detected in this study with long term accumulation and persistence of ivermectin in sediments under fish farms. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Ivermectin; Analytical method; Marine sediment; Salmon farm; Environmental impact
1. Introduction
Ž .
Lunestad 1992 argued that the majority of chemotherapeutants orally administered to fish in marine cage farms will leave the cages and enter the environment. Variations
in the bioavailability of therapeutic agents will alter the kinetics with which they leave the farms but will not necessarily alter the amount that does so. Unless an agent is
significantly metabolised by the fish, it can be assumed that the quasi-totality of the administered chemical will, ultimately, enter the environment. In the majority of studies
on the fate of orally administered chemotherapeutants, it has been a common assumption that a significant proportion of the agents will leave the cages associated with uneaten
Ž .
feed or with faeces Smith, 1996 . It has further been assumed that such particle associated chemicals will be deposited on the sediments under the farms. Therefore, the
majority of the studies of the fate of chemotherapeutic agents has focused on the Ž
. concentrations in these sediments that result from therapy. Smith 1996 has recently
reviewed the data that have been presented from the study of 17 administrations of oxytetracycline in marine fish farms and has questioned the validity of the assumption
that sediment deposition is the dominant fate of this agent.
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The data of Høie et al. 1990 indicated that ivermectin is excreted from fish in an unmodified form and therefore it is reasonable to assume that the quasi-totality of the
Ž .
agent administered will enter the environment of the farm. Davies et al. 1998 have used a variety of modelling approaches to attempt an estimate of the concentrations of
ivermectin that will accumulate in the sediment under fish farms. These estimates were, however, theoretical and there are at present no published data on the concentrations of
orally administered ivermectin that accumulate in the sediments under farms.
This paper reports the concentration of 22,23-dihydroavermectin B detected at a
1a
farm using orally administered ivermectin as an agent to control sea-lice infestations in Atlantic salmon. Preliminary studies of the biological impact of this treatment have been
Ž .
presented by Costelloe et al. 1998 .
2. Materials and methods
2.1. Farm stocking and treatment The site at which this study was performed was located approximately 1 km off-shore
on the west coast of Ireland. Between 7 and 12 March 1997, the site was stocked with Ž
. 350,000 Atlantic salmon post smolts av. wt. 64 g in cages. During the period 12 March
to 10 July 1997, ivermectin was orally administered to these fish twice each week. Administration was achieved by presenting the fish with commercial fish feed pellets
that were surface coated with sufficient ivermectin to provide a dose of 0.05 mgrkg. The amount of ivermectin added to the feed was calculated from weekly estimations of
fish weight. Farm records show that a total of 23 g of ivermectin were use at the farm between stocking and sampling.
2.2. Sample collection The cage block at the farm was 138 m = 35 m in area and was arranged with its
Ž longest axis lying in a north–south direction. Seven sites E3, E2, E1, C, W1, W2 and
. Ž
. W3 were arranged on an east–west axis through the centre of the cage block Fig. 1 . A
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further nine sites N4, N3, N2, N1, C, S1, S2, S3 and S4 were aligned on a north–south axis. In order to facilitate divers, the north–south axis did not pass through the centre of
Ž .
Fig. 1. Estimates of total amount of 22,23-dihydroavermectin B H B
detected at all depths in the
1a 2
1a
sediment, with respect to the location of the cages of the Atlantic salmon farm. The area covered by the cages Ž
.
2
35 m=138 m is shaded. Each square represents 25 m of sediment. Numerical values refer to mg H B
2 1a
estimated to be present in each 25-m square using data not corrected for analytical recovery. Numbers in bold Ž
. refer to squares from which samples were collected and analysed site numbers are in brackets . Numbers in
normal type are mathematically derived from those in bold. As H B was not detected in sample W3,
2 1a
estimates of the concentration present in the north–south column through this site are not shown.
the cage block but was offset 10 m to the west of the centre line. Sampling sites were 25 m apart and as the site C was common to both sampling axes a total of 15 sites were
used. Sample sites were located using a weighted, measured rope placed on the sediment surface. Sediment sample cores were collected on 10 July 1997 by divers using 25 cm
long plexiglass tubes of 4.5 cm internal diameter. A minimum of 15 cm sediment was collected in each core. Sediment cores were transported to the laboratory on ice and
were vertically sectioned on the day of collection. Homogenised material from each
Ž .
3-cm section of each core was stored at y208C until it was analysed 4–6 weeks . Three samples cores, collected from the sediment at this farm in 1992 and 1993, were
also analysed. These had been collected from a sample site approximately equivalent to the site C. These cores had been stored at y208C for 4 and 5 years prior to their
analysis. As a result of the changes in the ownership of the farm, no detailed records were available of the amounts of ivermectin being administered at the time these
samples were collected.
2.3. Current flow measurement Current flows over a 24-h period were assessed from hourly readings taken with a
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directional meter B7M 008, Mark III, Valeport Marine Scientific, Dartmouth, UK . On the manufacturers recommendation the limit of quantitation of the current flow meter
was taken to be 0.05 mrs. Current flow measurements were taken close to sample site N3 at 1 m below the surface and 1 m above the sediment.
2.4. Sediment density measurement Ž
To determine the density of the sediment, the vertical sections of sediment cores 3 .
Ž .
cm deep were homogenised and triplicate aliquots approximately 15 g were intro- duced into preweighed 50 ml volumetric flasks. The weight of each sediment aliquot
was determined by reweighing the flasks after the introduction of the sediment and then the sediment volume was determined by measuring the amount of water required to
bring the total flask contents to 50 ml. The mean of the three determinations of the
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sediment density weightrvolume was calculated for each 3-cm vertical section made from the cores taken at E1 and N1.
2.5. Chromatographic materials All solvents were of HPLC grade and other chemicals were of analytical reagent
Ž .
grade. Ivermectin was obtained from Merck Sharp and Dohme Hoddeston, UK . A Ž
. stock standard solution 1 mgrml was prepared in methanol and stored at y208C. A
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dilute standard 100 ngrml was prepared by dilution of the stock standard in methanol and was shown to be stable for at least 3 months when stored at 48C. Working standards
Ž .
were prepared with each batch of test samples by evaporating aliquots 500 ml of dilute standard solution to dryness at 708C under nitrogen. These were derivatised as described
in Section 2.9 below. The standard was equivalent to a concentration of 18.6 ng Ž
. 22,23-dihydroavermectin B
H B per g sample.
1a 2
1a
2.6. HPLC system The HPLC system consisted of a model L-6000 pump, AS 2000A autosampler,
Ž F-1050 fluorescence spectrophotometer, and D-2500 chromato-integrator Merck-Hitachi,
. Ž
. Ž
Tokyo, Japan , and a Partisil 5 ODS-3 25 = 4.6 cm column Whatman, Clifton, NJ, .
USA . The excitation and emission wavelengths were set to 365 and 470 nm, respec- Ž
. tively. The mobile phase was methanolrwater 95:5, vrv , degassed by filtration
through a 0.45-mm filter under vacuum. 2.7. Sample extraction
Ž .
Sediment samples 5 g were weighed into 50-ml plastic centrifuge tubes. Spiked samples were prepared by adding an appropriate amount of ivermectin to known
negative sediment samples taken from the core W5. The spiked samples were allowed to Ž
. stand for 10 min before extraction. Methanol 10 ml was added and the tubes were
capped and shaken vigorously on a mechanical shaker for 30 min. The tubes were Ž
. Ž
. centrifuged 600 = g, 48C for 10 min and aliquots 5 ml of the supernatants transferred
into 100 = 12 mm tubes. The extracts were evaporated to dryness at 708C under nitrogen Ž
. and the residues redissolved in acetonitrilerwater 30:70, vrv containing 0.1 trieth-
Ž .
ylamine 5 ml . 2.8. Sample clean-up
Ž .
Sample clean up was performed using Bond-Elut C 3 ml disposable extraction
8
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Ž .
columns Varian, Harbour City, CA, USA with a Vac-Elut vacuum manifold Varian fitted with stainless steel Luer stopcocks. Columns were conditioned immediately before
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Ž .
use with acetonitrile 4 ml followed by acetonitrilerwater 30:70, vrv containing Ž
. 0.1 triethylamine 5 ml and were not allowed to dry out before the application of
sample extracts. The tubes containing the extracts were washed with acetonitrilerwater Ž
. Ž
. 30:70, vrv containing 0.1 triethylamine 2 ml and this was also applied to the
columns. The columns were dried under vacuum for 5 min and ivermectin was eluted Ž
. with acetonitrile 3 ml .
2.9. DeriÕatization The eluates from the C columns were evaporated to dryness at 708C under nitrogen
8
Ž .
as were the working standards. The residues were dissolved in an aliquot 200 ml of Ž
. Ž
. 1-methylimidazole Aldrich Chemical, UK in acetonitrile 1:1, vrv . Aliquots of 300
Ž .
ml of trifluoroacetic acid anhydride in acetonitrile 1:2, vrv were added and the tubes Ž
. were capped and allowed to stand for a few minutes. Aliquots 200 ml were transferred
to autosampler vials for analysis. 2.10. HPLC analysis
Mobile phase was pumped through the system for 10 min to equilibrate the system Ž
. before beginning the analyses. Aliquots 25 ml of the derivatised standard were injected
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Ž .
until reproducible peaks were obtained usually three injections . Aliquots 25 ml of the samples were then injected, with a standard injection after every 4–5 samples. Results
