James T. Oris

CYP1A expression in liver and gill of rainbow trout following waterborne exposure: implications for biomarker determination

Abstract The purpose of this study was to compare the time-course for induction of cytochrome P4501A (CYP1A) mRNA levels between liver and gill tissue from rainbow trout (Oncorhyncus mykiss) following waterborne exposure to the polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP). Liver and gill CYP1A mRNA levels and CYP1A catalytic activity were rapidly induced following exposure to BaP concentrations of less than 1 μg l−1. The rise of CYP1A mRNA levels ahead of the rise of CYP1A catalytic activity demonstrated the typical pattern of a gene that is under transcriptional control. Nuclear run-on assays with control and BaP-exposed fish demonstrated that transcriptional activation of the CYP1A gene is an important regulator of CYP1A mRNA levels during the first 24 h of exposure. Liver CYP1A mRNA levels were induced between 6 and 72 h of exposure to 1.23±0.08 μg l−1 BaP but decreased to basal levels between 72 and 120 h of exposure. In comparison, gill CYP1A mRNA levels were induced after 6 h of exposure and remained at this high level of induction through 120 h of exposure to 0.78±0.11 μg l−1 BaP. Gill BaP-hydroxylase activity was maximally induced between 24 and 120 h of exposure, and this time period corresponded with the time period when liver CYP1A mRNA levels decreased back to basal levels. Extensive first-pass metabolism of BaP by the gill after 24 h of exposure may have prevented the majority of parent BaP that was cleared by the gill from entering circulation and maintaining induction of liver CYP1A mRNA levels. The comparison between liver and gill CYP1A mRNA levels and CYP1A catalytic activity demonstrated that CYP1A expression should be measured in tissues that are proximate to the environment when assessing fish from contaminated environments.

Anthracene reduces reproductive potential is maternally transferred during long-term exposure in fathead minnows

Abstract The long-term effects of anthracene (a phototoxic 3-ring polycyclic aromatic hydrocarbon (PAH) on the reproductive potential of fathead minnows (Pimephales promelas) were examined. Sexually mature adult fish (1 male and 2 females per 18-1 aquarium) were exposed in the absence of solar ultraviolet radiation (SUVR) to 0, 6, or 12 μg anthracene/l for 6 weeks, followed by concentration increases to 12 and 20 μg anthracene/l for the latter 2 treatment groups for 3 wk. Dosing was conducted via a flow through dosing system. Eggs were collected daily, placed into clean water (i.e., no anthracene) and divided into SUVR and no-SUVR exposure groups. Survivorship and development of eggs was followed until 96 h posthatch. Significant bioconcentration of anthracene was observed in the eggs laid, in the gonads and in the carcasses of the spawning fish in all treatments except controls. Decreased reproductive output (number of eggs laid) was observed in all anthracene exposed fish. Maternal exposure to anthracene (in the absence of SUVR) also caused reductions in the survivorship of those eggs (percent hatch) and fry (percent survival to 96 h posthatch) reared under SUVR. Teratogenic effects (internal hemorrhaging, edema, and eye and yolk deformities) were also observed in fry maternally exposed to anthracene with subsequent exposure to SUVR. Decreased survivorship of fry which were maternally exposed to anthracene but had no subsequent exposure to SUVR was also observed. These findings provide evidence that anthracene, previously thought to be non-toxic in the absence of SUVR, may cause reproductive impairment in fish.

Accumulation and food chain transfer of fluoranthene and benzo[a]pyrene in Chironomus riparius and Lepomis macrochirus

This research investigated the accumulation of polycyclic aromatic hydrocarbons (PAHs) by Chironomus riparius from sediments and transfer of these contaminants to bluegill, Lepomis macrochirus. Experiments were conducted in laboratory microcosms containing sediments spiked with either benzo[a]pyrene (BAP) or fluoranthene (FLU). Chironomids rapidly accumulated PAHs from sediments. Concentrations of BAP and FLU in chironomids increased with sediment concentration; however, FLU accumulated to a much greater extent. At sediment concentrations ranging from 47 to 4,040 μg/kg, levels of FLU in chironomids ranged from below detection to 181,000 μg/kg. In contrast, the maximum concentration of BAP measured in chironomids at similar sediment levels was 6,030 μg/kg. Levels of FLU and BAP in bluegill that were fed contaminated chironomids were generally low, indicating either low uptake or rapid metabolism of these compounds.Bioturbation of sediments by chironomids decreased water clarity and released sediment-associated BAP to overlying water. BAP in water and in C. riparius increased significantly with chironomid density. In experiments where bluegill were exposed to BAP from water, direct contact with sediments, and chironomids, each source contributed to total body burden. The results of the experiments indicated that PAHs in sediments may be mobilized and made available to benthic invertebrates and fish.

An ultrastructural examination of the mode of UV-induced toxic action of fluoranthene in the fathead minnow, Pimephales promelas

Juvenile fathead minnows (Pimephales promelas) were exposed to fluoranthene (< 0.2, 6.1, and 12.5 μg l−1) in the presence of solar ultraviolet radiation (SUVR) for 6, 12, 24, 48, and 96 h. Ultrastructural pathology of the secondary gill lamellae was examined using transmission electron microscopy. Following 12 h of exposure to 12.5 μg l−1 fluoranthene and SUVR (UV-A = 49.5 μW cm−2, UV-B = 4.2 μW cm−2), the outer mucosal layer of epithelial cells exhibited cellular swelling and dilation of endoplasmic reticulum and mitochondria. At 48 h, additional morphological alterations in mucosal cells included hypertrophy, blebbing of the plasma membrane, and the presence of cytolysosomes, myelinoid bodies, and lipid droplets. The staining characteristics of these droplets were consistent with that of neutral unsaturated lipids, which suggested that their formation was the result of rapid lipid peroxidation reactions. In addition, a second physiologically distinct reaction occurred within the secondary lamellae following 24 h of exposure. This reaction was inflammatory in nature, and as the exposure progressed, was characterized by edema, leukocyte infiltration, and vasoconstriction. In those fish exposed to 6.1 μg l−1 fluoranthene and SUVR, a similar progression compared to 12.5 μg l−1 exposure of mucosal cell damage and inflammatory-type reaction was evident beginning at 24 h of exposure. The net result of mucosal cell hypertrophy and the inflammatory-type reaction was a significant increase in the water-blood diffusion distance of 294% and 285% for those fish exposed to SUVR and the 12.5 and 6.1 μg l−1 fluoranthene treatments, respectively, suggesting that lethality was a consequence of respiratory stress due to the decreased oxygen diffusion capacity of the gills. These observations demonstrate that the mode of action of photo-induced fluoranthene toxicity in fish is a disruption of mucosal cell membrane function and integrity.

Effect of anthracene and solar ultraviolet radiation exposure on gill ATPase and selected hematologic measurements m the bluegill sunfish (Lepomis macrochirus)

Anthracene, a polycyclic aromatic hydrocarbon, is acutely toxic to fish when they are exposed simultaneously to solar ultraviolet radiation (SUVR). However, the physiological mechanism of acute anthracene photo-induced toxicity is not known. The purpose of this study was to investigate possible modes of action associated with simultaneous anthracene and SUVR exposure using blood and gill biochemistry in bluegill sunfish (Lepomis macrochirus). Fish were exposed to anthracene (< 0.01 and 7.04 μg/l) and SUVR in a flow-through system. Following 96 h of anthracene and SUVR exposure, fish exhibited significant increases in hematocrit and significant decreases in whole blood hemoglobin content when compared to no-anthracene controls. Evidence of hemolysis was also observed in anthracene and SUVR exposed fish. Using in vitro enzyme analysis, both Na,K-ATPase and Mg-ATPase were signficantly inhibited in gill tissue homogenates exposed to anthracene and SUVR when compared to homogenates exposed to SUVR alone. These blood and gill measurements provide evidence of osmotic stress in exposed fish. Given the results presented in this and in previous studies, we conclude that there are numerous sites of acute toxic action with anthracene and SUVR exposure in fish and that this toxicity appears to be associated with a general disruption of cell membrane function.

Mercury toxicity in livers of northern pike (Esox lucius) from Isle Royale, USA

Many laboratory studies have documented that mercury can be toxic to fish, but it is largely unknown if mercury is toxic to fish in their natural environments. The objective of our study was to investigate the toxic effects of mercury on northern pike (Esox lucius) at Isle Royale, Michigan. In 124 northern pike from eight inland lakes, concentrations of total mercury in skin-on fillets ranged from 0.069 to 0.622 microg/g wet mass (wet wt). Concentrations of total mercury in livers increased exponentially compared with concentrations in fillets, to a maximum of 3.1 microg/g wet wt. Methylmercury constituted a majority of the mercury in livers with total mercury concentrations <0.5 microg/g wet wt, but declined to 28-51% of the mercury in livers with total mercury concentrations >0.5 microg/g wet wt. Liver color (absorbance at 400 nm) varied among northern pike and was positively related to liver total mercury concentration. The pigment causing variation in liver color was identified as lipofuscin, which results from lipid peroxidation of membranous organelles. An analysis of covariance revealed lipofuscin accumulation was primarily associated with mercury exposure, and this association obscured any normal accumulation from aging. We also documented decreased lipid reserves in livers and poor condition factors of northern pike with high liver total mercury concentrations. Our results suggest (i) northern pike at Isle Royale are experiencing toxicity at concentrations of total mercury common for northern pike and other piscivorous fish elsewhere in North America and (ii) liver color may be useful for indicating mercury exposure and effects in northern pike at Isle Royale and possibly other aquatic ecosystems and other fish species.

Suspended C60 nanoparticles protect against short-term UV and fluoranthene photo-induced toxicity, but cause long-term cellular damage in Daphnia magna

The increased production of nanotechnology materials is a potential source of nano-sized particles (NSPs) in aquatic ecosystems. Meanwhile, polycyclic aromatic hydrocarbons (PAHs), in the presence of ecologically relevant levels of ultraviolet radiation (UV), can be acutely toxic to aquatic species including fish and invertebrates. Considering that suspended carbon-based NSPs (e.g., C60 fullerenes) may act in similar ways as dissolved organic matter (DOM) by altering the bioavailability of PAHs, the objective of this research was to determine the effect of suspended C60 on the photo-induced toxicity of fluoranthene. Transmission electron microscopy indicated that the presence of C60 protected cellular components (e.g., mitochondria, microvilli, and basal infoldings) in organisms exposed to UV and fluoranthene phototoxicity in short-term exposures. However, we found that long-term exposure (21d) of low-level C60 caused significant cellular damage in the Daphnia magna alimentary canal. This paper highlights the importance of examining the interactions between existing stressors and nanoparticles in the aquatic environment.

Regulatory aspects on the use of fish embryos in environmental toxicology

Animal alternative tests are gaining serious consideration in an array of environmental sciences, particularly as they relate to sound management of chemicals and wastewater discharges. The ILSI Health and Environmental Sciences Institute and the European Centre for Ecotoxicology and Toxicology of Chemicals (ECETOC) held an International Workshop on the Application of the Fish Embryo Test in March, 2008. This relatively young discipline is following advances in animal alternatives for human safety sciences, and it is advisable to develop a broad comparison of how animal alternative tests involving fish are viewed in a regulatory context over a wide array of authorities or advising bodies. These include OECD, Western Europe, North America, and Japan. This paper summarizes representative practices from these regions. Presently, the global regulatory environment has varying stances regarding the protection of fish for use as an experimental animal. Such differences have a long-term potential to lead to a lack of harmony in approaches to fish toxicity testing, especially for chemicals in commerce across multiple geographic regions. Implementation of alternative methods and approaches will be most successful if accepted globally, including methods of fish toxicity testing. An important area for harmonization would be in the interpretation of protected and nonprotected life stages of fish. Use of fish embryos represent a promising alternative and allow bridging to more technically challenging alternatives with longer prospective timelines, including cell-based assays, ecotoxicogenomics, and QSARs.

Patterns of spatial and temporal variability of UV transparency in Lake Tahoe, California‐Nevada

Lake Tahoe is an ultra-oligotrophic subalpine lake that is renowned for its clarity. The region experiences little cloud cover and is one of the most UV transparent lakes in the world. As such, it is an ideal environment to study the role of UV radiation in aquatic ecosystems. Long-term trends in Secchi depths showed that water transparency to visible light has decreased in recent decades, but limited data are available on the UV transparency of the lake. Here we examine how ultraviolet radiation varies relative to longer-wavelength photosynthetically active radiation (PAR, 400-700 nm, visible wavelengths) horizontally along inshore-offshore transects in the lake and vertically within the water column as well as temporally throughout 2007. UV transparency was more variable than PAR transparency horizontally across the lake and throughout the year. Seasonal patterns of Secchi transparency differed from both UV and PAR, indicating that different substances may be responsible for controlling transparency to UV, PAR, and Secchi. In surface waters, UVA (380 nm) often attenuated more slowly than PAR, a pattern visible in only exceptionally transparent waters with very low dissolved organic carbon. On many sampling dates, UV transparency decreased progressively with depth suggesting surface photobleaching, reductions in particulate matter, increasing chlorophyll a, or some combination of these increased during summer months. Combining these patterns of UV transparency with data on visible light provides a more comprehensive understanding of ecosystem structure, function, and effects of environmental change in highly transparent alpine and subalpine lakes such as Tahoe.

Effect of water temperature and dissolved oxygen concentration on the photo-induced toxicity of anthracene to juvenile bluegill sunfish (Lepomis macrochirus)

Abstract Polycyclic aromatic hydrocarbons (PAHs) are widespread pollutants in the aquatic environment and demonstrate a wide variety of toxic effects in freshwater and marine organisms. We have previously documented photo-induced toxic effects of a model PAH (i.e. anthracene) in fish. The goal of our ongoing studies is to examine environmental factors which may enhance or mitigate this toxicity in order to better estimate the risks of phototoxic PAHs to aquatic organisms. The purpose of the present study was to determine the effect of water temperature and oxygen concentration on the photo-induced toxicity of anthracene to juvenile bluegill sunfish (Lepomis macrochirus). Bluegill sunfish were exposed to anthracene ( μg l ) and solar ultraviolet radiation (SUVR) at different water temperatures (20 and 30°C) and oxygen concentrations (5.0, 6.9 and 8.1 mg O2/1 for 120 h in a laboratory flow-through system. A trend toward an inverse relationship was found between water temperature and LC50 values, although no significant temperature effect was observed. A nonlinear relationship was found between oxygen concentration and LC50 values, with significantly increased toxicity at the middle oxygen concentration. Opercular ventilation rates were inversely related to oxygen concentration and directly related to anthracene concentration. A statistical relationship was developed on the basis of oxygen concentration to predict LC50 values. We conclude that dissolved oxygen concentration is an important environmental factor in the assessment of photo-induced toxicity of anthracene to fish.