The lowest supplement of food (80 mg) in a static exposure regime was chosen as satisfactory for the chronic test.

Therefore, this procedure is proposed for the reduction of ammonia, prior a toxicity test is conducted with artificial sediment, in order to avoid the presence of this compound at lethal or sublethal levels that could act as a confounding factor in chronic toxicity tests. A combination of sediment aeration during two weeks followed by a renewal of water column reduced the levels of ammonia in the water column and interstitial water below 1 mg/l N-NH3. The weekly water renewal did not give better results in survival or reproduction at the end of the 28-day tests. The lowest peak of ammonia in water column and sediment pore-water was measured with 80 mg of Tetramin®. Two different quantities of rations (80 and 160 mg Tetramin® flakes) were compared, combined or not with a weekly renewal of the overlying water. The behaviour of the worms that escaped from sediment resulted in a reduction of their feeding activity, reproduction, and also mortality during the chronic test. The presence of ammonia at toxic levels in the artificial sediment during the first days after its preparation influences the quality of the water column and interstitial water, affecting the biological results of the test. The addition of supplementary food to the sediment at the beginning of the test is necessary to get satisfactory reproductive levels at the end of the 28-day chronic test, but this supplement of organic matter also provoked an additional increase of the levels of ammonia. In the present study, artificial sediment has been used with Tubifex tubifex (Tubificidae, Oligochaeta) in both culture and in 28-day toxicity tests with spiked toxicants, in order to avoid the presence of occasionally important concentrations of some pollutants in sediments from apparently clean sites. 'The formulation and preparation of artificial sediments to be used in chronic toxicity tests is an important issue in the standardisation process.

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