Combined effects of climate change and the herbicide diuron on the coral Acropora millepora (NESP TWQ 2.1.6 and NESP TWQ 5.2, AIMS)

This dataset shows the combined effects of climate change (ocean warming (OW) and ocean acidification (OA)) and the herbicide diuron (frequently detected in the Great Barrier Reef catchments) on the chlorophyll a fluorescence (effective quantum yield, Delta F/Fm'), net photosynthesis (photosynthetic oxygen production and respiration rates) and calcification rates (light and dark calcification) on the coral Acropora millepora during laboratory experiments conducted in 2018.

The aim of this project was to investigate the combined effects of climate change (OW and OA) and the herbicide diuron on the chlorophyll a fluorescence (effective quantum yield, Delta F/Fm'), net photosynthesis (oxygen production and respiration rates) and calcification rates (light and dark calcification) on the coral Acropora millepora. These toxicity data will enable improved assessment of the risks posed by PSII herbicides to coral in a changing climate. 

Methods:

Four adult colonies (30-40 cm diameter) of Acropora millepora were collected from Backnumbers Reef (18° 29.264 S, 147° 09.174 E), GBR under Great Barrier Reef Marine Park Authority (GBRMPA) permit G12/35236.1. Colonies were transported to the National Sea Simulator (SeaSim) at the Australian Institute of Marine Science (AIMS) in Townsville and maintained in 1700 L flow-through tanks. Terminal portions of A. millepora branches (~2 cm height) were affixed on marked glass tiles and maintained in flow-through filtered seawater at 27 ± 1 °C under 150 µmol m-2 s-1 for at least one week prior to experimentation. Coral fragments were fed daily with Artemia nauplii (0.5 nauplii mL-1) during the healing period; however, corals were not fed during the duration of the experiment due to possible effect on water quality within the experimental chambers.

Experimental setup
A. millepora were exposed to three climate change scenarios (OW + OA): 28.1 ± 0.3°C and 397 ± 64 ppm CO2, 29.1 ± 0.3°C and 680 ± 90 ppm CO2, 30.2 ± 0.3°C and 858 ± 149 ppm CO2, and six concentrations of diuron (measured: 0, 0.29, 0.96, 2.9, 9.6 and 29 µg L-1), including a solvent control, in an orthogonal design. Corals were placed in custom 3 L acrylic chambers (15 cm diameter x 19 cm height, working volume 2.5 L) in temperature-controlled water baths with the appropriate concentration of pCO2 gently bubbling directly in each chamber. Due to a limited number of experimental chambers, there were no seawater controls (only solvent controls) and there was a 7-day staggered start for the 29 µg L-1 corals to maximise the number of diuron treatments. pCO2 dosing was achieved via mass flow controllers (Aalborg GFC17) delivering a precise flow of CO2 to a series of membrane contactors (3M Liqui-Cel Extra-Flow 2.5X8). Analytical grade diuron (&gt; 98% purity) was purchased from Sigma-Aldrich (NSW, Australia) and stock solutions (10 mg L-1) were prepared in filtered seawater (0.4 µm) using ethanol as a solvent carrier (&lt; 0.03% v/v). Header tanks (60 L) were filled daily with filtered seawater (1 µm) and spiked with the appropriate volume of diuron stock to achieve target diuron concentrations. Diuron was delivered from the header tanks to the experimental chambers using peristaltic pumps (Masterflex L/S and Ismatec IPC 12) for a turnover rate of 1-1.5 times per day. Four coral fragments were placed in each experimental chamber and three independent replicate chambers were used for each Diuron concentration and each climate change scenario (a total of 54 chambers). Chambers were randomised within their respective water baths to minimise potential systematic effects of chamber position.

Corals were illuminated over 13 h cycles (Aqua Illumination LED Hydra) with ramping up of light intensity for the first three hours to approximately 200 µmol m-2 s-1 then down to darkness over the last three hours (equivalent to 7 mol m-2 d-1 daily light integral, DLI). Irradiance was measured with a Licor LI-250A meter with LI-190R quantum sensor. Parameters (pH, salinity and dissolved oxygen) were measured at least four times during the duration of the 15-d experiment (Table 1). Water temperature and pCO2 levels were controlled by a programmable logic controller (PLC) and measured every 10 minutes (Table 1). Salinity was measured via a handheld meter (Horiba LAQUAact PC110), pH was measured with a multimeter (HQ40d equipped with Intellical PHC301 pH electrode (Hach, USA)) and oxygen concentration was determined with a handheld meter (HQ30d equipped with Intellical LDO101 oxygen probe (Hach, USA)). 

Chemical analysis 
Analytical samples (two concentrations per climate scenario) were taken at initiation and termination of experiment. Aliquots (1 mL) were transferred into 1.5 mL liquid chromatography amber glass vials and spiked with a diuron surrogate standard (diuron-d6; stock solution of 1 µg mL-1), of which the final concentration of the surrogate standard was 10 ng mL-1. Herbicide concentrations were determined by HPLC-MS/MS using an SCIEX Triple QuadTM 6500 QTRAP® mass spectrometer (SCIEX, Concord, Ontario, Canada) equipped with an IonDriveTM Turbo V ion source using a TurboIonSpray® probe (Mercurio et al., 2015). The geometric mean from measured start and end of exposure concentrations was used as the measured concentration in that sample.

Seawater carbonate system parameters
Dissolved inorganic carbon (DIC) and total alkalinity (AT) were determined on a Vindta 3C system (Marianda, Kiel, Germany) from seawater samples taken at the end of the experiment. DIC and AT were used to calculate carbonate system parameters (calcite saturation state, omegaCa, and aragonite saturation state, omega arag) according to (Mehrbach et al., 1973) with modifications from (Dickson and Millero, 1987) using CO2sys software (Pierrot et al., 2006).

Chlorophyll fluorescence
Effective quantum yield (Delta F/Fm') measurements were taken just prior to the start of exposure and at 7 and 14 days exposure using a pulse amplitude modulation fluorometer (mini PAM, WALZ, Germany; settings: ML = 5, G = 2, D= 2, SI = 8). Two measurements per coral were obtained using a 6-mm fibre optic probe perpendicular to the surface of the coral. Minimum fluorescence was determined by applying a pulse-modulated red measuring light (650 nm, 0.15 µmol photons m-2s-1). A short pulse (800 ms) of saturating actinic light (&gt;3000 µmol photons m-2s-1) was applied to measure light adapted maximum fluorescence. Effective quantum yield was calculated based on F and Fm' (Genty et al., 1989): Delta F/Fm'=  = (Fm' – F)/Fm'. 

Physiological parameters 
Oxygen production and respiration rates, and light and dark calcification rates were determined after 12-15 d exposure for the 0, 0.29, 0.96 and 2.9 µg L-1 diuron treatments with three replicate coral fragments per treatment. Corals exposed to 9.6 µg L-1 diuron died after 9 days of exposure, irrespective of the climate scenario, therefore rates of oxygen production, respiration and calcification could not be determined for these fragments. Since there was a staggered start for the 29 µg L-1 corals, these physiological processes were measured after 7-8 days exposure in these corals. 

To assess net photosynthesis and calcification rates, coral fragments were incubated in the light and dark with respective pCO2 seawater and diuron treatments in 0.05 L clear acrylic chambers for 3 h under experimental light conditions and 4 h incubations in the dark. The incubation chambers were placed on custom underwater stirrer plates under the same light and water temperature as the experimental chambers. After incubation, dissolved oxygen concentration was measured in each incubation chamber including two blank incubations per treatment as per Strahl et al. (2015). Oxygen production and respiration rates were calculated from changes in dissolved oxygen concentration. Incubation water samples were collected in 50 mL conical centrifuge tubes and fixed with mercuric chloride and analysed for AT to determine calcification rates by the alkalinity anomaly technique (Chisholm and Gattuso, 1991). AT was measured on a Metrohm 855 robotic titrosampler (Metrohm, Switzerland) by gran titration using 0.5 mol L-1 hydrochloric acid using multiple seawater standards and certified reference material (CRM Batch 106, A. Dickson, Scripps Oceanographic Institute) as described in Uthicke and Fabricius (2012). 

After dark incubations, coral fragments were snap frozen in liquid nitrogen for determination of surface area and volume. Oxygen production and respiration rates (in mg O2 cm-2 h-1) and light and dark calcification rates (in µmol CaCO3 cm-2 h-1) were normalized to surface area. These rates were then used to calculate daily net photosynthesis and daily net calcification rates using 13 h of daylight and 11 h of darkness. Surface area of A. millepora fragments were determined via the wax dipping method (Stimson and Kinzie, 1991) and volume of coral was determined using water displacement as per Strahl et al. (2015). 

Mortality
Images were taken at initiation and termination of the experiment using a high-resolution digital camera (Nikon D810 with a 60-mm lens and a Nikon Speedlight SB-910 flash). Partial mortality was visually assessed and quantified using ImageJ2 (Rueden et al., 2017).

Format:
This dataset consists of one Excel spreadsheet:
Acropora millepora diuron climate change data_eAtlas.xlsx
There are six tabs in the spreadsheet:
Tab 1) Physiological parameters
Tab 2) PAM
Tab 3) SA and volume
Tab 4) Mortality 
Tab 5) WQ
Tab 6) Irradiance

Data Dictionary:

There are six tabs in the spreadsheet:
Tab 1) Physiological parameters:
Physiological parameters - physiological endpoints investigated in this study
Climate - three climate change scenarios:  2018, 2050, 2100
Diuron - measured diuron concentrations (in µg L-1) analysed at The University of Queensland: 0, 0.29, 0,96, 2.9, 9.6, 29
Chamber - three replicate chambers used per treatment: 1,2,3
Genotype - four genotypes were randomly distributed among the treatments used for the study: 1,2,3,4
IncubationDay - photosynthetic oxygen production/respiration rates, and light/dark calcification rates were conducted in 50 mL incubation chambers and these were performed at days 12-15 exposure; 29 µg L-1 corals were incubated at 7-8 days exposure
Production - oxygen production rate (in mg O2 cm-2 h-1); incubated corals in their respective temperature and pCO2 treatment water and under the light for 3 h and measured dissolved oxygen immediately afterwards
DarkResp - respiration rate (in mg O2 cm-2 h-1); incubated corals in their respective temperature and pCO2 treatment water and in the dark for 4 h and measured dissolved oxygen immediately afterwards
DailyNet - daily net photosynthesis (in mg O2 cm-2 d-1) = (Production rate * 13) + (Respiration rate * 11); corals were illuminated under a 13:11 h L:D cycle
LightCalc.umol - light calcification rate (in µmol CaCO3 cm-2 h-1)
LightCalc.mg - light calcification rate (in mg CaCO3 cm-2 h-1); used 100.0869 mg molecular weight for CaCO3
DarkCalc.umol - dark calcification rate (in µmol CaCO3 cm-2 h-1)
DarkCalc.mg - dark calcification rate (in mg CaCO3 cm-2 h-1); used 100.0869 mg molecular weight for CaCO3
DailyCalc.umol - daily calcification rate (in µmol CaCO3 cm-2 h-1) = [light calcification rate (in µmol) * 13] + [dark calcification rate (in µmol) * 11]
DailyCalc.mg - daily calcification rate (in mg CaCO3 cm-2 d-1) = [light calcification rate (in mg) * 13] + [dark calcification rate (in mg) * 11]
Blanks denote lost water sample

Tab 2) PAM
PAM = pulse amplitude modulation fluorometry to calculate effective quantum yield, DeltaF/Fm' (light adapted)
Nominal (µg L-1) = nominal diuron concentrations; SC denotes solvent control which is no herbicide and contains &lt; 0.03% v/v ethanol solvent carrier as per the treatments
T7_ - denotes day 7 data
T14_ - denotes day 14 data
T7_Climate/T14_Climate - three climate change scenarios:  2018, 2050, 2100
T7_Measured (µg L-1) = measured diuron concentrations analysed at The University of Queensland
T7_Chamber/T14_Chamber - three replicate chambers used per treatment: 1,2,3
T7_Coral Rep/T14_CoralRep - there were 4 replicate corals for treatments up to 9.6 µg L-1 and 3 replicate corals for 29 µg L-1 treatments: 1,2,3,(4)
T7_Yield/T14_Yield - effective quantum (light adapted) yield measured by a mini Pulse Amplitude Modulation (miniPAM) fluorometer at 7 days (or 14) exposure
T14_Diuron - four measured diuron concentrations (in µg L-1): 0, 0.29, 0,96, 2.9; corals at 9.6 and 29 µg L-1 all died after 9 days exposure so no 14-day Delta F/Fm' data

Tab 3) SA and Volume
Climate - three climate change scenarios: 2018, 2050, 2100
Diuron - measured diuron concentrations (in µg L-1) analysed at The University of Queensland: 0, 0.29, 0,96, 2.9, 9.6, 29
Genotype - four genotypes were randomly distributed among the treatments used for the study: 1,2,3,4; blank spaces denote we did not record genotype for those corals
SA - estimated surface area of corals (in cm2) using the wax dipping method; one coral replicate per chamber
Volume (cm3) - estimated volume of corals (in cm3) using the water displacement method; one coral replicate per chamber

Tab 4) Mortality 
Mortality - percent mortality post 14-day exposure
Climate - three climate change scenarios:  2018, 2050, 2100
Diuron - measured diuron concentrations (in µg L-1) analysed at The University of Queensland: 0, 0.29, 0,96, 2.9, 9.6, 29
Chamber - three replicate chambers used per treatment: 1,2,3
Coral Rep - four coral replicates in each chamber: 1,2,3,4

Tab 5) WQ
WQ – water quality parameters measured during the experiment
Date – date the water quality parameters were measured
Climate - three climate change scenarios: 2018, 2050, 2100
Diuron - measured diuron concentrations (in µg L-1) analysed at The University of Queensland: 0, 0.29, 0,96, 2.9, 9.6, 29
pH – measured pH in experimental chambers
Temp (°C) – measured temperature (in degree Celsius) in experimental chambers
Salinity (PSU) – measured salinity (in practical salinity units, PSU) in experimental chambers
DO (mg L-1 or %) - dissolved oxygen measured in mg L-1 and in % saturation in experimental chambers
blanks denote measurement of that parameter were not taken on that date

Tab 6) Irradiance
Irradiance - light intensity (in µmol photons m-2 s-1) measured by a Licor LI-250A meter with LI-190R quantum sensor

References:

DICKSON, A. G. &amp; MILLERO, F. J. 1987. A comparison of the equilibrium constants for the dissociation of carbonic acid in seawater media. Deep Sea Research Part A, Oceanographic Research Papers, 34, 1733-1743.

GENTY, B., BRIANTAIS, J. M. &amp; BAKER, N. R. 1989. The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochimica et Biophysica Acta (BBA) - General Subjects, 990, 87-92.

MEHRBACH, C., CULBERSON, C. H., HAWLEY, J. E. &amp; PYTKOWICZ, R. M. 1973. Constants of carbonic acid in seawater at atmospheric pressure. Limnology and Oceanography, 18, 897-907.

MERCURIO, P., MUELLER, J. F., EAGLESHAM, G., FLORES, F. &amp; NEGRI, A. P. 2015. Herbicide persistence in seawater simulation experiments. PLoS ONE, 10, e0136391.

PIERROT, D., LEWIS, E. &amp; WALLACE, D. W. R. 2006. S Excel Program Developed for CO2 System Calculations. ORNL/CDIAC-105a. Carbon Dioxide Information Analysis Center, Oak Ridge National Laboratory, U.S. Department of Energy, Oak Ridge, Tennessee.

RUEDEN, C. T., SCHINDELIN, J. &amp; HINER, M. C. 2017. ImageJ2: ImageJ for the next generation of scientific imaging data. BMC Bioinformatics, 18.

STIMSON, J. &amp; KINZIE, R. A. 1991. The temporal pattern and rate of release of zooxanthellae from the reef coral Pocilopora damicornis (Linnaeus) under nitrogen-enrichment and control conditions. Journal of Experimental Marine Biology and Ecology, 153, 63-67.

STRAHL, J., STOLZ, I., UTHICKE, S., VOGEL, N., NOONAN, S. H. C. &amp; FABRICIUS, K. E. 2015. Physiological and ecological performance differs in four coral taxa at a volcanic carbon dioxide seep. Comparative Biochemistry and Physiology, Part A, 179-186.

UTHICKE, S. &amp; FABRICIUS, K. E. 2012. Productivity gains do not compensate for reduced calcification under near-future ocean acidification in the photosynthetic benthic foraminifer species Marginopora vertebralis. Global Change Biology, 18, 2781-2791.

Data Location:

This dataset is filed in the eAtlas enduring data repository at: data\NERP-TE\4.2_Pesticide-effects\

Organization: Australian Ocean Data Network