Groundwater pre-treatment prevents the onset of chronic ulcerative dermatopathy in juvenile Murray cod, Maccullochella peelii peelii (Mitchell)

Aquaculture 312 (2011) 19–25

Contents lists available at ScienceDirect

Aquaculture j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

Groundwater pre-treatment prevents the onset of chronic ulcerative dermatopathy in juvenile Murray cod, Maccullochella peelii peelii (Mitchell) Aaron G. Schultz a,⁎, Sarah L. Shigdar b, Paul L. Jones c, Alister C. Ward b, Tes Toop a a b c

School of Life and Environmental Sciences, Deakin University, Pigdons Road, Geelong, 3217, Australia School of Medicine, Deakin University, Pigdons Road, Geelong, 3217, Australia School of Life and Environmental Sciences, Deakin University, PO Box 423, Warrnambool, 3280, Australia

a r t i c l e

i n f o

Article history: Received 22 July 2009 Received in revised form 8 December 2010 Accepted 9 December 2010 Available online 21 December 2010 Keywords: Murray cod Pathology Chronic ulcerative dermatopathy Biofilter Water treatment

a b s t r a c t Chronic ulcerative dermatopathy (CUD) is a disfiguring condition affecting Murray cod cultured in untreated groundwater. This study sought to further investigate the possible etiology of the syndrome and determine whether groundwater pre-treatment could suppress the development of CUD in juvenile Murray cod. Electrolyte enrichment or pre-treatment with UV irradiation delayed the onset of CUD. In contrast, preconditioning of groundwater either in a vegetated earthen pond or in the presence of artificial macrophytes drastically reduced both the incidence and severity of CUD, with more than 90% of fish exhibiting no visual signs. Haematology and blood parameters were assessed for future diagnostic potential, but no changes in blood parameters were observed, even in advanced CUD-affected fish. This paper identified that the treatment of groundwater via an earthen pond and in the presence of an artificial macrophyte are two highly effective methods of preventing CUD arising in juvenile Murray cod. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Chronic ulcerative dermatopathy, also termed chronic erosive dermatopathy, was first identified in Murray cod aquaculture in the mid to late 1990s (Ingram et al., 2004). Murray cod affected with CUD are grossly disfigured, with degeneration and necrosis of the epidermal layer surrounding the cephalic and trunk lateral line sensory canals and erosion of fins (Baily et al., 2005; Schultz et al., 2008). This condition currently remains idiopathic and despite extensive histological and bacterial culture investigations, no pathogenic organisms have been associated with the pathology of this disorder (Baily et al., 2005). Furthermore, the condition does not appear to be transmissible between fish as it was observed that unaffected juvenile Murray cod do not develop the condition after 2 months culture with CUD-affected fish (A.G. Schultz, unpublished data). The current consensus is that some component of the groundwater in which the fish are cultured is responsible for CUD. This was highlighted in a study by Baily et al. (2005) who discovered that lesions of affected Murray cod slowly resolved when they were cultured in Murray river water. Currently there have been no documented cases observed in wild Murray cod. This condition is a significant problem, as disfigurement of fish substantially reduces their marketability, and combined with decreased growth and increases in mortality, affected farms are faced ⁎ Corresponding author. Current address: Department of Biological Sciences, University of Alberta, Edmonton, AB, Canada T5G 2E9. Tel.: + 1 780 492 5879. E-mail address: [email protected] (A.G. Schultz). 0044-8486/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2010.12.013

with economical losses (Ingram et al., 2004). The condition has only been described in one other fish species, the goldfish, Carassius auratus, by Baily et al. (2005), who cultured fish in groundwater at an affected Murray cod farm and reported hyperplasia and necrosis of the epithelium in sensory canals after four weeks of exposure. However, there are reports of a similar condition arising in Taiwanese grouper, Epinephelus sp., (P. Hardy-Smith, pers. comm.) and sharp snout sea bream, Diplodus puntazzo, (J. Bailey, pers. comm.) cultured in saline groundwater. Recently, an apparently similar condition termed ‘lateral line depigmentation’ was reported in channel catfish, Ictalurus punctatus (Rafinesque) after exposure to chronic nutritional stress (Corrales et al., 2009). The gross lesions reported in the catfish were similar to those reported in CUD-affected Murray cod by Baily et al. (2005). In a previous study, we reported visual signs of CUD development in large (700 g) Murray cod after five months of culture in groundwater. In contrast, Baily et al. (2005) reported that six week old juvenile Murray cod developed lesions after only one month in groundwater. Thus, this condition appears to develop and manifest more rapidly in small, juvenile Murray cod. For that reason, juvenile Murray cod were used in the present study to enable early detection of the condition and enable a thorough assessment of the development of CUD in response to different water treatments. This study was designed in an attempt to identify the relative influence of various biotic and abiotic components of the groundwater on the physiology and development of CUD in juvenile Murray cod. Our primary aim was to identify an effective method of curing (preconditioning) the groundwater to make it suitable for culture. White

20

A.G. Schultz et al. / Aquaculture 312 (2011) 19–25

blood cell indices, plasma electrolyte concentrations and osmolality were analysed to establish whether blood sampling could be employed as a diagnostic test to detect the early onset of CUD in juvenile Murray cod. Blood sampling has been used as a valuable routine diagnostic technique, with specific haematological parameters and blood electrolyte concentrations capable of indicating early pathological changes or adding to our understanding of disease etiology in fish (Burrows, 2001; Chen et al., 2003; Nair and Nair, 1983). 2. Materials and methods 2.1. Animals and experimental protocols This study was conducted at Brimin Lodge Murray cod farm (BL), which is located in close proximity to the Murray River at Rutherglen, Victoria, Australia. Three thousand weaned Murray cod juveniles (2.29 ± 0.01 g), free from any outward signs of CUD were transferred to BL from the Deakin University Aquaculture Research Facility, Warrnambool, Victoria, Australia. At BL, these fish were cultured indoors in an insulated steel frame shed, in a flow-through aquaculture system in half-filled 1000 l plastic troughs (Reln Plastics, NSW, Australia) containing either untreated groundwater (bore; positive control) or four experimental groundwater treatments; pond, AquaMat, electrolyte-enriched and UV irradiation, with two hundred fish per tank and each treatment run in triplicate. In a previous pilot study, we observed that pumping groundwater to an earthen pond for approximately 72 h prevented 98% of the juvenile Murray cod from developing CUD (A. Schultz, results not shown). Therefore, this treatment method was modeled in this study, with the pond-water treatment tanks supplied with groundwater that was initially pumped into a 4 m × 4 m × 1 m deep vegetated earthen pond with a retention time of approximately 72 h. In the AquaMat treatment, groundwater was pumped into 2 × 5000 l plastic circular pre-treatment tanks (D 3.0 m × H 0.7 m) containing an artificial macrophyte material (AquaMats®, Meridian Applied Technology Systems, MD, USA) which promotes biofilm growth. Twenty two AquaMats® strips (D 0.9 m × L 2.0 m) were placed in both tanks and each comprised a weighted submerged cylinder, from which extended 200 cm × 1 cm positively buoyant strips that floated upwards in a fashion similar to a submerged aquatic macrophyte. The AquaMat pre-treatment tank was located outside the main facility where it was exposed to sunlight to promote biofilm growth and had an approximate water retention time of 72 h. Water was pumped into the AquaMat tank from the main groundwater supply and then gravity fed into the treatment tanks located inside the facility. Prior to use, the AquaMat pre-treatment tanks were fertilised with Miracle-Gro® (N:P:K = 15:13.1:12.4; Scotts® Aust. Pty. Ltd., NSW, Australia) on two occasions to facilitate the development of biofilms on the AquaMats® material and were allowed to condition for approximately 4 weeks prior to use in the experimental tanks. In the electrolyte-enriched treatment, groundwater was trickled over 100 l of crushed oyster shell contained in a 200 l plastic drum suspended over each treatment tank. In addition, 5 l of ground agricultural limestone was placed in fine mesh (nylon netting) and placed inside each treatment tank underneath the outflow of each oyster shell pre-treatment drum. Treatment of groundwater by UV irradiation involved passing the groundwater through a UV unit containing 2 × 40 W UV lamps with an approximate output of 950,000 μWs cm−2 (QL-80, Lifegard Aquatics, CA, USA; 254 nm), prior to entering the treatment tanks. Flow rate of water through the UV unit was approximately 3 l min−1. According to the manufacturer's instructions, the average output of one 40 W UV lamp at a flow rate of 5700 l h−1 is approximately 15,910 μWs cm−2 (Lifegard Aquatics, CA, USA). Therefore, with a flow rate of approximately 3 l min−1 (180 l h−1) through the UV

unit, the UV dose of two 40 W lamps would be 1,007,633 μWs cm−2 at 100% UV transmittance. However, according to Summerfelt (2003), spring water has a UV transmittance of 95–98%. Therefore, 0.95 × 1,007,633 μWs cm−2 = 957,251 μWs cm−2 or approximately 950,000 μWs cm−2. Throughout the study, juvenile Murray cod were hand fed 2 mm floating native grower pellets (Ridley, Aqua-Feed, Queensland, Australia) once daily to apparent satiation. Water flow into each of the experimental tanks was approximately 1 l min−1 (approximately 3 exchanges day−1) and was regularly checked to ensure uniform flow across the 15 tanks. The treatment tanks were supplied with air (via an airlift pump) from a centrifugal blower. The tanks were fitted with an external standpipe to enable easy cleaning and siphoning of accumulated solids from each tank, which was performed each morning after feeding. All animals in this study and experiments were approved by the Deakin University Animal Welfare Committee following the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (2004). 2.2. Sampling and analytical techniques The study commenced at the beginning of May 2006 and continued for 9 months so fish grew to an adequate size for sampling. At the completion of the study, 40 juveniles from each tank were anaesthetized with benzocaine (1.0 g l−1; Sigma, MO, USA), and assessed for CUD development (see Section 2.2.2). Six additional fish from each treatment were euthanized by an overdose of benzocaine (5.0 g l−1) and venous blood samples (2 ml) were collected via caudal puncture using 5 ml lithium-heparinised syringes (4 mg ml−1; Sigma). Blood smears were made for staining and haematological analysis and the blood samples were centrifuged for 15 min at 7500 ×g and 4 °C (Beckman Allegra™ 21R Centrifuge) to isolate plasma from the cells and were frozen on dry ice. Plasma samples were stored at −80 °C until analysis. 2.2.1. Water samples Water samples were collected in duplicate from the pre-treatment and culture tanks in May (day 0), June (day 50), September (day 142) and the completion of the study at the end of January (day 275). The water was analysed for pH, alkalinity, hardness, electrical conductivity, total organic carbon (TOC), total dissolved solids (TDS) and ion concentrations (Amdel Ltd., Victoria, Australia). Temperature data loggers (Thermodata Pty. Ltd., Victoria, Australia) were placed in the tanks, which continuously recorded the temperature every 4 h. Each week, water samples were also routinely analysed for: dissolved oxygen (Titron RJ20 pocket meter handheld oxygen probe), pH, ammonia, nitrite, nitrate, phosphate (Aquamerck® standard reagent test kits, Merck, Darmstadt, Germany) and total CO2 (via titration in carbonate). 2.2.2. CUD development Juvenile fish were closely monitored and notes were regularly made about their general health and CUD development, including lesion formation, skin coloration and fin erosion. A scoring scheme was developed to assess the development and severity of CUD in fish at the completion of the growth period (Fig. 1). This scoring scheme assessed the degree of epidermal erosion surrounding the cephalic and trunk lateral line canals and fin erosion. Each tank was sampled four times non-sequentially by the 3rd author, with groups of 10 fish scored by the 1st author who was unaware of the treatment from which they came. 2.2.3. Blood analysis Leucocyte differentials were performed on blood smears stained with May-Grűnwald Giesma (Fronine Laboratory Supplies Pty. Ltd.,

A.G. Schultz et al. / Aquaculture 312 (2011) 19–25

0

21

3. Results 3.1. Water quality

1

2

Chemical characteristics of the supply water and the water in culture tanks varied slightly between treatments during the course of the study (Tables 1 and 2). Water in the electrolyte treatment tanks appeared to be more alkaline, harder and had a higher Ca2+ and Mg2+ concentration compared to the other treatment tanks. Ammonia: 0.15 ± 0.03–0.20 ± 0.04 mg l −1 (bore and electrolyte-enriched, respectively); nitrite: 0.14 ± 0.05–0.40 ± 0.11 mg l−1 (bore and UV, respectively); nitrate: 57.73 ± 7.29–65.71 ± 8.58 mg l−1 (electrolyteenriched and AquaMat, respectively); phosphate: 0.35 ± 0.03–0.52 ± 0.03 (AquaMat and UV, respectively); and total CO2: 11.73 ± 0.83– 15.45 ± 0.88 mg l−1 (bore and electrolyte-enriched, respectively) levels in the tanks remained stable. 3.2. CUD development

3

4

All fish cultured in groundwater (bore treatment) developed CUD, although the severity of the condition varied among individuals, with scores ranging from 1 (mildly affected) to 4 (severe). A large number of fish in this treatment had acute signs of the condition, with 39 ± 17% receiving a score of 3 and 13 ± 11% receiving a score of 4 (Fig. 2). In the pond and AquaMat treatments, the majority of the fish (92 ± 4% and 94 ± 1%, respectively) showed no visual signs of the condition. The small percentage of individuals in these two treatments which received scores of 1 had slight erosion of epidermis surrounding infraorbital and lateral line canals. Fish cultured in the electrolyte-enriched and UV treatments were moderately affected, with 54± 23% and 24 ± 1%, respectively, receiving a score of 1 and 45± 23% and 76± 1%, respectively, receiving a score of 2. A small number of individuals (1± 1%) cultured in the electrolyte-enriched treatment received a score of 3. 3.3. Blood and ionic analysis

New South Wales, Australia), as described by Shigdar et al. (2007). Cytochemical analysis was also performed, as described by Shigdar et al. (2009). Plasma samples were thawed at room temperature and Na+, K+, Ca2+ and Mg2+ concentrations were determined using an atomic absorption spectrophotometer (model GBC 933; GBC Scientific, Victoria, Australia). Plasma Cl− concentrations were determined by a mercuric thiocyanate assay as described by Zall et al. (1956), using a LKB Biochrom Novaspec spectrophotometer (model 4049; Biochrom Ltd., Cambridge, UK). A VAPRO® vapor pressure osmometer was initially calibrated with known standards (model 5520; Wescor Inc., UT, USA) and was then used to determine the plasma osmolality.

Control and juvenile fish maintained in the four different water treatments did not show any significant differences in the majority of the blood indices measured with the exception of fish cultured in the electrolyte enriched treatment, which had a significantly reduced number of basophils compared to fish in the bore treatment (Table 3; F4,25 = 2.83, p b 0.05). In addition, no changes in enzyme activity, as demonstrated by cytochemical analysis, were detected. Murray cod plasma Na+ concentrations and osmolality were consistent across all of the water treatments (F4,25 = 0.79 and F4,25 = 1.62, p N 0.05, respectively), however, variations in plasma Ca2+ (F4,25 = 8.23, p b 0.05), Mg2+ (F4,25 = 4.16, p b 0.05) and Cl− (F4,25 = 3.17, p b 0.05) were observed (Table 3). Fish cultured in the pond treatment had a significantly lower plasma Ca2+ and higher plasma Mg2+ concentration compared to fish cultured in bore, AquaMat, electrolyte-enriched and UV treated water. Plasma K+ concentrations were significantly lower in Murray cod cultured in the UV treatment compared to the bore, pond, AquaMat and electrolyte treatments (F4,25 = 26.99, p b 0.05).

2.3. Statistical analysis

4. Discussion

Data are reported as the mean ± SE. Murray cod plasma electrolyte levels, osmolality and white cell indices were tested for normality via a normal probability plot using Minitab v15.0. Following this, data sets were tested for homogeneity of variance and compared via a one-way ANOVA using SPSS v15.0. If significant differences (p ≤ 0.05) were found, a post-hoc multiple comparisons Tukey's test was applied to determine these differences. No statistics were conducted on the scoring percentages.

Chronic ulcerative dermatopathy appears to be associated exclusively with aquaculture facilities utilising groundwater (Baily et al., 2005; Schultz et al., 2008). While a previous study showed that the lesions resolve when affected fish are transferred into river water, which suggests that some component of groundwater is responsible for the disease development, no apparent bacterial, viral, fungal, or parasitic infections were discovered (Baily et al., 2005). In addition, detailed chemical analyses of groundwater from affected sites

Fig. 1. Scoring scheme used to classify CUD development and severity in juvenile Murray cod. Score 0: no visual signs of pathology; 1: erosion of epidermis surrounding infraorbital ( ) and lateral line ( ) canals; 2: erosion of epidermis surrounding infraorbital, supraorbital ( ), preopercularmandibular ( ) and lateral line canals; 3: more advanced stage of 2, with pits joining; 4: more advanced stage of 3, with severe fin erosion.

22

A.G. Schultz et al. / Aquaculture 312 (2011) 19–25

Table 1 Supply and culture tank water parameters. Temp (°C)

pH

Alkalinity (mg l−1) as CaCO3

Hardness (mg l−1) as CaCO3

Elec. cond. (μS cm−1)

TOC (mg l−1)

TDS (mg l−1)

Supply Bore Pond AquaMat Electrolyte UV

– – – – –

7.57 ± 0.30 7.77 ± 0.32 7.60 ± 0.36 7.95 ± 0.15 7.55 ± 0.45

117.2 ± 8.7 116.5 ± 10.9 114.2 ± 10.0 123.3 ± 6.7 105.0 ± 5.0

123.5 ± 10.1 95.5 ± 5.3 112.0 ± 13.8 140.0 ± 5.8 110.0 ± 20

820 ± 14.7 710 ± 46.5 772 ± 69.7 867 ± 29.1 805 ± 25.0

1.20 ± 0.25 3.97 ± 0.66 2.13 ± 0.64 1.25 ± 0.25 1.75 ± 0.45

425 ± 15 365 ± 35 425 ± 15 435 ± 5 415 ± 5

Culture tank Bore Pond AquaMat Electrolyte UV

17.8 ± 0.02 16.0 ± 0.06 15.8 ± 0.06 17.6 ± 0.03 17.7 ± 0.02

7.97 ± 0.19 7.83 ± 0.09 7.97 ± 0.17 8.07 ± 0.13 7.87 ± 0.15

120.0 ± 10.8 119.7 ± 14.8 119.2 ± 11.3 140.0 ± 10.8 119.7 ± 11.0

114.7 ± 15.8 89.2 ± 16.1 104.7 ± 22.8 147.5 ± 4.8 125.0 ± 8.7

830 ± 25.5 712 ± 47.8 790 ± 71.3 880 ± 26.8 842 ± 24.6

2.30 ± 0.21 3.87 ± 1.44 2.17 ± 0.62 1.60 ± 0.35 2.07 ± 0.58

440 ± 10 365 ± 35 420 ± 10 440 ± 10 430 ± 20

Values represented as mean ± SE, n = 4, except for temp, measured continuously and TDS, n = 2. TOC: total organic carbon; TDS: total dissolved solids; Elec. cond.: electrical conductivity.

compared to unaffected sites have so far failed to identify any notable differences in ion, trace metal and total organic concentrations between each of the groundwater sources (Baily, 2003; Schultz et al., 2008). For example, Fe2+ was observed to be b0.1 mg l−1 and Cd2+, Pb2+, Cu2+ and Zn2+ were b0.005 mg l−1 in the groundwater source at BL (Schultz et al., 2008). Although CUD does not appear to affect the quality of the meat, it does cause mild growth reduction and gross disfigurement, and therefore, decreased marketability (Ingram et al., 2004). The robust development of the emerging Murray cod aquaculture industry remains constrained while the etiological factors that cause CUD remain unknown. Therefore, the primary aim of this study was to identify an effective method of “curing” groundwater, thus providing a benefit to farmers dealing with this disorder. In total, four different methods of curing groundwater were tested, with bore water used as a control. The severity of CUD in juvenile Murray cod cultured in groundwater varied among individuals, with some fish being more susceptible to the condition than others. Half of the individuals cultured in this treatment received a score of 3 or 4 as they showed a more advanced stage of CUD, with erosion of the epidermis surrounding the sensory canals being so severe that pits became joined; the extent of fin erosion was the only feature that separated these individuals receiving a score of 3 from a score of 4. CUD developed at a faster rate and was more pronounced in the juvenile Murray cod cultured in the groundwater in this study, compared to larger adults observed in our previous investigation (Schultz et al., 2008). This was consistent with the findings of Baily et al. (2005), who observed the development of CUD in juvenile Murray cod after one

month of culture in groundwater. Severity of CUD, therefore, appears to be linked to the rate of growth of Murray cod, as susceptibility appears to be higher in smaller fish and possibly indicates that CUD has a growth and maturation basis. In the electrolyte and UV water treatments, CUD in juvenile Murray cod was less pronounced compared to fish cultured in the groundwater control. Further investigations need to be conducted to assess whether increasing the contact time of the groundwater with UV and the crushed oyster shell biofilter or combining the two treatments, could prevent CUD development in juvenile Murray cod. Groundwater passed through the crushed oyster shell biofilter in the electrolyte treatment was harder, had a slightly higher pH and contained higher Ca2+, Mg2+ and Na+ concentrations compared to the other water treatments. It is well known that water hardness limits the toxicity of many metals (e.g. Cd2+, Cu2+, and Zn2+) to freshwater fish (Bradley and Sprague, 1985; Brinkman and Hansen, 2007; Everall et al., 1989; Niyogi et al., 2008; Welsh et al., 2000), which is thought to be due to Ca2+ competing with the metals for binding sites on the surface of fish gills (Welsh et al., 2000). Increases in water Ca2+ have been associated with decreased Cu2+ toxicity in chinook salmon, Oncorhynchus tshawytscha, and rainbow trout, Oncorhynchus mykiss (Welsh et al., 2000), and decreased Cd2+ toxicity in the latter (Niyogi et al., 2008). UV irradiation was used in this study primarily to disinfect the groundwater prior to its use, in order to confirm whether an infectious agent contributes to CUD development in Murray cod. In an aquaculture setting the recommended minimum UV dose required to kill common fish pathogens is 30,000 μWs cm−2 (Liltved, 2002;

Table 2 Mean concentrations of major and minor ions in the untreated groundwater (bore) and pre-treated groundwater supplies and water in treatment tanks. Cl− (mg l−1)

Ca2+ (mg l−1)

Na+ (mg l−1)

Mg2+ (mg l−1)

K+ (mg l−1)

Si4+ (mg l−1)

S (mg l−1)

Supply Bore Pond AquaMat Electrolyte UV

150 ± 14.1 108 ± 10.5 127 ± 15.1 150 ± 5.8 135 ± 15.0

15.7 ± 1.3 13.0 ± 1.1 14.7 ± 1.4 23.3 ± 0.9 14.0 ± 3.0

99.7 ± 7.3 91.5 ± 6.5 96.2 ± 8.5 100.7 ± 5.2 88.0 ± 12.0

20.0 ± 1.4 15.7 ± 0.8 18.0 ± 2.4 20.3 ± 0.9 17.5 ± 2.5

2.10 ± 0.21 1.93 ± 0.30 2.18 ± 0.19 2.20 ± 0.25 1.75 ± 0.15

15.0 ± 0.6 10.9 ± 0.7 14.0 ± 0.1 15.0 ± 1.0 14.0 ± 0.1

5.67 ± 0.12 5.77 ± 0.23 5.53 ± 0.23 5.75 ± 0.25 5.50 ± 0.11

Culture tank Bore Pond AquaMat Electrolyte UV

145 ± 9.6 111 ± 7.0 125 ± 9.6 135 ± 8.7 155 ± 12.6

15.7 ± 2.4 13.1 ± 2.7 14.0 ± 2.9 25.7 ± 0.7 17.0 ± 1.6

94.5 ± 12.4 84.2 ± 14.9 88.0 ± 16.5 99.5 ± 0.5 98.5 ± 5.4

18.7 ± 2.3 14.2 ± 2.5 16.6 ± 3.7 20.5 ± 0.5 20.2 ± 1.2

2.00 ± 0.28 1.88 ± 0.40 2.00 ± 0.32 2.30 ± 0.21 2.23 ± 0.23

15.0 ± 0.6 11.2 ± 1.0 14.3 ± 0.3 14.7 ± 0.7 14.7 ± 0.3

5.70 ± 0.12 5.97 ± 0.12 5.63 ± 0.09 5.80 ± 0.15 5.77 ± 0.09

Water treatment

Values represented as mean ± SE, n = 4. Fe2+ and P concentrations were below the detection limit (b 0.1 mg l−1) in all water samples tested.

A.G. Schultz et al. / Aquaculture 312 (2011) 19–25

Bore

Pond

23

Electrolyte

AquaMat

UV

1 ± 1%

6±

%

17

±1

1%

11

4%

±

8±

13

8%

24 ± 1% 45 ± 23%

39 ± 17%

31± 8%

92 ± 4%

Score 0

54 ± 23% 76 ± 1%

94 ± 1%

Score 1

Score 2

Score 3

Score 4

Fig. 2. Percentages of juvenile Murray cod showing varying degrees of CUD in each of the groundwater treatments. See Fig. 1 for the scoring scheme. (n = 40).

Wedemeyer, 1996). Thus, the UV irradiation of 950,000 μWs cm−2, used in this study should have eliminated these infectious agents (particularly considering the clear water conditions of the bore). It should also be noted that total organic carbon levels and total suspended solids have been measured in the groundwater at Brimin Lodge (1.20 ± 0.25 mg l−1 and b 5.0 mg l−1, respectively; A. G. Schultz, unpublished data) and are well below levels previously identified to impede UV irradiance (Liltved, 2002). Interestingly, all juvenile Murray cod cultured in the UV-treated water developed CUD and since pathogens have not previously been associated with the condition (Baily et al., 2005) we consider it unlikely that a pathogen is a necessary precondition for CUD development. In addition, a lack of infection is further supported by the absence of haematological changes. Also interesting was that the juvenile Murray cod in the UVtreatment developed less severe CUD than the fish cultured in bore water. This finding tends to indicate that the causative agent is only partially removed by UV treatment. It is worth considering that as well as destroying pathogens, UV-irradiation of water can remove organic compounds, including pharmaceuticals, by direct photolysis (Gagnon et al., 2008; Heberer, 2002; Kim and Tanaka, 2009; Snyder et al., 2003). Gagnon et al. (2008), reported a 25% and 40% removal efficiency of diclofenac and triclosan, respectively, from water using a UV dose of 25,000 μWs cm−2, while Kim and Tanaka (2009) reported 100% removal of diclofenac at 230,000 μWs cm−2. Ketoprofen, ceftiofur and sulfamethoxazole were also reported to have high photodegradation (b90%) by UV-irradiation (Kim and Tanaka, 2009). The UV dose used in the present study was considerably greater (× 38 and ×4, respectively) than the dose used in both of those studies and, thus, it is plausible that the groundwater contains an organic compound or other unknown agent that was partially degraded by

photolysis. Moreover, it cannot be ruled out that CUD is attributable to more than a single causative agent. Pumping groundwater into a vegetated earthen pond or a tank containing biofilms growing on an artificial macrophyte virtually eradicates CUD occurrence. Unlike the pond, the AquaMat treatment was devoid of an earthen substrate, but rather contained a large biomass of algal and bacterial biofilms growing on the artificial macrophyte substrate (AquaMats®). The use of aquatic macrophytes as biofilters for removal of nutrients and heavy metals from wastewater and water bodies is well documented (Dhote and Dixit, 2008; Jacinto et al., 2009; Loutseti et al., 2009; Marinho-Soriano et al., 2009; Mukherjee and Kumar, 2005; Reddy et al., 1982; Tripathi and Shukla, 1991; Wolverton et al., 1983). Bio-filtration is also commonly used in re-circulating aquaculture facilities to treat waste water for reuse (Gutierrez-Wing and Malone, 2006; Yang et al., 2001). Therefore, the use of the AquaMats® bio-filtration clearly provides a technology that would be beneficial to farmers relying on flow-through groundwater for their water supply. This method also enabled a greater control of biosecurity. Many factors have been identified that influence and alter haematological variables. These include sex, age, diet, season, temperature and stress of capture and handling (Flos et al., 1987; Tierney et al., 2004; Tripathi et al., 2004). Despite these variables, haematological indices can be an effective method for assessing the health of fish and monitoring physiological and pathological changes given that the haematopoietic system plays an integral role in normal biology and immunity. For example, changes in leucocytes are apparent with a range of infectious agents, such as parasites (Nair and Nair, 1983; Ruane et al., 2000), bacterial infections (Hine, 1992; Silveira-Coffigny et al., 2004), and viral infections (Novoa et al., 1995).

Table 3 White cell indices, plasma ion concentrations and osmolality for juvenile Murray cod cultured in treated groundwater. Index

Water treatments Bore

Pond

AquaMat

Electrolyte

UV

White cell indices % Lymphocytes % Heterophils % Basophils % Eosinophils % Monocytes % Myeloid precursor

82.1 ± 2.9 16.1 ± 2.5 0.8 ± 0.2B 0.0 1.0 ± 0.4 0.02 ± 0.01

84.1 ± 1.7 15.1 ± 1.7 0.4 ± 0.1A,B 0.0 0.3 ± 0.1 0.03 ± 0.02

83.3 ± 2.5 14.5 ± 1.7 0.6 ± 0.2A,B 0.0 1.6 ± 1.4 0.06 ± 0.04

91.6 ± 1.1 7.6 ± 0.9 0.2 ± 0.1A 0.0 0.5 ± 0.2 0.03 ± 0.02

84.7 ± 3.6 14.4 ± 3.5 0.4 ± 0.1A,B 0.0 0.5 ± 0.2 0.01 ± 0.01

Plasma values [Na+] (mM) [Ca2+] (mM) [K+] (mM) [Mg2+] (mM) [Cl−] (mM) Osmolality (mmol kg−1)

140.5 ± 9.4 4.23 ± 0.11A 2.97 ± 0.33B,C 0.64 ± 0.03B 105.5 ± 2.6A,B 271.3 ± 3.4

125.3 ± 7.9 3.09 ± 0.11B 3.81 ± 0.27A,B 0.81 ± 0.06A 107.2 ± 5.1A,B 270.0 ± 4.4

131.0 ± 8.7 4.08 ± 0.15A 4.19 ± 0.21A 0.73 ± 0.05A,B 101.2 ± 3.0A 268.4 ± 4.8

135.0 ± 2.0 4.12 ± 0.20A 2.14 ± 0.21C 0.62 ± 0.02B 117.3 ± 3.3B 275.3 ± 3.1

128.3 ± 3.1 3.97 ± 0.15A 1.00 ± 0.12D 0.75 ± 0.02A,B 109.4 ± 2.5A,B 280.2 ± 3.5

Values represented as mean ± SE, n = 6. Mean values in rows with different superscripts are significantly different to each other (ANOVA; p b 0.05).

24

A.G. Schultz et al. / Aquaculture 312 (2011) 19–25

Other environmental insults also lead to haematological changes in fish, including acute Cu2+ exposure (Mazon et al., 2002; Nussey et al., 1995; Svobodová et al., 1994) and pesticides (Ek et al., 2003; Satyanarayan et al., 2004). We have previously characterised the blood cells in healthy adult Murray cod cultured in a re-circulating aquaculture system and identified mean haematological reference intervals (Shigdar et al., 2007). The most abundant type of leucocyte was the lymphocytes, followed by heterophils and finally the basophils, monocytes and myeloid precursors, which were present in similar numbers. While a previous study has determined the Murray cod to have a well developed immunological system (Shigdar et al., 2009), no overt changes were observed in the leucocyte indices during this study that would be expected in the case of an infectious disease. In addition, no morphological or cytochemical changes in blood were observed in these fish. In our previous investigation, we hypothesised that osmoregulatory homeostasis may be compromised in large Murray cod (700 g) affected with CUD, due to ion losses across the damaged epidermis (Schultz et al., 2008). However, plasma ion concentrations and osmolality were identified to be consistent between CUD-affected and non CUD-affected Murray cod, indicating that there was no apparent osmoregulatory distress. Similarly, in the current study, no major differences were observed in plasma ion concentrations (Na+, Ca2+, K+, Mg2+, and Cl−) and osmolality values in severely CUDaffected juvenile Murray cod (bore) compared to juveniles that showed no apparent signs of the condition (pond and AquaMat). This study found that the retention of groundwater in an earthen pond or in tanks containing AquaMat for 72 h is an effective mechanism for the minimisation and prevention of CUD in juvenile Murray cod. However, we were unable to determine the causative agent of CUD. Specifically, the use of AquaMats® as a biofilter clearly provides a technology that would be beneficial to farmers in Australia and globally, relying on groundwater to support their aquaculture activities. Although, analysis of haematological parameters has been discounted as a simple diagnostic test for the early identification of CUD in young Murray cod, such analysis has provided support to the observation that no infectious agent or parasite load has been identified as a potential causative factor of CUD. Acknowledgements We are grateful to Simon and Phillipa Noble of Brimin Lodge, Victoria, who allowed us to carry out the study at their Murray cod aquaculture facility. Aaron Schultz and Sarah Shigdar were supported by Deakin University Postgraduate Research Awards during this study. This research was funded by an Australian and Pacific Science Foundation research grant (APSF 06/3). References Baily, J.E., 2003. A histopathology based investigation of chronic erosive dermatopathy in Murray cod, Maccullochella peelii peelii. MSc Thesis, Institute of Aquaculture, University of Stirling, Stirling. Baily, J.E., Bretherton, M.J., Gavine, F.M., Ferguson, H.W., Turnbull, J.F., 2005. The pathology of chronic erosive dermatopathy in Murray cod, Maccullochella peelii peelii (Mitchell). Journal of Fish Diseases 28, 3–12. Bradley, R.W., Sprague, J.B., 1985. The influence of pH, water total hardness, and alkalinity on the acute lethality of zinc to rainbow trout (Salmo gairdneri). Canadian Journal of Fisheries and Aquatic Sciences 42, 731–736. Brinkman, S.F., Hansen, D.L., 2007. Toxicity of cadmium to early life stages of brown trout, Salmo trutta, at multiple water hardnesses. Environmental Toxicology and Chemistry 26, 1666–1671. Burrows, F.M., 2001. Haematology of the turbot, Psetta maxima (L.): ultrastructural, cytochemical and morphological properties of peripheral blood leucocytes. Journal of Applied Ichthyology 17, 77–84. Chen, C.-Y., Wooster, G.A., Getchell, R.G., Bowser, P.R., Timmons, M.B., 2003. Blood chemistry of healthy, nephrocalcinosis-affected and ozone-treated tilapia in a recirculation system, with application of discriminant analysis. Aquaculture 218, 89–102. Corrales, J., Ullal, A., Noga, E.J., 2009. Lateral line depigmentation (LLD) in channel catfish, Ictalurus punctatus (Rafinesque). Journal of Fish Diseases 32, 705–712.

Dhote, S., Dixit, S., 2008. Water quality improvement through macrophytes—a review. Environmental Monitoring and Assessment. Ek, H., Dave, G., Birgersson, G., Förlin, L., 2003. Acute effects Of 2, 4, 6-trinitrotoluene (TNT) on haematology parameters and hepatic EROD-activity in rainbow trout (Oncorhynchus mykiss). Aquatic Ecosystem Health & Management 6, 415–421. Everall, N.C., MacFarlane, N.A.A., Sedgwick, R.W., 1989. The interactions of water hardness and pH with the acute toxicity of zinc to the brown trout, Salmo trutta L. Journal of Fish Biology 35, 26–36. Flos, R., Tort, L., Balasch, J., 1987. Effects of zinc sulphate on haematological parameters in the dogfish Scyliorhinus canicula and influences of MS222. Marine Environmental Research 21, 289–298. Gagnon, C., Lajeunesse, A., Cejka, P., Gagne, F., Hausler, R., 2008. Degradation of selected acidic and neutral pharmaceutical products in a primary-treated wastewater by disinfection processes. Ozone: Science & Engineering: The Journal of the International Ozone Association 30, 387–392. Gutierrez-Wing, M.T., Malone, R.F., 2006. Biological filters in aquaculture: trends and research directions for freshwater and marine applications. Aquacultural Engineering 34, 163–171. Heberer, T., 2002. Occurrence, fate, and removal of pharmaceutical residues in the aquatic environment: a review of recent research data. Toxicology Letters 131, 5–17. Hine, P.M., 1992. The granulocytes of fish. Fish & Shellfish Immunology 2, 79–98. Ingram, B.A., De Silva, S.S., Gooley, G.J., 2004. Murray cod aquaculture. In: Ingram, B.A., De Silva, S.S. (Eds.), Development of Intensive Commercial Aquaculture Production Technology for Murray cod. PIRVic. Department of Primary Industries, Snobs Creek, Victoria, pp. 1–23. Jacinto, M.L.J.A.J., David, C.P.C., Perez, T.R., De Jesus, B.R., 2009. Comparative efficiency of algal biofilters in the removal of chromium and copper from wastewater. Ecological Engineering 35, 856–860. Kim, I., Tanaka, H., 2009. Photodegradation characteristics of PPCPs in water with UV treatment. Environment International 35, 793–802. Liltved, H., 2002. Ozonation and UV-irradiation. In: Timmons, M.B., Ebeling, J.M., Wheaton, F.W., Summerfelt, S.T., Vinci, B.J. (Eds.), Recirculation Aquaculture Systems. Cayuga Aqua Ventures, Ithaca, pp. 393–426. Loutseti, S., Danielidis, D.B., Economou-Amilli, A., Katsaros, C., Santas, R., Santas, P., 2009. The application of a micro-algal/bacterial biofilter for the detoxification of copper and cadmium metal wastes. Bioresource Technology 100, 2099–2105. Marinho-Soriano, E., Nunes, S.O., Carneiro, M.A.A., Pereira, D.C., 2009. Nutrients' removal from aquaculture wastewater using the macroalgae Gracilaria birdiae. Biomass and Bioenergy 33, 327–331. Mazon, A.F., Monteiro, E.A.S., Pinheiro, G.H.D., Fernandes, M.N., 2002. Hematological and physiological changes induced by short-term exposure to copper in the freshwater fish, Prochilodus scrofa. Brazilian Journal of Biology 62, 621–631. Mukherjee, S., Kumar, S., 2005. Arsenic uptake potential of water lettuce (Pistia stratiotes L.). International Journal of Environmental Studies 62, 249–258. Nair, G.A., Nair, N.B., 1983. Effect of infestation with the isopod, Alitropus typus M. Edwards (Crustacea: Flabellifera: Aegidae) on the haematological parameters of the host fish, Channa striatus (Bloch). Aquaculture 30, 11–19. Niyogi, S., Kent, R., Wood, C.M., 2008. Effects of water chemistry variables on gill binding and acute toxicity of cadmium in rainbow trout (Oncorhynchus mykiss): a biotic ligand model (BLM) approach. Comparative Biochemistry and Physiology. C: Toxicology & Pharmacology 148, 305–314. Novoa, B., Barja, J.L., Figueras, A., 1995. Entry and sequential distribution of an aquatic birnavirus in turbot (Scophthalmus maximus). Aquaculture 131, 1–9. Nussey, G., van Vuren, J.H.J., du Preez, H.H., 1995. Effect of copper on the differential white blood cell counts of the Mozambique tilapia (Oreochromis mossambicus). Comparative Biochemistry and Physiology. C: Comparative Pharmacology and Toxicology 111, 381–388. Reddy, K.R., Campbell, K.L., Graetz, D.A., Portier, K.M., 1982. Use of biological filters for treating agricultural drainage effluents. Journal of Environmental Quality 11, 591–595. Ruane, N.M., Nolan, D.T., Rotllant, J., Costelloe, J., Wendelaar Bonga, S.E., 2000. Experimental exposure of rainbow trout Oncorhynchus mykiss (Walbaum) to the infective stages of the sea louse Lepeophtheirus salmonis (Krøyer) influences the physiological response to an acute stressor. Fish & Shellfish Immunology 10, 451–463. Satyanarayan, S., Bejankiwar, R.S., Chaudhari, P.R., Kotangale, J.P., Satyanarayan, A., 2004. Impact of some chlorinated pesticides on the haematology of the fish Cyprinus carpio and Puntius ticto. Journal of Environmental Sciences (China) 16, 631–634. Schultz, A.G., Healy, J.M., Jones, P.L., Toop, T., 2008. Osmoregulatory balance in Murray cod, Maccullochella peelii peelii (Mitchell), affected with chronic ulcerative dermatopathy. Aquaculture 280, 45–52. Shigdar, S., Cook, D., Jones, P., Harford, A., Ward, A.C., 2007. Blood cells of Murray cod Maccullochella peelii peelii (Mitchell). Journal of Fish Biology 70, 1–8. Shigdar, S., Harford, A., Ward, A.C., 2009. Cytochemical characterisation of the leucocytes and thrombocytes from Murray cod (Maccullochella peelii peelii, Mitchell). Fish & Shellfish Immunology 26, 731–736. Silveira-Coffigny, R., Prieto-Trujillo, A., Ascencio-Valle, F., 2004. Effects of different stressors in haematological variables in cultured Oreochromis aureus S. Comparative Biochemistry and Physiology. C: Toxicology & Pharmacology 139, 245–250. Snyder, S.A., Westerhoff, P., Yoon, Y., Sedlak, D.L., 2003. Pharmaceuticals, personal care products, and endocrine disruptors in water: implications for the water industry. Environmental Engineering Science 20, 449. Summerfelt, S.T., 2003. Ozonation and UV irradiation—an introduction and examples of current applications. Aquacultural Engineering 28, 21–36.

A.G. Schultz et al. / Aquaculture 312 (2011) 19–25 Svobodová, Z., Vykusová, B., Machová, J., 1994. The effects of pollutants on selected haematological and biochemical parameters in fish. In: Muller, R., Lloyd, R. (Eds.), Sublethal and Chronic Effects of Pollutants on Freshwater Fish. FAO Fishing New Books, London, pp. 39–52. Tierney, K.B., Farrell, A.P., Kennedy, C.J., 2004. The differential leucocyte landscape of four teleosts: juvenile Oncorhynchus kisutch, Clupea pallasi, Culaea inconstans and Pimephales promelas. Journal of Fish Biology 65, 906–919. Tripathi, B.D., Shukla, S.C., 1991. Biological treatment of wastewater by selected aquatic plants. Environmental Pollution 69, 69–78. Tripathi, N.K., Latimer, K.S., Burnley, V.V., 2004. Hematologic reference intervals for koi (Cyprinus carpio), including blood cell morphology, cytochemistry, and ultrastructure. Journal of Fish Biology 33, 74–83.

25

Wedemeyer, G.A., 1996. Physiology of Fish in Intensive Culture Systems. Chapman and Hall, New York. Welsh, P.G., Lipton, J., Chapman, G.A., Podrabsky, T.L., 2000. Relative importance of calcium and magnesium in hardness-based modification of copper toxicity. Environmental Toxicology and Chemistry 19, 1624–1631. Wolverton, B.C., McDonald, R.C., Duffer, W.R., 1983. Microorganisms and higher plants for waste water treatment. Journal of Environmental Quality 12, 236–242. Yang, L., Chou, L.-S., Shieh, W.K., 2001. Biofilter treatment of aquaculture water for reuse applications. Water Research 35, 3097–3108. Zall, D.M., Fischer, M.D., Garner, Q.M., 1956. Photometric determination of chloride in water. Analytical Chemistry 28, 1665–1668.