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Scientific Reports Volume 6, Article Number: 21153 (2016) Cite this article

The risk of oil spills to coral reefs is difficult to assess, partly because of the lack of adequate studies to assess the toxicity of related coral reef species. Here, we experimentally tested the acute toxicity of condensate (representing part of light crude oil) to coral (Acropora tenuis) and sponge (Rhopaloeides odorabile) larvae. The metamorphosis of coral larvae is suppressed under the water holding fraction (WAF) with a total petroleum aromatics (TPAH) concentration as low as 103 μg l-1, similar to the concentration detected in seawater after a large spill. When coral larvae are exposed to UV light that they may encounter in shallow reef systems, their sensitivity increases by 40%. By summarizing the toxicity of its main components (benzene, toluene, p-xylene, and naphthalene), the condensate WAF is more toxic to coral larvae than predicted. In contrast, the sensitivity of sponge larvae to condensed water WAF (>10,000 μg l−1 TPAH) is much lower than corals in the presence and absence of ultraviolet light, but is similar to other marine invertebrates. Although these results highlight the relative sensitivity of coral larvae to oil, further research is needed to better understand and predict the impact and risk of hydrocarbons on tropical coral reef systems.

Two major oil spills ("blowouts"), the Montara wellhead platform accident in northwestern Australia in 20091 and the 2010 Macondo (deep water Horizon) incident in the Gulf of Mexico shortly after2,3. The Montara incident released about 4,500 cubic meters of intermediate crude oil 1, 4, and 5 to a unique marine biogeographic province, which contained biodiversity hotspots such as carbonate reefs, river banks, and shoals, which are associated with activities and activities. Paleohydrocarbon leakage is spatially related (possibly causal)6, 7. Flooded banks and shoals formed by the green alga Halimeda spp. Coral algae and other coralline algae 8 are particularly abundant in the Timor Sea. The mountaintop areas where they are exposed to the sun are mainly photosynthetic autotrophic stony corals, eight corals, green algae and seagrass4, with deeper (> 25-30 m) flanks Mainly filter and pellet feeders, including sponges, soft corals and bryozoans4. The greater Deepwater Horizon event released approximately 780,000 cubic meters of light crude oil, and the underground plume was reported to have affected deep benthic communities, including cold-water coral populations9,10.

Tropical coral reefs are declining, and the additional impact of human activities, including hydrocarbon pollution, puts these ecosystems at greater risk11. In addition to blowouts, coral reefs may also be exposed to hydrocarbons due to shipping accidents12,13 and leaks from coastal processing facilities. One of the largest recorded oil spills in a shallow tropical coral reef environment (Galeta oil spill, 1968) involved the release of approximately 10,000 cubic meters of medium-sized crude oil from refinery storage into the offshore waters of the Caribbean coast of Panama14. The spill had extensive effects on mangroves, seagrass, and corals14, and after five years, little evidence of coral reef recovery was observed15.

Tropical creatures most vulnerable to oil are sessile benthic invertebrates that cannot be actively avoided. The impact of hydrocarbons on corals is particularly important because they are the main habitat-forming species on coral reefs, and several laboratory and field studies have described the lethal and sublethal effects of exposure12, 16, 17, 18. For other important taxa, such as sponges, they are the main filter-feeding taxa on coral reefs and are important links between benthic and pelagic habitats, but there is little research. In addition, little is known about the effects of hydrocarbons on the early life history stages of coral reef invertebrates. These stages are especially important because successful recruitment supports the recovery and resilience of coral reefs after disturbance20,21. Groundbreaking field studies in the Red Sea described the reproductive yield of corals and a significant reduction in the settlement and metamorphosis of pelagic coral larvae in locations long-term contaminated by crude oil22,23. These early observations were supplemented by about 12 laboratory studies that investigated the effects of hydrocarbons (gasoline, condensate, fuel oil, crude oil, and lubricants) on the different life stages of corals (including early release of larvae, fertilization, The effects of embryonic development, larval survival and metamorphosis) and juvenile growth are summarized in Table 1). Some laboratory studies indicate that the early life stages of corals may be sensitive to petroleum hydrocarbons, but the impact threshold concentration is more than three orders of magnitude between research, species, and life stage. The only equivalent study on sponges was the use of the shell sponge Crambe crambe and indicated that the larvae may be more sensitive to hydrocarbons than most coral larvae (Table 1).

In order to predict the risks of oil spills and blowouts to marine species, it is necessary to understand the potential exposures and possible effects of a range of different organisms. The extensive susceptibility of corals and sponges to hydrocarbons in the early life stages in Table 1 may be due to differences in oil types and experimental conditions, including exposure time. Differences may also be due to differences in the life history stages used and inherent differences in the sensitivity of different species or taxa. The latter source of variation is particularly important for the purpose of deriving environmental quality standards and risk assessment, because it is the basis of species sensitivity distribution (SSD) analysis (that is, the probability distribution of a certain toxicity measure of a certain chemical in a population) )twenty four. Comparing hydrocarbon exposure studies is not always possible, because the concentration is usually expressed in terms of water holding fraction (% WAF) or total hydrocarbons (THC), whose composition may vary depending on the source of the hydrocarbon and the amount used for WAF preparation. Agreements vary. 25. The exposure of coral reef organisms to hydrocarbons after uncontrolled release has not been reliably reported, but the comprehensive water column measurement performed during the Deepwater Horizon leak provides a suitable reference for the possible concentrations in the underground slick point. The spill released a variety of petroleum hydrocarbons (natural gas and petroleum), including normal alkanes, branched chain alkanes, monoaromatic hydrocarbons (MAH) and polycyclic aromatic hydrocarbons (PAH)26. Total underground polycyclic aromatic hydrocarbons (ΣPAH41) reached 189 μg l−1 (~1,300 meters deep, <5 kilometers from the platform) 27, while total benzene, toluene, ethylbenzene and xylene (collectively referred to as BTEX) reached 78 μg l−1 (In samples collected at a depth of about 1200 meters and 6 kilometers from the wellhead) 26.

Oil and gas exploration and production in Northwest Australia face unique protection challenges, that is, balancing the commercial value of potential oil and gas prospects with the possibility of accidental leakage during the exploration and production process and the potential loss of the protection value of these unique ecosystems. The toxicity of light crude oil in northwestern Australia has been studied on several temperate species as well as tropical fish, shrimp and sea urchin larvae28; however, the toxicity to sessile tropical reef invertebrates such as corals and sponges has not been described. For tropical ecotoxicology, the important environmental variables are water temperature and ultraviolet irradiance, both of which are higher in low-latitude environments. For example, ultraviolet radiation can increase the effectiveness of PAH through the formation of oxygen free radicals and the accompanying damage to membranes and DNA. In order to improve risk assessment and better provide information for oil and gas industry and government oil spill response decisions, we examined the acute toxicity of condensate from the Northwest Shelf to coral and sponge larvae. Condensate oil (also known as natural gasoline or distillate oil) is the hydrocarbon fraction of gas wells or light crude oil wells, which remain liquid at room temperature and 1 atmosphere. In addition, we examined the effects of simultaneous exposure to ultraviolet rays and the toxicity of the four main aromatic components in the condensed water portion to examine their potential contribution to toxicity.

The chemical properties of stable condensate are typical of relatively light petroleum products. The analysis of hydrocarbons suitable for GC shows that the condensate mainly contains odd and even normal alkanes (the most abundant in the range of n-C5 to n-C10), branched chain alkanes, and parent and alkyl-substituted cycloalkanes, benzene and PAH (Supplementary Figure S1). Compared with heavy oil, the molecular weight distribution characteristics of PAHs are lower, mainly including the parent and alkyl naphthalene, fluorene and phenanthrene (Supplementary Table S1). BTEX and PAH accounted for 2.8% and 0.6% (w/w) of the condensate, respectively.

Compared with the original condensate, the solvent extract of the newly prepared condensate WAF is very different in its hydrocarbon distribution. By extracting the three main ion characteristics (m/z 57, 71, 85) of these compounds from the GC-MS total ion current chromatogram, no normal alkanes or branched alkanes were detected in WAF. On the contrary, the hydrocarbon composition is dominated by BTEX and other alkyl-substituted benzene and naphthalene series (Supplementary Figure S2). Due to evaporation, some more volatile condensed hydrocarbons may be lost during mixing. The remaining condensate formed a visible immiscible slick on the surface of the sea after sedimentation. The total BTEX concentrations contained in the exposed seawater portion of corals and sponges were 12.7 and 27.8 mg l-1, respectively, while the total PAH concentrations were 157 and 224 μg l-1, respectively (Supplementary Table S2). The total BTEX and PAHs in the coral-exposed WAF were 55% and 30% lower than the sponge-exposed WAF, respectively. This change may be due to the greater loss of volatile and semi-volatile components during the preparation and/or sampling of the coral exposed WAF. Table 2 summarizes the individual BTEX and PAH concentrations in the condensate and the freshly prepared WAF used in the exposure experiment. During the larval exposure experiment, the headspace (~10%) in the sealed glass culture flask caused TPAH to evaporate 35-55% within 24 hours, and then the average of the initial and final TPAH concentrations was used as the exposure in the concentration-response curve quantity.

After 24 hours in uncontaminated seawater, 70-84% of "control" coral larvae successfully settled and metamorphosed in each experiment (Table 3). During this period, there was no difference in metamorphic success (performance) between control larvae (7-13 days old) (ANOVA F5 = 1.4, p = 0.24). Coral larvae exposed to condensate water WAF for more than 24 hours also show normal sedimentation and metamorphosis behavior at low concentrations (<100 μg l-1), but this development process becomes more and more severe at higher condensate concentrations The more restrained (Figure 1). Fitting% inhibition data to the logistic equation allows calculation of the concentration of inhibited metamorphosis 10% (IC10) = 103 μg l-1 and 50% (IC50) = 339 μg l-1 TPAH (Table 3). Under other same conditions, the coral larvae were exposed to high ultraviolet light for 2 hours, and the deformation was inhibited by 50% at a significantly lower IC50 = 132 μg l-1 TPAH (p <0.05, Figure 2b, Table 3) .

Concentration-response curve that inhibits the metamorphosis of coral larvae when the main aromatic components found in the condensed water WAF (μg l-1) are present.

The solid circles represent the average inhibition (%, relative to the control) of coral larvae metamorphosis on (a) benzene, (b) toluene, (c) p-xylene and (d) naphthalene. Open circles represent the average abnormality (%) of larvae in the same treatment. Mean ± SE of six repeated exposures. The summary results of these curves can be found in Table 3.

A photomicrograph of a 24-hour-old juvenile coral shows normal post-settlement metamorphosis, showing that the primary and secondary mesenteric formation of a single polyp is complete, with six tentacles around the mouth, which partially and destroy metamorphosis when exposed to PAH and WAF condensate. (A) Control, (b), 5,600 μg l-1 TPAH in stable condensate WAF and (c) 34,000 μg l-1 benzene WAF.

Concentration-response curve for inhibiting metamorphosis of coral and sponge larvae in the presence of condensed water WAF (μg l−1 TPAH).

The solid circles represent the average inhibition of coagulation WAF by coral larval metamorphosis (%) in the absence of ultraviolet light and (b) when ultraviolet light and sponge larvae metamorphose to condensed WAF (c) in the absence of ultraviolet light (%, relative to the control) ) (d) There is ultraviolet light. Open circles represent the average abnormality (%) of larvae in the same treatment. Mean ± SE of six repeated exposures. The summary results of these curves can be found in Table 3.

Coral larvae without metamorphosis remained intact within 24 hours until the maximum concentration tested was 11,100 μg l-1 TPAH. After the addition of CCA extract to induce metamorphosis and an additional 18 hours of development, 54% of larvae exposed to ~5,600 μg l-1 TPAH exhibited abnormal development (Figure 2a), characterized by partial metamorphosis without attachment in the water column (Figure. 1b). For coral larvae exposed to ≥3,900 μg l-1 TPAH, abnormal development of similar polyps in water bodies was observed (Figure 1b). Some of the abnormally floating polyps were separated and kept in uncontaminated seawater for another 48 hours, but did not show any attempts to attach or deform.

After 24 hours of treatment in uncontaminated seawater, 82% and 90% of the control sponge larvae were able to successfully attach and metamorphose (Figure 2c, d, Table 3). Sponge larvae are much less sensitive to condensing WAF within 24 hours than corals, and are not affected by condensing WAF until the concentration exceeds 11,000 μg l-1 TPAH (Table 3). Above this concentration, the larvae are either spherical or deformed, with mucus-like filaments falling off the surface. Successful metamorphosis was not observed in any larvae above 11,000 μg l-1 TPAH (Figure 2c, d) and the common exposure of sponge larvae to ultraviolet light had no additional effect on metamorphosis.

The four main aromatic components of the condensed water WAF, benzene, toluene, p-xylene and naphthalene, individually inhibited the sedimentation and metamorphosis of coral larvae after 24 hours of exposure (Figure 3). Fitting the inhibition percentage data to the logistic equation can calculate the IC50, indicating the relative order of toxicity: naphthalene> xylene> toluene> benzene (Table 3). In each case, abnormal development of polyps (floating and unattached, see Figure 1c) was observed at high concentrations (1,000-10,000 μg l-1, Figure 3), and total mortality was observed at higher concentrations.

In the absence and presence of ultraviolet light, the measured toxicity of the condensate treatment to coral larvae is estimated to be 39 and 93 times higher than the predicted toxicity by adding up the contribution of each measured component to the total toxicity. This estimate is obtained by summing the comprehensive toxicity of the tested components, including the assumed toxicity of the other main components m o-xylene (= p-xylene) and C1-alkylnaphthalene (= naphthalene) in WAF, The result is that the predicted toxicity of 100% WAF is 0.85 Toxicity Units (TU).

The close spatial connection between the coral reefs, river banks and shallows of Australia's northwestern continental shelf (Timor Sea) and offshore oil and gas exploration and production has brought many unique development and protection challenges. This is the first study to study the effect of the water holding part of the NW shelf condensate gas on the formation of corals and sponges in ecologically important habitats. It also represents one of the very few studies on coral hydrocarbon ecotoxicology, which incorporates important ecotoxicological principles, such as the generation of concentration-response curves (used to calculate IC10 and IC50 values) and measuring the concentration of 30, which will Allows to compare the types of toxic oil spill risk assessments between species, pollutants, and future research25.

After short-term exposure, the susceptibility of coral and sponge larvae to condensed water WAF varies greatly. Coral larvae are more sensitive, and the lowest concentration that inhibits metamorphosis (IC10) is similar to the concentration measured in groundwater after the Deepwater Horizon leak26,27. Sponge larvae are about 50 times less sensitive than coral larvae, but exhibit susceptibility comparable to other tropical/subtropical adult species (fish, shrimp), sea urchin larvae, and several temperate species28 that are exposed to the NW shelf Light crude oil (and WAF condensate tested here).

The comparison of the effects of hydrocarbons on corals and sponges hinders differences between studies due to differences in the composition of the tested oil, the protocol used for WAF preparation, the exposure regime and duration of the experiment, and the species tested and life history stage. The comparison is also challenging because most oil exposure tests performed with corals and sponges report nominal concentrations, and the individual components of the oil have not been measured (Table 1). Despite these limitations, some urgent generalizations are (i) the coral metamorphosis analysis described here shows an impact concentration similar to the previously reported effects of crude oil and production formation water on coral larvae 30, 31 and (ii) coral larval metamorphosis It seems to be increasingly becoming one of the life stages in which corals are more sensitive to hydrocarbon exposure. For example, the inhibition of metamorphosis of Acropora millepora larvae is twice as sensitive to crude oil WAF31 as fertilization, and the IC50 and LOEC of most reported WAF concentrations (Tables 1 and 3) for metamorphosis inhibition are one lower than the LC50 of coral larval mortality. Order of magnitude. Peters and colleagues33 reported that adult corals can survive for 12 weeks at a WAF total hydrocarbon concentration as high as 2,800 μg l−1, compared to short-term (48 hours) exposure to commercial lubricants. ​​WAF can cause adult corals to die at the following concentrations as low as 190 μg l−1 THC34. The metamorphosis of sponge C. crambe larvae is inhibited by the PAH mixture, and its concentration is much lower than the TPAH concentration (0.5 μg l-1) reported here for corals and sponges35. The apparent high sensitivity of C. crambe may be due to species-specific reactions or due to a series of methodological factors, including differences in pollutant components, especially PAH components that are more toxic.

The general toxicity pattern of hydrocarbons (BTEX and PAH) to animals is considered to be non-specific anesthesia36; however, the high sensitivity detected in the coral larval metamorphosis test indicates a more specific destruction of this critical life history transition. The larvae of many coral species, including A. tenuis and A. millepora, do not undergo metamorphosis without external chemical signals (cues), and the external chemical signals (cues) mainly come from the hard shell coral algae 37,38. The components of WAF may affect one of many key processes, from clue recognition to subsequent signal transduction or the regulation of genes and biochemistry that control metamorphic processes. The observation of some larvae undergoing partial metamorphosis (changing their body plan) without attachment indicates that the early process of this life cycle transition has been affected 39.

Exposure to ultraviolet (280–400 nm) light increased the sensitivity of coral larvae (but not sponge larvae) to condensed water WAF by approximately 43%. Ultraviolet radiation can help the degradation of toxic PAHs (photolysis), but it can also significantly enhance the toxicity of PAHs through the formation of oxygen free radicals (photoactivation)29. Previous studies have shown that polycyclic aromatic hydrocarbons have increased toxicity to marine invertebrates by 12-50,000 times. Why the impact on coral larvae is not so obvious, but it may be due to the low concentration of PAH in the tested WAF, which is mainly 2-ring naphthalene, whose photoactivation is much lower than many 3-ring and 4-ring polycyclic aromatic hydrocarbons29. It is believed that BTEX compounds will not be activated by UV29 light. Obviously, UV exposure is another important consideration. Future research needs to consider the toxicity of petroleum to tropical organisms in shallow and clear water environments.

The necrotic toxicity of BTEX and PAH can be based on their octanol/water partition coefficient log Kow (most water-soluble benzene = 1.94, toluene = 2.48, p-xylene = 3.05 and the least water-soluble naphthalene = 3.26) 36,41 predict. A strong negative linear correlation was observed between the log Kows of the individual pollutants tested here and their respective IC50s (r2 = 0.83, p = 0.089), indicating that similar modes of action and toxicity are close to the predicted anesthetic effects28, 41. However, the sensitivity of coral larvae to condensed water WAF is much higher than the main individual components of condensed water WAF (IC50s 2,000–80,000 μg l-1, Table 3). The difference between the measured and expected condensate toxicity is confirmed by adding the potency × concentration of each WAF component and calculating the Σi (Ci / IC50,i) = 0.85 TU of 100% WAF. The condensate WAF is then calculated to inhibit 50% of the metamorphosis at 0.026 TU, indicating that the observed efficacy is about 40 times higher than predicted (50% inhibition should occur at 1 TU25). Although the anesthetic effects of BTEX and PAH components are generally considered to be additive, non-additive effects including synergistic effects are often observed during the developmental stage of the organism. Undetected or unreported trace components in the WAF analysis (such as traces of 3-ring polycyclic aromatic hydrocarbons or hydrogen sulfide) may also be responsible for the destruction of the specific development process mentioned above.

The condensate used in this experiment is part of the refined components that may be found in light crude oil blowouts or leaks. If unrefined hydrocarbons leak from underground wells or the surface into water, are exposed to weathering, or if oil spill control agents are used to disperse hydrocarbons into the water column, their composition and toxicity will be different43. In this study, we used a 24-hour exposure, which may not have enough time to maximize the absorption of toxic ingredients into the young larvae. However, compared to the standardized 48 or 96 hours of chronic exposure usually recommended for testing early life-stage toxicity, 24 hours may represent the duration of environmentally relevant exposure to the highly volatile aromatics associated with light crude oil44.

Corals and sponges have important ecological significance. The habitats form tropical coral reef taxa. Exposure to uncontrolled release of hydrocarbons will adversely affect reproduction and replenishment, which will have a considerable long-term impact on coral reef maintenance or reef restoration after disturbance. Influence. In order to perform more environmentally relevant tests on the WAF of condensate from Australia’s northwestern shelf (ie using tropical rather than temperate species), we describe the effects on larval metamorphosis at environmentally relevant concentrations26,27, indicating that after exposure to ultraviolet light The toxicity is enhanced, and it proves that coral larvae are more sensitive to condensed water WAF toxicity than sponge larvae. Further testing of a series of related tropical taxa using the methods defined here will be able to generate species sensitivity distributions, which will enable a more comprehensive prediction and management of the risk of oil spills in tropical coral reef systems.

Stable condensate is typically condensate produced by bringing natural gas hydrocarbons from the browse basin of Australia's northwestern continental shelf to room temperature and atmospheric pressure. This type of stable condensate (also called natural gasoline or distillate) has similar behavior and composition to Type I light crude oil (Table S1). Samples were obtained in sealed steel drums and stored at room temperature until use. The density of the condensate is 0.75 g ml-1 and the viscosity is ~0.65 cP. Benzene, toluene, p-xylene and naphthalene were purchased from Sigma-Aldrich, and the purity was ≥99%.

According to recommendations 45,46, use fresh condensate, benzene, toluene, p-xylene or naphthalene to prepare the water contained fraction (WAF). In short, 800 ml of 0.45 μm filtered sea water (36 PSU, pH 8.1) was added to a solvent-washed Il glass suction bottle and mixed with a magnetic stirrer to generate a 20-25% vortex. Add condensate or pure aromatics to the center of the vortex at a ratio of 1:100 (8 ml), loosen the aspirator cap, and mix the fluid for 18 hours in the dark. No signs of emulsion or bubble formation were observed after 10 minutes of settling, so the WAF was allowed to settle for 1 hour, and then the water was immediately sampled for analysis and used for toxicity testing. The eight dilutions of 100% WAF were prepared using the same 0.45 μm filtered seawater to simulate the dilution in the water column47 instead of CROSERF's variable loading technology, which is designed to simulate the absorption of oil slick from the surface45. The serial dilution method was chosen to keep the proportions of each component in the ten treatment levels consistent, and to be able to test more treatment levels to improve the concentration-response model. The National Research Council considers both methods to be effective48.

Hydrocarbon analysis was performed at ChemCentre (Perth, Western Australia). BTEX analysis is based on USEPA Method 8260. In Selective Ion Monitoring (SIM) mode, use purge and trap (P&T) GC-MS to analyze WAF samples directly from sealed vials. The condensate was diluted in dichloromethane (DCM) and analyzed by P&T GC-MS in scan mode. Internal standards (chlorobenzene-d5, 2-fluorobenzene and 1,4-dichlorobenzene-d4) were added via the P&T system immediately before analysis. Each sample batch was run with a method blank and spiked control (deionized water with a known amount of BTEX added). Non-quantitative whole oil analysis was performed using GC-MS directly injected with 1 μL of undiluted condensate. The pre-characterized crude oil is used as a reference for hydrocarbon identification.

The analysis of PAH is based on USEPA Method 8270. The WAF sample (500 ml) was extracted 3 times with DCM, the combined extracts (80 ml) were dried with sodium sulfate, and an 8 ml aliquot was concentrated to 1 ml under nitrogen. Analyze the concentrated WAF extract and condensate (diluted in DCM) using GC-MS in scan mode. Before extraction, substitute standards (2-fluorobiphenyl, nitrobenzene-d5 and p-terphenyl-d14) were added to WAF samples, and internal standards (naphthalene-d8, acenaphthene-d10, phenanthrene-d10, Pyrene-d12 and perylene-d12) were added to the extract and diluted condensate before analysis. Run a method blank and spike control (deionized water containing known amounts of acenaphthene and pyrene) for each sample batch.

In November 2014, from the trunk reef on the central shelf of the Great Barrier Reef (GBR, 18.329°S, 146.846°E). The gestational colonies were transported to the National Ocean Simulator (SeaSim) of AIMS in Townsville and placed in a flow cell at approximately 28°C until spawning. As mentioned earlier, gametes were collected from 6 parent colonies, and fertilized and symbiont-free larvae were cultured in a flow tank with fewer than 500 larvae 1-1. A. millepora larvae reach their maximum colonization capacity after six days and maintain their capacity for more than one month (reviewed by Jones et al. 201549). Therefore, to ensure consistent sedimentation and metamorphosis, 7 to 13-day-old larvae were used in separate exposure experiments. Rhopaloeides odorabile (Thompson, Murphy, Bergquist & Evans, 1987) is a common, viviparous, gonorrhea, double-angled sponge (Demospongiiae), which incubates parenchyma larvae at 5-6 months of 50 months in the summer of GBR Released each year during the week, 51. On January 11, 2015, 10 complete female sponges were collected from Davis Reef (18.843°S, 147.627°E) in the middle of the Australian GBR and shipped to SeaSim of AIMS. The sponge is kept in a flow-through aquarium, which allows the controlled collection of larvae within a few hours during the afternoon release. According to the established method, use larval traps to collect brooding larvae. Since R. odorabile larvae reached their maximum sedimentation capacity after 1-2 d52, the 24-hour-old larvae were exposed to the experiment.

Coral and sponge larvae were respectively exposed to WAF of stable condensate water, while coral larvae were additionally exposed to WAF of pure aromatics. Static exposure was carried out in a 7 ml glass vial containing 8-10 coral larvae or 20-30 sponge larvae. These sponge larvae were treated with 10 WAF diluents (100%, 50%, 25%, 12.5%, 6.25%, 6.25%). 3.125%, 1.6%, 0.8%, 0.4%, 0% WAF), using six replicate vials for each WAF concentration. The vial is sealed with a cap, and about 0.5 ml of headspace allows oxygen exchange (exposure> 7.5 mg l-1 in 24 hours) and prevents the larvae from settling, which is often observed in experiments without headspace.

Transfer the vial to an incubator/shaker and incubate at a light level of 40 μmol quantum m-2 s-1 for a 12:12 hour L:D cycle to maintain gentle water movement. After 24 hours of exposure, the vial was taken out, and the larvae and WAF in a single vial were directly transferred to a single 6-well cell culture plate (12 ml, Nunc, NY, USA). Before the sponge sedimentation measurement, the culture plate was immersed in the water flowing through the aquarium for 48 hours to form the microbial biofilm 53 required for successful sedimentation. To test the potential UV activation of the toxicity of PAHs, a series of condensed water WAF of coral and sponge larvae were exposed to full sunlight and immersed in a water bath at 28°C for 2 hours (UVA UVB ranges between 4.5 and 6.8 mW cm -2 Use Solartech ultraviolet radiometer during this period).

For coral larvae, sedimentation and metamorphosis were initiated by adding a slightly sub-optimal (to maximize the sensitivity of the assay) concentration (10 μl) of Coral Coralella extract 54 prepared using 4 g of Coral Coralella Porolithon onkodes37. The metamorphosis is evaluated after 18 hours. If the larva changes from a free-swimming or freely attached pear-shaped form to a squatting, firmly attached disc-shaped structure, the oral-oral axis is significantly flattened, and the septal mesentery is present. Is normal. After 37. 48 hours of radiation from the central mouth area, the metamorphosis of the sponge is evaluated. If the larva is firmly attached to the surface and undergoes body flattening to form a disc-like shape, the center shows the residue of the posterior larval pole, then it is scored as normal.

Deformation inhibition (% inhibition relative to 0% WAF control) is calculated from the treatment data as inhibition (%) = 100 × [(% deformation control-% deformation treatment)/% deformation control]. Since larval toxicity is most likely due to BTEX and PAH25,44, a concentration-response curve was generated based on total petroleum aromatics (TPAH). In the context of this study, TPAH was defined as total BTEX and PAHs (ΣBTEX ΣPAHs). The concentration of TPAH (IC10 and IC50) that inhibited 10% and 50% metamorphosis was calculated from a concentration-response curve (four-parameter logistic model), which was fitted with GraphPad Prism program to fit the inhibition percentage and total aromatics data of each treatment (v6 , San Diego, USA). The model is limited between 0% and 100%, all curves are tested for residual normality, and repeated tests are applied to evaluate the goodness of fit. By applying the F test in Graph Pad Prism v6 to test the statistically different probability of IC50 values ​​generated by the logic curve. When p <0.05, IC50 is considered different. A one-way analysis of variance (ANOVA) was performed to determine the treatment that resulted in a significant (p <0.05) suppression of the metamorphosis compared to the control treatment (NCSS v9, Utah, USA).

The toxicity of aromatic hydrocarbons is considered as an additive 44. By combining the concentration and toxicity of individual compounds, the overall toxicity of the mixture can be predicted. The experimental toxicity is combined with the measured concentrations of the four main components of WAF (benzene, toluene, p-xylene, and naphthalene) to determine the contribution of these components to the measured toxicity of the condensate to coral larvae. This comparison is made by adding the concentration of each ingredient to its toxicity ratio TU = Σi (Ci/IC50,i), where TU = toxicity unit, Ci = 100% Concentration of each ingredient in WAF = Ci And calculate the IC50 from the concentration response curve. If the total TU of the mixture is 1 or more, the effect on metamorphosis is expected to reach 50% or more25.

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We thank ChemCentre Perth WA for the technical advice and the staff of the AIMS National Ocean Simulator for their expertise and help. Thanks to Jan Tebben for preparing the hard shell coral algae extract. New South Wales is funded by the Australian Research Council Future Scholarship FT120100480. The funder has no role in data analysis, publication decision or manuscript preparation.

Australian Institute of Marine Science, Townsville, 4810, Queensland, Perth, Western Australia, 6009, Australia

Andrew P. Negri, Diane L. Brinkman, Florita Flores, Emmanuelle S. Botté, Ross J. Jones, and Nicole S. Webster

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APN, NSW and DLB designed and conducted experiments. FF and EB also conducted experiments. APN, NSW, DLB, RJJ and FF analyzed the data. APN, NSW, and DLB wrote the manuscript, and all authors (FF, ESB, and RJJ) commented on the manuscript.

The author declares that there are no competing economic interests.

This work has been licensed under the Creative Commons Attribution 4.0 International License Agreement. The images or other third-party materials in this article are included in the Creative Commons license of the article, unless otherwise stated in the credit line; if the material is not included under the Creative Commons license, the user will need permission from the license holder to copy The material. To view a copy of this license, please visit http://creativecommons.org/licenses/by/4.0/

Negri, A., Brinkman, D., Flores, F. etc. The acute ecotoxicology of natural hydrocarbon condensate to coral reef larvae. Scientific Report 6, 21153 (2016). https://doi.org/10.1038/srep21153

DOI: https://doi.org/10.1038/srep21153

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