Nitro musks, nitro musk amino metabolites and polycyclic musks in sewage sludges. Quantitative determination by HRGC-ion-trap-MS/MS and mass spectral characterization of the amino metabolites

Article abstract of DOI:10.1016/S0045-6535(99)00325-2

Nitro- and polycyclic musks were quantified in sewage sludge samples from different catchment areas using high-resolution gas chromatography (HRGC) and ion-trap MS/MS. Collision induced dissociation (CID) turned out to be a useful tool for quantification

Full text of DOI:10.1016/S0045-6535(99)00325-2

Chemosphere 40 (2000) 565±574
Nitro musks, nitro musk amino metabolites and polycyclic
musks in sewage sludges
Quantitative determination by HRGC-ion-trap-MS/MS and
mass spectral characterization of the amino metabolites
D. Herrena, J.D. Bersetb,*
a
Interlabor Belp AG, Birkenweg 6, CH-3123 Belp, Switzerland
Institute of Environmental Protection and Agriculture (IUL), Department of Soil Protection and Ecotoxicology, Schwarzenburgstrasse
155, CH-3003 Berne, Switzerland
b
Received 11 June 1999; accepted 3 August 1999
Abstract
Nitro- and polycyclic musks were quanti®ed in sewage sludge samples from di€erent catchment areas using high-
resolution gas chromatography (HRGC) and ion-trap MS/MS. Collision induced dissociation (CID) turned out to be a
useful tool for quanti®cation of the analytes. Negative chemical ionization (NCI) quadrupole MS in the selected ion
mode (SIM) showed similar sensitivities compared to ion trap MS/MS. Among the nitro musks, musk ketone (MK) and
musk xylene (MX) were the main compounds in predominantly domestic sewage sludges, found at low lg/kg dry matter
(d.m.) whereas polycyclic musks were present in domestic as well as in industrial sludges up to 12 mg/kg d.m. Ga-
laxolide (HHCB) and Tonalide (AHTN) were the major polycyclic musks found in the sludges. Amino metabolites of
the nitro musks, amino musk xylene (AMA), amino musk moskene (AMM) and amino musk ketone (AMK) were
detected for the ®rst time in sewage sludges, and reached partly higher concentrations compared to the parent com-
pounds. Ó 1999 Elsevier Science Ltd. All rights reserved.
Keywords: Nitro musks; Polycyclic musks; Amino metabolites; Sewage sludge; HRGC-ion-trap MS/MS; HRGC-EI/PCI- and NCI
quadrupole MS
1. Introduction
in agriculture (Kettler, 1998). In the last couple of years
agriculture has moved towards more and more envi-
ronmental friendly techniques with the goal of pro-
ducing high quality food products. This also requires
the use of fertilizers of high quality. For sewage sludge
and compost limit values for heavy metals have been
The amendment of sewage sludge as fertilizer to
agricultural land represents an economical way to use
the high amounts of sludge produced by the wastewater
treatment plants. Currently, approximately 2.1 Â 108 kg
(dry matter) of sewage sludge are produced annually in
Switzerland, of which 1.07 Â 108 kg (51%) are applied


established (Verordnung uber umweltgefahrdende
Sto€e, 1996). These inorganic compounds are analyzed
on a routine basis. However, the characterization and
long-term observation of organic contaminants in
sludge has received little attention so far due to the
absence of limit values. It is well known that sewage
sludge contains many xenobiotic (anthropogenic)
* Tel.: +41-31-323-8398; fax: +41-31-323-8415.
E-mail address: jean-daniel.berset@iul.admin.ch
(J.D. Berset).
0045-6535/00/$ - see front matter Ó 1999 Elsevier Science Ltd. All rights reserved.
PII: S 0 0 4 5 - 6 5 3 5 ( 9 9 ) 0 0 3 2 5 - 2

566
D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
organic chemicals which might have a negative impact
on soil organisms and fertility (Tarradellas and Die-
rcxsens, 1983; Drescher et al., 1989; Demirjian et al.,
2. Experimental section
2.1. Laboratory contamination with musk compounds

1987; Klop€er, 1996; Lega et al., 1997; Halling-Soren-
sen et al., 1998). Among these, non-polar and highly
lipophilic compounds like polycyclic aromatic hydro-
carbons (PAHs), polychlorinated biphenyls (PCBs),
polychlorinated dioxins (PCDD) and dibenzofurans
(PCDF) are important contaminants as they persist for
a long time in the di€erent environmental compart-
ments. Quite recently, two other groups of compounds
exhibiting similar chemical and physical properties like
those mentioned above, have been described to occur as
ubiquitous contaminants: nitro musk and polycyclic
musk substances. The nitro musks include a group of
®ve synthetic alkylated nitro benzenes with a typical
musk odor: musk moskene (MM), musk tibetene (MT),
musk xylene (MX), musk ketone (MK) and musk am-
brette (MA). The polycyclic musks are chemically al-
kylated tetralin or indane systems. Main representatives
of the polycyclic musk are HHCB (Galaxolide), AHTN
(Tonalide), ADBI (Celestolide), AHMI (Phantolide)
and AITI (Traseolide). These compounds are widely
used as fragrance ingredients in washing and cleaning
agents, air fresheners, shampoos, perfumes and other
cosmetic products, as food additives in ®sh baits, and in
cigarettes. They have been detected in ®sh, surface and
sewage waters (Eschke et al., 1994a,b, 1995; Rimkus
and Wolf, 1993a), human milk and adipose tissue
(Rimkus and Wolf, 1996, 1993b; Ott et al., 1999), sed-
iment samples (Lach and Ste€en, 1997; Winkler et al.,
1998) as well as in the North Sea (Gatermann et al.,
1995). The lack of biological and chemical degradation
of these compounds as well as their bioaccumulation
potential has raised considerable attention in the ®eld of
environmental chemistry (Suter-Eichenberger et al.,
Due to the widespread use of musks in house
hold products it is important to remove all possible
sources from the laboratory (soaps, hand cream,
towels, etc.) which might contain these compounds in
order to eliminate contamination. Also, blank samples
should be run in parallel to every series of samples
extracted.
2.2. Sample characterization
Sewage sludges from di€erent catchment areas were
collected: seven samples containing mainly domestic
sewage obtained from separate sewer systems (A-type);
three samples containing a mixture of domestic sewage,
storm water runo€, and low amounts of industrial waste
water obtained from combined sewer systems (B-type)
and two samples as described for the B-type but
containing higher amounts of industrial waste water
(C-type). Fresh sewage sludge was sampled into pre-
cleaned (hexane and acetone) glass bottles, stored over
night at 4°C and extracted the next day.
2.3. Standards, chemicals and solvents
Nitro musks (musk tibetene, musk ketone, musk
xylene, musk moskene, musk ambrette) were obtained


from Essentia, Atherische Ole, Winterthur, Switzerland,
polycyclic musk compounds (Tonalide, Celestolide,
Phantolide, Cashmeran, Traseolide, Galaxolide) were
purchased from Promochem, Wesel, Germany.
Cyclohexane, chloroform, dichloromethane (DCM),
ethylacetate, hexane, methanol, isopropanol, toluene
and acetone, residue grade, were obtained from Merck,
Dietikon, Switzerland and Promochem, Wesel. Silica
gel 40 for column chromatography, anhydrous sodium
sulfate (Na2SO4), sodium chloride (NaCl) and sodium
hydrogen carbonate (HNaCO3) was from Merck,
Dietikon, palladium on activated alumina, celite
545 AW and ammonium formate was from Fluka,
Buchs, Switzerland. Waters Sep-Pak Silica cartridges
(0.5 g) were bought from Waters, Rupperswil,
Switzerland.

1998; Ka€erlein et al., 1998). Very recently, metabolites
of nitro musks have been described in the aquatic en-
vironment (Gatermann et al., 1998). These amino
transformation products have been shown to occur also

in mammals during metabolism (Ka€erlein et al., 1998).
From a toxicological point of view these metabolites
might well be more toxic than their parent compounds
(Gatermann et al., 1998).
To our knowledge very little is known about the
contamination of nitro- and polycyclic musk com-
pounds in sewage sludges. A study from the Nether-
lands has dealt with the analysis of musk fragrances in a
few sewage sludge samples (Blok, 1997). Due to the
importance of sludge use in Swiss agriculture, the aim
of this study was to determine nitro and polycyclic
musks in sludges from di€erent catchment areas and
also to look for the presence of amino metabolites of
the nitro musk compounds. Moreover mass spectral
data informations are provided for sensitive detection
of these metabolites.
2.4. Synthesis of the amino metabolites of the nitro musks
Amino metabolites were synthesized according to
Ram and Ehrenkaufer (1984). Brie¯y, the nitro musk
(100 mg) was diluted in 3.5 ml of dry methanol. To the
solution 20 mg of palladium on activated alumina and
106 mg of ammonium formate were added and the re-
action stirred at room temperature for 2±18 h depending
on the reactivity of the nitro group. After completion of

D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
567
the reaction the mixture was ®ltered over celite, con-
centrated, redissolved in chloroform, washed with
NaHCO3 and deionized water, dried over Na2SO4 and
concentrated to dryness. Puri®cation of the product was
performed using silica gel column chromatography
(100 Â 10 mm) and hexane/ethylacetate (2:1) as elution
solvent. After reconcentration, the pure product was
redissolved in hot hexane from which crystallization
occurred upon leaving at 4°C.
gas. The temperature program was 100°C, 1 min hold,
15°C/min to 157°C, 0.6°C/min to 178°C, 5°C/min to
220°C, 10°C/min to 300°C, 6.8 min hold. The injector
was set at 260°C. Injection (2 ll) was performed in the
splitless mode using a splitless time of 0.7 min. Stock
solutions (100 lg/ml) of the standard compounds were
prepared in toluene. The standard mixture of 500 ng/ml
was obtained by appropriate dilution of a mixture of the
stock solutions in toluene. Standard mixtures of lower
concentrations were prepared by suitably diluting the
500 ng/ml stock mixture with toluene. The mass range
for spectrum acquisition was 100±300 m/z, CID-RF m/z
90 and mass width 3 m/z for all substances. Table 2
shows MS/MS parameters used for the external stan-
dard quanti®cation.
2.5. Sample extraction
Wet sewage sludge (1 l, n ˆ 1) was extracted
with 600 ml of hexane at ambient temperature for 2 h
by agitating vigorously. Phase separation was im-
proved by adding NaCl and small amounts of iso-
propanol to the two-phase system. The organic phase
was decanted, dried over Na2SO4 and concentrated at
35°C under a slight vacuum to a volume of 5 ml.
Ethylacetate (5 ml) was added and the solution further
used for the gel permeation chromatography (GPC)
clean-up.
2.8. HRGC and electron impact (EI), positive chemical
ionization (PCI) and negative chemical ionization (NCI)
quadrupole MS of the amino metabolites of the nitro musk
compounds
HRGC was performed on a HP 6890 GC equipped
with a HP 6890 Series automatic injector (enhanced
parameters) and a HP 5973 mass spectrometer with PCI
and NCI option. Separation was reached on a 30 m HP
35 MS capillary column, 0.25 mm ID and 0.25 lm ®lm
(Hewlett-Packard, Wilmington, Delaware, USA) using
the following temperature program: 90°C, 1 min hold,
10°C/min to 300°C, 10 min hold. Helium was used as
carrier gas at a linear velocity of 37 cm/s (temperature
90°C). Standards were injected (2 ll) in the on-column
mode. In the CI mode (PCI and NCI) methane was
used as a reagent gas. A chemical ion gas puri®er for
methane was installed just before methane was entering
the MSD. The methane ¯ow was set at 20% for PCI
and 40% for NCI of a ¯ow of 5 ml/min. MS tempera-
ture parameters: transfer line 290°C, quadrupole 106°C
(PCI), 110°C (NCI), ion source 250°C (PCI), 110°C
(NCI).
2.6. Clean-up: gel permeation chromatography (GPC)
and solid phase extraction (SPE)
Gel permeation chromatography was carried out on
a Autoprep GPC Model 1002B from Gerber Instru-
ments, E€retikon, Switzerland. Cyclohexane/ethylace-
tate (1:1) was used as eluent with a ¯ow rate of 5 ml/min.
The column (300 mm  20 mm) was ®lled with 30 g of
Bio-Beads SX3 (Bio Rad Laboratories, Hercules, CA,
USA). The optimum sampling window was between 15
and 27 min (75 and 135 ml). The GPC eluates were
evaporated at 40°C to approximately 5 ml and concen-
trated to 1 ml by a gentle stream of nitrogen. After GPC,
extracts were submitted to a ®nal clean-up by use of a
silica cartridge (SPE). The column was conditioned with
10 ml of DCM. After loading the cartridge with the
extract, elution was carried out with 8 ml of DCM. The
eluate was concentrated nearly to dryness by a gentle
stream of nitrogen and the volume adjusted to exactly
1 ml with toluene. Careful evaporation was crucial and
could easily be monitored by the recoveries of Cash-
meran (DPMI).
2.9. Recovery experiments and detection limits
Due to the high loads of mainly the polycyclic musk
compounds in the investigated sludges, two di€erent
recovery experiments were carried out: (i) water was
spiked with 500 ng/l of the analytes, (ii) samples of
sewage sludge were spiked with 10 lg/l of the polycyclic
musk compounds (except Galaxolide and Tonalide) and
1 lg/l of the nitro musks and the amino metabolites of
the nitro musk compounds, respectively. Recovery rates
were satisfactory for most of the compounds, which
allowed for uncorrected calculation of the amounts
found (Table 1). The gas chromatographic method al-
lowed the detection of as little as 2 pg for most of the
compounds on the column, corresponding to 2 ng/l
in the sewage sludge. However, due to the high
2.7. HRGC and ion-trap mass spectrometry (MS)
quanti®cation using CID
HRGC was performed on a Varian 3400 GC equip-
ped with a Varian 1078 injector, a Varian 8200 auto-
sampler and a Varian Saturn 2000 ion-trap mass
spectrometer with MS/MS option. Separation was
achieved on a 60 m DB-1 capillary column, 0.25 mm
ID., 0.25 lm ®lm (J+W Scienti®c, supplied by MSP

Friedli, Koniz, Switzerland). Helium was used as carrier

568
D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
Table 1
Recovery data of the di€erent musk compounds from water
and sewage sludgea
3. Results and discussion
3.1. HRGC and ion-trap MS quanti®cation; mass spectral
characterization of the amino metabolites of the nitro
musks
Mean recovery
rate (%)
Compound class
Name
Water
Sewage
sludge
Due to the non-polar character of the musk com-
pounds ecient extraction was obtained using hexane.
Recovery rates of the di€erent compounds are summa-
rized in Table 1. Nitro-and polycyclic musks were re-
covered quantitatively whereas recoveries of the amino
metabolites were somewhat lower.
1
2
Polycyclic musk
Polycyclic musk
Polycyclic musk
Nitro musk
DPMI
ADBI
113
113
73
108
106
112
118
n.s.
n.q.
n.s.
102
82
3
AHMI 116
4
MA
MX
101
110
5
Nitro musk
6
Polycyclic musk
Polycyclic musk
Polycyclic musk
Nitro musk
HHCB 114
AITI 105
AHTN 113
Separation of most of the compounds could be
achieved by the chromatographic conditions described.
Coelution was observed for Galaxolide (compound 6)
and Traseolide (7) as well as for MM (9) and AMA
(10) as shown in Fig. 1. Solution to this problem was
o€ered by the multiple-reaction-monitoring tool
(MRM) of Saturn 2000, which allows to change the
precursor ion from scan to scan. MRM worked well for
the standard mixtures and for musk moskene and
amino musk ambrette in the samples, but as the con-
centrations of Galaxolide and Traseolide in the sludges
di€ered orders of magnitude and the precursor ion of
Traseolide was also present in the mass spectrum of
Galaxolide, a reliable quanti®cation of Traseolide was
not possible.
7
8
9
MM
AMA
MT
93
69
10
11
12
13
14
15
16
a.r.
a.r.
a.r.
Amino metabolite
Nitro musk
105
78
89
98
Amino metabolite
Nitro musk
AMM
MK
102
108
97
107
90
Amino metabolite
Amino metabolite
Amino metabolite
Nitro musks
AMT
AMK
AMX
50
76
91
102
89
106
80
Metabolites
Polycyclic musks
112
n.c.
a n ˆ 2, n.s.: not spiked, n.q.: not quantitated, a.r.: average re-
covery, n.c.: not calculated; compound identi®cation see also
Table 2.
background in the matrix, the real detection limit was
estimated to be 100 ng/l, depending on the sludge under
investigation.
2.10. Compound abbreviations and trade names
MT: 1-tert-Butyl-3,4,5-trimethyl-2,6-dinitrobenzene
(musk tibetene), MK: 4-tert-Butyl-2,6-dimethyl-3,
5-dinitroacetophenone (musk ketone), MX: 1-tert-Bu-
tyl-3,5-dimethyl-2,4,6-trinitrobenzene (musk xylene),
MM: 1,1,3,3,5-Pentamethyl-4,6-dinitroindane (musk
moskene), MA: 6-Methoxy-1-tert-butyl-2,4-dimethyl-
3,5-dinitrobenzene (musk ambrette), HHCB (1,3,4,6,7,8-
Hexahydro-4,6,6,7,8,8-hexamethylcyclopenta(g)-2-ben-
zopyran (Galaxolide), AHTN: 6-Acetyl-1,1,2,4,4,7-
hexamethyl tetralin (Tonalide), ADBI: 4-Acetyl-6-tert-
butyl-1,1-dimethylindane (Celestolide), AHMI: 6-Ace-
tyl-1,1,2,3,3,5-hexamethylindane (Phantolide), AITI:
5-Acetyl-3-isopropyl,1,1,2,6-tetramethylindane (Traseo-
lide), DPMI: 1,2,3,5,6,7-hexahydro-1,1,2,3,3-pentam-
ethyl-4H-inden-4-one (Cashmeran). Amino metabolites
of the nitro musks: AMA: amino musk ambrette,
AMM: amino musk moskene, AMT: amino musk
tibetene, AMK: amino musk ketone, AMX: amino
musk xylene.
Fig. 1. Full scan run (upper chromatogram) and MS/MS run
(lower chromatogram) of a standard mixture containing nitro
musks, amino metabolites and polycyclic musk compounds; for
compound identi®cation see Table 2.

D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
therefore the 16 mass units as part from the M
569
73
‡
Quanti®cation of the analytes was based on successor
ions after collision induced dissociation (CID) in the
ion-trap (Table 1). Full scan and MS/MS spectra of the
amino metabolites of the nitro musks and chemical
structures are shown in Figs. 2 and 3. All spectra exhibit
a molecular weight peak of 30 mass units lower com-
pared to the parent compounds indicating that one nitro
group had been reduced. Basically two groups of mass
spectra can be distinguished: one having the molecular
fragment observed originate from the NH2 group and
not of a cleavage of O from a nitro group. In no case
losses of m/z 57 or 16 were observed in these CID ex-
periments showing that cleavage of these groups oc-
curred initially.
AMA reveals a M
‡
17 fragment as base peak (m/z
221) which corresponds to the cleavage of an OH radical
ion (OH´). This reaction is frequently observed for or-
tho-nitro toluenes. The same type of cleavage can be
deduced from the MS/MS spectrum of AMX which
shows fragment ions at m/z 235 and 218 corresponding
‡
ion (M ) as base peak as for AMA, AMT and AMK,
‡
the other group having the M
CH3 fragment ion as
base peak as for AMM and AMX. CID experiments on
the precursor ion (see Table 2) resulted in mass spectra
with characteristic successor ions. Their presence can be
‡
‡
‡
‡
CH3 OH´ and M
to M
17 (M
15 17 (M
15 2 Á
CH3 ꢀOH´†2). In AMX this OH´ cleavage is
rationalized as follows: The MS/MS spectra of AMA,
‡
very pronounced as there are two nitro groups ortho to
a methyl group. Finally the MS/MS spectrum of AMM
shows strong fragmentation ions at m/z 216, 187 and 173
AMT and AMK show a strong, common M
73
fragment ion (AMA: m/z 165, AMT: m/z 163 and AMK:
m/z 191) presumably due to the cleavage of the tertiary
‡
which can be explained as follows: m/z 216 (M
corresponds to the cleavage of a CH3 group and an OH´
32)
‡
butyl group and the amino function (M
This hypothesis was supported by a second set of CID
57 16).
‡
(M
15 17) similarly to AMX, m/z 187 and 173 are
C2H5
‡
15 17 29) and C3H7 (M 15 17 43)
‡
experiments performed on the M
73 fragment ions of
successor ions by additional cleavage of a
‡
AMA, AMT and AMK (®gure not shown). For AMA
for example, loss of m/z 30 (NO) and m/z 58 (NO + CO)
clearly demonstrate that a nitro group is involved and
(M
originating from ring-opening and cleavage of the
tetramethylated cyclopentane ring.
With mass spectrometrical methods the location of
the reduction of the nitro group in the molecule cannot
be determined. In AMT and AMK the nitro groups are
chemically aequivalent whereas in AMA, AMM and
AMX the nitro groups have a di€erent chemical envi-
ronment. For the latter compounds 1H-NMR, 13C-
NMR, NOE and long-range coupling experiments
should resolve this problem. This aspect will be treated
elsewhere (Berset et al., 1999).
3.2. EI, PCI and NCI quadrupole MS of the amino
metabolites
Performing a comparison study applying EI, PCI and
NCI±MS useful information was further gathered re-
garding fragmentation and response sensitivity of the
‡
amino metabolites (Table 3). Besides the M peak nu-
merous fragment ions can be observed in EI±MS,
‡
whereas M
ones. PCI±MS shows less fragmentation, typical a mo-
CH3 and m/z 57 (C4H9) are dominant
‡
lecular ion MH due to proton transfer reactions and
‡
‡
adduct ions M +29 and M +41 can be identi®ed, well
known from PCI with methane. NCI±MS results in only
few fragmentation ions. In most cases the molecular ion
(M ) turned out to be the base peak besides m/z 46
characteristic for the presence of a nitro group in the
molecule. Regarding the sensitivity of the di€erent ion-
ization methods NCI was the most sensitive one, al-
lowing the detection of femtograms, followed by EI and
®nally PCI. Therefore, quadrupole NCI±MS might be a
valuable alternative to ion trap MS/MS for low level
quanti®cation of these metabolites.
Fig. 2. Full scan ion-trap mass spectra of the amino metabo-
lites of the nitro musks (left column) and MS/MS spectra (right
column) after collision induced dissociation (CID); P: precursor
‡
ion, M : molecular ion.

570
D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
Fig. 3. Structures of the amino metabolites of the nitro musks: 1: AMA, 2: AMM, 3: AMT, 4: AMK, 5: AMX; see also Table 2.
Table 2
Conditions for resonant CID MS/MS fragmentation
Musk compound
Precursor ion (m/z)
Successor ions for
quanti®cation (m/z)
CID res. (V)
tR (min)
1
2
Cashmeran
Celestolide
191
229
229
253
282
243
215
243
263
238
251
233
279
236
264
252
135, 163, 173
173, 187
173, 187
0.35
0.35
0.35
0.30
0.35
0.35
0.40
0.35
0.30
0.60
0.30
0.35
0.35
0.35
0.30
0.35
16.3
27.4
30.3
34.9
37.1
37.6
37.6
38.9
39.7
39.7
42.4
43.2
45.2
45.6
50.6
51.4
3
Phantolide
Musk ambrette
Musk xylene
Galaxolidea
Traseolidea
4
219, 251
265, 280
5
6
187, 213
171, 173
173, 187
7
8
Tonalide
9
Musk moskenea
Amino musk ambrettea
Musk tibetene
Amino musk moskene
Musk ketone
229, 245, 246
165, 221, 223
204, 234, 249
188, 216, 233
191, 262
10
11
12
13
14
15
16
Amino musk tibetene
Amino musk ketone
Amino musk xylene
162, 163
148, 191
218, 235
a These substances were separated in the mass spectrometer by multiple-reaction-monitoring (MRM).
3.3. Concentrations of musk compounds in the sewage
sludges of di€erent catchment areas
MK and 5 lg/kg d.m. for MX (Sauer et al., 1997; Blok,
1998). The concentrations found in our study re¯ect the
industrial production volume of these compounds, MX
and MK being the major nitro musks still used in dif-
ferent products. Also e€orts undertaken by the fra-
grance industry in Switzerland since 1994 to reduce the
amount of MX in detergents and cleaning agents are
well documented by these results.
Levels of nitro musks, polycyclic musks and metab-
olites of the nitro musks were determined in some se-
lected sewage sludges containing no (type A), low (type
B) or higher (type C) amounts of industrial waste water.
A typical chromatogram of an extract is shown in Fig. 4
and quantitative results are presented in Table 4. Among
the nitro musks MK was identi®ed in 7 out of 12 sludges
whereas MX was detected in just one sludge. Not sur-
prisingly these nitro musks were found mainly in A-type
sludges containing only domestic sewage water. Con-
centrations were rather low with an average concentra-
tion of 5 lg/kg d.m. for MK and 30 lg/kg d.m. for MX.
These results are similar to recent studies which found
values of 10±60 lg/kg d.m. (mean 30 lg/kg d.m.) for
Considerable research is focused on transformation
products of the nitro musks which have been described
to occur in the environment and biota. These metabo-
lites are formed by reduction of a nitro group to the
corresponding amine. Very recently, published data
have shown that the 4-amino-musk-xylene was much
more toxic for Daphnia magna than the parent com-
pound (Behechti et al., 1998) emphasizing the impor-
tance of these compounds for risk assessment

D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
571

572
D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
Fig. 4. Total ion chromatogram (TIC) of a sewage sludge extract and extracted ion chromatograms (EIC) of 12 (AMM), 13 (MK) and
16 (AMX); selected MS/MS spectra obtained from the sample and the corresponding standard.
considerations. In our sludge study three amino me-
tabolites could be identi®ed and quanti®ed: amino musk
xylene (AMX), amino musk moskene (AMM) and
amino musk ketone (AMK). Concentrations were be-
tween 1 and 50 lg/kg d.m. and therefore reached levels
in the same order as the parent compounds or even
higher. AMX turned out to be the major metabolite,
followed by AMM and AMK. Clearly, the two nitro
musks MX and MM are found mainly as their amino
metabolites in the sewage sludges. The decrease of the
concentrations of the parent compounds and the in-
crease of the amino metabolites strongly suggest, that
besides adsorption to sludge particles transformation of
nitro musks under anaerobic conditions is a relevant
process in the sewage treatment plant (STP).
4. Conclusions
HRGC and ion-trap MS/MS was used to identify
and quantify two groups of synthetic musks in sewage
sludge samples, namely the nitro and polycyclic musks.
Among the nitro musks mainly MK was detected
whereas MX was determined in just one sludge. These
nitro musks were measured in the low lg/kg d.m. in
sludges originating from domestic waste water. Amino
transformation products of MX, MM and MK were
detected for the ®rst time in some sewage sludge samples
which partly exceeded the concentrations of the parent
compounds. This is important, taking into account the
toxicity of some of these metabolites. Therefore, besides
adsorption to the sewage sludge particles, transforma-
tion processes in the STP seem to play an important role
with respect to the elimination of the nitro musks.
Polycyclic musks were the main representatives of these
synthetic compounds, found in all types of sewage
sludges up to 12 mg/kg d.m. The two most important
products detected were Galaxolide and Tonalide which,
at the same time, are the compounds produced in
highest quantities by the fragrance industry.
Based on the production volume, polycyclic musks
represent about 93% of the market in Europe for syn-
thetic musk compounds (OSPAR, 1998). As expected,
they could be found in all sewage sludges most often in
much higher concentrations than the corresponding ni-
tro compounds (Table 4). Galaxolide (HHCB) and
Tonalide (AHTN) were the main representatives reach-
ing concentrations of 0.7±12.1 mg/kg d.m., followed by
Phantolide (AHMI), Celestolide (ADBI) and Cashme-
ran (DPMI) which were present in concentrations below
1 mg/kg d.m.
Future work will focus on (i) ecotoxicological studies
of the amino-metabolites of the nitro musks as well as
the polycyclic musks, (ii) soil degradation experiments of

D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574
573
Table 4
Concentrations of nitro musks, amino metabolites of the nitro musks and polycyclic musks in sewage sludges of di€erent catchment
areasa
Type A
sludge
Musk
compound
lg/l
lg/kg
d.m.
lg/l
lg/kg lg/l
d.m.
lg/kg
d.m.
lg/l
lg/kg
d.m.
lg/l
lg/kg
d.m.
lg/l
lg /kg
d.m.
Nitro musks
Ambrette
Xylene
n.d.
n.d.
n.d.
n.d.
0.2
n.d.
n.d.
n.d.
n.d.
6.1
n.d.
n.d.
n.d.
n.d.
0.2
n.d.
n.d.
n.d.
n.d.
6.8
n.d.
n.d.
n.d.
n.d.
0.2
n.d.
n.d.
n.d.
n.d.
6.9
n.d.
n.d.
n.d.
n.d.
0.1
n.d.
n.d.
n.d.
n.d.
1.5
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.2
n.d.
n.d.
n.d.
n.d.
5.0
Moskene
Tibetene
Ketone
Amino
metabolites
AMA
AMX
n.d.
0.4
n.d.
10.9
7.9
n.d.
0.4
n.d.
15.3
n.d.
n.d.
n.d.
n.d.
1.2
n.d.
49.1
1.6
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.1
n.d.
n.d.
0.7
n.d.
0.9
n.d.
24.9
n.d.
n.d.
n.d.
AMM
AMT
0.3
n.d.
n.d.
n.d.
0.1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
AMK
Polycyclic
musks
Cashmeran
Celestolide
Phantolide
Galaxolide
Traseolide
Tonalide
1.8
3.7
55.6
113
1.7
3.3
72.3
140
1.8
1.5
74.7
61.6
210
1.7
4.7
47.2
131
2.7
6.2
51.4
116
2.2
2.4
60.9
65.4
103
14.5
178
n.q.
44.5
441
19.8
190
n.q.
34.9
843
5.1
9.2
256
7.3
139
3.7
5416
n.q.
1352
8068
n.q.
1485
145
n.q.
44.1
5922
n.q.
1800
158
n.q.
71.4
4374
n.q.
1983
124
n.q.
51.5
2347
n.q.
973
132
n.q.
42.7
3658
n.q.
1183
Type A
sludge
Type B
sludge
Type C
sludge
Musk
compound
lg/l
lg/kg
d.m.
lg/l
lg/kg lg/l
d.m.
lg/kg
d.m.
lg/l
lg/kg
d.m.
lg/l
lg/kg
d.m.
lg/l
lg/kg
d.m.
Nitro musks
Ambrette
Xylene
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.6
n.d.
32.5
n.d.
n.d.
7.0
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
0.2
n.d.
n.d.
n.d.
n.d.
3.1
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
Moskene
Tibetene
Ketone
n.d.
n.d.
0.4
Amino
metabolites
AMA
AMX
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
1.6
n.d.
31.5
n.d.
n.d.
13.1
n.d.
n.d.
0.3
n.d.
n.d.
7.2
n.d.
0.9
n.d.
15.6
n.d.
n.d.
n.d.
n.d.
n.d.
1.2
n.d.
n.d.
36.2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
AMM
AMT
n.d.
n.d.
0.7
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
AMK
Polycyclic
musks
Cashmeran
Celestolide
Phantolide
Galaxolide
Traseolide
Tonalide
4.3
3.2
332
245
3.7
73.9
330
3.8
4.3
93.3
108
2.2
2.4
38.4
41
2.1
3.1
64.1
96.6
180
10.3
7.9
147
113
266
2977
n.q.
784
16.3
6.1
10.7
158
n.q.
54.1
819
122
7.9
198
3.7
64.9
2293
n.q.
741
5.8
18.6
208
n.q.
54.9
12157
n.q.
4161
202
n.q.
62.8
4074
n.q.
1269
150
n.q.
57.0
3718
n.q.
1418
132
n.q.
42.7
104
n.q.
41.7
3229
n.q.
1290
a n.d.: not detected; n.q.: not quanti®ed.

574
D. Herren, J.D. Berset / Chemosphere 40 (2000) 565±574


Ka€erlein, H.U., Goen, T., Angerer, J., 1998. Musk xylene:
analysis occurrence kinetics and toxicology. Critical Rev.
Toxicol. 28, 431±476.
the chiral polycyclic musk compounds and (iii) the
identi®cation and spectroscopical characterization of
polar metabolites of these substances.
Kettler, R., 1998. Umwelt-Materialien Nr. 90. Abfall-Statistik1
1996, pp. 6±149.

Klop€er, W., 1996. Environmental hazard assessment of
Acknowledgements
chemicals and products. Part V: anthropogenic chemicals
in sewage sludge. Chemosphere 33, 1067±1081.
Lach, G., Ste€en, D., 1997. Orientierende Untersuchungen von
T. Candinas, G. Chassot, T. Kupper and W. Stau€er
(IUL) are highly acknowledged for their cooperation in
this project.

Gewassersedimenten auf Nitro-/Polymoschusverbindungen
und die Flammenschutzmittel TCEP und TCPP, Nie-

dersachsisches Landesamt fur Oekologie, vol. 3. Hi-
ldesheim, Germany, pp. 5±13.

References
Lega, R., Ladwig, G., Meresz, O., Clement, R.E., Crawford,
G., Salemi, R., Jones, Y., 1997. Quantitative determination
of organic priority pollutants in sewage sludges by GC-MS.
Chemosphere 34, 1705±1712.
Behechti, A., Schramm, K.W., Attar, A., Niederfellner, J.,
Kettrup, A., 1998. Acute aquatic toxicity of four musk
xylene derivatives on Daphnia Magna. Water Res. 32,
1704±1707.
OSPAR, 1998. Convention for the protection of the marine
environment of the North-East Atlantic, Working group
on di€use sources (DIFF), Helsinki, 6±9 October 1998:
Draft Background Document Concerning the Elaboration
of Programmes and Measures on Nitro Musks and
Polycyclic Musks, 1±38 Draft 11.9.1998.
Berset, J.D., Bigler, P., Herren, D., 1999. Analytical Chemistry,
in preparation.
Blok, H. 1997. Measurement of musk fragrances in sludges of
sewage treatment plants in the Netherlands. In: van de
Plassche, E.J. Balk, F. (Eds.), Report 601503 008, Envi-
ronmental risk assessment of the polycyclic musks AHTN
and HHCB according to EU-TGD, BKH Consulting
Engineers, pp. 1±121.
Ott, M., Failing, K., Lang, U., Schubring, Ch., Gent, H.J.,
Georgii, S., Brunn, H., 1999. Contamination of human milk
in middle Hesse, Germany ± A cross-sectional study on the
changing levels of chlorinated pesticides, PCB congeners
and recent levels of nitro musks. Chemosphere 38, 13±32.
Ram, Ehrenkaufer, 1984. A general procedure for mild a rapid
reduction of aliphatic and aromatic nitro compounds using
ammonium formate as a catalytic hydrogen transfer agent.
Tetrahedron Lett. 25, 3415±3418.
Blok, J. 1998. Measurements of polycyclic and nitro musks in
sludge of sewage treatment plants in the Netherlands;
Report to RIVM, BKH Consulting Engineers, January.
Demirjian, Y.A., Joshi, A.M., Westman, T.R., 1987. Fate of
organic compounds in land application of contaminated
municipal sludge. J. WPCF 59, 32±37.
Rimkus, G., Wolf, M., 1993a. Nachweis von Nitro-
moschusverbindungen in Frauenmilch und Humanfett.
Deutsche Lebensmittel-Rundschau 89, 103±107.

Drescher, U., Matthies, M., Bruggemann, R., 1989. Organische


Schadsto€e in Klarschlammen. GWF 130, 613±619.
Eschke, H.D., Traud, J., Dibowski, H.J., 1994a. Un-
tersuchungen zum Vorkommen polycyclischer Moschus-
Duftsto€e in verschiedenen Umweltkompartimenten,


Rimkus, G., Wolf, M., 1993b. Ruckstande und Ve-
runreinigungen in Fischen aus Aquakultur 2. Mitteilung:
Nachweis von Moschus Xylol und Moschus Keton in
Fischen. Deutsche Lebensmittel-Rundschau 89, 171±177.
Rimkus, G., Wolf, M., 1996. Polycyclic musk fragrances in
human adipose tissue and human milk. Chemosphere 33,
2033±2043.

Nachweis und Analytik mit GC-MS in Ober¯achen,
Abwassern und Fischen (1.Mitteilung), Z. Umweltchem.

Oekotox. 6, 183±189.
Eschke, H.D., Traud, J., Dibowski, H.J., 1994b. Arti®cial nitro
musk substances in surface water wastewater and ®sh. Vom
Wasser 83, 373±383.
Sauer, J., Antusch, E., Ripp, Ch., 1997. Monitoring lipophiler
organischer Schadsto€e im Kanalnetz mittels Si-
hlhautuntersuchungen. Vom Wasser 88, 49±69.
Eschke, H.D., Traud, J., Dibowski, H.J., 1995. Un-
tersuchungen zum Vorkommen polycyclischer Moschus-
Duftsto€e in verschiedenen Umweltkompartimenten, 2.
Suter-Eichenberger, R., Altorfer, H., Lichtensteiger, W., Sch-
lumpf, M., 1998. Bioaccumulation of musk xylene in
developing and adult rats of both sexes. Chemosphere 36,
2747±2762.


Mitteilung: Befunde in Ober¯achen-, Abwassern und
Fischen sowie in Waschmitteln und Kosmetika, Z.
Umweltchem. Oekotox. 7, 131±138.
Tarradellas, J., Diercxsens, P., 1983. Les micropolluants dans
ꢀ
les boues d'epuration. Schweiz. Arch. Tierheilk. 125, 589±

Gatermann, R., Huhnerfuss, H., Rimkus, G., Wolf, M.,
Franke, St., 1995. The distribution of nitrobenzene and
other nitroaromatic compounds in the North Sea. Marine
Pollut. Bull. 30, 221±227.
605.
Verordnung uber umweltgefahrdende Sto€e (Sto€verordnung

StoV), 1996. pp. 61±62.


Gatermann, R., Huhnerfuss, H., Rimkus, G., Attar, A.,
Winkler, M., Kopf, G., Hauptvogel, C., Neu, T., 1998. Fate of
arti®cial musk fragrances associated with suspended par-
ticulate matter (spm) from the river Elbe (Germany) in
comparison to other organic contaminants. Chemosphere
37, 1139±1156.
Kettrup, A., 1998. Occurrence of musk xylene and musk
ketone metabolites in the aquatic environment. Chemo-
sphere 36, 2535±2547.
Halling-Sorensen, B., Nors Nielsen, S., Lanzky, P.F., Ingerslev,

F., Holten Lutzhoft, H.C., Jorgensen, S.E., 1998. Occur-
rence, fate and e€ects of pharmaceutical substances in the
environment ± a review. Chemosphere 36, 357±393.