Manometric Respirometry Test Research Articles

How data on transformation products can support the redesign of sulfonamides towards better biodegradability in the environment

Sulfonamide antibiotics (SUAs) released into the environment can affect environmental und human health, e.g., by accelerating the development and selection of antimicrobial resistant bacteria. Benign by Design (BbD) of SUAs is an effective risk prevention approach. BbD principles aim for fast and complete mineralization or at least deactivation of the SUA after release into the aquatic environment. Main objective was to test if mixtures of transformation products (TPs) generated via photolysis of SUAs can be used as an efficient way to screen for similarly effective but better biodegradable SUA alternatives.Six SUAs were photolyzed (Hg ultraviolet (UV) light), and generated UV-mixtures analysed by high performance liquid chromatography coupled to an UV and tandem mass spectrometry detector. UV-mixtures were screened for antibiotic activity (luminescence bacteria test, LBT, on luminescence and growth inhibition of Aliivibrio Fischeri) and environmental biodegradability (manometric respirometry test, MRT, OECD 301F) using untreated parent SUAs in comparison. Additionally, ready environmental biodegradability of three commercially available hydroxylated sulfanilamide derivatives was investigated.SUA-TPs contributed to acute and chronic bacterial luminescence inhibition by UV-mixtures. LBT's third endpoint, growth inhibition, was not significant for UV-mixtures. However, it cannot be excluded for tested TPs as concentrations were lower than parents' concentrations and inhibition by most parental concentrations tested was also not significant. HPLC analysis of MRT samples revealed that one third of SUA-TPs was reduced during incubation. Three of these TPs, likely OH-SIX, OH-SMX and OH-STZ, were of interest for BbD because the sulfonamide moiety is still present. However, hydroxylated sulfanilamide derivatives, tested to investigate the effect of hydroxylation on biodegradability, were not readily biodegraded. Thus, improving mineralization through hydroxylation as a general rule couldn't be confirmed, and no BbD candidate could be identified. This study fills data gaps on bioactivity and environmental biodegradability of SUAs' TP-mixtures. Findings may support new redesign approaches.

Analysis of environmental biodegradability of cellulose-based pharmaceutical excipients in aqueous media

Pharmaceutical cellulosic polymers will inevitably reach natural water systems if they are not removed after entering wastewater. Biodegradation of organic chemicals in sewage or in the aquatic environment is an important removal mechanism. In this study, we investigated the environmental biodegradation of 14 cellulose derivatives commonly utilized as pharmaceutical excipients using three different test systems that are based on the closed bottle test (OECD 301D) and the manometric respirometry test (OECD 301F). For the different cellulose derivatives tested, we observed varying degrees of biodegradation ranging from 0 to 20.4 % chemical oxygen demand (COD). However, none met the criteria for classification as ‘readily biodegradable’. In addition, 10 out of 14 cellulose derivatives and/or their possible transformation products formed during the experiments, may exhibit possible toxic inhibitory effects on the inoculum. This includes one or several derivatives of hydroxy propyl methyl cellulose, hydroxy propyl cellulose, methyl cellulose, ethyl cellulose, and hydroxy ethyl cellulose. Based on the results obtained, we have developed a graded classification score (‘traffic light system’) for excipient biodegradation. This could help streamline the assessment and classification of cellulose derivatives concerning risk of persistence and potential adverse environmental effects, thereby assisting in the prioritization of more favorable compounds. In the long term, however, excipients should be designed from the very beginning to be biodegradable and mineralizable in the environment (‘benign by design’).

Determination of aerobic and anaerobic biological degradability of waste tyres

Environmental contamination of rubber from waste tyres poses a risk to the environment. Rubber particles from tyres enter the environment due to the abrasiveness of the road surface. The aim of the scientific work was to observe the biological degradability of waste tyres in aquatic environment and its ecotoxicity. Biodegradability was observed under aerobic and anaerobic conditions that simulate both aerobic and anaerobic conditions in the aquatic natural environment. Aerobic conditions in the aquatic environment take place in flowing fresh water, sea water, lakes. Leachate was prepared to simulate the behaviour of tyres in an aqueous environment. Aerobic degradability was evaluated through complete biodegradability using a 301 F manometric respirometry test. Anaerobic biodegradability was evaluated by measuring biogas production using OECD 311 Anaerobic Biodegradability of Organic Compounds in Digested Sludge. For a better simulation of the natural environment, the pH of the leachates from the tyres was adjusted to a neutral range. It should be noted that standard degradability tests were extended by 7 days due to low biodegradability. Adjusting the pH during the biodegradability test is also a modification of the original test. This modification was used to better simulate biodegradability when the pH of tyres in the natural environment is reduced by acid rain. An essential part of monitoring the behaviour of waste tyres was the assessment of ecotoxicity using standard tests. The contribution of the scientific article lies in the evaluation of the course of decomposition in aerobic and anaerobic conditions with and without pH adjustment and in the use of modified biodegradability tests. The benefit of the scientific work is in the determination of the biodegradability of waste tyres with and without pH treatment, which simulate a comparison of the degradability of tyres in an acid rain environment. Another benefit of the scientific work is the depiction of biodegradation using 3D modelling with calculations of 100% degradability at different input concentrations of waste tyres. Modelling was used for the time for the absolute decomposition of tyres without pH adjustment (outside the acid rain environment) and with pH adjustment (in the acid rain environment). By monitoring, it is possible to determine whether acid rain as an anthropogenic activity influences the degradability of waste tyres in the natural environment. Biodegradability tests confirmed the low biological degradability of waste tyres. The highest average rate of biological degradability—15% was recorded at the input concentration of waste tyres of 350 mg/L. The aerobic degradability test confirmed the improvement of tyre decomposition when adjusting the pH to the level of 6.5–7.5. On the contrary, the anaerobic degradability test confirmed the improvement of the decomposition in the alkaline region compared to the neutral pH values of the mixture. By mathematical–statistical evaluation of aerobic decomposition with preservation of degradability trends at three input concentrations, the time of absolute decomposition of waste tyre particles at a concentration of 370 mg/L was found to be approximately 336 days. By adjusting the pH to the neutral range during aerobic decomposition, the total decomposition time was reduced to 126 days. The ecotoxicity tests performed confirmed the toxic effect of tyre leachate on selected tested organisms. In the future, the authors propose to focus on a more detailed assessment of the ecotoxicity of the waste conditions and to modify the biodegradability tests by changing the conditions (wider range of input pH value, longer biodegradability time, temperature) for a better simulation of different types of environments. Adjusting the pH to a neutral environment increased aerobic degradability but had no significant effect on anaerobic degradability. Therefore, it is important to focus future research on the adjustment of various conditions to support the degradability of tyres, of which pH has clearly been confirmed as an important factor.

Ready Biodegradability Ring Testing of 4-Isopropylphenol in Different Laboratories for Critical Evaluation of a Biodegradable Reference Substance.

Ready biodegradability is a key property for studying the environmental and ecological risks posed by chemicals, and a reference substance is used to check the reliability of the test results. Traditional reference substances such as aniline, sodium benzoate, and ammonium acetate rapidly degrade even in test systems without inoculum. In this study, 4-isopropylphenol was investigated as a reference candidate. Aerobic biodegradation of 4-isopropylphenol was studied using different methods, and repeatability tests were performed in multiple laboratories. 4-Isopropylphenol met the ready biodegradability passing level in a closed bottle test (OECD 301D) and manometric respirometry test (OECD 301F). No obvious toxicity was found in activated sludge respiration inhibition and biodegradable toxicity control tests. The results of 301F tests performed in 15 Chinese laboratories were compared using statistical robustness analyses and Z-scores. The robust average 28-d biodegradability rate for 4-isopropylphenol was 80.3%, and the relative standard deviation was 9.20%. The data from the 15 laboratories consistently indicated that 4-isopropylphenol was readily biodegradable, except for an "unsatisfactory" outlier result from 1 laboratory. The degradation kinetics of 4-isopropylphenol were derived from a logistic model, which had a good fit for the degradation curve. The biodegradation lag phase was 2.3 d (range 2-5 d), and the pass level was attained at 6.5 d on average (range 2-9 d), which met the 10-d window requirement. The biodegradation rate at 28 d was 75.3% (range 64.0%-95.1%). The ready biodegradability of 4-isopropylphenol indicates that it will be more appropriate as a reference chemical than other reference compounds used for investigating the biodegradability of chemicals. Integr Environ Assess Manag 2021;17:562-572. © 2020 SETAC.

Application of seven different clay types in sorbent-modified biodegradability studies with cationic biocides.

The cationic surfactants cetyltrimethylammonium bromide (CTAB) and cetylpyridinium chloride (CPC) can exert inhibitory effects on micro-organisms responsible for their biodegradation. However, under environmentally relevant exposure scenarios the presence of and sorption to organic and inorganic matter can lead to significant reduction of inhibitory effects. In our studies we investigated silica gel and seven clays as inert sorbents to mitigate these inhibitory effects in a 28 day manometric respirometry biodegradation test. CTAB was not inhibitory to the used inoculum, but we did observe that seven out of eight sorbents increased maximum attainable biodegradation, and four out of eight decreased the lag phase. The strongly inhibitory effect of CPC was successfully mitigated by most sorbents, with five out of eight allowing >50% biodegradation within 28 days. Results further indicate that bioaccessibility of the sorbed fractions in the stirred manometric test systems was higher than in calmly shaken headspace test systems. Bioaccessibility might also be limited depending on characteristics of test chemical and sorbent type, with montmorillonite and bentonite apparently providing the lowest level of bioaccessibility with CPC. Clay sorbents can thus be used as environmentally relevant sorbents to mitigate potential inhibitory effects of test chemicals, but factors that impede bioaccessibility should be considered. In addition to apparently increased bioaccessibility due to stirring, the automated manometric respirometry test systems give valuable and highly cost-effective insights into lag phase and biodegradation kinetics; information that is especially relevant for test chemicals of gradual biodegradability.

Aeration and sawdust application effects as structural material in the bioremediation of clayey acid soils contaminated with diesel oil

Soil contamination, due to the leakage of petroleum or its by-products, is a reason for concern nowadays. Technologies, such as bioremediation, have been developed to remove these pollutants from the environment. This paper aims to evaluate the bioremediation of clayey acid soils contaminated with diesel oil, comparing the effect of aeration use and the addition of different sawdust ratios (5% and 10%) as bulking material. This research was developed in three phases, with experiments of (a) natural attenuation (exclusively natural processes), (b) sawdust-amended soil and (c) aeration combined with sawdust addition. The manometric respirometry test was applied to monitor CO2 production during biodegradation experiments and sodium hydroxide (NaOH) was used as an absorbent solution. The contamination of the samples was carried out by the addition of diesel oil (5% w/w (gg−1)) into the soil. The results indicated that natural attenuation presented a slower bioremediation process when compared to the treatments with sawdust addition. The addition of 5% of sawdust was beneficial to the microbial activity of the soil, but a proportion above 5% caused a negative effect on the samples, reducing the microbial activity. Statistical analysis (Kruskal-Wallis test) showed significant oil removals on experiments with the combination of sawdust and aeration. The aeration was considered the main agent in increasing oil biodegradation in the soil. The degradation rate reactions followed a zero-order linear model. The highest contaminant degradation rate was observed in the samples with 5% of sawdust and aeration (24.79 mgO2 kg−1dry soil day−1), which also improved microbial activity and diesel oil removal.

Biodegradation tests of mercaptocarboxylic acids, their esters, related divalent sulfur compounds and mercaptans

Mercaptocarboxylic acids and their esters, a class of difunctional compounds bearing both a mercapto and a carboxylic acid or ester functional group, are industrial chemicals of potential environmental concern. Biodegradation of such compounds was systematically investigated here, both by literature search and by experiments (Closed Bottle Test OECD 301D and Manometric Respirometry Test OECD 301F). These compounds were found either readily biodegradable or at least biodegradable to a significant extent. Some related compounds of divalent sulfur were tested for comparison (mercaptans, sulfides, disulfides). For the two relevant monofunctional compound classes, carboxylic acids/esters and mercaptans, literature data were compiled, and by comparison with structurally similar compounds without these functional groups, the influence of COOH/COOR’ and SH groups on biodegradability was evaluated. Thereby, an existing rule of thumb for biodegradation of carboxylic acids/esters was supported by experimental data, and a rule of thumb could be formulated for mercaptans. Concurrent to biodegradation, abiotic processes were observed in the experiments, rapid oxidative formation of disulfides (dimerisation of monomercaptans and cyclisation of dimercaptans) and hydrolysis of esters. Some problems that compromise the reproducibility of biodegradation test results were discussed.

Initial fate assessment of teratogenic drug trimipramine and its photo-transformation products – Role of pH, concentration and temperature

Trimipramine (TMP) is an antidepressant drug used for the treatment of a variety of depressive states and other psychiatric disorders. It has been already detected in the aquatic environment. Currently, no further knowledge is available on fate and effects of TMP in the aquatic environment. Therefore, we studied the biodegradability of TMP and of its photolysis transformation products (PTPs) generated by irradiation with polychromatic UV light in aqueous solution. Different conditions including initial drug concentration, pH, and temperature were applied during TMP photolysis. Subsequently, the time courses of TMP and dissolved organic carbon (DOC) concentrations were monitored throughout the whole photo-degradation process. Then, high-resolution mass spectrometry was used to identify and elucidate the structures of the resulting PTPs. After that, the two standardized biodegradation tests, Closed Bottle test (CBT; OECD 301 D) and Manometric Respirometry test (MRT; OECD 301 F), were performed for TMP and its photolytic mixtures to assess the biodegradability of TMP and its PTPs. Finally, the toxicity of TMP and its photolytic mixtures was predicted using different quantitative structure activity relationship (QSAR) software. It was found that after 128 min of UV-irradiation, 91.8% of TMP at the initial concentration of 100 mg L−1 was eliminated with only 23.9% removal in the DOC. So, it can be pointed out that more than 65% of the degraded TMP is transformed to new non-mineralized PTPs. 14 new PTPs were detected in TMP's photolytic mixtures. Their supposed structures indicate that the proposed photo-transformation pathway is mainly by hydroxylation. The statistical analysis confirms that the differences in the degradation rates of TMP as a function of concentration, pH, and temperature are statistically significant in most cases investigated here. In biodegradation testing, TMP and its PTPs are classified as not readily biodegradable, while LC-MS analysis revealed some PTPs to be eliminated more than TMP itself. Results from QSAR analysis confirmed that some of the PTPs could be biodegradable, and revealed that some of the non-biodegradable PTPs may be human and/or eco-toxic, posing a risk to the environment. Our findings show that TMP under UV-irradiation could lead to the formation of some more easily biodegradable PTPs and some others toxic and non-biodegradable PTPs. Therefore, further studies should be conducted regarding the fate and effects of TMP and its PTPs elucidated in this study on human health and on the environment.

Experimental and in silico assessment of fate and effects of the antipsychotic drug quetiapine and its bio- and phototransformation products in aquatic environments

The antipsychotic drug quetiapine (QUT) has been frequently detected in sewage treatment plants. However, information on the fate of QUT in aquatic environments and its behavior during UV treatment is limited. In this study, QUT is shown not to be readily biodegradable in the Closed Bottle Test and the Manometric Respirometry Test according to OECD guidelines. The main biotransformation product (BTP) formed in the tests, a carboxylic acid derivative, was identified by means of high-resolution mass spectrometry. This BTP is presumably a human metabolite and showed higher detection rates than QUT in a river sampling campaign conducted in northern Germany. UV elimination kinetics of QUT at different initial concentrations (226.5, 45.3, 11.3, and 2.3 μmol L−1) were faster at lower initial concentrations. All seven phototransformation products (PTPs) could be still identified at initial concentration of 11.3 μmol L−1. The photolytic mixture generated after 128 min of photolysis of QUT was not better biodegradable than QUT. Initial UV treatment of QUT led to the formation of several additional BTPs. Four of them were identified. The bacterial cytotoxicity and genotoxicity before and after phototransformation of QUT in a modified luminescent bacteria test (LBT) and the umu-test (ISO/FDIS 13829) showed cytotoxic effects in the LBT for QUT. Furthermore, PTPs had similar cytotoxic effects on luminescent bacteria. The umu-test did not reveal any genotoxic activity for QUT or PTPs. In conclusion, the release of QUT into sewage treatment plants and aquatic environments could result in the formation of a main BTP. Additional UV treatment of QUT would lead to the formation of additional BTPs. Moreover, treatment did not result in lower toxicity to tested organisms. In conclusion, UV treatment of QUT should be considered critically as a potential treatment for QUT in aquatic systems.

Elucidation of the upper pathway of alicyclic musk Romandolide® degradation in OECD screening tests with activated sludge

The degradation of Romandolide ([1-(3',3'-dimethyl-1'-cyclohexyl)ethoxycarbonyl] methyl propanoate), a synthetic alicyclic musk, by activated sludge inocula was investigated using both the manometric respirometry test OECD 301F and the CO₂ evolution test. In addition to measuring its biodegradability, key steps of the upper part of the metabolic pathway responsible for Romandolide degradation were identified using extracts at different time points of incubation. Early metabolism of Romandolide yielded ester hydrolysis products, including Cyclademol (1-(3,3-dimethylcyclohexyl)ethanol). The principal metabolites after 31 days were identified as 3,3-dimethyl cyclohexanone and 3,3-dimethyl cyclohexyl acetate. Formation of 3,3-dimethyl cyclohexanone from Cyclademol by sludge was confirmed in subsequent experiments using Cyclademol as a substrate, indicating the involvement of an oxygen insertion reminiscent of a Baeyer-Villiger oxidation. Further mineralization of 3,3-dimethyl cyclohexanone was also confirmed in subsequent studies. Three steps were thus required for complete biodegradation of the alicyclic musk: (1) successive ester hydrolyses leading to the formation of Cyclademol with concomitant degradation of the resulting acids, (2) conversion of Cyclademol into 3,3-dimethyl cyclohexanone, and (3) further mineralization via ring cleavage.

Aquatic photochemistry, abiotic and aerobic biodegradability of thalidomide: Identification of stable transformation products by LC–UV–MSn

Thalidomide (TD), besides being notorious for its teratogenicity, was shown to have immunomodulating and anti-inflammatory activities. This is why recently TD became a promising drug for the treatment of different cancers and inflammatory diseases. Yet nothing is known about the environmental fate of TD, which therefore was assessed experimentally and by in silico prediction programs (quantitative structure activity relationship (QSAR) models) within this study. Photolytic degradation was tested with two different light sources (medium-pressure mercury lamp; xenon lamp) and aerobic biodegradability was investigated with two OECD tests (Closed Bottle test (CBT), Manometric Respirometry test (MRT)). An additional CBT was performed for TD samples after 16min of UV-photolysis. The primary elimination of TD was monitored and the structures of its photo-, abiotic and biodegradation products were elucidated by HPLC–UV–Fluorescence–MSn. Furthermore, elimination of dissolved organic carbon was monitored in the photolysis experiment. LC–MS revealed that new photolytic transformation products (TPs) were identified, among them two isomers of TD with the same molecular mass. These TPs were different to the products formed by biodegradation. The experimental findings were compared with the results obtained from the in silico prediction programs where e.g. a good correlation for TD biodegradation in the CBT was confirmed. Moreover, some of the identified TPs were also structurally predicted by the MetaPC software. These results demonstrate that TD and its TPs are not readily biodegradable and not fully mineralized by photochemical treatment. They may therefore pose a risk to the aquatic environment due to the pharmacological activity of TD and unknown properties of its TPs. The applied techniques within this study emphasize the importance of QSAR models as a tool for estimating environmental risk assessments.

Photolysis of sulfamethoxypyridazine in various aqueous media: Aerobic biodegradation and identification of photoproducts by LC-UV–MS/MS

Sulfonamides are one of the most frequently used antibiotics worldwide. Therefore, mitigation processes such as abiotic or biotic degradation are of interest. Photodegradation and biodegradation are the potentially significant removal mechanisms for pharmaceuticals in aquatic environments. The photolysis of sulfamethoxypyridazine (SMP) using a medium pressure Hg-lamp was evaluated in three different media: Millipore water pH 6.1 (MW), effluent from sewage treatment plant pH 7.6 (STP), and buffered demineralized water pH 7.4 (BDW). Identification of transformation products (TPs) was performed by LC-UV–MS/MS. The biodegradation of SMP using two tests from the OECD series was studied: Closed Bottle test (OECD 301 D), and Manometric Respirometry test (OECD 301 F). In biodegradation tests, it was found that SMP was not readily biodegradable so it may pose a risk to the environment. The results showed that SMP was removed completely within 128min of irradiation in the three media, and the degradation rate was different for each investigated type of water. However, dissolved organic carbon (DOC) was not removed in BDW and only little DOC removal was observed in MW and STP, thus indicating the formation of TPs. Analysis by LC-UV–MS/MS revealed new TPs formed. The hydroxylation of SMP represents the main photodegradation pathway.

Captopril and its dimer captopril disulfide: Photodegradation, aerobic biodegradation and identification of transformation products by HPLC–UV and LC–ion trap-MSn

In some countries effluents from hospitals and households are directly emitted into open ditches without any further treatment and with very little dilution. Under such circumstances photo- and biodegradation in the environment can occur. However, these processes do not necessarily end up with the complete mineralization of a chemical. Therefore, the biodegradability of photoproduct(s) by environmental bacteria is of interest.Cardiovascular diseases are the number one cause of death globally. Captopril (CP) is used in this study as it is widely used in Egypt and stated as one of the essential drugs in Egypt for hypertension. Three tests from the OECD series were used for biodegradation testing: Closed Bottle test (CBT; OECD 301 D), Manometric Respirometry test (MRT; OECD 301 F) and the modified Zahn–Wellens test (ZWT; OECD 302 B). Photodegradation (150W medium-pressure Hg-lamp) of CP was studied. Also CBT was performed for captopril disulfide (CPDS) and samples received after 64min and 512min of photolysis.The primary elimination of CP and CPDS was monitored by LC–UV at 210nm and structures of photoproducts were assessed by LC–UV–MS/MS (ion trap). Analysis of photodegradation samples by LC–MS/MS revealed CP sulfonic acid as the major photodegradation product of CP. No biodegradation was observed for CP, CPDS and of the mixture resulting from photo-treatment after 64min in CBT. Partial biodegradation in the CBT and MRT was observed in samples taken after 512min photolysis and for CP itself in MRT. Complete biodegradation and mineralization of CP occurred in the ZWT.

Biodegradability and ecotoxicitiy of tramadol, ranitidine, and their photoderivatives in the aquatic environment

This study was designed to assess the fate and the overall potential impacts of the widely prescribed drugs ranitidine and tramadol after their introduction into the aquatic environment. The probability to detect these two drugs in the aquatic environment was studied by analyzing their abiotic and biotic degradation properties. For this purpose, samples were irradiated with different light sources, and three widely used biodegradability tests from the OECD series, the closed bottle test (OECD 301 D), the manometric respirometry test (OECD 301 F) and the Zahn-Wellens test (OECD 302 B), were conducted. The ecotoxicity of the photolytically formed transformation products was assessed by performing the bacterial growth inhibition test (EN ISO 10712). Furthermore, quantitative structure-activity relationship analysis and a risk analysis based on the calculation of the predicted environmental concentrations have also been conducted to assess the environmental risk potential of the transformation products. The possible formation of stable products by microbial or photolytical transformation has been investigated with DOC and LC-MS analytics. In the present study, neither ranitidine, nor tramadol, nor their photoderivatives were found to be readily or inherently biodegradable according to test guidelines. The photolytic transformation was faster under a UV lamp compared to the reaction under an Xe lamp with a spectrum that mimics sunlight. No chronic toxicity against bacteria was found for ranitidine or its photolytic decomposition products, but a low toxicity was detected for the resulting mixture of the photolytic transformation products of tramadol. The study demonstrates that transformation products may have a higher environmental risk potential than the respective parent compounds.

Persistency assessment and aerobic biodegradation of selected cyclic sesquiterpenes present in essential oils

Sesquiterpenes are ubiquitous in essential oils but an assessment of their environmental behavior is still required for their use as components of natural fragrance ingredients and oral care flavors. Persistency plays a key role in hazard and risk assessment, but the current knowledge on the biodegradation of sesquiterpenes in the aquatic environment is limited. This could have important consequences for the persistent, bioaccumulative and toxic (PBT) assessment of essential oils because most of the sesquiterpene components have a log K(OW) of >4.5 and are identified as potentially bioaccumulating according to REACH screening criteria. In the present study, a persistency screening assessment was conducted on 11 cyclic sesquiterpenes selected from 10 different families of sesquiterpenes characterized by their carbon skeleton. Current biodegradation prediction models (BioWin™, BioHCwin, and Catalogic) were found to be of limited use because most of the sesquiterpenes studied were outside the structural domain of the models. Aerobic biodegradation was measured in a standard or prolonged Organisation for Economic Co-operation and Development (OECD) 301F Manometric Respirometry test for ready biodegradability. α-Bisabolol, α-humulene, β-caryophyllene, α-cedrene, cedrol, longifolene, and δ-cadinene exceeded the pass level of 60% degradation and can be regarded as not persistent. Alpha-gurjunene, himachalenes (α, β, γ), and (-)-thujopsene almost achieved the pass level reaching between 51% and 56% ultimate biodegradation. Although germacrene D only achieved 24% ultimate biodegradation, specific analysis at the end of the test did indicate complete primary degradation. Given that the shape of the biodegradation curves indicates poor bioavailability and ready biodegradability tests are very stringent, it is expected that all the sesquiterpenes tested in the present study would be degraded under environmental conditions.

Synthesis of Room‐Temperature Ionic Liquids with the Weakly Coordinating [Al(ORF)4]− Anion (RF=C(H)(CF3)2) and the Determination of Their Principal Physical Properties

A large series of ionic liquids (ILs) based on the weakly coordinating alkoxyaluminate [Al(hfip)(4)](-) (hfip: hexafluoroisopropoxy) with classical as well as functionalized cations were prepared, and their principal physical properties determined. Melting points are between 0 ([C(4)MMIM][Al(hfip)(4)]) and 69 °C ([C(3)MPip][Al(hfip)(4)]); three qualify as room-temperature ILs (RTILs). Crystal structures for six ILs were determined; their structural parameters and anion-cation contacts are compared here with known ILs, with a special focus on their influence on physical properties. Moreover, the biodegradability of the compounds was investigated by using the closed-bottle and the manometric respirometry test. Temperature-dependent viscosities and conductivities were measured between 0 and 80 °C, and described by either the Vogel-Fulcher-Tammann (VFT) or the Arrhenius equations. Moreover, conductivities and viscosities were investigated in the context of the molecular volume, V(m). Physical property-V(m) correlations were carried out for various temperatures, and the temperature dependence of the molecular volume was analyzed by using crystal structure data and DFT calculations. The IL ionicity was investigated by Walden plots; according to this analysis, [Al(hfip)(4)](-) ILs may be classified as "very good to good ILs"; while [C(2)MIM][Al(hfip)(4)] is a better IL than [C(2)MIM][NTf(2)]. The dielectric constants of ten [Al(hfip)(4)](-) ILs were determined, and are unexpectedly high (ε(r)=11.5 to 16.8). This could be rationalized by considering additional calculated dipole moments of the structures frozen in the solid state by DFT. The determination of hydrogen gas solubility in [Al(hfip)(4)](-) RTILs by high-pressure NMR spectroscopy revealed very high hydrogen solubilities at 25 °C and 1 atm. These results indicate the significant potential of this class of ILs in manifold applications.

Degradation Studies of New Substitutes for Perfluorinated Surfactants

Perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) are manmade, stable perfluorosurfactants. The properties of perfluoroalkylated compounds that cause them to persist in the environment are also the properties that made them attractive compounds for industrial usage for over 50 years. Due to the unique properties of the carbon-fluorine bond and the polarity of perfluoroalkyl groups, potential substitutes to replace perfluorinated surfactants in most cases continue to be perfluoroalkyl based. Thus, issues of persistence in the environment remain. There is a need to test emerging new substitute surfactants for biodegradability. This study involved degradability measurements of emerging perfluorinated surfactant substitutes. The stability of the substitutes of perfluorinated surfactants was tested by employing advanced oxidation processes, which were based on degradation by ultraviolet lamp, hydrogen peroxide, or both, followed by conventional tests, among them an automated method based on the manometric respirometry test (OECD 301 F; OxiTop), closed-bottle test (OECD 301 D), and standardized fixed-bed bioreactor on perfluorobutane sulfonate, fluorosurfactant Zonyl, two fluoraliphatic esters (NOVEC FC-4430 and NOVEC FC-4432), and 10-(trifluoromethoxy) decane 1 sulfonate. Most of these new surfactants are well established in the marketplace and have been used in several applications as alternatives to PFOS- and PFOA-based surfactants. Ready biodegradation tests for fluoroaliphatic esters, the fluorosurfactant Zonyl, perfluorobutane sulfonate, and 10-(trifluoromethoxy) decane-1-sulfonate using the manometric respirometry test (OxiTop) did not meet the ready biodegradability test criteria. However, 10-(trifluoromethoxy) decane-1-sulfonate was observed to be degradable when a standardized fixed-bed bioreactor test was applied.

Application of the OECD 301F respirometric test for the biodegradability assessment of various potential endocrine disrupting chemicals

The biodegradability of several potential endocrine disrupting compounds, namely 4- n-nonylphenol (4- n-NP), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), bisphenol A (BPA), triclosan (TCS), di-(2-ethylhexyl)-phthalate (DEHP), perfluorooctanoate (PFOA) and perfluorononanoate (PFNA) was evaluated in this study, using OECD method 301F (manometric respirometry test) and activated sludge as inoculum. According to the results, 4- n-NP and BPA meet the strict definition of ready biodegradability and they are not expected to be persistent during the activated sludge process. Partial biodegradation was observed for DEHP (58.7 ± 5.7%, n = 3), TCS (52.1 ± 8.5%, n = 3) and NP1EO (25.9 ± 8.1%, n = 3), indicating their possible biodegradation in wastewater treatment systems, while no biodegradation was observed for NP2EO, PFOA and PFNA. Experiments in the co-presence of a readily biodegradable compound showed the absence of co-metabolic phenomena during 4- n-NP, BPA and TCS biodegradation. Using first order kinetics to describe biodegradation of the target compounds, half-lives of 4.3 ± 0.6, 1.3 ± 0.2, 1.8 ± 0.5, 6.9 ± 2.6 days were calculated for 4- n-NP, BPA, TCS and DEHP, respectively. Toxicity tests using marine bacterium Vibrio fischeri showed that biodegradation of 4- n-NP, NP1EO, BPA and TCS is a simultaneous detoxification process, while possible abiotic or biotic transformations of NP2EO, DEHP, PFOA and PFNA during respirometric test resulted to significant increase of their toxicities.

Evaluation and Modification of the OECD 301F Respirometry Biodegradation Test Method with Regard to Test Substance Concentration and Inoculum

The majority of OECD (Organisation for Economic Co-operation and Development) ready biodegradation test methods require a relatively high concentration of test substance. Such high dosage concentrations are unsuitable for screening of inhibitory or biocidal compounds. Unfortunately, the method with the lowest dosage concentration (Closed Bottle Test, 301D) also has the lowest biodegradation potential. Therefore, we have evaluated the ability of the Manometric Respirometry (301F) test method to study biodegradation of test substances at concentrations of a few mgl−1, using sodium acetate as a model substance. Results have been compared to those obtained from closed bottle tests (301D). Problems with using activated sewage as an inoculum have been demonstrated, as respirometry curves were biphasic, and the recorded biological oxygen demand (BOD) was higher than the calculated theoretical oxygen demand (ThOD). A consortium of bacteria isolated from activated sewage was used as an alternative inoculum. Using the alternative inoculum for respirometry tests, monophasic growth curves were observed with recorded BOD values at expected levels. Sensitivity thresholds for the respirometer tests were comparable to those in closed bottles, with biodegradation being detected at concentrations as low as 2.5 mgl-1 ThOD. Comparison of whole community carbon source utilisation profiles for both inocula (obtained using BIOLOG microplates) revealed their catabolic potential to be similar. Therefore, use of an activated sewage-derived inoculum is recommended as an alternative inoculum for respirometer-based biodegradation testing.

Biodegradation of All Stereoisomers of the EDTA Substitute Iminodisuccinate by Agrobacterium tumefaciens BY6 Requires an Epimerase and a Stereoselective C-N Lyase

Biodegradation tests according to Organization for Economic Cooperation and Development standard 301F (manometric respirometry test) with technical iminodisuccinate (IDS) revealed ready biodegradability for all stereoisomers of IDS. The IDS-degrading strain Agrobacterium tumefaciens BY6 was isolated from activated sludge. The strain was able to grow on each IDS isomer as well as on Fe(2+)-, Mg(2+)-, and Ca(2+)-IDS complexes as the sole carbon, nitrogen, and energy source. In contrast, biodegradation of and growth on Mn(2+)-IDS were rather scant and very slow on Cu(2+)-IDS. Growth and turnover experiments with A. tumefaciens BY6 indicated that the isomer R,S-IDS is the preferred substrate. The IDS-degrading enzyme system isolated from this organism consists of an IDS-epimerase and a C-N lyase. The C-N lyase is stereospecific for the cleavage of R,S-IDS, generating d-aspartic acid and fumaric acid. The decisive enzyme for S,S-IDS and R,R-IDS degradation is the epimerase. It transforms S,S-IDS and R,R-IDS into R,S-IDS. Both enzymes do not require any cofactors. The two enzymes were purified and characterized, and the N-termini were sequenced. The purified lyase and also the epimerase catalyzed the transformation of alkaline earth metal-IDS complexes, while heavy metal-IDS complexes were transformed rather slowly or not at all. The observed mechanism for the complete mineralization of all IDS isomers involving an epimerase offers an interesting possibility of funneling all stereoisomers into a catabolic pathway initiated by a stereoselective lyase.