Effects of herbicide exposure on growth and photosynthetic efficiency of the green algae Raphidocelis subcapitata (Chlorophyta) (NESP TWQ 3.1.5, AIMS and JCU)

This dataset shows the effects of herbicides (detected in the Great Barrier Reef catchments) on the growth rates (from cell density data) and photosynthesis (effective quantum yield) on the green algae Raphidocelis subcapitata during laboratory experiments conducted during 2019.

The aims of this project were to develop and apply standard ecotoxicology protocols to determine the effects of Photosystem II (PSII) and alternative herbicides on the growth and photosynthetic efficiency of the green algae Raphidocelis subcapitata. Growth bioassays were performed over 3-day exposures using herbicides that have been detected in the Great Barrier Reef catchment area (O’Brien et al. 2016). Effects of herbicides on the photophysiology of Raphidocelis subcapitata, measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm') were investigated using pulse amplitude modulation (PAM) fluorometry after 72 h herbicide exposure. These toxicity data will enable improved assessment of the risks posed by PSII and alternative herbicides to microalgae for both regulatory purposes and for comparison with other taxa.

Methods:
The chlorophyte Raphidocelis subcapitata (strain CS-327) was purchased from the Australian National Algae Supply Service, Hobart (CSIRO). Cultures of R. subcapitata were established in MLA medium (Bolch and Blackburn 1996). Cultures were maintained in sterile 250 mL Erlenmeyer flasks as batch cultures in exponential growth phase with weekly transfers of 1 - 3 mL of a 7 day-old R. subcapitata suspension to 100 mL MLA medium under sterile conditions. Clean culture solutions were maintained at 26 ± 2°C, and under a 12:12 h light:dark cycle (91 ± 12 µmol photons m–2 s–1).

Herbicide stock solutions were prepared using PESTANAL (Sigma-Aldrich) analytical grade products (HPLC greater than or equal to 98%): diuron (CAS 330-54-1), haloxyfop-p-methyl (CAS 72619-32-0) and imazapic (CAS 104098-48-8). The selection of herbicides was based on application rates and detection in coastal waters of the GBR (Grant et al. 2017, O’Brien et al. 2016). Stock solutions were prepared in sterile 100 - 500 mL glass volumetric flasks using milli-Q water. Diuron and haloxyfop-p-methyl were dissolved using analytical grade acetone (final concentration &lt; 0.01% (v/v) in exposures). Imazapic was dissolved in methanol (final concentration less than or equal to 0.01% (v/v) in exposure). 

Cultures of R. subcapitata were exposed to a range of herbicide concentrations over a period of 72 h. Inoculum was taken from cultures in exponential growth phase (4 – 7 day-old cultures). A R. subcapitata working suspension was prepared in a 100 mL volumetric flask. A 1:10 and 1:100 dilutions were prepared and counted using a haemocytometer under a compound microscope to determine appropriate dilution volumes. The pre-determined inoculum was added to 50 mL of each test and control treatment replicates to the required dilution (3 – 3.1 x 104 cells/ mL). In each toxicity test, a control (no herbicide) and solvent control (if used) treatments were added to support the validity of the test protocols and to monitor continued performance of the assays. All treatment concentrations were prepared in 0.5x strength MLA medium. Replicates were incubated at 26.6 ± 0.5 °C under a 12:12 h light:dark cycle (190 ± 14 µmol photons m–2 s–1). Sub-samples were taken from each replicate to measure cell densities of algal populations at 72 h using a haemocytometer and photographed under phase contrast conditions. Cell counts were done either manually or using ImageJ from microscope photographs (Rueden et al 2017). Specific growth rates (SGR) were expressed as the logarithmic increase in cell density from day i (ti) to day j (tj) as per equation (1), where SGRi-j is the specific growth rate from time i to j; Xj is the cell density at day j and Xi is the cell density at day i (OECD 2011).

SGR i-j = [(ln Xj - ln Xi )/(tj - ti )] (day-1)
                                                          (1)

SGR relative to the control treatment was used to derive chronic effect values for growth inhibition. A test was considered valid if the SGR of control replicates was greater than or equal to 0.92 day-1 (OECD 2011). Physical and chemical characteristics of each treatment were measured at 0 h and 72 h including pH, electrical conductivity and temperature. Chamber temperature was also logged in 15-min intervals over the total test duration. Analytical samples were taken at 0 h and 72 h.

Effects of herbicides on the photophysiology of R. subcapitata, measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm'), were investigated at 72 h using a mini-PAM fluorometer (mini-PAM, Walz, Germany). Light adapted minimum fluorescence (F) and maximum fluorescence (Fm') were determined and effective quantum yield was calculated for each treatment as per equation (2)(Schreiber et al. 2002). 

Delta F/Fm’ = (Fm’-F)/Fm’                                                         (2)

Mini- PAM settings were set to ETR-F = 0.84, F-Offset = 92, measuring light frequency = 3, measuring intensity = 4, gain = 3; damp = 3. Saturation pulse settings: intensity = 6, width = 0.6.
Mean percent inhibition in SGR and Delta F/Fm' of each treatment relative to the control treatment was calculated as per equation (3)(OECD 2011), where Xcontrol is the average SGR or Delta F/Fm' of control and Xtreatment is the average SGR or Delta F/Fm' of single treatments.

% Inhibition = [(X control - X treatment )/X control] x 100                                                         (3)

Format:
Raphidocelis subcapitata herbicide toxicity data_eAtlas.xlsx

Data Dictionary:

There are two tabs for diuron in the spreadsheet. The first tab corresponds to the specific growth rate (SGR) data; the second tab is the pulse amplitude modulation (PAM) fluorometry data. For haloxyfop and imazapic there is one tab, corresponding to specific growth rate (SGR) data. The last tab of the dataset shows the measured water quality (WQ) parameters (pH, electrical conductivity and temperature) of each herbicide test.

Diu – Diuron
Halo – Haloxyfop
Imaz – Imazapic

For each ‘herbicide’_SGR tab:
SGR = specific growth rate – the logarithmic increase from day 0 to day 3
Nominal (µg/L) = nominal herbicide concentrations used in the bioassays; SC denotes solvent control which is no herbicide and contains less than 0.01% v/v solvent carrier as per the treatments
Measured (µg/L) = measured concentrations analysed by The University of Queensland
Rep = Replicate: for SGR, notation is 1-3; for PAM data, notation is 1-3
T3_CellsPer_ml = cell density at day 3 
ln(day3) = natural logarithm of cell density at day 3
Average T0_CellsPer_ml = average cell density at day 0
ln(Day0) = natural logarithm of cell density at day 0

For Diuron_PAM tab:
PAM = pulse amplitude modulation fluorometry to calculate effective quantum yield (light adapted)
Nominal (µg/L) = nominal herbicide concentrations used in the bioassays; SC denotes solvent control which is no herbicide and contains less than 0.01% v/v solvent carrier as per the treatments 
Measured (µg/L) = measured concentrations analysed by The University of Queensland
Rep = Replicate: for SGR, notation is 1-3; for PAM data, notation is 1-3
Delta F/Fm' = effective quantum (light adapted) yield measured by a Pulse Amplitude Modulation (PAM) fluorometer

References:
Bolch, C. J. S. and Blackburn S. I. (1996). Isolation and purification of Australian isolates of the toxic cyanobacterium Microcystis aeruginosa Kütz. Journal of Applied Phycology 8, 5-13

Grant, S., Gallen, C., Thompson, K., Paxman, C., Tracey, D. and Mueller, J. (2017) Marine Monitoring Program: Annual Report for inshore pesticide monitoring 2015-2016. Report for the Great Barrier Reef Marine Park Authority, Great Barrier Reef Marine Park Authority, Townsville, Australia. 128 pp, http://dspace-prod.gbrmpa.gov.au/jspui/handle/11017/13325 

O’Brien, D., Lewis, S., Davis, A., Gallen, C., Smith, R., Turner, R., Warne, M., Turner, S., Caswell, S. and Mueller, J.F. (2016) Spatial and temporal variability in pesticide exposure downstream of a heavily irrigated cropping area: application of different monitoring techniques. Journal of Agricultural and Food Chemistry 64(20), 3975-3989.

OECD (2011) OECD guidelines for the testing of chemicals: freshwater alga and cyanobacteria, growth inhibition test, Test No. 201. https://search.oecd.org/env/test-no-201-alga-growth-inhibition-test-9789264069923-en.htm (accessed 28 August 2019).

Rueden, C.T., Schindelin, J., Hiner, M.C. et al. (2017) ImageJ2: ImageJ for the next  generation of scientific image data. BMC Bioinformatics 18:529, PMID 29187165, doi:10.1186/s12859-017-1934-z

Data Location:

This dataset is filed in the eAtlas enduring data repository at: data\nesp3\3.1.5_Pesticide-guidelines-GBR

Organization: Australian Ocean Data Network