BS EN ISO 15952:2018 pdf download – Soil quality – Effects of pollutants on juvenile land snails (Helicidae) – Determination of the effects on growth by soil contamination
4 Principle Juvenile land snails (usually Helix aspersa aspersa Müller) are exposed during a period of 28 d to test mixtures containing the test substance, preparation or matrix at different concentrations.
The test mixtures are freshly prepared and renewed every 7 d. According to the objectives of the study, the test mixtures may be prepared with artificial soil (see 6.3.2) or with a suitable natural soil (see 6.3.3). The snails are fed during the test with uncontaminated food. The effects on growth (biomass and shell diameter) and on survival are measured after 28 d of exposure (optionally, effects could be measured every 7 days during 28 d). The results obtained during testing are compared with those of a control to determine the NOEC or LOEC and to allow the estimation of the concentration which reduces the growth of the snails by 50 % within 28 d with respect to the fresh mass [EC 50,m (28 d)] and to the shell diameter [EC 50,d (28 days)] or other values of EC x .
If the concentrations selected result in lethal effects, the results obtained during testing are compared with those of a control and used for estimating the concentration which causes the death of 50 % of the snails [LC 50 (28 d)]. For particular applications, various parameters (EC x , NOEC, LOEC, LC 50 ) can be determined (optional) after exposure periods lower than 28 d (7 d, 14 d or 21 d). The test is conducted in two stages:
— a preliminary test intended to indicate both the non-observed effect concentration, NOEC, and the complete growth inhibition. The resulting dose-response relationship is important for the proper design of the definitive test;
— a definitive test specifying the concentrations which cause between 10 % and 90 % of growth inhibition. It is not necessary to perform a final test where the preliminary test has not revealed any inhibitory effects at the maximum concentration tested.
5 Test environment
The test shall be carried out at a temperature of (20 ± 2) °C under a day-night photoperiod of 18 h to 6 h. The illumination intensity (artificial light of daylight type), without any natural light in the test containers shall be 50 to 100 lx.
6 Reagents
6.1 Water, of purity at least deionized.
6.2 Biological material. Test organisms shall be juvenile snails. The recommended species is Helix aspersa aspersa Müller (also known as Cantareus aspersus and Cornu aspersum) which shall be 3 to 5 weeks old, having a mean fresh mass of (1 ± 0,3) g and a shell diameter of (15,5 ± 1) mm. NOTE The use of some other genus and/or species of Helicidae is possible (see examples and conditions in Annex G). The snails shall be selected from synchronous breeding in order to form a population as homogeneous as possible with respect to size, mass and age. The breeding techniques for snails are described in Annex B. After a nursery period (3 to 5 weeks, see Annex B), the young snails shall be used after at least 1 week of aestivation and no more than 5 months. The aestivation is carried out in round wooden boxes (approximately 12 cm in diameter and 4 cm in height), with the snails under dry conditions, at a temperature of 17 °C to 20 °C.
Two to three days before starting the test, snails shall be woken by spraying water (6.1) into the boxes used for aestivation. The proportion of snails not woken shall be less than 10 %. As soon as they have resumed activity (snails not stuck to the walls of the box and which are beginning to move about), the snails shall be transferred to a test container (7.1) that has been moistened with water (6.1). The bottom of this box either is covered with absorbent paper that has also been moistened, or can contain some test substrate (6.3) moistened to 50 % to 60 % of its water-holding capacity. Between waking and the start of the test (2 d to 3 d), the snails shall be fed (6.4).