Activated Sludge Respiration Inhibition Test Research Articles

Bacterial Toxicity Testing: Modification and Evaluation of the Luminescent Bacteria Test and the Respiration Inhibition Test

The activated sludge respiration inhibition test and the luminescent bacteria test with Vibrio fischeri are important bacterial test systems for evaluation of the toxicity of chemical compounds. These test systems were further optimized to result in better handling, reliability and sensitivity. Concerning the Vibrio fischeri test, media components such as yeast extract and bivalent cation concentrations like Ca2+ and Mg2+ were optimized. The cultivation, storage conditions and reactivation process of the stored bacteria were also improved, which enabled simpler handling and led to good reproducibility. Additionally, the respiration inhibition test with a prolonged incubation time was further analyzed using different chlorinated phenols as reference compounds. It could be stated that a longer incubation period significantly improved the sensitivity of the test system.

Development and Application of a Microplate Assay for Toxicity Testing on Aquatic Cyanobacteria.

Regulatory environmental risk assessment, applied to establish a protection limit for all bacterial diversity in surface waters, relies on a growth inhibition test performed on a single species of cyanobacteria and the activated sludge respiration inhibition test. Recently, the ability of this approach to protect adequately for bacteria that provide important ecosystem services has been questioned, and empirical data on additional species to further investigate the effectiveness of the environmental risk assessment are urgently required. We present the development and validation of a cost-effective and time-efficient microplate assay that is comparable to the traditional shake flask test for measurement of cyanobacteria growth rate after chemical exposure. The assay has been optimized to ensure that comparisons of cyanobacteria sensitivity under exponential growth are assessed across equivalent experimental conditions using phycocyanin fluorescence as a surrogate for cell density. The test system is validated using potassium dichromate, and the results are compared with those obtained in an Organisation for Economic Co-operation and Development (2011) test guideline 201 shake flask test system. This assay is suitable for the screening of new and legacy chemicals (including antibiotics) for which ecotoxicology data are lacking across a wide range of cyanobacteria, with the aim of developing more comprehensive environmental risk assessment. Environ Toxicol Chem 2020;39:705-720. © 2019 SETAC.

Sub- and super-critical water oxidation of wastewater containing amoxicillin and ciprofloxacin

Feasibility for removal of two commonly used antibiotics (amoxicillin and ciprofloxacin) from hospital wastewater was experimentally investigated using sub- and super-critical water oxidation. Effect of temperature and flow rate on the concentration of antibiotics was observed in the temperature range from 473K to 773K and at flow rates of 3 and 5mL/min. Samples were analyzed in terms of their total organic compound (TOC) and chemical oxygen demand (COD). Highest COD reduction was achieved at the highest temperature of 773K, where it was reduced for 76%. Furthermore, the highest decrease in TOC was 63% at 773K. Using liquid chromatography tandem-mass spectrometry (LC–MS/MS) amoxicillin and ciprofloxacin were identified and the structure of products was assigned by MS fragmentation patterns. An activated sludge respiration inhibition test demonstrated that samples after sub- and super- critical oxidation were less inhibitory than the stock solution.

Improving the sensitivity of the activated sludge respiration inhibition test and verifying the results using an oxidation-reduction potential electrode

The inhibition of activated sludge respiration is one of the most important parameters for monitoring wastewater toxicity. The main objective of this study was to improve respiration inhibition testing in order to protect the biological degradation within the aerobic process in a wastewater treatment plant more efficiently. In order to influence the sensitivity of the testing bacteria, two different nutrient solutions were selected for biological testing, synthetic wastewater according to ISO 8192 and NaAc (sodium acetate). The effects of the nutrient solutions on heavy metal speciation and their respiration inhibition were investigated. The toxicants Zn(II), Cu(II), Cr(VI) and 3,5 DCP (3,5-dichlorophenol) were used as standards to determine toxicities and to study the influence of nutrient solutions. Results have shown that NaAc as a nutrient solution sensitized the respiration inhibition test up to a factor of 7.7. Furthermore, an oxidation reduction potential electrode can be used as an alternative tool to verify the results obtained with an oxygen sensor.

Interference sources in ATP bioluminescence assay of silica nanoparticle toxicity to activated sludge

ATP measurement provides an overview of the general state of microbial activity, and thus it has proven useful for the evaluation of nanoparticle toxicity in activated sludge. ATP bioluminescence assay, however, is susceptible to interference by the components of activated sludge other than biomass. This paper presents the interference identified specific to the use of this assay after activated sludge respiration inhibition test of silica nanoparticles (OECD 209).We observed a high degree of interference (90%) in the presence of 100mg/L silica nanoparticles and a low level of ATP being measured (0.01μM); and 30% interference by the synthetic medium regardless of silica nanoparticle concentration and ATP level in the samples. ATP measurement in activated sludge with different MLSS concentrations revealed interference of high biomass content. In conclusion, silica nanoparticles, synthetic medium and activated sludge samples themselves interfere with ATP bioluminescence; this will need to be considered in the evaluation of silica nanoparticle toxicity to activated sludge when this type of assay is used.

Toxicity of carbon nanotubes to the activated sludge process

The discharge of carbon nanotubes (CNTs) from industrial waste or disposal of such materials from commercial and/or domestic use will inevitably occur with increasing production and enter into wastewater treatment facilities with unknown consequences. Therefore, a better knowledge of the toxicity of CNTs to biological processes in wastewater treatment will be critical. This study examined the toxicity of multi-walled carbon nanotubes (MWCNTs) on the microbial communities in activated sludge. A comparative study using the activated sludge respiration inhibition test was performed on both unsheared mixed liquor and sheared mixed liquor to demonstrate the potential toxicity posed by MWCNTs and to illustrate the extent of extracellular polymeric substances (EPS) in protecting the microorganisms from the toxicity of CNTs. Respiration inhibition was observed for both unsheared and sheared mixed liquor when MWCNTs were present, however, greater respiration inhibition was observed for the sheared mixed liquor. The toxicity observed by the respiration inhibition test was determined to be dose-dependent; the highest concentration of MWCNTs exhibited the highest respiration inhibition. Scanning Electron Microscopy (SEM) images demonstrated direct physical contact between MWCNTs and activated sludge flocs.

Toxicity assessment of chlorophenols using a mediated microbial toxicity assay

While direct toxicity assessment (DTA) is now widely recognised as a useful tool for environmental risk assessment, many existing tests fail to meet end-user needs. This article describes the significant progress made to the MICREDOX® DTA assay, developed at Lincoln Ventures Ltd, brought about by miniaturising this assay to a multi-well plate format combined with limiting current microelectrode transduction. The benefits have been reduced: preparation time, reduced assay time, lower material costs and a higher level of replication achieved. To validate the precision of the miniaturised format, the concentrations required to cause a 50% decrease in signal (EC50) by an archetypal group of toxicants, the chlorophenols, were determined using two terrestrial bacterial strains, Escherichia coli K12 and Klebsiella oxytoca 13183. The assay time was then reduced by stepwise adjustment of the incubation time, from 60 down to 5 min, and the EC50s reported by E. coli to each of the toxicants after 45, 30, 15 and 5 min incubations were determined. The results obtained match closely with those reported by the Activated Sludge Respiration Inhibition Test and confirm the miniaturised multi-well plate MICREDOX® DTA assay reliably reports representative EC50 values for these toxicants. The previously described trends of increasing toxicity with increasing chlorine substitution and the observation that meta-substituted chlorophenols are more toxic than their ortho-substituted counterparts are also confirmed. The ability to monitor toxicity using terrestrial organisms, in volumes amenable to multi-well microtitre plates and incubations requiring only a few minutes, facilitates the rapid generation of highly reproducible, easy to operate and inexpensive DTA measurements.

Rapid Activated Sludge Respiration Inhibition Test Performed by CO2 Producing Rate Using a Carbon Dioxide Sensor

The rapid activated sludge inhibition test (rapid ASRI) is one of the promising bioassays to evaluate environmental risk to the ecosystem caused by various pollutants. To improve the sensitivity and stability of ASRI, the CO2 producing rate (CPR) using a carbon dioxide gas sensor was employed to examine the respiration activity of activated sludge and to compare it to that by the conventional activated sludge respiration inhibition test using oxygen uptake rate (OUR) by EC50 values derived from dose response curve. Detection of respiratory activity based on CPR has higher reliability and sensitivity than that of OUR. It should be noted that the sensitivity of CPR for evaluating toxicity is sufficiently high in terms of derived dose response curve of some model environmental pollutants and the resultant EC50 values.

Evaluation and further development of the activated sludge respiration inhibition test

The activated sludge respiration inhibition test is an important bacterial test system for the determination of bacterial toxicity of chemical compounds. The exposure period recommended by OECD 209 and ISO 8192 protocols is 30 and 180 min. A modified version of the test was developed which allowed a prolonged incubation period of 27 h to enhance the possibilities of the test system. The test system with the prolonged incubation time was evaluated by the recommended reference compound 3.5-dichlorophenol and showed an EC50 of 6.3 mgl−1 with a coefficient of variation of 12.7%. Furthermore, the use of an open test system was evaluated showing a comparable toxicity but a higher coefficient of variation than the closed test system. A storage of activated sludge for several days accompanied by a daily feed with OECD nutrient solution should be avoided, for it can cause a decreased sensitivity of the inoculum. Different statistical fit procedures were tested indicating that Weibull fit procedures were superior for extended data sets covering a wide range of concentrations whereas Gamma and Probit fits were appropriate for smaller data sets mainly restricted to the linear part of the dose response curve.

Biosensors for the measurement of toxicity of wastewaters to activated sludge†

When wastewaters from chemical manufacturing are discharged for biological treatment it is important to ensure that the presence of potentially toxic chemicals is below concentrations that may impair the performance of the treatment plant. This report describes the use of mediated amperometric biosensors incorporating activated sludge to provide rapid determination of toxicity in wastewaters to activated-sludge-based water-treatment plants. Redox-mediated interrogation of the microbial consortium comprising the biosensor biocatalyst allowed real-time monitoring of metabolic activity and the detection of perturbation due to toxic challenge. A time-independent inhibition value was determined within 30 min of exposure, using a mathematical model based on a hyperbolic relationship between exposure time and biosensor inhibition. Comparative testing of the biosensors and an activated sludge respiration inhibition test (ASRIT) against 35 wastewater samples, from a wide variety of industrial discharges, showed the close correlation coefficient of 0·924 between the two test methods. © 1998 Society of Chemical Industry

Acid mine-drainage toxicity testing

The BOD inhibition test, the activated sludge respiration inhibition test, and the Microtox® Bioassay procedure were examined for their potential as rapid toxicity screening methods for acid mine drainage (AMD) and for setting toxicity threshold levels in receiving waters. The BOD inhibition test proved to be unsuitable due to the high chemical oxygen demand of the toxicant. The long incubation time (minimum 5 d at 20°C) allowed growth of chemo-autotrophic bacteria and resulted in excessive flocculation and precipitation of metals, making accurate and meaningful measurements of inhibition impossible. The activated sludge respiration inhibition test was successful in measuring AMD toxicity, and for deriving toxicity threshold levels for the basic heterotrophic community in surface waters. However, it is a very time-consuming technique and is not sensitive enough to be used as a routine screening technique. The variability in toxicity was found to be due to the availability of metals as well as the effect of pH on metabolism. Simple linear models were derived to estimate inhibition within surface waters. Prediction of the inhibition caused by AMD can be made as long as the pH of the river water after mixing and the dilution of the AMD are known. Most accurate predictions are made using equations for specific pH ranges, but a useful estimation of inhibition can be obtained using the general equation: inhibition (%) = -2.34 pH + 6.41 AMD (%) + 22.1. The Microtox® Bioassay method, although expensive, was rapid and simple to use. It was the most sensitive test of the three, with the toxic response of marine bacteria increasing notably from 5 to 15 min due to the presence of bivalent metals in the toxicant. The problem of repeatability and reproducibility encountered with the activated sludge inhibition test was overcome with the highly standardised Microtox® method, making it an ideal screening method for AMD. However, the advantage of the activated sludge inhibition test is that the types of heterotrophic micro-organisms found in activated sludge are similar to those found in receiving waters such as the Avoca River and so more meaningful toxicity threshold values can be obtained.

The toxicity of four organic solvents commonly used in the pharmaceutical industry to activated sludge

The ISO activated sludge respiration inhibition test was evaluated as a tool for assessing the treatability of pharmaceutical wastewaters. The EC 50 values of four test chemicals: ethylene glycol, methanol, isopropanol and acetone, were determined using different combinations of sludge and substrate types. The coefficient of variation of EC 50 results within-batch was found to be 3.7% and between-batch variations ranged from 7.8 to 13.5% using sludge from a municipal treatment plant. Between-source variations were 6.72–54.05% depending on the combination of sludge and substrate used and the test chemical. The method was found to be inexpensive, rapid and accurate in terms of reproducibility and variability.

Comparison of three rapid toxicity test procedures: Microtox,® polytox,® and activated sludge respiration inhibition

AbstractMethods for measuring respiratory activity can detect adverse effects on heterotrophic microorganisms. These methods are useful tools for assessing the impact of chemicals on biological wastewater treatment processes. They are also well suited for evaluating the treatability potential of individual wastes. However, varying results are often reported when using municipal or industrial activated sludge substrate in toxicity assessments. The lack of reproducibility of these tests has also greatly limited efforts to measure the effectiveness of treatment plants in removing toxic contaminants. The toxicity of waste‐waters and chemicals to biological treatment systems can be determined in 30 min with the new Polytox® toxicity procedure. The Polytox® kit utilizes a specialized blend of bacterial cultures. The objective of this paper is to evaluate the relative sensitivity of the new Polytox® toxicity procedure with two accepted microbial assay procedures; namely, the Microtox® bioluminescence assay and the Organization for Economic Cooperation and Development's Activated Sludge Respiration Inhibition test. This paper compares toxicity values for selected inorganic and organic chemicals as determined in the three microbial assay systems. The sensitivity and reliability of the new Polytox® toxicity procedure is discussed.