The effects of sediments contaminated with antifoulant paint on coral larvae, coral recuitment, coral recruits and adult corals from the Great Barrier Reef

On 2 November 2000, the 184 m cargo ship, Bunga Teratai Satu, ran aground on Sudbury Reef, on the Great Barrier Reef and remained grounded for 12 days. The ship was re-floated only 3 days prior to the November 2000 mass coral spawning. No cargo or fuel was lost but the impact resulted in significant contamination of the reef with antifoulant paint containing tributyltin (TBT), copper (Cu) and zinc (Zn). Sediment was collected from the grounding scar site and a control site 1 km from the grounding site in a similar habitat zone. Sediments were ground and sieved and the 1-4 mm fraction retained. Sediments from the control site were used to dilute contaminated sediment to the concentrations required for experimental treatments.Larvae were raised from gametes released by adult Acropora microphthalma, which had been collected from Pelorus Island and spawned in the outdoor aquaria at the Orpheus Island Research Station on November 17 2000. Live crustose coralline algae (Hydrolithon sp.) was collected from Pelorus Island, ground and extracted with methanol to produce a crude inducer stock, which was used to initiate attachment, settlement and metamorphosis of larvae. The influence of contaminated sediment on larval survival and competency was examined by firstly exposing groups of 200, 7 day old larvae for 24 hours to one of 4 contaminated sediment treatments (0, 5, 25 and 100%). Each 500 ml container contained 0.4 g of experimental sediment in 400 ml of filtered seawater. Larvae were then transferred to well cell culture plates (10-20 per well) containing inducer and filtered sea water. After 24 hours, counts of larvae and newly metamorphosed polyps were made using a dissecting microscope. The effect of contaminated sediment upon larval recruitment was examined by exposing previously unexposed 8 day old larvae to 0, 5, 25 and 100% contaminated sediment treatments. Approximately 660 larvae were placed in each 10 l aquaria containing a pre-conditioned terracotta tile and 6 g of experimental sediment in 6 l of filtered seawater. Tiles were raised 2cm above the sediment to allow larvae to settle on the top and bottom of the tiles. Recruitment was scored on each tile after 40 h using a dissecting microscope.The effect of contaminated sediments on previously unexposed coral recruits was examined by firstly placing 16 pre-conditioned tiles in a 500 l outdoor aquarium holding 8 day old larvae at a density of approximately 100 larvae/l. Tiles were elevated 2 cm from the bottom and left for 3 days after which the tiles were removed and the number of recruits counted. Each tile was then placed into individual 10 l aquaria containing 5 l of filtered seawater and one of four contaminated sediment treatments (0, 0.5, 5 and 25%). The number of coral recruits were counted after 38 and 72 hours.Six colonies of Acropora formosa were collected from Magnetic Island and transferred to the outdoor aquaria at the Orpheus Island Research Station. Branchlets, 4-6 cm long were cut from the colonies, mounted on cylindrical holders and acclimatised for 10 days prior to experiments. Five branchlets were randomly placed in each of twenty five 10 l aquaria. Five aquaria were exposed to each of 4 contaminated sediment treatments (0, 5, 25 and 100%) and 5 were used as controls (no sediment). Branchlets were visually inspected every 24 or 48 hours and categorised according to the degree of bleaching that had occurred. Branchlet mortality was also recorded at each census point.The maximum effective quantum yield (Fv/Fm') of each illuminated branchlet was determined using a DIVING-PAM fluorometer. Fv/Fm' ratios were recorded at 15 random locations over the surface of each coral branchlet at 3 mm distance to the coral surface. Zooxanthellae density counts were undertaken on a random subset of experimental branchlets (eight per treatment).The concentrations of TBT, DBT, MBT, Cu and Zn in sediment and water samples from the larval competency experiments and the coral recruit and branchlet toxicity experiments were measured.
This study was undertaken to test the effects of sediments contaminated with anti-foulant paint on survival, competency and recruitment of coral larvae, coral recruits and adult corals.

2000年11月2日，总长度184米的货轮"Bunga Teratai Satu"号在大堡礁（Great Barrier Reef）的萨德伯里礁（Sudbury Reef）搁浅，搁浅时长共计12天。该船于2000年11月珊瑚大规模产卵事件前3天才得以脱浅。本次事故未发生货物或燃油泄漏，但防污漆中含有的三丁基锡（tributyltin, TBT）、铜（copper, Cu）与锌（zinc, Zn）对该礁体造成了严重污染。

研究人员从搁浅疤痕区域以及距离搁浅点1公里、生境相似的对照区域采集了沉积物样本。将沉积物研磨过筛后，保留1-4 mm粒径的组分。使用对照区域的沉积物对污染沉积物进行稀释，以配制实验所需的各浓度处理组。

实验所用幼虫由成年小眼鹿角珊瑚（Acropora microphthalma）释放的配子培育获得，该成年个体采集自佩洛鲁斯岛（Pelorus Island），并于2000年11月17日在奥费斯岛研究站（Orpheus Island Research Station）的室外水族箱内完成产卵。从佩洛鲁斯岛采集鲜活的壳状珊瑚藻（crustose coralline algae），经研磨后以甲醇萃取得到粗诱导剂原液，用于启动幼虫的附着、沉降与变态过程。

为探究污染沉积物对幼虫存活与附着能力的影响，研究人员首先将200只7日龄幼虫分为4组，分别置于浓度为0%、5%、25%与100%的污染沉积物处理体系中暴露24小时。每个500 mL容器内装有400 mL过滤海水与0.4 g实验用沉积物。随后将幼虫转移至含有诱导剂与过滤海水的多孔细胞培养板（每孔接种10-20只幼虫）中。24小时后，采用体视显微镜对幼虫与新变态水螅体进行计数。

为探究污染沉积物对幼虫招募的影响，研究人员将未经过预处理的8日龄幼虫分别暴露于0%、5%、25%与100%浓度的污染沉积物处理体系中。每个10 L水族箱内装有6 L过滤海水、6 g实验用沉积物与一块预调理陶土砖，共投放约660只幼虫。将陶土砖抬升至沉积物上方2 cm处，以便幼虫在砖的上下表面均可附着。40小时后，采用体视显微镜对每块陶土砖上的幼虫招募情况进行评分。

为探究污染沉积物对未暴露过的珊瑚幼体的影响，研究人员首先将16块预调理陶土砖放入500 L室外水族箱中，该水族箱内8日龄幼虫的密度约为100只/L。将陶土砖抬升至水族箱底部上方2 cm处，放置3天后取出，计数其上的珊瑚幼体数量。随后将每块陶土砖单独放入10 L水族箱中，每个水族箱内装有5 L过滤海水，并分别加入0%、0.5%、5%与25%浓度的污染沉积物处理液。分别在38小时与72小时后计数珊瑚幼体的数量。

研究人员从马格尼特岛（Magnetic Island）采集6株美丽鹿角珊瑚（Acropora formosa），转移至奥费斯岛研究站的室外水族箱中暂养。从珊瑚株体上剪取4-6 cm长的分枝，固定于圆柱形支架上，于实验前驯化10天。将5根分枝随机放入25个10 L水族箱中，每个处理组设置5个水族箱，分别对应0%、5%、25%与100%浓度的污染沉积物处理组，另设5个水族箱作为无沉积物对照组。每24或48小时对珊瑚分枝进行目视检查，根据其白化程度进行分级，并在每次普查时点记录分枝的死亡率。

采用DIVING-PAM荧光计测定每块受光照珊瑚分枝的最大有效量子产率（Fv/Fm'）。在每块珊瑚分枝表面随机选取15个测点（距珊瑚表面3 mm）记录Fv/Fm'比值。从各处理组的实验分枝中随机选取子集（每个处理组8根）进行虫黄藻密度计数。

对幼虫附着能力实验、珊瑚幼体与分枝毒性实验中的沉积物与水样中的TBT、二丁基锡（dibutyltin, DBT）、一丁基锡（monobutyltin, MBT）、Cu与Zn浓度进行了测定。

本研究旨在检验防污漆污染沉积物对珊瑚幼虫存活、附着能力与招募，以及珊瑚幼体与成年珊瑚的影响。