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Hypoxia impairs embryo development and survival in black bream (Acanthopagrus butcheri)
Marine Pollution Bulletin 57 (2008) 302–306
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Hypoxia impairs embryo development and survival in black bream (Acanthopagrus butcheri) Kathryn L. Hassell a,*, Patrick C. Coutin b, Dayanthi Nugegoda a a b
Biotechnology and Environmental Biology, School of Applied Sciences, RMIT University, P.O. Box 71, Bundoora, Vic 3083, Australia Department of Primary Industries, Fisheries Research Branch, P.O. Box 114, Queenscliff, Vic 3225, Australia
a r t i c l e Keywords: Dissolved oxygen Early life stage Reproduction Sparidae Seabream Porgies Australia
i n f o
a b s t r a c t Coastal environments are threatened by the increasing frequency, extent and severity of hypoxic events. Hypoxia affects vast areas around the world and often causes fish kills, reduced abundance, altered distribution, low benthic biomass and declines in fisheries. In Australia, many fisheries are based on sparid fishes and in the southern states black bream (Acanthopagrus butcheri) is important to both the recreational and commercial sectors. This species completes its entire life cycle in estuaries and annual recruitment is highly variable and very likely influenced by environmental conditions during the spawning season. In a laboratory-based experiment, fertilised black bream eggs (embryos) were exposed to five different levels of dissolved oxygen (DO). The DO levels were maintained in small test wells using nitrogen gas in a novel chamber design. Embryo development was assessed over a 2-day period and hatched larvae were observed until Day 2 post-hatch. Significant differences (p < 0.05) were observed in embryonic development and survival as a function of DO level. In severely hypoxic conditions (30% saturation) survival to 1 day was reduced and no hatching occurred. In moderately hypoxic conditions (45–55%S), both precocious and delayed hatching was observed and hatch rates were reduced, whilst the number of hatched larvae with deformities increased, resulting in reduced larval lengths. No larvae survived to Day 2 post-hatch when held in hypoxic conditions (<55%S). This study demonstrates the detrimental effect that severe hypoxia can have on the early development of black bream which could result in reduced recruitment and lowered abundance. Other species that share similar early life histories may also be at risk. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Hypoxia describes the condition whereby dissolved oxygen (DO) levels within water fall below 2.0 ml O2/l, while in conditions of no oxygen (0.0 ml O2/l), the environment is termed anoxic (Diaz and Rosenberg, 1995; Wu, 2002). Various units for describing dissolved oxygen have been developed, and under conditions of 20 ppt salinity, 25 °C and 1 atm pressure, 1.0 ml O2/l is equivalent to 1.4 mg/l, 23.9 mmHg, 45.7 mM and 14% air saturation (Diaz and Rosenberg, 1995). Percentage saturation (%S) is expressed relative to air saturation, which is 100% (Diaz and Rosenberg, 1995). Hypoxia can occur naturally due to processes such as low water flow or limited water–atmosphere gas exchange and may be intensified by the presence of vertical stratifications due to haloclines or thermoclines (Diaz and Rosenberg, 1995; Wu, 2002; Breitberg et al., 2003). Hypoxia may also occur when systems become over-
* Corresponding author. Tel.: +61 3 9925 7155; fax: +61 3 9925 7110. E-mail address: [email protected] (K.L. Hassell). 0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.02.045
loaded with organic matter by the process of eutrophication (Wu, 2002; Breitberg et al., 2003). Hypoxia in the marine environment is a global issue that is responsible for a range of environmental problems that result in reduced species abundance and distribution and fisheries declines (Wu, 2002; Breitberg et al., 2003). It has been suggested that hypoxia in the marine environment may be the most widespread environmental stressor responsible for mortality of benthic fauna (Diaz and Rosenberg, 1995). Whilst a number of ecological studies have observed changes in community structures and faunal distributions in association with hypoxia (Diaz and Rosenberg, 1995; Breitberg et al., 2003; Ekau and Verheye, 2005), only limited information is available on individual level effects, particularly in relation to early life histories. In a study of larval and juvenile fishes from the East and Gulf coasts of the United States of America, Miller et al. (2002) reported 4-day LC50 values ranging from 0.6 to 2.4 mg/l (10–40%S). Aside from mortality, another effect that has recently been reported is a shift in sex ratios as a result of exposure to hypoxia. Shang et al. (2006) reported male-biased populations of zebrafish (Danio rerio)
303
K.L. Hassell et al. / Marine Pollution Bulletin 57 (2008) 302–306
following a 4-month exposure of embryos and subsequently, larvae to hypoxia, concluding that this would have serious impacts on reproductive success and population sustainability in this species. The black bream (Acanthopagrus butcheri) is a member of the Sparidae family and is widely distributed throughout southern Australia. In studies of eastern Victorian populations, it has been reported that recruitment is variable from year to year, possibly attributable to unfavourable environmental conditions (Morison et al., 1998; Walker and Neira, 2001). Like other sparids, black bream are pelagic serial spawners, and have been documented to release more than 1,000,000 eggs per season (Sarre and Potter, 1999). Black bream spawn in estuaries, utilising the salt-wedge to find spawning locations with suitable salinities (15–35 ppt) (Haddy and Pankhurst, 2000). By spawning near the halocline, black bream eggs are at risk of exposure to hypoxia. The aims of this study were to assess the effects of moderate (<55%S) and severe hypoxia (30%S) on the hatchability and survival of black bream eggs.
2. Materials and methods
chamber. Once the required levels were achieved, the gas inlet and outlet valves were closed. Hence the chambers were airtight and gas exchange between the chamber atmosphere and the water surface of each well achieved equilibrium at the desired level of dissolved oxygen. 2.4. Water quality Water quality was recorded every 12 h using a WP-91 dissolved oxygen meter and a WP-81 conductivity and pH meter (TPS Ionode, Australia). A mercury-filled thermometer was used for temperature measurements. 2.5. Statistical analyses Differences between treatments were evaluated using one-way ANOVA and Tukey’s multiple comparisons tests to detect significantly different treatment pairs. If the requirements of parametric tests were not satisfied (i.e. homogeneity of variance), Kruskal– Wallis and Mann–Whitney tests were used. All statistical analyses were performed using SPSS for Windows (version 14.0; SPSS Inc., Chicago, IL USA). The significance level in all tests was 0.05.
2.1. Test organisms 3. Results Black bream eggs were obtained from broodstock held in the Department of Primary Industries aquaculture facility in Queenscliff, Victoria. Ovulation in female black bream was induced using the chorionic gonadotrophin hormone Chorulon (Intervet, Australia). The fish were anaesthetised in a 30 ppm AQUI-S solution (Crop & Food Research, Lower Hutt, New Zealand), then given an intramuscular injection of Chorulon (500 IU/kg). The ratio of males to females was 1:1 throughout the spawning period. 2.2. Embryo collection Spawning fish were held in a tank that was fitted with an overflow that led into a 500 lm mesh egg collector, allowing for collection of buoyant, fertilised eggs (embryos) only. Good quality black bream embryos are positively buoyant at 36 ppt salinity and lose buoyancy between 25 and 30 ppt (personal observation). At the time of collection, all embryos were in the 8–16 cell stage (in accordance with OECD Guidelines for conducting short term toxicity tests on embryo and sac-fry stages (#212)).
3.1. 24 h survival Embryo survival to 24 h post-fertilisation was significantly reduced (p < 0.05) in severely hypoxic conditions (30%S) (Fig. 1). No significant differences were observed in 24 h survival of embryos in 45%S, 55%S, 65%S or 85%S treatments. Associated with the increased mortality of embryos held in 30%S was the observation of disrupted, uncoordinated development, whereby the normal organisation of cells forming the cephalic region during late gastrulation did not occur (Fig. 2). This lack of cephalisation represents abnormal development of black bream embryos and always resulted in poor survival. 3.2. Time to hatching The time to hatching was significantly different between dissolved oxygen treatments (p < 0.05). At 45%S, mean time to hatching for embryos was 43 h, whereas embryos held in other treatments had mean hatching times between 33 and 35 h (Table 1).
2.3. Controlled atmosphere chambers 3.3. Hatch rates Hatch rates were significantly affected by dissolved oxygen levels (p < 0.05). Mean hatch rates at 45%S were 46%, whereas at 55%S
100
b
24 h survival (%)
Embryos were randomly distributed in plastic six-well plates (Iwama, Australia) containing 10 ml of filtered sea water (36 ppt) at five different dissolved oxygen concentrations in a laboratory with an ambient temperature of 23 °C. Each treatment consisted of six replicates, each containing 69.7 (±4.2 SEM) embryos. Dissolved oxygen levels were classed as: severely hypoxic (30%S 2.1 mg/l), moderately hypoxic (45%S, 55%S and 65%S 3.1, 3.8 and 4.5 mg/l, respectively) and normoxic 85%S 5.9 mg/l). Dissolved oxygen levels in seawater were attained by bubbling nitrogen gas through the water. All plates were then placed inside airtight Perspex containers (controlled atmosphere chambers). Nitrogen gas (BOC Gases, Australia) was introduced to the controlled atmosphere chambers through an inlet valve and the nitrogen–air gas mixture flowed out via an outlet valve. The oxygen content of the outlet gas was analysed using a DiveTek Monitor oxygen analyser (DiveTek, Australia), calibrated at 21% using compressed air. The five chambers were set up so that the outlet gas readings corresponded to pre-determined values that matched the levels of dissolved oxygen within the well plates inside each
b
b
b
55
65
85
80 60 40
a
20 0 30
45
Dissolved Oxygen (%S) Fig. 1. Survival to 24 h post-fertilisation in black bream embryos exposed to five different levels of dissolved oxygen (mean ± SEM). Different letters indicate statistically significant differences between treatments (p < 0.05).
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Fig. 2. Disrupted embryonic development in black bream following exposure to hypoxia: (a) normal appearance of embryo at 24 h post-fertilisation (maintained at 85%S) and (b) abnormal appearance of embryo at 24 h post-fertilisation (maintained at 30%S). Bar = 200 lm.
Table 1 Percentage of embryos hatched, percentage deformed and time to hatching in treatment groups exposed to different levels of dissolved oxygen (mean ± SEM) DO concentration (%S)
Hatched (%)
Deformed (%)
Time to hatching (h)
30 45 55 65 85 (control)
0.0 ± 0.0 46.1 ± 7.4a 59.9 ± 3.1ab 72.8 ± 3.0b 71.2 ± 4.6b
NA 100 ± 0.0a 12.3 ± 3.1b 7.7 ± 2.9b 9.6 ± 2.6b
NA 43.0 ± 0.9a 33.4 ± 0.1b 35.0 ± 0.3c 35.7 ± 0.2c
(p < 0.001) at 45%S (100%) compared to all other treatments (7.7– 12.3%) (Table 1). Furthermore, all embryos that hatched at 45%S died within 24 h. 3.5. Survival Day 2 post-hatch
Different letters indicate statistically significant differences between treatments of each variable (p < 0.05).
No embryos that hatched at 45%S survived to Day 2 post-hatch (72 h post-fertilisation) (Fig. 4). There were no significant differences in survival to Day 2 post-hatch for embryos hatched at 55%S, 65%S or 85%S (p > 0.05). 3.6. Larval length at Day 2 post-hatch
3.4. Deformities Upon hatching larvae were observed to be normal or deformed. Deformed larvae displayed severe curvature of the spine and an inability to swim. Deformed larvae were observed in all treatments that hatched, however, the occurrence was significantly higher
Hatch Rate (%)
100 b
80
b
ab a
60 40 20 0
Upon completion of the experiment a sub-sample (n = 50) of surviving larvae from each treatment were preserved in formalin and then total length (mm) was measured under a dissecting microscope fitted with an eyepiece graticule. At 55%S a greater percentage of embryos hatched with spinal deformities as compared to the 65%S and 85%S treatments, resulting in shorter mean total lengths and higher variability in the lengths (Table 1 and Fig. 5).
72 h (Day 2) survival (%)
mean hatch rates were 60% and at 65%S and 85%S mean hatch rates were >70% (Fig. 3).
100
a 80
a
a
60 40 20
DNH 0
30
45
55
65
85
Dissolved Oxygen (%S) Fig. 3. Mean hatch rates for black bream embryos exposed to five different levels of dissolved oxygen (mean ± SEM). Different letters indicate statistically significant differences between treatments (p < 0.05).
30
45
55
65
85
Dissolved Oxygen (%S) Fig. 4. Survival to Day 2 post-hatch (72 h post-fertilisation) in black bream embryos exposed to five different levels of dissolved oxygen (mean ± SEM). Letters indicate no significant differences between treatments (p > 0.05). DNH – did not hatch.
K.L. Hassell et al. / Marine Pollution Bulletin 57 (2008) 302–306
Larval length (mm)
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 45
50
55
60
65
70
75
80
85
90
Dissolved Oxygen (%S) Fig. 5. Lengths (mm) of black bream larvae on Day 2 post-hatch (72 h post-fertilisation). n = 50.
3.7. Water quality Mean dissolved oxygen concentrations (± SEM) in the different treatments were: 86.34 ± 1.54 (85%S), 66.13 ± 1.23 (65%S), 55.26 ± 2.61 (55%S), 46.20 ± 5.8 (45%S) and 31.9 ± 7.60 (30%S). Salinity, pH and temperature were constant across treatments throughout the experiment. Salinity (ppt) 36.05 ± 0.03, pH 8.09 ± 0.03 and temperature (°C) 23.10 ± 0.09. 4. Discussion This study assessed the effects of varying degrees of hypoxia on embryonic development and yolk-sac larvae survival in the black bream. Significant differences were observed in a range of variables as the dissolved oxygen levels were decreased, including disruptions and delays to development, altered hatch rates and reduced survival. These findings indicate that the early life stages of black bream are very sensitive to hypoxia, and the likely consequences of spawning in hypoxic estuaries would include reduced recruitment and lowered population survival. Our results show that in severely hypoxic conditions (30%S) all black bream embryos developed abnormally, resulting in an undifferentiated mass of cells towards the animal pole and a lack of cephalisation. The normal developmental processes that occur late in the gastrula period (neural plate thickening) were not observed (Kimmel et al., 1995). Lack of cephalisation in response to hypoxia has not been previously reported in sparid fishes. However, developmental disturbances have been demonstrated following exposure to toxicants in marine sparid species (Pagrus major and Sparus aurata), and observations of embryonic development have shown defects such as reductions in the perivitelline space, irregular chorion shape and smaller head size (Yamauchi et al., 2006; Oliva et al., 2007). Embryonic development appeared normal at 45%S but was significantly slower than at 55%S, 65%S or 85%S. The embryos required a longer period of time to develop sufficiently to hatch, resulting in an increase in the mean time to hatching. Furthermore, when embryos did hatch at 45%S, all displayed spinal deformities. In severely hypoxic conditions (30%S), embryonic development was abnormal and no hatching occurred. Unfavourable environmental conditions can reduce rates of myogenesis in fish larvae and may retard growth and cause spinal deformities (see Johnston, 2006 for review). Our results clearly demonstrate that low dissolved oxygen levels interfere with these developmental processes and hence influence the hatch rates and survival of black bream eggs and larvae. Furthermore, subsequent growth of those larvae that do survive may also be impaired if embryonic development is abnormal but does not cause mortality. Differences in hatching times in fishes in response to hypoxia may manifest as either premature or delayed hatching. Early (precocious) hatching has been observed in two freshwater salmonids
305
(Coregonus lavaretus, Coregonus albula) in response to low dissolved oxygen, and the incidence increased as the duration of exposure to hypoxia increased (Czerkies et al., 2001). Ciuhandu et al. (2005) observed slower growth as well as both early and delayed hatching in rainbow trout (Oncorhynchus mykiss) embryos exposed to hypoxic conditions. Likewise, black bream in the present study displayed both premature and delayed hatching times depending on the severity of the hypoxic stress. Hypoxia obviously affects multiple developmental processes in teleost embryos and further work is needed to investigate the specific mechanisms involved. Survival to Day 2 post-hatch was similar in all larvae that survived the hatching process without significant levels of deformity. However, none of the deformed larvae that hatched in 45%S survived to Day 2 post-hatch. Yamauchi et al. (2006) observed increased larval mortality in red seabream (P. major), a marine sparid, following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was associated with under-development of the heart, bradycardia and circulation failure. It is quite possible that hypoxia may cause high mortality from similar malformations in the organs of black bream embryos, however this was not examined in our study and needs further research. Hypoxia is an important, widespread environmental stressor that occurs throughout aquatic environments. This study has demonstrated the significant influences that hypoxia has to the hatching success and early larval survival of an estuarine sparid, and based on these results it is reasonable to assume that hypoxia would subsequently affect other developmental stages and ultimately recruitment. Furthermore, if hypoxic events continue to occur and expand in geographic extent, and increase in frequency, high levels of larval morality will be reflected in the age structure, size and growth performance of each year class. Other species that utilise estuaries for spawning and share similar early life histories to black bream are also likely to be at risk from the effects of hypoxia which clearly threatens recruitment to fish stocks and may impact species composition of the fish fauna. Ultimately fisheries productivity will be adversely impacted by deteriorating conditions in aquatic environments unless catchment management strategies can effectively address the environmental threat of hypoxia. Acknowledgements We thank Nathan O’Mahony for technical assistance and the Victorian Environmental Protection Authority for providing funding for the study. The study was also supported by an Australian Postgraduate Award to Kathryn Hassell. The experiments were conducted in the laboratories of the Victorian Marine Science Consortium at Queenscliff, Victoria, Australia. References Breitberg, D.L., Adamack, A., Rose, K.A., Kolesar, S.E., Decker, M.B., Purcell, J.E., Keister, J.E., Cowan Jr., J.H., 2003. The pattern and influence of low dissolved oxygen in the Patuxent River, a seasonally hypoxic estuary. Estuaries 26, 280– 297. Ciuhandu, C.S., Stevens, E.D., Wright, P.A., 2005. The effect of oxygen on the growth of Oncorhynchus mykiss embryos with and without a chorion. Journal of Fish Biology 67, 1544–1551. Czerkies, P., Brzuzan, P., Kordalski, K., Luczynski, M., 2001. Critical partial pressures of oxygen causing precocious hatching in Coregonus lavaretus and C. albula embryos. Aquaculture 196, 151–158. Diaz, R.J., Rosenberg, R., 1995. Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanography and Marine Biology: An Annual Review 33, 245–303. Ekau, W., Verheye, H.M., 2005. Influence of oceanographic fronts and low dissolved oxygen on the distribution of ichthyoplankton in the Benguela and southern Angola currents. African Journal of Marine Science 27, 629–639. Haddy, J.A., Pankhurst, N.W., 2000. The effects of salinity on reproductive development, plasma steroid levels, fertilisation and egg survival in black bream Acanthopagrus butcheri. Aquaculture 188, 115–131.
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Johnston, I.A., 2006. Environmental plasticity of myogenesis in teleost fish. Journal of Experimental Biology 209, 2249–2264. Kimmel, C.B., Ballard, W.W., Kimmel, S.R., Ullmann, B., Schilling, T.F., 1995. Stages of embryonic development of the zebrafish. Developmental Dynamics 203, 253– 310. Miller, D.C., Poucher, S.L., Coiro, L., 2002. Determination of lethal dissolved oxygen levels for selected marine and estuarine fishes, crustaceans, and a bivalve. Marine Biology 140, 287–296. Morison, A.K., Coutin, P.C., Robertson, S.G., 1998. Age determination of black bream, Acanthopagrus butcheri (Sparidae), from the Gippsland Lakes of south-eastern Australia indicates slow growth and episodic recruitment. Marine and Freshwater Research 49, 491–498. Oliva, M., Garrido, M.D.C., Perez, E., Gonzalez de Canales, M.L., 2007. Evaluation of acute copper toxicity during early life stages of gilthead seabream, Sparus aurata. Journal of Environmental Science and Health 42A, 525–533.
Sarre, G.A., Potter, I.C., 1999. Comparisons between the reproductive biology of black bream Acanthopagrus butcheri (Teleostei: Sparidae) in four estuaries with widely differing characteristics. International Journal of Salt Lake Research 8, 179–210. Shang, E.H.H., Yu, R.M.K., Wu, R.S.S., 2006. Hypoxia affects sex differentiation and development, leading to a male-dominated population in Zebrafish (Danio rerio). Environmental Science and Technology 40, 3118–3122. Walker, S., Neira, F.J., 2001. Aspects of the reproductive biology and early life history of black bream, Acanthopagrus butcheri (Sparidae), in a brackish lagoon system in southeastern Australia. Journal of Ichthyology and Aquatic Biology 4, 135–142. Wu, R.S.S., 2002. Hypoxia: from molecular responses to ecosystem responses. Marine Pollution Bulletin 45, 35–45. Yamauchi, M., Kim, E.-Y., Iwata, H., Shima, Y., Tanabe, S., 2006. Toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in developing red seabream (Pagrus major) embryo: an association of morphological deformities with AHR1, AHR2 and CYP1A expressions. Aquatic Toxicology 80, 166–179.
Contents lists available at ScienceDirect
Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul
Hypoxia impairs embryo development and survival in black bream (Acanthopagrus butcheri) Kathryn L. Hassell a,*, Patrick C. Coutin b, Dayanthi Nugegoda a a b
Biotechnology and Environmental Biology, School of Applied Sciences, RMIT University, P.O. Box 71, Bundoora, Vic 3083, Australia Department of Primary Industries, Fisheries Research Branch, P.O. Box 114, Queenscliff, Vic 3225, Australia
a r t i c l e Keywords: Dissolved oxygen Early life stage Reproduction Sparidae Seabream Porgies Australia
i n f o
a b s t r a c t Coastal environments are threatened by the increasing frequency, extent and severity of hypoxic events. Hypoxia affects vast areas around the world and often causes fish kills, reduced abundance, altered distribution, low benthic biomass and declines in fisheries. In Australia, many fisheries are based on sparid fishes and in the southern states black bream (Acanthopagrus butcheri) is important to both the recreational and commercial sectors. This species completes its entire life cycle in estuaries and annual recruitment is highly variable and very likely influenced by environmental conditions during the spawning season. In a laboratory-based experiment, fertilised black bream eggs (embryos) were exposed to five different levels of dissolved oxygen (DO). The DO levels were maintained in small test wells using nitrogen gas in a novel chamber design. Embryo development was assessed over a 2-day period and hatched larvae were observed until Day 2 post-hatch. Significant differences (p < 0.05) were observed in embryonic development and survival as a function of DO level. In severely hypoxic conditions (30% saturation) survival to 1 day was reduced and no hatching occurred. In moderately hypoxic conditions (45–55%S), both precocious and delayed hatching was observed and hatch rates were reduced, whilst the number of hatched larvae with deformities increased, resulting in reduced larval lengths. No larvae survived to Day 2 post-hatch when held in hypoxic conditions (<55%S). This study demonstrates the detrimental effect that severe hypoxia can have on the early development of black bream which could result in reduced recruitment and lowered abundance. Other species that share similar early life histories may also be at risk. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Hypoxia describes the condition whereby dissolved oxygen (DO) levels within water fall below 2.0 ml O2/l, while in conditions of no oxygen (0.0 ml O2/l), the environment is termed anoxic (Diaz and Rosenberg, 1995; Wu, 2002). Various units for describing dissolved oxygen have been developed, and under conditions of 20 ppt salinity, 25 °C and 1 atm pressure, 1.0 ml O2/l is equivalent to 1.4 mg/l, 23.9 mmHg, 45.7 mM and 14% air saturation (Diaz and Rosenberg, 1995). Percentage saturation (%S) is expressed relative to air saturation, which is 100% (Diaz and Rosenberg, 1995). Hypoxia can occur naturally due to processes such as low water flow or limited water–atmosphere gas exchange and may be intensified by the presence of vertical stratifications due to haloclines or thermoclines (Diaz and Rosenberg, 1995; Wu, 2002; Breitberg et al., 2003). Hypoxia may also occur when systems become over-
* Corresponding author. Tel.: +61 3 9925 7155; fax: +61 3 9925 7110. E-mail address: [email protected] (K.L. Hassell). 0025-326X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2008.02.045
loaded with organic matter by the process of eutrophication (Wu, 2002; Breitberg et al., 2003). Hypoxia in the marine environment is a global issue that is responsible for a range of environmental problems that result in reduced species abundance and distribution and fisheries declines (Wu, 2002; Breitberg et al., 2003). It has been suggested that hypoxia in the marine environment may be the most widespread environmental stressor responsible for mortality of benthic fauna (Diaz and Rosenberg, 1995). Whilst a number of ecological studies have observed changes in community structures and faunal distributions in association with hypoxia (Diaz and Rosenberg, 1995; Breitberg et al., 2003; Ekau and Verheye, 2005), only limited information is available on individual level effects, particularly in relation to early life histories. In a study of larval and juvenile fishes from the East and Gulf coasts of the United States of America, Miller et al. (2002) reported 4-day LC50 values ranging from 0.6 to 2.4 mg/l (10–40%S). Aside from mortality, another effect that has recently been reported is a shift in sex ratios as a result of exposure to hypoxia. Shang et al. (2006) reported male-biased populations of zebrafish (Danio rerio)
303
K.L. Hassell et al. / Marine Pollution Bulletin 57 (2008) 302–306
following a 4-month exposure of embryos and subsequently, larvae to hypoxia, concluding that this would have serious impacts on reproductive success and population sustainability in this species. The black bream (Acanthopagrus butcheri) is a member of the Sparidae family and is widely distributed throughout southern Australia. In studies of eastern Victorian populations, it has been reported that recruitment is variable from year to year, possibly attributable to unfavourable environmental conditions (Morison et al., 1998; Walker and Neira, 2001). Like other sparids, black bream are pelagic serial spawners, and have been documented to release more than 1,000,000 eggs per season (Sarre and Potter, 1999). Black bream spawn in estuaries, utilising the salt-wedge to find spawning locations with suitable salinities (15–35 ppt) (Haddy and Pankhurst, 2000). By spawning near the halocline, black bream eggs are at risk of exposure to hypoxia. The aims of this study were to assess the effects of moderate (<55%S) and severe hypoxia (30%S) on the hatchability and survival of black bream eggs.
2. Materials and methods
chamber. Once the required levels were achieved, the gas inlet and outlet valves were closed. Hence the chambers were airtight and gas exchange between the chamber atmosphere and the water surface of each well achieved equilibrium at the desired level of dissolved oxygen. 2.4. Water quality Water quality was recorded every 12 h using a WP-91 dissolved oxygen meter and a WP-81 conductivity and pH meter (TPS Ionode, Australia). A mercury-filled thermometer was used for temperature measurements. 2.5. Statistical analyses Differences between treatments were evaluated using one-way ANOVA and Tukey’s multiple comparisons tests to detect significantly different treatment pairs. If the requirements of parametric tests were not satisfied (i.e. homogeneity of variance), Kruskal– Wallis and Mann–Whitney tests were used. All statistical analyses were performed using SPSS for Windows (version 14.0; SPSS Inc., Chicago, IL USA). The significance level in all tests was 0.05.
2.1. Test organisms 3. Results Black bream eggs were obtained from broodstock held in the Department of Primary Industries aquaculture facility in Queenscliff, Victoria. Ovulation in female black bream was induced using the chorionic gonadotrophin hormone Chorulon (Intervet, Australia). The fish were anaesthetised in a 30 ppm AQUI-S solution (Crop & Food Research, Lower Hutt, New Zealand), then given an intramuscular injection of Chorulon (500 IU/kg). The ratio of males to females was 1:1 throughout the spawning period. 2.2. Embryo collection Spawning fish were held in a tank that was fitted with an overflow that led into a 500 lm mesh egg collector, allowing for collection of buoyant, fertilised eggs (embryos) only. Good quality black bream embryos are positively buoyant at 36 ppt salinity and lose buoyancy between 25 and 30 ppt (personal observation). At the time of collection, all embryos were in the 8–16 cell stage (in accordance with OECD Guidelines for conducting short term toxicity tests on embryo and sac-fry stages (#212)).
3.1. 24 h survival Embryo survival to 24 h post-fertilisation was significantly reduced (p < 0.05) in severely hypoxic conditions (30%S) (Fig. 1). No significant differences were observed in 24 h survival of embryos in 45%S, 55%S, 65%S or 85%S treatments. Associated with the increased mortality of embryos held in 30%S was the observation of disrupted, uncoordinated development, whereby the normal organisation of cells forming the cephalic region during late gastrulation did not occur (Fig. 2). This lack of cephalisation represents abnormal development of black bream embryos and always resulted in poor survival. 3.2. Time to hatching The time to hatching was significantly different between dissolved oxygen treatments (p < 0.05). At 45%S, mean time to hatching for embryos was 43 h, whereas embryos held in other treatments had mean hatching times between 33 and 35 h (Table 1).
2.3. Controlled atmosphere chambers 3.3. Hatch rates Hatch rates were significantly affected by dissolved oxygen levels (p < 0.05). Mean hatch rates at 45%S were 46%, whereas at 55%S
100
b
24 h survival (%)
Embryos were randomly distributed in plastic six-well plates (Iwama, Australia) containing 10 ml of filtered sea water (36 ppt) at five different dissolved oxygen concentrations in a laboratory with an ambient temperature of 23 °C. Each treatment consisted of six replicates, each containing 69.7 (±4.2 SEM) embryos. Dissolved oxygen levels were classed as: severely hypoxic (30%S 2.1 mg/l), moderately hypoxic (45%S, 55%S and 65%S 3.1, 3.8 and 4.5 mg/l, respectively) and normoxic 85%S 5.9 mg/l). Dissolved oxygen levels in seawater were attained by bubbling nitrogen gas through the water. All plates were then placed inside airtight Perspex containers (controlled atmosphere chambers). Nitrogen gas (BOC Gases, Australia) was introduced to the controlled atmosphere chambers through an inlet valve and the nitrogen–air gas mixture flowed out via an outlet valve. The oxygen content of the outlet gas was analysed using a DiveTek Monitor oxygen analyser (DiveTek, Australia), calibrated at 21% using compressed air. The five chambers were set up so that the outlet gas readings corresponded to pre-determined values that matched the levels of dissolved oxygen within the well plates inside each
b
b
b
55
65
85
80 60 40
a
20 0 30
45
Dissolved Oxygen (%S) Fig. 1. Survival to 24 h post-fertilisation in black bream embryos exposed to five different levels of dissolved oxygen (mean ± SEM). Different letters indicate statistically significant differences between treatments (p < 0.05).
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Fig. 2. Disrupted embryonic development in black bream following exposure to hypoxia: (a) normal appearance of embryo at 24 h post-fertilisation (maintained at 85%S) and (b) abnormal appearance of embryo at 24 h post-fertilisation (maintained at 30%S). Bar = 200 lm.
Table 1 Percentage of embryos hatched, percentage deformed and time to hatching in treatment groups exposed to different levels of dissolved oxygen (mean ± SEM) DO concentration (%S)
Hatched (%)
Deformed (%)
Time to hatching (h)
30 45 55 65 85 (control)
0.0 ± 0.0 46.1 ± 7.4a 59.9 ± 3.1ab 72.8 ± 3.0b 71.2 ± 4.6b
NA 100 ± 0.0a 12.3 ± 3.1b 7.7 ± 2.9b 9.6 ± 2.6b
NA 43.0 ± 0.9a 33.4 ± 0.1b 35.0 ± 0.3c 35.7 ± 0.2c
(p < 0.001) at 45%S (100%) compared to all other treatments (7.7– 12.3%) (Table 1). Furthermore, all embryos that hatched at 45%S died within 24 h. 3.5. Survival Day 2 post-hatch
Different letters indicate statistically significant differences between treatments of each variable (p < 0.05).
No embryos that hatched at 45%S survived to Day 2 post-hatch (72 h post-fertilisation) (Fig. 4). There were no significant differences in survival to Day 2 post-hatch for embryos hatched at 55%S, 65%S or 85%S (p > 0.05). 3.6. Larval length at Day 2 post-hatch
3.4. Deformities Upon hatching larvae were observed to be normal or deformed. Deformed larvae displayed severe curvature of the spine and an inability to swim. Deformed larvae were observed in all treatments that hatched, however, the occurrence was significantly higher
Hatch Rate (%)
100 b
80
b
ab a
60 40 20 0
Upon completion of the experiment a sub-sample (n = 50) of surviving larvae from each treatment were preserved in formalin and then total length (mm) was measured under a dissecting microscope fitted with an eyepiece graticule. At 55%S a greater percentage of embryos hatched with spinal deformities as compared to the 65%S and 85%S treatments, resulting in shorter mean total lengths and higher variability in the lengths (Table 1 and Fig. 5).
72 h (Day 2) survival (%)
mean hatch rates were 60% and at 65%S and 85%S mean hatch rates were >70% (Fig. 3).
100
a 80
a
a
60 40 20
DNH 0
30
45
55
65
85
Dissolved Oxygen (%S) Fig. 3. Mean hatch rates for black bream embryos exposed to five different levels of dissolved oxygen (mean ± SEM). Different letters indicate statistically significant differences between treatments (p < 0.05).
30
45
55
65
85
Dissolved Oxygen (%S) Fig. 4. Survival to Day 2 post-hatch (72 h post-fertilisation) in black bream embryos exposed to five different levels of dissolved oxygen (mean ± SEM). Letters indicate no significant differences between treatments (p > 0.05). DNH – did not hatch.
K.L. Hassell et al. / Marine Pollution Bulletin 57 (2008) 302–306
Larval length (mm)
3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 45
50
55
60
65
70
75
80
85
90
Dissolved Oxygen (%S) Fig. 5. Lengths (mm) of black bream larvae on Day 2 post-hatch (72 h post-fertilisation). n = 50.
3.7. Water quality Mean dissolved oxygen concentrations (± SEM) in the different treatments were: 86.34 ± 1.54 (85%S), 66.13 ± 1.23 (65%S), 55.26 ± 2.61 (55%S), 46.20 ± 5.8 (45%S) and 31.9 ± 7.60 (30%S). Salinity, pH and temperature were constant across treatments throughout the experiment. Salinity (ppt) 36.05 ± 0.03, pH 8.09 ± 0.03 and temperature (°C) 23.10 ± 0.09. 4. Discussion This study assessed the effects of varying degrees of hypoxia on embryonic development and yolk-sac larvae survival in the black bream. Significant differences were observed in a range of variables as the dissolved oxygen levels were decreased, including disruptions and delays to development, altered hatch rates and reduced survival. These findings indicate that the early life stages of black bream are very sensitive to hypoxia, and the likely consequences of spawning in hypoxic estuaries would include reduced recruitment and lowered population survival. Our results show that in severely hypoxic conditions (30%S) all black bream embryos developed abnormally, resulting in an undifferentiated mass of cells towards the animal pole and a lack of cephalisation. The normal developmental processes that occur late in the gastrula period (neural plate thickening) were not observed (Kimmel et al., 1995). Lack of cephalisation in response to hypoxia has not been previously reported in sparid fishes. However, developmental disturbances have been demonstrated following exposure to toxicants in marine sparid species (Pagrus major and Sparus aurata), and observations of embryonic development have shown defects such as reductions in the perivitelline space, irregular chorion shape and smaller head size (Yamauchi et al., 2006; Oliva et al., 2007). Embryonic development appeared normal at 45%S but was significantly slower than at 55%S, 65%S or 85%S. The embryos required a longer period of time to develop sufficiently to hatch, resulting in an increase in the mean time to hatching. Furthermore, when embryos did hatch at 45%S, all displayed spinal deformities. In severely hypoxic conditions (30%S), embryonic development was abnormal and no hatching occurred. Unfavourable environmental conditions can reduce rates of myogenesis in fish larvae and may retard growth and cause spinal deformities (see Johnston, 2006 for review). Our results clearly demonstrate that low dissolved oxygen levels interfere with these developmental processes and hence influence the hatch rates and survival of black bream eggs and larvae. Furthermore, subsequent growth of those larvae that do survive may also be impaired if embryonic development is abnormal but does not cause mortality. Differences in hatching times in fishes in response to hypoxia may manifest as either premature or delayed hatching. Early (precocious) hatching has been observed in two freshwater salmonids
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(Coregonus lavaretus, Coregonus albula) in response to low dissolved oxygen, and the incidence increased as the duration of exposure to hypoxia increased (Czerkies et al., 2001). Ciuhandu et al. (2005) observed slower growth as well as both early and delayed hatching in rainbow trout (Oncorhynchus mykiss) embryos exposed to hypoxic conditions. Likewise, black bream in the present study displayed both premature and delayed hatching times depending on the severity of the hypoxic stress. Hypoxia obviously affects multiple developmental processes in teleost embryos and further work is needed to investigate the specific mechanisms involved. Survival to Day 2 post-hatch was similar in all larvae that survived the hatching process without significant levels of deformity. However, none of the deformed larvae that hatched in 45%S survived to Day 2 post-hatch. Yamauchi et al. (2006) observed increased larval mortality in red seabream (P. major), a marine sparid, following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), which was associated with under-development of the heart, bradycardia and circulation failure. It is quite possible that hypoxia may cause high mortality from similar malformations in the organs of black bream embryos, however this was not examined in our study and needs further research. Hypoxia is an important, widespread environmental stressor that occurs throughout aquatic environments. This study has demonstrated the significant influences that hypoxia has to the hatching success and early larval survival of an estuarine sparid, and based on these results it is reasonable to assume that hypoxia would subsequently affect other developmental stages and ultimately recruitment. Furthermore, if hypoxic events continue to occur and expand in geographic extent, and increase in frequency, high levels of larval morality will be reflected in the age structure, size and growth performance of each year class. Other species that utilise estuaries for spawning and share similar early life histories to black bream are also likely to be at risk from the effects of hypoxia which clearly threatens recruitment to fish stocks and may impact species composition of the fish fauna. Ultimately fisheries productivity will be adversely impacted by deteriorating conditions in aquatic environments unless catchment management strategies can effectively address the environmental threat of hypoxia. Acknowledgements We thank Nathan O’Mahony for technical assistance and the Victorian Environmental Protection Authority for providing funding for the study. The study was also supported by an Australian Postgraduate Award to Kathryn Hassell. The experiments were conducted in the laboratories of the Victorian Marine Science Consortium at Queenscliff, Victoria, Australia. References Breitberg, D.L., Adamack, A., Rose, K.A., Kolesar, S.E., Decker, M.B., Purcell, J.E., Keister, J.E., Cowan Jr., J.H., 2003. The pattern and influence of low dissolved oxygen in the Patuxent River, a seasonally hypoxic estuary. Estuaries 26, 280– 297. Ciuhandu, C.S., Stevens, E.D., Wright, P.A., 2005. The effect of oxygen on the growth of Oncorhynchus mykiss embryos with and without a chorion. Journal of Fish Biology 67, 1544–1551. Czerkies, P., Brzuzan, P., Kordalski, K., Luczynski, M., 2001. Critical partial pressures of oxygen causing precocious hatching in Coregonus lavaretus and C. albula embryos. Aquaculture 196, 151–158. Diaz, R.J., Rosenberg, R., 1995. Marine benthic hypoxia: a review of its ecological effects and the behavioural responses of benthic macrofauna. Oceanography and Marine Biology: An Annual Review 33, 245–303. Ekau, W., Verheye, H.M., 2005. Influence of oceanographic fronts and low dissolved oxygen on the distribution of ichthyoplankton in the Benguela and southern Angola currents. African Journal of Marine Science 27, 629–639. Haddy, J.A., Pankhurst, N.W., 2000. The effects of salinity on reproductive development, plasma steroid levels, fertilisation and egg survival in black bream Acanthopagrus butcheri. Aquaculture 188, 115–131.
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