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Evaluation of the safety of new anti-fouling agents for use in Australian temperate waters

Final Report for the Department of the Environment and Heritage
S Duda, JH Myers and S Hoffman
Victorian Department of Natural Resources and Environment, 2003


2 Methods

2.1 Collection of Test Species

2.1.1 Algae

The brown alga Hormosira banksii (Figure 1), was collected from the intertidal sandstone rock platforms near 13th Beach, Barwon Heads Victoria, Australia (Figure 2) The collection was undertaken one day prior to conduction of the tests. At low tide, H. banksii individuals, or portions thereof, were collected haphazardly by hand from the rock platform and placed into an inert plastic bag for transport to the laboratory. At the time of collection, temperature of a tidal pool on the intertidal reef was recorded and a 250mL sample of seawater was taken for physico-chemical measurements (pH, temperature and salinity). In the laboratory, H. banksii were immediately placed flat onto paper towel and allowed to dry for 2h at 18-20oC. Algae were then placed into brown paper bags and stored overnight at 4oC, until use the following day (maximum of 24h storage).

Figure 1

Figure 1: Hormosira banksii on rock platforms at Barwon Heads, Victoria

2.1.2 Amphipods

C. insidiosum were obtained, by sieving, from the intertidal zone on the western shores of Port Phillip Bay. The Bay (DMSO)is a large marine embayment about 1,930 km2 in area, with a coastline length of 264 km. Located in Victoria, south-eastern Australia (Figure 2). Sediment was collected using a shovel, and the top 10cm layer was sieved through 1mm mesh into clean plastic buckets containing fresh seawater. The amphipods were then transported back to MAFRI, where they were sorted and placed into holding tanks containing clean sediment until time of exposure. Experimentation followed a two-week acclimation period.

Figure 2

Figure 2: Port Phillip Bay: Sampling site for C.insidiosum, H, banksii, mussels and fish

2.1.3 Mussels and Fish

The Sand flathead, Platycephalus bassensis, were collected from DMSO on the south side of the south channel marker by hook and line. Black bream, Ancanthopagrus butcherii were obtained from brood stock (Swan bay) at MAFRI's aquaculture compound. Mussels, Mytilus edulis were obtained from Mr Mussel, mussel farm located in Portarlington, DMSO (Figure 2)

2.2 Dilution water

Seawater was pumped from Port Phillip Bay through 100mm diameter pipes into 1000 000L concrete settlement tanks. This then passed through a high pressure sand filter (Permutit), which retained particles of 30mm size, to 1890L insulated holding tanks. From these holding tanks the water was pumped throughout the MAFRI facilities.

In preparation for each bioassay approximately 25L of natural seawater from Port Phillip Bay was filtered to 0.2mm, using a CUNOÒ filter system, to remove all debris, small organisms and suspended particles. Before water was used in bioassays it was aerated overnight. These processes ensured the seawater supply was uniform for testing. Unless otherwise stated, this filtered and aerated seawater was used throughout the study and is referred to as seawater.

2.3 Cleaning

Beakers, glassware and equipment used in all toxicity bioassays were cleaned prior to use to remove any possible contaminants. Equipment used for bioassay preparation (measuring cylinders, collection bowls, beakers) were rinsed 2 times with hot water, soaked in a 2% detergent bath (Decon 90Ò Selby Biolab Scientific Ltd.) for at least 24h, rinsed 3 times with hot water, then rinsed 3 times with purified reverse osmosis water (Milli - RO Water SystemÒ). Clean glassware was then placed into a drying cabinet for at least 24h then covered with an inert plastic film and stored until required.

Glassware used for conducting bioassays were washed using a Gallay dishwasher (Gallay ScientificÒ, LAB 901) at 80oC, with a detergent wash cycle, rinse stage and 2 distilled rinse stages. Any disposable items were hot water rinsed and disposed of in accordance with MAFRI protocols.

2.4 Preparation of stock solutions

Unless otherwise stated, all toxicants used were of Analytical Reagent (AR) Grade. On the first day of testing stock concentrations of each toxicant to be tested were prepared, unless otherwise stated. All stock concentrations were stored at 18oC in a fume cupboard in sealed volumetric flasks. All stock solutions were prepared with purified reverse osmosis water, unless otherwise stated.

Zineb, zinc pyrithion (ZPT), seanine 211 and diuron were obtained from International Paints (Britain) dealing as Akzo-nobel Paints Ltd in Australia. Tributyltin oxide (TBT) was obtained from Tokyo Kasei (TCI). Dimethylsulphoxide (DMSO) was of AR spectrometry grade and was obtained from Aldrich Company. Zineb, ZPT and diuron were supplied in the form of a powder and seanine 211 and TBTO were in a liquid form. As each biocide degraded at a different rate, new working stock solutions were prepared on the day of each new test setup.

Working stock solutions of zineb and ZPT were prepared by dissolving 0.025g of each biocide in 7 and 5mLs of DMSO respectively, then making them up to 500mL with seawater. Diuron was prepared by dissolving 0.035g in 5mL of DMSO then making up to 500mL with seawater. This produced a 50, 50 and 126 mgL-1 stock solutions for each of zineb, ZPT and Diuron respectively. Seanine 211 was prepared by dissolving 39 mL of the biocide in 3mL of DMSO then making up to 500mL with seawater to produce a 100 mgL-1 standard solution. Dimethylsulphoxide was used as a solvent to dissolve each of the biocides due to their low solubilities in water. Tributyltin-oxide was prepared by dissolving 85mL of TBTO in 5mL of methanol then making up to 100mL with seawater. This produced a 1gL-1 stock solution. A working standard was then produced by diluting 100mL of 1gL-1 stock to 1L with seawater to produce a 0.1mgL-1 standard solution. Methanol was found to be the appropriate solvent for dissolving and keeping the TBTO in solution. Stock solutions of DMSO and methanol were prepared and used as solvent controls in each test. These were prepared by dissolving the same amount of each solvent used in preparation of test toxicant stocks to the same volume.

2.5 Test Initiation and Preparation

2.5.1 Hormosira banksii

The H. banksii germination inhibition bioassay is adapted from the procedures of Kevekordes and Clayton (1996). Female gametes were collected from H. banksii plants and approximately 2000 eggs mL-1 were added to each 80mL test solution twenty minutes prior to the addition of sperm. An Internal Visual Optical System (IVOS) was used to calculate the density of sperm in the stock suspension and the volume of sperm required to achieve an egg to sperm ratio of 1:200 was then determined. Sperm was introduced to test solutions, and incubated under optimal conditions.

Subsequent development of the fertilised eggs in the toxicant solutions was assessed for germination and rhizoid elongation at 48h and 72h. For rhizoid elongation endpoints, rhizoid lengths were measured microscopically and results were compared with control rhizoid lengths.

Nominal concentrations for 48h and 72h exposures for ZPT, seanine 211, diuron and TBTO were based on sensitivities of related species found in the literature. Range find tests were therefore conducted using concentrations of 0, 1, 10, 15, 20, 25, 30mgL-1 for diuron, 0, 0.001, 0.005, 0.04, 2 and 4mgL-1 for seanine 211, 0, 0.0005, 0.008, 0.15, 1 and 2mgL-1 for ZPT and 0, 10-7, 10-6, 10-5, 10-4, 10-2 and 1mgL-1 for TBTO. Literature relating to the toxicity of zineb was unattainable and therefore a scale of 1-10mgL-1 was used for the concentrations.

Definitive test concentrations were then determined and tests repeated following the range finders. Diuron nominal concentrations were 0, 0.5, 1.0, 2.5, 5.0, 7.5 and 10.0mgL-1, for ZPT 0, 0.0005, 0.008, 0.05, 0.15, 0.5 and 1.0mgL-1. Seanine 211 nominal concentrations were 0, 0.001, 0.005, 0.04, 0.25, 0.5 and 1.0mgL-1 and TBTO were 0, 10-8, 10-7, 10-6, 10-5, 10-4, 10-3 mgL-1. Zineb showed the characteristic concentration-response relationship desired, therefore concentrations used in range find tests were used in definitive tests.

Solvent concentrations used in solvent controls followed that of the concentration of solvent in the highest test concentration. The maximum percent concentration of each solvent in each toxicant tested was 0.079% for diuron, 0.02% for ZPT and 0.006%, 0.28% and 0.5% for seanine 211, zineb and TBTO respectively.

2.5.2 Corophium insidiosum

Acute LC50 experiments using C. insidiosum were conducted in a temperature control room at 12.5 ± 1ºC. This approximated DMSO water temperatures at the time of the experiments. The pH was 8.0 ± 0.6 and aeration, at a rate of 3-5 bubbles per second, maintained dissolved oxygen above 90% saturation. The airlines, constructed of non-toxic tubing with pasteur pipettes attached, were secured within the test chambers. These parameters were measured before termination of the experiments and test chambers were monitored daily to ensure sufficient airflow. Twenty amphipods were placed in each test chamber (0.8L Beaker). Each concentration filled the test chamber to 0.5L and had 4 replicates. Each experiment had up to eight concentrations with one control, one solvent control and one reference toxicant concentration and five test concentrations (ASTM, 1999).

The test began when C. insidiosum were added to the test chambers. After the 96hr exposure C. insidiosum were individually pipetted onto a white tray. C. insidiosum that did not move were gently stimulated by rinsing with a plastic pipette. If no further response was observed, animals were considered to be dead.

Reference toxicant tests were conducted with each 96hr C. insidiosum test. This ensured that C. insidiosum was responding normally under test conditions. The acute response of C. insidiosum to the biocides was compared to that of a previously established LC50 for CuSO4 (0.280 mgL-1) (Stokie, 2000 unpublished)

Organisms were not fed during exposures according to ASTM protocol. Food may influence the bioavailability of toxins and is also likely to interfere the route of contaminant exposure ie. if food is being consumed less energy is being exerted to excrete toxins (ASTM, 1999).

Survival of C. insidiosum in the control replicate was required to be greater than or equal to 80% for a test to be acceptable. Control survival reflects the health of test organisms and acceptability of test methods. If mortality is greater than 20% other factors may be affecting survival. One of these factors may include bacterial diseases, (Environment Canada, 1995)

2.5.3 Mussels and Fish

The flow-through exposures were conducted using 12 large glass aquaria (80L). Each exposure tank had 12 fish (A. butcherii) or for the mussels (Mytilus edulis) 20 specimens. The P. bassensis exposures were undertaken in 8 x 800 L grey plastic tanks, each tank contained 20 fish. For each biocide 2 replicates were run. Dosing was undertaken from a reservoir by peristaltic pump into a mixing chamber at the rate of 0.5 mL min-1 (described in Goulan and Yong, 1995). The exception was for P. bassensis, in which dosing was administered by injection every 2 weeks. Volumes of 200µl were used at the concentration of 4.6 x 10-3 mgL-1 for all biocides and solvent controls. A. butcherii were fed an aquaculture pellet mixture for juvenile fish. P. bassensis were fed frozen puree of squid, Australian pilchards, pellet food and gelatine.

Concentrations used were: Diuron- 2.3 x10-1 mgL-1, Seanine 211- 1.4 x10-2 mgL-1, ZPT- 4.6 x10-3 mgL-1, Zineb- 1.3x10-2 mgL-1, TBT- 2.0x10-8 mgL-1.

On completion of the 6-week exposure A. butcherii were anethsetised using 20 mL of 10 mgL-1 benzocaine dissolved into 10 L of filtered seawater. Fish were then dissected for their gills, muscle tissue, liver, spleen and a sample of their blood was taken. These were then labelled with wax paper wrapped in tin foil and frozen in liquid nitrogen or on dry ice, then transferred to a -80°C freezer to await analysis.

M. edulis were removed from aquaria and frozen in labelled snap-lock bags and placed in a -20°C freezer to await analysis.

2.6 Statistical Analysis

Toxicity of each contaminant was expressed as an EC50 value, the concentration that affected 50% of organisms. EC50 values and their 95% confidence intervals for the test endpoints were calculated using the maximum likelihood probit or Trimmed spearman-karber method and Linear Interpolation with Bootstrapping (ICp) or Non-Linear Interpolation respectively. The ICp method was developed by the EPA to generate a point estimate, standard error and confidence intervals for quantitative data (ie. growth). If the data were not able to be analysed using ICp then non-linear interpolation was used.

Data was analysed for normality and homogeneity of variances using Shapiro-Wilk's test for normality and Bartlett's test of homogeneity of variances. After testing for normality and homogeneity of variances in the data set, hypothesis testing was performed to determine which treatments significantly differed from the controls. If both assumptions were met, Dunnett's Multiple Comparison Test was used. If data failed to meet the ANOVA assumptions, Steel's Many One Rank Test was performed.

Statistical analysis for the determination of NOEC, LOEC and EC50 values and 95% confidence intervals (CI) were conducted using the statistical package Toxcalc Version 5.0, Tidepool Software