Environmental Toxicology and Chemistry, Vol. 33, No. 4, pp. 768–775, 2014 # 2014 SETAC Printed in the USA
EFFECTS OF 17b-ESTRADIOL POLLUTION ON WATER MICROBIAL METHANE OXIDATION ACTIVITY AIDONG RUAN,*y FENGJIAO ZONG,y YING ZHAO,y CHENXIAO LIU,y and JING CHENz yState Key Laboratory of Hydrology-water Resources and Hydraulic Engineering, Hohai University, Nanjing, China zState Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Beijing Normal University, Beijing, China (Submitted 6 August 2013; Returned for Revision 26 November 2013; Accepted 20 December 2013) Abstract: 17b-estradiol (17b-E2), a widespread and natural estrogen in the environment, has imposed a serious threat to the safety and function of aquatic ecosystems because of worsening pollution and high potential toxicity. In the present study, the authors focus on the impact of 17b-E2 pollution on water microbial methane oxidation function. The authors investigated the mechanism of its influence on water microbial activity and discuss the growth rate of methane-oxidizing bacteria. The results showed that 17b-E2 could significantly inhibit the function of water microbial methane oxidation. When 17b-E2 concentration was 5 ng L1, the methane oxidation rate increased with increasing 17b-E2 and finally settled to a constant value. Furthermore, the authors found no significant linear correlation between 17b-E2 concentrations and its methane oxidation rate. However, increasing 17b-E2 dramatically improved water microbial community activity, because a significant or highly significant promotion in the generation rate of CO2 was measured. Moreover, within a certain period of time and at certain concentrations, positive linear correlation existed between water CO2 generation rate and 17b-E2 concentrations. In addition, the growth rate of culturable methane-oxidizing bacteria was promoted when 17b-E2 pollution concentration from 2 ng L1 to 20 ng L1. Therefore, 17b-E2 pollution can inhibit microbial methane oxidation function in water, which indirectly promotes the release of water methane and directly contributes to the rate of water-generated and released CO2. Specifically, 17b-E2 pollution can promote water emissions of greenhouse gases. Environ Toxicol Chem 2014;33:768–775. # 2014 SETAC Keywords: 17b-estradiol
Methane
Carbon dioxide
Methane-oxidizing bacteria
Methane oxidation function
of 0.1 ng L1 to 88 ng L1. Finlay-Moore et al. [17] measured concentrations of 17b-E2 as high as 2530 ng L1 in runoff water from fields fertilized with poultry litter. Kolodziej and Sedlak [18] detected estrone in 78% of collected samples with concentrations as high as 38 ng L1 and measured 17b-E2 in 18% of samples with concentrations as high as 1.7 ng L1. In monitoring 256 water samples from 109 rivers in Japan, Tabata et al. [11] reported that 222 water samples contained 17b-E2 with an average concentration of 2.1 ng L1. Kolpin et al. [19] surveyed America surface water in 30 states; in the 139 extracted water samples, nearly 40% registered pollution levels of 17b-E2. Lee et al. [20] detected total estrogen material of 260 ng L1 to 300 ng L1 in sewage outfalls of the sea in Shenzhen, China. Therefore, 17b-E2 has become a significant and widespread pollutant in water environments and has received international academic attention. Currently, 17b-E2 research is focused mainly on animal toxicology. It has been reported that 17b-E2 can impact humans and animals significantly [21–22] at concentrations of 1.0 ng L1 and through synergy with other substances 17b-E2 activity strengthens [23]. However, research focused on ecological toxicology is rarely seen in literature. Recently, we [24] explored the regularity effects of 17b-E2 on methane emissions in anaerobic sediments. The production of methane and methane oxidation is a contradiction, so we assumed 17b-E2 impacted methane oxidation. In the present study, we explored the impact of 17b-E2 pollution on the water methane generation and the release from the perspective of microbial methane oxidation. Methane is a natural energy and greenhouse gas accounting for more than 15% of global atmospheric warming [28]. The greenhouse effects it produces are 20 to 30 times the quality of carbon dioxide [25], and the atmospheric ozone layer destruction can reach 7 times that of CO2 [26–27]. According to a report from the
INTRODUCTION
Environmental estrogens are exogenous substances that can enter the human body. Their characteristics interfere with the normal secretion in vivo synthesis, release, transport, metabolism, combine and other processes, and activate or inhibit the endocrine system function. This undermines the body’s collective ability to maintain stability and regulatory effects [1]. The pollution of environmental estrogens has long been a major global environmental issue following discovery of the greenhouse effects and ozone depletion. Even very small amounts of estrogen [2–3] can cause hormone imbalance, genital malformations, and even cancer [4–5] in organisms. Environmental estrogens are also difficult to degrade and toxicity has long-term potential; indeed, certain species or even regional ecosystems have caused disastrous damage when harmful symptoms appear [6]. Over time, environmental estrogens (especially in water environments) have been significant environmental pollutants; the scientific issues of migration, conversion, distribution, toxicological effects, and degradation mechanisms have gradually become an international hot field for research [7]. The 17b-estradiol (17b-E2) is the most active, strongest, and most widely distributed environmental estrogen and is considered one of the natural steroidal estrogens that is a direct threat to human survival. It is listed as a carcinogen by the US Department of Health and Human Services [8]. 17b-estradiol is widely distributed in waste, animal waste, sludge, soil, surface water, and groundwater environments [9–15]. In the continual effluent discharge of wastewater treatment plants (WWTP) containing 17b-E2 concentrations, Bradley et al. [16] found concentrations * Address correspondence to [email protected]. Published online 10 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2516 768
E2 impacts on water microbial methane oxidation activity
Intergovernmental Panel on Climate Change [29], approximately 60 Tg global methane was released in 2002; this number tended to increase yearly. Our research questions in the present study are: 1) How is the pollution of environmental estrogen related to the methane-producing bacteria and methaneoxidizing bacteria community structure? and 2) How do these contribute to methane emissions? Our research indicated that 17b-E2 pollution in the aquatic microbial system has influences on regularity, and low concentrations of 17b-E2 pollution inhibit anaerobic microorganism group activity and promote the generation and release of methane [24]. However, methaneoxidizing bacteria based on methane as its sole carbon and energy source and oxidized methane to methanol at room temperature [30] (which has been the most important biological barrier for preventing methane discharge) has played a major role in the global methane balance. In view of this, the present study analyzed the 17b-E2 effect of water body sediment microbial activity of methane oxidation under aerobic conditions. Furthermore, from the perspectives of water microorganism group activity and culturable methaneoxidizing bacteria growth rate, we explored the impact of 17b-E2 pollution on the aquatic microbial methane oxidation function mechanism. MATERIALS AND METHODS
Laboratory water microcosms construction of simulation system
Test samples of sediment and water were collected from China’s Yangtze river in Nanjing. Among them, the sediment samples were collected from 5 cm to 15 cm of mud below the interface [31]. Sediment samples were stored in the laboratory in a dark, well-ventilated location. After air-drying, we evaluated the average moisture content of the samples after grinding, sieving, and mixing. All laboratory system incubations were performed in 150 mL serum bottles, capped with 6 layers of gauze, using 40 g powdery sediment and 100 mL water samples. The headspace was maintained at 30 mL. In this way, the laboratory water micro-ecological simulation system was constructed and used to study the effects of 17b-E2 pollution in the aquatic microbial activity of methane oxidation. All of the samples were stationary cultured in an incubator (DK-GJ003; Memmert) for 72 h at 28 8C. Enrichment of methane-oxidizing bacteria in simulation system
The 72-h-culture-activated simulation systems were sealed by the destroyed bacteria of an isobutyl rubber plug under sterile conditions. The gases in the system were pumped in 5 mL portions by syringe, then injected with 5 mL methane gas (99.90%; Nanjing Special Gas). Finally, we put it in the incubator under 120 rpm shake culture at 28 8C. Next, 2 mL of gas was extracted from the system every 24 h, which was applied to analyze the upper gas concentrations of CH4 and CO2. The gas phase of the bottle was refreshed by air in a sterile desk for 20 min, corked tightly after replacing a volume of 5 mL of CH4, and returned to the shake culture at 28 8C. For 7 consecutive days (pre-experiments confirmed that the methane oxidation rate was relatively stable for 7 d), methane oxidative bacteria was enriched. 17b-E2 pollution treatment scheme
In every system we used a blank control group (n ¼ 3) without 17b-E2 (99%; J&K) along with 6 concentrations of 17b-E2 solution (0.5 ng L1, 1.0 ng L1, 5.0 ng L1, 10.0 ng L1, 50.0 ng L1, and 100.0 ng L1) respectively, in addition to creating 3 parallel
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samples in each dosage group. Each group was replaced in the 28 8C incubator for shaking. We collected processing system gas every 24 h and analyzed its system of CH4 and CO2 concentration, gas pressure, and temperature. Following the gas phase, each bottle was refreshed with fresh air, then corked tightly to a replaced volume of 5 mL of CH4. We then continued shaking the culture at 28 8C. Operations were thus continuous for 8 d (the first day was without 17b-E2), with the objective of analyzing 17b-E2 pollution in the aquatic microbial communities activity and the impact of methane oxidizing bacteria methane oxidation function. Determining CH4, CO2, and analysis method
The CH4 and CO2 concentrations were measured by gas chromatograph (GC-7890A; Alilent). The system was equipped with a capillary gas chromatography column (a stainless steel -2mm 2m; stationary phase, HayeSep Q80/100) and a flame ionization detector. Pure N2 (99.999%), pure H2, and pure air served as the carrier gas, fuel gas, and supporting gas at a flow rate of 3.0 mL min1, 40 mL min1, and 400 mL min1, respectively. The temperature of the column, detector, and injector were 40 8C, 300 8C, and 100 8C, respectively. The retention times of CH4 and CO2 were 2.2 min and 4.4 min, respectively. The detection limits of CH4 and CO2 were 0.2 mL L1 and 0.03 mL L1, respectively. OriginPro 8 and Microsoft Office Excel 2007 software were used with the test data for statistical analysis. The rate of CH4 oxidized and CO2 produced the formula v¼
M 22:4
273 P ppm 273þT 101325 103 V mt
where v is the gas production rate (mg g1 h1); M is the molar mass of CH4 or CO2 (g mol1); 22.4 L mol 1 indicates the molar volume of gas under standard conditions (standard temperature and pressure). The notation ppm (parts per million) is the gas volume concentration; T is the gas temperature (8C); P is the gas pressure (Pa); V is the headspace volume (mL); m is the dry weight of the sediment (g); and t is the cumulative gas oxidation or production time (h). Methane-oxidizing bacteria cultivation and enrichment
We took 1 mL mixture of the laboratory micro-ecological simulation system in the control group, which was evenly diluted 1000-fold via the sterilization of saline solution. We then accessed the methane-oxidizing bacteria inorganic salt liquid medium with methane as the sole carbon and energy source. Finally, we put it under 120 rpm shake culture for 72h at 28 8C. Every 24 h we replaced fresh air in the sterile console, and the upper gas volume of 10 mL was replaced with methane. Cultures then continued to be diverted and cultured another 3 times, which thus enriched the methane-oxidizing bacteria mixed strains of methane as a sole carbon source and energy. The main components for methane-oxidizing bacteria inorganic salt medium were: KH2PO4, 1.0 g L1; Na2HPO4 12H2O, 2.9 g L1; MgSO4 7H2O, 0.32 g L1; (NH4)2SO4, 3.0 g L1; 10 mL mineral elements solution [32]; and 990 mL distilled water, pH 6.8. The configuration of a solution of mineral elements was: MgSO4·7H2O 3 g; MnSO4·2H2O 0.5 g; NaCl 1g; FeSO4·7H2O 0.1g; CoCl2 0.1g; CaCl2·2H2O 0.1g; ZnSO4·7H2O 0.1g; CuSO4·5H2O 0.01g; AlK(SO4)2 0.01g; H3BO3 0.01g; and Na2MoO4·2H2O 0.01g in 1000 mL volumetric flask. This was fully dissolved in distilled water and diluted to 1000 mL, then readied to use after high-pressure steam sterilization.
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60.79% lower than system water receiving the 17b-E2 treatment compared to system water receiving no 17b-E2. When c (17bE2) equaled 5.0 ng L1, methane oxidation rate was 83.18% lower; when c (17b-E2) equaled 50.0 ng L1, it was 68.23% lower; and when c (17b-E2) equaled 100.0 ng L1, it was 56.98% lower. The linear regression analysis results among various concentrations of 17b-E2 pollution and methane oxidation rate showed no significantly linear correlation in each time period. However, in the low concentration range, that is. c (17b-E2) < 5.0 ng L1, 17b-E2 inhibition methane oxidation effects in the system increased with increasing concentrations of 17b-E2 additions, showing a linear dose– effect relationship (Figure 2). The methane oxidation activity of the system as it changed over time is shown in Figure 3 under concentrations of 17b-E2 pollution. The system of microbial methane oxidation activity exhibited the cyclical process of change, and there was a significant difference between the treatment group and the control group, as seen in Figure 3. The change of microbial methane oxidation activity was larger than water receiving no 17b-E2 treatment, with a longer period of change; whereas the change was significantly faster from water receiving 17b-E2 treatment that has been caused shorten in the change cycle, and the changes were basically the same in all treatment groups. In a future study, we will focus on exploring why a sharp increase exists in the 96-h time period. The above analysis shows a significant inhibition of 17b-E2 pollution exists on a water microbial system methane oxidation function. Within all the pollution concentrations range of the experimental design, there was no significant linear correlation between the methane oxidation rate and concentrations of 17bE2. Hence, the inhibition mechanism of 17b-E2 pollution on water microbial methane oxidation function is more complex. Further research is warranted to explore the mechanisms of 17bE2 pollution on water microbial methane oxidation function, so we have been studying the impact of 17b-E2 pollution on water microbial community activity and cultures of methane-oxidizing bacteria growth rate.
17b-E2 pollution effects of growing methane-oxidizing bacteria
Sterilized methane-oxidizing bacteria inorganic liquid medium was dispensed into 21 sterile 200 mL serum bottles with 50 mL per bottle. There was a blank control group (n ¼ 3) without the 17b-E2 (control group rather than the solvent), and 6 concentrations (0.2 ng L1, 1 ng L1, 2 ng L1, 10 ng L1, 20 ng L1, 100 ng L1) of 17b-E2 solution in every system in addition to 3 parallel samples of each dosage group. Groups of medium were uniformly accessed to 200 mL of methaneoxidizing bacteria cultures. After sealing the serum bottle with a silica gel plug, 15 mL of training system overhead gas was replaced with methane. It was placed in the shake culture at 30 8C for 24 h under 120 rpm and then uniformly sampled 1 mL in aseptic conditions. Thereafter, the bacterial fresh weights were harvested after centrifugation at 8000 g for 6 min at 4 8C. Finally, we calculated the methane-oxidizing bacteria growth rate. While it was re-injected with methane, we continued the thermostat shaking. Measuring occurred for 14 d with the objective of analyzing 17b-E2 pollution effects of methane-oxidizing bacteria growth rate. RESULTS
17b-E2 effect on the aquatic microbial methane oxidation function
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The concentration effect of 17b-E2’s pollution impact on the water microbial methane oxidation function is illustrated in Figure 1. Although the 17b-E2 polluted concentrations varied, the results show significant or extremely significant lower methane oxidation activity in water microbial than the blank control group, with its trends basically identical with each time concentration effect. When the concentration of 17b-E2 was 5ng L1, the rate of water microbial methane oxidation decreased with increasing 17b-E2 concentrations; otherwise, methane oxidation rate slowly rebounded with an increasing concentration of 17b-E2 and trended to a constant value. Further analysis showed that during the experimental time (7 d), with c (17b-E2) equal to 1.0 ng L1, methane oxidation rate was 1.6
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Figure 1. 17b-estradiol (17b-E2) concentration effect of pollution on the water microbial methane oxidation function. Statistical analysis was completed to compare experimental groups with control groups; denotes significant differences (p < 0.05); denotes extremely significant differences (p < 0.01). Error bars represent standard deviations.
E2 impacts on water microbial methane oxidation activity
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Figure 2. Linear regression analysis between methane oxidation rate and concentrations of 17b-estradiol (17b-E2) pollution. Error bars represent standard deviations.
17b-E2 pollution on water microbial community activity
Carbon dioxide is the major end product of aerobic microbial respiration; moreover, the strength of microbial respiration to some extent reflects the activity of the environmental microbiology group [33]. Therefore, the carbon dioxide production rate of the water was the main analysis indicator by which we judged the activity of microbial groups in the system. The analysis results of 17b-E2 pollution on water microbial community activity are shown in Figure 4. Concentrations of
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17b-E2 pollution on the CO2 generation rate of the water microbial have a significant role in promoting this process. That is, within a range of concentrations in the experimental design, 17b-E2 pollution significantly improved the activity of water microbial groups. Further analysis indicated that the CO2 generation rate of the control group changed slowly, and there was no significant difference in each time period. Notably, the CO2 generation rate significantly increased with time in the experimental group systems (within 120 h), and the microbial community activity in the system was enhanced significantly
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Figure 3. 17b-estradiol (17b-E2) time (h) effect of pollution on water microbial methane oxidation function. Error bars represent standard deviations.
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Figure 4. CO2 changes with concentrations of 17b-estradiol (17b-E2). Statistical analysis was completed to compare experimental groups with control groups; denotes significant differences (p < 0.05); denotes extremely significant differences (p < 0.01). Error bars represent standard deviations.
after 120 h incubation. As such, besides the high concentrations (17b-E2 concentrations for 50 ng L1 or 100 ng L1) of the experimental groups, the CO2 generation rate continued to increase, while the low concentrations of 17b-E2 pollution CO2 generation rate began to decrease. This may be a result of the system’s 17b-E2 being subject to partial degradation by microorganisms. In addition, in each time period, the concentrations of 17b-E2 pollution and CO2 generation rate appeared to have an obvious dose–effect relationship. In the groups, pairwise comparisons were shown to have a significant difference; CO2 production rate increased with increasing 17b-E2 additions. Linear regression analysis (parameters shown in Table 1) showed a significant linear correlation of r2 > 0.9 before 120 h between the water microbial CO2 production rate and 17b-E2 pollution concentrations. Hence, we believed that 17b-E2 pollution on water microbial groups activity had a stimulating effect and caused the stress response of water microbial physiology. The extent of its response was related linearly by concentrations of 17b-E2 pollution, thereby reducing the function of water microbials on methane oxidation. Its mechanism is not clear and needs further investigation.
Low, middle, and high concentrations of 17b-E2 pollution in the experimental groups (1.0 ng L1, 10.0 ng L1, and 100.0 ng L1) were compared with the blank control group. The growth curve of methane-oxidizing bacteria is shown in Figure 5 respectively. Figure 5 indicates the methane-oxidizing bacteria growth was slow cultured in laboratory conditions; furthermore, the growth change between each treatment group and control group on time effect was basically the same. In each treatment group, methane-oxidizing bacteria had significant growth on the third day, entered the fast-growing period on the sixth day, showed the highest growth rate on the seventh day, and by the ninth day the methane-oxidizing bacteria growth rate was reduced significantly. Based on the above analysis, we chose the systems methane-oxidizing bacteria growth rate of day 3, day 6, day 7, and day 9 for concentrations of 17b-E2 pollution effects analysis (Figure 6). Figure 6 shows the different concentration–effect relationships among the methane-oxidizing bacteria growth rate and concentrations of 17b-E2 pollution in each growing season. In the growth adaptation period (third day) with high concentrations of the 17b-E2 pollution group (100 ng L1), the remaining 17b-E2 pollution treatment groups of the methaneoxidizing bacteria growth rate were significantly higher than the blank control group. Among them, the methane-oxidizing bacteria growth rate was the fastest when 17b-E2 was 2.0 ng L1, whereas there was no significant difference between the treatment group and the control group when the concentration of 17b-E2 pollution was 100.0 ng L1. The rapid growth period (sixth day), except for the concentration of 1.0 ng L1, group was slightly lower than the control group, whereas the rest of the methane-oxidizing bacteria growth rate of the treatment groups were significantly or extremely significantly higher than the control group. Among them, the growth rate was the highest when 17b-E2 was 10.0 ng L1. After each treatment group system achieved the highest cell concentration (seventh day and ninth day), the dose–effect relationship was mostly the same among all treatment groups of methane-oxidizing bacteria growth rate and concentrations of 17b-E2 pollution. When the concentration of 17b-E2 was from 2.0 ng L1 to 10.0 ng L1, the treatment groups with methane-oxidizing bacteria the growth rate was higher than that of the control group, while the growth rate of the remainder were significantly lower than the control group.
Table 1. Fitting straight line parameters in concentrations of 17b-E2 conditionsa Model summaryb Time (h) 24 48 72 96 120 144 168
Parameter estimates
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0.9678 0.9805 0.8568 0.9962 0.9426 0.7872 0.5770
4.05E-05 1.15E-05 1.75E-03 1.98E-07 1.73E-04 0.00481 0.02906
0.36194 0.35254 0.40454 0.42887 0.46480 0.57683 0.34816
0.00513 0.00350 0.00501 0.00377 0.00433 0.00589 0.01576
0.00513 0.00350 0.01307 0.00717 0.00198 0.01411 0.00655
0.00010 0.00019 0.00031 0.00007 0.00012 0.00069 0.00095
Fitting a straight line equation as y ¼ a þ bx. r for goodness of fit; p for groups’ one-way ANOVA of t-test. Standard error for standard deviation of constant a. d Standard error for standard deviation of slope b. 17b-E2 ¼ 17b-estradiol a
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E2 impacts on water microbial methane oxidation activity
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Figure 5. The flora growth curve of methane-oxidizing bacteria under 17b-estradiol (17b-E2) pollution conditions. Error bars represent standard deviations.
The analysis results and laboratory finding of the 17b-E2 pollution effect on water methane oxidation function were not completely consistent. One explantation for this is that the proportion of culturable methane-oxidizing bacteria in the environment of methane-oxidizing bacteria populations is rare and there may be differences between the 17b-E2 pollution response mechanism and the groups. A second explantation for this is that the growth and reproduction of the methane-oxidizing bacteria class in the different 17b-E2 pollution conditions may differ. Therefore, the mechanism of 17b-E2 pollution methane-
oxidizing bacteria in the environment is very complex. This mechanism is under further investigation. DISCUSSION
Methane was an important chemical raw material and chemical energy, but is also an extremely important greenhouse gas. Methane-oxidizing bacteria are a class of microorganisms under aerobic conditions with methane as their sole carbon and energy source for growth and reproduction. Methane-oxidizing
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Figure 6. Methane-oxidizing bacteria growth rate changes with concentrations of 17b-estradiol (17b-E2). Statistical analysis was completed to compare experimental groups with control groups; denotes significant differences (p < 0.05); denotes extremely significant differences (p < 0.01). Error bars represent standard deviations.
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bacteria are widely distributed in soil, marshes, rice fields, lakes, rivers, and the ocean, which are the most important biological barriers to preventing methane emissions into the atmosphere. In the experimental system designed for the present study, all remaining conditions were kept the same except for different concentrations of 17b-E2. Therefore, the results reflect only environmental hormone pollution on microbial methane oxidation activity in the aquatic environment. The CH4 and CO2 will dissolve in the water; however, the amount of dissolved methane is very small. After every 24-h ventilation during the experiment and prior to collecting the gas samples, we shook the bottles and then measured the difference values of gas concentration (CH4 for decrease; CO2 for increase). As such, the experimental error (concentration of CH4 and CO2 in the liquid phase) was reduced to its minimum. Judged from the enzymatic reaction of microbial methane oxidation pathway, methane monooxygenase was a key enzyme in the pathway. We investigated which activity and content of methane oxidation rate in the system is related to the system of microbial methane oxidation activity [30]. The results of the present study indicated that 17b-E2 pollution showed a statistically significant inhibition of methane oxidation activity. Low concentrations of 17b-E2 pollution played a role in promoting the growth of methane-oxidizing bacteria. Therefore, we believe that the existence of methane monooxygenase activity was likely to be inhibited by the degradation products of 17b-E2 or was 17b-E2 directly, either of which acted as a disincentive to generating methane monooxygenase. The mechanism of 17b-E2 pollution inhibition of water microbial methane oxidation still needs further research. The 17b-estradiol is a fourth-ring structure, molecular formula for C18H24O2, which has limited solubility in water and is difficult to decompose naturally. However, prior studies have shown that 17b-E2 can be degraded by a small number of microbials [34–35]. The present study resulted in concentrations of 17b-E2 pollution and water microbial activity appearing as a significant positive linear correlation (r2 > 0.9). Methaneoxidizing bacteria in the system used methane as a carbon source for growth of synthetic material itself. It adopted respiration to oxide methane and thereby produced CO2, which was used to obtain the energy required for their metabolism. This explains why the total carbon dioxide production rate in Figure 4 is lower than the methane oxidation rate in Figure 1. Overall 17b-E2 inhibited water microbial methane oxidation activity, while at the same time promoted total microbial activity. We believe that there may be 2 reasons for these findings. First, added 17b-E2 could harm the normal physiological function of water microorganisms. Even when microbial populations dominated by methane oxidation flora were given the stress effect, respiratory metabolism speeded up; therefore, the methane oxidation function was reduced. Second, adding 17b-E2 provided a carbon source for water microbial systems and promoted their growth, thus reduced the use of methane. Its mechanism is still under study. In summary, 17b-E2 pollution demonstrated a significant inhibitory effect on water microbial methane oxidation activity, which indirectly promoted the release of water methane. In addition, 17b-E2 pollution played a significant role in promoting water production and release of CO2. Therefore, we believe that environmental estrogen pollution’s effect on water greenhouse gas discharge exists with regularity; that is, 17b-E2 pollution on water methane and carbon dioxide release creates the presence of a catalytic role. Furthermore, research regarding 17b-E2 pollution’s impact on the methane oxidation function indeed
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is reported. The present study was the first to explore the effect of environmental estrogen pollution on water greenhouse gas emissions, while the mechanism of microbial ecology is still under investigation. Acknowledgment—A. Ruan and F. Zong contributed equally to this paper. This work was supported financially by the National Natural Science Foundation of China (Grant 51378175), the Special Fund of State Key Laboratory of China (2011585112), and the open fund project of State Key Laboratory of Hydrology-Water Resources and Hydraulic Engineering (2011490211). The authors thank the Hohai University State Key Laboratory of Hydrology and Water Resources for allowing us to use their laboratory space and equipment. REFERENCES 1. Jiang XZ. 1997. The potential impact of environmental estrogens on human health. Chinese Journal of Public Health 13:251–252. 2. Hansen PD, Dizer H, Hock B, Marx A, Sherry J, McMaster M, Blaise CH. 1998. Vitellogenin-a biomarker for endocrine disruptors. Trends Anal Chem 17:448–451. 3. Nogueira JMF. 2003. Desreguladores endócrinos: Efeitos adversos e estrategias para monitoração dos sistemas aquáticos. Química 88:65–71. 4. Rahman MF, Yanful EK, Jasim SY. 2009. Endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs) in the aquatic environment: Implications for the drinking water industry and global environmental health. J Water Health 7:224–243. 5. Isidori M, Bellotta M, Cangiano M, Parrella A. 2009. Estrogenic activity of pharmaceuticals in the aquatic environment. Environ Int 35:826–829. 6. Chen WX, Xie GJ, Wang YY, Zhang J, Zhang JH. 2008. Review of studies on treatment of 17b-estradiol. Environmental Science Tribune 27:1–3. 7. World Resources Institute, United Nations Environment Programme, the United Nations Development Programme, and the World Bank. 1999. The World Resources Report: Environmental change and Human Health (1998–1999). China Environmental Science Press, Beijing, China. 8. US Department of Health and Human Services, National Institute of Environmental Health Sciences. 1994. Seventh Annual Report on Carcinogens Summary. Research Triangle Park, NC. 9. Ji SL, Liu ZP, Ren HY. 2005. Separation of steroidal estrogens advantage degrading bacteria in the wastewater and characteristics. China Environmental Science 5:585–588. 10. Ren HY, Ji SL, Cui CW, Liu ZP, Wang D, Chen S. 2004. Vehicle pollution condition and remove way of estrogen. China Water & Wastewater 20:24–26. 11. Tabata A, Kashiwa S, Ohnishi Y. 2001. Estrogenic influence of ertradiol-17b, pnonylphenol and bisphenol environmental concentrations. Water Sci Technol 43:109–116. 12. Lguchi T, Watanabe H, Katsu Y. 2001. Developmental effects of estrogenic agents on mice, fish, and frogs. Horm Behav 40:248– 251. 13. Tilton F, Benson WH, Schlenk D. 2002. Evaluation of estrogenic activity from a municipal wastewater treatment plant with predominantly domestic input. Aquat Toxicol 61:211–224. 14. Chiu TY, Koh YKK, Paterakis NL, Boobis AR, Cartmell E, Richards KH, Lester JN. 2009. The significance of sample mass in the analysis of steroid estrogen in sewage sludge and the derivation of partition coefficients in wastewater. Chromatography 1216:4923–4926. 15. Kjaer J, Olsen P, Bach K, Barlebo HC, Ingerslev F, Hansen F, Sorensen BH. 2007. Leaching of estrogenic hormones from manure-treated structured soils. Environ Sci Technol 41:3911–3917. 16. Bradley PM, Barber LB, Chapelle FH, Gray JL, Kolpin DW, McMahon PB. 2009. Biodegradation of 17b-estradiol, estrone, and testosterone in stream sediments. Environ Sci Technol 43:1902–10. 17. Finlay-Moore O, Hartel PG, Cabrera ML. 2000. 17b-estradiol and testosterone in soil and run-off from grasslands amended with broiler litter. J Environ Qual 29:1604–11. 18. Kolodziej EP, Sedlak DL. 2007. Range land grazing as a source of steroid hormones to surface waters. Environ Sci Technol 41:3514–20. 19. Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999–2000: A national reconnaissance. Environ Sci Technol 36:1202–1211. 20. Lee YC, Wang LM, Xue YH, Ge NC, Yang XM, Chen GH. 2006. Natural estrogens in the surface water of Shenzhen and the sewage discharge of Hong Kong. Hum Ecol Risk Assess 12:301–312.
E2 impacts on water microbial methane oxidation activity 21. Kavlock RJ. 1999. Overview of endocrine disruptor research activity in the United States. Chemosphere 39:1227–1236. 22. Purdom CE, Hardiman PA, Bye VJ, Eno NC, Tyler CR, Sumpter JP. 1994. Estrogenic effects of effluents from sewage treatment works. Chem Ecol 8:275–285. 23. Rajapakse N, Ong D, Kortenkamp A. 2001. Defining the impact of weakly estrogenic chemicals on the action of steroidal estrogens. Toxicol Sci 60:296–304. 24. Aidong R, Ying Z, Chenxiao L, Yuan W, Xianchuan X. 2013. Effect of low concentration 17b-estradiol on the emissions of CH4 and CO2 in anaerobic sediments. Environ Toxicol Chem 32:2672– 2677. 25. Zheng J. 2003. The greenhouse effect impact on the environment. Shandong Environment 1:51–52. 26. Dickinson RE, Cicerone RJ. 1986. Future global warming from atmospheric trace gases. Nature 319:109–115. 27. Amato FD, Mazzinghi P, Castagnoli F. 2002. Methane analyzer based on TDL’s for measurements in the lower stratosphere: Design and laboratory tests. Appl Phys B 75:195–202.
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28. Cicerone RJ, Oremland RS. 1988. Biogeochemical aspects of atmospheric methane. Glob Biogeochem Cycles 2:299–327. 29. Nakicenovic N, Swart R, eds. 2000. Special Report on Emissions Scenarios. Intergovernmental Panel on Climate Change, Cambridge University Press, Cambridge, UK. 30. Hanson RS, Hanson TE. 1996. Methanotrophic bacteria. Microbiol Rev 60:439–471. 31. Ji SL, Han L, Ren HY, Cui CW, Liu ZP, Zhang GJ. A Strain of Raoultella sp. 2006. ZY3 for degrading sex steroid hormones. J Beijing University of Technology 32:196–202. 32. Steinkamp R, Zimmer W, Papen H. 2001. Improved method for detection of methanotrophic bacteria in forest soils by PCR. Curr Microbiol 42:316–322. 33. Institute of Soil Science, Chinese Academy of Sciences. 1985. Soil microbial method. Science Press, Beijing, China. 34. Kou S, Lang L, Ji YB. 2012. Toxic effect of estradiol on endocrine. Safe Environ Engin 19:15–18. 35. Li FS, He YL. 2003. Study on Biodegradation Characteristics of 17betaestradiol (E2). China Water & Wastwater 19:9–12.
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EFFECTS OF 17b-ESTRADIOL POLLUTION ON WATER MICROBIAL METHANE OXIDATION ACTIVITY AIDONG RUAN,*y FENGJIAO ZONG,y YING ZHAO,y CHENXIAO LIU,y and JING CHENz yState Key Laboratory of Hydrology-water Resources and Hydraulic Engineering, Hohai University, Nanjing, China zState Key Joint Laboratory of Environment Simulation and Pollution Control, School of Environment, Beijing Normal University, Beijing, China (Submitted 6 August 2013; Returned for Revision 26 November 2013; Accepted 20 December 2013) Abstract: 17b-estradiol (17b-E2), a widespread and natural estrogen in the environment, has imposed a serious threat to the safety and function of aquatic ecosystems because of worsening pollution and high potential toxicity. In the present study, the authors focus on the impact of 17b-E2 pollution on water microbial methane oxidation function. The authors investigated the mechanism of its influence on water microbial activity and discuss the growth rate of methane-oxidizing bacteria. The results showed that 17b-E2 could significantly inhibit the function of water microbial methane oxidation. When 17b-E2 concentration was 5 ng L1, the methane oxidation rate increased with increasing 17b-E2 and finally settled to a constant value. Furthermore, the authors found no significant linear correlation between 17b-E2 concentrations and its methane oxidation rate. However, increasing 17b-E2 dramatically improved water microbial community activity, because a significant or highly significant promotion in the generation rate of CO2 was measured. Moreover, within a certain period of time and at certain concentrations, positive linear correlation existed between water CO2 generation rate and 17b-E2 concentrations. In addition, the growth rate of culturable methane-oxidizing bacteria was promoted when 17b-E2 pollution concentration from 2 ng L1 to 20 ng L1. Therefore, 17b-E2 pollution can inhibit microbial methane oxidation function in water, which indirectly promotes the release of water methane and directly contributes to the rate of water-generated and released CO2. Specifically, 17b-E2 pollution can promote water emissions of greenhouse gases. Environ Toxicol Chem 2014;33:768–775. # 2014 SETAC Keywords: 17b-estradiol
Methane
Carbon dioxide
Methane-oxidizing bacteria
Methane oxidation function
of 0.1 ng L1 to 88 ng L1. Finlay-Moore et al. [17] measured concentrations of 17b-E2 as high as 2530 ng L1 in runoff water from fields fertilized with poultry litter. Kolodziej and Sedlak [18] detected estrone in 78% of collected samples with concentrations as high as 38 ng L1 and measured 17b-E2 in 18% of samples with concentrations as high as 1.7 ng L1. In monitoring 256 water samples from 109 rivers in Japan, Tabata et al. [11] reported that 222 water samples contained 17b-E2 with an average concentration of 2.1 ng L1. Kolpin et al. [19] surveyed America surface water in 30 states; in the 139 extracted water samples, nearly 40% registered pollution levels of 17b-E2. Lee et al. [20] detected total estrogen material of 260 ng L1 to 300 ng L1 in sewage outfalls of the sea in Shenzhen, China. Therefore, 17b-E2 has become a significant and widespread pollutant in water environments and has received international academic attention. Currently, 17b-E2 research is focused mainly on animal toxicology. It has been reported that 17b-E2 can impact humans and animals significantly [21–22] at concentrations of 1.0 ng L1 and through synergy with other substances 17b-E2 activity strengthens [23]. However, research focused on ecological toxicology is rarely seen in literature. Recently, we [24] explored the regularity effects of 17b-E2 on methane emissions in anaerobic sediments. The production of methane and methane oxidation is a contradiction, so we assumed 17b-E2 impacted methane oxidation. In the present study, we explored the impact of 17b-E2 pollution on the water methane generation and the release from the perspective of microbial methane oxidation. Methane is a natural energy and greenhouse gas accounting for more than 15% of global atmospheric warming [28]. The greenhouse effects it produces are 20 to 30 times the quality of carbon dioxide [25], and the atmospheric ozone layer destruction can reach 7 times that of CO2 [26–27]. According to a report from the
INTRODUCTION
Environmental estrogens are exogenous substances that can enter the human body. Their characteristics interfere with the normal secretion in vivo synthesis, release, transport, metabolism, combine and other processes, and activate or inhibit the endocrine system function. This undermines the body’s collective ability to maintain stability and regulatory effects [1]. The pollution of environmental estrogens has long been a major global environmental issue following discovery of the greenhouse effects and ozone depletion. Even very small amounts of estrogen [2–3] can cause hormone imbalance, genital malformations, and even cancer [4–5] in organisms. Environmental estrogens are also difficult to degrade and toxicity has long-term potential; indeed, certain species or even regional ecosystems have caused disastrous damage when harmful symptoms appear [6]. Over time, environmental estrogens (especially in water environments) have been significant environmental pollutants; the scientific issues of migration, conversion, distribution, toxicological effects, and degradation mechanisms have gradually become an international hot field for research [7]. The 17b-estradiol (17b-E2) is the most active, strongest, and most widely distributed environmental estrogen and is considered one of the natural steroidal estrogens that is a direct threat to human survival. It is listed as a carcinogen by the US Department of Health and Human Services [8]. 17b-estradiol is widely distributed in waste, animal waste, sludge, soil, surface water, and groundwater environments [9–15]. In the continual effluent discharge of wastewater treatment plants (WWTP) containing 17b-E2 concentrations, Bradley et al. [16] found concentrations * Address correspondence to [email protected]. Published online 10 January 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/etc.2516 768
E2 impacts on water microbial methane oxidation activity
Intergovernmental Panel on Climate Change [29], approximately 60 Tg global methane was released in 2002; this number tended to increase yearly. Our research questions in the present study are: 1) How is the pollution of environmental estrogen related to the methane-producing bacteria and methaneoxidizing bacteria community structure? and 2) How do these contribute to methane emissions? Our research indicated that 17b-E2 pollution in the aquatic microbial system has influences on regularity, and low concentrations of 17b-E2 pollution inhibit anaerobic microorganism group activity and promote the generation and release of methane [24]. However, methaneoxidizing bacteria based on methane as its sole carbon and energy source and oxidized methane to methanol at room temperature [30] (which has been the most important biological barrier for preventing methane discharge) has played a major role in the global methane balance. In view of this, the present study analyzed the 17b-E2 effect of water body sediment microbial activity of methane oxidation under aerobic conditions. Furthermore, from the perspectives of water microorganism group activity and culturable methaneoxidizing bacteria growth rate, we explored the impact of 17b-E2 pollution on the aquatic microbial methane oxidation function mechanism. MATERIALS AND METHODS
Laboratory water microcosms construction of simulation system
Test samples of sediment and water were collected from China’s Yangtze river in Nanjing. Among them, the sediment samples were collected from 5 cm to 15 cm of mud below the interface [31]. Sediment samples were stored in the laboratory in a dark, well-ventilated location. After air-drying, we evaluated the average moisture content of the samples after grinding, sieving, and mixing. All laboratory system incubations were performed in 150 mL serum bottles, capped with 6 layers of gauze, using 40 g powdery sediment and 100 mL water samples. The headspace was maintained at 30 mL. In this way, the laboratory water micro-ecological simulation system was constructed and used to study the effects of 17b-E2 pollution in the aquatic microbial activity of methane oxidation. All of the samples were stationary cultured in an incubator (DK-GJ003; Memmert) for 72 h at 28 8C. Enrichment of methane-oxidizing bacteria in simulation system
The 72-h-culture-activated simulation systems were sealed by the destroyed bacteria of an isobutyl rubber plug under sterile conditions. The gases in the system were pumped in 5 mL portions by syringe, then injected with 5 mL methane gas (99.90%; Nanjing Special Gas). Finally, we put it in the incubator under 120 rpm shake culture at 28 8C. Next, 2 mL of gas was extracted from the system every 24 h, which was applied to analyze the upper gas concentrations of CH4 and CO2. The gas phase of the bottle was refreshed by air in a sterile desk for 20 min, corked tightly after replacing a volume of 5 mL of CH4, and returned to the shake culture at 28 8C. For 7 consecutive days (pre-experiments confirmed that the methane oxidation rate was relatively stable for 7 d), methane oxidative bacteria was enriched. 17b-E2 pollution treatment scheme
In every system we used a blank control group (n ¼ 3) without 17b-E2 (99%; J&K) along with 6 concentrations of 17b-E2 solution (0.5 ng L1, 1.0 ng L1, 5.0 ng L1, 10.0 ng L1, 50.0 ng L1, and 100.0 ng L1) respectively, in addition to creating 3 parallel
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samples in each dosage group. Each group was replaced in the 28 8C incubator for shaking. We collected processing system gas every 24 h and analyzed its system of CH4 and CO2 concentration, gas pressure, and temperature. Following the gas phase, each bottle was refreshed with fresh air, then corked tightly to a replaced volume of 5 mL of CH4. We then continued shaking the culture at 28 8C. Operations were thus continuous for 8 d (the first day was without 17b-E2), with the objective of analyzing 17b-E2 pollution in the aquatic microbial communities activity and the impact of methane oxidizing bacteria methane oxidation function. Determining CH4, CO2, and analysis method
The CH4 and CO2 concentrations were measured by gas chromatograph (GC-7890A; Alilent). The system was equipped with a capillary gas chromatography column (a stainless steel -2mm 2m; stationary phase, HayeSep Q80/100) and a flame ionization detector. Pure N2 (99.999%), pure H2, and pure air served as the carrier gas, fuel gas, and supporting gas at a flow rate of 3.0 mL min1, 40 mL min1, and 400 mL min1, respectively. The temperature of the column, detector, and injector were 40 8C, 300 8C, and 100 8C, respectively. The retention times of CH4 and CO2 were 2.2 min and 4.4 min, respectively. The detection limits of CH4 and CO2 were 0.2 mL L1 and 0.03 mL L1, respectively. OriginPro 8 and Microsoft Office Excel 2007 software were used with the test data for statistical analysis. The rate of CH4 oxidized and CO2 produced the formula v¼
M 22:4
273 P ppm 273þT 101325 103 V mt
where v is the gas production rate (mg g1 h1); M is the molar mass of CH4 or CO2 (g mol1); 22.4 L mol 1 indicates the molar volume of gas under standard conditions (standard temperature and pressure). The notation ppm (parts per million) is the gas volume concentration; T is the gas temperature (8C); P is the gas pressure (Pa); V is the headspace volume (mL); m is the dry weight of the sediment (g); and t is the cumulative gas oxidation or production time (h). Methane-oxidizing bacteria cultivation and enrichment
We took 1 mL mixture of the laboratory micro-ecological simulation system in the control group, which was evenly diluted 1000-fold via the sterilization of saline solution. We then accessed the methane-oxidizing bacteria inorganic salt liquid medium with methane as the sole carbon and energy source. Finally, we put it under 120 rpm shake culture for 72h at 28 8C. Every 24 h we replaced fresh air in the sterile console, and the upper gas volume of 10 mL was replaced with methane. Cultures then continued to be diverted and cultured another 3 times, which thus enriched the methane-oxidizing bacteria mixed strains of methane as a sole carbon source and energy. The main components for methane-oxidizing bacteria inorganic salt medium were: KH2PO4, 1.0 g L1; Na2HPO4 12H2O, 2.9 g L1; MgSO4 7H2O, 0.32 g L1; (NH4)2SO4, 3.0 g L1; 10 mL mineral elements solution [32]; and 990 mL distilled water, pH 6.8. The configuration of a solution of mineral elements was: MgSO4·7H2O 3 g; MnSO4·2H2O 0.5 g; NaCl 1g; FeSO4·7H2O 0.1g; CoCl2 0.1g; CaCl2·2H2O 0.1g; ZnSO4·7H2O 0.1g; CuSO4·5H2O 0.01g; AlK(SO4)2 0.01g; H3BO3 0.01g; and Na2MoO4·2H2O 0.01g in 1000 mL volumetric flask. This was fully dissolved in distilled water and diluted to 1000 mL, then readied to use after high-pressure steam sterilization.
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60.79% lower than system water receiving the 17b-E2 treatment compared to system water receiving no 17b-E2. When c (17bE2) equaled 5.0 ng L1, methane oxidation rate was 83.18% lower; when c (17b-E2) equaled 50.0 ng L1, it was 68.23% lower; and when c (17b-E2) equaled 100.0 ng L1, it was 56.98% lower. The linear regression analysis results among various concentrations of 17b-E2 pollution and methane oxidation rate showed no significantly linear correlation in each time period. However, in the low concentration range, that is. c (17b-E2) < 5.0 ng L1, 17b-E2 inhibition methane oxidation effects in the system increased with increasing concentrations of 17b-E2 additions, showing a linear dose– effect relationship (Figure 2). The methane oxidation activity of the system as it changed over time is shown in Figure 3 under concentrations of 17b-E2 pollution. The system of microbial methane oxidation activity exhibited the cyclical process of change, and there was a significant difference between the treatment group and the control group, as seen in Figure 3. The change of microbial methane oxidation activity was larger than water receiving no 17b-E2 treatment, with a longer period of change; whereas the change was significantly faster from water receiving 17b-E2 treatment that has been caused shorten in the change cycle, and the changes were basically the same in all treatment groups. In a future study, we will focus on exploring why a sharp increase exists in the 96-h time period. The above analysis shows a significant inhibition of 17b-E2 pollution exists on a water microbial system methane oxidation function. Within all the pollution concentrations range of the experimental design, there was no significant linear correlation between the methane oxidation rate and concentrations of 17bE2. Hence, the inhibition mechanism of 17b-E2 pollution on water microbial methane oxidation function is more complex. Further research is warranted to explore the mechanisms of 17bE2 pollution on water microbial methane oxidation function, so we have been studying the impact of 17b-E2 pollution on water microbial community activity and cultures of methane-oxidizing bacteria growth rate.
17b-E2 pollution effects of growing methane-oxidizing bacteria
Sterilized methane-oxidizing bacteria inorganic liquid medium was dispensed into 21 sterile 200 mL serum bottles with 50 mL per bottle. There was a blank control group (n ¼ 3) without the 17b-E2 (control group rather than the solvent), and 6 concentrations (0.2 ng L1, 1 ng L1, 2 ng L1, 10 ng L1, 20 ng L1, 100 ng L1) of 17b-E2 solution in every system in addition to 3 parallel samples of each dosage group. Groups of medium were uniformly accessed to 200 mL of methaneoxidizing bacteria cultures. After sealing the serum bottle with a silica gel plug, 15 mL of training system overhead gas was replaced with methane. It was placed in the shake culture at 30 8C for 24 h under 120 rpm and then uniformly sampled 1 mL in aseptic conditions. Thereafter, the bacterial fresh weights were harvested after centrifugation at 8000 g for 6 min at 4 8C. Finally, we calculated the methane-oxidizing bacteria growth rate. While it was re-injected with methane, we continued the thermostat shaking. Measuring occurred for 14 d with the objective of analyzing 17b-E2 pollution effects of methane-oxidizing bacteria growth rate. RESULTS
17b-E2 effect on the aquatic microbial methane oxidation function
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The concentration effect of 17b-E2’s pollution impact on the water microbial methane oxidation function is illustrated in Figure 1. Although the 17b-E2 polluted concentrations varied, the results show significant or extremely significant lower methane oxidation activity in water microbial than the blank control group, with its trends basically identical with each time concentration effect. When the concentration of 17b-E2 was 5ng L1, the rate of water microbial methane oxidation decreased with increasing 17b-E2 concentrations; otherwise, methane oxidation rate slowly rebounded with an increasing concentration of 17b-E2 and trended to a constant value. Further analysis showed that during the experimental time (7 d), with c (17b-E2) equal to 1.0 ng L1, methane oxidation rate was 1.6
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Figure 1. 17b-estradiol (17b-E2) concentration effect of pollution on the water microbial methane oxidation function. Statistical analysis was completed to compare experimental groups with control groups; denotes significant differences (p < 0.05); denotes extremely significant differences (p < 0.01). Error bars represent standard deviations.
E2 impacts on water microbial methane oxidation activity
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Figure 2. Linear regression analysis between methane oxidation rate and concentrations of 17b-estradiol (17b-E2) pollution. Error bars represent standard deviations.
17b-E2 pollution on water microbial community activity
Carbon dioxide is the major end product of aerobic microbial respiration; moreover, the strength of microbial respiration to some extent reflects the activity of the environmental microbiology group [33]. Therefore, the carbon dioxide production rate of the water was the main analysis indicator by which we judged the activity of microbial groups in the system. The analysis results of 17b-E2 pollution on water microbial community activity are shown in Figure 4. Concentrations of
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Figure 3. 17b-estradiol (17b-E2) time (h) effect of pollution on water microbial methane oxidation function. Error bars represent standard deviations.
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0.0 24
48
72
96
120
144
168
Time (h)
Figure 4. CO2 changes with concentrations of 17b-estradiol (17b-E2). Statistical analysis was completed to compare experimental groups with control groups; denotes significant differences (p < 0.05); denotes extremely significant differences (p < 0.01). Error bars represent standard deviations.
after 120 h incubation. As such, besides the high concentrations (17b-E2 concentrations for 50 ng L1 or 100 ng L1) of the experimental groups, the CO2 generation rate continued to increase, while the low concentrations of 17b-E2 pollution CO2 generation rate began to decrease. This may be a result of the system’s 17b-E2 being subject to partial degradation by microorganisms. In addition, in each time period, the concentrations of 17b-E2 pollution and CO2 generation rate appeared to have an obvious dose–effect relationship. In the groups, pairwise comparisons were shown to have a significant difference; CO2 production rate increased with increasing 17b-E2 additions. Linear regression analysis (parameters shown in Table 1) showed a significant linear correlation of r2 > 0.9 before 120 h between the water microbial CO2 production rate and 17b-E2 pollution concentrations. Hence, we believed that 17b-E2 pollution on water microbial groups activity had a stimulating effect and caused the stress response of water microbial physiology. The extent of its response was related linearly by concentrations of 17b-E2 pollution, thereby reducing the function of water microbials on methane oxidation. Its mechanism is not clear and needs further investigation.
Low, middle, and high concentrations of 17b-E2 pollution in the experimental groups (1.0 ng L1, 10.0 ng L1, and 100.0 ng L1) were compared with the blank control group. The growth curve of methane-oxidizing bacteria is shown in Figure 5 respectively. Figure 5 indicates the methane-oxidizing bacteria growth was slow cultured in laboratory conditions; furthermore, the growth change between each treatment group and control group on time effect was basically the same. In each treatment group, methane-oxidizing bacteria had significant growth on the third day, entered the fast-growing period on the sixth day, showed the highest growth rate on the seventh day, and by the ninth day the methane-oxidizing bacteria growth rate was reduced significantly. Based on the above analysis, we chose the systems methane-oxidizing bacteria growth rate of day 3, day 6, day 7, and day 9 for concentrations of 17b-E2 pollution effects analysis (Figure 6). Figure 6 shows the different concentration–effect relationships among the methane-oxidizing bacteria growth rate and concentrations of 17b-E2 pollution in each growing season. In the growth adaptation period (third day) with high concentrations of the 17b-E2 pollution group (100 ng L1), the remaining 17b-E2 pollution treatment groups of the methaneoxidizing bacteria growth rate were significantly higher than the blank control group. Among them, the methane-oxidizing bacteria growth rate was the fastest when 17b-E2 was 2.0 ng L1, whereas there was no significant difference between the treatment group and the control group when the concentration of 17b-E2 pollution was 100.0 ng L1. The rapid growth period (sixth day), except for the concentration of 1.0 ng L1, group was slightly lower than the control group, whereas the rest of the methane-oxidizing bacteria growth rate of the treatment groups were significantly or extremely significantly higher than the control group. Among them, the growth rate was the highest when 17b-E2 was 10.0 ng L1. After each treatment group system achieved the highest cell concentration (seventh day and ninth day), the dose–effect relationship was mostly the same among all treatment groups of methane-oxidizing bacteria growth rate and concentrations of 17b-E2 pollution. When the concentration of 17b-E2 was from 2.0 ng L1 to 10.0 ng L1, the treatment groups with methane-oxidizing bacteria the growth rate was higher than that of the control group, while the growth rate of the remainder were significantly lower than the control group.
Table 1. Fitting straight line parameters in concentrations of 17b-E2 conditionsa Model summaryb Time (h) 24 48 72 96 120 144 168
Parameter estimates
r2
p
Constant a
Slope b
SEc
SEd
0.9678 0.9805 0.8568 0.9962 0.9426 0.7872 0.5770
4.05E-05 1.15E-05 1.75E-03 1.98E-07 1.73E-04 0.00481 0.02906
0.36194 0.35254 0.40454 0.42887 0.46480 0.57683 0.34816
0.00513 0.00350 0.00501 0.00377 0.00433 0.00589 0.01576
0.00513 0.00350 0.01307 0.00717 0.00198 0.01411 0.00655
0.00010 0.00019 0.00031 0.00007 0.00012 0.00069 0.00095
Fitting a straight line equation as y ¼ a þ bx. r for goodness of fit; p for groups’ one-way ANOVA of t-test. Standard error for standard deviation of constant a. d Standard error for standard deviation of slope b. 17b-E2 ¼ 17b-estradiol a
b 2 c
E2 impacts on water microbial methane oxidation activity
Environ Toxicol Chem 33, 2014 0.04
0.05 0.0 ng/L
1.0 ng/L
0.04
0.03
−1
The concentration of methanotrophs (g mL )
773
0.03 0.02 0.02 0.01
0.01 0.00 0.06
0
2
4
6
8
10
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14
0.00
0 0.04
2
4
6
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14
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10
0.03 0.04 0.03
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0
2
4
6 8 10 Time (d)
12
14
0.00
0
2
4
6 8 10 Time (d)
12
14
Figure 5. The flora growth curve of methane-oxidizing bacteria under 17b-estradiol (17b-E2) pollution conditions. Error bars represent standard deviations.
The analysis results and laboratory finding of the 17b-E2 pollution effect on water methane oxidation function were not completely consistent. One explantation for this is that the proportion of culturable methane-oxidizing bacteria in the environment of methane-oxidizing bacteria populations is rare and there may be differences between the 17b-E2 pollution response mechanism and the groups. A second explantation for this is that the growth and reproduction of the methane-oxidizing bacteria class in the different 17b-E2 pollution conditions may differ. Therefore, the mechanism of 17b-E2 pollution methane-
oxidizing bacteria in the environment is very complex. This mechanism is under further investigation. DISCUSSION
Methane was an important chemical raw material and chemical energy, but is also an extremely important greenhouse gas. Methane-oxidizing bacteria are a class of microorganisms under aerobic conditions with methane as their sole carbon and energy source for growth and reproduction. Methane-oxidizing
0.014
0.012 3d
**
−1
0.010 0.008
0.045
*
0.010
-**
0.006 0.004
6d
0.011
*
−1
The growth rate of methanotrophs (g mL d )
0.012
* 0
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0.040
* **
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*
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*
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0.035
-- *
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*
0.025 0.030 0.020 0.025 0.020
*
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20
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100 −1
Concentration of 17β-E2 (ng L )
0.010
* 0
**
** 20
40
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Concentration of 17β-E2 (ng L )
Figure 6. Methane-oxidizing bacteria growth rate changes with concentrations of 17b-estradiol (17b-E2). Statistical analysis was completed to compare experimental groups with control groups; denotes significant differences (p < 0.05); denotes extremely significant differences (p < 0.01). Error bars represent standard deviations.
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Environ Toxicol Chem 33, 2014
bacteria are widely distributed in soil, marshes, rice fields, lakes, rivers, and the ocean, which are the most important biological barriers to preventing methane emissions into the atmosphere. In the experimental system designed for the present study, all remaining conditions were kept the same except for different concentrations of 17b-E2. Therefore, the results reflect only environmental hormone pollution on microbial methane oxidation activity in the aquatic environment. The CH4 and CO2 will dissolve in the water; however, the amount of dissolved methane is very small. After every 24-h ventilation during the experiment and prior to collecting the gas samples, we shook the bottles and then measured the difference values of gas concentration (CH4 for decrease; CO2 for increase). As such, the experimental error (concentration of CH4 and CO2 in the liquid phase) was reduced to its minimum. Judged from the enzymatic reaction of microbial methane oxidation pathway, methane monooxygenase was a key enzyme in the pathway. We investigated which activity and content of methane oxidation rate in the system is related to the system of microbial methane oxidation activity [30]. The results of the present study indicated that 17b-E2 pollution showed a statistically significant inhibition of methane oxidation activity. Low concentrations of 17b-E2 pollution played a role in promoting the growth of methane-oxidizing bacteria. Therefore, we believe that the existence of methane monooxygenase activity was likely to be inhibited by the degradation products of 17b-E2 or was 17b-E2 directly, either of which acted as a disincentive to generating methane monooxygenase. The mechanism of 17b-E2 pollution inhibition of water microbial methane oxidation still needs further research. The 17b-estradiol is a fourth-ring structure, molecular formula for C18H24O2, which has limited solubility in water and is difficult to decompose naturally. However, prior studies have shown that 17b-E2 can be degraded by a small number of microbials [34–35]. The present study resulted in concentrations of 17b-E2 pollution and water microbial activity appearing as a significant positive linear correlation (r2 > 0.9). Methaneoxidizing bacteria in the system used methane as a carbon source for growth of synthetic material itself. It adopted respiration to oxide methane and thereby produced CO2, which was used to obtain the energy required for their metabolism. This explains why the total carbon dioxide production rate in Figure 4 is lower than the methane oxidation rate in Figure 1. Overall 17b-E2 inhibited water microbial methane oxidation activity, while at the same time promoted total microbial activity. We believe that there may be 2 reasons for these findings. First, added 17b-E2 could harm the normal physiological function of water microorganisms. Even when microbial populations dominated by methane oxidation flora were given the stress effect, respiratory metabolism speeded up; therefore, the methane oxidation function was reduced. Second, adding 17b-E2 provided a carbon source for water microbial systems and promoted their growth, thus reduced the use of methane. Its mechanism is still under study. In summary, 17b-E2 pollution demonstrated a significant inhibitory effect on water microbial methane oxidation activity, which indirectly promoted the release of water methane. In addition, 17b-E2 pollution played a significant role in promoting water production and release of CO2. Therefore, we believe that environmental estrogen pollution’s effect on water greenhouse gas discharge exists with regularity; that is, 17b-E2 pollution on water methane and carbon dioxide release creates the presence of a catalytic role. Furthermore, research regarding 17b-E2 pollution’s impact on the methane oxidation function indeed
A. Ruan et al.
is reported. The present study was the first to explore the effect of environmental estrogen pollution on water greenhouse gas emissions, while the mechanism of microbial ecology is still under investigation. Acknowledgment—A. Ruan and F. Zong contributed equally to this paper. This work was supported financially by the National Natural Science Foundation of China (Grant 51378175), the Special Fund of State Key Laboratory of China (2011585112), and the open fund project of State Key Laboratory of Hydrology-Water Resources and Hydraulic Engineering (2011490211). The authors thank the Hohai University State Key Laboratory of Hydrology and Water Resources for allowing us to use their laboratory space and equipment. REFERENCES 1. Jiang XZ. 1997. The potential impact of environmental estrogens on human health. Chinese Journal of Public Health 13:251–252. 2. Hansen PD, Dizer H, Hock B, Marx A, Sherry J, McMaster M, Blaise CH. 1998. Vitellogenin-a biomarker for endocrine disruptors. Trends Anal Chem 17:448–451. 3. Nogueira JMF. 2003. Desreguladores endócrinos: Efeitos adversos e estrategias para monitoração dos sistemas aquáticos. Química 88:65–71. 4. Rahman MF, Yanful EK, Jasim SY. 2009. Endocrine disrupting compounds (EDCs) and pharmaceuticals and personal care products (PPCPs) in the aquatic environment: Implications for the drinking water industry and global environmental health. J Water Health 7:224–243. 5. Isidori M, Bellotta M, Cangiano M, Parrella A. 2009. Estrogenic activity of pharmaceuticals in the aquatic environment. Environ Int 35:826–829. 6. Chen WX, Xie GJ, Wang YY, Zhang J, Zhang JH. 2008. Review of studies on treatment of 17b-estradiol. Environmental Science Tribune 27:1–3. 7. World Resources Institute, United Nations Environment Programme, the United Nations Development Programme, and the World Bank. 1999. The World Resources Report: Environmental change and Human Health (1998–1999). China Environmental Science Press, Beijing, China. 8. US Department of Health and Human Services, National Institute of Environmental Health Sciences. 1994. Seventh Annual Report on Carcinogens Summary. Research Triangle Park, NC. 9. Ji SL, Liu ZP, Ren HY. 2005. Separation of steroidal estrogens advantage degrading bacteria in the wastewater and characteristics. China Environmental Science 5:585–588. 10. Ren HY, Ji SL, Cui CW, Liu ZP, Wang D, Chen S. 2004. Vehicle pollution condition and remove way of estrogen. China Water & Wastewater 20:24–26. 11. Tabata A, Kashiwa S, Ohnishi Y. 2001. Estrogenic influence of ertradiol-17b, pnonylphenol and bisphenol environmental concentrations. Water Sci Technol 43:109–116. 12. Lguchi T, Watanabe H, Katsu Y. 2001. Developmental effects of estrogenic agents on mice, fish, and frogs. Horm Behav 40:248– 251. 13. Tilton F, Benson WH, Schlenk D. 2002. Evaluation of estrogenic activity from a municipal wastewater treatment plant with predominantly domestic input. Aquat Toxicol 61:211–224. 14. Chiu TY, Koh YKK, Paterakis NL, Boobis AR, Cartmell E, Richards KH, Lester JN. 2009. The significance of sample mass in the analysis of steroid estrogen in sewage sludge and the derivation of partition coefficients in wastewater. Chromatography 1216:4923–4926. 15. Kjaer J, Olsen P, Bach K, Barlebo HC, Ingerslev F, Hansen F, Sorensen BH. 2007. Leaching of estrogenic hormones from manure-treated structured soils. Environ Sci Technol 41:3911–3917. 16. Bradley PM, Barber LB, Chapelle FH, Gray JL, Kolpin DW, McMahon PB. 2009. Biodegradation of 17b-estradiol, estrone, and testosterone in stream sediments. Environ Sci Technol 43:1902–10. 17. Finlay-Moore O, Hartel PG, Cabrera ML. 2000. 17b-estradiol and testosterone in soil and run-off from grasslands amended with broiler litter. J Environ Qual 29:1604–11. 18. Kolodziej EP, Sedlak DL. 2007. Range land grazing as a source of steroid hormones to surface waters. Environ Sci Technol 41:3514–20. 19. Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD, Barber LB, Buxton HT. 2002. Pharmaceuticals, hormones, and other organic wastewater contaminants in U.S. streams, 1999–2000: A national reconnaissance. Environ Sci Technol 36:1202–1211. 20. Lee YC, Wang LM, Xue YH, Ge NC, Yang XM, Chen GH. 2006. Natural estrogens in the surface water of Shenzhen and the sewage discharge of Hong Kong. Hum Ecol Risk Assess 12:301–312.
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