Micro-computed tomography for analysis of heavy metal accumulation in the opercula.

Micro-computed tomography (micro-CT) provides numerous opportunities in biomedical research. It allows the examination of samples in a non-destructive manner and visualization of the inner structures of various biological and nonbiological objects. This study was conducted to evaluate the potential of micro-CT scanner in the assessment of heavy metal accumulation in the opercula. The samples were taken from Prussian carp (Carassius gibelio) exposed to waterborne Cd (4.0 mg/L), Zn (4.0 mg/L), and the mixture of these two metals (4.0 mg Cd/L and 4.0 mg Zn/L) for 28 days. Heavy metal concentrations were determined using atomic absorption spectrometry. The results demonstrated higher concentrations of Cd and Zn in the treatment group opercula samples compared with the control group opercula samples. A simple micro-CT scan was performed to verify whether heavy metal accumulation could be determined in the reconstructed images. The results showed that micro-CT is potentially a powerful tool for metal accumulation detection. Moreover, it allowed visualization of the examined samples, revealing regions of heavy metal accumulation and providing the opportunity to compare samples exposed to different types of heavy metals.

Effect of a Diet Supplemented with Nettle (Urtica dioica L.) or Fenugreek (Trigonella Foenum-Graecum L.) on the Content of Selected Heavy Metals in Liver and Rabbit Meat.

The literature on herbal additives for rabbit feed offers little information on the use of nettle and fenugreek. Both of these herbs are valuable sources of vitamins and minerals. These herbs affect the growth, health, and meat quality of rabbits. They regulate the digestive system, stimulate the appetite, have a positive effect on the functioning of the immune system, and exhibit antibacterial activity. The purpose of the present study was to determine the effect of nettle (Urtica dioica L.) leaves or fenugreek (Trigonella foenum-graecum L.) seeds in the feed on the content of selected heavy metals in the liver and meat of the rabbit. The rabbits were divided into three groups: group C (n = 20; 10♂ and 10♀) was fed ad libitum with a complete feed, N group (n = 20; 10♂ and 10♀) was fed a complete mixture with 1% added nettle, and group F (n = 20; 10♂ and 10♀) was fed with a complete mixture with 1% added fenugreek. The experiment lasted 7 weeks (from the 35th to the 84th day of the rabbits' lives). All the rabbits were slaughtered on the 84th day of age, with a body weight of about 2.6 kg. The concentration of heavy metals (Zn, Cu, Ni, Mn, Fe, Pb, Cd) was determined by the atomic absorption spectrometry (AAS). The additives to the feed significantly affected the content of elements in both the liver and the meat of rabbits (p < 0.05). The highest level of the heavy metals, regardless of the used diet, was recorded in the liver (p < 0.05). The meat (m. longissimus lumborum) and the liver of rabbits fed with herbal fodder contained less tested metals than in animals fed with fodder without additives (p < 0.05). Moreover, more essential metals were found in the liver of rabbits fed with fenugreek than rabbits fed with nettle. In the meat and liver of rabbits, the permissible content of cadmium and lead was not exceeded. Additionally, male livers had a significantly higher content of copper and manganese compared to female livers (p < 0.05). It is important to study the content of heavy metals in the used animal herbal feed additives and their interaction with each other, as they affect the distribution of elements in tissues and organs.

Genotoxic Effect of Cadmium and Zinc in the Peripheral Erythrocytes of Prussian Carp (Carassius Gibelio B.).

Introduction: Cadmium and zinc are often found in aquatic environment and may accumulate in living organisms. The aim of this study was to evaluate the genotoxic effect of Cd, Zn, and their binary mixture on the peripheral blood erythrocytes of Prussian carp (Carassius gibelio B.). Material and Methods: The fish were exposed to 4.0 mg/L Cd, 4.0 mg/L Zn or a mixture of 4.0 mg/L Cd and 4.0 mg/L Zn for a period of 14, 21 or 28 days. Genotoxic effects were investigated in peripheral blood cells using the comet assay and the erythrocyte micronucleus assay. Results: The results demonstrated that the frequencies of micronuclei (MN) and both nuclear and cellular abnormalities in erythrocytes were significantly higher in all exposure groups as compared to the control group. The fish exposed to the mixture of Cd and Zn presented the highest frequency of MN. Furthermore, there was a decrease in the frequency of MN and an increase in the occurrence of DNA integrity defects (DNA damage) with longer time of exposure to the metals studied. Conclusion: Erythrocyte micronucleus and comet assays confirmed the genotoxicity of Cd and Zn. The results of the tests applied (which showed considerable variability) suggest the involvement of various toxicity mechanisms. Therefore, an integrative and comprehensive approach, using a set of assays for toxicity profile determination, should be adopted during ecotoxicological studies and environmental risk assessment pertaining to these elements.

Roundup in the Reproduction of Crucian Carp (Carassius carassius): An In Vitro Effect on the Pituitary Gland and Ovary.

Roundup, the most popular herbicide in global agriculture, is regarded as an endocrine disruptor causing alterations of important hormones at the hypothalamic-pituitary-gonadal axis as well as impairment of gametogenesis. The whole pituitary glands of crucian carp (Carassius carassius) were incubated for 3 h in the medium containing Roundup (0-control, 1 and 10 ng/mL). The level of luteinizing hormone (LH), and mRNA transcript abundance of kisspeptin (kiss-1) and its receptor (gpr54), were determined. The isolated ovarian fragments were incubated for 24 h in the presence of Roundup and the following effects on reproductive parameters were determined: the final oocyte maturation and ovulation, structural changes in follicles, secretion of 17,20ß-progesterone (17,20ß-P) as well as mRNA transcript abundance of the luteinizing hormone receptor (lhr), estrogen receptors (erα, erß1, erß2), and zona radiata (chorion) proteins (zp2 and zp3). Roundup inhibited final oocyte maturation and decreased the percentage of ovulated eggs, and furthermore, caused structural changes in the ovarian follicular components. There were no significant changes in the measured hormone levels and analyzed genes mRNA transcript abundance. Summing up, obtained results indicate that Roundup may adversely affect oocyte maturation and the quality of eggs, suggesting that exposure to this herbicide can lead to reproductive disorders in fish.

Cadmium Accumulation and Depuration in the Muscle of Prussian Carp (Carassius gibelio Bloch) after Sub-Chronic Cadmium Exposure: Ameliorating Effect of Melatonin.

The aim of this study was to investigate the bioaccumulation of cadmium in the muscle tissue of Prussian carp during 7 and 13 weeks of exposure to different concentrations of this metal in water (0.4 and 4.0 mg/L), and the depuration of cadmium from muscle during the following 6-week depuration period in the presence of melatonin implants. Furthermore, the relationship between cadmium accumulation and the levels of essential bioelements (copper, zinc, iron) in muscle was evaluated, as well as the bioconcentration factor of cadmium. Heavy metal concentration was determined using atomic absorption spectrometry. Cadmium accumulation in fish muscle increased with the duration of exposure. Cd concentrations exceeded the permissible levels for human consumption in groups exposed to the higher concentration of this metal. Moreover, a significant increase of Zn and Fe levels in the muscle was observed. In the fish that received melatonin implants and were exposed to Cd, its level in the muscle was significantly lower. The depuration of accumulated cadmium depended mainly on the duration of the elimination period. This is the first study to report that melatonin co-administration can effectively protect the fish from the accumulation of cadmium in muscle tissue and changes in trace metal levels.

Cadmium-induced oxidative stress in Prussian carp (Carassius gibelio Bloch) hepatopancreas: ameliorating effect of melatonin.

The oxidative status of the hepatopancreas of Prussian carp females (Carassius gibelio) co-exposed to sublethal cadmium in water and melatonin was studied. The activities of antioxidant enzymes such as glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD) as well as the concentration of reduced glutathione (GSH) were measured in homogenates of the hepatopancreas. Furthermore, concentrations of cadmium (Cd), zinc (Zn), copper (Cu), and iron (Fe) in the hepatopancreas were assayed. These females received melatonin implants and were exposed to 0.4 mg/L or 4.0 mg/L Cd in water for either a 13- or a 7-week period, followed by further 6 weeks of purification in clear water. Exposure to Cd influenced the increase in this metal concentration in fish hepatopancreas. In contrast, the fish exposed to cadmium with additional administration of melatonin had a lower accumulation of this metal. Exposure to Cd caused the increase in GSH content and the activity of GR, and a reduction in GPx activity, whereas the SOD activity varies depending on the exposure time on cadmium. In the hepatopancreas of fish treated with Cd alone, the content of Cu and Zn were increased and that of Fe was changed. After melatonin administration to Cd-exposed fish, a decrease in copper and zinc hepatopancreas content was noted. The present findings imply that melatonin co-treatment can effectively protect the fish against the toxic effects of cadmium on endogenous antioxidant status in hepatopancreas tissues and variations in metal concentration, such as Zn, Cu, and Fe.

Protective effect of melatonin on cadmium-induced changes in some maturation and reproductive parameters of female Prussian carp (Carassius gibelio B.).

The aim of this study was to determine whether melatonin (Mel), which is a known antioxidant and free radical scavenger, could perform the role of a preventive agent against the toxic effects of cadmium (Cd2+) on mortality, fish growth, gonadosomatic index (GSI), luteinizing hormone (LH) secretion, the response to hormonal stimulation of spawning, and also tissue accumulation of Cd in Prussian carp females. These females received melatonin implants and were exposed to 0.4 or 4.0 mg/L of Cd (as CdCl2·2.5H2O) over either a 5- or 3-month period, followed by further 2 months of purification in clear water. Negative changes caused by exposure to cadmium in the water were as follows: higher fish mortality, lower body weight, increased accumulation of cadmium in the brain and ovary, lowered GSI, impaired spontaneous LH secretion during exposure, and impaired LH secretion during stimulation of spawning. All of these effects were observed in the group of fish exposed to 0.4 and/or 4.0 mg Cd/L but did not occur or were less pronounced in the groups exposed to cadmium in the presence of melatonin released from the implants. During depuration, in the group of fish which had been exposed to the highest Cd concentration, we observed a significant improvement in fish survival rate, body growth, inhibition of further cadmium accumulation in tissues, and gradual return of spontaneous LH secretion as well as normalization of the GSI value to the control group levels. In conclusion, these findings indicate that melatonin can be a preventive agent for some toxic effects on fish reproduction induced by environmental cadmium contamination.

Effect of long-term dietary lead exposure on some maturation and reproductive parameters of a female Prussian carp (Carassius gibelio B.).

Lead is reported to be an endocrine disruptor. In the current study, we exposed female Prussian carp to artificial feed, supplemented with five doses of dietary Pb (0, 1 (control), 8, 13, 24, and 49 mg/kg) over either a 24-month period or a 12-month period, followed by further 12 months where the fish were fed the control diet. Periodically, oocyte maturation, gonadosomatic index (GSI), ability to secret luteinizing hormone (LH) as well as gonad Pb concentrations were measured. It was found that the reproductive system of the female Prussian carp is not indifferent to chronic exposure to lead. The negative effect was manifested by a decrease in the GSI after 12 months despite the fact that a higher proportion of oocytes at more advanced maturity stages were concurrently observed. After 12 months of exposure, the effect on LH secretion varied according to the dose. In the group exposed to the lowest dose (8 mg/kg), LH decreased spontaneously, and in the groups exposed to the highest two doses (24 and 48 mg/kg), a significantly higher LHRH-A-stimulated LH secretion was observed. After 24 months of lead exposure, the effects on oocyte maturation and size and on GSI values were not pronounced. Analysis of the effect of lead exposure on LH secretion showed that the relationships were similar to those observed after 12 months but nonsignificant. During chronic a 24-month period exposure to Pb, Prussian carp female appears to acclimate to Pb doses used in the experiment. After 12 months of exposure and 12 months of depuration, the levels of spontaneous and stimulated LH secretion observed in all the groups were similar to the control, which is evidence that depuration eliminates the previously observed effects of exposure to lead. Lead is easily accumulated in the ovary, reaching a fivefold higher level (0.8 mg/kg tissue) compared to the control(0.15 mg/kg tissue), but after discontinuation of exposure, this organ is quickly depurated. The results indicate that environmental Pb can be a potent endocrine disruptor affecting ovarian steroidogenesis, gametogenesis, and ovulation, which may lead to adverse impacts on fish reproduction and population density and that female Prussian carp become resistant to the negative effects of lead with advancing age, and their organs cope by reaching a state of homeostasis.

Lead accumulation and elimination in tissues of Prussian carp, Carassius gibelio (Bloch, 1782), after long-term dietary exposure, and depuration periods.

We studied the bioaccumulation of lead in selected tissues of Prussian carp Carassius gibelio (Bloch, 1782) during 12 and 24 months exposure to different doses of this metal in feed and the elimination of lead from tissues during the following 12-month depuration period. Lead concentration was determined using atomic absorption spectrometry method. The highest lead concentrations were observed at 2.0 ± 0.54 to 7.4 ± 1.1 mg kg(-1) in the kidney, 3.0 ± 0.13 to 5.2 ± 0.17 mg kg(-1) in the bone, and 4.5 (±0.4) mg kg(-1) in the hepatopancreas of fish from groups exposed to lead dietary concentration from 8 to 49 mg kg(-1) for 24 months. The rate of accumulation were generally the highest at the beginning of exposure as evidenced by the highest monthly increments of bioaccumulation observed after 3 months of contamination for muscles, hepatopancreatic gland, intestine, and gills. Also analysis of the monthly increments of lead bioaccumulation in bone tissue and the highly significant coefficients of correlation indicate that the dynamics of accumulation are clearly dependent on dose of exposure. Depuration of accumulated lead from the organs depended mainly on tissue and duration of elimination period. Very rapid depuration was observed in soft tissues such as the intestine or muscles. Very low elimination was observed for scales and bones where until the end of the experiment highly significant lead concentration differences were observed in all groups in relation to the control group. Chronic dietary exposure in the range of 8-49 mg Pb kg(-1) resulted in no significant effects on the growth and survival of Prussian carp females.

Influence of heavy metals and 4-nonylphenol on reproductive function in fish.

Many industrial and agricultural chemicals (including heavy metals and alkylphenols) present in the environment have adverse effects on the reproductive function in fish. Three studies were conducted to assess toxicity of these chemicals towards reproduction of freshwater fish. It was shown that heavy metals added to the diets accumulate in brain tissue of carp, and this accumulation results in inhibition of the secretion of noradrenaline and stimulation of the secretion of dopamine in the hypothalamus. These processes results in a disturbance of hormonal equilibrium of the hypothalamo-pituitary system, which can unfavorably influence the efficiency of artificial spawning in fish. Quality of salmonid and sturgeon sperm was impaired after in vitro exposure to heavy metals. The degree of this toxic effect was species-specific. It was demonstrated that sperm motility parameters appeared to be good indicators of adverse effects of heavy metals fish sperm. The protection role of seminal plasma against toxic effects of heavy metals was suggested for salmonid fish. Oral application of 4-nonylphenol (NP) disrupted reproduction in pikeperch. In juvenile fish a decrease in the percentage of males and an increase of intersex fish was observed in relation to dose of NP and time of exposure to this alkylphenol. Exposure of adult males to the NP led to the reduction in fecundity, milt quality and fertility.