## 3 Results and Discussion
### Chlorophyll-A Estimation  
_A. platensis_ has only chlorophyll type \"a\" content [56; 57]. It is a qualitative factor of _A. platensis_ cultures as it depends on cultivation characteristics such as light, the medium of cultivation, the age of the cells, and the nitrogen concentration [58]. Chlorophyll-a in _A. platensis_ is located within the thylakoid lamellae, which are membrane-bound structures in the cytoplasm. The thylakoid membranes are very important for the photosynthetic process of the organism since light energy is absorbed by chlorophyll and converted into chemical energy during photosynthesis. In these lamellae, chlorophyll is packed in such a way as to maximize light capture and enhance the efficiency of the photosynthetic process for _A. platensis_ to flourish in different aquatic conditions and purify water [59]. The chlorophyll-a results (Figure 5) seem to support the quantitative and qualitative characteristics of the cultures, as presented from the previous measurements. Figure 5: Chlorophyll-a content on Days 0 and 7 of cultivation of all cultures (Control and MT). Each value is average of two measurements, with uncertainties less than 5% over each one. * Control culture: The chlorophyll-a increase relates to the biomass increase through cultivation. * Cu and Ni mono-MT cultures: Low concentrations of 1 ppm seem to elevate the pigment's content, while higher concentrations exhibit toxic effects, drastically reducing it. This is in agreement with [PERSON] et al. [5], although we found similar toxicity of Ni in comparison to Cu. The toxicity of Cu to _A. platensis_ relates to its role in increasing reactive oxygen groups. This affects lipids, proteins, and DNA, while it also substitutes Mg from chlorophyll. The authors [5] also showed that up to Day 7 of cultivation, low Ni concentrations, up to 1 ppm, act positively to the cultivations, surpassing the Control sample, while higher concentrations of Ni act negatively. * Zn and Pb mono-MT cultures: Again, the low Zn concentration, that of 1 ppm, elevates the chlorophyll-a content. [PERSON] et al. [5] showed that up to Day 7 of cultivation, 1 ppm of Zn had similar results with their Control sample, but higher concentrations had a negative effect. * Cd mono-MT cultures: All metal concentrations seem to elevate the chlorophyll-a content of the microalga, in comparison to the Control culture. This suggests an adaptive response and may be connected to the induction of stress-related metabolic pathways that affect growth. This hormetic response to low concentrations of Cd was observed with two other microalgae strains, _Spirulina indica_[60] and _Chromochloris zofingiensis_[61], and in peppermint plants [62]. * Multi-MT cultures: Their chlorophyll-a content reduces when metal concentration increases. While the 5 and 10 ppm cultures present similar characteristics with the mono-MT cultures, the Mix 1 ppm presents the lowest chlorophyll-a content, in comparison to all the mono-MT cultures. It should be noted that the differences that seem to appear between Figures 3 and 5 are due to the applied methods. NIR spectroscopy directly estimates _A. platensis_ biomass/overall growth. In contrast, chlorophyll-a content serves primarily as an indicator of cell health and photosynthetic activity. Under heavy metal stress, chlorophyll is a cell health indicator, and shows the damage of photosynthetic systems, before the detection of significant reductions in biomass. This is consistent with the microscopy observations (Figures 1 and 2), which show visible morphological changes and cellular stress despite a relatively stable biomass in some conditions. The enhancement and the reduction in chlorophyll-a content of _A. platensis_ upon heavy metal exposure can be attributed to several factors. The oxidative stress due to metals--such as Cu and Ni--results in the generation of reactive oxygen species (ROS), which are capable of cellular damage including chlorophyll and thylakoid membranes, thus resulting in the reduction in pigment content [5].