Effects of herbicide exposure on growth of the stonewort Ceratophyllum demersum (Tracheophyta – Magnoliopsida) (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 stem length and biomass) on the stonewort Ceratophyllum demersum during laboratory experiments conducted in 2019.

The aims of this project were to develop and apply standard ecotoxicology protocols to determine the effects of non-PSII herbicides on the growth of the stonewort Ceratophyllum demersum. Growth bioassays were performed over 7-day exposures using herbicides that have been detected in the Great Barrier Reef catchment area (O’Brien et al. 2016). This toxicity data will enable improved assessment of the risks posed by non-PSII herbicides to aquatic macrophytes for both regulatory purposes and for comparison with other taxa.

Methods:
The stonewort Ceratophyllum demersum was supplied by Watergarden Paradise Aquatic Nursery, Bass Hill, NSW. Cultures were maintained in 500 L outdoor plastic tanks in recirculating dechlorinated tap water, aerated and maintained at ambient outdoor temperature and lighting. Test replicates selected 48 h in advance and acclimated in dechlorinated tap water, 26 ± 2 °C, under a 12:12 hr light:dark cycle (102 ± 9 µmol photons m–2 s–1).

Herbicide stock solutions were prepared using PESTANAL (Sigma-Aldrich) analytical grade products (HPLC less than or equal to 98%): haloxyfop-p-methyl (CAS 72619-32-0), imazapic (CAS 104098-48-8) 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 500 mL glass volumetric flasks using milli-Q water. Haloxyfop-p-methyl was 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 triclopyr.

Cultures of Ceratophyllum demersum were exposed to a range of herbicide concentrations over a period of 7 days. Plants were sourced from actively growing cultures free of overt disease or deformity. Individual plants approximately 35 mm long with 5 whorls and an apical tip were added to 150 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 autoclaved, recirculating dechlorinated tap water. Five 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 (90 ± 6 µmol photons m–2 s–1). Each replicate treatment was photographed at a standard height to measure stem length at Day 0 and Day 7. Biomass of a representative numbers of fronds were weighed at Day 0 to 3 significant figures using an analytical balance after blotting for 15 seconds to remove excess moisture. Fronds from each treatment replicate were weighed at Day 7 using the same technique. Specific growth rates (SGR) were expressed as the logarithmic increase in stem length (mm) or biomass (g) 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 stem length or biomass at day j and Xi is the stem length or biomass at day i (OECD 2006).

SGRi-j =[(ln Xj - ln X i) / (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 0.0495 day-1 determined from (OECD 2006 and Riethmuller et al 2003). Physical and chemical characteristics of each treatment were measured at 0 and 7 days 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 and 7 days.

Format:
Ceratophyllum demersum herbicide toxicity data_eAtlas.xlsx

Data Dictionary:

There are one or two tabs for each herbicide in the spreadsheet. The first tab corresponds to the specific growth rate – biomass (SGR-B) data; the second tab is stem length (SGR-L) data. The last tab of the dataset shows the measured water quality (WQ) parameters (pH, electrical conductivity and temperature) of each herbicide test.

Halo – Haloxyfop
Imaz – Imazapic
Tri – Triclopyr

For each ‘herbicide’_SGR tab:
SGR = specific growth rate – the logarithmic increase from day 0 to day 7 as either biomass (B) (g/day) or stem length (L) (mm/day)
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 1-5 
T7_Biomass or Length = Biomass or Stem Length at Day 7
ln(day7) = natural logarithm of biomass (g) or stem length (mm) at Day 7
T0_Surface Area or Frond = Biomass (g) or Stem Length (mm) at Day 0
ln(day0) = natural logarithm of biomass (g) or stem length (mm) at Day 0

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 

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) Current approaches in the statistical analysis of ecotoxicity data. OECD Publishing.

OECD (2014) Test Guideline 238: Sediment-free Myriophyllum spicatum toxicity test, OECD Publishing, Paris.

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