This work was presented at "EPRW 2002". It illustrates the identification of 3-sec-butyl-6-methyluracil, a metabolite isolated in groundwater, probably derived from herbicide "Bromacil" (3-sec-butyl-5-bromo-6-methyluracil).

1. IDENTIFICATION OF A “METABOLITE” IN GROUNDWATER, WITH STRUCTURE
RELATED TO BROMACIL: 3-SEC-BUTYL-6-METHYLURACIL
Tiziano VENDRAME, Alfredo MUSSATO
ARPAV - Agenzia Regionale per la Prevenzione e Protezione Ambientale del Veneto -
Servizio Laboratori del Dipartimento Provinciale ARPAV di Treviso - Via D’Annunzio 12,
31100 Treviso - Italy
e-mail: tvendrame@arpa.veneto.it - amussato@arpa.veneto.it
During groundwater controls it is not unusual detect compounds that are different from
analysis’ object. Most of analytical “artifacts” (e.g. reagent contaminants, release from contact
materials, stationary phase degradation in GC) are well known interferences. Typical
examples are phthalates and silicon derivatives.
A lot of other compounds really present in sample are usually overlooked, if they are
not analysis’ targets, especially in routine analyses that are conditioned from time and cost.
However, for an effective aquifer control, the simple search of compounds from “rigid”
lists is inadequate. The appearance of new “pollutants” in known aquifers deserves a deeper
study.
In our case, attention was attracted from a new chromatographic peak (Fig. 1a - 1b),
noted in routine analyses of triazinic herbicides (SPE technique, C8 phase), for two main
reasons:
• The presence of an unusual herbicide/pesticide nitrogen containing was suspected from
positive response to NPD detector;
• The distribution in a delimited area, near a small town, in repeated samples (this excluded
an analytical artifact).
Last peculiarity, since the town is aqueduct lacking, spurred the research on this
unknown compound.
Initial GC-MS screening was a poor aid: our library (Wiley 275.l) is lacking of wanted
spectra (Fig. 1c). Other libraries also gave no useful data.
The feeling on a possible new pesticide press to extend sampling, and then
circumscribe “polluted” area, until exhausted gravel quarry. The lacking of upstream wells
stopped the research for long time.
Later the starting point of pollution was identified in a closed landfill, six kilometers
away. The “unknown” compound was detected only in downstream groundwater wells and in
landfill leachate. In spite of a well-known leak from this landfill, the finding of a pollutant,
although in trace only, so remote from origin, was an unexpected finding (Fig. 2).
Probably the landfill leak started two year ago, at least, with peaks of 100 mg/l of
ammonia in the nearest wells. However, the “common” pollutants (e.g. ammonia, chloride
etc.) are not detectable in aquifer some hundreds meters downstream, notwithstanding the
gravel ground.
After source’s discovery, it was urgent try compound identification, characterized from:
• mass spectra with feeble molecular ion, mass = 182 (1-2%), near other peaks, sources of
possible confusion;
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2. • base ion mass = 127 (100%)
• characteristic masses: 167 (5-6%), 153 (10%), 110 (23%), 84 (30%), 70 (8-9%);
• halogen lacking from spectra and ECD detector behaviour.
Structure assignation started from a molecule with spectra similar to unknown
compound, the 3-propyl-6-methyluracil, selected (visually) among those singled from
research program of our library (Fig. 3).
This have MW (molecular weight) 168, whereas unknown have MW 182, but shows
similar fragmentation feature, at lower mass. Then it was hypotized an additional CH3 or CH2
in “unknown”, and it was outlined some possible isomers and keto-enolic tautomers (12 with
MW 182 ad 4 with MW 168 for comparison). From “Beilstein Handbuch der Organische
Chemie” (“on-line” version), resulted 5 known compounds, but the only one with right
spectrum is 3-sec-butyl-6-methyluracil (ref. 1 for spectrum description - Fig. 5 for structure)
This compound is not commercially available, but starting from few articles, which
cited this substance in last years, it was contacted at last Prof. J. Suflita (Univ. Oklahoma).
He supplied kindly a sample of this compound, which shows chromatographic behaviour and
spectra identical with “unknown” (Fig. 4).
Later, the agreement of fragmentation pattern with specialized literature (2-3) was
checked.
With this standard was possible to measure concentrations in groundwater which
increase from about 0,1 µg/l near the town (six kilometers away from landfill), to 10 µg/l in
wells near the landfill, and 5000 µg/l in landfill leachate (see ref. 5 for comparison standards).
The origin of this compound in landfill is still obscure. From patent literature results a
possible use as intermediate in “Bromacil” synthesis (3-sec-butyl-5-bromo-6-methyluracil), via
bromuration. Bromacil is a well-known herbicide, but with limited use in North Italy. No other
industrial use was quoted in available literature. A possible source is also microbiological
debromination of Bromacil, in anoxic conditions (see ref. 4 - A. Neal, J. Suflita), similar to
landfill interior.
Then, the compound can be buried directly in landfill, or it can derive from Bromacil
residues degraded in landfill. The Bromacil research gives negative results; however could
not be ruled out the second hypothesis, because of possible fast degradation of Bromacil in
anoxic environment.
The topical problem is to decide if polluted water (at low level) is allowable as drinking
water.
Bibliography and notes:
1. A. Acher, C. Hapeman J. Agric. Food Chem. 1994, 42, pp. 2040-2047: Comparison of
formation and biodegradation of bromacil oxidation products in aqueous solutions;
2. J. M. Rice J. of the American Chemical Society, (1965) pp. 4569-4576: Mass
spectra of nucleic acid derivatives. Pyrimidines;
3. E. Falch Acta Chemica Scandinavica, 24 (1970) pp.137-144: Mass spectra of
Pyrimidines (Part I. N-Alkiluracils);
4. A. Neal, J. Suflita Appl. Environ. Microbiol. 56 (1), pp. 292-294: Reductive
dehalogenation of a nitrogen heterocyclic herbicide in anoxic aquifer slurries.
5. A second sample of standard was synthesized at laboratory of "Istituto Tecnico Industriale
Statale E. Fermi" in Treviso, from R. Scandiuzzi (Laboratory Teacher) and his students.
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3. Chromatographic behaviour and spectrum was identical with sample from Prof. J. Suflita
(Fig. 7).
Illustration Index:
Fig. 1 a: chromatogram of typical water well extract (SPE technique, C8 phase).
Fig. 1 b: as 1 a, enlarged; the "unknown" peak is at 8,9 min (red).
Fig. 1 c: spectrum of 3-sec-butyl-6-methyluracil from the same extract of 1 a.
Fig. 2: spatial distribution of 3-sec-butyl-6-methyluracil (red: positive; blue: negative).
Fig. 3: spectrum of 3-propyl-6-methyluracil (Wiley 275.l).
Fig. 4: spectrum of 3-sec-butyl-6-methyluracil (sample from Prof. J. Suflita).
Fig. 5: structures of 3-sec-butyl-6-methyluracil and "Bromacil" (5-bromo3-sec-butyl-6-
methyluracil).
Fig. 6: possible relations between 3-sec-butyl-6-methyluracil and "Bromacil" .
Fig. 7: spectrum of 3-sec-butyl-6-methyluracil (sample from I.T.I.S. "E. Fermi" -
Treviso).
Fig 1a Index
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4. Fig 1b Index
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5. Fig1c Index
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6. 6

7. Fig 2 Index
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8. Fig 3 Index
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9. Fig 4 Index
Fig 5 Index
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10. Fig 6 Index
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11. Fig 7 Index
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12. 12

13. 13