Identification of Photosynthesis Inhibitors of Pelagic Marine Algae Using 96-Well Plate Microfractionation for Enhanced Throughput in Effect-Directed Analysis

Abstract

Because of large-scale production and use of an increasing diversity of chemicals in modern society, estuarine and coastal waters may be contaminated with numerous substances. Some of these compounds have the potential to affect microalgae at the base of the pelagic food chain. Therefore, we identified the main chemical stressors that negatively affect the effective photosystem II efficiency (ϕPSII) in marine microalgae of the Dutch estuarine and coastal waters. An enhanced effect-directed analysis (EDA) was carried out by combining reversed-phase ultra performance liquid chromatography fractionation of extracts from passive samplers, followed by effect assessment using the pulse amplitude modulation fluorometry assay and chemical analysis of biologically active fractions using high-resolution mass spectrometry. This study focuses on a novel microfractionation technique using 96-well plates to enhance throughput in EDA, structure elucidation, and the analytical and effect confirmation of the compounds that are identified. Although there are numerous unknown compounds present in estuarine and coastal waters, our EDA study shows that atrazine, diuron, irgarol, isoproturon, terbutryn, and terbutylazine are the main contributors to the observed effect on the ϕPSII of marine microalgae.

Recent trends in water analysis triggering future monitoring of organic micropollutants

“…This approach is very time-consuming, and therefore an interesting alternative is a microfractionation system that combines LC or even LC×LC separations with a simultaneous collection of fractions in microtiter plates for effect analysis that allows for linking of effect and chemical identity [42–44]. The applicability of such a system has already been demonstrated for high-throughput screening of several end points relevant in aquatic systems.…”

Toxic pressure of herbicides on microalgae in Dutch estuarine and coastal waters

“…From these plots (data not shown) the CF at EC 10 level was determined (CF EC 10 ). Assuming, as demonstrated by Booij et al (2014), that the 6 key contaminants are responsible for the observed effect, the ΣTU EC 10 in the PAM assay is calculated with Eq. (3).…”

How to Improve Exposure Assessment

“…Typical endpoints in EDA include, but are not limited to, endocrine disruption (Chapter 15), fish embryo toxicity (Zielke et al, 2011) mutagenicity, and aryl hydrocarbon receptor effects (Brack et al, 2005). Recent examples of EDA applications have been published by Booij et al (2014b) and Fetter et al (2014). EDA has also been applied in the context of the EU WFD (Brack et al, 2007(Brack et al, , 2015.…”