死鱼事件中污染源废水对鱼类急性毒性及诱变微核的研究

为了研究2006年9月广州郊区某鱼塘因化工企业污染源废水进入而导致大量死鱼事件中鱼类死亡原因,用斑马鱼(Brachydanio rerio)和蚕豆(Vicia faba)根尖对污染源废水及鱼塘水分别进行了鱼类急性毒性试验和蚕豆根尖微核试验。污染源废水和鱼塘水的96hLC₅₀分别为1.69%、23.95%,污染源废水和鱼塘水的微核率分别为27.8‰、13.7‰,污染源废水和鱼塘水的COD、BOD、石油类、氨氮、总磷等均出现超标情况,其中氨氮、苯胺、石油类污染物严重超标。监测结果表明,以氨氮污染为主的污染源废水是导致死鱼事件的主要原因,废水对蚕豆根尖微核率的形成具有显著性影响,可能存在潜在的致突变性危险。     

A description of ammonium content of output waters from trout farms in relation to stocking density and flow rates

Daily levels of ammonium content in the outflow water of an intensive trout farm and of a mountain pond stocked with rainbow trout were monitored. Output water was sampled for 30 days and analysed every 30 min by an automatic ion selective electrode system (Applikon ADI 2013); input water was also monitored. The ammonium output level was influenced more by atmospheric events than nitrogen excretion by fish. Observed data confirmed an overall ammonium excretion model previously estimated in both laboratory and field conditions. The high water flow, that characterises the intensive trout farm where the observations were made induced a high dilution of metabolites. Consequently, if the peaks of ammonium output did not reach values of 0.35–0.40 mg l⁻¹ the environmental impact was limited and not easily detected. Our results allow us to affirm that the optimal level of water flow rate for effective zeolite-operated filtration is around 10.3 l t⁻¹ s⁻¹.     

Biochemical and cytogenetic biomarkers of pollutant exposure in marine fish (Aldrichetta forsteri Valenciennes and Sillago schomburgkii Peters) near industrial and metropolitan centres in South Australia

This project aimed to measure biochemical and cytogenetic biomarkers in marine fish (Aldrichetta forsteri and Sillago schomburgkii) associated with industrial and urban centres in South Australia. These sites were Port Pirie (affected by metal-contaminated outflows), Barker Inlet (adjacent to Metropolitan Adelaide), and Wills Creek (reference site). The biochemical biomarkers included sorbitol dehydrogenase (SDH) and alanine aminotransferase (ALAT) in serum, adenylate levels (ATP, ADP and AMP) and adenylate energy charge (AEC) in gill and liver, and sodium/potassium ATPase (Na⁺, K⁺-ATPase) in gill. Erythrocyte micronucleus frequency was a marker of cytogenetic effect. Serum enzyme levels were generally higher in fish from Port Pirie and Barker Inlet than in those from Wills Creek, with SDH demonstrating the clearest site-associated differences. Tissue adenylates were consistently lower at Port Pirie than elsewhere, suggesting a greater metabolic strain in fish at this site. AEC in gill and liver were consistently lower at Port Pirie than at Wills Creek, with Barker Inlet generally between these two. The reversed rank order was observed with erythrocyte micronucleus frequencies. Seasonal variations in the biomarkers may be attributed either to seasonal physiological changes in fish or changes in pollutant input levels or compositions. Na⁺, K⁺-ATPase did not differ between sites nor seasons in this study. This work shows that biochemical and cytogenetic differences occur in marine fish at specific locations in South Australia. It also shows that of these tests, serum SDH and erythrocyte micronuclei are potentially the most sensitive and reliable biomarkers of pollutants effects on marine fish. The results also suggest that these data may be used as a baseline against which future changes in marine water quality, and their consequent biological effects, can be compared.     

Biochemical and cytogenetic biomarkers of pollutant exposure in marine fish (Aldrichetta forsteri Valenciennes and Sillago schomburgkii Peters) near industrial and metropolitan centres in South Australia

This project aimed to measure biochemical and cytogenetic biomarkers in marine fish (Aldrichetta forsteri and Sillago schomburgkii) associated with industrial and urban centres in South Australia. These sites were Port Pirie (affected by metal-contaminated outflows), Barker Inlet (adjacent to Metropolitan Adelaide), and Wills Creek (reference site). The biochemical biomarkers included sorbitol dehydrogenase (SDH) and alanine aminotransferase (ALAT) in serum, adenylate levels (ATP, ADP and AMP) and adenylate energy charge (AEC) in gill and liver, and sodium/potassium ATPase (Na⁺, K⁺-ATPase) in gill. Erythrocyte micronucleus frequency was a marker of cytogenetic effect. Serum enzyme levels were generally higher in fish from Port Pirie and Barker Inlet than in those from Wills Creek, with SDH demonstrating the clearest site-associated differences. Tissue adenylates were consistently lower at Port Pirie than elsewhere, suggesting a greater metabolic strain in fish at this site. AEC in gill and liver were consistently lower at Port Pirie than at Wills Creek, with Barker Inlet generally between these two. The reversed rank order was observed with erythrocyte micronucleus frequencies. Seasonal variations in the biomarkers may be attributed either to seasonal physiological changes in fish or changes in pollutant input levels or compositions. Na⁺, K⁺-ATPase did not differ between sites nor seasons in this study. This work shows that biochemical and cytogenetic differences occur in marine fish at specific locations in South Australia. It also shows that of these tests, serum SDH and erythrocyte micronuclei are potentially the most sensitive and reliable biomarkers of pollutants effects on marine fish. The results also suggest that these data may be used as a baseline against which future changes in marine water quality, and their consequent biological effects, can be compared.     

养殖乌鳢类立克次体感染的组织病理学研究

本文系统报道了养殖乌鳢类立克次体(Rickettsia-like organism,RLO)感染的各器官(脑、眼、鳃、心脏、头肾、肝、胰腺组织、脾、肾、肠和卵巢)的组织病理变化,探讨了炎症发展的基本规律。感染乌鳢病理解剖学特征和最具病理诊断意义的是体内各器官普遍出现的白色结节。这些结节的显微结构为肉芽肿炎症即一种慢性增生性炎症。在严重病变的肾脏,由于组织坏死区域较大和周围明显的细胞增生形成了境界较为清楚的巨大“肉瘤”状肿物。内脏器官的血管(特别是造血器官的血管)出现明显纤维素性血栓、混合血栓、弥散性血管内凝血(disseminated intravascu-lar coagulation,DIC)和组织细胞大范围的变性或坏死、溶解。大多数器官的组织细胞主要是上皮性细胞、吞噬性细胞,胞质内富含嗜酸性包涵体(eosinophilic intracytoplasmic inclusion),这种细胞多位于鳃上皮处、鳃部和体内器官血管内皮及血管周边结缔组织。血管内皮细胞及周边细胞内大量包涵体的出现导致血管内皮细胞的肿胀和破坏。     

鱼微核试验筛检水体诱变物的应用与研究

对五种试验污染物的遗传毒性采用鱼微核试验技术进行了筛检,试验结果表明鱼微核试验用作筛检诱变物的遗传毒性有效的,对监测水体的质量也具有潜在的价值;同时,对国内外学将各种各样的技术应用到鱼微核式试验进行了总结,概述了微核形成的机理;在此基础上,提出应用鱼微核试验筛检水体诱变物时应注意的问题。     

Cadmium induced bioaccumulation,histopathology, gene regulation in fish and its amelioration – A review

Different anthropogenic activities as well as natural sources contribute enormously towards various heavy metal contaminations in aquatic habitats. Cadmium (Cd) is one of most prevalent and toxic heavy metals with a long half life. Unlike terrestrial animals, exposure of Cd in fishes may happen not only through feeds but also from its habitat water. Bioaccumulation of Cd in fishes occurs in many tissues, but mainly in gill, liver, kidney, skin, and muscle. The concentrations of Cd in fish tissues depend upon the extent and duration of Cd exposure, species and age of fishes, dietary minerals and antioxidant concentrations, and habitat water quality. Specific histopathological observations in liver, kidney, and gill are useful to understand the effects of Cd, which could help to determine the ameliorating methods to be adopted. Exposure of Cd exerts several adverse effects on general growth and development, reproductive processes, osmoregulation, morphological and histological structures, stress tolerance, and endocrine system, mainly due to changes in biological functions induced by differential expressions of several genes related to oxidative stress, apoptosis, inflammation, immunosuppressions, genotoxicity, Cd chelation and carbohydrate metabolism. Chronic biomagnifications of Cd exceeding the permitted level may be harmful not only to the fishes itself but also to humans through food chains. Amelioration of such toxic heavy metal that has been categorized as a potent carcinogenic in humans is of utmost importance. Main modes of amelioration encompas reducing oxidative damages by promoting the antioxidative defenses, decreasing Cd absorption, increasing excretion through excretory system and improving the tolerance of fishes to Cd toxicity. Many amelioration measures such as use of minerals (for example, zinc, calcium, and iron), vitamins (vitamin C, A, and E), different herbs, probiotics and other agents (taurine, bentonite, chitosan, zeolite, and metallothionein) have been explored for their effective roles to reduce Cd bioaccumulation and toxicity symptoms in fishes. The present review discusses bioaccumulation of Cd, histopathological alterations, oxidative stress, synergism-antagonism, and gene regulation in different tissues, and its amelioration measures in fishes.     

Effects of trace metals on growth of yellow perch (Perca flavescens) as measured by RNA-DNA ratios

Synopsis Relationships between sublethal concentrations of cadmium and zinc in natural water and metal uptake by and growth of fish were investigated. RNA-DNA ratios and weight gain were used to assess seasonal growth differences between yellow perch populations from contaminated and control sites. Whole-body concentrations of cadmium and zinc in young-of-the-year perch (Perca flavescens) were significantly different between sites. Measurable growth differences did occur and were significantly correlated with cadmium levels. Growth differences that were prominent during mid-summer were reduced by late summer. RNA-DNA ratios were sensitive indicators of fish growth.     

长江下游重点江段水质污染及对鱼类的毒性影响

通过对长江下游安庆,南京,镇江和长江口4江段水质及其对鱼类毒性影响的调查发现,长江下游江段主航道的水质符合国家地面II类标准,但近岸的污染带水质受到一定程度的污染,其主要污染物为石油烃类和挥发酚等,这些污染物对鱼类有一定的毒性影响,可引起鱼类的急性中毒,并可诱导鱼类产生微核,同时在上述几个江段内的鱼体内可检出较高的污染物残留,这表明长江下游江段已受到不同程度的污染,这种污染对区域性渔业有影响。     

Environmentally induced nephrotoxicity and histopathological alternations in Wallago attu and Cirrhinus mrigla

Fish kidneys are sensitive to chemical changes in the freshwater ecosystem because they are directly and constantly exposed to chemicals dissolved in the water. This study evaluated nephrotoxicity in Wallago attu and Cirrhinus mrigala harvested from the Chenab River in an area of industrial and sewage waste disposal. Induced histological alternation data were correlated to the severity of environmental degradation in order to determine whether this biological system can be used as a tool for environmental monitoring programs. Kidneys from two fish species occupying different niches were collected and stored for 24 h in 10% formalin. Control fish were collected upstream of the polluted river area. Specimens were processed using topical histological methods. The major histological alterations observed in both species were renal tubule myxospora, hyperemia, glomerulonephritis, degeneration of renal tubule cells, dilation of glomerular capillaries, presence of pycnotic nuclei in the hematopoietic tissue, epithelial hypertrophy, vacuolization, reduced lumen of renal tubules, and shrinkage of glomeruli. Renal tubular atrophy, degeneration due to extensive degranulation, necrosis of glomeruli, glomerular expansion, absence of Bowman’s space, hypertrophied nucleus, necrosis and hyalinization of the interstitium, clogging of tubules, and regeneration of tubules was also observed. Wallago attu exhibited the maximum incidence of moderate to severe changes and was defined as having the highest “histopathologic alteration index”. These severe alterations were found to be related to environmental degradation, indicating the presence of stressors in freshwater. Control groups showed normal tissue morphology in the kidneys.     

Accumulation, distribution and toxicology of dietary nickel in lake whitefish (Coregonus clupeaformis) and lake trout (Salvelinus namaycush)

An 18-day experiment was conducted to investigate the uptake and sublethal toxicity of dietary Ni in adult lake whitefish (LWF, Coregonus clupeaformis) and lake trout (LT, Salvelinus namaycush) fed diets containing 0, 1000 and 10000 microg Ni/g, prepared with and without brine shrimp. The results of this experiment were used to design an experiment of longer duration in which one of the fish species was selected and exposed to lower dietary Ni doses. In the present study feed refusal was observed in LT and LWF fed 10000 microg Ni/g, after three and 4-5 feedings, respectively. LT fed Ni-contaminated diets exhibited different patterns of Ni accumulation than LWF. Increased Ni concentrations in all LWF tissues, except the intestine, were associated with increased doses of Ni. Copper and Zn concentrations in kidney and liver of LWF were altered. Metallothionein concentrations in kidneys of LT fed 1000 microg Ni/g and 10000 microg Ni/g and LWF fed 10000 microg Ni/g and in livers of LWF fed 10000 microg Ni/g (diet without shrimp only) increased significantly. Increased lipid peroxide production in the plasma of LT fed 10000 microg Ni/g was observed. Blood glucose and electrolytes were affected by Ni exposure. Histopathological alterations were observed in kidneys of LWF fed low and high dose diets, livers of whitefish fed high dose diets, and intestines of LWF fed high dose diets and LT fed low and high dose diets. LT fed high dose diets exhibited significant decreases in weight.     

Insights into the drivers of histopathological changes and potential as bio-indicator of riverine health of an aquatic apex predator from a premier conservation area: A multiple lines of evidence and multivariate statistics approach

There is a need for sensitive bio-monitoring and indicator tools in toxicant impact assessment to show the effect on fish health. Histopathological assessment of fish tissue is a bio-monitoring tool allowing for early warning signs of disease and detection of long term injury in cells, tissues or organs. The aims of this study were firstly to determine and compare the health status of the tigerfish (Hydrocynus vittatus) from two lowland rivers in Kruger National Park (KNP), secondly to attempt to explain the histological changes observed, through the application of a suite of multivariate statistics to relate changes to biotic levels of selected metals and organochlorine pesticides (OCPs) and thirdly to determine the suitability of H. vittatus as a bio-indicator of riverine health. Tigerfish were caught using rod and reel from the Olifants (n = 37) and Luvuvhu Rivers (n = 34) between 2009 and 2011. The histology-based fish health assessment (liver, kidney, gills and gonads) indicated that fish were in relatively good health. Even though fish were considered to be healthy, the general fish health in both rivers improved over time, corresponding to an overall decrease in river pollution, most notably in terms of metals. The incorporation of multiple lines of evidence in tigerfish, including histopathological changes and organ and whole organism indices, proved to be a valuable tool in using a bio-indicator approach toward river monitoring. These histopathological changes serve as an early warning system to more serious health concerns arising if the pollution in the rivers of the KNP is not dealt with. The use of a suite of uni- and multivariate statistics proved helpful in determining the links between fish health and river contamination and further proved to be a valuable tool in assessing spatial and temporal differences in river pollution and the effects thereof on the selected bioindicator.     

The use of innate immune responses as biomarkers in a programme of integrated biological effects monitoring on flounder (Platichthys flesus) from the southern North Sea

Immunological biomarkers that reflect the effects of exposure to environmental contaminants in coastal marine habitats were sought in European flounder (Platichthys flesus) from five locations in the German Bight with different anthropogenic impacts. During a 2-year period of sampling, innate immune responses were monitored from a total of 331 individual flounder of a body length of 18 to 25 cm. From the fish, plasma lysozyme, phagocytosis and respiratory burst activity of head kidney leucocytes were analysed and implemented as part of an integrated biological effects monitoring programme. As the measurements of the parameters applied here varied within wide ranges at some locations, spatial differences could not always be established, but some general trends could be drawn: plasma lysozyme activity was decreased in flounder contaminated with DDT adducts and some PCBs, while cellular functions such as phagocytosis and respiratory burst were stimulated by some chlorinated hydrocarbons. Correlation analysis also revealed connections not only between the parameters applied here and some contaminants but also with some biochemical parameters used as biomarkers in pollution monitoring: in flounder with decreased integrity of hepatocyte lysosomal membranes, immune functions also were impaired, and plasma lysozyme as well as phagocytosis activity of head kidney cells were impaired when the activity of cytochrome P450 1A was induced. The data presented here indicate that innate immune responses may be useful parameters to monitor cellular functions in a battery of biomarkers of different levels of biological organisation. Communicated by H. v. Westernhagen, A. Diamant     

Impact of municipal effluents and hydrological regime on myxozoan parasite communities of fish

Increased productivity from sewage effluents can enhance species richness locally. Results from a study of spottail shiners (Notropis hudsonius) in 1999 showed that prevalence and the mean number of myxozoan parasite species per host were higher downstream of the wastewater outflow from the Island of Montreal than upstream in the St. Lawrence River, Quebec, Canada. This was attributed to organic enrichment of the sediments which presumably lead to increased densities of oligochaetes, the alternate hosts, downstream of Montreal. Spottail shiners subsequently were collected every August/early September in 2001–2004 to examine the stability and repeatability of these patterns. Prevalence and mean number of myxozoan species per fish typically were again higher downstream of the sewage source each year compared to upstream, although there was no significant difference in these measurements pooled across years between localities immediately upstream and downstream of the effluent outflow. Density of the oligochaete Limnodrilus hoffmeistereri, a common alternate host of myxozoans, was much higher at two downstream localities than at an upstream one. At a larger spatial scale, mean myxozoan infracommunity richness across sites in the St. Lawrence River was negatively correlated with mean water levels measured in the 3 months prior to fish sampling. Results suggest that on a local scale, variations in prevalence and diversity among localities are influenced by municipal effluents, but that at a landscape scale annual variations across sites are affected by the hydrological regime and climate. In effect, water level fluctuation had a landscape-wide impact that was superimposed over pollution-induced local variations.     

Assessment of some innate immune responses in dab (Limanda limanda L.) from the North Sea as part of an integrated biological effects monitoring

The marine flatfish dab (Limanda limanda), which lives in direct contact with contaminated sediments, is frequently used as a sentinel species in international monitoring programmes on the biological effects of contaminants. In this study, immune responses were recorded as indicators of sublethal chronic effects of contaminants, in addition to measurement of the induction of mono-oxygenase ethoxyresorufin O-deethylase (EROD) in liver cells, the inhibition of acetylcholin esterase (AChE) in muscle and a quantification of grossly visible diseases and parasites. In total, 336 dab were analysed from five sampling areas in the North Sea, including the German Bight, the Dogger Bank, the Firth of Forth, and two locations close to oil and gas platforms (Ekofisk and Danfield). When considering plasma lysozyme levels, pinocytosis and respiratory burst activity of head kidney leucocytes, a clear gradient could be observed with decreased levels in individuals collected from the Firth of Forth and locations near the oil or gas platforms compared with dab from the Dogger Bank or the German Bight. Individuals with induced EROD activity displayed reduced lysozyme and respiratory burst activities. Lysozyme levels were also reduced in dab with lymphocystis or with nematodes. The data obtained indicate that the assessment of innate immune parameters in a monitoring programme provides supplementary information about immunomodulatory effects associated with the exposure of fish to contaminants. In particular, concentrations of plasma lysozyme, which can be analysed in an easy and inexpensive assay, are considered to be an appropriate parameter for use in a battery of other bioindicators. Communicated by H. von Westernhagen and A. Diamant     

Differences in catalytic activity between rat testicular and ovarian carbonyl reductases are due to two amino acids

The sequences of rat testis carbonyl reductase (rCR1) and rat ovary carbonyl reductase (rCR2) are 98% identical, differing only at amino acids 140, 141, 143, 235 and 238. Despite such strong sequence identity, we find that rCR1 and rCR2 have different catalytic constants for metabolism of menadione and 4-benzoyl-pyridine. Compared to rCR1, rCR2 has a 20-fold lower K(m) and 5-fold lower k(cat) towards menadione and a 7-fold lower K(m) and 7-fold lower k(cat) towards 4-benzoyl-pyridine. We constructed hybrids of rCR1 and rCR2 that were changed at either residues 140, 141 and 143 or residues 235 and 238. rCR1 with residues 140, 141 and 143 of rCR2 has similar catalytic efficiency for menadione and 4-benzoyl-pyridine as rCR1. rCR1 with Thr-235 and Glu-238 of rCR2 has the catalytic constants of rCR2, indicating that it is this part of rCR2 that contributes to its lower K(m) for menadione and 4-benzoyl-pyridine. Comparisons of three-dimensional models of rCR1 and rCR2 show how Thr-235 and Glu-238 stabilize rCR2 binding of NADPH and menadione.     

Antioxidant and detoxifying fish enzymes as biomarkers of river pollution

The activity of several antioxidant and detoxifying enzymes, superoxide dismutase SOD , GSH peroxidase GSHPx , GSSG reductase GSR and GSH S transferase GST , the contents of thiobarbituric acid reactive substances TBARS , and the SOD and GST isoenzyme patterns were studied in the livers of chubs Leuciscus cephalus from reference river areas and polluted urban sites. Livers of polluted fish contained higher concentrations of transition metals, especially copper and iron. Total GSHPx activity was 1.8 fold higher in the polluted fish than in reference animals, while the SOD and GSR activities and the TBARS content were not significantly changed. Three new SOD isoforms pI 4.45, 5.1, 5.2 and a higher intensity of the band pI 4.2 were observed after isoelectrofocusing of polluted fish extracts. Total GST activity was higher in fish from polluted areas. The GST isoenzyme pattern was studied using subunit specific substrates DCNB, EPNP, EA, NPB, NBC and by Western blot using antibodies specific to rat GST subunits 1, 8 Alpha class , 3 Mu class and 7 Pi class . Reference and polluted fish lacked cross reactivity towards Alpha class GSTs. Reference fish displayed weaker cross reactivity towards CST 7 and 2.3 fold lower activity with EA, while higher cross reaction with GST 3 was observed in polluted fish.