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Environmental impact of sunscreeens and personal care products in the aquatic environment Karl Fent, Sara Zucchi & Verena Christen University of Applied.

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Presentation on theme: "Environmental impact of sunscreeens and personal care products in the aquatic environment Karl Fent, Sara Zucchi & Verena Christen University of Applied."— Presentation transcript:

1 Environmental impact of sunscreeens and personal care products in the aquatic environment Karl Fent, Sara Zucchi & Verena Christen University of Applied Sciences Northwestern Switzerland Muttenz, Switzerland and Swiss Federal Institute of Technology (ETHZ), Zürich

2 UV-filters Physically acting filters: TiO2, ZnO nanoparticles –Scatter and reflect UV rays Organic UV-absorbing chemicals (contain one or more aromatic rings) –Absorb energetic solar photons and returning to the ground state by thermally emitting absorbed energy –UVA: nm, UVB: nm Currently 28 UV-filters permitted for sunscreens and cosmetics in Europe

3 Organic UV-filters in sunscreens and cosmetics Benzophenone-3 (BP3) Benzophenone-4 (BP4) Deo EHMC Aftershave Shampoo BP4 Hairspray BP4 Lotions BP OC, EHMC Octocrylene (OC) Ethylhexyl-methoxy cinnamate (EHMC) BP1, BP3 Nail Polish Sunscreens

4 How do UV-filters enter the aquatic environment? directly indirectly

5 Many UV-filters are lipophilic and tend to bioaccumulate in biota and in human milk

6 Estrogenic UV-filters Polar: BP4 and BP3 Lipophilic: 4MBC and EHMC POCIS and fate modelling show that wastewater is a significant source Glatt May 2007

7 Swiss rivers: BP-4 and EHMC most important Water: Passive sampling (POCIS) BP-4 (log P = 0.89)up to 2402 ng BP-3 (log P = 3.64)up to 178 ng 4-MBC (log P = 4.95)up to 106 ng EHMC (log P = 5.66)up to 31 ng Fish and macroinvertebrates BP-4not detected BP-3up to 151 ng/g lipids 4-MBCrarely detected EHMCup to 337 ng/g lipids Zenker et al. 2008, J. Chromatogr. A: 1202: 64-74, Fent et al. (2010) Environ. Pollut. 158: 1817 BP-4 in Spain: up to 850 ng/L in rivers and up to 1480 ng/L in untreated wastewater (Rodil et al., 2008).

8 EHMC in all 53 biota, residues higher in cormorants but no significant biomagnification (BMF max. 1.5) Fent et al. (2010) Environ. Pollut.158:

9 BP4 is most prominent in water and EHMC in biota, others are also present Is there an ecological concern? What is their hormonal activity? What is their mode of action?

10 Effects on zooplankton Daphnia magna Toxicity increases with lipophilicity UV filterLog PowLC50 (48 h) (mg/L) Benzophenone-4 (BP-4) Benzophenone-3 (BP-3) Methylbenzylidene camphor (4-MBC) Ethyl-hexyl-4- trimethoxycinnamate (EHMC) Chronic (reproduction 21 d) 0.05 Fent et al. 2010, Mar.Env.Res. 69: S4-S6

11 Analysis of endocrine effects In vitro hormonal activity (YES, YAS) –Estrogenicity, antiestrogenicity –Androgenicity, antiandrogenicity In vivo estrogenicity in fish: vitellogenin induction –BP-1, BP-2, 3-BC, Et-PABA, BP-4, EHMC In vivo reproduction effects: decreased fertility –3-BC, BP-2, EHMC Mixture activity in vitro and in vivo in fish Molecular analysis Gene expression analysis: BP4 Transcriptomics: EHMC

12 19 UV-Filters Estrogenic Antiestrogenic Androgenic Antiandrogenic _______________________________9__________14_____________6___________10_____ Benzophenone Benzophenone ,4-Dihydroxybenzophenone Ethyl-4-aminobenzoate Benzophenone-3 (BP-3) nd Benzophenone-4 (BP-4) nd Benzylsalicylate (BS) nd Phenylsalicylate (PS) nd 3-Benzylidene camphor (3-BC) Ethylhexyl methoxy cinnamate (EHMC = OMC) Octyl salicylate (OSC) Methylbenzylidene camphor (4-MBC) Homosalate (HMS) Isopentyl-4-methylcinnamate (IMC) Octocrylene (OC) nd 4-Aminobenzoate (PABA) nd (N,N')-dimethyl-octyl-4- aminobenzoate (OD-PABA) nd Ethyl 4-aminobenzoate (Peg25 PABA) -+- nd Kunz & Fent (2006), Aquat. Toxicol.79: Hormonal activity in vitro

13 Estrogenic activity in vitro – in vivo Kunz et al. (2006). Toxicological Sciences 93:

14 Kunz & Fent (2006) Tox. Appl Pharmacol 217: Activity of UV-filter mixtures in vitro Four and eigth UV filters show marked activity at NOEC They act synergistically in vitro Recombinant yeast YES

15 Mixture activity of 3 estrogenic UV-filters BP1, BP2, 3BC in fish in vivo UV filters show concentation addition for estrogenicity in mixtures Tendency to antagonism at low concentrations Kunz and Fent, Toxicol.Appl.Pharmacol. 234: 77-88, 2009

16 Benzophenone 2 Estrogenic activity (vitellogenin induction) and effects on fertility and reproduction in fathead minnows

17 Effects of BP-2 on fertility in fathead minnows: egg production reduced at 1.2 mg/L and higher

18 Secondary sexual characteristics in males Dose-dependent feminization and effects on fertility (decrease in egg production): LOEC for 3BC: 3 ug/L, for BP-2: 1.2 mg/L Kunz et al. (2006) Tox Sci.; Weisbrod et al. (2007), Toxicol. Appl. Pharmacol.. 3-Benzylidene camphorBenzophenone-2

19 Zebrafish: embryo study 5 days exposure in 24-well microtiter plates, 6 replicates per dose 30 µg/L (worst case environmental level) and 3 mg/L after 120 hpf, 15 larvae were pooled for qRT-PCR Effects of benzophenone-4: gene expression analysis with focus on hormonal pathways in zebrafish 14 d exposure to 3 concentrations, 5 replicates each 3 concentrations: 30 µg/L, 300 µg/L and 3 mg/L after 14 d dissection of brain, liver and testis, RNA extraction qRT-PCR Targeted gene expression approach Zebrafish: male adults Zucchi et al. (2011), Tox. Appl. Pharmacol. 250:


21 Expression of genes involved in 4 different hormonal pathways investigated by qRT-PCR Embryo: 10 gene transcripts Vtg1 Vtg3 Estrogen receptor alpha (esr1) Estrogen receptor beta1 (esr2b) Androgen receptor 17 ß-HSD (hydroxsteroid dehydr.) P450aromA (CYP19A) P450aromB (CYP19B) paired box gene 8 (pax8) hematopoietically expressed homeobox (hhex) Adult males: 11 transcripts Vtg1 Vtg3 Estrogen receptor alpha (esr1) Estrogen receptor beta1 (esr2b) igfbp1a (liver) Androgen receptor 17 ß-HSD (hydroxsteroid dehydr.) P450aromA (CYP19A) (testis) P450aromB (CYP19B) (brain) StAR (testis) 3 ß–HSD (testis) Estrogenic, androgenic, steroidogenesis, thyroid hormone system develop.

22 P450aromB (cyp19b) P450aromA (cyp19a) Effects on genes involved in steroidogenesis Hydroxysteroid 17-ß dehydrogenase-3 (hsd17ß3)

23 Liver Insulin-like growth factor binding protein 1 (igfbp1) Estrogen responsive genes (Hoffman et al., 2006) *** ** * *** P450aromB (cyp19b) Brain ** * P450aromA (cyp19a) Testis

24 BP-4: Summary BP4 leads to alteration of gene expression in adult fish and embryos. Effects in adults occur already at 30 µg/L, in embryos mainly at 3 mg/L. Expressional changes differ between different tissues, and also between embryos and adults. Embryos: Estrogen-like activity –Up-regulation of vtg1 and vtg3 at 3 mg/L, esr1, esr2b (at 30 µg/L ) Embryos: Upregulation of genes involved in steroidogenesis –aromatases cyp19a, cyp19b and hsd17ß3 Adults: –Liver: Antiestrogen-like activity by down-regulation of vtg, esr, but up-regulation of igfbp1 –Brain and testis: Induction of vtg > Consequence: Interference with hormonal pathways in embryos and adults BP-4 has low activity: alterations occur mainly at 3 mg/L Effects on physiology, steroid levels, sex determination and fertility? Zucchi et al. 2011, TAAP 250:

25 Effects of EHMC on gene expression, vitellogenin induction and histology of gonads in fathead minnows Christen et al. (2011)

26 Down-regulation of androgen receptor in liver after 14 d of exposure of fathead minnows E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: g/L EHMC H: 394 g/L EHMC

27 Down-regulation of 3-beta hydroxysteroid dehydrogenase Conversion of pregnenolone > progesterone E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: g/L EHMC H: 394 g/L EHMC

28 Induction of plasma vitellogenin in males E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: g/L EHMC H: 394 g/L EHMC

29 Testis A: water B: solvent C: 5.4 g/L EHMC D: 37.5 g/L EHMC E: g/L EHMC F: 394 g/L EHMC Histology of gonads: fathead minnows Sg: spermatogonia Sc: spermatocytes M: spermatides Christen et al. (2011)

30 Histological effects on gonads Testes Decreased number of spermatocytes increase in spermatides (sperms) E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: g/L EHMC H: 394 g/L EHMC Ovaries Increase in vitellogenic (and in atretic) oocytes

31 What is the mode of action of EHMC? What toxicological pathways are affected? In vitro study showed antiestrogenic and antiandrogenic activity

32 Quantitative real time PCR Microarrays Analysis of molecular effects and modes of action of EHMC in zebrafish: Transcriptomics (Agilent 4×44 K Zebrafish) Aim: Determine molecular effects and mode of action in whole body of fish after 14 d exposure to 2.2 (environmental) and 890 µg/L (toxicological relevant) (measured concentrations). Whole body (RNA extraction) for microarray analysis 3 tissue collected for qRT-PCR validation analysis (liver, brain and testis).

33 Venn Diagramm: Whole body transcription profile GeneSpring GX genes altered in common 2.2 µg/L890 µg/L 594 (54.2%) 699 (61.48%) 502 (45.8%) 438 (38.52 %) 4 replicates for each exposure groups 3000 µg/L (ANOVA; p<0.05, fold change 2, TukeyHSD as Posthoc Test, FDR = 0.05) At 2.2 µg/L: 1096 genes At 890 µg/L: 1137 genes Environmental Concentration (ANOVA; p<0.05, und fold change 2, TukeyHSD as Posthoc Test, FDR = 0.05) All genes Zucchi et al. (2011) Env. Poll. 159: 3086

34 Selected transcripts among altered pathways GO process Investigated transcripts (qPCR) MetaCore software (FDR< 0.05) Apoptosis/cell differentiation Response to stress growth arrest and DNA-damage-inducible beta (gadd45b) prostaglandin D2 synthase (ptgds) vitellogenin 1 (vtg1) retinol binding protein 2 (rbp2) Insulin-like growth factor binding protein 1 (igfbp1) Lipid Biosynthesis and regulation vitamin A metabolic process/ retinol metabolic process regulation of cell growth insulin receptor signaling pathway tissue regeneration Lipid metabolism/ Response to estrogen stimulus

35 After confirmation of microarray data with qRT-PCR: Whole body vs. liver brain and testis qRT-PCR validation Whole body LiverBrainTestisWhole body LiverBrainTestis Zucchi et al. (2011) Env. Poll. 159: 3086 prostaglandin D2 synthase

36 EHMC: Summary EHMC leads to differential gene expression in liver, gonads and brain. Changes differ between tissues, and between males and females of zebrafish Expressional changes point to multiple hormonal activities –Estrogen-like Induction of vitellogenin protein in plasma Down-regulation of ar in liver –Interference with genes involved in steroidogenesis Down-regulation of hsd3 Histological effects: lower spermatocytes and altered oocyte maturation (ovary) in fathead minnows (fhm) Expressional changes at 2.2 and 890 g/L (zebrafish). Histology: g/L, VTG induction: 245 g/L (fhm). Highest level in raw wastewater 19, in surface water 3 g/L Christen et al. 2011, Aquat. Toxicol. 102:

37 MEC / PNEC Measured environ conc.(MEC) (Langford 2007; Rodil 2008,2009) (own data) BP4: ug/L EHMC: 0.238, 3 ug/L 6 ng/L 4MBC: 1.14 ug/L 17 ng/L BP3: ug/L 68 ng/L 3BC: det. BP1, BP2: n.d. Et-PABA: n.d. Effect concentration (PNEC) Ref: fish: literature, own data, Daphnia: own data BP4: 0.03 ug/L (fish), 50 ug/L (Daphnia) EHMC: ug/L (fish), 0.29 ug/L (Daphnia) 4MBC: 0.74 ug/L (fish), 0.56 ug/L (Daphnia) BP3: 0.62 ug/L VTG, decreased fecundity in fish 3BC: 30 ng/L (Kunz et al. 2006) (SF 100) Safety factor (SF) 1000 (acute tox., gene expr.) Environmental risk for BP4, EHMC, 4MBC. 3BC and BP2: Risk probable, however not yet detected in environment Environmental risk assessment for UV filters see also Fent et al. 2008, Chimia 62: for 3BC, BP1, BP2, Et-PABA Mixture activity must be considered: additive action of EHMC, BP3, OC Fent et al. 2010, Mar.Env.Res. 69: S3-S6

38 Conclusions Chemicals in cosmetics and personal care products lead to chronic exposure of humans and environment UV-filters 3BC, Et-PABA, BP2, BP4 and EHMC affect hormonal system of fish, although some at higher than environmental concentrations BP4, EHMC: Targeted gene expression and transcriptomics reveals molecular effects and modes of action: –More pathways than hormonal signalling are affected –Gene expression analysis is more sensitive than physiological or histological effects –Transcriptional changes are complex, tissue-dependent and different in males and females EHMC shows multiple hormonal activities and affects many different cellular, biochemical and physiological pathways

39 Cosmetics containing phthalates, disinfectans (triclosan) Phthalates (DEP) Disinfectants Shampoos Tooth paste Fragrances Hair styling Conditioner Anti ageing products Shaving foam After shave Shaving foam Lotions Cleansing Deo

40 Some phthalates have anti-androgenic activity in vitro (MDA-kb2 cells) benzylbutyl phthalate, dibutyl phthalate, diethyl phthalate Christen et al. Chemosphere 2010

41 Binary mixtures of phthalates BBP, DBP and DEP show concentration addition

42 Binary mixtures show concentration addition with tendency to antagonism at low and synergism at high levels (toxic unit approach) EC50 mixture EC25 mixture no lines EC10 mixture

43 Antimicrobials/disinfectants triclosan and triclocarban Triclocarban (TCC) used in PCPs since 1957 Triclosan (TCS) widespread use Widespread in environment. Found in surface waters: –TCC: ng/L, median 95 ng/L –TCS: ng/L, median 48 ng/L Algae have high sensitivity to TCS through disruption of lipid synthesis, membrane destabilization, uncoupling of oxidative phosphorylation Acute ffects in invertebrates and fish in the hundreds of ug/L through unspecific narcosis Hormonal effect of TCS in fish (Raut & Angus, Env.Tox.Chem. 2010, 29:1287) –Vitellogenin mRNA expression significantly elevated at ug/L (350 nM) TCS. Sperm counts significantly decreased in western mosquitofish, Gambusia affinis)

44 Enhancement of androgenic activity by biocides triclosan (TCS) and triclocarban (TCC) Christen et al. 2010, Chemosphere 81: LOEC of TCS 10 nM=2.9 ug/L

45 Financial support


47 Endocrine activity shown mammals…

48 UV filters accumulate in human milk 54 samples analysed between Schlumpf et al. 2010, Chemosphere 81: UV filterNumber of positive samples (%) Mean level (ng/g) lipids Ethylhexyl-methoxy cinnamate (EHMC) 42 (78%)27.5 Octocrylene (OC)36 (67%) Methylbenzylidene camphor (4MBC) 11 (20%)22.1 Benzophenone-3 (BP3) 7 (13%)52.2 Homosalate3 (6%)29.4 Octyldimethyl-PABA1 (2%)49.0

49 Mixtures of 2 UV filters show mainly synergistic activity in vitro Kunz & Fent (2006) Tox. Appl Pharmacol 217: Recombinant yeast YES

50 3BC (3-Benzylidene camphor) BP2 (Benzophenone-2) Kunz et al. (2006). Toxicol.Sci. 90 (2): Yeast in vitro Fish in vivo BP2 estrogenic (full agonist)estrogenic (VTG induction) 3BC estrogenic (partial agonist)estrogenic (VTG inductionI Effects on fecundity and reproduction: 3BC and BP2 Kunz & Fent (2006). Aquat. Toxicol. 79:

51 Histology of gonads Ovary A: water B: solvent C: 5.4 g/L EHMC D: 37.5 g/L EHMC E: g/L EHMC F: 394 g/L EHMC 1: primary oocyte 2: previtellogenic oocyte 3: vitellogenic oocyte

52 Expression of estrogen receptor Males Females E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: g/L EHMC H: 394 g/L EHMC

53 Expression of vitellogenin (qPCR) Males Females E: 5.4 g/L EHMC L: 37.5 g/L EHMC M: g/L EHMC H: 394 g/L EHMC

54 cyp19b fold change is 2.4 Whole body Liver BrainTestis Whole body Liver BrainTestis Brain

55 14 d exposure: whole body (RNA extraction) for microarray analysis For transcriptome analysis, 16 microarrays (Agilent 4 × 44 K) were analysed 3 tissue collected for qRT-PCR validation analysis (liver, brain and testis) Exposure of male zebrafish to 3 µg/L and 3000 µg/L Scan and quantification of the raw data GeneSpring GX GO analysis Fish exposure mRNA extracted from whole body Cy3 labeled Cy3 cDNA RT Hybridisation Microarray Wash Danio rerio

56 Hormonal activity in vitro of phthalates and disinfectants in personal care products Aims –Determination of antiandrogenic activity of single compounds in personal care products Method: in vitro activity in MDA-kb2 cells - Human AR/luciferase cell line from mammary gland - express androgen responsive firefly luciferase reporter gene

57 2-ethyl-hexyl-4-trimethoxycinnamate (EHMC) untreated municipal wastewater up to 19 μg/L EHMC detected in human milk (Hany and Nagel,1995) and tap water; found in all biota in Swiss rivers, EHMC tend to bioaccumulate in the food chain (Fent et al., 2010) (logP=5.66). estrogenic activity in male medaka at high concentrations (Inui et al., 2003). Aim Determine the modes of action of this wide use UV filter compound in zebrafish (Danio rerio) adult males by analyzing the global gene expression pattern (microarray) and by investigating mRNA expression (qRT-PCR) of more classical genes involved in hormonal pathways.

58 Expression of target genes involved in 3 different hormonal pathways in liver, gonads, brain after exposure for 14 days and comparison with plasma vitellogenin and gonad histology Adult males and females: 8 transcripts Estrogen receptor alpha (esr1) Vitellogenin (vtg1) Prostaglandin D2 synthase (ptgds): induced by estradiol gluthation S-transferase alpha (gst): induced by estradiol, phase II enzyme Androgen receptor (ar) 3 ß–hydroxysteroid dehydrogenase ( converts pregnelolone > progesterone ) P450aromA (cyp19a1a) P450aromB (cyp19a1b) Estrogenic, androgenic, steroidogenesis

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