Evaluation of wastewater toxicity: comparative study between Microtox and activated sludge oxygen uptake inhibition

Microtox is a frequent toxicity tool for the screening of wastewaters discharged into wastewater treatment plants. There is currently an increasing controversy between this test and others using activated sludge. A Microtox and electrolytic respirometry comparative study for toxicity determination has been performed. Seven organic and five inorganic toxic compounds have been assessed for comparing both methods. Microtox proved to have a higher sensitivity to toxicants but was less representative of effects on activated sludge compared to respirometry. For instance, assays accomplished with LAS, a biodegradable reference surfactant, showed a toxic effect by Microtox but good biodegradability and no toxicity in respirometry. This could be explained by the different nature of the biological material used, as Microtox utilises the seawater Vibrio fischeri, whereas respirometry uses the bacterial consortium in activated sludge. For the evaluation of the potential toxicity of a compound on a WWTP, the preferred biological material be used should be activated sludge itself. Results obtained with any other biological material would be just an approach to reality.     

Solar driven production of toxic halogenated and nitroaromatic compounds in natural seawater

Natural seawater (NSW) sampled in March and June 2007 in the Gulf of Trieste, Italy, has been spiked with phenol and irradiated in a device simulating solar light spectrum and intensity. Opposite to the case of artificial seawater, for which phenol is slightly degraded by direct photolysis, in NSW the phenol degradation mediated by natural photosensitizers occurs, forming several secondary pollutants, including hydroxyderivatives (1,4-benzoquinone, resorcinol), three chlorophenol isomers, 2,3-dichlorophenol, 2- and 4-bromophenol, 2- and 4-nitrophenol, and several condensed products (2 and 4-phenoxyphenol, 2,2'-, 4,4'- and 2,4-bisphenol). These compounds are toxic to bacteria and other living organisms. Ecotoxicologic effect has been evaluated by using the Vibrio Fischeri luminescent bacteria assay. This technique uses marine organisms, and it is therefore well suited for the study on marine samples. A correlation exists between the intermediates evolution and the toxicity profile, as the largest toxicity is observed when compounds with the lower EC50 (halophenols, phenoxyphenols) are formed at higher concentration.     

Estimating the toxicities of organic chemicals to bioluminescent bacteria and activated sludge

Toxicity assays based on bioluminescent bacteria have several advantages including a quick response and an easily measured signal. The Shk1 assay is a procedure for wastewater toxicity testing based on the bioluminescent bacterium Shk1. Using the Shk1 assay, the toxicity of 98 organic chemicals were measured and EC50 values were obtained. Quantitative structure-activity relationship (QSAR) models based on the logarithm of the octanol-water partition coefficient (log(Kow)) were developed for individual groups of organic chemicals with different functional groups. The correlation coefficients for different groups of organic compounds varied between 0.69 and 0.99. An overall QSAR model without discriminating the functional groups, which can be used for a quick estimate of the toxicities of organic chemicals, was also developed and model predictions were compared to experimental data. The model accuracy was found to be one order of magnitude from the observed values.     

A rapid method for determining the toxicity of chemicals to activated sludge

This paper reports the development of a method to determine the toxicity of chemicals to activated sludge. In the method, activated sludge was exposed to various concentrations of test chemicals and the inhibition of [¹⁴C]glucose uptake was measured after 15 min. Data for the decrease in glucose uptake as a function of the log of the test chemical concentration was analyzed by a nonlinear regression model to determine the concentration of chemical inhibiting uptake by 50% (IC₅₀ value). In control experiments, glucose uptake in the absence of test chemicals was rapid, specific and totally dependent on the presence of metabolically-active activated sludge. However, uptake in the presence of inorganic and organic test chemicals showed significant, dose-dependent decreases as the concentration of test material increased. Inhibition of uptake was accurately described by the nonlinear regression model, and calculated IC₅₀ values for two chemicals, mercuric chloride and 3,5-dichlorophenol, agreed well with values reported in the literature to adversely affect wastewater treatment plant operation. Based on the results of our studies, inhibition of glucose uptake proved to be a rapid, accurate and reproducible method of determining the toxicity of chemicals to activated sludge.     

A Mixture Toxicity Study Employing Binary Combinations Of Dinitrobenzene And Trinitrobenzene Using The Bioluminescent Marine Bacterium Vibrio Harveyi As The Test Organism

A combination mixture study was conducted with two organic compounds, dinitrobenzene and trinitrobenzene, using the marine bioluminescent bacterium Vibrio harveyi . This assay employs the photogenic properties of this organism for determination of toxicity in a direct procedure. Evaluation of toxicity was performed using an additive index methodology, mixture toxicity index and graphic representations. Graphic representations employed were isobole plot and isobologram, both isopleths. The additive index procedure employed both chemicals at their estimated median effective concentration (EC 50 ) for determination of a toxicity value. Isopleths employed combinations of both chemicals, using an equitoxic concentration, at 20% intervals of their EC 50 . An additive index value for this mixture was statistically antagonistic. Mixture toxicity index suggests that these chemical in various combinations are antagonistic. Combinations for both isopleths were similar and exhibited additivity for three mixture values and statistical synergism for one mixture. Statistical analysis of combinations using the z test found some mixtures to be statistically different. This analysis, in part, supports findings for isopleths. These results suggest that different mixtures of the two chemicals may have different interaction outcomes.     

Adverse effects of organic arsenical compounds towards Vibrio fischeri bacteria

The most frequently encountered forms of organic arsenic, namely, dimethylarsinic acid, monomethylarsonic acid, arsenobetaine, arsenocholine and Roxarsone (4-hydroxy-3-nitrobenzene arsonic acid) were tested for toxicity either by using the Microtox bioassay, based on the rapid (within 15 min) fading of luminescence emitted by Vibrio fischeri marine bacteria, or by monitoring growth rate of the same bacteria for 3 days. Organic arsenic was generally found to be less toxic to these biological models than inorganic arsenic. In many cases, EC50 values for DMA, MMA or HNAA when using luminescence or growth inhibition assays could not be determined because of the low toxicity of the compounds. Nevertheless, results from the luminescence inhibition assay, which was found to be more sensitive than the growth inhibition assay, allowed to rank toxicity as follows: arsenate at pH 8>HNAA at pH 5>arsenate at pH 5>MMA at pH 5>HNAA at pH 8>DMA at pH 5. Arsenobetaine and monomethylarsonic acid were unexpectedly found to stimulate bacterial growth (hormesis effect). pH was found to have a strong influence on the observed toxicity as a consequence of the pH-induced changes in the chemical speciation of the tested molecules. In most cases it appeared that negatively charged forms were less toxic than the uncharged ones.     

The combined toxic effects of nonpolar narcotic chemicals to Pseudokirchneriella subcapitata

This paper presents the toxicity data of 10 nonpolar narcotic chemicals on Pseudokirchneriella subcapitata (green algae) assessed by a new algal toxicity testing technique conducted under air-tight environment. Based on DO production, median effective concentration (EC50) varies from 1.73 mg/L (1-octanol) to 8,040 mg/L (2-propanol). The endpoint of algal growth rate reveals similar sensitivity as that from DO production. Compared to literature data, Pseudokirchneriella subcapitata and Nitrosomonas are apparently more sensitive to nonpolar narcotics than other organisms such as minnow, daphnia, and Tetrahymena pyriformis. Furthermore, good correlations between toxic effects observed from Pseudokirchneriella subcapitata and other aquatic organisms were found. Hence, algal toxicity test can be considered as a surrogate test for estimating the toxicity of nonpolar chemicals to fathead minnow, Microtox, activated sludge, Daphina magna, and Tetrahymena pyriformis. The combined effects of 13 binary mixtures of nonpolar chemicals were investigated using both additive-index method and isobologram analysis. Overall speaking, the joint actions between these chemicals are strictly additive. Model analyses indicate that these compounds act on identical reaction sites or receptors, which verify that these chemicals are of the same toxicity mechanism (narcosis).     

An investigation into the acute and chronic toxicity of eleven pharmaceuticals (and their solvents) found in wastewater effluent on the cnidarian, Hydra attenuata

Pharmaceuticals previously identified in the effluent from the wastewater treatment plant (WWTP) in Montreal discharging into the St. Lawrence river, were tested for acute and chronic toxicity using the cnidarian Hydra attenuata. Acute toxicity was based on the established technique looking at morphological changes in the Hydra, while recently developed endpoints of feeding behaviour, attachment and growth (hydranth number) were used to measure chronic effects. The compounds under investigation (ibuprofen, naproxen, gemfibrozil, bezafibrate, carbamazepine, sulfamethoxazole, sulfapyridine, oxytetracycline, novobiocin, trimethoprim and caffeine) were tested individually in controlled laboratory exposures with LC(50) and EC(50) results calculated. All compounds tested had relatively high LC(50) values with gemfibrozil, ibuprofen and naproxen having the lowest at 22.36 mg/L and EC(50) values based on morphology of 1.18 to 2.62 mg/L (all concentrations are nominal). The EC(50) values based on feeding were similar to those based on morphology but with increased sensitivity for carbamazepine, bezafibrate and novobiocin. A trend of a reduction in feeding with deterioration in morphology was observed in the Hydra, with the exception of novobiocin, where a lower than expected EC(50) of 13.53 mg/L was found with no negative effect on morphology. Significant reductions in attachment and hydranth number were seen at concentrations of 1 and 5 mg/L for gemfibrozil and ibuprofen respectively. A toxicity threshold (TT) of 320 microg/L was calculated for ibuprofen, only a factor of 10(2) or 10 higher than the concentration found in the effluent in the present study (1.19 mug/L) and in other Canadian effluents studied (22 microg/L [Brun GL, Bernier M, Losier R, Doe K, Jackman P, Lee HB, Pharmaceutically active compounds in Atlantic Canadian sewage treatment plant effluents and receiving waters and potential for environmental effects as measured by acute and chronic aquatic toxicity. Environ Toxicol Chem 2006; 25(8): 2163-2176.] respectively. Using EU directive 93/67/EEC the pharmaceuticals under investigation can be classified as toxic (gemfibrozil, ibuprofen and naproxen), harmful (carbamazepine, bezafibrate, sulfapyridine, oxytetracycline and novobiocin) and non-toxic (sulfamethoxazole, trimethoprim and caffeine) and their potential toxicity for the aquatic environment is discussed.     

Comparison of several microbiological toxicity screening tests

Four short-term microbiological toxicity screening tests were compared using the following test chemicals: 3,5 dichlorophenol, cetyl trimethyl ammonium chloride, sodium lauryl sulfate, phenol, copper(II) sulfate, mercury(II) chloride and zinc(II) sulfate. These seven chemicals represent a wide range of toxicity. The methods examined were Beckman's Microtox system, the Spirillum volutans motility test, inhibition of respiratory activity of activated sludge, and inhibition of activated sludge TTC-dehydrogenase activity.The results obtained indicate that each method has its own toxicity sensitivity pattern, and among the substances tested, only mercury(II) chloride and phenol were ranked equally by the four methods as the most and the least toxic chemical, respectively. In a tentative ranking of the methods, according to sensitivity, the Microtox test came out as the most sensitive test, followed by the Spirillum test, which in turn appeared more sensitive than the two sludge tests. This ranking has meaning only in a statistical sense, however, and the variable nature of the results support the philosophy that for assessing toxicity more thoroughly, a battery of several tests is required.     

Assessing chemical toxicity with the bioluminescent photobacterium (Vibrio fischeri): a comparison of three commercial systems

The inhibition of light emitted by the bioluminescent bacterium, Vibrio fischeri, is the basis for several toxicity bioassays. The inhibitory effects of 81 chemicals, after 5 min contact time, were studied at eight concentrations using reagents from three commercial assay systems (ToxAlert 10, Microtox and LUMIStox). Solubility in water was the limiting factor in determining the selection of chemicals for study. The effective nominal concentrations (EC) resulting in 20, 50 and 80% inhibition were determined using Ln dose/Ln gamma plots and the results obtained for each system were compared by linear regression. The chemical concentrations producing 10-90% inhibition extended over 9 orders of magnitude and ranged from a minimum of 0.001 ppm to a maximum of 1,000,000 ppm. The toxicity of many chemicals was apparently related to their pH in solution and at high chemical concentrations, to osmotic imbalance. The fact that the same operator tested the same solutions simultaneously on three different systems reduced sources of error and variability and improved the consistency and reliability of the results. Only five compounds gave EC 50s that varied more than three-fold between assays. These data provide comparisons of toxicity that have not been previously available and demonstrate that, when used under standardised conditions, these bioluminescence-based toxicity assays produce very similar results.     

Toxicity of the 13 priority pollutant metals to Vibrio fisheri in the Microtox chronic toxicity test

The Microtox Acute Toxicity Test has been successfully used to measure the toxicity of metals and other pollutants at high concentrations (ppm) in selected environmental samples. However, metals and other toxicants are often found in much lower concentrations (ppb) in many municipal wastewaters and receiving waters. In order to assess the toxicity of these pollutants in these samples, a more sensitive toxicity assay is needed. The Microtox chronic toxicity test has been developed to measure the sublethal effect of toxicants over multiple generations of the test species, Vibrio fisheri. In this study, the toxicity of the 13 priority pollutant metals [i.e. As, Se, Cd, Cr (III and VI), Cu, Pb, Sb, Ag, Tl, Zn, Be, Hg and Ni] to V. fisheri was evaluated using the Microtox chronic toxicity test. In this test, the inhibitory concentration (IC), lowest observable effect concentration (LOEC), and no observable effect concentration (NOEC) were obtained after 22-h of incubation at 27+/-1 degrees C, by comparing the light output of the control to that of the test sample. Among the 13 priority pollutant metals, beryllium (Be) was found to be the most toxic in the test (LOEC=0.742-1.49 microg/l) while thallium (Tl) was the least toxic (LOEC=3840-15300 microg/l). The LOECs for copper (as Cu) and lead (Pb) in reagent (ASTM Type I) water were 6.78-13.6 microg/l and 626-1251 microg/l, respectively. The toxicity of copper sulfate (as Cu) in reagent water was shown and significantly reduced with the addition of natural organic matter (fulvic acid) or EDTA to the sample. The LOEC values for the 13 priority pollutant metals in this test were comparable to or lower than those reported for commonly used aquatic toxicity tests, such as the Ceriodaphnia dubia assay.     

Toxicity potential of disinfection agent in tannery wastewater

Wastewater from a tannery was investigated using chemical-specific analyses and assessment of the acute toxicity of the whole effluent over a 2-year period. The wastewater samples were overloaded with organic and inorganic compounds, and measured concentrations of the chemical parameters as well as dilution factors estimating acute toxicity, frequently exceeded the permissible limits for the discharge of wastewater from a tannery into the receiving stream. In the later part of the monitoring programme, the toxicity of the samples was significantly increased in comparison to the previous samples. The agent for hide disinfection was assumed to be the reason for the increased toxicity of the wastewater samples, and the extremely high acute and chronic toxicity of the agent to bacteria, algae, daphnids, and fish confirmed this suspicion. The most sensitive species was Daphnia magna; the 48 h EC50 was 0.70 x 10(-5)v/v% and the 21d IC25 was 0.40 x 10(-6)v/v% of the agent. After withdrawal of this highly toxic agent for hide disinfection from the technological process in the tannery, the toxicity of the wastewater declined to the previous level.     

Decolorizing and detoxifying textile wastewater, containing both soluble and insoluble dyes, in a full scale combined anaerobic/aerobic system

The wastewater originating from the bleaching and dyeing processes in the textile factory Ten Cate Protect in Nijverdal (the Netherlands) was successfully treated in a sequential anaerobic/aerobic system. In the system, a combination of an anaerobic 70-m3 fluidized bed reactor and a 450-m3 aerobic basin with integrated tilted plate settlers, 80-95% of the color was removed. The color was largely removed in the preacidification basin and the anaerobic reactor. Color, deriving from both reactive as well as disperse, was anaerobically removed, indicating that these type of dyes were reduced to colorless products. Interestingly, the vat dyes, the anthraquinones and indigoids, which were thought to be removed mainly aerobically, were largely anaerobically decolorized. Apparently the anaerobic system is capable of effectively removing the color of both soluble as insoluble dyes. The treated effluent of the sequential anaerobic/aerobic treatment showed no toxicity towards the bioluminescent bacterium Vibrio fisheri (EC20 (95%) > 45%). Partially bypassing the anaerobic stage resulted in increased toxicity (EC20 (95%) of 9% and 14%) in the effluent of the aerobic treatment and caused significant decrease of color removal. The results of this study show a main contribution of anaerobic treatment in decolorizing and detoxifying the textile wastewater in the sequential anaerobic/aerobic system.     

Ozonation and biological activated carbon filtration of wastewater treatment plant effluents

This study investigates the fate of trace organic chemicals (TrOCs) in three full-scale reclamation plants using ozonation followed by biological activated carbon (BAC) filtration to treat wastewater treatment plant effluents. Chemical analysis was used to quantify a wide range of TrOCs and combined with bioanalytical tools to assess non-specific toxicity (Microtox assay) and estrogenicity (E-SCREEN assay). Limited dissolved organic carbon (DOC) removal (<10%) was observed in the ozonation stages showing that oxidation leads to the formation of transformation products rather than mineralization. The quantified TrOCs were removed to a degree highly dependent on the compounds’ structures and the specific ozone dose (mg_O3 mg_DOC⁻¹). Non-specific toxicity was reduced by 31-39%, demonstrating that the mixture of remaining parent compounds and their transformation products as well as newly formed oxidation by-products had an overall lower toxic potential than the mixture of parent compounds. Estrogenicity was reduced by more than 87% indicating that the transformation products of the estrogenic chemicals lost their specific toxicity potential. The subsequent BAC filtration removed between 20 and 50% of the DOC depending on the plant configuration, likely due to biodegradation of organic matter. The filtration was also able to reduce the concentrations of most of the remaining TrOCs by up to 99%, and reduce non-specific toxicity by 33-54%. Overall, the combination of ozonation and BAC filtration can achieve removals of 50% for DOC and more than 90% for a wide range of TrOCs as well as a reduction of 70% of non-specific toxicity and more than 95% of estrogenicity. This process combination is therefore suggested as an effective barrier to reduce the discharge of TrOCs into the environment or their presence in water recycling schemes.     

An ATP luminescence method for direct toxicity assessment of pollutants impacting on the activated sewage sludge process

An ATP luminescence method was used to determine the toxicity of three reference toxicants to two sources of domestic activated sludge, and an activated sludge from a laboratory model plant. Repeatability in the ATP test was demonstrated for Cr (as K2Cr2O7), Zn (as ZnSO4 x 7H2O), and 3,5-dichlorophenol (3,5-DCP) using each source of activated sludge. The three sources of sludge showed sensitivity to Cr and 3,5-DCP, and insensitivity to Zn using the ATP luminescence method. Sludge source did not appear to effect test response. The toxic response to 3,5-DCP in model and domestic activated sludge was shown to be dependent on sludge solid concentration (measured as total suspended solids, gTSS(-1). It is recommended that a standard solids concentration is used during toxicity evaluation.     

Aerobic storage by activated sludge on real wastewater

Activated sludge processes are often operated under dynamic conditions, where the microbial response can include, besides of growth, several COD removal mechanisms, and particularly the storage in form of polymers. While abundant evidence of aerobic storage under dynamic conditions with synthetic substrates can be found (Majone et al., Water Sci. Technol. 39(1) (1999) 61), there is still little knowledge about COD removal mechanisms with real activated sludge and wastewater. The aim of the present paper is therefore to give a direct evidence of storage phenomena occurring when a real sludge is mixed with influent wastewater and of their influence onto OUR profiles in typical respirometric batch tests. For this purpose, respirometric batch tests were performed on the same sludge by using acetate, filtered wastewater and raw wastewater as carbon source along with determination of acetate uptake and storage polymer formation. Comparison of results obtained has shown that poly-3-hydroxybutyrate (PHB) storage gives always the main contribution to acetate removal and that in the case of wastewater PHB is also formed from other substrates. PHB formation clearly occurs during the high-rate RBCOD-phase, however for wastewater it accounts for only a fraction (18-22%) of overall RBCOD removal, so calling for other unidentified storage compounds or other non-storage phenomena. In the low-rate SBCOD phase of respirogram PHB is clearly utilised in tests with acetate as internal reserve material once the acetate is depleted. In tests with filtered and raw wastewater the PHB concentration decreases much slower, probably because more PHB is formed due to the availability of external SBCOD (soluble and not). Moreover, reported OUR in the SBCOD-phase from filtered or raw wastewater are quite higher than those reported in batch tests with acetate, so confirming a main contribution of external SBCOD. However, the respective contributions for utilisation of previously stored compounds and of external SBCOD cannot be easily separated by the comparison of tests on filtered and raw wastewater, because both substrates are simultaneously present also in tests with the filtered wastewater. As a side consequence, the chemical-physical method for evaluation of true soluble and biodegradable COD tends to overestimate the respirometry-based RBCOD, at least for the wastewater under observation. Even though modelling by ASM3 (Gujer et al., Water Sci. Technol. 39(1) (1999) 183) makes it possible to well describe the whole experimental behaviour, it requires that much more storage compounds are formed than the experimentally observed PHB. These compounds have still to be identified and quantified in order to confirm the conceptual structure of ASM3.     

A comparison of microbial bioassays for the detection of aquatic toxicants

The toxicity of 35 test chemicals was analyzed using two microbial bioassay systems. The commercially available Microtox Toxicity Analyzer System™ and the two-organism procedure of Tchan were used to determine the concentration of test chemicals resulting in a 50% reduction in response (EC₅₀). Both of the tests employed a luminescent bacterium while the procedure of Tchan also utilized an alga. Results from the two microbial tests were compared with available data obtained with fish toxicity bioassays and with each other. The Microtox™ procedure was somewhat more sensitive than the Tchan bioassay in detecting most of the test chemicals and fish bioassays were generally more sensitive than either of the microbial tests. As a notable exception, photosynthesis-inhibiting herbicides were detected at remarkably lower concentrations with the procedure of Tchan than any of the other bioassays. Potential applications for these tests are discussed.     

Selective removal of resin and fatty acids from mechanical pulp effluents by ozone

Fresh chemithermomechanical (CTMP) mill primary clarifier (combined) effluents were treated using a system capable of accurately applying small measured charges of ozone. It was shown that the efficiency of ozone transfer into effluent was much better than into water, and that there was no residual dissolved ozone. Small ozone charges destroyed high proportions of the total resin and fatty acids (RFAs) and juvabiones (JBs) present in the effluents. This effect diminished with increasing ozone charges. Likewise, effluent toxicity, as measured in the Microtox assay, decreased rapidly with the low ozone charges. The low charges of ozone were also shown to be effective in destroying both soluble and particulate RFAs. It was shown that there was a strong positive correlation between the RFA content and the acute toxicity of an effluent sample, and that all the acute toxicity was extractable with methyl-t-butyl ether. Both Microtox and Daphnia magna acute toxicity assays correlated with effluent RFA content, but the Microtox was somewhat more sensitive. Thus, ozone may be a useful economical treatment agent for selectively removing RFAs and toxicity from mill effluent streams.     

Toxicity of benzotriazole and benzotriazole derivatives to three aquatic species

Benzotriazole and its derivatives comprise an important class of corrosion inhibitors, typically used as trace additives in industrial chemical mixtures such as coolants, deicers, surface coatings, cutting fluids, and hydraulic fluids. Recent studies have shown that benzotriazole derivatives are a major component of aircraft deicing fluids (ADFs) responsible for toxicity to bacteria (Microtox). Our current research compared the toxicity of benzotriazole (BT), two methylbenzotriazole (MeBT) isomers, and butylbenzotriazole (BBT). Acute toxicity assays were used to model the response of three common test organisms: Microtox bacteria (Vibrio fischeri), fathead minnow (Pimephales promelas) and water flea (Ceriodaphnia dubia). The response of all the three organisms varied over two orders of magnitude among all compounds. Vibrio fischeri was more sensitive than either C. dubia or P. promelas to all the test materials, while C. dubia was less sensitive than P. promelas. The response of test organisms to unmethylated benzotriazole and 4-methylbenzotriazole was similar, whereas 5-methylbenzotriazole was more toxic than either of these two compounds. BBT was the most toxic benzotriazole derivative tested, inducing acute toxicity at a concentration of < or = 3.3 mg/l to all organisms.     

Effects of carbon dioxide deficiency and metal toxicity on biological oxygen demand

In this study, the effects of carbon dioxide (CO₂) deficiency on the biological growth under respirometric conditions, first‐ and second‐order biological oxygen demand (BOD) progressions using two different BOD measurement techniques and metal toxicity effects on the respirometric BOD are investigated. The effects of CO₂ deficiency in the growth of bacteria and related effects on the first‐ and second‐stage BOD progressions are investigated using various media with respirometers in comparison with the BOD dilution method. CO₂ deficiency causes significant retardations on the growth of bacteria and the second‐stage respirometric BOD values are suppressed. CO₂ seems to be an essential nutrient for the growth of microorganisms and for the oxygen uptake progressing rates. HgCl₂, HgSO₄, CuSO₄, K₂Cr₂O₇, ZnSO4 and Al₂(SO₄)₃ inorganic metal compounds cause significant retardations in the respirometric BOD values obtained from a synthetic autotrophic medium. Effects are found to be dependent on the applied concentrations of these chemicals in the medium.