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  • The methods for animal housing, handling and experimental protocols were assessed and approved by the Biological Sciences Animal Ethics Committee at Monash University (permit number: BSCI/2011/07). Because mosquitofish are a noxious species under State laws, the terms of the collecting permit (Department of Primary Industries Victoria, permit number NP191) did not allow them to be returned to the wild and hence fish were euthanised. Mosquitofish were collected from Brodies Lake in Victoria, Australia. This is a relatively pristine site located adjacent to a reservoir that supplies drinking water to parts of suburban Melbourne. Fish were caught during the breeding season (February) using dip nets and transported in coolers back to the laboratory. In total, 280 fish were collected, of which 140 were females and 140 males. Fish were separated by sex and acclimated to laboratory conditions (12∶12 h light regime) for 10 days in 54 L tanks (20 fish per tank). After acclimation, fish were randomly placed into separate-sex ‘exposure’ tanks (60 cm×30 cm×24 cm; 20 fish per tank), the set up of which followed the design of Saaristo et al. [17] with a few modifications. Briefly, 14 tanks were assigned to one of two treatments, namely, (1) a 17β-trenbolone exposed treatment (TB), and (2) a freshwater control. In total, 280 fish were exposed: seven tanks were allocated to the TB treatment (4 tanks for males and 3 tanks for females) and seven tanks were allocated to the control treatment (4 tanks for males and 3 tanks for females). We randomly took four fish from each of the holding tanks and placed them into each of the exposure tanks. This was continued until all of the fish from the holding tanks had been assigned to an exposure tank. Thus, each exposure tank had fish from several holding tanks. Male and female mosquitofish tanks in the TB treatment were exposed to trenbolone at a nominal concentration of 15 ng/L (measured concentration = 6 ng/L; see below for details on how trenbolone levels were monitored) via a flow-through system for 21-days. Mosquitofish tanks in the control treatment were connected to an identical, but separate, flow through system over the same period but, in contrast to the TB tanks, the flow through system supplied only freshwater to the fish during the exposure period. The water supplied to these fish tanks was fed through a mixing tank into which either trenbolone from a stock solution (in the case of the TB treatment) or freshwater (in the case of the control treatment) was pumped using a peristaltic pump (Watson Marlow 323 U/MC). From the mixing tanks, the water was channelled into the fish tanks using silicon tubing. The flow rate was kept constant (2.25 L/h) or all tanks using flow meters (BES Flowmeters, MPB Series 1200) and adjustable valves. For the trenbolone exposure, a fresh stock solution was prepared once a week and the stock solution tank was changed every third day to minimize the possible deterioration of TB. Water temperature in the tanks was monitored daily and ranged from 19–23°C. Fish were fed ab litium with commercial fish flakes (Otohime Hirame, Aquasonic) once a day during the exposure period. The level of trenbolone used was achieved by firstly dissolving 17β-trenbolone (4,9,11-estratrien-17-ol-3-one; Novachem, Germany) in 100% ethanol (600 ug/L, 1% of ethanol) to create a stock solution, which was then diluted in the flow-through system to achieve the desired concentration. The final solvent concentration was 0.00006% in the exposure tanks. The concentration of trenbolone in the exposure and control tanks was monitored by enzyme-linked immunosorbent assay (ELISA). To do this, a 100 mL water sample was taken from each exposure tank once a week. Water samples were acidified by adding a mixture of 1% acetic acid methanol, then loaded onto a conditioned solid phase cartridges (Strata×33 u, 500 mg,/6 mL; Phenomenex, Torrance, CA, USA). The cartridge was then eluted with methanol (2×4 mL), with the eluate dried under nitrogen stream. Samples were reconstituted with 100 uL methanol and 900 uL of deionised water. Measurement of trenbolone was undertaken using commercial ELISA kits in accordance with the manufacturer’s instructions with a minor modification (Trenbolone ELISA kit; EuroProxima, Arnhem, The Netherlands). In short, a total of thirty samples and trenbolone calibration standards (freshly made in 10% methanol water) were dispensed (50 uL) in duplicate into an antibody coated 96 well plate by an auto dispenser (epMotion 5070, eppendorf, Hamburg, Germany). Thereafter, 25 uL of HRPO conjugate and 25 uL of antibody were dispensed into the wells. After 1 hour incubation at room temperature in the dark, the plate was washed three times with wash buffer by a microplate washer (Atlantis, ASYS HITECH, Eugendorf, Austria), and 100 uL of substrate was added to all wells. The plate was then incubated for a further 30 minutes at room temperature in the dark. Finally, 100 uL of stop solution was dispensed into all wells, and the absorbance of the solutions in the wells measured at 450 nm by a microplate reader (UVM40, ASYS HITECH, Eugendorf, Austria). Calculation of sample concentrations was undertaken by 4 parameter logistics method after creating a calibration curve using a series of standard calibration solutions (0, 0.125, 0.25, 0.5, 1.0, 5.0 ug/L) made up in 10% methanol. In order to verify calibration accuracy, check standards (i.e. standards from the kit run as samples) were run in duplicate on each ELISA plate during each ELISA test. The detection limit of trenbolone ELISA was 2.0 ng/L. The ratio of nominal concentrations and measured values were 90%, which indicates that the calibration curve provided good (accurate and precise) sample concentration values provided the ELISA response was within the upper and lower bounds of the calibration curve. A spike recovery experiment was conducted in triplicate using a 5 ng/L 17β-trenbolone solution. The average recovery was 97%, providing confidence that trenbolone in water samples was efficiently extracted, and that measured values were neither under nor over estimates of sample concentrations. All behavioural trials were conducted in tanks (60 cm×30 cm×24 cm) containing freshwater with a 2 cm layer of gravel on the bottom as substrate. One male and one female from the same treatment group (i.e. either TB or control) were randomly assigned to an experimental tank and allowed to freely interact. We specifically paired fish from the same treatment groups because, in the wild, both sexes would typically be exposed to the same environmental contaminants simultaneously. Male and female behaviours were recorded with a video camera. Filming began when the male and female were released into the tank. Fish were filmed for 15 minutes and the behaviour of each sex was analyzed using JWatcher software, which calculates the total time and frequency of each quantified behaviour during this period. For females, we quantified whether or not the female was interacting with the male and, if so, whether she was actively associating with the male (i.e. swimming towards him), exhibiting aggressive behaviour (i.e. biting and performing tale beats), or trying to avoid the male by swimming away from him. For males, we quantified whether or not the male was showing an interest in the female. If so, we noted whether the male was orienting towards the female (within 5 cm of her body), chasing her, or engaging in gonopodial thrusts. We also noted the time the male took to perform the first chase. Trials were replicated 19 times for the trenbolone treatment and 18 times for the control. Each trial had a new pair of fish. We used 12 tanks for behavioural trials and ran 12 trials per day (6×TB and 6×control). For each tank, we alternated between TB and control trials to avoid tank effects. After each behavioural trial, fish were euthanized with an overdose (40 mg/L) of anaesthetic clove oil [39]. Fish were then weighted and measured from the tip of the snout to the end of the caudal fin, and preserved in 70% ethanol for further anal fin measurements. The male gonopodium and female anal fin were analysed using the morphometric analysis described by Angus et al. [34]. The anal fin was photographed using a moticam 3.0 mounted on a Motic SMZ-168 stereomicroscope. From these images, ray 4 and ray 6 were measured to the nearest 0.001 mm using Motic Digilab II (Motic Instruments Inc., Hong Kong). The R4:R6 ratio is known to be influenced by EDCs in both the male gonopodium [40], [41] and the female anal fin [35], and was thus calculated (dividing length of R4 by R6) for both sexes. Data was checked for normality and heterogeneity of variance. In our analyses of female and male behaviour, the data did not conform to a normal distribution and we were unable to render them normal with transformation. Therefore, the effect of treatment on behavioural variables were analysed using Mann-Whitney tests. For the morphological (length and anal fin) data, independent-sample t-tests were used to test differences between TB-exposed and control fish. SE = standard error of mean. All statistical analyses were performed using SPSS (19.0).
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