5.1 Applicability of the OECD test guideline 303A for testing nanoscale particles
The use of laboratory sewage treatment plants (LSTP) is one option for assessing the fate and behaviour of nanoparticles in STPs, but few data about its applicability to nanomaterials is available so far. Therefore, the OECD simulation test according to OECD Guideline 303A (2001) was evaluated using nanoscale TiO2. The results indicate that the OECD guidance on testing the efficiency of sewage treatment plants is in principle applicable to assess the behaviour of nanoscale particles. The test guideline allows enough modifications to adapt the design to the question. Still certain criteria specific for nanomaterials have to be set to allow for comparability and interpretability of the results obtained.
The test should be run under nitrifying conditions as in most technical STP, in order to assess the impact of nanomaterials to activated sludge. Here, the nitrifying bacteria are among the most sensitive of activated sludge. Hence the recoding of corresponding data should be recommended.
A clear statement should be included that other stages of the biological wastewater treatment, such as primary sedimentation, denitrification, and/or the filtration of the effluent from the clarifier is not simulated with this test system and might be influenced by the nanomaterial.
The use of Synthetic Drinking Water (SDW) instead of tab water is recommended in order to have reproducible test conditions and to achieve better comparability between laboratories.
The dosage of nanoscale particles should be made separately from that of the organic synthetic wastewater in order to avoid any agglomeration of the particles. The use of a suitable dispersant may be considered, but its potential impact on the treatment process has to be assessed.
When the impact of the dispersant has been determined in a pre-test to be acceptable, the reference LSTP unit may also be fed with the same concentration of the dispersant as the test LSTP unit. However, a negative impact of the dispersant on the clearing efficiency might counteract the overall validity of the test.
The determination of the filterable solids in the effluents of the LSTP is recommended offering an additional tool for describing the influence of the dispersant. Also at least indicative analyses on the nature and partitioning of the nanoscale particles in the effluent, whether adsorbed to filterable solids or not, should be conducted.
The main sampling points for subsequent chemical analysis include the activated sludge next to the effluents of the LSTP. The calculation of an overall balance of the nanomaterial in question is recommended as a quality control. The OECD 303 A should include a paragraph describing the principle of calculating such a balance and achievable recovery rates. So far the possibility to establish a mass balance is only referred to in Annex III of OECD “guideline 303 A” for poorly water soluble or volatile test substances. OECD guideline 314 “Simulation tests to assess the biodegradability of chemicals discharged in wastewater” might serve as an example for the description of a mass balance.
We recommend that the above mentioned points be added to the OECD test guideline 303 A for use with nanomaterials. Provided this addition we find the test guideline to be applicable for nanomaterials, here specifically Titanium Dioxide.
5.2 Applicability of the OECD test guidelines 312 for testing nanoscale particles
The OECD test guideline 312 tests the mobility of chemicals, here specifically nanomaterials in soil columns to derive information on possible environmental transport. We found the OECD test guideline 312 to be principally also applicable to assess the behaviour of nanomaterials. The test guideline allows a series of proposed modifications making it appropriate. Certainly at some stages of application difficulties occur, and adaptations should be considered for following points:
Soil selection
Concentration and detection
Application of the nanomaterial Soil selection
In the test guideline several criteria for soil selection are provided with regard to pH values, texture and organic content. But no limitations concerning their water permeability are made.
In this study a backwater soil (Gleyic Podsol A04) was used whereupon difficulties occurred due to the lack of breakthrough by gravity for water. A once applied suction power overcame this problem and triggered a run off of the material. Unluckily the transport was mainly along the glass column wall, as could be seen by the deposition of TiO2 along the column, which in turn made a statement about the mobility of a material in soils difficult.
We recommend limiting the use to soil types with normal to low retention potential in soil column tests which allow water transport by gravity. If other soil types are employed, needing the aid of suction power, all data should be carefully evaluated for transport mainly along the soil column walls.
Concentration and detection
The amount of the substance to be applied in the test is defined in the guideline to be high enough to enable detection of at least 0.5% of the applied dose in any single segment.
Alternatively, the dose may correspond to the maximum recommended use rate – real exposure concentration (single application).
The latter recommendation could not be followed due to missing information on additional, anthropogenic TiO2 nanomaterial concentrations in the environment. Only modelled concentration (Gottschalk et al., 2010) are available, which predict an increase of anthropogenic Ti in soils of around 1.3 µg/kg*a. For soils treated with sewage treatment plant sludge an increase of around 89 µg/kg*a is predicted. The corresponding soil concentrations cannot be used for the soil column experiments considering the high Titanium background and the needed detection limit.
To ensure that the added Ti could be detected in any segment of the soil column an amount of 500 mg TiO2 was used based on the assumption of equal distribution. This corresponds to a concentration of 5 g/L of the stock suspension, whereof 100 mL were applied to the soil column. It is conceivable that the used concentration of the material may affect the mobility (increased agglomeration, increased filtration and pore clogging).
We recommend the test scenario to be clearly defined for better comparison between different nanomaterials. In the here chosen case of TiO2 relatively high, worst case scenario concentrations had to be employed possibly leading to high agglomeration and pore clogging. If another nanomaterial with significantly lower concentration is used possibly a higher transport rate could be determined. Hence information on concentration dependent effects in soil column test for nanomaterials is needed.
We recommend that for the simulation of a more realistic scenario the test design should be adapted for the application of lower concentrated suspensions over a longer time
We recommendet that the sampling of different allliquots of the eluate can be necessary, to achieve a higher concentration in the sample.
We recommend some detailed tests using also other detection methods in the eluate, like Field Flow Fractionation coupled with a mass spectrometer (FFF-MS) or Surface-Enhanced Raman Spectroscopy (SERS) which are able to detect nanomaterials in matrices with a high background of other materials. However these detection methods are expensive and the detailed studies should be conducted mainly for method evaluation purposes. More information about detection methods can be found in Tiede et al., 2008.
SEM / EDX analysis is a useful tool to detect the transport of isolated TiO2 agglomerates and tiny amounts of Ti and their shape.
Application of the nanomaterial
No specific recommendation can be given with regard to the way the nanomaterial should be applied to the soil column.
In this study the material was applied to the soil columns in form of a suspension, a likely path of entry into the environment. The use of a suspension allows reproducible conditions of the applied particle size distribution. With a suspension a homogeneous application could be warranted, whereas reproducible homogeneous spiking with a dry powder may be challenging. Still, premixing of the dry powder with a soil section may also be used.
5.3 Applicability of the OECD test guidelines 106 for testing nanoscale particles
The OECD test guideline 106 was designed for the testing of the adsorption behaviour of soluble chemicals to a soil matrix. By testing nanomaterials following difficulties occurred during the test:
- Agglomeration of the nanomaterial during the test
- No separation between adsorbed and non adsorbed materials
No adsorption isotherms could be determined for nanomaterials with this method.
Influence of agglomeration on the results
Agglomeration of the test material may occur when mixing the suspension with the soils due to changes in the ionic strength, pH and destabilising compounds (chapter 2.1). This may also be the case in natural conditions. Increases in particle size may result in the separation of the suspended particulate nanomaterial along with the soil fraction using filtration or centrifugation. Both centrifugation and filtration remove nanomaterial agglomerates from the supernatant. Hence no differentiation of adsorbed or agglomerated nanomaterial can be made using the OECD test guideline 106 and no information can be correctly obtained for adsorption coefficients and isotherms.
We conclude that the OECD test guideline 106 is principally not applicable for nanomaterial testing.