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The tests on Daphnia magna were conducted over a 48-hour period, using a static limit test methodology. As with the fish test above the test solution was prepared using dimethylformamide. Four replicate tests were conducted using ten Daphnia in each test vessel, together with a solvent control, using ten test animals. During the tests the temperature was controlled at 21± 1°C, while the pH was always between 7.9 and 8.1, dissolved oxygen between 7.7 and 8.2 mg/L and water hardness around 100 mg/L as CaCO3. Analysis of duplicate samples of the BDP in water after the 48-hour test period found concentrations of 1.03 and 1.06 mg/L, which was around 105% of the nominal concentration. No immobilisation of any of the 40 test Daphnia was observed during the course of the test, nor was any other aberrant behaviour observed. These results indicate that the BDP is not toxic to this species up to the limits of its water solubility.

A limit test on the inhibition of algal growth was also conducted on Selanastrum subspicatus over a 72-hour incubation period at 21 ± 1with nominal concentration of the test material of 1.0 mg/L. The test material was introduced to the solutions in the manner indicated in the studies above. Six replicate tests were conducted in 250 mL Erlenmeyer flasks, together with three control flasks containing no chemical. Three solvent controls (i.e. water and the appropriate amount of dimethylformamide) were also run. Each flask contained 100 mL of the test medium, and the flasks were continuously agitated to maintain the algal cells in suspension. The pH of the test solutions increased from around 8.0 at the start of the period, to around 10 after 72 hours. The growth of algal biomass was determined over the test period by measurement of cell density using absorbance at 665 nm. At 0 hours the mean cell density in the control was 1.85 x 104 cell/mL, increasing to 2.17 x 106 cells/mL after 72 hours. The corresponding values for the solvent control were 2.17 x 104 and 1.3 x 106 cell/mL, respectively. While the report did not detail the actual cell densities for the test media, the absorbance closely paralleled that of the controls (and solvent control), and consequently it was concluded that the BDP is not inhibitory to growth of algae up to the limits of its water solubility.

A test on the inhibition of bacterial respiration was also conducted. The test substance was suspended in artificial sewage at nominal loadings of 20, 500 and 1,000 mg/L using a 30 minute sonication to assist dispersion. The test flasks were inoculated with sewage sludge bacteria and aerated for 30 minutes. Following aeration the contents of the flasks were poured into darkened 300 mL BOD bottles fitted with oxygen sensing electrodes. The rate of oxygen consumption was measured for the dispersions, and compared with the vessel. None of the tests indicated any significant inhibition of bacterial respiration compared with the controls, and it was concluded that the BDP is not toxic to sewage bacteria up to the limits of its water solubility.

The acute test on rainbow trout was a Static Limit Test (no replacement of test water) performed over 96 hours using a solution of the test substance made up at a nominal concentration of 1 mg/L in charcoal filtered dechlorinated tap water. The test was performed in duplicate using ten fish in each test vessel. A solvent control was also run in parallel, also using ten fish. During the tests the temperature was controlled at 14 ± 1oC, while the pH was always between 7.7 and 8.0, dissolved oxygen between 9.4 and 10.1 mg/L and water hardness around 100 mg/L as CaCO3. All test specimens survived over the 96 hour test period. Further, no physical or behavioural anomalies were observed during the test period, and accordingly it was concluded that the BDP is non toxic to rainbow trout up to the limits of its water solubility.