Fisheries Research Services Contract Report No 01/04

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1 Not to be quoted without prior reference to the authors Fisheries Research Services Contract Report No 01/04 DETERMINATION OF THE SEMI-SYNTHETIC AVERMECTIN, EMAMECTIN BENZOATE, BY LIQUID CHROMATOGRAPHY ATMOSPHERIC PRESSURE CHEMICAL IONISATION MASS SPECTROMETRY (LC-APCI-MS) IN SEDIMENT FROM LOCH SUNART AND LOCH KISHORN Pamela Walsham and Lynda Webster January 2004 Fisheries Research Services Marine Laboratory Victoria Road Aberdeen AB11 9DB

2 DETERMINATION OF THE SEMI-SYNTHETIC AVERMECTIN, EMAMECTIN BENZOATE, BY LIQUID CHROMATOGRAPHY ATMOSPHERIC PRESSURE CHEMICAL IONISATION MASS SPECTROMETRY (LC-APCI-MS) IN SEDIMENT FROM LOCH SUNART AND LOCH KISHORN Pamela Walsham and Lynda Webster Fisheries Research Services, Marine Laboratory 375 Victoria Road, Aberdeen, AB11 9DB SUMMARY 1. In 2000 the UK Veterinary Medicines Directorate (VMD) issued a Marketing Authorisation for the treatment of sea lice in salmon aquaculture using Slice, which has the active ingredient emamectin benzoate. 2. A liquid chromatography atmospheric pressure chemical ionisation mass spectrometry (LC-APCI-MS) method was developed and validated for the determination of the semisynthetic avermectin, emamectin benzoate, in marine sediment. Recoveries from sediment samples ranged from 66.50% to % (mean = 85.7%, n= 7, standard deviation [SD] = 11.97). 3. A total of thirty-two sediment samples were collected from Loch Kishorn and Loch Sunart and analysed for emamectin benzoate. Sediment samples were collected both pre and post treatment of a fish farm with Slice. 4. Twenty-five sediment samples were collected from Loch Kishorn, between June 2001 and January 2002, of which three were from reference sites. 5. Emamectin benzoate concentrations were found to be either below the limit of quantification or not detected for twelve samples analysed from Loch Kishorn. Concentrations in the remaining samples ranged from 1.01 ng g -1 dry weight to ng g -1 dry weight. 6. Seven sediment samples were collected from Loch Sunart during May Emamectin benzoate concentrations in all but one sample from Loch Sunart were either below the limit of quantification or not detected. INTRODUCTION Annual Atlantic salmon (Salmo salar) production has increased from 10,337 tonnes in 1986 to an estimated 159,060 tonnes in The rapid increase in intensive production practices has resulted in a number of fish health problems including parasitic sea lice damage. In Scotland alone it is estimated that the problems caused by sea lice cost the salmon farming industry between 20 and 30 million per annum 2. 1

3 The sea louse, a small copepod, damages the host fish by eating the mucus, epithelium, scales and, in severe cases, the underlying dermis. There are two species of sea lice, which specifically attack fish; Lepeophtherius salmonis (Krøyer) and Caligus elongatus (Nordmann). A number of chemicals have been used in formulations to combat sea lice in fish farms including the bath treatments dichlorvos, hydrogen peroxide and cypermethrin. The use of bath treatments however, is often impractical during bad weather and is very stressful to the fish. To eliminate these potential problems in-feed treatments have been developed including the semisynthetic avermectin, emamectin benzoate, which consists of the two-avermectin homologs B1a and B1b (Fig. 1). Emamectin benzoate has been found to be effective against both the immature and adult stages of sea lice when administered at a dose of 50 µg kg -1 body weight day -1, for seven days 3,4,5,6,7. At high doses however, it has been found to be toxic to fish and planktonic marine copepods and induce molting in American lobster, Homarus americanus 8, 9,10. In 2000 the UK Veterinary Medicines Directorate (VMD) issued a Marketing Authorisation for the treatment of sea lice in salmon aquaculture using Slice, which has the active ingredient emamectin benzoate. An environmental risk assessment of emamectin benzoate was undertaken by The Scottish Environment Protection Agency (SEPA) prior to approval for use. Results from this study indicate that the fish will absorb 90% of emamectin benzoate while 10% will be immediately excreted in the faeces 11. The potential impact on the marine environment will, therefore, either be as a result of indirect input from excreted material or the direct input from waste feed. SEPA have set predicted no effect concentrations (PNEC) of 7.76 ng g -1 dry weight for sediment within 25 m of the edge of the cage (the allowable zone of effect) and ng g -1 dry weight for sediment beyond the allowable zone of effect 11. The allowable zone of effect is defined as: the area of sea-bed or receiving water body in which SEPA will allow some exceedence of a relevant Environmental Quality Standard (EQS) or some damage to the environment 12. Emamectin benzoate has a low seawater solubility (5.5 mg l -1 ) and a relatively high octanol:water partition coefficient (log K ow = 5.0), indicating that it is lipophillic and has the potential to be adsorbed and bound to particulate material and surfaces. A number of techniques have been developed for the analysis of emamectin benzoate in different matrices. These include milk, fish feed and fish muscle by high performance liquid chromatography (HPLC) with fluorescence detection and soil by HPLC with ionspray mass spectrometry (MS)/MS 13,14,15,16,17. A government-funded project was commissioned to assess the ecological effects of sea lice treatments, post authorisation. Fisheries Research Services (FRS), Analytical Investigations Group was contracted to undertake chemical analysis in support of biological sampling for the project. To investigate the possible accumulation of emamectin benzoate in sediments following treatment with Slice, samples were collected from both Loch Kishorn and Loch Sunart pre and post treatment, in the vicinity of the fish farm. 2

4 EXPERIMENTAL Materials Dichloromethane (DCM), iso-hexane, methanol and acetone were purchased from Rathburn Chemicals Ltd (Walkerburn, Scotland). Anhydrous sodium sulphate (Na 2 SO 4 ) was of analytical grade and was supplied by Fisher Chemicals, Loughborough, UK. Isolute C 18, (5 g x 25 ml) SPE cartridges were supplied by Crawford Scientific, Strathaven, UK. Emamectin benzoate standard was of Pestanal analytical standard grade and supplied by Sigma-Aldrich Comapny Ltd, Dorset, UK. Equipment and Sodium Sulphate Preparation 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 o C. Anhydrous sodium sulphate was washed twice with DCM followed by iso-hexane prior to drying overnight (85 o C ± 10 o C). Extraction and Analysis of Emamectin Benzoate in Sediment Samples Each sediment sample was thoroughly mixed and an aliquot (~ 10 g) removed for the determination of water content by oven drying at 80 o C for 22 (±2) hrs 18,19. 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 Na 2 SO 4. The extract was solvent exchanged to iso-hexane and concentrated (~ 1 ml) by rotary evaporation (water bath temperature, < 30ºC). Samples were transferred to pre-conditioned (iso-hexane, 10 ml) C 18 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 the mixture shaken before being transferred to a crimp top vial for analysis by liquid chromatography mass-spectrometry (LC-APCI-MS). A PE Sciex API 150 (Perkin Elmer, Macclesfield, UK) single quadropole mass spectrometer equipped with an atmospheric pressure chemical ionisation (APCI) source, in positive ion mode, 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 with a run time of 12 minutes. A 150 x 2.00 mm ID column packed with 3 µm particles coated with a C 18 stationary phase was used. A method was set up for quantification purposes in single ion monitoring (SIM) mode 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 seven-point calibration curve was used for quantification. The ions monitored were m/z 886.6, 872.7, and with a dwell time of 150 msecs. Quantification was based on the area of the summed (total) ion chromatogram (TIC). 3

5 Determination of Particle Size Distribution (PSA) and Total Organic Carbon (TOC) in Sediments The particle size analysis was carried out on freeze-dried sediments by laser granulometry using a Malvern Mastersizer E Particle Size Analyser. The TOC was determined on freeze-dried and ground sediment, following acid treatment to remove carbonate. The organic carbon was measured using a combustion method on a Perkin Elmer CHN 2400 elemental analyser. Method Validation RESULTS AND DISCUSSION Emamectin is synthesised from abamectin (avermectin B 1a and B 1b ) by substitution of an epimethylamino group or a hydroxyl group at the 4 - position on the disaccharide and is isolated as a benzoate salt. Emamectin benzoate is composed of a mixture of two homologues, 4 - epimethylamino-4 -deoxyavermectin B1a and B1b benzoate with a ratio of B1a to B1b of approximately 9:1. The components differ only with B1a having an extra methylene group on one of the side chains (Fig. 1). Emamectin benzoate standard was initially scanned to identify the major characteristic ions for quantification purposes. The molecular weights of the emamectin benzoate homologues are 1008 and 994 daltons. The base ion in the mass chromatogram was from the emamectin part of the B1a homologue and gave a peak at m/z with B1b giving a smaller peak at m/z 872.7, with the ratio of 9:1, as expected. A selective ion monitoring (SIM) quantification method was developed using these ions along with two qualifier ions (m/z and 776.6) (Fig. 2). Quantification of the area of the summed (total) ion chromatogram (TIC) was plotted against amount injected on column, using Analyst software, to give the calibration curve and correlation coefficient. The 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 (Table1). The linearity of the response of the LC-APCI-MS was assessed for emamectin benzoate over the standard calibration range ~5 ng ml -1 to 1,500 ng ml -1. The detector response for emamectin benzoate was linear, with a correlation coefficient of at least An external standard method was used for the quantification of emamectin benzoate in sediment. Calibration standards covering the range 5 ng ml -1 to 1,500 ng ml -1 were analysed with each batch of samples. Instrument Precision for the Analysis of Emamectin Benzoate In-series precision of the standards by replicate analysis (n = 8) of 90% ( ng ml -1 ) and 10% (111.2 ng ml -1 ) of the stock standard was determined. The coefficient of variance (CV%) was 2.1% at the higher concentration and 1.6% at the lower concentration indicating precision was better for the instrument at lower concentrations (Table 2). The limit of quantification for the instrument was determined as 1 ng g -1 for a 10 g sample of sediment, using the equation: Limit of detection = (4.65 x standard deviation of the low standard/ weight of sample used for extraction) 20. 4

6 Emamectin Benzoate Recovery from Sediment Reference sediment, collected from a site within a sea loch but distant from any fish farms, was analysed and was found not to contain emamectin benzoate above the limit of detection. Seven aliquots (~ 10 g) of this sediment were spiked with 1112 ng (~ ng g -1 ) of emamectin benzoate and extracted and the subsequent extract analysed by LC-MS. Recoveries of between 66.50% and % (mean = 85.7%, n=7, standard deviation [SD] = 11.97, CV% = 13.97%) were achieved (Table 3). Data Summary Thirty-two sediment samples were analysed for emamectin benzoate the results of which are presented in Tables 4 and 5. Twenty-five of the sediment samples were collected from Loch Kishorn, between June 2001 and January Eight of the sediment samples were found to contain emamectin benzoate concentrations at trace level (detected but below the limit of quantification of 1 ng g -1 dry weight, Table 4). Concentrations of emamectin benzoate in the remaining, Loch Kishorn, sediment samples ranged from 1.01 ng g -1 to ng g -1 and included the reference sediment collected on 26 June 2001 (Table 4). Emamectin benzoate concentrations found in the seven sediment samples, collected from Loch Sunart during May 2002, were either trace or not detected in all but one sample (Table 5). Loch Kishorn Twenty-five sediment samples were collected from seven sampling stations, including the reference sediment samples, within Loch Kishorn, between June 2001 and January 2002 (Fig. 3, Table 4). Sediment samples were collected along two legs each consisting of three sampling stations, either by a diver collected core or using a hand held Van veen Grab (Table 4). Sampling stations were located at 25 m intervals from the edge of the cage on leg 1 (T1) and at 20 m intervals on leg 2 (T2), Figure 3. One sampling station from each of the legs (Station A) was located within the zone of effect. The reference sediment samples were collected south west of the cage away from the predicted current flow (Fig. 3) 21. Two sediment samples were collected from each of the sampling stations during June 2001, prior to the first Slice treatment in July A diver using a corer collected one set of samples while the second set of samples was collected from a boat by means of a hand held Van veen Grab (Table 4). All but two, of the twelve, sediment samples collected contained emamectin benzoate. Six of the sediment samples contained trace amounts (detected but below the limit of quantification of 1 ng g -1, dry weight) of emamectin benzoate. Concentrations in the remaining four ranged from 1.77 ng g -1 dry weight (T1A1) to 4.09 ng g -1 dry weight (Kishorn 4V V1, Table 4). These sediment samples were collected closest to the cage, Station A (Table 4). However, concentrations were all within SEPA s PNEC for sediment within 25 m of the edge of the cage of 7.76 ng g -1 dry weight. A license for the use of Slice at the fish farm was issued prior to the first treatment in July The sample collection in June 2001 was expected to be pre-treatment, however it has not been possible to establish whether the operators undertook a treatment prior to this date. Two sediments were collected from the reference site, one of which was collected by a diver with a corer and the other from a boat by means of a hand held Van veen Grab (Table 4). Emamectin benzoate was not detected in the reference sediment, which had been collected by the grab. The reference sediment collected by diver core contained 1.78 ng g -1 dry weight, 5

7 emamectin benzoate, which exceeds SEPA s PNEC concentration of ng g -1 dry weight for sediment collected outwith the 25 m allowable zone of effect. The reference sample was collected following the collection of transect sediment samples. However, between each sampling the core tubes were rinsed with solvent to prevent possible cross contamination and therefore, the emamectin benzoate observed in the reference sample can not be attributed to contamination from the core tube. The fish farm at Loch Kishorn underwent two applications with the in-feed treatment, Slice following the collection of sediment samples in June, the first in July 2001 and the second in October The amount of active ingredient used in each treatment was reported as g and g, respectively. Further sediment samples collections were made from the stations during August 2001 and January 2002, resulting in analysis of sediment ~1 month and ~3 months post treatment. A diver using a corer collected one sediment sample from each of the stations, apart from T1B in August 2001 (~ 1 month post treatment, Table 4). Emamectin concentrations in sediment collected, approximately one month after treatment, ranged from not detected in Kishorn T2C 13/08/01 to ng g -1 dry weight in Kishorn T2A 13/08/01 (Table 4). Kishorn T2A 13/08/01 was collected at a distance of 20 m from the edge of the cage, within the allowable zone of effect, however the concentration observed exceeded the PNEC concentration of 7.76 ng g -1 dry weight. The sediment collected from the site closest to the cage on the first leg (T1A) fell within the PNEC (Kishorn T1A 13/08/01, 3.54 ng g -1 ; Table 4). Of the remaining three sediment samples collected outwith the 25m zone effect only one sample, Kishorn T2C 13/08/01 contained no detectable levels of emamectin benzoate (Table 4). Kishorn T2B 13/08/01 and Kishorn T1C 13/08/01 contained 2.04 ng g -1 dry weight and 1.08 ng g -1 dry weight emamectin benzoate, respectively. Emamectin benzoate concentrations in these samples were above the PNEC of emamectin benzoate (0.763 ng g -1 dry weight) for sediment collected outwith the 25 m, zone of effect. The reference sample, which was collected along with these samples contained trace concentrations of emamectin benzoate. Four leg stations were sampled in January 2002 (~ 3 months post treatment), of which only one sediment was collected from leg 1 (T1A, Table 4). Concentrations of emamectin benzoate ranged from 1.01 ng g -1 dry weight, Kishorn T2B 24/01/02, to 4.83 ng g -1 dry weight, Kishorn T2A 24/01/02 (Table 4). Emamectin benzoate concentrations from sediment collected from within the allowable zone of effect, Station A, were below the PNEC. However, the two sediments collected outwith the allowable zone of effect contained emamectin benzoate concentrations in excess of ng g -1 dry weight, the PNEC. Kishorn T2B 24/01/02 and Kishorn T2C 24/01/02 contained 1.01 ng g -1 dry weight and 1.59 ng g -1 dry weight of emamectin benzoate respectively (Table 4). The reference sample, which was collected along with these samples, contained trace concentrations of emamectin benzoate. Only one sample on the first leg (T1A) was sampled both one month and 3 months post treatment. Concentrations of emamectin benzoate were similar for both 3.54 ng g -1 dry weight for the ~ 1 month and 3.26 ng g -1 dry weight for the ~ 3 month sample (Table 4). Only one further sample was collected from the first leg (T1C, 13/08/01), therefore no further comparison was possible. The largest decrease in emamectin benzoate concentration was observed in leg 2 station A where T2A 13/08/01 (~1 month post treatment) contained ng g -1 dry weight while T2A 24/01/02 (~3 month post treatment) contained 4.83 ng g -1 dry weight (Table 4). Transect 2 station B contained 2.04 ng g -1 dry weight and 1.01 ng g -1 dry weight emamectin benzoate for the 1 and 3 month post treatment samples, respectively (Table 4). Emamectin benzoate was not observed in transect 2 station C 1 month post treatment, however a 6

8 concentration of 1.59 ng g -1 dry weight was determined in the 3 month post treatment sample (Table 4). Four reference sediment samples were collected from a single site located to the south west of the cage, at the time of transect sampling, between June 2001 and January 2002 (Fig. 3). Only the reference sediment sample, which had been collected by hand held Van veen grab contained no trace of emamectin benzoate (Table 4). The core tubes, used by the diver for sediment collection, were rinsed between samples with solvent to prevent possible cross contamination therefore, the emamectin benzoate observed cannot be attributed to core tube contamination. A procedural blank was analysed along with each batch of samples to monitor for any contamination within the laboratory. The procedural blanks analysed with these sediments did not contain emamectin benzoate and therefore, the emamectin benzoate observed in the reference samples cannot be attributed to background laboratory contamination. Loch Sunart The fish farm at Loch Sunart underwent a treatment with Slice, on 6 May The amount of active ingredient used in the treatment was reported as g. Following this treatment seven sediment samples, including a reference sediment, were collected on 14 May 2002 (Fig. 4). Sediment samples were collected along two transects running south easterly from the farm towards the shore. Sampling stations were located at intervals of 25 m, from the cage, on transect 1 and at intervals of 20 m, from the cage, on transect 2. One sampling station from each of transect (Station A) was located within the zone of effect. A reference sediment sample was also collected at a distance of 400 m from the cage. In all but one of these samples, Sunart S2A 14/5/02, concentrations observed for emamectin benzoate were either trace, (detected but below the limit of quantification of 1 ng g -1 dry weight), or not detected (Table 5). Loch Sunart sample S2A 14/5/02, which was the closest station to the cage, and collected within 25m of the cage, contained 7.38 ng g -1 dry weight of emamectin benzoate. However, it should be noted that between sample variance determined during method validation was 13.97% and thus the results should be considered as 7.38 ± 1.03 ng g -1 dry weight. The Scottish Environment Protection Agency (SEPA), predicted no effect concentrations (PNEC) for sediment within 25 m of the edge of the cage (the allowable zone of effect) is 7.76 ng g -1 dry weight. Particle size analysis (PSA), Total organic carbon (TOC) and Total nitrogen Finer sediments (those with a high % percentage of particles < 63 µm) tend to accumulate higher loads of hydrophobic contaminants because of their greater surface area per unit of volume. Sediments with higher organic carbon contents also have the potential to accumulate significant concentrations of the sea lice treatments. The detailed results for PSA are presented in Appendix 1 and a summary is included in Table 6. The percentages for < 63 µm fraction in the Loch Kishorn samples ranged from 19.2% (Kishorn T2B 24/01/02) to 71.5% (Kishorn T214 13/08/01) while in Loch Sunart they ranged from 25.0% (Sunart S1B 14/05/02) to 41.2% (Sunart Ref 14/05/02). The percentages of the TOC are presented in Table 6. The TOC from Loch Kishorn samples ranged from 1.432% (Kishorn T2C 13/08/01) to 5.903% (Kishorn T2A 24/01/02) while in Loch Sunart they ranged from 0.98% (Sunart S1B 14/05/02) to 2.39% (Sunart Ref 14/05/02). PSA and TOC were found not to be correlated (p > 0.05, ANNOVA). 7

9 CONCLUSIONS 1. A LC-APCI-MS method was developed and validated for the analysis of emamectin benzoate in marine sediments. 2. Emamectin benzoate recoveries from sediment ranged from 66.50% to %, mean = 85.7%, n = 7, SD = 11.97, CV = 13.97%. 3. Emamectin benzoate was detected in sediment collected from Loch Kishorn pre and post Slice treatment. The highest concentration found was in a sample collected within 25 m of the edge of the cage. 4. All but two of the sediments collected in June 2001 contained emamectin benzoate. This collection was expected to be pre-treatment, however it has not been possible to establish whether the fish farm operators had undertaken a treatment prior to this date. 5. One sample collected from Loch Sunart, post Slice treatment, was found to contained detectable levels of emamectin benzoate. The sediment sample containing emamectin benzoate was collected within 25 m of the edge of the cage. 6. One sediment sample from Loch Kishorn and one from Loch Sunart, which had been collected within 25 m of the edge of the cage, contained emamectin benzoate at concentrations in excess of the PNEC (7.76 ng g -1 dry weight, within 25 m of the cage). 7. Five sediment samples collected from Loch Kishorn, outwith the 25 m zone of effect, contained concentrations of emamectin benzoate in excess of the PNEC (0.763 ng g -1 dry weight). REFERENCES 1. Fisheries Research Services Scottish Fish Farms Annual Production Survey, The Scottish Executive Environment and Rural Affairs Department. 2. Rae, G.H Sea louse control in Scotland, past and present. Pest Manage. Sci., 58, Stone, J., Sutherland, I.H., Sommerville, C.S., Richards, R.H. and Varma, K.J The efficacy of emamectin benzoate as an oral treatment of sea lice, Lepeophtherius salmonis (Krøyer), infestations in Atlantic salmon, Salmo salar L. J. Fish Dis., 22, Stone, J., Sutherland, I.H., Sommerville, C.S., Richards, R.H. and Varma, K.J Commercial trials using emamectin benzoate to control sea lice, Lepeophtherius salmonis infestations in Atlantic salmon Salmo salar. Dis. Aquat. Org., 41, Stone, J., Sutherland, I.H., Sommerville, C.S., Richards, R.H. and Varma, K.J Field trials to evaluate the efficacy of emamectin benzoate in the control of sea lice, Lepeophtherius salmonis (Krøyer) and Caligus elongatus (Nordmann), infestations in Atlantic salmon, Salmo salar L. Aquaculture, 186,

10 6. Armstrong, R., Macphee, D., Katz, T. and Endris, R A field efficiacy evaluation of emamectin benzoate for the control of sea lice on Atlantic salmon. Can. Vet. J., 41, Treasurer, J.W., Wallace, C. and Dear, G Control of sea lice on farmed Atlantic salmon S.salar L. With the oral treatment emamectin benzoate (SLICE). Bull. Eur. Ass. Fish. Pathol., 22, Roy, W.J., Sutherland, I.H., Rodger, H.D.M. and Varma, K.J Tolerance of Atlantic salmon, Salmo salar L., and rainbow trout, Oncorhynchus mykiss (Walbaum), to emamectin benzoate, a new orally administered treatment for sea lice. Aquaculture, 184, Willis, K.J. and Ling, N The toxicity of emamectin benzoate, an aquaculture pesticide, to planktonic marine copepods. Aquaculture, 221, Waddy, S.L., Burridge, L.E., Hamilton, M.N.,, Mercer, S.M., Aiken, D.E. and Haya, K Emamectin benzoate induces molting in American lobster, Homarus americanus. Can. J. Fish. Aquatic. Sci., 59, Scottish Environment Protection Agency Emamectin Benzoate an Environmental Risk Assessment. 12. Scottish Environmental Protection Agency Regulation and monitoring of marine cage fish farming in Scotland- a procedural manual. 13. Mushtaq, M, Allen, L.R.S., Crouch, L.S. and Wislocki, P.G Fate of 3 H- and 14 C- labeled Emamectin benzoate in lactating goats. J. Agri. Food. Chem., 45, Farer, L.J., Hayes, J., Rosen, J. and Knight, P Determination of emamectin benzoate in medicated fish feed. J. AOAC International, 82, VAN DE Riet, J.M., Brothers, N.N., Pearce, J.N. and Burns, B.G Simultaneous determination of Emamectin and Ivermectin residues in Atlantic salmon muscle by liquid chromatography with fluoresence detection. J. AOAC International, 84, Chukwudebe, A.C., Atkins, R.H. and Wislocki, P.G Metabolic fate of Emamectin benzoate in soil. J. Agric. Food Chem., 45, Kim-Kang, H., Crouch, L.S., Bova, A., Robinson, R.A. and Wu, J Determination of emamectin benzoate residues in tissue of Atlantic salmon (Salmo salar L.) using HPLC with fluorescence detection. J. Agric. and Food Chemistry, 49, Webster, L., McIntosh, A.D., Moffat, C.F., Dalgarno, E.J., Brown, N.A. and Fryer, R.J Analysis of sediments from Shetland Island voes for polycyclic aromatic hydrocarbons, steranes and triterpanes. J. Environ. Monit., 2, Webster, L., Fryer, R.J., Dalgarno, E.J., Megginson, C. and Moffat, C.F The polycyclic aromatic hydrocarbon and geochemical biomarker composition of sediments from voes and coastal area in the Shetland and Orkney Islands. J. Environ. Monit., 3,

11 20. Cheesman, R.V. and Wilson, A.L A manual on quality control for the water industry. WRc Technical Report No PAMP Ecological effects of sea lice medicines in Scottish sea lochs. Draft report April

12 TABLE 1 Optimised parameters for the analysis of emamectin benzoate by LC-MS fitted with the atmospheric pressure chemical ionisation (APCI) source Parameter Setting Nebuliser Gas (N 2 ) 9 Curtain Gas (N 2 ) 8 Nebuliser Temperature Nebuliser Current Declustering Potential (DP) Focusing Potential (FP) 350 o C 5 Å 15 V 200 V 11

13 TABLE 2 Method precision for emamectin benzoate analysed on a PE Sciex API 150 LC-MS 10% Std (111.2 ng ml -1 ) 90% Std ( ng ml -1 ) Peak Area Mean SD a CV % a Standard deviation 12

14 TABLE 3 Recovery data of seven sediment samples, each spiked with ~111.2 ng g -1 Benzoate. of Emamectin Sample Recovery (ng) % Recovery A B C D E F G Mean Median SD a CV% a Standard Deviation 13

15 TABLE 4 Sample sites, sampling dates, collection method and emamectin benzoate concentrations for sediment collected from Loch Kishorn between June 2001 and January Where samples A were collected within the 25 m zone of effect and samples B and C were outwith this zone. Lab ID Field ID/Collection Date Station Code/ Collection method Concentration (ng g -1 dry weight) June /02 Kishorn Reference 26/06/01 Reference/Diver core /02 Kishorn T1A1 25/6/01 T1A/Diver core /02 Kishorn T1B1 25/6/01 T1B/ Diver core Trace 10075/02 Kishorn T1C1 25/6/01 T1C/ Diver core Trace 10076/02 Kishorn T2A1 26/6/01 T2A/ Diver core /02 Kishorn T2B1 26/6/01 T2B/ Diver core Trace 10078/02 Kishorn T2C1 26/6/01 T2C/ Diver core ND 10085/02 Kishorn 7V V1 28/06/01 Reference/Vanveen Grab ND 10079/02 Kishorn 1V V2 JUNE 01 T1A/Vanveen Grab /02 Kishorn 2V V1 25/6/01 T1B/Vanveen Grab ND 10081/02 Kishorn 3V V1 JUNE 01 T1C/Vanveen Grab Trace 10082/02 Kishorn 4V V1 27/6/01 T2A/Vanveen Grab /02 Kishorn 5V V2 28/6/01 T2B/Vanveen Grab Trace 10084/02 Kishorn 6V V2 28/6/01 T2C/Vanveen Grab Trace August /02 Kishorn Reference 13/8/01 Reference/ Diver core Trace 7268/02 Kishorn T1A 13/8/01 T1A/ Diver core /02 Kishorn T1C 13/8/01 T1C/ Diver core /02 Kishorn T2A 13/8/01 T2A/ Diver core /02 Kishorn T2B 13/8/01 T2B/ Diver core /02 Kishorn T2C 13/8/01 T2C/ Diver core ND January /02 Kishorn Reference 24/1/02 Reference/ Diver core Trace 10094/02 Kishorn T1A 24/1/02 T1A/ Diver core /02 Kishorn T2A 24/1/02 T2A/ Diver core /02 Kishorn T2B 24/1/02 T2B/ Diver core /02 Kishorn T2C 24/1/02 T2C/ Diver core 1.59 Trace = < 1 ng g -1 ND = Not detected 14

16 TABLE 5 Emamectin Benzoate concentrations (ng g -1 dry weight) observed in sediment samples collected from Loch Sunart during May Laboratory ID Field ID Concentration (ng g -1 ), dry weight Trace = < 1 ng g -1 ND = Not detected 10067/02 Sunart S2A 14/05/ /02 Sunart Reference Trace 10069/02 Sunart S1C 14/05/02 Trace 10070/02 Sunart S2B 14/05/02 Trace 10071/02 Sunart S1B 14/05/02 ND 10073/02 Sunart S1A 14/05/02 Trace 10073/02 Sunart S2C 14/05/02 Trace 15

17 TABLE 6 Results of total organic carbon and particle size analysis (PSA) for sediments collected from Loch Kishorn and Loch Sunart. UKAS ID Loch Kishorn UKAS YEAR FIELD ID/ COLLECTION DATE %TOTAL ORGANIC CARBON PSA < 63 µm Kishorn 1VV2 Jun Kishorn 2VV1 Jun Kishorn 3VV1 Jun Kishorn 4VV1 Jun Kishorn 5VV1 Jun Kishorn 6VV1 Jun Kishorn 7VV1 Jun Kishorn T1B1 25/6/ Kishorn REF 26/6/ * Kishorn T1 A1 25/6/ Kishorn T1 C1 25/6/ Kishorn T2 A1 26/6/ Kishorn T2 B1 26/6/ Kishorn T2 C1 26/6/ Kishorn, Ref 13/8/ Kishorn T213 13/8/ Kishorn T214 13/8/ Kishorn T2C 13/8/ Kishorn Stn3 21/8/ Kishorn T1A 13/8/ Kishorn T1B 13/8/ * Kishorn T1C 13/8/ Kishorn Stn 1 Aug Kishorn Stn 2 Aug Kishorn Stn 4 Aug Kishorn Stn 5 Aug

18 Kishorn Stn 6 Aug Kishorn Stn 7 Aug Kishorn Ref 24/1/ * Kishorn T1A 24/1/ Kishorn T2A 24/1/ Kishorn T2B 24/1/ Kishorn T2C 24/1/ * Loch Sunart Sunart S2A 14/5/ * Sunart Ref 14/05/ Sunart S1C 14/05/ Sunart S2B 14/05/ Sunart S1B 14/05/ Sunart S1A 14/05/ Sunart S2C 14/05/ *Sample not analysed 17

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23 APPENDIX 1 SAMPLE IDENTIFICATION NUMBERS AND THE MEAN RESULTS FOR PARTICLE SIZE ANALYSIS (PSA) FOR SEDIMENTS COLLECTED FROM LOCH KISHORN AND LOCH SUNART

24 UKAS ID UKAS YEAR FIELD ID Loch Kishorn Kishorn 1VV2 Jun Kishorn 2VV1 Jun Kishorn 3VV1 Jun Kishorn 4VV1 Jun Kishorn 5VV1 Jun Kishorn 6VV1 Jun Kishorn 7VV1 Jun Kishorn T1B1 25/6/ Kishorn REF 26/6/ Kishorn T1 A1 25/6/ Kishorn T1 C1 25/6/ Kishorn T2 A1 26/6/ Kishorn T2 B1 26/6/ Kishorn T2 C1 26/6/ Kishorn, Ref 13/8/ Kishorn T213 13/8/ Kishorn T214 13/8/ Kishorn T2C 13/8/ Kishorn Stn3 21/8/ Kishorn T1A 13/8/ Kishorn T1B 13/8/ Kishorn T1C 13/8/ Kishorn Stn 1 Aug Kishorn Stn 2 Aug Kishorn Stn 4 Aug Kishorn Stn 5 Aug Kishorn Stn 6 Aug Kishorn Stn 7 Aug Kishorn Ref 24/1/ Kishorn T1A 24/1/ Kishorn T2A 24/1/ Kishorn T2B 24/1/ Kishorn T2C 24/1/02 Loch Sunart Sunart S2A 14/5/ Sunart Ref 14/5/ Sunart S1C 14/5/ Sunart S2B 14/5/ Sunart S1B 14/5/ Sunart S1A 14/5/ Sunart S2C 14/5/02

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