. M.,and Dwrir,P.M., WoterRer~orch.10.65 119761, 1241 Grant. W. D..Seimea, 193.1137 119761. (251 Gjerring, E . . a n d L e , C . F.,Enuiron. Sci. Tech., 1.681 119671. 1261 Walnel, R. G.,Aiosri~ncs.19,539 119691. 1271 Su. K. U.. Staba, E. J., and Abul-Hajj H.. Lloydio. 36.72 119731 and previous pa-
"-"..".
1281 Murphy,T. R.,hsn D. R. %and Nalewajko,C.,Seienca. 192.900(19761. 1291 Jacob. J.. These #448L Faculte Science. Univ. of Paris, 1961. 18111 Pitt, W. W., Jr., Jolley. R. L., and Lott, C. D.. Enuiron. S r i Terhnol., 9, 1068 il9,Sl. 1111 (alGach, J. M.,andThakore.A.N..TAPPI,59.l29119751:1b~Keith.L.H..Enuiron. Sci Teehnol., 10,555 119761. 1121 la1 Page, T., Harris, R. H.. and Epatein, S. E., Scimcr, 193. 55 119761. lbl Rook, J., "Drinbiunterleidzng Rotterdam." P.O. Box 1166. Rotterdam. The Nethcrlsndr. 183) Rebhun. M..and Manks. J..Enuron. Sei. a n d Teeh.. 5,W5 119711. 1:141 Spikor, E. C.,snd Ruhin.M..Sciencr. 187.61 11971).
Volume
54. Number 10. October 1977 1 603
