Effects of acute crude oil exposure on basic physiological functions of Persian sturgeon, Acipenser persicus

[Research]

Effects of acute crude oil exposure on basic physiological functions of Persian sturgeon, Acipenser persicus

H. Khoshbavar Rostami¹*, M. Soltani²

1- Inland Water Aquatic Stocks Research Center, Gorgan. Iran.

2- Department of Aquatic Animal Health, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran.

* Corresponding author’s E-mail: hkr_rostami@yahoo.com

ABSTRACT

Hematological parameters are suitable biomarkers for evaluating the potential risk of the chemicals. The aim of this study was to investigate of acute crude oil exposure on basic physiological functions of Persian sturgeon, Acipenser persicus. 210 juvenile Persian sturgeon (9.4 ± 1g) were supplied by the Rajaei fish farm in Mazandaran Province, Iran.  Juveniles were exposed to the crude oil (15, 16, 17, 18 and 19 ppm). The 96h-LC₅₀ were detected under static condition by SPSS software. Hematological and biochemical parameters were compared between control group and treatment exposed to 96h-LC₅₀. The median lethal concentration was 16.5 ppm in 96 h toxicity test. WBC, RBC, Hb and MCHC decreased, while MCV, MCH and PCV were significantly higher in the treatment which exposed to LC₅₀ concentration (P<0.01). Results of differential leukocyte count showed that after treatment with LC₅₀ concentration, neutrophils and monocytes increased, while lymphocytes and eosinophils decreased (P<0.01). Biochemical parameters showed an increase in serum glucose (p<001). Other parameters including total protein, ALT, AST, ALP and LDH enzymes decreased in treatment group significantly (p<0.01). Our results provides evidences that crude oil may have disruptive action on the erythropoietic tissue which may be due to its influence on the viability of the cells. Crude oil also inhibited all of the enzymes activities leading to hyperglycemia due to stress inoculation.

INTRODUCTION

The Caspian Sea is the biggest land-locked body of water bordered by five countries: Azerbaijan, Iran, the Russian Federation, Kazakhstan and Turkmenistan (Kosarev & Yablonskaya 1994). It has five major inlet rivers but no outlets and acts as a watershed reservoir for the region. The key biological issues in recent years relate to the decline of fisheries and caviar harvesting, the massive mortality among seal populations and the introduction of invasive species in the Caspian Sea. There are several important fisheries in the Caspian Sea, but the greatest effect and hazard has always been placed on the sturgeons. Several anthropogenic factors, including both land-based and offshore pollution, threaten the survival of all fisheries, but especially sturgeon populations in the Caspian Sea (De Mora & Turner 2004). Among these, chemical contamination seems to be one of the most significant factors influencing the population of sturgeons in the Caspian Sea (Ivanov 2000; Pourkazemi 2006). Many potentially toxic contaminants released into the Caspian Sea, are lipophilic and insoluble in water. These properties increase their availability for uptake and accumulation by aquatic organisms. Previous investigations have demonstrated the occurrence of polycyclic aromatic hydrocarbons (PAHs) in fishes, water and sediments of the Caspian Sea (Tolosa et al. 2004; Khoshbavar Rostami et al. 2012; Eghtesadi Araghi et al. 2014; Mashroofeh et al. 2015). European Union and U.S Environmental Protection Agency (US EPA) put these compounds in the priority pollutant list because of their mutagenic, carcinogenic, tetratogenic and toxic properties, environmental persistence, bioaccumulation and trophic transfer of PAHs in aquatic ecosystem (ATSDR, 1995; European Commission, 2011), and for this reason, the increase in levels of PAHs contaminations that has occurred in recent decades in the Caspian Sea, a landlocked system where the PAHs cannot be flushed out, is a cause for concern (Mashroofeh et al. 2015).

Furthermore, the life history of sturgeons may leave them particularly sensitive to effects of these pollutants. As an opportunistic bottom feeders, these fish frequently be in contact with sediments that may contain sediment adsorbed hydrophobic pollutants (Billard & Lecointre 2001; Kajiwara et al. 2003). Also, sturgeons are particularly long lived animals (up to 100 years in the wild) that take 5-30 years to reach sexual maturity (Mashroofeh et al. 2013; Billard & Lecointre 2001). These characteristics put the sturgeons at a high potential risk for accumulating persistent organic and inorganic contaminants in their tissues.

Previous studies reported induction of hepatic lesions, physiological and biochemical disorders in these fish (Ribeiro et al. 2005); and high levels of tumors or abnormalities in gonad development and gametogenesis and disturbances in the morphogenesis of organs have been noticed in the Caspian sturgeons since the late 1980s (Kajiwara et al. 2003). Acute toxicity data can help to determine the mode of toxic mechanism of a substance and may provide information on doses associated with target organ toxicity and lethality that can be used in setting dose levels for repeated dose studies.

This information may also be extrapolated for diagnosis and treatment of toxic reactions in humans.

The results of acute toxicity tests can provide information for comparison of toxicity and dose-response among numbers of chemical classes and application in selection of candidate material for future work (Hedayati et al. 2010).

However the environmental pollutions, such as spills of oil distillate products into the coastal waters are growing in the recent years in Caspian Sea, There are only few papers concerning the effects of crude oil exposure on physiological indices in sturgeons and there is a real need for information about the effects of this fuel oil on these fish species. Fish hematological indices are very sensitive to water contaminants, and its alternation in the hematological and immunological parameters can be used as toxicity indices of xenobiotics (Sancho et al. 2000). The aim of the present study was to investigate hematological and biochemical indices of the Persian sturgeon, Acipenser persicus, exposed to crude oil, as potential biomarkers, in order to assess pollution through these petroleum products and getting information on the threat imposed by these spills to this valuable fish species.

MATERIAL AND METHODS

The experiment was conducted on the juvenile specimens of Persian sturgeon (N = 210) with average weight of 9.4 ± 1g,  supplied by Rajai fish farm in Mazandaran Province, Iran. Prior to the toxicity tests, fish were acclimated to laboratory conditions for at least two weeks in 160-L fiberglass tanks.

During acclimatization, fish were fed with commercial trout pellet (protein 36%, lipid 14%, ash 11%, fiber 3.5%, phosphorous 1%, wet 11%, carbohydrate 22.5%) and fish meal 50% twice a day. During the tests period, water was continuously monitored for temperature, dissolved oxygen, pH, and conductivity (Hedayati et al. 2010). Average temperature, dissolved oxygen, pH and total hardness were measured 22 ± 1°C, 8.2 ± 0.8 mg.L^-1, 7.5 ± 0.1 & 145 ± 5 mg.L^-1 respectively. Other water quality parameters were ammonia < 0.02 mg.L^-1, nitrite < 0.1 mg.L^-1, nitrate < 0.503 mg.L^-1 and phosphate < 0.285 mg.L^-1. Also fish were maintained under natural photoperiod (L: D=14:10). Range Finding Test values indicated that there is no mortality up to 15 ppm crude oil exposure while 100% mortality in 19 ppm. Then fish were transferred into the treatments with the different crude oil concentrations (15, 16, 17, 18 and 19 ppm) as a static exposure test (test medium was not renewed during the assay and no food was provided for animals), in tanks of 160 L, each containing nine fish. Three replicates were performed for each dose. Acute toxicity tests were done in order to calculate the 96-h LC₅₀ for crude diesel oil, based on OCED (1984). Mortality was recorded after 24, 48, 72 and 96 h. Dead fish were immediately removed with special plastic forceps to avoid possible deterioration of the water quality. Value of LC₅₀ was calculated from the data obtained in acute toxicity bioassays by SPSS statistical software.

After acute toxicity bioassay, two groups including ten specimens exposed to 96-h LC₅₀ and one control group, exposed only to water (the same as that used for acclimation) were selected to determine the effect of acute crude oil exposure on hematological and biochemical indices of the Persian sturgeon.

Immediately after removing the fish from the tank, they were anesthetized with clove powder (200 ppm), and blood samples were taken from the caudal vein by means of heparinized plastic syringes (Hedayati & Safahieh, 2011). Subsequently, fish were killed by struck on head.

Red blood cells (RBCs) and white blood cell (WBCs) were measured immediately on fresh blood by diluting heparinized blood with Merck Giemsa stain at 1:30 dilution and cells were counted using a hemocytometer Neubauer under the light microscope (Banaee et al. 2008).

Blood smears were prepared, and leukocytes were categorized into lymphocytes, monocytes, neutrophils and eosinophils (Banaee et al. 2008). Hematocrit (Ht) was immediately determined after sampling by placing fresh blood in glass capillary tubes and centrifugation for 5 min at 10,000 rpm in a microhematocrit centrifuge (Hettich, Germany) then measured the packed cell volume (PCV) (Goldenfarb et al. 1971). Hemoglobin (Hb) levels were determined colorimetrically by measuring the formation of cyanomethemoglobin according to Lee et al. (1998). Mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) were calculated from RBC, PCV and Hb according to Lee et al. (1998). Ultrapure water was used for all serum dilutions and standard preparations and duplicate readings were recorded for standards and serum samples. The quantitative determination of serum glucose was carried out using commercially available diagnostic Experimental Protocols kits (Pars Azmoon, Iran), at 546 nm and 37 °C by the glucose oxidase method (Hedayati & Safahieh 2011). Serum total protein level was determined using kits provided by Pars Azmoon Co., Iran, with bovine serum albumin serving as standard by the method of Canli (1996) at 546 nm and 37°C. Aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined with Pars-Azmoon Diagnostics Infinity AST reagent kit and Sigma Diagnostics Infinity ALT reagent kit, respectively, by enzymatic methods with a Technicon Ra1000 auto-analyzer. Alkaline phosphatase (ALP) was determined by enzymatic method with the automate apparatus auto-analyzer, a Technicon Ra1000, with Darman Kave kit at 37°C and 410 nm. Lactate dehydrogenase (LDH) was determined with Pars-Azmoon Diagnostics Infinity kits with a Technicon Ra1000 auto-analyzer.

For each index, the data were tested for normality and homogeneity of variances by Kolmogorov-Smirnov test. T test was used to determine significant differences to evaluate the effect of crude oil on blood/serum parameters. The differences between means were analyzed at the 5% significance level. Data are reported as means ± standard deviation. The software SPSS, version 17 (SPSS, Richmond, VA, USA) was used as described by Dytham (1999).

RESULTS

All controls resulted in low mortalities fewer than 5%, which indicated the acceptability of the experiments. The mortality of Persian sturgeon due to crude oil was examined during the exposure times at 24, 48, 72 and 96 h in Table 1.

Fish exposed during the period of 24-96h had significantly increased number of dead individual with increasing concentration (P<0.05).

There were significant differences in number of dead fish between the duration of 24 and 96h. Considering the crude oil bioassay, LC₅ and LC₅₀ of 24, 48, 72 and 96h were 0.0, 15.6, 14.9 and 14.3 ppm and also 0.0, 22.1, 18.2 and 16.5 ppm respectively. Fish exposed to crude oil for 96h showed a significant change in most hematological indices (P<0.01).

Results showed that PCV and MCV increased, while Hb, RBC, WBC and MCHC decreased significantly after crude oil exposure.

Only Mean corpuscular hemoglobin (MCH) had no significant difference (P>0.05) compared to control group (Fig. 1A-B).

 Table 1. Cumulative mortality of Persian sturgeon during acute exposure to crude oil (n = 10, each concentration).

                                                                                                                                                               A

                                                                                                                                                                                            B

Fig. 1. Hematological indices of Persian sturgeon after acute exposing to crude oil (N = 10, concentration =16.5 ppm, ٭٭shows significant differences at α=0.01).

Differential leucocyte counting showed that lymphocytes and eosinophils decreased in group exposed to LC50 (96 h) of crude oil in comparison with control group (P<0.01). In contrast, mean frequency of neutrophils and monocytes were higher in treatment group significantly (P<0.01, Fig. 2).

Some serum biochemical indices showed a significant decline in fish exposed to LC50-(96 h) (P<0.01) including total protein, ALP, ALT, AST, and LDH enzyme activities, whereas serum glucose was significantly higher in control group (P<0.01, Table 2).

Fig.2. Leukocyte count of Persian sturgeon after acute exposing to crude oil (N = 10, concentration = 16.5 ppm, ٭٭shows significant differences at α=0.01.).

Table 2. Biochemical parameters of Persian sturgeon after acute exposing to crude oil (N=10, concentration=16.5 ppm).

    Data presented as mean ± standard deviation. ٭٭shows significant differences at α=0.01.