Acinetobacter baumannii is an opportunistic Gram-negative pathogen that causes a wide range of infections including pneumonia, septicemia, necrotizing fasciitis and severe wound and urinary tract infections. Analysis of A. baumannii representative strains grown in Chelex 100-treated medium for hemolytic activity demonstrated that this pathogen is increasingly hemolytic to sheep, human and horse erythrocytes, which interestingly contain increasing amounts of phosphatidylcholine in their membranes. Bioinformatic, genetic and functional analyses of 19 A. baumannii isolates showed that the genomes of each strain contained two phosphatidylcholine-specific phospholipase C (PC-PLC) genes, which were named plc1 and plc2. Accordingly, all of these strains were significantly hemolytic to horse erythrocytes and their culture supernatants tested positive for PC-PLC activity. Further analyses showed that the transcriptional expression of plc1 and plc2 and the production of phospholipase and thus hemolytic activity increased when bacteria were cultured under iron-chelation as compared to iron-rich conditions. Testing of the A. baumannii ATCC 19606T plc1::aph-FRT and plc2::aph isogenic insertion derivatives showed that these mutants had a significantly reduced PC-PLC activity as compared to the parental strain, while testing of plc1::ermAM/plc2::aph demonstrated that this double PC-PLC isogenic mutant expressed significantly reduced cytolytic and hemolytic activity. Interestingly, only plc1 was shown to contribute significantly to A. baumannii virulence using the Galleria mellonella infection model. Taken together, our data demonstrate that both PLC1 and PLC2, which have diverged from a common ancestor, play a concerted role in hemolytic and cytolytic activities; although PLC1 seems to play a more critical role in the virulence of A. baumannii when tested in an invertebrate model. These activities would provide access to intracellular iron stores this pathogen could use during growth in the infected host.

An increased demand for chemical toxicity evaluations has resulted in the need for alternative testing strategies that address animal welfare concerns. The fish embryo toxicity (FET) test developed for zebrafish (Danio rerio) is one such alternative, and the application of the FET test to other species such as the fathead minnow (Pimephales promelas) has been proposed. In the present study, the performances of the FET test and the larval growth and survival (LGS; a standard toxicity testing method) test in zebrafish and fathead minnows were evaluated. This required that testing methods for the fathead minnow FET and zebrafish LGS tests be harmonized with existing test methods and that the performance of these testing strategies be evaluated by comparing the median lethal concentrations of 2 reference toxicants, 3,4-dicholoraniline and ammonia, obtained via each of the test types. The results showed that procedures for the zebrafish FET test can be adapted and applied to the fathead minnow. Differences in test sensitivity were observed for 3,4-dicholoraniline but not ammonia; therefore, conclusions regarding which test types offer the least or most sensitivity could not be made. Overall, these results show that the fathead minnow FET test has potential as an alternative toxicity testing strategy and that further analysis with other toxicants is warranted in an effort to better characterize the sensitivity and feasibility of this testing strategy.

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Phototoxicity occurs when exposure to ultraviolet radiation increases the toxicity of certain contaminants, including polycyclic aromatic hydrocarbons (PAHs). This study aimed to (1) develop a quantitative model to predict the risk of PAH phototoxicity to fish, (2) assess the predictive value of the model, and (3) estimate the risk of PAH phototoxicity to larval and young of year Pacific herring (Clupea pallasi) following the Exxon Valdez oil spill (EVOS) in Prince William Sound, Alaska. The model, in which median lethal times (LT50 values) are estimated from whole-body phototoxic PAH concentrations and ultraviolet A (UVA) exposure, was constructed from previously reported PAH phototoxicity data. The predictive value of this model was confirmed by the overlap of model-predicted and experimentally derived LT50 values. The model, along with UVA characterization data, was used to generate estimates for depths of de minimiz risk for PAH phototoxicity in young herring in 2003/2004 and immediately following the 1989 EVOS, assuming average and worst case conditions. Depths of de minimiz risk were estimated to be between 0 and 2 m deep when worst case UVA and PAH conditions were considered. A post hoc assessment determined that <1% of the young herring population would have been present at depths associated with significant risk of PAH phototoxicity in 2003/2004 and 1989.

The goal of the current study was to determine whether sediments from agriculturally-intense watersheds can act as a potential source of anti-estrogenic endocrine-disrupting compounds. The specific objectives of the current study were to determine 1) whether female fathead minnows (Pimephales promelas) experience alterations in endocrine function when exposed to sediments collected from agriculturally-intense watersheds and 2) if these sediments display anti-estrogenic activity in an in vitro assay. In addition, sediment samples were analyzed for the presence of steroid hormones and pesticides associated with local agricultural practices. To accomplish this, sediments and water were collected from three sites within two agriculturally-intense Nebraska watersheds (Bow Creek and the Elkhorn River). In 2009, minnows were exposed to sediment and/or water collected from the two Bow Creek sites (East Bow Creek and the Confluence) in the laboratory, while in 2010, minnows were exposed to sediment and/or water from East Bow Creek, the Confluence and the Elkhorn River. Following the 7-d exposure period, the hepatic mRNA expression of two-estrogen responsive genes, estrogen receptor α (ERα) and vitellogenin (Vtg) was determined. In 2009, females exposed to Confluence sediments, in the presence of laboratory water or Confluence water, experienced significant reductions in ERα expression relative to unexposed and Confluence water-exposed females. The defeminization of these females suggests the presence of a biologically-available anti-estrogenic compound in sediments collected from this site. In 2010, sediments were assessed for anti-estrogenic activity on days 0 and 7 of the exposure period using a four-hour yeast estrogen screen. Lipophilic extracts (LEs) of day 0 sediments collected from the Confluence and the Elkhorn River induced significant reductions in the estrogenic reporter activity of treated yeast cultures suggesting the presence of a lipophilic anti-estrogenic compound in these extracts. Chemical analysis revealed the presence of a variety of steroid hormones, including those associated with the production of beef cattle (ie: β-trenbolone, α-zearalanol and α-zearalenol), in sediments indicating that compounds utilized by local beef cattle operations are capable of entering nearby watersheds. Overall, the results of this study indicate that an environmentally-relevant anti-estrogenic compound is present in sediments from agriculturally-intense watersheds and that this compound is bioavailable to fish. Furthermore, the presence of steroid hormones in sediments from these watersheds provides evidence indicating that steroids are capable of sorbing to sediments. Clearly, sediments are capable of acting as a source of endocrine-disrupting compounds in the aquatic environment.

Recent research has suggested that the fate of biologically active compounds (BACs) originating from point sources such as wastewater treatment plants is fundamentally different from that of similar compounds released from nonpoint sources through runoff from agricultural landscapes. Downstream from wastewater treatment plants, BACs will degrade via a variety of mechanisms; however, their concentration in the water adjacent to the point of discharge may not decrease over time, as the compounds are continually released. In contrast, in agricultural systems, BACs are episodically introduced to surface water during snowmelt and rainstorm events, and under these circumstances, may be found in water for only hours or days after a storm event. Recent research in our laboratories as well as others, has suggested that sediments play an important role in the persistence of herbicides and steroids in watersheds after nonpoint source loading events. Conceptually, the sediment serves as both a sink and a source, equilibrating with BACs during storm events then slowly releasing them back into the water over time, long after the initial pulse of chemicals has moved downstream.

Since the 1940s, effluent toxicity testing has been used to assess potential ecological impacts of effluents and help determine necessary treatment options for environmental protection prior to release. Strategic combinations of toxicity tests, analytical tools, and biological monitoring have been developed. Because the number of vertebrates utilized in effluent testing is thought to be much greater than that used for individual chemical testing, there is a new need to develop strategies to reduce the numbers of vertebrates (i.e., fish) used. This need will become more critical as developing nations begin to use vertebrates in toxicity tests to assess effluent quality. A workshop was held to 1) assess the state of science in effluent toxicity testing globally; 2) determine current practices of regulators, industry, private laboratories, and academia; and 3) explore alternatives to vertebrate (fish) testing options and the inclusion of modified/new methods and approaches in the regulatory environment. No single approach was identified, because of a range of factors including regulatory concerns, validity criteria, and wider acceptability of alternatives. However, a suite of strategies in a weight‐of‐evidence approach would provide the flexibility to meet the needs of the environment, regulators, and the regulated community; and this “toolbox” approach would also support reduced reliance on in vivo fish tests. The present Focus article provides a brief overview of wastewater regulation and effluent testing approaches. Alternative methodologies under development and some of the limitations and barriers to regulatory approaches that can be selected to suit individual country and regional requirements are described and discussed. Environ Toxicol Chem 2018;37:2745–2757. © 2018 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals, Inc. on behalf of SETAC.

The fish embryo toxicity (FET) test has been proposed as an alternative to the larval growth and survival (LGS) test. The objectives of the present study were to evaluate the sensitivity of the FET and LGS tests in fathead minnows (Pimephales promelas) and zebrafish (Danio rerio) and to determine if the inclusion of sublethal metrics as test endpoints could enhance test utility. In both species, LGS and FET tests were conducted using 2 simulated effluents. A comparison of median lethal concentrations determined via each test revealed significant differences between test types; however, it could not be determined which test was the least and/or most sensitive. At the conclusion of each test, developmental abnormalities and the expression of genes related to growth and toxicity were evaluated. Fathead minnows and zebrafish exposed to mock municipal wastewater-treatment plant effluent in a FET test experienced an increased incidence of pericardial edema and significant alterations in the expression of genes including insulin-like growth factors 1 and 2, heat shock protein 70, and cytochrome P4501A, suggesting that the inclusion of these endpoints could enhance test utility. The results not only show the utility of the fathead minnow FET test as a replacement for the LGS test but also provide evidence that inclusion of additional endpoints could improve the predictive power of the FET test.