Sterechinus neumayeri fertilisation and larval development toxicity tests

Brief description

This dataset contains results of toxicity tests with early life stages of the sea urchin Sterechinus neumayeri as part of the AAS Project 3054 'Ecological risks from oil products used in Antarctica: characterising hydrocarbon behaviour and assessing toxicity on sensitive early life stages of Antarctic marine invertebrates.' Dataset consists of excel spreadsheets with separate spreadsheets for each test. Test details are outlined on worksheets 'Test conditions' and results of test in worksheet 'Counts'. This metadata record contains the results of toxicity tests conducted to characterise the response of Antarctic nearshore marine invertebrates to hydrocarbon contaminants in fuels commonly used in Antarctica as part of AAS Project 3054. This dataset contains results of toxicity tests conducted at Davis Station in 2010/11 summer season to test the sensitivity of fertilisation and early life stages of the sea urchin Sterechinus neumayeri to fuels in seawater. The three fuel types used were: Special Antarctic Blend diesel (SAB), Marine Gas Oil diesel (MGO) and an intermediate grade (180) of marine bunker Fuel Oil (IFO). Test treatments were obtained by experimentally mixing fuel and seawater in temperature controlled cabinets at -1 degrees C to prepare a mixture of fuel hydrocarbons in filtered seawater (FSW) termed the water accommodated fraction (WAF). WAF was produced by adding fuel to seawater in Pyrex glass bottles using a ratio of 1:25 fuel : FSW. This mixture was stirred at slow speed with minimal vortex for 18 h on a magnetic stirrer then settled for 6 h before the water portion was drawn from beneath the fuel. Mature S. neumayeri were collected from the outlet of Ellis Fjord, East Antarctica (68.62°S, 77.99°E) in December and early January 2010/11. Sea urchins were collected from shallow nearshore waters less than 1m deep, placed in 20 L buckets of seawater and transported to Davis station. They were held for 1–2 d in a flow-through aquarium at -1 plus or minus 1°C, with macroalgae from the collection site as a food source, before being used for testing. Seawater for experiments was collected ~20 m from the shoreline north of Davis station (68°34’ S, 77°57’ E). Collected seawater was filtered to 0.45 µm (FSW) and stored in 30 L polyethylene containers at 0°C. Fertilisation and early embryo toxicity tests. Effects of WAFs on fertilisation and on development to the 2 cell stage were determined in static tests in which both eggs and sperm were pre-exposed to SAB, MGO and IFO 180 WAFs, fertilised within treatments and developed to the 2 cell stage (G1, G2, G3). Gamete exposure and fertilisation was done in a temperature controlled room at 0°C. Test vessels were 22 mL borosilicate glass vials with foil lined lids holding 20 mL of test solution. There were 10 vials for each treatment; 5 replicates for fertilisation and 5 replicates for the 2 cell endpoint. To pre-expose eggs, 5 mL of prepared egg solution was added to vials that contained 5 mL of 2, 20 and 100% WAFs and FSW controls, to give final treatment concentrations of 1, 10 and 50% WAF dilutions and FSW controls. Vials were sealed, swirled gently to mix and left standing for 20 min. To pre-expose sperm, pooled sperm were activated by dilution in FSW to the density required for a sperm to egg ratio of 800:1. One µL of sperm solution was added to vials containing 5 mL of FSW and gently mixed. Five mL of this solution was then added to vials containing 5 mL of 2%, 20% and 100% WAFs (final treatments of 1, 10 and 50% WAF dilutions) and FSW controls. The vials were sealed, swirled gently to mix and left for 15 mins. After the gamete exposure period was complete, for each treatment the contents of the sperm vials were added to the egg vials with a final target concentration of ~10 eggs per mL. Vials were sealed and placed into temperature-controlled cabinets set at -1 plus or minus 1°C. Temperature was recorded at 10 min intervals using a data logger (Maxim ibutton) and averaged -1.3 plus or minus 0.5°C. Tests were terminated at 4 h for the fertilisation endpoint, and at 11 h for the 2 cell endpoint by the addition of 1 mL of 2.5% (v/v) buffered glutaraldehyde. Samples were viewed in a Sedgewick Rafter counting cell under a compound microscope at 10 times magnification. Fertilisation was assessed according to the presence or absence of a fertilisation membrane in the first 100 eggs counted, to obtain the percentage of eggs fertilised in each replicate. The 2 cell endpoint was assessed in the first 100 embryos counted, as the percentage of embryos in each replicate with normal first cleavage. Embryonic and larval toxicity tests. Effects of fuel WAFs on embryonic and larval development were tested with 1, 10, and 100% WAFs of SAB, MGO and IFO 180 and FSW control, with 5 replicates per treatment. Eggs and sperm were collected and density of solutions adjusted as described above to obtain the optimal sperm to egg ratio of 800:1. Two semi-static tests (EL1, EL2) were done to test effects of WAFs on embryos and larvae when first exposed as zygotes (eggs fertilised in FSW then exposed to treatments before the first cleavage). To fertilise eggs, sperm were activated by their addition to 10 mL of FSW, and 1 µL of this sperm solution was added to beakers containing 700 mL of egg solution and gently mixed. After two hours, the mixture was stirred with a glass rod to maintain a homogeneous suspension while aliquots were transferred into 100 mL glass vials filled with 80 mL of test treatment, to a final density of ~10 zygotes per mL. Three tests (GL1, GL2, GLP) were done to test effects of WAFs on larval development with exposure commencing as gametes. One mL aliquots of egg mixture were added to vials containing 80 mL of test solution (to a density of ~10 eggs per mL) and left for 20 min. Sperm were activated in 10 mL of FSW and 0.1 mL aliquots added to the vials to fertilise eggs within treatments at a sperm to egg ratio of 800:1. Two exposure regimes were used; continuous semi-static WAF renewal (GL1 and GL2) and a single static pulse of WAF exposure up to the 4 d unhatched blastula stage, followed by post exposure recovery in FSW up to the 21 d pluteus stage (GLP). Vials were left uncovered and placed in a temperature controlled cabinet at -1 plus or minus 1°C with an 18 h light, 6 h dark photoperiod. Tests were under semi-static conditions, with test solutions renewed every 4 d. Water quality data was collected at each water change. Treatment renewals were done by removing and replacing approximately 90% of test solution. Disposable syringes with silicon tubing attached to the nozzle, and with the end of the tubing covered with plankton mesh, were used to withdraw test solution while preventing embryos/larvae from being removed. The vials were then refilled to the 80 mL mark with fresh test solutions. Treatment renewals for tests EL1, EL2 and GL1, GL2 were with freshly made WAFs every 4 d. For the single pulse WAF exposure test (GLP) on the first treatment renewal at 4 d, treatment solutions were removed as described above, and replaced with FSW. All subsequent 4 d renewals for test GLP were with FSW. To maintain the volume and salinity of test treatments a small volume of purified and deionised (Milli-Q) water at -1°C was stirred into the vials to the 80 mL mark every 2 d between water changes. Water quality measurements were made at the start of tests and pre and post treatment renewals. Mean water quality parameter measurements were pH 8.08 plus or minus 0.10, salinity 36.6 plus or minus 0.9‰ and dissolved oxygen 11.1 plus or minus 0.61 mg/L. Temperature was recorded at 10 min intervals using a data logger (Maxim ibutton) and averaged -1.0 plus or minus 1.0°C. In tests where exposure commenced as zygotes, endpoints were the embryonic 4-8 cell (20 h) and unhatched blastula (48 h) stages, and the larval blastula (6–7 d) and gastrula (14–15 d) stages. In tests with exposure commencing as gametes, endpoints were the larval blastula, gastrula and early 4-arm pluteus (21–24 d) stages. At each endpoint a sample was taken from each replicate by drawing an aliquot with a glass pipette and transferring it to a vial, to which 1 mL of 2.5% (v/v) buffered glutaraldehyde was added. Embryo and larvae were viewed in a Sedgewick Rafter counting cell under a compound microscope at 10 times magnification. The first 30 individuals in each sample at the 4-8 cell and unhatched blastula endpoints, and the first 100 individuals at the blastula, gastrula and pluteus endpoints, were assessed for normality. Test EL1 ended at the blastula stage and tests EL2 and GL2 at the gastrula stage as there were insufficient numbers of larvae remaining to continue the test beyond these stages. All remaining larvae were counted at the final endpoint. Chemical analysis of water accommodated fractions Total hydrocarbon content (THC) in WAFs were derived from replicate tests conducted under the same conditions but without test organisms. In these tests at 0°C, the concentrations of freshly made WAFs of each of the three fuels, and the depletion of hydrocarbons from 100%, 50%, 10% and 1% WAFs at multiple time points over 7 d were measured. Extracts were analysed for THC with GC-FID. Total hydrocarbon content was reported as the sum of hydrocarbons (µg/L) in the range less than n-C9 to C28 (Dataset AAS_3054_THC_WAF). For fertilisation, and 2 cell embryonic development assays that were done in sealed vials, measured values in freshly decanted 50% and 10% WAF dilutions were used as the exposure concentrations. For the embryonic and larval toxicity tests that were done in open vials, the exposure concentrations of THC in WAFs were modelled from the measured concentrations in WAF depletion tests. Exposure concentrations used to model sensitivity estimates were derived by calculating the time weighted mean THC between pairs of successive measurements in the 100% WAFs and dilutions to give overall exposure concentrations for each time point. These modelled concentrations integrated the loss of hydrocarbons over time, and renewal of test solutions at 4 d intervals.

Lineage

Progress Code: completed

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Purpose
In this study we use S. neumayeri to assess the risk from fuel spills to Antarctic nearshore marine ecosystems. We investigate the effects of the water accommodated fractions (WAFs) of three fuels: Special Antarctic Blend diesel (SAB), marine gas oil (MGO) and intermediate fuel oil (IFO 180), on fertilisation and normal embryonic and larval development of S. neumayeri.