Prefertilization Exposure of Rainbow Trout Eggs to Per- and Polyfluoroalkyl Substances to Simulate Accumulation During Oogenesis.

Aqueous film-forming foams (AFFFs) are used in firefighting and are sources of per- and polyfluoroalkyl substances (PFAS) to the environment through surface runoff and groundwater contamination at defense and transportation sites. Little is known regarding the toxicity and bioaccumulation of newer AFFF formulations containing novel PFAS. To mimic maternal transfer of PFAS, prefertilization rainbow trout eggs were exposed to three PFAS using novel methodologies. Batches of unfertilized oocytes were exposed for 3 h to 0, 0.01, 0.1, 1, or 10 µg/ml separately to perfluorooctanoic acid, perfluorohexanoic acid, or perfluorooctanesulfonic acid in either coelomic fluid or Cortland's solution. After exposure, the gametes were fertilized and rinsed with dechlorinated water. Egg yolk was aspirated from a subset of fertilized eggs for PFAS quantification. Each PFAS was detected in yolks of eggs exposed to the respective PFAS, and yolk concentrations were directly proportional to concentrations in aqueous media to which they were exposed. Exposure in coelomic fluid or Cortland's solution resulted in similar concentrations of PFAS in egg yolks. Ratios of PFAS concentrations in oocytes to concentrations in exposure media (oocyte fluid ratios) were <0.99 when exposed from 0.01 to 10 µg/ml and <0.45 when exposed from 0.1 to 10 µg/ml for both media and all three PFAS, demonstrating that the water solubility of the chemicals was relatively great. Prefertilization exposure of eggs effectively introduced PFAS into unfertilized egg yolk. This method provided a means of mimicking maternal transfer to evaluate toxicity to developing embryos from an early stage. This method is more rapid and efficient than injection of individual fertilized eggs and avoids trauma from inserting needles into eggs. Environ Toxicol Chem 2021;40:3159-3165. © 2021 SETAC.

Chronic toxicity of oil sands tailings pond sediments to early life stages of fathead minnow (Pimephales promelas).

In this study fathead minnow (Pimephales promelas) embryo-larval stages were exposed to two oil sands tailings pond sediments which had previously been shown to decrease the survival of embryo-larval larval stages of walleye (Sander vitreus) and northern pike (Esox lucius). Fathead minnow are standard test species and we wanted to compare their sensitivity to the other two species. Fathead minnow larvae were exposed for 20 days (5 days in the egg stage and 15 days in the larval stage) with daily renewal of sediments and waters. Sediments contained polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs (APAHs). Results from an earlier study showed that Sediment 1 contained 173 µg/g total PAHs + APAHs (97 % alkylated), and sediment 2 contained 401 µg/g total PAHs + APAHs (95 % alkylated). Fathead minnow larvae exposed to oil sands tailings pond sediments had decreased survival, decreased weight, and increased deformities. Fathead minnow survival was unaffected at the embryo stage and at hatch. Most deaths occurred at the larval stages 1-8 days after hatching, showing the importance of exposing the fish for at least a week after hatch. Toxicity was seen at 0.2 g/L of sediment, which was equivalent to the addition of 35 and 80 µg total PAHs + APAHs to 1 L of overlying water for sediment 1 and 2, respectively. When compared to embryo-larval northern pike and walleye results from previous studies, all three species of fish responded more strongly to sediment 2 compared to sediment 1. For effects on lethality, fathead minnow were equally sensitive to pike, but walleye were 5-28 times more sensitive to the lethal effects of the sediments compared to both fathead minnow and pike. The study (and comparisons to our previous studies) shows the difference in sensitivity between a model laboratory species (fathead minnow) and some species of wild fish that are highly relevant to the oil sands area of Alberta.

Oil sands tailings pond sediment toxicity to early life stages of northern pike (Esox lucius).

The Athabasca River in Alberta flows through natural sources of eroding oil sands bitumen and oil sands mining operations that may result in low level contamination of surface waters. Northern pike (Esox lucius) are apex predators and important food and game fish species native to the Athabasca River system. This species has the potential to be exposed to both natural and anthropogenic sources of contamination from oil sands related materials throughout its life cycle. Pike are difficult to rear in the laboratory and little information exists on the toxicity of oil sands related materials to this key indigenous fish species. In this study, the potential effects of two sediment samples collected from different areas of one tailings pond in the Athabasca oil sands area are assessed in a daily renewal bioassay on early life stages of northern pike. Gametes were collected from spawning wild pike captured from a reference site outside of the oil sands area. Fertilized eggs were exposed to control water or increasing concentrations of tailings pond sediments for 21days, coinciding with initiation of exogenous feeding and completion of yolk absorption. Developing fish were examined for survival and changes in body weight, length, and development. Embryos exhibited increased developmental abnormalities and decreased growth and survival with increasing sediment concentration. Both sediment samples had similar levels of naphthenic acids and similar types of PAHs, with alkylated PAHs dominating. However, concentrations of total and alkylated PAHs differed between sediment samples and were related to increasing developmental abnormalities and decreased growth and survival. This is consistent with developmental changes observed with exposure to PAHs in other fish species. These results provide information on the effects of tailings pond sediments comprising mixtures of PAHs and alkylated PAHs on the development and survival of a key species in the northern aquatic ecosystem.

The effect of oil sands tailings pond sediments on embryo-larval walleye (Sander vitreus).

Walleye (Sander vitreus) are a commercially important North American fish species that inhabit the Athabasca River. This river flows through the Athabasca oil sands where natural sources of bitumen erode from the McMurray formation. Little information is available on responses of walleye embryos to oil sands tailings pond sediments in a laboratory setting. The current study describes the design and implementation of a daily-renewal bioassay to assess the potential effects of tailings pond sediments from the Athabasca oil sands area on walleye development. Developing walleye embryos were exposed to increasing concentrations of two tailings pond sediments (collected in the Athabasca oil sands area) until the completion of yolk absorption in control fish. Sediments from the tailings pond represent a mixture of polycyclic aromatic hydrocarbons (PAHs) and alkylated PAHs. During the 31 day exposure, the walleye were examined for mortalities, weight, length and developmental abnormalities to provide an initial evaluation of the effects of the oil sands tailings pond sediments. Walleye embryo survival differed between the tailings pond sediments, and survival decreased with increasing sediment concentration. Alkylated PAH content differed between the two tailings pond sediments and lower embryo survival corresponded to higher total and alkylated PAH content. Tailings pond sediment-exposed walleye exhibited a delay in development, as well as increased percentages of larvae with heart and yolk sac edema, and cranial and spinal malformations. These abnormalities in development are often associated with PAH and alkylated PAH exposure. This study provides an exposure design that can be used to assess sediment toxicity to early developmental stages of a fish species not commonly tested in the lab, and lays the groundwork for future studies with this and other difficult-to-culture species. These results offer information on the potential effects of tailings pond sediments containing PAH/alkylated PAH mixtures on walleye development and survival.

Parasitological Analysis and Gill Histopathology of Pearl Dace (Semotilus Margarita) and Brook Stickleback (Culaea Inconstans) Collected from the Athabasca Oil Sands Area (Canada).

Pearl dace (Semotilus margarita) and brook stickleback (Culaea inconstans) were collected from tributaries of the Athabasca River (Alberta, Canada), upstream (reference site) and downstream of oil sands deposits where fish were expected to be exposed to naturally occurring oil sands constituents. The objective was to determine if fish collected from these sites exhibited differences in the prevalence or intensity of infection by parasites or in gill histology. Dace did not display significant differences in these parameters. Alternately, upstream stickleback were predominantly infected by complex life history parasites, while downstream fish were primarily infected by parasites with simpler life histories. Moreover, downstream stickleback exhibited significantly more clubbing and aneurysms in secondary gill lamellae relative to upstream fish. This suggested a difference in habitat quality between upstream and downstream sites. However, based on basic body condition parameters of the fish, it would appear that any impacts upon the health of the fish due to the presence of naturally occurring oil sands associated chemical constituents would have been minor.

Systemic thyroid hormone is necessary and sufficient to induce ultraviolet-sensitive cone loss in the juvenile rainbow trout retina.

Rainbow trout possess ultraviolet-sensitive (UVS) cones in their retina that degenerate naturally during development. This phenomenon can be induced with exogenous thyroxine [T4, a thyroid hormone (TH)] treatment. However, the previous T4 exposure experiments employed static water immersion; a method that could introduce confounding stress effects on the fish. Because of this, it was uncertain if T4 alone was sufficient to induce retinal changes or if stress-related hormones were also necessary to initiate this process. Furthermore, it was unclear whether endogenous T4 was the factor responsible for initiating natural UVS cone loss during development. The current study examined the role of systemic T4 on the juvenile rainbow trout retina using a slow-release implant. Exogenous T4 treatment resulted in SWS1 opsin downregulation and UVS cone loss after four weeks of exposure, signifying that T4 is sufficient to induce this process. Blocking endogenous T4 production with propylthiouracil (PTU, an anti-thyroid agent) attenuated SWS1 downregulation and UVS cone loss in the retina of naturally developing rainbow trout, suggesting that endogenous T4 is necessary to initiate retinal remodelling during development. Quantitative real-time RT-PCR analysis demonstrated that several TH-regulating components are expressed in the trout retina, and that expression levels of the TH receptor isoform TRbeta and the type 2 deiodinase (D2) change with T4 treatment. This suggests that T4 may act directly on the retina to induce UVS cone loss. Taken together, these results demonstrate that systemic TH is necessary and sufficient to induce SWS1 opsin downregulation and UVS cone loss in the retina of juvenile rainbow trout.

Changes in thyroid hormone reception precede SWS1 opsin downregulation in trout retina.

Rainbow trout undergo natural cone degeneration and thus are interesting models for examining mechanisms of neural degeneration. They have ultraviolet-sensitive (UVS) cones that are lost over most of the retina during development; only a small functional population remains in the dorsal retina. How this spatial distribution of UVS cones is maintained is unclear. Thyroxine (T4) induces UVS cone loss, and local thyroid hormone regulation was hypothesized to control UVS cone distribution. Thyroid hormone receptor alpha (TRalpha), thyroid hormone receptor beta (TRbeta) and Type 2 deiodinase (D2) regulate thyroid hormone exposure to target cells. Regional retinal expression of these genes was investigated during exogenous T4 treatment and natural smoltification of rainbow trout. Each retina from dark-adapted parr, T4-treated parr and natural smolts was divided into four quadrants, and total RNA was isolated. Quantitative real-time RT-PCR analysis demonstrated that all retinal quadrants had increased accumulation of TRbeta transcripts 2 days post-T4 treatment, corresponding to initiation of SWS1 opsin downregulation. Smolts exhibited decreased accumulation of TRalpha and TRbeta transcripts in all quadrants, but this effect was most pronounced in the dorso-temporal (DT) retinal quadrant where UVS cones persist. By contrast, in 2 day T4-treated parr, the DT quadrant showed increased expression of TRalpha and TRbeta. Furthermore, D2 transcripts decreased in the DT quadrant of T4-treated parr but increased in the DT quadrant of smolts. These results suggest that T4 upregulates TRbeta expression to initiate SWS1 opsin downregulation, while TRalpha and TRbeta downregulation occurs to prevent natural loss of UVS cones from the DT retina.

Distribution and regional stressor-induced regulation of corticotrophin-releasing factor binding protein in rainbow trout (Oncorhynchus mykiss).

The corticotrophin-releasing factor (CRF) system plays a key role in the co-ordination of the physiological response to stress in vertebrates. Although the binding protein (BP) for CRF-related peptides, CRF-BP, is an important player in the many functions of the CRF system, the distribution of CRF-BP and the impact of stressors on its expression in fish are poorly understood. In the present study, we describe the distribution of CRF-BP in the brain and peripheral tissues of rainbow trout (Oncorhynchus mykiss) using a combination of real-time reverse transcriptase-polymerase chain reaction, in situ hybridisation and immunohistochemistry. Our results indicate a widespread and highly localised distribution of CRF-BP in the central nervous system, but do not support a significant peripheral production of the protein. Major expression sites in the brain include the area ventralis telencephali, nucleus preopticus, anterior and lateral tuberal nuclei, and the posterior region of the pituitary pars distalis. We further characterise changes in CRF-BP gene expression in three discrete brain regions after exposure to 8 h and 24 h of social stress or hypoxia. The plasma cortisol concentration in subordinate fish was much higher than in dominant fish and controls, and was indicative of a relatively severe stressor. By contrast, the increase in plasma cortisol concentration in fish exposed to hypoxia was characteristic of the response to a mild stressor. Changes in CRF-BP gene expression were only observed after 24 h of either stressor, and were region-specific. CRF-BP mRNA in the telencephalon increased in both subordinate fish and fish exposed to hypoxia, but CRF-BP in the preoptic area only increased after 24 h of hypoxia exposure. In the hypothalamus, CRF-BP mRNA levels decreased in dominant fish relative to controls after 24 h. Taken together, our results support a diverse role for CRF-BP in the central actions of the fish CRF system, but a negligible role in the peripheral functions of circulating CRF-related peptides. Furthermore, the differential changes in forebrain CRF-BP mRNA appear to occur independently of the hypothalamic-pituitary-inter-renal axis.

Effect of fasting on thyroid hormone levels, and TR(alpha) and TR(beta) mRNA accumulation in late-stage embryo and juvenile rainbow trout, Oncorhynchus mykiss.

The accumulation of mRNA encoding for hepatic and intestinal T3-receptor (TR) and body and liver masses were measured in fed and 3-week fasted juvenile and swim up stage rainbow trout embryos. Plasma and total body thyroid hormone (TH) levels were measured for juvenile and swim up stages, respectively. Fasted juveniles exhibited a lower hepatosomatic index (HSI), liver mass and plasma T4 and T3 concentrations than fed animals, but there were no changes in body mass or the accumulation of mRNA encoding for either of the TR(alpha) or TR(beta) isoforms in liver or intestine. TR(beta) mRNA accumulation was greater than TR(alpha) mRNA accumulation in both tissues. Fasted embryos had lower whole body TH levels and body, liver and intestinal tract masses, in addition to a lower intestinosomatic index. However, there was no change in HSI. Fasting did not affect whole body or hepatic TR(alpha) and TR(beta) mRNA accumulation, although intestinal tract TR(alpha) and TR(beta) mRNA accumulation was lower in the fasted embryos. The HSI and body mass changes in fasted juvenile and embryo stages, respectively, indicated that both developmental stages were impacted by fasting. Both stages also showed evidence of decreased TH production. The lower TR gene expression in the intestinal tract of fasted embryos may suggest a role for THs in the transitional stage of intestinal development during this period of development.

Assessment of thyroid function in adult medaka (Oryzias latipes) and juvenile rainbow trout (Oncorhynchus mykiss) using immunostaining methods.

The effect of injected bovine TSH on the pattern of anti-T(4) and anti-T(3) immunostaining of the thyroid tissue was examined in rainbow trout (Oncorhynchus mykiss) and medaka (Oryzias latipes) to determine if the previously reported immunostaining of the cytoplasm of thyrocytes is due to the presence of colloid pinocytotic vesicles and is thus indicative of thyroid hormone release. We hypothesized that the number of immunostained thyrocytes should increase following a TSH challenge, and this should parallel other indicators of increased thyroid activity. In medaka, immunostained thyrocytes were only found following the TSH challenge, and were most marked after 24 to 72 hours; the immunostaining was associated with large colloid-filled cytoplasmic vesicles. In trout, the number and staining intensity of immunostained thyrocytes were increased after the TSH challenge; the immunostaining was present throughout the cytoplasm of the thyrocytes. These findings support the working hypothesis that the immunostaining of the thyrocytes is associated with the pinocytosis of thyroglobulin by the thyrocytes in parallel with an increase in release of thyroid hormone, and that this investigational approach provides a reliable indicator of thyroid hormone release activity.

Morphological and functional development of the thyroid tissue in rainbow trout (Oncorhynchus mykiss) embryos.

The development of the thyroid tissue in rainbow trout (Oncorhynchus mykiss) embryos between 20 and 88 days postfertilization (dpf) was studied using traditional histological methods in combination with immunostaining using antibodies raised against L-thyroxine (T4) and triiodo-L-thyronine (T3). In 20 dpf embryos, the thyroid primordium appeared as a simple tube aligned with the dorsal surface of the ventral aorta. The tubular primordium becomes progressively bifurcated at each end and follicles are formed by pinching off balls of cells from the tips of the bifurcations. The secretory activity of the thyroid tissue appears first as a synthesis phase, evident at 30 dpf, characterized by T4 and T3 immunostaining present only in the follicle lumen. A later hormone release or secretory phase was first evident for T4 immunostaining at 43 dpf and for T3 immunostaining at 46 dpf and was characterized by the presence of immunostaining both within the follicle lumen and also in the cytoplasm of some (but not all) thyrocytes. This stage was also coincident with a marked increase in the number of pituitary thyrotrophs. The proportion of embryos exhibiting immunostained thyrocytes increased progressively from 43 to 52 dpf, at which stage all embryos had T4- and T3-immunostained thyrocytes.