Development and evaluation of an online CO(2) evolution test and a multicomponent biodegradation test system

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO(2)) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO(2) method with existing CO(2) evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation--i.e., CO(2) evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)--in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO(2) and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.     

On the fate of LAS, NPEOs and DEHP in municipal sewage sludge during composting

The fate of hydrophobic xenobiotic pollutants such as linear alkylbenzene sulfonates (LAS), nonylphenol ethoxylates (NPEO) and di-ethyl-hexyl phthalate (DEHP) during sewage sludge composting was addressed in this work. The experiments were conducted in a fully automated in-vessel autothermal composting system which was fed with a mixture of primary and secondary sludge and manure. The mixture composition was determined to achieve satisfactory humidity, C/N ratio and free air space (FAS). The effect of various parameters, such as the initial xenobiotic concentration, the presence of multiple xenobiotic compounds and the temperature of composting material sustained during the process on the xenobiotics biodegradation kinetics was investigated. It was generally established that significant xenobiotic reduction is achievable through composting under all conditions tested. According to the obtained results, the presence of LAS, NPEO and DEHP even at higher concentrations was not inhibitory to the bioprocess. However, the presence of multiple xenobiotic compounds such as NPEO, NP and DEHP in the sludge can influence LAS removal during LAS composting.     

Inhibition of Biogas Production by Alkyl Benzene Sulfonates (LAS) in a Screening Test for Anaerobic Biodegradability

The effect of the inoculum source on the digestion of linear alkylbenzene sulfonates (LAS) under anaerobic conditions has been investigated. The potential for primary and ultimate LAS biodegradation of anaerobic sludge samples obtained from wastewater treatment plants (WWTPs) of different geographical locations was studied applying a batch test system. It was found that only 4–22% of the LAS added to the batch anaerobic digesters was primarily transformed suggesting a poor primary degradation of the LAS molecule in anaerobic discontinuous systems. Regarding ultimate biodegradation, the addition of LAS to the batch anaerobic digesters caused a reduction on the extent of biogas production. Significant differences in the inhibition extent of the biogas production were observed (4–26%) depending on the sludge used as inoculum. Effect of the surfactant on the anaerobic microorganisms was correlated with its concentration in the aqueous phase. Sorption of LAS on anaerobic sludge affects its toxicity by depletion of the available fraction of the surfactant. LAS content on sludge was related to the total amount of calcium and magnesium extractable ions. The presence of divalent cations promote the association of LAS with anaerobic sludge reducing its bioavailability and the extent of its inhibitory effect on the biogas production.     

Biodegradation of tert-butylphenyl diphenyl phosphate

The biodegradation of tert-butylphenyl diphenyl phosphate (BPDP) was examined in microcosms containing sediment and water from five different ecosystems as part of our studies to elucidate the environmental fate of phosphate ester flame retardants. Biodegradation of [14C]BPDP was monitored in the environmental microcosms by measuring the evolution of 14CO2. Over 37% of BPDP was mineralized after 8 weeks in microcosms from an ecosystem which had chronic exposure to agricultural chemicals. In contrast, only 1.7% of BPDP was degraded to 14CO2 in samples collected from a noncontaminated site. The exposure concentration of BPDP affected the percentage which was degraded to 14CO2 in microcosms from the two most active ecosystems. Mineralization was highest at a concentration of 0.1 mg of BPDP and was inhibited with 10- and 100-fold higher concentrations of BPDP in these microcosms. Indigenous heterotrophic and BPDP-utilizing microbial populations and phosphoesterase enzyme activities were highest in sediments which had the highest biodegradation of BPDP. We observed adaptive increases in both microbial populations and phosphoesterase enzymes in some sediments acclimated to BPDP. Chemical analyses of the residues in the microcosms indicated undegraded BPDP and minor amounts of phenol, tert-butylphenol, diphenyl phosphate, and triphenyl phosphate as biodegradation products. These data suggest that the microbial degradation of BPDP results from at least three catabolic processes and is highest when low concentrations of BPDP are exposed to sediment microorganisms of eutrophic ecosystems which have high phosphotri- and diesterase activities and previous exposure to anthropogenic chemicals.     

Biodegradation of tert-butylphenyl diphenyl phosphate.

The biodegradation of tert-butylphenyl diphenyl phosphate (BPDP) was examined in microcosms containing sediment and water from five different ecosystems as part of our studies to elucidate the environmental fate of phosphate ester flame retardants. Biodegradation of [14C]BPDP was monitored in the environmental microcosms by measuring the evolution of 14CO2. Over 37% of BPDP was mineralized after 8 weeks in microcosms from an ecosystem which had chronic exposure to agricultural chemicals. In contrast, only 1.7% of BPDP was degraded to 14CO2 in samples collected from a noncontaminated site. The exposure concentration of BPDP affected the percentage which was degraded to 14CO2 in microcosms from the two most active ecosystems. Mineralization was highest at a concentration of 0.1 mg of BPDP and was inhibited with 10- and 100-fold higher concentrations of BPDP in these microcosms. Indigenous heterotrophic and BPDP-utilizing microbial populations and phosphoesterase enzyme activities were highest in sediments which had the highest biodegradation of BPDP. We observed adaptive increases in both microbial populations and phosphoesterase enzymes in some sediments acclimated to BPDP. Chemical analyses of the residues in the microcosms indicated undegraded BPDP and minor amounts of phenol, tert-butylphenol, diphenyl phosphate, and triphenyl phosphate as biodegradation products. These data suggest that the microbial degradation of BPDP results from at least three catabolic processes and is highest when low concentrations of BPDP are exposed to sediment microorganisms of eutrophic ecosystems which have high phosphotri- and diesterase activities and previous exposure to anthropogenic chemicals.     

Testing of some assumptions about biodegradability in soil as measured by carbon dioxide evolution.

Conversion to CO2 upon incubation in aerobic soil is one of the standard test procedures to assess biodegradability. It may be measured with unlabeled test compounds in biometer flasks. In this case, the background CO2 evolution by unamended soil is subtracted from the CO2 evolution by the amended soil and the resulting net CO2 evolution becomes the measure of biodegradation. Alternately, 14CO2 release from radiocarbon substrates is measured to assess biodegradability. Both approaches measure ultimate (complete) biodegradation and bypass the theoretical and technical limitations of residue analysis. This report examines the underlying assumptions that, except for carbon content, conversion percentage to CO2 is relatively independent of chemical composition, that CO2 production is proportional to the amount of added test compound, and that the background CO2 evolution of the soil is not influenced by the test substance. Work with unlabeled and radiolabeled substrates proved the first two assumptions to be essentially correct. However, more than half of net CO2 production may represent the mineralization of biomass and soil organic matter, some of it unrelated to the test compound. The soil microbial community in its nongrowing steady state appears to convert a much lower percentage of a radiocarbon substrate to 14CO2 than a growing soil community that responds to a substantial substrate addition. These findings may help to improve test methods and may aid in the interpretation of test results.     

Aerobic biodegradation potential of subsurface microorganisms from a jet fuel-contaminated aquifer.

In 1975, a leak of 83,000 gallons (314,189 liters) of jet fuel (JP-4) contaminated a shallow water-table aquifer near North Charleston, S.C. Laboratory experiments were conducted with contaminated sediments to assess the aerobic biodegradation potential of the in situ microbial community. Sediments were incubated with 14C-labeled organic compounds, and the evolution of 14CO2 was measured over time. Gas chromatographic analyses were used to monitor CO2 production and O2 consumption under aerobic conditions. Results indicated that the microbes from contaminated sediments remained active despite the potentially toxic effects of JP-4. 14CO2 was measured from [14C]glucose respiration in unamended and nitrate-amended samples after 1 day of incubation. Total [14C]glucose metabolism was greater in 1 mM nitrate-amended than in unamended samples because of increased cellular incorporation of 14C label. [14C]benzene and [14C]toluene were not significantly respired after 3 months of incubation. With the addition of 1 mM NO3, CO2 production measured by gas chromatographic analysis increased linearly during 2 months of incubation at a rate of 0.099 mumol g-1 (dry weight) day-1 while oxygen concentration decreased at a rate of 0.124 mumol g-1 (dry weight) day-1. With no added nitrate, CO2 production was not different from that in metabolically inhibited control vials. From the examination of selected components of JP-4, the n-alkane hexane appeared to be degraded as opposed to the branched alkanes of similar molecular weight. The results suggest that the in situ microbial community is active despite the JP-4 jet fuel contamination and that biodegradation may be compound specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Aerobic biodegradation potential of subsurface microorganisms from a jet fuel-contaminated aquifer

In 1975, a leak of 83,000 gallons (314,189 liters) of jet fuel (JP-4) contaminated a shallow water-table aquifer near North Charleston, S.C. Laboratory experiments were conducted with contaminated sediments to assess the aerobic biodegradation potential of the in situ microbial community. Sediments were incubated with 14C-labeled organic compounds, and the evolution of 14CO2 was measured over time. Gas chromatographic analyses were used to monitor CO2 production and O2 consumption under aerobic conditions. Results indicated that the microbes from contaminated sediments remained active despite the potentially toxic effects of JP-4. 14CO2 was measured from [14C]glucose respiration in unamended and nitrate-amended samples after 1 day of incubation. Total [14C]glucose metabolism was greater in 1 mM nitrate-amended than in unamended samples because of increased cellular incorporation of 14C label. [14C]benzene and [14C]toluene were not significantly respired after 3 months of incubation. With the addition of 1 mM NO3, CO2 production measured by gas chromatographic analysis increased linearly during 2 months of incubation at a rate of 0.099 mumol g-1 (dry weight) day-1 while oxygen concentration decreased at a rate of 0.124 mumol g-1 (dry weight) day-1. With no added nitrate, CO2 production was not different from that in metabolically inhibited control vials. From the examination of selected components of JP-4, the n-alkane hexane appeared to be degraded as opposed to the branched alkanes of similar molecular weight. The results suggest that the in situ microbial community is active despite the JP-4 jet fuel contamination and that biodegradation may be compound specific.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biodegradation of phthalic acid esters in river water and activated sludge.

The primary and ultimate biodegradability of phthalic acid, monobutyl phthalate, and five structurally diverse phthalic acid ester plasticizers in river water and activated sludge samples were determined via ultraviolet spectrophotometry, gas chromatography, and CO2 evolution. The compounds studied underwent rapid primary biodegradation in both unacclimated river water and acclimated activated sludge. When activated sludge acclimated to phthalic acid esters was used as the inoculum for the CO2 evolution procedure, greater than 85% of the total theoretical CO2 was evolved. These studies demonstrate that the phthalic acid ester plasticizers and intermediate degradation products readily undergo ultimate degradation in different mixed microbial systems at concentrations ranging from 1 to 83 mg/liter.     

A dynamic river model for biodegradability studies

The objective of this publication is to present a new dynamic aerobic biodegradation test method simulating a river. A laboratory cascade test system and standardized batch shake flask tests were used for biodegradation studies with the non-volatile and non-sorbing model compounds 2,4-dinitrophenol, naphthalene-1-sulphonic acid and sulphanilic acid. To be closer to the often very low concentrations of substances in the environment the concentrations of the compounds used were standard test concentrations and lower. ¹⁴C labelled compounds were measured at 50 μg/l, capillary electrophoresis at 5000 μg/l and the removal of dissolved organic carbon at 50000 μg/l. The test results obtained confirmed the known ultimate biodegradability of the test compounds and showed that biodegradation degrees, rates and degradation durations depended on the test systems, the concentrations of test compounds and the inocula. The river model is a suitable simulation test for natural dynamic surface waters which can be used to perform biodegradability studies at low test concentrations if adequate analytical tools, preferably radioactive-labelled substances, are available.     

Mineralization of Surfactants by the Microbiota of Submerged Plant Detritus

In wetlands and canopied bodies of water, plant detritus is an important source of carbon and energy. Detrital materials possess a large surface area for sorption of dissolved organics and are colonized by a large and diverse microbiota. To examine the biodegradation of surfactants by these microorganisms, submerged oak leaves were obtained from a laundromat wastewater pond, its overflow, and a pristine control pond. Leaves were cut into disks and incubated in sterile water amended with 50 μg of ¹⁴C-labeled linear alkylbenzene sulfonate (LAS), linear alcohol ethoxylate, stearyltrimethyl ammonium chloride, distearyldimethyl ammonium chloride, benzoic acid, or mixed amino acids per liter. Sorption of the test compounds to the detritus and evolution of ¹⁴CO₂ were followed with time. All of the compounds sorbed to the detritus to various degrees, with LAS and stearyltrimethyl ammonium chloride the most sorptive and benzoic acid the least. All compounds were mineralized without a lag. With leaves from the laundromat wastewater pond, half-lives were 12.6 days for LAS, 8.4 days for linear alcohol ethoxylate, 14.2 days for stearyltrimethyl ammonium chloride, 1.0 days for benzoic acid, and 2.7 days for mixed amino acids. Mineralization of LAS and linear alcohol ethoxylate by control pond leaves was slower and exhibited an S-shaped rather than a typical first-order pattern. This study shows that detritus represents a significant site of surfactant removal in detritus-rich systems.     

Priming effect of substrate addition in soil-based biodegradation tests.

To test whether substrate addition changes background CO2 evolution of soil, we measured both 14CO2 and net CO2 evolution from various test compounds. Glucose caused a priming effect, defined as substrate-stimulated soil organic matter mineralization. Formate, benzoate, n-hexadecane, and bis(2-ethylhexyl)phthalate caused no priming, and phenol caused only a transient one. The priming effect of glucose appears to be unusual and does not require a general rejection of net CO2 evolution measurements in biodegradability testing.     

Diffusive exchange of linear alkylbenze suifonates (LAS) between overlying water and bottom sediment

Linear alkylbenzenesulfonate (LAS) was detected in a 0–30 cm deep sediment column collected in Lake Teganuma (one of the most polluted lakes in Japan). The range of the LAS concentration in sediments was between 0.1 and 500 µg g^–1 (C₁₁-C₁₄ homologs per dry solid) and its vertical profile showed a seasonal variation. A mathematical model, which includes a diffusion term and a biodegradation term, was used to simulate the temporal variation of LAS in the sediment column and to calculate the diffusive flux rate of LAS across the sediment/water interface. An averaged diffusion coefficient of 2.4 × 10^–5 cm² s^–1 for the sediment interstitial water was obtained from sediment core samples located in Lake Teganuma. The biodegradation rate constant (0.002 d^–1) of LAS in the sediment obtained from the model analysis was considerably less than that reported for LAS in anaerobic waters. These results confirm that a model describing diffusive transport and biodegradation of LAS in the sediments can simulate the temporal variation of LAS in near surface sediments. The diffusive flux rate from overlying water to bottom sediment was calculated to be between –0.20 and 0.52 (C₁₁-C₁₄ LAS) mg m^–2 h^–1 and the annual net flux rate was 0.7 g m^–2 y^–1.     

Priming effect as determined by adding 14C-glucose to modified controlled composting test

The development of new biodegradable packaging materials, especially biodegradable plastics, has created a need for biodegradability testing. The European standard for controlled composting test was used in this study for assessing if the addition of a test material results in excess CO₂ production in compost. This effect, designated as the priming effect, would give an erroneous result for biodegradation, which is based on CO₂ formation from the test material. Glucose was selected as a test substrate because it is the degradation product of starch and cellulose, which are major compounds of many packaging materials. Both ¹⁴C-glucose and non-labelled glucose was applied to nine compost samples of variable stability and agefrom two weeks to 1.5 years. CO₂ and ¹⁴CO₂ evolution were measured during the incubation. Biodegradation of glucose in unstable composts (age leq6 months) was negative and ¹⁴CO₂ evolution was poor, although the respective composts without glucose produced relatively high amounts of CO₂. It was concluded that a negative priming effect was observed in unstable composts, in which glucose remained mostly non-degraded and apparently inhibited the mineralization of native organic matter in the compost. In stable composts (age 6 months), biodegradation of glucose was high and approximately equal to ¹⁴C-glucose mineralization, i.e., the composts showed no priming effect. Young composts were unsuitable for controlled composting test due to lack of stability. It is important to ensure that the compost inoculum used for the test is sufficiently stable.     

Shake Flask Biodegradation of 14 Commercial Phthalate Esters

An acclimated shake flask CO₂ evolution test was used to study the biodegradability of 14 commercial phthalate esters that are commonly used as plasticizers. Both CO₂ evolution (ultimate biodegradation) and loss of parent phthalate esters (primary biodegradation) were measured. With only a few exceptions, primary biodegradation was 90% or higher, and ultimate biodegradation was in excess of 55% of theoretical results in 28 days. The results showed that all of the commercial phthalate esters were susceptible to biodegradation by mixed populations of microorganisms from natural sources. The results also provide considerable insight into the utility and reproducibility of a standard biodegradation test that is being recommended for widespread screening of chemicals.     

Toxic effects of linear alkylbenzene sulfonate on metabolic activity, growth rate, and microcolony formation of Nitrosomonas and Nitrosospira strains

Strong inhibitory effects of the anionic surfactant linear alkylbenzene sulfonate (LAS) on four strains of autotrophic ammonia-oxidizing bacteria (AOB) are reported. Two Nitrosospira strains were considerably more sensitive to LAS than two Nitrosomonas strains were. Interestingly, the two Nitrosospira strains showed a weak capacity to remove LAS from the medium. This could not be attributed to adsorption or any other known physical or chemical process, suggesting that biodegradation of LAS took place. In each strain, the metabolic activity (50% effective concentration [EC(50)], 6 to 38 mg liter(-1)) was affected much less by LAS than the growth rate and viability (EC(50), 3 to 14 mg liter(-1)) were. However, at LAS levels that inhibited growth, metabolic activity took place only for 1 to 5 days, after which metabolic activity also ceased. The potential for adaptation to LAS exposure was investigated with Nitrosomonas europaea grown at a sublethal LAS level (10 mg liter(-1)); compared to control cells, preexposed cells showed severely affected cell functions (cessation of growth, loss of viability, and reduced NH(4)(+) oxidation activity), demonstrating that long-term incubation at sublethal LAS levels was also detrimental. Our data strongly suggest that AOB are more sensitive to LAS than most heterotrophic bacteria are, and we hypothesize that thermodynamic constraints make AOB more susceptible to surfactant-induced stress than heterotrophic bacteria are. We further suggest that AOB may comprise a sensitive indicator group which can be used to determine the impact of LAS on microbial communities.     

Metabolic efficiency and turnover of soil microbial communities in biodegradation tests.

Biodegradability screening tests of soil commonly measure 14CO2 evolution from radiolabeled test compounds, and glucose has often served as a positive control. When constant amounts of radiolabel were added to soil in combination with increasing amounts of unlabeled substrates, glucose and some related hexoses behaved in an anomalous manner. In contrast to that of formate, benzoate, n-hexadecane, or bis(2-ethylhexyl) phthalate, dilution of glucose radiocarbon with unlabeled glucose increased rather than decreased the rate and extent of 14CO2 evolution. [14C]glucose incorporation into biomass and Vmax values were consistent with the interpretation that application of relatively high concentrations of glucose to soil shifts the balance of the soil microbial community from the autochthonous (humus-degrading) to the zymogeneous (opportunistic) segment. The higher growth and turnover rates that define zymogeneous microorganisms, combined with a lower level of carbon incorporation into their biomass, result in the evolution of disproportionate percentages of 14CO2. When used as positive controls, glucose and related hexoses may raise the expectations for percent 14CO2 evolution to levels that are not realistic for other biodegradable compounds.     

A dynamic river model for biodegradability studies

The objective of this publication is to present a new dynamic aerobic biodegradation test method simulating a river. A laboratory cascade test system and standardized batch shake flask tests were used for biodegradation studies with the non-volatile and non-sorbing model compounds 2,4-dinitrophenol, naphthalene-1-sulphonic acid and sulphanilic acid. To be closer to the often very low concentrations of substances in the environment the concentrations of the compounds used were standard test concentrations and lower. ¹⁴C labelled compounds were measured at 50 g/l, capillary electrophoresis at 5000 g/l and the removal of dissolved organic carbon at 50000 g/l. The test results obtained confirmed the known ultimate biodegradability of the test compounds and showed that biodegradation degrees, rates and degradation durations depended on the test systems, the concentrations of test compounds and the inocula. The river model is a suitable simulation test for natural dynamic surface waters which can be used to perform biodegradability studies at low test concentrations if adequate analytical tools, preferably radioactive-labelled substances, are available.Abbreviations BOD biochemical oxygen demand - DAWT DOC-die-away test - DNP 2,4-dinitrophenol - DOC dissolved organic carbon - E effluent of laboratory wastewater treatment plants - MOST modified OECD screening test - NSA naphthalene-1-sulphonic acid - P pond water - SAA sulphanilic acid (=4-amino benzene sulphonic acid)     

Development and Evaluation of an Online CO2 Evolution Test and a Multicomponent Biodegradation Test System

Well-established biodegradation tests use biogenously evolved carbon dioxide (CO₂) as an analytical parameter to determine the ultimate biodegradability of substances. A newly developed analytical technique based on the continuous online measurement of conductivity showed its suitability over other techniques. It could be demonstrated that the method met all criteria of established biodegradation tests, gave continuous biodegradation curves, and was more reliable than other tests. In parallel experiments, only small variations in the biodegradation pattern occurred. When comparing the new online CO₂ method with existing CO₂ evolution tests, growth rates and lag periods were similar and only the final degree of biodegradation of aniline was slightly lower. A further test development was the unification and parallel measurement of all three important summary parameters for biodegradation—i.e., CO₂ evolution, determination of the biochemical oxygen demand (BOD), and removal of dissolved organic carbon (DOC)—in a multicomponent biodegradation test system (MCBTS). The practicability of this test method was demonstrated with aniline. This test system had advantages for poorly water-soluble and highly volatile compounds and allowed the determination of the carbon fraction integrated into biomass (heterotrophic yield). The integrated online measurements of CO₂ and BOD systems produced continuous degradation curves, which better met the stringent criteria of ready biodegradability (60% biodegradation in a 10-day window). Furthermore the data could be used to calculate maximal growth rates for the modeling of biodegradation processes.     

Physical and biological parameters that determine the fate of p-chlorophenol in laboratory test systems.

Shake-flask and microcosm studies were conducted to determine the fate of para-chlorophenol (p-CP) in water and sediment systems and the role of sediment and nonsediment surfaces in the biodegradation process. Biodegradation of p-CP in estuarine water samples in shake flasks was slow over incubation periods of 300 h. The addition of detrital sediment resulted in immediate and rapid degradation evidenced by the production of 14CO2 from [14C]p-CP. The addition of sterile sediment, glass beads, or sand resulted in approximately four to six times more CO2 evolution than observed in the water alone. Densities of p-CP-degrading bacteria associated with the detrital sediment were 100 times greater than those enumerated in water. Bacteria in the water and associated with the sediment after preexposure of both water and sediment of p-CP demonstrated enhanced biodegradation. In some microcosms, p-CP was degraded completely in the top 1.0 cm of intact sediment beds. Sediment reworking activities by benthic invertebrates from one site were sufficient to mix p-CP deep into the sediment bed faster than biodegradation or molecular diffusion. p-CP was persistent at lower depths of the sediment, possibly a result of reduced oxygen conditions preventing aerobic biodegradation.