Reaction mass of 2,4,6-tris(1-phenylethyl)phenol and bis(1-phenylethyl)phenol and o-(1-phenylethyl)phenol

Biodegradation in soil

Description of key information

The rate and route of transformation of [14C]tristyrenated phenol (TSP) was studied in four European soils: a sandy loam (18 Acres, from the United Kingdom), a silt loam (Gartenacker, from Switzerland), a silt loam (Krone, from the United Kingdom), and a loam (Vetroz, from Switzerland). The radiolabeled carbon atom in [14C]TSP is located randomly at one of the 6 possible locations in the central phenol ring. The study was performed according to OECD TG 307 and in compliance to GLP.

The test was run in the dark, under aerobic conditions, at two test temperatures (12 ± 2 °C and 20 ± 2 °C) using a nominal concentration of 0.6 μg/g applied to 100 g of soil. The test duration was ca. 120 days for all soils except 18 Acres, which was continued up to ca. 180d. Tables compiling the results for the different soils are available in the RSS. The main findings are described below.

Ultimate degradation of the test item was monitored by means of 14CO2 evolution, which increased in all soil test systems over the course of the incubation period, up to a maximum of 14.3% of applied radioactivity in the test at 12°C and up to 22% of applied radioactivity in the test at 20°C. Small but nevertheless significant mineralization of the test item thus is demonstrated.

Decrease of TSP concentration was monitored by means of mild extraction of the soil samples. At incubation start, the radioactivity was (almost) completely extractable from the soil samples (ranging from 94.8% to 105.8%). The radioactive material was confirmed to be the parent chemical, TSP, thus demonstrating that the (mild) extraction protocol does not lead to conversion / transformation of the parent substance. At the end of the incubation period, 11.3 -20.6% and 3.9-­15.6% of applied radioactivity was still present as parent TSP in the test system at 12°C and 20°C, respectively.

Primary degradation of the test item was also monitored by means of examination of the mild extraction fractions. Metabolites that were formed at concentrations >10% of applied radioactivity, or >5% in at least 2 consecutive intervals, were identified. The identified primary metabolites are in general oxidation products, i.e. containing additional hydroxyl groups. At day 3 after incubation, up to 22.3% and 22.0% of applied radioactivity was identified as primary metabolites in the 12°C and 20°C experiment, respectively. This indicates that primary degradation of TSP starts quickly after application of the test item to the soil. In total, 9 metabolites were investigated and potential chemical structures were proposed where possible.

Any radioactivity not removed during the (mild) extraction process was considered ‘Non-Extractable Residue’ (NER). The extracted soil samples were divided in two parts and submitted either to Soxhlet extraction or to Organic Matter Fractionation (OMF).

Soxhleting allowed to further extract 5.8-­11.5% of applied radioactivity from the soil samples. Characterisation of this fraction identified it as polar transformation product. However, as Soxhlet is a harsh technique, it cannot be excluded that conversion of parent chemical occurred during the process.

The organic matter fractionation separates the fulvic acid, humic acid and humin fractions of the soil organic matter. For the TSP soil samples, the radioactivity was found to be mainly (present in the insoluble humin fraction: 19.9-­31.1% of applied radioactivity. This indicates that it is not bioavailable.

Finally, kinetic modelling was performed in order to obtain the transformation/dissipation rate of [14C]TSP and its major transformation products. All calculations were carried out by means of the TESSELLA model CAKE v3.3 (Computational Assisted Kinetic Evaluation). Three kinetic models were used: i) Single first-­order (SFO), ii) Biphasic double first-­order (DFOP) and iii) First-order multi-­compartment (FOMC).

[14C]Tristyrenated phenol was found to transform rapidly over the incubation period under the conditions of this aerobic soil study, with DT50 ranging from 3.13 to 12.5 days at 12 °C and 2.88 to 8.46 days at 20 °C.

The DT50 values of the identified transformation products were also determined by means of the same kinetic model, and ranged from 14.7 to 110 days at 12°C and from 4.78 to 83 days at 20°C.

Conclusion:

The rapid degradation of [14C]Tristyrenated phenol, with the formation of several metabolites, combined with a significant amount (≤ 22% of applied radioactivity) of 14CO2 generated over the course of the incubation period, is an indication of significant mineralization and non-persistent nature of the parent compound.

The amount of non-­extractable residues increased to a maximum of 51.6% AR. Characterization by organic matter fractionation demonstrated that most of the non-extractable residue is associated with the insoluble humin fraction of the SOM; therefore, it is not bioavailable.

The DT50 of TSP is determined to be ranging from 3.13 to 12.5 days at 12 °C and 2.88 to 8.46 days at 20 °C. The DT50 values of the identified transformation products ranged from 14.7 to 110 days at 12°C and from 4.78 to 83 days at 20°C.

Additional information

For 2,4,6­tristyryl phenol (TSP), an aerobic mineralization test in surface water (OECD 309) was requested, if technically feasible. If the OECD 309 test is found to be not feasible, ECHA’s Final Decision Letter provides a soil simulation test (OECD 307) or a sediment simulation test (OECD

308) as alternative options.

The registrants have assessed the technical feasibility of the OECD 309 test, and concluded on the grounds described below, that such a test is not technically feasible, and that, as a result of the anticipated technical difficulties, the outcome of such an OECD 309 test would not contribute to a better understanding of the persistence properties of TSP.

According to OECD Guideline 309, “the principal objective of the simulation test is to determine the mineralisation of the test substance in surface water, and mineralisation constitutes the basis for expressing degradation kinetics. However, an optional secondary objective of the test is to obtain information on the primary degradation and the formation of major transformation products. Identification of transformation products, and if possible quantification of their concentrations, are especially important for substances that are very slowly mineralised (e.g. with half-­lives for total residual 14C exceeding 60 days). Higher concentrations of the test substance (e.g., >100 μg/l) should normally be used for identification and quantification of major transformation products due to analytical limitations.”

In the registration dossier, a ready biodegradation test in accordance with OECD Guideline 301B (modified Sturm test) is available. The test item used for this ready biodegradation test consists of ca. 80% TSP and 20% DSP. In this test, less than 1% mineralization (CO2 evoluation) was noted after 28 days. These results clearly indicate the slow mineralization rate for TSP. As quoted above, the OECD 309 Guideline in this case advises that it is important to assess primary degradation, by means of identification and – where possible – quantification of major transformation products.

For the assessment of (ultimate) degradation kinetics, the OECD 309 guideline prescribes a test concentration of < 1 μg/L in order to ensure that the biodegradation follows first order kinetics. However, when intending for the identification of major transformation products in an OECD 309 test, the test should be run at a higher test concentration. According to the guideline, a concentration > 100 μg/L or sometimes even > 1000 μg/L should be used, due to the analytical limitations related to chemical structure identification techniques. However, 2,4,6-­Tristyrenated phenol has an experimentally determined water solubility of 7.07 μg/L. Thus, it is not feasible to obtain sufficiently high test item concentrations in order to allow for the evaluation of primary degradation by means of an OECD 309 test.

The PBT criteria as described in Annex XIII refer to the degradation half-­life of a substance in the different compartments. As further clarified in ECHA Guidance R11: “Degradation may be biotic and/or abiotic (e.g. hydrolysis) and result in complete mineralisation, or simply in the transformation of the parent substance (primary degradation). Where only primary degradation is observed, it is necessary to identify the degradation products and to assess whether they possess PBT/vPvB properties.”

Hence, it is clear that the goal of the requested persistence testing should be to identify a degradation half-­life for the parent substance (TSP), and – in case primary degradation is observed, but not complete mineralization – to identify the degradation products and examine their persistence.

Based on the above, the registrants concluded that:

i) It is technically impossible to assess the primary degradation potential of the parent substance TSP by means of an aqueous OECD 309 test due to the insufficient solubility of the test item in water.

ii) Assessment of the ultimate degradation potential of TSP by means of an OECD 309 test at low test concentration – although maybe technically possible – will not be of added value to the PBT assessment. The slow mineralization of the test item has already been confirmed in the available ready biodegradation test (OECD 301B).