Evidence-Based Framework to Manage Cyanobacteria and Cyanotoxins in Water and Sludge from Drinking Water Treatment Plants

Freshwater bodies and, consequently, drinking water treatment plants (DWTPs) sources are increasingly facing toxic cyanobacterial blooms. Even though conventional treatment processes including coagulation, flocculation, sedimentation, and filtration can control cyanobacteria and cell-bound cyanotoxins, these processes may encounter challenges such as inefficient removal of dissolved metabolites and cyanobacterial cell breakthrough. Furthermore, conventional treatment processes may lead to the accumulation of cyanobacteria cells and cyanotoxins in sludge. Pre-oxidation can enhance coagulation efficiency as it provides the first barrier against cyanobacteria and cyanotoxins and it decreases cell accumulation in DWTP sludge. This critical review aims to: (i) evaluate the state of the science of cyanobacteria and cyanotoxin management throughout DWTPs, as well as their associated sludge, and (ii) develop a decision framework to manage cyanobacteria and cyanotoxins in DWTPs and sludge. The review identified that lab-cultured-based pre-oxidation studies may not represent the real bloom pre-oxidation efficacy. Moreover, the application of a common exposure unit CT (residual concentration × contact time) provides a proper understanding of cyanobacteria pre-oxidation efficiency. Recently, reported challenges on cyanobacterial survival and growth in sludge alongside the cell lysis and cyanotoxin release raised health and technical concerns with regards to sludge storage and sludge supernatant recycling to the head of DWTPs. According to the review, oxidation has not been identified as a feasible option to handle cyanobacterial-laden sludge due to low cell and cyanotoxin removal efficacy. Based on the reviewed literature, a decision framework is proposed to manage cyanobacteria and cyanotoxins and their associated sludge in DWTPs.

Conventional treatment processes, including coagulation, flocculation, sedimentation, and filtration, are widely applied to remove cyanobacterial cells and cell-bound cyanotoxins [11][12][13][14]. However, conventional treatment processes may not be able to remove observed in the sludge of the DWTP with pre-ozonation ( Figure 1).
In many DWTPs, the supernatant of the stored sludge is recycled to the head of the plant as the spent filter backwash water [45,57,62]. A full-scale study on a low-risk DWTP (3400 cells/mL at the intake water) documented that cyanobacterial cell counts in intake water increased by up to 43% after recycling the supernatant. Surprisingly, 80% of the transferred cells from the supernatant water were viable [60]. A recent laboratory investigation on intake water that contained 1 × 10 6 cells/mL of cultured M. aeruginosa reported that although conventional treatment maintained the treated effluent parameters at below WHO and USEPA guidelines, recycling of the sludge supernatant resulted in an additional increase in cells and cyanotoxins levels in the influent by up to 7 × 10 4 cells/mL and 0.26 μg/L MC-LR, respectively [63].  [43].

Impact of Pre-Oxidation on Cyanobacteria Cell Counts
Cyanobacterial entry into DWTPs can be dampened by using pre-oxidation. Pre-oxidation may cause cell lysis, damage, and cyanotoxin release and degradation. Several studies were conducted to evaluate the pre-oxidation impact on cyanobacteria (e.g., cell viability and lysis) and cyanotoxins (release and degradation). A recent study tried to map the treatment barriers against cyanobacteria cells and cyanotoxins in drinking water facilities [10]. The results showed that the efficiency of the multi-barrier approach depends on the species present, metabolite concentration, and pre-oxidation dose [10]. Tables 1-4 summarize the literature on the impact of pre-oxidation on cyanobacteria (cultured-based and natural blooms) for four common oxidants (chlorine, ozone, potassium In many DWTPs, the supernatant of the stored sludge is recycled to the head of the plant as the spent filter backwash water [45,57,62]. A full-scale study on a low-risk DWTP (3400 cells/mL at the intake water) documented that cyanobacterial cell counts in intake water increased by up to 43% after recycling the supernatant. Surprisingly, 80% of the transferred cells from the supernatant water were viable [60]. A recent laboratory investigation on intake water that contained 1 × 10 6 cells/mL of cultured M. aeruginosa reported that although conventional treatment maintained the treated effluent parameters at below WHO and USEPA guidelines, recycling of the sludge supernatant resulted in an additional increase in cells and cyanotoxins levels in the influent by up to 7 × 10 4 cells/mL and 0.26 µg/L MC-LR, respectively [63].

Impact of Pre-Oxidation on Cyanobacteria Cell Counts
Cyanobacterial entry into DWTPs can be dampened by using pre-oxidation. Preoxidation may cause cell lysis, damage, and cyanotoxin release and degradation. Several studies were conducted to evaluate the pre-oxidation impact on cyanobacteria (e.g., cell viability and lysis) and cyanotoxins (release and degradation). A recent study tried to map the treatment barriers against cyanobacteria cells and cyanotoxins in drinking water facilities [10]. The results showed that the efficiency of the multi-barrier approach depends on the species present, metabolite concentration, and pre-oxidation dose [10]. Tables 1-4 summarize the literature on the impact of pre-oxidation on cyanobacteria (cultured-based and natural blooms) for four common oxidants (chlorine, ozone, potassium permanganate,  Tables 1-4 show that CT (residual concentration × contact time) is a main driver of cyanobacterial cell lysis, damage, cyanotoxin release, and degradation.
Although some studies have reported a reduction of more than 90% in taxonomic cell counts following pre-oxidation in the lab-cultured cells, Zamyadi, et al. [35] reported a cell reduction of 70% at high chlorine exposure (CT 296 mg min/L). Fan, et al. [37] showed a limited impact of chlorine exposure (CT 104 mg min/L) on the taxonomic cell counts of Microcystis aeruginosa (logarithmic phase). These observations might be related to the cyanobacteria stage of life and agglomeration. Furthermore, comparing the taxonomic cell count percentage in cultured-based and natural bloom studies demonstrates the lower impact of pre-oxidation during natural blooms. Figure 2 exhibits a lower impact of preozonation (2 mg/L) on cell number reduction in a natural bloom in comparison with lab-cultured cyanobacteria.
permanganate, and hydrogen peroxide). Tables 1-4 show that CT (residual concentration × contact time) is a main driver of cyanobacterial cell lysis, damage, cyanotoxin release, and degradation.
Although some studies have reported a reduction of more than 90% in taxonomic cell counts following pre-oxidation in the lab-cultured cells, Zamyadi, et al. [35] reported a cell reduction of 70% at high chlorine exposure (CT 296 mg min/L). Fan, et al. [37] showed a limited impact of chlorine exposure (CT 104 mg min/L) on the taxonomic cell counts of Microcystis aeruginosa (logarithmic phase). These observations might be related to the cyanobacteria stage of life and agglomeration. Furthermore, comparing the taxonomic cell count percentage in cultured-based and natural bloom studies demonstrates the lower impact of pre-oxidation during natural blooms. Figure 2 exhibits a lower impact of pre-ozonation (2 mg/L) on cell number reduction in a natural bloom in comparison with lab-cultured cyanobacteria.
Tables 1-4 and Figure 2 show that pre-oxidation, even at high CTs, may not be able to cause complete cell lysis. Consequently, it is important to clarify how far the pre-oxidation can cause viability loss and cyanotoxin release.  [64], (b) Natural bloom [65]. Tables 1-4 and Figure 2 show that pre-oxidation, even at high CTs, may not be able to cause complete cell lysis. Consequently, it is important to clarify how far the pre-oxidation can cause viability loss and cyanotoxin release.      Figure 3 is a reconstructed graph from the cell viability results following pre-chlorination based on the oxidant exposure (CT). Parameters such as background water quality (e.g., pH and dissolved organic carbon (DOC)) which have an impact on the oxidant demand are included in the CT concept. Therefore, a comparable level of damage should be found by comparing cell viability results using oxidant exposure (CT) for lab-cultured and natural blooms. Figure 3 demonstrates that at the same level of chlorine exposure, the natural bloom is more resistant to pre-chlorination as compared to lab-cultured cells. In other words, lab-cultured studies are not representative of natural bloom pre-chlorination. Fan, et al. [73] reported that the level of cell damage and toxin release depends on the colony size. Figure 3b shows the cultured-based studies fitted with the Chick-Watson equation. Although the results from different unicellular studies are aligned with each other, the colonial Microcystis chlorination shows a different cell damage rate. This could be related to the agglomeration of cyanobacteria cells and increasing the mucilage sheath in colonial cyanobacteria [73]. Despite using the CT calculation to compare the results, Figure 3c demonstrates different cell damage rates for each study of real bloom after chlorination, and the same level of chlorine exposure may not result in the same level of cell damage. Figure 3c shows that cyanobacterial bloom oxidation could be site-and bloom-specific, depending on the agglomeration, cyanobacteria (bloom) stage of life, and metabolic functions. Higher cell damage following pre-oxidation (especially with higher CTs) can lead to higher cyanotoxin release, which cannot be removed during conventional treatment. Soft chlorination showed cell damage by up to 45% and total microcystin (MC) degraded by up to 23%, while no cyanotoxin release was observed [76]. In addition, soft chlorination may cause lower disinfection by-products as a lower oxidant concentration is used in this approach.  [37,68,71,72,85] and natural cyanobacterial blooms [74,76,77] following pre-chlorination. [74] used Chl-a as a proxy for cell viability. (b) Cell viability experimental data and fitted model of unicellular [37,68,71,72,85] and colonial Microcystis [73] following pre-chlorination. (c) Cell viability experimental data and fitted model for three different cyanobacterial blooms following pre-chlorination. Figure 4 shows the impact of pre-ozonation on cyanobacteria cell damage for culturedbased and natural bloom studies. Figure 4a demonstrates lower cyanobacteria cell damage for a specific ozone exposure for natural blooms as compared to the lab-cultured cyanobacteria. The model fit results (Figure 4b) show a higher cell damage rate for the lab-cultured cyanobacteria in comparison to the natural bloom. As per soft oxidation, soft pre-ozonation was reported to cause up to 21% of cell damage and 14% of MC degradation, while no MC release was observed simultaneously [76]. Such an observation implies the effectiveness of soft pre-ozonation to damage the cells without cyanotoxin release. Figure 5 demonstrates that the viability loss of the lab-cultured studies harvested in the logarithmic phase is lower than those that harvested in the stationary phase. This observation implies the impact of the cyanobacteria stage of life on pre-oxidation efficiency. A comparison of the cell viability results of the lab-cultured with natural bloom studies following potassium permanganate pre-oxidation confirms the higher resistance of real cyanobacterial bloom cells ( Figure 5). In addition, the degradation rate constant of dissolved MCs was higher than that released by MCs for high potassium permanganate doses [36,81].

Potassium Permengeanate
tured-based and natural bloom studies. Figure 4a demonstrates lower cyanobacteria cell damage for a specific ozone exposure for natural blooms as compared to the lab-cultured cyanobacteria. The model fit results (Figure 4b) show a higher cell damage rate for the labcultured cyanobacteria in comparison to the natural bloom. As per soft oxidation, soft preozonation was reported to cause up to 21% of cell damage and 14% of MC degradation, while no MC release was observed simultaneously [76]. Such an observation implies the effectiveness of soft pre-ozonation to damage the cells without cyanotoxin release.  [37,64,68,78,79], and natural blooms [65,76] following pre-ozonation. (b) Cell viability experimental data and fitted model for cultured-based and natural bloom samples following pre-ozonation. Figure 5 demonstrates that the viability loss of the lab-cultured studies harvested in the logarithmic phase is lower than those that harvested in the stationary phase. This observation implies the impact of the cyanobacteria stage of life on pre-oxidation efficiency. A comparison of the cell viability results of the lab-cultured with natural bloom studies following potassium permanganate pre-oxidation confirms the higher resistance of real cyanobacterial bloom cells ( Figure 5). In addition, the degradation rate constant of dissolved MCs was higher than that released by MCs for high potassium permanganate doses [36,81].

Hydrogen Peroxide
Matthijs, et al. [86] reported that a concentration of 2 mg/L H2O2 was able to decrease cyanobacteria (natural bloom) by two logs within 3 days. In addition, cyanobacteria remained at a low abundance level for 7 weeks following H2O2 addition. Figure 6 demonstrates that natural blooms are more resistant to H2O2 than the lab-cultured cyanobacteria, as observed for other oxidants (Figures 2-5). Foo, et al. [87] reported that the impact of H2O2 on cyanobacteria is dependent on the residual concentration (C) and contact time (T). In addition, the authors concluded that toxic and non-toxic Microcystis aeruginosa are impacted by H2O2 with the same trend. Zhou, et al. [84] stated that a low dose of H2O2 (<5 mg/L) would have a low and recoverable impact on the lab-cultured Microcystis. On the other hand, the higher the H2O2 dose (> 8 mg/L), the higher necrosis, cell death, and consequent cyanotoxin release. A medium dosage of H2O2 with low to medium contact time can activate apoptosis-like programmed cell death (AL-PCD) [84]. The cellular energy required for AL-PCD is provided from the transcriptional, biochemical, and structural changes. Zhou, et al. [84] documented the maximum cell death with low MC production by AL-PCD activation. Zamyadi, et al. [17] studied the impact of H2O2 on blooms and labcultured cyanobacteria (Microcystis aeruginosa). The results highlighted a delayed impact of H2O2 on cyanobacteria cells after complete depletion of H2O2 during stagnation (up to one week) [17]. Chl-a and phycocyanin (PC) fluorescence were significantly declined by

Hydrogen Peroxide
Matthijs, et al. [86] reported that a concentration of 2 mg/L H 2 O 2 was able to decrease cyanobacteria (natural bloom) by two logs within 3 days. In addition, cyanobacteria remained at a low abundance level for 7 weeks following H 2 O 2 addition. Figure 6 demonstrates that natural blooms are more resistant to H 2 O 2 than the lab-cultured cyanobacteria, as observed for other oxidants (Figures 2-5). Foo, et al. [87] reported that the impact of H 2 O 2 on cyanobacteria is dependent on the residual concentration (C) and contact time (T). In addition, the authors concluded that toxic and non-toxic Microcystis aeruginosa are impacted by H 2 O 2 with the same trend. Zhou, et al. [84] stated that a low dose of H 2 O 2 (<5 mg/L) would have a low and recoverable impact on the lab-cultured Microcystis. On the other hand, the higher the H 2 O 2 dose (>8 mg/L), the higher necrosis, cell death, and consequent cyanotoxin release. A medium dosage of H 2 O 2 with low to medium contact time can activate apoptosis-like programmed cell death (AL-PCD) [84]. The cellular energy required for AL-PCD is provided from the transcriptional, biochemical, and structural changes. Zhou, et al. [84] documented the maximum cell death with low MC production by AL-PCD activation. Zamyadi, et al. [17] studied the impact of H 2 O 2 on blooms and labcultured cyanobacteria (Microcystis aeruginosa). The results highlighted a delayed impact of H 2 O 2 on cyanobacteria cells after complete depletion of H 2 O 2 during stagnation (up to one week) [17]. Chl-a and phycocyanin (PC) fluorescence were significantly declined by 93% and 74% in natural bloom and lab-cultured samples, respectively. Additionally, the lab-cultured results revealed delayed MC release during stagnation [17]. 93% and 74% in natural bloom and lab-cultured samples, respectively. Additionally, the lab-cultured results revealed delayed MC release during stagnation [17].
Besides the current oxidants, peracetic acid (PAA) has been used in wastewater treatment facilities as a disinfection alternative for chlorine [88]. Almuhtaram and Hofmann [89] studied the impact of PAA and PAA/UV on cyanobacteria and cyanotoxin removal. The results show that 10 mg/L of PAA with 60 min contact time was able to degrade MC-LR by 80% (3.46 M −1 s −1 lower reaction rate as compared to HOCl 1.2 × 10 2 M −1 s −1 ). In addition, the results elaborated that PAA alone can barely remove cyanobacteria, except at a high dose (10 mg/L) and with lower cyanobacterial cell counts (10 5 cells/mL).  [37,68,83,84] and natural bloom cells [76,86] after oxidation by hydrogen peroxide.

Considerations on the Impact of Pre-Oxidation on Downflow Processes
The impact of pre-oxidation on downflow processes should also be considered as it may influence the removal of cyanobacteria by coagulation, flocculation, and sedimentation. Previous studies have been reported that pre-oxidation has a positive impact on enhancing cyanobacterial removal through coagulation/flocculation and sedimentation [27,28,31,32,34,90]. Pre-oxidation can cause morphological deformation [82] and changes in the surface charge of the cells, leading to increased cell removal efficiency during coagulation/flocculation [37].
KMnO4 increases the binding potential to the coagulant by oxidizing organic matter (extracellular and released cell-bound) to lower molecular weight fractions, as well as forming colloids (by MnO2) to be adsorbed to the cyanobacterial cells and forming larger flocs [32,34,81]. Xie, et al. [27] reported that KMnO4 exposure (CT: 10 mg min/L, estimated) could increase cyanobacteria cell removal by 22% during coagulation/flocculation. In addition, pre-ozonation with CT: 4, 10, and 20 mg min/L (estimated) led to an increase in cyanobacteria cell removal during coagulation by 14%, 20%, and 24%, respectively [27]. Cyanobacteria cell removal during coagulation was improved in a full-scale DWTP equipped by pre-ozonation systems (CT: 2.52-3.78 mg min/L (estimated)) [43]. Pre-oxidation may cause metabolite release (organic matter and cell-bound cyanotoxins) following cyanobacterial cell damage. Besides the challenge to remove dissolved cyanotoxins, coagulation efficiency can be compromised by high algal organic matter release following preoxidation. Xie, et al. [27] showed that due to pre-ozonation with CT > 4 mg min/L (estimated), cyanobacteria cell viability was completely degraded, and consequently, organic matter concertation increased. Further, Barešová, et al. [91] demonstrated that pre-ozonation (CT < 40 mg min/L (estimated)) could interrupt the coagulation (Al/Fe-based) Besides the current oxidants, peracetic acid (PAA) has been used in wastewater treatment facilities as a disinfection alternative for chlorine [88]. Almuhtaram and Hofmann [89] studied the impact of PAA and PAA/UV on cyanobacteria and cyanotoxin removal. The results show that 10 mg/L of PAA with 60 min contact time was able to degrade MC-LR by 80% (3.46 M −1 s −1 lower reaction rate as compared to HOCl 1.2 × 10 2 M −1 s −1 ). In addition, the results elaborated that PAA alone can barely remove cyanobacteria, except at a high dose (10 mg/L) and with lower cyanobacterial cell counts (10 5 cells/mL).

Considerations on the Impact of Pre-Oxidation on Downflow Processes
The impact of pre-oxidation on downflow processes should also be considered as it may influence the removal of cyanobacteria by coagulation, flocculation, and sedimentation. Previous studies have been reported that pre-oxidation has a positive impact on enhancing cyanobacterial removal through coagulation/flocculation and sedimentation [27,28,31,32,34,90]. Pre-oxidation can cause morphological deformation [82] and changes in the surface charge of the cells, leading to increased cell removal efficiency during coagulation/flocculation [37].
KMnO 4 increases the binding potential to the coagulant by oxidizing organic matter (extracellular and released cell-bound) to lower molecular weight fractions, as well as forming colloids (by MnO 2 ) to be adsorbed to the cyanobacterial cells and forming larger flocs [32,34,81]. Xie, et al. [27] reported that KMnO 4 exposure (CT: 10 mg min/L, estimated) could increase cyanobacteria cell removal by 22% during coagulation/flocculation. In addition, pre-ozonation with CT: 4, 10, and 20 mg min/L (estimated) led to an increase in cyanobacteria cell removal during coagulation by 14%, 20%, and 24%, respectively [27]. Cyanobacteria cell removal during coagulation was improved in a full-scale DWTP equipped by pre-ozonation systems (CT: 2.52-3.78 mg min/L (estimated)) [43]. Pre-oxidation may cause metabolite release (organic matter and cell-bound cyanotoxins) following cyanobacterial cell damage. Besides the challenge to remove dissolved cyanotoxins, coagulation efficiency can be compromised by high algal organic matter release following pre-oxidation. Xie, et al. [27] showed that due to pre-ozonation with CT > 4 mg min/L (estimated), cyanobacteria cell viability was completely degraded, and consequently, organic matter concertation increased. Further, Barešová,et al. [91] demonstrated that preozonation (CT < 40 mg min/L (estimated)) could interrupt the coagulation (Al/Fe-based) efficiency of DOC removal (in comparison with higher CTs) due to the degradation of high molecular weight algal organic matter to low molecular weight compounds.
It is noteworthy to recall that H 2 O 2 can have a delayed impact on cyanobacteria and, potentially, cyanotoxin release after complete degradation of the oxidant [17]. This delayed cyanotoxin release should be considered in the downstream processes, as well as in sludge handling.
The oxidant exposure must be adjusted to maximize cell damage and cyanobacteria cell removal (directly or after coagulation) and minimize cyanotoxin release and cell accumulation in the sludge, simultaneously. Figure 7 summarizes the pre-oxidation (soft and normal) advantages/disadvantages of cyanobacteria and cyanotoxins during water treatment. In fact, soft pre-oxidation (low CT of Cl 2 and O 3 ) can (1) partially degrade cyanobacteria cells, (2) cause low cyanotoxin release, (3) improve coagulation efficiency to remove cells, and (4) cause low cell accumulation in the downflow processes. efficiency of DOC removal (in comparison with higher CTs) due to the degradation of high molecular weight algal organic matter to low molecular weight compounds. It is noteworthy to recall that H2O2 can have a delayed impact on cyanobacteria and, potentially, cyanotoxin release after complete degradation of the oxidant [17]. This delayed cyanotoxin release should be considered in the downstream processes, as well as in sludge handling.
The oxidant exposure must be adjusted to maximize cell damage and cyanobacteria cell removal (directly or after coagulation) and minimize cyanotoxin release and cell accumulation in the sludge, simultaneously. Figure 7 summarizes the pre-oxidation (soft and normal) advantages/disadvantages of cyanobacteria and cyanotoxins during water treatment. In fact, soft pre-oxidation (low CT of Cl2 and O3) can (1) partially degrade cyanobacteria cells, (2) cause low cyanotoxin release, (3) improve coagulation efficiency to remove cells, and (4) cause low cell accumulation in the downflow processes.

Sludge Storage, Oxidation, and Handling
Cyanobacteria and cyanotoxins (cell-bound) accumulate in the sludge of clarifiers throughout the flocculation/coagulation/sedimentation processes. This cyanobacterialaden sludge remains in the sludge holding tank before disposal. In addition, potential options to treat cyanobacteria-laden sludge need to be considered. Furthermore, safe (healthy, both operationally and environmentally) cyanobacteria-laden sludge handling approaches are required.
Besides cell survival potential during sludge storage, some studies have hypothesized that cyanobacteria can also grow in stored sludge [53][54][55]. Water Research Foundation (WRF) and Water Research Australia [53] documented that concentrations of DOC, MC-LR, and cylindrospermopsin in stored coagulated sludge contained M. aeruginosa and C. raciborskii exceeded the expected concentrations by 4-10-fold based on the cell quota (if all cell-bound metabolites are released) within 7-16 days, respectively. Dreyfus, et al. [55] studied the fate of stored sludge that contained cultured M. aeruginosa, D. circinale, and C. raciborskii within 18 days. The authors demonstrated that DOC, MC-LR, MC-LA, and CYN concentrations increased by up to 5-, 2.2-, 1.2-, and 2.5-fold during storage, respectively. Another investigation on stored sludge containing cultured M. aeruginosa and D. circinale reported that taxonomic cell counts increased by up to 4.2-fold in sludge stored in a lagoon within 7 days [54]. The authors also reported that the concentrations of cyanobacterial metabolites increased by up to 5 times in the sludge supernatant within 20 days. In the worst case, cyanobacteria could survive by up to 35 days in the stored sludge [54]. Despite the important findings of the previous studies on cell survival and metabolite release during sludge storage, cyanobacterial cell growth during sludge storage is yet to be explored in detail. In these studies, cell and metabolite increase during sludge storage might be due to the cell growth or to either the (1) underestimation of cell quota, (2) increase of metabolite production per cell during storage, or (3) additional cell settling from the supernatant to the sludge during the storage [53][54][55].
Our recent study on the cyanobacteria-laden sludge of a DWTP documented cell depletion, survival, and growth in different sludge samples [58]. Cell growth was observed in four out of eight sludge samples (different sampling dates) stored in the dark for 7-38 days. In the worst-case scenario, taxonomic cell counts increased from 2.7 × 10 6 to 5.3 × 10 6 cells/mL within 16 days (96% cell growth). Cell growth was also confirmed by increasing cyanobacterial biomarkers such as the "Pentose phosphate pathway" marker, which is responsible for the heterotrophic growth of cyanobacteria [93]. Table 5. Impact of sludge storage on cyanobacteria and cyanotoxins. STX: saxitoxin, PACl: colyaluminium chloride, CTSAC: Chitosan-aluminum chloride.  Microcystis flos aquae

Cyanobacteria-Laden Sludge Treatment
A summary of studies on the treatment of cyanobacteria-laden sludge is presented in Table 6. The available data demonstrated that sludge oxidation could not completely remove cyanobacteria cells and metabolites from the sludge [57,59]. Sludge is often stored after oxidation, while its supernatant can be recycled to the head of the DWTP. Thus, the impact of oxidation on sludge storage should be investigated. Recent findings showed no remarkable benefits in sludge oxidation followed by sludge storage as compared to only sludge storage [59]. The maximum additional taxonomic cell count decreased by a combination of oxidation (KMnO 4 or H 2 O 2 ) and storage was 32% as compared to storage only. However, oxidation/storage could cause a remarkable cell growth (by up to 145%) and toxic gene copy numbers of mcyD increase (by up to 13.0×) in some sludge samples [59]. This phenomenon can be attributed to gene expression regulation due to the presence of oxidative stresses [58,59,95,96]. Similarly, sludge oxidation could not completely remove cyanobacteria and cyanotoxins from the supernatant sludge [59]. Finally, the costs and by-product formation during the oxidation of organic-matter-rich sludge should be considered [58].

Sludge Handling Challenges
In general, the sludge supernatant is recycled to the head of the DWTP or is discharged into the source [13,56,97]. The solid phase either can be transferred to the WWTP or is ap-plied for landfilling [98][99][100]. Less environmentally friendly approaches such as untreated residual discharge into lakes or ponds can be also applied in some circumstances [101]. The re-use of DWTPs' residuals is growing [99,[102][103][104]. However, an investigation demonstrated that the half-life of MC analogs varies from 8 to 18 days in soil [105]. Since there is a risk of soil and groundwater contamination, landfill and field applications of cyanobacterialaden sludge should be avoided. Overall, cyanobacteria-laden sludge should be treated before disposal either in situ or via sending it to wastewater treatment plants.
Flocs may have a protective role during sludge storage for M. aeruginosa [40,[46][47][48]52]. In contrast, Li, et al. [52] documented that polyaluminium ferric chloride (PAFC) can stimulate the lysis of C. raciborskii and CYN release by up to 94% during sludge storage. This may occur in the sludge and lead to cyanotoxin release. However, all studies have been conducted in laboratory conditions and on cultured-based cyanobacteria. In fact, due to complex parameters such as the presence of various cyanobacterial cells in various forms (aggregated, multicellular), ages, and viabilities, the design of such experiments in full scale is complex.
Stresses such as oxidation and storage can shift cyanobacterial communities towards resistant genera (e.g., Microcystis and Aphanocapsa), which can produce MCs [56,58,59]. Thus, the survival probability of MC producer species can increase during sludge oxidation or storage. The fate of cyanotoxins in the sludge is complex due to the simultaneous occurrence of various phenomena such as cell survival, growth, lysis, cell-bound cyanotoxin release, and released cyanotoxin degradation [53][54][55][56]59,106,107]. Based on the increased risk of cell lysis and cyanotoxin release during sludge storage, some studies have suggested that cyanobacteria-laden sludge should be disposed of prior to 4 days to avoid metabolite release [47,51,108]. However, these studies only focused on metabolite release and not on cell survival/growth phenomena. Additionally, the possibility of sludge disposal can be a technical and financial challenge in large DWTPs.

Framework Basis
Since cyanobacterial cells and their associated metabolites, including cyanotoxins, as well as taste and odor agents such as geosmin and 2-Methylisoborneol (MIB), affect water and sludge quality, monitoring should be applied for the evaluation of the water treatment chain and sludge handling.
Microscopy taxonomic cell count techniques have been widely applied to evaluate the water and sludge in previous studies [12,18,43,44,56,77]. Previous cyanobacterial monitoring guidelines were prepared based on taxonomic cell counts and biovolumes [109][110][111]. A recent study suggested 0.3 mm 3 /L biovolumes as the vigilance level [109]. However, bias related to human error [112], the negative impact of Lugol's iodine on biovolumes [113], cell underestimation/overestimation due to the presence of aggregated cells [114], and the presence of debris and sediments, especially in the sludge samples [56], may affect the results. More importantly, the significant time required for taxonomic cell counts is a major barrier in using them for a real-time/practical approach.
In situ fluorometry using on-line probes is a compromising technique for measurement of PC based on relative fluorescence units (RFU) in water resources [18,[115][116][117][118][119]. However, the correlation between RFU and biovolume is complex and site-specific [119][120][121]. Previous investigations have reported that various RFUs ranging from 0.7 to 1.8 could correlate with a 0.3 mm 3 /L biovolume in different sources and bloom events [116,119]. Therefore, it is recommended to perform a correlation between on-line probe readings (RFU) and biovolumes for each water resource. It is noteworthy that the limits of detection and quantification of on-line probes should be considered [116,118].
MC concentration has been introduced in several guidelines such as those of the WHO (1.0 µg/L MC-LR) [122,123] and Health Canada (1.5 µg/L) [124]. Geosmin and MIB negatively affect water quality, raise complaints about taste and odor, and decrease the public's confidence about the treated water safety [125][126][127][128][129]. Thus, they should be monitored throughout the treatment chain and the recycled sludge supernatant. Using enzyme-linked immunoassay (ELISA) tests for cyanotoxins measurement has been accepted for cyanotoxin monitoring [44,[130][131][132]. The reported thresholds for geosmin and MIB are 1.3-4.0 ng/L and 6.3-15 ng/L, respectively [133][134][135]. Since the taste and odor agents are not harmful, but increase complaints and concerns about the water quality [126,127,129,134], olfactory detection can be considered for monitoring and detection [128,136].

Decision Framework
A decision framework to manage cyanobacteria and cyanotoxins in DWTPs is presented in Figure 8. The objective of this framework is to minimize cell breakthrough and accumulation throughout DWTPs and sludge. The three steps, (i) source water risk assessment, (ii) treatment breakthrough assessment and management, and (iii) sludge and supernatant risk assessment and management, should be taken for cyanobacteria and cyanotoxin control in DWTPs and sludge. This framework can help water utilities to understand appropriate approaches/strategies against cyanobacteria and cyanotoxins in DWTPs.   Overall, taxonomic cell counts, MCs, and taste and odor agents should be monitored in the (i) intake water, (ii) treatment chain, and (iii) sludge supernatant. These points are subjected to cyanobacteria and cyanotoxin accumulation, leading to a negative impact on water quality.
The optimization of conventional processes may include coagulant dose adjustment, applying aid-flocculants, and lowering the sedimentation and filtration rate during cell breakthrough [12,[137][138][139]. Secondary barriers such as pre-oxidation, PAC injection, and GAC, in case of metabolite breakthrough, should be applied [15,25,39,65,[140][141][142][143][144]. The impact of supernatant recycling or discharging on the source/intake water quality should be considered during toxic cyanobacterial blooms. Supernatant treatment may be required in the presence of cyanotoxins or taste and odor agents. MC concentration levels should be monitored in the sludge (solids) in case of landfilling or land application. In the case of elevated concentrations of MCs, sludge treatment is required.

Conclusions
Using the exposure unit (CT) is recommended for cyanobacteria and cyanotoxins oxidation studies, rather than using dose or contact time individually. Regardless of the oxidant type, lab-cultured studies cannot depict the complete picture of natural cyanobacterial bloom behavior during oxidation and may overestimate the oxidation efficiency. In addition, cyanobacterial bloom oxidation is site-and bloom-specific, which could be related to the level of agglomeration, cyanobacteria (bloom) stage of life, and metabolic functions. Soft pre-chlorination and pre-ozonation can compromise cell viability with no or limited cyanotoxin release. Overall, soft pre-oxidation may cause lower disinfection by-products compared to normal pre-oxidation. The cyanobacteria in stored sludge can not only survive, but also grow and release cyanotoxins, even in the dark. Although dissolved cyanotoxins can be degraded during sludge storage, the potential risk of growth and cyanotoxin release should be considered. In fact, the cell growth/depletion in stored sludge is complex and not easy to predict. Therefore, the worst-case scenario should be considered during sludge handling. Due to the low efficacy of sludge oxidation as compared to only stored sludge, as well as the occurrence of cell growth, and gene expression regulation during oxidation/storage, oxidation cannot be a reliable approach in sludge treatment and management. Management of cyanobacteria and cyanotoxins in sludge should be initiated with the minimization of cyanobacteria and cyanotoxin accumulation throughout DWTPs. To control the negative impacts of cyanobacterial accumulation in DWTPs, recycling sludge supernatant to the head of the DWTPs should be regulated during cyanobacterial seasons. Suitable treatment and disposal approaches should be set into guidance and regulations for sludge-containing cyanotoxins.