3.1.2. Diurnal Changes of Algal *Fv/F<sup>m</sup>*

The diurnal changes of *Fv/F<sup>m</sup>* were similar for three species in the PAR and UV-B treatments (Figure 3). During the incubation, diurnal *Fv/F<sup>m</sup>* did not change significantly after 4 h of PAR treatment (*p* > 0.05), irrespective of species. In comparison, algal diurnal *Fv/F<sup>m</sup>* decreased after 4 h of UV-B radiation (*p* < 0.05), which could then increase after the withdrawal of UV-B radiation on each day. However, the decline degree and recovery of *Fv/F<sup>m</sup>* was dependent on the incubation time and species.

For three species, their *Fv/F<sup>m</sup>* decreased to 15.2–40.6% of the initial values after UV-B radiation on Day 2 and they recovered to 75.2–83.3% of the initial values within 20 h, when the decline was lower for toxic *M. aeruginosa* (*p* < 0.05). On Days 6 and 8, the decline of algal *Fv/F<sup>m</sup>* worsened after UV-B radiation and algal *Fv/F<sup>m</sup>* could all recover to the initial values within 20 h. Meanwhile, the recovery rate increased with the development of incubation, and the recovery rate was highest for toxic *M. aeruginosa* (*p* < 0.05). In comparison, the inhibition of UV-B radiation on *Fv/F<sup>m</sup>* was maximum for *C. pyrenoidosa* on each day and the recovery rate of its *Fv/F<sup>m</sup>* was lower.

*Int. J. Environ. Res. Public Health* **2022**, *19*, x FOR PEER REVIEW 6 of 23

**Figure 1.** Cell density (line and scatter) and *Fv/Fm* (vertical bar) of three species in the PAR and UV‐ B treatments under normal growth conditions (the arrow indicates the initial value of *Fv/Fm*). **Figure 1.** Cell density (line and scatter) and *Fv/Fm* (vertical bar) of three species in the PAR and UV-B treatments under normal growth conditions (the arrow indicates the initial value of *Fv/Fm*). **Figure 1.** Cell density (line and scatter) and *Fv/Fm* (vertical bar) of three species in the PAR and UV‐ B treatments under normal growth conditions (the arrow indicates the initial value of *Fv/Fm*).

**Figure 2.** Maximum growth rates of three species in the PAR and UV‐B treatment under normal growth conditions and nutrient enrichment conditions. **Figure 2.** Maximum growth rates of three species in the PAR and UV‐B treatment under normal growth conditions and nutrient enrichment conditions. **Figure 2.** Maximum growth rates of three species in the PAR and UV-B treatment under normal growth conditions and nutrient enrichment conditions.

The diurnal changes of *Fv/Fm* were similar for three species in the PAR and UV‐B treatments (Figure 3). During the incubation, diurnal *Fv/Fm* did not change significantly

The diurnal changes of *Fv/Fm* were similar for three species in the PAR and UV‐B treatments (Figure 3). During the incubation, diurnal *Fv/Fm* did not change significantly

3.1.2. Diurnal Changes of Algal *Fv/Fm*

3.1.2. Diurnal Changes of Algal *Fv/Fm*

after 4 h of PAR treatment (*p* > 0.05), irrespective of species. In comparison, algal diurnal *Fv/Fm* decreased after 4 h of UV‐B radiation (*p* < 0.05), which could then increase after the withdrawal of UV‐B radiation on each day. However, the decline degree and recovery of

For three species, their *Fv/Fm* decreased to 15.2–40.6% of the initial values after UV‐B radiation on Day 2 and they recovered to 75.2–83.3% of the initial values within 20 h, when the decline was lower for toxic *M. aeruginosa* (*p* < 0.05). On Days 6 and 8, the decline of algal *Fv/Fm* worsened after UV‐B radiation and algal *Fv/Fm* could all recover to the initial values within 20 h. Meanwhile, the recovery rate increased with the development of incu‐

*Fv/Fm* was dependent on the incubation time and species.

and the recovery rate of its *Fv/Fm* was lower.

**Figure 3.** Diurnal changes of *Fv/Fm* of three species in the PAR and UV‐B treatments under normal growth conditions on Day 2, Day 6, and Day 8. **Figure 3.** Diurnal changes of *Fv/F<sup>m</sup>* of three species in the PAR and UV-B treatments under normal growth conditions on Day 2, Day 6, and Day 8.

#### *3.2. K+ Contents in the Algal Cultures 3.2. K<sup>+</sup> Contents in the Algal Cultures*

The release rates of K+ by three species were all less than 5% before Day 6 in the PAR treatment (Figure 4), which could indicate the integrity of cells. Moreover, algal release The release rates of K<sup>+</sup> by three species were all less than 5% before Day 6 in the PAR treatment (Figure 4), which could indicate the integrity of cells. Moreover, algal release rates of K<sup>+</sup> did not differ significantly between PAR and UV-B treatments during this period (*p* > 0.05), when the death and propagation of algal cells might be in a state of balance. After reaching the exponential growth stage in the PAR treatment, algal metabolism was enhanced with higher cell density, leading to the gradual increase of K<sup>+</sup> in the cultures (9.38–10.22% on Day 12). In comparison, the cell rupture of three species and algal release rates of K<sup>+</sup> were significantly promoted in the UV-B treatment (*p* < 0.05), indicating the greater damage of UV-B radiation on algal cells during this period.

greater damage of UV‐B radiation on algal cells during this period.

rates of K+ did not differ significantly between PAR and UV‐B treatments during this pe‐ riod (*p* > 0.05), when the death and propagation of algal cells might be in a state of balance. After reaching the exponential growth stage in the PAR treatment, algal metabolism was enhanced with higher cell density, leading to the gradual increase of K+ in the cultures

**Figure 4. (a−c)** The release rates of K+ by three species in the PAR and UV‐B treatments in the mono‐ cultures. **(d)** A comparison of the initial and ultrasonic disrupted cells of three species. **Figure 4.** (**a**–**c**) The release rates of K<sup>+</sup> by three species in the PAR and UV-B treatments in the mono-cultures. (**d**) A comparison of the initial and ultrasonic disrupted cells of three species.

#### *3.3. EPS Determination of Algal Cells 3.3. EPS Determination of Algal Cells*

At the beginning of incubation, 3‐D EEM spectra of algal EPS were determined and EEM contours were depicted (Figure S3). Results showed that EEM contours of BEPS and SEPS were similar for *C. pyrenoidosa* and non‐toxic and toxic *M. aeruginosa*, indicating the similar metabolism patterns of three species. For BEPS, two peaks were presented near Ex/Em of 225/325 nm (peak T2) and 280/325 nm (peak T1), which belonged to the low‐ molecular aromatic protein and soluble microbial by‐product like protein (such as tyro‐ sine and tryptophan‐like substances), respectively [44]. In contrast, three peaks were pre‐ sented near Ex/Em of 280/325 nm (peak T1), 340/430 nm (peak C, humic‐acid like sub‐ stances), and 275/435 nm (peak A, fulvic‐acid like substances) in SEPS. At the beginning of incubation, 3-D EEM spectra of algal EPS were determined and EEM contours were depicted (Figure S3). Results showed that EEM contours of BEPS and SEPS were similar for *C. pyrenoidosa* and non-toxic and toxic *M. aeruginosa*, indicating the similar metabolism patterns of three species. For BEPS, two peaks were presented near Ex/Em of 225/325 nm (peak T2) and 280/325 nm (peak T1), which belonged to the low-molecular aromatic protein and soluble microbial by-product like protein (such as tyrosine and tryptophan-like substances), respectively [44]. In contrast, three peaks were presented near Ex/Em of 280/325 nm (peak T1), 340/430 nm (peak C, humic-acid like substances), and 275/435 nm (peak A, fulvic-acid like substances) in SEPS.

During the incubation, EPS contents increased for all three species in the PAR and UV‐B treatments, and SEPS and BEPS had distinct changing trends during the incubation (Figure 5). For two *M. aeruginosa* species in the PAR treatment, SEPS content gradually increased and then remained constant, but BEPS content both increased in the early stage and decreased with increasing cell density. Meanwhile, the production of BEPS and SEPS was stronger for toxic *M. aeruginosa* (*p* < 0.05) during the incubation. In comparison, algal production of BEPS and SEPS by *M. aeruginosa* species was enhanced (*p* < 0.05) before Day 8 in the UV‐B treatment. With the decline of cell densities, BEPS contents of two *M. aeru‐* During the incubation, EPS contents increased for all three species in the PAR and UV-B treatments, and SEPS and BEPS had distinct changing trends during the incubation (Figure 5). For two *M. aeruginosa* species in the PAR treatment, SEPS content gradually increased and then remained constant, but BEPS content both increased in the early stage and decreased with increasing cell density. Meanwhile, the production of BEPS and SEPS was stronger for toxic *M. aeruginosa* (*p* < 0.05) during the incubation. In comparison, algal production of BEPS and SEPS by *M. aeruginosa* species was enhanced (*p* < 0.05) before Day 8 in the UV-B treatment. With the decline of cell densities, BEPS contents of two *M. aeruginosa* species decreased and their SEPS contents increased greatly after Day 10. In contrast, EPS production by *C. pyrenoidosa* was weaker in the PAR treatment (*p* < 0.05) and UV-B radiation did not significantly promote BEPS production before Day 8.

UV‐B radiation did not significantly promote BEPS production before Day 8.

**Figure 5.** Contents of BEPS and SEPS produced by **(a)** *C. pyrenoidosa*, **(b)** non‐toxic *M. aeruginosa* and **(c)** toxic *M. aeruginosa* cells in the PAR and UV‐B treatments. **Figure 5.** Contents of BEPS and SEPS produced by (**a**) *C. pyrenoidosa*, (**b**) non-toxic *M. aeruginosa* and (**c**) toxic *M. aeruginosa* cells in the PAR and UV-B treatments.

#### *3.4. Antioxidant Responses of Algal Species under Normal Growth Conditions 3.4. Antioxidant Responses of Algal Species under Normal Growth Conditions*

3.4.1. ROS in Algal Cells and SOD Activity 3.4.1. ROS in Algal Cells and SOD Activity

The variation patterns of ROS in algal cells and algal SOD activity were similar for three species (Figure 6). In the PAR treatment, ROS and algal SOD activities were constant (*p* > 0.05) before Day 4 compared to the initial values, indicating that PAR treatment did not cause evident oxidative stresses on three species. However, ROS gradually increased after Day 6 in the PAR treatment, and algal SOD activity was elevated for all three species. Compared with PAR treatment, ROS of two *M. aeruginosa* species were comparable in the UV‐B treatment on Day 2 and they were higher (*p* < 0.05) after Day 6. In comparison, ROS of *C. pyrenoidosa* was higher in the UV‐B treatment on Day 2 and it was further promoted after Day 6, which was higher than that in the cells of two *M. aeruginosa* species. Irrespec‐ tive of species, algal SOD activities were significantly higher (*p* < 0.05) in the UV‐B treat‐ ment before Day 8, which could provide effective antioxidant protection. However, as the The variation patterns of ROS in algal cells and algal SOD activity were similar for three species (Figure 6). In the PAR treatment, ROS and algal SOD activities were constant (*p* > 0.05) before Day 4 compared to the initial values, indicating that PAR treatment did not cause evident oxidative stresses on three species. However, ROS gradually increased after Day 6 in the PAR treatment, and algal SOD activity was elevated for all three species. Compared with PAR treatment, ROS of two *M. aeruginosa* species were comparable in the UV-B treatment on Day 2 and they were higher (*p* < 0.05) after Day 6. In comparison, ROS of *C. pyrenoidosa* was higher in the UV-B treatment on Day 2 and it was further promoted after Day 6, which was higher than that in the cells of two *M. aeruginosa* species. Irrespective of species, algal SOD activities were significantly higher (*p* < 0.05) in the UV-B treatment before Day 8, which could provide effective antioxidant protection. However, as the incubation progressed, algal SOD activity decreased sharply and leveled off until the end of incubation.

of incubation.

**Figure 6.** ROS in the cells of three species (vertical bar) and algal SOD activity (line and scatter) during the incubation in the PAR and UV‐B treatments under normal growth conditions (the arrow indicates the initial value of ROS contents). **Figure 6.** ROS in the cells of three species (vertical bar) and algal SOD activity (line and scatter) during the incubation in the PAR and UV-B treatments under normal growth conditions (the arrow indicates the initial value of ROS contents).

incubation progressed, algal SOD activity decreased sharply and leveled off until the end

#### 3.4.2. Contents of Photosynthetic Pigments 3.4.2. Contents of Photosynthetic Pigments

For all three species, the whole‐cell absorption spectra indicated that they have Chl‐ a (two absorption peaks in the blue and red parts of the spectra at around 440 and 680 nm) and CAR with an absorption peak at around 495 nm (Figure S4). Moreover, non‐toxic and toxic *M. aeruginosa* had an extra absorption peak at around 620 nm, which was regarded to phycocyanin in cyanobacterial cells [45]. For all three species, the whole-cell absorption spectra indicated that they have Chl-a (two absorption peaks in the blue and red parts of the spectra at around 440 and 680 nm) and CAR with an absorption peak at around 495 nm (Figure S4). Moreover, non-toxic and toxic *M. aeruginosa* had an extra absorption peak at around 620 nm, which was regarded to phycocyanin in cyanobacterial cells [45].

Similar Chl‐a contents of algal single cells were observed in the PAR and UV‐B treat‐ ments on Day 1 (*p* > 0.05, Table 1). However, CAR of three species and PC of two *Micro‐ cystis* species were significantly promoted (*p* < 0.05) in the UV‐B treatment on Day 1. More‐ over, algal CAR/Chl‐a and PC/Chl‐a ratios in the UV‐B treatment were higher (*p* < 0.05) Similar Chl-a contents of algal single cells were observed in the PAR and UV-B treatments on Day 1 (*p* > 0.05, Table 1). However, CAR of three species and PC of two *Microcystis* species were significantly promoted (*p* < 0.05) in the UV-B treatment on Day 1. Moreover, algal CAR/Chl-a and PC/Chl-a ratios in the UV-B treatment were higher (*p* < 0.05) than those in the PAR treatment at this moment. As incubation progressed on Day 8, Chl-a in algal cells were lower (*p* < 0.05) in the UV-B treatment compared to those in the PAR treatment, which could indicate the damage to chlorophyll synthesis. Meanwhile, algal

CAR and PC contents in the UV-B treatment decreased greatly (*p* < 0.05) compared with Day 1, which were lower (*p* < 0.05) than those in the PAR treatment. Compared with PAR treatment, the CAR/Chl-a ratio of *C. pyrenoidosa* on Day 8 was lower in the UV-B treatment (*p* < 0.05), whereas CAR/Chl-a and PC/Chl-a ratios of two *M. aeruginosa* species were significantly higher on Day 8 in the UV-B treatment (*p* < 0.05).

**Table 1.** Contents of photosynthetic pigments of three species in the PAR and UV-B treatments on Day 1 and Day 8 under normal growth conditions).


\* Bold values with \* indicated significant higher contents in the UV-B treatment compared with PAR treatment at *p* < 0.05, while those bold and underlined values with \* indicated significant lower contents in the UV-B treatment compared with PAR treatment at *p* < 0.05.
