doi:10.1111/jfd.12218
Journal of Fish Diseases 2014, 37, 1003–1011
Low concentrations of sodium hypochlorite affect population dynamics in Gyrodactylus salaris (Malmberg, 1957): practical guidelines for the treatment of the Atlantic salmon, Salmo salar L. parasite A G Hagen1, S Hytterød2 and K Olstad3 1 Norwegian Institute for Water Research, Oslo, Norway 2 Norwegian Veterinary Institute, Oslo, Norway 3 Norwegian Institute for Nature Research, Lillehammer, Norway
Abstract
Introduction
Atlantic salmon, Salmo salar L. parr (age 1+), infected by the monogenean ectoparasite Gyrodactylus salaris (Malmberg, 1957), were exposed to chlorine (Cl)-enriched water at three different concentrations: Cllow (0–5 lg Cl L1), Clmedium (18 lg Cl L1) and Clhigh (50 lg Cl L1). There was a negative correlation between G. salaris infections and the hypochlorite concentrations added. The parasite infection was eliminated by day 6–8 and day 2–4 in the groups Clmedium and Clhigh, respectively, while inhibition of G. salaris population growth was observed in the Cllow group. An important note to this matter, however, is that the G. salaris specimens observed at day 6 in Clmedium and at day 2 in Clhigh were all considered dead by subjective judgement. No mortality in the salmon parr was observed during the first 8 days of the experiment, demonstrating that Cl has a stronger effect on G. salaris than on the salmonid host. The differences in sensitivity between the parasite and the Atlantic salmon indicate that hypochlorite has a potential use as a parasiticide with a therapeutic margin. The low-dose sensitivity may imply that Cl pollution in urban areas may pose a greater risk towards biodiversity than previously assumed.
Species of the monogenean genus Gyrodactylus von Nordmann, 1832 are principally ectoparasites of fish. Currently, some 400 Gyrodactylus species have been described (Harris et al. 2004). A straightforward extrapolation of the total number of teleost fish worldwide, however, suggests that the total number of species in this group may be some 20 000 (Bakke, Cable & Harris 2007). Some cause severe gyrodactylosis, such as Gyrodactylus salaris Malmberg, 1957, which has had a devastating effect on East Atlantic salmon (Salmo salar L.) populations since the accidental introduction to Norway during the 1970s (Johnsen, Møkkelgjerd & Jensen 1999; see also reviews by Bakke, Harris & Cable 2002; Bakke et al. 2007). Gyrodactylus salaris thus is considered to constitute one of the main threats towards wild European Atlantic salmon populations, and it is listed as a ‘notifiable disease’ by the World Organization for Animal Health (OIE) in the Aquatic Animal Health Code (Chapter 10.3). Several countries have taken precautionary measures to avoid the introduction of the parasite. Accordingly, the European Commission (EC) has restricted the import of salmonids that are susceptible to G. salaris to areas with an equivalent health status (Peeler et al. 2006). Norwegian management authorities spend vast amounts every year on control measures in rivers and fish hatcheries in attempts to eliminate the parasite and to prevent further spread (Bakke et al. 2007).
Keywords: biodiversity, disinfection, Monogenea, parasite, treatment. Correspondence A G Hagen, Norwegian Institute for Water Research, Gaustadalle en 21, NO-0349 Oslo, Norway (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Journal of Fish Diseases 2014, 37, 1003–1011
Chlorine in different forms is the most frequently used disinfectant worldwide. It is used as a disinfectant for drinking water, for treatment of runoff water and sewage, and for treatment against unwanted growth of bacteria and algae in different contexts. It is well established that chlorine kills waterborne pathogens (World Health Organization 2006). Mostly, this has been taken into account in the numerous aquarium-based Gyrodactylus studies that have been conducted: as a precautionary measure, the water used in the experimental tanks in many fish holding departments is thus routinely dechlorinated. The quantitative sensitivity of these parasites, however, is not known. The only published work known to the authors on the sensitivity of Gyrodactylus to hypochlorite is the study by Lewis & Ulrich (1967), exploring quick-dip treatment with chlorine for control of gyrodactylids parasitizing Golden shiner, Notemigonus crysoleucas (Mitchill, 1814). Dissolved in freshwater, hypochlorite will make a hypochloric acid equilibrium. The fractions in this equilibrium (HOCl and OCl), together with Cl2 and ClO2, constitute what is frequently annotated free chlorine. If the water contains ammonia (NH3), chloramines (mono-, di- and tri-) will be formed. Chloramines constitute the major part of the fraction frequently annotated bound chlorine. Together, these two fractions constitute total residual chlorine (TRC). A pilot-experiment exposing G. salaris-infected Atlantic salmon to dechlorinated water (using sodium thiosulphate) and to non-dechlorinated water gave marked differences in G. salaris population growth between the two groups (Hytterød & Olstad, unpublished data). No negative effects were observed on the Atlantic salmon in this experiment. Accordingly, based on these findings, it was hypothesized that G. salaris has a sensitivity towards hypochlorite such that typical concentrations of free chlorine found in chlorinated Oslo tap water can remove the infection within days. The current experimental approach set out to investigate the dose dependency in hypochlorite toxicity towards G. salaris. Materials and methods
The experiments were performed at the fish holding department at The Norwegian Veterinary Institute in Oslo. The department source of water is the Oslo city tap water. Before being distributed Ó 2014 John Wiley & Sons Ltd
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A G Hagen et al. Chlorine eradicate G. salaris
to the fish holding tanks, the water is run through a particle filter (Hydrex II, model GX05-20, length 20″, 5 lm polypropylene) and an activated charcoal filter (20 lm/20″ – GAC-20BB, granular activated carbon filter cartridge). A summary of chemical characteristics of the filtered and dechlorinated tap water is given in Table 1. Experimental animals The animal work presented was approved by the Norwegian Animal Research Authority (NARA; no. 3473/2011). Atlantic salmon parr (age 1+) were obtained from Ljøsne hatchery near Lærdal, western Norway. The salmon was of the river Lærdalselva stock, ranging from 7.7 to 12.0 cm in total fork length (9.4 1.1 cm, mean SD). The fish were transported from the hatchery to the Veterinary Institute of Oslo in oxygenated water. Upon arrival at the fish holding department, the fish were immediately transferred to a holding tank (60 9 60 9 70 cm) and acclimated in laboratory water at 9 °C for 10 days prior to the experiment. The fish were not fed during the experimental period. The experimental fish were naive to gyrodactylids and were prophylactically disinfected (with formalin) against ectoparasites and fungal (oomycete) pathogens in the Ljøsne hatchery at a quarterly basis. Gyrodactylus salaris used in the experiments originated from wild-caught salmon parr from river Lierelva. The parasites were kept on donor fish in separate fish tanks in the Table 1 Water quality parameters of filtered and dechlorinated Oslo city tap water (means SE, n, number of measurements) from water chemistry analyses run at the laboratory at Oslo vann og avløpsetat (VAV) in the period 2 May 2011–26 September 2011
pH Turbidity (FTU) Conductivity (mS m1) Colour Ammonium (mg L1 NH 4N ) Aluminium (mg L1 Al) Alkalinity (mM) 1 NO NO 3 N ðmg L 3 NÞ TOC (mg L1 C) 1 Cl (mg L Cl) Sulphate (mg L1 SO4) 1 NO NO 2 N ðmg L 2 NÞ Tot-P (mg L1 P) 1 Tot-N (mg L N) KOF-Mn (mg L1 O2)
AVG
SE
n
7.77 0.11 12.00 4.10 0.01 0.03 0.75 0.25 2.00 9.74 2.87 0.05). Together, the significant increase in the control group and the Ó 2014 John Wiley & Sons Ltd
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Clmedium
Clhigh
1210 553 3 4 0
1221 35 0 0
(18/18) (18/18) (2/18) (2/18) (0/18)
(18/18) (12/18) (0/18) (0/18)
lack of significant increase in the Cllow group reveal a significant effect of the treatment on the parasite population development in the latter. In
Journal of Fish Diseases 2014, 37, 1003–1011
A G Hagen et al. Chlorine eradicate G. salaris
No fish mortality was observed in any group the first 5 days of the experiment. The onset of mortality was day 6 for the groups Cllow and Clmedium and day 10 in the Clhigh and the control group. In total, 7 fish in the control group, 4 fish in the Cllow group, 3 fish in the Clmedium group and 13 fish in the Clhigh group died (Fig. 5). There were no observations of clinical signs that could reveal the cause of death. The mortality in the groups Cllow and Clmedium was lower than in the control group. This indicates that Cl was at least not the single cause of the mortality in these groups. The mortality in the Clhigh group was higher than in the other groups, indicating that Cl concentrations of 50 lg L1 have adverse effects on the fish in this experiment. Figure 4 Relative (%) temporal development in total number of Gyrodactylus salaris pooled from individual fish exposed to charcoal filtered tap water (control) (solid line); 0–5 lg Cl L1 added (dotted line); 18 lg Cl L1 added (fine broken line); 50 lg Cl L1 added (broken/dotted line). Sums based on n = 18 fish in all groups.
the groups Clhigh and Clmedium, the infection was eliminated between days 2 and 4 and between days 6 and 8, respectively. An important note to this matter, however, is that the G. salaris specimens observed at day 2 in Clhigh and at day 6 in Clmedium were all considered dead by subjective judgement.
Figure 5 Temporal development in mortality of experimental fish exposed to charcoal filtered tap water (control; solid line); 0–5 lg Cl L1 added (dotted line); 18 lg Cl L1 added (fine broken line); 50 lg Cl L1 added (broken/dotted line). Ó 2014 John Wiley & Sons Ltd
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Discussion
The results from the present study demonstrate a dose–response relationship of hypochlorite on development of infections with G. salaris. The traditional general trajectory of infection development for G. salaris parasitising on Norwegian Atlantic salmon is exponential (Bakke, Jansen & Hansen 1990; Bakke et al. 2002). The control group, with Atlantic salmon from river Lærdalselva, infected with the river Lierelva strain of G. salaris (haplotype F according to Hansen, Bachmann & Bakke 2003) displayed a developing number of parasites similar to what has previously been reported to be the general trend. When compared to the control group, there was a significant effect from the chlorine enrichment in all three experimental groups. At the lowest added concentration of hypochlorite (Cllow), the effect was basically a prevention of population growth. This concentration was not sufficient to reduce the infection within the experimental period. In the Clmedium exposure group, there was a reduction to extinction of the infection within 8 days, whereas in the Clhigh exposure group, the infection was removed within 4 days of exposure. Due to the 2-day interval in monitoring, 8 and 4 days represent overestimates of time until total removal of the parasites: hence, maximum survival at the different hypochlorite treatments is within 6–8 days and 2–4 for Clmedium and Clhigh, respectively. The observed effects on G. salaris infrapopulations development do not give conclusive indications as to the causal mechanism. The alterations of trajectory could be explained by parasite
Journal of Fish Diseases 2014, 37, 1003–1011
mortality, by the parasites leaving their hosts or by a combination of the two. A third alternative, reduction in reproductive rate is not likely: according to Jansen & Bakke (1991), G. salaris have an expected life span exceeding 20 days at 9–10 °C. Thus, a reduction in reproductive rate can be excluded as an explanation for the elimination of infections in Clmedium and Clhigh. Chlorine is a powerful oxidizing agent with a broad spectre of toxic actions due to the highly reactive oxidative property of the chemical. It has a number of biochemical targets in cells, with the potential effect of inhibiting membrane transports as well as damaging respiratory enzymes (e.g. reviewed by Singleton & Bio 1989). However, the exact mechanism by which it is toxic to monogeneans is not fully understood. Furthermore, the physiological processes of respiration and membrane ion transport in G. salaris have not been studied. The animal is quite simple, both physiologically and anatomically, and it is reason to believe that gas exchange and ion transport take place over the entire body surface, as tegumental diffusion and/or active uptake. Hence, a powerful oxidizing chemical such as chlorine is likely to have a considerable potential to inflict injury to the G. salaris specimens on a systemic level. Considering this, we suggest parasite mortality to be the cause of the altered infection trajectory observed when exposing G. salaris to chlorine. Freshwater fish are considered sensitive to hypochlorite. The reported 96-h LC50 levels, however, are varying widely from 10 to 132 lg L1 among salmonids (Singleton & Bio 1989). According to the same authors, salmonids are the most sensitive group, with an average 96-h LC50 of 70 lg L1. In contrast, average 96-h LC50 for cyprinids is reported to be 128 lg L1. The effects reported on G. salaris in our study, however, is due to Cl concentrations lower than many of the levels reported as lethal to fish (Singleton & Bio 1989). Nevertheless, 13 fish that were exposed to Clhigh water died during the later phase of the experiment. Fish mortality was observed from day 7 in the experiment for the groups Cllow and Clmedium. In the control group and Clhigh, mortality was observed from day 10. The populations of G. salaris were eliminated at days 8 and 6 for the groups Clmedium and Clhigh, respectively. The similar mortality pattern observed between the control group with no addition of Cl and the groups Ó 2014 John Wiley & Sons Ltd
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A G Hagen et al. Chlorine eradicate G. salaris
Cllow/Clmedium indicates that the mortality in the latter groups was not related to the exposure towards Cl. These findings imply that there is a therapeutic margin when eliminating G. salaris from Atlantic salmon, using sodium hypochlorite. Implications of the findings The main characteristics of our findings are the effects on G. salaris from prolonged exposure to low concentrations of chlorine, combined with survival of the fish host. The low-concentration sensitivity of G. salaris towards Cl has to our knowledge not earlier been reported. The only previously published work known to the authors is the one by Lewis & Ulrich (1967), exploring quick-dip treatment with chlorine for control of gyrodactylids parasitising golden shiner (N. crysoleucas), fathead minnow, Pimephales promelas (Rafinesque, 1820), and green sunfish, Lepomis cyanellus (Rafinesque, 1819). Lewis & Ulrich (1967) reported that a therapeutic margin between the parasite and the fish does exist. Our findings also support that chlorine may be efficient as a quick-dip in vivo treatment against G. salaris in controlled conditions as described by Lewis & Ulrich (1967). Based on our findings, however, a revised approach can be suggested for gyrodactylid control: a prolonged time of exposure, combined with a low concentration of chlorine. As the free Cl fraction was 10 lg L1 or less in all three treatments in the present experiments, and as the detection limits in the analyses used was 10 lg L1, the exact concentration of this fraction remains unknown. The concentration of bound Cl, herein for example chloramines, also remains unknown. However, taken the amount of ammonia in the water (Table 1), the chloramine fraction cannot be ruled out to have an effect on or contribute to the effect on G. salaris. The difference in sensitivity between the parasite and the host is interesting in the context of hypochlorite as a parasiticide in large-scale treatments in natural river systems. Since the early 1980s, Norwegian authorities have engaged a programme aiming for eradication of the parasite G. salaris from Norwegian territory. The control measures include chemical treatment of full-river systems, aiming for eradication of the parasite. Despite the simple and direct life cycle of G. salaris (for a thorough review on the biology and ecology of G. salaris, see Bakke et al. 2007),
Journal of Fish Diseases 2014, 37, 1003–1011
and despite the fact that Norwegian authorities have spent vast amounts of money in the programme against this parasite, a remedy with a sufficient therapeutic window (parasite sensitivity versus host sensitivity to the chemical) appropriate for treatment of whole-rivers systems has proved hard to find. The main strategy has therefore been to remove all potential hosts by chemical treatment using rotenone (i.e. the commercial product CFT-Legumin; containing 2.5% rotenone; VESO AS). Of 48 rivers to which the parasite has been spread (Linaker et al. 2012), 34 have been treated using rotenone (Miljødirektoratet 2013). Among these, 20 have been declared free of the parasite (Linaker et al. 2012). The remaining 14 were treated within the last 5 years and have not to date been officially declared free (Linaker et al. 2012). Use of rotenone has been questioned due to its effects on non-target species of fish and also gill breathing invertebrates, but is still widely used and recommended as a fish management tool (Finlayson et al. 2005). Only one alternative chemical treatment providing a therapeutic window proven fruitful has been tested in full-river treatments: replicating chemical conditions in the river similar to those resulting from acid precipitation, by continuous dosing of aluminium sulphate (AlS) and sulphuric acid (AlS-treatment). In a range of doses, AlS allows for the removal of parasites and leaving the host unharmed (for further details, see Soleng et al. 1999; Poleo et al. 2004; Soleng, Poleo & Bakke 2005). Exposed to water containing 200 lg Al L1 at pH 6.1, the G. salaris infection was removed in 4–6 days without Al-related fish mortality (Poleo et al. 2004). The chlorine concentration in the present study was 16, 33 and 65 lg Cl L1 for the groups Cllow, Clmedium and Clhigh, respectively. Maximum registered survival of G. salaris at the different Cl-treatments was within 6–8 and 2–4 days for Clmedium and Clhigh, respectively. In the Clmedium group, only 0.3% of the initial parasite population remained at day 6. The results thus indicate that Cl might have at least a similar therapeutic window compared with AlS. Hence, further studies to investigate the suitability of chlorine in large-scale chemical treatments against G. salaris are called for. The low-concentration tolerance of G. salaris to hypochlorite should also have implications in a conservation context other than those concerning Ó 2014 John Wiley & Sons Ltd
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the host. Chlorine in different forms is the most frequently used disinfectant worldwide. It has been used as a disinfectant for drinking water, for treatment of runoff water and sewage, and for treatment against unwanted growth of bacteria and algae in different contexts. The widespread use has led management authorities and scientists to conduct thorough investigations of the effects both individually and on an ecological scale, both from direct exposure to high doses and from exposure to residual chlorine products in run-off waters (Brungs 1973; Larson et al. 1978; Singleton & Bio 1989). At present, about 400 species in the genus Gyrodactylus have been described to science (marine and aquatic), but only a few are considered pathogenic to their hosts (Bakke et al. 2007). Taken the widespread use of Cl worldwide, this is likely to have had consequences, at least as a limiting factor in the population dynamics of these organisms, but also potentially to have eradicated whole subpopulations of gyrodactylids. Acknowledgements The experimental work was funded by the Norwegian Institute for Water Research. Additional funding was provided by the Norwegian Veterinary Institute, and the Norwegian Institute for Nature Research. We thank Oslo vann og avløpsetat (VAV) for providing analyses of water samples. References Bakke T.A., Jansen P.A. & Hansen L.P. (1990) Differences in the host-resistance of Atlantic salmon, Salmo salar L, stocks to the monogenean Gyrodactylus salaris Malmberg, 1957. Journal of Fish Biology 37, 577–587. Bakke T.A., Harris P.D. & Cable J. (2002) Host specificity dynamics: observations on gyrodactylid monogeneans. International Journal for Parasitology 32, 281–308. Bakke T.A., Cable J. & Harris P.D. (2007) The biology of gyrodactylid monogeneans: the “Russian-doll killers”. Advances in Parasitology 64, 161–376. Brungs W.A. (1973) Effects of residual chlorine on aquatic life. Journal (Water Pollution Control Federation) 45, 2180–2193. Finlayson B., Somer W., Duffield D., Propst D., Mellison C., Pettengill T., Sexauer H., Nesler T., Gurtin S., Elliot J., Partridge F. & Skaar D. (2005) Native inland trout restoration on national forests in the western United States: time for improvement? Fisheries 30, 10–19. Hammer Ø., Harper D.A.T. & Ryan P.D. (2001) PAST: paleontological Statistics Software Package for Education and Data Analysis. Palaeontologia Electronica 4, 9 pp.
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Hansen H., Bachmann L. & Bakke T.A. (2003) Mitochondrial DNA variation of Gyrodactylus spp. (Monogenea, Gyrodactylidae) populations infecting Atlantic salmon, grayling and rainbow trout in Norway and Sweden. International Journal for Parasitology 33, 1471–1478. Harris P.D., Shinn A.P., Cable J. & Bakke T.A. (2004) Nominal species of the genus Gyrodactylus vonNordmann 1832 (Monogenea: Gyrodactylidae), with a list of principal host species. Systematic Parasitology 59, 1–27. Jansen P.A. & Bakke T.A. (1991) Temperature-dependent reproduction and survival of Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes – Monogenea) on Atlantic salmon (Salmo salar L.). Parasitology 102, 105–112. Johnsen B.O., Møkkelgjerd P.I. & Jensen A.J. (1999) The parasite Gyrodactylus salaris on salmon parr in Norwegian rivers, status report at the beginning of year 2000. NINA Oppdragsmelding 617, 1–129. Larson G.L., Warren C.E., Hutchins F.E., Lamperti L.P., Schlesinger D.A. & Seim W.K. (1978) Toxicity of Residual Chlorine Compounds to Aquatic Organisms. United States Environmental Protection Agency, Ecological Research Series EPA-600/3/78/023, 105 pp. Lewis W.M. & Ulrich M.G. (1967) Chlorine as a quick-dip treatment for the control of gyrodactylids on the golden shiner. The Progressive Fish-Culturist 29, 229–231. Linaker M.L., Hansen H., Mo T.A. & Jensen B.B. (2012) The surveillance and control programme for Gyrodactylus salaris in Atlantic salmon and rainbow trout in Norway 2012. Surveillance and control programmes for terrestrial and aquatic animals in Norway. Annual report 2012. Norwegian Veterinary Institute, 7 pp. Miljødirektoratet (2013) Regioner infisert av Gyrodactylus salaris [online]. Available at: http://www.miljodirektoratet. no/no/Tema/Arter-og-naturtyper/Villaksportalen/
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Hva-pavirker-laksefiskene/Gyrodactylus-salaris/ Regioner-infisert-av-Gyrodactylus-salaris/ [Accessed 28 October 2013]. Peeler E., Thrush M., Paisley L. & Rodgers C. (2006) An assessment of the risk of spreading the fish parasite Gyrodactylus salaris to uninfected territories in the European Union with the movement of live Atlantic salmon (Salmo salar) from coastal waters. Aquaculture 258, 187–197. Poleo A.B.S., Schjolden J., Hansen H., Bakke T.A., Mo T.A., Rosseland B.O. & Lydersen E. (2004) The effect of various metals on Gyrodactylus salaris (Platyhelminthes, Monogenea) infections in Atlantic salmon (Salmo salar). Parasitology 128, 169–177. Singleton H.J. & Bio R.P. (1989) Ambient water quality criteria for chlorine. Technical appendix. Resource Quality Section, Water Management Branch, Ministry of Environment, Province of British Columbia, Victoria, Canada. Soleng A., Poleo A.B.S., Alstad N.E.W. & Bakke T.A. (1999) Aqueous aluminum eliminates Gyrodactylus salaris (Platyhelminthes, Monogenea) infections in Atlantic salmon. Parasitology 119, 19–25. Soleng A., Poleo A.B.S. & Bakke T.A. (2005) Toxicity of aqueous aluminium to the ectoparasitic monogenean Gyrodactylus salaris. Aquaculture 250, 616–620. World Health Organization (2006) Guidelines for DrinkingWater Quality, Vol. 1, Recommendations, 3rd edn. World Health Organization. Available at: http://www.who.int/ water_sanitation_health/dwq/gdwq0506.pdf [Accessed 11 November 2013]. Received: 19 September 2013 Revision received: 15 November 2013 Accepted: 16 November 2013
Journal of Fish Diseases 2014, 37, 1003–1011
Low concentrations of sodium hypochlorite affect population dynamics in Gyrodactylus salaris (Malmberg, 1957): practical guidelines for the treatment of the Atlantic salmon, Salmo salar L. parasite A G Hagen1, S Hytterød2 and K Olstad3 1 Norwegian Institute for Water Research, Oslo, Norway 2 Norwegian Veterinary Institute, Oslo, Norway 3 Norwegian Institute for Nature Research, Lillehammer, Norway
Abstract
Introduction
Atlantic salmon, Salmo salar L. parr (age 1+), infected by the monogenean ectoparasite Gyrodactylus salaris (Malmberg, 1957), were exposed to chlorine (Cl)-enriched water at three different concentrations: Cllow (0–5 lg Cl L1), Clmedium (18 lg Cl L1) and Clhigh (50 lg Cl L1). There was a negative correlation between G. salaris infections and the hypochlorite concentrations added. The parasite infection was eliminated by day 6–8 and day 2–4 in the groups Clmedium and Clhigh, respectively, while inhibition of G. salaris population growth was observed in the Cllow group. An important note to this matter, however, is that the G. salaris specimens observed at day 6 in Clmedium and at day 2 in Clhigh were all considered dead by subjective judgement. No mortality in the salmon parr was observed during the first 8 days of the experiment, demonstrating that Cl has a stronger effect on G. salaris than on the salmonid host. The differences in sensitivity between the parasite and the Atlantic salmon indicate that hypochlorite has a potential use as a parasiticide with a therapeutic margin. The low-dose sensitivity may imply that Cl pollution in urban areas may pose a greater risk towards biodiversity than previously assumed.
Species of the monogenean genus Gyrodactylus von Nordmann, 1832 are principally ectoparasites of fish. Currently, some 400 Gyrodactylus species have been described (Harris et al. 2004). A straightforward extrapolation of the total number of teleost fish worldwide, however, suggests that the total number of species in this group may be some 20 000 (Bakke, Cable & Harris 2007). Some cause severe gyrodactylosis, such as Gyrodactylus salaris Malmberg, 1957, which has had a devastating effect on East Atlantic salmon (Salmo salar L.) populations since the accidental introduction to Norway during the 1970s (Johnsen, Møkkelgjerd & Jensen 1999; see also reviews by Bakke, Harris & Cable 2002; Bakke et al. 2007). Gyrodactylus salaris thus is considered to constitute one of the main threats towards wild European Atlantic salmon populations, and it is listed as a ‘notifiable disease’ by the World Organization for Animal Health (OIE) in the Aquatic Animal Health Code (Chapter 10.3). Several countries have taken precautionary measures to avoid the introduction of the parasite. Accordingly, the European Commission (EC) has restricted the import of salmonids that are susceptible to G. salaris to areas with an equivalent health status (Peeler et al. 2006). Norwegian management authorities spend vast amounts every year on control measures in rivers and fish hatcheries in attempts to eliminate the parasite and to prevent further spread (Bakke et al. 2007).
Keywords: biodiversity, disinfection, Monogenea, parasite, treatment. Correspondence A G Hagen, Norwegian Institute for Water Research, Gaustadalle en 21, NO-0349 Oslo, Norway (e-mail: [email protected]) Ó 2014 John Wiley & Sons Ltd
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Journal of Fish Diseases 2014, 37, 1003–1011
Chlorine in different forms is the most frequently used disinfectant worldwide. It is used as a disinfectant for drinking water, for treatment of runoff water and sewage, and for treatment against unwanted growth of bacteria and algae in different contexts. It is well established that chlorine kills waterborne pathogens (World Health Organization 2006). Mostly, this has been taken into account in the numerous aquarium-based Gyrodactylus studies that have been conducted: as a precautionary measure, the water used in the experimental tanks in many fish holding departments is thus routinely dechlorinated. The quantitative sensitivity of these parasites, however, is not known. The only published work known to the authors on the sensitivity of Gyrodactylus to hypochlorite is the study by Lewis & Ulrich (1967), exploring quick-dip treatment with chlorine for control of gyrodactylids parasitizing Golden shiner, Notemigonus crysoleucas (Mitchill, 1814). Dissolved in freshwater, hypochlorite will make a hypochloric acid equilibrium. The fractions in this equilibrium (HOCl and OCl), together with Cl2 and ClO2, constitute what is frequently annotated free chlorine. If the water contains ammonia (NH3), chloramines (mono-, di- and tri-) will be formed. Chloramines constitute the major part of the fraction frequently annotated bound chlorine. Together, these two fractions constitute total residual chlorine (TRC). A pilot-experiment exposing G. salaris-infected Atlantic salmon to dechlorinated water (using sodium thiosulphate) and to non-dechlorinated water gave marked differences in G. salaris population growth between the two groups (Hytterød & Olstad, unpublished data). No negative effects were observed on the Atlantic salmon in this experiment. Accordingly, based on these findings, it was hypothesized that G. salaris has a sensitivity towards hypochlorite such that typical concentrations of free chlorine found in chlorinated Oslo tap water can remove the infection within days. The current experimental approach set out to investigate the dose dependency in hypochlorite toxicity towards G. salaris. Materials and methods
The experiments were performed at the fish holding department at The Norwegian Veterinary Institute in Oslo. The department source of water is the Oslo city tap water. Before being distributed Ó 2014 John Wiley & Sons Ltd
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A G Hagen et al. Chlorine eradicate G. salaris
to the fish holding tanks, the water is run through a particle filter (Hydrex II, model GX05-20, length 20″, 5 lm polypropylene) and an activated charcoal filter (20 lm/20″ – GAC-20BB, granular activated carbon filter cartridge). A summary of chemical characteristics of the filtered and dechlorinated tap water is given in Table 1. Experimental animals The animal work presented was approved by the Norwegian Animal Research Authority (NARA; no. 3473/2011). Atlantic salmon parr (age 1+) were obtained from Ljøsne hatchery near Lærdal, western Norway. The salmon was of the river Lærdalselva stock, ranging from 7.7 to 12.0 cm in total fork length (9.4 1.1 cm, mean SD). The fish were transported from the hatchery to the Veterinary Institute of Oslo in oxygenated water. Upon arrival at the fish holding department, the fish were immediately transferred to a holding tank (60 9 60 9 70 cm) and acclimated in laboratory water at 9 °C for 10 days prior to the experiment. The fish were not fed during the experimental period. The experimental fish were naive to gyrodactylids and were prophylactically disinfected (with formalin) against ectoparasites and fungal (oomycete) pathogens in the Ljøsne hatchery at a quarterly basis. Gyrodactylus salaris used in the experiments originated from wild-caught salmon parr from river Lierelva. The parasites were kept on donor fish in separate fish tanks in the Table 1 Water quality parameters of filtered and dechlorinated Oslo city tap water (means SE, n, number of measurements) from water chemistry analyses run at the laboratory at Oslo vann og avløpsetat (VAV) in the period 2 May 2011–26 September 2011
pH Turbidity (FTU) Conductivity (mS m1) Colour Ammonium (mg L1 NH 4N ) Aluminium (mg L1 Al) Alkalinity (mM) 1 NO NO 3 N ðmg L 3 NÞ TOC (mg L1 C) 1 Cl (mg L Cl) Sulphate (mg L1 SO4) 1 NO NO 2 N ðmg L 2 NÞ Tot-P (mg L1 P) 1 Tot-N (mg L N) KOF-Mn (mg L1 O2)
AVG
SE
n
7.77 0.11 12.00 4.10 0.01 0.03 0.75 0.25 2.00 9.74 2.87 0.05). Together, the significant increase in the control group and the Ó 2014 John Wiley & Sons Ltd
1007
Clmedium
Clhigh
1210 553 3 4 0
1221 35 0 0
(18/18) (18/18) (2/18) (2/18) (0/18)
(18/18) (12/18) (0/18) (0/18)
lack of significant increase in the Cllow group reveal a significant effect of the treatment on the parasite population development in the latter. In
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A G Hagen et al. Chlorine eradicate G. salaris
No fish mortality was observed in any group the first 5 days of the experiment. The onset of mortality was day 6 for the groups Cllow and Clmedium and day 10 in the Clhigh and the control group. In total, 7 fish in the control group, 4 fish in the Cllow group, 3 fish in the Clmedium group and 13 fish in the Clhigh group died (Fig. 5). There were no observations of clinical signs that could reveal the cause of death. The mortality in the groups Cllow and Clmedium was lower than in the control group. This indicates that Cl was at least not the single cause of the mortality in these groups. The mortality in the Clhigh group was higher than in the other groups, indicating that Cl concentrations of 50 lg L1 have adverse effects on the fish in this experiment. Figure 4 Relative (%) temporal development in total number of Gyrodactylus salaris pooled from individual fish exposed to charcoal filtered tap water (control) (solid line); 0–5 lg Cl L1 added (dotted line); 18 lg Cl L1 added (fine broken line); 50 lg Cl L1 added (broken/dotted line). Sums based on n = 18 fish in all groups.
the groups Clhigh and Clmedium, the infection was eliminated between days 2 and 4 and between days 6 and 8, respectively. An important note to this matter, however, is that the G. salaris specimens observed at day 2 in Clhigh and at day 6 in Clmedium were all considered dead by subjective judgement.
Figure 5 Temporal development in mortality of experimental fish exposed to charcoal filtered tap water (control; solid line); 0–5 lg Cl L1 added (dotted line); 18 lg Cl L1 added (fine broken line); 50 lg Cl L1 added (broken/dotted line). Ó 2014 John Wiley & Sons Ltd
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Discussion
The results from the present study demonstrate a dose–response relationship of hypochlorite on development of infections with G. salaris. The traditional general trajectory of infection development for G. salaris parasitising on Norwegian Atlantic salmon is exponential (Bakke, Jansen & Hansen 1990; Bakke et al. 2002). The control group, with Atlantic salmon from river Lærdalselva, infected with the river Lierelva strain of G. salaris (haplotype F according to Hansen, Bachmann & Bakke 2003) displayed a developing number of parasites similar to what has previously been reported to be the general trend. When compared to the control group, there was a significant effect from the chlorine enrichment in all three experimental groups. At the lowest added concentration of hypochlorite (Cllow), the effect was basically a prevention of population growth. This concentration was not sufficient to reduce the infection within the experimental period. In the Clmedium exposure group, there was a reduction to extinction of the infection within 8 days, whereas in the Clhigh exposure group, the infection was removed within 4 days of exposure. Due to the 2-day interval in monitoring, 8 and 4 days represent overestimates of time until total removal of the parasites: hence, maximum survival at the different hypochlorite treatments is within 6–8 days and 2–4 for Clmedium and Clhigh, respectively. The observed effects on G. salaris infrapopulations development do not give conclusive indications as to the causal mechanism. The alterations of trajectory could be explained by parasite
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mortality, by the parasites leaving their hosts or by a combination of the two. A third alternative, reduction in reproductive rate is not likely: according to Jansen & Bakke (1991), G. salaris have an expected life span exceeding 20 days at 9–10 °C. Thus, a reduction in reproductive rate can be excluded as an explanation for the elimination of infections in Clmedium and Clhigh. Chlorine is a powerful oxidizing agent with a broad spectre of toxic actions due to the highly reactive oxidative property of the chemical. It has a number of biochemical targets in cells, with the potential effect of inhibiting membrane transports as well as damaging respiratory enzymes (e.g. reviewed by Singleton & Bio 1989). However, the exact mechanism by which it is toxic to monogeneans is not fully understood. Furthermore, the physiological processes of respiration and membrane ion transport in G. salaris have not been studied. The animal is quite simple, both physiologically and anatomically, and it is reason to believe that gas exchange and ion transport take place over the entire body surface, as tegumental diffusion and/or active uptake. Hence, a powerful oxidizing chemical such as chlorine is likely to have a considerable potential to inflict injury to the G. salaris specimens on a systemic level. Considering this, we suggest parasite mortality to be the cause of the altered infection trajectory observed when exposing G. salaris to chlorine. Freshwater fish are considered sensitive to hypochlorite. The reported 96-h LC50 levels, however, are varying widely from 10 to 132 lg L1 among salmonids (Singleton & Bio 1989). According to the same authors, salmonids are the most sensitive group, with an average 96-h LC50 of 70 lg L1. In contrast, average 96-h LC50 for cyprinids is reported to be 128 lg L1. The effects reported on G. salaris in our study, however, is due to Cl concentrations lower than many of the levels reported as lethal to fish (Singleton & Bio 1989). Nevertheless, 13 fish that were exposed to Clhigh water died during the later phase of the experiment. Fish mortality was observed from day 7 in the experiment for the groups Cllow and Clmedium. In the control group and Clhigh, mortality was observed from day 10. The populations of G. salaris were eliminated at days 8 and 6 for the groups Clmedium and Clhigh, respectively. The similar mortality pattern observed between the control group with no addition of Cl and the groups Ó 2014 John Wiley & Sons Ltd
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Cllow/Clmedium indicates that the mortality in the latter groups was not related to the exposure towards Cl. These findings imply that there is a therapeutic margin when eliminating G. salaris from Atlantic salmon, using sodium hypochlorite. Implications of the findings The main characteristics of our findings are the effects on G. salaris from prolonged exposure to low concentrations of chlorine, combined with survival of the fish host. The low-concentration sensitivity of G. salaris towards Cl has to our knowledge not earlier been reported. The only previously published work known to the authors is the one by Lewis & Ulrich (1967), exploring quick-dip treatment with chlorine for control of gyrodactylids parasitising golden shiner (N. crysoleucas), fathead minnow, Pimephales promelas (Rafinesque, 1820), and green sunfish, Lepomis cyanellus (Rafinesque, 1819). Lewis & Ulrich (1967) reported that a therapeutic margin between the parasite and the fish does exist. Our findings also support that chlorine may be efficient as a quick-dip in vivo treatment against G. salaris in controlled conditions as described by Lewis & Ulrich (1967). Based on our findings, however, a revised approach can be suggested for gyrodactylid control: a prolonged time of exposure, combined with a low concentration of chlorine. As the free Cl fraction was 10 lg L1 or less in all three treatments in the present experiments, and as the detection limits in the analyses used was 10 lg L1, the exact concentration of this fraction remains unknown. The concentration of bound Cl, herein for example chloramines, also remains unknown. However, taken the amount of ammonia in the water (Table 1), the chloramine fraction cannot be ruled out to have an effect on or contribute to the effect on G. salaris. The difference in sensitivity between the parasite and the host is interesting in the context of hypochlorite as a parasiticide in large-scale treatments in natural river systems. Since the early 1980s, Norwegian authorities have engaged a programme aiming for eradication of the parasite G. salaris from Norwegian territory. The control measures include chemical treatment of full-river systems, aiming for eradication of the parasite. Despite the simple and direct life cycle of G. salaris (for a thorough review on the biology and ecology of G. salaris, see Bakke et al. 2007),
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and despite the fact that Norwegian authorities have spent vast amounts of money in the programme against this parasite, a remedy with a sufficient therapeutic window (parasite sensitivity versus host sensitivity to the chemical) appropriate for treatment of whole-rivers systems has proved hard to find. The main strategy has therefore been to remove all potential hosts by chemical treatment using rotenone (i.e. the commercial product CFT-Legumin; containing 2.5% rotenone; VESO AS). Of 48 rivers to which the parasite has been spread (Linaker et al. 2012), 34 have been treated using rotenone (Miljødirektoratet 2013). Among these, 20 have been declared free of the parasite (Linaker et al. 2012). The remaining 14 were treated within the last 5 years and have not to date been officially declared free (Linaker et al. 2012). Use of rotenone has been questioned due to its effects on non-target species of fish and also gill breathing invertebrates, but is still widely used and recommended as a fish management tool (Finlayson et al. 2005). Only one alternative chemical treatment providing a therapeutic window proven fruitful has been tested in full-river treatments: replicating chemical conditions in the river similar to those resulting from acid precipitation, by continuous dosing of aluminium sulphate (AlS) and sulphuric acid (AlS-treatment). In a range of doses, AlS allows for the removal of parasites and leaving the host unharmed (for further details, see Soleng et al. 1999; Poleo et al. 2004; Soleng, Poleo & Bakke 2005). Exposed to water containing 200 lg Al L1 at pH 6.1, the G. salaris infection was removed in 4–6 days without Al-related fish mortality (Poleo et al. 2004). The chlorine concentration in the present study was 16, 33 and 65 lg Cl L1 for the groups Cllow, Clmedium and Clhigh, respectively. Maximum registered survival of G. salaris at the different Cl-treatments was within 6–8 and 2–4 days for Clmedium and Clhigh, respectively. In the Clmedium group, only 0.3% of the initial parasite population remained at day 6. The results thus indicate that Cl might have at least a similar therapeutic window compared with AlS. Hence, further studies to investigate the suitability of chlorine in large-scale chemical treatments against G. salaris are called for. The low-concentration tolerance of G. salaris to hypochlorite should also have implications in a conservation context other than those concerning Ó 2014 John Wiley & Sons Ltd
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the host. Chlorine in different forms is the most frequently used disinfectant worldwide. It has been used as a disinfectant for drinking water, for treatment of runoff water and sewage, and for treatment against unwanted growth of bacteria and algae in different contexts. The widespread use has led management authorities and scientists to conduct thorough investigations of the effects both individually and on an ecological scale, both from direct exposure to high doses and from exposure to residual chlorine products in run-off waters (Brungs 1973; Larson et al. 1978; Singleton & Bio 1989). At present, about 400 species in the genus Gyrodactylus have been described to science (marine and aquatic), but only a few are considered pathogenic to their hosts (Bakke et al. 2007). Taken the widespread use of Cl worldwide, this is likely to have had consequences, at least as a limiting factor in the population dynamics of these organisms, but also potentially to have eradicated whole subpopulations of gyrodactylids. Acknowledgements The experimental work was funded by the Norwegian Institute for Water Research. Additional funding was provided by the Norwegian Veterinary Institute, and the Norwegian Institute for Nature Research. We thank Oslo vann og avløpsetat (VAV) for providing analyses of water samples. References Bakke T.A., Jansen P.A. & Hansen L.P. (1990) Differences in the host-resistance of Atlantic salmon, Salmo salar L, stocks to the monogenean Gyrodactylus salaris Malmberg, 1957. Journal of Fish Biology 37, 577–587. Bakke T.A., Harris P.D. & Cable J. (2002) Host specificity dynamics: observations on gyrodactylid monogeneans. International Journal for Parasitology 32, 281–308. Bakke T.A., Cable J. & Harris P.D. (2007) The biology of gyrodactylid monogeneans: the “Russian-doll killers”. Advances in Parasitology 64, 161–376. Brungs W.A. (1973) Effects of residual chlorine on aquatic life. Journal (Water Pollution Control Federation) 45, 2180–2193. Finlayson B., Somer W., Duffield D., Propst D., Mellison C., Pettengill T., Sexauer H., Nesler T., Gurtin S., Elliot J., Partridge F. & Skaar D. (2005) Native inland trout restoration on national forests in the western United States: time for improvement? Fisheries 30, 10–19. Hammer Ø., Harper D.A.T. & Ryan P.D. (2001) PAST: paleontological Statistics Software Package for Education and Data Analysis. Palaeontologia Electronica 4, 9 pp.
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Hansen H., Bachmann L. & Bakke T.A. (2003) Mitochondrial DNA variation of Gyrodactylus spp. (Monogenea, Gyrodactylidae) populations infecting Atlantic salmon, grayling and rainbow trout in Norway and Sweden. International Journal for Parasitology 33, 1471–1478. Harris P.D., Shinn A.P., Cable J. & Bakke T.A. (2004) Nominal species of the genus Gyrodactylus vonNordmann 1832 (Monogenea: Gyrodactylidae), with a list of principal host species. Systematic Parasitology 59, 1–27. Jansen P.A. & Bakke T.A. (1991) Temperature-dependent reproduction and survival of Gyrodactylus salaris Malmberg, 1957 (Platyhelminthes – Monogenea) on Atlantic salmon (Salmo salar L.). Parasitology 102, 105–112. Johnsen B.O., Møkkelgjerd P.I. & Jensen A.J. (1999) The parasite Gyrodactylus salaris on salmon parr in Norwegian rivers, status report at the beginning of year 2000. NINA Oppdragsmelding 617, 1–129. Larson G.L., Warren C.E., Hutchins F.E., Lamperti L.P., Schlesinger D.A. & Seim W.K. (1978) Toxicity of Residual Chlorine Compounds to Aquatic Organisms. United States Environmental Protection Agency, Ecological Research Series EPA-600/3/78/023, 105 pp. Lewis W.M. & Ulrich M.G. (1967) Chlorine as a quick-dip treatment for the control of gyrodactylids on the golden shiner. The Progressive Fish-Culturist 29, 229–231. Linaker M.L., Hansen H., Mo T.A. & Jensen B.B. (2012) The surveillance and control programme for Gyrodactylus salaris in Atlantic salmon and rainbow trout in Norway 2012. Surveillance and control programmes for terrestrial and aquatic animals in Norway. Annual report 2012. Norwegian Veterinary Institute, 7 pp. Miljødirektoratet (2013) Regioner infisert av Gyrodactylus salaris [online]. Available at: http://www.miljodirektoratet. no/no/Tema/Arter-og-naturtyper/Villaksportalen/
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Hva-pavirker-laksefiskene/Gyrodactylus-salaris/ Regioner-infisert-av-Gyrodactylus-salaris/ [Accessed 28 October 2013]. Peeler E., Thrush M., Paisley L. & Rodgers C. (2006) An assessment of the risk of spreading the fish parasite Gyrodactylus salaris to uninfected territories in the European Union with the movement of live Atlantic salmon (Salmo salar) from coastal waters. Aquaculture 258, 187–197. Poleo A.B.S., Schjolden J., Hansen H., Bakke T.A., Mo T.A., Rosseland B.O. & Lydersen E. (2004) The effect of various metals on Gyrodactylus salaris (Platyhelminthes, Monogenea) infections in Atlantic salmon (Salmo salar). Parasitology 128, 169–177. Singleton H.J. & Bio R.P. (1989) Ambient water quality criteria for chlorine. Technical appendix. Resource Quality Section, Water Management Branch, Ministry of Environment, Province of British Columbia, Victoria, Canada. Soleng A., Poleo A.B.S., Alstad N.E.W. & Bakke T.A. (1999) Aqueous aluminum eliminates Gyrodactylus salaris (Platyhelminthes, Monogenea) infections in Atlantic salmon. Parasitology 119, 19–25. Soleng A., Poleo A.B.S. & Bakke T.A. (2005) Toxicity of aqueous aluminium to the ectoparasitic monogenean Gyrodactylus salaris. Aquaculture 250, 616–620. World Health Organization (2006) Guidelines for DrinkingWater Quality, Vol. 1, Recommendations, 3rd edn. World Health Organization. Available at: http://www.who.int/ water_sanitation_health/dwq/gdwq0506.pdf [Accessed 11 November 2013]. Received: 19 September 2013 Revision received: 15 November 2013 Accepted: 16 November 2013
