摘要
本研究进行了野外调查和实验室观测两个系列的实验。
系列一是调查多环芳烃(PAHs)在长江上游鱼类体内的含量和分布情况。于2010年9一11月在长江朱杨江段采集到10种鱼类标本,分别为中华倒刺鲃(Spinilbarbus sinensis Linnaeus)、瓦氏黄颡鱼(Pelteobagrus vachelli Richardson)、圆筒吻鮈(Rhinogobio cylindricus Gunther)、鲤(Cyprinus carpio Linnaeus)、鱼即(Carassius auratus Linnaeus)、大眼鳜(Siniperca kneri Garman)、铜鱼(Coreius heterodon Bleeker)、圆口铜鱼(Coreius guichenoti Sauvage et Dabry)、大鳍鳠(Mystus macropterus B leeker)和鲇(Silurus asotus Linnaeus);在该江段上游支流沱江富顺江段采集到6种鱼类标本,分别为鲫、黄颡鱼(Pelteobagrus fulvidraco Richardson)、鲤、大眼鳜、大鳍鳠和鲇。每种鱼的样本量为3—11尾,共计195尾。采用快速溶剂萃取及气相色谱法对鱼体中的16种多环芳烃含量进行检测。系列二是实验室条件下含菲(PAHs物质之一)食物暴露实验。以中华倒刺鲃(Spinibarbus sinensis)为实验对象(该种鱼在系列一实验中被发现含PAHs总量在所测种类中最多,且为长江上游特有珍稀鱼类),以菲(Phenanthrene, PHE)为毒物,配制含PHE的人上饲料进行投喂。在水温27.5℃下的条件下,进行了两个部分的实验：1、饲料中不同浓度PHE暴露实验。PHE含量分别为0、500、1000和1500μg/g的饲料,喂养中华倒刺鳃16周后取样；2、饲料PHE不同暴露时间实验。以PHE含量为0和1000μg/g的饲料,分别喂养中华倒刺鲃0、1、3、7、14、28、42、56、84和112天后取样。分别观测了上述两类处理的实验鱼体的抗氧化胁迫能力、代谢能力、生长状态和体内PHE累积等方面的指标。
由实验系列一所得结果如下：
1、在长江干流朱杨江段所采集到的10种鱼体样本中均检测到了多种PAHs的存在,其中中华倒刺鲃鱼体内PAHs总量(ΣPAHs)最高,其含量为(2.91±0.45)μg/g;鲇体内ΣPAHs最低,其含量为(0.52±0.04)μg/g。
2、在沱江富顺江段采集到的6种鱼均检测到多种PAHs存在,其中鲫体内ΣPAHs最高,为(8.50±1.13)μg/g;鲇体内ΣPAHs最低,其含量为(1.30±0.07)μg/g。
3、采集自长江朱杨段和沱江富顺段两采集地的相同种类有5种,富顺段5种鱼体内ΣPAHs均分别高于朱杨段同种类鱼体内的含量；除大眼鳜外,其余4种鱼的差异均达到显著水平。富顺江段鱼类体内高分子量PAHs比例较高,而朱杨江段鱼类体内富集的低分子量PAHs和中分子量PAHs的比例较高。
由实验系列二所得结果如下：
1、饲料中PHE含量水平未对血清中的红细胞数目、血红蛋白和总蛋白造成显著差异；血糖含量随饲料中PHE浓度含量水平的升高表现出先升高后降低的趋势,且各染毒组均显著高于空白对照组(p<0.05)；球蛋白含量在500μgPHE/g组显著高于对照组(p<0.05),白蛋白含量则在500μg PHE/g组显著低于对照组(p<0.05),而这两个指标在其余含毒饲料组与空白对照组间均无显著差异。
2、血清谷丙转氨酶活性随饲料PHE含量水平的升高表现出升高的趋势,仅在最高浓度组(1500μg PHE/g组)时与空白对照组的差异达到显著水平(p<0.05)；谷草转氨酶也随饲料中PHE含量水平的升高而升高,1000μg PHE/g和1500μgPHE/g组与空白对照组之间差异达到显著水平(p<0.05)。
3、肝糖原随饲料中PHE浓度含量水平的升高而降低,且各组间的差异均达到了显著水平(p<0.05)；各实验处理组的肌糖原含量均显著低于对照组(p<0.05),而3个染毒饲料组之间无显著差异。
4、脑组织中乙酰胆碱酯酶(TChE)活性随着饲料中PHE含量水平增高而降低,且各实验处理组之间的差异显著；该活性还随着PHE食物暴露时间的延长而降低,从染毒的第7天开始,染毒组活性显著低于空白对照组(p<0.05)。
5、肝胰脏、肠和鳃组织中丙二醛(MDA)含量随着饲料中PHE含量水平的增高而增高,在肠和鳃组织中,各处理组之间的差异达到显著；在肝胰脏组织中,500μg PHE/g组与空白组之间差异不显著,其余各组间差异显著。这3种组织的MDA含量还随着PHE食物暴露时间的延长而增高。其中,肝胰脏和肠组织中MDA含量均于染毒第3天开始显著高于空白对照组(p<0.05)；鳃组织中MDA含量水平从第84天开始显著高于空白对照组(p<0.05)。
6、随着饲料中PHE含量水平的增高,实验鱼肝胰脏和肠组织中总抗氧化能力(T-AOC)随之降低。各染毒组的肝胰脏组织中T-AOC水平与空白对照组之间的差异显著(p<0.05)。各染毒组肠组织中T-AOC水平差异显著(p<0.05)。随着染毒时间的延长,实验鱼体的肝胰脏组织T-AOC水平呈“降低-升高-降低”趋势；肠组织T-AOC水平呈“升高-降低”趋势；鳃组织T-AOC水平呈一直升高的趋势。
7、实验鱼肝胰脏、肠和鳃组织中谷胱甘肽-S-转移酶(GST)活性随着饲料中PHE含量水平的增高而降低。各染毒组肝胰脏GST活性均显著低于空白对照组(p<0.05)。肠组织GST活性在各处理组间的差异均达到显著水平(p<0.05)。1500μg PHE/g组鳃组织GST活性显著低于其余3组(p<0.05),其余3组间无显著差异。随着染毒时间的延长,染毒组肝胰脏组织GST活性呈“降低-升高-降低”趋势；肠组织GST活性呈“升高-降低”趋势。
8、实验鱼体的标准体重代谢率(MS)随着饲料中PHE含量水平的升高而升高,1000和1500μg PHE/g组的MS显著高于空白对照组,且二者间差异显著(p<0.05)。MS还随着PHE食物暴露时间的延长而升高,从染毒第7天开始显著高于空白对照组(p<0.05)。
9、实验鱼肝胰脏组织线粒体的状态3呼吸率随着饲料中PHE含量水平的增高而增高。1000和1500μg PHE/g组肝胰脏组织线粒体呼吸率显著高于空白对照组,且二者间差异显著(p<0.05)。该呼吸率还随染毒时间的延长而增高,从染毒的第56天开始,染毒组肝胰脏组织线粒体呼吸率显著高于空白对照组(p<0.05)。
10、实验鱼的摄食量和饲料效率随着饲料中PHE含量水平的升高而降低;其中,500、1000和1500μg PHE/g组摄食量之间的差异不显著,但三者均显著低于空白对照组(p<0.05);各染毒组饲料效率差异显著(p<0.05)。这两个指标还随着染毒时间的延长而降低,分别从染毒的第14和28天开始显著低于空白对照组(p<0.05)。
11、实验鱼的器官指数随着饲料中PHE含量水平增高而升高；肝胰脏指数在500和1000μg PHE/g组之间无显著差异,二者与空白对照及1500μg PHE/g组之间的差异达到显著水平(p<0.05)。500μg PHE/g组肠指数与空白对照组差异不显著,其余各组间差异均显著(p<0.05)。
12、实验鱼体的特定体重生长率随着饲料中PHE含量水平的增高而降低,各实验处理组之间的差异显著(p<0.05)。该值也随着染毒时间的延长而降低,从14天开始与空白组间的差异显著(p<0.05)。
13、鱼体肥满度随着饲料中PHE水平的升高而降低。其中500和1000μg PHE/g组差异不显著,但二者均显著高于1500μg PHE/g组而显著低于空白对照组(p<0.05)。
14、实验鱼体对饲料中PHE的摄入量、日粮水平、累积量和累积率均随着饲料中PHE含量水平的升高而增高,月.各组间差异均显著(p<0.05)。
15、肝胰脏、肠和肌肉组织对于饲料中PHE的累积量PHE/g)随着饲料中PHE含量水平的升高而升高。同一染毒组个体不同器官组织的PHE含量从高到低依次均为：肝胰脏>肠>肌肉；其中,肌肉的PHE含量与饲料PHE含量水平的相关关系为：y=0.038x-2.900(r2=0.978)。这3种组织的PHE含量也随着染毒时间的延长而增高；在1000μg PHE/g饲料条件下,肌肉的PHE含量与食物PHE暴露时间的相关关系为：y=0.341x+0.791(r2=0.942)。
通过讨论得出以下结论：
1、长江朱杨段及其支流沱江富顺段的鱼类均受到了PAHs的污染,而沱江鱼类受PAHs的污染程度高于长江干流朱杨段鱼类。
2、沱江富顺段鱼体所含的高分子量PAHs组成百分比高于长江干流朱杨段的鱼体,而中、低分子量PAHs组成百分比相对于干流鱼类较低,表明两个江段PAHs的污染源不同。
3、中华倒刺鲃血液学指标、肝胰脏指数和糖原含量受到饲料中PHE含量水平的影响,PHE对鱼体的肝胰脏组织造成了组织损伤,并干扰了鱼体正常的碳水化合物代谢过程。
4、PHE食物暴露会导致鱼体组织器官的氧化损伤并抑制鱼体相应解毒酶系统的活性,可引起鱼类肝脏和肠道出现组织增大等形态结构特征的改变；且这些毒性胁迫会随着PHE暴露的浓度增长或时间延长而加剧。
5、中华倒刺鳃静止代谢率和肝胰脏组织线粒体呼吸耗氧率随着暴露浓度的增高和染毒时间的延长而增高,表明鱼体可通过生理性调节,提高其代谢强度,以满足机体抵抗PHE食物暴露胁迫的有关生理过程对能量的额外需求。
6、染毒组中华倒刺鳃的生长率、摄食率、饲料效率等生长状态指标的下降与鱼体的代谢增强,糖原含量降低,解毒酶系统功能调整等现象相伴而发生,表明染毒鱼体生长率较低的主要原因是外源能量摄入不足和额外代谢能量消耗。
7、在食物中一定浓度的PHE暴露下,中华倒刺鳃鱼体各器官所累积的PHE浓度不同,表明PHE在鱼体内的累积与分布具有器官及组织的特异性。
8、根据我国食品安全标准进行估算,为保证鱼体肌肉中PHE含量不超标,对中华倒刺鲃饲喂16周时所采用的食物中PHE含量不得高于207.90μg/g;若PHE食物暴露的浓度达到1000μg/g时,则暴露天数最多不得超过12天。
Two series of experiments were carried out in this study:field investigation and observation in laboratory.
The experiment I was conducted on the contents and distribution of polycyclic aromatic hydrocarbons (PAHs) in the fishes from the upper reaches of the Yangtze River. From September to November in2010, the samples of10species were collected from the Zhuyang section of the Yangtze River, including Spinilbarbus sinensis, Pelteobagrus vachelli, Rhinogobio cylindricus, Cyprinus carpio, Carassius auratus, Siniperca kneri, Coreius heterodon, Coreius guichenoti, Mystus macropterus, and Silurus asotus. And the samples of6species were collected from the Fushun section of the Tuo River, a tributary of the Yangtze River, including Carassius auratus, Pelteobagrus fulvidraco, Cyprinus carpio, Siniperca kneri, Mystus macropterus, and Silurus asotus. The sample size for each fish species was3-11, totaling195. The contents of16polycyclic aromatic hydrocarbons (PAHs) in each sample were measured by the accelerated solvent extraction and gas chromatography. The experiment Ⅱ was conducted on effects of deitary phenanthrene (PHE, one of PAHs) on Spinibarbus sinensis in the laboratory conditions. Two treatments were carried out in this experiment. One was different levels of dietary PHE exposure. The tested fish was fed by experimental diets with different PHE contents (0,500,1000and1500μg/g) for16weeks. The other was different exposure time to dietary PHE. The diets containing0and1000PHE μg/g were used to feed the fish for0,1,3,7,14,28,42,56,84and112 days, respectively. The ability of anti-oxidative stress, hematological index, energy metabolism, growth performance and PHE accumulation were measured in the tested fish at each sampling time.
The main results from experiment I were as follows:
1. Various kinds of PAHs were detected in the10species of fish collected from the Zhuyang section of the Yangtze River. Spinibarbus sinensis had the highest total content of PAHs (ΣPAHs)[(2.91±0.45) μg/g], and Silurus asotus had the lowest ΣPAHs [(0.52±0.04) μg/g].
2. Various kinds of PAHs were detected in the6species of fish collected from the Fushun section of the Tuo River. Carassius auratus had the highest ΣPAHs [(8.50±1.13) μg/g], and Silurus asotus had the lowest ΣPAHs [(1.30±0.07) μg/g].
3. Comparing the five same species collected from the Zhuyang section to those from the Fushun section, each of the latter showed higher contents of ΣPAHs than its counterpart of the former, with significant differences in all pairs except Siniperca kneri. The fishes from the Fushun section contained a higher percentage of high-molecular-weight PAHs (HMW-PAHs), while those from the Zhuyang section contained higher percentages of low-molecular-weight PAHs (LMW-PAHs) and medium-molecular-weight PAHs (MMW-PAHs).
The main results from experiment II were as follows:
1. There were no significant differences in the number of red blood cells, hemoglobin and total protein in the serum among the different groups. Glucose content in the tested groups showed a "up-down" trend with increasing of the dietary PHE concentration, and that in each group exposed to dietary PHE was significantly higher than that in the control group (p<0.05), respectively. The contents of the globulin in the group of500μg PHE/g were significantly higher than that in the control group; the contents of the albumin in the group of500μg PHE/g were significantly lower than that in the control group (p<0.05); there were no significant differences of the two values in the other two groups with dietary PHE (1000and1500μg PHE/g) compared with the control.
2. The activity of alanine aminotransferase increased with the concentration of dietary PHE increasing, but the difference reached significant only between the group of1500μg PHE/g and the control (p<0.05). The activity of aspartate aminotransferase also increased with the concentration of PHE increasing, and the values in the groups at1000and1500μg PHE/g were significantly higher than that in the control group (p<0.05).
3. The content of glycogen in hepatopancreas decreased with the dietary PHE increasing, all the differences between the every two tested groups showed significant (p<0.05). The muscle glycogen in the three groups with dietary PHE exposure were significantly lower than that in the control group (p<0.05), but there was no significant difference among them.
4. The activity of acetylcholinesterase (TChE) in the brain decreased with the increasing of the dietary PHE level. With the exposure time extension, the activity of TChE in the PHE exposed group was significantly lower than that of the control groups at the7th day and the latter(p<0.05).
5. The malonaldehyde (MDA) contents in hepatopancreas, gut and gill increased with the increasing of dietary PHE level. All the differences in guts and gills between the every two tested groups showed significant (p<0.05). In hepatopancreas there was no significant difference betweent500μg PHE/g group and the control, but there were significant differences among the other groups (p<0.05). The MDA contents in hepatopancreas, gut and gill also increased with the exposure time extension. The MDA contents of the hepatopancreas and gut in PHE exposed group were significantly higher than that in the control group at the3rd day and the latter (p<0.05); the MDA content in gill was significantly higher than the control groupat the84th day and the latter (p<0.05).
6. With the increasing of dietay PHE level, the total antioxidant capacity (T-AOC) in the hepatopancreas and gut decreased. There was a significant difference in T-AOC level of hepatopancreas between each of the PHE exposed groups and the control group; there was no significant difference in the value between500and1000μg PHE/g group, but those in both groups were significantly higher than that in1500μg PHE/g group (p<0.05), respectively. With extension of the exposure time, T-AOC level in the hepatopancreas in the PHE exposed group showed a "down-up-down" trend, that of gut showed an "up-down" trend, and that of gill showed an increasing trend.
7. The activity of glutathione-S-transferase (GST) in the hepatopancreas, gut and gill decreased with dietary PHE level increasing. The GST activity in the hepatopancreas in each of PHE exposed groups were significantly lower than that in the control group (p<0.05). And there were significant differences in the activities of the gut among the different groups (p<0.05). The GST activity of the gill in1500μg PHE/g group was significantly lower than that in each of the other three groups (p<0.05), and there was no significant difference among the other three groups. With the exposure time extension, GST activity in the hepatopancreas of the exposure groups showed a "down-up-down" trend; that in gut showed an "up-down" trend.
8. The resting metabolic rate for the standard body weight (MS) increased with the increasing of the dietary PHE level. MS in1500μg PHE/g group was significant higher than that in1000μg PHE/g group (p<0.05), each of them was significantly higher than the control (p<0.05). MS increased with the exposure extension, that in the PHE exposed group was significantly higher than that in the control group at the7th day and the latter (p<0.05).
9. The mitochondrial State3respiration rate in the hepatopancreas increased with the increasing of the dietary PHE level. The respiration rates in1000and1500μg PHE/g group were significantly higher than that in the control, and difference was significant between the two groups(p<0.05). With the extension of exposure time, State3respiration rates in the PHE exposed group decreased and it was significantly higher than that of the control group at the56th day and the latter (p<0.05).
10. The feed intake and feed efficiency decreased with the increasing of dietary PHE level. There were no significantly differences among the feed intakes in500,1000and1500μg PHE/g groups, but those in each of them was significantly lower than that in the control group (p<0.05); the feed efficiencies among each of the exposure groups was significantly different (p<0.05). With the extension of exposure time, the feed intake and feed efficiency of the PHE exposed group were significantly lower than those in the control group (p<0.05) at the14th and28th days, respectively.
11. The organ index increased with the increasing of the dietary PHE level. Hepatopancreas index between500and1000μg PHE/g groups was not significantly different. But those in the two groups were significantly higher than that in the control and lower than that in1500μg PHE/g group (p<0.05). The intestinal index in500μg PHE/g group was not significantly different from the control group, but there were significant difference among the other groups (p<0.05).
12. The specific growth rates of body weight (SGR) of the tested fish decreased with the increasing of the dietary PHE level. SGR among the different groups were significantly different (p<0.05). With the exposure time extension, SGRs in the exposure groups were significantly lower than that in the control group at the14th day and the latter(p<0.05).
13. The condition factor of fish decreased with the increasing of the dietary PHE level. The condition factor between500and1000μg PHE/g groups were not significantly different (p<0.05), but they were significantly higher than that in1500μg PHE/g group (p<0.05) and also were significantly lower than that in the control (p <0.05)。
14. The PHE intake, dietary PHE ration level, PHE accumulation and the ratio of accumulation in the bodies increased with the increasing of the dietary PHE level. All the values among the different groups were different significantly (p<0.05).
15. The PHE accumulation in the hepatopancreas, intestine and muscle increased with the increasing of the dietary PHE level. The PHE contents of different organs in the same exposure groups declined in the following order:hepatopancreas> intestine> muscle. The regression relationship of PHE contents in the muscle with the dietary PHE level could be described as:y=0.038x-2.900(r=0.978, p<0.05). The regression relationship of PHE content in the muscle with the exposure time could be described as: y=0.341x+0.791(r2=0.942, p<0.05).
The conclusions suggested by the discussion were as follows:
1. The fishes from the Zhuyang section of the Yangtze River and those from the Fushun section of the Tuo River are contaminated by PAHs, more serious in the latter.
2. The fishes from the Fushun section contained a higher percentage of HMW-PAHs but lower percentages of MMW-PAHs and LMW-PAHs, as compared with those from the Zhuyang section, which may be due to the difference in pollution source of PAHs between the two river sections.
3. The hematological index, hepatopancreas index and glycogen content in Spinilbarbus sinensis were influenced by the dietary PHE level. PHE damaged the normal function of the hepatopancreas as well as the carbohydrate metabolism.
4. PHE exposure could cause peroxidation damage to the tissues and organs, impairment of detoxification enzyme system, and histological changes in this fish such as enlargement of the hepatopancreas and intestinal tract. All of the toxicological stress would be worse with the increasing of dietary PHE level or extension of PHE exposure time.
5. The resting metabolic rate and respiratory rate of mitochondria in the hepatopancreas tissue increased with the increasing of dietary PHE exposure concentration and extension of its exposure time, which suggested that the metabolic rate would increase by physiological adjustment in the fish with dietary PHE exposure to supply extra energy for resisting the toxicological stress.
6. The descend of growth performance (growth rate, feed efficiency, etc.) were accompanied with the metabolism boosting, glycogen contents descend, adjustment of detoxification enzyme activity and so on, which suggested that the lower growth rate in the tested fish was due to insufficiency of its exogenous energy intake and extra cost of the energy for resisting toxico logical stress.
7. Under a certain concentration of dietary PHE exposure, the PHE accumulations in various organs of Spinilbarbus sinensis were different, which suggested that PHE accumulation and distribution in this fish should be tissue and organ-specific.
8. Based on the national food safety standard in P.R.China, to control the PHE content of the muscle in Spinilbarbus sinensis below the permitted level, the dietary PHE concentration should not be over207.90μg/g when the fish was fed no longer for16weeks, and it should be fed no longer for12days when the dietary PHE is1000μg/g.
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