In situ evaluation of the genotoxic potential of the river Nile: II. Detection of DNA strand-breakage and apoptosis in Oreochromis niloticus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822)

This work is part of a wider eco-toxicological study proposed to evaluate the biological impact of contaminants along the whole course of the river Nile, Egypt. Here we present data on the presence of DNA strand-breaks and apoptotic cells assessed by use of comet and diffusion assays in erythrocytes of Nile tilapia (Oreochromis niloticus niloticus) and African catfish (Clarias gariepinus). The results showed high degrees of DNA damage and increased frequencies of apoptotic nuclei in blood of fish collected from downstream compared with those sampled from upstream river Nile. Qualitative analysis revealed a shift in the frequency of DNA-damage classes towards higher damage levels correlating with the increasing pollution gradient. The degree of DNA damage measured by use of comet assay and diffusion assay exhibited seasonal variations. Both fish species showed significant increases in DNA damage during the summer. The results of our study indicated that the alkaline comet assay seems to be a useful technique for in situ genotoxic monitoring. At the same time the diffusion assay is sensitive enough to detect low frequencies of apoptotic nuclei. The results reveal species-specific differences in sensitivities, suggesting that Nile tilapia may serve as a more sensitive test species compared with the African catfish. Based on the outcome of the comet and diffusion assays, it can be concluded that the water quality of the river Nile with respect to the presence of genotoxic compounds needs to be improved, especially in its estuaries. As far as we know this is the first time that the comet and diffusion assays are used for genotoxic monitoring of the river Nile.     

Anesthesia of fish with benzocaine does not interfere with comet assay results

Fish blood erythrocytes are frequently used as sentinels in biomonitoring studies. Usually, fish blood is collected by painful cardiac or caudal vein punctures. Previous anesthesia could decrease animal suffering but it is not known at present whether anesthesia can cause confounding effects. Therefore, using the alkaline single cell gel (SCG)/comet assay with blood erythrocytes of the cichlid fish Nile tilapia, we tested for a possible modulation of induced DNA damage (methyl methanesulfonate; MMS) by the anesthetic benzocaine administered by bath exposure (80mg/l for approximately 10min). Furthermore, benzocaine (80-600mg/l) was tested for its genotoxic potential on fish erythrocytes in vitro and for potential interactions with two known genotoxins (MMS and hydrogen peroxide). Our results did neither indicate a significant increase in the amount of DNA damage (even after a 48h follow-up), nor indicated interactions with MMS-induced DNA damage when fish were exposed to benzocaine in vivo. There was also no increase in DNA damage after in vitro exposure of fish erythrocytes to benzocaine. Clear concentration-related effects were observed for the two genotoxins in vitro, which were not significantly altered by the presence of benzocaine. These results suggest that anesthesia of fish does not confound comet assay results and the use of blood samples from anesthetized fish can be recommended with regard to animal welfare.     

Metabolic and molecular responses in Nile tilapia,Oreochromis niloticus during short and prolonged hypoxia

The strictly aquatic breathing Nile tilapia, Oreochromis niloticus is an extremely hypoxia-tolerant fish. To augment our understanding of the effects of hypoxia on anaerobic glycolysis in the Nile tilapia, we studied the effect of short-term for 1 day (trial 1) and long-term for 30 days (trial 2) hypoxia on a selected glycolytic enzymes activity and mRNA expression in liver and white muscle. The hypoxic oxygen concentrations used in the two trials were 2, 1, and 0.5 mg O₂ L^?1 for comparison with a control normoxic group 8 mg O₂ L^?1. The activity of phosphofructokinase (PFK), pyruvate kinase (PK), and lactate dehydrogenase (LDH) in liver and white muscle except liver LDH decreased in trial 1 and increased in trial 2. Assessments of mRNA levels in trial 1 revealed that PFK was downregulated and LDH was upregulated in liver and white muscle, while PK fluctuated between upregulation in liver and downregulation in white muscle. Meanwhile, PK and LDH were upregulated while PFK was similar to control values in both tissues in trial 2. Comet assay results demonstrated an increase in DNA damage that was directly proportional to increasing hypoxic concentrations. This damage was more pronounced in trial 1. This suggests that the Nile tilapia cope better with long-term hypoxic conditions, possibly as an adaptive response.     

Cyto-histopathological and protein polymorphism alterations in five populations of Nile tilapia (Oreochromis niloticus) as biomonitor for water heavy metal pollution

Cytological, histopathological and sodium dodecyl sulphate polyacrylamide gel analyses were carried out on five populations of common Nile tilapia fish (Oreochromis niloticus) occurring in five sites – River Nile (reference site), Bahr Yusef canal, Ibrahimia canal, Irrigation drain and El Moheet drain of El Minia Province, Egypt – to evaluate the usability of Nile tilapia as a biomonitor for water heavy metal contaminants. Water surface samples were collected from the five sites, and lead (Pb) concentration was shown to surpass the limits defined by WHO. Ni and Cd levels were shown to be elevated in Ibrahimia canal samples. Moreover, the concentration of heavy metals in fish muscles collected from Bahr Yusef canal and El Moheet drain was the highest in comparison with those of the other water sites. Cytological examinations of blood smears showed not only a significant percentage of micronuclei in Irrigation drain population but also a significant percentage of binucleated cells in Ibrahimia canal and El Moheet drain populations. In addition, pathological alteration was observed in blood cells, especially in samples collected from Irrigation drain and El Moheet drain. Histopathological changes were strongly observed in the liver and the kidneys of El Moheet and Irrigation drain population. Moreover, total protein band pattern profiles showed extra bands in both Ibrahimia canal and Irrigation drain more than that recorded for the River Nile population. In conclusion, cyto-histopathological and total protein band pattern results confirmed that O. niloticus responds sensitively to the excess of heavy metals present in the water.     

Non-lethal detection of DNA from Cichlidogyrus spp. (Monogenea, Ancyrocephalinae) in gill mucus of the Nile tilapia Oreochromis niloticus

Infection of Nile tilapia Oreochromis niloticus by monogeneans of the genus Cichlidogyrus is harmful. Currently, diagnosis of this infection is based on invasive techniques and the identification of isolated parasites by their morphology. To facilitate diagnosis, we have developed a non-lethal polymerase chain reaction (PCR) test for detection of Cichlidogyrus spp. DNA in the gill mucus of O. niloticus, using 5 pairs of specific primers based on Cichlidogyrus sclerosus 28S rRNA (Cicly 1 to Cicly 5) which generate fragments of approximately 188, 180, 150, 159 and 189 bp, respectively. PCR specificity was tested using genomic DNA extracted individually from 175 isolated Cichlidogyrus spp., 75 Gyrodactylus cichlidarum and 75 endopararasitic Enterogyrus spp., as well as from 75 protozoans Trichodina spp. The Cicly primers were used to detect Cichlidogyrus spp. DNA in mucus from the gills of 23 Nile tilapia confirmed to be infected with the parasite. Negative controls consisted of 45 uninfected Nile tilapia. The limit of sensitivity of the assay was 1.2 ng of purified parasite DNA. The Cicly primers did not amplify DNA from the mucus of non-infected Nile tilapia, G. cichlidarum, Trichodina spp. or Enterogyrus spp. In all cases, the sensitivity and specificity of the test were 100%. The sequences of all the amplified fragments showed a high similarity to that of the 28S rRNA region of C. sclerosus (93 to 100% identical to GenBank Accession No. DQ157660.1). We provide evidence for a safe and non-invasive DNA-based diagnostic method for the presence of Cichlidogyrus in the gill mucus of O. niloticus.     

The use of cyprinodont fish, Aphanius fasciatus, as a sentinel organism to detect complex genotoxic mixtures in the coastal lagoon ecosystem

In the present work we aimed to standardise the alkaline comet assay with erythrocytes of the cyprinodont, Mediterranean Killifish, Aphanius fasciatus. The aims of the study were to explore the suitability of this fish to assess biomarkers of genotoxic effects and as a sentinel organism to detect complex genotoxic mixtures in coastal lagoon ecosystems. Following proper optimisation, the application and effectiveness of the comet assay in erythrocytes of A. fasciatus were tested by measuring the tail DNA (%) induced by (a) in vivo exposure of individual fish to X-rays (dose, 3Gy) and (b) following in vitro challenge of erythrocytes with restriction endonucleases Fok-I and Eco-RI, which selectively induce double-strand breaks with cohesive and blunt termini, respectively. Furthermore, in order to evaluate whether circulating fish blood contained actively proliferating cells that could influence the extent of DNA damage in control (untreated) fish, we measured the number of "comets" positive for 5-bromo-2'-deoxyuridine (BrdU) by the use of anti-BrdU antibody and immuno-histochemical methods. Both treatments (i.e. with X-rays and restriction endonucleases) induced statistically significant increases in tail DNA (%) values compared with the relevant untreated controls, indicating the effectiveness of the comet assay in the erythrocytes of A. fasciatus to detect different types of DNA lesions. Results from anti-BrdU antibody labelling of erythrocytes indicated a very low percentage (5%) of "comets" positive for BrdU. Following optimisation and validation of the assay under laboratory conditions, fish were collected in the Orbetello lagoon (Tuscany, Italy), considered to be a significantly polluted site. The results showed statistically significant increases for tail DNA (%) compared with corresponding values observed in erythrocytes of fish caught in the unpolluted reference site "Saline di Tarquinia". The effects of physico-chemical parameters of the water (i.e., salinity, pH and oxygen content) did not significantly influence the induction of DNA damage. These results indicate that the comet assay provides a reliable parameter and that A. fasciatus is a promising "sentinel organism" to detect the genotoxic impact of complex mixtures in coastal lagoon ecosystems.     

Assessment of the genotoxicity in toad Bufo raddei exposed to petrochemical contaminants in Lanzhou Region, China

Single cell gel electrophoresis or comet assay, micronucleus (MN) test and global DNA methylation detection were used to assess the genotoxicity in toad Bufo raddei exposed to the petrochemical (mainly oil and phenol) polluted area in Lanzhou Region (LZR) comparing with a relatively unpolluted area in Liujiaxia Region (LJXR). The results from the present study indicated that DNA damage and MN frequency in toad from LZR were significantly higher than those from LJXR at the same sampling month, whereas the degree of global DNA methylation was lower, which implies that the petrochemical contaminants at environmental level in LZR were genotoxic to B. raddei. The degree of genotoxic damage was obviously related with the extent of pollution among the three sampling months in LZR. The significantly positive correlations between DNA damage and concentrations of oil and/or phenol existed in liver cells but erythrocytes, implying that liver is more suitable as a sentinel tissue for the assessment of genotoxic impact of low-level contamination. The results from both comet assay and global DNA methylation detection on liver cells showed that the genotoxicity varied significantly with oil and/or phenol concentrations, suggesting that these two methods are relatively sensitive and suitable for monitoring the genotoxicity of petrochemical pollutants on amphibians.     

Genotoxicity of the Kishon River,Israel: the application of an in vitro cellular assay

The alkaline comet assay, a sensitive method for DNA strand breaks and alkali labile site detection in individual cells, was employed here as an ecotoxicological monitoring tool for evaluation of genotoxicity in the Kishon River, Israel. This river, the most polluted river in Israel, has recently elicited major public concern with regard to cancer incidences in people who have dived there over many years. Five water samples were collected every odd month throughout the year 2001, from four localities. The comet assay was employed on fish hepatoma cell line RTH-149. Cells were exposed for 2h, in triplicate, to Kishon water: medium (1:1) samples that were pre-adjusted for pH and salinity percentage levels. Three DNA damage parameters (comet percentages, score of damage, and cumulative tail lengths of the comet), revealed significantly higher genotoxic values in Kishon water-treated cells as compared with the controls (up to 2.4, 3.2, and 3.6-fold, respectively). Part of the sampling sites revealed higher genotoxicity than other polluted sites. The results of this study demonstrate that the comet assay with RTH-149 cells can be successfully applied to a variety of aquatic samples revealing freshwater, marine and estuary conditions. The method found to be fast, sensitive, and suitable for monitoring programs.     

Intraspecific variation in gill morphology of juvenile Nile perch, Lates niloticus, in Lake Nabugabo, Uganda

Several studies have demonstrated intraspecific variation in fish gill size that relates to variation in dissolved oxygen (DO) availability across habitats. In Lake Nabugabo, East Africa, ecological change over the past 12 years has coincided with a shift in the distribution of introduced Nile perch such that a larger proportion of the population now inhabits waters in or near wetland ecotones where DO is lower than in open waters of the lake. In this study, we compared gill size of juvenile Nile perch between wetland and exposed (open-water) habitats of Lake Nabugabo in 2007, as well as between Nile perch collected in 1996 and 2007. For Nile perch of Lake Nabugabo [<20 cm total length (TL)], there was a significant habitat effect on some gill traits. In general, fish from wetland habitats were characterized by a longer total gill filament length and average gill filament length than conspecifics from exposed habitats. Nile perch collected from wetland areas in 2007 had significantly larger gills (total gill filament length) than Nile perch collected in 1996, but there was no difference detected between Nile perch collected from exposed sites in 2007 and conspecifics collected in 1996.     

Ecogenotoxicological studies for an early toxicity screening and monitoring in Epinephalus chlorostigma and Scamberomorus commerson

The study was planned to investigate DNA fragmentation in fish to screen aquatic toxicity and in Epinephalus chlorostigma and Scamberomorus commerson collected from Red sea near Jizan, Saudi Arabia from three locations “(Corniche North park: “16.92161, 42.54631; Jizan Port: 16.874, 42.54952” N and Jizan Economic City: 17.26589, 42.34738“ ”)“ were used as a case study for the application of comet assay. The study area of the Red Sea is polluted due to anthropogenic activities and the disposal of wastes from multiple sources. Comet and micronucleus assays were used to detect genotoxicity in these fish species harvested from three sites. The concentration of Pb, Cr, Zn, Mn, Cu, Cd, Sn, and Hg was higher in the water samples collected from the polluted site compared to the non-polluted site of the Red sea. Comet assay for S. commerson showed significant (p < 0.05) genetic damage about 44.33 ± 3.03% DNA in comet tail at site S1. It was subsequently reduced to 31.71 ± 3.52% and 22.11 ± 2.52% at sites S2 and S3. E. chlorostigma also showed significant DNA in comet tail as 17.34 ± 2.19%, 11.87 ± 3.01%, and 36.41 ± 3.98% at site S1-S3, respectively. Significant (p < 0.05) DNA damage was observed in the fishes procured from non-polluted locations and upstream locations. The micronucleus induction in E. chlorostigma was recorded as 23.20 ± 4.19 and 2.20 ± 0.58%, respectively, non-polluted and polluted sites. S. commerson exhibited significant differences between polluted and non-polluted sites (44.80 ± 3.73 and 8.20 ± 2.20‰) polluted and upstream (44.80 ± 3.73 and 20.60 ± 4.02‰), respectively. A significant difference was obtained between E. chlorostigma and S. commerson for nuclear abnormalities S. commerson showed higher frequencies for nuclear deformities than E. chlorostigma. S. commerson showed substantial micronucleus induction frequencies collected from an area of low pollution intensity (upstream). This study showed that E. clorostigma and S. commerson could be successfully used as a bioindicator to determine the health of the Red Sea through the most specific assays such as comet and micronucleus tests as an early warning and to devise the monitoring strategies to ensure a safe supply of fish for human consumption.     

Genotoxicity biomarkers in M. galloprovincialis as indicators of marine pollutants

Many substances pollute the marine environment. There is today a growing evidence on the increased risk of disease in marine organisms, especially fish, that inhabit contaminated waters. Different types of tumours have been evidenced in fish and shellfish populations. Different short-term biomarkers are available to predict the impact of carcinogens on marine organisms. Their endpoints are different effects at the molecular and cellular level such as gene mutation, chromosome alteration and induction of DNA damage and repair. We have applied two different assays: alkaline elution to measure DNA single strand breaks and micronucleus assay as an index of a chromosomal damage. In order to select an aquatic organism as an indicator of water pollution by carcinogenic agents, we have focused on the mussel. A program of validation of genotoxicity was conducted in aquarium using DMBA. A time-dependence increase of micronuclei was evident after the exposure to 100 ppb/animal. For alkaline elution the effect was 4 times the level of the controls. Experiments in the fields were conducted on adult specimens of Mytilus gdlloprovincialis collected from natural substrates. Our sampling stations were located in the La Spezia gulf, Ligurian sea. Genotoxic effects were evaluated in gill cells. A significant increment of the two parameters in polluted, in comparison with the unpolluted sites has been observed. High frequencies of micronuclei (the highest value was 42 ± 13 with respect to control value 3 ± 2) were scored in mussels from polluted stations. The extent of DNA damage was also relevant with respect to clastogenic damage as revealed by micronucleus test. The greatest value of K (constant of elution) was 8-fold higher with respect to the value of K obtained in the same tissue of mussel from reference areas. Evidence of DNA damage could reflect a recent pollution status, since DNA strand breaks can be rapidly repaired by different mechanisms. On the contrary animals exposed to clastogenic compounds may exhibit elevated micronucleus frequency long after the exposure has ceased. The evaluation of both parameters could provide information of great significance about the pollution status of the water.     

Cichlidogyrus sclerosus (Monogenea: Ancyrocephalinae) and its host, the Nile tilapia (Oreochromis niloticus), as bioindicators of chemical pollution

Experimental results showed that the gill monogenean Cichlidogyrus sclerosus and its host, the Nile tilapia Oreochromis niloticus, exhibited significant numerical and physiological responses after exposure to sediments polluted with polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls, and heavy metals in comparison with control fishes. After 15 days of exposure, C. sclerosus abundance significantly increased in treatments with low to fairly high sediment pollutant concentrations, but declined at high sediment pollutant concentrations. Hypertrophy and hyperplasia in secondary gill lamellae and the spleen melanomacrophage centers were significantly higher at extremely high sediment pollutant concentrations compared with the controls. Spleen lymphocyte and monocyte counts were significantly lower at extremely high sediment pollutant concentrations and were significantly correlated with high fluorescent aromatic compound concentrations measured as PAH exposure indicators. A multivariate redundancy analysis showed significant statistical association between sediment pollutant concentration, C. sclerosus abundance, and tilapia physiological variables. The polluted sediments negatively affected monogenean abundance and induced immunosuppression in hosts, consequently increasing histological damage in hosts and allowing persistent C. sclerosus infection. This study documents evidence suggesting that C. sclerosus and its host are indeed excellent models to test environmental quality in tropical freshwater ecosystems.     

The protective influence of ascorbic acid against the genotoxicity of waterborne lead exposure in Nile tilapia Oreochromis niloticus (L.)

The present study investigated the effects of lead (Pb) and ascorbic acid (AA) on Nile tilapia Oreochromis niloticus using the micronucleus (MN) and nuclear abnormality (NA) tests for periods of 7, 14 and 21 days. The MN frequencies in the erythrocytes, gill, liver and fin cells were analysed comparatively to evaluate the sensitivity and suitability of these different cell types. The NA shapes in erythrocytes were scored into blebbed nuclei (BL), lobed nuclei (LB), notched nuclei (NT) and binuclei (BN). It was observed that fish showed significant sensitivity to the different treatments. In general, the highest value of both MN and NA cells were significantly increased in the Pb-treated group followed by the combination of the Pb and AA-treated group. On the other hand, the MN and NA frequencies in erythrocytes were the most sensitive to the treatment and could provide more valuable information than those in gill, liver and fin cells. The frequencies of each NA shape in erythrocytes of all treatments were observed in the following ranked order NT > LB > BN > BL. The results demonstrated the efficacy of AA in reducing genotoxicity in fish induced by Pb. They showed the sensitivity and suitability of MN and NA frequencies in erythrocytes as pollution biomarkers.     

Circular DNA intermediate in the duplication of Nile tilapia vasa genes

vasa is a highly conserved RNA helicase involved in animal germ cell development. Among vertebrate species, it is typically present as a single copy per genome. Here we report the isolation and sequencing of BAC clones for Nile tilapia vasa genes. Contrary to a previous report that Nile tilapia have a single copy of the vasa gene, we find evidence for at least three vasa gene loci. The vasa gene locus was duplicated from the original site and integrated into two distant novel sites. For one of these insertions we find evidence that the duplication was mediated by a circular DNA intermediate. This mechanism of gene duplication may explain the origin of isolated gene duplicates during the evolution of fish genomes. These data provide a foundation for studying the role of multiple vasa genes in the development of tilapia gonads, and will contribute to investigations of the molecular mechanisms of sex determination and evolution in cichlid fishes.     

Ultrastructural biomarkers as tools to characterize the health status of fish in contaminated streams

Ultrastructural biomarkers in gill, liver, andkidney of brown trout (Salmo truttaf. fario) and stone loach (Barbatulabarbatula) were investigated over afive year period. The cellular damage of theorgans was assessed semi-quantitatively basedon a three-step classification ofultrastructural responses. Data obtained forfish exposed under semi-field conditions in twodifferently polluted test streams and in thelaboratory demonstrated that the ultrastructureof the organs can be correlated with differentpollutant exposure conditions. Cellular damagewas generally more severe in fish exposed to acomplexly polluted stream than in those exposedto a moderately polluted stream or to tap waterin the laboratory. Histopathological effects inliver and gill of trout were more pronounced inferal fish than in transplanted fish, whereasresponses in the kidney in both species, and inliver and gill of loach, were similar forintroduced and feral fish. In a laboratoryexperiment where trout were exposed todifferent mixtures of pollutants includingpesticides, PAH, and ammonia, only theultrastructure of kidney and liver showedsignificant differences between the threeexperimental set-ups. In a recovery experiment,where trout were transferred from thesemi-field condition back to the laboratory,ultrastructural investigations showed adifferential capacity of the respective organsto recover from stress under field conditions.Kidney and liver fully recovered after threemonths under control condition whereas gillsdemonstrated only partial recovery.     

Potential value of the comet assay and DNA adduct measurement in dab (Limanda limanda) for assessment of in situ exposure to genotoxic compounds

An in situ study of the relationship between marine contamination and genotoxic effects was performed on female dab (Limanda limanda) collected from different sites in the eastern English Channel (France) known to be contaminated by polycyclic aromatic hydrocarbons (PAHs) and polychlorobiphenyls (PCBs). DNA adducts in liver and DNA strand breaks in blood cells were determined respectively by the nuclease P1-enhanced post-labelling technique and an alkaline version of the comet assay. The extent of DNA base oxidation was also assessed for three of the six sampling sites in the study, using a comet assay in combination with a specific DNA repair enzyme, formamidopyrimidine glycosylase (Fpg).With Comet data, two groups of sites that seem in accordance with the pollution level have been distinguished. The extent of DNA strand breaks was higher in adult than juvenile female dab. From a technical point of view, comet assay sensitivity was affected by high intra-individual variability that accounted for nearly 70% of total variance (the site factor represented no more than 26%). The combined use of the comet assay and Fpg showed the presence of DNA oxidised bases in environmentally exposed dab.Although qualitative differences between the sampling sites were observed in DNA adduct profiles, no significant differences were found for total DNA adduct levels. DNA adducts did not appear to be associated with PAH exposure.Histopathological studies showed hepatic steatosis in most of the animals examined. Only one pre-cancerous lesion (an early stage of hyperplasia) was detected (associated frequency of 0.8%).     

尼罗罗非鱼早期发育形态及其在珠江水系的空间分布

【背景】罗非鱼作为联合国粮农组织（FAO）向全世界推广养殖的优良品种之一,有多个品系,其养殖范围已遍布85个国家和地区。1956年我国从越南引进莫桑比克罗非鱼,经过养殖及推广,2006年我国罗非鱼产量达到100万t。但是,该外来物种在给我国带来良好经济价值的同时,对土著种类及水环境造成了极大的影响。【方法】对近几年珠江水系渔获物的调查数据进行整理,并观察尼罗罗非鱼早期发育形态,统计尼罗罗非鱼苗对其他鱼苗的最大捕食量及捕食规格,以分析罗非鱼生物学特性及其在珠江水系的入侵现状。【结果】尼罗罗非鱼早期发育快,卵黄营养非常丰富,比珠江水系土著种类更有竞争性;罗非鱼苗呈现很强的攻击性与捕食性;尼罗罗非鱼已经扩散到珠江水系各主要河流,并在部分江段形成优势种群。【结论与意义】尼罗罗非鱼种群快速扩张,对土著种已构成严重威胁,有必要将其列为珠江水系高危入侵种。     

Profenofos induced DNA damage in freshwater fish, Channa punctatus (Bloch) using alkaline single cell gel electrophoresis

The aim of the present study was to evaluate the induced genotoxicity (DNA damage) due to organophosphate pesticide profenofos (PFF) in gill cells of freshwater fish Channa punctatus using single cell gel electrophoresis (SCGE)/Comet assay. The 96h LC(50) value of PFF (50% EC) was estimated for the fish species in a semistatic system and then three sub-lethal of LC(50) concentrations viz the sub-lethal 1, sub-lethal 2 and sub-lethal 3 concentrations were determined as 0.58ppb, 1.16ppb and 1.74ppb, respectively. The fish specimens were exposed to these concentrations of the pesticide and the gill tissue samplings were done on 24h, 48h, 72h and 96h post exposure for assessment of DNA damage in terms of percentage of DNA in comet tails. In general, a concentration dependent response was observed in the gill cells with induction of maximum DNA damage at the highest concentration of PFF. The results of the present investigation indicated that PFF could potentially induce genotoxic effect in fish, even in sub-lethal concentrations and SCGE as a sensitive and reliable tool for in vivo assessment of DNA damage caused by the genotoxic agents.     

DNA damage in eelpout (Zoarces viviparus) from Göteborg harbour

The relationship between DNA damage and the exposure of marine organisms to environmental contaminants was examined in the G?teborg harbour area. This research is part of a wider ecotoxicological study planned to evaluate the biological impact of chemical contamination in the River G?ta estuary, following a bunker oil (10-100 tonnes) spill occurred in June 2003. Here we present data on the DNA strand breaks derived using the comet assay and the presence of apoptotic cells using the diffusion assay in nucleated erythrocytes of the eelpout (Zoarces viviparus) from the study area and at a clean reference site. Polycyclic aromatic hydrocarbon metabolites were also analyzed in the bile of exposed fish. The results showed a high level of damaged DNA, paralleled by a peak in bile PAH metabolites, in fish from the most impacted site, 3 weeks after the oil spill. A significant recovery was observed in specimens from the spill site, 5 months later, but not in fish caught in the middle part of G?teborg harbour, which is chronically subjected to heavy chemical pollution. The levels of apoptic cells did not show any marked variations, but a significant recovery was observed in fish from the oil impacted site 5 months after the spill.     

Intracytoplasmic Sperm Injection Using Cryopreserved, Fixed, and Freeze-dried Sperm in Eggs of Nile Tilapia

Gamete preservation techniques are essential in animal husbandry as well as in assisted reproduction for humans. In this research we attempted to use 3 different sperm preservation techniques in combination with newly developed techniques for intracytoplasmic sperm injection (ICSI) to fertilize eggs of a teleost fish, the Nile tilapia (Oreochromis niloticus). Of 47 eggs injected with fresh sperm, 11 (23%) were fertilized, 5 developed abnormally, and 4 developed normally and hatched; from these, one grew to adulthood. Nuclear DNA content of 4 of the abnormal embryos indicated that they were diploid. Flow cytometric analysis of a blood sample from the surviving ICSI fish collected 2 months after fertilization indicated that the fish was diploid. Of 45 eggs injected with cryopreserved sperm, 9 (20%) developed to the blastula stage. Of 40 eggs injected with sperm preserved in 70% methanol, none were fertilized. No injections were possible with freeze-dried Nile tilapia sperm owing to technical difficulties during manipulation. Although the findings described here are limited, they provide the first steps toward using sperm preservation methods in addition to cryopreservation for fertilization in fishes.