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- Therapy of hemorrhagic shock with following resuscitation-induced liver injury : in vivo study (2010)
- Shock resulting from life-threatening blood-loss (hemorrhagic shock) represents the most frequent injury pattern after a traumatic insult. Hemorrhagic shock induces inflammatory changes, characterized by highly complex pathophysiological pathways often resulting in death. In this study, we establish an experimental in vivo model of H/R in rats and study the mechanisms which determine the hepatic injury after H/R. Furthermore, we show that hemorrhagic shock with following resuscitation is accompanied with release of systemic and local pro-inflammatory mediators, increased infiltration of hepatic neutrophils in the liver, increased oxidative and nitrosative stress, enhanced cell death of both types, apoptosis and necrosis, conspicuous cytoskeletal rearrangements, loss of hepatic integrity and finally high general mortality rates, up to 80%. In addition, the effects of two potential therapeutic interventions to prevent the H/R induced liver injury are explored in a model of H/R in rats. First, the role of JNK and its inhibition by D-JNKI-1 in preservation of hepatic integrity following H/R was analyzed. Second, we investigated the potential of simvastatin to prevent the disturbed inflammatory response and hepatic injury after H/R. The effects of both therapeutic interventions were studied by looking at several inflammatory parameters, markers of oxidative and nitrosative stress, cytoskeleton integrity, microcirculatory parameters, underlying signaling cascades, liver damage and mortality. Highly specific blockade of JNK with the potent, inhibitory peptide D-JNKI-1 revealed the crucial role of the JNK signaling pathway in the H/R induced pathophysiology and strong protective effects of DJNKI- 1 in H/R induced liver injury, when the peptide was applied before and even after hemorrhagic shock. The other therapeutic intervention tested in this study was the use of simvastatin which also revealed protective effects after H/R and even a remarkable improvement in survival after H/R. We show that H/R induced release of pro-inflammatory cytokines, hepatic PMNL infiltration, increased oxidative and nitrosative stress, apoptosis and necrosis can be diminished by treatment with D-JNKI-1 but also with simvastatin in vivo. Furthermore, simvastatin reduces H/R induced cytoskelatal rearrangements, loss of liver integrity and the mortality rate after H/R. The key pathway which underlies these beneficial effects of simvastatin is the Rho kinase pathway. Identification of both mechanisms as well as the effectiveness of both substances provide new insights in the close interaction between hypoxia and the immune system and present a promising basis for the anti-inflammatory, hepatoprotective treatment after H/R.
- Ecotoxicological assessment of small surface waters with emphasis on sediments : a case study in Hesse, Germany (2012)
- Chemical contamination of the environment and thus of aquatic ecosystems is steadily increasing. Whenever environmental pollutants enter a water body, they affect not only the water, but also the sediment. Substances that bind to sediment particles can be stored for a long time, whereby sediments act as sinks for some contaminants. Therefore, sediment assessments often more accurately describe the contamination of a water body than investigations of the water itself. Among environmental chemicals, endocrine disrupting compounds (EDCs) have gained more and more attention in recent years. Since they interfere with endocrine systems and may disturb reproduction, they endanger the survival of populations or even species. Hazardous substances enter the aquatic environment by different pathways, with sewage treatment plants (STPs) belonging to the most important contamination sources.The main objective of this work is a comprehensive sediment assessment of predominantly small surface waters in the German federal state of Hesse. The 50 study sites, located in 44 different creeks and small rivers, are situated in the densely populated and economically important Frankfurt/Rhine-Main area, as well as in rural and less urbanized regions. Chemical analytical data, provided by the Hessian Agency for the Environment and Geology (HLUG), indicated different contamination levels of the study sites. In order to investigate the general toxicity of the sediment samples, the oligochaete Lumbriculus variegatus and the midge Chironomus riparius were exposed to whole sediments and apical endpoints regarding biomass, survival, and reproduction were determined. In further experiments, special attention was paid to the contamination with endocrine active compounds. For this purpose, the reproductive success of the New Zealand mudsnail Potamopyrgus antipodarum was analyzed after exposure to whole sediments. Additionally, a yeast-based reporter gene assay was applied with sediment eluates to assess the estrogenic and androgenic activity of the samples. Biotest results were compared with chemical analysis data to investigate whether the test organisms reflect the measured pollution of the study sites and if the observed effects can be explained by chemical contamination. Five study sites, all located less than 1 km downstream of a STP discharger, were selected for further investigations based on the results of the sediment monitoring. The sediments from these sites were conspicuous due to their general toxic and/or estrogenic activity. In order to investigate whether the observed effects can be ascribed to the effluents, an active biomonitoring study was conducted with the mudsnail P. antipodarum and the zebra mussel Dreissena polymorpha, exposed at study sites located up- and downstream of the discharger. In addition to endocrine activity, genotoxic effects were investigated using the comet assay and the micronucleus assay. Endocrine activity was examined based on the reproductive output of P. antipodarum and the content of vitellogenin-like proteins in D. polymorpha. Yeast-based reporter gene assays were used to estimate the endocrine potential (estrogen, anti-estrogen, anti-androgen, dioxin-like) of sediment and water samples. 22% of the 50 sediments showed ecologically relevant effects in the biotests with L. variegatus and C. riparius. Only one sediment caused a relevant effect on both test organisms, while the other ten positively tested sediments affected either L. variegatus or C. riparius, probably due to differences in inter-species sensitivities. This suggests that a combination of different biotests is necessary for a comprehensive evaluation of sediment toxicity. 78% of the sediments caused a significantly increased number of embryos in P. antipodarum, which could be ascribed to estrogenic contamination of the sediment samples. An increase in the number of embryos by 60%, as observed in this study, and an associated increase in population size may result in the displacement of other, less competitive species. In the in vitro tests, 66% of the sediments showed estrogenic activity and 68% showed androgenic activity. Maximum observed values were 40.9 ng EEQ/kg sediment (EEQ = estradiol equivalent) for estrogenic and 93.4 ng TEQ/kg sediment (TEQ = testosterone equivalent) for androgenic activity. Natural and synthetic hormones as well as alkylphenols were the major contributors to the total estrogenicity of environmental samples in several other studies, and are likely responsible for a large part of the estrogenic activity in this case as well. Similarly, androgenic activity is mainly due to natural steroids and their metabolites. Bioassay results reflect the analytically measured contamination levels at the study sites only very infrequently. This can be ascribed to the occurrence of integrated effects of chemical mixtures present in the sediments. Additionally, effects of substances not included in the analytical program or of substances present in concentrations below the detection limit of the chemical analytical investigations as well as varying bioavailabilities might be relevant. The fact that a large part of the observed effects cannot be explained by the chemical contamination demonstrates the need for effect studies in ecotoxicological sediment assessments. In order to identify possible causes for the effects observed in the sediment monitoring, e.g. contamination sources, the area types (urban fabrics, arable lands, pasturages, etc.) of the catchment areas belonging to the study sites were analyzed. No significant differences were found between the area profiles of the sampling sites with and without effects in the biotests. The results indicate that the contamination responsible for the observed effects can be ascribed to different sources. Furthermore, study sites whose sediments exerted significant effects in biotests were located in anthropogenic as well as in predominantly natural areas. The active biomonitoring study at STPs revealed genotoxic and endocrine effects only sporadically. However, in the in vitro tests considerable endocrine activities of sediment and water samples were determined. No conclusive picture emerges as to whether the observed effects occur more frequently downstream of the dischargers, and thus could be attributed to a contamination by sewage. This indicates that contamination sources other than STP dischargers, for example agricultural runoff, may contribute to the observed effects. Weaker effects and biological activities downstream of a discharger compared to an upstream site might be ascribed to a dilution effect by the effluents. A comparison of the measured in vitro estrogenicity with exposure studies described in the literature shows that adverse effects in aquatic organisms can be expected at the EEQ concentrations determined in the present study. The results of the sediment monitoring and the STP study revealed a widespread endocrine pollution of small surface waters in Hesse. The fact that the bioassay results only rarely reflect study site contamination as determined by chemical analysis demonstrates the need for effect studies in comprehensive sediment assessments. In some cases STP dischargers increased, in other cases they decreased the observed in vivo effects and in vitro activity of environmental samples. Transferring the results obtained in laboratory studies to the field, adverse effects on aquatic ecosystems can be expected. The study illustrates the need for restrictive measures that contribute to the removal or reduction of environmental pollutants. For the identification of substances that have so far not been linked to adverse effects on the environment, methods such as effect-directed analyses (EDA) or toxicity identification evaluation (TIE) should be increasingly applied in future studies. Furthermore, bioassays for the assessment of endocrine activity should be implemented in standardized monitoring programs.
- Ecotoxicological assessment of advanced wastewater treatment technologies (2010)
- Wastewater treatment plants (WWTPs) do not eliminate micropollutants completely and are thus important point sources for these substances. In particular, concerns about en-docrine disrupting compounds in WWTP effluents give rise to the implementation of advanced treatment steps for the elimination of trace organic contaminants. The present study investigated ozonation (O3) and activated carbon treatment (AC) at two WWTPs. For an ecotoxicological assessment at WWTP Regensdorf, conventionally treated wastewater, wastewater after ozonation, and ozonated wastewater after sand filtration were evaluated in parallel via the fish early life stage toxicity test (FELST) using rainbow trout (Oncorhynchus mykiss). Additionally, a comparative toxicity evalu-ation of ozonated and activated carbon treated effluents was performed at the pilot scale treatment plant in Neuss (WWTP Neuss). For this purpose, four invertebrate tests and one higher plant toxicity test were selected to assess potential biological effects on or-ganisms [Lemna minor growth inhibition test, chironomid toxicity test with Chironomus riparius, Lumbriculus variegatus toxicity test, comet assay with haemolymph of the zebra mussel (Dreissena polymorpha), reproduction test with Potamopyrgus antipo-darum]. All in vivo assays were performed on site at the treatment plants in flow-through test systems. Furthermore, the present study investigated the effects of ozona-tion and activated carbon treatment on endocrine activities [estrogenicity, anti-estrogenicity, androgenicity, anti-androgenicity, aryl-hydrocarbon receptor (AhR) agonistic activity] with yeast based bioassays using solid phase extracted water samples. To evaluate the removal of in vitro non-specific toxicity, a cytotoxicity assay using a rat cell line was applied. The FELST at WWTP Regensdorf revealed a considerable developmental retardation of test organisms exposed to ozonated WW. This was accompanied by a significant decrease in body weight and length compared to reference water, to the conventionally treated WW, and to the ozonated water after sand filtration. Hence sand filtration obvi-ously prevents from adverse ecotoxicological effects of ozonation. An additional test – starting with yolk-sac larvae – resulted in a significant reduction of vitellogenin levels in fish exposed to ozonated wastewater compared to fish reared in conventionally treat-ed wastewater. This demonstrates the effective removal of estrogenic activity by ozonation. At WWTP Neuss, the reproduction test with the mudsnail P. antipodarum exhibited a decreased reproductive output after advanced treatment compared to conventional treatment. This indicates an effective estrogenicity removal by ozonation and activated carbon treatment and is confirmed by results of the yeast estrogen screen with a reduc-tion of in vitro estrogenic activity by > 75%. The L. variegatus test revealed a signifi-cantly enhanced toxicity after ozonation compared to conventional treatment, whereas this effect was reduced following subsequent sand filtration. When ozonation was applied, a significantly increased genotoxicity was observed, detected with the comet assay using haemolymph of the zebra mussel. Again, this effect was removed by subsequent sand filtration to the level of conventional treatment. Activated carbon treatment even resulted in a significant reduction of genotoxicity. At both treatment plants, adverse effects after ozonation may have been a result of the formation of toxic oxidation by-products. However, sand filtration reduced toxication effects, indicating that these oxidation by-products are readily degradable or adsorbable. The results point out that, in any case, ozonation should not be applied without subsequent biologically active post treatment appropriate for oxidation by-products removal (e.g. sand filtration). However, only activated carbon achieved a toxicity reduction compared to the conventional treated wastewater. Thus, it cannot be excluded that po-tential beneficial effects due to ozonation might be masked by residual toxic oxidation by-products passing the sand filter or ozonation is not as effective in toxicity removal as PAC treatment. The yeast based assays with solid phase extracted samples revealed an effective endo-crine activity removal during ozonation and activated carbon filtration (estrogenicity: 77 – 99%, anti-androgenicity: 63 – 96%, AhR agonistic activity: 79 – 82%). The cyto-toxicity assay exhibited a 32% removal of non-specific toxicity after ozonation com-pared to conventional treatment. Ozonation in combination with sand filtration reduced cytotoxic effects by 49%, indicating that sand filtration contributes to the removal of toxicants. Activated carbon treatment was the most effective technology for cytotoxici-ty removal (61%). Sample evaporation reduced cytotoxic effects by 52% (after activated carbon treatment) to 73% (after ozonation), demonstrating that volatile substances contribute considerably to toxic effects, particularly after ozone treatment. These results confirm an effective removal or transformation of toxicants with receptor mediated mode of action and non-specific toxicants during both investigated treatment steps. However, due to the limited extractability, polar ozonation by-products were neglected for toxicity analysis, and hence non-specific toxicity after O3 is underestimated. In the long run, only on-site comparisons at WW receiving water bodies (e.g. communi-ty analysis of fish, macroinvertebrates, plants, microorganisms) – before and after up-grading WWTPs – allow drawing environmentally relevant conclusions regarding bene-fits and risks of advanced WW treatment methods. Conclusively, the benefits and possible negative impacts have to be carefully evaluated to prove that not more environmental impact will be induced than removed by advanced treatment technologies as each additional treatment requires considerable amounts of energy, resources, and infrastructure facilities. Accordingly, comprehensive sustainable approaches for pollution prevention and wastewater treatment (e.g. source control and source separation) are preferable compared to end-of-pipe treatment systems.
- Biodegradation and elimination of industrial wastewater in the context of whole effluent assessment (2010)
- The focus of this thesis is on the assessment of the degradability of indirectly discharged wastewater in municipal treatment plants and on assessing indirectly discharged effluents by coupling the Zahn-Wellens test with effect-based bioassays. With this approach persistent toxicity of an indirectly discharged effluent can be detected and attributed to the respective emission source. In the first study 8 wastewater samples from different industrial sectors were analysed according to the “Whole-Effluent Assessment“ (WEA) approach developed by OSPAR. In another study this concept has been applied with 20 wastewater samples each from paper manufacturing and metal surface treating industry. In the first study generally low to moderate ecotoxic effects of wastewater samples have been determined. One textile wastewater sample was mutagenic in the Ames test and genotoxic in the umu test. The source of these effects could not be identified. After treatment in the Zahn-Wellens test the mutagenicity in the Ames test was eliminated completely while in the umu test genotoxicity could still be observed. Another wastewater sample from chemical industry was mutagenic in the Ames test. The mutagenicity with this wastewater sample was investigated by additional chemical analysis and backtracking. A nitro-aromatic compound (2-methoxy-4-nitroaniline) used for batchwise azo dye synthesis and its transformation products are the probable cause for the mutagenic effects analysed. Testing the mother liquor from dye production confirmed that this partial wastewater stream was mutagenic in the Ames test. The wasteweater samples from paper manufacturing industry of the second study were not toxic or genotoxic in the acute Daphnia test, fish egg test and umu test. In the luminescent bacteria test, moderate toxicity was observed. Wastewater of four paper mills demonstrated elevated or high algae toxicity, which was in line with the results of the Lemna test, which mostly was less sensitive than the algae test. The colouration of the wastewater samples in the visible band did not correlate with algae toxicity and thus is not considered as its primary origin. The algae toxicity in wastewater of the respective paper factory could also not be explained with the thermomechanically produced groundwood pulp (TMP) partial stream. Presumably other raw materials such as biocides might be the source of algae toxicity. In the algae test, often flat dose–response relationships and growth promotion at higher dilution factors have been observed, indicating that several effects are overlapping. The wastewater samples from the printed circuit board and electroplating industries (all indirectly discharged) were biologically pre-treated for 7 days in the Zahn–Wellens test before ecotoxicity testing. Thus, persistent toxicity could be discriminated from non-persistent toxicity caused, e.g. by ammonium or readily biodegradable compounds. With respect to the metal concentrations, all samples were not heavily polluted. The maximum conductivity of the samples was 43,700 micro S cm -1 and indicates that salts might contribute to the overall toxicity. Half of the wastewater samples proved to be biologically well treatable in the Zahn–Wellens test with COD elimination above 80%, whilst the others were insufficiently biodegraded (COD elimination 28–74%). After the pre-treatment in the Zahn–Wellens test, wastewater samples from four companies were extremely ecotoxic especially to algae. Three wastewater samples were genotoxic in the umu test. Applying the rules for salt correction to the test results following the German Wastewater Ordinance, only a small part of toxicity could be attributed to salts. In one factory, the origin of ecotoxicity has been attributed to the organosulphide dimethyldithiocarbamate (DMDTC) used as a water treatment chemical for metal precipitation. The assumption, based on rough calculation of input of the organosulphide into the wastewater, was confirmed in practice by testing its ecotoxicity at the corresponding dilution ratio after pre-treatment in the Zahn–Wellens test. The results show that bioassays are a suitable tool for assessing the ecotoxicological relevance of these complex organic mixtures. The combination of the Zahn–Wellens test followed by the performance of ecotoxicity tests turned out to be a cost-efficient suitable instrument for the evaluation of indirect dischargers and considers the requirements of the IPPC Directive.
- Assessing the combined effects of xenobiotics, climate change and predators on aquatic organisms in multiple stressor experiments : a case study with pyrimethanil (2012)
- The environmental impact of climate change is meanwhile not only discussed in the scientific community but also in the general public. However, little is known about the interaction between climate change and pollutants like pesticides. A combination of multiple stressors (e.g. temperature, pollutants, predators) may lead to severe alterations for organisms such as changes in time of reproduction, reproductive success and growth performance, mortality and geographic distribution. The questions if aquatic organisms tend to react more sensitive towards incidents under climate change conditions remains. Therefore, within the present thesis the aquatic ecotoxicological profile of the fungicide pyrimethanil, as an exemplarily anthropogenic used contaminant, was examined. A large test battery of ecotoxicological standard tests and supplement bioassays with non-model species was conducted to investigate if species-specific or life stage-specific differences occur or if temperature alteration may change the impact of the fungicide. Two of the most sensitive species (Chironomus riparius and Daphnia magna) were used to investigate the acute and chronic thermal dependence of pyrimethanil effects. The results clearly depict that the ecotoxicity of pyrimethanil at optimal thermal conditions did not depend on the trophic level, but was species-specific. With regard to EC10 values the acute pyrimethanil toxicity on C. riparius increased with higher temperature (6.78 mg L-1 at 14°C and 3.06 mg L-1 at 26°C). The chronic response of D. magna to the NOEC (no observed effect concentration) of the fungicide (0.5 mg L-1) was examined in an experiment which lasted for several generations under three simulated near-natural temperature regimes (‘cold year, today’ (11 to 22.7°C), ‘warm year, today’ (14 to 25.2°C) and ‘warm year, 2080’ (16.5 to 28.1°C)). A pyrimethanil-induced mortality increase was buffered by the strongly related increase of the general reproductive capacity, while population growth was stronger influenced by temperature than by the fungicide. At a further pyrimethanil concentration (LOEC – lowest observed effect concentration: 1 mg L-1), a second generation could not be established by D. magna under all thermal regimes. Besides daphnids, the midge C. riparius was used for a second multigeneration study. In a bifactorial test design it was tested if climate change conditions alter or affect the impact of a low fungicide concentration on life history and genetic diversity. The NOAEC/2 (half of the no observed adverse effect concentration derived from a standard toxicity test) was used as a low pyrimethanil concentration to which laboratory populations of the midges were chronically exposed under the mentioned temperature scenarios. During the 140-day-multigeneration study, survival, emergence, reproduction, population growth, and genetic diversity of C. riparius were analyzed. The results reveal that high temperatures and pyrimethanil act synergistically on life history parameters of C. riparius. In simulated present-day scenarios, a NOAEC/2 of pyrimethanil provoked only slight to moderate beneficial or adverse effects. In contrast, an exposure to a NOAEC/2 concentration of pyrimethanil at a thermal situation likely for a summer under the future expactations uncovered adverse effects on mortality and population growth rate. In addition, genetic diversity was considerably reduced by pyrimethanil in the ‘warm year, 2080’ scenario, but only slightly under current climatic conditions. The multigeneration studies under near-natural thermal conditions indicate that not only the impact of climate change, but also low concentrations of pesticides may pose a reasonable risk for aquatic invertebrates in the future. This clearly shows that thermal and multigenerational effects should be considered when appraising the ecotoxicity of pesticides and assessing their future risk for the environment. In addition to temperature further multiple abiotic and biotic stressors alterate pollutant effects. Moreover, to better discriminate and understand the intrinsic and environmental correlates of changing aquatic ecosystems, it was experimentally unraveled how the effects of a low-dose of pyrimethanil on daphnids becomes modified by different temperatures (15°C, 20°C, 25°C) and in the presence/ absence of predator kairomones of Chaoborus flavicans larvae. The usage of a fractional multifactorial test design provided the possibility to investigate the individual growth, reproduction and population growth rate of Daphnia pulex via different exposure routes to the fungicide pyrimethanil at an environmentally relevant concentration (0.05 mg L-1) - either directly (via the water phase), indirectly (via algae food), dually (via water and food) or for multiple generations (fungicide treated source population). The number of neonates increased with increasing temperatures. At a temperature of 25°C no significant differences between the individual treatment groups were observed although the growth was overall inhibited due to pyrimethanil. Besides, at 15 and 20°C it is obvious that daphnids which were fed with contaminated algae had the lowest reproduction and growth rate. The obtained results clearly demonstrate that multiple stress factors can modify the response of daphnids to pollutants. The exposure routes of the contaminant are of minor importance, while temperature and the presence of a predator are the dominant factors impacting the reproduction of D. pulex. It can be concluded that low concentrations of pyrimethanil may disturb the zooplankton community at suboptimal temperature conditions, but the effects will become masked if chaoborid larvae are present. Therefore it seems necessary to observe prospectively if the combination of several stress factors like pesticide exposure and suboptimal temperature may influence the life history and sensitivity of several aquatic invertebrates differently. Besides standard test organisms it is inevitable to conduct test with aquatic invertebrate which are not yet considered regularly in ecotoxicological experiments. For example molluscs represent one of the largest phyla of macroinvertebrates with more than 100.000 species, being ecologically and economically important. Therefore, within the present study embryo, juvenile, half- and full-life cycle toxicity tests with the snail Physella acuta were performed to investigate the impact of pollutants on various life stages. Different concentrations of pyrimethanil (0.06-0.5 or 1.0 mg L-1) assessed at three temperatures (15°C, 20°C, 25°C) revealed that pyrimethanil caused concentration-dependent effects independent of temperature. Interestingly, the ecotoxicity of pyrimethanil was higher at lower temperature for the embryo hatching and F1 reproduction, but its ecotoxicity for the growth of juveniles and the F0 reproduction increased with increasing temperature. More specifically, it could have been observed that especially during the reproduction test high mortality rates occurred at the highest concentration of 1 mg L-1 at all temperatures. Due to high mortality rates no snails were available for the F1 at the highest concentrations (0.5 and 1.0 mg L-1). Compared to the F0, overall more egg masses were produced in the F1, being all fertile and no mortality occurred. For the F1-generation the strongest pyrimethanil effects were detected at 15°C. A comparison of effect concentrations between both generations showed that the F1 is more sensitive than the F0. These results indicate that an exposure over more than one generation may give a better overview of the impact of xenobiotics. With the establishment of an embryo and reproduction test under different temperatures and various concentrations of pyrimethanil with P. acuta we could successfully show that molluscs can respond more sensitive than model organisms and that both, chemical and thermal stressor strongly influence the behaviour of the pulmonates. It can be concluded that the high susceptibility for the fungicide observed in gastropods clearly demonstrates the complexity of pesticide-temperature interactions and the challenge to draw conclusions for the ecotoxicological risk assessment of pesticides under the impact of global climate change.
- Occurrence and sources of 2,4,7,9-tetramethyl-5-decyne-4,7-diol (TMDD) in the aquatic environment (2011)
- The aim of the present study was to identify the sources of 2,4,7,9-tetramethyl-5-decyne-4,7- diol (TMDD) into the aquatic environment and to investigate its occurrence in rivers and wastewater treatment plants (WWTPs). Therefore, TMDD was analyzed in 441 wastewater samples from influents and effluents of 27 municipal WWTPs, in 6 sludge samples, in 52 wastewater samples from 3 sewage systems of municipal WWTPs, in 489 surface samples from 24 rivers, in 9 wastewater samples of 3 paper-recycling industries and in 65 groundwater samples. TMDD was also analyzed in household paper products, in 23 samples of toilet papers, in 5 types of paper towels and in 12 types of paper tissues. The samples were collected between 2007 and 2011. The water samples were extracted with solid phase extraction (SPE) and the household paper samples with Soxhlet extraction. Gas chromatography-mass spectrometry (GC-MS) was used for quantification purposes. Between November 2007 and January 2008, TMDD was detected in the river Rhine at Worms with permanent high concentrations (up to 1330 ng/L). The results showed that TMDD is uniformly distributed across the river at Worms. An increase of the mean TMDD concentration from approximately 500 ng/L to 1000 ng/L was registered in January 2008. Due to the minor fluctuations of the TMDD concentration during the sampling period it is expected that the input of TMDD into the river is continuous. Therefore, TMDD might rather originate from effluents of municipal WWTPs than from temporal sources. The mean TMDD load based on the analysis of 147 water samples collected in the River Rhine was 62.8 kg/d which is equivalent to 23 t/a suggesting that TMDD must be used and/or produced in high quantities in order to be found in those high concentrations. To determine if TMDD is discharged by effluents of municipal WWTPs into the rivers, 24 hours influent and effluent samples of four municipal WWTPs in the Frankfurt/Rhine-Main metropolitan region were collected during November 2008 and February 2010 and analyzed for TMDD. The TMDD influent concentrations varied between 134 ng/L and 5846 ng/L and the effluent concentrations between <LOQ (limit of quantitation) and 3539 ng/L. The TMDD elimination rates in the four WWTPs varied between 33% and 68%. The results showed that effluents of municipal WWTPs are an important source of TMDD in the aquatic environment because TMDD is not completely removed from the sewage during the wastewater treatment. Weekly and daily variations of the TMDD concentration in the influents of two municipal WWTPs indicated that both private households and indirect industrial dischargers contribute to the introduction of TMDD into the municipal sewage systems. A more detailed study of the TMDD elimination rate in the different wastewater treatment stages was carried out in the WWTP Niederrad/Griesheim in Frankfurt am Main. The results showed that the removal of TMDD is mainly carried out during the aerobic biological treatments, where the elimination rate was 46%. In contrast, during the anoxic treatment the removal efficiency was only 1.4% and during the mechanical treatment the elimination rate was 19%. To determine the sources of TMDD in the sewage, household paper products (paper tissues, toilet papers and paper towels) were analyzed for TMDD using Soxhlet extraction. TMDD was detected in 83% of the samples (n=40). The highest mean TMDD concentrations were found in recycled toilet paper (0.20 μg/g) and in paper towels (0.11 μg/g). In paper tissues and non-recycled toilet paper the mean TMDD concentrations were lower 0.080 μg/g and 0.025 μg/g respectively. According to these results the high TMDD influent concentrations found previously in municipal WWTPs (mean 1.20 μg/L) cannot be explained due to migration of TMDD from the household paper products into the sewage. Thus indirect industrial dischargers are the cause of the high influent TMDD concentrations. Effluents of municipal WWTPs with different indirect industrial dischargers (textile-, metal processing-, food processing-, electroplating-, paper-recycling- and printing ink factories) were analyzed. The highest mean TMDD concentrations were found in the effluents of municipal WWTPs that have paper-recycling (71.3 μg/L) and printing ink factories (138 μg/L) as indirect industrial dischargers. These results were confirmed by analyzing process wastewater of three paper-recycling factories located in Germany. High TMDD concentrations were detected and fluctuated between 1.83 μg/L and 113 μg/L. TMDD was also analyzed in the wastewater of a non-recycling-paper factory but its concentration was much lower (0.066 μg/L) indicating that TMDD is introduced into the processing water during the papermaking process due to the use of waste paper. Analyses of wastewater samples from different parts of the sewage pipes of a municipal WWTP in Hesse, which receives the wastewater from a printing ink factory, were carried out. The TMDD concentration in the wastewater sample from the sewage pipe of the printing ink factory was much higher (3,300 μg/L) than the TMDD concentration detected in the other wastewater samples from the sewage system (0.030 μg/L – 0.89 g/L). These results confirm the printing ink production as one of the principal sources of TMDD in the sewage. Analysis of surface water samples of the River Modau downstream from the effluent of the WWTP Nieder-Ramstadt showed TMDD concentrations of up to 28.0 μg/L. These high TMDD concentrations might be caused by the indirect wastewater discharges of a paint factory connected to the municipal sewage system. These results indicate that TMDD is introduced into the municipal WWTPs principally by indirect industrial dischargers and they are mainly paint and printing ink factories. The paper-recycling factories also represent an important source of TMDD in municipal WWTPs but indirectly. According to statements given by the representatives of two paper recycling factories neither TMDD or any other TMDD containing product is used or added during the papermaking process. Therefore, TMDD is washed out from the printing inks of the coloured waste paper and concentrated in the process wastewater in the closed water circuits of paper-recycling factories reaching rivers and municipal WWTPs. The occurrence and distribution of TMDD in surface waters in Germany was also studied. The results showed that TMDD is widely distributed across different rivers systems in the federal states of Hesse, North-Rhine-Westphalia, Bavaria, Baden-Wuerttemberg and Rhineland-Palatinate. In Hesse, TMDD was detected in the some of main rivers with mean concentrations of 812 ng/L (Schwarzbach, Hessian Ried), 374 ng/L (Kinzig), 393 ng/L (Main, at Frankfurt), 539 ng/L (Werra), 326 ng/L (Fulda), 151 ng/L (Emsbach) and 161 ng/L (Nidda). In small rivers (creeks) the mean TMDD concentrations varied between <LOQ (Diemel, Urselbach) and 1890 ng/L (Darmbach). The results showed that the TMDD concentrations in creeks are highly influenced by both effluents of WWTPs and by the distance between the sampling point and the nearest WWTP. Surface samples from sampling locations downstream from WWTPs dischargers showed higher TMDD concentrations (mean 518 ng/L) than sampling locations upstream from WWTPs dischargers (mean 35.1 ng/L). The behavior of TMDD during bank filtration was investigated at two locations, at a water utility company at the Lower River Rhine (urban area) and at the Oderbruch polder (rural area). The results indicated that TMDD is removed from the surface water by bank filtration at both sampling locations. The removal process is probably carried out in the first meters of the aquifer (hyporheic zone) by biodegradation processes, since TMDD does not tend to be absorbed by sediments and it was not found in the groundwater of monitoring wells. In groundwater samples from the Hessian Ried (n=23) TMDD was found only in five samples and the highest TMDD concentration was 135 ng/L. According to these results, TMDD does not represent a concern for drinking water in Germany, since it does not reach the groundwater with high concentrations and it has a low toxicity potential. The input of TMDD into the North Sea was estimated to be 60.7 t/a by considering the mean transported loads of TMDD by the River Rhine at Wesel (58.3 t/a) and Meuse in the Netherlands (2.40 t/a). The estimated discharge of TMDD by German municipal WWTPs (8.19 t/a) and paper-recycling factories (9.24 t/a) into rivers seems to be too low considering that the mean TMDD load in the River Rhine downstream from Wesel is 58.3 t/a. However, due to the high density of population and industries at the Lower Rhine it is expected that more relevant sources of TMDD are located along the Rhine River increasing the transported load. According to the results of this PhD project TMDD is a non-ionic surfactant contained in products, which are applied on surfaces (printing inks and paints) and has the potential to reach the aquatic environment. Therefore, TMDD should fulfill the requirement of a biodegradability of 80% established by the “Law on the Environmental Impact of Detergents and Cleaning Products” in Germany. However, due to the partial elimination rates of TMDD obtained in municipal WWTPs (between 33% and 68%) and to the absence of information about the execution of the biodegradation test on TMDD, it is unknown if TMDD is in accordance with this law. Otherwise, its use as surfactant in such products is questionable.