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Environmental fate & pathways

Bioaccumulation: terrestrial

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bioaccumulation: terrestrial
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Peer-reviewed study comparing laboratory-based bioaccumulation in earthworms with in silico modelling predictions. Bioaccumulation study was carried out in accordance with OECD 317 guidelines by the National Research Council Canada and by the regulator Environment Canada.
according to guideline
other: OECD Guideline 317
GLP compliance:
not specified
Specific details on test material used for the study:
Details on properties of test surrogate or analogue material (migrated information):
Not applicable
Details on sampling:
The purpose of the study was to compare in silico-based model predictions with laboratory derived data from bioaccumulation studies on earthworms (Eisenia andrei) for two organic chemicals; the test substance for this dossier, 2,2',6,6'-tetra-tert-butyl-4,4'-methylenediphenol (CAS 118-82-1) [known as Binox] and a xanthene dye 2', 4', 5', 7'-tetrabromo-4,5,6,7-tetrachloro-3', 6'-dihydroxy-, disodium salt [Phloxine B]. Only the details and results of the test substance CAS 118-82-1 are relevant to this dossier and reported here.

The bioaccumulation test design followed OECD Guideline 317 for assessing bioaccumulation in terrestrial oligochaetes. The test consisted of two phases; an uptake phase whereby earthworms were exposed to a concentration in the soil lower than the 25% inhibiting concentration and an elimination phase where earthworms were transferred to uncontaminated reference soil.

Two soils were used:
- Field-collected clay loam soil comprising 10% clay, 46% silt, 44% sand and 5.4% organic carbon with pH 5.6.
- Sandy soil comprising 2% clay, 4% silt, 94% sand and 0.36% organic carbon with pH 6.5.

Design details for the clay loam soil:
Nominal concentration = 50 mg/kg dry soil
Duration of each phase = 28 days
Number of organisms per replicate = 5
Soil mass per organism = 50 g dry soil
Sampling frequency (test day for each phase) = 0, 1, 2, 3, 6, 8, 12, 15, 19, 22, 26 and 28.

Design details for the sandy soil:
Nominal concentration = 10 mg/kg dry soil
Duration of each phase = 21 days
Number of organisms per replicate = 3
Soil mass per organism = 67 g dry soil
Sampling frequency (test day for each phase) = 0, 1, 2, 3, 7, 14 and 21.

Test vessels = 250 mL glass jars with perforated lids.
Weighed = weekly to maintain soil moisture conditions.
Details on preparation and application of test substrate:
Acetone was used as a carrier due to the water insolubility of the test substance. The solvent was then evaporated from the soils for 24 hours. A solvent control was included in the test design.

Soils were then hydrated with deionised water to an optimal moisture content of the soil's water holding capacity (60% for the clay loam and 56% for the sandy soil).
Test organisms (species):
Eisenia sp.
Details on test organisms:
Test organism = Eisenia andrei
Established laboratory cultures of the Soil Toxicology Laboratory (Environment Canada) were used for all testing.
Total exposure / uptake duration:
>= 21 - <= 28 d
Total depuration duration:
>= 21 - <= 28 d
Test temperature:
Not reported.
- Field-collected clay loam soil = pH 5.6.
- Sandy soil = pH 6.5.
- Field-collected clay loam soil comprised 5.4% organic carbon.
- Sandy soil comprised 0.36% organic carbon.
Water holding capacities of the clay loam soil = 73% and sandy soil = 29%.
Details on test conditions:
Test vessels consisted of 250-mL glass jars with perforated lids, which were weighed weekly so that the soil moisture content could be maintained through the addition of deionized water. Test organisms were fed cooked oatmeal (one-half of teaspoon on day 0 and one-fourth to one-half of teaspoon thereafter, based on quantity of food remaining in vessel) on day 0, day 14, and day 28 of the uptake and elimination phases. Test measurements included wet mass of earthworms before and after depuration at each sampling date to determine mean mass loss for test validity (i.e., no less than 20%); test validity also required no more than 10% mortality of test organisms.
Nominal and measured concentrations:
Nominal concentrations of 10 and 50 mg/kg dry soil were applied to the sandy soil and clay loam soil, respectively.
> 0.13 - < 0.32 other: g organic carbon/g lipid
other: Normalised for lipid and soil organic carbon content
Time of plateau:
6 d
Calculation basis:
steady state
Remarks on result:
other: Results for the clay loam soil
> 0.067 - < 3.5 other: g organic carbon/g lipid
other: Normalised for lipid content and soil organic carbon content
Remarks on result:
other: Results for the sandy soil. Note that a steady state was not reached in the uptake phase.
Depuration time (DT):
7.3 d
Depuration time (DT):
14 d
Kinetic parameters:
The steady state BAFk for earthworms in the clay loam soil was calculated to be 0.71. When normalized for lipid and soil organic carbon content, the BAFk was 0.23. For the sandy soil, the calculated BAFk was 2.6 and when normalized for lipid and soil organic carbon content, it was 0.86.
No data
Details on results:
The test substance was accumulated within earthworm tissue, such that there was no significant change in tissue concentrations after 6 d of exposure in the clay loam soil, after which steady state was achieved. Although there was no significant (p = 0.11) loss of the test substance from the clay loam soil, a degradation rate was nonetheless estimated to derive the half-life in clay loam soil of 46 days. Similarly, a half-life for the sandy soil was estimated to be 11 days.

For the clay loam soil, the mean BSAF for each sampling date was also determined and there was no significant difference (p = 0.15) in the BSAFs after day 1; when normalized for lipid and soil organic carbon content, the mean BSAF ranged from 0.13 ± 0.034 g organic carbon/g lipid to 0.32 ± 0.070 g organic carbon/g lipid (the mean 28-d BSAF was 0.22 ± 0.17 g organic carbon/g lipid).

The results from the sandy soil test contrasted with those of the clay loam test in that there was a rapid and consistent accumulation of the test substance, and steady state was not reached by the end of the uptake phase (i.e., day 21). The test substance was eliminated by the earthworms, but 30 ± 4.6% remained within the tissue at the end of the elimination phase, relative to that observed at the end of the uptake phase; this was similar to the non-eliminated residues observed in the clay loam soil at the end of the test (29% ± 7.3%).

The BSAF for each sampling date was also determined forthe sandy soil and there was a significant rise (p < 0.05) in the BSAFs after 3 d. When normalized for lipid and soil organic carbon content, the mean BSAFc ranged from 0.067 ± 0.020 g organic carbon/g lipid on day 1 to 3.5 ± 0.25 g organic carbon/g lipid on day 21 in the sandy soil experiment.
Reported statistics:
No data

Visual examination of the test substance 2D structure reveals that it can be classified as a hindered phenol, as the phenolic structure might be expected to cause membrane disruption via a polar narcosis or respiratory uncoupling mode of toxic action; however, the specific activity is likely to be substantially mitigated or hindered by the t-butyl functional groups. The reported log Kow (Princz et al, 2014) for this neutral phenol is 9.0 (KOWWIN Ver 1.6), which suggests a very high lipophilicity but little or low bioavailability in environmental matrices and possibly in organism tissues. Results of the structural profiling using the OECD QSAR Toolbox (Ver 3.0) revealed that the test substance has the potential to have phenol-like ecotoxicity (OASIS MOA and ECOSAR profilers) and was classified as having an intermediate to high toxicity potential according to Cramer Rules, largely because of the non-hydrolysable substituted aromatic nature of this compound. Molecular cross-sectional dimensions of 30 predicted conformers of the test substance from MOPAC calculations revealed that it has an average maximum diameter (Dmaxof 1.6 nm and an average effective diameter (DEFF) of 1.1 nm, which suggests that the molecular dimensions may be a mitigating factor for the rate of permeation of the chemical across dermal tissues; however, it is uncertain whether this can be directly linked to invertebrate tissues. The test substance was profiled to have a 92% probability of blood plasma lipoprotein binding potential, but the estimation was considered not reliable by the model's reliability statistics. Nonetheless, a 94% structurally similar chemical (Tanimoto Similarity Index) to the test substance, Probucal (CAS no. 23288-49-5), has an 80% observed protein plasma binding potential and is used in the training set of the model. The test substance was profiled to have a 100% transcellular passive route of uptake, but is predicted to be poorly permeable in Caco-2 intestinal cell assays.

Validity criteria fulfilled:
The results of this study demonstrated that the test substance, a neutral organic compound, had low bioaccumulation potential, which is consistent with the mechanistic reasoning for a slow rate or poor permeability yet good elimination in earthworms and low bioavailability of this compound in soils. The test substance can be said to follow a classic neutral passive diffusion paradigm driven by hydrophobicity. It is unlikely to bioaccumulate in terrestrial organisms.
Executive summary:

In silico-based model predictions, originating from structural and mechanistic (e.g., transport, bioavailability, reactivity, and binding potential) profiling, were compared against laboratory-derived data to estimate the bioaccumulation potential in earthworms of two organic substances. The results for the test substance (CAS 118-82-1) are reported here. Soil bioaccumulation studies were conducted using Eisenia andrei and two field-collected soils (a clay loam and a sandy soil). In general, the in silico structural and mechanistic profiling was consistent with the observed soil bioaccumulation tests. The test substance did not bioaccumulate to a significant extent in E. andrei in either soil type.

Description of key information

A peer-reviewed study (Princz et al, 2014) comparing laboratory-bioaccumulation in earthworms with in silico modelling predictions is available and presented as a key study. This shows no significant bioaccumulation for the test substance in the terrestrial species Eisenia Andrei.
Three papers, Kelly & Gobas, 2003; Kelly et al, 2007; and Gobas et al, 2003, demonstrate that, based on theoretical QSAR modelling, biomagnification may occur in terrestrial food chains. However, these papers are not supported by data on the test substance in question, and therefore remain a theory based on modelling numbers and general trends across similar substances.
A further detailed literature review adds a number of additional data sources, however the literature shows conflicting results. Some studies demonstrate a calculated BCF in excess of 5000, which would result in classification of the test substance as B and vB under the PBT assessment criteria. The models used to calculate these BCFs are criticized in a paper, which claims the models show a number of inaccurate results and classification of chemicals as B and vB may be inaccurate when used.
Ultimately, the Princz paper, which includes solid, validated laboratory data, provides the most useful and reliable information on the test substance. This shows that bioaccumulation does not occur in terrestrial species. The papers by Kelly & Gobas, 2003, Kelly et al, 2007, and Gobas et al, 2003, suggest biomagnification, but this is not supported by the Princz data which shows that bioaccumulation will not occur in terrestrial soil organisms and therefore making it unlikely that the substance would enter the food chain.

Key value for chemical safety assessment

Additional information

A single study (Princz et al, 2014), Klimisch score of 1, is presented as a key study. This has been conducted to OECD guideline 317 guidelines and also includes comparison within silicodata, and the results show no significant bioaccumulation of the test item in the earthworm speciesEisenia Andrei. The study results were comparable to thein silicomodelling, and resulted in a BSAF of 0.13 – 0.32 g organic carbon/g lipid in a clay soil and 0.067 – 3.5 g organic carbon/g lipid in a sandy soil.


Papers were also highlighted by ECHA in their draft decision of May 6th, 2015), including Kelly & Gobas (2003); Kelly et al (2007); and Gobas et al (2003). An overview of the data is presented below.


These three literature sources usein-silicomethods in conjunction with select field studies to study the potential bioaccumulation of Persistent Organic Pollutants (POPs). The specific test material in question was not included. The papers all provide similar conclusions on potential bioaccumulation of POPs.POPs with an octanol-water partition coefficient, logKOW, in excess 2 and an octanol-air partition coefficient, logKOA,­ in excess of 5 show potential for bioaccumulation in terrestrial food chains. Based on this criteria, TBMD could be considered bioaccumulative, as it has a measured logKOWof >6.5 and a calculated logKOA­­value of 17.015. This conclusion is purely speculative, as no measured data for the target substance is provided within the studies. As detailed in the key study byPrincz et al, 2014, measuredin vivodata showed that bioaccumulation in a terrestrial earthworm species does not occur. As such, the data provided in these three papers by Kelly and Gobas are considered only supplementary and of lesser dependability that actual measuredin vivodata. These studies are therefore not considered as key studies for the submission.


There are four additional studies which predict the bioconcentration factor for substances using a variety of methods. Tyle et al (2002) have identified a number of PBT and vPvB substances using QSAR technology which shows that it has a calculated BCF of 5623 using the Connell calculation approach, which classifies the substance as both B and vB. A PHD dissertation by Inoue (2012) further supports the high BCF value for the target substance, showing a 5% lipid normalized BCF of 8100. This dissertation also suggests that biomagnification would occur in the food chain, supporting the findings of the three above referenced Kelly and Gobas studies.


However, a further QSAR study on bioaccumulation potential undertaken by Dimitrov et al (2003) offers conflicting results, discrediting the Connell, Meylan and Dimitrov models as they show a number of incorrect B and vB classifications, thereby making them inadequate for legislative purposes. Ultimately, it suggests that the target substance is not classified as B or vB, but only P, given the classification in the CATABOL and BCFMAX­models.


There is a disparity within the literature available as to the actual BCF value of the target substance, 2,2’,6,6’-tetra-tert-butyl-4,4’-methylenediphenol, and whether this substance should be classified as B, vB or not at all. The Princz et al study (2014)shows that no bioaccumulation occurs in earthworms. As this is the only available formal study data on the test material, with other papers either using a QSAR model on the test substance or its analogues and therefore only operating in a theoretical sphere, it is believed that the results of the Princz study should be taken as the most pertinent and reliable results.


Reference list:


Tyle, H. et al (2002), Identification of potential PBTs and vPvBs by use of QSARs,Danish EPA summary report

Dimitrov, S.D. et al (2003), Bioconcentration potential predictions based on molecular attributes—an early warning approach for chemicals found in humans, birds, fish and wildlife,QSAR Comb. Sci. 22

Gobas, F.A.P.C. et al (2003), Quantitative Structure Activity Relationships for Predicting the Bioaccumulation of POPs in Terrestrial Food-Webs,QSAR Comb Sci, 22:329-336.

Inoue, Y. (2012), Studies on an evaluation method for bioaccumulation of chemicals in fish,Kyushu University, Japan, February 2012

Kelly, B.C. et al (2007), Food Web-Specific Biomagnification of Persistent Organic Pollutants,Science 317:236-239

Kelly, B.C. and Gobas, F.A.P.C. (2003), An Arctic Terrestrial Food-Chain Bioaccumulation Model for Persistent Organic Pollutants,Environ Sci Technol, 37:2966-2974