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Biochemical analysis

At the each sampling time, three fish per tank were sampled and whole body of the fish were used to ectract the ions (Postlethwaite and Mcdonald.,1995). Samples were homogenized using DDW and then centrifuged for 15 min at 10,000 rpm and plasma was collected for the estimation of plasma electrolytes (Na+, K+ and Cl-).

Calculation of chloride cells

Fish per each concentration were sampled and sacrificed after 24 and 96 h of exposure in both experiments. Tissues were fixed in 10% buffered formalin (Roberts, 1989) , processed, placed in paraffin blocks, Sections of 4 |im were prepared from paraffin blocks by using a Reichert microtome, and stained with haematoxylin and eosin. Photos were taken using Nikon EC 600 Eclipse microscope. The number of chloride cell in the basal and between five primary and secondary lamella was counted.

Statistical analysis

Statistical analyses were performed using SPSS (version 19) software. All the data were tested for normality (Kolmogorov-Smirnov test). Data were analyzed by one-way of variance analysis (ANOVA). The significant means were compared by Duncan test and a p < 0.05 was considered statistically significant.

Result

Caspian roach fingerlings were exposed to sublethal concentrations of diazinon according to 96 h LC50 value (Mohammadnejad shamoshaki and Shahkar, 2010). Sublethal toxicity of diazinon on plasma biochemical parameters Mean values of plasma biochemical parameters and the number of chloride cell of caspian roach fingerlings exposed to sublethal concentration of diazinon are presented in Table 1 and Fig 1 respectively.

Table 1. Cjhanges in Plasma biochemical parameters in Caspian roach (Rutilus rutilus) during and after exposure to sub-lethal concentrations of diazinon

Biochemical parameter!

 

Exposure period (hour)

 

Located in sea water period (hour)

 

Oh

241

48 h

721

96 h

01

481

96 h

Sodium (mmolL -)

 

 

 

 

 

 

 

 

1 -lill"

 

138.66*0.57*

142.33*1.52rf

141.31*1.15^

140.66*0.57^

140.66tO.571

145.66*3.51'

134.бб±2.51а

Diazinon (1 rngL-1)

139.66i 1.52м

Ш.ООіі.ОО"

139.rjOirj.51*1

138.33il.52*

140.00i0.53M

140.O0iO.531

142.00il.001'

145.33il.521

DiaHnoat^mgL1)

139.66* 1.І21"

140.66*1.15*

140.66*1.15*

137.66*0.57'

13P.66iO.571"!

139.66*0.57"

132.66*3.21'

142.33i2.511-

Diazinon (3 mg I/1)

139.66il.52lu:

Ошибка! Недопустимый объект гиперссылки.

142.66il.15'

138.33il.15*

138.33i0.53*

138.33i0.531

141.33i3.211

141.33i2.081

Potassium (uuhdLL1)

 

 

 

 

 

 

 

 

Control

4.86*0.11*

4.90*0.10і1

4.83*0.05=

4.83*0.05'

4.89±0.09>'

4.89±0.09>

5.63*0254

5.70*0.10!

Diazinon (1 rngL-1)

4.86*0.11»

4.96*0.15'"

5.03*0.05""

5.00*0.17'"

5.03*0.05c"

5.03*0.05*

5.26*0.28"

5.30*010"

Diazinon f2mgL_1)

4.86i0.11*

4.96*0.05*'

5.13*0.11=*

5.10*0.01™

5.10*0.17™

5.10*0.17*'

5.33*0.05"

5.40*0.26"

Diazinon (3 mg L.-1)

4.16*0.11*

5.26±0.11-

5.2O±0.O4<l<

5.26*0.11'

5.23*0.05'

5.23*0.05'=

510*0 20*=

5.20*0.10*'

Chloride (шщі/1)

 

 

 

 

 

 

 

 

Control

100.33i2.081

99.33i2.511

101.66i7.091

100.66i4.721

97.33*5.681

97.33*5.68а

114.33*1.52'

116.00i2.001

Diazinon [1 mgl/1)

100.33i2.081

85.00i3.6O"

110.66iL52«

117.66i5.50"

113.33i2.08"

113.33*2.08'

105.66*1.52'

88.O0il.OO1

Diazinon (2 ffigL-1)

100.33i2.081

ІВббИШР

US.OOil.OO"

122.00^.35"

122.66i2.3il

122.66*2.3-'

114.33i2.08'

76.66i2.081

Diazinon [3 tnt* L"1)

100.33i2.081

84.33i2.08-

119.66*1.15d=

119.00i5.19*

125.33il.52r

125.33il.52-'

114.33*2.51'

70.00i5.001

Changes in Plasma biochemical parameters in caspian roach (Rutilus rutilus) during and after exposure to sub-lethal concentrations of diazinon

:6

0  4— r— —r— —,

0 24 96

l ime period fish exposed to pesticide (hour)

Fig. 1. The number of chloride cells in the basal primary lamella and in five bases secondary lamella

Values are given as means ±S.D. (n= 9)

The life of juvenile fish that entered to sea is related to previous stage of their life in fresh water (Waring and Moore, 2004). Qare Soo river estuary is the restocking place of caspian roach fingerlings might be polluted with different concentration of diazinon (SHAYEGHI et al., 2006).

Alterations in ionic regulation in animals can be affected by environmental stressors. In this study potassium and chloride (except 24 h) levels were significantly increased compared with the control group during the exposure due to haemodilution occurred. Sodium changes were irregularly but more changes were observed in the high concentrations of toxin. Suvetha and Ramesh (2010) found that electrolytes levels decreased in fish that exposed to pesticide. Cause of the rise electrolyte levels may be due to raised passive efflux of ions across the gills and also the restraint of active ion uptake by the chloride cells of the gills. However, it is not clear exactly why it happens but probably related to the particular stage of fingerlings life, because in this course of life they live in a hypo-osmotic environment but their bodies are physiologically ready to adapt to sea water conditions.. Watson and Beamish (1980) reported electrolyte levels in fish exposed to pesticides have been able to return to their initial concentrations. In our experiments we observed that after transferring of fish to brackish water chloride levels decreased.

In this study chloride cell proliferation was examined too. Increase in chloride cells were observed in fish exposed to diazinon, so that at 96 hours in group 3, 30% more than control group. Increases in cell numbers were found in many studies. Wendelaar Boonga (1992) and mallatt (1985) were found increased chloride cells after exposure to mercuric chloride and organic toxicants respectively. Increased numbers of chloride cells may reimburse for the failure of ion uptake capacity caused by the toxic actions of contaminant on these cells.

In summary, capacity of osmoregulation in fish that were exposed to the toxin was reduced in both the river and the sea environments. This degradation is due to tissue destruction and chaos on the electrolytes regulation of blood.

Coad, B.W. (1980). Environmental change and its impact on the freshwater fishes of Iran. Biological Conservation, 19: 51-80.

Fairchild, W.L.. Swansburg, E.O.. Arsenault, J.T..Brown, S.B. (1999). Does an association between pesticide use and subsequent declines in catch of Atlantic salmon (Salmo salar) represent a case of endocrine disruption? Environmental Health Perspectives, 107: 349.

Keyvanshokooh, S..Kalbassi, M.R. (2006). Genetic variation of Rutilus rutilus caspicus (Jakowlew 1870) populations in Iran based on random amplified polymorphic DNA markers: a preliminary study. Aquaculture Research, 37: 1437-1440.

Kiabi, B.H.. Abdoli, A..Naderi, M. (1999). Status of the fish fauna in the south Caspian Basin of Iran. Zoology in the Middle East, 18: 57-65.

Mohammad nejad shamoshaki, M..Shahkar, E. (2010). Determine the lethal concentration (LC50 96) Insecticide clorpirifos and diazinon on Juvenile fish Caspian roach. Iranian journal fisheries, Number 4: 1-7.

Postlethwaite, E..Mcdonald, D. (1995). Mechanisms of Na+ and C-regulation in freshwater-adapted rainbow trout (Oncorhynchus mykiss) during exercise and stress. Journal of Experimental Biology, 198: 295-304.

Roberts, R. (1989). Fish Pathology, 2nd edition. 467.

Shayeghi, M.. HOSSEINI, M..ABTAHI, M. (2006). The determination of dimethoate

insecticide residues upon the cucumber product (Fars Province). Journal of Environmental Science and Technology, 27: 30-35.

Waring, C.P..Moore, A. (2004). The effect of atrazine on Atlantic salmon (< i> Salmo salar</i>) smolts in fresh water and after sea water transfer. Aquatic toxicology, 66: 93-104.

Wendelaar Bonga, S..Lock, R. (1991). Toxicants and osmoregulation in fish. Journal of

Zoology, 42: 2-3.

Histopathological Changes in the Gill of Caspian roach (Rutilus rutilus) Exposed to Diazinon

Kheyrollah Khosravi Katuli*1, BagherMojaziAmiri1 SmaeylSmaeylzadegan1, SaeedYelghi2

1 Department of Fisheries, Faculty of Natural Resource, University of Tehran, Karaj, 31585-4314 Iran

2 Iranian Fisheries Research Organization (IFRO), Inland Waters Fisheries Research, Gorgan, Iran

* E-mail address: khosravLkh@ut.acir

Introduction

The development and optimization of agricultural have led to increase use of pesticides in agriculture which caused in high pollution of aquatic environments. Diazinon are widely used to control the pest population in the rice form of Gillan, Mazandaran, Golestan and other areas in Iran(Shayeghi et al., 2006). This pesticide in Qare Soo rivers estuaries in southeast Caspian sea that is site release of juveniles caspian roach (Rutilusrutilus) , there is reported (Bagheri, 2007).

Fish gills are sensitive to pollutants in water because of their great surface area and outside position. Although many researchers have reported to effect of organophosphate pesticides on gill organ in fish, however there is no information about effects of toxins in the Qare Soo river in north Iran.

The R.rutilus is an economically important species in the Caspian Sea (Coad, 1980) and is also a main food sources for sturgeon species.Such as other Caspian sea fishes (e.g. sturgeons) this species is considered at the risk (Kiabi et al., 1999). There for Iranian Shilat organization attempted to semi-synthetic replication and release of juveniles to the estuary of Qare soo river and while the high levels of diazinon had been reported in this river (Bagheri, 2007). The importance of both marine and freshwater environments in the life cycle of the fishes that have reproductive migration, in the numerous studies has been demonstrated. So that specified, among the fingerlings who have been exposed to the

contaminant and returning their adults there is a correlation (Fairchild et al., 1999). Salinity tolerance and increased hypo-osmoregulatory ability are necessary if fingerlings are to success survive in the marine environment, thus, the histological changes of fish which transferred to the sea water were also evaluated.

The present study was conducted to determine histological changes in fish gills which located to sub-lethal levels of diazinon in fresh water and diazinon-free sea water.

Materials and methods

Caspian roach fingerlings (average weight of 0.770 ± 0.05 g), used in this experiment originated from the Sijowal Caspian sea teleost fish reproduction center (Golestan province, Iran), and were acclimatized for at least 1 week under laboratory conditions. The tanks werecontinuously aerated and 10% of the water was changed daily. The nominal concentrations that the fish were exposed to they were 0 (control), 1, 2 and 3 mg L-1 and triplicate fiberglass tanks (100 L) were concentrated for each concentration. This concentrations was determined according to 1/12th, 1/6th and 1/4th 96 h LC50 value for Caspian roach fingerling that

reported 12 mg L-1 (Mohammad nejadshamoshaki and Shahkar, 2010).

Fish per concentration were sampled and sacrificed after 24 and 96 h of exposure for histological analyses of the gills. Tissues were fixed in 10% buffered formalin (Roberts, 1989), processed, placed in paraffin blocks, Sections of 4 were prepared from paraffin blocks by using a Reichert microtome, and stained with haematoxylin and eosin. Photos were taken whit using Nikon EC 600 Eclipse microscope. All fish surviving 96 h of exposure at various concentrations of diazizon were transferred to separate aquaria with clean seawater (12 mg L-1) and maintained for 96 h.Experimental conditions were similar for the four groups. Fish per concentration were sampled at the end of the 96 h period for histological analyses of the tissues.

No changes were observed in the control fish gill tissue. The structural details of the gill of control group Caspian roachis shown in Fig 1.

At 24 h of exposure to 1mg L-1 diazinon, the gills of experimental fish showed shortening of secondary lamellae and oedema in many areas of secondary lamellae with destruction of epithelial lamella.Epithelial hyperplasia and lamellar fusion, Curling of secondary lamellae, Oedema, Epithelial lifting and Haemorrhage at primary lamella noticed after 96 h of exposure to diazinon (Fig 2). The most change that observed in all three 1 mg L-1, 2 mg L-1 and 3 mg L-1 concentrations of diazinon were collapsed secondary lamellae, oedema, epithelial lifting and shortening of secondary lamellae. After 96 h of entry fish to sea water histopathological change was slightly reduced. The histological changes affected by diazinon and sea water free diazinon are shown in Table1.

Table 1

Siimnianzedlii&topaHiologicaleffecLEin thegil& ofRuiUus rul'ilus expo&edto diazinon and control fi&li

 

 

Shori^oima of

Destruction

- :r:::::":":.= iT

Oedema, and

Collapsed of

Epithelial

Cmlin^ of

Chib-

Concentration (nigL ])

. erni:

s econdary

of eprrhelial

at primary

 

secondary

hyperplasia and

secondary

ec

 

 

Ism diss

lamella

lamella

lifting

lamellae

lamellar fusion

lamellae

lamella

Control

 

 

 

 

 

 

 

 

 

:

24h

-

-

 

-

-

 

 

 

 

96 b

-

-

-

 

-

 

2

24 h

-

 

-

-

 

 

-

 

96 b

 

-

-

-

3

24h

-

 

 

96 h

-

-

Diazinon free sea water

Control

 

 

 

 

 

 

 

 

-

Croup 1

96 b

-

-

-

-

 

-

-

C-rrup -

96 h

-

-

-

-

 

-

Group 3

96b

++

+

+

++

++

 

+

+

None (), mild (), moderate (), and severe(+++).

Discussion

In fish, gills are vital organs for their respiratory, osmoregulatory and secretory functions and histopathological results showed that gill was the main target tissue influenced by diazinon. In this study histological changes include: shortening of secondary lamellae, oedema, destruction of epithelial lamella, Shortening of secondary lamellae, epithelial lifting, curling of secondary lamellae, epithelial hyperplasia and lamellar fusion were observed in the gills after exposure to diazinon.

Res. 1-2. {1) Gill stiuchag of control £He {a) jail filaoi€Et 01 priraary lamella, {□} н^XlKмг-■ lmallaa. £0 ^ttralial call, {d} pilljirf-jll anri {,=■) r"hlf»firfa -.-jII h.-ef tjTO PHll ti am jif p; fjjrjfij.-j arpriijf?t-r. l nrT-: diaziEOE for 9o" t: fa) Epittalial frpatplaE-ia ar_r- lamallai ioioL. ,ti) CuLiee: of E^OLiar-' lEoallaa. |c) Oa^aoia. {c) Epittalial liftinE sd& {a) Haaaontaaa at jMiELar>- lamalla K&.E. x ICO.

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