70N J. Environ. Monit., 1999, 1 Viewpoint Synthetic musks in environmental samples: indicator compounds with relevant properties for environmental monitoring Synthetic musks (nitro and polycyclic musks) are a group of chemicals oVering a wide range of important properties for environmental monitoring programs. They are produced as odorous chemicals and added to a wide variety of perfumes, toiletry products and other household products.As such, they are directly applied in cosmetic products or in washed textiles to the human body in considerable concentrations and accumulate owing to dermal resorption. In addition, synthetic musks also enter the environment via waste water treatment. Several polycyclic musks are chiral. By using chiral gas chromatographic methods, it is possible to determine the enantiomeric ratio and assess their bioavailability. Although an comprehensive quality assurance program must be followed during the analysis of synthetic musks in environmental samples, the determination of these compounds is not very demanding and can be carried out by a standard analytical laboratory specialising in trace analysis of organic pollutants.Owing to the pheromone-like behavior of some synthetic musks, the induction of receptors in olfactory systems should be investigated. For HHCB (1,3,4,6,7,8-hexahydro- 4,6,6,7,8,8-hexamethylcyclopenta[ g]- 2-benzopyran, e.g., GalaxolideA), threedimensional structural similarities with androstenone (5a-androst-16-en-3-one), a mammalian steroid pheromone, were found, which support the hypothesis of HHCB as an artificial pheromone.Owing to their environmental abundance, their relation to human activities and their potential for pheromone-like environmental behavior, synthetic musks are especially valuable as future indicator chemicals for environmental monitoring. Introduction Synthetic musks are important artificial fragrances added in large amounts to perfumes, toiletry products, laundry detergents, fabric softeners, etc.Owing to their chemical structures, synthetic musks can roughly be divided in twomain groups: nitro musks and polycyclic musks. Nitro musks were discovered in 1891 as a by-product during the development of new explosives.1 The main constituents of this group aremusk ketone andmusk xylene (Table 1). In Europe at least, owing to voluntary restrictions by the IFRA (International Fragrance Association), the use of nitro musks in toiletry products is decreasing.Recently, in various types of environmental samples the dominant occurrence of polycyclicmusks was demonstrated. However, in Canadian fish samples musk ketone still dominates.2 A comprehensive review of the occurrence and fate of polycyclic musks has been published.3 In the early 1980s, the occurrence of nitro musks in biota and river water was reported for the first time in Japan.4 Relatively high levels were found in freshwater fish from the Tama river near Tokyo.Ten years after this first Japanese report, new results were presented for the occurrence of this compound group in Europe.5,6 Today, the presence of nitro and polycyclic musks has been reported in a large number of environmental samples, including human tissue, and summarized in three recently published overviews.6–8 Owing to the dermal application of synthetic musks, uptake via the skin is considered to be the most important entrance route into the human body.However, in 1998, nitro and polycyclic musks were determined for the first time in Norwegian air samples from Kjeller.9 In one indoor air sample analyzed during the same sampling campaign, 10 times higher concentrations than detected in outdoor air were found, raising the suspicion that air as a transport and transfer medium for synthetic musk is still underestimated.Therefore, inhalation should also be included in considerations concerning exposure pathways for humans.Synthetic musks as indicator compounds for environmental monitoring Based on scientific considerations and oYcial control demands, for all industrialized countries priority lists of pollutants are developed and used for investigations in long-term national monitoring programs. In addition, international monitoring programs such as the Arctic Monitoring and Assessment Programme (AMAP) and the European Monitoring and Evaluation Programme (EMEP) and also the Oslo and Paris Commission (OSPARCOM) and the Helsinki Commission (HELCOM) have developed priority lists covering a representative number of environmental pollutants.Compounds on these lists are chosen according to applications and environmental properties. Persistent organic substances such as polychlorinated biphenyls (PCBs) as typical industrial chemicals, chlorinated pesticides as indicators of agricultural applications, chlorinated benzenes as indicators of unwanted industrial byproducts and polycyclic aromatic hydrocarbons (PAHs), chlorinated dioxins and furans produced during incineration processes are usually represented.All these indicator compounds chosen for monitoring purposes have several common properties: (1) they are highly persistent and accumulate in the food web; (2) they can be transported over long distances via the atmosphere and/or water currents into remote pristine regions; and (3) consequently, top predators (including humans) in the food web are often confronted with high concentrations of these compounds owing to biomagnification in food items.Recently, some nitro musks have joining these groups of monitoring target compounds. In 1997, MX and MK were added to the list of chemicals for priority action of the EU and in 1998 MX was added to the corresponding list of OSPARCOM. However, all synthetic musks, including the polycyclic compounds, have some specific properties in the environment which make them worth considering as a group of indicator chemicals for environmental monitoring programs.The following should illustrate the applicability of synthetic musks as priority compounds for environmental monitoring. 1. Direct relation to individual human traditions and habits Owing to their use as fragrances, synthetic musks are exclusively related to individual human use and can give a direct measure of human input into theJ.Environ. Monit., 1999, 1 71N Viewpoint Table 1 Structures, names and abbreviations of the most abundant synthetic musks Structure Chemical nomenclature Trivial name CAS number Abbreviation 1-tert-Butyl-3,5-dimethyl- Musk xylene 81-15-2 MX 2,4,6-trinitrobenzene O2N NO2 NO2 MK 4-Acetyl-1-tert-butyl-3,5- Musk ketone 81-14-1 dimethyl-2,6-dinitrobenzene O2N NO2 O GalaxolideA, AbbalideA, 1,3,4,6,7,8-Hexahydro-4,6,6,7,8,8- 1222-05-5 HHCB hexamethylcyclo[g]-2-benzopyran PearlideA O AHTN 1-(5,6,7,8-Tetrahydro-3,5,5,6,8,8- TonalideA, FixolideA 1506-02-1 hexamethyl-2-naphthalenyl)ethanone O 1-[2,3-Dihydro-1,1,2,6-tetramethyl- TraselideA 68140-48-7 ATII 3-(1-methylethyl)-1H-inden-5-yl]ethanone O environment.As reported for polycyclic musks, in 1995, an average use of about 15.5 mg d-1 for a ‘standard’ person in Europe was estimated.10 For nitro musks in 1996 the annual production rate was 2000 t a-1. According to the Research Institute of Fragrance Material (RIFM), for HHCB and AHTN annual use volumes were 3285 and 2067 t a-1 for 1992 and 1995, respectively.3 Hence synthetic musks must be considered at least in Europe as high volume chemicals for human usage.Synthetic musk does not undergo food web-related biomagnification (as documented for PCBs and pesticides) before reaching humans, because these compounds are usually applied directly to the human skin in high concentrations.11 2. High volume entry into the environment via households and waste water Direct application to the human skin from the use of washing agents and toiletry products and from washed textiles is assumed to be the most important step for human exposure.Subsequently, this application leads to the release of these compounds into the environment via households and sewage plants. In this context it is worth noting that high bioconcentration factors were calculated for freshwater fish via gills.3,7,12 Thus, in addition to the direct exposure to synthetic musks via toiletry products, an uptake via consumption of freshwater fish can be assumed for humans. However, this exposure will not be of the same order of magnitude as estimated for direct dermal application. 3. Indicator compounds for indoor air quality and ambient air levels The first investigations of indoor air levels of synthetic musks revealed another possible source of synthetic musks.For HHCB a concentration of 2.5 ng m-3 was determined in Norwegian laboratory air, whereas for ambient air samples an average value of 140 pg m-3 was found.9 Hence the importance of air as a possible impact route should not be neglected in the discussion of the environmental significance of synthetic musks. 4. Analytical accessibility Although special care has to be taken when analyzing environmental samples for synthetic musks,13 the analysis of this compound group is not diYcult and does not require particularly sophisticated analytical instrumentation. Usually, a standard trace analytical laboratory is able to determine synthetic musk levels by multi-residue methods if an eVective quality assurance program is followed which includes eliminating laboratory contamination, defining background levels and analyzing representative amounts of field, transport and laboratory blank samples.72N J.Environ. Monit., 1999, 1 Viewpoint investigated in numerous types of environmental samples, the documentation about concentration levels in the environment is not yet suYcient to be able to draw general conclusions.However, in freshwater systems, it could be shown that MK and MX are microbiologically degraded into their amino metabolites (Fig. 1). High concentrations of these metabolites were detected in sewage eZuent and river water in the River Elbe (Germany).16 These metabolites again have a much higher ecotoxicological risk potential than the parent compounds.17 Therefore, these metabolites must be included in ecotoxicological risk evaluation of nitro musks.Polycyclic musks For polycyclic musks, a first study of their ecotoxicological risk was published recently.10 Based on standard test parameters (e.g., EC50 calculations), the authors concluded that the ecotoxicological risk for these compounds is low, but their calculations did not consider the metabolites of the polycyclcic compounds.3 However, polycyclic musks are ideal examples to describe the limitations of parameters and scientific approaches used for standard ecotoxicological considerations.Odorous compounds such as synthetic musks are obviously produced to induce the human olfactory system as a messenger substance, and should therefore be considered as artificial pheromones for humans.In general, the receptors of the olfactory system belong to the same membrane binding receptor type as the hormone receptors. Therefore, the fact that pheromones can trigger the release of hormones is not very surprising.18 This connection between pheromone and related endocrine induction is well documented in fresh water fish for several pheromone–hormone interactions.Although no scientific investigations concerning pheromone–endocrine interactions have yet been carried out for polycyclic musks, the possible connection between pheromone and hormonal eVects is striking for this compound group. This hypothesis is also supported by the results presented by Frater et al.,19 who revealed interesting three-dimensional structure similarities between 5a-androst-16-en- 3-one (androstenone) and the two stereoisomers of HHCB with a musky HHCB Androstenone A: B: O O Fig. 2 Structural similarity between androstenone and HHCB, leading to comparable stimulation of the human olfactory systems. The oxygen atoms are marked as large black circles in (A).(A) Three-dimensional surface comparison; (B) chemical structures. O2N NO2 NO2 O2N NH2 NO2 O2N NO2 NH2 MX 4–amino–MX 2–amino–MX Fig. 1 The parent compound musk xylene (MX) and its main metabolites 2-aminomusk xylene (2-amino-MX) and 4-aminomusk xylene (4-amino-MX) which were detected in high concentrations in the River Elbe (Germany).16 Thus, for environmental monitoring, owing to their direct linkage to individual human tradition and habits, synthetic musks oVer the unique opportunity to investigate the role of chemical substances which are directly applied to humans via toiletry and perfume products.Dermal resorption of lipophilic cosmetic ingredients seems to be a new and relevant aspect concerning consumer health protection. In addition, the significance of biomagnification for uptake via the food web can be investigated and evaluated compared with direct application. For ecotoxicological considerations, the environmental input via both air and waste water should be considered and estimated in monitoring campaigns.Based on these first results and owing to the obvious environmental relevance, synthetic musks should be included in national and international food control measures as high priority chemicals and not only on a voluntary basis.For example, in 1994 MX and MK were included in the German Food Monitoring System7 and subsequently also in several German river and human milk monitoring systems. Toxicological aspects Nitro musks For nitro musks, the toxicological potential is still under discussion.In several publications, the low toxicity of nitro musks is documented and the conclusion is drawn that nitro musks in general do not represent an environmental threat at reported concentration levels.14,15 Although the occurrence of nitro musks has been odor. Androstenone is a steroid (Fig. 2), which can be found in secretions of various mammal species (e.g., domestic pig). In 1987 Gilbert and Wysocki20 argued for a non-independent relationship of androstenone-anosmia and HHCB-anosmia.Baydar et al.21 tested the ability and disability of a human test group to smell androstenone and HHCB. They found a sexmodulated linkage between traits of androstenone and HHCB smelling. Hence possible pheromone-like interactions cannot be neglected for polycyclic musks when estimating the ecotoxicological risk of these compounds.However, these aspects have not yet been considered in any ecotoxicological assessments available for polycyclic musks. Hence a more comprehensive discussion including pheromone- and hormone-like eVects is needed for polycyclic musks. Chirality as an important monitoring property All biochemical processes are based on chiral molecules (amino acids, proteins, nucleic acids).Therefore, a chiral pollutant, released into the environment as racemate (enantiomer ratio 151), can be metabolized enantioselectively. In this case, an enantiomeric ratio (ER) diVerent from unity is found in an environmental sample. Evidence for such chiral degradation based on theJ. Environ. Monit., 1999, 1 73N Viewpoint compounds can provide important information concerning ecotoxicological eVects at much lower concentration levels, as shown for enzymatic and endocrine ecotoxicological eVects.24 Therefore, these trace analytical results for crucian carps in combination with the enantioselective odorous properties of polycyclic musks indicate that the quantitative induction of the olfactory system can be a highly sensitive bioindicator for possible pheromone–hormone interactions.Perspectives and challenges Today, the need for sensitive evaluation systems for pollution eVects is often expressed. However, for such purposes, special biochemical properties of pollutants must be considered. A new, interesting approach is the identification of endocrine disrupting properties as an eVective measure of possible environmental harmful eVects.However, first investigations on the proportion of hormone disrupting xenobiotics in the total endocrine eVects in sewage treatment work eZuents have shown that the eVects of anthropogenic endocrine disruptors might be minimal compared with natural and synthetic steroid hormones (17b-estradiol, estrone and 17aethynylestradiol). 25 It is always diYcult to define ecotoxicological eVects for low concentration levels. Therefore, very sensitive indication systems are needed for such low-level monitoring purposes. The use of ‘biomarkers’ is gaining more and more importance in the field of biological monitoring. According to a recently published ecotoxicological textbook, biomarkers are defined as ‘a biological response to a chemical or chemicals that gives a measure of exposure and sometimes, also, of toxic eVect’.24 Ecotoxicologists prefer in this context the quantitative expression of a toxic eVect.However, only a few examples are known where environmental pollutants express measurable ecotoxic eVects at low concentration levels. Owing to the known low induction level for the olfactory system concerning natural pheromones, a scenario can be envisaged, where artificial pheromones might be able to induce endocrine responses at ultra-trace concentration levels in higher organisms.In addition, induced by artificial pheromones, the normal behavioral pattern of an organism might be disturbed and, thus, the reproduction rate could be reduced. However, these possible scenarios are only scientific speculations so far and need more data evaluation (e.g., during comprehensive monitoring programs).Therefore, strong interdisciplinary research eVorts are needed to confirm or falsify the aspects and suspicions raised here. Acknowledgements Robert Gatermann thanks the Norwegian Research Council (NFR) for a guest researcher fellowship (125745/720), ‘Ecotoxicological considerations concerning nitro and polycyclic musk compounds in the Norwegian Environment’.This work was partially funded by the Strategic Institute Programme ‘Ecotoxicology’ of the Polar Environmental Centre, Tromsø, Norway. We thank Frank HoVmann (University of Hamburg, Germany) for his help during the preparation of this publication. References 1 A. Baur, Ber.Dtsch. Chem. Ges., 1891, 24, 2832. 2 R. Gatermann, J. Hellou, H. Hu� hnerfuss, G. G. Rimkus and V. Zitko, Chemosphere, 1999, 38, 3431. 3 G. G. Rimkus, T oxicol. L ett., in the press. 4 T. Yamagishi, T. Myazaki, S. Horii and S Kaneko, Arch. Environ. Contam. T oxicol., 1983, 12, 83. 5 B. Liebl and S. Ehrensdorfer, Chemosphere, 1993, 27, 2253. 6 G. G. Rimkus and M.Wolf, Chemosphere, 1995, 30, 641. 7 G. G. Rimkus and H. Brunn, Erna�hrungs- Umschau, 1996, 43, 442. 8 H. Brunn and G. G. Rimkus, Erna�hrungs- Umschau, 1996, 44, 4. 9 R. Kallenborn, R. Gatermann, S. Planting, G. G. Rimkus, M. Lund, M. Schlabach and I. C. Burkow, J. Chromatogr., in the press. 10 E. J. van de Plassche and F. Balk, RIVM Report No. 601503008, RIVM, Bilthoven, 1997. 11 G. G. Rimkus, in Fragrances: Beneficial and Adverse EVects, ed.P. J. Frosch, J. D. Johansen and I. R. White, Springer, Berlin, 1998, p. 136. 12 G. G. Rimkus, W. Butte and H. J. Geyer, Chemosphere, 1997, 35, 1497. 13 K. Kokot-Helbling, P. Schmitt and C. Schlatter, Mitt. Geb. L ebensmittelunters Hyg., 1995, 86, 1. 14 D. E. Rickert, B. E. Butterworth and J. A. Popp, CRC Crit. Rev. T oxicol., 1984, 13, 217. 15 L. D. Lehman-McKeeman, D. R. Johnson and D. Caudill, T oxicol. Appl. Pharmacol., 1997, 142, 169. 16 R. Gatermann, H. Hu� hnerfuss, G. Rimkus, A. Attar and A. Kettrup, Fig. 3 Capillary gas chromatographie/mass spectrometric (electron ionization mode) determination of polycyclic musk enantiomers according to the method described.23 (A) Crucian carp muscle (Carassius carassius); (B) standard technical mixture.determination of ER has been found for a large number of chiral persistent pollutants.22 Several members of the polycyclic musk group are chiral and hence oVer the opportunity to study enantioselective processes in the environment. Chiral gas chromatographic methods using modified cyclodextrin stationary phases have been successfully employed for the enantioelective separation of chiral polycyclic musks (Fig. 3). A recent presentation on the chiral properties of polycyclic musks in freshwater biota was given by Gatermann et al.23 The enantiomeric distribution of chiral polycyclic musks has been described in freshwater biota (Fig. 3).23 For HHCB and ATII, two asymmetric carbon atoms can be defined and therefore two diastereomers and four enantiomers exist for these polycyclic musks (Fig. 3). Frater et al.19 proved that only the 4S,7R/S enantiomers carry the musky odor of HHCB. The remaining enantiomers do not smell at all, at least for humans. This finding proved that only two of four galaxolide enantiomers bind to the olfactory receptor, causing the musky odor impression. In addition, Gatermann et al.23 were able to demonstrate that in highly contaminated crucian carps mainly the odorous enantiomers of HHCB were degraded.It is known that the induction of pheromone eVects is often induced by much lower concentrations, as for example hormone induction. Therefore, the induction of the olfactory system by pheromone-like74N J. Environ. Monit., 1999, 1 Viewpoint Chemosphere, 1998, 36, 2535. 17 L. D. Lehman-McKeeman, D. R. Johnson, D. Caudill and S. B. Stuard, Drug Metab. Dispos., 1997, 25, 384. 18 A. N. Gilbert, Perfumer Flavorist, 1992, 17, 17. 19 G. Frater, U. Mu� ller, J. A. Bajgrowicz and M. Petrzilka, in Proceedings of the 13th International Congress of Flavours, Fragrances and Essential Oils, Istanbul, T urkey, October 15–19, 1995, ed. K. H. C. Baser, 1995, p. 151. 20 A. N. Gilbert and C. J. Wysocki, Nat. Geogr., 1987, 172, 514. 21 A. Baydar, M. Petrzilka and M. P. Schott, Chem. Senses, 1993, 18, 661. 22 W. Vetter and V. Schurig, J. Chromatogr., 1997, 774, 143. 23 R. Gatermann, S. Biselli, M. Hecker, G. G. Rimkus, L. Karbe, R. Kallenborn, W. A. Ko�nig and H. Hu� hnerfuss, Environ. Sci. T echnol., submitted for publication. 24 C. H. Walker, S. P. Hopkin, R. M. Sibly and D. B. Peakall, Principles of Ecotoxicology, Taylor and Francis, London, 1997. 25 C. Desbrow, E. J. Routledge, G. C. Brightly, J. P. Sumpter and M. Waldock, Environ. Sci. T echnol., 1998, 32, 1549. Roland Kallenborn, R. Gatermann Norwegian Institute for Air Research The Polar Environmental Centre, 9296 Tromsø, Norway G. G. Rimkus OYcial Food and Veterinary Institute Schleswig-Holstein, D-24517 Neumu