Ecological Effects of Sea Lice Treatments Scottish Association for Marine Science The Marine Laboratory, Aberdeen Plymouth Marine Laboratory SEAS Ltd Summary This report presents the results of a five year project investigating the ecological effects of medicines used for controlling sea lice on farmed salmon. The objective of this report is to explain the background to the project, describe the research methodology, and provide a synopsis of the project results and conclusions. Measurement and sampling programmes were undertaken at four active salmon farm sites on the west coast of Scotland in Lochs Diabaig, Craignish, Kishorn and Sunart. Each of these sites initially had access to the bath-treatment Excis as the main medicine for sea lice control but in 2001 the in-feed medicine Slice became available for use and was used at three of the sites. Aspects studied include: examination of littoral and sub-littoral settlement panels to assess whether settlements of flora and fauna are affected by chemical usage; sampling of sediments around and away from the farms for meiofauna, macrofauna and the presence of Slice; zooplankton sampling before during and after medications and as a time-series to assess natural variability; time-series measurements of phytoplankton populations and nutrient concentrations; acoustic ground discrimination to determine substrate type around each farm; experiments to measure hydrographic parameters using Differential Global Positioning System (DGPS) drifters and current meter arrays to allow modelling of the dominant water movement processes and simulate dispersion of the treatments used at each site. The project's primary aim was not to determine local effects of sea lice medicines: all discharges to the marine environment have some effect on the receiving environment and it is a general principle of pollution control that such perturbations should be confined to the immediate environment, the mixing zone. We attempted to look beyond ephemeral, local effects and concentrated on trying to detect long-term changes beyond the immediate mixing zone. This was a difficult task and, taking all the results together, we did not detect any clear effect of medicine usage, or indeed other farm activities, beyond the local scale. The processes of species succession and population dynamics that we observed were well within the range of what might be expected or predicted for fjordic sea loch systems. The project has achieved much by helping to improve our understanding of natural variability in relatively unstudied systems and, most especially, by demonstrating that wide-scale ecosystem-level effects from medicine use, if they exist at all, are likely to be of the same order of magnitude as natural variability and, therefore, inherently difficult to detect. 1 Introduction Sea lice are a major problem for salmon farmers in terms of fish health costing the industry in the region of £15-30 million per annum. Anglers and conservationists are also concerned as excessive background lice larval numbers may be one factor in the decline in wild sea trout and salmon on the west coast of Scotland. Several strategies are pursued to reduce sea lice on farmed fish but the availability of an effective range of sea lice treatment medicines is crucial. Although these products have passed a variety of regulatory tests, only when they are used on a commercial scale over long periods can the large-scale, long-term effects of these products be accurately assessed. A number of new treatments have recently been granted Marketing Authorisations. Before they can be used, these treatments also require discharge Consents and much effort has gone into ecotoxicity testing to establish Environmental Quality Standards (EQS). Discharge consents are granted so that after discharge the concentration in the receiving water at the edge of the allowable zone of mixing does not exceed the EQS, thus avoiding acute or chronic harm to the environment. Ecotoxicity tests are generally performed on indicator or sentinel species that give a good indication of the likely environmental risk. However, any wider effects can only be ascertained once the new treatments are in use at production scale. Therefore, this project was designed specifically to look for any medium to long-term (1-4+ years) ecosystem responses attributable to the use of some of these veterinary medicines. The objective of the study from the outset was to detect changes in the natural species assemblages in Scottish sea lochs and determine whether such changes might result from the use of sea lice treatments. To this end, the study was designed to assess changes in the plant and animal assemblages from a range of habitats in the vicinity of the farm locations selected for study by measuring a wide range of ecosystem components. Sea Lice Figure 1. Adult Lepeophtheirus salmonis. Photo courtesy of Alan Pike. 2 Salmon farms in Scotland are affected by sea lice, Lepeophtheirus salmonis (Figure 1) and to a lesser extent Caligus elongatus (Figure 2, Figure 3), ectoparasitic copepods that feed on the skin of the salmon. Sea lice, in common with other copepods, have a free-living life stage called the nauplius. After developing into the copepodid (at which stage the lice are actively searching for a fish host), the sea lice metamorphose into the chalimus stage on contact with a fish. Lepeophtheirus salmonis will only infect salmonids, while C. elongatus have been found on a range of marine fish. The sea lice undergo four chalimus stages while they develop (Figure 4) attached to the host fish before they metamorphose into the pre-adult and adult stages, which are unattached stages, allowing the sea lice access to the whole of the fish’s skin surface (Figure 5). Figure 2. Caligus elongatus. Photo courtesy of Paul Tatner. Figure 3. Adult Caligus females. Photo courtesy of Gordon Rae. Caligus may be found all over the body of the fish, while the larger Lepeophtheirus show some preference for the head region. The open wounds caused by untreated sea lice weaken the fish and allow the proliferation of secondary bacterial and viral infections that can lead to mortality. 3 Figure 4. Life cycle of sea lice. Drawing courtesy of Gordon Rae. Figure 5. Damage to fish from sea lice. Photo courtesy of Jim Treasurer. 4 Veterinary Medicines for Sea Lice Recently eleven compounds representing five modes of action were identified as being used worldwide on commercial salmon farms in the period 1997 to 1998, although the substances used in individual countries varies. These included two organophosphates (dichlorvos, azamethiphos); three natural pyrethrin/pyrethroid compounds (pyrethrum, cypermethrin, deltamethrin); one oxidizing agent (hydrogen peroxide); three avermectins (ivermectin, emamectin benzoate, doramectin) and two benzoylphenyl ureas (teflubenzuron, diflubenzuron). In the EU, a substance for which a Maximum Residue Limit has been established can only be used as a veterinary medicine following the granting of a Marketing Authorisation. In the period 1999 to 2004 (the duration of this project), only Salmosan, Excis, Salartect, Slice and Calicide were licensed for use in Scotland (Table 1). Table 1. Sea lice medicines used on commercial salmon farms in Scotland. Active Ingredient Cypermethrin Emamectin benzoate Azamethiphos Teflubenzuron Hydrogen peroxide Trade Name Excis Slice Salartect Calicide Salmosan Medicine Type Pyrethrin/Pyrethroid Avermectin Organophosphate Benzoylphenyl urea Oxidizing agent Calicide (teflubenzuron) has not been widely used in Scotland and Salartect (hydrogen peroxide), which degrades rapidly to water and oxygen, is not considered to be a hazard to marine life. Excis and Slice were the two most commonly used sea lice medicines in Scotland during this project: Excis (Cypermethrin) Cypermethrin is the active ingredient in Excis. Cypermethrin is a synthetic pyrethroid which acts on the nervous system by increasing sodium permeability of the nerve membrane during excitation, resulting in prolonged nerve stimulation. It has low water solubility, degrades rapidly and is applied as a bath treatment. Cypermethrin released following a bath treatment is rapidly diluted in the sea and the majority is eventually adsorbed onto particulate material, which will settle to the sea bed. The main degradation products of cypermethrin are approximately 1000 times less toxic than the parent compound. It does not accumulate in fish as they readily metabolise pyrethroids. Bioconcentration factors are greater in shellfish, which can be exposed through feeding on particulates or though direct uptake, apparently due to slower rates of metabolism and depuration. Slice (Emamectin benzoate) Although all the appropriate ecotoxicological testing has been carried out to allow this product to be licensed for use, there is relatively little information available in the published literature on the fate and ecological effects of Slice in the marine environment. Emamectin benzoate is the active ingredient of Slice. Emamectin benzoate is a semi-synthetic avermectin that acts by increasing membrane permeability to chloride ions and disrupting physiological processes. Emamectin benzoate is administered as a component of salmon feed and enters the marine 5 environment associated with uneaten feed and fish faeces. Salmon readily assimilate emamectin benzoate and excretion continues for an extended time period after administration. Given its association with particulate material and low water solubility most emamectin benzoate will settle to the sea bed. The organisms most likely to be exposed to this chemical are, therefore, sediment dwelling animals in the vicinity of fish farm cages. Slice use is increasing in Scotland and, in many loch systems, strategic treatments are being undertaken simultaneously at several farm sites. At the outset of the project Excis became widely available to fish farmers including the farms selected for study. Discharge consents for Slice were delayed for a variety of regulatory reasons and it was not until the summer of 2001 that we were able to identify a suitable site with a Slice discharge consent. The Study Sites During the first three years of the project four fish farms on the Scottish west coast (one each in Lochs Craignish, Sunart, Diabaig and Kishorn) were identified as being appropriate for the project. One problem encountered at the start of the project was that much of the industry around the Oban area (where the lead institute SAMS is based) was closed owing to an outbreak of ISA (Infectious Salmon Anaemia). This meant that farms had to be selected from further away than was logistically optimal. The longest time-series of data was collected from a fish farm at the head of Loch Sunart, with samples collected from late 1999 through to mid-2004. This site initially used Excis with occasional use of Salmosan, but in 2002 gained access to and used Slice. Sampling also began at Loch Diabaig in 1999, but most sampling ceased there in 2001. The Loch Diabaig farm used Excis, Salmosan and Slice but proved generally unsatisfactory as a result of its high winter exposure that resulted in damage to moored equipment. The third site in Loch Craignish was intensively sampled in 2000, but most sampling was discontinued in 2001 as only one lice treatment was reported during the 12 month period and the site had not applied for a Slice consent. In 2001 we transferred our attention from Loch Craignish to a site in Loch Kishorn that had consent for Slice and planned to use it. The work in Kishorn continued until the end of 2002, when the site became fallow. Study Components and Results In order to determine the broad range of basin-scale effects from sea lice treatment medicines over the medium to long term, we focussed on the main components of the ecosystem that were likely to be affected: • • • • • • Benthic meiofauna - animals < 1.0 mm in size, living on or in the sea bed Benthic macrofauna - animals > 1.0 mm in size, living on or in the sea bed Inter-tidal organisms - plants and animals settling on the sea shore Sublittoral organisms - plants and animals settling on hard substrates deployed in the water column Zooplankton - small animals drifting in the water column Phytoplankton - microscopic plants drifting in the water column 6 Each sampling programme involved multiple stations at varying distances from the fish farm in order to examine whether there were any differences that might be attributed to sea lice treatments. Fish farms have some well known local ecological effects as a consequence of organic enrichment so particular attention was paid to Crustacea as the component of the community most susceptible to effects from sea lice medicines. In addition to the ecological work, the study was complemented by measurements of sediment medicine concentrations and the development and use of computer models to simulate the dispersion of medicines. Each of these components is discussed below: Meiofauna in Sediments Meiofauna is a collective name given to animals that are small enough to pass through the sieve meshes (1 or 0.5 mm) which are used to separate larger animals (macrofauna) from sediments. Specialised techniques are used to separate the meiofauna from sediments, but essentially they are collected on a fine mesh with apertures 63 µm across. In sediments such as occur in sea lochs the most abundant meiofaunal groups would be expected to fall into two major categories, namely freeliving nematodes (roundworms) and tiny crustaceans in a group called harpacticoid copepods. Although generally too small to be seen with the naked eye, meiofaunal animals are useful indicators of environmental change for several reasons. Their small size and high densities mean that smaller volumes of sediment are required, and the majority of the sediment processing may be done under the controlled conditions of the laboratory. Apart from such practical advantages, there are also sound scientific reasons for expecting meiofaunal assemblages to be sensitive to environmental change. Unlike the macrofauna, many species of which have a planktonic larval stage and lifespans in excess of a year, meiofauna have no specific dispersal phase and spend their whole lives in one place, living, breeding and feeding. Meiofaunal reproduction tends to be rapid, with generation times of a few months, and continuous, with juveniles occurring throughout the year. This means that they are far more likely to react rapidly to changes in environmental conditions. We initially looked at nematode and copepod assemblages in the vicinity of salmon farms where bath treatments for sea lice using Excis, were being used. In Loch Sunart, meiofauna samples were collected from the same sites, and at the same time, as macrofauna samples in October 1999 and March 2001. Excis was used on several occasions between these two surveys. Samples were also collected in Loch Diabaig in November 2000 and August 2001, again before and after Excis treatments. Slice was also used at this site during the period, but as the spatial dispersal and potential impacts of in-feed treatments are expected to be rather different to those of the bath treatment Excis, a separate intensive study on the effects of Slice was undertaken at a farm in Loch Kishorn in 2001. Nemotode community composition close to the fish farm cages was similar in Lochs Kishorn, Diabaig and Craignish, and differed from assemblages at the reference stations (Figure 6). Nemotode assemblages at Loch Sunart were generally different from those observed in the other lochs. Copepod community composition displayed a similar, if less clear, pattern (Figure 7). 7 At Loch Kishorn, where samples were collected from very close to the farm, strong gradients in meiofaunal assemblage structure were apparent, and there was evidence of greater differences in copepod community structure between stations in posttreatment surveys. Meiofaunal community structure was most related to station distances from the cages and it is likely that deposition from the cages impacted nematode and copepod assemblages, with a combination of changes in sediment composition and the presence of in-feed treatment chemicals causing the observed changes in copepod community structure. At Loch Diabaig, there was no evidence that sea lice treatments were a major driver of patterns in meiofaunal community structure, instead most of the differences between stations can be explained by variation in sediment type, and to a lesser extent other factors (distance from the cages, depth, % total organic carbon or nitrogen) which may or may not reflect organic enrichment effects from the cages. Figure 6. MDS ordination of similarities between abundances of nematode genera averaged within stations from different surveys. Contours group samples at a level of 40 % similarity. The stations are denoted as follows: K11A = Kishorn, Survey 1 (June 2000), transect 1, station A; D22 = Diabaig, Survey 2 (August 2001), Station 2; C13 = Craignish, Survey 1 (October 2000), Station 3; S11 = Sunart, Survey 1 (October 1999), Station 1; K3R = Kishorn, Survey 3 (January 2001), Reference station; and so forth. All stations represented in the plot were sampled with a van Veen grab except the reference stations at Kishorn. At Lochs Sunart and Craignish, changes in meiofaunal assemblages were also unrelated to sea lice treatments. Only small differences in meiofaunal community structure were observed at Loch Sunart, which were related primarily to site location, with differences in depth and sediment structure rather than fish-farming activity driving the differences seen. Seasonality and different sampling methods may also have caused the shift in pattern. At Loch Craignish, species known to be indicators of disturbance and organic enrichment contributed to differences between stations, leading to the conclusion that the observed differences between stations reflect the normal organic enrichment gradient associated with fish-farming activity. 8 Figure 7. MDS ordination of similarities between abundances of copepod taxa averaged within stations from different surveys. Contours group samples at a level of 25 % similarity. The stations are denoted as follows: K11A = Kishorn, Survey 1 (June 2000), transect 1, station A; D22 = Diabaig, Survey 2 (August 2001), Station 2; C13 = Craignish, Survey 1 (October 2000), Station 3; S11 = Sunart, Survey 1 (October 1999), Station 1; K3R = Kishorn, Survey 3 (January 2001), Reference station; and so forth. All stations represented in the plot were sampled with a van Veen grab except the reference stations at Kishorn. The comparative studies between sites have proved to be useful, with the results indicating that, at some level, the effects of fish-farming on meiofaunal communities are similar in different sea lochs. Therefore, the results of in-depth studies in one sea loch may provide useful information for the purposes of regulation at another. The results also suggest that it may be possible to select a subset of species which could act as indicators of aquaculture impacts. Macrofauna in Sediments The identification and enumeration of bottom-living macro-invertebrates (macrobenthos) and subsequent analysis of the population data is a widely-recognised means of assessing impacts of discharges of materials on the marine environment, and is usually an important component of monitoring requirements stipulated by regulators when granting consents to discharge. The macrofauna studies were designed to detect any medium term (up to 3-4 year) effects on benthic populations (meiofaunal and zooplankton studies detect more rapid changes). Macrobenthic invertebrate populations (retained on a 1 mm mesh after sieving samples of sea-bed sediments) were sampled along transects in the vicinity of each fish farm and at reference sites anticipated to be well beyond the zone of impact of the fish farms in each sea loch. Where possible, samples were collected before and after sea lice medicine treatments. Additional samples were collected at annual intervals in an attempt to quantify the influence of natural seasonal effects on the populations being compared. 9 The effects of aquaculture on the macrobenthic communities in this study were broadly comparable between the different sea lochs, although macrofaunal assemblages at Lochs Kishorn and Diabaig were more similar than those at Loch Sunart (Figure 8). Figure 8. MDS plot of similarities between macrofaunal abundances averaged within survey/station combinations from Lochs Sunart, Diabaig and Kishorn. The first letter indicates the site (S, Sunart; D, Diabaig; K, Kishorn). The first numeral indicates surveys (For Sunart 1, October 1999; 2, March 2000; 3, November 2000; 4, March 2001; 5, November 2003. For Diabaig 1, July/August 2000; 2, November 2000; 3, August 2001. For Kishorn 1, June 2001; 2, August 2001) and the second numeral indicates stations (1 to 4 at Sunart, 1 to 3 at Diabaig, 1 to 3 and R at Kishorn). Differences in macrofaunal community composition between sample stations in each loch indicated a gradient of organic enrichment originating at the fish farm cages, with no evidence to indicate that sea lice treatments adversely affected the macrobenthic communities in any of the lochs studied. At Loch Sunart, although macrobenthic assemblages close to the fish cages differed from the reference station, they did not change over time indicating that sea lice treatments between surveys did not cause species abundance or composition to change. The gradient of organic enrichment was considerably stronger at Loch Kishorn and differences in community composition were observed between surveys undertaken before and after a sea lice treatment with Slice. In particular, reductions in the number of crustacean and echinoderm species and their abundance were observed at one station close to the cages after the Slice treatment. These groups are sensitive to both organic enrichment and sea lice treatment agents, thus either (or both) could have been responsible for the differences seen, and overall there was no evidence that Slice adversely affected the macrobenthic community at Loch Kishorn. Similarly, at Loch Diabaig, differences in macrofaunal assemblages between stations and surveys were more likely the result of seasonal variability and organic enrichment. Species diversity was lowest closest to the fish cages and dominated by nematodes and small polychaetes, while species typical of normal shell/muddy sediments dominated at the 10 reference station. Following a Slice treatment, dominance increased at all stations and crustaceans declined closest to the cages. Shore Flora and Fauna At all four fish farm sites studied the inter-tidal zone consisted mainly of bedrock and/or boulders. Following initial ecological surveys, three or four shore stations were established in each area, two stations at locations where information on current flows suggests that any effects of the treatment chemicals would be most likely to be detected, and one or two reference stations, situated beyond the anticipated zone of influence of activities associated with the fish farms. All of these shores were characterised by abundant growths of brown algae mixed with abundant invertebrate populations of attached barnacles and mussels, limpets and mobile snails: mainly Littorina species and the dog whelk Nucella lapillus, which feeds on barnacles. Barnacles (Semibalanus balanoides) are the only sedentary crustaceans that settle predictably on these shores in significant amounts. Barnacles belong to the same group as sea lice, and are therefore appropriate ‘target’ organisms for studies aimed at detection of any effects of the sea lice treatments. In each area, sets of four slate panels were bolted to the rock at approximately Mid Tide Level to receive any settlements (Figure 9). The barnacles and other biota on the surrounding rocky shores were also studied and photographed at each visit (approximately three times per year at each location). Settlement on the slates revealed that barnacle densities and subsequent growth varied considerably between sea lochs, and between study sites in each sea loch. Figure 9. Slates showing heavy barnacle settlement. 11 The highest barnacle settlement densities occurred at Loch Diabaig where exposure to wave action was the highest of the four lochs. There was also evidence of a relationship between reduced barnacle egg production and predicted impact at this site. At Loch Diabaig, barnacles at the predicted high impact station (Station C) produced fewer egg masses than barnacles at the predicted intermediate impact (Stations B) and reference stations (Station A; Figure 10). The difference was greatest in 2000 and 2001 with 60 - 70 % of barnacles without eggs at the high impact station. In 2002 and 2003, this had fallen to 30 - 50% at the high impact station, but had increased at the other two stations. Thus, the overall trend was of increasing frequency of egg mass occurrence at the predicted high impact station and decreasing frequency at the intermediate and reference stations (Figure 10). Figure 10. Reproductive failure of littoral barnacles (Semibalanus balanoides) at Loch Diabaig. Error bars are 95% confidence limits. The reproductive failure of barnacles observed in this study could have been caused by a variety of natural factors such as degree of exposure, and not just fish farm activities such as sea lice treatments. The trend diminished with time over a period when fish farm activity and the use of sea lice treatments also declined at the site, but we do not know whether this is correlated or not. However, the results indicate that the influence of environmental factors on egg production in littoral barnacles merits further investigation, including study areas remote from fish farm activities. 12 Sublittoral Fauna Settlement The study of organism settlement on the sublittoral arrays of slate panels suspended at three different depths and at three or four stations in Lochs Sunart, Diabaig and Craignish yielded a clear picture of natural seasonal and annual successions, and fauna and flora abundances. Overall, species diversity of fauna settling on the sublittoral slates was limited, but similar at all sites studied. At all sites and stations the most significant ecological event each year was the spring settlement of the dominant sublittoral barnacle Balanus crenatus. At all stations the slate panels were heavily colonised, with the barnacles growing quickly to attain 100% cover, especially at the optimal upper and mid-water depths. The results clearly show that barnacle settlement was similar at all stations, regardless of distance from the fish farm cages. It follows that sea lice treatments at the salmon farms did not affect barnacle settlement at any of the sites studied. Other crustacean species settled on the panels only rarely, such as Balanus balanus at Lochs Sunart and Diabaig and the small crab Porcellana longicornis at Loch Sunart. In December 2003, the non-native (alien) caprellid amphipod Caprella mutica was observed for the first time at two of the sample stations in Loch Sunart. Macroscopic algae (seaweeds) scarcely featured on the sublittoral slates, although they often colonised the near-surface ropes and buoys; and mussel spat (Mytilus edulis) settled readily on ropes, hydroids and any fine-branched attached seaweeds. The main settlement events observed are summarised in the following figures (Figure 11 to Figure 14): In April to June heavy settlements of barnacles (Balanus crenatus) usually occur followed, through the summer months, by mussels (Mytilus) and ascidians (seasquirts) and lesser numbers of a variety of other species (a total of around 40). Meantime, the main predators of barnacles and mussels, namely the sea-slug Onchidoris bilamellata, followed by the even more voracious starfish, Asterias rubens, settle from the plankton, grow rapidly and kill all the barnacles. The strong empty shells of the barnacles then often remained for many months, greatly altering the character of the surface for all later settling animals through the late summer and autumn. These included increasing numbers of ascidians and often polychaete worms including Pomatoceros and Hydroides in calcareous tubes and those in soft muddy tubes such as Sabella pavonina (peacock worm) (Figure 11). 13 Figure 11. Loch Sunart Station 1, middle array. 12 Dec 2001, showing abundant polychaete worms Pomatoceros, Hydroides, and Sabella pavonina. Figure 12. 6 July 2000. Massive settlement of barnacle Balanus crenatus (superabundant; 100% cover), with young ascidians Ascidiella aspersa and Ciona intestinalis and hydroid Obelia longissima all common. The barnacles were being eaten by nudibranch Onchidoris fusca (frequent) settled directly from the plankton. 14 Figure 13. 7 Nov 2000. The barnacles are now mostly empty shells having been eaten by the predators Onchidoris fusca and the starfish Asterias rubens (common), which also settles (later) from the plankton. The ascidians Ciona intestinalis (common) are now much larger. Slate #2 (deployed 6/7/00) has extensive cover (~30%) of the gelatinous prostrate ascidian Diplosoma listerianum, also being browsed by Asterias; also present occasional young tube worms Pomatoceros triqueter. Note mussels Mytilus edulis abundant on the upper bar and Asterias feeding on them. Figure 14. 3 March 2001. The empty barnacle shells are mostly still present; some loss of the mature ascidians; loss of Mytilus from top bar and predator Asterias. 15 Figure 15. Massive growth of ascidians overloading settlement arrays. In several lochs, the settlement of ascidians was massive and completely obscured everything else present on the slates, the mooring ropes and the buoys. At one location, the over-loaded arrays had to be removed at the end of one season (Figure 15). Zooplankton Copepods are the main constituent of the zooplankton and are the major herbivores in sea loch ecosystems, playing a fundamental role both as consumers of phytoplankton and as a food resource for larger animals. Because zooplankton have limited mobility they are largely dependent on currents and tides for their location, consequently they are unable to avoid unfavourable water conditions. The response of zooplankton to chemical exposure is generally considered to be informative of the relative impacts on the whole ecosystem. Significant declines in copepod abundance and consequent reductions in their grazing rates could potentially result in serious environmental problems such as algal blooms. Planktonic copepods have a similar life cycle to ectoparasitic copepods (sea lice) and are likely to be adversely affected by sea lice medicines, which are highly toxic to crustaceans. 16 Zooplankton are most likely to be exposed to cypermethrin in soluble form given its mode of administration as a bath treatment. In the immediate post-treatment period, zooplankton entrained within a cypermethrin treatment plume may be exposed to potentially lethal concentrations for several hours. Cypermethrin is then predicted to adhere to particulate material in the water column providing a further route of exposure to zooplankton through ingestion. Zooplankton have a lower risk of exposure to emamectin benzoate as it is administered as a component of the salmon feed. The most plausible exposure route is associated with ingestion of feed and faecal particles. Excretion of emamectin benzoate by salmon continues for an extended period post-treatment, increasing the duration of any exposure to particulate associated emamectin benzoate. Resuspension of freshly deposited material will also reintroduce emamectin benzoate to the water column for a considerable period post-treatment thus making it potentially available. To determine the effects of sea lice medicines on the zooplankton community, sampling campaigns were undertaken at fish farms using the bath treatments Excis and Salmosan, and the in-feed treatment Slice. Initially, sampling campaigns were of relatively short duration with plankton samples collected before, during and after sea lice treatments. However, because of difficulties distinguishing treatment-related changes in zooplankton community composition from naturally occurring changes observed during the short-term surveys, a long-term fortnightly sampling campaign was subsequently undertaken in Loch Sunart, during which Excis and Slice were the sea lice medicines predominantly used. At each site, zooplankton samples were collected from four or five sample stations positioned around the farm cages. Three or four sample stations were located within 100 m of the salmon cages, and a reference station was located at a distance from the cages. At each sample station, five samples were collected using a small boat using a plankton net (120 µm mesh) hauled from a maximum depth of 24 m. For all sea lice treatments undertaken during this study no adverse affects on zooplankton were detected at either the species or community level. Changes observed were naturally occurring, with patchiness in distribution, life history characteristics, and water currents being the most influential factors affecting zooplankton distribution and community composition. The long-term sampling programme undertaken in Loch Sunart from November 2001 to April 2004 allowed us to identify the natural seasonal cycles in abundance and species composition and confirmed that sea lice treatment events did not significantly alter the seasonal trends. The seasonal cycles of species and abundance were similar in 2002 and 2003, with peaks in abundance during the summer months between May and September in both years, although total numbers were higher in late summer 2003 (Figure 16). Declines in abundance in late summer (September/October) could have mistakenly been attributed to sea lice treatments with Slice (2002) and Salmosan (2003) had a two year data set not been collected to identify the seasonal cycles of abundance. 17 Figure 16. Total zooplankton abundance (number m-3) at Loch Sunart during the long-term monitoring program (November 2001 to May 2004). Vertical bars are the stations (A, B, D) positioned close to the fish cages (0 and 100 m). Closed circles are the reference station. Timing of sea lice treatments is indicated by the arrows; Excis (C), Salartect (HP), Salmosan (A), Slice (EB1, EB2, EB3). The small copepods which dominate zooplankton communities in Scottish sea lochs are the group most likely to be affected by sea lice treatment agents because of their toxicity to crustaceans. Nonetheless, it is perhaps unsurprising that we did not detect treatment related affects on the zooplankton communities in this study. Notwithstanding the patchy nature of zooplankton distribution, which makes detection of treatment related effects difficult, it is unlikely that water column concentrations of sea lice treatment agents were maintained at sufficiently high levels to cause discernable or widespread changes in the zooplankton communities. In the case of Excis, measured and predicted cypermethrin water column concentrations following a single cage treatment at Loch Sunart were considerably lower than concentrations shown to cause acute toxicity to copepods in laboratory studies. The results also indicated that the 3-hour EQS of 16 ng l-1 was unlikely to have been breached following single cage treatments with Excis. It is possible that there may have been localised affects on zooplankton within dispersing treatment plumes. However, the wider zooplankton community within the loch was not adversely impacted at a detectable level following any sea lice treatments in this study. 18 Phytoplankton Phytoplankton play an important role in marine ecosystems. Their growth is directly influenced by physical factors such as temperature, salinity, light and nutrients, and they are first link in the marine food chain. Information on seasonal changes in phytoplankton species and abundance is vital as it allows identification of responses that may be caused by the use of sea lice treatment agents, as opposed to those caused by physical factors such as changing salinity, temperature, light and nutrients, or biological variables such as grazing pressure. Phytoplankton and supporting salinity and nutrient data were collected in high frequency sampling programmes so that effect(s) of sea lice treatments on phytoplankton communities could be separated from the effects of continually changing environmental conditions. Despite differing physical conditions and sea lice treatment histories, comparison of the four sea loch phytoplankton communities revealed significant similarity between them. This reflects the widespread distribution of many of the phytoplankton species in Scottish coastal waters. In all lochs, the phytoplankton community was typically dominated by a relatively small number of species with many other species present in low numbers at different times of the year. Some of the more common (and bloomforming) species were observed year-round at all sites (e.g. small Chaetoceros sp., Skeletonema costatum, Gymnodinium sp., and unidentified cryptophytes). Phytoplankton blooms occurred with normal frequency and duration for Scottish coastal waters and were caused by species commonly observed at all sites. Summer populations were more species-rich than winter populations, and some typical species observed include the diatoms Eucampia zodiacus, Leptocylindrus minimus, Pseudo-nitzschia sp., Asterionellopsis glacialis, a variety of pennate diatoms, and Cylindrotheca closterium. Some representative dinoflagellates observed during summer months include Dinophysis (e.g. D. acuta, D. acuminata), Protoperidinium sp. and Heterocapsa sp. Cell abundance during the winter months (November to February) was lower than during summer and dominated by microflagellates, with low numbers of diatoms and dinoflagellates (Figure 17). Species typical of winter populations included the dinoflagellates Gymnodinium sp. and Gyrodinium sp., and the diatoms Cylindrotheca closterium, unidentified naviculoids, Skeletonema costatum and Pseudo-nitzschia sp. The presence or absence of any phytoplankton species could not be attributed to the use of sea lice treatments in any of the four lochs. Instead, changes in phytoplankton community composition and abundance were more closely related to season, temperature, salinity and nutrient concentrations, and to a lesser extent zooplankton density. 19 Population percentage 100% 80% Dinoflagellates Diatoms Microflagellates Others * 60% 40% 20% Dec-03 Aug-03 Jun-03 Apr-03 Mar-03 Jan-03 Oct-02 Aug-02 Jun-02 May-02 Feb-02 Dec-01 Oct-01 Aug-01 Jun-01 Mar-01 Nov-00 Aug-00 Jun-00 0% Date Figure 17. Gross phytoplankton community composition at Loch Sunart between June 2000 and April 2004. A Skeletonema costatum bloom (17.2 x 106 cells l-1) in March 2004 is included to illustrate its dominance at this time. “Others*” includes other flagellates, silicoflagellates, cryptophytes, ciliates, tintinnids and resting stages/cysts. Modelling Chemical Dispersion In order to fully understand the ecological fate of sea lice medicines it is important to be able to predict where these substances might go when released into the environment and at what concentrations. It was by modelling that we hoped to improve our understanding of physical, chemical and biological processes and to allow predictions of impact at other sites. Sea lice medicines in bath form disperse in the water current after being released from salmon cages. Designing sampling protocols for medicines and their impacts is made much easier by the use of computer models that can predict how water moves around in the vicinity of the cages. Information on current movements was collected using current meters deployed at each site, and acoustic surveys provided information on the sea bed topography and sediment types in each loch basin. Drifting buoys were also used to track water movements during sea lice treatments to determine the horizontal dispersion characteristics at each site and thus give an idea of the rate of dispersion of the treatment chemicals. The drifters have sophisticated electronics for receiving satellite Global Positioning System (GPS) signals and radioed GPS corrections from a land base. The differentially corrected positions (DGPS) are consistently accurate to ±2 m with a maximum error of ±4 m. During a deployment, the position of the drifters was recorded every 30 seconds at the shore base. The drifters were attached to socks hanging in the water column at a depth of 6 m thus allowing water movement at that depth to be tracked at the surface. This novel technology is a vast improvement on more traditional methods as more frequent and accurate position fixing can be obtained. 20 The data from the current meters and drifters were then used in mathematical models to simulate dispersion of sea lice medicines following a treatment. The models predicted chemical concentrations in the dispersing treatment plume and final sediment concentrations following settlement. The results from the model simulations were then compared with chemical concentrations measured in water and sediment samples. At Loch Sunart, cypermethrin water column concentrations were measured in an effluent plume following a single cage treatment with Excis in January 2002. The treatment plume was tracked for a period of three hours post release using drifter buoys so that water samples could be collected from the plume. Figure 18. Observed and predicted cypermethrin concentrations in the effluent plume following a single cage treatment at Loch Sunart in January 2002. The drifter buoy track is shown in northings and eastings. Sample collection times post-release are shown in brackets; bars show predicted and observed (means of duplicate samples) concentrations (ng l-1). Cypermethrin concentrations measured in the dispersing treatment plume in the first 43 minutes post release ranged from 2.2 ng l-1 to 21 ng l-1 (Figure 18). Cypermethrin was not detected in the majority of samples collected after this. Only four samples taken after 43 minutes contained cypermethrin concentrations higher than the detection limit (1 ng l-1). Predicted cypermethrin water column concentrations were 21 within the same order of magnitude as measured concentrations for the same times and locations (Figure 18). Given that concentrations decreased 1000 times from the treatment concentration of 5000 ng l-1 over a short period of time (within 30 minutes), this is considered an acceptable level of accuracy for the model. The model was used to predict sediment cypermethrin concentrations following a farm wide treatment (Figure 19). The centre of deposition was positioned to the west of the cage group, with peak concentrations of about 20 ng g-1 dry weight. The area of sediment predicted to contain concentrations above the detection limit of 2.25 ng g1 was about 108,000 m2, with a length and breadth of roughly 530 and 260 m respectively. 600 D ISTA N C E O FFS HO R E (m ) 500 400 300 200 100 Concentration (ng g 1900 20 16 18 12 14 1700 10 8 4 6 0 2 0 1500 -1 dry weight) 2100 2300 2500 2700 2900 DISTANCE ALONGSHORE (m) Figure 19. Predicted sediment cypermethrin concentrations (ng g-1) following a full farm treatment at Loch Sunart, 30 January to 6 February 2002. The hatched rectangle represents the cage group. The results suggest that multiple cage treatments may result in the deposition of detectable quantities of cypermethrin on the seabed in the vicinity of fish farms. However, because cypermethrin may remain in the water column for several hours, whether in soluble or adsorbed form, the location of peak deposition will be determined by the prevailing wind-driven currents during and after treatment, in addition to the tidal current direction. The dependence on wind-driven currents makes prediction of cypermethrin dispersion, and selection of sample sites, more difficult. This is in contrast to in-feed medicines, whose settlement is primarily determined by tidal currents, allowing more accurate prediction of deposition patterns. The sediment concentrations depicted in Figure 19 are due to direct deposition of cypermethrin; chemical decay was not included in the simulations. Decay of the compound in high organic marine sediments is thought to halve concentrations after 35 days. Processes of sediment resuspension and redistribution, which were not included in the model, are also likely to reduce the predicted concentrations. Emamectin benzoate deposition was modelled using the particle tracking model DEPOMOD, which is used by SEPA in consenting Slice applications. Prediction of sediment emamectin benozoate deposition footprints below the fish farm cages assisted positioning of sample stations for benthic fauna and sediment chemistry. The 22 predicted post-treatment concentrations were also compared with measured concentrations from sediment samples and have helped with further validation of the model. Figure 20. Predicted emamectin benzoate sediment concentrations at Loch Sunart on days 9, 114 and 269 post-treatment. 23 At Loch Sunart, the predicted footprint of sediment emamectin benzoate deposition was below and slightly to the west of the cage group as a result of the residual current (Figure 20). Following sea lice treatments with Slice in 2002, predicted sediment emamectin benzoate concentrations were considerably higher than measured concentrations of the chemical in sediment samples collected from the stations along transects S1 and S2 (indicated in Figure 20). In most of the sediment samples collected after Slice treatments at Loch Sunart, emamectin benzoate concentrations were either trace or not detected. Stations where emamectin benzoate was detected were directly below the cages and concentrations were generally two orders of magnitude lower than predicted concentrations. The chemical was also not detected in samples collected from the macrofauna stations following two Slice treatments at the farm. Given that the DEPOMOD model has been validated for different environments and farmed fish species, possible reasons for the large differences between predicted and measured sediment emamectin benzoate concentrations during this project are: 1) inaccurate or insufficient representation of emamectin benzoate behaviour or properties in the model; 2) inaccurate model input data, particularly use of default data; 3) unreliable or insufficient measured concentrations to test the model across a range of temporal and spatial scales. Discussion and Conclusions This project was conducted in the real world of regulated commercial salmon farming. In many cases this created difficulties in planning the field sampling campaigns. For example, because of the lengthy discharge consenting process for Slice, work was unable to start on this medicine until the summer of 2001 nearly two years after the start of the project. Our philosophy had been to establish long term sample collection programmes at three sites and to continue them for the duration of the project but this strategy had to be abandoned when the risk that we might not get adequate time to sample a site using Slice became too great. At that point we transferred our attention from Loch Craignish to Loch Kishorn. It is important to emphasise the dynamic nature of the marine ecosystem, which is driven by changes in season, weather and biological factors (e.g. in variations in the timing of larval supply), leading to natural differences between years and even decades. These natural changes can obscure more subtle changes caused by the discharge of medicines and other substances into the sea. Long-term zooplankton and phytoplankton sampling campaigns confirmed that sea lice treatments did not alter natural seasonal trends as the processes of species succession and population dynamics were well within the range of what might be expected or predicted for fjordic sea loch systems. Similarly, the study of organism settlement on sublittoral arrays yielded a clear picture of natural seasonal and annual species successions and abundances, beginning with the spring settlement of barnacles each year. 24 Gradients in meiofaunal and macrofaunal community structure were observed at most of the sites, however, the differences observed between stations generally reflected the normal organic enrichment gradient associated with fish farming activity. At Loch Kishorn, it is possible that a combination of changes in sediment composition and the presence of in-feed treatment chemicals may have impacted meiofaunal assemblages, however, these variables are co-linear with the strong organic enrichment gradient at this site. In contrast to the other sites, stations at Kishorn were deliberately positioned very close to the cages to try to detect some effect. Barnacles are the only sedentary organisms that settle predictably on Scottish rocky shores in high numbers. The highest barnacle densities occurred at Loch Diabaig, the most exposed site. It is possible that the low frequency of egg masses observed at this site may have been related to sea lice treatments at the fish farm and this aspect of the study merits further investigation to determine the effects of natural and anthropogenic processes on barnacle reproduction. The broad objective of the project was to determine the ecological effects of sea lice treatments in Scottish sea lochs, and in those terms that objective has been met, with no gross effects of medicines on the receiving environment distinguished. The project has achieved much by helping to improve our understanding of natural variability in relatively unstudied systems and, most especially, by demonstrating that wide-scale ecosystem-level effects from medicine use, if they exist at all, are likely to be of the same order of magnitude as natural variability and, therefore, inherently difficult to detect. Acknowledgements The scientific consortium responsible for this work are pleased to acknowledge the assistance we have received from the farm managers and their staff at Lochs Sunart, Diabaig, Kishorn and Craignish. Without their logistical support this project would not have been possible. Thanks also to Jim Treasurer, the late Gordon Rae, and Alan Pike for photos. Paul Tatner supplied the photo of Caligus elongatus from the Biomedia Museum Project - a joint funded project originally compiled by Dr Andrea Fidgett and directed by Dr Douglas Neil, University of Glasgow in association with the Universities of Paisley and Strathclyde. This project was sponsored by: The Department for Environment, Food, and Rural Affairs Veterinary Medicines Directorate and Chemicals and Genetic Modification Policy Directorate; Scottish Executive Environment and Rural Affairs Department; Scottish Natural Heritage; The Scotland and Northern Ireland Forum for Environmental Research and Scottish Quality Salmon. 25 Questions or Comments Any questions or comments on this work should be emailed to the scientific coordinator, Dr Kenny Black: [email protected] and/or to the sponsors co-ordinator Dr Nick Renn: [email protected] Glossary of Technical Terms Ectoparasitic: External parasites Transect: A line along which samples are taken. Reference station: Control sample station positioned beyond the area where impacts are expected. Environmental Quality Standard (EQS): A value, generally defined by regulation, which specifies the maximum allowable concentration of a potentially hazardous chemical in an environmental sample such as water or air. ng l-1: 1/1,000,000,000 gram per litre = 1 part per trillion. Crustaceans: A group of animals that have an external skeleton, jointed legs, and a segmented body. Most crustaceans live in water. Copepods: Small shrimp-like crustaceans. Echinoderms: Spiny-skinned invertebrates that live on the sea floor such as sea urchins and starfish. Polychaetes: Marine segmented worms, usually with bristles on each segment. Hydroids: Small animals with stinging cells that build colonies of polyps. Related to jellyfish. 26
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