Developmental alterations and endocrine-disruptive responses in farmed Nile crocodiles (Crocodylus niloticus) exposed to contaminants from the Crocodile River, South Africa

Aquatic Toxicology 173 (2016) 83–93

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Developmental alterations and endocrine-disruptive responses in farmed Nile crocodiles (Crocodylus niloticus) exposed to contaminants from the Crocodile River, South Africa Augustine Arukwe a,∗ , Jan Myburgh b , Håkon A. Langberg a , Aina O. Adeogun c , Idunn Godal Braa a , Monika Moeder d , Daniel Schlenk e , Jordan Paul Crago e , Francesco Regoli f , Christo Botha b a

Department of Biology, Norwegian University of Science and Technology (NTNU), 7491 Trondheim, Norway Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort 0110, South Africa c Department of Zoology, University of Ibadan, Ibadan, Nigeria d Helmholtz center for Environmental Research UFZ, Department of Analytical Chemistry, Leipzig, Germany e Department of Environmental Sciences, University of California, Riverside, CA 92521, United States f Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche, Ancona, Italy b

a r t i c l e

i n f o

Article history: Received 26 June 2015 Received in revised form 26 December 2015 Accepted 31 December 2015 Available online 6 January 2016 Keywords: Endocrine-disruption Reproductive effects Crocodylus niloticus Environmental contaminants Biomarkers Crocodile River (Limpopo)

a b s t r a c t In the present study, the developmental (including fertility) and endocrine-disruptive effects in relation to chemical burden in male and female Nile crocodiles (Crocodylus niloticus), from a commercial crocodile farm in the Brits district, South Africa, exposed to various anthropogenic aquatic contaminants from the natural environment was investigated. Hepatic transcript levels for vitellogenin (Vtg), zona pellucida (ZP) and ER˛ (also in gonads) were analyzed using real-time PCR. Plasma estradiol-17␤ (E2), testosterone (T) and 11-ketotestosterone (11-KT) were analyzed using enzyme immunoassay. Gonadal aromatase and hepatic testosterone metabolism (6␤-hydroxylase (6␤-OHase)) were analyzed using biochemical methods. Overall, there is high and abnormal number (%) of infertile and banded eggs during the studied reproductive seasons, showing up to 57 and 34% of infertile eggs in the 2009/2010 and 2013/2014 seasons, respectively. In addition, the percentage of banded eggs ranged between 10 and 19% during the period of 2009–2014 seasons. While hepatic ER˛, Vtg, ZP mRNA and testosterone 6␤-OHase, were equally expressed in female and male crocodiles, gonadal ER˛ mRNA and aromatase activity were significantly higher in females compared to male crocodiles. On the other hand, plasma T and 11-KT levels were significantly higher in males, compared to female crocodiles. Principal component analysis (PCA) produced significant grouping that revealed correlative relationships between reproductive/endocrinedisruptive variables and liver contaminant burden, that further relates to measured contaminants in the natural environment. The overall results suggest that these captive pre-slaughter farm crocodiles exhibited responses to anthropogenic aquatic contaminants with potentially relevant consequences on key reproductive and endocrine pathways and these responses may be established as relevant species endocrine disruptor biomarkers of exposure and effects in this threatened species. © 2016 Elsevier B.V. All rights reserved.

1. Introduction The extent and severity by which anthropogenic chemicals in the environment produce adverse effects on the health of wildlife species, as well as humans and domestic animals through interactions with the endocrine system have received increased attention

∗ Corresponding author. Fax: +47 73591309. E-mail address: [email protected] (A. Arukwe). http://dx.doi.org/10.1016/j.aquatox.2015.12.027 0166-445X/© 2016 Elsevier B.V. All rights reserved.

since the 1990s (Bogi et al., 2003; Kavlock et al., 1996). While a vast majority of endocrine and reproductive effects studies have concentrated on fish species, there is limited number of such studies in reptiles. Despite the fact that wild Nile crocodile (Crocodylus niloticus) numbers have been decreasing significantly (Ashton, 2010; Combrink et al., 2011), with reports on body contaminant burden throughout tropical and subtropical areas worldwide (Campbell, 2003; Rainwater et al., 2007), endocrine disruptive studies are almost non-existent in this species and there is a need for information on whether environmental toxicants are contributing to

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their decline (Gibbons et al., 2000). Environmental contaminants, including organochlorine (OC) pesticides were shown to produce deleterious reproductive and developmental effects on American alligators (Alligator mississippiensis) (Milnes et al., 2005). In addition, Western Australian freshwater and estuarine crocodiles (Crocodylus johnstoni and Crocodylus porosus) (Yoshikane et al., 2006) and American crocodiles (Crocodylus acutus) from Costa Rica (Rainwater et al., 2007) were reported to have high tissue burdens of OC pesticides. Elsewhere, in Morelet’s crocodiles (Crocodylus moreletii) from northern Belize, OC pesticides in the eggs and tail scutes have been reported in these populations (Pepper et al., 2004; Wu et al., 2006). Bouwman et al. (2014) reported high concentrations of chlorinated, brominated and fluorinated organic pollutants in Nile crocodile eggs collected from the lower Olifants River, in the Kruger National Park, South Africa. Previous studies have reported high concentrations of metals and other environmental organic pollutants in selected tissues of C. niloticus (Sparling et al., 2010), but little is known about sublethal effects from exposure of C. niloticus to environmental contaminants through the analysis of endocrine-disruptor biomarkers directly related to reproductive alterations. Successful reproduction in oviparous animals requires accumulation of yolk materials into oocytes during oogenesis, and the mobilization of these proteins during embryogenesis is an integral process in oocyte assembly. Most oocyte yolk proteins and lipids are derived from the enzymatic cleavage of complex precursors, predominantly vitellogenin (Vtg) and very low-density lipoprotein (Tyler and Sumpter, 1996). Hepatic synthesis of Vtg (vitellogenesis) and eggshell zona pellucida proteins (ZP: zonagenesis) are estrogen-induced maturation processes through the estrogen receptor-␣ (ER␣). Toxicologists have used the induction of Vtg and ZP proteins in male and juvenile oviparous vertebrates as an effective and sensitive biomarker for xenoestrogen exposure (Arukwe et al., 1997; Palmer et al., 1998). Together with the hepatic induction of Vtg and ZP expression, hydroxylation of steroids, and androgen to estrogen conversion through the aromatase (CYP19) enzyme have also been used as specific endocrine disruption biomarkers (Gunderson et al., 2001; Lyssimachou et al., 2006). Steroid hormones serve as endogenous substrates for cytochrome P450 enzymes belonging to the CYP3A subfamily and 6␤-position is one of the major site of hydroxylation, where the capacity to hydroxylate steroids is often related to gender (Gunderson et al., 2001; Waxman et al., 1983). Surface water is often utilized as drinking water and in the preparation of food, without prior and suitable purification in most parts of Africa. Furthermore, aquatic species form a large part of the diet of rural populations in Africa (Chigor et al., 2012). Thus, tissue residues in the flesh contribute to the daily intake of contaminants by humans (Oberholster et al., 2008) and also represent health consequences for biota (Zhou et al., 2014). Treated sewage water or other wastewater from urban or agricultural sources are linked to environmental release of chemicals such as persistent organic pollutants (POPs), pharmaceutical agents and industrial by-products (Bouwman et al., 2014; Swanepoel et al., 2000). The present study was initiated after reduced fertility and other egg developmental problems were observed at the Le Croc crocodile farm by the management. Our aims were to evaluate the expression of molecular and biochemical endocrine disruption responses in the liver and gonads of farmed Nile crocodiles exposed to contaminated water in the breeding dams. Furthermore, these responses in relation to liver and environmental contaminant levels were evaluated and the usefulness of these responses as biomarkers of exposure and reproductive effect in this threatened species were assessed. Due to the decline in egg quality at Le Croc farm, we hypothesized that the deteriorating egg quality parameters at this farm is caused by exposure of the breeding crocodiles to environmental contami-

Fig. 1. Map of the outflow water from the Hartbeespoort Dam through the Crocodile River-West in a northern direction, passing through Le Croc farm (study site) to the Limpopo River (border between South Africa, Botswana and Zimbabwe).

nants with potential endocrine disrupting activities, through the breeding dam water that is supplied from the nearby Crocodile River. 2. Materials and methods 2.1. Chemicals and reagents Bovine serum albumin (BSA), NADPH and Trizol reagent were purchased from Gibco-Invitrogen Life Technologies (Carlsbad, CA, USA). iScriptTM cDNA synthesis kit, iTaq DNA polymerase, dNTP mix, iTaqTM Sybr® Green supermix with ROX and EZ Load 100 bp Molecular Ruler were purchased from Bio-Rad Laboratories (Hercules, CA, USA). GelRedTM Nucleic Acid Gel Stain was purchased from Biotium (Hayward, CA, USA). All organic solvents were of HPLC- or GC-purity grade and purchased from Sigma Aldrich. All other chemicals were of the highest commercially available grade. 2.2. Crocodile tissue sampling The study animals were sampled at Le Croc farm, which is located downstream of the Hartbeespoort dam and the sewage treatment plant (STP) in the town of Brits (about 40 km West of Pretoria) South Africa. The Hartbeespoort Dam is the primary recipient of all sewage and wastewater outflow from Johannesburg and surrounding towns. The outflow water of the Hartbeespoort Dam flows in the Crocodile River (Limpopo) in a northern direction, passing the crocodile farm on its eastern border, and then to the Limpopo River (border between South Africa and Botswana as well as Zimbabwe: Fig. 1). Thus, Le Croc farm is next to the Crocodile River in the Northwest Province of South Africa; 20 km north of Brits town, which is situated in the Northwest Province of South Africa. GPS location is: S 25 29.548, E 027 40.827. Water for the Le Croc commercial

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crocodile farm is obtained from an irrigation canal coming from from the Crocodile River (Limpopo). The crocodiles used in this study were approximately 2 years old premature animals, and total weight (minimum 1.5 and maximum 2.2 kg) and length did not differ between sexes. The crocodiles were given electro shock and thereafter pithing, prior to decapitation. The tissues (gonad, liver, plasma, adipose tissue and kidney) from 14 male and 15 female crocodiles were collected during a routine slaughtering at Le Croc farms with permission from the management, labeled and snap-frozen in liquid nitrogen and transported to the laboratory at the Department of Paraclinical Sciences, Faculty of Veterinary Science, University of Pretoria at Onderstepoort, and was shipped to Trondheim (Norway) thereafter. The liver and gonad samples were only used in this report. The procedures described in our paper are conducted in accordance with the laws and regulations controlling experiments with live animals in South Africa (where the experiment was conducted). Given that the tissue samples, that otherwise would have gone to waste, were collected during routine slaughtering at Le Croc farms, no other approval by an Institutional Animal Care and Use Committee (IACUC) or equivalent animal ethics committee was needed. In addition, all sampling procedures were reviewed or specifically approved as part of obtaining the field study permit.

2.3. Egg quality evaluations The male and female breeding crocodiles are kept in captivity on the farm. There was no egg harvesting from the wild. Crocodilians are strictly seasonal breeders laying only one clutch per season. Second clutches have been reported but for all practical reasons it is one clutch per season (Grigg and Kirshner, 2015). In general, egglaying season starts in September through to the end of October. There are geographical differences showing that in southern Africa such as in KwaZulu Natal egg-laying happens earlier than in Pretoria area. Hatching at Le Croc occurs after ± 76 days incubation period at 31.5 ◦ C and 100% humidity—from mid-November to midJanuary. Individual females are not marked, as it is unlikely to get more than one egg clutch per female for a specific season. During the egg-laying season, clutches of eggs are removed from the sand on a daily basis. The eggs from each clutch are kept together throughout the whole incubation process. Polystyrene boxes were used with one or two boxes per clutch (depending on the number of eggs per clutch). Vermiculite was used inside the boxes as filler. Each morning, eggs were removed from the sand (i.e. eggs laid during the previous night). The number of eggs collected and date was recorded on the box and for bookkeeping. The polystyrene containers were then transferred to incubator. During hatching, each box was handled separately and in isolation. Number of hatchlings was counted relative to the total number of eggs. The percentage (%) hatching rate is expressed as live hatchlings per clutch (total eggs). “Dead in egg” is expressed as the occurrence of dead hatchling during the hatching process and is relatively easy to see as a crack in the egg or hatchling in the process of getting out of the egg. Egg is counted as not hatched, when it did not hatch or did not show any sign of activity (egg still intact). These variables were classified by farm workers and are standardized as either not fertilized (nothing changed inside egg) or fertilized (dead embryo inside or rotten or banding still visible). Hatching rate (%) is an accurate estimation expressed as the number of eggs per nest (per box) vs. number of live hatchlings from that specific box or nest. Within 24 h after the egg has been laid, the embryo attaches its membranes to the inside wall of the eggshell. By displacing the watery albumin toward the poles of the egg, it causes the shell to dry out somewhat, changing the optical properties of the shell and produces the visible

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opaque banding. Consequently, a banded egg can be regarded as a fertilized egg (Huchzermeyer, 2003). 2.4. Chemical sampling device and deployment Contaminant sampling in the ambient environment was performed using Exposmeter Hydrophilic (EWH) and Lipophilic (EWL) for water samplers that are based semi-permeable sampling device (SPMD) technology supplied by ExposMeter AB, (Trehorningen, Sweden) that were dismounted and separated in membrane and sorbent parts. Both materials were separately extracted and finally combined to one extract that was analyzed. The EWH and EWL device series (2 per canister/location) are used to monitor respective lipophilic and hydrophilic contaminants with possible endocrine disrupting effects or acutely toxic effects in the natural environment. The devices were assembled in a canister according to the manufacturer´ıs instructions and deployed at 3 different locations as follows: (1) in the Crocodile River that is flowing past Le Croc farm, (2) at the inflow area where the canal water enters the farm, and (3) in the crocodile breeder dam within Le Croc farm. The EWH and EWL samplers were deployed in-situ, and upwardly concentrated ambient contaminants in the above mentioned locations for 28 days. Therefore, the samplers were able to capture contaminant transport and episodic flow at the locations during the exposure period. At the end of the 28-days deployment period, the devices were removed and transported in a cooler to the laboratory, then frozen at −80 ◦ C until analyzed. 2.5. Chemical analyses The EWH and EWL membranes were air-dried and extracted twice with 5 mL methanol in the ultrasonic bath (Branson 5500) for 15 min. The unified extracts were evaporated using inert gas (TurboVap II, Zymark, Idstein, Germany) and reconstituted in n-hexane (1 mL) that was added later to the extract of the sorbent material. After drying the sorbent in a stream of nitrogen, elution was performed with 12 mL of methanol and 12 mL of ethylacetate/nhexane (v/v, 1/1). The extract of the membrane material was combined with the sorbent extract. After evaporation, the analysis was carried out using a final volume of 200 ␮L solution. Gas chromatography-mass spectrometry (GC–MS) was applied at target and non-target screening mode using an Agilent 6890 GC coupled to an Agilent 5973 N Mass spectrometer (Agilent Technologies, Waldbronn, Ger many). One microliter of the extract was injected at 280 ◦ C injector temperature at splitless mode (vent time: 1 min). Helium served as carrier gas at a flow of 1 mL/min. The substances were separated on a 30 m-long GC capillary (HP 5MS, 0.25 mm i.d., 0.25 ␮m film thickness, Agilent Technologies). The initial GC-oven temperature of 60 ◦ C was held for 1 min and increased by 10 K/min to 280 ◦ C holding for 15 min. The transfer line temperature was set at 280 ◦ C and the mass spectrometer operated at electron ionization at 70 eV—electron energy. Full scan (nontarget) analysis covered the mass range from 50 u to 500 u. The target ions used for target analysis and detailed analytical methods and procedures for quality assurance/quality control are given in Supplementary material (S1). Aliphatic hydrocarbons (C10–C40), polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), organohalogenated pesticides (OCPs), chlorophenols, monoaromatic compounds (BTEX: benzene, toluene, ethylbenzene and xylene congeners), brominated flame retardants and trace metal were analyzed in crocodile liver by conventional procedures based on gas chromatography with flame ionization detector, electron capture detector and mass detector, high performance liquid chromatography (HPLC) with diode array and fluorimetric detection, atomic absorption spectrophotometry (Regoli and Giuliani,

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2014). Details on analytical methods and procedures for quality assurance/quality control are given in Supplementary material (S2). 2.6. Aromatase and testosterone metabolism Cytosolic and microsomal fractions were prepared as described in Lavado et al. (2004). Aromatase activity was determined by the tritiated-water release method as described in Lavado et al. (2004). Testosterone oxidation was assayed by incubating 0.4 mg of sample hepatic microsomal protein with [14 C] testosterone (500 pmol, 0.07 ␮Ci), 7.5 mM NADPH in a final volume of 0.25 mL of 50 mM Tris–HCl, 10 mM MgCl2 , pH 7.4. Samples were incubated for 1 h in a shaking water bath maintained at 30 ◦ C. Details on analytical methods and procedures for quality assurance/quality control are given in Supplementary material (S1). 2.7. Quantitative (real-time) PCR Liver and gonad samples were homogenized in Trizol reagent for total RNA isolation (Gibco-Invitrogen Life Technologies). Total cDNA was generated from 1 ␮g total RNA using a combination of oligo(dT) and random hexamer primers from iScript cDNA synthesis kit, as described by the manufacturer (Bio-Rad). Real-time PCR were performed with primers (Table 1 in S2) designed from alligator genes on the NCBI GenBank, using the Mx3000 P realtime PCR system (Stratagene, La Jolla, CA) as previously described (Arukwe, 2006). The primer pairs amplified single DNA fragment of the expected size in all cases. Details on analytical method is given in Supplementary material (S2) 2.8. Steroid hormone extraction and analysis Plasma estradiol-17␤ (E2), testosterone (T) and 11ketotestosterone (11-KT) concentrations were measured using enzyme immunoassay (EIA) kits from Cayman Chemical Company (Ann Arbor, MI, USA). Steroid hormones were first extracted in plasma with organic solvent prior to analysis. Details on analytical method are given in Supplementary Material (S2).

nearly all the deployed passive sampler extracts. Most of the detected pesticides are banned in the EU and USA due to their toxic properties and negative environmental effects. In particular, desethylatrazine, simazine, atrazine and carbamazepine were detected at all deployed locations and at respective 233, 146.4, 273.4 and 76 ng/L in the crocodile breeder dam, demonstrating strong accumulation in the dam, compared to the river and inflow source (Table 1). In addition, pharmaceuticals and personal care products (PPCPs) including—EE2, carbamazepine, galaxolide and tonalide with known toxicity and suspected endocrine disrupting effects were also detected. Particularly, carbamazepine, an antiepileptic drug, was measured in the river (probably coming from Brits sewage treatment plant (STP)) and at the crocodile breeder dam at high concentration. For the liver, all chemical classes were analyzed in both male and female crocodiles. Total aliphatic hydrocarbons did not exhibit significant differences between males and females with concentrations of 5920 ± 4488 and 4214 ± 2904 ␮g/g dry weight (dw: mean ± standard deviation), respectively. Trace metals did not differ between male and female crocodiles in mean average values. A comprehensive overview of the analyzed tissue contaminants was recently published in Arukwe et al. (2015). 3.2. Eggs, fertilization and hatching The percentage of infertile, hatched, banded, dead and rotten eggs, relative to the total number collected between the periods of 2009–2014 is shown in Fig. 2. Overall, there is high and abnormal number (%) of infertile and banded eggs during these reproductive seasons, up to 57 and 34% of infertile eggs in the 2009/2010 and 2013/2014 seasons (Fig. 2). In addition, the percentage of banded eggs ranged between 10 and 19% during the periods of 2009–2014 seasons (Fig. 2), compared to previous seasons from Le Croc records. 3.3. Plasma steroid levels Plasma sex steroids (E2, T and 11-KT) were measured in male and female crocodiles showing apparently higher concentration of all the steroids in males, compared to females (albeit not significant, Fig. 3).

2.9. Statistical analyses 3.4. Transcriptional changes Statistical analyses of enzyme activities were performed using SPSS® statistical software, version 20. Normality within each group was tested using the Shapiro–Wilk test. Possible single outliers were detected and assessed using box and whiskers plot provided by SPSS combined with Grubbs’ test for outliers. Non-normal datasets were treated to approach normality using power transformations. As normal distribution within groups was achieved, datasets were tested for homoscedasticity using Levene’s test. Given homoscedasticity, group means for males and females was tested using student t-test to detect significant differences between sexes. Chemical data together with estrogenic and physiological (hormones) parameters were analyzed using principal component analysis (PCA). Possibly correlated variables were evaluated using Spearman’s Rank-Order Correlation in SPSS. PCA analyses were performed using Umetrics SIMCA-P+ software version 12.01. Significance level was set to ˛ = 0.05. Graphs were made using Systat Software® SigmaPlot® , version 12.5.

In the liver, the ER˛ mRNA expression pattern was not significantly (p < 0.05) different between female and male crocodiles, while in the gonad, the females expressed significantly (p < 0.01) higher ER˛ mRNA, compared to male crocodiles (Fig. 4A and B, respectively). On the other hand, hepatic ZP and Vtg mRNA levels did not differ between male and female crocodiles (Fig. 4C and D, respectively). 3.5. Gonadal aromatase and hepatic steroid metabolism Gonadal aromatase activity was significantly (p < 0.01) higher in female crocodiles, compared to males (Fig. 5A). On the other hand, hepatic sex steroid metabolism using testosterone as substrate was performed, showing the absence of significant sex-related differences in the activity testosterone 6␤-hydroxylase (6␤-OHase: Fig. 5B).

3. Results

3.6. Multivariate data analysis

3.1. Contaminant levels in liver and environment

PCA analysis of chemicals, gene expression, plasma steroid levels and biochemical analysis in male and female crocodiles was performed showing unique grouping of parameters in both males and females. In male tissues (i.e. liver and gonad), metals, aliphatic

A general analysis of environmental contaminants showed the detection of a series of pesticides and pharmaceuticals in

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Table 1 Concentration of contaminants (ng/L) in water estimated from one individual lipophilic or hydrophilic passive sampler data (instrumentation limit of detection (LOD) are listed in Table S1). Sampling rates given in brackets were used to estimate the water concentration for contaminants with unknown experimental sampling rates.

TM

AHTN (Tonalide ) Atrazine BPA Caffeine Carbamazepine Chlofenvinphos Desethylatrazine Ethinylestradiol (EE2) HHCB (GalaxolideTM ) Lindane Metazachlor Metolachlor Metoxuron Prometrine Propazine Sebutylazine Simazine Terbutylazine ␣-HCH ␤-HCH ␦-HCH Sorbent weight of passive sampler (mg)

River1

Inflow2

Dam3

Sampling rate (L/d)

0.1 4.3
0.7 24.6 0.3 3.4 0.5 3.8 4.6 2.5 0.2 0.5 0.9 38.7 36.9 0.8 60.2

(0.85c ) 0.12a , b 0.033e 0.13c 0.56c 0.35f 0.10a 0.85c (0.85c ) (0.35f ) (0.12a ) 0.22d 0.13c (0.13a ) (0.12a ) (0.13a ) (0.12a ) (0.13a ) (0.35f ) (0.35f ) (0.35f )

1

At the Crocodile River, flowing past Le Croc farm. At the inflow area of the Crocodile River to Le Croc farm. 3 At the crocodile breeder dam. a Vermeirssen E.L.M., Mramaz N., Hollender J., Singer H., Escher B.I. (2009) Passive sampling combined with ecotoxicological and chemical analysis of pharmaceuticals and biocides-evaluation of three Chemcatcher configurations. Water Res. 43: 903-914. b Zhang Z., Hibberd A., Zhou J.L. (2008) Analysis of emerging contaminats in sewage effluent and river water: Comparison between spot and passive sampling. Anal. Chim. Acta 607: 37–44. c Li H., Helm P.A., Metcalfe C.D. (2010) Sampling in the great lakes for pharmaceuticals, personal care products, and endocrine disrupting substances using the passive polar organic chemical integrative sampler. Environ. Chem. 29: 751–762. d Mazella N., Lissalde S., Moreira S., Delmas F., Mazellier P., Buckins J.N. (2010) Evaluation of the Use of Performance Reference Compounds in an Oasis-HLB Adsorbent Based Pasive Sampler for Improving Water Concentration Estimates of Polar Herbicides in Freshwater. Environ. Sci. Technol. 44: 1713–1719. e Vallejo A., Prieto A., Moeder M., Usobiaga A., Zuloaga O., Etxebarria N., Paschke A. (2013) Calibration and field test of the Polar Organic Chemical Integrative Samplers for the determination of 15 endocrine disrupting compounds in wastewater and river water with special focus on performance reference compounds (PRC). Water Res. 47: 2851–2862. f Schäfer R.B., Ökologische Risikoabschätzung von Pestiziden in kleinen Fließgewässern anhand von Felduntersuchungen in Mittel- und Nordeuropa“, 2008, PhD thesis, Leuphana University Lüneburg, p.71 (English) Helm P.A., Howell E.T., Li H., Metcalfe T.L., Chomicki K.M. (2012) Influence of nearshore dynamics on the distribution of organic wastewater-associated chemicals in Lake Ontario determined using passive samplers. J. Great Lakes Res. 38: 105–115. 2

Fig. 2. Seasonal egg collection data at Le Croc farm with associated egg quality parameters that include—relative number hatched, infertile, banded, dead and rotten eggs. Different letter denotes significant differences (p < 0.05) between seasons analyzed using student t-test.

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50 b

40

150 T (pg/ml)

E2 (pg/ml)

200

B

A

100 50

30 20 10 a

0

Female

0

Male

11-KT (pg/ml)

40

Female

Male

C b

30 20 a

10 0

Female

Male

Fig. 3. Plasma concentration of 17␤-estradiol (E2: A), testosterone (T: B) and 11-ketotestosterone (11-KT; C) in female and male Nile crocodiles (Crocodylus niloticus) from a commercial crocodile farm (Le Croc) in Pretoria, South Africa exposed to various anthropogenic aquatic pollutants. Steroid hormones were analyzed in plasma samples using enzyme immunoassay (EIA) technique. All values represent the mean (n = 14 per sex) ± standard error of the mean (SEM). Different letters denote significant differences (p < 0.05) between females and males, analyzed using student t-test.

hydrocarbons and PAH levels were separately grouped in the PCA plot (Fig. 6A). Particularly, significant positive correlations were observed between testosterone 6␤-OHase, 11-KT, ER˛ (liver), that further showed significant negative correlation with heavy metals (Ni, As) and PAHs (Fig. 6A). In addition, mRNA expression for liver oocyte maturation factor (OMF), transthyretin (TTR), high-density lipoprotein binding protein (HDLBP), gonadal ER˛, and plasma T and DHT (a testosterone metabolite) were grouped together with aliphatic hydrocarbons and BaP in males, showing positive correlation (Fig. 6A). Overall, PAHs and plasma E2, liver OMF and gonadal hepatocyte growth factor-like protein (HGFP) were grouped at opposite ends of the plot showing significant negative relationship between these variables (Fig. 6A). The two principal components (PCs) accounted for 50% of the total variation in the data set in males. For the female crocodiles, PCA plots showed that aliphatic hydrocarbons were positively grouped on the same side of the plot with liver OMF, gonadal aromatase activity, liver testosterone 6␤-OHase, and HDLBP mRNA (Fig. 6B), and showed a negative relationship with liver ER˛ and other (unidentified) steroid metabolites that were grouped on the opposite end of the plot. Further, some PAHs and responses that include Vtg, E2, TTR, gonadal OMF and HGFP were grouped on the same end of the plot showing positively correlation (Fig. 6B), and these variables were further away from 11-KT and some metals (e.g. Pb) and PAHs, showing negative correlation in female crocodiles (Fig. 6B). The two principal components (PCs) accounted for 44% of the total variation in the data set in females.

4. Discussion The endocrine disruptive and reproductive effects in relation to chemical burden in organs (liver and gonad) of male and female

Nile crocodiles with varying degrees of anthropogenic chemical contamination have been investigated in the present study. Organ analysis of chemicals that include metals and PAHs was performed showing the presence of these chemicals in the liver of crocodiles. In addition, analysis of polychlorinated biphenyls (PCBs), organohalogenated pesticides (OCPs) and brominated flame retardants in liver, indicate that these chemicals were generally present at lower concentration compared to those previously reported in alligators from China and Florida (Guillette et al., 1999a,b; Semenza et al., 1997; Wu et al., 2014; Wu et al., 2006). Furthermore, analysis of passive samplers deployed in the natural environment of the crocodiles and in dam water supplied from the natural environment show that several chemicals with purported endocrine disruptive and reproductive effects were present in the natural environments of these captive crocodiles. Trace metal concentrations were comparable to those reported in Nile crocodiles from Zambia and American alligators (Almli et al., 2005; Horai et al., 2014) and the concentrations of aliphatic hydrocarbons, polycyclic aromatic hydrocarbons and benzene (among the mono-aromatic hydrocarbons) exhibited relatively high levels in the crocodile tissues, compared to those normally present in aquatic vertebrates (Neff, 2002). Overall, these variables correlated with biological responses in the estrogenic pathways of these captive crocodiles.

4.1. Fertilization and reproductive effects Previous studies (Crain et al., 1995; Guillette et al., 1994; Markey et al., 2001) have reported that decreased in fertility, hatching rates and offspring viability, as well as altered plasma hormone levels and/or sexual behavior have been associated with exposure to EDCs. It should be noted that the present study was initiated after reduced fertility and other egg developmental problems

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Liver

Gonad 525

0.8

ERα copies x 10 -5

ERα copies x 10 -5

A

0.6 0.4 0.2 0.0

Female

89

420 315 210 105 0

Male

B

a

b Female

Male

8

ZP copies x 10 -5

C

6 4 2 0

D

Female

Male

Vtg copies x 10 -5

1.50 1.25 1.00 0.75 0.50 0.25 0.00

Female

Male

Fig. 4. Transcriptional responses of liver estrogen receptor-˛ (ER˛: A), gonadal ER˛ (B); liver zona pellucida (ZP: D) and vitellogenin (Vtg: D) in female and male Nile crocodiles (Crocodylus niloticus) from a commercial crocodile farm (Le Croc) in Pretoria, South Africa exposed to various anthropogenic aquatic pollutants. Messenger RNA (mRNA) levels were analyzed by real-time PCR with gene-specific primer pairs and presented as mRNA copies (ng/␮L). All values represent the mean (n = 14 per sex) ± standard error of the mean (SEM). Different letters denote significant differences (p < 0.05) between females and males, analyzed using student t-test.

were observed at the Le Croc crocodile farm by the management. Reduction in fertilization due to exposure of crocodilian species to environmental contaminants increased significantly in the late 1990s and early 2000s, and this was generally attributed to observations showing population declines and reproductive impairment in American alligators (A. mississippiensis) inhabiting Florida contaminated lakes in USA (Guillette, 2000). However, there has not been similar data on Nile crocodile, despite the declining population and reoccurring massive mortalities at the Kruger National Park in South Africa (Bouwman et al., 2014). Herein, significant variability in the number of hatched, infertile, banded and rotten eggs, have been recorded between 2009/2010–2013/2014, and these effect could be explained by the levels of contaminants in the liver and gonads of these crocodiles and their natural environ-

ment. The present findings are consistent with previous studies showing the detection of organochlorines (OCs) in eggs of crocodilian species including American alligators, American crocodiles (C. acutus), Morelet’s crocodiles (C. moreletii), and Nile crocodiles (Campbell, 2003; Pepper et al., 2004; Sepulveda et al., 2004). Reduction in intra-clutch variability in OC type and levels in American crocodile eggs was reported previously (Hall et al., 1979), and was attributed to the consistency in reproductive pattern of yolk deposition in reptiles. Elsewhere, residues of OC between pairs of alligator eggs from the same nests were reported (Heinz et al., 1991). Clutches of Morelet’s crocodile eggs sampled in northern Belize and examined for OC pesticide residues, showed that p,p-DDE, p,p-DDT, p,p-DDD, methoxychlor, aldrin, and endosulfan I were detected in all seven (7) clutches of eggs at concentra-

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Gonad

Liver

B

0.15

a 0.10

0.05

b 0.00

Female

Male

6β-OHase (pmol/h/mg prot.)

Aromatase (pmol/h/mg prot.)

A 4 3 2 1 0

Female

Male

Fig. 5. Gonadal aromatase (A) and hepatic testosterone 6␤-hydroxylase (6␤-OHase: B) activities, expressed as pmol/h/mg protein in female and male Nile crocodiles (Crocodylus niloticus) from a commercial crocodile farm (Le Croc) in Pretoria, South Africa exposed to various anthropogenic aquatic pollutants. All values represent the mean (n = 14 per sex) ± standard error of the mean (SEM). Different letters denote significant differences (p < 0.05) between females and males, analyzed using student t-test.

Fig. 6. Principal component analysis (PCA) showing individual contaminant and variable loadings according to liver chemical burden and gonadal/liver variables in females (A) and male (B) Nile crocodiles (Crocodylus niloticus) from a commercial crocodile farm (Le Croc) in Pretoria, South Africa exposed to various anthropogenic aquatic pollutants.

tions ranging from 4 ppb to >500 ppb (Wu et al., 2006). Although environmental contaminants were not analyzed in eggs from the present study, given the level of contaminants previously reported in crocodilian eggs, it is possible that the fertility and egg developmental problems observed in the present study are attributable to the contaminants burden observed in liver, gonads and environmental samples from the present.

4.2. Endocrine disruptive effects Vtg and eggsell ZP are female-specific oogenetic proteins and their induction in juveniles and males have been widely used as a biomarker of endocrine disruption in wildlife species (Arukwe and Goksoyr, 2003; Palmer and Palmer, 1995), although only a limited number of studies have used these responses in crocodilian

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from contaminated habitats. Estrogens are known to play integral roles in sex determination of crocodilians, as in most nonmammalian vertebrates. Under normal conditions, crocodilians exhibit temperature-dependent sex determination, showing that during a thermo-sensitive period (TSP) the temperature of the incubated egg, determines the sex of the offspring (Kohno et al., 2015). Indeed, a male to female sex reversal during the TSP was induced by the administration of estrogens at a male-producing temperature (MPT) in crocodilians and some turtles, amphibians (Dournon et al., 1990; Smith and Sinclair, 2004). Since the development of the reproductive systems is a continuous process throughout ontogeny, these processes may therefore be susceptible to chemical insult from anthropogenic sources. In the present study, Vtg and ZP mRNA were induced in female and male crocodiles, and these responses did not differ between males and females. Estrogens and their mimics may control the recruitment of ER and possibly other co-activators, in controlling hepatic Vtg and ZP synthesis. In the present study, the female crocodiles significantly expressed higher gonadal estrogen receptor (ER˛), compared to the males, while ER˛ was equally expressed in the liver of both males and females. The expression of hepatic ER˛, Vtg and ZP in males, corresponds to measured plasma steroid hormones (E2, T and 11-KT) in males. While hepatic expression of ZP in crocodilian species under environmental contaminant stress has not been previously reported, Vtg detection in plasma of male broad-snouted caiman (Caiman latirostris) is a valuable tool in biomonitoring xenoestrogen exposure in a polluted environment (Rey et al., 2006). Given that the use of reproductive and physiological markers, such as E2 -induced proteins, as indicators of early response to environmental contamination with endocrine disruptive capability may provide important information regarding the potential effects of these contaminants on ecosystem’s health, the induction of genes encoding Vtg and ZP proteins in male crocodiles with associated changes in plasma steroid hormone levels clearly suggest that these crocodiles are experiencing exposure to EDCs. However, we showed herein that males produced higher T and 11-KT plasma concentrations than females, and this may be normal for the Nile crocodiles, at this time of the year, in addition to other factors. The molecular data supports an estrogenic exposure of males, while the hormonal profile should be better studied in the absence of normal reproductive endocrinology of this species. Male alligators produce E2 under normal physiological condition (Lance et al., 2003), and the observed lower E2 concentration in males, may represent normal physiology for the Nile crocodile. Several pesticides that are banned in the EU and USA due to their toxic properties and negative environmental effects, including endocrine disruptive effects, have been detected in the environment. These chemicals include desethylatrazine, simazine, atrazine, propazine, ␤-HCH and terbutylazine that were detected at all deployed locations, but at very high concentrations in the crocodile breeder dam. In addition, emerging PPCPs (EE2, carbamazepine, galaxolide and tonalide) with reported endocrine disruptive effects were also measured at high concentrations in passive samplers deployed in the crocodile breeder dam. Overall, the concentration of PPCPs measured in the crocodile breeder dam is several orders of magnitude higher than the concentrations measured in the river and the inflow point. For example, atrazine, carbamazepine, desethylatrazine and simazine had 80, 50 and 10 times higher concentration, respectively, in the breeder dam, compared to the river. These differences may be attributed to the fact that water in the breeder dam is completely replaced once every year, or otherwise filled up when volume is below critical level due to evaporation. In addition, the high half-life of these emerging PPCPs in the environment may also have contributed to the observed differences. The calculated environmental half-life of several PPCPs was recently determined (Walters et al., 2010),

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showing that carbamazepine was persistent in soils, biosolids, and soil-biosolid mixtures (Monteiro and Boxall, 2009), with a calculated half-life of 495 ± 36 days (Walters et al., 2010). In addition, heavy metals, PAHs, PCBs and OCs were detected in organ (liver and gonad) samples. While the detection of these potential EDCs in the environment and biota samples does not directly prove causality, it does provide direct correlation between observed fertility, egg quality and endocrine disruptive effects and contaminant burden in these captive crocodiles as demonstrated with the PCA. These findings are consistent with previous studies that have linked reproductive and endocrine disruptive effects in wild America alligators and contaminant burden in several Florida lakes (Crain et al., 1997; Guillette et al., 1999a,b, 1996; Gunderson et al., 2001). 4.3. Effects on steroid hormone synthesis and metabolism In addition to their roles in xenobiotic metabolism, CYP enzymes play significant roles in the synthesis (CYP19) and metabolism (CYP3A) of endogenous compounds, including steroid hormones (Zimniak and Waxman, 1993). Herein, we observed sexual dimorphism in gonadal aromatase activity, with higher activity being observed in female crocodiles. No such sexual dimorphic differences were observed in hepatic steroid hydroxylation. In vertebrate liver, steroid hormones serve as endogenous substrates for CYP3A subfamily of metabolic enzymes. Steroid metabolism in lower vertebrates is catalyzed by specific CYP3A enzymes (Klotz et al., 1986; Miranda et al., 1991; Stegeman, 1993) and the ability of CYP3A to hydroxylate steroids often shows sexual dimorphism (Zimniak and Waxman, 1993). For example, it has been suggested that CYP3A proteins are constitutively expressed, regulated during sexual maturation (with males showing higher protein levels than females) and metabolize endogenous substrates like testosterone and progesterone at the 6␤-position (Klotz et al., 1986; Miranda et al., 1991; Stegeman, 1993). Previous findings have demonstrated that steroid hormone mimics (EDCs) modulate CYP3A-mediated catalytic activities in similar manner as natural steroid hormones (Klotz et al., 1986; Miranda et al., 1991; Stegeman, 1993). Endocrine toxicology research has mainly focused on estrogenicity that involves direct estrogen receptor mediated effects. As a result, studies on endocrine disruption, on steroidogenic pathways, have focused mainly on reproductive steroids. The CYP19 enzyme is a crucial steroidogenic enzyme in vertebrates by catalyzing the final step in the conversion of androgens to estrogens (Callard et al., 2001; Kishida and Callard, 2001). In vertebrates, P450aromA and P450aromB are two structurally distinct CYP19 isoforms, where the P450aromA is predominantly expressed in the ovary and plays important roles in sex differentiation and oocyte growth, while P450aromB is expressed in neural tissues such as brain and retina and is involved in the developing central nervous system and sex behaviors (Callard et al., 2001; Kishida and Callard, 2001). In accordance with previous study showing that environmental contaminants altered gonadal steroidogenesis by targeting the aromatase enzyme (Crain et al., 1997), we observed that gonadal aromatase activity was higher in female crocodiles, compared to males, and this effect negatively correlated with the changes in plasma E2 and T levels, showing significantly higher levels in male crocodiles, compared to females. Overall, these findings suggest that increases and decreases of aromatase and steroid hydroxylating enzymes are potential mechanisms of action for EDC induced alteration of steroidogenesis (Crain et al., 1997). In conclusion, the relationship between the measured endocrine disruptor responses and complex interactions between contaminant levels (both in crocodile liver and the environment) that can be modulated at different levels, from gene transcription to biochemical levels indicate that the captive crocodiles are experiencing contaminant-induced endocrine disruptive responses. It

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should be noted that Le Croc farm is experiencing significant reproduction problems (poor hatching rate), most likely due to either decreased fertility or increased in-egg mortality. While variations in transcript levels may be considered as biomarkers of exposure, those occurring on the physiological (plasma steroid hormones) and enzymatic activities (aromatase and testosterone 6␤-OHase) better reflect functional alterations. Therefore, we suggest that the captive Nile crocodiles are experiencing endocrine disruptive effects due to anthropogenic contaminants in the rearing water directly supplied from a river recipient of several sewage effluents and agricultural run-offs. Acknowledgement This work is based upon research supported by the National Research Foundation of South Africa (CB and JM) and NTNU internal research support (AA). The authors thank Anette Venter, Randi Røsbak and Maura Bennetti for technical assistance during sampling and analysis. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.aquatox.2015.12. 027. References Almli, B., Mwase, M., Sivertsen, T., Musonda, M.M., Flaoyen, A., 2005. Hepatic and renal concentrations of 10 trace elements in crocodiles (Crocodylus niloticus) in the Kafue and Luangwa rivers in Zambia. The. Sci. Total Environ. 337, 75–82. Arukwe, A., 2006. Toxicological housekeeping genes: do they really keep the house? Environ. Sci. Technol. 40, 7944–7949. Arukwe, A., Goksoyr, A., 2003. Eggshell and egg yolk proteins in fish: hepatic proteins for the next generation: oogenetic, population, and evolutionary implications of endocrine disruption. Comp. Hepatol. 2, 4. Arukwe, A., Knudsen, F.R., Goksoyr, A., 1997. Fish zona radiata (eggshell) protein: a sensitive biomarker for environmental estrogens. Environ. Health Perspect. 105, 418–422. Arukwe, A., Røsbak, R., Adeogun, A.O., Langberg, H.A., Venter, A., Myburgh, J., Botha, C., Benedetti, M., Regoli, F., 2015. Biotransformation and oxidative stress responses in captive Nile crocodile (Crocodylus niloticus) exposed to organic contaminants from the natural environment in South Africa. PLoS One 10 (6), e0130002. Ashton, P.J., 2010. The demise of the Nile crocodile (Crocodylus niloticus) as a keystone species for aquatic ecosystem conservation in South Africa: the case of the Olifants River. Aquat. Conserv. 20, 489–493. Bogi, C., Schwaiger, J., Ferling, H., Mallow, U., Steineck, C., Sinowatz, F., Kalbfus, W., Negele, R.D., Lutz, I., Kloas, W., 2003. Endocrine effects of environmental pollution on Xenopus laevis and Rana temporaria. Environ. Res. 93, 195–201. Bouwman, H., Booyens, P., Govender, D., Pienaar, D., Polder, A., 2014. Chlorinated, brominated, and fluorinated organic pollutants in Nile crocodile eggs from the Kruger National Park, South Africa. Ecotoxicol. Environ. Saf. 104, 393–402. Callard, G.V., Tchoudakova, A.V., Kishida, M., Wood, E., 2001. Differential tissue distribution, developmental programming, estrogen regulation and promoter characteristics of cyp19 genes in teleost fish. J. Steroid Biochem. Mol. Biol. 79, 305–314. Campbell, K.R., 2003. Ecotoxicology of crocodilians. Appl. Herpetol. 1, 45–163. Chigor, V.N., Umoh, V.J., Okuofu, C.A., Ameh, J.B., Igbinosa, E.O., Okoh, A.I., 2012. Water quality assessment: surface water sources used for drinking and irrigation in Zaria, Nigeria are a public health hazard. Environ. Monit. Assess. 184, 3389–3400. Combrink, X., KorrÛbel, J.L., Kyle, R., Taylor, R., Ross, P., 2011. Evidence of a declining Nile crocodile (Crocodylus niloticus) population at Lake Sibaya, South Africa. S. Afr. J. Wildl. Res. 41, 145–157. Crain, D.A., Gross, T.S., Cox, M.C., Guillette Jr., L.J., 1995. Insulin-like growth factor-I in the plasma of two reptiles: assay development and validations. Gen. Comp. Endocrinol. 98, 26–34. Crain, D.A., Guillette Jr., L.J., Rooney, A.A., Pickford, D.B., 1997. Alterations in steroidogenesis in alligators (Alligator mississippiensis) exposed naturally and experimentally to environmental contaminants. Environ. Health Perspect. 105, 528–533. Dournon, C., Houillon, C., Pieau, C., 1990. Temperature sex-reversal in amphibians and reptiles. Int. J. Dev. Biol. 34, 81–92. Gibbons, J.W., Scott, D.E., Ryan, T.J., Buhlmann, K.A., Tuberville, T.D., Metts, B.S., Greene, J.L., Mills, T., Leiden, Y., Poppy, S., Winne, C.T., 2000. The global decline of reptiles, de´ıja’vu amphibians. Bioscience 50, 653–666.

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