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Immune responses and behavior alterations of Persian sturgeon fingerlingsAcipenser persicus exposed to sublethal concentrations of diazinonZ. Padash-Barmchia; A. Safahieha; M. Bahmanib; A. Savaria; R. Kazemib

a Marine Biology Faculty, Khorramshahr Marine Science University, Khorramshahr, Iran b

International Sturgeon Research Institute, Rasht, Iran

First published on: 13 November 2009

To cite this Article Padash-Barmchi, Z. , Safahieh, A. , Bahmani, M. , Savari, A. and Kazemi, R.(2010) 'Immune responsesand behavior alterations of Persian sturgeon fingerlings Acipenser persicus exposed to sublethal concentrations ofdiazinon', Toxicological & Environmental Chemistry, 92: 1, 159 — 167, First published on: 13 November 2009 (iFirst)To link to this Article: DOI: 10.1080/02772240902927577URL: http://dx.doi.org/10.1080/02772240902927577

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Toxicological & Environmental Chemistry

Vol. 92, No. 1, January 2010, 159–167

Immune responses and behavior alterations of Persian sturgeon

fingerlings Acipenser persicus exposed to sublethal concentrations

of diazinon

Z. Padash-Barmchia*, A. Safahieha, M. Bahmanib, A. Savaria and R. Kazemib

aMarine Biology Faculty, Khorramshahr Marine Science University, P.O. Box 64199–43175,Khorramshahr, Iran; bInternational Sturgeon Research Institute, P.O. Box 41635–3464,Rasht, Iran

(Received 1 February 2009; final version received 27 March 2009)

Diazinon is an organophosphate pesticide widely used in agricultural areas(especially paddy fields) in Iran. Similar to other pesticides, diazinon mayenter aquatic systems due to run-off and affect various organisms living inrivers and estuaries. Persian sturgeon Acipenser persicus is one of the mosteconomically important species in the Caspian Sea, which enters Iranianrivers, such as the Sefidroud River, for breeding. Fish eggs hatch in theriver and fingerlings migrate to the sea after a period of time. TheInternational Sturgeon Research Institute in Rasht, Iran, produces andreleases millions of fingerling sturgeons into the coastal area of the CaspianSea annually and the fingerlings may thus be affected by this pesticide. Thisstudy was carried out to determine sublethal effects of diazinon on somehematological indices of Persian sturgeon under lab conditions. Fish wereexposed to concentrations of 0, 0.01, 1.095 (0.25 LC50), 2.19 (0.5 LC50) or3.285 ppm (0.75 LC50) diazinon and responses were studied after 12, 24 and96 h. After 12 h exposure, the fingerlings exposed to 2.19 and 3.285 ppmrevealed a series of abnormal responses such as muscle paralysis, change ingills and abdominal color, erratic swimming, rapid operculum movement,and loss of body balance. The exposed sturgeons also exhibited a signifi-cantly lower number of erythrocyte (RBC) and leukocyte count (WBC)after 24 and 96 h exposure. Similarly, after 96 h, there was a significantdecrease in hematocrit (Htc). Results also revealed a decrease inlymphocyte numbers and an increase of meta neutrophils, band neutrophilsand total neutrophils after 24 and 96 h diazinon. Data suggest that diazinonaffects physiological and hematological functions of A. persicus, which mayfinally affect survival of the fish. Diazinon concentration of 0.01 ppm hasbeen recorded in water of the Sefidroud River, where about five millionPersian sturgeon fries are released annually by the Iranian department offishery hatcheries. Since this level of pesticide was found to be toxic for fishfries, it is critical to avoid any toxic impact by regular monitoring ofdiazinon levels in the river water.

Keywords: diazinon; Persian sturgeon Acipenser persicus; hematologicalindices; toxicology

*Corresponding author. Email: zhrpadash@gmail.com

ISSN 0277–2248 print/ISSN 1029–0486 online

� 2010 Taylor & Francis

DOI: 10.1080/02772240902927577

http://www.informaworld.com

Downloaded At: 10:15 8 January 2011

Introduction

Diazinon is an organophosphate (OP) insecticide widely used to control insects inagricultural areas. Diazinon is transported and persistent in the environment(Phonon and Zhang 2002). Due to its chemical properties, widespread use andapplication, diazinon is frequently found at both point sources (wastewatertreatment plant effluent) and nonpoint sources (storm water run-off) in urban andagricultural areas. Diazinon is known to be extremely toxic to birds and aquatic life(Environmental Protection Agency (EPA) 2003).

Fish like sturgeons are particularly sensitive to environmental contamination, whichmay result in certain physiological and biochemical changes associated with exposureto pollutants when they enter the fish body (Lang et al. 1997). Therefore, analysis ofhematological and biochemical parameters of fish can contribute to the assessment offish health (Thrall 2004), and enable one to determine the extent of pollution.

Many studies showed that diazinon alters hematological indices in several fishspecies such as Silurus glanis (Koprucu et al. 2006), Acipenser nudiventris(Khoshbavar-Rostami and Soltani 2005), Acipenser stellatus (Khoshbavar-Rostami, Soltani, and Yalghi 2005), Acipenser guldenstadti (Soltani andKhoshbavar-Rostami 2002), and Huso huso (Shamloofar et al. 2007). Diazinon iswidely used in agricultural areas (especially paddy fields) in Iran. However, theeffects of diazinon on Persian sturgeon Acipenser persicus, which is one of the mosteconomically important species in the Caspian Sea and enters Iranian rivers such asthe Sefidroud River for breeding, are not known. Fish eggs hatch in the river andfingerlings migrate to the sea after a period of time. The International SturgeonResearch Institute located in Rasht produces and releases millions of fingerlingsturgeons into the coastal area of the Caspian Sea annually. Studies conducted by theInternational Sturgeon Research Institute indicated the presence of considerableamounts of diazinon in water samples of the Sefidroud River (Arshad et al. 2006).Thus, the aim of the present study was to investigate the effect of diazinon on thebehavior and hematological parameters of A. persicus.

Materials and methods

Test fish

Acipenser persicus fingerlings (weight 6� 2 g) were obtained from the fish propagationand rearing unit of the International Sturgeon Research Institute in Rasht, Iran andtransferred to the lab. The fish were acclimatized to lab conditions in maintenancetanks a week before starting the experiments. Five hundred liter fiberglass tanks wereused for the experiments. The experimental tanks contained 300L of dechlorinatedtap water. Water quality parameters were regularly checked during experiments.Throughout the experiment, the water temperature was maintained at 24.7–26.8�C,dissolved oxygen level was maintained at 7.2–7.5mgL�1, pH ranged from 7.3 to 7.7and total hardness was 180–182 ppm measured with CaCO3. Control and experi-mental fish were not fed for 24 h before and throughout the toxicity tests.

Toxicant preparation

Commercial diazinon [O,O-diethyl-O-(2-isopropyl-6-methyl-4-pyrimidinyl) phos-phorothioate], Basudin 60EC, dissolved in 40% acetone was used to prepare

160 Z. Padash-Barmchi et al.

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test solutions. The stock solution of 104 ppm was prepared using double-distilled

water in a volumetric flask. Appropriate amounts of stock solution were added to

obtain the final desired concentrations.

Experimental protocol

Fish were divided into five different groups each containing 15 individuals in three

replicates. Exceeding aeration was applied to the aquariums for 2 h in order to obtain

a homogeneous concentration of the toxic compound by air-stones. Sublethal

concentrations were selected based on 96 h LC50 value for A. persicus (4.38 ppm)

determined by Pajand, Esmaeli-Sari, and Piri-Zirkoohi (2003). The first group was

held in freshwater without toxicant as control. Other groups were exposed to 0.25,

0.5, or 0.75 diazinon 96 h LC50 (1.095, 2.19, and 3.285 ppm, respectively) and

0.01 ppm diazinon (toxicant concentration in the Sefidroud River) for 24 and 96 h.

Observations

During the toxicity tests fish behavior including active swimming, loss of balance,

abnormal swimming, and some visible changes in physiological activities such as

breathing frequency and color alterations in skin were observed and recorded.

Blood sampling and hematological assay

At the end of each exposure period (24 and 96 h), three to four fish from each

experimental tank were removed, and 1.5mL blood samples were collected from the

caudal peduncle and immediately analyzed for erythrocyte count (RBC,� 106 mL�1),total leukocytes (WBC,� 103 dL�1), hematocrit (packed cell volume, %), and

differential leukocyte count. The numbers of erythrocytes and leukocytes were

determined by the hemocytometry method (Stevens 1997); hematocrit was

determined by the microhematocrit method (Goldenfarb et al. 1971). All procedures

used were based on the hematological techniques described by Campbell (1990).

Statistical analysis

The replicates were used for calculation of the mean values. The hematological

parameters were expressed as mean� SD. Hematological differences between

various concentrations, and between different exposure times were statistically

compared using one way analysis of variance (ANOVA) followed by Duncan’s

multiple comparison test. Significant differences were determined at the level of

�¼ 0.05. Statistical analyses were performed using SPSS program version 13.

Results

Effects of diazinon on behavior

After exposure of A. persicus to different diazinon concentrations, the fish exhibited a

series of abnormal behavioral responses including muscle paralysis, changes in

operculum and abdominal color, erratic swimming, rapid operculum movement, and

Toxicological & Environmental Chemistry 161

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loss of body balance after 12 h of exposure to 2.19 ppm and 3.285 ppm. The testorganisms were also more aggressive and exhibited excessive mucus secretion after12 h in all the exposed groups. There were no fish mortalities in the control groupduring the experiment.

Effects of diazinon on hematological indices

Table 1 shows the hematological parameters of the test organisms under control andexperimental conditions. After exposure to diazinon (0.01, 1.095, 2.19, or3.285 ppm), erythrocyte and leukocyte density decreased significantly after 24 h.The number of lymphocytes decreased significantly, while meta neutrophil, bandneutrophil, and total neutrophil counts increased significantly after 24 h of diazinonexposure.

After 96 h of diazinon exposure erythrocyte and leukocyte density, lymphocyteand segment neutrophil levels significantly decreased, while meta neutrophil andtotal neutrophil levels increased. After 96 h exposure to all diazinon concentrations,the levels of band neutrophil, meta eosinophil, band eosinophil, segmentedeosinophil, total eosinophil, and monocyte in exposed organisms were notsignificantly different from control. The results of average differential leukocytecount after 96 h are shown in Figure 1.

Discussion

Organophosphate insecticides produced marked alterations in blood parameters offish, which threatens their survival. Measurement of biochemical and physiologicalparameters is a commonly used diagnostic tool in aquatic toxicology andbiomonitoring (McDonald and Milligan 1992; Folmar 1993; Soimasuo et al. 1995;Kang, Lee, and Jee 1999, Kang, Kim, and Jee 2003). In this study, diazinon wasfound to markedly affect hematological parameters in sturgeon fingerlingssuggesting that the fingerling may be used for biomonitoring for exposure to OPin Iran.

When the Persian sturgeon fingerlings were exposed to diazinon, fish exhibiteda series of abnormal behavioral responses. Erratic swimming and convulsions wereobserved after 12 h exposure to diazinon in A. persicus. In general, fish poisoned withOP insecticides show signs of muscle paralysis, hyperactivity, and loss of balance(Sancho, Ceron, and Ferrando 1998). Dembele, Haubruge, and Gaspar (2000) foundthat abnormalities in fish behavior occurred with OP insecticides (such aschlorfenvinphos, chlorpyrifos, and diazinon). Similar abnormal behavioral responsesof fish were also observed in European catfish S. glanis exposed to diazinon(Koprucu et al. 2006); Anguilla anguilla exposed to carbamate molinate (Sancho,Ceron, and Ferrando 1999); rainbow trout Oncorhynchus mykiss exposed toendosulfan (Capkin, Altinok, and Karahan 2005); A. stellatus exposed to diazinon(Khoshbavar-Rostami, Soltani, and Yalghi 2005), and freshwater catfishHeteropneustes fossilis exposed to cypermethrin (Saha and Kaviraj 2003). Theabnormal behavior of A. persicus exposed to diazinon may be due to the permanentneurological impairment induced by this insecticide.

The present study revealed that exposure to diazinon at low concentrationsresults in significant hematological alterations. The decrease in RBC count and Htc

162 Z. Padash-Barmchi et al.

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Table

1.Hem

atologicalparametersofPersiansturgeonafter

exposure

todiazinon.

Hem

atologicalparameter

Exposure

time(h)

Diazinonconcentration(ppm)

Control

0.01

1.095

2.19

3.285

Erythrocyte

(106cellmL�1)

24

1.168�0.10a

0.922�0.04b

0.887�0.02b

0.857�0.05bc

0.801�0.02c

96

1.168�0.10a

0.904�0.04b

0.847�0.06b,c

0.795�0.02c

_____________

Leukocyte

(103celldL�1)

24

1.250�0.16a

0.785�0.03b

0.740�0.03b

0.690�0.03b

0.685�0.01b

96

1.250�0.16a

0.745�0.01b

0.710�0.01b

0.650�0.02b

_____________

Hem

atocrit(%

)24

24.50�0.50a

24.00�1.32a

22.67�5.80a

22.00�0.87a

20.12�5.22a

96

24.50�0.50a

21.33�1.53b

19.67�1.53b,c

18.50�1.50c

_____________

Lymphocyte

(%)

24

82.67�2.52a

79.33�1.53a

73.00�2.00b

69.67�1.53b

65.67�2.08c

96

82.67�2.52a

77.67�3.06b

69.67�1.53c

65.33�1.53d

_____

Meta

neutrophil(%

)24

1.00�1.00b

2.00�1.00b

5.33�1.53a

7.33�0.58a

7.00�2.00a

96

1.00�1.00c

1.67�1.53bc

4.33�2.08a,b

5.67�1.53a

Band

neutrophil(%

)24

12.33�1.53b

14.00�1.00b

16.00�2.00a,b

14.67�2.08b

18.33�2.52a

96

12.33�1.53b

15.00�4.00a

18.67�1.53a

18.33�3.06a

_______________

Segment

neutrophil(%

)24

2.00�1.00a

1.67�0.58a

1.33�1.53a

3.67�0.58a

2.67�1.53a

96

2.00�1.00b

1.33�0.58b

2.00�1.00b

4.67�1.15a

____________

Total

neutrophil(%

)24

15.33�1.53c

17.67�1.53c

22.67�4.73b

25.67�2.08ab

28.00�1.73a

96

15.33�1.53b

18.00�5.29b

25.00�1.73a

28.67�3.51a

Meta

eosinophil(%

)24

1.00�1.00a

0.33�0.58a

0.67�1.15a

0.67�0.58a

1.00�1.00a

96

1.00�1.00a

1.33�1.15a

1.00�1.00a

1.33�0.58a

_____________

Band

eosinophil(%

)24

0.67�0.58a

1.33�0.58a

1.67�1.53a

1.67�0.58a

2.33�0.58a

96

0.67�0.58a

1.33�1.15a

1.67�0.58a

1.33�0.58a

_____________

Segment

eosinophil(%

)24

0.33�0.58a

1.00�1.00a

1.33�0.58a

1.33�1.15a

1.67�0.58a

96

0.33�0.58a

1.00�1.00a

1.67�0.58a

2.00�1.00a

Total

eosinophil(%

)24

2.00�1.00a

2.67�0.58a

3.67�2.31a

3.67�1.15a

5.00�1.00a

96

2.00�1.00a

3.67�2.08a

4.33�2.08a

4.67�1.53a

_____________

Monocyte

(%)

24

0.00�0.00a

0.33�0.58a

0.67�0.58a

1.00�1.00a

1.33�0.58a

96

0.00�0.00a

0.67�0.58a

1.00�1.00a

1.33�0.58a

_____________

Notes:Bloodparameter

values

inrowswithdifferentletterssignificantlydiffer(p5

0.05).Each

valueisamean�standard

deviationoffiveindividual

observations.

Toxicological & Environmental Chemistry 163

Downloaded At: 10:15 8 January 2011

level may be due to the disruptive action of diazinon on the erythropoietic tissue andaffects viability of the cells (Svoboda et al. 2001). Other OP pesticides were alsofound to induce such changes indicative of decreased hematopoiesis followed byanemia. For example, changes in erythrocyte profile induced by acute effect ofdichlorvos in Clarias batrachus (Benarji and Rajendranath 1990), formothion inHeteropneustes fossilis (Singh and Srivastava 1994), malathion in Cyprinion watsoni(Khattak and Hafeez 1996), and trichlorphon in Piaractus mesopotamicus (Tavares,Martins, and Nascimento 1999). The impacts on hematological factors areparticularly likely to affect the productivity of stocks from contaminated sitesbecause of their potential to reduce fecundity and inhibition of age of sexualmaturation. Hemolytic anemia, a reduction in red blood cells due to theirdestruction, is a possible problem for productivity reduction (Jee, Kim, and Kang2004). Morgan et al. (1980) found that alterations in the hematological parameterswere produced by diazinon, as evidenced by an anemic condition due to decreasedsynthesis of erythrocyte in bone marrow equivalents. Svoboda et al. (2001) reporteda decrease of nonspecific immunity in Cyprinus carpio after acute exposure to OPpesticides due to decreased leukocyte count. These changes in differential leukocytecount also provide evidence for decreased levels of nonspecific immunity in fish afteracute exposure to toxic substances.

There are differences in the acute toxicity of diazinon for various fish species andorganisms. Giddings et al. (1996) reported a 96 h LC50 value of diazinon for fishranged in tenths to several tens of mgL�1. The Office of Pesticide Programs (2000)reported 96 hLC50 value of diazinon for fry bluegill Lepomis macrochirus as0.14–0.46mgL�1, for American oyster Crassostrea virginica as 0.88mgL�1, forsheepshead minnow Cyprinodon variegates as 1.47mgL�1, for fry rainbow troutO. mykiss as 1.65mgL�1, and for fathead minnow Pimephales minnow as 7.8mgL�1.These different values indicate that diazinon toxicity varies for different species.Oh et al. (1991) presented three factors producing selective toxicity of diazinon forvarious fish species: (1) differences in inhibition of acetylcholinesterase,(2) differences in detoxification, and (3) variation in absorption. In general, this

0.00

10.00

20.00

30.00

40.00

50.00

60.00

70.00

80.00

90.00

0 0.01 1.095 2.19

Diazinon concentration (ppm)

Lym

ph

ocy

te p

erce

nt

(96

h)

–2.00

1.00

4.00

7.00

10.00

13.00

16.00

19.00

22.00

25.00

28.00

Neu

tro

ph

il, E

osi

no

ph

il an

dM

on

ocy

te p

erce

nt

(96

h)

Lymphocyte 96 h

Neutrophil 96 h

Eosinophil 96 h

Monocyte 96 h

*

**

*

*

Figure 1. Effects of different concentrations of diazinon on differential leukocyte count ofA. persicus for 96 h. The columns and vertical bars represent the means of threereplicates� S.E. *Different from control group.

164 Z. Padash-Barmchi et al.

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toxic effect changes with respect to species, size of fish, and duration of exposure

(Oh et al. 1991; Dutta et al. 1995).

Conclusions

Data indicated that diazinon is highly toxic to fingerling Persian sturgeon A. persicus.

Exposure to low concentrations of diazinon resulted in significant hematological

alterations and behavioral changes. These changes might be potentially disruptive for

survival of Persian sturgeon fingerlings in their natural environment. This fact needs

to be taken into consideration when diazinon is used for pest control in the

agricultural fields surrounding their natural freshwater reservoirs.

Acknowledgments

This study was supported by a research grant of the International Sturgeon Research Institutein Rasht, Iran and Abadan Oil Refinery in Iran.

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