Elsevier

Water Research

Volume 47, Issue 9, 1 June 2013, Pages 3211-3219
Water Research

Oxidation by-products formation of microcystin-LR exposed to UV/H2O2: Toward the generative mechanism and biological toxicity

https://doi.org/10.1016/j.watres.2013.03.037Get rights and content

Highlights

  • Exposed to UV/H2O2, MCLR was quickly transformed but formed several MCLR-OBPs.

  • Adda aromatic ring/conjugated diene and Mdha Cdouble bondC bond were the major target sites.

  • Oxidation mechanism involved substitution, addition and dehydration reactions.

  • Most MCLR-OBPs still possessed certain biological toxicity on PP1 and PP2A.

  • MCLR-OBPs pollution also deserved further attention even though MCLR was destroyed.

Abstract

The presence of microcystins (MCs) in water sources is of concern due to their direct threats to human health and potential to form oxidation by-products (OBPs) in finished water. To control the environmental risk of MCs related OBPs, we evaluated their generative mechanisms and biological toxicity by mass spectrometry technology and molecular toxicity experiment. Exposed to UV/H2O2, model toxin microcystin-LR (MCLR) in clean water was quickly transformed but successively generated seven types of MCLR-OBPs with the chemical formulas of C49H74N10O13, C49H76N10O14, C49H78N10O16, C49H76N10O15, C37H58N10O12, C33H54N10O12, and C34H54N10O12. Probable isomers for each MCLR-OBP type were then separated and identified, indicating the aromatic ring and conjugated diene in Adda and the Cdouble bondC bond in Mdha were the major target sites of oxidation. Though subsequent toxicology data showed the toxicity of MCLR-OBPs on protein phosphatases 1 and 2A decreased with the extending of treatment by and large, they still possessed considerable biological toxicity (especially for product d). Influenced by MCLR-OBP distribution, concentration and residual toxicity, the secondary pollution of MCLR-OBPs in drinking water also deserved further attention even though MCLR was totally destroyed.

Introduction

The increasing frequency and intensity of cyanobacterial blooms are a growing environmental and human health concern (de Figueiredo et al., 2004; Zamyadi et al., 2012). The most common toxins produced by cyanobacteria are microcystins (MCs), a class of hepatotoxic monocyclic heptapeptides (Merel et al., 2009; Zamyadi et al., 2012). Based on their methylation pattern and the two variable amino acids at positions 2 and 4 of the structures, more than 80 structural analogs have been identified (Diehnelt et al., 2005; Kubwabo et al., 2005). Among these variants, microcystin-LR (MCLR) is found to be one of the most abundant and toxic. MCs have been the cause of several poisonings of livestock and wildlife around the world, and they also posed a health hazard for humans through the use of drinking water (de Figueiredo et al., 2004; Adam and Tadeusz, 2007). When orally ingested MCs are actively absorbed to hepatic cells, they irreversibly inhibit protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), subsequently leading to disruption of cell structures, intrahepatic hemorrhage and death (Gulledge et al., 2002; Campos and Vasconcelos, 2010).

Since MCs are potent hepatotoxins, controls of their levels in drinking water became of great importance (Gilroy et al., 2000; Yan et al., 2006). A recommended guideline value of 1 μg/L for total MCLR has been established by World Health Organization (Antoniou et al., 2005). Previous studies have suggested various techniques for the control of MCs in drinking water, such as coagulation, flocculation, and filtration (Chow et al., 1999; Antoniou et al., 2005). These treatment processes were efficient in removing cyanobacteria cells and internal MCs, but not very effective for dissolved MCs. Nanofiltration and activated carbon adsorption were reported to be able to decrease dissolved MCs (Antoniou et al., 2005; Yan et al., 2006). However, these costly techniques only worked as transfer media and could not eliminate the adverse effects of MCs. Consequently, there was still urgent need for water treatment methods resulting in a complete decomposition of these toxins, to nontoxic end-products (de la Cruz et al., 2011). Being able to destroy the crucial structures of organic matter by oxidizing species, chemical oxidation has been proposed as an effective treatment method for dissolved MCs (Antoniou et al., 2005; de la Cruz et al., 2011). Traditional treatment processes mainly used ClO2, HClO, and KMnO4 to oxidize dissolved MCs. Recently, more research has focused on using advanced oxidation processes (AOPs, for instance UV/H2O2, Fenton regents, and photocatalysis) to eliminate these toxins in raw water (Song et al., 2006; Miao et al., 2010). AOPs involve the generation of nonselective •OH, •O, and HO2• species and have shown considerable promise for the remediation of MCs.

However, a major concern with oxidation treatment (whether traditional processes or AOPs) is the formation of oxidation byproducts (OBPs) that result in secondary pollution (Sohn et al., 2004; de la Cruz et al., 2011). Influenced by substrate structure and concentration, coexisting organic compounds, and water quality, the complete mineralization of MCs was often infeasible. Oxidation against MCs inevitably produced undesired MC-OBPs that might retain the original toxic groups and toxicity. Partial research gradually revealed that MC samples subject to oxidants also showed certain biological toxicity (Harada, 1999; Hoger et al., 2002). There was an ongoing debate on the relevance of MC-OBPs compared to hepatic diseases. To date, this issue has not been thoroughly investigated, and no concrete conclusion has been made. Pursuit of these issues is urged by the need to improve the separation and identification methods for MC-OBPs; to evaluate their generative mechanism; and to verify the toxicity of diverse MC-OBPs using different techniques.

The principal objective of this work was to provide an evaluation of the generative mechanism and biological toxicity of MC-OBPs involved in UV/H2O2 treatment. Compared with other treatment methods (including AOPs), UV/H2O2 had many advantages such as direct formation of •OH without phase transfer problems, simplicity of operation, no impurity substance formation (less Interference on MS analysis), and lower investment costs (Qiao et al., 2005; Song et al., 2009). The widespread and dangerous microcystin, MCLR, was selected as the target of oxidation treatment and its primary OBPs were identified by mass spectrometry (MS), liquid chromatography/mass spectrometry (LC/MS) and tandem mass spectrometry (MS/MS). In addition to the generative mechanism studies, the biological toxicity of MCLR-OBPs on PP1 and PP2A was evaluated by molecular toxicity experiments. These results can be further used to regulate and improve oxidation techniques and thus have a great significance to control the environmental risk induced by MCs and their OBPs.

Section snippets

Reactants

Ascorbic acid, bovine serum albumin, dithiothreitol, diethyl-p-phenylen-diamin, ethylene glycol tetraacetic acid, glycerol, 2-mercaptoethanol, p-nitrophenyl disodium orthophorphate, sodium thiosulfate, tris(hydroxymethyl)aminomethane (Tris), H2O2, MnCl2, and MgCl2 were purchased from Sinopharm (Shanghai, China). MCLR and microsystin-YR (MCYR) were purchased from Sigma (Saint-Quentin Fallavier, France). PP1 (2500 U/mL) from rabbit skeletal muscle and PP2A (catalytic subunit, 100 U/mL) from human

Candidate MCLR-OBPs identification by MS

Though MCLR might be oxidized into diversified MCLR-OBPs, the primary candidates could be precisely probed by mass spectrography according to the changed molecular weights (Antoniou et al., 2008a, b). In the mass spectrum for native sample (Fig. 1A), two cluster m/z signals could be fixed around 995.5554 and 1045.5347, corresponding to MCLR and internal standard MCYR. For an oxidized sample terminated at 25 min, MCLR still exist in the mass spectrum but had much lower intensity than internal

Conclusion

This research investigated the generative mechanism as well as biological toxicity of typical MCLR-OBPs formed in UV/H2O2 treatment. MCLR have shown to be efficiently removed through UV/H2O2 oxidation, but to form several MCLR-OBPs. Partial MCLR-OBPs with stable m/z signals were tentatively identified as C49H74N10O13, C49H76N10O14, C49H78N10O16, C49H76N10O15, C37H58N10O12, C33H54N10O12, and C34H54N10O12. Based on the specific LC retention times and fragment ions, possible isomers for each

Acknowledgments

The authors acknowledge financial support from the National Natural Science Foundation of China (21207082) and Graduate Innovation Foundation of Shandong University (yyx10035).

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