This dissertation thesis is focus on using toxicity tests with testing organism - terrestrial isopod Porcellio scaber. This organism is use for toxicity assessment of selected anorganic compunds which could contaminate terrestrial ecosystem. This organism was selected for several reasons - sophisticated test methodology; well known biology of these animals and their ecological relevance. Besides classical endpoints such as mortality, change in mass of test specimens and the effect of the presence and concentration of contaminants onto food consumption; we monitored changes in the morphometric characteristics at the level of tissues and cells, the cytotoxicity and possibly bioaccumulation in various parts of the body of the organism. Selected test substance in this thesis were nanoparticles and inorganic salts. Wherein for chosen gold nanoparticles was carried out a complete study presents several levels of organization of living matter (organism, tissue, cell). For selected salts compound, this study was carried out on the behavior of organisms and classical endpoints.

<p>Perfluoroalkyl substances are synthetic organic chemicals of environmental concern because they have been associated with adverse effects in both human epidemiological studies and standard laboratory animals. In the environment, PFAS occur as mixtures, especially in areas with a history of PFAS application, such as aqueous film forming foam (AFFF) sites. Among the PFAS, perfluorooctanesulfonic acid (PFOS) and perfluorohexanesulfonic acid (PFHxS) are the most common, and occur at the highest concentrations. Thus, amphibian populations at or near AFFF sites are at risk of exposure to known bioaccumulative and persistent chemicals, likely compromising the physiology and body condition of the animals. Here, we exposed northern leopard frogs to environmentally relevant concentrations of 0.5 and 1 ppb PFOS and PFHxS, alone or as a mixture comprised of 0.5 ppb PFOS and 0.5 ppb PFHxS. Univariate analyses showed that in the larval stages, tadpoles exposed to PFAS had significantly reduced scaled mass indexes (SMI’s) relative to the control, and only the organisms exposed to PFHxS 0.5 ppb were significantly larger. Sex did not significantly influence toxicity in the later stages (GS 42 & 46), indicating no sex-related effects. Altered body condition (i.e., fat stores) in the larval stages indicate potential effects to energy balance. There is a need to assess fitness-related effects as amphibians’ transition into the terrestrial environment, and include endpoints such as: reproductive, developmental, immunological, mating, feeding, competition, and survival. Early developmental effects in the larval stages also suggests that earlier developmental endpoints may be of interest. Establishing ecological risk assessments for PFAS are necessary, as they are toxic, persistent, and bioaccumulative.</p>

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The mode of feeding exhibited by different organisms may influence the form or quantity of copper that is bioaccumulated. This hypothesis has been tested by exposing 2 species of sea cucumbers, Pentacta anceps and Stichopus chloronotus, which possess different feeding modes, to varying concentrations of copper. The digestive tract and body wall were dissected and analyzed for copper concentration using atomic absorption spectroscopy. A trend was present, exhibiting a small dose dependent curve. The results did not show a significant difference between species or treatments. This study indicates that feeding mode may influence the amount of copper accumulated but it could not be concluded because of a small sample size. The model used in this study demonstrated that copper was being actively precipitated out of the water and deposited into sediment. Future studies focusing on pollutant uptake may find this useful when evaluating the role of feeding mode or habitat.

Nitrogen-, sulfur-, and oxygen-substituted heterocyclic polyaromatic hydrocarbons (heterocyclic PAHs) are ubiquitous in all environmental compartments, often co-occurring with their unsubstituted carbocyclic analogues. However, they have received little attention in the hazard assessment of contaminated sites and little is known about their fate and impacts. Due to their higher polarity resulting from the presence of electronegative atoms possessing lone electron pairs, most heterocycles are expected to be less toxic and bioaccumulative than their homocyclic counterparts, which is perhaps why research on their hazard and fate is much more limited. If this assumption is not true (e.g., because factors other than polarity/hydrophobicity drive their hazard), this may lead to an underestimation of the risks posed by contaminated sites or emissions. Therefore, the main objectives of this thesis are (1) to improve the understanding of the physicochemical properties, biodegradability, ecotoxicity, bioaccumulation, and mobility of these compounds in environmental contexts, (2) to evaluate the effectiveness of different modeling approaches in accurately predicting their properties, and (3) to tackle experimental barriers by designing robust techniques that can produce reliable and reproducible data output in the long run. From the large family of heterocycles, structurally comparable representative compounds with log KOW values (as a measure of hydrophobicity) ranging over four orders of magnitude were selected as model structures. First, the most environmentally relevant physicochemical properties of heterocyclic PAHs were investigated, including water solubility (SW), n-octanol-water partition coefficient (KOW), and organic carbon-water partition coefficient (KOC). The effect of molecular size, type and number of heteroatoms on the solubility and partition coefficients was investigated. These properties were then used to test the performance of in silico models, including Quantitative Structure-Property Relationship (QSPR), Polyparameter Linear Free Energy Relationship (pp-LFER), and Conductor-like Screening Model for Realistic Solvation (COSMO-RS), that predict these properties. A screening-level environmental hazard assessment was performed by integrating the data and evidence from multiple sources. The screening results revealed (i) high hazard for 4- and 5-ring compounds mainly due to high persistence and bioaccumulation potential, (ii) limited availability of experimental data, and (iii) notable uncertainty due to the limited applicability domain of the models, stressing the urgent need for a detailed hazard assessment for this group of compounds. The persistence of heterocyclic PAHs in the environment was assessed experimentally through a series of biodegradation tests under aerobic conditions. These included ultimate biodegradation, primary biodegradation and inoculum toxicity tests at different exposure concentrations. None of the test substances (≥3 rings) were found to be readily biodegradable. Therefore, to stimulate the biodegradation process, the microbial community from a wastewater treatment plant was adapted to a mixture of heterocyclic PAHs and biodegradation tests were performed with either adapted or non-adapted bacteria. Surprisingly, there were trade-offs in bacterial adaptation: a positive outcome (degradation of carbazole) is accompanied by a less desirable outcome (increased sensitivity to toxic effects of benzo[c]carbazole). Four of the compounds were identified as primarily biodegradable, while five compounds showed no evidence of biodegradation in any of the tests, indicating a high potential for environmental persistence. Predictions from mathematical models were also compared with measured results and it was found that the models were only partially successful in predicting the degradation timeframe of the heterocycles tested. Furthermore, the short-term acute aquatic toxicity of heterocyclic PAHs was investigated using three surrogate species from different trophic levels of the aquatic food web, including bacteria (Aliivibrio fischeri), unicellular algae (Raphidocelis subcapitata), and water fleas (Daphnia magna). In order to maintain a stable exposure and to avoid loss of concentration caused mainly due to sorption, the passive dosing method was adapted to routine test protocols. Most of the compounds were identified as highly toxic to the tested organisms, and non-polar narcosis (baseline toxicity) was found to be the most likely mode of toxic action for the tested chemicals. Membrane-water partition coefficients (KMW) of heterocyclic PAHs were also determined by solid-supported lipid membrane binding experiments and modeling. The measured KMW values were closely aligned with the KOW values, suggesting that both may be good descriptors for predicting the baseline toxicity and bioaccumulation potential of the tested heterocyclic PAHs. Lastly, the long-term chronic reproductive toxicity and bioaccumulation of large heterocyclic PAHs were investigated using Daphnia magna as an aquatic model organism. The passive dosing method ensured constant exposure even at very low concentration levels (70 ng L-1 to 68 µg L-1) in large and complex experimental systems. Given the enormous time and effort required to conduct long-term toxicity tests with minimal data output, a method was developed to assess chronic toxicity and bioaccumulation potential in a single test. All four substances tested were highly toxic and bioaccumulative in Daphnia magna. However, daphnids’ fecundity recovered rapidly from the toxic effects of the heterocycles during the depuration period in the absence of chemicals. The Predicted No Effect Concentrations (PNECs) of the test compounds were also estimated from the chronic toxicity data and ranged from 1 to 150 ng L-1 in a freshwater environment. Due to the lack of regulation and attention to these compounds, no systematic environmental monitoring data are available for their concentrations in freshwater. However, environmental concentrations of heterocyclic PAHs appear to be higher than the PNECs, particularly at contaminated sites. Thus, the tested heterocycles are of high concern in terms of risks to ecosystems and human health. Overall, the results of this research not only contribute to a comprehensive understanding and progress in the environmental hazard assessment of heterocyclic PAHs, but also present several methodological advances that can be applied to future testing of other challenging substances commonly referred to as 'difficult-to-test'. / Stickstoff-, schwefel- und sauerstoffsubstituierte heterocyclische polyaromatische Kohlenwasserstoffe (heterocyclische PAK) sind in allen Umweltkompartimenten gegenwärtig und treten häufig zusammen mit ihren homocyclischen Analoga auf. Bei der Gefährdungsbeurteilung kontaminierter Standorte wurde ihnen bisher verhältnismäßig wenig Aufmerksamkeit geschenkt, zudem ist über ihr Verhalten und ihre Ökotoxizität nur wenig bekannt. Aufgrund ihrer höheren Polarität, die auf das Vorhandensein elektronegativer Atome zurückzuführen ist, ging man bislang davon aus, dass die meisten heterocyclischen PAK mobiler und weniger toxisch sind als ihre homocyclischen Pendants, weshalb sie wahrscheinlich bislang vergleichsweise wenig studiert wurden. Sollte die Annahme einer geringeren Toxizität nicht zutreffen, weil beispielsweise andere Mechanismen als die Polarität/Hydrophobie für ihre Gefährdung verantwortlich sind, könnte dies zu einer Unterschätzung der Risiken, die von kontaminierten Standorten oder anderen Emissionen ausgehen, führen. Die Hauptziele dieser Arbeit bestehen daher darin, (1) ein umfassenderes Verständnis der physikalisch-chemischen Eigenschaften, der biologischen Abbaubarkeit, der Ökotoxizität, der Bioakkumulation und der Mobilität dieser Verbindungen in der Umwelt zu erlangen; (2) eine Bewertung verschiedener Modellierungsansätze zur Vorhersage dieser Eigenschaften durchzuführen und (3) experimentelle Hindernisse zu überwinden, indem robustere Techniken entwickelt werden, die langfristig zuverlässige und reproduzierbare Daten liefern können. Aus der sehr breiten Gruppe der heterocyclischen Verbindungen wurden strukturell vergleichbare repräsentative Verbindungen mit unterschiedlichen log KOW-Werten (als Maß für die Hydrophobie), die vier Größenordnungen abdecken, als Modellstrukturen ausgewählt. Zunächst erfolgte eine Untersuchung umweltrelevanter physikalisch-chemischer Eigenschaften dieser heterocyclischen PAK, wie die Wasserlöslichkeit (SW), der n-Octanol-Wasser-Verteilungskoeffizient (KOW) und der Verteilungskoeffizient bezogen auf den organischen Kohlenstoff und Wasser (KOC). Dabei wurden die Auswirkungen von Molekülgröße, Art und Anzahl der Heteroatome auf die Löslichkeit und die Verteilungskoeffizienten systematisch untersucht. Diese experimentell ermittelten Parameter wurden dann verwendet, um die Leistung von In-silico-Modellen zu testen, einschließlich der quantitativen Struktur-Eigenschafts-Beziehung (QSPR), der linearen Polyparameter-Energie-Beziehung (pp-LFER) und des Conductor-like Screening Model for Realistic Solvation (COSMO-RS), die diese Eigenschaften vorhersagen. Zudem wurde eine Umweltverträglichkeitsprüfung auf Screening-Ebene durchgeführt, bei der Daten aus verschiedenen Quellen berücksichtigt wurden. Den Screening-Ergebnissen zufolge ist davon auszugehen, dass kleine Verbindungen mit zwei oder drei Ringen in der Umwelt mobil sind, während größere heterocyclische PAK im Allgemeinen als bioakkumulierbare Stoffe identifiziert wurden, was auf ein erhebliches Gefahrenpotenzial hinweist. Die Persistenz von heterocyclischen PAK wurde experimentell durch eine Reihe von Tests zum biologischen Abbau unter aeroben Bedingungen untersucht. Dazu gehörten Tests zur Mineralisierung, zum biologischen Primärabbau und zur Inokulumtoxizität bei unterschiedlichen Expositionskonzentrationen. Keine der Prüfsubstanzen mit drei oder mehr Kohlenstoffringen erwies sich als biologisch gut/schnell abbaubar. Um den biologischen Abbauprozess zu stimulieren, wurde daher die mikrobielle Gemeinschaft aus einer Kläranlage an das Gemisch heterocyclischer PAK angepasst, wobei die Tests zum biologischen Abbau entweder mit angepassten oder nicht angepassten Bakterien durchgeführt wurden. Überraschenderweise gab es bei der bakteriellen Anpassung Widersprüche: positive Effekte (wie der bessere Abbau von Carbazol) gehen mit weniger wünschenswerten Ergebnissen (erhöhte Toxizität gegenüber Benzo[c]carbazol) einher. Für vier der betrachteten Verbindungen wurde ein biologischer Primärabbau festgestellt, während fünf Substanzen in keinem der Tests Anzeichen für eine mikrobielle Umsetzung zeigten, was auf ein hohes Potenzial für eine Persistenz in der Umwelt hindeutet. Die Vorhersagen softwaregestützter Modelle wurden ebenfalls mit den Messergebnissen verglichen, und es zeigte sich, dass die Modelle bei der Vorhersage der Abbaukinetik der getesteten Heterocyclen nur teilweise übereinstimmende Ergebnisse lieferten. Darüber hinaus wurde die akute Kurzzeittoxizität heterocyclischer PAK anhand von drei Arten aus verschiedenen trophischen Ebenen des aquatischen Nahrungsnetzes untersucht: Bakterien (Aliivibrio fischeri), einzellige Algen (Raphidocelis subcapitata) und Wasserflöhe (Daphnia magna). Um eine stabile Exposition aufrechtzuerhalten und Verluste, zum Beispiel durch Sorption an Gefäßwandungen, zu vermeiden, wurde die Methode des „passive dosing“ an Routinetestprotokolle angepasst. Die meisten Verbindungen wurden in diesen Tests als hochgiftig für die getesteten Organismen identifiziert, wobei festgestellt werden konnte, dass unpolare Narkose (Basistoxizität) die wahrscheinlichste Art der toxischen Wirkung der getesteten Chemikalien ist. Darüber hinaus wurden die Membran-Wasser-Verteilungskoeffizienten (KMW) der heterocyclischen PAK durch Lipidmembran-Bindungsexperimente auf festem Trägermaterial sowie mit Hilfe von Vorausberechnungen (Modellierung) bestimmt. Die gemessenen KMW-Werte stimmten eng mit den KOW-Werten überein, was darauf hindeutet, dass beide Werte gute Deskriptoren für die Vorhersage der Basistoxizität bzw. des Bioakkumulationspotenzials der getesteten heterocyclischen PAK sein können. Schließlich wurden die langfristige chronische Reproduktionstoxizität und die Bioakkumulation großer heterocyclischer PAK mit Daphnia magna als aquatischer Modellorganismus untersucht. Die „passive dosing method“ gewährleistete eine konstante Exposition selbst bei sehr niedrigen Konzentrationen (70 ng L-1 bis 68 µg L-1) in groß angelegten und komplexen Versuchssystemen. Angesichts des enormen Zeit- und Arbeitsaufwands, der für die Durchführung von Langzeit-Toxizitätstests erforderlich ist, wurde eine Methode entwickelt, um die chronische Toxizität sowie das Bioakkumulationspotenzial in einem einzigen Test zu bewerten. Alle vier getesteten Substanzen waren gegenüber Daphnia magna hochtoxisch und stark bioakkumulierend. Unerwarteterweise erholte sich die Reproduktionsfähigkeit der Daphnien jedoch schnell von den toxischen Wirkungen der Heterocyclen, wenn die Chemikalienexposition endete. Die PNECs (Predicted No Effect Concentrations) der Testsubstanzen wurden ebenfalls anhand der Daten zur chronischen Toxizität verglichen. Die Werte lagen in einer Süßwasserumgebung zwischen 1 und 150 ng L-1 (PNEC). Für die meisten Testverbindungen gibt es keine Informationen zu Monitoringdaten in der Umwelt. Die Umweltkonzentrationen heterocyclischer PAK scheinen jedoch höher zu sein als die PNEC-Werte, insbesondere an kontaminierten Standorten, was eine Gefahr für die Ökosysteme und die menschliche Gesundheit darstellt. Insgesamt tragen die Ergebnisse dieser Forschungsarbeit nicht nur zu einem umfassenderen Verständnis und zu Fortschritten bei der Bewertung der von heterocyclischen PAK ausgehenden Umweltgefahren bei, sondern liefern auch mehrere methodische Verbesserungen, die in Zukunft bei der Prüfung anderer Stoffe, die gemeinhin als 'schwierig zu testen' bezeichnet werden, angewendet werden können.

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<p>Recently, the focus of metal toxicity has shifted from concentrations within the aqueous environment to bioaccumulation within the organism. In this regard, the Biotic Ligand Model (BLM) relates the binding of metal at specific toxic sites (“ligands”) to eventual toxicity, whereas the Tissue Residue Approach (TRA) relates metal burdens at whole body, tissue, or subcellular levels to eventual toxicity. However, much less is currently known regarding Ni in comparison to other metals. This thesis addresses this knowledge gap by evaluating the use of Ni bioaccumulation as a predictor of toxicity in a number of fish and invertebrate species; bioaccumulation endpoints examined included Michaelis-Menten uptake parameters (K_d, B_max), a BLM parameter (log K_NIBL values) and critical body residues (CBR50 values)</p> <p>More sensitive species exhibited higher binding affinities and lower binding capacities for Ni. In invertebrates, a strong overall correlation was observed between log K_NiBL values for whole organism binding and acute toxicity to the extent that measurement of toxicity was an acceptable alternative to measurement of binding affinity, and vice versa. However, in two teleosts, the same comparison showed that a Ni BLM built on bioaccumulation would be more protective than one built on toxicity. The results further validated a central concept of the BLM - that short term metal bioaccumulation is predictive of longer term toxicity. Acute (96-h) Ni bioaccumulation predicted chronic (15 or 30-day) mortality in both salt and fresh water. In the latter, acute (96-h) sub-cellular bioaccumulations of Ni in either biologically active (BAM) or biologically inactive metal pools (BIM) of one fish species (round goby) were also predictive of 30-d mortality. However, goby were more sensitive to Ni bioaccumulation in the BAM versus BIM fraction.</p> <p>This thesis advances the use of bioaccumulation as a predictor of Ni toxicity and may have implications for metal toxicity frameworks such as the BLM and TRA.</p> / Doctor of Philosophy (PhD)

Freshwater rainbow trout were exposed to 9.2 µg/L total Ag (as AgNO3, a level approximately equal to the 168 h LC50) for 144 h to clarify the toxic mechanism of silver in freshwater teleosts. It was found that silver inhibits active Na+ and Cl- uptake at the gills, resulting in a net loss of both ions from the fish and creating a metabolic acidosis. This leads to a classic stress response (mobilization of cortisol and glucose into the blood plasma), and hyperventilation as a respiratory response to decreased blood pH. Plasma ammonia levels rise without any decrease in ammonia excretion; ammonia excretion later increases. This suggests that the increased plasma levels are due to increased metabolic production. Increased [H+] (decreased pH) results in excess H+ ions in the internal fluids, which are either complexed with ammonia to form NH4+ or are buffered in muscle tissue. The latter results in increased movement of K+ ions into the plasma, which are then excreted at the gills, preventing hyperkalemia. In the end, freshwater teleosts probably die from iono-and osmo-regulatory failure and associated cardiovascular collapse. Seawater teleosts (rainbow trout, tidepool sculpins, English sole, and plainfin midshipmen) and elasmobranchs (Pacific spiny dogfish and long nose skate) were exposed to constant concentrations on total Ag (as AgNO3) ranging from 1.5 to 50.0 µg/L for periods of up to 21 d at salinities of 18 ppt or 30 ppt. These exposure levels are well below those causing acute toxicity in seawater. Silver appears to enter marine teleosts and marine elasmobranchs differently. Seawater teleosts drink the seawater, so the intestines are a major site of silver uptake, along with the gills. Since marine elasmobranchs do not drink, the gills appear to be the sole site of silver uptake from the water. As in freshwater, the liver is the main site for silver accumulation in all marine fish studied. Despite similar terminal liver silver concentrations, marine elasmobranchs have a higher rate of silver accumulation since the livers in elasmobranchs are 10-20 fold larger than in teleosts. Both environmental salinity and exposure concentration play direct roles in determining silver bioaccumulation in marine teleosts. Increasing salinity alters the speciation of silver in the water, which decreases the amount of silver able to enter the fish. Increased silver concentrations mean more silver is available to enter the fish and subsequently cause sublethal toxic effects. Oxygen consumption decreased during the first 7 d of chronic exposure to sublethal silver levels in marine teleosts, with the decrease being more pronounced at higher (still sublethal) silver levels. Ammonia excretion, unaltered during acute exposure (48 h) to high silver levels (250 µg/L), was decreased during the first 7 d of exposure to sublethal silver levels (14.5-50.0 µg/L). This suggests that silver interferes with energy demanding processes such as protein synthesis or iono-regulation. Activity levels of the main enzyme involved in iono-regulation, namely Na+/K+ -ATPase, was affected differently in different fish. In a marine teleost that lives solely in seawater (plainfin midshipmen), silver inhibited the gill ATPase activity after 7 d of exposure, with the inhibition being more effective at higher silver levels. In the tidepool sculpin, a truly euryhaline species, gill ATPase activity increased as the silver levels increased, the latter probably representing a compensatory strategy. Similarly, intestinal ATPase activity was unchanged in the midshipmen, but was increased in the sculpins. Drinking rate in tidepool sculpins, which is involved with both iono-and osmo-regulation, was unaffected by salinity, but was decreased in fish exposed to silver for 8 d. Overall, Ag is far less toxic in seawater than in freshwater, but the mechanisms of toxicity are similar. In both waters, Ag interferes with iono- and osmo-regulation. In seawater, Ag exhibits a significant potential for bioaccumulation and interference with physiological processes during long term low level exposures of marine fish, especially at lower salinity levels. / Thesis / Master of Science (MSc)