Alaa G. M. Osman

Use of hematological parameters to assess the efficiency of quince (Cydonia oblonga Miller) leaf extract in alleviation of the effect of ultraviolet - a radiation on African catfish Clarias gariepinus (Burchell, 1822).

The present study aimed to elucidate the negative impacts of UVA on some biochemical and hematological variables of the economically important African catfish, Clarias gariepinus and investigates the putative role of quince (Cydonia oblonga Miller) leaf extract in protection and/or alleviation of such negative impacts. Changes in the hematological and blood biochemical values often reflect alteration of physiological state. Blood parameters can be useful for the measurement of physiological disturbances in stressed fish and thus provide information about the level of damage in the fish. We found a significant (P<0.05) decrease in the red blood cell counts, hemoglobin and hematocrit in the groups exposed to UVA compared to the control groups. Exposure to UVA induced marked red cell shrinkage (increased mean cell hemoglobin concentration) and showed an elevation in mean cell volume and mean cell hemoglobin in the blood of the exposed fish compared to the control. A significant (P<0.05) reduction in the total white blood cells was recorded in the exposed fish compared to the control. The biochemical parameters (blood glucose, total plasma protein, blood cholesterol, plasma creatinine, aspartic amino transferase and alanine amino transferase) exhibited a significant increase in the blood of fish exposed to UVA. Methanolic extract of quince leaf before ripening of the fruits was analyzed by GC/MS. To investigate the biological impact of this extract and its biologically active components, this extract was tested for its putative role in alleviation of UVA effect on catfish. Quince leaf extract had the ability to prevent hematotoxic stress induced by UVA and resulted in enhancement of the immune system of catfish represented by significant (P<0.05) increase in the number of white blood cells and lymphocytes of the catfish. Quince extract also protected the red blood cells from UVA damage. To our knowledge this is the first report of the effect of quince leaf extract on an aquatic organism.

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.

In situ evaluation of the genotoxic potential of the river Nile: I. Micronucleus and nuclear lesion tests of erythrocytes of Oreochromis niloticus niloticus (Linnaeus, 1758) and Clarias gariepinus (Burchell, 1822)

This study aimed to investigate the genotoxic potential of chemicals present along the course of the river Nile using frequencies of micronuclei (MN) and nuclear lesions (NL) in erythrocytes of Nile tilapia Oreochromis niloticus niloticus and African catfish Clarias gariepinus, as biomarkers. Results showed that most of the physicochemical parameters detected and heavy metal concentrations were significantly higher in the water collected from the estuaries of the river Nile compared to other sites of the upper Nile. The frequencies of MN and NL in peripheral blood erythrocytes of Nile tilapia and African catfish were significantly higher in estuary sites in Damietta and Rosetta compared to upper sites. The lowest level of genotoxicity was observed at two sites (Aswan and Kena), considered to be less contaminated. Our results suggested that higher frequencies of MN and NL determined at Damietta and Rosetta sites may be indicative of damage produced by pollutants in these areas. The most remarkable result was that MN and NL frequencies appear to be strongly related to water quality at different sites examined, indicating that MN frequencies may serve as a reliable biomarker for testing genotoxicity in situ. The positive correlation between MN and NL induction suggested that NL may be a useful complementary assay for genotoxicity analyses when fish are used as experimental animals. It was also found that seasonal variations in MN and NL frequencies might contribute to a better understanding of genotoxic responses in the field. The use of fish as indicator organisms for monitoring the presence of genotoxic-inducing contaminants in the environment seemed justified because the effects of exposure to a “complex mixture” such as river water were obtained. Nile tilapia appears to be a more suitable bioindicator species than African catfish in studying genotoxic chemical pollution in the river Nile attributed to a higher sensitivity.

Monitoring of DNA breakage in embryonic stages of the African catfish Clarias gariepinus (Burchell, 1822) after exposure to lead nitrate using alkaline comet assay

Increasing lead contamination in Egyptian ecosystems and high lead concentrations in food items have raised concern for human health and stimulated studies on monitoring ecotoxicological impact of lead‐caused genotoxicity. In this work, the alkaline comet assay was modified for monitoring DNA strand breakage in sensitive early life stages of the African catfish Clarias gariepinus. Following exposure to 100, 300, and 500 μg/L lead nitrate, DNA strand breakage was quantified in embryos at 30, 48, 96, 144, and 168 h post‐fertilization (PFS). For quantitative analysis, four commonly used parameters (tail % DNA, %TDNA; head % DNA, %HDNA; tail length, TL; tail moment, TM) were analyzed in 96 nuclei (in triplicates) at each sampling point. The parameter %TDNA revealed highest resolution and lowest variation. A strong correlation between lead concentration, time of exposure, and DNA strand breakage was observed. Here, genotoxicity detected by comet assay preceded the manifested malformations assessed with conventional histology. Qualitative evaluation was carried out using five categories are as follows: undamaged (%TDNA ≤ 10%), low damaged (10% < %TDNA ≤ 25%), median damaged (25 < %TDNA ≤ 50%), highly damaged (50 < %TDNA ≤ 75%), and extremely damaged (%TDNA > 75%) nuclei confirming a dose and time‐dependent shift towards increased frequencies of highly and extremely damaged nuclei. A protective capacity provided by a hardened chorion is a an interesting finding in this study as DNA damage in the prehatching stages 30 h‐PFS and 48 h‐PFS was low in all treatments (qualitative and quantitative analyses). These results clearly show that the comet assay is a sensitive tool for the detection of genotoxicity in vulnerable early life stages of the African catfish and is a method more sensitive than histological parameters for monitoring genotoxic effects.

Genotoxicity of two pathogenic strains of zoosporic fungi (Achlya klebsiana and Aphanomyces laevis) on erythrocytes of Nile tilapia Oreochromis niloticus niloticus

In the present work we have described the genotoxic potential of two pathogenic strains of zoosporic fungi (Achlya klebsiana and Aphanomyces laevis) on erythrocytes of Nile tilapia Oreochromis niloticus niloticus for the first time by three complementary tests: micronucleus test (MN), nuclear lesions (NL) and comet assay (CA). The groups exposed to the zoosporic fungi subjected to the MN and NL test showed statistically significant differences in MN and NL frequencies with respect to the control one. Also, a significant increase (p<0.001) in micronuclei and nuclear lesions frequencies were recorded with the increase in exposure time. A correlation was observed between the frequencies of MN and NL, suggesting the importance for recording this anomaly in order to improve the information obtained with the MN test. Therefore, our results suggest that the nuclear lesions found here should be considered indicators of genotoxicity, in addition to the typical micronuclei forms. The result of the comet assay showed a significant difference in the percentages of the damaged DNA in the comet tail (%TDNA) of the treated groups comparing to the control. A significant (p<0.001) increase in the DNA damage of the treated groups with the increase in exposure time was recorded, confirming the results of the MN and the NL tests. The results of the current study will be useful for future work involving the biomonitoring of regions where Nile tilapia survive. These data allow us to consider O. niloticus niloticus as a good bioindicator of the effects of genotoxic agents that might be present in the aquatic habitat.

Reproductive Biology of Round Herring Etrumeus teres (Dekay, 1842) from the Egyptian Mediterranean Water at Alexandria

Maturity stages of round herring Etrumeus teres were divided anatomically into six stages. Monthly distribution of such maturity stages showed that E. teres had prolonged spawning season extended from December to May for females and to July for males. The observed sex ratio was 1 : 2 (male: female). Gonadosomatic index (GSI) values were high during breeding season for both sexes. Hepatosomatic index (HIS) values for females increased from December to April and from December to July for males. Both sexes of E. teres showed the same correlation between GSI and HSI during their breeding season. The absolute and relative fecundity increased with increasing of length and weight. Histologically, the ovarian cycle of E. teres was classified into six stages and the testicular cycle into five stages. Female of E. teres was a multiple spawner and had prolonged spawning season with a group-synchronous ovarian which contained oocytes at all stages of development. Also, different sizes of spermatocytes at different developmental stages of maturation were observed during the same season. This may reflect the suitability of the specific reproductive behavior of male and its adaptation to the prolonged spawning season with females.

Screening of multiple hormonal activities in water and sediment from the river Nile, Egypt, using in vitro bioassay and gonadal histology

In Egypt, until yet no records are available regarding possible multiple hormonal activities in the aquatic systems and especially in the river Nile. In this paper, in vitro yeast estrogen screen (YES) and yeast androgen screen (YAS) were used to assess (for the first time) the multiple hormonal activities in surface waters and sediments of the river Nile. This study aimed to determine whether river Nile water can cause changes in gonadal histology of Nile tilapia (Oreochromis niloticus niloticus). All water samples exhibited extremely low levels of estrogenicity. Estrogenicity was nearly not detected in any of the sediment samples. Unlike the estrogenicity, significant androgenic activities were recorded in the water and sediment samples along the course of the river Nile. The present study reports for the first time quantification anti-estrogenic and anti-androgenic activities with high levels in both water and sediment of the river Nile. The greatest anti-estrogenic and anti-androgenic activities were observed in samples from downstream river Nile. These results indicated that the anti-estrogenic and anti-androgenic activities along the Nile course were great and the pollution of the sites at downstream was more serious than the upstream sites due to industrial and anthropogenic activities at these sites. Good correlations were observed among some hormonal activities, suggesting coexistence of these contaminants in the environmental matrices. There were no signs of sexual disruption in any of the gonads analyzed from either male or female Nile tilapia, demonstrating that no hormonal activity present along the Nile course was sufficient to induce adverse effects on reproductive development. Further investigation is necessary to identify the compounds responsible for the hormonal activities in the river Nile and to examine effects of very low levels of hormonally active compounds on gonadal histology, as well as in the development of more sensitive biomarkers.

Blood Biomarkers in Nile tilapia Oreochromis niloticus niloticus and African Catfish Clarias gariepinus to Evaluate Water Quality of the River Nile

In this work, we explore new sights of biomarker research and establish the use of blood parameters and erythron profile in wild fish. Water quality assessment along the Nile course concluded higher mean values of the detected physicochemical parameters in water collected from downstream sites. The mean values of nearly all the detected haematological parameters were significantly lower in the blood of Nile tilapia and African catfish collected from downstream (contaminated) compared to upstream sites. Lower cellular and nuclear areas and cellular and nuclear shape factor were recorded in the erythrocytes of fish collected from downstream compared to those caught from upstream sites. This was confirmed by higher immature ratios of red cells in the blood of fish sampled from downstream sites. Karyorrhectic and enucleated erythrocytes were recorded to be higher in the blood of fish collected from downstream sites. The levels of all the selected biochemical variables in the blood of both fish species were recorded to be significantly higher in downstream sites. The detected blood variables and erythrocyte malformations were proven to be suitable for bio-monitoring of aquatic pollution. The results revealed speciesspecific differences in sensitivities, suggesting that Nile tilapia may serve as a more sensitive test species compared to African catfish.