Recommendations for the inclusion of targeted testing to improve the regulatory environmental risk assessment of veterinary medicines used in aquaculture

Environment International 85 (2015) 1–4

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Recommendations for the inclusion of targeted testing to improve the regulatory environmental risk assessment of veterinary medicines used in aquaculture Adam Lillicrap ⁎, Ailbhe Macken, Kevin V. Thomas Norwegian Institute for Water Research (NIVA), Gaustadaléen 21, NO-0349 Oslo, Norway

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Article history: Received 23 April 2015 Received in revised form 27 July 2015 Accepted 27 July 2015 Available online xxxx Keywords: Acute to chronic ratios Chitin synthesis inhibitors Salmon lice Aquaculture

Aquaculture is a globally important food production industry which has over the past years shown major growth. Data from the Food and Agriculture Organization of the United Nations (FAO) indicate that the value of global aquaculture production has increased from approximately $50 billion to nearly $140 billion in the years between 2003 and 2012. The biggest aquaculture producer is China with approximately 61% of the global market with other Asian countries accounting for 27% and other countries making up the remaining 12%. Unsurprisingly, due to the vast scale of aquaculture in Asia, carp is the most produced fish in the world followed by salmonids that are the most produced mariculture fish (marine fish farms). The salmonid farming industry comprises much of the remaining 12% of the global aquaculture market and of these countries Norway is the largest producer (33% of the salmonid world market) followed by Chile (31%) and then other European countries (19%) including Scotland which is the largest producer in the European Union (FAO, 2008). Production volumes of salmonids within Scotland have remained relatively consistent since 2003, producing approximately 150,000 tonnes per annum. The aim in Scotland is to increase production to 210,000 tonnes by 2020 (Scotland's National Marine Plan Consultation document). Conversely, Norway has seen a steady increase from just over 0.5 to 1.2 million tonnes between 2003 and 2013. Export sales in Norway equate to an annual value of approximately $5 billion, and as a consequence aquaculture is the second biggest Norwegian export industry after oil and gas (Statistisk Sentralbyrå, 2014). ⁎ Corresponding author.

http://dx.doi.org/10.1016/j.envint.2015.07.019 0160-4120/© 2015 Elsevier Ltd. All rights reserved.

A major challenge for salmonid aquaculture is the infestation of salmon lice, and the use of pesticides is often vital to control these ectoparasites. Since 2009, there has been a noticeable increase in the use of salmon lice treatments in Norway (see Fig. 1), with more than 12 tonnes of active ingredients being used in 2014 (Norwegian Institute of Public Health, 2014). Surprisingly, the increase in salmon production in Norway is not paralleled by the increase in the use of salmon lice pesticides that has increased over 120 times compared to the amount used in 2003 (see Table 1 and Fig. 1). Salmon lice infestations do not just have economic consequences due to the loss of sales, but there is also a requirement to avoid transmission from fish farms to wild salmonid stocks. In addition, to meet the increasing demand for salmon, the future expansion of fish farms is dependent on salmon lice levels being kept below 0.1 lice per fish. In 2014, the average salmon lice levels in Norwegian fish farms were just over 0.2 per fish (Ramsden, 2015) suggesting that current salmon lice control practices are not adequately controlling the situation. The problem is further exacerbated by the rise of resistant strains to many of the products that are approved for use in aquaculture. In combination with the attempt to control salmon lice levels, is the possible environmental impact of the treatments where potential effects on non-target organisms may occur during and after treatment (Macken et al., 2015; Langford et al., 2014; Samuelsen et al., 2014). This is partly due to aquaculture medicines often being toxic to non-target species in order to elicit the desired effect on sea lice. Veterinary medicines used in aquaculture are strictly controlled with marketing authorisations issued on a country by country basis. To obtain a marketing authorisation, guidelines developed by the International Cooperation on Harmonisation of Technical Requirements for Registration of Veterinary Medicinal Products (VICH) need to be followed. VICH is a trilateral (EU-Japan-USA) programme aimed at harmonising technical requirements for veterinary product registration. Although the guidelines relating specifically to environmental hazard and risk assessments are a strict obligation for regulatory authorities, the ecotoxicity tests that are required may not always be completely relevant for specific substances. For example, chitin synthesis inhibitors are one group of chemicals that are typically not inherently acutely toxic to non-target organisms such as crustaceans but may cause long term developmental effects. Although the exact mode of action (MoA) of these substances is currently unknown, it is understood they affect reproduction and development of chitin synthesizing organisms, and typically induce malformations whilst reducing chitin production

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Fig. 1. Quantities (kg of active ingredient) of different aquaculture medicines used to treat salmon lice in Norway since 2005.

(Merzendorfer, 2013). Since these chemicals appear to have a very specific MoA on chitin producing organisms, these substances are likely to have chronic effects at concentrations significantly lower than data from standard ecotoxicity tests indicate. Many chitin synthesis inhibitors (e.g. benzoylurea substances) were first developed for plant protection purposes. More recently, two of these substances (diflubenzuron and teflubenzuron) have been used to treat salmonids for salmon lice infestations. The use of diflubenzuron and teflubenzuron has increased significantly over the last 5 years in Norway even though a voluntary ban was introduced in the country in the late 1990s. This is partly due to the emergence of resistance to the other commonly used pesticides (Wholesaler-based Drug Statistics and Norwegian Institute of Public Health, 2014; Langford et al., 2014). However, the use of benzoylurea substances in aquaculture has received media scrutiny for possible effects to non-target commercially relevant species such as prawns and lobsters. Treatment of fish for salmon lice using the benzoylurea substances normally occurs at the larval and pre-adult stage of the ectoparasites life cycle. Due to the low water solubility of these substances they are administered to the fish via the feed, and uneaten food and faeces

are considered to be the main routes for the benzoylureas to enter into the aquatic environment. Environmental monitoring data for diflubenzuron and teflubenzuron have indicated concentrations at nanogram per litre levels (e.g. up to 0.295 μg/L) in the vicinity of fish farms off the coast of Norway (Langford et al., 2014; Samuelsen and Ervik, 2010). Standard 48 h acute toxicity test (ISO 14669) data for these two substances, using the copepodite stage of a potentially at risk non-target organism Tisbe battagliai, indicated no acute effects up to a concentration of 1000 μg/L. In comparison, naupliar larval developmental effect data of these two substances, using T. battagliai, resulted in No Observed Effect Concentrations (NOEC) of 0.010 μg/L and 0.0032 μg/L for diflubenzuron and teflubenzuron, respectively (Macken et al., 2015). Long term developmental effects on the European lobster (Homarus gammarus) have also been reported, post exposure to teflubenzuron at therapeutic dose levels (Samuelsen et al., 2014). The deformities that were observed during the post exposure period of this study were present even after multiple moult cycles and some were considered to be permanent deformities. The exposure scenario in the lobster test was designed to reflect in situ exposure during a fish farm treatment cycle. However, non-target organisms, located proximally to fish farms being treated, are likely to be exposed for longer than the actual treatment cycle by foraging on uneaten exposed food or faeces. Thus, developmental effects are likely to be more pronounced in situ than that seen within the laboratory experiments described above due to the possible prolonged exposure. To define the possible risks of the use of diflubenzuron and teflubenzuron on non-target organisms, risk quotients (RQ) have been derived based on the predicted no effect data (PNEC) from the larval development studies (Macken et al., 2015) in combination with measured environmental concentrations (MEC) (Langford et al., 2014). These data indicate that certain sites around the coast of Norway have a RQ value N 1 demonstrating that there is a significant risk to non-target organisms from the use of these benzoylurea substances to treat salmon lice. A more important concern however, is the difference between the acute ecotoxicity values and the larval developmental chronic toxicity data that have been reported (Macken et al., 2015). For example, based on the data of Macken et al. (2015) the acute to chronic ratios (ACRs) of these substances are extremely high (e.g. N300,000 for teflubenzuron). These ACRs are in the same order of magnitude as some of the most potent endocrine disrupting substances (e.g. 17α-ethinylestradiol and 17β-ethinylestradiol have reported ACRs of 150,000 and 390,000, respectively (Cunningham et al., 2006)) and as such may be considered as substances of very high concern. In comparison, other veterinary medicines to treat salmonids, such as the broad spectrum organophosphorus insecticides (e.g. azamethiphos), avermectins (e.g. emamectin benzoate) and synthetic pyrethroids (e.g. cypermethrin and deltamethrin), are typically more acutely

Table 1 Active ingredients of veterinary medicines and quantities used (kg) to treat salmon lice within Norway and Scotland. Active ingredient

Country

2005

2006

2007

2008

2009

2010

2011

2012

2013

2014

Azamethiphos

Nor Sco Nor Sco Nor Sco Nor Sco Nor Sco Nor Sco Nor⁎

0 0 45 6 16 0 0 0 39 34 0 0 0

0 0 49 9 23 0 0 0 60 36 0 0 0

0 0 30 37 29 0 0 0 73 62 0 96 0

66 100 32 21 39 3 0 0 81 63 0 0 0

1884 204 88 12 62 13 1413 0 41 52 2028 62 308

3346 158 107 3 61 14 1839 0 22 61 1080 75 3071

2437 407 48 1 54 42 704 0 105 144 26 316 3144

4059 195 232 0 121 21 1611 0 36 73 751 225 2538

3037 154 211 0 136 12 3264 0 51 60 1704 262 8262

4630 253 162 0 158 17 5016 0 172 64 2674 0 31,577

Cypermethrin Deltamethrin Diflubenzuron Emamectin Teflubenzuron Hydrogen-peroxide 100% (tonne)

Nor — Norway (source Public Health Institute Norway http://www.fhi.no/artikler/?id=114175); Sco — Scotland (source data supplied by SEPA on 03/03/2015 http:// aquaculture.scotland.gov.uk/data/fish_farms_monthly_biomass_and_treatment_reports.aspx). ⁎ No use data for Scotland.

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toxic than the benzoylurea compounds. In addition, the ACRs of many of these substances are generally much smaller. For example, azamethiphos is acutely toxic (LC50 or EC50: the lethal or effect concentration affecting 50% of the test population) to both the naupliar and copepodite stages of the marine copepod T. battagliai, at concentrations of 7 and 8 μg/L, respectively (Macken et al., 2015). Employing the same 7 day naupliar development test design (described previously for the benzoylureas) resulted in a chronic NOEC value at a concentration of 4 μg/L azamethiphos (Macken et al., 2015). Using these data, an ACR for T. battagliai exposed to azamethiphos is equivalent to approximately 2. In addition, cypermethrin is acutely toxic to the mysid shrimp (Mysidopsis bahia) at a concentration of 0.0059 μg/L with corresponding chronic data (28 day NOEC) at a concentration of 0.0015 μg/L (EU RAR , 2013). Similarly, this equates to an ACR of approximately 4. Considering emamectin benzoate, which is acutely toxic to Daphnia magna at a concentration of 1.0 μg/L in comparison to a 21 day NOEC (based on reproduction) at a concentration of 0.088 μg/L, results in a corresponding ACR of 11 (EU DAR, 2011). It should be noted that these comparisons are based on standardised acute and chronic ecotoxicity tests performed according to regulatory guidelines and do not consider any specific MoA or endpoint for derivation of the NOECs. However, for these substances where the specific MoAs are well characterised, the adverse effect will ultimately be death rather than sub-lethal endpoints (e.g. morphological or reproductive). For example, azamethiphos acts by inhibiting acetylcholinesterase; cypermethrin is a sodium channel modulator and emamectin benzoate is a GABA gated chloride channel antagonists. These are all neurotoxic endpoints where the effects are likely to be terminal dependent on the dose, and when compared to the benzoylureas are more likely to have a short term acutely toxic effect on the salmon lice. Therefore, the current regulatory ecotoxicity testing requirements for risk assessing these neurotoxicants may be considered sufficient. It should be emphasised that it is not the intention of providing these comparisons to advocate the use of neurotoxic pesticides within aquaculture as there are equally far reaching socio/environmental impacts from substances that are specifically aimed at killing salmon lice during treatment. For example, at therapeutic dose levels of these pesticides, there is a possibility for pronounced effects on non-target organisms in the immediate area of the treated fish farms. These areas are designated by regulatory authorities in some countries as allowable zones of effect (AZE). However, with the emergence of resistant salmon lice strains there may be a greater risk to non-target organisms from treatment regimens needing to be more aggressive in order to delouse the fish. From a public perception, the prospect of dead organisms being discovered close to fish farms is just one of the many reasons why fish farming has received significant (negative) press coverage over recent years. From a pragmatic point of view however, there may be justification for short term localised impacts rather than potentially long term more dispersed effects. An example could be the use of hydrogen peroxide, which has also increased significantly over the past few years within Norway. This may be considered more environmentally sound, albeit not necessarily environmentally friendly, since hydrogen peroxide dissipates relatively swiftly to water and oxygen through abiotic degradation. However, the use of hydrogen peroxide needs to be carefully controlled to avoid effects on the treated fish. For example, Arndt and Wagner (1997) suggested that concentrations of hydrogen peroxide used to treat trout should not exceed 280 ppm for 30 min however current practices employ concentrations ranging between 1250 and 1500 ppm (Arthur and Mackie, 2014). Since hydrogen peroxide is highly aversive to fish this provides an additional ethical conundrum for treatment regimes. Clearly, the use of chemicals to treat salmon lice is an extremely important issue, particularly with the emergence of resistant strains meaning that some of these veterinary medicines become less effective against the primary target organism. This means that the development of new strategies is constantly required to control salmon lice, which is ultimately providing a significant cost to industry.

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Encouragingly, the Ministry of Trade, Industry and Fisheries of Norway in collaboration with the Norwegian Seafood Federation and the Norwegian Seafood Association, recognise the challenges within aquaculture and the potential environmental consequences of the use of veterinary medicines. Subsequently, in an attempt to alleviate the environmental impact of aquaculture processes within Norway, the following three measures have been implemented: whole or partial withdrawal of the licensing approval in locations where the problems are most pronounced; review and monitoring of internal systems for salmon lice and veterinary medicines used by industry; and increased supervision of the use of veterinary medicines. How these measures will be implemented in practise or the affect they will have on the environmental impact of veterinary medicines is currently not clear. It is therefore also our responsibility as environmental scientists to help protect the environment from adverse effects of chemicals and the question is whether current environmental risk assessment data requirements are sufficient for all aquaculture medicines? In answer, standard acute and chronic ecotoxicity data may be appropriate for assessing the environmental hazards of a large number of substances. However, it is clearly not sufficient for substances that specifically affect certain organisms or where hazards may not be predicted based on standard environmental hazard assessments alone. Therefore, substances such as chitin synthesis inhibitors with potentially high ACRs, or substances with a specific MoA should be hazard and subsequently risk assessed based on chronic ecotoxicity data with specific endpoints relevant to the substance. This does not preclude other substances that are released into the environment through aquaculture processes in large volumes. These also need to be regulated or restricted more appropriately and more intelligent/targeted treatment strategies are necessary to be developed in the future to reduce the potential environmental impact from aquaculture. Forthcoming improvements in environmental safety of aquaculture medicines needs research initiatives towards the new paradigm for risk assessments. This involves incorporating higher-tier and subindividual testing, development of toxicokinetic/toxicodynamic models, understanding ecologically relevant mechanisms and generation of ecological predictive tools (SCHER, Scientific Committee on Health and Environmental Risks et al., 2013). However, these are seen as long term goals which may be achieved within the next 5 to 10 years dependent upon species and ecosystems of concern. In terms of the short term ecological effects from the current practices within aquaculture, it is apparent that these efforts may be too late to provide sufficient protection to the environment. Henceforth, it may be more relevant for regulatory authorities to consider risk assessing substances not only based on the traditional battery of test organisms and methods that fulfil the Klimisch criteria for data acceptability (Klimisch et al., 1997). A problem with the Klimisch criteria assessment scheme is that it is only relevant for retrospective data analysis. Currently, regulatory authorities may consider data from non-standardised ecotoxicity tests to be assigned a Klimisch score of 3 or lower (i.e. not reliable or not assignable). However, if the tests have been designed and performed appropriately (for example according to the “12 principles of sound ecotoxicology”; Harris et al., 2014) data from non-standardised tests may be more relevant for environmental risk assessment purposes and could be ranked with a higher reliability factor. A new classification scheme for assessing data reliability may be warranted in the future to encompass any non-standardised ecotoxicity data. It should be stressed that these opinions are not intended to discredit current environmental risk assessment practices or the standard test requirements that are stipulated in regulatory guidance/legislation. Moreover, we feel these are a bare minimum requirement, and a more holistic, case by case approach focussing on specific endpoints relevant to the species of concern should be adopted in the future. In addition, there may be sufficient justification for revising the current VICH guidelines to be more in line with other regulatory frameworks (e.g. EFSA PPR Panel, EFSA Panel on Plant Protection Products and their Residues, 2013). This may result in supplementary non-

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