Toxic cyanobacterial blooms in a shallow artificially mixed urban lake in Colorado, US

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Selenastrum capricornutum biotest

In this study we used a 96-hour growth, standard freshwater algal toxicity test with the unicellular, crescent-shaped, green alga Selenastrum capricornutum (40-60 mm3) that can be found in both eutrophic and oligotrophic freshwater environments. Methods used for culture and testing were as described by the Environmental Protection Agency (EPA)(U.S. EPA 1994). This method was originally developed for use with effluents, receiving waters, and pure compounds, but it is also suitable for testing sediment. The test was used to measure changes in cell density and chlorophyll content. Starter algae cultures were obtained from Aquatic Bio Systems Inc. Fort Collins, Colorado. Instructions for the preparation of glassware and nutrient stock solution were as detailed by the EPA (U.S. EPA 1994). Cultures were grown in a nutrient medium that include EDTA and then tested in a medium without EDTA. The cultures were grown in 250-mL glass Erlenmeyer flasks containing 100 mL culture medium. New cultures were started each week. The flasks were sealed with foam plugs to prevent contamination and placed in an incubation chamber at 25 ± 1 °C under continuous illumination (60-80 μE/m2/s) supplied by cool light fluorescent lamps. The toxicity test was initiated after the stock cultures have reached a cell density of 1 x 102 cells/mL and were entering a log-phase growth stage. The same amount of sediment pore water obtained at the different sampling sites, while sampling macroinvertebrates with a core sampler, was filtered through a 0.45 mm filter to remove particle material. The test was conducted as a screen (100 % concentration). Nutrients without EDTA were added to ensure that reduction in growth was not due to nutrient limitation. The toxicity test was conducted in 250-mL Erlenmeyer flasks, with a test volume of 100 mL and three replicates per treatment. The negative control consisted of three replicates of the culture medium, prepared without EDTA. The inoculum density was adjusted to 1 x 106 cells/mL and each flask was inoculated with 1 mL stock culture. Each of the flasks were sealed with a foam plug and placed in an incubation chamber at 25 ± 1 °C with a continuous photoperiod.
The flasks were swirled twice daily by hand and randomly repositioned in the incubation chamber. Cell density and chlorophylla (Chla) contents were measured of the triplicate samples at 24, 48, 72 and 96 hours and the mean count (cells/mL) was calculated for each treatment. Chla was extracted from lyophilized GF filters using N,N-dimethylformamide for 2 h at room temperature. Chla was measured photospectrometrically at 647 and 664 nm and calculated according to Porra et al. (1989). The following water quality measures: pH, alkalinity, hardness and temperature were taken at test initiation and termination.

Daphnia magna test

Standardized techniques involving whole sediment exposures, with acute or sublethal endpoints, have been used in the 48-hour toxicity test (American Society for testing and Materials, ASTM 2000). Test organisms Daphnia magna < 24 hour neonates were obtained from Aquatic Bio Systems Inc. Fort Collins, Colorado. They were cultured at 22 °C, under a photoperiod of 16:8 h light:darkness and a light intensity of 10 Ue/m2/s. The culture medium was deionized water, with a pH 7.99, total hardness of 250 mg CaCO3/L and total alkalinity of 96 mg CaCO3/L. A static 48-hour acute test, with whole sediment samples, was conducted as a screen (100 % concentration).
Each test container was a 250-mL glass beaker, and there were five replicates per treatment. Approximately 50 g sediment (wet weight) was placed in each beaker, and then 200 mL culture water was added, taking care not to disturb the sediments. After a settling period of 3 days as described (ASTM 2000), ten neonates were added to each container, and care was taken to release them below the water surface. For the negative control, the Daphnia were kept in pure water. Dissolved Oxygen (DO) and temperature was measured daily, while pH, ammonia, conductivity, alkalinity, and hardness were determined at test initiation and termination. After 24 h and 48 h the number of immobilized Daphnia was determined visually. Daphnia that sank to the bottom of the vessels within 15 s of having been slightly shaken were considered immobile. Data from the replicate beakers were pooled and treatments were compared to the negative control and reference sites.

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Chironomus tentans test 

The assessment of whole sediment toxicity involves a 10-day exposure of Chironomus tentans to the contaminated sediment. We conducted the toxicity test in 300-mL beakers with 8 replicate chambers. The sediment volume for the test was 100 mL with 175 mL of overlying water. Sediment of the different sampling sites were prepared the day before test initiation and allowed to equilibrate overnight following the instructions of the U.S. EPA (2000). The following day 10 second-to third-instars larvae were added per chamber under a 16:8 h light; dark photoperiod at 23 ± 1 °C.
Overlying water in the test containers were renewed, with two volume replacements per day (U.S. EPA 2000). Hardness, alkalinity, conductivity, pH and ammonia were monitored at the beginning and end of the test while temperature and DO were monitored daily. After 10 days the mean survival of larvae exposed to test samples were compared to the mean survival of larvae exposed to controls and reference samples.

Chapter 1 Introduction 
Outline of Chapters 
-Chapter 2
-Chapter 3
-Chapter 4
-Chapter 5
-Chapter 6
Chapter 2 An overview of the impact of anthropogenic activity on water quality with special reference to human health, risks and detection of toxic compounds present in water bodies
2.1. Introduction
2.2 Water quality
2.3 Urban runoff
2.4 Eutrophication
2.5 Human health risks of long-term exposure to toxic compounds
2.6 Methods for detection of toxicity as a result of chemical pollutants and biological toxic compounds in raw water
2.7 Raw water treatment processes
2.8 Conclusion
Chapter 3 Using a battery of bioassays, Benthic Phytoplankton and the AUSRIVAS method to monitor long-term coal tar contaminated sediment in the Cache la Poudre River, Colorado 
3.1 Introduction
3.2 Material and methods
3.3 Results
3.4 Discussion
3.5 Conclusion
Chapter 4 Population dynamics and ecological changes in an urban artificially mixed shallow lake Colorado, one year after restoration 
4.1 Introduction
4.2 Materials and methods
4.3 Results
4.4 Discussion
4.5 Conclusion
Chapter 5 Toxic cyanobacterial blooms in a shallow artificially mixed urban lake in Colorado, US. 
5.1 Introduction
5.2 Study site description and background
5.3 Materials and methods
5.4 Results
5.5 Discussion
5.6 Conclusion
Chapter 6 Assessment of the genetic diversity of geographical unrelated Microcystis aeruginosa strains using Amplified fragment length polymorphisms (AFLPs) 
6.1 Introduction
6.2 Material and Methods
6.3 Results 183
6.4 Discussion


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