溢油及表面活性剂成分对刺参幼参的毒理效应研究

发布时间：2019-05-21 19:28

【摘要】：随着经济社会的发展，表面活性剂成分的应用延伸到生产和生活的各个方面，用途和用量也不断增加，并伴随生产和生活污水的排放进入环境中；除此之外，规模较大的突发性溢油事故不断频发，也给环境带来严重危害。本文重点研究常见的表面活性剂成分十二烷基磺酸钠（SDS）、十二烷基苯磺酸钠（SDBS）及消油剂处理海上溢油事故对海水养殖刺参（Stichopus japonicas）的影响，运用毒理学研究手段进行相应的毒理效应研究，开展了如下研究：为探究表面活性剂和消油剂处理溢油对海洋珍品刺参的毒理学效应，SDS、SDBS、消油剂、机械分散原油（water accommodated fraction,WAF）和化学分散原油（chemical enhanced water accommodated fraction,CEWAF）对刺参幼参的急性毒性。结果显示，SDS对4月龄刺参的72h-LC50和96h-LC50分别为15.03和10.89mg·L-1；SDBS对4月龄刺参的72h-LC50和96h-LC50分别为2.50和1.71mg·L-1；消油剂对4月龄刺参的96h-LC50为7498.94mg·L-1。由此可见，试验中所用消油剂属微毒性，毒性远远小于SDBS和SDS。原油、消油剂的96h大剂量表面涂抹并未使刺参幼参出现死亡，仅消油剂对刺参幼参产生较小的刺激，表现为躯体短时间的收缩。原油CEWAF对3月龄和4月龄刺参幼参的96h-LC50分别为246.09mg·L-1和399.15mg·L-1。为了优化原油WAF和CEWAF毒性试验中的暴露过程，探究了紫外分光光度法测定总石油烃浓度、实验室条件下的风化过程和组分的分析。结果显示，为了更准确地的测定试验原油CEWAF的TPH质量浓度，确定采用原油+消油剂的浓度-吸光度曲线作为原油CEWAF的总石油烃质量浓度标准曲线，消油剂在正己烷中有白色絮状物生成，在制备标准储备液后须充分静置（或以2000r/min离心5min），吸取上清液进行稀释，制备标准使用液，以免带入絮状物而干扰吸光度读数；在实验室条件下，80h内，机械分散的原油中总石油烃呈现线性风化的趋势，随着时间的推移风化速率减小。实验室条件下，化学分散的原油在120h内的风化趋势可以看出，开始的12h内总石油烃含量基本没有变化，但是12h到36h内出现了急剧的风化，36h后基本稳定下来，但仍存在波动。可通过一些方法减小误差，例如可以考虑在保证对受试生物无伤害的情况下频繁的更换试验液，WAF建议2～3小时换液一次，CEWAF建议10～12小时换液一次，也可测定试验起始时总石油烃含量，并做相关说明。试验原油WAF中的多环芳烃类物质丰度较高，而原油和原油CEWAF中正构烷烃的丰度较高，原油WAF中的烷烃所占比例虽远小于原油和原油CEWAF中的比例，试验原油CEWAF中除C11，C12和C15以外其它正构烷烃相对比例均低于试验原油WAF，特别是姥鲛烷在试验原油CEWAF中的相对比例明显低于在试验原油WAF中的相对比例，多环芳烃总比例在试验原油WAF中高达44.44%，而试验原油CEWAF中仅占10.35%，烷烃类总比例占试验原油WAF中的16.16%，消油剂大大增加了烷烃类在水相中的比例，而机械搅拌溶解的多是原油中的芳烃类物质，萘和菲及其取代物在试验原油WAF和CEWAF中所占比例较高，试验原油WAF中的萘含量达到13.36%。为探讨表面活性剂成分对刺参幼参的亚急性毒性，以超氧化物歧化酶（SOD）和过氧化氢酶（CAT）为生物标志物，实验室模拟刺参幼参污染暴露于十二烷基磺酸钠（SDS）和消油剂7天，随后再进行7天的免疫恢复，采用羟胺法和可见光分光光度法测定了4月龄刺参体壁肌和内脏中SOD、CAT活性。结果表明，在SDS暴露期间比较SOD和CAT的活力变化趋势大体相近，这也反映了在抗氧化反应中两者合作进行活性氧（ROS）氧化分解的关系。SDS胁迫下对比刺参幼参体壁肌中的SOD和CAT活性变化发现，SDS污染暴露对刺参幼参体壁肌中SOD和CAT活性的诱导效应存在时间依赖性，对内脏中SOD活性的诱导效应存在时间依赖性，与SDS暴露浓度无关，内脏中CAT活性则没有这样的表现。消油剂胁迫下对比刺参幼参体壁肌中的SOD和CAT活性变化发现，消油剂对刺参幼参体壁肌中的SOD和CAT活性的影响没有明显的时间和剂量依赖性，这样的现象比较异常。考虑到试验中SDS和消油剂的浓度并不在一个量级上，推测消油剂对刺参幼参SOD和CAT活性的影响较SDS小，但消油剂在处理溢油事故中一般是大量使用，，且可导致大量刺参幼参“排脏”，因此似乎具有较深远的影响，这一点值得关注。为探讨消油剂处理溢油事故的安全性，以SOD和CAT作为生物标志物，实验室模拟刺参幼参污染暴露于原油CEWAF和原油WAF7天，随后再进行7天的免疫恢复，测定刺参幼参体壁肌和内脏中的SOD和CAT活性。结果显示，原油WAF和CEWAF暴露对刺参幼参体壁肌和内脏中的抗氧化酶活性均有影响，但原油WAF免疫恢复后，无论是体壁肌还是内脏中的抗氧化酶活性均未能恢复正常，而原油CEWAF免疫恢复后有所恢复，表明原油WAF污染暴露对刺参幼参的长期影响较原油CEWAF严重，尽管原油CEWAF中的总石油烃浓度高于原油WAF中总石油烃浓度，结合对原油WAF和CEWAF的GC-MS分析，发现原油WAF和原油CEWAF中组分存在差异，多环芳烃总比例在试验原油WAF中高达44.44%，而试验原油CEWAF中仅占10.35%，相反烷烃类总比例占试验原油WAF中的16.16%，而占试验原油CEWAF中的39.37%，有研究认为多环芳烃的生物毒性较烷烃类大，这可能是导致原油WAF污染暴露对刺参幼参的长期影响较原油CEWAF严重的原因。通过比较我们发现，内脏中CAT活性明显比体壁肌中高，对于刺参这样较为低等的生物，以内脏作为抗氧化酶测定的组织是比较合适的。
[Abstract]:With the development of the economic and social development, the application of the surfactant component extends to various aspects of production and life, and the application and the dosage are also continuously increased, and the emission of production and domestic sewage enters the environment; besides, the large-scale sudden oil spill accidents are frequent, And also brings serious harm to the environment. In this paper, the effects of common surfactant component sodium dodecyl sulfonate (SDS), sodium dodecyl benzene sulfonate (SDBS) and oil-removing agent on the sea oil spill were studied. The following studies have been carried out: In order to study the toxicological effects of surfactants and oil-removing agents on the marine treasures, such as the toxicological effects of the oil spill on the marine treasures, SDS, SDBS, oil-removing agent, water-dispersed crude oil (WAF) and chemical-dispersed crude oil (CEWAF), the acute toxicity of the sea-oil-oil-oil spill to the juvenile sea cucumber (Apostichopus japonicus) was studied. The results showed that the 72 h-LC50 and 96 h-LC50 of the four-month-old Apostichopus japonicus were 15.03 and 10.89 mg 路 L-1, respectively. The 72h-LC50 and 96h-LC50 of the four-month-old stichopus japonicus were 2.50 and 1.71 mg 路 L-1, respectively. The 96h-LC50 of the four-month-old Apostichopus japonicus was 7498.94 mg 路 L-1. 1. It can be seen that the deoiling agent used in the test is of micro-toxicity, and the toxicity is much smaller than that of SDBS and SD. S. The application of 96-h large-dose surface of crude oil and oil-removing agent does not cause the young ginseng of the stichopus japonicus to die, and only the oil-removing agent can generate small stimulation to the young ginseng of the stichopus japonicus, and the oil-removing agent can be collected for a short period of time. The 96 h-LC50 of the crude oil CEWAF for the 3-month-old and 4-month-old stichopus japonicus was 246.09 mg 路 L-1 and 399.15 mg 路 L-, respectively. 1. In order to optimize the exposure process of WAF and CEWAF toxicity test of crude oil, an ultraviolet spectrophotometry was used to determine the total petroleum hydrocarbon concentration and the weathering process and composition under laboratory conditions. The results show that, in order to determine the TPH mass concentration of the crude oil CEWAF more accurately, it is determined that the concentration-absorbance curve of the crude oil + deoiling agent is used as the standard curve of the total petroleum hydrocarbon mass concentration of the crude oil CEWAF, and the deoiling agent has a white floccus in n-hexane. The product is generated. After the standard stock solution is prepared, it shall be allowed to stand sufficiently (or be centrifuged at 2000r/ min for 5 min), the supernatant is extracted for dilution, and the standard solution is prepared so as not to bring the floccule to interfere with the absorbance reading; in laboratory conditions, the total petroleum hydrocarbon in the mechanically dispersed crude oil exhibits linear weathering. The trend of the weathering rate over time The rate is reduced. Under the laboratory conditions, the weathering tendency of the chemically dispersed crude oil in 120 h can be seen, the total petroleum hydrocarbon content in the beginning of the 12 h is basically unchanged, but there is a sharp weathering within 12 h to 36 h, which is basically stabilized after 36 h, but still remains The error can be reduced by some methods, such as frequent replacement of the test solution in the event of no harm to the subject to be tested, WAF recommended for 2 to 3 hours of change, the CEWAF is recommended for 10 to 12 hours to be changed once, and the total petroleum hydrocarbon content at the start of the test can also be determined, and the phase can be made The results show that the abundance of the polycyclic aromatic hydrocarbons in the WAF of the crude oil and the crude oil is high, while the proportion of the paraffin in the crude oil WAF is much smaller than that of the crude oil and the crude oil CEWAF, and the test crude oil CEWAF The relative proportion of the other n-alkanes except C11, C12 and C15 is lower than that of the test crude oil WAF, especially the relative proportion of the basestalk in the test crude oil CEWAF. The total proportion of the polycyclic aromatic hydrocarbons is as high as 44.44% in the experimental crude oil WAF, and only 1 of the test crude oil CEWAF is 1. The total proportion of alkane is 16.16% in WAF of the test crude oil, and the oil-removing agent greatly increases the proportion of the alkane in the water phase, while the mechanical stirring and dissolution are the aromatic substances in the crude oil, and the amount of the oil and the phenanthrene and the substitute thereof in the experimental crude oil WAF and CEWAF The proportion is high, and the oxygen content in the WAF of the test crude oil reaches 1. 3.36%. In order to study the sub-acute toxicity of the surfactant component to the young ginseng of the stichopus japonicus, the superoxide dismutase (SOD) and catalase (CAT) were used as biomarkers, and the laboratory simulated stichopus japonicus was exposed to sodium dodecyl sulfonate (SDS). And 7 days after the deoiling agent,7 days of immune recovery is carried out, and a hydroxylamine method and a visible light spectrophotometry are adopted to determine the SO in the body wall and the internal organs of the body wall of the stichopus japonicus at the age of 4 months. The results showed that the changes of the activity of SOD and CAT were similar in the course of SDS exposure, which also reflected the active oxygen species (ROS) in the anti-oxidation reaction. The changes of SOD and CAT activity in the body wall of the stichopus japonicus were found by SDS under the stress of SDS. The time-dependent effect of SDS-contaminated exposure on the activity of SOD and CAT in the body wall of the stichopus japonicus was found. The S-exposure concentration is independent, and the CAT activity in the viscera The activity of SOD and CAT in the body wall of the stichopus japonicus was found to have no significant time and dose-dependent effect on the activity of SOD and CAT in the body wall of the stichopus japonicus. The results show that the concentration of the SDS and the deoiling agent in the test is not on the order of one order, the effect of the deoiling agent on the SOD and CAT activity of the stichopus japonicus is less than that of the SDS, but the oil-removing agent is generally used in a large amount in the treatment of the oil-spill accident, and can lead to a large number of stichopus japonicus. ", so it seems to have a far-reaching impact, In order to study the safety of oil spill accident, SOD and CAT were used as biomarkers, and the laboratory simulated stichopus japonicus was exposed to the crude oil CEWAF and the crude oil WAF for 7 days, and then the immune recovery was carried out for 7 days, and the body wall and the internal organs of the stichopus japonicus were determined. The activities of SOD and CAT showed that the exposure of WAF and CEWAF of crude oil had an effect on the activities of antioxidant enzymes in the body wall and the internal organs of the stichopus japonicus, but the activity of the antioxidant enzymes in the body wall and the internal organs of the crude oil did not return to normal after the recovery of the WAF of the crude oil, and the CEWAF of the crude oil The recovery of the immune recovery indicates that the long-term effect of WAF contamination exposure on the crude oil WAF is more severe than that of the crude oil CEWAF, although the total petroleum hydrocarbon concentration in the crude oil CEWAF is higher than the total petroleum hydrocarbon concentration in the crude oil WAF, the crude oil WAF and the crude oil CEW are found in combination with the GC-MS analysis of the crude oil WAF and CEWAF. The total proportion of the polycyclic aromatic hydrocarbons in the AF was 44.44% in the pilot crude WAF, and only 10.35% of the test crude oil CEWAF, while the total proportion of the opposite alkanes was 16.16% in the experimental crude oil WAF, accounting for 39.37% of the test crude oil CEWAF, and it was considered that the polycyclic aromatic hydrocarbons Biotoxicity is large, which may result in a long-term effect of WAF contamination exposure of crude oil on the juvenile of the stichopus japonicus compared to the crude oil C EWAF is a serious cause. By comparison we have found that the activity of CAT in the viscera is significantly higher than that in the body wall muscle, and for the lower-grade organisms such as the stichopus japonicus, the internal organs are determined as the antioxidant enzymes.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：X55;X171.5

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