FE-SEM images reveal that the size of the nTiO2 was around 20 nm, where PSMPs measured around 1.9 µm. The comparable increase in the surface area to volume ratio of nTiO2 may lead to more pronounced toxic effects than PSMPs. A previous study (Li et al. 2020c) observed that 0.1 and 0.55 µm PSMPs caused enhanced toxic effects than 5 µm PSMPs in algal cells. Additionally, comparing the size of the PSMPs (1.9 µm) and Chlorella sp (3–4 µm) it is noted that PSMPs could adsorb less onto the surface of algal cells. These findings align with the observations of (Thiagarajan et al. 2021), where they interacted 6 µm PSMPs with marine Chlorella sp. ranging from 3–4 µm. Furthermore, the surface charge of NPs/MPs can also influence their adsorption onto the algal cells and subsequent internalization (Awashra and Młynarz 2023). It is expected that nTiO2 (+ 13.95 mV) would have a stronger electrostatic attraction towards the algae compared to NH2 (+ 4.75 mV) and COOH (-15.45 mV) PSMPs. In agreement with these results, (Thiagarajan et al. 2019a) reported surface charge-based attraction of nTiO2, NH2, and COOH functionalized PSMPs towards marine Chlorella sp.
Growth inhibition can be considered a major physiological indicator of various biological processes within the cell. As expected, the pristine nTiO2 significantly enhanced the suppression of algal growth under UVAR compared to the VL conditions. (Sendra et al. 2017a) reported that pristine nTiO2 showed more toxicity towards marine algae (Phaeodactylum tricornutum) under UVAR compared to VL conditions. However, pristine PSMPs did not exhibit a significant toxicity difference between UVAR and VL conditions. This indicates that UVAR presumably had no influence on the toxic effects of PSMPs. Furthermore, it is to be noted that the aging process of MPs typically spans several months, and within a 72-hour period, no significant change in the MNPs could occur. Our finding through FTIR analysis confirmed that there was no degradation of PSMPs under both the light conditions. Similarly, (Li et al., 2023) investigated the toxic effect of pristine polymethyl methacrylate (PMMA) MPs towards marine diatom Thalassiosira pseudonana under UV-B radiation. They suggested that a 96-h interaction under UV-B radiation did not cause any degradation of PMMA MPs.
Compared to pristine test groups the algal growth inhibition was substantially increased for the mixtures of nTiO2 + PSMPs. The increased toxicity of the mixtures can primarily be attributed to the interactive toxic effects of nTiO2 and PSMPs. These findings align with the results from the independent action modeling (Table 1). The mode of interaction was found to be additive for the mixtures of nTiO2 + PSMPS (10 mg/L). However, antagonistic mode interaction was observed for the mixtures nTiO2 + COOH PSMPs (2.5 mg/L). This could be attributed to the competition among toxicants to occupy binding sites for cellular entry (Zhu et al. 2019). These results are in accordance with the findings of (Thiagarajan et al. 2019a), where the mixtures of nTiO2 (37.56/12.09/112.68 (µM)) with 6 µm NH2 MPs (1000 mg/L) exhibited an additive mode of interaction. On the other hand, the mixture of nTiO2 + COOH MPs showed an antagonistic mode of interaction towards marine algae chlorella sp. Our findings reveal that under UVAR conditions, the mixture toxicity was significantly increased compared to the VL conditions. This can be attributed to the enhanced photoactivity of TiO2. On the contrary, (Gunasekaran et al. 2020) reported that plain PSMPs (1mg/L) reduced the toxic effect of photocatalytic ZnO NPs on marine algae Dunaliella salina under UVAR conditions. The reason behind this toxicity decrement could be the presence of uncharged plain PSMPs surrounding the algal cells serving as a barrier, hindering the interaction between the negatively charged algal cells and positively charged ZnO NPs.
The abnormal chrematistics of Chlorella sp., including cell shrinkage, aggregation, cellular distortion, and ruptures on exposure to the mixtures of nTiO2 and PSMPs under both light conditions were validated through FE-SEM images. Under UVAR conditions, the control algal cells exhibited increased homo-aggregation compared to the VL conditions. Studies indicate that algal cells can undergo homo-aggregation as a response to stress (Mao et al. 2018). This highlights the potential influence of UVAR alone on the toxicity of algal cells. Additionally, the mixtures of nTiO2 + NH2 PSMPs displayed enhanced cellular damage than the mixtures of nTiO2 + COOH PSMPs under both the light conditions. This difference can result from favorable electrostatic attraction exhibited by nTiO2 + NH2 PSMPs mixture (+ 18.4 mV) compared to the nTiO2 + COOH PSMPs mixture (-8.87 mV) towards the Chlorella sp (-28.6 mV).
The hetero aggregation between the algae and NPs/MPs can lead to cell membrane damage (Lagarde et al. 2016; Li et al. 2023b). Consequently, the compromised cell wall can facilitate the internalization of the NPs, potentially intensifying the toxic effects (Sendra et al. 2017b). Additionally, PSMPs can be adsorbed to the caveolae present on the surface of algal cells. This adsorption could result in the closure of caveolae, limiting the uptake of nutrients, CO2, and O2 (Zhang et al. 2017). The impact of UV radiation on the aggregation of algal cells was emphasized by (Dong et al. 2021). Under UVAR conditions, the aggregation rate among the algae (homo-aggregation) and algae with the pollutants (hetero-aggregation) was observed to be enhanced than in VL conditions. This can be attributed to the extracellular polymeric substance secretion by the algal cells. UV conditions can stimulate the production of EPS that may facilitate the aggregation of algal cells (Xiao and Zheng 2016; Zhu et al. 2022). This was validated through FE-SEM images in the current work, which clearly depicts the excessive layer of EPS forming around the algal cells in the test groups exposed under UVAR conditions. Another group of researchers (Zhu et al. 2020) documented comparable SEM observations when studying the effects of PSNPs on the marine algae Chlorella vulgaris. It was observed that the rate of hetero aggregation in the mixture of nTiO2 and PSMPs was significantly enhanced compared to their pristine forms. The increased rate of hetero aggregation can be attributed to the combined interaction of nTiO2 and PSMPs on algal cells. Similarly, (Thiagarajan et al. 2019a) reported that hetero-aggregation of nTiO2 and NH2 MPs with marine algae Chlorella sp. resulted in cellular damage.
The generation of ROS and the ensuing oxidative stress has long been recognized as a principal mode of action underpinning the toxicity associated with NPs (Zhu et al. 2022). These reactive species possess the ability to induce cellular damage, lipid peroxidation, and to impede photosynthesis (Liu et al. 2018; Middepogu et al. 2018). Upon exposure to UVAR, nTiO2 exhibits photocatalytic activity as a result of the combined impact of electron-hole pair formation and subsequent ROS generation (Pattanaik and Sahoo 2014; Moma and Baloyi 2019). The physical adsorption of MPs may also damage the cell membrane, and also hinder the transport of light and nutrients (Zhang et al. 2023). This disruption in cellular processes also can trigger ROS production (Prata et al. 2018). Under UVAR conditions, pristine nTiO2 exhibited exacerbated ROS production than in VL conditions. A comparable pattern of increased ROS levels was documented by (Thiagarajan et al. 2019b) in marine Chlorella sp. under UVAR conditions. Another related study (Zhu et al. 2022) also noted that the exposure of nTiO2 under UV-B radiation enhanced ROS production in marine algae Chlorella pyrenoidosa. However, pristine PSMPs did not exhibit such kind of differences between the light conditions. In comparison to the pristine toxicants (nTiO2 and PSMPs), the mixture of nTiO2 + PSMPs resulted in enhanced ROS production. Specifically, the mixture of nTiO2 + NH2 PSMPs showed a greater increase in ROS production compared to the mixture of nTiO2 + COOH PSMPs. This difference in ROS production again can be linked to the surface charge-based attraction of PSMPs to the algal cells. In agreement with this study (Zhang et al. 2020b) reported that NH2-MPs showed increased ROS production than COOH-MPs in C. pyrenoidosa.
The correlation between ROS generation and LPO implies that oxidative radicals can damage the lipid cell membrane of algae (Rezayian et al. 2019). As a consequence, membrane permeability increases, while membrane fluidity and integrity decrease (Huang et al. 2021). The ROS can target the unsaturated fatty acids found in the membranes of algae cells, thus initiating the process of lipid peroxidation (Zhang et al. 2020a). This results in the production of MDA as one of the final byproducts. MDA is a highly reactive aldehyde and is commonly used as a biomarker to indicate oxidative stress (Yang et al. 2020). In our study, the generation of MDA levels in relation to both pristine and mixtures (nTiO2 + PSMPs) exhibited a comparable increase with increased ROS production. This trend was also reported by (Wan et al. 2021) in two different freshwater algae Chlorella sp. and Pseudokirchneriella subcapitata in response to the combined toxicity of Cu and PSMPs.
The measurement of chlorophyll content can serve as an indicator of the physiological health status of algae (Mo et al. 2022). The Chl A and Chl B can serve as an indicator of the sensitivity of the light-harvesting complex II (LHCII) enzyme system in chloroplasts in response to external factors (Negi et al. 2020). A modification in chlorophyll production signifies a change in the stoichiometric balance between the reaction center complexes of both photosystems and the light-collecting complex of photosystem II (Arrojo et al. 2022). In this study, photosynthetic pigment production was reduced considerably for the test groups under UVAR conditions than the VL conditions, which can be attributed to the increased accumulation of ROS in algal cells. This enhanced ROS production can leads to peroxidation and subsequent damage to the stoichiometry of the photosynthetic apparatus (Sachdev et al. 2023). Compared to pristine nTiO2, there was not much reduction in pigment production for the pristine PSMPs. The reduced particle size of nTiO2 can lead to heightened cellular damage, including the internalization of particles and subsequent damage to chloroplasts (Li et al. 2020b). Whereas, PSMPs tend to attach to the cell membrane, inhibiting light penetration and causing comparatively less damage (Fig. S3) (Nava and Leoni 2021). Under UVAR conditions, the mixture of nTiO2 with NH2 PSMPs (10 mg/L) showed more reduction in the photosynthetic pigments than nTiO2 with COOH PSMPs (10 mg/L) when compared to VL conditions. Similarly, (Zhu et al. 2022) reported the photosynthetic pigments reduction in marine algae Chlorella pyrenoidosa on exposure to nTiO2 under UV-B radiation. These observations are in accordance with the findings of (Khalifeh et al. 2022), where ROS generation affected the pigment production in marine algae Chlorella sorokiniana on exposure to MnO2 nanorods. Similarly, (Zhu et al. 2022) reported a decline in photosynthetic pigment production in marine algae C. pyrenoidosa on exposure to nTiO2 under UV-B radiation.
Carotenoids play a substantial role in the overall antioxidant potential of microalgae as they are vital components of the non-enzymatic defense system. They effectively neutralize radicals, acting as potent quenchers, thus averting free radical reactions (Sirohi et al. 2022). Algal cells utilize carotenoids to effectively counteract and eliminate ROS (Maltsev et al. 2021; Song et al. 2023). However, it is evident from our results that for the mixture, nTiO2 + PSMPs (10 mg/L) carotenoid production drastically reduced, mainly under UVAR conditions. This implies that the algal cells may experience a reduction in the ability to effectively counteract the impact of free radicals, which can be indirectly associated with decreased activity of antioxidant enzymes and exacerbated oxidative damage. The effect of different light conditions on the combined toxic effects of nTiO2 and MPs towards Chlorella sp, as well as their underlying mode of action, are concisely summarized using a schematic diagram (Fig. 8).