Recognizing The Groundwater Related To Chronic Kidney Disease Of Unknown Etiology By Humic-like Organic Matter

INTRODUCTION 

Chronic kidney disease affects ~15% of the population worldwide and is one of the global public health problems1. Hypertension, diabetes, and chronic nephritis are usually the main causes of chronic kidney disease2. However, a chronic kidney disease that is unrelated to the above-mentioned causes, namely chronic kidney disease of unknown etiology (CKDu), has been reported in Central America, India, Sri Lanka, and other places. In Sri Lanka, the health of more than 400,000 people is threatened by CKDu, and the incidence of CKDu is as high as 15–23% in North Central Province4,5. Most patients suffering from CKDu have a low standard of living in agricultural communities in arid regions6. In recent years, many studies have shown that CKDu is associated with groundwater quality5,7, and there are not enough infrastructures to provide purified drinking water. According to a survey, in CKDu-prevalent areas, the occurrence of CKDu is dependent on the quality of drinking groundwater for local residents, and the distributions of clean groundwater sources and CKDu-related groundwater sources were uneven8. Therefore, the recognition of clean groundwater sources has become an urgent action taken for the residents in CKDu-prevalent areas in developing countries such as Sri Lanka5.

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The inorganic chemicals of groundwater have been identified to be related to the prevalence of CKDu in many studies9,10. For example, F− concentrations in groundwater samples from CKDuprevalent areas in Sri Lanka, mostly exceed the threshold level (0.6 mg L−1 ) defined for World Health Organization9; the hardness and the ratio of Ca2+ to Na+ in groundwater from CKDu-endemic areas are also higher than those in non-CKDu areas10,11. In addition, CKDu is associated with the concentrations of heavy metals with nephrotoxicity (e.g., Cd and Pb) and Si in groundwater12–14. A recent study has shown that organic compounds can interact with Ca2+ and SO4 2− through complexation, and the complex is harmful to human kidneys15. The formation of certain kidney diseases is related to dissolved organic carbon (DOC) in groundwaters16. Excessive humic substances can cause the death of human kidney cells based on a study in endemic areas of kidney disease of Balka17. However, few studies have investigated the difference in the key components of dissolved organic matter (DOM) between CKDu and non-CKDu water sources. The studies available showed that DOM in groundwater from CKDu-endemic areas was more refractory, higher aromatic, and lower bioavailable than that from non-CKDu areas18,19. However, it is still unknown how significant the sensitive components of DOM are in recognizing CKDu-related water sources.

Excitation-mission matrix spectroscopy (EEM) has the advantages of fast measurement, high sensitivity, and easy preprocessing. It has been widely used to characterize the fluorescent DOM (FDOM)20. EEM coupled with parallel factor analysis (EEMPARAFAC) can effectively reveal the optical characteristics of DOM, identify the fluorescent components and sources of DOM, and clarify the transformation process of DOM21,22. Furthermore, it also can be applied to explore the dynamic changes of DOM affected by natural and human factors23,24. EEM−PARAFAC has the potential feasibility to investigate the association between DOM and groundwater quality20.

Therefore, this study aimed to (1) determine the optical characteristics, composition, and sources of FDOM in CKDu-related and CKDu-unrelated groundwater from Sri Lanka by EEMPARAFAC technique, (2) verify the feasibility of using key fluorescent components to identify CKDu-related water sources by combining with inorganic sensitive chemicals, and (3) propose an early warning and screening tool for CKDu-related groundwater sources utilizing key fluorescent components. This study will provide an effective method for ensuring the safety of drinking water, which is helpful for the prevention and traceability of CKDu.

Fig. 1 The spectral indices of DOM. The upper point of the black box is the maximum and the lower is the minimum; the upper boundary of the black box is the 75th percentile and the lower is the 25th percentile, and the middle white square is the average value. The density diagram outside the box diagram represents the density probability. The significant difference of p < 0.01 and the significant difference of p < 0.05 have been marked with two asterisks and an asterisk in spectral indices between two different groups, respectively.

RESULTS AND DISCUSSION

DOC and EEMs of DOM in CKDu-related groundwater 

DOC concentrations (3.25 ± 0.73 mgC L−1 ) in CKDu groundwater were significantly lower than those in surface water (4.65 ± 0.89 mgC L−1 ), but significantly higher than those in non-CKDu groundwater (2.81 ± 0.76 mg L−1 ) (p < 0.05) (Supplementary Fig. 2). Although DOC concentrations reported by Cooray et al.25 were greater than the observed data in this study, they also found relatively higher DOC concentrations (6.4 mgC L−1 ) in CKDuprevalent regions than in non-CKDu-prevalent regions (3.7 mgC L−1 ). There may be two reasons for the difference in DOC concentrations between CKDu groundwater and non-CKDu groundwater. One is that CKDu groundwaters are likely to receive more surface water recharge11,26, which increases DOC concentration27,28. The other is that stronger microbial activity in non-CKDu groundwater (Fig. 1E, Supplementary Fig. 3) would degrade more organic substances into inorganic substances29, which is discussed below.

The a254 values in CKDu groundwater (median: 5.07 m−1 ) were higher than those in non-CKDu groundwater (median: 4.38 m−1 ) (Supplementary Fig. 2), indicating that DOM of CKDu groundwater consists of more unsaturated DOM. Generally, the concentrations of unsaturated DOM (a254) in natural groundwater were less than 1 m−1 30–32. In this study, the lowest a254 value in all groundwater samples was 1.15 m−1 (Supplementary Table 1), indicating that both CKDu and non-CKDu groundwater may be contaminated to some extent. Compared with groundwater, surface water had higher a254 values (median:10.48 m−1 ), which would result from anthropogenic activities. In addition, there is a significant positive correlation between DOC concentrations and a254 (Supplementary Fig. 4), indicating that the increase of DOC might be attributed to anthropogenic input.

The EEM features of DOM from non-CKDu groundwater were different from those from CKDu groundwater and surface water (Supplementary Fig. 5). The EEM of non-CKDu groundwater presented a relatively stronger T peak signal but weaker A peak signal (Supplementary Table 2) relative to CKDu groundwater, which indicated that non-CKDu groundwater contained more bioactive tryptophan-like substances33,34. Conversely, the peak A region in the EEMs of DOM from CKDu groundwater and surface water was extended into a plateau between peaks M and C, indicating the dominant role of humic-like organic matter in CKDu groundwater and surface water. Furthermore, the similar EEMs feature of DOM between CKDu groundwater and surface water indicated that groundwater-surface water interaction may have occurred and surface water was recharging groundwater.

Spectral indices of DOM in CKDu-related groundwater As shown in Fig. 1, in terms of DOM aromaticity (SUVA254), CKDu groundwater (mean: 0.78 ± 0.37) was similar to non-CKDu groundwater (mean: 0.78 ± 0.33) (p > 0.05). However, several CKDu groundwater samples exhibited high DOM aromaticity, which is related to their high a254 (Supplementary Fig. 4). The significant positive correlation between a254 and SUVA254 indicated that the aromatic DOM dominated over the unsaturated DOM. The spectral slope (S275–295) of CKDu groundwater (mean: 20.5 ± 6.7) was relatively lower than non-CKDu groundwater (mean: 23.7 ± 9.4), indicating that CKDu groundwater are enriched with high-molecular-weight organic matter. It can be attributed to the stronger microbial activities35 in non-CKDu groundwater with relatively higher biological index (BIX) values (Fig. 1E). Microorganisms might decompose high-molecularweight OM-like lignin into low-molecular-weight OM-like aromatic compounds36,37

The fluorescence index (FI) of DOM in all samples ranged from 1.26 to 1.88, indicating the weaker autochthonous feature and a mixture of terrestrial and microbial origins. However, the FI of DOM in non-CKDu groundwater (mean: 1.64 ± 0.12) was relatively greater than that in CKDu groundwater (mean: 1.52 ± 0.17), indicating that DOM of CKDu groundwater contains more allochthonous organic matter and less indigenous organic matter. In general, flowing groundwater has a stronger terrestrial signal than stagnant groundwater 38,39. Consequently, CKDu groundwater with lower FI might be of better-flowing condition. There were no differences between non-CKDu groundwater and surface water in terms of FI. As shown in Fig. 1E, the BIX values in CKDu groundwater (mean: 0.81 ± 0.09) and surface water (mean: 0.79 ± 0.08) were significantly lower than those in non-CKDu groundwater (mean: 0.86 ± 0.08) (p < 0.05), suggesting that DOM of nonCKDu groundwater performs a feature of relatively higher biological activity40,41, which was in accordance with the FI. There is a positive correlation between FI and BIX, which is consistent with observations by Dalmagro et al.42 and Qi et al.43. BIX was preferred in discriminating the biological activity of DOM between groundwater and surface water44.

The humification index (HIX) of DOM in CKDu groundwater (mean: 4.36 ± 2.16) was significantly higher than that in non-CKDu groundwater (mean: 3.15 ± 1.49) (p < 0.01), showing the greater humification degree of DOM in CKDu groundwater. This may be caused by surface water replenishing the CKDu groundwater or the stronger microbial activity of non-CKDu groundwater40,45. The increase in humification degree corresponds with the decrease in H/C44. These organic matter with low H/C mainly consisted of hydrophobic aromatic compounds or unsaturated compounds46. Therefore, DOM of CKDu groundwater would contain more hydrophobic aromatic or unsaturated compounds, which tend to be stable and persistent. Zheng et al.18 and Makehelwala et al.19 also showed that DOM in groundwater from CKDu-endemic areas contained more fulvic acid and other macromolecular hydrophobic humus.

The correlation between FI and HIX was not significant in this study (Supplementary Fig. 6). Therefore, source identification based on the HIX was not in accordant with that based on FI and BIX (Supplementary Fig. 3). The decoupling between the FI and HIX might be caused by the complex hydrogeological conditions and anthropogenic factors20. For instance, endogenous organic matter might be released from sediments and/or rocks due to water-rock interaction 47.

Although the spectral indices can help to quickly assess the composition of DOM, all indices are calculated using fixed wavelengths13, which are susceptible to the influence of multiple interacting fluorophores, leading to “distortion” when evaluating the composition of complex organic compounds from mixed sources48. Thus, the interpretation of fluorescence indicators should be cautious, and PARAFAC needs to be used for further analysis.

Sensitive FDOM components in CKDu-related groundwater Four fluorescent components were extracted and passed half-split validation (Supplementary Fig. 7, Supplementary Table 3). C1 was terrestrial humic-like components produced through biogeochemical processing of terrestrial particulate organic matter49, which resembled a combination of Coble A and C peaks50; C2 was similar to Coble M peak, microbial humic-like components with low molecular weight, and might dominate the fluorescence DOM of wastewater51,52; C3 was humic-like components with high molecular weight, which is derivatives of terrestrial organic material51,53; C4 was autochthonous protein-like components attributed to microbial activity54,55.

FDOM was predominated by C1 and C2 with an abundance of 68.7% and 62.9% in CKDu and non-CKDu groundwater, respectively (Fig. 2), indicating the dominative role of humic-like substances. The relative abundance of C1 (C1%, 35.1%) in CKDu groundwater was significantly higher than that in non-CKDu groundwater (28.0%, p < 0.01), while the C2% and C3% in CKDu and non-CKDu groundwater were similar (p > 0.05). Due to the similar geological and irrigation conditions in the study area, C2 is likely to be a by-product of organic matter produced by in-situ microbial activities or some humic substances that were fractionated by minerals easily56, and C3 may be related to the input of agricultural materials57. Furthermore, significantly higher C4% was detected in the DOM from non-CKDu groundwater (23.8%) than that of the DOM in CKDu groundwater (17.3%, p < 0.01), indicating the greater bioavailability of DOM from non-CKDu groundwater. The lower pH values of non-CKDu groundwater support this speculation (Supplementary Table 4), because the proton is produced during microbial degradation of organic matter58,59. C1% in surface water was significantly higher than that in non-CKDu groundwater (p < 0.01), but similar to that in CKDu groundwater (p > 0.05), indicating that the increase in C1% in CKDu groundwater may be related to the input of surface water.

The two-component principal component analysis showed that the loadings of C1% were located in the positive direction of PC1, which accounted for 43.9% of the variances, and the loadings of C4% were laid on the negative direction of PC1 (Fig. 3). Samples close to the positive direction of PC1 generally had high abundance of humic-like substances, and samples close to the negative axis of PC1 generally had high proportion of protein-like components related to microbial activities. Notably, around 50% CKDu groundwater samples and only around 22% of non-CKDu groundwater samples were located on the positive direction of PC1. It showed that CKDu groundwater and non-CKDu groundwater could be distinguished effectively by C1% and C4%. However, C4 seemed to be susceptible to interference from microbial background in aquifers resulting in deviations of screening20. Thus, C1 with better stability and persistence in the environment60, can be preferably used to recognize CKDu groundwater. Meanwhile, HIX, a254, and SUVA254 presented positive loadings to PC1, indicating their association with humic-like substances (C1). For PC2 (22.2%), C2% and C3% were placed in the positive direction. Around 53% of CKDu groundwater samples and 28% of non-CKDu groundwater samples were located in the negative direction of PC2. However, considering the similar C2% and C3% between CKDu groundwater and non-CKDu groundwater, the recognition of CKDu groundwater by C2% and C3% can be misleading.

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