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.

Cationic ester-containing gemini surfactants: chemical hydrolysis and biodegradation.

Two cationic gemini surfactants having ester bonds between the hydrophobic tail and the cationic moiety have been synthesized. The ester bonds were either with the ester carbonyl group away from the positive charge (esterquat type arrangement) or facing the positive charge (betaine ester type arrangement). The chemical hydrolysis of the surfactants was investigated and compared with the hydrolysis of the corresponding monomeric surfactants. The betaine ester type of surfactants was found to hydrolyze much faster than the esterquat surfactants. It was also seen that above the critical micelle concentration the gemini surfactants were much more susceptible to alkaline hydrolysis than the corresponding monomeric surfactants. The biodegradation of the geminis and the monomeric surfactants were also studied. It was found that whereas the monomeric surfactants were rapidly degraded, the two gemini surfactants were more resistant to biodegradation and could not be classified as readily biodegradable. The 60% biodegradation was reached after 35-40 days. Thus, there was no correlation between rate of chemical hydrolysis and rate of biodegradation.

Environmental exposure assessment of ethinyl estradiol (EE) from a combined hormonal vaginal contraceptive ring after disposal; leaching from landfills.

In this study an environmental exposure assessment and experiments were carried out to identify the leaching potential of ethinyl estradiol (EE) present in a vaginal contraceptive (NuvaRing) when disposed of in landfills. Landfill material and a sandy soil were used to investigate the mobility of EE. Log K(oc) values determined in the range of 3 to 4 indicate that EE does not have a high mobility in landfills and soils. Column experiments were used to estimate that it takes approximately 40 years before EE leaches from a column of 1 m of landfill material or sandy soil. This column experiment, which was performed with an EE concentration based on worst-case assumptions, demonstrates that the emission of EE from landfills is negligible. Sandy soils below landfills also act as a strong sorbent of EE, thereby further reducing the potential for groundwater contamination.

Read-across of ready biodegradability based on the substrate specificity of N-alkyl polypropylene polyamine-degrading microorganisms.

The biodegradation of N-alkyl polypropylene polyamines (NAPPs) was studied using pure and mixed cultures to enable read-across of ready biodegradability test results. Two Pseudomonas spp. were isolated from activated sludge with N-oleyl alkyl propylene diamine and N-coco alkyl dipropylene triamine, respectively. Both strains utilized all NAPPs tested as the sole source of carbon, nitrogen and energy for growth. Mineralization of NAPPs was independent of the alkyl chain length and the size of the polyamine moiety. NAPPs degraded in closed bottle tests (CBTs) using both river water and activated sludge. However, ready biodegradability of NAPPs with alkyl chain lengths of 16-18 carbon atoms and polyamine moieties with three and four nitrogen atoms could not be demonstrated. Biodegradation in the CBT was hampered by their limited bioavailability, making assessment of the true ready biodegradability of these highly adsorptive surfactants impossible. All NAPPs are therefore classified as readily biodegradable through read-across. Read-across is justified by the broad substrate specificity of NAPP-degrading microorganisms, their omnipresence and the mineralization of NAPPs.

Pretreatment and biotreatment of saline industrial wastewaters.

Wastewater from an Akzo Nobel production site contains more than 50 g/l total dissolved salts, mainly chlorides and sulphates, and is currently being treated after 10-20 x dilution. Biological treatment of undiluted or less diluted wastewater is very desirable for environmental and economic reasons. Possibilities were investigated in laboratory scale reactors to treat this highly saline and high strength wastewater aerobically, either after long adaptation or after removing part of the salts by a pretreatment. Adaptation and selection from mixed activated sludge populations took approximately 40 days to finally achieve a COD removal in aerobic treatment of 55-65% at two times dilution (11-16 g/l chloride and 5-7 g/l sulphate). Undiluted wastewater was not treatable. A higher removal percentage (> 80%) was possible at the original high salt concentration only when the sludge load was limited to approximately 0.4-0.5 kg COD/kg sludge/day. A longer adaptation time was required. Nanofiltration (NF) and crystallization could be used as a pretreatment to remove and recover up to 80% of the sulphate in the form of crystallized Glauber salt. Recovery strongly depended on the sulphate and chloride concentration in the NF concentrate and on crystallization temperature. The salt (sulphate) reduction through the NF improved the removal efficiency of a consecutive biotreatment only at a relatively low chloride level, demonstrating that the combination of nanofiltration-crystallization-aerobic biodegradation is less feasible for very saline wastewaters. Anaerobic pretreatment of saline waters turned out to be rather sensitive to high salinities. Only wastewater diluted to 10 g/l chloride could be treated well: sulphate concentration decreased by 80% and COD by 40%. Removal efficiencies of the combined anaerobic-aerobic treatment were approximately 80-85%, proving that this was a feasible route for 2-3 x diluted wastewater. The study has shown that several alternatives are available for treatment of the very saline wastewaters at a much lower degree of dilution than currently practiced.

Reduction of bromate to bromide coupled to acetate oxidation by anaerobic mixed microbial cultures.

Bromate, a weakly mutagenic oxidizing agent, exists in surface waters. The biodegradation of bromate was investigated by assessing the ability of mixed cultures of micro-organisms for utilization of bromate as electron acceptor and acetate as electron donor. Reduction of bromate was only observed at relatively low concentrations (<3.0 mM) in the absence of molecular oxygen. Under these conditions bromate was reduced stoichiometrically to bromide. Unadapted sludge from an activated sludge treatment plant and a digester reduced bromate without lag period at a constant rate. Using an enrichment culture adapted to bromate, it was demonstrated that bromate was a terminal electron acceptor for anaerobic growth. Approximately 50% of the acetate was utilized for growth with bromate by the enrichment culture. A doubling of 20 h was estimated from a logarithmic growth curve. Other electron acceptors, like perchlorate, chlorate and nitrate, were not reduced or at negligible rates by bromate-utilizing microorganisms.

Cationic gemini surfactants with cleavable spacer: chemical hydrolysis, biodegradation, and toxicity.

The paper describes synthesis and characterization of a new type of cationic gemini surfactant, which has dodecyl tails and a spacer that contains an ester bond. The nomenclature used to describe the structure is 12Q2OCO1Q12, with Q being a quaternary ammonium group and the numbers indicating the number of methylene or methyl groups. Due to the close proximity to the two quaternary ammonium groups, the ester bond is very stable on the acid side and very labile already at slightly alkaline conditions. The hydrolysis products are two single chain surfactants (i.e. 12Q2OH and 12Q1COOH) which are less surface active than the intact gemini surfactant. 12Q2OCO1Q12 was found to be readily biodegradable, i.e. it gave more than 60% biodegradation after 28 days. This is interesting because similar gemini surfactants but with ester bonds in the tails instead of the spacer, have previously been found not to be readily biodegradable. The gemini surfactant was found to be toxic to aquatic organisms (ErC50 value of 0.27 mg/l), although less toxic than the two hydrolysis products.

Persistence assessment of cyclohexyl- and norbornyl-derived ketones and their degradation products in different OECD screening tests.

The persistence of synthetic cyclohexyl- and norbornyl-derived ketones was assessed by using OECD 301F and 301D biodegradation tests. While cyclohexyl-derived ketones either reached or came close to the pass level (60%) after 60 d, the corresponding norbornyl derivatives yielded significantly less biodegradation (<40%). By analyzing extracts at 60 d, the key degradation products of four norbornyl derivatives were identified. Consistently, 2-bicyclo[2.2.1]heptane carboxylic acid was found as a principal degradation product with minor quantities of bicyclo[2.2.1]heptan-2-one and 2-bicyclo[2.2.1]heptane acetic acid. When the three degradation products were re-synthesized and tested individually for biodegradability, the former two were found to be ultimately biodegradable after 60 d in OECD 301D tests, thus proving non-persistence. Similarly, 2-bicyclo[2.2.1]heptane acetic acid was found to be degraded significantly, albeit with long lag phases exceeding 60 d in the case of freshwater inoculum, then ultimately reaching the pass level. On the other hand, norbornyl ketones were still only partially biodegradable in the same test. We conclude that despite the potential for ultimate biodegradation of norbornyl-derived ketones, current screening tests yield an incomplete picture of their biodegradability, particularly when applying strict OECD criteria. The appearance of long lag phases when re-testing norbornyl ketone degradation products underlines the importance of extending tests to well beyond 28 and even 60 d in the case of freshwater inocula.

Biodegradation of p-toluenesulphonamide by a Pseudomonas sp.

A bacterium capable of utilising p-toluenesulphonamide was isolated from activated sludge. The isolated strain designated PTSA was identified as a Pseudomonas sp. using chemotaxonomic and genetic studies. Pseudomonas PTSA grew on p-toluenesulphonamide in a chemostat with approximately 90% release of sulphate and 80% release of ammonium. The isolate was also able to grow on 4-carboxybenzenesulphonamide and 3,4-dihydroxybenzoate but did not grow on p-toluenesulphonate. The transient appearance of 4-hydroxymethylbenzenesulphonamide and 4-carboxybenzenesulphonamide during p-toluenesulphonamide degradation proves oxidation of the methyl group is the initial attack in the biodegradation pathway. Both metabolites of p-toluenesulphonamide degradation were identified by high-performance liquid chromatography-mass spectrometry. 4-Carboxybenzenesulphonamide is probably converted into 3,4-dihydroxybenzoate and amidosulphurous acid. The latter is a chemically unstable compound in aqueous solutions and immediately converted into sulphite and ammonium. Both sulphite and ammonium were formed during degradation of 4-carboxybenzenesulphonamide.

Biodegradability of ethoxylated fatty amines: detoxification through a central fission of these surfactants.

Non-ionic surfactants are initially degraded by a central fission of the molecule or by the oxidation of the far end of the polyoxyethylene (EO) chain. Alcohol ethoxylates are metabolized via a central fission, whereas alkylphenol ethoxylates are degraded by a stepwise shortening of the polyoxyethylene moiety. The biodegradation curves of ethoxylated fatty amines suggest a 'rapid' mineralization via an oxidation of the alkyl chain. The intermediates formed, viz., secondary ethoxylated amines, are 'slowly' biodegraded. This 'total' mineralization of the ethoxylated fatty amines was demonstrated in 'prolonged' Closed-Bottle tests. Decisive evidence for a central fission of ethoxylated fatty amines was obtained in a pure culture study. An isolated Pseudomonas sp. cleaved the C(alkyl)-N bond of octadecyl-bis(2-hydroxyethyl) amine and utilized the alkyl chain as sole carbon and energy source. Biodegradation products of both alcohol ethoxylates and ethoxylated fatty amines, formed through a central fission of the molecule, are non-toxic.

Degradation of ethinyl estradiol by nitrifying activated sludge.

Degradation of ethinyl estradiol (EE2) by nitrifying activated sludge was studied with micro-organisms grown in a reactor with feedback of sludge fed with only a mineral salts medium containing ammonium as the sole energy source. Ammonium was oxidised by this sludge at a rate of 50 mg NH4+ g(-1) DW h(-1). This activated sludge was also capable of degrading EE2 at a maximum rate of 1 microg g(-1) DW h(-1). Using sludge with an insignificant nitrifying capacity of 1 mg NH4+ g(-1) DW h(-1), no degradation of EE2 was detected. Oxidation of EE2 by nitrifying sludge resulted in the formation of hydrophilic compounds, which were not further identified. Most probably degradation by nitrifying sludge results in a loss of estrogenic activity, as hydroxylated derivatives of EE2 are known to have a substantially lower pharmacological activity than EE2.

Biological reduction of chlorate in a gas-lift reactor using hydrogen as an energy source.

Chlorate release into the environment occurs with its manufacture and use. Biological reduction of chlorate offers an attractive option to decrease this release. A hydrogen gas-lift reactor with microorganisms attached to pumice particles was used for the treatment of wastewater containing high concentrations of chlorate. The microorganisms used chlorate as an electron acceptor and hydrogen gas as a reducing agent. After a start-up period of only a few weeks, chlorate reduction rates of 3.2 mmol L(-1) h(-1) were achieved during continuous operation. During this period, a hydrogen consumption rate of 14.5 mmol L(-1) h(-1) was observed. Complete removal of chlorate was maintained at hydraulic retention times of 6 h. This study clearly demonstrates the potential of hydrogen gas-lift bioreactors for the treatment of chlorate-containing waste streams.

Cometabolic reduction of bromate by a mixed culture of microorganisms using hydrogen gas in a gas-lift reactor.

The discharge of bromate, a suspected carcinogen, will be restricted in the near future. To assess the possibility of biotechnological treatment of bromate-containing wastewaters, the removal of bromate by chlorate-reducing microorganisms was studied. The removal of bromate and chlorate was studied in laboratory gas-lift bioreactors supplied with hydrogen gas as electron donor in the absence of molecular oxygen. In these reactors, bromate was reduced cometabolically by chlorate-respiring microorganisms. To allow the cometabolic reduction of bromate, a chlorate:bromate molar ratio of at least 3:1 was required. The cometabolic conversion permitted almost complete reduction of bromate into bromide at hydraulic retention times of at least 6 h. Optimal bromate reduction activity was observed at approximately 35 degrees C. The pH optimum was between 7 and 8. Bromate reduction in excess of 80% and a maximum bromate reduction rate of 2.3 g l(-1) day(-1) in a pilot-scale gas-lift bioreactor demonstrates that the process is sustainable.

Complete degradation of xenobiotic surfactants by consortia of aerobic microorganisms.

Linear alkylbenzene sulphonates are primarily attacked via a hydroxylation of the alkyl chain from the methyl group followed by beta-oxidation. The alkyl chain is metabolized by pure cultures to give sulphophenyl carboxylates which accumulate in the medium. In mixed culture, other microorganisms are capable of degrading sulphophenyl carboxylates. Formation of ethylene glycol monosulphates as major products of alkyl ethoxy sulphates demonstrates that the ether bonds are cleaved. The bacteria involved in growing on the alkyl chain are unable to utilize the hydrophilic moiety. This hydrophilic moiety, in turn, is degraded by other microorganisms. The degradation of alkylphenol ethoxylates and highly branched alcohol ethoxylates proceeds by shortening the polyoxyethylene chain leaving the hydrophobic part of the molecule. The biodegradation of linear alcohol ethoxylates and ethoxylated fatty amines is initiated by a central cleavage or omega-oxidation. Subsequent oxidation of the alkyl chains results in the production of polyethylene glycols and secondary ethoxylated amines. Both polar moieties are metabolized by other microorganisms. Degradation of alkyltrimethylammonium salts and alkylamines is initiated by a cleavage of the Calkyl-N bond. The central fission leads to the formation of alkanals which are readily converted by beta-oxidation. The alkyl chain-utilizing bacteria are not able to degrade the methylamines. The methylamines, in turn, are subject to biodegradation by methylotrophs. The limited metabolic capacities of pure cultures of microorganisms utilizing surfactants point to the requirement of consortia to degrade surfactants completely. Complete degradation of surfactants is accomplished by mixed cultures of microorganisms constructed on the basis of synergistic and commensalistic relationships. However, degradation of a surfactant by one member of a commensalistic consortium may lead to the production of toxic or non-toxic metabolites. Waste water treatment without the build up of such metabolites can be achieved in plants operated with sludge retention times that are suitable for maintaining all microorganisms of the consortium. In contrast, in natural ecosystems the introduction of a surfactant may result in a transient formation of a metabolite.

Simple method to prolong the closed bottle test for the determination of the inherent biodegradability.

A method to prolong the closed bottle test up to 200 days is described and validated. This prolonged closed bottle test has been used to determine the biodegradability of "recalcitrant" and toxic organic compounds. The results obtained in the prolonged closed bottle test are in accordance with those mentioned in the literature on biodegradation of organic compounds in waste water purification plants and on the isolation of microorganisms capable of utilizing these compounds as carbon and energy source. Furthermore, this test may prevent discrepancies and unexplainable results obtained in a 28-day test. The test has the potential to be used as an inherent biodegradability test when recognized by the authorities.

Complete mineralization of dodecyldimethylamine using a two-membered bacterial culture.

Complete degradation of dodecyldimethylamine was achieved using a two-membered bacterial culture isolated from activated sludge. One member, identified as Burkholderia cepacia, was capable of degrading the alkyl chain of the molecule. The other member, identified as Stenotrophomonas maltophilia, was able to degrade dimethylamine, the product of the former. Batch culture experiments revealed that the two-membered culture consisting of B. cepacia and S. maltophilia was based on a commensalistic relationship under carbon-limited conditions. Under nitrogen-limited conditions, the relationship of this culture was transformed from a commensalistic to a mutualistic one. A two-membered culture was therefore imperative for growth on dodecyldimethylamine under nitrogen-limited conditions, whereas a pure culture of B. cepacia was capable of growth on dodecyldimethylamine under carbon-limited conditions.

Metabolism of dodecyldimethylamine by Pseudomonas MA3.

Pseudomonas MA3 was isolated from activated sludge on the basis of its capacity to use dodecyldimethylamine as a sole carbon (C) and energy source. Dodecylamine, dodecanal, dodecanoic acid and acetic acid also supported growth of Pseudomonas MA3. Dodecyldimethylamine-grown cells oxidized a wide range of alkylamine derivatives, dodecanal, dodecanoic acid and acetic acid. Degradation of the alkyl chain of dodecyldimethylamine by Pseudomonas MA3 appeared from the stoichiometric liberation of dimethylamine. A dehydrogenase catalysed the cleavage of the Calkyl-N bond. The first intermediate of the proposed degradation pathway, dodecanal, accumulated in the presence of decanal used as a competitive inhibitor. The second intermediate, dodecanoic acid, was formed in the presence of acrylic acid during the degradation of dodecyldimethylamine. Dodecanal was converted into dodecanoic acid by a dehydrogenase and dodecanoic acid was then degraded via the beta oxidation pathway.

Assessment of the biodegradability of dialkyldimethylammonium salts in flow through systems.

A bacterium capable of utilizing the alkyl chains of didecyldimethylammonium salt was isolated from activated sludge. In addition, the isolate also utilized didodecyldimethylammonium salt, ditetradecyldimethylammonium salt and alkyltrimethylammonium salts (C10 to C18) as sole source of carbon and energy. The broad substrate with respect to the alkyl chain length was also demonstrated with oxidation rates of various quaternary ammonium salts by didecyldimethylammonium chloride-grown cells. The oxidation rate decreased with increasing alkyl chain lengths. The main factor impeding the biodegradation of dialkyldimethylammonium salts with long alkyl chains is probably the low bioavailability of water-insoluble chemicals. The biodegradability of dialkyldimethylammonium salts was therefore determined in flow-through columns at concentrations below their aqueous solubility. Dialkyldimethylammonium salts adsorbed on silica gel particles packed in flow-through columns were immediately metabolized by the isolate when dissolved. Microorganisms present in river water pumped through a sterile column degraded dissolved dicocodimethylammonium salts within a week.

Influence of the size and source of the inoculum on biodegradation curves in closed-bottle tests.

Test variables influencing the results of the closed-bottle test include size and source of the inoculum. The inoculum originates from activated sludge plants maintained at various sludge retention times. The lag period prior to biodegradation, which is influenced primarily by acclimation, decreased with increasing numbers of competent microorganisms. The rate of biodegradation in the closed-bottle test is influenced by the sludge retention time by which the inocula are maintained. Acclimated sludges maintained at low sludge retention times used as inocula gave biodegradation curves with steep slopes, indicating degradation at high rates. Therefore, unacclimated ready biodegradability tests enable only conservative assessments of the biodegradation potential of wastewater purification plants. The results reported here demonstrate that other tests should be used in risk assessment to estimate the full biodegradation potential in wastewater treatment plants.

Purification and characterization of chlorite dismutase: a novel oxygen-generating enzyme.

A novel enzyme that catalyzes the disproportionation of chlorite into chloride and oxygen was purified from a gram-negative bacterium, strain GR-1 to homogeneity. A four-step purification procedure comprising Q-Sepharose, hydroxyapatite, and phenyl-Superose chromatography and ultrafiltration resulted in a 13.7-fold purified enzyme with a final specific activity of 2.0 mmol min-1 (mg protein)-1. The dismutase obeyed Michaelis-Menten kinetics. The Vmax and Km calculated for chlorite were 2,200 U (mg protein)-1 and 170 microM, respectively. Dismutase activity was inhibited by hydroxylamine, cyanide, and azide, but not by 3-amino-1,2,4-triazole. Chlorite dismutase had a molecular mass of 140 kDa and consisted of four 32-kDa subunits. The enzyme was red-colored and had a Soret peak at 392 nm. Per subunit, it contained 0.9 molecule of protoheme IX and 0.7 molecule of iron. Chlorite dismutase displayed maxima for activity at pH 6.0 and 30 degrees C.