To Örebro University

1,2-dibromo-4-(1,2-dibromoethyl)-cyclohexane (DBE-DBCH) is a brominated flame retardant used in commercial and industrial applications. The use of DBE-DBCH containing products has resulted in an increased release into the environment. However, limited information is available on the long-term effects of DBE-DBCH and its effects in aquatic invertebrates. Thus, the present study was aimed at determining how DBE-DBCH diastereomers (αβ and γδ) affects aquatic invertebrates using Daphnia magna as a model organism. Survival, reproduction, feeding, swimming behavior and toxicogenomic responses to environmental relevant concentrations of DBE-DBCH were analyzed. Chronic exposure to DBE-DBCH resulted in decreased lifespan, and reduced fecundity. Expression of genes involved in reproductive processes, vtg1 and jhe, were also inhibited. DBE-DBCH also induced hypoxia by inhibiting the transcription of genes involved in heme biosynthesis and oxygen transport. Furthermore, DBE-DBCH also inhibited feeding resulting in emptiness of the alimentary canal. Increased expression of the stress response biomarkers was observed following DBE-DBCH exposure. In addition, DBE-DBCH diastereomers also altered the swimming behavior of Daphnia magna. The present study demonstrates that DBE-DBCH cause multiple deleterious effects on Daphnia magna, including effects on reproduction and hormonal systems. These endocrine disrupting effects are in agreement with effects observed on vertebrates. Furthermore, as is the case in vertebrates, DBE-DBCH γδ exerted stronger effects than DBE-DBCH αβ on Daphnia magna. This indicate that DBE-DBCH γδ has properties making it more toxic to all so far studied animals than DBE-DBCH αβ.

Detection of various organic contaminants in the aquatic environment at low concentrations, has raised concerns for animal and human health. Structural similarities of these compounds to estrogens and ecdysones suggests that organic pollutants may interfere with hormonal system of aquatic organisms. Techniques of traditional toxicity tests that are based on observable physiological defects may fail to identify sub-lethal / subobservable effects. Under these techniques the mode of action of pollutants cannot be investigated. Thus, there is a need for more sensitive techniques that are capable to evaluate effects at molecular level and provide an early warning to environmental pollution. Toxicogenomic is proposed to be a useful tool to meet this goal. However, there is a limited data that compare toxicogenomic and traditional toxicity test approaches. The aim of this thesis is to evaluate the utility of qRT-PCR based toxicogenomic analysis in risk assessment. To achieve this goal, we first analyzed toxicogenomic and physiological responses of Daphnia manga exposed to three class of widely detected organic pollutant sphthalates, perfluorinated alkylated substances and brominated flame retardant in Paper I-III. Investigated endpoints included effects on hatching, acute toxicity, survival, developmental abnormalities, reproduction success, swimming and feeding behavior, fat metabolism, and life span. Result of these studies demonstrated a high degree of correlation between transcriptional data and the traditional physiological data. Besides, toxicogenomic approach was able to identify effects of sublethal concentrations. Thus, in Paper IV we analyzed environmental sample from Akaki river in Ethiopia using transcriptional approach and showed toxicogenomic tools utility in environmental risk assessment. Overall, the finding of these studies showed that the use of toxicogenomic can improve traditional physiological based toxicity assay to determine environmental risk assessment. Furthermore, toxicogenomic analysis can be performed with relatively short time and limited volume of samples.

Per- and polyfluorinated alkyl substances (PFASs) are synthetic organofluorine compounds with unique stability accompanied with hydrophobic and lipophobic properties. Perfluorooctane sulfonate (PFOS) and Perfluorooctanoic acid (PFOA) are of high concern due to their wide application in consumer and industrial products, extreme persistence, abundant occurrence in the environment and their toxic effect to humans and animals. However, knowledge on the molecular mechanisms of toxicity and the effects on reproduction output remain scarce. In this study, we analyzed the effects of PFOS and PFOA on Daphnia magna. Acute toxicity, development, reproduction, lipid metabolism (lipid-accumulation) and lifespan was investigated, as well as the expression of genes related to these endpoints. Exposure of PFOS and PFOA at 1, 10 and 25 μM did not cause acute lethality. Hatching was reduced following exposure to both compounds, and lifespan was decreased following exposure to 25 μM PFOS. Body length of Daphnia magna was reduced significantly by 25 μM PFOS following 7 days exposure. Lipid staining revealed that all PFAS exposures increased lipid accumulation. qRT-PCR analysis of genes involved in lipid metabolism suggests that the increase in lipid content could be due to inhibition of genes involved on absorption and catabolism of fatty acids. Exposure to both PFOA and PFOS reduced the fecundity significantly. Downregulation of genes involved in development and reproductive process, including vtg2, vasa, EcRA, EcRB, usp, jhe, HR3, ftz-F1, E74 and E75 were observed. The alterations in developmental and reproductive genes as well as the disturbed lipid metabolism provides mechanistic insight into the possible causes for decreased fecundity and lifespan observed following exposure to both PFOS and PFOA.

Phthalates are used as plasticizers to increase durability, resistivity and flexibility of plastic materials. The commonly used phthalate, diethylhexyl phthalate (DEHP) is used in different plastic materials like food packaging, toys and medical devices. DEHP has been linked to different toxicities in humans as well as in animals, and as a consequence other phthalates, including dibutyl phthalate (DBP) and diethyl phthalate (DEP) are being introduced. The increased use of phthalates has resulted in contamination of aquatic ecosystem and it directly threatens the aquatic life. In this study, we analyzed the effects of three phthalates DEHP, DEP and DBP using freshwater organism Daphnia magna. Although, exposure of the three phthalates at 1 and 10 μM did not result any lethality and hatching delay, the chronic exposure for 14 days resulted in reduction of body length. There was enhanced fat accumulation on exposure to all the phthalates, as indicated by oil red O staining. qRT-PCR analysis of genes involved in fat metabolism suggests that the increase in fat content could be due to inhibition of absorption and catabolism of fatty acids. Reproduction analysis showed that DBP and DEP did not alter fecundity but surprisingly, DEHP at 1 μM increased reproduction by 1.5 fold compared to control group. Phthalates also showed negative effect on lifespan as DEP at 10 μM and DBP at both 1 and 10 μM significantly reduced the lifespan. Our data indicates that along with the banned phthalate DEHP, the other substitute phthalates DEP and DBP could also have detrimental effect on aquatic organisms.

The increasing contamination of freshwater with pharmaceuticals, surfactants, pesticides and other organic compounds are of major concern. As these contaminants are detected at trace levels in the environment it is important to determine if they elicit biological responses at the observed levels. In addition to chemical pollutants, there is also a concern for increasing levels of bacteria and other microorganisms in freshwater systems. In an earlier study, we observed the activation of inflammatory systems downstream of a wastewater treatment plant (WWTP) in southern Sweden. We also observed that the water contained unidentified components that were pro-inflammatory and potentiated the immune response in human urinary bladder epithelial cells. In order to determine if these effects were unique for the studied site or represent a common response in Swedish water, we have now performed a study on three WWTPs and their recipient waters in central Sweden. Analysis of immune responses in urinary bladder epithelial cells, monocyte-like cells and blood mononuclear cells confirm that these waters activate the immune system as well as induce pro-inflammatory responses. The results indicate that the cytokine profiles correlate to the endotoxin load of the waters rather than to the levels of pharmaceuticals or culturable bacteria load, suggesting that measurements of endotoxin levels and immune responses would be a valuable addition to the analysis of inland waters.