environmental impact of sunscreeens and personal care products in the aquatic environment karl fent,...
Post on 27-Mar-2015
212 Views
Preview:
TRANSCRIPT
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
karl.fent@fhnw.ch, kfent@ethz.ch
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
E: 5.4 g/L EHMCL: 37.5 g/L EHMCM: 244.5 g/L EHMCH: 394 g/L EHMC
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)
E: 5.4 g/L EHMCL: 37.5 g/L EHMCM: 244.5 g/L EHMCH: 394 g/L EHMC
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
(%
)
E: 5.4 g/L EHMCL: 37.5 g/L EHMCM: 244.5 g/L EHMCH: 394 g/L EHMC
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
E: 5.4 g/L EHMCL: 37.5 g/L EHMCM: 244.5 g/L EHMCH: 394 g/L EHMC
Expression of vitellogenin (qPCR)
MalesFemales
E: 5.4 g/L EHMCL: 37.5 g/L EHMCM: 244.5 g/L EHMCH: 394 g/L EHMC
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
top related