parabens. from environmental studies to human...

Environment International 67 (2014) 27–42

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Parabens. From environmental studies to human health

Dorota Błędzka ⁎, Jolanta Gromadzińska, Wojciech WąsowiczDepartment of Toxicology and Carcinogenesis, Nofer Institute of Occupational Medicine, ul. św. Teresy od Dzieciątka Jezus 8, 91-348 Łódź, Poland

⁎ Corresponding author at: Department of ToxicologyE-mail address: [email protected] (D. Błędzka).

http://dx.doi.org/10.1016/j.envint.2014.02.0070160-4120/© 2014 Elsevier Ltd. All rights reserved.

a b s t r a c t
a r t i c l e i n f o
Article history:Received 7 November 2013Accepted 24 February 2014Available online 19 March 2014

Keywords:ParabenPreservativeOccurrence in the environmentEndocrine disruptorCancerOxidative stress

Parabens are a group of substances commonly employed as preservatives, mainly in personal care products,pharmaceuticals and food. Scientific reports concerning their endocrine disrupting potential and the possiblelink with breast cancer raised wide discussion about parabens' impact and safety. This paper provides holisticoverview of paraben usage, occurrence in the environment, methods of their degradation and removal fromaqueous solution, as well as hazards related to their endocrine disrupting potential and possible involvementin carcinogenesis.

© 2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.1. Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.2. Usage of parabens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.3. Legislation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281.4. Human exposure. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

2. Occurrence in environment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.1. Naturally occurring parabens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.2. Water resources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.3. Soils, sediments and sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 292.4. Air and dust . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 302.5. Biota . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312.6. Pathways and sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

3. Methods of removal and degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.1. Wastewater treatment plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.2. Biodegradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313.3. Advanced oxidation processes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

4. Influence on organisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 314.1. In vitro studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 334.2. In vivo studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

4.2.1. Oral administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 344.2.2. Subcutaneous administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354.2.3. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

4.3. Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.4. Studies involving humans. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364.5. Additive and synergistic effect. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38

and Carcinogenesis, Nofer Institute of Occupational Medicine, 8 Teresy St., 91-348 Lodz, Poland. Tel.: +48 42 6314634.

http://dx.doi.org/10.1016/j.envint.2014.02.007

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28 D. Błędzka et al. / Environment International 67 (2014) 27–42

1. Introduction

1.1. Properties

In terms of chemical structure, parabens (PBs) are esters of p-hydroxybenzoic acid (pHBA), with alkyl substituents ranging frommethyl to butyl or benzyl groups (Jonkers et al., 2010). Namely, we candistinguishmethylparaben (MePB), ethylparaben (EtPB), propylparaben(PrPB), isopropylparaben (iPrPB), butylparaben (BuPB), isobutylparaben(iBuPB) and benzylparaben (BePB). Among them, methylparaben andpropylparaben are the most commonly used and often present in theproducts together (Núñez et al., 2008).Main physicochemical propertiesof parabens are shown in Table 1.

Commercially, parabens are produced by esterification of p-hydroxybenzoic acid with an appropriate alcohol in the presence of acatalyst (e.g. concentrated sulfuric acid or p-toluenesulfonic acid)(Liao et al., 2002).

In acidic aqueous solutions parabens are stable. In alkaline solutionsparabens are hydrolyzed to p-hydroxybenzoic acid and the correspond-ing alcohol. In general, with the increase in the alkyl chain length, theresistance of aqueous solutions of parabens to hydrolysis increases(Masten, 2005). PB antibacterial properties are directly proportional tothe chain length of the ester group, and so, for example, butylparabenhas 4-fold greater ability to inhibit microbial growth than ethylparaben.However, simultaneously with the increase of the length of the alkylchain, the value of octanol–water partition coefficient rises, whichresults in decrease of water solubility (Table 1; Jewell et al., 2007; Soniet al., 2005). At concentrations as low as those used forwater treatment,chlorine reacts with parabens to produce chlorinated derivatives(Canosa et al., 2006b). Canosa et al. (2006b) observed that even a fewminutes of contact between cosmetics containing PB (e.g. bath gel)and chlorinated tap water results in the formation of chlorinatedand brominated by-products. The reaction rate increases with tempe-rature. This phenomenon is alarming due to the high stability of theresulting di-chlorinated derivatives and unknown estrogenic potential(Canosa et al., 2006b; Terasaki andMakino, 2008). Moreover, chlorinat-ed derivatives are considerably more toxic to aquatic organisms thanthe respective parent compounds (Terasaki et al., 2009).

The numerous properties predisposing parabens for usage aspreserving agents have contributed to their considerable popularity.The features determining PB high utility include, among others(Aguilar-Bernier et al., 2012; Guadarrama et al., 2008; Rastogi et al.,1995; Soni et al., 2001, 2005; Terasaki et al., 2012):

• broad spectrum of activity against yeasts, molds and bacteria,• chemical stability (for a wide temperature interval and pHs rangingfrom 4.5 to 7.5),

• inertness,• low degree of systemic toxicity,• low frequency of sensitization,• sufficientwater solubility (enabling to obtain effective concentration),• relatively safe use,• low costs of production,• no perceptible odor or taste,• not causing changes in consistency or coloration of products.

Table 1Physical and chemical characteristics of parabens.

Characteristic MePB EtPB

Chemical formula C8H8O3 C9H10O3

Molecular weight (g/mol) 152.16 166.18pKa 8.17 8.22Log octanol–water partition coefficients (log kOW) 1.66 2.19Solubility in water at 25 °C (g/100 ml) 2.00 0.86

The combination of these properties makes it relatively difficult tofind a preservative, which will be a satisfactory replacement forparabens.

Parabens are classified as “generally regarded as safe” (GRAS)compounds and approved for use in foods by the US Food and DrugAdministration (FDA) and the European Union (EU) regulations(Soni et al., 2001).

1.2. Usage of parabens

Parabens were first introduced in mid 1920s as preservatives indrug products (Liebert, 1984). Currently, they are widely used pre-servatives, mainly in cosmetics and pharmaceuticals, but also infood commodities and industrial products. Besides water, they areregarded as the most common ingredient of cosmetics (Cashmanand Warshaw, 2005; Janjua et al., 2007). Parabens are present in ap-proximately 80% of personal care products (Pouillot et al., 2006). In a1995 study, parabens were found in 77% of rinse-off and 99% ofleave-on cosmetics (Rastogi et al., 1995). However, in a report fromthe Danish market, 36% of the considered 751 commoditiescontained parabens. A Norwegian study revealed that parabenswere present in 32% of 117 baby care products (Eriksson et al.,2008). Other estimation shows that butylparaben is present in 13%,while propylparaben and/or methylparaben in 48% of cosmeticsand personal care products (Masten, 2005).

At the turn of the millennium, several studies were publishedsuggesting PB estrogenic activity (Oishi, 2001; Routledge et al., 1998)and carcinogenic potential (Darbre et al., 2004). As a result, someman-ufacturers altered composition of their cosmetic products by replacingparabens with other preservative system and introducing so called“paraben free” formulae. Even though, the Cosmetic Ingredient ReviewExpert Panel (CIR, 2008) reported, on the basis of USA Food and DrugAdministration (FDA) database, that the number of cosmetic formula-tions in which PB was used was 1.7 times higher in 2006 compared to1981. Namely, the use in 1981 was equal to 13,200 (Liebert, 1984),whereas in 2006 it was as high as 22,000 (CIR, 2008). However, the PBcontent in cosmetics seems to decrease. According to the industry'svoluntary submission to the FDA in 1981, the concentration for a singleparaben was up to 25% for MePB and PrPB, 5% for BuPB and 1% for EtPB.The usual content of parabens was up to 1% (Liebert, 1984). About14 years later, the content of PBs in 215 tested products from theDanishmarket ranged from 0.01% to 0.87% (Rastogi et al., 1995). Nevertheless,the difference may also arise from regional disparities between UnitedStates and Europe.

1.3. Legislation

In European Union (EU) countries, the allowable content of PB incosmetic products is 0.4% for single ester and 0.8% for mixtures of allparabens (Official Journal of the European Union, 2009). The govern-mental units of the United States (Food and Drug Administration,FDA) and Canada (Health Canada) have recommended the samethreshold for PB as that of EU.However, there is no legislation regulatingconcentrations of parabens in cosmetics in any of those countries

PrPB BuPB BePB References

C10H12O3 C11H14O3 C14H12O3 –

180.21 194.23 228.25 CIR (2008)8.35 8.37 – Soni et al. (2005)2.71 3.24 3.56 Golden et al. (2005)0.30 0.15 0.05 Jewell et al. (2007)

29D. Błędzka et al. / Environment International 67 (2014) 27–42

(Kirchhof and de Gannes, 2013). The maximum total paraben concen-tration allowed in Japan is equal to 1.0% (Masten, 2005). In 2011, theDanish government decided to introduce additional restrictions,banning the use of some parabens (propyl-, isopropyl-, butyl- andisobutyl-parabens) in personal care products intended for childrenyounger than 3 years (SCCS, 2011).

In their opinion on parabens, the Scientific Committee on ConsumerSafety has evaluated that the consumers' safety is ensured as long as thetotal content of butylparaben and propylparaben in finished cosmeticproducts does not exceed 0.19% (SCCS, 2010). However, the opiniondid not result in a modification of UE legislation so far.

1.4. Human exposure

Industry estimates of the daily use of cosmetic products that maycontain parabens were 17.76 g for adults and 0.378 g for infants (CIR,2008). From that information, assuming that PB are used at the highestpermissible concentration (0.8%), the daily dose of total parabens fromcosmetics has been estimated at 142.08 mg and 3.024 mg, for adultsand infants respectively. The PB dose for an adult person weighing60 kg is equal to 2.368 mg/kg body weight (bw)/day. The averagedaily total personal paraben exposure estimated by Soni et al. (2005)was lower and equaled to 76 mg (1.26 mg/kg bw/day), with cosmeticsand personal care products (PCPs) accounting for 50 mg (0.833 mg/kgbw/day), pharmaceutical products for 25 mg (0.417 mg/kg bw/day),and food accounting approximately for 1 mg (10–13 μg/kg bw/day).In their calculations, those authors assumed that the total parabencontent in PCPs is equal to 1%, while in our estimations we used thetotal PB concentration of 0.8%. Despite employing for their calculationof higher PB percentage (1%) Soni et al. (2005) obtained lower doses.The difference arises from the assumption made by Soni et al. (2005)that the daily use of cosmetics containing parabens was 5 g.

The mean daily usage of body lotion and face cream, estimated byLoretz et al. (2005) in a group of 360 women, was equal to 10.75 g(median value: 9.16 g). Although not all PCPs contain PB, it is worth tomention that the estimation did not include rinse-off products.

Recently, Liao et al. (2013) determined concentrations of sixparabens in various foodstuffs from China. The detection rate was veryhigh (99%) and the total paraben concentrations reached up to2530 ng/g fresh weight (mean: 39.3 ng/g). However, in comparisonwith exposure to PBs originating from personal care products, thevalue of the estimated daily intake of parabens from food was low,about 1 μg/kg bw/day (mean) and 3 μg/kg bw/day (95th percentile)(Liao et al., 2013).

2. Occurrence in environment

2.1. Naturally occurring parabens

Although commercially used parabens are of synthetic origin, it hasbeen found that some organisms are able to produce them naturally.Peng et al. (2006) has reported that a marine bacterium biosynthesizespHBA and its alkyl esters. A4B-17 strain of the bacterium belongingto the genus Microbulbifer was found to produce surprisingly highamounts of these compounds: 10 mg/L of pHBA, 24 mg/L of butylester, 0.4 mg/L of heptyl ester and 6 mg/L of nonyl ester. The pHBAesters produced by the bacterium were found to be effective inpreventing the growth of yeasts, molds, and Gram-positive bacteria(Peng et al., 2006). The observed exudation of β-carbolines along withmethyl paraben in herbaceous plant: Oxalis tuberosa hairy roots wastriggered upon fungal cell wall elicitation (Bais et al., 2003).Methylparaben was also detected at a trace concentration (0.8 μg/g)in Andrographis paniculata herbs, but the origin of the compound inthe plant tissue was unknown (Li et al., 2003).

2.2. Water resources

Widespread production and use of parabens can result in theirleaking into environment. Surface waters, due to, among others, theirlocation in the lowest points of the landscape, are often most suscepti-ble to contamination. Parabens can be released into the aqueous envi-ronment mainly through wastewater treatment discharges, but also asa runoff from non-point sources and deposition of particles from theatmosphere.

The scarce accessible data concerning presence of parabens insurface waters are shown in Table 2. In general, among all measuredparabens, the highest frequency of detection and concentration valueswere recorded for the species most commonly used in cosmetics:metyl- and/or propylparaben (Table 2). The concentrations of MePBand PrPB in Chinese rivers reached the value of 1062 ng/L and3142 ng/L, respectively (Peng et al., 2008). The maximum concentra-tions detected in European rivers were lower, up to 400 ng/L forMePb (Kasprzyk-Hordern et al., 2008) and 69 ng/L for PrPB (González-Mariño et al., 2009). The presence of EtPB and BuPB in water sampleswas recorded less frequently. Measured concentrations of the com-pounds were relatively low in comparison with MePB and PrPB, andequaled up to 147 ng/L (EtPB) (Ramaswamy et al., 2011b) and163 ng/L (BuPb) (Yamamoto et al., 2011). There are few studies regard-ing BePB presence in surface waters. BePB was rarely detected in watersamples, with very low concentrations reaching maximally 4.4 ng/L(Jonkers et al., 2009).

Concentration and occurrence of the pollutants were highly season-dependent. The maximum measured paraben concentrations werecorrelated with low flow conditions (Loraine and Pettigrove, 2006;Peng et al., 2008). During low flow seasons, the loads of the releasedcompounds are dissolved in smaller volume of water, which results inan increase of their concentrations. Jonkers et al. (2009), however,surprisingly recorded higher values of parabens during high flowperiod. The reason for that was the discharge of some of the rawwaste-water from wastewater treatment plants (WWTPs) into the river.During heavy rainfall, the capacities of the WWTPs were exceededand, as a result, some portion of the untreated influents was releaseddirectly into the environment.

The seasonal variability should be taken into accountwhile estimatingpharmaceutical and personal care products impact on the environment,taking carefully under consideration local conditions and applied tech-nological solutions.

The results concerning paraben presence in drinking water arecontradictory. Ferreira et al. (2011a) reported occurrence of most com-monly used methylparaben in tap water at concentrations of around15 ng/L (17 ± 4 ng/L), while Loraine and Pettigrove (2006) did notdetect MePB in treated drinking water from water filtration plants.

2.3. Soils, sediments and sludge

The solubility of the compounds and the values of octanol–waterpartition coefficient determining affinity to organic matter indicatethat the possibility of accumulation of parabens in sediments increasesproportionally to chain length of alkyl substituent. Some results indicatethat parabens, aswell as their chlorinated derivatives preferentially par-tition into the suspended solid phase in river water. The concentrationsof parabens and their chlorinated derivatives are several times higher inthe suspended solid phase compared to the dissolved phase (Terasakiet al., 2012).

Núñez et al. (2008) analyzed series of parabens in forestry andagricultural soils and sediments from different areas of Spain. Thehighest concentrations in the majority of the samples were recordedfor MePB and PrPB (the most often used preservatives in health careand beauty products). The recorded values were up to 6.35, 5.10,0.29, 4.03, 0.45, 0.71 ng/g dry weight (dw) for MePB, EtPB, iPrPB,PrPB, BePB and BuPB, respectively. It is worth mentioning that, in

Table 2Concentration ranges of parabens detected in surface waters (ng/L).

Sampling area (number of sites) MePB EtPB PrPB BuPB BePB References

Riverine water Pearl River Delta, South China(9 sites)

NQ-1062 – NQ-3142 ND – Peng et al. (2008)

South Wales, UK(10 sites)

b0.3–400 b0.5–15 b0.2–24 b0.3–52 – Kasprzyk-Hordern et al. (2008)

Galicia, Spain(2 sites)

1.8–17.3 NQ-3.0 NQ-69 NQ-7.0 NQ-1.2 González-Mariño et al.(2009)

North-eastern part of Switzerland(3 sites)

3.1–17 b0.3–1.6 b0.5–5.8 b0.2–2.8 b0.2–4.4 Jonkers et al. (2009)

Not specified ND–NQ(LOD = 17)

ND–NQ(LOD = 8.8)

NQ-23.8(LOD = 4.0)

ND-54.1(LOD = 5.7)

– Regueiro et al. (2009)

Southern India(29 sites)

ND-22.8 2.47–147 ND-57.0 NQ – Ramaswamy et al. (2011b)

Urban streams in Tokushima and Osaka, Japan(12 sites)

25–676 b1.3–64 b0.8–207 b0.6–163 b0.2–2.3 Yamamoto et al. (2011)

Central Pacific region of Japan(4 sites)

2.1–5.4 NQ 4.9–25 NQ-12 – Terasaki et al. (2012)

Greater Pittsburgh Area, USA(6 sites)

2.2–17.3 ND ND-12 ND-0.2 – Renz et al. (2013)

Estuarine system Ria de Aveiro area, Portugal(N50 sites)

b1.6–62 b0.3–6.7 b0.5–64 b0.2–42 b0.2–0.3 Jonkers et al. (2010)

NQ — detected but too low to be quantified; ND — not detected; LOD — limit of detection.


general, the highest concentrations were detected in sediments(Núñez et al., 2008). This may arise from the fact that sediments typ-ically contain a lot of organic fraction and may have continuous con-tact with pollutants dissolved in aqueous solution, which leads toparaben deposition. Highly elevated concentration of BuPB(377 ng/g dw) was found in marine sediment samples from contam-inated area in Tenerife (Delgado et al., 2012).

Pérez et al. (2012) reported the presence of several parabens in ag-ricultural soils, soils amendedwith treated sewage sludge and industrialsoils, with the higher concentration of MePB (up to 8.04 ng/g dw),followed by EtPB (up to 1.23 ng/g dw) and two forms of BuPB (about1 ng/g dw each). Ferreira et al. (2011b) found PrPB to be present inthe garden soil at 1.5 ng/g dw. Relatively high concentrations ofparabens, up to 127 ng/g dw for MePB and up to 15–23 ng/g dw forEtPB, PrPB and BuPB, were found in sediments and soils from Canada(Viglino et al., 2011).

The effect of effluent discharges from wastewater treatment plantson water ecosystems is clearly visible when comparing concentrationsof parabens in sediments from parts of the river situated below thedispersion plume of the wastewater effluents (19–56 ng/g dw), tosediments from a pristinemountain stream, where no PBs were detect-ed (Viglino et al., 2011).

Several studies focused on occurrence of parabens in sewage sludge(Albero et al., 2012; Nieto et al., 2009; Yu et al., 2011) and biosolids(Viglino et al., 2011).

Methylparaben and propylparaben were found in most of theWWTP samples, at levels between 5–202 ng/g dw and 4–44 ng/d dw, respectively (Albero et al., 2012; Nieto et al., 2009; Yu et al.,2011). The concentrations of MePB and PrPB in municipal sludgecakes (biosolids) were similar, up to 91 ng/d dw and 8 ng/d dw, re-spectively. However, EtPB and BuPB were not detected (Viglinoet al., 2011). None of the six tested parabens was present in the veg-etables harvested from soil fertilized with municipal biosolids(Sabourin et al., 2012).

Albero et al. (2012) estimated that parabens found in sewagesludges from WWTPs were at concentrations which do not pose thetoxicological risk for humans or the environment when the sludge isapplied as fertilizer or amendment in agricultural soil. However, furtherstudies to establish the endocrine disrupting potential would berecommended.

2.4. Air and dust

Parabens present in indoor dust and air presumably originate frompersonal care products used in households (Canosa et al., 2007a;Rudel et al., 2003). The route of personal exposure to contaminantsfrom indoor dust and air is through inhalation and oral ingestion(Canosa et al., 2007a).

The concentrations of parabens detected in indoor air were up to21 ng/m3 for MePB, 4.0 ng/m3 for EtPB and 3.2 ng/m3 for BuPB (Rudelet al., 2003). The estimated daily mean breathing rates for adults(16–70 years) and children (0–2 years) are equal to 185 dm3/kgbw/day and 658 dm3/kg bw/day, respectively (Blaisdell, 2012). Assum-ing that most commonly detected (67%) MePB is present in indoor air atmedian concentration (2.9 ng/m3) (Rudel et al., 2003) and employingmean breathing rates, the daily MePB intake through inhalation is equalto 0.547 ng/kg bw/day and 1.91 ng/kg bw/day for adults and children,respectively. When maximal MePB concentration (21 ng/m3) is used inthe calculations (Rudel et al., 2003), the estimated daily intake is equalto 3.89 ng/kg bw/day (adults) and 13.8 ng/kg bw/day (children).

The values of concentrations of parabens in indoor dust showed highregional and personal variability, probably due to different per capitaconsumption of personal care products. Methyl- and propylparabenwere the most abundant. The maximal measured concentrations were14,300 ng/g for MeP, 3110 ng/g for EtPB, 110,800 ng/g for PrPB, 3920ng/g for BuPB and 190 ng/g for BePB (Canosa et al., 2007a, 2007b;Ramírez et al., 2011; Rudel et al., 2003;Wang et al., 2012). Surprisingly,the concentrations of parabens in dustweremuchhigher than those de-tected in the sewage sludge.

The following total geometric mean concentrations of six parabensin dust were found, in the decreasing order, in Korea (2320 ng/g) N

Japan (2300 ng/g) N USA (1390 ng/g) N China (418 ng/g), while theaverage concentration of the sum of four parabens in Europe (Spain)was 1399 ng/g.Methylparaben accounted for 42–73%of the total parabenconcentrations (Canosa et al., 2007a, 2007b; Ramírez et al., 2011; Rudelet al., 2003; Wang et al., 2012).

The mean estimated daily intakes (EDIs) of total parabens via dustingestion were relatively high for infants (5.57 ng/kg bw/day) andtoddlers (6.63 ng/kg bw/day). The mean EDIs for children rangedfrom 0.98 ng/kg bw/day to 5.42 ng/kg bw/day, depending on theregion. The mean EDIs of parabens via dust ingestion were 5–10 times


lower in adults than in children, 0.2–1.18 ng/kg bw/day (Wang et al.,2012).

2.5. Biota

The data concerning paraben presence in organisms are scarce.Parabens were detected in fish tissue (20 species) with great frequency(Kim et al., 2011; Ramaswamy et al., 2011a).MePB, PrPB and BuPBwerefound in over 90% of the analyzed fish samples, whereas EtPB in about70%. The highest concentrations were measured for most ubiquitouslyused MePB (up to 3600 ng/g). EtPB, PrPB and BuPB concentrationsreached values of 840 ng/g, 1100 ng/g and 70 ng/g, respectively. Thevalues of total concentrations of parabens in adult fish (coral grouper)were over twice as high (4700 ng/g) as in juvenile form (2200 ng/g),which may indicate growth-dependent compound accumulation(Ramaswamy et al., 2011a). However, concentrations of parabensfound in fish tissue by Jakimska et al. (2013) were much lower andequaled maximally 84.69 ± 6.58 ng/g for methylparaben and 0.19 ±0.04 ng/g for propylparaben. Moreover, Renz et al. (2013) did not findany parabens in the fish brain tissue.

The estimated dietary human exposure to the four parabens in thePhilippines through fish meat intake was 2 μg/kg/day (Ramaswamyet al., 2011a), and was 5000-times lower than the acceptable dailyintake (10 mg/kg/day).

2.6. Pathways and sources

Worldwidewidespread use of parabens results in their ubiquitous oc-currence in the environment. The main point sources of pollution arewastewater treatment plants (WWTPs). PBswere detected in surfacewa-ters, soils and sediments, biota, aswell as in indoor air and dust. However,themain sources of human exposure to parabens are personal care prod-ucts andpharmaceuticals. The fate of parabens in the environment aswellas sources and pathways of human exposure are compiled in Fig. 1.

3. Methods of removal and degradation

3.1. Wastewater treatment plants

Parabens were detected in all of the tested influent samples fromwastewater treatment plants (WWTP), at concentrations reaching79,600 ng/L. Data on concentrations of parabens in influents andeffluents fromWWTP are shown in Table 3.

PBswere predominantly (N97%) present in the aqueous phase of theinfluent. During anaerobic processes performed at the first treatmentsystem in a sewage treatment plant, concentrations of all the parabensdecreased significantly and continued to decrease in the followingbiological treatment. The mass load ending up in the dewatered sludgeindicates little relevance of compound sorption during wastewatertreatment process in comparison to biodegradation mechanism. Themass loads, in relation to the mass flows entering WWTP, were equalto 0–1.6% and 0–0.4% for final effluents and dewatered sludge, respec-tively (Yu et al., 2011). The average efficiency of the removal of parabensin WWTPs was high and equal to 96.1–99.9% (González-Mariño et al.,2011; Jonkers et al., 2009). However, parabens were still detected inmost of the effluents, at concentrations as high as about 4000 ng/L(Table 3). Therefore, WWTPs were considered as the major pointsources of PBs in the environment due to incomplete elimination ofthe contaminants (Yu et al., 2011).

3.2. Biodegradation

MePB was reported to undergo hydrolysis to p-hydroxybenzoicacid in water, with a half-life (t1/2) of several tens of hours for bioticdegradation, to several tens of days for abiotic hydrolysis (Blauget al., 1974; Valeanu et al., 2001). Parabens have been described as

readily biodegradable under aerobic conditions, with a biodegrad-ability of about 90% of theoretical oxygen demand (for MePB, EtPBand PrPB) (Madsen et al., 2001). However, González-Mariño et al.(2011) reported that an activated sludge biodegradation was per-formed via biological routes rather than abiotic processes or ad-sorption. The studied parabens (MePB, EtPB, PrPB, BuPB andiBuPB) were nearly completely biodegraded (99%) in less than5 days, with half-lives not exceeding 3 days. The persistence ofthe compounds slightly increased with the length of the alkylchain and the chlorination degree. The half-lives of dihalogenatedderivatives of MePB were up to 10 days (González-Mariño et al.,2011; Jonkers et al., 2009).

3.3. Advanced oxidation processes

Since parabens were detected in effluents from WWTPs (Table 3),riverine water (Table 2) and even in drinking water (Ferreira et al.,2011a), the implementation of more efficient degradation systemsseems advisable. Due to cost efficiency, simplicity and no post-treatment problems, advanced oxidation processes seem to provide asatisfactory solution. Recently, several studies evaluating efficiencyand optimal conditions for removal of parabens from aqueous modelsolutions were performed.

The results of batch sunlight photolysis revealed low and moderateefficiency in terms of BuPB and BePB degradation, respectively. Half-life times and quantum yields (ϕ) of the compounds equaled 410 h forBuPB (ϕ = 7.6–8.6 × 10−6), 580 h for iBuPB (ϕ = 2.3–2.4 × 10−5)and 15 h for BePB (ϕ= 3.9–6.2 × 10−4) (Yamamoto et al., 2007). Pho-tolysis of aqueous MePB and BuPB standard solution by UVC irradiation(254 nm) also had poor performance, with a quantum yield equal to6.07 × 10−3 and 3.3 × 10−3, respectively (Błędzka et al., 2009;Sánchez-Martín et al., 2013). Far better results were obtained by apply-ing advanced oxidation processes, such as ozonation (Tay et al., 2010),photocatalytic degradation by TiO2 (Lin et al., 2011) or combinedH2O2/UV system (Błędzka et al., 2010a, 2010b).

In H2O2/UV system, the rate constant for hydroxyl radical reactionwith BuPB (kOH) slightly depended on solution pH and temperature(3.84 × 109 M−1 s−1–8.56 × 109 M−1 s−1) (Błędzka et al., 2012b),while second-order rate constants for the reaction between PB andozone (kO3) were strongly decreased in acid solution (pH = 2; kO3 ∈b3.3 × 102M−1 s−1–4.2 × 102M−1 s−1N) compared to alkaline solution(pH=12; kO3∈ b1.02× 109M−1 s−1–1.38× 109M−1 s−1N) (Tay et al.,2010). Alkaline pH was also beneficial for BePB photocatalysis by TiO2

(Lin et al., 2011).Photosensitized oxidation of BuPB and BePB under visible-light

irradiation in homogeneous aqueous solution leads to slow targetcompound degradation, mainly as a result of reaction with singletoxygen (Gmurek and Miller, 2012; Gmurek et al., 2012; Gryglik et al.,2009). Photosensitized oxidation in natural water spiked with BuPBwas increased compared to degradation in pure, buffered aqueoussolution (Błędzka et al., 2012a).

4. Influence on organisms

For a long time parabenswere considered as harmless and thereforethey have been widely used as preservatives, mainly in personal careproducts. Over the last two decades, a vivid discussion on paraben safetyis ongoing (Boberg et al., 2010; Darbre and Harvey, 2008; SCCP, 2005;Soni et al., 2005). The main concern arises from their endocrinedisrupting potential.

Endocrine disruption effects include alteration of endogenoushormone action as well as modification of the hormone synthesis,metabolism and transport. Though itwas initially believed that endocrinedisrupting compounds (EDCs) act via binding competitively to nuclearreceptors (estrogen, androgen, thyroid, progesterone and retinoid) asagonists or antagonists, it is now known that the involved mechanism


is considerably more complex. It also includes influencing the productionand break-down of endogenous steroids and receptor synthesis (Waringand Harris, 2011; Whitehead and Rice, 2006).

Fig. 1. Sources and pathways of human exposure and fate of parabens in environment (maxbw — body weight, dw— dry weight, fw — fresh weight).

The reproductive system is vulnerable and therefore it is highlyaffected by EDCs. Additionally, EDCs can also influence various steroidsensitive tissues, thereby disturbing the functioning of the central

imum concentration values are shown; based on data discussed inparagraphs 1.4, 2, 3.1;

Table 3Concentration range of parabens determined in influents and effluents of wastewater treatment plants (ng/L).

Sampling region(number of sites)

MePB EtPB PrPB BuPB BePB References

Wastewater treatment plant Effluent Sweden(3 largest WWTPs)

ND-300 b100–200 b100–300 – ND-1000 Paxeus (1996)

Influent Southern Ontario, Canada(8 WWTPs)

100–1470 20–270 200–2430 20–260 – Lee et al. (2005)

Effluent Southern Ontario, Canada(8 WWTPs)

20–30 b10 b10–40 b10–10 – Lee et al. (2005)

Influent Southern California, USA(1 WWTP)

12,500–79,600 – – – – Loraine and Pettigrove (2006)

Effluent Southern California, USA(1 WWTP)

ND-3830 – – – – Loraine and Pettigrove (2006)

Influent Spain(1 WWTP)

430–2920 52–210 230–810 20–86 – Canosa et al. (2006a)

Effluent Spain(1 WWTP)

ND ND ND ND – Canosa et al. (2006a)

Influent Galicia, Spain(2 WWTPs)

1926–5138 452–549 1147–1302 150–181 NQ González-Mariño et al. (2009)

Effluent Galicia, Spain(2 WWTPs)

NQ-1.5 NQ NQ NQ-3.6 NQ González-Mariño et al. (2009)

Influent North-eastern part of Switzerland(7 WWTPs)

65–9880 2.2–719 43–1540 9.7–864 b0.2–4.1 Jonkers et al. (2009)

Effluent North-eastern part of Switzerland(7 WWTPs)

4.6–423 b0.3–17 b0.5–28 b0.2–12 0.2–16 Jonkers et al. (2009)

Effluent Ria de Aveiro area, Portugal(3 WWTPs)

13–31 b0.3–6.6 b0.5–21 0.2–3.9 b0.2 Jonkers et al. (2010)

Influent North-western Spain(3 WWTPs)

290–10,000 250–1600 520–2800 39–270 – González-Mariño et al. (2011)

Effluent North-western Spain(3 WWTPs)

6.1–50 ND-9.8 ND-21 ND – González-Mariño et al. (2011)

Influent Central Pacific region of Japan(1 WWTP)

1400–2400 52–57 2200–2600 370–450 – Terasaki et al. (2012)

NQ — detected but too low to be quantified; ND — not detected.


nervous system (Colborn, 2004; Kim et al., 2009; Miodovnik et al.,2011), immune system (Chalubinski and Kowalski, 2006; Milla et al.,2011), lipid homeostasis (Baillie-Hamilton, 2002; Newbold et al.,2009; Smink et al., 2008), glucose levels (Alonso-Magdalena et al.,2005; Hectors et al., 2011), thyroid function and act as epigenetic mod-ulators causing transgenerational effects (Waring and Harris, 2011).

4.1. In vitro studies

Paraben cytotoxicity was studied in freshly isolated rat hepatocytes,bymeasurements of cell viability, levels of ATP and total adenine nucle-otides, and mitochondrial membrane potential. Cytotoxicity was deter-mined at 60 min of incubation. MePB and EtPB exhibited very lowtoxicities. PrPB and iPrPB were moderately toxic, inducing decrease incell viability and mitochondrial membrane potential by 50%, about25% reduction of total adenine nucleotides and fall in the levels of ATPto 2–3 nmol/106 cells. BuPB and iBuPB highly altered the cells, causingdeath of 98% of cells and almost total loss of ATP. They also causedreduction in the total adenine nucleotide pool and mitochondrialmembrane potential. However, the PB concentrations used in thisexperiment were very high (0.5 to 2.0 × 10−3 M; equivalent to 90 to360 mg/L) (Nakagawa and Moldéus, 1998).

Parabens were shown to exhibit estrogenic activity as full agonists,though their effect was considered as “weak” due to relatively low es-trogen receptor binding affinity in comparison with natural hormonesor some other EDCs.

Compared to 17β-estradiol, concentrations of MePB, EtPB, PrPB andBuPB required to result in similar response of the yeast estrogen screenwere approximately 2,500,000-fold, 150,000-fold, 30,000-fold, and10,000-fold higher, respectively. Butylparaben was found to be able tocompete with 17β-estradiol for binding to the rat estrogen receptor(ER). However, BuPB affinity to the receptor was approximately 5orders of magnitude lower than that of diethylstilboestrol, and between

1 and 2 orders of magnitude less than that of nonylphenol (Routledgeet al., 1998).

Themean half maximal inhibitory concentration (IC50), causing 50%inhibition of 17β-estradiol in competitive binding assays ranged from2.45 × 10−4 M (37.28 mg/L) for MePB to 1.05 × 10−4 M (20.39 mg/L)for BuPB (Blair et al., 2000). Okubo et al. (2001) obtained similar valuesranging from 2.7 × 10−4 M (44.87 mg/L) for EtPB to 2.7 × 10−5 M(5.244 mg/L) for iBuPB. The weak estrogenic effect of parabens in vitrowas also found by other authors (Byford et al., 2002; Miller et al.,2001). In sensitive transactivation assays using mammalian cells,MePB at concentrations up to 10−5 M (15 mg/L) showed no effect.Other parabens were found to exhibit agonistic activities toward ERαand ERβ (20% relative effective concentration), at a concentrationrange from 4.3 × 10−6 M (714.5 μg/L) to 1.5 × 10−7 M (29.13 μg/L),for commonly used EtPB, PrPB and BuPB (Watanabe et al., 2013).PBs' activity was also tested by employing a model for 17β-estradiolreplacement by paraben, evaluated by conformational analyses andinteraction energy calculations. MePB, which exhibited a relativelyweak effect in vitro, was found to show high estrogenic activity in silico(Guadarrama et al., 2008).

Parabens, at concentrations of ≥10−6 M (≥180 μg/L), were able toincrease expression of both ERE-CAT reporter gene and endogenousestrogen-regulated pS2 gene in the MCF7 human breast cancer cells(Byford et al., 2002). However, in terms of global gene expressionpatterns, the effect of parabens was not identical to the action of 17β-estradiol, and varied also within the parabens (Pugazhendhi et al.,2007; Terasaka et al., 2006).

Parabens were found to activate both ERα and ERβ receptors, withsimilar or stronger effect versus ERβ receptors (Gomez et al., 2005;Okubo et al., 2001; Watanabe et al., 2013). In a recent study, parabenswere observed to be able to bind competitively to the humanestrogen-related receptor γ (ERRγ), which is a diagnostic biomarkerand a treatment target of breast cancer. Tested parabens exhibitedinverse antagonist activities to ERRγ, at concentrations from 10−7 M


(lowest observed effect level) to 3.09 × 10−7–5.88 × 10−7 M (50%relative effective concentrations, corresponding to 60 μg/L for BuPB and134 μg/L for BePB) (Zhang et al., 2013).

It was also suggested that parabens can alter homeostasis by ER-independent mechanism of action. MCF-10A immortalized, non-transformed human breast epithelial cells, which have no detectableERα or ERβ, were stimulated to growbyparabens present in suspensionat concentrations as low as 10−6 M (MePB; 152.2 μg/L) and 10−7 M(PrPB; 18.0 μg/L and BuPB; 19.4 μg/L) (Khanna and Darbre, 2013).

In addition to estrogenic properties, some parabens were reportedto display antiandrogenic activity, by binding to human androgenreceptors (AR) (Satoh et al., 2005) and causing inhibition of testoster-one (T)-induced transcription (Chen et al., 2007). While MePB andEtPB showed no AR-binding affinity, PrPB and BuPB at 1.9 × 10−4 Minhibited testosterone binding by about 40% (Satoh et al., 2005).Surprisingly, in the study of Chen et al. (2007)MePB happened to exhib-it more potent antagonizing action toward testosterone (40% reductionof hormone activity at 10−5 M) than butyl (19%) and propyl (33%)paraben. Watanabe et al. (2013) observed only a slight AR-mediatedtranscriptional activity induced by MePB and pHBA at concentrationsof 10−5M (about 1.52mg/L). Kjærstad et al. (2010) reported antagonis-tic effects only of iBuPB (25 × 10−6 M and above). However, a mixtureof five parabens (MePB, EtPB, PrPB, BuPB, iBuPR) antagonized the AR atlower concentrations of 2 × 10−6 M (Kjærstad et al., 2010).

Nishizawa et al. (2006) conducted studies which indicated a pro-oxidant potential of parabens dermally. Namely, in the presence ofsinglet oxygen, hydroquinone and 1,4-benzoquinone were producedfrom parabens and, consequently, multi-substituted glutathione (GSH)conjugateswere formed. The biologically relevant effect of that process-es would be the consumption of an important antioxidant (GSH) andintensified hydrogen peroxide generation, which both can induceoxidative stress (Nishizawa et al., 2006). The decrease of glutathioneand protein-sulfhydryl groups in rat hepatocytes were observed afterBuPB exposure (Masten, 2005).

Most often used as a preservative and considered as least potent tocause adverse effect MePB, was found to exhibit detrimental impacton human normal skin keratinocytes, while combined with UVB lightexposure (Handa et al., 2006). Handa et al. (2006) simulated the naturalexposure conditions by adjusting for MePB content in personalcare products and its efficiency in reaching the basal layer of the skinepidermis. UVB doses were selected so as to simulate 30–60 s ofsunlight exposure. MePB or UVB alone slightly, but not significantly,induced reactive oxygen species (ROS) and nitric oxide (NO) production.No effect on redox-sensitive nuclear transcription factors (NFκB and AP-1) activation was observed when HaCaT keratinocytes were exposedtoMePB or UVB separately. However, UVB-induced ROS andNO produc-tion, as well as NFκB and AP-1 activation in keratinocytes, weresignificantly enhanced by MePB. Moreover, a significant increase oflipid peroxidation of HaCaT keratinocytes took place after combinedUVB and MePB exposure. Alterations of those factors were likely tocontribute to an increase of cell death which was observed followingsimultaneous MePB and UVB exposure (Handa et al., 2006).

Altogether, these findings indicate a potentially harmful effect ofapplication of PB-containing cosmetics on human skin, particularlywhen exposed to sunlight.

4.2. In vivo studies

Widespread usage of parabens results in contamination of waterresources (Table 2). Therefore, the potential effect on aquatic wildlifewas examined.

In general, acute and chronic toxicity of parabens toward aquaticorganisms increases in the following order: MePB b EtPB b PrPB b

BuPB b BePB (Bazin et al., 2010; Dobbins et al., 2009; Yamamoto et al.,2011). In acute toxicity tests, the lowest-observed-effect concentration(LOEC) values ranged from 0.02 mg/L BePB toward Vibrio fischeri

(bacteria) to 15 mg/L MePB toward Daphnia magna (crustacean)(Bazin et al., 2010). In chronic toxicity tests, the measured LOEC valuesof parabens ranged from 0.1 mg/L BePB toward D. magna to 25 mg/LMePB toward fathead growth (fish) (Dobbins et al., 2009). All measuredLOEC concentrations were below PB levels detected in samples fromsurface water and WWTPs (Tables 2, 3).

The sensitive biomarkers of compound estrogenic activity in fishesare: vitellogenin — a protein produced in the females' liver, used tobuild the egg yolk and choriogenin — a precursor of the inner layersubunits of the egg envelope (Chen et al., 2008). Exposure to PrPB ofadult male Japanese medaka fish caused a dose-dependent increase invitellogenin plasma concentration. Additionally, an increase in mRNAexpression levels of estrogen-responsive gene products: vitellogeninand choriogenin was observed (Inui et al., 2003). Even though the pres-ence of PrPB in aqueous solution strongly influenced mRNA expressionlevels of vitellogenin in exposed fish liver, PrPB concentrations(0.055 mM ≈ 10 mg/L or above) used in the experiment (Inui et al.,2003) exceeded the maximum concentrations detected in surfacewaters over 3000 times (Table 2).

Exposure to lower concentration of the contaminant (225 μg/L)resulted in an increase of vitellogenin synthesis in a rainbow troutOncorhynchus mykiss. However, at PrPB concentration equal to 50 μg/L,which is nearly 16-times higher than the maximum concentrationrecorded in surface waters (Table 2), no effect was observed(Bjerregaard et al., 2003).

Exposure to 201 μg/L butylparaben caused an increase in vitellogen-in plasma concentration in rainbow trout. However, BuPB present inwater solution at concentrations of 35 μg/L, which is about 40 timeshigher than maximal concentration recorded in WWTP influent(Table 3), did not cause an increased vitellogenin synthesis (Alslevet al., 2005).

Though an in vitro study revealed that paraben exposure had similaror stronger effect on ERβ in comparison to ERα (Gomez et al., 2005;Okubo et al., 2001; Watanabe et al., 2013), an in vivo study did notconfirm these findings (Inui et al., 2003). While exposure to PrPB atconcentrations of ~100 mg/L increased mRNA expression levels ofestrogen receptor ERα, the expression levels of ERβ and AR were notsignificantly influenced at concentrations as high as 1 g/L and 10 g/L,respectively (Inui et al., 2003).

4.2.1. Oral administrationThe lowest dose of PrPB andBuPB administered per os tofish resulting

in an increase in vitellogenin level was equal to 7 mg/kg/2 days(Bjerregaard et al., 2003) and 9 mg/kg/2 days (Alslev et al., 2005),respectively. It is noteworthy that the concentration required toproduce an effect was decreasing with a longer exposure time (Alslevet al., 2005; Bjerregaard et al., 2003), indicating a potentially enhancedeffect of endocrine disruption during chronic exposure.

The effect of the oral administration of parabens in rodents wasstudied by various authors (Boberg et al., 2008; Daston, 2004;Hoberman et al., 2008; Hossaini et al., 2000; Oishi, 2001, 2002a,2002b, 2004; Routledge et al., 1998; Vo et al., 2010). The estrogeniceffect of the compound can be determined from the increase in uterineweight, as it is indirectly controlled by estrogens (Sato et al., 2002).

MePB and BuPB administered via oral gavage were found to beinactive in the rat uterotropic assay, at doses as high as 800 mg/kg/dayand 1200 mg/kg/day, respectively (Routledge et al., 1998). Similarresults were obtained in uterotropic assay conducted on immatureB6D2F1 mice (Hossaini et al., 2000). Oral application of MePB, PrPB,BuPB and pHBA at doses up to 100 mg/kg bw/day and EtPB at adose level of 1000 mg/kg bw/day did not result in estrogenic response(Hossaini et al., 2000).

Daston (2004) studied BuPB influence on Sprague–Dawley rats'fetus development. In utero exposure to BuPB at concentrations up to1000 mg/kg/day did not significantly alter examined developmentalparameters, such as implantations, litter size, number of alive and


dead fetuses, early and late resorptions, percent resorbed conceptuses,fetal body weight, sex ratio or malformations. However, at the highestdose level (1000 mg/kg/day) maternal food consumption and weightgain during some of the measurement intervals were lower (Daston,2004).

Vo et al. (2010), by employing the same rat model during othervulnerable period of life, the juvenile-peripubertal period, observedvarious disruptions induced by parabens. MePB, EtPB, PrPB, iPrPB,BuPB, iBuPB were applied to female rats by oral gavage at doses of62.5, 250 and 1000 mg/kg bw/day. A significant delay in the date ofvaginal opening and a decrease in length of the estrous cycle wererecorded after exposure to high doses of methyl- and isopropylparaben(1000 mg/kg bw/day). Similar to results obtained by Daston (2004),the body weight was not altered following paraben treatment. Never-theless, the significant organ weight changes were noted in ovaries,adrenal glands, thyroid glands, liver and kidneys (Vo et al., 2010).Additionally, alterations in uterus morphology (myometrial hypertro-phy) and histopathological abnormities in ovaries (decrease of corporalutea, increase in the number of cystic follicles, and thinning of thefollicular epithelium) were observed (Vo et al., 2010). However, thenumber of corpora lutea in mature females was not influenced byBuPB exposure (Daston, 2004). Moreover, paraben treatment caused asignificant decrease in serum estradiol and thyroxine concentrations(Vo et al., 2010).

A series of studies on the effect of various parabens (MePB,EtPB, PrPB, BuPB) on male rodents were performed by Oishi (2001,2002a, 2002b, 2004). Methyl- and ethylparaben at doses up to1000 mg/kg bw/day, applied for a period of 8 weeks, did not affecteither reproductive function or secretion of hormones (testosterone,LH and FSH) in male rats (Oishi, 2004). None of the tested parabens atdoses up to 1000–1500 mg/kg bw/day influenced the weight of mostof the male reproductive system organs (testes, ventral prostates,seminal vesicles and preputial glands) (Oishi, 2001, 2002a, 2002b,2004). Epididymis weight was altered only after exposure to BuPB.Surprisingly, contradictory results were obtained for different rodentspecies. While BuPB administration to Wistar rats resulted in a dose-dependent decrease of relative and absolute weights of epididymides(Oishi, 2001), high doses of BuPB (1500 mg/kg bw/day) caused anincrease of epididymis weight in mice (Oishi, 2002a). A significantdecrease of body weight was noted only for rats treated with PrPB at adose equal to about 1300 mg/kg bw/day (Oishi, 2002b).

The decrease of serum testosterone level was observed after ratexposure to 1300 mg/kg bw/day of PrPB and application of BuPBat doses approximately equal to 100 mg/kg bw/day and 1500 mg/kgbw/day to rats and mice, respectively (Oishi, 2001, 2002a, 2004).

Importantly, daily sperm production was altered by BuPB and PrPBapplied at relatively low doses, similar to the upper limit of acceptabledaily intake level (10 mg/kg/day) (Oishi, 2001, 2002a, 2002b). Theseresults may indicate the substantial potential of parabens for disruptionof reproductive system functioning. Hoberman et al. (2008) aimed toreproduce, as closely as possible, the conditions of Oishi experiments(Oishi, 2001), employing the same rat model and dosing procedure.However, they did not obtain any statistically significant dependencebetween BuPB exposure of male Wistar rats and reproductive systemparameters, even after compound application at doses as high as1088 mg/kg bw/day (Hoberman et al., 2008).

In utero tests revealed that oral application of BuPB at a dose of100 mg/kg bw/day, altered neither maternal ovarian estradiol levelsnor fetus testosterone production. Also steroidogenesis-related geneexpression was not influenced by BuPB. No changes in fetus testicularanogenital distance or testicles histology were observed. However, inutero exposure to BuPB resulted in reduced plasma leptin levels inmale and female offspring (Boberg et al., 2008). Since altered leptinlevels in gestation can affect the development of obesity and insulinresistance (Vickers, 2007), reducing plasma leptin levels in fetusesmay indicate an impact of parabens on that process.

Parabens were found to induce oxidative stress in rodents followingoral administration. Rat exposure to 250 mg/kg bw/day ofMePB result-ed in lipid peroxidation. However, at the same time, MePBwas found todecrease the concentration of in vivo hydroxyl radical productionmark-er (2,3-dihydroxybenzoic acid) (Popa et al., 2011). BuPB application,even at much lower doses (13.33 mg/0.2 mL olive oil/kg bw/day),caused an increase of malondialdehyde level in mice, indicating hightissue injury caused by lipid peroxidation. Moreover, significant anddose-dependent reduction in non-enzymatic (glutathione and ascorbicacid) and enzymatic (superoxide dismutase, catalase, glutathioneperoxidase, glutathione transferase) antioxidant concentrations wasnoted (Shah and Verma, 2011).

4.2.2. Subcutaneous administrationImmature rainbow trout were subcutaneously administered with

parabens (EtPB, PrPB and BuPB). Estrogenic response via significantelevation of vitellogenin concentration was induced by all testedparabens applied at doses of 100–300 mg/kg (Pedersen et al., 2000).

Several investigators conducted rodent uterotropic assay followingsubcutaneous (sc.) paraben administration for 3 consecutive days.BuPB sc. injection to immature female rats resulted in uterotropicresponse at a dose of 600 mg/kg/day (Hossaini et al., 2000; Routledgeet al., 1998). The administration of iBuPB at 230.9 mg/kg bw/day wasrelated to 10% increase in rat uterine mass (Koda et al., 2005). Darbreet al. (2002) observed a dose-dependent estrogenic response followingtreatment with about 92 mg/kg bw/day iBuPB to CD1 mice. However,BuPB subcutaneous application to ovariectomized mice (CF-1 and CD-1 strains) at doses up to 950 mg/kg bw/day did not result in statisticallysignificant uterine weight alteration (Shaw and deCatanzaro, 2009).Lemini et al. (2003) studied the influence of MePB, EtPB, PrPB andBuPB on Wistar rats and CD1 mice. Statistically significant uterineweight alteration was recorded for relatively low paraben doses. Thestrongest effect was observed in immature mice, with the lowestobserved effect level (LOEL) 6–60 mg/kg bw/day, followed by ovar-iectomized mice (LOEL 18–165 mg/kg bw/day) and immature rats(LOEL 55–210 mg/kg bw/day). No adverse effect of exposure to parabenmixture, including MePB, EtPB and PrPB, at a total dose 100 mg/kgbw/day was observed (Hossaini et al., 2000).

In addition to uterine weight alteration (Darbre et al., 2002; Kodaet al., 2005; Lemini et al., 2003), parabenswere found to induce changesin uterine histomorphological structure when applied to ovariecto-mized mice at doses of 55–70 mg/kg bw/day (Lemini et al., 2004).

Subcutaneous administration of butylparaben to pregnant rats(100 and 200 mg/kg) resulted in a decrease in the proportion of live-born young and those surviving the first period of development. Onthe day 49 after the birth, young females had significantly lower bodyweight. Male reproductive organs (testes, seminal vesicles, epididymis,prostate gland) were significantly reduced. Moreover, the quantity andmotility of sperm in the epididymis have been significantly reduced.BuPB did not affect the weight of the female genital organs (Kanget al., 2002). These results indicate that maternal exposure to BuPBmay adversely affect the development of the offspring, in particularmale. Still, in utero studies on CF-1 mice exposure to BuPB, at doses upto 950 mg/kg bw/day, did not reveal any influence on litter size andsurvival. Furthermore, no impact of PrPB (1000 mg/kg bw/day) on thenumber of implantation sites was observed (Shaw and deCatanzaro,2009).

The influence of parabens on males was examined by employingrelatively low doses (2 mg/kg/day). Therefore, the effect on testisweight and structure or function of testicular excurrent ducts in neona-tal rats was not recorded (Fisher et al., 1999).

No treatment-related effects of parabens on rodent body weightwere observed (Koda et al., 2005; Shaw and deCatanzaro, 2009).

Endocrine disruptors were proven to alter neurodevelopment,influencing learning abilities and behavior (Colborn, 2004; Miodovniket al., 2011). The effect of maternal exposure to iBuPB released from


subcutaneous silastic capsule on offspring was examined. Therelease rate from the capsules, estimated from in vitro incubationin saline, was equal to 4.36 mg/L/day (Kawaguchi et al., 2009).However, the study design did not allow for a precise determination ofa daily dose of maternal exposure on a daily dose (authors report thevalue of about 4.36 mg/kg/day). There is no information availablewhether the release from the capsule during the period of experimentwas relatively constant or varied in time. Offspring were exposed inutero through placenta and then through milk till weaning. Thetreatment-induced disruptions were observed only for the male off-spring, suggesting iBuPB ability to influence rat anxiety-related andlearning behavior (Kawaguchi et al., 2009).

4.2.3. SummaryThe results of the in vivo studies are equivocal in terms of the poten-

tial of parabens to exhibit harmful effect in the animals. Despite the useof very high PB doses of 100–1000 mg/kg bw/day, a number of studieshave failed to observe any adverse effect. In most of the other cases,PB doses required to cause disruptions were much above the ADIlevel (10 mg/kg bw/day). Only a few studies managed to reveal PBs'influence on reproductive or nervous system at doses similar to ADI.PBs at doses as low as around 10 mg/kg bw/day were found to induceoxidative stress via lipid peroxidation (Shah and Verma, 2011), alterfemale mice uterine weight (Lemini et al., 2003) and male rodents'daily sperm production (Oishi, 2001, 2002a, 2002b). However, theresponse obtained by Oishi (2002a) was not replicable (Hobermanet al., 2008). Moreover, maternal exposure to iBuPB at presumably lowdoses altered male offspring behavior in adulthood (Kawaguchi et al.,2009). Still, the estimated average daily dose of 4.36 mg/kg bw/daywas calculated on the basis of substance release into saline in vitro.Therefore, the estimated value might fail to reflect the real amount anddynamics of release from the subcutaneous silastic capsule in vivo.

Finally, the daily dose of PBs from personal care products, which arethemain sources of human exposure (Fig. 1), was estimated to be equalto about 2.4 mg/kg bw/day (see paragraph 1.4). None of the discussedstudies observed adverse effect at such low PB concentrations.

4.3. Metabolism

Following oral administration, parabens are rapidly absorbed fromthe gastrointestinal tract and blood. Both oral and dermal administra-tions most likely lead to hydrolysis of parabens by non-specificesterases, widely distributed in the body and abundant at sites ofentry such as skin, subcutaneous fat tissue and digestive system. Themain product of the hydrolysis of parabens is p-hydroxybenzoic acid(Soni et al., 2005; Ye et al., 2006).

Laboratory tests on rats revealed that over 50% of the paraben dosewas unabsorbed following dermal application (Aubert et al., 2012).Orally or subcutaneously administered parabens were predominantlyexcreted in the urine, mainly during the first 24 h. However, 2% ofapplied doses were retained in the tissues and carcasses, while lessthan 4% were removed with the feces (Aubert et al., 2012). Parabensand their hydrolysates are excreted in urine as free form or glycine,glucuronide and sulfate conjugates (Janjua et al., 2008).

Efficiency and pattern of hydrolysis of parabens in the organismvaryconsiderably depending on alkyl chain length and tissue (Imai et al.,2006; Ozaki et al., 2013). Though human skin contains carboxylesteraseisoforms, which are able to metabolize parabens to pHBA, esteraseslevels and activities could be insufficient for a complete hydrolysis ofdermally applied parabens (Darbre, 2006). Interestingly, in vitro studiesrevealed that parabens undergo much slower hydrolysis in human skinthan in human liver, rat liver and rat skin (Harville et al., 2007). Follow-ing dermal administration, part of MePB does not undergo hydrolysisand therefore a certain amount of unmetabolized compound mayremain systemically available (Pažoureková et al., 2013). Additionally,skin damage can result in an increase of MePB absorption rate.

Pažoureková et al. (2013) estimated that up to 923 μg/kg bw/day ofunhydrolized MePB can become systemically available, followingapplication of leave-on emulsion containing PB to damaged skin. More-over, continuous PB exposure may lead to compound persistence andaccumulation (Ishiwatari et al., 2007).

4.4. Studies involving humans

The study of Ishiwatari et al. (2007) revealed that continuous der-mal exposure to PBs may result in the compound accumulation. Re-peated application of MePB-containing topical formulations byhuman volunteers was accompanied by the increase of the com-pound concentration remaining in the stratum corneum of the skin(Ishiwatari et al., 2007).

Janjua et al. (2007) studied the effect of the whole-body topicalapplication of a cream with 2% (w/w) of BuPB. The PB dose equaledapproximately 10 mg/kg bw/day. The BuPB-containing formulationwas applied daily for a period of one week by young male volunteers.A rapid skin penetration and systemic uptake of BuPB in humans wereconfirmed by the pronounced increase of compound concentration inserum, reaching 135 μg/L 3 h after application. Although 24 h afterexposure BuPB concentration decreased to 18 μg/L, it still remainedabove the control level (Janjua et al., 2007). Exposure did not alter levelsof reproductive hormones (follicle stimulating hormone (FSH), luteiniz-ing hormone (LH), testosterone, estradiol, inhibin B) and thyroid hor-mones (thyroid stimulating hormone (TSH), free thyroxine (FT4),total triiodothyroxine (T3), and total thyroxine (T4)). Nevertheless,the effects were studied on a relatively small group (n = 24) for ashort period of time (1 week) (Janjua et al., 2007).

No relationship between urinary concentrations of MePB, PrPB andBuPB and hormone levels or conventional semen quality parametersof men attending an infertility clinic (n ≥ 132) was found. However, astatistically significant positive association between BuPB concentrationand sperm DNA damage (measured by employing a comet assay) wasobserved (Meeker et al., 2011). Furthermore, suggestive relationshipsbetween MePB and TSH and MePB or PrPB and DNA damage markersindicate that (for such subtle interactions) the sample size might bestill too small to provide statistical significance.

Koeppe et al. (2013) conducted a study employing the samplescollected from 1831 subjects from the US population. An inverse associ-ation between urinary levels of parabens and serum thyroid hormoneconcentration was observed. The strongest and most consistent associ-ations were obtained in adult female subgroup (Koeppe et al., 2013).This can be a result of significantly elevated PB urinary concentrationsin women compared to men (Calafat et al., 2010; Koeppe et al., 2013).

Parabens were commonly detected in human serum samples(Table 4). Median total paraben concentrations measured in serumwere equal to 10.9 μg/L, 0.2 μg/L and 1.4 μg/L for MePB, EtPB and PrPB,respectively. Detection frequency ranged from 53% (EtPB) to 100%(MePB) (Ye et al., 2008b). The median PB concentrations measured byFrederiksen et al. (2011) were much lower (0.89 μg/L for MePB,0.23 μg/L — PrPB and bLOD for EtPB, BuPB and BePB). However, thedifference may have arisen from a difference in exposure between sex,as a tested group consisted of men only (Frederiksen et al., 2011).BuPB serum levels were below detection limit in most of the cases,with a maximum concentration not exceeding 1.0 μg/L (Frederiksenet al., 2011; Janjua et al., 2007).

Parabens were also detected in human milk samples. Ye et al.(2008a) developed a method for analysis of five parabens: MePB,EtPB, PrPB, BuPB and BePB. However, only MePB and PrPB were detect-ed in some of the tested milk samples. The study conducted bySchlumpf et al. (2010) revealed the presence of MePB, EtPB and PrPBin 34.1%, 19.5% and 14.6% of defatted milk samples, respectively. Lackof detection of BuPB in the milk samples could have arisen from thesample preparation process, including defatting, rather than its absencein human milk. BuPB, being the most lipophilic molecule among the

Table 4Urinary and serum total (free form plus conjugates) concentrations (μg/L) of parabens and frequency of paraben detection (%; italics in brackets); unless noted otherwise, the specifiedfigures represent minimum and maximum.

MePB EtPB PrPB BuPB BePB References

Urine n = 100 4.2–680a

(99)ND-47.5a

(58)0.2–279a

(96)ND-29.5a

(69)ND-0.5a

(39)Ye et al. (2006)

n = 2548≥6 years

5.60–974b

(99)ND-57.2b

(42)0.30–299b

(93)ND-19.6b

(47)– Calafat et al. (2010)

n = 60male

ND-2002(98)

ND-564(80)

ND-256(98)

ND-67.6(83)

ND-2.06(7)

Frederiksen et al. (2011)

n = 120pregnant women

191.0 (415.5)c

(100)8.8 (25.7)c

(98)29.8 (61.3)c

(88)2.4 (10.3)c

(90)– Casas et al. (2011)

n = 30children

150.0 (427.8)c

(100)8.1 (26.2)c

(100)21.5 (56.4)c

(80)1.2 (3.7)c

(83)– Casas et al. (2011)

n = 194male

5.1–1,080d

(100)– 0.4–294d

(92)ND-64.5d

(32)– Meeker et al. (2011)

n = 860≥6 years

0.5–14,900 0.5–1110 0.1–7210 0.1–1240 – Savage et al. (2012)

n = 653 ND-23,200(99.7)

– ND-2870(96.5)

ND-998(65.4)

– Smith et al. (2012)

n = 30adults

0.778–240(100)

0.098–23.5(100)

– – – Wang and Kannan (2013)

n = 40children

1.69–5240(100)

NQ-7.60(60)

– – – Wang and Kannan (2013)

n = 879female ≥12 years

ND-4282(99.9)

ND-3010(60)

ND-1002(98)

ND-309(65)

– Koeppe et al. (2013)

n = 970male ≥12 years

ND-7909(99.5)

ND-771(38)

ND-1486(91)

ND-723(28)

– Koeppe et al. (2013)


ND-1238(94)

ND-2022(81)

ND-5380(89)

ND-81.5(54)

– Shirai et al. (2013)


60.6–451.5e

(98)16.9–202.8e

(100)0.94–65.4e

(98)ND-0.47e

(28)– Kang et al. (2013)

n = 46newborn infants

39.9–272.3e

(100)1.0–8.0e

(98)0.84–15.2e

(100)ND-1.7e

(41)– Kang et al. (2013)

n = 10050 males50 females2.5–87 years

1.2–803(100)

NQ-61.0(87)

NQ-575(72)

NQ-113(46)

NQ-0.8(6)

Asimakopoulos et al. (2014)

Serum n = 154 males 11 females

0.4–301(100)

ND-5.4(53)

ND-67.4(80)

ND ND Ye et al. (2008b)

n = 60male

ND-59.6(95)

ND-20.8(30)

ND-5.50(93)

ND-0.87(3)

ND-0.29(3)

Frederiksen et al. (2011)

ND — not detected; NQ — not quantified.a 5th–95th percentiles.b 10th–95th percentiles.c Median and inter quartile range (in brackets).d 10th — maximum.e 25th–75th percentiles (specific gravity adjusted paraben concentrations).


analyzed parabens, could have been retained in lipids and separatedfollowing the centrifugation. Detected median concentrations of PBs inmilk were equal to 1–1.5 μg/L. An estimated median daily intake ofparabens with human milk by infants was 192.5–301.3 ng/kg bw/day.The maximum intake amounted to 1314.9 ng/kg bw/day for MePBand 381.1 ng/kg bw/day for EtPB and PrPB (Schlumpf et al., 2010).Even at the highest doses, the estimated infant intakewas over three or-ders of magnitude lower than the ADI level of 10 mg/kg bw/day.Nevertheless, the removal of the lipid fraction from milk could lead toa loss of the compounds during analytical procedure and significantunderestimation of the PB dose.

Frequent detection of parabens in the urine (Table 4) confirms thewidespread exposure to parabens, also among pregnant women andchildren. The concentrations of MePB and PrPB, which were detectedin most of the analyzed urine samples (N80%), were strongly correlatedwith each other (Koeppe et al., 2013;Meeker et al., 2011; Ye et al., 2006)indicating a common source of exposure.

As parabens were commonly found in the human placental tissue(Jiménez-Díaz et al., 2011) and were suggested to accumulate in theamniotic fluid (Frederiksen et al., 2008), theymay pose in utero adverseeffects in humans. A significant correlation between urinary parabenconcentrations of pregnantwomen and theirmatching newborn infantsindicates the transfer of the compound from the mother to the fetus(Kang et al., 2013).

Although employing a relatively small group (n = 46) Kang et al.(2013) were able to note a significant relationship between the PBexposure and oxidative stress biomarkers, even after the adjustmentof relevant covariates (e.g. maternal age, mode of delivery, pre-pregnancy BMI). Similar to the effect observed in rodents (Popa et al.,2011; Shah and Verma, 2011), infant and maternal urinary MePB orEtPB concentrations were associated with malonyldialdehyde levels.Suggestive relationships between urinary levels of EtPB and marker ofoxidative DNA damage (8-oxo-2′-deoxyguanosine) in pregnantwomen were also observed (Kang et al., 2013). Moreover, PrPB andBuPB levels were significantly associated with aeroallergenic sensitiza-tion (Savage et al., 2012).

No association between maternal paraben levels and anogenitaldistance in male infants (Shirai et al., 2013) was consistent with resultsobtained in the animal experimental study (Vickers, 2007).

Althoughmany reports confirmed that parabens undergo hydrolysisin organism (Aubert et al., 2012; Ye et al., 2006), they were found to becompletely stable inMCF7 breast cancer cell homogenates. The stabilityof parabens may lead to their accumulation in breast tumor tissue(Dagher et al., 2012). However, Barr et al. (2012) did not find any signif-icant correlation between the level of parabens in the breast tissue andage of the patient. Recently, parabens have been found to be ubiquitousin the tissue samples of human breast tumors. The mean value of thetotal PB concentration in breast tumor tissues equaled 20.6 ± 4.2 ng/g


(n= 20) and 9794 ng/g (n= 9) for samples fromUK and India, respec-tively (Darbre et al., 2004; Shanmugam et al., 2010). The highest detect-ed concentration in tumors was equal to 26,469 ng/g for BuPB(Shanmugam et al., 2010). Moreover, the occurrence of parabens inthe unaffected breast tissue adjacent to cancer was found in nearly allof the studied samples (99%, n = 160), at a total median PB concentra-tion of 85.5 ng/g (range 0–5134.5 ng/g) (Barr et al., 2012). Darbre et al.(2004) suggested that the presence of PB in breast tumor tissue mightbe related to carcinogenesis. Some authors have contested the validityof that hypothesis by arguing that the mere presence of PB in breastcancer tissue does not provide sufficient evidence for PB's harmful effect(Golden et al., 2005; Witorsch and Thomas, 2010).

Although, according to some in vitro reports, relatively high concen-trations (10−4–10−5M) of parabens are required to produce estrogeniceffect (Blair et al., 2000; Okubo et al., 2001), other authors observed PBactivity at lower levels of the order of 10−7 M, which correspond toabout 15.2–19.4 ppb for MePB–BuPB (Watanabe et al., 2013; Zhanget al., 2013). As the concentrations found in breast tumor tissue,which is an estrogen-responsive tissue (Pugazhendhi et al., 2005),were up to 26,469 ppb (Shanmugam et al., 2010), it is not inconceivablethat parabens may induce disruptions via binding to ERRγ or ERreceptors.

Charles and Darbre (2013) tested whether PBs at concentrationlevels detected in human breast tissue (Barr et al., 2012) were ableto stimulate the proliferation of MCF-7 cells in vitro. The calculationsof the PBs' lowest-observed-effect concentrations (LOEC) and no-observed-effect concentrations (NOEC) were based on the differencebetween the number of cell doublings with and without paraben. Itwas found that the content of at least one paraben in human breasttissue was ≥LOEC in 27% of the tissue samples and NNOEC in 40%.Importantly, it was shown in the in vitro study that, even if none ofthe separately tested five parabens caused a significant increase ofMCF-7 cell proliferation, combined exposure to all 5 parabens at thesame concentrations could result in a significant increase of prolifera-tion of the MCF-7 cells. Moreover, long lasting (about 2–4 months)MCF-7 cell exposure to a mixture of 5 parabens present in the testedbreast tumor tissue at the concentrations below LOEL resulted in anincreased cell proliferation in 5/6 cases (Charles and Darbre, 2013). Ashumans are likely to be continuously exposed to a number of variousparabens, the potentially adverse effect of the preservatives might notbe negligible.

4.5. Additive and synergistic effect

Environmental endocrine disruption is most often an effect ofexposure not to a single compound, but to a number of substances atlow concentrations (Sumpter and Johnson, 2005). Despite being at theconcentrations below observed effect levels (called no-observed-effectconcentrations), a mixture of EDCs can cause an estrogenic response(Brian et al., 2005; Rajapakse et al., 2002).

Darbre (2009) tested the effect of PBs on the proliferation of breastcancer cell MCF 7. A series of analyseswere performed using: separatelyfive parabens at no-observed-effect concentrations (NOEC), a mixtureof these parabens at NOEC, 10−11 M solution of estradiol (E2) and 10−11 M E2 together with a mixture of five parabens at NOEC. The resultsindicate that parabens mixed at no-observed-effect concentrations canproduce estrogenic response by increasing cell proliferation in MCF 7breast cancer cells. Moreover, estrogenic response of estradiol can beenhanced by a mixture of parabens at NOEC. In the organism, thiscould take place in periods when estradiol is present at low levels,occurring during a menstrual cycle, before puberty or after menopause(Darbre, 2009).

Kim et al. (2012a) reported the effect of combined doses ofbisphenol A (BPA) and isobutylparaben by measuring the expressionof calbindin-D9k (CaBP-9k) in rat pituitary cancer GH3 cells. The resultsindicate that BPA and iBuPB may have additionally an increased

estrogenic potential via an estrogen receptor-mediated pathway(Kim et al., 2012a). The synergistic increase in luciferase activityand CaBP-9k expression was also observed for simultaneous GH3rat pituitary cell treatment with octylphenol and isobutylparaben(Kim et al., 2012b). However, in terms of lipid peroxidation, the effectof simultaneous treatment of animals with MePB and BPA was notsignificantly different from the effect of exposure to a single compound(Popa et al., 2011). Also the study of Meeker at al. (2011) failed to findany evidence of BuPB and BPA interaction in terms of DNA damage in-duction. Nevertheless, the authors stress that the statistical powerof the test was low due to insufficient size of the study group(Meeker et al., 2011).

The possible synergic or additive effect of PBs and other EDCs wouldbe of great importance, taking into account that humans are most oftenexposed to a mixture of substances rather than a single compound.

5. Conclusions

The global widespread use of parabens has resulted in their ubiqui-tous occurrence in the environment. Themain point sources of pollutionare wastewater treatment plants. Concentrations in influents reachabout 80,000 ng/L for the most common MePB. Although PBs' removalpercentage in WWTPs is high (on average 96.1–99.9%), the contami-nants are still present in effluents (at concentrations up to about4000 ng/L), which results in their leaking into the environment. PBswere detected in water resources, soil and sediments, air and dust, aswell as in biota. Nevertheless, the concentration values observed innatural environment seem to be too low to produce adverse effects.

The main sources of human exposure to parabens are personal careproducts and pharmaceuticals. The exposure level is reflected by thefrequent detection of the compounds in urine. At least one of the PBswas found in nearly 100% of tested urine samples.Moreover, the preser-vativeswere detected in human serum,milk, placental tissue and breasttumor tissue. However, the question arises whether the PB concentra-tions to which we are exposed are high enough to cause a threat tohuman health.

In general, the in vivo studies suggest that, at the current levels ofexposure, PB impact on the organism reproduction, development andhomeostasis seems to be of marginal importance. However, few studiesinvolving human populations managed to find weak yet significantrelationship between urinary paraben concentration and oxidativestress biomarker, sperm DNA damage or serum thyroid hormones.Although the correlations might be coincidental, it does not seemreasonable to exclude the chance that parabens, even at very lowconcentrations, influence organism homeostasis.

Moreover, parabens at concentration levels detected in humanbreast tissue were found to induce proliferation of cells in vitro, inparticular when present in mixtures. Therefore, it does not seemreasonable to neglect the possible involvement of parabens in the pro-cess of carcinogenesis.

Further research, including large sample size studies involvinghumans are necessary to assess possible adverse effects of parabens.Additionally, the influence of parabens on other steroid sensitivetissues, in particular those within the immune and the central nervoussystem should be tested.

Acknowledgment

The study was partially supported by the Nofer Institute of Occupa-tional Medicine fund no. IMP 1.28/2014.

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