Demonstrating the need for chemical exposure characterisation in a microplate test system: toxicity screening of sixteen pesticides on two marine microalgae

Abstract

Pesticides used in viticulture create a potential risk for the aquatic environment due to drift during application, runoff and soil leaching. The toxicity of sixteen pesticides and one metabolite were evaluated on the growth of two marine microalgae, Tisochrysis lutea and Skeletonema marinoi, in 96-h exposure assays conducted in microplates. For each substance, concentrations of stock solutions were analytically measured and abiotic assays were performed to evaluate the chemical stability of pesticides in microplates. For two chemicals, microalgae exposures were run simultaneously in microplates and culture flasks to compare EC 50 calculated from the two exposure systems. Results from chemical analyses demonstrated the low stability of hydrophobic pesticides (log K OW > 3). For such chemicals, EC 50 values calculated using measured pesticide concentrations were twofold lower than those first estimated using nominal concentrations. Photosystem II inhibitors were the most toxic herbicides, with EC 50 values below 10 μg L −1 for diuron and around double this for isoproturon. Chlorpyrifos-methyl was the only insecticide to significantly affect the growth of T. lutea, with an EC 50 around 400 μg L −1. All fungicides tested were significantly toxic to both species: strobilurins showed low overall toxicity, with EC 50 values around 400 μg L −1 , whereas quinoxyfen, and spiroxamine, showed high toxicity to both species, especially to T. lutea, with an EC 50 below 1 μg L −1 measured for spiroxamine in culture flasks. This study highlights the need to perform chemical analyses for reliable toxicity assessment and discusses the advantages and disadvantages of using microplates as a toxicity screening tool. 2 Please note that this is an author-produced PDF of an article accepted for publication following peer review. The definitive publisher-authenticated version is available on the publisher Web site. Highlights ► The toxicity of 17 pesticides was tested on two microalgae species. ► A microplate assay was developed for accurate determination of EC 50 values. ► Analytical measurement of exposure concentrations was performed. ► Significant differences between nominal and measured concentrations were found. ► Four herbicides, one insecticide and four fungicides showed significant toxicity.

Time-Resolved Freely Dissolved Concentrations of Semivolatile and Hydrophobic Test Chemicals in In Vitro Assays—Measuring High Losses and Crossover by Headspace Solid-Phase Microextraction

“…1,2,6,8 Therefore, the actual exposure of the cells to the test substances is often not known and decreases with time, which can lead to reduced toxicity and assay sensitivity. 1,2,9,10 Changes in assay setup affect the losses and thereby alter the toxicity parameter based on nominal concentrations. 11 This hampers comparisons of effect data generated using different in vitro bioassay setups and between different chemicals.…”

“…The test results are normally based on the nominal concentration of a test chemical added to the culture medium at the start of the test. However, several studies have found that volatile and/or hydrophobic chemicals evaporate from wells, − crossover to adjacent wells, sorb to microtiter plates, ,,, and sorb to medium constituents. ,,, Therefore, the actual exposure of the cells to the test substances is often not known and decreases with time, which can lead to reduced toxicity and assay sensitivity. ,,, Changes in assay setup affect the losses and thereby alter the toxicity parameter based on nominal concentrations . This hampers comparisons of effect data generated using different in vitro bioassay setups and between different chemicals. , These exposure issues can limit the applicability, value, and relevance of in vitro toxicological data because even a well-characterized toxicological response is of limited value if it cannot be linked to a well-defined exposure level.…”

Dynamic Mass Balance Modeling for Chemical Distribution Over Time in In Vitro Systems With Repeated Dosing

“…The IV-MBM DP v1.0 model was parameterized and applied to simulate the chemicals and single dose scenarios described in Tanneberger et al (2013) and Dupraz et al (2019) The observations being compared to are the ratios of measured medium concentration at the end of the 24 or 96 h exposure period (C24 or C96) to the initial medium concentration (C0) (i.e., C24/C0 or C96/C0). Cellular concentrations were not measured in these studies.…”

“…The chemicals simulated for the Tanneberger et al (2013) study ( n = 27) and the Dupraz et al (2019) study ( n = 13) along with the required property data and initial nominal doses are compiled in the Supplementary Material . The simulated chemicals cover a wide range of hydrophobicity and volatility [e.g., the octanol-water partition coefficient (log K OW ) and the air-water partition coefficient (log K AW ) estimates span more than ten orders of magnitude] and thus are expected to behave very differently in in vitro test systems.…”

Toxicity of binary mixtures of pesticides to the marine microalgae Tisochrysis lutea and Skeletonema marinoi: Substance interactions and physiological impacts

“…As pointed out in the results section, EC50s determined in culture flasks (supplementary material: Table S6) were two-to six-fold lower than those in microplates, indicating a higher toxicity for exposed microalgae (Table 1 and supplementary material: Table S9). These discrepancies are probably due to the different characteristics of the materials composing microplates and culture flasks (plastic vs. glass), as well as to the different growth endpoints used to assess impacts on growth in the two exposure systems (cell density in culture flasks vs chlorophyll fluorescence in microplates) as previously discussed in Dupraz et al (2019).…”