Ecological Effects of Sea Lice Medicines in Scottish Sea Lochs

Ecological effects of sea lice medicines in Scottish sea lochs

Final report 9 February 2005

Scottish Association for Marine Science Plymouth Marine Laboratory Fisheries Research Services, Aberdeen SEAS Ltd Contributors

Meiofauna: Mike Gee (PML); Hazel Needham (PML); Paul Somerfield (PML) Macrofauna: Tom Pearson (SEAS); John Blackstock (SEAS); Janet Duncan (SAMS) Littoral/sublittoral: Harry Powell; John Blackstock Phytoplankton: Pippa Sammes (FRS) Zooplankton: Kate Willis (SAMS) Chemistry: Pam Walsham (FRS); Linda Webster (FRS) Physical Modelling: Chris Cromey (SAMS); Phil Gillibrand (SAMS) Project Coordinator: Kenny Black (SAMS) Editing: Chris Cromey; Thom Nickell (SAMS); Kate Willis

Ecological effects of sea lice medicines in Scottish sea lochs 2 of 286 TABLE OF CONTENTS

1 EXECUTIVE SUMMARY ...... 6

2 INTRODUCTION...... 12

2.1 THE SEA LOCH ENVIRONMENT: PHYSICAL, BIOGEOCHEMICAL AND ECOLOGICAL BACKGROUND...12 2.2 AQUACULTURE IN SCOTLAND ...... 13 2.3 SEA LICE...... 13 2.4 SHORT REVIEW OF ECOLOGICAL EFFECTS OF AQUACULTURE...... 14 2.4.1 The discharges of waste nutrients...... 14 2.4.2 Medicines and chemicals ...... 15 2.5 SEA LICE BIOLOGY AND MEDICINES USED TO CONTROL SEA LICE ...... 15 2.6 ECOTOXICOLOGY OF SEA LICE MEDICINES...... 15 2.6.1 Azamethiphos (Salmosan) ...... 15 2.6.2 Cypermethrin (Excis)...... 16 2.6.3 Emamectin benzoate (Slice) ...... 17 2.6.4 Teflubenzuron (Calicide) ...... 18 2.7 POTENTIAL EFFECTS IN ZOOPLANKTON...... 19 2.8 POTENTIAL EFFECTS IN PHYTOPLANKTON...... 19 2.9 POTENTIAL EFFECTS IN MEIOFAUNA...... 20 2.10 POTENTIAL EFFECTS IN LITTORAL SETTLEMENT ...... 20 2.11 POTENTIAL EFFECTS IN SUBLITTORAL SETTLEMENT...... 21 2.12 POTENTIAL EFFECTS IN MACROFAUNA...... 21 3 METHODS...... 22

3.1 SITE SELECTION PHILOSOPHY ...... 22 3.2 SAMPLE COLLECTION AND ANALYSIS...... 22 3.2.1 Zooplankton...... 22 3.2.2 Phytoplankton, nutrients and salinity ...... 23 3.2.3 Meiofauna...... 24 3.2.4 Macrofauna...... 24 3.2.5 Uni- and multivariate analyses of faunal community structure...... 25 3.2.6 Sublittoral settlement on suspended panels...... 27 3.2.7 Littoral site assessment - shore fauna and flora ...... 27 3.2.8 Sediment chemistry...... 28 3.2.9 Hydrodynamic surveys...... 31 3.2.10 Bathymetric surveys...... 32 3.2.11 DGPS drifting buoy surveys...... 32 3.2.12 Modelling...... 33 4 LOCH SUNART...... 36

4.1 SITE DESCRIPTION...... 36 4.2 FISH FARM HISTORY, BIOMASS, CAGE POSITIONING AND MEDICINE USE ...... 38 4.3 HYDROGRAPHY...... 43 4.4 CYPERMETHRIN WATER COLUMN CONCENTRATIONS: EXCIS TREATMENT JANUARY 2002...... 44 4.5 PREDICTED POST-TREATMENT CYPERMETHRIN WATER COLUMN CONCENTRATIONS...... 46 4.6 PREDICTED POST-TREATMENT SEDIMENT CYPERMETHRIN CONCENTRATIONS ...... 48 4.6 EMAMECTIN BENZOATE SEDIMENT CONCENTRATIONS: SLICE TREATMENTS MAY AND SEPTEMBER 2002 ...... 54 4.7 PREDICTED POST-TREATMENT SEDIMENT EMAMECTIN BENZOATE CONCENTRATIONS...... 56 4.8 ZOOPLANKTON...... 60 4.8.1 November 2000 Excis and Salmosan treatments...... 60 4.8.2 Zooplankton long-term monitoring ...... 64 4.9 PHYTOPLANKTON ...... 69 4.9.1 November 2000 Excis and Salmosan treatment...... 69 4.9.2 Phytoplankton long-term monitoring ...... 71 4.10 MEIOFAUNA ...... 75 4.10.1 Nematodes ...... 76 4.10.2 Meiobenthic copepods ...... 78 4.11 MACROBENTHOS...... 79 4.11.1 Sediment conditions at Loch Sunart fish farm...... 79 4.11.2 Macrofaunal distribution, abundance and structure...... 79 4.11.3 Analysis across surveys ...... 86

Ecological effects of sea lice medicines in Scottish sea lochs 3 of 286 4.11.4 Conclusions ...... 87 4.12 SUBLITTORAL SETTLEMENT ON SUSPENDED PANELS...... 87 4.13 LITTORAL SITE ASSESSMENT - SHORE FAUNA AND FLORA...... 93 4.13.1 Barnacle (Semibalanus balanoides) populations and development...... 96 5 LOCH DIABAIG ...... 100

5.1 SITE DESCRIPTION...... 100 5.2 FISH FARM HISTORY, CAGE POSITIONING, BIOMASS, AND MEDICINE USE...... 102 5.3 HYDROGRAPHY...... 107 5.4 ZOOPLANKTON...... 107 5.5 PHYTOPLANKTON RESULTS ...... 107 5.6 SUBLITTORAL STUDIES ...... 110 5.6.1 Meiofauna...... 110 5.6.2 Macrobenthos ...... 114 5.6.3 Sublittoral settlement panels...... 117 5.7 LITTORAL STUDIES: INTERTIDAL PANELS...... 120 5.7.1 Populations and development of barnacles, Semibalanus balanoides ...... 121 6 LOCH KISHORN ...... 125

6.1 SITE DESCRIPTION...... 125 6.2 FISH FARM HISTORY, CAGE POSITIONING, BIOMASS, AND MEDICINE USE...... 127 6.3 HYDROGRAPHY...... 132 6.3.1 Dispersion data...... 133 6.4 EMAMECTIN BENZOATE SEDIMENT CONCENTRATIONS: SLICE TREATMENTS JULY AND OCTOBER 2001 ...... 135 6.5 PREDICTED POST-TREATMENT SEDIMENT EMAMECTIN BENZOATE CONCENTRATIONS...... 139 6.6 ZOOPLANKTON...... 142 6.7 PHYTOPLANKTON ...... 147 6.8 SUBLITTORAL...... 150 6.8.1 Meiofauna...... 151 6.8.2 Comparison of sampling methods ...... 154 6.8.3 Relationships with environmental variables ...... 156 6.8.4 Macrobenthos ...... 157 6.8.5 Sublittoral settlement panels...... 167 6.9 LITTORAL STUDIES: INTERTIDAL PANELS...... 167 6.9.1 Populations and development of barnacles, Semibalanus balanoides...... 168 7 LOCH CRAIGNISH ...... 172

7.1 SITE DESCRIPTION...... 172 7.2 FISH FARM HISTORY, CAGE POSITIONING, BIOMASS, AND MEDICINE USE...... 174 7.3 HYDROGRAPHY...... 178 7.4 ZOOPLANKTON...... 179 7.5 PHYTOPLANKTON ...... 181 7.6 MEIOFAUNA AND MACROFAUNA...... 184 7.7 SUBLITTORAL SETTLEMENT ON SUSPENDED PANELS...... 185 7.8 LITTORAL SITE ASSESSMENT - SHORE FAUNA AND FLORA...... 188 8 ANALYSIS ACROSS SITES ...... 192

8.1 HYDROGRAPHY...... 192 8.2 PREDICTED SEDIMENT EMAMECTIN BENZOATE CONCENTRATIONS - SLICE MODELLING ...... 193 8.2.1 Emamectin benzoate behaviour/properties ...... 194 8.2.2 Model input data...... 195 8.2.3 Measured data...... 195 8.3 SEDIMENT CHEMISTRY...... 195 8.4 ZOOPLANKTON...... 197 8.5 PHYTOPLANKTON ...... 197 8.6 MEIOFAUNA ...... 200 8.7 MACROFAUNA...... 202 8.8 SUBLITTORAL...... 203 8.9 LITTORAL ...... 203 9 CONCLUSIONS...... 206

9.1 CHEMISTRY ...... 206 9.2 IMPACTS OF SEA LICE TREATMENT AGENTS ON ZOOPLANKTON ASSEMBLAGES ...... 206

Ecological effects of sea lice medicines in Scottish sea lochs 4 of 286 9.3 IMPACT OF SEA LICE TREATMENT AGENTS ON PHYTOPLANKTON ASSEMBLAGES ...... 208 9.4 MEIOFAUNA ...... 208 9.4.1 Loch Sunart ...... 208 9.4.2 Loch Diabaig ...... 209 9.4.3 Loch Craignish ...... 209 9.4.4 Loch Kishorn...... 209 9.5 MACROFAUNA...... 210 9.5.1 Loch Sunart ...... 210 9.5.2 Loch Kishorn...... 210 9.5.3 Loch Diabaig ...... 211 9.6 SUBLITTORAL SETTLEMENT ARRAYS ...... 211 9.7 LITTORAL SETTLEMENT PANELS...... 211 9.8 PROJECT SUMMARY ...... 212 9.9 FINAL COMMENTS...... 214 10 REFERENCES...... 216

11 GLOSSARY...... 224

12 APPENDIX I - HYDROGRAPHY ...... 225

13 APPENDIX II - CHEMISTRY...... 227

13.1 CYPERMETHRIN ...... 227 13.1.1 Methods ...... 227 13.2 RP-HPLC ANALYSIS WITH FLUORESCENCE DETECTION ...... 230 13.2.1 Results...... 230 13.3 EMAMECTIN BENZOATE...... 231 13.3.1 Method 1...... 231 13.4 METHOD 2...... 232 13.4.1 Method Summary...... 233 13.4.2 References ...... 234 13.5 EMAMECTIN BENZOATE IN SEDIMENT: GORSTEN EXPERIMENT...... 235 13.5.1 Introduction ...... 235 13.5.2 Materials and methods ...... 235 13.5.3 Results...... 235 14 APPENDIX III - PHYTOPLANKTON ...... 239

15 APPENDIX IV - MACROFAUNA ...... 254

16 APPENDIX V - LITTORAL SETTLEMENT ...... 274

17 APPENDIX VI - SEDIMENT PROPERTIES...... 282

Ecological effects of sea lice medicines in Scottish sea lochs 5 of 286 1 Executive summary The salmon farming industry uses medicines to control infestations of parasitic sea lice on its stock. The amount of medicine and the frequency of application are controlled by regulation to ensure that the potential for negative effects on the ecosystem is minimised. In this project, which began in September 1999 and was completed in August 2004, our aim was to determine whether there were any measurable, long-term or wide-scale ecological consequences of the use of such medicines at commercially operating fish farms on the west coast of Scotland.

An experimental approach was not possible as the researchers had no control on medicine useage. Instead, our philosophy was to measure ecological parameters that might be influenced by any use of these substances. Thus, advanced information on the use of medicines was not essential to the project, although this was generaly made available by the farmers.

We set up sampling programmes at four active salmon farm sites on the west coast of Scotland in Lochs Sunart, Diabaig, Craignish and Kishorn. Each of these sites initially had access to bath-treatments, such as Excis (active ingredient cypermethrin), Salartect (active ingredient hydrogen peroxide) and Salmosan (active ingredient azamethiphos) for sea lice control but in 2001 the in-feed medicine Slice (active ingredient emamectin benzoate) became available and was used at three of the sites. Site selection was complex as at the beginning of the project many sites were under threat from an Infectious Salmon Anemia outbreak. Additionally, it was hard to predict when and which sites would receive discharge consent for Slice. Thus initial work began in Lochs Sunart, Diabaig and Craignish with a subsequent re-focus on the Sunart and Kishorn systems. These lochs cover a spectrum of sea loch types varying widely in scale, exposure, current speed, salinity, coastal exchange and latitude. They are thus representative systems that allow generalization of the projects results and conclusions.

At each site we examined hydrographic parameters using Differential Global Positioning System drifters and current meter arrays to allow modelling of effluent dispersion and acoustic ground discrimination to determine substrate type and bathymetry. Ecological sampling programmes were carried out each focusing on separate ecosystem ‘compartments’. This involved: • examination of littoral and sublittoral 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 and macrofauna (and the presence of emamectin); • zooplankton sampling before, during and after sea lice treatments and also as a time-series to assess successional processes (at Lochs Craignish and Sunart); • time-series measurements of phytoplankton communities (together with nutrient concentrations).

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.

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. Planktonic copepods have a similar life cycle to ectoparasitic copepods (sea lice) and are thus the group most likely to be adversely

Ecological effects of sea lice medicines in Scottish sea lochs 6 of 286 affected by sea lice medicines, which are highly toxic to crustaceans. 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 changes to phytoplankton populations.

Zooplankton sampling campaigns undertaken during this project monitored the effects of sea lice treatments with Excis, Salmosan, Salartect and Slice. For all sea lice treatment events, no adverse effects on zooplankton were detected at either the species or community level and observed changes in community composition were unrelated to treatment events. Initially, sampling campaigns were of short duration, with intensive sampling pre- and post-treatment. Changes observed during these campaigns were naturally occurring, with patchiness in distribution, life history characteristics, and advection being the most influential factors affecting zooplankton distribution and community composition. To improve our ability to separate treatment effects from seasonal changes in abundance, a long-term sampling programme was undertaken in Loch Sunart from November 2001 to May 2004. This allowed identification of the natural seasonal cycles in abundance and species composition in Loch Sunart and confirmed that sea lice treatment events did not significantly alter the observed seasonal trends.

Phytoplankton Sea lice treatment medicines are designed to interfere with some aspect of the metabolism of the sea louse. The physiology of plants and animals differs in many fundamental respects, however the in-feed chemical teflubenzuron (Calicide) has the potential to directly affect phytoplankton because it targets chitin synthesis. A number of important diatoms (e.g. Cyclotella sp., Thalassiosira sp.) produce chitin as an integral component of the cellular frustule structure, and many other phytoplanktonic organisms produce or metabolise chitin during normal growth. However, Calicide is not widely used in the UK and no farm participating in this study used this product. The most likely mode of impact on phytoplankton is indirect via a change in grazing pressure. Monitoring phytoplankton is, therefore, a good way of assessing the wider scale effects of lice treatments on zooplankton populations.

Despite highly variable hydrographical features and sea lice treatment histories, comparison of the four sea loch phytoplankton communities revealed significant similarity (> 76 %) between them. This reflects the ubiquitous presence of many of the phytoplankton species in Scottish coastal waters. At all lochs, the phytoplankton was typically dominated by a relatively small number of taxa with many other species maintaining low threshold concentrations (e.g. < 1000 cells l-1). These low-biomass species typically exhibited highly variable distributions both temporally and spatially, whereas a number of the more common (and bloom-forming) 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.

The presence or absence of any phytoplankton species could not be attributed to the application of sea lice treatment chemicals 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 normal patterns of zooplankton density.

Ecological effects of sea lice medicines in Scottish sea lochs 7 of 286 Meiobenthos Meiofauna have evoked considerable interest as potential indicators of anthropogenic perturbation in aquatic ecosystems aquatic ecosystems. They have several potential advantages over macrofauna, which have traditionally been the component of the benthos examined in pollution monitoring surveys. These advantages include their small size and high densities (allowing smaller samples to be collected), shorter generation times and the absence of a planktonic phase in their life-cycles. These factors suggest a shorter response time and higher sensitivity to pollution.

In initial samples (1999) at Loch Sunart, a weak gradient in meiofaunal community structure was observed. This could not be related unequivocally to normal organic enrichment from aquaculture activities although the presence of the fish cages close to Station 1 (ca. 200 m west of the cage group) was considered to be the probable cause of the observed differences. In March 2001, no obvious gradient effect was detected, but combined analysis of data from both surveys suggested a mild organic enrichment impact at Station 1.

Abundances, particularly of copepods, were low at Loch Sunart. Sub-sampling of grabs can be an inefficient method for extraction of meiofaunal copepods. Adequate data on copepod abundances are still lacking for this loch and further sampling both here and at other sites would be required to determine whether observed abundances are genuinely low in the area. From the analysis across sites, Sunart did not appear to be highly affected.

Evidence emerged from the first survey data (July 2000) that meiofauna community composition varied between stations at Loch Diabaig, and was obviously different at Station 2 (ca. 300 m NW from the cages), located in the deeper part of the loch. In particular, the copepod assemblage at this station was extremely impoverished and the suite of nematode species found in greater numbers at this site, such as Spirinia parasitifera and Dorylaimopsis punctata, were indicative of muddier sediments. Hydrographic surveys indicated that clockwise rotation of water in the Loch may concentrate material settling from the cages, and any associated contaminants, in the vicinity of this station. There were clear differences in copepod community structure between stations within each survey, and abundance and diversity declined at all stations, particularly Stations 1 (near the farm) and 3 (the reference), between the two sampling periods. The differences observed were most likely caused by natural seasonal variation. On both sampling occasions, Station 2 was dominated by the copepod Typhlamphiascus confusus, suggesting that seasonality may not affect this species, but that it is taking advantage of other conditions such as sediment type or food source. Much of the observed pattern in meiofaunal community structure could be explained by variation in sediment type (proportions of coarse silts for nematodes, medium sands and coarse silt for copepods), and there was little evidence to support the view that sea lice treatments were a major driver of patterns in meiofaunal community structure.

At Loch Craignish, species contributing to differences between stations included species from groups known to be indicators of disturbance and organic enrichment, leading to the conclusion that the observed differences between stations reflect a normal organic enrichment gradient associated with fish farming activity.

At Loch Kishorn, stations were selected on transects very close to the cages in order to cross the normal enrichment gradient where maximum concentrations of in-feed medicine were predicted. Very strong gradients in assemblage structure were apparent in both nematode and copepod assemblages. The impact on nematode assemblages from the cages extended to beyond 50 m, and was relatively stable compared to the impact on

Ecological effects of sea lice medicines in Scottish sea lochs 8 of 286 copepods. There was evidence of a significant and spreading impact on copepod community structure in post-treatment surveys. Using variables measured at each station, there were no significant relationships between variation in nematode community structure and environmental variables. There is evidence that deposition from the adjacent cages impacted nematode and copepod assemblages, and that a combination of changes in sediment composition and the presence of in-feed treatment chemicals were related to observed changes in copepod community structure, although these variables are co-linear with the strong organic enrichment gradient.

Macrobenthos The infaunal element of the sublittoral macrofauna inhabit burrow or tube structures below the sediment surface and, therefore, is relatively sedentary. The epifaunal element either move across the sediment surface or use the upper layers of the sediment as a refuge from predators between feeding excursions into the lower water column. Many such epifaunal groups are small crustaceans with similar taxonomic origins and physiological properties to sea lice and are thus likely to be more responsive to contact with the treatment medicines than other invertebrate groups. However, they are mobile and thus capable of moving out of contaminated areas. On the other hand infaunal populations are dominated by polychaetes, molluscs and echinoderms whose structure and physiology differ markedly from the crustaceans. Thus they might be less susceptible to the treatment effects. However, as they are unable to move rapidly from contaminated areas they may be subjected to longer contact times and higher contamination levels following treatments. In general, we would anticipate declines in the populations of small crustacea in areas downstream of any treatments as the most likely immediate effect that might result from the treatment. Long-term effects might be seen in reductions in those populations of infaunal animals that feed extensively at the sediment surface (surface deposit feeders) and may ingest contaminated particles sedimenting out following treatments.

Macrofaunal communities were analysed from repeated sampling at Lochs Sunart, Diabaig and Kishorn. At Diabaig and Kishorn, the stations formed a gradient away from the cages and there was evidence of a shift in the community structure typical of that on organic enrichment gradients. There was no change in species composition that could be attributed to treatment with sea lice medicines. At Loch Sunart, where the stations were located a little further from the cages there was no strong evidence of any influence by the farm.

Sublittoral settlement and succession The release of sea lice treatment medicines into the environment from sea cages might potentially impact sublittoral communities on hard substrates in those areas downstream from the cages. However, there was little, if any, sublittoral hard ground substrate in the vicinity of any of the farm sites. It was, therefore, decided to provide artificial sublittoral hard substrates in the form of slates suspended at three depths from buoyed arrays. If sea lice medicines were having some effect on communities living on hard substrates, comparison of the composition and successive changes in communities settling on the slates prior to, and after sea lice treatments, would be expected to show lower settlements of certain species (particularly crustacean species such as barnacles) after treatments.

The study of the settlement of organisms on the sublittoral arrays of slate panels suspended at three different depths and at three or four stations in Lochs Sunart, Diabaig and Craignish has yielded a clear picture of the natural seasonal and annual successions and abundances of the fauna and flora involved. At all of these sites and stations the most significant ecological event of every year was the spring settlement of the dominant sublittoral barnacle Balanus crenatus. Barnacle larvae spend about three weeks as

Ecological effects of sea lice medicines in Scottish sea lochs 9 of 286 zooplankton passing through several typically crustacean developmental stages, ending with the larval cyprid stage that settles on solid substrates mainly in the period April to June. At all of the stations the slate panels became heavily colonised and, as the barnacles grew, quickly achieved 100% cover especially at the optimal upper and mid-water depths. Nowhere was the settlement of barnacles at the stations of ‘Highest predicted impact’ different from the reference stations. It follows that any chemicals originating from sea lice treatments at the salmon cages were not affecting the abundant barnacle settlements at any of the sites studied. The overall species diversity of the fauna settled on the sublittoral slates was limited, very similar at all sites studied and mainly determined by the initial heavy settlement of barnacles.

Littoral larval settlement and succession The release of sea lice treatment medicines into the environment from sea cages might potentially impact littoral communities on hard substrates in those areas downstream from the cages. In such areas those organisms firmly attached to the substrate and unable to escape from contaminants are most at risk. Barnacles and mussels (Mytilus edulis) are two of the most abundant invertebrates on Scottish rocky shores. The former are known to be sensitive to many biocides and have long been used as target organisms in antifouling tests. The latter are known to accumulate pollutants such as metals and pesticides and have been used as target organisms in many pollution monitoring studies. Both have juvenile larval stages that are initially planktonic prior to settlement and taking up fixed positions.

Barnacles (mainly Semibalanus balanoides) are the only sedentary crustaceans that settle predictably on all Scottish rocky shores in high numbers and thus were chosen as the appropriate target organism for studies aimed at detection of any effects of the sea lice treatments. The highest densities of barnacle settlement occurred, predictably, at Loch Diabaig reference Station A (the most exposed to wave action) with up to 100 % cover. Conversely, the very poor settlement of barnacles at Loch Sunart Stations A and B, Loch Craignish Station A and Loch Kishorn Station A were the result of the very sheltered nature of these locations, with dominance by fucoids and mussels on the adjacent boulders and the very limited areas of bedrock.

The low frequency of developed egg masses in 2000 and 2001 cohorts of S. balanoides on settlement panels at the predicted high impact station at Loch Diabaig was possibly related to discharge from the fish farm but a number of natural processes could also produce the same effects. At Lochs Sunart, Kishorn and Craignish there were no consistent trends which could reflect impacts of the fish farms.

Conclusions 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 have attempted to look beyond ephemeral, local effects and have concentrated on trying to detect long-term changes beyond the immediate mixing zone. This has been a difficult task and, taking all the results together, we have not been able to 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 have observed are well within the range of what might be expected or predicted for fjordic sea loch systems.

The question should be asked as to whether our experimental design leant itself to detecting subtle effects over long time-scales against inherent natural variability in marine ecosystems. The answer is that, in order to reliably detect such subtle changes, longer

Ecological effects of sea lice medicines in Scottish sea lochs 10 of 286 time-series of data would be required from systems where there was greater coupling between the commercial management of the farms and the scientific needs of the project. Nevertheless, the project has achieved much by helping to improve our understanding of natural variability in relatively unresearched 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.

Ecological effects of sea lice medicines in Scottish sea lochs 11 of 286 2 Introduction 2.1 The sea loch environment: physical, biogeochemical and ecological background Aquaculture activity is ubiquitous on the west coast of Scotland. The predominant physical coastal form is the fjordic sea loch. These indentations offer shelter from the shelf seas and, therefore, have been the locations of choice for the expanding industry. New sites in sea lochs have become more difficult to obtain so the Sounds and more exposed coastal areas have begun to be used for aquaculture, but the vast majority of aquaculture sites are still located within sea loch environments.

Sea lochs were formed during the last period of mid-latitude glaciation that ended around 12000 BP. Many have shallow sills dividing the loch interior into basins. Silled lochs restrict the exchange of deep water for periods from days to years - for many lochs the period of deep water renewal, and the driving processes that contribute to this, have not been determined, although much can be deduced from basic geographical information such as catchment area and bathymetry. These basic statistics have been conveniently catalogued by Edwards and Sharples (1986) for 110 sea loch systems.

Sea lochs provide shelter from waves and wind but this shelter itself results in restrictions in the exchange of water with the coastal environment that may be detrimental to aquaculture activity and the ecosystem it interacts with. Organic inputs to sediments, either directly or through stimulated primary production, could enhance organic mater flux to isolated bottom-waters thus increasing the rate of oxygen depletion. Nutrients from farms may be able to stimulate local primary production and the microbial loop if they are retained long enough in the system. The rapid dilution of medicines and other chemical effluents, such as anti-foulants, may be restricted allowing them to remain at ecotoxicologically significant concentrations. These issues and others are briefly discussed below and, more fully, in a recent review for the Scottish Executive and Parliament Committee (Black et al., 2002) and the last of these issues is the subject of the research project reported here.

The typical sea loch ecosystem can be simply divided into 3 areas: littoral, benthic and pelagic although each is interconnected by transfers of materials, biota and genes. Compared to many other estuary types, sea lochs typically have a rather low proportion of intertidal area to total surface area although there are some exceptions to this where sandy mud areas are exposed at low tide. Substrata range from bedrock through boulders, cobbles and gravel to sand depending on the degree of exposure to physical energy from waves and tides and the predominant life-forms vary accordingly. The substrate at the mouth of the loch is commonly hard, composed of bedrock outcrops and boulders dominated by barnacles with submarine kelp forests, changing progressively to soft muddy sand at the sheltered head dominated by sea weeds with a community highly adapted to low and rapidly varying salinity.

The deep areas of sea lochs are typically dominated by soft sediment communities. Maximum depths vary between about 50 m to nearly 200 m. Large areas may be sheltered from high currents allowing fine particulate material to accumulate providing refuge for a large number of epi- and infaunal species. These range in size from the so-called megafauna, which through foraging and burrowing may interact with their environment at a scale of metres both vertically in the sediment and horizontally, through to the meiofauna - small animals that typically occupy a narrow depth range near the sediment surface and interact with sediments at the millimetre scale. Sediments are typically rich in organic material and are thus usually anoxic within a few millimetres of the sediment

Ecological effects of sea lice medicines in Scottish sea lochs 12 of 286 surface thus at depth being dominated by bacteria capable of utilising other electron acceptors such as sulphate.

The sea loch water-column is typically high in nutrients during the winter as a consequence of inflows from coastal waters, mixing of deeper waters, nutrient flux from sediments and runoff from the land. In spring, increasing light energy allows utilisation of these nutrients by phytoplankton. The spring bloom is typically dominated by fast growing populations of diatoms. These require silicate for construction of their external skeleton and, when this silicate is depleted, they are replaced by phytoplankton groups not requiring silicate such as dinoflagellates. High levels of primary production can deplete the surface waters of sea lochs of nutrients in the summer although nutrients are often not the major factor limiting production. As lochs are typically highly turbid with low light penetration, light may often be the limiting factor. Nutrients are thought to be efficiently recycled through the microbial loop comprised of bacteria and protozoa that can process dead cells, cell fragments and dissolved organic material. Some species can also use light as an energy source when available.

Above the microplankton in the trophic web are the meso-zooplankton, small (200 - 2000 µm) animals capable of grazing large amounts of algae and thought to control phytoplankton abundance in many systems (e.g. Ross et al. 1993). In addition to these permanent members of the zooplankton (holoplankton) are the many species with transient planktonic larval stages (meroplankton). These include larvae of such diverse species as many polychaete worms and crustacea e.g. barnacles and parasitic sea lice.

The complex sea loch food chain is completed by a diverse range of fish, birds and mammals including otters, seals and cetaceans. Each component of the ecosystem from bacteria to dolphins has the potential for direct or indirect interaction with aquaculture activities.

2.2 Aquaculture in Scotland Aquaculture in Scotland has developed most rapidly in the past 2 decades and, although a variety of species are cultivated, production is dominated by Atlantic Salmon. In 2001 salmon production was estimated at 139000 tonnes with 5500 tonnes of rainbow trout and 3000 tonnes of cultivated shellfish (Scottish Executive, 2003). Salmon production is split between a freshwater juvenile phase followed by a transfer to sea water - almost exclusively to net cage farms in coastal waters. In 2001 a total of 318 fish farm sites were active, concentrated on the west coast mainland, the Hebrides and northern Isles (FRS, 2002).

2.3 Sea lice 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 high numbers of lice larvae are likely to 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). The SEPA website has considerable background information on these tests. Discharge Consents are granted such that the concentration that is discharged is kept

Ecological effects of sea lice medicines in Scottish sea lochs 13 of 286 below these EQS levels 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. This project, therefore, has been 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.

2.4 Short review of ecological effects of aquaculture Research on the interactions of aquaculture with the environment have been reviewed on several occasions (e.g. Pearson and Black, 2001; Davenport et al., 2003). A critical analysis of the relative importance of the various impact types has also been attempted (Black et al., 2002).

2.4.1 The discharges of waste nutrients An important concept is that of the Allowable Zone of Effects (AZE). For any discharge, however slight, some effect will be observable very close to the source. The AZE concept acknowledges that discharges may have some impact but that this should be constrained by a quality standard which will be less stringent within this zone. For organic impacts on sediments this zone is defined as the area within 25 m from around the perimeter of the fish cages in a farm. Effects within this zone are monitored to ensure that they meet pre- defined sediment quality criteria. Sediments outside this zone are monitored to ensure that they are close to the ‘quality’ of distant reference stations. Thus some deterioration of quality is permitted but only within a small distinct zone. For dissolved constituents, the same process is applied but for some effluents the criteria may be defined in terms of a time after discharge rather than an area.

Organic wastes from fish farming (faeces and uneaten food) can affect the sediments beneath fish cages causing changes in the seabed ecology that are typically found across any organic enrichment gradient. Farms are closely regulated to ensure that the spread of wastes and their effects on sediments are limited in scale to the area immediately around the farm. This is done by setting an upper limit for the biomass of fish to be held on a site, from which can be made predictions of the maximum amount of waste that will be generated. Hydrographic data on current speeds and bathymetry are entered into simple models that can predict the scale of likely effect and thus allow tailoring of the biomass limit to the assimilative capacity of the site. Benthic communities exposed to organic enrichment show decreases in diversity and increases in abundance of smaller, opportunistic organisms. The process is reversible (Pereira et al., 2004) and recovery will be essentially complete within a few months to years after inputs cease depending on site conditions.

Dissolved nutrients can be dispersed over a wide area but, on the basis of current understanding, it appears that nutrients from fish farms currently make only a small contribution to algal production and probably do not directly affect toxicity either by promoting toxic strains or increasing the toxicity of toxic strains. These conclusions are based to a large extent on the results of modelling studies, which need to be supported by the collection of appropriate long term data.

Ecological effects of sea lice medicines in Scottish sea lochs 14 of 286 Shellfish farms produce much more limited local waste than finfish farms and the issue of carrying capacity revolves around establishing that there are sufficient planktonic organisms in the water to grow a given biomass without seriously depleting the resource. For many areas of Scotland this is unlikely to be a major problem even should there be a major expansion of the shellfish farming industry.

2.4.2 Medicines and chemicals A variety of medicines are used on fish farms. The most important in terms of potential impacts are thought to be sea lice treatment medicines and metal-based anti-foulant. Although these products are used under controlled conditions such as to protect the environment using the Ecological Quality Standards concept, there are still many important research gaps. The EQS, however, is used widely to overcome limited knowledge of any substance through the application of high safety factors in a range of discharge types.

2.5 Sea lice biology and medicines used to control sea lice Salmon farms in Scotland are affected by sea lice, Lepeophtheirus salmonis and to a lesser extent Caligus elongatus, 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 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. 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.

2.6 Ecotoxicology of sea lice medicines 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, 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 Scotland, Discharge Consents are only being granted for azamethiphos, cypermethrin, hydrogen peroxide, emamectin benzoate and teflubenzuron.

2.6.1 Azamethiphos (Salmosan) Azamethiphos, an organophosphate pesticide, is the active of ingredient (47.5 % w/w) of Salmosan, and acts as a cholinesterase inhibitor. The recommended application rate for lice control is 0.1 mg l-1 as a bath immersion treatment for 30 minutes to 1 hour, depending on temperature. Azamethiphos is likely to remain in the aqueous phase on entering the environment as a consequence of its high water solubility and half life (Table 2.1). Bioaccumulation of azamethiphos is unlikely to be a problem and its degradation products are not toxic. Dispersion modelling predicts that concentrations around a treated cage would fall below 0.2 µg l-1 within 24 h in a poorly flushed site, and 0.02 µg l-1 in a more energetic system (Turrell, 1994). Modelling of the long-term distribution of single azamethiphos treatments in 54 Scottish sea lochs predicted annual mean concentrations in the surface waters of 0.02 to 4.5 ng l-1. This suggests that the PEC (Predicted

Ecological effects of sea lice medicines in Scottish sea lochs 15 of 286 Environmental Concentration derived by modelling dispersion) could approach or exceed the PNEC (Predicted No Effect Concentration derived from toxicity data; Table 2.1) in approximately 10 % of surface waters in Scottish sea lochs. It is considered unlikely that azamethiphos would accumulate in sediments in detectable concentrations as it is likely to remain in the aqueous phase due to its high water solubility, and dispersion studies indicate treatment plumes would disperse within the upper 10 m of the water column.

Field studies in Scotland using deployed mussels and lobster larvae indicate that effects on marine organisms in the vicinity of treated cages are unlikely (SEPA, 1997). A dispersion and toxicity study undertaken in the Lower Bay of Fundy, New Brunswick, at sites displaying a range of dispersive energy conditions, concluded that azamethiphos presented a low to moderate environmental risk (Ernst et al., 2001). In the Canadian study, water samples were collected from dispersing treatment plumes over a five hour period and their toxicity to the benthic amphipod Eohaustorius estuaris tested in 48 h exposures. Very few of the water samples displayed any toxicity.

Table 2.1. Treatment rates, Environmental Quality Standards (EQS) and physical properties of the sea lice treatment chemicals cypermethrin, emamectin benzoate, azamethiphos and teflubenzuron. Cypermethrin Emamectin benzoate Azamethiphos Teflubenzuron Trade name Excis Slice Salmosan Calicide Compound group Pyrethroid Avermectin Organophosphate Benzoyl urea Treatment dosage 5 µg l-1 for 60 min 50 µg kg-1 for 7 d 0.1 mg l-1 for 30-60 min 10 mg kg-1 for 7 d Water column EQS PNEC: No EQS 0.22 ng l-1 5 ng l-1 (72 h) No EQS Annual average: 0.05 ng l-1 No EQS No EQS 6 ng l-1 MAC: 0.5 ng l-1 (24 h) No EQS 150 ng l-1 (24 h) 30 ng l-1 16 ng l-1 (3 h) 250 ng l-1 (3 h) Sediment EQS Far-field PNEC1: No EQS 0.763 µg kg-1 wet wt No EQS 2 µg kg-1 dry wt (5 cm sediment depth) 2 Near field PNEC : No EQS 7.63 µg kg-1 wet wt No EQS 10 mg kg-1 dry wt (5 cm sediment depth) Half life 35 - 80 d 164 - 175 d 9 d 104 - 123 d Water solubility 5-10 µg l-1 550 µg l-1 1.1 g l-1 3 µg l-1 Log Kow 6.3 5.0 1.05 4.56 References: SEPA, 1997; 1998; 1999a,b 1“ a consent-limiting concentration of chemical permitted within the sea bed sediment” (SEPA, 2003a) 2“a non consent-limiting concentration of chemical permitted within the sea bed sediment which, if exceeded, will trigger a requirement for enhanced monitoring” (SEPA, 2003a)

Current azamethiphos use for sea lice control on salmon farms is limited and will probably continue to decline as the use of in-feed treatments increases. At present, azamethiphos is most often used in conjunction with cypermethrin treatments when lice numbers necessitate control measures but farms have reached their discharge consent limits for cypermethrin.

2.6.2 Cypermethrin (Excis) Cypermethrin, a synthetic pyrethroid pesticide, is the active ingredient (1 % w/v) of Excis and acts on the nervous system by increasing sodium permeability of the nerve membrane during excitation, resulting in prolonged nerve stimulation (WHO, 1989). Cypermethrin is applied as a bath immersion treatment to control sea lice at a concentration of 5 µg l-1 for one hour. Cypermethrin has low water solubility and degrades rapidly (Table 2.1). It binds strongly to organic particles and other solids, and is rapidly adsorbed by sediments, reducing its biological availability and hence its toxicity to benthic organisms. The main degradation products of cypermethrin are approximately 1000 times less toxic than the parent compound (WHO, 1989). Cypermethrin is unlikely to accumulate in fish as they readily metabolised pyrethroids (Bradbury & Coats, 1989). Bioconcentration factors are greater in shellfish, apparently due to slower rates of metabolism and depuration or exposures through feeding and aqueous uptake (Clark et al., 1989). A concentration

Ecological effects of sea lice medicines in Scottish sea lochs 16 of 286 factor of 26.5 was calculated for mussels exposed to Excis in a treated cage, and the time to 50 % depuration for mussels exposed over seven treatments was 34.6 h (SEPA, 1998).

Cypermethrin released following a bath treatment will be rapidly diluted in the receiving environment and the majority will be adsorbed onto particulate material, which will settle to the sea bed. Dispersion modelling of a single cage treatment incorporating a water borne phase followed by adsorption to particulate material, estimated that after 3.2 h, maximum cypermethrin concentrations in the dispersing treatment plume would be 20 ng l-1, and that settlement would be complete after 14.5 h (Turrell & Gillibrand, 1995). Hence, the PNEC (Table 2.1) would be exceeded in the dispersing plume for a short period after treatment, creating the potential for short term toxic effects. In terms of the sediment, cypermethrin concentrations of 0.3 to 1.0 µg kg-1 (dry sediment) are estimated, which are unlikely to result in toxic effects on sediment dwelling organisms.

Cypermethrin has been shown to be extremely toxic to crustaceans. In laboratory studies, mortality following 96 h exposures to cypermethrin has been reported at concentrations as low as 5 ng l-1 for the shrimp Mysidopsis bahia (Hill, 1985) and nauplii of the copepod Acartia tonsa (Medina et al., 2002), three orders of magnitude lower than treatment concentrations, but still lower than the 3 h EQS (Table 2.1).

Several studies have monitored treatment plume dispersion from salmon cages. In Scotland, cypermethrin dispersed rapidly upon release from salmon cages in Loch Eil (Hunter & Fraser, 1995). At a distance of 25 m from the cages the highest concentration measured was 187 ng l-1 25 min after release. No mortality was observed in shrimps (Crangon crangon) deployed near the cages and attached to drogues released with the treatment plume. Ernst et al. (2001) investigated the dispersion and toxicity of cypermethrin released from salmon pens at four sites with different dispersive characteristics in the Lower Bay of Fundy, New Brunswick. They concluded that even a single cage application of cypermethrin has the potential to create a lethal plume of up to 1 km2, and that the plume may retain its toxicity for substantial periods of time. In their study, water samples collected up to five hours post-treatment were toxic to the benthic amphipod, E. estuarius, causing immobilisation during 48 h exposures.

Dispersion modelling and field based studies have focussed on single treatment releases. In reality, cypermethrin treatments involve multiple releases daily, usually over several consecutive days. Furthermore, there may be several fish farms treating in a system at the same time. The dispersion and cumulative effects of multiple treatment releases on the marine environment are less well understood than that of single treatments. The potential for cypermethrin concentrations to exceed water and sediment EQS is increased with multiple treatment events, with the consequent ecological effects unknown.

2.6.3 Emamectin benzoate (Slice) Emamectin benzoate, a semi-synthetic avermectin, is the active ingredient of Slice, and acts by increasing membrane permeability to chloride ions and disrupting physiological processes. Emamectin is administered orally as an in-feed additive at a rate of 50 µg kg-1 body weight per day, for seven days. Approximately 90 % of orally administered emamectin is absorbed by the salmon, with the remaining 10 % excreted immediately in the faeces. Excretion of emamectin continues for a considerable time post-treatment, with the depuration half-life in salmon estimated as 36 d at 10 ºC. Metabolites of emamectin benzoate, which may display similar or reduced toxicity, will also be excreted for a considerable time post-treatment (SEPA, 1999a).

Emamectin benzoate has low sea water solubility and a high potential to be adsorbed and bound to suspended particulate material and sediments, with a relatively long half-life in

Ecological effects of sea lice medicines in Scottish sea lochs 17 of 286 anaerobic sediments (Table 2.1). Given the treatment method (in-feed) and the physical properties of emamectin benzoate, most of the emamectin reaching the sediments will be associated with particulate material in the form of fish faeces and uneaten food pellets. Therefore, the organisms most likely to be affected by the chemical are those closely associated with the sediment.

Benthic communities in the organically enriched sediments below fish farm cages are generally dominated by capitellid polychaetes, which play a vital role in remineralising waste products. A recent study (Nickell et al., 2004) on the effects of emamectin benzoate on infaunal polychaetes indicates that sediment emamectin concentrations predicted by DEPOMOD are unlikely to adversely affect polychaete communities below fish farm cages. There were significantly fewer surviving worms in the 10000 µg kg-1 nominal (1120 µg kg-1 measured) treatment than in the vehicle controls, and it was concluded that the NOEC value was 1000 µg kg-1 nominal (460 µg kg-1 measured), considerably higher than sediment PNECs (Table 2.1).

Water column concentrations are expected to be considerably lower than sediment concentrations and are unlikely to pose a risk to planktonic organisms. Results from laboratory toxicity tests support this conclusion, with acute toxicity values orders of magnitude higher than the water column MAC (Table 2.1). However, American lobsters have been shown to moult prematurely following exposure to 1 µg g-1 body weight emamectin benzoate (Waddy et al., 2002).

Emamectin benzoate use for sea lice control is increasing in Scotland. In many loch systems, strategic treatments are being undertaken simultaneously at several farm sites within a loch system.

2.6.4 Teflubenzuron (Calicide) Teflubenzuron, a benzoyl urea insecticide, is the active ingredient of Calicide (0.2 % w/w), and acts as a moult inhibitor by preventing the formation of chitin, the predominant component of arthropod and crustacean exoskeletons. The specific mode of action of teflubenzuron means it is highly toxic to aquatic crustacean invertbrates, but low in toxicity to fish, mammals and birds. Teflubenzuron is administered orally as an in-feed additive at a rate of 10 mg kg-1 body weight per day, for seven days (SEPA, 1999b). It has been estimated that salmon retain only around 10 % of the administered dose, the remaining 90 % is excreted via the faeces in the period immediately following treatment. Teflubenzuron may also enter the marine environment with uneaten feed. Teflubenzuron has low water solubility, a relatively long half life in sediments (Table 2.1), with a high potential to be adsorbed and bound to suspended particulate material and sediments. As with emamectin benzoate, it is likely that the sediments will act as a sink for teflubenzuron, therefore sediment associated organisms are most likely to be affected by this chemical.

Field trials undertaken in Scotland (Loch Eil) have examined the fate and dispersion of teflubenzuron under treatment conditions. Measurable concentrations were generally not present in the water column after treatment, and sediment levels followed the predicted dispersion with measurable levels initially extending about 50 m from the cages in the direction of current flow. Measurable levels of teflubenzuron were found up to 1000 m from the cages during the study but it was estimated that by 645 d, most (98 %) of the teflubenzuron had been degraded or dispersed from the site.

A recent study investigating the toxicity of sea lice chemotherapeutants to non-target -1 planktonic copepods determined 48 h EC50 values ranging between 0.12 and 2.44 µg l teflubenzuron (nominal concentrations) for naupliar and pre-adult stages (Willis & Ling,

Ecological effects of sea lice medicines in Scottish sea lochs 18 of 286 unpubl. data). Adult copepods were unaffected. These values are orders of magnitude higher than water column EQS (Table 2.1). Discharge consents are being granted for the use of teflubenzuron as a sea lice medicine in Scotland, but it is not being widely used, primarily because it is not effective against adult sea lice. Consequently, if adult lice are present, numbers of lice may quickly return to pre-treatment levels, necessitating further treatments with teflubenzuron or another treatment medicine.

2.7 Potential effects in 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. Planktonic copepods have a similar life cycle to ectoparasitic copepods (sea lice) and are thus the group most likely to be adversely affected by sea lice medicines, which are highly toxic to crustaceans. 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.

Zooplankton are most likely to be exposed to cypermethrin and azamethiphos in their soluble forms given their mode of administration as bath treatments. In the immediate post-treatment period, zooplankton entrained within cypermethrin and azamethiphos treatment plumes may be exposed to potentially lethal concentrations for several hours. Azamethiphos is likely to remain in aqueous phase due to its high water solubility, whereas cypermethrin is predicted to adhere to particulate material in the water column providing a further route of exposure to zooplankton through ingestion. In principle, zooplankton are only likely to be exposed to toxic levels of these medicines in a relatively small area around the farm and for only a short period around relatively infrequent treatment events.

Zooplankton have a lower risk of exposure to emamectin benzoate and teflubenzuron as they are administered as components of the salmon feed. The most plausible exposure route is associated with ingestion of feed and faecal particles. Ninety percent of teflubenzuron is excreted immediately following treatment, whereas excretion of emamectin benzoate by salmon continues for an extended period post-treatment, increasing the duration of any exposure to particulate associated emamectin. Resuspension of freshly deposited material will also reintroduce emamectin and teflubenzuron to the water column for a considerable period post-treatment thus making them potentially available.

2.8 Potential effects in phytoplankton A number of important diatoms (e.g. Cyclotella sp., Thalassiosira sp.) produce chitin as an integral component of the cellular frustule structure, and many other phytoplanktonic organisms produce or metabolise chitin during normal growth (Smucker, 1991). In addition, teflubenzuron remains largely bound to organic matter and sediment particles following release, so if a treatment coincided with high phytoplankton biomass (e.g. a bloom) then cells could be exposed to the chemical for an extended period of time. Sedimentation of the bloom biomass could rapidly transport teflubenzuron to the benthos, where other groups of organisms (primarily animals) could potentially be directly affected by the chitin inhibitor.

Bath treatments of sea lice treatment medicines (e.g. organophosphates and pyrethroids) expose phytoplankton cells directly to relatively higher concentrations of chemical than

Ecological effects of sea lice medicines in Scottish sea lochs 19 of 286 in-feed treatments. Generally, however, impacts of sea lice treatments on the phytoplankton are more likely to be indirect due to interaction of the treatment chemical with zooplanktonic grazing organisms, such as copepods. Phytoplankton community structure and cell abundance may be affected if certain elements of the zooplankton are affected by the treatment chemicals when they are released to the environment and thereby grazing dynamics are altered. However, a complex suite of environmental factors (e.g. light availability and nutrient fluctuations) directly affect the growth of individual phytoplankton species, and hence alter the phytoplankton community structure and size on continual short- and long-term bases. Under such circumstances, it was anticipated that separating a possibly small effect of altered grazing pressure from the influence of other significant environmental factors would require a robust and reproducible dataset encompassing a wide range of environmental conditions and phytoplankton community composition characteristics.

2.9 Potential effects in meiofauna Meiofauna have evoked considerable interest as potential indicators of anthropogenic perturbation in aquatic ecosystems (see review by Coull & Chandler 1992) as they have several potential advantages over macrofauna, which have traditionally been the component of the benthos examined in pollution monitoring surveys. These include their small size and high densities (allowing smaller samples to be collected), shorter generation times and no planktonic phase in their life-cycles. These factors suggest a potentially shorter response time and higher sensitivity to pollution (Heip et al. 1988; Warwick 1993). Although the responses of different groups of organisms to certain types of perturbation might be expected to differ, there are few studies in which the impact of anthropogenic disturbance on more than one component of the biota has been examined directly. Meiofaunal communities in shallow sublittoral sediments are generally dominated by nematodes and copepods. The analyses here, therefore, are confined to these two taxa.

Although some studies have described variation in meiofaunal communities in the vicinity of aquacultural structures these have tended to focus on organic enrichment effects, and to the authors’ knowledge none have specifically examined the effects of sea lice control agents. The authors do not, therefore, have any data or studies with which to compare their own.

2.10 Potential effects in littoral settlement The release of sea lice treatment medicines into the environment from sea cages might potentially impact littoral populations on hard substrates in those areas downstream from the cages. In such areas those organisms firmly attached to the substrate and unable to escape from contaminants are most at risk. Barnacles and mussels (Mytilus edulis) are two of the most abundant invertebrates on Scottish rocky shores. The former are known to be sensitive to many biocides and have long been used as target organisms in antifouling tests. The latter are known to accumulate pollutants such as metals and pesticides and have been used as target organisms in many pollution monitoring studies. Both have juvenile larval stages that are initially planktonic prior to settlement and taking up fixed positions.

In view of their ubiquity on the rocky shores in the vicinity of the sea cage sites, it was thought that the health of populations of the intertidal barnacle, Semibalanus balanoides, in areas that might be subject to contamination by the sea lice treatment agents, would be a good indicator of any effects on hard ground organisms. As barnacles and sea lice are crustaceans, the selection of the former as a target organism seemed appropriate.

Ecological effects of sea lice medicines in Scottish sea lochs 20 of 286 Semibalanus balanoides has predictable and usually abundant annual settlements of spat. It was thought that such settlements might be particularly vulnerable to the effects of the farm treatments, possibly through effects on the planktonic juvenile stages, and affecting settlement rates in impacted areas. In addition the treatment chemicals could possibly affect the growth and development of the sessile adults after settlement. Thus the treatments may also have an effect on the reproductive capacity of this species. The settlement and development of the populations, and the frequency of occurrence of fully developed fertilised egg masses in individuals were therefore monitored in each of the study areas.

2.11 Potential effects in sublittoral settlement The release of sea lice treatment medicines into the environment from sea cages might potentially impact sublittoral populations on hard substrates in those areas downstream from the cages. However, there were little, if any, sublittoral hard ground substrates in the vicinity of any of the farm sites. It was, therefore, decided to provide artificial sublittoral hard substrates in the form of slates suspended at three depths from buoyed arrays. Comparison of the composition and successive changes in communities settling on the slates prior to, and after sea lice treatments, may have shown lower settlements of certain species (particularly crustacean species such as barnacles) after treatments, or on arrays situated closest to the cages.

2.12 Potential effects in macrofauna The infaunal element of the sublittoral macrofauna inhabit burrow or tube structures below the sediment surface and, therefore, is relatively sedentary. The epifaunal element either move across the sediment surface or use the upper layers of the sediment as a refuge from predators between feeding excursions into the lower water column. Many such epifaunal groups are small crustaceans with similar taxonomic origins and physiological properties to sea lice and are thus likely to be more responsive to contact with the treatment medicines than other invertebrate groups. However, they are mobile and thus capable of moving out of contaminated areas. On the other hand infaunal populations are dominated by polychaetes, molluscs and echinoderms whose structure and physiology differ markedly from the crustaceans. Thus they might be less susceptible to the treatment effects. However, as they are unable to move rapidly from contaminated areas they may be subjected to longer contact times and higher contamination levels following treatments. In general we would anticipate declines in the populations of small Crustacea in areas downstream of any treatments as the most likely immediate effect that might result from the treatment. Long-term effects might be seen in reductions in those populations of infaunal animals that feed extensively at the sediment surface (surface deposit feeders) and may ingest contaminated particles sedimenting out following treatments.

Ecological effects of sea lice medicines in Scottish sea lochs 21 of 286 3 Methods 3.1 Site selection philosophy The researchers had no influence on the use of sea lice medicines at fish farms: decisions on treatments are based on fish health grounds. An experimental approach was, therefore, not possible. Instead, our philosophy was to measure ecological parameters that might be influenced by any use of these substances. Thus, advanced information on the use of medicines was not essential to the project, although this was generaly made available by the farmers. The initial selection process was influenced by the restrictions on access and uncertainties in farm management plans as a consequence of the ISA outbreak in 2000. As a consequence, some of the study sites chosen were further away than logistically optimal for the range of sampling activities undertaken. In order for the study to provide representative results, it was important to choose sites that had access (or planned to have access) to the major medicines available, that they were taken from hydrodynamically distinct water bodies, that they covered a reasonable spectrum of site size and loch size, and that they covered a range of ambient current regimes and exposures. In addition, logistical constraints such as having the appropriate depth for diver studies, access to boats and distance from the main contractor in Oban were important. Ultimately, four fish farms on the Scottish west coast were identified as being appropriate for study (one each in Lochs Sunart, Diabaig, Kishorn and Craignish).

Sampling at the fish farm near the head of Loch Sunart between 1999 and 2004 resulted in the longest time series of all sites. This site initially used Excis (cypermethrin) with occasional use of Salmosan (azamethiphos). In 2002, Slice (emamectin benzoate) was used twice and Salartect (hydrogen peroxide) once. The Loch Diabaig site was sampled between 1999 and 2002, but most sampling ceased by the end of 2001. This site used Excis, Salmosan and Slice but the high level of winter exposure caused damage to moored equipment which subsequently influenced sampling programmes. The site in Loch Craignish was intensively sampled in 2000, however, most sampling was discontinued in 2001 as relatively few sea lice treatments were carried out on this small site. During this period there were only three treatments, two with Salartect and one with Excis, and no consent application had been made for Slice. As a result, sampling effort was transferred to Loch Kishorn in 2001 as a Slice consent had been granted. Sampling continued until the end of 2002 when the site became fallow. Time lines were constructed for each site showing information on sampling and management events (Loch Sunart - Table 4.2, page 16, Loch Diabaig - Table 2.1, page 104, Loch Kishorn - Table 6.2, page 16, Loch Craignish - Table 7.2, page 176).

3.2 Sample collection and analysis 3.2.1 Zooplankton To determine the effects of sea lice medicines on the zooplankton community, sampling campaigns were undertaken at fish farms using bath treatments of Excis and Salmosan, and the in-feed treatment Slice. Zooplankton samples were collected using a small vessel before, during, and after sea lice treatments from four or five sample stations at increasing distances from the cages. In Lochs Sunart and Kishorn, stations were positioned at distances 0 and 100 m to the east and west of the cage group at fixed moorings. A reference station was also positioned at a fixed mooring approximately 400-500 m from the cage group (Loch Sunart, Figure 4.16, page 29; Loch Kishorn, Figure 6.10, page 29). In Loch Craignish, zooplankton samples were collected from four stations positioned at increasing distance from the two cage groups, which were positioned in the narrow channel on the south side of Loch Craignish (Figure 7.4, page 179).

Zooplankton samples were collected using a 120 µm mesh plankton net hauled by hand from a maximum depth of 24 m. To account for the patchy distribution of zooplankton

Ecological effects of sea lice medicines in Scottish sea lochs 22 of 286 communities, five replicate samples were collected and pooled for analysis at each station. Pooled samples were concentrated and preserved in 5 % buffered formalin.

During the early stages of this study, sampling campaigns were of short duration and included pre- and post-treatment sampling periods. Intensive sampling during the treatment period attempted to establish whether sea lice treatments adversely affect zooplankton communities during and immediately after treatment. A year-long fortnightly sampling campaign was undertaken in Loch Craignish between 2000 and 2001 incorporating two sea lice treatments with Salartect and one with Excis at the very end of the sampling programme (August 2001). To undertake sampling which incorporated more treatments, a long term zooplankton monitoring programme was established in Loch Sunart in November 2001. This involved fortnightly sample collection over a period of 2.5 years until May 2004. During this period, sea lice treatments at the site included Excis, Salmosan, Salartect and Slice (Table 4.1, page 16). The emphasis of the monitoring programme was to investigate whether sea lice treatments cause long-term changes in zooplankton communities in Scottish sea lochs.

In the laboratory, zooplankton samples were resuspended with distilled water and made up to known volumes in a measuring cylinder. Appropriate dilutions were chosen to give counts of 50 to 1000 individuals of the dominant taxa per sub-sample. After thorough mixing, 5 ml sub-samples were transferred to a gridded Perspex counting tray mounted on the movable stage of a model SZX9 Olympus stereomicroscope. Two sub-samples were counted as a greater dilution was generally required to count Oithona similis, often the most abundant species. Copepod adults (C6) and copepodites (C1 to C5) were separated according to species and life stage and separated into two groups C1 to C3 and C4 to C5. Copepod nauplii were not separated according to species, cladocerans were identified to genus. Texts consulted for copepod species and life stage identification were Sars (1903, 1918), Corkett (1967), Uchima (1979), Klein Breteler (1982) and Nishida (1985). Sea lice (Lepeophtheirus salmonis) nauplii and copepodites were identified according to Johnson & Albright (1991).

3.2.2 Phytoplankton, nutrients and salinity Long-term monitoring programmes were established at Lochs Sunart, Diabaig and Craignish to sample a range of environments, improve the probability of identifying intermittent impacts that may be associated with sea lice treatment events, and to provide information on the natural background variation of phytoplankton communities. These high resolution datasets were essential if changes in phytoplankton communities from sea lice treatment events were to be distinguished from variation in seasonal and environmental factors (e.g. nutrients and salinity). Essential supporting trophic information was also provided for the zooplankton dataset. Several intensive short-term monitoring campaigns were also undertaken during sea lice treatment events at Loch Sunart (November 2000 - Excis) and Loch Kishorn (July 2001 - Slice).

A weighted plastic hose (10 m) with valves at both ends was deployed using either a vessel or personnel standing on the cages. This was slowly lowered into the loch to collect an integrated water column sample (4.5 l) covering the top 10 m. Well-mixed aliquots were removed for the analyses of dissolved inorganic nutrients, salinity, and for the identification and enumeration of phytoplankton species.

A phytoplankton sample (1 l) was transferred to an opaque plastic Nalgene bottle and preserved with Lugol's iodine (5 ml). The contents were transferred to amber glass bottles at the laboratory in order to minimise leaching of iodine into the plastic. Nutrient samples (75 ml) were filtered (sterile 0.2 µm filters) into either glass bottles (ToxN (nitrate plus nitrite), phosphate and ammonium; 100 ml) or plastic bottles (silicate; 100 ml). The

Ecological effects of sea lice medicines in Scottish sea lochs 23 of 286 ToxN/ phosphate/ammonium sample was frozen (-20 ºC) on return to the laboratory, while the silicate sample was kept cool (5 to 15 ºC depending on season) in the dark until analysis. A salinity sample (150 ml) was collected into a glass bottle.

Phytoplankton samples were prepared for analysis by settling well-mixed 50 ml aliquots in graduated cylinders for at least 40 h, then resettling the bottom 10 ml in bespoke sample pots for at least 4 h. Analysis consisted of the identification of the first 500 live/healthy (at the time of preservation) phytoplankton cells to the lowest taxonomic level possible using a Zeiss Axiovert 10 inverted light microscope. Texts consulted for phytoplankton identification were Bérard-Therriault et al. (1999), Dodge (1982), Drebes (1974), Hallegraeff (1988), Horner (2002), Ricard (1987), Round et al. (1990), Sournia (1986) and Tomas (1997a,b; 1993).

Nutrients were analysed as described by Shaw et al. (2003) using methods accredited under ISO 17025 by the United Kingdom Accreditation Service (UKAS). The standard deviation of the nutrient analyses was typically 2 to 4 %, with detection limits of 0.01 to 0.1 µM.

Salinity analysis followed established procedures using a calibrated pumped Guildline Portasal Salinometer 8410A. The method has an accuracy of ± 0.003.

3.2.3 Meiofauna At Loch Sunart, various test deployments were undertaken at benthic sampling stations using a Craib corer. In general, sample quality by this method was inadequate becasue of stones in the sediment preventing penetration and closure of the equipment. To resolve this problem and maintain consistency across sites, van Veen grab samples (0.1 m2) were taken and a standard Craib core tube inserted into the open inspection hatch of the grab to take sub-samples (Perspex, inner diameter = 57 mm, length > 100 mm). Core samples for meiofauna were preserved in 4 % buffered formalin in sea-water.

Sub-sampled cores collected this way can be of reasonable quality under specific and highly controlled circumstances. Generally, however, this can be an unreliable method for collecting meiofauna, especially taxa which live in or on the sediment surface layers (e.g. copepods). In order to address concerns about the quality of samples collected by van Veen grab, parallel samples were collected by grab and SCUBA at Loch Kishorn. Several stations at the other sites were too deep to be sampled using SCUBA. All meiofauna samples were taken at macrofauna stations.

Extraction methods followed Somerfield & Warwick (1996). Samples were washed on a 63 µm sieve to remove formalin and most of the silt/clay sediment fraction. The meiofauna were then extracted using elutriation in fresh water and decantion through a 63 µm sieve. Flotation extraction using a colloidal silica solution (Ludox from DuPont) with a specific gravity of 1.15 was then used. Sub-sampling was employed at this stage. Each (sub)sample was washed into a lined petri dish, the copepods were picked out under a binocular microscope and identified to species using a compound microscope with Nomarski differential interference contrast illumination. Sub-samples from the remaining sample were then slowly evaporated to glycerol, evenly spread on microscope slides and the coverslips ringed with Bioseal. Nematodes were identified to species or putative species using a compound microscope with conventional bright-field illumination.

3.2.4 Macrofauna A van Veen grab sampler was used to collect sediment samples containing sufficient macrobenthic infauna for full analyses of the fauna. The van Veen grab retains a sediment

Ecological effects of sea lice medicines in Scottish sea lochs 24 of 286 sample of 0.1 m2 surface area and a volume of up to 20 l, of which the retained volume is a function of sediment compaction and grab weight.

Each sample was carefully sieved on a 1 mm mesh. The sample residues were preserved in 4 % buffered formalin in sea-water, containing Rose Bengal indicator to aid subsequent retrieval of the fauna from residual sediment and detritus.

In the laboratory, fauna were retrieved from each sample by hand sorting and identified to the lowest possible taxon. For each sample, the abundance (A, total number of individuals), total number of species/taxa (S), and biomass (B, g wet weight) were recorded for each major taxonomic group. The samples were stored in 70 % v/v ethanol.

3.2.5 Uni- and multivariate analyses of faunal community structure 3.2.5.1 Zooplankton Effects at the community level were analysed by correspondence analysis (CA) using CANOCO version 4 (ter Braak & Smilauer, 1999). Correspondence analysis is based on a unimodal response model in which the abundance of species rises and falls within a limited range of values of an environmental variable (van Wijngaarden et al., 1995).

The results are presented as ordination plots of species and sites using species-centred CA in which each species is implicitly weighted by the variance of its abundance values. Species with high variance, often the abundant ones, therefore dominate the CA solution, whereas species with low variance, often the rare ones, have only minor influence (ter Braak, 1987). Euclidean scaling was used so that resulting ordination plots were optimal for interpreting distances between sites (sample stations and times). Species abundance was not transformed and each sample station at each sample time was considered to be a site (indicated as a point on the ordination plot).

The CA ordination plots summarise changes in the zooplankton community over the sample period and the goodness-of-fit of the axes is indicated by the eigenvalues. The higher the eigenvalue, the more variation is explained by the axes. In the ordination plots, sites with similar species composition lie close together while sites with dissimilar species composition lie far apart. The origin (centre) of a species ordination diagram represents the mean abundance of the individual species at all sites. Species closely associated with a site and present in higher than average abundance, occur on the same side of the origin, while sites on the opposite side of the origin from a species point contain less than average abundance of that species.

3.2.5.2 Meiofauna Non-parametric multivariate techniques were employed (Clarke, 1993) using Primer v6β R3 (PRIMER-E Ltd, Plymouth, UK). Lower triangular similarity matrices were constructed using a range of data transformations and the Bray-Curtis similarity measure. Transformations were used to reduce contributions to similarity by abundant species, and therefore to increase the importance of the less abundant species in the analyses. Nematodes and copepods varied between single specimens and hundreds of specimens at most, so a square root transformation was generally used. Ordination was by non-metric multidimensional scaling (MDS) (Kruskal & Wish, 1978; Clarke & Green, 1988) and formal significance tests for differences between stations were performed using the ANOSIM permutation test (Clarke & Green, 1988, Clarke, 1993). The species contributing to dissimilarities between stations were investigated using the similarities percentages procedure (SIMPER) (Clarke, 1993). K-dominance plots were also constructed (Lambshead et al. 1983).

Ecological effects of sea lice medicines in Scottish sea lochs 25 of 286 3.2.5.3 Macrofauna For each sampling date and station, population data from the five replicate grab samples collected at each station were pooled. The number of taxa (S), abundance (A) and biomass (B) per nominal 0.5 m2 of sediment surface were calculated for each station so that:

A B Average no. of individuals per taxon = , Average mass of individuals (mg) = *1000 S A

The abundances of the five most numerous taxa found for each sampling date and station were calculated. Interpretation of the changes in populations between successive samplings was based on pairwise comparisons of the pooled data for each station, using Student’s t-test at the 95 % level of probability. Temporal changes in the dominant taxa and conditions in the sediments (Section 3.2.5.4) were also taken into account when interpreting these changes.

SHANNON-WIENER DIVERSITY INDEX (H') This index is based upon the observed distribution of individuals among species/taxa and provides a measure relating to dominance in the populations. H' is influenced by both the number of species/taxa and their relative abundance in the sample, so that a high H’ is indicative of a diverse community.

S abundance of ith species H' = − p log p , where p = ∑ i 2 i i total abundance i=1

PIELOU’S MEASURE OF EVENNESS (J) This index based on Shannon-Weiner index and the number of taxa is a measure of the population sizes of respective species. J is maximised as the population size of each species’ approaches equality.

H' J = log2 S

INFAUNAL TROPHIC INDEX UK (ITI) This index is based on grouping the taxa by the type of food consumed and where the food was obtained (Codling and Ashley, 1991) and has values between 0 and 100. For example, a community dominated by group four deposit feeders indicative of a pollution effect results in a low value of ITI:

0n + n + 2n + 3n ITI =100 - [33.33 1 2 3 4 ] n1 + n 2 + n 3 + n 4 where n1-4 = the number of individuals in trophic groups 1 - 4.

3.2.5.4 Sediment conditions at sublittoral sampling stations During sublittoral sampling surveys, sediment nature and appearance, and physico- chemical conditions were assessed at each sampling station from February 2000 to assess the level of organic enrichment effects on the seabed sediments as a consequence of normal fish farm operations.

Two intact sediment core samples (60 mm to 120 mm in depth) were extracted from the grab sampler at each station. The sediment type and changes in the appearance with depth

Ecological effects of sea lice medicines in Scottish sea lochs 26 of 286 in the core samples were assessed and the sediments visually categorised on a scale of 1 (finest) to 4 (coarsest).

Redox potential/depth profiles were recorded. Prior to the samples being obtained, all probes were calibrated in the laboratory using Zobell’s ferrocyanide-ferricyanide redox standard solution. All probes reading outwith the range 240-260 mV were rejected. This test was undertaken after each series of measurements for individual cores as well as electrode rinsing in distilled water before and after use. On board the vessel, profiles for each sediment core sample were measured immediately after extraction using a WTW Microprocessor pH meter (model-pH 196). A platinum electrode with a silver/silver chloride reference system measured redox through stepwise penetration, mainly at 10 mm increments. After a stabilising period of 60 seconds, readings were taken in the overlying water (+10 mm), sediment/water interface and at 5, 10, 15, 20, 30, 40, 50 and where possible 75 mm depth in the sediment column. A correction factor of +198 mV for a Normal Hydrogen Electrode (NHE) was used to normalise the data.

3.2.6 Sublittoral settlement on suspended panels To quantify animal and plant settlement from the water column onto hard substrates, arrays of four slate settlement panels (each 40 cm x 20 cm) were suspended at three depths at each macrofauna station. These depths were typically 2 m below the surface, mid-water depth and 2 m above the bed. The stations were at three or four locations at varying distances (and potential impact) from the cages in Lochs Sunart (2000-2003), Diabaig (2000) and Craignish (2001). The arrays were winched onto a survey vessel at intervals of three or four months for examination. The organisms on the slates were identified and assessed in situ using SACFOR abundance scales (MNCR, JNCC; in Connor et al., 1997). The plates were then photographed and one slate from each set of four was returned fresh to the laboratory for more detailed study. New slates were placed on the arrays to replace those removed or lost.

3.2.7 Littoral site assessment - shore fauna and flora Following initial ecological surveys at the four sites, the intertidal (littoral) zone was found to consist of mainly bedrock and/or boulders. All shores were characterised by abundant growths of perennial brown fucoid algae mixed with invertebrate populations of attached barnacles and mussels, limpets, mobile snails (mainly Littorina spp.) and the dog whelk Nucella lapillus that feeds on barnacles. Overall exposure to wave action largely determines whether rocky shores are dominated by fauna (e.g. barnacles, limpets) indicative of high wave action, or fucoid seaweeds (Fucus spp., Ascophyllum, Pelvetia) indicative of reduced wave action. Following initial surveys, three or four littoral stations were established in each area. Two stations were located where hydrographic and modelling information suggested effects of the treatment chemicals were possible. Where effects were most likely to occur, stations were designated as ‘predicted high impact’. Where effects were less likely, stations were designated as ‘predicted intermediate impact’. One or two reference stations were situated beyond the anticipated zone of influence. Summary descriptions of the principal communities present at each shore station are presented for each loch studied. A summary classification based on a biological exposure scale defined by Lewis (1964) is given for the littoral sites surveyed (Table 8.5).

The barnacle Semibalanus balanoides is the only sedentary crustacean that settles predictably on these shores in large numbers. Barnacles belong to the same phylum as sea lice and are therefore, appropriate ‘target’ organisms for studies aimed at detection of sea lice treatment effects. Sets of four slate panels were bolted to the rock at approximately mid-tide level to receive any settlements at predicted high impact, intermediate impact and reference sites. The barnacles and other biota on the surrounding rocky shores were

Ecological effects of sea lice medicines in Scottish sea lochs 27 of 286 studied and photographed at each visit (approximately three times per year at each location). Barnacle abundance on each panel was estimated by enumerating individuals within six square sectors of accurately known area on each slate (usually 400 or 2500 mm2 per sector). Approximate shell diameters were also noted.

After 2001, the first annual inspection preceded the release of barnacle larvae into the water column so that mature egg masses were still retained within the barnacles. During these visits in late January to early March, slates with settled barnacles were removed and returned to the laboratory where the maximum diameters of fifty individuals on each slate were measured. The same individuals were then detached from the slates and the egg masses, whole bodies and valves were removed separately and weighed on a Perkin- Elmer AD 2Z microbalance. The frequency of occurrence of egg masses in the individuals was also recorded. By late summer, recently settled populations could not be distinguished with certainty from the previous year’s settlement (cohort). Thus, comparisons between shore sites of the cohort from the previous spring settlement were made early in the year, before the subsequent spring settlement.

On the sheltered shores of Loch Sunart, settlement of S. balanoides was too sparse to allow detailed comparisons of populations prior to 2002. Natural bedrock at the sites was wetter and attracted higher densities of S. balanoides than the smoother, drier slates. Consequently, areas of rock were cleared in the vicinity of the slates to monitor barnacle settlement. This improved approach was extended to shore sites at Lochs Kishorn and Diabaig and enabled comparison of population settlement on slates and natural rock surfaces.

Settlement and development data of S. balanoides populations were collated to determine mean densities of settled populations, mean size attained after each settlement season, mean mass of body tissue and fully developed egg lamellae, and the frequency of occurrence of egg lamellae in populations. Where settlement panel and bare rock assessments were undertaken, mean population densities, size of individuals, and frequency of occurrence of developed egg lamellae were measured.

3.2.8 Sediment chemistry Cores sub-sampled from van Veen grab samples as detailed in Section 3.2.3 were extruded and sliced such that the surface 4 cm of sediment was retained. The samples were placed in a cold box (5-10 ºC) and frozen (-20 ºC) on return to the laboratory. The sediment core slices were used for emamectin benzoate analyses, particle size analysis, and total organic carbon and nitrogen determinations.

With the introduction of in-feed treatments by the aquaculture industry, the sediment sampling protocol was altered to reflect the specific requirements of locating in-feed medication in sediments. Divers sampled along fixed seabed transects along fixed seabed transects at marked stations, ensuring strict repeatability of sample location (Figure 3.1). Diver-obtained cores were processed as described above.

Ecological effects of sea lice medicines in Scottish sea lochs 28 of 286 Figure 3.1. A SCUBA diver taking a sediment core near fish farm.

3.2.8.1 Cypermethrin Cypermethrin [(RS)-α-cyano-3-phenoxybenzyl (1RS)-cis, trans-3-(2,2-dichlorvinyl) -2,2- dimethylcyclopropane carboxylate] is a mixture of the four diasteroisomers each consisting of two enantiomers. Two have a cis-configuration and two a trans- configuration about the cyclopropane ring (Figure 3.2). The ratio of the cis to trans isomers varies from 50:50 to 40:60. The n-octanol/water partition coefficient of cypermethrin is high (log Kow 6.3), therefore this medicine has the potential to accumulate in muddy sediment.

Figure 3.2. Cypermethrin [(RS)-α-cyano-3-phenoxybenzyl (1RS)-cis, trans-3-(2,2- dichlorvinyl) -2,2-dimethylcyclopropane carboxylate] is a mixture of the four diasteroisomers each consisting of two enantiomers.

For water column measurements, duplicate water samples were collected in Winchester bottles (2.5 l) at each sampling station from a depth of 6 m. Dichloromethane (DCM) (100 ml) was added to each sample before storing in the dark between 5-10 ºC. A reference water sample was collected (ca. 500 m from cages) and spiked with a known concentration of cypermethrin.

Detailed methods on cypermethrin extraction, analysis by gas chromatography, method validation and quality control is in Appendix II - Chemistry. Preparation stages required for water column and sediment samples, and measurement of cypermethrin concentrations using gas chromatography electron capture detection methods (GC-ECD) are detailed. Method validation and quality control resulted in recoveries of greater than

Ecological effects of sea lice medicines in Scottish sea lochs 29 of 286 80 % from water samples spiked with cypermethrin at concentrations of 500 ng l-1 (limit of determination = 1 ng l-1). Samples prepared from sediment were spiked with ~300 ng (~ 15 ng g-1, dry weight) of cypermethrin and recoveries of between 82.70 % and 112.18 % (mean = 91.46 %, n = 8, standard deviation [SD] = 11.82) were obtained. The limit of determination for the method was 2.25 ng g-1 dry weight.

3.2.8.2 Emamectin benzoate Emamectin benzoate is a mixture of two avermectin homologues of 4’’-epimethylamino- 4’’-deoxyavermectin B1a and B1b benzoate with the B1a to B1b ratio being approximately 9:1. The B1a homologue has an additional methylene group on the iso- butyl side chain (Figure 3.3). It has a low seawater solubility (5.5 mg l-1) and a relatively high octanol:water partition coefficient (log Kow = 5.0), indicating that it has a potential to be adsorbed and bound to particulate material and surfaces.

Figure 3.3. The semi-synthetic avermectin, emamectin benzoate is formed from the benzoate salt and consists of a mixture of two avermectin homologues of 4”- epimethylamino-4”-deoxyavermectin B1a and B1b benzoate with a ratio of B1a to B2b of approximately 9:1.

Method 1 Detailed methods on emamectin benzoate sample preparation, extraction, analysis by liquid chromatography mass spectrometry (LC-MS), method validation and quality control is in Appendix II - Chemistry (Section 13.3.1). For quality control and method validation, calibration curves over the range 5 ng ml-1 to 1500 ng ml-1 were linear with correlation figures of at least 0.999. Replicate analysis of 90 % and 10 % solutions of the stock solution (1112 ng ml-1) resulted in coefficients of variation of 2.1 % and 1.6 % respectively. The limit of detection was calculated using 4.65 x standard deviation of the low standard/weight of sample used for extraction and was equal to 1 µg kg-1 for sediment (10 g used).

Reference sediment collected from a site distant from any fish farms, was analysed and found to contain no emamectin benzoate above the limit of detection. Seven aliquots (~ 10 g) of this sediment were spiked with emamectin benzoate, extracted and analysed by LC-MS. Recoveries of 76.3 % to 102.5 % (mean = 85.7 %, n = 7, CV % = 13.97 %) were achieved.

Method 2 Two sediment samples collected on each of August 2002 and January 2003 were spiked at the time of collection with emamectin benzoate. Approximately 37 g of sediment was spiked with 1 ml of 1112 ng ml-1 solution (30.05 µg kg-1 wet weight). Samples of

Ecological effects of sea lice medicines in Scottish sea lochs 30 of 286 sediment were freeze dried prior to extraction. The extraction and analysis was based on the method described by Heasook et al. (2001). Brief detail is given in Appendix II - Chemistry (Section 13.4.1).

Calibration curves over the range 0 ng g-1 to 20 ng g-1 (equivalent) were linear, with correlation figures of at least 0.99 for both emamectin benzoate and its metabolite (N- demethylated emamectin). The intercept was forced through zero because, as commonly found with fluorescence detection, the majority of calibration curves were observed to have a very small negative value for the intercept. The limit of detection for the method was 1 ng g-1.

Reference sediment was spiked with emamectin benzoate (10 ng g-1). The average recovery was 70 %. Analysis results were corrected for the purity of the standard but not for the recovery of emamectin benzoate and its metabolite from sediment.

3.2.8.3 Particle size analysis (PSA), total organic carbon (TOC) and total organic nitrogen (TON) PSA was carried out on freeze-dried sediments by laser granulometry using a Malvern Mastersizer E Particle Size Analyser, which measures particles from 0.1 to 600 µm. Any larger particles present were analysed by sieving, with the two data sets combined to give an overall PSA of the bulk sediment.

TOC and TON was determined on freeze-dried and ground sediment, using a ThermoQuest FlashEA 1112 elemental analyser. The working range for carbon is 0.06 - 55.6 mg per sample, for nitrogen 0.005 - 6.07 mg per sample. Samples were acidified with HCl in silver cups, prior to analysis, to remove the inorganic carbon fraction. The CHN analyser uses a combustion method to convert the sample elements to simple gases (CO2, H2O and N2). The sample is first oxidised in a pure oxygen environment; the resulting gases are then controlled at exact conditions of pressure, temperature and volume. Finally, the product gases are separated. Then, under steady state conditions, the gases are measured as a function of thermal conductivity. The Relative Standard Deviation of analyses of sediment containing 0.18% N is 12%. The mean recovery of acetanilide standard material added to sediment samples is 94.6% with an RSD of 4.6%.

3.2.9 Hydrodynamic surveys Hydrographic data were required primarily for modelling studies, but were also used to provide background information to assist sampling station selection for shoreline and benthic surveys. Surveys varied in length from a few days to several months depending on requirements. Shorter intensive surveys had higher temporal or spatial sampling and were often undertaken at the same time as other surveys, such as during the Excis treatment at Loch Sunart in January 2002. Hydrographic information collected over longer periods was required for post treatment periods and long term modelling of Slice (Lochs Sunart and Kishorn).

Current meter surveys were undertaken using a number of different instrument types, mostly of the low threshold type. The majority of surveys were undertaken with electromagnetic Interocean S4 current meters, which have a high degree of accuracy and dependency (Interocean, San Diego, USA) and measure water current according to the level of distortion of an electromagnetic field. Other instruments used included Sontek current meters which acoustically measure current according to the velocity of planktonic material and other scattering particles in the water column (Sontek, San Diego, USA). Aanderra current meters were used occasionally and measure current via a spinning rotor (Aanderra, Bergen, Norway). The latter current meter was the only instrument used with

Ecological effects of sea lice medicines in Scottish sea lochs 31 of 286 moving parts, which make it susceptible to fouling and thus less reliable in terms of data return. Current meters were typically deployed at several depths on either a standard or U-shaped mooring. Ten or twenty minute sampling intervals were used for long term deployments, while five minute intervals were used for short term deployments. Instruments were deployed close to the cages but beyond the hydrodynamic ‘shadow’ of the cages as required by SEPA (SEPA, 2003b). Summary statistics of data were used to compare sites and assess the potential for resuspension of pollutants.

A meteorological station with similar sampling intervals was generally deployed concurrently on either the cage group or at the fish farm land base. On longer deployments meteorological stations occasionally had longer sampling intervals due to memory and battery limitations. The amount of variance in current data attributed to different processes of tidal and wind driven flows at Loch Kishorn was determined by Wilson (2004).

3.2.10 Bathymetric surveys Bathymetric and sediment type surveys were undertaken to determine depth, sediment types, and cage and sampling station positions (Figure 12.1) (Appendix I - Hydrography). A RoxAnn acoustic ground discrimination system operating at 200 kHz with single beam echo sounders was used to examine the strength and decay of the signal emitted from the sounder as it is reflected back off the sea bed (Stenmar, Aberdeen, UK). Both sediment roughness and hardness were measured, with the system giving DGPS position, depth and strengths of first and second echoes. The echo strengths are given numerical values which were verified for each sediment type with a hand held van Veen grab (0.025 m-2). Data were filtered for outliers, adjusted for tidal height to obtain depth at lowest astronomical tide, and contoured using the kriging algorithm. Bathymetric and cage position information was also used to generate model grids.

3.2.11 DGPS drifting buoy surveys Drifting buoy (drogue) surveys were used to determine general circulation and dispersion characteristics for a site, and to track effluent plumes following sea lice treatments (Figure 3.4).

Ecological effects of sea lice medicines in Scottish sea lochs 32 of 286 Figure 3.4. Differential Global Positioning System (DGPS) drifting buoy system used at all sites generated differential corrections independently of external DGPS sources (SAMS, Oban, UK).

Six Differential Global Positioning System (DGPS) drifting buoys with sock depths of 6 m were deployed, each logging its position every 30 s (horizontal accuracy ± 4 m). Drifters deployed close to fish farm cages were advected in the current for varying periods up to 5 h. As general values of dispersion for the area around a farm were sought under different tidal conditions, multiple, relatively short deployments were undertaken. Longer deployments measuring dispersion beyond the immediate area were not necessary.

Dispersion coefficients calculated from the degree of drifter spread over time were used in modelling. In addition, general circulation patterns were measured around the farm by this lagrangian means. Complex circulation patterns are not easily measured with single point current meter deployments, i.e. eulerian type measurements.

3.2.12 Modelling Predicting and hindcasting the dispersion of soluble and particulate effluents from aquaculture sites requires: a) knowledge of prevailing current flow fields both at the time of discharge and for a period following release appropriate to the time scale of dispersion, and b) a model that uses these flow fields to simulate effluent transport and dispersion. Within this study, some effort was expended on the first objective, but most was spent on developing and applying the dispersion models.

3.2.12.1 Excis (cypermethrin) Initially, modelling work focussed on developing a model capable of simulating and reproducing the hydrodynamic conditions in the surface layers of the selected field study sites. Effort concentrated on applying the inverse model WOLF (Dr Graham Copeland, University of Strathclyde, UK, pers. comm.). However, in this study, data requirements

Ecological effects of sea lice medicines in Scottish sea lochs 33 of 286 for WOLF to be validated satisfactorily were not achievable. Consequently, modelling effort was switched to dispersion models driven by the collected current meter data.

Cypermethrin in Excis applied as a bath treatment is released from cages as a dissolved effluent. However, due to its physical and chemical characteristics, cypermethrin has a low water solubility and is strongly adsorbed by solid surfaces and organic matter (McHenery, 1995). The limited data available suggest that at concentrations of 50 ng l-1 and below (i.e. at dilutions of 100 times the treatment concentration of 5 µg l-1), cypermethrin will not remain in solution and will be more liable to bind to particulate material. As a result of this behaviour, both dissolved and particulate stages of dispersal were modelled. The key difference between these two phases is that during the former, the effluent was considered to be in suspension behaving in a passive manner relative to the ambient flow field. During the latter phase however, the effluent had a definite downward velocity due to binding to particulate material. In a previous modelling study, Turrell and Gillibrand (1995) found that under typical conditions of dispersion in a Scottish sea loch environment, the dissolved phase of dispersion is likely to last about 3 h (treatment concentration = 5 µg l-1). That finding was utilised in the present study.

In order to be able to simulate both phases of dispersal, a particle tracking approach was used. Particle tracking modelling is an established tool for predicting the dispersion of dissolved and particulate contaminants in the marine environment (e.g. Allen, 1982; Hunter, 1987; Elliott et al., 1992a; Gillibrand et al., 1995; Gillibrand and Turrell, 1997; Cromey et al., 2002a). The method employs a large number of particles to represent the dissolved effluent, with each individual particle representing a theoretical ‘droplet’ of fluid. Both dissolved and particulate phases were simulated by initially setting the particles to behave passively in the water column, and then by ‘switching on’ a settling velocity after a prescribed period to represent the particulate phase. This approach was used by Turrell and Gillibrand (1995).

3.2.12.2 Slice (emamectin benzoate-EmBz) The particle tracking model DEPOMOD (Cromey et al., 2002a) has a validated resuspension component and is used by SEPA in consenting Slice applications (SEPA 2003a). In the model, emamectin benzoate bound to settling waste particles (i.e. faecal and uneaten feed material) is advected in the current until contact with the sea bed occurs. A random walk model also simulates dispersion of particles in the horizontal and vertical plane. Post deposition, particles may be eroded if a critical near bed current speed is exceeded or, consolidated into the bed over a period of time. Sediment concentrations of emamectin benzoate are then predicted. An important aspect of the model is excretion of emamectin benzoate by fish over an extended period of time (Table 3.1). This complex behaviour, as well as chemical decay, is included in the model.

Changes were not made to the DEPOMOD code, so the model used was the same version as that used by SEPA for consenting sea lice medicines. However, significant improvements were made to data input, detailed later. Both preliminary and detailed modelling of the farms pre- and post- Slice treatment were undertaken. Pre-treatment predicted sediment emamectin benzoate concentrations assisted positioning of sampling stations for benthic fauna and sediment chemistry. Post-treatment concentrations were modelled in detail and compared with observations for model validation purposes.

Standard procedures for running the model as given by SEPA (2003a) were followed. Default data from the SEPA guidelines were used, including feed water content, digestibility, and percentage uneaten feed. Default feed and faecal settling velocity data were also used as well as SEPA default data on bulk sediment density and mixing depth

Ecological effects of sea lice medicines in Scottish sea lochs 34 of 286 of emamectin benzoate. These latter data enable the necessary conversion of model predictions of emamectin benzoate mass per unit area of bed (µg m-2) to sediment concentration (µg kg-1) to allow comparison with observed concentrations. The resuspension model within DEPOMOD was used to simulate erosion-transport- deposition-consolidation processes, which were validated by Cromey et al. (2002b).

Each Slice treatment was assumed to last seven days, during which emamectin benzoate enters the environment via both pathways of uneaten feed and faecal material. After 7 d, emamectin benzoate enters via excretion from the fish in faecal material only (Table 3.1). Decay of emamectin benzoate was incorporated as a first order decay model where Gt is the concentration of emamectin benzoate at time t (years), G0 is the initial concentration and k is a first order decay constant (1.0127 yr-1) (half life = 250 d):

(−kt) G t = G 0 exp

Loss of emamectin benzoate mass via solubility was not included as this information was not available. Predicted and observed sediment concentrations were compared for all sampling events after treatment. Multiple Slice treatments were included in the modelling runs.

Table 3.1. Emamectin benzoate excretion from salmon used in the DEPOMOD model. Period (days from start of % of emamectin benzoate remaining in treatment) fish excreted as faeces during period 1 to 7 10 8 to 43 50 44 to 79 50 80 to 115 50 116 to 151 50 152 to 187 50 188 to 223 50

Ecological effects of sea lice medicines in Scottish sea lochs 35 of 286 4 Loch Sunart 4.1 Site description Loch Sunart is located on the west coast of Scotland, the entrance to the loch connecting to the Sound of Mull, which separates the island of Mull from the mainland (Figure 4.1). The loch is Scotland’s second longest at 31 km, with an average width of 1.5 km, a maximum depth of 124 m, and consisting nominally of six sills and basins (Edwards and Sharples, 1986). The tidal regime in the loch is predominantly semidiurnal, with a range of 4 m at spring tides and 1 m at neap tides. The catchment area is 299 km2, with mountainous ground to the north and east, and approximately half the freshwater enters the system in the upper basin. Loch Sunart has a small freshwater discharge to tidal flow ratio (1:189) (Edwards and Sharples, 1986), which probably contributes to the relatively frequent renewal of its bottom waters (Gillibrand et al., 1995).

The study site for this project lies in the upper basin of the loch, which is separated from the main loch by a sill 500 m long, 600 m wide and 8 m deep (below Mean Sea Level) at the Laudale Narrows. Mixing across the inner sill is intense and manifested as a ‘boiling’ of the water surface. The maximum water depth in the upper basin is 91 m.

Previous studies in the loch have shown that the upper basin is a very dynamic region, with strong vertical velocity gradients indicating the presence of a strong progressive internal tide (Elliott et al., 1992b). The deep water in the upper basin is subject to considerable vertical exchange and this contributes to the regular renewal of the basin water (Gillibrand et al., 1995). Simple tidal prism calculations of flushing, using the physical characteristics of the upper basin given by Edwards and Sharples (1986), suggest that the surface 10 m layer of the basin will have a flushing time of about 3 d.

Ecological effects of sea lice medicines in Scottish sea lochs 36 of 286 A) B)

Figure 4.1. A) Location of Loch Sunart on the Scottish west coast and B) Loch Sunart and location of study area (inset box). Grid is OS.

Ecological effects of sea lice medicines in Scottish sea lochs 37 of 286 4.2 Fish farm history, biomass, cage positioning and medicine use Marine Harvest Sunart Farm (also known as Laudale Farm) was established in 1979. This first year of production saw 60000 fish introduced, leading to a harvest biomass of 200 tonnes after the two year growing cycle. The biomass remained fairly static until 1989, when 1.2 million fish were introduced, harvesting out at 1400 tonnes at the end of the cycle. By the late 1990s the biomass had increased markedly to 2300 t (1997), dropping to 2000 t in 1999 and 1800 t in 2002 (pers. comm. J. Muckart, Marine Harvest).

Medicine use at Sunart fish farm during this study comprised many of the treatments available to the aquaculture industry. Since 1999, the farm has used Excis, Salartect, Salmosan and Slice. Treatment dates and amounts used are presented in Table 4.1. Cage positions at Sunart fish farm are shown in Figure 4.2.

Table 4.1. Sea lice medicine use at Sunart fish farm since 1999. Date Treatment Amount active Active ingredient ingredient 26-29/10/99 Excis 8 l cypermethrin 23-26/11/99 Excis 12 l cypermethrin 17-18/1/00 Excis 6.4 l cypermethrin 6-8/3/00 Excis 8 l cypermethrin 16-20/3/00 Excis 5.6 l cypermethrin 24-26/4/00 Excis 14.4 l cypermethrin 30/5-2/6/00 Excis 11.6 l cypermethrin 19-23/6/00 Excis 18 l cypermethrin 3-6/8/00 Salmosan 600 g azamethiphos 3-6/8/00 Excis 13.8 l cypermethrin 21-25/8/00 Salmosan 1920 g azamethiphos 21-25/8/00 Excis 18 l cypermethrin 18-22/9/00 Salmosan 2320 g azamethiphos 18-22/9/00 Excis 13.8 l cypermethrin 28-30/11/00 Salmosan 1520 g azamethiphos 28-30/11/00 Excis 7.8 l cypermethrin 16-17/1/01 Excis 4.8 l cypermethrin 12-13/9/01 Salmosan 1080 g azamethiphos 30/1/02-4/2/02 Excis 7.8 l cypermethrin 27-29/3/02 Salartect 19000 l hydrogen peroxide 6/5/02 Slice 315 g emamectin benzoate 3/9/02 Slice 315 g emamectin benzoate 8-13/10/03 Salmosan 2880 g azamethiphos 29/2 - 7/3/04 Slice 316.6 g emamectin benzoate

Ecological effects of sea lice medicines in Scottish sea lochs 38 of 286 1)

2)

Figure 4.2. 1) Positions of cage groups (grey rectangles) at Sunart fish farm with sampling stations. Littoral stations are shown as , zooplankton stations as , sediment chemistry stations as , and combined macrofaunal/meiofaunal/sublittoral stations as . Original (A) and post-move March 2001 (B) cage positions are shown; 2) Bathymetry of Sunart study area derived from Admiralty chart and RoxAnn survey

Farm management details (fish movements, treatments) and project details are arranged chronologically in Table 4.2.

Ecological effects of sea lice medicines in Scottish sea lochs 39 of 286 Table 4.2. Timeline of sampling and management events at Loch Sunart fish farm. Figs. in kg are feed input; Aza = Salmosan, Ema = Slice, Cyper = Excis, H2O2 = Salartect Key: S: Sampling D: Deployed R: Retrieved Scientific events 24 - Shore slates (S) Day - Event 31 - Prelim. shore survey 28 - Shore slates (D) 8 - Shore slates (D) 18 - Macro/meiofauna (S) 7 - Shore slates (S)

1999 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 2025 kg 16975 kg 66725 kg 31975 kg 43525 kg 26 -Cyper 295975 kg 224575 kg 212100 kg 232623 kg 207550 kg 23 - Cyper 15 - Fish in Management (’99 year class)

Zoopl 28 Feb - 10 Mar (S) Zooplankton 26 Oct - 23 Dec (S) 21 - Shore slates (S) Scientific events Day - Event 28 -Macro/meiofauna (S) 2 - Current meters (D) 3 - Sublittoral arrays (D) 14 -Sublittoral arrays (D) 20 - Shore slates (S) 1 - Sublittoral arrays (S) 21 - Shore slates (S) 2 - Shore slates (S) 6 - Drogues (D) 6 - Sublittoral arrays (S) 8 - RoxAnn (D) 6 - Macro/meiofauna (S) 7 - Sublittoral arrays (S)

2000 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 3 - Aza 18 - Aza 21 - Aza 28 - Aza 99200 kg 87100 kg 62175 kg 6 - Cyper 3 - Cyper 21- Cyper 203853 kg 171225 kg 249600 kg 224600 kg 292800 kg 204750 kg 220100 kg 133825 kg 140575 kg 24 - Cyper 18 - Cyper 17 - Cyper 16 - Cyper 19 - Cyper 28 - Cyper 3 0 - Cyper Management

Ecological effects of sea lice medicines in Scottish sea lochs 40 of 286 Table 4.2 (cont.) Key: S: Sampling D: Deployed R: Retrieved Zoo/phytoplankton (S) Scientific events Day - Event 7 - Sublittoral arrays (S) 8 - Macro/meiofauna (S) 7 - Shore slates (S) 12 - Sublittoral arrays (S) 9 - Shore slates (S) 25 - Shore slates (S) 18 - Sublittoral arrays (S) 13 - Macro/meiofauna (S)

2001 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 4400 events Fish out 12 - Aza 21175 kg 40450 kg 97725 kg 11699 kg 27127 kg 53275 kg 96925 kg 93525 kg 2 - Fish in 105850 kg 16 - Cyper repositioned Cage groups Management (‘01 year class) (‘99 year class)

Zoo/phytoplankton (S) (S)

30 - Water chemistry (S) 14 - Sediment Chemistry (S) 27 - Sediment Chemistry (S) 1 - Macro/meiofauna (S) 6 - Sublittoral arrays (S) Scientific events Day - Event 18 - Current meters (D) 30 - Drogues (D) 1 - Shore slates (S) 17 - Sediment Chemistry 4 -Sublittoral arrays (S) 27 - Sore slates (S) 4 - Sublittoral arrays (S)

2002 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2 O 2 events 6 - Ema 3 - Ema 9671 kg Fish out 58450 kg 68250 kg 27 - H 200827 kg 185254 kg 122458 kg 168189 kg 192022 kg 207886 kg 176497 kg 198529 kg 146310 kg 30 - Cyper Management (’01 year class)

Ecological effects of sea lice medicines in Scottish sea lochs 41 of 286 Table 4.2. (cont.) Key: S: Sampling D: Deployed R: Retrieved Timeline of Zoo/phytoplankton (S) sampling and management events at Loch Sunart fish farm. Scientific events 26 - Macrofauna (S) Day - Event 29 - Sediment Chemistry (S) 10 - Sublittoral arrays (D) 19 - Sublittoral arrays (D) 3 - Sublittoral arrays (S) 22 - Sediment Chemistry (S) 12 - Sublittoral arrays (R)

2003 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 8 - Aza 7881 kg 14381 kg 40791 kg 86609 kg 24670 kg 73969 kg 86700 kg 130879 kg 162456 kg 135363 kg 124698 kg 23 - Fish in Management (’03 year class)

Zoo/phytoplankton (S) Scientific events Day - Event

2004 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 29 - Ema 84932 kg 157149 kg 122262 kg 136162 kg 187990 kg Management

Ecological effects of sea lice medicines in Scottish sea lochs 42 of 286 4.3 Hydrography Two current meter deployments (S4 current meters) were undertaken in March 2000, one each to the east and west of the cage groups in water depths of 25 and 35 m respectively. The current meters were deployed near-surface, mid-water and near-bed. Surface currents were generally quite high with a mean and maximum of approximately 8 and 30 cm s-1 respectively, with the residual current flowing seaward to the west. Mid-water flows and near-bed flows were different to surface flows as they were generally quiescent with minimal residual speed. Both deployments showed similar patterns to those in Figure 4.3.

A)

B)

C)

Figure 4.3. Time series plots of current speed and vector displacement for Loch Sunart (sampling interval 10 mins., total length 17 days). (A) Near-surface and (B) mid-water speed, (C) near-surface and mid-water displacement.

The strong surface current at the site is likely to rapidly disperse treatment plumes in the upper surface layers, with reduced dispersion in deeper layers. The weak near-bed currents are likely to result in little resuspension of particulate bound chemicals at the sea bed.

Ecological effects of sea lice medicines in Scottish sea lochs 43 of 286 Table 4.3. Horizontal dispersion coefficients calculated from the drifter release on 30 January 2002. Calculated variables are along-loch dispersion coefficient (KX) and transverse dispersion coefficient (KY). 2 -1 2 -1 Time Time after KX (m s ) KY (m s ) Release (s) 11:10:00 600 0.12 0.049 11:15:00 1200 0.075 0.043 11:20:00 1800 0.066 0.027 11:35:00 3600 0.042 0.031 12:05:00 7200 0.043 0.018 12:35:00 10800 0.029 0.014 12:42:30 11700 0.014 0.015

Drifting buoys were released during two Excis treatment events (March 2000 and January 2002) to track movement of the discharged effluent plumes, and calculate water velocities and dispersion coefficients. Dispersion coefficients calculated for the January 2002 Excis treatment are given in Table 4.3. Drifter releases at Loch Sunart during an Excis treatment in 2000 also confirm the surface layers to be quite dynamic (Figure 4.4), with dispersion 2 -1 2 -1 coefficients of Kx of 0.68 m s and Ky of 0.01 m s calculated. For comparison, the default horizontal dispersion coefficient recommended by SEPA for all sea lice medicine consent modelling is 0.1 m2 s-1.

Figure 4.4. DGPS drifter buoy survey on 6th March 2000 in Loch Sunart showing buoy tracks, current meter and cage group positions. Drifter release was at 11:45, tide turned at 13:30, release ended at 15:45.

4.4 Cypermethrin water column concentrations: Excis treatment January 2002 Cypermethrin water column concentrations were measured in the effluent plume following a single cage treatment during the January 2002 Excis treatment. 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 effluent plume.

Prior to and immediately after release of the treatment plume, duplicate water samples were collected from the water surface in the treatment cage. Samples were also collected from a depth of 6 m at the west end of the cage group (down-current of the treatment cage) five minutes after the plume was released. All further samples were collected at a depth of 6 m in the dispersing treatment plume (as tracked by the drifter buoys) using a

Ecological effects of sea lice medicines in Scottish sea lochs 44 of 286 small boat (Figure 4.5). Duplicate samples (2.5 l) were collected ca. every 10 minutes using a remote opening device. Immediately after collection, dichloromethane (DCM) was added (100 ml) and samples were stored in the dark for analysis on return to the laboratory. A reference sample collected at distance (ca. 500 m) from the fish farm was spiked with cypermethrin (500 ng l-1).

Figure 4.5 Positions of water samples collected from Loch Sunart fish farm during the Excis treatment in January 2002. The grey rectangle represents the fish farm.

Cypermethrin concentrations measured in the treatment cage before release of the effluent plume were 3250 ng l-1 and 2960 ng l-1. These concentrations were considerably lower than the estimated treatment concentration of 5000 ng l-1. (Note they are also outside the calibration range). Cypermethrin concentrations in water samples taken from the treatment cage 1 min after removal of the tarpaulin were significantly lower (9.7 and 3.2 ng l-1), while samples collected at the end of the cage group 5 min after release of the effluent plume contained 21.1 and 7.0 ng cypermethrin l-1 (Figure 4.6).

Ecological effects of sea lice medicines in Scottish sea lochs 45 of 286 Cypermethrin concentrations measured in the dispersing treatment plume in the first 43 min post release ranged from 2.2 ng l-1 to 21.0 ng l-1 (Figure 4.6). Cypermethrin could not be detected in the majority of samples collected after this, although in some cases this was due to the cypermethrin peaks being masked by co-eluting peaks. Only four samples taken after 43 min contained cypermethrin concentrations higher than the detection limit (1 ng l-1).

Figure 4.6. 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.

4.5 Predicted post-treatment cypermethrin water column concentrations Water column cypermethrin concentrations measured during the January 2002 Excis treatment at Loch Sunart were used to calibrate the dispersion model and establish confidence in subsequent predictions of sediment cypermethrin concentrations.

The model was driven by current data collected during the field survey. These data were provided by both a deployment of current meters and by deriving current velocities from drogue deployments. Both options were used for the January treatment simulations. The model did not incorporate any spatial heterogeneity in the current field; current velocities derived from the data were considered spatially uniform. The time increment of velocity data derived from the drifter buoys was 10 min.

Ecological effects of sea lice medicines in Scottish sea lochs 46 of 286 Within the model, particles representing the effluent are advected by the measured current field and are also subject to turbulent diffusion in both the horizontal and vertical. Eddy diffusion coefficients were taken from the drogue deployments made at the time of the release. Twelve thousand particles were released to simulate the patch of cypermethrin that was tracked during the field survey. The position of each particle was updated every minute over the simulation period. Concentrations of cypermethrin in the water column were calculated at every particle time step by gridding the distribution of particles and summing the number of particles in each grid cell. Since each particle represents a known mass of cypermethrin (calculated from the mass of cypermethrin used during the treatment divided by the number of particles used in the simulation), the sum of particles divided by the volume of the grid cell provides a predicted concentration for that cell.

The model was run for a 3 h period following the release of cypermethrin at 11:00 on 30 January 2002. Predicted cypermethrin water column concentrations for the same times and locations as measured concentrations were within the same order of magnitude (Figure 4.6). Given that concentrations fell by three orders of magnitude from the treatment concentration of 5000 ng l-1 over a short period of time (within 30 min), this is considered an acceptable level of accuracy for the model.

The difficulties in sampling an effluent plume emanating from aquaculture cages mean that it is impossible to be certain that the drogues were deployed in the centre of the plume; therefore the measured concentrations do not necessarily reflect peak concentrations in the water column at the time of observation. In order to investigate this possibility, time series of predicted peak concentrations, at various depth intervals were compared to measured concentrations (Figure 4.7). The predicted peak concentrations were consistently much higher than the measured values, suggesting that either a) the model is not accurately incorporating all processes affecting the dispersion and loss of cypermethrin, and/or b) that the measured data were not collected in the centre of the plume where concentrations were highest. It is likely that the behaviour of cypermethrin in the marine environment is more complex than simulated by the model. However, while samples were probably not collected from the centre of the plume, the broad agreement between model and observed concentrations gives confidence in the model predictions (Figure 4.6).

Ecological effects of sea lice medicines in Scottish sea lochs 47 of 286 Figure 4.7. Predicted time series of peak water column cypermethrin concentrations at various depths following release at 11:00 on 30 January 2002. The observed data, collected at 6 m, are also shown. The dotted line indicates the 3 h EQS value stipulated by SEPA. The time series are plotted on a logarithmic scale for clarity.

The model results indicate that significant vertical gradients of cypermethrin exist in the water column in the initial stages following release, with much higher concentrations (almost two orders of magnitude) occurring at shallower depths. Horizontal and vertical mixing then rapidly reduced concentrations in the upper layers. However, the same vertical mixing resulted in increasing concentrations at depths below 6 m during the first half of the simulation (1.5 h) before concentrations began to decline.

Predicted dispersion of cypermethrin was rapid with surface (0-3 m) concentrations falling by 99.7 % over the 3 h period of the simulation, a dilution factor of about 300 from the treatment concentration of 5000 ng l-1. After the first hour of release, concentrations at the surface fell to 70 ng l-1, a dilution factor of 70. The 3 h EQS specified by SEPA of 16 ng l-1 (SEPA, 1998) was not substantially breached within the water column layer at the end of the simulation, as the highest predicted concentration was 16.25 ng l-1 in the 3-6 m layer. Given the low diffusion coefficients used in this study, these results suggest that the 3 h EQS is unlikely to be breached following a single cage treatment.

4.6 Predicted post-treatment sediment cypermethrin concentrations Following calibration against measured water column concentrations, the model was used to predict sediment cypermethrin concentrations following the January 2002 treatment. For these simulations, the model was run from 11:00 30 January 2002, when treatment began, until 6 February when all released cypermethrin had settled on the seabed following the final cage treatment on 4 February. In all, 13 individual cage treatments took place over the period. Therefore, these simulations predict the cumulative seabed impact of a full farm treatment rather than a single cage treatment. Results from a single cage treatment are presented for comparative purposes.

Ecological effects of sea lice medicines in Scottish sea lochs 48 of 286 The attachment of cypermethrin to particulate material in the water column, following the dissolved phase, is simulated by ‘switching on’ a positive settling velocity for each particle after a specified interval following release. This method follows Turrell and Gillibrand (1995). It is not clear as to what particulate material in the water column cypermethrin might attach, but the most likely options are fish farm particulate wastes (i.e. fish faeces), planktonic material (e.g. zooplankton faecal pellets), or organic riverine particulates. Assuming that the cypermethrin remains soluble for a period following release, it is most likely that the effluent will attach to planktonic material in the water column away from the farm. A literature search gave approximate settling rates of zooplankton faecal material in the range 35 - 200 m d-1 (Turrell and Gillibrand, 1995).

For the present model simulations (1-4 below), the following behaviour patterns of the discharged cypermethrin were used, and their effects on sediment concentrations investigated:

1. Cypermethrin attaches immediately to particulate fish farm waste with settling -1 speeds in the range 0.015 < ws < 0.063 m s (Cromey et al., 2002a). 2. Cypermethrin attaches immediately to planktonic material in the water column -1 with settling speeds 35 < ws < 200 m d (Turrell and Gillibrand, 1995). 3. Cypermethrin remains soluble for 3 h (i.e. particles have a settling speed of zero) and then attaches to planktonic material in the water column (i.e. settling speeds are ‘switched on’). 4. Cypermethrin gradually attaches to planktonic material in the water column over a period of about 6 h as shown in Figure 4.8. This is an arbitrary adsorption curve, designed to smooth the abrupt transition at 3 h.

Simulations were also performed to investigate the effect of varying the coefficients of horizontal and vertical diffusion. For the above simulations, the horizontal diffusion 2 -1 2 -1 coefficients were KX = 0.056 m s and KY = 0.028 m s (mean values of those calculated from drogue releases [Table 4.3] at the time of the treatment), and the vertical 2 -1 diffusion coefficient (Kz) was 0.0010 m s . Simulation 3 above was repeated with varying horizontal and vertical diffusion as follows:

2 -1 2 -1 5. As Run 3, but KX = 0.100 m s , KY = 0.050 m s . 2 -1 2 -1 6. As Run 3, but KX = 0.500 m s , KY = 0.250 m s . 2 -1 2 -1 7. As Run 3, but KX = 1.000 m s , KY = 0.500 m s . 2 -1 8. As Run 3, but KZ = 0.0001 m s . 2 -1 9. As Run 3, but KZ = 0.00001 m s .

Ecological effects of sea lice medicines in Scottish sea lochs 49 of 286 Figure 4.8. The profile of adsorption of cypermethrin onto organic particulate material used in Run 4.

For the 9 simulations described above, particle deposition on the seabed was monitored and total cypermethrin deposition per unit area of seabed calculated. Predicted deposition rates were then converted to sediment concentrations assuming the following: (1) sediment samples collected following the treatment were taken to a depth of 4 cm and this material was thoroughly mixed; (2) dry sediment densities were 1216 kg m-3 (SEPA, 2003a); (3) the mass of cypermethrin settled on a unit area of seabed was mixed into a sediment volume of 0.04 m3 with mass 48.64 kg.

Predicted sediment cypermethrin concentrations from the simulation following a full farm treatment are shown in Figure 4.9. Peak concentrations were ca. 20 ng g-1 dry weight. The area of sediment predicted to contain concentrations above the detection limit of 2.25 ng g-1 (Section 3.2.12.1) was about 108000 m2, with a length and breadth of roughly 530 and 260 m respectively. The centre of deposition was positioned to the west of the cage group.

Ecological effects of sea lice medicines in Scottish sea lochs 50 of 286 600

500) m (

E R 400 O H S F F O

300 E C N A

200T S I D

100 Concentration (ng g -1dry weight)

0 2 4 6 8 1 1 1 1 1 2 0 2 4 6 8 0 0 1500 1700 1900 2100 2300 2500 2700 2900 DISTANCE ALONGSHORE (m) Figure 4.9. 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.

Predicted cypermethrin sediment concentrations on the alongshore and offshore transects in Figure 4.9 are presented in Figure 4.10. Results from simulations 2 to 9 are shown (Run 1 was considered a less realistic scenario and not included). For all simulations where cypermethrin behaviour included a soluble phase (Runs 3 to 9), concentrations along both transects peaked at ca. 14 ng g-1. The results demonstrate that predicted concentrations are most sensitive to changes in diffusion coefficients, both vertical and horizontal. The smoother adsorption of cypermethrin onto particulates (Run 4) had only a minor effect on concentrations predicted by the abrupt switching on of settling velocities (Run 3).

For comparison, predicted sediment cypermethrin concentrations following a single cage treatment on 30 January 2002 are shown in Figure 4.11. Cypermethrin was deposited to the southwest of the cage group, with a peak concentration of approximately 2.6 ng g-1. The area where concentrations exceeded detectable limits was ca. 1500 m2. This simulation used the same diffusion coefficients as Run 3 above.

Ecological effects of sea lice medicines in Scottish sea lochs 51 of 286 25 (A) Run 2 )

Run 3 1 - g Run 4 g n

( 20

Run 5 N

O Run 6 I

T Run 7 A

R Run 8 T 15 N Run 9 E C N O C 10 D E T C I D E

R 5 P

0

-200 -100 0 100 200 ALONGSHORE DISTANCE (m) 20 (B) Run 2 )

Run 3 1 - g Run 4 g n

( 16

Run 5 N

O Run 6 I

T Run 7 A

R Run 8 T 12 N Run 9 E C N O C 8 D E T C I D E

R 4 P

0

-200 -100 0 100 200 OFFSHORE DISTANCE (m) Figure 4.10. Predicted sediment cypermethrin concentrations on alongshore (A) and across-shore (B) transects following a full treatment at Loch Sunart, 30 January to 6 February 2002. Transect locations are shown in Figure 4.9. The origin of the horizontal axes is the centre of the salmon farm.

Ecological effects of sea lice medicines in Scottish sea lochs 52 of 286 600

500) m (

E R 400 O H S F F O

300 E C N A

200T S I D -1 100 Concentration (ng g dry weight)

0 0 1 1 1 2 2 .2 .6 .0 .4 . 8 .2 .6 0 1500 1700 1900 2100 2300 2500 2700 2900 DISTANCE ALONGSHORE (m) Figure 4.11. Predicted sediment cypermethrin concentrations (ng g-1) following a single cage treatment at Loch Sunart on 30 January 2002. The hatched rectangle represents the cage group.

The results indicate 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 model results suggest that sample sites selected parallel and perpendicular to the main tidal flow would not have picked up the peak in cypermethrin deposition, which may partly explain why cypermethrin has been difficult to detect in the sediment in previous studies. Furthermore, the diffusion coefficients used here were relatively low, particularly considering the length of the simulation. The coefficients used in Run 3 were derived from drogue data obtained over a period of a few hours, which are probably not appropriate for a simulation lasting several days since diffusion is generally considered to increase as patch size increases. The simulations using larger diffusion coefficients (e.g. Runs 6 and 7) resulted in much lower sediment concentrations (Figure 4.10) with a much reduced area of sediment containing detectable cypermethrin concentrations.

Sediment resuspension, redistribution, and chemical degradation processes were not included in these simulations, but would further lower predicted concentrations. Cypermethrin is thought to decay in sediments with a half-life of 2 to 4 weeks, and would probably not significantly influence the results presented here. Resuspension and redistribution may be more important processes, although near-bed current velocities during the treatment period were relatively weak, so resuspension of large quantities of settled material is unlikely.

Predicted sediment concentrations following a single cage treatment were much lower and mostly below analytical detection limits, despite the low diffusion coefficients used. Therefore, for all the above reasons, it is perhaps unsurprising that previous field studies have either not detected cypermethrin in sea loch sediments (Hunter and Fraser, 1995) or have found only relatively low concentrations (SEPA, 2004a).

Ecological effects of sea lice medicines in Scottish sea lochs 53 of 286 4.6 Emamectin benzoate sediment concentrations: Slice treatments May and September 2002 Sediment samples were collected on five occasions between April 2002 and January 2003 for analysis of sediment emamectin benzoate concentrations incorporating Slice treatments in May and September 2002. Sediment samples were collected 0, 30 and 60 m along two transects starting from the south east end of the cages (Figure 4.12). These transects were selected on the basis of depositional contours predicted by DEPOMOD, and situated at the southeastern end of the cage group to permit diver collected samples. Reference samples were collected ca. 400 m from the farm cages. Pre-treatment samples were collected in April 2002 prior to the Slice treatment in May (Table 4.4). None of the pre-treatment sediment samples contained emamectin benzoate or its metabolite, N- demethylated emamectin. Post-treatment sediment samples were collected in May, August and November 2002, and January 2003 (Table 4.4).

Figure 4.12. Positions of sediment chemistry samples collected from Loch Sunart fish farm in May 2002. The grey rectangles represent the cage groups.

In all but one sample collected in May 2002, nine days after the first Slice treatment, emamectin benzoate concentrations were either trace, (detected but below the limit of quantification [<1 µg kg-1]), or not detected. One of the samples collected directly below the cages contained 7.38 ±1.03 µg kg-1 dry weight emamectin benzoate (Table 4.4).

Only one sediment sample collected in August 2002, three months after the May treatment, contained a detectable concentration of emamectin benzoate (1.64 µg kg-1 dry weight). Concentrations in the remaining samples were below the detection limit (1 µg kg-1).

Emamectin benzoate was not detected in any of the samples collected from the macrofauna stations in November 2002 (six months after the May treatment and two months after the September treatment).

Ecological effects of sea lice medicines in Scottish sea lochs 54 of 286 Emamectin benzoate concentrations were below the detection limit in most of the sediment samples collected in January 2003. The two samples containing emamectin benzoate (1.41 and 1.61 µg kg-1 dry weight) were collected within a 25 m distance from the cages. The metabolite N-demethylated emamectin was not detected in any of the samples collected.

Reference sediment samples collected in August 2002 and January 2003 were spiked with emamectin benzoate (30.05 ng g-1 wet weight) at the time of collection. Assuming recovery of 70 % (as determined during method validation), concentrations in the spiked samples should therefore be in the region of 21 µg kg-1 dry weight. Concentrations detected in the spiked reference samples were lower than this ranging from 6.82 to 14.59 µg kg-1 dry weight, but indicate that concentrations recovered from the field collected samples are within acceptable limits.

Ecological effects of sea lice medicines in Scottish sea lochs 55 of 286 Table 4.4. Loch Sunart sediment chemistry sample collection dates, sample codes, and emamectin benzoate concentrations. Samples with A in the station code were collected with diver cores within the near-field (25 m) AZE; those with B and C were collected by diver cores inside the far-field (100 m) AZE. Samples from macrofauna stations (Figure 4.31) were collected by van Veen grab. ND = Not detected (<1 µg kg-1). Sample collection date and code Emamectin benzoate (µg kg-1 dry weight) 17 APRIL 2002 S1A ND S1B ND S1C ND S2A ND S2B ND S2C ND Reference ND 14 MAY 2002 S1A Trace S1B ND S1C Trace S2A 7.38 S2B Trace S2C Trace Reference Trace 27 AUGUST 2002 S1A ND S1B ND S1C ND S2A 1.64 S2B ND S2C ND S1A spike* 11.53 Reference spike* 14.59 Reference ND NOVEMBER 2002 Macrofauna 1 ND Macrofauna 2 ND Macrofauna 3 ND Macrofauna 4 ND 29 JANUARY 2003 S1A 1.61 S1B 1.18 S1C ND S1C ND S2A 1.41 S2B ND S2C ND S1A spike* 19.03 Reference spike* 6.82 Reference ND * spiked with 30.05 ng emamectin benzoate g-1 wet weight. The metabolite N-demethylated emamectin was not detected in any of the samples collected.

4.7 Predicted post-treatment sediment emamectin benzoate concentrations Sediment emamectin benzoate concentrations measured following the May and September 2002 Slice treatments were compared to predicted concentrations from the Slice module in DEPOMOD (Cromey et al., 2002a).

The model was driven by current meter data collected during the field survey in 2000, (Section 4.3). The current data were assumed to be representative of a spring-neap cycle

Ecological effects of sea lice medicines in Scottish sea lochs 56 of 286 and were repeated within the model for a period of 270 d. A similar assumption is made for sea lice medicine consent modelling (SEPA, 2003a), and limitations of this approach are discussed later. During the modelled period, the farm fish biomass fluctuated (Table 4.2), and all fish were harvested by December 2002. Thus, release of emamectin benzoate from the farm ceased post-harvest. However, continued decay and erosion of emamectin benzoate in deposited wastes would have occurred and this was included in the resuspension model. The total number of particles in the simulation was optimised until increasing numbers did not improve accuracy and only increased computational time (1.6 x 106).

A total of 315 g emamectin benzoate was used for each Slice treatment (Table 4.1). The release of emamectin benzoate adhered to faecal material emanating from the farm was predicted to peak immediately after treatment with Slice and decline exponentially thereafter (Figure 4.13). Release of emamectin benzoate adhered to waste feed was predicted for the treatment period only.

Figure 4.13. Release of emamectin benzoate adhered to uneaten feed and faecal material from Loch Sunart fish farm after Slice treatments in May and September 2002.

Predicted sediment emamectin benzoate concentrations were considerably higher than measured concentrations at most sample stations (Figure 4.14). For those samples in which emamectin benzoate was detected, predicted concentrations were generally two orders of magnitude higher. For example, measured and predicted concentrations below the cages (station S2A) on day 269 post-treatment were 1.4 and 146.7 µg kg-1, respectively (Figure 4.14). On day 9 post-treatment, measured and predicted concentrations at station S2A were 7.4 and 20.5 µg kg-1, respectively.

The predicted footprint of emamectin benzoate deposition was below and slightly to the west of the cage group as a result of the residual current (Figure 4.15). As Transect S2 was perpendicular to the main flow direction and deposition footprint, predictions between S2A and S2C decreased by two orders of magnitude for all sampling events. Predicted concentrations at S2C furthest from the cage group, were less than 1 µg kg-1, in agreement with measured concentrations, which were either trace or not detected (Table

Ecological effects of sea lice medicines in Scottish sea lochs 57 of 286 4.4). Predicted concentrations declined less markedly along the length of Transect 1, which was parallel to the main flow direction and the deposition footprint.

Figure 4.14. Predicted and measured sediment emamectin benzoate concentrations at Loch Sunart on 14 May, 27 August 2002 and 29 January 2003 (9, 114 and 269 days after May Slice treatment). Slice treatments were on 6 May and 3 September 2002 (days 1 and 120 respectively).

Ecological effects of sea lice medicines in Scottish sea lochs 58 of 286 Figure 4.15. Predicted emamectin benzoate sediment concentrations at Loch Sunart on days 9, 114 and 269 post-treatment.

Ecological effects of sea lice medicines in Scottish sea lochs 59 of 286 4.8 Zooplankton A short-term sampling campaign was undertaken at Loch Sunart fish farm to investigate the effects of the bath treatment Excis on the zooplankton community. Zooplankton samples were collected pre- and post-treatment to incorporate the Excis treatment in November 2000. With the introduction of in-feed treatments (Slice) resulting in prolonged release of treatment chemicals, a long-term monitoring programme was established at Loch Sunart. Zooplankton samples were collected fortnightly from five sample stations (Figure 4.16). Regular sampling began in November 2001 and finished in May 2004.

Figure 4.16. Zooplankton sampling station locations at Loch Sunart. Station A is 50 m W of cage group, Station B = 0 m W, Station C = 0 m E, and Station D = 50 m E. Cage groups are indicated by grey rectangles.

4.8.1 November 2000 Excis and Salmosan treatments The zooplankton community in Loch Sunart was sampled over an eight week period in the vicinity of Sunart fish farm to determine the effects of Excis and Salmosan treatments over a period of four days from 28 November to 1 December 2000. During the treatment period, 18 salmon cages were dosed with a total of 1520 g azamethiphos and 7.8 l cypermethrin.

Oithona similis dominated the zooplankton community during the sample period (Figure 4.17). Abundance fluctuated between 3000 and 7000 animals per m3 in a similar manner at the reference station and the four stations close to the salmon cages. Abundance of the

Ecological effects of sea lice medicines in Scottish sea lochs 60 of 286 other zooplankton species was considerably lower and also varied over time (Figure 4.17). In the case of Pseudocalanus elongatus, abundance fluctuated during the sample period and abundance of the later stages (CIV to CV) was often greater at the stations around the salmon cages than at the reference station. Diaixis hibernica abundance was generally greater at the reference station for all life stages. Copepod nauplii decreased in abundance over time at both the reference and cage stations.

A treatment effect was not detected at the community level. In Figure 4.18, there is no obvious relationship between community composition and sample day or distance from the salmon pens. There is a shift from right to left and back again along the first axis, indicating a change in species composition over time. For example, stations on pre- treatment days -4 and -3 are located in the right quadrants while most stations from day -2 pre-treatment to day 1 post-treatment are located towards the left of the ordination plot. There is then a shift back towards the right on day 2 post-treatment. On each sample day, at least one station has a slightly different species composition compared to the other stations, as indicated by its position on the ordination plot. For example, on post- treatment day 23 (+), sample station D is positioned in the top right quadrant, whereas the other four stations (including reference station E) are located together in the bottom right quadrant.

Ecological effects of sea lice medicines in Scottish sea lochs 61 of 286 Figure 4.17. Abundance (number m-3) of the predominant zooplankton species at Loch Sunart during November 2000 Excis and Salmosan treatments at Sunart fish farm over a period of 32 days pre-treatment and 23 days post-treatment. Sea lice treatments were on days 1 to 4. For each sample day, abundance data for the four sample stations within 50 m of the salmon cages were combined and are presented as the mean (± S.E.). CI-CIII and CIV-CV are pre-adult copepod stages 1 to 5. Note: y-axis scales are unequal.

Ecological effects of sea lice medicines in Scottish sea lochs 62 of 286 Figure 4.18. Zooplankton sample stations CA plot for November 2000 Excis and Salmosan treatments at Loch Sunart fish farm. Of the variance, 26% is explained by the first two axes. Open symbols are pre-treatment sample days -32 (), -25 (), -18 (), - 10 (), -4 () and -1 (). Closed symbols and crosses are post-treatment sample days 1 (), 2 (), 3 (), 4 (), 10 (X) and 23 (+). Letters indicate fish farm sample stations A to D, and reference station E.

The species associated with the sample stations are shown in Figure 4.19. All life stages of Diaixis hibernica are positioned in the top left quadrant, indicating greater abundance of this species at sample stations located in this quadrant, which were samples collected primarily on pre-treatment day -25, and post-treatment days 2 and 10 (Figure 4.18). Whereas Pseudocalanus are positioned in the bottom right quadrant, indicating higher abundance in samples collected on pre-treatment day -18 and post-treatment day 23.

There was no relationship between fluctuations in zooplankton abundance and the release of sea lice treatments. Instead, changes in abundance during the sample period were most likely due to advection and natural patchiness.

Ecological effects of sea lice medicines in Scottish sea lochs 63 of 286 Figure 4.19. Zooplankton species CA plot for November 2000 Excis and Salmosan treatments at Loch Sunart fish farm.

4.8.2 Zooplankton long-term monitoring A long-term zooplankton monitoring programme in Loch Sunart involving fortnightly sampling was undertaken from November 2001 to May 2004. Unfortunately, breaks in sampling occurred during March, April and December 2002. The sampling campaign incorporated Excis, Salmosan and Salartect sea lice treatments, and three Slice treatments (Table 4.1).

Trends in zooplankton abundance over the 31 month sampling period are shown in Figure 4.20 to Figure 4.23, with sea lice treatments indicated on the uppermost graphs in each figure. The seasonal patterns of abundance were similar over both 2002 and 2003 with peaks in abundance during the summer months (May to September), and low densities during the winter months. In the summer of 2003, zooplankton abundance peaked at considerably higher densities than in 2002. The greatest contributors to total zooplankton abundance were cyclopoid copepods belonging to the genus Oithona, which were present all year round, with particularly high numbers between June and November in both years (Figure 4.20). The other non-calanoid copepods, Microsetella norvegica and Monothula subtilis were also present throughout most of the year with peaks in abundance between July and November. The calanoid copepods (Pseudocalanus, Acartia, Temora; Figure 4.21 to Figure 4.23) and the cladoceran Evadne (Figure 4.20) generally displayed two peaks in abundance; the first in early summer (April/May), followed by a second peak in July/August. High numbers of copepod nauplii were observed in early summer in 2002, whereas a considerably larger peak occurred in July 2003 (Figure 4.20).

Ecological effects of sea lice medicines in Scottish sea lochs 64 of 286 Figure 4.20. Abundance (number m-3) of zooplankton at Loch Sunart during the long- term monitoring program starting November 2001. 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 are indicated by arrows on the uppermost graphs; Excis (C), Salartect (HP), Salmosan (A), Slice (EB1, EB2, EB3). Note: y-axis scales are unequal.

Ecological effects of sea lice medicines in Scottish sea lochs 65 of 286 Figure 4.21. Abundance (number m-3) of Pseudocalanus life stages at Loch Sunart during the long-term monitoring program starting November 2001. 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 are indicated by arrows on the uppermost graphs; Excis (C), Salartect (HP), Salmosan (A), Slice (EB1, EB2, EB3).

Seasonal progression through the developmental stages can be seen for Pseudocalanus, Acartia and Temora (Figure 4.21 to Figure 4.23). Peaks in abundance of the earlier life stages (CI-CIII) preceded peaks of later stage copepodites (CIV-CV) and finally smaller peaks of adults. In the case of Pseudocalanus (Figure 4.21), stages CI-CIII peaked twice in 2002 (in April/May and July/August), with smaller peaks of CIV-CV in May and October. Abundance of adult females peaked in October 2002, and in June 2003 following peaks in the earlier stages from April to June. In 2003 and 2004, major peaks in abundance of Pseudocalanus developmental stage occurred only once each year.

Acartia CI-CIII peaked in abundance from May to July 2002, and in July 2003 (Figure 4.22). Smaller peaks in abundance of CIV-CV and adults followed in July and August.

Ecological effects of sea lice medicines in Scottish sea lochs 66 of 286 Figure 4.22. Abundance (number m-3) of Acartia life stages at Loch Sunart during the long-term monitoring program starting November 2001. 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 are indicated by arrows on the uppermost graphs; Excis (C), Salartect (HP), Salmosan (A), Slice (EB1, EB2, EB3).

Ecological effects of sea lice medicines in Scottish sea lochs 67 of 286 Figure 4.23. Abundance (number m-3) of Temora life stages at Loch Sunart during the long-term monitoring program starting November 2001. 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 are indicated by arrows on the uppermost graphs; Excis (C), Salartect (HP), Salmosan (A), Slice (EB1, EB2, EB3).

Temora CI-CIII densities were highest between April and July in 2002 and 2003 (Figure 4.23). Lower densities of CIV-CV were also present during this period, with even lower densities of adults present from May to August of each year.

The seasonal zooplankton data clearly indicate that the various sea lice treatments at Loch Sunart fish farm did not adversely impact the zooplankton community as the seasonal trends in species composition and abundance were similar in 2002 and 2003, and followed seasonal cycles. It is unlikely that differences in zooplankton densities in the summers of 2002 and 2003 were related to sea lice treatments. Instead, inter-annual variation in rainfall may have contributed to the observed trends. In July 2002, a sharp drop in surface salinity was observed (Figure 4.24) due to high rainfall. A similar decline in salinity was not observed in 2003 when total zooplankton abundance peaked at greater than 2 x 104 individuals m-3 (Figure 4.20).

Ecological effects of sea lice medicines in Scottish sea lochs 68 of 286 Figure 4.24. Salinity (A) and temperature (B) at different depths from December 2001 to June 2004. The data were measured by a Seabird SBE19 CTD at zooplankton sample station B in Loch Sunart.

Furthermore, zooplankton abundance at the reference and cage stations was generally similar and displayed the same seasonal patterns. There were differences in species abundance between the stations located close to the fish farm, but they were unpredictable, illustrating natural variability in zooplankton distribution resulting from patchiness and current flow around the cages, rather than treatment related differences in abundance.

It is possible that declines in zooplankton abundance in late summer in both 2002 and 2003 could have mistakenly been attributed to sea lice treatments of Slice (2002) and Salmosan (2003) had a long term data set not been collected to show the repeating seasonal cycles.

4.9 Phytoplankton 4.9.1 November 2000 Excis and Salmosan treatment The phytoplankton community was sampled over an eight week period in the vicinity of Sunart fish farm to examine the potential effects of Excis and Salmosan treatments over a four-day period from 28 November to 1 December 2000 (Table 4.1). Samples for phytoplankton, nutrients and salinity were collected from zooplankton station D (Figure 4.16).

Ecological effects of sea lice medicines in Scottish sea lochs 69 of 286 Salinity was relatively low (30.72, ±0.36 S.E.) during the sampling period. Dissolved nutrient concentrations (ToxN) were relatively high and within normal ranges for winter nutrients in Scottish coastal waters (Figure 4.25) (e.g. Gubbins et al., 2003). Total phytoplankton cell abundance was relatively low (1.5 x 105 to 3.5 x 105 cells l-1), again representative of winter conditions (Figure 4.25). Slightly elevated ToxN concentrations during the four-day sea lice treatment period (hatched bars Figure 4.25) corresponded with slightly lower phytoplankton cell numbers. These trends are consistent with the natural variability of a low biomass winter population and are reproduced in other winters sampled during this project (Section 4.9.2).

Figure 4.25. Total phytoplankton cell abundance (cells l-1) and ToxN concentration (nitrate plus nitrite, µM) at Loch Sunart from 17 November to 14 December 2000. The hatched bars indicate the sea lice treatment period (Excis and Salmosan).

Proportions of the four main types of phytoplankton (diatoms, dinoflagellates, microflagellates and others) were relatively similar throughout the sampling period (Figure 4.26), and species composition was representative of Scottish winter communities (e.g. Dodge et al., 1995; Gubbins et al., 2003). Microflagellates (<15 µm) dominated the community numerically, while diatoms and dinoflagellates each contributed ca. 1 x 104 cells l-1 to the population at any time. Diatom species included Skeletonema costatum, Pseudo-nitzschia sp., Chaetoceros sp. (<15 µm), Cylindrotheca closterium and Thalassiosira sp. (<20 µm). Dinoflagellate species included small (<15 µm) armoured dinoflagellates, Gymnodinium, and a Heterocapsa-type species. A total of 49 phytoplankton taxa were observed, however 87 to 96 % of the population comprised only eleven of these. The remaining 38 taxa never individually exceeded 2 x 103 cells l-1.

Neither phytoplankton community composition or cell abundances observed in winter 2000 differed significantly from winter communities and abundances when there were no

Ecological effects of sea lice medicines in Scottish sea lochs 70 of 286 sea lice treatments (in 2003) (Section 4.9.2). In addition, species successional characteristics in November and December were similar each year. As a result, no direct relationship between applications of sea lice treatment medicines in November/December 2000 and adverse affects on the phytoplankton community could be discerned.

100%

80%

60% Dinoflagellates Diatoms Microflagellates 40% Others *

Population percentage 20%

0% 7-Dec 1-Dec 14-Dec 30-Nov 29-Nov 28-Nov 27-Nov 23-Nov 17-Nov Date (2000) Figure 4.26. Gross phytoplankton community composition at Loch Sunart between 17 November and 14 December 2000. Sea lice treatments (Excis and Salmosan) were from 28 November to 1 December. *“Others” includes other flagellates, cryptophytes, ciliates, cysts/resting stages and silicoflagellates.

4.9.2 Phytoplankton long-term monitoring A long-term phytoplankton monitoring programme was undertaken at Loch Sunart from June 2000 to April 2004. Samples for phytoplankton, salinity and nutrients were collected from the top 10 m of the water column at either zooplankton station D (Figure 4.16) or from the farm cages. This long-term dataset forms a comprehensive and important information resource with a high resolution of sample collection events.

The relatively low salinity average over the four years at Loch Sunart (31.6 ±0.16 S.E.) was due to higher relative freshwater input from two substantial rivers (the Strontian and the Carnoch) and from a burn next to the farm site which can spate considerably in spring months. No regular seasonal fluctuation could be observed in the salinity samples collected from the top 10 m of the water column.

Total phytoplankton cell abundance and ToxN concentrations (nitrate plus nitrite) at Loch Sunart over the four-year sampling programme show classical seasonal phytoplankton abundance and nutrient uptake characteristics, e.g. relatively high [ToxN] during autumn/winter months correlated with relatively low phytoplankton cell abundance, and vice versa during the spring/summer months (Figure 4.27). Maximum and minimum ToxN concentrations were relatively similar each year.

Ecological effects of sea lice medicines in Scottish sea lochs 71 of 286 Figure 4.27. Total phytoplankton cell abundance (cells l-1) and dissolved inorganic ToxN (nitrate plus nitrite; µM) at Loch Sunart between June 2000 and April 2004. A significant bloom of the chain-forming diatom Skeletonema costatum (17.2 x 106 cells l-1) in March 2004 has been omitted from the graph as its inclusion distorted the representation of other values.

Successive annual phytoplankton cell abundance trends at Loch Sunart are superimposed on a Julian Day scale, where January 1 = 1 and December 25 = 365, in Figure 4.28. Summer phytoplankton population maxima occurred between May and July, with a general increase in cell abundance occurring with successive years during the sampling programme. During 2000 and 2001, maximum phytoplankton standing stock was typically just under 4 x 106 cells l-1, however this increased to about 8 x 106 cells l-1 in 2002, and in 2003 the maximum standing stock was 10 x 106 cells l-1 in May. The increased cell abundance in the two later years was primarily due to an increased frequency of diatom blooms between May and August in 2002 and to a significant bloom of the small chain-forming diatom Skeletonema costatum (5.3 x 106 cells l-1) during May 2003 (Figure 4.29).

Ecological effects of sea lice medicines in Scottish sea lochs 72 of 286 Figure 4.28. Total phytoplankton cell abundance at Loch Sunart using the Julian Day scale (where January 1 = 1 and December 31 = 365). A significant bloom of the chain- forming diatom Skeletonema costatum (17.2 x 106 cells l-1) in March 2004, has been omitted from the graph as its inclusion distorted the representation of other values.

Twelve significant phytoplankton blooms (>9 x 105 cells l-1) were observed at Loch Sunart during the four-year sampling programme. This relatively high occurrence of blooms may reflect the sheltered geography of the loch. For presentational reasons, the S. costatum bloom in March 2004 has been omitted from Figure 4.27 and Figure 4.28 as its inclusion distorted representation of the phytoplankton population at other times. At its peak, the bloom constituted 94 % of the entire phytoplankton population (Figure 4.29).

Ecological effects of sea lice medicines in Scottish sea lochs 73 of 286 100%

80%

Dinoflagellates 60% Diatoms Microflagellates 40% Others *

Population percentage 20%

0% Jan-03 Jun-03 Jun-01 Jun-02 Jun-00 Oct-01 Oct-02 Feb-02 Apr-03 Dec-03 Dec-01 Mar-03 Mar-01 Aug-03 Aug-02 Nov-00 Aug-00 Aug-01 May-02 Date Figure 4.29. Gross phytoplankton community composition at Loch Sunart between June 2000 and April 2004. The 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.

Cell abundance during winter months (November to February) in 2000/01 and 2001/02 was consistently low at typically 2.5 x 105 cells l-1. However, during the winter months of 2002/03 and 2003/04 higher total cell abundances (typically 8.5 x 105 cells l-1) were due to higher numbers of microflagellates (<15 µm). Winter samples were always dominated by microflagellates, with diatoms and dinoflagellates each contributing typically 1 x 104 to 1.5 x 104 cells l-1. Some 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.

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.

Sea lice treatments undertaken during the long-term monitoring programme did not affect the phytoplankton community. Furthermore, if sea lice treatments had affected the zooplankton community, phytoplankton cell abundance should have increased as grazing pressure decreased as differential zooplankton grazing is likely to be a factor controlling the abundance of phytoplankton populations at certain times of the year. In 2002, density increases of both phytoplankton and zooplankton occurred simultaneously and fluctuations in abundance throughout the summer did not suggest close coupling between the two populations (Figure 4.30). In 2003, there was some indication of coupling between zooplankton and phytoplankton populations. Spring growth of zooplankton preceded the phytoplankton bloom, which was subsequently followed by a second larger peak in zooplankton abundance in July 2003, with a concomitant decline in phytoplankton abundance, suggesting grazing pressure.

However, sea lice treatments did not appear to be a causal factor in any of the abundance trends observed. For example, following the Slice treatment in May 2002, zooplankton abundance increased while phytoplankton abundance decreased (Figure 4.30). Whereas

Ecological effects of sea lice medicines in Scottish sea lochs 74 of 286 after the September 2002 Slice treatment, both zooplankton (primarily Oithona) and phytoplankton (primarily microflagellates) abundance increased initially before decreasing with the onset of winter. The S. costatum bloom in March 2004 occurred not long after the third Slice treatment, and the spring increase in zooplankton abundance also began following this treatment.

Thus, at Loch Sunart, any potential effects of sea lice treatment events on the phytoplankton community could not be separated from natural factors such as inherent patchiness, bloom characteristics, the natural variability of species succession and grazing dynamics.

Figure 4.30. Total zooplankton (animals m-3) and phytoplankton abundance (cells l-1) from integrated water column samples at Loch Sunart between November 2001 and May 2004. Total zooplankton is presented as the mean of four sample stations (± S.E.). Timing of sea lice treatments are indicated by arrows; Excis (C), Salartect (HP), Slice (EB1, EB2, EB3), Salmosan (A). Note different y axes.

4.10 Meiofauna Meiofaunal analyses were conducted on samples collected from van Veen grabs at Loch Sunart in November 1999, February 2000 and March 2001.

Ecological effects of sea lice medicines in Scottish sea lochs 75 of 286 Figure 4.31. Locations of combined macro-, meiofaunal and sublittoral sampling stations in Loch Sunart; (A) shows cage position pre- and (B) post-move.

Five samples were collected from each of four stations in 1999 and 2001 (Figure 4.31). In 2000, two sub-samples were collected from a single grab at each location. The amount of sediment collected varied between sampling occasions. The average sample volume in 1999 was 264 cm3, 46 cm3 in 2000, and 142 cm3 in 2001. Differing sub-samples of sediment (20 % for 1999 samples, 100 % for 2000 samples, 5 % for 2001 samples) were processed from each set of samples. Nematodes and copepods in each sub-sample were identified and counted. Replicate samples were not collected in 2000, so no formal statistical analysis was attempted.

4.10.1 Nematodes The nematode community in Loch Sunart is typical of muddy sediments. 259 putative species were recognised. Species occurring included Comesa interrupta, Desmodora pontica, Dorylaimopsis punctata, Leptolaimus elegans, L. venustus, Leptolaimus juveniles, Metalinhomoeus typicus, Metalinhomoeus longiseta, Paramonhystera sp., Thalassamonhystera sp., Richtersia inequalis, Sabatieria ornata, Sabatieria punctata, Spirinia parasitifera, Stephanolaimus sp., Terschellingia longicaudata, Terschellingia spp., Daptonema spp., Theristus sp. and Viscosia elegans.

Non-metric multidimensional scaling (MDS) ordinations and ANOSIM tests of inter- sample similarities calculated using the Bray-Curtis similarity coefficient based on mildly transformed nematode abundance data revealed subtle, albeit significant, differences in nematode community structure between stations in both the November 1999 and March 2001 samples. Although these differences suggested a minor effect of fish farming in 1999, in that the assemblage at Station 1 differed from those at other stations further from

Ecological effects of sea lice medicines in Scottish sea lochs 76 of 286 the cages, differences between stations in 2001 appeared to be more influenced by depth. In neither case did the pattern of inter-station differences suggest that the farm was having a strong influence on nematode community structure.

Data from the three surveys were combined. In order to minimise seasonal and taxonomic errors, abundances were aggregated to the genus level and summed to emphasize inter- stations differences and to remove noise resulting from inter-sample variability within stations. The data were then mildly transformed (square-root transformation) to downweight the influence of numerically dominant species, and used to calculate Bray- Curtis similarities. The MDS plot based on these similarities shows that there were clear differences between surveys (Figure 4.32). The within-survey variability in 2000 was very much higher than in 1999 or 2000. This is, in large part, a probable consequence of sampling error. These were small samples collected from single grabs, whereas the other points represent centroids from groups of five samples. Differences between stations within the 1999 and 2001 surveys are smaller than differences between the 1999 and 2001 surveys. The observed differences resulted from small differences in abundances of common taxa, and do not suggest a clear cause related to fish-farming activity.

Figure 4.32. MDS plot of Bray-Curtis similarities in nematode abundances from the 3 surveys at Loch Sunart. Nematode abundances were aggregated to genus, summed within each station, and square-root transformed prior to calculating similarities. Numbers (1- 4) indicate stations, where Station 4 is the reference. Symbols (, 1999; , 2000; , 2001) represent surveys.

Within each of the 1999 and 2001 surveys, inter-station variability was small, and the patterns of differences between stations do not suggest a strong influence from fish- farming activity in the vicinity of Station 1. That being said, it does appear that in 2001 Station 1 is more clearly separated from other stations than in 1999. This is reinforced by K-dominance curves (Figure 4.33), which show that in Station 1 has a slightly lower nematode diversity, and was more dominated, than other stations in 2001 and all stations in 1999. This suggests a mild effect, of a type commonly associated with organic enrichment.

Ecological effects of sea lice medicines in Scottish sea lochs 77 of 286 Figure 4.33. K-dominance curves (cumulative dominance % against species rank) plotted from adjusted summed nematode abundances from each survey/site combination in the 1999 and 2001 surveys from Loch Sunart. In the key the first numeral denotes the survey (1, 1999; 3, 2001) and the second (1-4) denotes the station. Station 4 is the reference.

4.10.2 Meiobenthic copepods The copepod community in Loch Sunart was typical of muddy sediments. 41 taxa were identified. Species included Bradya scotti, Halectinosoma spp., Stenhelia (Delavalia) longicaudatus, Stenhelia (D.) reflexa, Typhlamphiascus confusus, Haloschizopera pygmaea, Pseudameira mixta, an unidentified species of Ameirid, Cletodes longicaudatus, Normanella mucronata and Normanella sarsi.

In samples from the 1999 survey, copepod abundances ranged from 0 to 5 individuals, distributed among 0 to 4 species, per sub-sample identified and enumerated. Thus the data were too sparse for a meaningful analysis. The samples from the 2000 survey were not sub-sampled, but only contained 0 to 2 individuals in 0 or 1 species. In samples from the 2001 survey, abundances ranged from 1 to 70 individuals, distributed among 1 to 18 species, per sub-sample. Although better, these data are still too sparse for a meaningful analysis, with high abundances and numbers of species being limited to a small subset of samples. While it might have been possible to enhance the data within the 1999 and 2001 surveys by counting and identifying all individuals, these findings suggest that the low numbers of specimens present in the samples precluded inter-comparisons of data from the different surveys.

This is unfortunate, as it is probable that any affects of sea lice treatments on meiofaunal assemblages would be expected to primarily impact the copepods, which are crustaceans. While the possibility cannot be discounted that the general lack of copepods in these samples may be linked to the use of sea lice treatment medicines at the farm, neither can other possible causes be discounted. It is not known what the expected numbers of

Ecological effects of sea lice medicines in Scottish sea lochs 78 of 286 copepod individuals and species would be in samples from this Loch. Samples from elsewhere in Scottish waters have produced much higher numbers, however no background studies have been identified from Loch Sunart. More importantly, the sampling method employed (sub-sampling from a van Veen grab) can be an unreliable way to sample copepods. If the sediments are cohesive and the grab is in excellent condition, then it is sometimes possible to get adequate samples. It is possible this was not the case, and given the stochastic nature of the data, problems associated with sample quality cannot be discounted as the cause of the lack of copepods in samples from Loch Sunart.

4.11 Macrobenthos Macrobenthic invertebrate populations at Loch Sunart were sampled on 19 October 1999 (pre-treatment), 28 February, 6 November 2000, 8 March 2001 and 26 November 2003 (post-treatment) to determine whether the effects of sea lice treatments could be detected against the background of organic enrichment normally associated with fish farm activities.

4.11.1 Sediment conditions at Loch Sunart fish farm Sediment nature and appearance, and physico-chemical conditions at each site were assessed. Visual description of sediment type (Appendix IV - Macrofauna; Table 15.3) and measurements of redox potential (Eh) values down the sediment column (Appendix IV - Macrofauna; Table 15.2) can provide an indication of organic enrichment effects on seabed sediments and are of assistance when attempting to resolve any effects of sea lice treatment agents on the macrofauna from effects of organic enrichment. Sandy mud sediments were recorded at Stations 1 to 3 throughout the sampling period, and mud sediments at station 4. Eh values at a sediment depth of 40 mm depth were generally positive in all surveys, apart from a low negative value in two core samples in November 2000 and four in November 2002. This suggests that sedimentary oxidation in the sampling area was satisfactory throughout the sampling period.

4.11.2 Macrofaunal distribution, abundance and structure There was a considerable difference between macrofauna populations at Stations 1-3 and those at Station 4 (Table 4.5). The latter had half the number of species and less than half the number of individuals and biomass recorded at Stations 1-3. Community structure was also different at Station 4 (Table 15.3). On the initial sampling in October 1999, Station 4 was characterised by a sea slug, Cylichna, the small bivalve molluscs Nuculoma and Corbula, the polychaetes Minuspio and the brittle star Amphiura filiformis. Cylichna did not recur in high numbers during the ensuing four years sampling but Nuculoma was among the dominants in the following year, Corbula in the following two years and Minuspio in the following four years. A. chiajei was not among the dominants in 2000/1 but recurred in 2002/3. Another brittle star, A. filiformis appeared in the dominants at Station 4 in 2000 and 2003.

The other three stations were dominated by a different suite of small bivalves and polychaetes including Thyasira, Mysella, Melinna, Prionospio and Magelona (Table 15.3). Amphiura filiformis was always present among the dominants at Station 3, and in 1999 and 2000 at Station 2. At Station 1, however it was among the dominants only in 2003.

Ecological effects of sea lice medicines in Scottish sea lochs 79 of 286 Table 4.5. Macrofaunal population statistics and indices at Loch Sunart (1999 - 2003).

(i) October 1999

Station number 1 2 3 4

Total no. of taxa 100 118 104 51 Mean no. of taxa per sample (S) 46 ± 5 54 ± 14 50 ± 7 25 ± 4 Total Abundance (A) 1241 1078 1044 367 Mean Abundance (A) 248 ± 101 216 ± 98 209 ± 43 73 ± 28 Total biomass (g) (B) 30.71 36.41 33.47 19.48 Mean biomass per sample (g) (B) 6.14 ± 1.03 7.28 ± 2.72 6.69 ± 3.49 3.90 ± 0.49 A/S 12 9 10 7 B/A (mg) 25 34 32 53 H´ 4.32 - 4.83 5.46 - 4.81 3.88 - 5.08 3.90 - 4.44 J 0.78 - 0.87 0.84 - 0.90 0.72 - 0.90 0.87 - 0.93 ITI 55 - 63 61 - 76 65 - 83 54 - 64

(ii) February 2000

Station number 1 2 3 4

Total no. of taxa 103 70 91 55 Mean no. of taxa per sample (S) 55 ± 5 34 ± 5 52 ± 4 23 ± 2 Total Abundance (A) 2176 795 1061 19 Mean Abundance (A) 435 ± 68 159 ± 23 212 ± 24 4 ± 16 Total biomass (g) (B) 82.34 31.43 49.33 8.69 Mean biomass per sample (g) (B) 16.47 ± 7.19 6.29 ± 1.23 2.33 ± 0.45 .16 ± 0.71 A/S 21 11 12 6 B/A (mg) 38 40 47 59 H´ 3.59 - 4.26 3.86 - 4.40 4.81 - 5.22 3.87 - 4.23 J 0.63 - 0.72 0.81 - 0.85 0.86 - 0.89 0.84 - 0.93 ITI 46 - 53 54 - 62 58 - 68 59 - 67

(iii) November 2000

Station number 1 2 3 4

Total no. of taxa 88 143 76 69 Mean no. of taxa per sample (S) 45 ± 4 75 ± 11 37 ± 4 33 ± 4 Total Abundance (A) 1536 2177 1322 528 Mean Abundance (A) 307 ± 78 435 ± 68 364 ± 62 106 ± 19 Total biomass (g) (B) 34.54 92.85 77.70 22.28 Mean biomass per sample (g) (B) 3.49 ± 1.52 3.04 ± 1.64 3.48 ± 1.22 1.53 ± 0.84 A/S 18 15 17 8 B/A (mg) 23 43 59 42 H´ 3.99 - 4.46 4.80 - 5.75 3.41 - 4.03 4.08 - 4.79 J 0.71 - 0.80 0.80 - 0.89 0.65 - 0.79 0.84 - 0.91 ITI 59 - 63 54 - 59 62 - 65 61 - 66

Ecological effects of sea lice medicines in Scottish sea lochs 80 of 286 Table 4.5 (cont.)

(iv) March 2001

Station number 1 2 3 4

Total no. of taxa 83 99 103 45 Mean no. of taxa per sample (S) 45 ± 3 56 ± 7 50 ± 5 25 ± 5 Total Abundance (A) 1689 1497 901 533 Mean Abundance (A) 338 ± 77 299 ± 42 180 ± 34 107 ± 17 Total biomass (g) (B) 33.52 53.67 49.62 18.94 Mean biomass per sample (g) (B) 6.70 ± 1.40 10.73 ± 4.44 9.92 ± 2.98 3.79 ± 1.46 A/S 20 15 9 12 B/A (mg) 20 36 55 36 H´ 3.90 - 4.17 4.34 - 5.08 4.72 - 5.05 3.53 - 4.47 J 0.73 - 0.78 0.77 - 0.86 0.85 - 0.89 0.80 - 0.89 ITI 55 - 59 57 - 66 65 - 68 57 - 68

(v) November 2003

Station number 1 2 3 4

Total no. of taxa 88 104 74 51 Mean no. of taxa per sample (S) 43 ± 5 45 ± 14 34 ± 7 23 ± 5 Total Abundance (A) 1067 1067 797 599 Mean Abundance (A) 213 ± 41 213 ± 126 159 ± 26 120 ± 45 Total biomass (g) (B) 50.18 50.52 72.10 47.12 Mean biomass per sample (g) (B) 10.04 ± 4.30 10.10 ± 7.32 14.42 ± 7.76 9.42 ± 2.39 A/S 12 10 11 5 B/A (mg) 47 47 90 83 H´ 4.34 - 4.90 4.35 - 4.91 3.85 - 4.77 2.50 - 4.20 J 0.83 - 0.88 0.82 - 0.91 0.80 - 0.88 0.60 - 0.89 ITI 40 - 45 39 - 45 64 - 77 55 - 68

The differences between Station 4 and the others are probably related to the presence of finer sediments at Station 4 (Appendix IV - Macrofauna; Table 15.1), but it has also been suggested that the somewhat impoverished community at Station 4 might be the result of trawling disturbance in that area. However, the differences between station 4 and the other stations are consistent and therefore should not influence conclusions drawn from a comparison of changes in the fauna observed over the two years. The results of pair-wise significance tests of differences between the faunal groups in each survey are presented in Table 15.4.

There were no significant differences between annelid, crustacean and echinoderm taxa numbers at station 1 in the first survey in October 1999 and surveys in 2000-2003. However there was a significant difference in the number of annelid taxa in March 2001 and November 2003 compared to February 2000. There were significantly lower numbers of mollusc taxa in the 2000-2001 surveys, but no significant difference in 2003. Significantly higher abundances of annelids were found in February 2000. There were no significant differences in numbers of crustacean and echinoderm taxa between surveys. In March 2001, echinoderm biomass was significantly lower than in the two previous surveys, however biomass recovered to previous levels by 2003.

At Station 2, numbers of annelid, mollusc and echinoderm taxa were significantly lower in February 2000, but by November 2000 had recovered to levels higher than observed in October 1999. In March 2001, taxon numbers were the same as in October 1999, but were significantly lower in November 2003. Annelid and mollusc abundance was significantly higher in 2000-2001, but there were no significant differences in the two later surveys.

Ecological effects of sea lice medicines in Scottish sea lochs 81 of 286 Biomass was significantly lower in November 2000 than in the first survey, recovered during 2001-2002, but declined significantly in 2002-2003. No significant differences were found in crustacean abundance between surveys, but biomass was significantly higher in February 2000, March 2001 and November 2003.

At Station 3, numbers of annelid, mollusc and crustacean taxa were significantly lower in November 2000. Numbers of annelid and crustacean taxa had recovered in March 2001, but numbers of mollusc taxa remained significantly lower than in October 1999, in all subsequent surveys. Numbers of echinoderm taxa were significantly lower in February 2000 than in October 1999, but no significant difference was detected in any of the later surveys. Mollusc abundance was significantly higher in November 2000 and significantly lower in March 2001 but there were no significant differences in November 2003. There were no significant differences in abundance of annelids, crustaceans or echinoderms between surveys. Annelid biomass was significantly higher in February 2000 than in October 1999 as was echinoderm biomass in November 2000 and November 2003. There were no significant differences in biomass of any other taxa between surveys.

Annelid taxa numbers and abundance were significantly higher in February 2000 but no significant differences were seen in any subsequent surveys. No significant differences in taxon numbers, abundance or biomass were detected between surveys with the exception of significantly higher crustacean and echinoderm biomass in March 2001 and November 2003 respectively.

It should be noted that Station 2 was initially sited 200 m E from the cages and Station 3 400 m E. The cages were repositioned following the March 2001 survey such that Station 2 was adjacent to the cages and Station 3 was 200 m E of the cages. Because the residual flow through the cages was easterly the move would have increased the likelihood of released contaminants affecting these two stations between the sampling periods. Station 4 was 1500 m NE of the cage group and Station 1 200 m W. These positions make it less likely that effects would be seen in these areas. Stations 2 and 3 were shallower (16-20 m) than the other two stations (32-40 m).

The higher numbers and biomass of annelids and molluscs at Station 1 in February 2000 may be attributed to a normal spring increase in settlement and growth, particularly of annual species, e.g. the small annelid worms Prionospio and Magelona. Conversely the significant decrease in annelid, mollusc and echinoderm species in February 2000, accompanied by no changes in abundance in these groups at Station 2 suggests inhibition in settlement and growth at station 1. Significant decreases in mollusc and echinoderm species at Station 3 might be attributable to contaminant effects, however concomitant increases in crustacean species and numbers casts doubt on such a conclusion as crustaceans are the group most vulnerable to cypermethrin effects. No significant changes were seen at Station 4 (reference station). The lack of spring increases in these areas might also indicate an inhibition effect.

Ecological effects of sea lice medicines in Scottish sea lochs 82 of 286 Figure 4.34. K-dominance curves based on macrofaunal abundances from Loch Sunart in October 1999.

To assess changes in community diversity over the sampling period significant differences in K-dominance curves between groups of samples were determined (Figure 4.34). The curve for Station 4 (reference) in October 1999 was significantly different from the other three stations, in particular stations 1 and 2 closest to the cages differed significantly. However, there was no clear evidence of reduced diversity or increased dominance in areas close to the cages. In subsequent surveys (2000 and 2001) there was some evidence of lower diversity and increased dominance at the three stations closest to the cages. However, in 2003, diversity was reduced at the reference station.

Changes in community structure over the sampling period were examined by ordinating inter-sample similarities of transformed macrofaunal abundance data using MDS analysis.

Ecological effects of sea lice medicines in Scottish sea lochs 83 of 286 Figure 4.35. MDS plot based on Bray-Curtis similarities calculated from square-root transformed macrofaunal abundances from Loch Sunart in October 1999.

In October 1999, inter-replicate variability was quite high at all stations (Figure 4.35), with samples from the reference station (4) grouped together to the left of the plot, and the three other stations positioned to the right. Samples from Station 1 are grouped to the centre left whereas those from stations 2 and 3 overlap on the left hand side of the plot. The ANOSIM gave a Global R (measure of overall difference between samples) of 0.826, p<0.001, with all pairwise comparisons p=0.008. Note, for this and all subsequent tests a value of p=0.008 was the highest significance (lowest p value) achievable from two groups of five replicates (126 possible permutations). This indicates that community structure was significantly different between all stations.

Figure 4.36. MDS plot based on Bray-Curtis similarities calculated from square-root transformed macrofaunal abundances from Loch Sunart in March 2000.

In March 2000, the distribution of samples from the four stations was similar to that seen in the previous survey, although separation of the three stations closest to the cages is considerably clearer (Figure 4.36). Inter-replicate variability increased from Stations 1 to 4, but was generally less than in October 1999 and samples from stations 1, 2 and 3 are clearly separated from each other on the left of the plot. The ANOSIM gave a Global R of 0.900, p<0.001, and for all pairwise comparisons p=0.008. Thus, community structure was significantly different between all stations.

Ecological effects of sea lice medicines in Scottish sea lochs 84 of 286 Figure 4.37. MDS plot based on Bray-Curtis similarities calculated from square-root transformed macrofaunal abundances from Loch Sunart in November 2000.

In November 2000, inter-replicate variability was higher at Station 4 than the other three stations (Figure 4.37). The ANOSIM Global R was 0.902, p<0.001 and for all pairwise comparisons p=0.008. Thus community structure was significantly different between stations, with stations 2 and 4 clearly separated from each other and from Stations 1 and 3, which were very similar.

Figure 4.38. MDS plot based on Bray-Curtis similarities calculated from square-root transformed macrofaunal abundances from Loch Sunart in March 2001.

In March 2001, inter-replicate variability was highest at Station 3, followed by Station 4 (Figure 4.38). The ANOSIM Global R was 0.976, p<0.001 and for all pairwise comparisons p=0.008, thus community structure was significantly different between all stations, with Stations 1, 2 and 3 clearly separated.

Ecological effects of sea lice medicines in Scottish sea lochs 85 of 286 Figure 4.39. MDS plot based on Bray-Curtis similarities calculated from square-root transformed macrofaunal abundances from Loch Sunart in November 2003.

In November 2003, inter-replicate variability was highest at Station 2, followed by Station 4 (Figure 4.39). The ANOSIM Global R was 0.706, p<0.001 and for all pairwise comparisons p=0.008. Community structure was significantly different between all stations, although as in October 1999 (Figure 4.35), separation between the stations closest to the cages is not as clear as at other times. There was evidence of a gradient in community structure from Station 2, through 1 and 3 (which were similar), to station 4.

4.11.3 Analysis across surveys The averaged assemblage structure within each site/station combination shows that variability at individual stations across time was greatest at Station 2, followed by stations 4, 3 and 1 (Figure 4.40).

There were no significant differences in average community structure between Stations 1, 2 and 3, and Station 4 (reference) that differed in any way from patterns seen in the individual surveys (ANOSIM p=0.008).

Figure 4.40. MDS plot based on Bray-Curtis similarities calculated from square-root transformed macrofaunal abundances averaged within survey/station combinations from Loch Sunart. The numerals indicate surveys (1, October 1999; 2, March 2000; 3, November 2000; 4, March 2001; 5, November 2003).

Ecological effects of sea lice medicines in Scottish sea lochs 86 of 286 4.11.4 Conclusions Between surveys the Sunart fish farm treated with Excis in October and November 1999, eight further times in 2000 and again in January 2001 (Table 4.1). It is possible that declines in species and animal densities at Station 2, and the lack of a seasonal increase at Stations 3 and 4 in February 2000 could be responses to these treatments. Such changes might also be attributable to other disturbance effects of farm activities, such as carbon enrichment. However, moderate levels of detrital carbon would be expected to increase growth and benthic production and there was no evidence of high inputs to the area (Table 17.1). Therefore, evidence for a direct influence of sea lice treatments on benthic fauna based on a univariate statistical approach is equivocal.

There were significant differences between stations in each survey at Loch Sunart, both in community structure, and the dominance/diversity structure of assemblages. However, these differences were often subtle, and difficult to relate to the effects of aquaculture at the site. It is probable that the differences were attributable to a gradient in organic enrichment originating at the cages and influencing all but the reference station communities.

The K-dominance plots for individual surveys, and for all surveys combined (not shown), did not show evidence of strong decreases in diversity or increases in dominance at any station. While differences were significant, they may well reflect natural variability in the loch. The combined MDS ordination indicated that the macrobenthic community at Station 4 differed from the other stations, but over time the assemblages at Stations 1, 2 and 3 remained the same. Differences within individual surveys may therefore reflect the spatial dispersion of replicates, with differences in assemblages between replicates at each station varying between surveys. Differences between stations may simply reflect a gradient of organic enrichment across the site related to cage feed and faecal inputs to the sediments. Taken overall, there is no evidence that sea lice treatments affected macrofaunal community composition.

4.12 Sublittoral settlement on suspended panels Sublittoral arrays of slate panels were deployed at four stations in Loch Sunart (Figure 4.31) on 14 January 2000, LS2 (predicted high impact), LS1 and LS3 (intermediate) and LS4 (reference). A brief inspection of the arrays on 1 March 2000 showed occasional thin films of microalgae (diatoms and cyanobacteria) on the upper slates (~ 2 m depth).

The first full inspection of the arrays carried out on 6 July 2000 (Table 4.6) revealed array LS1 was missing as well as the upper sets of panels at LS2 and LS3. Occasional loss of slates occurred at all loch study sites, but this was less of a problem at this study site compared to Lochs Diabaig and Craignish. However, the upper set of four slates at Station LS4 was the most vulnerable and was missing on several occasions.

Heavy settlements of common ‘fouling’ organisms had occurred on most of the slates and all of the ropes, buoys and spars used in each assemblage. The most abundant biota on the slates were barnacles Balanus crenatus, two species of ascidians Ascidiella aspersa and Ciona intestinalis, and the wiry, much branched hydroid Obelia longissima. This itself was covered with superabundant small spat of the mussel Mytilus edulis. The hydroid with adhering mussel spat also occurred abundantly on all the connecting ropes of the arrays. In general, the barnacles had settled heavily on the slates at all three depths while the two ascidians were most abundant on the middle and lower sets of slates, often growing on top of (and thus obscuring) the barnacles already there.

The superabundant barnacles (B. crenatus) with up to 100 % cover had grown rapidly into large individuals. Where settlement was very dense, pressure hummocks had formed

Ecological effects of sea lice medicines in Scottish sea lochs 87 of 286 (elongated individuals). However, the frequent presence of small B. crenatus (2-5 mm diameter) on several panels indicated that there had been a prolonged period of settlement (since April).

Occasional nudibranchs Onchidoris bilamellata and very small Asterias rubens (starfish) had settled on some panels and were actively feeding on the barnacles. The ascidians A. aspersa and C. intestinalis were co-dominants on many panels. Also present were ascidians Corella parallelogramma (occasional) and Ascidiella scabra (rare). Occasional young Pomatoceros triqueter and Hydroides norvegica (tubeworms) occurred on a few clear areas of slate and bristle worms Harmothoe impar were rare. The only seaweed found was young Laminaria saccharina on a few upper panels.

Table 4.6. Biota on sublittoral arrays at Loch Sunart on 6 July 2000 (arrays deployed 14 January 2000). Station/Impact LS1 I LS2 H LS3 I LS4 R Level Um Mm Lm Um M L Um M L U M L Balanus crenatus 1 1 1 1 4 5 2 Obelia longissima 4 5 3 5 4 4 5 Mytilus edulis 4 4 3 4 3 5 6 Onchidoris bilamellata 5 5 Ascidiella aspersa 2 3 3 3 3 2 4 Ciona intestinalis 3 2 3 2 5 4 4 Corella parallelogramma 5 6 5 5 5 5 Pomatoceros triqueter 5 4 5 5 4 Hydroides norvegica 5 5 6 5 5 6 Electra pilosa Tubularia larynx Balanus balanus Harmothoe impar 6 6 Psammechinus miliaris Asterias rubens 6 6 4 5 6 Caprella mutica Diplosoma listerianum Anomia ephippium Lepidochiton asellus Aequipecten opercularis Eupolymnia nebulosa Metridium senile Ascidiella scabra 5 6 Porcellana longicornis Musculus marmoratus Sabella pavonina Capitella capitata Antedon bifida Facelina auriculata Laminaria saccharina 5 Polysiphonia elongata Ceramium rubrum *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: Level: Predicted impact: 1 = S-Superabundant U = Upper, ~2 m below surface H = High 2 = A-Abundant M = Mid, Midwater I= Intermediate 3 = C-Common L = Lower, 2 m above seabed R = Reference 4 = F-Frequent 5 = O-Occasional 6 = R-Rare Missingm: Array LS1 and the upper sets of panels at LS2 and LS3 replaced

The summary of biota and abundance (Table 4.6) shows close similarities between Stations LS2 (predicted high impact) and LS3 (predicted intermediate impact). The reference Station (LS4) had lighter settlements particularly of B. crenatus and C.

Ecological effects of sea lice medicines in Scottish sea lochs 88 of 286 intestinalis, but otherwise a similar suite of species. It should be noted that LS4 was situated in more open water (Figure 4.31).

On 7 November 2000 (Table 4.7) the arrays were intact with the exception of middle and lower sets of panels at LS4. By then, most of the heavy settlements of B. crenatus had been predated, particularly by O. bilamellata and A. rubens. The ascidians C. intestinalis and A. aspersa were still abundant and now large. On many of the replacement panels (only out since 6 July or 8 August) there was abundant cover (up to 75 %) by the encrusting gelatinous ascidian Diplosoma listerianum. The tube worms P. triqueter and H. norvegica were frequent and now large, particularly on the lower sets of panels. Young Psammechinus miliaris (frequent) and queen scallop Aequipecten (Chlamys) opercularis (occasional) were present. There had been a heavy settlement of mussels M. edulis at all of the arrays, followed by rapid predation by large A. rubens. Most surviving M. edulis occurred at the edges of the panels and on the ropes and spars supporting them.

Ecological effects of sea lice medicines in Scottish sea lochs 89 of 286 Table 4.7. Biota on sublittoral arrays at Loch Sunart on 7 November 2000. Arrays LS2, LS3, LS4 deployed 14 January 2000; LS1 deployed (as replacement) 3 August 2000. See data sheets (Table 16.6) for further details of panel losses and replacements. Station/Impact LS1* I LS2 H LS3 I LS4 R Level U M L U M L U M L U Mm Lm 1 Balanus crenatus 1(d) 1(d) 1(d) 3 Obelia longissima 5 4 5 4 3 Mytilus edulis 5 6 4 5 4 4 3 Onchidoris bilamellata 5 4 6 Ascidiella aspersa 5 6 5 3 2 5 2 3 4 Ciona intestinalis 5 4 5 1 2 3 1 5 Corella parallelogramma 6 4 Pomatoceros triqueter 5 5 4 4 5 5 5 Hydroides norvegica 5 5 Electra pilosa 4 5 Tubularia larynx 5 5 5 Balanus balanus 6 Harmothoe impar 5 5 Psammechinus miliaris 5 5 5 4 Asterias rubens 4 3 5 4 Caprella mutica Diplosoma listerianum 2 2 4 4 4 3 Anomia ephippium 6 Lepidochiton asellus Aequipecten opercularis 4 6 5 Eupolymnia nebulosa 6 6 Metridium senile 6 6 6 Ascidiella scabra 6 5 Porcellana longicornis 6 Musculus marmoratus Sabella pavonina Capitella capitata 5 6 Antedon bifida 5 6 Facelina auriculata 5 Laminaria saccharina Polysiphonia elongata Ceramium rubrum 6 *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: 1 = S-Superabundant Level: Predicted impact: 2 = A-Abundant U = Upper, ~2 m below surface H = High 3 = C-Common M = Mid, Midwater I= Intermediate 4 = F-Frequent L = Lower, 2 m above seabed R = Reference 5 = O-Occasional 6 = R-Rare *LS1 deployed (as replacement) 03/08/00 Balanus crenatus1 (1d) = Superabundant (dead), but the strong barnacle shells remained firmly attached to the panels Missingm: middle and lower sets of panels at LS4 replaced

Qualitative and semi-quantitative observations of settled species in July indicate that an abundant settlement of various taxa, particularly barnacles, ascidians and mussel spat, had occurred between early March and early July. Between July and November there had been further settlement by ascidians and mussels, and the mussels and remaining barnacles had been heavily predated by A. rubens. However, the dead shells of the barnacles remained firmly fixed to the slates, materially changing the nature of most of the surface for subsequent organisms to settle on.

The study of the settlement of organisms on the sublittoral slate arrays at the four stations continued through seasons 2001 (7 March, 18 June, 12 December) and 2002 (4 April, 4 July and 6 December). On each visit, any lost or sampled slates were replaced. Typically 12 slates were brought back to the laboratory for closer examination of the biota.

Ecological effects of sea lice medicines in Scottish sea lochs 90 of 286 The sequences and abundances of the dominant organisms through both years were very similar to the year 2000, previously described. Thus, abundant spring settlements of barnacles Balanus crenatus were again followed by summer settlements of abundant ascidians (mainly Ascidiella aspersa and Ciona intestinalis) on and between the dense barnacles. Other species included Obelia longissima, Mytilus edulis and Pomatoceros triqueter.

The main predators Onchidoris bilamellata and Asterias rubens (settling out from the plankton in spring and summer, respectively) predated most of the barnacles. The ascidians however, persisted and became the dominant organisms on the middle and lower sets of slates. Similar to 2000, soft black mud accumulated on the slates beneath the dense populations of Ascidiella in which several species of polychaete worms flourished, including Sabella pavonina (sometimes common).

The summer settlements of Mytilus spat from the plankton were again most successful where prior growth of the hydroid Obelia was present. The spat settles first and preferentially on finely branched organisms before moving down and attaching firmly by byssus threads to more solid surfaces. Again there were close similarities between Station LS2 (predicted high impact) and LS3 (predicted intermediate impact), especially in that the B. crenatus cohorts were regularly abundant and dominant at these stations.

The sublittoral array study in Loch Sunart continued for a final year in 2003, with inspections carried out on 3 July and 12 December 2003 (Table 4.8, Table 4.9). The dominant settled fauna at all stations was similar to that in the preceding years with superabundant B. crenatus (up to 100 %), mostly still alive, but its predator Onchidoris bilamellata (young, frequent) had arrived as expected. The hydroid Obelia was well established and often covered with abundant mussel spat, M. edulis. Ascidiella aspersa (young) was increasingly common. The other faunal species present (occasional or rare) were the same as in previous years at this time. At Station LS1, unusually Laminaria saccharina (sugar kelp) was growing strongly especially on the upper set of panels, accompanied by a number of smaller seaweeds. Barnacles were also abundant on the panels.

Ecological effects of sea lice medicines in Scottish sea lochs 91 of 286 Table 4.8. Abundance of biota on sublittoral arrays at Loch Sunart on 3 July 2003. Arrays LS1, LS2 and LS4 deployed 10 March 2003; LS3 on 19 March 2003. Station/Impact LS1 I LS2 H LS3 I LS4 R Level U M L U M L U M L U M L Balanus crenatus 1 2 2 1 1 1 1 1 1 1 3 3 Obelia longissima 3 4 5 1 3 3 2 4 4 2 4 Mytilus edulis (spat) 2 4 6 1 2 5 1 1 5 1 5 Onchidoris bilamellata 4 3 5 5 5 6 5 6 6 5 6 Ascidiella aspersa 3 3 5 3 5 3 4 4 3 3 Ciona intestinalis 5 6 6 5 5 Corella parallelogramma 6 6 Pomatoceros triqueter 5 6 6 5 5 4 Hydroides norvegica 5 5 5 5 5 Electra pilosa 5 4 5 Tubularia larynx 6 6 Balanus balanus 6 Harmothoe impar 6 6 6 6 Psammechinus miliaris 6 Laminaria saccharina 3 4 5 5 2 + Lacuna vincta 5 Scytosiphon lomentaria 5 Enteromorpha compressa 5 5 Rhodomela subfusca 5 4 Polysiphonia elongata 6 Polysiphonia fibrata 6 5 Ceramium rubrum 6 Cutleria multifida 4 Ectocarpus sp. 5 5 *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: 1 = S-Superabundant Level: Predicted impact: 2 = A-Abundant U = Upper, ~2 m below surface H = High 3 = C-Common M = Mid, Midwater I= Intermediate 4 = F-Frequent L = Lower, 2 m above seabed R = Reference 5 = O-Occasional 6 = R-Rare

On the final inspection on 12 December (Table 4.9), two arrays (LS1 and LS4) were missing, array LS2 (predicted high impact) had one slate missing and LS3 (intermediate impact) had four slates missing. All of the slate panels remaining on LS2 and LS3 were retrieved for study.

In December, the earlier superabundant settlements of B. crenatus, now virtually all dead with only their shells present, were being scavenged mainly by the large starfish, Asterias rubens. Mytilus edulis was common on upper and mid-level panels and was also being predated by A. rubens.

Ascidiella aspersa was abundant on all mid-depth panels on top of barnacles (dead) and mussels but not growing as vigorously as measured at Loch Craignish in December 2001. Ciona intestinalis was again more common than A. aspersa on the lower panels. The tube worm P. triqueter was present mainly on the cleaner panels (deployed since July), up to common on lower panel 2L1, but was also being predated by A. rubens.

Ecological effects of sea lice medicines in Scottish sea lochs 92 of 286 Table 4.9. Loch Sunart: summary of abundances of biota settled on the sublittoral arrays at Stations LS1-4; second (final) inspection 12 December 2003. Station/Impact LS1 I LS2 H LS3 I LS4 R Level Um Mm Lm U M L U M L Um Mm Lm

1 Balanus crenatus 1 (d) 1 (d) 2 (d) 6 1 1 Obelia longissima 6 4 5 5 4 Mytilus edulis 2 4 3 3 6 Onchidoris bilamellata 3 6 5 4 Ascidiella aspersa 6 2 4 6 2 3 Ciona intestinalis 5 5 4 3 2 Corella parallelogramma 6 Pomatoceros triqueter 5 5 5 (d) 5 5 Hydroides norvegica 6 5 6 5 Electra pilosa 5 4 5 Tubularia larynx Balanus balanus Harmothoe impar 6 6 6 Psammechinus miliaris 6 6 6 Asterias rubens 3 4 4 4 4 Caprella mutica2 5 5 4 5 4 Diplosoma listerianum 4 3 Anomia ephippium 5 Lepidopleurus asellus 6 Aequipecten opercularis 5 4 6 5 Eupolymnia nebulosa 4 5 5 4 Metridium senile 5 5 5 5 Ascidiella scabra 6 6 Porcellana longicornis 6 5 5 5 Musculus marmoratus 5 Sabella pavonina 5 (d) Polysiphonia elongata 5 Ceramium rubrum 5 *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: 1 = S-Superabundant Level: Predicted impact: 2 = A-Abundant U = Upper, ~2 m below surface H = High 3 = C-Common M = Mid, Midwater I= Intermediate 4 = F-Frequent L = Lower, 2 m above seabed R = Reference 5 = O-Occasional 6 = R-Rare Missing: arrays LS1 and LS4. Numbers in bold indicate presence only on panels deployed as replacements on 6 July 2003. Balanus crenatus1 1 (d) = Superabundant (dead), but the strong barnacle shells remained, firmly attached to the panels. Caprella mutica2: these are the only records of this alien species found on panels during this study.

In 2003 the number of invertebrate taxa recorded on the panels increased from 16 in July to 25 in December. Apart from barnacles, the only other crustaceans recorded were small motile crabs Porcellana longicornis (occasional), and the alien caprellid amphipod Caprella mutica. This species was present (occasional to frequent) at both LS2 and LS3 and is of special interest, being a relatively recent introduction to British waters (Willis et al., 2004). Caprella mutica was not observed anywhere else during this project.

The results from 2003 again show close similarities between the biota measured at Stations LS2 (predicted high impact) and LS3 (predicted intermediate impact).

4.13 Littoral site assessment - shore fauna and flora Three sampling stations (A, B and C) were established on the loch shore on the basis of initial model predictions of cypermethrin dispersion (not the model runs discussed in Section 4.6). Station B was approximately 200 m W of the slip and 500m SE of the cage group, A was approximately 1000 m E of the cages and reference station C approximately 1000 m WSW of the cage group (Figure 4.41).

Ecological effects of sea lice medicines in Scottish sea lochs 93 of 286 Figure 4.41. Location of littoral sample stations at Loch Sunart (). Grey rectangles represent cage groups.

At Station B (high impact, sheltered) the midlittoral zone consisted of medium sized boulders, densely covered with the large brown fucoids Ascophyllum nodosum and Fucus vesiculosus. One very large prominent boulder had mixed populations of large mussels, Mytilus edulis, and frequent barnacles, Semibalanus balanoides, and afforded the only suitable substratum on this extensive beach on which to fix the four slate panels.

At Station A (intermediate impact, sheltered) most of the midlittoral zone was fucoid dominated, but an area of bedrock of moderate slope dominated by large Mytilus also had frequent barnacles, limpets (Patella vulgata) and snails (Nucella lapillus and Littorina spp.). The four slates were fixed to the bedrock.

Farther west at reference Station C the dominant midlittoral species were similar to Stations A and B but the aspect (facing NW) was more open and exposed, with good patches of mussels and barnacles on bedrock on which to fix the slates.

Slates were first installed at the shore sites in September-October 1999, and were examined during late winter/early spring, early to mid summer, and in late summer/early autumn each year. There was very little settlement of biota on the slates in the first two years.

In early 2000, greenish-brown microalgal films developed on the slates, especially at Station B. Microscopic examination showed these to consist of mixed blue-green Cyanobacteria and littoral diatoms. During the year the algal films were browsed on by small and medium sized Littorina littorea (occasional), and small patches of brown algae Ralfsia verrucosa appeared. At Station C only, there was a very sparse settlement of barnacle cyprids, which only survived at or near the rough edges of the slates.

In 2001, there was a late, but small, settlement of Semibalanus balanoides on the slates at Station C and on nearby rock surfaces at all three locations. By 1 March 2002, there were

Ecological effects of sea lice medicines in Scottish sea lochs 94 of 286 75 S. balanoides of up to 7 mm shell diameter settled on Slate C1, 71 on Slate C3, and 108 on Slate C4. As the area of each slate surface was 0.08 m-2 the settlement density approximated to 1060 individuals m-2. On the previously cleared rock surface below Slate C3, the settled barnacle density was equivalent to 30000 m-2. Thus, barnacle settlement at Station C was approximately eight times greater on the wetter, more pitted rock surface than on the slates. Slate C2 was missing and was replaced by Slate C5.

At Station A, only five S. balanoides were present on the slates. Dog whelks (Nucella lapillus) and snails (Littorina littorea) were common on adjacent rock. Occasional limpets (Patella vulgata) were present. Barnacle densities on previously cleared adjacent rock surfaces averaged 6800 m-2.

At Station B, there was no settlement of S. balanoides on Slates B1-B4, which were situated slightly above the MLWN on the western side of the large boulder. Two additional slates (B5 and B6) were fixed on the southern (shoreward) side and were sited approximately 1m higher than the original slates. The densities of barnacles on previously cleared areas of the face of the boulder, at a similar level to Slates B5 and B6, were also assessed and averaged approximately 25800 m-2.

Before leaving the Loch Sunart shore stations on 1 March 2002, all barnacles were cleared from the same areas of rock surfaces, which had first been cleared in 2001, so that any new settlement could be monitored. In March there were few biota present on the panels at Stations A (predicted intermediate impact) and B (predicted high impact).

The shore plates at all three locations were re-examined on 27 May and 10 September 2002. When the sites were revisited on 27 May 2002, a light new settlement had occurred on all the cleared areas of rock and barnacle densities of approximately 3400 m-2 at Station A, 2010 m-2 at Station B and 9500 m-2 at Station C were noted. The sites were revisited on 10 September 2002, by which time barnacle densities (on the previously cleared rock faces) had declined to approximately 1970 m-2 at Station A, 1170 m-2 at Station B and 7800 m-2 at Station C. Between May and September 2002, barnacle populations settled on the rock surfaces had therefore declined by approximately 42 % at Stations A and B and by 18 % at Station C. Predation by Nucella lapillus (common at A and B; frequent at C) was probably the main cause.

By 10 September 2002, the slates at the Reference Station C had received storm damage. Only small parts of Slates C1 and C5 remained, and these were replaced by Slates C6 and C7. Slate C3 was split, but otherwise intact, and the top half of Slate C4 remained in place. The attachments securing these slates to the rock were replaced.

Low barnacle densities on slates at Stations A and B meant that populations could not be compared. On the next visit to Loch Sunart (4 March 2003), Slate C3 and the remains of Slate C4 were retrieved and returned to the laboratory for analyses. At each location, densities of S. balanoides on the rock surface cleared previously were estimated, the barnacles were measured on site and examined for development of egg masses.

On completion of the examination and sampling of S. balanoides on site, all of the areas of rock surface cleared in 2002 were again cleared so that the settlement and development of any populations could be followed at all three stations until early 2004.

The stations were visited again on 15 May and 28 September 2003, and on 22 February 2004. By 15 May, there was a slightly heavier settlement of S. balanoides on the slates at Station C compared with earlier years. There were very few barnacles settled on slates at Station A (a total of 18 individuals on four slates). There were 57 individuals on Slate B6

Ecological effects of sea lice medicines in Scottish sea lochs 95 of 286 and occasional spat on Slate B5. Slate B1 had received a somewhat heavier settlement approximating to 35000 m-2, but some of the cyprids appeared to be dead. Lighter settlements totalling 105 and 109 individuals/slate were found on Slates B2 and B3, and Slate B4 was reasonably well settled, but again many of the barnacles appeared to be dead. By 28 September, there was only one individual on Slate B6, and none remaining on any of the other panels at Station B, while there were 17 individuals on the slates at Station A. Thus, the lack of surviving barnacles on slates at Stations A and B meant that again populations at each station could not be compared, and detailed analyses of populations in March 2004 once again utilised comparisons of populations on slates at Station C, and on the adjacent rock surfaces, which had been cleared previously at all three stations.

4.13.1 Barnacle (Semibalanus balanoides) populations and development The results of detailed laboratory analyses of barnacle populations on slates removed in February 2003 and February 2004 are summarised in Table 16.1 and Figure 4.42, and compared with populations on adjacent rock surfaces cleared previously.

Ecological effects of sea lice medicines in Scottish sea lochs 96 of 286 Figure 4.42. Growth and development of barnacles, Semibalanus balanoides on shore panels at Loch Sunart (2002-2003).

Ecological effects of sea lice medicines in Scottish sea lochs 97 of 286 Table 4.10. Barnacle populations on settlement panels and adjacent rock surfaces cleared the previous year at Loch Sunart (2002 - 2003). Cohort 2002 Cohort 2003 Station Predicted S. balanoides (m-2) S. balanoides (m-2) Impact On slates On rock On slates On rock

B High 0 880 0 6800 A Intermediate 22 580 390 10300 C Reference 1200 6000 18400 33100

NB: Densities > 1000 were rounded to nearest 100

In 2003, higher population densities on the rock surfaces indicated preferential settlement on the more pitted, wetter rock, than on the slates (Table 4.10). The counts made in 2004 indicated denser settlement on the slates at Stations A and C, than in the previous year, and population densities on the rock surfaces were again higher than on the slates.

During visits to Loch Sunart in May and September 2003 and February 2004, growth of barnacle populations on slates and the adjacent bedrock faces, which were cleared in March 2003, was assessed. The most extensive data set was obtained for Station C (Table 4.11).

Table 4.11. Seasonal variations in Semibalanus balanoides populations on settlement panels and previously cleared adjacent rock surfaces at Loch Sunart reference Station C (2003). Populations on slates Populations on No. of 95% Confidence slates (n) Mean Limits adjacent rocks 15 May 03 Total m-2 4 216000.0 20400.0 71000.0 % Live 4 100.0 0.0 100.0 % Cover 4 9.0 15.0 Not noted Max. diameter (mm) 4 1.5 0.0 1.5 28 Sep 03 Total m-2 4 17600.0 16200.0 44500.0 % Live 4 100.0 1.0 100.0 % Cover 4 34.0 32.0 Not noted Max. diameter (mm) 4 7.5 0.8 7.0 22 Feb 04 Total m-2 4 18400.0 16100.0 33100.0 % Live 4 95.0 8.0 91.0 % Cover 4 29.0 29.0 Not noted Max. diameter (mm) 4 8.6 0.7 8.0 NB: Densities > 1000 were rounded to nearest 100

There were large numbers of recently settled individuals in May 2003, although settlement at Station B was light (predicted high impact) and some of the newly settled cyprids were thought to be dead (Table 4.11). Maximum shell diameters were ca. 1.5 mm at all sites. By 28 September 2003, there were no barnacles on slates at Station B, and only 22 individuals remaining on slates at Station A. At the Reference Station C, population densities had decreased by approximately 20 % on slates and 40 % on adjacent rock surfaces, but very few were dead (c.f. Loch Kishorn - Section 6.9.1) and maximum shell diameters were approximately 7 to 8mm. In February 2004, mortality was slightly

Ecological effects of sea lice medicines in Scottish sea lochs 98 of 286 higher (9 %) on the bedrock than the slates (5 %), but overall growth and mortality of the populations was very similar on both surfaces at this location.

It was obvious from the 2004 settlement data, that light settlement of barnacles occurred at Station B, but none survived to the end of September. There were consistently lighter settlements at Station A, which was situated nearest to the head of the loch. The more sheltered locations of Stations A and B on shores that are fucoid dominated may explain the lower settlement densities at these stations, which were nearer to the head of the upper basin of Loch Sunart than the more exposed Reference Station C (see Jenkins & Hawkins, 2003 for a recent investigation of ‘Barnacle larval supply to sheltered rocky shores’). Other factors, e.g. different densities of predators, may also be important. However, the high mortality of settled spat at Station B (predicted high impact) cannot be explained, and some effects of fish farm activities therefore cannot be completely ruled out at this location.

Table 4.12. Barnacles without fertilised egg masses (%) at Loch Sunart (2002 - 2003). Station B A C Impact High Intermediate Reference Substrate Rocks Rocks Rocks Rocks Slates Slates Rocks Rocks Rocks % no Individuals % no Individuals Mean% Slates 95% % no Individuals eggs (n) eggs (n) no eggs (n) C.Ls eggs (n)

2002 24 25 10 19 6 2 18 8 40 2003 13 40 13 40 26 4 22 8 40

In the 2002 barnacle cohort, 24 % of barnacles at Station B (predicted high impact) did not contain egg lamellae (Table 4.12). At the predicted intermediate impact site (Station A), 10 % had no egg lamellae, and at Reference Site C, 8 % of barnacles from the rock surfaces, and 6 % of those attached to Slates C3 and the remaining part of Slate C4 were without egg lamellae. Thus in 2002-2003 there was a slight trend towards the lower frequency of egg lamellae present in S. balanoides from the location predicted to receive the highest impact from fish farming activities. However, by February 2004, the lowest frequency of occurrence of egg masses was found in the 2003 cohort barnacles on slates at reference Station C, where only 50 % of individuals on one slate (C6) and 75-82 % of the individuals on the other three slates contained egg masses. At this time, 13 % of individuals examined on the rock surfaces at both Stations A and B did not contain egg- masses. Thus from March 2003 to February 2004, the frequency of occurrence of egg- masses in barnacles on the slates had increased at Station A (predicted intermediate impact), and decreased at Reference Station C. It was also noted that at Station C in both years, egg-masses were absent in only 8 % of individuals settled on the rock faces.

On 4 March 2003, mussels (Mytilus edulis) with attached S. balanoides were also collected from each site and 20 S. balanoides settled on the mussel shells from each site were removed and analysed. On each set of mussels, the settled barnacles belonged to several year groups, so could not be reliably distinguished, and the mean shell diameters, valve weights and weights of egg masses were all higher than barnacles from the slates at Station C. All of the animals settled on the mussels contained egg lamellae, possibly indicating that differences in the occurrence of egg lamellae in barnacles from different sites may only be seen up to 1 or 2 years after settlement.

Because of the high variability in the frequency of occurrence of egg masses, within and among sites, and between populations settled on slates and rocks at Station C in 2004, there were no consistent trends to suggest sea lice treatments at Sunart fish farm were impacting barnacle egg development.

Ecological effects of sea lice medicines in Scottish sea lochs 99 of 286 5 Loch Diabaig 5.1 Site description Loch Diabaig, in Wester Ross, lies on the northern shore of outer Loch Torridon, roughly level in latitude with the northern tip of the Applecross peninsula (Figure 5.1). In their catalogue of Scottish sea lochs, Edwards and Sharples (1986) considered Loch Diabaig as part of Loch Torridon, and did not present separate data for it. Loch Diabaig is located inside Loch Torridon sill 2 (Torridon sill 1 depth 64 m; Torridon sill 2 depth 79 m). The length from the head of Loch Diabaig to the middle of the sill is 1800 m, and the loch width of 712 m at the widest point gives an aspect ratio of 2.52. The sill depth is ca 12 m, and the maximum depth recorded in the present work was 51 m.

The flushing time for the Torridon system is given by Edwards and Sharples (1986) as nine days; for Loch Diabaig this should be less than one day, given the water depth over the two sills seaward of it. The watershed of the Torridon system is 242 km2, which will contribute to the individual embayments such as Loch Diabaig. The burn arising from Loch Mhullaich would appear to be the only significant source of freshwater entering Loch Diabaig; the burn enters the loch by the fish farm jetty. The tidal range given by Edwards and Sharples (1986) is 4.9 m, and the fresh/tidal ratio is very low (1:400). Bottom water renewal would thus appear to be frequent.

The farm site is located in the southeastern part of the loch, making use of the limited shelter afforded by the Rubha na h-Airde. The fetch is in excess of 60 km, making this site very exposed during northwesterlies.

No published or grey literature has been found on the physical environment of Loch Diabaig, and the bathymetric survey performed during the present project appears to be the first since initial Admiralty soundings.

Ecological effects of sea lice medicines in Scottish sea lochs 100 of 286 A) B)

Figure 5.1. A) Location of Loch Diabaig on the Scottish west coast and B) Loch Diabaig and location of study area (inset box). Grid is OS.

Ecological effects of sea lice medicines in Scottish sea lochs 101 of 286 5.2 Fish farm history, cage positioning, biomass, and medicine use Diabaig production figures have been supplied by Marine Harvest from 1996. The peak biomass for the first growing cycle was 1024 t; this figure had declined to 787 t in the next cycle (1999), rising again in 2001 to 1141 t, and decreasing again to 626 t as of August 2003 (pers. comm. D. Runcieman, MH) (Table 5.2).

The cage positions did not changed a great deal during the project; this is not surprising given the restricted area available in the loch. Cage positions, sampling stations and bathymetry for Loch Diabaig are shown in Figure 5.2.

Table 5.1. Sea lice medicine use at Loch Diabaig fish farm. Date Treatment Amount Active Ingredient active ingredient 27-29/1/99 Salartect 24100 l hydrogen peroxide 17-19/3/99 Excis 6.2 l cypermethrin 29-30/4/99 Excis 7.1 l cypermethrin 30/4/99 Salmosan 200 g azamethiphos 30/4/99 Aquagard 1.5 l dichlorvos 4-11/6/99 Excis 8.3 l cypermethrin 14-16/7/99 Excis 8.7 l cypermethrin 16-18/8/99 Excis 7.5 l cypermethrin 27-29/9/99 Excis 10 l cypermethrin 21-27/10/99 Excis 10 l cypermethrin 6-9/12/99 Excis 9.5 l cypermethrin 26-27/1/00 Excis 4.5 l cypermethrin 29-31/8/00 Excis 8.3 l cypermethrin 30/10-2/11/00 Excis 10.9 l cypermethrin 1-9/12/00 Salmosan 2600 g azamethiphos 8-12/1/01 Excis 8.5 l cypermethrin 29/1-2/2/01 Excis 8.5 l cypermethrin 26/2-2/301 Excis 8.5 l cypermethrin 8-11/5/01 Excis 12.5 l cypermethrin 7-13/6/01 Slice 335 g emamectin benzoate 15-18/10/01 Excis 6 l cypermethrin 3-8/12/01 Excis 13.2 l cypermethrin 8-12/1/02 Excis 10.8 l cypermethrin 14-16/1/03 Excis 3.5 l cypermethrin 14-21/3/03 Slice 127 g emamectin benzoate 26-29/8/03 Excis 7.6 l cypermethrin 18-22/1/04 Excis 8.4 l cypermethrin 17-23/2/04 Slice 266 g emamectin benzoate

Ecological effects of sea lice medicines in Scottish sea lochs 102 of 286 A)

B)

Figure 5.2. A) Cage group position (grey rectangle) at Loch Diabaig with sampling stations. Littoral stations are shown as , combined macro- and meiofaunal stations as ; and B) bathymetry for Loch Diabaig from SAMS RoxAnn survey.

Ecological effects of sea lice medicines in Scottish sea lochs 103 of 286 Table 5.2. Timeline of sampling and management events at Loch Diabaig. Figs. in kg are feed input; Aza = Salmosan, Ema = Slice, Cyper = Excis, H2O2 = Salartect, dichlorvos = Aquagard Key: S: Sampling D: Deployed R: Retrieved Scientific events Day - Event 28 - Prelim shore survey

1999 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2 O 2 events 30 - Aza 97325 kg 70625 kg 77475 kg 70350 kg 6 - Cyper 4 - Cyper 27 - H 117825 kg 128275 kg 108625 kg 140550 kg 162450 kg 135200 kg 117625 kg 102425 kg 29 - Cyper 17 - Cyper 14 - Cyper 16 - Cyper 27 - Cyper 21 - Cyper Management 30 - Dichlorvos

Phytoplankton Scientific events Day - Event 8 -Shore slates (D) 20 -Sublittoral arrays (D) 22 - Shore slates (D) 2- Shore slates (S) 17 - Shore slates (S) 3 - Sublittoral arrays (S) 11 - Macro/meiofauna (S) 16 - Macro/meiofauna (S) 14 - Sublittoral arrays (S)

2000 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 300 kg Fish in events 1 - Aza Fish out 2400 kg 62250 kg 13575 kg 19788 kg 28450 kg 49713 kg 79675 kg 24750 kg 90700 kg 83200 kg 107100 kg 26 - Cyper 29 - Cyper 30 - Cyper Management (’98 year class) (’00 year class)

Ecological effects of sea lice medicines in Scottish sea lochs 104 of 286 Table 5.2 (cont.) Key: S: Sampling D: Deployed R: Retrieved Phytoplankton Shore slates (S) Shore slates (S) Shore slates (S) Shore slates (S) Macro/meiofaunal (S) Scientific events Day - Event 11 - 20 - 26 - Check for lodt sublittoral array 7 - 1 - 17 -

2001 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 7 - Ema 74550 kg 92900 kg 84950 kg 8 - Cyper 8 - Cyper 3 - Cyper 113850 kg 136025 kg 119675 kg 120950 kg 104325 kg 123450 kg 167451 kg 238050 kg 237275 kg 29 - Cyper 26 - Cyper 15 - Cyper Management

Phytoplankton Shore slates (S) 29 - Scientific events Day - Event

2002 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec class) class) 14338 Fish in 225 kg events Fish out 2273 kg 8168 kg (’00 year (’02 year 18793 kg 37800 kg 70975 kg 27657 kg 8 - Cyper Management

Ecological effects of sea lice medicines in Scottish sea lochs 105 of 286 Table 5.2 (cont.) Key: S: Sampling D: Deployed R: Retrieved Shore slates (S) Shore slates (S) 14 - Scientific events 16 - Day - Event

2003 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 14 - Ema 49124 kg 65160 kg 21235 kg 34750 kg 59663 kg 94062 kg 84876 kg 105373 kg 116888 kg 107870 kg 114438 kg 127738 kg 14 - Cyper 26 - Cyper Management Scientific events Day - Event 20 - Shore slates (R)

2004 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 17 - Ema 75300 kg 94637 kg 78980 kg 58525 kg 47275 kg 18 - Cyper Management

Ecological effects of sea lice medicines in Scottish sea lochs 106 of 286 5.3 Hydrography Current meter measurements were not made at Loch Diabaig during this project for several reasons. A data set measured by Marine Harvest already existed for regulatory purposes, although this was only four days in length. Towards the mid-point of the project as emphasis was shifting away from further research at Loch Diabaig, resources were put into undertaking DGPS drifter surveys rather than current meter surveys. In addition, long-term deployments of current meters at Loch Diabaig were seen as medium to high risk due to the site’s exposure, especially in light of the loss of sublittoral settlement arrays.

The existing data for Loch Diabaig show relatively low mean and maximum speeds. 2 -1 Drifter surveys resulted in dispersion coefficients of 0.10 and 0.31 m s for kx and ky respectively. Interestingly, a clockwise circulation was shown for the loch, which would have been missed using current meters deployed in a single position (Figure 5.3).

Figure 5.3. DGPS drifter release at Loch Diabaig showing the position of the cage group and the clockwise circulation in the loch.

5.4 Zooplankton Due to the distance of Loch Diabaig from the laboratory, it was decided after consultation with the sponsors that for logistical reasons an intensive zooplankton sampling programme would not be undertaken.

5.5 Phytoplankton results Phytoplankton community composition was monitored between 28 July 2000 and 8 October 2001. Samples for phytoplankton, salinity and nutrients were collected from the top 10 m of the water column from the fish farm cages. Salinity and dissolved nutrient concentrations are presented in Table 14.7 and phytoplankton community composition is summarised in Table 14.8.

The phytoplankton standing stock during summer 2001 was 2 to 3 times higher than during summer 2000. Grazing by zooplankton is likely to be the most significant factor controlling the phytoplankton population size in Loch Diabaig as nutrient and salinity

Ecological effects of sea lice medicines in Scottish sea lochs 107 of 286 regimes were almost identical between the two seasons. Salinity in particular, varied little (33.81 ± 0.51) throughout the 15-month sampling programme.

The gross contributions of the four main types of phytoplankton (diatoms, dinoflagellates, microflagellates and others) to the total community are shown in Figure 5.4. Numerically, the biomass was dominated by microflagellates (< 15 mm) and cryptophytes. All phytoplankton observed at Diabaig (full species list given in Appendix III - Phytoplankton) have been noted previously in Scottish coastal waters (e.g. SAHFOS, 2001; McKinney et al. 1997; Dodge, 1995; etc.). Populations were generally dominated by one or two ubiquitous marine species (e.g. diatoms Rhizosolenia spp. and Chaetoceros spp., and dinoflagellates Heterocapsa triquetra and Ceratium lineatum), with a variety of other species present in low numbers (several thousand cells per litre).

Figure 5.4. Gross phytoplankton community composition at Loch Diabaig between July 2000 and October 2001. “Others” includes other flagellates, silicoflagellates, cryptophytes, ciliates, tintinnids and resting stages/cysts.

There were no phytoplankton blooms between July and December 2000. The small increase in autumn diatom abundance in September 2000 was due to increased growth of at least 18 different species. Loch Diabaig did not exhibit a classical spring bloom in 2001, although there was a small bloom (4.12 x 103 cells l-1) of the chain-forming diatom Skeletonema costatum in March 2001. Concentrations of dissolved inorganic nitrate (ToxN) declined rapidly during May 2001, and phytoplankton increased linearly in cell abundance and cell size from April to August (Figure 5.5). In August/September 2001 there was a bloom of the diatom Leptocylindrus minimus (9 x 105 cells l-1). The timing of the eight sea lice treatments (Table 5.1) undertaken during the phytoplankton sampling programme at Loch Diabaig is shown in Figure 5.5. There were no obvious changes in phytoplankton abundance that could be related to any of the sea lice treatments.

Ecological effects of sea lice medicines in Scottish sea lochs 108 of 286 Figure 5.5. Total phytoplankton cell abundance (cells l-1) and dissolved inorganic ToxN (nitrate and nitrite, µM) at Loch Diabaig between July 2000 and October 2001. Sea lice treatments are depicted by dashed vertical lines: C = Excis, A = Salmosan and E = Slice.

Nine consecutive days in early September 2001 were isolated from Figure 5.5 to demonstrate the high spatio-temporal variability (patchiness) of phytoplankton at Loch Diabaig (Figure 5.6). All samples were collected at high tide, the weather was uniformly overcast and rainy/dull and the winds were prevailing westerlies during this time. A degree of water column mixing is indicated by decreased phytoplankton numbers and increased nutrient concentrations towards the end of the nine-day period. However, the 10+ fold variability in cell abundance was driven primarily by changes in diatoms, and in particular by a bloom of L. minimus and fluctuations in cell abundances of Pseudo- nitzschia sp., Lennoxia faveolata, Chaetoceros spp. and five unidentified pennate diatoms.

The most likely impact of sea lice treatments on phytoplankton would be indirectly, via top-down control by zooplankton grazers. The phytoplankton community at Loch Diabaig was naturally very dynamic and observed fluctuations were due to natural processes such as seasonality, patchiness and advection, rather than sea lice treatments.

Ecological effects of sea lice medicines in Scottish sea lochs 109 of 286 Figure 5.6. Close-up analysis of nine consecutive days in August/September 2001 showing high variability of phytoplankton cell abundance and ToxN concentrations at Loch Diabaig.

5.6 Sublittoral studies Sublittoral sampling at Loch Diabaig was initially intended to follow the model established for Loch Sunart, ie. a pre-treatment survey (completed in August 2000) and annual surveys thereafter. One subsequent survey was completed in August 2001, after which the sublittoral sampling at Loch Diabaig was terminated following consultation with the sponsors. Adverse weather conditions prevented the continued deployment of permanent station markers and their associated sublittoral settlement arrays.

5.6.1 Meiofauna Meiofaunal analyses were conducted on samples collected from Loch Diabaig in July/August 2000 and August 2001. Five samples were collected from each of three stations in each survey (Figure 5.7). The amount of sediment collected was similar (approximately 160 cm3) on both sampling occasions. For each sample, all copepods and a 10 % sub-sample of the nematodes were enumerated and identified.

Ecological effects of sea lice medicines in Scottish sea lochs 110 of 286 Figure 5.7. Combined meio- and macrofaunal sample stations () in Loch Diabaig. The cage group is shown as a grey rectangle.

5.6.1.1 Nematodes There was little similarity in nematode community structure between stations on either survey. In July/August 2000, Station 2 had lower species diversity and a more dominated assemblage, than Stations 1 or 3. In August 2001, Station 1, closest to the cages, had the highest dominance and lowest diversity.

Nematode abundance data from both surveys were combined, and aggregated to genus to reduce the influence of seasonal factors and identification biases. MDS of the data shows clear differences between surveys (Figure 5.8). Shifts in nematode community structure between surveys at Stations 2 and 3 were comparable in magnitude, while the shift at Station 1 was greater. This pattern was also reflected in the diversity/dominance structure of the nematode assemblages, which show that community composition at Station 1 in 2001 was very different to Stations 1 and 2 in 2000, and that while Station 2 shows an

Ecological effects of sea lice medicines in Scottish sea lochs 111 of 286 increase in diversity and a decrease in dominance between surveys, both Stations 1 and 3 show an opposite trend (Figure 5.9).

Figure 5.8. MDS ordinations based on Bray-Curtis similarities between samples calculated from square-root transformed nematode genus (upper panel) and copepod species (lower panel) abundances. Symbols indicate the survey (, July/August 2000; , August 2001) and numerals (1-3) denote stations

5.6.1.2 Meiobenthic copepods There were clear differences in copepod community structure between stations within each of the two surveys. In both cases, Station 2 had low numbers of species and the copepod assemblage was highly dominated by a single species, Typhlamphiasus confusus. Data from the two surveys were combined, and the MDS shows that changes in copepod community structure between surveys at Station 2 were minor, while relatively large changes occurred at Station 3, and the greatest change (least overlap between points) occurred at Station 1 (Figure 5.8). K-dominance curves show that diversity tended to decrease, and dominance to increase, at Stations 1 and 3 (Figure 5.9). The average number of individuals per sample decreased from 37.6 to 7.6 at Station 1, 26.2 to 11.2 at Station 2, and 31.2 to 24.8 at Station 3. The average number of species per sample remained approximately the same at Stations 2 and 3, but decreased by approximately 50 % at Station 1.

Ecological effects of sea lice medicines in Scottish sea lochs 112 of 286 Figure 5.9. K-dominance curves (cumulative dominance % against species rank) plotted from average abundances of nematode genera (upper panel) and copepod species (lower panel) from each survey/site combination in the 2000 and 2001 surveys from Loch Diabaig. In the key the first numeral denotes the survey (1, 2000; 2, 2001) and the second (1-3) denotes the station.

5.6.1.3 Relationships with environmental variables Physical/environmental variables (particle size analysis, Table 17.5; total organic carbon and nitrogen, Table 17.2; and depth) were combined with meiofaunal community composition data to identify factors contributing to the patterns observed. As a proxy for the effects of the farm an additional variable, distance from the cage group, was added to the analysis, along with a variable denoting the year. As environmental variables were not replicated within each survey/site combination, meiofaunal abundances were averaged within stations, prior to being square-root transformed. Environmental variables were normalised and the Bio-env procedure was used to isolate subsets of environmental variables which provided the best match (defined here as the highest Spearman rank correlation between a biotic resemblance matrix and a euclidean distance matrix derived from the subset of normalised variables) with Bray-Curtis similarity matrices derived from the transformed nematode and copepod abundances.

Ecological effects of sea lice medicines in Scottish sea lochs 113 of 286 For nematodes the closest match (ρ = 0.69) was with a combination of 4, 4.5 and 5 ϕ sediment fractions, distance and depth. For copepods the closest match (ρ = 0.71) was with a combination of 0.5, 1 and 4 ϕ sediment fractions, and % total organic nitrogen. For both biotic components a number of other subsets provided matches that were almost as good (ρ>0.67). Many of the environmental variables were highly correlated (e.g. and % TOC, ρ = 0.96, 4 ϕ sediment fraction and % TON ρ = 0.97) and the one used in the analysis is in part determined by chance. Much of the observed pattern in meiofaunal community structure could be explained by variation in sediment type (proportions of coarse silts for nematodes, medium sands and coarse silt for copepods), and to a lesser extent by other factors (distance from the cages, depth, % total organic carbon or nitrogen) which may or may not reflect organic enrichment effects from the cages.

The K-dominance curves showed an extremely impoverished copepod assemblage at Station 2 (Figure 5.9). Hydrographic studies indicated that clockwise rotation of water in the Loch may concentrate material settling from the cages, and associated contaminants, in the vicinity of this station. Whilst abundances at all stations, and particularly those nearest the cages (1 and 2), declined between the two sampling periods, it is likely that the differences observed between the two surveys were within the natural range of variation caused by changing environmental/seasonal conditions. Thus, there was little evidence that sea lice treatments were a major driver of patterns in meiofaunal community structure at Loch Diabaig.

5.6.2 Macrobenthos Sampling was undertaken on 1 August 2001, very soon after a Slice treatment. A comparative assessment of the macrobenthic populations sampled was undertaken and a summary of the main features of this assessment follows with details provided in (Table 5.3 and Table 5.5).

5.6.2.1 Distribution, abundance and faunal structure The first survey (July 2000) data reveal the presence of a steep carbon enrichment gradient across the three sampling stations. Table 5.3 lists the five most abundant taxa found at each of the sampling stations in each survey. The populations at Station 1, closest to the cages (100 m SE) are dominated by large nematodes and small polychaete worms, e.g. Capitella capitata, Mediomastus fragilis, Prionospio fallax and Scalibregma inflatum, all of which are frequently associated with areas of high carbon enrichment. At Station 2, situated 300 m NW from the cages, the populations are dominated by small bivalve molluscs, brittle stars and larger polychaetes typical of normal soft sediments in sheltered sea lochs. The furthermost station, Station 3, is dominated by high populations of the gastropod mollusc Turritella communis, with a mixture of polychaetes, bivalve molluscs and amphipod crustaceans typical of sea loch associations on shell/muddy sand grounds.

Ecological effects of sea lice medicines in Scottish sea lochs 114 of 286 Table 5.3. Loch Diabaig July 2000 and August 2001. The five most abundant taxa at each station on each sampling occasion (No.= Total No. of organisms from nominally 0.5 m2 sediment surface area). July 2000 Station 1 No. Station 2 No. Station 3 No. Nematoda 2818 Mysella bidentata 182 Turritella communis 777 Capitella capitata 592 Amphiura chiajei 97 Nemertea T1 194 Prionospio fallax 331 Amphiura filiformis 80 Prionospio fallax 111 Mediomastus fragilis 134 Nephtys kersivalensis 57 Diplocirrus glaucus 88 Scalibregma inflatum 119 Spiophanes kroyeri 35 Pholoe inornata 77 August 2001 Station 1 No. Station 2 No. Station 3 No. Thyasira flexuosa 636 Scalibregma inflatum 346 Turritella communis 300 Abra alba 285 Mysella bidentata 85 Nephtys kersivalensis 41 Mysella bidentata 232 Amphiura filiformis 29 Melita bergensis 30 Mediomastus fragilis 121 Nephtys incisa 27 Mysella bidentata 19 Nematoda sp. 107 Cylinchna cylindracea 26 Amphiura chiajei 18

The three most dominant taxa at Station 1 in the second (August 2001) survey were small bivalve molluscs (Thyasira flexuosa, Abra alba, Mysella bidentata). These are frequently found in areas of moderately high carbon enrichment suggesting an improvement in conditions close to the cages although the presence of Mediomastus fragilis and nematodes amongst the dominants indicates the presence of some carbon related disturbance. The population structure at Stations 2 and 3 in 2001 remained diverse, with polychaetes, molluscs, crustacea and echinoderms all present among the dominant taxa.

Table 5.4 and Table 5.5 compare the major population statistics recorded from each of the surveys. It can be seen that at Station 1 the number of annelid taxa were the same in both surveys, but their abundance and biomass declined markedly in the second (August 2001) survey. The number of crustacean taxa and their abundance were halved in the second (August 2001) survey, but the numbers, biomass and abundance of molluscs and echinoderms increased significantly. At Station 2 significant increases in the number of taxa found in the second (August 2001) survey were recorded for all groups and large increase in the abundance and biomass of annelids and crustaceans were also recorded. The abundance of molluscs and echinoderms declined however, although the biomass of molluscs increased, implying an increase in the numbers of larger species. At Station 3 in the second (August 2001) survey significantly fewer taxa were found in all groups and both abundances and biomass were also significantly lower.

Ecological effects of sea lice medicines in Scottish sea lochs 115 of 286 Table 5.4. Loch Diabaig, July 2000 and August 2001. Population statistics for major faunal groups at each station on each of the two sampling occasions. (Total No. of Species, Abundance and Biomass nominally 0.5 m2 of sediment surface area). Number of taxa (S) Date Group Station 1 Station 2 Station 3 07/2000 Annelids 37 26 74 08/2001 38 36 26 07/2000 Crustacea 12 1 12 08/2001 6 6 5 07/2000 Molluscs 0 8 18 08/2001 14 19 10 07/2000 Echinoderms 0 4 7 08/2001 3 8 3 Abundance (A) Date Group Station 1 Station 2 Station 3 07/2000 Annelids 1403 225 803 08/2001 349 506 135 07/2000 Crustacea 16 1 50 08/2001 7 12 41 07/2000 Molluscs 62 298 1086 08/2001 1274 187 351 07/2000 Echinoderms 2 203 175 08/2001 5 57 27 Biomass (B (g)) Date Group Station 1 Station 2 Station 3 07/2000 Annelids 8.60 9.50 13.30 08/2001 1.10 13.69 11.50 07/2000 Crustacea 0.01 <0.01 0.50 08/2001 0.02 0.06 0.15 07/2000 Molluscs 4.10 3.70 248.10 08/2001 21.10 11.0 47.40 07/2000 Echinoderms <0.01 65.10 10.50 08/2001 0.01 8.30 6.70

Interpretation of these changes in the second (August 2001) population survey is difficult since the effects of carbon enrichment in the area are probably masking any additional disturbance to the populations caused by the release of other contaminants. However the decrease in the crustacean populations at Station 1 may be of significance as this group is expected to be most sensitive to the release of treatment agents. It is noted that the site was treated with emamectin in July 2001, less than 1 month before the second survey. The general increase in populations at Station 2 may be linked to a decrease in carbon enrichment close to the cages; the position of Station 2 at 300 m NW of the cage group would put it directly downstream of the surface circulation (down to 7 m) illustrated from the drogue study in Figure 5.3, but the bottom deposition of any enrichment from waste food and faeces remains unknown for this site, as near-bed current meters were not deployed.

Ecological effects of sea lice medicines in Scottish sea lochs 116 of 286 Table 5.5. Loch Diabaig, July 2000 and August 2001. Macrofauna pre- and post- Excis treatment data. S, total number of species; A, total number of animals; B, total biomass of animals. T-test at 95% level of significance. SD, significant difference; NSD, not significant. + values higher in 2000; - values lower in 2000; = values the same. Annelida Group Station 1 Station 2 Station 3 S -NSD -NSD +SD A +NSD -NSD +SD B -SD -NSD +NSD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43 Crustacea Group Station 1 Station 2 Station 3 S +NSD -SD +SD A +NSD -NSD +NSD B -NSD -NSD +NSD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43 Mollusca Group Station 1 Station 2 Station 3 S -SD -NSD +SD A -SD +NSD +SD B -SD -NSD +SD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43 Echinodermata Group Station 1 Station 2 Station 3 S +NSD -NSD +SD A +NSD +SD +SD B +NSD +SD +NSD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43

5.6.3 Sublittoral settlement panels Sublittoral arrays of slate panels were deployed at three stations in Loch Diabaig (Figure 5.7) on 21 March 2000: LT1 (predicted high impact)), LT2 (intermediate) and LT3 (reference) were situated at the same locations as the macrofaunal collections. The arrays had sets of four slates at each of three depths: ~2 m below the surface, mid-depth and 2 m above the seabed.

The arrays were first inspected on 12 July 2000; both upper sets of panels at LT2 and LT3 were missing. The organisms on the slates were identified and assessed in situ using SACFOR abundance scales. The slates were photographed and one from each set of four brought back fresh to the laboratory for more detailed study. New slates were placed on the arrays to replace those removed or lost.

The results for each station are summarised in Table 5.6. The main features of the biota present on the panels were as follows:

Ecological effects of sea lice medicines in Scottish sea lochs 117 of 286 Table 5.6. Loch Diabaig: summary of biota settled on the sublittoral arrays at Stations LT1-3, inspected 12 July 2000 (deployed 21 March 2000). Station/Impact LT1 H LT2 I LT3 R Level U M L Um M L Um M L Balanus crenatus 1 2 5 2 5 5 4(d) Obelia longissima 5 4 4 5 5 1 5 Mytilus edulis (spat) 4 5 6 Onchidoris bilamellata 3 3 4 5 5 Ascidiella aspersa 4 5 4 5 4 Ciona intestinalis 4 3 5 3 5 5 3 Corella parallelogramma 6 6 6 Ascidiella scabra Pomatoceros triqueter (juv.) 5 5 4 5 3 3 1 Hydroides norvegica 6 5 5 5 5 Electra pilosa 4 4 5 5 5 4 Balanus balanus 6 6 Harmothoe impar 6 Psammechinus miliaris Asterias rubens Antedon bifida Anomia ephippium Aequipecten opercularis 6 Metridium senile Enteromorpha compressa 5 Cutleria multifida 5 *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: 1 = S-Superabundant Level: Predicted impact: 2 = A-Abundant U = Upper, ~2 m below surface H = High 3 = C-Common M = Mid, Midwater I = Intermediate 4 = F-Frequent L = Lower, 2 m above seabed R = Reference 5 = O-Occasional 6 = R-Rare Missingm: the upper set of panels at LT2 and LT3/replaced

Upper panels (at LT1): superabundant large barnacles Balanus crenatus (up to 100 % cover and hummocking) dominated the single surviving top set of panels, with frequent young ascidians Ciona intestinalis, Ascidiella aspersa and mussels Mytilus edulis (small) on and among the barnacles. The sea slug Onchidoris bilamellata was common and large, actively feeding on the barnacles and producing egg-masses. The hydroid Obelia longissima (mostly young) was frequent and two macroalgae were present - Cutleria multifida and Enteromorpha compressa (occasional).

Middle panels (LT1, LT2): large B. crenatus were again abundant (50-80 % cover) with O. bilamellata feeding on them; young C. intestinalis (up to 60 % cover) and A. aspersa (occasional); hydroid O. longissima (common, young); tube worms Pomatoceros triqueter (frequent); and Hydroides norvegica (occasional); also the encrusting bryozoan Electra pilosa was occasionally present on otherwise bare areas of slate.

Middle panels (LT3, out in more open water): were much more lightly colonised with only occasional B. crenatus and C. intestinalis, but with young hydroid O. longissima abundant. Occasional Balanus balanus; P. triqueter (juvenile, common); the polychaete Harmothoe impar and queen scallop Aequipecten opercularis (juvenile, rare).

Lower panels (LT1, LT2, LT3): these also were relatively lightly colonised with scattered barnacles, ascidians and hydroid, but some panels had superabundant P. triqueter (mostly very juvenile). Electra pilosa encrusted up to 10 - 20 % of the surface areas of other panels. Balanus balanus and the ascidian Corella parellogramma were both rare.

Ecological effects of sea lice medicines in Scottish sea lochs 118 of 286 Comparison of the data between LT1 (high) and LT2 (intermediate), and particularly comparing the middle and lower sets of panels, shows close similarity between them, with reference to both the species present and their abundances.

In comparison with the Loch Sunart arrays (6 July 2000): • The settlement of Mytilus spat was only just starting in Loch Diabaig. • The ascidians and the hydroid Obelia were mostly smaller (younger). • Pomatoceros and Electra were more abundant. • The presence of a few small B. crenatus again indicated a prolonged settling period. • Predation of B. crenatus by the sea slug Onchidoris was more advanced/severe.

The next array inspection in Loch Diabaig was on 15 November 2000, following a period of strong NW gales (pers. comm., G. Macdonald, MH). Arrays LT1 and LT2 were missing and no evidence could be found of the surface buoys within Loch Diabaig. Array LT3 had dragged some distance into more open water; the upper set of panels was missing, Slate 3M2 (damaged) was the only middle level panel recovered, but the lower set of panels (3L1 - 3L4) was recovered intact.

The biota present on panels 3L1 - 3L4 from Station LT3 are recorded in Table 5.7. The main features (compared with July data) were: the ascidian C. intestinalis was now abundant; P. triqueter was still common but being eaten by starfish Asterias rubens; additional species (since July) were A. aspersa, A. scabra, the green urchin Psammechinus miliaris and featherstar Antedon bifida (all occasional).

Table 5.7. Loch Diabaig: summary of biota settled on the only remaining sublittoral panels at Station LT3, inspected 15 November 2000 (deployed 28 March 2000). Station/Impact LT1 H LT2 I LT3 R Level Um Mm Lm Um Mm Lm Um Mm L Balanus crenatus 5 Obelia longissima 5 Mytilus edulis (spat) Onchidoris bilamellata Ascidiella aspersa 5 Ciona intestinalis 2 Corella parallelogramma 6 Ascidiella scabra 5 Pomatoceros triqueter (juv.) 3 Hydroides norvegica 5 Electra pilosa 5 Balanus balanus Harmothoe impar Psammechinus miliaris 5 Asterias rubens 5 Antedon bifida 5 Anomia ephippium Aequipecten opercularis Metridium senile Enteromorpha compressa Cutleria multifida *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: 1 = S-Superabundant Level: Predicted impact: 2 = A-Abundant U = Upper, ~2 m below surface H = High 3 = C-Common M = Mid, Midwater I = Intermediate 4 = F-Frequent L = Lower, ~2 m above seabed R = Reference 5 = O-Occasional 6 = R-Rare Missingm: arrays LT1 and LT2, and the upper and middle set of panels at LT3

Ecological effects of sea lice medicines in Scottish sea lochs 119 of 286 The November 2000 gales had also caused damage to fish farm structures (moorings broken and several cages left adrift). Earlier losses here such as the upper sets of panels missing in July at LT2 and LT3, could also have been as a result of storm damage, or from them being inadvertently trawled by local creel boats. It was concluded by sponsors and collaborators that the use of substantial further resources to re-install and maintain sublittoral arrays at Loch Diabaig during 2001 would not be justified, particularly since a continuous record of settlement and succession of sublittoral fauna over two successive years could not be obtained.

5.7 Littoral studies: intertidal panels Three littoral sampling stations were established on the loch shore in areas where any impacts of cage treatments were predicted to be high (Station C), intermediate (Station B) and low (Station A). Figure 5.10 shows the positions of Stations C (approximately 450 m S of the cages), B (approximately 350 m ESE of the cages) and the reference Station A (approximately 250 m NE of cages).

Figure 5.10. Littoral sample stations () at Loch Diabaig. The cage group is shown as a grey rectangle.

Ecological effects of sea lice medicines in Scottish sea lochs 120 of 286 Shore slates were installed at each station in the late winter to early spring each year from 2000 until 2003. Selected slates were retrieved approximately one year later, transported to the laboratory for detailed examination of the settled populations, and replaced by new slates. During the first two years of the study (2000-01 and 2001-02) some slates were left in situ with the intention of following the development of the barnacle populations on the slates over more than one settlement season. In 2000, 2001 and 2003, the settled populations were examined during the summer and early autumn, to monitor the growth of the populations. By August 2001, it was becoming difficult to distinguish the two year groups, and subsequent detailed analyses of the settled populations in the laboratory confirmed that the different year groups were not clearly distinguishable. From January 2002, all 4 slates were replaced at each shore station and retrieved the following year, so that all subsequent analyses were of a single generation (cohort). Analyses of the results of the settlement studies at Loch Diabaig have only used the data from slates with populations that had settled during the preceding year. Figure 5.11 shows the results of the study for each station in each year. The mean values and statistics have been compiled from the means for the slates retrieved from each station each year and analysed in the laboratory.

During the first two years only 1 or 2 slates were removed annually from each station. By January 2003, one slate at each station had been in situ for three years and therefore contained three generations. Very few (26) barnacles remained on one slate, C9 at Station C. This population was examined on site for the presence/absence of fertilised egg masses and the diameters of the barnacle shells were measured. A slate was missing from Station B and the remaining slates were examined in detail in the laboratory. In February 2004, four slates were retrieved and examined in the laboratory.

5.7.1 Populations and development of barnacles, Semibalanus balanoides Results of population and growth studies of S. balanoides in Loch Diabaig are summarised below in Figure 5.11 and fully in Table 16.4. Shell diameters, body weights, and valve weights are all size related parameters. The body weights, however, change considerably over the annual cycle and are near their annual minimum in the late winter/early spring when the slates were retrieved for detailed examination in the laboratory. Maximum body weights are attained towards October to November when the bodies contain mature sperm, prior to copulation. In densely populated areas of the slates, shell diameters are influenced by crowding, but in the detailed laboratory examination attempts were made to measure individuals with undistorted shells. Diameters have also been measured in the field, when appropriate, to provide an indication of size. When the shells are not grossly distorted, Barnes et al. (1963) found that the most reliable and consistent measure of growth was the weights of the opercular valves. However, accurate dissection and weighing of the valves is difficult when the individuals are very small. It is also acknowledged that selection of individuals for dissection and weighing will tend to be biased towards selection of animals above the average size for the populations. Thus the averages taken from measurements of 50 individuals from each slate are likely to be higher than the true averages for the settled populations. Because of the wide variation among populations on different slates at each station few of the observed differences among mean values are statistically significant at the 95 % level of confidence. However, at Station C the mean size of individuals, as represented by valve weights, tended to be higher in 2003 and 2004 than in 2002, while at the reference Station A the animals tended to be smaller in 2002 and 2003.

Ecological effects of sea lice medicines in Scottish sea lochs 121 of 286 Figure 5.11. Loch Diabaig, cohorts 2000 - 2003: Growth and development of the barnacle, Semibalanus balanoides on shore panels. (Error bars are 95 % confidence limits).

Ecological effects of sea lice medicines in Scottish sea lochs 122 of 286 Semibalanus balanoides population densities were highly variable among the slates from each station, among stations and from year to year at each station. In general the lowest populations settled on the slates at Station C, but there is a trend towards an increase in population densities on the slates at this location over the 4 year period. It is not known if this trend reflects changes in population densities on the natural rock substrates at Diabaig.

There was a trend towards increased mean weights of egg masses in individuals from Station C from 1.9 mg in the 2001 group to 4.5 mg in 2003, but from the very wide confidence limits this difference is not statistically significant at the 95 % level. There were slight decreases in mean weights of egg masses in individuals from Stations B (intermediate impact) and A (reference) over the same period.

When assessing the absence of fertilised egg masses, the variability among populations settled on different slates, together with the relatively small numbers of slates examined in detail each year, means that many of the observed trends are not statistically significant at the 95 % level. However there have been some notable trends at all stations and these are summarised in Table 5.8.

Table 5.8. Loch Diabaig, cohorts 2000 - 2003: percent of individuals with no fertilised egg masses. Cohort Station C Station B Station A (High impact) (Intermediate impact) (Reference) 95% 95% 95% Mean% Slates Mean% Slates Mean% Slates Conf. Conf. Conf. no eggs (n) no eggs (n) no eggs (n) Limits Limits Limits 2000 72 2 90 16 1 - 0 1 - 2001 63 2 117 0 1 - 1 2 9 2002 31 3 40 27 3 22 10 3 19 2003 47 4 19 23 4 13 17 4 16

At Station C (high impact) in 2000-2002 60 - 70 % of individuals were without egg masses). At Station B (intermediate impact) 16 % were found without egg masses during this period whereas at reference Station A only 1 % of individuals were found without eggs. However in the period 2003-2004 10-20 % of the individuals at Stations A and B were found to lack egg masses whereas at Station C the numbers without eggs had fallen to 30 - 50 %. Thus the overall trend appears to be an increase in the frequency of occurrence of egg masses at Station C in 2002-3 and 2003-04, and a tendency towards a decrease at the other two stations.

As noted in Section 2.10, littoral biota are subject to a wide range of natural environmental variations, and the failure of individuals to reproduce could be caused by a wide range of natural causes. If the trends observed here are a response to fish farm effects, one would expect to find some correlation between the observed frequency of occurrence of egg masses at Station C and changes in fish farm production or sea lice treatments.

Ecological effects of sea lice medicines in Scottish sea lochs 123 of 286 At Loch Diabaig fish farm the peak biomass in 2001 was 1141 t, and declined to 626 t in August 2003. In 2000 there were 3 treatments with Excis (cypermethrin) and 1 with Salmosan (azimethipos). In 2001, there were 6 treatments with Excis and one with Slice (emamectin). Only 1 treatment with Excis was recorded in 2002, and two with Excis and 1 with Slice in 2003. In addition the quantities of both treatment agents used were much lower in 2003 than in 2001 (Table 5.1). Thus, the intensity of fish farming and sea lice treatments both appear to have declined from 2001 to 2003. If the observed changes in egg production in S. balanoides are fish farm related, the trend towards decrease in intensity of the fish farm operation seems to be consistent with the decline in observed impact at Station C.

To address the rise in the proportion of individuals with no egg masses at the reference Station A, and the similar level found at Station B over the last two years, some salient aspects of the reproductive cycle of S. balanoides must be considered. At spawning, free swimming larvae (nauplii) are released into the water column, usually in March. After metamorphosis to the non-feeding, free swimming cypris stage the larvae eventually settle, usually 20-30 days later. After settlement the barnacles grow and sperm and ovarian tissue develop over the summer and early autumn. Copulation takes place in October-November, and although apparently fully developed embryos are present in January the larvae are not released until March (Barnes and Blackstock, 1975). Thus the larval stages are in the water column for sufficient time to allow considerable dispersion, and mixing with cyprids originating from spawning throughout the littoral zone of the loch and beyond. The possibility of the observed changes at Stations A and B being attributable to some input of cyprids from the vicinity of Station C cannot be ruled out, but we have no information on which to test this hypothesis.

It is emphasised that a large number of natural factors could produce the effects suggested here. It is noted that similar effects have not been observed in some of the other areas studied in this project. From the data gathered, it is not possible to determine which stage of the reproductive cycle may be affected to reduce the frequency of occurrence of fertilised egg masses, and we have no information on the concentrations of fish-farm related materials in the vicinity of the shore sites. In addition no data are available on the ‘normal’ frequency of occurrence of mature fertilised egg masses in barnacle populations in non-impacted areas. Other possible natural environmental factors e.g. degree of exposure of the sites will be discussed later in Section 8 (Analysis across sites). However, the results indicate that the environmental factors influencing egg production in littoral barnacles merit further more detailed investigation, including study in areas, which could not possibly be affected by fish farming activities.

Ecological effects of sea lice medicines in Scottish sea lochs 124 of 286 6 Loch Kishorn 6.1 Site description Loch Kishorn, Wester Ross, lies south of the Applecross peninsula, opening on to the Inner Sound separating the Isle of Skye from the mainland, and runs roughly southwest to northeast (Figure 6.1). The loch lies within the Kishorn Thrust (an offshoot of the Moine Thrust), a major geological fault zone separating the Torridonian red sandstone formations on the northern Applecross shore from the Sleat formation of the southern shore (Johnstone and Mykura, 1989).

Loch Kishorn is a short (4.1 km) sea loch, with no sills or basins (sensu Edwards and Sharples, 1986), and has a flushing time of three days. The mean low water depth is 22.2 m, with a watershed of 66 km2 and tidal range of 4.7 m. Maximum depth is 61 m. The loch has the highest proportion of intertidal area (24 %) of any of the lochs investigated in the present project (the above data from Edwards and Sharples, 1986).

The Camas Doun farm site investigated during this project is near the head of the loch, on the southern side near the village of Achintraid, and is given some shelter from westerlies by Kishorn Island (Figure 6.2). The loch has a low fresh/tidal ratio of 1:125; the major freshwater sources are the River Kishorn entering at the head of the loch, and the Abhainn Cumhang a’ Ghlinne, which enters the loch at Achintraid.

Loch Kishorn has not been the subject of any detailed physical surveys; a mean current speed 0.05 m s-1 was the only reported hydrographic datum found (Karayucel & Karayucel, 1998).

Ecological effects of sea lice medicines in Scottish sea lochs 125 of 286 A) B)

Figure 6.1. (A) Location of Loch Kishorn on the Scottish west coast and (B) Loch Kishorn and location of study area. Grid is OS.

Ecological effects of sea lice medicines in Scottish sea lochs 126 of 286 6.2 Fish farm history, cage positioning, biomass, and medicine use Scottish Sea Farms (previously Golden Sea Produce) acquired the Kishorn sites Achintraid and Camas Doun in 1988. Company records do not differentiate between Achintraid and Camas Doun production until 1996, thus the following data may apply to either of the sites. Estimated biomass at Achintraid/Camas Doun was roughly 100 t in the first year (1988), equating to 1000 t at harvest in 1990. Harvest biomass remained constant at ca. 1200 t for each production cycle until 2002, when 1411 t were produced. The Camas Doun site was then fallowed in September 2002 (pers. comm., Sally Davies, Scottish Sea Farms).

Medicine use at Kishorn fish farm between May 1999 and March 2002 comprised Excis and Slice treatments only (Table 6.1). Farm management details (fish movements, treatments) and project details are arranged chronologically in Table 6.2.

Table 6.1. Sea lice medicine use at Camas Doun (Kishorn) Farm since 1999. Date Treatment Amount active Active ingredient ingredient May 1999 Excis 7.7 l cypermethrin Aug 1999 Excis 8.0 l cypermethrin Nov 2000 Excis 0.8 l cypermethrin Feb 2001 Excis 4.8 l cypermethrin April 2001 Excis 5.0 l cypermethrin May 2001 Excis 5.0 l cypermethrin June 2001 Excis 4.8 l cypermethrin July 2001 Slice 114.6 g emamectin benzoate Oct 2001 Slice 246.4 g emamectin benzoate Nov 2001 Excis 8.8 l cypermethrin Mar 2002 Slice 430.0 g emamectin benzoate Jan 2004 Slice 335.9 g emamectin benzoate Jan 2004 Excis 4.8 l cypermethrin Mar 2004 Excis 2.4 l cypermethrin

Ecological effects of sea lice medicines in Scottish sea lochs 127 of 286 Figure 6.2. Cage group position (grey rectangle) at Loch Kishorn and sampling stations. Littoral stations are shown as , zooplankton stations as , meiofaunal stations as , and combined macro/meiofaunal stations as .

Ecological effects of sea lice medicines in Scottish sea lochs 128 of 286 Table 6.2. Timeline of sampling and management events at Loch Kishorn fish farm. Figs. in kg are feed input; Aza = Salmosan, Ema = Slice, Cyper = Excis, H2O2 = Salartect Key: S: Sampling D: Deployed R: Retrieved Day - Event Scientific events

1999 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Cyper Cyper events 73325 kg 304967 kg 216105 kg 228964 kg 334275 kg 298772 kg 470778 kg 870954 kg 524924 kg 452514 kg 425931 kg 199792 kg Management Scientific events Day - Event

2000 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Cyper 453 kg events Fish out 3765 kg 4645 kg 41621 kg 10525 kg Camasdoun Management

Ecological effects of sea lice medicines in Scottish sea lochs 129 of 286 Table 6.2 (cont.) Key: S: Sampling D: Deployed R: Retrieved Zoopl (S) Day - Event 28 - Macrofauna (S) 22 - Current meters (D) 28 - Drogues (D) 30 - RoxAnn (S) 7 - Shore slates (S) Scientific events Day - Event 20 - Prelim. shore survey 25 - Meioofauna (S) 13 - Meiofauna (S) 20 - Macrofauna (S) 21 - Shore slates (D) 17 - Shore slates (D)

2001 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Ema Ema Cyper Cyper Cyper Cyper Cyper Cyper events 37475 kg 51200 kg 13485 kg 20485 kg 40000 kg 52275 kg 63471 kg 130236 kg 137380 kg 318334 kg 221675 kg 263972 kg Management Day - Event 24 - Meiofauna (S) Scientific events 14 - Current meters (R) 3 - Macrofauna (S)

2002 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Ema Fish in events Fish out 36115 kg 75600 kg 73500 kg 19000 kg 16225 kg 45575 kg 187300 kg 150650 kg 222175 kg 112800 kg Achintraid Management

Ecological effects of sea lice medicines in Scottish sea lochs 130 of 286 Table 6.2 (cont.) Key: S: Sampling D: Deployed R: Retrieved 15 - Shore slates (S) Scientific events Day - Event 28 - Shore slates (S)

2003 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 93050 kg 62761 kg 85490 kg 45800 kg 76800 kg 113855 kg 202915 kg 291700 kg 138445 kg 230750 kg 166750 kg 186829 kg Management Day - Event Scientific events 19 - Shore slates (R)

2004 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec events 17 - Ema 96605 kg 10- Cyper 164175 kg 132364 kg 169600 kg 27 - Cyper 22 - Cyper Management

Ecological effects of sea lice medicines in Scottish sea lochs 131 of 286 6.3 Hydrography Three long-term current meter deployments at Loch Kishorn ranging in duration from 35 to 69 d gave current meter data spanning a period of 168 d (Table 6.3). The current meter data collected were for durations ten times longer than those usually collected at sites for sea lice medicine consents. The current meter data were used for modelling to predict emamectin benzoate deposition footprints following Slice treatments, and to make predictions of hydrographic conditions during periods where current meters were not deployed.

Maximum and mean surface current speeds were quite high during all deployment periods. In particular, deployment I measured maximum and mean speeds of 55 and 14 -1 cm s respectively. The longitudinal axis of orientation of surface current was along 070º- 250º axis (Figure 6.3). For surface current, there was a strong residual current seawards (> 2.9 cm s-1), as expected. Near-bed currents had a weaker residual current, less than 1.5 cm s-1, but had high maximum and mean speeds. The main axis of flow was more variable for near-bed currents, with a longitudinal axis for each deployment of 090º-270º or 045º-235º respectively. For deployments II and III, 0.6 and 1.6 % of the data were above 10 cm s-1, the critical threshold for resuspension in the model. This is significant as resuspension events will erode and redistribute deposited solids and associated chemicals in the model grid area.

Table 6.3. Current meter summary statisitics. Deployment durations were: deployment I 22/08/01 to 30/10/01; deployment II 06/11/01 to 13/12/01; deployment III 13/12/01 to 14/02/02. No. Instrument Meter Max spd. Mean spd. Residual Residual Record length type position (cm s-1) (cm s-1) spd. (cm s-1) direction (ºT) (d) I Acoustic Surface 55.3 14.3 3.6 223 60.6 I Acoustic Near-bed 50.2 8.9 0.9 242 69.3 II S4 Surface 43.7 9.2 3.6 223 35.4 II S4 Near-bed 18.3 3.8 1.2 187 36.9 III S4 Surface 35.5 7.6 2.9 230 62.9 III S4 Near-bed 29.9 2.9 0.2 121 62.9

Ecological effects of sea lice medicines in Scottish sea lochs 132 of 286 Figure 6.3. Time series plots of current speed and vector displacement for Loch Kishorn (sampling interval 10 minutes, total length 36 days). (a) Near-surface and (b) near-bed water speed, (c) near-surface and near-bed water displacement.

6.3.1 Dispersion data Six DGPS drifting buoys were deployed at the site over two days during a neap tide. Drifters were deployed next to the farm and were advected in the current for approximately four hours so that dispersion values could be calculated for the area around the farm under different tidal conditions. Drifter tracks are shown for one release at the farm (Figure 6.4).

Ecological effects of sea lice medicines in Scottish sea lochs 133 of 286 Figure 6.4. DGPS drifter buoy survey at Loch Kishorn showing buoy tracks and cage position.

The calculated dispersion coefficients indicate reasonable dispersion at the site (especially as the survey was undertaken during a neap tide) and demonstrate changes over time (Table 6.4). Although drifter dispersion was initially quite low near the farm, dispersion rates increased with distance from the farm. Horizontal dispersion coefficients were higher than the default value (0.1 m2 s-1) recommended by SEPA for sea lice medicine consent modelling. The values are very much dependent on tidal and wind conditions at the time of survey, thus can only be used to give a general indication of dispersion potential. For example, if the drifter release ended after 2 h rather than 4.2 h, the calculated ky value would have been five times smaller. Given the cost of undertaking drifter surveys incorporating different tidal and wind states, several deployments over two days are normally taken as being appropriate providing the limitations are taken into account.

Ecological effects of sea lice medicines in Scottish sea lochs 134 of 286 Table 6.4. Horizontal dispersion coefficients (Kx and Ky) calculated for Loch Kishorn on 29/08/01. The centroid indicates the mean position of all drifters.

Time after release kx ky Centroid speed Centroid direction 2 -1 2 -1 -1 (m s ) (m s ) (cm s ) (ºT) 10 min 0.003 0.011 3.1 40 20 min 0.008 0.028 3.4 29 30 min 0.019 0.027 3.5 28 1 h 0.060 0.047 4.0 22 2 h 0.254 0.025 5.0 23 3 h 0.331 0.002 6.0 30 4 h 0.327 0.073 6.0 38 4.2 h 0.278 0.108 5.9 40

6.4 Emamectin benzoate sediment concentrations: Slice treatments July and October 2001 Sediment samples were collected on three occasions between June 2001 and January 2002 for analysis of emamectin benzoate concentrations incorporating Slice treatments in July and October 2001 (Table 6.1). Sediment samples were collected by van Veen grab or by a diver using a hand held corer along two transects starting at the south east end of the cages (Figure 6.5).

Figure 6.5. Positions of sediment sample stations in Loch Kishorn, June 2001 and January 2002. The cage group is marked by the grey rectangle.

Sample station positions on the transects were determined using model predictions of deposition foot prints for Slice post-treatment days 7 (at end of administration), 49 and 105 (14 weeks post treatment) (Figure 6.6), and were arranged at the eastern end to permit sampling by diver. Post-treatment day 105 was considered the time of maximum emamectin benzoate sediment concentrations without incorporating resuspension in the model.

Ecological effects of sea lice medicines in Scottish sea lochs 135 of 286 Differences in predicted sediment emamectin benzoate concentrations beneath the cages after administration (day 7) and at maximum bed concentration (day 105) were an order of magnitude (Figure 6.6). At a distance of 50 m from the cages, concentrations were also an order of magnitude higher on day 105 compared to day 7. Using the predictions, the two transects were positioned so that an order of magnitude difference in emamectin concentrations was covered. Stations were spaced 25 m apart on transect 1 to incorporate an order of magnitude difference between station T1A at 0 m and T1C at 50 m. The main direction of flow is along the length of the farm (longitudinal axis), so the predicted footprint did not extend far along the transverse axis of the farm. Consequently, stations on transect 2 (T2A, T2B, T2C) were only 20 m apart. The reference station was positioned beyond the predicted footprint area (ca. 500 m SW of the cages).

Pre-treatment samples were collected in June 2001, prior to the first Slice treatment in July. Of these, all but three samples contained emamectin benzoate at either trace (detected but below the limit of quantification; < 1.9 µg kg-1 dry weight) or quantifiable concentrations (Table 6.5). Sediment samples with quantifiable concentrations (2.3 and 4.1 µg kg-1 dry weight) were from the stations closest to the cages (Station A) on both transects, but were collected prior to any Slice treatments at the farm. The pre-treatment reference sediment collected by diver contained trace amounts of emamectin benzoate although none was detectable in a van Veen grab sample from the same station taken two days later.

Post-treatment sediment samples were collected in August 2001 and January 2002, 22 and 186 days after the July treatment (Table 6.5).

In all but one sample collected in August 2001, emamectin benzoate was detected in trace or quantifiable concentrations. The highest concentration (13.4 µg kg-1 dry weight) was detected in sample T2A, next to the cages on transect 2, whilst the sample collected next to the cages on transect 1 contained 3.5 µg kg-1 dry weight. Only one sample on the transects (T2C) did not contain detectable emamectin benzoate (Table 6.5). The reference sample also contained trace concentrations of emamectin benzoate.

Ecological effects of sea lice medicines in Scottish sea lochs 136 of 286 Figure 6.6. Emamectin benzoate deposition predictions for days 7, 49 and 105 post Slice treatment at Loch Kishorn fish farm (cage centres shown as ).

Concentrations of emamectin benzoate samples collected 186 days post treatment (January 2002) ranged from trace to 4.8 µg kg-1 dry weight (Table 6.5). The reference sample also contained trace concentrations of emamectin benzoate.

Emamectin benzoate was detected in all but one of the reference samples collected from Loch Kishorn between June 2001 and January 2002 (Table 6.5). Procedural blanks

Ecological effects of sea lice medicines in Scottish sea lochs 137 of 286 analysed with each batch of samples to monitor potential laboratory contamination did not contain emamectin benzoate. Therefore, emamectin benzoate measured in the reference and pre-treatment samples cannot be attributed to background laboratory contamination.

In assessing the quality of these data several factors should be borne in mind:

• both the farmer and the manufacturer have provided documentary evidence that they did not have access to Slice until after the samples in June were taken – farmers and manufacturers are obliged to keep careful and detailed records of the supply and use of emamectin

• it was the farmer who selected this site as a candidate for this study knowing that pre-treatment sediment samples would be taken and analysed for emamectin

• there is no satisfactory explanation for the detection of emamectin at the reference station either before or after treatment

• there must be some degree of doubt about the reliability of field data close to the instrumental detection limit.

Taking these factors together we conclude that the results of this survey should be interpreted with great caution. We conclude that these samples have been exposed to contamination at some point in the process, which we have been unable to identify. The following discussion on modelled and observed concentrations of emamectin should be read with these reservations on the quality of these data in mind.

Ecological effects of sea lice medicines in Scottish sea lochs 138 of 286 Table 6.5. Loch Kishorn sediment chemistry sample collection dates, collection method and emamectin benzoate concentrations. Samples with A in the station code were collected within the near-field (25 m) AZE; those with B and C were within the far-field (100 m) AZE. Field ID/ Collection Date Station Code/Collection method Concentration (µg kg-1 dry weight) 25-28 JUNE 2001 Kishorn Reference Reference/Diver core Trace Kishorn T1A1 T1A/Diver core Trace Kishorn T1B1 T1B/ Diver core Trace Kishorn T1C1 T1C/ Diver core Trace Kishorn T2A1 T2A/ Diver core 2.3 Kishorn T2B1 T2B/ Diver core Trace Kishorn T2C1 T2C/ Diver core ND Kishorn 1V V2 T1A/ van Veen grab Trace Kishorn 2V V1 T1B/ van Veen grab ND Kishorn 3V V1 T1C/van Veen grab Trace Kishorn 4V V1 T2A/van Veen grab 4.1 Kishorn 5V V2 T2B/van Veen grab Trace Kishorn 6V V2 T2C/van Veen grab Trace Kishorn 7V V1 Reference/van Veen grab ND 13 AUGUST 2001 Kishorn Reference Reference/Diver core Trace Kishorn T1A T1A/ Diver core 3.5 Kishorn T1C T1C/ Diver core Trace Kishorn T2A T2A/ Diver core 13.4 Kishorn T2B T2B/ Diver core 2.0 Kishorn T2C T2C/ Diver core ND 24 JANUARY 2002 Kishorn Reference Reference/Diver core Trace Kishorn T1A T1A/ Diver core 3.3 Kishorn T2A T2A/ Diver core 4.8 Kishorn T2B T2B/ Diver core Trace Kishorn T2C T2C/ Diver core Trace Trace = 0.03 - 1.9 µg kg-1 dry weight ND (not detected) = < 0.03 µg kg-1 dry weight

6.5 Predicted post-treatment sediment emamectin benzoate concentrations Sediment emamectin benzoate concentrations measured following Slice treatments in July and October 2001 were compared with predicted concentrations from the Slice module in DEPOMOD (Cromey et al., 2002a).

The model was driven by bathymetry and current meter data collected during field surveys in August 2001 and February 2002 (Section 6.3). The current meter data encompassed most of the treatment dates (see Section 6.2). Any minor gaps present in the current meter data (i.e. close to the first treatment and during mooring servicing when no meters were in place) were simulated using measurements extracted from an equivalent period in another spring-neap cycle (i.e. phase aligned). The current meter data used in the model represented a significant improvement on sea lice medicine consent modelling methods, which use repeated 15 day data sets. Unmedicated feed input data were also used in the model, where the farm biomass increased from 234 t to 1154 t between July 2001 and January 2002 (Table 6.2). The total number of particles in the simulation was

Ecological effects of sea lice medicines in Scottish sea lochs 139 of 286 optimised until increasing numbers did not improve accuracy and only increased computational time (7.9 x 105).

Quantities of emamectin benzoate used for each Slice treatment are given in Table 6.1. The release of emamectin benzoate adhered to faecal material emanating from the farm was predicted to peak immediately after treatment with Slice and decline exponentially thereafter, with a much greater release occurring after the second treatment (Figure 6.7). A higher release rate of emamectin benzoate adhered to waste feed during the second treatment was also predicted.

Figure 6.7. Release of emamectin benzoate adhered to uneaten feed and faecal material from Loch Kishorn fish farm after Slice treatments in July and October 2001.

Predicted sediment emamectin benzoate concentrations were similar to measured concentrations on day 22 post-treatment, with the exception of station T2A (Figure 6.8), which had a considerably higher concentration (13.4 µg kg-1) than that predicted by the model. With the exception of station T2A, both measured and predicted concentrations were less than 5 µg kg-1 however, predicted concentrations were generally lower than those measured.

In contrast, predicted sediment emamectin benzoate concentrations on day 186 post- treatment were significantly higher than those measured, with some of the predictions an order of magnitude higher than measured values. The inaccuracy of the model here is likely to be caused by absence of processes in the model such as particle burial by benthic fauna. Additionally, the potential for a particle to be resuspended is likely to vary over time as the particle properties change and this will effect its fate. Section 8.2 discusses issues relating to model performance.

Ecological effects of sea lice medicines in Scottish sea lochs 140 of 286 Figure 6.8. Predicted and measured emamectin benzoate sediment concentrations at Loch Kishorn on 13 August 2001 and 24 January 2002 (22 and 186 days after July Slice treatment). Slice treatments were on 23 July and 1 October 2001 (days 1 and 71 respectively).

Measured sediment emamectin benzoate concentrations were higher along transect 2 (SE transect) compared to transect 1 (NE transect) (Figure 6.9). Although the main axis of flow at Loch Kishorn is NE to SW, a residual current to the SW and SE was measured during the long term current meter deployments. This is reflected in the predicted footprint of emamectin benzoate deposition, which is displaced to the SW and SE (Figure 6.9). Consequently higher emamectin benzoate concentrations were predicted for transect 2, which is consistent with the measured data.

Ecological effects of sea lice medicines in Scottish sea lochs 141 of 286 Figure 6.9. Predicted emamectin benzoate sediment concentration at Loch Kishorn on days 22 and 186 post-treatment.

6.6 Zooplankton A sampling campaign was undertaken at Scottish Sea Farms’ Loch Kishorn fish farm to determine the effects of the in-feed treatment Slice on the plankton community. The fish were fed with emamectin benzoate coated feed (total of 114.6 g active ingredient) for a

Ecological effects of sea lice medicines in Scottish sea lochs 142 of 286 period of seven days from 23 to 29 July 2001. Plankton samples were collected over a five week period pre-treatment, daily during the treatment week and over a four week period post-treatment at the sample stations shown in Figure 6.10.

Figure 6.10. Zooplankton sampling stations at Loch Kishorn. Station A is 50 m W of cage group, Station B = 0 m W, Station C = 0 m E, and Station D = 50 m E. Cage groups are marked by grey rectangles.

Zooplankton community composition changed during the sample period, as indicated by the shift in sample stations from left to right along the first axis, and bottom to top along the second axis in the CA plot (Figure 6.11). Pre- and post-treatment samples are located to the left of the plot, while samples taken during the treatment week (day 1 to 6) are located to the right. Community composition at the reference station and the sample stations close to the fish cages was generally similar, as indicated by the proximity of the two data points for each sample day.

Ecological effects of sea lice medicines in Scottish sea lochs 143 of 286 Figure 6.11. Zooplankton sample stations CA plot for July 2001 Slice treatment at Loch Kishorn. Of the variance, 17 % is explained by the first two axes. For each sample day, the open circles are the reference sample station and the closed circles are the combined data for the four stations located within 100 m of the cages.

The species associated with the sample stations are shown in Figure 6.12, with trends in abundance for total zooplankton and five of the more abundant taxa shown in Figure 6.13. Zooplankton abundance peaked in the week (day -6) before the treatment then decreased. Copepod nauplii maintained high numbers throughout the treatment week and post- treatment, whereas Oithona abundance remained low. Microcalanus subtilis numbers fluctuated as did the pre-adult stages (C1 to C3) of Acartia and Temora. Trends in species abundance at the reference and cage stations were similar, although on most sample days abundance was higher at the reference station. The composition of the zooplankton community at Loch Kishorn fish farm changed during the sample period however, there were no obvious changes in abundance of any of the zooplankton species present that could be correlated with the sea lice treatment at Loch Kishorn. Instead, changes in abundance during the sample period were most likely due to advection and natural patchiness. Thus, we were unable to detect a treatment related effect at the zooplankton community level or species level.

Ecological effects of sea lice medicines in Scottish sea lochs 144 of 286 Figure 6.12. Zooplankton species CA plot for July 2001 Slice treatment at Loch Kishorn.

Ecological effects of sea lice medicines in Scottish sea lochs 145 of 286 Figure 6.13. Abundance (number m-3) of the predominant zooplankton taxa at Loch Kishorn during July 2001 Slice treatment. For each sample day, abundance data for the four sample stations within 100 m of the salmon cages were combined and are presented as the mean (± S.E). Days -41 to -6 are pre-treatments sample days. Day 1 is the first of seven treatment days. Days 9 to 30 are days post-treatment. CI-CIII are pre-adult copepod stages 1 to 3. Note: y-axis scales are unequal.

Ecological effects of sea lice medicines in Scottish sea lochs 146 of 286 6.7 Phytoplankton The phytoplankton community at Loch Kishorn was monitored from June to August 2001 to investigate the potential effects the July 2001 Slice treatment. Samples for phytoplankton, salinity and dissolved inorganic nutrients were collected from the top 10 m of the water column from at least two of the five zooplankton sample stations on each sampling occasion (Figure 6.10). Samples were collected over a five week pre-treatment period, daily during the seven-day treatment, and over a four week post-treatment period at roughly the same time of day, e.g. within two hours of midday.

From June to August 2001, salinity at Kishorn was relatively constant (33.53 ± 0.21), and low nutrient concentrations were representative of summer conditions in Scottish coastal waters (typically 0.5 µM ToxN (nitrate plus nitrite), 0.3 µM phosphate, 0.6 µM ammonium and 1.6 µM silicate) (Appendix III - Phytoplankton). Differences in phytoplankton abundance during the sampling period were most likely due to patchiness as stations C and D displayed similar values (Figure 6.14a).

Prior to and following the Slice treatment (shaded area in Figure 6.14a), total phytoplankton cell abundance was relatively constant at about 1.5 x 106 cells l-1. However, during the treatment period (23 July to 31 August 2001), cell abundance was lower (8.4 x 105 cells l-1). The decrease in abundance during the treatment may be partly explained by the weather conditions; strong easterly and southwest/westerly winds and rain were reported at Kishorn between 17 and 24 July (pers. comm., G. Cushnie SSF). Grazing by zooplankton may also be a factor in the decrease in cell abundance, as relative abundances of the copepod species Oithona and Temora (CI-CIII) increased the week before the treatment (Figure 6.13).

Phytoplankton abundance trends at stations A-E during the treatment week are shown in Figure 6.14b. With the exception of the first and last samples, cell abundance was relatively constant regardless of sample station or sample day

Two microflagellate blooms (< 15 µm cell size; > 1 x 106 cells l-1) were observed in early July and mid August (Figure 6.15). Numerically, diatoms were dominated by Chaetoceros spp. (10-30 µm) and by Leptocylindrus danicus, with smaller threshold populations of L. minimus, Guinardia flaccida, Dactyliosolen fragillismus and Skeletonema costatum (typically a few thousand cells per litre). Dinoflagellates were more species-rich than diatoms, although only rarely did a dinoflagellate species exceed 1 x 104 cells l-1. Some representative dinoflagellate species observed include Amphidinium sp., Ceratium furca, C. fusus and C. lineatum, Gymnodinium sp., Gyrodinium sp. and Prorocentrum micans. All of these species have previously been documented as occurring naturally in UK coastal waters (e.g. Gubbins et al., 2003; Dodge, 1982). The phytoplankton community at Loch Kishorn exhibited a classic mid to late summer population shift during the sampling programme.

Ecological effects of sea lice medicines in Scottish sea lochs 147 of 286 Figure 6.14. Total phytoplankton cell abundance (cells l-1) at Loch Kishorn from June to August 2001. (a) Cell abundance at stations C and D, the longest time series. The shaded area is the Slice treatment period. (b) Cell abundance at stations A-E during the Slice treatment period (23 to 29 July 2001).

Ecological effects of sea lice medicines in Scottish sea lochs 148 of 286 Figure 6.15. Gross phytoplankton community composition at Loch Kishorn between 12 June and 21 August 2001. “Others” classification includes other flagellates, cryptophytes, ciliates, and tintinnids.

Phytoplankton and zooplankton abundance was generally homogeneously distributed (as indicated by the small error) at Loch Kishorn, although zooplankton abundance was always slightly higher at the reference station than at the stations close to the cages (Figure 6.16). The variability in phytoplankton and zooplankton abundance during the ten-week sampling programme is to be expected as a consequence of patchiness, advection and normal seasonal progression from early to late summer.

Ecological effects of sea lice medicines in Scottish sea lochs 149 of 286 Figure 6.16. Total zooplankton (animals m-3) and phytoplankton abundance (cells l-1) from integrated water column samples at Loch Kishorn between June and August 2001. The shaded area represents the Slice treatment period. Total zooplankton is presented for the reference station () and as a mean of the four sample stations close to the cages (, ± S.E.). Total phytoplankton is presented as the average of all sample stations visited (n = 2-5, ± S.E.). Note different y axes.

Both phytoplankton and zooplankton abundance increased immediately before and after the Slice treatment and decreased during the treatment week. The increase in phytoplankton cell abundance in the four weeks following the Slice treatment may have been due primarily to an improvement in weather conditions and light availability to the surface waters. The simultaneous and slightly off-set increase in zooplankton abundance suggests a degree of coupling between phytoplankton and zooplankton and also that were there any effects of the Slice treatment, they were very short-lived and did not significantly impact the reproductive capacity of the zooplankton community.

6.8 Sublittoral Sublittoral sampling at Loch Kishorn was designed at the outset to concentrate on the effects of Slice treatments, and with the different generation times of macro- and meiofauna in mind, slightly different sampling regimes were initiated. Macrofauna were sampled pre- and immediately post-treatment, and one year after initial Slice treatment. This one year post-treatment sample was not analysed, after discussion with the sponsors, in order to concentrate effort on Loch Sunart samples. Meiofauna were originally intended to be sampled pre-, 10 days post-, 14 weeks post- and 28 weeks post-treatment, however the cage group was moved from the site without notification to the collaborators before the 28 week post-treatment meiofaunal sample could be taken.

Ecological effects of sea lice medicines in Scottish sea lochs 150 of 286 Figure 6.17. Positions of macro- and meiofaunal sample stations in Loch Kishorn. Grey rectangle is the fish cage.

6.8.1 Meiofauna Meiofaunal studies were designed to determine whether the effects of in-feed chemicals could be detected against a background of organic enrichment normally associated with the cage-culture of fin-fish. For this purpose core samples were collected by SCUBA divers from the stations where chemistry samples were collected (Figure 6.17). Station location and spacing was determined by preliminary modelling, so that equivalent stations on each transect were predicted to have similar emamectin benzoate bed-concentrations, and different stations on each transect were expected to have concentrations differing by orders of magnitude (Section 6.4).

Slice was administered from the 23 to the 29 of July (Table 6.1). Meiofaunal samples were collected pre-treatment (25 and 26 June 2001) and post-treatment on 13 August. A further Slice treatment was administered in October 2001 and a second set of post- treatment samples collected on 24 January 2002. Core samples had an average volume of 340 cm3, and nematodes and copepods in a sub-sample from each were extracted, enumerated and identified.

6.8.1.1 Nematodes There were clear and significant differences in nematode community structure between stations near to the cages and reference samples, indicating that the effects of culture were largely confined to the vicinity of the cages. There were clear and parallel gradients in nematode community structure along the transects reflecting strong organic enrichment gradients. Equivalent stations on the two transects had noticeable (albeit small) differences in nematode community structure.

Ecological effects of sea lice medicines in Scottish sea lochs 151 of 286 Figure 6.18. MDS based on Bray-Curtis similarity calculated from square-root transformed average abundances of nematode genera from each Loch Kishorn survey/station combination. Numerals (1, 2) indicate transects, letters and symbols (A: ,,; B: ,,; C: ,,; R: ,,) indicate stations (R being the reference station), and the colours of the symbols indicate the surveys (: June 2001 pre- treatment; : August 2001 post-treatment; : January 2002, post-treatment).

In order to compare changes in nematode community structure across surveys, nematode abundances were aggregated to genus, to reduce any potential seasonal or identification biases. Analyses showed that differences between stations, transects and surveys were significant. To view better the relative changes at each station, aggregated nematode abundances were averaged within stations, mildly (square-root) transformed to downweight the influence of dominant species, and used to calculate Bray-Curtis similarities.

Changes in nematode community structure at the reference station between surveys were smaller than differences between reference stations and stations close to the cages (Figure 6.18). The gradient in nematode assemblage structure along each transect (from A to C) was maintained across all surveys, with stations furthest from the cages being most similar to the reference station. Relative changes in nematode community structure at all (including the references) stations were similar in magnitude. Thus, there was no evidence that Slice treatments impacted nematode assemblages at Loch Kishorn.

6.8.1.2 Meiobenthic copepods There were clear and significant differences in copepod community structure between stations near the cages and reference samples. In contrast to nematodes, differences in copepod community structure between the reference stations and stations at the distal ends of the transects (C) were similar in magnitude to differences between stations near the cages in June and August. Thus, the effects of fish farming activity were largely confined to the vicinity of the cages. There were clear and parallel gradients in community structure along the transects reflecting strong organic enrichment gradients, although equivalent stations on the two transects had noticeable (albeit small) differences in copepod community structure.

Analyses of the copepod abundance data from the three surveys combined showed that differences between stations, transects and surveys were significant. Relative changes at each station were examined by averaging copepod abundances within stations, mildly (square-root) transforming them to downweight the influence of dominant species, and

Ecological effects of sea lice medicines in Scottish sea lochs 152 of 286 using them to calculate Bray-Curtis similarities which were then ordinated using MDS (Figure 6.19). The patterns revealed by this plot contrast strongly with those revealed by the plot derived from nematode abundances (Figure 6.18).

Figure 6.19. MDS based on Bray-Curtis similarity calculated from square-root transformed average abundances of copepod species from each Loch Kishorn survey/station combination. Numerals (1, 2) indicate transects, letters and symbols (A: ,,; B, ,,; C, ,,; R, ,,) indicate stations (R being the reference station), and the colours of the symbols indicate the surveys (, June 2001 pre-treatment; , August 2001 post-treatment; , January 2002, post-treatment).

While there was a clear gradient in copepod community structure along both transects in each survey from close to the cages (A) to the distal ends of the transects (C), with stations closest to the cages being most different to the reference stations, there was also a clear interaction with time. The gradient of change in copepod community structure along both transects decreased with time, and assemblages at stations B, and then C became less like those at the reference site and more like those closest to the cages (A). This indicates a spreading impact from the cages with time. Although less easy to visualise, the K- dominance plot (Figure 6.20) derived from average copepod abundances at different distance from the cages shows the spreading impact, as assemblages become less diverse and more highly dominated.

Ecological effects of sea lice medicines in Scottish sea lochs 153 of 286 Figure 6.20. Cumulative dominance (%) plotted against species rank, for average copepod abundances at different distances from the cages in the three Loch Kishorn surveys. In the key symbols and letters indicate distances from the cages (A, ,,; B, ,,; C, ,,; R, ,,, reference station) and numbers and colours indicate surveys (1, ,,,: June 2001; 2, ,,,: August 2001; 3, ,,,: January 2002).

6.8.2 Comparison of sampling methods Relative recoveries of benthic copepods from van Veen grabs and diver collected cores were compared in pre-treatment (June 2000) samples collected from the transect stations.

It should be remembered that such comparisons are not straightforward, as the sizes of samples differ between methods (average sample volume 340 cm3 for cores, 160 cm3 for grab sub-samples). It may be possible to adjust raw abundances to account for differing sample sizes, as copepods tend to live in the surface layers of the sediment; however, relationships between copepod abundances and sample area or sample volume were not straightforward. Also the relationship between the number of species recovered and sample size tends to be non-linear.

Both abundances and the numbers of species recovered were very much lower in the grab sub-samples (Figure 6.21). Although grab sub-sample volumes were, on average, almost 50 % those of the diver cores, the average number of copepods recovered in the grab sub- samples ranged from 8 to 21 % of that within the diver cores within stations, with an average across stations of only 12 %. The average number of species within stations ranged from 18 to 69 % of that in the diver cores, with an average across stations of 35 %. Declining, albeit insignificant (α ≤ 0.05), trends in abundance with distance from the cages were apparent in both diver cores and grab sub-samples. The number of copepod species in diver cores was significantly related to station distance from the cages (R2 = 0.79, ν = 26, p < 0.001), as were the number of species in grab sub-samples (R2 = 0.19, ν = 28, p < 0.02), but the details of the relationships differ.

Ecological effects of sea lice medicines in Scottish sea lochs 154 of 286 Figure 6.21. Scatter plots of copepod abundance and number of species in Loch Kishorn diver cores () and grab sub-samples () at transect stations A, B and C (note, transects not identified in the graph).

To visualise the overall relationship in variations in copepod community structure in samples collected by the two sampling methods, the data were averaged within station/gear combinations, square-root transformed to downweight the influence of dominant species, and used to calculate Bray-Curtis similarities which were ordinated using MDS (Figure 6.22). The clear gradient in community structure in the diver core samples from stations closest to the cages (1A and 2A) to stations further away (1C, 2C, and R), and the division between Stations 2A, 2B, 1C and 2C, was less clear in the grab sub-samples. The variability between equivalent stations on the two transects is also greater.

These findings support those of previous studies (c.f. Fleeger, et al., 1988), which show that grab sub-sampling can be an unreliable method of sampling meiofauna, especially copepods, quantitatively. However, where there are clear, strong, patterns in the benthos

Ecological effects of sea lice medicines in Scottish sea lochs 155 of 286 (as at this site) the results from grab sub-samples can still give an indication of relative changes between samples.

Figure 6.22. MDS based on Bray-Curtis similarities calculated from square-root transformed average abundances of copepods at each Loch Kishorn station by coring by SCUBA divers () and sub-sampling from a van Veen grab ().

6.8.3 Relationships with environmental variables Particle Size Analysis (PSA) data (Table 17.5) were analysed using principal components analysis. Principal components 1 and 2 explained 84 % of the variability between stations, and the scores from this analysis were used in subsequent analyses. PC1 reflected the amount of very coarse material (≤ 1.0ϕ) at each station, while PC2 reflected a gradient from sands to silts. Total organic carbon (TOC) and nitrogen (TON) data was also included. Emamectin benzoate concentrations were not available for a subset of stations: 2C from the June survey, 1B from the August survey, and 1B and 1C from the January survey. A variable representing the ranked order of stations according to modelled emamectin benzoate concentrations (matching A, B, and C on the two transects, and the reference station) was included.

As environmental variables were not replicated within each survey/site combination, meiofaunal abundances were averaged within stations, prior to being square-root transformed. Stations for which emamectin benzoate concentrations were unavailable were omitted. Environmental variables were normalised and the Bio-env procedure was used to pick out subsets of environmental variables which provided the best match (defined here as the highest Spearman rank correlation between a biotic resemblance matrix and a euclidean distance matrix derived from the subset of normalised variables) with Bray-Curtis similarity matrices derived from the transformed nematode and copepod abundances. A new permutation test was employed to determine whether the observed highest correlations could have arisen by chance.

For both nematodes and copepods the best match was with the variable denoting distance from the cages chosen on the basis of modelled emamectin concentrations (nematodes, ρ = 0.79, p < 0.01; copepods, ρ = 0.81, p < 0.01). When omitting this variable, and using only variables measured at the stations, there was no significant relationship between variation in nematode community structure and measured environmental variables (best match ρ = 0.24, with a combination of %TOC, concentrations of emamectin, and PC2, p = 0.3). The relationship for copepods was significant (ρ = 0.45, p < 0.01) with a combination of PC2 (representing the sand-silt gradient) and measured sediment emamectin benzoate concentrations.

Ecological effects of sea lice medicines in Scottish sea lochs 156 of 286 Thus, there was evidence that deposition from the cages impacted nematode and copepod assemblages, and that a combination of changes in sediment composition and emamectin benzoate concentrations were related to the observed changes in copepod community structure at Loch Kishorn. However, these variables lie on the same strong organic enrichment gradient, and are co-linear, so it was not possible to determine which variable was most responsible for changes in community response.

6.8.4 Macrobenthos Grab sampling of macrobenthic fauna and collection and examination of sediment cores was carried out at 6 sampling stations on 27 June 2001, and 7 sampling stations on 21 August 2001 (see Figure 6.17 for locations) and September 2002. Analyses of the pre- treatment and the one month post-treatment samples and a comparative description of the differences between the macrofaunal populations found in the two sets of samples are given below.

6.8.4.1 Conditions in the sediments in the vicinity of Loch Kishorn fish farm During sublittoral sampling surveys the nature and appearance of the sediments and physico-chemical conditions were assessed. The sediments at most stations were of soft muddy sand with the exception of Station K2 where there was an admixture of clay (Table 15.6). In the pre-treatment survey the redox values (Eh) at 40 mm depth in the sediments were negative at stations K1 and K3 but positive at Station 2 (Table 15.6). In the post-treatment survey Eh values at 40 mm were negative at K1 and K2 but positive at K3. At station K7 sited at 500 m from the cages and sampled only in the post treatment survey the Eh values at 40 mm in the sediments were positive. Negative values are indicative of anoxic sedimentary conditions.

6.8.4.2 Distribution, abundance and faunal structure of the macrobenthos at the stations sampled during the pre- and post-treatment surveys. During the pre-treatment survey three stations sited at the NE corner of the cages and at 25 and 50 m NE from the cages were sampled. During the post treatment survey these stations were re-sampled and samples were also taken at a station 500 m SW from the cages (reference K7). Water depth was similar at all sampling stations (26-32 m).

Table 6.6 and Figure 6.23 summarise the major population statistics from the stations sampled and Table 6.7 lists the five most abundant taxa found at each station during each survey. These demonstrate that there is a very strong organic enrichment gradient in the populations emanating from the cage group on each occasion.

Station 1, adjacent to the cages, was densely populated by a suite of species commonly found in areas of very high organic enrichment. The most numerous of these were the polychaete Capitella and a group of large macrofaunal nematodes. High numbers of three other polychaetes associated with carbon enrichment, Malacoceros, Mediomastus and Phyllodoce also occurred in large numbers. The area was species poor with only 11-16 annelid and five mollusc taxa recorded, although the total abundance and biomass of organisms were very high. There was, however, a significant increase in the number of annelid taxa recorded in the post-treatment survey and a significant decline in annelid abundance (Table 6.7). This, together with the presence of echinoderms in the post- treatment survey that were absent in the pre-treatment survey, suggests a slight improvement in conditions close to the cages during the period between surveys.

Ecological effects of sea lice medicines in Scottish sea lochs 157 of 286 Table 6.6. Loch Kishorn, June and August 2001. Basic macrofaunal population statistics and indices for each of the four* sampling stations and for each sampling occasion.

(i) June 2001

Station number 1 2 3 7*

Total no. of taxa 17 81 109 Mean no. of taxa per sample (S) 9 ± 3 43 ± 6 55 ± 8 Total abundance (A) 20445 2060 2224 Mean Abundance (A) 4089 ± 2728 412 ± 132 445 ± 92 Total biomass (g) (B) 1454.75 24.14 42.30 Mean biomass per sample (g) 290.95 ± 136.38 4.83 ± 1.68 8.46 ± 2.33 A/S 1203 25 20 B/A (mg) 71 12 19

Hlog2 (range over 5 samples) 1.20 - 1.52 3.04 - 4.03 3.75 - 4.85 J (range over 5 samples) 0.39 - 0.51 0.55 - 0.72 0.66 - 0.82 ITI 0.1 - 3 19 - 35 45 - 57 *Station 7 (ref) not sampled as grab was lost

(ii) August 2001

Station number 1 2 3 7

Total no. of taxa 24 45 123 88 Mean no. of taxa per sample (S) 14 ± 7 19 ± 6 66 ± 6 44 ± 5 Total abundance (A) 20383 10804 3176 2494 Mean Abundance (A) 4077 ± 1962 2161 ± 841 635 ± 88 499 ± 75 Total biomass (g) (B) 491.03 28.14 51.39 352.57 Mean biomass per sample (g) 98.21 ± 107.68 5.63 ± 2.27 10.28 ± 2.87 70.51 ± 29.53 A/S 849 240 26 28 B/A (mg) 24 3 16 141

Hlog2 (range over 5 samples) 1.25 - 1.70 1.26 - 2.13 3.94 - 4.62 3.12 - 4.03 J (range over 5 samples) 0.34 - 0.48 0.29 - 0.45 0.65 - 0.76 0.60 - 0.72 ITI 0.01 - 2 0.01 - 5 47 - 53 67 - 76

Ecological effects of sea lice medicines in Scottish sea lochs 158 of 286 Figure 6.23. Loch Kishorn, June and August 2001. Pre- and post-Excis treatment surveys: mean and 1 S.D. for each major phyla at each station. Total no. of species, abundance and biomass nominally per 0.5 m2 of sediment surface area. (a) Annelida

Ecological effects of sea lice medicines in Scottish sea lochs 159 of 286 Figure 6.23 (cont.). (b) Crustacea

Ecological effects of sea lice medicines in Scottish sea lochs 160 of 286 Figure 6.23 (cont.) (c) Mollusca.

Ecological effects of sea lice medicines in Scottish sea lochs 161 of 286 Figure 6.23 (cont.) (d) Echinodermata.

Ecological effects of sea lice medicines in Scottish sea lochs 162 of 286 Table 6.7. Loch Kishorn, June and August 2001: the five most abundant taxa at each station. A, abundance 0.5 m-2.

(a) June 2001 Station 1 No. Station 2 No. Station 3 No. Capitella capitata 8639 Mediomastus fragilis 706 Mediomastus fragilis 671 Nematoda sp. 5518 Nematoda sp. 386 Scalibregma inflatum 171 Malacoceros fuliginosus 949 Scalibregma inflatum 186 Polycirrus plumosus 146 Mediomastus fragilis 222 Melinna palmate 117 Ophiura affinis 144 Phyllodoce mucosa 165 Mysella bidentata 50 Melinna palmata 132

(b) August 2001 Station 1 No. Station 2 No. Capitella capitata 9938 Capitella capitata 5472 Nematoda 8506 Nematoda 907 Malacoceros fuliginosus 845 Mediomastus fragilis 657 Mediomastus fragilis 635 Phyllodoce mucosa 164 Mysella bidentata 305 Scalibregma inflatum 94

Station 3 No. Station 7 No. Mediomastus fragilis 932 Amphiura filiformis 573 Ophiura affinis 273 Mysella bidentata 516 Scalibregma inflatum 265 Amphiura chiajei 270 Melinna palmata 129 Pholoe inornata 179 Phascolion strombus 108 Amphiura sp. 151

Ecological effects of sea lice medicines in Scottish sea lochs 163 of 286 Table 6.8. Loch Kishorn, June and August 2001: Basic macrofaunal population statistics for each major phyla, (a) Annelida, (b) Crustacea, (c) Mollusca, (d) Echinodermata, at each station. A, abundance 0.5 m-2.

June 2001 Number of taxa (S) Group Station 1 Station 2 Station 3 Annelids 9 56 69 Crustacea 0 6 9 Molluscs 5 5 9 Echinoderms 0 8 10

Abundance (A) Group Station 1 Station 2 Station 3 Annelids 13768 1452 1598 Crustacea 0 7 29 Molluscs 45 83 112 Echinoderms 0 98 363

Biomass (B(g)) Group Station 1 Station 2 Station 3 Annelids 181.44 22.9 26.3 Crustacea 0 0.04 0.07 Molluscs 3.21 0.4 11.2 Echinoderms 0 0.5 3.44

August 2001 Number of taxa (S) Group Station 1 Station 2 Station 3 Station 7 Annelids 18 27 75 48 Crustacea 0 2 14 12 Molluscs 5 5 12 13 Echinoderms 1 5 9 6

Abundance (A) Group Station 1 Station 2 Station 3 Station 7 Annelids 11519 9399 2044 637 Crustacea 0 2 117 57 Molluscs 345 92 108 653 Echinoderms 8 15 537 1063

Biomass (g) Group Station 1 Station 2 Station 3 Station 7 Annelids 117.80 26.85 19.56 8.65 Crustacea 0 0.01 0.21 0.07 Molluscs 1.18 0.13 1.02 22.09 Echinoderms 0.08 0.23 8.11 141.43

The populations at Stations 2 and 3, situated at 25 and 50 m from the cages, were much more diverse but with lower abundance and biomass. Eighty-one taxa were recorded at Station 2 in the first survey and 45 in the second. At Station 3, 109 taxa were recorded in the first survey and 123 were found in the second survey. However, both areas showed signs of disturbance from carbon enrichment, with populations dominated by polychaetes associated with enriched conditions, e.g. Mediomastus and Scalibregma. Macrofaunal nematodes were also numerous at Station 2. On the other hand crustaceans, molluscs and echinoderms were present in moderate numbers at these stations suggesting a lesser degree of disturbance than that found at Station 1. At Station 7 (reference), 500 m from the cages 88 taxa were recorded and the populations were dominated by amphiurid

Ecological effects of sea lice medicines in Scottish sea lochs 164 of 286 echinoderms and small bivalve molluscs characteristic of undisturbed sediments in the area (Table 15.4).

To assess changes in community diversity over the sampling period significant differences between K-dominance curves between groups of samples were determined (Figure 6.24). A clear gradient of increased diversity and reduced dominance is evident from Station 1 to 3, away from the cages, in June. Taking inter-replicate variability into account the difference in the curves from Stations 2 and 3 is marginally insignificant (p=0.063). There is no difference between the curves from June and August at Station 1 (p=0.611) or Station 3 (p=0.421), or between the reference Station in August and Station 2 in June (p=0.738).

Figure 6.24. K-dominance curves based on macrofaunal abundances from Loch Kishorn. In the key the first numeral indicates the station (1, 2, 3, equivalent to K1, K2 and K3, and R, the reference station K7) and the second numeral indicates the time of sampling (1, June 2001; 2, August 2001).

Changes in community structure over the sampling period were examined by ordinating inter-sample similarities in transformed macrofaunal abundance data using MDS analysis. The ordination plot of data from the two surveys is shown in Figure 6.25.

Ecological effects of sea lice medicines in Scottish sea lochs 165 of 286 Figure 6.25. MDS plot based on Bray-Curtis similarities calculated from square-root transformed macrofaunal abundances from Loch Kishorn. In the key the first numeral indicates the station (1, 2, 3, equivalent to K1, K2 and K3, and R, the Reference Station K7) and the second numeral indicates the time of sampling (1, June 2001; 2, August 2001).

This shows a clear gradient in community structure from Station 1 (nearest the cages) through 2 to 3 in June, and from 1 to the reference Station in August. All stations are significantly different from each other (Global R = 0.87, p<0.001). Pairwise comparisons are significant (p=0.008) for all, except Station 2 from August, Station 1 from June, p=0.016) and Station 1 from both surveys (p=0.222). There is obvious clustering of Station 2 with Station 3 in June, and with Station 1 in August.

6.8.4.3 Relationship with environmental variables Analysis using %TOC, %TON (Table 17.3), emamectin concentration (Table 6.5), and scores from the first two principal components of the sediment data (representing 84 % of the variation in sediment composition) from all available stations (environmental variables were missing from Station 2 in the August survey) showed that the best match with average macrofaunal community composition (ρ=0.875, p=0.023) was with a combination of organic enrichment variables (carbon and nitrogen) and sediment variables. It should be noted that the highly impacted stations were dominated by large nematodes, Ophyotrocha and Capitella. Removing these from the analyses had no effect on the overall conclusions in terms of MDS and K-dominance curves, but the best match with environmental variables (ρ=0.9, p=0.018) was with a combination of carbon, nitrogen and emamectin concentration.

6.8.4.4 Conclusions The presence of a strong carbon enrichment gradient in the proximity of the cages was obviously the most important influence on the distribution of benthic organisms in areas up to 50 m from the cages, masking any additional influence of treatment related effects. However the reduction in the number of crustacean and echinoderm taxa and their abundance at Station 2 in the second survey may suggest that this was a treatment related effect. These two groups are thought to be potentially most vulnerable to the action of the treatments. However, they are also known to be affected by organic enrichment, and it is not possible to separate the impact of one or other from the data obtained. The ordination analysis showed that all stations are distinct from the reference Station, although a severe impact was only apparent at Station 1 in June, and Stations 1 and 2 in August. There was no difference in assemblage structure at Station 1 between the two surveys. However a clear increase in impact was evident at Station 2, both in terms of changes in community

Ecological effects of sea lice medicines in Scottish sea lochs 166 of 286 composition and in terms of changes in dominance and diversity. This could be interpreted as evidence of the effects of the Slice treatment applied between the two surveys, but in the absence of correlative evidence it seems more likely that the difference results from small differences in the location of the Station (bearing in mind that Stations 1 to 3 were only tens of metres apart in both surveys) or patchiness of severe enrichment (faecal material and waste food) around the cages. There was no evidence that any changes could be related unequivocally to the use of Slice. The fact that chemical concentrations were included in the suite of environmental variables most closely matching changes in average macrofaunal composition (excluding the dominant nematodes and annelids) probably suggests that it was the distribution of food waste on the seabed that influenced observed changes in community structure.

6.8.5 Sublittoral settlement panels In order to concentrate effort at Loch Sunart, sponsors and collaborators agreed that there would be no sublittoral settlement arrays deployed in Loch Kishorn.

6.9 Littoral studies: intertidal panels Three sampling stations were established on the loch shore (Stations A, B and C). Figure 6.26 shows the positions of Stations B (approximately 750 m S of the cage group) and A (approximately 1000 m E of the cages). The reference Station C is approximately 750 m SW of the cage group.

Figure 6.26. Map showing positions of littoral sample stations at Loch Kishorn (). Grey rectangle represents cage group.

Slates were first installed at the shore stations in August and September 2001. No further inspections were carried out until October 2002, when Slate C5 was installed to replace Slate C3, which was missing at the Reference Station. All of the slates installed previously at Station B and Station A had remained in position since installation. At this time moderate settlements of S. balanoides were evident on three slates at each of Stations B and C, but the settlements on the slates at Station A were very light. The

Ecological effects of sea lice medicines in Scottish sea lochs 167 of 286 stations were visited again on 16 and 17 February 2003, to retrieve the slates and return them to the laboratory for detailed analyses of the settled populations of barnacles. There were very few barnacles remaining on the slates at Station A, and they were mainly on the pitted edges of the slates. A total of 16 individual S. balanoides were found, the shell diameters were measured on site and the animals were removed from the slates and examined to record the presence or absence of egg lamellae. Slates B1, B2, B3, C2 and C4 were retrieved and returned to the laboratory for detailed analyses of the barnacle populations. Very few barnacles had settled on Slates B4 and C1 and these populations were assessed on site. Population densities were also measured, using 2 cm2 or 5 cm2 quadrats, on previously cleared rock surfaces adjacent to Slates A1 and A2, below Slate B4 and between Slates C1 and C2, C2 and C5, and C5 and C4.

All slates removed from the site, were replaced, and the settlement and development of S. balanoides were checked in May and September 2003, and on 19 February 2004 all of the slates were removed from the site for detailed examination in the laboratory. As in February 2003, population densities were also measured, using 2 cm2 or 5 cm2 quadrats, on previously cleared rock surfaces adjacent to Slates A1 and A2, below Slate B4 and between Slates C1 and C6, C6 and C5, and C5 and C7.

6.9.1 Populations and development of barnacles, Semibalanus balanoides The results of the detailed laboratory analyses of populations on slates removed in February 2003 (cohort 2002) and February 2004 (cohort 2003) are summarised in Table 16.3 and Figure 6.27 and are discussed in this section, together with further data from examinations of populations on adjacent rock surfaces cleared previously.

Examination of barnacle shell diameters, body weights and valve weights revealed a slight tendency towards increase in animal size from 2003 to 2004 at all three stations. This is suggested by increases in the mean shell diameters and valve weights at Station B, in the shell diameters at Station A, and in valve weights at Station C (reference).

Ecological effects of sea lice medicines in Scottish sea lochs 168 of 286 Figure 6.27. Loch Kishorn, cohorts 2002 and 2003: summary of growth and development of the barnacle, Semibalanus balanoides on shore panels.

In 2003 the much higher population densities on the natural rock surfaces were considered to indicate preferential settlement on the more pitted, moister rock, than on the slates (see also Loch Sunart, Section 4.13.1). The counts made in 2004 showed a much denser settlement on the slates than in the previous year, while the population densities on the rock surfaces were again higher but reasonably similar in both years (Table 6.9). Clearly the differences between the population densities on the slates and those on the rocks are greatest when there are very low population densities on the slates.

Ecological effects of sea lice medicines in Scottish sea lochs 169 of 286 Table 6.9. Loch Kishorn, cohorts 2002 and 2003: estimated average population densities of Semibalanus balanoides on the slates and adjacent rock faces cleared the previous year. Cohort 2002 Cohort 2003 Station S. balanoides m-2 S. balanoides m-2 on slates on rock on slates on rock A 67 22800 15800 27000 B 14800 41300 20500 32500 C 650 30400 28100 27100 NB: Densities > 1000 have been rounded to nearest 100

During periodic visits to the Loch Kishorn site in May and September 2003 and February 2004 the growth of the populations on slates and the adjacent bedrock faces, which were cleared in February 2003, was examined. The most extensive data set was obtained for Station C and a summary of the results is contained in Table 6.10.

Table 6.10. Loch Kishorn cohort 2003: Littoral reference Station C. Seasonal variations in Semibalanus balanoides populations on slates and previously cleared adjacent rock surfaces. Populations on slates Populations on No. of Mean 95 % adjacent rocks slates (n) Confidence Limits 15 May 03 Total m-2 4 21600.0 20400.0 71000.0 % live 4 100.0 0.0 100.0 % cover 4 9.0 15.0 Max. diameter (mm) 4 1.5 0.0 1.5

28-Sep-03 Total m-2 4 17600.0 16200.0 44500.0 % live 4 100.0 1.0 100.0 % cover 4 34.0 32.0 Not noted Max. diameter (mm) 4 7.5 0.8 7.0

22 Feb 04 Total m-2 4 18400.0 16100.0 33100.0 % live 4 95.0 8.0 91.0 % cover 4 29.0 29.0 Max. diameter (mm) 4 8.6 0.7 8.0 NB: Densities > 1000 have been rounded to nearest 100

The results show that in May 2003, there were large numbers of recently settled individuals with very few dead (<1 % on slates, not recorded on rock) and a mean maximum diameter of 1.5 mm. By September, the population densities had decreased by approximately 20 % on slates and 60 % on bedrock, but still <1 % of animals were dead, and the mean maximum shell diameters were 7 - 7.5 mm. In February 2004 there was a slightly higher mortality (9 %) on the bedrock than on the slates (5 %), but overall the growth and mortality of the populations was very similar on both surfaces at this location, which is the most exposed of the three stations.

Mean weights of barnacle egg masses (Table 16.3) were higher in February 2004 than in February 2003 in individuals at Station B and Station C (reference). There were also some notable changes in the absence of fertilised egg masses, and the data are summarised in Table 6.11.

Ecological effects of sea lice medicines in Scottish sea lochs 170 of 286 Table 6.11. Loch Kishorn, cohorts 2002 and 2003: percent of individuals with no fertilised egg masses Cohort Station A Station B Station C 95 % 95 % Mean 95 % Mean% Slates Mean % Slates Slates Conf. Conf. % no Conf. no eggs (n) no eggs (n) (n) Limits Limits eggs Limits 2002 67 3 52 15 4 2 17 3 33 2003 13 3 6 6 4 3 32 4 7

In contrast to the data obtained at Loch Diabaig, most of the differences among the population means were statistically significant at or above the 95 % confidence level. The only exceptions were the differences between the mean value for Station C (reference) in 2002, and the means at Station B in 2002 and Station C in 2003.

The results show that in February 2003 the lowest frequency of occurrence of egg lamellae occurred in the population from the predicted intermediate impact Station A, while at Stations B and C the frequencies of occurrence of egg lamellae were higher. Almost all of the individuals without egg lamellae from the reference Station C had settled on a single slate (C2), while only 1 individual of 94 examined on Slates C1 and C4 lacked egg lamellae.

The results for February 2004, however (Table 6.11), clearly show that the highest proportion of individuals without egg masses were settled on the slates from the reference Station C, and 87 % of individuals from Station A contained egg masses. In addition 10 individuals settled on the bedrock surface cleared previously below Slate A3 were examined on site and all contained fertilised egg masses.

At the Loch Kishorn (Camas Doun) site peak food input was 222 t in March 2002 (see Section 6.2), but the site was fallowed in September 2002. By February 2003 the site contained 200 t and there were plans to increase stock biomass to 300-400 t later that winter.

All of the sampling stations at Loch Kishorn were well beyond the range of the predicted 105 day range for the deposition of emamectin (Figure 6.9), although a trace of emamectin was found in the sediments in January 2002 from the sublittoral reference station situated some 500 m SW of the cages.

The changes in the frequency of occurrence of egg masses in the populations at Loch Kishorn are difficult to interpret, and may indicate that natural events have had a greater influence on egg mass production than the fish farm activities at this site. Some natural and anthropogenic factors, which may possibly have influenced the frequency of egg distribution in the various sea lochs studied in this project, are compared in Section 8 (Analysis across sites).

Ecological effects of sea lice medicines in Scottish sea lochs 171 of 286 7 Loch Craignish 7.1 Site description Loch Craignish is a relatively short sea loch on the Scottish west coast, 8.8 km long on a NNE-SSW axis and opening into the Sound of Jura (Figure 7.1). There are a total of five basins with an outer sill 7 m deep (Edwards and Sharples, 1986). It is relatively shallow (mean low water depth = 15.5 m, max. depth = 59 m) and with a flushing time of five days. Tidal range is a moderate 2.1 m and the intertidal represents 8 % of the loch area. The watershed is 73 km2 and the fresh water/tidal flow ratio is low (1:175).

Near the entrance to Loch Craignish between Garbh Rèisa and Craignish Point (water depth = 5.5 m) there is a strong tidal stream called the Dorus Mòr. This race runs at 4 m s-1 on both the ebb and flood spring tide. The dominant hydrographic feature of the area is the Corryvreckan, a very large whirlpool between the Isles of Jura and Scarba. The west going stream (ebb) from Loch Craignish sets towards this (ebb begins + 0330 h Oban) (West Coast Pilot, 1980).

No published data on the physical environment were found for Loch Craignish. However, an unpublished 21 d hydrographic record east of Liath-sgeir Mhor and some 2 km to the south of the study area exists (moored in 30 m, 56º 07.14 N, 5º 33.96 W). This deployment recorded maxima in excess of 50 cm s-1 and a residual current speed of 7 cm s-1 SSW (Ezzi et al., 1993). Thus, these data suggest the current at the entrance to the loch at this location is generally strongly tidal, oscillating semi-diurnally and the water column well-mixed.

The farm site lies on the eastern side of the loch in the channel between Eilean Righ and the mainland, north of the jetty at Port na Mòine (Figure 7.1). The site is moderately sheltered from wind and wave effects.

Ecological effects of sea lice medicines in Scottish sea lochs 172 of 286 A) B)

Figure 7.1 A) Location of Loch Craignish on the Scottish west coast and B) Loch Craignish and location of study area (inset box). The grid is OS.

Ecological effects of sea lice medicines in Scottish sea lochs 173 of 286 7.2 Fish farm history, cage positioning, biomass, and medicine use The earliest production data supplied by Lakeland Marine Farms Ltd. for Loch Craignish start in 1999. The farm site operated from Port na Mòine was split during the present study into two cage groups. The southern group at Port na Mòine (56º 09.300 N, 5º 32.100 W) was the group under study, whereas the northern group was located near Port a’ Bheachan (56º 09.690N, 5º 31.550W).

The Port na Mòine group’s biomass figures were small by the industry’s current standards (1999-2002 = 387, 708, 261, 417 tonnes; pers. comm., Colin Blair, Lakeland Marine). The Port a’ Bheachan group’s production was even more modest (1999-2002 = 291, 261, 104 and 77 tonnes). Sea lice medicine usage at the site was also low over the study duration (Table 7.1). Management and scientific events for both sites are shown in Table 7.2 and bathymetry and sampling station locations in Figure 7.2.

Table 7.1. Sea lice medicine use at Port na Mòine (study site) and Port a’ Bheachan, Loch Craignish (source: SEPA). Group Date Treatment Amount active Active ingredient ingredient Port na Mòine Aug. 2000 Salartect 10800 l hydrogen peroxide (study site) May 2001 Salartect 9000 l hydrogen peroxide Aug. 2001 Excis 2.17 l cypermethrin

Port a’ Aug. 2001 Excis 0.43 l cypermethrin Bheachan May 2002 Excis 3.44 l cypermethrin Sep. 2003 Salmosan 480 g azamethiphos

Ecological effects of sea lice medicines in Scottish sea lochs 174 of 286 A)

B)

Figure 7.2. A) Positions of cages groups (grey rectangles) at Loch Craignish (study group Port na Mòine to the south, Port a’ Bheachan to the north) with sampling stations and B) Bathymethry from RoxAnn survey. Littoral stations (), zooplankton stations (), combined meio and macrofaunal stations ().

Ecological effects of sea lice medicines in Scottish sea lochs 175 of 286 Table 7.2. Timeline of sampling and management events at Loch Craignish fish farm. Figs. in kg are feed input; Aza = Salmosan, Ema = Slice, Cyper = Excis, H2O2 = Salartect Key: S: Sampling D: Deployed R: Retrieved Zoo/phytoplankton (S) 17 - Macrofauna (S) 17 - Meiofauna (S) Scientific events Day - Event 5 - Prelim shore survey 9 - Current meters (D) 10 - RoxAnn (S) 25 - Current meters (R) 1 - Shore survey (D) 5 - Drogues (D, R) 13 - Shoreslate (S)

2000 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2 O 2 H events (Mòine) Management

Zoo/phytoplankton (S) 8 - Sublittoral arrays (S, D) 17 - Shore slates (S) Scientific events Day - Event 17 - Sublittoral arrays (D) 8 - Shore slates (S) 24 - Shore slates (S) 18 - Macrofauna (S) 18 - Sublittoral arrays (S)

2001 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec 2 O 2 H Cyper Cyper events (Mòine) (Mòine) (Bheacan) Management

Ecological effects of sea lice medicines in Scottish sea lochs 176 of 286 Table 7.2 (cont.) Key: S: Sampling D: Deployed R: Retrieved Scientific events Day - Event 28 - Shore slates (S,R)

2002 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Cyper events (Bheacan) Management

Ecological effects of sea lice medicines in Scottish sea lochs 177 of 286 7.3 Hydrography Surface and mid-water flows measured for the Port na Mòine site had a strong SSW residual current towards the Sound of Jura (Figure 7.3). Mean and maximum speeds measured were also relatively high with speeds of approximately 6 and 8 cm s-1 respectively. This site was also dominated by freshwater flow at the surface in a SSW (seaward) direction, often apparent during periods of rainfall. In general, the current data were consistent with data previously measured near the loch entrance.

DGPS drifter surveys were undertaken during a quiescent period around a neap tide and during a dry period. As a result, very low dispersion coefficients of 0.03 and 0.09 m2 s-1 were calculated for kx and ky respectively. Although these were worst case scenario conditions in terms of dispersion potential, dispersion coefficients were not expected to be much greater than 0.1 m2 s-1 during normal conditions. The value of 0.1 m2 s-1 is used by SEPA as a default value in Slice consent modelling studies of all Scottish sites in the absence of site specific data (SEPA, 2003a).

Figure 7.3. Time series plots of current speed and vector displacement for Loch Craignish (sampling interval 10 min, total length 16 d). (A) Near-surface and (B) mid- water speed, (C) near- surface and mid-water displacement.

Ecological effects of sea lice medicines in Scottish sea lochs 178 of 286 7.4 Zooplankton Fortnightly sampling at Loch Craignish began in August 2000 and ended in late September 2001. During this period, the farm treated for sea lice with Salartect in August 2000 and May 2001, and Excis in August 2001. Zooplankton samples were collected at four stations (Figure 7.4) and species trends in abundance during the sampling period are shown for stations B and D only.

Figure 7.4. Zooplankton sampling station locations at Loch Craignish. Station A ≈ 1500 m SW of the cage groups, Station B is 50 m SW, Station D = 200 m NE, Station C ≈ 1200 m NE. Cage groups are marked by grey rectangles.

Total zooplankton abundance was highest during August and September 2000 with most species showing a major peak in abundance during this time (Figure 7.5 and Figure 7.6). In 2001, total zooplankton abundance was considerably lower, with much smaller peaks in abundance in May and September. Copepod nauplii contributed most to zooplankton densities in September 2000, followed by the cyclopoid copepod Oithona spp., the cladoceran Evadne, and the calanoid copepods Centropages, Temora, Acartia and Paracalanus (Figure 7.6). The cladoceran Evadne also exhibited a second peak in abundance in May/June 2001 and planktonic barnacle stages (Balanus; Figure 7.5) peaked in February/March 2001.

Ecological effects of sea lice medicines in Scottish sea lochs 179 of 286 Figure 7.5. Abundance (number m-3) of zooplankton at Loch Craignish during the long- term monitoring program August 2000 to September 2001. Sea lice treatments are indicated by arrows on the uppermost graphs. Note: y-axis scales are unequal.

Ecological effects of sea lice medicines in Scottish sea lochs 180 of 286 Figure 7.6. Abundance (number m-3) of total Acartia, Temora, Paracalanus and Centropages at Loch Craignish during the long-term monitoring program August 2000 to September 2001. Sea lice treatments are indicated by arrows on the uppermost graphs. Note: y-axis scales are unequal.

Zooplankton abundance was generally similar at the two sample stations and followed similar seasonal patterns to other study sites (see Section 8.4). As a result, the observed changes in zooplankton community composition and abundance were as expected and the result of natural seasonal changes, advection and life history. The zooplankton community was not affected by the predominant use of Salartect at this site.

7.5 Phytoplankton Phytoplankton, nutrient, and salinity samples were collected at Loch Craignish between June 2000 and September 2001, from sample station B (Figure 7.4). Salinity was very constant at 33.53 ± 0.37 during the sampling period. Nutrient concentrations from May to September 2001 were relatively low (e.g. 0.9 µM ToxN, 1.8 µM silicate, 0.3 µM phosphate and 0.9 µM ammonium) (Appendix III - Phytoplankton).

All phytoplankton species observed at Craignish have been previously reported from UK coastal waters (e.g. SAHFOS, 2001; McKinney et al. 1997; Dodge, 1995; 1982) and exhibited normal seasonal variability and biomass ranges (see Appendix III -

Ecological effects of sea lice medicines in Scottish sea lochs 181 of 286 Phytoplankton). In summer 2000, phytoplankton densities fluctuated between 1 x 106 and 2 x 106 cells l-1 (Figure 7.7) and the gross contributions of the four main types of phytoplankton to the total community are shown in Figure 7.8. The phytoplankton community was relatively species-rich and abundance of individual species generally did not exceed 15 x 104 cells l-1. A small bloom of Chaetoceros sp. (probably C. socialis) peaked at 45.9 x 104 cells l-1 during August 2000.

During the winter months (November 2000 to March 2001), low phytoplankton biomass (total ≤ 27 x 104 cells l-1) coincided with relatively high nutrient concentrations (typically 7.2 µM ToxN, 6.1 µM silicate, 0.7 µM phosphate and 1.2 µM ammonium - see Appendix III - Phytoplankton. Microflagellates (< 15 µm size) and cryptophytes comprised 79 % ± 7 % of the winter phytoplankton community, but throughout the winter there were also a variety of low-biomass populations (< 9 x 103 cells l-1) of diatoms (e.g. Skeletonema costatum, Thalassiosira sp., Pseudo-nitzschia sp.) and dinoflagellates (e.g. Gonyaulax sp., Gymnodinium sp., Gyrodinium sp.), as well as ciliates and cysts/resting stages (Figure 7.8).

Throughout most of May and June 2001 there was a significant and prolonged bloom of Chaetoceros sp. (mainly C. socialis-type, peaking at 2.8 x 106 cells l-1), combined with a small bloom of a larger Chaetoceros sp. (30 x 104 cells l-1; 20-30 µm). Numbers of Chaetoceros sp. remained relatively high (10 x 104 to 20 x 104 cells l-1) for the remainder of the summer.

Figure 7.7. Total phytoplankton cell abundance (cells l-1) and dissolved inorganic ToxN (nitrate and nitrite, µM) at Loch Craignish.

Ecological effects of sea lice medicines in Scottish sea lochs 182 of 286 Figure 7.8. Gross phytoplankton community composition at Loch Craignish. “Others” includes other flagellates, silicoflagellates, cryptophytes, ciliates, tintinnids and resting stages/cysts.

The phytoplankton community at Loch Craignish exhibited classical seasonal population dynamics and contained species routinely observed in UK coastal waters. No correlation with sea lice treatments at the fish farm could be ascertained.

Between June 2000 and October 2001, the zooplankton and phytoplankton communities at Loch Craignish exhibited normal seasonal cycles with respect to species composition and abundance. An assessment of the degree to which coupling existed between these communities was hindered by the incomplete phytoplankton time series with breaks in autumn 2000 and spring 2001 (Figure 7.9). The significant peak in zooplankton abundance in August 2000 was not repeated during summer 2001, despite a similar phytoplankton standing stock in 2001, and may have been in response to a spring phytoplankton bloom which was not recorded in 2000. While the increase in zooplankton abundance in May 2001 could have been maintained by grazing on the bloom of the diatom Chaetoceros sp., it is likely to have been stimulated initially by an earlier spring bloom which was not recorded.

Three sea lice treatment events occurred at Loch Craignish during the plankton sampling programme (Salartect in August 2000 and May 2001, and Excis in August 2001). Abundances of both phytoplankton and zooplankton increased immediately after each of the 2001 treatment events. It is unlikely the Salartect treatment in 2000 contributed to the subsequent decline in zooplankton abundance, which was undergoing a natural seasonal decline in late summer.

Ecological effects of sea lice medicines in Scottish sea lochs 183 of 286 Figure 7.9. Total zooplankton (animals m-3) and phytoplankton abundance (cells l-1) from integrated water column samples at Loch Craignish between June 2000 and October 2001. Total zooplankton is presented as the average of two sample stations (B and D, ± S.E.). Sea lice treatments are indicated by arrows; Salartect (HP) and Excis (C). Note different y axes.

7.6 Meiofauna and macrofauna Sublittoral sampling at Loch Craignish was undertaken in October 2000 and December 2001. However, as detailed in site selection philosophy (Section 3.1), with the agreement of the sponsors, effort was transferred to Loch Kishorn due to the use of Slice at the fish farm. As a result, the October 2000 samples were analysed for meiofauna, but no samples were analysed for macrofauna. Five samples were collected from each of three stations in the survey (Figure 7.10). As the amount of sediment collected was large (approximately 360 cm3 on average) compared with other surveys, a sub-sample was taken for meiofauna analysis.

Ecological effects of sea lice medicines in Scottish sea lochs 184 of 286 Figure 7.10. Location of meiofaunal sample stations (♦) at Loch Craignish. Cage groups are represented by grey rectangles.

Nematodes from meiofauna samples were enumerated and identified and multivariate analyses showed clear and significant differences in community structure between stations. A gradient existed in these analyses from Station LC1 through to Station LC3, reflecting distances from the cage group. Dominance/diversity structure and species composition of the assemblages also indicated a gradient existed, with the most dominated and least diverse nematode assemblage at LC1. The gradient in meiofauna community structure suggested an organic enrichment gradient and disturbance associated with fish farm related activity.

7.7 Sublittoral settlement on suspended panels Sublittoral arrays of slate panels were deployed at three stations in Loch Craignish on 18 January 2001, LCI (predicted high impact), LC2 (intermediate) and LC3 (reference) (Figure 7.10). After inspections on June and December 2001, overwhelming settlements

Ecological effects of sea lice medicines in Scottish sea lochs 185 of 286 of Ascidiella aspersa (ascidians) were expected to be a regular event in Loch Cragnish, so effort was concentrated on studies in Loch Sunart.

The arrays were first raised on 8 June 2001 (Table 7.3) for inspection and unfortunately the upper sets of panels at LC2 and LC3 were missing. These panels were the most vulnerable to accidental damage from local boat traffic including yachts, active commercial creel fishing and fish farm boats. Also missing were the middle set of panels at LC3 and three of the lower set of panels at LC1.

Table 7.3. Loch Craignish: summary of biota settled on the sublittoral arrays at Stations LC1-3, inspected 8 June 2001 (deployed 18 January 2001). Station/Impact LC1 H LC2 I LC3 R m m m Level U M L U M L U M L Balanus crenatus 4 1 1 1 1 1 Obelia longissima 3 5 5 5 Mytilus edulis Onchidoris bilamellata Ascidiella aspersa (juv.) 4 5 5 4 Ciona intestinalis 5 Corella parallelogramma Pomatoceros triqueter Hydroides norvegica Electra pilosa 5 Asterias rubens 5 5 Diplosoma listerianum 5 Anomia ephippium Lepidopleurus asellus Aequipecten opercularis Antedon bifida Sabella pavonina Eupolymnia nebulosa Gammaridea 5 Laminaria saccharina 5 Desmarestia aculeata 4 Petalonia fascia 4 Cutleria multifida 6 Ceramium rubrum Brown organic detritus 3 *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: 1 = S-Superabundant Level: Predicted impact: 2 = A-Abundant U = Upper, ~2 m below surface H = High 3 = C-Common M = Mid, Midwater I= Intermediate 4 = F-Frequent L = Lower, 2 m above seabed R = Reference 5 = O-Occasional 6 = R-Rare Missingm: panels L1-L3 at LC1, upper set of panels at LC2, upper and middle sets - all replaced at LC3

Heavy settlements of a few common fouling organisms had occurred on most of the slates and all of the ropes, buoys and spars used in each assemblage. The most abundant biota on the slates were barnacles Balanus crenatus (up to 90 % cover), young ascidians A. aspersa (frequent) and the hydroid Obelia longissima (frequent). The few other invertebrate species listed in Table 7.3 occurred only occasionally, with Asterias rubens (common starfish) feeding on the barnacles as expected. The expected annual settlement of mussels Mytilus edulis had not yet started.

The biota on the upper set of panels at Station LC1 (H) situated 150 m SW of the fish cages were interestingly different to other sets of panels. As well as having much smaller settlements of B. crenatus (only frequent), they had good growths of several species of

Ecological effects of sea lice medicines in Scottish sea lochs 186 of 286 brown seaweeds including Laminaria saccharina, Desmarestia aculeata (frequent and up to 70 cm long) and rare Cutleria multifida. Additionally, quantities of brown organic detritus had settled on the panels which probably originated from the nearby fish cages.

The next inspection of the arrays 18 December 2001 revealed array LC1 and the upper set of panels at LC3 missing. A selection of seven slates from the remaining arrays LC2 and LC3 was brought back to the laboratory for closer study (Table 7.4).

Table 7.4. Loch Craignish: summary of biota settled on the sublittoral arrays at Stations LC1-3, inspected 18 December 2001 (all deployed 18 January 2001 except LC2 U which was deployed on 8 June 2001). Station/Impact LC1 H LC2 I LC3 R Level Um Mm Lm U M L Um M L Balanus crenatus 1(d) 1(d) 6 1(d) Obelia longissima 5 4 5 Mytilus edulis 1 5 5 Onchidoris bilamellata 5 Ascidiella aspersa 1 1 1 1 1 Ciona intestinalis 5 5 Corella parallelogramma 6 Pomatoceros triqueter 4 1 5 Hydroides norvegica 5 Electra pilosa 5 Asterias rubens 5 Diplosoma listerianum Anomia ephippium 5 Lepidopleurus asellus Aequipecten opercularis 5 Antedon bifida 5 6 5 Sabella pavonina 5 5 Eupolymnia nebulosa 5 4 3 4 Gammaridea Laminaria saccharina Desmarestia aculeata Petalonia fascia Cutleria multifida Ceramium rubrum 6 Dark organic detritus 4 4 4 5 4 *The abundances of species settled are ranked according to MNCR SACFOR scales as follows: 1 = S-Superabundant Level: Predicted impact: 2 = A-Abundant U = Upper, ~2 m below surface H = High 3 = C-Common M = Mid, Midwater I= Intermediate 4 = F-Frequent L = Lower, 2 m above seabed R = Reference 5 = O-Occasional 6 = R-Rare Missingm: array LC1 and upper set of panels at LC3; Balanus crenatus 1(d) = Superabundant (dead) but strong barnacle shells remained firmly attached to the panels

The major feature on all panels (including those deployed only in June) was a blanketing 100 % (plus) cover, of superabundant ascidians, predominantly A. aspersa with occasional Ciona intestinalis. On all of the older panels that had received spring settlements of B. crenatus (up to 100 % cover), the barnacles were still present beneath the ascidian cover. However, these were mostly empty shells eaten by predators Onchidoris bilamellata (sea slug) and A. rubens. In addition to cover by the dominant A. aspersa, the upper set of panels on station LC2 (deployed on 8 June) also had superabundant mussels M. edulis, many of which had also been eaten by predators. During 2001, the number of invertebrate taxa recorded increased from 8 in June to 16 in December.

Ecological effects of sea lice medicines in Scottish sea lochs 187 of 286 Many of the A. aspersa had developed long attachment stalks so that high densities could be achieved and appeared as rounded, heavy bunches on the slates. On retrieval of the heavily loaded slates, many of these bunches peeled off including dead barnacle shells to which they were attached.

Beneath the dense ascidian cover on the surfaces of the slates and dead barnacles, a considerable amount of dark coloured muddy detritus had accumulated. This was probably composed of mostly ascidian faecal material. In this detritus, polychaete tube worms Eupolymnia nebulosa (frequent) and Sabella pavonina (occasional) were present at all three depths.

Examination of these results for both June and December (2001) indicates that invertebrate populations were very similar at all three stations for panels that had been deployed for the same periods of time.

7.8 Littoral site assessment - shore fauna and flora Four sampling stations were established along the loch shore (Figure 4.41). Station A was selected as a reference station approximately 1700 m NNE from the Port na Mòine site. Station B was some 250 m NE of this site, but could have possibly been impacted by operations at the Port a’ Bheachan site to the north of the Port na Mòine site. Station C was some 100 m SE of the Port na Mòine site and was the predicted high impact station. A further reference Station D was situated some 1300 m SSW of this site.

The site was moderately sheltered with a relatively small spring tide range (2.2 m). In general, the rocky shores are fairly steep and mid-shore zone dominance by the barnacle Semibalanus balanoides was well developed. The individual barnacles were large and likely to be long-lived. Their main predator the dog whelk Nucella lapillus, was absent from most of the loch. In the more sheltered inner parts of the loch (Station A), brown fucoid algae dominate the mid-littoral zone so that patches of rock with barnacles extensive enough to accommodate four slates were difficult to find.

Ecological effects of sea lice medicines in Scottish sea lochs 188 of 286 Figure 7.11. Location of littoral sample stations at Loch Craignish (). Grey rectangle represents Port na Mòine site to the south (main study group) and Port a’ Bheachan site to the north.

The four shore stations established late in 2000 were revisited and examined on 8 February, 24 May and 17 October 2001. By 8 February there were thin films of algal growth on several slates from Stations B (possible impact station from northerly cage group) and D (southerly reference station). The growth was greatest on Slate D4. Samples of the algae on Slates B2, B3, B4, D3 and D4 were returned to the laboratory and identified.

By 24 May there had been a moderate settlement of S. balanoides on the slates at Stations C and D and a lighter settlement at Station B (possible high impact). Large areas of the green alga Enteromorpha compressa were also present on Slates B3 and B4. Only a single S. balanoides had settled at Station A (northerly reference station).

Ecological effects of sea lice medicines in Scottish sea lochs 189 of 286 On re-inspection on 17 October there were very few barnacles visible on the pitted edges of slates at Station A and between 4 and 95 individuals on Station B slates. On three of the remaining slates at Station C, 35-60 % cover was observed compared to 10-20 % cover on Station D slates. Microscopic examinations of scrapes of these primary colonising micro-algae showed varying mixtures of Cyanobacteria (mainly Entophysalis deusta), littoral diatoms (Acnanthes, Melosira, Navicula, etc.) and juvenile annual green and brown algae (Enteromorpha compressa, Ulothrix flacca, Pilayella littoralis).

On 28 February 2002, all shore slates were removed for examination of barnacles on 11 slates (B1-B4, C1, C2 and C4 and D1-D4) for shell diameter, weights of opercular valves, total body parts and egg lamellae (up to 50 individuals per slate) (Figure 7.12).

There was high variability among and within the barnacle populations recovered from each station (Figure 7.12). There were some statistically significant (at or above the 95 % confidence level) differences in size related measurements, indicating that individuals from Station C (predicted high impact) were slightly larger than those from Stations B and D. These results are not, however, considered to have biological significance. Egg lamellae were present in a large majority of the individuals, 99 % of those from reference Station D, 100% from Station C (predicted high impact) and 83 % from Station B (possible high impact from northerly cage group). In general, individuals were larger than those found at the much more exposed Loch Diabaig site and were reasonably similar in size to those found at Loch Sunart,

The stock biomass held at the sites was small by industry standards (2001 data: 261 t at Port na Mòine site, 104 t at Port a’ Bheachan) and sea lice treatments were also few. In conclusion, there was no evidence to suggest that growth and reproductive development of S. balanoides at Loch Craignish in 2001-02 were influenced by fish farm related activity.

Ecological effects of sea lice medicines in Scottish sea lochs 190 of 286 Figure 7.12. Growth and development of barnacles, Semibalanus balanoides on shore settlement panels at Loch Craignish, 2001 cohort. Error bars are 95 % confidence levels.

Ecological effects of sea lice medicines in Scottish sea lochs 191 of 286 8 Analysis across sites In this report, analysis of the ecological effects of sea lice treatments has concentrated on individual sea lochs and the components of the project in identifying any effects. To examine whether any global trends are identifiable, across-site analysis for each project component is presented.

8.1 Hydrography Current speed and direction summary statistics are shown for the four sites in Table 8.1. Lochs Sunart and Kishorn were the most dynamic in terms of surface current, and the highest dispersion was measured at these two lochs with drifter surveys. Surface currents were more quiescent at Lochs Craignish and Loch Diabaig. Lochs Sunart and Kishorn also showed a decrease in current speed with depth, resulting from current shear in the water column. This was not obvious at Loch Craignish, where the mean mid-water depth current speed was greater than at the surface. The four lochs were ranked in order of dispersion characteristics as follows (most dispersive first):

Surface water Loch Kishorn > Loch Sunart > Loch Diabaig > Loch Craignish

Near-bed water Loch Kishorn > Loch Craignish > Loch Sunart > Loch Diabaig

The separation of these rankings into surface and near-bed is important, as the mechanism for dispersion will affect pollutant behaviour in the environment. Dispersion in the surface layers is an important mechanism for dilution of dissolved chemicals, whereas near-bed currents generate erosion/resuspension events causing the redistribution of particulate bound chemicals. These ranking are only tentative as the effect of different weather conditions on dispersion potential was not assessed. For example had drifter surveys been undertaken in Loch Diabaig during rough weather, increased wave action would have increased the dispersion ranking of this site.

Table 8.1 Hydrography summary statistics for the four sites, primarily from current meter surveys. Data are 10 min sampling intervals with the exception of Loch Diabaig (5 min). Meter Max Speed Mean Speed Residual Residual Speed Length Site position (cm s-1) (cm s-1) Direction (cm s-1) (d) Loch Sunart Surface 32.0 8.1 245 3.0 17 Loch Sunart Mid 17.5 3.2 253 1.3 17 Loch Diabaiga Surface 9.8 2.8 - - 0.5 Loch Kishorn Surface 43.7 9.2 223 3.6 37 Bed 18.3 3.8 187 1.2 37 Loch Craignish Surface 17.8 3.7 243 1.8 16 Mid 16.9 5.3 234 4.2 16 a Data from drifter surveys

Mechanisms of resuspension are recognised as important when consenting sites for sea lice treatment medicines (SEPA, 2003a). If models predict significant resuspension and export of the released chemical from an area, then a subjective assessment is made as to the ultimate fate of the chemical. Erosion and resuspension causes particles to be transported at near-bed current speeds until the current drops below the deposition speed, resulting in net loss of material from an area (Cromey et al., 2002b). Bathymetry, topography and residual current speeds are used in further assessments when large amounts of exported chemical are predicted due to resuspension. In particular,

Ecological effects of sea lice medicines in Scottish sea lochs 192 of 286 redeposition in areas of reduced current flow and the dispersion potential of the receiving area are assessed.

Dispersion coefficients measured during this project were compared with data collected from other areas using the same methods (Table 8.2). All sites, with the exception of Loch Craignish, exceeded the SEPA management model coefficient value (SEPA, 2003a). Generally, low dispersion was measured at all sites, although dispersion in the Sound of Mull far exceeded those measured at the four loch sites, but this is not unexpected given the tidal nature of the Sound. Wind driven sites in the Mediterranean have similar levels of dispersion (Table 8.2).

Hydrographic data used for sea lice medicine consent modelling only provide snap shots of conditions. For example, drifter studies are only undertaken when conditions are below Beaufort Scale 5 (BF 5), so dispersion potential is not evaluated during windier periods. Consequently, Loch Diabaig was ranked as having quite low dispersion by drifters, despite it being an exposed site subject to large swells during windy periods. Extensive current meter deployments were not undertaken at this site so hydrographic conditions during these windy periods were unknown.

Table 8.2. Horizontal dispersion coefficients (Kx and Ky) from DGPS drifting buoy surveys (BF = Beaufort Scale). All sites were tidal with the exception of the inland freshwater loch and eastern Mediterranean sites, which are primarily wind-driven sites. 2 -1 2 -1 General site description Run length kx (m s ) ky (m s ) (h) Loch Sunart (BF 5) 2.7 0.68 0.01 Loch Diabaig 3.3 0.10 0.31 Loch Kishorn 4.2 0.28 0.11 Loch Craignish 2.9 0.03 0.09 Sea loch narrows above sill 1.6 0.02 0.69 Sound of Mull (strait) 1.7 0.25 0.19 Sound of Mull (inshore eddy) 2.1 0.79 0.32 Sound of Mull (main channel) 1.6 14.80 0.46 Inland fresh water locha - calm (BF 2) 1.1 0.02 0.00 - windy (BF 4) 1.0 0.07 0.12 Eastern Mediterranean - Simi 2.5 0.00 0.03 Eastern Mediterranean - Chios 2.9 0.17 0.00 Eastern Mediterranean - Korinthiakos 1.4 0.42 0.15 SEPA management model value NA 0.10 0.10 (theoretical) a Primarily wind driven or non-tidal

8.2 Predicted sediment emamectin benzoate concentrations - Slice modelling The DEPOMOD modelling suite was developed to model sediment emamectin benzoate concentrations following Slice treatments. The model requires input of emamectin benzoate chemical properties and fish excretion rates of the compound. The excretion of Slice is complex; a seven day treatment period is modelled initially followed by several 36 d periods of excretion post-treatment. Decay of emamectin benzoate uses a first order degradation model with rate constants supplied by the manufacturer.

Given that emamectin benzoate emanating from fish farms is bound to faecal and uneaten feed material, it is reasonable to assume that it should have the same environmental fate

Ecological effects of sea lice medicines in Scottish sea lochs 193 of 286 as the particulate material it is bound to. Validation of the particle tracking and resuspension components of the models detailed by Cromey et al. (2002) provided the basis for modelling these wastes and associated bound materials. Validation studies were undertaken at Loch Duich to compare modelled and measured emamectin benzoate concentrations (www.sepa.or.uk/aquaculture/modelling). Reasonable agreement was obtained on days 9, 32, 125 and 370 post-treatment, allowing additional confidence to be established in the resuspension model predictions. Particle tracking components of the model have been developed and validated for different environments and farmed species (e.g. wind driven eastern Mediterranean Sea containing farmed sea bass/bream). Given that the model has been validated, possible explanations for reduced model performance during this project can be grouped into the following categories: (1) inaccurate or insufficient representation of emamectin benzoate behaviour/properties in the model; (2) inaccurate model input data, particularly use of default data; (3) unreliable or insufficient measured data to test the model across a range of temporal/spatial scales, as discussed below.

8.2.1 Emamectin benzoate behaviour/properties The model most likely represents the excretion and decay of emamectin benzoate appropriately as it uses the best available information supplied by the manufacturer of Slice. However, other critical properties of emamectin benzoate may be missing in the model, such as the mechanism for emamectin benzoate loss over time, or dissolution of the waste material over time resulting in separation of emamectin benzoate from the particulate material. Evidence does exist for dissagregation (Stewart and Grant, 2002) and nutrient leaching (Chen et al., 2003) from uneaten feed pellets. This potential pathway for dispersion of emamectin benzoate is not currently incorporated in the model.

Under-prediction of sediment concentrations during early sampling events and over- prediction during later events at Loch Kishorn might be explained by consolidation and erosion mechanisms in the DEPOMOD resuspension model. Given the reasonably high near-bed current at this site, numerous resuspension events were modelled. The consolidation component of the model assumes that a particle remains in the resuspension layer for a finite time (four days), after which it is resuspended. In development of the DEPOMOD benthic response model, this parameter was validated by optimising the relationship between predicted solids flux and benthic effect. The time a particle remains in the resuspension layer reflects bioturbation rates, which are dependent on infaunal benthic community composition, sediment type and quality/quantity of food available. All of which vary significantly below fish farm cages over small spatial scales of tens of metres, similar to the distances between sampling stations. Bioturbation enhances vertical flux of waste material and associated emamectin benzoate, resulting in its burial or reintroduction into the resuspension layer. Bioturbation also decreases particle size, increasing the probability of dissolution of the waste components. The overall effect of these mechanisms would be greater dispersion of emamectin benzoate.

As an adjunct to the main study on the ecological effects of sea lice chemicals, a small study was undertaken at the Marine Harvest Gorsten site to assess the small scale horizontal and vertical sediment distribution of emamectin benzoate (see Appendix II - Chemistry) (Emamectin benzoate in sediment: Gorsten Experiment). Emamectin was measured at sediment depths down to 6 cm with peak concentrations at 2-4 cm depth and this burial below the sediment surface is likely to be caused by bioturbation. These measurements emphasize the importance of including bioturbation when modelling the fate pathway of this chemical. In the modelling studies presented for Lochs Kishorn and Sunart, a 5 cm sediment depth is utilised over which the chemical is mixed evenly.

Ecological effects of sea lice medicines in Scottish sea lochs 194 of 286 Sufficient bed shear stress above the erosion threshold results in erosion of bed particles. This is typically included as a constant parameter in resuspension models. However, in reality this threshold varies with sediment cohesive properties and size, as well as the properties of the discharged waste. Freshly deposited biogenic material may initially be cohesive, but this decreases over time with a resultant increase in resuspension potential.

A constant erosion threshold and a finite time in the resuspension layer will almost certainly affect model predictions. Increased dispersion of particles could be achieved by altering three mechanisms in the resuspension model: (1) increasing erosion potential over time; (2) increasing bioturbation activity; and (3) removal of a finite resuspension time. Incorporating these parameters in the model may well improve predictions of the dispersion of emamectin benzoate.

8.2.2 Model input data Default data were used in the model for feed water content, digestibility, wastage, and sediment density and mixing depth. Of these, the latter two are most likely to effect model predictions. These variables are required to make the conversion from mass (µg emamectin benzoate m-2 sediment) to sediment concentration (µg emamectin benzoate kg-1 sediment) for comparison of predicted and measured concentrations. In reality, sediment density will vary over small distances given the differences in ratios between discharged and natural material in sediments between stations. The necessity to make this conversion using default data may potentially introduce errors in the model predictions, however, they are not inherent to shortcomings in the model.

Other data were mostly site specific, such as husbandry data, dispersion coefficients from drogue studies, bathymetric and hydrographic data. At Loch Sunart, extensive hydrographic data were not available for the treatment periods, which was a limitation for the site. In particular, negligible erosion was predicted as current data above the 9 cm s-1 erosion threshold were not recorded. For example, significant differences in dispersion footprints were predicted with different 15 d current records at Loch Kishorn (Wilson, 2003). Thus, short-term current data sets are likely to have contributed to over-predicted sediment emamectin benzoate concentrations at Loch Sunart.

8.2.3 Measured data Uncertainty exists when discrete samples are used to validate models in a patchy environment. For example, sediment traps deployed below Mediterranean fish farms showed differences in solids flux to the sea bed by a factor of five or more for traps that were positioned only a few metres apart. Unsurprisingly, patchy deposition cannot be predicted by present models, given that complex centimetre-scale flow patterns around the cages are likely to be the cause. Fish behaviour and net fouling are also influential factors affecting particle distributions.

8.3 Sediment chemistry Two treatments with Slice were made in Lochs Sunart and Kishorn during the present study. Sediment sample collections from Loch Kishorn were ca. 1 and 6 months post Slice treatment, while sediment collections from Loch Sunart were ca. 1 week, 2, 4 and 5 months post Slice treatment. No reliable direct temporal comparisons can therefore be made between sites.

The highest concentrations of emamectin benzoate at both sites, however, were found in sediment collected from transect stations closest to the cages, which were located within the 25 m allowable zone of effect. With the exception of the sediment collected in Loch

Ecological effects of sea lice medicines in Scottish sea lochs 195 of 286 Sunart two months post treatment, emamectin benzoate was detected in both lochs within the 25 m allowable zones of effect.

One sediment sample from each loch, collected just outside the allowable zone of effect, 30 m from the cage, contained emamectin benzoate at concentrations above the limit of quantification. The sediment from Loch Kishorn had been collected 1 month post Slice treatment, while the sediment from Loch Sunart had been collected 5 months post Slice treatment. Emamectin benzoate was not detected, above the limit of quantification, in sediment from either loch collected 60 m from the cage. This is not unexpected, as in both lochs the relatively weak water currents close to the sea-bed will have caused limited sediment re-suspension.

The highest concentration of emamectin benzoate was measured in sediment collected directly below the cage one week post Slice treatment (Sunart S2A 14/05/02, 7.4 µg kg-1, dry weight) and one month post Slice treatment (Kishorn T2A 13/08/01, 13.4 µg kg-1, dry weight). The amount of active ingredient used in Loch Kishorn treatments (114.6 g, 246.4 g) was less than Loch Sunart treatments (315 g), however highest emamectin benzoate concentrations were found in sediment from Loch Kishorn. The distribution of emamectin benzoate under the cages would be expected to be patchy, arising from a mixture of excess (non-ingested) feed pellets and faeces. Hydographic data indicated that in both surface and near bed waters there was a higher degree of dispersion in Loch Kishorn than in Loch Sunart. It would therefore be predicted that dispersion of wastes would be more effective in Loch Kishorn than in Loch Sunart. However concentrations of emamectin benzoate were higher and more frequently detected in Loch Kishorn than Loch Sunart.

The sediment sample with the highest emamectin benzoate concentration from Loch Kishorn had a higher TOC (5.9 %) than that of the sediment sample with the highest emamectin benzoate concentration from Loch Sunart sediment (1.7 %). Therefore it is more likely to accumulate higher concentrations of hydrophobic contaminants. It may therefore be that the processes determining the distribution of emamectin benzoate in sediments from the two lochs are influenced by TOC distribution.

The majority of the sediments from all four Lochs were categorised as either mud or fine sandy mud. Exceptions were the samples Kishorn 7VV1 June 2001, Kishorn Stn 7 August 2001, Kishorn T2B, Diabaig Stn 1 and Diabaig Stn 2 which were coarse sand (Wentworth, 1922).

TOC means were similar for the four Lochs (Table 8.3). PSA and TOC were found not to be correlated (p > 0.05, ANOVA). Input of organic matter from fish farms in the form of excess (waste) feed pellets and faeces to the sediment will contribute directly to the measured TOC concentrations, and therefore close correlation between TOC and PSA would not be expected in sediments close to the farms.

Table 8.3. Total Organic Carbon (%) summary statistics for Loch Sunart, Loch Diabaig, Loch Kishorn and Loch Craignish.

Samples analysed TOC (mean) TOC (median) Loch Sunart 22 2.78 2.10 Loch Diabaig 6 2.54 2.55 Loch Kishorn 33 2.64 2.35 Loch Craignish 6 2.98 3.37

Ecological effects of sea lice medicines in Scottish sea lochs 196 of 286 8.4 Zooplankton Zooplankton community composition in Lochs Craignish, Kishorn and Sunart was similar. Although abundance of the various species varied between the lochs, the same species were present in all three loch systems. The seasonal cycles of abundance for the various species were also similar, with peaks occurring at approximately the same time of year in each loch. Overall, zooplankton abundance was greatest in Loch Sunart where total abundance peaked with 25000 individuals m-3 in July 2003. In 2002, abundance in Loch Sunart peaked in May with 15000 individuals m-3. The cyclopoid copepod, Oithona dominated the zooplankton community. In Loch Craignish, total abundance was highest in August 2000 with 13000 individuals m-3, whereas in 2001, numbers were considerably lower, with a maximum of 2500 individuals m-3 in May. At Loch Kishorn, the zooplankton community was sampled for three months in 2001 from June through to August. During this time abundance peaked in July with 8500 individuals m-3. Copepod nauplii were most abundant during the sampling period. The cyclopoid copepods Oithona and Monothula, the cladoceran Evadne and the calanoid copepods Acartia, Temora, and Pseudocalanus were also present in high numbers. Although zooplankton abundance was considerably lower in Loch Craignish compared to Loch Sunart, the two lochs with time series data, the timing of seasonal abundance peaks was similar. Copepod nauplii, calanoid copepods, Evadne, and Balanus nauplii peaked in spring and again in mid to late summer. In the case of Evadne in Loch Sunart, the two abundance peaks each year (spring and summer) were particularly pronounced in 2002 and 2003. At Loch Craignish, the second abundance peak was not observed in summer 2001. It is possible that the dispersive characteristics at Lochs Sunart and Craignish might explain the differences in zooplankton abundance observed between the two lochs. Near bed currents at Loch Craignish are more dynamic than those at Loch Sunart, which would reduce zooplankton residence times and abundance in deeper water.

8.5 Phytoplankton Seasonal concentrations of dissolved ToxN (nitrate plus nitrite) were similar at all four sites (Lochs Craignish, Diabaig, Sunart and Kishorn) between June 2000 and October 2001, with higher concentrations during winter months (Figure 8.1). Salinity (0-10 m) at Lochs Diabaig, Craignish and Kishorn was generally constant, whereas Loch Sunart exhibited lower and more changeable salinity (Figure 8.2).

Phytoplankton cell abundance and seasonal trends were very similar at Lochs Craignish, Diabaig and Sunart between June 2000 and April 2001 (Figure 8.3), with several blooms resulting in highly variable phytoplankton standing stocks between April and October 2001 (Table 8.4). Blooms were not observed at Loch Kishorn from June to August 2001, and the phytoplankton standing stock at this site, while species-rich, was always relatively low compared to the other lochs.

Ecological effects of sea lice medicines in Scottish sea lochs 197 of 286 Figure 8.1. Concentrations of dissolved ToxN (nitrate plus nitrite) at Lochs Sunart, Diabaig, Craignish and Kishorn between June 2000 and October 2001.

Figure 8.2. Salinity at Lochs Sunart, Diabaig, Craignish and Kishorn between June 2000 and October 2001.

A total of 88 phytoplankton taxa were observed at the four lochs (Appendix III - Phytoplankton; Table 14.13). Of these, 74 were observed at Lochs Diabaig and Craignish, 82 at Loch Sunart, and 54 at Loch Kishorn. In all lochs, summer phytoplankton populations were always more species-rich than spring, winter or autumn populations.

Ecological effects of sea lice medicines in Scottish sea lochs 198 of 286 Table 8.4. Phytoplankton blooms at Lochs Sunart, Diabaig and Craignish. With the exception of two blooms of unidentified cryptophytes (Diabaig in June 2001 and Sunart in June 2002), all blooms were of chain-forming diatom species. Sea Loch Date Species Maximum Biomass Sunart August 2000 Chaetoceros spp. (10-30 µm) 1.1 x 106 cells l-1 April/May 2001 Skeletonema costatum 1.8 x 106 cells l-1 June 2001 Chaetoceros spp. (10-30 µm) 941 000 cells l-1 September 2001 Chaetoceros spp. (10-30 µm) 1.3 x 106 cells l-1 March 2002 Skeletonema costatum 926 000 cells l-1 May 2002 Chaetoceros spp. (10-30 µm) 1.7 x 106 cells l-1 June 2002 Unidentified cryptophyte 2.6 x 106 cells l-1 July 2002 Chaetoceros sp. (< 15 µm) 2.0 x 106 cells l-1 August 2002 Rhizosolenia fragillisma 2.9 x 106 cells l-1 May 2003 Skeletonema costatum 5.3 x 106 cells l-1 July 2003 Chaetoceros spp. (10-30 µm) 972 000 cells l-1 March 2004 Skeletonema costatum 17.2 x 106 cells l-1 Diabaig June 2001 Unidentified cryptophyte 1.1 x 106 cells l-1 August 2001 Leptocylindrus minimus 836 000 cells l-1 Craignish May/June 2001 Chaetoceros sp. (< 15 µm) 2.8 x 106 cells l-1 August 2001 Chaetoceros spp. (10-30 µm) 1.0 x 106 cells l-1

A dendrogram of site similarity was constructed using phytoplankton community composition at each loch (Figure 8.4). Based on this, Lochs Craignish and Sunart were most similar (95 % similarity) while Lochs Kishorn and Sunart were least similar (76 %). The relatively high similarity, despite the inherent geographical and hydrographical variability of the sea lochs, illustrates the ubiquity of the majority of phytoplankton species identified through the course of this study, and found in Scottish sea lochs.

Figure 8.3. Total phytoplankton cell abundance (cells l-1) at Lochs Sunart, Diabaig, Craignish and Kishorn between June 2000 and October 2001.

Ecological effects of sea lice medicines in Scottish sea lochs 199 of 286 Similarity (%)

Figure 8.4. Relative phytoplankton community composition similarity at Lochs Sunart, Diabaig, Kishorn and Craignish. Plots were constructed from full presence/absence lists of phytoplankton taxa observed at each loch.

8.6 Meiofauna To compare the relative magnitude and direction of changes in meiofaunal assemblage structure across sites, and to assess the relative effects of site differences and the effects of aquaculture, data from all surveys were merged. Prior to this abundances were 1 averaged within stations, adjusted to a standard sample-proportion of 20 , square-root transformed and then used to calculate Bray-Curtis similarities. These similarities were then ordinated by MDS. Interestingly, after this heavy treatment of the data, analyses indicated that samples collected by van Veen grab and by divers may be comparable. As the majority of sites were sampled using grabs, the diver cores were all collected close to the cages at Loch Kishorn where patterns tended to match (loosely) those revealed by grab samples, and the focus was on comparisons between sites. The following discussion is primarily limited to stations sampled by grabs. The reference station at Kishorn, sampled on three occasions by divers, is included for comparison.

Relative changes in nematode community structure were similar at several of the sites, although some sites differed from the rest (Figure 8.5). Stations close to the cages in surveys at Kishorn, Diabaig and Craignish clustered together in the upper part of the plot, and the reference stations from Kishorn (collected by divers) and stations from Diabaig (with the exception of D21) in the lower part of the plot. If the primary axis of relative change in community composition runs from top to bottom of the plot (MDS plots do not have true axes) it may be seen that the relative change at Diabaig in November 2000 was slight compared to other surveys, whereas stations 1 and 2 at Craignish were very different from station 3. Stations at Sunart were generally different from the other lochs, although station 3 furthest from the cages at Craignish clustered with stations from Sunart and, if the top-bottom axis truly reflects relative change, community composition at Sunart was relatively similar at all stations.

For copepods a similar, if less clear, pattern emerges (Figure 8.6). Stations close to the cages at Loch Kishorn (station A on each transect) cluster with station 1 at Diabaig in August 2001. Stations further from the cages at Kishorn (stations B and C) cluster

Ecological effects of sea lice medicines in Scottish sea lochs 200 of 286 together, while the reference stations from all three surveys (collected by divers) form a tight cluster with station 3 from Diabaig (both surveys) and all stations from Sunart in March 2001. Again it may be surmised that an axis of relative change runs from top to bottom of the plot, but with an added complication that stations close to the cages at Craignish form a cluster to the left of the plot, as do those from Diabaig (with Station 4 from Sunart in 1999 and Station 3 from Craignish). These are generally samples with very low abundances. Thus differences in copepod assemblages may be manifest as changes in community structure, but in severe cases few individuals are left and the pattern of relative change indicated by the vertical axis breaks down. Again stations from the first survey at Sunart were different from the rest, but on the basis of the axis of change, community composition was relatively similar at these stations.

Figure 8.5. MDS ordination of Bray-Curtis similarities between square-root transformed adjusted 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.

From these results we conclude that comparative studies between sites may be useful. The results suggest that, at some level, the effects of fish-farming on meiofaunal communities are similar in different sea lochs. Thus the results of in-depth studies at one place 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.

Ecological effects of sea lice medicines in Scottish sea lochs 201 of 286 Figure 8.6. MDS ordination of Bray-Curtis similarities between square-root transformed adjusted 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.

8.7 Macrofauna Macrofaunal abundances from each survey/station combination at each site were averaged and the datasets combined. The resulting matrix was square-root transformed to downweight the influence of highly dominant species on intersample similarities. The resulting MDS plot based on Bray-Curtis similarities allows us to compare differences in macrofauna community composition across lochs and surveys, and indicates that differences in community composition between stations at each site were broadly comparable (Figure 8.7). In the MDS plot, it can be surmised that the axis of relative change in macrofauna community composition runs from left to right, with stations at Lochs Kishorn and Diabaig clearly separated from stations at Loch Sunart, which are closely grouped and towards the right of the plot. The gradient in community composition at Lochs Diabaig and Kishorn runs from left to right. Stations close to the cages at Diabaig and Kishorn are positioned towards the left of the plot and those further from the cages to the right, and closer to the stations at Loch Sunart indicating similarity in community composition. These results confirm those from individual surveys, and lend support to the hypothesis that the effects of aquaculture on macrobenthic communities are comparable between sites.

Ecological effects of sea lice medicines in Scottish sea lochs 202 of 286 Figure 8.7. MDS plot based on Bray-Curtis similarities calculated from square-root transformed 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).

8.8 Sublittoral The main annual settlement events in the sea lochs studied can be summarised as follows:

In April to June heavy settlements of barnacles (Balanus crenatus) usually occurred, followed by mussels (Mytilus edulis) and ascidians (sea squirts) and lower densities of a variety of other species (a total of around 40) throughout the summer months. Following the spring settlement, the main predators of barnacles and mussels, namely the sea slug, Onchidoris bilamellata, followed by the even more voracious starfish, Asterias rubens, settled from the plankton, grew rapidly and killed all the barnacles. The strong empty barnacle shells generally remained for many months, greatly altering the character of the surface for later settling animals in late summer and autumn. These included ascidians and often polychaete worms including Pomatoceros triqueter and Hydroides norvegica in calcareous tubes and those in soft muddy tubes such as Sabella pavonina (peacock worm).

In several of the lochs the growth of ascidian settlements in just one season was considerable and completely obscured everything else on the slates, also on the ropes and the buoys. In Loch Craignish it was necessary to remove the overloaded arrays at the end of one season.

8.9 Littoral At all four sea loch salmon farm sites (Lochs Sunart, Diabaig, Craignish and Kishorn) the intertidal zone consisted mainly of bedrock and/or boulders. All the shores were characterised by abundant growths of perennial brown fucoid algae mixed with good invertebrate populations (frequent to abundant) of attached barnacles and mussels, limpets and motile snails - mainly Littorina spp. and the dog whelk Nucella lapillus that feeds on barnacles.

Ecological effects of sea lice medicines in Scottish sea lochs 203 of 286 On all British rocky shores, the overall degree of exposure to wave action largely determines whether such littoral habitats are dominated by fauna (barnacles, limpets etc.) where there is considerable annual wave action, or mainly by fucoid seaweeds which limit the abundance of fauna throughout the littoral zone, where there is shelter from wave action.

A summary classification of the littoral sites and stations used for deployment of settlement slates, based on a biological exposure scale devised by Lewis (1964) is given in Table 8.5. Thus the sites ranged from overall sheltered (Sunart), through semi-exposed (Craignish and Kishorn) to overall exposed (Diabaig). Also, the mean range of spring tides which determines the vertical extent of rock space available for settlement varied widely from 4.9 m at Loch Diabaig to only 2.2 m at Loch Craignish (Table 8.5). Thus the exposed Loch Diabaig also had the widest mid-littoral zones available for colonisation, compared with less exposed and narrow zones in Loch Craignish.

Table 8.5. Summary classification of sea loch sites surveyed. Loch Sunart Loch Craignish Loch Kishorn Loch Diabaig (inner basin) (E. shore) (Achintraid) Wave Exposure (overall) Sheltered Exposed Semi-exposed Semi-exposed Biological Exposure Scale 4-5 2-3 3-4 3-4 1-5 (Lewis, 1964) LS A - 4 LD A - 2 LC A - 4 LK A - 4 Exposure at intertidal LS B - 5 LD B - 3 LC B - 4 LK B - 3 stations LS C - 4 LD C - 3 LC C - 3 LK C - 3 LC D - 3 All intertidal panel sites are bedrock (except LSB - huge boulder), in mid- Substratum, tide level and littoral zone, with varying abundances of Semibalanus balanoides, Patella principal biota vulgata, Mytilus edulis, Nucella lapillus, Littorina spp., and seaweeds Fucus vesiculosus and Ascophyllum nodosum, etc. Mean range spring tides 4.0 m 4.9 m 2.2 m 4.8 m Mean range neap tides 1.7 m 2.0 m 0.8 m 1.8 m *Key to biological exposure scale: 1 - Very exposed; 2 - Exposed; 3 - Semi-exposed; 4 - Sheltered; 5 - Very sheltered (Taken from Lewis, 1964, p 288).

Barnacles (mainly Semibalanus balanoides) are the only sedentary crustaceans that settle predictably on all Scottish rocky shores in high numbers and thus were chosen as the appropriate target organism for studies aimed at detection of any effects of the sea lice treatments.

The highest densities of barnacle settlement occurred, predictably, at Loch Diabaig reference Station A (the most exposed to wave action) with up to 100 % cover by S. balanoides, although it is emphasised that the settlement densities varied considerably over the four years of settlement studies there. Conversely, the very poor settlement of S. balanoides at Loch Sunart Stations A and B, Loch Craignish Station A and Loch Kishorn Station A were the result of the very sheltered nature of these locations, with dominance by fucoids and mussels on the adjacent boulders and the very limited areas of bedrock. The possible limiting factors to successful establishment of barnacle communities on sheltered rocky shores dominated by fucoid algae were examined by Jenkins and Hawkins (2003). Existing stands of fucoids and mussels were in fact removed in the present study, along with limpets and snails, from the proximity of the panels.

It is possible that the low frequency of developed egg masses in 2000 and 2001 cohorts of S. balanoides on settlement panels at the predicted high impact station at Loch Diabaig is related to discharge from the fish farm. However, it is emphasised that a number of natural factors could possibly produce the same effects, but at present the nature of the

Ecological effects of sea lice medicines in Scottish sea lochs 204 of 286 influences of many natural and anthropogenic factors on growth and development of S. balanoides are unknown. At Lochs Sunart, Kishorn and Craignish there were no consistent trends found that could be attributed to potential effects of fish farms.

Ecological effects of sea lice medicines in Scottish sea lochs 205 of 286 9 Conclusions 9.1 Chemistry Water samples collected from the effluent plume following an Excis treatment were analysed for cypermethrin and concentrations compared with those predicted by a model. At all the sampling stations the concentration of cypermethrin in water was below that predicted by the model. As expected, the highest concentrations were from samples collected within 5 min of the release of the tarpaulin and adjacent to the cage. Only one water sample collected adjacent to the cage exceeded the 3 h EQS of 16 ng l-1 (SEPA, 1998). The 3 h EQS was not breached in any other samples with cypermethrin concentrations falling below 4 ng l-1 after 15 min. Dispersion of cypermethrin in Loch Sunart was rapid with only four water samples collected from the effluent plume after 43 min having concentrations greater than the detection limit.

The measurement and dispersion of emamectin benzoate in sediment from Loch Sunart and Loch Kishorn provides information to support the interpretation of the ecological assessment. The water currents close to the seabed in Loch Sunart and Loch Kishorn were relatively weak, therefore, as expected dispersion of emamectin benzoate in sediment was limited, with no emamectin benzoate being detected in sediment collected at stations 60 m from the cages. Predictably, the highest emamectin benzoate concentrations, in both lochs, were detected in sediment collected from stations within 25 m of the cages. Emamectin benzoate was detected in sediment collected five months post-treatment, and therefore complete degradation had not occurred in that time. Metabolites of emamectin benzoate were not detected in any of the samples. These observations are consistent with the long half-life of emamectin benzoate in sediment (around 175 d).

9.2 Impacts of sea lice treatment agents on zooplankton assemblages Zooplankton sampling campaigns undertaken during this project monitored the effects of sea lice treatments with Excis, Salmosan, Salartect and Slice at Loch Sunart, and Slice at Loch Kishorn. For all sea lice treatment events no adverse affects on zooplankton were detected at either the species or community level (Table 9.1), and observed changes in community composition were unrelated to treatment events. Initially, sampling campaigns were of short duration, with intensive sampling pre- and post-treatment. Changes observed during these campaigns were naturally occurring, with patchiness in distribution, life history characteristics, and advection being the most influential factors affecting zooplankton distribution and community composition. To improve our ability to separate treatment effects from seasonal changes in abundance, a long-term sampling programme was undertaken in Loch Sunart from November 2001 to April 2004. This allowed identification of the natural seasonal cycles in abundance and species composition in Loch Sunart 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. 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.

Ecological effects of sea lice medicines in Scottish sea lochs 206 of 286 Table 9.1. Zooplankton community responses to sea lice treatments. Treatment agent Location Treatment date Zooplankton community response Excis Loch Sunart March 2000 No treatment effect detected Excis / Salmosan Loch Sunart November 2000 No treatment effect detected Salartect Loch Craignish August 2000, May 2001 No treatment effect detected Slice Loch Kishorn July 2001 No treatment effect detected Excis Loch Sunart February 2002 No treatment effect detected Salartect Loch Sunart March 2002 No treatment effect detected Slice Loch Sunart May 2002, Sept 2002 No treatment effect detected Salmosan Loch Sunart October 2003 No treatment effect detected Slice Loch Sunart March 2004 No treatment effect detected

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, water column cypermethrin concentrations at Loch Sunart were measured in the treatment plume released from one of the cages during the February 2002 Excis treatment and compared with model predictions. The highest measured cypermethrin concentrations (ca. 14 ng l-1) occurred immediately after release of the treatment plume in Loch Sunart and had fallen to 1.4 ng l-1 after 2.5 h. Predicted concentrations were within the same order of magnitude as the measured concentrations, and at the surface (0-3 m) fell by 99.7 % within 3 hours post release. This suggests that the 3 h EQS of 16 ng l-1 (SEPA, 1998) is unlikely to be breached following single cage treatments and, although there may have been localised affects on zooplankton within the dispersing treatment plume, the wider zooplankton community within the loch was not adversely impacted at a detectable level. The measured and predicted cypermethrin water column concentrations following a single cage treatment at Loch Sunart were also considerably lower than concentrations shown to cause acute toxicity to copepods in laboratory studies (Willis & Ling, 2004). The results of laboratory studies provide additional confirmation that environmental concentrations are unlikely to adversely impact zooplankton communities in the field. Cypermethrin concentrations causing acute toxicity to planktonic copepods, although lower than the treatment concentration of 5000 -1 ng l , were considerably higher than the 3 h EQS (Willis & Ling, 2004). The 48 h EC50 values for the most sensitive species, Oithona, ranged from 140 to 240 ng l-1, and in repeated short term (3 h) exposures, A. clausi egg production increased significantly at a cypermethrin concentration of 1580 ng l-1. Bath treatments with Excis involve multiple releases during the treatment period, which introduce increasing levels of cypermethrin to the loch, whereas models only predict concentrations following a single release, and so underestimate final water column concentrations. However, it is clear that cypermethrin concentrations arising from multiple releases do not adversely impact zooplankton communities.

Emamectin benzoate water column concentrations were not measured during this study (primarily because they would almost certainly have been below the limits of quantification), however, concentrations causing toxicity to planktonic copepods in laboratory tests (Willis & Ling, 2003) were several orders of magnitude higher than the PNEC for soluble emamectin (2.2 x 10-4 µg l-1). In laboratory tests, reproductive output of A. clausi was significantly reduced following 96 h exposure to 158 ng l-1 emamectin

Ecological effects of sea lice medicines in Scottish sea lochs 207 of 286 benzoate, however, it is unlikely that copepods would be exposed to such high concentrations in the field. Given that emamectin benzoate is administered as a component of salmon feed, zooplankton are more likely to be exposed to the compound via ingestion of feed and faecal particles. The average total concentration of soluble and particulate emamectin benzoate estimated for the water column by McHenery & Mackie (1999) is 8.28 x 10-6 µg l-1. This value is also orders of magnitude lower than concentrations causing acute and sublethal toxicity in laboratory tests, and thus very unlikely to adversely affect zooplankton communities, as confirmed in this study.

9.3 Impact of sea lice treatment agents on phytoplankton assemblages The phytoplankton datasets reflect a high-resolution sampling frequency at four Scottish sea lochs. Supporting salinity and nutrient information was collected so that effect(s) of sea lice treatments on phytoplankton communities could be separated from the effects of continually fluctuating environmental factors. A long-term dataset is essential if specific responses of the phytoplankton are to be confidently assigned to a single abiotic variable such as the impact of a sea lice treatment medicine. Scottish sea lochs are highly dynamic environments and phytoplankton are continually subject to fluctuating grazing pressure as well as changing nutrient, salinity, temperature and light conditions.

Classical seasonal trends in phytoplankton and zooplankton community composition and abundance were apparent at Loch Sunart between November 2001 and May 2004, however, no obvious relationships could be distinguished for any of the individual phytoplankton species or taxa and the dominant zooplankton species (calanoid copepods, non-calanoid copepods, Oithona, Monothula and Microsetella).

Despite highly variable hydrographical features and sea lice treatment histories, comparison of the four sea loch phytoplankton communities revealed significant similarity (> 76 %) between them. This reflects the ubiquitous presence of many of the phytoplankton species in Scottish coastal waters. At all lochs, the phytoplankton was typically dominated by a relatively small number of taxa with many other species maintaining low threshold concentrations (e.g. < 1000 cells l-1). These low-biomass species typically exhibited highly variable distributions both temporally and spatially, whereas a number of the more common (and bloom-forming) 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.

The presence or absence of any phytoplankton species could not be attributed to the application of sea lice treatment chemicals 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.

9.4 Meiofauna 9.4.1 Loch Sunart In initial samples (1999) at Loch Sunart, a weak gradient in meiofaunal community structure was observed. This could not be related unequivocally even to normal organic enrichment from aquaculture activities although the presence of the fish cages close to station 1 was considered to be the probable cause of the observed differences. In March 2001, no obvious gradient effect was detected, but combined analysis of data from both surveys suggested a mild organic enrichment impact at Station 1 in 2001.

Ecological effects of sea lice medicines in Scottish sea lochs 208 of 286 The variability in the data suggests that site location, depth and sediment structure may be influencing the observed variation in community structure rather than fish farming activity. Seasonality is possibly a factor in this shift in pattern. Another likely explanation is a difference in sampling methods between the two surveys.

Abundances, particularly of copepods, were low at Loch Sunart. Studies of organically enriched sites (Moore & Somerfield, 1997) and sites subjected to anthropogenic disturbance (Somerfield et al., 1995), in which the relative responses of nematodes, copepods and macrofauna were investigated, were based on samples containing many more specimens than were found in the grab samples in this study. Sub-sampling of grabs can be an inefficient method for extraction of meiofaunal copepods (Somerfield et al., 1995, Somerfield & Clarke, 1997). Adequate data on copepod abundances are still lacking for this loch and further sampling both here and at other sites would be required to determine whether abundances are genuinely low in the area. In the cross-site comparison this site did not appear to be highly impacted.

9.4.2 Loch Diabaig Evidence emerged from the first survey data (July 2000) that meiofauna community composition varied between stations at Loch Diabaig, and was obviously different at Station 2, located in the deeper part of the loch. In particular, the copepod assemblage at this station was extremely impoverished and the suite of nematode species found in greater numbers at this site, such as Spirinia parasitifera and Dorylaimopsis punctata, were indicative of muddier sediments. Hydrographic surveys indicated that clockwise rotation of water in the Loch may concentrate material settling from the cages, and associated contaminants, in the vicinity of this station.

There were clear differences in copepod community structure between stations within each survey, and abundance and diversity declined at all stations, particularly Stations 1 and 3, between the two sampling periods. The differences observed were most likely caused by natural seasonal variation. On both sampling occasions, Station 2 was dominated by the copepod Typhlamphiascus confusus, suggesting that seasonality may not affect this species, but that it is taking advantage of other conditions such as sediment type or food source.

Much of the observed pattern in meiofaunal community structure can be explained by variation in sediment type (proportions of coarse silts for nematodes, medium sands and coarse silt for copepods), 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. Thus, there was no evidence that sea lice treatments were a major driver of patterns in meiofaunal community structure at this site.

9.4.3 Loch Craignish At Loch Craignish, species contributing to differences between stations included species from groups known to be indicators of disturbance and organic enrichment, leading to the conclusion that the observed differences between stations reflect a normal organic enrichment gradient associated with fish-farming activity.

9.4.4 Loch Kishorn Very strong gradients in assemblage structure were apparent in both nematode and copepod assemblages. Differences in nematode assemblages with distance from the cages extended beyond 50 m, and were relatively stable compared to differences in copepod assemblages. There was evidence of greater differences in copepod community structure between stations in post-treatment surveys. Both nematode and copepod community

Ecological effects of sea lice medicines in Scottish sea lochs 209 of 286 structure were most highly correlated with station distances from the cages, which were chosen on the basis of modelled emamectin benzoate concentrations. Using variables measured at each station, there were no significant relationships between differences in nematode community structure and environmental variables. However, there was a significant relationship for copepods, the sand-silt gradient and measured sediment emamectin benzoate concentrations. We conclude that there is evidence that deposition from the cages is impacting nematode and copepod assemblages, and that a combination of changes in sediment composition and the presence of in-feed treatment chemicals are related to observed changes in copepod community structure.

Findings from the comparison of grab and diver-collected samples support those of previous studies (Fleeger et al., 1988; Somerfield et al., 1995; Somerfield & Clarke, 1997) showing that grab sub-sampling can be an unreliable method of sampling meiofauna, especially copepods, quantitatively. However, in situations where there are clear, strong, patterns in the benthos (as at Loch Kishorn), the results may give an indication of relative changes between sets of samples. The comparison did not shed adequate light on the relative abundances of copepods to be able to explain the lack of copepods in samples from Loch Sunart.

9.5 Macrofauna 9.5.1 Loch Sunart There were significant differences in community composition between stations in each survey at Loch Sunart, both in community structure, and the dominance/diversity structure of assemblages. However, the differences were often subtle, and difficult to relate to the effects of aquaculture at the site. Macrobenthic community composition at the stations closest to the fish farm remained the same over time but differed from the reference station. Thus, it is probable that the differences were caused by the gradient in organic enrichment originating at the cages, which influenced all but the reference station community. There were no obvious decreases in diversity or increases in dominance at any station within each survey or over time that could be related to sea lice treatments and differences within individual surveys probably reflected the spatial distribution of replicates, with differences in assemblages between replicates at each station varying between surveys. Therefore there was no evidence to suggest that sea lice treatments affected macrofaunal community composition at Loch Sunart.

9.5.2 Loch Kishorn The presence of a strong carbon enrichment gradient in the proximity of the cages at Loch Kishorn influenced benthic community composition within 50 m of the cages making it difficult to clearly discern any additional treatment related effects. Reductions in the number of crustacean and echinoderm taxa and their abundance at Station 2 in the second survey may have been treatment related as crustaceans in particular are the group most vulnerable to sea lice treatment agents. However, these groups are also adversely affected by organic enrichment. Macrofaunal community composition at the stations close to the cages differed from the reference Station, although differences were only apparent at Station 1 in June, and Stations 1 and 2 in August. Community composition at Station 1 was similar in both surveys, but differed at Station 2, both in community composition and in dominance and diversity. The Slice treatment undertaken between the two surveys may have been responsible for the differences, but it is more likely that the spatial distribution of stations and patchiness in enrichment (faecal material and waste food) around the cages contributed to the patterns seen. Thus, there was no evidence that Slice adversely affected the macrobenthic community at Loch Kishorn. Instead the gradient in organic enrichment was the most likely factor influencing changes in community composition.

Ecological effects of sea lice medicines in Scottish sea lochs 210 of 286 9.5.3 Loch Diabaig As at Lochs Sunart and Kishorn, significant differences in macrobenthic community structure between stations at Loch Diabaig indicated a gradient of organic enrichment originating at the fish farm cages. Differences in community composition between stations were significant in 2000 but not in 2001. In 2000, species diversity was lowest at the station closest to the cages and dominated by large nematodes and small polychaetes. Community composition at the station furthest from the cages was typical of normal shell/muddy sediments. Following the Slice treatment in 2001, there was a general increase in dominance at all stations and crustaceans declined at the station closest to the cages. However, it was not possible to ascertain whether changes in community composition between surveys were caused by the Slice treatment or were the result of seasonal variability and the gradient of organic enrichment.

9.6 Sublittoral settlement arrays The study of the settlement of organisms on the sublittoral arrays of slate panels suspended at three different depths and at three or four stations in Lochs Sunart, Diabaig and Craignish has yielded a clear picture of the natural seasonal and annual successions and abundances of the fauna and flora involved.

At all of these sites and stations the most significant ecological event of every year was the spring settlement of the dominant sublittoral barnacle Balanus crenatus. Barnacle larvae spend about three weeks as zooplankton passing through several typically crustacean developmental stages, ending with the larval cyprid stage that settles on solid substrates mainly in the period April to June. At all of the stations the slate panels became heavily colonised and, as the barnacles grew, quickly achieved 100 % cover especially at the optimal upper and mid-water depths. The above results show clearly that nowhere was the settlement of barnacles at the stations of ‘Highest predicted impact’ - LS2 (Sunart), LT1 (Diabaig) and LC1 (Craignish) - different from the ‘reference’ (I and R) stations. It follows that any chemicals originating from sea lice treatments at the salmon cages were not affecting the abundant barnacle settlements at any of the sites studied.

The subsequent death of most of the B. crenatus at most stations, caused by predation by Onchidoris bilamellata and Asterias rubens settling from the plankton in spring and summer, respectively, is a well documented natural event (Barnes & Powell, 1951, 1954; and review by Barnes, 1999).

Other species of crustacea settled on the panels only rarely, e.g. Balanus balanus (Sunart and Diabaig) and the small crab Porcellana longicornis (Sunart). However, in December 2003, we recorded the alien caprellid amphipod Caprella mutica (occasional to frequent) at both Stations LS2 (H) and LS3 (I) in Loch Sunart.

The overall species diversity of the fauna settled on the sublittoral slates was limited and closely similar at all sites studied and mainly determined by the initial heavy settlement of barnacles.

Macroscopic algae (seaweeds) scarcely featured in the settlements on the sublittoral slates, although they often colonised the near-surface ropework and buoys; and mussel spat (Mytilus edulis) settles readily on ropes, hydroids and any fine-branched attached seaweeds.

9.7 Littoral settlement panels In the intertidal zone, several miles of rocky shore adjacent to each of the fish farms studied in all four sea lochs were carefully inspected for any unusual features in the over-

Ecological effects of sea lice medicines in Scottish sea lochs 211 of 286 all ecology of the seaweeds and fauna. However, nothing was found that could not be related to natural causes or localised anthropogenic effects: e.g. wave action (exposure/shelter effects), substrate (rock, boulders, sand, mud), localised freshwater run- off or domestic waste water, winter frost or summer sunshine effects (can kill young seaweeds on the lower shore - observed at Loch Diabaig), biotic effects (predation, competition for limited space), etc. Detailed follow-up surveys of these shores were not necessary for this study. Effort was concentrated on selecting good shore stations for the study of the only attached crustacean, Semibalanus balanoides, although in Loch Sunart particularly the very sheltered boulder shores offered limited choice.

It is not known which stages of the reproductive cycle of S. balanoides may be affected to reduce the frequency of fertilised egg masses observed in the present study; no information has been obtained concommitantly on the concentrations of sea lice medicines in the vicinity of the shore stations. In addition, the ‘normal’ range for the frequency of occurrence of mature fertilised egg masses in barnacle populations in non- impacted areas remote from fish farms has not been studied.

It is emphasised that almost all of the data were derived from studies of the growth and development of barnacle cohorts which had only developed for one growth season (settlement in the late spring until late winter the following year, prior to spawning). In March 2003, limited examination of older, larger barnacles settled on mussels at Loch Sunart showed that all of the individuals examined (20 from each shore station) contained mature egg masses. It is also known that the planktonic larval stages of S. balanoides remain in the water column for sufficient time (ca. 3 weeks) to allow dispersion and mixing of populations from a wide area. Thus it is highly unlikely that any localised changes in egg production found in this study have general ecological significance.

Nevertheless, the results indicate that the effects of natural and anthropogenic factors on egg production in littoral barnacles merit further more detailed investigation, including study in areas which are unaffected by fish farming.

9.8 Project summary The salmon farming industry uses medicines to control infestations of parasitic sea lice on its stock. The amount of medicine and the frequency of application are controlled by regulation to ensure that the potential negative effects on the ecosystem are minimised. In this project, which began in September 1999 and was completed in August 2004, our objective was to determine whether there were any measurable, long-term or wide-scale ecological consequences of the use of such medicines at commercially operating fish farms on the west-coast of Scotland.

We set up sampling programmes 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 bath-treatments, such as Excis (active ingredient cypermethrin), Salartect (active ingredient hydrogen peroxide) and Salmosan (active ingredient azamethiphos) for sea lice control but in 2001 the in-feed medicine Slice (active ingredient emamectin benzoate) became available and was used at 3 of the sites. Site selection was complex as at the beginning of the project many sites were under threat from an Infectious Salmon Anemia outbreak. Additionally, it was hard to predict when and which sites would receive discharge consent for Slice. Thus initial work began in Lochs Sunart, Diabaig and Craignish with a subsequent re-focus on the Sunart and Kishorn systems.

Ecological effects of sea lice medicines in Scottish sea lochs 212 of 286 At each site we examined hydrographic parameters using Differential Global Positioning System drifters and current meter arrays to allow modelling of the dominant water movement processes at each site, and acoustic ground discrimination to determine substrate type and bathymetry around each farm. Ecological sampling programmes were carried out each focusing on separate ecosystem ‘compartments’. This involved examination of littoral and sublittoral 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 and macrofauna (and the presence of emamectin); zooplankton sampling before, during and after medications and also as a time series to assess successional processes (at Lochs Craignish and Sunart); and time-series measurements of phytoplankton populations (together with nutrient concentrations). 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 farm activity. Fish farms have some well known local ecological effects as a consequence of organic enrichment so we paid particular attention to Crustacea as the part of the community most susceptible to effects from sea lice medicines, which are by definition designed to be toxic to Crustacea.

Loch Sunart. The site in Loch Sunart had the longest and most complete sampling programme. Modelled concentrations of medicines were always higher than those measured in both the water-column and sediments. Both short-term (around a treatment event) and long-term (31 months) studies of zooplankton and phytoplankton failed to detect any link between community structure and medicine usage. In the meiobenthos, copepod numbers were low at all stations, possibly a sampling artifact, and nematodes showed mild effects consistent with organic enrichment but which could not be associated with treatments. Similarly, there was no evidence that macrofaunal community structure was influenced by medicine use. Successional processes on sublittoral settlement panels were broadly similar at all stations and there were no consistent trends that might indicate that barnacle fecundity on shore settlement panels was affected by the treatments.

Loch Diabaig. The site in Loch Diabaig was studied over several years but less intensively than the Loch Sunart site. The site is highly exposed and many of the sublittoral settlement panels were lost during poor weather. Phytoplankton were sampled over a 15 month period and showed no change in community structure that could be related to fish farming. Meio- and macrofauna were sampled in July 2000, 6 months after an Excis treatment and again in August 2001, about 2 months after a Slice treatment. Meiofaunal copepods were impoverished at station 2 (predicted to be intermediate in exposure compared to station 1, at the farm, and station 3, the reference) on both sampling events, whereas at the same station all groups of macrofauna showed increases in diversity and all except molluscs showed increased abundance. A decrease in macrofaunal crustacea abundance and diversity was observed at station 1 nearest the farm but, as this also occurred at the reference station; no strong conclusion can be drawn from this. The sublittoral panels that survived the storm damage showed no evidence of any colonisation or succession feature that could be regarded as unusual. Littoral panels, examined over 4 years, were dominated by barnacles and there was evidence of a relationship between reduced fecundity and predicted impact across the three stations. This trend diminished with time over a period where fish farm activity also diminished. While this is sufficiently interesting to warrant further study, there are many other factors

Ecological effects of sea lice medicines in Scottish sea lochs 213 of 286 that could have contributed to this trend and no firm conclusion can be drawn from this result with respect to medicine use.

Loch Kishorn. The benthic sampling design at the Loch Kishorn site was deliberately different from that employed at the other sites. Stations were selected very close to the cages in 2 transects immediately adjacent to the farm in areas predicted to receive high emamectin loads after treatment. In this way we hoped to see definite changes in the benthic community before and after treatment in the area immediately around the farm. Samples for meiofauna and macrofauna were taken immediately before and after the first planned treatment of Slice. A further sample for meiofauna only was collected 6 months later. Strong gradients in macrofaunal and meiofaunal community structure were observed across the farm footprint. Although the community at station 2 (intermediate distance) appeared to be more impacted between first and second samples, overall there is insufficient evidence to show that this was caused by the medicine as opposed to natural variability or imprecise station re-location given that these stations were only a few metres apart on a steep enrichment gradient. Changes in the fecundity of barnacles on shore panels over a 2.5 y sampling programme are difficult to interpret but do not show a clear influence of the fish farm. Comparisons of emamectin sediment concentration predictions and measured post-treatment concentrations revealed that the model under- predicted the measured concentration immediately after treatment but strongly over- predicted the concentration 186 d after treatment.

Loch Craignish. The Loch Craignish fish farm was small by industry standards and, during the sampling programme, it became apparent that sea lice treatments would be relatively infrequent and that the farmers did not have access to Slice nor were they likely to apply for it. Thus, sampling at the site was started in May 2000 and completed in early 2002 when efforts were transferred to the Kishorn site, which had a larger biomass consent and had then recently received a discharge consent for Slice. Nevertheless, detailed long-term (15 months) datasets for phytoplankton and zooplankton were collected revealing typical patterns of species and population dynamics with no evidence of effect from the relatively few sea lice treatments used by the farm. These datasets were useful for comparison with the subsequent long-time series collected in Loch Sunart. Although macrofaunal samples were collected, as priorities changed these were archived for possible future study and analytical effort transferred to samples from other sites. Analysis of one set of meiofaunal nematode samples showed a gradient in dominance/diversity typical of an organic enrichment gradient. Sublittoral settlement panels were deployed for one year only and there was a relatively low rate of recovery, possibly owing to damage from boat traffic (the site is close to a large marina). All panels recovered at the end of the deployment showed overwhelming dominance by ascidians. There was no evidence of a fish farm effect. Shore panels revealed no evidence to suggest that growth or reproductive development of barnacles was influenced by the fish farm.

9.9 Final comments The project's primary objective was not to determine local effects of sea lice medicines. All discharges to the marine environment, including fish farms, 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 or AZE. We have attempted to look beyond ephemeral, local effects and have concentrated on

Ecological effects of sea lice medicines in Scottish sea lochs 214 of 286 trying to detect long-term changes beyond the immediate mixing zone. This has been a difficult task and, taking all the results together, we have not been able to detect any clear effect of medicine usage, or indeed other farm activities, in sea lochs beyond the local scale. As far as we currently understand ecological processes in marine ecosystems, the processes of species succession and population dynamics that we have observed are well within the range of what might be expected or predicted for fjordic sea loch systems.

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 question may be asked as to whether our experimental design leant itself to detecting subtle effects over long time-scales against inherent natural variability in marine ecosystems. The answer is that, in order to detect such subtle changes reliably, longer time-series of data would be required from systems where there was better coupling between the commercial management of the farms and the scientific needs of the project. Also, the project would need to be transparently developed to avoid the potential for accusations to be made of data being hidden or withheld. Nevertheless, the project has achieved much by helping to improve our understanding of natural variability in relatively unresearched 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.

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Ecological effects of sea lice medicines in Scottish sea lochs 223 of 286 11 Glossary

ANOSIM - Routine in PRIMER statistical package for permutation-based hypothesis testing, an analogue of univariate ANOVA which tests for differences between groups of (multivariate) samples from different times, locations, experimental treatments etc. Benthic - On or in the sea bed CA - Correspondence Analysis. Statistical technique of factoring categorical variables and displaying them in an ordination space which maps their association in two or more dimensions

EC50 - Median effective concentration. Derived concentration of a substance expected to produce an effect in 50 % of test organisms EQS - Ecological Quality Standards Granulometry - Sediment particle size analysis Ground truth - the process of calibrating remote observations by in situ measurements Littoral - Intertidal MAC: Maximum Allowable Concentration Macrofauna - Benthic animals retained on a 0.5 or 1.0 mm sieve Meiofauna - Small benthic animals retained on a 0.063 or 0.045 mm sieve MDS - Multi-dimensional Scaling. Multivariate statistical technique used to represent visually the proximity of samples NOEC - No Observed Effect Concentration PEC - Predicted Environmental Concentration Pelagic - In or associated with the water column Phytoplankton - Free floating plants capable of converting inorganic nutrients into complex organic compounds PNEC - Predicted No Effect Concentration SIMPER - Routine in PRIMER statistical package for identifying the species primarily providing the discrimination between two observed sample clusters Sublittoral - Below the range of tide Zooplankton - Small animals that are carried by water currents, as they are incapable of vigourous movement

Ecological effects of sea lice medicines in Scottish sea lochs 224 of 286 12 Appendix I - Hydrography The RoxAnn acoustic ground discrimination system was used to survey the area around the studied farms. In Loch Kishorn, a total area of 1.5 km2 was surveyed and 12 grabs were taken to ground truth the sediment type information (Figure 12.1). The instrument was also used to position cage groups.

A low E1 value indicated that sediment was smooth in most of the area surveyed, with increasing E1 values indicating a rougher surface (Figure 12.1). A low E2 value indicated a soft sediment, with harder sediment as E2 increased. However, the sediment types sampled during this survey did not vary significantly, with mud being a major component of each sample. To the SE side of the survey area in shallower water, this area was rougher than the majority of the deeper areas. This is likely to be caused by some shelly material mixed in with mud and kelp and in less than 10 m depth. Some harder sediments were found to the NW of the grid and this is most likely to be caused by cohesive mud in these areas greater than 50 m deep. Although this survey was useful for bathymetry which will be used in modelling, sediment types across the grid were fairly homogeneous.

Ecological effects of sea lice medicines in Scottish sea lochs 225 of 286 Figure 12.1 Loch Kishorn bathymetry and sediment characteristics, with survey tracks shown in the bathymetry plot (cage group in centre of plot). Crosses mark ground truthing of sediment type undertaken with a hand held grab.

Ecological effects of sea lice medicines in Scottish sea lochs 226 of 286 13 Appendix II - Chemistry

13.1 Cypermethrin

13.1.1 Methods

Materials and equipment preparation:

Dichloromethane (DCM), iso-hexane, methanol, acetone, hexane and methyl-tertiary- butyl-ether (MTBE) were purchased from Rathburn Chemicals Ltd., Walkerburn, Scotland. The copper powder and the stock standard - cypermethrin were purchased from Sigma-Aldrich Company Ltd., Dorset, UK. Analytical grade hydrochloric acid was supplied by Merck Ltd., Lutterworth, UK. Anhydrous sodium sulphate was of analytical grade from Fisher Chemicals, Loughborough, UK. Bond Elut phenyl solid phase extraction cartridges (SPE) were purchased from Varian, Surrey, UK. All glassware was soaked in Decon 90 (Decon Laboratories Ltd., Hove, Sussex, UK) before being thoroughly scrubbed and then rinsed with water. The glassware was then further rinsed and dried in an oven at 85 ºC. Cellulose extraction thimbles (28 x 80 mm; Whatman International Ltd., Kent, UK) were cleaned by soxhlet extraction with MTBE. Copper powder was activated by washing with hydrochloric acid (1 % v/v) in distilled water (100 ml). This was followed by washing with distilled water (3 x 100 ml), acetone (3 x 100 ml) and hexane (3 x 100 ml), the latter remaining until the copper was required for use. Anhydrous sodium sulphate was washed twice with DCM followed by iso-hexane prior to drying overnight (85 ºC ± 10 ºC).

Extraction of cypermethrin from water samples:

The volume of each water sample was recorded (~2.5 l) and decanted into separating flasks. The sample was thoroughly shaken with an aliquot of 100 ml and allowed to partition from the water. The lower DCM layer was then collected in a conical flask. This extraction was repeated with a second aliquot of DCM (100 ml) and the pooled extracts were dried over anhydrous sodium sulphate. The DCM extract was transferred with washings (2 x 10 ml iso-hexane) to a 250 ml round bottomed flask and concentrated by rotary evaporation to approximately 2 ml. The solution was solvent exchanged to iso- hexane (25 ml) and concentrated to < 1 ml by rotary evaporation. This step was repeated. Samples were transferred to pre-conditioned (iso-hexane, 4 ± 1 ml) phenyl SPE cartridges and eluted with iso-hexane (15 ± 2 ml). The eluent was transferred and concentrated to < 1 ml by rotary evaporation. The samples were transferred with washings (iso-hexane, 2 x 250 µl) to a pre-weighed GC vial. The vials were re-weighed and the sample volume calculated, correcting for the density of iso-hexane (piso-hexane = 0.6532).

Extraction of cypermethrin from sediment samples:

On arrival at the laboratory the sediment samples were immediately placed in cold storage (-25 ºC ± 5 ºC). Samples remained frozen until they could be freeze dried using a Heto CD8 freeze drier (Heto Holten Ltd., Surrey, UK). The freeze dried sediment (20 g) was accurately weighed into solvent cleaned cellulose thimbles. Each batch of six samples contained a blank reference sediment (20 g), to which 500 µl of cypermethrin (1000 ng ml-1) had been added, and a procedural blank. The samples were soxhlet extracted overnight (12 ± 4 hrs) with MTBE (180 ml), containing anti bumping granules (4-20) and activated copper powder (10-20 g). The MTBE extract was transferred with washings (2 x 10 ml iso-hexane) to a 250 ml round bottomed flask and concentrated by rotary evaporation (water bath temperature ≤ 30 ºC) to approximately 2 ml. The solution

Ecological effects of sea lice medicines in Scottish sea lochs 227 of 286 was solvent exchanged to iso-hexane (25 ml) and concentrated to < 2 ml by rotary evaporation. This step was repeated. The solution was transferred, with washings, to a volumetric flask (2 ml) and diluted to the mark with iso-hexane. The cypermethrin was isolated from the lipid fraction by gel permeation chromatography (GPC). A phenogel guard column (50 x 7.8 mm, 50 Å) and two phenogel columns (300 x 21.2 mm, 50 Å) were connected in series (Phenomenex Ltd., Cheshire, UK). An aliquot (1 ml) of the iso- hexane extract was injected via a rheodyne injector onto the GPC column, using DCM: iso-hexane (1:1, v/v) as the mobile phase, with a flow rate of 2 ml min-1. Cypermethrin eluted in the 70-140 ml fraction. The specific volumes were pre-determined, after column cleaning, by injecting 1 ml of stock solution and collecting fractions for analysis by gas chromatography with an electron capture detector (GC-ECD). The column eluent was transferred with washings (iso-hexane, 2 x 10 ± 1 ml) to a 250 ml round bottom flask. The solution was solvent exchanged to iso-hexane (20 ± 2 ml) and concentrated to ~1 ml by rotary evaporation. This step was repeated.

An SPE clean-up step, as per the determination of cypermethrin in water, was required before analysis. The samples were transferred with washings (iso-hexane, 2 x 250 µl) to a pre-weighed GC vial. The vials were re-weighed and the sample volume calculated, correcting for the density of iso-hexane.

Cypermethrin analysis by Gas Chromatography - Electron Capture Detection (GC-ECD):

Cypermethrin was analysed by GC-ECD using an HP 6890 Series II GC (Agilent Technologies, Berkshire, UK) fitted with an electron capture detector (300 ºC) and an HP-5 fused silica capillary column coated with a 0.25 µm film of 5 % phenyl-substituted methylpolysiloxane (Agilent Technologies, Berkshire, UK). Data was collected via a PE Nelson 610 link box and processed using Perkin-Elmer Turbochrom Navigator version 6.1.0.2:GO7 software (Perkin-Elmer Ltd., Cheshire, UK). A stock solution of cypermethrin was prepared (~ 1000 ng ml-1) and from this, aliquots were removed and seven dilutions prepared covering the concentration range ~ 0.4 - 100 ng ml-1. Each of the prepared solutions, including the stock solution, was analysed in triplicate by GC-ECD and the mean of the three runs determined. These standard solutions were used to prepare a calibration curve, corrected to allow for the certified concentration.

Cypermethrin method validation and quality control:

The mean total area of the four diastereoisomer peaks (Figure 13.1) in the eight cypermethrin standards was plotted against the amount injected on-column. The GC-ECD response for cypermethrin was linear within the range 0.4 to 1000 ng ml-1, with a correlation coefficient (r) of at least 0.999. In-series precision of standards by replicate analysis (n = 8) of 90 % (902.7 ng ml-1) and 10 % (100.3 ng ml-1) of the stock standard was determined. The coefficient of variation was 6.66 % at the lower concentration and 10.21 % at the higher concentration, indicating precision was better for the instrument at lower concentrations. An external standard method was used for the quantification of cypermethrin. iso-Hexane blanks were routinely analysed by GC-ECD after every five samples and following the calibration standards so as to ensure no cypermethrin carryover was occurring.

Ecological effects of sea lice medicines in Scottish sea lochs 228 of 286 Figure 13.1. Gas Chromatographic separation of the cypermethrin disteroisomers (A,B,C,D). Each peak consists of equal amounts of the two enantiomers, which are assigned as follows: A - cis 1R, 3R, R and 1S, 3S, S; B - trans 1R, 3S, R and 1S, 3R, S; C - cis 1R, 3R, S and 1S, 3S, R; D - trans 1R, 3S, S and 1S, 3R, R. Injections were on to an HP- 5 column at 16 ºC, the oven temperature was immediately increased at 50 ºC min-1 to 260 ºC where it was held for 42 min prior to final elevation, at 30 ºC min-1, to 270 ºC where it remained for 10 min.

Ecological effects of sea lice medicines in Scottish sea lochs 229 of 286 13.2 RP-HPLC analysis with fluorescence detection

13.2.1 Results

Table 13.1. Cypermethrin concentration (ng l-1) in water samples following a single cage treatment with Excis at Loch Sunart Fish farm in January 2002. Water samples were taken from the water surface in the pen before (C1a and b) and after the release of the tarpaulin (C2a and b), and from west of the cage group at a depth of 6 m (F1-2). Treatment release time was 10:58. Time Decimal Decimal Concentration Sample Code Longitude Latitude (ng l-1) C1a 10:32 3250* C1b -5.62230 56.67929 2960* -5.62239 56.67966 C2a 10:59 9.7 -5.61873 56.67974 C2b -5.61874 56.67937 3.2 F1 11:04 -5.62230 56.67929 21.1 F2 7.0 SN1 11:16 -5.62354 56.67953 2.2 SN2 2.9 SN3 11:28 -5.62429 56.67958 2.2 SN4 3.9 SN5 11:41 -5.62481 56.67935 3.6 SN6 2.9 SN7 11:52 -5.62559 56.67954 Masked SN8 Not Detected SN9 12:03 -5.62699 56.67936 Not Detected SN10 1.2 SN11 12:15 -5.62690 56.67930 2.4 SN12 Not Detected SN13 12:27 -5.62819, 56.67913, Trace SN14 -5.62833 56.67910 Masked SN15 12:40 -5.62892 56.67945 4.1 SN16 Masked SN17 12:52 -5.62883 56.67967 Masked SN18 Not Detected SN19 13:15 -5.62978, 56.67996, Not Detected -5.63007, 56.67972, SN20 -5.63003 56.67964 Masked SN21 13:23 -5.62947 56.67992 Not Detected SN22 Trace SN23 13:31 -5.62995 56.68016 1.4 SN24 Not Detected SN25 13:40 -5.62957 56.67977 Not Detected SN26 Not Detected SN27 13:49 -5.63044, 56.68016, Sample lost -5.63057, 56.68003, SN28 Masked -5.63031 56.67993 SN29 14:01 -5.62926, 56.68010, Not Detected SN30 -5.62955 56.68001 Not Detected *: Outside calibration range Masked: Peak hidden and not quantifiable, if present Trace: Small peak probably present, but less than detection limit ND: No peak present. Concentration less than detection limit

Ecological effects of sea lice medicines in Scottish sea lochs 230 of 286 13.3 Emamectin benzoate

13.3.1 Method 1

Materials, equipment and sodium sulphate preparation:

DCM, iso-hexane, methanol and acetone were purchased from Rathburn Chemicals Ltd. (Walkerburn, Scotland). Anhydrous sodium sulphate (Na2SO4) was of analytical grade and was supplied by Fisher Chemicals, Loughborough, UK. All analytical standards were greater than 95 % pure and were supplied by Thames Restek Ltd.,Windsor, UK or Sigma- Aldrich Company Ltd., Dorset, UK. Glassware was prepared as previously described in the cypermethrin method. Anhydrous sodium sulphate was washed twice with DCM followed by iso-hexane prior to drying overnight (85 ºC ± 10 ºC).

Extraction and analysis of emamectin benzoate in sediment samples (FRS Method):

Each sediment sample was thoroughly mixed and an aliquot (~ 10 g) removed for the determination of water content by oven drying at 80 ºC for 22 hrs (Webster et al., 2000a and Webster et al., 2000b). An aliquot of sediment (10 g) was accurately weighed into a solvent washed centrifuge tube, to which was added DCM (20 ml) and methanol (20 ml). The sample was sonicated and centrifuged before the addition of water (18 ml). The halogenated solvent was isolated and the sample re-extracted with a further aliquot of DCM (20 ml). The halogenated solvent was combined and dried over Na2SO4. The extract was solvent exchanged to iso-hexane and concentrated (~ 1 ml) by rotary evaporation (water bath temperature, <30 ºC). Samples were transferred to pre- conditioned (iso-hexane 10 ml) C18 solid phase extraction cartridges and washed with iso- hexane (15 ml) followed by DCM (15 ml). Emamectin benzoate was eluted from the cartridge with methanol (15 ml). The eluent was transferred, with washings, to a pear shaped flask and concentrated to dryness by rotary evaporation. Methanol (1 ml) was added and transferred to a crimp top vial for analysis by liquid chromatography mass- spectrometry (LC-MS).

A PE Sciex API 150 (Perkin Elmer, Macclesfield, UK) single quadropole mass spectrometer equipped with an atmospheric pressure chemical ionisation (APCI) source was utilised for the analysis. The LC mobile phase used was methanol/ water, using a linear gradient (8:2, 1:0, 8:2 v/v). The flow rate was set at 200 µl min-1 using an HP1100 quaternary pump. The run time was 12 minutes. A 150 x 2.00 mm ID column packed with 3 µm particles coated with a C18 stationary phase was used.

The tuning parameters were optimised in the positive ion mode by injecting 10 µl of a 1 µg ml-1 emamectin benzoate stock solution directly onto the mass spectrometer. A method was also set up for quantification purposes in single ion monitoring (SIM) mode. A seven-point calibration curve was used for quantification. The ions monitored were m/z 886.6, 872.7, 869.6 and 776.6 with a dwell time of 150 msec. Quantification was based on the area of the summed (total) ion chromatogram (TIC).

Emamectin method validation (by liquid chromatography-mass spectrometry LC-MS) and quality control:

Emamectin benzoate is a mixture of two avermectin homologues, B1a and B1b benzoate. The emamectin ion is the base ion for both homologues giving peaks at m/z 886.6 and 872.7. Correlation figures and limits of detection are detailed in the main text.

Ecological effects of sea lice medicines in Scottish sea lochs 231 of 286 13.4 Method 2

The avermectins are a family of macrocyclic lactones produced by the soil microorganism, Streptomyces avermitilis. The major fermentation product is avermectin B1 (abamectin) which is a potent broad-spectrum acaricide/insecticide registered for use worldwide (Campbell, 1989). Ivermectin, the 22,23-dehydro derivative of abamectin, is chemically modified from abamectin and has found wide use as an antiparasitic agent in animals (Campbell et al., 1983). A semi-synthetic second-generation avermectin, 4’’- deoxy-4’’-epi-methylamino-avermectin B1 benzoate salt, or emamectin benzoate, has been developed for its different spectrum of activity against lepidopteran larvae (Dybas et al., 1989).

Emamectin benzoate is currently used as an in-feed, sea lice treatment for use in aquaculture and is marketed under the brand name Slice. While emamectin is easy to administer, non-weather-dependant and effective against all parasitical stages of the louse, it has been classified as a List II dangerous substance. SEPA are therefore obliged to establish safe environmental levels and ensure that those levels are not exceeded.

Similar to abamectin, emamectin benzoate is a mixture of two homologs designated B1a and B1b (Figure 1). These homologs differ by only one methylene unit (CH2) at the 25 position, where B1a contains a sec-butyl group and B1b an isopropyl group. The ratio of these homologs in emamectin benzoate is specified as containing a minimum of 90% B1a and maximum of 10% B1b. Emamectin benzoate differs from abamectin in that the 4’’- hydroxy group of abamectin is replaced by a 4’’-epimethyl amino and it is isolated as its benzoate salt.

Environmental fate and metabolism work using radiolabelled emamectin benzoate applied to soil indicates that the parent compound is the major product present under both anaerobic and aerobic conditions.

The avermectins are not volatile enough for gas chromatographic determinations so the chromatographic separation of choice is high pressure liquid chromatography. Methods using ultraviolet detection have not been sensitive enough to detect low levels of the avermectins in complex matrices (Vuik, 1991; Pvinchny et al., 1983; Fox and Fink, 1985). Fluorescence derivatisation, taking advantage of the inherent structure of the avermectins, has been the most successful techniques (Tolan et al., 1980; Tway et al., 1981, de Montigny et al., 1990; Prabhu et al., 1991; Prabhu et al.,1992; Wehrner et al., 1993).

The method used here is largely based on that developed by Schering-Plough Animal Health Corporation, Report No. A-28923, (SCH 58854), Validation of an Analytical Method for the Quantitation of Emamectin Benzoate (SCH58854) and its Desmethylamino Metabolite (L-653,649) in Sediment Flucculent, Water, Fish, Mussels and Crustaceans.

Ecological effects of sea lice medicines in Scottish sea lochs 232 of 286 13.4.1 Method Summary

SEDIMENT (10 g)

Extract with 1% NH4OAc

COMBINED METHANOLIC EXTRACT

Concentrate and extract with EtOAc.

COMBINED EtOAc EXTRACTS

Solid phase extraction using PRS column.

SOLID PHASE EXTRACTION ELUATE

Concentrate and extract with EtOAc.

COMBINED EtOAc EXTRACTS

Concentrate and derivatise (TFAA + NMIM)

Ecological effects of sea lice medicines in Scottish sea lochs 233 of 286 13.4.2 References

Campbell, W.C., Fisher, M.H., Stapley, E.O., Albers-Schonberg, G. and Jacob, T.A. (1983). Ivermectin - a potent new anti-parasitic agent. Science, 221: 823-828.

Campbell, W.C. (Ed.) (1989). Ivermectin and abamectin. Springer-Verlag, New York. 369 pp.

De Montigny, P., Shim, J.-S.K. and Pivnichny, J.V. (1990). Liquid-chromatographic determination of ivermectin in animal plasma with trifluoroacetic-anhydride and n- methylimidazole as the derivatization reagent. Journal of Pharmaceutical and Biomedical Analysis, 8: 607-511.

Dybas, R.A., Hilton, N.J., Babu, J.R., Preiser, F.A. and Dolce, G.J. (1989). Novel second- generation avermectin insecticides and miticides for crop protection. In: Novel microbial products for medicine and agriculture. Demain, A.L., Somkuti, G.A., Hunter-Cevera, J.C. and Rossmoore, H.W. (Eds.). Elsevier, Amsterdam, Netherlands, pp. 203-212.

Fox, A. and Fink, D.W. (1985), Determination of ivermectin in feeds by high performance liquid chromatography. Analyst, 110: 259-261.

Pivnichny, J.V., Shim, J.-S.K. and Zimmerman, L.A. (1983). Direct determination of avermectins in plasma at nanogram levels by high performance liquid chromatography. Journal of Pharmaceutical Sciences, 72: 1447-1450.

Prabhu, S.V., Wehner, T.A., Egan, R.S. and Tway, P.C. (1991). Determination of 4''- deoxy-4''-(epimethylamino)avermectin B1 benzoate (MK-0244) and its delta 8,9-isomer in celery and lettuce by HPLC with fluorescence detection. Journal of Agricultural and Food Chemistry, 39: 2226-2230.

Tolan, J.W., Eskola, P., Fink, D.W., Morzik, H. and Zimmerman, L.A. (1980). Determination of avermectins in plasma at nanogram levels using high performance liquid chromatography. Journal of Chromatography, 190: 367-376.

Tway, P.C., Wood, J.S. and Downing, G.V. (1981). Determination of ivermectin in cattle and sheep tissues using high performance liquid chromatography with fluorescence detection. Journal of Agricultural and Food Chemistry, 29: 1059-1063.

Vuik, J. (1991). Rapid determination of abamectin in lettuce and cucumber using high performance liquid chromatography. Journal of Agricultural and Food Chemistry, 39: 303-305.

Wehner, T.A., Lasota, J. and Demchak, R. (1993). In: Comprehensive Analytical Profiles of Important Pesticides, Vol. 2 of Modern Methods of Pesticide Analysis. Sherma, J. and Cairns, T. (Ed.). CRC Press, Inc., Littleton, MA. pp. 75-106.

Ecological effects of sea lice medicines in Scottish sea lochs 234 of 286 13.5 Emamectin benzoate in sediment: Gorsten Experiment

13.5.1 Introduction As an adjunct to the main study on the ecological effects of sea lice chemicals, a small study was undertaken at the Marine Harvest Gorsten site to assess the small scale horizontal and vertical sediment distribution of emamectin benzoate.

The farm treated with Slice at the beginning of August 2003, with 182.5 g of active ingredient (pers. comm., D. MacGillivray, MH). The fish were harvested three months later and the farm was fallowed from November 2003. The farm was still fallow when the samples were taken in January 2004.

13.5.2 Materials and methods Sea bed core samples were obtained by diver from Gorsten fish farm, Loch Linnhe (56º 48.40N 5º 4.80W) on 14 January 2004. The cores were obtained from the cage edge in a water depth of 26 m, using perspex cores (57 mm inner diameter). All cores were taken from a 2 x 2 m grid, and sliced on shore into 2 cm slices down to a depth 8 cm. Slices were kept chilled, returned to the laboratory and frozen (nominal -20 ºC). These samples were then transported to UHI Thurso for analysis (the analytical methods are described in Section 3.2.8).

13.5.3 Results Analysis of the sediment core slices is shown below in Table 13.2. The sediment was a soft silty mud, organically enriched in the surface - 4 cm zone. Descriptively, the peak concentration (3.63 µg kg-1) was not in the surface 0-2 cm, but at 2-4 cm. Additionally, there were quantifiable amounts of the chemical at the 4-6 cm horizon. No emamectin benzoate was detected in any 6-8 cm slice (Figure 13.2), nor was any metabolite detected in any slice.

Ecological effects of sea lice medicines in Scottish sea lochs 235 of 286 Figure 13.2. Vertical distribution of emamectin benzoate in sediment from Gorsten fish farm, Loch Linnhe on 14 January 2004.

It is interesting to note the relatively even horizontal dispersion of the chemical in the 4 m2 sample grid (Figure 13.3), along with the small variability between samples (apart from one sample from the 2-4 cm horizon, in which no emamectin benzoate was detected). The relatively even horizontal distribution may be a function of the small grid size (2 x 2 m), and/or the proximity of the sampled area relative to the original source of the chemical.

Ecological effects of sea lice medicines in Scottish sea lochs 236 of 286 Figure 13.3. Mean sediment concentration of emamectin benzoate by depth, from Gorsten fish farm, Loch Linnhe on 14 January 2004. Error bars are standard deviation.

Ecological effects of sea lice medicines in Scottish sea lochs 237 of 286 Table 13.2. Sediment emamectin benzoate concentration by depth from core slices taken at Gorsten fish farm, Loch Linnhe on 14 January 2004, approx. 4 months post-treatment. No metabolite was detected in any sample. Sample Emamectin (µg kg-1) Core 1 0-2 cm 2.92 2-4 cm 2.98 4-6 cm 1.31 6-8 cm ND

Core 2 0-2 cm 2.53 2-4 cm 3.63 4-6 cm 2.21 6-8 cm ND

Core 3 0-2 cm 2.63 2-4 cm 3.41 4-6 cm 1.80 6-8 cm ND

Core 4 0-2 cm 2.28 2-4 cm 2.35 4-6 cm 1.54 6-8 cm ND

Core 5 0-2 cm 2.46 2-4 cm 2.59 4-6 cm 1.48 6-8 cm ND

Core 6 0-2 cm 1.87 2-4 cm 3.08 4-6 cm 2.84 6-8 cm ND

Core 7 0-2 cm 2.42 2-4 cm ND 4-6 cm 1.64

Core 8 0-2 cm 2.75 2-4 cm 3.58 4-6 cm 1.94 6-8 cm ND

Core 9 0-2 cm 2.45 2-4 cm 2.53 4-6 cm 1.30 6-8 cm ND

Ecological effects of sea lice medicines in Scottish sea lochs 238 of 286 14 Appendix III - Phytoplankton

Table 14.1. Physico-chemical data at Loch Sunart (top 10 m): 17 November to 14 December 2000. Salinity and dissolved inorganic nutrients (µM). ToxN = nitrate plus nitrite; NS = No Sample. Treatment Salinity ToxN Phosphate Ammonium Silicate Date Day (no units) (µM) (µM) (µM) (µM) 17 Nov 2000 - 10 31.11 7.1 0.75 1.2 8.6 23 Nov 2000 - 4 31.18 6.7 0.63 0.9 8.4 27 Nov 2000 - 1 31.12 NS NS NS 8.9 28 Nov 2000 * T1 31.61 7.8 0.70 0.6 8.1 29 Nov 2000 * T2 31.76 8.0 0.74 0.9 8.7 30 Nov 2000 * T3 29.99 NS NS NS 8.5 01 Dec 2000 * T4 31.28 8.4 0.71 0.5 8.0 07 Dec 2000 10 30.01 7.4 0.68 0.8 8.2 14 Dec 2000 23 28.39 7.1 0.64 0.4 8.9 * Sea lice treatment days.

Table 14.2. Phytoplankton community composition (cells l-1) at Loch Sunart: 17 November to 14 December 2000. Treatment Diatoms Dinoflagellates Microflagellates Others a Date Day (cells l-1 x 103) (cells l-1 x 103) (< 15 µm) (cells l-1 x 103) (cells l-1 x 103) 17 Nov 2000 - 10 15 11 164 49 23 Nov 2000 - 4 62 12 233 43 27 Nov 2000 - 1 24 9 132 56 28 Nov 2000 * T1 13 5 128 40 29 Nov 2000 * T2 8 8 76 42 30 Nov 2000 * T3 8 8 141 26 01 Dec 2000 * T4 7 10 90 16 07 Dec 2000 10 7 6 113 15 14 Dec 2000 23 17 13 123 37 * Sea lice treatment days. a.“Others” includes other flagellates, cryptophytes, ciliates, cysts/ resting stages and silicoflagellates (e.g. Dictyocha speculum).

Ecological effects of sea lice medicines in Scottish sea lochs 239 of 286 Table 14.3. Dominant phytoplankton taxa at Loch Sunart: 17 November to 14 December 2000. 1 = most abundant; 5 = fifth most abundant; % = contribution to total phytoplankton population. Treatment 1 (Most 2 3 4 5 % Date Day Abundant) 17 Nov - 10 Micro-flagellate Cryptophyte Small Skeletonema Flagellate 92 2000 (< 30 µm) Dinoflagellate costatum (30-60 µm) (< 15 µm) 23 Nov - 4 Micro-flagellate Pseudo- Cryptophyte Pennate Small 87 2000 (< 30 µm) nitzschia sp. Diatom Dinoflagellate Var. 3 (< 15 µm) 27 Nov - 1 Micro-flagellate Cryptophyte Pseudo- Skeletonema Small 89 2000 (< 30 µm) nitzschia sp. costatum Dinoflagellate (< 15 µm) 28 Nov T1 Micro-flagellate Cryptophyte Skeletonema Small Cyst/spore 95 2000 * (< 30 µm) costatum Dinoflagellate (< 15 µm) 29 Nov T2 Micro-flagellate Cryptophyte Cyst/spore Skeletonema Small 89 2000 * (< 30 µm) costatum Dinoflagellate (< 15 µm) 30 Nov T3 Micro-flagellate Cryptophyte Small Pseudo- Cyst/spore 95 2000 * (< 30 µm) Dinoflagellate nitzschia sp. (< 15 µm) 01 Dec T4 Micro-flagellate Cryptophyte Small Ciliate Asterionellopsis 89 2000 * (< 30 µm) Dinoflagellate glacialis (< 15 µm) 07 Dec 10 Micro-flagellate Small Ciliate Cryptophyte Skeletonema 96 2000 (< 30 µm) Dinoflagellate costatum (< 15 µm) 14 Dec 23 Micro-flagellate Cryptophyte Ciliate Small Naviculoid 87 2000 (< 30 µm) Dinoflagellate Diatom (< 15 µm) * Sea lice treatment day.

Table 14.4. Phytoplankton blooms at Loch Sunart between June 2000 and April 2004. Date Taxa responsible Maximum cell abundance (cells l-1) August 2000 Chaetoceros sp. 1.1 x 106 cells l-1 April/May 2001 Skeletonema costatum 1.8 x 106 cells l-1 June 2001 Chaetoceros sp. 9.4 x 103 cells l-1 September 2001 Chaetoceros sp. 1.3 x 106 cells l-1 March 2002 S. costatum 9.3 x 103 cells l-1 May 2002 Chaetoceros sp. 1.7 x 106 cells l-1 June 2002 Unidentified cryptophyte 2.6 x 106 cells l-1 July 2002 Chaetoceros sp. 2.0 x 106 cells l-1 August 2002 Dactyliosolen fragillismus 2.9 x 106 cells l-1 May 2003 S. costatum 5.3 x 106 cells l-1 July 2003 Chaetoceros sp. 9.7 x 103 cells l-1 March 2004 S. costatum 17.2 x 106 cells l-1

Ecological effects of sea lice medicines in Scottish sea lochs 240 of 286 Table 14.5. Physico-chemical characteristics at Loch Sunart (top 10 m) between June 2000 and April 2004. Salinity (no units) and dissolved inorganic nutrients (µM). ToxN = nitrate plus nitrite;

Ecological effects of sea lice medicines in Scottish sea lochs 241 of 286 Table 14.5 (cont.) Date Salinity ToxN Phosphate Ammonium Silicate (µM) (µM) (µM) (µM) 5 Oct 01 31.19 3.4 0.65 5.2 NS 12 Oct 01 28.13 2.4 1.41 5.3 NS 19 Oct 01 31.56 2.8 0.69 3.0 NS 26 Oct 01 31.65 4.8 0.69 4.6 NS 2 Nov 01 28.44 5.3 0.58 0.9 NS 9 Nov 01 26.44 5.4 0.52 1.5 NS 15 Nov 01 30.20 6.1 0.64 1.6 NS 20 Nov 01 31.48 6.8 0.63 1.1 9.1 23 Nov 01 31.09 6.8 0.64 3.9 NS 29 Nov 01 27.82 6.3 0.82 2.8 NS 4 Dec 01 26.81 6.3 0.56 1.0 10.4 10 Dec 01 31.13 7.4 0.73 2.1 NS 14 Dec 01 NS 7.5 0.73 3.0 NS 20 Dec 01 32.54 7.3 0.70 1.2 8.6 21 Dec 01 32.33 7.1 0.62 1.8 NS 8 Jan 02 32.76 7.1 0.69 2.1 8.3 11 Jan 02 32.87 7.0 0.63 1.1 NS 18 Jan 02 32.32 5.2 0.67 3.0 NS 25 Jan 02 29.36 6.9 0.30 3.3 NS 27 Jan 02 30.32 NS NS NS 8.3 29 Jan 02 NS NS NS NS NS 2 Feb 02 NS 6.7 0.89 3.1 NS 6 Feb 02 28.79 6.8 0.58 2.4 9.0 8 Feb 02 28.59 6.9 0.60 9.2 NS 12 Feb 02 27.35 6.6 0.15 9.0 NS 26 Feb 02 28.33 6.8 1.26 3.0 9.1 8 Mar 02 26.84 5.6 0.13 2.3 NS 21 Mar 02 31.74 5.2 0.53 2.8 NS 12 Apr 02 33.31 1.9 0.40 5.9 NS 1 May 02 31.27 0.1 0.61 0.8 2.1 10 May 02 32.89 0.04 0.04 1.8 1.0 15 May 02 NS 0.1 0.28 3.5 3.9 17 May 02 33.24 0.4 0.10 3.7 2.2 24 May 02 28.84 0.6 0.04 4.3 3.1 1 Jun 02 32.01 0.3 0.03 4.3 2.5 5 Jun 02 31.97

Ecological effects of sea lice medicines in Scottish sea lochs 242 of 286 Table 14.5 (cont.) Date Salinity ToxN Phosphate Ammonium Silicate (µM) (µM) (µM) (µM) 22 Jan 03 31.08 7.3 0.65 NS 10.5 27 Jan 03 29.35 6.73 0.83 NS 10.41 5 Feb 03 29.79 NS NS NS NS 14 Feb 03 31.18 7.80 0.18 NS 11.47 20 Feb 03 32.73 7.16 1.13 NS 2.72 20 Feb 03 32.72 6.25 0.19 NS 9.30 27 Feb 03 33.03 6.43 0.31 NS 8.70 6 Mar 03 32.36 6.05 0.57 NS 10.17 6 Mar 03 32.11 6.60 0.23 NS 8.54 14 Mar 03 31.40 6.71 0.26 NS 8.72 19 Mar 03 32.58 3.76 0.66 NS NS 27 Mar 03 33.50 6.97 0.29 NS 7.63 4 Apr 03 32.93 NS NS NS 2.71 8 Apr 03 33.80

Ecological effects of sea lice medicines in Scottish sea lochs 243 of 286 Table 14.6. Phytoplankton community composition at Loch Sunart (cells l-1) between June 2000 and April 2004. NS = No Sample. Date Diatoms Dinoflagellates Microflagellates Others * (cells l-1 x 103) (cells l-1 x 103) (< 15 µm) (cells l-1 x 103) (cells l-1 x 103) 5 Jun 00 121 138 513 133 16 Jun 00 1299 87 328 143 23 Jun 00 681 91 735 129 30 Jun 00 802 201 279 154 7 Jul 00 1157 217 407 302 21 Jul 00 1408 152 1601 554 28 Jul 00 277 44 502 187 4 Aug 00 162 75 596 458 18 Aug 00 269 77 319 277 25 Aug 00 NS NS NS NS 2 Sep 00 594 139 514 89 9 Sep 00 265 43 209 65 23 Sep 00 27 29 252 73 17 Nov 00 15 11 164 49 23 Nov 00 62 12 233 43 27 Nov 00 24 9 132 56 28 Nov 00 13 5 128 40 29 Nov 00 8 8 76 42 30 Nov 00 8 8 141 26 1 Dec 00 7 10 90 16 7 Dec 00 7 6 113 15 14 Dec 00 17 13 123 37 11 Feb 01 15 12 272 30 23 Feb 01 35 2 432 81 2 Mar 01 63 16 547 101 12 Mar 01 74 8 206 52 26 Mar 01 143 22 311 60 4 Apr 01 75 8 1506 45 13 Apr 01 2045 11 1559 23 19 Apr 01 NS NS NS NS 25 Apr 01 825 20 445 23 26 Apr 01 NS NS NS NS 1 May 01 1647 56 876 155 9 May 01 288 186 2542 108 18 May 01 NS NS NS NS 25 May 01 34 79 2378 249 30 May 01 413 67 498 80 8 Jun 01 17 235 1812 317 14 Jun 01 159 136 678 211 22 Jun 01 998 18 582 123 29 Jun 01 988 38 561 69 5 Jul 01 105 60 1747 94 13 Jul 01 144 83 730 132 19 Jul 01 314 50 974 101 26 Jul 01 254 20 286 63 4 Aug 01 NS NS NS NS 10 Aug 01 NS NS NS NS 17 Aug 01 NS NS NS NS 23 Aug 01 70 40 679 102 30 Aug 01 640 58 712 393 6 Sep 01 1524 83 1152 423 14 Sep 01 418 105 523 147 20 Sep 01 71 26 497 78 28 Sep 01 308 56 543 91 5 Oct 01 45 85 2503 102 12 Oct 01 19 38 283 63 19 Oct 01 7 30 144 12 26 Oct 01 21 39 236 26

Ecological effects of sea lice medicines in Scottish sea lochs 244 of 286 Table 14.6 (cont.) Date Diatoms Dinoflagellates Microflagellates Others * (cells l-1 x 103) (cells l-1 x 103) (< 15 µm) (cells l-1 x 103) (cells l-1 x 103) 2 Nov 01 9 42 2020 61 9 Nov 01 NS NS NS NS 15 Nov 01 3 23 604 45 20 Nov 01 3 2 657 21 23 Nov 01 10 15 441 23 29 Nov 01 5 7 220 68 4 Dec 01 0 9 845 38 10 Dec 01 5 19 81 8 14 Dec 01 2 14 51 10 20 Dec 01 13 9 248 15 21 Dec 01 9 8 427 23 8 Jan 02 3 3 31 8 11 Jan 02 4 5 126 15 18 Jan 02 8 3 76 6 25 Jan 02 3 3 66 27 27 Jan 02 3 2 113 17 29 Jan 02 NS NS NS NS 2 Feb 02 1 0 81 4 6 Feb 02 6 9 77 26 8 Feb 02 6 5 132 13 12 Feb 02 3 2 28 10 26 Feb 02 50 3 98 12 8 Mar 02 253 6 200 21 21 Mar 02 1008 30 638 174 12 Apr 02 298 56 1235 132 1 May 02 463 18 NS 131 10 May 02 1695 56 4305 45 15 May 02 970 54 284 130 17 May 02 610 85 2717 141 24 May 02 99 106 3107 99 1 Jun 02 17 203 3090 311 5 Jun 02 17 213 664 448 11 Jun 02 45 102 3740 1164 15 Jun 02 23 102 2655 2655 18 Jun 02 670 121 808 316 1 Jul 02 508 102 2881 266 03 Jul 02 766 209 409 258 18 Jul 02 3053 116 717 350 31 Jul 02 39 153 533 45 10 Aug 02 83 41 1943 132 14 Aug 02 680 53 894 218 16 Aug 02 378 17 3259 141 22 Aug 02 2011 23 4519 192 28 Aug 02 3888 188 3314 132 6 Sep 02 288 31 1438 37 11 Sep 02 249 85 2525 23 17 Sep 02 742 81 3301 210 23 Sep 02 994 56 2164 79 27 Sep 02 480 56 2678 28 11 Oct 02 144 31 2013 41 15 Oct 02 33 37 666 37 18 Oct 02 108 38 1859 80 24 Oct 02 17 28 2186 40 30 Oct 02 8 12 489 65 11 Nov 02 34 15 1770 41 13 Nov 02 2 14 442 12 27 Nov 02 3 5 322 6 9 Jan 03 6 0 654 11 22 Jan 03 23 11 836 41 27 Jan 03 17 6 623 29 5 Feb 03 9 23 740 121 14 Feb 03 10 11 535 58

Ecological effects of sea lice medicines in Scottish sea lochs 245 of 286 Table 14.6 (cont.) Date Diatoms Dinoflagellates Microflagellates Others * (cells l-1 x 103) (cells l-1 x 103) (< 15 µm) (cells l-1 x 103) (cells l-1 x 103) 20 Feb 03 7 6 624 82 20 Feb 03 10 10 778 48 27 Feb 03 13 13 791 149 6 Mar 03 30 20 745 152 6 Mar 03 27 6 654 59 14 Mar 03 55 15 1257 215 19 Mar 03 223 93 1322 121 27 Mar 03 154 38 1793 143 4 Apr 03 738 2 201 6 8 Apr 03 NS NS NS NS 9 Apr 03 1206 35 1046 45 14 Apr 03 706 32 1408 106 24 Apr 03 140 45 804 23 25 Apr 03 24 6 627 49 9 May 03 13 0 1290 15 9 May 03 0 0 10187 38 21 May 03 82 9 1216 38 21 May 03 61 29 1189 95 29 May 03 5378 124 1627 158 5 Jun 03 18 132 1480 407 13 Jun 03 105 26 1359 55 18 Jun 03 19 72 1759 64 26 Jun 03 51 108 4576 226 7 Jul 03 655 90 5728 181 16 Jul 03 638 62 2904 85 24 Jul 03 1124 350 2887 260 30 Jul 03 40 141 2503 277 4 Aug 03 NS NS NS NS 19 Aug 03 378 215 2678 294 29 Aug 03 178 169 5649 202 29-Aug-03 367 198 3084 277 30-Sep-03 350 73 2288 171 1-Oct-03 212 27 965 50 22-Oct-03 45 45 1328 121 30-Oct-03 370 20 1429 184 5-Nov-03 47 27 706 57 10-Nov-03 52 15 542 59 3-Dec-03 2 10 464 36 12-Jan-04 400 2 161 13 10-Feb-04 1 2 191 19 24-Feb-04 21 32 335 161 10-Mar-04 2873 85 972 266 25-Mar-04 17794 282 418 271 13-Apr-04 75 83 1039 897 28-Apr-04 9 38 847 232 * “Others” includes other flagellates, silicoflagellates, cryptophytes, ciliates, tintinnids, and resting stages/cysts.

Ecological effects of sea lice medicines in Scottish sea lochs 246 of 286 Table 14.7. Physico-chemical characteristics at Loch Diabaig (top 10 m) from July 2000 to October 2001. Salinity (no units) and dissolved inorganic nutrients (µM). ToxN = nitrate plus nitrite; NS = No Sample. Date Salinity ToxN Phosphate Ammonium Silicate (µM) (µM) (µM) (µM) 28 Jul 00 34.29 0.3 0.18 0.7 0.7 02 Aug 00 34.26 0.2 0.17 0.4 0.5 11 Aug 00 34.23 0.2 0.14 0.3 1.5 22 Aug 00 34.09 0.3 0.20 0.2 3.0 30 Aug 00 34.31 0.7 0.19 1.2 2.8 08 Sep 00 34.33 0.2 0.23 0.5 0.3 18 Sep 00 34.26 NS NS 6.2 0.6 24 Jan 01 NS NS NS NS NS 01 Feb 01 33.76 5.4 0.58 1.1 5.4 13 Feb 01 33.84 5.9 0.59 0.6 5.3 27 Mar 01 34.10 4.4 0.52 0.5 4.4 11 Apr 01 34.08 6.3 0.60 1.2 3.6 11 May 01 34.23 7.6 0.73 1.0 3.1 14 May 01 34.27 1.4 0.36 1.3 2.3 06 Jun 01 33.64 1.3 0.22 1.5 3.1 18 Jun 01 34.04 0.7 0.17 1.1 1.7 21 Jun 01 32.66 0.4 0.16 1.1 2.1 22 Jun 01 33.23 0.5 0.21 1.2 1.9 26 Jun 01 33.70 1.2 0.27 1.4 2.3 27 Jun 01 33.77 0.9 0.25 1.6 2.5 02 Jul 01 NS 1.1 0.25 0.8 NS 11 Jul 01 33.30 0.4 0.24 0.7 2.2 12 Jul 01 32.39 0.3 0.18 0.9 2.4 01 Aug 01 34.01 0.6 0.27 3.2 2.0 29 Aug 01 33.98 0.2 0.15 0.9 0.5 30 Aug 01 34.00 0.7 0.24 0.4 0.8 31 Aug 01 33.97 0.4 0.24 1.3 1.2 01 Sep 01 34.09 0.8 0.28 1.5 1.3 02 Sep 01 33.76 0.5 1.23 1.3 1.0 03 Sep 01 32.48 0.8 0.25 1.4 2.3 04 Sep 01 33.73 0.7 0.21 1.0 1.2 05 Sep 01 33.75 0.9 0.24 1.1 1.5 18 Sep 01 33.68 2.1 0.58 4.8 2.9 06 Oct 01 NS NS NS NS NS 08 Oct 01 33.72 3.6 0.50 3.0 3.7

Ecological effects of sea lice medicines in Scottish sea lochs 247 of 286 Table 14.8. Phytoplankton community composition at Loch Diabaig from July 2000 to October 2001. NS = No Sample. Date Diatoms Dinoflagellates Microflagellates Others* (cells l-1 x 103) (cells l-1 x 103) (<15 µm)(cells l-1 x 103) (cells l-1 x 103) 28 Jul 00 412 40 469 88 02 Aug 00 NS NS NS NS 11 Aug 00 1 212 151 33 22 Aug 00 5 382 258 107 30 Aug 00 72 222 244 42 08 Sep 00 305 250 385 65 18 Sep 00 347 202 349 63 24 Jan 01 20 17 440 35 01 Feb 01 23 13 424 24 13 Feb 01 5 5 334 9 27 Mar 01 647 4 313 89 11 Apr 01 279 3 211 81 11 May 01 129 24 725 21 14 May 01 241 79 1405 38 06 Jun 01 111 174 597 219 18 Jun 01 NS NS NS NS 21 Jun 01 68 68 3254 1073 22 Jun 01 56 113 3662 668 26 Jun 01 10 105 660 342 27 Jun 01 NS NS NS NS 02 Jul 01 NS NS NS NS 11 Jul 01 21 89 1280 61 12 Jul 01 NS NS NS NS 01 Aug 01 NS NS NS NS 29 Aug 01 879 71 718 107 30 Aug 01 1654 72 937 180 31 Aug 01 621 34 760 175 01 Sep 01 1210 138 742 165 02 Sep 01 671 44 867 97 03 Sep 01 1525 108 885 328 04 Sep 01 296 40 778 104 05 Sep 01 131 30 495 46 18 Sep 01 NS NS NS NS 06 Oct 01 14 17 435 105 08 Oct 01 NS NS NS NS * “Others” includes other flagellates, silicoflagellates, cryptophytes, ciliates, tintinnids and resting stages/cysts.

Ecological effects of sea lice medicines in Scottish sea lochs 248 of 286 Table 14.9. Physico-chemical characteristics at Loch Kishorn (top 10 m): June, July and August 2001. Salinity (no units) and dissolved inorganic nutrients (µM). ToxN = nitrate plus nitrite; NS = No Sample. Date Station Salinity ToxN Phosphate Ammonium Silicate (µM) (µM) (µM) (µM) 12 Jun 01 C 33.65 NS NS NS NS 12 Jun 01 D 33.68 NS NS NS NS 28 Jun 01 C 33.73 1.5 0.31 1.1 2.6 28 Jun 01 D 33.67 1.4 0.29 0.7 2.6 04 Jul 01 C 33.35 NS NS NS 2.1 04 Jul 01 D 33.45 1.4 0.32 1.7 2.2 17 Jul 01 C 33.82 0.3 0.21 0.9 0.7 17 Jul 01 D 33.79 0.4 0.27 1.3 0.7 23 Jul 01 * A 33.71 0.3 0.48 0.6 1.5 23 Jul 01 * B 33.64 0.4 0.36 0.3 2.3 23 Jul 01 * C 33.65 0.3 0.48 0.4 1.6 23 Jul 01 * D 33.62 0.3 0.38 0.7 1.9 23 Jul 01 * E 33.61 0.2 0.42 0.3 1.6 24 Jul 01 * C 33.53 0.4 0.40 0.6 1.4 24 Jul 01 * D 33.55 0.2 0.49 0.2 1.3 25 Jul 01 * C 33.64 0.8 0.70 0.7 1.6 25 Jul 01 * D 33.46 0.5 0.38 0.4 1.8 26 Jul 01 * A 33.66 0.6 0.39 0.5 1.4 26 Jul 01 * B 33.57 0.7 0.20 0.6 1.4 26 Jul 01 * C 33.53 0.5 0.19 0.8 1.5 26 Jul 01 * D 33.49 0.5 0.21 0.5 1.5 26 Jul 01 * E 33.54 0.5 0.19 0.5 1.4 27 Jul 01 * C 33.53 0.7 0.22 0.4 1.5 27 Jul 01 * D 33.49 0.4 0.20 0.5 1.3 28 Jul 01 * C 33.38 0.5 0.19 0.7 1.3 28 Jul 01 * D 33.19 0.3 0.58 0.2 1.8 29 Jul 01 * D 32.90 0.2 0.17 0.4 1.4 29 Jul 01 * E 32.98 0.3 0.16 0.4 1.6 31 Jul 01 C 33.52 0.2 0.21 0.7 0.9 31 Jul 01 D 33.38 0.2 0.15 0.5 0.9 08 Aug 01 C 33.67 0.7 0.31 0.8 2.0 08 Aug 01 D 33.68 0.9 0.27 0.9 2.3 21 Aug 01 C 33.29 0.5 0.03 0.8 2.5 * Slice treatment day

Ecological effects of sea lice medicines in Scottish sea lochs 249 of 286 Table 14.10. Phytoplankton community composition at Loch Kishorn: June, July and August 2001. Date Site Diatoms Dinoflagellates Microflagellates Others A Station (cells l-1 x 103) (cells l-1 x 103) (< 15 µm) (cells l-1 x 103) (cells l-1 x 103) 12 Jun 01 C 109 16 610 84 12 Jun 01 D 124 20 576 37 28 Jun 01 C 587 98 805 118 28 Jun 01 D 416 75 786 74 04 Jul 01 C 214 90 911 97 04 Jul 01 D 229 84 1064 143 17 Jul 01 C 114 38 1274 84 17 Jul 01 D 187 63 1135 93 23 Jul 01 * A 168 27 436 108 23 Jul 01 * B 165 24 487 80 23 Jul 01 * C 189 28 512 66 23 Jul 01 * D 136 13 473 47 23 Jul 01 * E 210 31 741 86 24 Jul 01 * C 119 24 674 78 24 Jul 01 * D 175 83 618 96 25 Jul 01 * C 157 48 598 75 25 Jul 01 * D 165 35 601 31 26 Jul 01 * A 147 44 587 43 26 Jul 01 * B 131 47 512 84 26 Jul 01 * C 172 46 604 32 26 Jul 01 * D 138 31 578 45 26 Jul 01 * E 155 54 647 58 27 Jul 01 * C 169 49 532 103 27 Jul 01 * D 181 55 639 56 28 Jul 01 * C 130 47 577 62 28 Jul 01 * D 226 48 499 81 29 Jul 01 * D 153 66 598 90 29 Jul 01 * E 99 37 437 46 31 Jul 01 C 128 122 628 83 31 Jul 01 D 159 201 527 71 08 Aug 01 C 132 229 914 49 08 Aug 01 D 96 178 868 42 21 Aug 01 C 28 425 1049 211 * Slice treatment day. A. “Others” includes two further classes of flagellates, cryptophytes, ciliates, and tintinnids.

Ecological effects of sea lice medicines in Scottish sea lochs 250 of 286 Table 14.11. Physico-chemical characteristics at Loch Craignish (top 10 m) from November 2000 to September 2001. NS = No Sample. Date Salinity ToxN Phosphate Ammonium Silicate (psu) (µM) (µM) (µM) (µM) 22 Nov 00 33.65 6.1 0.64 1.3 5.1 06 Dec 00 32.57 6.8 0.69 2.4 6.4 20 Dec 00 33.17 7.1 0.81 1.1 6.3 10 Jan 01 33.27 7.4 0.75 1.2 6.0 24 Jan 01 33.33 8.0 0.79 NS 6.2 07 Feb 01 33.33 7.8 0.80 1.2 6.6 16 May 01 33.85 2.1 0.32 1.2 1.3 30 May 01 33.97 0.5 0.25 0.7 0.8 13 Jul 01 33.89 1.0 0.35 1.7 2.1 01 Aug 01 33.77 0.3 0.17 0.4 1.3 15 Aug 01 33.35 1.3 0.34 2.0 3.4 29 Aug 01 33.56 0.4 0.20 0.5 1.0 12 Sep 01 33.63 0.4 0.26 0.7 2.2 26 Sep 01 33.90 1.4 0.31 0.6 2.6

Table 14.12. Phytoplankton community composition at Loch Craignish (cells l-1) from June 2000 to September 2001. NS = No Sample. Date Diatoms Dinoflagellates Microflagellates Others * (cells l-1 x 103) (cells l-1 x 103) (< 15 µm size) (cells l-1 x 103) (cells l-1 x 103) 06 Jun 00 605 39 716 218 16 Jun 00 369 37 982 220 23 Jun 00 329 60 778 111 30 Jun 00 767 50 974 221 28 Jul 00 291 32 609 105 04 Aug 00 246 30 1107 101 16 Aug 00 817 65 975 152 08 Nov 00 14 9 167 21 22 Nov 00 45 17 140 62 06 Dec 00 14 7 126 73 20 Dec 00 10 4 100 17 10 Jan 01 6 4 91 44 24 Jan 01 18 3 112 18 07 Feb 01 30 3 169 30 16 May 01 2294 11 878 262 30 May 01 3817 55 816 217 14 Jun 01 2195 61 2164 350 13 Jul 01 842 25 1023 73 01 Aug 01 944 96 763 187 15 Aug 01 415 45 1264 77 29 Aug 01 1627 121 915 243 12 Sep 01 1091 62 823 100 26 Sep 01 695 61 939 136 * “Others” includes other flagellates, silicoflagellates, cryptophytes, ciliates, tintinnids and resting stages/cysts.

Ecological effects of sea lice medicines in Scottish sea lochs 251 of 286 Table 14.13. Phytoplankton taxa observed in each loch. Sampling period is given in row 2. CRAIGNISH DIABAIG KISHORN SUNART 6/6/0-26/9/1 28/7/0-8/10/1 12/6/1-21/8/1 5/6/0-28/4/4 Dinoflagellate categories UI dinoflagellate (< 15 µm)     UI dinoflagellate (> 15 µm)     Alexandrium spp.     Amphidinium spp.     Amylax tricantha -  - - Ceratium furca     Ceratium fusus     Ceratium lineatum     Ceratium tripos - - - - Dinophysis acuminata     Dinophysis acuta     Dinophysis norvegica     Diplopsalopsis sp. - -  - Dissodinium spp.  -  Gonyaulax spp.     Gymnodinium spp.     Gymnodinium cf. mikimotoi   -  Gyrodinium spp.     Heterocapsa spp.     Heterocapsa triquetra   -  Katodinium spp.   -  Oxytoxum spp.   -  Polykrikos spp. -   - Prorocentrum gracile -    Prorocentrum micans     Prorocentrum minimum - -   Protoperidinium spp.     Scrippsiella spp.     Diatom categories UI Diatom Auxospore  - -  UI Naviculoid Diatom     UI Centric Diatom     UI Pennate diatom (variant 1)     UI Pennate diatom (variant 2)     UI Pennate diatom (variant 3)   -  UI Pennate diatom (variant 4)     UI Pennate diatom (variant 5)     UI Pennate diatom (variant 6)   -  Asterionellopsis glacialis   -  Attheya longicornus  - -  Bacteriastrum spp.  - -  Cerataulina pelagica     Chaetoceros spp. (< 15 µm)     Chaetoceros spp. (> 15 µm)     Corethron cf. criophilum  - -  Coscinodiscus spp. -  -  Cylindrotheca closterium /     Nitzschia longissma-type Dactyliosolen fragillismus     Delphineus spp. - - - 

Ecological effects of sea lice medicines in Scottish sea lochs 252 of 286 Table 14.13 (cont.) CRAIGNISH DIABAIG KISHORN SUNART Detonula spp.   -  Ditylum brightwellii   -  Entomoneis spp. - - -  Eucampia zodiacus     Fragillariopsis spp.   -  Guinardia delicatula     Guinardia flaccida     Gyrosigma/Pleurosigma-type     Lauderia annulata     Lennoxia faveolata     Leptocylindrus danicus     Leptocylindrus mediterraneus   -  Leptocylindrus minimus     Licmophora spp.   -  Melosira spp.  - -  Paralia sulcata   -  Porosira spp. -  -  Proboscia alata   -  Pseudo-nitzschia spp.     Rhizosolenia hebetata - - -  Rhizosolenia imbricata -  - - Rhizosolenia setigera     Rhizosolenia shrubsolei   -  Rhizosolenia stolterfothii     Rhizosolenia styliformis   -  Scenedesmus spp. - - -  Skeletonema costatum     Stephanopyxis turris  - -  Striatella unipunctata - - -  Thalassionema spp.     Thalassiosira spp.     Other categories UI Flagellate (< 30 µm)     UI Flagellate (30-60 µm)     UI Flagellate (> 60 µm)     UI Cryptophyte     UI Ciliate     UI Tintinnid     UI Cyst / resting stage / spore   -  Dictyocha fibula -  - - Dictyocha speculum    

Ecological effects of sea lice medicines in Scottish sea lochs 253 of 286 15 Appendix IV - Macrofauna

Table 15.1. Descriptions of sediment core samples at Loch Sunart (1999 - 2002). (a) Station LSB1 Position rel. Sediment Sediment Date Core Surface Column to fish farm classification type Length: 85 mm. Water column clear. 0-55 mm: light brown. 200 m E 18/10/99 1 2 Sand/mud Sediment surface undisturbed. 55-85 mm: dark brown Several worms & worm tubes. 45 mm: a single worm. Length: 110 mm. Water column disturbed. 2 2 Sand/mud Brown throughout. Sediment surface obscured. No fauna observed. Length: 115 mm. Water column disturbed. 29/02/00 1 2 Sand/mud Light brown throughout. Sediment surface obscured. No fauna observed. Length: 95 mm. Water column clear. 0- 5 mm: very soft mud. 2 2 Sand/mud Sediment surface undisturbed. 5-95 mm: light brown A few worm tubes. sand/mud. 50 mm: a single worm. Water column disturbed. Length: 120 mm. 06/11/00 1 2 Sand/mud Sediment surface disturbed. Dark brown throughout. Several worm tubes. No fauna observed. Water column clear. Length: 95 mm. 2 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. Several worm tubes. No fauna observed. Length: 110mm. Water column disturbed. 08/03/01 1 2 Sand/mud Light grey throughout. Sediment surface flocculant. No fauna observed. Length: 140mm. No overlying water Light grey throughout. 2 2 Sand/mud Sediment surface flocculant. 80-140 mm: black streaks No fauna observed. Length: 70 mm. Water column clear. Light brown throughout. 01/11/02 1 2 Sand/mud Sediment surface undisturbed. 2 burrows observed at 30 A single worm tube. and 40 mm. Water column clear. Length: 70 mm. 2 2 Sand/mud Sediment surface undisturbed. Light brown throughout. A few worm tubes observed. No fauna observed.

Ecological effects of sea lice medicines in Scottish sea lochs 254 of 286 Table 15.1 (cont.) (b) Station LSB2 Position rel. Sediment Sediment Date Core Surface Column to fish farm classification type Length: 100 mm. Water column disturbed. 0-70 mm: light brown. 200 m N 18/10/99 1 2 Sand/mud Sediment surface obscured. 70-100 mm: dark brown. No fauna observed. Length: 85 mm. 60-70 mm: dark brown Water column disturbed. 2 2 Sand/mud patch. Sediment surface obscured. Brown throughout. No fauna observed. Water column disturbed. Length: 110 mm. 29/02/00 1 2 Sand/mud Sediment surface disturbed. Light brown throughout. A few worms. 65 mm: 1 worm Length: 105 mm. Water column disturbed. Light brown throughout. 2 2 Sand/mud Sediment surface disturbed. 60 mm: dark brown A few worm tubes. patch. No fauna observed. Water column clear. Length: 80 mm. 06/11/00 1 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. A few worm tubes. 35 mm: A single worm. Water column clear. Length: 55 mm. 2 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. A few worm tubes. 15 mm: A single worm. No overlying water. Length: 75 mm. Sediment surface undisturbed Dark brown throughout. 08/03/01 1 2 Sand/mud and uneven. 15 mm: A single worm. A single worm tube. Length: 90 mm. No overlying water. 0-30 mm: light brown 2 2 Sand/mud Sediment surface undisturbed. 30-90 mm: dark brown. No fauna observed No fauna observed. Water column clear. Length: 90 mm. Sediment surface undisturbed. Dark brown throughout. 01/11/02 1 2 Sand/mud 1 worm and 1 worm tube No fauna observed. observed. Water column clear. Length: 80 mm. 2 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. A few worm tubes observed. No fauna observed.

(c) Station LSB3 Position rel. Sediment Sediment Date Core Surface Column to fish farm classification type Water column disturbed. Length: 110 mm. 400 m E 18/10/99 1 2 Sand/mud Sediment surface obscured. Brown throughout. 20 mm: a single worm. Water column disturbed. Length: 100 mm. 2 2 Sand/mud Sediment surface obscured. Brown throughout. No fauna observed. Water column disturbed. Length: 80 mm. 29/02/00 1 2 Sand/mud Sediment surface undisturbed. Light brown throughout. A single worm tube. No fauna observed. Length: 95 mm. Water column disturbed. Light brown throughout. 2 2 Sand/mud Sediment surface uneven. 0-5 mm: very soft mud. A few worm tubes. No fauna observed. No overlying water. Length: 110 mm. 06/11/00 1 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. No fauna observed. No fauna observed. No overlying water. Length: 120 mm. 2 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. No fauna observed. No fauna observed. No overlying water. Length: 60 mm. 08/03/01 1 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed. Water column disturbed. Length: 70 mm. 2 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed. Water column cloudy. Length: 100 mm. 01/11/02 1 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed. Water column cloudy. Length: 100 mm. 2 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed.

Ecological effects of sea lice medicines in Scottish sea lochs 255 of 286 Table 15.1 (cont.) d) Station LSB4 Position rel. Sediment Sediment Date Core Surface Column to fish farm classification type Water column disturbed. Length: 110 mm. 1500m NE 18/10/99 1 2 Sand/mud Sediment surface obscured. Brown throughout. 20 mm: a single worm. Water column disturbed. Length: 100 mm. 2 2 Sand/mud Sediment surface obscured. Brown throughout. No fauna observed. Water column disturbed. Length: 80 mm. 29/02/00 1 2 Sand/mud Sediment surface undisturbed. Light brown throughout. A single worm tube. No fauna observed. Length: 95 mm. Water column disturbed. Light brown throughout. 2 2 Sand/mud Sediment surface uneven. 0-5 mm: very soft mud. A few worm tubes. No fauna observed. No overlying water. Length: 110 mm. 06/11/00 1 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. No fauna observed. No fauna observed. No overlying water. Length: 120 mm. 2 2 Sand/mud Sediment surface undisturbed. Dark brown throughout. No fauna observed. No fauna observed. No overlying water. Length: 60 mm. 08/03/01 1 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed. Water column disturbed. Length: 70 mm. 2 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed. Water column clear. Length: 170 mm. 01/11/02 1 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed. Water column cloudy. Length: 150 mm. 2 2 Sand/mud Sediment surface undisturbed. Light brown throughout. No fauna observed. No fauna observed.

Table 15.2. Redox potential (Eh in mV) profiles of sediment cores at Loch Sunart (1999-2002).

February 2000 Station LSB1 LSB2 LSB3 LSB4 Core Sample 1 2 1 2 1 2 1 2 + 10 mm + 457 + 293 + 375 + 293 + 424 + 304 + 108 - Surface 0 mm + 213 + 178 + 172 + 208 + 403 + 83 + 52 - - 5 mm + 255 + 168 + 132 + 177 + 211 + 45 + 36 - - 10 mm + 228 + 130 + 116 + 144 + 111 + 25 + 33 - - 20 mm + 114 + 95 + 72 + 153 + 58 + 26 + 33 - - 30 mm + 80 + 82 + 69 + 147 + 28 + 22 + 35 - - 40 mm + 81 + 56 + 51 + 137 + 24 + 21 + 39 - - 50 mm + 92 + 45 + 48 + 133 + 24 + 21 + 43 - - 60 mm + 78 - + 42 + 130 + 23 + 19 + 47 - - 75 mm + 80 - + 45 + 125 + 29 + 17 + 51 -

November 2000 Station LSB1 LSB2 LSB3 LSB4 Core Sample 1 2 1 2 1 2 1 2 + 10 mm + 217 + 212 + 256 + 398 - - - - Surface 0 mm + 95 + 134 + 119 + 222 + 92 + 71 + 78 + 63 - 5 mm + 60 + 101 + 63 + 150 + 30 + 44 + 66 + 56 - 10 mm + 46 + 70 + 40 + 138 + 26 + 4 + 63 + 54 - 20 mm + 50 + 47 + 34 + 119 + 30 - 4 + 58 + 52 - 30 mm + 35 + 33 + 32 + 93 + 25 - 50 + 43 + 48 - 40 mm + 24 + 23 + 36 - 7 + 26 - 15 + 36 + 49 - 50 mm + 22 + 15 + 36 - + 28 - 15 + 37 + 47 - 60 mm + 14 + 8 + 31 - + 17 - 14 + 39 + 40 - 75 mm + 8 - 5 + 27 - + 15 - 16 + 38 + 38

Ecological effects of sea lice medicines in Scottish sea lochs 256 of 286 Table 15.2 (cont.)

March 2001 Station LSB1 LSB2 LSB3 LSB4 Core Sample 1 2 1 2 1 2 1 2 + 10 mm +139 - - - - + 250 - - Surface 0 mm +122 +82 + 101 + 157 + 250 + 377 + 126 + 90 - 5 mm +110 +58 + 95 + 129 + 185 + 352 + 83 + 63 - 10 mm +103 +50 + 91 + 106 + 161 + 277 + 67 + 48 - 20 mm +104 +53 + 84 + 98 + 146 + 127 + 63 + 47 - 30 mm +101 +54 + 78 + 86 + 135 + 48 + 58 + 45 - 40 mm + 81 +41 + 78 + 75 + 126 + 50 + 59 + 43 - 50 mm + 48 +25 + 28 + 64 + 95 + 51 + 61 + 47 - 60 mm + 13 +23 - + 62 - + 50 + 61 + 47 - 75 mm + 15 + 5 - + 39 - - - -

November 2002 Station LSB1 LSB2 LSB3 LSB4 Core Sample 1 2 1 2 1 2 1 2 + 10 mm + 422 + 279 + 430 + 289 + 94 - - - Surface 0 mm + 126 + 94 + 214 + 178 + 72 + 5 + 12 + 24 - 5 mm + 74 + 78 + 153 + 137 + 32 - 10 + 5 + 17 - 10 mm + 52 + 42 + 124 + 84 + 28 - 16 + 1 + 14 - 20 mm + 48 + 10 + 95 + 2 + 19 - 19 - 14 + 10 - 30 mm + 38 - 1 + 82 + 2 + 17 - 11 - 17 + 8 - 40 mm + 32 - 8 + 67 - 42 + 18 - 20 - 16 + 8 - 50 mm + 28 - 17 + 68 - 30 + 16 - 16 - 16 + 9 - 60 mm + 18 - 16 + 64 - 29 + 12 - 7 - 15 + 9 - 75 mm + 56 - 47 + 14 - 13 - 13 + 32 * No data from November, 2003 due to equipment malfunction.

Table 15.3. Dominant macrofauna taxa (first 5) at Loch Sunart (1999 - 2003). (No. = total no. of organisms from nominally 0.5 m2 sediment surface area) October 1999 Station 1 No. Station 2 No. Thyasira flexuosa 113 Astrorhizidae 90 Scalibregma inflatum 110 Mysella bidentata 76 Magelona minuta 106 Thyasira flexuosa 59 Prionospio fallax 100 Melinna palmata 53 Melinna palmata 65 Amphiura filiformis 52

Station 3 No. Station 4 No. Astrorhizidae 193 Cylichna cylindracea 42 Thyasira flexuosa 82 Nuculoma tenuis 33 Corbula gibba 72 Amphiura chiajei 32 Melinna palmata 52 Corbula gibba 31 Amphiura filiformis 45 Minuspio cf. multibranchiata 28

Ecological effects of sea lice medicines in Scottish sea lochs 257 of 286 Table 15.3 (cont.)

February 2000 Station 1 No. Station 2 No. Melinna palmata 582 Thyasira flexuosa 181 Prionospio fallax 397 Prionospio fallax 66 Thyasira flexuosa 182 Corbula gibba 55 Mysella bidentata 129 Amphiura filiformis 53 Magelona minuta 124 Pholoe inornata 45

Station 3 No. Station 4 No. Melinna palmata 110 Nephtys incisa 31 Thyasira flexuosa 80 Cylichna cylindracea 28 Trichobranchus roseus 61 Amphiura filiformis 28 Amphiura filiformis 57 Corbula gibba 24 Phoronis muelleri 52 Minuspio cf. multibranchiata 24

November 2000 Station 1 No. Station 2 No. Magelona minuta 265 Melinna palmata 259 Prionospio fallax 235 Scalibregma inflatum 208 Mysella bidentata 177 Mysella bidentata 134 Thyasira flexuosa 130 Nemertea T1 100 Corbula gibba 69 Owenia fusiformis 99

Station 3 No. Station 4 No. Mysella bidentata 468 Minuspio cf. multibranchiata 77 Prionospio fallax 106 Corbula gibba 49 Magelona minuta 99 Nephtys incisa 40 Amphiura filiformis 97 Magelona minuta 28 Pholoe inornata 93 Mysella bidentata 23

March 2001 Station 1 No. Station 2 No. Prionospio fallax 322 Melinna palmata 238 Magelona minuta 224 Mysella bidentata 152 Mysella bidentata 192 Owenia fusiformis 111 Melinna palmata 149 Thyasira flexuosa 86 Thyasira flexuosa 111 Phoronis muelleri 70

Station 3 No. Station 4 No. Phoronis muelleri 139 Minuspio cf. multibranchiata 72 Mysella bidentata 40 Amphiura chiajei 39 Lumbrineris latreilli 39 Mysella bidentata 39 Amphiura filiformis 34 Nucula sulcata 34 Melinna palmata 34 Nephtys incisa 31

Ecological effects of sea lice medicines in Scottish sea lochs 258 of 286 Table 15.3 (cont.)

November 2003 Station 1 No. Station 2 No. Mysella bidentata 116 Thyasira flexuosa 92 Amphiura filiformis 97 Prionospio fallax 85 Thyasira flexuosa 89 Prionospio cirrifera 71 Magelona minuta 69 Scalibregma inflatum 67 Nucula nucleus 59 Mysella bidentata 64

Station 3 No. Station 4 No. Amphiura filiformis 86 Litocorsa stremma 112 Mysella bidentata 84 Nucula nucleus 76 Thyasira flexuosa 70 Amphiura filiformis 61 Nephtys incisa 67 Amphiura chiajei 41 Turritella communis 63 Nephtys incisa 35

Ecological effects of sea lice medicines in Scottish sea lochs 259 of 286 Table 15.4. Taxa numbers, abundance and biomass for each of the major macrofaunal groups at Loch Sunart (1999 - 2003). Results of pair-wise significance tests. S, total number of species found; A, total number of animals found; B, total biomass of animals found. T-test at 95% level of significance. SD, significant difference; NSD, not significant. + values higher than previous; - values lower than previous; = values the same.

Annelida Station 1 Station 2 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 +NSD +NSD +NSD -NSD -SD +SD +NSD -NSD Feb-00 -NSD -SD -SD +SD +SD -NSD Nov-00 -NSD -NSD -SD -SD Mar-01 -NSD =NSD Nov-03 A Oct-99 +SD +NSD +NSD -NSD -NSD +SD +NSD +NSD Feb-00 -SD -SD -SD +SD +SD +NSD Nov-00 +NSD -SD -SD -SD Mar-01 -SD -NSD Nov-03 B Oct-99 +SD -NSD +SD +NSD -NSD +SD +NSD -NSD Feb-00 -SD -SD -SD +SD +SD +NSD Nov-00 +SD +SD -SD -NSD Mar-01 -NSD -NSD Nov-03 Distance 200 m W 200 m E Depth 32 20 (m)

Station 3 Station 4 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 +NSD -SD +NSD -NSD -NSD +SD +NSD -NSD Feb-00 -SD =NSD -NSD +SD +NSD =NSD Nov-00 +SD =NSD -SD -SD Mar-01 -NSD -NSD Nov-03 A Oct-99 +NSD +NSD +NSD -NSD +NSD +SD +NSD +NSD Feb-00 -NSD -NSD -SD +SD +SD +NSD Nov-00 -NSD -SD -NSD -NSD Mar-01 -SD +NSD Nov-03 B Oct-99 +SD +NSD +NSD +NSD -NSD +NSD -NSD +NSD Feb-00 -SD +NSD -NSD +NSD =NSD +NSD Nov-00 +NSD +NSD -NSD +NSD Mar-01 -NSD +NSD Nov-03 Distance 400 m E 1500 m NE Depth 16 40 (m)

Ecological effects of sea lice medicines in Scottish sea lochs 260 of 286 Table 15.4 (cont.)

Crustacea Station 1 Station 2 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 +NSD =NSD -NSD +NSD -NSD +NSD -NSD -NSD Feb-00 -NSD -SD +NSD +SD +SD +NSD Nov-00 -NSD +NSD -NSD -NSD Mar-01 +SD =NSD Nov-03 A Oct-99 +NSD -NSD -NSD +NSD -NSD +NSD +NSD =NSD Feb-00 -NSD -SD +NSD +SD +SD +SD Nov-00 -NSD =NSD -NSD -SD Mar-01 +SD -NSD Nov-03 B Oct-99 +SD +NSD +NSD +NSD -NSD +NSD -NSD -NSD Feb-00 +NSD -NSD +NSD +NSD +NSD +NSD Nov-00 -NSD -NSD -NSD -NSD Mar-01 =NSD +NSD Nov-03 Distance 200 m W 200 m E Depth 32 20 (m)

Station 3 Station 4 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 +SD -SD -NSD +NSD -NSD -NSD =NSD -NSD Feb-00 -SD -NSD -NSD +NSD +NSD +NSD Nov-00 +NSD +SD +NSD +NSD Mar-01 +NSD -NSD Nov-03 A Oct-99 +NSD -NSD +NSD -NSD -NSD -NSD +NSD -NSD Feb-00 -SD -NSD +NSD +NSD +NSD +NSD Nov-00 +NSD +NSD +NSD +NSD Mar-01 -NSD -NSD Nov-03 B Oct-99 +NSD -NSD +NSD -NSD -NSD +NSD +NSD +NSD Feb-00 -NSD +NSD +NSD +NSD +SD +NSD Nov-00 +NSD +NSD +NSD +NSD Mar-01 -NSD +NSD Nov-03 Distance 400 m E 1500 m NE Depth 16 40 (m)

Ecological effects of sea lice medicines in Scottish sea lochs 261 of 286 Table 15.4 (cont.)

Mollusca Station 1 Station 2 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 -SD -SD -SD -NSD -SD -SD -NSD -SD Feb-00 =NSD -NSD +NSD +NSD +SD +NSD Nov-00 -NSD +NSD +NSD +NSD Mar-01 +NSD -NSD Nov-03 A Oct-99 -NSD +NSD +NSD -NSD +NSD -NSD +NSD -NSD Feb-00 +NSD +NSD -NSD -NSD +NSD -NSD Nov-00 =NSD -NSD +SD +NSD Mar-01 -NSD -NSD Nov-03 B Oct-99 +NSD +NSD -NSD -NSD -NSD +NSD +SD +SD Feb-00 -NSD -NSD -NSD +NSD +SD +SD Nov-00 -NSD -NSD -NSD -NSD Mar-01 +NSD -NSD Nov-03 Distance 200 m W 200 m E Depth 32 20 (m)

Station 3 Station 4 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 -NSD -SD -SD -SD -NSD +NSD =NSD =NSD Feb-00 -NSD -NSD -SD +NSD +NSD +NSD Nov-00 -NSD -SD -NSD -NSD Mar-01 -NSD -NSD Nov-03 A Oct-99 +NSD +SD -SD +NSD -NSD +NSD +NSD +NSD Feb-00 +SD -SD -NSD +NSD +NSD +NSD Nov-00 -SD -SD +NSD +NSD Mar-01 +SD +NSD Nov-03 B Oct-99 +NSD +NSD -NSD +NSD -NSD +NSD +NSD +NSD Feb-00 +NSD -NSD -NSD +NSD +NSD +SD Nov-00 -NSD -SD +NSD +NSD Mar-01 +SD +NSD Nov-03 Distance 400 m E 1500 m NE Depth 16 40 (m)

Ecological effects of sea lice medicines in Scottish sea lochs 262 of 286 Table 15.4 (cont.)

Echinodermata Station 1 Station 2 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 +NSD =NSD -NSD -NSD -SD +NSD -SD -SD Feb-00 =NSD -NSD -NSD +SD -SD -NSD Nov-00 -NSD -NSD -SD -SD Mar-01 =NSD +NSD Nov-03 A Oct-99 -NSD -NSD -NSD +NSD -NSD +NSD -SD -SD Feb-00 -NSD -NSD +NSD +NSD -NSD -NSD Nov-00 -NSD +SD -SD -SD Mar-01 +SD -NSD Nov-03 B Oct-99 +NSD +NSD -SD +SD +NSD +NSD +NSD =NSD Feb-00 -NSD -SD +NSD -NSD -NSD -NSD Nov-00 -SD +NSD -NSD -NSD Mar-01 +SD -NSD Nov-03 Distance 200 m W 200 m E Depth 32 20 (m)

Station 3 Station 4 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 Feb-00 Nov-00 Mar-01 Nov-03 Oct-99 -SD =NSD -NSD -NSD =NSD +NSD +NSD +NSD Feb-00 +SD +NSD +SD +NSD +NSD +NSD Nov-00 -NSD -SD =NSD +NSD Mar-01 +NSD +NSD Nov-03 A Oct-99 +NSD +NSD -NSD +NSD -NSD -NSD +NSD +NSD Feb-00 +NSD -NSD +NSD =NSD +SD +SD Nov-00 -SD -NSD +NSD +SD Mar-01 +NSD +NSD Nov-03 B Oct-99 +NSD +SD -NSD +SD -NSD +NSD -NSD +SD Feb-00 +SD -SD +NSD +NSD +NSD +SD Nov-00 -SD -SD -NSD +SD Mar-01 +SD +SD Nov-03 Distance 400 m E 1500 m NE Depth 16 40 (m)

Ecological effects of sea lice medicines in Scottish sea lochs 263 of 286 Table 15.5. Descriptions of sediment cores at Loch Kishorn, June and August 2001

(i) June 2001

Station K1

Approximate Date Core Sediment Sediment Description of Sediments posn. relative classification type to cage group Surface Column

Adjacent to 26/06/01 1 2 Sand/mud Water column disturbed. Length: 120 mm. NE corner Sediment surface undisturbed 0-60 mm: Dark brown soft mud A few worm tubes observed 60-120mm:Dark brown mud/sand 0-60 mm: many worms observed

2 2 Sand/mud Water column disturbed Length: 75 mm. Sediment surface undisturbed 0-25 mm: Dark brown mud A few worm tubes observed. 25-60mm: Light brown mud/sand 1 worm observed at 40 mm.

Station K2

Approximate Date Core Sediment Sediment Description of Sediments posn. relative classification type to cage group Surface Column

25 m NE 26/06/01 1 2 Sand/mud/ Water column disturbed. Length: 95 mm. clay Sediment surface undisturbed 0-25 mm: Dark brown mud/sand A few worm tubes observed 25-95 mm: Light brown clay/mud 1 animal observed at 50 mm.

2 2 Sand/mud/ Water column undisturbed Length: 95 mm. clay Sediment surface undisturbed 0-40 mm: Dark brown sand/mud A few worm tubes observed. with dead shell 40-95 mm: Light brown clay/mud Fauna observed at 40 mm.

Station K3

Approximate Date Core Sediment Sediment Description of Sediments posn. relative classification type to cage group Surface Column

50 m NE 26/06/01 1 2 Sand/mud Water column disturbed. Length: 95 mm. Sediment surface undisturbed 0-30 mm: light brown mud No fauna observed 30-95mm: light brown mud/sand No fauna observed

2 2 Sand/mud Water column disturbed Length: 110 mm. Sediment surface undisturbed 0-20 mm: light brown mud. A single worm tube observed. 20-110mm:Light brown sand/mud No fauna observed.

Ecological effects of sea lice medicines in Scottish sea lochs 264 of 286 Table 15.5 (cont.)

(ii) August 2001

Station K1

Approximate Date Core Sediment Sediment Description of Sediments posn. relative classification type to cage group Surface Column

Adjacent to 21/08/01 1 2 Sand/mud Water column disturbed. Length: 100 mm. NE corner Sediment surface undisturbed 0-40 mm: Dark brown soft mud No fauna observed 40-100 mm: Dark brown mud/sand No fauna observed Slight smell of H2S.

2 2 Sand/mud Water column disturbed Length: 60 mm. Sediment surface undisturbed 0-25 mm: Dark brown mud No fauna observed 25-60 mm: Dark brown mud/sand 1 worm observed at 50 mm.

Station K2

Approximate Date Core Sediment Sediment Description of Sediments posn. relative classification type to cage group Surface Column

25 m NE 21/08/01 1 2 Sand/mud Water column clear. Length: 80 mm. Sediment surface undisturbed Light brown throughout with Dead shell on sediment surface occasional black streaks. No fauna observed 0-30mm: numerous annelida observed

2 2 Sand/mud Water column disturbed Length: 90 mm. Sediment surface undisturbed 0-20 mm: light brown mud. 1 worm tube observed. 20-90 mm: light brown mud/clay. Fauna observed at 10 mm.

Station K3

Approximate Date Core Sediment Sediment Description of Sediments posn. relative classification type to cage group Surface Column

50 m NE 21/08/01 1 2 Sand/mud Water column disturbed. Length: 75 mm. Sediment surface undisturbed 0-20 mm: light brown mud No fauna observed 20-75 mm: light brown mud/sand 0-40 mm: numerous annelida observed

2 2 Sand/mud Water column disturbed Length: 90 mm. Sediment surface undisturbed Light brown throughout A few worm tubes observed. Annelida observed at 20, 30 and 45 mm.

Ecological effects of sea lice medicines in Scottish sea lochs 265 of 286 Table 15.5 (cont.)

Station K7

Position relative Date Core Sediment Sediment Description of Sediments to cage group classification type Surface Column

~ 500 m SSW 21/08/01 1 2 Sand/mud Water column disturbed. Length: 65 mm. Sediment surface undisturbed 0-10 mm: light brown mud No fauna observed 10-65 mm: light brown mud/sand 1 worm observed at 10 mm. 1 burrow observed at 35 mm.

2 2 Sand/mud Water column disturbed Length: 70 mm. Sediment surface undisturbed Light brown mud/sand throughout. No fauna observed. No fauna observed

Table 15.6. Redox potential (Eh in mV) profiles of sediment cores at Loch Kishorn, June and August 2001.

(i) June 2001

Station K1 K2 K3

Core Sample 1 2 1 2 1 2

+ 10 mm + 455 + 185 + 180 + 160 + 215 + 120 Surface 0 mm + 378 + 20 + 114 + 106 - 152 + 104 - 5 mm + 254 - 10 + 95 + 75 - 15 + 87 - 10 mm + 211 - 15 + 94 + 55 - 21 + 81 - 20 mm + 164 - 53 + 81 + 43 - 32 + 57 - 30 mm + 143 - 64 + 67 + 36 - 36 + 45 - 40 mm - 52 - 68 + 62 + 29 - 38 + 29 - 50 mm - 77 - 71 + 70 + 20 - 50 + 21 - 60 mm - 90 - 74 + 63 + 15 - 65 + 15 - 75 mm

(ii) August 2001

Station K1 K2 K3 K7

Core Sample 1 2 1 2 1 2 1 2

+ 10 mm + 26 + 4 + 437 + 100 + 141 + 191 + 465 + 138 Surface 0 mm + 3 - 29 + 42 - 8 + 166 + 103 + 139 + 76 - 5 mm 0 - 28 + 25 - 17 + 161 + 77 + 139 + 39 - 10 mm 0 - 33 + 12 - 23 + 123 + 65 + 138 + 53 - 20 mm - 2 - 36 - 12 - 32 + 92 + 43 + 147 + 42 - 30 mm - 5 - 39 - 19 - 36 + 56 + 24 + 132 + 42 - 40 mm - 8 - 41 - 20 - 44 + 44 + 8 + 131 + 54 - 50 mm - 12 - 42 - 19 - 53 + 35 - 1 + 112 + 52 - 60 mm - 14 - 44 - 15 - 58 + 19 0 + 96 + 47 - 75 mm - 19 - 70 + 7 - 7

Ecological effects of sea lice medicines in Scottish sea lochs 266 of 286 Table 15.7. Loch Kishorn, August 2001. Comparison of the distribution of the ten most numerous macrofauna taxa: (a) the five most abundant taxa at each station; (b) a ranked comparison of the most abundant species at each station. A, abundance/0.5 m2.

(a)

Taxon Taxon

Station 1 A Station 2 A

Capitella capitata 9938 Capitella capitata 5472 Nematoda 8506 Nematoda 907 Malacoceros fuliginosus 845 Mediomastus fragilis 657 Mediomastus fragilis 635 Phyllodoce mucosa 164 Mysella bidentata 305 Scalibregma inflatum 94

Station 3 Station 7

Mediomastus fragilis 932 Amphiura filiformis 573 Ophiura affinis 273 Mysella bidentata 516 Scalibregma inflatum 265 Amphiura chiajei 270 Melinna palmata 129 Pholoe inornata 179 Phascolion strombus 108 Amphiura sp. 151

(b)

Taxon Ranking score (1, highest:38, lowest) Station 1 Station 2 Station 3 Station 7 Capitella capitata 1 1 34 36 Nematoda 2 2 8 12 Malacoceros fuliginosus 3 13 - - Mediomastus fragilis 4 3 1 29 Mysella bidentata 5 7 19 2 Phyllodoce mucosa 14 4 30 - Scalibregma inflatum 14 5 3 17 Ophiura affinis - 6 2 - Melinna palmata 14 6 4 25 Phascolion strombus - 15 5 - Amphiura filiformis - 38 37 1 Amphiura chiajei - 23 13 3 Pholoe inornata 8 24 22 4 Amphiura sp. 10 - 6 5

Ecological effects of sea lice medicines in Scottish sea lochs 267 of 286 Table 15.8. Loch Kishorn, June and August 2001. Comparison of the population statistics for each major macrofaunal group found at each station on each of the two sampling occasions. (Total No. of Species, Abundance and Biomass nominally 0.5 m2 of sediment surface area).

Number of taxa (S)

Date Group Station 1 Station 2 Station 3 Station 7 06/2001 11 55 69 - 08/2001 Annelids 16 27 75 48 06/2001 0 6 9 - 08/2001 Crustacea 0 2 14 12 06/2001 5 5 9 - 08/2001 Molluscs 5 5 12 13 06/2001 0 8 10 - 08/2001 Echinoderms 1 5 10 6

Abundance (A)

Date Group Station 1 Station 2 Station 3 Station 7 06/2001 13768 1425 1598 - 08/2001 Annelids 11519 9399 2044 637 06/2001 0 7 29 - 08/2001 Crustacea 0 2 117 57 06/2001 45 83 112 - 08/2001 Molluscs 345 92 108 653 06/2001 0 98 363 - 08/2001 Echinoderms 10 15 535 1051

Biomass (B (g))

Date Group Station 1 Station 2 Station 3 Station 7 06/2001 1451.52 22.91 26.35 - 08/2001 Annelids 942.40 26.85 19.56 8.65 06/2001 0 0.03 0.06 - 08/2001 Crustacea 0 0.32 0.20 0.07 06/2001 3.23 0.38 11.20 - 08/2001 Molluscs 9.40 0.11 1.02 22.09 06/2001 0 0.54 3.44 - 08/2001 Echinoderms 0.64 0.22 8.11 141.43

Ecological effects of sea lice medicines in Scottish sea lochs 268 of 286 Table 15.9. Loch Kishorn, June and August 2001. Comparison of pre- and post- treatment data on macrofaunal samples. S = total number of species found; A = total number of animals found; B = total biomass of animals found in nominally 0.5m2 sediment surface area. T- test at 95% level of significance. SD = significant difference; NSD = not significant. +values higher in June; - values lower in June; = values the same.

Annelida Group Station 1 Station 2 Station 3 S +NSD +NSD +NSD A -NSD +SD +NSD B -SD +NSD -NSD Distance 0 25 50 Depth (m) 32 28 26

Mollusca Group Station 1 Station 2 Station 3 S +SD -NSD +NSD A +SD +NSD -NSD B -NSD -NSD -SD Distance 0 25 50 Depth (m) 32 28 26

Crustacea Group Station 1 Station 2 Station 3 S * -NSD +SD A * -NSD +SD B * -NSD +NSD Distance 0 25 50 Depth (m) 32 28 26

Echinodermata Group Station 1 Station 2 Station 3 S * -SD =NSD A * -SD +NSD B * -NSD +NSD Distance 0 25 50 Depth (m) 32 28 26 *Phyla not present.

Ecological effects of sea lice medicines in Scottish sea lochs 269 of 286 Table 15.10. Loch Diabaig macrofauna July 2000 and August 2001. The five most abundant taxa found at each station on each sampling occasion (No.= Total No. of organisms from nominally 0.5 m2 sediment surface area)

July 2000

Station 1 No. Station 2 No. Station 3 No. Nematoda 2818 Mysella bidentata 182 Turritella communis 777 Capitella capitata 592 Amphiura chiajei 97 Nemertea T1 194 Prionospio fallax 331 Amphiura filiformis 80 Prionospio fallax 111 Mediomastus fragilis 134 Nephtys kersivalensis 57 Diplocirrus glaucus 88 Scalibregma inflatum 119 Spiophanes kroyeri 35 Pholoe inornata 77

August 2001

Station 1 No. Station 2 No. Station 3 No. Thyasira flexuosa 636 Scalibregma inflatum 346 Turritella communis 300 Abra alba 285 Mysella bidentata 85 Nephtys kersivalensis 41 Mysella bidentata 232 Amphiura filiformis 29 Melita bergensis 30 Mediomastus fragilis 121 Nephtys incisa 27 Mysella bidentata 19 Nematoda sp. 107 Cylinchna cylindracea 26 Amphiura chiajei 18

Ecological effects of sea lice medicines in Scottish sea lochs 270 of 286 Table 15.11. Loch Diabaig macrofauna population statistics July 2000 and August 2001.

Number of taxa (S) Date Group Station 1 Station 2 Station 3 07/2000 Annelids 37 26 74 08/2001 38 36 26 07/2000 Crustacea 12 1 12 08/2001 6 6 5 07/2000 Molluscs 0 8 18 08/2001 14 19 10 07/2000 Echinoderms 0 4 7 08/2001 3 8 3

Abundance (A) Date Group Station 1 Station 2 Station 3 07/2000 Annelids 1403 225 803 08/2001 349 506 135 07/2000 Crustacea 16 1 50 08/2001 7 12 41 07/2000 Molluscs 62 298 1086 08/2001 1274 187 351 07/2000 Echinoderms 2 203 175 08/2001 5 57 27

Biomass (B (g)) Date Group Station 1 Station 2 Station 3 07/2000 Annelids 8.6 9.5 13.3 08/2001 1.1 13.69 11.5 07/2000 Crustacea 0.01 <0.01 0.5 08/2001 0.02 0.06 0.15 07/2000 Molluscs 4.1 3.7 248.1 08/2001 21.1 11.0 47.4 07/2000 Echinoderms <0.01 65.1 10.5 08/2001 0.01 8.3 6.7

Ecological effects of sea lice medicines in Scottish sea lochs 271 of 286 Table 15.12. Loch Diabaig macrofauna pre- and post Excis treatment (July 2000 and August 2001). S, total number of species found; A, total number of animals found; B, total biomass of animals found. T-test at 95% level of significance. SD, significant difference; NSD, not significant. + values higher in 2000; - values lower in 2000; = values the same.

Annelida Group Station 1 Station 2 Station 3 S -NSD -NSD +SD A +NSD -NSD +SD B -SD -NSD +NSD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43

Crustacea Group Station 1 Station 2 Station 3 S +NSD -SD +SD A +NSD -NSD +NSD B -NSD -NSD +NSD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43

Mollusca Group Station 1 Station 2 Station 3 S -SD -NSD +SD A -SD +NSD +SD B -SD -NSD +SD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43

Echinodermata Group Station 1 Station 2 Station 3 S +NSD -NSD +SD A +NSD +SD +SD B +NSD +SD +NSD Distance 100 m S 300 m WSW 850 m WNW Depth (m) 23 40 43

Ecological effects of sea lice medicines in Scottish sea lochs 272 of 286 Table 15.13 (i - ii). Loch Kishorn macrofauna population statistics June 2001.

(i) Population statistics

Number of taxa (S) Group Station 1 Station 2 Station 3 Annelids 9 56 69 Crustacea 0 6 9 Molluscs 5 5 9 Echinoderms 0 8 10

Abundance (A) Group Station 1 Station 2 Station 3 Annelids 13768 1452 1598 Crustacea 0 7 29 Molluscs 45 83 112 Echinoderms 0 98 363

Biomass (B(g)) Group Station 1 Station 2 Station 3 Annelids 181.44 22.9 26.3 Crustacea 0 0.04 0.07 Molluscs 3.21 0.4 11.2 Echinoderms 0 0.5 3.44

(ii) The five most abundant taxa found at each station

Station 1 No. Station 2 No. Station 3 No. Capitella capitata 8639 Mediomastus fragilis 706 Mediomastus fragilis 671 Nematoda sp. 5518 Nematoda sp. 386 Scalibregma inflatum 171 Malacoceros fuliginosus 949 Scalibregma inflatum 186 Polycirrus plumosus 146 Mediomastus fragilis 222 Melinna palmate 117 Ophiura affinis 144 Phyllodoce mucosa 165 Mysella bidentata 50 Melinna palmata 132

Ecological effects of sea lice medicines in Scottish sea lochs 273 of 286 16 Appendix V - Littoral Settlement

Table 16.1. Growth and development of barnacles, Semibalanus balanoides at Loch Sunart (2002 - 2003). Predicted high impact - Station B Installed/Cleared 01.03.02 01.03.03 Retrieved/Examined 04.03.03 22.02.04 Comments No settlement on slates B1-B6 No settlement on slates B1-B6 Cohort 2002 2003

Predicted intermediate impact - Station A Installed/Cleared 01.03.02 04.03.03 Retrieved/Examined 04.03.03 22.02.04 Cohort 2002 2003 Total on slates (4) 7 Mean Slates(n) Diameter (mm) 4.9 1 Eggs (mg) 2.1 1 Bodies (mg) 1.6 1 Valves (mg) 2.8 1 % no eggs 17 1

Population.m-2 22 391 4

Predicted minimum impact - Station C Installed/Cleared 01.03.02 04.03.03 Retrieved/Examined 04.03.03 22.02.04 Cohort 2002 2003 Mean No. 95% Conf Mean Slates(n) 95%Conf Diameter (mm) 9.9 2 1.9 6.7 4 0.4 Eggs (mg) 27.6 2 21.5 5.9 4 1.6 Bodies (mg) 6.4 2 4.0 3 4 1 Valves (mg) 12.7 2 10.5 5 4 1 % no eggs 6.0 2 18.0 26 4 22

Population.m-2 1200 1 18500 4 16100 % live 0 1 95 4 8 % cover Less than 1 1 29 4 29 Max diameter (mm) Not noted 8.6 4 0.7 NB: Densities > 1000 were rounded to nearest 100

Ecological effects of sea lice medicines in Scottish sea lochs 274 of 286 Table 16.2. Growth and development of barnacles, Semibalanus balanoides at Loch Craignish, 2001 cohort. Predicted impact from northern cage group - Station B Installed/Cleared 13.10.00 Retrieved/Examined 28.02.02 Cohort 2001 Mean Slates(n) 95%Conf Diameter (mm) 8.1 4 0.8 Eggs (mg) 10.8 4 7.8 Bodies (mg) 3.8 4 1.8 Valves (mg) 9.4 4 3.9 % no eggs 17.0 4 35.9

Population (Ind m-2) 31 4 34 % live 98 4 7 % cover Not estimated Predicted impact from southern cage group - Station C Installed/Cleared 13.12.00 Retrieved/Examined 28.02.02 Cohort 2001 Mean Slates(n) 95%Conf Diameter (mm) 9.5 3 1.6 Eggs (mg) 25.7 3 15.2 Bodies (mg) 8.6 3 3.4 Valves (mg) 15.7 3 5.6 % no eggs 0.0 3 0.0

Population (Ind m-2) 745 3 716 % live 95.3 3 1.2 % cover Not estimated Predicted minimum impact - Station D Installed/Cleared 13.10.00-08.02.01 Retrieved/Examined 28.02.02 Cohort 2001 Mean Slates(n) 95%Conf Diameter (mm) 7.7 4 0.8 Eggs (mg) 14.7 4 5.5 Bodies (mg) 4.2 4 1.5 Valves (mg) 9.8 4 1.4 % no eggs 1.0 4 1.6

Population (Ind m-2) 155 4 222 % live 95 4 7 % cover Not estimated

Ecological effects of sea lice medicines in Scottish sea lochs 275 of 286 Table 16.3. Growth and development of barnacles, Semibalanus balanoides at Loch Kishorn (2002 and 2003). Predicted high impact - Station B Installed/Cleared 22/08/01 - 19/09/01 17/02/03 Retrieved/Examined 17/02/03 19/02/04 Cohort 2002 2003 Mean Slates (n) 95 % Conf Mean Slates (n) 95 % Conf Diameter (mm) 6.9 4 1.6 8.0 4 2.4 Eggs (mg) 8.5 3 1.9 11.6 4 7.2 Bodies (mg) 4.6 3 4.0 4.7 4 2.9 Valves (mg) 6.8 3 1.7 7.5 4 5.4 % no eggs 14.5 4 1.6 6 4 3

Population m-2 14800 4 18100 20500 4 21000 % Live 98 3 2 84 4 10 % Cover 43 4 49 34 4 32 Predicted intermediate impact - Station A Installed/Cleared 22/08/01 - 19/09/01 19/02/03 Retrieved/Examined 17/02/03 19/02/04 Mean Slates (n) 95 % Conf Mean Slates (n) 95 % Conf Diameter (mm) 4.9 3 4.0 5.3 3 0.2 Eggs (mg) 3.1 3 0.6 Bodies (mg) 1.4 3 0.5 Valves (mg) 2.1 3 0.5 % no eggs 67 3 52 13 3 6 Counted on site- A3 no settlement Population m-2 67 3 15 15800 3 15000 % Live 90 3 17 % Cover < 1 % 18 3 12 Predicted minimum impact - Station C Installed/Cleared 19/03/00 17/02/03 Retrieved/Examined 17/02/03 19/02/04 Mean Slates (n) 95 % Conf Mean Slates (n) 95 % Conf Diameter (mm) 6.1 3 0.5 6.1 4 0.1 Eggs (mg) 4.0 2 8.8 5.2 4 0.4 Bodies (mg) 2.1 2 0.0 3.1 4 0.1 Valves (mg) 3.9 2 0.0 4.3 4 0.2 % no eggs 17 3 33 32 4 7

Population m-2 650 3 907 28100 4 5900 % Live 99 3 2 89 4 5 % Cover 1 35 4 10 NB: Densities > 1000 were rounded to nearest 100

Ecological effects of sea lice medicines in Scottish sea lochs 276 of 286 Table 16.4 (A-C). Loch Diabaig, cohorts 2000 - 2003: Growth and development of the barnacle, Semibalanus balanoides at different impact levels on shore panels.

(A) Predicted high impact - Station C

Installed/Cleared 17/04/00 12/03/01 Retrieved/Examined 12/03/01 01/03/02 Cohort 2000 2001

95 % Mean Slates (n) 95 % Conf Mean Slates (n) Conf Diameter (mm) 5.0 2 6.7 5.1 2 10.0 Eggs (mg) 1.9 2 7.2 2.4 2 11.7 Bodies (mg) 1.7 2 7.2 1.5 2 8.1 Valves (mg) not measured 3.2 2 16.2 % no eggs 72 2 90 63 2 117

Population m-2 2053 2 17794 3564 2 30871 % live 77 2 207 88 2 18 % cover 36 2 310 41 2 351 Installed/Cleared 30/01/02 16/02/03 Retrieved/Examined 18/02/03 20/02/04 Cohort 2002 2003

95 % Mean Slates (n) 95 % Conf Mean Slates (n) Conf Diameter (mm) 5.4 3 1.7 5.6 4 0.5 Eggs (mg) 4.5 2 1.4 4.1 4 1.5 Bodies (mg) 2.3 2 0.9 2.3 4 0.4 Valves (mg) 4.6 2 9.8 4 0.7 % no eggs 31 3 40 47 4 19

Population m-2 19552 3 33872 35104 4 5290 % live 85 3 24 67 4 13 % cover

Ecological effects of sea lice medicines in Scottish sea lochs 277 of 286 Table 16.4 (A-C). (Cont.).

(B) Predicted intermediate impact - Station B

Installed/Cleared 21/03/00 12/03/01 Retrieved/Examined 12/03/01 01/03/02 Cohort 2000 2001 Single slate only Single slate only 95 % Mean Slates (n) 95 % Conf Mean Slates (n) Conf Diameter (mm) 5.2 1 6.4 1 Eggs (mg) 4.4 1 6.3 1 Bodies (mg) 2.4 1 2.6 1 not Valves (mg) 1 4.7 1 measured % no eggs 16 1 0 1

Not Population m-2 1 5200 1 countable % live 45 1 97 1 % cover 65 1 55 1 Installed/Cleared 29/01/02 16/02/03 Retrieved/Examined 18/02/03 20/02/04 Cohort 2002 2003

95 % Mean Slates (n) 95 % Conf Mean Slates (n) Conf Diameter (mm) 4.8 2 3.6 5.2 4 0.5 Eggs (mg) 2.3 2 2.6 3.6 4 1.1 Bodies (mg) 1.2 2 1.9 1.7 4 0.6 Valves (mg) 2.2 2 3.4 3.3 4 1.3 % no eggs 27 2 22 23 4 13

Population m-2 72916 2 110037 43333 4 11662 % live 62 2 34 80 4 7 % cover 57 2 128 53 4 20

Ecological effects of sea lice medicines in Scottish sea lochs 278 of 286 Table 16.4 (A-C). (Cont.).

(C) Reference site - predicted minimum impact - Station A

Installed/Cleared 19/03/00 11/03/01 Retrieved/Examined 11/03/01 29/02/02 Cohort 2000 2001 Single slate only 95 % Mean Slates (n) 95 % Conf Mean Slates (n) Conf Diameter (mm) 7.3 1 6.95 2 13.0 Eggs (mg) 9.2 1 7.9 2 41.3 Bodies (mg) 3.7 1 3.15 2 14.8 not Valves (mg) 1 6.55 2 33.7 measured % no eggs 0 1 1 2 9

Population m-2 2200 1 4400 2 17969 % live 91 1 93 2 9 % cover 25 1 55 2 180 Installed/Cleared 29/01/02 16/02/03 Retrieved/Examined 18/02/03 20/02/04 Cohort 2002 2003

95 % Mean Slates (n) 95 % Conf Mean Slates (n) Conf Diameter (mm) 5.2 3 0.8 5.3 4 0.5 Eggs (mg) 2.5 3 1.3 3.2 4 1.0 Bodies (mg) 1.0 3 0.8 1.6 4 0.4 Valves (mg) 2.1 3 1.2 2.7 4 0.4 % no eggs 10 3 19 17 4 16

Population m-2 52152 3 74312 57333 3 58040 % live 85 3 14 78 3 41 % cover 50 3 61 63 3 6

Ecological effects of sea lice medicines in Scottish sea lochs 279 of 286 Table 16.5. Loch Sunart. Semibalanus balanoides on shore sites exposed on previously cleared rock surfaces for a single settlement season to 4 March 2003. For comparison, data for Semibalanus balanoides which settled on Slates C3 & C4 over 2 settlement seasons are included below.

Semibalanus balanoides data collected on retrieval date 4 March 2003 Site A B C C* Predicted impact Intermediate High Low-Ref Low-Ref Rock Surface Rock Surface Rock Surface Slates C3&C4

No. S. balanoides.m-2 580 880 6000 1440

No. examined 19 25 40 100

% live 100 91 98 96

% without egg lamellae 11 24 8 6

% empty (dead) 0 9 2 4

Mean shell diameter(mm) 7.8 9.1 7.7 9.9

Mean valve wt. (mg) ND ND ND 7.3

Mean body wt. (mg) ND ND ND 3.6

Mean wt. of egg lamellae (mg) ND ND ND 21.0 *S. balanoides populations recovered on Slates C3 & C4 included a small proportion of individuals which settled in the summer of 2001 (up to 25%). By late summer the two year groups cannot be distinguished with certainty.

Ecological effects of sea lice medicines in Scottish sea lochs 280 of 286 Table 16.6. Summary of biota settled on sublittoral arrays at Loch Sunart (Inner Basin), station numbers LS 1-4, 3 July 2003.

Station LS1 I LS2 H LS3 I LS4 R Impact Level U M L U M L U M L U M L Balanus crenatus 1 2 2 1 1 1 1 1 1 1 3 3 Obelia longissima 3 4 5 1 3 3 2 4 4 2 4 Mytilus edulis (spat) 2 4 6 1 5 5 1 1 5 1 5 Onchidoris bilamellata 4 3 5 5 5 6 5 6 5 6 Ascidiella aspersa 3 3 5 3 5 3 4 4 3 3 Ciona intestinalis 5 6 6 5 5 Corella parallelogramma 6 6 Pomatoceros triqueter 5 6 6 5 5 4 Hydroides norvegica 5 5 5 5 5 Electra pilosa 5 4 5 Tubularia larynx 6 6 Balanus balanus 6 Harmothoe impar 6 6 6 6 Psammechinus miliaris 6 Laminaria saccharina 3 4 5 5 2 + Lacuna vincta 5 Scytosiphon lomentaria 5 Enteromorpha compressa 5 Rhodomela subfusca 5 4 Polysiphonia elongata 6 Polysiphonia fibrata 6 5 Ceramium rubrum 6 Cutleria multifida 4 Ectocarpus sp. 5 5

*The abundance of species settled are ranked according to MNCR SACFOR scale as follows: Level: Predicted impact: 1 = S-Superabundant U = Upper, ~2 m below surface H = High 2 = A-Abundant M = Mid, Midwater I = Intermediate 3 = C-Common L = Lower, 2 m above seabed R = Reference 4 = F-Frequent 5 = O-Occasional 6 = R-Rare

Ecological effects of sea lice medicines in Scottish sea lochs 281 of 286 17 Appendix VI - Sediment Properties

Table 17.1. Loch Sunart sediment total organic carbon and nitrogen: February 2000 to May 2002. Sample Collection date Total Organic Carbon (%) Total Nitrogen (%) Sunart Stn1 LSB1 Feb/March 2000 1.115 0.668 Sunart Stn2 LSB2 Feb/March 2000 1.895 0.110 Sunart Stn3 LSB3 Feb/March 2000 0.954 0.176 Sunart Stn4 LSB4 Feb/March 2000 7.216 - Sunart Stn2 LSB2 March 2000 5.564 0.438 Loch Sunart Stn 1 March 2001 3.758 0.348 Loch Sunart LS B3-2 2.677 0.245 Loch Sunart LS B2-2 2.057 0.206 Sunart Stn 2 March 2001 0.770 0.275 Sunart LSB 4 March 2001 7.535 0.079 Sunart Stn 3 March 2001 2.140 0.224 Sunart Stn 2 TBF December 2001 2.468 0.208 Sunart Stn 1 TBF December 2001 2.952 0.288 Sunart Stn 4.1 December 2001 7.649 0.288 Sunart Stn 3 TBF December 2001 0.868 0.679 Sunart S2A May 2002 1.673 0.204 Sunart Ref May 2002 2.391 0.207 Sunart S1C May 2002 1.511 0.159 Sunart S2B May 2002 1.413 0.152 Sunart S1B May 2002 0.982 0.108 Sunart S1A May 2002 2.245 0.252 Sunart S2C May 2002 1.297 0.149

Table 17.2. Loch Diabaig sediment total organic carbon and nitrogen: November 2000 and August 2001.

Station/ %Total Organic %Total Collection Date Carbon Nitrogen Diabaig Station 1 15/11/00 2.932 0.438 Diabaig Station 2 15/11/00 3.611 0.455 Diabaig Station 3 15/11/00 1.109 0.149 Diabaig Station 1 2/8/01 2.170 0.342 Diabaig Station 2 2/8/01 3.870 0.499 Diabaig Station 3 2/8/01 1.555 -

Ecological effects of sea lice medicines in Scottish sea lochs 282 of 286 Table 17.3. Loch Kishorn sediment total organic carbon and nitrogen: June 2001 to January 2002. Field Id/ Collection Date % Total Organic % Total Carbon Nitrogen Kishorn 1VV2 Jun 01 2.478 0.414 Kishorn 2VV1 Jun 01 2.336 0.256 Kishorn 3VV1 Jun 01 2.482 0.389 Kishorn 4VV1 Jun 01 3.595 0.585 Kishorn 5VV1 Jun 01 1.592 0.295 Kishorn 6VV1 Jun 01 1.722 0.295 Kishorn 7VV1 Jun 01 2.529 0.316 Kishorn T1B1 25/6/01 2.442 0.339 Kishorn REF 26/6/01 1.977 0.271 Kishorn T1 A1 25/6/01 3.886 0.545 Kishorn T1 C1 25/6/01 2.761 0.354 Kishorn T2 A1 26/6/01 3.827 0.531 Kishorn T2 B1 26/6/01 2.016 0.265 Kishorn T2 C1 26/6/01 1.558 0.140 Kishorn, Ref 13/8/01 1.740 0.235 Kishorn T213 13/8/01 1.940 0.273 Kishorn T214 13/8/01 4.424 0.605 Kishorn T2C 13/8/01 1.432 0.186 Kishorn Stn3 21/8/01 2.355 0.297 Kishorn T1A 13/8/01 3.439 0.467 Kishorn T1B 13/8/01 2.201 0.248 Kishorn T1C 13/8/01 2.382 0.236 Kishorn Stn 1 Aug 01 3.661 0.505 Kishorn Stn 2 Aug 01 2.559 0.361 Kishorn Stn 4 Aug 01 4.110 0.583 Kishorn Stn 5 Aug 01 1.578 0.232 Kishorn Stn 6 Aug 01 1.856 0.291 Kishorn Stn 7 Aug 01 2.306 0.355 Kishorn Ref 24/1/02 1.700 0.268 Kishorn T1A 24/1/02 4.548 0.648 Kishorn T2A 24/1/02 5.903 0.881 Kishorn T2B 24/1/02 2.206 0.300 Kishorn T2C 24/1/02 1.754 0.286

Table 17.4. Percentages of total organic carbon and total nitrogen for sediments collected from Loch Craignish between October 2000 and December 2001. Field ID/ %Total %Total Collection Date Organic Nitrogen Carbon LC1 1 Oct 00 3.578 0.615 LC2 Oct 00 3.902 - LC3 Oct 00 4.360 0.630 LC1 18/12/01 3.158 0.473 LC2 18/12/01 1.707 0.254 LC3 18/12/01 1.135 0.232

Ecological effects of sea lice medicines in Scottish sea lochs 283 of 286 Table 17.5 Particle Size Analysis data Identifiers Phi Summary Stats Phi Percentiles Phi Quart. Dev. Sort. Coeff. Diameter (mm) summary stats Fract Description lab field mean var skew kurt 16% 25% 50% 75% 84% devn. coeff. mean median 63um Kishorn Ref 13/8/01 3.3 8.406 0.046 0.651 0.095 0.422 3.32 5.763 6.44 2.67 2.871 0.102 0.1 45.4 A,B,C Kishorn T2B 13/8/01 3.8 7.02 -0.116 0.7 0.507 0.935 4.1 5.809 6.505 2.437 2.76 0.072 0.058 51.1 D,E,F Kishorn T2A 13/8/01 4.84 4.107 -0.063 1.333 2.931 3.75 4.91 6.055 6.78 1.153 2.099 0.035 0.033 71.5 D,B,G Kishorn T2C 13/8/01 3.26 7.887 0.02 0.731 -0.21 0.692 3.2 5.557 6.292 2.432 2.941 0.105 0.109 42.2 A,B,F Kishorn Stn3 21/8/01 3.4 8.353 -0.129 0.701 0.248 0.586 3.93 5.723 6.409 2.569 2.871 0.094 0.066 49.4 A,E,F Sunart Stn 1 03/01 4.14 4.664 -0.029 1.143 2.126 2.786 4.21 5.529 6.2 1.371 2.177 0.057 0.054 53.8 D,B,G Kishorn T1A 13/8/01 4.36 5.588 -0.131 1.223 2.049 3.139 4.62 5.844 6.551 1.353 2.349 0.049 0.041 62.8 D,E,G Kishorn T1B1 25/6/01 4.37 6.499 -0.225 0.949 0.831 2.657 4.79 6.176 6.847 1.759 2.738 0.048 0.036 61.6 D,E,H Sunart LS B3-2 3.75 5.09 -0.07 1.132 0.831 2.524 3.79 5.261 5.926 1.368 2.419 0.074 0.072 45.5 A,B,G Sunart LS B2-2 3.89 4.946 0.008 1.075 0.955 2.611 3.74 5.458 6.153 1.424 2.431 0.067 0.075 44.9 A,B,H Kishorn REF 26/6/01 3.98 7.946 -0.238 0.681 0.451 0.902 4.63 6.123 6.796 2.611 2.901 0.064 0.04 56.5 D,E,F Kishorn T1B 13/8/01 3.73 8.396 -0.199 0.699 0.287 0.705 4.32 5.963 6.659 2.629 2.952 0.075 0.05 53.6 D,E,F Sunart Stn 2 7/3/01 3.26 3.598 0.197 1.457 1.594 2.173 3.04 4.042 5.139 0.934 1.893 0.104 0.122 25.4 J,I,G Sunart LSB 4 8/3/01 4.93 4.457 -0.105 1.705 3.507 4.156 5.11 6.096 6.764 0.97 2.037 0.033 0.029 78.2 K,E,L Stn1 LSB1 Sunart 2.59 4.992 0.209 0.962 0.371 0.772 2.32 3.899 5.008 1.564 2.272 0.166 0.2 23.8 A,I,H Feb/March 2000 Stn2 LSB2 Sunart 2.92 6.039 0.081 0.803 0.273 0.688 2.89 4.677 5.565 1.994 2.508 0.132 0.135 32.4 A,B,F Feb/March 2000 Stn3 LSB3 Sunart 2.85 5.095 0.212 0.849 0.564 0.881 2.59 4.341 5.357 1.73 2.284 0.138 0.166 28.2 A,I,F Feb/March 2000 Stn4 LSB4 Sunart 3.1 8.285 -0.177 0.712 -0.162 0.371 3.74 5.359 5.979 2.494 2.848 0.117 0.075 47.6 A,E,F Feb/March 2000 Stn2 LSB2 March Sunart 2.47 7.517 0.058 0.746 -0.365 0.054 2.54 4.658 5.456 2.302 2.725 0.18 0.172 32.9 A,B,F 2000 Sunart Stn 3 March 2001 2.98 6.736 0.041 0.728 0.084 0.549 3.01 5.064 5.771 2.258 2.637 0.127 0.124 37.8 A,B,F Sunart Stn 2 TBF Dec 01 3.87 5.137 0.057 1.287 2.115 2.77 3.81 5.345 6.047 1.288 2.208 0.069 0.071 45.4 A,B,G

Ecological effects of sea lice medicines in Scottish sea lochs 284 of 286 Table 17.5 (cont.) Identifiers Phi Summary Stats Phi Percentiles Phi Quart. Dev. Sort. Coeff. Diameter (mm) summary stats Fract Description lab field mean var skew kurt 16% 25% 50% 75% 84% devn. coeff. mean median 63um Stn 1 TBF Dec 01 Sunart 3.58 5.817 -0.052 1.06 0.714 2.08 3.61 5.276 5.971 1.598 2.567 0.083 0.082 43.3 A,B,H Sunart Stn 4.1 Dec 01 3.89 7.51 -0.289 0.7 0.422 0.818 4.61 5.803 6.456 2.493 2.798 0.067 0.041 60.1 D,E,F Stn 3 TBF Dec 01 Sunart 3.21 4.38 0.131 0.977 0.884 1.518 3.05 4.452 5.432 1.467 2.197 0.108 0.121 30.3 A,I,H Sunart Ref 14/5/02 3.58 5.678 -0.013 1.028 0.683 2.082 3.54 5.285 6.027 1.601 2.553 0.083 0.086 41.2 A,B,H Sunart S1C 14/5/02 2.64 7.555 0.038 0.79 -0.564 0.179 2.66 4.709 5.7 2.265 2.889 0.161 0.158 31.1 A,B,F Sunart S2B 14/5/02 2.39 7.067 0.062 0.858 -0.667 0.141 2.37 4.21 5.33 2.034 2.79 0.191 0.193 26.7 M,B,F Sunart S1B 14/5/02 2.59 5.813 0.137 0.968 0.128 0.621 2.46 4 5.266 1.69 2.494 0.166 0.182 25 M,I,H Sunart S1A 14/5/02 3.23 3.66 0.178 1.299 1.461 2.055 3.01 4.165 5.057 1.055 1.912 0.106 0.124 27 J,I,G Sunart S2C 14/5/02 3.16 5.283 0.185 0.857 0.737 1.246 2.9 4.774 5.729 1.764 2.366 0.112 0.134 31.9 A,I,F Kishorn T1 A1 25/6/01 4.54 6.667 -0.261 0.986 0.904 2.909 5.01 6.389 7.056 1.74 2.806 0.043 0.031 64 K,E,H Kishorn T1 C1 25/6/01 3.01 10.19 0.091 0.649 -0.419 0.041 2.93 5.862 6.616 2.91 3.156 0.124 0.131 44.2 M,B,C Kishorn T2 A1 26/6/01 4.44 6.456 -0.259 1.119 0.909 3.039 4.86 6.155 6.849 1.558 2.774 0.046 0.034 64.4 D,E,G Kishorn T2 B1 26/6/01 2.49 10.346 0.505 0.651 -0.878 -0.334 0.94 5.521 6.375 2.928 3.223 0.178 0.52 36.1 N,O,C Kishorn T2 C1 26/6/01 2.59 9.847 0.099 0.654 -1.07 -0.419 2.47 5.366 6.205 2.892 3.218 0.166 0.181 36.1 A,B,C Kishorn 1VV2 Jun 01 4.3 7.314 -0.203 0.98 0.816 2.533 4.64 6.302 6.992 1.884 2.909 0.051 0.04 58.5 D,E,H Kishorn 2VV1 Jun 01 3.57 9.686 -0.196 0.684 -0.224 0.438 4.19 6.085 6.791 2.823 3.181 0.084 0.055 51.8 D,E,F Kishorn 3VV1 Jun 01 3.33 8.719 -0.112 0.686 -0.039 0.42 3.8 5.707 6.424 2.644 2.956 0.099 0.072 48.2 A,E,F Kishorn 4VV1 Jun 01 3.6 10.058 -0.157 0.676 -0.119 0.511 4.13 6.294 6.999 2.892 3.225 0.082 0.057 51.1 D,E,F Kishorn 5VV1 Jun 01 2.82 9.988 0.066 0.646 -0.77 -0.285 2.78 5.599 6.386 2.942 3.194 0.141 0.146 39.6 M,B,C Kishorn 6VV1 Jun 01 3.45 8.879 -0.095 0.716 -0.066 0.596 3.73 5.884 6.593 2.644 3.065 0.091 0.075 47.5 A,B,F Kishorn 7VV1 Jun 01 2.48 9.377 0.614 0.663 -0.378 -0.037 0.81 5.419 6.194 2.728 2.98 0.18 0.57 36.5 N,O,C Kishorn Ref 24/1/02 3.71 9.962 -0.111 0.647 0.032 0.567 4.11 6.46 7.168 2.947 3.194 0.076 0.058 50.9 D,E,C Kishorn T1C 13/8/01 4.09 8.785 -0.29 0.67 0.253 0.791 4.84 6.341 6.99 2.775 3.058 0.059 0.035 59.8 D,E,C Kishorn Stn 1 Aug 01 3.92 7.93 -0.202 0.701 0.384 0.913 4.43 6.053 6.768 2.57 2.928 0.066 0.046 55.1 D,E,F Kishorn Stn 2 Aug 01 3.05 10.805 -0.11 0.639 -1.019 -0.237 3.53 5.866 6.615 3.051 3.349 0.121 0.086 46.2 A,E,C Kishorn Stn 4 Aug 01 3.78 8.55 -0.23 0.686 0.116 0.709 4.46 6.022 6.74 2.657 3.003 0.073 0.046 55.2 D,E,F Kishorn Stn 5 Aug 01 3.47 7.924 -0.023 0.712 0.116 0.708 3.55 5.735 6.474 2.513 2.913 0.09 0.085 44.9 A,B,F Kishorn Stn 6 Aug 01 2.9 9.6 0.05 0.661 -0.55 -0.045 2.94 5.624 6.406 2.834 3.125 0.134 0.13 40.4 M,B,C Kishorn Stn 7 Aug 01 2.66 9.52 0.567 0.664 -0.417 -0.016 0.96 5.523 6.273 2.769 3.033 0.158 0.513 38.6 N,O,C

Ecological effects of sea lice medicines in Scottish sea lochs 285 of 286 Table 17.5 (cont.) Identifiers Phi Summary Stats Phi Percentiles Phi Quart. Dev. Sort. Coeff. Diameter (mm) summary stats Fract Description lab field mean var skew kurt 16% 25% 50% 75% 84% devn. coeff. mean median 63um Kishorn T1A 24/1/02 4.5 5.954 -0.18 1.129 1.593 3.11 4.79 6.125 6.858 1.508 2.574 0.044 0.036 64 D,E,G Kishorn T2A 24/1/02 4.56 6.84 -0.249 0.931 1.345 2.686 5.04 6.47 7.133 1.892 2.75 0.042 0.03 63.9 K,E,H Kishorn T2B 24/1/02 0.74 9.124 0.864 1.703 -1.331 -1.236 -0.87 0.894 5.028 1.065 2.93 0.599 1.833 19.2 P,O,L Kishorn T2C 24/1/02 3.45 8.22 0.001 0.739 0.253 0.784 3.45 5.798 6.51 2.507 2.934 0.091 0.091 45.7 A,B,F Craignish Stn 1 Oct 00 2.79 10.503 0.308 0.642 -0.611 -0.198 1.9 5.716 6.442 2.957 3.166 0.145 0.268 43.4 Q,O,C Craignish Stn 2 Oct 00 4.39 7.581 -0.303 0.799 0.656 1.925 5.06 6.355 7.033 2.215 2.903 0.048 0.03 64.5 K,O,F Craignish Stn 3 Oct 00 2.88 11.379 0.551 0.622 -0.658 -0.207 0.96 6.029 6.767 3.118 3.291 0.135 0.514 44.4 N,O,C Craignish Stn 1 18/12/01 4.33 8.53 -0.342 0.666 0.449 0.941 5.17 6.465 7.136 2.762 3.031 0.05 0.028 64 K,R,C Craignish Stn 2 18/12/01 3.11 10.849 0.066 0.62 -0.608 -0.092 3.05 6.083 6.816 3.088 3.271 0.116 0.121 45.7 A,B,C Craignish Stn 3 18/12/01 2.54 10.112 0.424 0.656 -0.785 -0.252 1.32 5.546 6.395 2.899 3.202 0.171 0.399 36.5 Q,O,C Diabaig Stn 1 15/11/00 2.05 11.257 0.551 0.598 -1.209 -1.045 0.57 5.349 6.27 3.197 3.283 0.241 0.675 33.3 N,O,C Diabaig Stn 2 15/11/00 2.78 12.331 0.549 0.604 -0.926 -0.399 0.85 6.171 6.927 3.285 3.431 0.145 0.554 43.3 N,O,C Diabaig Stn 3 15/11/00 4.04 7.512 0.055 0.849 0.749 2.108 3.73 6.335 7.026 2.113 2.896 0.061 0.075 47.2 A,B,F Diabaig T1 Aug 01 4.15 8.417 -0.302 0.849 0.525 1.647 4.87 6.243 6.967 2.298 3.053 0.056 0.034 61.1 D,R,F Diabaig T2 Aug 01 4.25 9.032 -0.346 0.628 0.426 0.755 5.25 6.497 7.173 2.871 3.019 0.053 0.026 63.5 K,R,C Diabaig T3 Aug 01 3.95 7.63 -0.208 0.691 0.451 0.902 4.47 6.011 6.706 2.555 2.87 0.065 0.045 55.9 D,E,F

*A - Very poorly sorted very fine sand B - Symmetrical C - Very platykurtic D - Very poorly sorted coarse sand E - Coarse skewed F - Platykurtic G - Leptokurtic H - Mesokurtic I - Fine sand J - Poorly sorted very fine sand K - Very poorly sorted medium silt L - Very leptokurtic M - Very poorly sorted fine sand N - Very poorly sorted coarse sand O - Strongly fine skewed P - Very poorly sorted very coarse sand Q - Very poorly sorted medium sand R - Strongly coarse skewed

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