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Jun 18, 2015 - After flowing about 560 km, it joins River Chenab. ... Shahdara Bridge to Balloki Headworks has adversely affected the quality of river water and.
Accepted Manuscript Original article Assessment of differential trace metals accumulation in tissues of Labeo rohita (rohu) from River Ravi Shahid Mahboob, K.A. Al-Ghanim PII: DOI: Reference:

S1319-562X(15)00157-6 http://dx.doi.org/10.1016/j.sjbs.2015.06.024 SJBS 509

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Saudi Journal of Biological Sciences

Received Date: Revised Date: Accepted Date:

4 May 2015 18 June 2015 22 June 2015

Please cite this article as: S. Mahboob, K.A. Al-Ghanim, Assessment of differential trace metals accumulation in tissues of Labeo rohita (rohu) from River Ravi, Saudi Journal of Biological Sciences (2015), doi: http://dx.doi.org/ 10.1016/j.sjbs.2015.06.024

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ASSESSMENT OF DIFFERENTIAL TRACE METALS ACCUMULATION IN TISSUES OF LABEO ROHITA (ROHU) FROM RIVER RAVI Shahid Mahboob and K. A. Al-Ghanim Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh-11451, Saudi Arabia. *Corresponding author email: [email protected] Tele No. of corresponding author: +966-1-4675925; Fax No: +966-1-4678514

ASSESSMENT OF DIFFERENTIAL TRACE METALS ACCUMULATION IN TISSUES OF LABEO ROHITA (ROHU) FROM RIVER RAVI Abstract In the present study, concentrations of As, Cd, Co, Cr, Cu, Fe, Hg, Mn, Ni, Pb, Se, and Zn in the muscle, gills, liver, and intestine of the Labeo rohita from the Indus River were analysed by atomic absorption spectrophotometry. The objective of the study was to assess whether in complex muscle/skin, gill filament/gill arch, proximal/distal liver and proximal/median/distal intestine samples, and particular components variation in concentrations of detected elements. Results indicated that there were no differences in elemental accumulation between the proximal and distal liver segments, as well as between the proximal and median intestine sections. Contrarily, muscle and skin significantly differed based on their elemental accumulation patterns. Significant differences were also noticed between the gill arch and filaments, as well as between the distal and the two upper intestine segments. These results express the importance of detailed reporting of tissue sampling, i.e. whether the skin was included with the muscle sample, as well as if the gill arch and filaments were analyzed together. We are of the view a potential bias that can be arised by different muscle/skin or gill arch/filament ratios included in the sample; we strongly recommend that they should not be analyzed together. The findings of this study might be of interest for the other workers working in aquatic ecosystem monitoring programs. Keywords: metal; assessment; tissues; Labeo rohita; Atomic absorption. 1. Introduction Trace metals are regarded to be most poisonous of the aquatic environment (Alkan et al. 2013). Fish are among the most susceptible aquatic organisms to water and sediment pollution (Alibabi et al. 2007). At the same time, as species positioned at the top of the food chain, they can accumulate large metal levels (Yilmaz et al. 2007). The presence of metals in fish tissues is therefore of considerable importance for environmental and food safety, as well as for public health (Azevedo et al. 2012). Metal pollution in fish has become a worldwide concern, and numerous studies and monitoring programs on metal accumulation in fish have been conducted (Erdo rul and Erbilir, 2007; Begum et al. 2013; Zhuang et al.,2013; Mahboob et al. 2014).

Studies related to metal pollution in fish have been generally focused on the fish muscle, being the main portion of fish onsumed by humans, as well as on the gills, liver, kidneys, and intestine, which represent either major accumulation centers in fish or main metaluptake routes (Storelli et al., 2006; Golovanova, 2008; Begum et al., 2013; Mahboob et al. 2014). “The River Ravi is the smallest among the five main rivers originated from India and enters in Pakistan. After flowing about 560 km, it joins River Chenab. Raw domestic sewage, un-treated industrial effluents originating from Lahore and adjacent cities are dumped into the River Ravi by main tributaries of Hudiara drain and Degh Fall. River Ravi between it stretches from Shahdara Bridge to Balloki Headworks has adversely affected the quality of river water and freshwater fisheries. The water of the River is used for agriculture and is the main source of drinking water and industrial water in this basin. River Ravi is a classic example of pollutants due to dumping of untreated domestic and industrial waste, resulting in a high level of impurities in the water. It is estimated that about 48 percent of the overall pollution discharged into the River Indus comes from the River Ravi. It is considered view of the scientist; the River Ravi is no more a river but a sludge carrier. A large number fish species have been extinct due to heavy pollution load in this river (Mahboob et al. 2015)”. However, there is an apparent lack of a standardized approach regarding fish tissue sampling. To assess this issue in a greater detail, we conducted a small literature survey. According to Crafford and Avenant-Oldewage (2010), authors either include or remove the skin from the muscle sample, although they often fail to report which approach was used. None of the authors specified which part of the liver was included in the sample. In aquatic ecosystem fish can be successfully use as a bio-indicator for heavy metals pollution. The distribution of metals between different tissues depends on the way of exposure (environmental or dietary) (Alam et al. 2007). From water fish accumulates large amounts of metals which may be harmful for human consumption (Malakootiani et al. 2011). Yousafzi et al. (2010) reported that level of heavy metal intake in aquatic system causes an extra stress on fish that in turn concentrate metals in metabolically active organs and tissues. Labeo rohita has undergone a significant decline due to overexploitation as a food fish throughout its range. Unfortunately, no attention has been given by the concerned authorities in the country in order to control anthropogenic activities in this river to conserve this this commercially important fish species in the region. In the present study, we assessed elemental

concentrations in different segments of muscle, gills, liver, and intestine of the Labeo rohita (rohu) from the River Ravi, in order to determine possible differences between them. Such information could indicate the importance of the sampling procedure standardization and reporting in studies dealing with elemental accumulation in fish. This issue has not received a proper attention so far, and the results of the present study might therefore be of interest for both the scientific community and the stakeholders involved in aquatic ecosystem monitoring programs. 2. Material and methods 2.1. Sampling site specifications: A 72 km stretch of the Ravi River from Lahore Siphon to Bulloki Headworks was selected based on earlier report about the heavy pollution of the water, sediment and fish with different pollutants in this river (Jabeen and Javed 2011). The effect of pollution was assessed on fish samples of Labeo rohita. This study was conducted at two sites Shahdrah (31o 36‘ 54.92’’ N 74o 18‘ 50.09’’E upstream) and Ballouki Headworks (downstream) along the stretch of the River Ravi, which were about 90 km apart from each other. These sites were receiving domestic and municipal wastes and agricultural runoffs. During its course, some pollutants from agricultural runoffs and domestic and municipal wastes enter into the river 2.2 Sample collection Seven Labeo rohita of same weight about (1600 ± 30.45 g) were collected by a professional fishermen during April 2012 from the River Ravi from Shahdrah (SH) and Baulloki (BU). Specimens were sacrificed with a quick blow to the head, measured for their total body length (cm) and total body weight (g). Samples of the muscle (right dorsal muscle), skin, gill filaments, gill arch, liver, and intestine were collected. Each liver sample was separated into two sections, proximal and distal. Given that intestine of catfish species is clearly differentiated into three principal regions - proximal, median and distal (Bosi et al. 2006), samples from each region were sectioned. All samples were washed with distilled water and stored at −20 °C for analysis. 2.3. Sample preparation and analysis The samples were freeze-dried using a rotary vacuum concentrator Christ, model GAMMA 116LSC (Osterode am Harz, Germany). Analytical portions of approximately 0.3 g (dry weight) were accurately weighted and subsequently processed in a microwave digestion system. Samples

were mineralized by adding 6 mL of 65% HNO3 and 4 mL of 30% H2O2 (Merck, Germany). Microwave assisted digestion was performed in Speed wave TM MWS3+ oven (Berghof, Germany). The following temperature schedule was used (default food program): 5 min – 160 °C; 15 min – 190 °C; 20 min. – 100 °C. The digested samples were transferred into 100 mL polypropylene volumetric flasks. In order to assess the possible presence of trace elements in reagents or carry-over effects of digestion vessels, five reagent blank samples were prepared as well, one per each session, according to the described procedure. These samples were detected in each analytical batch. All reagents used in the analysis are analytical were purchased from Merck (Germany). Heavy metal contaminants in water and fish samples were assessed by using Atomic Absorption Spectrophotometer (Hitachi Polarized Zeeman AAS, Z-8200, Japan). The following metals were measured: chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), copper (Cu), zinc (Zn), arsenic (As), selenium (Se), cadmium (Cd), and lead (Pb). The blanks and calibration standard solution were also assessed in the same manner as for the samples. The instrument calibration standards were made by diluting standard (1000 ppm) supplied by Merck, Germany. A known 1000 mg/l concentration of all the above mentioned metal solution was prepared from their salts. All chemicals used were of analytical grade. The precision of the applied analytical method was validated through analysis of the standard reference material (National Institute of Standards and Technology, USA). Concentrations of each measured metals were corrected for response factors of both higher and lower mass internal standards using the interpolation method. Mercury (Hg) was measured using cold vapour technique by atomic absorption spectrometer SpectrAA 220 (Varian, Palo Alto, USA) with VGA 77 hydride system and SnCl2 in HCl as a reductant. Calibration was performed in five points, standard concentration range was 0.5 – 15.0 ng/mL. 2.5. Statistical analysis The data was analyzed for comparisons of metal and trace element concentrations between the skin and the muscle, the gill arch and the filaments, the proximal and distal liver sections, and the three intestine sections (proximal, median and distal). The normality of distribution of analyzed samples was tested by the Kolmogorov- Smirnov test. Since the variables lacked normality of distribution, nonparametric tests were applied. The groups were compared by the DMR test. 3. Results and Discussions

The average body weight of the analyzed Labeo rohita specimens were 1600 ± 30.45 g. All specimens were immature, with the males representing the majority of specimens (85%). Elemental accumulation in the muscle and the skin significantly differed (p Zn >Fe > Hg > Cu > Se > Mn > Co > Ni >Cr > As > Pb. There were no differences between the two tissues regarding Cr, Cd, Ni, Pb, and Se concentrations. Order of bioaccumulation of these metals was Shahdrah (upstream) followed by Balluoki (downstream). The gill filaments and the gill arch also differed significantly (P0.05) between elemental concentrations in the two studied liver sections (Table 2). Fe was most abundant in distal proximal and sections of liver (688.52±7.88 and 671.42±11.42 µg/g dry weight) in fish collected from Shahdra (upstream). Cd was recorded in least quantity as 0.44±0.17 and 0.52-±0.22 µg/g dry weight recorded in the proximal and distal section of liver of Labeo rohita collected from downstream (Ballouki), respectively. The order of bioaccumulation of studied metals in proximal and distal section of liver of Labeo rohita was > Fe >Zn > Cu > Se > Mn > Hg > Ni > Co >Pb > Cd > As (Table 2). The proximal and the median intestine segments had the same elemental accumulation levels, while they both had significantly higher Co and Zn concentrations and lower Mn concentrations than the posterior segment (Table 3). Fe was most abundant in the posterior section closely followed by middle and anterior section of and distal sections of intestine of fish (Table 3). Cd was recorded in minimum quantity as 0.30±0.10 in the middle section of intestine of Labeo rohita collected from downstream. The order of bioaccumulation of studied metals in anterior, middle and posterior sections of intestine

of Labeo rohita was > Fe >Zn > Cu > Se > Mn > Hg > Ni > Co >Pb > As > Cd (Table 3). A more accumulation of metals in gill filaments and the gill arch of Labeo rohita collected from River Ravi at Shahdrah. No differences in elemental accumulation were observed between the two studied liver segments, as well as between the two upper intestine sections. On the other hand, the muscle and the skin significantly differed based on their elemental accumulation patterns. Significant differences were noted between the gill arch and the filaments, as well as between the distal and the two upper intestine segments. Higher accumulation of metals in gill filaments and the gill arch of Labeo rohita collected from Shahdrah. Higher accumulation of As, Co, Cu, Fe, Mn, and Zn in the skin than in the muscle was also observed by other authors (Al-Yousuf et al. 2000; Storelli et al. 2006; Al-Weher 2008; Schenone et al. 2014). Higher concentrations in the skin could be the result of the metal complexion with the mucus (Al-Weher 2008). Metal ions from water are able to bind to the mucus layer present on the body surface, which can lead to a higher uptake and absorption in the skin (Tao et al., 2000). This is particularly the case with fishes without scales, where mucus layer serves as a shield against permeation of environmental chemicals (Rose et al. 1999). The results of this study are line with previous findings reported by Canli and Atli (2003), and Monday and Nsikak (2007). However, Zn is essential micronutrients, which comprises nearly 300 enzymes in marine organisms and is responsible for certain biological functions that require relatively higher Zn (Al-Yousaf et al. 2000). Zn is also critical for aquatic organisms, including fishes; however, Zn becomes poisonous when it exceeds its maximum value. Many researchers have stated that dietary Zn is the fundamental reason for increased Zn in marine fish (Canli and Atli 203). The findings of above mentioned workers are not in line as Labeo rohita is fish with scales. On the other hand, the muscle has a weak accumulation potential, and it often represents the tissue with the lowest elemental concentrations in fish (Erdo rul and Erbilir 2007; Lenhardt et al. 2012). Uysal et al. (2009) observed a lack of clear accumulation patterns between the two tissues, since different species had maximum concentrations in either muscle or skin. In the present study, higher Hg concentrations were detected in the muscle (Table 3), while Storelli et al. (2006) have not observed any differences between these two tissues. According to Fu et al. (2010), skin is not an active tissue for Hg bioaccumulation. The inclusion of skin in the sample can actually reduce resulting concentrations detected in the muscle sample (EPA 2000), and consequently present a false image of the acceptable metal levels in fish meat. Other authors also

found differences between the two tissues regarding Cd, Cr, Ni, Pb, and Se accumulation (AlYousuf et al. 2000; Storelli et al, 2006; Al-Weher 2008; Uysal et al. 2009; Schenone et al. 2014), which was not observed in the present study. Fish sampling protocols (UNEP 1984; EPA 2000; Sharma et al. 2009) commonly recommend removal of skin from the muscle sample for metal analyses. Potential differences between the gill arch and the filaments regarding the elemental accumulation was rarely determined. Crafford and Avenant-Oldewage (2010) reported higher Ni and Pb accumulation in the gill arch, which was not observed in the present study. However, bony tissues is considered a major Pb accumulation center, where it accumulates due to its similarity to calcium (Castro-González and Méndez-Armenta 2008). Our results indicated a higher Mn concentration in the gill arch, while most of the other studied elements had lower concentrations than those found in the gill filaments (Table 2). Mn tends to accumulate at the highest levels in bony tissues and it represents a normal constituent of vertebrate skeletal tissues (Castro-González and Méndez-Armenta 2008). Higher Cd, Co, Cr, Cu, Fe, Hg, and Se accumulation levels in the gill filaments are probably a result of the direct uptake from the water, since gills represent the main accumulation route of waterborne pollution (Storelli et al. 2006; Golovanova 2008). Some metals also tend to accumulate at higher concentrations in gills due to their slow excretion rate (Qadir and Malik 2011). Our results exhibited that there were no significant differences in elemental accumulation between the two studied liver sections (Table 3). To our knowledge, this issue was not determined in any of the previous studies. Assessment of metal accumulation in the intestine indicated that Co, Mn, and Zn concentrations in the distal section differed from those in the two upper intestine sections, while there were no differences observed between the latter two. The observed differential accumulation among the studied intestine sections could be caused by the differences in their activity. According to the literature survey, the present study was the first that addressed this issue. The results of present study emphasize the need of a detailed reporting how the fish tissue was sampled. It is especially important to report whether the skin was included with the muscle sample as well as if the gill arch and filaments were analyzed together. Moreover, information on the exact intestine section that was sampled should also be provided, especially if the study is focused on the elements for which differences in the accumulation level have been observed in the present study. It is important to note that a potential bias can occur if

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Figure 1: Map of the study area

Table: 1: Metal and trace element concentration concentrations (µg/g dry weight) ± S.E in muscle and skin of the Labeo rohita from Shahdra and Ballouki stations River Ravi

Zn

Se

Pb

Ni

Mn

Hg

Fe

Cu

Cr

Co

Cd

As

52.94±3.77c

1.83±0.46c

0.16±0.01c

0.45±0.05c

1.17±0.31c

1.97±0.41a

39.26±3.25b

1.90±0.31c

0.36±0.18c

0.44±0.03c

0.23±0.01c

0.31±0.03c

43.85±1.95d

1.67±0.48d

0.11±0.01c

0.32±0.04d

1.12±0.22c

1.85±0.35b

32.65±2.84c

1.44±0.24d

0.34±0.11c

0.32±0.02d

0.18±0.02d

0.22±0.01d

85.92±4.44a

3.05±0.60a

0.49±0.02a

0.90±0.07a

2.36±0.52a

0.98±0.28c

47.51±3.80a

4.95±0.74a

0.63±0.27a

0.77±0.07a

0.45±0.04a

0.58±0.09a

77.42±3.60b

2.77±0.51b

0.38.±0.02b

0.72±0.04b

2.05±0.41b

0.92±0.21c

41.66±3.18b

3.14±0.65b

0.52±0.24b

0.54±0.08b

0.29±0.03b

0.47±0.07b

Muscle Shahdra Ballouki SKIN Shahdra Ballouki

The value with a different letters in the same column is different (p

0.05)

Table: 2: Metal and trace element concentration concentrations (µg/g dry weight) ± S.E in two liver section (proximal and distal) of the Labeo rohita from Shahdra and Ballouki stations River Ravi

Zn Se Pb Proximal Section 115.81±4.25a 8.75±1.71b 0.98±0.23 Shahdra 85.44±5.61b 5.22±1.10c 0.64±0.31 Ballouki Distal Section 119.62±6.75a 10.12±1.12a 1.08±0.14a Shahdra 91.62±7.33b 6.20±1.43c 0.73±0.24 Ballouki

Ni

Mn

Hg

Fe

Cu

Cr

Co

Cd

As

1.77±0.42b

6.79±1.65b

2.42±0.72a

671.42±11.45b

23.95±3.85b

0.95±0.29a

1.41±0.42b

0.66±0.11b

0.69±0.22b

1.11±0.37d

5.12±1.52c

1.80±0.60c

543.21±20.28d

18.91±2.77d

0.74±0.16b

1.05±0.28c

0.44±0.17d

0.46±0.18d

1.96±0.33a

7.42±1.21a

2.65±0.51a

688.52±7.88a

25.60±2.44a

1.04±0.36a

1.57±0.36a

0.77±0.26a

0.79±0.25a

1.42±0.22c

5.90±1.61c

1.95±0.44c

568.22±9.41c

20.77±3.05c

0.82±0.39b

1.14±0.44c

0.52±0.22c

0.57±0.17c

The value with a different letters in the same column is different (p

0.05)

Table: 3: Metal and trace element concentration concentrations (µg/g dry weight) ± S.E in three section (proximal, median and distal) of intestine of the Labeo rohita from from Shahdra and Ballouki stations River Ravi

Anterior Section Shahdhra Ballouki Middle Section Shahdhra Ballouki Posterior Section Shahdhra Ballouki

Zn

Se

Pb

Ni

Mn

Hg

Fe

Cu

Cr

Co

Cd

As

91.90±4.75a

6.85±1.41a

0.54±0.09b

1.10±0.33b

5.41±0.76b

2.05±0.52a

142.65±7.78a

14.60±1.92b

0.59±0.18b

0.69±0.18a

0.42±0.11a

0.50±0.10a

80.95±6.60d

5.22±1.62c

0.43±0.06d

0.92±0.20d

4.90±0.88d

1.83±0.66d

122.72±6.90d

11.23±2.80c

0.45±0.12d

0.54±0.20c

0.33±0.08c

0.42±0.08c

88.52±4.90b

6.11±1.58b

0.47.±0.07c

1.02±0.22c

5.15±0.70c

1.96±0.72c

140.61±5.90c

13.70±2.45b

0.52±0.24c

0.63±0.14b

0.40±0.06b

0.46±0.05b

78.22±5.66d

4.97±1.42c

0.40±0.08d

0.80±0.18e

4.60±0.60e

1.76±0.63e

120.81±6.42d

10.02±3.11d

0.40±0.18c

0.50±0.16d

0.30±0.10c

0.39±0.07c

94.82±6.70a

7.05±2.22a

0.68.±0.10a

1.26±0.35a

5.9±0.74a

2.42±0.64a

145.90±7.65a

15.85±3.44a

0.67±0.18a

0.74±0.21a

0.47±0.09a

0.54±0.09a

82.77±7.66c

5.88±1.90b

0.51±0.11b

1.06±0.26c

4.98±0.88d

1.97±0.52c

124.62±6.77d

12.42±2.33b

0.49±0.21d

0.59±0.18c

0.38±0.07b

0.47±0.05b

The value with a different letters in the same column is different (p

0.05)