Proceedings of The Fourth International Iran & Russia Conference 1370

Determination of Optimal Dietary Protein Level for Juvenile Beluga (Huso huso)

Abedian. A.M1., Mohammadi. M2., Shariatmadari. F3 Assi. Prof. Fisheries Department. Marine Science Faculty. Tarbiat Modares Univ. Noor. 46414-356, Mazandaran. Iran. Tel: 0122- 6253101-3, E- mail: [email protected] and [email protected] M. Sc. Graduate. Fisheries Department. Marine Science Faculty. Tarbiat Modares Univ. Asso. Prof. Animal Science Department. Agriculture Faculty. Tarbiat Modares Univ.

Abstract: This study was conducted to determine dietary protein requirement of juvenile Beluga (Huso huso) with emphasis on amino acids balance. In this experiment six iso caloric diets with different levels of protein (25, 30, 35, 40, 45 and 50 % ) prepared by using accessible materials. Juvenile Beluga with an initial body weight of 100±10 g were fed satiation way during an 8-week experiment. Analysis of variance (One Way ANOVA) and Duncan’s multiple range test suggested that growth performances were significantly affected by the different levels of dietary protein (P<0.01). The growth performances involve body weight increase percentage, specific growth rate and yield increase, further the volume of feed and protein consumption per day increase significantly (P<0.01) as dietary protein increased from 25 to 50%, whereas there was no significant effect on cost index, feed conversion ratio, protein efficiency ratio and net protein utilization. The maximum and minimum mean of body weight increase percentage, specific growth rate and yield occurred at 50% and 25% dietary protein levels, respectively. As there was no significant difference (P<0.01) in feed conversion ratio and cost index between diets with different protein levels and high levels of protein (45-50) was the best, therefore volume nearly 23 gr protein (dry matter)/day was suggested for growth of juvenile Beluga (100-300 gr) based on used materials in this experiment.

Key words: Diet, Protein, Amino Acid, Beluga (Huso huso).

Introduction: Considering the growing rate of world population, humans, need of various and healthy protein resources, aquaculture plays an important role in providing proteins. Meanwhile, sturgeon beneficially can be developed because they have high ecological adaptation and can coexist with teleostei fish and use variant biotopes. Being succeeded in sturgeon production before 1980’s, their rearing developed in many countries (although some reports have been existed that this began since 1980’s 80). Main production occurred in 1996 that resulted in production of 600t fish and less than 1t caviar from White sturgeon (Acipenser transmontanus) in Italy and United States; 235t fish and little caviar from Siberian sturgeon (Acipenser baerii) in Belgium, France, Germany, Italy and Poland; 60t Adriatic sturgeon (Acipenser naccari) in Spain and 67t hybrid of Bester in Austria and Hungary (Bronzi, etal, 1999). Caspian Sea is the largest habitat of sturgeon in the world, as more than 90% of their resources live there. Suitable climatic situation, existence of five sturgeon species and valuable experiments in last years have been some of the important parameters to start rearing fish and producing caviar of sturgeon. The improvement of sturgeon rearing techniques in the one hand and sufficient potentialities on the other, especially in the Caspian coasts have caused the formation of rearing fish plan in condensed circumstances, and using artificial food in Iran. Proceedings of The Fourth International Iran & Russia Conference 1371

In fish culture, food forms 50 percent of costs and it is an important factor in growth rate. Further, protein is a main nutrient in diets and a principal matter in tissue of fish and it forms 65-75% of dry body weight (Halver, 1989). Protein reduction violently affect fish growth, therefore protein is a very important composition in fish diets. Determination of protein requirement for gaining the maximum growth in sturgeon, is the first stage for the improvement of a cheap but growth effective way of feeding fish (Moore, etal, 1988). Many researchers have used pure and semi-pure diets to determine the fish protein requirement, but most rate of growth is the best. Different protein requirement in a fish depends on the three following factors: The amount of diet energy, amino acid composition and digestibility of consumer protein (Halver, 1989). Optimum protein for fish like other animals is affected by optimum protein for balance diet energy, amino acid composition and digestibility of consumer protein and energy of non-protein sources. Most of the researchers used iso-energetic diet for determination of optimum protein requirement (Halver, 1989). The Purpose of this experiment is determination of optimum protein requirement for juvenile beluga as well as finding a physiological and economical diet.

Materials and methods: 2-1- design of experiment In this experiment Lindo software was used (copy right 1999, release 6.1) for formulating. 6 iso-energetic dry diets with different levels of protein including (25, 30, 35, 40, 45 and 50 %) were made. The Diets were made with the use of inland and available materials. Since knowing the amount of the essential amino acids of the animal body is necessary in order to gain a suitable diet (Ng, 1994); and, considering the fact that one of the methods of meeting the aquatic needs is to adjust the diets on the basis of the amount of the essential amino acids of juvenile Beluga (Tacon, 1990), therefore the diets have been formulated on the basis of the essential amino acids of the juvenile Beluga bodies (nearly 80 gr)(table 2-1). For making diets, primarily, ingredients were mixed; then the produced pastes was passed through a mincer with a 2.5 mm cranny diameter and were put in the handmade drier for 24 hours. After diets dried, they were broken in suitable sizes then packaged and frozen in –20 º C . Table 2-2 show composition of ingredients in experimental diets.

2-2-2- biometry and feeding:

Once in two weeks all the fish were weighed with a scale with the precision of 0.01 gr. First the fish were enervated with 6gr pink powder per 30 l water, for biometry. Feeding Primarily was based on 2% biomass and then fed as satiation (Santiago, 1996). Fish were fed 4 meals per day at 8, 12, 18 and 24 o’clock. Every day before 12 o’clock bottom of tanks were siphoned and considering the remainder of meals tomorrow’s food was determined.

2-2-3- measuring the chemical and organic compositions:

Approximate analysis of consuming initial materials, and diets at first and juvenile beluga body compositions at the end of the experiment moisture, crude protein, crude lipid, crude fiber and ash were measured with the standard method of AOAC (1985). Total energy was measured by calorimetric bomb equipment. All the experiment done in poultry and livestock nutrition lab of Dr. Miralami, Tarbiat Modarres University (the school of natural resources) and sturgeon international research institute of Dr. Dadman.

2-2-4- growth indexes:

Proceedings of The Fourth International Iran & Russia Conference 1372

In order to review fish growth and to compare the treatments, growth indexes including increase body weight percentage, specific growth rate (SGR), feed conversion ratio (FCR), yield, cost index, protein efficiency ratio (PER) and net protein utilization (NPU) were used.

2-2-5- statistical method:

In this experiment 6 different level of protein, from 25 to 50 percent with equal energy were used. Therefore there were 6 treatments in this experiment for every one of which, three plicate was also considered, and altogether 18 tanks were used. The tanks distributed in one row under indoor and outdoor, on the basis of completely randomized design. Data analysis was done with soft ward package of Excel and Spss. Average treatments were compared with Duncan Multiple Range tests and the existence or non-existence of significant different was determined in 1 percent level (p= 0.01).

Results: 3-1- diets approximate analysis:

Fish meal and meat meal as protein resources contained 70.41 and 64.96 percent protein, respectively. In this experiment commercial and initial materials were used. Meat meal as a secondary protein resource was fixed and inconstant factor was fish meal. The protein of wheat flour, molasses and lecithin weren’t considerable. Table 3-1 and 3-2 shows ingredients and diets’ food value.

3-2- results of protein level on growth index:

Table of 3-3 show the effects of protein level on juvenile beluga growth index. The growth performance including weight gain percentage, SGR and yield have been increased significantly (P<0.01) as dietary protein increased from 25 to 50% but no increase was observed in FCR, cost index, PER and NPU. Max and min of BWI% were 186.18 and 53.59% in diets with 50% and 25% protein respectively, and similarly max and min of yield were in diets with 50% and 25% protein respectively. FCR and cost index did not significantly differ (P>0.01) when dietary protein increased, but their value decreased when the dietary protein increased. As a result, the most suitable FCR and cost index were related to 50% Protein. Food and protein consume per day (based on dry matter) increased significantly (P<0.01) as dietary protein increased from 25 to 50%, as their max were 45.94 and 22.89 gr respectively in diet with 50% protein and their min were 21.66 and 5.57 gr respectively in diet with 25% protein (Table 3-2). PER and NPU did not show much difference (P>0.01), PER’s max and min were in diets with 25 and 30% protein respectively and NPU’s max and min were in diets with 25 and 45% protein respectively. Charts 3-1 and 3-2 show growth situation of juvenile Beluga in different levels of protein.

4-3- results of protein levels on biochemical body composition:

The table of 3-4 shows the effects of different protein levels on biochemical body composition of juvenile beluga. Through different protein levels, body composition didn’t differ significantly (P>0.01). Max and min of body protein were 79.52 and 66.66 in diets with 50 and 35% protein, respectively. Max and min of energy were 6664.49 and 6115.07 calorie Proceedings of The Fourth International Iran & Russia Conference 1373 per gram in diets with 25 and 35% protein, respectively, and the max and min of fat were 35.64 and 21.03 in diets with 35% and 50%, respectively. discussion : 4-1- juvenile beluga growth index:

Many studies were done about the affect of the level of protein on different spices of aquatic animals. In most of these studies, it’s been specified that in an iso-energetic diet, the increase of protein can significantly affect the growth index. There is a direct relation between the BWI, SGR and yield with an increase in the ratio of protein to energy; but there is an inverse relation between the FCR, PER and NPU in one hand and pure protein on the other (Davis and Arnold, 1997; Das, etal, 1991; Hajra, etal, 1988; Bautista, 1986).

In this study on the basis of the data resulted from Dankan’s multiple range test, different protein levels significantly (P<0.01) affected BWI%, SGR and yield, and like what the above-mentioned researchers had concluded, the increase of these indexes, as well as the amount of food and protein consume were followed by the increase of protein. But, no significant difference (P>0.01) was seen in FCR, PER, NPU and cost index. However, FCR and cost index decreased with the increase of protein percentage of diets. In one study, increasing the protein levels from 20 to 52.7 % Moor, etal (1988) obtained a significant (P<0.05) effect on BWI%, FCR, PER and NPU in juvenile White sturgeon (Acipenser teransmontanus). As a result, rate of FCR, PER and NPU decreased and rate of weight gain percentage increased all of which coincide with our results. In this experiment, based on Dankan’s multiple range test volume of protein requirement for juvenile White sturgeon (A.teransmontanus) with 140–300 gr weight was estimated 38.4-43 %. Also based on Second – Order Polunomial Regression method, the minimum protein requirement was assessed 36.5-40.5 % and based on curve break point, volume of protein requirement was measured 40.5±1.6 %. In another study, Kaushik, etal (1991), with the increase of protein levels from 29 to 52 % obtained a significant (P<0.05) effect on BWI and SGR in juvenile Siberian sturgeon (Acipenser baerii). As a result, their rate increased, that coincide with our results. In this experiment, on the basis of Polynomial and break point methods, the rate of protein requirement for juvenile Siberian sturgeon (A.baerii) with 20-40 gr weight was estimated 40±2%. Considering the above-mentioned results and the significant differences between the protein levels in BWI%, SGR and yield, and the fact that maximum values were in diet with 50% protein and that in FCR and cost index there weren’t significant difference between protein levels and the minimum value was in diet with 50% protein, therefore the best growth was in fish that have been fed with the diet with maximum protein and this level had a better capability in comparison with the others. Therefore considering the feed intake per day and protein intake per day (based on dry matter), protein requirement for juvenile beluga (100-300 gr) was suggested 22.89 gr/day with materials that have same digestibility to matters used in this experiment. It’s noticeable that although decreasing the diet’s protein should cause the increasing of the feed consumption; but, in this experiment, its volume has been decreased. Considering PER and NPU, the reason can not be the low digestibility of the diets with less protein. Perhaps the reason of the feed consumption decrease with the decrease of the diet protein percentage, can be related to the lack of fatness in the diets with low protein in order to regulate the energy. A general review of this research show that protein levels affect on growth index of juvenile beluga significantly and increasing protein level can improve their growth indexes, Proceedings of The Fourth International Iran & Russia Conference 1374 the result which is similar to other researchers results. But the main difference is in optimum protein volume which is different from that of the research on sturgeon. This can be related to many reasons as: fish species, initial materials and breeding conditions.

4-2- Approximate analysis of juvenile beluga body:

The volume of the protein of diets did not significantly (P>0.01) affect on protein, lipid and energy of juvenile beluga body. Same effect was suggested by Moore, etal (1988), so as Farabi. M. V (1377) who has said diets with different volume of protein don’t affect on body composition (such as protein and lipid) of Beluga (Huso huso), Russian sturgeon (Acipenser guldenstadtii) and White sturgeon (A.transmontanus). However, in the experiment of Kaushik, etal (1989) who used two protein levels (36 and 42 percent) and four different resources of carbohydrate for Siberian sturgeon (A.baerii), their body protein decreased and their fat increased at the end of the test; the results of which, perhaps for the use of different carbohydrate resources, are different from that of ours. In another study, Ng, etal (2001) reported that different volume of protein from 20 to 50 % don’t affect on body protein of Catfish (Mystus nemurus) significantly (P>0.05), similarly Nandeeshe, etal (1994) reported the volume of diet’s protein doesn’t have significant effect on protein and ash of Rohu carp (Labeo rohita) body but with a slight increase in protein of the diet, body moisture increases partially, and this coincide with our results.

Acknowledgement: Financial support of Dr. Dadman International Sturgeon Research Institute is gratefully acknowledged. Thanks to misters A.Djahedi, S.Keyvanshokooh, R.Aminzade, A.Gharaei and madames M.Ghaemi and S.Ansari that during the total stages of the experiment supported us and also thanks to total engineers and workers of Dr. Dadman International Sturgeon Research Institute that helped us in the accomplishment of this research.

References: Abedian. A. M. ( 2001). The effects of dietary protein and energy levels on growth performances and body composition of white Indian shrimp (Penaeus indicus) under different salinity. Ph.D thesis of aquaculture. Faculty of natural resources and marine science. Tarbiat modares university. 131 p. AOAC (Association of official Analytical chemists ), 1990 . Official methods of analysis AOAC . Washington , DC, 1263 PP. Bautista, M. N. ( 1986 ). The response of Penaeus monodon juveniles to varying protein / energy ratios in test diets. Aquaculture, 53 : 229 – 242. Bronzi, p. , Rosenthal, H. , Arlati, g. and williot, p. (1999). A brief overview on the status and prospects of sturgeon farming in western and central Europe . journal of Applied Ichthyology 15 , 224-227 . Das, K. M., Mohanty, S. N. and Sarkar, S. ( 1991 ). Optimum dietary protein to energy ratio for Labeo rohita fingerlings. Fish Nutrition Research in Asia. Editor S. S. De Silva, Proceedings of the third asian fish nutrition network meeting. Pp : 69 – 73. Davis, A. D. and Aold, C. R. ( 1997 ). Response of atlantic croaker fingerlings of practical diet formulations with varying protein and energy contents. Journal of the world aquaculture Society, 28 ( 3 ) , pp : 241 – 247. Fakhraei. J. (2001). Effects of different levels of energy and protein on the performance of commercial laying Hens. Ms.c thesis of animal science. Faculty of agriculture. Tarbiat modares university. 90 p. Farabi. M. V. (1998). Investigation of four diets on the growth and body composition of (Acipenser gueldenstaedti) and beluga (Huso huso) in second years culture. Ms.c thesis of fisheries. Faculty of natural resources and marine science. Tarbiat modares university. 43 p. Proceedings of The Fourth International Iran & Russia Conference 1375

Halver, J. E. (1989). Fish Nutrition. Academic Press. INC. 798p. Kaushik, s. J.˭ Luquet, p. ˭ Blanc, D. and Paba, A. 1989. Studies on the nutrition of Siberian Sturgeon , Acipenser baerii. I. Utilization of digestible carbohydrates by sturgeon . Aquaculture , 76 (1989) 97 – 107 . Kaushik, S. J. ; Breque, J. ; Blanc, D. 1991. Requirements for protein and essential amino acids and their utilization by Siberian Sturgeon ( Acipenser baerii ). In : Acipenser actes du fer colloque international sur I`esturgeon. Williot, P., ed., France. CEMAGREE-DICOVA, Anthony, 25 – 39. Moore, B. J., Hung. s. s. o. and Medrano, J. F. (1988 ). Protein requirement of htchery – produced juvenile White Sturgeon (Acipenser transmontanus) . Aquaculture , 71 (1988) 235-245. Nandeesha, M. C., Dathathri, K., Krishnamurthy, D., Varghese, T. J., Gangadhar, B. and Umesh, N. R. ( 1994 ). Effect of varied levels of protein on the growth and tissue biochemistry of stunted yearlings of Rohu, Labeo rohita , in the absence and presence of natural food. In : De Silva, S. S. ( ed ). Fish nutrition research in Asia. Procceedings of the fifth Asian fish nutrition workshop. Published by Asian fisheries society in association with the DRC ( Canada ). Pp: 93 – 99. Ng, W. K., Soon, S. C. and Hashim, R. ( 2001 ). The dietary protein requirement of a Bagrid Catfish, Mystus nemurus ( Cuvier and Valenciennes ), determined using semipurified diets of varying protein level. Aquaculture Nutrition 7: 45 – 51. Paghe.E. (2002). Effects of salinity on growth and survival of Indian white shrimp (Penaeus indicus). Ms.c thesis of fisheries. Faculty of natural resources and marine science. Tarbiat modares university. 70 p. Santiago, C. B. (1996). Approches and design of fish nutrition experiments. Training course on fish nutrition .SEAFDEC , philippines , pp,17 . Silva, s. s. (ed). fish nutrition research in Asia . Procceedings of the fifth Asian fish nutrition workshop . Published by Asian fisheries society in association with the IDRC (Canada ) . pp: 1-7. Tacon, A. G. J. (1990). Standard methods for the nutrition and feeding of famed fish and shrimp . Argent Laboratories Press . pp. 4,24 .

Proceedings of The Fourth International Iran & Russia Conference 1376

Table 2-1- essential and non essential amino acids profile of juvenile beluga (mgr/gr sample) 1 amino acids Volume Aspartic acid 5.36 Glutamic acid 15.43 Serine 4.4 Glycine 10.21 Histidine 3.2 Arginine 7.69 Threonine 4.13 Alanine 5.79 Proline 6.76 Valine 3.52 Tyrosine 1.4 Methionine 0.8 Iso-Leucine 2.54 Leucine 4.85 Phenylalanine 4.43 Lysine 9.43 Cysteine 3.2 1 mean weight of juveniles beluga were 80gr. Body protein was 70%.

Table 2-2- Type and composition of ingredients in experimental diets Number of 1 2 3 4 5 6 diets/ ingredients (%) Fish meal 17.9 26.2 34.9 42.03 49.94 57.6 Wheat flour 37.5 30 20 20 15 11.44 Meat meal 20 20 20 20 20 20 Molasses 2.6 1 1 1 1 1 Fish oil 8.3 8 7 5.2 4 3 Soybean oil 8 6.84 7.13 5.01 3.89 2.1 Coline 0.7 0.7 0.7 0.7 0.7 0.7 Lecithin 0.5 0.5 0.5 0.5 0.5 0.5 L- Carnitine 0.06 0.06 0.06 0.06 0.06 0.06 Salt 0.5 0.5 0.5 0.5 0.5 0.5 Vitamin C 0.06 0.06 0.06 0.06 0.06 0.06 Vitamin mix 1.5 1.5 1.5 1.5 1.5 1.5 Mineral mix 0.5 0.5 0.5 0.5 0.5 0.5 Lime 3.42 4.11 2.12 2.9 3.35 1 Proceedings of The Fourth International Iran & Russia Conference 1377

Table 3-1- approximate analysis and food value of ingredients Initial matter Protein % Lipid % Carbohydrate % Gross energy (cal/gr) Fish meal 70.41 9.8 0.4 4909 Wheat flour 11.32 0.5 72.47 3657 Meat meal 64.96 11.7 3.48 4900 Lecithin 22.73 5.3 50.74 3857 Molasses 3.273 0.2 67.76 2980 Vegetable oil - 95 - 9000 Fish oil - 98.7 - 8885

Table 3-2- approximate analysis and food value of diets Number Protein Lipid Carbohydrate Ash Gross P/E of diets % % % energy (cal/gr) 1 25.71 32.08 31.40 10.8 5635.79 45.62 2 29.56 30.99 25.25 14.2 5590.26 52.87 3 34.53 31.44 22.31 13.4 5858.38 58.94 4 38.38 27.75 18.99 14.9 5734.38 66.93 5 44.43 25.96 14.51 15.1 5802.81 76.57 6 49.77 24.92 10.40 14.9 5636.95 88.29

Table 3-3- comparison average growth index toward the influence of protein level 1 Protein% / index BWI % SGR Yield (gr) 25 53.59±24.74a 0.75±0.30a 599.43±272.98a 30 53.94±0.31a 0.77±0.00a 622.90±8.02a 35 86.66±12.10b 1.11±0.11b 961.14±59.99b 40 108.87±7.27bc 1.31±0.06bc 1247.47±83.95bc 45 133.11±4.21c 1.51±0.03c 1525.30±36.34c 50 186.18±4.77d 1.88±0.03d 2188.50±2.14d 1 average ± S.D, numbers in a column with different letters have significant difference (P<0.01). BWI%: body weight gain percentage (Moore, etal, 1988) SGR: specific growth rate= 100*[(LnW2 – LnW1)/days of culture] (Tacon, 1990) W2: secondary weight W1: initial weight Yield: growth * survival (Paghe, 1380) Proceedings of The Fourth International Iran & Russia Conference 1378

Continuance table 3-3- comparison of the average growth index toward the influence of protein level1 Protein% / index FCR PER NPU% 25 2.77±1.93a 1.83±0.91 a 170.48±75.67 a 30 2.51±0.02a 1.35±0.01 a 152.46±31.97 a 35 2.19±0.37a 1.35±0.25 a 123.85±28.84 a 40 1.86±0.09 a 1.40±0.07 a 144.31±2.29 a 45 1.63±0.05 a 1.38±0.04 a 120.99±5.99 a 50 1.23±0.02 a 1.63±0.03 a 157.74±6.17 a 1 average ± S.D, numbers in a column with different letters have significant difference (P<0.01). FCR: feed conversion ratio: used food (gr) / body weight increase (gr) (Tacon, 1990) PER: protein efficiency ratio: body weights increase (gr) / used protein (gr) (Moore, etal, 1988) NPU: net protein utilization: body proteins increase (gr) / used protein (gr)* 100 (Tacon, 1990)

Continuance table 3-3- comparison of the average growth index toward the influence of protein level1 Protein% / index Used food (gr dry Used protein (gr Cost index matter/day) dry matter/day) 25 21.66±1.61a 5.57±0.41a 1233.9±860.3a 30 26.31±0.14b 7.78±0.04b 1307.4±11.7 a 35 37.03±2.02c 12.79±0.70c 1297.5±217.5 a 40 39.52±0.67cd 15.16±0.26d 1173.6±59.5 a 45 41.90±0.40d 18.62±0.18e 1224.1±32.9 a 50 45.94±0.71e 22.89±0.29f 913.3±14.5 a 1 average ± S.D, numbers in a column with different letters have significant difference (P<0.01). Used protein: used food * %protein Cost index: cost of a kilogram diet * FCR (Fakhraii, 1380)

Chart 3-1- Weight gain percentag toward protein levels affect

250 min of weight gain 200 percentag

150 mean of weight gain percentag 100 Weight gain % Weight 50 max of weight gain percentag 0 25 30 35 40 45 50 Percentage of diets protein Proceedings of The Fourth International Iran & Russia Conference 1379

Chart 3-2- The growth situation of juvenile beluga in different protein level during culture period 400

350

300 25%protein 30%protein 250 35%protein 200 40%protein 45%protein

weight ( gr ) weight 150 50%protein 100

50

0 0 14284256 culture daies

Table 3-4- comparison average of biochemical body composition of juvenile beluga toward the influence of protein level 1 Composition Moisture% Protein% Lipid% Total energy (cal/gr)

Protein% 25 81.81±0.64a 69.18±0.49 a 32.03±1.57 a 6664.49±95.69 a 30 83.05±2.64 a 71.12±8.92 a 31.07±8.00 a 6367.45±302.48 a 35 81.23±0.11 a 66.66±2.71 a 35.64±6.72 a 6115.07±971.83 a 40 83.23±0.77 a 76.15±2.16 a 26.38±3.55 a 6193.68±138.66 a 45 82.49±0.30 a 70.95±3.86 a 27.68±4.84 a 6310.40±262.37 a 50 85.69±0.99 a 79.52±2.76 a 21.03±3.03 a 6246.05±94.32 a 1 average ± S.D, numbers in a column with different letter contain significantly difference (P<0.01). Proceedings of The Fourth International Iran & Russia Conference 1380

Biological control of intermediate host parasites (snails) by the use of Tinca.tinca fish.

M.abou, A.A.Saeedi , A.Rezai, Z.Rezvani ,S.F Mirhashemi. 1,2,3,4:Ecology Research center of Caspian sea ,Ecology Research station of Caspian sea ,Nowshahr,P.O.Box 498 ,Telefax . 0192-3672657 . IRAN 5-Bony fish Research center of Caspian sea ,Research station of Astane Ashrafieh Address: aliasgharsaeedi@ yahoo.com

Abstract The snails evolution in the process of fishes and other chordatae are playing most important role as a first intermediate host and they are effective in distribution of parasite infestation in the nature . Now days their control in the most parts of the word seems to be difficult and sometimes impossible .The control of these parasites present in fish farms can be done through medical method by the use of copper sulfate. in order to control the parasite infestation of human pathogenic like schistosoma, this substance can be use by 30ppm Dose . The other physical and mechanical control method can be used by drainage ,snail collecting and to drain the living environments of snails .All these methods had not a permanent and continued results. These days the most suitable control method is called biologic method because it dosent have abuse effect of copper sulfate and on the other hand it has a minimum risk to ecological changes in the nature (water environment).Thus pathogenic parasites of fish (parasitic blindness or diplostomiasis) occurred in warm water fish farms are called as a widespread disease and caused a high reduction in fish production amount .Hence for achievement the positive results regarding biological control of these pathogenic diseases ,on Autumn and spring 2000,the number of three fiberglass tank (1.5 × 1.5 × 0.7 m , volume of 1.5m3 ) was prepared and filled with water (by fish farm water supply ).In each tank about 450 snails in different sizes were released .Then 5 Tinca tinca fish in sizes of >25g ,50-100g and <30g in each tank were also released.

Key words: Tinca – tinca fish – snails – parasite – feeding – fish farm- water resources

Introduction Now days the development important once of fish culture from the view point of occupation ( getting job ) , protein supply , quality and quantity of food security for human society, foreign currency income and economical progress on the basis of constant extension were paid attention to . In recent years the poor development process of fish farming caused more special attention to increase and enlargement of fish production . for instance in Iran the total yield amount of fisheries production were increased ( 2001) from 73000 tons to 89000 tons and in 2002 it reached 110000 tons . But out break and spreading of various diseases in fish population particularly parasitic diseases ( diplostomiasis ) occurred in carp fishes caused partial omission of fish production process annually . Effects of diseases cause fish reduction in production to 10 percent . The aim.of this study is acquiring the standard control ( economically performable ) of diplostomiasis diseases and prevention of reduction in fish quantity production . Diplostomiasis is one of the most widespread diseases of parasites in carp fishes . The diseases are parasitic settlement in eye lens of fishes and cause visional disorder and opaque ( blindness) . Proceedings of The Fourth International Iran & Russia Conference 1381

In this case the fish is unable to touch the food . This phenomena is observed in most of warm water fish breeding and hatchery centers ( Ghoroghy , 1996) .Diplostomum parasites select snails in their life cycle as an intermediate host ( jalali , 1998 ) . In order to control diseases , the best method is omission and interruption of snails ( intermediate host ) form life cycle process. In this connection there are various methods for controlling the snails . one of the methods is exposure of fish to 1-15 ppm of copper sulphate . This process is called chemical control of diseases ( fish diseases in Africa , FAO Journal , 1980) . The other chemical substances are named as linden and carbaryl , 2,4,6 Tri – chlorophenil . Another methods is named as physical and ecological control of diseases like collection snails , biological charges in snails life ( changing temperature , PH , to construct drainage system . And drying of swamps and stagnant waters ( Ralf – Muller , 1979 ). Hydro – electric is another method ( Jabin – Laracuente , 1984 ) . Biological control is the best method applied against diseases . In this method the snail feeder fishes can be used namely geophagus – braeilliansis ( Duck – lo w , 1980 ) or black carp (mylopharyngodon – piceus ) , tilapia fish ( Hemichrem is binacupatus ) .But the most important and valuable fish against the snails is called Tinca- tinca fish which lives in south basin of Caspian sea. This fish has most efficiency to control the diseases by feeding snails in warm water fish farms.

Materials and methods In order to do the experiments , 4 fiber glass tanks in dimension of 2m × 2 m × 0.6 m ( 1.4 m 3 ) for two work stage were selected . Tanks were antisepticize by the use of cholorine solution , Then rinsed , after that filled with clean and fresh water and left for 48 hours. The experimental parameters have been done at equal situation as bellows : Temperature ranges : 17- 20 oc Amount of oxygen dissolved in water : 6.4 – 7.6 mg/lit PH : 7.7- 8.2 Water temperature was checked thrice per day ( morning , non , night ) . Dissolved oxygen measured twise per day ( morning , after non ) . PH checked once in a week . All the necessary water data were recorded in special record book. The other specification regarding water tanks containers are as follows : Care stage 1. 1- -Fiber glass tanks : 2 in number 2- Tinca – tinca fish : 10 in number and each in 250 gr weight 3- Snails ( Limnea , physa, planorbis ) : 100 in each water tank with different size. Care stage 2 . 1-Fiber glass tanks : 2 in number 2- Tinca – tinca fish : 10 in number , each in 250 gr weight . 3- Snails (Limnea , physa, planorbis) : 100 in each water tanks with different size . 4- Concentrated fish food GFC : Growth stage food for carp fish were given daily about 4% of Tinca- tinca bio-mass. In order to prevent the snails from going out of tanks , the water exit and top space of tanks were covered by nets in small mesh size . The experiments of each care stage were repeated for three phases . Along the time of experiments a number of Tinca – tinca fish were caught from each care stage for the purpose of intestinal container consideration by the use of microscope . At the same time the snails of each tank were counted perfectly. Proceedings of The Fourth International Iran & Russia Conference 1382

In order to prove this matter that the snails are the main food of Tinca- tinca fish , the ratio comparison test ( z test ) has been used . On the other side the averages comparison have been determined by the use of t test ( student ) .

Results The parameters of water temperature , dissolved oxygen , pH during the experiment were measured as follows :

Table 1 : mean variations of water parameters in care stage 1 .

Parameters Water temperature Dissolved oxygen PH Months (oc) ( mg/ L ) April 17.34 7025 7.75 May 19.11 7.68 8.05 June 19.87 6.91 8.21 July 20.95 6.44 7.97 Average 19.31 7.07 7.99

Table 2 : mean variations of water parameters in care stage 2 .

Parameters Water temperature Dissolved oxygen PH Months (oc) ( mg/ L ) April 17.35 7.27 7.55 May 19.18 7.66 7.91 June 19.84 6.94 8.71 July 20.98 6.40 7.88 Average 19.33 7.06 7.87

Differences between water temperature , dissolved oxygen and pH in both of the care stages were statistically significant ( P>0.05 ) . t- test . The results of snail feeding of Tinca- tinca fish in first care stage tanks were taken place in three repetition. According to this assessment daily 200 snails ( Lymnea auricularia, physa, planorbis ) were released into the first care stage tanks. These snails were fed by 10 Tinca- tinca fish . The absence of snails in water tanks and also its presence along with shells in the fish intestines proved this matter. In second care stage tanks the concentration food along with snails were used . This experiment showed that initially Tincai tinca fish fed on snails but very seldom they touched the concentrated food because the remains and residue of concentrated unused food presented in water tanks proved this subject . In comparison it can be stated that about 100% of snails were utilized by Tinca-tinca fish but only 30% of concentration food were consumed by fish in each care stage tank. In statistical comparison and also by the use of relations test ( z test ) the equality between ratios were failed .

Discussion On the basis of obtained results , the mean average of water temperature in first care stage tanks were 19.31 oc and the same in second care stage tanks were recorded as 19.23 oc . on the Proceedings of The Fourth International Iran & Russia Conference 1383 other side the amount of dissolved oxygen in first care stage tanks were 7.07 mg/lit and the same in second care stage tanks were recorded as 7.06 mg/lit . The average PH in first and second care stage tanks were recorded as 7.99 and 7.87 respectively . The variations between the parameters of water temperature, dissolved oxygen and PH regarding both the care stage tanks ( first and second ) in case of statistical calculations were not significant .P>0.05 ( t- test ) . Each one of these parameter amounts in comparison to the amounts submitted by faridpak- 1981 , Aniarovich – 1986 and Azari takami – 1993 in case of Tinca – tinca fish farming ( syprinidae family ) were conformable . In case of nourishment results of Tinca – tinca fish in first care stage tank it was already proved that the fish was snail feeder . This result was also conformed in second care stage tanks on the base of 0.04 % of total body weight of fish feeding with concentration food . In this case only 30 gr of concentrated food were consumed by fish . But on the opposite side all the snails released into the water tanks at the second stage were utilized by fish . In case of attention to obtained results it seem that Tinca- tinca fish as a biological factor could obviously reduce and control the biomass of intermediate host snails which are called as origin of epidemic and widespread of diplostomiasis in fish farm pools . Hence it is possible to introduce Tinca-tinca fish as a biological controller to challenge snails particularly against intermediate host snails . Mean while this fish is an endemic fish adapted fully to ecological condition of southern basins of Caspian sea. It is possible to release 500-600 of this fish per hectare in body weight of more than 200 gr to fish farming pools for the purpose of snails control ( Rohani- 1993 ) . It is very important that using this method is quite safe and we need not worry to use chemical substances which are lethal and destroy some useful water organisms by making water toxicity .

References 1- Azari.T, F.,1993, Health management and the methods of prophylaxy and treatment of fish diseases , 199-205. ( In Persian ) 2- FAO , 1980, African salmonidae fish diseases . 3- groughi, A, 1996 , Study on diplostomiasis parasite infestation in young Acipenser fish , fisheries science publication , NO.2 , fifth year , summer , 1996, 11-12, 20-21 ( In Persian ). 4- Jalali J,B, 1998 parasites and parasite diseases of fresh water fish of Iran , 304-305 , 318-321 ( In Persian ). 5- Jobin.W.R, Laracuenta.A,1984 , Blueline health project in sudan world health organization c /5 UNDP, one un plaza, Newyork, NY , 10017 , snail Marisa cornvarieties in tropical hydro-electric. 6-Mirhashemi.S.F. 2002 , Biological study of Tinca-tinca fish and its role in life cycle control of diplostomum parasite, fisheries research center of Gillan . IRAN( In Persian ) 7- Ralf – Muller , 1975 , worms and diseases department of medical helminthlogy , London , school of hygiene and tropical medicine . 8- Rohani .sh , 1993 , Assessment on diagnosis , prophylaxy treatment and fish toxicity , 116-158- 159( In Persian ). 9-Saeedi .A.A, 2000, Assessment on identification of intermediate host snails and methods of their control fisheries research center of mazandaran , IRAN, ( In Persian ) . Proceedings of The Fourth International Iran & Russia Conference 1384

Reproductive phenology and breeding success on the Whiskered Tern (Chlidonias hybridus) in South Caspian Sea Ab-bandans (Artificial Wetlands) in the North of Iran

Seyed Mehdi Amini Nasab 1, Behrooz Behroozi Rad 2 , Ali Reza riahi Bakhtiari 3

1- Senior expert (M.Sc.) of Environmental Science, Department of Environmental Science, Tarbiat Modarres University ,Noor, Iran Phone:+98-123-2240042 , E-mail:[email protected] , Ad: Mazandaran province, Ghaemshahr, Darzikola, zip code: 47617-38775; 2- academic Staff (Ph.D) ,Department of Environmental Science,Tarbiat Modarres University, Noor, Iran Phone:+98-122-6253101 ; 3- Academic Staff (M.Sc) ,Department of Environmental Science,Tarbiat Modarres university, Noor, Iran Phone:+98-122-6253101

Abstract This study was done during May-September, 2003. Zarrinkola and Marzoonabad Ab-bandans (Artificial Wetlands) were selected for this study. There were one (930 pairs) and two (1390 pairs) breeder groups of Whiskered Tern (Chlidonias hybridus) in Zarrinkola Ab-bandan and Marzoonabad Ab-bandan respectively. Clutch sizes were 1-4 eggs in Zarrinkola and 1-5 eggs Marzoonabad. Brood size included 1-3 chicks. Breeding success in zarrinkola, first and second breeder group was %57/08, %82/96 and %75/87, respectively. There is not any significant difference between clutch size and brood size with breeding success (p>0/05). There is significant difference (p<0/05) between the rate of losses among the reproductive phenology stages. Because of intense rainfall, the maximum losses were observed during the incubation time (egg stage).

Keywords: Breeding, Whiskered tern, Artificial Wetlands, Ab-bandan, Caspian Sea, Mazandaran

Introduction Wetlands are the most complicated ecosystems which faced with settle many threats (Baldi et al.,1998 ; Baldi & Kisbenedek, 2000). They are suitable forecaster for habitat changes (Baldi et al., 1999). Artificial Wetlands are designated Ab-bandan in Farsi. They are places for resting, nesting, wintering, feeding and breeding of birds. Islamic Republic of Iran has 105 important and first rank zone for birds populations in middle east. There fore, studies about key regions are very important (Evans, 1994). Birds in breeding season have high sensitivity against environment changes and reproductive success indicates habitat suitability (Schjorring & Bregnball, 1999). Whiskered Tern (Chlidonias hybridus) belong to migratory birds of Laridae family and Charadriiformes order. For reproductive phenology studies, colonies are Considered as the best index (Scarton & Valle, 1996). Therefore, C.hybridus was selected for this study.

Materials and Methods Study area Zarrinkola and Marzoonabad Ab-bandans are located in Jooybar and Babol city in the Southern region of Caspian Sea in Mazandaran province (The north of Iran) and have 327/2 and, 220 ha area, respectively. Proceedings of The Fourth International Iran & Russia Conference 1385

Nest select field activities and search for nests were started with nesting behavior time (end of May) to maximum chicks fledged time (The end of July) and conducted with local boat in two day time interval (Bacon & Rotella, 1998 ; Scarton et al., 1994) and selected 30 random nests per region were being selected. Population survey For C.hybridus population survey, we used sampling method in zarrinkola Ab-bandan and total count of nests in Marzoonabad Ab-bandan. Eggs and chicks characteristics After egg lying in selected nests (figure1), the eggs were counted and after the hatching, chicks divided in to categories in basis of (Allan, 1988): 1-Nestling: new born chicks and downy bodies that can not move in the nest (figure2). 2-Post-nestling: body downs obliterated and new feathers growth being complete and they can not fly but move and swim on the water level in neighbour nests. 3-flight age: the chicks can fly and leave the nest. This observation was recorded between clutch size and brood size (The number of chicks that born synchronously). Incurred determination of Losses in different reproduction phenology stages Losses were determined in egg, Nestling, Post-nestling to chick fledged time in basis of (Hovis & Gore, 2000; Reynolds, 1990): 1-Human effects: nests, eggs and chicks that disappeared after local boat movements. 2- Water level fluctuations: nests, eggs and chicks that disappeared intermediate water depth increased. 3- In the first nestling stage: new born chicks (age< 2 day) that found dead per nest. 4-Other natural factors: nests, eggs and chicks that were lost and did not covered by previous categories. Reproductive phenology (duration) Reproductive phenology time was duration determined in the basis of observation of start nesting behavior to chick fledged time. Statistical methods Kruskal-Wallis Test was used for determining clutch size and brood size effect on the success and losses between reproductive stages ( Fasola & Canova, 1992) and Friedman Test used for difference determination between egg, Nestling and Post-nestling stages ( Spendelow & Zingo, 1997).

Results One breeder group of C.hybridus was in Zarrinkola Ab-bandan, Where as, two breeder groups were selected in Marzoonabad Ab-bandan. Therefore, first and second breeder group terms are being used here in after.

Population survey There were 930 nests in Zarrinkola and 1155 and 235 nest in first and second breeder groups (Totally, 1390 nest) in Marzoonabad Ab-bandan, respectively.

Eggs and chicks characteristics Proceedings of The Fourth International Iran & Russia Conference 1386

There were 80, 92, 76 eggs in Zarrinkola, first and second breeder group of Marzoonabad (of 30 nests studied) respectively. Clutch size was 1-4 egg in Zarrinkola and second breeder group of Marzoonabad, where as 1-5 egg was observed in first breeder group. Brood size of 42, 57 and 50 chicks in zarrinkola, first and second breeder group of Marzoonabad were sen, respectively. Reproductive phenology (duration) Reproductive phenology durations (day) are shown in table1. Breeding success Breeding success percent ages are shown in table2. There are no significant differences between breeding success with clutch size in zarrinkola, first and second breeder groups of Marzoonabad Ab-bandan (P= 0/573, P=0/118, P=0/802), respectively. Losses determination C.hybridus losses in reproductive phenology stages are shown in table 3. In basis of Friedman Test, there are significant differences between Egg, Nestling and Post-nestling stages in Zarrinkola, first and second breeder groups of Marzoonabad (P= 0/001, P=0/002, P=0/000), respectively. Effective factor percent ages in per stage losses are shown in table 4.

Discussion There was only one Breeder group of C.hybridus in Zarrinkola Ab-bandan, where as, because of conservative and secure conditions and unoccupied spatial niches, there were two breeder groups in breeding season in Marzoonabad. Breeding success was higher in clutch size with 4-5 eggs rates than 2-3 eggs because clutch size with 4-5 eggs was laid by 2 female (Cramp et al., 1985). The maximum losses was seen in egg stage that there was significant difference in basis of Friedman Test. Because of Their static nature, eggs exposed to higher threats than Nestling and Post-nestling against water level fluctuations, predation and etc (Soon bok et al., 1998). Therefore, the maximum losses are in egg stage. Because of was intense rainfall and water level fluctuations, the maximum losses among egg stage occurred in Zarrinkola and the first breeder group of Marzoonabad Ab-bandan. because, C.hybridus in breeding season is dependent to climate conditions (Cramp et al., 1985) and there is high correlation between breeding success with intense rainfall (Allan, 1988), but in second breeder group of Marzoonabad ab-bandan, beholden to temporal maters down water level and light rainfall, this agent dose not role in eggs losses. Nestling losses occurred in first days are exposed, because, bin first 2-3 days after hatching, chicks can not move and leave the nest and many threats, as intense rainfall and nest disappear, too (Coburn et al., 2001; Nisbet et al., 1998), but in mid Nestling to end Post-nestling stage There are lesser exposure than egg stage, because chicks can move and escape of natural and human agents (Calado, 1996).

Reference Allan DG (1988) Whiskered Tern ( Chlidonias hybridus) breeding in the Southeastern Transvaal Highveld, South Africa, Cormorant, Vo1.16, NO.1: 3-6. Bacon LM and Rotella J (1998) Breeding ecology of interior Least Terns on the unregulated Yellowstone river, Montana, J.Field Ornithol, 69(3) :391-401. Baldi A, Moskat C and Zagon A (1998) Faunal mapping of birds in a riparian area of river Danube after construction of a hydroelectric power station, Folia zool, 47(3): 173-180. Baldi A, Moskat C and Zagon A (1999) Evaluating the effectiveness of Of faunal mapping, forest and marshland bird censuses for monitoring Environmental changes, vogelwelt 120, suppl.:131-134. Proceedings of The Fourth International Iran & Russia Conference 1387

Baldi A and Kisbenedek T (2000) Bird species number in an archipelago of reeds at lake Velence, Hungary, Global Ecology & Biogeography, 9:451-461.

Calado M (1996) Little Tern (Sterna albifrons) status and conservation at RIA Formosa natural park, Algavve, Portugal, colonial waterbirds, Vol.19, No.special:78-80. Cramp S , Simmons K, Gillmor R , Hollom P, Hudson R , Nichelson E , Ogilvie M , Olney P , Roselaar C , Voous K , Wallace D, Wattel J Brooks D.J and Dunn E (1985) Handbook of the birds of Europe the Middle East and North Africa, the birds of the western palearctic, volume IV ,Terns to Woodpeckers, Oxford University Press. Coburn LM, Cobb DT and Gore JA (2001) Management opportunities and techniques for roof and ground-nesting Black Skimmers, wildlife society bulletin, 29(1):342-348. Evans MI (1994) important bird areas in the Middle East, Birdlife International Inc, Cambridge. Eyler TB, Erwin RM, Stotts DB and Hatfield J (1999) Aspects of hatching success and chick survival in Gull-billed Terns in coastal Virginia, Waterbirds, 22(1): 54-59. Fasola M and Canova L (1992) Nest habitat selection by eight syntopic species of Mediterranean Gulls and Terns, Colonial Waterbirds , 15(2):169-178 . Hovis J and Gore J (2000) Nesting shorebird survey, Florida fish and wildlife conservation commission, 79pp. Nisbet IC (1996) Post- fledging survival in Common Terns in relation to brood order, hatching date and parental age, colinial waterbirds, 19 (2): 253-255.

Nisbet IT, Spendelow JA and Hatfied JS, Zingo JM and Gough GA (1998) Variations in growth of Roseate Tern chicks: II.Early growth as an index of parental quality, the condor 100: 305-315. Reynolds JV (1990) the breeding Gulls and Terns of the Islands of Lough Derg, Irish Birds, 4:217-226.

Scarton F, Valle R and Borella S (1994) Some comparative aspects of the breeding biology if Black- headed Gull (Larus ridibundus), Common Tern ( Sterna hirundo) and Little Tern ( Sterna albifrons) in the lagoon of Venice, NE Italy, Avocetta, No.18:119-123.

Scarton F and Valle R (1996) Colony and nest-site selection of Yellow- legged Gulls (Larus cachinnans michahellis) on Barrier islands of the delta (NE Italy), Vogelwelt, 117: 9- 13.

Schjorring S and Bregnball T (1999) Prespecting enhance breeding Success of first time breeder in the Great Cormorant, Animal Behavior, Vol. 57, and No 3: 664-674.

Soon bok H , Yong W and Seigo H (1998) Effects of clutch size and egg-laying order on the breeding success in the Little Tern ( Sterna albifrons) on the Nakdong estuary, Republic of korea, Ibis, 140 (3): 408- 414.

Spendelow JA and Zingo JM (1997) Female Roseate Tern Fledges a chick following the death of her mate during the incubation period, colonial waterbird 20 (3): 552- 555.

Smith JW and Renken RB (1993) Reproductive success of Least Terns in the Mississippi river valley, Colonial Waterbirds, 16(1):39-44. Proceedings of The Fourth International Iran & Russia Conference 1388

Table1- Reproduction phenology duration (day) in zarrinkola and Marzoonabad Ab-bandan (numbers in brackets show the shifting range). Ab-bandan nesting egg laying incubation Nestling Post- end Post- name time nestling nestling to chick fledged time Zarrinkola 6/31 ± 0/86 2 18 ± 1/29 5/15 ± 1/22 13/89 ± 1/33 3/26 ± 1/74 (5-8) (16-20) (4-8) (10-15) (2-10) Marzoonabad 4/66 ± 0/94 2/66 ± 1/08 19/03 ± 1/13 6/25 ± 2/10 15/85 ± 1/7 3 ± 2/72 first breeder (4-6) (2-6) (18-22) (4-12) (12-18) (1-10) group Marzoonabad 5/62± 0/61 2/25 ± 0/58 19/11 ± 1/47 5/92± 1/38 15/51 ± 1/7 2/44 ± 0/99 second (4-7) (2-4) (18-22) (4-10) (12-18) (1-6) breeder group

Table2- Breeding success percent ages of Whiskered Tern (Chlidonias hybridus) in Zarrinkola and Marzoonabad Ab-bandan (numbers in brackets are frequency). Ab-bandan hatched primary eggs primary eggs Breeding name primary eggs after Nestling after Post- success stage nestling stage Zarrinkola 66/25(53) 55(44) 50(40) 57/08 Marzoonabad 85/86(79) 82/60(76) 80/43(74) 82/96 first breeder group Marzoonabad 81/57(62) 73/68(56) 72/36(55) 75/87 second breeder group

Table3-Losses percent in per reproduction phenology (numbers in bracket are frequency). region before egg hatching Nestling Post-nestling Zarrinkola 33/75(27) 11/25(9) 5(4) Marzoonabad first 14/13(13) 3/26(3) 2/17(2) breeder group Marzoonabad 18/42(14) 7/89(6) 1/31(1) second breeder group Proceedings of The Fourth International Iran & Russia Conference 1389

Table 4- effective factors (percentage) in egg, Nestling and Post-nestling losses. region reproductive human water level first other total phenology factors fluctuations Nestling natural losses stage factors Zarrinkola egg 6/94 18/40 0 6/59 31/93

Nestling 0/52 1/04 1/91 4/51 7/98

Post-nestling 1/38 0 0 3/99 5/38 Marzoonabad egg 0 20 0 10/75 30/75 first breeder group Nestling 0 0 0/66 0/33 0/99 Post-nestling 0 0 0 0/66 0/66 Marzoonabad egg 0 0 0 14/82 14/82 second breeder Nestling 0 0 8/37 1/75 10/12 group Post-nestling 0 0 0 1/75 1/75

Figure1-Whiskered Tern (Chlidonias hybridus ) nest with 4 eggs.

Figure 2- Whiskered Tern (C.hybridus) chick in Nestling stage. Proceedings of The Fourth International Iran & Russia Conference 1390

Metal Ion-mediated oxidative stress associated with increased cell membrane and DNA damage in Carp gill suspensions Mehran Arabi Department of Biology , Animal Physiology lab. , Shahrekord University , Shahrekord , Iran .Telefax : ++ 98- 381- 4424419 . , [email protected]

Abstract Reduction in the number of fish for the sake of water contamination is a paradox. Due to direct exposure of gills in the water, it is dominantly accepted that they are the main sites for water contaminants (e.g. metal ions). In the present study the attempt was made to investigate the metal ion contamination impact on the functional capacity of Carp gill cells with antioxidants interactions as a in vitro study. The extent of cellular membrane damage/LPO (as TBARS levels), GSH content, were investigated after addition of two metal ion compounds i.e. CuSO4 and HgCl2 in various concentrations of 300, 500, 700, 1000 and 3000 µM to gill cells preparation belong to freshwater Fish Carp (Cyprinus carpio) with modulations by BSA (0.5 & 1.0 %) and DMSO (0.5 %) as free radical scavengers. The Comet assay technique was also performed for the highest concentrations of two mentioned metal ions as an index to DNA breaks. The outcomes were: (a) Copper and mercury increased the rate of LPO dose- dependently (r= +0.995 and r= +0.993 respectively, p<0.001) but the GSH content was only marginally affected (r=-0.787 and r=-0.844 respectively, p<0.05). (b) Depleting of GSH molecules by copper showed a wider range than mercury. (c) In at highest concentration of metal ions (3000 µM) both DMSO and 1.0% BSA showed a pro-oxidative potential to elevate the levels of TBARS (p<0.001) but for other concentrations when supplemented with three scavengers, it was found a fall in the levels of the latter. (d) Addition of 1.0 % BSA to medium containing 3000 µM of metal ions caused a significant decline in GSH content (p<0.01). (e) Copper and mercury could imposed high rate of DNA breaks (single-stranded) in Carp gill cell suspensions as a Comet appearance. These findings indicate that, copper and mercury have deleterious influence on membrane integrity and GSH content as well in a relative dose-dependent manner. The complexes of metal ions and thiol (-SH) residues of cell proteins could also act as a more potential cell toxicant leading to disturbances in cell functions towards cell death. DNA fragmentation is frequent in metal ion contamination.

Keywords: Metal ions, Carp, membrane integrity, GSH, oxidation, DNA breaks.

Introduction Common Carp (Cyprinus carpio L.) is an important commercial species around the world and is as an economic rather than an ornamental fish. As a group, Carp provide 4 million metric tons of fish annually-over a quarter of all fish cultured worldwide. Reducing the number of these precious animals for water contamination (e.g. metal ion toxicity) is as a paradox to heavy demands. Teleosts, functionally, have four pairs of gill arches furnished with tiny structures called gill lamellae. The latter, are rich in capillary networks and covered with a simple squamous epithelial cells which are responsible for gas exchanges in aquatic media. Due to direct exposure of gills in the water medium, it has been dominantly accepted that they are the main site to water contamination and toxicity (1&2). Coping with to fish is very crucial. The water companies are routinely consuming metal ions in aquatic media e.g. Iron sulfate is used to combat algal blooms in reservoirs (3).Copper sulfate is one of the most widely used algicides for the control of phytoplanktons in lakes, reservoirs, and ponds, it is also used for aquatic weed control (1). Mercuric chloride is also mainly used to control the Proceedings of The Fourth International Iran & Russia Conference 1391 mass of other fish partners as mollucicide (4) . It has been shown that some metal ions in fish express deleterious impacts on some organs such as liver, gill, gonads and components of blood as well (1,5-7). A number of researchers have observed that the reactive oxygen species ( ROS ) such as . . superoxide anion ( O2-) and hydroxyl radical ( OH) through their own generating systems can stimulate the peroxidation of polyunsaturated fatty acids (PUFAs) of the biological membranes which is called lipid peroxidation , LPO (5&8). Continued fragmentation of fatty acid side chains to produce further more aldehyde and hydrocarbons will eventually leads to LPO propagation and complete loss of membrane integrity and cell functions. Therefore , LPO is an useful index to measure the membrane integrity and relative lesions. To overcome this degenerative process cells are well- equipped with a powerful battery of defense systems (Antioxidants) to mop up and then neutralize the ROS (9). The ubiquitous tripeptide, reduced glutathione (L-gamma-glutamyl-L-Cysteinylglycine ,GSH ) , most prevalent intracellular thiol (-SH) functions as a quick and vibrant antioxidant in cells by donating one hydrogen atom from two molecules to a toxic substance(10).Glutathione is present in the oxidized (GSSG) form, which is readily converted to the GSH form by the enzyme glutathione reductase (GRD). It has been reported that glutathione is present mainly in its reduced form in biological tissues, at concentrations as high as 2180 µg/g of tissue and in form of GSSG to be present in much smaller concentrations, ranging from 0 to 288 µg/g of tissue (11). A shift to a more oxidative state or any imbalance between production and degradation of ROS in animal tissues may causes LPO, plasma membrane alternations, inactivation of enzymes, ect. ( 5,7,12&13 ) . The current study was conducted to investigate the influence of different concentrations of two metal ion compounds viz. copper sulfate (CuSO4) and mercuric chloride (HgCl2) on some properties of carp gill cells; membrane integrity ,GSH content , and DNA integrity. We also estimated the scavenging activity of 0.5 & 1.0 % small molecule protein, bovine serum albumin (BSA) , and 0.5 % dimethylsulfoxide (DMSO) for possibly generateing ROS in different media.

METHODS AND MATERIALS Reagents Thiobarbituric acid (TBA), DTNB (5,5'-dithio-bis [2-nitrobenzoic acid]), LMPA, NMPA, DMSO, and Acridine orange were obtained from Sigma Chemical Co. (St. Louis, MO ,USA). All other chemicals were from Merk Co. ( Darmstadt , Germany) . All solutions were made with triple-distilled water. Collection of Samples Twelve fresh Carps were collected from local fish farm. Immediately branchial arches were gently taken out and kept in chilled Sorenson's buffer (pH 7.4) and then transferred to lab and kept at 4 oC for a short period of time till final processing . Protocol for the Estimation of Lipid Peroxidation and GSH Content a) Preparation of Tissue Homogenate After careful removal of the cartilaginous portions supporting gill arches, the gill filaments were gently cleared of other parts and washed several times with chilled 0.154 M NaCl solution (0.308 osmolar) to remove blood. After that, grinding the bulk of gill filaments resulting in bared ones and free bands of lamellae. The latter, separated from bony parts by means of ordinary tea strainer. Consequently, the resulting filtrate homogenized in 0.05 M phosphate buffer (pH 7.4) using Polytron homogenizer at a speed of 10,000 rpm in ice bath . The homogenate was then centrifuged (3000 rpm ,10 min).The supernatant was Proceedings of The Fourth International Iran & Russia Conference 1392 used for assays throughout the work. All the above steps commencing from dissection of the tissue till the preparation of homogenate were performed at 0-4 oC. b) Estimation of Proteins Cell protein content was estimated by the modified sodium Dodecyl Sulphate-Lowry method of Lees and Paxman (14) using BSA as standards. Activities of measured parameters were calculated and expressed per mg Protein. c) Assay of Lipid Peroxidation by Spectrophotometric method The LPO was estimated in terms of thiobarbituric acid reactive substances (TBARS), particularly Malondialdehyde (MDA) by the method of Ohkawa et al. (15) with slight modifications. The reaction mixture (3ml) contained 0.1 ml of homogenate supernatant, 0.2 ml of 8.1 % sodium dodecyl sulphate, 1.25 ml of 20 % glacial acetic acid (pH 3.5), 1.25 ml of 1.2% aqueous solution of TBA and 0.2 ml distilled water in control group instead of adding 0.1 ml of metal ions or antioxidants in treated one. Finally, after heating, adding 3 ml of n- butanol-pyridine mixture and centrifuging at 2200 xg, the amount of MDA formed was measured by the absorbance of the upper organic layer at 532 nm which is the λmax of MDA (Extinction coefficient of 1.56x105 M-1 cm-1) using appropriate controls. The LPO rate was finally expressed as n moles MDA. mg prot –1. min –1. d) GSH Content Measurement Reduced glutathione residue after treatments as well as in control group was assayed according to the method of Sedlak and Lindsay (16) using DTNB with minor modification. The unit used was µ moles -SH . mg prot -1.min-1.

Single Cell Gel Eelectrophoresis ( Comet ) Assay The Comet assay is a sensitive and amenable technique in order to detect the presence of DNA strand breaks and alkali labile damages in the individual cells .The DNA fragments so produced migrate towards the anode pole , at a rate inversely proportional to the size of the fragment during electrophoresis . Consequently, each cell with damaged DNA gives the appearance of a Comet .Hence, this assay is known as Comet assay .The alkaline single cell gel electrophoresis (Comet) assay was based on existing methods which first described by Singh et al. (17), modified as indicated below. Pre-cleaned microscope slides were dipped in a solution of 1% normal melting point agarose (NMPA) dissolved in 0.2 M PBS ( pH 7.2 ) and air-dried overnight . Again , 200 µl of NMPA put on slides and covered with a cover slip and left to solidify at room temperature . 25 µl of sample (suspension ) plus 75 µl of 1 % low melting point agarose (LMPA) was added to slide . A final layer of 1 % LMPA was added to slides and allowed to solidify at 4 oC at least one hour . Following removal of the cover slip, slides then were immersed in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-HCl, 10% DMSO, Triton X-100) for 1 hour at 4 oC. This treatment lyses the nuclear and the cell walls and permits DNA to unfold. Slides were immersed in a horizontal gel tank filled with alkaline buffer (300mM NaOH/ 1mM EDTA, pH 12.3) for 20 min to allow DNA to unwind . Electrophoresis was carried out for 15 min at 25 V (0.862 V cm –1). The slides, then, were placed in Coplin jars containing fresh neutralizing solution ( 0.4 M Tris-HCl , pH 7.4 ) for 5 min (repeated 3 times). Acridine orange (AO) as a fluorescence dye (20 µl ) was used to stain sperm DNA which were imaged by fluorescent microscope (Excitation filter 515-560 nm ; Dichroic filter 580 nm ; and Suppresion filter 580 nm) . AO bound to single-stranded fluoresces red and to double- stranded DNA fluoresces green . For each sample, five replicate slides were prepared and 50 randomly selected microscopic fields were scored on each slide . The percentage of categories of DNA damages : undamaged (no migration) and damaged (migrated), were recorded . Proceedings of The Fourth International Iran & Russia Conference 1393

Statistical Analysis The data reported in the paper are means of three assays . All measurements were performed in triplicate. The data are given as means ± SD. The comparison of the control and treated series was statistically analyzed by Student's t-test using the SPSS software (SPSS 8.0,1998). Correlation was evaluated using linear regression (LR) analysis between parameters assayed in the present study. RESULTS Influence on TBARS levels As shown in Table 1, production of MDA/TBARS in Carp gill cells preparation was induced by the presence of various concentrations of copper in a dose-dependent and linear manner ( p<0.001) .There was a positive and strong correlation (r = +0.995) between the elevated CuSO4 concentrations and TBARS levels. When ROS scavengers (DMSO & BSA) were supplemented to the media a sharp significant decrease was observed in TBARS levels (p<0.001) for all concentration of CuSO4 as compared to treated ones with same concentration of metal ion (alone), but here at the concentration of 3000 µM for DMSO and 1.0% BSA instead of decreasing there was a dramatic increase in TBARS production (p<0.001) which is the result of their pro-oxidant activities. Also all three scavengers exerted a significant fall in TBARS levels as compared to control group (no metal ion treatment and supplementation) which expressed a spontaneously LPO production in medium (Table 1, p<0.01 & p<0.001, cf. different data). The data in Table 2 show that the addition of upgraded concentrations of HgCl2 caused highly significant elevation in TBRAS production (p<0.001). The strength of association between metal ion effect and LPO generation was found to be positive and considerable (r = +0.993). As soon as supplementation was initiated, it was clearly observed that BSA (0.5 & 1.0 %) can inhibit the extent of MDA/TBARS production (p<0.001) when compared to the same samples treated with metal ions (alone) but DMSO affected the LPO rate only marginally (p<0.05 & p<0.01, cf. The listed data). Here again we found out that when 0.5% DMSO and 1.0% BSA added to media with 3000 µM metal ion a drastic increase in TBARS levels was occurred (p<0.01& p<0.001, respectively). Addition of the all three scavengers to control group resulted in a fall in TBARS levels (Table 2 , p<0.01 & p<0.001). The protective effect of 0.5% BSA for holding the membrane integrity was found much more better than DMSO & 1.0 % BSA ( p<0.001). Influence on reduced glutathione (GSH) content Table 3 summarizes the effect of CuSO4 concentrations on the GSH pool in Carp gill cells preparation. This evaluation showed only 3 concentration of copper (700-3000 µM) could change the GSH content which was a slightly significant decrease (p<0.05).A negative correlation occurred between metal ion influence and GSH levels in preparation (r = -0.787). No significant change was observed in the levels of GSH when DMSO & 0.5% BSA were added (p>0.05). There was an interesting finding when 1.0% BSA was added to the assay mixture which was a significant diminution for GSH content as compared to control value (p<0.05 & p<0.01, cf. different data). Moreover, supplementation of the ROS scavenger 0.5% BSA to control group (alone) led to an elevation in GSH levels (p<0.05) and no significant alteration following addition of two others (p>0.05). The data in Table 4 reveal that addition of various concentration of mercury (HgCl2) to gill cell preparations of Carp could lower the levels of GSH dose-dependently but slightly significant (p<0.05) for two higher concentrations namely 1000 & 3000 µM. A negative significant correlation existed between this parameter and metal ion concentrations (r = - 0.844). Supplementation of DMSO to assay mixture containing metal ion resulted in a slight Proceedings of The Fourth International Iran & Russia Conference 1394 diminution but non-significant (p>0.05) in GSH content. 0.5% BSA caused an almost significant elevation (p<0.05) to GSH content when compared to respective samples treated with same metal ion concentration (alone) (1000 & 3000 µM).The addition of 1.0% BSA could not change the GSH levels significantly in series treated with mercury except at the highest concentration which led to a sharp significant decrease (p<0.01) as compared to control group( Table 4 ).Among the ROS scavengers used in this experiment only 0.5% BSA could change and elevate the levels of GSH when compared to the control data ( p<0.05 ).

Influence on DNA integrity In order to determine whether metal ions could also induce DNA damage in suspensions of Carp gill cells , only the highest dose , 3000 µM concentration , of both copper and mercury was added . It has been found that metal ion contamination could impose single-stranded DNA breaks , almost significantly, in the genetic materials of Carp gill cells after one hour incubation as compared to control groups from 2.41 ± 0.842 % to 7.32 ± 0.764% and 8.50 ± 0.654%, respectively (p< 0.05) (see Fig. 1). Interestingly, no significant difference was found after antioxidant additions to metal ion-treated samples when compared to control groups (data not shown).

DISCUSSION As mentioned above, there is a growing body of evidence to indicating that metal ions can exert some deleterious impacts on a number of tissues of fish Carp (Cyprinus carpio L.). For instances, mercury concentration effect the in vivo model is believed to be associated with lowered succinate dehydrogenase activity and O2 consumption (4) and also an induction of severe proteolysis in Carp gill cell metabolism (18). Radi and his co-worker (5) reported a sharp fall in antioxidant activity of some enzymes e.g. glutathione peroxidase (GPx) and an elevation in the rate of LPO and also protein contents after mercury treatments in liver, white muscles and gill of Carp. On the other hand , copper increases the activity of some key metabolism enzymes such as alkaline phosphotase, alanine and aspartate aminoteransferases and showed induction of some lesions in Carp gill e.g. epithelial hyperplasia and chloride cells dysfunction (1). In addition Kurant et al. (19) reported that copper treatment in Carp liver cells induces a drop in the content of low molecular weight protein thiols. The reports on the effect of metal ions toxicity as an in vitro model are not too many, this kind of work is able to open new lines of intracellular compartments when exposed to pollutants. Owing to the role of metal ions as important catalysts in living organisms finding the knowledge regarding to other facets of these compounds sounds much vital. Under the present experimental conditions, the results presented here have clearly demonstrated that the elevated metal ion concentrations for copper and mercury are associated with high production of the TBARS (Table 1&2 , p<0.001). We observed a strong link between concentration effect of metal ions used and extent of TBARS. The correlation coefficients for copper and mercury amounted to + 0.995 and + 0.993 at 37 0C respectively (p<0.001). Here, copper and mercury acted as potent oxidants to biological membranes , both plasma and intracellular membranes . Therefore , these results indicate that in spite of physiological role of metal ions in maintenance of cell functions , could also function as toxic agents when present in excess. The metal ions as transition metals cause cellular damages via formation of highly reactive oxygen free radical viz .OH . LPO initiation phase results in the formation of lipid . . - hydroperoxides (Lipid-OOH) in the presence of OH (9) which is derived from O2 and . - hydrogen peroxide (H2O2) , generally metabolically generated in medium. Neither O2 nor H2O2 is energetic enough to initiate LPO directly, but in presence of catalytic amounts of Proceedings of The Fourth International Iran & Russia Conference 1395 metal ions , they can react and form .OH units under a net equation , Harber-Weiss reaction (8) : metal ion . - . . - O2 + H2O2 O2 + OH + OH (catalyst)

Here, the reduction of membrane integrity and fluidity is as a consequence of enhancement of LPO process. On the other hand, with commencing LPO cascade, the release of significant quantities of lipid-OOH in medium will be occurred , which is presumably a consequence of the activity of membranous enzyme phospholipase A2 (PLA2) by cleaving oxidized PUFAs from the 2-position of phospholipid glycerol backbone so that they can then be metabolized by GPx to the corresponding alcohols ( 8&20 ). It has been determined that metal ions like mercury can alleviate the rate of GPx activity in Carp gill and liver resulting in accumulation of lipid-OOH in cell and LPO propagation throughout the cell membranes which injures the membrane integrity (5). Furthermore, as Huang et al. (21) and Halliwel (22) suggested it is possible that lipid-OOH formed in the biological membrane . systems by the effect of metal ions convert to the form of peroxyl radical (Lipid-O2 ) which is able to re-attack the membrane. It is of great interest to know that MDA formed during LPO could conceivably reacts as bifunctional reagent to cross-link through Schiff ’s base formation between some classes of phospholipids (23). Lack of the uniformity to these cross-links in membranes will be led to physical forces which may disturb the membrane lipid distributions (24). We found here that GSH was marginally affected by the elevated metal ion concentration effect (Table 3&4, p<0.05) to a lesser content .GSH can react with peroxyl radicals to achieve a steady state for themselves and itself converts to thiyl radical (GS.) which is not rapid as oxidative attack at polyunsaturated membrane lipids: . . GSH + Lipid – O2 lipid – OOH + GS It seems that the metal ion extrusion from cells presumably involves movement of diffusible complexes such as Hg-GSH , it also gives rise to alleviating in the level of thiol groups pool including GSH in suspensions . Due to more reactivity of copper with GSH molecules than mercury, it can be assumed that copper could present more oxidative stress on living organism making its cells being more susceptible to damages. Despite the extensive evidence implicating the depletion and / or oxidation of GSH in a wide variety of experimental toxicities (25), here it has been shown that GSH content was not much altered under the influence of metal ion stress (p<0.05). An explanation here is that relative inhibition following metal ion impact on some enzymatic defense systems such as GPx (5), converts lipid-OOH to respective alcohol through oxidation of GSH to GSSG, causes no remarkable alteration in GSH content due to less GSH consumption by inhibited enzyme. Supplementations were carried out to check the protective role of BSA (0.5 & 1.0%) and DMSO (0.5%) in media. DMSO as a lipophilic OH. scavenger normally is used to mop up ROS and protect biological membranes against oxidation . On the other hand, BSA also as a OH. scavenger might play a far more important role in protection of plasma membrane from peroxidative injuries by binding to and neutralizing the lipid hydroperoxides liberated by PLA2 (20) .BSA can impair the LPO cascade either by acting as a sacrificial antioxidant or as a chelator of the metal ions that promote this process (25). Another intriguing possibility from Cha and Kim (26) is that BSA is able to exhibit peroxidase activity , providing that thiol-reducing equivalents are available. Addition of 1.0 % BSA and DMSO caused a significant fall in the level of TBARS up to concentration of 1000 µM of metal ions and after that led an increase in TBARS/MDA Proceedings of The Fourth International Iran & Russia Conference 1396 production (Table 1&2), this is due to of the fact that thiol groups which exist in these . - antioxidants could play a multifaceted role as pro-oxidants by autoxidizing to produce O2 in the presence of metal ions (27). R-SH + metal ion (oxidized) RS + metal ion (reduced). . - metal ion (reduced) + O2 metal ion (oxidized) + O2 Collectively ,our results showed that 0.5% BSA exerts a positive influence in protection against fatty acid peroxidation than 1.0 % BSA and DMSO as well (Table 1 & 2) in control and treated suspensions . The intracellular compartmentation of GSH molecules including the nucleus , mitochondrial matrix and endoplasmic reticulum have many important implications for cells that are exposed to toxic compounds or to other stresses (25). It is noteworthy that there is a kinetically distinct pool of GSH in the nucleus, estimated to be 5 to 10% of the total GSH and may be concentrated at the nuclear membrane surface (28&29) when an adequate stimulation is made, it might be released and added to cytoplasmic pool. We found that among the antioxidants tested only 0.5% BSA yielded an alteration in the GSH content of the control group of preparations (Table 3 & 4, p<0.05 ). It seems that BSA functioned as a stimulation either for releasing the stored GSH molecules from their cellular compartments or to detach the masked GSH molecules from trapping points in the preparation . However the exact mechanism of this phenomenon remains to be elucidated. As mentioned earlier, thiol groups in cases of DMSO and 1.0% BSA could imposed a severe peroxidation on cell membranes, however 1.0 % BSA has been found to be more toxic than DMSO to reduce the GSH content when accompanied with metal ions particularly in higher concentrations. More analysis needs to be conducted. Oxidative stress can disrupt the functional capacity of gill cells by attacking the integrity of their DNA in the nucleus and mitochondria . As indicated earlier, due to direct exposure of gills in the water , it is assumed that they are the main targets for any contamination in external environment .By-products of LPO like MDA and other lipid radicals, generated in metal ion- treated samples are able to enter the DNA ladder and induce base oxidation towards dysfunction e.g. ion pumps inhibition in plasmatic membranes which governing the gas exchanges in water medium. Work on other animal experimental models revealed that metal ions treatments can enhance persistence of DNA damage (30&31) . It has been also suggested that metal ions may enhance the production of tumor necrosis factor (TNF) alpha and other stress proteins (32) with a key role in the induction of DNA damages and subsequently cell death . These data support the use of DNA strand breakage as an indictor / a biomarker of chemical contamination.Our results showed that the percentage of damaged nuclei/Comets increased after application of highest dose of metal ions . Almost all DNA breaks were under category of single-stranded ones which is in agreement with the work by Bucio et al. on fetal hepatic cell line (31) showing that these metal ions have no lethal impact on Carp gill cells as single-stranded DNA breaks have capacity to be repaired when compared to double-stranded ones which are generated under severe oxidative stress .On the other hand , here the Comet assay shown to be considered as an useful application in biomonitoring studies for determining the potential genotoxicity of water pollutants such as metal ions . Taken together, these findings revealed that used metal ions used i.e. copper and mercury are very harmful to gill cells of fish Carp when particularly are used in excess. These metal ions not only act as anti-fluidity agents for membrane lipids and as DNA cutters but also in making a complex with cell protein thiols will be able to express more toxic effects to push the cell toward dysfunction and ,ultimately, death .

ACKNOWLEDGEMENTS Proceedings of The Fourth International Iran & Russia Conference 1397

The author is grateful to Dr. Behzad Shareghi (PhD), Dean of faculty of Sciences , Shahrekord University , Iran , for providing necessary laboratory facilities and materials ; Dr. (Mrs) Noha Eftekhari (PhD) , Maths. Dept. , Shahrekord University , for the management of statistical analyses ; and Mr. Rasool Seidaei (MSc.) , Biology Dept. , Shahrekord University , for his endless laboratory assistance .

EFERENCES 1 . V. Karan , S. Vitorovic , V. Tutundzic and V. Poleksic , Functional enzymes activity and gill histology of Carp after Copper sulfate exposure and recovery, Ecotoxicol. Environ. Saf. 40(1/2) , 49-55 ( 1998 ). 2 . D.J.B. Dalzell and N.A.A. Macfarlane , The toxicity of iron to brown trout and effects on the gills: a comparison of two grades of iron sulphate, J. Fish Biol. 55 , 301 - 315 ( 1999 ) . 3 . C. Hayes , R.G. Clark , R.F. Stent and C.J. Red show , The control of algae by chemical treatment in a eutrophic water supply reservoir , J. Inst. Water Engin. Sci. 35 , 149-162 ( 1984 ) . 4 . K. Radhakrishnaiah , A. Suresh and B. Sivaramkrishna , Effect of sublethal concentration of mercury and zinc on the energetics of a freshwater fish Cyprinus carpio (Linnaeus), Acta. Biol. Hung. 44(4) , 375-385 ( 1993 ) . 5 . A.A. Radi and B. Matkovics , Effects of metal ions on the antioxidant enzyme activities, protein content and lipid peroxidation of Carp tissues , Comp. Biochem. Physiol. (C) 90(1) , 69 -72 ( 1988 ) .6 . D. Mukherjee , V. Kumar and Chakraborti P , Effect of mercuric chloride and cadmium chloride on gonadal function and its regulation in sexually mature common Carp Cyprinus carpio , Biomed. Environ. Sci. 7(1) , 13-24 ( 1994 ). 7. A. Akahori , Z. Jozwiak , T. Gabryelak and Gondko R , Effect of zinc on Carp (Cyprinus Carpio L.) erythrocytes , Comp. Biochem. Physiol. (C) 123 , 209-215 (1999). 8 . B. Halliwel and J.M.C. Gutteridge , Oxygen toxicity, oxygen radicals, transition metals and disease , Biochem. J. 219 , 1-14 (1984) . 9 . R.K. Sharma and A. Agarwal , Role of reactive Oxygen species in male infertility, Urology 98(6) , 835-850 (1996 ). 10 . A. Meister and M.E. Anderson , Glutathione , Ann. Rev. Biochem. 52 , 711-760 (1983) . 11 . P.J. Hissin and R. Hilf , A fluorometric method for determination of oxidized and reduced glutathione in tissues, Anal. Biochem. 74 , 214-226 (1976) . 12 . J. Matta , R. Hanger and T. Tosteson ,Heavy metal, lipid Peroxidation and ciguatera toxicity in the liver of the carbibean barracuda (Sphyraena, barracuda), Biol. Trace. Elem. Res. 70 (1) , 69- 79 (1999) . 13 . R.J.K. Anand , M. Arabi , K.S. Rana and U. Kanwar , Role of vitamins C and E with GSH in checking the peroxidative damage to human ejaculated spermatozoa, Int. J. Urol. , Volume 7 , Supplementation : S1 - S98 (2000) . 14 . M. Lees and J. Paxman , Modification of Lowry procedure for the analysis of proteolipid protein , Anal. Biochem. 47 ,184-192 (1972) . 15 . H. Ohkawa , N. Ohishi and K. Yagi , Assay for Lipid Peroxides in animal tissues by thiobarbituric acid reaction , Anal. Biochem. 95: 351-358 (1979) . 16 . J. Sedlak and R.H. Lindsay , Estimation of total, protein-bound and non- protein sulfhydryl groups in tissue with Ellman’s reagent , Anal. Biochem. 25 ,192-205 (1968). 17 . N.P. Singh , M.T. McCoy , R.R. Tice and E.L. Schneider , A simple technique for quantitiation of low levels of DNA damage in individual cells , Exp. Cell. Res. 175 , 184- 191 (1988 ). 18 . A. Suresh , B. Sivaramakrishna , P.C. Victoriamma and K. Radhakrishnaiah , Shifts in protein metabolism in some organs of freshwater fish, Cyprinus Carpio under mercury stress , Biochem. Int. 24(2) , 379- 89 (1991) . 19 . V.Z. Kurant , O.B. Stolyar , R.B. Balaban and V.O. Homechivck , Effect of Copper and lead on the nucleoproteins and thiols in tissues of Carp (Cyprinus Carpio L.). The society for experimental biology annual meeting , ( 7-11 April ).University of Kent at Canterbury 1997 , Online. Proceedings of The Fourth International Iran & Russia Conference 1398

20 . J.G. Alvarez and B.T. Storey , Differential incorporation of fatty acids into and peroxidative loss of fatty acids from phospholipid of human spermatozoa, Mol. Reprod. Dev. 42 , 334-346 (1995) . 21 . C-H Huang , H.O. Hutlin and S.S. Jafar , Some aspects of Fe2+- catalyzed oxidation of fish sarcoplasmic reticular lipid, J. Agricul. Food Chem. 41 , 1886-1892 (1993) . 22 . B. Halliwel , Oxidation of low-density lipoproteins: questions of initiation, propagation , and the effect of antioxidants , Ameri. J. Clin. Nutr. 61(Suppl): 760 S-677 S (1995) . 23 . K. Eichenberger , P. Bohni , K.H. Winterholter , S. Kawato and C. Richter, Microsomal lipid peroxidation causes an increase in the order of membrane lipid domain , FEBS Lette. 142 , 59 - 65 (1982) . 24 . I. Yuli , W. Wilbrandt and M. Shinitzky , Glucose transport through cell membrane of modified lipid fluidity , Biochem. 20 , 4250-4258 (1981) . 25 . C.V. Smith , D.P. Jones , T.M. Guentiner and B.H. Lauterbury , Compartmentation of Glutathione: implications for the study of toxicity and disease, Toxicol. Appl. Pharmaco. 140 , 1- 12 (1996) . 26 . M.K. Cha and L.H. Kim , Glutathione - link thiol peroxidase activity of human serum albumin-a possible antioxidant role of serum albumin in blood plasma , Biochem. Biophys. Res. Commun. 222 , 619 - 625 (1996). 27 . M. Tien , J.R. Bucher and S.D. Aust , Thiol-dependent lipid peroxidation, Biochem. Biophys. Res. Commun. 107(1) , 279-285 (1982). 28 . S.N. Loh , L.A. Dethlefsen and R.C. Fahey , Nuclear thiols: Thechnical limitations on the determination of endogenous nuclear glutathione and the potential importance of sulfhydryl proteins , Radiat. Res. 121 , 98-108 (1990). 29 . R.A. Britten , J.A. Green and H.M. Winenius , The relationship between nuclear glutathione levels and resistance to mephlalan in human ovarian tumor cells , Biochem. Pharmacol. 41 , 647 - 649 (1991). 30 . T. Guecheva , J.A. Henriques , B. Erdtmann , Genotoxic effects of copper sulphate in freshwater planarian in vivo studied with the single-cell gel test (Comet assay) , Mutat. Res. , 497 (1-2) : 19-27 (2001) . 31 . L. Bucio , C. Garcia , V. Souza , E. Hernandez , C. Gonzalez , M. Betancourt , M.C. Gutierrez-Ruiz , Uptake , cellular distribution and DNA damage produced by mercuric chloride in a human fetal hepatic cell line , Mutat. Res., 423 (1-2): 65-72 (1999) . 32 . S.J. Stohs , D. Baghchi , Oxidative mechanisms in the toxicity of metal ions , Free Radic. Biol. Med. , 18 (2) : 321-326 (1995) . Proceedings of The Fourth International Iran & Russia Conference 1399

EFFECT OF PHOTOPERIOD ON GROWTH INDICES , STRESS AND BIOCHEMICALS LEVELS IN ONE YEAR OLD REARED BELUGA STURGEON (Huso huso)

F. Askarian 1 , M. Bahmani 2 ,R. Kazemi 2 ,M. Mohseni 2 1-Islamic Azad university,Science and Research Branch,Agriculture and Natural Resources, faculty,Fisheries Group, Ph.D Course and member of Young Researchers Club.E-mail:[email protected];2-,International Sturgeon Research Institute,P.O.BOX:41635-3464, Rasht, Iran.

Introduction The first trials in sturgeon farming were carried out almost simultaneously in mid 19th century in Russia , Germany and North America to compensate for declines in harvest from wild sturgeon.The present sturgeon farming described is performed to provide meat and caviar for human consumption plus recreational purposes such as for aquariom fish and garden ponds as well as for recreational fishing market.Sturgeon ( noteworthy that even semianadromous species ) is farmed in different types of freshwater systems, utilising surface water , well water ( geothermal ) , and industrial wastewaters ( including thermal efluents ). Beluga ( Huso huso ) is one of the most important species of sturgeon in Caspian sea . The production of them for meat has been increasing recently in Iran (Bahmani, 1998) because of their high growth rate (Keyvan, 2003). Potoperiod has been considered one of the most important growth promoting factor in several fish species (Boeuf and Le Bail, 1999 ) . More information is required about the different techniques and physiological condition of its cult ure to contribute to a better management and development . This study was design to examine the effect of photoperiod on growth rate in one year old reared beluga and their physiological responses to different light regimes for the first time .

Materials and Methods One year old reared beluga (475± 2.91 g and 49.13± 0.22 cm) were kept in reared tanks (2m×1.5m×1.5m) on artificial diet at International Sturgeon Research Institute and were subjected to four different light regimes including natural photoperiod (NP), continuos dark (0L:24D), continuos light (24L:0D) and long day regime (16L:8D) from April to October of 2003. Growth was checked every 20 days and blood samples were taken from caudal vein with unheparinized syringe every 60 days. Serume cortisol (RIA) , glucose, triglycerid and cholesterol (specterophotometer ) were assayed. Data were analysed using a one-way variance and the results were expressed as the mean ± standard error of the mean (S.E.M.)

Results

The results reveal that extended of daylength have positive effect on the growth rate of beluga and their growth were progressively faster under continuous light ( Table I ).

Table I. Growth indices of one year old reared beluga sturgeon ( n=14 in each treatment) Treatment TL (cm) Wt (g) Cf (%) FCR GR (g) SGR (%) Control 54.51±0.53 719.02±23.91 0.71±0.02 3.05±0.10 4.71±0.72 0.67±0.11 0L:24D 54.43±0.48 707.39±20.82 0.73±0.07 3.23±0.10 4.12±0.63 0.59±0.08 Proceedings of The Fourth International Iran & Russia Conference 1400

24L:0L 56.13±0.62 816.61±33.34 0.75±0.007 1.98±0.09 6.18±0.77 0.78±0.09 16L:8D 56.12±0.56 811.47±27.02 0.74±0.008 2.08±0.09 5.63±0.93 0.70±.0.13

Furthermore there was significant difference in either growh indices of beluga between continuos light and long day regime with control and continuos dark treatment (p<0.05).

Table II. Serum stress and biochemical levels in in one year old reared beluga sturgeon (n=14 in each treatment) Treatment Cortisol(nmol/dl) Glucose(mg/dl) Triglyceride(mg/dl) Cholesterol(mg/dl) Control 16.26±2.79 44.17±2.75 347.68±24.79 76.11±4.95 0L:24D 18.00±1.81 54.39±2.89 443.48±32.99 84.55±5.71 24L:0D 18.72±2.21 49.18±2.12 367.30±19.43 67.21±3.80 16L:8D 23.84±3.06 53.01±2.62 397.60±23.18 91.41±6.51

Elevation of serum cortisol , glucose and triglyceride concentrations were reported in continuos dark ,continuos light and long day regime treatments (Table II). There was no significant difference in the serum corrtisol level between the treatments (p>0.05). Significant elevation in serum glucose and triglyceride occurred in response to continuos dark regime (p<0.0 ). We observed that increased secretion of cortisol is correlated with elevation of serum gelucose and triglyceride concentrations but it did not affect the cholesterol levels.

Discussion Beluga reacts to photoperiod treatments and long daylength stimulates their growth through a better food conversion . Changes of daylength and light regimes were involved in primary and secondary stress responses in beluga but they catch physiological acclimatization after a longtime . A few projects conducted in this regard can be cited (Trenkler and Semenkova, 1995) and this aspect will need more investigation.

References Bahmani M., 1998. Phylogenic and systematic study on sturgeon . Presented in: Association of the universities of the Caspian Region States. Astrakhan, Russia. Boeuf G., Le Bail P., 1999. Does light have an influence on fish growth ? . Aquaculture 177:129-152. Keyvan A., 2003. Iran’s sturgeons. Naghshemehr, Iran. 400pp. ( in Persian ). Trenkler I.V., Semenkova T.B., 1995. The influence of photoperiod , pinealectomy and pharmacological prepration on growth rate and metabolism in young sturgeon. Proc. Intern. Sturg. Symp. Vniro publ. 34-42.

Acknowledgments The authors would like to thanks colleagues International sturgeon Research Institute and Dr. Tooraj Valinasab for their assistance and Islamic Azad university ,Science and Research branch, for its supports. Proceedings of The Fourth International Iran & Russia Conference 1401

Effects of potassium permanganate treatment on fungal Infection, eyed eggs, hatching percentage and larval deformities of Rainbow Trout in West Mazandaran conditions.

Banavreh, A.¹*, Abtahi, B.² ,Mirzakhani, M.K.³ 1,3- M.Sc. In Fisheries, Faculty of Marine Scinces, Tarbiat Modarres University. Noor. Mazandaran, [email protected] 2-Assist. Prof. Of Fisheries Dept, Faculty of Marine Scinces, Tarbiat Modarres University, Noor Mazandaran.

Abstract Fungal infection may decrease hatching rate of salmonid eggs. Historically culturists to control fungus of eggs used Malachite Green. However, the U.S. Food and Drug Administration (FDA) withdrew its approval in 1991 because of its teratogenicity. In Iran, it is still in use for aquaculture practices. Potassium Permanganate could be considered as the alternative choice within salmonid farms, taking into account that E.P.A. Potassium Permanganate knew it as oxidant and detoxifier. In this study Potassium Permanganate (50 and 100 mg/l in 15 min bath) Was compared with Malachite Green (1.5 mg/l in 45 min bath) and natural control (without drugs). Each treatment group consisted of 3 replicates, which covered 1000 Rainbow Trout eggs. After one week, fungal infection, eyed eggs and hatching percentage, and larval deformities rate were determined. The results indicated the low fungal infection rate in Malachite Green treated group in comparison with natural control (p<0.05), although the former and lather revealed no significant difference with Potassium Permanganate treated groups. Comparing with other treatments, Potassium Permanganate (100 mg/l), showed an increment in rate of eyed eggs (p<0.05).

Keywords: Potassium Permanganate, Malachite Green, Fungal infection, Rainbow Trout, Mazandaran.

Introduction: Saprolegniasis is a kind of fungal disease of fish and their eggs cause by saprolegniacea (Noga, 2000). These fungies live in freshwater but some of them can grow in brackish water up to 2.8 ppt, (Azari, 1997). Handling fish, fluctuation of temperature, presence of parasites and increasing the organic matter, increase the chance of affection of this disease (Bruno and Wood., 1994). Dead eggs are a very good medium for developing the saprolegnia fungi. A saprolegnia zoospore can grow on dead egg and produce a mass of mycelium. These myceliums surround live eggs and suffocate them so another mass of eggs died and invaded directly by the fungi. This cycle continues until all the eggs will die. Fungal growth on dead eggs produces too many zoospores. These zoospores invade another masses of eggs and kill them. Malachite green use to control this disease. But its harmful effects i.e. carcinogenic and teratogenic effects and slow decomposition in the nature using of this drug was prohibited by F.D.A (Food And Drug Administration) (Marking et al., 1994). Potassium permanganate is used for ectoparasites, bacteria, fungal disinfections on the skin and gill of fishes. It is accepted by E.P.A (Environment Protection Agency) as an oxidant. It decreases BOD of water by oxidize the organic matter and the amount of oxygen will be increase by using potassium permanganate (Noga, 2000). Marking (1994) examined two concentrations 50 and 100 mg/l of potassium permanganate for prohibition of fungal infection on Rainbow Trout eggs and find out that although it decreased fungal infection of Rainbow Trout eggs but rate of hatching didn’t increase. Proceedings of The Fourth International Iran & Russia Conference 1402

Shafizadeh (2002) examined the effect of potassium permanganate concentrations in prevention of infection in Persian sturgeon eggs (Acipenser persicus) and showed that potassium permanganate at 5.37 mg/l can prevent fungal disease. Malachite green is used in most breeding and cultivation farms of Iran and research and recommendation of some new ant fungal drugs is needed.

Methods This research had been taken in Shahid Bahonar breeding and cultivation farm in Kalardasht from 11/13/2003 to 2/10/2004. The Fertilized eggs were remained in a container (in a dark place) to absorb water. Then the amount of 1000 Rainbow Trout eggs were selected by graduated jar and counting voluminally and were placed in a traph (each traph consist of three trays and each treatment consist three replicates). Drug treatment began 48 hour after fertilization. Potassium permanganate treatment by two concentration of 50 and 100 mg/l for 15 min were implemented every other day until 4 day before hatching. Malachite green was treated by 1.5 mg/l (usual concentration in farms) every other day until 4 day before hatching too. We had also a natural control, received no treatment. Physico-chemical characters of hatchery water were determined by suitable eqiupments. Measured parameters are temprature, pH, dissolved oxygen, conductivity and hardness. To determine the fertilization rate, the eggs were entered into stocard solution seven days after blastopor formation (Barnes et al.,1998). Determining factors in this research were as fallows: A) Fungal infection: Amount of infection from fertilization to eyed egg stage, number of infection eggs, infection masses and number of eggs in each mass were determined (Barnes et al., 1998).

Number of infected eggs I = ĜΙ ×100Ύ╢ Έ΄ Ŋėŋẃħ Total number of eggs

B) Rate of eyed eggs (Arndt et al.,2001):

Number of eyed eggs E = ×100 Total number of eggs- Primitive mortality

C) Hatching rate (Arndt et al.,2001):

Number of hatched eggs H= ĜΙ ×100Ύ╢ Έ΄ Ŋėŋẃħ Number of eyed eggs

D) Deformity rate: Finally, deformited larvae were determined 6 days after hatching. They were as fallow: Fry deformities included joined eggs, curved and bent spines and deformed yolk sacs.

Deformed larvae M= ĜΙ ×100Ύ╢ Έ΄ Ŋėŋẃħ Hatching eggs Proceedings of The Fourth International Iran & Russia Conference 1403

The SPSS software and one-way ANOVA were used to analysis the data and comparision of averages were taken by DUNCAN test.

Results: Physico-chemical parameters during incubation of eggs (November-December) are shown in table 1.

Table 1: Physico-chemicals parameters during incubation of eggs. Parameter Max Min Medium Dissolved oxygen 8.4 7.5 8.05 (D.O) Temprature (T°C) 11 8 8.8 PH 8 7.9 7.95 Hardness (mg/l) 112 142 132 Conductivity µs/cm 353 312 336

Infection, infected mass and the number of eggs in each mass in eyed egg stage were shown in Table 2.

Table 2: Infection, infected mass and the number of eggs in each mass Number of infected Average number of Treatments Percent of infection mass eggs in each mass Potassium permanganate 15 6.3 ±1.9 ab 4.2 (50 mg/l) Potassium permanganate 16 7±0.66ab 4.45 (100 mg/l) Malachite green 2 1.1±1.5a 5.4 Control 23 9.7±1.9b 4.3

Results by Duncan test shown significant difference between malachite green and natural control treatment but there is no significant difference between potassium permanganate and other treatments. The result of average percent of eyed egg in different treatments was shown in table 3.

Table 3: Average percent of eyed egg in different treatments Treatment Percent of eyed egg Potassium permanganate 67.6±3.4b (50 mg/l) Potassium permanganate 73.9± 0.86 a (100 mg/l) Malachite green 62.2±2.9b Control 64.6±0.3b

Duncan test indicated that the percentage of eyed egg increase in potassium permanganate by 100 mg/l concentration treatment and have significant difference in the other treatments, but there is no significant difference in the other treatments. Proceedings of The Fourth International Iran & Russia Conference 1404

Hatching period of eggs takes place 43 days and average percent of hatching in different treatments has shown in table 4.

Table 4: average hatching percent of treatments Treatment Percent of hatching Potassium permanganate 98±0.96a (50 mg/l) Potassium permanganate 98.5±0.45a (100 mg/l) Malachite green 98.2±1.1a Control 98.3±0.24a

Result of Duncan test indicate hatching percent have no significant difference in different treatments. At least, average percent of deformed larvae was determined 1 week after hatching that was shown in table 5. Deformed Rainbow Trout larvae were shown in figure 2.

Table 5: average percent of deformed larvae 1 week after hatching Treatment percent of deformity Potassium permanganate 0.09±0.085a (50 mg/l) Potassium permanganate 0.13±0.13a (100 mg/l) Malachite green 0.2±0.08a Control 0.1±0.14a

Although malachite green had most deformity percent, no significant difference was shown between treatments in Duncan test.

Discussion: Increasing aquatic fungal (Saprolegniacea) cause problems on farmed fish eggs (Schreier et al., 1996). However the sensitively of eggs to fungal disease depends on some things such as water quality, flow rate of water and density of eggs in incubators. Dead eggs and organic matter in aquaculture are suitable substances for growing fungal on them. One way to control of Saprolegniasis is using chemical drugs. But should be noticed that chemical drugs always be can’t used as the some way because their efficacy and toxicity varies by existing of organic matter and physico-chemical quality parameters of water (Rach et al., 1998). Chemotrophy by common drugs is a usual way to treatment this disease especially in eggs of fish and malachite green is one of the most important chemicals for treatment and prevention of disease. Although it is widely used for its suitable effects in Iran but it is prohibited in some countries because of its teratogenic, carcinogenic, toxic effects and environmental pollution (Marking et al.,1994). Proceedings of The Fourth International Iran & Russia Conference 1405

Permanganate is an active oxidant ion against parasites that kills them by oxidizing their cellular walls. Another reports indicate that magnate dioxide construct protein complex on the outer surface that caused brown color and producing this protein complex effect on respiratory structure of parasites and kill them (Anonymous, 1994). It used as a aquatic anti- fungal but it isn’t used anymore now. Potassium permanganate is toxic in high pH because magnate dioxide sediment on fish gills. So it can’t use in salt water effectively. It shouldn’t be also used combined with formalin (Noga, 2000). During treatments with potassium permanganate in 15 min bath dissolved oxygen and pH were measured every 5, 10 and 15 min and no significant difference was indicated. Results of potassium permanganate treatments for Rainbow trout eggs indicate that it has weaker effect than malachite green to prevent fungal infection but the rate of eyed egg in potassium permanganate treatments by 100 mg/l are more than all other treatments and have significant difference by other ones and it’s because of increasing bacteria density in eyed egg stage and so potassium permanganate increased the amount of eyed eggs by killing the bacteria on the eggs surface (Barnes et al., 2000). The rate of hatching and the rate of deformities have no significant difference between treatments.

Reference Arndt, E. R., Wagner, E. J and Routledge, M. D. 2001. Reducing or Withholding hydrogen peroxide treatment During a critical stage Rainbow Trout development: effects on eyed eggs, hatch, deformities, and fungal control, North American Journal of Aquaculture, 63:161-166. Azari takami, G. 1997. Health Management, Prevention and Treatment Methods of Fish Diseases, pub. parivar. p. 304. Barnes, M. E., Ewing, D. E., Cordes, R. J and Young, G. L. 1998. Observation on hydrogen peroxide control of Saprolegnia spp. during Rainbow Trout egg incubation, The Progressive Fish-Cuturist, 60: 67-70. Barnes, M. E., Gabel, A. C and Cordes, R. J. 2000. Bacterial population during Rainbow Trout egg culture in vertical-flow tray incubators, North American Journal of Aquaculture, 62: 48-53. Barnes, M. E and Stephenson, H. 2003. Use of hydrogen peroxide and formalin treatments durring incubation of Landlocked fall Chinook Salmon eyed eggs. North American Journal of Aquaculture, 65: 151-154. Barnes, M. E., Wintersteen, K., sayler, W and Cordes, R. 2000. Use of formalin during incubation of Rainbow Trout eyed eggs, North American Journal of Aquaculture, 62: 54-59. Bruno, D. V and Wood, B. P. 1994. Saprolegnia and other Oomycets. In Fish Disease, CABI pub. UK. pp. 599-659. Czeczuga, B and Godlewska, A. 2001. Aquatic insects as vectors of aquatic zoosporic fungi parasitic on fishes, Acta Ichthyol. Piscat, 2: 87-104. Czeczuga, B and Muszynska, E. 1999. Aquatic fungi growing on the eggs of fishes representing 33 cyprinid taxa (Cyprinidae) in laboratory conditions, Acta Ichthyol. Piscat, 2: 53-72. Gaikowski, M. P., Rach, J. J., Olson, J. J and Ramsay, R. T. 1998. Toxicity of hydrogen peroxide treatmentds to Rainbow Trout, Journal of Aquatic Animal Health, 10: 241-251. Gaikowski, M. P., Rach, J. J and Ramsay, R. T. 1999. Acute toxicity of hydrogen peroxide treatments to selected life stage of cold-, cool-, and warm water fish, Aquaculture, 178: 191-207. Howe, G. E., Gingerich, W. H., Dawson, V. K and Olson, J. J. 1999. Efficacy of hydrogen peroxide for treating saprolegniasis in channel cat fish, Journal of Aquatic Animal Healths, 11: 222-230. Proceedings of The Fourth International Iran & Russia Conference 1406

Jeffery, G. H.1989. Textbook of QualitatieCemical Analysis, 5th ED. pp. 272- 273. Jones, C. W. 1999. Application of Hydrogen Peroxide and Derivatives, RS. C, pub. UK. P.341. lilley, J. H and Inglis, V. 1997. Comparative effects of various antibiotic, fungicides and disinfectants on Aphanimyces invaderies and other saprolegniaceous fungi, Aquaculture Research, 28: 462-496. Marking, L. L., Rach, J. J and Schreier, T. M. 1994. Evaluation of antifungal agents for fish culture, The Progressie Fish-Culturist, 59: 225-232. Meyer, F. P., Yorgensen, T. A. 1983. Teratological and other effects of malachite green on development of Rainbow Trout and Rabbits, Trans Am Fish Soc, 112: 818-824. Noga, E. J. 2000. Fish Disease: Diagnosis and Treatment. Mosby-Yearbook, Inc, St. Louis, Mo. pp. 367. Piper, R. J., McElwain, I. B., Orme, L. E., McCraren, J. P., Flower, L. G and Leonard, J. R. 1992. Fish Hatchery Management, U S Fish and Wildlife service. pp. 517. Pottinger, T. G and Day, J. G. 1999. A saprolegnia parasitica challenge system for Rainbow Trout: assessment of pyceze as an anti-fungal agent for both fish and ova, Dis Aquat Org, 36: 129-141. Rach, J. J., Gaikowski, M. P., Howe, G. E and Schreier, T. M. 1998. Evaluation of toxicity and efficacy of hydrogen peroxide treatments on eggs of warm and cool water fishes, Aquaculture, 165: 11-25. Rach, J. J., Howe, G. E and Schreier, T. M. 1995. A miniature hatching system for evaluating chemical treatments on fish eggs. Elsevier Science, Vol. 29, No. 9, pp. 2103-2107. Rach, J. J and Ramsay, R. T. 2000. Analytical verification of waterborn chemical treatment regiments in hatchery raceway, North American Journal of Aquaculture, 62: 60-66. Sattari, M. Rousaii, M. 1999. Textbook of Fish Health (1) , Gilan University publication, p. 284. Proceedings of The Fourth International Iran & Russia Conference 1407 Comparison of sensitivity to oil pollution in two Caspian Sea zooplankton; Mnemiopsis leidyi (Ctenophora) and Acartia clausi (Crustacea)

Barazandeh M.1, Abtahi B2, Javanshir A.3, Esmaeili Sari A.4, Khodabandeh S.5

1- Marine Sciences Department, Natural Resources and Marine Sciences Faculty, Tarbiat Modarres University, Noor, Iran, phone: +98-1226253901, Email: [email protected]; 2- Marine Sciences Department, Natural Resources and Marine Sciences Faculty, Tarbiat Modarres University, Noor, Iran, phone: +98-1226253901, Email: [email protected]; 3- Caspian Ecology Research Center, Sari, Iran, , phone:+981523462498, Email: [email protected]; 4- Marine Sciences Department, Natural Resources and Marine Sciences Faculty, Tarbiat Modarres University, Noor, Iran; 5- Marine Sciences Department, Natural Resources and Marine Sciences Faculty, Tarbiat Modarres University, Noor, Iran and University of Montpellier II, France.

Abstract Mnemiopsis leidyi that belongs to the Phylum Ctenophora has caused many ecological problems after invasion into the Caspian Sea. Oil and gas extraction along with transportation and industrial production, bring air, water and soil pollution into the Caspian Sea region. Study on different species sensitivity to pollutants, is an important biological issue in the Caspian Sea. To this end, LC50 method with WAF (Water Accommodated Fraction) of crude oil was used in this experiment. The determined sensitivity by this method at the same conditions can be used to compare various species sensitivity to different pollutants and their survival rate and compatibility in the sea. So in this study the acute toxicity of the WAF of the crude oil to Mnemiopsis leidyi and Acartia clausi were determined and their LC50 for 96h were compared. The experiment was carried out in identical bottles containing 1litre 55 µm filtered seawater and there were 10 individuals in each bottle. Toxicity tests were carried out under static conditions and consisted of 3 stages (Survival test, lethal range determination and main toxicity test). After calculating the mortality during 96 h in the main toxicity test, LC50 for these 2 species were determined by Pharmacologic Calculation System software and the obtained results are: 3.311 and 0.148 ml/l for M.leidyi and A.clausi respectively. The results showed that Mnemiopsis leidyi seemed to be more resistant against WAF of the crude oil than Acartia clausi. This difference was probably caused mainly by the different composition of these 2 species structures. This study indicates that high resistance of Mnemiopsis leidyi to the marine pollutants in comparison with other marine zooplankton, may be the main result of its highly distribution in the Caspian Sea and it is strongly recommended that further studies on the tolerance of M.leidyi population to various marine pollutants including other oil products, heavy metals and etc. be carried out and also can be compared with other marine organisms.

Keywords: Caspian Sea, Crude oil, LC50, Mnemiopsis leidy, Acartia clausi.

Introduction Ctenophores are marine invertebrates that develop rapidly and directly into juvenile adults. They are likely to be the simplest metazoans possessing definitive muscle cells and are possibly the sister group to the Bilateria (Martindale and Henry, 1999). According to Boltovskoy(1999), Mnemiopsis leidyi belongs to the Class Tentaculata, Order Lobata and Family Bolinopsidae. In 1999, Mnemiopsis was first identified in the Caspian Sea, presumably after being introduced a few years earlier with ballast waters. The Caspian Sea is a completely isolated basin with mostly favorable conditions for Mnemiopsis development throughout the year. Mnemiopsis has expanded in the Caspian Sea, in 2001 at a rate sufficient to reach levels that could critically endanger the current function of the ecosystem and pose grave risks of extinction to a range of species, mainly invertebrates, and even fish (kilka and other species, including beluga sturgeon). Loss of Proceedings of The Fourth International Iran & Russia Conference 1408

biodiversity as well as economic loss are beginning to result there from (The first Mnemiopsis adversary group workshop, 2001). Mnemiopsis leidyi reproduces rapidly and has a high ability to adapt new situations and places. It can survive without any food for 3 weeks or even more by decreasing the body size. It can also live in many marine habitats with various salinities, temperatures and water qualities. However its ecological features may intensively vary in different ecosystems around the world (Esmaeili et al., 1380). The pollutants are enormously varied. The pollutants accumulation in marine organisms depends on ecological, physiological, chemical and biological characteristics of water which also consists of the organism position in the food-chain (Esmaeili, 1368). So entrance of industrial, agricultural and other pollution to the Caspian Sea is of great importance. For example the total Oil load to the Caspian Sea is 122350 t/y (CEP (1, 2), 2001). Oil and gas extraction, along with transportation and industrial production, are the source of severe air, water, and soil pollution in the Caspian region. Systematic water sampling in different parts of the Caspian basin showed contamination to phenols, oil products, and other sources. Mineral deposit exploration, particularly oil extraction and pipeline construction, have contributed to the pollution of about 30,000 hectares of land (Internet Search, URL: http://www.eia.doe.gov/emeu/cabs/caspenv.html, 2003). Iran has a small share from polluting point of view, but it gets a much extensive part of pollution created by other countries because of the sea currents in the Caspian Sea (Aghaei, 2002). Study of different species sensitivity to pollutants, is an important biological subject in the Caspian Sea to research and to this purpose we used LC50 method in this experiment. The determined sensitivity by this method at the same conditions can be used to compare various species sensitivity to different pollutants and as a result of it obtain their survival rate and competition ability in the sea. Whereas no significant study about the effect of these pollutions on Mnemiopsis leidyi has been done since its entrance to the Caspian Sea, it seemed necessary to know more about its resistance to Caspian Sea circumstances and the comparison with Acartia clausi which is both its main food and the most common pelagic invertebrate in the Caspian Sea at the present, was done at the same conditions for both of them as the same as their habitat’s.

Materials and Methods The Mnemiopsis leidyi and Acartia clausi were caught from the Caspian Sea at a distance of 3 to 4 Km from Khazar abad coast. A Plankton-net with a mesh of 500µ and a diameter of 54 cm was used to collect M. leidyi from a depth of 2 m from the surface area. The A. clausi were also caught form the surface water by a zooplankton-net with a mesh of 100µ. After being transferred to the laboratory, the individuals were kept at the sea water, at the same conditions as their habitat. The experiment containers consisted of 1.5 litre bottles containing 1 litre 55 µm filtered sea water. For the Survival experiment, 6 of them with 10 8-12 mm (most size frequency) M.leidyi in each 3 ones and 10 A.clausi in copepodity stage in the other 3 ones were used. The experiment time was 8 days (twice as the main experiment time) and the samples were checked out every 24 hours to determine their mortality. During the 8 days, the mortality should be less than 10% in the total 3 repetition. If it become more, the experiment conditions must be changed and then the survival experiment for another 8 days should be repeated. In the present study no mortality was observed between the M.leidyi individuals and the Acartia mortality rate was 6.6%. So we could continue the experiment at the same conditions. The second stage was determination of the lethal range which involves 2 concentrations of the poison, one of them the highest one with no observed mortality and the other the lowest one with 100% mortality. WAF of the crude oil was prepared according to the method of Anderson et al. One part of Aghajari crude oil was mixed with nine parts of filtered sea water in a specific container prepared Proceedings of The Fourth International Iran & Russia Conference 1409

for this purpose for 23 h, and then was left to settle for 1 hour. The aqueous phase was collected and used as the stock solution for subsequent experiments. Toxicity test was carried out under static conditions in which the test circumstances were the same during the whole experiment. The recently caught Mnemiopsis and Acartia were first placed in the filtered sea water for 24 hours to avoid their nutrition. Then they were assigned to prepared containers for the lethal range determination test. In this stage, 10 concentrations with 3 controls were used for each series of experiments. WAF used concentrations were 0.25, 0.5, 1, 2, 4, 8, 16 ml/l and 0.1, 0.25, 0.5, 1, 2, 4, 8 ml/l for M.leidyi and A.clausi, respectively. The animals were not fed during the experiment. The mortality was recorded daily. Physico-chemical parameters were also recorded in each container daily; temperature, salinity, pH dissolved Oxygen ranged from 18- 20ºC, 12.5-12.7 ppt, 8.38-8.48, 10.9-11.4 mg/L, respectively. After 96 hours, concentration ranges of CdSO4 and WAF for the final toxicity test were determined. These ranges were divided to 7 equal parts and then the animals were exposed to these 7 concentrations with 3 replicates for each of them by the same method as the lethal range determination (10 individuals in each container). 3 controls were also used for each experiment. Concentrations used for each experiment consist in: WAF for M.leidyi: 4, 3.5, 3, 2.5, 2, 1.5, 1 ml/l; WAF for A.clausi: 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 ml/l. The mortality rates were recorded every 24 hours and the final results were obtained after 96 hours. Each pollutant LC50 for Mnemiopsis and Acartia was earned by Probit Value Analysis and Pharmacologic Calculation System software, the charts were prepared and then the animals sensitivity were compared according to obtained LC50s.

Results Result of this experiments, showed that A.clausi is more sensitive to WAF of the crude oil than M.leidyi. Tables 1 and 2 contain the data of mortality caused by WAF of crude oil in M.leidyi and A.clausi in every 24 hours (charts 1 and 2). WAF LC50 for the under experiment animals were determined by Pharmacologic Calculation System software; also the upper and the lower confidence limits were obtained for both 2 species by the same software. For WAF of the Crude Oil the LC50s for 24, 48, 72 and 96 hours could be calculated. The results are seen in tables 3. By comparing both LC50s and their confidence limits, it is concluded that M.leidyi is more resistant to WAF of the crude oil than A.clausi. Also the chart 3 contains the same data. As shown there is no overlapping between standard errors of both species charts. So their differences are statistically meaningful.

Discussion

Toxicity tests The present study indicates that WAF of the crude oil could affect soon on both 2 species; the mortality was relatively high after the first 24 hours and it increased regularly over 96 h. Sensitivity of M.leidyi and A.clausi to toxicants The results show that Mnemiopsis leidyi seemed to be more resistant to WAF of the crude oil than Acartia clausi. This difference was probably caused mainly by the different composition of these 2 species structures. Some other comb jellies like Mnemiopsis mccradyi was used in acute toxicity testing of chemicals such as Mercuric chloride, Copper sulfate, etc; however, this is the first study on Mnemiopsis leidyi which expose petroleum hydrocarbons as a toxicant and to compare with zooplankton like Acartia clausi and it indicates that high resistance of Mnemiopsis leidyi to the marine pollutants in comparison with other marine zooplankton may be the main result of its highly distribution in the Caspian Sea. Proceedings of The Fourth International Iran & Russia Conference 1410

Conclusions This study is the first to quantify the toxicity of WAF of crude oil to Mnemiopsis leidyi and Acartia clausi. Also there were no definite experiments on Mnemiopsis leidyi resistance to environmental pollutions, especially in the Caspian Sea. So further studies on the tolerance of Mnemiopsis leidyi population to various marine pollutants including other oil products, heavy metals and etc. and comparison with other marine zooplankton are strongly recommended.

References -Aghaei Diba B (2002) Pollution in the Caspian Sea. http://www.payvand.com/news/02/jul/1073.html - Amini Rad A (1997) Determination of Diazinon’s Lethal Concentration(LC50) in Caspian Salmon (Salmo trutta caspius). Master thesis (Fisheries). Faculty of Natural Resources and Marine sciences, Tarbiat Modarres University. P: 112. (In Persian). - Bayrami A (1381) Study of Ion Regulation and Osmotic Pressure of Mnemiopsis leidyi. Master thesis (Marine Biology). Faculty of Natural Resources and Marine sciences, Tarbiat Modarres University. P: 45. (In Persian). - Boltovskoy D (1999) South Atlantic Zooplankton. Bckhuys publishers. Netherlands. Vol: 1: 561-573. -CEP1(Caspian Environment Programme) (2001, a) Pollution of the Caspian Sea (coastal and offshore industry). CRTC for pollution control. http://www.caspianenvironment.org/pollution/Coastal and Off-shore industry.htm. -CEP2(Caspian Environment Programme) (2001, b) Pollution of the Caspian Sea (Pollution sources), CRTC for pollution control. http://www.caspianenvironment.org/pollution/sources.htm. - Esmaeili A (1989) Cycle of Heavy Elements as Pb, Hg, Cd, …, Their Assimilation by Marine Organisms and their effects on them: 267-277. Articles Collection of the National Conference “Proper Use of Marine Resources of Persian Gulf and Oman Sea”. Fisheries Department, Tehran, Iran. - Esmaeili A, Abtahi B, Seifabadi J, Khodabandeh S, Talaei R, Darvishi F, Ershad H (2001) Invasive Comb Jelly, Mnemiopsis leidyi, and the Future of the Caspian Sea. Naghshe Mehr publications: 8-13. (In Persian). - Esmaeili A, Seifabadi J, Khodabandeh S, Abtahi B, Talaei R (2000) Study of Nutrition of the Caspian Sea Ctenophore (Mnemiopsis leidyi). Daneshvar Journal 31: 139- 144. (In Persian). - Fathollahi B (1998) Determination of Endosulfan’s Lethal Concentration (LC50) and its Effects on Common Carp (Cyprinus carpio). Master thesis (Fisheries). Faculty of Natural Resources and Marine sciences, Tarbiat Modarres University. P: 101. (In Persian). - Fini DJ (1964) Statistical Methods in Biostatistics. Translated by: Nahaptian V, Malek Afzali H. Tehran University Publication (Iran, Tehran): 439-450. (In Persian). -Henry J, Martindale MQ (2001) Multiple Inductive Signals Are Involved in The Development of the Ctenophore Mnemiopsis leidyi. Developmental Biology 238:40-46. - Javadi M (1999) Determination of Endosulfan’s Lethal Concentration (LC50) and its Effects on Huso huso. Master thesis (Fisheries). Faculty of Natural Resources and Marine sciences, Tarbiat Modarres University. P: 67. (In Persian). -Jeffress D, Frank S (1990) Common Jellyfish of the Mid. Atlantic. Underwater naturalist.Vol:19. No: 2. http://www.littoralsociety.org/jelly.htm. - Kasimov AG (2001) New Introduced Species in the Caspian Sea, Mnemiopsis leidyi. First International Meeting “The Invasive of the Caspian Sea by the Comb jelly, Proceedings of The Fourth International Iran & Russia Conference 1411

Mnemiopsis Problems, Perspectives, Need for Action”, 24-26 April, 2001, Baku, Azerbaijan. - Labunska I, Stringer R, Brigden K (2000) Metal and organic pollution associated With the Bayer facility in Nova Iguaçu, Rio de Janeiro, Brazil. Greenpeace Research Laboratories. Department of Biological Sciences. University of Exeter,Exteter UK: 46-48. - Lee WY, Nicol JAC (1997) The Effects of the Water Soluble Fractions of No.2 Fuel Oil on the Survival and behavior of Coastal and Oceanic Zooplankton. Environmental Pollution. V: 12. Issue: 4: 279-292. - Long S, Douglas AH (2001) Acute toxicity of crude and dispersed oil to Octopus pallidus hatchings. Water Research. Vol: 36. No: 11: 2769-2776. - Martidale MQ, Henry JQ (1999) Intracellular Fate Mapping in a Basal Metazoan, the Ctenophore Mnemiopisis leidyi, Reveals the Origins of Mesoderm and the Existence Of Indeterminate Cell Lineages. Developmental Biology. V: 214: 243-257. - Mellanby K (1961) Pollution threat of heavy metals in Aquatic environments. Monkwood Experimental Station. Abbots Ripton. Huntington. P: 437. - Movahedinia A, Esmaeili A, Abtahi B, Ershad H (2002) Study of seasonal Density And Biomass changes of Mnemiopsis leidyi Along the Southern Beach of the Caspian Sea (Noor). Marine Sciences Journal (Iran,Tehran) 3 : 57-64. (In Persian). - Reeve MR, Grice GD Gibson VR, Walter MA, Darcy K, Ikeda T (1976) A Controlled Environmental Pollution Experiment (CEPEX) and its Usefulness in the Study of Larger Marine Zooplankton under Toxic Stress. Orme S, Kegley S. (Ed). Effects of Pollutants on Aquatic Organisms. Vol: 2:145-162. - URL: http://www.eia.doe.gov/emeu/cabs/caspenv.html (2003) Caspian Sea Region: Environmental Issues. -Zamini A (1996) Determination of Pb and Cd’s Lethal Concentrations (LC50) on Amur (Ctenopharyngodon idella) and Phytophag (Hypophthalmichthys molitrix). Master Thesis (Fisheries). Lahijan Azad University. P: 57. (In Persian).

Tables Legends

Table 1. Total mortality rate of M.leidyi exposed to WAF of crude oil in all 3 repetition minus the controls mortality. Hours

WAF 24h 48h 72h 96h concentrations (ml/l) 1 3 4 4 5 1.5 3 4 4 5 2 5 8 9 9 2.5 7 13 12 12 3 12 14 13 13 3.5 12 16 14 14 4 14 15 18 20

Table 2. Total mortality rate of A.clausi exposed to WAF of crude oil in all 3 repetition minus the controls mortality. Proceedings of The Fourth International Iran & Russia Conference 1412

Hours

WAF 24h 48h 72h 96h concentrations (ml/l) 0.1 3 5 11 12 0.2 3 11 17 18 0.3 7 12 20 20 0.4 11 14 20 20 0.5 11 17 20 21 0.6 14 19 24 24 0.7 14 19 23 24

Table 3. Crude Oil LC50 M.leidyi A.clausi Lower Upper Lower Upper LC50(ml/l) LC50(ml/l) C.L. C.L. C.L. C.L. 24h 4.633 3.677 5.837 0.77 0.578 1.024 48h 3.5 2.85 4.3 0.401 0.301 0.532 72h 3.466 2.832 4.241 0.167 0.117 0.236 96h 3.311 2.676 4.098 0.148 0.104 0.212

Figure Legends

25

20 24h 15 48h 72h 10 96h Mortality Rate Mortality 5

0 1 1.5 2 2.5 3 3.5 4 ml/Lit Oil Co nce ntratio n( )

Figure 1. Total Mortality Rate of Mnemiopsis leidyi Exposed to WAF of the Crude Oil in all 3 repetition. Proceedings of The Fourth International Iran & Russia Conference 1413

30

25 24h 20 48h 15 72h 96h 10

Mortality R ate 5

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 ml/Lit Oil Co nce ntartion( )

Figure 2. Total Mortality Rate of Acartia clausi Exposed to WAF of the Crude Oil in all 3 repetition.

6

5

0 4

M.leidyi 3 A.clausi (ml/Lit)

Crude oil LC5 2

1

0 24h 48h 72h 96h

Figure 3. Crude Oil LC50 Proceedings of The Fourth International Iran & Russia Conference 1414

Fish-eating bird's diversity of south coast of the Caspian Sea

Dr. Behrouz Behrouzirad 1-Assist. Prof. ,Faculty of Natural Resources and Marine Science. Tarbiat modarres university . E-Mail: [email protected]

Abstract There are 61 species of 14 families of fish-eating birds in Iran. The fish-eating birds of the Iranian coast in Caspian Sea belong to 41 species in 10 families, which include 61 percent of total fish eating bird species and 70 percent of bird family of Iran. Resident birds constitute 13 percent of the fish -eating birds of the Caspian Sea coast and the rest are migratory in autumn and winter. 3 threatened species of fish-eating birds exist at the Iranian coast of Caspian Sea, which are Oxyura leucocephala, Phalacrocorax pygmeus, and Pelecanus crispus wintering in the south coast wetlands of the region. 17, 5 and 78 percent of the fish- eating birds of the Iranian coast of Caspian Sea are aquatic, terrestrial and wadding respectively. The Phalacrocorax carbo is the wintering and breeding migratory in the wetlands of the region, which feed in the fishponds. The main habitat of these birds is Miankaleh and Amirkelayeh wildlife refuge, Anzaly marsh and Mouth of Sefidrood River of the Ramsar Site. Oxyura lecucephala, Phalacrocorax pygmeus and Pelecanus crispus are protected species and the rest are not allowed to be eaten in accordance with Islamic canons, for the same reason man is not threatening them.

Keywords: Breeding, Caspian Sea, Fish-eating bird, Wintering, Iran.

Introduction South coast of the Caspian Sea from Astara in west to Gomishan to east possesses suitable habitats for fish-eating birds. Fresh, brackish and salt marshes with permanent water and rivers, estuaries and good condition of weather during four seasons provide desirable circumstance for tow-third of resident or migratory fish-eating birds observed in Iran. Main habitats of these birds are Miankaleh and Amirkelaieh wildlife refuge, Anzaly marsh, mouth of Sefidoord, Ramsar sites, Alalan, Ramsar wetland, Rivers, Siahkeshim protected area and Selkeh wildlife refuge. Key sites for surviving of fish- eating birds are those habitats that abound with fish.

Materials and Methods Identification of fish-eating birds of the Iranian coast of the Caspian Sea has been carried out for several years using binocular and telescope and some species of the birds have been shot.

Result Forty-one species of fish eating birds belonging to 10 families have been identified along the Iranian coast of Caspian Sea (Table 1). Fish-eating birds live in marshes, wetlands, rivers mouth and seacoast. There are the following three groups of fish- eating birds at Caspian coast, (Fig 1 and 2): 1.1- Migration-based grouping a- Migratory breeders: Fish eating birds migrate to the region in spring to breed in wetlands, marshes, and Ab- Bandans (Man made wetlands). Caledonians hybrid and Sterna albifrons represent this group. b- Wintering Proceedings of The Fourth International Iran & Russia Conference 1415

These birds migrate to the region in autumn and winter. The Saw-bill Ducks represent this group. c- Residents Some species of fish-eating birds are resident of the Iranian coast of the Caspian Sea. Thachybaptus ruficollis and Egretta garzetta are represent this group. 1.2- Habitat preference- based grouping Habitat preference-based of fish-eating birds of the Iranian coast of the Caspian Sea are divided into the three following groups, (Fig 3): a- Terrestrial species These species live on land and are not swimmers, they only depend on water bodies for feeding. Alcedenidae species represent this group. b- Wadding birds: These species live along the edges of water bodies, but do not swim. Egretta alba and Egretta garzetta represent this group. c-Aquatic birds: These swimming species feed in various water bodies. Phalacrocorax carbo and Podicepcidae species represent this group. 1.3- Population based grouping Based on population fish-eating birds of the Caspian coast can be divided into the following tow groups, (Fig. 4) a-Threatened species: These species are threatened and their population is very low in the region. They have been listed on Red Data Book of IUCN, 2004 Dalmation pelican Pelecanus crispus, Pygmy Cormorant Phalacrocorax pygmeus and White-headed Duck Oxyura leucocephala belong to this group. b-Common species: Population of these species is common in the region. Herons Ardeidea species represent this group. 1.4- Based on feeding behavior Based on feeding behavior Fish- eating bird are divided into the following four groups (Fig 5): a- Group moving, moving feeders, these birds move in group and feed while moving. Cormorant Phalacrocorax carbo represent this group. b- Individual, moving and still- feeder, these birds move individually and feed while still, Great White Egret Egretta Alba and Grey Heron Ardea cinerea represent this group. c- Combined moving and feeding behavior, these species show a combined feeding and moving behavior as the earlier two categories. Ardeidae species represent this group. d- Individual moving and moving feeder, these birds move individually and feed while moving, White Headed Duck, Oxyura leucocephala represent this group. 2- Sensitive habitats Caspian shoreline, Miankaleh, and Gorgan Bay wildlife refuge, Ajygol, Olmagol, Alagol, mouth of rivers, Ab-Bandans (man made wetlands) in Mazandaran province, Anzaly marsh, Siahkeshim protected area, Selkeh and Amirkelaieh wildlife refuge, mouth of sefidrood river Ramsar site, Abas-abad marsh in Gilan province are regarded as sensitive area for fish-eating birds.

Discussion Fourteen fish-eating birds family exist in Iran that constitute about 13 percent of bird species in the country (Behrouzirad, 1997). Ten families including 41 identified fish-eating bird species exist along the Iranian coast of the Caspian Sea, which constitute 67 percent of total Proceedings of The Fourth International Iran & Russia Conference 1416 fish-eating and 8 percent of total bird species observed in Iran. Two species Alcedo atthis, a resident and Pandion haliaetus a winter migratory are terrestrial birds, but feed on fish. Eight species of Herons live along the margin of water bodies, of which Ardea cinerea, Egretta Alba, Egretta garzetta and Nycticorax nycticorax can be observed throughout the year and breed with Cormorants in Alalan and Ramsar. Breeding population of all above mentioned species were disturbed in Abas-abad marsh due to heavy deforestation. Their breeding is currently restricted to Alalan and Ramsar with very limited breeding in Anzaly marsh. The Podicepcidae species are common wintering birds in Anzaly marsh, Amirkelaieh and Miankaleh, but the Tachybaptus ruficollis breed in Ab-bandans, Anzaly marsh and Miankaleh. About 15000 pairs of Phalacrocorax carbo with Ardeidae species were reportedly bred in Abas-abad and nearby marshes during 1970s (Behrouzirad, 1978), but the number is limited to 5000 pairs in Ramsar and Alalan. All Gull species Larus sp. are wintering birds throughout the Caspian region of Iran. Two Tern species Sterna albifrons and Chlidonias hybrida breed in the varius wetlands of the region. Bitren Butarus stelaris is rare species that breed in reed-beds of the region. Pygmy Cormorant Phalacrocorax pygmeus regard as a threatened species (IUCN 2002) used to breed in Anzaly marsh during 1970s (Scott, 1970), but no longer found to breed in the area, although limited wintering is reported. Two species of Skua, Stercoraridae, Stercorarius pomarinus and Stercorarius parasiticus and two species of Divers, Gavidae Gavia arctic and Gavia stelata are only observed in the Caspian Sea and not in the wetlands. No information on population status of these species is currently available. Abundance and diversity of fish-eating birds in various habitat of the Caspian region depend on the security and food abundance. From breeding point of view Anzaly marsh, Ramsar and Alalan are regarded as sensitive areas and from wintering point of view; Miankaleh, Anzaly and Amirkelaieh are regarded as sensitive areas.

Reference 1- Behrouzirad, B.(1984): Feeding behavior of Cormorant in fish pond in Khozestan province ( in Farsi)- unpublished report,15 pp. 2- Behrouzirad, B.( 1977-1991): Pelecans ringing report in Uromieh Lake (in Farsi), unpublished reports . 3- Behrouzirad, B.(1977-1979): Cormorants ringing report in Gilan and Mazandaran Province (in Farsi),unpublished reports. 4- Behrouzirad, B.(1977-1984): Terns ringing reports in Persian Gulf ( In Farsi), unpublished reports. 5- Behrouzira, B. (1987): Dalmation Pelecan as a Threatened species ,(in Farsi), seasonal Journal of Environment,D.O.E.Spring 1997, 6- Behrouzirad, B. (1997): Animal migration,(in Farsi),Seasonal Journal of Environment, D.O.E.Winter 1997. 7- Barber ,S,G.M.&T.J.Wassenorey (1989): Feeding ecology of the picivorus birds ,Marin Biology,1989,Astralia. 8- Carss,D.N.& M.Marqivs (1991): Avian peredation at farmland and natural fisheries ,Institute of terrestrial ecology, Scotland. 9- Doorbons,J. (1984): Picivorus birds on the saline lake, Journal of the sea research group, The Netherlands. Proceedings of The Fourth International Iran & Russia Conference 1417

Table No. 1 Fish eating birds of south Caspian coast

IUCN Sientfic name Family National law Migration Pupulation criteria Tachybaptus Podicepcidae Non protected - Resident Common ruficollis Podiceps nigricollis Podicepcidae Non protected - Resident Common Podiceps auritus Podicepcidae Non protected - Wintering Common Podiceps grisegena Podicepcidae Non protected - Resident Common Podiceps cristatus Podicepcidae Non protected - Resident Common Botarus stellaris Ardeidae Non protected - Resident Common Ixobrychus Ardeidae Non protected - Resident Common minutus Nycticorax Ardeidae Non protected - Resident Common nycticorax Ardeola ralloides Ardeidae Non protected - Resident Common Egretta alba Ardeidae Non protected - Resident Common Egretta garzetta Ardeidae Non protected - Resident Common Ardea cinerea Ardeidae Non protected - Resident Common Pelecanus Pelecanidae Non protected - Wintering Common onocrotalus Pelecanus crispus Pelecanidae Protected Vu Wintering Rare Larus argentatus Laridae Non protected - Wintering Common Larus genei Laridae Non protected - Wintering Common Larus ridibundus Laridae Non protected - Wintering Common Larus minutus Laridae Non protected - Wintering Common Larus marinus Laridae Non protected - Common Common Larus canus Laridae Non protected - Wintering Common Larus fuscus Laridae Non protected - Wintering Common Chlidonias hybrida Laridae Non protected - Breeding Common Proceedings of The Fourth International Iran & Russia Conference 1418

Table 1: continued IUCN Sientfic name Family National law Migration Pupulation criteria Chelidonias Laridae Non protected - Wintering Common leucopterus Sterna hirundo Laridae Non protected - Breeding Common Sterna albifrons Laridae Non protected - Breeding Common Gelochelidon Laridae Non protected - Wintering Common nilotica Hydroprogne Laridae Non protected - Wintering Common tschgrava Chelidonias nigra Laridae Non protected - Wintering Common Sterna sandivensis Laridae Non protected - Wintering Common Phalacrocorax carbo Phalacrocoracidae Non protected Wintering Common Phalacrocorax Phalacrocoracidae protected Vu Wintering Rare pygmeus Pandion haliaetus Alcedenidae protected - Wintering common Alcedo atthis Anatidae Non protected - Resident Common Oxyura leucocephala Anatidae protected Vu Wintering Rare Rare in Mergus merganser Anatidae Non protected Wintering Rare in Iran Iran Rare in Mergus serattor Anatidae Non protected Wintering Rare in Iran Iran Rare in Mergus albelus Anatidae Non protected Wintering Rare in Iran Iran Stercorarius Rare in Stercoraridae Non protected Wintering Rare in Iran pomarinus Iran Stercorarius Rare in Stercoraridae Non protected Wintering Rare in Iran parasiticus Iran Rare in Gavia arctic Gavidae Non protected Wintering Rare in Iran Iran Rare in Gavia stelata Gavidae Non protected Wintering Rare in Iran Iran

Table. 2 breeders of fish eating birds of Caspian coast. Breeding sites Name Alalan marsh, Abas abad marsh before 1980s , Phalacrocorax Carbo Ramsar marsh Anzaly marsh before 1970s. it isnot breed now Phalacrocorax pygmeus Anzaly marsh ,Khoshkeh daran wood wetland Ardea cinerea before 1990s Alalan marsh, Ramsar marsh Anzaly marsh, Khoshkeh daran wood wetland Egretta alba befor 1990s, Alalan marsh, Ramsar marsh Anzaly marsh, Khoshkeh daran wood wetland Egretta garzetta before 1990s,Alalan marsh, Anzaly marsh, Khoshkeh daran before 1990s, Nycticorax nycticorax Alalan marsh,Ramsar marsh Anzaly marsh Thachybaptus ruficollis Proceedings of The Fourth International Iran & Russia Conference 1419

Anzaly marsh Ixobrychus minutus Miankaleh Sterna albifrons Anzaly marsh, Ab-bandans of Mazandaran. Chlidonias hybrida

Fig 2-Relative seasonal Fig 1- Relative temporal aboundance of migratory Fish- aboundance of Fish-eating birds eating birds resident Breeding 31% 39% migrator Wintering y 61% 69%

Fig 4-Aboundance percentage of Fig 3-Relative aboundance of Fish-eating birds population Fish-eating birds on habitat preference Shoreline Rare 29% 17% Common 71% Water 78% Terresterial 5%

Fig 5- Aboundance percentage of Fish- eating birds feeding behavior individualy. Group Still moving 15% 58%

Individualy Individualy, moving moving ,still 20% 7% Proceedings of The Fourth International Iran & Russia Conference 1420

Pain sensitivity of fishes and analgesia induced by opioid and nonopioid agents

Lilia S. Chervova1, Dmitii N. Lapshin2. 1- Department of Ichthyology, Moscow State University, Moscow, Russia. Phone: +007(095)9394092. Email: [email protected]. 2 - Institute for Information Transmission Problems, Moscow, Russia. Phone: +007(095)9523303 Email: [email protected]

Abstract. Experiments were performed on carp Cyprinus carpio, rainbow trout Oncorhynchus mykiss, cod Gadus morhua and sturgeon Acipenser ruthenus. An originally designed optico-mechanical system was used to record the response to painful electrical stimulation. before and after administration of analgetic agents. Drugs used were mu agonists tramadol, dermorphine and beta-casomorphine, kappa agonist U-50488, delta agonist DADLE, and nonopioid agents sydnophenum, analginum, novocainum. Drugs were administrated by different ways - peritoneally, subcutaneously, intranasally. Administration of drugs produced dose-dependent and lasting for at least 1 h increase of NT in 1.5-3 times. In rainbow trout, intranasal administration of dermorphine 0.20-0.75 mg/kg caused a concentration-dependent decrease in the pain sensitivity by 12-55%. The analgesic effect was usually observed within 10 min after administration and it lasted for at least 1 h (up to 2-3 h in some fish). In cod, intranasal administration of beta-casomorphine 2.5-12.5 mg/kg and peritoneal one 10-30 mg/kg decreased the pain sensitivity by 15-37% and 14-35%, respectively. In carp, nociceptive thresholds significantly increasd following the intramuscular injection of agonists mu, delta, and kappa opioid receptors, tramadol 10-100 nmol/g, DADLE 10-50 nmol/g, and U-50488 30-80 nmol/g, respectively. Antinociceptive effects of opioid agents were blocked or significantly reduced by pretreatment with naloxone. In cod, injected peritoneally sydnophenum 15-100mg/kg decreased the pain sensititivity by 15-89%. Intraperitoneal injection of 50% solution of analginum 0.5-2.5 ml/100g and subcutaneous one 0.25-1 ml/100g decrease the pain sensitivity by 16-21% and 29-45%, respectively. Local subcutaneous injections of 2% solution of novocainum blocked the nociceptive reactions. Stress significantly reduced nociceptive responses.

Key Words: analgesia, fish, nonopioids, opioids, pain.

Introduction Growth of the role of fish farm industry in the food yield of people requests the knowledge of biology and physiology of subjects of aquaculture. In fishes, the system of pain sensitivity and behavior induced by aversion stimuli are almost never investigated. The information available is fragmentary and scarce. The sensation of pain as a factor for protection has been formed during natural salection from the fundamental property of animals - to identify unfavorable external stimuli and respond to them. This property is inherent, to a certain degree, in representatives of all phylogenetic (Kavaliers, 1988). Pain as a type of sensory modality is interpreted as several complex physiological processes: information, sensation, emotion, or neurophysiological phenomenon; therefore, neigher the nature of pain, nor the range of sensations involved in this concept has a definite determination, even for people. Especially pain is difficult for study and analysis in nonmammalian and voiseless species. However, the main anatomical, physiological, and biochemical components of nociception in fish are similar to those in mammals. Pain receptors in fish, i.e. nociceptors, as with other vertebrates, represent free nerve endings of spinal (on the trunk) and trigeminal (on the head) nerve fibers, are localized over the entire body surface including fins (Chervova et al., 1992; Chervova et al., Proceedings of The Fourth International Iran & Russia Conference 1421

1994; Chervova, 1997). Recently, the basic part of antinociceptive system (mu, delta and kappa opioid receptors) was found also in the brain of some species of fish. Although the monitoring and estimations of analgesic efficacy of drugs in fishes is of important value, no criteria have been established for the study of their nociception (pain sensation) and analgesia.

Materials and Methods Experiments were carried out on carp Cyprinus carpio weighting 50-100 g, rainbow trout Oncorhynchus mykiss (200-400 g), cod Gadus morhua (100-300 g) and sturgeon Acipenser ruthenus (60-70 g). For the investigation of responses of fish to painful stimuli, an originally designed optico-mechanical system was elaborated on based on the motor-locomotory reaction aimed at the removal of aversive stimulus. The fish was semirigidly fixed in a flow chamber (in the region of the mouth and pectoral fins). The gills were continuously moistened with water. The stimulating electrodes were inserted into the caudal fin blade in order to exclude the direct stimulation of muscle fibers. The recording apparatus was a movable wire “fork” embracing the caudal peduncle in the posterior third of the body. In response to painful stimulation (bursts of short pulses 0.5 ms of current 0.5-2.0 mA, with frequency 300/s), the fish moved its caudal peduncle and deviated “the fork” from the zero point. The stimulation and registration of the locomotor reaction were made by computer control, the recording system was synchrinized with the painful (nociceptive) stimulus. Amplitude and duration of locomotor response was visualized on display (Fig. 1). The setup allowed to mesure the nociceptive thresholds (NT) to an approximation of 10%. NT were measured at 5-min intervals for 1 h before and 1-2 h after administration of analgesic agents. Drugs used were mu agonists tramadol, dermorphine and beta-casomorphine, kappa agonist U-50488, delta agonist DADLE, and nonopioid analgesic agents sydnophenum, analginum, novocainum. Sodium chloride (0,9%) served as the solvent and a control solution. Drugs were administrated by different ways - peritoneally, subcutaneously, intranasally. The data were processed using the Wilcoxon-Mann-Whitney test.

Results It was found that the caudal, dorsal and pectoral fins, the skin surface around the eyes, and the epithelium of olfactory sacs were the most sensitive nociceptive zones; the skin on the head and trunk was less sensitive. The fishes reacted to nociceptive stimuli with a jerk of a tail. The response was the highest to the first of the applied stimuli. To Fig. 1. (a) A scheme of an electron opticomechanical system for the measurement of nociceptive thresholds in fish. (b) An example of fish response to a painful stimulus as displayed on the monitor. Designations: 1, a chamber with current water; 2, sensor; 3, analog-to-digital converter; 4, computer; 5, stimulator; 6, amplifier; 7, electrodes; 8, the monitor screen; 9, period of stimulation (s); i, the amplitude of electric stimulus (mA).

the following stimuli the responses were less intensive but, as a rule, were stable for two hours Proceedings of The Fourth International Iran & Russia Conference 1422 or longer. Administration of drugs produced dose-dependent and lasting for at least 1 h increase of NT in 1.5-3 times. In cod, injected peritoneally sydnophenum 15-100 mg/kg decreased the pain sensititivity by 15-89%. Intraperitoneal injection of 50% solution of analginum 0.5-2.5 ml/100g and subcutaneous one 0.25-1 ml/100g decrease the pain sensitivity by 16-21% and 29-45%, respectively. Local subcutaneous injections of 2% solution of novocainum blocked the nociceptive reactions. Stress significantly reduced nociceptive responses in fish. In rainbow trout, intranasal administration of mu opioid agent dermorphine 0.20-0.75 mg/kg caused a concentration-dependent decrease in the pain sensitivity by 12-55%. The analgesic effect was usually observed within 10 min after administration and it lasted for at least 1 h (up to 2-3 h in some fish). In cod, intranasal administration of beta-casomorphine 2.5-12.5 mg/kg and peritoneal one 10-30 mg/kg decreased the pain sensitivity by 15-37% and 14-35%, respectively. In carp, nociceptive thresholds significantly increased following the intramuscular injection of agonists mu, delta, and kappa opioid receptors, tramadol 10-100 nmol/g (Fig. 2), DADLE 10-50 nmol/g, and U-50488 30-80 nmol/g, respectively. The result obtained indicate that individual nociceptive thresholds in intact fish ranged within 10% (p<0,01) and remainded stable for 1-2 h or even longer. Five to fiteen minutes after the administration of tramadol, changes in fish sensitivity to painful stimuli were observed. The analgetic effect was dose-dependent; the higher the dose, the more quickly it acted (Fig. 2). In some experiments, the overall time of analgesia was more than 2 h. The lack of responses to increasing pain could not be blamed on tramadol immobilizing the fish, because the same fish placed into an aquarium showed normal swimming and behavior. Administration of the highest concentrations of tramadol (100-50 nmol/g ) elicited the decrease of the latency for first 10 min, than latency was increased. Tramadol had no analgetic effect if naloxone, an antagonist of opioid receptors, was administered before (p<0.05). After the administration of a control solution fish showed no changes in their

Fig. 2. Changes in the nociceptive thresholds of carp to painful stimuli after administration of either tramadol solutions (1-5: 100, 80, 50, 30, and 10 nmol/g, respectively) or 0,9% NaCl (6, control). All experimental values differ significantly from control values (1-5: P<0,001, <0,001, <0,001, <0,01, <0,05, respectively). Abscissa shows the time after injection of a solution; ordinate shows the degree of analgesia (A). 0,8 100nmol/g f 0,6 80nmol/g

/lgT 50nmol/g

i 0,4 30nmol/g 0,2 lgT 10nmol/g Analgesia 0 0,9%NaCl 0 20 40 60 80 100 min

Fig. 3. Changes in the latency of the nociceptive responses of carp to painful stimuli after administration of either tramadol solutions (1-5: 100, 80, 50, 30, and 10 nmol/g, respectively). Proceedings of The Fourth International Iran & Russia Conference 1423

f 0,4 100 nmol/g 0,3 /lgL

i 80nmol/g 0,2 50nmol/g 0,1 30nmol/g 0 10nmol/g

Latency lgL Latency -0,1 0 20406080100min

response to stimuli (p<0.01). Preliminary experiments have shown that sturgeon Acipenser ruthenus possess of nociception as well as bony fishes. Its reacted to painful electrical stimuli with the same behavior – a jerk of the tail. Their nociceptive thresholds were comparable to that of carp. The pattern of response was the same after administration of the 100 nmol/g tramadol solution to carp and sturgeon. However, the period of recovery in sturgenn lasted more than in carp, for at least 3-5 days – fish demonstrated slowly swimming and prefered to lie at the bottom.

Discussion Studies performed on Cyprinus carpio, Parasalmo mykiss, Gadus morhua, and Acipenser ruthenus indicated that the fishes possess a developed system of pain sensitivity with receptors (nociceptors) presented on the whole body. The most sensitive to noxious stimuli in these species were the blade of the caudal fin, dorsal and pectoral fins, skin around eyes, and epithelium of the olfactory sac; the skin of the head and body surface was less sensitive. NT of fish under this condition was comparable with human’s one.The high density of nociceptors on fins is likely to be related to the fact, in particular, that fins are damaged in fish during their nest-building activity or agressive interactions (Lorenz, 1984). The information from nociceptors comes to the neurons of the spinal cord or the trigeminal caudal nucleus of the brain stem; then, it is distributed in the central nervous system to provide for the formation of a rapid behavioral response. In fishes as in other vertebrates, the behavioral defensive response to a painful stimulus is manifestated – movement aimed to the removal of the aversion sensation. In terrestrial animals this may be the whithdrawal of a limb, the drawing in of the tail or the body, escape, jumping ets. Fish attempts to swim away starting with movement of caudal peduncle. Processes that occur in the central nervous system in responce to nociceptive stimulation have not been studied in fish. However, substance P, a neuropeptide that is a mediator of nociception in mammals, was found in the peripheral nerve endings of Gadus morhua and Parasalmo mykiss (Johnsson et al., 1998), and in different regions of the brain Salmo salar (Vecino and Ecstrοm,1991). In the spinal cord and trigeminal ganglion of Petromizon marinus, neurons were found that respond by pulsed discharges to a strong squeezing of skin that leaves traces of pinching, punctures with an acute needle, and cauterization up to appearance of white Matthews and Wickelgren, 1978). Behavioral responses that in mammals are considered to be markers of primary pain sensitivity and are controlled, as is supposed, by the limbic system are primary motor start responses, a simple nonspecific avoidance (Wall, 1992). These reactions are observed also in fish in response to pain stimuli (Rekubratskii, 1967; Jansen and Green, 1970; Sickarulidze and Kadagishvili, 1974; Ehrensing et al., 1982). In mammals, nociceptor stimulation is Proceedings of The Fourth International Iran & Russia Conference 1424 accompanied by an affective response, i.e., vocalization. Special sounds that are emitted at wounding were also recorded in Misgurnus fossilis, these sounds were generated by the swimming bladder and were characterized by a frequency spectrum of 0 to 4000 Hz with maxima of 500, 1500, and 3000 Hz and continued for 490 ms, on average. According to these parameters, this pain “shout” differed from sounds that accompanied food seizure, feeding, or spawning behavior (Nikol’skii et al., 1968). In bony fish the conditioned reflex of avoiding the aversive stimulus is easily formed if rapid impetuous swimming is peculiar to these fish in nature. Thus, Trachurus mediterraneus ponticus was taught to avoid a light signal that was followed by a pain electric stimulus after 12 to 13 combinations. In slow-moving fish, "ambuscaders", as with Neogobius melanostomus and Symphodus roissali, a reflex of avoidance failed to form in response to an conditional stimulus. In response to an electric stimulus, these fish shuddered, performed a short dart aside, and came to a standstill. They responded to the subsequent pain stimuli by an increase in respiration rate (Rekubratskii, 1967). There are facts causing doubts as to whether there is any pain sensitivity in fishes, e.g., a shark’s devouring its own viscera falling out of its open belly. Probably, in hungry animals, as it was shown in mammals, the feeding motivation may be so strong that a functional blockade with a feeding excitation of the mechanisms to the central structures takes place. Increasing of the pain threshold seem to be adaptive, permitting the animals in case of need to perform the vital requirements (foraging or defensive) in spite of damaging stimuli. Our results indicate that, like higher vertebrates, fish also develop a prolonged analgesia in response to agonists of the opioid mu receptors. Hence, fish have an antinociceptive system consisting of the opioid receptors similar to those in terrestrial vertebrates. The opioid receptors were first found in mammals and shown to mediate the effects of morphine and its derivates (analgesia, addiction, etc.). They are also targets for endogenous opioid peptides: enkephalins and endorphins (Dhavan et al., 1996). Opioid receptors were later found in lower vertebrates. In the bony fish Catostomus commersony and Brachydanio rerio, cDNA for opioid receptors were isolated to clone the mu, delta, and kappa receptors (G proteins). All fish’s and mammalian’s opioid receptors showed a high degree of amino acid homology (Darlison et al., 1997; Barallo et al., 1998). In mammals, the analgesic effects are primarily mediated through mu opioid receptors. Our results indicate that in fish, the same receptors are responsible for increasing the pain threshold. The decrease in pain sensitivity under the action of nonopioid preparations analginum and sidnophenum as well as analgesy caused by stress, illustrates the presence in fishes of other endogenous analgesic systems in addition to the opioid system. It is reasonably safe to suggest that fish similar to higher vertebrates have nociceptive and both opioid and nonopioid antinociceptive systems that take part in the control of fish behavior and physiological status on the level of central mechanisms. This should be considered when surgical treatment is performed on fish, in particular, on sturgeons which often suffer from sex products extraction. The use of analgesics may minimize a harm and accelerate the wound healing.

Acnowledgments The study was supported with Russian Foundation for Basic Research ʋ 03-04-49230, ʋ NSh-1334.2003.4, and the Program "Universities of Russia"

References Barrallo A, Gonzalez-Sarmiento R, Porteros A, et al. (1998) Cloning, molecular characterization, and distribution of a gene homologous to delta-opioid receptor from zebrafish (Danio rerio). Biochemical and Biophysical Research Communication 245: 544-548. Chervova LS (1997) Pain sensitivity and behavior of fishes. J. of Ichthyology 37: 98- 102. Proceedings of The Fourth International Iran & Russia Conference 1425

Chervova LS, Kamenskii AA, Malyukina GA, et al. (1992) Investigation of the mechanism of intranasal effect of dermorphine in representatives of two classes of vertebrates. Zh. Evol. Biokhim. Fiziol. 28: 45-48. Chervova LS, Lapshin DN, Kamenskii AA (1994) Pain sensitivity of trout and analgesia caused by intranasal administration of dermorphine. Dokl. Biol. Scienc. 338: 424-425. Darlison MG, Greten FR, Harvey RJ, et al. (1997) Opioid receptors from a lower vertebrate (Catostomus commersoni): sequence, pharmacology, coupling to a G-protein-gated inward-rectifying potassium channel (GIRK1) and evolution. Proc. Natl. Acad. Sci. USA. Neurobiology 94: 8214-8219. Dhawan BN, Cesselin F, Raghubir R, et al. (1996) International union of pharmacology. X11. Classification of opioid receptors. Pharmacological Reviews. 48: 567-592. Ehrensing RH, Michell GF, Kastin FJ (1982) Similar antagonism of morphine analgesia by MIF-1 and naloxone in Carassius auratus. Pharmacol. Biochem. and Behav. 17: 757-761. Jansen GA, Greene NM (1970) Morphine metabolism and morphine tolerance in goldfish. Anesthesiology 32: 231-235. Johnsson M, Axelsson M, Holmgren S (1998) Localization of Neuropeptides in Perivascular Nerves Innervating Veins in the Atlantic Cod (Gadus morhua) and the Rainbow Trout (Oncorhynchus mykiss). Abstr. of Vlll Intern. Symp. on Fish Physiol., 15-18 August, Uppsala, Sweden p. 34. Kavaliers M (1988) Evolutionary and comparative aspects of nociception. Brain Research Bull. 21(6): 923-931. Lorenz K (1984) Das sogenannte bοse. Zur naturgeschichte der agression. Mυnchen: Piper. Translated under the title “Agressia”, Moscow: Progress, 1994. Matthews G, Wickelgren WO (1978) Trigeminal sensory neurons of the sea lamprey. J. Comp. Physiol. 123: 329-333. Nikolskii ID, Protasov VR, Romanenko EV, Shishkova EV (1968) Zvuki ryb. Atlas (Sounds of fishes. Atlas), Moscow: Nauka. Rekubratskii VA (1967) Ekologicheskie stereotipy pischedobyvatelnogo i zaschitnogo povedenija ryb (Ecological stereotypes of foraging and defensive behavior of fishes), Povedenie i recepcii ryb (Behavior and receptions of fishes), Moscow: Nauka,121-125. Vecino E, Ekstrom P (1991) Distribution of met-Enkephalin, leu-Enkephalin, Substsnce-P, neuropeptide Y, FMRFamide and Serotonin Immunoreactivities in the Optic Tectum of the Atlantic Salmon (Salmo salar L.). J. of Comp. Neurol. 299: 229-241. Wall PD (1992) Defining ‘Pain in Animals’. Animal Pain. Short CE, Van Poznak A eds. New-York: Churchill Livingstone: 63-79. Proceedings of The Fourth International Iran & Russia Conference 1426

The occurrence of fat liver disease (FLD) in Rainbow trout due to side effects of dietary lipids at cultured salmonids farms in North of Iran Ebrahimzadeh Musavi H*. Rostami.M. Ahmadi, M.R. Department of Fish Health. Faculty of Veterinary Medicine, Tehran University, Email;[email protected]

Abstract Industrially cultured fish of rainbow trout (Oncorhynchus mykiss) are the most sensitive fish of salmonids to the fat degenerative disease of the liver, which may be the result of feeding with high-fat diets or inadequate nutritional dietary regimens at low temperatures. The particular important problem contributed to the provision of fat requirements in fishes is the prevention of O2 auto oxidation of lipids or unsaturated fatty acids chemically comprised chains of hydrogenated carbon atoms with long chains (4 to 6 carbon atoms in the chain). In this work, the qualitative histopathological tests were performed on liver, kidney, intestines and muscle tissues specimens of the rainbow trout to revealed fat accumulation. The severity of fatty infiltrations was judged by the relative quantities of intracellular fat droplets. The cellular changes in the following cells were obvious; sinusoidal endothelial lining cells, hepatocytes, the epithelial cells of bilitary, and hepatic ducts. The histopathological examination in rainbow trout after using the lipids which are the most effective source of energies in fish after proteins, showed steatites in the visceral fat area. Increasing the fat rate up to %17 in the dietary regimens is economical and will save protein usage, will reduces N2 excretion and water pollution.

Keywords: Fatty Liver Disease (FLD), Rainbow trout, liver

Introduction: A salmonids are susceptible to lipid liver degeneration but it is a particularly Significant problem in Rainbow trout culture and, recently Rainbow trout as an important farming cold water fish, has been cultured in large number. Since, the dietary regimens have a great real role in the costs for a breeding system of fish, changing of the regimens and having a proper formula is very important and essential, since it could provide the nutritional requirements. Using the lipids in a proper percentage, as the most significant source of energy in fish, is very important. In this study, the fishes collected by the laboratory of fish pathology were examined histopathologically for the occurrence of fat liver disease. The supplementary of the fish feed usually contained (more than 17% by the weight) either Kilka Oil (K.O) or Soy Oil (S.O) were used, the lipids comprised of polyunsaturated fatty acids (PUFA) (with four or more double bands) at 17% of diet for K.O and S.O respectively. In fish culture, using a proper rate of fat is very important because of the quality of the product and with a view of containing fat in muscles and making histopathological alterations in the fish liver, so the fat should be used in rates with definite limits to prevent mortality in fishes. The purpose of this study was to show the histopathological changes of the fatty liver disease in Rainbow trout, due to dietary lipids. The rate of 17% fat could be recommended regarding growth which not produce damages or mortality in fish.

Materials &Methods; Proceedings of The Fourth International Iran & Russia Conference 1427

In 1999, the occurrence of FLD in the specimens of 100 Rainbow trout at cultured salmonids farms from northern of Iran, received by the laboratory of fish histopathology, was examined microscopically. The autopsied samples of the liver, kidney, intestine, muscle and spleen were fixed in 10% formaline and then the Hematoxylen & Eosine (H&E) staining method was performed.

Results: In contribution to the histopatological effects of fat in diets of fishes various investigations have been conducted. As Randall et al reported in 1997 that the hybrids of white bass were fed an experimental fat diet up to 205% by weight; there were no significant effects from lipid storage point of view in the tissue of fish. Guarda, Hellou et al (1997) reviewed the effects of lipids in fish. In this work the purpose of study was to show the histopathological lesions of (FLD) Fatty liver Disease and to demonstrate the liver changes. The rate of IDF (intra peritoneal fat) was significantly high. The FLD effects on the normal metabolism of liver, the liver enlarged and showed microscopic changes. The lesions were as follows, 1. Liver, In the liver vacualation was increased, predominantly due to accumulation of fat. Cellular edema and infiltration of fat in the hepatocytes were occurred because of the lack of the metabolism and cellular oxidation resulted from existence of toxicants in food and anemia produced by disease. In some hepatic cells coagulation necrosis induced by the effects of toxicants were observed and finally substitution by fats were obvious. Accordingly, histopahtologically, the Main feature was the extreme infiltration of hepatocytes by lipid, which caused loss of cytoplasm staining and distortion of hepatic muralia. Kidneys, Hemosidrine was present. The hematopoetic tissue was degenerated. Spleen, Congestion , hemosidrosis and pale – staining fragment in the melanomacrophage centers were observed. Intestines, Congestion and infiltration of monocytes in the parrine mucusal section were noticed. Also, Steatites or infiltration and substitution by the inflammatory cells and the extensive deposits of hyaline intercellular material and thickening of the cell membrane were cleared, which was due to the high rate of lipids or the effects of toxicants resulted from the food or the fat oxidation consumed. Proceedings of The Fourth International Iran & Russia Conference 1428

Fig.1: Gross lesions of FLD in Rain bow trout at the necropsy examination time. Proceedings of The Fourth International Iran & Russia Conference 1429

Fig 2: Microscopic views of the liver in the FLD.

Fig 3: Cross section of liver bile ducts with melanomacrophagh around them and the accumulation of hemosidrine in hepatic tissue.

Fig 4: The vacuolated hepatocytesƍ s cytoplasm’s, precipitation of hemosidrine in liver tissue. Proceedings of The Fourth International Iran & Russia Conference 1430

Fig 5: In kidney, the existence of hemosidrine and proliferation hematopoetic tissue and vacuolated cytoplasm of urinary ducts epithelial cells.

Fig6: In spleen, hemosidrine and presence of light pink cerroid liquids in the macrophages of some areas. Proceedings of The Fourth International Iran & Russia Conference 1431

Fig7: Proliferation of inter peritoneal stocked fat tissues .

Fig 8: Steatitis of IPF. Proceedings of The Fourth International Iran & Russia Conference 1432

Fig. 9: Infiltration of lymphocyte cells into the IPF.

Fig 10: Increasing the thickness of the fatty cell membranes and substitution with the inflammation cells.

Discussion: The main changes were noticed in the liver, and the IPF (intaperitoneal fat tissue) . In this study the aim of the work was to show the microscopically features of FLD in Rainbow trout fish. Invasion and infiltration of lipids followed by hepatocyte cell swelling occurred. Proceedings of The Fourth International Iran & Russia Conference 1433

Anemia after failure of the liver to secrete hemopoetine was induced and at last mortality in fish could be seen. In IPF thickening of the cell membrane and substitution of fat tissue by hyaline intercellular material and inflammatory cells were seen. Also, steatitis or infiltration and substitution by the inflammatory cells and the extensive deposits of hyaline intercellular material and thickening of the cell membrane were cleared, which was due to the high rate of lipids or the effects of toxicants resulted from the nutritional effects of lipid dietary and was related to the consumed fat oxidation. In conclusion, it can be said that increasing the fat rate in the regimens is partly cast benefited and will save protein usage, reduce N2 exertion and water pollution. The results show that the fishes whit nutritional problems, show edema of hepatocytes and after lipid invasion and after impairment of liver in secretion of hemopoetin, the anemia will occur. In IPF (Intaperitoneal Fat tissue), the increase of intracellular hyaline and thickening of membrane cells will be seen. The fishes show anemia, the gills are pale, and the liver with fatty vacuoles is yellow and pale in color. Microscopically in FLD the infiltration of fat into hepatocytes caused pale cytoplasm staining and the wary maralia. The changes spleen and the hematopoetic tissue of kidneys were present. The macrophages containing ceroid liquid produced by metabolism and ligation of phospholipids, were different. The other changes such as steatitis of fat in the IPF, darkening, hemosidrosis of spleen, atrophy of muscles, the substitution by fat tissue, the degeneration of hematopoetic tissue in kidneys were seen, compared with controls. Accordingly, it can be emphasized that increasing the rate of fat in dietary can improve the growth in a limited rates and adding inappropriate rate of fat in diet can make the diet economical and inhibit water pollution due to reduction of N2 excretion but can causes mortality in fish.

References: 1-Amold-Reed,D.E, Bentley.-P.J.,Phan,-C.T., Redgrawe.-T.G. The clearance of lipids from the plasma of a teleost fish, the black bream (Acanthopagus butcher). COMP-BIOCHEMI-PHYSIOL- , -A. , 1997 .vol. 116A, no. 2, pp. 167-172 2- AOAC , 1990 . Official Methods of Analysis. Association of Official Analytical Chemists. 15th end. Arlington. Arlati, G., Bronzi,P., 1994. Principali aspetti tecnici sull, allevamento degli storioni in Italia ( Main technical aspects of sturgeon culture in Itali) . In: Proceedings of the Symposium “ Parliamo di ….Acquacoltura " . Fussano, Cuneo, Itali, pp. 113-122. 3-Aya,-Z, Barlasm-N, Kolankaya,-D, Determination organochlorine pesticides residues in varios enviroments and organisms in Goksu Delta, Turkey. AQUAT,-TOXICOL., 1997 , vol. 39, no,. 2 m, pp.171- 181. 4-,Anderson.J.W.,Shanks,A.M, Bell, J.C,B 1985. Some effects of vitaminee Eand selenium deprivation on tissue enzyme levels and induces tissue peroxidation in rainbow trout (Salmo gairdneri) .Br.J.Nutri.53,149- 157. 5- Cowey, C.B.,Adron, J.W., Youngson, A., 1983. Vitamin E requirements of rainbow trout given diets containing polyunsaturated fatty acids derived from fisoil . Aquaculture 30,85-93. 6-FAO, 1995. FAO world fishery production , 1993. FAO, Roma. 7-Fowler, L.G., J., 1969. Test of vitamin supplement and formulation changes in the Abernathy salmon diet . Tech. Pap.U.S.,Bur.Sport Fish. Wild .26.,3-19. 8- Roberts. Fish pathology, Bailier Tindall. 1997, pp. 340-345. Proceedings of The Fourth International Iran & Russia Conference 1434

Karyological study on Bighead gobie (Neogobius kessleri) in Mahmoudabad Area (South Caspian Sea)

Amirhossein Esmaily*- Mohammadreza Kalbasi** Ø&ØØ Faculty of Natural Resources and Marine Sciences, University of Tarbiat Modares. P.O.Box: 46414-356. Nour, Iran. Correspondence to: M. R. Kalbassi, E-mail: [email protected]

Abstract Karyological characters of Neogobius kessleri , in Caspian sea were studied by examining metaphase chromosome spreads taken from the kidney tissues. The examination of 30 metaphase spread prepared from 10 specimens indicated that the chromosome numbers of this species was found 2n=46 and the arm number was determined as NF=46. The prepared karyotype of this species was consisted of 23 pairs acrotelocentric (at) chromosomes. The chromosome formula can be stated as 2n=46(at). Karyological parameters showed that relative length and length variation range of chromosomes were between 2.34-7.04, 1.67-5.01 respectively and total length is 71.16µ. In this study, it was found that the best chromosomal spread quality were obtained from 40 µg/gr Colchicine injection, height dropping of 120 cm, cooled slide with flame and %1 Tri-sodium Citrat as a hypotonic solution in 4ºC.

Keywords: Chromosome, Karyology, Bighead gobie, Neogobius kessleri, South Caspian Sea.

Introduction Gobies are the most abundant fish in freshwater in oceanic islands. The smallest fishes (and vertebrates) in the world belong to this family which mostly live in shallow coastal waters and around coral reefs. Some species have symbiotic relationships with invertebrates. Neogobius is found in the Black and Caspian seas in where there are about 11 species, some large enough to be the object of commercial fisheries. The general Farsi name for fishes in this genus is gav mahi. This species is separated from other Caspian gobies in Iran by having a completely scaled nape. Systematically Neogobius kessleri belongs to teleostei class, Perciformes order, Gobidae family and Neogobius genus. Which found in the inshore Caspian Sea and tributary rivers. In Iran, it is reported from a wide range of rivers from Astara to the Gorgan and probably Atrak, the Aras River, the Anzali Mordab and Gorgan Bay, the southeast Caspian Sea, southwest Caspian Sea and south-central Caspian Sea (Holþík and Oláh, 1992).That this fish is endemic in Caspian sea. Since 1960s, karyological studies in teleost fishes have made noteworthy contributions to increasing knowledge in the fields of genetics, taxonomy and environmental toxicology (Cucchi & Baruffaldi, 1990). The progress in increasing such knowledge has been closely related to the evolution of application methodologies (Rivlin et al., 1985). Studies of the chromosomes of fishes have not been as successful or widespread as in other vertebrate groups. Standard karyotypes are reported for less than 10% of more than 20000 extant species of fishes (Gold et al., 1990). The study of fish chromosome has become an active area of research in recent years (Thorgaard, 1983). Chromosomal analysis is important for fish breeding from the viewpoint of genetic control, pants of rapid production of inbred lines, taxonomy and evolutionary studies (Hosseini 2003). Karyological studies have provided basic information about the number, size and morphology of chromosomes Proceedings of The Fourth International Iran & Russia Conference 1435 that is important to undertake chromosome manipulations in fish (khan et al., 2000). Genetic divergences of populations and their local adaptation are a potential source for breeding programs in aquaculture and for fishery management (Philips & Rab, 2001). However, as happened in the Iranian Cyprinids, such as Rutilus frisii kutum(Nowruzfashkhami and Khosroshahi, 1995), Abramis brama(Nahavandi et al., 2001), Ctenopharyngodon idella(Nowruzfashkhami et al., 2002) and Hypophthalmichthys molitrix(Varasteh et al., 2002), cytogenetic studies in fish have not been comprehensive when compared to other vertebrate grows. The aim of this study was to investigate the chromosomes and karyotype of Neogobius kessleri in Iran

Materials and methods Ten, Neogobius kessleri weight 100-150 gr, were caught in Mahmood abad shores(Caspian sea) in north of Iran. The fishes were transported live to the laboratory, and kept in a well-aerated aquarium at15 - 20ºC before analysis. 2.1. Mitotic inhibitors The stock solution of colchicine was made by dissolving 10 mg colchicine and 100 mg NaCl in 20 ml distilled water. The colchicine was administrated at dose of 25 and 40 µg/gr body weight (BW) and slowly injected into the intraperitoneal muscle. Then, fishes left in aquaria at 15-20˚C for 5-10 hours before sacrificing. then, the fish were killed and their anterior kidneys removed, suspensioned and placed in hypotonic treatment(0.075M KCl and 1 % Sodium citrate solution) at two different temperature 4˚C and 25˚C. Lasting time for hypotonisation treatment was 45-50 min.

2.2. Fixation The swollen cell suspensions were fixed in 3:1 cooled Carnoy’s fluid (3 parts methanol and 1 part glacial acetic acid) for 30 min, then, the old fixative was replaced with the fresh Carnoy’s. Lasting time for fixation treatment was 60 min.

2.3. Spreading The slides, previously washed in alcohol and ether and kept at -1˚C, were prepared by letting two drops of the fixing solution containing the cell suspension fall onto the cooled slide with flame and warm slide(40ºC) in different heights (60, 90 and 120cm),. Immediately thereafter the fixative was burned off, using the technique developed by Mellman(1965), for obtaining better cell spread.The slides were stained in series of concentrations of Gimsa Merck solution in distilled water(5, 10 and 15%) and buffered by phosphate(40 mol Na2HPO4 and 26.6 mol K H2PO4) at PH 6.8 and were assessed at 7, 8, 9 and 10 min exposure times to determine optimum staining conditions. Slides were dipped into distilled water to wash out extra Giemsa solution and then were allowed to airdry at 25ºC for 2–3 h.

2.4. Chromosome examinations and morphometric measurements Metaphases were examined under a microscope (Leca SER. NO. 990398, Equipted with a green filter and digital camera) with an oil immersion lens at 1000 magnification. The chromosomes at the metaphase were photographed with a digital camera (Sony SSC-DC 58 AP) onto Kodak colour films(ASA 25). In the course of the microscopic examinations the chromosome sets of 30 cells were counted and 10 of the best mitotic metaphases were used to measure karyotypes. The morphometric measurements of chromosome pictures were conducted with photographic software Proceedings of The Fourth International Iran & Russia Conference 1436

Photoshop 6.0 (Adobe Systems). Each chromosome was tagged with a reference number. The data were transferred to the Excel 2000 (Microsoft) for analysis.

2.5. Chromosome pairing To increase differences between the homologous chromosomes the total length of chromosome was computed by summing up the average chromatid lengths of each diploid complement. The length recorded in pixels by the Colour Image Analysis System Video Pro 32 (Leading Edge) was converted into micrometers after the scale factor was calibrated with a stage micrometer. The chromosome pairs were classified following the recommendations of Macgregor (1993). The pair numbers were definitely attributed following this classification and the decreasing length order within each class. Finally, the karyotype was constructed by first dividing arranging the homologous pairs in the decreasing length order within each group.

Results Relatively small and high numbers of chromosomes were observed in Neogobius kessleri. The counts of chromosome was 46 in per metaphases. In 30 metaphases from the anterior kidney cells of 10 N.kessleri specimens, the diploid chromosome number was 2n=46 (Fig.1). All chromosomes in the karyotype have a homologous pair. Homologous pairs of chromosomes were arranged in decreasing size. The investigation of metaphases showed notable difference in size of chromosomes in N.kessleri but in type there is no difference between chromosomes. In addition, the sex chromosomes could not be distinguished in this species. The representative karyotype for N.kessleri is shown in Fig2. The karyotype of N.kessleri (Fig.2) has 23 pairs acro-telocentric chromosomes. The number of chromosome arms were determined NF=46 and chromosome formula can be expressed as 2n = 46(a-t). The morphological and numerical data are summarized in Table 1. Other data are represented in Table 2. According to this table, relative length and length variation range of chromosomes are between 2.34-7.04 and 1.67-5.01 respectively. Total length of chromosomes was 71.16µ. The ideogram of the N.kessleri was made on the basis of the karyotype(Fig.3). In this study, the optimum colchicine concentration for N.kessleri was determined to be 40 µg/gr BW of colchicine solution for five hours. This concentration have effectively arrested dividing cells in metaphase. In addition the best chromosomal spread quality(well-spread metaphase) were obtained from treatment of cells with 1% Sodium citrate solution at 4oC for 45-50min and height dropping 120 cm ,cooled slide with flame. The other hypotonic solution tested, 0.075M KCl, did not result in many scorable metaphases. Proceedings of The Fourth International Iran & Russia Conference 1437

Figure 1- metaphase chromosomes of bighead goby(Neogobius kessleri)

Table 1- centromeric index of bighead goby(Neogobius kessleri) chromosome total length centromer arms relative Chromosome Pair arm(µm) (µm) index ratio length type 1 5.01 5.01 0 ∞ 7.04 A* 2 4.34 4.34 0 ∞ 6.09 A 3 4.01 4.01 0 ∞ 5.63 A 4 3.84 3.84 0 ∞ 5.39 A 5 3.84 3.84 0 ∞ 5.39 A 6 3.51 3.51 0 ∞ 4.93 A 7 3.34 3.34 0 ∞ 4.69 A 8 3.34 3.34 0 ∞ 4.69 A 9 3.17 3.17 0 ∞ 4.45 A 10 3.01 3.01 0 ∞ 4.22 A 11 3.01 3.01 0∞ 4.22 A 12 3.01 3.01 0∞ 4.22 A 13 3.01 3.01 0∞ 4.22 A 14 2.84 2.84 0 ∞ 3.99 A 15 2.84 2.84 0 ∞ 3.99 A 16 2.67 2.67 0 ∞ 3.75 A 17 2.67 2.67 0 ∞ 3.75 A 18 2.67 2.67 0 ∞ 3.75 A 19 2.67 2.67 0 ∞ 3.75 A 20 2.51 2.51 0 ∞ 3.52 A 21 2.51 2.51 0 ∞ 3.52 A 22 1.67 1.67 0 ∞ 2.34 A 23 1.67 1.67 0 ∞ 2.34 A *A: acrocentric

Table 2- Karyotype characters of bighead goby(Neogobius kessleri) Number of Chromosome Total length Haploid total chromosome number (2n) chromosomes chromosome Proceedings of The Fourth International Iran & Russia Conference 1438

arms(NF) (µm) length (µm) 46 46 71.16 71.16

Figure 2- Karyogram of bighead goby(Neogobius kessleri)

Figure 3- Idiogram of bighead goby(Neogobius kessleri) Discussion Rapidly growing tissues are required to obtain a large number of chromosome spreads in metaphase for karyotypical studies(Tan et al., 2004). Several techniques have been developed to examine chromosomes in tissues of adult fish. These include squashed(Ojima et al., 1963; Roberts, 1967; Al-Sabti et al., 1983), blood leucocyte culture(barker, 1972; Al-Sabti, 1985; Hartley & Hornne, 1985) and cell suspensions from tissues such as gill, kidney, intestine(McPhail & Jones, 1966; Gold, 1974; Klingerman & Bloom, 1977), cornea(drewery, 1964) and scales(Denton & Howell, 1969). Each of these procedures was optimized to obtain large numbers of well- spread metaphases and was used regularly for karyotypeic analysis that in present lecture we utilized anterior kidney. Karyological study of teleost fishes presents Proceedings of The Fourth International Iran & Russia Conference 1439 technical difficulties which are not encountered in the study of other vertebrates, and these difficulties are due to the small size and high number of chromosomes(Cucchi & Baruffaldi, 1990). Different techniques are presently being used to perform such studies: direct, in vivo; and indirect, in vitro. With those forms employing direct techniques, the preparation of slides for optical microscopy is quite easy. Furthermore, these techniques are rather inexpensive and results are obtained relatively quickly. Such techniques are based on the use of Colchicine to block quickly-proliferating cell populations at the metaphase. Karyological study has in different steps. Each of the steps involves in the preparation of tissues and slides for cytogenetic analysis is which important in attaining large number of well-spread metaphases. The first step in the procedure is the treatment of the cells with Colchicine, which arrests cell division at metaphase (Baski & Means, 1988). High concentration and long period of Colchicine treatment affect chromosome, causes to aggregate and shrink of chromosome and their arms, so it is difficult to identify short arm of acrocentric chromosomes and other chromosomes. This study suggests that colchicines concentrations of 50 µg/gr BW can effectively arrest dividing cells in metaphase in kidney tissues. But the maintenance periods may vary according to species. Type of hypotonic treatment and the length of exposure affect on the degree of chromosome spreading. In this study, 0.075M KCl hypotonic treatment was ineffective in obtaining well-spread metaphases. Although condensed chromosomes could be observed, they were often seen inside an intact cell or only slightly spread. Fixative treatment was not found to be as important as hypotonic treatment in obtaining well-spread metaphases. The main difficulty in working with fish chromosomes is in obtaining high quality metaphase spreads. A few studies have been used fish standard karyotypes to examine taxonomic or systematic problems (Bolla, 1987). The major difficulty encountered is the current morphological variation even between homologous chromosomes in the same nucleus (Al-Sabti,1991 & Levan et al., 1964). Sometimes it could happen that some chromosomes get more contracted than others, so chromosome measurements are very small compared to those of man and mammals. Another problem is that fish karyotypes are not identical, as in human being or other animal species, so we cannot have a standard karyotype for fish not only because there are differences between species, but also polymorphism often occurs within the same fish species (Al-Sabti, 1991). Several incomplete metaphases were encountered in the preparation, and these probably resulted from hypotonic over treatment (Nanda et al., 1995). The majority of authors classify uni-armed and bi-armed chromosomes according to the guidelines of Macgregor (1993) where differences in the number of chromosome arms were seen, aril usually on the result of a difference in the scoring of subtelocentric chromosomes by different authors (Philips & Rab, 2001).The majority of Gobidae species have 2n = 46 chromosomes while Neogobius fluviatilis and Neogobius melanostomum have 2n= 42-46 (Klinkhardt, M,1995) and fishes which have normal chromosome series (2n = 50) are called diploids fish. Until now, karyotype of some member of Neogobius genus were determined such as Neogobius melanostomus affinis (2n=46, NF=46 2n=46a-t) (Klinkhardt, M, 1995). The karyotype analysis is a key step toward the stock improvement by polyploidy manipulation, hybridisation and related genetic engineering (Tan et al., 2004). Therefore like to other animal species, comprehensive genetic researches are required this fish too.

Refrences: Proceedings of The Fourth International Iran & Russia Conference 1440

Al-Sabti, K., Fijan, N., Kureiec, B., 1983. A simple and fast technique for the chromosome preparation in the fish. Vet. Arch. 54, 83-89. Al-Sabti, k., 1985. The karyotype of Cyprinus carpio and Leuciscus cephalus Cytobios 47, 19-25. Al-Sabti, K., 1985. Frequency of chromosomal a aberrations in the rainbow trout, salmo gairdneri Rich., exposed to five pollutants. J. Fish Biol. 26, 13-19. Al-Sabti, k., 1991. Handbook of Genotoxic effects and fish chromosome. Ljubljana, 97. Anonym., 1982. Baksi, S.M., Means, J.C., 1988. Preparation of chromosomes from early stages of fish for cytogenetic analysis. J. Fish Biol. 32, 321-325. Barker, C.J., 1972. A new method for the display of chromosomes of plaice, Pleuronectes platessa, and other marine species. Copeia 1972, 365-368. Beck, M. L. ;Bigger, C. J. and Dupree, H. K. 1980. Karyological analysis of Ctenopharyngodon idella, Aristichthys nobilis, and their F1 Hybrid. Transact. Amer. Fish. Soc. Vol. 109, pp.133-438. Bianco, P.G., Banarescu, P., 1982. A contirbution to the knowledge of the cyprinidae of Iran (Pisces, Cypriniformes). Cybium 6(2), 75-96. Bolla, S., 1987. Cytogenetic studies in Atlantic salmon and rainbow trout embryos. Hereditas 106, 11-17. Cucchi, C., Baruffaldi, A., 1990. Anew method for karyological studies in teleost fishes. J. Fish Biol. 37, 71-75. Demirok, N.K., Ünlü, E., 2001. Karyotype of cyprinid fish capoeta trutta and capoeta capoeta umbla(Cyprinidae) from the Tigris River .Turk. J. Zool. 25, 389-393. Denton, T.E., Howll, W.M., A technique for obtaining chromosomes from the seale epithelium of teleost fishes. Copeia, 392-394. Drewry, G. 1964. Chromosom number bull. Thx. Mem. Mus. 5-72. Firouz, E., 2000. A guide to the fauna of Iran. Iran University Press. Tehran. 491. Gold, J.R., 1974. A fast and easy method for chromosome karyotyping in adult teleosts. Progve Fish Cult. 36, 169-171. Gold, J.R., Li, Y.C., Shipley, N.S., Power, P.K., 1990. Improved methods for working with fish chromosomes with a review of metaphase chromosome banding. J. Fish Biol. 37, 563- 575. Gül, S., çolak, A., KaloƧlu, B., 2004. Karyotype analysis in Alburnus heckeli (Battalgil, 1943) from lake Hazer. Turk. J. Vet. Anim. Sci. 28, 309-314. Hartley S.E., Horne, M.T., 1985. Cytogenetic techniques in fish genetics. J. Fish Biol. 26, 575-582. Holþík and Oláh, 1992 . http :// www.briancoad. com/species%20accounts/ Neogobius .htm Hosseini, S.V., 2003. Karyological study in Schizothorax zarudnyi in Zahak-Iran. … Khan, T.A., Bhise, M.P., Lakra, W.S., 2000. Chromosome manipulation in fish—a review. Indian J. Anim. Sci. 70, 213–221. Klingerman, A.D., Bloom, S.E., 1977. Rapid chromosome preparation from solid tissues of fish. J. Fish. Res. Bd Can. 34, 266-269. Klinkhardt, M., Tesche, M. & Greven, H. (1995). Database of Fish Chromosomes. Magdeburg: Westarp Wissenschaften Levan, A., Fredge, K., Sandberg, A.A., 1964. Nomenclature for centrometric position on chromosome. Hereditas 52, 201-202. Macgregor, U.C., 1993. Chromosome preparation and analysis. Chapter 6, 177-186. McPhail J.D., Jones, R.L., 1966. A simple technique for obtaining chromosomes from teleost fishes. J. Fish. Res. Bd Can. 23, 767-768. Nahavandi, R., Amini, F., Rezvani, S., 2001. Karyology of Abramis brama in southern waters of Caspian sea. Journal of Fisheries Sciences. vol. 10(3), 89-100. Nanda, l., Schartl, M., Feichtinger, W., Schlupp, I., Parzefall, J., Schmid, M., 1995. Chromosomal evidence for laboratory synthesis of a triploid hybrid between the gynogenetic teleost Poecilia formosa and host species .J. Fish. Biol. 47, 221-227. Proceedings of The Fourth International Iran & Russia Conference 1441

Nowruzfashkhami, M.R., Khosroshahi, M., 1995. Karyology of the Caspian sea kutum roach by with blood cells culture. Journal of Fisheries Sciences. vol. 4(1),64-71. Ojima, Y., Makei, K., Takayama, S., Nogusa, S., 1963. A cytotaxonomic study of the Salmonidae. Nucleus (Calcutta) 6, 91-98. Philips, R., Rab, P., 2001. Chromosome evolution in the Salmonidae(Pisces): an update. Biol. Rev. 76, 1-25. Rivlin, K., Rachlin, J.W., Dale, G., 1985. A simple method for the preparation of fish chromosomes applicable to field work teaching and banding. J. Fish Biol. 26, 267-272. Roberts, E. L., 1967. Chromosome cytology of the Osteichthyes. Progve Fish Cult. 29, 75- 83. Tan, X., Jian G.Q., Chen, B., Chen, L., Li, X., 2004. Karyological analyses on redclaw crayfish Cherax quadricarinatus (Decapoda: Parastacidae). Aquaculture 234, 65-76. Thorgaard, G.H. Disney, J.E., 1993. Chromosome preparation and analysis. Chapter 6, 171- 186. Varasteh, A., Hossienzadeh, M.M., Pourkazemi, M., 2002. Karyotyping and number of chromosomes of silver carp(Hypophtalmichthys molitrix). Journal of Fisheries Sciences. vol. 11(1), 107-114. Proceedings of The Fourth International Iran & Russia Conference 1442

Survival of Sturgeon Fries by Controlling Submerged Weeds

Y. FILIZADEH1* and H. AHMADI2 1 Department of Agronomy, Shaded University, Ramsar, Iran. 2Mirza Koochak Khan Higher Fisheries Education Centre, Rasht, Iran.

Abstract Sturgeons are the most valuable aquatic animals in the Caspian Sea. Sturgeon fries at 100 mg weight easily trapped in heavy dense stands of submerged aquatic vegetation. Due to the often detrimental impacts of submerged weeds on growth and mortality of young fish, removal of nuisance vegetation with minimal harm to sturgeon fries is a desirable goal. Paraquat (1,1´-dimethyl-4, 4´-bipyridinium ion) was evaluated at rates of 0, 0.5, 1, 2, 5, and 10 mg l-1 in the fish ponds for control of Potamogeton crispus, Ceratophyllum demersum, Myriophyllum spicatum and Hydrilla verticillata in the International Institute of Caspian Sea, Shahid Ansari and Shahid Beheshti fish research stations, Rasht, Iran during 1999-2000. Paraquat treatments were conducted on May 25, 1999 and June 10, 2000. The 5 mg l-1 treatments and above reduced biomass of all aquatic weeds by > 75%. Treatment of 10 mg l-1 reduced the biomass of aquatic weeds by > 90%; however this application rate also significantly reduced growth several desirable and non-target species such as Chara by > 90%. Biomass of submerged weeds following the 0.5, 1, 2 mg l-1 application of paraquat were reduced 8, 12 and 26% respectively. Results showed the removal of competitive, canopy forming weeds such as Potamogeton crispus, Ceratophyllum demersum and Myriophyllum spicatum open new areas and increased the survivability rate of sturgeon fries by 45%.

Keywords: Sturgeons fries, Fish ponds, Submerged weeds, Paraquat.

Introduction In aquatic habitats plant density is an important factor, which can make plants wanted or unwanted. Aquatic plants are usually useful plants which become weeds when their growth becomes excessive, and some type of control or management become necessary to ensure continued use of water body (Pieterse and Murphy, 1990; Nichols, 1991). Weed problems in aquatic habitats are generally caused by the growth of dense vegetation, which hampers the use of water bodies. When massive growths of submerged weeds occur, they can have an influence on water quality because oxygen is depleted by plant respiration, interfere with fish movement, which often causing fish kills (Mitchell, 1974; Riemer, 1984; Murphy and Pieterse, 1990). Control methods are usually based on physical removal or herbicides (Wade 1993; Murphy and Barrett, 1993). Various methods have been used in the last 30 years to control of unwanted aquatic plants. Although herbicides and plant growth regulators can control many weed species, there is limitation on using them in water bodies. The success of a chemical treatment against submerged weeds depends on the concentration of the herbicide that comes into contact with target plant, the length of time a target plant is exposed to the herbicide, and timing of application. The response is also related to the properties of individual herbicides and the sensivity of the target species to each herbicide (Langeland and Laroche, 1994). Information on herbicide uptake and lethal concentration in plant tissues is extremely limited for aquatic macrophytes, especially submerged species (Van and Conant, 1988). Rapid dilution and dispersal of herbicide residues from the treatment area Proceedings of The Fourth International Iran & Russia Conference 1443 following herbicide application (due to diffusion and water movement) can reduce both concentration and exposure time to a level less is required for complete control. Previous studies have focused on the use of contact herbicide like diquat for control of submerged weeds, to provide a temporary weed-free period in the target area for up to one year (Fox et al. 1986; Van and Conant, 1988; Caffrey, 1990). The Potamogeton crispus, Ceratophyllum demersum, Myriophyllum spicatum and Hydrilla verticillata and Potamogeton pectinatus are problematic submersed weeds of rivers, irrigation networks and drainage channels and fish ponds throughout temperate and subtropical regions of the world. (Caffrey, 1990; Sabbatini and Murphy, 1996; Spencer, 1986; van Wijk, 1988; Kantrud, 1990). Sturgeon fries easily trapped in heavy dense stands of submerged aquatic vegetation. Due to the often detrimental impacts of submerged weeds on growth and mortality of young fish, removal of nuisance vegetation with minimal harm to sturgeon fries is a desirable goal. Paraquat (1,1´-dimethyl-4, 4´-bipyridinium ion) is widely used to control these submerged weeds, and is typically applied at 1 mg l-1 active ingredient, with a minimum exposure period of 24 hours (Barrett and Murphy, 1982), to provide control for up to 1 year (Fox et al. 1986; Van and Conant, 1988; Caffrey, 1990). Increasing concern about use of herbicides in aquatic ecosystems has produced pressure to reduce the loadings of herbicides used for aquatic weed management. One possible approach is to combine reduced concentrations of herbicide treatments with other control techniques in integrated management programs. Van Vierssen and Hootsmans (1990) suggested manipulation of underwater light regime, i.e., using turbidity promoting benthic feeding fish coupled with a low dose of herbicide to cause chronic stress to the target weeds, and followed, where necessary, by mechanical removal. This approach was used in channel systems in Argentina (Sabbatini et al. 1998; Sidorkewicj et al. 1998), with good results. In this study we evaluated the effectiveness of low doses of diquat to control of submerged weeds in the fish ponds at the north of Iran.

Materials and Methods In order to evaluate the potential of paraquat for controlling the growth and spread of submerged weeds such as Potamogeton crispus, Ceratophyllum demersum, Myriophyllum spicatum, Hydrilla verticillata and P. pectinatus an experiment was conducted between 1999-2000, in the experimental fish pond facility, located at the International Institute of Caspian Sea, Shahid Ansari and Shahid Beheshti Fish Research Stations, Rasht, Iran. Six small fish ponds (12m length, 10m width, 1.5m water depth) which covered by natural vegetation such as Hydrilla verticillata, Potamogeton pectinatus, P. crispus, Ceratophyllum demersum, and Myriphyllum spicatum were selected. Stock rate in each fish pond was 125 Sturgeon fries. A pond, without paraquat application acted as control. Mean chemical characteristics of ponds water were calcium 4.6 mg l-1, pH 8.26, nitrate 0.63 mg l-1; reactive phosphate 0.53 mg l-1 and chlorine 0.31 mg l-1. When plants height reached an average of 40 cm, paraquat treatments were applied on May 25, 1999 and June 10, 2000. Paraquat concentrations were 0, 0.5, 1, 2, 5, and 10 mg l-1. Each treatment was replicated 2 times in a complete randomized block design. Treatments were made by injecting the herbicide solution into the water with hypodermic syringes. This experiment took place in 90 days. Samples were taken by using 50 cm2 quadrat frame with all plants within the quadrat removed to evaluate a percentage of visual damage, plant dry weight (g), shoot length (cm) and Proceedings of The Fourth International Iran & Russia Conference 1444 compared with controls. Also, sturgeon survival (%) at each pond at the end of experiment was measured. In this experiment data were analyzed for treatment effects by standard ANOVA procedures with subsequent use of Tukey’s Least Significant Difference test to separate means (Little and Hills, 1978).

Results Injury to submerged weeds occurred at most concentrations. Dry weight (g) of submerged weeds following the 0.5, 1, 2 mg l-1 application of paraquat were reduced 8, 12 and 26% respectively. The 5 mg l-1 treatments and above reduced biomass of all aquatic weeds by > 75%. Treatment of 10 mg l-1 reduced the biomass of aquatic weeds by > 90%; however this application rate also significantly reduced growth several desirable and non-target species such as Chara by > 90% (Figure 1a). Paraquat at 0.5 and 1 mg l-1 were ineffective to decrease significantly submerged weeds dry weight and shoot length. Results showed the removal of competitive, canopy forming weeds such as Potamogeton crispus, Ceratophyllum demersum and Myriophyllum spicatum open new areas and increased the survivability rate of sturgeon fries by 45% (Figure 1d). Most shoots in the 1, 2, 5 and 10 mg l-1 paraquat concentrations were brown, necrotic and appeared dead. Shoot lengths were significantly reduced by increasing the paraquat concentrations. Despite a 22 and 27% reduction in shoot length compared with untreated plants, there were no significant differences between 0.5 and 1 mg l-1 paraquat with controls. Greater reductions of plant length were observed at high doses of paraquat (Figure 1b). The inhibition of tuber and rhizome production persisted long after the plants had recovered from initial herbicidal effects. The duration of belowground organs suppression increased with increasing dose. The 0.5, 1, 2, 5 and 10 mg l-1 paraquat reduced tuber and rhizome production 33, 48, 58, 72 and 96% respectively. The tubers produced in the treated ponds were also much smaller than those of untreated plants (Figure 1c).

Discussion Increasing effects on plants were observed, as expected, with increasing paraquat concentrations. However, rapid regrowth occurred at low concentrations treatments (0.5 and 1 mg l-1 paraquat) were inefficient in significantly reducing submerged weeds dry weight. New growth of submerged weeds at the treated ponds remained bleached and necrotic while in contact with paraquat. Regrowth of treated weeds depended on the paraquat concentrations and exposure times. When paraquat was removed and deactivated, submerged weeds began to regrow from rhizome and tubers. Regrowth from tuber and rhizomes suggests a lack of herbicide transport to tubers (Van and Stewart, 1985), in keeping with the well-known poor ability of paraquat for translocation within the plant (Murphy and Barrett, 1993). The results suggest that low doses of paraquat can control submerged weeds, but must be in contact for >24 hours. Long exposure periods are difficult to achieve for paraquat in ambient conditions, even using formulations with slow-release properties (Murphy and Pieterse, 1990; Murphy and Barrett, 1993). Residue loss in flowing water, herbicide adsorption to organic and clay particles in water and sediment, and antagonistic action from Ca++ ions in water are all known problems affecting paraquat (Murphy and Barrett, 1982, 1993). Our results showed that submerged weeds response to paraquat were unable to survive by concentration of 2 mg l-1 and above. Proceedings of The Fourth International Iran & Russia Conference 1445

a 400

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Figure 1. Effects of paraquat concentrations on plant dry weight (g), shoot length (cm), belowground biomass (g) and (d) sturgeon survival in experimental fish ponds. Separate bars represent least significant difference: L.S.D. (p<0.05). Acknowledgments This study was supported by Guilan Research and Training Fishery. We are deeply grateful to Dr. Pourkazwmi, Dr. Piri, Eng. Danesh and Eng. Tooloi for their valuable assistance.

References 1- Barrett, P. R. F. and K. J. Murphy 1982. The use of diquat-alginate for weed control in flowing waters. Proc. EWRS 6th Int. Symp. on Aquatic Weeds 1982, European Weed Research Society, Wageningen, The Netherlands. pp. 200-208. 2- Caffrey, J.M. 1990. Problems relating to the management of Potamogeton pectinatus L. in Irish rivers. Proc. EWRS 8th Int. Symp. on Aquatic Weeds European Weed Research Society, Wageningen, The Netherlands. pp. 61-68. 3- Fox, A.M., K.J. Murphy and D. Westlake 1986. Effects of diquat alginate and cutting on the submerged macrophyte community of a Ranunculus stream in Northern England. Proc. EWRS 7th Int. Symp. on Aquatic Weeds 1986, European Weed Research Society, Wageningen, The Netherlands. pp. 105-111. 4- Kantrud, H.A. 1990. Sago pondweed (Potamogeton pectinatus L.): a literature review. U.S. Fish Wildl. Serv., Resourc. Publ. 176. 89 pp. Little, T.M. and F.J. Hills 1978. Agricultural experimentation: design and analysis. Wiley, Chichester. 5- Langeland, K.E. and Laroche, F.B. 1994. Persistence of bensulfuron methyl and control of hydrilla in shallow ponds. J. Aquatic Plant Manage. 32: 12-14. 6- Little, T.M. and F.J. Hills. 1978. Agricultural experimentations: design and analysis. Wiley, Chichester. 7- Mitchell, D.S. 1974. Aquatic vegetation and its use and control. UNESCO, Paris. 135 pp. 8- Murphy, K.J. and A.H. Pieterse 1990. Present status and prospects of integrated control of aquatic weeds. In: A.H. Pieterse and K.J. Murphy, eds. Aquatic Weeds: 2nd Ed., Oxford University Press, Oxford. pp. 222-227. 9- Murphy, K.J. and P.R.F. Barrett 1990. Controlled-release aquatic herbicides. In: R. M. Wilkins, ed. Controlled Delivery of Crop Protection Agents. Taylor and Francis, London. pp. 193-211. 10- Murphy, K.J. and P.R.F. Barrett 1993. Chemical control of aquatic weeds. In: A.H. Pieterse and K.J. Murphy, eds. Aquatic Weeds: 2nd Ed., Oxford University Press, Oxford. pp. 136-173. 11- Nichols, S.A. 1991. The interaction between biology and the management of aquatic macrophytes. Aquat. Bot. 41: 225-252. 12- Riemer, D.N. 1984. Introduction to freshwater vegetation. AVI Publishing, Connecticut, 255 pp. 13- Pieterse, A.H. and K.J. Murphy. 1990. Aquatic weeds. Oxford Univ. Press, Oxford, UK, 593 pp. 14- Sabbatini, M.R. and K.J. Murphy 1996. Submerged plant survival strategy in relation to management and environmental pressures in drainage channel habitats. Hydrobiologia. 340: 191-195 15- Sabbatini M.R., K.J. Murphy and J.H. Irigoyen. 1998. Vegetation environment relationships in irrigation channel systems of Southern Argentina. Aquat. Bot. 60: 119-133 16- Sidorkewicj, N.S., A.C. Lo˴pez Cazorla, K.J. Murphy, M.R. Sabbatini, O.A. Fernandez and J.C.J. Domaniewski. 1998. Interaction of common carp with aquatic weeds in Argentine drainage channels. J. Aquatic Plant Manage. 36: 5-10. 17- Spencer, D.F. 1986. Early growth of Potamogeton pectinatus L. in response to temperature and irradiance: morphology and pigment composition. Aquat. Bot. 26: 1-8. 18- Van, T.K. and R.D. Conant. 1988. Chemical control of hydrilla in flowing water: Herbicide uptake characteristics and concentration versus exposure. Tech. Rep. A-88-2, US Army Engineer Waterways Experiment Station, Vicksburg, MS. 33 pp. 19- Van, T.K. and K.K. Stewart. 1985. The use of controlled-release fluridone fibers for control of hydrilla (Hydrilla verticillata). Weed Sci. 34: 70-76. 20- Van Vierssen, W. and M.J.M. Hootsmans. 1990. Weed control strategies for Potamogeton pectinatus L. based on computer simulations. Proc. EWRS 8th Int. Symp. on Aquatic Weeds 1990, European Weed Research Society, Wageningen, The Netherlands. pp. 231-236. 21- Van Wijk, R.J. 1988. Ecological studies on Potamogeton pectinatus L. I. General characteristics, biomass production and life cycles under field conditions. Aquat. Bot. 31: 211-258. 22- Wade, P.M. 1993. Physical control of aquatic weeds. In: A. H. Pieterse and K. J. Murphy, eds. Aquatic Weeds: 2nd ed. Oxford University Press, Oxford. pp. 93-135. Proceedings of The Fourth International Iran & Russia Conference 1447

The Feeding Preferences of Grass Carp (Ctenopharyngodon idella Val.) For Ten Aquatic Plants

Y. FILIZADEH1*, H. AHMADI2, K. ZOLFINEJAD3 1 Department of Agronomy, Shahed University, Ramsar, Iran. 2Mirza Koochak Khan Higher Fisheries Education Centre, Rasht, Iran. 3 Guilan Research and Training Fishery, Bandar-Anzali, Iran.

Abstract Heavy dense stands of submerged and free floating weeds causes numerous problems in the Iranian freshwater systems. Aquatic weeds interfere with agricultural irrigation, restrict recreation and fish production, and has a detrimental effect on dissolved oxygen concentrations. A widely used method of controlling aquatic weeds in through the use of herbivorous fish, including grass carp (Ctenopharyngodon idella). The feeding preferences of the grass carp (Ctenopharyngodon idella) for 10 aquatic plants were examined in 2 replicate experiments under field and fiberglass conditions. Individual grass carp (45-70 g) were placed in aerated, large outdoor tanks that hold approximately 2500 liter of water with randomly assigned individual plants during the 168-hr period and fish ponds for 90 days. Results showed that grass carp preferring succulent young plans and will not control all types of aquatic weeds. The grass carp preferred plants in the following order in both experiments: [1] Lemna minor., [2] Chara sp., [3] Najas guadalupensis. [4] Hydrilla verticillata., [5] Potamogeton pectinatus., [6] P. perfoliatus., [7] P. crispus., [8] Azolla filiculoThe problemides., [9] Ceratophyllum demersum., and [10] Myriophyllum spicatum.

Keywords: Ctenopharyngodon idella, Biological control, Aquatic plant management.

Introduction Emergent, submerged, free-floating and floating-leaved macrophytic plants are common and integral componenet of many ponds and lakes. Excessive growth of aquatic weeds causes numerous problems in Iranian freshwater systems (Filizadeh, 1996). These weeds interfere with agricultural irrigation, restrict recreation, reduce fish harvest and have detrimental effects on fish populations, interferes with receational activities, and causes water quality to deteriorate (Allen and Wattendrof, 1987). The problems are particularly acute in the shallow, warm environments of farm ponds during the summer months (Miller and Decell, 1984). The macrophyte cover of Iranian freshwater system is generally dominated by ten species, Lemna minor, Chara sp, Najas guadalupensis, Hydrilla verticillata, Potamogeton pectinatus, P. perfoliatus, P. crispus, Azolla filiculoides, Ceratophyllum demersum, and Myriophyllum spicatum. These plants and filamentous algae were indicated as the most troublesome weeds in a recent national inquiry to water managers. Mechanical removal of plants such as hand harvesting, weed cutters, chaining and raking, the only weed control method extensively used in Iran, is often labor expensive and costly. Mechanical method is generally ineffective because fragments of all major weeds form new shoots and roots, and reinfestation rapidly occur (Mitchell, 1980; Opuszynski, 1972). Chemical treatments are also expensive and can directly or indirectly harm other aquatic life and water quality; despite a good experiemntal results were obtained against Azolla filiculoides and Typha latifolia (Catarino, et al. 1997). However submerged weeds such as Potamogeton pectinatus, P. crispus, Ceratophyllum demersum, and Myriophyllum spicatum the species presenting the largest weed problems, showed weak response to several herbicides such as diquat, paraquat and glyphosate (Zweerde, 1990). Some experimental results of aquatic weeds control by grass carp in irrigation canals, ditches, drinage channel and fish ponds are available (Fowler and Robson, 1978; Opuszynski, 1972; Pine and Proceedings of The Fourth International Iran & Russia Conference 1448

Anderson, 1991). Although it has been reported that grass carp prefers filamentous algae and duckweed to macrophytes, there are little published data on the feeding preferences of the species for macrophytes (Swanson and Bergersen, 1988). Iranian freshwater systems have favourable water temperature (12-32 ºC) for grass carp suitable for the maintenance of feeding throughout most of the year. Also grass carp are highly tolerant to adverse limnetic conditions such as low oxygen, high salinity and chemical hazards (Catarino, et al. 1997). The composition and relative abundance of the existing plant assemblages are important in determining the control efficacy, the most favourable stocking rates and potential changes in local vegetation. The objective of this study was to determine the feeding preferences of grass carp for Lemna minor, Chara sp, Najas guadalupensis, Hydrilla verticillata, Potamogeton pectinatus, P. perfoliatus, P. crispus, Azolla filiculoides, Ceratophyllum demersum, and Myriophyllum spicatum. The results were used in conjunction with data from a field study to determine the effectiveness of Ctenopharyngodon idella as a biological vegetation control agent.

Materials and Methods The feeding preferences of grass carp for Lemna minor, Chara sp, Najas guadalupensis, Hydrilla verticillata, Potamogeton pectinatus, P. perfoliatus, P. crispus, Azolla filiculoides, Ceratophyllum demersum, and Myriophyllum spicatum were tested in two replicated experiments.

Fiberglass tank experiment: Twelve 2500 l fiberglass tanks (200 cm long, 110 cm wide, 115 cm water depth), placed outdoor at Guilan Research and Training Fishery, Bandar-Anzali, Iran, in 2000 and 2001. The water in the tanks were aerated by air pump, warmed to 25 ± 0.5 ºC, and had an open circuit water exchange system that completely replaced the water volume every 24 hours. Preference trials were conducted in June 2000 and July 2001 using grass carp with an average weight of 55 grams (SD, 10.5 g). Each individual fish was used in only one feeding trial. New fishes were used in each replication. Four tanks were used as replicates. Mature plants for fiberglass experiment were collected daily from Anzali lagoon. In each trial at duration of 168 hours, the experimental plants, previosly weighted, were placed in the fish tanks. By the end of first day, if any plants were nearly or totally consumed, additional plants were added. At the end of test period, all uneaten plants and plant fragments were removed and weighted to determine the amount ingested. There was plant growth in the test figerglass tanks during the 196 hours of experiment. No grass carp mortality occurred during the experiemnt. Temperature (ºC), water pH, conductivity and dissolved oxygen were measured daily in experimental tanks.

Pond experiment: To assess the potential of grass carp to consume the aquatic weeds an additional experiment was conducted in the Shahid Ansari Research Station, Rasht, Iran during June 2000 and July 2001. This experiment took place in 90 days. Experimental ponds were covered by natural vegetation such as Chara sp, Najas guadalupensis, Hydrilla verticillata, Potamogeton pectinatus, P. perfoliatus, P. crispus, P. natans, Azolla filiculoides, Ceratophyllum demersum, and Myriophyllum spicatum. Grass carp with an average weight of 60 grams were transferred to two small fish ponds (18m length, 10m width, 2.5m water depth). Each pond was divided in two parts by a plastic net, providing four replicates, and preventing the passage of grass carp. Stock rate in each part was 18 grass carp. A third pond, without grass carp acted as control, with two replicates. At the end of experiment, total and individual plant Proceedings of The Fourth International Iran & Russia Conference 1449

cover in each half pond were measured and compared with controls. Also, grass carp were weighted at the end of experiment. In both experiments data were analyzed for treatment effects by standard ANOVA procedures with subsequent use of Tukey's Least Significant Difference test (Little and Hills, 1978) to separate means.

Results and Discussions The mean of water quality parameters in the tank and pond were water temperature 25 ºC, pH 7.7, dissolved oxygen 8.9 mgl-1 and conductivity 435 µS.cm-1 . During the two years of the experiments in the fiberglass tanks, grass carp showed generally small growth rate, although an increase occurred after carp release within pond with values above 0.65% per day. Grass carp growth rate are strongly dependent on several factors such as the quantity and nutritional value of plant food, the salinity, age and density of carp population, size location, temperature and the dissolved oxegen in the water. The high carp densities used in pond experiment and the typed of food were likely responsible for the reduce growth rates. In tank experiment, a mean consumption of Lemna minor (504 g), Chara sp (485 g), Najas guadalupensis (480 g) and Hydrilla verticillata (473 g) at 168 hours were not significantly different and have eaten significantly greater quantities than any other plant (Table. 1). Preference declined significantly (P<0.05) for consumption of Potamogeton pectinatus (155 g), P. perfoliatus (146 g) P. crispus (135 g), Azolla filiculoides (128 g), Ceratophyllum demersum (109 g) and Myriophyllum spicatum (85 g) respectively. At the end of experiment in tank conditions, grass carp fed on nearly all parts of Lemna minor, Chara sp, Najas guadalupensis and Hydrilla verticillata. However the soft and tender plant tissue such as young leaves of Potamogeton pectinatus, P. perfoliatus, P. crispus, Azolla filiculoides, Ceratophyllum demersum and Myriophyllum spicatum were preferred.

Table 1. Mean consumption of ten macrophytic plants by grass carp (Ctenopharyngodon idella) in tank conditions at duration of 196 hours.* Species Mean consumption (g) Lemna minor 504a Chara sp 485a Najas guadalupensis 480a Hydrilla verticillata 473a Potamogeton pectinatus 155b P. perfoliatus 146b P. crispus 135b Azolla filiculoides 128bc Ceratophyllum demersum 109c Myriophyllum spicatum 85c *Means with a common superscript were not significantly different at P<0.05 as determined by Tukey's Least Significant Difference test to separate means. The variation of plant biomass and cover in the ponds are presented in Table 2. At the end of experiment, the total vegetation cover was significantly higher in the control pond (P<0.05). P. crispus, Azolla filiculoides, Ceratophyllum demersum, and Myriphyllum spicatum cover and weight in the trail ponds were no different from the control. These results suggested that grass carp with the average weight of 60 grams did not eat properly these species. The final biomass and cover of Lemna minor, Chara sp, Najas guadalupensis and Hydrilla verticillata showed a great difference between test and control ponds (P<0.05). Proceedings of The Fourth International Iran & Russia Conference 1450

Table 2. Mean biomass (kg m-2) and cover (%) of experimental macrophytes at the beginning and the end of pond experiments with grass carp (Ctenopharyngodon idella) Species Final Final Beginning (Control) (Test)

Biomass (kg m-2)Cover (%)Biomass(kg m-2)Cover (%)Biomass (kg m-2)Cover (%) Lemna minor 0.12 6 0.10 8 0.04 2.5 Chara sp. 0.105 8 0.09 8 0.01 3 Najas guadalupensis 0.346 11 0.305 10 0.06 3.5 Hydrilla verticillata 0.146 9 0.150 16 0.08 5.5 Potamogeton pectinatus 0.189 8 0.210 11 0.165 9 P. perfoliatus 0.445 11 0.555 10 0.405 10 P. crispus 0.505 13 0.495 12 0.210 8 Azolla filiculoides 0.235 7 0.260 9 0.143 7.5 Ceratophyllum demersum 0.40 11 0.385 10 0.195 8 Myriophyllum spicatum 0.245 8 0.230 7 0.195 7

Several researches had showed the types of macrophytes preferred by grass carp (Cross, 1969; George, 1983; Pine and Anderson, 1988). However, large variations of food preference can occur, when pond or field conditions are used (Fowler and Robson, 1978; Mitchell 1980; Zweerde, 1990). Results showed that smaller fish had selected softer plant tissue and youngest plants, while bigger fish eat a wide variety of tough and fibrous plants. Lemna minor, Chara sp, Najas guadalupensis and Hydrilla verticillata had shown as preferred whilst Potamogeton pectinatus, P. perfoliatus, P. crispus, Azolla filiculoides, Ceratophyllum demersum and Myriophyllum spicatum are generally avoided. Despite variety results which showed a large variation, from 100 percent body weight and more to as low as 1%, daily consumption rates of grass carp for the Potamogeton pectinatus, P. perfoliatus, P. crispus, Azolla filiculoides, Ceratophyllum demersum and Myriphyllum spicatum were relatively low (Bonar et al. 1990; Cai and Curtis, 1989). Some studies showed that the rate of grass carp consumption and its growth, are related to the nutritive value of the plants, such as the gross energy content of the diet (Cassani and Caton, 1983, 1986; Bonar et al. 1990; Cai and Curtis, 1989). The rate of calcium and cellulose in macrophyte tissue were the best predictors of consumption rates and palatability. Data on macrophyte consumption in the tank experiment was in close agreement with the results of a pond study in which grass carp was tested as a biological agent for vegetation control. Agreement with Duthu and Kilgen (1975), results showed that Lemna minor, Chara sp, Najas guadalupensis and Hydrilla verticillata were controlled successfully, while Potamogeton pectinatus, P. perfoliatus, P. crispus, Azolla filiculoides, Ceratophyllum demersum and Myriophyllum spicatum persisted in the test ponds throughout the study. Results obtained indicate that grass carp can effectively control the aquatic weeds of Iranian fresh water systems. Although, grass carp will preferably consume some beneficial native plants (Dekozlowski, 1994; Kirk and et al. 1996). Water managers and farmers must be practiced caution to choose this type of weed control to avoid the escape of carp in non-target areas to feed on native plants, which may favor the spread of undesirable species. In many Iranian fresh water systems, wetlands and natural rivers, the native plant community Lemna minor, Chara sp, Najas guadalupensis, Hydrilla verticillata and P. natans already being substituted by exotic plants such as Azolla filiculoides, Potamogeton pectinatus, P. crispus, Ceratophyllum demersum and Myriophyllum spicatum (Filizadeh, 1996). The feeding of Proceedings of The Fourth International Iran & Russia Conference 1451 grass carp if enhance for the indigenous plants, decreasing the plant diversity of these ecosystems will occurs (Santha and et al. 1991; Schramm and Jirka, 1986).

Acknowledgments This study was supported by Shahed University and Guilan Research and Training Fishery. We are deeply grateful to Dr. Piri, Eng. Danesh and Eng. Tooloi for their valuable assistance.

References 1- Allen, S. K., and R.J. Wattendrof. 1987. Triploid grass carp: Status and management implication. Fisheries, 12: 20-24. 2- Bonar, S.A., Sehgal, S.H., Pauley, G.B, and Thomas. 1990. Relationship between the chemical composition of aquatic macrophytes and their consumption by grass carp (Ctenopharyngodon idella). J. Fish Biol., 38:149-157. 3- Cai, Z. and L.R. Curtis. 1989. Effects of diet on consumption, growth and fatty acid composition in young grass carp. Aquaculture, 81: 47-60. 4- Cassani, J.R., and W.E. Caton. 1983. Feeding behaviour of yearling and older hybrid grass carp. J. Fish Biol., 22:35-41. 5- Cassani, J.R., and Caton, W.E. 1986. Efficient production of triploid grass carp (Ctenopharyngodon idella) using hydrostatic pressure. Aquaculture, 55: 43-50. 6- Catarino, L.F., M.T. Ferreira., and I.S., Moreira. 1997. Preferences of grass carp for macrophytes in Iberian drainage channels. J. Aquat. Plant. Manage., 36: 79-83. 7- Cross, D.G. 1969. Aquatic wed control using grass carp. J. Fish Biol. 1:27-30. 8- Dekozlowski, S.J. 1994. Stocking update and vegetation changes in Lake Marison, South Carolina. Proceedings of the Grass Carp Symposium. U.S. Army Engineer Waterways Experiment Station, Vicksburg, MS. 186-187. 9- Duthu, G.S. and R.H. Kilgen. 1975. Aquarium studies on the selectivity of 16 aquatic plants as food by fingerling hybrids of the cross between Ctenopharyngodon idella and Cyprinus carpio. J. Fish Biol., 7:203-208. 10- Filizadeh, Y. 1996. The management ecology of aquatic weeds which cause problems in Iranian freshwater systems. Ph.D. thesis, University of Glasgow, 155 pp. 11- Fowler, M.C. and T.O. Robson. 1978. The effects of food preferences and stocking rates of grass carp (Ctenopharyngodon idella Val.) on mixed plant communities. Aquatic Botany. 5: 261-276. 12- George, T.T. 1983. The Chinese grass carp, Ctenopharyngodon idella, its biology, introduction, control of aquatic macrophytes and breeding in the Sudan. Aquaculture. 27: 317-327. 13- Kirk, J.P., K.J. Killgore., J.V. Morrow., and , J.W. Folyz. 1996. Triploid grass carp in Lake Marison, South Carolina, Miscellaneous Paper A-96-2, U.S. Army Engineer Waterways Experiment Station, Vicksburg, MS. 14- Mitchell, C.P. 1980. Control of water weeds by grass carp in two small lakes. N.Z.J. Marine and Freshwater Research. 14: 381-390. 15- Miller, A.C., and J.L. Decell. 1984. Use of the white amur for aquatic plant management. Instruction Report A-84-1, U.S.Army Engineer Waterways Experiment Station, Vicksburg, MS. 16- Opuszynski, K. 1972. Use of phytophagous fish to control aquatic plants. Aquaculture. 1: 61-74. 17- Pine, R.T. and L.W.J. Anderson. 1991. Plant preferences of triploid grass carp. J. Aquat. Plant. Manage. 29: 80-82. 18- Schramm, H.L. and K.J. Jirka. 1986. Evaluation of methods for capturing grass carp in agricultural canals. J. Aquat. Plant. Manage. 24: 57-59. 19- Santha, C.R., W.E. Grant., W.H. Neil., and R.K. Strawn. 1991. Biological control of aquatic vegetation using grass carp: Simulation of alternative strategies. Ecological Modelling. 59: 229-249. 20- Swanson, E.D. and E.P. Bergersen. 1988. Grass carp stocking model for cold water lakes. North American J. Fishery Manage. 8: 284-291. 21- Zweerde, W. van. 1990. Biological control of aquatic weeds by means of phytophagous fish. In Aquatic Weeds. Edited by A.H.Pieterse and K.J.Murphy. Oxford Science Publications. Oxford. Pp. 201-221. Proceedings of The Fourth International Iran & Russia Conference 1452

Reproduction and Embryonic Development of Comb jelly Mnemiopsis leidyi in the Southeast Caspian Sea Conditions

Ghabooli S.1, Abtahi B.2, Javanshir A.3, Seifabadi J.4, Shiganova TA.5

1- Marine Sciences Department, Natural Resources and Marine Sciences Faculty, Tarbiat Modarres University, Noor, Iran, phone: +98-1226253901, Email: [email protected]; 2- Marine Sciences Department, Natural Resources and Marine Sciences Faculty, Tarbiat Modarres University, Noor, Iran, phone: +98- 1226253901, Email: [email protected]; 3- Caspian Ecology Research Center, Sari, Iran, , phone:+981523462498, Email: [email protected]; 4- Marine Sciences Department, Natural Resources and Marine Sciences Faculty, Tarbiat Modarres University, Noor, Iran, phone: +98-1226253901 ; 5- Institute of Oceanography of Russian Academy of Sciences,Moscow, Russia, phone: +7951292327, Email: [email protected].

Abstract Among so many studies on distribution and population dynamics of Mnemiopsis leidyi, only a few were carried out on fecundity rate and early life stages of M.leidyi. In this study the fecundity rate, individually (according to Individual Biological Methods, IBM) and some early life stages of M.leidyi in southeast Caspian Sea conditions (higher salinity compared to north and middle regions). Three stages of the larvae before hatching were observed. In compliance with pervious studies several categories and wide range of hatching time variability, fecundity rates (the fecundity rate was much lower than one reported from the Black Sea, with the maximum of 558 eggs/day), reproduction size and temperature dependant mortality were revealed too.

Keywords: Caspian Sea, Early life stages, Fecundity, Mnemiopsis leidyi.

Introduction Eastern coastline of North and South America is the native habitat of Mnemiopsis leidyi. It is adapted for high population, growth and reproduction rates (Finenko et al., 1995), therefore it has the ability to colonize rapidly (Kremer, 1976). Mnemiopsis could be found along American estuaries in 40 °N – 46 °N and can get along with salinity ranges from 3.4- 75 ppt and temperatures from 1.3-32 °C (Ivanov et al., 2000). Successful reproduction is one of the most important facts that support the survival of the organism. Although Mnemiopsis leidyi can tolerate different ranges of temperature and salinity, but its ecological characteristics in different ecosystems around the world or even in parts of Mediterranean could be completely different from those in Black Sea. For example Mnemiopsis leidyi can reproduce in the most parts of the Black Sea through the year, but in Azov Sea reproduction is reported only in warm months of the year (Esmaili et al., 2002).In a study on Mnemiopsis mccradyi it is shown that some of them can reproduce before Lobate stage and very often these larvae can also reproduce after Lobate stage (Martindale,1987). In another study by Martindale and Henry in 1999 the comb plate lineage of Mnemiopsis leidyi is shown, but little works have been done about the reproduction of Mnemiopsis leidyi in Iran coastlines and most of the studies are limited to morphological and distribution aspects of it. The invasion of Mnemiopsis leidyi to the Caspian Sea was first reported in February of 2000 (Esmaili et al., 2000). Ecological characteristics of the Caspian Sea as a closed basin differ from north to the south and also from Black Sea where Mnemiopsis was introduced through ballast waters. Salinity from north to south changes from 0.1‰ in north to 10-11‰ in the middle and 12.6-13‰ in the south. In this study we tried to figure out the fecundity rate of Proceedings of The Fourth International Iran & Russia Conference 1453

Mnemiopsis for different length groups and to do a brief study on early life stages of this invasive species in southeast Caspian Sea conditions.

Materials and Methods Experiments were done during Sep-Oct, 2003. Mnemiopsis leidyi were obtained from the southeast waters of Caspian Sea by the shore of Farah-Abad. We caught the comb-jellies from the surface layer of the sea water by a transparent jar. Zooplankton was also gathered by zoo-net (30 µm), for feeding and adjusting Mnemiopsis leidyi with the lab conditions (22- 24 ºC). Cached Mnemiopsis were divided in three size groups (15±5 mm, 25±5 mm, 35±5 mm) considering the lobes, it is very important to have undamaged individuals because some of the eggs are formed along each auricular canal (Zaika and Revkov, 1995). It is also proved that short-lobed ctenophores (due to any reason) have reduced fecundity (Purcell & Cowan, 1995). After keeping the Mnemiopsis in the sea water with zooplankton in order to adjust the lab conditions for 3-4 hours, the individuals were put inside glass cylinders contained 2 liters of filtered sea water and darkened at about 16-18 pm, since we know that Mnemiopsis produce naturally at nights (Zaika & Revkov, 1995). First we tried to figure out the time of spawning, so we prepared the individuals as previously described, then every two hours we checked the water, after that the eggs are laid down in small glass tanks made out of slides (microscope slides).This way we could easily observe and take pictures of them under the microscope. In order to find out the fecundity rate we counted the eggs by Bugarov slide and invert microscope.

RESULTS Between the combs, along the 8 meridional canals the gonads are situated and the rows of testis are faced to the other side. The results showed the spawning time between 23pm-1am, means about 7 hours after Mnemiopsis were darkened. We could observe three distinct stages before hatching:

1- Newly fertilized egg: which has absorbed water and had a thick wall only few minutes after spawning, these eggs are spherical (figure 1) and sometimes oval shaped (figure 2). The diameter was measured 226.6-484.1 µm.

2- Ctenes Appearance: In this stage the comb plates and tentacles are easily seen (figures3- 5), also the beating of ctenes and movement of tentacles through their bulbs, of course the ctenes and their beating can be seen sooner than the tentacles. These tentacles are contractile and covered with numerous colloblasts.We could observe the beating of ctenes from about 5 o’clock in the morning and from 7 the movement of tentacles was often clear. The embryon diameter in this stage was measured 144.2-175.1 µm.

3- Gastrulation(end): About 8 hours after spawning means at about 9 o’clock in the morning gastro-vascular system forming has been seen (figure 6), then the stomadeum, the large pharynx and endodermal canals also the movement of muscles were seen. We could see the larvae move in all directions. The diameter of organism in the egg wall was 175.1- 226.6 µm, so the more the larvae grows, the less space remains in the egg capsule. Therefore near hatching the beating of ctenes seemed to help the larvae get out of the egg (figure 7).

4- Cydippid Larvae: Newly hatched cydippid larvae which resemble the adults move in all directions and we could see the tentacles contract or elongate easily in response to any slight stimuli (figure 8-10). In most cases we could observe this stage 20-24 hrs after spawning but Proceedings of The Fourth International Iran & Russia Conference 1454 in some cases we had cydippid larvae 12-15 hrs after spawning although our lab temperature was always kept 22-24 ºC. Now the cydippid larvae has a size between 206-278.1 µm

In order to find out the fecundity rate of Mnemiopsis leidyi in the southeast Caspian conditions we studied 3 length dependent groups of them and in each group we tested about 40 organisms. In our first group (15±5 mm), spawning was observed in only five individuals out of 36 ones. The maximum fecundity was for an 18 mm one (0.90 gr, 0.9 cm³) with 66 eggs counted and the minimum (16 eggs) was counted for a 13 mm one (0.78 gr, 0.6 cm³).The scatter chart is shown in figure 11. The average wet weight and volume in this group is 0.7879(gr) and 0.755(cm3).

The second group (25±5 mm) showed better results. Among 52 Mnemiopsis of this group 75% spawned. The highest and lowest fecundity among them was respectively 558 eggs/day (from a 29 mm one, 4.21 gr, 4.5 cm³) and 12 eggs/day (23 mm, 1.89 gr and 1.9 cm³). (figure13). We could see that individuals with bigger length had higher fecundity rate in this group. The average wet weight and volume of this group is measured respectively 2.11(gr) and 2.10(cm3).

In the third group (35±5 mm) 78.04% of the individuals spawned. The maximum and minimum fecundity among this group(40 Mnemiopsis ) was measured respectively 336 egg/day(39 mm,5.93 gr,6 cm³) and 10 eggs/day(30 mm,4.21 gr,4.2 cm³).(figure 15). This group had lower fecundity compared to the second one but the reproduction percent was more. It seems although longer individual has more reproduction potential, but the southeast Caspian conditions (specially the food and salinity conditions) is not good enough for them.

In figures 12, 14 and 16 the volume (cm³) and wet weight (gr) of the Mnemiopsis in each group is shown. In another experiment we put the individuals in higher temperature (30-32 ºC) but we had high mortality rate and very few of them reproduced, actually the gathered eggs didn’t seem to have continued any more divisions (figure 17). In most cases they couldn’t continue till morning. We could see that they could survive in lower temperature much better, for instance they stayed alive with out any food for 25 days, the temperature was 10-11ºC and each of them was put in a jar with 1.5 lit of filtered sea water.

Discussion Egg numbers depends greatly on temperature and feeding conditions (Zaika and Revkov, 1995), also the body size. Large specimens produce between 2 to 8x1000 eggs during spawning. The largest animals in native habitat can produce from 10 to 14 x 1000 eggs (Baker and Reeve, 1974). Experimental studies on Mnemiopsis have indicated that Mnemiopsis begins to produce eggs in the Caspian Sea when it reaches length about 15 mm, the smallest organism spawned in our experiments was a specimen 12 mm, 0.39 gr, 0.4 cm³, so the size of reproductive maturity is much less in the Caspian Sea (about 15-16 mm) than in the Black Sea which is about 30 mm (Shiganova, 1997). Reproduction starts in the Southern Caspian in June when temperature reaches 21ºC. In July Mnemiopsis reproduces also in the whole Middle Caspian, and in August with penetration to the Northern Caspian it reproduces there as well. Peak of reproduction occurs in August and continues until October similar the Black Sea pattern. Average fecundity of Mnemiopsis in the Caspian Sea was 1174 eggs/day with maximal 2824 eggs/day for specimens 30-39(Data of Tamara Proceedings of The Fourth International Iran & Russia Conference 1455

Shiganova), as mentioned the maximum eggs number we counted was 558 for specimen 29 mm. We could also see that the more the Mnemiopsis length is increased we have higher fecundity rate, but for Mnemiopsis bigger than 35 mm the Caspian Sea condition seems to be not suitable enough because we have a slight decrease in fecundity for this group. The comparison of the egg diameter shows that this factor doesn’t relate to the Mnemiopsis length and all the eggs from all the individuals in the three length group had similar diameter of about 356.864 µ. The maximal size of Mnemiopsis in the Caspian Sea is also less (6.4 mm) than that it was recorded for the Black Sea, where Mnemiopsis reaches 10-12 cm with maximal 18 cm (Shiganova, 1997), we found a Mnemiopsis with 68mm length. The embryonic development takes about 20-24 hours in the Black Sea upper water layers with 23°C and the size of hatched larvae was measured 0.3-0.4 mm. Our experiments took place in similar temperature (22-24 ºC) and in most cases we had the same time for development of the embryo, but although we didn’t change the temperature during our experiments, in some cases we had cydippid larvae12-15 hrs after spawning.

References Baker JD, MR, Reeve (1974), Laboratory culture of lobate ctenophore Mnemiopsis mccradyi With notes on feeding and fecundity. Marine Biol.V.31.N1.p.61-100 Esmaili A, Khodabandeh S, Abtahi B, Sifabadi J, Arshad H, (2000), First Report on Occurance of a Comb jelly in the Caspian Sea Dep. Marine Biology, Faculty of Natural Resources and Marine Sciences, University of Tarbiat Modarres, Iran Esmaili A , Abtahi B, Sifabadi J, Khodabandeh S, Talaei R, Darvishi F, Arshad H, (2002) Invasive Comb jelly Mnemiopsis leidyi and the Future of the Caspian Sea, Naghsh Mehr Publishers, Iran, Finenko GA, Abolmasova GI, Romanova ZA, (1995), Consumption, respiration and growth rates of Mnemiopsis mccradyi in relation to food conditions. Biologia Morya 21:315-320 (in Russian). Kremer, P. (1976), Population dynamics and ecological energetics of a pulsed zooplankton predator, the ctenophore Mnemiopsis leidyi. In M. L. Wiley (ed.), Estuarine Processes. Academic Press, NewYork 1: 197-215 Martindale MQ, (1998), Reproduction in the Ctenphore Mnemiopsis mccradyi, J.Marine Biology, vol:94,no:3,p.p.667-682 Martindale MQ., Henry JQ, (1999), Interacellular Fate Mapping in a Basal Metazoan,the Ctenophore Mnemiopsis leidyi, Reveals the Origin of Mesoderm and Existance of Indeterminate Cell Lineage,Developmental Biology, 1999, vol:214, p.p:243-257 Purcell JE, Cowan JH, (1995), Predation by the Scyphomedusan Chrysaora quinquerriha on Mnemiopsis leidyi Ctenophores, Marine Ecology, vol:129, pp:63-70 Shiganova, TA, (1998), Invasion of the Black Sea by the Ctenophore Mnemiopsis leidyi and Recent Change in Pelagic Community Structure, .Fisheries Oceanography, vol:7, no: 3- 4,p.p:305-310. Shiganova TA, Bulgakova JV, Volovik SP, Mirzoyan ZA, Dudkin SI., (2000). The new Invader Beroe ovata Esch and its effect on the ecosystem in the northeastern Black Seain August- September 1999 In S. P. Volovik (ed.), Ctenophore Mnemiopsis leidyi (A.Agassiz) in the Azov and Black Seas: its biology and consequences of its intrusion, pp. 432-449 (in Russian) Shiganova TA, (2000), Effect of Glatinous Plankton on Black Sea and Sea of Azov Fish and Their Food Resources, J.Marine Science, vol: 57, p.p.641-648. Shiganova TA, Mirzoyan ZA, Studenikina EA, Volovik SP, Siokoi-Frangou I, Zervoudaki S, Christou ED, Skirta AY, Dumont H, (2001), a review of the invader Proceedings of The Fourth International Iran & Russia Conference 1456

Ctenophore Mnemiopsis leidyi ( A.Agassiz) population development in the Black Sea and in the Other seas of the Mediterranean basin. Marine biology, 139:431-445

Figure Legends

70 60 50 40 30

egg/day/ind 20 10 0 10 12 14 16 18 20 length(mm) Figure 12

600 500 400 300 200 egg/day/ind 100 0 20 22 24 26 28 30 length(mm) Figure 14 Proceedings of The Fourth International Iran & Russia Conference 1457

600 500 400 300 egg 200 100 0 0.5 1.5 2.5 3.5 4.5 wet wigth(gr) Figure15

400 350 300 250 200 150 egg/day/ind 100 50 0 30 32 34 36 38 40 length(mm) Figure16

400 350 300 250 200 egg 150 100 50 0 2468 wet weigth(gr) Figure17 Proceedings of The Fourth International Iran & Russia Conference 1458

Food Ecology of Eurasian Coot Fulica atra (L.) in Caspian Sea Region in Golestan Wetlands, Iran

Seyed Mahmoud Ghasempouri 1, Hamid Reza Rezaee 2, Samira Dantism 3

1-Department of Environmental Science, Natural Resources Faculty of Tarbiat Modares University, Mazandaran, Noor 46414, Iran. Phone: +98-122-6253101 Email: [email protected] 2-Department of Fisheries and Environment, Gorgan University, Iran. Email: [email protected] 3-Department of Environmental Science, Allameh Mohaddes Noori University, Noor 46415, Iran

Abstract In order to primary study of Eurasian Coot Fulica atra (L.) in the wetland of Golestan province, after study of the general situation of wetlands and ecology of this species, during scientific visit of region, from the beginning of the fall until beginning of the winter (2000); hunting of Coot did during four months period and 21 Coot collected. This entire Coot studied morphologically. In this way that, collection of appearance characteristics including: biometry, measurement of body weight; and biologic characteristics including: intestine length, intestine weight and contents of gizzard (in order study of food habits) studied. After collecting of primary data, with using of two statistical methods, “Multiple regression” and “One way analysis of variance test” different variations compared with each other. The results show that correlation between volumes of undigested contents with volume of gravel, weight with body length, volume of digested contents with weight of digested contents, orderly for the first and second cases weren’t meaningful, and it was meaningful for the last one (used method: Multiple regression). The results of compared variations including: weight with regional code, volume of digested contents with regional code, intestine length with regional code, for the first and second case, show that there isn’t meaningful difference; But the last one shows a possibility of meaningful difference (used method: one way analysis of variance test).

Key Words: Coot, Caspian Sea region, Ecology, Food habit

Introduction There are more than 250 small and large wetlands in Iran that are habitat of 140 migratory and native birds. Among this, 77 species use wetland as wintering area and 63 ones have nestlings. One of them is Eurasian Coot (Fulica atra). In Europe there are 46 000 individuals in Britain but absent from the Scottish Highlands. A further 8600 found in Ireland. Winter immigrants push the winter population up to 114100 birds. More than a million breeding pairs found over many areas of Europe but populations become more sporadic in southern France and Spain. Its northern limits are at about 60 degrees north. In Iran Coot is seen in great number in various wetlands during fall and winter throughout the country. They incubate in some inland waters. In addition to incubatory they exist in wetlands of Golestan province and in the south of Caspian Sea region, Alagule, Ajigule, Almagule, Gomishan and protected wetlands of the Gorgan gulf and Incheh wetlands as spending winter and passing migrant. Coot is waterfowl that is hunted more in Iran, but there is a little information about its ecology, feeding and population in spite of a lot of hunting and highly economical value (Dantism, 2000). An investigation had done on the ecosystems of wetlands in Golestan Province and they referred to Coot situation in animal checklist in this area (Kiabi et al., 1999). It was discussed on inter specific variations of Coot in Anzali, Gomishan and Proceedings of The Fourth International Iran & Russia Conference 1459

Fereydoonkenar wetlands (Yazdandad, 2000). Japanese studies shown their nestling on artificial objects, some evidence is available that aquatic ecosystem changes by humans have made the condition of its different growth and distribution both desirable and undesirable (Hiraoka, 1996). It was studied its winter spending in Ireland (Irwin and Halloran, 1997). The studies in Poland determined food habits of 3+ Coots: macrophytes of Characeae family and Lemna sp and Zebra Muscle (Dressena Polymorpha L.) (Dabrowolski et al., 1996). Nutrition of Coot nestlings an dry and wet years was studied in Spain during 10 years. Parents feed chickens in dry years and by themselves in wet years (Amat, 1995). The goal of this study is to determine the differences of food items of Coot wetlands of the south of Caspian Sea region and it has been tried to be studied relationship between habitat conditions and feeding behavior and body size variations.

Materials and Methods a. Area: Alagule (2500 ha) 37˚, 20ƍ north latitude and 54˚, 35ƍ east longitude and –6 m altitude under oceans level. Ajigule (320 ha) is located in north west of Alagule with 37˚, 26ƍ north latitude and 54˚, 40ƍ east longitude and –4 m altitude under oceans level [Figure 2-1].

Fig. 2-1 Map of study area, two wetlands in the north east of Gorgan city b. Fauna and Flora: These plants are 9 families and consist of at least 12 aquatic plants. Some dominant genuses are Typha, Ceratophylum, Juncus, Potamogeton and Myriophyllum. Potamogeton pectinatus resistance against brackish water is the most ones. The density of Alagule aquatic plants is almost ¼ Ajigule. In spite of other animals phylum or classes, reptiles, amphibians, birds and mammals. It has been reported 13 fish species of 4 families and 12 genuses, 11 genus and 12 species are of Cyprinidae family. There are 7 exotic species, which are released in these wetlands with aquaculture approach or entered there accidentally. There is the considerable population of fish in the wetlands. c. Sampling: 21 samples of Coot were hunted by gun during 4 month in fall and winter 2000. They were carried out biometry and were studied the characteristics of morphometric and meristic. Apparent characters of bird (6 characteristic) were measured with accuracy 0.01 cm. Proceedings of The Fourth International Iran & Russia Conference 1460

Biological characters (4 characters) consisted of intestine length, intestine weight, and food items, chemical analyses of dead bodies of Coot were performed. The investigation of food behavior was done by considering gizzard contents with binoculars 40X and was studied digested and indigested items, plants, animals, gravel stones and volume of stomach contents.

Results The following crude data obtained after studies of apparent characters different biological characters [table 3-1 and 3-2].

Table 3-1 The result of volumetric and weight study of food contains

No. Intestine Digested Undigested Digested Undigested Sand Sample Length Weight Weight. Volume Vol. Plants Volume No. 1 106.20 1.19 13.10 1.90 0.00 5.60 6 2 119.10 1.63 44.67 2.00 0.00 4.10 7 3 97.10 0.00 6.08 0.00 0.00 2.30 8 4 114.00 0.53 7.70 0.50 0.60 3.20 9 5 98.00 0.88 6.97 1.50 0.00 2.70 10 6 94.40 0.12 4.71 0.50 0.00 1.60 11 7 ...... 18 8 ...... 19 9 ...... 20 10 117.80 4.96 6.32 5.00 1.50 1.60 1 11 88.30 0.00 0.54 0.00 0.41 0.00 2 12 98.20 0.00 9.27 0.00 0.10 3.70 3 13 150.60 4.12 6.18 4.50 0.00 3.30 4 14 107.20 5.78 4.98 4.00 0.30 2.60 5 15 88.00 1.23 3.04 2.00 1.50 0.30 12 16 152.10 0.19 8.67 0.50 0.80 2.70 13 17 153.40 0.34 3.45 0.50 0.40 1.00 14 18 133.80 0.75 7.41 2.00 0.35 2.70 15 19 141.80 0.34 8.33 1.00 3.75 0.85 16 20 140.30 0.66 5.60 1.50 0.70 1.80 17

Table 3-2 Biometric data of weight and length determination

No. Code Weight Body Paired Wing Tarsus Beak Intestine Length Wing Length Length Length Weight 1 1 840 41.50 76.50 22.10 . 3.55 37.14 2 2 500 37.70 63.50 20.00 . 2.78 35.76 3 2 600 39.30 70.00 21.40 . 2.86 25.80 4 2 500 38.30 67.00 19.20 . 2.77 25.82 5 2 400 39.20 67.30 18.50 . 2.72 28.89 6 2 700 37.40 77.00 22.10 . . 22.98 7 1 450 36.50 70.00 20.30 . 2.85 . Proceedings of The Fourth International Iran & Russia Conference 1461

8 1 700 44.50 73.20 20.20 . 2.76 . 9 1 560 38.70 70.40 22.40 . 2.78 . 10 1 575 40.20 73.30 21.50 6.10 2.72 40.71 11 1 325 37.80 69.80 19.00 6.19 2.93 20.16 12 1 500 36.80 71.20 20.40 6.39 2.85 28.64 13 2 450 37.20 68.20 19.30 6.03 2.98 51.10 14 1 400 38.10 71.20 20.10 6.16 2.69 31.28 15 1 725 41.10 71.10 19.10 6.78 2.63 55.27 16 1 700 41.60 75.10 29.00 7.16 2.69 63.19 17 1 525 41.30 70.30 19.20 6.75 2.79 58.89 18 1 700 40.50 72.10 20.20 7.13 . 61.38 19 1 700 41.40 70.20 21.10 7.32 2.85 55.86 20 1 710 41.20 71.30 21.40 6.60 2.96 57.99

Discussion Comparison between the volume of indigested herbal food contents and the volume of gravel stones: This relation was studied by forward regression. It seems that there will be % 85 errors if we accept any relation. Therefore the rate of stones in gizzard depends on other factors [table 4- 1].

Table 4-1 One-way analysis of variance test between the volume of indigested herbal food contents and the volume of gravel stones

D.F Sum of Square Square Mean

Regression 1 3.46692 3.46692 Multiple R 0.49362 Residual 15 10.76159 0.71744 R Square 0.24366 Adjusted R S 0.19324 F = 4.83235 Sig. F = 0.0440 Standard Error 0.84702

Comparison between weight and area code: The weight of hunted samples from Alagule and Ajigule wetland (code: 1 and 2) were compared by one-way ANOVA method, because table F (0.2366) is smaller than calculated F (1.4993) in 0.05 level. Null hypothesis, H0 means that equivalence between the weight of hunted samples in two wetlands is not accepted. Perhaps the habitat Quality and genetic are important factors [table 4-2].

Table 4-2 One-way analysis of variance test between weight of Coot in Alagule and Ajigule

Source of Var. D.F Sum of Square Square Mean F F table

Between Groups 1 283339.28 28339.28 1.4993 0.2366 Inter Groups 18 340235.71 18901.98 Total 19 368575.00 Proceedings of The Fourth International Iran & Russia Conference 1462

Intestinal length changes with area code have significant differences in 0.01 levels too. Relations between weight and body lengths are not considerable for total specimens and this can show the lot of variation in the Coot’s population. Philopatry evidence shows the birds’ return to its nestling colonies so may be it can be said the differences are related to habitat conditions between Alagule and Ajigule wetland. Literature review has shown Coots have a Good flexibility related to urban ecology and meddling in natural ecosystems. It may happen in two wetlands genetics and population studies are highly required with more samples and monitoring for many years.

Acknowledgment This study was made in the laboratory of Allameh Mohades University, so I appreciate university panel especially Dr. M.Ghanbarzadeh and M.Salar.

References Amat, J.A., 1995, Parent – Offspring Feeding Relationship of Coot (Fulica atra) in a varying Environment, Behavior 132 (7-8): 519-528 Dantism, S., 2000, An Introduction to Coot (Fulica atra) Study in Golestan Wetlands, B.Sc Thesis, Allameh Mohades University Dobrowolski, K.A., B., Leznicka, R., Halba, 1996 Natural Food of Duck and Coots in Shallow Macrophyte Dominated Lake: Lake Luknajno (Masurian Lakeland, Poland), Ecol. Polska 44 (3-4): 217-287 Hiraoka, T., 1996, Utilization of Artificial Floating Objects as Nest Platforms by Little Grebs and Eurasian Coot in Lake Tegnuma, Central Japan, Journal of the Yamashina Institute for Ornithology 28 (2): 108–112 Irwin, S., O., Halloran John, 1997, The Wintering Behaviour of Coot Fulica atra (L.) Cork Lough, South- West Ireland, Biology and Environment. Oct. 97B (2) 157-162 Kiabi, B., R., Ghaemi, A., Abdoli, Wetland and Riverain Ecosystem of Golestan Province Department of Environment (DOE), Golestan, Iran Yazdandad,?., 2000, ???? M.Sc Thesis of Environment. Tehran University Proceedings of The Fourth International Iran & Russia Conference 1463

Determination of heavy metals content in water, sediments and muscle of Crayfish, Astacus leptodactylus in Southern Coasts of Caspian Sea (Abbasa river of Nour city) Seyyed V. Hosseini1, Seyyed M. Amininasab2, Reza Tahergorabi3, Abbas E. Sari4 and Sadegh Bor5 1-5: - Fishery Department, Faculty of Natural Resources and Marine Sciences, Tarbiat Modares University, Iran. Phone: +98-122-6253102 Email:[email protected]

Abstract Rivers are the sensitive ecosystems due to their conditions. Determination of pollutant such as heavy metals in benthic samples is one of the most important indices for studying of rivers pollution. In this study, sampling of water, sediments and muscle of Crayfish, Astacus leptodactylus, was carried out in six stations of river. Then, the heavy metals chemical analysis of the samples was done by Atomic Absorption Spectrophotometer (A.A.S ). Mean rate of Cu, Pb, Cd, Cr, Zn, Ba and Fe in water of inspected stations was 6.95, 1.33, 2.62, 3.28, 124.2, 134.96 and 5.06 mg/lit, respectively, that aren’t suitable to drink (in the base of global standards), although this water can be used for agriculture and aquaculture. Also, heavy metals content in the sediments had been in the tolerance ranges for aquatic animals. Mean contents of heavy metals in muscle of Crayfish, Astacus leptodactylus, inspected stations are 45.73, 2.44, 3.27, 184.20, 145.12 and 9.66 for Cu, Pb, Cd, Cr, Zn, Ba and Fe respectively (mg/kg dry weight) that is less than range of sanitary standards for human consumes. There is significant correlation between heavy metals contents in water, sediments and Crayfish, Astacus leptodactylus in Abbasa river.

Keywords: Astacus leptodactylus, Heavy metals, Crayfish, Caspian Sea, Abbasa River.

Introduction Rivers are valuable aquatic ecosystems because of their conditions, plant and animal diversity and their importance (Bilgrami et al., 1996). Due to industrialization, the number of factories and population have been increased rapidly (Merian, 1991; Chen and Chen, 2001). That is way, massive amounts of domestic wastewater and industrial effluents, with heavy metals, are entered in rivers (Gibbs and Miskiewicz, 1995). These contaminants entering the aquatic ecosystem may not directly damage the organisms; however, they can be deposited into aquatic organisms through the effects of bioconcentration, bioaccumulation and the food chain process (Chernoff and Dooley, 1979; Eromosele et al., 1995) and eventually threaten the health of humans by seafood consumption. Heavy metals include a great variety of chemical elements that typically occur in low or trace amounts in the environment and all have the potential to provoke toxic effects in organisms. Heavy metals occur in nature in very low concentrations, yet are capable of exerting biological effects at concentrations within a few orders of magnitude. In systems as heavily disturbed as rivers, the use of biological sentinels is a most interesting way of obtaining continues assessment to environmental quality (Anton et al., 2000). Bioindicators should be relatively sedentary or resident in the area of interest, easy to identify, abundant, long-lived, available for sampling all year, and with a wide distribution. They should be relative tolerant to environmental stressors, be net accumulators of the pollutants in question, and provide sufficient tissue for individual analysis (MacFarlane et al., 2000). Crayfish is an index to show the probable risk factors of pollution in rivers (Stinson and Eaton, 1983; Anderson and Brower, 1978; Evans, 1980; Madigosky et al., 1991; Schilderman et al., 1999; AntÓn et al., 2000; Payedar et al., 2001). So it was decided to Proceedings of The Fourth International Iran & Russia Conference 1464 analyses these factors to know the quantity of them and adopting some strategies against the risk factors. In this study, heavy metal concentrations, Barium (Ba), Cadmium (Ca), Chromium (Cr), Copper (Cu), Iron (Fe), Lead (Pb) and Zinc (Zn), were analyzed on the eatable portion of A. lepthoducthylus, water and sediments. The result could evaluate the seafood consumption safety in Abbassa River. In addition, it could establish a baseline for the heavy metals deposited in the crustaceans of this region in order to monitor heavy metal pollution trends in the future.

Materials and methods Samples were captured at the end of summer 2003 in six points on the Abbassa River in southern of Caspian sea. Because there isn’t any significant correlation between in size or weight of crayfish with amount of metal concentrations in its body (Martin et al., 1998-9; Payedar et al., 2001), the crayfishes with weight of 20±1gr. At each sampling point, 20 individuals were selected. In addition, in all sampling areas, samples of sediments and water were taken for analysis too. We analysis all samples for heavy metal contents i.e., Ba, Ca, Cr, Cu, Fe, Pb and Zn. Muscle of crayfish burned at 450˚C for 16h. Each sample was treated with 2ml of 1:1 mixture of concentrated HNO3 and distilled water and subsequently boiled for 10 min on a hot plate. The remaining solutions were filled up to 10 ml with distilled water. Samples of sediment were dried at 90˚C to a constant weight and subsequently ground to powder using an agate mortar. Samples (0/5 gr) were subsequently boiled under reflux for 2h using Aqua Regia. After filtration the filtrate was diluted with 0/1% HNO3. Water samples were acidified with concentrated nitric acid to PH<2 (Van, 1980). Atomic absorption spectrometry standards for heavy metals (Faculty of Natural Recourse and Marin Science, Noor-Iran) were used in specimen analysis.

Results Each sample was processed and analyzed three times. Table 1, 2 and 3 present the average content of Ba, Ca, Cr, Cu, Fe, Pb and Zn in water, sediments and crayfish muscle at the six different sampling sites, respectively. Sample analysises were shown that the highest concentrations of heavy metals are found in sixth station (downstream) and lowest concentrations for Cd, Cr, Cu, Fe and Zn is found first station, but lowest concentrations for Ba and Pb is found second station. There was significant difference between concentration each of elements in the water, sediments and muscle of crayfish (p<0/05). According to heavy metals concentration in the water and results of LSD, there was more significant difference between Cd, Cu, and Pb with Ba and Zn (p<0/05). This difference was significant between Ba with Cd, Cr and Zn with Cd, Cr, Cu, Fe & Pb and Fe with Ba & Zn, but there isn’t any significant difference between their remainder (P>0/05). In sediment samples, there was significant difference between Cu with Ba, Cd, Cr, Pb & Zn and Zn with Ca, Cu & Pb and Cd with Cu, Cr & Fe and Fe with Ba & Zn (p<0/05), where as, there isn’t any significant difference between their remainder (P>0/05). In crayfish specimens, except for Ba and Pb, for other elements highest pollution levels were observed in downstream, where as lowest pollution levels were observed in upstream for all of elements. There was significant difference between Pb with Ba, Cu & Zn and Cd with Ba, Cu & Zn and Cr with Ba, Cu & Zn and too Ba, Cu and Zn with other elements (p<0/05). But there isn’t any significant difference between their remainder (P>0/05).

Discussion Proceedings of The Fourth International Iran & Russia Conference 1465

Water quality with respect both organic and inorganic pollution can be assessed on the basis of chemical measurements, but this often yields limited information about the bioavailability of these substances (Kraak et al., 1991). In biomonitoring studies it may, therefore, be more reliable to analyze indigenous organisms such as crayfish, since they have been exposed their entire lifetime and may therefore give a reflection of the contamination history of a particular location (Kraak et al., 1991) General pattern between the metals in water at the six different locations was Ba > Zn > Cu > Fe > Cr > Ca > Pb. In this study, highest pollution levels were observed in downstream and lowest pollution levels were observed in up and midstream. This condition created because of human different activities like to residential regions development, agriculture, transport activities and etc. In this region, we distinguished that the main resource of pollution and effective parameters on rate of Pb and Cd is settle 80 percent of river in neighbor with rod and car transports [Pb is predominantly derived from automobile emissions (Hoffmann and Wynder, 1977; Merian, 1991)]. Cr and Pb have less solvent than other elements (Bowen, 1979), therefore, their amount were less of other elements in Abbasa river but amount of Ba and Zn were higher than of othere elements because they have more solubility and more find in earth shell (Schilderman et al., 1999). Comparing mean concentration of heavy metals in Abbassa river with World Health Organization (WHO) and Environmental Conservation Organization (ECO) standards (Fusher et al., 1996; Mierzykowski and Carr, 2000) indicated that water of river isn’t suitable for human drinking but it can be used for agriculture and aquaculture. Sediments of bed are the most important part of river for storing the pollution, especially for heavy metals, in aquatic ecosystem (King et al., 1999). In this study, general pattern between the metals in Sediments at the six different locations was Zn> Ba> Cu> Fe> Cr> Ca> Pb. Like to condition of disturbed about water of Abbasa river, this condition created because of human different activities like to residential regions development, agriculture, transport activities and etc. That is way, the maximum of heavy metals is found in downstyream (stations 5 and 6). Comparison of mean concentration of heavy metals in sediments of Abbassa river with suitable standards for benthoses, showed that only content of Zn was higher than of acceptance range for benthoses. In our studies, there was high significance and positive correlation between metal concentrations in water and sediments. Metal concentration in crayfish muscles related to the pollution status of the regions, so that, the lowest and highest observed in stations 1 and 6, respectively. At six different locations the following pattern was seen: Zn > Ba > Cu > Fe > Cr > Ca > Pb, which was similar to heavy metals concentration in sediments. Similar to sediments, condition created because of human different activities such as to residential regions development, agriculture, transport activities and etc. This results conclude of other researcher Blevins and Pancorbo, 1986; Creswell, 1993; Batlcy, 1996; Martin et al., 1998-9; Mierzykowski and Carr, 2000; Payedar et al., 2001. Significant relationships were found between river conditions (water and sediment) and heavy metal contents in crayfish, so that, with increase of heavy metals concentration in downstream of Abbasa river, heavy metals concentration in crayfish muscle increased. The comparison of heavy metals in crayfish muscle of Abbasa river with FAD standard, shown that heavy metals concentration in crayfish muscle is at acceptable range for human consumption. Usually it is not a single substance that penetrates into water bodies but a number of chemical substances belonging to the same or different groups. Thus, aquatic organisms are being affected by mixtures of toxic substances of different origins (Mickơnienơ and Proceedings of The Fourth International Iran & Russia Conference 1466

Šyvokienơ, 1999). Due to this, benthic invertebrate, such as crayfish, live and feed directly on deposited sediments, are in direct contact with pollutants, for example heavy metal. Therefore, it appears that crayfish could be used bioindicator or for biomonitoring of pollution in river systems.

Reference Anderson MB, Preslan JE, Jolibois L, Bollinger JE, George WJ (1997) Bioacumulation of lead nitrate in Red Swamp crayfish (Procambarus clarkii). J. Hazard. Mater. 54, 15–29. AntÓn A, Serrano T, Angulo E, Ferrero G, Rallo A (2000) The use of tow species of crayfish as environmental quality sentinels: the relationship between heavy metal content, cell and tissue biomarkers and physico-chemical characteristics of the vironment. The Science of the Total Environment 247: 239-251 Chen Y, Chen M (2001) Heavy metal concentrations in nine species of fishes caught in costal waters off Ann-Ping, S.W. Taiwan. Journal of Food and Drug Analysis 9(2): 107-114 Chernoff B, Dooley JK (1979) Heavy metals in relation to the biology of the mummichog Fundulus heteroclitus. J. Fish Biol. 14: 309-328 Eromosele CO, Eromosele IC, Muktar SLA, Birdling SA (1995) Metals in fish from the upper Benue river and lakes Geriyo and Njuwa in northeastern Nigeria. Bull. Environ Contam. Toxicol. 54: 8-14 Evans ML (1980) Copper accumulation in the crayfish (Oeconectus rusticus). Bull. Environ. Contam. Toxicol. 24: 916-920 Gibbs PJ, Miskiewicz AZ (1995) heavy metal in fish near a major primary treatment sewage plant outfall. Mar. Pollut. Bull. 30: 667-674 Hoffmann D, Wynder EL (1997) Organic particulate pollutants: chemical analysis and bioassays for carcinogenicity. In: Air pollution, Vol. 2 (A. C. Stern, Ed.). Academic press, New York. Kraak MHS, Scholten MCT, Peeters WHM, Kock WCH (1991) Biomonitoring of heavy metals in the western European rivers Rhine and Meuse using the freshwater mussel, Dreissena polymorpha. Environ. Pollut. 74: 101-114 MacFarlanne GR, Booth DJ, Brown KR (2000) The semaphore crab, Heloecius cordiformis: bio-indication potential for heavy metals in estuarine systems. Aquat. Toxicol 50: 153-166 Madigosky SR, Alvarez-Hernandez X, Glass J(1991). Lead, cadmium and aluminum accumulation in the red swamp crayfish, Procambarus clarkia G. collected from roadside drainage ditches in Louisiana. Aech. Environ. Contam. Toxicol. 20: 253-258 Martin G, Monica AJ, Insidor J (1998-9) Heavy metals in the Rock oyster, Crassostrea iridescens, from Mazaltan, Sinaloa. Mixisco Ronson-Paulim, 8 Merian E (1999) Metal and their compounds in the environment: occurrence, Analysis and biological relevance. VCH Verlaggesellschaft, Weinham. Mickơnienơ L, Šyvokienơ J (1999) The effect of heavy metals on microorganisms of digestive trace element of crayfish. Acta Zoologica Lituanica. Hydrobiologia 9(2): 37-39 Payedar M, Saeif FM, Riahi AR (2003) Determination of heavy metals content in Astacus leptodactylous caspicus in Anzali lagon. Iranian Journal of Fisheries Sciences 12: 1- 14 Schilderman PAEL, Moonen EJC, Maas ML, Welle I, Kleinjans JCS (1999) Use of crayfish in biomonitoring studies of environmental pollution of the river Meuse. Ecotoxicology and Environmental Safety 44: 241-252 Stinson MD, Eaton DL (1983) Concentrations of lead, cadmium and copper in crayfish, Pacifasticus leniusculus, obtained from a lake receiving urban runoff. Aech. Environ. Contam. Toxicol. 12: 693-700

Table1. The means of heavy metal concentrations in water of Abbasa river (mg/li). Station Fe Ba Zn Cr Cd Pb Cu No. Proceedings of The Fourth International Iran & Russia Conference 1467

1 3.51 83.16 76.12 2.50 1. 58 1.05 4.52 2 4.36 57.97 105.23 2.88 1.85 0.78 6.28 3 5.00 118.13 170.58 3.15 3.52 1.13 7.18 4 5.24 147.89 183.18 3.41 2.91 1.21 7.33 5 5.91 187.11 191.12 3.72 3.05 1.8 7.96 6 6.37 215.52 199.05 4.02 3.85 2.05 8.47 Minimum 3.51 57.97 76.12 2.50 1.58 0.78 4.52 Maximum 6.37 215.52 199.05 4.02 3.85 2.05 8.47 Mean 5.06 134.96 124.05 3.28 2.62 1.33 6.95 Standard 1.03 60.44 50.94 0.55 0.83 0.48 1.40 Deviation

Table 2. The means of heavy metal concentrations in sediments from Abbasa river (mg/li) Station Fe Ba Zn Cr Cd Pb Cu No. 1 5.12 141.82 113.16 5.05 2.63 2.87 37.12 2 8.83 107.27 193.73 5.64 2.91 2.35 51.01 3 11.97 194.82 218.12 6.17 3.39 2.97 58.43 4 14.18 221.28 305.02 6.93 4.08 3.16 66.18 5 17.05 265.95 328.51 7.32 5.11 3.27 81.74 6 18.64 301.12 394.36 8.71 6.92 3.93 90.51 Minimum 5.12 107.27 113.26 5.05 2.63 2.35 37.12 Maximum 18.64 301.11 394.36 8.71 6.92 3.93 90.51 Mean 12.63 205.37 258.81 6.63 4.17 3.09 64.16 Standard 5.08 73.29 102.44 1.31 1.61 0.52 19.71 Deviation

Table 3. The means of heavy metal concentrations in muscle of crayfish from Abbasa river (mg/kg dry weight). Station Fe Ba Zn Cr Cd Pb Cu No. 1 4.05 102.05 94.05 2.14 2.05 1.83 21.63 2 5.89 89.63 127.27 3.26 2.21 1.64 33.08 3 7.37 129.63 185.83 2.38 2.83 2.09 40.83 4 10.11 161.05 212.41 3.85 3.42 2.47 49.12 5 14.27 187.13 232.05 4.14 4.18 2.93 58.63 6 15.92 201.27 253.61 4.63 4.95 3.71 71.12 Minimum 4.05 89.63 94.05 2.14 2.05 1.64 21.63 Maximum 15.92 201.27 253.61 4.63 4.90 3.71 71.12 Mean 9.66 145.12 184.20 3.40 3.27 2.44 45.73 Standard 4.72 45.48 62.08 0.99 1.13 0.77 17.81 Deviation