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Marine Environmental Research 92 (2013) 79e86

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Marine Environmental Research

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Vitellogenin as biomarker for estrogenicity in flounder Platichthysflesus in the field and exposed to 17a-ethinylestradiol via food andwater in the laboratory

Louise Leonharder Madsen, Bodil Korsgaard, Knud Ladegaard Pedersen,Lisette Bachmann Bjerregaard, Thomas Aagaard, Poul Bjerregaard*

Institute of Biology, University of Southern Denmark, Odense, Campusvej 55, DK-5230 Odense M, Denmark

a r t i c l e i n f o

Article history:Received 9 July 2013Received in revised form4 September 2013Accepted 9 September 2013

Keywords:FlounderPlatichthys flesusAccumulationVitellogeninDietary exposureEstrogenic effect17a-Ethinylestradiol

* Corresponding author. Tel.: þ45 65502456; fax: þE-mail address: poul@biology.sdu.dk (P. Bjerregaa

0141-1136/$ e see front matter � 2013 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.marenvres.2013.09.002

a b s t r a c t

The ability of 17a-ethinylestradiol (EE2) to elevate vitellogenin levels were investigated in male flounderPlatichthys flesus and vitellogenin concentrations in flounders from the Danish coastal environment weredetermined. Male flounders were exposed to 17a-ethinylestradiol (EE2) via food or water. Averagevitellogenin concentrations in the control fish ranged between 25 and 100 ng mL�1. Exposure to 5.1, 8.1and 16.8 ng EE2 L�1 in water and 500 and 5000 ng EE2 kg�1 body weight (bw) every second day in thefood increased the plasma vitellogenin concentration in a concentration and time dependent manner,whereas exposure to 2.7 ng EE2 L�1 in water for 21 d and 5 and 50 ng EE2 kg�1 bw for 12 days in the fooddid not. EE2 could be detected in liver and testes (but not in muscle) after exposure to 8.1 and 16.8 ng EE2L�1 in the water and 5000 ng EE2 kg�1 bw in the food; the highest concentration was 6 ng g�1 wetweight in liver. The majority of the male flounders collected from nine coastal Danish sites from 1999 to2004 had vitellogenin concentrations below 100 ng mL�1, and only at two sites moderate estrogenicinputs were indicated.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Wild populations of freshwater and marine fish have beenshown to exhibit signs of endocrine disruption in the form ofelevated vitellogenin levels, altered sex steroid levels, skewed sexratios and abnormal gonad development (reviewed by Desforgeset al., 2010; Jobling and Tyler, 2003; Matthiessen, 2003; Sumpter,2005).

In the marine and estuarine environment, especially the floun-der Platichthys flesus has been used as a biomonitoring organism insuch studies (Allen et al., 1999a,b; Kleinkauf et al., 2004; Lye et al.,1997, 1998, 1999; Matthiessen et al., 2002; Matthiessen, 2003;Matthiessen and Law, 2002; Scott et al., 2006; Vethaak et al., 2002,2005) and the synthesis of the egg yolk protein (vitellogenin) injuvenile and male fish has been used as one of the most wellestablished endpoints for demonstration of estrogenic effects ofenvironmental chemicals (Harries et al., 1997; Sumpter and Jobling,1995).

45 6550 2786.rd).

ll rights reserved.

Estrogenic constituents in effluents from sewage treatmentplants are the main causative agents responsible for the inductionof vitellogenin synthesis in male and juvenile fish (Desbrow et al.,1998). The compounds causing the majority of the estrogenic ef-fects have been identified as the natural (17b-estradiol [E2] andestrone [E1]) and synthetic hormones (EE2) (Desbrow et al., 1998;Larsson et al., 1999). EE2 is the major component of oral contra-ceptives and is excreted mainly as glucoronide conjugates in theurine of women. When released into water these EE2 glucoronideconjugates are unconjugated and the EE2 enters the aquatic envi-ronment via untreated sewage and the effluent from sewagetreatment plants (Lopez de Alda and Barcelo, 2001). EE2 has beendetected in concentrations up to 15 ng L�1 in effluents from sewagetreatment plants and up to 5.1 ng L�1 in surface water (Kuch andBallschmiter, 2001; Langston et al., 2005; Ternes, 1998; Terneset al., 1999). EE2 is a more potent inducer of vitellogenin synthe-sis in male and juvenile fish than either E2 and E1 (Bjerregaardet al., 2008; Holbech et al., 2006; Thorpe et al., 2003) and thehalf-life for EE2 in the aquatic environment is longer than that of E2(Robinson and Hellou, 2009). EE2 will thus have a risk of exertingestrogenic effects in the coastal environment. Exposure to 14.5 ngEE2 L�1 for three weeks induced vitellogenin synthesis in male

L.L. Madsen et al. / Marine Environmental Research 92 (2013) 79e8680

flounders whereas exposure to a nominal concentration of 1 ng EE2L�1 did not (Allen et al., 1999b).

The most important exposure route for EE2 in fish is probablyfrom water. However, EE2 has been shown to accumulate in sedi-ments (de Alda and Barcelo, 2001) and with the benthic feedinghabits of the flounder, uptake of EE2 from food might also add tothe estrogenic effect as we have demonstrated in experiments with4-tert-octylphenol (Madsen et al., 2003, 2006).

In laboratory experiments, EE2 has been shown to accumulatein rainbow trout (Oncorhyncos mykiss) upon water exposure(Skillman et al., 2006) and EE2 has been detected in redhorsesuckers (Moxostoma macrolepidotum) living downstream dis-charges from waste water treatment plants (Al-Ansari et al., 2010),but EE2 uptake in flounders has never been investigated.

Elevated levels of plasma vitellogenin in male flounders werefound at many sites in English estuaries and coastal marine areas(Allen et al., 1999a,b) and also in a similar Japanese study of vitel-logenin in male flounders Pleuronectes yokohamae collected inTokyo Bay (Hashimoto et al., 2000). In a large-scale field study inThe Netherlands, the vitellogenin levels of P. flesuswere lowatmostsites except at two sites where moderately elevated vitellogenin

Fig. 1. Map showing the sampling locations, names of loc

levels were found. These stations were situated in the same in-dustrial harbour zone also receiving effluent from sewage treat-ment works (Vethaak et al., 2002). Danish waste water treatmentplants generally quite efficiently remove estrogens from the wastewater (Stuer-Lauridsen et al., 2005) but the potential occurrence ofestrogenic effects in the Danish coastal environment has not beeninvestigated.

The aim of this study was to produce dose-relationships for theestrogenic effect of EE2 administered to male flounders in food andwater, to assess uptake of EE2 and to carry out a preliminaryinvestigation on the presence of estrogenic effect in the Danishmarine environment.

2. Materials and methods

2.1. Laboratory experiments

2.1.1. Experimental animals and experimental setupSexually mature male flounders P. flesus were caught in

November at Gabet, Denmark (Fig. 1); fifty fish were used for boththe feeding experiment (421.6 � 10 g; mean � SEM) and the water

ations and sampling times in the field investigations.

L.L. Madsen et al. / Marine Environmental Research 92 (2013) 79e86 81

exposure experiment (357.3 � 8.5 g; mean � SEM). The fish weretransported to the laboratory (Marine Biological ResearchCentre, Kerteminde) and acclimated for a week, during whichthey were not fed. Fish were held in a 12:12 h lightedarkphotoperiod and kept in 5 large fibre glass tanks (470e960 L)with aerated running seawater pumped in directly from theKerteminde Fjord into the tanks via PVC pipes at a flow rate of205 L per day. The sewage outlet closest to the Marine BiologicalResearch Centre is approximately 6 km away. The temperature inthe tanks followed the temperature of the seawater at Kerte-minde (8e10 �C).

Prior to experiments, fish were weighed and individuallymarked with an ink gun. The fish in each of the two experimentswere divided into 5 groups of 10 fish, and distributed to the avail-able tanks. The control group of each experiment was kept in a470 L tank, whereas the four exposure groups were kept in 960 Ltanks.

2.1.2. Feeding experimentIn a feeding experiment, the flounders were fed a food-paste

consisting of crushed commercial trout food (BioMar A/S, Brande,Denmark). The trout food was crushed in a mortar and mixed witha seawater volume in the proportion 2:3. Acetone (control) ordifferent concentrations of EE2 (SigmaeAldrich, Denmark) dis-solved in acetone (1 mL 100 mL�1 food) were added to the foodhomogenate under rapid stirring. The amount of food-pasteadministered to each fish was assigned according to body weight(bw) for the individual fish to receive the nominal doses of 5, 50,500 and 5000 ng EE2 kg�1 bw. The fish were fed by direct gavageinto the stomach every second day for a period of 12 days (day 0, 2,4, 6, 8, 10, 12) by means of a silicone tube attached to a 5 mldisposable syringe. A sample of water (1 L) for EE2 determinationwas taken from each tank at day 0, 6 and 13. Blood samples forvitellogenin measurements were taken on day 0, 6 and finally onday 13 prior to sampling of the fish.

2.1.3. Water exposure experimentFive groups of 10 male flounders were exposed to nominal

concentrations of 0, 2.5, 5, 10 and 25 ng L�1 EE2 for 21 days in acontinuous flow through system. The fish were not fed during theexperiment. Peristaltic pumps (Ole Dich Instrument Makers,Copenhagen, Denmark) supplied stock solution to each tank. TheEE2 was dissolved in 100% isopropanol and applied directly to thetanks at a rate of 72 mL day�1; isopropanol was also added to thecontrol water. The addition of EE2 to the tanks were initiated 10days before the addition of the fish and water samples werecollected for analyses of actual EE2 concentrations prior to theexperiment (10 and 6 days before) and throughout the experimentat days 0, 3, 4, 8, 11, 14 and 20. Blood was collected from all fish atonset of the experiment and at day 7 and 14 during the experimentand then finally at day 21 when all the fish were taken out foranalyses.

2.1.4. Sampling proceduresAt the end of the experiments fish were weighed, measured

(total length) and blood was collected from the caudal artery intoheparinized eppendorf tubes. Blood was centrifuged at10,000 rpm for 4 min and aliquots of plasma were storedat �80 �C until vitellogenin analyses. After the fish were eutha-nized by decapitation, the liver, testes and a piece of muscle werequickly excised, weighed and frozen in liquid nitrogen. The sam-ples were stored at �80 �C until analysis for EE2. Liver and testisweights were used to express their size as a percentage of totalbody mass (hepatosomatic index (HSI) and gonadosomatic index(GSI)).

2.2. Field investigations

Male flounders were caught at the 6 different locations indi-cated in Fig. 1 between January 1999 and November 2000. Fiveadditional samplings at the station Gabet from 2000, 2001, 2002,2003 and 2004 were also included (blood samples taken from fishused in experiments). Finally, two samplings from two additionalstations in 2002 were also included (Seden Strand and Odense ydreFjord).

The flounders were transported to the laboratory and wereeither sampled the same day or left over night in large indoor tankscontaining 250 L of aerated, recirculated seawater at 9 �C andsampled the following day as described in Section 2.1.4.

2.3. Chemical and biochemical analyses

2.3.1. Tissue and water extractionSamples of frozen liver, muscle or testis tissue were placed in a

mortar filled with liquid nitrogen and crushed. Aliquots (ca. 0.2 g)were allowed to partly thaw and transferred to extraction glass-ware and internal standard was added (10 mL of a 10 mg mL�1 so-lution of 17a-ethinylestradiol-2,4,16,16-d4 (EE2-d4) (CDN Isotopes,Quebec, Canada)).

The tissue was digested with 1 mL 1.0 M NaOH and the sampleswere left at room temperature overnight. The following day thesamples were sonicated for 5 s and EE2/EE2-d4 was extracted twicewith 5 mL of dichloromethane. After each of the two extractions,the dichloromethane phases were removed and then combinedprior to evaporation to dryness (TurpoVap). The samples wereredissolved in 4 mL of methanol:hexane (1:20) and applied to Sep-Pak CN 500-mg cartridges, previously conditioned with 6 mL ofmethanol and 6 mL of methanol:hexane (1:20). Before desorption,the cartridges were washed with 6 mL of hexane and after drying,the samples were desorbed by 4 mL ethylacetat:hexan (2:3).Finally, the extract was evaporated to dryness (TurpoVap) andredissolved in 300 mL of 50% methanol and 0.2% HCOOH in MilliQwater and used for the analysis of EE2.

Samples for recovery and analytical precision (inter/intra assayvariability) were spiked with EE2 and then treated as normalsamples. Depending on the amount of tissue available, the detec-tion limit was approximately 0.2 ng EE2 g�1 wet weight. Some ofthe samples from the experiments were used in the development ofthe method and the final analysis was carried out on tissues frombetween 5 and 10 fish in each group.

Water samples were extracted by the method described by Roseet al. (2002).

2.3.2. LC-MS-MS and LC-MS analysisHigh-performance liquid chromatographyetandem mass spec-

trometry (a 1200 Series HPLC and a 6410 Triple Quad LC/MS, bothAgilent Technologies) was used to analyse for EE2 in tissues, usingEE2-d4 as internal standard. Conditions were as follows: Column:C18 150 � 2.00 mm, 5 mm. Luna (Phenomenex). Column tempera-ture: 20 �C. Eluents: A: 0.2% formic acid in H2O and B: 0.2% formicacid in methanol. Flow: 0.4 mL min�1. Stoptime: 13 min. Posttime:7 min. Injection: 30 mL. Mode: APCI, positive. Selected ion moni-toring (SIM) was performed in positive mode for the ions m/z 279(EE2) andm/z 283 (EE2-d4). Samples were analysed at a fragmentorvoltage of 70 V. Drying gas flow: 5.0 L min�1. Nebulizer pressure:30 psig. Drying gas temperature: 350 �C. Vaporizer temperature:475 �C. Capillary Voltage: 2500 V. Corona Current: 5 mA.

In water samples, EE2 was quantified with E2 as internal stan-dard on an A HewlettePackard LC-MSD system consisting of a se-ries 1100 HPLC (solvent degasser, binary pump, autosampler,thermostatted column department) and a G1946AMSD quadrupole

-10 -5 0 5 10 15 20

Time (days)

0

5

10

15

20

25

30

EE

2 co

ncen

tratio

n (n

g l-1

)

L.L. Madsen et al. / Marine Environmental Research 92 (2013) 79e8682

mass spectrometer equipped with an atmospheric pressurechemical ionisation (APCI) interface was used for separation andquantification of EE2 and E2 as described by Rose et al. (2002).

2.3.3. VitellogeninA direct non-competitive sandwich ELISA using polyclonal rab-

bit antibodies raised against flounder vitellogenin was used tomeasure plasma vitellogenin concentration as described forrainbow trout by (Christiansen et al., 2000), with the modificationsthat in flounder ELISA Dextran-peroxidase (Dex-HRP, AMDEX A/S,Copenhagen, Denmark) conjugated antibodies were used instead ofthe horseradish peroxidase conjugated antibodies and Dextran-HRP conjugation was performed as described for zebra fish byHolbech et al. (2001). The detection limit for the purified standardvitellogenin by the ELISA method was 0.2 ng mL�1. Plasma sampleswere diluted a minimum of 100-fold, and the detection limit was20 ng vitellogenin mL�1.

Fig. 2. Concentrations of ethinylestradiol in the tanks of the water exposure experi-ment with nominal concentrations of 2.5 (;), 5 (A), 10 (:) and 25 (-) ng L�1. Thebroken vertical line indicates the start of the exposure at time 0, when the flounderswere added to the tanks. Each of the horizontal lines indicates the mean actual waterconcentration during the exposure period (2.7, 5.1, 8.1 and 16.8 ng L�1).

2.4. Data handling and statistical treatment

Datawere tested for normality and variance homogeneity and, ifnecessary, log10-transformed prior to analysis. If normality andvariance homogeneity could not be obtained, non-parametricmethods were used. In the laboratory experiments, two wayrepeated measures ANOVA followed by a Fisher LSD test were usedto test the effects of the treatment on the vitellogenin concentra-tion. The P value of 0.05 was corrected for the number of compar-isons. Values of vitellogenin lying below the detection limit wereset to 20 ng mL�1. In the laboratory experiments, a one way ANOVAfollowed by a Tukey test was used to test the effects of treatmentson HSI and GSI. In the field studies, KruskaleWallis ANOVA onRanks test were used to test if there were any significant differencein vitellogenin concentration and in the values of HSI/GSI. All sta-tistics were calculated using the software program SigmaStat 2.03with P < 0.05 as the level of significance.

0 7 14 21

Time (days)

10

102

103

104

105

106

107

108

Vitello

gen

in

(n

g m

l-1

)

*

*

*

*

0

2.7

5.1

8.1

16.8

*

*

*

*

Fig. 3. Vitellogenin concentrations in the blood plasma of male flounder P. flesus afterexposure to various concentrations of ethinylestradiol in the water (2.7, 5.1, 8.1and16.8 ng EE2 L�1). Mean � SEM for 9 or 10 fish. *: Significantly different from controls onthe respective day (P < 0.05).

3. Results

3.1. Laboratory experiments

3.1.1. EE2 in waterIn the water exposure experiment, the concentration of EE2 in

the tanks was measured from 10 days before the fish were addedand throughout the experiment (Fig. 2). The actual mean concen-tration of EE2 in the two lowest exposure groups of 2.7 and5.1 ng L�1 were very close to the nominal concentrations of 2.5 and5.0 ng L�1. In the two highest exposure groups with nominal con-centrations of 10 and 25 ng L�1, the actual average concentrationsduring the exposure of the flounders were 8.1 and 16.8 ng L�1

(Fig. 2). No EE2 was detected in the water containing control fish.In the feeding exposure experiment, EE2 could not be detected

in any of the water samples from day 0, 6 and 13.

3.1.2. Plasma vitellogeninIn the water exposure experiment mortality was low (2/50),

with no increase in mortality with increasing dose. There were nosignificant difference between plasma vitellogenin in control fishand fish exposed to 2.7 ng EE2 L�1 on both day 7, 14 and 21. Sta-tistically significant increases in plasma vitellogenin levels wereobserved in fish exposed to 8.1 and 16.8 ng EE2 L�1 on day 7 and infish exposed to 5.1e16.8 ng EE2 L�1 on both day 14 and 21 (Fig. 3).Three weeks of exposure to 5.1, 8.1 and 16.8 ng EE2 L�1 increasedthe concentration of vitellogenin from less than 100 ng mL�1 to0.01, 2.1 and 17 mg mL�1, respectively.

In the feeding experiment, 10 of the 50 fish died or were dis-carded due to apparent illness; mortality was not related to expo-sure dose. No regurgitation of food was observed. The plasmaconcentration of vitellogenin was significantly higher in floundersorally exposed to 500 and 5000 ng EE2 kg�1 bw both on day 6 and13, when compared to the control group (Fig. 4). The vitellogeninconcentration increased from less than 100 ng mL�1 to0.093mgmL�1 and 31.5 mgmL�1 in the groups exposed to 500 and5000 mg EE2 kg�1 bw, respectively. The flounders exposed to 5 and50 ng EE2 kg�1 bw had mean vitellogenin concentrations below100 ng mL�1 and there were no significant increases compared tothe control group.

3.1.3. Gonadosomatic and hepatosomatic indexThe GSI ranged from 0.4 to 6.5% in individual fish with a mean of

3.3 � 0.2% (mean � SEM) in groups of the feeding experiment and3.1 � 0.2% (mean � SEM) in the water exposure experiment (datanot shown). No significant difference in GSI was observed in any ofthe EE2 treated groups when compared with controls.

0 7 14

Time (days)

10

102

103

104

105

106

107

108

Vitello

gen

in

(n

g m

l-1

) *

*

*

*

Fig. 4. Vitellogenin concentrations in the blood plasma of male flounder P. flesus afteroral exposure to various doses of ethinylestradiol: 0 (B), 5 (:), 50 (A), 500 (;) and5000 (-) ng EE2 kg�1 body weight). Mean � SEM for 7 to 10 fish. *: Significantlydifferent from controls on the respective day (P < 0.05).

L.L. Madsen et al. / Marine Environmental Research 92 (2013) 79e86 83

The HSI (data not shown) ranged from 1.0 to 3.5% in individualfish with a mean of 1.8 � 0.1% (mean � SEM) for all groups in thewater exposure experiment. In the feeding experiment, the groupexposed to 500 ng EE2 kg�1 bw had a significantly higher HSI(2.6� 0.1%) than the controls (1.9� 0.2%); there were no significantincreases in any of the other exposure groups (5, 50 and 5000 ngEE2 kg�1 bw) in the feeding experiment.

3.1.4. EE2 in tissuesEE2 was detectable in testes and liver after exposure to the

highest dose in the food (Fig. 5A) and to the two highest concen-trations in the water (Fig. 5B), whereas no EE2 could be detected in

0 2.7 5.1 8.1 16.8

Exposure concentration (ng EE2 l-1

)

0

500

1000

1500

0

4000

8000

EE

2 in

tissu

es (

ng

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2 k

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0 5 50 500 5000

Exposure dose (ng EE2 kg-1

)

A

B

b.d.b.d.

b.d.

n.a.n.a.

b.d. b.d.

3/5 4/5

8/9 10/103/5 5/5

Fig. 5. EE2 concentrations (mean � SEM for 5e10 fish) in liver (hatched bars) andtestes (solid bars) of male flounders exposed to EE2 in food for 12 days (A) or water (B)for 21 days. b.d.: below detection limit. n.a.: not analysed. Numbers inside the barsindicate how many of the analysed fish that had detectable EE2 concentrations; fornon-detectable values the detection limit (0.2 ng g�1) was used.

muscle. Liver concentrations were generally 2e3 times higher thantestes concentrations (Fig. 5).

3.2. Field investigation

The male flounders collected from the stations A-G were similarin size (Table 1), whereas the fish from the two stations H and Iwere smaller, immature flounders (Table 1).

The majority (83.3%) of the flounders had plasma vitellogeninlevels below 100 ng mL�1 and at 3 sampling stations no individualfish exceeded this value (Table 1). Vitellogenin levels between 100and 1000 ng mL�1 were found in 12.1% of the flounders and 4.6%had vitellogenin levels above 1000 ng mL�1; the latter were allfound at 5 sampling stations/times (A, B and G). The highest valueof vitellogenin of 85.9 mg Vtg mL�1 measured in this investigationwas found in a single fish from station A. At station B in January1999 only 5% of the fish had plasma vitellogenin concentrations lessthan 100 ng mL�1. At station F, 100% of the fish had vitellogeninlevels below 100 ng mL�1.

At the station G (Table 1) there was a difference betweenvitellogenin levels in flounders sampled at different years. Thevitellogenin levels in year 2001 and 2004 were significantlydifferent from the levels in year 2000.

4. Discussion

NOEC and LOEC values for induction of vitellogenin afterexposure to EE2 in thewater were 2.7 and 5.1 ng L�1 and this makesthe flounder equally sensitive to the estrogenic effects of EE2 as anumber of other species tested. The values are also in accordancewith the LOEC of 14.5 ng L�1 found by (Allen et al., 1999b), as theirNOEC was 1 ng L�1 (nominal). For comparison, water exposure toEE2 induces vitellogenin in male zebrafish Danio rerio at 3.0 ng L�1

(8 days exposure) (Rose et al., 2002), in rainbow troutOncorhynchusmykiss and carp Cyprinus carpio at 10 ng L�1 (10 days exposure)(Purdom et al., 1994) and in male fathead minnow Pimephalespromelas at 1 ng L�1 (3 weeks exposure) (Jobling et al., 2003). NOECand LOEC for elevated plasma vitellogenin concentrations in browntrout Salmo trutta after 8 days’ exposure are 1.1 and 5.1 ng L�1,respectively, whereas the LOEC for elevated liver concentrations is1.1 ng L�1 (Bjerregaard et al., 2008).

Vitellogeninwas induced in flounders orally exposed to EE2, andthe lowest observed effect dose (LOED) and the no observed effectdose (NOED) were 500 ng kg�1 bw and 50 ng kg�1 bw after 14 daysof exposure, respectively. This indicates that EE2 is approximately10,000 times as potent as 4-tert-octylphenol, where the LOED in asimilar experiment was 5 mg kg�1 after 10 days of exposure(Madsen et al., 2003). This is in good accordance with the fact that4-tert-octylphenol is 1000 times less potent compared to E2(vitellogenin gene expression) and E2 is 11e27 times less potentthan EE2 (vitellogenin induction) (Thorpe et al., 2003; White et al.,1994).

The route of exposure affected the estrogenic response of 4-tert-nonylphenol in male fathead minnows P. promelas, where a 10-foldgreater sensitivity was found in fish exposed via the watercompared to fish exposed orally (Pickford et al., 2003). In rainbowtrout O. mykiss, propylparaben (Bjerregaard et al., 2003) andbutylparaben (Alslev et al., 2005) were, however, slightly morepotent when administered orally than dosed via the water.

EE2 could be detected in liver and gonads e but not in muscle e

at the two highest water exposure concentrations and the highestfood exposure dose. Rainbow trout O. mykiss rapidly accumulateEE2 in the plasma when exposed to EE2 in the water and equilib-rium is reached within 16 h resulting in concentration factors in theplasma between 480 and 720 (Skillman et al., 2006) and the plasma

Table 1Data for male flounder Platichthys flesus collected at different locations around Denmark during 1999e2004. Mean � SEM.

Station Area Time ofsampling

n Length (cm) Weight (g) Conditionfactor

GSI HSI Vitellogenin(ng mL�1)

Percentage of fish (%) in three groups of vitellogenin(ng mL�1) concentrations

Median <100 100e1000 >1000

A Kerteminde Jan 1999 49 33.1 � 0.8 420 � 28 1.14 � 0.03 3.21 � 0.22 1.45 � 0.06 64 80 7 13B Vejle Jan 1999 22 31.1 � 0.3 334 � 12 1.11 � 0.03 2.90 � 0.18 1.78 � 0.09 384 5 69 27

Sep 1999 37 a 312 � 13 a 0.54 � 0.16 2.50 � 0.19 35 68 6 27C Ringkøbing Nov 1999 49 a 403 � 18 a 3.70 � 0.25 1.69 � 0.06 51 82 18 0D Århus Oct 2000 13 31.1 � 1.0 329 � 24 1.07 � 0.02 0.88 � 0.12 1.81 � 0.11 78 69 31 0E Seden Strand Oct 2002 10 31.1 � 0.8 393 � 33 1.29 � 0.03 2.72 � 0.30 2.83 � 0.28 20 64 36 0F Odense Fjord Nov 2002 7 29.9 � 1.0 360 � 33 1.31 � 0.06 3.13 � 0.37 2.36 � 0.28 150 29 71 0G Gabet Nov 2000 55 b 337 � 09 b b b 20 96 2 2

Nov 2001 53 b 365 � 12 b b b 74 83 17 0Nov 2002 86 b 351 � 09 b b b 26 94 5 1Nov 2003 38 b 422 � 10 b b b 35 97 3 0Nov 2004 48 b 357 � 09 b b b 45 90 10 0

H Ålborg Sep 2000 35 19.3 � 0.4 75 � 5 1.00 � 0.01 0.33 � 0.07 1.48 � 0.05 24 100 0 0I Sebbersund Sep 2000 13 21.3 � 0.5 110 � 8 1.07 � 0.01 0.27 � 0.04 1.49 � 0.06 13 100 0 0

a Lacking data on length.b Only blood samples were taken immediately after catch, because the fish were used in experiments.

L.L. Madsen et al. / Marine Environmental Research 92 (2013) 79e8684

to liver partitioning is approximately one. In the present experi-ments, male flounders exposed to 8.1 and 16.8 ng L�1 concentratedEE2 in the liver with concentrations factors of 382 and 356,respectively, reaching concentration factors in a range only slightlylower than the rainbow trout (Skillman et al., 2006). Bile andplasma concentrations of free EE2 in the rainbow trout werecomparable but already after 16 h, concentrations of conjugatedEE2 was 4.5 times higher than the concentrations of free EE2(Skillman et al., 2006), indicating a rapid metabolisation; efficientelimination of EE2 was also observed in goldfish Carassius auratus(Al-Ansari et al., 2013).

The fish in the food exposure groupwere sacrificed one day afterthe last feeding and it cannot be excluded that the metabolism ofEE2 may proceed so rapidly (Al-Ansari et al., 2013; Skillman et al.,2006) that any EE2 accumulated in the liver and gonads in thelowest food exposure groups might have disappeared between thefeeding and the sampling time.

Al-Ansari et al. (2010) found 1.6� 0.6 and 1.43� 0.96 ng EE2 g�1

whole body wet weight, respectively, in male and female shortheadredhorse suckers M. macrolepidotum collected near a wastewatertreatment plant in the St. Clair River, Ontario, Canada. This showsthat it is possible to detect EE2 in fish caught in the wild, butalthough Al-Ansari et al.’s (2010) values are in the same range as thevalue for the highest of our exposures, direct comparison with thepresent results on flounders is hampered by the fact that we onlydetected EE2 in liver and gonads (and not in muscle) whereas Al-Ansari et al. (2010) used the whole fish in their analysis.

In flounders orally exposed to mg kg�1 BW quantitites of 4-tert-octylphenol, 4-tert-octylphenol accumulates in both liver, muscleand testes both after continued (Madsen et al., 2003) and singlepulse (Madsen et al., 2006) exposure, with the highest concentra-tions in liver followed by testes andmuscle. The lack of detection ofEE2 in muscle in the present experiment e contrasted with theresults on 4-tert-octylphenol e may be a simple question ofdetection limits for the 106 times lower doses of EE2 or a differenthandling of the synthetic steroid in the organism compared to thealkylphenol.

Vitellogenin levels were elevated in the groups exposed to5.1 ng L�1 EE2 in the water and 500 ng kg�1 BW in the food, butEE2 was still not detectable in the liver in any of these groups,showing that vitellogenin synthesis may be induced without anydetectable accumulation of EE2 in the target organ for vitellogeninsynthesis.

At the time of the experiments, the testes of the flounders werewell-developed and the GSI did not change in any of the two ex-periments. This is in accordance with the findings of (Allen et al.,1999b), where exposure to 14.5 ng EE2 L�1 for three weeks inP. flesus did not lead to any change in GSI.

In groups with a high plasma vitellogenin concentration, liverhypertrophy leading to increased HSI in fish from both the feedingand thewater exposure experiments couldbeexpected.However, theonly increase in HSI in the present investigation was seen in fish fed500 ng EE2 kg�1 BW. Flounders exposed to EE2 in the water did nothave increasedHSI,which is in contrast to thefindings by (Allen et al.,1999b) where HSI was increased after exposure to 14.5 ng EE2 L�1.

The pattern in plasma vitellogenin concentrations demon-strated among Danish male flounders collected in the coastal areasbetween 1999 and 2004 indicates that there is only a moderatedegree of contamination with estrogenic compounds aroundDenmark and there is no reason to believe that the impact shouldhave increased during the latest decade.

Plasma or liver vitellogenin levels in male or juvenile fish havebeen used as efficient biomarkers to detect oestrogenic contami-nation in both freshwater and marine areas (e.g. Bjerregaard et al.,2008; Harries et al., 1996, 1997; Kirby et al., 2004; Vethaak et al.,2005) and a number of studies (Harries et al., 1997; Kirby et al.,2004) have presented vitellogenin levels that are markedly andunambiguously elevated - maybe several orders of magnitude -compared to background levels. Less effort has been devoted todefining when less severe increases in plasma vitellogenin levelsactually indicate a difference relative to unaffected backgroundlevels. For most species investigated, the natural backgroundplasma vitellogenin level in male and juvenile fish seems to be inthe range from approximately 50e100 ng mL�1 (when given asmean or median for groups). The question, whether or not values inindividual fish exceeding 100 ng mL�1 indicate some kind of es-trogenic contamination is, however, not straight forward to answer,since our knowledge about the distribution of vitellogenin con-centrations in populations living in pristine areas is limited as is ourknowledge about seasonal variations.

At most investigated Danish sites the majority of the fish (60%e100%) had vitellogenin concentrations less than 100 ng vitellogeninmL�1, a value which probably does not indicate major estrogenicinput. However, at most stations some of the flounders had highervitellogenin levels; this is similar to the findings in male floundersfrom the open sea and estuaries of the Netherlands, where a similar

L.L. Madsen et al. / Marine Environmental Research 92 (2013) 79e86 85

wide variation in vitellogenin levels was found (Vethaak et al.,2005). The highest value of vitellogenin (85.9 mg vitellogeninmL�1) among Danish male flounders was measured in a single fishfrom station A, and this value is still fairly low compared to a meanvalue of 59 mg vitellogenin mL�1 of plasma in male flounders fromthe contaminated Mersey Estuary in United Kingdom (Allen et al.,1999b).

The moderate degree of estrogenic contamination of the Danishcoastal environment is in accordancewith the knowledge about thestate of the Danish waste water treatment facilities. The vast ma-jority of the Danish waste water treatment plants perform tertiarywaste water treatment, including nutrient removal, and this type oftreatment is known to be fairly efficient in the removal of estro-genic compounds from the wastewater (Ternes et al., 1999). Dis-charges of estrogenic activity to the freshwater environment fromDanish waste water treatment plants has been shown to be rela-tively low (Stuer-Lauridsen et al., 2005).

Although the experimental part of this work concerns the es-trogenic effects of EE2, we have no information about the exactnature of the compounds that exert the e albeit moderate e es-trogenic effects in the male flounders in the Danish coastal envi-ronment; also industrial chemicals with estrogenic properties (e.g.nonylphenol) have been detected in flounders and their sur-roundings in other parts of Europe (Lye et al., 1999; Thomas et al.,2001). In the UK, the reduction in vitellogenin levels in maleflounders at some of the investigated sites during the late 1990’swere attributede at least partlye to reductions in the discharges ofnonylphenols to the rivers and estuaries (e.g.Sheahan et al., 2002).In Denmark, nonylphenol is less likely to have had a major impactin the estuarine environment, since a voluntary agreement fromthe 1980’s between the Danish Environmental Protection Agencyand The Association of Danish Cosmetics, Toiletries, Soap andDetergent Industries to reduce as much as possible the use ofnonylphenolpolyethoxylates has resulted in a lower degree ofcontamination with these compounds in Denmark compared tomost other countries (TemaNord, 1996).

The mean HSI varies from 1.5 to 2.8% among the Danish maleflounders; this variability may be explained by the difference in thesampling times at the different stations; there is a tendency for theHSI to be lower in winter time. The seasonal variation is also thepossible explanation for the range of GSI (1.1e3.2%), with the lowestvalues in the autumn and the highest in the winter, implying thatthe male flounders were in different stages of spermatogenesis.

The collection of flounders in this investigation was carried outat different times of year, which is not optimal for field surveys. Inmale flounder P. yokohamae a seasonal variation in serum vitello-genin levels has been demonstrated, with the highest values in thespawning season, which is explained by endogenous, circulatingestrogen during this period (Hashimoto et al., 2000). Even if thecollection schedule in the present field survey cannot be consideredto be optimal, major estrogenic impacts in the Danish coastalenvironment comparable to the UK conditions of the early to mid1990s are not likely to have been missed if present, despite theshortcomings in the sampling schedule. As previously stated thepattern in plasma vitellogenin concentrations demonstratedamong Danish male flounders collected in the coastal areas duringdifferent times of the year between 1999 and 2004 indicates thatthere is only a moderate degree of contamination with estrogeniccompounds in Danish waters.

The moderate contamination with estrogenic activity in theDanish coastal environment indicated in the present investigationon flounders is in accordance with the presence of intersex e albeitat a low frequency e among the male eelpout Zoarces viviparus(Velasco-Santamaria et al., 2010; Velasco-Santamaría et al., 2013) inthe Danish populations.

Acknowledgement

This project was supported by grants from the Danish NaturalScience Research Council. The experiments were carried out underpermission from the Danish Experimental Animal Board under theDanish Ministry of Justice.

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