Responses of Wild Finescale Dace (Phoxinus neogaeus) to Experimental Spills of Cold Lake Blend Diluted Bitumen at the International Institute for Sustainable Development-Experimental Lakes Area, Northwestern Ontario.

Pipelines carrying diluted bitumen (dilbit) traverse North America and may result in dilbit release into sensitive freshwater ecosystems. To better understand the potential effects of a freshwater oil release, the Boreal-lake Oil Release Experiment by Additions to Limnocorrals project at the International Institute for Sustainable Development-Experimental Lakes Area (Ontario, Canada) modeled seven dilbit spills contained within a 10-m diameter of littoral limnocorrals in a boreal lake. Wild finescale dace (Phoxinus neogaeus) were released in the limnocorrals 21 days after oil addition and remained there for 70 days. Dilbit volumes covered a large range representing a regression of real spill sizes and total polycyclic aromatic compounds (TPAC) between 167 ng L-1 day-1 and 1989 ng L-1 day-1 . We report the effects of chronic exposure on reproductive potential as well as physiological responses in the gallbladder and liver. In exposures >1000 ng L-1 day-1 , there was a significant decrease in fish retrieval, culminating in zero recapture from the three highest treatments. Among the fish from the limnocorrals with lower levels of TPAC (<500 ng L-1 day-1 ), effects were inconsistent. Gallbladder bile fluorescence for a naphthalene metabolite was significantly different in fish from the oil-exposed limnocorrals when compared to the lake and reference corral, indicating that fish in these lower exposures were interacting with dilbit-derived polycyclic aromatic compounds. There were no significant differences in condition factor, somatic indices, or hepatocyte volume indices. There were also no significant changes in the development of testes or ovaries of exposed dace. The results from the present study may serve to orient policymakers and emergency responders to the range of TPAC exposures that may not significantly affect wild fish. Environ Toxicol Chem 2022;41:2745-2757. © 2022 SETAC.

Thyroid disruption and oxidative stress in American kestrels following embryonic exposure to the alternative flame retardants, EHTBB and TBPH.

Brominated flame retardant chemicals, such as 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EHTBB) (CAS #: 183658-27-7) and bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (TBPH) (CAS #: 26040-51-7), have been detected in avian tissues and eggs from remote regions. Exposure to EHTBB and TBPH has been shown to cause oxidative stress and altered thyroid function in rodents and fish, yet no controlled studies have examined potential adverse effects of exposure in birds. Because flame retardants have been detected in wild raptors, we used American kestrels (Falco sparverius) as a model raptor to determine whether in ovo exposure to EHTBB or TBPH affected growth, hatching success, oxidative stress, or thyroid function. We exposed kestrel embryos to nominal concentrations (10, 50, or 100 ng g-1 egg weight) of EHTBB and TBPH via egg-injection on embryonic day 5. Embryonic exposure (~23 d) to EHTBB increased thyroid gland mass, reduced glandular colloid and total thyroxine (T4) in hatchling males and females, whereas deiodinase enzyme activity increased in males but decreased in females. Hatchlings exposed to TBPH in ovo exhibited reduced colloid and increased oxidative stress. Although exposure to EHTBB and TBPH caused several physiological effects (e.g., heart and brain mass), only exposure to 50 ng g-1 EHTBB appeared to reduce hatching success. Our results suggest these flame retardants may be hazardous for predatory birds. Future research should evaluate long-term survival and fitness consequences in birds exposed to these chemicals.

Effects of Environmentally Relevant Residual Levels of Diluted Bitumen on Wild Fathead Minnows (Pimephales promelas).

Transportation of crude oil across North America's boreal ecozone creates the potential for spills in freshwater where less is known about the sensitivity of resident fish than for marine systems. The sensitivity of wild fathead minnows (FHM) to residual concentrations (ppb range) of the water accommodated fraction (WAF) of diluted bitumen (dilbit) was assessed by exposing them for 21 days followed by a 14 days depuration. Target concentrations were well below detection limits for GC-MS, but were estimated by dilution factor (1:100,000 and 1:1,000,000 WAF:water) to contain less than 0.0003 µg/L of polycyclic aromatic compounds. Confinement and handling stress caused by transfer of wild fish into tanks much smaller than their natural range resulted in mortality and lower body condition among all groups, but interactive effects of oil exposures still resulted in females with smaller cortical alveolar oocytes, and males with larger testicular lobe lumen sizes. Additional studies examining the compounded effects of stress and environmentally relevant oil exposures in wild fishes are needed.

In ovo exposure to brominated flame retardants Part II: Assessment of effects of TBBPA-BDBPE and BTBPE on hatching success, morphometric and physiological endpoints in American kestrels.

Tetrabromobisphenol A bis(2,3-dibromopropyl ether) (TBBPA-BDBPE) and 1,2-bis(2,4,6-tribromophenoxy)ethane (BTPBE) are both brominated flame retardants (BFRs) that have been detected in birds; however, their potential biological effects are largely unknown. We assessed the effects of embryonic exposure to TBBPA-BDBPE and BTBPE in a model avian predator, the American kestrel (Falco sparverius). Fertile eggs from a captive population of kestrels were injected on embryonic day 5 (ED5) with a vehicle control or one of three doses within the range of concentrations that have been detected in biota (nominal concentrations of 0, 10, 50 or 100 ng/g egg; measured concentrations 0, 3.0, 13.7 or 33.5 ng TBBPA-BDBPE/g egg and 0, 5.3, 26.8 or 58.1 ng BTBPE/g egg). Eggs were artificially incubated until hatching (ED28), at which point blood and tissues were collected to measure morphological and physiological endpoints, including organ somatic indices, circulating and glandular thyroid hormone concentrations, thyroid gland histology, hepatic deiodinase activity, and markers of oxidative stress. Neither compound had any effects on embryo survival through 90% of the incubation period or on hatching success, body mass, organ size, or oxidative stress of hatchlings. There was evidence of sex-specific effects in the thyroid system responses to the BTBPE exposures, with type 2 deiodinase (D2) activity decreasing at higher doses in female, but not in male hatchlings, suggesting that females may be more sensitive to BTBPE. However, there were no effects of TBBPA-BDBPE on the thyroid system in kestrels. For the BTPBE study, a subset of high-dose eggs was collected throughout the incubation period to measure changes in BTBPE concentrations. There was no decrease in BTBPE over the incubation period, suggesting that BTBPE is slowly metabolized by kestrel embryos throughout their ∼28-d development. These two compounds, therefore, do not appear to be particularly toxic to embryos of the American kestrel.

Sex-specific responses in neuroanatomy of hatchling American kestrels in response to embryonic exposure to the flame retardants bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate.

Bis(2-ethylhexyl)-2,3,4,5-tetrabromophthalate (BEH-TEBP) and 2-ethylhexyl-2,3,4,5-tetrabromobenzoate (EH-TBB), flame retardant components of FireMaster 550® and 600® have been detected in tissues of wild birds. To address the paucity of information regarding potential impacts of flame retardants on the brain, brain volume regions of hatchling American kestrels (Falco sparverius) were evaluated following in ovo injection at embryonic day 5 with safflower oil or to 1 of 3 doses of either BEH-TEBP (12, 60, or 107 ng/g egg) or EH-TBB (11, 55, or 137 ng/g egg). The doses for both chemicals reflected concentrations reported in wild birds. The volumes of the hippocampus and telencephalon and volumetric differences between left and right hemispheres were measured in hatchlings (embryonic day 28). A sex-specific effect of BEH-TEBP on relative hippocampus volume was evident: the hippocampus was significantly enlarged in high-dose females compared to control females but smaller in low-dose females than the other females. There was no significant effect of EH-TBB on hippocampus volume in female kestrel hatchlings or of either chemical in male hatchlings and no effects of these concentrations of EH-TBB or BEH-TEBP on telencephalon volume or the level of symmetry between the hemispheres of the brain. In sum, embryonic exposure of female kestrels to these BEH-TEBP concentrations altered hippocampus volume, having the potential to affect spatial memory relating to ecologically relevant behavior such as prey capture, predator avoidance, and migration. Environ Toxicol Chem 2018;37:3032-3040. © 2018 SETAC.

Female hatchling American kestrels have a larger hippocampus than males: A link with sexual size dimorphism?

The brain and underlying cognition may vary adaptively according to an organism's ecology. As with all raptor species, adult American kestrels (Falco sparverius) are sexually dimorphic with females being larger than males. Related to this sexual dimorphism, kestrels display sex differences in hunting and migration, with females ranging more widely than males, suggesting possible sex differences in spatial cognition. However, hippocampus volume, the brain region responsible for spatial cognition, has not been investigated in raptors. Here, we measured hippocampus and telencephalon volumes in American kestrel hatchlings. Female hatchlings had a significantly larger hippocampus relative to the telencephalon and brain weight than males (∼12% larger), although telencephalon volume relative to brain weight and body size was similar between the sexes. The magnitude of this hippocampal sex difference is similar to that reported between male and female polygynous Microtus voles and migratory and non-migratory subspecies of Zonotrichia sparrows. Future research should determine if this sex difference in relative hippocampus volume of hatchling kestrels persists into adulthood and if similar patterns exist in other raptor species, thus potentially linking sex differences in the brain to sex differences of space use of adults in the wild.

Investigating endocrine and physiological parameters of captive American kestrels exposed by diet to selected organophosphate flame retardants.

Organophosphate triesters are high production volume additive flame retardants (OPFRs) and plasticizers. Shown to accumulate in abiotic and biotic environmental compartments, little is known about the risks they pose. Captive adult male American kestrels (Falco sparverius) were fed the same dose (22 ng OPFR/g kestrel/d) daily (21 d) of tris(2-butoxyethyl) phosphate (TBOEP), tris(2-chloroethyl) phosphate (TCEP), tris(2-chloroisopropyl) phosphate (TCIPP), or tris(1,2-dichloro-2-propyl) phosphate (TDCIPP). Concentrations were undetected in tissues (renal, hepatic), suggesting rapid metabolism. There were no changes in glutathione status, indicators of hepatic oxidative status, or the cholinergic system (i.e., cerebrum, plasma cholinesterases; cerebrum muscarinic, nicotinic receptors). Modest changes occurred in hepatocyte integrity and function (clinical chemistry). Significant effects on plasma free triiodothyronine (FT3) concentrations occurred with exposure to TBOEP, TCEP, TCIPP, and TDCIPP; TBOEP and TCEP had additional overall effects on free thyroxine (FT4), whereas TDCIPP also influenced total thyroxine (TT4). Relative increases (32%-96%) in circulating FT3, TT3, FT4, and/or TT4 were variable with each OPFR at 7 d exposure, but limited thereafter, which was likely maintained through decreased thyroid gland activity and increased hepatic deiodinase activity. The observed physiological and endocrine effects occurred at environmentally relevant concentrations and suggest parent OPFRs or metabolites may have been present despite rapid degradation.

Changes in population, growth, and physiological indices of Longnose dace (Rhinichthys cataractae) in the Red Deer River, Alberta, Canada.

The Red Deer River, Alberta, Canada is a prairie river that is impacted by the point-source input of Red Deer's municipal wastewater effluent and non-point- source agricultural runoff. We used population, growth, and physiological performance end points in longnose dace (Rhinichthys cataractae), an endemic and abundant minnow, to evaluate changes in fish health over a 220 km section of the Red Deer River. Longnose dace immediately downstream of Red Deer had elevated catch per unit effort with larger body and liver sizes compared to upstream of Red Deer sites, likely due to elevated riverine productivity from Red Deer's wastewater effluent. Longnose dace immediately downstream of Red Deer showed depressed testosterone production capacity and elevated ethoxyresorufin-O-deethylase (EROD) activity, which is consistent with exposure to endocrine-disrupting compounds and aromatic hydrocarbons, respectively. Longnose dace 150-180 km downstream of Red Deer had reduced liver and gonad sizes, elevated EROD, and increased 11-ketotestosterone production capacity compared to upstream of Red Deer sites, possibly related to a non-point-source agricultural influence on water quality. Longnose dace populations at the most downstream sites were missing the oldest age classes and might reach sexual maturity faster than at upstream sites, which is consistent with a younger age structure. Our results highlight the importance of assessing multiple performance end points to reveal physiological or reproductive effects in natural fish populations. We have demonstrated how longnose dace populations change over a river impacted by municipal wastewater and agricultural runoff; further studies are required to determine if these changes will influence the long-term viability of longnose dace in the Red Deer River.

Effects of oil sands process-affected waters and naphthenic acids on yellow perch (Perca flavescens) and Japanese medaka (Orizias latipes) embryonic development.

Syncrude Canada Ltd. is currently developing environmentally acceptable oil sands process-affected water management methods as part of their land reclamation strategy. Surface waters of the "wet landscape" reclamation option characteristically have elevated concentrations of sodium sulphate and naphthenic acids (NAs), with low levels of PAHs. The following experiment compared early-life stage responses of yellow perch (Perca flavescens) to those of Japanese medaka (Oryzias latipes) when exposed to Mildred Lake settling basin (MLSB) surface water and a commercial sodium naphthenate (Na-NA) standard. Perch eggs were fertilized and incubated in: 100%, 50%, 20%, 4%, 0.8%, and 0.16% dilutions of MLSB water, as well as 20, 10, 5, 2.5, and 1.25 mg/l solutions of the commercial standard. Medaka embryos were exposed to the same treatments, post-fertilization. Both species demonstrated an increase in the incidence of deformity, and a decrease in length at hatch as NA concentrations increased. MLSB surface water contained higher levels of NAs than the commercial standard, however, showed consistently higher NA threshold effect concentrations for both species. Significant differences between the MLSB water and the Na-NA standard suggest that they contain NA congeners with different toxicity, or other compounds such as PAHs. Species differences in thresholds could be explained by the difference in developmental stage in which the exposures were initiated.

Biochemical and histopathological effects in pearl dace (Margariscus margarita) chronically exposed to a synthetic estrogen in a whole lake experiment.

Potential effects of exposure to the synthetic estrogen 17alpha-ethynylestradiol (EE2) were examined in several species of fish from a lake experimentally treated with environmentally relevant concentrations of the contaminant. Ethynylestradiol was added to Lake 260, a small Precambrian shield lake at the Experimental Lakes Area in northwestern Ontario, Canada, from May to October of 2001, 2002, and 2003. Mean concentrations of EE2 in epilimnetic waters ranged between 4.5 and 8.1 ng/L during the three years, with overall means of 6.1 (+/- 2.8), 5.0 (+/- 1.8), and 4.8 (+/- 1.0) ng/L for the three years, respectively. Male and female pearl dace (Margariscus margarita) captured after EE2 additions began contained up to 4,000-fold higher concentrations of the egg yolk precursor vitellogenin than fish captured from the same lake before the EE2 additions or when compared to fish from reference lakes. Edema in the ovaries, inhibited development of testicular tissue, intersex, and histopathological kidney lesions were all evident in fish exposed to EE2. Some indications that EE2 exposure affected in vitro steroidogenic capacity of the ovaries and the testes existed, although results were not always consistent between years. Pearl dace abundance was similar in the lake treated with EE2 and the reference lake. A trend exists toward a reduced overall population of pearl dace from the treated and reference lakes, as do indications that young-of-the-year size classes are less abundant in the EE2-treated lake. Biochemical and histopathological impacts observed in fish exposed to EE2 in this study have not yet been linked to clear population level impacts in pearl dace. Monitoring of these populations is ongoing.