Removal of cyantraniliprole from aquatic environments by Chlamydomonas reinhardtii

Alrowais, R., Ibrahim, E. S., El-Hefny, D. E., Helmy, R. M. A., Yousef, R. S., Mottale, R. A., Abdel daiem, M. M., Ounaies, W., Alwushayh, B., and Mahmoud-Aly, M. (2024). “Removal of cyantraniliprole from aquatic environments by Chlamydomonas reinhardtii,” BioResources 19(3), 6653-6669.

Abstract

This paper reports the first study of phyco-remediation of cyantraniliprole, a second-generation diamide insecticide with high toxicity and persistence in aquatic environments, using the green microalga Chlamydomonas reinhardtii. Cultures of C. reinhardtii were treated with four concentrations of cyantraniliprole (0, 25, 50, and 100 ppm). The removal efficiency, antioxidant responses, and biomass composition of the microalga were measured after 1 h and one week of exposures. C. reinhardtii was able to remove cyantraniliprole from the medium by biodegradation, biotransformation, bioaccumulation, and bio-adsorption mechanisms, achieving up to 87.0% removal within 1 h and 84.5% after one week. The microalga also maintained acceptable levels of enzymatic and non-enzymatic antioxidants, indicating its tolerance to cyantraniliprole stress. Moreover, some treated cultures (especially those with 25 and 50 ppm cyantraniliprole) showed enhanced specific growth rate, and biomass productivity compared to control cultures. In addition, those with 50 and 100 ppm cyantraniliprole showed enhanced carbohydrate and lipid concentrations compared to the control cultures. These results suggest that C. reinhardtii is a promising candidate for bioremediation of cyantraniliprole-contaminated water and biofuel production.

Download PDF

Full Article

Removal of Cyantraniliprole from Aquatic Environments by Chlamydomonas reinhardtii

Raid Alrowais,^a,h Eldesoky Sabri Ibrahim,^b Dalia E. El-Hefny,^c Rania M. A. Helmy,^c Rania Saber Yousef,^d Shady Abdel Mottaleb,^eMahmoud M. Abdel daiem,^f,g,* Wassef Ounaies,^a Bandar Alwushayh,^a and Mohamed Mahmoud-Aly ^e

DOI: 10.15376/biores.19.3.6653-6669

Keywords: Phyco-remediation; Cyantraniliprole; Chlamydomonas reinhardtii; Antioxidant responses; Biomass composition

Contact information: a: Department of Civil Engineering, College of Engineering, Jouf University, Sakakah 72388, Saudi Arabia; b: Department of Economic Entomology and Pesticides, Faculty of Agriculture, Cairo University, Giza, 12613, Egypt; c: Pesticides Residues and Environmental Pollution Department, Central Agricultural Pesticide Laboratory, Agricultural Research Center, Giza, 12618, Egypt; d: Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, 12613, Egypt; e: Plant Physiology Division, Department of Agricultural Botany, Faculty of Agriculture, Cairo University, Giza, 12613, Egypt; f: Environmental Engineering Department, Faculty of Engineering, Zagazig University, Zagazig, 44519, Egypt; g: Civil Engineering Department, College of Engineering, Shaqra University, 11911, Al-Duwadmi, Ar-Riyadh, Saudi Arabia; h: Sustainable Development Research and Innovation Center, Deanship of Graduate Studies and Scientific Research, Jouf University, Sakaka, Saudi Arabia;

*Corresponding author: mmabdeldaiem@eng.zu.edu.eg

INTRODUCTION

Cyantraniliprole, a member of the anthranilic diamide category of insecticides, has found widespread use in the agricultural industry for combating various insect pests, including Coleoptera, Lepidoptera, Diptera, Homoptera, and Thysanoptera (Dong et al. 2017; Ananthi et al. 2019; Yan et al. 2023). Its approval in various countries, such as the USA, select EU nations, and China underscores its significance (EFSA 2014; Wang et al. 2018; MDA 2024). However, despite its efficacy, cyantraniliprole poses a significant threat to aquatic ecosystems, particularly invertebrates. Both surface water bodies (such as rivers and lakes) and benthic habitats (inhabited by sediment-dwelling organisms) are at major risk (EFSA 2014; MDA 2014; Wang et al. 2018)

Phyco-remediation is a bioremediation technique that focuses on harnessing algal diversity to address hazardous contaminants such as hydrocarbons, pesticides, radioactive substances, and heavy metals (Alrowais et al. 2023a,b, 2024a,b). Simultaneously, it aims to utilize the treated algal biomass for creating value-added products such as fertilizers and biofuels. Thus, phyco-remediation is considered safe, cost-effective, and environment-friendly technology. Beyond acting as a carbon sink, algae possess a significant surface-to-volume ratio, enabling processes such as bio-adsorption (facilitated by metal-binding groups on cell surfaces), bioaccumulation (storing pollutants inside their cells’ compartments), biotransformation, and biodegradation (facilitated by algal enzymes to transform and degrade pollutants to produce other metabolites) of pollutants (Méndez-Díaz et al. 2012; El-Shatoury et al. 2014; Abdel daiem et al. 2019; Kaloudas et al. 2021; Koul et al. 2022; Morais et al. 2022; Ummalyma and Singh 2022).

Chlamydomonas reinhardtii (hereafter Chlamy) is a model green microalga widely studied in several fields such as physiology, genetics, biochemistry, and environmental science (Sehrawat et al. 2021; Dupuis and Merchant 2023). While Chlamy is not traditionally associated with direct insecticide remediation, it possesses intriguing properties that could contribute to environmental detoxification. Notably, Chlamy demonstrates a remarkable ability to remediate water contaminated with phenols and pesticides (Nazos et al. 2017; Wan et al. 2020; Sehrawat et al. 2021).

Cyantraniliprole has been shown to increase the activities of several oxidative stress-related enzymes in Procambarus clarkii, as evidenced by the upregulation of related genes (Liang et al. 2020). Among these enzymes, catalase (CAT) and superoxide dismutase (SOD) play crucial roles in detoxification and antioxidant defense. They help metabolize toxic substances and protect living cells from oxidative damage in zebrafish, making them valuable markers of antioxidant response (Paravani et al. 2019).

In this study, the authors explore the potential of Chlamy for safeguarding aquatic environments from leaching toxic insecticides, specifically cyantraniliprole. This research is the first reported instance of Chlamy’s involvement in cyantraniliprole removal. Additionally, various remediation strategies were discussed, including the roles of enzymatic and non-enzymatic antioxidants. Through bridging the gap between insecticide science and algal physiology, this study opens new avenues for sustainable environmental protection.

EXPERIMENTAL

Materials and Methods

Chemical and biological materials

The source of cyantraniliprole used in this study was Benevia^®(10.26% cyantraniliprole) purchased from FMC India Pvt. Ltd (India). The green microalga used in this study was Chlamydomonas reinhardtii CC1690 (Chlamy) purchased from the Chlamydomonas resource center (CRC, University of Minnesota, Minneapolis, MN, USA).

Experimental setup and growth conditions

Seed culture was grown mixotrophically in tris-acetate-phosphate medium (TAP) prepared based on a standard recipe (Gorman and Levine 1965) for 4 days and then inoculated into fresh media after harvesting and washing twice with TAP as described in (Mahmoud-Aly et al. 2018).

Two separate experiments were applied. First, a preliminary drop-test experiment on TAP-agar plates containing (0, 25, 50, and 100 ppm cyantraniliprole) was executed. Nine separate 5 µL drops (OD₇₅₀ = 0.39) of Chlamy inoculum were cultured in each plate, then the plates were incubated under low light intensity 32 ± 2 µmol photons m^-2 s^-1 and 16:8 h light: dark cycles at 20 ± 1 °C for 96 h.

Second, the main experiment, Fig. 1a, involved growing Chlamy in 1-L bioreactors containing (0, 25, 50, and 100 ppm cyantraniliprole; green reactors) and cyantraniliprole in TAP medium without Chlamy (25, 50, and 100 ppm; white reactors). All experiments were executed in three separate biological replicates following completely randomized design (CRD).

The initial cell density after inoculation was nearly 1.23 ± 0.001 × 10⁶ cells/mL and OD₇₅₀ = 0.078 ± 0.002. Under fully aseptic conditions, cultures were mixotrophically grown 500 mL each in 1-L bioreactor units, as shown in Figs. 1a and 1b, under low light intensity 32 ± 2 µmol photons m^-2 s^-1 and 16:8 h light: dark cycles at 20 ± 1 °C. All reactors containing Chlamy cultures (green reactors) or containing cyantraniliprole in TAP medium without Chlamy (white reactors) were handled at the exact same conditions under continuous atmospheric air pumping 1.75 L/min through 0.22-µm filters.

Algal growth analyses and pH determination

The growth was monitored in three different ways; determination of cell density (CD) using a hemocytometer (Hausser Scientific, Horsham, PA, USA), measuring optical density at 750 nm (OD₇₅₀) with a Helios gamma spectrophotometer (ThermoSpectronic, Allentown, PA , USA), and cell dry weight (CDW) by weighing dried 8 mL-containing cells harvested through 0.45 µm cellulose nitrate filters (Sartorius, Göttingen, Germany). All the following parameters, specific growth rate (SGR), division time (DivT), generation time (GenT), and biomass productivity (BP), were calculated as mentioned before by Morgan et al. (2023). The pH values were determined in algal cultures and cyantraniliprole-TAP by HANNA® pH meter (Padova, Italy).

Chemical and biochemical analyses

For quantification of photosynthetic pigments (Chlorophyll a, Chlorophyll b, and total carotenoids), 1 mL from each algal culture was used (at zero time, after 1 h, and 7 days of cyantraniliprole treatment). For all other parameters (algal main chemical components, oxidative stress indicators, and cyantraniliprole residues), 10 mL of each culture (at zero time, after 1 h, and 7 days of cyantraniliprole treatment) were centrifuged at 1500 ×g for 5 min at 20 °C, then supernatants were used for cyantraniliprole residual quantification.

Photosynthetic pigments

Chlorophyll a (Chl-a), Chlorophyll b (Chl-b), total Chlorophylls, Chl-a/Chl-b ratio, and total Carotenoids were determined in a 1.0 mL algal culture sample using dimethyl sulfoxide (DMSO) according to Wellburn (1994) following Eqs. 1 through 4,

Fig. 1. a) The experimental setup of the main experiment where TAP medium treated with cyantraniliprole 0, 25, 50, and 100 ppm (T0, T25, T50, and T100, respectively) in 1-L bioreactors in triplicates divided into C. reinhardtii containing reactors (+ Chlamy = green reactors) and no Chlamy containing reactors (Chlamy = White reactors); b) The fully aseptic system of bioreactors used in this study. It was composed of separate 1-L bioreactors, each bioreactor contained submerged inlet stainless-steel pipe for continuous atmospheric air pumping through 0.22-µm filters and outlet short stainless-steel pipe connected with 0.22-µm filters for gas exhausts; and c) Hierarchy diagram shows removal fate of Cyantraniliprole

where A₆₆₅, A₆₄₉, and A₄₈₀ stand for absorption at 665, 649, and 480 nm respectively. All equations were calculated as mg/L considering the dilution factor.

Chemical composition of algal cells

Using a modified micro-Kjeldahl method as described in Latimer (2023), total nitrogen was determined; then the nitrogen percentage was multiplied by 6.25 for crude protein estimation. For extraction of total lipids, the rapid chloroform: methanol (2:1) method (Bligh and Dyer 1959) was used then total lipids were quantified by dry lipid weighing method as mentioned in Abomohra et al. (2018). Total carbohydrates were quantified using phenol-sulfuric method (Dubois et al. 1956).

Some oxidative stress indicators and enzymatic and non-enzymatic antioxidants

As an indicator of lipid peroxidation, malondialdehyde (MDA) levels were determined according to Senthilkumar et al. (2021). Ascorbic acid and tocopherols were quantified by colorimetric methods in Chlamy cells as detailed in Tütem et al. (1997) and Gómez Ruiz et al. (2016), respectively. The total phenolic contents in samples were quantified using Folin-Ciocalteau method at 765 nm compared to a standard curve generated with gallic acid standard solutions (Kupina et al. 2019). Total flavonoids content was quantified as described by Ayele et al. (2022). Both CAT and SOD were determined according to Cohen et al. (1970) and Levine et al. (1994), respectively.

Calculations of Cyantraniliprole total removal percentage (TR%) and its divisions as shown in Fig. 1c are given below:

Statistical analyses

The reported data are mathematical means of three separate biological repeats with standard deviations (SD) as error values. Significant differences among treatments were analyzed by one way analysis of variance (ANOVA) at probability level P ≤ 0.05. Duncan’s multiple range post hoc tests were applied. All data were analyzed and plotted using MS Excel 365 (Microsoft Corp., Redmond, WA, USA), IBM SPSS package 27.0.1.1 (IBM Corp., Armonk, NY, USA), and GraphPad Prism 9.0.2 (University of California San Diego, San Diego, CA, USA).

RESULTS AND DISCUSSION

Preliminary Experiment

Cyantraniliprole, known for its high toxicity to aquatic invertebrates, poses an environmental threat (EFSA 2014). Remarkably, Chlamy is a model green microalga that thrives naturally in freshwater and soil and exhibits potential for bioremediation of xenobiotics-contaminated water (Nazos et al. 2017; Sehrawat et al. 2021). Inspired by this, the authors formulated a hypothesis: could Chlamy cells be harnessed for bioremediating agricultural wastewater and aquatic environments?

To test the hypothesis, two experiments were conducted. The first one was a drop test experiment using Chlamy cells on TAP-agar plates. These plates contained varying concentrations of cyantraniliprole: T0 (0 ppm), T25 (25 ppm), T50 (50 ppm), and T100 (100 ppm) (Fig. 2). After 96 h of inoculation, the results revealed important information about the effects of various cyantraniliprole concentrations on cellular growth. T0 drops were coherent, dark, and uniformly colored. T25 drops were slightly less dense, with a green hue showing a moderate impact on chlorophyll content. T50 drops had an even lighter green color, indicating further cyantraniliprole effect. Finally, T100 drops were very light in color and highly dispersed, showing a significant impact from that high concentration of cyantraniliprole.

Fig. 2. The drop test of C. reinhardtii grown for 96 h on TAP agar medium contains 0, 25, 50, and 100 ppm Cyantraniliprole (T0, T25, T50, and T100), respectively

These observations suggest that cyantraniliprole affects Chlamy growth, chlorophyll content, and cell coherence. Interestingly, previous study on Chlamy CC125 cells (similar to Chlamy CC1690 cells used in this study) revealed that smaller, slower-growing cells tend to aggregate (Sathe and Durand 2016). The current findings align with this; high concentration of cyantraniliprole (i.e., 100 ppm) appeared to hinder cell division and promote larger dispersed cells. Intrigued by these preliminary results, the authors then aimed to delve deeper into cyantraniliprole’s ecological implications for Chlamy cell physiology in the second experiment using liquid culture bioreactors.

Cyantraniliprole Removal Strategies by Chlamy

It could be inferred from Wan et al. (2020) that Chlamy cells, like other microalgal cells, have different ways to deal with water chemical-contaminants. In this study (Fig. 1c) when Chlamy cells were grown in a cyantraniliprole contaminated medium, part of the insecticide will be removed (Total removal; TR) by algal cells (Phyco-removal; PhycoR), and the other part by physical and chemical factors (Physical-chemical transformation and degradation; PCTD). PhycoR of the contaminant can happen by two main processes: 1) bioadsorption and bioaccumulation (Phyco-adsorption and -accumulation; PhycoSA) as well as 2) biotransformation and biodegradation (Phyco-transformation and -degradation; PhycoTD).

Table 1 describes the behavior of cyantraniliprole when exposed to green microalga Chlamy. Cyantraniliprole was effectively removed from water in Chlamy green reactors, with higher removal percentages observed at longer exposure time (7 days), where Chlamy absorbed and accumulated cyantraniliprole. Physical and chemical transformations contribute to the overall removal process as well because some degradation of cyantraniliprole occurred within the microalgal cells and the surrounding water.

These results provide valuable insights into the interactions between cyantraniliprole and Chlamy, which can have implications for environmental remediation and risk assessment.

Table 1. Percentages of Cyantraniliprole’s Removal, Transformation, Bio-adsorption, and Bioaccumulation by C. reinhardtii Grown for 1 h and 7 days on TAP Medium Treated with Various Cyantraniliprole Concentrations

C_i: Initial concentration; TR%: Cyantraniliprole total removal % from water; RS%: Residue % in water; PCTD%: Physical-chemical transformation and degradation %; PhycoR%: Phyco-removal % from water; PhycoSA%: Phyco-adsorption and accumulation%; PhycoTD: Phyco-transformation and degradation in (cells and water) %; SD: standard deviation. Data are presented as means of three biological replicates ± SD. The same letter in the same column indicates insignificant differences at P ≤ 0.05

Chlamy Growth and Photosynthetic Patterns Under Cyantraniliprole Treatments

Table 2 and Fig. 3 present growth parameters for Chlamy algal cells grown under different cyantraniliprole concentrations for 7 days. In T0, the algae exhibited a specific growth rate of 0.187 d^-1, a division number of 0.270 div.d^-1, and a generation time of 3.709 days. The biomass productivity was 38.7 mg CDW.L^-1.d^-1. Interestingly, in T25 and T50, they showed significantly higher growth parameters compared to T0. In T100, their specific growth rate was lower (0.110 d^-1), division number was less (0.159 div.d^-1), and generation time was longer (6.286 days). In addition, their biomass productivity was significantly lower (16.667 mg CDW.L^-1.d^-1).

Table 2. Percentages of Cyantraniliprole’s Removal, Transformation, Bio-adsorption, and Bioaccumulation by C. reinhardtii Grown for 1 h and 7 days on TAP Medium Treated with Various Cyantraniliprole Concentrations

SGR: Specific growth rate; DivT: Cell division time; GenT: Generation time; BP: Biomass productivity; CDW: Cell dry weight and SD: Standard deviation. Data are presented as means of three biological replicates ± SD. The same letter in the same column indicates insignificant differences at P ≤ 0.05.

Fig. 3. Growth monitoring as cell dry weight (CDW), and cell density (CD) of C. reinhardtii cells grown for 7 days on TAP medium treated with cyantraniliprole at T0, T25, T50, and T100 in 1-L photobioreactors. Data are presented as means of three biological replicates ± standard deviation (SD). The same letter in the same data series indicates insignificant differences at P ≤ 0.05.

In summary, cyantraniliprole affects Chlamy growth parameters, with higher concentrations leading to altered growth rates and biomass productivity. These findings are crucial for understanding the impact of cyantraniliprole on algal populations and ecosystem dynamics.

Figure 4 shows the status of the photosynthetic pigments of Chlamy cells grown under different cyantraniliprole concentrations. It is well known that Chlorophyll a (Chl-a) is the primary pigment involved in capturing light energy during photosynthesis, while Chlorophyll b (Chl-b) is an accessory pigment that complements Chl-a in light absorption. The sum of both Chl-a and Chl-b form the total Chlorophylls (Total Chls). In contrast, total carotenoids are pigments that assist in light harvesting and photoprotection. It is clear from the results that the concentrations of Chl-a and Chl-b decreased when the concentrations of cyantraniliprole were increased.

Nevertheless, carotenoids concentrations were positively correlated with cyantraniliprole concentrations until T50 only. Early reports document that biochemical reactions displayed by the photosynthetic system can differ depending on the insecticide (Haile et al. 1999). This is also the case between insecticides of the same family such as diamides. For instance, Vukovic et al. (2021) reported a high reduction in Chl-a relative percentage caused by chlorantraniliprole (compared to control) to 58.7% in the leaves of peach trees. In contrast, a smaller decrease to only 81.2% was caused by cyantraniliprole. Moreover, the effect of one pesticide has distinct impacts between different photosynthetic microalgae genera, such as Chlorella green alga and Anabaena cyanobacterium (Mostafa and Helling 2002).

The specific causes of these differences are still unclear. However, in addition to chlorophyll degradation, it is assumed that insecticides decrease photosynthetic rates in microalgae by affecting Photosystem II (PSII) machinery in terms of PSII quantum yields, photochemical quenching, and electron transport rate (Chalifour et al. 2009). Based on chlorophyll fluorescence methodology, the authors also speculate that a blocking in the flow of electrons occurs at the QB binding site, a plastoquinone molecule that plays a crucial role in the electron transport chain of photosynthesis. All these factors lead to a significant decrease in photosynthetic rates.

In contrast, the same holds true regarding the differential response of carotenoids content to different types of insecticides, where it increases with increasing levels of insecticides, but with different percentages. For chlorantraniliprole, it had a higher stimulatory effect and led to a higher relative accumulation of carotenoids than cyantraniliprole, being 170% and 153%, respectively (Vukovic et al. 2021). This is expected, as carotenoids increase to high levels when the stressor is more harmful. In the case of the authors’ experiments, increases were detected between T25 and T50, but higher levels (e.g., T100 in this study) become detrimental and carotenoids levels drop below control levels.

Carotenoids’ primary antioxidant protective function is to shield membranes from damage caused by reactive oxygen species (ROS). Peroxyl (ROO·), alkoxyl (RO·), hydroxyl (·OH), and superoxide anion radicals (O₂^·–) can all be effectively scavenged by carotenoids. Conjugated double bonds, which can accept unpaired electrons and widely delocalize them over the conjugated system of double bonds, are linked to the antioxidant qualities of carotenoids (Mortensen et al. 2001).

Fig. 4. Photosynthetic pigments (Chl-a, Chl-b, Total Chls, and total Carotenoids) of C. reinhardtii cells grown for 7 days on TAP medium treated with cyantraniliprole (T0, T25, T50, and T100 respectively): Chl-a: Chlorophyll a; Chl-b: Chlorophyll b; Total Chls: total Chlorophylls and Chl-a/b: Chlorophyll a/ Chlorophyll b. All values are presented as means of three biological replicates ± standard deviation (SD) except the values of total chlorophylls are presented as minimum to maximum values. The same letter in the same data series indicates insignificant differences at P ≤ 0.05.

Implications of Cyantraniliprole in Changing Biochemical Composition and Oxidative Stress Response of Chlamy Cells

Metabolism plays a crucial role in sustaining biological activities within organisms. It enables growth, reproduction, and interaction with the external environment. Researchers have found that pollutants can inhibit sensitive microorganisms, while, interestingly, the metabolic capacity of degrading microorganisms can be enhanced (Huang et al. 2022; Chen et al. 2023).

The results of chemical composition are presented in Table 3 for Chlamy biomass grown under different cyantraniliprole concentrations. At zero time, the initial composition of the algal biomass showed 15.1% proteins, 31.7% carbohydrates, and 11.8% lipids. After 7 days, in T0 cells, the concentrations of proteins and carbohydrates decreased slightly to 13.6% and 30.1%, respectively. The concentration of lipids increased to 12.9% in T25, T50, and T100. Similar trends are observed with a decrease in the protein concentrations (13.2%, 12.7%, and 9.2%, respectively). However, gradual increases in concentrations of carbohydrates (33%, 38.4%, and 43.8%, respectively) and lipids (15.5%, 16.4%, and 20.1%, respectively) were observed. In summary, cyantraniliprole exposure affected the algal biomass composition, with alterations in protein, carbohydrate, and lipid levels. These changes may have implications for algal growth, metabolism, and overall health.

As the concentrations and exposure time of cyantraniliprole are increased, all studied oxidative parameters and antioxidative parameters (MDA, total phenolic compounds, total flavonoids, SOD, CAT, and Tocopherols) increased, as shown in Fig. 5, except for ascorbic acid (Vit C). There is a negative correlation usually between Vit C and Tocopherol (Vit E) as shown in a previous study (Morgan et al. 2023). Normal levels of ROS play a crucial role in various cellular activities, including enhancing enzyme function and boosting immunity (Li and Kataoka 2020). However, exposure to pesticides can lead to toxic effects by elevating ROS levels (Moniruzzaman et al. 2020).

Table 3. The Chemical Composition at Zero Time and after 7 days of C. reinhardtii Biomass Grown on TAP Medium Treated with Cyantraniliprole 0, 25, 50, and 100 ppm

Data are presented as means of three biological replicates ± standard deviation (SD). The same letter in the same column indicates insignificant differences at P ≤ 0.05.

In this study, the authors observed an upward trend in ROS levels after exposure to cyantraniliprole throughout the exposure period. Elevated ROS levels can harm cells and macromolecules such as DNA and lipids (Song et al. 2019). Notably, significant increases in MDA contents were found (Fig. 5a) after 1 h and seven days of exposure to all concentrations of cyantraniliprole. These findings align with Yan et al. (2023)’s work, which demonstrated increased MDA content during the late stage of insecticide exposure.

To counter excess ROS, cells rely on both enzymatic and non-enzymatic antioxidant defenses. Catalase (CAT) and SOD activities must maintain a dynamic balance with ROS levels to support organismal survival and manage environmental stress. Additionally, Ca²⁺ directly activates antioxidant enzymes like glutathione reductase, CAT, and SOD, facilitating ROS clearance (Thompson et al. 2014).

In the current study, CAT and SOD activities were enhanced in cyantraniliprole-exposed algae cells from time zero to the seventh day compared to the control group (T0). These results confirm that cyantraniliprole induces oxidative stress and detoxification effects. Furthermore, Yan et al. (2023) reported a positive correlation between the SOD gene and SOD activity, suggesting that cyantraniliprole has subchronic toxic impacts related to oxidative stress and DNA changes in the earthworm Eisenia fetida.

An essential line of defense for cells against ROS is represented by low molecular weight antioxidants. A subset of water-soluble low molecular weight antioxidants exists within the cytoplasmic matrix or cytosol. Another class of lipid-soluble antioxidants functions within the membranes. The most prominent instances of these antioxidants include flavonoids, carotenoids, glutathione, and vitamins C, E, and A (Pinchuk et al. 2021).

Vitamin C’s pro-oxidant properties in the presence of oxygen and redox-active metal ions have primarily been demonstrated in vitro; evidence for their occurrence in vivo has not been sufficiently demonstrated (Halliwell 1996). This could be because redox metal homeostasis is strictly regulated and metal sequestration results in minimal amounts of free metals. In vitro, ascorbate can regenerate vitamin E from its radical tocopheryl form. Fat-soluble vitamin E, also known as α-tocopherol, shields cellular membranes from ROS-caused lipid peroxidation (NIH 2021). The hydroxyl group on vitamin E’s six-membered ring is outside the membrane and faces the aqueous environment, where it scavenges ROS. Vitamin E is anchored in a lipid membrane. α-Tocopheryl radical (α-TO·) is created when α-Tocopherol (α-TOH) reacts with radicals, such as·OH.

Phenolic compounds and flavonoids have antioxidant properties that are able to terminate free radicals. Additionally, they can stimulate the production of antioxidant enzymes and regenerate certain vitamins, such vitamin E, that have a higher electron reduction potential (Simunkova et al. 2019). Hydroxyl groups of flavonoids are directly involved in the scavenging activity of radicals, forming flavonoid radicals that can be regenerated by Glutathione (GSH).

Fig. 5. Oxidative stress indicators and certain defense factors in C. reinhardtii cells grown for 7 days on TAP medium treated with cyantraniliprole at 0, 25, 50, and 100 ppm: MDA: Malondialdehyde; CAT: Catalase enzyme and SOD: Superoxide dismutase enzyme. Data are presented as means of three biological replicates ± standard deviation (SD). The same letter in the same data series indicates insignificant differences at P ≤ 0.05.

CONCLUSION

This study demonstrated the potential of Chlamydomonas reinhardtii in the bioremediation of cyantraniliprole-contaminated water, achieving up to 87.0% removal within 1 h.
The microalga exhibited resilience to cyantraniliprole stress, maintaining antioxidant levels.
Remarkably, certain treated cultures showed enhanced growth rate, biomass productivity, and increased carbohydrate and lipid concentrations, suggesting potential for biofuel production.
Therefore, C. reinhardtii not only offers a solution for cyantraniliprole pollution, but also contributes to sustainable bioenergy production. Further research could explore optimizing these processes for large-scale applications.

ACKNOWLEDGMENTS

This work was funded by the Deanship of Graduate Studies and Scientific Research at Jouf University under grant No. (DGSSR-2023-02-02229).

REFERENCES CITED

Abdel daiem, M. M., Sánchez-Polo, M., Rashed, A. S., Kamal, N., and Said, N. (2019). “Adsorption mechanism and modelling of hydrocarbon contaminants onto rice straw activated carbons,” Polish Journal of Chemical Technology 21(4), 1-12. DOI: 10.2478/pjct-2019-0032

Abomohra, A. E. -F., Eladel, H., El-Esawi, M., Wang, S., Wang, Q., He, Z., Feng, Y., Shang, H., and Hanelt, D. (2018). “Effect of lipid-free microalgal biomass and waste glycerol on growth and lipid production of Scenedesmus obliquus: Innovative waste recycling for extraordinary lipid production,” Bioresource Technology 249, 992-999. DOI: 10.1016/j.biortech.2017.10.102

Alrowais, R., Abdel daiem, M. M., Nasef, B. M., and Said, N. (2024a). “Activated carbon fabricated from biomass for adsorption/bio-adsorption of 2, 4-D and MCPA: Kinetics, isotherms, and artificial neural network modeling,” Sustainability 16(1), article 299. DOI: 10.3390/su16010299

Alrowais, R., Bashir, M. T., Khan, A. A., Bashir, M., Abbas, I., and Abdel Daiem, M. M. (2024b). “Adsorption and kinetics modelling for chromium (cr6+) uptake from contaminated water by quaternized date palm waste,” Water 16(2), article 294. DOI: 10.3390/w16020294

Alrowais, R., Said, N., Bashir, M. T., Ghazy, A., Alwushayh, B., and Abdel daiem, M. M. (2023a). “Adsorption of diphenolic acid from contaminated water onto commercial and prepared activated carbons from wheat straw,” Water 15(3), article 555. DOI: 10.3390/w15030555

Alrowais, R., Yousef, R. S., Ahmed, O. K., Mahmoud-Aly, M., Abdel Daiem, M. M., and Said, N. (2023b). “Enhanced detoxification methods for the safe reuse of treated olive mill wastewater in irrigation,” Environmental Sciences Europe 35(1), article 95. DOI: 10.1186/s12302-023-00797-2

Ananthi, V., Siva Prakash, G., Chang, S. W., Ravindran, B., Nguyen, D. D., Vo, D.-V. N., La, D. D., Bach, Q.-V., Wong, J. W. C., Kumar Gupta, S., et al. (2019). “Enhanced microbial biodiesel production from lignocellulosic hydrolysates using yeast isolates,” Fuel 256, article ID 115932. DOI: 10.1016/j.fuel.2019.115932

Ayele, D. T., Akele, M. L., and Melese, A. T. (2022). “Analysis of total phenolic contents, flavonoids, antioxidant and antibacterial activities of Croton macrostachyus root extracts,” BMC Chemistry 16(1), article 30. DOI: 10.1186/s13065-022-00822-0

Bligh, E. G., and Dyer, W. J. (1959). “A rapid method of total lipid extraction and purification,” Canadian Journal of Biochemistry and Physiology 37(8), 911-917. DOI: 10.1139/o59-099

Chalifour, A., Scarpellino, L., Back, J., Brodin, P., Devèvre, E., Gros, F., Lévy, F., Leclercq, G., Höglund, P., and Beermann, F. (2009). “A role for cis interaction between the inhibitory Ly49A receptor and MHC class I for natural killer cell education,” Immunity 30(3), 337-347. DOI: 10.1016/j.immuni.2008.12.019

Chen, H., Jiang, H., Nazhafati, M., Li, L., and Jiang, J. (2023). “Biochar: An effective measure to strengthen phosphorus solubilizing microorganisms for remediation of heavy metal pollution in soil,” Frontiers in Bioengineering and Biotechnology 11, article ID 1127166. DOI: 10.3389/fbioe.2023.1127166

Cohen, G., Dembiec, D., and Marcus, J. (1970). “Measurement of catalase activity in tissue extracts,” Analytical Biochemistry 34(1), 30-38. DOI: 10.1016/0003-2697(70)90083-7

Dong, J., Wang, K., Li, Y., and Wang, S. (2017). “Lethal and sublethal effects of cyantraniliprole on Helicoverpa assulta (Lepidoptera: Noctuidae),” Pesticide Biochemistry and Physiology 136, 58-63. DOI: 10.1016/j.pestbp.2016.08.003

Dubois, M., Gilles, K. A., Hamilton, J. K., Rebers, P. A., and Smith, F. (1956). “Colorimetric method for determination of sugars and related substances,” Analytical Chemistry 28(3), 350-356. DOI: 10.1021/ac60111a017

Dupuis, S., and Merchant, S. S. (2023). “Chlamydomonas reinhardtii: A model for photosynthesis and so much more,” Nature Methods 20(10), 1441-1442. DOI: 10.1038/s41592-023-02023-6

EFSA (2014). “Conclusion on the peer review of the pesticide risk assessment of the active substance cyantraniliprole,” European Food Safety Authority Journal 12(9), article ID 3814. DOI: 10.2903/j.efsa.2014.3814

El-Shatoury, S., El-Baz, A., Abdel Daiem, M., and El-Monayeri, D. (2014). “Enhancing wastewater treatment by commercial and native microbial Inocula with factorial design,” Life Science Journal 11(7), 736-742.

Gómez Ruiz, B., Roux, S., Courtois, F., and Bonazzi, C. (2016). “Spectrophotometric method for fast quantification of ascorbic acid and dehydroascorbic acid in simple matrix for kinetics measurements,” Food Chemistry 211, 583-589. DOI: 10.1016/j.foodchem.2016.05.107

Gorman, D. S., and Levine, R. P. (1965). “Cytochrome f and plastocyanin: Their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi,” Proceedings of the National Academy of Sciences of the United States of America 54(6), 1665-1669. DOI: 10.1073/pnas.54.6.1665

Haile, F. J., Peterson, R. K. D., and Higley, L. G. (1999). “Gas-exchange responses of alfalfa and soybean treated with insecticides,” Journal of Economic Entomology 92(4), 954-959.

Halliwell, B. (1996). “Antioxidants in human health and disease,” Annual Review of Nutrition, Annual Reviews 16(1), 33-50.

Latimer, G. W. (2023). Official Methods of Analysis: 22^nd Edition, G. W. Latimer (Ed.), AOAC Publications, New York, NY, USA. DOI: 10.1093/9780197610145.002.001

Huang, J., Fan, C.-M., Chen, J.-H., and Yan, J. (2022). “Meshless generalized finite difference method for the propagation of nonlinear water waves under complex wave conditions,” Mathematics 10(6), article 1007. DOI: 10.3390/math10061007

Kaloudas, D., Pavlova, N., and Penchovsky, R. (2021). “Phycoremediation of wastewater by microalgae: A review,” Environmental Chemistry Letters 19(4), 2905-2920. DOI: 10.1007/s10311-021-01203-0

Koul, B., Sharma, K., and Shah, M. P. (2022). “Phycoremediation: A sustainable alternative in wastewater treatment (WWT) regime,” Environmental Technology & Innovation 25, article ID 102040. DOI: 10.1016/j.eti.2021.102040

Kupina, S., Fields, C., Roman, M. C., and Brunelle, S. L. (2019). “Determination of total phenolic content using the Folin-C assay: Single-laboratory validation, first action 2017.13,” Journal of AOAC International 102(1), 320-321. DOI: 10.5740/jaoacint.18-0031

Levine, A., Tenhaken, R., Dixon, R., and Lamb, C. (1994). “H₂O₂ from the oxidative burst orchestrates the plant hypersensitive disease resistance response,” Cell 79(4), 583-593. DOI: 10.1016/0092-8674(94)90544-4

Li, J., and Kataoka, K. (2020). “Chemo-physical strategies to advance the in vivo functionality of targeted nanomedicine: The next generation,” Journal of the American Chemical Society 143(2), 538-559. DOI: 10.1021/jacs.0c09029

Liang, M., Chen, L., Zhao, G., and Guo, Y. (2020). “Effects of solution treatment on the microstructure and mechanical properties of naturally aged EN AW 2024 Al alloy sheet,” Journal of Alloys and Compounds 824, article ID 153943. DOI: 10.1016/j.jallcom.2020.153943

Mahmoud-Aly, M., Li, Y., Shanab, S. M. M., Amin, A. Y., and Ahmed, A. H. H. (2018). “Physiological characterization of a Chlamydomonas reinhardtii vacuolar transporter chaperon1 mutant under phosphorus deprivation condition,” Bioscience Research 15, 4532-4539. DOI: 10.14720/aas.2023.119.4.13421

MDA (2014). “New active ingredient and new use special registration reviews,” Minnesota Department of Agriculture, (https://www.mda.state.mn.us/chemicals/pesticides/regs/newreviews), Accessed 10 May 2024.

Méndez-Díaz, J. D., Abdel daiem, M. M., Rivera-Utrilla, J., Sánchez-Polo, M., and Bautista-Toledo, I. (2012). “Adsorption/bioadsorption of phthalic acid, an organic micropollutant present in landfill leachates, on activated carbons,” Journal of Colloid and Interface Science 369(1), 358-365. DOI: 10.1016/j.jcis.2011.11.073

Moniruzzaman, M., Mukherjee, M., Das, D., and Chakraborty, S. B. (2020). “Effectiveness of melatonin to restore fish brain activity in face of permethrin induced toxicity,” Environmental Pollution 266, article ID 115230. DOI: 10.1016/j.envpol.2020.115230

de Morais, M. G., Zaparoli, M., Lucas, B. F., and Costa, J. A. V. (2022). “Chapter 4 – Microalgae for bioremediation of pesticides: Overview, challenges, and future trends,” in: Algal Biotechnology, A. Ahmad, F. Banat, and H. B. T.-A. B. Taher (Eds.), Elsevier Ltd., Amsterdam, Netherlands, pp. 63-78. DOI: 10.1016/B978-0-323-90476-6.00010-8

Morgan, S. H., Mahmoud, M. A. W., Mottaleb, S. A., El-Bahbohy, R. M., and Mahmoud-Aly, M. (2023). “A sustainable nanotechnology producing high-quality remediated sewage wastewater used for microalgal protein-rich biomass and biodiesel production,” Biomass Conversion and Biorefinery 3, Published Online. DOI: 10.1007/s13399-023-05016-9

Mortensen, A., Skibsted, L. H., and Truscott, T. G. (2001). “The interaction of dietary carotenoids with radical species,” Archives of biochemistry and Biophysics 385(1), 13-19. DOI: 10.1006/abbi.2000.2172

Mostafa, F. I. Y., and Helling, C. S. (2002). “Impact of four pesticides on the growth and metabolic activities of two photosynthetic algae,” Journal of Environmental Science and Health, Part B 37(5), 417-444. DOI: 10.1081/PFC-120014873

Nazos, T. T., Kokarakis, E. J., and Ghanotakis, D. F. (2017). “Metabolism of xenobiotics by Chlamydomonas reinhardtii: Phenol degradation under conditions affecting photosynthesis,” Photosynthesis Research 131(1), 31-40. DOI: 10.1007/s11120-016-0294-2

NIH (2021). “Vitamin E: Fact sheet for health professionals,” National Institutes of Health, (https://ods.od.nih.gov/factsheets/VitaminE-HealthProfessional), Accessed 10 May 2024.

Paravani, E. V., Simoniello, M. F., Poletta, G. L., and Casco, V. H. (2019). “Cypermethrin induction of DNA damage and oxidative stress in zebrafish gill cells,” Ecotoxicology and Environmental Safety 173, 1-7. DOI: 10.1016/j.ecoenv.2019.02.004

Pinchuk, I., Kohen, R., Stuetz, W., Weber, D., Franceschi, C., Capri, M., Hurme, M., Grubeck-Loebenstein, B., Schön, C., and Bernhardt, J. (2021). “Do low molecular weight antioxidants contribute to the Protection against oxidative damage? The interrelation between oxidative stress and low molecular weight antioxidants based on data from the MARK-AGE study,” Archives of Biochemistry and Biophysics 713, article ID 109061. DOI: 10.1016/j.abb.2021.109061

Sathe, S., and Durand, P. M. (2016). “Cellular aggregation in Chlamydomonas (Chlorophyceae) is chimaeric and depends on traits like cell size and motility,” European Journal of Phycology 51(2), 129-138. DOI: 10.1080/09670262.2015.1107759

Sehrawat, A., Phour, M., Kumar, R., and Sindhu, S. S. (2021). “Bioremediation of pesticides: An eco-friendly approach for environment sustainability,” in: Microbial Rejuvenation of Polluted Environment, D. G. Panpatte, and Y. K. Jhala (eds.), Springer Singapore, Singapore, pp. 23-84. DOI: 10.1007/978-981-15-7447-4_2

Senthilkumar, M., Amaresan, N., and Sankaranarayanan, A. (2021). Estimation of Malondialdehyde (MDA) by Thiobarbituric Acid (TBA) Assay, Springer Protocols Handbooks, Humana, New York, NY, USA. DOI: 10.1007/978-1-0716-1080-0_25

Simunkova, M., Alwasel, S. H., Alhazza, I. M., Jomova, K., Kollar, V., Rusko, M., and Valko, M. (2019). “Management of oxidative stress and other pathologies in Alzheimer’s disease,” Archives of Toxicology 93, 2491-2513. DOI: 10.1007/s00204-019-02538-y

Song, P., Gao, J., Li, X., Zhang, C., Zhu, L., Wang, J., and Wang, J. (2019). “Phthalate induced oxidative stress and DNA damage in earthworms (Eisenia fetida),” Environment International 129, 10-17. DOI: 10.1016/j.envint.2019.04.074

Sun, J., Feng, N., Tang, C., and Qin, D. (2012). “Determination of cyantraniliprole and its major metabolite residues in pakchoi and soil using ultra-performance liquid chromatography-tandem mass spectrometry,” Bulletin of Environmental Contamination and Toxicology 89(4), 845-852. DOI: 10.1007/s00128-012-0752-2

Thompson, M. D., Mei, Y., Weisbrod, R. M., Silver, M., Shukla, P. C., Bolotina, V. M., Cohen, R. A., and Tong, X. (2014). “Glutathione adducts on sarcoplasmic/ endoplasmic reticulum Ca²⁺ ATPase Cys-674 regulate endothelial cell calcium stores and angiogenic function as well as promote ischemic blood flow recovery,” Journal of Biological Chemistry 289(29), 19907-19916. DOI: 10.1074/jbc.M114.554451

Tütem, E., Apak, R., Günaydı, E., and Sözgen, K. (1997). “Spectrophotometric determination of vitamin E (α-tocopherol) using copper(II)-neocuproine reagent,” Talanta 44(2), 249-255. DOI: 10.1016/S0039-9140(96)02041-3

Ummalyma, S. B., and Singh, A. (2022). “Biomass production and phycoremediation of microalgae cultivated in polluted river water,” Bioresource Technology 351, article ID 126948. DOI: 10.1016/j.biortech.2022.126948

Vukovic, S., Žunic, A., Maksimovic, I., Lazic, S., Šunjka, D., Žunic, V., and Putnik-Delic, M. (2021). “Insecticide-induced changes of photosynthetic pigments content in peach leaves,” Pakistan Journal of Agricultural Sciences 58(6), 1705-1710.

Wan, L., Wu, Y., Ding, H., and Zhang, W. (2020). “Toxicity, biodegradation, and metabolic fate of organophosphorus pesticide trichlorfon on the freshwater algae Chlamydomonas reinhardtii,” Journal of Agricultural and Food Chemistry 68(6), 1645-1653. DOI: 10.1021/acs.jafc.9b05765

Wang, R., Wang, J.-D., Che, W.-N., and Luo, C. (2018). “First report of field resistance to cyantraniliprole, a new anthranilic diamide insecticide, on Bemisia tabaci MED in China,” Journal of Integrative Agriculture 17(1), 158-163. DOI: 10.1016/S2095-3119(16)61613-1

Wellburn, A. R. (1994). “The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrophotometers of different resolution,” Journal of Plant Physiology 144(3), 307-313. DOI: 10.1016/S0176-1617(11)81192-2

Yan, S., Ren, X., Zheng, L., Wang, X., and Liu, T. (2023). “A systematic analysis of residue and risk of cyantraniliprole in the water-sediment system: Does metabolism reduce its environmental risk?,” Environment International 179, article ID 108185. DOI: 10.1016/j.envint.2023.108185

Article submitted: May 13, 2024; Peer review completed: June 20, 2024; Revised version received and accepted: June 27, 2024; Published: July 28, 2024.

DOI: 10.15376/biores.19.3.6653-6669