الفهرس 
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Abstract
The widespread use of TiO2 NPs in many applications like (medicines, personal care products, plastic products, paints, paper…) has led to its significant release to the aquatic systems. TiO2 NPs have adverse negative effects on biosystems. Subsequently this study aimed to find out the negative effects of TiO2 NPs on the biological aspects of Caelatura nilotica clams including bioaccumulation, morphometrics, biometrics, DNA damage of gills, haemocytes count, Neutral red uptake and the histological structure of gills, digestive gland and gonads. As well as the possible chelating/ ameliorative role of DMSA against the same nanoparticles. C. nilotica clams were divided into eight groups (15 clams/group/3 replicates) and the experiment lasted for 4 successives weeks as the following;
a- Control group,
b- Two groups were exposed to 25 and 150 µg/L of TiO2,
c- Two groups were exposed to 25 and 150 µg/L of TiO2 NPs,
d- One group was exposed to 200 µg/L of DMSA and
e- Two groups were exposed to 25 and 150 µg/L of TiO2 NPs + 200 µg/L of DMSA.
The results of the present study could be summarized as follows:
1- Titanium concentrations in the water and Caelatura nilotica clams` tissues
The concentration of both Ti (TiO2 and TiO2 NPs) was significantly higher in water of groups exposed to 150 µg/l compared to those exposed to 25 µg/l and control (P= 0.004). While the highest concentration was 161.68±1.56 µg/l (150 μg/l TiO2 group exposed) and the lowest concentration was (10.06±0.22 µg/l) (200 µg/l DMSA group exposed) in the experimental water at the begening of exposure. In C. nilotica clams’s tissues, Ti concentrations significantly increased over time in TiO2 / TiO2 NPs group exposeds (P= 0.0001). At the end of experiment, the lowest Ti concentration was (14.04±3.70 μg/gm dry weight) in clams exposed to 150 µg/l TiO2 NPs + 200 µg/l DMSA. The clams exposed to 150 µg/l TiO2 NPs had significantly high Ti concentration (78.5±28.93 μg/g/ dry weight). It was noted that, after 4 weeks, exposure to 200 µg/l DMSA led to a decrease in TiO2 NPs concentrations in all clams group exposed to DMSA.
2- Effects on Morphometrics/Biometrics
The body length of C. nilotica clams increased significantly over time across all groups (P = 0.0001), with clams exposed to 150 µg/l TiO2, 25, and 150 µg/l TiO2 NPs showing the highest body lengths after 4 weeks. The body width of control clams increased from 17.83 ± 1.65 mm at zero time to 21.00 ± 2.64 mm after 4 weeks, with significant increases observed in clams exposed to 25 and 150 µg/l TiO2, 25, 150 µg/l TiO2 NPs, and 200 µg/l DMSA after 1 day and 2 weeks (P ≤ 0.05). However, clams exposed to 25 and 150 µg/l TiO2 NPs + 200 µg/l DMSA showed a significant reduction in body width after 2 weeks (P ≤ 0.05). Body height increased significantly in all groups except those exposed to 25 µg/l TiO2, with control clams rising from 28.5 ± 1.73 mm to 31.00 ± 1.73 mm after 4 weeks. The 150 µg/l TiO2 and 150 µg/l TiO2 NPs groups showed significant body height increases after 2 weeks, while the 200 µg/l DMSA group showed a slight but non-significant increase (P > 0.05).
The shell weight of clams exposed to 25 µg/l TiO2 NPs increased significantly after 2 weeks, and the 150 µg/l TiO2 group showed a significant increase after 4 weeks (P ≤ 0.05). The flesh weight increased significantly across all groups, with the control group starting at 5.14 ± 1.14 gm and the lowest value in clams exposed to 25 µg/l TiO2 NPs + 200 µg/l DMSA (3.50 ± 0.31 gm, P=0.0001). Total body weight of clams exposed to 25 µg/l TiO2 NPs was higher than other groups after 4 weeks, while clams exposed to 25 µg/l TiO2 showed a slight decrease from 17.01 ± 1.01 to 16.46 ± 2.03 gm (P ≤ 0.05). Clams exposed to 25 and 150 µg/l TiO2 NPs + 200 µg/l DMSA had a notable increase in total body weight. Condition index significantly increased in clams exposed to 25, 150 µg/l TiO2, and 200 µg/l DMSA (P=0.0001), with the lowest index in clams exposed to 150 µg/l TiO2 NPs (38.04 ± 1.45, P=0.001). Groups exposed to combinations of TiO2 NPs and DMSA showed significant changes in shell and flesh weights, and body condition indices over the exposure period.
The gonadal weight of clams exposed to 200 µg/l DMSA and 150 µg/l TiO2 NPs + 200 µg/l DMSA significantly increased over time (P= 0.0001). Clams exposed to 25 µg/l TiO2 NPs had an initial high gonadal weight (1.50 ± 0.40 gm) at zero time, followed by a decrease until the second week, and a subsequent significant increase after four weeks (1.61 ± 0.46 gm, P ≤ 0.05). Conversely, clams exposed to 150 µg/l TiO2 NPs showed a significant decrease in gonadal weight over the experimental period, ending at 1.01 ± 0.57 gm (P ≤ 0.05). The Gonado Somatic Index decreased non-significantly in clams exposed to 25 and 150 µg/l TiO2 NPs (from 32.07 ± 13.20 to 26.93 ± 4.40 and 32.63 ± 17.01 to 19.15 ± 7.75 gm, respectively), while clams exposed to 200 µg/l DMSA showed an increase, peaking at 34.33 ± 7.39 gm after four weeks (P ≥ 0.05).
3- Effects on DNA integrity
DNA damage in the gills of C. nilotica after one day of exposure to TiO2 NPs was assessed using the Comet assay. A significant concentration-dependent increase in DNA damage was observed (P ≥ 0.05). Control clams exhibited the lowest tail length (1.42 ± 0.02 µm), also the group exposed to 200 µg/l DMSA had the lowest values for DNA in Tail (1.43 %), tail moment (2.05 ± 0.02 units), and tailed cells (2 %). Clams exposed to 150 µg/l TiO2 NPs showed significantly higher DNA damage, with a tail length of 5.49 ± 0.07 µm, 4.20 % DNA in Tail, a tail moment of 23.1 ± 0.20 units, and 28.33 % tailed cells. The lowest percentage of untailed cells (71.67 %) was found in clams exposed to 150 µg/l TiO2 NPs, while the 200 µg/l DMSA group had the highest percentage of untailed cells (98 %), followed by the 25 µg/l TiO2 NPs + 200 µg/l DMSA group (97.67 %) and the control group (96.67 %).
4- Effects on immunological responses
Exposure of C. nilotica clams to various concentrations of TiO2 NPs significantly reduced the total haemocytes count (P= 0.00001). Clams exposed to 25 µg/l TiO2 showed a significant decrease in haemocytes recording 79.70 x 104 ± 21.50 x 104 cells/ml, and those exposed to 150 µg/l TiO2 showed an even greater reduction of haemocytes count to 27.33 x 104 ± 4.72 x 104 cells/ml (P= 0.003). Co-administration of DMSA with TiO2 NPs mitigated this impact, with total haemocytes counts observed at 393.00 x 104 ± 105.71 x 104 for 200 µg/l DMSA alone, 268.33 x 104 ± 87.64 x 104 for 25 µg/l TiO2 NPs + 200 µg/l DMSA, and 347.00 x 104 ± 52.40 x 104 for 150 µg/l TiO2 NPs + 200 µg/l DMSA (P≤ 0.05). Additionally, there was a significant reduction in large granulocytes count in clams exposed to 25 and 150 µg/l TiO2 NPs, recording 52.00 x 102 ± 45.83 x 102 and 56.00 x 102 ± 32.74 x 102 cells/ml, respectively (P= 0.004). However, the count of small granulocytes in clams exposed to 150 µg/l TiO2 NPs was the lowest being 23.33 x 102 ± 30.09 x 102 cells/ml, although this reduction was not statistically significant (P> 0.05).
After one day of exposure, the neutral red uptake (NRU) in haemocytes of clams varied across different groups. The lowest NRU value was observed in clams exposed to 25 µg/l TiO2 (0.059 ± 0.004), while the highest values were recorded in clams exposed to 25 and 150 µg/l TiO2 NPs + 200 µg/l DMSA (both 0.084 ± 0.01 and 0.084 ± 0.005, respectively). NRU values in haemocytes of clams exposed to TiO2 alone showed a non-significant decrease with increasing concentration, whereas those exposed to TiO2 NPs showed a non-significant increase (P > 0.05). Clams exposed to 200 µg/l DMSA had an NRU value of 0.068 ± 0.0046, significantly higher than the control group’s value of 0.066 ± 0.0041 (P = 0.002). Notably, exposure to 25 µg/l and 150 µg/l TiO2 NPs + 200 µg/l DMSA resulted in a significant increase in NRU in haemocytes compared to controls (P ≤ 0.05).
5- Effects on the histological structures of C. nilotica tissues
The histology of gills in the clams exposed to TiO2 NPs showed severe histological alterations including blood deprivation of the gills and cilia erosion, inflammatory response, and more thikning of the chitinous rods. TEM analysis supported the histological findings of the same concentration. But co-administration with DMSA showed notable improvements in gill histology and ultrastructure.
The digestive gland of C. nilotica exposed to 25 and 150 µg/l TiO2 NPs showed some histological changes including haemocytic infiltration, hyperplasia in some tubules, and an increase in secretory cells. Fibrous tissue was detected between tubules, necrotic tubules, enlarged lumens, and deteriorated connective tissue. Clams exposed to DMSA exhibited a healthier digestive gland structure compared to controls, with well-preserved cellular architecture and clear cell boundaries. Ultrastructural examination showed abnormalities of some digestive tubules that were irregularly shaped and detached secretory and digestive cells of the clams exposed. Also, damage to some organelles like Golgi bodies, and disrupted chromatin condensation inside the nuclei of those groups. However, DMSA administration shows a promised mitigation of the adverse effects of TiO2 NPs, leading to histological improvements.
The histological examination of male gonads in the clams exposed to TiO2 NPs induced histopathological changes including convoluted epithelial layers, necrosis in the concretion layer, and diminished muscle layers, with a reduction in mature sperms. Exposure to DMSA resulted in improved gonadal health, with ciliated epithelial cells, increased muscle layer size, and well-formed, densely packed testicular follicles.
Exposure of C. nilotica clams to to TiO2 NPs resulted in histological changes of female gonads as a reduced space occupied by gonadal layers, fibrous tissue between follicles, and fewer mature ova, with necrosis in some areas. However, the exposure to 150 µg/l TiO2 NPs enlarged the gonadal wall, fused ovarian follicles, and further reduced mature ova, while exposure to 200 µg/l DMSA improved gonadal health, with enhanced oogonia development and improved mature ova quality.
In conclusion, this study demonstrates that C. nilotica is a promising model for assessing the adverse effects of TiO2 NPs on aquatic organisms. TiO2 NPs have more severe negative effects on C. nilotica clams than bulk TiO2. Furthermore, DMSA emerges as a valuable chelating agent that effectively mitigates the negative impacts of TiO2 NPs, highlighting its potential use against nanoparticles induced toxicity.
It is recommended that to mitigate the adverse effects of TiO2 NPs on aquatic systems and organisms, it is essential to implement strict regulations on nanoparticles discharge into ecosystems and conduct regular environmental monitoring. Educate industrial sector on safe nanoparticle handling and disposal and promote the use of DMSA as a chelating agent.