Thiacloprid (THI) is a neonicotinoid insecticide of interest to the USEPA due to its low absorption by crops, greater distribution into the surrounding areas, and potential for adverse effect to terrestrial and aquatic organisms. Prior to this report, there was very limited information addressing the ex vivo metabolism of THI by fish species and the metabolic pathways regulating its potential adverse effects. The in vitro and ex vivo biotransformation pathway of THI is defined by the formation of three primary metabolites (TM1, TM2 and TM3) via separate paths differentiated by reductive decyanation, reductive dechlorination with hydration and dealkylation processes, respectively. Kinetic rates were calculated for the rat microsomal decyanation of THI into TM1 (Km=299.2 µM and Vmax=5.3 pmol/min/mg), and for the dealkylation of THI into TM3 (Km=368.9 µM µM and Vmax=3.95 pmol/min/mg). Formation confirmation and identity inference of THI metabolites in absence of standards was achieved by LC-UV and High Resolution-MS strategies. It was concluded that the in vitro and ex vivo metabolic products of THI are conserved both across species (rat and RBT) and levels of biological organization (microsomes and liver slices), as previously reported for the neonicotinoid insecticides Imidacloprid and Acetamiprid. This dataset is associated with the following publication: Serrano, J., R. Kolanczyk, B. Blackwell, B. Sheedy, and M. Tapper. In vitro metabolism assessment of thiacloprid in rainbow trout and rat by LC-UV and high resolution-mass spectrometry. XENOBIOTICA. Taylor & Francis, Inc., Philadelphia, PA, USA, 51(5): 536-548, (2021).

This dataset shows the effects of herbicides and one fungicide (detected in Great Barrier Reef catchments) on the specific growth rates (from cell density data) of the microalgae Tisochrysis lutea during laboratory experiments conducted from 2018-2019. The aim of this project was to apply standard ecotoxicology protocols to determine the effects of Photosystem II (PSII), alternative herbicides and one fungicide on the growth of the marine microalgae Tisochrysis lutea. Growth bioassays were performed over 3-day exposures using pesticides that have been detected in the Great Barrier Reef catchment area (O'Brien et al., 2016). These toxicity data will enable improved assessment of the risks posed by PSII and alternative herbicides as well as the fungicide propiconazole to microalgae for both regulatory purposes and for comparison with other taxa. Methods: The haptophyte Tisochrysis lutea (formerly known as Isochrysis galbana)(Grant etal. 2017) (strain CS-177) was purchased from the Australian National Algae Supply Service, Hobart (CSIRO). Cultures of T. lutea were established in EDTA-free Guillard’s f/2 marine medium (Trenfield et al. 2015) (1 ml L-1 of f/2 medium in autoclaved natural seawater). Cultures were maintained in sterile 500 ml Erlenmeyer flasks as batch cultures in exponential growth phase with weekly aseptically transfers of 10 ml T. lutea suspension to 300 ml f/2 medium. Culture were maintained at 28 ± 1°C, 33 ± 1.5 psu and under a 12:12 h light:dark cycle (80 – 100 µmol photons m–2 s–1). Pesticide stock solutions were prepared using PESTANAL (Merck) analytical grade products (purity greater than or equal to 98%): diuron (CAS 330-54-1), metribuzin (CAS 21087-64-9), tebuthiuron (CAS 34014-18-1), bromacil (CAS 314-40-9), propazine (CAS 139-40-2), simazine (122-34-9), imazapic (CAS 104098-48-8), haloxyfop-p-methyl (CAS 72619-32-0), 2,4-D (CAS 94-75-7), MCPA (CAS 94-74-6), fluroxypyr (CAS 69377-81-7) and propiconazole (CAS 60207-90-1). The selection of pesticides was based on application rates and detection in coastal waters of the GBR (Grant et al. 2017, O’Brien et al. 2016). Pesticide stock solutions (100 – 1,000 mg L-1) were prepared by dissolving aliquots of the pure compounds in ultrapure water using clean, acid-washed (5% nitric acid) glass screw-top containers. Simazine, tebuthiuron and haloxyfop-p-methyl were dissolved using the carrier dimethyl sulfoxide (DMSO) (less than or equal to 0.02 % (v/v) in exposure solutions). Diuron, imazapic, metribuzin, bromacil, 2,4-D, propazine, MCPA, fluroxypyr and propiconazole were dissolved in acetone (less than or equal to 0.01 % (v/v) in exposure). Stock solutions were stored refrigerated and in the dark. Tests were conducted as previously described (Trenfield et al. 2015). Cultures of T. lutea were exposed to increasing concentrations of individual pesticides over a period of 72 h. Inoculum was taken from cultures in exponential growth phase (4-d old culture) and starting cell density assessed using a haemocytometer. For each treatment, a total volume of 250 mL test media was prepared in a clean, acid-washed 500 mL Schott bottle. Test media consisted of filtered (0.45 µm) seawater spiked with the respective pesticide stock, quarter strength EDTA-free f/2 media as nutrient source and T. lutea at a starting density of 3x103 or 1x104 cells mL-1. In each toxicity test, the response (specific growth rate of the culture) of the treatments exposed to pesticide were assessed against a seawater control group (no herbicide). For each test, 2 – 3 replicate 125 mL Erlenmeyer flasks (50 mL test volume) were assessed. Flasks were incubated at 27 – 29.0°C under a 12:12 h light:dark cycle (80 – 100 µmol photons m–2 s–1). After 72h, sub-samples (7 ml) were taken from each flask and cell densities measured using a flow cytometer (BD Accuri C6, BD Biosciences, CA, USA). Specific growth rates (SGR) were expressed as the logarithmic increase in cell density from day i (ti) to day j (tj) as per

This dataset shows the effects of eight herbicides (detected in Great Barrier Reef catchments) on the specific growth rates (from cell density data) of the microalgae Tetraselmis sp. during laboratory experiments conducted in 2019. The aim of this project was to apply standard ecotoxicology protocols to determine the effects of Photosystem II (PSII) and alternative herbicides on the growth of the marine microalgae Tetraselmis sp. Growth bioassays were performed over 3-day exposures using pesticides that have been detected in the Great Barrier Reef catchment area (O’Brien et al. 2016). 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 Tetraselmis sp. (strain CS-317) was purchased from the Australian National Algae Supply Service, Hobart (CSIRO). Cultures of Tetraselmis sp. were established in EDTA-free Guillard’s f/2 marine medium (Guillard and Ryther 1962) (1 ml L-1 of f/2 medium in autoclaved natural seawater). Cultures were maintained in sterile 500 ml Erlenmeyer flasks as batch cultures in exponential growth phase with weekly aseptically transfers of 10 ml Tetraselmis sp. suspension to 300 ml f/2 medium. Culture were maintained at 28 ± 1°C, 33 ± 1.5 psu and under a 12:12 h light:dark cycle (80 – 100 µmol photons m–2 s–1). Pesticide stock solutions were prepared using PESTANAL (Merck) analytical grade products (purity greater than or equal to 98%): diuron (CAS 330-54-1), metribuzin (CAS 21087-64-9), tebuthiuron (CAS 34014-18-1), bromacil (CAS 314-40-9), propazine (CAS 139-40-2), simazine (122-34-9), imazapic (CAS 104098-48-8) and haloxyfop-p-methyl (CAS 72619-32-0). The selection of pesticides was based on application rates and detection in coastal waters of the GBR (Grant et al. 2017, O’Brien et al. 2016). Pesticide stock solutions (100 – 1,000 mg L-1) were prepared by dissolving aliquots of the pure compounds in ultrapure water using clean, acid-washed (5% nitric acid) glass screw-top containers. Simazine, tebuthiuron and haloxyfop-p-methyl were dissolved using the carrier dimethyl sulfoxide (DMSO) (less than or equal to 0.02 % (v/v) in exposure solutions). Diuron, imazapic, metribuzin, bromacil and propazine were dissolved in acetone (less than or equal to 0.01 % (v/v) in exposure). Stock solutions were stored refrigerated and in the dark. Test protocols were based on previously published methods (Trenfield et al. 2015, OECD, 2011). Cultures of Tetraselmis sp were exposed to increasing concentrations of individual pesticides over a period of 72 h. Inoculum was taken from cultures in exponential growth phase (5-d old culture) and starting cell density assessed using a haemocytometer. For each treatment, a total volume of 250 mL test media was prepared in a clean, acid-washed 500 mL Schott bottle. Test media consisted of filtered (0.45 µm) seawater spiked with the respective pesticide stock, quarter strength EDTA-free f/2 media as nutrient source and Tetraselmis sp at a starting density of 2.5x103 cells mL-1. In each toxicity test, the response (specific growth rate of the culture) of the treatments exposed to pesticide were assessed against a seawater control group (no herbicide). For each test, 2 – 3 replicate 125 mL Erlenmeyer flasks (50 mL test volume) were assessed. Flasks were incubated at 27 – 29.0°C under a 12:12 h light:dark cycle (80 – 100 µmol photons m–2 s–1). After 72h, sub-samples (7 ml) were taken from each flask and cell densities measured using a flow cytometer (BD Accuri C6, BD Biosciences, CA, USA). 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 6. SGR i-j = [(ln Xj - ln Xi )/(tj - ti )] (day-1) (1) (1) Mean SGR for a pesticide

This dataset shows the effects of of the insecticide imidacloprid and the fungicide propiconazole on larval development of the acorn barnacle Amphibalanus amphitrite experiments conducted in 2018 and 2019. The aim of this project was to apply standard ecotoxicology protocols to determine the effects of the insecticide imidacloprid and the fungicide propiconazole on larval development rate of the acorn barnacle Amphibalanus Amphitrite. Larval development bioassays(4-d exposures) were conducted using a fungicide and insecticide that have been detected in the Great Barrier Reef catchment area (O'Brien et al., 2016). These toxicity data will enable improved assessment of the risks posed by pesticides to marine crustaceans for both regulatory purposes and for comparison with other taxa. Methods: Pesticide stock solutions were prepared using PESTANAL (Merck) analytical grade products (purity greater than or equal to 98%): imidacloprid (CAS 138261-41-3) and propiconazole (CAS 60207-90-1). This selection was based on application rates and detection in coastal waters of the GBR (O’Brien et al., 2016; Grant 2017). Pesticide stock solutions (100 – 1,000 mg L-1) were prepared by dissolving aliquots of the pure compounds in ultrapure water using clean, acid-washed (5% nitric acid) glass screw-top containers. Acetone was used to dissolve the imidacloprid and propiconazole (less than or equal to 0.01 % (v/v) in exposure solutions). Stock solutions were stored refrigerated and in the dark. Broodstock barnacles had been grown for several generations in the AIMS-NT aquaria facility (originally sourced from Darwin Harbour – 12°26'57.48"S, 130°51'7.51"E). Broodstock were fed freshly hatched brine shrimp (Artemia salina) and live rotifers daily. Broodstock were spawned as previously described (van Dam et al., 2016) and nauplii collected. Tests were conducted as previously described (van Dam et al., 2016). Nauplii were exposed in a custom-designed experimental test system that allowed for constant movement of the exposure media. The system consisted of a series of silanized glass funnels in which nauplii were exposed to increasing concentrations of imidacloprid or propiconazole and tested against control nauplii. Generally, a total of 24 funnels were used for 7 treatment concentrations and a control group, thus allowing for 3 replicate funnels per treatment. Each treatment vessel contained 100 mL exposure media, 50 newly released stage II nauplii and 1 x 107 cells of rinsed Chaetoceros muelleri. Every 24 h, 1 x 107 cells of rinsed C. muelleri were added to each funnel. After 96 h exposure, funnel contents were drained over a 150 µm nitrile mesh. The mesh was examined under a stereomicroscope and the number of cyprids and settled larvae scored. Quality control criteria (> 70% survival in control group) for test acceptability were met for each test used to derive toxicity estimates. Treatment effects were quantified by the percentage successful transition to cyprid in treatment groups relative to controls. Following prescribed statistical procedures (OECD 2006) the R package DRC (R-project 2015, Ritz & Streibig 2005), was used to model the test data and calculate toxicity estimates. Regression models evaluated included log-logistic and Weibull models of different levels of parametrisation. Model comparisons were conducted using the Akaike Information Criterion (AIC) and models that best described the data were applied to approximate pesticide concentrations eliciting 10 and 50% inhibition of successful transition relative to control animals (EC10 and EC50, respectively). The associated 95% confidence limits were estimated using the delta method. Format: The dataset is summarised in one file named ‘Amphibalanus amphitrite pesticide toxicity data_eAtlas.xlsx’ Data Dictionary: The excel spreadsheet has one tab for each pesticide. The last tab of the dataset shows the measured (start and end of test) water quality (WQ) parameters (pH, salinity, dissolved

Neonicotinoids have become the most widely used insecticides in world with rapid growth in applications as seed coatings. Nontarget organisms are exposed to concentrated levels of pesticidal active ingredients through ingestion of treated seeds. To better understand pesticide fate, analytical methods are necessary to rapidly screen and accurately quantitate contaminants in environmental and biological matrices. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is commonly employed for neonicotinoid analyses but requires expensive analytical instrumentation and potentially laborious sample preparation. Enzyme-linked immunosorbent assays (ELISAs) are efficient and sensitive alternative methods for neonicotinoid analyses, that does not require costly equipment. In this study, application of ELISA was compared to LC-MS/MS in the quantitation of imidacloprid in plasma, liver, and fecal matter from Japanese quail (Coturnix japonica) orally exposed to imidacloprid-treated wheat seeds. Two major imidacloprid metabolites, 5-OH-imidacloprid and imidacloprid-olefin, were found to cross-react within the immunoassay, confounding results. Imidacloprid concentrations from ELISA analysis ranged from 90.8-181 ng/mL, 7.90-40.4 ng/g, and 989-4404 ng/g in plasma, liver, and fecal matter, respectively. In comparison, LC-MS/MS imidacloprid concentrations ranged from 32.8-188 ng/mL for plasma, 18.7-116 ng/g for liver, and 51.3-427 ng/g for fecal matter. Imidacloprid concentrations determined by ELISA were generally higher in plasma and exceptionally higher in fecal matter compared to LC-MS/MS, Liver imidacloprid concentrations were higher when analyzed by LC-MS/MS. Good correlation was observed between imidacloprid concentrations determined in plasma (r2 = 0.93) and fecal matter (r2 = 0.89) by ELISA and LC-MS/MS, whereas a poor correlation (r2 = 0.35) was found in liver. In the analysis of these biological matrices, ELISA was found to be a good screening tool to measure active ingredients along with their metabolites. However, accurate quantitation should be completed with chromatographic based techniques such as LC-MS/MS.

Neonicotinoids, a widely used class of insecticide, have been found in surface waters globally. They pose a risk to non-target species found in aquatic environments such as aquatic macroinvertebrates. To better understand the effects of the neonicotinoids imidacloprid, clothianidin and their mixtures on aquatic communities we ran a 30 day mesocosm test. Rock trays were colonized with natural benthic communities in the Cache La Poudre River, located in the mountains of Northern Colorado, and relocated to a laboratory experimental stream setting. In total there were 33 experimental streams: 3 controls and 30 treatments consisting of both single compound and binary compound exposures with 5 treatment levels for each exposure series. Water quality and chemistry samples were collected throughout the experiment. Larval invertebrates remaining in each experimental stream at the end of the experiment were collected, enumerated and identified to the lowest taxonomic unit practical, typically genus or species. Emergent insects were collected each day of the experiment and identified to lowest taxonomic unit. Chlorophyll a was measured in each experimental stream 3 times throughout the 30 day experiment.

This dataset shows the effects of three insecticides (diazinon, fipronil, imidacloprid) and two fungicides (chlorothalonil, propiconazole) on larval metamorphosis in the coral Acropora tenuis. These five pesticides have been detected in the Great Barrier Reef lagoon and/or catchments. Settlement assays were conducted in Nov-Dec 2016 and Nov 2017. The aim of this research is to add toxicity data for inclusion into water quality guidelines. In order to improve water quality guidelines and subsequent risk assessments for pesticides in tropical marine ecosystems, the current study investigated the effects of three insecticides (diazinon, fipronil, imidacloprid) and two fungicides (chlorothalonil and propiconazole) on larval settlement and metamorphosis of the common reef-building coral Acropora tenuis larvae following 48 h exposures. Concentration-response curves were plotted to estimate no effect concentration (NEC) and effect concentration (ECx) values that inhibited larval settlement by 10% and 50% (EC10 and EC50, respectively). NEC is the concentration below which the pesticides are not expected to cause a reduction in larval metamorphosis. Methods: Gravid colonies (25-40 cm diameter) of the coral Acropora tenuis (Dana, 1846) were collected from 4 – 8 m depth in November 2016 from Trunk Reef (18°18.2’ S, 146°52.2’ E) and in November 2017 from Falcon Island (18°46’ S, 146°32’ E), GBR under Great Barrier Reef Marine Park Authority Permit G12/35236.1. Colonies were transported to the National Sea Simulator at the Australian Institute of Marine Science (AIMS) in Townsville and maintained in 1700 l flow-through holding tanks until spawning. Temperatures were held at 26-27°C, which was equivalent to the water temperature at the collection site. Gametes were collected from 8 parental colonies on each occasion, fertilised and symbiont-free larvae were cultured at approximately 500 larvae L-1 in 500 L flow-through tanks (Negri and Heyward, 2001, Nordborg et al., 2018). Larvae were competent to settle after 5 d and we used 7-10-day old A. tenuis larvae, each 800-1000 µm in length for consistency in the pesticide exposure experiments. The five pesticides in this study were > 98% pure and purchased from Sigma-Aldrich (NSW, Australia). Stock solutions (5 mg l-1) of all pesticides were dissolved in dimethyl sulfoxide (DMSO, final concentration < 0.01% (v/v) in exposures) and prepared in milli-Q water. A. tenuis larvae were exposed to diazinon (2.62 – 638 µg l-1), fipronil (1.57 – 1144 µg l-1), imidacloprid (3.88 – 947 µg l-1), chlorothalonil (0.69 – 507 µg l-1) and propiconazole (8.42 – 2053 µg l-1). Pesticide analyses were done by The University of Queensland, Queensland Alliance for Environmental Health Sciences (QAEHS), Woolloongabba, Australia. Static exposures were conducted in 20 mL glass scintillation vials containing 12-14 larvae made up to 10 mL filtered seawater (0.5 µm) with 6-7 concentrations (per pesticide) and 6 replicate vials per concentration. All tests included solvent controls containing identical concentrations of DMSO carrier. Seawater and solvent carrier controls were run in 12-18 replicate vials. Copper (CuCl2) was used as a reference toxicant at 6 concentrations between 1.12 – 36 µg L-1 and 6 replicate vials per concentration. Glass vials were transferred in random positions within a refrigerated shaking incubator (TLM-530, Thermoline Scientific) at 70 RPM to maintain gentle water movement which prevents larvae from attaching and undergoing metamorphosis in the containers (Negri et al., 2016). Larvae were exposed under a light intensity of approximately 60 µmol photons m-2 s-1 (12:12 h L:D cycle) and at 26.7 ± 0.7 °C (range). Vials were re-randomised at 24 h. After 48 h exposure larvae and treatment water were transferred into 6-well polystyrene culture plates (Nunc, NY, USA) and returned to the incubator but without water movement. Metamorphosis was initiated by the addition of crustose coralline algae (CCA) extract

The phenylpyrazole insecticide fipronil and its degradates are a potential surface-water contaminant and toxicant to nontarget species such as aquatic macroinvertebrates. To better understand how fipronil, fipronil sulfide, fipronil sulfone, desulfinyl fipronil, and fipronil amide affect aquatic communities, a 30-day mesocosm experiment was run. Rock trays were colonized with natural benthic communities in the Cache La Poudre River in the mountains of northern Colorado and transplanted into a laboratory experimental stream setting. In total, there were 36 experimental streams: 3 controls, 3 solvent controls, and 30 treatments. Water quality metrics and samples for pesticide analysis were collected throughout the experiment. At the end of the experiment, larval invertebrates remaining in each experimental stream were collected, enumerated, and identified. Emergent insects were collected each day of the experiment and identified to lowest taxonomic unit. These data were used to derive species-specific effect concentrations and, along with published data, derive species sensitivity distributions for fipronil(s) and hazard concentrations for the 5th percentile of affected species (HC5). The resulting HC5 values were used to convert fipronil compound concentrations in field samples to the sum of toxic units (∑TUFipronils), and the field invertebrate data were converted into a Species at Risk (SPEAR) pesticides metric (SPEAR_pesticide) and used to explore the relationship between the invertebrate community and ∑TUFipronils.

A simple quantitative bioassay was developed using widely available technology to determine pre-emergent phytotoxicity. Digitaria didactyla (Queensland Blue Couch Grass) was assessed by analysing digital images of the plants growing in microtitre plates for the effects of herbicides on germination. The herbicides tested were metsulfuron, triasulfuron, fluometuron atrazine, simazine and prometryn. Images of each 24-well microtitre plate (taken with a digital camera 7-10 days after planting), were analysed using a public domain image processing program, and a filter (developed using plugins available within the program) selected pixels within the green spectrum. Pixel counting took place using a spreadsheet function. To investigate the use of microtitre plates for the discovery of phytotoxic compounds within higher plants. This bioassay system allows rapid throughput of test samples with minimum handling.