导读 | Introduction
多环芳烃（PAHs）因其毒性高、持久性强、迁移范围广、生物蓄积性强等特点引起了人们的广泛关注。萘（Nap）和蒽（Ant）作为低环多环芳烃的典型代表，在石油产品加工过程中排放量最大。此外，Nap和Ant对微生物和土壤酶有严重危害，破坏了土壤中正常的物质循环。荧光素二乙酸酯水解酶（FDA水解酶）是土壤微生物活性和质量变化的可靠生化生物标志物。然而，低环多环芳烃对土壤FDA水解酶的影响及其机制尚不清楚。本文研究了萘（Nap）和蒽（Ant）两种典型的低环多环芳烃对6种不同性质土壤中FDA水解酶活性和动力学特性的影响。
Polycyclic aromatic hydrocarbons (PAHs) attracted researchers' interest due to their high toxicity, strong persistence, wide migration range, and bioaccumulation. As typical representatives of lower-ring PAHs, naphthalene (Nap) and anthracene (Ant) have the most significant emissions in the processing of petroleum products. Furthermore, Nap and Ant pose a severe hazard to microorganisms and soil enzymes, disrupting the normal circulation of substances in the soil. Fluorescein diacetate hydrolase (FDA hydrolase) is a reliable biochemical biomarker of changes in soil microbial activity and quality. However, the effect and mechanism of lower-ring PAHs on soil FDA hydrolase are still unclear. In this work, we investigated the effects of two typical lower-ring PAHs, naphthalene (Nap) and anthracene (Ant), on the activity and kinetic characteristics of FDA hydrolases in six soils differing in their properties.
一、FDA水解酶对多环芳烃胁迫的响应
| Response of FDA hydrolase activity to PAHs stress
Nap和Ant在不同浓度下对供试土壤FDA水解酶活性有抑制作用。无Nap处理土壤的FDA水解酶活性（9.76 ~ 39.01 μg−1 h−1）是1000 mg kg−1 Nap处理土壤（3.62 ~ 38.11 μg−1 h−1）的1.02 ~ 3.12倍。无Ant处理（10.09 ~ 37.16 μg−1 h−1）土壤酶活性是100 mg kg−1 Ant处理（2.87 ~ 22.66 μg−1 h−1）土壤酶活性的1.64 ~ 3.87倍。因此，Ant对土壤FDA水解酶活性的抑制率远高于Nap。当Nap浓度从50 mg kg−1增加到100 mg kg−1时，FDA水解酶活性的抑制率从2.62% ~ 10.96%增加到8.19% ~ 24.52%。当Ant浓度从2.5 mg kg−1增加到5 mg kg−1时，FDA水解酶活性的抑制率从4.45 ~ 15.30%增加到8.29 ~ 34.19%。在Nap和Ant的最大浓度下，6种土壤的FDA水解酶活性分别下降了2.31 ~ 67.96%和39.01 ~ 74.15%。
Nap and Ant inhibited soil FDA hydrolase activity at different concentrations in tested soils. The FDA hydrolase activities in soils without Nap treatment (9.76–39.01 μg−1 h−1) were 1.02–3.12 times greater than that under the 1000 mg kg mg kg−1 Nap treatment (3.62–38.11 μg−1 h−1). Similarly, the enzyme activities in soil without Ant treatment (10.09–37.16 μg−1 h−1) were 1.64–3.87 times greater than that under the 100 mg kg mg kg−1 Ant treatment (2.87–22.66 μg−1 h−1). Thus, the inhibition rate of soil FDA hydrolase activity by Ant was much higher than that by Nap. When the Nap concentration increased from 50 to 100 mg kg−1, the inhibition rate of the FDA hydrolase activity changed from 2.62 to 10.96 % to 8.19 to 24.52 %. When the Ant concentration increased from 2.5 to 5 mg kg−1, the inhibition rate of the FDA hydrolase activity changed from 4.45 to 15.30% to 8.29 to 34.19 %. At the maximum concentration of Nap and Ant, the FDA hydrolase activity in the 6 soils decreased by 2.31–67.96% and 39.01–74.15%, respectively.
图1 萘和蒽对土壤荧光素二乙酸酯水解酶活性的抑制率
Fig. 1 Inhibition rate of naphthalene and anthracene on soil fluorescein diacetate hydrolase activity
二、多环芳烃对土壤FDA水解酶动力学特性的影响
| Kinetic properties of soil FDA hydrolase as affected by PAHs
结果表明，6种土壤的FDA水解酶Vmax值都随着Nap和Ant浓度的增加而降低。除S6外，FDA水解酶的Km值也随PAHs浓度的增加而降低。当Nap和Ant浓度达到最大值时，土壤Km分别减少了35.84 ~ 74.47%和74.00 ~ 91.61%。随着Nap浓度的增加，FDA水解酶的Vmax /Km几乎没有变化，因为Vmax和Km具有相似的下降速率。而随着Ant的添加，只有S2、S4和S6的Vmax/Km有降低趋势，当Ant浓度为100 mg kg−1时，其Vmax/Km分别降低19.45%、15.70%和37.61%。结合Km和Vmax的变化发现，Nap和Ant对供试土壤FDA水解酶的影响相似，表现为反竞争性抑制（土壤S6除外）。土壤S6在Ant污染下Vmax降低，Km不变，表现为非竞争性抑制。
For Vmax, the result showed that the values of FDA hydrolase in 6 soils decreased as the concentration of Nap and Ant increased. The Km of FDA hydrolase decreased with an increase in PAHs concentration, except S6 under Ant contamination. When Nap and Ant concentrations increased to the maximum, Km decreased by 35.84–74.47% and 74.00–91.61% among tested soils. The Vmax/Km of the FDA hydrolase is almost unchanged with the increasing Nap concentration since both Vmax and Km had a similar decline rate. Whereas, with the addition of Ant, only the Vmax/Km in S2, S4, and S6 showed a decline of 19.45%, 15.70%, and 37.61%, respectively, when the Ant concentration increased to 100 mg kg−1. Combining the changes of Km and Vmax, it was found that the effect of Nap and Ant on FDA hydrolase was similar, which exhibited an uncompetitive inhibition due to the decrease of Km and Vmax under the Nap and Ant stress (except for soil S6). For soil S6, the Vmax decreased and the Km unchanged under Ant contamination, indicating noncompetitive inhibition.
Ki是酶-底物-抑制剂（ESI）复合物的解离常数，表示PAHs对酶-底物复合物的亲和力。较低的Ki表明ESI复合物的结合能力更强。NAP和Ant对FDA水解酶的Ki分别为0.192-1.051和0.019-0.087 mM。在Ant污染下，除土壤S6外，Ki小于无污染的Km（2.34–6.03×10-2 mM），表明ESI复合物的亲和力强于ES复合物。
The Ki, the dissociation constant for the enzyme-substrate-inhibitor (ESI) complex, denotes PAHs' affinity to the enzyme-substrate complex. A lower Ki suggests a stronger combining capacity of the ESI complex. The Ki of the Nap and Ant on FDA hydrolase were 0.192–1.051 and 0.019–0.087 mM, respectively. With Ant contamination, the Ki was smaller than Km without pollution (2.34–6.03×10−2 mM) except for soil S6, indicating that the affinity of the ESI complex was stronger than the ES complex.
图2 萘和蒽对土壤荧光素二乙酸酯水解酶动力学参数的影响
Fig. 2 Effect of naphthalene and anthracene on soil fluorescein diacetate hydrolase kinetic parameters
三、生态剂量值
| Ecological doses
为了评估Nap和Ant对FDA水解酶的生态毒性，采用完全抑制模型拟合PAHs浓度与酶活性的关系及其与Vmax的关系。利用该模型计算了Nap和Ant污染下土壤FDA水解酶的ED10和ED50。根据FDA水解酶活性的降低，Nap污染下的ED10和ED50分别为43.92 ~ 203.55 mg kg−1和395.26 ~ 917.43 mg kg−1。相比之下，对于Ant污染，ED10和ED50分别为2.19 ~ 17.53 mg kg−1和19.75 ~ 69.40 mg kg−1。对于Vmax，Nap污染下的ED10和ED50分别为20.02 ~ 139.14 mg kg−1、180.18 ~ 684.93 mg kg−1，Ant污染下的ED10和ED50分别为1.30 ~ 20.81 mg kg−1、11.74 ~ 27.62 mg kg−1。通过Vmax计算的ED值小于酶活性计算得到的ED值。
To assess the ecological toxicity of Nap and Ant on the FDA hydrolase, the relationship between PAH concentrations and the enzyme activity and its relationship with Vmax were fitted by a complete inhibition model. Using this model, the ED10 and ED50 of soil FDA hydrolase under Nap and Ant contamination were obtained. Based on the reduction of FDA hydrolase activity, the ED10 and ED50 under Nap contamination ranged from 43.92 to 203.55 mg kg−1 and 395.26 to 917.43 mg kg−1, respectively. In contrast, for Ant contamination, the ED10 and ED50 ranged from 2.19 to 17.53 mg kg−1 and 19.75 to 69.40 mg kg−1, respectively. For Vmax, the ED10 and ED50 ranged from 20.02 to 139.14 mg kg−1, 180.18 to 684.93 mg kg−1 under Nap contamination, and 1.30 to 20.81 mg kg−1，11.74 to 27.62 mg kg−1 under Ant contamination, respectively. The soil FDA hydrolase exhibited lower ED values calculated from Vmax than from enzyme activity.
四、影响Nap和Ant对土壤FDA水解酶毒性的主要因素
| Toxic effects of nanoplastics on aquatic organismsThe major factors influencing Nap and Ant toxicity to soil FDA hydrolase
Pearson相关分析进一步阐明了ED10（由Vmax计算）、Ki和土壤性质之间的关系。结果表明，Nap的ED10和Ki与SOM含量呈正相关（p＜0.05）。这表明Nap对土壤FDA水解酶的毒性随有机质含量的增加而降低。此外，Ant的ED10和Ki与SOM和TN呈显著正相关，表明随着SOM和TN含量的增加，Ant与FDA水解酶-底物复合物的结合能力下降。此外，ED10与Ki呈极显著正相关。
The Pearson correlation analysis was implemented to clarify further the relationships between ED10 (calculated from Vmax), Ki, and soil properties. The results showed that ED10 and Ki of Nap were positively correlated with the content of SOM (p < 0.05). It indicated that the toxicity of Nap to soil FDA hydrolase decreased with the increase of organic matter content. Additionally, ED10 and Ki of Ant were significantly positively correlated with the SOM and TN, suggesting that the binding ability between Ant and FDA hydrolase-substrate complexes decreased with the increase of SOM and TN content. In addition, the ED10 exhibited a highly significant positive correlation with Ki.
图3 土壤荧光素二乙酸酯水解酶抑制参数与土壤性质的相关性研究
Fig. 3 Correlations between inhibition parameters of soil fluorescein diacetate hydrolase and soil properties
主成分分析（PCA）中，保留用于PCA分析的两个成分分别解释了总方差的72.8%和70.8%，其中PC1和PC2分别解释了39.8%、43.7%和33.0%、27.1%。在Nap污染下，土壤ED10、Ki、SOM、粉粒、NH4+-N和CEC的PC1特征值均为正值（＞0.50）。在Ant污染下，土壤ED10、Ki、SOM、黏粒、NH4+-N、TN和CEC的特征值为正值（＞0.50）。土壤性质与参数指标间的余弦值表明，SOM与ED10、Ki呈正相关。
Regarding principal component analysis (PCA), the two components retained for PCA analysis explained 72.8% and 70.8% of the total variance, respectively, with PC1 and PC2 explaining 39.8%, 43.7% and 33.0%, 27.1% respectively. Under Nap contamination, PC1 showed a positive Eigenvalue (>0.50) for ED10, Ki, SOM, silt, NH4+-N and CEC. Under Ant contamination, PC1 showed a positive eigenvalue (>0.50) for ED10, Ki, SOM, clay, NH4+-N, TN and CEC. Results showed that SOM was positively correlated with ED10 and Ki according to the cosine value between soil properties and parameter indexes.
图4 纳米塑料诱导水生生物的氧化损伤示意图
Fig. 4 Effects of nanoplastic exposure on oxidative damage induction in aquatic organisms
总结 | Conclusions
本研究揭示了FDA水解酶对低环多环芳烃急性污染的响应及其机制。结果表明，Nap和Ant对土壤FDA水解酶活性均有显著抑制作用，且抑制类型以反竞争性抑制为主。土壤FDA水解酶活性和Vmax均可作为评价多环芳烃毒性的指标，其中Vmax更为敏感。相关分析、逐步回归分析和主成分分析表明，SOM是影响两种多环芳烃对FDA水解酶毒性的主要因素，其主要通过影响多环芳烃与酶-底物复合物的结合，从而影响其对FDA水解酶的毒性。本研究为评价多环芳烃污染土壤的潜在环境风险提供了理论依据。
This study demonstrated the response and mechanism of FDA hydrolase to acute pollution by the lower-ring PAHs. The results showed that Nap and Ant significantly inhibited the activity of soil FDA hydrolase, and the type of inhibition was mainly uncompetitive in tested soils. Both soil FDA hydrolase activity and Vmax can be used as indicators to evaluate PAHs toxicity, between which Vmax is more sensitive. Correlation analysis, stepwise regression analysis, and principal component analysis indicated that SOM was the major controlling factor influencing the two PAHs' toxicity to FDA hydrolase via affecting the combination of PAHs with the enzyme-substrate complex. This study provides a theoretical basis for evaluating the potential environmental risks of PAH-contaminated soils.
https://www.sciencedirect.com/science/
article/pii/S0048969723011373
本文内容来自ELSEVIER旗舰期刊Sci Total Environ第874卷发表的论文：
Li, Y., Wang, Z., Tian, H., Megharaj, M., Jia, H., He, W., 2023. Using soil enzyme Vmax as an indicator to evaluate the ecotoxicity of lower-ring polycyclic aromatic hydrocarbons in soil: Evidence from fluorescein diacetate hydrolase kinetics. Sci Total Environ 874, 162521.
DOI: https://doi.org/10.1016/j.scitotenv.2023.162521
第一作者：李岩 博士
西北农林科技大学资源环境学院
现为西北农林科技大学资源环境学院博士研究生。主要研究方向为有机污染物对土壤酶的生态毒理。
通讯作者：和文祥 教授
西北农林科技大学资源环境学院
西北农林科技大学资源环境学院教授，主要从事土壤生物化学、生态毒理和环境微生物学方面的研究工作，近年来更关注土壤酶的应用、有机和无机污染物的生态毒理、环境中有机污染物降解等。主持或参与多项国家自然科学基金项目及国家重点研发项目。
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