Laboratory simulation of biodegradation of chemicals in surface waters: closed bottle and respirometric test

Microbial degradation is the most dominant elimination mechanism of organics from the environment. For evaluation of biodegradability of pure chemicals many standardized tests are available, but no standardized procedure for assessment of biodegradability of chemicals in surface water is agreed upon. Rates of in-situ biodegradation are usually estimated in laboratory simulation where environmental factors are reproduced to some extent. The aim of our study was to compare standardised ready biodegradability assessment, test (Closed bottle test) and its modifications employing the basic agreements on test conditions to simulate biodegradation in surface water. Standard test was modified using various natural river waters to simulate the natural environment in a simplified way. The impact of different types and amounts of nutrients and microorganisms on biodegradation was confirmed. The conditions in the recipient should be examined to extrapolate the results from ready biodegradability tests to real surface water.     

Improving ready biodegradability testing of fatty amine derivatives

This study assesses the biodegradation potential of a number of fatty amine derivatives in tests following the OECD guidelines for ready biodegradability. A number of methods are used to reduce toxicity and improve the bioavailability of the fatty amine derivatives in these tests. Alkyl-1,3-diaminopropanes and octadecyltrimethylammonium chloride are toxic to microorganisms at concentrations used in OECD ready biodegradability tests. The concentration of these fatty amine derivatives in the aqueous phase can be reduced by reacting humic, or lignosulphonic acids with the derivatives or through the addition of silica gel to the test bottles. Using these non-biodegradable substances, ready biodegradability test results were obtained with tallow-1,3-diaminopropane and octadecyltrimethylammonium chloride. Demonstration of the ready biodegradability of the water-insoluble dioctadecylamine under the prescribed standard conditions is almost impossible due to the limited bioavailability of this compound. However, ready biodegradability results were achieved by using very low initial test substance concentrations and by introducing an organic phase. The contents of the bottles used to assess the biodegradability of dioctadecylamine were always mixed. False negative biodegradability results obtained with the fatty amine derivatives studied are the result of toxic effects and/or limited bioavailability. The aids investigated therefore improve ready biodegradability testing.     

Effect of the alkyl chain length on the anaerobic biodegradability and toxicity of quaternary ammonium based surfactants

The anaerobic biodegradability and toxicity to methanogenic gas production of different alkyl chain length homologs of quaternary ammonium based surfactants were examinated. Two series of these cationic surfactants were selected: alkyl trimethyl ammonium and alkyl benzyl dimethyl ammonium compounds. A simple anaerobic gas production test containing municipal digester solids as a source of anaerobic bacteria was used. Under the applied methanogenic conditions, the cationic surfactants tested showed a very poor primary biodegradation and no evidence of any extent of ultimate biodegradation was observed. The toxicity of quaternary ammonium based surfactants to methanogenic gas production decreased with increasing the alkyl chain length.     

Biodegradation of chemicals in a standardized test and in environmental conditions

The estimation of biodegradation rates is an important source of uncertainty in chemical risk assessment. The existing OECD tests for ready biodegradability have been developed to devise screening methods to determine whether a chemical is potentially easily biodegradable, rather than to predict the actual rate, of biodegradation in the environment. However, risk assessment needs degradation rates. In practice these rates are often estimated (default values) from ready biodegradability tests. These tests have many compromising arbitrary features compared to the situation in the real environment. One important difference is the concentration of the chemical. In wastewater treatment or in the environment many chemicals are present at ng l(-1) to microg l(-1) levels whereas in the tests the concentrations exceed 10-400 mg carbon per litre. These different concentrations of the chemical will lead to different growth kinetics and hence different biodegradation rates. At high concentrations the chemical, if it is degradable, can serve as a primary substrate and competent microorganisms will grow exponentially, resulting in a sigmoid biodegradation curve. At low environmental concentrations the chemical does not serve as a primary substrate, and therefore does not support significant growth of the degraders, and the substrate has a linear biodegradation rate. In this study the biodegradation rates of two reference chemicals, aniline and 4-chloroaniline, were compared in a standard method and in more realistic conditions at low concentrations, using 14C-labelled substances and different sources of inocula. Biomass evolution during the tests was monitored by adenosine triphosphate measurement and also on the basis of the residual 14C-activity in the particulate matter. The results partly support the thesis that low concentrations lead to different biodegradation kinetics compared to the concentrations used in the standard tests. Furthermore the biodegradation rates of the chemicals studied, particularly of 4-chloroaniline, in Finnish natural waters appeared to be lower than those reported in some other countries.     

Biodegradability testing of synthetic ester lubricants--effects of additives and usage

The optimised biodegradability test system "O2/CO2 Headspace Test with GC-TCD" is used for the assessment of synthetic ester lubricants. The effects of both additives and usage on biodegradability are examined and discussed. Ester based cutting fluids and hydraulic fluids with and without additives are used under defined conditions at machine tools and hydraulic and plain bearing test benches. The lubricants are characterised additionally with respect to kinematic viscosity, acidity and elemental composition. Furthermore, a formulated mineral oil is characterised before and after usage at an hydraulic test bench. The results clearly show that the mineral oil is far less biodegradable than the ester oils and that their biodegradability is not affected by usage. Biodegradability of the ester oils is mainly depending on the characteristics of the base fluids and not affected by the additives. Antioxidants are influencing stability respectively biodegradability indirectly, since they prevent oxopolymerisation effects. Other effects of usage on biodegradation are not detected. In this context, the antioxidants ensure ready biodegradability and have a positive effect on the environmental fate of synthetic ester lubricants.     

A new bioseed for determination of wastewater biodegradability: analysis of the experimental procedure

A new bioassay proposed in the patent P201300029 was applied to a pre-treated wastewater containing a mixture of commercial pesticides to simulate a recalcitrant industrial wastewater in order to determine its biodegradability. The test uses a mixture of standardized inoculum of the lyophilized bacteria Pseudomonas putida with the proper proportion of salts and minerals. The results highlight that biodegradation efficiency can be calculated using a gross parameter (chemical oxygen demand (COD)) which facilitates the biodegradability determination for routine water biodegradability analysis. The same trend was observed throughout the assay with the dehydrated and fresh inoculums, and only a difference of 5 % in biodegradation efficiency (E _f) was observed. The obtained results showed that the P. putida biodegradability assay can be used as a commercial test with a lyophilized inoculum in order to monitor the ready biodegradability of an organic pollutant or a WWTP influent. Moreover, a combination of the BOD₅/COD ratio and the P. putida biodegradability test is an attractive alternative in order to evaluate the biodegradability enhancement in water pre-treated with advanced oxidation processes (AOPs).     

Application of the headspace CO2 method (ISO 14 593) to the assessment of the ultimate biodegradability of surfactants: results of a calibration exercise

In the recent review of the control of marketing surfactants used in detergents, the EU decided to increase the severity of the testing procedure by using the criterion of ultimate biodegradability (mineralization) rather than primary biodegradation (removal of the parent molecule) to ensure that possible harmful organic metabolites do not reach the environment. The relatively new ISO headspace CO2 test, considered to be an improvement on the OECD 301B (Sturm CO2) test was chosen. The method was subjected to a ring test by 11 laboratories using one of each of four classes of surfactants plus a poorly degradable reference surfactant; all laboratories satisfactorily applied the method. The necessary addition of silica gel to the medium containing the cationic surfactant, known as a class to be more inhibitory than other classes, was confirmed as a technique for avoiding inhibition of the inoculum. The biodegradability of the surfactants was in general agreement with results reported in the literature and the often reported variable values of % inorganic carbon (IC) produced of the theoretical was found. The anionic and cationic surfactants were readily biodegradable (%IC > 60), the non-ionic surfactant was well below the pass value, while the amphoteric was borderline. The IC production by the blank controls, one of the validity criteria, was about 0.3 mg C/100 ml test medium, equivalent to 3 mg C/l, as recommended in the ISO text. Mild conditions of pre-exposure of the inoculum to the test surfactant did not produce consistent worthwhile effects on either the percentage biodegradation or on its variability.     

The use of inert carriers in regulatory biodegradation tests of low density poorly water-soluble substances

Many poorly water-soluble compounds fail regulatory ready biodegradation tests as the method of test material preparation limits the bioavailability of the chemical. The recognised method for delivery of poorly soluble materials into biodegradability tests consists of coating test material inside the test vessel or onto inert substrates (i.e., glass cover slide, boiling beads, filter paper, or Teflon stir bar) that are placed inside the vessels. Volatile solvents are often used to augment this process. Although these substrates work fairly well for delivering many poorly soluble materials into biodegradability tests, they have not been effective in keeping low density, poorly water-soluble substances in the test medium. Soon after medium is added to the test vessels, these chemicals break loose from the substrates and float on the surface where they have limited contact with micro-organisms in the test medium. Hence, there is a reduced potential for measuring substantial biodegradability in the test. This paper describes the work undertaken to establish a standard method of adding low density, poorly water-soluble substances into test vessels of biodegradability studies to ensure these materials remain in contact with micro-organisms in the test medium. The substances are prepared for testing by adsorption onto silica gel followed by dispersion into the culture medium. This method of delivery may provide greater intra- and inter-laboratory consistency in biodegradability test results for low density, poorly water-soluble substances and it may more closely mimic the probable transport and fate of these substances in the environment.     

Biodegradability and ecotoxicity of amine oxide based surfactants

The aerobic and anaerobic biodegradability as well as the aquatic toxicity of two fatty amine oxides and one fatty amido amine oxide were investigated. Aerobic biodegradation was evaluated using the CO(2) headspace test (ISO 14593) and biodegradation under anaerobic conditions was assessed employing a standardised batch test. The three amine oxide based surfactants tested were readily biodegradable under aerobic conditions but only the alkyl amido amine oxide was found to be easily biodegradable under anaerobic conditions. Toxicity to Photobacterium phosphoreum and Daphnia magna was evaluated. Bacteria (EC(50) from 0.11 to 11 mg l(-1)) proved to be more sensitive to the toxic effects of the amine oxide based surfactants than crustacea (IC(50) from 6.8 to 45 mg l(-1)). The fatty amido amine oxide showed the lowest aquatic toxicity.     

Standard inocula preparations reduce the bacterial diversity and reliability of regulatory biodegradation tests

OECD ready biodegradability tests have been central to understanding the biodegradation of chemicals from a regulatory perspective for many decades. They are not fit for contemporary prioritisation of chemicals based on persistence, however, due to the low concentration of inocula used, short duration and high variability between tests. Two OECD standard inoculum pretreatment methods (settlement and filtration) were investigated to observe their effect on the probability of biodegradation and associated changes in bacterial community structure and diversity of inocula sourced from the activated sludge process of wastewater treatment plants. Both settlement and filtration were shown to dramatically and significantly reduce the probability and increase the variability of biodegradation of 4-nitrophenol compared to the use of unprocessed inocula. These differences were associated with a significant hundred-fold reduction in cell numbers and solids content and a significant shift in bacterial community structure that was sometimes accompanied by significant reductions in detectable operational taxonomic unit richness and evenness. The natural variation (between different environments) and variation due to differential selection of bacterial communities (by different pretreatment methods) is offered as an explanation for the historical high variability in standard OECD ready biodegradability tests.     

Kinetic study of the anaerobic biodegradation of alkyl polyglucosides and the influence of their structural parameters

This paper reports a study of the anaerobic biodegradation of non-ionic surfactants alkyl polyglucosides applying the method by measurement of the biogas production in digested sludge. Three alkyl polyglucosides with different length alkyl chain and degree of polymerization of the glucose units were tested. The influence of their structural parameters was evaluated, and the characteristics parameters of the anaerobic biodegradation were determined. Results show that alkyl polyglucosides, at the standard initial concentration of 100 mgC L^?1, are not completely biodegradable in anaerobic conditions because they inhibit the biogas production. The alkyl polyglucoside having the shortest alkyl chain showed the fastest biodegradability and reached the higher percentage of final mineralization. The anaerobic process was well adjusted to a pseudo first-order equation using the carbon produced as gas during the test; also, kinetics parameters and a global rate constant for all the involved metabolic process were determined. This modeling is helpful to evaluate the biodegradation or the persistence of alkyl polyglucosides under anaerobic conditions in the environment and in the wastewater treatment.     

Fate and effect of monoalkyl quaternary ammonium surfactants in the aquatic environment

The effect of the alkyl chain of quaternary ammonium-based surfactants on their aquatic toxicity and aerobic biodegradability has been studied. Two families of monoalkylquats surfactants were selected: alkyl trimethyl ammonium and alkyl benzyl dimethyl ammonium halides. Acute toxicity tests on Daphnia magna and Photobacterium phosphoreum were carried out and EC50 values in the range of 0.1-1 mg/l were obtained for the two series of cationic surfactants. Although the substitution of a benzyl group for a methyl group increases the toxicity, an incremental difference in toxicity between homologs of different chain length were not observed. Biodegradability of the different homologs was determined not only in standard conditions but also in coastal water, both tests yielding similar results. An increase in the alkyl chain length or the substitution of a benzyl group for a methyl group reduces the biodegradation rate. The degradation of these compounds in coastal waters was associated with an increase in bacterioplankton density, suggesting that the degradation takes place because the compound is used as a growth substrate.     

生物降解实验中的接种物标准化研究

针对降解实验中接种物采集困难、活性差异大、实验可重复性及结果可比性差的问题展开接种物标准化研究。对多来源的接种物在实验室可控条件下进行驯养,驯养后生长状态良好的活性污泥分别固定于灭菌麸皮和藻酸钙凝胶等2种微生物载体上,形成2种固定化接种物(分别简称为Bran及Ball)。对上述固定化接种物进行微生物活性、适应性、降解能力以及稳定性研究:通过2种固定化接种物与商品化接种物Polyseed对葡萄糖-谷氨酸(GGA)五日生化需氧量(BOD5)的结果与活菌细胞数的关系,确认3种接种物在降解实验中的用量可控制在相同数量级;比较不同批次及保存时间的2种固定化接种物、相同批次不同保存时间的Polyseed以及污水处理厂曝气池污泥为接种物对苯胺及二甘醇的快速生物降解实验(OECD301F)结果,发现Ball及Bran较Polyseed及新鲜采集活性污泥的微生物适应性及降解能力更佳。此外,在较长保存期内2种固定化接种物活性相对稳定。2种固定化接种物初具成为标准化接种物的必要条件。     

Biodegradation and toxicity of wastewater from industry producing mineral fibres for thermal insulation

The water chemistry, toxicity, and biodegradation of wastewater from an industry producing mineral fibres for thermal insulation were studied. Values for COD, BOD5, suspended solids, and phenol exceeded permissible values for the wastewater discharged into a nearby river and acute toxicity was also detected. Consequently, the effluent should be treated in a municipal wastewater treatment plant so its ready biodegradability was investigated. We found that the wastewater was readily biodegradable, therefore we assume it can be treated in the wastewater treatment plant as the ratio of the wastewater flow rate and the minimal total inflow into the sewage treatment plant would be one to at least 30.     

A kinetic distribution model of evaporation, biosorption and biodegradation of polychlorinated biphenyls (PCBs) in the suspension of Pseudomonas stutzeri

Kinetics of distribution of PCBs in an active bacterial suspension of Pseudomonas stutzeri was studied by monitoring the evaporated amounts and the concentration remaining in the liquid medium with the biomass. To determine the biodegradation rate constants of the individual congeners of the PCB formulation Delor 103, a model considering biosorption, evaporation, and primary biodegradation constructed previously was used. Rate constants of biodegradation imply that biodegradation of individual congeners is structure-dependent process. Biodegradability decreases with increasing number of chlorine substituents in the molecule, especially if they are in the ortho and para positions. On the other hand, the increasing number of free ortho and meta positions in the biphenyl molecule leads to better biodegradability. For a simple empirical determination of the influence of the chlorine substitution pattern on biodegradability, the di- and trichlorobiphenyl rate constants of biodegradation were analysed.     

Biodegradability of the antioxidant diaryl-p-phenylene diamine using a modified inherent biodegradation method at an environmentally relevant concentration

The chemical product diaryl-p-phenylene diamine (DAPD), produced by The Goodyear Tire & Rubber Company as POLYSTAY 100® (CAS 68953-84-4), is employed as an antidegradant in polymers used in tires and industrial rubber products. Previous evaluations pertaining to the ecological fate of DAPD indicated a lack of biodegradative activity in aquatic media. In order to further pursue the biodegradation potential of DAPD, it was deemed necessary to enhance the sensitivity of the aquatic biodegradation assay through (a) employment of a radiotracer of the test substance, and (b) optimisation of conditions for achieving maximal solubilisation of test material in the aquatic media of the incubation vessels. Test vessels were prepared according to the OECD ready biodegradability test guidelines, with DAPD added on silica gel at concentrations of 10 or 100 μg L⁻¹, together with a surfactant to aid solubilisation. After 63 d incubation up to 37% mineralisation was measured and up to 29% of the applied radioactivity was incorporated into cell biomass. Also, after 28 d no DAPD could be measured in solution by radio-TLC and HPLC–MS. These three results demonstrate that the antioxidant DAPD undergoes microbiologically mediated biodegradation and is highly unlikely to persist in the environment.     

Environmental applications for biosurfactants

Biosurfactants are surfactants that are produced extracellularly or as part of the cell membrane by bacteria, yeasts and fungi. Examples include Pseudomonas aeruginosa which produces rhamnolipids, Candida (formerly Torulopsis) bombicola, one of the few yeasts to produce biosurfactants, which produces high yields of sophorolipids from vegetable oils and sugars and Bacillus subtilis which produces a lipopeptide called surfactin. This review includes environmental applications of these biosurfactants for soil and water treatment. Biosurfactant applications in the environmental industries are promising due to their biodegradability, low toxicity and effectiveness in enhancing biodegradation and solubilization of low solubility compounds. However, more information is needed to be able to predict and model their behaviour. Full scale tests will be required. The role of biosurfactants in natural attenuation processes has not been determined. Very little information is available concerning the influence of soil components on the remediation process with biosurfactants. As most of the research until now has been performed with rhamnolipids, other biosurfactants need to be investigated as they may have more promising properties.     

Environmental features of two commercial surfactants widely used in soil remediation

One of the main limitations for a wider application of surfactants in soil remediation is the lack of knowledge about environmental fate and toxicity of surfactant itself especially for in situ application. Sorption behaviour, biodegradability, toxicity of parent compound and its metabolites are important processes that affect environmental fate of surfactants in site remediation applications. Tween 80 (poly(oxyethylene)(20)-sorbitane monooleate) and Aerosol MA+80 (dihexyl sodium sulfosuccinate) are surfactants that have been tested in laboratory and field scale remediation of soil and groundwater. In this work, the sorption and biodegradability of these surfactants were assessed to provide conditions and limitations for their use. The soil used in this experimentation was analysed for organic carbon content, soil bacteria, and size fraction and resulted to be a good model because is characterised by mean values for almost all considered parameters. Tween 80 showed high degree of biodegradability but a high affinity for soil matrix. Results suggest that Tween 80 could find its best application in ex situ solid phase remediation like ex situ bioremediation; its high affinity to soil could limit in situ applications. Biodegradation tests for Aerosol MA+80 show low degree of biodegradability and mineralisation. Biodegradation experiments, coupled with analysis of toxicity, could support the hypothesis that degradation of Aerosol MA+80 is not complete and leads to an accumulation of intermediates with at least the same toxicity of the parental compound. Therefore, aquifer remediation application with Aerosol MA+80 has to be conducted with necessary precautions to avoid product loss and excess surfactant should be flushed from the soil.     

脂肪酰基氨基酸对矿物润滑油生物降解性的影响研究

通过生物降解试验研究了N-月桂酰基谷氨酸、N-月桂酰基甘氨酸、N-月桂酰基丙氨酸和N-油酰基甘氨酸4种脂肪酰基氨基酸对HVI 350矿物润滑油生物降解性的影响,采用高倍电子显微镜分析了生物降解过程中微生物数量和形态变化。结果表明,在矿物润滑油中添加少量脂肪酰基氨基酸后,矿物润滑油的生物降解性能明显改善,且生物降解过程中微生物数量增多、形态发生变化,这可能是脂肪酰基氨基酸增加了微生物的营养,且具有表面活性,有利于细胞的吸收,从而促进了矿物润滑油生物降解。