Effects of the sea lice bath treatment pharmaceuticals hydrogen peroxide, azamethiphos and deltamethrin on egg-carrying shrimp (Pandalus borealis)
Graphical abstract
Introduction
The continued growth of the aquaculture industry in Norway has led to environmental and production challenges, and one of the major challenges is related to the ectoparasitic salmon louse (Lepeophtheirus salmonis) (Torrissen et al., 2013). A method to control sea lice within farm cages is treatment by various pharmaceutical delousing agents (Lillicrap et al., 2015). In Norway, the delousing agents are used either as bath treatments or in-feed drugs. The four bath treatments used are cypermethrin, deltamethrin (DEL), azamethiphos (AZA) or hydrogen peroxide (H2O2). Diflubenzuron, teflubenzuron and emamectin benzoate are used as in-feed drugs. Due to development of resistance in sea lice and implementation of alternative methods for lice control (mechanical and biological), the use of pharmaceuticals has decreased since 2015. The dynamic arms race of controlling sea lice levels in salmon aquaculture has seen some regional differences in bath pharmaceutical treatment regimes, and in many production areas (e.g. Northern-Norway) the total amounts of bath treatments applied is still high (Remen and Sæther, 2018). Due to resistance challenges (sea lice developing resistance towards the most commonly used delousing pharmaceuticals) different sequential combinations of bath treatment pharmaceuticals are also being used to improve treatment efficiency.
Delousing agents used against sea lice are designed to be toxic to a crustacean parasite. Although the delousing agents are approved for use in aquaculture, the large amounts of medicated feed and the large volumes of bath pharmaceuticals used have raised concerns over the survival and wellbeing of populations of non-target crustaceans, which represent key elements of many marine food webs (Langford et al., 2014). In Norway, there is a strong focus on deep-water shrimp (Pandalus borealis), which is one of the most important commercial crustacean species. After a bath treatment of salmon with delousing agents added to the water, in the cage, or in a well-boat, the treatment water is discharged to the surrounding environment as long as the discharge point is at least 500 m away from any registered shrimp ground or fish spawning ground. Hydrodynamic dispersion modelling indicates that 100–1000 times diluted concentrations of the pharmaceuticals can be present more than one km away from the discharge point (Ernst et al., 2001, 2014; Page et al., 2014; Refseth et al., 2016).
H2O2 is considered the most environmentally friendly pharmaceutical used for sea lice control because it rapidly breaks down to oxygen and water. Large volumes of this pharmaceutical have therefore been used by the Norwegian aquaculture industry (ca. 43 000 metric tonnes in 2015, ca. 9000 metric tonnes in 2017, Remen and Sæther, 2018). However, recent research has shown that H2O2 can stay long enough in the environment in areas from 0 to 1 km from the release site to induce mortality in shrimp after the treatment water is released (Refseth et al., 2016). As H2O2 is heavier than seawater it will sink, and when there is no stratification of the water column (e.g. during winter) it may reach the seabed a few minutes after release. Sinking of H2O2 can coincide with the time of year when deep-water shrimp carry eggs. It is known that H2O2 affects reproduction in sea lice by reducing hatching success and development of early life stages (McAndrew et al., 1998; Toovey and Lyndon, 2000; Aaen et al., 2014), and there is also concern that embryo development and hatching success of deep-water shrimp may be similarly impacted. There is limited knowledge of the effects of H2O2 on egg-bearing shrimp or on eggs and embryos. It has been demonstrated that H2O2 can induce sub-lethal effects in non-target species, e.g. through the production of reactive oxygen species, which can induce DNA damage, including base oxidation, and DNA strand breaks (El-Bibany et al., 2014; Valavanidis et al., 2006; Azqueta et al., 2009). Maintenance of DNA integrity is essential for proper cell and organismal function, and prevention of disease and mutations (Reinardy and Bodnar, 2015; Wurgler and Kramers, 1992). Unrepaired DNA damage may also be transferred to offspring via affected parents and lead to long-term effects in populations (Barber et al., 2006; Jha, 2004).
Bath treatments such as azamethiphos (AZA; trade name Salmosan/Azasure) and deltamethrin (DEL; trade name AlphaMax), can also potentially impact survival and the reproduction of shrimp. DEL is a pyrethroid and acts on nerve transmission by interfering with sodium channels (Miller and Adams, 1982), which results in the depolarization of motor neurons and repetitive discharges at nerve endings, leading to eventual paralysis and death (Crane et al., 2011; Haya et al., 2005). It has a low water solubility, and the half-life of DEL in the water column is 2–4 h (Muir et al., 1985). Laboratory and field studies have shown that DEL is highly toxic to crustaceans (e.g. Crane et al., 2011; Burridge and Van Geest, 2014), and that the recommended aquaculture user dose of DEL for control of sea lice is above the LC50-values reported for most crustaceans (reviewed by Urbina et al., 2019). AZA is a water-soluble organophosphate that can also have negative effects on e.g. crustaceans (Burridge et al., 2000; Ernst et al., 2014), but toxicity occurs at higher concentrations than for DEL (Burridge and Van Geest, 2014). Organophosphates are neurotoxic and inhibit the activity of acetylcholinesterase, the enzyme responsible for acetylcholine hydrolysis (Intorre et al., 2004; Kaur et al., 2017). As such, both DEL and AZA are known to affect locomotion capability and behavior in exposed individuals (Burridge and Van Geest, 2014; Urbina et al., 2019).
Given the sensitivity of crustaceans and the significant use of delousing agents in Norway, there is a need to assess effects of bath treatments and combinations of bath treatments on deep-water shrimp. Since the delousing agents are rapidly diluted in the sea, it is important to study both lethal and relevant sub-lethal effects at low concentrations. The aim of our study was to examine acute and delayed lethal and sub-lethal effects on egg-carrying deep-water shrimp exposed to environmentally realistic concentrations of three bath treatments alone (H2O2, AZA, DEL), or in sequential use with each other (H2O2 and AZA, H2O2 and DEL, AZA and DEL). All exposures lasted for 2 h to simulate a realistic environmental exposure time at a given distance from the release point. Exposure concentrations were therefore defined as dilutions of recommended treatment dose of the different bath pharmaceuticals. Effects were assessed in terms of shrimp behavior and mortality, egg loss, embryo development and -mortality. Further, acute DNA-damage was assessed in shrimp tissue and eggs after exposure to a high (16 mg/L) H2O2 concentration.
Section snippets
Exposure scenarios
Three exposure experiments on egg-carrying shrimp were carried out between January 25th – February 22nd, 2018 (experiment one; Exp1), February 14th – March 14th, 2018 (experiment two; Exp2) and February 21st – March 12th, 2019 (experiment three; Exp3). In Exp1, lethal and sub-lethal effects of exposure to 1000, 500 and 100-times dilutions of the recommended treatment dose of H2O2 (1600 mg/L) were investigated (Table 1). In Exp2, lethal and sub-lethal effects of the three different bath
Shrimp behavior and mortality
In Exp1, there was no significant difference in behavior or mortality between treatments. Percentage of shrimp performing normal behavior ranged between 90.0 and 100% in the different tanks throughout the experiment. Overall, more shrimp were laying on their side in the period T1d – T5d (range 2.0–10.4% per tank; average 6.8 ± 2.8%) post-exposure compared to any other periods of the experiment (range 0–7.5% per tank; average 1.1 ± 1.8%). Overall, few incidences of stress swimming were observed
Experimental conditions
In the present study P. borealis was exposed to H2O2, DEL and AZA in water in short (2 h) single-pulse exposures to reflect the acute nature of treatment effluent plumes from aquaculture sites. In addition, sequential exposures with H2O2 and AZA, H2O2 and DEL and AZA and DEL were performed. The concentrations selected represent environmentally realistic concentrations. Field studies of dispersion of AZA and DEL from aquaculture sites have found that effluent plumes are detectable 2–5.5 h after
Conclusions
DEL was shown to be highly toxic to shrimp, emphasizing the need for specific precautions regarding the use of this bath pharmaceutical. The concentration limit for no lethal or sub-lethal effects of DEL on egg-carrying shrimp remains to be established. Further, the combined effect of H2O2 and AZA, inducing >40% mortality at 500 times dilution, is of concern and emphasizes the need for more knowledge regarding the effects of combined/sequential treatments in general.
The results from the
Ethics statement
Permission to carry out experiments was granted by the Norwegian Animal Welfare Authority in 2017 (ID 13353).
CRediT authorship contribution statement
Marianne Frantzen: Data curation, Writing - original draft. Jenny Bytingsvik: Data curation, Writing - original draft. Luca Tassara: Formal analysis, Writing - original draft. Helena C. Reinardy: Formal analysis, Data curation, Writing - original draft. Gro Harlaug Refseth: Writing - original draft. Ellie J. Watts: Writing - original draft. Anita Evenset: Data curation, Writing - original draft.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgement
This study was funded by Norwegian Seafood Research fund (project no. 901425) and Akvaplan-niva AS. We thank the staff from the Akvaplan-niva FISK facility for support during the experiments, as well as personnel onboard RV Hyas from UiT, the Arctic University of Norway for providing shrimp. Perrine Geraudie and Lisa Torske are thanked for technical assistance with DNA extractions and lipid analyses.
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