P. Booij

Biomonitoring of estrogenic exposure and identification of responsible compounds in bream from Dutch surface waters.

The exposure to and effects of estrogenic compounds in male breams from Dutch freshwater locations were investigated. Ovotestis was observed infrequently (maximum frequency 16%). However, plasma vitellogenin (VTG) concentration was elevated highly at some locations. Estrogenic activities in male bream plasma, liver, and in gastrointestinal content were measured in the estrogen-responsive chemical-activated luciferase gene expression (ER-CALUX) assay. Plasma concentrations of vitellogenin correlated very well with the estrogenic activities in gastrointestinal content. The ER-CALUX activity in gastrointestinal content thus could provide a biomarker for recent exposure to estrogenic compounds, and the gastrointestinal content was chosen as investigative matrix for the toxicity identification and evaluation ([TIE]; bioassay-directed fractionation) of estrogenic compounds in bream. The approach consisted of a reversed-phase high-performance liquid chromatography fractionation of gastrointestinal content extract, directed by ER-CALUX and followed by gas chromatography analysis. The estrogenic hormones 17beta-estradiol and its metabolite estrone were identified as major contributors to the activity at all locations (except the reference location), independent of the presence or absence of a known source of estrogenic activity, such as a sewage treatment plant. Chemical screening showed the presence of other pollutants, such as a lower chlorinated dioxin and the disinfectants clorophene and triclosan. However, these compounds did not have high estrogenic potencies and their concentrations were not high enough to contribute significantly to the observed estrogenic activity.

Mixtures of estrogenic chemicals enhance vitellogenic response in sea bass.

Background The potential impact of natural and synthetic estrogens on aquatic ecosystems has attracted considerable attention because it is currently accepted that their joint effects are more severe when they are present in mixtures. Although it is well-known that they occur as mixtures in the marine environment, there is little information about the combined effects of estrogenic chemicals on marine biota. Objective In 14-day tests with juvenile sea bass, we analyzed singly and in combination the estrogenic activity of estradiol (E2), ethynylestradiol (EE2), and bisphenol A (BPA) using vitellogenin induction as an end point. Methods Fish were exposed to each compound, and on the basis of these concentration–response data, we predicted mixture effects by applying the model of concentration addition. The mixtures were tested using a fixed-ratio design, and the resulting mixture effects were compared to the predictions. Results EE2 was the most potent steroid, with an EC50 (median effective concentration) of 0.029 μg/L, 3.6 times more potent than E2 (EC50 = 0.104 μg/L); BPA was the least potent chemical, with an EC50 of 77.94 μg/L. The comparative assessment yielded a good agreement between observed and predicted mixture effects. Conclusions This study demonstrates the potential hazard of these compounds to seawater life by their ability to act together in an additive manner. It provides evidence that concentration addition can be used as a predictive tool for assessing the combined effects of estrogenic chemicals in marine ecosystems.

Hazard and risk of herbicides for marine microalgae

Due to their specific effect on photosynthesis, herbicides pose a potential threat to coastal and estuarine microalgae. However, comprehensive understanding of the hazard and risk of these contaminants is currently lacking. Therefore the aim of the present study was to investigate the toxic effects of four ubiquitous herbicides (atrazine, diuron, Irgarol(®)1051 and isoproturon) and herbicide mixtures on marine microalgae. Using a Pulse Amplitude Modulation (PAM) fluorometry based bioassay we demonstrated a clear species and herbicide specific toxicity and showed that the current environmental legislation does not protect algae sufficiently against diuron and isoproturon. Although a low actual risk of herbicides in the field was demonstrated, monitoring data revealed that concentrations occasionally reach potential effect levels. Hence it cannot be excluded that herbicides contribute to observed changes in phytoplankton species composition in coastal waters, but this is likely to occur only occasionally.

Laboratory algal bioassays using PAM fluorometry: Effects of test conditions on the determination of herbicide and field sample toxicity

Pulse Amplitude Modulation (PAM) fluorometry, based on chlorophyll a fluorescence, is a frequently used technique in algal bioassays to assess toxicity of single compounds or complex field samples. Several test conditions can influence the test results, and because a standardized test protocol is currently lacking, linking the results of different studies is difficult. Therefore, the aim of the present study was to gain insight into the effects of test conditions of laboratory algal bioassays using PAM fluorometry on the outcome of toxicity tests. To this purpose, we described the results from several pilot studies on test development in which information is provided on the effects of the main test factors during the pretest phase, the test preparation, the exposure period, and the actual measurement. The experiments were focused on individual herbicides and complex field samples and included the effects of culturing conditions, cell density, solvent concentration, exposure time, and the presence of actinic light. Several of these test conditions were found to influence the outcome of the toxicity test, and the presented information provides important background information for the interpretation of toxicity results and describes which test conditions should be taken into account when using an algal bioassay with PAM fluorometry. Finally, the application of PAM fluorometry in algal toxicity testing is discussed.

Extraction tools for identification of chemical contaminants in estuarine and coastal waters to determine toxic pressure on primary producers

The extent to which chemical stressors affect primary producers in estuarine and coastal waters is largely unknown. However, given the large number of legacy pollutants and chemicals of emerging concern present in the environment, this is an important and relevant issue that requires further study. The purpose of our study was to extract and identify compounds which are inhibitors of photosystem II activity in microalgae from estuarine and coastal waters. Field sampling was conducted in the Western Scheldt estuary (Hansweert, The Netherlands). We compared four different commonly used extraction methods: passive sampling with silicone rubber sheets, polar organic integrative samplers (POCIS) and spot water sampling using two different solid phase extraction (SPE) cartridges. Toxic effects of extracts prepared from spot water samples and passive samplers were determined in the Pulse Amplitude Modulation (PAM) fluorometry bioassay. With target chemical analysis using LC-MS and GC-MS, a set of PAHs, PCBs and pesticides was determined in field samples. These compound classes are listed as priority substances for the marine environment by the OSPAR convention. In addition, recovery experiments with both SPE cartridges were performed to evaluate the extraction suitability of these methods. Passive sampling using silicone rubber sheets and POCIS can be applied to determine compounds with different structures and polarities for further identification and determination of toxic pressure on primary producers. The added value of SPE lies in its suitability for quantitative analysis; calibration of passive samplers still needs further investigation for quantification of field concentrations of contaminants.

Development of a polydimethylsiloxane film-based passive dosing method in the in vitro DR-CALUX® assay.

In bioassays, exposure concentrations of test compounds are usually expressed as nominal concentrations. As a result of various processes, such as adsorption, degradation, or uptake, the actual freely dissolved concentration of the test compound may differ from the nominal concentration. The goal of the present study was to develop a method to dose passively the freely dissolved fraction of organic chemicals in an in vitro bioassay with adherent cells. To this end, a polydimethylsiloxane (PDMS) film-based method was developed for a reporter gene assay for dioxin-like compounds in a rat liver cell line. Polydimethylsiloxane films loaded with test compounds ensure that the concentration during exposure is in equilibrium and that the ratio between the concentration on the film and the concentration in medium is constant. Benzo[k]fluoranthene (BkF) was used as a model compound to develop the passive dosing method in transwell plates, which was further tested with a complex mixture, i.e., an extract prepared from a contaminated sediment. A higher dioxin-like activity was found when extracts were dosed by passive dosing with PDMS than when directly added to medium. Comparison with analysis of the concentration of BkF in medium shows that passive dosing of individual chemicals may not be necessary if freely dissolved concentrations are known. Use of PDMS for passive dosing of complex samples may represent a more realistic method for exposure in in vitro bioassays.

Non-target metabolomic profiling of the marine microalgae Dunaliella tertiolecta after exposure to diuron using complementary high-resolution analytical techniques

Traditionally, bioassays are used to assess the toxicity of chemicals. Bioassays often focus on one specific mode of action or end point and their responses offer a limited understanding of the health status and underlying pathways of the species under consideration. Metabolomics can be used to detect hundreds of metabolites in which each metabolite, or set of metabolites, represents short term and long term changes, indicating the status of the organism. The effects of the herbicide diuron, one of the compounds of concern for European water bodies, on the marine microalgae Dunaliella tertiolecta were investigated through non-target metabolomic profiling and bioassay testing. The pulse amplitude modulation (PAM) fluorometry bioassay was employed to measure the effective photosystem II efficiency (ϕPSII), while non-target metabolomic profiling using complementary analytical techniques characterized the metabolomic response in the algae during diuron exposure. The use of complementary analytical techniques was necessary to identify a broad range of metabolites. Twenty-eight compounds were identified as metabolites affected by diuron exposure, including several amino acids, adenosine, lactic acid, and citric acid. Collectively, these metabolites indicated that diuron negatively affects energy processes in the algae both at the citric acid cycle pathway as well as on the amino acid metabolism at realistic environmental concentrations. In addition, dose-response relationships were found between a number of affected metabolites and the inhibition of the ΦPSII of D. tertiolecta. Non-target metabolomic profiling using complementary analytical techniques proved to have additional and complementary benefits to traditional toxicology tests.