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Toxicity to soil macroorganisms except arthropods

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Information requirement: Toxicity testing on invertebrates ECHA Decision No. TPE-D-21 14300033 -75-01/F.

Proposed test guideline: OECD Guideline 222 (Earthworm Reproduction Test (Eisenia fetida/Eisenia andrei))

The registrants recommended that the stability of test substance concentrations in the soil under realistic test conditions be explored as part of method development. Subsequent toxicity testing was to be carried out subject to satisfactory results from the stability studies (i.e. adequate maintenance of test concentrations for a meaningful duration, under conditions relevant to OECD TG 222). The stability test under OECD TG 222 conditions with the related substance L3 demonstrated significant loss of test item from the test system. As discussed in IUCLID Section 6.0, HMDS is more volatile and also more rapidly degradable than L3, meaning that similar or greater losses from the test system would be expected under equivalent test conditions. Additionally, in an OECD TG 216 (Soil Microorganisms: Nitrogen Transformation Test) for the effects of hexamethyldisiloxane on nitrate formation rate of soil microflora, analysis of the test substance concentrations show that test material was lost by day three of the test (see IUCLID Section 6.3.4). Based on these experimental findings, the registrants believe it is not technically feasible to proceed with the OECD TG 222 test for the registration substance on the basis that the test substance is too volatile to maintain adequate concentrations in the test system.

Stability study using the related substance octamethyltrisiloxane (L3) under OECD TG 222 conditions without test organisms:

A stability/recovery test was conducted in preparation for terrestrial ecotoxicology studies with the related substance L3. HMDS and L3 are within the siloxanes analogue group of compounds, and have high vapour pressures (5500 Pa and 530 Pa), high log Kow (5.1 and 6.6), high log Koc (3.0 and 4.3) and low water solubility (0.93 mg/l and 0.034 mg/l). In addition, the substances have a slow hydrolysis rate relative to the time-scale of ecotoxicity testing (t1/2 = 116 h and 329 h at pH 7 and 25°C, respectively). In the context of terrestrial toxicity, L3 is more likely to remain in the soil as the parent substance during the terrestrial toxicity studies. Therefore it is considered valid to read-across the results of the soil stability study with L3.

The study demonstrated a method of introducing neat 14C-octamethyltrisiloxane (14C-L3) into natural soil with subsequent mixing to distribute the test article throughout the soil uniformly. 

The second phase of the study investigated the stability of 14C-L3 in the same soil under conditions representative of those used for the OECD TG 222 Earthworm Reproduction Test. The system was partially open to allow for respiration during a planned future toxicity experiment. 

The effect of headspace on loss of test material during dosing of soil was examined. The main experiment test vessel was selected in order to minimise headspace as much as possible, while still allowing for enough tumbling to ensure homogeneity. However, even the container with the best recovery only observed an average recovery of 69.7% (or 22.9 mg/kg soil (d.w.)).

Measures were taken to avoid loss of test substance through volatilisation during the homogeneity experiment. These included:

- fitting the test vessel jar with a polytetrafluoroethylene (PTFE) lined lid and covering the threads of the jar with PTFE tape;

- spiking the soil with a calibrated gastight syringe;

- spiking with a concentration 34% above the saturation concentration, to attempt to account for test substance losses;

- taping the lid closed to avoid inadvertent opening.

The stability experiment was then carried out using the spiked soil from the homogeneity experiment, which was divided into five beakers. Each beaker had 10 mL of headspace and was covered with plastic film having five small holes (approximately 3 mm in diameter) to allow for air exchange. The plastic film was secured with rubber bands. 

By day 3 in the stability experiment, only 6.3% of the initially observed radioactivity was detected, and sampling was stopped. However, the average of the homogeneity determination was taken as the initial concentration for each beaker in the stability test. The soil was not analysed again after dividing into the beakers, therefore the potential loss from moving the test soil from the container used in the homogeneity experiment to the 5 test beakers used in the stability study, was not determined.

A single peak was observed consistent with 14C-L3, showing that the primary mechanism of loss in the initial recovery experiment was volatilisation.

The absence of degradation products in the vast majority of samples, coupled with the rapid loss of 14C activity, shows that the primary mechanism of test article loss was volatilisation of 14C-L3 from the simulated OECD TG 222 test setup.