Effect of Metal Cations on Colloids-Microcystin-LR Interaction

Colloidal particles, mixture with continuous molecular weight distribution and multiple organic components, is widespread in lake and have significant impact on the retention, migration, transportation, and fate of contaminants in lake ecosystems. Here we extract sedimentary colloids from algal growth dominant area (AD) in Taihu Lake and further separated into four different particle size ranges by cross-flow ultra-filtration (CFUF). The interaction mechanism between colloids and Microcystin-LR (MC-LR) was investigated under different cation conditions by dialysis equilibrium experiment method. Adsorption kinetics research shows the adsorption of MC-LR by colloids follows second-order kinetics and can be simulated by Freundlich isotherms. The effects of different cations on colloids-MC-LR interaction shows the addition of Mg(II) decreased colloids-MC-LR interaction, while Cu(II) increased colloids-MC-LR binding. MC-LR also increased Cu(II) binding to colloids, while MC-LR decreased Mg(II) binding. Therefore, different effect of cations to colloids-MC-LR interaction was proposed.

During the outbreak of cyanobacterial blooms, cyanobacterial cells such as Microcystis aeruginosa, Fritillaria spp., Chlamydomonas nodosus, Phytoplankton and Candida spp. produce large amounts of microcystins. Due to the combined effects of eutrophication and global warming, the risk of algal toxin release in water bodies has increased in freshwater systems worldwide (Paerl and Otten 2013). Microcystins (MCs) are cyclic heptapeptide compounds with about 1000 Da molecular weight, which are the most frequent, productive and harmful algal toxins in cyanobacterial blooms. More than two hundred and fifty variants of MCs have been identified (Christophoridis et al. 2018). In the past decades, MCs have been frequently detected in food chain and freshwater around the world. Among the MCs, MC-LR being the most common microcystin in environment (Kim et al. 2021), and it poses serious harm to water safety and human health (Shi et al. 2015). Therefore, it is urgent to reveal the migration and transformation mechanism of MC-LR in environment.
MC-LR is mostly found in sediments, with the highest affinity for montmorillonite. pH and ion may affect the distribution behavior of MC-LR (Chen et al. 2006;Liu et al. 2019). In the mean time, hydrophobic properties of microcystins themselves also cause changes in distribution characteristics. Previous studies have shown that the highly hydrophobic Adda-side chains on MC-LR cause MC-LR to adsorb on polyethersulfone (PES) membranes. However, MC-LR can be easily desorbed from PES owing to lower activation energy of hydrophobic interactions Walker 2006, 2008). Affected by both polar and non-polar functional groups, the influence of sediments on MC-LR fate cannot be ignored (Chen et al. 2006;Rapala et al. 1994;Munoz et al. 2017). However, the colloidal fraction has been neglected in traditional environment studies, numerous studies have found colloidal particles have strong adsorption complexation on organic pollutants due to their huge 1 3 28 Page 2 of 8 surface energy and abundant organic functional groups (Xu et al. , 2018. Sediment colloids contain large amounts of low molecular weight and high molecular weight natural organic matter (NOM), for example substances, proteins, and other substances (Yan et al. 2013;Rong et al. 2022). The physical and chemical properties of NOM colloids with different molecular weights may also vary, affecting the dispersion/ aggregation of NOM colloids (Tamamura et al. 2013). On one hand, colloids provide organic pollutants with abundant surface reaction sites, which can enhance the interaction between colloids and pollutants, and promote the migration of organic pollutants (Grant et al. 2011); on the other hand, through the competition of ions, the surface binding sites of sediment colloid can inhibit the migration of organic pollutants (Pan et al. 2012). Among the research of lake environmental conditions, effect of cations on the behavior of organic pollutants attracted extensive attention . So far, there is no explicit conclusion on the roles of cations in colloids-MC-LR interaction behavior. Therefore, detailed studies are warranted to determine the role of catios in colloid-organic pollutants interaction mechanism.
In this study, the interaction mechanism between algalderived colloids (algal growth dominant area, AD) and MC-LR was investigated under different cation conditions by dialysis equilibrium experiment method. The results can provide theoretical basis for endogenous pollution deterrence and remediation in eutrophic lakes.

Materials and Methods
MC-LR (> 95%, Enzo Life Science) was used without further purification. Mg(NO 3 ) 2 and Cu(NO 3 ) 2 were were purchased from Aladdin Co. All the other reagents were analytical grade. The mobile phase (Merck Co., Germany) for LC-MS were gradient grade.
Weigh 50 g of sedimentary from algae-dominant area (AD) in beaker, and add ultra-pure water to adjust the sediment to water ratio to be 1:10. The suspend were shake under 25°C for 6 h at 200 r/min (Yasutaka et al. 2017). After shaking, the suspends centrifuged at 3000 r/min for 20 min were passed through 1 μm and 0.45 μm membranes. Then, the solution are cut into colloids (ADCs) with different molecular weights by cross-flow ultra-filtration (CFUF) (Si et al. 2019). Finally, the colloids with four molecular weight ranges obtained, including ADCs-A ~ D (A: 0.45 μm ~ 1 μm; B: 1000KDa ~ 0.45 μm; C: 100 KDa ~ 1000 KDa; D: 10 KDa ~ 100 KDa (Cheng et al. 2018). The colloidal solutions of different molecular weights were freeze-dried for the following study.
Dialysis equilibrium system is used to research the interaction between colloids and MC-LR. In experiment system, the dialysis membranes were place in 80 mL brown vials with one end tied tightly. MC-LR solution were added into brown vials outside the dialysis bag, 20 mL colloids (50 mg C/L) with different molecular weight were injected into the dialysis bag. As MC-LR molecules can diffuse through membrane, the final concentration of MC-LR was calculated according to the volume of solution. All the sets mentioned above were shaked for 48 h under 25°C to attain adsorption equilibrium. The concentrations of MC-LR were quantified by LC-MS.
To study the effects of cations on colloids-MC-LR interaction, Mg(II) and Cu(II) were selected as typical cations, and the cation solutions were added outside the dialysis bag. 20 mL colloids (50 mg C/L) with 10 KDa ~ 100 KDa (ADCs-D) molecular weight were added inside the bag. The concentrations of cations were quantified by atomic absorption spectrometer (AAS).
The interaction mechanism between colloids, MC-LR and cations were revealed by IR spectra. Spectrums including colloids、colloids/Cu, colloids/Mg, colloids /MC-LR, colloids/Cu/MC-LR and colloids/Mg/MC-LR. The concentrations of colloids, MC-LR, Mg(II) and Cu(II) were as bellow: 50 mgC/L, 50 ug/L, 0.1 mM and 0.01 mM, respectively. The solid powder sample were obtained by freeze drying. IR spectra were gathered with 32 scans in the range of 4000-400 cm −1 at 4 cm −1 resolution on Bruker Vertex 70 spectroscopy.

Results and Discussion
The adsorption behavior of MC-LR on colloids is research by adsorption kinetics models. Adsorption efficiency is an important parameter to characterize absorption kinetics. Models of first-order (Eqs. (1)) and second-order kinetic (Eqs. (2)) (Prasanna Kumar et al. 2006) were selected to fit the experimental data. To elucidate the influencing factors of adsorption process, we investigated the relationship between adsorption capacity and adsorption time.
Q t represent the adsorption amount at time t; Q e represent the saturated adsorption amount; K 1 /K 2 is pseudo-first/ second-order kinetic adsorption reaction rate constant.
The adsorption kinetics of MC-LR by colloids (ADCs-A, ADCs-B, ADCs-C, ADCs-D) was evaluated and calculated in Fig. 1. To verify the applicability of adsorption models, the kinetic parameters calculated according to models were Table 1. According from Table 1, the R 2 values obtained in pseudo-second-order kinetic model were all greater than 0.985, which imply the kinetic model can better fit the experimental data. Also, in kinetic model, the K 2 values of MC-LR on ADCs-A ~ D were 0.00247, 0.00209, 0.00191, 0.00179 h/(ug/g) respectively. In summary, the adsorption process of MC-LR on colloids followed the pseudo-second-order kinetic model, and the adsorption of MC-LR onto colloids may be through chemisorption valence forces at specific colloids sites (Prasanna Kumar et al. 2006;Zhao et al. 2011). The adsorption isotherm is a common method to study the adsorption properties between MC-LR and colloids (Oladoja et al. 2008). Freundlich (Eqs. 3) and Linear model (Eqs. 4) were select to fit the experimental data respectively (Naiya et al. 2009;Langmuir 1915).  Freundlich index (n), which represent the intensity of adsorption, indicate favorable adsorption when n values between 0 and 1 (Mittal et al. 2009).The n values in Table 2 represented a beneficial MC-LR adsorption on colloids (Naiya et al. 2009). Furthermore, the n of ADCs-D (0.176) is smaller than that of ADCs-C (0.228), ADCs-B (0.226), ADCs-A (0.315), what made ADCs-D (0.176) having a preferential sorption for MC-LR. Also comparing four different molecular weights colloids, the magnitude of K value indicates the strength of adsorption, and according to the data in the table, K ADCs-A (55.28) < K ADCs-B (151.9) < K ADCs-C (155.7) < K ADCs-D (214.1), which shows that the smaller the molecular weight colloids have stronger and larger adsorption. The Freundlich correlation coefficients R 2 for MC-LR and ADCs-A ~ D were 0.926, 0.951, 0.945 and 0.938, respectively, the result indicated Freundlich equations can well describe MC-LR adsorption isotherms (Fig. 2).
To further elucidate the effects of different cations on colloids-MC-LR interactions, ADCs-D (molecular weight from 10 to100 kDa) were selected for further study. With the addition of different metal ion, there are significant difference in interaction mode between colloids-MC-LR. With the addition of Mg(II), colloids-MC-LR binding were significantly inhibited, while the addition of Cu(II) greatly enhanced their interactions. The proportion between colloids-MC-LR binding constants (K NOC ) and binding constants with cations added (K' NOC ) were shown in Fig. 3b. For Cu 2+ , its value is greater than 1, and the value increases with the addition of Cu 2+ concentration, which indicates that high concentration of Cu 2+ can positively enhance the interaction between colloid and MC-LR; meanwhile, for Mg 2+ , its value are less than 1, especially high concentrations of Mg 2+ weaken the interaction between colloids and MC-LR. This opposite interaction between Cu 2+ and Mg 2+ deserves to be considered. On one hand, the positively charged cations can adsorb on the surface of colloids by coulomb forces, reducing their effective specific surface area. On the other hand, Mg 2+ can form a certain competition with MC-LR, robbing the binding sites of colloids and forming insoluble complex masses with CO 3 2− , HCO 3 − and OH − in colloids, which eventually leads to the presence of Mg 2+ inhibiting the interaction. As for Cu 2+ , Cu(II) can interact with colloids by catechol and/or ethylenediamine, the complexes change the surface properties of the colloids and improve the attraction of colloids to MC-LR. The more in-depth mechanism of the interaction needs to be investigated through a series of further studies.
To further investigate the interaction relationship among cations, colloids and MC-LR, the changes of cations in the dialysis equilibrium system were measured by AAS. From  Fig. 4, it can be seen that the presence of colloids promoted the interaction between Cu 2+ and MC-LR, on the contrary, colloids inhibited the complexation between Mg 2+ and MC-LR. With the addition of the cation, the value of cation binding (C) increased gradually. The products of binding cations and MC-LR gradually increased, due to the stability of colloids, there was no obvious flocculent precipitation throughout the experiment,. Therefore, the cation concentrations in this section are lower than the concentrations that  ). An interesting phenomenon is that the addition of MC-LR significantly change the behavior of metals in metal-colloids interaction system. In Fig. 5, the presence of MC-LR decreased Mg-colloids binding, but significantly enhanced Cu-colloids interaction. Previous research have shown the difference roles of cations in MC-LR-colloids interaction. Different binding mechanisms can be identified by molarity-based analysis. The binding change of MC-LR-colloids (△C b , the difference between MC-LR binding colloids with/ without cations) was caused by cation binding colloids (C).
When Cu(II) concentration is below 0.01 mM, the values of C/△C b were keep between 1 and 4, and then with the continuous addition of Cu(II), the values of C/△C b increased to more than 5. Obviously, from the view of stoichiometric point, The increase of MC-LR binding (△C b ) was always lower than that of Cu (II) binding (C). The increasing MC-LR binding probably due to the cationic bridging of Cu(II) to form colloids-Cu-MC-LR ternary complex. In other words, Cu(II) can be used as a bridge between colloids and MC-LR. Theoretically, the formation of colloids-Cu-MC-LR ternary complex could lead to C/△C b ratios about 1, it can be easily inferred the higher C/△C b ratios must involve other processes. Our analysis of metal behavior showed that MC-LR could also increase the binding of Cu(II) to colloids. Obviously, colloids-Cu-MC-LR ternary complex could not explain this phenomenon.  It can be speculated colloids-bound MC-LR can bind with Cu(II) to form colloids-MC-LR-Cu(II) ternary complex. The formation of colloids-Cu(II)-MC-LR and colloids-MC-LR-Cu(II) complexes leads to the synergetic increasing Cu(II) and MC-LR binding. It is difficult to distinguish the contribution of colloids-Cu(II)-MC-LR and colloids-MC-LR-Cu(II) in the ternary system. However, the proportion of C/△C b around 4, indicating that colloids-MC-LR-Cu(II) may have greater effect than colloids-Cu-MC-LR especially under high Cu(II) concentration.
With the addition of Mg(II), the binding amount of MC-LR decreased, therefore the values of C/△C b were negative. When Mg(II) concentrations below 1 mM, values of C/△C b were always around -5, indicating Mg(II) was responsible for the reducing bounded MC-LR molecule. Take molecular weight, size and probable reaction sites of MC-LR and Mg(II) for consider, Mg(II) is impossible to replace MC-LR at exact 1:1 value. Overall, the interaction between colloids-Mg(II) and colloids-MC-LR is reduced in ternary system. As mentioned above, there is a certain competitive relationship between Mg 2+ and MC-LR, the colloid-Mg complexation product would wrap its own binding site, so that it could not carry out the exchange with cations through the hydrogen bonding by oxygen-containing functional groups, which would also hinder the binding effect of MC-LR. With the increase of Mg(II) concentration, values of C/△C b decreased to − 10. The result did not necessarily imply that one MC-LR molecule was replaced by ten Mg(II) ion, Mg(II) ions may interact with sites other than MC-LR in colloids. In other word, the increase of C may be related to △C b especially at high Mg(II) concentrations. According to previous research, the addition of metal ions probably change the conformation of colloids, resulting in the inability to portion binding sites.
The difference between Cu(II)/Mg(II) and MC-LRcolloids shows that these two cations may bind with different sites in colloids, therefore they have their own binding mechanism in altering MC-LR-colloids interaction. In our study, FTIR spectra was used to clarify the different binding mechanism for cations in the cation-colloids-MC-LR and colloids-cation-MC-LR system. Several typical absorption peaks could be found in Fig. 6, for example, 1300-1250 cm −1 region were attributed to C-O stretch and phenolic C-OH stretch, the peak in the 1650-1600 cm −1 region was assigned to the C=O stretch and C=C stretch (Trivedi and Vasudevan 2007).
Interestingly, although MC-LR itself has a strong carboxyl group, the signal of carboxyl group greatly inhibited   (Kang and Xing 2007).
In view of the above analysis, MC-LR can interact with colloids through cation exchange or hydrogen bonding with carboxyl groups. Due to the synergistic effect of colloidal-bound MC-LR and colloidal-bound Cu(II), Cu (II) can bind to the catechol and/or ethylenediamine components in colloids with very high affinity constants, which may change the surface characteristics of the colloids and increase the attractiveness of colloids to MC-LR, the addition of Cu(II) promotes the formation of colloid-MC-LR-Cu and colloid-Cu-MC-LR compounds. However, Mg(II) may bind to carboxyl through cation exchange, resulting in remarkable competition with MC-LR or occupate the binding sites of MC-LR.
Effects of cations on colloids-MC-LR interaction were studied, specifically, Mg(II) decrease the binding between colloids and MC-LR, while Cu(II) increase their binding. The addition of cation in colloids-MC-LR-cation and colloids-cation showed that MC-LR promote colloids-Cu interaction and inhibit colloids-Mg(II) interaction. Combined with FTIR results, we suggest that increased colloids binding of both MC-LR and Cu(II) in colloids-MC-LR-Cu and colloids-Cu-MC-LR. Mg(II) may compete the binding sites with MC-LR or shield binding sites of MC-LR in colloids. From the view of dissolved concentration, the risks of MC-LR to aquatic organisms in colloids-MC-LR-Cu(II) could be reduced, while the bioavailability of MC-LR may be improved in colloids-MC-LR-Mg(II) systems.
Author Contributions XH, HH and SL contributed to the conception and design of the study. XH and HH wrote the draft of the manuscript. XH, FZ, LY, HHu and SL analyzed the data. All author contributed to the manuscript revision and approved the submitted version.
Funding This work was supported by the National Natural Science Foundation of China (Grant No. 42007332 and 51979137) and Natural Science Foundation of Jiangsu Province (20KJB610001).

Data Availability
The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Declarations
Competing interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Ethical Approval and Consent to Participate Not applicable.