The measured concentrations of nonylphenol in the exposure tanks after 7 days, were approximately one third of the nominal concentrations in all the exposure concentrations. Similar studies have reported actual concentrations which were approximately 1/3 to 1/5 of the nominal concentrations (Nimrod and Benson, 1998; Nichols et al., 2001; Schoenfuss et al., 2008). The reduced concentrations could be due to several reasons such as degradation of nonylphenol in the exposure tanks. Alternatively the lower measured concentrations may have been related to an incomplete solubilisation of nonylphenol in the stock solution (aqueous solubility limit of NP = 5.24 ± 0.11 mg NP/l at 14 °C, Ahel and Giger, 1993) although unfortunately we did not measure the stock solution concentration. Other potential factors include volatilization of nonylphenol, adsorption to tubing or the glass tanks. Adsorption to organic matter present in the tanks can also occur (Villeneuve et al., 2002), and fish themselves may absorb some nonylphenol (Pickford et al., 2003). However, in our study we have used a flow through system which makes these factors unlikely to happen. Furthermore, the pump’s flow rate was not checked for accuracy during and after the study to ensure the delivery of the proper concentration required for the study. Since the loss was around 70 % in all the doses the pump’s flow rate may also be a factor.
124 Other studies that have exposed fish to nonylphenol have found widely differing recoveries of nonylphenol in the water. Pickford et al., (2003) exposed male fathead minnows (Pimephales promelas) to nonylphenol through waterborne exposure for 2 weeks at nominal concentrations of 1, 10 or 50 µg/l, and the measured concentrations in the dosed tanks were between 72-129 % of nominals. One possible reason why the measured concentrations of nonylphenol were more closely aligned with nominals in that study was that their exposure tanks were dosed for 7 days with nonylphenol prior to addition of the fish, whereas in our study we pre-dosed the system for only 4 days, that longer period of exposure might have helped. This suggestion is further supported by another study, where dosing of nonylphenol (nominal concentrations 1, 10 and 100 µg/l) were initiated 3 weeks prior to adding fish, and the measured concentrations were between 70 % and 85 % of nominals (Harris et al., 2001). However, although lower levels of nonylphenol were obtained in the exposure tanks than anticipated, they are still close to the relevant environmental concentrations which should not be considered as a total loss.
5.2 Plasma vitellogenin
The exposure to nonylphenol for 7 days revealed no vitellogenin induction in the plasma of rainbow trout. The results showed no vitellogenin induction which could be due to the low exposure level and short exposure period. Also, it turned out that the nominal exposure levels did not reach to the exposure tanks. Several studies have exposed fish to nonylphenol at concentrations between 1 and 50 µg NP/l and have reported either a non-significant effect on plasma vitellogenin concentrations and/or great variability among fish in the same treatment (Pickford et al., 2003; Van den Belt et al., 2003). Similar to our result in terms of
125 vitellogenin induction, Atlantic salmon (Salmo salar L.) smolts were exposed in freshwater to 5, 10, 15, and 20 µg NP/l for a 30-day period where no vitellogenin induction detected (Moore et al., 2003). There was also no detectable vitellogenin in juvenile Atlantic salmon treated with 10 µg NP/l for 21 days in freshwater (Lerner et al., 2007).
It is not known why there was an elevated level of vitellogenin in all fish after the seawater challenge in the present study. One possible reason is a consequence of a stress response after being subjected to the accidentally reduced oxygen level, but this is speculation only. Indeed, generally, stress is known to reduce plasma vitellogenin levels in fish (Carragher et al., 1989), rather than increase them, so this seems an unlikely explanation. However, the increase observed after seawater challenge was actually rather small relative to the orders of magnitude changes commonly observed in response to significant estrogenic stimulation. It is therefore wise to interpret these data with caution, and it is quite likely that the small difference may reflect different performance of the ELISA assays for samples analysed on these different experimental days.
On the other hand, several studies have documented that hypoxia (low oxygen) acts as an endocrine disruptor of fish reproduction by affecting hormones secreted by the hypothalamus-pituitary-gonadal-liver system (Wu et al., 2003; Landry et al., 2007; Thomas et al., 2007). But, there is no direct evidence that hypoxia can affect the vitellogenin level. Male fathead minnows (Pimephales promelas) were exposed to a mixture of estrogenic chemicals under hypoxic conditions (< 2 mg of O2/l) and the results revealed no effect of hypoxia on vitellogenin response (Brian et al., 2009).
126 One possible reason might explain the difference in the plasma vitellogenin levels in seawater from the freshwater is the difference sensitivity between different ELISAs carried out. As the plasma samples used to measure the vitellogenin were divided for different ELISAs in different days.
5.3 Osmoregulatory variables
The reduction in oxygen that occurred due to the accidental loss of aeration in the exposure tanks would have added an additional stress to the exposed fish besides the exposure to nonylphenol. The gills carry out multiple tasks such as gas exchange and ion transport (Evans et al., 2005). In general, freshwater fish face the problem of gaining water and losing ions, and the major ions in plasma, Na+ and Cl-, have particularly high rates of loss via branchial diffusion, even under normoxic conditions (Wood, 1992). Data showed a significant decrease in these ions in the tanks affected by oxygen depletion in comparison to their unaffected replicate tanks within each treatment (Table 2A). That can be explained by the extra stress, as fish would normally increase their ventilation as a normal physiological response to lower oxygen levels (Evans et al., 2005). Higher ventilation helps to assist in the extraction of enough oxygen from the water to satisfy the normal oxygen demand. However, a consequence of elevated ventilation could also be increased diffusive ion loss across the gills (Gonzalez and McDonald, 1992, 1994) and may result in decreasing the level in the plasma detected in the affected tanks. An increase in the gill functional surface area, as during hyper-ventilation, promotes oxygenuptake and increases the Na+ efflux rate, whereas reduction in surface areas cause the opposite. This relationship between oxygen uptake and diffusive ion fluxes is called the osmo-respiratory compromise.
127 A study on freshwater adapted rainbow trout experimentally showed a relationship between oxygen consumption and Na+ loss, where Na+ loss increased whenever oxygen consumption increased (Gonzalez and McDonald, 1992). Another reason could be the down regulation of the uptake channels and/or Na+-K+-ATPase activity to make energetic savings during hypoxia (Wood et al., 2007). In freshwater fish hyperventilation (due to low oxygen) is expected to cause reduced plasma ions (especially Na+ and Cl-), whereas hyperventilation in seawater fish will cause the opposite (increased plasma ions, especially Na+, Cl-, and Mg2+ that have the biggest gradients across the gills). The results support the above ideas on low oxygen supply and decrease in the plasma osmolality, Na+ and Cl- level in freshwater fish accordingly.
Due to the loss of aeration at a key stage of the experiment, the data obtained were not complete in terms of a complete set from replicate tanks for each treatment. To study the potential effects of nonylphenol on the osmoregulatory variables was the major aim of this study and this was clearly hampered by this problem. Therefore, the investigation of the potential physiological effects of low oxygen level on the osmoregulatory variables has been the focus in the latter part of the discussion.
Version to the chapter submitted to: The Journal of Experimental Biology
Noura J. Al-Jandal, Jonathan M. Whittamore, Eduarda M. Santos, and Rod W. Wilson 128