Chemical and toxicological assessment of leachates from UV-degraded plastic materials using in-vitro bioassays.

The broad use of plastics and the persistence of the material results in plastic residues being found practically everywhere in the environment. If plastics remain in the (aquatic) environment, natural weathering leads to degradation processes and compounds may leach from plastic into the environment. To investigate the impact of degradation process on toxicity of leachates, different types of UV irradiation (UV-C, UV-A/B) were used to simulate weathering processes of different plastic material containing virgin as well as recyclate material and biodegradable polymers. The leached substances were investigated toxicologically using in-vitro bioassays. Cytotoxicity was determined by the MTT-assay, genotoxicity by using the p53-CALUX and Umu-assay, and estrogenic effects by the ERα-CALUX. Genotoxic as well as estrogenic effects were detected in different samples depending on the material and the irradiation type. In four leachates of 12 plastic species estrogenic effects were detected above the recommended safety level of 0.4 ng 17ß-estradiol equivalents/L for surface water samples. In the p53-CALUX and in the Umu-assay leachates from three and two, respectively, of 12 plastic species were found to be genotoxic. The results of the chemical analysis show that plastic material releases a variety of known and unknown substances especially under UV radiation, leading to a complex mixture with potentially harmful effects. In order to investigate these aspects further and to be able to give recommendations for the use of additives in plastics, further effect-related investigations are advisable.

Photolytic degradation of novel polymeric and monomeric brominated flame retardants: Investigation of endocrine disruption, physiological and ecotoxicological effects.

Ecotoxicological effects of photolytic degradation mixtures of the two brominated flame retardants PolymericFR and Tetrabromobisphenol A-bis (2,3-dibrom-2-methyl-propyl) Ether (TBBPA-BDBMPE) have been studied in vitro and in vivo. Both substances were experimentally degraded separately by exposure to artificial UV-light and the resulting degradation mixtures from different time points during the UV-exposure were applied in ecotoxicological tests. The in vitro investigation showed no effects of the degraded flame retardants on the estrogenic and androgenic receptors via the CALUX (chemically activated luciferase gene expression) assay. Short-term exposures (up to 96 h) of Lumbriculus variegatus lead to temporary physiological reactions of the annelid. The exposure to degraded PolymericFR lead to an increased activity of Catalase, while the degradation mixture of TBBPA-BDBMPE caused increases of Glutathione-S-transferase and Acetylcholine esterase activities. Following a chronic exposure (28 d) of L. variegatus, no effects on the growth, reproduction, fragmentation and energy storage of the annelid were detected. The results indicate that the experimental degradation of the two flame retardants causes changes in their ecotoxicological potential. This might lead to acute physiological effects on aquatic annelids, which, however, do not affect the animals chronically according to our results.

Effect-based evaluation of ozone treatment for removal of micropollutants and their transformation products in waste water.

The aim of this interdisciplinary research project in North Rhine-Westphalia (NRW), Germany, entitled "Elimination of pharmaceuticals and organic micropollutants from waste water" involved the conception of cost-effective and innovative waste-water cleaning methods. In this project in vitro assays, in vivo assays and chemical analyses were performed on three municipal waste-water treatment plants (WWTP). This publication focuses on the study of the in vitro bioassays. Cytotoxic, estrogenic, genotoxic and mutagenic effects of the original as well as enriched water samples were monitored before and after wastewater treatment steps using MTT and PAN I, ER Calux and A-YES, micronucleus and Comet assays as well as AMES test. In most cases, the measured effects were reduced after ozonation, but in general, the biological response depended upon the water composition of the WWTP, in particular on the formed by-products and concentration of micropollutants. In order to be able to assess the genotoxic and/or mutagenic potential of waste-water samples using bioassays like Ames test, Comet assay or micronucleus test an enrichment of the water sample via solid-phase extraction is recommended. This is in agreement with previous studies such as the "ToxBox"-Project of the Environmental Agency in Germany.

Accumulation pattern and possible adverse effects of organic pollutants in sediments downstream of combined sewer overflows.

The present study was conducted to investigate sediment accumulation patterns of PAHs, PCBs, flame retardants and pesticides along 100 m transects downstream from three different combined sewer overflows (CSOs). Additionally, the concentrations of PAHs and PCBs were quantified to allow a characterization of the sediment quality. The suspect screening revealed that usually more substances, especially pesticides and flame retardants, were detected in sediments located in the vicinity of CSOs. Except for PAHs at Location 1 and 3, all substances followed the same accumulation pattern, showing higher contaminations in sediments sampled downstream from the CSOs compared to an upstream located reference site. With increasing distance to the respective input, sediment concentrations decreased. Different accumulation patterns of PAHs at Locations 1 and 3 were related to high background concentrations of the receiving creek. Although the general contamination patterns were similar, the level of contamination was different at each location. PAH concentrations are indicative for the occurrence of sediment toxicity at Location 3. However, higher background concentrations in the receiving creek compared to concentrations present in the retention zone indicate a higher probability for sediment toxicity due to inherited waste or contamination from upper reaches. PCBs introduced by the sewer outfall at Location 1 significantly increased the toxic potential of sediments located in the vicinity of the CSO. The retention zones at Locations 2 and 3 appeared to be a good measure to trap particles and their associated pollutants before they accumulate in the creek sediments where they became potentially harmful for the aquatic biota. Based on this study recommendations for sampling strategies were concluded to harmonize sampling designs in studies analyzing the impact of point sources on the sediment quality and to minimize misinterpretation of results.

Predicted sediment toxicity downstream of combined sewer overflows corresponds with effects measured in two sediment contact bioassays.

The present study investigated the sediment toxicity along 100 m transects downstream from three different combined sewer overflows (CSOs) using a coupled substance risk assessment and bioassay approach. As a first step, a risk assessment according to the consensus based sediment quality guideline was performed to estimate the probability for an incidence of toxicity to benthic organisms in freshwater sediments using their pollutant concentrations of metals, PAHs and PCBs. Secondly, two sediment contact assays (SCAs) using the nematode Caenorhabditis elegans and embryos of the fish Danio rerio were conducted. Sediments downstream from CSOs were strongly influenced by discharged particles in terms of pollution, due to associated contaminants as well as oxygen depletion caused by oxygen consuming matter. In general, the calculated probability for sediment toxicity corresponded with results of the SCAs. With increasing distance from the CSOs both the toxic potential of the sediments as well as their effects detected in the SCAs decreased. However, clear correlations between concentrations of metals as well as PCBs and toxic effects were found for the nematode SCA. The toxicity assay with embryos of D. rerio was strongly influenced by oxygen depletion in the sediments. Therefore, it was not possible to differentiate between responses mediated by pollution or oxygen depletion using the D. rerio embryo test. The results of the present study demonstrated a clear effect of CSOs on the toxicity of downstream located sediments. As particles were identified as vector for pollutants and as source for oxygen depletion, the retention of particles within the sewer network is of high importance to minimize both stressors. The present study shows that a retention zone located between the CSO and the receiving creek is an efficient measure to enhance sediment and water quality downstream of CSOs.

Metal accumulation in sediments and amphipods downstream of combined sewer overflows.

The aim of this study was to investigate the concentrations of the metals Ag, Cd, Cu, Mn, Mo, Pb, Pt and Zn in sediments and amphipods along a 100m transect downstream of three different combined sewer overflows (CSOs). Moreover, the study was implemented to better understand the biological availability of metals downstream of CSOs as well as to identify a potential risk of CSOs for benthic or pelagic biota. Samples were taken at downstream sampling sites, which were 1, 5, 10, 20, 50 and 100m away from the outlets of the CSOs. An upstream-located sampling site each was used as reference for the respective transect. Additionally, sediments in two retention zones, located between the CSOs and the receiving creeks were analyzed. All downstream located creek sediments showed a similar metal pattern. Metal concentrations were found to be highest within the first 20m of the creek sediments. Elevated metal concentrations were also detected in the sediments of the retention zones. Metal accumulation in the amphipods was different at all three locations, but highest levels were always found downstream of the sediment accumulation hot spots. This might indicate that the metals were remobilized and became available for the amphipods further downstream of the CSOs. Although the general contamination pattern with respect to each reference site was similar, the degree of contamination was different at each location as demonstrated by anthropogenic enrichment factors. The discharge frequency as well as anthropogenic influences from upper reaches appear to be the most important factors for high metal concentrations in the sediments. Accordingly, in one case high background concentrations in combination with the CSO lead to an exceedance of the predicted effect level of Zn for aquatic organisms. Therefore, sediment contamination should be included in risk assessment when constructing CSOs to protect aquatic life.

Assessment of sublethal endpoints after chronic exposure of the nematode Caenorhabditis elegans to palladium, platinum and rhodium.

The aim of this study was to investigate chronic effects of the platinum-group elements (PGE) palladium (Pd), platinum (Pt) and rhodium (Rh) on the nematode Caenorhabditis elegans. Aquatic toxicity testing was carried out according to ISO 10872 by determining 96 h EC50 values for sublethal endpoints, including growth, fertility and reproduction. Single PGE standard solutions were used as metal source. Based on the EC50 values for Pt, reproduction (96 h EC50 = 497 µg/L) was the most sensitive endpoint followed by fertility (96 h EC50 = 726 µg/L) and growth (96 h EC50 = 808 µg/L). For Pd, no precise EC50 values could be calculated due to bell-shaped concentration response curves, but the 96 h EC50 for reproduction ranged between 10 and 100 µg/L. Pd and Pt had effects on all endpoints. With raising element concentrations reproduction was inhibited first. At a certain concentration, fertility was also affected, which in turn had an additional effect on reproduction. Growth inhibition can also lead to a loss of fertility if the worms do not reach an appropriate body size to become fertile. Rhodium showed no inhibition of any endpoint between concentrations of 100 to 10,000 µg Rh/L. The results of this study allow the following order of PGE with respect to decreasing toxicity to C. elegans: Pd > Pt ¼ Rh.

Effect of multiple microsporidian infections and temperature stress on the heat shock protein 70 (hsp70) response of the amphipod Gammarus pulex.

BACKGROUND: Increasing temperatures can be a significant stressor for aquatic organisms. Amphipods are one of the most abundant and functionally important groups of freshwater macroinvertebrates. Therefore, we conducted a laboratory experiment with Gammarus pulex, naturally infected with microsporidians. METHODS: In each group, 42 gammarids were exposed to 15°C and 25°C for 24 h. Sex of gammarids was determined and microsporidian infections were detected by specific PCR. To quantify stress levels of the amphipods, the 70 kDa heat shock proteins (hsp70) were analyzed by western blot. RESULTS: More males than females were detected in the randomized population sample (ratio of females/males: 0.87). No mortality occurred at 15°C, while 42.9% of gammarids died at 25°C. Sequences of three microsporidians (M1, M2, M3) were detected in this G. pulex population (99.7%-100% sequence identity to Microsporidium spp. from GenBank). Previous studies showed that M3 is vertically transmitted, while M1 and M2 are presumably horizontally transmitted. Prevalences, according to PCR, were 27.0%, 37.8% and 64.9% for Microsporidium sp. M1, M2 and M3, respectively. Cumulative prevalence was 82.4%. Multiple infections with all three microsporidians in single gammarids were detected with a prevalence of 8.1%, and bi-infections ranged between 12.2% and 25.7%. In dead gammarids, comparatively low prevalences were noted for M1 (males and females: 11.1%) and M2 (females: 11.1%; males 0%), while prevalence of M3 was higher (females: 66.7%; males: 88.9%). No significant effect of host sex on microsporidian infection was found.Significant effects of temperature and bi-infection with Microsporidium spp. M2 + M3 on hsp70 response were detected by analysis of the whole sample (15°C and 25°C group) and of M2 + M3 bi-infection and gammarid weight when analyzing the 25°C group separately. None of the parameters had a significant effect on hsp70 levels in the 15°C group. CONCLUSION: This study shows that some microsporidian infections in amphipods can cause an increase in stress protein level, in addition to other stressors. Although more harmful effects of combined stressors can be expected, experimental evidence suggests that such an increase might possibly have a protective effect for the host against acute temperature stress.

Comparison of the metal accumulation capacity between the acanthocephalan Pomphorhynchus laevis and larval nematodes of the genus Eustrongylides sp. infecting barbel (Barbus barbus).

BACKGROUND: Metal uptake and accumulation in fish parasites largely depends on the parasite group with acanthocephalans showing the highest accumulation rates. Additionally, developmental stage (larvae or adult) as well as parasite location in the host are suggested to be decisive factors for metal bioconcentration in parasites. By using barbel (Barbus barbus) simultaneously infected with nematode larvae in the body cavity and adult acanthocephalans in the intestine, the relative importance of all of these factors was compared in the same host. METHODS: Eleven elements Arsenic (As), Cadmium (Cd), Cobalt (Co), Copper (Cu), Iron (Fe), Manganese (Mn), Lead (Pb), Selenium (Se), Tin (Sn), Vanadium (V) and Zinc (Zn) were analyzed in barbel tissues (muscle, intestine, liver) as well as in their acanthocephalan parasites Pomphorhynchus laevis and the larval nematode Eustrongylides sp. (L4) using inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: Nine elements were detected in significantly higher levels in the parasites compared to host tissues. The element composition among parasites was found to be strongly dependent on parasite taxa/developmental stage and localization within the host. Intestinal acanthocephalans accumulated mainly toxic elements (As, Cd, Pb), whereas the intraperitoneal nematodes bioconcentrated essential elements (Co, Cu, Fe, Se, Zn). CONCLUSION: Our results suggest that in addition to acanthocephalans, nematodes such as Eustrongylides sp. can also be applied as bioindicators for metal pollution. Using both parasite taxa simultaneously levels of a wide variety of elements (essential and non essential) can easily be obtained. Therefore this host-parasite system can be suggested as an appropriate tool for future metal monitoring studies, if double infected fish hosts are available.