Acute aquatic toxicities of four musk xylene derivatives on Daphnia magna

Article abstract of DOI:10.1016/S0043-1354(97)00361-8

Musk xylene derivatives were obtained by hydration of the parent compound. These materials were assayed for acute toxicity on Daphnia magna. In contrast to the parent compound all these reduction products showed acute toxicities below their water solubili

Full text of DOI:10.1016/S0043-1354(97)00361-8

Wat. Res. Vol. 32, No. 5, pp. 1704±1707, 1998
# 1998 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(97)00361-8
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RESEARCH NOTE
ACUTE AQUATIC TOXICITIES OF FOUR MUSK XYLENE
DERIVATIVES ON DAPHNIA MAGNA
A. BEHECHTI1*, K.-W. SCHRAMM1, A. ATTAR1, J. NIEDERFELLNER2 and
A. KETTRUP1
1GSF-National Research Center for Environment and Health, Institute of Ecological Chemistry,
Ingolstadter Landstr. 1, D-85764 Neuherberg, Germany and Institute of Biomathematics and
Biometrics, Ingolstadter Landstr. 1, D-85764 Neuherberg, Germany
2
(First received December 1996; accepted in revised form August 1997)
AbstractÐMusk xylene derivatives were obtained by hydration of the parent compound. These ma-
terials were assayed for acute toxicity on Daphnia magna. In contrast to the parent compound all these
reduction products showed acute toxicities below their water solubility. The para-amino-dinitro-musk
xylene was highly toxic (EC50=0.00025 mg L 1). Ortho- and ortho-para and triamino-musk-xylene
1
were less toxic and showed EC50-values of 1.07, 23.3, 58.8 mg L respectively. A 1:1:1:1 mixture of all
1
derivatives, each in a concentration corresponding to of EC50, exhibited a strong concentration-addi-
4
tivity relationship. # 1998 Elsevier Science Ltd. All rights reserved
Key wordsÐnitromusk derivatives, acute toxicity, Daphnia magna, octanol/water partition coecient,
water solubility
INTRODUCTION
musk xylene is studied on Daphnia magna as an
aquatic target organism.
Musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trini-
trobenzene/CAS 81-15-2) is a synthetic variant of
natural musk and belongs to the group of nitroben-
MATERIAL AND METHODS
zenes. The chemical structure is shown in Fig. 1.
Synthesis of musk xylene derivatives by hydrogenation of
musk xylene (1-tert-butyl-3,5-dimethyl-2,4,6-trinitroben-
zene)
Musk xylene is used as an additive in soaps,
detergents, lotions and other scented products
(Ford et al., 1990). Geyer et al. (1994) reviewed the
signi®cance of nitromusks in the environment. They
were found in fresh-water ®sh, shell®sh, in saliva of
tobacco chewers (Nair et al., 1986), in human adi-
pose tissues, breast milk (Maekawa et al., 1990) and
in food.
297,30 mg (1 mmol) of musk xylene is added to a round
bottom ¯ask ®lled with 600 mg palladium on activated
charcoal (10% Pd) and 3 ml absolute ethanol. It is heated
at 458±508C on an oil bath. In a stream of H2 80% of p-
amino, 20% of o-amino or 2-NH2-musk xylene and 4-
NH2-musk xylene were formed within 30 min. The mixture
was ®ltered, the ethanol was evaporated and the products
were puri®ed on a silicagel column. The column was ®lled
with silicagel type 60 70±230 mesh (Aldrich Chemie,
39.148.4) and chloroform was used as eluant. After
increasing the reaction time up to 60 min in addition 2,4
diamino-musk xylene was formed. Increasing further to
180 min 2,4,6-triamino musk xylene could be isolated. The
recovery of the above products was 90% and the purity
was found to be >98.5% in the total ion current obtained
from GC-MS technique in scan-mode. The synthesised de-
rivatives are shown in Fig. 2.
The use pattern of musk xylene causes its occur-
rence in sewage treatment facilities and in the
aquatic environment. During anaerobic sewage
sludge digestion some or all of the nitro groups in
musk xylene are likely to be reduced, yielding the
corresponding aminobenzenes. In addition amino-
benzenes are expected to form azo-compounds in
the presence of oxyreductases (Simmons et al.,
1987). Afterwards these derivatives might be
released to agricultural systems via sewage sludge
or by the e‚uents of treatment facilities to the sur-
face or groundwater aquatic environment. With this
Daphnia magna acute toxicity test
In the acute immobilisation test musk xylene and its de-
rivatives were tested within a range of concentrations.
background the toxicity of amino-derivatives of They showed di€erent toxicities on the swimming capa-
bility of Daphnia magna under identical test conditions.
The test procedure followed the OECD guideline 202 for
*Author to whom all correspondence should be addressed testing of chemicals (OECD, 1981). A Daphnia magna
[Tel: +49-899-31873-1874531; Fax: +49-899-31873- monoclonal culture was used. The four nitromusk xylene
1873371; E-mail: akbar.behechti@gsf.de].
derivatives were dissolved in ethanol which is known to be
1704

Research Note
1705
1-tert-butyl-3,5-dimethyl-4-amino-2,6-dinitrobenzene
exhibits far increased toxicity to Daphnia magna
(Table 1). The other derivatives are less toxic. The
result of the mixture study indicates a concentration
additivity of the mixture constituents according to
equation 1.
n
Ci
ˆ 0:98
ꢀ1†
Fig. 1. Chemical structure of musk xylene (1-tert-butyl-
3,5-dimethyl-2,4,6-trinitrobenzene).
EC50i
iˆ1
If we apply the formula of Konemann (1981) which
overcomes some of the problems of devising a
widely applicable scale we found
nontoxic at the applied amount. The test was performed
in 20 ml of water saturated with oxygen. In each of the
20 ml ¯asks 10 individuals not older than 24 h were used.
With each test concentration at least 20 animals were
used. In addition, an experiment was performed using a
1:1:1:1 mixture of four derivatives, each in a concentration
n
log
Ci=EC50i
iˆ1
log n
1
ˆ 1:01
ꢀ2†
1
corresponding to of their EC50. The test was carried out
4
in the dark at 208C. The water ¯eas were not fed during
the test.
The test compounds were visually completely dissolved
at the di€erent test concentrations. The chosen concen-
trations in the main-test were based on the results of pre-
Both results con®rm a strong concentration additiv-
ity of the nitromusk derivatives.
Regarding e€ect additivity by applying equation 3
we found a value of 0.03. Thus e€ect additivity can-
not be observed for this mixture.
testing. The pretests con®rm the elaborated range of EC50
.
Statistical treatment of the raw data was performed
using probit analysis.
n
ECxi ˆ 0:03,
ꢀ3†
iˆ1
ECxi is the proposed e€ect x of chemical i obtained
from the dose-response curve of the single chemical.
Unfortunately, actual concentrations of amino
RESULTS AND DISCUSSION
In contrast to ®ndings on nitromusk xylene
(Schramm et al., 1996) the reduced amino derivative derivatives in the environment are not known.
Fig. 2. Synthesised and tested derivatives of musk xylene.

1706
Research Note
Table 1. EC50 and EC10 values for Daphnia magna of amino musk xylene derivatives, their equivalent mixture and reference compound
K2Cr2O7
1-tert-butyl-3,5-dimethyl-
4-amino-2,6-dinitrobenzene
(Compound A)
1-tert-butyl-3,5-dimethyl-
2-amino-4,6-dinitrobenzene
(Compound B)
1-tert-butyl-3,5-dimethyl-
2,4-diamino-6-nitrobenzene
(Compound C)
Chemical
EC50 [mg L 1] (95% conf. lim.)
EC10 [mg L 1] (95% conf. lim.)
0.00025 (0.00023±0.00028)
0.00018 (0.00015±0.00022)
1.07 (0.89±1.28)
0.60 (0.42±0.85)
23.3 (20.8±26.1)
14.7 (11.9±18.1)
1-tert-butyl-3,5-dimethyl-2,4,6-
triaminobenzene (Compound D)
Mixture of all four derivatives
1
Reference K2Cr2O7
Positive control
Chemical
(1:1:1:1 at of their EC50
)
4
58.8 (51.2±67.6)
31.8 (24.2±41.7)
A: 0.00006, B: 0.25, C: 6.1, D:
14.9
A: 0.00004, B: 0.15, C: 3.6, D: 8.8
EC50 [mg L 1] (95% conf. lim.)
EC10 [mg L 1] (95% conf. lim.)
0.61 (0.57±0.64)
0.38 (0.35±0.42)
Table 2. Physicochemical properties of nitromusk derivatives
log Kow (SRC-WSKOW)
(Meylan and Howard, 1995)
log Kow (C log P)
(Leo, 1993)
S (mg/L) (SRC-WSKOW)
(Meylan et al., 1996)
Chemical
1-tert-butyl-3,5-dimethyl-2,4,6-trinitrobenzene
1-tert-butyl-3,5-dimethyl-4-amino-2,6-dinitrobenzene
1-tert-butyl-3,5-dimethyl-2-amino-4,6-dinitrobenzene
1-tert-butyl-3,5-dimethyl-2,4-diamino-6-nitrobenzene
1-tert-butyl-3,5-dimethyl-2,4,6-triaminobenzene
4.45
4.29
4.29
3.01
1.70
3.72
3.59
2.68
2.68
0.99
0.82
4.08
4.08
74.9
1428
However, due to their structure and our obser- of amino groups were much smaller than found for
the nitromusk derivatives.
vation the Kow of amino-musk xylenes is much
lower than that of musk xylene. Table 2 shows esti-
mated octanol-water partition coecients (log Kow)
and water solubilities (S) of the compounds investi-
gated. As expected amino groups contribute to the
hydrophilic properties of the chemicals. The esti-
mate of the water solubility is based on log Kow-
values. For the estimation of log Kow-values (see
Table 2) the WSKOW program of the Syracuse
Research Company and alternatively the C log P
program from Biobyte were used. Using log Kow
values from C log P result in elevated solubilities.
Measured values for log Kow and S of 1-tert-butyl-
3,5-dimethyl-2,4,6-trinitrobenzene have been already
Amino groups are reactive and can easily bind
covalently to organic matter, which corresponds to
a certain detoxi®cation. Furthermore it is to some
extent likely that amino-benzenes can form toxic
azobenzenes.
CONCLUSION
The test results are showing a toxicity of reduced
derivatives of musk xylene. Musk xylene with one
amino group in para position is highly toxic, the
other compounds are less toxic. However it is likely
that the toxic e€ect of this toxic compound is
decreased in the environment by the presence of po-
tential competitors.
The extent of toxicity is following the concept of
concentration additivity which means that single
compounds contribute to the observed e€ect below
the no e€ect concentration (NOEC) levels.
1
reported to be 4.5 and 0.15 mg L (Schramm et al.,
1996), respectively. All determined toxicological
values (EC50) are below the corresponding solubili-
ties. So far, uncertainties due to solubility problems
can be excluded.
Correlations between EC50 and 1/log Kow,
log Kow and (log Kow)2 did not yield signi®cant re-
lationships between these properties. Unspeci®c nar-
cotic interaction should lead to signi®cant
correlation between toxic concentration and hydro-
philicity. In conclusion, the toxicity of the nitro-
musk derivatives, in particular the toxic action of 1-
tert-butyl-3,5-dimethyl-4-amino-2,6-dinitrobenzene,
must be based on a speci®c mechanism. A general
mode of toxic action of an amino group in para
position is not described in the literature. Johnson
et al. (1994) found para-amino-dinitrotoluene to be
more toxic in a 96 h planarian acute toxicity test
than ortho-amino-dinitrotoluene. The same author
observed opposite e€ects in a 96 h acute toxicity
test with Daphnia. However, the di€erences in tox-
icity of aromatic para and ortho or meta position
Although the nitromusk xylene is replaced par-
tially by polycyclic musk compounds (Eschke et al.,
1995), the toxicity of its amino derivatives must be
considered as a severe ecotoxicological problem.
Thus the ecotoxicological e€ects of amino musks
on the environment deserve a careful investigation.
AcknowledgementsÐThe authors sincerely thank Ina-
Ursula Grande for technical assistance.
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Research Note
1707
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