Photooxidation Products of a Fuel Oil and Their Antimicrobial Activity Richard A. Larson”’, Thomas L. Bott, Laura L. Hunt, and Kurt Rogenmuser Stroud Water Research Center of the Academy of Natural Sciences of Philadelphia, R.D. # l . Box 512, Avondale, Pa. 19311 H Photooxidation products of a no. 2 fuel oil were fraction-
ated and examined by gas chromatography-mass spectrometry. Among the compound types identified were hydroperoxides (alkylated derivatives of tetralin hydroperoxide), phenolic compounds (highly alkylated phenols and tetrahydronaphthols), and carboxylic acids (substituted benzoic and naphthoic acids). Oil fractions and pure compounds characteristic of the photooxidation products were tested for toxicity to a yeast and several filamentous algae. ‘The species tested varied in their tolerance toward the photoproducts, but hydroperoxides generally showed the highest toxicity: tetralin hydroperoxide, a t 4 X lo-,-’ M. significantly reduced yeast growth. Carboxylic acids had less activity and phenolics were still less active. Tetralin hydroperoxide, a t concentrations greater than 8 X l W 5 M. reduced photosynthetic carbon fixation by Vaucht’ria and increased excretion of fixed car hon. It has been suggested that aging or weathering of petroleum surface films might lessen their toxicity to aquatic organisms because of the evaporation of toxic, low- boiling hydrocarbons (1) or transformation of hydrocarbons into oxygenated derivatives easily leached from slicks (2). However, thermal treatment or long-wavelength ultra\Violet (UV) irradiation of some petroleum products affords water-soluble fractions with greatly increased toxicity to bacteria (Y), algae ( 4 ) , marine invertebrates, and fish ( 5 ) .A preliminary study of the photooxidation of a no. 2 fuel oil showed that hydroperoxides, carbonyl compounds, phenols, and acids were produced ( 6 ) . Some photoproducts (hydroperoxides in particular) displayed considerable toxicity to a yeast. Succhciromyces cerecisiae. We report additional work on the molecular identification and antimicrobial activity of some constituents of the acidic fraction of the no. 2 fuel oil photoproducts. This fraction includes hydroperoxides (very weak acids having pK, = 12), phenols (weak acids, pK, = lo),and carboxylic acids (pK;, 3.51.
MatcJrials arid Methods Separation of Acidic Fractions. T h e characteristics of the no. 2 fuel oil and the conditions for its irradiation with a Pyrex-filtered mercury arc were reported previously ( 6 ) .Irradiated oil (25 mL) was extracted three times with twice its volume of 1 M KOH containing 6% NaCi. T h e combined aqueous layers were washed with hexane, brought to p H 11.0, and reextracted with diethyl ether t o afford a n ether-soluble “very weak acid fraction”. The p H of the residual water layer was adjusted successively to 7.0 and 4.0 and extracted similarly a t each p H to afford a “weak acid” and a “strong acid” fraction, respectively. T h e concentrated ether extracts were separated by preparative TLC (the very weak acid fraction was first treated with thiacyclopentane ( 7 ) to convert hydroperoxides to alcohols) and the constituents of the principal bands were examined by GC-MS using a double-focusing instrument (Shrader Laboratories, Detroit, Mich.). Preparation of Water Extracts. T h e no. 2 fuel oil was layered on deionized water in a 1:40 oil-water ratio. T h e oil
I
Present address. Institute l o r Environmental Studies, University Urbana, Ill. 61801.
of Illinois,
was extracted by the method of Hoylan and Tripp ( 8 )for 96 h, and exposed t o Pyres-l’iltrredrnercurv arc light or sunlight (irradiated extracts: sunlight ivas used exclusively for extracts used in algal studies) or in !‘oil-covered vessels (dark extracts). Extracts were membrane t’iltcwd (0.45jim pore size, Millipore Corp., Redford, Mass.) 1)ct’oreuse in algal studies. Nutrients were added to provide coiicentrations present in Cladophora medium 2 (9).The perositle content of the oil and water layers was determined iotlomet r i w l l y . Determination of Hydrocarbons and Phenols Extractable by Water. Dttpliccite 5(K)-niI,water samples were extracted twice with 1r11111,O ~ ’ L ‘ C ’ I , ~‘I‘he . CCI.1 was dried with MgSOl and concentrittioris were determined l’roni infrared absorbances a t 2930 (aliphatics),:3040 (aromatics),and 3640 cni-1 (phenolics) usin; ~ ~ - c nwlls. i I)ilutions of’ Nigerian Crude, No. 2 , or crankciist~oil.; in ( T I , were used for standard curves. Oils were sup~~ltinetited Lvith known quantities of benzene and toluene t o piteprc, st;iiidard curves for aromatic hydrocarbons. Antimicrobial Activity: Yeast. Yeast growth was determined turt)idinietric,ally :is previously described (6). T h e conipo~nidsto he tested ~ s \ ~adtied ~ i ~ tt o ~ the cultures in aqueous or ethereal soluti~ii.S i x rt.plic;rtti flasks were iiicubated with each concentration 01’ dl ~ ~ ~ ~ n ~ p exiimined. ~ i i t i d s T h e 48-h culturrs were compared optical densities of’the t~spcritnt~iital with those of six coiitrolk (run at the m i l e time) and tested for statistical significance u 3 i i - i Studelit‘s ~ t test. Controls always grew well. with a d O U ~ J ~ ~tlitne ’ I $ of’ca. ( j h. Most of the indi\~idualo j n i l m u i i c l s ivtare obtained commercially and recry5t;tlliztd or tedistilled. ‘I’etralin hydroperoxid{>was synthtsii/cd ti\. air osidiition of tetralin and recrystallimd from toluene . i t -7;) ’(.’~ (’uiiiene and tcrt-butyl hydroperoxides were ptirititd t)! precipitating their sodium salts at 0 “C, washing the > < i l t h with cold toluene, carefully acidifying to pH 5, m d k.xt r*irtitig t h e purified hydroperoxides into diethyl ether. Algal Cultures. l ’ ( i i i 1 / : ( I , . \ l i c . r o c * o l t s l i a , and Spirogyra species collected f’roin Ii’hitt C’hy reek. C‘hestrr Co., Pa., were maintained i r i c.iiltiiro> 1 h ; r t \vert’ unialgal with respect to filamentous gentIra, .ilthough diatom epiphytes were present. Stigr’oc,loiiiiciii a i i d 1 ‘/‘,,thrir spec,ieh \vere ohtained commercially and intiint,iined uninlgnlly. Antialgal Activity of Weak arid Very Weak Acid Fractions and Pure Compounds. Plates of (‘icidophora medium 2 (inorganic, s;alt. with added vitamins) (9) with added prepurif’ied agar ( 1.5 g i l 0 0 m1,: Halt itnore Hiological, Baltimore, Md.) were poured. Froin u.1 t o 1.0 nil, of the oil fractions (dissolved i n et her) \vas added to depressions made in the agar and the ether \viis t-vaporxted. Kther alone served as a control. A t u f t o f ’ d g i i e (15-25 ing \vet weight) was inoculated in the deprt.. +.ion. ’ Pure compounds wtart’ d i s s d v t d i n ether to provide concentrations ranging f r o m (iX 10. ,-’ to 9 X M.Antibiotic sensitivity disks (1.27 cni diameter) were impregnated with 100-jiL volurnes and t h e tit her was evaporated. Ether alone was the control. ‘I’he disk5 were pliiced on algal lawns developed on C’ludophorci 2 nit~tliuniand the plates were sealed with tape. All plates were incut)ated for 5 days with illumination (“Vita Lites” I h i r c i Test Corp., North Hergen, N.J., 12-h photoperiod) at :%O“C‘. \\‘htw disks were used, plates were illuminated from t)eneath Algae were examined for evidence of growth, cellular disrupt ion, ; ~ n dchlorosis. Effects of Hydroperoxides and Water Extracts on
0013-936X/79/0913-0965$01 .OO/O @ 1979 American Chemical Society
Volume 13.Number 8. August 1979
965
150 164
C10H140 CllH160
178
C12H180
possible s ructures
MW
+
160
16
Ion composition
(CH3)4_6
Ion composition
possible structures
174 188
178
162
‘llH14’
176
‘12%’
190
c13H1@
204
14??0°
196
C14H120
), ob +
192
‘12 96O2
(CH3)1-4
1
or
+ (CH3)2-5 n
Figure 2. GC-MS analysis of methyl esters of strong acid fraction of photooxidized fuel oil
1,2,3,4-tetrahydronaphthol, although many features of the spectra were similar (12, 13). Figure 1. GC-MS analysis of weak acid fraction of photooxidized fuel oil. Ion composition determined by high-resolution MS
Algal Photosynthesis. Vaucheria was cleaned superficially by agitation in water. Samples (approximately 40 mg wet weight) were spread out in petri dishes containing 35 mL of culture medium (controls) to which tetralin hydroperoxide was added or oil extracts with added nutrients (experimental treatments). For each treatment, three to five replicates were incubated in the light and two in the dark. Isotope (NaH14C03, sp act. 8.4 mCi mM-l) was added to provide a final concentration of 1 pCi in 35 mL. Samples for dark incubation were covered immediately with aluminum foil. After 2-h incubation a t ambient water temperature under “Vita Lites” providing an intensity of 42 pEinsteins m-2 s-l, incorporation was terminated with 1 mL of 37% CH20. Medium (10 mL) from each light incubation was membrane filtered (0.45 pm pore size) a t 0.5 atm, acidified to p H 3.0, bubbled for 30 min to drive off unincorporated inorganic 14C, and neutralized. The 14Cremaining (organic material excreted by the algae) was determined by liquid scintillation counting. The algae were recovered by centrifugation a t 11400g for 10 min a t 4 “C, air dried, and exposed to HC1 fumes to remove absorbed 14C.Incorporated radioactivity was determined by liquid scintillation counting after combustion in a sample oxidizer. Data reported have been corrected for counting efficieqcies, dark uptake, and excreted 14C. From 20 to 30 additional replicates (approximately 40 mg) were extracted in 7.0 mL of acetone overnight a t 4 “C and centrifuged a t 4 “C, and the chlorophyll a content was determined, with correction for phaeophytin ( I O ) .
OH
I 4
1
Further work must be performed to characterize the alcohol series fully; it is possible that each of the alcohols derived from the oxidized oil may be a mixture of closely related isomers, inseparable by the GC conditions employed. Weak Acid Fraction. Analysis of phenols derived from the weak acid fraction showed a complex mixture of highly alkylated compounds (Figure l).Some possible structural assignments are shown. A series of phenols was identified, having the same empirical formulas as the alkylated tetrahydronaphthols derived from the hydroperoxide fraction. The fragmentation patterns of the phenols were quite different, however. In particular, the intensities of the M - 17 and M - 18 ions were notably weaker than in the tetrahydronaphthol series, as would be expected for phenols (11). The antioxidant 2,6-di-tert-butyl-p-cresol (BHT) was also identified in the weak acid fraction; presumably, this was an oil additive. Strong Acid Fraction. The methyl esters of the carboxylic acid fraction were analyzed by GC-MS; several structural types were tentatively identified (Figure 2). In addition to highly alkylated benzoic and benzenedicarboxylic acids, naphthoic acids were identified, having either no alkyl substitution or only one methyl group. In addition, compounds which could be formulated as alkylcoumarins were present. Such compounds may have been formed from phenolic acids, known to be produced in oil photooxidation (6,14). Results Peroxide Extraction by Water. Irradiation of a film of no. 2 fuel oil over deionized water afforded the results sumVery Weak Acid Fraction. After reduction of the hydromarized in Figure 3.The total peroxide formed by irradiation peroxides of the very weak acid fraction, the derived alcohols increased linearly for ca. 100 h to about 0.07 mol/L of oil and were examined by GC-MS, revealing a series of compounds differing by -CH2 units (mol wt 162,176,190,and 204). The then declined slowly. The concentration of peroxides in the fragmentation patterns of these alcohols suggested that they water reached a maximum of 3 X M a t 168 h. Peroxides formed initially were largely retained within the oil layer, but were methylated benzylic alcohols (11); moderately strong as irradiation proceeded they were either gradually extracted molecular ions, weaker M - 1 ions, and strong M - 15,M into the water layer or transformed into peroxides more sol17, and M - 18 fragments were observed. A likely partial uble in water. The former interpretation appears more likely structure for the alcohol series is 1; the implication would be on the basis of TLC analyses; the water-soluble peroxides t h a t the original hydroperoxides were methyl-substituted derivatives of l-hydroperoxy-1,2,3,4-tetrahydronaphthalene formed by irradiation were less polar than tetralin hydroperoxide, in keeping with their tentative formulation as al(tetralin hydroperoxide). The mass spectra did not exactly kylated derivatives of this compound. match those of any of the isomeric methylated tetrahydroAfter 48 h of irradiation, the ratio of the concentrations (oil naphthols previously reported, nor those of 1- or 2-methyl966
Environmental Science & Technology
0
30
00
90
120 150 TIME Ihr )
180
?IO
240
270
Figure 3. Peroxide formation and yeast growth inhibition in fuel oil ir-
radiated over water: (A)growth inhibition by water extract: ( 0 )peroxide peroxide concentration in water; (a)total concentration in oil; (0) peroxide concentration peroxide)/(water peroxide) was 270; after 168 h, the ratio had decreased to 140. Even in the early stages of the experiment, however, a sufficient quantity of toxic material had been extracted to exert inhibitory effects on yeast growth, as Figure 3 shows. Individual Compound Toxicity. Yeast. Individual compounds related to the compound types identified by GC-MS from the photooxidized oil were tested for their activity in Saccharomyces growth inhibition. In addition, p-toluidine was tested, since this compound has been demonstrated to occur in the water extract of an unweathered no. 2 fuel oil and is toxic to several unicellular algae ( 1 5 ) . Tetralin hydroperoxide inhibited yeast growth significantly (82%,P I 0.01) a t 3 X M (5 mg/L). A similar concentraM) of peroxides in the water extract from no. tion (5.8 X 2 fuel oil exposed to light for 24 h was associated with 86% inhibition of growth. Other hydroperoxides tested were less toxic; cumene hydroperoxide required a concentration of 2.5 X M (88 mg/L) for virtually complete growth inhibition, and tert-butyl hydroperoxide inhibited growth only 50% a t M (90 mg/L). Naphthoic acids (both the 1- and 2-isomers) showed sigM (17 mg/L), but nificant ( P I 0.01) growth inhibition a t
the absolute inhibition of 25-50% was less than t h a t caused by tetralin hydroperoxide. 2,4,6-Trimethylbenzoic acid was not significantly toxic a t or below 5 X M (82 mg/L). Phenolic compounds and p-toluidine showed little or no growth-inhibitory activity; significant growth promotion was observed a t all concentrations of 2,3,5,6-tetramethylphenol tested. Algae. Weak acid (phenolic, 1.2 ppt) and very weak acid M) and (peroxide) fractions prepared in the dark (5.3 X in the light (1.77 X M) were tested for toxicity to four genera: Vaucheria (Chrysophyta), Microcoleus (Cyanobacteria), Stigeoclonium, and Ulothrix (Chlorophyta). Cultures were examined after 4- and 7-days exposure. Controls were all healthy and exhibited good growth. Stigeocloniurn was most tolerant of the algae tested to both fractions. After 7 days, it was healthy and grew when exposed to 1 mL of the phenol-containing fraction, whereas 0.25 mL was lethal to Ulothrix and 0.1 mL was lethal to Vaucheria and Microcoleus. Stigeoclonium also showed no cell destruction on exposure to 0.25 mL of peroxide fraction from irradiated extract, whereas other algae were killed by 0.1 mL. The peroxidecontaining fraction was more toxic than the phenol-containing fraction to all the algae tested, and extracts from irradiated oil containing photooxidation products were more toxic than fractions from oil held in the dark (e.g., Stigeoclonium withstood 0.25 mL of the peroxide fraction from irradiated extract with no cell destruction, but grew when exposed to 0.50 mL of this fraction from extracts held in the dark). Three genera, Vaucheria, Spirogyra (Chlorophyta), and Microcoleus, were screened for sensitivity to individual compounds typical of photooxidation products. Observations a t 1 day and 4-5 days are presented in Table I. Tetralin hydroperoxide was acutely toxic to Vaucheria (effects were noted a t 1 day), but slight recovery was evident by 5 days. Toxicity to the other algae was exhibited more slowly. Microcoleus showed greater susceptibility to hydroperoxides than Vaucheria. At 4-5 days, 1-naphthoic acid was more toxic to Vaucheria than 2-naphthoic acid, but the reverse was true with Spirogyra and Microcoleus. These compounds exerted effects slowly. Toxicity of p-toluidine and 2,4,6-trimethylphenol to all three algae was evident only a t the higher concentrations tested, although loss of pigmentation was sometimes observed a t lower concentrations. Effects of Hydroperoxides on Algal Photosynthesis. Photosynthetic incorporation by Vaucheria and closely attached epiphytes was tested in culture medium with or without tetralin hydroperoxide additions. The results of five experiments performed on different days are presented in Table 11. Hydroperoxide concentrations 18 X M depressed photosynthetic activity compared to the unexposed controls in all experiments, but statistical significance could not always be attached to this observation because the variance in the data was sometimes large. At 5 x 10-5 M, stimu~~
~
~~~~~~~~~~
Table 1. Growth Inhibition of Selected Filamentous Algae by Oil Associated Compounds or Photooxidized Derivativesa effectlve dose (pg) lor cellular disruption and no growthb 1 day 4-5 days SPlrogyra Mlcrocoleus Vaucherla S~lrogyra
compound
Vaucherla
1-naphthoic acid 2-naphthoic acid tetralin hydroperoxide curnene hydroperoxide 2,4,6-trimethylphenoI p-toluidine
200 (50) 400 (200) 50 (25)
200 200 200 (100)
500 (50) 500
500 (100) 500 (100)
a Compounds applied in 0, 1, 10, 25,50, 100, 200, 400, or 0, 1, in parentheses.
200 >400 100 (50)
500 500
IO, . . ,100, 500, 1000 fig
amounts.
50 (10) 100 (25) 100 (50)
400 50 50
100 500 500
500 500 (100)
Mlcrocoleus
400 100 25 25 500 500
Or pigment bleaching: if concentration differed, shown
Volume 13, Number 8, August 1979 967
Table II. Effect of Tetralin Hydroperoxide on Photosynthetic I4C Incorporation by Vaucheria and Closely Attached Epiphytesa tetralin hydroperoxide exposure. pmol/mL
expt
dpm 14C lncorpl
pg chlorophyll a , X i SD
2372 f 386 1086 f 320b 68 f 44O 28 f g b 1904 f 196 730 f 430b 491 f 53 94 f 996 3654 f 2529 5936 f 2274 2576 f 1195 2022f 1460 607 f 529' 2692 f 1001 2707 f 2525 1354 f 1431 256 f 76 63 k 31
control
1
0.082 0.164 0.328 2
control
3
control
0.291 0.291 control
4
0.05 0.10 0.20 0.40 control
5
0.05 0.10 0.20 0.40
Significantly a Data corrected for excretion, dark uptake, and sorption. different from control, Dunnett's test or t test (each at p = 0 05) used when n > 2 or n = 2, respectively, where n = number of treatments
-
~
_
_
_ _ _ _ ~ _ _
-
lation occurred in experiment 4. In other experiments to identify toxicants in complex mixtures,14C incorporation was assayed in light and dark water extracts of no. 2 fuel oil with and without tetralin hydroperoxide additions. Because light extracts had higher concentrations of aliphatic and aromatic hydrocarbons as well as hydroperoxides, assays were also conducted in light extracts that were diluted to approximate the aliphatic hydrocarbon concentrations found in dark extracts. Results of' a representative experiment are presented in 'I'able I l l . Activity was depressed significantly in all treatments hut dark cxtract exposure, in which activity was significantly elevated. 'I'etralin hydroperoxide additions significantly depressed incorporation over the treatment with no added hydroperoxides. except where the light-exposed water soluble extract was tested. 'l'he excretion of fixed l*C increased in all instanws on hydroperoxide addition. Samples grouping into suhset H (uhen photosynthetic activity was tested) show that toxicity may result from compounds other than hydroperoxides. h u t subset A samples emphasize the importance of hydroperoxides i n toxic mixtures. Discussion
Photooxidation of Hydrocarbons in the Environment.
Petroleum-derived hydrocarbons occur in most polluted environments and are typical constituents of industrial and municipal wastes. Previous work on the fates of hydrocarbons and related lipids in the environment has estahlished two abiotic mechanisms for their oxidation. In the first mechanism. free radicals (generated thermally or by the action of light, metal ions, etc.) combine with ground-state triplet oxygen ( . ' ' 0 2 ) to afford peroxy radicals: these abstract hydrogen atoms (with the formation of hydroperoxides) from reactive substrates to continue the radical chain reaction. Hydroperoxides are also formed by the second pathway: direct addition of excited singlet oxygen (IO.) molecules to reactive acceptors such as substituted olefins and polyunsaturated fatty acids. In either case, hydroperoxides are subject to further thermal and photochemical transformations, with ultimate formation of acids, ketones, alcohols, and other oxygenated products. We showed earlier, in experiments in which reactive acceptors and quenchers of 1 0 2 were added to fuel oil, that both processes appear to be involved in petroleum photooxidation. Singlet oxygen, generated by transfer of energy from excited triplet sensitizers (anthracene, pyrene, etc.) present in the oil, reacts with acceptors to produce hydroperoxides; further photodegradation leads t o a mixture of other oxygenated products ( 1 6 ) . A mechanism for the photooxidation of a retined oil, incorporating structural types of hydrocarbons known to occur in petroleum and leading to some ofthe oxygenated products identified in the present investigation, is sunimarized in Figure 4. The molecules which initially accept IO2 in petroleum to form hydroperoxides are not certain. but it is unlikely that substituted tetralins are reactive enough toward '0. to be the initial acceptors. Substances more likely to react rapidly with IO2 include highly alkylated olefins such as ??:3-dimethylindene ( I 7) or substituted phenols (18).The substit uted tetralin hydroperoxides probably represent products of' radical attack; the tertiary benzylic hydroget1 atom of 1methyltetrahydronaphthalene, for example, would be highly reactive toward abstraction and a particularly stable radical would be formed. The hydroxyl radical, a key intermediate in this mechanism, is known to be produced during the pho' of hydroperoxides ( 1 9 ) and is very reactive toward extraction of aromatic and benzylic hydrogens and addition t o aromatic rings (20). I t is plausible that the highly alkylated phenols and henzoic acids identified in this study were produced by such mechanisms. Mechanisms of Toxicity. Incorporation o f oxygen into hydrocarbons results in large increases in water solubility. For example. the solubility of naphthalene in water is only ca. 35 rng/L, whereas 2-naphthol is soluble to the extent of 1000 mg/I,. Accordingly, some of the hydrocarbons in oil films on water will probably gradually enter the water column in the
Table 111. Photosynthetic I4C Incorporation by Vaucheria Species and Epiphytes Exposed to Irradiated and Unirradiated Water Extracts of No. 2 Fuel Oil with and without Additions of Tetralin Hydroperoxide treatment
control control, Tb WE-NI WE-NI, T WE-I WE-I, T WE-I, dil. WE-I, dil., T
hydrocarbons, mg/L aliphatic aromm
0 0 1.50 1 .so 7.94 7.94 1.50 1.50
0
0 0 0 4.86 4.86 0.92 0.92
'IC incorp
% fixed
hydroperoxides, pnol/mL
dpm/ pg of Chi a
excreted
0 0.291 0.025 0.291 0.291 0.580 0.055 0.291
491 f 53 94 f 99 892 f 97 199 f 39 186 f 96 10 f 156 261 f 50 4 3 f 10
43 80 34 62 71 95 59 85
140
statist slgnii
=
C A 0
D A 0 A 0 A
0 A 0
Treatments not significantly different from each other are given same letter (Scheffe multiple range test, p = 0.05). WE, water extract: NI. nonirradiated; 1, irradiated; T, tetralin hydroperoxide added. a
968
Environmental Science & Technology
I
carboxylic orlds
t to2
A
t
Figure 4. Proposed mkchanism for fuel oil photooxidation
form of oxygenated derivatives during photooxidation. Although the absolute concentrations of petroleum photooxidation products in water may be quite low, it is likely t h a t they could be extensively concentrated by organisms, as in the case of synthetic detergent molecules, which have similar polar and nonpolar regions and are readily taken up by the gills of fish and other hydrophobic tissues (21). Once incorporated into cell or organelle membranes, detergent-like molecules may exert disruptive effects by means of their surface-active and solubilizing properties toward the membrane constituents. In extreme cases, changes in the permeability of membranes result in leakage of essential metabolites, disorganization of enzyme systems, and death of the cell. Hydroperoxides derived from petroleum hydrocarbons may also react with membrane lipids by radical pathways or by direct oxygen transfer (epoxidation). Evidence of membrane damage due to hydroperoxides was obtained in algal 14C uptake experiments; the ratio ( 14Cexcreted/14C in cells 14Cexcreted) was always greater where tetralin hydroperoxide was added. In photosynthetic organisms chlorophyll destruction by hydroperoxides may also be important (22); this mechanism may explain the loss of pigmentation observed in some of our experiments. The algal species used in our experiments possessed differing tolerances to the toxicants tested. The literature shows clearly that responses to oils and their constituents are species specific. Hydrocarbons, p-toluidine and other substituted anilines, substituted phenols and cresols, and phenalen-1-one have all been shown to be toxic in varying degrees to given algal Species (15, 23, 24). In our experiments, toxicity was observed in both phenolic and peroxide-containing fractions. The latter were more toxic, however, and additions of tetralin hydroperoxide increased the toxicity of fuel oil extracts in 14C incorporation experiments. The organisms used were selected because of their importance in algal communities observed a t field sites in the upper Delaware estuary or their filamen-
+
tous growth; our results are not directly comparable to studies using other species. The observations noted here may help explain some of the species alterations in algal communities that occur as a result of hydrocarbon pollution acting synergistically with other stresses on individual populations. Community alterations will affect the flow of energy through the food web. In our studies, inhibition of growth of a heterotrophic microorganism (a yeast) by some hydrocarbon oxidation products was also observed. Oxygenated compounds of these types may conceivably have deleterious effects on the metabolism of a microbial community if they are toxic or inhibitory to a significant fraction of its heterotrophic microflora. Alternatively, they may exert selective pressures leading t o the development of alternate communities capable of metabolizing them. Acknowledgments
We thank Dr. Michael L. Gross for helpful discussions, D. W. Blankenship, S. Roberts, and K. Goehl for experimental assistance, and the National Science Foundation (RANN program) for financial support (Grant No. G42282). Literature Cited
-
(1) Anderson. J. W.. Neff. J. M.. Cox. B. A.. Tatem. H. E.. Hiehtower. G. M., Mar Biol , 27,75 (1974). (2) Freegarde, M., Hatchard, C. G., Parker, C. A,, Lab Pract , 20,35
11971). (3)-Griffin,L. F., Calder, J. A,, Appl. Enuiron. Microbiol., 33, 1092 (1977). (4) Lacaze, J. C., Villedon de Naide, O., Mar. Pollut. Bull., 7, '73 (1976). 15) Scheier. A,. Gominner. D.. Bull. Enuiron. Contam. Toricol.. 16. ,595 (1976). (6) Larson. R. A,. Hunt. L. L.. Blankenshim D. W.. Enuiron Sci Technol , 11,492 (1977). ( 7 ) Drushel, H. V.. Sommers, A. L., Anal Chem, 39.1819 (1967). (8) Boylan, D. B., Tripp, B. W., Nature ( L o n d o n ) ,230,44 (1971). (9) Fitzgerald, G. P., Gerloff, G. C., U.S.N.T.I.S., PBRep., PB-253, 343/8WP (1976). (10) Lorenzen, C. J., Lirnnol. Oceanogr., 12,343 (1967). (11) Aczel, T., Lumpkin, H. E., Anal. Chem., 32,1819 (1960). (12) Gross. M. L., DeRoos, F. L., J . A m . Chem. Soc., 98, 7128 (1976). (13) Heimgartner, H., Weibel, P. A,, Hesse, M., Helu. Chirn. Acta, 57,1510 (1974). (14) Hansen. H. P.. Mar. Chern.. 3.183 11975). (15) Winters, K., O'Donnell, R., Batterton, J. C:, Van Baalen, C.,Mar. Biol., 36, 269 (1976). (16) Larson, R. A,, Hunt, L. L., Photochem. Photobiol., 28, 553 (1978). (17) Fenical, W., Kearns, D. R., Radlick, P., J . A m . Chern. Soc., 91, 3396 (1969). (18) Pfoertner, K., Bose, D., Helu. Chirn. Acta, 53, 1553 (1970). (19) Carlsson, D. J., Wiles, D. M., Macromolecules, 2,597 (1969). (20) Doyle, G. J., Lloyd, A. D., Darnall, K. R., Winer, A.M., Pitts, J. N., Enuiron. Sci. Technol., 9, 237 (1975). (21) Bardach, J. E., Fujiya, M., Holl, A., Science, 148, 1605 (1965). (22) Peiser, G. D., Yang, S. F., Phytochemistry, 17,79 (1978). (23) Kauss, P., Hutchinson, T. C., Soto, C., Hellebust, J., Griffiths, M., Proceedings API/EPA/USCG Conference on Prevention and Control of Oil Spills, American Petroleum Institute, Washington, D.C., 1973, p 703. (24) Winters, K., Batterton, J. C., Van Baalen, C., Enuiron. Sci. Technol., 11,270 (1977). u
Received for reuieu) September 26, 1978. Accepted April 24, 1979.
Volume 13, Number 8, August 1979 969
