Donat-P. Häder

Daphniatox – Online monitoring of aquatic pollution and toxic substances

The microcrustacean Daphnia is sensitive to many toxic substances and can be cultured easily. The Daphniatox instrument is based on computerized image analysis tracking swimming organisms in real time. The software evaluates 14 endpoints including motility, swimming velocity, orientation with respect to light and gravity as well as cell form and size. The system determines movement vectors of a large number of organisms to warrant high statistical significance and calculates mean values as well as standard deviation. Tests with K dichromate show that the toxin inhibits motility (EC50 0.75xa0mg/L), swimming velocity (EC50 0.70xa0mg/L) and even causes a significant decrease in length (16% at 4xa0mg/L) and changes the form of the animals, This bioassay can be used to monitor the toxicity of a large number of dissolved pollutants and toxic substances such as arsenic, dichromate and persistent organic pollutants.

18 – Ecotoxicological monitoring of wastewater

Abstract Many bioassays have been used to monitor toxicity of wastewater from households and industry. The efficiency of wastewater treatment plants can be determined by taking samples from the influx and after each treatment step (physical, biological, chemical), as well as from the output of the plant. Measurements using ECOTOX have shown a significant decrease in toxicity after each step. Industrial wastewaters are usually pretreated before being transferred to a municipal wastewater treatment plant. Toxicities of several industries such as a producer of catalytic car converters, a special wastewater treatment plant, a textile factory, an industrial services plant, a technical paper plant, and a car recycling plant have been determined using their effects on the motility, orientation, and form parameters of Euglena gracilis. A number of comparisons have been carried out between the ECOTOX, the Lemna bioassay, and the cress germination test using pollutants from various industrial oils, anonymized samples obtained from a company determining toxicity of wastewater, and copper granulate.

7 – Toxicity testing using the marine macroalga Ulva pertusa: Method development and application

Abstract Ulva pertusa Kjellman is reported from the Mediterranean, Pacific, and Indian Ocean. Ulva spp. are important representatives of macroalgal communities in coastal waters that are under threat from the frequent inundations of toxic waste derived from industrial and municipal sources. These algae are primary producers, constituting the basis of the coastal ecosystem and, moreover, providing shelter and habitats for other marine organisms. They are also important natural sources of food and pharmaceutical products. Reproduction is a critical process by which populations perpetuate, and disturbance of this process can cause failure in recruitment, leading to the disappearance of the population and ultimately to modifications in community structure and dynamics. Members of Ulva have a wide range of geographic distributions and undergo similar reproductive processes. Therefore, the test method presented in this standard has the potential for application to other species belonging to the same genus reported from coastal waters in most parts of the world. The proposed macroalgal toxicity test provides many practical advantages over other currently employed techniques. No specialist expertise is required. The test is cost- and time-effective, since it only requires a cell plate and a small volume of water and takes a total of approximately 3xa0h to conduct around a 96xa0h incubation period. The sensitivity of the Ulva method is similar to—and in many cases better than—commonly available or well-established bioassay methods, and since reproduction is the means by which population recruitment is facilitated, the measured endpoint being ecologically significant cannot be disputed. In addition, field-collected samples of unialgal Ulva plants can easily be held and acclimated in a holding tank for 1 to 2 month(s), and artificial induction of reproduction in a laboratory is easily achieved using vegetative thalli, allowing year-round testing to be performed. The Ulva reproduction bioassay would be a valuable addition to the existing suite of tests routinely used to monitor coastal and estuarine ecosystems. The Ulva test can also be applicable to the TIE/TRE procedures for wastewaters. The TIE/TRE technique using Ulva may signify a breakthrough in wastewater management technology since no such technique has been developed so far based on the established ISO methods. The Ulva kit and automated UlvaTox have been developed to facilitate the use of the Ulva method.

Environmental monitoring using bioassays

Abstract Scientific knowledge regarding the effects of toxic compounds in aquatic ecosystems has increased over recent years. But still there is a need to establish relevant monitoring tools in order to generate an early warning signal of environmental conditions. In the aquatic ecosystems microalgae are the first step in the ecological food chain and as they are very sensitive to toxic elements they can be used as an early warning system when monitoring pollution. The most common parameters when using microalgae in bioassays are photosynthetic parameters and growth. However, there is an increasing need for monitoring tools that are fast and sensitive to a broad range of toxicants. As a complement to more common methods, swimming behavior of microalgae is a promising tool in order to get accurate and rapid analyses. Bioassay methods based on the response of living microorganisms could therefore provide important information concerning the ecological status of aquatic environments. The ECOTOX bioassay calculates the number of motile cells, percentage of cells moving upwards (gravitaxis), the mean velocity, the compactness (form factor) and the precision of orientation (r-value) (cf. Chapter 10: Ecotox, this volume). Studies testing the effects of wastewater and heavy metals on the flagellate Euglena gracilis using the ECOTOX system demonstrate that upward swimming and precision of gravitactic orientation are the most sensitive parameters. The effects show differences between short- and long-term tests but the effects from herbicides showed pronounced effects on velocity and upward swimming of E. gracilis already after 30 and 60xa0s. ECOTOX measurements from Egyptian lakes which are polluted by industrial and domestic wastewaters showed strong inhibition on upward swimming and r-value of E. gracilis. Samples taken from the wastewater treatment plant west of Alexandria showed that the toxicity is not reduced by the treatment. The results from studies using the ECOTOX system indicate that this bioassay could be very useful in the future for short- and long-term risk assessments.

5 – Image analysis for bioassays – the basics

Abstract Many modern bioassays utilize computerized automatic image analysis of color or black-and-white images and video sequences. This chapter discusses modern hardware and describes the details of image improvement by contrast enhancement, background removal, and adaptive thresholding. Mathematical filters and look-up tables are essential tools for augmentation. Binarization is used to separate objects of interest from the background. Techniques to find objects in an image and extract parameters such as position, area, and form factors are described. The next step is organism tracking by movement analysis through time to determine the percentage of motile objects, their movement velocity, directionality, and precision of orientation with respect to external stimuli such as light or gravity. Another useful tool is pattern recognition, which identifies objects according to stored images.

Historical development of bioassays

Abstract Bioassays have been developed—since long before the availability of computers—to detect water, air, and soil toxicity. While today ethical considerations prevent more and more the use of vertebrate species as bioindicators, early bioassays employed fish and rodents to a large extent. Early bioassays have also been used to indicate insecticidal and antibacterial activity such as antibiotic substances excreted by Penicillium. Since maintaining a number of cultures for different bioassays in the laboratory may be difficult, a set of microbiotests have been developed which are provided as kits which contain the bioindicator organisms as spores, seeds, or dormant eggs or cysts. These tests have also been used to achieve comparability between different bioassays and to classify toxicity. Bioassays have been used to determine human health hazards. They allow not only to determine the type of tumor but also to quantify the effects of irradiation and anticancer drugs. Cyanobacterial and algal toxins in drinking water may directly result in poisoning of humans and animals or are concentrated in invertebrates and vertebrates by bioaccumulation. Hazards from these toxins have been determined using bioassays involving rodents and larger animals.

17 – A comparison of commonly used and commercially available bioassays for aquatic ecosystems

Abstract Bioassays are common alternatives to chemical analysis for assessing toxicity and detecting potential pollutants in aquatic ecosystems. A large number of bioassays are in use for this purpose and many of them are commercially available on the market. Mostly these bioassays are not restricted to specific groups of toxins or pollutants but rather respond to heavy metals, persistent organic pollutants, organic toxic substances, pesticides, and other toxicants—however with different sensitivity to the different groups of chemicals. Sensitivity, response time, and cost are the major factors in choosing a bioassay. This chapter focuses on the comparison of the most common bioassays used in aquatic ecotoxicology in terms of their sensitivity, cost, and response time.

4 – Regulations, political and societal aspects, toxicity limits

Abstract Archeological records show that 40 centuries ago humankind took steps to obtain water appropriate for various everyday uses. Over time, desirable water quality conditions have been established with an emphasis on use for human consumption (potability), which we deem important due to the concern to preserve our species. Global economic development has increased the generation of contaminants in the air and soil, and has led consequently to the contamination of groundwater. The use of bioassay tests to monitor aquatic environments has been established for many years in a number of countries, including the United States, Canada, France, Germany, Korea, Japan, China, Brazil, and virtually all developed economies, with economies in transition and developing economies and countries. The aspect that undoubtedly deserves to be emphasized is that the legislation was motivated by the recognition of the pertinence of the topic, over time, by scientists from different countries, with different academic backgrounds, working on the protection of resources (water, air, and soil). The legal base and its forms of presentation for ecotoxicological tests vary between countries and/or associations of countries, such as the European Union. These regulations are defined by laws written by the respective congress of each country and provide the authority for the environmental control bodies to write the regulations. The regulations explain the technical, operational, and legal details needed to implement laws. This chapter demonstrates some examples of regulations, social aspects, and definitions of toxicity limits.

Bioassays for solar UV radiation

A number of bioassays have been developed to determine exposure to solar and artificial UV on Earth and in space. These dosimeters are based on several organisms such as phages, bacteria, cyanobacteria, flagellates, higher plants, and animal cell lines. The DLR biofilm using immobilized, dry spores of Bacillus subtilis is a robust, easy to use and reliable dosimeter which can be worn outside the clothes to determine reliable UV exposure of humans during outdoor activities. It has been used in many campaigns in Europe, Japan, Antarctica, and the high Arctic.