- Laboratory experiments
- Field experiments
- Assessment system (for soil toxicity test)
1. Laboratory experiments
1.1. Acute toxicity
- Endpoints: Mortality.
- Evaluations: LC50, LD50.
1.2. Chlonic toxicity
1.2.1. Behavioral endpoints
- Ingestion rate, respiration rate, reworking activity, swimming activity, sediment avoidance, and et al.
[Notes] There are advantages to using behavior as an endpoint for assessing aquatic toxicity. Behavioral responses are usually very rapid, occurring in minutes rather than days as for traditional endpoints. Advances in video microscopy and computer interfacing have made automated data collection for behavioral endpoints feasible and quite cost-effective when compared to traditional toxicity tests. The disadvantages of behavioral endpoints are that it is often unclear whether the effect is truly adverse. (ref. ID; 1560)
- Evaluation: EC50, IC50, ICG50, NOEC, and et al.
- Abundance of stress protein (electrophoretic separation-Western blotting, mRBA), esterase enzyme activity, and et al.
[Notes] Several approaches have been taken with aquatic animals and two have yielded promising results. The first characterizes the abundance of stress proteins and the second quantifies in vivo enzyme activity. The stress response is a highly conserved alteration in gene expression associated with a variety of stressors (Sanders 1993). (ref. ID; 1560)
The term biomarker was originally defined as "a detectable biochemical or physiological alteration or cellular manifestation brought about by environmental stress". (ref. ID; 4938)
- Evaluation: NOEC, EC50.
The classical biosensor concept involves the existence of two components; a bioreceptor (biological material) and a physicochemical transducer. The bioreceptor might be a biomolecule or a whole cell that recognises the target (heavy metal), whereas the transducer converts the recognition event into a measurable signal.
A whole-cell biosensor uses the whole prokaryotic or eukaryotic cell as a single reporter incorporating both bioreceptor and transducer elements. Two types of bioassays using whole-cell biosensors may be considered: "turn off" and "turn on" assays.
- "Turn off" assay: The sample toxicity is estimated from the degree of inhibition of a cellular activity that is a normally continuous (e.g. inhibition of growth, respiration or metabolism, motility or the biosynthesis of a specific molecule), and is based on the measurement of a decrease in growth rate, light (fluorescence/bioluminescence) emission, colour-less cell population, mortality, etc. as a function of sample toxic concentration.
- "Turn on" assay: A quantifiable molecular reporter is fused to a specific gene promoter (like metallothionein promoter), known to be activated by the target chemical or environmental pollutant (such as a heavy metal). (ref. ID; 4938)
Metabolomics is the study of the complete set of metabolites/low molecular weight intermediates, which are context dependent, varying according to the physiology, developmetal or pathological state of the cell, tissue, organ or organism (Oliver 2002). This approach has proven to be highly sensitive for the detection of effects associated with both drugs and environmental toxins/toxicants, in that metabolic perturbations often present much earlier than pollutant induced histopathological changes (Griffin et al. 2000). For many years a major analytical method for metabolomic studies has been nuclear magnetic resonance (NMR) spectroscopy. This has a number of advantages in that it requires minimal sample preparation and is fast and a robust technique, which allows a wide range of small molecule metabolite to be measured simultaenously. Its major disadvantage is a lack of sensitivity. For this reason many metabolomic studies also additional analytical techniques such as use gas chromatography mass spectrometry (GC-MS). This has the advantage of greatly enhanced sensitivity compared to NMR, but the disadvantages of increased sample preparation time and the fact that some large and/or very polar metabolites, such as some hormones, cannot be analysed. (ref. ID; 6708)
1.2.5. Reproduction endpoints
- Growth rate, intrinsitic population growth rate (r), fecundity (F), life table, carrying capacity (K), net reproduction (Ro), life expectancy, generation time (T), reproductive value (Vx), residual reproductive value (V*x), and et al.
- Evaluation: NOEC, EC50.
Microcosm & Mesocosm
Abundance, biomass, productivity, species diversity, and et al.
Integrated soil microcosm (ISM) test protocol (ref. ID; 4959)
Microcosms, set up in a greenhouse, consisted of cylinders made from high-density polyethylene pipe, 7.5 cm (i.d.) x 15 cm high. A fine nylon mesh was placed across the bottom of each microcosm for leachate collection. Field soil, (silty clay loam), collected from Florsheim, Germany, was sieved through a 5 mm screen and mixed thoroughly. Earthworms, enchytraeids, and microarthropods were added to each microcosm. Each microcosm contained five wheat seedlings, and was maintained at a 12-12 hr light-dark cycle. Artificial rainwater was used to water microcosms as required.
Permeable Pavement Structures (PPS) (ref. ID; 4941)
PPS are a form of urban paving designed to simulate the drainage characteristics of soil. The system comprises, from the surface down, porous block, a gravel bedding layer, a permeable geotextile membrane and a granite sub-base. Their environmental benefits include flood prevention through increased infiltration capacity, retention of urban-derived pollution such as heavy metals or hydrocarbons and a source of 'grey water' for reuse.
Two mesocosms 113 mm x 113 mm in area and 270 mm deep, which were small-scale replicas of field-based PPS made using standard air-dried components encased in sealed glass tanks with steel supports.
Artificial rainfall, in the form of distilled water, was applied to the pavement surface at 1.6 mm hr-1, which is approximately the average rate at which rain water falls on pavements during rainfall in lowland England.
- Run 1: By the indigenous community of micro-organisms derived from resting stages on the construction materials.
- Run 2: Inoculated with 30 ml of the commercial oil-degrading microbial suspensions Biotreat HD (Bio-industries Ltd, Dublin, Lreland), containing a mixture of gram-positive and gram-negative bacteria having a concentration of 10E7 cells ml-1.
Oil was applied to the pavement surface at a rate of 0.04 g hr-1 using a purpose built dripper to mimic the leaking of a vehicle engine.
Nutrient application was made by placing 2.20 g of Osmocote Plus (Scotts Europe B.V., The Netherlands) slow release plant fertiliser onto the mesocosm surface and allowing it to dissolve gradually in the applied rainwater.
Effluent was collected from beneath the mesocosms in sterile glass bottles each week for a period of 6 weeks.
Total direct counts were made under phase contrast microscopy using a haemocytometer with Thoma chamber.
Terrestrial Model Ecosystem (TME) (ref. ID; 4960)
The principle of the (specific) TME is to excise soil cores (17.5 cm diameter, 40 cm depth), including plants, animals and microbes present, from an uncontaminated natural or managed site and to place them under controlled conditions in the laboratory. After an acclimatisation period (2-4 weeks) typically 4-8 replicates are treated with increasing amounts of chemical or left untreated as controls to investigate concentration-response relationships for various endpoints. TMEs are sampled at intervals of 4-8 weeks for structural (e.g. plant biomass, animal abundance) or functional (e.g. litter breakdown, microbial activity, nutrient cycling) parameters. Test duration from application to end of experiment is normally up to 16 weeks, but can be extended. Thus the TME, by design, is a complex system developed to obtain ecotoxicological data on higher levels of biological organization (population, community and ecosystem). In addition chemical fate data can be obtained. The basic method was developed in an earlier project (Knacker et al., 1990, 1991), with the suggestion of a subsequent workshop (Knacker and Morgan 1994) to use the TME for level 2 in the notification of new substances and for plant protection products' registration.