Occurrence of endocrine disruptors in wastewater from combined sewers in Paris and Beirut: what about parabens?

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1 Occurrence of endocrine disruptors in wastewater from combined sewers in Paris and Beirut: what about parabens? D. Geara-Matta* 1,2, C. Lorgeoux 3, V. Rocher 4, A. ElSamrani 5, G. Chebbo 1,6 and R. Moilleron 3 1 Leesu, Université Paris-Est, AgroParisTech, 6-8 avenue Blaise Pascal, Cité Descartes, Champs sur Marne, Marne la Vallée Cedex 2 (France) 2 Lebanese Atomique Energy Commission, Airport Highway BP , Riad El-Solh , Beirut (Lebanon) gearad@cereve.enpc.fr 3 Leesu, Université Paris-Est, AgroParisTech, 61 avenue du Général de Gaulle, Créteil Cedex (France) 4 SIAAP, Direction de Développement et de la Prospective, 82 Avenue Kleber, Colombes (France) 5 Sciences-EDST-PRASE Departement, Lebanese University, Campus Hadath-BP5, Beirut (Lebanon) 6 Engineering Departement, Lebanese University, Campus Hadath, Beirut (Lebanon) ABSTRACT Parabens are widely employed as preservative agents in many personal care products (PPCPs). In 2004, a possible link has been reported between breast tumors and the use of cosmetics containing parabens involving a controversy over parabens (Darbre et al. 2004). As a consequence, a new range of PPCPs, free of paraben, appeared on the market. However, about 60,000 PPCPs still contain parabens, mainly methylparaben (). Besides, parabens are known to be endocrine disrupting compounds. They are introduced into the receiving environment mainly via wastewater treatment plant. The aim of this study was to determine their levels in urban areas of two different conurbations, Paris and Beirut, during dry weather. For both cities, parabens followed the same uniform distribution: methylparaben () > ethylparaben (), propylparaben () > butylparaben () > isobutylparaben (Iso). However, their levels in France were higher than those in Lebanon, and they were correlated with PPCP references, containing parabens, available on the market in both countries. To our knowledge, this was the first study allowing the monitoring of these compounds in raw wastewater in France and in Lebanon. Our results will provide reference levels for the contamination of wastewater in both countries for paraben congeners. KEYWORDS Parabens; monitoring; France; Lebanon; wastewater Geara-Matta et al. 1

2 INTRODUCTION Societies throughout history have employed various forms of cosmetics to improve appearance, scent and health. To maintain the integrity of the personal care products, a preservative is required such as parabens, for example. Esters of p-hydroxybenzoic acid with alkyl and aryl subsistent (parabens) including methyl- (), ethyl- (), propyl- (), isobutyl- (Iso), butyl- () and benzylparaben (BzP) are mostly applied as preservative agents in Pharmaceuticals and Personal Care Products (PPCPs). They are allowed in concentrations of up to 1% in cosmetics (Tavares et al. 2009). In addition, they are added to canned foods and beverages, as preservatives. Parabens have probably been present in water and the environment for as long as humans have been using them. Indeed, their extensive consumption leads to continuous discharges into the environment mainly via wastewater treatment plant effluents and urban discharges during wet weather (Agüera et al. 2003) and it may lead to a pseudo-persistent situation (Jonkers et al. 2009). Recently, particular attention was giving to these chemicals due to their ecotoxicological potencies on human health and on the fauna and flora (Bazin et al. 2010). In fact, they have been identified as endocrine disrupters (Routledge et al. 1998). Moreover, possible synergistic effects of PPCPs with other chemicals, even at trace levels, may also become an issue (Canosa et al. 2006). In 2004, a possible link has been reported between breast tumors and the use of cosmetics containing parabens involving a controversy over parabens (Darbre et al. 2004). As a consequence, a new range of PPCPs, free of paraben, appeared on the market. However, about 60,000 PPCP still contain parabens, mainly (Mintel, report 2010). Besides, parabens have been identified as emergent pollutants by the NORMAN network in the category of PPCPs, meaning they should be monitored. The handful of studies that have examined paraben concentrations in wastewater provide data about their identification in the dissolved phase. Indeed, parabens have been detected in raw wastewater in Canada (Lee et al. 2005) and some European countries like Spain (González-Mariño et al. 2009; Pedrouzo et al. 2009), Denmark (Andersen et al. 2007) and Switzerland (Jonkers et al. 2009). To date, there is no data available about their environmental occurrence both in France and Lebanon. The main reason is that there is no regulation on parabens, since they are not officially identified or listed as endocrine disrupting chemicals by any government or regulation. However, other compounds such as bisphenol A which presents a similar EC50 than parabens for Daphnia magna (10 and 5-30 mg/l, respectively) have been recently banned in Europe. Therefore, the aim of this study was to determine their levels in urban areas of two different conurbations, Paris (France) and Beirut (Lebanon), during dry-weather. To our knowledge, this was the first study allowing the monitoring of parabens (,,, BzP, IsoBup and ) in raw wastewater in sewer trunks in both countries for the dissolved phase. The analytical procedure applied for the determination of those compounds in the dissolved phase of raw wastewater samples was based on a preconcentration, using solid phase extraction (SPE), followed by a quantification by ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS). METHODS Sampling strategy In Paris, four campaigns, during dry-weather, in April 2010 (without weekend days), were conducted on the five tributaries - namely, Clichy Bezon (CLB), Clichy Argenteuil (CLA), Saint Denis (StD), Sèvres Nanterre (SN) and Sèvres Reuil (SR) - of the largest WWTP in Paris, Seine Aval, that treats 1,780,000 m 3 /d of wastewater. A flow proportional 24-h composite sample was analyzed for parabens and water quality parameters (SS, COD, 2 Occurrence of endocrine disruptors in WW

3 conductivity, NTK, nitrates and nitrites). In Lebanon, six campaigns were performed on the unique tributary of the only existing and working WWTP, ElGhadir (Beirut), which treats 224,640 m 3 /d of wastewater. Due to technical aspects, it was not possible to get a 24-h composite sample unlike Paris. So, samples were collected by means of automatic sampler for different time periods of the day taking into consideration human activities (5-11 h, h, h and 23-5 h) on two consecutive days of the week (Monday 25 and Tuesday 26 October 2010) during dry-weather also. Sample treatment Upon arrival to laboratory, the samples were immediately filtered through glass fiber filters (GF/D 2.7 µm and GF/F 0.7 µm) to get the filtrate or dissolved phase. The suspended solids (i.e., the particulate phase) retained on filters were lyophilized prior to analysis. The filtrate was acidified to ph 2-3 with sulfuric acid, then spiked with a surrogate standard (deuterated -d4) and finally extracted on Oasis HLB (200 mg) according to Geara-Matta et al. (2010). Paraben analysis Parabens were analyzed by UHPLC-MS/MS in negative mode (Geara-Matta et al. 2010). UHPLC analysis was carried out using an Acquity UPLC system (Waters), coupled to a triple quadrupole TQD (Waters). An ACQUITY UPLC BEH C18 column (1.7 µm x 2.1 mm x 100 mm), fitted with a guard column (4 mm x 2.0 mm C18; Phenomenex), was used to achieve the chromatographic separation. The injected volume was 10 μl. A binary mobile phase was used. Solvant A was Milli-Q water with ammonium acetate (5 mm) and solvent B was methanol with ammonium acetate (5 mm). Quality control The quantification of all compounds was assessed by internal calibration as follows: for a direct adjustment was made by its deuterated internal standard (-d4), added to the sample just before chromatographic analysis, while for the remaining parabens (,,, Iso and BzP), the concentrations were corrected systematically by the surrogate standard, -d4 (result validated by the extraction recovery on real matrix). The linearity range was evaluated by injecting standard solutions at increasing concentrations up to 20 mg/l, according to the French AFNOR XP T standard (December 2008). The calibration models displayed linear behavior for parabens with coefficients of determination (R 2 ) ranging between and The method quantification limit (MQL) for each compound was calculated based on the instrumental quantification limit (determined according to the XP T standard), the overall recovery method and the volume of sample extracted. They ranged between 46 and 56 ng/l. According to our results, the overall method recoveries ranged from 94 ± 1 % to 108 ± 4 % (Geara-Matta et al. 2010). RESULTS AND DISCUSSION Paris (France) Temporal variability. The temporal variability was studied for each site via four campaigns conducted on four days of the week (Tuesday 06, Wednesday 14, Monday 19 and Tuesday 27 April 2010). The paraben concentrations appeared to be homogeneous, since RSD was < 20 % in most cases for four sites out of five: CLA, CLB, SN and SR (Table 1). However, the last site StD exhibited a slightly different trend, since the RSD ranged between 20 and 45 %. It might be related to the nature of the wastewater discharged. Geara-Matta et al. 3

4 It is interesting to note that for all samples, whatever the site or the day, BzP was never observed, its concentration was always < MQL. As a consequence, it will be no more considered. Our results indicated that was the most abundant congeners identified with a maximal concentration of 29,874 ng/l (for StD tributary) followed by and. These results are consistent with the occurrence of the different paraben congeners in cosmetics. Indeed,, and are the most used due to their higher solubility compared to that of their congeners with longer alkyl chains (Routledge et al. 1998). Table 1. Paraben levels (mean (ng/l) and RSD (%)) for the five tributaries in April 2010 (n=4) Iso Σparabens CLA 17,016 (7) 3,818 (5) 3,412 (6) 130 (11) 972 (8) 25,346 (16) CLB 14,302 (18) 3,412 (13) 3,345 (9) 203 (31) 1,065 (29) 22,326 (6) SN 11,727 (15) 3,169 (19) 3,018 (13) 122 (14) 674 (22) 18,710 (26) SR 11,774 (22) 3,267 (13) 3,108 (12) 131 (24) 708 (32) 18,988 (15) StD 21,321 (27) 4,778 (26) 3,913 (20) 213 (45) 1,221 (25) 31,445 (19) Spatial variability. The levels of parabens identified in the dissolved phase for the five tributaries analyzed are summarized in Table 2. By comparing the results for all tributaries, it can be observed that StD was the most contaminated site, whatever the paraben. Indeed, the highest concentrations were identified at this site: 29,874 (for ), 6,485 (), 5,049 (), 1,646 () and 347 ng/l (Iso). As can be seen from the Table 2, the study of the spatial variability confirmed the previous observation about StD. For example, the statistical treatment of our data showed that, excluding StD, the spatial variability, expressed as RSD, decreased from 30 to 21 %. The same trend was observed for all congeners but. No significant differences were found between temporal and spatial variabilities. Table 2. Paraben concentrations (ng/l) for all the tributaries with or without (*) considering StD tributary (n = 20 and 16, respectively) * * * Iso Iso* * Min. 8,243 8,243 2,423 2,423 2,493 2, Max. 29,874 17,994 6,485 4,013 5,049 3, ,646 1,423 Median 14,942 13,398 3,606 3,558 3,327 3, Mean 15,228 13,704 3,689 3,416 3,359 3, SD 4,692 2, RSD % Beirut (Lebanon) For Beirut, the paraben concentrations determined in wastewater are presented in Table 3. As previously observed for Paris, BzP was never observed. was the most abundant compound with a maximal concentration of 10,547 ng/l (17-23 h (Monday 25/10/10)). The concentrations for and were in the same range ( ng/l), whereas Iso and exhibited the lowest levels observed, less than 100 and 250 ng/l, respectively. The sample strategy followed for Beirut allowed us to observe that the levels of parabens in wastewater vary within the day. As a consequence, the highest levels were observed between 11 and 23 h, whereas the lowest were observed during in the 23 5 h sampling period. This present findings can be explained by human activities taking place in the morning (before going to work) and in the early evening (after coming back from work), and by the time needed for wastewater once discharged in the sewer network to reach the wastewater 4 Occurrence of endocrine disruptors in WW

5 treatment plant. During the night, the paraben concentration decreased by a third up to a quarter depending on the congener. This observation highlights the absence of human activities, implying PPCPs, during the night, as expected. Table 3. Concentration of parabens (ng/l) identified in ElGhadir tributary on 25 and 26 October 2010 for the four time slot (5-11 h; h; h; 23-5 h) Iso Σparabens 5-11 h (25/10) 7, , <MQL 9, h (25/10) 10,412 1,129 1, , h (25/10) 10, , , h (26/10) 8, , , h (25/10) 7, <MQL 113 9, h (25/10) 2, <MQL <MQL 3,640 Comparison of paraben concentrations between France and Lebanon and worldwide Comparison between France and Lebanon. These campaigns led to the identification of all parabens but BzP, with the highest occurrence for followed by and, at the same levels, in all raw wastewater samples in both countries (Figure 1). However, the levels recorded for Beirut were significantly smaller than those for Paris. Indeed, in Beirut,, and concentration represented 54 %, 25 % and 40 % respectively of the concentration determined in Paris. There are several possible explanations for this result, either the quality of wastewater or the nature and quantity of cosmetics used/available in both countries (Figure 2). In fact, the wastewater in Beirut, unlike Paris, is a mixture of industrial, including catering sector, and domestic wastewater, as confirmed by the observed levels of COD (1049 mgo 2 /L in Lebanon and 552 mgo 2 /L in France). 25,000 20,000 France Lebanon Concentration ng/l 15,000 10,000 5,000 0 Iso Figure 1. Comparison of paraben concentrations (mean ± SD) between Lebanon (n = 6) and France (n = 20) Comparison with other countries. Though parabens have been recently monitored in wastewater owing to an increasing concern about them, only a few numbers of studies provide data about their identification either in the dissolved phase or the raw sample. For the dissolved phase, our concentrations in wastewater both for France and Lebanon were much higher than those reported for Spain (5,138 (for ), 549 (), 1,302 (), 181 () and 89 ng/l (Iso) (Gonzalez-Marino et al. 2009)). However, the paraben concentrations in Geara-Matta et al. 5

6 Lebanon are close to those reported for Switzerland by Jonkers et al. (2009), especially for (9,880 ng/l), (719 ng/l) and (540 ng/l). Furthermore, as for France and Lebanon, the same trend was observed in Spain (Gonzalez-Marino et al.2009), Switzerland (Jonkers et al. 2009) and Canada (Lee et al. 2005), that is to say that presented the highest concentration followed by and. Correlation between paraben concentration and availability on cosmetic market Based on the database from Mintel (Mintel, report 2010), parabens are still used in a great numbers of cosmetics. Their occurrence in wastewater was in good agreement with their ubiquitous presence in the formulations of cosmetics on both the French and Middle East markets (Figure 2). Therefore, they were identified in wastewater in the following order: > ~ > >Iso. The comparison between Lebanon and France in terms of concentration and references emphasizes the presence of the same trend. 16,000 12,000 France Lebanon Concenration ng/l 8,000 4,000 y = 2.99x R 2 = 0.67 y = 3.20x R 2 = Iso Iso 0 2,000 4,000 6,000 8,000 References Figure 2. Paraben concentrations with respect to references on French ( ) and Lebanese (--) cosmetic markets. Besides, as shown Figure 3a, the ratio of the available references on market to paraben concentrations in wastewater for France and Lebanon (i.e., (Ref/[X]) France vs (Ref/[X]) Lebanon ; where [X] is the median concentration for all parabens but Iso) followed a linear regression emphasing a good correlation between the occurrence of parabens in raw wastewater and their availibility on French and Lebanese cosmetic markets (R 2 = ). Taking into account Iso (Figure 4b), the correlation decreased but still remained significant (R 2 = ). This could be explained by the fact that the short-chain parabens (, and ), frequently applied in aqueous suspensions such as shampoos and soaps, reflect the daily use in both countries, whereas the long-chain parabens, like Iso, are mostly used in oil-based skin creams whose frequency of uses could be strongly linked to lifestyle of each country. 6 Occurrence of endocrine disruptors in WW

7 20 20 y = x R 2 = y = x R 2 = Lebanon 10 Lebanon 10 Iso France Figure 3a. Comparison of the ratio (Ref/[X]) for France and Lebanon for all parabens but Iso France Figure 4b. Comparison of the ratio (Ref/[X]) for France and Lebanon for all parabens Distribution Paris (France) Beirut (Lebanon) 64(±3)% 16(±1)% 15(±2)% 4(±0.4)% Iso 1(±0.1)% 78(±2)% 8(±1)% 1 11(±1)% 1(±0.1)% Iso 1(±0.58)% Figure 5. Contribution (in percentage (±SD)) of each congener relative to the total level of parabens identified in Paris and Beirut The Figure 5 illustrates the paraben distribution. For a more reliable representation, the distribution was calculated using all samples except the one corresponding to night time (23-5h) in Lebanon. As previously stated, the same tendency was observed in Lebanon and France. is the most abundant congeners identified in all samples. It represented 64 ± 3 % of the sum of all the congeners in France and 78 ± 2 % in Lebanon. and present similar distributions, about 15 % in France and 10 % in Lebanon (Figure 5) that correlate with their use in cosmetics. Iso has the lowest proportion (1 % in both countries) since it is less used due to its poor water solubility (Routledge et al. 1998). CONCLUSION This is the first study to be conducted in France and Lebanon highlighting the presence of high concentrations of, and which correlate with their occurrences in PPCPs. The level of contamination followed a uniform distribution at the studied tributaries in France (except StD tributary) revealing the frequent use of personal care products containing these compounds in France. In Lebanon, parabens were determined at concentrations lower than in France but with a similar tendency ( > ~ > >Iso). These levels were correlated with references available on both PPCP markets. This work will be complemented by the quantification of these compounds in the particulate matters in order to assess their partition between the two phases (particulate and dissolved) and to state whether they are mainly in the dissolved phase or sorbed to particulate matter. Geara-Matta et al. 7

8 ACKNOWLEDMENT This study has been performed within the framework of the OPUR research program. The authors gratefully acknowledge the Seine-Normandy Water Agency (AESN), the Interdepartmental Association for Sewage Disposal in Paris Conurbation (SIAAP), the Municipality of Paris, the Water and Sewage Disposal Departmental Administrations of Seine Saint Denis (DEA93), the Water and Sewage Disposal Departmental Administration of Val de Marne (DSEA94) and Regional Council of "Ile de France" (CRIF) for their financial support. The authors would also like to thank Mrs Céline Briand (SIAAP, France) and Mr Ralph Salameh (PRASE, Lebanon) for their valuable technical assistance and Mrs Robbe-Tomine (Yves Rocher, France) for having helped us in understanding the cosmetic field. REFERENCES Agüera A., Fernandez-Alba A.R., Piedra L., Mezcua M. and Gomez M.J. (2003). "Evaluation of triclosan and biphenylol in marine sediments and urban wastewaters by pressurized liquid extraction and solid phase extraction followed by gas chromatography mass spectrometry and liquid chromatography mass spectrometry." Analytica Chimica Acta 480(2): Andersen H. R., Lundsbye M., Wedel H. V., Eriksson E. and Ledin A. (2007). "Estrogenic personal care products in greywater reuse system." Water Science & Technology 56(12): Bazin I., A. Gadal, Touraud E. and Roig B. (2010). Hydroxy Benzoate Preservatives (Parabens) in the Environment: Data for Environmental Toxicity Assessment. Xenobiotics in the Urban Water Cycle, Springer Netherlands. 16: Canosa P., Rodríguez I., Rubí E., Bollaín M. H. and Cela R. (2006). "Optimisation of a solid-phase microextraction method for the determination of parabens in water samples at the low ng per litre level." Journal of Chromatography A 1124(1-2): Darbre P. D., Aljarrah A., Miller W. R., Coldham N. G., Sauer M. J. and Pope G. S. (2004). "Concentrations of parabens in human breast tumours." Journal of Applied Toxicology 24(1): Geara-Matta, D., Lorgeoux C., Naah M., ElSamrani A., Chebbo G. and Moilleron R. (2010) Triclosan, triclocarban et parabenes, des perturbateurs endocriniens dans les eaux usées en France: approches analytiques pour déterminer les niveaux de concentrations. Proc. 19 th edition of Journées Information Eau, Poitiers, France September (2): González-Mariño I., Quintana J. B., Rodríguez I. and Cela R. (2009). "Simultaneous determination of parabens, triclosan and triclocarban in water by liquid chromatography/electrospray ionisation tandem mass spectrometry." Rapid Communications in Mass Spectrometry 23(12): Jonkers N., Kohler H.-P. E., Dammshäuser A. and Giger W. (2009). "Mass flows of endocrine disruptors in the Glatt River during varying weather conditions." Environmental Pollution 157(3): Lee H.-B., Peart T. E. and Svoboda M. L. (2005). "Determination of endocrine-disrupting phenols, acidic pharmaceuticals, and personal-care products in sewage by solid-phase extraction and gas chromatography-mass spectrometry." Journal of Chromatography A 1094(1-2): Pedrouzo M., Borrull F., Marcé R. M. and Pocurull E. (2009). "Ultra-high-performance liquid chromatographytandem mass spectrometry for determining the presence of eleven personal care products in surface and wastewaters." Journal of Chromatography A 1216(42): Routledge E. J., Parker J., Odum J., Ashby J. and Sumpter J. P. (1998). "Some Alkyl Hydroxy Benzoate Preservatives (Parabens) Are Estrogenic." Toxicology and Applied Pharmacology 153(1): Tavares R. S., Martins F. C., Oliveira P. J., Ramalho-Santos J. and Peixoto F. P. (2009). "Parabens in male infertility--is there a mitochondrial connection?" Reproductive Toxicology 27(1): Occurrence of endocrine disruptors in WW