The impacts of chemical discharges on the reproductive biology of the bullhead Cottus gobio and the dipper Cinclus cinclus in the Tamar catchment
Fowler, Vivienne Frances
Thesis or dissertation
University of Exeter
It is now well established that a wide range of natural and anthropogenic chemicals present in the aquatic environment have the potential to disrupt the endocrine system of many organisms. In fish, many of these effects appear to be of a feminising nature, including stimulation of vitellogenin production and induction of intersex. In piscivorous birds these so called endocrine disrupting contaminants have been shown to impair reproduction, influencing reproductive behaviour, sex ratio, eggshell thickness and reproductive success. The effects seen in fish have been associated with high levels of oestrogenic activity in the effluent from waste water treatments works (WwTWs), but few studies have focused on the effects of WwTWs effluents on birds. In this thesis, the effects of effluents from WwTWs on fish and birds were investigated in the Tamar catchment, SW England. The work spanned making detailed assessment on the oestrogenic and anti-androgenic activity of 3 WwTWs effluents, using a variety of water sampling techniques and applying both recombinant yeast oestrogen screen (YES) and recombinant yeast androgen screen (anti-YAS) bioassays to quantify the different hormonal activities. A survey was undertaken of the hormonal activities at 13 sites to determine concentrations of contaminants in the surface waters throughout the Tamar catchment, using both recombinant yeast screens and targeted analytical chemistry for specific pollutants (LC/MS-TOF and GCMS). An ELISA was developed to quantify vitellogenin (VTG) in the bullhead (our study fish sentinel) as a biomarker of oestrogen exposure, and evidence of endocrine disruption was investigated in wild populations of the bullhead, Cottus gobio and the dipper, Cinclus cinclus. Macroinvertebrates from upstream and downstream of three WwTW's effluent discharges and from three sampling sites were also sampled as an index of overall water quality in the Tamar catchment, and as an assessment of food availability for the bullheads and dippers. For the studies on the hormonal activities in three WwTWs in the Tamar catchment, samples were collected by both spot and passive sampling; passive samplers (in replicate) were placed in the effluent discharges for a three week period, and collected on days 7, 14 and 21, spot samples were taken simultaneously. Measurement of total oestrogenic and total anti-androgenic activity was conducted using the YES and anti-YAS, respectively. Spot and passive samples were collected from 13 sites within the Tamar catchment (sampling sites were >2 km downstream of effluent discharges). Additionally, liquid chromatography mass spectrometry time-of-flight (LC/MS-TOF) was used to measure the concentration of oestrone (E1), 17β-oestrodiol (E2) and 17α-ethinylestradiol (EE2) in each sample. Gas chromatography mass spectrometry (GCMS) was used to measure the concentration of individual PBDE and PCB congeners in the spot samples only. Levels of oestrogenic and anti-androgenic activity observed in the WwTWs effluent were comparable with those measured in effluents in the UK and in other countries. Surface waters of the Tamar, away from the WwTWs effluent discharges, contained very little oestrogenic activity (<1.1 ng E2 EQs L-1), and anti-androgenic activity was undetectable. Quantification of oestrogenic activity using passive samplers showed an increasing amount of total oestrogenic activity between days 7 and 21 when measured by the both the YES and LC/MS-TOF. Low levels of PBDE congeners 47, 99, 100, 138 and 153 were detected in the spot samples taken from the Tamar catchment, with BDE 47 being the most abundant. In contrast PCBs were undetectable. Neither PBDEs nor PCBs were detected in any of the extracts from the passive samples. No assay was available to measure VTG (one of the most widely used biomarkers of oestrogen exposure in fish) in the bullhead and so an enzyme linked immunosorbant assay (ELISA) was developed for application to studies on wild bullheads in the Tamar catchment. The bullhead vitellogenin (bh-VTG) ELISA was developed successfully, and proved to be sensitive and robust, with a detection range between 10.5 and 300 ng bh-VTG mL-1 (undiluted), comparing favourably with other fish VTG ELISAs. Plasma VTG concentrations measured in male bullheads (collected from the same sites as for the water samples) ranged from below the limit of detection to 990 ng bh-VTG mL-1. Whether these upper levels in the range reflected VTG induction was difficult to conclude. Because of this controlled caged exposures with bullheads and trout were used to assess the relative levels of oestrogenicity in two key WwTWs effluent discharges and to determine the response sensitivity of the bullheads (and trout) to those effluents. These controlled exposures found no responses in plasma VTG in bullheads (ranging between 126 and 934 ng bh-VTG mL-1) suggesting a lack of sensitivity for VTG induction. This was supported by the inability to induce VTG in fish held in the laboratory and treated with steroidal oestrogens. For the effluent exposures on the caged rainbow trout, it was also found that there was no significant induction of VTG, a species normally sensitive to oestrogens. These findings may indicate that the fish were highly stressed due to the river being in spate and the movement of the cages during the controlled exposures. It may also be the case, however, that the use of immature female rainbow trout with a highly variable baseline plasma VTG concentration may prevent any detection of a response. There were no signs of sexual disruption in any of the gonads analysed from either male or female wild bullheads, demonstrating that any hormonal activity present in the catchment away from the WwTWs effluents was not sufficient to induce adverse effects on reproductive development. An interesting feature noted in the male testes of the bullheads was the presence of spermatid masses, which have been recorded in 10 other Cottidae species, but not previously in the bullhead. For the studies on dippers, eggs were collected from the nests of breeding dippers to measure for sperm numbers and morphology from sperm trapped in the perivitelline membrane (PVM), and the yolks were analysed for PBDEs, PCBs and organochlorine pesticides (OCPs) by GCMS, for E1, E2, and EE2 by LC/MS-TOF. Eggs of the dipper were collected from nests at the 13 sampling sites, plus an additional three sites and over three years of field study. The number of sperm trapped in the PVM ranged between three and 188, with a mean of 68.78 ± 8.78 SE. Dipper sperm had not previously been characterised, and was found to be similar to other passerine sperm, in that the head was helical, complemented by a mitochondrial helix or keel, which continued in a spiral around the flagellum. Sperm were classed as ‘abnormal’ if they did not adhere to this typical structure. No assessment of motility could be made in relation to the structural abnormalities seen. Contaminants in the dipper eggs were dominated by BDE 99, an unusual result considering the dippers aquatic lifestyle. PCB 153 was the most common PCB, and p,p’-DDE was the most abundant OCP; all other pesticides tested were below the limit of detection, as were the levels of all three steroid oestrogens. There was inter- and intra-nest variability between contaminant burdens in all eggs as well as the number of sperm trapped in the PVM, but there was no relationship between sperm number and the level of contaminant loadings in the eggs. There were no correlations between contaminants and oestrogenic activity measured in the water samples, and plasma VTG concentrations in bullheads or contaminant loadings in eggs, or indeed sperm number. Analysis of macroinvertebrate assemblages proved that the surface waters of the Tamar catchment were of ‘very good’ quality, even in close proximity to WwTWs effluent discharges. Indeed the oestrogenicity and contaminant loadings in both eggs and surface waters were very low, and this study agrees with a national risk assessment that there appears to be no risk of intersex in fish in the Tamar catchment.
National Environment Research Council, AstraZeneca
PhD in Biological Sciences