Bull Environ Contam Toxicol (2015) 94:549–553 DOI 10.1007/s00128-015-1507-7
Effects of Crude Oil and Dispersed Crude Oil on the Critical Swimming Speed of Puffer Fish, Takifugu rubripes Xiaoming Yu • Chuancai Xu • Haiying Liu Binbin Xing • Lei Chen • Guosheng Zhang
•
Received: 9 July 2014 / Accepted: 25 February 2015 / Published online: 3 March 2015 Ó Springer Science+Business Media New York 2015
Abstract In order to examine the effects of crude oil and dispersed crude oil (DCO) on the swimming ability of puffer fish, Takifugu rubripes, the critical swimming speeds (Ucrit) of fish exposed to different concentrations of water-soluble fraction (WSF) of crude oil and DCO solution were determined in a swimming flume. WSF and DCO significantly affected the Ucrit of puffer fish (p \ 0.05). The Ucrit of puffer fish exposed to 136 mg L-1 WSF and 56.4 mg L-1 DCO decreased 48.7 % and 43.4 %, respectively. DCO was more toxic to puffer fish than WSF. These results suggested that crude oil and chemically dispersed oil could weaken the swimming ability of puffer fish. Keywords Crude oil Dispersed crude oil Critical swimming speed Takifugu rubripes With the rapid development of oil and petroleum exploration, production, and transportation, many catastrophic oil spill accidents have occurred around the world (Agamy 2012; Jiang et al. 2012). The adverse effects of oil spills include not only serious acute but also chronic damage to marine ecosystems, especially to marine wildlife (Gagnon and Holdway 2000; Peterson et al. 2003; Esler et al. 2010). Mechanical containment and recovery equipment is used to capture and store the spilled oil. Chemical dispersants can be used in conjunction with mechanical means to break up oil slicks and to prevent impacts on coastlines, air breathing marine animals and benthic resources (Hook and Osborn 2012; Lee et al. 2013). The dispersants used are surfactants with a chemical affinity for both oil and water, X. Yu C. Xu H. Liu B. Xing L. Chen G. Zhang (&) Center for Marine Ranching Engineering Science Research of Liaoning, Dalian Ocean University, Dalian 116023, China e-mail: [email protected]; [email protected]
enabling the petroleum to be mixed into the water column in small mixed oil-surfactant micelles (Canevari 1978). Dispersants not only dilute the crude oil but also enhance the rate of oil degradation by physical and chemical processes. However, the chemical dispersant itself is potentially toxic to aquatic organisms, and oil dispersed following the action of a dispersant can lead to an increase in toxicity of the water accommodated hydrocarbon fraction (Vindimian et al. 1992; Anderson et al. 2014; Cohen et al. 2014). Critical swimming speed (Ucrit) can be used to quantify and compare the physical status, evaluate the maximal swimming speed, and reflect the maximum oxygen consumption capability of fish (Farrell and Steffensen 1987; Brauner et al. 1994; Plaut 2001). It is also used to evaluate the effects of environmental conditions and pollutants on fish performance (Landman et al. 2006; Guan et al. 2008). However, little research has been done on the effects of water-soluble fraction (WSF) of crude oil and dispersed crude oil (DCO) on the Ucrit of fish (Kennedy and Farrell 2006; Milinkovitch et al. 2012). The puffer fish, Takifugu rubripes, is an extremely important species in China’s fishery and mariculture industries, especially in northern. Several oil spill accidents occurred in Yellow Sea and Bohai Sea (e.g., Dalian oil pipelines exploded in 2010, Bohai Bay accidents in 2010 and 2011, respectively). After oil spill accidents, the crude oil and DCO could pose risks to the mariculture and stock of puffer fish in this region. The primary objective of the present study was to examine the effects of exposure to crude oil and DCO on the swimming ability of puffer fish in a laboratory experiment, simulating possible exposures to these chemicals following an oil spill accident. The Ucrit of puffer fish exposed to different concentrations of WSF and DCO was determined.
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Results can be useful in assessing the toxicity and ecological effects of crude oil and DCO on fish.
Materials and Methods The puffer fish (wet mass: 16.29 ± 3.90 g, body length: 8.52 ± 0.61 cm) used in the experiment were purchased from Tianzheng Co. Ltd. (Dalian, China). The fish were acclimated in a 20 m3 tank for 7 days before the experiment. During this acclimation period, fish were fed twice daily with commercial pellets, but were fasted for 24 h before the experiment. Filtered sea water (drawn from Heishijiao Bay) in the tank was maintained at a temperature of 19.0–22.0°C, salinity of 32.0 % ± 1.0 %, and dissolved oxygen [6.0 mg L-1. The crude oil was from Liaohe oil field (Panjin, China) and stored in a dark, sealed storage bottle before being used to prepare the stock solution. The chemical dispersant used was Shuangxiang Oil Spill Dispersant produced by Longquan chemical plant (Dalian, China). The Ucrit test was conducted in a cylindrical swimming flume with a 7 9 50 cm (diameter 9 length) swimming chamber (Fig. 1). The current in the swimming flume was generated by a centrifugal pump (Huakang, 102-4, China), the motor of which was controlled by a frequency converter (Xilin, EH640S, China). The current velocity was determined by a miniature propeller current meter (KENEK, VR-101, Japan). To obtain the WSF solution, 10 mL crude oil was mixed with 1 L filtered sea water (Tian et al. 2008). The mixture was sonicated for 120 min with energy of 150 W in an ultrasonic bath and then left to settle for 1 h. The aqueous phase was collected and stored at 4°C, and used as the stock solution for subsequent experiments. To obtain the DCO solution, 100 mL crude oil was mixed with 900 mL filtered sea water in a 1 L glass mixing chamber, and a vortex was created using a magnetic stirrer (Agamy 2012). The chemical dispersant was then added
Bull Environ Contam Toxicol (2015) 94:549–553
drop by drop to the floating oil during mixing, at a ratio of 1:5 (dispersant:oil). The solution was capped, mixed for 24 h, and allowed to settle for 1 h to separate the water and oil phases. The aqueous phase was collected and stored at 4°C, and used as the stock solution for subsequent experiments. Total petroleum hydrocarbons (TPH) were measured in WSF and DCO test solutions using the triple peak method (Chinese National Environmental Protection Standard HJ 637-2012). The sample was extracted with the solvent, carbon tetrachloride followed by analysis with an infrared spectrometer (Beiguang, JDS-107U, China) at three spectral regions: 2930, 2960 and 3030 cm-1. The detection limit of the method was 0.08 mg L-1. All analytical procedures were monitored with strict quality assurance and control measures. The instrument was calibrated with calibration standard. Each set of samples was accompanied by a blank and a duplicate sample, which were carried throughout the entire analytical procedure in a manner identical to the samples. The determined concentrations of the procedural blanks were no more than the method detection limit. The relative percent difference of the duplicate samples was\5 %. TPH in WSF test solutions were 0, 34.0, 68.1, 102 and 136 mg L-1, and the water temperature was 21.2 ± 0.5°C. TPH in DCO test solutions were 0, 14.1, 28.2, 42.3, and 56.4 mg L-1, and the water temperature was 21.0 ± 0.7°C. Five puffer fish were tested for each concentration. The water in the swimming flume was adjusted to the experimental concentration before the swimming test and dissolved oxygen was maintained at [6.0 mg L-1. One puffer fish was tested in each swimming trial. Before the experiment, the puffer fish was allowed to acclimate for 10 min at a speed of 10.5 ± 0.3 cm s-1 to orient it to the current. The water velocity of the swimming chamber was then increased by 4 cm s-1 every 10 min until fatigue occurred. Swimming fatigue was considered to have been reached when a fish fell against the downstream screen and did not respond to gentle prodding. The swimming time at the highest velocity was recorded for each puffer fish. The fatigued fish was immediately removed from the swimming chamber and weighed to the nearest 0.01 g. No feed was given during the experimental period. Ucrit (cm s-1) was calculated using the equation (Brett 1964) Ucrit ¼ U1 þ ðT1 =T2 Þ U2
Fig. 1 Diagram of the swimming flume
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where U1 is the highest velocity attained for an entire 10 min interval, T1 is the time taken until fatigue is reached during the final speed interval, T2 denotes the time interval (10 min), and U2 represents the speed increment (4 cm s-1). Relative critical swimming speed (Ucrit0 , BL s-1, body lengths per second) was calculated as critical
Bull Environ Contam Toxicol (2015) 94:549–553
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swimming speed (cm s-1) divided by the body length (cm) of test fish. The difference in Ucrit of puffer fish exposed to different concentrations of WSF or DCO was analyzed using oneway ANOVA and Duncan’s multiple range tests. Statistical analyses were performed using PASW Statistics 18 (SPSS Inc., Chicago, USA), and differences were considered significant when p \ 0.05 for all analyses.
Results and Discussion The Ucrit of puffer fish exposed to different concentrations of WSF solution is shown in Table 1. The Ucrit decreased as the concentration of WSF solution increased from 0 to 136 mg L-1 (TPH). The mean Ucrit values for fish at all WSF TPH treatment levels were significantly reduced relative to control fish (p \ 0.05). The Ucrit of puffer fish exposed to different concentrations of DCO solution is shown in Table 2. The Ucrit decreased as the concentration of DCO solution increased from 0 to 56.4 mg L-1 (TPH). The mean Ucrit values for fish exposed to 28.2, 42.3 and 56.4 mg L-1 DCO TPH were significantly reduced relative to fish in the control group (p \ 0.05). Locomotion in fish is an integrated physiological process, and its performance may be affected by contaminants in sea water. Swimming performance has been used as an important criterion in the determination of sublethal effects of toxicants on fish. Ucrit has been used previously to define tolerance limits to various pollutants and environmental parameters, including pulp and paper effluent (Landman et al. 2006), copper (De Boeck et al. 2006), and temperature (Yu et al. 2010). Swimming performance of fish can be reduced by the toxic effects of WSF. Kennedy and Farrell (2006) reported that decreases in Ucrit were dependent on both exposure time and concentration. They found that the Ucrit of juvenile Pacific herring, Clupea pallasi, exposed to 100 lg L-1 WSF for 24 h and 40 lg L-1 for 96 h were significantly
lower compared to control fish. Exposure of coho salmon, Oncorhynchus kisutch, to the WSF affected swimming performance at [2.5 mg L-1 in exposures of 48 h and at 0.66 mg L-1 in exposures of 5–13 d (Thomas and Rice 1987). In the present study, the Ucrit of puffer fish decreased from 27.0 to 13.9 cm s-1 as the concentration of WSF solution increased from 0 to 136 mg L-1 (TPH). These results showed that reductions in swimming performance of fish occurred in a concentration-dependent and time-dependent manner after exposure to WSF. At present, however, little research has been published on the effect of DCO on the Ucrit of fish. A study by Milinkovitch et al. (2012) showed that the Ucrit of juvenile golden grey mullet, Liza aurata, was not altered by 44.0 mg L-1 (TPH) DCO. In the present study, the Ucrit was significantly reduced in puffer fish exposed to 28.2 mg L-1 (TPH) DCO. These results indicated that the puffer fish may be more sensitive to DCO than juvenile golden grey mullet. Impaired Ucrit often is related to an inability to supply enough oxygen to the gills, to deliver enough oxygen to the tissues, to remove metabolic products, to provide adequate substrates, or to activate enzymatic processes (Kennedy and Farrell 2006). Gills are the major route of uptake of waterborne pollutants by fish. WSF and DCO have been found to promote structural damage in the branchial respiratory epithelium, affecting gas exchange processes and limiting oxygen transfer in fish (McKeown and March 1978; Nikl and Farrell 1993; Duarte et al. 2010). Kennedy and Farrell (2005) reported that WSF impairs respiratory gas exchange based on observations of gill hyperplasia, which also could adversely affect extrarenal excretion and ionic exchange at the gills. Evidence suggests that the DCO is significantly more toxic than the WSF. Chemical dispersion increased both the total polycyclic aromatic hydrocarbon (PAH) concentrations and the proportion of high-molecular-weight PAHs in the water-accommodated fraction (WAF) of oil (Couillard et al. 2005). Chemical dispersants can also greatly increase the toxicity of crude oil by increasing the
Table 1 Ucrit and Ucrit0 of T. rubripes exposed to different concentration of water-soluble fraction solution Concentration (mg L-1 TPH)
Number of fish
Ucrit (cm s-1) Mean ± SE
0
5
Ucrit0 (BL s-1) Minimum
a
27.0 ± 3.09
b
19.3
Maximum 37.8
Mean ± SE
Minimum
Maximum
a
2.54
4.73
b
3.22 ± 0.39
34.0
5
18.5 ± 2.99
10.5
27.5
2.16 ± 0.35
1.18
3.06
68.1
5
17.3 ± 2.55b
11.3
25.7
1.97 ± 0.28b
1.36
2.86
102
5
13.9 ± 1.46b
11.3
19.3
1.65 ± 0.19b
1.34
2.35
136
5
13.9 ± 1.61b
10.5
18.5
1.78 ± 0.25b
1.26
2.43
Values with different letters in the same column indicate a significant difference from each other (p \ 0.05)
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Bull Environ Contam Toxicol (2015) 94:549–553
Table 2 Ucrit and Ucrit0 of T. rubripes exposed to different concentration of dispersed crude oil solution Concentration (mg L-1 TPH)
Number of fish
Ucrit (cm s-1) Mean ± SE 27.0 ± 3.09a
Ucrit0 (BL s-1) Minimum
Maximum
Mean ± SE
Minimum
Maximum
19.3
37.8
3.22 ± 0.39a
ab
2.54
4.73
14.1
5
21.7 ± 1.37
18.5
26.7
2.63 ± 0.21ab
2.18
3.34
28.2
5
20.3 ± 2.32bc
14.5
26.5
2.46 ± 0.24bc
1.75
3.15
42.3
5
bc
16.5 ± 1.71
12.1
21.7
bc
2.00 ± 0.21
1.49
2.58
56.4
5
15.3 ± 0.67c
13.7
17.7
1.81 ± 0.06c
1.59
1.97
0
5
Values with different letters in the same column indicate a significant difference from each other (p \ 0.05)
chemical oxygen demand (COD) of the dispersant enhanced WAF (Radniecki et al. 2013). Rico-Martı´nez et al. (2013) reported that Corexit 9500A and Macondo crude oil are similar in their toxicity. However, when Corexit 9500A and oil are mixed, toxicity to the marine rotifer, Brachionus plicatilis, increases up to 52-fold. In the current study, the concentrations of WSF and DCO at which the Ucrit of puffer fish decreased about 37 % were found to be 68.1 and 42.3 mg L-1 (TPH), respectively. The results showed that DCO was more toxic to puffer fish than the WSF. In conclusion, WSF and DCO significantly affected the Ucrit of puffer fish. DCO was more toxic to puffer fish than WSF. The results suggested that crude oil and chemically dispersed oil could weaken the swimming ability of puffer fish. Acknowledgments The authors would like to thank the three anonymous reviewers for their helpful comments. This study was supported by funds from Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology (201202), Public Science and Technology Research Funds Projects of Ocean (201205023-2, 201305002) and Special Fund for Agro-scientific Research in the Public Interest (201203018).
References Agamy E (2012) Histopathological liver alterations in juvenile rabbit fish (Siganus canaliculatus) exposed to light Arabian crude oil, dispersed oil and dispersant. Ecotoxicol Environ Saf 75:171–179 Anderson JA, Kuhl AJ, Anderson AN (2014) Toxicity of oil and dispersed oil on juvenile mud crabs, Rhithropanopeus harrisii. Bull Environ Contam Toxicol 92:375–380 Brauner CJ, Iwama GK, Randall DJ (1994) The effect of short-duration seawater exposure on the swimming performance of wild and hatchery-reared juvenile coho salmon (Oncorhynchus kisutch) during smoltification. Can J Fish Aquat Sci 51:2188–2194 Brett JR (1964) The respiratory metabolism and swimming performance of young sockeye salmon. J Fish Res Board Can 21:1183–1226 Canevari GP (1978) Some observations on the mechanism and chemistry aspects of chemical dispersion. In: McCarthy LT, Lindblom GP, Walter HF (eds) Chemical dispersants for the control of oil spills. American Society for Testing and Materials, Philadelphia, pp 2–5
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Chinese National Environmental Protection Standard-Water qualityDetermination of petroleum oils and animal and vegetable oilsInfrared spectrophotometry (HJ 637-2012). Ministry of Environmental Protection of the People’s Republic of China. Beijing China Cohen JH, McCormick LR, Burkhardt SM (2014) Effects of dispersant and oil on survival and swimming activity in a marine copepod. Bull Environ Contam Toxicol 92:381–387 Couillard CM, Lee K, Le´gare´ B, King TL (2005) Effect of dispersant on the composition of the water-accommodated fraction of crude oil and its toxicity to larval marine fish. Environ Toxicol Chem 24:1496–1504 De Boeck G, van der Ven K, Hattink J, Blust R (2006) Swimming performance and energy metabolism of rainbow trout, common carp and gibel carp respond differently to sublethal copper exposure. Aquat Toxicol 80:92–100 Duarte RM, Honda RT, Val AL (2010) Acute effects of chemically dispersed crude oil on gill ion regulation, plasma ion levels and haematological parameters in tambaqui (Colossoma macropomum). Aquat Toxicol 97:134–141 Esler D, Trust KA, Ballachey BE, Iverson SA, Lewis TL, Rizzolo DJ, Mulcahy DM, Miles A, Woodin BR, Stegeman JJ, Henderson JD, Wilson BW (2010) Cytochrome P4501A biomarker indication of oil exposure in harlequin ducks up to 20 years after the Exxon Valdez oil spill. Environ Toxicol Chem 29:1138–1145 Farrell AP, Steffensen JF (1987) An analysis of the energetic cost of the branchial and cardiac pumps during sustained swimming. Fish Physiol Biochem 4:73–79 Gagnon MM, Holdway DA (2000) EROD induction and biliary metabolite excretion following exposure to the water accommodated fraction of crude oil and to chemically dispersed crude oil. Arch Environ Contam Toxicol 38:70–77 Guan L, Snelgrove PVR, Gamperl AK (2008) Ontogenetic changes in the critical swimming speed of Gadus morhua (Atlantic cod) and Myoxocephalus scorpius (shorthorn sculpin) larvae and the role of temperature. J Exp Mar Biol Ecol 360:31–38 Hook SE, Osborn HL (2012) Comparison of toxicity and transcriptomic profiles in a diatom exposed to oil, dispersants, dispersed oil. Aquat Toxicol 124–125:139–151 Jiang Z, Huang Y, Chen Q, Zeng J, Xu X (2012) Acute toxicity of crude oil water accommodated fraction on marine copepods: the relative importance of acclimatization temperature and body size. Mar Environ Res 81:12–17 Kennedy CJ, Farrell AP (2005) Ion homeostasis and interrenal stress responses in juvenile Pacific herring, Clupea pallasi, exposed to the water-soluble fraction of crude oil. J Exp Mar Biol Ecol 323:43–56 Kennedy CJ, Farrell AP (2006) Effects of exposure to the watersoluble fraction of crude oil on the swimming performance and the metabolic and ionic recovery postexercise in Pacific herring (Clupea pallasi). Environ Toxicol Chem 25:2715–2724
Bull Environ Contam Toxicol (2015) 94:549–553 Landman MJ, Heuvel MR, Finley M, Bannon HJ, Ling N (2006) Combined effects of pulp and paper effluent, dehydroabietic acid, and hypoxia on swimming performance, metabolism, and hematology of rainbow trout. Ecotoxicol Environ Saf 65:314–322 Lee KW, Shim WJ, Yim UH, Kang JH (2013) Acute and chronic toxicity study of the water accommodated fraction (WAF), chemically enhanced WAF (CEWAF) of crude oil and dispersant in the rock pool copepod Tigriopus japonicus. Chemosphere 92:1161–1168 McKeown BA, March GL (1978) The acute effect of bunker C oil and an oil dispersant on: 1 Serum glucose, serum sodium and gill morphology in both freshwater and seawater acclimated rainbow trout (Salmo gairdneri). Water Res 12:157–163 Milinkovitch T, Lucas J, Floch SL, Thomas-Guyon H, Lefranc¸ois C (2012) Effect of dispersed crude oil exposure upon the aerobic metabolic scope in juvenile golden grey mullet (Liza aurata). Mar Pollut Bull 64:865–871 Nikl DL, Farrell AP (1993) Reduced swimming performance and gill structural changes in juvenile salmonids exposed to 2-(thiocyanomethylthio) benzothiazole. Aquat Toxicol 27:245–263 Peterson CH, Rice SD, Short JW, Elser D, Bodkin JL, Ballachey BE, Irons DB (2003) Long-term ecosystem response to the Exxon Valdez oil spill. Science 302:2082–2086 Plaut I (2001) Critical swimming speed: its ecological relevance. Comp Biochem Physiol 131A:41–50
553 Radniecki TS, Schneider MC, Semprini L (2013) The influence of Corexit 9500A and weathering on Alaska North Slope crude oil toxicity to the ammonia oxidizing bacterium, Nitrosomonas europaea. Mar Pollut Bull 68:64–70 Rico-Martı´nez R, Snell TW, Shearer TL (2013) Synergistic toxicity of Macondo crude oil and dispersant Corexit 9500AÒ to the Brachionus plicatilis species complex (Rotifera). Environ Pollut 173:5–10 Thomas RE, Rice SD (1987) Effect of water-soluble fraction of cook inlet crude oil on swimming performance and plasma cortisol in juvenile coho salmon (Oncorhynchus kisutch). Comp Biochem Physiol 87C:177–180 Tian LF, Ren Z, Cui Y, Chen BJ, Jiao GB, Wang XD, Zhang XG (2008) Acute toxicity of Shengli crude oil and its impact on AKP activity of Paralichthys olivaceus. Mar Fish Res 29:95–100 (in Chinese) Vindimian E, Vollat B, Garric J (1992) Effect of the dispersion of oil in freshwater based on time-dependent Daphnia magna toxicity tests. Bull Environ Contam Toxicol 48:209–215 Yu XM, Zhang XM, Duan Y, Zhang PD, Miao ZQ (2010) Effects of temperature, salinity, body length, and starvation on the critical swimming speed of whiteleg shrimp, Litopenaeus vannamei. Comp Biochem Physiol 157A:392–397
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Effects of Crude Oil and Dispersed Crude Oil on the Critical Swimming Speed of Puffer Fish, Takifugu rubripes Xiaoming Yu • Chuancai Xu • Haiying Liu Binbin Xing • Lei Chen • Guosheng Zhang
•
Received: 9 July 2014 / Accepted: 25 February 2015 / Published online: 3 March 2015 Ó Springer Science+Business Media New York 2015
Abstract In order to examine the effects of crude oil and dispersed crude oil (DCO) on the swimming ability of puffer fish, Takifugu rubripes, the critical swimming speeds (Ucrit) of fish exposed to different concentrations of water-soluble fraction (WSF) of crude oil and DCO solution were determined in a swimming flume. WSF and DCO significantly affected the Ucrit of puffer fish (p \ 0.05). The Ucrit of puffer fish exposed to 136 mg L-1 WSF and 56.4 mg L-1 DCO decreased 48.7 % and 43.4 %, respectively. DCO was more toxic to puffer fish than WSF. These results suggested that crude oil and chemically dispersed oil could weaken the swimming ability of puffer fish. Keywords Crude oil Dispersed crude oil Critical swimming speed Takifugu rubripes With the rapid development of oil and petroleum exploration, production, and transportation, many catastrophic oil spill accidents have occurred around the world (Agamy 2012; Jiang et al. 2012). The adverse effects of oil spills include not only serious acute but also chronic damage to marine ecosystems, especially to marine wildlife (Gagnon and Holdway 2000; Peterson et al. 2003; Esler et al. 2010). Mechanical containment and recovery equipment is used to capture and store the spilled oil. Chemical dispersants can be used in conjunction with mechanical means to break up oil slicks and to prevent impacts on coastlines, air breathing marine animals and benthic resources (Hook and Osborn 2012; Lee et al. 2013). The dispersants used are surfactants with a chemical affinity for both oil and water, X. Yu C. Xu H. Liu B. Xing L. Chen G. Zhang (&) Center for Marine Ranching Engineering Science Research of Liaoning, Dalian Ocean University, Dalian 116023, China e-mail: [email protected]; [email protected]
enabling the petroleum to be mixed into the water column in small mixed oil-surfactant micelles (Canevari 1978). Dispersants not only dilute the crude oil but also enhance the rate of oil degradation by physical and chemical processes. However, the chemical dispersant itself is potentially toxic to aquatic organisms, and oil dispersed following the action of a dispersant can lead to an increase in toxicity of the water accommodated hydrocarbon fraction (Vindimian et al. 1992; Anderson et al. 2014; Cohen et al. 2014). Critical swimming speed (Ucrit) can be used to quantify and compare the physical status, evaluate the maximal swimming speed, and reflect the maximum oxygen consumption capability of fish (Farrell and Steffensen 1987; Brauner et al. 1994; Plaut 2001). It is also used to evaluate the effects of environmental conditions and pollutants on fish performance (Landman et al. 2006; Guan et al. 2008). However, little research has been done on the effects of water-soluble fraction (WSF) of crude oil and dispersed crude oil (DCO) on the Ucrit of fish (Kennedy and Farrell 2006; Milinkovitch et al. 2012). The puffer fish, Takifugu rubripes, is an extremely important species in China’s fishery and mariculture industries, especially in northern. Several oil spill accidents occurred in Yellow Sea and Bohai Sea (e.g., Dalian oil pipelines exploded in 2010, Bohai Bay accidents in 2010 and 2011, respectively). After oil spill accidents, the crude oil and DCO could pose risks to the mariculture and stock of puffer fish in this region. The primary objective of the present study was to examine the effects of exposure to crude oil and DCO on the swimming ability of puffer fish in a laboratory experiment, simulating possible exposures to these chemicals following an oil spill accident. The Ucrit of puffer fish exposed to different concentrations of WSF and DCO was determined.
123
550
Results can be useful in assessing the toxicity and ecological effects of crude oil and DCO on fish.
Materials and Methods The puffer fish (wet mass: 16.29 ± 3.90 g, body length: 8.52 ± 0.61 cm) used in the experiment were purchased from Tianzheng Co. Ltd. (Dalian, China). The fish were acclimated in a 20 m3 tank for 7 days before the experiment. During this acclimation period, fish were fed twice daily with commercial pellets, but were fasted for 24 h before the experiment. Filtered sea water (drawn from Heishijiao Bay) in the tank was maintained at a temperature of 19.0–22.0°C, salinity of 32.0 % ± 1.0 %, and dissolved oxygen [6.0 mg L-1. The crude oil was from Liaohe oil field (Panjin, China) and stored in a dark, sealed storage bottle before being used to prepare the stock solution. The chemical dispersant used was Shuangxiang Oil Spill Dispersant produced by Longquan chemical plant (Dalian, China). The Ucrit test was conducted in a cylindrical swimming flume with a 7 9 50 cm (diameter 9 length) swimming chamber (Fig. 1). The current in the swimming flume was generated by a centrifugal pump (Huakang, 102-4, China), the motor of which was controlled by a frequency converter (Xilin, EH640S, China). The current velocity was determined by a miniature propeller current meter (KENEK, VR-101, Japan). To obtain the WSF solution, 10 mL crude oil was mixed with 1 L filtered sea water (Tian et al. 2008). The mixture was sonicated for 120 min with energy of 150 W in an ultrasonic bath and then left to settle for 1 h. The aqueous phase was collected and stored at 4°C, and used as the stock solution for subsequent experiments. To obtain the DCO solution, 100 mL crude oil was mixed with 900 mL filtered sea water in a 1 L glass mixing chamber, and a vortex was created using a magnetic stirrer (Agamy 2012). The chemical dispersant was then added
Bull Environ Contam Toxicol (2015) 94:549–553
drop by drop to the floating oil during mixing, at a ratio of 1:5 (dispersant:oil). The solution was capped, mixed for 24 h, and allowed to settle for 1 h to separate the water and oil phases. The aqueous phase was collected and stored at 4°C, and used as the stock solution for subsequent experiments. Total petroleum hydrocarbons (TPH) were measured in WSF and DCO test solutions using the triple peak method (Chinese National Environmental Protection Standard HJ 637-2012). The sample was extracted with the solvent, carbon tetrachloride followed by analysis with an infrared spectrometer (Beiguang, JDS-107U, China) at three spectral regions: 2930, 2960 and 3030 cm-1. The detection limit of the method was 0.08 mg L-1. All analytical procedures were monitored with strict quality assurance and control measures. The instrument was calibrated with calibration standard. Each set of samples was accompanied by a blank and a duplicate sample, which were carried throughout the entire analytical procedure in a manner identical to the samples. The determined concentrations of the procedural blanks were no more than the method detection limit. The relative percent difference of the duplicate samples was\5 %. TPH in WSF test solutions were 0, 34.0, 68.1, 102 and 136 mg L-1, and the water temperature was 21.2 ± 0.5°C. TPH in DCO test solutions were 0, 14.1, 28.2, 42.3, and 56.4 mg L-1, and the water temperature was 21.0 ± 0.7°C. Five puffer fish were tested for each concentration. The water in the swimming flume was adjusted to the experimental concentration before the swimming test and dissolved oxygen was maintained at [6.0 mg L-1. One puffer fish was tested in each swimming trial. Before the experiment, the puffer fish was allowed to acclimate for 10 min at a speed of 10.5 ± 0.3 cm s-1 to orient it to the current. The water velocity of the swimming chamber was then increased by 4 cm s-1 every 10 min until fatigue occurred. Swimming fatigue was considered to have been reached when a fish fell against the downstream screen and did not respond to gentle prodding. The swimming time at the highest velocity was recorded for each puffer fish. The fatigued fish was immediately removed from the swimming chamber and weighed to the nearest 0.01 g. No feed was given during the experimental period. Ucrit (cm s-1) was calculated using the equation (Brett 1964) Ucrit ¼ U1 þ ðT1 =T2 Þ U2
Fig. 1 Diagram of the swimming flume
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where U1 is the highest velocity attained for an entire 10 min interval, T1 is the time taken until fatigue is reached during the final speed interval, T2 denotes the time interval (10 min), and U2 represents the speed increment (4 cm s-1). Relative critical swimming speed (Ucrit0 , BL s-1, body lengths per second) was calculated as critical
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swimming speed (cm s-1) divided by the body length (cm) of test fish. The difference in Ucrit of puffer fish exposed to different concentrations of WSF or DCO was analyzed using oneway ANOVA and Duncan’s multiple range tests. Statistical analyses were performed using PASW Statistics 18 (SPSS Inc., Chicago, USA), and differences were considered significant when p \ 0.05 for all analyses.
Results and Discussion The Ucrit of puffer fish exposed to different concentrations of WSF solution is shown in Table 1. The Ucrit decreased as the concentration of WSF solution increased from 0 to 136 mg L-1 (TPH). The mean Ucrit values for fish at all WSF TPH treatment levels were significantly reduced relative to control fish (p \ 0.05). The Ucrit of puffer fish exposed to different concentrations of DCO solution is shown in Table 2. The Ucrit decreased as the concentration of DCO solution increased from 0 to 56.4 mg L-1 (TPH). The mean Ucrit values for fish exposed to 28.2, 42.3 and 56.4 mg L-1 DCO TPH were significantly reduced relative to fish in the control group (p \ 0.05). Locomotion in fish is an integrated physiological process, and its performance may be affected by contaminants in sea water. Swimming performance has been used as an important criterion in the determination of sublethal effects of toxicants on fish. Ucrit has been used previously to define tolerance limits to various pollutants and environmental parameters, including pulp and paper effluent (Landman et al. 2006), copper (De Boeck et al. 2006), and temperature (Yu et al. 2010). Swimming performance of fish can be reduced by the toxic effects of WSF. Kennedy and Farrell (2006) reported that decreases in Ucrit were dependent on both exposure time and concentration. They found that the Ucrit of juvenile Pacific herring, Clupea pallasi, exposed to 100 lg L-1 WSF for 24 h and 40 lg L-1 for 96 h were significantly
lower compared to control fish. Exposure of coho salmon, Oncorhynchus kisutch, to the WSF affected swimming performance at [2.5 mg L-1 in exposures of 48 h and at 0.66 mg L-1 in exposures of 5–13 d (Thomas and Rice 1987). In the present study, the Ucrit of puffer fish decreased from 27.0 to 13.9 cm s-1 as the concentration of WSF solution increased from 0 to 136 mg L-1 (TPH). These results showed that reductions in swimming performance of fish occurred in a concentration-dependent and time-dependent manner after exposure to WSF. At present, however, little research has been published on the effect of DCO on the Ucrit of fish. A study by Milinkovitch et al. (2012) showed that the Ucrit of juvenile golden grey mullet, Liza aurata, was not altered by 44.0 mg L-1 (TPH) DCO. In the present study, the Ucrit was significantly reduced in puffer fish exposed to 28.2 mg L-1 (TPH) DCO. These results indicated that the puffer fish may be more sensitive to DCO than juvenile golden grey mullet. Impaired Ucrit often is related to an inability to supply enough oxygen to the gills, to deliver enough oxygen to the tissues, to remove metabolic products, to provide adequate substrates, or to activate enzymatic processes (Kennedy and Farrell 2006). Gills are the major route of uptake of waterborne pollutants by fish. WSF and DCO have been found to promote structural damage in the branchial respiratory epithelium, affecting gas exchange processes and limiting oxygen transfer in fish (McKeown and March 1978; Nikl and Farrell 1993; Duarte et al. 2010). Kennedy and Farrell (2005) reported that WSF impairs respiratory gas exchange based on observations of gill hyperplasia, which also could adversely affect extrarenal excretion and ionic exchange at the gills. Evidence suggests that the DCO is significantly more toxic than the WSF. Chemical dispersion increased both the total polycyclic aromatic hydrocarbon (PAH) concentrations and the proportion of high-molecular-weight PAHs in the water-accommodated fraction (WAF) of oil (Couillard et al. 2005). Chemical dispersants can also greatly increase the toxicity of crude oil by increasing the
Table 1 Ucrit and Ucrit0 of T. rubripes exposed to different concentration of water-soluble fraction solution Concentration (mg L-1 TPH)
Number of fish
Ucrit (cm s-1) Mean ± SE
0
5
Ucrit0 (BL s-1) Minimum
a
27.0 ± 3.09
b
19.3
Maximum 37.8
Mean ± SE
Minimum
Maximum
a
2.54
4.73
b
3.22 ± 0.39
34.0
5
18.5 ± 2.99
10.5
27.5
2.16 ± 0.35
1.18
3.06
68.1
5
17.3 ± 2.55b
11.3
25.7
1.97 ± 0.28b
1.36
2.86
102
5
13.9 ± 1.46b
11.3
19.3
1.65 ± 0.19b
1.34
2.35
136
5
13.9 ± 1.61b
10.5
18.5
1.78 ± 0.25b
1.26
2.43
Values with different letters in the same column indicate a significant difference from each other (p \ 0.05)
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Bull Environ Contam Toxicol (2015) 94:549–553
Table 2 Ucrit and Ucrit0 of T. rubripes exposed to different concentration of dispersed crude oil solution Concentration (mg L-1 TPH)
Number of fish
Ucrit (cm s-1) Mean ± SE 27.0 ± 3.09a
Ucrit0 (BL s-1) Minimum
Maximum
Mean ± SE
Minimum
Maximum
19.3
37.8
3.22 ± 0.39a
ab
2.54
4.73
14.1
5
21.7 ± 1.37
18.5
26.7
2.63 ± 0.21ab
2.18
3.34
28.2
5
20.3 ± 2.32bc
14.5
26.5
2.46 ± 0.24bc
1.75
3.15
42.3
5
bc
16.5 ± 1.71
12.1
21.7
bc
2.00 ± 0.21
1.49
2.58
56.4
5
15.3 ± 0.67c
13.7
17.7
1.81 ± 0.06c
1.59
1.97
0
5
Values with different letters in the same column indicate a significant difference from each other (p \ 0.05)
chemical oxygen demand (COD) of the dispersant enhanced WAF (Radniecki et al. 2013). Rico-Martı´nez et al. (2013) reported that Corexit 9500A and Macondo crude oil are similar in their toxicity. However, when Corexit 9500A and oil are mixed, toxicity to the marine rotifer, Brachionus plicatilis, increases up to 52-fold. In the current study, the concentrations of WSF and DCO at which the Ucrit of puffer fish decreased about 37 % were found to be 68.1 and 42.3 mg L-1 (TPH), respectively. The results showed that DCO was more toxic to puffer fish than the WSF. In conclusion, WSF and DCO significantly affected the Ucrit of puffer fish. DCO was more toxic to puffer fish than WSF. The results suggested that crude oil and chemically dispersed oil could weaken the swimming ability of puffer fish. Acknowledgments The authors would like to thank the three anonymous reviewers for their helpful comments. This study was supported by funds from Key Laboratory of Marine Spill Oil Identification and Damage Assessment Technology (201202), Public Science and Technology Research Funds Projects of Ocean (201205023-2, 201305002) and Special Fund for Agro-scientific Research in the Public Interest (201203018).
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