The biodegradability testing of poorly water soluble compounds

A few prerequisites for the biodegradability evaluation of poorly water soluble compounds such as the selection of suitable biological and analytical parameters, dispersion techniques, introduction of the test compound into the culture medium, and the choice of a suitable inoculum were elucidated. Furthermore, various published tests were scrutinized for their suitability for the purpose under discussion. It is felt that a modified Blok method holds presently the most promise.     

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.     

Evaluation of the toxicity of substances to be assessed for biodegradability

The ability to distinguish between true non-biodegradability, and inhibition of biodegradability due to the toxic effects of the compound tested present problems. It results in possible false identification of substances as non-biodegradable.Six chemicals were evaluated in a variety of tests to assess their toxicity to aerobic sludge organisms. The tests employed were: BOD₅ and Closed Bottle inhibition tests, inhibition of respiration of activated sludge, growth inhibition of activated sludge, light emission from (MICROTOX test) and the repetitive die-away test (RDA). Results from these tests were compared with results obtained from a number of ready biodegradability tests using the compounds listed above at anticipated non-toxic and toxic concentrations.No test method evaluated consistently forecast toxicity due to the chemicals tested. The MICROTOX and nitrification inhibition tests were too sensitive. A combination of sludge respiration rate and/or growth tests seem most appropriate. To avoid toxicity to the sludge, biodegradability testing should be made at 10% of the EC₅₀ value. Compounds with an EC₅₀ value greater than 300 mg/l are unlikely to be toxic in ready biodegradability tests. Compounds with an EC₅₀ value of less than 20 mg/l may pose problems necessitating the use of the stringent closed bottle test, or the use of C¹⁴ labelled test materials. Compounds with an intermediate EC₅₀ that is between 20–300 mg/l, need to be evaluated at a range of concentrations in biodegradability tests, or may need to be evaluated carefully to define the precise no-effect level. Procedure recommendations to distinguish between the inhibition of biodegradability and inertness of the test substance are made.     

Biodegradability of wheat gluten based bioplastics

A large variety of wheat gluten based bioplastics, which were plasticized with glycerol, were subjected to biodegradation. The materials covered the total range available for the biochemical control parameter Fi, which expresses the percentage of aggregated proteins. This quantity can be related to the density of covalent crosslinks in the wheat gluten network, which are induced by technological treatments. The biodegradability tests were performed in liquid medium (modified Sturm test) and in farmland soil. All gluten materials were fully degraded after 36 days in aerobic fermentation and within 50 days in farmland soil. No significant differences were observed between the samples. The mineralization half-life time of 3.8 days in the modified Sturm test situated gluten materials among fast degrading polymers. The tests of microbial inhibition experiments revealed no toxic effects of the modified gluten or of its metabolites. Thus, the protein bulk of wheat gluten materials is non-toxic and fully biodegradable, whatever the technological process applied.     

Acid-base properties of water-soluble organic matter of forest soils, studied by the pK-spectroscopy method

Using the potentiometric titration and pK spectroscopy method, acid-base properties of water-soluble organic matter of forest soils have been studied. Five acidic classes composed of different substances with pK(a) values around 3.6; 4.8; 6.7; 8.7 and 9.7 have been identified. Testing the properties of soluble soil fraction, it is to be taken into account that when it is isolated from non-soluble soil matter, some water-soluble substances remain in soil and do not pass into the solution. Most firmly adsorbed in soil are water-soluble components with pK(a) 9.6-9.8.     

Modification of the Zahn-Wellens test: determination of the biodegradability of poorly soluble, adsorbing and volatile substances by measurement of oxygen consumption and carbon dioxide production

Based on the Zahn-Wellens test (OECD 302 B, 1992; DIN EN ISO 9888, 1999), a test system has been developed which enables a continuous and parallel determination of oxygen consumption (pressure measurement) and carbon dioxide production (conductivity measurement). It is a closed test system consisting of a culture flask, a carbon dioxide absorption flask, a pump as well as integrated measuring and control instruments. The air circulating within the test system causes the carbon dioxide present in the test solution to be stripped out completely and directly absorbed by the absorption solution. Avoiding costly thermostatting of the test apparatus, the results of the pressure measurements were temperature-corrected arithmetically. The functional reliability of the measuring apparatus has been demonstrated exemplary in degradation experiments with selected test substances. This new test system also facilitates to test poorly soluble, adsorbing and volatile substances for inherent biodegradability and constitutes an appropriate complement to the standardised Zahn-Wellens test. The Federal Environmental Agency will use it as input to international standardisation activities ongoing within the OECD, as a draft standard.     

An automated test for measuring polymer biodegradation

The biodegradability of polymer materials as evaluated by the modified Sturm test is labor-intensive, cumbersome and costly and also tends to cumulate errors. An automated system for the measurement of carbon dioxide would overcome many of these disadvantages. We describe here a method in which CO2 was determined by IR spectroscopy. We compared the results with those from trapping CO2 in a solution of barium hydroxide (Ba(OH)2) followed by manual titration. The automated system was more reproducible, less costly and more compact. The automated system could also be employed to measure the biodegradability of other substances such as oils and detergents.     

Genotoxicological effects of some phenoxyherbicides and their metabolites on Bacillus subtilis M45 Rec- and H17 Rec+ strains

Because they are used in a number of commercial preparations phenoxyacetic acids and their salts can occur in wastewater. During their degradation genotoxic substances may be created. The results of investigations of biodegradability and genotoxicity of some phenoxyherbicides are presented. Commercial formulations of 2,4-D (Aminopielik 720) and MCPA (Chwastox Extra) were the substrates studied. Biodegradation tests were conducted according to OECD guidelines for testing of chemicals--confirmatory test (OECD Method 303 A). Genotoxicity tests were conducted with Bacillus subtilis strains according to the method of [Chemical Mutagens, vol. 6, Plenum Press. New York, 1980, p. 149]. Genotoxicity of biodegradation products was also studied. Both commercial formulations were biodegradable. Aminopielik 720 was potentially genotoxic but only at great concentrations while Chwastox Extra was not genotoxic. Biodegradation products of neither compound were genotoxic.     

An 'inherent' biodegradability test for oil products: description and results of an international ring test. CONCAWE Biodegradation Task Force.

Current test guidelines for assessing 'inherent' (potential) biodegradability were designed for water-soluble, organic compounds of low volatility and are unsuitable for most oil products. It was against this background, that CONCAWE (the oil companies' European organisation for environment, health and safety) formed a task force to develop a standard test protocol for assessing the 'inherent' biodegradability of oil products.     

Biodegradability testing of poorly soluble compounds by means of manometric respirometry

Manometric respirometry (UK-MITI test) was found convenient for biodegradability testing of poorly soluble chemicals. Various dispersion techniques were evaluated with anthraquinone (solid) and di-isooctylphtalate (liquid). Application on silica gel via a solvent (dichloromethane) was found generally suitable. Ultrasonic dispersion was found suitable only if a stable emulsion forms.     

Aerobic biodegradability of the calcium channel antagonist verapamil and identification of a microbial dead-end transformation product studied by LC-MS/MS

In recent years pharmaceuticals and personal care products have been detected in increasing concentrations in hospital effluents, sewage treatment plants (STP) as well as in different environmental compartments such as surface water, groundwater and soil. Little is known about the elimination of these substances during sewage treatment or about the formation of potential metabolites in the environment caused by bacterial biotransformation. To assess the biodegradability of the popular cardiovascular drug verapamil and the possible formation of potential microbial degradation products, two tests from the OECD series were used in the present study: the widely used Closed Bottle test (OECD 301 D) and the modified Zahn-Wellens test (OECD 302 B). In the Closed Bottle test, a screening test that simulates the conditions of an environmental surface water compartment, no biological degradation was observed for verapamil at concentrations of 2.33mgl(-1). In the Zahn-Wellens test, a test for inherent biodegradability which allows evaluation of aerobic degradation at high bacterial density, only a partial biological degradation was found. Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass spectrometry (HPLC-MSn) revealed 2-(3,4-dimethoxyphenyl)-2-isopropyl-5-(methylamino)pentane nitrile, already known as D617 (Knoll nomenclature), a metabolite of mammalian metabolism, which is the major degradation product and dead-end transformation product of aerobic degradation of verapamil.     

牛粪堆肥腐殖质的动态变化研究

牛粪堆肥实质是有机物质分解的同时形成腐殖质的过程。本研究通过向牛粪中接种发酵微生物进行堆肥,研究分析了总有机碳含量、总腐殖酸、游离腐殖酸和水溶性腐殖酸等的腐殖质物质含量的动态变化。研究结果表明：在牛粪发酵过程中,总有机碳含量呈明显降低趋势,发酵微生物处理下的下降幅度明显大于不接种发酵微生物的处理;总腐殖酸、游离腐殖酸和水溶性腐殖酸含量在发酵前期呈明显的降低趋势,但至发酵腐熟期又逐渐升高。发酵前期,外源微生物的处理,使其下降幅度明显大于不接种外源微生物的处理,但在发酵腐熟期变化不大。     

Mixtures of quaternary ammonium compounds and anionic organic compounds in the aquatic environment: Elimination and biodegradability in the closed bottle test monitored by LC-MS/MS

Quaternary ammonium compounds (QACs) are widely used as disinfectants, detergents and fabric softeners. Anionic detergents are one of the most widely used chemical substances. QACs and anionic surfactants can form ionic pairs. In the present study we investigated the biodegradability of QACs in the presence of different anionic surfactants. The biodegradability of three QACs, namely benzalkonium chloride (BAC), didecyldimethylammonium chloride (DDMAC) and ethacridine lactate (EL), when applied as single substances and in combination with anionic surfactants such as benzene sulfonic acid (BSA), LAS, naphthalene sulfonic acid (NSA) and sodium dodecylsulfonate (SDS) was studied applying the closed bottle test (CBT) [OECD 301D, 1992. Guidelines for Testing of Chemicals. Closed bottle test. Organisation of Economic Cooperation and Development, Paris] at a ratio of 1:1 (mol:mol). Biodegradation was monitored by measuring oxygen concentration in the test vessels with an oxygen electrode in accordance with international standard methods [ISO 5414, 1990. Water quality - determination of dissolved oxygen. In: German Standard Methods for the Examination of Water, Wastewater and Sludge. VCH Verlagsgesellschaft, Weinheim, New York, Basel Cambridge]. Primary elimination of the QACs and of LAS was monitored by LC-MS/MS. There was little biodegradability of the QACs as either single compounds or in the presence of organic counter ions. The biodegradability of the organic counter ions was lower in the presence of QACs as compared to the single substances. Primary elimination of the QACs by sorption took place.     

Incomplete aerobic degradation of the antidiabetic drug Metformin and identification of the bacterial dead-end transformation product Guanylurea

Active pharmaceutical ingredients as well as personal care products are detected in increasing prevalence in different environmental compartments such as surface water, groundwater and soil. Still little is known about the environmental fate of these substances. The type II antidiabetic drug Metformin has already been detected in different surface waters worldwide, but concentrations were significantly lower than the corresponding predicted environmental concentration (PEC). In human and mammal metabolism so far no metabolites of Metformin have been identified, so the expected environmental concentrations should be very high.To assess the aerobic biodegradability of Metformin and the possible formation of degradation products, three Organisation of Economic Cooperation and Development (OECD) test series were performed in the present study.In the Closed Bottle test (OECD 301 D), a screening test that simulates the conditions of an environmental surface water compartment, Metformin was classified as not readily biodegradable (no biodegradation). In the Manometric Respiratory test (OEDC 301 F) working with high bacterial density, Metformin was biodegraded in one of three test bottles to 48.7% and in the toxicity control bottle to 57.5%. In the Zahn-Wellens test (OECD 302 B) using activated sludge, Metformin was biodegraded in both test vessels to an extent of 51.3% and 49.9%, respectively.Analysis of test samples by high performance liquid chromatography coupled to multiple stage mass spectrometry (HPLC-MS(n)) showed in the tests vessels were biodegradation was observed full elimination of Metformin and revealed Guanylurea (Amidinourea, Dicyandiamidine) as single and stable aerobic bacterial degradation product. In another Manometric Respiratory test Guanylurea showed no more transformation. Photodegradation of Guanylurea was also negative.A first screening in one of the greatest sewage treatment plant in southern Germany found Metformin with high concentrations (56.8 μg L⁻¹) in the influent (PEC = 79.8 μg L⁻¹), but effluent concentration was much lower (0.76 μg L⁻¹) whereas Guanylurea was detected in a low influent and high effluent concentration (1.86 μg L⁻¹). These data support the experimental findings in the OECD tests and analytical results of other studies, that Metformin under aerobic conditions can bacterially be degraded to the stable dead-end transformation product Guanylurea.     

A method for measuring the anoxic biodegradability under denitrifying conditions

A test for assessing the anoxic biodegradability of organic compounds under denitrifying conditions is proposed. The method is based on the recovery and quantification of the CO2 produced, which is evidence of complete biodegradation of the test compound (added as the sole carbon source). The tests were carried out in a mineral medium, with nitrate as electron acceptor. Whole lake sediments, sediment extracts and a commercial inoculum were assayed as a possible inoculum source by means of glucose biodegradability tests. It was found that the sediment extracts constitute a suitable and environmentally-relevant inoculum source, since they add non-significant amounts of carbon to the tests. Two xenobiotic compounds, namely, aniline and phenol, were tested in the aforementioned conditions as well as in a standard aerobic biodegradability test. Both aniline and phenol attained a biodegradation level higher than 60% in a short time period (<28 days) and thus can be considered as readily biodegradable in denitrifying environments. Nevertheless, the kinetics obtained in the anoxic test were slower than in aerobic conditions, and even suggested the accumulation of intermediate metabolites in the case of phenol. The results of this study indicate that the fate of xenobiotic compounds under anoxic conditions differs from that observed in an oxic environment, and therefore it should be considered by standard biodegradability testing procedures.     

Evaluation of laboratory-made sludge for an anaerobic biodegradability test and its use for assessment of 13 chemicals

Laboratory-made sludge for a biogas based anaerobic biodegradability test was prepared as an alternative for digested sludge from wastewater treatment plants (WWTPs). Biodegradation activities and background gas productions of digested sludge from various WWTPs were found to vary significantly depending on the source, which adversely affected test reliability. Subsequently, test conditions such as sludge concentration and sludge washing were examined with the laboratory-made sludge and a sludge concentration of 1.0 g-SS/L without washing was determined to be most suitable. Under these conditions, biodegradability tests were conducted for 13 select chemicals and their relative toxicities to methanogenic bacteria were evaluated. The results of biodegradability tests showed that chemicals with -OH and -CH2OH radicals were readily biodegraded and those with -Cl, -NO2, -NH2, -SO3H and -CH3 had inhibited degradation responses m-nitriphenol and 2,4,6-trichlorophenol were highly toxic to methanogenic bacteria, with m-nitrophenol completely inhibiting methane fermentation as low as 20 mg/L.     

Determination of the aerobic biodegradability of polymeric material in aquatic batch tests

Results of an international ring-test of two laboratory methods are presented for investigating the biodegradability of organic polymeric test materials in aquatic test systems based on respirometry and the evolution of carbon dioxide. These methods are developed further from the well-known standardized biodegradation tests ISO 9408 (1999) and ISO 9439 (1999), which have been successfully used for many years. The most important improvements are the extension of the test period up to six months, the increase of the buffer capacity and nutrient supply of the inorganic medium, an optimization of the inoculation, and optionally, the possibility of a carbon balance. A ring test, organized by the International Biodeterioration Research Group (IBRG), was run using a poly(,-caprolactone)-starch blend and an aliphatic-aromatic co-polyester as test materials and a microcrystalline cellulose powder as a reference material. The test results and the experience gained by the participants showed that the methods are suitable and practicable. The test methods have been meanwhile established as standards ISO 14851 (1999) and ISO 14852 (1999).     

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.     

Direct VUV photodegradation of gaseous α-pinene in a spiral quartz reactor: intermediates, mechanism, and toxicity/biodegradability assessment

Photodegradation of gaseous α-pinene by a vacuum ultraviolet (VUV) in a spiral reactor was investigated under various gaseous reaction media and residence time, and their respective effects on types and biodegradability of the intermediates were studied. Analysis of carbon amounts showed that about 33% and 43% of total carbon were converted to soluble organic carbon in the air medium with a relative humidity (RH) of 35-40% at empty bed residence times (EBRTs) of 18 and 45 s. Based on the identified intermediates by GC/MS and IC, a photodegradation pathway was proposed by the combined roles of photolysis, OH. photooxidation and O? photooxidation. Biodegradability to active sludge, toxicity to Chlorella vulgaris and 96-well microplates showed that α-pinene could be largely converted to more biodegradable and less toxic compounds through photodegradation in the air reaction medium with a RH of 35-40% at an EBRT of 18s, in which the initial concentration was 600 mg m?3. Therefore, VUV photodegradation could be applied as an effective pre-treatment method for detoxification and biodegradability improvement under the optimized photodegradation conditions. Such results supported the potential use of VUV photodegradation to improve the removal capacity of conventional biological treatments for hydrophobic and poorly biodegradable compounds.