LEVELS of TRACE METALS in FISH (Euthynnus Affinis) from the GULF of AQABA, JORDAN

LEVELS OF TRACE METALS IN FISH (Euthynnus affinis) FROM THE GULF OF AQABA, JORDAN

Tariq Al-Najjar1,*, Khalid Abu Khadra2, Omar Rawashdeh2, Maroof Khalaf1 and Mohammad Wahsha3

1 Department of Marine Biology, The University of Jordan, Aqaba Branch, Jordan 2 Department of Biological Sciences, Yarmouk University, Irbid, Jordan 3 Marine Science Station, The University of Jordan, Aqaba Branch, Jordan

ABSTRACT metal bioaccumulation in Tilapia Zilli and Clarias Gariepi- nus, intestine was the tissue with the second highest metal The aim of this study is to provide knowledge and es- bioaccumulation after gills due to the mucus on the gills tablish data about the levels of some trace metals in the Eu- which was nearly impossible to reove completely and con- thynnus affinis fish. The Euthynnus affinis is an important tained high metals levels. It was found by Al-Najjar et al. commercial migratory fish consumed by the locals in the [8] that bones of Caesio varilineata and Caesio lunaris Gulf of Aqaba, Red Sea, during its seasonal period. The contain very low concentrations of Cu, Ni, Pb, Cd, Zn and levels of these heavy metals (magnesium, manganese, Fe, and a high concentration of Mg, which might be related nickel, chromium, cobalt, cadmium and copper) were de- to the significance and importance of Mg in absorbance of termined in the liver, heart, spleen, muscle, kidney, gills, Ca from blood to the bones. gonads and stomach of forty Euthynnus affinis fish col- Euthynnus affinis (common name kawakawa or lected from the Gulf of Aqaba, Red Sea. Significant differences in the levels of trace metals were found among dif- mackerel tuna) belong to the superclass Osteichthyes, class ferent organs and high variations in the concentrations and Actinopterygii, Perciformes order and the family Scombri- dae. E. affinis may grow up to 1 meter in fork length and the levels of the analyzed metals were obtained. Our study reveals that the length of the samples did not affect the lev- about 13.5 Kg in weight [9]. E. affinis is considered as an els of most of the analyze metals, and for those metals that important commercial fish in Aqaba, as well as one of the favorite meals for the locals. At ecological level the im- were affected there is a negative relationships with the length. portance of E. affinis and other fish species comes from their feces that form a natural fertilizer for the producers to get nutrition from, thus signifying their rule in balancing the ecosystems. For these reasons, studying and examining KEYWORDS: trace metals, levels, concentration, E. affinis, Red Sea, Aqaba. the metals level in the organs of E. affinis and the effect of other factors such as the body size on the bioaccumulation of trace metals is very significant and important for the

monitoring and assessment of the ecosystem. 1. INTRODUCTION Several studies reveal a high susceptibility of the Gulf Toxic elements, or more generally trace elements, are of Aqaba to metal pollution. These studies stated that the now considered to be among the most effective environ- Gulf of Aqaba is surrounded mostly by dry desert lands, mental contaminants. The releasing of the trace elements thus, it has a great chance to get polluted with metals car- into the environment is increasing in the last decades, ried by air currents [10]. However, the aim of this study is threatening invertebrates, fish, and humans [1]. Fish are to establish background data and knowledge about the lev- relatively situated at the top of the aquatic food chain; els of some trace metals in the organs of the E. affinis fish, therefore, they normally accumulate heavy metals from which is an important commercial migratory fish, con- food, water and sediments. Therefore, many international sumed by the locals during its seasonal period. monitoring programs have been established in order to as- sess the quality of fish for human consumption and to mon- itor the quality, and suitability as well as the health of the 2. MATERIAL AND METHODS aquatic ecosystem [2]. In the last few decades, the concentrations of heavy metals in fish have been extensively stud- 2.1 Study Area ied in different parts of the world [3-6]. In a study [7] on The Gulf of Aqaba is located at the east fork of the Red Sea (Figure 1). Its coasts are shared by Jordan, Palestine, * Corresponding author Egypt and Saudi Arabia. The Gulf contains the only port

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for Jordan, Aqaba port, which the Gulf is named after. The muffle furnace at 450 °C for 8 hours. The ash was digested Gulf biodiversity is unique, and some species are endemic using wet digestion method. Wet digestion method was to the area. It gained a high and unique biodiversity due to carried out using 10 ml diluted Hydrochloric acid (2X pre- its semi enclosed nature, which also makes it more suscep- pared from stock of 12X HCl), the Teflon beakers contain- tible to pollution with these metals. After the year 2000, ing the samples were heated on a hot plate under the fume Aqaba was declared as a special economic zone. The hood until the solution started to boil. Samples solutions chance for a pollution to occur has increased in the Jorda- were filtered using Whatman No. 2 filter paper. Teflon nian sector of the Gulf of Aqaba, especially the chance for beakers were then washed with small amounts of distilled a metal pollution due to the developments that were made water and the washed water was collected and filtered us- along the coastline of the Gulf, represented by the projects ing the same filter paper and then added to the samples. that were carried out in different activities such as industry This process was repeated until the total volume of the and tourism [3, 5]. samples reached 25 ml. Finally filtered solution were transferred to plastic bottles and stored at room temperature to be analyzed for metals concentrations. Blanks were pre- pared for each metal using the same method but without a sample (only heated the 10 ml HCl on the hot plate until it starts to boil, then filtered using the same method). The fi- nal concentration of trace metals in each sample was meas- ured using Analytikjena Flame Atomic Absorption Spec- trophotometer novAA 400, available at the Marine Science Station.

2.3 Statistical analysis All statistical analysis was carried out using Minitab (developed by researchers from the Pennsylvania State University). The concentrations of trace metals among different organs were compared with weights. Analysis of Variance (ANOVA) was performed to compare the means between different organs. Regression test was used to ver- ify if a relationship between the weight and the Metals levels in the organs exist.

FIGURE 1 - Gulf of Aqaba, with its semi-enclosed nature. 3. RESULTS

2.2 Samples collection and preparations 3.1 Metal concentrations among the different organs Forty fish sample were collected for this study. Fresh Figure 2 shows mean concentrations of magnesium in samples were bought from the local fishermen at the mini- all sampled organs ± standard deviations. The clear differ- port in Aqaba. The collected fish were placed in clean plas- ences shown by Figure 2 were found to be significant (P tic bags and transferred to the laboratory at the Marine Sci- Value = 0.000, R-Sq = 79.33%). Gills were found to be the ence Station for further study and analysis were performed major site of magnesium bioaccumulation, with mean of according to the procedures recommended by Abu Hilal 1.8366 g/Kg, Tukey’s pairwise comparison test showed and Ismail [3]. Each fish was washed by distilled water, to that the liver is the second bioaccumulation site for magne- get rid of any remnants of trace metals on the outer surface sium, with mean concentration of 1.2989 g/Kg, followed of the fish. For each fish sample, morphometric data in- by gonads, muscle, stomach and the spleen, with means cluding weight and length were recorded, the fork length concentrations of 1.1107, 1.0569, 1.0484 and 1.0484 g/Kg ranges from 66.5 to 45 cm wear as the weight ranges from respectively, and no significant differences between them, 3907 to 943 g. Then samples were dissected using a stain- and both the kidney (0.8483 g/Kg) and the heart (0.7747 g/ less steel knife. Samples of each of the following organs Kg) had the least concentrations of magnesium per dry were taken: liver, heart, spleen, muscle, kidney, gills, gon- weight. ads and stomach. The organs were placed in glass Petri The variation of nickel levels that was resulted from o dishes, before they were placed into an oven at 85 C for the tissue type (different organ) was found to be weak (R- 24 hours to obtain dry samples. Dry samples were grinded Sq = 6.01%), however it is statistically significant (P value using a mechanical homogenizer, and stored in plastic bags = 0.015). Tukey’s test could not detect any significant dif- for storage at room temperature until further use. ferences between the organs. Because ANOVA rejected About 0.1 mg of dry weight of each sampled organ was the H0, it is safe to use fisher test to do the pairwise com- placed in a crucible and dry ashed ????? using Carbolite parison. Fisher test showed that nickel had three major sites

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FIGURE 2 - Magnesium concentrations in E. affinis fish organs (± SD).

FIGURE 3 - Nickel and chromium levels in E. affinis fish organs (± SD).

for bioaccumulation with no significant differences be- For cadmium results, even though the P value for the tween them, spleen, kidney and the gonads, in order (Fig- ANOVA were significant at confidence level 95% (P ure 3). Value = 0.022, R-Sq = 5.21%), Tukey’s test did not report Chromium levels were found to be significantly differ any of the organs levels as significantly different from the from one organ to the other (P Value = 0.008, R-Sq = others, again fisher was used since H0 was rejected by 7.57%), with the highest level in gills, with a mean of ANOVA. Fisher test showed that cadmium readings were 0.039468 g/Kg. However, Tukey’s test did not detect any the highest in the gills too (0.006412 g/Kg), followed by significant differences between the gills and the following the liver with mean concentration of 0.006282 g/Kg, then organs, stomach, spleen, heart and gonads (Figure 3). the rest of organs (Figure 4).

Manganese was found to accumulate in the gills in a Cobalt had similar results to manganese, H0 rejected (P very large amounts (0.01392 g/Kg) (P Value = 0.000, R-Sq Value = 0.000, R-Sq = 13.14%), gills had the highest lev- = 27.83%) compared to the other organs, that had no sig- els, with mean of 0.012218 g/Kg, and all the rest of the nificant differences between the levels of manganese bio- organs had no significant differences between the level of accumulation in them. (Figure 4). cobalt accumulation in them (Figure 4).

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FIGURE 4 - Manganese, Cadmium, Cobalt and Copper concentrations in E. affinis fish organs (± SD).

TABLE 1 - Regression results for Sample total length in (cm) VS the levels of metals in all eight in (g/kg) in E. affinis fish organs.

Metal/organ P R-Sq PCF Metal/organ P R-Sq PCF Liver 0.455 1.8% -0.135 Liver 0.822 0.1% -0.037 Heart 0.304 3.9% -0.198 Heart 0.475 1.5% -0.121 Spleen 0.184 6.0% -0.245 Spleen 0.442 1.6% -0.127 Muscle 0.078 10.0% -0.316 Muscle 0.224 4.0% -0.199 Mn Cd Kidney 0.612 0.9% -0.097 Kidney 0.846 0.1% 0.032 Gills 0.443 1.6% -0.126 Gills 0.406 1.9% -0.137 Gonads 0.041* 13.1% -0.362 Gonads 0.985 0.0% -0.003 Stomach 0.245 4.8% -0.219 Stomach 0.419 1.7% -0.131 Liver 0.573 0.9% 0.094 Liver 0.536 1.1% -0.105 Heart 0.075 9.0% -0.301 Heart 0.928 0.0% 0.015 Spleen 0.003* 20.8% -0.457 Spleen 0.646 0.6% -0.079 Muscle 0.153 5.8% -0.240 Muscle 0.463 1.6% -0.128 Mg Co Kidney 0.007* 19.9% -0.446 Kidney 0.896 0.0% 0.022 Gills 0.320 2.8% -0.168 Gills 0.085 8.0% 0.283 Gonads 0.016 15.6% -0.395 Gonads 0.806 0.2% -0.042 Stomach 0.069 8.9% -0.299 Stomach 0.937 0.0% -0.013 Liver 0.916 0.0% -0.017 Liver 0.373 2.9% -0.172 Heart 0.956 0.0% -0.010 Heart 0.177 5.6% -0.237 Spleen 0.628 0.8% 0.091 Spleen 0.006* 24.9% -0.499 Muscle 0.395 2.0% -0.140 Muscle 0.537 1.4% -0.119 Ni Cu Kidney 0.839 0.1% 0.038 Kidney 0.740 0.3% -0.056 Gills 0.698 0.4% -0.063 Gills 0.785 0.2% 0.044 Gonads 0.680 0.6% 0.077 Gonads 0.259 3.7% -0.193 Stomach 0.928 0.0% -0.015 Stomach 0.188 4.6% -0.215 Liver 0.442 2.0% 0.141 Heart 0.447 2.1% 0.144 Spleen 0.739 0.4% 0.061 Muscle 0.682 0.6% 0.682 P: P Value, R-Sq: R Square, PCF: Person’s correlation factor. Cr Kidney 0.839 0.1% 0.038 * P Value with * next to them are significant at confidence level = 95% Gills 0.805 0.2% -0.046 Gonads 0.958 0.0% 0.010 Stomach 0.476 1.8% 0.133

Copper levels were found to be statistically different tion reactions in the liver, as a cofactor for enzymes that from one organ to another (P Value = 0.000, R-Sq = catalyze phase II detoxification reactions [13]. 54.89%). Unlike most metals, the heart had the highest Ni had three major bioaccumulation sites in E. affinis, level of copper accumulation (0.01586 g/Kg), even signif- spleen, kidney and the gonads. It’s accumulation in the icantly higher than the copper level in the gills (0.006425 spleen could be explained by a very recent finding, in g/Kg), which was second in order after the heart. The liver which a binding site for the Nickel was found in the hemo- had slightly lower levels than the gills (0.005400 g/Kg) and globin. Nickel bioaccumulation in the gonads in large no significant difference between them was detected. The amounts might affect the gonads function [14]. It was no- rest of the organs had no significant differences between ticed that nickel reduces the number of the sperms in rats, them as reported by Tukey’s test (Figure 4). or in a positive way as a necessary nutrient for a healthy reproduction. Nickel bioaccumulation in the kidneys 3.2 Effect of fish length on the metals concentration might be interpreted by the need for the Ultrafilterable Ni+2 Table 1 summarize all the results for the regressions binding protein in the kidney to eliminate the excess Ni tests for all the seven metals, fish length had no effect on from the blood [15]. This result is not similar to what Al- most of the levels. All levels that were affected by the fish Najjar et al. [8] and Nussey et al. [12] have found. In their length had a negative relationship with it. R-Sqs were gen- studies the gills had the highest level, except for the D. rus- erally found to be low, even for the levels that show a sig- selli, where Al-Najjar findings [8] point to the kidney as nificant association with the fish length. The strength of the major site of bioaccumulation. negative relationship between the length and the levels of In a review by Klevay [16], it was reported that copper metals varied from weak to medium. deficiency can affect the heart on different aspects, and not just the heart but the whole cardiovascular system, the current study add one more reason to signify the copper role 4. DISCUSSION in the heart health, since the bioaccumulation of copper in the heart was shown to be very high even compared to the Magnesium readings were the highest among the se- gills which is the most expose organ to the environment, lected metals, similar results for magnesium were obtained and that should indicate how large and significant is the by [8], as it is the case in most fishes, and that is due to its role that the copper plays in keeping the heart muscle large abundance in the sea water, which allows for a larger healthy. level of bioaccumulation inside the fish organs, either The fish length had no effect on most of the metals through respiration or as a result of biomagnification effect concentrations, only Mg, Cu and Mn bioaccumulation lev- from the food chains. els in some organs were affected by the length of the fish. The high bioaccumulation level of Mn in the gills is All the associations between the length of the fish and the related to the important role of Mn in the development and level of the affected metals were negative, big fish samples growth of the connective tissue as previously mentioned contained less metals concentrations per dry weight. The [11]. Thus it is expected that the bones in the gills will have negative relationship between the length and the metals plenty of Mn supply, this results also in consistent with the levels can be explained by the higher specific metabolic findings of Al-Najjar et al. [8] and Nussey et al. [12]. Sim- rate for smaller organisms, which lead to faster respiration ilarly, the high bioaccumulation of Mg in the gills could be rate and thus higher uptake rate for metals. The lack of a explained according to the role of Mg in the development significant relationship between the length and the Cr, Cd, of healthy connective tissues, in addition to the fact that Co and Ni levels might be due to factors not under study, bone surface acts as storage place for about 60% of total errors or weak effect of the weight on their levels (low R body Mg. square). In agreement with our finding, Nussey et al. [12] Higher bioaccumulation of Cr, Cd and Co in the gills found similar result for these metals that affected by the compared to the other organs is probably only due to the fish length were they got negative relationships between fact the gills is the most exposed organ with and in direct the length and the metal concentration. contact with the environment which allows for faster un- regulated uptake of the metals from the environment. This reason may also contribute to the high readings for Mn and 5. CONCLUSION Mg too. Nussey et al. [12] reported similar results for Cr in the gills of Labeo umbratus. For Cd Al-Najjar et al. [8], The high bioaccumulation for certain metal in an organ found different results were the Cd levels were the lowest can be due its function in that organ because binding pro- in the gills of D. macarosoma and D.macarellus, and sec- teins or enzymes with site for these metals to bind with. ond lowest after the muscles in D.russelli. This might indi- This data point to how large is the bioaccumulation for the cate that E. affinis gets most of the Cd through respiration metals in the E. affinis, which is expected since E. affinis while these three Decapterus species get the most from is located in the top layers of the food chain, thus, raising food sources. Liver had the highest level of Mg, and that the potential for them to be used as bioindicators, however, could be related to the role that Mg plays in the detoxifica- there are few limitations that can affect their use, such as

its large migration route, which eliminates the possibility Decapterus macrosoms and Decapteru srusselli of the family to be used as a bioindicator for the Gulf of Aqaba region or carangidae from the Gulf of Aqaba, Red Sea, Jordan. Natural Science. 4: 362-367. any localized area, but this does not affect its chance to be used as a global indicator for metals levels especially to [6] Kojadinovic, J., potier, M., Corre, M., Cosson, R. & Busta- mante, P. (2007) Bioaccumulation of trace elements in pelagic detect any changes which might accompany the persistence fish from the western Indian Ocean. Environmental Pollution. issue of global warming. 146: 548-566.

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