Effect of fluazifop-P-butyl herbicide on growth, pigment, and ultrastructure of Auxenochlorella pyrenoidosa and Raphidocelis subcapitata

Abstract

The terrible intensification in global food demand predictably results in an increase in agricultural practices, with a focus on the usage of herbicides. Hence, a deeper understanding of the impact of this herbicide on aquatic ecosystems is required. The characterization of nano-emulsion of fluazifop-P-butyl (FL) and the toxic effects in both traditional and nanoforms on the growth, pigmentation and ultrastructure of the freshwater microalgae: Auxenochlorella pyrenoidosa and Raphidocelis subcapitata were studied. The prepared nano-emulsion has a particle size of 52-94 nm by transmission electron microscopy (TEM). Zeta potential recorded (38.5 mV) and polydispersity index recorded (0.394). Traditional fluazifop-P-butyl (TFL) in the case of A. pyrenoidosa exhibited median effective concentration (EC₅₀) (10.909 ± 3.635 mg/L) (P= 0.235), while nano fluazifop-P-butyl (NFL) recorded EC₅₀ (14.281 ± 6.251 mg/L) (P= 0.205). In the case of R. subcapitata, TFL recorded EC₅₀ (1.174 ± 0.501 mg/L) (P= 0.105), while NFL exhibited EC₅₀ (4.757 ± 1.755 mg/L) (P= 0.150). Exposure of R. subcapitata cells to 0.117 mg/L of TFL significantly decreased chlorophyll a level by (-)15.51% (P= 0.002) relative to control. Also, chlorophyll b content in A. pyrenoidosa cells significantly decreased upon treatment with 1.43 mg/L of NFL by (-)67.16% (P= 0.001). Carotenoid content in A. pyrenoidosa cells significantly increased after exposure to 1.09 mg/L of TFL by 108.88% (P= 0.001) compared to the control. Likewise, exposure of R. subcapitata cells to 0.117 mg/L of TFL elevated carotenoid levels by 200.57% (P < 0.001). Destroyed chloroplasts and compacted cell walls were observed through TEM when A. pyrenoidosa was exposed to 1.09 mg/L of TFL and 1.43 mg/L of NFL. Likewise, R. subcapitata cells were exposed to 0.117 mg/L of TFL and 0.476 mg/L of NFL. This study demonstrates that both conventional and nano-forms of FL endanger the integrity of the ecosystem and have adverse impacts on non-target organisms. Thus, it's crucial to follow biosafety protocols concerning non-target species before deciding to use this herbicide.

Introduction

In recent decades, pesticides have played a significant role in boosting agricultural productivity by protecting seeds and crops from a wide variety of pests and diseases [1]. But overuse of them leads to contamination of water, soil, and air and threatens sustainability and biodiversity. Additionally, it has a negative effect on both the environment and human health [2, 3]. Zhao et al. [4] stated that less than 10% of traditional pesticides influence the plant. Only 0.1% of traditional pesticide is long enough to produce a harmful effect on the intended pest, while the mainstream dissolves into the environment [5, 6].

Herbicides' possible impacts on aquatic primary producers must be assessed via ecotoxicological testing. These chemicals have the potential to disrupt the composition and functioning of aquatic ecosystems by amending the species structure of populations, including algae. Generally, pesticides can either accumulate in organisms or be degraded by microorganisms [7]. The majority of the pesticides under examination are herbicides, which are poisonous, because they prevent photosynthesis and seriously damage phototrophic microbes. For algal toxicity, growth rate is used to determine the median effective concentration (EC₅₀). This growth rate can be determined either by direct cell count or optical density [7, 8].

Fluazifop-P-butyl (FL) is a post-emergence phenoxy herbicide [9]. According to Horbowicz et al. [10], it inhibits acetyl-CoA carboxylase, an enzyme involved in producing malonyl-CoA during lipid metabolism. After being rapidly absorbed through leaf surfaces, FL is quickly hydrolyzed into fluazifop acid. In susceptible species, this acid is mainly transported via the phloem and gathers in the meristems, where it hinders lipid synthesis [11]. Horbowicz et al. [10] investigated the effects of FL on maize plants and found that chlorophyll a and chlorophyll b decreased at the highest concentrations, while both pigments increased at lower concentrations of this herbicide.

The use of nanotechnology in pesticides, such as nano-gels, nano-emulsions, nano-encapsulates and electrospun nanofibers, was recently reviewed by Abdollahdokht et al. [12]. For example, nano-emulsions which are biphasic dispersion systems, are created by using surfactants. The kinetic stability and capacity of these nano-emulsions to break down weakly water-soluble pesticides into tiny oil droplets, increasing their bioavailability and efficacy, make them very valuable [13].

Algae are crucial in the primary production of aquatic ecosystems. Their sensitivity to various toxins differs, making them valuable bio-indicators for assessing the impact of industrial waste and other pollutants [14]. Certain chlorophytes have become model organisms in ecological effect assessments, helping to shed light on the ecological consequences of herbicide exposure [15]. For example, Auxenochlorella pyrenoidosa, is one of the most prevalent single-celled microalgae, and it is well-known for its remarkable photosynthetic efficiency. It has been utilized as a biological model to evaluate environmental contaminants due to its sensitivity to toxins and rapid reproduction [16, 17] . Additionally, Raphidocelis subcapitata is highly regarded for its extreme sensitivity to a range of chemicals, leading to its endorsement by various international organizations for ecotoxicity evaluations [18].

The purpose of this study was to: (1) compare the toxicity of FL's nano-emulsion to its traditional formulation by using the freshwater algae Auxenochlorella pyrenoidosa and R. subcapitata and (2) assess the pigment content, the ultra-structure, and cytotoxic effects of both microalgae at sub-lethal concentrations. Notably, this study is the first to investigate the cytotoxicity of the traditional and nano-form of FL on A. pyrenoidosa and R. subcapitata.

Materials and Methods

Microalgae

The unicellular freshwater algae A. pyrenoidosa (H. Chick) Molinari & Calvo-Pérez and R. subcapitata (Korshikov) Nygaard, Komárek, J. Kristiansen & O. M. Skulberg were used in this study. The Institute of Oceanography and Fisheries in Alexandria, Egypt is the source of A. pyrenoidosa, whereas the Department of Botany, Faculty of Science, Mansoura University, Mansoura, Egypt is the source of R. subcapitata. Both microalgae were cultivated in Allen and Arnon's medium (AAM) [19], at 25 ± 3 °C with continuous "Cool-White" fluorescent lighting (195.08 μmol.m^-2. s^-1) and pH (7.5).

Herbicide and synthesis of nano-emulsions

Fluazifop-P-butyl (FL) (High class penta^® 15% EC); IUPAC Name: Butyl (R)-2-[4-(5-trifluoromethyl-2-pyridyloxy) phenoxy] propionate was supplied by Shoura for Chemicals Co., Egypt. Conversely, the FL technical (purity 90%) active ingredient (a. i) which used in the process of creating nano-emulsions was supplied by the Central Agricultural Pesticides Laboratory (CAPL), Agricultural Research Center (ARC), Cairo, Egypt. Nano-emulsion of FL (5%) was prepared by underneath high energy mode using sonication technique at 60 HZ for 10 min and rest time 30 sec for each cycle underneath cooling. The a. i of FL was dissolved in vegetable oil and applied to disperse into the liquid phase (water) at a ratio of (1:20 v/v). The main surfactant (Tween 80) was dropped into the mixture at percent 5% of the total volume. So, the addition of polymer (polyvinyl pyriddinol, PVP) was used at 0.05% to enhance the hydrophobic/lipophilic balance (HLB). Such method was described previously by Abdel-Halim et al. [20].

Characterization of nano-emulsion of Fluazifop-P-butyl

The nano-characters of nano-formulated FL were assayed using thermo-dynamic stability following ICH guidelines Q1A (ICH) [21] methodology, transmission electron microscope (TEM) (JOEL 1400 Plus, Japan), Fourier transform infrared (FTIR) using TENSOR 27 Buker, Germany-FTIR L203/12887 [22], and Zeta sizer (Malvern, ZS Nano, UK) as the values were estimated by dynamic light scattering (DLS) technique [23].

Acute toxicity test

Microalgal cells of A. pyrenoidosa and R. subcapitata were exposed to a serious of concentrations (0.005, 0.01, 0.1, 1, 5, 10 and 20 mg/L) of traditional FL and its nano-form examined for 96 h in a static system according to USEPA protocol [24]. The growth of the microalgae studied was determined in terms of changes in cell density (cell counts/ml using hemocytometer slide under light microscope). The average growth rate and percent inhibition were determined by the following two equations 1 and 2, respectively [25].

$${\mu }_{i-j}=\frac{\ln {\text{B}}_j-\ln {\text{B}}_i}{{\text{t}}_j-{\text{t}}_i}\cdot {\text{d}}^{-1}$$ (1)

$$\text{I\%}=\frac{{\mu }_c-{\mu }_T}{{\mu }_c}\times 100$$ (2)

For equation 1, μ_i-j represents the growth rate from the initial time of the experiment i to the end time j, Bj is the cell number/ml after 96 h, B_i is the initial cell number/ml, t_i is the zero time, t_j is the end time of the test (96 h), I% stands for percent inhibition, μ_c denotes mean growth rate in the control, and μ_T represents growth rate in the treatment in equation 2.

Sub-lethal toxicity

Two levels: 1/10 and 1/40 of calculated value of EC₅₀ for traditional and nano-form FL. A. pyrenoidosa was treated with 0.237 and 1.09 mg/L from traditional-FL (TFL) and 0.357 and 1.43 mg/l from nano-FL (NFL). Also, R. subcapitata was treated with 0.029 and 0.117 mg/L from TFL and 0.119 and 0.476 mg/L from NFL. The harvesting process for both microalgal cells was performed after 96 h.

Algal pigments

The content of carotenoids, chlorophyll a, and chlorophyll b in A. pyrenoidosa and R. subcapitata cells were determined using a spectrophotometer (Spectronic 21D, Milton Roy, USA) at 666, 653, and 470 nm in accordance with the method of Lichtenthaler and Wellburn [26] by using 96% methanol. The following formulas were used to estimate the pigment amounts:

C_a =15.65 A₆₆₆-7.340A₆₅₃, C_b=27.05 A₆₅₃-11.21 A₆₆₆ and C_c+x=1000A₄₇₀-2.860 C_a-129.2 C_b/245

Where: C_a, C_b and C_c+x are Chlorophyll a, Chlorophyll b and Total carotenoids, respectively.

Ultrastructural investigation

After 96 h exposure of A. pyrenoidosa to 1.09 mg/L of TFL and 1.43 mg/L of NFL and exposure of R. subcapitata to 0.117 mg/L of TFL and 0.437 mg/L of NFL herbicide, 5 ml from each microalgal culture were centrifuged (500 xg, for 5 min.). After discarding the supernatant, 10 ml of deionized water were used to wash the pellets. Two ml of 2.5% glutaraldehyde (0.1 M phosphate buffer, pH 7.2) were used to fix the quickly cleaned pellets, which were then kept at 4 °C until they were needed. A pH 7.2, 0.1 M phosphate buffer was used to wash these fixative samples. After 1-2 h at 4 °C in 1% osmium tetra oxide (OSO₄), the pellets were washed for 2 min in buffer. The samples were dehydrated using acetone at progressively higher concentrations (25, 50, 75, and 100%) for 5-min. After the dehydration process, the tissues were infiltrated using propylene oxide. Using an Ultratome machine, capsuled samples were sectioned at a thickness of 20 to 30 nm. To help with orientation, the sections were gathered on metal mesh "grids" and stained with toluidine blue. After 5 min of uranyl acetate (4%) staining, the grids were repeatedly rinsed in four beakers of pure water. Following a 5-min staining process with 1% lead acetate, the grids were rinsed with water once more and kept in a grid box until they were examined. TEM (JOEL 1400 Plus, Japan) was applied to monitor the cells as reported by Reynolds [27].

Statistical analysis

Results were given as the mean of three replicates in each treatment. Using Origin Pro^® 2024b software, EC₅₀ values were estimated. For these analyses, all the test series of FL on both the studied microalgae were combined to obtain sigmoidal fit of the concentration-response curve. The upper and lower parameters were fixed at 0 and 100 to represent the percentage range for inhibition, and the center of the distribution is the estimated EC₅₀ value. Comparison of treatment groups was performed using t-test from the statistical package (Minitab-17). According to the t-test, the p-value was deemed significant at the P ≤ 0.05 probability level.

Results

Characterization of nano-emulsion of fluazifop-P-butyl

During the storage period of the freeze-thaw cycle, the nano-form investigated remained stable. There were no floating or creaming stages formed. Additionally, following the centrifugation and shaking procedures, no separation phase developed. Images of TEM showed accurate data about morphology (Figure 1a & b). The prepared nano-emulsion of FL appeared as spherical shape with size mainly in the range of 52-94 nm. Droplet size and particle dispersion index (PDI) are illustrated in Figure 1c. The droplet size distribution curve of the nano-form by dynamic light scattering (DLS) had a droplet size average of 370.4 nm and PDI value, 0.394. Zeta potential recorded 38.5 mV (Figure 1d).

Figure 1.

Transmission Electron Microscope (TEM) photographs (a&b), (a) at 40000X and (b) at 60000X, (c) Droplet size distribution measurement and (d) Zeta potential profile of the nano-emulsion of nano-form of fluazifop-P-butyl.

The output diagram for Fourier Transform Infrared (FTIR) profiles of TFL and NFL is illustrated in Figure 2. In FTIR pattern of TFL (spectrum 1, blue color) indicates O-H stretching and H-bonded in phenyl rings at 3424.88 cm^-1 with broad peak. Medium peaks are indicated at 2863.0-2875.0 cm^-1 for H-C=O stretching in aldehydes. Stretching C-C in aromatic rings is indicated in the range 1752-1487 cm^-1 with sharp peaks. Stretching of C-N is indicated at 1390 cm^-1 for pyridine ring. Small peaks are noted in range 1320-1079 cm^-1 for C-O in esters. Strong peak is noted at 433 cm^-1 for C-F group as alkyl halide. In the case of NFL (spectrum 2, red color) similar profile of the traditional one is indicated, especially for C-C stretching in aromatic rings at 1642.26 cm^-1 as sharp peak. Also, C-O and C-N stretching are indicated in the same wavelengths. Moreover, shifting of C-F group is noted at 457.92-429.07 cm^-1. However, O-H stretching and H-bonded is significantly absorbed at 3448.54 cm^-1 with shifting pattern in association with the used polymer in nano-preparation.

Figure 2.

Fourier Transform Infrared (FTIR) pattern (1, Blue) traditional form of fluazifop-P-butyl and (2, Red) its nano-form accomplished at absorption range 400-4000 cm^-1.

Effect of the traditional and nanoform of fluazifop-P-butyl on the growth of Auxenochlorella pyrenoidosa and Raphidocelis subcapitata

Results obtained for cell number, growth rate and inhibition ratio of A. pyrenoidosa and R. subcapitata cultured for 96 h under the effect of different concentrations of TFL and NFL are shown in Figure 3 (a, b, c & d). These results showed that cell number and growth rate gradually decreased with increasing the concentration of both TFL and NFL from 0.005 to 20 mg/L in both the microalgae studied. It is also clear that, the cell number and growth rate recorded for all the concentrations were less than the control even at the lower concentration of 0.005 mg/L. Meanwhile, the order of maximum values in A. pyrenoidosa is as follows: control > (0.005 mg/L) TEL > (0.005 mg/L) NFL. These highest values of cell number and growth rate were (2.80 × 10⁶/ml & 1.853), (2.35 × 10⁶/ml & 1.794) and (1.78 × 10⁶/ml & 1.700), respectively. Regarding R. subcapitata, the following order of the maximum values of cell number and growth rate was control> (0.005 mg/L) TFL = (0.005 mg/L) NFL. These greatest values of cell number and growth rate were (2.25 × 10⁶/ml & 1.789), (1.79 × 10⁶/ml & 1.713) and (1.79 × 10⁶/ml & 1.713), respectively. On the other hand, the inhibition ratio increased by increasing the concentration of both TFL and NFL in both microalgae. The highest inhibition rate (100%) was recorded by using 20 mg/l of TFL on R. subcapitata.

Figure 3.

Effect of different concentrations of traditional form of fluazifop-P-butyl and its nano-form on cell number, growth rate and inhibition ratio of Auxenochlorella pyrenoidosa (a & b) and Raphidocelis subcapitata (c & d), respectively after exposure time (96 h). Values are mean of three replicates.

Acute cytotoxicity

Herbicide, TFL and NFL were evaluated on microalgae A. pyrenoidosa and R. subcapitata to obtain 96 h toxicities as shown in Figure 4. Traditional form (TFL) exhibited EC₅₀ (10.909 ± 3.635 mg/L) (P= 0.235) on A. pyrenoidosa while, NFL recorded the value 14.281 ± 6.251 mg/L (P= 0.205). In the case of R. subcapitata, TFL recorded EC₅₀ value (1.174 ± 0.501 mg/L) (P= 0.105), while NFL exhibited EC₅₀ value (4.757 ± 1.755 mg/L) (P= 0.150).

Figure 4.

The median effective concentration (EC₅₀) of traditional and nano-form of fluazifop-P-butyl on Auxenochlorella pyrenoidosa and Raphidocelis subcapitata after exposure time (96 h). NFL: Nano fluazifop-P- butyl, TFL: Traditional fluazifop-P- butyl.

Chlorophyll a (Chl. a) content in A. pyrenoidosa cells significantly decreased upon treatment with TFL and NFL. Exposure to 0.273 and 1.09 mg/l of TFL decreased chlorophyll a level by (-)1.08% (P= 0.114) and (-)5.80% (P= 0.009), respectively, with respect to control. Likewise, treatment with 0.357 and 1.43 mg/l of NFL induced significant decreases of (-)3.51% (P= 0.023) and (-)3.49% (P= 0.020), respectively. The lowest chlorophyll a content (544.42 μg/g fresh weight) was observed in cells treated with 1.09 mg/L of TFL (Figure 5a).

Figure 5.

Effects of different concentrations of fluazifop-P-butyl on pigments of Auxenochlorella pyrenoidosa and Raphidocelis subcapitata. (a) traditional fluazifop-P-butyl on pigments of A. pyrenoidosa, (b) nano fluazifop-P-butyl on pigments of A. pyrenoidosa, (c) traditional fluazifop-P-butyl on pigments of R. subcapitata, (d) nano fluazifop-P-butyl on pigments of R. subcapitata. Data are mean of three replicates. Error bars refer to standard deviation.

Chlorophyll b (Chl. b) content in A. pyrenoidosa cells significantly decreased upon treatment with TFL and NFL. Exposure to 0.273 and 1.09 mg/L of TFL decreased Chl. b levels by (-)11.55% (P= 0.030) and (-)28.12% (P= 0.006), respectively, compared with control. Likewise, treatment with 0.357 and 1.43 mg/L of NFL induced significant decreases of (-)10.56% (P= 0.041) and (-)67.16% (P= 0.001), respectively. The lowest Chl. b content (113.25 μg/g fresh weight) was observed in cells treated with 1.43 mg/L NFL (Figure 5b).

Carotenoids content in A. pyrenoidosa cells significantly increased upon treatment with TFL and NFL. Exposure to 0.273 and 1.09 mg/L of TFL elevated carotenoids levels by 62.47% (P= 0.003) and 108.88% (P= 0.001), respectively, in comparison with the control. Likewise, treatment with 0.357 and 1.43 mg/L of NFL induced significant increases of 13.49% (P= 0.056) and 124.89% (P= 0.001), respectively. The highest carotenoids content (298.97 μg/g fresh weight) was observed in cells treated with 1.43 mg/L of NFL (Figure 5b).

In case of R. subcapitata, Chl. a content in the cells significantly decreased upon treatment with TFL and NFL. Exposure to 0.029 and 0.117 mg/L of TFL decreased Chl. a level by (-)3.15% (P= 0.102) and (-)15.51% (P= 0.002), respectively, relative to control. Likewise, treatment with 0.119 and 0.476 mg/l of NFL induced significant decreases of (-)2.76% (P= 0.141) and (-)11.63% (P= 0.010), respectively. The lowest Chl. a content (633.86 μg/g fresh weight) was observed in cells treated with 0.117 mg/L of TFL (Figure 5c). Regarding Chl. b content, significant decreases upon treatment with TFL and NFL were noted. Exposure to 0.029 and 0.117 mg/L of TFL decreased Chl. b levels by (-)10.51% (P= 0.011) and (-)13.75% (P= 0.005), respectively, with respect to control. Likewise, treatment with 0.119 and 0.476 mg/L of NFL induced significant decreases of (-)19.08% (P= 0.005) and (-)31.93% (P< 0.001), respectively. The lowest Chl. b content (241.45 μg/g fresh weight) was observed in cells treated with 0.476 mg/L of NFL (Figure 5d). On the other hand, carotenoids content in R. subcapitata cells significantly increased upon treatment with TFL and NFL. Exposure to 0.029 and 0.117 mg/L of TFL elevated carotenoids levels by 190.90% (P< 0.001) and 200.57% (P< 0.001), respectively, relative to the control. Likewise, treatment with 0.119 and 0.476 mg/L of NFL induced significant increases of 8.41% (P= 0.029) and 24.39% (P= 0.006), respectively. The highest carotenoids content (468.90 μg/g fresh weight) was observed in cells treated with 0.117 mg/L of TFL (Figure 5c).

Effect of fluazifop-P-butyl on the ultrastructure of Auxenochlorella pyrenoidosa and Raphidocelis subcapitata

Alga, A. pyrenoidosa is a spherical, single-cell microalga. Ultrastructure of untreated cells (Fig. 6a) showed marked regular and concentric cell wall system and homologous distribution of cellular organelles. The algal cells treated with 1.09 mg/L of TFL (Figure 6b) had compacted intracellular organelles with different shapes and marked concentric cell wall system, starch-pyrenoid complex, destroyed and disorganized chloroplast. Also, A. pyrenoidosa treated with 1.43 mg/L of NFL (Figure 6c) obtained an obvious concentric cell wall system, compacted intracellular organelles, destroyed starch grains and chloroplast with denser grana, in cup-shaped, surrounding starch-pyrenoid complex. The thylakoids and mitochondria were noted.

Figure 6.

Electron micrograph of Auxenochlorella pyrenoidosa and Raphidocelis subcapitata treated with herbicide fluazifop-P-butyl (FL) for 96 h. (a) untreated cell of A. pyrenoidosa, (b) A. pyrenoidosa treated with 1.09 mg/L of traditional FL (c) A. pyrenoidosa treated with 1.43 mg/L of nano-form FL (d) untreated cell of R. subcapitata, (e) R. subcapitata treated with 0.117 mg/L of traditional FL (f) R. subcapitata treated with 0.476 mg/L nano-form FL., chloroplast (Ch), starch-pyrenoid complex (SPC), nucleus (N), lipid vesicles (L), black-colored particles (BCP), pyrenoid (P) and vacuoles (V) (Notched Right arrow) [F4G1-OsO4 fixed-uranyl acetate lead citrate stained preparation, 6000X].

Alga, R. subcapitata is a half-moon or C-shaped single-cell microalga. The ultrastructure of untreated cells (Figure 6d) indicated marked cellular organelles. While the cells treated with 0.117 mg/l of TFL (Figure 6e) had many starch granules, destroyed and disorganized chloroplast, lack of nucleus, black-colored particles and polyphosphate granules. Also, the cells treated with 0.437 mg/L of NFL had double cell walls and destroyed chloroplast (Figure 6f).

Discussion

One significant class of pesticides, herbicides, can have both direct and indirect effects on the composition and operation of aquatic ecosystems [28]. In the present study, we probe the effect of FL herbicide, and its nano-form on growth, pigment and ultrastructure of microalgae A. pyrenoidosa and R. subcapitata.

The prepared nano-emulsion of FL appeared as spherical shape with size mainly in the range of 52-94 nm. This was in the range of many previous studies as Somala et al. [29] who reported TEM analysis of a Cymbopogon nardus essential oil nano-emulsion with spherical droplets of 50-120 nm and the spherical droplets of thymol nano-emulsion with size range of 80 –150 nm [22]. Transmission electron microscope (TEM) and the DLS analysis are complementary methods to determine the shape and size of particle size within a system [30]. In our investigation the range of particle sizes estimated by TEM (52-94 nm) is less than DLS value (370.4 nm). This decrease could be explained as a liquid droplet in TEM shows a certain degree of shrinkage in dried form and tends to deteriorate under vacuum during the analysis of samples. On the other hand, the sample for DLS is examined in its hydrated forms in solvent and water [31].

Polydispersity Index (PDI) is a key parameter when evaluating particle dispersion, homogeneity and stability of nano-emulsion. An optimum PDI value for creating nano-emulsions is close to zero, which denotes high stability and uniformity [32]. A slightly higher polydispersity was indicated by the study's PDI value of 0.394, which is still within allowable bounds for nano-emulsion stability. A zeta potential of > +30 or < −30 mV confirms that the nano-emulsion is stable [29]. According to our investigation, zeta potential recorded 38.5 mV, which confirmed the stability of the nano-emulsion formed. The output diagram for FTIR profiles of TFL and NFL results are agrees with Coates [33].

One of the most sensitive parameters for assessing the toxicity of chemical compounds is the microalgal population's growth rate [34]. In this study the inhibition ratio increased by increasing the concentration of both FL and its nano-form in both microalgae. The highest inhibition rate (100%) was recorded by using 20 mg/L of FL on R. subcapitata. The exposure duration (96 h) for both micro-algae was selected as recommended by EPA authority to be applied in the OECD protocol, which is specific for acute toxicity of substances on freshwater algae. In contrast, acute toxicity and sub-lethal exposure experiments were evaluated independently using a standard protocol cited by EPA. Similar findings were illustrated by Lu et al. [35] who recounted that pyraclostrobin (a fungicide) caused growth inhibition of both C. vulgaris and Microcystis aeruginosa. The same fungicide showed growth inhibition of R. subcapitata [34].

Matters were divided into three categories according to their EC₅₀ values by the EU-Directive 93/67/EEC (Commission of the European Communities, 1996). These categories of EC₅₀ values: less than 1.0 mg/L (very toxic to aquatic organisms); from 1.0 to 10.0 mg/L (toxic to aquatic organisms); from 10.0 to 100.0 mg/L (harmful to aquatic organism) [36]. Accordingly, the TFL and NFL could be considered harmful to A. pyrenoidosa and toxic to R. subcapitata after 96 h exposure.

Planktonic algae contain a large amount of chlorophyll pigment [37]. The concentrations of this pigment can objectively reflect the growth of plant and photosynthesis levels. In addition, this pigment is used as an important indicator of algal response to pollutants [38]. In our investigation Chl. a and Chl. b significantly decreased in both microalgae within all treatments of FL when compared to the control. Parallel results are noted by Noaman et al. [7] who found that both Chl. a and b were decreased when the cells of C. vulgaris were treated with 0.025EC₅₀ and 0.1EC₅₀ of herbicide pendimethalin and its nano-form. Also, Zheng et al. [39] reported that Chl. a and b declined in Navicula species after exposure to different concentrations of acetochlor, atrazine and glyphosate for 96 h. During photosynthesis, carotenoids provide photoprotection, and the production of these compounds directly reflects photosynthetic capacity [40]. According to our results carotenoid content significantly increased in both microalgae studied that were treated with herbicide compared with the control. This increase could be interpreted as a defense mechanism, because carotenoids have antioxidant properties that prevent excessive intracellular accumulation of reactive oxygen species (ROS). Also, they defend against photo-damage by quenching singlet oxygen and trilinear chlorophyll and disperse excess absorbed energy. This dispersion is done by reacting with excited chlorophyll molecules to decrease the degree of cellular lipid peroxidation (LPO) [41,42].

This study showed that exposure of A. pyrenoidosa to 1.09 mg/l of TFL and 1.43 mg/L of NFL and exposure of R. subcapitata to 0.117 mg/L of TFL and 0.476 mg/L of NFL led to the destruction of the chloroplast and the cell walls. These findings were in harmony with that obtained by Noaman et al. [7] who found that C. vulgaris had been shown a destroyed chloroplast and marked fatty bodies when treated with herbicide pendimethalin and its nano-form. Xu et al. [43] showed a significant change in cellular structures of Scenedesmus obliquus after exposure to sulfamethoxazole. Such as a destroyed grana lamellar in the chloroplast, partially disintegration of the nucleus, and a rise in the number of mitochondria in the cells. Hernández-García and Martínez-Jerónimo [44] found that as the concentration of glyphosate increased, the chloroplasts of P. subcapitata, C. vulgaris, Ankistrodesmus falcatus and S. incrassatulus decreased, became disorganized and gradually degraded.

Our study highlights that risk assessment based on comparative toxicity between traditional-formulation of the herbicide and its nano-derived form and sub-lethal concentrations exposure is inadequate issue for framework of decision-making, specifically for nanopesticides [45,46,47].

Conclusions

This study demonstrates that conventional agricultural herbicides may have a real influence on microalgae. Fluazifop-P-butyl (FL), both in its traditional and nano-form, influenced the growth, the pigmentation, and the ultrastructure of the microalgae: A. pyrenoidosa and R. subcapitata. Furthermore, R. subcapitata was more sensitive to herbicide than A. pyrenoidosa and the potency of traditional form was greater than the nano one. With a focus on NFL, this study provides valuable insights on the ecological effects of FL formulation overall. This finding is significant since it contributes to the rising abundance of data required to assess the ecological safety of nanopesticides, which are increasingly being investigated as substitutes to their traditional formulations due to their potential for lower dosages and targeted delivery. The outcomes reported here are based on laboratory-scale exposure scenario, which may differ under changing settings. Additionally, the assessments of TFL and NFL at concentrations approaching that can be found in the environment. The outcomes reported here are based on laboratory-scale exposure scenario, which may differ under changing settings. Additionally, the assessments of TFL and NFL at concentrations approaching that can be found in the environment.