Bioassays with Daphnia

The freshwater micro-crustacean Daphnia magna is the most often used organism for the standardised methods OECD 202 [45] and DIN 38412-30 [50]. For a short-term test, five animals of a defined physiological state are added to about 25 cm of an aqueous sample or its dilutions. The survival of the Daphnia is observed over a period of 48 hours and compared with the survivors of the control test. For a long-term test the same set up is used but the animals are fed with algae and exposed over a period of up to four weeks. The number of young Daphnia (due to reproduction) is counted and compared with those of the control test. Defined concentrations of potassium chromate are used as a positive reference to validate the required sensitivity of the animals. 

Currently no research results are available for systematic investigations concerning specific influences of typical soluble substances in compost samples. Looking at the results from our own research [64] it is to be expected, that dissolved humic substances in high concentrations may inhibit Daphnia in a short-term test. Since those elutriates have contained remarkable concentrations of some heavy metals too, no generally valid interpretation could be given. 

7 Evaiuation of Bioassay Resuits Obtained from Sampies of Compiex Composition 

Most of the standardised ecotoxicity methods do recommend the use of a synthetic or other well-known and fully defined control. That control is needed to detect the behaviour (growth, survival) of the test organisms in the absence of toxic or harmful agents. Those results are the reference values, defined as zero percent inhibition or 100% vitality and are the basis for the calculation of inhibition from the samples. 

Complex samples, such as compost, soil and sediment could either inhibit or support the growth or reproduction rate of the test organisms compared to a synthetic control. The 

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We completed acute bioassays with Daphnia pulex for 35 representative and related chemicals from the following top-ranked classes for which available toxicity data were deemed inadequate to evaluate the hazard of the classes (National Fisheries Center - Great Lakes 1986 Perry 1985) polyaromatic hydrocarbons (PAHs), alkyl halides, mono- and polycyclic alkanes, heterocyclic nitrogen compounds, other nitrogen-containing compounds, and silicon-containing compounds (alkylether silanes). The acute bioassays with Daphnia pulex followed the methods of National Fisheries Center - Great Lakes (1986) with the temperature at 20° C. 

The use of bioassays in environmental monitoring has not been developed in Chile [191]. In 1998 the Ministry of Agriculture started to set up a bioassay laboratory for evaluation of the presence of toxic substances in water for irrigation and animal consumption. This ministry is now in the process of implementation of EPA standardized crustacean and algal tests with Daphnia and Selenastrum capricomutum, respectively. There is no governmental wastewater bioassay monitoring. 

Invertebrate species have been widely used in toxicity studies of pesticides [61]. Zooplankton play a key role in the food chain because they occupy a central position. Therefore, their responses to natural and anthropogenic stresses are intimately linked with other food predator organisms. The most widely accepted bioassays employ species such as Ceriodaphnia dubia, Daphnia magna, Artemia salina, or Thamnocephalus platyurus [62-64]. D. magna has been used for many years as a standard aquatic test species and formally endorsed by the major international organizations such as the EEC, OECD, and ASTM [65-67]. Its choice is mainly because it represents the zooplankton community and is a species of worldwide occurrence. In addition, it has a greater sensitivity to toxicants, particularly pesticides, compared with other aquatic species [61,68] (Table 1). 

Standardized ecotoxicity tests (bioassays) have been developed and optimized over the last few years and encompass the effects on bacteria, daphnia and fish (DIN 38 412, parts 30, 31 and 34). These tests are designed to assess the toxicity on aquatic organisms. They are quick to perform, easy to handle and comparatively inexpensive, with the goal of allowing the toxicity of a complex water matrix to be estimated. However, they use pre-concentration steps so that it is possible that not all byproducts are recovered (which itself is hard to prove). 

The pT-index scale appraises the relative hazard of aquatic environmental samples by assigning them to a numerical class. As bioassays within a test battery are considered equal in rank, the most sensitive test with its pTmax-value defines the toxicity class of the test material. If, for example, an effluent yields pT-values of 7, 2, 8 and 0 for the bacterial, algal, daphnid and fish tests, respectively, its pT-index is therefore assigned to toxicity class 8, based on the most sensitive response obtained with the Daphnia test. Since, by convention, sample toxicity classes are designated by Roman numerals, the test material is then assigned to toxicity class Vm. A toxicity class is not a strictly defined value, but rather the consequence of the... 

Two bioassays a) 48h plate incorporation AMES test with Salmonella typhimurium his- (TA 98, 100, 1535, 1537 et 1538) b) 24h acute crustacean test (Daphnia magna) MSW landfill leachates Ashes and slags from MSW incinerator leachates Centrifuged and 0.45 pm filtered only for AMES test... 

Four bioassays a) 30 min acute bacterial test (Vibrio fischeri) b) 72h algal test (Pseudokirchneriella subcapitata) c) Acute (24h) and chronic (28d) crustacean test (Daphnia magna) d) 48h plate incorporation AMES test (Salmonella typhimurium his-with TA 97a, 98, 100, 102) Industrial solid waste leachates Batch leaching test with demineralized water followed by paper filtration (crude leachate), Liquid/liquid extraction (organic extract), lyophilization (lyophilized extract. pH adjusted according to tolerance of organisms and 0.22 pm filtration for AMES test... 

The Microtox test system utilizes a strain of naturally occurring luminescent bacteria - Vibrio fischeri. Exposure to a toxic substance causes a disruption of the respiratory process of the bacteria resulting in reduced light output. The effective concentration (EC50) is determined as the concentration of a toxicant that causes a 50% reduction in light output over a prescribed period of time (typically 5, 15, or 30 min). The test is fast, fairly simple to conduct, uses small sample sizes, and is relatively inexpensive. Results correlate well with those from other toxicity bioassays such as fish and Daphnia. The test is used... 

Different types of organisms such as daphnia, mussels, algae, and fish have been extensively incorporated in toxicity tests for water assessment systems [65], Most of these assays are developed as test systems with few as laboratory-based sensor systems. Membranes with their active enzyme system have also been implemented in the development of toxicity kits and sensors. An example is the MitoScan Kit (Harvard BioScience, Inc., Holliston, MA), which uses fragmented inner mitochondrial membrane vesicles isolated from beef heart (EPA, 2005 [9]). The submito-chondrial particles contain complexes of enzymes responsible for electron transport and oxidative phosphorylation. When specific toxins are in the sample, the enzyme reactions are slowed or inhibited, and these are monitored spectophotometrically at 340 mn. This is still in a bioassay test kit format but may be developed to optical sensor system. 

Step 4 Compost analysis for conventional parameters determining any quality change compared with the blind and ecotoxicity test using at least two plant species if negative influences are detected additional bioassays should be performed using more plant species, daphnia, water lens, luminescent bacteria or special local species. 

Step 4 Determination of ecotoxic effects using bioassays compatible with the degradation environment, e.g., 3 plant species for soil applications, or daphnia and... 

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