Environmental protection has become a global issue of concern. To properly establish legislation regulating wastewater effluent discharge and monitoring water quality, novel risk-based approaches and test methods adopted to assess water body status, including ecological safety, chemical quality, and biological impact, become essential (Bodini et al. 2018). As the representative index of biological impact, water toxicity is determined to assess the hazardous effects of pollutants, chemicals, or heavy metals on ecosystems or environments. Ecotoxicity tests are the most frequently used tools for assessing water toxicity by detecting the biological response produced by microorganisms or higher organisms affected by the toxic chemicals (García-Gómez et al. 2015, Petric et al. 2016). Compared with physiochemical analyses to understand the water quality, ecotoxicity tests can overcome the limits of physiochemical analyses in demonstrating the biochemical influence of toxic chemicals on living organisms, bioavailability toward toxic chemicals, and the antagonistic and synergetic interactions (Bolan et al. 2015, Rosado et al. 2016, Rosal et al. 2010). In ecotoxicity tests, organisms' trophic levels, including mammalian cells, algae, plants, fish, zooplankton, phytoplankton, and bacteria, are examined as model and target organisms. (Hund-Rinke &Simon 2006, Wang et al. 2015, Yao et al. 2018). In contrast to multi-cellular eukaryotic organisms, bacteria have rapid rates of growth. Ecotoxicity test based on bacteria as model organisms are easily applicable for widely and routinely toxicity screening because it has advantages of relatively short assay time, high sensitivity, cost-effectiveness, and less ethical responsibility (Muneeswaran et al. 2021, Parvez et al. 2006, Wang et al. 2010).
Various kinds of biochemical responses have established the bacteria-based toxicity assessment method. For instance, luminescent bacteria, such as Vibrio fischeri (formerly known as Photobacterium phosphoreum) (Venancio et al. 2021) or Photobacterium leiognathid (Muneeswaran et al. 2021, Neale et al. 2017) are the most widely used method for evaluating and monitoring ecotoxicity. They have been applied to assess the ecotoxicity of petroleum hydrocarbon, pesticide-contaminated soils, contaminated river sediments, nanoparticles, and industrially processed wastewater (Jarque et al. 2016, Moi et al. 2017, Zhang et al. 2020). The luminescent bacteria can naturally produce bioluminescence by expressing their luciferase gene and the bioluminescent signals can be monitored using a luminometer. After the bacterial cells are exposed to the target chemicals for 15 or 30 min, the bioluminescent signal is subsequently determined. The amount of the target chemicals to cause a 50% luminescence inhibition is called the median effect concentration (EC50) (Froehner et al. 2000). Alike the principle of monitoring the bioluminescent signal, other biochemical indicators, such as nitrification, electron transfer, respiration, or unique enzyme expression, have also been applied to ecotoxicity tests, which are related to nitrifying bacteria (Gernaey et al. 1997), iron-oxidizing bacteria (Yang et al. 2017), sulfur-oxidizing bacteria (Eom et al. 2019), electroactive bacteria (Chu et al. 2021), and fermentative bacteria (Eom et al. 2020). However, detecting or monitoring all the above biochemical responses requires their specific equipment; thus, extending the generalization of those ecotoxicity methods is restricted. Besides, the primary purpose of those ecotoxicity assays is to collect and establish the toxicity profile and database of interesting chemicals as much as possible based on one reliable microbiological system. Those methods are unable to adapt the requirement for an opposite purpose, for instance, to evaluate toxicity response of regulation concerning chemicals to prospective bioremediation bacteria (Kang &Park 2010, Ruggiero et al. 2005), specific plant growth-promoting bacteria (Mubeen et al. 2006, Verma et al. 2016), and sewage bacteria (Strotmann et al. 1994). Therefore, ecotoxicity assessment determined based on the inhibition of bacterial growth is still the simplest method and applicable for these purposes (Baek &An 2011, Giri &Golder 2015).
For quantifying bacterial growth and inhibition after toxic chemical exposure, the viable-count or spread plate method is one of the most commonly used techniques by counting the number of forming colonies on an agar plate and evaluating the difference between samples with or without adding chemicals. However, the main disadvantage of the spread plate method is that it takes a relatively long time (at least overnight) for incubation before the results are obtained. Alternatively, a Start Growth Time (SGT) method for high throughput determination of viable bacterial cell counts in 96-well plates has been established (Hazan et al. 2012). The SGT method prepares a series dilution of the bacterial liquid culture, monitors the growth curve of each diluted culture, and sets up an optical density (O.D.) threshold around 0.15 to 0.20 to point as the SGT value. Then, it establishes a linear correlation between the SGT values and the cell density to quantify the viable bacterial cell counts for the other samples. Because the SGT value represents the bacterial growth during the early exponential phase, the duration of the overall monitoring process is relatively short. For instance, the time required to establish the SGT correlation for Pseudomonas aeruginosa strain PA14 only took 11.5 hours to reach the SGT for the most diluted culture (Hazan et al. 2012). Xia et al. (2020) applied the SGT analysis for four common pathogenic aquaculture bacteria, e.g., Aeromonas hydrophila, Edwardsiella tarda, Vibrio alginolyticus, and Vibrio harveyi, and their highest SGT values were from 4 to 10 hr. Although using the SGT method to quantify the viable bacterial cell count has the advantages of high throughput and short test duration, its applicability and stability to gram-negative and gram-positive bacteria are unclear. The target bacteria in the previous studies, which have applied the SGT method, were almost gram-negative bacteria, such as the genera of Pseudomonas, Aeromonas, Edwardsiella, Vibrio, Burkholderia, and Coxiella (Ahn et al. 2017, Khan et al. 2019, Maura et al. 2016, Xia et al. 2020). There were only two studies applying the SGT method to investigate the gram-positive bacteria, Bacillus megaterium and Enterococcus faecalis, but no detailed SGT correlation was established in their study (Li et al. 2018, Oyama et al. 2017). Besides, the SGT method has only been applied in rare studies for investigating the ecotoxic effects on bacteria, e.g. chlorhexidine gluconate and benzalkonium chloride toward Burkholderia cenocepacia (Ahn et al. 2017) and ruminal and antimicrobial peptide toward E. faecalis (Oyama et al. 2017). Its applicability for ecotoxicity assessment and the effect of existence of dead/injured bacterial cells is still unclear.
Therefore, this study aims to evaluate the applicability and stability of the SGT method when it is applying to gram-positive and gram-negative bacteria. Besides, the applicability of the SGT method as an alternative ecotoxicity assessment method was also detailed verified in this study. This study established the SGT correlations of three gram-positive and three gram-negative bacteria with their cells collected from the middle exponential and the early stationary phases. Besides, various live and dead cell mixture ratios were prepared and verified by the flow cytometry measurement. The effects of the existence of dead bacterial cells on the SGT correlation establishment were examined. Finally, the ecotoxicity and inhibitory impact of copper ions on Escherichia coli cells was evaluated using its SGT correlation established with the different live-to-dead cell ratios.