environmental impact of sunscreeens and personal care products in the aquatic environment karl fent,...

Environmental impact of sunscreeens and personal care products in the aquatic

environment

Karl Fent, Sara Zucchi & Verena Christen

University of Applied Sciences Northwestern SwitzerlandMuttenz, Switzerland

andSwiss Federal Institute of Technology (ETHZ), Zürich

[email protected], [email protected]

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: 400-320 nm, UVB: 320-280 nm

• Currently 28 UV-filters permitted for sunscreens and cosmetics in Europe

Organic UV-filters in sunscreens and cosmetics

Benzophenone-3 (BP3) Benzophenone-4 (BP4)

Deo

EHMC

Aftershave

Shampoo

BP4

Hairspray

BP4

Lotions

BP3

www.codecheck.info, 2011

OC, EHMC

Octocrylene (OC) Ethylhexyl-methoxy cinnamate (EHMC)

BP1, BP3

Nail Polish

Sunscreens

How do UV-filters enter the aquatic environment?

directlyindirectly

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

Estrogenic UV-filters

Polar: BP4 and BP3Lipophilic: 4MBC and EHMC

POCIS and fate modellingshow that wastewater is a significant source

GlattMay 2007

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 ng4-MBC (log P = 4.95) up to 106 ngEHMC (log P = 5.66) up to 31 ng

Fish and macroinvertebratesBP-4 not detectedBP-3 up to 151 ng/g lipids4-MBC rarely detected EHMC up 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).

EHMC in all 53 biota, residues higher in cormorantsbut no significant biomagnification (BMF max. 1.5)

Fent et al. (2010) Environ. Pollut.158: 1817-1824

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?

Effects on zooplankton Daphnia magnaToxicity increases with lipophilicity

UV filter Log Pow LC50 (48 h)

(mg/L)

Benzophenone-4

(BP-4)

0.89 50

Benzophenone-3

(BP-3)

3.64 1.9

4-Methylbenzylidene camphor (4-MBC)

4.95 0.56

2-Ethyl-hexyl-4-trimethoxycinnamate

(EHMC)

5.66 0.29

Chronic (reproduction 21 d)0.05

Fent et al. 2010, Mar.Env.Res. 69: S4-S6

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

19 UV-Filters Estrogenic Antiestrogenic Androgenic Antiandrogenic_______________________________9__________14_____________6___________10_____

Benzophenone-1 +++ - - +++ Benzophenone-2 +++ - +++ + 4,4‘-Dihydroxybenzophenone +++ - - +++ Ethyl-4-aminobenzoate +++ - - + Benzophenone-3 (BP-3) + +++ - ndBenzophenone-4 (BP-4) + +++ - ndBenzylsalicylate (BS) + +++ - ndPhenylsalicylate (PS) + +++ - nd3-Benzylidene camphor (3-BC) + +++ - +++Ethylhexyl methoxy cinnamate (EHMC = OMC) - +++ ++ +++ Octyl salicylate (OSC) - +++ +++ +++4-Methylbenzylidene camphor (4-MBC) - +++ - +++Homosalate (HMS) - +++ +++ +++Isopentyl-4-methylcinnamate (IMC) - +++ ++ +++Octocrylene (OC) - +++ + nd4-Aminobenzoate (PABA) - +++ - nd(N,N')-dimethyl-octyl-4-aminobenzoate (OD-PABA) - +++ - nd Ethyl 4-aminobenzoate (Peg25 PABA) - + - nd

Kunz & Fent (2006), Aquat. Toxicol.79: 305-324

Hormonal activity Hormonal activity in vitroin vitro

Estrogenic activity Estrogenic activity in vitroin vitro – – in vivoin vivo

4MBC

* ***

*

Wat

erS

olv

ent

E2 9

100

435

953 9

100

500

981

4919 10 100

500

1067

8783 9

100

415

7531

10

100

1000

10000

100000

1000000

10000000

3BC BP2BP1

Concentration (g/L)

Vit

ello

gen

in (

ng

/mL

)

*

4DHB

Wat

erS

olv

ent

E2 8

100

500

889

5025 12 100

500

766

3900 11

100

500

1048

4897 10 100

500

900

5011

1

10

100

1000

10000

100000

1000000

10000000

OMC BP4BP3

Concentration (g/L)

Vit

ello

gen

in (

ng

/mL

)

10-10 10-8 10-6 10-4 10-2-25

0

25

50

75

100

125E2

Blank

4MBC

3BC

BP2

BP-1

Concentration (M)

Eff

ect

(%)

10-10 10-8 10-6 10-4 10-2-25

0

25

50

75

100

125E2

BP3

BP4

Blank

4DHB

OMC

Concentration (M)

Eff

ect

(%)

Kunz et al. (2006). Toxicological Sciences 93: 311-321.

Kunz & Fent (2006) Tox. Appl Pharmacol 217: 86-99

0.025

0.042

0.128

0.021

0.004

0.009

0.034

0.011

-2.5 -1.5 -0.5 0.5 1.5 2.5 3.5 4.5

4 pure hERαagonists

4 pure hERαagonists + E2

4 pure and 4 partialhERα agonists

4 pure and 4 partialhERα agonists +E2

1Synergism Additivity line Antagonism

BC10

NOEC

Activity of UV-filter mixtures in vitroFour and eigth UV filters show marked activity at NOECThey act synergistically in vitro

Recombinant yeast YES

10-7 10-6 10-5 10-4-10

0

10

20

30

40

50

60

70

80

90

100 3BC

BP2BP1

EC30

EC20

EC10

EC-NOECEC1EC5

Concentration (M)

Vit

ello

ge

nin

In

du

cti

on

(%

)

Mixture activity of 3 estrogenic UV-filters BP1, BP2, 3BC in fish in vivo

UV filters show concentation addition for estrogenicity in mixturesTendency to antagonism at low concentrations

10-11-10

0

10

20

30

40

50

60

70

80

90

100

3BC

BP2BP1

EC30EC20EC10

EC-NOECControls

10 -7 10 -6 10 -5

EC1

EC5Mixture

EE2

SC

Concentration (M)

Vit

ello

gen

in I

nd

uct

ion

(%

)

Kunz and Fent, Toxicol.Appl.Pharmacol. 234: 77-88, 2009

Benzophenone 2 Estrogenic activity (vitellogenin induction) and effects

on fertility and reproduction in fathead minnows

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

Secondary sexual characteristics in malesDose-dependent feminization and effects on fertility (decrease in eggproduction): LOEC for 3BC: 3 ug/L, for BP-2: 1.2 mg/L

H20 Solvent 0.5 3 33 74 2850

5

10

15

20

3BC (g/L)

Nu

mb

er o

fN

up

tial

Tu

ber

cule

s

Kunz et al. (2006) Tox Sci.; Weisbrod et al. (2007), Toxicol. Appl. Pharmacol..

3-Benzylidene camphor Benzophenone-2

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: 137-146

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.

cyp19b

Control

g/L

30

g/L

3000

0.0

0.5

1.0

1.5

2.0

2.5*

Fo

ld c

han

ge

cyp19a

0

1

2

3 **

Fo

ld c

han

ge

P450aromB (cyp19b) P450aromA (cyp19a)

Effects on genes involved in steroidogenesis

hsd173

Control

g/L

30

g/L

3000

0.0

0.5

1.0

1.5 *

Fo

ld c

han

ge

Hydroxysteroid 17-ß dehydrogenase-3

(hsd17ß3)

LiverInsulin-like growth

factor binding protein 1 (igfbp1)

Estrogen responsive genes (Hoffman et al., 2006)

0

1

2

3

4

Log

2

***

***

-2

0

2

4

6

Log

2

***

P450aromB (cyp19b)

Brain

-6

-4

-2

0

2

Log

2

** *

P450aromA (cyp19a)

Testis

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: 137-146

Effects of EHMC on gene expression, vitellogenin induction and histology of

gonads in fathead minnows

Christen et al. (2011)

Down-regulation of androgen receptor in liverafter 14 d of exposure of fathead minnows

E: 5.4 g/L EHMCL: 37.5 g/L EHMCM: 244.5 g/L EHMCH: 394 g/L EHMC

Down-regulation of 3-beta hydroxysteroid dehydrogenaseConversion of pregnenolone > progesterone


Induction of plasma vitellogenin in males

Males

water

Solven

t E L M H0

5

10

15

20*

VT

G ( g

/ml)

Females

Wat

er

Solven

t E L M H0

1000

2000

3000

4000

5000

VT

G ( g

/ml)


Testis

A: waterB: solventC: 5.4 g/L EHMCD: 37.5 g/L EHMCE: 244.5 g/L EHMCF: 394 g/L EHMC

Histology of gonads: fathead minnows

Sg: spermatogoniaSc: spermatocytesM: spermatides

Christen et al. (2011)

Histological effects on gonads

Testes

Decreased number of spermatocytesincrease in spermatides (sperms)

0

20

40

60

80

100

SpermatogoniaSpermatocytesSpermatides

*Fre

qu

ency

(%

)


Wat

er

Solven

t E L M H0

20

40

60

80

100

primary oocytes previtellogenic oocytesvitellogenic oocytes atretic follicles

*

*

*

Fre

qu

ency

(%

)

Ovaries

Increase in vitellogenic (and in atretic) oocytes

What is the mode of action of EHMC?

What toxicological pathways are affected?

In vitro study showed antiestrogenic and antiandrogenic activity

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).

Venn Diagramm: Whole body transcription profile

GeneSpring GX11708 genes altered in common

2.2 µg/L 890 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

Selected transcripts among altered pathways

GO processInvestigated transcripts

(qPCR)

MetaCore software (FDR< 0.05)

Apoptosis/cell differentiationResponse 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 growthinsulin receptor signaling pathway

tissue regeneration

Lipid metabolism/Response to estrogen stimulus

After confirmation of microarray data with qRT-PCR:Whole body vs. liver brain and testis qRT-PCR validation

vtg1

DMSO g/L

2.

2 g/L

890

DMSO g/L

2.

2 g/L

890

DMSO g/L

2.

2 g/L

890

DMSO g/L

2.

2 g/L

890

-4

-2

0

2

**

* *

**

****

Fo

ld C

ha

ng

e (

log

2)

Wholebody

Liver Brain Testis

ptgds

-2

-1

0

1

2

3

*

**

**

*

Fold

Cha

nge

(log2

)

Wholebody

Liver Brain Testis

Zucchi et al. (2011) Env. Poll. 159: 3086

prostaglandin D2 synthase

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: 38-394 g/L, VTG induction: 245 g/L (fhm).

Highest level in raw wastewater 19, in surface water 3 g/LChristen et al. 2011, Aquat. Toxicol. 102: 167-176

MEC / PNEC

Measured environ conc.(MEC) (Langford 2007; Rodil 2008,2009) (own data)

BP4: 0.894 ug/L

EHMC: 0.238, 3 ug/L 6 ng/L 4MBC: 1.14 ug/L 17 ng/LBP3: 0.268 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: 0.002-0.890 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 filterssee also Fent et al. 2008, Chimia 62:368-375 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

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

Cosmetics containing phthalates, disinfectans (triclosan)

Phthalates (DEP) Disinfectants

www.codecheck.info

Shampoos Tooth paste

Fragrances

Hair styling

ConditionerAnti ageing productsShaving foam

After shaveShaving foam

Lotions

Cleansing

Deo

Some phthalates have anti-androgenic activityin vitro (MDA-kb2 cells)

benzylbutyl phthalate, dibutyl phthalate, diethyl phthalate

Christen et al. Chemosphere 2010

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

Binary mixtures show concentration addition with tendency to antagonism at low and synergism at high levels (toxic unit approach)

EC50 mixture

EC25 mixtureno lines EC10 mixture

Antimicrobials/disinfectants triclosan and triclocarban

• Triclocarban (TCC) used in PCPs since 1957• Triclosan (TCS) widespread use• Widespread in environment. Found in surface waters:

– TCC: 19-6750 ng/L, median 95 ng/L– TCS: 0.1-2300 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 101.3 ug/L (350 nM)

TCS. Sperm counts significantly decreased in western mosquitofish, Gambusia affinis)

Enhancement of androgenic activity by biocidestriclosan (TCS) and triclocarban (TCC)

Christen et al. 2010, Chemosphere 81: 1245-1252

LOEC of TCS 10 nM=2.9 ug/L

Financial support

Endocrine activityshown mammals…

UV filters accumulate in human milk54 samples analysed between 2004-2006

Schlumpf et al. 2010, Chemosphere 81: 1171-1183

UV filter Number of positive samples (%)

Mean level

(ng/g) lipids

Ethylhexyl-methoxy cinnamate (EHMC)

42 (78%) 27.5

Octocrylene (OC) 36 (67%) 30.2

4-Methylbenzylidene camphor (4MBC)

11 (20%) 22.1

Benzophenone-3 (BP3)

7 (13%) 52.2

Homosalate 3 (6%) 29.4

Octyldimethyl-PABA 1 (2%) 49.0

Mixtures of 2 UV filters show mainly synergistic activity in vitro

Kunz & Fent (2006) Tox. Appl Pharmacol 217: 86-99

Recombinant yeast YES

3BC (3-Benzylidene camphor)

BP2 (Benzophenone-2)

Kunz et al. (2006). Toxicol.Sci. 90 (2): 349-361.

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: 305-324.

Histology of gonads

Ovary

A: waterB: solventC: 5.4 g/L EHMCD: 37.5 g/L EHMCE: 244.5 g/L EHMCF: 394 g/L EHMC

1: primary oocyte2: previtellogenic oocyte3: vitellogenic oocyte

Expression of estrogen receptor

Males Females


Expression of vitellogenin (qPCR)

MalesFemales


cyp19b fold change is 2.4

Wholebody

Liver

Brain Testis

Wholebody Liver

Brain Testis

Brain

• 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 GXGO analysis

Fish exposure mRNA extracted from whole body Cy3 labeled

Cy3

Cy3

Cy3

cDNART

Hybridisation

Microarray

Wash

Danio rerio

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

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 .

Expression of target genes involved in 3 different hormonal pathways in liver, gonads, brain after exposure for 14 daysand 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

environmental impact of sunscreeens and personal care products in the aquatic environment karl fent,...

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