Sediment amino acids as indicators of anthropogenic activities and potential environmental risk in Erhai Lake, Southwest China

Science of the Total Environment 551–552 (2016) 217–227

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Sediment amino acids as indicators of anthropogenic activities and potential environmental risk in Erhai Lake, Southwest China Zhaokui Ni a,b,c,d, Shengrui Wang a,b,c,d,⁎, Mianmian Zhang a a

State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China State Environmental Protection Key Laboratory For Lake Pollution Control, Research Center of Lake Environment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China Yunnan Key Laboratory of Pollution Process and Management of Plateau Lake-Watershed, Kunming, 650000, China d Dongting Lake Ecological Observation and Research Station, Yueyang, 414000, China b c

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Relationship between sediment THAAs and anthropogenic activities and lake environment. • Potential sediment THAA release based on changes in environmental conditions. • Sediment THAA could effective reflects anthropogenic activities and aquatic environmental characteristics. • Sediment THAA is important to influence lake water quality.

a r t i c l e

i n f o

Article history: Received 27 October 2015 Received in revised form 28 December 2015 Accepted 1 February 2016 Available online xxxx Editor: F.M. Tack Keywords: Amino acids Component indication Risk Sediment

a b s t r a c t Total hydrolysable amino acids (THAAs) constitute the most important fraction of labile nitrogen. Anthropogenic activities directly influence various biogeochemical cycles and then accelerate lake ecosystem deterioration. This is the first study that has established the relationship between sediment THAAs and anthropogenic activities using dated sediment cores, and evaluated the possibility of THAAs release at the sediment interface based on changes in environmental conditions in Erhai Lake. The results showed that historical distribution and fractions of THAAs could be divided into three stages: a stable period before the 1970s, a clear increasing period from the 1970s to 1990s, and a gradually steady period that started after the 1990s. The chemical fraction, aromatic and sulfur amino acids (AAs) accounted for only ≤ 3% of THAAs. Basic AAs accounted for 5–17% of THAAs, and remained at a relatively stable level. However, acidic and neutral AAs, which accounted for 19–44% and 35–69% of THAAs, respectively, were the predominant factors causing THAAs to increase due to rapid agricultural intensification and intensification of contemporary sedimentation of phytoplankton or macrophytes since the 1970s. These trends were closely related to both anthropogenic activities and natural processes, which implied that sediment THAAs could act as an effective indicator that reflects anthropogenic activities and aquatic environmental characteristics. The current contributions of sediment THAAs on TN and TOC were b5% and 1.5%, respectively. However, the dramatic increase in THAAs in the sediment cores indicated that there was a huge potential

⁎ Corresponding author at: State Key Laboratory of Environmental Criteria and Risk Assessment, Chinese Research Academy of Environmental Sciences, Beijing 100012, China. E-mail address: [email protected] (S. Wang).

http://dx.doi.org/10.1016/j.scitotenv.2016.02.005 0048-9697/© 2016 Elsevier B.V. All rights reserved.

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source of labile nitrogen for the overlying water under certain environmental conditions. Correlation analysis suggested that the release of THAAs was negatively correlated with pH, whereas positively correlated with bacterial number and degree of OM mineralization, which particularly depend on the stability of HFOM. Therefore, the risk of sediment THAAs release might increase when the sediment environment continuously changes. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Dissolved organic nitrogen (DON) has been recognized as an important component of fixed nitrogen (N) in aquatic ecosystems and plays an important role in phytoplankton growth and eutrophication status (Lu et al., 2014; Berman and Bronk, 2003). Amino acids (AAs), the building blocks of proteins, are the predominant component of DON in both terrestrial and marine organisms (Fernandes et al., 2014). Total hydrolysable amino acids (THAAs) and its components serve essential functions in lake ecosystems because these could be released into the overlying water and be utilized as an important N and carbon source for phytoplankton and microorganism growth (Stepanauskas et al., 1999). Evidence has suggested that THAAs account for only a small proportion of the total DON pool, but these significantly contribute to the DON flux in aquatic ecosystems (Tada et al., 1998; Lu et al., 2014). Therefore, the role of THAAs in aquatic ecosystems has recently become an important research topic (Fernandes et al., 2014; Gupta and Kawahata, 2000). Previous studies have partly proven that THAAs properties, including content, forms, and their availability could be utilized as an effective tool that could identify the source, decomposition, and remineralization of organic matter (OM) and N (Pantoja and Lee, 2003; Cowie et al., 1992; Dauwe and Middelburg, 1998; Thomas and Eaton, 1996; Zhao et al., 2013a). Sediments are the major repository of THAAs and play an important role in biogeochemical cycles that occur in lake ecosystems (Bourgoin and Tremblay, 2010). On one hand, sediments are an important sink: THAAs are adsorbed on suspended particulates that eventually reach the lake floor and are continuously covered by successive sediment layers (Ni and Wang, 2015), indicating that the record of temporal variations in THAAs in sediments directly reflects the natural and anthropogenic activities over time. However, our understanding of the relationship between THAAs characteristics and environmental dynamics as well as anthropogenic activities in a specific aquatic ecosystem is limited. Therefore, detailed information on THAAs characteristics relative to environmental dynamics and anthropogenic activities is important in guiding anthropogenic production and life style, as well as in protecting the aquatic ecological environment. On the other hand, sediments also act as a potential source of THAAs that are released into the overlying water as sedimentary environmental conditions change. Hence, elucidating the effect of environmental parameters on the potential release of THAAs at the sediment interface is important in controlling water quality and preventing the occurrence of algae blooms. Erhai Lake is the second largest freshwater lake in Yunnan Province, which is located in the southwest region of China. This region plays a crucial role in local socioeconomic development as a source for irrigation, drinking water, tourism, and fisheries. Monitoring data have shown that the mean concentrations of total nitrogen (TN), dissolved TN, and DON in water in 2013 were approximately 0.57 ± 0.06, 0.41 ± 0.02, and 0.15 ± 0.03 mg·L− 1, respectively (Wang et al., 2015). The water quality has been graded to be of classes II to III, which is mainly based on the overlying water environment quality standard (GB3838-2002) of China (Wang et al., 2012a). However, the contents of TN, dissolved TN, DON, and OM in sediments were much higher than most eutrophicated lakes in China (Wang et al., 2015), reaching 4207 ± 1867, 89 ± 19, 27 ± 9 mg·kg− 1, and 5.2%, respectively. THAAs are the major component of labile DON in the sediments of Erhai Lake, and accounts for approximately 31% of DON (Zhang,

2015). These large amounts of THAAs represent a massive reservoir for N and C that is released to the overlying water under certain geochemical environments. Unfortunately, its environmental conditions (including pH, microorganisms and OM mineralization) have continuously changed over the past decades (Wang et al., 2015), thus increasing the risk for THAAs release from sediments. On the other hand, due to agricultural intensification in the upper area of the watershed since the 1970s, the ecosystems of Erhai Lake have severely deteriorated and its water quality has changed from mesotrophic to eutrophic (Zhao et al., 2013b). Therefore, the effect of the endogenous labile N (THAAs) on the water quality of Erhai Lake should be extensively evaluated. Accordingly, the main objectives of the present study were as follows: (1) to establish the relationship between anthropogenic activities and changes in THAAs using dated sediment cores, and (2) to evaluate the potential risk for surface sediment THAAs release based on changes in environmental conditions (e.g., pH, microorganisms and OM mineralization) of Erhai Lake. 2. Materials and methods 2.1. Study area Erhai Lake is located in Dali City, Yunnan Province, with a surface area of approximately 249 km2 and a watershed area of 2,565 km2. The lake has a mean depth of 10.5 m and a volume of 2.8 × 109 m3 (Fig. 1). As one of the typical agricultural regional lakes in the Yunnan-Guizhou Plateau of China, exogenous N is primarily derived from agriculture by river and ditch from the northern and western areas such as farmland, aquaculture, and livestock, particularly due to the extensive use of artificial N fertilizers in the upper area of the watershed. The rapid intensification of agricultural activities in the last few years, especially since the opening of agriculture jobs in the late 1970s, has resulted in a significant deterioration of the lake's ecosystem and water quality (Ni et al., 2011). Therefore, to select the Erhai Lake as a case study area was critical and necessary. 2.2. Sample collection 2.2.1. Surface sediments Ten surface sediments (0–5 cm) from Erhai Lake were collected using a core sampler (HL-CN, Xihuayi Technology, Beijing, China) in October 2012, which was a high-risk period for algal blooms and decline in water quality. Samples were selected from the northern (E1, E2, E3, and E4), central (E5, E6, and E7), and southern (E8, E9, and E10) areas of the lake, which was based on the topography of the lake bottom (Li et al., 1999). These areas were assumed to represent the entire lake based on the overall characteristics of different regions and to reflect the true situation of Erhai Lake. 2.2.2. Core sediments Three sediment cores (E1, E6, and E8) were collected from different areas of Erhai Lake in October 2012. Core E1 (northern site: 100°09′13″E, 25°54′00″N) is located at the lower edge of the alluvial fan of the Yongan River, a major inflow river. Water exchange is this particular region occurs at a faster rate relative to that of other areas. The area has no aquatic plant growth due to severe agricultural nonpoint source pollution and livestock manure. Core E6 (central site: 100°11′49″E, 25°47′59″N) is located at the deepest region (21.1 m)

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Fig. 1. Map of China and the Erhai Lake (inset) showing the study area.

of Erhai Lake, where water exchange is slow and no aquatic plant growth occurs. Core E8 (southern site: 100°13′23″E, 25°39′30″N) is located in the underwater platform of south Erhai. A large amount of submerged macrophytes in this area nearly disappeared, which was probably due to the increase in water level and pollution since 2003. Sampling was performed using a core sampler (HL-CN, Hengling Technology Ltd., Corp., China), with a length of 24 cm and an internal diameter of 2 cm. All samples were stored in a sealable and sterile plastic bag, and then transported to the laboratory in the dark at 4 °C for 24 h. The samples were freeze-dried and then passed through a 100-mesh sieve for homogeneity prior to analysis.

2.3. Sediment analyses 2.3.1. DON DON pertains to the difference in dissolved total nitrogen (DTN), − − ammonium (NH+ 4 ), ambient nitrate (NO3 ), and nitrite (NO2 ). The ana+ − lytical technique for DTN, NH4 and NO3 , was conducted by using the alkaline potassium sulfate digestion method, Nessler reagent colorimetric method, and HCl–H3NO3S method, respectively. A dry sample and CaCl2 solution (0.01 mol/L) were mixed in a colorimetric tube at a ratio of 10:1. The mixture was then oscillated for 10 min, and then centrifuged at 10,000 g for 10 min. The supernatant was passed through a 0.45-μm fiberglass membrane and used in the analyses.

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2.3.2. THAAs analysis Instrumentation: high-performance liquid chromatography (Agilent 1200, American Agilent Technologies Inc., Silicon Valley, CA, USA), Agilent eclipse equipped with a C18 chromatographic column (5 μm, 4.6 mm × 250 mm), Kjeldahl apparatus, and other laboratory equipment. THAAs standards (a total of 18 AA) solution configuration: Included aspartic acid (Asp), glutamate (Glu), serine (Ser), histidine (His), glycine (Gly), threonine (Thr), arginine (Arg), alanine (Ala), tyrosine (Tyr), valine (Val), methionine (Met), phenylalanine (Phe), isoleucine (Iso), leucine (Leu), proline (Pro), cysteine (Cys), tryptophan (Try), and lysine (Lys). The mixed standard solution was diluted, aliquoted to a series of standard solutions, and then refrigerated until laboratory analysis. Derivative reagent configuration: 13.4 g of o-phthalaldehyde was dissolved in 25 mL of methanol. Approximately 5 mL of the solution was mixed with 20 μL of 3–mercaptopropionic acid, and then brought to a total volume of 25 mL using boric acid buffer (pH 9.9 ± 0.5). The solutions were kept in the dark for at least 90 min and then stored in a refrigerator (4 °C) for less than 9 days. Mobile phase configuration included mobile A and mobile B. Mobile A: 1.4 g of anhydrous Na2HPO4 and 3.8 g of Na2B4O7·10H2O were dissolved in 1 L of deionized water, and concentrated HCl was added until the pH reached 8.2. Mobile B: acetonitrile, methanol, and deionized water were mixed at a ratio of 45:45:10. Pretreatment of sediment sample: THAAs were measured using the acid hydrolysis method. Briefly, 0.25 g of the freeze-dried sample was placed in an ampule (performed in triplicate), to which 6 mol/L of HCl added, and then sealed after filling with N for 2 min. The treated samples were hydrolyzed at 110 °C for 24 h, centrifuged at 8,000 g for 30 min, filtered, then a 3-mL aliquot of the supernatant was incubated in an oven at 50 °C until totally dried, to which 3 mL of 0.1 M borate buffer (pH 10.2) was added, filtered, sealed, and cryopreserved. Derivative reaction: Approximately 200 μL of the derivative reagent was added to 0.5 mL of AA standard solution or samples for 7 min, with an injection volume of 20 μL. Chromatographic conditions: velocity: 1.0 mL/min; column temperature: 40 °C; excitation wavelength: 340 nm; and emission wavelength: 450 nm. The gradient elution program is presented in Table 1. Due to limitations of the experimental method, Try, Cys, and Pro were not measured as these rapidly disintegrate during hydrolysis. Cys showed no absorption wavelength using the fluorescence detector. Pro could not be derived using o-phthalaldehyde. All correlation coefficients of standard curve of the rest individual THAAs were N 0.99, and the percent recovery, except for methionine, ranged from 80% to 95%. 2.3.3. OM component analysis Sediment OM could be divided into the following categories based on sediment volume-weight: light fraction organic matter (LFOM), and heavy fraction organic matter (HFOM) (Skjemstad et al., 1988; Yang et al., 2009). Active fraction organic matter (AFOM) was also measured in this study. OM content was measured by using the dichromate external heating method (Nanjing Institute of Soil, Chinese Academy of Science, 1978). The total organic carbon (TOC) content was measured by using a TOC analyzer (Shimadzu TOC-500, Japan). LFOM and HFOM contents:

Table 1 Check of Gradient table of liquid chromatography. Time Organic mobile (min) phase (%)

Buffer mobile phase (%)

Time Organic mobile (min) phase (%)

Buffer mobile phase (%)

0 12 20 22

90 62 70 65

31 35 40 45

63 53 45 45

10 28 30 35

37 47 55 55

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Approximately 5 g of each sample was placed in a 100-mL centrifuge tube, and mixed with 20 mL of 1.7 kg/L sodium iodide. The mixture was subjected to ultrasonication for 10 min, and then centrifuged at 10,000 g for 10 min. The supernatant was then passed through a 5mm copper mesh (repeated four or five times), and the material that collected in the copper mesh was used in the analysis. LFOM content was measured by using the dichromate external heating method. The HFOM was calculated as the difference between OM and LFOM. AFOM was measured by the oxidation method using 333 mol/L of potassium permanganate (Lefroy et al., 1993). 2.3.4. Microorganisms number analysis Microorganisms were divided into actinomycetes, bacteria, and fungi in this study. The number of viable actinomycetes, bacteria, and fungi was measured by using the plate dilution method (Zhang et al., 2015a). 2.3.5. Core chronology Sediment cores were dated by measuring the 210Pb and 137Cs in each sediment layer. The core sediment sites of the present study were the same as those described by Zhang et al. (1993) and Ni et al. (2011). Therefore, the sedimentation rates of the northern (E1), central (E6), and southern (E8) areas were 0.24, 0.16, and 0.20 cm·yr−1, respectively. 3. Results 3.1. Spatial distribution of THAAs The DON content of surface sediments from Erhai Lake ranged from 25.3 to 68.4 mg·kg−1, with an average of 38.4 mg·kg−1 (accounting for 45 ± 3.5% of DTN). THAAs concentrations ranged from 8.6 to 17.9 mmol·kg−1, with an average of 12.5 mmol·kg−1 (accounting for 28 ± 6% of DTN). THAAs distribution showed a decreasing pattern as follows: southern N northern N central, with averages values of 16.3 ± 1.3, 11.2 ± 0.6, and 8.3 ± 0.2, respectively. Similar trends for DON and DTN were also observed in the surface sediment (Fig. 2). THAAs content was positively corrected with DTN, DON, and OM, with correlation coefficients of 0.687 (n = 10, p b 0.05), 0.782 (n = 10, p b 0.01), and 0.738 (n = 10, p b 0.01), respectively. The contribution of THAAs on TN and TOC in surface sediments is presented in Fig. 3. The results showed that the sediment THAAs/TN and THAAs/TOC were b5% and 1.5%, respectively. The THAAs components in surface sediments were generally similar among various regions of Erhai Lake (Fig. 4). The Gly, Als, Ser, Asp, and Glu comprised the predominant THAAs fraction in Erhai Lake; each AA accounted for 10–18% of the THAAs, reaching a total of 60% of sediment THAAs. Lys, Arg, Thr, Met, and Tyr comprised the second group of predominant THAAs; each of these AAs accounted for 4–10% of the THAAs, and in total accounted for approximately 30% of sediment THAAs. Leu, Ile, Val, His, and Phe accounted for only ≤4% of the THAAs in the sediment. Sediment THAAs could be divided into acidic, basic, aromatic, sulfurcontaining, and neutral AAs based on its attributes (functional groups).

Fig. 5 presents various attributes of THAAs in the surface sediments of Erhai Lake. The attribute of sediment THAAs in the entire areas were in the order of neutral N acidic N basic AA. The aromatic and sulfur AAs were barely detected in the surface sediment. 3.2. Temporal distribution of THAAs The relationship between THAAs content and sample age (sediment cores were established based on sedimentation rate) of the three cores collected from different areas was analyzed to reconstruct the historical record of THAAs and elucidate the effect of natural and anthropogenic processes on different areas of Erhai Lake in the past decades (Fig. 6). In general, the temporal distribution of THAAs could be divided into three periods that were most closely related to the history of local natural and anthropogenic activities. Before the 1970s, the THAAs content was low and showed no obvious trend, with a mean content of 6.6 μmol·g− 1. These contents later showed a dramatic increase, reaching 14.2, 8.9, and 8.7 in the southern, northern, and central areas, respectively from the 1970s to the 1990s. After the 1990s, sediment THAAs content gradually remained stable throughout the entire area. The contribution of THAAs on TN in the cores is presented in Fig. 7. The ratio of THAAs/TN dramatically increased since the 1970s, and then increased once in the past 40 years. THAAs attributes also displayed variability in different periods. The aromatic (Phe, Tyr) and sulfur-containing (Met) AAs accounted for only ≤ 3% on average in the three sediment cores. The basic AAs, including Lys, Arg, and His, which represented the non-labile AA in the sediment, accounted for 5%–17% of THAAs in the three sediment cores. The acidic AAs, including Asp and Glu, which represented the labile AAs, were the second group of predominant THAAs in the sediment, accounting for 19–44% of the THAAs. The neutral AAs, which included the rest of the THAAs, were the predominant THAAs in the sediments, accounting for 35–69% of the THAAs. These findings thus indicate that THAAs fractions underwent a minimal to negligible increase before the 1970s. However, the contents of acidic, neutral, aromatic, and sulfur-containing AAs in the southern and northern regions began to increase since the 1970s. The basic AAs in the sediments throughout the entire area showed relatively small changes in content. 3.3. Relationship of OM to THAAs With the increase in the sediment depth, the OM content decreased (Fig. 8). The trend that was similar to that of THAAs in the sediment cores. Before the 1970s, the content of OM was approximately 31.4 g·kg−1 on average in all sediment cores, with the lowest value at 12.6 g·kg−1, thus showing a clear increasing trend after the 1970s, when the contents reached as high as 145.2, 125.8, and 106.3 g·kg− 1 in the northern, southern, and central areas, respectively. OM was divided into the categories of AFOM, LFOM, and HFOM in the present study. AFOM, which represented labile OM in sediments, accounted for 7–34% of the OM. LFOM accounted for 1–12% of the OM

Fig. 2. Spatial distributions of THAAs, DON and DTN in surface sediments of the Erhai Lake.

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Fig. 3. Spatial distributions of ratio of THAAs–N to TN and THAAs–C to total organic carbon (TOC) in surface sediment of Erhai Lake.

in the sediment cores. HFOM consists of humic acid (Yang et al., 2009), and was the predominant OM in the sediments cores (accounted for 58–91% of the OM). With the increase in sediment depth, the contents of the AFOM, LFOM and HFOM all showed increasing trend, which was similar to that OM and THAAs. The relationship between THAAs and OM in the three sediment cores was analyzed by using Pearson's correlation (Table 2). The neutral, acidic, basic, aromatic, and sulfur-containing AAs were significantly positively correlated with THAAs, with correlative coefficients of 0.97, 0.732, 0.812, 0.831, and 0.911 (p b 0.01, n = 34), respectively. In addition, THAAs were significantly positively correlated with OM (r = 0.608, p b 0.01, n = 34) and HFOM (r = 0.473, p b 0.01, n = 34) in all sediment cores.

3.4. Relationship between microorganisms and THAAs Fig. 9 presents the number of actinomycetes, fungi, and bacteria in the surface sediments. Actinomycetes were determined to be the predominant microorganisms in the sediment, reaching 67 × 106 colony forming units (CFU)·g−1, whereas fungi showed the lowest density, with an average of 40 × 103 CFU·g−1. Bacterial density was on average 13 × 106 CFU·g−1. A similar spatial distribution was observed between THAAs and bacteria, whereas that of actinomycetes and fungi varied. Table 3 presents the relationship between microorganisms and THAAs as well as OM in the surface sediment. The number of bacteria was negatively correlated with THAAs content, namely, basic and sulfur-containing AAs, with correlation coefficients of − 0.635, −0.652, and −0.667 (n = 10, p b 0.05), respectively. In addition, bacteria were significantly negatively correlated with OM and HFOM, with correlation coefficients of − 0.762 and −0.858 (n = 10, p b 0.05), respectively. Fungal density was positively correlated with neutral AAs (r = 0.63, n = 10, p b 0.05), and significantly positively correlated with aromatic (r = 0.852, p b 0.01) and sulfur-containing AAs (r = 0.744, p b 0.01).

4. Discussion 4.1. Relationship between anthropogenic activities and records of sediment THAAs The distribution, components, and source of sediments THAAs are largely influenced by biological and non-biological factors (Wang et al., 2012b). Non-biological factors include light, temperature, salt, nutrients, and hydrodynamic condition. Except for hydrological nonbiological factors, others indirectly affect biological activities, which could in turn influence the features of various THAAs. Biological factors mainly include aquatic plants, plankton, and microorganisms. In addition, anthropogenic activities also significantly influence the distribution of THAAs. Previous studies have shown that the AAs content of offshore and bay areas are relatively high because of frequent anthropogenic activities, whereas aquatic regions farther from the coast have relatively low AAs levels (Thomas and Eaton, 1996). Agricultural intensification, which rapidly developed in the upper area of Erhai Lake since the 1970s, caused the water quality, organisms (aquatic plant and phytoplankton community structure, distribution of microorganisms) and environmental conditions to significant change at almost the same time. This study observed a strong relationship between historical records of sediment THAAs and anthropogenic activities, as well as aquatic ecosystem dynamics in Erhai Lake (Fig. 10). Before the 1970s, the content of THAAs were relatively small and showed no obvious trend, and the THAAs components also had minimal differences in the three sediment cores, which suggested that the source and component characteristics of THAAs were controlled by natural processes. Monitoring data has shown that the watershed at a closed stage of natural economy and the lake had good water quality with lower nutrient concentrations during the period (Ni et al., 2011). However, the content of THAAs, acidic and neutral AAs, underwent a significant increase since the 1970s. This trend was likely due to the rapid population and agricultural intensification since China's opening and reform in 1978. Agricultural intensification has resulted in a dramatic

Fig. 4. Concentrations of individual sediment THAAs in the three regions of Erhai Lake.

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Fig. 5. Spatial distribution of attribute property of THAAs in three regions of Erhai Lake.

increase in artificial nitrogenous fertilizers usage in the upper area of Erhai Lake. The mean dosage of organic nitrogenous fertilizers increased nearly 10 times, whereas crop productivity only increased 3 times from 1970 to 1999 (Ni and Wang, 2015). This implied that a large amount of organic nitrogenous fertilizers was lost to the lake, thereby resulting in an increase in THAAs content in the sediment, particularly in the southern and northern areas due to the major rivers in the two parts of the lake. In addition, the increase in THAAs content was strongly correlated

with the increase in distribution and biomass of submerged macrophytes. The submerged macrophyte communities had rapidly spread throughout the lake due to the decrease in the water level, which mainly resulted from the operation of the Xi'erhe hydropower station in 1972 (Pan et al., 1999). No submerged macrophytes were detected at depths of N3 m, with coverage of about 20% before the 1970s. Thereafter, macrophyte growth further extended to the water depths of 6–10 m, with coverage reaching at least 60% from 1970s to 1990s (Ni et al., 2011). In particular, the distribution of submerged macrophytes mainly appeared in the southern and northern areas, whereas the central area showed no increase because this region contained relatively deep water. These changes also enhanced the accumulation of plantderived sediment THAAs in the southern and northern areas. Moreover, the increase in nutrient content led to an increase in population size of phytoplankton in the southern and northern areas, the number and densities reaching 5.6 × 106·L−1 and 4.65 mg·L−1 in 1990, respectively, which was nearly 5 times higher than that in the past 20 years. Phytoplankton death and sedimentation also enhanced the accumulation of THAAs in the sediment. After the 1990s, the contents of THAAs in the southern and northern areas gradually stabilized, which could be largely attributed to the largescale deterioration of submerged macrophyte communities. The coverage of submerged macrophytes decreased from 40% to 5% (700%

Fig. 6. Temporal distributions of TN, THAAs and fractions of THAAs in cores E1, E6 and E8 sediment of the Erhai Lake.

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Fig. 7. Temporal distributions of ratio of THAAs–N to TN in sediment cores of Erhai Lake.

decrease), and its biomass decreased from 3.9 to 0.7 km·m−1 (450% decrease), the population size of submerged macrophyte communities had fallen to historic lows. Severe degradation of submerged macrophytes has resulted in large amounts of OM deposited into the sediment. The decomposition processes of OM consumes massive amounts of dissolved oxygenin the local area, which enhanced the mineralization of organic N in sediments (Zhao et al., 2013a). Because THAAs are the labile component of DON, these were easily released to the overlying water, which caused the release of formerly accumulated sediment THAAs into the water and the content gradually maintained

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stability. Faced the risk of serious ecological degradation and accelerated eutrophication, the Chinese State Council and local government conducted a series of load reduction projects and have issued fiveyear plans since the 1990s (Wang et al., 2015). These schemes restricted the external input of N loading, which also partly inhibited the increase of THAAs content in the sediment. The content of THAAs at the top of the cores were stable or even less than those in deeper depths, but these were still higher than the baseline values before the 1970s. The massive reservoir of sediment THAAs could act as a potential source of N and C to the overlying water column in various geochemical environments. THAAs fractions also displayed variability during various periods. Neutral AAs were the predominant THAAs form in the sediment cores, which steadily increased in the southern and northern areas after the 1970s. This could mostly be attributed to contemporary sedimentation of submerged macrophytes and phytoplankton, as well as intensified exogenous N input such as agricultural and domestic sewage. Acidic AAs was the second predominant THAAs form in the sediment cores that served as a potential source that could be released into the overlying water under certain geochemical conditions (Zhang et al., 2015b). Unfortunately, the levels of acidic AAs dramatically increased in the three sediment cores since the 1970s. On one hand, this was strongly associated with the increase in exogenous N input and the sedimentation of submerged macrophytes and phytoplankton. On the other hand, this could be attributed to its own characteristics, i.e., rapid decomposition during the early mineralization process in the deeper sediments, which would reduce the content of acidic AAs (Burdige and Zheng, 1998; Wu and Fu, 2005). The content of basic AAs was relatively low and minimal changes were observed in the sediment cores, which could be attributable to its nonlabile chemical characteristics such as its rapid binding to clay minerals and humus as well as being fixed in the sediment. Aromatic AAs mainly exist in the cytoplasm, and its content is mainly influenced by the degradation of OM in the sediment (Dauwe and Middelburg, 1998). In this study, the content of aromatic AAs showed an increasing trend in the sediment core from the southern

Fig. 8. Temporal distributions of OM and fractions of OM in cores E1, E6 and E8 sediments of the Erhai Lake.

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Table 2 Pearson correlation coefficients for the relationship between THAAs and OM of sediment cores (n = 36).

THAAs Neutral AAs Alkaline AAs Acidic AAs Aromatic AAs Sulfur AAs OM AFOM LFOM HFOM a b

THAAs

Neutral AAs

Alkaline AAs

Acidic AAs

Aromatic AAs

Sulfur AAs

OM

AFOM

LFOM

HFOM

1 0.97a 0.812a 0.732a 0.831a 0.911a 0.608a 0.264 0.254 0.473a

1.000 0.763a 0.588a 0.898a 0.883a 0.485a 0.311 0.061 0.325b

1 0.419 0.742 a 0.831 a 0.565 a 0.291 0.193 0.484a

1 0.316 0.528 a 0.596a 0.525a 0.556a 0.556a

1 0.821a 0.354b −0.061 −0.075 0.235

1 0.621 a 0.282 0.288 0.48a

1 0.862a 0.644a 0.945a

1 0.756a 0.895a

1 0.635a

1

Correlation is significant at the 0.01 level (2-tailed). Correlation is significant at the 0.05 level (2-tailed).

area. This was consistent with the evolution of submerged macrophytes in the southern area over the past 40 years. The sulfur-containing AAs also showed an increasing trend in the southern area. This was strongly associated with the increase in the population size of sulfate-reducing bacteria and sulfur-oxidizing bacteria in the sediment that is influence by changes in the distribution of submerged macrophytes in the southern area (Wang et al., 2007). 4.2. Environmental risks of sediment THAAs 4.2.1. Contribution of THAAs on N and C levels Amino acids are important N and C sources. A previous study proved that the contribution of THAAs on N (AA–N) and C (AA–C) could be better understood based on the capacity to regenerate amino acid N in sediments (Fernandes et al., 2014). Compared to the contribution of AAs to N and C from other stream sediments (Suthhof et al., 2000; Ingalls et al., 2003; Yao et al., 2012; Zhao et., 2013a), the results of this study showed that the levels of TN and TOC in surface sediments from Erhai Lake were significantly larger than those of the other stream sediments, but the contribution of THAAs on TN and TOC were generally less than those of selected stream sediments. This phenomenon suggested that the THAAs minimally contributed to the observed N and C levels. However, OM and TN were significantly positively correlated with THAAs in the surface sediment, which reflected that sediment THAAs were an important source of labile N in situations of N deficiency in overlying water. In addition, the steady increase in THAA–N/TN content in the sediment cores indicated that the contribution of THAAs to TN annually increased (Fig. 7). Therefore, the release of sediment THAAs should be further investigated in future studies. 4.2.2. Potential release risk of THAAs 4.2.2.1. pH effects. In general, the ratio of acidic/neutral AAs is 0.167 in a neutral environment, and 0.333 in an alkaline environment. The acidic/ neutral AA ratio in contemporary sediments of Erhai Lake ranged from 0.37 to 0.46, which was indicative of an alkaline environment. This was consistent with the real situation (sediment pH: 7.9–8.8) of Erhai Lake (Zhang et al., 2014). Thus, THAAs composition can be used as an effective tool in the assessment of the sedimentary environment. Acidic AAs could be fully ionized to form anions in an alkaline environment,

whereas these reactions could not be conducted in an acidic environment (Ma et al., 1999). In Erhai Lake sediment, the alkaline environment would be beneficial for the binding of acidic AAs and clay as well as humus, and then enhance the stability of sediment acidic AAs. Finally, the acidic AAs are easily enriched in the sediment rather than released to overlying waters in its free state. However, due to the sustained increase in red soil loss from the southern mountainous region of Erhai Lake, the pH of sediment showed a clear decreasing trend (Zhao et al., 2013b). These findings indicate that sediment acidic AAs probably sustained the activation and release to overlying unless the input of red soil from the upper area was controlled. The level of acidic AAs in the southern area was higher than those of the other areas (Fig. 3), which indicated that the southern area has a higher release rate for THAAs from the sediments and a higher risk for algal blooms in water. 4.2.2.2. The effect of microorganism activity. Sediment microorganisms play an important role in the breakdown OM and in the remineralization of nutrients, thus strongly affecting nutrient cycles and energy flux in aquatic ecosystems (Muylaert et al., 2002; Eiler and Bertilsson, 2004; Fernandes et al., 2014). Correlation analysis (Table 3) showed that the number of bacteria was negatively correlated with THAAs, basic and sulfur-containing AAs, OM as well as HFOM, which indicated that a high bacterial count enhanced the release of THAAs and decomposition of OM in the sediment, thereby advancing the decomposition of nonlabile molecules such as basic AAs and HFOM. However, number of bacteria in the sediments of Erhai Lake showed an clearly increase in the past decades, the mean number of bacteria increased from 15 × 106 CFU·g−1 to 32 × 106 CFU·g−1 during the 30-year period because contamination continuously aggravated the ecosystem, thereby inducing its deterioration (Zhang et al., 2015a). Therefore, the gradual increase in bacteria resulted in an increase in the release risk for sediment THAAs. 4.2.2.3. Effects of OM mineralization. Correlation analysis showed that THAAs and their forms were positively correlated with HFOM (Table 2), which suggested that the sediment THAAs of Erhai Lake mainly existed in the humus. Humus, which represents nonlabile OM, was the dominant OM form in the sediment. The results of the present study suggested that sediment OM degradation and THAAs release

Fig. 9. Numbers of actinomycetes, fungi and bacteria population at different sampling sites.

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Table 3 Pearson correlation coefficients for the relationship between THAAs and microorganism of surface sediments (n = 10).

THAAs Neutral AAs Alkaline AAs Acidic AAs Aromatic AAs Sulfur AAs OM AFOM LFOM HFOM a b

Total population

Actinomycetes

Fungi

Bacteria

0.079 0.046 0.014 0.209 0.282 0.155 0.293 −0.539 0.31 0.524

0.162 0.127 0.09 0.238 0.332 0.25 0.395 −0.451 0.342 0.628b

0.551 0.63b 0.142 0.239 0.852a 0.744a 0.404 −0.333 0.472 0.495

−0.635b −0.537 −0.652b −0.258 −0.446 −0.667b −0.762a −0.368 −0.312 −0.858a

Correlation is significant at the 0.01 level (2-tailed). Correlation is significant at the 0.05 level (2-tailed).

may be relatively difficult in Erhai Lake. However, the observed high density of bacteria and high AFOM content in surface sediments suggested that the sediment partly has a high potential for OM degradation and THAAs release. OM degradation results in changes in pH and redox, thus further affecting the release processes of various N compounds (Wang et al., 2009). The differences in THAAs composition reflects the degree of OM degradation. The degradation index (DI) is the relationship between each AA and the degree of OM degradation, and is calculated by principal components analysis (PCA) (Dauwe and Middelburg, 1998) as follows: DI ¼

X vari−AVG vari  fac: Coe: f i ; i LSTD vari

where vari is the molar ratio of some kind of AAs and THAAs; AVG vari and STD vari are the mean value and standard deviation of vari,

Fig. 11. DI of surface sediment in different sites of Erhai Lake.

respectively; and fac.Coe.fi is the corrected parameter of each specific substances. The lower value of DI represents the higher degradation degree of sediment OM. In contrast, the higher DI means fresher sediment OM. Fig. 11 shows the variation of DI in the surface sediment of Erhai Lake. In general, DI was positive in the southern and northern areas, but negative in the central area, except for site E6. These results suggested that the degree of OM degradation in the southern and northern areas were generally much higher than that in the central area. In the southern and northern areas, the aquatic plants suffered serious degradation and large amounts of OM accumulated in the areas since 2003. Meanwhile, the sediment from the two areas has high number of bacteria and high enzyme activity due to the shallow water and sufficient amount of nutrients, as well as frequent anthropogenic activities (Zhang et al., 2015b). The superimposed factor finally enhanced the degradation of sediment OM in the northern and southern areas. These results also imply that the southern and northern areas have higher a THAAs release risk when OM degradation continuously occurs.

Fig. 10. Temporal variation of watershed, aquatic ecosystem, and water quality as well as sediment THAA content in Erhai Lake.

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Fig. 12. Liner coefficients for the relationship between DI and THAAs, acidic, neutral and basic AAs contents in surface sediment (n = 10).

In contrast, the OM in the central part of the lake was relatively fresh, and this is because the central area accumulated less OM and barely had microorganisms that underwent OM degradation. Correlation analysis showed that DI was negatively correlated with acidic and basic AAs (Fig. 12), which suggested that OM degradation would enhance the release of acidic and basic AAs in Erhai Lake. Because basic AAs represent nonlabile AAs, future studies should mainly focus on the release of acidic AAs. In summary, sediment THAAs is an important factor that influences water quality and the aquatic ecosystem. However, the risk of THAAs release from sediments might increase when the sediment environmental conditions of Erhai Lake continuously change such as a decrease in pH, or an increase in bacterial density, enzyme activity, and OM degradation. Therefore, the local government and scientists should further investigate measures for the ecological restoration, control of exogenous pollution, and maintenance of reasonable water habitats to prevent the release of sediment THAAs.

suggested that the release of THAAs was negatively correlated with pH, whereas positively correlated with bacterial density and OM mineralization degree, which mainly depend on the stability of HFOM. However, the risk for sediment THAAs release might increase when the sediment environmental conditions continuously change (i.e., decrease in pH and increase in bacterial density, enzyme availability, as well as OM degradation). Therefore, maintaining reasonable sediment environmental conditions is essential to protect the water quality and ecosystem of Erhai Lake.

Acknowledgements The National Natural Science Foundation of China (No. U1202235), National High-level personnel of special support program (People Plan No. 2012002001), and the National Key Science and Technology Special Program “Water Pollution Control and Treatment” (2012ZX07102-004) supported this study.

5. Conclusions This study reconstructed the relationship between sediment THAAs dynamics and anthropogenic activities, as well as lake ecosystem processes using dated sediment cores. The results showed that the historical distribution and fractions of THAAs could be divided into three stages: a stable period before 1970s, a clear increasing period from the 1970s to 1990s, and a gradually steady or declining period after the 1990s to 2010. The chemical fraction, aromatic, and sulfur-containing AAs, accounted for only ≤3% of the AA. Basic AAs accounted for 5–17% of THAAs, and stayed at a relatively stable level in the sediment cores. However, acidic and neutral AAs, which accounted for 19–44% and 35–69% of the THAAs, respectively, was the predominant factor that caused an increase in THAAs due to the rapid development of agricultural intensification and increase in contemporary sedimentation of phytoplankton or macrophytes in Erhai Lake. These trends were strongly associated with both anthropogenic activities and natural processes, which implied that sediment THAAs could act as an effective indicator that reflects the anthropogenic activities and aquatic environmental characteristics. This study also analyzed the effects of environmental conditions on AA release at the sediment interface of Erhai Lake. The results showed the present contribution of THAAs on N and OM in sediments was b5% and 1.5%, respectively, but the increase in the content of THAAs indicated that there was a huge potential source of labile DON to the overlying waters, which in turn may cause continuous deterioration of the ecosystem once environmental conditions change. Correlation analysis

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