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Effects of 4-Nonylphenol on the Endocrine System of the Shore Crab, Carcinus maenas Christina M. Lye,1 Matthew G. Bentley,1 Tamara Galloway2 1

School of Marine Science & Technology, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE1 7RU, United Kingdom 2

School of Biological Sciences, University of Plymouth, Plymouth, Devon, PL4 8AA, United Kingdom

Received 17 November 2006; revised 18 September 2007; accepted 20 September 2007 ABSTRACT: There is a considerable body of evidence to suggest that many anthropogenic chemicals, most notably xeno-estrogens, are able to disrupt the endocrine system of vertebrates. There have been few comparable studies on the effects of exposure to these chemicals that may serve as biomarkers of endocrine disruption in aquatic invertebrate species. In addition, the evidence available is complex, conflicting, and far from conclusive. The present study aimed to investigate the impact of the xeno-estrogen 4-nonylphenol (4-NP, nominal concentrations 10–100 lg L21) on the regulation and functioning of the endocrine system of the shore crab Carcinus maenas. It also set out to establish whether 4-NP are causing the effects (i.e., changes of exoskeletons including secondary sexual characteristics, pheromonally mediated behavior and ecdysone levels, and the presence of vt in the male hepatopancreas) found recently in wild shore crabs (Lye et al. (2005) Mar Ecol Prog Ser 288:221–232). The study utilizes morphological (e.g., gonadosomatic and hepatosomatic indices) and hormonal (ecdysteroid moulting hormone levels and the induction of female specific proteins, vitellins) biomarkers using radioimmunoassay and an indirect enzyme linked immunosorbent assay applied to the soluble protein fraction of adult male hepatopancreatic homogenates. Exposure of C. maenas to an effective concentration as low as 1.5 lg L21 4-NP resulted in a reduced testis weight, increased liver weight, and altered levels of ecdysone equivalents compared to controls. Induction of vitellin-like proteins was absent in all samples tested. The ecological implications and the possible mechanisms for the action of 4-NP on the response of the shore crab to xeno-estrogen exposure are discussed. # 2007 Wiley Periodicals, Inc. Environ Toxicol 00: 000–000, 2007. Keywords: endocrine disruption; invertebrates; Carcinus maenas; 4-nonylphenol; ecdysteroids; vitellin; gonadosomatic index; hepatosomatic index

INTRODUCTION The endocrine systems of animals coordinate and regulate growth, development, reproduction, and other physiological processes. In recent years, a number of scientific studies have shown that exogenous chemicals can interfere with the normal functioning of these systems and thus have the Correspondence to: C. M. Lye; e-mail: [email protected] Contract grant sponsor: Natural Environment Research Council (NERC). Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.20344 C

potential to adversely affect populations and communities (IEH, 1999; Pait and Nelson, 2002). Despite the fact that each of the diverse endocrine systems could potentially be affected by environmental chemicals concern has focused in the United Kingdom on systems regulated by estrogens because of the apparent feminization of several fish species in rivers and estuaries, as evidenced by the presence of a female yolk protein, vitellogenin (vtg) in male serum and occurrence of intersex (presence of female tissue in male testes) (Allen et al., 2002). A range of natural and synthetic estrogens have been detected in the estuaries where the most marked effects in

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LYE, BENTLEY, AND GALLOWAY

fish have been observed. These include the natural human hormone 17b-estradiol, the synthetic hormone 17a-ethinyloestradiol (the component of contraceptive pill), and alkylphenol polyethoxylates (APnEOs), which have been shown to trigger significant estrogenic effects in fish (Jobling and Sumpter, 1993; Harries et al., 1995; Jobling et al., 1996). APnEOs are synthetic surfactants commonly used in industrial detergents and plastics manufacturing (Blackburn et al., 1999). Approximately 80% of all manufactured APnEOs are nonylphenol ethoxylate (NPnEOs) (Naylor, 1998), which in sewage treatment works (STWs) degrade to nonylphenol (NP) (Ahel et al., 1994). NP concentrations in UK rivers range between \0.2 and 12 lg L21, although concentrations as high as 180 lg L21 have been reported in water receiving effluent directly from STWs (Blackburn and Waldock, 1995; Allen et al., 2002). In the marine environment, concentrations are normally lower, ranging between 0.03 and 5.2 lg L21 (Blackburn and Waldock, 1995; Blackburn et al., 1999; Lye et al., 1999; Allen et al., 2002; Sanders et al., 2005). Most of the estrogenic activity detected in marine waters is strongly adsorbed on sediment particles (Allen et al., 2002). It also appears that fish species like the flounder, Platichthys flesus, a bottom-living fish often burrowing into and ingesting sediment while feeding (Bryan et al., 1985), obtains some estrogenic exposure through feeding on benthic invertebrates. Whether crustaceans, such as the shore crab, Carcinus maenas, inhabitants of the benthic communities and sediment dwellers themselves, are susceptible to estrogenic exposure remains however an open question. Nevertheless, because sediments may concentrate these compounds by perhaps hundreds to thousands of times higher than in the water column, biological availability of only a small proportion of the particulate EDC loading could result in significant uptake in benthic invertebrate organisms including crustaceans, which are in direct contact with sediments (Langston et al., 2005). There is some evidence suggesting that crustaceans are not affected or at least are less vulnerable than other invertebrates to endocrine disruption (Allen et al., 2002). For example, neither vtg or its corresponding egg yolk protein vitellin (vt) was detected in the hemolymph of male crabs, Carcinus maenas or in brown shrimp Crangon crangon collected from several UK estuaries as part of a national sampling program, or following a 21-day exposure to known estrogens (diethylstilbestrol, 4-NP) (Allen et al., 2002). A number of laboratory studies on marine crustaceans (e.g., copepods Tisbe battagliai, Nitocra spinipes, and glass prawn Palaemon elegans) have also been reported, where a range of natural and synthetic estrogens including 17a-ethinyloestradiol, 17b-estradiol, estrone, and 4-NP exhibited no effects on several population relevant parameters including survival, development, reproduction, and sex ratio (Hutchinson et al., 1999; Breiholtz and Bengtsson, 2001; Allen et al., 2002; Sanders et al., 2005), although one study did

show that the ecdysteroid (steroid moulting hormone) 20hydroxyecdysone and the reference estrogen diethylstilbestrol (DES) caused significant inhibition of development and reproduction of Tisbe battagliai (Allen et al., 2002). In contrast to the earlier, evidence is beginning to emerge of endocrine disrupting effects occurring in the field. Ford et al. (2003a,b, 2004, 2005) recently reported several effects indicative of endocrine disruption in marine/ estuarine amphipod Echinogammarus marinus from fieldbased research in East Scotland. These included (i) intersexuality with separate male and female intersex forms identified, (ii) a higher incidence of intersex at sites receiving chemical discharges compared to reference sites, (iii) a reduction of fecundity, fertility, paring success, and delayed maturity in intersex Crustacea, and (iv) a link between the degree of endocrine dysfunction and the degree of maleness in intersex females. A further study investigating the effects of EDCs on the induction of secondary sexual characteristics in wild freshwater crabs (Geothelphusa dehaani) from 10 Japanese rivers in Sasebo City, Nagasaki have found that 8–32% of males collected at all sites developed female genital openings (Ayaki et al., 2005). This abnormality was thought to be caused by the presence of agricultural chemicals and chemicals present in waste gases from cars. Similarly, field studies in the United Kingdom have indicated apparent changes of shore crab, Carcinus maenas exoskeletons including secondary sexual characteristics, pheromonally mediated behavior and ecdysone levels, and the presence of vt in the hepatopancreas of male shore crabs, from estrogen-contaminated UK estuaries (i.e., the Mersey, Tyne, Tees, Tamar, Dee, and Clyde) compared to reference sites (Allen et al., 2002; Clare et al., 2003; Brian, 2005; Lye et al., 2005). Although these effects are not conclusive and warrant further investigation, an increasing number of laboratory studies have also implicated endocrine disrupting contaminants including estrogens, xeno-estrogens, PCBs, PAHs, a number of pesticides, and sewage outfalls as having detrimental effect on moulting, development, and the reproduction of a number of crustacean species (for review see Oehlmann and Schulte-Oehlmann, 2003). For example, induction of larval storage protein in the glass prawn larvae (Palaemon elegans) and cypris major protein in the barnacle larvae (Balanus amphitrite) as well as inhibition of barnacle settlement have been reported after exposure to 0.01–20 lg L21 4-NP and 0.2–20 lg L21 17bestradiol (Billinghurst et al., 1998, 2000; ; Sanders et al., 2005). Increased antenna length has been observed in Corophium volutator amphipods exposed to 4-NP (Brown et al., 1999). Vandenbergh et al. (2003) recently reported indications of hermaphroditism, disturbed maturation of germ cells and spermatogenesis, reduced gnathopod size, and female biased sex ratios in the amphipod, Hyallela azteca exposed to 0.1–10 lg L21 17a-ethinylestradiol. In Daphnia magna, disturbances in testosterone metabolism and the development of secondary sex characteristics have been

Environmental Toxicology DOI 10.1002/tox

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IMPACT OF 4-NONYLPHENOL ON MARINE CRUSTACEANS

reported to occur after exposure to different endocrine active substances (Baldwin and LeBlanc, 1994; Baldwin et al., 1995, 1997; Olmstead and LeBlanc, 2000). Thus, it appears that the scientific evidence about ED in crustaceans is complex, conflicting, and far from conclusive and need further investigations. The hypothesis of this study is that xeno-estrogenic substances alter endocrine regulated processes in adult male crabs. The objective of the study was therefore to evaluate the impact of a wellknown xeno-estrogen, 4-NP, on the regulation and functioning of the endocrine system of the male shore crab, C. maenas. The sediment-dwelling shore crab was chosen as it inhabits benthic communities of estuaries in addition to open coastlines, a habitat often susceptible to contamination from sediment-associated lipophilic endocrine disrupting compounds such as 4-NP. More specifically, this study set out to investigate whether compounds such as 4-NP could induce some of the effects (e.g., changed steroid moulting hormone, induction of vitellin-like protein, and morphometric abnormalities) recently found in wild male shore crabs (Lye et al., 2005). To this end, the study employed a number of morphological (e.g., gonadosomatic/ hepatosomatic index) and hormonal (ecdysone and hepatopancreatic vt levels) biomarkers.

MATERIALS AND METHODS Chemicals and Test Solution 4-n-nonylphenol (4-NP, [99.9%) was supplied by Lancaster Chemicals, (UK). Unless otherwise stated all other chemicals were of molecular analytical grade and were obtained from Sigma-Aldrich Chemical Company Ltd (Dorset, UK). Solvents were of glass distilled or high-performance liquid chromatography (HPLC) grade. Stock solution of 4-NP was prepared in acetone at 1 mg mL21 with working solutions prepared every second day by dilution with acetone. Control treatment contained seawater and acetone at 10–100 lL L21.

Analytical Methods

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lg L21 4-NP with and without shore crabs (4 crabs in 25 L). At t 5 0, 24, and 48 h, 3 3 500 mL of each test solution was subjected to SPE to isolate 4-NP. 4-tert-octylphenol (4-OP: Fluka, UK, [90%) and 2,4,6-tribromophenol (TBP) were used as surrogate (Ss) and internal standards (Is), respectively. Seven ions, m/z 107, 135, 141, 206, 220, 330, and 332 were monitored with the selected ions m/z 107, 135, and 332 corresponding to 4-NP, 4-OP, and TBP, respectively. Concentrations were determined by integrating the peak areas in the m/z 107 (4-NP), 135 (4-OP), and 332 (TBP) mass chromatograms and rationing the analyte peak areas against the Is, using a response factor from the Ss. Procedural blanks were run for each sample period and a mean extraction recovery (% standard added/% sample recovered) of 93.7% 6 3.2% was determined for this method.

Test Organisms and Chronic Laboratory Exposure Intermoult male shore crabs, Carcinus maenas (n 5 110) collected from reference site at Lindisfarne Island, NE of England, where distributed between 13 glass aquaria after an acclimatization period of 7 days. Separate groups of 8 crabs with similar size ranges (carapace width 35–85 mm) in 50 L of aerated UV treated, filtered seawater, salinity 34–36 psu were exposed to technical grade 4-NP at concentrations of 10 and 100 lg L21. Each treatment and control was run in triplicate (except 100 lg L21 which was run in quadruple). Test solutions were changed every 2 days and all crabs were fed the muscle tissue of white fish to satiation every 2 days prior to water exchange. Tanks were maintained at 158C throughout the experiment. After 12 weeks, 1 mL of hemolymph was taken and hepatopancreas extract cleanly dissected from individual animals and immediately frozen in liquid nitrogen before being transferred to a 2808C freezer. Hemolymph samples were subsequently analyzed for ecdysone using radioimmunoassay (RIA) and hepatopancreas samples for vt using the vt enzyme linked immunoassay (ELISA) as described later.

Morphological Index

To determine the measured experimental concentrations of 4-NP, experimental solutions underwent solid phase extraction (SPE) using C18 Isolute trifunctional (nonendcapped) cartridges and GC-MS using a Hewlett Packard 5973 MSD fitted with a 30 m DB5MS column (0.25 lg coating with 0.25 mm internal diameter, J&W Scientific) adapted from the methods of Lye et al. (1999) and Billinghurst et al. (1998), see also Sanders et al. (2005). Test solutions of 4-NP was prepared in seawater to give nominal concentrations of 10 and 100 lg L21. 4-NPs persistence/adsorption in the tanks was determined by preparing extracts from tanks containing 100

For each crab the following measurements were made; body weight, carapace width, hepatopancreas weight, and gonad weight, and from these the gonadosomatic index (GSI) and the hepatosomatic index (HSI) were determined.

Identification and Quantification of Ecdysteroids Hemolymph samples were prepared and ecdysteroids were identified and quantified according to Lye et al. (2005). Hemolymph samples taken with a 1-mL syringe were


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prepared by adding 100% ethanol to precipitate protein. Samples were incubated at 48C for 18 h and then centrifuged at 2000g for 20 min at 48C. The supernatant was removed and evaporated to dryness. Samples were purified by SPE using Varian-Bond-Elut LRC-C18, 500 mg cartridges. The crude extract residue was re-suspended in 4 mL 10% (v/v) methanol, and applied to the cartridge. The C18Sep-Pak cartridges were sequentially eluted with 6 mL of 10% methanol, 6 mL 25% methanol, 10 mL 60% methanol, and 6 mL 100% methanol. The free ecdysteroids were eluted in the 60% (v/v) methanol fraction and collected in silanized glass vials. All RIA were carried out in triplicate and used ecdysone as standard, with bound and unbound [23, 24-3H] ecdysone (NEN) being separated by ammonium sulfate precipitation (Mendis et al., 1983). The antiserum employed was H-22 which was produced by immunization with ecdysone 22succinylthyroglobulin amide and shows greatest specificity toward the ecdysone nucleus (Warren and Gilbert, 1986). A typical 50% binding occurred at about 125 pg of 25 deoxyecdysone, 150 pg of 3-dehydroecdysone, 175 pg of ecdysone, and 600 pg of 20-hydroxyecdysone (Chang and O’Connor, 1979; E.S. Chang pers. comm.).

Determination of vt by Enzyme Linked Immunosorbent Assay An indirect well-established ELISA (Allen et al., 2002; Dr Shaw Bamber, RF-Rogaland Research, Randaberg, Norway, pers. comm.) was used to quantify vitellin-like proteins in Carcinus maenas hepatopancreas extracts. For each samples, 40 lg of total soluble protein in 100 lL of coating buffer (50 mM carbonate, pH 9.6) was added to three wells of a 96-well polystyrene micro plate (Elkay Laboratory products, UK), which was left to incubate for 2 h at room temperature. The wells were washed four times with Tween-20 phosphate buffered saline (TPBS) (10 mM sodium phosphate, pH 7.3, 0.05% Tween-20) and incubated for 1 h at 218C with 200 lL blocking buffer (10 mM sodium phosphate, pH 7.3, 1.75% powered milk). After washing with TPBS four times, antiserum to Carcinus maenas, diluted 1:3000 in blocking buffer was added to each well and incubated for 1 h at 378C within the plate reader incubator. The plate was covered throughout. Wells were subse-

quently rinsed four times with TPBS and incubated with 100 lL of PBS (10 mM sodium phosphate, pH 7.3), containing horseradish peroxidase conjugated antirabbit IgG (Sigma, A6154), for 1 h at 378C. Following a further five washes in TPBS, 100 lL of citrate buffer (0.1 M citrate buffer, 0.03% H2O2, pH 4.2) containing 0.5 mg mL21 of 2,20 azinobis[3-ethylbenzothiazo line-6-sulfonic acid] diammonium salt, was added to each well. After incubating for 15 min in darkness at room temperature, the absorbance was recorded at 405 nm using a microplate reader (Molecular Devices). A range of vitellin standards (2–100 ng) was added to each plate, in triplicate, to obtain a standard curve. From this, the amount of vitellin-like protein present in Carcinus maenas homogenates could be expressed as vt equivalents.

Statistical Analyses For the analysis, data for seawater and solvent controls were pooled. Statistical analyses were performed using Minitab Statistical Package (Zar, 1999). Statistial significance was accepted at P \ 0.05 for all comparisons. Intergroup differences were assessed using one-way analysis ANOVA (parametric, normalized data after checking for normality and homogeneity of variance with Bartlett’s test).

RESULTS Chemical Analyses Measured concentrations of freshly prepared test solutions in both treatments were either in close agreement with the nominal values or slightly higher (Table I). Measured concentrations after 48 h were 80% lower than initial levels when no shore crabs were present. A greater loss of 90.5% (after 24 h) and 96.1% (after 48 h) between initial and the final measurement of 4-NP was recorded when shore crabs were present. The effective concentrations were calculated by taking the geometric mean from all measured concentrations. This lead to an effective concentration of 15.7 lg L21 4-NP (14.7% of initial measured concentration) in the treatment with crabs and nominal concentration of 100 lg L21 4-NP over the whole exposure duration. It was

TABLE I. Nominal and measured concentrations of 4-NP in test solution over 48 h as determined by GC/MS Measured Nominal 100 lL21 100 lL21 1 shore crabs

0 (h)

24 (h)

48 (h)

Effective

107.3 6 6.3 (17.3%) 106.2 6 2.6 (16.2%)

– 9.5 6 0.7 (290.5%)

19.7 6 8.5 (280.2%) 3.8 6 1.6 (296.1%)

46.0 lL21 (242.8%) 15.7 lL21 (14.7%)

Concentrations (lL21) presented as mean values (n 5 3) with standard error. Percentage differences relative to nominal concentration are presented in brackets. Shore crabs present at 4 crabs per 25 L.


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Fig. 1. Gonadosomatic index (GSI; gonad weight [g]/body weight [g]) of Carcinus maenas following a 12-week exposure to nominal concentrations of 10 and 100 lg L21 4-NP.

assumed that a similar loss occurred at nominal 10 lg L21 4-NP, leading to an approximated effective concentration of about 1.5 lg L21 4-NP over the whole exposure duration.

Morphological Index Gonadosomatic Index and Hepatosomatic Index

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Mortality was restricted to two crabs, one from control and one from exposure treatment during the exposure period. No other effects of the condition of the animals were observed. As no significant difference could be detected between the solvent and water controls (ANOVA, P [ 0.05), the data for these treatments were pooled. Figure 1 compares the mean GSI from 10 and 100 lg L21 4-NP treated crabs to those of control male crabs. The results show a significant reduction by an average of 46.5% in gonad weight of male crabs exposed to 10 lg L21 and a significant reduction by an average of 36% in gonad weight of male crabs exposed to 100 lg L21 4-NP compared to control males (ANOVA, P \ 0.05). In the control group, the testes made up about 1.2% of the body weight, whereas in crabs treated with 10 and 100 lg L21 4-NP the equivalent values were 0.6% and 0.7%, respectively. As no significant difference could be detected between the solvent and water controls (ANOVA, P [ 0.05), the data for these treatments were pooled. Figure 2 compares the mean HSI from 10 and 100 lg L21 4-NP treated crabs to those of control male crabs. The results show a significant increase by 21% in liver weight of male crabs exposed to 10 lg L21 4-NP and a significant increase by 25% in liver weight of male crabs exposed to 100 lg L21 compared to control males (ANOVA, P \ 0.05).

Ecdysteroid Levels The amount of free ecdysteroid detected in the hemolymph samples by RIA with the H-22 antiserum are shown in

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Fig. 2. Hepatosomatic index (HSI; hepatopancreas weight [g]/body weight [g]) of Carcinus maenas following a 12week exposure to nominal concentrations of 10 and 100 lg L21 4-NP.

Figure 3. It was decided to analyze the free ecdysteroids collectively, as separating the different ecdysteroid compounds would have required all samples to be subjected to HPLC followed by RIA of multiple fractions. This would have significantly restricted the number of crabs that could efficiently be analyzed. The standard unlabeled ligand was ecdysone. Thus, RIA activity was expressed in ecdysone equivalents (ng mL21 hemolymph). In reality the change in ecdysone equivalents between treatments probably represents a change in a mixture of ecdysteroids including ecdysone and 20-hydroxy-ecdysone. As no significant difference could be detected between controls (Mann–Whitney ANOVA, P [ 0.05) the data for these tanks were pooled. The results in Figure 3 indicated that levels of ecdysone equivalents were significantly lower in crabs exposed to 100 lg L214-NP compared to those from controls (ANOVA, P \ 0.05). The levels of ecdysone

Fig. 3. Carcinus maenas. Quantification of free ecdysteroids extracted from hemolymph samples of males following a 12-week exposure to nominal concentrations of 10 and 100 lg L21 4-NP, determined by radioimmunoassay of extracts with H-22 antiserum. Values (mean 6 SE) are ecdysone equivalents (ng mL21) hemolymph.


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equivalents, although not statistically significant, tended to be higher in crabs exposed to 10 lg L21 4-NP compared to control. This pattern was further emphasized by a significant difference in levels between crabs exposed to 10 and 100 lg L214-NP, so that significantly higher levels were found in crabs exposed to low levels of 4-NP compared to those from the high 4-NP level treatment (Mann–Whitney, P \ 0.05).

Vitellin Analyses Hepatopancreas extracts from male crabs from all treatments was analyzed for the presence or absence of vitellinlike proteins using the ELISA as described earlier. Of all samples analyzed (n 5 102) none of the samples gave a positive indication of vt.

DISCUSSION Many anthropogenic compounds, most notably xeno-estrogens are known to have the potential to disrupt vertebrate endocrine systems. There have been few comparable studies of the effects of these compounds on marine invertebrates (Oehlmann and Schulte-Oehlmann, 2003). This study has provided evidence that some reproductive (e.g., GSI), morphological (e.g., HSI), and hormonal (ecdysone equivalent levels) parameters in male shore crab, Carcinus maenas are significantly modified by exposure to the xeno-estrogen. Studies focusing on morphological parameters such as GSI and HSI provide a crude indication of the potential effects of EDCs and are therefore useful in identifying any specific target areas (e.g., gonads, hepatopancreas) worthy of further investigation. In this study, the testis weight was reduced by an average of 46.5% in crabs exposed to an effective concentration of 1.5 lg L21 4-NP (e.g., nominal concentration of 10 lg L21 4-NP) and an average of 36% in crabs exposed to an effective concentration of 15.7 lg L21 4-NP (i.e., nominal concentration of 100 lg L21 4-NP) compared to controls (Fig. 1) and although no histological analyses accompanied this study there was evidence of possible testicular growth retardation in exposed males. There appear to be no comparative studies on GSI and HSI in crustaceans following exposure to EDCs in the literature. In contrast, numerous laboratory studies on fish have clearly demonstrated reduced gonad weight following exposure to a range of EDCs including several alkylphenols e.g., 4-tertpentylphenol (Gimeno et al., 1998a), octylphenol (Mills et al., 2001), and 4-NP (Jobling et al., 1996). Furthermore, in fish it has been shown that reduction in testis weight following estrogenic and antiandrogenic exposure in most instances is accompanied by abnormalities in testis histology and/or a reduction in sperm count potentially affecting

reproductive success (Jobling et al., 1998; Baatrup and Junge, 2001). It is also known that abnormalities of the reproductive tract of other aquatic crustaceans, i.e., the male amphipod Hyalella azteca including hermaphroditism, disruption of maturation of germ cells, and disturbed spermatozoa do take place following chronic estrogen exposure (Vandenbergh et al., 2003). Thus, this suggests that similar scenarios as seen in fish and other aquatic crustaceans may occur also in the shore crab, and that this group of organisms are sensitive to endocrine disrupting effects of estrogens. Although there is little literature on HSI in invertebrates, increases in HSI, as observed in male shore crabs exposed to effective concentrations of 1.5–15.7 lg L21 4-NP (Fig. 2), have previously been reported for several fish species exposed to xeno-estrogens. Vtg, the major precursor of the egg yolk proteins vt are normally synthesized in the liver (or equivalent organs) of mature females in response to endogenous estrogens, such as 17b-estradiol, released into the blood stream and stored in developing oocytes. Thus, in fish, an increase in HSI normally occurs in females during vtg synthesis (Johnson et al., 1991) but can also be induced in male fish in response to exogenous estrogen treatment (Pait and Nelson, 2002). The observed increases in HSI of shore crabs exposed to 4-NP in this study are unlikely to be attributed to a shift in metabolism toward production of vtg as no vt was found in hepatopancreas (provided this is the organ where vtg is normally produced in shore crabs). Increases in liver weight (HSI) are also associated with enhanced detoxification activities in response to the presence of toxic compounds e.g., PAHs, PCBs etc. (Janssen, 1996). The acute toxicity (48 h LC50) of 4-NP to the larval stages of Carcinus maenas has been shown to vary between 45 and 100 lg L21 with the later larval stages generally less sensitive (Sanders, 2004). The finding of this study suggests, therefore, that although chemicals like alkylphenols may be toxic to crustaceans, it remains to be determined whether such effects are mediated via the endocrine system. Exposure to 4-NP in this study was conducted by static renewal. Under these conditions, the concentrations of 4NP declined by 90% over 24 h. Similar time dependant losses (60–86.4%) have previously been attributed to sorption of 4-NP to test containers, algae or Artemia (present as food) or the test subjects (Daphnia magna and glass prawn Palaemon elegans) themselves (Comber et al., 1993; Sanders et al., 2005). In the current study, the concentrations of 4-NP declined by 80% over 48 h in the absence of biological material. In the presence of biological material losses were greater with concentrations declining to 4% of nominal. The same tendency has been reported by Sanders et al. (2005) who demonstrated a loss of 86.4% of 4-NP in test media containing live Artemia sp., compared with only 17.8% in the absence of Artemia sp. over 24 h. Constant exposure levels were not maintained in this study. It is nevertheless possible to determine exposure


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concentrations over the whole duration of exposure time on the basis of effective concentrations (as calculated based on three different time points during 48 h exposure). In general, the effective concentrations provide a more accurate reflection of the exposure conditions than the nominal exposure concentrations. Thus, in this study the effective concentrations provide the basis for the concentration effect relationship. The presence of vtg in male plasma has been proposed as a powerful biomarker in evaluating estrogenic effects of various contaminants in animal, fish in particular (Sumpter and Jobling, 1995). Since the process of vitellogenesis in crustaceans is very similar to that in fishes (Pinder et al., 1999), there is the potential for crustaceans to be affected following exposure to environmental estrogens and likewise, the potential to adopt a biomarker approach for crustacean analogs to that for vertebrates. This study has demonstrated the absence of vt induction in mature male shore crabs by exposure to effective concentrations of 1.5–15.7 lg L21 4-NP, suggesting that 4-NP does not interfere with the process of vitellogenesis in mature shore crabs. The negative response were corroborated by Allen et al. (2002) who investigated the impact of 4-NP (100 lg L21, 21 days) and DES on the hemolymph concentration of vtg in mature female and male shore crabs (Carcinus maenas) using the same ELISA assay as used in this study and found no obvious pattern of effect (Allen et al., 2002). In contrast, the findings are not in agreement with a recent study clearly showing the presence of vt in hepatopancreas of male shore crabs collected from the Tyne and Tees estuaries (NE of England), where effects of exposure to estrogenic contamination have been observed in fishes (Lye et al., 2005). Neither does the finding corroborate recent preliminary observations showing the expression of vtg mRNA in mature male edible crabs (Cancer cancer) from polluted waters near Scarborough, East of England (pers. comm. Dr Elaine Sefton, Liverpool University, School of Biological Sciences, Liverpool, UK). There are several plausible explanations to the discrepancy in results. One could be that the timing of estrogenic exposure is critical to the adverse effects of estrogenic compounds in crustacean. In fish, it is well known that the period of sexual differentiation and likewise of sexual maturation are the stages in the reproductive physiology where the endocrine system plays a key role in regulating essential physiological and morphological processes, and are particularly sensitive to endocrine disruption (Gimeno et al., 1998a; Nash et al., 2004). For example while exposure of adult male carp to the alkylphenol 4-tert-pentylphenol (100, 320, and 1000 lg L21) for 3 months did not result in intersex fish, the same exposure of young male carp resulted in severely inhibited spermatogenesis of the testes (Gimeno et al., 1998b), suggesting a similar situation may be valid also in crustaceans. Indeed, evidence show that exposing Elminius modestus barnacle larvae within

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the first 4 days of development to 1 lg L21 4-NP had no effect on settlement success (Billinghurst et al., 2000) whereas a single exposure for 24 h to 1 lg L21 4-NP during the 3 days prior to the naupliar-cyprid moult did. Similar critical windows have been reported for the oyster Crassostrea gigas, where exposure to 4-NP at 1 lg L21, 7–8 days post fertilization significantly affected growth, survival, sexual differentiation (Nice et al., 2000, 2003) whereas these parameters were unaffected following exposure to 4-NP at 1 and 100 lg L21 23 day post fertilization (Nice et al., 2001). In this and the study of Allen et al. (2002) shore crabs were exposed to 4-NP after sexual maturation. Conversely, the shore crabs from UK estuaries showing vt in their hepatopancreas (Lye et al., 2005), were likely to have been exposed to EDCs at early stages of male development perhaps in response to a long-term exposure to chemical(s) during the whole life cycle of males. This could explain the discrepancy in results and further suggests that the vt phenomenon is induced at early stages of male development. Although it is clear, therefore, that the shore crab do not respond to estrogenic exposure in a similar way to fish, whether vt induction occurs in response to xeno-estrogens at certain more susceptible developmental stages, or indeed in response to other EDCs (in particular androgens and antiandrogens) present in the environment remains to be investigated. While it is unclear whether Crustacea are susceptible to vertebrate steroids it is clear that there are similarities between estrogens and ecdysteroid in that they bind to nuclear receptor (Chung et al., 1998). This has supported the suggestion that estrogenic chemicals have the ability to impact on moulting in Crustacean by interacting with the nuclear ecdysteroid receptor (EcR) (Zou and Fingerman, 1997a,b). More recent evidence from an ecdysone receptor assay has indicated that neither natural nor synthetic estrogens/androgens bind to the EcR (Pounds et al., 2002). However, certain xeno-estrogens such as bisphenol-A, diethylphthalate, lindane, and 4-NP have been shown to be weak EcR antagonists via the same in vitro assay, which imply a different mechanistic action to other EDCs, possibly via interaction with the EcR ligand-binding site (Dinan, 2001). The present study provide evidence on the effects of 4-NP on the whole animal level taking into account the scope for metabolism of xenobiotic compounds within an exposed animal. The results show a significant reduction in ecdysteroid levels in male shore crabs exposed to effective concentrations of 15.7 lg L21 4-NP (Fig. 3). The findings therefore suggest that the antagonistic activity reported in vitro at relatively high concentrations are supported by antagonistic interactions also in vivo. The mechanistic action behind this effect is currently not known and further work is needed to understand adequately the biochemical and physiological factors, which influence this effect. However, it was noted that at


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the lowest concentration (effective concentration of 1.5 lg L21 4-NP) there was no decline in ecdysteroid levels but an (although not significant statistically) increase. This does not give a sigmoidal dose-response curve as might be expected when a population of organisms is exposed to sublethal stressors rather the dose response appears to be an inverted U-shape curve (or so-called hormesis effect). Nonmonotonic curves such as this is often reported in the study of endocrine disruption (Jobling et al., 2003) and tend to occur when a secondary mechanism of action is suspected that is mediating an otherwise normal dose-response. It must be stressed however, that there are several arguments why this may not be a real U-shape curve. This include the fact that the increase of ecdysteroid level at effective 1.5 lg L21 4-NP was not statistically significant compared to control and thus, could be within the natural variability of this endpoint in these animals, and further that it was observed at one single concentration. The ecological relevance of the current findings is dependent on the environmental levels of 4-NP. Alkylphenols such as 4-NP, are generally detected at concentrations below 10 lg L21 4-NP in sewage effluents and below 1 lg L21 in surface waters (see Introduction). The calculated effective concentration (i.e., 15.7 lg L21 4-NP) at nominal exposure concentration of 100 lg L21 4-NP is close to the environmental levels. In addition, for most of the endpoints investigated in this study, there was no significant difference in effects between the low and high exposure treatments. Thus, assuming (as stated in methodology) that a similar loss occurred at nominal 10 lg L21 4-NP as nominal 100 lg L21 4-NP, this suggests that crabs were experiencing effects at effective concentrations as low as 1.5 lg L21 4-NP. Moreover, it is well known that most of the estrogenic activity identified in UK estuaries is strongly adsorbed on sediment particles (Allen et al., 2002). Thus, although the role of estuarine sediments in mediating the fate and bioavailability of environmental xeno-estrogens such as 4-NP remains a key area of uncertainty in the assessment of risk (Langston et al., 2005) it is clear that the accumulative effect of estrogens such as 4-NP in the sediment is nevertheless likely to represent a risk to sedimentbottom-dwellers such as the shore crab. Furthermore, while the bioconcentration factor (BCF) for 4-NP is not known for C. maenas a BCF of 90–100 has been obtained from common shrimp, Crangon Crangon (Ekelund et al., 1990) suggesting that 4-NP has the potential to bioaccumulate in marine crustacean.

CONCLUSION This study has demonstrated significantly altered reproductive, morphological, and hormonal effects in C. maenas exposed to 4-NP including reduced testis weight, increased hepatopancreas weight, and reduced ecdysone levels. These

effects have the potential to impact on reproduction and fertility, and on growth, and moulting, respectively. However, several pressing questions remain unanswered. For example, whether or not the measured changes are actually translated into impaired reproduction and/or growth and development in shore crabs requires investigation. Also, the possible effects of 4-NP on female fertility should be examined to establish whether effects found in this study were antiandrogenic (e.g., decreased testes weight) and/or estrogenic (hypothesis: increase in ovarian weight). Further pressing studies include long-term exposure to environmentally realistic concentrations of 4-NP involving all life stages, a study using 4-NP spiked sediment, and a study establishing the BCF of C. maenas. Finally, as there is a growing body of evidence that other endocrine disrupting compounds are present in the aquatic environment (for example the extensive presence of antiandrogenic chemicals derived from sewage effluents across the UK, Dr Melanie Gross-Sorokin, Environment Agency, UK), the impact of other ED compounds on marine crustaceans also warrants further investigation. The authors gratefully acknowledge Professor H. Rees, School of Biological Sciences, University of Liverpool and Dr Martin Jones, Fossil Fuels & Geochemistry, University of Newcastle, for the generous use of analytical equipment and other research facilities, Dr Shaw Bamber (RF-Rogaland Research, Randaberg, Norway) for producing the vt-antiserum employed and for assistance with the vt-ELISA, Dr E.M. Sefton School of Biological Sciences, University of Liverpool for valuable technical assistance with the RIA and discussions on ecdysone, Dr W.E. Bollenbacher, University of North Carolina, Chapel Hill for producing the ecdysone antiserum employed, and Dr E.S. Chang, Bodega Marine Laboratory, Bodega Bay, California for providing it. The authors also thank Mr Ian Harrison and Mr Bernie Bowler for assistance with the analytical procedures. Thanks finally to the anonymous referees for constructive comments.

REFERENCES Ahel M, Giger W, Koch M. 1994. Behavior of alkylphenol polyethoxylate surfactants in the aquatic environment. I. Occurrence and transformation in sewage-treatment. Water Res 23:1131– 1142. Allen Y, Balaam J, Bamber S, Bates H, et al. 2002. Endocrine disruption in the marine environment (EDMAR). The Centre for Environment, Fisheries and Aquaculture Sciences CEFAS, Contract Report A1127, London, UK: EDMAR Secretariat, Department for Environment, Food and Rural Affairs (Defra). Ayaki T, Kawauchino Y, Nishimura C, Ishibashi H, Arizono K. 2005. Sexual disruption in the freshwater crab (Geothelphusa dehaani). Integr Comp Biol 45:39–42. Baldwin WS, LeBlanc GA. 1994. Identification of multiple steroid hydroxylases in Daphnia magna and their modulation by xenobiotics. Environ Toxicol Chem 13:1013–1021.


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Baldwin WS, Milam DL, LeBlanc GA. 1995. Physiological and biochemical perturbations in Daphnia magna following exposure to the model environmental estrogen diethylstilbestrol. Environ Toxicol Chem 14:945–952. Baldwin WS, Graham SE, Shea D, LeBlanc GA. 1997. Metabolic androgenization of female Daphnia magna by the xenoestrogen 4-nonylphenol. Environ Toxicol Chem 16:1905–1911. Baatrup E, Junge M. 2001. Antiandrogenic pesticides disrupt sexual characteristics in the adult male guppy (Poecilia reticulate). Environ Health Perspect 109:1063–1070. Billinghurst Z, Clare AS, Fileman T, McEvoy J, Readman J, Depledge MH. 1998. Inhibition of barnacle settlement by the environmental estrogen 4-nonlphenol and the natural estrogen 17b-estradiol. Mar Pollut Bull 36:833–839. Billinghurst Z, Clare AS, Matsumura K, Depledge MH. 2000. Induction of cypris major protein in barnacle larvae by exposure to 4-n-nonylphenol and 17b-estradiol. Aquat Toxicol 47:203–212. Blackburn MA, Waldock MJ. 1995. Concentrations of alkylphenols in rivers and estuaries in England and Wales. Water Res 29:1623–1629. Blackburn MA, Kirby SJ, Waldock MJ. 1999. Concentrations of alkylphenol polyethoxylates entering UK estuaries. Mar Pollut Bull 38:109–118. Breitholtz M, Bengtsson BE. 2001. Estrogens have no hormonal effect on the development and reproduction of the harpacticoid copepod Nitocra spinipes. Mar Pollut Bull 42:879–886. Brian JV. 2005. Interpopulation variability in the reproductive morphology of the shore crab (Carcinus maenas): Evidence of endocrine disruption in a marine crustacean? Mar Poll Bull 50:410–416. Brown RJ, Conradi M, Depledge M. 1999. Long-term exposure to 4-nonylphenol affects sexual differentiation and growth of the amphipod Corophium volutator (Pallas, 1766). Sci Tot Environ 233:77–88. Bryan GW, Langston WJ, Hummerstone LG, Burt GR. 1985. A guide to the assessment of heavy-metal contamination in estuaries using biological indicators. Mar Biol Ass UK Occ Publ 4:1–92. Chang ES, O’Connor JD. 1979. Arthropod molting hormones. In: Jaffe BM, Behrman HR, editors. Methods of Hormone Radioimmunoassay, 2nd ed. New York: Academic Press. pp797–814. Chung ACK, Durica DS, Hopkins PM. 1998. Tissue-specific patterns and steady-state concentrations of ecdysteroid receptor and retinoid-X-receptor mRNA during the molt cycle of the fiddler crab, Uca pugilator. Gen Comp Endocrinol 109:375–389. Clare AS, Bentley MG, Ladle RJ. 2003. Multilevel analyses of endocrine disruption in shore crabs form UK estuarine and coastal marine environments. Report to Department for Environment, Food and Rural Affairs (Defra), Contract No CDEP/84/5/309. London, UK: Defra. Comber MHI, Williams TD, Stewart KM. 1993. The effects of nonylphenol on Daphnia magna. Water Res 27:273–276. Dinan L, Bourne P, Whiting P, Dhadialla TS, Hutchinson TH. 2001. Screening of environmental contaminants for ecdysteroid agonist and antagonist activity using the Drosophila melanogaster B-II cell in vitro assay. Environ Toxicol Chem 20:2038–2046.

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status in juvenile male summer flounder (Paralichthys dentatus). Aquatic Toxicol 52:157–176. Oehlmann J, Schulte-Oehlmann U. 2003. Endocrine disruption in invertebrates. Pure Appl Chem 75:2207–2218. Olmstead AW, LeBlanc GA. 2000. Effects of endocrine-active chemicals on the development of sex characteristics of Daphnia magna. Environ Toxicol Chem 19:2107–2113. Pait AS, Nelson JO. 2002. Endocrine disruption in fish: An assessment of recent research and results. NOAA Tech Memo NOS NCCOS CCMA 149. Silver Spring, MD: National Oceanic Service, Centre for Coastal Monitoring and Assessment, p55. Panter GH, Thompson RS, Sumpter JP. 2000. Intermittent exposure of fish to estradiol. Environ Sci Technol 34:2756–2760. Pinder LCV, Pottinger TG, Billinghurst Z, Depledge MH. 1999. Endocrine function in aquatic invertebrates and evidence for disruption by environmental pollutants, Technical Report E67. Swindon, UK: UK Environment Agency. Pounds NA, Hutchinson TH, Williams TD, Whiting P, Dinan L. 2002. Assessment of putative endocrine disrupters in an in vivo crustacean assay and an in vitro insect assay. Mar Environ Res 54:709–713. Sanders MB. 2004. Biomarkers of xeno-estrogen exposure in decapod crustaceans. PhD Thesis, Plymouth University, Plymouth, UK. Sanders MB, Billinghurst Z, Depledge MH, Clare AS. 2005. Larval development and vitellin-like protein expression in Palaemon elegans larvae following xeno-estrogen exposure. Integr Comp Biol 45:51–60. Sumpter JP, Jobling S. 1995. Vitellogenesis as a biomarker for estrogenic contamination of the aquatic environment. Environ Health Perspec 103:173–178. Vandenbergh GF, Adriaens D, Verslycke T, Janssen CR. 2003. Effects pf 17a-ethinylestradiol on sexual development of the amphipod Hyalella azteca. Ecotoxicol Environ Saf 54:216–222. Warren JT, Gilbert LI. 1986. Ecdysone metabolism and distribution during the pupal-adult development of Manduca sexta. Insect Biochem 16:65–82. Zar JH. 1999. Biostatistical Analysis, 4th ed. Upper Saddle River, NJ: Prentice-Hall. Zou E, Fingerman M. 1997a. Synthetic estrogenic agents do not interfere with sex differentiation but do inhibit molting of the cladoceran Daphnia magna. Bull Environ Contam Toxicol 58:596–602. Zou EM, Fingerman M. 1997b. Effects of estrogenic xenobiotics on molting of the water flea, Daphnia magna. Ecotox Environ Saf 38:281–285.


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AQ1: Kindly provide the location of Company NEN (city/state if within the US, and city/country if outside the US). AQ2: Please note that the year in reference ‘‘Jobling et al 2004’’ has been changed to ‘‘2003’’ in accordance with that given in the reference list. OK? AQ3: Journal style is to generally list out all authors. Kindly replace ‘‘et al.’’ in this reference with the missing author names. AQ4: Kindly provide the journal title for this reference. AQ5: This reference is not cited anywhere in the text. Kindly insert its citation at an appropriate place or delete it from the reference list. AQ6: This reference is not cited anywhere in the text. Kindly insert its citation at an appropriate place or delete it from the reference list.

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Date: 8/10/07

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