Environ. Sci. Technol. 1992, 26, 107-116
15N and 13C NMR Investigation of Hydroxylamine-Derivatized Humic Substances Kevin A. Thorn" US. Geological Survey, Water Resources Division, Denver Federal Center, Mail Stop 408, Box 25046, Denver, Colorado 80225
Jeffrey B. Arterburn Laboratorium fur Organische Chemie, ETH-Zentrum, Zurich, Switzerland
Mlchael A. Mlkita" Department of Chemistry, California State University, Bakersfield, Bakersfield, California 933 11
Five fulvic and humic acid samples of diverse origins were derivatized with 15N-labeledhydroxylamine and analyzed by liquid-phase 15NNMR spectrometry. The 15N NMR spectra indicated that hydroxylamine reacted similarly with all samples and could discriminate among carbonyl functional groups. Oximes were the major derivatives; resonances attributable to hydroxamic acids, the reaction products of hydroxylamine with esters, and resonances attributable to the tautomeric equilibrium position between the nitrosophenol and monoxime derivatives of quinones, the first direct spectroscopic evidence for quinones, also were evident. The 15N NMR spectra also suggested the presence of nitriles, oxazoles, oxazolines, isocyanides, amides, and lactams, which may all be explained in terms of Beckmann reactions of the initial oxime derivatives. INEPT and ACOUSTIC 15NNMR spectra provided complementary information on the derivatized samples. 13C NMR spectra of derivatized samples indicated that the ketone/quinone functionality is incompletely derivatized with hydroxylamine. Introduction It is the oxygen-containing functionality which is responsible for many of the properties of humic substances in the environment. This study was undertaken both to learn more about the carbonyl functionality of humic and fulvic acids and to examine the utility of using 15NNMR spectrometry to analyze humic substances specifically derivatized or reacted with 15N-labeledreagents. There are a number of important processes in which nitrogencontaining compounds react with humic substances in the environment. For example, ammonia applied to agricultural soils as a nitrogenous fertilizer may react chemically with the soil organic matter in what is known as ammonia fixation (1). Under conditions of heavy application of ammonia to soils, nitrite can accumulate due to inhibition of nitrification and also become fixated by the soil humic substances (2). The reaction of nitrite with humic substances, in the process known as chemodenitrification, can lead to the formation of nitrogen gases, including nitrous oxide, a greenhouse gas and scavenger of stratospheric ozone (2). Many pesticides, dyes, explosives, and other industrial chemicals may be converted to aromatic amine compounds in the environment (3). There is evidence that aromatic amines form covalent bonds with humic substances, either through abiotic condensation reactions (3, 4 ) or xenobiotic coupling reactions (5). Monochloramine is currently being used as an alternative drinking water disinfectant to chlorine. It has been hypothesized that under basic conditions, chloramine could add nitrogen to the aldehydic and ketonic functionalities of aquatic fulvic acids (6). We anticipated that 15N NMR could play a 0013-936X/92/0926-0107$03.00/0
significant role in elucidating the reaction chemistry of these and other processes. Hydroxylamine itself is considered an intermediate in the nitrification of ammonium ion to nitrite and nitrate (7), and one can speculate that hydroxylamine reacts with humic substances under natural conditions in soils, sediments, and waters. In this regard, the uptake by organic matter of 15N-labeledhydroxylamine added to soils has been measured in at least two studies (8,9). Hydroxylamine has been used in a number of previous studies to determine the carbonyl content of humic substances (10-12). It has never been known, however, to what extent the hydroxylamine reacts with groups other than ketones, quinones, and aldehydes to form derivatives other than oximes, or how completely these functionalities are derivatized (13). For example, it may be anticipated that hydroxylamine can react with naturally occurring esters in humic or fulvic acid molecules to form hydroxamic acids. 15N NMR can be used to directly observe and quantitate the various derivatives formed from the reaction of hydroxylamine with the humic substances. In this report, we present a more detailed interpretation of the 15NNMR spectra of the hydroxylamine-derivatized Suwannee River fulvic acid than reported previously (14) and extend the analyses to four other samples, the Armadale soil fulvic acid and the International Humic Substances Society (IHSS) reference soil, peat, and leonardite humic acids. Natural-abundance quantitative and DEPTGL liquid-phase 13C NMR spectra of the underivatized samples also are presented, as well as quantitative 13C NMR spectra of samples derivatized with unlabeled hydroxylamine. Experimental Procedures Suwannee River fulvic acid was obtained from J. A. Leenheer, and Armadale soil fulvic acid, isolated from a Bh podzolic soil, Prince Edward Island, Canada, was purchased from C. Langford, Concordia University. Reference soil, peat, and leonardite humic acids were purchased from the IHSS. Elemental analysis, molecular weight, and potentiometric titration data for the samples are listed in Table I. The derivatizations of the humic samples with hydroxylamine were performed by adding 0.17-0.19 g of hydroxylamine hydrochloride (99 atom % 15N)and 0.60-0.81 g of the humic or fulvic acid (H-saturated form) in 100 mL of deionized and distilled water, titrating to pH 5 with 1 N NaOH, and refluxing from 4 to 12 h at 88 "C. The derivatized samples were then H-saturated on a cationexchange column and freeze-dried. The derivatized samples were dissolved in DMSO-d6 for 15N NMR analysis. The Suwannee fulvic and leonardite humic acids were
0 1991 American Chemical Society
Environ. Sci. Technol., Vol. 26, No. 1, 1992
107
Table I. Elemental Composition of Humic and Fulvic Acids (Weight Percent; Ash-Free and Moisture-FreeBasis), Number-Average Molecular Weights, and Potentiometric Titration Data
sample
C
H
0
N
S
P
ash
M,,, Da
COOH, mequiv/g
PhOH, mequiv/g
Suwannee River FA Armadale FA' IHSS Soil HA IHSS Peat HA IHSS Leonardite HA
53.6 50.9 57.99 56.82 63.08
3.93 3.35 3.78 4.06 3.72
40.9 44.8 33.69 34.91 30.96
0.87 0.75
0.66 0.25 0.41 0.68 0.71
ND" ND" 0.32 0.11 0.05
0.58 0.8 0.90 1.92 1.66
8006 951
6.0b 7.7
3.3
a
ND, not determined.
4.18
3.74 1.15
From ref 56. All Armadale data from ref 57.
similarly derivatized for 13C NMR analyses, using approximately 200 mg of sample and 9.4 mmol of unlabeled hydroxylamine hydrochloride. The method of Fritz et al. (15) was used to prepare model compound oximes. NMR spectra were recorded on a Varian XL-300 NMR spectrometer at 13Cand 15Nresonant frequencies of 75.4 and 30.4 MHz, respectively. (Use of trade names in this report is for identification purposes only and does not constitute endorsement by the U.S. Geological Survey.) Natural-abundance 13C NMR spectra were recorded on 200-375 mg of underivatized sample (H-saturated) in 2.0 g of DMSO-d6, 99.9 atom % 12C (10-mm NMR tubes). Acquisition parameters for the quantitative 13C NMR spectra included a 50 000-Hz spectral window, 45' pulse angle, 0.2-s acquisition time, 8.0-10.0-s pulse delay (sufficient for complete relaxation of all nuclei), and inverse gated decoupling (to eliminate NOE); line broadenings (LB) applied to the FIDs are listed with the figures. The same acquisition parameters were used to record quantitative 13CNMR spectra of the Suwannee fulvic and leonardite humic acids derivatized with unlabeled hydroxylamine. DEPTGL 13CNMR spectra were recorded using the pulse sequence of Sorensen et al. (16);delays were set by assuming maximum and minimum l J C H values of 200 and 125 Hz, respectively. The spectral window was 30000 Hz, acquisition time 0.2 s, and delay for proton relaxation 2.0 s. Only the spectra showing all protonated carbons (e = 38") are presented in this report. 13C spectra were referenced on the DMSO-d6peaks. Additional lH and 13C NMR analyses of these samples have been reported elsewhere (17-19). 15NNMR spectra were recorded on 400-680 mg of derivatized samples dissolved in 2-3 g of DMSO-d6 (10-mm NMR tubes). Three sets of spectra were recorded. Normal pulsed spectra were acquired using a 21276.6-Hz (700 ppm) spectral window, 0.5-s acquisition time, 45" pulse angle, 2.0-s pulse delay, and inverse gated decoupling. These spectra should be close to quantitative, as the 2.0-s pulse delay was found to be adequate for complete relaxation of all nitrogen nuclei, based upon comparison with spectra recorded using longer delays. ACOUSTIC (20) spectra were recorded using a 15649.5-Hz spectral window, 0.5-9 acquisition time, 90° pulse angle, 2.0-s pulse delay, and T delay of 0.1 ms. Refocused INEPT (21) spectra (proton decoupled) were acquired using a spectral window of 14992.5 Hz, acquisition time of 0.5 s, and delay for proton relaxation of 2.0 s. The polarization transfer time and refocusing delay were set equal to 1/4 J, or 2.78 ms (assuming lJNH= 90.0 Hz), values which have been reported optimal for signal enhancement of singly protonated nitrogens (22). Neat formamide in a 5-mm NMR tube, assumed to be 112.4 ppm, was used as an internal reference standard. 15NNMR chemical shifts are reported in ppm downfield of ammonia, taken as 0.0 ppm. This is the chemical shift scale used by Levy and Lichter (23). The DEPTGL sequence was used in the 13C NMR analyses to obtain the most accurate spectral editing (24). 108
1.26
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The objective in using polarization transfer in the I5N
NMR analyses was to maximize signal enhancement, and for this reason INEPT was chosen. INEPT has been reported slightly superior to DEPT for signal enhancement of 15N nuclei (25).
Results and Discussion Reaction of Hydroxylamine with Organic Functional Groups. Hydroxylamine reacts with aldehydes and ketones to form aldoximes and ketoximes, respectively (26-30). Either the mono- or the dioxime can be obtained upon reaction of hydroxylamine with monosubstituted or 2,5-disubstituted 1,4-benzoquinones (31). The monoximes of the less hindered carbonyl group are obtained in the monosubstituted case and are the only product with 2,6disubstituted and trisubstituted 1,4-benzoquinones (32). Hydroxylamine also reacts with 1,2-benzoquinonesto form either the mono- or dioxime derivative. Quinone monoximes undergo a tautomeric equilibrium with their corresponding nitrosophenol forms (32,33). Hydroxylamine condenses with the carbonyl group of a,P-unsaturated ketones under acidic conditions to form the oxime (28). Hydroxylamine reacts with P-diketones to form isoxazoles (26, 34, 35), with P-keto esters to form isoxazolones (26, 28), and with esters to form hydroxamic acids (29). Additional details of hydroxylamine chemistry have been reported recently (36-39). Oximes undergo Beckmann reactions under acidic conditions, including rearrangements, fragmentations, and rearrangement-cyclizations (40,41). Products from these secondary reactions include secondary amides, lactams, and isocyanides (isonitriles), (rearrangements); nitriles and primary amides (fragmentations); and dihydroisoquinolines, imidazoles, isoquinolines, oxazoles, oxazolines, pyridines, and Apyrrolines (rearrangement-cyclizations). 15N NMR Spectroscopy. Nitrogen-15 has a natural abundance of 0.37%, and compared to 13C,a receptivity of 2.19 x While the chemical shift range of 15N extends to approximately 900 ppm, most nitrogens in organic compounds lie within the range from 0 to -500 ppm. Table I1 lists chemical shift ranges of nitrogen compounds pertinent to this study. Several experimental difficulties may arise in liquid-phase 15N NMR spectrometry. In contrast to 13C,15Nhas a negative magnetogyric ratio, and therefore negative nuclear Overhauser enhancement (NOE) factors. The maximum value of the NOE factor (7) for 15Nis -4.93, and this is realized when spin-lattice relaxation occurs solely through the dipolar relaxation mechanism and within the extreme narrowing limit of fast molecular motion. Since signal enhancement is equal to 1 + 7, the maximum signal enhancement obtained when protons are decoupled from nitrogens is approximately -4. However, when 15NJH dipolar spin-lattice relaxation is not the dominant relaxation mechanism, a range of NOE factors from 0 to -4.93 may be observed. Any NOE factor between 0 and -2.0 lowers the signal intensity, and a NOE factor of 1.0 eliminates the signal entirely (22, 23). The
Table 11. I5N NMR Chemical Shift Ranges for Nitrogen Compounds (pprn)" Nitrosophenols, 425-418 ppm 1-nitroso-2-naphthol 2-nitroso-1-naphthol
425 418
Oximes, 390-320 ppm benzoquinone dioxime chalcone oxime benzoylacetone oxime benzophenone oxime D-mannose oxime l-(aminoxy)-l-phenyl-4,4-dimethylpentan-3-one oxime benzoin oxime acetophenone oxime levulinic acid oxime phenyl-2-propanone oxime
377 371, 366* 370 362 326, 361b 35LIc 357 356, 325' 348, 343' 346
Isoxazoles, 388-377 ppm 388 382 381 377
1,2-benzisoxazole isoxazole 5-methylisoxazole 2,1-benzisoxazole Isoxazolone
367
3-phenyl-5-isoxazolone Pyridine pyridine
318
A-Pyrroline 3,3-diphenyl-P-ethyl-l-pyrroline
317
Dihydroisoquinoline 3,4-dihydroisoquinoline
319
Nitriles, 267-239 ppm benzonitrile acetonitrile
256 243
Oxazole, 259-249 ppm 2,4,5-trimethyloxazole oxazole 2,5-diphenyloxazole benzoxazole
259 257 253 249
Oxazoline 2,4,4-trimethyl-2-oxazoline
246
Imidate ethyl N-phenylacetimidate benzyl 2,2,2-trichloracetimidate
249 215
Isocyanide (Isonitrile), 199-161 ppm 1,1,3,3-tetramethylbutylisocyanide benzyl isocyanide
195 170
Hydroxamic Acid, 168-166 ppm hydrocinnamohydroxamic acid 2-(4-methoxyphenyl)acetohydroxamicacid benzohydroxamic acid acetohydroxamic acid
168 167 166 166
Secondary Amides, 133-105 ppm 131
acetanilide Primary Amides, 109-102 ppm
105
benzamide Lactams, 126-112 ppm caprolactam
117
Ranges are for all solvents. Chemical shifts listed for specific compounds are in DMSO-d6. *Separate resonances are observed for the 2 and E isomers of oximes. In general, the 2 isomers of aldoximes are deshielded with respect to the E isomers, whereas the reverse is true for ketoximes. From ref 37.
use of inverse-gated decoupling is commonly employed to eliminate NOE and thereby prevent signal degradation or cancellation for 15Nnuclei with unfavorable NOE factors. Polarization transfer offers two important advantages for nuclei such as 15N,which have low sensitivities,negative
magnetogyric ratios, and oftentimes long spin-lattice relaxation times, Tqs. First, an enhancement factor of Y ~ / Y N (7, gyromagnetic ratio) is obtained and is independent of the relaxation processes that contribute to the relaxation of lH and 15N. The theoretical maximum signal enhancement for 15Nis approximately 10 (22,25). Second, the pulse sequence repetition rate is a function of TI .for the protons, which typically have much shorter relaxation times than 15Nnuclei (22,25). In the present case of I5N nuclei incorporated into fulvic or humic acid molecules of molecular weights greater than or equal to -800 Da, however, long Tl's do not appear to be a problem. The longest 15N Ti's in the derivatized humic samples were estimated to be 0.5 s or less. The INEPT (21) pulse sequence was used in this study to enhance the signal intensities of protonated nitrogens, and to distinguish between protonated and nonprotonated nitrogens, in the derivatized humic and fulvic acids. A limitation of polarization transfer is that 15Nnuclei attached to protons which are undergoing rapid exchange w ill not be polarized. Acoustic ringing can be a problem with low resonant frequency nuclei such as I5N,which may result in severe distortions of the base line (42). The problem is especially acute in spectra which consist of broad bands of low intensity spread out over a wide chemical shift range, which is the case with 15N NMR spectra of derivatized humic substances. The ACOUSTIC pulse sequence (20)was used in this study to reduce ringing in the 15N NMR spectra. Since NOE is retained in this sequence, some signals may be lost because of unfavorable NOE factors. However, the NOE can be quenched by addition of a paramagnetic relaxation reagent if necessary. There is a significant solvent dependency of chemical shift in 15NNMR spectrometry. The largest effects occur in the oxime region. For example, the 15NNMR chemical shift of benzophenone oxime is 346 ppm in CHC13and 362 ppm in DMSO-$. In order to make the most accurate assignments in the 15N NMR spectra of the derivatized humic samples, which were recorded in DMSO-&, it was necessary to generate 15N NMR chemical shift data of model compounds in DMSO-d6,to supplement the literature. I3C NMR Spectra of Underivatized Fulvic and Humic Acids. The quantitative 13CNMR spectra of the underivatized samples in Figure 1exhibit the five major bands characteristic of humic substances. Aliphatic carbons (sp3hybridized) bonded to other carbons occur in the region from 0 to 60 ppm; carbons bonded to naturally abundant nitrogen and sulfur also occur in this region, Methoxyl carbons occur in the range from 50 to 56 ppm. Aliphatic carbons bonded to oxygens, i.e., ether and alcohol carbons, including carbohydrates, occur in the region from 60 to 90 ppm. Aromatic carbons occur from 90 to 165ppm, with phenolic carbons in the range from 135 to 165 ppm, and olefinic carbons from 110 to 150 ppm. Acetal and hemiacetal carbons, including the anomeric carbons of carbohydrates, may overlap with aromatic carbons in the region from about 90 to 110 ppm. The 13C NMR chemical shift ranges of carbonyl functional groups in DMSO-d, are listed in Table 111. The peaks in the spectra in Figure 1from about 160 to 180 ppm represent primarily carboxylic acid carbons; esters, amides, and lactones also may overlap in this region. A splitting of the carboxylic acid peaks is evident in the Suwannee and Armadale fulvic acids, and the leonardite humic acid, with peak maxima at about 167 and 172 ppm. The peak at 167 ppm represents benzenecarboxylic and cr,p-unsaturated carboxylic acids, and the peak at 172 ppm aliphatic, Environ. Sci. Technol., Vol. 26,No. 1, 1992
109
LE=2o
compd class
chem shift range, ppm
compd class
chem shift range, ppm
ketone quinone carboxyl ester
220-189 190-178 175-159 172-164
amide lactone phenolic carbon
174-165 178-159 165-135 chem shift, PPm 218.0b 216.8b 215.0b 209.6 208.0 207.3 205.5 205.4 199.3 197.3 197.2 197.2 195.6 192.6 189.2 176.0
ketone 2,2,4,4-tetramethyl-3-pentanone 2-norbornanone cycloheptanone 4-heptanone methyl ethyl ketone levulinic acid butyl levulinate phenyl-2-propanone 2,2',4,4'-tetrahydroxybenzophenone 3-penten-2-one acetophenone 2,4,4'-trihydroxybenzophenone benzophenone benzophenonetetracarboxylic acid anhydride chalcone quercetin
1
Suwannee River FA
Table 111. 13CNMR Chemical Shift Ranges of Carbonyl and Phenolic Carbons and Specific Chemical Shifts of Ketone Carbonyls in DMSO-d$
1-DMSO-d.
i~-
Armadale Soil FA 1 1 LB=30
I
1
Ji
"From ref 17. bFrom ref 58. solvent unsaecified.
benzylic, and o-hydroxybenzenecarboxylic acids. The peaks from about 180 to 220 ppm represent ketone and quinone carbons. DEPTGL 13CNMR spectra of the samples are presented in Figure 2. Since only protonated carbons are observed in these spectra, the absence of resonances in the region from about 192 to 202 ppm indicates that aldehydes are not present in the samples. The DEPTGL spectra also separate the nonprotonated from the protonated aromatic carbons, indicating that the latter occur in the range from 90 to -140 ppm. Peak areas and aromaticities determined from the quantitative 13C NMR spectra in Figure 1 are listed in Table IV. The ketone/quinone functionality is significant in all five samples, ranging from 6 to 7% of the total carbon. These values translate into concentrations of 2.7-3.4 mmol/g when combined with elemental carbon contents. Published 13CNMR spectra indicate that the ketone and quinone functionality is barely detectable in marine and algal-derived humic substances (43). Chemical shift data in Table I11 indicate that dialkyl ketones occur downfield of alkyl aryl ketones, which in turn are downfield of diaryl ketones. It is noteworthy that the ketone/quinone peaks in the quantitative spectra in Figure 1encompass the range from about 180 to 220 ppm, indicating that dialkyl, alkyl aryl, and diaryl ketones are all present in the
DMSO-d,
I
i!
I
I
I
I
I
300
200
100
0
-1 00
PPm
Flgure 1. Quantitative liquid-phase I3C NMR spectra of underivatized humic and fulvic acids.
samples. The peak maxima (193-195 ppm) occur in the alkyl aryl and diaryl range. The overlap of various functional groups in the carbonyl region from 160 to 220 ppm is a limitation of 13C NMR spectra, but one that can be partially resolved in the 15N NMR spectra. 15NNMR Spectra of Hydroxylamine-Derivatized Samples. 15NNMR spectra of the derivatized Suwannee fulvic acid are presented in Figure 3. In the quantitative
Table IV. Peak Areas of Quantitative 13C NMR Spectra of Underivatized Humic and Fulvic Acids as Percent of Total Area: and Total Ketone + Quinone Contentsb sample
220-180 ppm
Suwannee FA Armadale FA soil HA peat HA Leonardite HA
6 6 7 7 6
180-160 ppm
160-90 ppm
90-60 ppm
60-0 ppm
19
31 48 56 51 62
15 10 10
27 14 13 14 14
22
16 16 14
11
3
ketone + quinone content, mmol/g 2.7
2.5' 3.4 3.3 3.2
Peak areas measured by electronic integration. Error approximately *5%. Ketone + quinone contents calculated from combination of elemental carbon contents (Table I) and peak areas from 220 to 180 ppm. "he total ketone + quinone content for the Armadale as determined by wet chemical methods has been determined to be 3.1 mmol/g (57). 110
Environ. Sci. Technol., Vol. 26, No. 1, 1992
A
Suwannee River FA
--DMSO-d,
Inverse gated decoupled LB = 5
I
Ib
Suwannee River FA
Armadale Soil FA I
B
I
I
I
366.8
I
1
1
Suwannee River FA Acoustic
IHSS Soil HA
IHSS Peat HA LB = 60
Suwannee River FA
172.2-
IHSS Leonardite
86.5
%
I
5o; I
I
I
l
300
200
100
0
l
-50
PPm Figure 2. DEPTGL I3C NMR spectra of underivatized humic and fulvlc acids. Spectra show all protonated carbons.
spectrum (3A; inverse gated decoupled), the major peak is the oxime resonance, which occurs from about 390 to 330 ppm, and is centered at 360 ppm. Isoxazole and isoxazolone resonances also would occur in this region. Other resonances occur from 270 to 255 ppm, 255 to 240 ppm, 216 to 201 ppm, 201 to 191 ppm, 191 to 160 ppm, 150 to 115 ppm, and 115 to 90 ppm. The reagent peaks at 84.8 and 24.2 ppm correspond to hydroxylamine hydrochloride and free hydroxylamine, respectively. (The peak at 307 ppm does not show up in any of the other spectra and appears to be an artifact.) The base-line roll in the quantitative spectrum, a result of acoustic ringing, is substantially alleviated in the ACOUSTIC spectrum (3B). The most important feature of the ACOUSTIC spectrum is the presence of a shoulder on the downfield side of the oxime peak. This shoulder, centered at 395 ppm, and extending from about 430 to 390 ppm, represents the monoxime derivative of quinones, in tautomeric equilibrium with their corresponding nitrosophenol forms. This shoulder is lost in the distortion of the base line in
4d0
1
3d0
2d0
Id0
,
0 -50
PPm
Figure 3. Liquid-phase "N NMR spectra of Suwannee River fuhric acid derivatlzed with ''N-labeled hydroxylamine.
the quantitative spectrum (3A). Continuous-wave 14N NMR has been used previously to detect the quinone monoxime-nitrosophenol equilibrium in model compounds (33). The upfield portion of the 191-160 ppm peak in the quantitative spectrum, from about 170 to 160 ppm, assigned as hydroxamic acid nitrogen, is absent in the ACOUSTIC spectrum, indicating that the hydroxamic acid nitrogens have unfavorable NOE factors. Negative nuclear Overhauser enhancement also is indicated in the ACOUSTIC spectrum by the negative inversion of both the peaks from 150 through 90 ppm and the reagent peaks. (The free hydroxylamine peak is slightly out of phase,) Most of the resonances besides the oxime and hydroxamic acid peaks in the quantitative 15NNMR spectrum can be assigned most plausibly as secondary reaction products resulting from Beckmann reactions of the initial oxime derivatives. The peaks from 270 to 255 ppm and 255 to 240 ppm correspond primarily to nitriles. Oxazoles (259-249 ppm), oxazolines, and imidates (intermediates in Beckmann reactions; 250-215 ppm) also may occur in this region. The identity of the peak from 216 to 201 ppm, centered at 210 ppm, is uncertain. Imidates may extend into this range; the chemical shifts of amidines, which have Environ. Sci. Technol., Vol. 26, No. 1, 1992
111
Table V. Summary of Assignments for 15NNMR Spectra of Hydroxylamine Derivatized Humic and Fulvic Acids"
chem shift range, ppm
assignment
430-390 390-330 270-240 216-201 201-191 191-170 170-160 150-115 115-90
nitrosophenol: quinone monoxime, furazan oxime, isoxazole, isoxazolone nitrile, oxazole, oxazoline imidate, amidine, imidazole isocyanide isocyanide, imide hydroxamic acid, imide secondary amide primary amide, lactam
Most probable assignments listed in italics.
been reported to result from Beckmann rearrangements of aromatic ketoximes (40) have been reported to occur in this range as well (44). Imidazoles are another possibility. The peak from 201 to 191 ppm, and the downfield region of the peak from 191 to 160 ppm-191 to 170 ppm-most likely correspond to isocyanide nitrogens. The chemical shift range of imides extends from about 175 to 155 ppm, and it is possible that imides contribute to some of the resonances in this region of the spectrum. The peaks from 150 to 115 ppm and 115 to 90 ppm encompass amides and lactams. Secondary amides occur in the range from 133 to 105 ppm, primary amides from 109 to 102 ppm, and lactams from 120 to 113 ppm. Assignments for the 15N NMR spectra are summarized in Table V. These are the best assignments we are able to make at this time. Complete 15NNMR chemical shift ranges are not available for all classes of nitrogen compounds relevant to this study, and this limits the ability to correlate spectral peaks with classes of nitrogen compounds. The INEPT spectrum shown in Figure 3C was acquired using polarization-transfer times and refocusing delays optimized for signal enhancement of singly protonated nitrogens; only protonated nitrogens show up in the spectrum. The peaks in the quantitative spectrum (3A) assigned as amides and lactams are significantly enhanced in intensity in the INEPT spectrum. The decrease in intensity of these peaks in the ACOUSTIC spectrum (3B) compared to the quantitative spectrum (3A) indicates that these 15Nnuclei have unfavorable NOE factors. Thus, in order to observe these peaks in any detail, polarization transfer must be used. In the quantitative spectrum (3A), isocyanide resonances merge into the hydroxamic acid resonances in the peak from 191 to 160 ppm; in the INEPT spectrum, the hydroxamic acid nitrogens, corresponding to the peak centered at 172 ppm, are resolved from the isocyanide nitrogens. The INEPT spectrum also indicates that nitrogens downfield of 180 ppm in the quantitative spectrum are nonprotonated, assuming none of these nitrogens are bonded to protons exchanging too fast for polarization transfer to occur. There is a minor peak at 42 ppm in the INEPT spectrum, which is not evident in the quantitative or ACOUSTIC spectra. This peak most likely represents primary amine nitrogen, which again can be explained as a Beckmann reaction product (40). As a final observation, the spin echo in the INEPT pulse sequence apparently serves to alleviate acoustic ringing as evidenced by the flat base line in the INEPT spectrum. DEPTGL 15NNMR spectra also were recorded on the Suwannee fulvic acid, but not shown here, to discriminate between singly and doubly protonated nitrogens in the polarization-transfer spectra. These spectra indicated that the peak from about 115 to 90 ppm consisted mainly of doubly protonated nitrogens with some contribution from
-
112
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singly protonated nitrogens. This peak therefore comprises mainly primary amides with some overlap of lactams. This corrects our earlier report in which the peak from 115 to 90 ppm was assigned as primarily lactam nitrogen (14). The DEPTGL I5N NMR spectra also confirmed that the peaks downfield of -115 ppm are singly protonated nitrogens, which is consistent with their assignments as secondary amides (150-1 15 ppm) and hydroxamic acids (170-160 ppm). Peak areas were measured on an ACOUSTIC spectrum of the Suwannee fulvic acid (not presented here) recorded with the addition of the paramagnetic relaxation reagent Cr(Acac)3. The nitrosophenol-oxime peak (430-330 ppm) comprised approximately 67 %, the nitrile peaks (270-240 ppm) lo%, and the peaks from 216 to 160 ppm (imidates, amidines, imidazoles, isocyanides, and hydroxamic acids) 23% of the total nitrogen in the spectrum. The nitrosophenol-oxime peak was integrated from 430 to 390 ppm and from 390 to 330 ppm, indicating that of the total amount of ketone and quinone groups derivatized, approximately 23% represent quinone monoximes and 77% represent ketoximes or quinone dioximes. ACOUSTIC and INEPT I5N NMR spectra of the hydroxylamine-derivatized Armadale fulvic and soil, peat, and leonardite humic acids are presented in Figure 4. All four sets of spectra show the same resonances that are present in the Suwannee River fulvic acid spectra. There are differences in the chemical shift positions of peak maxima and in the relative intensities of the various peaks. For example, the three distinct peaks in the region from about 216 to 170 ppm in the ACOUSTIC spectra of the Armadale fulvic and soil humic acid differ in their relative intensities. The relative intensities of the peaks assigned as lactam, amide, and hydroxamic acid nitrogen in the INEPT spectrum of the leonardite humic acid differ from the other four samples. The quinone monoxime-nitrosophenol shoulder is not as clearly resolved in the ACOUSTIC spectrum of the soil humic acid compared to the Armadale fulvic acid. Primary amine peaks of minor intensity are evident in the INEPT spectra of the Armadale and peat samples. The overall similarity of the 15NNMR spectra, however, suggests that, despite the diverse nature and origins of the five humic and fulvic acid samples, carbonyl groups must occur in structural configurations which are common to all five samples. Trace quantities of hydroxamic acids have been reported in marine fulvic acids (45). Naturally abundant 15Nnuclei were not observed in ACOUSTIC and INEPT 15NNMR spectra recorded on underivatized Suwannee fulvic and soil and peat humic acids. This confirms the fact that the signals assigned as hydroxamic acids in the spectra of the oximated samples arise from the reaction of hydroxylamine with ester groups in the samples and do not represent naturally occurring hydroxamic acids which might possibly exist in the samples. Finally, the quantitative I3C NMR spectra of the Suwannee fulvic acid and leonardite humic acid derivatized with unlabeled hydroxylamine are presented in Figure 5. The 13CNMR chemical shift range of the carbon-nitrogen double bond in the oxime group is about 155-167 ppm; the increase in intensity in this region is apparent compared to the spectra of the underivatized samples in Figure 1. The persistence of the peaks from 220 to 180 ppm in the spectra of Figure 5 indicates that not all of the ketone and quinone carbonyl functional groups are converted to oximes under the conditions of the derivatization reaction. The presence of hindered ketones and quinones is therefore indicated. This result suggests that previous studies
B
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Armadale Soil FA
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I
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IHSS Soil HA
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I
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I
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I
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500
400
300
200
100
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400
300
200
100
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Figure 4. Liquid-phase I5N NMR spectra of Armadale fulvic, soil humic, peat humic, and leonardlte humic acids derivatized with '5N-labeled hydroxylamine: (A) ACOUSTIC spectra; (8) INEPT spectra.
which have measured the carbonyl content of humic substances based upon derivatization with hydroxylamine have probably underestimated the carbonyl content, notwithstanding error introduced through reaction of hydroxylamine with esters. Implication of 15N NMR Spectra. The 15N NMR spectra have provided evidence for the reaction of hydroxylamine with ketone, quinone, and ester functional groups present in the humic and fulvic acid samples. The amount of hydroxamic acid formed from the reaction of hydroxylamine with esters in the samples appears to be minor. That hydroxylamine does react with esters in the samples, however, is consistent with observations we have made in a study on the fixation of ammonia by humic substances. Primary amides resulting from aminolysis of
esters were observed in 15NNMR spectra of the Suwannee, peat, and leonardite samples, all of which had been allowed to react with 15NH40H(46). The presence of quinone groups in humic substances has always been the subject of some contention (13). For example, conflicting views regarding the detection of quinones in IR spectra of both acetylated and underivatized humic substances can be found in the literature (13). Reports in which quinone groups were measured by reduction with tin chloride have determined that quinones comprise a significant fraction of the total ketone plus quinone functionality as measured by oximation (45,47). Studies which have examined the condensation of aniline compounds with humic substances have implicated quinone groups as major sites for nucleophilic addition by the Environ. Sci. Technol., Vol. 26, No. 1, 1992
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Suwannee River FA
quinones. Numerous examples of cyclic ketoximes which have been converted to lactams via the Beckmann rearrangement have been documented (41); the monoximes of anthraquinone and phenanthraquinone have been reported to rearrange to their corresponding lactam and imide, respectively (53). The largest class of oximes that undergo fragmentation to nitriles are oximes that have quaternary carbon centers adjacent to the oxime carbon, because of the stability of the carbonium ion which is cleaved (27,41, 54). Examples include monoximes of quinones (53))and oximes of a-diketones, a-keto acids, a-hydroxy ketones, etc. (27, 41, 54). Thus the presence of nitriles in the ACOUSTIC 15N NMR spectra suggests the presence of quinones and of ketones a to quaternary carbons, in both cyclic and alicyclic structures, in the humic samples. As discussed above, the incomplete derivatization of the samples indicated by the spectra in Figure 5 suggests the presence of ketone and quinone groups in the humic samples that are unreactive to hydroxylamine either through steric hindrance, hydrogen-bonding, or other electronic effects. Examples of ketones unreactive to hydroxylamine have been reported (30,55). Hydroxyquinones of the types 1-111 are also known not to react completely with hydroxylamine (13).
-DMSO-dG
Hydroxylamine derivatized LB=50
I
-DMSO-dG
Leonardite HA Hydroxylamine derivatized LB=80
300
200
100
0
-100
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Flgure 5. Quantitative liquid-phase I3C NMR spectra of Suwannee River fulvic acid and leonardite humic acid derivatized with unlabeled hydroxylamine.
anilines ( 3 , 4 ) . Probably the most convincing evidence in favor of the presence of quinone groups in humic substances has come from ESR studies (48-51). The results of the present study provide strong confirmatory evidence for the presence of quinones in humic and fulvic acids through the observation of the quinone monoxime-nitrosophenol shoulder in the ACOUSTIC 15NNMR spectra. The monoximenitrosophenol shoulder represents only the monoxime derivatives of quinones. Dioxime derivatives of quinones would overlap with the oximes of ketones in the range from 330 to 390 ppm. Thus it is not possible to differentiate between the total amount of quinone versus ketone from the 15NNMR spectra. Interestingly, Porter (12) suggested that the quantities and distributions of nitrogen gases produced upon reaction of sodium nitrite with hydroxylamine-derivatizedsoil and leonardite humic acids were consistent with the presence of quinone monoximes and dioximes. Further 15NNMR evidence for the addition of amine compounds to quinone groups in our samples has been provided in two of our other studies. The Michael addition products of aniline with quinone groups were detected in Suwannee fulvic acid which had been reacted with aniline at pH 6 and room temperature (52). Amino hydroquinone nitrogen, also the result of Michael addition of ammonia to quinone groups, was observed in the ammonia-fixated Suwannee and peat samples (46). Although a thorough analysis is beyond the scope of this paper, some further information on the structural nature of the ketone and quinone functionality present in the humic and fulvic acids may be inferred from a consideration of the factors which dictate the course of the Beckmann reactions. For example, the sources of lactams, the possible presence of which is indicated by the peaks in the region from 115 to 90 ppm in the INEPT I5N NMR spectra, are both oximes of cyclic ketones and oximes of 114
Environ. Sci. Technol.. Vol. 26, No. 1, 1992
I
II
Ill
Conclusion Derivatization with 15N-labeledhydroxylamine followed by 15N NMR analysis has provided new insight into the carbonyl functionality of the humic and fulvic acids examined in this study. Both 13C and 15N NMR analyses have provided complementary information on the nature of the carbonyl groups. Chemical shift considerations in the quantitative 13C NMR spectra of the underivatized samples indicate that diaryl, alkyl aryl, and dialkyl ketones all occur in the samples. DEPTGL 13CNMR spectra of the underivatized humic substances indicate that aldehydes are not present in the samples, or at least are not present in significant enough concentrations to be detected by NMR. The 15NNMR spectra have demonstrated that quinones and esters occur in the samples, information not discernible from the 13C NMR spectra. The 13C NMR spectra of the oximated samples indicate that a significant fraction of the ketone and quinone groups do not react with hydroxylamine, either because of steric hindrance, hydrogen-bonding, or other electronic effects. The formation of nitriles and lactams through Beckmann reactions, as demonstrated in the 15NNMR spectra, provides additional but indirect evidence for the presence of quinones and also suggests the possible presence of cyclic ketones and ketones a to quaternary carbons. All this information reaffirms the structural complexity of the humic and fulvic acid samples by demonstrating the large multiplicity of individual structural configurations that occurs in the carbonyl functionality alone. The experimental difficulties associated with liquidphase 15NNMR were discussed at the outset. These include unfavorable NOE factors, long Ti's, acoustic ringing, and nitrogens bonded to protons exchanging too rapidly so as to prevent polarization transfer. The problems of unfavorable NOE factors and acoustic ringing have been
encountered in the 15N NMR spectra of the hydroxylamine-derivatized samples. In the ammonia fixation study (46))we noted that resonances assigned as amino hydroquinone nitrogens could not be observed in INEPT 15N NMR spectra because of exchange and were barely detected in spectra recorded under quantitative conditions. In this instance, retention of the NOE was necessary to observe the resonances in full detail. Thus it appears that when 15N NMR is used to analyze humic substances reacted with 15N-labeledreagents, each case must be treated individually, and precautions must be taken for all complications that can arise. Registry No. HONH2, 7803-49-8.
Literature Cited (1) Nommik, H.; Vahtras, K. In Nitrogen in Agricultural Soils; Stevenson, F. J., Ed. Agronomy 1982,22, Chapter 4. (2) Stevenson, F. J. Cycles of Soil-Carbon, Nitrogen, Phosphorus, Sulfur, Micronutrients; John Wiley and Sons: New York, 1986; Chapter 4. (3) Parris, G. E. Environ. Sci. Technol. 1980,14, 1099-1106. (4) Hsu, T. S.; Bartha, R. Soil Sci. 1974, 116, 444-452. (5) Simmons, K. E.; Minard, R. D.; Bollag, J.-M. Environ. Sci. Technol. 1989,23, 115-121. (6) Jensen, J. N.; Johnson, J. D.; St. Aubin, J.; Christman, R. F. Org. Geochem. 1985, 8(1), 71-76. (7) Denitrification, Nitrification, and Atmospheric Nitrous Oxide;Delwiche, C. C., Ed.; Wiley-Interscience: New York, 1980. (8) Nelson, D. W. Proc. Indiana Acad. Sci. 1978,87,409-413. (9) Markelova, V. N. Dokl. Vses. Akad. Skh. Nauk im. V.I. Lenina 1979, 12, 32-35. (10) Schnitzer, M.; Gupta, U. C. Soil Sci. SOC.Am. Proc. 1964, 28,374-377. (11) Schnitzer, M.; Skinner, S. I. M. Soil Sci. 1966,101,120-124. (12) Porter, L. K. Soil Sci. SOC.Am. Proc. 1969, 33, 696-702. (13) Stevenson, F. J. Humus Chemistry-Genesis, Composition, Reactions;John Wiley and Sons: New York, 1982; Chapter 9. (14) Thorn, K. A.; Folan, D. W.; Arterburn, J. B.; Mikita, M. A.; MacCarthy, P. Sci. Total Environ. 1989,81/82,209-218. (15) Fritz, J. A.; Yamamara, S. S.; Bradford, E. C. Anal. Chem. 1959, 31, 260-262. (16) Sorensen, 0. W.; Donstrup, S.; Bildsoe, H.; Jakobsen, H. J. J . Magn. Reson. 1983,55, 347-354. (17) Thorn, K. A. In Humic Substances in the Suwannee Riuer,
Georgia: Interactions, Properties, and Proposed Structures; Averett, R. C., Leenheer, J. A., McKnight, D. M., Thorn, K. A,, Eds. Open-File Rep.-U.S. Geol. Surv. 1989, NO.87-557, 251-309. (18) Thorn, K. A.; Folan, D. W.; MacCarthy, P. Characterization
(19) (20) (21) (22)
(23) (24) (25) (26)
of the IHSS Standard and Reference Fulvic and Humic Acids By Solution State 13Cand 'H NMR. Water-Resour. Invest. (U.S. Geol. Suru.) 1991, No. 89-4196. Thorn, K. A.; Steelink, C.; Wershaw, R. L. Org. Geochem. 1987, 11(3), 123-137. Patt, S . L. J . Magn. Reson. 1982, 49, 161-163. Morris, G. A.; Freeman, R. J . Am. Chem. SOC.1979,101, 760-762. Witanowski, M.; Stefaniak, L.; Webb, G. A. In Annual Reports on NMR Spectrometry; Webb, G. A., Ed.; Academic Press, Harcourt Brace Jovanovich London, 1986; voi. 18. Levy, G. C.; Lichter, R. L. Nitrogen-15Nuclear Magnetic Resonance Spectrometry;John Wiley and Sons: New York, 1979. Sorensen, 0. W.; Jakobsen, H. J. In Pulse Methods in ID and 2 0 Liquid-Phase NMR; Brey, W. S., Ed.; Academic Press: New York, 1988; Chapter 3. von Philipsborn, W.; Muller, R. Angew. Chem., Int. Ed. Engl. 1986,25,383-486. Sandler, S. R.; Karo, W. Organic Functional Group Preparations, 2nd ed.; Academic Press, Harcourt Brace Jovanovich: New York, 1989.
(27) March, J. Advanced Organic Chemistry; John Wiley and Sons: New York, 1985. (28) Dayagi, S.; Degani, Y. In The Chemistry of the CarbonNitrogen Double Bond; Patai, s.,Ed.; John Wiley: New York, 1970; Chapter 2. (29) Smith, P. A. S. The Chemistry of Open-Chain Organic
Nitrogen Compounds, VolumeII. Derivatives of Oxidized Nitrogen: Hydrazines to Nitrates; W. A. Benjamin: New York, 1966. (30) Pearson, D. E.; Keaton, 0. D. J. Org. Chem. 1962, 28, 1557-1558. (31) Finley, K. T. In Chemistry of the Quinonoid Compounds, Part 2; Patai, S., Ed.; John Wiley and Sons: London, 1974; Chapter 17. (32) Finley, K. T.; Tong, L. K. J. In The Chemistry of the Carbon-Nitrogen Double Bond; Patai, S.,Ed.; John Wiley and Sons: New York, 1970; Chapter 14. (33) Witanowski, M.; Stefaniak, L.; Januszewski, H.; Szymanski, S.; Webb, G. A. Tetrahedron 1973,29, 2833-2836. (34) Fitton, A. 0.; Smalley, R. X. Practical Heterocyclic Chemistry; Academic Press: New York, 1968. (35) Katritaky, A. R.; Bird, C. W.; Boulton, A. J.; Cheeseman, G. W. H.; Lagowski, J. M.; Lwowski, W.; McKillop, A,; Potts, K. T.; Rees, C. W. Handbook of Heterocyclic Chemistry; Pergamon: New York, 1985. (36) Weller, D. D.; Luellen, G. R.; Weller, D. L. J.Org. Chem. 1982,42,4803-4806. (37) Unterhalt, B.; Reinhold, H. J. Arch. Pharm. 1983,316(1), 68-75. (38) Diaz, E. d; Barrios, H.; Ortiz, B.; Sanchez-Obregon, R.; Yuste, F. Magn. Reson. Chem. 1989, 719-724. (39) Chantegrel, B.; Nabi, A. I.; Gelan, S. J. Org. Chem. 1984, 49,4419-4424. (40) Donaruma, L. G.; Heldt, W. Z. In Organic Reactions; Cope, A. C., Ed.; John Wiley & Sons: New York, 1960; Vol. 11, pp 1-156. (41) Gawley, R. E. In Organic Reactions;Kende, A. S., Ed.; John Wiley: New York, 1987; Vol. 35, pp 1-420. (42) Fukushima, E.; Roeder, S. B. W. Experimental Pulse NMR. A Nuts and Bolts Approach; Addison-Wesley: Reading, MA, 1981. (43) Gillam, A. H.; Wilson, M. A. In Aquatic and Terrestrial Humic Materials; Christman, R. F., Gjessing, E. T., Eds.; Ann Arbor Science: Ann Arbor, MI, 1983; pp 25-35. (44) Martin, G. J.; Martin, M. L.; Gouesnard, J. P. 15N-NMR Spectroscopy; Springer-Verlag: New York, 1981. (45) Gillam, A. H.; Riley, J. Anal. Chin. Acta 1982,141,287-299. (46) Thorn, K. A.; Mikita, M. A. Sci. Total Environ., in press. (47) Griffith, S. M.; Schnitzer, M. Soil Sci. SOC.Am. Proc. 1975, 39,861-867. (48) Steelink, C. In NMR of Humic Substances and CoalTechniques, Problems, and Solutions; Wershaw, R. L., Mikita, M. A., Eds.; Lewis Publishers: Chelsea, MI, 1987; pp 47-72. (49) Senesi, N.; Steelink, C. In Humic Substances II. In Search of Structure; Hayes, M. H. B., MacCarthy, P., Malcolm, R. L., Swift, R. S., Eds.; John Wiley and Sons: West Sussex, England; 1989, pp 373-408. (50) Steelink, C. Geochim. Cosmochim. Acta 1964, 28, 1615-1622. (51) Saleh, F. Y.; Theriot, L. J.; Amani, S. K.; Kim, I. In Humic
Substances in the Suwannee River, Georgia: Interactions, Properties, and Proposed Structures; Averett, R. C., Leenheer, J. A., McKnight, D. M., Thorn, K. A., Eds. Open-File R e p . 4 J . S . Geol. Surv. 1989, No. 87-557, 119-133. (52) Weber, E. J.; Baughman, G. L.; Clodfelter, K. H.; Thorn, K. A. 5th International Meeting of the International Humic Substances Society, Nagoya, Japan, August 6-10, 1990. (53) Hodge, P. In The Chemistry of the Quinonoid Compounds, Part 2; Patai, S., Ed.; John Wiley and Sons: New York, 1974; pp 579-616. (54) McCarty, C. G. In The Chemistry of the Carbon-Nitrogen Double Bond; Patai, S., Ed.; John Wiley and Sons: New York, 1970; pp 363-464. (55) Kadesch, R. G. J . Am. Chem. SOC.1944,66, 1207-1213. Environ. Sci. Technol., Vol. 26, No. 1, 1992
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(56) Humic Substances in the Suwannee River, Georgia: Znteractions, Properties, and Proposed Structures; Averett, R. C., Leenheer, J. A., McKnight, D. M., Thorn, K. A., Eds. Open-File Rep.-U.S. Geol. Surv. 1989, No. 87-557. (57) Underdown, A. W.; Langford, C. H.; Gamble, D. S. Anal. Chem. 1981,53, 2139-2140.
(58) Breitmaier, E.; VoeIter, W. Carbon-13 NMR Spectroscopy, 3rd ed.; VCH: New York, 1987; pp 218-219. Received for review February 12, 1991. Revised manuscript received June 17, 1991. Accepted July 18, 1991.
Mass Spectrometric Analysis for Aromatic Compounds in Bile of Fish Sampled after the Exxon Valdez Oil Spill Margaret M. Krahn," Douglas G. Burrows, Gina M. Ylitaio, Donald W. Brown, Catherine A. Wlgren, Tracy K. Collier, Sin-Lam Chan, and Usha Varanasi
Environmental Conservation Division, Northwest Fisheries Science Center, National Marine Fisheries Service, National Oceanic and Atmospheric Administration, 2725 Montlake Boulevard East, Seattle, Washington 98 1 12 After the Exxon Valdez oil spill, the exposure of marine organisms to petroleum had to be determined. Gas chromatography/mass spectrometry was used to identify metabolites of aromatic compounds (ACs), such as alkylated naphthols, phenanthrols, and dibenzothiophenols, in the hydrolyzed bile of five salmon (Oncorhynchus gorbuscha) and four pollock (Theragra chalcogramma) captured in Prince William Sound several months after the oil spill. These metabolites were not found in control fish sampled from areas not impacted by the oil. The metabolites were identified by comparison to those from the hydrolyzed bile of a halibut (Hippoglossus stenolepis) which had been injected with weathered Prudhoe Bay crude oil. The dibenzothiophenols are proposed as promising marker compounds for identifying the exposure of fish to certain crude oils. In addition, a high-performance liquid chromatographic method to screen bile for metabolites of ACs was validated for use in estimating the exposure of fish to petroleum. Introduction
Following the spill of 11million gallons of Prudhoe Bay crude oil (PBCO) from the Exxon Valdez into Prince William Sound (PWS), AK, in March 1989, analyses to determine oil exposure in the biota along the path of the spill were essential. The degree of exposure of marine organisms to oil is often assessed by measuring their body burden of petroleum-related aromatic compounds (ACs), because ACs are potentially harmful to the animals ( I ) . However, fish and marine mammals extensively metabolize most ACs in their livers and then the metabolites are excreted, predominantly into bile (2-6). A rapid screening method for bile, which determines the metabolites as fluorescent ACs (FACs), has proven useful in estimating the exposure of fish and marine mammals to petroleum (7,B). But this screening method is limited to providing relative concentrations of FACs in bile; individual metabolites are not identified and quantitated. Accordingly, detailed chemical analyses are needed to determine the concentrations of individual metabolites of petroleum-related ACs in selected bile samples and, thus, to support the results of the semiquantitative bile screening method showing exposure of marine organisms to spilled oil. The specific individual metabolites that result from the uptake and metabolic conversion of petroleum ACs in fish have not been well-characterized. Two previous studies identified only a few individual AC metabolites in the livers (2) or bile ( 3 )of fish that had been exposed to no. 2 fuel oil, a distillate fraction of petroleum that contains only a portion of the ACs found in crude oil. In a more detailed 116
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study, several individual metabolites were identified by gas chromatography/mass spectrometry (GC/MS) in the bile of fish captured from urban sites (9, 10). However, urban sediments generally contain high proportions of products from the combustion of fossil fuels, such as unsubstituted, four- to six-ring ACs. In contrast, PBCO contains a variety of one- to three-ring alkylated ACs, as well as the alkylated dibenzothiophenes typical of the North Slope crude oils (11-14). But because spilled oil is degraded in time by physical, chemical, and microbial processes, the aromatic fraction of the weathered oil will be dominated by those ACs (e.g., highly alkylated naphthalenes, phenanthrenes, and dibenzothiophenes) that are most resistant to weathering (15,16). Therefore, metabolites of these resistant ACs should be found in bile of fish exposed to weathered crude oil; in this paper, we report the results of our efforts to characterize such compounds. Initially, the products resulting from the metabolism of PBCO by fish were identified by GC/MS in the enzymatically hydrolyzed bile of a halibut (Hippoglossus stenolepis) and a Dolly Varden (Salvelinus malma) injected with weathered PBCO. Subsequently, many of these metabolites were determined in the hydrolyzed bile of five pink salmon (Oncorhynchus gorbuscha) and four pollock (Theragra chalcogramma) collected in PWS several months after the oil spill. Finally, the bile screening method was validated by demonstrating a strong statistical correlation between concentrations of FACs determined by screening and the sums of metabolite concentrations determined by GC/MS in oil-exposed fish. Experimental Section
Chemicals. A sample of PBCO was obtained from the oil remaining in the hold of the Exxon Valdez, and the weathered PBCO sample was collected from PWS 11days 6after the spill occurred. 2,6-Dimethyl-3-naphthol, methyl-2-naphthalenemethanol, and trans-3,4-dihydroxy-3,4-dihydro-2,6-dimethylnaphthalene were prepared in our laboratories (17). Reference Standard. A GC/MS standard, containing reference compounds dissolved in methanol, was prepared (listed in order of GC elution, ng/pL): 2,6-dibromophenol (surrogate standard, 7.38), hexamethylbenzene (GC internal standard, 7.56), 1-naphthol (15.66), 2-hydroxybiphenyl (15.72), 6-methyl-%naphthalenemethanol (2.76), 4-methyl-l-naphthol (15.42), 2,6-dimethyl-3-naphthol (2.82), trans-3,4-dihydroxy-3,4-dihydro-2,6-dimethylnaphthalene (3.36), 9-fluorenol (15.54), phenanthrene-d,, (HPLC internal standard, 6.00), 9-phenanthrol (15.06), 9-anthracenemethanol (14.76), and l-pyrenol (13.80).
Not subject to U.S. Copyright. Published 1991 by the American Chemical Society
