The Combined Algae Test for the Evaluation of Mixture Toxicity in Environmental Samples

Abstract

The combined algae test (CAT) is a 96-well plate-based algal toxicity assay with the green algae Raphidocelis subcapitata that combines inhibition of 24h population growth rate with inhibition of photosynthesis detected after 2h and 24h with pulse-amplitude modulated (PAM) fluorometry using a Maxi-Imaging PAM. The CAT has been in use for more than a decade but had limitations due to incompatibilities of the measurements of the two biological endpoints on the same microtiter plates. These limitations could be overcome by increasing growth rates and doubling times on black, clear-bottom 96-well plates by application of dichromatic red/blue LED illumination. Different robotic dosing approaches and additional data evaluation approaches helped to further expand the applicability domain of the assay. The CAT differentiates between non-specifically acting compounds and photosynthesis inhibitors, such as photosystem II (PSII)-herbicides. PSII-herbicides acted immediately on photosynthesis and showed growth rate inhibition at higher concentrations. If growth was a similar or more sensitive endpoint than photosynthesis inhibition, this was an indication that the tested chemical acted non-specifically or a mixture or a water sample was dominated by chemicals other than PSII-herbicides acting on algal growth. We fingerprinted the effects of 45 chemicals on photosynthesis inhibition and growth rate and related the effects of the single compounds to designed mixtures of these chemicals detected in water samples and to Running Head: Combined algae test and mixtures This article is protected by copyright. All rights reserved. Accepted Article the effects directly measured in water samples. Most of the observed effects in the water samples could be explained by known photosystem II inhibitors such as triazines and phenylurea herbicides. The improved setup of the CAT gave consistent results with the previous method but has lower cost, higher throughput and higher precision. This article includes online-only Supplemental Data.

Bioanalytical Tools in Water Quality Assessment

“…For whole organism tests, photosynthesis inhibition in algae or immobilisation of Daphnia magna by insecticides are good examples. Herbicides are 10,000 to 100,000 times more potent than other chemicals for inhibition of photosynthesis and hence the mixture effect is often completely dominated by herbicides unless a sample does not contain any herbicides de Baat et al, 2018;Glauch and Escher 2020). Herbicides are not only used in agriculture but also in urban applications, so they are quite ubiquitous and general mixture risk drivers.…”

“…Although not directly relevant for human health, several studies have applied algal assays to assess photosystem II (PSII) inhibition in a range of water matrices Hamers et al, 2018). Most studies have assessed PSII inhibition using the combined algae test (CAT) with PSII inhibition measured after 2 h using imaging pulse-amplitude modulated (PAM) fluorometry using green microalgae Raphidocelis subcapitata (formerly named Selenastrum capricornutum and Pseudokirchneriella subcapitata) (Escher et al, 2008a;Glauch and Escher 2020).…”

Estrogenicity of chemical mixtures revealed by a panel of bioassays

“…The applicability of bioassays as screening methods to complement chemical analysis for water quality monitoring is currently being investigated given their potential advantages over chemical methods ( Brack et al, 2019 ; Escher et al, 2014 ; Glauch and Escher, 2020 ; Neale et al, 2017 ; Wernersson et al, 2015 ). First, bioassays cover a broad range of modes of action (MoA).…”

Resilience of Micropollutant and Biological Effect Removal in an Aerated Horizontal Flow Treatment Wetland

“…Final extracts were stored at -18 • C until the bioassays were performed. All cell-based bioassays were applied to quantify the mixture effects using the method described by König et al [11], Neale et al [30], and Glauch & Escher [31] and are summarized in Table 2.…”

“…Furthermore, by using the BEQ from the influent (BEQ in ) and the effluent (BEQ out ) the removal efficacy was calculated according to Equation (3). For a more comprehensive description of the data evaluation the reader is referred to Kahl et al [8] for the micropollutants data analysis and to Escher et al [34] and Glauch and Escher [31] for bioassay data evaluation.…”