Testing particles using the algal growth inhibition test (OECD 201): the suitability of in vivo chlorophyll fluorescence measurements

Abstract

Background The freshwater algae and cyanobacteria growth inhibition test (OECD test guideline 201) is frequently used to assess the ecotoxicity of chemicals or particles. A central issue is the measurement of algal growth by quantifying algal biomass over time. Chlorophyll fluorescence measurements are recommended for the testing of particles. The analysis of in vivo fluorescence is the simplest and fastest approach, but is only suitable if there is no interference with the materials. Therefore, in vitro fluorescence analysis is often preferred. We carried out a comprehensive comparison of chlorophyll fluorescence measurements in vitro and in vivo to evaluate the suitability of rapid in vivo testing for the determination of Raphidocelis subcapitata biomass in the presence of diverse particles. Results For the in vitro measurement, we applied a method that separates particles from chlorophyll using locust bean gum. We tested inorganic and organic particles (including alloys and polymers), ion-releasing and non-releasing materials, and particle sizes in the nanometer to micrometer range with a variety of shapes (spherical, flaky and fibrous). Some of the materials were nontoxic, whereas others showed varying degrees of toxicity (ErC50 = 0.2–100 mg/L in both methods). There were only minor differences between the methods in ErC50 values and the percent inhibition at various test concentrations, but the confidence intervals for the ErC50 values in vivo were narrower and were covered by the range observed in vitro. The in vivo approach showed no limitations, whereas the validity criteria listed in OECD test guideline 201 were not always fulfilled by the in vitro measurements. Conclusion The in vivo approach was a suitable and time-saving method for a wide range of particles, although we cannot completely exclude the possibility that some particles may interfere with fluorescence measurement. To avoid false assessments, pre-tests with simple measurements are therefore recommended. Graphical abstract

Prioritising nano- and microparticles: identification of physicochemical properties relevant for toxicity to Raphidocelis subcapitata and Daphnia magna

“…During the tests, all vessels were placed on an Incubation Shaker Multitron (INFORS-HT, Switzerland) and shaken continuously at 150 rpm. Algae biomass was determined via chlorophyll fluorescence [26] followed by calculation of the growth rate. In vitro and in vivo data from valid fluorescence measurements were used.…”

“…For the in vitro measurement, we applied a method that separated particles from chlorophyll by using locust bean gum. Both applied methods resulted in comparable effect data [26]. Every material was tested at several concentrations (see "Preparation of suspensions" Section).…”

Applicability of OECD TG 201, 202, 203 for the aquatic toxicity testing and assessment of 2D Graphene material nanoforms to meet regulatory needs

“…Also test setups using double-vial systems to distinguish direct toxic effects and indirect physical effects have been used (Sørensen et al, 2015). Alternative method for chl measurement that have been described include modified versions of ISO 10260:1992 incorporating an algal filtration step (Farkas and Booth, 2017), as well as a locust bean gum separation technique (Hund-Rinke et al, 2022). Such approaches may not be applicable in all cases and thus prior validation studies would be needed.…”

Enantioselective Toxic Effects of Prothioconazole toward Scenedesmus obliquus

“…S. obliquus was purchased from the Institute of Hydrobiology, Chinese Academy of Sciences, and cultured in a BG-11 medium at 24 ± 1 °C in an incubator under illumination at 4500 lx with daily cycles of 14:10 h light: dark cycle. An algal growth inhibition test was conducted according to the updated OECD guideline 201 [ 36 ]. Exponential growth phase algal cells were cultivated in 150 mL of a culture medium with a series of Rac -PTC or PTC enantiomer dilution concentrations (0, 0.5, 1, 2.5, 5, and 10 mg·L −1 ) in 250 mL Erlenmeyer flasks with an initial cell density of 4.33 × 10 5 cells·mL −1 (OD 685 = 0.018) for 72 h. To ensure optimum growth, the flasks were shaken five times, and their position in the incubator was randomly changed per day.…”