3.1.1. Toxicity to M. aeruginosa PCC7028
Figure 1 shows the change in concentration of H
2O
2 for the studied water with and without 10
6 cells/mL of
M. aeruginosa. In the experiments, 20 mg·L
−1 of H
2O
2 was dosed into the reactor, with a light intensity of 2.3 W·m
−2 and the spectrum shown in
Figure S1 of the Supplementary Information. The figure shows that after 72 h, the amount of H
2O
2 was reduced by 75% and 24.4% for the cases of with and without
Microcystis addition, respectively. Clearly, the presence of cyanobacteria led to a faster decay of H
2O
2. Mikula et al. (2012) [
52] observed that light (140 µmol·m
−2 ·s
−1~30.4 W·m
−2) is a sine qua non-condition for H
2O
2 decomposition and for its toxicity to
M. aeruginosa. They also reported that in dark conditions, H
2O
2 decomposes very slowly over 72 h. Zepp et al. (1987) [
53] reported that algae might have a role in natural waters for the production of H
2O
2. They also suggested that H
2O
2 degradation follows a second order reaction in the dark. However, when exposed to sunlight, the algae may both produce and degrade H
2O
2 [
53]. In the current study, the oxidant degraded for more than 72 h, and led to the long-term low toxicity of H
2O
2 with regard to the algae. The present study shows that under the condition of light intensity = 2.3 W·m
−2 and H
2O
2 dose = 20 mg·L
−1, degradation of H
2O
2 was three times faster in the sample with
M. aeruginosa (2.3 × 10
6 cells/mL) than that in deionized water (
Figure 1). Huo et al. (2015) [
54] reported that H
2O
2 remained stable for up to 3.5 h when 60 mg·L
−1 H
2O
2 was incubated in the dark along with
Microcystis cells. In addition, the degradation of H
2O
2 is known to follow a pseudo-first order reaction when incubated with UV light [
55], and this supports the importance of light in H
2O
2 degradation.
Figure 2 shows the effect of copper sulfate and H
2O
2 on the growth of
M. aeruginosa. Within the exposure time (14 days), copper sulfate efficiently inhibited the growth of
Microcystis cells at doses greater than 1 mg·L
−1. Compared with the controlled sample, at 14 days the copper sulfate suppressed cell growth by 99%, 97% and 90%, respectively at the doses of 2, 1.5, and 1 mg·L
−1 for
M. aeruginosa with an initial concentration of 3 × 10
6 cells/mL (
Figure 2a). The cell concentrations at all the applied copper doses showed statistically significant differences (
p < 0.05) if compared with the controlled case. Tsai (2015) [
56] reported that 0.16 mg·copper·L
−1 (= 0.62 mg copper sulfate pentahydrate·L
−1 in this study) may cause a 90% reduction in
M. aeruginosa cells (initial concentration = 10
7 cells/mL) within eight days. McKnight et al. (1983) [
57] reported a general copper dose ranging from 0.025 to 1 mg·L
−1 that can be used to achieve control of algae blooms. With lower copper sulfate doses, although slight inhibition was observed if compared with controlled samples, cells still grew within 14 days of the experiments (
Figure 2a). Gibson (1972) [
58] observed that 0.25 mg·L
−1 copper only led to a growth depression followed by a recovery of nine days for an aged
Anabaena flos-aquae culture. However, the same dosage killed a freshly cultured
Anabaena flos-aquae. It has been reported that some cyanobacteria might develop a resistance to algaecides, and can therefore colonize the lake environment. For instance, Garcı́a-Villada et al. (2004) [
59] reported a copper resistant
M. aeruginosa mutants, with Cu
2+ resistance to concentrations greater than 5.8 µM (1.44 mg·L
−1 copper sulfate pentahydrate in this study). Erickson et al. (1994) [
60] reported that high values of pH affect both adsorption and absorption of the metal-based algaecides (toxic chemicals) by the cell, thus reducing their toxicity. In this study, with the addition of copper sulfate, the pH increased from 9.1 to 10.6 after eight days of incubation when 1 mg·L
−1 copper sulfate was dosed. However, the pH decreased again until pH ~ 8 at the 12th day of culture for 2 mg·L
−1 copper sulfate (
Figure S5a of the Supplementary Information), and the calculated alkalinity was 130 mg·L
−1 leading to a safe maximum copper sulfate dose of 1 mg·L
−1 [
61] for algae growth control.
Figure 2b demonstrates that exposure of
Microcystis may inhibit cell growth by 9%, 46%, 58%, and 95%, respectively at day 7 of exposure to 3, 5, 10, and 20 mg·L
−1 doses, with statistically significant differences (
p < 0.05) between the samples of all the H
2O
2 dosed samples and the controlled sample. After seven days,
Microcystis cells regrew and increased to 197%, 174%, 141%, and 125% of their initial concentrations, respectively for 3, 5, 10, and 20 mg·L
−1 of H
2O
2 doses. For the cases of lower H
2O
2 doses (1 and 2 mg·L
−1), although lower inhibitions were observed compared to those for the controlled samples, cells continued to grow. This kind of inhibition followed by regrowth of cyanobacteria during the application of H
2O
2 has been reported by Qian et al (2010) [
62], where
M. aeruginosa grew after 96 h of exposure to a dose of 100 µM (3.4 mg·L
−1) H
2O
2. In addition, Huo et al., (2015) [
54] reported a two-stage in
M. aeruginosa cell integrity change when exposed to H
2O
2 under light illumination, with cell rupturing following the Delayed Chick−Watson Model, where before the lag time all cells remained integrated and after the lag time the cells started to be ruptured. Although the experiments in that study were only conducted for 6 h, much less than in the current work, their results demonstrated that
Microcystis cells are not resistant to H
2O
2 exposure with 99% of the
Microcystis cells damaged within 3 h when exposed to 22.34 W·m
−2 (solar irradiance at the surface of the water). In the present study, attempts were made to obtain the rate constants for
Microcystis cells degradation using the commonly known degradation models, but they did not fit the degradation pattern. pH is a very important parameter to consider for photo-degradation because it causes differences for the chemical adsorption by the cell. In the present study, it was observed that with H
2O
2 the pH increased to reach 11.4 at day 8 under 5 mg·L
−1 H
2O
2. The increase in the pH is due to the depletion in CO
2 through the high photosynthesis by
Microcystis cells, but it may also be due to the production of hydroxyl anions. The sudden change in pH has been reported to be lethal to some aquatic animals, such as the catfish, which cannot tolerate a rapid pH change of 1 unit. As the growth period of the cells increased, the pH decreased to reach a value of 9.8 on the 12th day (
Figure S5b of the Supplementary Information).
3.1.2. Toxicity to Bacillus sp.
Figure 3 shows the impact of copper sulfate on
Bacillus sp. growth under different doses. It was observed that copper sulfate ≥1 mg·L
−1 was enough to kill
Bacillus sp. The mortality of
Bacillus sp. under exposure to copper sulfate followed a first-order reaction, with rate constants = 0.07 h
−1, 0.05 h
−1 and 0.04 h
−1, respectively, for 2, 1.5 and 1 mg·L
−1 of copper sulfate doses when incubated with only
Bacillus sp., and = 0.05 h
−1, 0.05 h
−1 and 0.04 h
−1, respectively, when the bacteria were incubated together with MC-LR, under the same copper sulfate doses. The results show that the mortality rates were not influenced by the presence of crude MC-LR in the water. However, higher copper doses led to larger bacteria mortality rates (
p = 0.001). For the conditions of copper sulfate doses <0.5 mg·L
−1, the mortality rate constants were all less than 10
−3 h
−1, suggesting that the effect on the studied bacterium is negligible. Sani et al. (2001) [
63] reported an IC
50 of 13.3 µM copper (3.3 mg copper sulfate pentahydrate·L
−1) to sulfate-reducing bacteria (SRB)
Desulfovibrio desulfuricans G20. When a higher dose was applied, 30 µM copper (7.4 mg copper sulfate pentahydrate·L
−1), 100% of the SRB were killed in 25 h and no bacteria were detected after 384 h of incubation. In addition, Zevenhuizen et al. (1979) [
64] observed a
Pseudomonas bacterium very tolerant to cupric ions Cu
2+ for up to 10
−3 M (250 mg·L
−1 copper sulfate pentahydrate). Our study showed 100% mortality for
Bacillus sp. at 1 mg copper sulfate pentahydrate·L
−1, and this is lower than the concentrations for the SRB and
Pseudomonas, and may suggest that different bacteria may have different resistances to copper.
The change in pH was monitored for the experiments at copper sulfate pentahydrate doses of 1 mg·L
−1, 1.5 mg·L
−1, and 2 mg·L
−1, and the results are shown in
Figure S6 of the Supplementary Information. The pHs were found to reduce from an initial 7.4 to 6.2 at the end of the experiments for all the studied cases. Yu-Sen et al. (2002) [
65] observed that at pH 9 cupric ions led to only a 10-fold reduction of
Legionella sp. in 24 h while, a million-fold decrease was observed for pH 7.0, with the precipitation of insoluble copper complexes observed at pH > 6.0, suggesting that pH is an important factor in determining the efficiency of copper ionization for killing
Legionella species in water. Water chemistry varies with many parameters, such as pH, and a decrease in copper toxicity has been reported with an increase in pH [
66]. In addition, numerous studies have been conducted to assess copper toxicity in water environments [
67], and it was found to be due to free cupric ion Cu
2+ in Sunda (1975) [
68]. The chemical speciation of copper may thus enable us to estimate the toxicity of the metal. To estimate the species of copper in the solution, a water chemistry software package, Visual MINTEQ V3.1 [
69], was used to predict the speciation.
Table 1 summarizes the model’s results for the copper species in the experimental solution at different pHs. It is clear that at pH = 7.4 the copper was initially in the form of 50.9% Cu
2+ and 39.7% CuOH
+, and at the end of the experiment (pH = 6.2) Cu
2+ was the predominant copper species (94.54%) in the solution. Yu-sen et al. (2002) [
65] reported that at pH 9 a copper concentration of 4 mg·L
−1 was not able to kill
Legionella pneumophila, even when the bacteria were exposed to this for 72 h. However, they observed that at pH 7, only 0.4 mg·L
−1 copper led to a 10
6-fold bacteria reduction within 1.5 h. In this study, the pH = 7.4 (initial value) decreased to pH = 6.2 at day 12 at the end of the experiments, with this decrease due to the water chemistry of copper, because OH
− anions are consumed by the metal and this leads to precipitation as Cu(OH)
2, with the pH decreasing as the copper sulfate concentration increases. The variations in pH (see
Figure S6) indicate that this is neither influenced by the presence of crude MC-LR (
p = 0.824), nor by the bacteria (
p = 0.066).
Figure 4 shows the effects of six different H
2O
2 doses on
Bacillus sp. viability. It was observed that H
2O
2 at doses ≥5 mg·L
−1 was lethal to the bacterium, with the mortality rate following the first order reaction and with rate constants of 0.03 h
−1, 0.1 h
−1 and 0.14 h
−1, respectively, for H
2O
2 doses of 5, 10 and 20 mg·L
−1. For lower H
2O
2 doses, negligible inhibition of the bacterium was observed, with the rate constants all less than 2 × 10
−3 h
−1. The effect of H
2O
2 on the
Bacillus sp. viability became lessened when crude MC-LR was added into the experimental water matrix. Lower H
2O
2 doses and the presence of crude MC-LR in the water may lead to slower mortality rates for the studied bacterium (
p < 0.05). During the experimental period, 288 h, the
Bacillus sp. population decreased by 90%, 75%, and 5% when exposed to 10, 5 and 3 mg·L
−1 H
2O
2, respectively.
Figure S7 of the Supplementary Information shows the concentration of residual OH radicals over 2.25 h (8100 s). The hydroxyl radical concentrations were very low, at 0.58 × 10
−19 M, 1.86 × 10
−19 M, and 0.27 × 10
−19 M, respectively, with MC-LR,
Bacillus sp., and for the control (without bacteria nor MC-LR), and the statistical analysis indicates no significant difference (
p = 0.069) among the three tested cases. The low concentration of OH radicals is reasonable since the irradiance used was very low (2.3 W·m
−2). Huo et al. (2015) [
54] reported 1.54 × 10
−15 M of OH radical concentration in their experimental system, when
M. aeruginosa PCC7820 was incubated with 10 mg·L
−1 H
2O
2 under 22.34 W·m
−2 (9.7 times higher than in this study) solar irradiance. In addition, under dark conditions, no hydroxyl radical production was detected. Thomas et al. (1994) [
70] showed that both doses of and exposure time to H
2O
2 were essential parameters for H
2O
2 to kill
Streptococcus mutans, serotype c (Strain GS-5), in which 6, 10, 0.3 and 7 × 10
−3 g·L
−1 H
2O
2 were required when the exposure time was 15 s, 2 min, 1 h and 24 h, respectively. The organic matters present in the water, including cells and associated metabolites in this studied system, may react with hydrogen peroxide [
71], decreasing the efficiency with which H
2O
2 oxidizes the cyanotoxins, which is similar to our observation that slower mortality rates were found for the cases with crude MC-LR addition. Additionally, with 5, 10 and 20 mg·L
−1 H
2O
2 doses, the pH generally increased from 6.8 to 7.6 (
Figure S6 of the Supplementary Information). Variations were statistically significant between crude MC-LR,
Bacillus sp. and MC-LR/
Bacillus sp. solutions (
p = 0.013), but there was no statistical difference for different H
2O
2 concentrations (
p = 0.271). Jung et al. (2009) [
72] observed that with 5% (50 g·L
−1) H
2O
2, the pH increased from 9.0 to 9.8 within 88 h, and the increase in pH is due to H
2O
2 decomposition, since there is consumption of H
+ or production of OH
− [
73], and the change in the pH value may affect the adsorption or the effect of H
2O
2 (via hydroxyl radicals) on the targeted cells.
Figure 5 summarizes the degradation of H
2O
2 under 2.3 W·m
−2 visible light illumination, at 25 °C during 12 days (288 h) of experiments. H
2O
2 was observed to degrade and reached a non-detectable limit within 50 h. The H
2O
2 degradation rate constants were 0.97 h
−1, 0.88 h
−1 and 0.22 h
−1, when 10 mg·L
−1 was incubated with MC-LR-
Bacillus sp.,
Bacillus sp., and MC-LR, respectively, according to a first order degradation reaction simulation. Schmidt et al. (2006) [
74] reported that in eutrophic to somewhat oligotrophic fresh water, the half-life of naturally occurring H
2O
2 is around 2–8 h, although this could be up to several days in natural water without microorganisms. H
2O
2 degrades quickly when it is inoculated with organic compound in natural waters [
75], and such degradation is enhanced mainly by bacteria, UV-light, pigments and humic substances.