Effects of herbicide exposure on growth and photosynthetic efficiency of the lesser duckweed Lemna aequinoctialis (Tracheophyta – Liliopsida) (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 frond number and surface area) and photosynthesis (effective quantum yield) on the duckweed Lemna aequinoctialis during laboratory experiments conducted from 2017-2019.

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 lesser duckweed Lemna aequinoctialis. Growth bioassays were performed over 4-day exposures using herbicides that have been detected in the Great Barrier Reef catchment area (O’Brien et al. 2016). Chronic effects of herbicides on the photophysiology of L. aequinoctialis, measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm') were investigated using mini-PAM fluorometry after 4-day herbicide exposure. These toxicity data will enable improved assessment of the risks posed by PSII and alternative herbicides to aquatic macrophytes for both regulatory purposes and for comparison with other taxa.

Methods:
The lesser duckweed Lemna aequinoctialis was supplied by the Supervising Scientist, Department Agriculture, Water &amp; Environment, Darwin, N.T.). Cultures were established in 0.5x strength CAAC medium (Riethmuller et al 2003; Pease et al 2016). Cultures were maintained in sterile 250 mL Erlenmeyer flasks as batch cultures with weekly transfers of 5-10 triplicate fronds to 100 mL 0.5x strength CAAC medium under sterile conditions. Clean culture solutions were maintained at 26 ± 2°C under a 12:12 h light:dark cycle (41 ± 5 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%): bromacil (CAS 314-40-9); diuron (CAS 330-54-1), fluroxypyr (CAS 69377-81-7), haloxyfop-p-methyl (CAS 72619-32-0), hexazinone (CAS 51235-04-2), imazapic (CAS 104098-48-8), isoxaflutole (CAS 141112-29-0), prometryn (CAS 7287-19-6), propazine (CAS 139-40-2) and triclopyr (CAS 5535-06-3). 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 1 L glass volumetric flasks using milli-Q water. Diuron, haloxyfop-p-methyl, hexazinone, isoxaflutole and prometryn were dissolved using analytical grade acetone (&lt; 0.01% (v/v) in exposures). Imazapic was dissolved in methanol (less than or equal to 0.01% (v/v) in exposure). No solvent carrier was used for the preparation of the remaining herbicide stock solutions.

Cultures of L. aequinoctialis were exposed to a range of herbicide concentrations over a period of 96 h. Inoculum was taken from actively growing cultures free of overt disease or deformity. Four triplicate fronds were added to 100 mL of each herbicide solution concentration and control treatment. 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. Experiments were conducted in either 0.25 CAAC with no added sucrose (Riethmuller et al 2003) or synthetic soft water (SSW) (Pease et al 2016). Three replicates of each treatment solution and control  were prepared and incubated at 26.6 ± 0.5 °C under a 12:12 h light:dark cycle (110 ± 13 µmol photons m–2 s–1). Each replicate treatment was photographed at a standard height (Riethmuller et al 2003; Pease et al 2016) to estimate surface area and frond number at Day 0 and Day 4. Specific growth rates (SGR) were expressed as the logarithmic increase in surface area or frond number 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 surface area or frond number at day j and Xi is the surface area or frond number at day i (OECD 2006).

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

SGR relative to the control / solvent control treatment was used to derive chronic effect values for growth inhibition. A test was considered valid, if the SGR  for frond number or surface area of control replicates was greater than or equal to 0.325 (frond number) or 0.305 (surface area) day-1 determined from a linear interpolation of  respective SGR from (OECD 2006 and Riethmuller et al 2003). Physical and chemical characteristics of each treatment were measured at 0 h and 96 h including pH, electrical conductivity and temperature. Temperature was also logged in 15-min intervals over the total test duration. Analytical samples were taken at 0 h and 96 h.

Chronic effects of herbicides on the photophysiology of L. aequinoctialis, measured by chlorophyll fluorescence as the effective quantum yield (Delta F/Fm'), were investigated at 96 h using PAM fluorometry (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 = 46, measuring light frequency = 3, measuring intensity = 4, gain = 2; 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 2006), 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:
Lemna aequinoctialis herbicide toxicity data_eAtlas.xlsx

Data Dictionary:

There are three tabs for each herbicide in the spreadsheet. The first tab corresponds to the frond number specific growth rate (SGR-FC) data; the second tab is the surface area specific growth rate (SGR-SA); the third is pulse amplitude modulation (PAM) fluorometry data. The last tab of the dataset shows the measured water quality (WQ) parameters (pH, electrical conductivity and temperature) of each herbicide test.

Brom - Bromacil
Diu – Diuron
Flur - Fluroxypyr
Halo – Haloxyfop
Hex - Hexazinone
Imaz – Imazapic
Isox - Isoxaflutole
Prom - Prometryn
Prop - Propazine
Tri - Triclopyr

For each ‘herbicide’_SGR tab:
SGR = specific growth rate – the logarithmic increase from day 0 to day 4 (as either frond number (FC) or surface area (mm2) (SA)
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
T4_Surface Area or Frond = Surface area (mm2)  or frond number at Day 4
ln(day4) = natural logarithm of surface area (mm2)  or frond number at Day 4
T0_Surface Area or Frond = Surface area (mm2)  or frond number at Day 0
ln(day0) = natural logarithm of surface area (mm2)  or frond number at Day 0

For each ‘herbicide’_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
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:
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 

Mercurio, P., Eaglesham, G., Parks, S., Kenway, M., Beltran, V., Flores, F., Mueller, J.F. and Negri, A.P. (2018) Contribution of transformation products towards the total herbicide toxicity to tropical marine organisms. Scientific Reports 8(1), 4808.

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 (2006) Test No. 221: Lemna sp. Growth Inhibition Test, OECD Publishing, Paris.

Pease, C., Trenfield, M., Cheng, K., Harford, A., Hogan, A., Costello, C., Mooney, T. and van Dam, R. (2016) Refinement of the reference toxicity test protocol for the tropical duckweed Lemna aequinoctialis. Internal Report 644, June, Supervising Scientist, Darwin.

Riethmuller, N., Camilleri, C., Franklin, N., Hogan, A., King, A., Koch, A., Markich, S.J., Turley, C. and van Dam, R. (2003) Ecotoxicological testing protocols for Australian tropical freshwater ecosystems. Supervising Scientist Report 173, Supervising Scientist, Darwin NT.

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