'From bench to field': The development of a fish gill cell culture system for site specific water monitoring.

欧洲联盟水框架指令（WFD）于2000年12月推出，其目的是提供一个连贯的监管机制，以保护和改善我们的沃茨质量。为了实现这一目标，WFD需要一个三级监测方法：监测，其目的是发现长期趋势;操作，这将有助于对那些有可能无法达到良好生态状态的水体进行分类，以及调查，这将确定某些水体没有达到良好生态状态的原因。目前，为我们提供水质信息的监测计划包括评估生态（例如生活在水中的动植物的多样性和数量）和生境质量，其中包括测量污染物。如果调查性监测表明生态质量差，则需要进行一系列化学分析，以确定是否存在毒性升高的物质（例如金属、杀虫剂或除草剂、多氯联苯）。然后评估这些污染物的浓度，看看这是否可以解释这个问题。这些化学测量都需要单独的分析，因此可能很昂贵。鱼鳃是氧气摄取的部位，并参与离子调节。鳃经常浸泡在水中，因此总是暴露在水生污染物中。在以前的工作中，我们已经开发了一个鱼鳃细胞培养系统，这是独一无二的，因为它可以容忍淡水在外面，并具有功能，像一个完整的鳃。这项工作，在实验室中进行，已经证明，在鳃细胞培养系统中的基因表达的增加测量的毒性的终点准确地模仿整个生物体的金属毒性的反应。结果表明，该系统中的基因表达是水中有毒金属的良好指示剂。本项目的目的是测试这种鱼类原代鳃细胞培养系统在现场污染物监测中的多功能性。与传统的污染物浓度测量相比，我们的方法的重要区别在于，我们将允许生物学，鳃细胞，告诉我们是否存在生物活性化合物。这是监测程序中的一个范式转变，因为它使我们能够确定在自然沃茨中确实引起生物反应的污染物，而不是根据水化学测量结果推断影响。这个为期一年的项目将使用康沃尔郡受金属污染的河流中的沃茨水测试我们的系统。以前的工作表明，这种细胞培养系统可以区分金属和一些有机污染物，如除草剂，并且可以区分不同的金属。因此，这一系统在区分不同污染物类别方面具有很大的潜力，从而更有可能采集含有多种污染物的单一天然水样，并确定那些存在的环境污染物。这项研究的直接受益者将是那些对测量水质感兴趣的人，其中包括环境署，环境顾问和工业界。该项目将包括一个项目结束研讨会，与这些最终用户讨论我们的结果，并为未来的发展制定路线图。然而，改善水质具有更广泛的社会效益。淡水目前受到严重威胁，据估计，这些生态系统中的物种损失最大。确定环境关注的污染物可以就需要立即注意的污染物向政策制定者提出建议。由此产生的水质改善将增加这些生态系统为人类提供的服务，并产生更优质的饮用水，有利于我们的健康。

The European Union Water Framework Directive (WFD) was introduced in December 2000 and its aim is to provide a cohesive regulatory mechanism to protect and improve the quality of our waters. To achieve this the WFD requires a 3-tiered monitoring approach: surveillance, whose aim is to detect long-term trends; operational, which will help classify those water bodies that are at risk of failing to reach good ecological status and investigative, that will ascertain the reasons why a certain water body is not achieving good ecological status. The monitoring programmes that inform us of the quality of the water currently include assessing the ecology (e.g. the diversity and numbers of plants and animals living in the water), and quality of the habitat, which includes the measurement of pollutants. If investigative monitoring indicates poor ecological quality then a battery of chemical analyses are required to determine the presence of elevated toxicants (e.g. metals, pesticides or herbicides, polychlorinated biphenyls (PCBs)). The concentrations of these pollutants are then assessed to see if this can explain the problem. These chemical measurements all require separate analysis and can thus be expensive. The fish gill is the site of oxygen uptake and is involved in ion regulation. The gill is constantly bathed in water and thus is always exposed to aquatic pollutants. In previous work we have developed a fish gill cell culture system that is unique because it can tolerate freshwater on the outside and has features, and functions, like an intact gill. This work, conducted in the laboratory, has demonstrated that end-points of toxicity measured as an increase in gene expression in the gill cell culture system accurately mimics the response of the whole organism to metal toxicity. The results demonstrate that gene expression in this system is an excellent indicator of the toxic metals in the water. The aim of this project is to test the versatility of this fish primary gill cell culture system for pollutant monitoring in the field. The important difference in our approach compared to traditional pollutant concentration measurements, is that we will allow biology, the gill cells, to inform us whether there are biological active compounds present. This is a paradigm shift in monitoring procedures because it enables us to identify pollutants that definitely induce a biological response in natural waters, rather than inferring effects based on water chemistry measurements. This 1-year project will test our system with waters from metal contaminated rivers in Cornwall. Previous works has shown that this cell culture system can distinguish between metal and some organic pollutants, such as herbicides, and can distinguish between different metals. Thus, there is great potential for this system to distinguish between different groups of pollutants; bringing nearer the possibility of taking a single natural water sample with a mixture of pollutants and identifying those pollutants present that are of environmental concern. The immediate beneficiaries of this research would be those interested in measuring water quality, which include the Environment Agency, environmental consultants, and industry. The project will include an end-of-project workshop to discuss our results with these end users and develop a roadmap for future development. However, improving water quality has a far wider societal benefit. Freshwaters are currently under great threat and it is estimated that the greatest loss of species is occurring in these ecosystems. Identifying pollutants that are of environmental concern can advise policy makers on those pollutants that require immediate attention. The resulting improved water quality will increase the services that these ecosystems provide for humans, and generate better quality drinking water that will benefit our health.