Tissue slices can be made from organs, such as liver, kidney, brain, and heart, and from various species including humans, dogs, non-human primates, rats and mice. It has been demonstrated that human and rat liver slices are viable for up to 2 days, and liver slices have been extensively used as an in vitro method to study hepatic drug metabolism and toxicity in humans. The objective of this study was to determine the utility of canine hepatic slices as an in vitro model for studying drug metabolism and hepatotoxicity in dogs. Canine hepatic slices were incubated in media containing various drugs to determine the hepatotoxicity of the agents and the ability of the slices to metabolize the drugs. The toxicity of phenobarbital, primidone, lidocaine and carprofen to canine hepatic slices was assessed by determining changes in supernatant concentrations of potassium ions and adenosine triphosphate (ATP); histologic lesions were determined as necrosis, extent of vacuolation and severity of vacuolation. Xenobiotic drug metabolizing enzymatic activity was investigated by determining the metabolism of lidocaine to monoethylglycinexylidide (MEGX), and administration of phenobarbital plus primidone was used as a positive control for hepatotoxicity in dogs. The function of drug-metabolizing enzymes was demonstrated by the successful metabolism of lidocaine to MEGX. Carprofen, a drug which causes idiosyncratic hepatic disease in dogs, did not show any hepatotoxicity at concentrations of 10, 50 and 100 µg/ml using potassium ion levels, ATP concentrations and histology as indicators of hepatotoxicity. Slices incubated in media without drug showed no toxicity over 24 hours based on potassium ion and ATP supernatant concentrations while significant increases in histologic lesions were noted at 8, 12 and 24 hours. Canine hepatic slices were a useful model for examining drug metabolism and toxicity for up to 24 hours.

Alzheimer’s disease (AD) is a neurodegenerative disorder that is one of such diseases associated with protein aggregation. Aβ is the main protein component of senile plaques in AD, and is neurotoxic when aggregated. In particular, soluble oligomeric forms of Aβ are closely related to neurotoxicity. In this dissertation, we examine the differences in Aβ aggregation intermediates, and final structures formed when only a simple modification in Aβ aggregation conditions is made, the presence or absence of mixing during aggregation. We show that intermediates in the aggregation pathway show significantly different structural rearrangements. The protein stabilities of Αβ species show that spherical aggregates corresponding to the most toxic Αβ species change their structure the most rapidly in denaturant, and that in general, increased toxicity correlated with decreased aggregate stability. In Alzheimer’s disease, even delaying Aβ aggregation onset or slowing its progression might be therapeutically useful, as disease onset is late in life. Small heat shock proteins (sHsps) may be useful for prevention of Αβ aggregation, since sHsps can interact with partly folded intermediate states of proteins to prevent incorrect folding and aggregation. In this research, several small heat shock proteins (sHsps) are tested to prevent Aβ aggregation and toxicity. sHsps used in this research are Hsp17.7, Hsp27, and Hsp20. All types of Hsp20, Hsp20-MBP, His-Hsp20 and His-Hsp20 without 11 residues in C-terminus, can prevent Aβ1-40 aggregation. Hsp20 also prevents Aβ toxicity in the same concentration ranges of it aggregation prevention activity. Hsp17.7 and Hsp27, however, can inhibit Αβ1-40 aggregation but not toxicity. A number of experiments to examine the mechanism of Hsp20 suggest that multivalent binding of sHsp to Aβ is necessary for the toxicity prevention activity. Conclusively, different Aβ incubation conditions in vitro can affect the rate of Aβ fibril formation, the morphology, the toxicity and the conformation of intermediates in the aggregation pathway. Hsp20 rather than other sHsps may be a useful molecular model for the drug design of the next generation of Aβ aggregation inhibitors to be used in the treatment of AD.

Plastic debris are accumulating in our oceans and are degraded into smaller pieces which eventually becomes small enough to be available to lower thropic level organisms. Microplastics, commonly defined as plastic particles <5 mm, are globally distributed and found at remote locations far away from industrialized and populated areas. The effects of macro sized plastics is well understood whilst the effects of microplastics is hard to predict. It is known that microplastics act as transfer vectors for a wide range of toxic chemicals into organisms, and it is also known that the particle itself can cause toxic responses such as increased immune response and endocrine disruption. Researchers utilize virgin plastic pellets in order to determine the toxicological effect of the plastic particle itself, but resent research suggest that these virgin plastics may release chemicals that contribute to the toxic response and thus complicates the interpretation of the results. In present study, five different virgin plastics were allowed to leach in artificial seawater under conditions that mimic those used in particle toxicity studies. Plastics included were polyethylene, polypropylene, polyvinyl chloride, high-density polyethylene and polystyrene. Leachable monomers and oligomers were found in three of the five plastics tested: polyvinyl chloride, high-density polyethylene and polystyrene. Leached compounds from polyvinyl chloride were not identified due to time limitations. Aliphatic hydrocarbons in the size C14-C22 were leached out from high-density polyethylene in the concentration range 0.47 × 10-3 – 1.13 × 10-3 μg ml-1 within 24 hours. Polystyrene was found to leach styrene monomer which reached a concentration of 0.17 μg ml-1 within 24 hours.

The use of biomarkers is considered a promising alternative, or complement, to traditional ecotoxicological assays. Toxic effects are often initially manifested at the molecular or biochemical level, biomarkers are therefore used as sensitive indicators of toxic exposure. Ideally, biomarkers would also indicate reduced fitness and possible later effects at the individual or population levels. However, implementing biomarkers in ecotoxicology is challenging and few biomarkers have an established connection to reduced individual fitness. The aim of this thesis was to increase the value and improve the interpretation of biomarker responses in ecotoxicological studies by examining the impact of confounding factors and the relationship between oxidative biomarkers and reproductive effects in crustaceans. The sensitivity of biomarkers was confirmed in paper I as toxic effects of pharmaceuticals with conserved drug target orthologs were observed at the molecular and biochemical levels both earlier and at lower concentrations than effects on mortality and reproduction. No toxic effects were observed for the pharmaceutical without identified drug target orthologs, thus stressing the importance of considering toxic mechanisms and being aware of the most likely target when evaluating toxic effects also in non-target species. Many xenobiotics and environmental stressors interfere with oxidative processes, making oxidative biomarkers interesting to study in ecotoxicology and stress ecology. Still, feeding rate was identified as a confounding factor for antioxidant capacity (assayed as oxygen radical absorbance capacity, ORAC) and lipid peroxidation in ecotoxicological studies (paper II). However, ORAC normalized to protein was independent of altered feeding rates, hence it can be applied as a suitable exposure biomarker without considering alterations and effects of feeding rate. The connection between reproduction and oxidative stress is dual, as reproduction both can be inhibited by oxidative stress and induce pro-oxidative processes. Further, a positive association was found between ORAC and the occurrence of embryo aberrations in the benthic amphipod Monoporeia affinis (paper III). An association between antioxidant defense and reproduction was also observed for Daphnia magna (paper IV). Threshold values for identification of exposed individuals and prediction of possible later reproductive effects were established for ORAC. This thesis has contributed to diminishing some of the knowledge gaps limiting the use of oxidative biomarkers in ecotoxicology, by contributing to increased understanding of how oxidative biomarkers relate to important life-traits. Moreover, ORAC has been identified as a suitable biomarker of not only exposure, but also reproductive effects. Future research should continue to establish connections between biomarker responses and effects at higher levels, and focus on providing defined threshold values to enable predictions about later effects. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: Manuscript. Paper 4: Manuscript.</p>

Tiriant poveikį vandens ekosistemoms labai svarbu yra įvertinti ne tik nuotekų cheminius rodiklius, bet taip pat įvertinti kompleksinį toksinių medžiagų poveikį jautriems organizmams, kurie reprezentuotų tiriamai ekosistemai sukeliamą poveikį. Šiame darbe buvo atliktas Vilniaus miesto nuotekų kokybės tyrimas, nustatant nuotekų cheminę kokybę, bei toksiškumą vertinant sausumos ir vandens augalais, sėjamąja salota (Lactuca sativa L.), mažąja plūdena (Lemna minor L.) bei dafnijų (Daphnia magna) judrumo slopinimo testu. Darbo tikslas buvo palyginti skirtingų biotestų: sausumos augalų – sėjamosios salotos (Lactuca sativa L.) ir vandens augalų – mažosios plūdenos (Lemna minor L.) augimo slopinimo ir dafnijų (Daphnia magna) judrumo slopinimo testų jautrumą ir tinkamumą bendram nuotekų toksiškumui vertinti. Atlikus cheminę analizę gauta, kad nevalytose nuotekose pH= 7, NH4+ – 100 mg/l, NO2- – 0,03 mg/l, NO3- – 1 mg/l , PO4-3 – 18 mg/l, o nuotekose po išvalymo pH = 7,5, amonio koncentracija sumažėjo 10 kartų (NH4+ – 10 mg/l), nitritų koncentracija padidėjo 16,7 karto (NO2- - 0,5 mg/l), nitratų 20 kartų (NO3- –20 mg/l), o fosfatų koncentracija sumažėjo 30 kartų (PO4-3 – 0,6), tačiau nors ir amonio jonų koncentracija valytose nuotekose sumažėjo, ji viršijo nuotekoms taikomą DLK 1,5 karto. Atlikus sėjamosios salotos (Lactuca sativa L.) biotestavimą, nustatyta, kad šaknelės augimo slopinimas buvo jautriausias galutinis rodiklis. Mažosios plūdenos (Lemna minor L.) testas parodė, jog... [toliau žr. visą tekstą] / The examination of the impact on aquatic life is very important to evaluate not only the water chemical parameters, but also to assess a complex toxic effects on sensitive organisms that are representative of the investigated ecosystem effects. This work was carried out in Vilnius water quality test for chemical water quality and toxicity evaluation of terrestrial and aquatic plants, lettuce (Lactuca sativa L.), the duckweed (Lemna minor L.) and Daphnia (Daphnia magna) acute immobilisation tests. After physicochemical analysis revealed that untreated wastewater pH = 7, NH4+ – 100 mg/l, NO2- – 0.03 mg /l, NO3- – 1 mg /l, PO4-3 – 18 mg/l, and after treatment the effluent pH were 7.5, ammonium concentrations decreased by a factor of 10 (NH4+ – 10 mg /l), nitrite levels increased 16.7 times (NO2- – 0.5 mg / l), 20 times the nitrate (NO3- – 20 mg /l) and phosphate concentration decreased by 30 times (PO4-3 – 0.6), but while ammonium ion concentration in treated wastewater has decreased, it exceeded the limits applicable to discharges of 1.5. The lettuce (Lactuca sativa L.) bioassay showed that root growth inhibition was the most sensitive end point. Duckweed (Lemna minor L.) test showed that the most sensitive indicator was the mass of duckweed. Daphnia (Daphnia magna) mortality undiluted untreated wastewater after 48h was 100% and in treated wastewater – 40%. After a comparative study found that the main agent causing the effluent toxicity to Daphnia was ammonium. The... [to full text]

There is increasing global recognition of the effects of large scale land disturbance by wildfire on a wide range of water and ecosystem services. In 2003, the Lost Creek wildfire burned a contiguous area of 21,000 ha on the eastern slopes of the Rocky Mountains in southern Alberta. This disturbance had a significant and prolonged impact on the water quantity and quality of downstream river reaches and reservoirs in the Oldman watershed. Previous research in this watershed demonstrates that dissolved metal concentrations in rivers draining burned landscapes were 2 to 15 times greater than in unburned reference streams (Silins et al. 2009a). Currently there is no information on the effects of wildfire on the bioaccumulation and toxicity of sediment-associated metals in fire-impacted streams in Alberta. This study was designed to evaluate the linear downstream disturbance effects of wildfire in the Crowsnest River located in southern Alberta. The toxicity and bioaccumulation of particulate-associated metals from wildfire impacted tributaries to the Crowsnest River on freshwater amphipod Hyalella azteca were evaluated. Phillips samplers were deployed to collect suspended solids in streams draining burned zone impacted by the Lost Creek wildfire and reference (unburned) zones within the area. Metal toxicity and bioaccumulation were determined in the laboratory by exposing the epi-benthic freshwater amphipod Hyalella azteca to particulates collected from the Crowsnest River. A metal effects addition model (MEAM) was used to assess the impact of metal mixtures and to predict chronic mortality (Norwood et al. 2013). Increased concentrations of Al, Ba, Co, Cr, Mn and Zn were found in the tissues of H. azteca exposed to particulates from burned watersheds in comparison to the unburned watersheds. H. azteca mean survival was similar when exposed to the particulates samples from both burned and unburned sites indicating that 9 years after this landscape disturbance, there was little impact due to the wildfire. However, at burned site (B1), the observed survival was lower than survival predicted by MEAM. The data suggests that factors other than the metals examined in this study were influencing the survival of H. azteca. The concentrations of sediment-associated metals have decreased in the nine years since the wildfire, and minimal metal toxicity was observed in H. azteca. Although metal toxicity in H. azteca was minimal 9 years after the Lost Creek Fire, the short term effects of wildfire on metal toxicity remain largely unknown. In addition, other factors such as burn severity, stream size and hydroclimatic conditions can influence the effects of wildfire on abundance and diversity of aquatic invertebrates (Minshall et al. 2001). Therefore, the influences of those factors on metal toxicity as a result of wildfire should be rigorously assessed in future studies.

The risk of crude oil exposure to the early life stages (ELS) of fish is difficult to assess, given the complexity of the chemical composition of different oils. The aromatic portion of crude oil contains polycyclic aromatic hydrocarbons (PAH), which are known to be toxic. In 2004, an “effects-driven” fractionation research program was initiated in an attempt to better identify toxic constituents of two unique crude oils, Alaskan North Slope Crude (ANSC; medium-heavy crude) and Scotia Light (SCOT; light crude). The ANSC contained much more PAH than SCOT. These oils induced cytochrome P4501A1 (CYP1A) enzymes in juvenile rainbow trout (Oncorhynchus mykiss) and caused blue-sac disease (BSD) and mortality in larval Japanese medaka (Oryzias latipes). Four unique fractions (F1-F4) were produced from both oils via low temperature vacuum distillation. The F3 contained an array of unsubstituted and alkyl-PAH, and was responsible for most of the CYP1A induction and chronic toxicity associated with whole oil. Cold acetone extraction (CAE) of F3 produced 2 new sub-fractions (F3-1 and F3-2). The F3-1 was rich in alkyl-PAH, was a potent CYP1A inducer, and was chronically toxic to ELS of fish. The F3-1 was further separated into five more sub-fractions via normal phase HPLC (F3-1-1 – F3-1-5). Neither F3-1-1 nor F3-1-2 induced CYP1A or produced BSD, but F3-1-2 was lethal. The F3-1-3, 4, and 5 were all potent CYP1A inducers and were all chronically toxic. Induction of CYP1A proved to be an effective tool for tracing potentially toxic PAH throughout fractionation (Chapter 2), and sub-fractions rich in alkyl-PAH caused the most BSD and mortality (Chapter 3). Alkyl-homologues of phenanthrene, fluorene, naphthobenzothiophene (NBT), pyrene, and chrysene are perhaps the most toxic of the known constituents present in crude oil. The ANSC sub-fractions were more toxic than the SCOT ones, indicating that heavier crude oils with a higher proportion of intermediate-sized alkyl-PAH may be more toxic than lighter crude oils that are comprised of fewer of these compounds. / Thesis (Master, Biology) -- Queen's University, 2007-12-20 13:18:50.794 / This research was accomplished with funding assistance from the Nationmal Oceanic and Atmospheric Administration (NOAA), Petroleum Research Atlantic Canada (PRAC), Environment Canada, and the Department of Fisheries and Oceans.

To minimize the damage caused by oil spills, responders may chemically disperse floating oil into the underlying water before it contacts shorelines and wildlife. Quantifying this strategy’s net ecological and commercial benefits requires an analysis of its effects on subsurface ecosystems and biota. Unfortunately, spill-responders have little empirical data on which to base such an analysis. Herein I report the effects of dispersed oil to early life stages of Atlantic herring (Clupea harengus). Medium South American crude oil (MESA) dispersed with Corexit 9500 caused blue sac disease (BSD) in embryos, but not in free-swimming embryos. The ages of embryos were negatively correlated with their sensitivity to oil, making those that were freshly fertilized the most sensitive. However, sensitivity was also high after hatch. Free-swimming embryos displayed signs of narcosis following brief exposure to dispersed oil. Gametes were also tested; dispersed oil dramatically impaired fertilization success. Toxicity was a function of concentration and duration of exposure, as well as of the life stage exposed. When the duration of exposure was < 24 h, gametes and free-swimming embryos were the most sensitive life stages (i.e. responded to the lowest concentrations). For durations > 24 h, young embryos (< 1 day old) were most sensitive. The results are presented as toxicity models that incorporate developmental stage, oil concentration, and exposure duration. Current effects-forecasting models for oil dispersion are based on published chronic toxicity data, which do not account for the effects of exposure duration and developmental events on toxicity. Thus, the results will better-enable modelers to estimate the effects of realistic exposures to dispersed oil in various locations, including spawning shoals. / Thesis (Master, Biology) -- Queen's University, 2009-04-26 12:55:12.266

Worldwide, estuaries are under increasing pressure from numerous contaminants. There is a need to develop reliable bioassay methodologies to assess the effects of these stressors on estuary health. This thesis aimed to develop and validate toxicity tests in a New Zealand marine harpacticoid copepod species for use in monitoring and evaluating the effects of estuarine pollution. A survey and toxicological assessment of a range of native copepod species resulted in the selection of Quinquelaophonte sp. as the ideal bioassay species. This selection was based on a broad regional distribution, ease of culture and high reproductive rate in the laboratory, sexual dimorphism, and sensitivity to contaminants. To validate the bioassay, spiked sediments were used to expose Quinquelaophonte sp. to three reference compounds representing important categories of estuarine chemical stressors: zinc (a metal), atrazine (a pesticide), and phenanthrene (a polyaromatic hydrocarbon). A method for spiking sediments that Quinquelaophonte sp. inhabit was developed to ensure even contaminant distribution in sediments. Two sediment bioassays using lethal and sublethal endpoints were validated, one acute (96 h) and one chronic (14 d). These assays incorporated both lethal and sublethal endpoints, which included reproductive output and mobility. Acute-to-chronic ratios were calculated for use in environmental risk assessment and to provide insight into the mode of action of the reference contaminants. The chronic sediment bioassay was used to assess sediment quality in three estuaries across New Zealand: Napier, Christchurch and Invercargill. This validated the bioassay for use with naturally-contaminated field sediments with varying mixtures of pollutants and sediment types (coarse sandy to fine silty organic rich sediments). Quinquelaophonte sp. was also tested to assess whether it can be used to characterise multi–generation impacts. After four generations of exposure to zinc, there were changes in acute sensitivity, indicating this species possesses mechanisms for acclimating or adapting to toxic stressors. Sediment bioassays in Quinquelaophonte sp. were successfully developed and validated, offering significant promise as a tool for monitoring effects of pollution in New Zealand estuaries.

Phthalate esters (phthalates) such as dibutylphthalate (DBP) are commonly used as plasticisers and pesticides in a variety of products such as children‟s plastic toys, food packaging, cosmetics, medical equipment (including surgical equipment), and acaricides. Because of their widespread use phthalates are ubiquitous environmental contaminants that humans are commonly exposed to. Phthalates are known endocrine-disrupting chemicals (EDCs) that are well known to cause male reproductive defects such as cryptorchidism (failed descent of the testes) and hypospadias (malformations in the urethra) in a range of different species if they are exposed in utero. They do this by reducing testosterone production in Leydig cells, which are the primary site of testosterone biosynthesis in the male. Because phthalates are dose-additive they are considered to share the same mechanism of toxicity. However, the details of phthalates mechanism of toxicity are not fully understood. The aim of this research was to investigate the effects of DBP on the steroidogenesis pathway using the cultured rat Leydig cell cancer line R2C as a Leydig cell model. R2C cells were exposed to a range of DBP concentrations (10 μg/mL, 5 μg/mL, 1 μg/mL, and 0.1 μg/mL) and their steroid hormone production was analysed using reverse phase HPLC. R2C cells did not synthesise testosterone at detectable levels. However, DBP exposure stimulated cortisol biosynthesis at all concentrations but caused no change in progesterone biosynthesis. This cortisol stimulation in Leydig cells has not been observed before. Because cortisol and testosterone compete for precursors an increase in cortisol synthesis could starve testosterone synthesis of precursors. On top of this it has been shown that glucocorticoids including cortisol have an adverse effect on Leydig cell development reducing steroid production and even causing apoptosis. This could explain how DBP and other phthalates can cause male developmental defects such as cryptorchidism and hypospadias.