The use of herbicides for weed control is expanding every year in agricultural production areas. However, their application may lead to a series of adverse consequences, such as their persistence in the soil, which can affect the subsequent crops in the rotation, or the possible presence of residual herbicides in the fruits, leaves, and roots of crops. Furthermore, cultivation fields are often located in close proximity to watercourses, floodplains, river deltas, or marshy areas, thereby heightening the potential for herbicide usage to adversely affect natural water bodies, thus increasing the likelihood of a detrimental environmental impact [[1], [2], [3]].

This impact is particularly evident for aquatic plants and microalgae, leading to a decline in primary producers and an alteration of the entire ecosystem [4,5]. Atrazine is particularly harmful and is frequently used for canola, corn and sugarcane crops [6]. This herbicide is moderately soluble in water and weakly absorbed by soils, which reduces its persistence in the land but increases the ability to reach groundwater [7].

In March 2004, the European Union banned the use of Atrazine due to the high toxicity to aquatic biota [8]. However, it is still found in European groundwater and it remains widely used in the United States and in developing countries, where its use is increasing, primarily due to weed resistance to other herbicides such as glyphosate [9].

Atrazine is an endocrine disruptor, and it has been shown to cause demasculinization and feminization of male gonads [[10], [11], [12]]. Although its half-life in soils is five days, it can persist for extended periods in water, ranging from three days to eight months [[13], [14], [15]]. According to the Environmental Protection Agency, the maximum contaminant level for Atrazine in drinking water is 3 μg/L [16,17]. Nevertheless, concentrations well above this limit have been reported in natural bodies of water. In the United States, several watersheds have displayed peak maximum concentrations exceeding 100 ppb. Moreover, at Little Pigeon Creek in Indiana, a maximum concentration of 237.5 ppb was detected in May 2005 [18]. In South Africa, a concentration as high as 298 ppb has been found in surface water [19]. In Argentina, a typical agricultural country, a maximum value of 86 μg. L−1 was detected in the surface water of Entre Ríos Province [1,20] and values around 43 μg.L−1 were found in groundwater in Brazil [21]. Atrazine has even been found in rainwater, with a maximum value of 26.9 μg. L−1 in Argentina [20] and maximum concentrations of 40 μg. L−1 in USA [22].

Atrazine belongs to the group of photosynthesis-inhibiting herbicides and works by blocking the electron transport chain between PSII and PSI, which prevents the formation of NADPH and ATP. Both NADPH and ATP are necessary for the fixation of CO2 and other biochemical processes in plants and algae [23] (see Fig. 1).

When algae are exposed to Atrazine or high-intensity radiation, there is an accumulation of excited states of chlorophyll that triggers the formation of the chlorophyll triplet state (3Chl*). This triplet state can lead to the formation of reactive oxygen species (ROS), particularly singlet oxygen [23] which can damage lipids, proteins, and nucleic acids, as well as the oxygen-evolving complex and the electron acceptors QA and/or QB in the photosynthetic chain.

Currently, there is significant interest in analyzing the specific impact of Atrazine on photosynthetic matter in natural aqueous environments, as demonstrated by recently published articles [24]. In the present study, our hypothesis is that the inhibitory effect of Atrazine on the electron transport chain may lead to changes in the photosynthetic parameters and the distribution of absorbed energy within the photosynthetic organism, affecting processes such as photosynthesis, fluorescence, and heat dissipation. To test this hypothesis, we exposed the microalga Parachlorella kessleri to various concentrations of Atrazine for different durations, and conducted comprehensive analyses of chlorophyll fluorescence. The freshwater alga Parachlorella kessleri was selected for this study because it grows rapidly, is easy to cultivate, and adapts well to diverse light and temperature conditions. Additionally, its high biomass production and elevated pigment concentration make it particularly well-suited for highly sensitive chlorophyll fluorescence measurements. The selected microalga shows strong potential as a biosensor for herbicide detection in aquatic environments. However, existing studies have not provided a comprehensive analysis of its fluorescence properties. In this work, we address this gap by performing an in-depth fluorescence assessment using multiple complementary methodologies and by comparing the outcomes to offer a more integrated and complete understanding.

Variable chlorophyll fluorescence (Kautsky kinetics) was recorded over time [25], and the rapid fluorescence induction curve (OJIP transient) was examined in detail [26]. Photosynthetic parameters derived from these fluorescence measurements were evaluated as effective indicators for ecotoxicological assessment. Furthermore, this study investigated energy partitioning to determine how the absorbed light energy is distributed among photosynthesis, regulated heat dissipation (non-photochemical quenching), and physical dissipation, including fluorescence emission and unregulated heat loss.

Furthermore, steady-state fluorescence spectra were obtained for the dark-adapted state (F0). To enhance the analysis, reflectance measurements were included to correct the spectral distribution of fluorescence, accounting for light re-absorption effects (inner filter), which are often overlooked in the literature [27].