Effects of Sublethal Copper Concentrations on Gills of White Shrimp (Litopenaeus vannamei, Boone 1931)
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- Soegianto, A., Irawan, B. & Usman, N. Bull Environ Contam Toxicol (2013) 91: 630. doi:10.1007/s00128-013-1113-5
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The objective of this study was to measure the copper (Cu) concentration in gills of juveniles Litopenaeus vannamei after exposure to Cu at sublethal concentrations, and to evaluate its effect upon the structure of gill tissue. The Cu concentration in gills of control shrimp was 0.075 mg/kg. Copper concentrations increased significantly by 147 %, 180 % and 205 % in gills of shrimp exposed to 0.675, 1.325 and 2.010 mg Cu/L, respectively. After exposure to 0.675 mg Cu/L for 15 days, gill tissue hyperplasia was observed, with a narrowing of the hemolymphatic lacunae. Necrosis and loss of hemolymphatic lacunae were observed at exposures of 1.325 and 2.010 mg Cu/L.
Copper exists in trace quantities in the marine environment. Its average concentration in unpolluted waters is <0.05 μg/L (Saager et al. 1997). However, due to anthropogenic input and local geological conditions, concentrations of Cu in coastal and estuarine waters may be considerably higher. For example, a Cu concentration of 0.85 mg/L was reported in coastal waters of Hong Kong (Wong 1993). Copper concentrations of 0.53–0.74 mg/L were reported in Santong Bay, Indonesia (Edward and Marasabessy 2003). In areas near industrial discharges, concentrations may be even higher, reaching levels of 1.4–2 mg/L (Stauber et al. 2005; Dan’azumi and Bichi 2010).
Copper is an essential element required by crustaceans for synthesis of the respiratory pigment haemocyanin, but it is potentially toxic if present in excess (Brouwer et al. 2002). Tissues of crustaceans may accumulate copper depending on external copper concentrations (Rainbow 2002). Uptake occurs through the epithelial surfaces related to ion absorption and excretion, such as the gills (Soegianto et al. 1999). The gills are important organs of respiration, as well as of osmoregulation (Romano and Zeng 2012). Crustacean gills have been shown to be damaged by Cu (Soegianto et al. 1999; Frias-Espericueta et al. 2008). In a recent study from our laboratory (Usman et al. 2013), it was shown that sublethal concentrations of Cu perturbed the osmoregulatory capacity of white shrimp, Litopenaeus vannamei. In view of these studies, the objectives of the present study were to determine the accumulation of Cu and its bioconcentration factor (BCF) in gill tissue of L. vannamei following exposure to sublethal concentrations, and to evaluate any histological effects upon gill tissue.
Materials and Methods
Juveniles of L. vannamei (mean body weight: 9.09 ± 0.19 g) were obtained from a commercial shrimp farm located in Sidoarjo, East Java, Indonesia. After being transported to the laboratory, juveniles were kept for 2 days before the experiments at a salinity of 15 ppt, based on local shrimp farming practices. Other conditions were: temperature of 28–29 °C, pH of 7.5–8.0, dissolved oxygen of 6.5–7.1 mg/L, continuous water aeration and filtration with a 12-h light and 12-h dark light cycle. The background dissolved Cu concentration, as determined using a Shimadzu type AA-6200 (Kyoto, Japan) flame atomic absorption spectrophotometer (AAS), was 0.0025 ± 0.0008 mg/L. Shrimp were fed with commercial fish food (35 % protein, 3 % fat and 4 % fiber) during the acclimation. Uneaten food and feces were removed every day to avoid the diminution of water quality. Only healthy juveniles were used for experiments. A healthy shrimp has natural body color, complete appendages and constant motion as it looks for food in the tank.
A stock solution (1,000 mg Cu/L) was prepared from Cu(CH3COO)2·H2O (Merck, Pro Analysi, Darmstadt, Germany) in deionized water (ultrapure). Selected experimental concentrations were made by addition of adequate volumes of the stock solution to dilute seawater (15 ppt).
In previous work, Usman et al. (2013) reported that the 96-h LC50 (lethal concentration for 50 % of the exposed individuals) for Cu was 2.1 mg/L in juvenile L. vannamei, and that their osmoregulatory capacity was significantly reduced after 15 days exposure to 0.675, 1.325 and 2.010 mg Cu/L at salinity of 15 ppt. Based on these values, we conducted this study.
Eight groups of 10 juveniles of L. vannamei were exposed for 15 days to sublethal concentrations of copper (nominal concentrations: 0.7, 1.4 and 2.1 mg Cu/L ≈ measured concentrations: 0.675, 1.325 and 2.010 mg Cu/L) at salinity of 15 ppt, while no metal was used for the control group. Each treatment group was in duplicate. Aeration was provided and the test media were renewed every 48 h. The Cu concentration was checked every 48 h in each medium just after renewal. During the test, shrimp were fed ad libitum with commercial fish food two times each day. Uneaten food and feces were removed during the exposure period.
After 15 days, 10 shrimp from each treatment group were dissected and the gills of each organism were removed, dried at 65 °C for 48 h to constant weight and homogenized. Approximately 1 g of homogenized tissue sample was transferred to 3 ml concentrated nitric acid (Merck, Suprapur, Darmstadt, Germany) and digested at 90 °C for 4 h. After cooling, samples were filtered and diluted to 50 ml with deionized water; copper concentrations were measured as described in an earlier study (Soegianto et al. 1999). Copper concentrations of samples were expressed as mg/kg dry weight (dw).
Analytical blanks were run in the same way as the samples, and concentrations were determined using standard solutions prepared in the same acid matrix. Validity of analytical methods was checked using dogfish muscle reference material (DORM-2) provided by the National Research Council of Canada (Ottawa), with Cu recovery of 106 %–108 % (certified value: 2.34 ± 0.16 mg/kg dw, measured value: 2.52 ± 0.03 mg/kg dw, n = 3). The detection limits of Cu were 0.0015 mg/L for seawater and 0.02 mg/kg dw for tissue.
For histological study, on the last day of the experiment, gills were carefully excised and fixed in 4 % buffered formalin, embedded in paraffin, sectioned at 8 μm thickness on a microtome (Microm HM 315, Walldorf, Germany), stained with hematoxylin and eosin, and examined with a microscope (Olympus CX41, Tokyo, Japan). Gills were histologically examined in at least 6 samples at each treatment.
All data were normally distributed according to the Kolmogorov–Smirnov test (Steel and Torrie 1981). Therefore, comparisons of the effects of different treatment on concentration, BCF and accumulation rate of Cu in gills were analysed by one way analysis of variance respectively and, when significant differences were detected (p < 0.05), the different means were separated with Duncan’s test.
Results and Discussion
Copper concentrations (mean ± SD) in gill tissues of juvenile L. vannamei after animals were exposed to various Cu concentrations for 15 days at 15 ppt salinity
Treatment (mg Cu/L)
Copper concentration in gill tissues (mg Cu/kg)
0.075 ± 0.020a
0.1101 ± 0.0066b
0.1359 ± 0.0091c
0.1534 ± 0.0074c
Bioconcentration factor of copper in gills of juvenile L. vannamei after animals were exposed to various Cu concentrations for 15 days at 15 ppt salinity
Treatment (mg Cu/L)
0.163 ± 0.010a
0.103 ± 0.007b
0.076 ± 0.004c
Accumulation rate of copper from media to gills of juvenile L. vannamei after animals were exposed to various Cu concentrations for 15 days at 15 ppt salinity
Treatment (mg Cu/L)
Accumulation rate (mg/kg/day)
0.003 ± 0.002
0.004 ± 0.002
0.005 ± 0.001
This study shows that Cu is accumulated significantly in gills of juveniles L. vannamei after sublethal exposures for 15 days. The exposure to Cu causes the level of Cu in gills to rise successively in direct proportion to external Cu concentration. The BCF values in gills decrease with increasing Cu concentrations in media. This may indicate that the tissue concentration of Cu in the body of the shrimp is being regulated and has not reached a maximum level. Our findings are in agreement with the results of Soegianto et al. (1999). An increase in gill Cu concentration was observed in the shrimp Penaeus japonicus following exposure to Cu between 0.5 and 1.0 mg/L for 4 days, with BCF values decreasing substantially with increasing Cu concentration in media (Soegianto et al. 1999). The mean Cu concentration in the juveniles of the present study surviving in the high Cu levels (0.675–2.010 mg Cu/L) suggests that L. vannamei can either tolerate high tissue Cu levels or may be capable of effectively removing the metal from general circulation by depositing it in particular tissues. The hepatopancreas was identified as a site of deposition in Crangon vulgaris (Djangmah 1970). Metals are known to have a short biological half-life in the gills, and are progressively transferred from the gills to the hepatopancreas via the hemolymph (Caceci et al. 1988).
This present study reported that accumulation of Cu in gills caused alterations to the gill structure, and that the histological damage increased with increasing Cu concentration in gills, resulting in gill hyperplasia, necrosis, narrowing and even loss of hemolympathic lacunae. Similar alterations have been observed in other crustaceans exposed to heavy metals (Soegianto et al. 1999; Frias-Espericueta et al. 2008; Wu et al. 2009). These authors demonstrated that when crustaceans were exposed to different levels of metals, alterations consisted of a blackened appearance of the gills, hyperplasia and necrosis of gill cells that resulted in narrowed or obstructed hemolymphatic vessels, loss of regular structure of the epithelium and even fragmentation of nuclei within gill cells. In view of these serious structural damages, copper could inhibit the physiological gill functions, as shown in a previous work by the 70.3 %, 77.2 %, and 77.8 % loss of osmoregulatory capacity of shrimp exposed to 0.675, 1.325 and 2.010 mg Cu/L, respectively (Usman et al. 2013). Similar findings have been reported in other species. An impairment of osmoregulation by Cu was reported in P. japonicus juveniles after 4 and 7 days of exposure to 500 and 1,000 μg Cu/L (Bambang et al. 1995). The mechanism responsible for the perturbation of osmoregulatory capacity of this species (Bambang et al. 1995) was in agreement with the gill structure alterations of the exposed shrimp in the present study.
Other authors have reported that the perturbation of osmoregulation in crustaceans exposed to metals correlated with reductions in the concentrations of major hemolymph ions (Na+, Ca2+, K+, Mg2+, Cl−), and with the inhibition of gill Na+–K+-ATPase activity (Bjerregaard and Vislie 1986; Weeks et al. 1993). We report here that structural degradation of gill tissue in L. vannamei following exposure to Cu likely contributed to the decreased osmoregulatory ability of the shrimp, as reported by Usman et al. (2013).
Special thanks are due to Mrs. Setiyanto and Soewarni for their technical assistance during the experiments. We also wish to thank anonymous reviewers for valuable advice and constructive comments to improve the manuscript.