Aquatic Toxicology 99 (2010) 360���369 Contents lists available at ScienceDirect Aquatic Toxicology j o u r n a l h o m e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a q u a t o x Mechanisms of toxicity of di(2-ethylhexyl) phthalate on the reproductive health of male zebrafish Tamsyn M. Uren-Webster, Ceri Lewis, Amy L. Filby, Gregory C. Paull, Eduarda M. Santos ��� Hatherly Laboratories, School of Biosciences, University of Exeter, Prince of Wales Road, Exeter, Devon EX4 4PS, UK a r t i c l e i n f o Article history: Received 22 February 2010 Received in revised form 17 May 2010 Accepted 19 May 2010 Keywords: Estrogen DEHP Danio rerio PPAR Peroxisome proliferation Testes a b s t r a c t Phthalates are ubiquitous in the aquatic environment and are known to adversely affect male repro- ductive health in mammals through interactions with multiple receptor systems. However, little is known about the risks they pose to fish. This project investigated the effects of di(2-ethylhexyl) phtha- late (DEHP), the most commonly used phthalate, on the reproductive health of male zebrafish (Danio rerio). Males were treated with 0.5, 50 and 5000 mg DEHP kg���1 (body weight) for a period of 10 days via intraperitoneal injection. The effects of the exposure were assessed by analysing fertilisation success, testis histology, sperm DNA integrity and transcript profiles of the liver and testis. A significant increase in the hepatosomatic index and levels of hepatic vitellogenin transcript were observed following expo- sure to 5000 mg DEHP kg���1. Exposure to 5000 mg DEHP kg���1 also resulted in a reduction in fertilisation success of oocytes spawned by untreated females. However, survival and development of the resulting embryos were unaffected by all treatments, and no evidence of DEHP-induced sperm DNA damage was observed. Exposure to 50 and 5000 mg DEHP kg���1 caused alterations in the proportion of germ cells at specific stages of spermatogenesis in the testis, including a reduction in the proportion of spermatozoa and an increase in the proportion of spermatocytes, suggesting that DEHP may inhibit the progression of meiosis. In parallel, exposure to 5000 mg DEHP kg���1 increased the levels of two peroxisome proliferator- activated receptor (PPAR) responsive genes (acyl-coenzyme A oxidase 1 (acox1) and enoyl-coenzyme A, hydratase/3-hydroxyacyl coenzyme A dehydrogenase (ehhadh). These data demonstrated that exposure to high concentrations of DEHP disrupts spermatogenesis in adult zebrafish with a consequent decrease in their ability to fertilise oocytes spawned by untreated females. Furthermore, our data suggest that the adverse effects caused by exposure to DEHP are likely to occur preferentially via PPAR signalling path- ways in the testis and oestrogen signalling pathways in the liver. We found no evidence of adverse effects on zebrafish reproductive health following exposure to the concentrations occurring in most aquatic sys- tems, indicating that DEHP alone may not be a causative agent of the reproductive abnormalities seen in wildlife, at least as a result of short-term exposures. �� 2010 Elsevier B.V. All rights reserved. 1. Introduction Phthalates are extensively used as plasticisers in many mass- produced products including food packaging, toys, electrical equipment, medical devices, paints and cosmetics (Jobling et al., 1995 Bauer and Herrmann, 1997). Global production is now over 4 million tonnes per year with the most widely used phthalate, di(2-ethylhexyl) phthalate (DEHP), accounting for at least a quar- ter of this production (Bauer and Herrmann, 1997 Petrovic et al., 2001). Phthalates are not chemically bound to plastic mate- rials, and so are easily leached into the environment with time and use (Bauer and Herrmann, 1997). Phthalates also enter sur- face waters via waste-water treatment works effluents and from ��� Corresponding author. Tel.: +44 01392 264607 fax: +44 01392 263700. E-mail address: e.santos@exeter.ac.uk (E.M. Santos). the atmosphere via plastic manufacture and burning (Jobling et al., 1995 Bauer and Herrmann, 1997 Staples et al., 1997). Although phthalates readily undergo microbial and abiotic degradation, and are therefore not persistent in the aquatic environment (Staples et al., 1997), continual release of large volumes means they are found very widely and often at substantial concentrations. DEHP is the most widespread phthalate in the aquatic environment which reflects its highest rate of production. The reported con- centrations of DEHP are up to 100 g L���1 in surface waters and 200 mg kg���1 (wet weight) in sediments, although hotspots of con- tamination occur in heavily industrialised areas (Petrovic et al., 2001 Fromme et al., 2002). Fish are exposed to phthalates present in the water column and sediment, and also via their diet, and the concentration of DEHP in wild freshwater fish tissue ranges widely. For example, a comprehensive survey of DEHP in fish in Austrian rivers found concentrations ranging up to 1 mg kg���1 (wet weight) in most cases, and the maximum value measured 0166-445X/$ ��� see front matter �� 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.05.015

T.M. Uren-Webster et al. / Aquatic Toxicology 99 (2010) 360���369 361 was 2.6 mg kg���1 in carp (Cyprinus carpio) (European Commission, 2003). Phthalates are generally classed as oestrogenic chemicals due to their ability to bind and activate oestrogen receptors (ER) in vitro (Harris et al., 1995 Jobling et al., 1995 Takeuchi et al., 2005). However, in vivo studies have consistently reported that phthalates are extremely weak oestrogens in fish. For example, a concentra- tion of 1500 mg DEHP kg���1 dosed via the diet was required to induce a small incidence of intersex in juvenile salmon (Salmo salar) (Norrgren et al., 1999 Norman et al., 2007), and 500 mg kg���1 (dosed via intraperitoneal injection) of butylbenzyl phthalate (BBP) was required to induce a 3-fold increase in vitellogenin (VTG) in male rainbow trout (Oncorhynchus mykiss) (Christiansen et al., 2000). Other studies have found no evidence that phthalates induce any oestrogenic effects at the concentrations tested (Harries et al., 2000 Metcalfe et al., 2001 Patyna et al., 2006). Phthalates have been widely reported to disrupt the male repro- ductive system in fish. Dibutyl phthalate (DBP) and DEHP disrupted the activity of various enzymes involved in the metabolism and syn- thesis of testosterone in carp in vitro (Thibaut and Porte, 2004). A high concentration of diethyl phthalate (DEP) (1 mg L���1) induced testicular atrophy in carp (Barse et al., 2007). Recently, exposure to an environmentally relevant concentration of BBP (6 g L���1) was found to induce changes in sperm motility, an important com- ponent of male reproductive health, in the zebrafish (Danio rerio) (Oehlmann et al., 2009). The exact mechanisms by which phthalates induce these effects are not clear, and may involve interaction with multiple receptor systems including those responsible for mediat- ing the effects of sex steroids and peroxisome proliferator-activated receptors (PPARs) (Lampen et al., 2003 Takeuchi et al., 2005). Mammalian studies have provided considerable evidence that phthalates induce a range of reproductive effects in males includ- ing disruption of reproductive development, alteration of steroid hormone balance, testicular lesions and atrophy, disruption of sper- matogenesis and infertility (Sharpe et al., 1995 Bhattacharya et al., 2005 Corton and Lapinskas, 2005 Howdeshell et al., 2008). These effects are distinct from an oestrogenic mechanism of action, but are also considered to be independent of the androgen receptor (AR) (Miura et al., 2007 Onorato et al., 2008 Pant et al., 2008). It is unlikely that one exclusive mechanism is responsible for the com- plex effects seen, but activation of PPARs is widely recognised as an important mechanism by which phthalates induce some of these effects (Bhattacharya et al., 2005 Corton and Lapinskas, 2005). Specifically, disruption of spermatogenesis by phthalates in mam- mals has been attributed to disturbance of oxidative balance in the testis, and this has been suggested to occur via PPAR activation (Kasahara et al., 2002 Corton and Lapinskas, 2005 Onorato et al., 2008), potentially resulting in lower sperm quality/quantity, fertil- isation ability and embryo survival (Park et al., 2002 Agarwal and Said, 2005 Pant et al., 2008). There is a significant lack of research investigating the effects of phthalates on the reproductive health of fish, particularly consid- ering the multiple mechanisms of action of these environmentally relevant compounds. This information is essential to establish the mechanisms of toxicity of phthalates in lower vertebrates and will help to provide a more comprehensive understanding of the poten- tial threat phthalates pose to the reproductive health of fish in the environment. In this study, we investigated the effects of exposure to a range of concentrations of DEHP, including those occurring in the aquatic environment, on the reproductive health of male zebrafish (D. rerio). Males were exposed via intraperitoneal injec- tion and allowed to breed with untreated females for a period of 10 days. The effects of the exposure on spermatogenesis, fertilisa- tion success and embryo survival were investigated, together with measurements of transcript profiles for 13 genes, to elucidate the pathways of disruption mediating the effects seen. 2. Materials and methods 2.1. Fish maintenance Adult zebrafish (wild-type WIK strain, originally from the Max Planck Institute, Tubingen, Germany) were bred and maintained in the specialist zebrafish aquaria facility at the University of Exeter in 140 L mixed sex stock tanks before experimentation. The aquarium water supply was reverse-osmosis treated tap water reconstituted with analytical grade salts to produce a standardised synthetic freshwater, as described in Paull et al. (2008), the temperature was maintained at 28 �� 1 ���C and the photoperiod was set at 12:12 h light:dark with a 30 min gradual transition period at dawn and dusk. Fish were fed with freshly hatched Artemia nauplii every morning and Tetramin tropical flake food (Tetra Melle, Germany) every afternoon to satiation. Sexually mature males and females were identified visually and allocated into colonies of two male and two female fish (2 �� 2 colonies) which were allowed to breed naturally in 15 L glass aquaria. Each aquaria was aerated and supplied with a water flow rate of 48 L day���1. In small colonies of zebrafish, dominance hier- archies typically develop between males, and aggression from dominant males can cause stress of subordinate males (Paull et al., 2008). To avoid this, in this study males and females in each colony were evenly matched in size to limit the chance of domi- nant individuals becoming overly aggressive. Spawning occurred daily at dawn, with all males and females within each tank breed- ing as a group, using artificial weed placed at the base of the tank as a spawning substrate. Eggs were collected from each tank 1 h post-fertilisation (hpf) before morning feeding, transferred to Petri dishes and maintained in tank water. Immediately after collection, eggs were washed with tank water to remove waste food and fae- ces and dead eggs were removed, while the remaining eggs were left to incubate in tank water at 28 ���C. The number of fertilised and unfertilised eggs was determined at 3 hpf by visual inspec- tion using light microscopy (Kyowa Optical SDZ PL, Kyowa Optical, Kanagawa, Japan). The unfertilised eggs were removed, while the fertilised eggs were left to incubate until 24 hpf, under the same conditions. Embryo mortalities were assessed and removed at 6, 8 and 24 hpf. Embryos were regularly monitored at 24 hpf, using light microscopy, to assess if normal development was occurring. 2.2. Pre-exposure At the start of the experiment, 18 colonies were set up and allowed to acclimate for a period of 5 days. Following this initial acclimation, egg number, fertilisation success and embryo survival at 6, 8 and 24 hpf were monitored daily during a pre-exposure period of 10 days. Colony egg production tends to follow a number of patterns including consistent daily spawning, regular intermit- tent peaks in spawning, and irregular spawning including several days without spawning (Paull et al., 2008). The aim of this pre- exposure period was to select 16 colonies with consistent egg production and fertilisation success for use in the DEHP exposure. 2.3. Exposure experiment Although exposure via water or diet is advocated as the most environmentally relevant route of phthalate exposure (Patyna et al., 2006 Norman et al., 2007), intraperitoneal injection was used to deliver DEHP in this study because it allowed males to be targeted, and the effects of DEHP on male reproduction to be specifically investigated, in isolation from possible effects on females. Addi- tionally, dietary exposure does not guarantee that an equal dose is delivered to each fish, and there are significant practical difficul- ties in maintaining the required exposure concentration of DEHP