Development of flow cytometry‐based algal bioassays for assessing toxicity of copper in natural waters

Abstract

Copper toxicity to the freshwater algae Selenastrum capricornutum and Chlorella sp. and the marine algae Phaeodactylum tricornutum and Dunaliella tertiolecta was investigated using different parameters measured by flow cytometry: cell division rate inhibition, chlorophyll a fluorescence, cell size (i.e., light-scattering), and enzyme activity. These parameters were assessed regarding their usefulness as alternative endpoints for acute (1-24 h) and chronic (48-72 h) toxicity tests. At copper concentrations of 10 micrograms/L or less, significant inhibition (50%) of the cell division rate was observed after 48- and 72-h exposures for Chlorella sp., S. capricornutum, and P. tricornutum. Bioassays based on increases in algal cell size were also sensitive for Chlorella sp. and P. tricornutum. Copper caused both chlorophyll a fluorescence stimulation (48-h EC50 of 10 +/- 1 micrograms Cu/L for P. tricornutum) and inhibition (48-h EC50 of 14 +/- 6 micrograms Cu/L for S. capricornutum). For acute toxicity over short exposure periods, esterase activity in S. capricornutum using fluorescein diacetate offered a rapid alternative (3-h EC50 of 90 +/- 40 micrograms Cu/L) to growth inhibition tests for monitoring copper toxicity in mine-impacted waters. For all the effect parameters measured, D. tertiolecta was tolerant to copper at concentrations up to its solubility limit in seawater. These results demonstrate that flow cytometry is a useful technique for toxicity testing with microalgae and provide additional information regarding the general mode of action of copper (II) to algal species.

Time-averaged copper concentrations from continuous exposures predicts pulsed exposure toxicity to the marine diatom, Phaeodactylum tricornutum: Importance of uptake and elimination

“…Although P. tricornutum has been shown to produce phytochelatins in response to dissolved copper exposure (Smith et al, 2014), the sharp increase in biomass inhibition as intracellular copper increased above approximately 10 ×10 -16 g/cell (Figure 4), suggests that P. tricornutum was not able to store significant quantities of intracellular copper without experiencing toxic effects. P. tricornutum is also a relatively sensitive species to copper exposure (Franklin et al, 2001) meaning detoxification mechanisms may be overwhelmed at relatively low dissolved copper concentrations. Levy et al (2008) also measured negligible complexation of dissolved copper by algal exudates, ruling out P. tricornutum using this form of detoxification.…”

“…It was chosen because it is sensitive to copper (72-h growth rate IC50 = 10 ± 4 µg/L), it is easy to count, and does not clump or adsorb to the walls of the test containers (Angel et al, 2013;Levy et al, 2008). All cultures were maintained in f 2 growth medium (half-strength f medium) at 21±2ºC on a 12:12 h light: dark cycle (Philips TL 40 W fluorescence daylight, 72 µmol photons/m/s) (Franklin et al, 2001). Cultures were frequently checked microscopically for the presence of bacteria after incubation of a streaked sub-sample on an agar plate (2% Bacto agar, 0.1% pepsin, and 0.1% yeast; Oxoid) in the dark.…”

“…Sub-cultures of P. tricornutum cells in the exponential phase of growth (3-to 6-d old) were washed three times in 0.45 µm filtered clean seawater (Port Hacking, Sydney, Australia) by centrifuging (Spintron GT-175BR, 7 mins × 700 g) before inoculation (Franklin et al, 2001). Cells were inoculated into 250 mL borosilicate glass Erlenmeyer flasks (silanised with Coatasil to reduce metal adsorption) containing 50 or 100 mL of clean filtered seawater supplemented with 15 mg NO 3 − /L and 1.5 mg PO 4 3− /L.…”

“…Algal cell densities were measured using a flow cytometer (FACSCalibur, Becton Dickinson, Biosciences) calibrated using TruCount fluorescent beads (BD Trucount TM tubes, BD Biosciences) and gates in chlorophyll a fluorescence (Franklin et al, 2001). The inhibition of biomass yield for treatments was expressed as a percentage of the control biomass yield and was calculated using equation 1.…”

“…While this only represented approximately 1% of the copper pulse concentration, it was significant because of the high sensitivity of the algal species, i.e. the inter-pulse copper concentrations could be expected to exert significant biomass inhibition (Franklin et al, 2001). The inclusion of this dissolved copper in the TAC calculation more accurately accounted for the toxicity.…”