Rik I. L. Eggen

Reducing the Discharge of Micropollutants in the Aquatic Environment: The Benefits of Upgrading Wastewater Treatment Plants

Micropollutants (MPs) as individual compounds or in complex mixtures are relevant for water quality and may trigger unwanted ecological effects. MPs originate from different point and diffuse sources and enter water bodies via different flow paths. Effluents from conventional wastewater treatment plants (WWTPs), in which various MPs are not or not completely removed, is one major source. To improve the water quality and avoid potential negative ecological effects by micropollutants, various measures to reduce the discharge should be taken. In this feature we discuss one of these measures; the benefits of upgrading WWTPs toward reduced MP loads and toxicities from wastewater effluents, using the recently decided Swiss strategy as an example. Based on (i) full-scale case studies using ozonation or powder activated carbon treatment, showing substantial reduction of MP discharges and concomitant reduced toxicities, (ii) social and political acceptance, (iii) technical feasibility and sufficient cost-effectiveness, the Swiss authorities recently decided to implement additional wastewater treatment steps as mitigation strategy to improve water quality. Since MPs are of growing global concern, the concepts and considerations behind the Swiss strategy are explained in this feature, which could be of use for other countries as well. It should be realized that upgrading WWTPs is not the only solution to reduce the discharge of MPs entering the environment, but is part of a broader, multipronged mitigation strategy.

Comparative analysis of estrogenic activity in sewage treatment plant effluents involving three in vitro assays and chemical analysis of steroids

In this study, we assessed and compared the suitability of three in vitro screening tools for the measurement of estrogenic activity in sewage treatment plant effluents (STPEs). These assays were the yeast estrogen screen (YES), production of zona radiata proteins (ZRPs) in trout hepatocytes, and the induction of reporter gene expression in the transfected rainbow trout gonad cell line RTG-2. Data obtained with the YES were additionally compared with calculated estrogenicity, based on steroid analysis data of the effluents. For comparison purposes, the response of the in vitro systems toward the estrogenic chemicals beta-estradiol, ethinyl estradiol, bisphenol-A, nonylphenol, and octylphenol was assessed. All three assays showed sensitivities in the same order of magnitude in response to the steroid compounds tested, with ZRP production being the least sensitive. Regarding the estrogenic environmental chemicals tested, the RTG-2 assay was more than an order of magnitude more sensitive than the other two assays. Despite their different sensitivities toward selected test chemicals, the three in vitro systems indicated estrogenic activity in the same concentration range for the tested STPEs. Calculated estrogenicity (chemical analysis) and measured estrogenicity (YES) were of the same order of magnitude for the STPEs tested. The present study indicates that all three in vitro systems, with the yeast-based system being the easiest and most robust, are applicable for the screening of estrogenic activity in effluent samples.

Role of singlet oxygen in chloroplast to nucleus retrograde signaling in Chlamydomonas reinhardtii

High light illumination of photosynthetic organisms stimulates the production of singlet oxygen by photosystem II and causes photooxidative stress. In Chlamydomonas reinhardtii, singlet oxygen also induces the expression of the nuclear‐encoded glutathione peroxidase homologous gene GPXH. We provide evidence that singlet oxygen stimulates GPXH expression by activating a signaling mechanism outside the thylakoid membrane. Singlet oxygen from photosystem II could be detected with specific probes in the aqueous phase of isolated thylakoid suspensions and the cytoplasm of high light stressed cells. This indicates that singlet oxygen can stimulate a response farther from its production site than generally believed.

The glutathione peroxidase homologous gene from Chlamydomonas reinhardtii is transcriptionally up-regulated by singlet oxygen

The glutathione peroxidase homologous gene (Gpxh gene) in Chlamydomonas reinhardtii is up-regulated under oxidative stress conditions. The Gpxh gene showed a remarkably strong and fast induction by the singlet oxygen-generating photosensitizers neutral red, methylene blue and rose Bengal. The Gpxh mRNA levels strongly increased, albeit much more slowly, upon exposure to the organic hydroperoxides tert-butyl hydroperoxide (t-BOOH) and cumene hydroperoxide. In contrast, the Gpxh mRNA levels were only weakly induced by exposure to the superoxide-generating compound paraquat and by hydrogen peroxide. A comparison of the Gpxh mRNA levels with those of the heat shock protein HSP70A and the iron superoxide dismutase gene showed qualitative and quantitative differences for the three genes under oxidative stress conditions tested. The Gpxh gene is specifically induced by singlet-oxygen photosensitizers and the relative induction by other compounds is much weaker for Gpxh than for the other genes investigated. Using Gpxh promoter fusions with the arylsulfatase reporter gene, we have shown that the Gpxh was transcriptionally up-regulated by singlet-oxygen photosensitizers. It is also shown that the Gpxh promoter contains a region between 104 and 179 bp upstream of the transcription start that is responsible for the mRNA up-regulation upon exposure to 1O2 but not t-BOOH. Within this region a regulatory sequence homologous to the mammalian cAMP response element (CRE) and activator protein 1 (AP-1) binding site was identified within a 16 bp palindrome.

Zebrafish (Danio rerio) neuromast: Promising biological endpoint linking developmental and toxicological studies

Aquatic toxicology is facing the challenge to assess the impact of complex mixtures of compounds on diverse biological endpoints. So far, ecotoxicology focuses mainly on apical endpoints such as growth, lethality and reproduction, but does not consider sublethal toxic effects that may indirectly cause ecological effects. One such sublethal effect is toxicant-induced impairment of neurosensory functions which will affect important behavioural traits of exposed organisms. Here, we critically review the mechanosensory lateral line (LL) system of zebrafish as a model to screen for chemical effects on neurosensory function of fish in particular and vertebrates in general. The LL system consists of so-called neuromasts, composed of centrally located sensory hair cells, and surrounding supporting cells. The function of neuromasts is the detection of water movements that is essential for the fishs ability to detect prey, to escape predator, to socially interact or to show rheotactic behaviour. Recent advances in the study of these organs provided researchers with a broad area of molecular tools for easy and rapid detection of neuromasts dysfunction and/or disturbed development. Further, genes involved in neuromasts differentiation have been identified using auditory/mechanosensory mutants and morphants. A number of environmental toxicants including metals and pharmaceuticals have been shown to affect neuromasts development and/or function. The use of the LL organ for toxicological studies offers the advantage to integrate the available profound knowledge on developmental biology of the neuromasts with the study of chemical toxicity. This combination may provide a powerful tool in environmental risk assessment.

The Peroxiredoxin and Glutathione Peroxidase Families in Chlamydomonas reinhardtii

Thiol/selenol peroxidases are ubiquitous nonheme peroxidases. They are divided into two major subfamilies: peroxiredoxins (PRXs) and glutathione peroxidases (GPXs). PRXs are present in diverse subcellular compartments and divided into four types: 2-cys PRX, 1-cys PRX, PRX-Q, and type II PRX (PRXII). In mammals, most GPXs are selenoenzymes containing a highly reactive selenocysteine in their active site while yeast and land plants are devoid of selenoproteins but contain nonselenium GPXs. The presence of a chloroplastic 2-cys PRX, a nonselenium GPX, and two selenium-dependent GPXs has been reported in the unicellular green alga Chlamydomonas reinhardtii. The availability of the Chlamydomonas genome sequence offers the opportunity to complete our knowledge on thiol/selenol peroxidases in this organism. In this article, Chlamydomonas PRX and GPX families are presented and compared to their counterparts in Arabidopsis, human, yeast, and Synechocystis sp. A summary of the current knowledge on each family of peroxidases, especially in photosynthetic organisms, phylogenetic analyses, and investigations of the putative subcellular localization of each protein and its relative expression level, on the basis of EST data, are presented. We show that Chlamydomonas PRX and GPX families share some similarities with other photosynthetic organisms but also with human cells. The data are discussed in view of recent results suggesting that these enzymes are important scavengers of reactive oxygen species (ROS) and reactive nitrogen species (RNS) but also play a role in ROS signaling.

A European perspective on alternatives to animal testing for environmental hazard identification and risk assessment

Tests with vertebrates are an integral part of environmental hazard identification and risk assessment of chemicals, plant protection products, pharmaceuticals, biocides, feed additives and effluents. These tests raise ethical and economic concerns and are considered as inappropriate for assessing all of the substances and effluents that require regulatory testing. Hence, there is a strong demand for replacement, reduction and refinement strategies and methods. However, until now alternative approaches have only rarely been used in regulatory settings. This review provides an overview on current regulations of chemicals and the requirements for animal tests in environmental hazard and risk assessment. It aims to highlight the potential areas for alternative approaches in environmental hazard identification and risk assessment. Perspectives and limitations of alternative approaches to animal tests using vertebrates in environmental toxicology, i.e. mainly fish and amphibians, are discussed. Free access to existing (proprietary) animal test data, availability of validated alternative methods and a practical implementation of conceptual approaches such as the Adverse Outcome Pathways and Integrated Testing Strategies were identified as major requirements towards the successful development and implementation of alternative approaches. Although this article focusses on European regulations, its considerations and conclusions are of global relevance.

Isolation and Characterization of the Hyperthermostable Serine Protease, Pyrolysin, and Its Gene from the Hyperthermophilic Archaeon Pyrococcus furiosus

The hyperthermostable serine protease pyrolysin from the hyperthermophilic archaeon Pyrococcus furiosus was purified from membrane fractions. Two proteolytically active fractions were obtained, designated high (HMW) and low (LMW) molecular weight pyrolysin, that showed immunological cross-reaction and identical NH2-terminal sequences in which the third residue could be glycosylated. The HMW pyrolysin showed a subunit mass of 150 kDa after acid denaturation. Incubation of HMW pyrolysin at 95°C resulted in the formation of LMW pyrolysin, probably as a consequence of COOH-terminal autoproteolysis. The 4194-base pair pls gene encoding pyrolysin was isolated and characterized, and its transcription initiation site was identified. The deduced pyrolysin sequence indicated a prepro-enzyme organization, with a 1249-residue mature protein composed of an NH2-terminal catalytic domain with considerable homology to subtilisin-like serine proteases and a COOH-terminal domain that contained most of the 32 possible N-glycosylation sites. The archaeal pyrolysin showed highest homology with eucaryal tripeptidyl peptidases II on the amino acid level but a different cleavage specificity as shown by its endopeptidase activity toward caseins, casein fragments including αS1-casein and synthetic peptides.

The glutamate dehydrogenase-encoding gene of the hyperthermophilic archaeon Pyrococcus furiosus: sequence, transcription and analysis of the deduced amino acid sequence.

Glutamate dehydrogenase (GDH) from the hyperthermophilic archaeon, Pyrococcus woesei, has been isolated, characterized and found to be very similar if not identical to the recently purified GDH from P. furiosus. Using a polymerase chain reaction, based on the N-terminal amino acid sequences of GDH, the P. furiosus gdh gene was identified, cloned into Escherichia coli and sequenced. The transcription start point of gdh has been mapped 1 nucleotide upstream from the ribosome-binding site. Using antiserum raised against purified GDH, expression of gdh was observed in E. coli. The deduced primary sequence of the P. furiosus GDH has been compared to various bacterial, archaeal and eukaryal GDHs and showed a high degree of similarity (32-52%).

Effect of pulse herbicidal exposure on Scenedesmus vacuolatus: A comparison of two photosystem II inhibitors

Herbicide concentrations fluctuate in rivers following crop application and can reach high levels after rain events, yet the duration of these pulses is short. In the present study, we assessed the effect of atrazine and isoproturon pulse exposure on Scenedesmus vacuolatus (Chlorophyceae; strain 211-8b, Kessler) as well as the recovery in the postexposure period. We further explored whether the time-dependent toxicity is similar for herbicides inhibiting the photosystem II (PSII). The growth rate was assessed for different exposure durations, and in addition the inhibition of the effective quantum yield of PSII was measured to monitor the response at the target site. Atrazine and isoproturon did not have similar time-dependent effects on growth rate, despite their same primary mode of action on PSII. Atrazine was less toxic than isoproturon after 10 h of exposure, but the toxicity of both herbicides was similar after 48 h of exposure. However, both compounds inhibited the PSII effective quantum yield within 1 h following exposure. Similarly, the effective quantum yield recovered completely within 4 h after removal of the toxicants, leading to rapid recovery of algal growth. The rapid onset of effects of isoproturon on the growth of the alga during exposure suggests that a single pulse to this herbicide is likely to induce greater effects than an atrazine pulse at the same concentration, even if these effects are reversible. The information gained in the present study should support the effect assessment of sequential exposures as well as the risk evaluation of fluctuating herbicidal exposure.