# Introduction ecently there are many research about the acute toxicity of Tributyltin Chloride (TBTCl) on marine organisms. In this research studying the acute toxicity tests on Artemia sp. in previous studies focused on a nauplii of Artemia sp. This research aims to investigate the toxicities of (TBTCl) of the hatching stage of dry cysts and morphological changes of newly hatching nauplii of Artemia salina. In most scientific research widely used Artemia sp. as a model marine organism for ecotoxicity test, due to it is large geographical distribution. Despite it is popularity, the use of Artemia sp. in toxicity check is subjected to a wide discussion, at the global level, more often than not due to a number of criticisms about low sensitivity and lack of protocol standardization. (George-Ares et al., 2003; Mayorga et al., 2010;Leis et al., 2014;Libralato, 2014 andRotini et al., 2015). Biological influences of TBTCl on A. salina may additionally furnish clues for of the accumulation mechanisms in coastal ecosystems as nicely as of the mode of action of TBT in these organisms. A. salina and different Artemia species have been used in the literature for the screening of acute toxicities of booster biocides (Bartolomé and Sánchez-Fortún, 2005; Koutsaftis andAoyama, 2008 andRotini et al., 2015). There are many advantages to use Artemia for example, adaptability to high temperature, adaptability to wide ranges of salinity, adaptability to varied nutrient resources, ease of culture, small body size and short life cycle (Nunes et al., 2006 and Koutsaftis and Aoyama, 2008). In addation, Artemia is low cost and can use it anywhere at any time. Tributyltin chloriad is environmental hazards. The half of lives of tributyltin in the marine surroundings had been reported as nearly a number of days to weeks in water and frome one to ten years in sediments (Huang et al., 2004 andAl-Rashdi, 2011). In the previous studies toxicities of booster biocides have been reported on embryos of some marine organisms such as freshwater mussels, zebra mussels, blue mussels, sea urchins, oysters, and sea squirts (Bellas et al., 2007;Fent, 1996 andWang et al., 2012). High concentrations of BTs have been detected in lower trophic animals such as caprellids. It appears that TBT accumulates specially in caprellids in the marine ecosystem, irrespective of the trophic level in the food chain, and that it may additionally establish breakpoint for disturbance in the herbal meals chain structure, therefore inflicting different organisms to accumulate TBT at increased concentrations due to their lower metabolic capacities to degrade TBT (Ohji et al., 2002a). in most of study about TBTCl was recognized as an environmental hazard and can effects on the early embryonic development of Artemia salina. They have proven toxic to embryos and larval stages of numerous aquatic organisms even at environmentally concentrations (Bryan and Gibbs, 1991). TBTCl a biocide added to antifouling paints to prevent the accumulation and attachment of algae and barnacles on the bottom of boats. TBTCl has become popular because it is effective, for a long time and causes environmental hazards to non-targeted estuarine organisms. This study aimed to investigate the effect toxicities of tributyltin chloride on hatchability cysts and study morphological changes of newly hatching nauplii of A. salina. And observe the morphological abnormalities of A. salina newly nauplii of completely hatched in each concentration of TBTCl toxicant and measurement the total length and width of the body (head width, abdominal width and tail width). # II. # Materials and Methods # a) Hatching procedure and acute toxicity tests The tributyltin chloride (TBTCl) used in toxicity tests was kindly provided by Sigma-Aldrich, USA (purity 96%). Stock solutions of TBTCl were prepared by diluting with artificial seawater up to 35? salinity. The range of concentration TBTCl was selected as (5, 10, 15, 20, 25, 50, 75, 100, 150, 200 ngl -1 ). The experiments were performed in 50 ml test tube within tube racks that were submerged in water up to the midpoints of the tubes. Constant aeration, illumination (1000 Lux), and temperature (28 ± 1 °C) were maintained and the replicate number was three in the experiments each replicate 100 cysts of A. salina cysts. For each test group, added 40 ml from the test solution of different concentration TBTCl to test tubes, and then a 24 hours hatching period was initiated. After the hatching period, the number of newly hatched nauplii, viable hatched, cysts, and malformation newly hatched nauplii were counted. Hatching percentages (HPs) of cysts were determined by counting the number of completely hatched of nauplii. Hatching failure (found by subtracting the number of completely hatched nauplii from total group size). After that account hatchability (%), Deformity (%) and viable hatchability (%) by using the following formulae (Revathi and Munuswamy, 2010). Hatchability (%) = 100* (no. of hatched larvae) / (no. of total cyst in test) Deformity (%) = 100*(no. of deformed larvae)/ (no. of hatched larvae) Viable hatchability (%) = 100*(no. of viable hatchability larvae in test)/ (no. Total cyst in test). # b) Median effective concentration (EC 50 ) The data on the hatchability inhibitor % of cysts was used in the estimation a 50% effective concentration (EC 50 ) in different concentration of TBTCl. The effective concentration EC 50 values were determined by using probit analysis in XL TEST-Pro (version 2014.5.03). And each end point was calculated by using the following formulae (Shimasaki et al., 2003). # c) Morphological abnormalities The morphological abnormalities of A. salina newly hatched nauplii of completely hatched in each concentration exposed of TBTCl toxicant were observed under magnification (10x) using a Leica M 205 stereomicroscopy attached to a camera with an aid of software (Easy-Grab; Noldus Information Technology) and the total length and width of the body (head width, abdominal width and tail width) have been measured (Alyuruk and Cavas, 2013). # III. # Results and Discussion # Effects of Tributyltin Chloride on Cysts Hatchability in Artemia salina # Hatching Percentages (%) Hatchability of the exposed A. salina cysts to different concentrations of TBTCl observed in this study is presented in . The hatching percentages were shown to be affected by TBTCl concentrations. TBTCl significantly reduced hatching percentages of A. salina cysts at the various concentrations by using the following formulae (Revathi and Munuswamy, 2010). Hatchability (%) = 100* (no. of hatched larvae) / (no. of total cyst in test). The hatching percentages in the control were 97%, which is within the reported value of the manufacturer (minimum of 90% hatchability). Among these completely hatched cysts, 76% were active and 21% were viable hatched (completely hatched, but still not active). The remaining 3% cysts were found hatched after hatching period was prolonged for more than 24 to 48hr. From the results observed a significantly decrease hatching percentages from the 5 to 200 ngl -1 and was TBTCl had varying effects on the hatching percentages of A. salina cysts (Figure 1). In 200 ngl -1 showed completle hatching inhibition percentages of A. salina cysts. The cysts exposed to TBTCl within 24hr were unable to hatch even the hatching period was prolonged until 48hr. This result confermed TBTCl can kill a embryo and inhibit hatchability of A. salina cysts. 2006) studied estimated the median high-quality concentrations (EC 50 s) for metallic salts, suggesting that the hatching end point for A. franciscana is the most touchy examined to date for steel salts in saline environments and same in sensitivity with the most touchy tested to date for Cu. But in present finding A. salina cysts are more sensitive to TBTCl at lower concentration 5 ngl -1 it was 25% cysts comparative with the control samples 3% cysts, and that mean TBTCl can inhibits hatching process and can kills dormant embryo in the low concentration and also when increasing the TBTCl concentration. Revathi and Munuswamy, (2010) investigated the effects of TBT on the embryonic development, and hatching success of eggs uncovered to TBT in the freshwater prawn brooder Macrobrachium rosenbergii, and observed TBT at 3.12 ppm, delayed the embryonic development and significantly reduced the hatchability of eggs as well. two on the different hand, the treated businesses showed impaired embryonic development with reduced body growth. Thus, TBT has appreciably retarded the embryonic improvement in the freshwater prawn M. rosenbergii. These studies clearly demonstrated the possible effects of toxicants particularly TBTCl on unhatched eggs or cysts of crustaceans, including A. salina was more sensitive to TBTCl at 5 ngl -1 , and the possible reason that TBT is more toxic to A. salina because the body size is small and it is life cycle is very short Figure 2 shows effect of different concentrations of TBTCl on the performances hatching of A. salina cysts percentages. Figure 2 (a) shows the completely hatching (%) that mean the newly hatching nauplii is active and healthy as shown in control. Several nauplii exposed to 10, 25 and 50 ngl -1 wereviable hatching (%) that mean the newly hatching nauplii completely hatching, but still not activeand in the embryonic membrane (Figure 2 (b), (c) and (d)), while A. salina cysts exposed to 75 ngl -1 TBTCl concentration was unable to completely break the cyst wall (Figure 2 ), while the A. salina cysts in the 100 ngl -1 unable to hatching (Figure 2 (f)). This sequence of effects relatively showing the severity of TBTCl as its concentration increase in the aquatic environment. # Median Effective Concentration (EC 50 ). The median effective concentration EC 50 of TBTCl as shown in (Figure 3), at different concentration of TBTCl the A. salina nauplii completely hatching after 24hr was (EC 50 46.48 ngl -1 ), this is mean the TBTCl impacted the process of hatchability A. salina cysts and significantly reduced the hatchability cysts when increasing the concentration of TBTCl. Since there is a confined research on the inhibitory effects of the hatchabilty share cysts of A. salina it was once two examine EC 50 of TBTCl with one of a kind toxicants such as metals have been pronounced on the hatchability percentage of cysts for instance (Caldwell et al., 2003) studied A. salina, have been observed to inhibit hatching success of A. salina cysts in dose. A higher sensitivity was once discovered in the 24 and 72hr publicity EC 50 for 24hr was once 2.14 and 72hr was 0.023 µg ml -1 . This result is an settlement with (Brix et al., 2006) studied estimated the EC 50 of metallic salts are suggesting that the hatching endpoint for A. franciscana is the most sensitive examined to metals in marine environments. Meanwhile, Aly?r?k and Çava?, (2013) mentioned their investigation related to the toxicities of diuron to the hatching stage of A. salina, their results showed that diuron should be a attainable hatching enzyme inhibitor and used to be substantially lowered the hatching proportion of A. salina cysts and prevented the hatching of cysts. Rotini et al., 2015 said in their learn about Artemia sp hatching assay is a touchy choice device to acute toxicity take a look at and the hatching test resulted extra touchy than acute mortality tests. The outcomes show the reliability and excessive sensitivity of this hatching assay on a short time and guide it is a useful application of first tier risk assessment techniques in the marine environment. 1. The higher rate of deformities of newly hatching nauplii at 5 ngl -1 TBTCl concentration was 32.00 ± 4.62. Because in this concentration is a chance to newly nauplii stay a longer period survival, which gives greater opportunity to appear changes in shape and deformities. While the lower morphological deformities (%) were1.00 ± 0.00 at concentration 75 ngl -1 . Because the chance to survival newly nauplii is very weak and continued growth and development to the body is slowly and it is difficult to note the deformities clearly. As for the other concentration of TBTCl the deformities (%) was between this means. That means the TBTCl different concentration can impact the morphological changes in newly hatching nauplii when exposure the dry cysts to artificial sea water contaminated with TBTCl. Because in this concentration is a chance to newly nauplii stay a longer period survival, which gives greater opportunity to appear changes in shape and deformities. While the lower morphological deformities (%) were1.00 ± 0.00 at concentration 75 ngl -1 . Because the chance to survival newly nauplii is very weak and continued growth and development to the body is slowly and it is difficult to note the deformities clearly. As for the other concentration of TBTCl the deformities (%) was between this means. That means the TBTCl different concentration can impact the morphological changes in newly hatching nauplii when exposure the dry cysts to group was (350.9 ± 49.6) µm, but the means for total lengths in different concentration of TBTCl 5,10,15,20,25, The morphological deformities such as total length in the newly hatching nauplii as can be seen in the increase concentration of TBTCl, the total length of newly hatching nauplii A. salina will significantly decrease in general total length in newly hatching nauplii were represented in Table 2. This table is shown means of total length newly hatching nauplii shows the control The morphological changes such as total length and width of body in the newly hatching nauplii as can be seen in Figures 4,5,6 and 7. In general the regression analysis (r) shown the high regression values and this demonstrates a strong inverse relationship between morphological measurements and increase TBTCl concentration. The head width is more affected compared to the total length r = 53 %. And the head is more caricatures and more sensitive to increasing of TBTCl r was 95%. While the abdomen and tail width of body shown moderately affected r = 89 % and decrease when the increasing concentration of TBTCl. In present study need to mention there are not enough studies about effects of TBTCl on the morphological changes in newly hatching nauplii so will be compare these findings with similar studies about nauplii exposed to different types of toxins. For example, Abushaala et al., (2015a) study effect of TBTCl on nauplii stage of A, salina and reported in their results the TBTCl had effect the morphology changes of nauplii A. salina. On the # Conclusion In this study increased TBTCl concentration in solution could significantly decreased the hatchability percentage of A. salina cysts and prevented the hatching of larvae. And the EC 50 value of TBTCl was once recognized as 46.48 ngl -1 after 24hr exposure. Early nauplii of A. salina is sensitive to TBTCl contamination to reflect hatchablity cysts and early life stage effects of toxicant. In addation, the TBTCl effect on morphological abnormalitiy active newly nauplii A. salina. And significant morphological differences were observed in all nauplii exposed to the different concentration of TBTCl are used in this research. These results indicate that in this system TBTCl, it is proven environmentally toxic substances. In general, these results indicated that when increasing TBTCl concentration affected the total body length and the body width of A. salina newly hatched nauplii. In spite this result indicated that the system TBTCl is acutely toxic. ![Figure 1](image-2.png "") 1![Figure 1: The cysts hatchability percentage of A. salina after exposure for 24-h in different TBTCl concentrations [Completely hatching = active nauplii, Viable hatching = nauplii in membrane embryo, Cysts = unable to hatch]A. salina cysts are a barrier to withstand external environmental condition. Disruption of these activities by certain concentration of TBTCl bioaccumulated into the cysts may cause death to the dormant embryo, and this finding is relative agreement with the results of this research have shown that the toxicity of TBTCl can impact the hatching process of A. salina cysts. This result is supported throughBrix et al., (2006) studied estimated the median high-quality concentrations (EC 50 s) for metallic salts, suggesting that the hatching end point for A. franciscana is the most touchy examined to date for steel salts in saline environments and same in sensitivity with the most touchy tested to date for Cu. But in present finding A. salina cysts are more sensitive to TBTCl at lower concentration 5 ngl -1 it was 25% cysts comparative with the control samples 3% cysts, and that mean TBTCl can inhibits hatching process and can kills dormant embryo in the low concentration and also when increasing the TBTCl concentration. Revathi and Munuswamy, (2010) investigated the effects of TBT on the embryonic development, and hatching success of eggs uncovered to TBT in the freshwater prawn brooder Macrobrachium rosenbergii, and observed TBT at 3.12 ppm, delayed the embryonic development and significantly reduced the hatchability of eggs as well. two on the different hand, the treated businesses showed impaired embryonic development with reduced body growth. Thus, TBT has appreciably retarded the embryonic improvement in the freshwater prawn M. rosenbergii. These studies clearly demonstrated the possible effects of toxicants particularly TBTCl on unhatched eggs or cysts of crustaceans, including A. salina was more sensitive to](image-3.png "Figure 1 :") 2![Figure 2: Effects of Increasing TBTCl Concentration on the Performances Hatching Percentages (%) HPs of A. salina Cysts. [Remark: (a) control = Completely hatching, b) 10, c) 25, and d) 50 ngl -1 = Viable hatching, e) 75 ngl -1 = unable to complete break the cysts wall, and f) 100 ngl -1 = unable to hatch A. salina cyst. [Bar: 100 µm].](image-4.png "Figure 2 :") 3![Figure 3: Median Effective Concentration 50% (EC 50 = 46.48 ngl -1 ) between TBTCl Concentration and Hatchability Inhibition (%) of A. salina Cysts 3. Morphological Effects of A. salina Newly Hatching Nauplii after the Exposed the Cysts to Different Concentration of TBTCl Analysis of the morphological deformities (%) of the means A. salina newly hatching nauplii after the exposed the cysts to different concentration of TBTCl as shown in Table1. The higher rate of deformities of newly hatching nauplii at 5 ngl -1 TBTCl concentration was 32.00 ± 4.62. Because in this concentration is a chance to newly nauplii stay a longer period survival, which gives greater opportunity to appear changes in shape and](image-5.png "Figure 3 :") ![contaminated with TBTCl in the low concentrations.](image-6.png "") 4![Figure 4: Morophological of Total Length A. salina Nauplii after Exposed the Cysts to Different Concentration of TBTCl](image-7.png "Figure 4 :") 567![Figure 5: Morphological of Head Width A. salina Nauplii after Exposed the Cysts to Different Concentration of TBTCl](image-8.png "Figure 5 :Figure 6 :Figure 7 :") 1TBTCl (ngl -1 )NDeformity (%)03000.00 ± 0.00 a530032.00 ± 4.62 c1030016.33 ± 3.76 b153009.00 ± 0.58 a, b203009.67 ± 1.46 a, b253006.33 ± 1.76 a, b503002.33 ± 0.33 a753001.00 ± 0.00 a 2Hatchability Dry Cysts and Morphological Effects of Newly Hatching Nauplii of Artemia Salina (Linnaeus,1758) after Exposed to Tributyltin ChlorideYear 2020TBTCl (ngl -1 )NMean ± SEM (µm)Minimum (µm)Maximum (µm)32 Volume XX Issue III Version I0 5 10 15 20 2535 35 35 35 35 35350.9 ± 49.6 d 284.6 ± 51.6 c 266.8 ± 54.6 a,b,c 282.2 ± 59.3 a,b 294.9 ± 40.6 c 288.8 ± 45.7 b,c314.3 221.6 202.6 212.5 223.8 216.5451.7 377.8 364.2 398.9 389.9 352.1D D D D ) B5035274.8 ± 39.7 b,c244.8393.3(Medical Research7535269.8 ± 54.6 a other hand, (Rao et al., 2007) studies toxicity of 201.6 304.2 Organophosphates on morphology changes in nauplii A. salina and significant morphological alteration were noticed in nauplii. In under study Abushaala et al.,Global Journal of(2015b) studied effect of Diorun on nauplii stage of A. salina their results shown the Diorun had effect the morphology changes in the nauplii stage of A. salina. Also Anderson, (2009) his find out about confirmed the impact of alcohol proportion on the improvement rate of A. salina nauplii.© 2020 Global Journals © 2020 Global JournalsHatchability Dry Cysts and Morphological Effects of Newly Hatching Nauplii of Artemia Salina(Linnaeus, ## Acknowledgements This work was once supported by using the one of a kind Fundamental Research Grant Scheme (FRGS) Reference No. KPT.P.(S) 400-7/2/29-4 (65) for matching fund lookup between JSPS Asian CORE Program and Universiti Putra Malaysia (UPM), and FRGS (Reference No. FRGS/1/2014/STWN01/UPM/02/4) from the Ministry of Education Malaysia. ## This page is intentionally left blank * Selected Morphological Changes in Nauplii of Brine Shrimp (Artemia salina) after Tributyltin Chloride NMAbushaala SZZulkifli AIsmail MN AAzmai FMohamat-Yusuff TBTCl) Exposure. 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