Effects of chronic nitrite exposure on growth in juvenile Atlantic cod, Gadus morhua

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1 Aquaculture 255 (2006) Effects of chronic nitrite exposure on growth in juvenile Atlantic cod, Gadus morhua Sten I. Siikavuopio, Bjørn-Steinar Sæther Norwegian Institute of Fisheries and Aquaculture Research (Fiskeriforskning), Tromsø N-9291, Norway Received 8 July 2005; received in revised form 28 November 2005; accepted 30 November 2005 Abstract The effects of nitrite concentration on growth and feed intake in juvenile cod, Gadus morhua, were examined. In the experiment, juvenile cod with a mean (S.D.) live weight of 7.0 (1.9) g were exposed to four concentrations of nitrite [0, (control), 1.0 (low), 2.5 (medium) and 5 mg NO 2 Nl 1 (high)] for 96 days at 8.0 C, salinity 33 ppt and ph 8.0. No mortality occurred in any of the experimental groups throughout the study. Growth was significantly reduced in fish exposed to all treatment levels, with reduced growth in the high concentration group in the first period of the experiment (day 1 31) and in all experimental concentrations during later periods. No significant difference in feed intake or food conversion efficiency was observed between treatment groups. The fish from the exposed groups displayed an acclimatory response to nitrite. These results indicate that management of intensive aquaculture systems of G. morhua, should avoid levels of nitrite even at 1.0 mg NO 2 Nl 1 not to impair growth Elsevier B.V. All rights reserved. Keywords: Juvenile Atlantic cod; Chronic nitrite exposure; Growth 1. Introduction Atlantic cod (Gadus morhua) is an important commercial fishery species. Declining catches have led to increased market value, and now once again cod is considered a promising candidate for aquaculture (Foss et al., 2004). High construction and running costs of intensive land-based facilities require effective utilisation of pumped water. In intensive culture rearing systems it is common to observe accumulation of inorganic nitrogen, such as ammonia, nitrite and nitrate (Colt and Armstrong, 1981; U.S. EPA, 1984, 1989; Hargreaves, Corresponding author. Tel.: ; fax: address: Sten.Siikavuopio@fiskeriforskning.no (S.I. Siikavuopio). 1998; Jensen, 2003). Nitrite (NO 2 ) is the most common pollutant in culture systems (Grosell and Jensen, 2000; Wang et al., 2004). Nitrite is formed from ammonia and may accumulate in aquatic systems as a result of imbalance of nitrifying bacterial activity (Nitrosomas sp. and Nitrobacter sp.) (Colt and Armstrong, 1981; Handy and Poxton, 1993; Masser et al., 1999). High levels of nitrite in the water is a potential factor triggering stress and may even cause high mortality in aquatic organisms (Lewis and Morris, 1986; Martinez and Souza, 2002; Jensen, 2003; Wang et al., 2004; Ferreira da Costa et al., 2004). Nitrite is generally much more toxic to freshwater organisms (Jensen, 1995, 2003). In freshwater, nitrite is actively taken up across the gill in competition with chloride (Eddy and Williams, 1987). The principal /$ - see front matter 2005 Elsevier B.V. All rights reserved. doi: /j.aquaculture

2 352 S.I. Siikavuopio, B.-S. Sæther / Aquaculture 255 (2006) effect of such nitrite loading is a progressive oxidation of haemoglobin to methaemoglobin, but several other physiological changes occur (Jensen, 1990, 2003). Seawater fish are less susceptible but take up nitrite across intestine and gills (Jensen, 2003). The role of NO 2 uptake in marine fish and subsequent physiological disturbances are poorly described (Grosell and Jensen, 2000). Detailed knowledge of the impact of water quality factors on fundamental production characteristics such as growth performance, food conversion efficiency, and animal welfare is therefore needed in order to effectively exploit such systems. Acute toxicity of nitrite has been investigated in a number of fish species (Colt and Armstrong, 1981; Handy and Poxton, 1993; Grosell and Jensen, 2000; Martinez and Souza, 2002; Ferreira da Costa et al., 2004; Das et al., 2004), but the effects of chronic nitrite exposure on survival and growth of juvenile Atlantic cod have not been investigated. Hence, the present study was designed to investigate the long-term effects of chronic exposure to sub-lethal levels of nitrite, ranging from 1 to 5 mg N NO 2 l 1, on growth and feed intake in juvenile Atlantic cod. 2. Materials and methods 2.1. Experimental conditions and origin of fish Commercially produced cod (Troms Marin Yngel AS, Tromsø), were brought to the Tromsø Aquaculture Research Station in April The fish were kept in a 400 l fibre glass tank provided with an abundant supply of running seawater until the experiment was initiated. The fish were fed on commercially formulated feed; 1.8 mm granulate from day 0 until day 32 (DAN-EX 1362, containing 13% crude oil and 62% protein), and 2.0 mm pellets from day 33 until the experiment was terminated (DAN-EX 1562, containing 15% crude oil and 58% protein). Water flow was set to 5 l min 1 per tank. The fish were exposed to simulated natural photoperiod (70 N) during the experiment, using two timer-controlled 15 W light bulbs Experimental design The experiment began 20 April A total of 520 cod were distributed into eight 100 l fibre glass tanks with 65 fish in each tank. The cod were gradually acclimated to nitrite concentrations of 1.0, 2.5 and 5 mg NO 2 Nl 1, by increasing the concentration of nitrite 0.5 mg l 1 per day (Table 1). The nitrite concentration of the control group was 0.02 mg NO 2 Nl 1 (no addition) and all groups were replicated. Table 1 Treatment levels of nitrite given as mg NO 2 Nl 1 (nitrite nitrogen) with the corresponding levels of NO 2 (mg l 1 ), temperature and stocking densities (kg m 3 ) in the control and three experimental groups of juvenile Atlantic cod Treatment mg NO 2 Nl 1 At the start of the experiment the cod were anaesthetised (metacaine, 0.1 g l 1 seawater), individual weight and length were recorded to the nearest 0.1 g and 0.1 cm, respectively, with follow-up samplings at days 32, 63 and 93. The initial mean weights (S.D.) was 6.9 g (1.8) and did not differ significantly between groups. Oxygen saturation was measured weekly in the effluent water of all tanks and was above 90% at all times. The water temperature was measured daily and maintained at 8.0 C throughout the experiment (Table 1). The required nitrite concentrations were obtained by adding a concentrated solution of sodium nitrite (technical grade, VWR International AS, no un 1500, adr kl. 5-1,23c, eec number , classification code 5121,6141) by three electromagnetic metering pumps (EH/W, Iwaki Co. Ltd.) to header tanks that supplied the experimental tanks with water. Levels of nitrite (NO 2 N, nitrite nitrogen) were determined spectrophotometrically every morning (Spectroquant Nova 60, Merck, Lab business). Nitrite was calculated using nitrite reagent test, art , method number: 036 (Griess reaction). Feed was provided in excess for 6 h (between, 0300 and 0900) except for Saturday and Sundays (not fed). Feed intake at tank level was determined on a daily basis using waste feed collectors, as described by Bendiksen et al. (2002). Due to the small particle size, feed intake measurements were only made from day 33 and until the experiment was terminated Measured parameters mg NO 2 l 1 Temperature Stocking density Day 0 Day 69 Control 0.02 (0.00) 0.07 (0.00) 8.0 (0.1) Low 1.01 (0.11) 3.32 (0.36) 8.0 (0.1) Medium 2.44 (0.43) 8.02 (1.41) 8.0 (0.1) High 4.99 (0.62) 16.4 (2.04) 8.0 (0.1) Values are given as mean (S.D.). All growth estimates, results on daily feed consumption and feed conversion efficiency in the present study are based on tank biomass, but fish were weighed individually at samplings. Feed conversion efficiency (FCE) was calculated as: FCE=(W 2 W 1 ) C 1, where C is total food intake in an experimental period.

3 S.I. Siikavuopio, B.-S. Sæther / Aquaculture 255 (2006) Ten fish from each group were terminally sampled at monthly intervals. From these individuals, blood was collected from the caudal vessels using heparinised syringes, and analysed with an i-stat Portable Clinical Analyzer. The analyzer was used in conjunction with EC8+ disposable cartridges, measuring plasma sodium (Na + ) and potassium (K + ) content, urea N content, glucose, haematocrit (Hct) and carbon dioxide (PCO 2 ) (Table 3) Statistical methods Data are presented as mean ± standard deviation (S.D.). Differences between groups were tested by a General Linear Method (GLM) (Zar, 1984; Kleinbaum et al., 1988) with treatment as a categorical factor, followed by a Tukey post hoc test if significant. Significance was assumed when Pb Results With the exception of terminally sampled fish, no mortality occurred in any of the experimental groups throughout the experimental period. Mean weight of the fish in the various treatment groups was significantly influenced by nitrite concentration, with decreased growth in groups exposed to experimentally increased NO 2 N levels. The initial weights were similar between groups, but at day 32 the mean weight was significantly lower in the high concentration group compared to all others (Fig. 1). From day 63 until termination of the experiment all treatment groups had lower mean body Mean weight (g) Control Low Medium High aaab a b b b a b b b Time (days) Fig. 1. Effect of different nitrite levels on the increase in body weight in juvenile Atlantic cod, Gadus morhua. The columns show mean bodyweight in grams, before during and after a feeding period of 93 days, with variability given as standard deviation. Table 2 Effect of different nitrite levels on feed intake (FI) and feed conversion efficiency (FCE) in juvenile Atlantic cod, Gadus morhua Treatment FI1 FI2 FCE1 FCE2 Control 1.78 (0.33) 1.50 (0.37) 1.04 (0.07) 0.82 (0.16) Low 2.02 (0.54) 1.59 (0.35) 0.94 (0.09) 0.85 (0.11) Medium 1.77 (0.19) 1.54 (0.11) 0.95 (0.01) 0.74 (0.08) High 1.79 (0.09) 1.47 (0.04) 0.82 (0.16) 0.72 (0.17) Feed intake is calculated as percent of mean body weight based on tank biomass and gross feed intake. FCE is calculated as gram biomass change per gram ingested feed. weight as compared to the control (Fig. 1). Feed intake in percent of mean body weight, based on daily growth adjusted tank biomass, number of fish and gross feed intake, did not differ between groups (F 3, 4 =1.142; PN0.05), but increased with time (F 1, 4 =27.2; P=0.006). There were no differences in FCE between the groups at any time (F 3, 8 = 0.376; PN0.05), or pooled between times (F 3, 8 =1.281; PN0.05), but FCE pooled between groups declined during the experiment (F 1, 14 =7.292; Pb0.05, Table 2). Total blood glucose, urea, Na +,K +, Hct and PCO 2 concentrations at day 32, 63 and 93, are shown in Table 3. Significant differences between groups occurred only on day 32. Nitrite concentration had a marked effect on urea, with medium and high concentration groups showing significantly lower blood urea levels compared to low and control. The concentration of glucose tended to increase with increasing treatment level, and the high concentration group had higher level than the low concentration group, however, the control group was intermediate to treatment groups and not significantly different to either. PCO 2 was significantly higher in the group exposed to the highest nitrite concentration compared to all other treatments. 4. Discussion The results show clear negative effects of moderate concentrations of nitrite on growth in young cod. During the first 32 days growth rate declined only in the group exposed to the highest nitrite concentration. This changed, however, and from day 63 and onwards all treatment groups had lower mean weight than the control. Feed intake did not differ significantly between treatments, and as such does not seem a good candidate to assess negative effects of what may be referred to as moderately elevated nitrite levels. Feed conversion was similar in all groups, and did not reveal any increase in energy demand to support physiological changes that accompanies nitrite exposure, e.g. ion-regulatory, respiratory and cardiovascular challenges (Grosell and

4 354 S.I. Siikavuopio, B.-S. Sæther / Aquaculture 255 (2006) Table 3 Measured and calculated blood plasma parameters at day 32, 63 and 93 of the experiment in juvenile Atlantic cod reared at different concentrations of nitrite Urea (mmol l 1 ) Glucose (mg dl 1 ) Na (mmol/l) K (mmol/l) Hct (% PCV) PCO 2 (mm Hg) Day 32 Control 2.7 (0.7)a 3.0 (0.6)ab (4.5) 5.9 (1.5) 13.8 (1.8) 2.5 (0.9)a Low 2.2 (0.6)a 2.4 (1.6)a (4.5) 4.2 (0.8) 12.6 (1.5) 2.8 (0.3)a Medium 1.5 (0.5)b 3.5 (0.5)ab (1.8) 4.7 (1.1) 13.4 (2.1) 3.0 (2.6)a High 1.6 (0.6)b 4.1 (0.5)b (3.2) 4.7 (0.7) 14.2 (3.6) 5.6 (2.2)b Day 63 Control 1.7 (0.5) 3.2 (0.8) (2.5) 3.9 (0.4) 17.7 (2.3) 3.1 (0.3) Low 2.0 (0.4) 3.5 (0.8) (2.7) 4.3 (1.2) 16.3 (3.2) 2.9 (0.3) Medium 2.2 (0.5) 3.9 (1.1) (2.3) 4.0 (0.3) 16.8 (1.9) 2.9 (0.3) High 2.2 (0.6) 3.3 (0.7) (2.9) 4.2 (0.7) 16.2 (2.4) 3.0 (0.3) Day 93 Control 1.4 (0.3) 3.8 (0.8) (2.4) 4.1 (1.0) 17.6 (2.9) 3.4 (0.4) Low 1.6 (0.5) 4.4 (0.7) (2.9) 4.8 (1.2) 16.8 (3.3) 3.2 (0.5) Medium 1.3 (0.4) 4.2 (1.3) (2.5) 4.2 (0.5) 18.0 (4.0) 3.3 (0.2) High 1.9 (0.6) 3.7 (0.7) (2.7) 4.1 (0.3) 17.9 (1.4) 3.0 (0.5) Values are given as mean (S.D.). n=8 for all groups. Different letters denote significant differences (Pb0.05) between treatments. Jensen, 2000; Jensen, 2003). However, the groups did grow differently, thus, either feed intake, feed conversion efficiency or both were affected, but according to the applied methods and design not significantly so. The method chosen for feed intake studies is well suited for rapid feedback on factors that may affect the fish's appetite (Sørum and Damsgård, 2004). However, it does not allow for individual recording of feed intake and this reduce the accuracy of the data as interindividual variability is neglected. Information on safe levels of nitrite in seawater is scarce, and in particular long term effects are poorly described. For Atlantic cod the present study is, to the best of our knowledge, the only one providing this kind of data. Timmons et al. (2001) gave water quality criteria for aquaculture in freshwater, and refers to acceptable levels of nitrite below 1.0 mg NO 2 N l 1. Handy and Poxton (1993) refers to NO 2 N levels in the range mg l 1 as acutely toxic to freshwater adapted salmonids. In the present study none of the nitrite levels tested was below NOEC level (No Observable Effect Concentration) on growth. Nitrite is much more toxic to freshwater organisms than to seawater organisms (Grosell and Jensen, 2000; Jensen, 2003), and several possible explanations have been put forward to explain this difference (see e.g. Handy and Poxton, 1993 for details). Jensen (2003) relates this to the higher chloride concentration in seawater and the different osmotic gradients that fish living in seawater and freshwater experience. Seawater teleosts are hypoosmotic to the environment and face the problems of passive efflux of water and passive influx of Cl and Na + across the gills (Jensen, 2003). The uptake, toxicity and effects of nitrite vary between fish species and life stages within species (Lewis and Morris, 1986; Martinez and Souza, 2002; Jensen, 2003), and with temperature (Saroglia et al., 1981). Thus, comparison of toxicity values between different studies is difficult. Nitrite induces a large variety of physiological disturbances in fish and the toxicity may result from a combination of effects, nevertheless, the most studied is the formation of methaemoglobin (methb) (Martinez and Souza, 2002; Jensen, 2003). Nitrite binds competitively to haemoglobin oxidising it to methb, a variant causing the blood to appear brown in colour (hence the name brown blood disease ) and vastly reduce the ability to bind and transport oxygen (Hargreaves, 1998; Martinez and Souza, 2002; Jensen, 2003). In their work on sea bass, Dicentrarchus labrax, held at 36 ppt salinity, Scarano et al. (1984) found that 12.9 mg NO 2 Nl 1 induced methb formation during a 96 h exposure study. Chinook salmon in seawater had a threshold level of 37.5 mg NO 2 N l 1 before methb formation took place (Crawford and Allen, 1977). Whether the cod in this study had formed methb is unknown as it was never measured; a major weakness of the present work. Compared to the above mentioned threshold levels, the cod in the present study was exposed to levels well below these. As such, there may be long term effects on growth caused by nitrite levels that would not elicit any increase in methb. Based on these

5 S.I. Siikavuopio, B.-S. Sæther / Aquaculture 255 (2006) assumptions, use of elevated levels of methb as an indicator of poor water quality due to aggregated nitrite cannot be envisaged. The blood parameters measured during this study did not reveal any dramatic change in treatment groups as compared to control. Reviewed in Jensen (2003), marine shrimps have been shown to increase excretion of urea upon nitrite exposure. The cod in the present study showed a tendency for declined blood urea concentrations with increasing nitrite concentration at the sampling on day 32. This had changed on day 63, as the urea concentration showed a slight increase with increasing nitrite in the environment, but at the last sampling (day 93) no consistent trend could be observed. Nitrite was expected to have a critical influence on the potassium balance (K + ) resulting in an extra cellular elevation (Jensen, 2003), but no consistent changes occurred in this study. K + is involved in cell volume regulation via the K + /CL cotransporter, and changes in K + should be followed by shrinkage of red blood cells due to withdrawal of osmotically obligated water (Jensen, 1990). Shrinkage of RBCs should then lead to reduced haematocrit (Hct) values, but although Hct to some extent varies with K +, there is no consistent drop with increased nitrite concentration. In the present study Na + varied very little between treatments and showed no consistent changes with increased nitrite level in the water. According to Jensen (1990) and Grosell and Jensen (2000) Na + is expected to remain unchanged or slightly decline in nitrite exposed fish. Nitrite exposure severe enough to elicit hyperventilation, due to reduced O 2 carrying capacity of oxidised haemoglobin, would lead to reduced plasma concentrations of CO 2 (Jensen, 2003). However, owing to the linkage between O 2 and CO 2 transport in the blood, modifications of the blood O 2 carrying capacity or haemoglobin O 2 binding affinity may in turn have repercussions for blood CO 2 transport (Gilmour, 1998). The long-term exposure to ambient nitrite resulted only in minor physiological disturbances characterised by a temporal increase of blood CO 2 in the high level group at day 32. Common to all blood parameters measured is that differences only appeared at day 32, possibly indicating adaptation to the environment enabling the fish to compensate for the unfavourable conditions. However, judging by the reduced growth performance, such adaptation occurs at a cost. Stress response in fish involves energy demanding processes and glucose is an important energy source to the metabolism in tissue as the brain, heart, blood cells, and gills (Mommsen et al., 1999). Increased plasma glucose is an adaptive response to a stressor (Barton and Iwama, 1991), and environmental conditions that impose stress to the fish should therefore be expected to give increased blood plasma levels of glucose. Staurnes et al. (1994) reported that unstressed cod at 8 C had a plasma glucose level at 3.8 mg l 1 and that this had increased to 4.9 mg l 1 after 17 days in fish exposed to low temperature. Plasma glucose concentrations in the present experiment gave inconsistent indications as to whether the treatment imposed stress to the fish. The intermediate groups had lower glucose levels than the high concentration group, but were also lower than the control. Water quality modelling should be an integrated part in the design and building of aquaculture facilities in order to ensure that economical as well as ethical aspects of intensive high density culture are accounted for. When considering the various threshold limits that exist for different species, and also for different life stages within species, it becomes clear that comprehensive studies are necessary in order to facilitate safe rearing procedures in intensive culture facilities. This should preferably be done using a multifactor approach, which far more efficiently may provide information that reflects a true culture situation, where several parameters interacts to affect juvenile cod performance simultaneously. Acknowledgements This study was supported by Norwegian Institute of Fishery and Aquaculture Research, the EU project SEAFOODplus, contract and the Norwegian Research Council, project no /140. We would like to thank Ivar Nevermo for his technical assistance and advice on this work. References Barton, B.A., Iwama, G.K., Physiological changes in fish from stress in aquaculture with emphasis on the response and effects of corticosteroids. Annu. Rev. 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