Introduction

Antibiotics, a class of pharmaceuticals that are extensively used worldwide, have raised significant concerns (Shen et al. 2015; Zhang et al. 2015a, b; Ling 2010) in recent decades due to their potential threat to the ecological environment and human health. Antibiotics are widely used to treat or prevent human and animal diseases, as well as to promote the growth of animals in the livestock and aquacultural operations (Shea 2003; Xu et al. 2015). They are often partially metabolized in organisms and may be excreted as the parent compounds or metabolites into environment via urine and feces, resulting in higher concentrations of antibiotics in the sewage, treated urban wastewater, and wastewater from the livestock, poultry, and aquaculture activities. As a result of the incomplete removal of antibiotics during wastewater treatment and the direct discharge of the wastewater into receiving water (Xu et al. 2014), the detection frequencies of antibiotics in aquatic environments are high.

Although the concentrations of antibiotics in water range from nanograms per liter to micrograms per liter, and although many antibiotics are unstable, having short half-life periods (Christen et al. 2010), antibiotics cause long-term potential harm to the environment and health due to their continuous input. Inducing and spreading antibiotic-resistant bacterial strains is the most serious risk to the ecological environment and human health (Zhang et al. 2014). Therefore, the environmental risk of antibiotics should not be ignored. The consumption and production of antibiotics in China are the highest in the world. According to the study results of Zhang et al. (2015a, b), the total antibiotic usage in 2013 was estimated to be approximately 1.60 × 105 t, and human consumption accounts for approximately 48%, with the rest shared by animals. The antibiotic emissions into water are up to 5.0 × 104 t/year, which results in the high detection frequency of antibiotics in water, such as Pearl (Liang et al. 2013), Haihe (Luo et al. 2011), and Yangtze Rivers (Yan et al. 2014; Shi et al. 2014). In addition, the concentrations of some antibiotics are determined to be thousands of nanograms per liter, which are higher than those in the water of the developed countries.

Dongting Lake, the second largest freshwater lake in China, plays an important role in the water supply for fisheries and agriculture, and it is also a water source for drinking water production (Yuan et al. 2015; Hu et al. 2015). However, there are a large number of highly polluting industries including paper mills and chemical plants within the Dongting Lake basin, and their discharge causes the deterioration of the quality of water of the Dongting Lake. In addition, Dongting Lake receives a large quantity of sewage, treated urban wastewater, and wastewater from the livestock, poultry, and aquaculture activities, which are the main sources of antibiotics and could pose a substantial threat to the environment and human health (Yang et al. 2015). According to the research results of Zhang et al. (2015a, b), the emissions of antibiotics (2190–3560 t year−1) in the Dongting Lake basin are the highest in China, with an emission density of 22.2–32.4 kg (km2·year−1). The results of Wu et al. (2014) show that there are various antibiotics (trimethoprim, lincomycin, clindamycin, and erythromycin) in the surface water of Dongting Lake. However, the pollution level of antibiotics in the lake and its ecological risk are not well known due to limited sampling locations and types of antibiotics studied, which could not comprehensively reflect the antibiotic pollution status of Dongting Lake.

The main objectives of this study are the following: (1) to investigate the pollution levels of antibiotics in Dongting Lake, (2) to assess the potential ecological risk associated with the antibiotics present by the risk quotient (RQ) method, (3) to examine the seasonal and spatial trends. The results could provide reliable information regarding the pollution characteristics, sources, and risk levels of antibiotic contamination in Dongting Lake.

Materials and methods

Chemicals

A total of 12 antibiotics, classified into four classes, were selected as target compounds and are given in Table 1. The selection of the target analytes was based on their high detection frequencies in other lakes and rivers and their high consumption. Analytical standards were purchased from Dr. Ehrenstorfer GmbH (Germany). The internal standards (ciprofloxacin (CIP)-D8, sulfamethoxazole (SMX)-D4, trimethoprim (TMP)-13C3, and demeclocycline (DMC)) were obtained from Sigma-Aldrich (St. Louis, MO, USA), except for sulfamethazine-13C6 (SMZ-13C6) which was from Cambridge Isotope Laboratories (Andover, MA, USA). All other chemicals and solvents were purchased from Fisher Chemicals (Fair Lawn, NJ, USA). The SPE cartridges used were Oasis HLB (500 mg/6 mL) purchased from Waters Corporation (Milford, MA, USA). Glass fiber filters were from Sigma-Aldrich (St. Louis, MO, USA). Stock standards and internal standard solutions were prepared in methanol at 1 g L-1 and stored at − 20 °C, which gave a shelf-life of at least 3 months and were diluted before use.

Table 1 Selected antibiotics and their classification
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Study area and sample collection

Dongting Lake (27° 39′–29° 51′ N, 111° 19′–113° 34′ E), located in Hu’nan Province and Hubei Province China, is the second largest freshwater lake in China. Currently, the lake is split into several parts, including West Dongting Lake, South Dongting Lake, and East Dongting Lake. Dongting Lake covers a large surface water area of 2691km2, with the watershed area of 2.57 × 105 km2. The annual water level of the lake changes by as much as 15 m (Li et al. 2000). The area, whose total capacity is 1.78 × 1010 m3, receives an annual average rainfall of 1302 mm, and the average water depth is 6.39 m. Dongting Lake plays an important role in regulating the amount of water in the Yangtze River.

To better reflect the antibiotic pollution in the aquatic environment of the Dongting Lake, 18 samples (Fig.1 and Table S1) covering the entire lake were collected in December 2015 (dry season) and August 2016 (wet season). The surface water samples were collected at a depth of 1.0 m and placed in pre-rinsed brown glass bottles. Three parallel samples were collected from each sample location, then kept in the brown glass bottle at 4 °C before laboratory analysis. The surface water samples were treated within 12 h after being transported to the laboratory.

Fig. 1
figure 1

Distribution of the total antibiotics in the water from Dongting Lake, ng L−1

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Sample preparation and instrumental analysis

The water samples (2 L) were filtered through glass microfiber filters (0.45 μm) (Whatman GF/F, 0.45 μm, USA) to remove suspended particles. Then, the pH value was adjusted to 3 using 0.1 mol L−1 sulfuric acid, Na2EDTA (0.5 g), and 100 μL of 1 mg L−1 surrogate standards (CIP-D8, SMX-D4, TMP-13C3, DMC, SMZ-13C6) was added to water samples. The treated solutions were extracted using Waters Oasis HLB cartridges (500 mg, 6 mL), which were sequentially preconditioned with 10 mL of methanol and 10 mL of ultrapure water, after which the water samples were loaded into the cartridges at a flow rate of 3–5 mL min−1. The cartridges were rinsed with 10 mL of a 1% methanol aqueous solution and 10 mL of ultrapure water and dried for 2 h under vacuum. The antibiotics retained on the Oasis HLB cartridges were eluted with 6 mL of methanol and 6 mL of a 5% NH4–H2O methanol solution, and the eluents were concentrated to near dryness under a gentle stream of nitrogen, after which the resulting residues were re-dissolved in 1 mL of a 10% methanol aqueous solution. After filtration through a 0.22-μm membrane to remove particles, the final extracts were transferred to 2-mL amber glass vials in preparation for analysis.

The analysis of the antibiotics was performed by ultra-performance liquid chromatography–tandem mass spectrometry (ACQUITY UPLC-XEVO-TQMS) (USA, Waters) equipped with an UPLC BEH–C18 column (50 × 2.1 mm, 1.7 μm). The column was maintained at 40 °C during sample analysis. The mobile phase consisted of eluent A (0.3% formic acid and 0.1% ammonium formated in ultrapure water) and eluent B (acetonitrile/methanol = 1:1). The separation of the antibiotics was achieved with the gradient program given in Table S2.

The results of the mass spectrometric analyses are provided in Table S3.

Quality assurance and quality control

Quantification using internal standard was the approach to determine the concentration of antibiotics. The calibration curves (0.1~500 μg L−1 concentrations) for the antibiotic detection exhibited good linear relationship (R2 > 0.98). The recoveries of the antibiotics from the surface water samples were ranged from 80 to 121% (Table S4). The limit of quantification calculated with a signal/noise ratio of 10 was 0.1 to 0.92 ng L−1. Duplicates, method blanks, and solvent blanks were used for quality control. The target antibiotics were not detected in the method blanks and solvent blanks.

Ecological risk

The ecological risks of the antibiotics were based on the RQ method (Ma et al. 2016; Vryzas et al. 2011). The RQ values were calculated using the formula below:

$$ \mathrm{RQ}=\frac{\mathrm{MEC}}{\mathrm{PNEC}} $$
(1)
$$ \mathrm{PNEC}=\frac{\mathrm{TD}}{\mathrm{AF}}\kern0.5em $$
(2)
$$ \kern1.50em {\mathrm{RQ}}_{\mathrm{com}}=\sum \mathrm{RQ} $$
(3)

where MEC is the measured environmental concentration, nanograms per liter. PNEC is the predicted no effect concentration in water, nanograms per liter. PNECs were based on the reported acute or chronic toxicity data. The TD is toxicity data, milligrams per liter. AF is assessment factor. According to the calculated RQ values, the environmental risks were classified into four levels: no risk (RQ < 0.01), low risk (0.01–0.1), medium risk (0.1–1), and high risk (> 1) (Zheng et al. 2012; Sassman and Lee 2005). The RQcom is the combined risk quotient at each point.

Results and discussion

Occurrence of antibiotics in lake water and comparisons between areas

The concentrations of antibiotics in surface water of Dongting Lake are provided in Table 2. Eleven of the 12 antibiotics were detected in the 18 sampling sites, where oxytetracycline was the only antibiotic not detected. The antibiotic concentrations ranged from ND (not detected) to 61.28 ng L−1. The sulfonamides (SAs) were the main components among the tested analytes in Dongting Lake. There was a distinct seasonal variation in the antibiotic concentration. Overall, the contamination by antibiotics in Dongting Lake was relatively low compared to that in other lakes in China. As the Yangtze River-connected Lake and the second largest freshwater lake in China, the fluidity of the water body is large in Dongting Lake, which has strong dilution effect.

Table 2 Statistical characteristics of the concentrations of antibiotics in surface water in wet season and dry season of Dongting Lake (ng L−1)
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The concentration of SAs was in the range of ND–61.8 ng L−1, with detection frequencies of over 60%. Sulfamethazole was the most frequently detected compound. The concentration of sulfamethoxazole was in the range of 0.47–47.41 ng L−1 (11.8 ng L−1) in the dry season and ND–5.63 ng L−1 (1.36 ng L−1) in the wet season, with detection frequencies of over 80%.The average concentration of sulfadiazine was 24.35 ng L−1 in the dry season which was nearly 23 times as high as that in the wet season. Among the three SAs, the average concentration and detection frequencies of sulfamethazine were relatively low. Compared to other lakes in China (see Table 3), the pollution level of sulfamethazine and sulfamethoxazole in Dongting Lake was lower. However, the pollution level of sulfadiazine was much higher than that in Bosten Lake, Chaohu Lake, and Wulungu Lake, except Baiyangdian Lake. Trimethoprim was in a range of ND–2.25 ng L−1. Trimethoprim, an antibacterial synergists, was most commonly used in combination with sulfa-drugs, especially sulfamethoxazole and sulfadiazine, in a ratio of 1:5 (Grossman and Remington 1979; Qin et al. 2015), which led to the relatively high detection frequency in detected antibiotics. The mean concentrations of TMP in both dry and wet seasons are quite close.

Table 3 The concentration of antibiotics in typical lakes, China (ng·L−1)
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Among the five QNs, the detection frequency of sarafloxacin was higher in both dry and wet seasons. The concentration of sarafloxacin was in the range of ND–21.29 ng L−1. As a class of dedicated animal veterinary products, sarafloxacin is becoming increasingly widespread in clinical animal applications (Pulgarín et al. 2012), which might be one of the major reasons for its high concentration and detection frequency in Dongting Lake. Although the detection frequency of ciprofloxacin was low, its concentration was higher than that of some of the detected antibiotics. Ciprofloxacin was widely used in aquaculture, livestock, and poultry, and the usage in the medical field is at the top (Zhang and Liu. 2010). Although the concentrations of norfloxacin and enrofloxacin were low, the concentration and detection frequencies of these two antibiotics were higher in the wet season than in the dry season, especially for enrofloxacin, which was similar to the trend observed in Hongze Lake (Sun 2015). In the wet season, the fish and livestock were in a period of rapid growth, which corresponds to an increase in the usage of norfloxacin and enrofloxacin to treat or prevent diseases and promote the growth of animals. However, norfloxacin and ofloxacin were prohibited from being produced and used in food animals after September 1, 2015, according to the regulations of the Ministry of Agriculture, and these antibiotics were no longer circulated after December 1, 2015. The detection frequencies of norfloxacin rebounded during the wet season, which may indicate that the norfloxacin in the water of Dongting Lake was mainly from human medication. The lower concentration of norfloxacin may be related to the prohibited use in juveniles (Li et al. 2016), which limits its usage. Generally, the pollution levels of QNs in Dongting Lake were much lower than those reported for other lakes of China.

In the dry season, the concentration of tetracycline ranged from ND to 21.51 ng L−1 with a mean value of 2.17 ng L−1, and chlortetracycline ranged from ND to 6.5 ng L−1 with a mean value of 1.79 ng L−1. Tetracyclines are widely used to treat diseases and protect the health of animals. They are also added at subtherapeutic levels to animal feed to prevent infection and act as growth promoters. Tetracyclines are also the most widely used veterinary drugs and food additives in the aquacultural and livestock industries of China. In 2010, the chlortetracycline usage reached approximately 7.19 × 104 t, which was 22 times the annual usage of tetracycline antibiotics in America (Guo et al. 2014). The usage of tetracycline antibiotics is enormous in Dongting Lake which is as an important aquacultural, livestock, and poultry breeding base. Therefore, the detection frequencies of tetracycline (26.32%) and chlortetracycline (47.37%) were higher than those of the other antibiotics except for the SAs. The concentrations of chlortetracycline and tetracycline were much lower than those of Taihu Lake, and of the same order of magnitude as those of Chaohu Lake, Bosten Lake, and Wulungu Lake. In the wet season, tetracycline and oxytetracycline were not detected. As the only detectable tetracycline antibiotic, the concentration of chlortetracycline declined drastically in the wet season, compared to the dry season.

Overall, the concentrations of antibiotics in the dry season were significantly higher than in the wet season. This phenomenon is similar to the Poyang Lake (Ding et al. 2017), the Victoria Harbor and the Pearl River (Xu et al. 2006), the Honghu Lake (Wang et al. 2017), and the Xiang River (Fan et al. 2015). The conclusions of these studies reached a consensus that the significantly lower concentrations of antibiotics in the wet season mainly resulted from the dilution effect of water flow. The average water level of Dongting Lake has been increasing two times between December 2015 and August 2016 (http://www.yymsa.gov.cn/yymsa/xxgk/xxgkml/aqxx/swgg/).On the other hand, the cold water temperatures, low microbial activity, and low dissolved oxygen might enhance the persistence of antibiotics (Yan et al. 2013; Kümmerer. 2009). In addition, winter was the peak incidence of diseases, which resulted in larger usage of antibiotics.

Spatial distribution and source identification

The total concentrations of the antibiotics in Dongting Lake are shown in Fig.1. The total concentrations of the detected antibiotics at each sampling site ranged from 6.35 to 135.40 ng L−1 in the dry season and 1.06 to 17.42 ng L−1 in the wet season. In Nandu, the total concentration was the highest. The difference in the distribution of the antibiotics was obvious, in which the highest pollution level of antibiotics was observed in East Dongting Lake (mean value of 65.14 ng L−1 in the dry season and 8.76 ng L−1in the wet season), followed by South Dongting Lake (mean value of 54.35 ng L−1 in the dry season and 6.72 ng L−1in the wet season) and West Dongting Lake (mean value of 41.85 ng L−1 in the dry season and 3.36 ng L−1in the wet season) (see Fig.2). The spatial distribution of antibiotics in Dongting Lake was similar to the distribution of Pharmaceuticals and Personal Care Products (PPCPs) (Ma et al. 2016). The objective reasons were as follows: (1) as the convergence site of lake water, a large number of pollutants gathered in East Dongting Lake (Bu 1991); (2) the outlet of Dongting Lake near East Dongting Lake was easily affected by the backwater effect of the Yangtze river (Wang et al. 2012), which facilitated the accumulation of pollutants; (3) the populations around East Dongting Lake was 1,783,000, which had high population density, and the two wastewater treatment plants of Yueyang City were very close to East Dongting Lake (Yang et al. 2016). The spatial distribution pattern of the antibiotics in Dongting Lake was closely related to the distribution of the pollution sources. Sulfadiazine, sulfamethoxazole, sulfamethazine, and sarafloxacin, the major pollutants of the Dongting Lake, are used in aquaculture, livestock, and poultry. From 2003 to 2013, the use of veterinary drugs increased from 46 to 52% of the total usage. Furthermore, more than 60 to 90% of the ingested antibiotics may be excreted by animals without modification, resulting in a potential pollution risk for the water environment (Boundj and Voulvoulis 2004).

Fig. 2
figure 2

Spatial distribution in the dry (A) and wet season (B) between East Dongting, West Dongting, and South Dongting Lake

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The key distribution areas of aquaculture, livestock, and poultry are seen in Fig.3, and Fig. 4. In the West Dongting Lake basin, the key distribution areas of livestock and poultry are located in Taoyuan county, Anhua county, and Lixian. However, as an important risk source of antibiotics, this area is far from West Dongting Lake. During the migration of antibiotics, photodegradation and hydrolysis become important environmental factors, which caused lower antibiotic concentrations in West Dongting Lake. In the East and South Dongting Lake basins, the key distribution areas are in Xiaoyin county, the downtown area, Miluo City, and the Pingjiang county of Yueyang City. As the second largest producer of livestock and poultry in the Dongting Lake Basin, Yueyang City is an important risk source of antibiotics. However, the phenomenon of direct discharge of aquaculture, livestock, and poultry wastewater still existed (http://www.hbzhan.com/news/detail/109718.html).

Fig. 3
figure 3

Key distribution area of livestock and poultry in Dongting Lake

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Fig. 4
figure 4

Key distribution area of aquaculture in Dongting Lake

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The regulatory controls are weak and antibiotics are often overused in aquaculture in China. In 2014, the total aquacultural production of Dongting Lake was 1.31 × 106 t. The key distribution areas of aquaculture are located in Hanshou county (3.0 × 105 acres), Xiangyin county (1.5 × 105 acres), Huarong county (the aquaculturing areas are 2.0 × 105–3.0 × 105 acres), and Nan county (the aquaculturing areas are over 1.0 × 105–2.0 × 105 acres), which are mainly located in East Dongting Lake.

Ecological risk

Based on the worst case scenario, the RQs were estimated based on the PNECs of the most sensitive species (see Table 4), and conducted, using the maximum concentrations of the antibiotics according to Yan et al. (2013) and Xue et al. (2013). The results are displayed in Table 5. Due to their trace residue, the RQs for most of the antibiotics were below 0.1 in the dry season. However, the RQs of sulfamethoxazole, ciprofloxacin, and sarafloxacin were over 1, indicating that they pose a significant environmental risk to Dongting Lake. Furthermore, ciprofloxacin had the highest RQ (up to 7.23) due to its low PNEC, which may pose adverse ecological effects. Sulfadiazine, the target compound with the highest concentration and detection frequency, showed the low risk. The rest of the antibiotics exhibited low or no environment risk, except for ofloxacin (medium risk). In the wet season, ciprofloxacin (RQ = 0.54) had the highest risk factor as in the dry season, followed by sarafloxacin (RQ = 0.37) and sulfamethoxazole (RQ = 0.21) with these antibiotics exerting medium ecological risk. The RQ of rest antibiotics were in the range of 10−2~10−4, which would not cause obvious risk to ecological environment.

Table 4 Aquatic toxicity data of antibiotics to the most sensitive aquatic species
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Table 5 RQs and RQcom for the antibiotics in surface water from the Dongting Lake
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Furthermore, antibiotics in the actual environment often exist as mixtures, which can enhance the effect on the environment due to cocktail effects (Cleuvers 2004). However, most of the toxicity researches on antibiotics only analyze individual compounds. Therefore, it is assumed that the effects of antibiotics on biological system fail to exhibit antagonistic or synergistic relationship, and the simple accumulation model was used to calculate the combined risk in this study, see Formula (3). The calculated results of the combined risk assessment are shown in Table 5. In the dry season, the combined risk index at the different sites were significantly different, ranging from 0.05 to 8.73. Of the sites, S7 (Nandu) had the highest combined risk (up to 8.73), followed by S4 (Hanshouyuanshui bridge) (up to 6.09), which posed a substantial threat to the ecological security of Dongting Lake. The combined risk for S1, S6, S8, S10, S13, and S17, which were all located in the East and South Dongting Lake, ranged from 1 to 2, whereas the combined risk indexes in the rest of the sites were below 1, which corresponded to a medium or low risk. Compared to the dry season, the combined risk index of all sites were lower in the wet season, with values below 1. Of the sites, S7 (Nandu) had the highest combined risk as dry season (0.89), followed by the sites in the order of S8 > S10 > S1 > S9, which showed a medium risk. The risk index of other sites exhibited low or no environmental risk. In the cumulative risk for S7, ciprofloxacin accounted for over 80%. Although the detection frequency of ciprofloxacin was low, its risk to the Dongting Lake was not ignored.

Although the ecological risk of most of the antibiotics was low, the antibiotic risk to human health should be not ignored. Antibiotics could lead to a selective pressure on the water bacteria and induce the formation of antibiotic-resistant bacteria. Additionally, antibiotic-resistant genes (ARGs) can be transferred between non-pathogens, pathogens, and even distantly related organisms, such as Gram-positive and Gram-negative bacteria, through horizontal gene transfer, and the resulting antibiotic-resistant bacteria are considered a potential threat to human health through the food chain (Pruden et al. 2006). In Peking Union Medical College Hospital, the fungal infection rate was 0% in the 1980s and increased to 7–8% in 2000, which was caused by the appearance of pandrug-resistant nonfermenters (ChinaIRN, 2012). Xiao (2008) found that the mortality rate (11.7%) of patients infected by drug-resistant bacteria was more than twice that in patients with a common infection (5.4%). In addition, related studies showed that the highest resistance rates of tetracycline, sulfamethoxazole, and cefalotin sodium have reached 57, 13, and 35%, respectively (Dai et al. 2009). The antibiotic-resistant bacteria and genes are abundant in the Pearl (Tao et al. 2010), Haihe (Luo et al. 2010), and Huangpu rivers (Shen et al. 2012). However, relevant studies on the risk assessment of ARGs are limited. In Dongting Lake, the pollution characteristics and ecological risk of ARGs in water were still absent, which relevant research should be conducted.

Conclusions and suggestions

This study reported the occurrence of 10 individual antibiotics in the surface water of the second largest freshwater lake in China. The results showed that the concentrations of sulfonamides were relatively higher than other antibiotics in Dongting Lake. Compared to other lakes in China, the pollution level of antibiotics was relatively low. The pollution level of antibiotics in the dry season was significantly higher than in the wet season except for trimethoprim, enrofloxacin, and norfloxacin. The concentrations showed an obvious spatial difference, which followed the trend of East Dongting Lake> South Dongting Lake > West Dongting Lake. The spatial distribution of antibiotics in Dongting Lake may be related to the distance of risk sources (aquaculture, livestock, and poultry) from Dongting Lake and their scale. The risk assessment revealed that sulfamethoxazole, ciprofloxacin, and sarafloxacin had higher ecological risk to the Dongting Lake. Therefore, a risk warning system should be built. Further study is needed to investigate the usage and emission of typical antibiotics in Dongting Lake basin, and the rates of contribution from different sources should be quantified in detail, especially in the aquacultural, livestock, and poultry industries. The occurrence, distribution, and environmental risk in multimedia samples from Dongting Lake should be researched. In addition, the mechanisms and routes of the migration and transformation of the antibiotics from West Dongting Lake to East Dongting Lake, and the types and toxic effects of the derivatives and transformation products should be analyzed. These properties have important theoretical and practical significance for evaluating and diagnosing the environmental safety of Dongting Lake.