Contaminant pathways in Port Curtis: Final report - OzCoasts

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Contaminant pathways in Port Curtis: Final report8: Effect of pulse eventset al. 1998; Jarvinen et al. 1988; Kallander et al. 1997; Mancini 1983; Marr et al.1995; Pascoe & Shazili 1986). In these cases it appears that the exposureduration has a greater influence on the toxic response of the organism than thecontaminant concentration (Reinert et al. 2002), allowing adequate time foruptake during longer exposures.The aim of this work was to compare the effects of pulse exposures to dissolvedcopper with continuous exposures, using a metal-sensitive microalga. Thepennate marine diatom Phaeodactylum tricornutum was selected for study as it iswidely distributed in temperate and tropical environments and is an important foodsource for invertebrates and fish. Copper was an obvious choice as a modelcontaminant given its elevated concentration in waters and biota in Port Curtis(Apte et al. 2005).8.2 Experimental8.2.1 Chemical analysisGeneral trace metal sample treatment and analysis procedures described by theauthors in previous publications (Apte et al. 2005; Simpson et al. 2003) were usedin this study. Dissolved copper concentrations in saline solutions were determinedby inductively coupled argon plasma atomic emission spectroscopy (ICP-AES,Spectroflame EOP) calibrated using matrix-matched standard solutions. Thedetection limit for copper was 2 µg/L. Intracellular copper analyses were made bygraphite furnace atomic absorption spectroscopy (GFAAS, Perkin Elmer 4100ZL)using Zeeman effect background correction and operating conditionsrecommended by the manufacturer. Extracellular copper analyses were made bysquare-wave anodic stripping voltammetry (SW-ASV) (Metrohm 646 VoltammetricAnalyser) with a hanging mercury drop electrode. Samples were stirred andde-aerated with nitrogen for 300 s before deposition for 300 s at -0.6V vs SCE.A potential scan was initiated (scan rate 3.3 mV/s, pulse height 50mV, pulse step2 mV) and the copper oxidation peak areas recorded between -0.2 and 0.2V.A calibration curve was constructed using matrix-matched standards.8.2.2 Algal bioassay procedureThe algal bioassay measured the decrease in growth rate and cell yield of themarine unicellular alga Phaeodactylum tricornutum. P. tricornutum was chosen forthe study because it has previously been shown to be sensitive to copper (growthrate 72-h IC50 = 10 ± 4 µg/L, Franklin et al. 2001b), it is easy to count and does97
Contaminant pathways in Port Curtis: Final report8: Effect of pulse eventsnot clump or adsorb to the walls of the test containers. The bioassay protocol wasbased on the OECD Guideline 201 (2005) and the protocol of Stauber et al.(1994). P. tricornutum cultures between 3–5 days were used for inoculation toensure that the algae were in the exponential phase of growth. Cultures werewashed three times in clean sea water to ensure the complete removal of anyculture medium and cellular exudates from the algal solution (Stauber & Florence1987). Cells were inoculated into 250 mL borosilicate glass Erlenmeyer flasks(pre-silanised with Coatasil to reduce metal adsorption to flask walls) containing50 or 100 ml of clean filtered sea water. The initial cell density was typically4–6 ×10 3 cells/mL. The flask contents were supplemented with 0.5 mL of 26 mMsodium nitrate (15 mg NO − 3 /L) and 0.5 mL of 1.3 mM potassium dihydrogenphosphate (1.5 mg PO 3− 4 /L) in order to maintain exponential growth over 72 h.Copper was added to treatment flasks (Day 0) and cell densities determined dailyover the next 3 days. Flasks were shaken twice daily by hand to avoid CO 2limitation. Cell density measurements were made daily using a FACSCalibur flowcytometer (BD Biosciences). All tests included a reference toxicant copper (testedat five concentrations, 0–40 µg Cu/L) to determine an IC50 value (i.e. the inhibitoryconcentration to cause a 50% decrease in growth rate or cell yield). Bioassay testresults were processed using standard statistical procedures described elsewhere(Franklin et al. 2001a; ToxCalc 1984; Sprague & Fogels 1977).8.2.3 Pulsed exposures to dissolved copperTo generate contaminant pulses, the algae needed to be repeatedly cycledbetween seawater solutions containing different amounts of copper. A gentlecentrifugation method (3500 rpm for 4 min, ~1500 g) was developed for isolatingalgal cells post-exposure which did not damage algal cells or affect growth. Threetypes of copper exposure scenarios were investigated (Figure 8.1):(i)(ii)(iii)exposures of equivalent copper ‘dose’, but varying pulse duration andmagnitude (four experiments with different copper dose, each comprisingcontinuous, 1-, 2-, 4-, and 8-h pulse exposures)equivalent copper ‘dose’ and concentration, but dose applied at varyingpulse frequency (two experiments with varying pulse frequency)high copper concentration pulses of decreasing pulse duration. Thecopper concentrations for each exposure were calculated so that thealgae would receive an equal dose of copper per day and this wasgenerally equivalent to the IC50 copper concentration for the continuousexposure (7–10 µg/L).98
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