Performance of a miniaturized algal bioassay in phytotoxicity screening

Abstract

A miniaturized and low-cost algal growth-inhibition assay, with Pseudokirchneriella subcapitata, based on the standard ISO 8692 and using 96-well microplates, was tested and optimized in this work, to be used as a useful tool for pollutant phytotoxicity screening. For validation, the performance of the microplate algal growth-inhibition assay was first compared with the standard flask assay for the toxicity testing of five reference toxicants (copper(II) sulfate, zinc sulfate, potassium permanganate, potassium dichromate and 3,5-Dichlorophenol) and six wastewater samples. Statistical evaluation of EC(50) results from both methods demonstrated a good agreement between microplate and flask assays either in testing chemicals (r (2) = 0.975, p < 0.0017) or environmental samples toxicity (r (2) = 0.984, p < 0.0001). In addition, the performance of this algal microplate bioassay was also evaluated in comparison with Lemna test, ISO 20079, for phytotoxicity assessment of 27 wastewater samples from industries and treatment plants. The results showed that the algal test was more sensitive for most of the samples, but a significant agreement between both tests was observed (r (2) = 0.644, p < 0.0001). In conclusion, this miniaturized test can be a good tool to include in a battery of tests for phytotoxicity screening of a wide range of chemicals and environmental samples, with the advantage of requiring low sample volumes for the test, allowing large numbers of samples to be tested, and generating low volumes of waste.

A Novel Miniature Culture System to Screen CO2-Sequestering Microalgae

“…Compared with flask culture, miniature screening systems occupy less space and allow a large increase in experimental throughput. Compared with previous miniature microalgal culture systems [9][10][11], agreement of results between M96SS and flask culture growth (in response to different CO 2 supply conditions) was statistically proven. Moreover, M96SS uses moderate vertical mixing.…”

“…The high frequency shaking generates excess heat and causes serious sample evaporation [23]. Additional cooling devices have been used to prevent excess heat and sample evaporation for algal bioassays in toxin screening [10]. Without additional cooling devices, a sealed microplate can reach 32 °C (room temperature 25 °C) when using a 600 rpm microplate shaker for mixing ( Figure 5).…”

“…Lukavský [9] cultivated microalgae for toxin bioassays in 96-well plates in a closed flat box containing CO 2 . Later researchers [5,10] verified the agreement between Lukavský's [9] system and the flask culture system for bioassays. However, these authors did not address similarities and differences between microalgal growth characteristics in miniature and flask culture.…”

Probabilistic risk assessment of diuron and prometryn in the Gwydir River catchment, Australia, with the input of a novel bioassay based on algal growth

“…However, the experimental formats are labor intensive, specifying large sample volumes, requiring large areas of benchspace, and generating large volumes of waste. Since the 1990s, many researchers have attempted to optimize the procedure, performing tests at microscale using cuvettes, scintillation tubes, or microplates (Arensberg et al, 1995;Nguyen-Ngoc et al, 2009;Paixao et al, 2008;Rojiekova et al, 1998). The 24-or 96-well algal growth inhibition assay performed in a microplate, gave comparable results to standard flask bioassay for the tested algae (Thellen et al, 1990;Rojiekova et al, 1998;Eisentraeger et al, 2003;Horvatic et al, 2007;Riedl and Altenburger, 2007).…”

“…This simpler method has been used in routine toxicity testing, pollutant phytotoxicity screening, and screening of sensitivity of algae. In terms of pollutants, many heavy metals, and organic pollutants, have been tested with these micro-scale methods, however literature reports of assessments for the herbicides diuron and prometryn are rare (Gomez De Barreda Ferraz et al, 2004;Gregor and Maralek, 2005;Paixao et al, 2008).…”