抗生素类污染物对活性污泥生物毒性测定方法的筛选与评价

尽管衡量化学物质生物毒性的标准方法很多,但关于环境中抗生素类污染物的生物毒性及其对污泥活性影响的科学数据较少。以磺胺和四环素两类作用机理和作用谱带不同的抗生素为研究对象,分别用发光细菌法、脱氢酶活性法、生长抑制法及呼吸速率法进行了抗生素类污染物生物毒性测定方法的筛选与评价。结果表明,发光细菌标准方法中30min的作用时间太短,延长作用时间不仅导致各种抗生素的EC50值大幅度降低,同时毒性排列顺序也发生了改变;以活性污泥为对象的脱氢酶活性和呼吸速率抑制实验的EC50值与抗生素类物质对敏感致病菌的MICs相比异常高,不适宜于作为单独方法准确评估抗生素类污染物的生物毒性。生长抑制实验中,活性污泥混合菌种增殖生长对磺胺类抗生素敏感,而假单胞杆菌对四环素类抗生素敏感。不同方法测定抗生素毒性的灵敏度顺序是发光细菌(24 h)>生长抑制(7 h)>呼吸速率(24 h)>脱氢酶活性(24 h)。用标准方法评价抗生素类污染物的生物毒性,可能导致对抗生素排放到水环境中所带来的风险估计不足。     

Individual and combined inhibition of phenol and thiocyanate on microbial activity of partial nitritation

This study evaluated the individual and interactive effect of phenol and thiocyanate (SCN⁻) on partial nitritation (PN) activity using batch test and response surface methodology. The IC₅₀ of phenol and SCN⁻ on PN sludge were 5.6 and 351 mg L⁻¹, respectively. The PN sludge was insensitive to phenol and SCN⁻ at levels lower than 1.77 and 43.3 mg L⁻¹, respectively. A regression model equation was developed and validated to predict the relative specific respiration rate (RSRR) of PN sludge exposed to different phenol and SCN⁻ concentrations. In the range of independent variables, the most severe inhibition was observed with a valley value (17%) for RSRR, when the phenol and SCN⁻ concentrations were 4.08 and 198 mg L⁻¹, respectively. An isobole plot was used to judge the combined toxicity of phenol and SCN⁻, and the joint inhibitory effect was variable depending on the composition and concentration of the toxic components. Furthermore, the toxic compounds showed independent effects, which is the most common type of combined toxicity.

     

Anaerobic inhibition and biodegradation of antibiotics in ISO test schemes

Municipal sewage is the main exposure route for antibiotics that are used in human medical care. Antibiotics that adsorb to the primary sludge and/or sur-plus activated sludge will enter the anaerobic digesters of municipal sewage treatment plants. Here anaerobic biodegradation or inhibition of anaerobic bacteria resulting in a disturbance of the process might occur. ISO standards 13641 (2003) and 11734 (1999) were used for assessing the anaerobic inhibition of 16 and the anaerobic biodegradability of 9 antibiotics respectively. Digestion sludge from a municipal sewage treatment plant (1g/l d.s.) was used as inoculum in both tests. In ISO 13641 (2003) most antibiotics showed only moderate inhibition effects after a 7 day incubation period, with EC50 values between 24 mg/l and more than 1000 mg/l (equal to mg/g d.s.). In contrast, metronidazol was decisively toxic to anaerobic bacteria with an EC50 of 0.7 mg/l. In the anaerobic degradation tests according to ISO standard 11734 (1995), only benzylpenicillin showed certain ultimate biodegradation after 60 days and most antibiotics inhibited the digesting sludge in the respective parallel tested inhibition controls. Thus the inhibition of anaerobic bacteria by antibiotics observed in the degradation tests was higher than expected from the results of the inhibition tests. The possible explanations are that distinct substrates are used (yeast extract versus sodium benzoate), that the digestion sludge loses activity during the washing steps performed for the degradation tests and that the exposure time in the degradation tests was 8 times longer than in the inhibition test.     

Acute toxicity of 353-nonylphenol and its metabolites for zebrafish embryos

Background, aim and scope  Nonylphenol (NP) can be detected in the aquatic environment all over the world. It is applied as a technical mixture of isomers of which 353-NP is the most relevant both in terms of abundance (about 20% of total mass) and endocrine potential. 353-NP is metabolised in sewage sludge. The aims of the present study were to determine and to compare the acute toxicity of t-NP, 353-NP and its metabolites as well as to discuss if the toxicity of 353-NP changes during degradation. Materials and methods  353-NP and two of its metabolites were synthesised. The zebrafish embryo test was performed according to standard protocols. Several lethal and non-lethal endpoints during embryonal development were reported. NOEL, LOEL and EC₅₀ were calculated. Results  All tested compounds caused lethal as well as non-lethal malformations during embryo development. 353-NP showed a higher toxicity (EC₅₀ for lethal endpoints 6.7 mg/L) compared to its metabolites 4-(3.5-dimethyl-3-heptyl)-2-nitrophenol (EC₅₀ 13.3 mg/L) and 4-(3,5-dimethyl-3-heptyl)-2-bromophenol (EC₅₀ 27.1 mg/L). Discussion  In surface water, concentrations of NP are far below the NOEC identified by the zebrafish embryo test. However, in soils and sewage sludge, concentrations may reach or even exceed these concentrations. Therefore, sludge-treated sites close to surface waters should be analysed for NP and its metabolites in order to detect an unduly high contamination due to runoff events. Conclusions  The results of the present study point out that the toxicity of 353-NP probably declines during metabolisation in water, sediment and soil, but does not vanish since the major metabolites exhibit a clear toxic potential for zebrafish embryos. Recommendations and perspectives  Metabolites of environmental pollutants should be included in the ecotoxicological test strategy for a proper risk assessment. An erratum to this article can be found at     

Monitoring the toxicity of phenolic chemicals to activated sludge using a novel optical scanning respirometer.

This paper reports the development of optical scanning respirometer for determining the toxicity of chemicals to activated sludge. The respirometer is used to measure the dissolved oxygen concentration by monitoring the luminescence intensity of ruthenium dye immobilized in a polymer film in contact with the wastewater sample. The intensity is related to the extent of oxygen quenching of luminescence. The toxicity of chemicals can be evaluated by measuring the inhibition on respiration rate of microorganism using the scanning respirometer. The IC50 values (concentration of a chemical that exhibits 50% respiration inhibition) of various phenolic chemicals in activated sludge were determined. The performance of this method is compared with other toxicity methods. The experimental results indicate that the reproducibility and sensitivity of this respirometer are reasonably good.     

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.     

苯酚及其衍生物对氨氮生物硝化的抑制研究

利用富集培养的硝化污泥研究了苯酚及其衍生物2,4-二氯苯酚、对氨基酚、邻甲基酚和对硝基酚对氨氮生物硝化过程的抑制特性。结果表明,上述5种酚类化合物对硝化细菌的抑制类型均为非竞争性抑制,抑制常数K_I和EC₅₀值相等,分别为2.61、1.92、8.50、1.18和6.65 mg/L。上述5种酚类化合物对硝化细菌的抑制程度由大到小的顺序为：邻甲基酚>2,4-二氯苯酚>苯酚>对硝基酚>对氨基酚。酚类化合物对硝化细菌的抑制是可逆的,通过简单的抑制剂稀释或洗涤可以使硝化细菌恢复活性。在保证出水水质的前提下,抑制剂存在时硝化工艺的泥龄被迫延长,且抑制程度越大,泥龄延长的程度越大。当系统受到有毒物质冲击时,和调控泥龄相比,调节工艺的进水流量或改变容积负荷是缓冲冲击的更简捷、快速和有效的途径。     

Toxicity of 56 substances to trees

Toxicity data of substances to higher plants is needed for the purpose of risk assessment, site evaluation, phytoremediation, and plant protection. However, the results from the most common phytotoxicity tests, like the OECD algae and Lemna test, are not necessarily valid for higher terrestrial plants. The willow tree toxicity test uses inhibition of transpiration (aside of growth and water use efficiency) of willow cuttings grown in spiked solutions or soils as end point to quantify toxicity. This overview presents results from 60 studies including 24 new unpublished experiments for 56 different chemicals or substrates. Highest toxicity (EC₅₀ < 1 mg/L) was observed from exposure to heavy metals like copper and cadmium. Also, organotins and free cyanide showed very high toxicity. The toxic effect of chlorophenols on willows was comparable to that on duck weed (Lemna) and green algae, while volatile compounds like chlorinated solvents or benzene, toluene, ethylbenzene, and xylene had less effect on trees than on these aquatic plants, due to volatilization from leaves and test media. In particular low (g/L range) toxicity was observed for tested nanomaterials. Effects of pharmaceuticals (typically weak acids or bases) depended strongly of the solution pH. Like for algae, baseline toxicity was observed for willows, which is related to the water solubility of the compounds, with absolute chemical activity ranging from 0.01 to 0.1, but with several exceptions. We conclude that the willow tree toxicity test is a robust method for relating uptake, accumulation, and metabolism of substances to the toxicity to trees.     

Discrimination of excess toxicity from narcotic effect: Comparison of toxicity of class-based organic chemicals to Daphnia magna and Tetrahymena pyriformis

The discrimination of excess toxicity from narcotic effect plays a crucial role in the study of modes of toxic action for organic compounds. In this paper, the toxicity data of 758 chemicals to Daphnia magna and 993 chemicals to Tetrahymena pyriformis were used to investigate the excess toxicity. The result showed that mode of toxic action of chemicals is species dependent. The toxic ratio (TR) calculated from baseline model over the experimentally determined values showed that some classes (e.g. alkanes, alcohols, ethers, aldehydes, esters and benzenes) shared same modes of toxic action to both D. magna and T. pyriformis. However, some classes may share different modes of toxic action to T. pyriformis and D. magna (e.g. anilines and their derivatives). For the interspecies comparison, same reference threshold need to be used between species toxicity. The excess toxicity indicates that toxicity enhancement is driven by reactive or specific toxicity. However, not all the reactive compounds exhibit excess toxicity. In theory, the TR threshold should not be related with the experimental uncertainty. The experimental uncertainty only brings the difficulty for discriminating the toxic category of chemicals. The real threshold of excess toxicity which is used to identify baseline from reactive chemicals should be based on the critical concentration difference inside body, rather than critical concentration outside body (i.e. EC₅₀ or IGC₅₀). The experimental bioconcentration factors can be greatly different from predicted bioconcentration factors, resulting in different toxic ratios and leading to mis-classification of toxic category and outliers.     

Evaluation of laboratory-made sludge for an anaerobic biodegradability test and its use for assessment of 13 chemicals

Laboratory-made sludge for a biogas based anaerobic biodegradability test was prepared as an alternative for digested sludge from wastewater treatment plants (WWTPs). Biodegradation activities and background gas productions of digested sludge from various WWTPs were found to vary significantly depending on the source, which adversely affected test reliability. Subsequently, test conditions such as sludge concentration and sludge washing were examined with the laboratory-made sludge and a sludge concentration of 1.0 g-SS/L without washing was determined to be most suitable. Under these conditions, biodegradability tests were conducted for 13 select chemicals and their relative toxicities to methanogenic bacteria were evaluated. The results of biodegradability tests showed that chemicals with -OH and -CH2OH radicals were readily biodegraded and those with -Cl, -NO2, -NH2, -SO3H and -CH3 had inhibited degradation responses m-nitriphenol and 2,4,6-trichlorophenol were highly toxic to methanogenic bacteria, with m-nitrophenol completely inhibiting methane fermentation as low as 20 mg/L.     

Ionic liquids as lubricants or lubrication additives: An ecotoxicity and biodegradability assessment

This paper reports on the (eco)toxicity and biodegradability of ionic liquids considered for application as lubricants or lubrication additives. Ammonium- and pyrrolidinium-based cations combined with methylsulphate, methylsulphonate and/or (CF₃SO₂)₂N⁻ anions were investigated in tests to determine their aquatic toxicity using water fleas Daphnia magna, green algae Selenastrum capricornutum and marine bacteria (Vibrio fischeri). Additional test systems with an isolated enzyme (acetylcholinesterase) and isolated leukaemia cells from rats (IPC-81) were used to assess the biological activity of the ionic liquids. These compounds generally exhibit low acute toxicity and biological activity. Their biodegradability was screened according to OECD test procedures 301 B and 301 F. For choline and methoxy-choline ionic liquids ready biodegradability was observed within 5 or 10 d, respectively. Some of the compounds selected have a considerable potential to contribute to the development of more sustainable products and processes.     

Relationship between water solubility of chlorobenzenes and their effects on a freshwater green alga

The effective concentrations of benzene and 12 chlorobenzenes that reduced 50% of the primary productivity (EC₅₀) of a freshwater green alga, $\text{Ankistrodesmus falcatus}$, were determined. Benzene was the least toxic chemical and the toxicity increased as the degree of chlorine substitution in the aromatic ring increased. No EC₅₀ value could be obtained for HCB. A quantitative relationship was found to exist between water solubility, lipophilicity and the EC₅₀. A good correlation was also observed between the EC₅₀ for this alga and other toxicity data for various aquatic biota.     

Phytotoxicity and antioxidative enzymes of green microalga (Desmodesmus subspicatus) and duckweed (Lemna minor) exposed to herbicides MCPA,chloridazon and their mixtures

In this study, we evaluate the toxicity of MCPA (auxin-like growth inhibitor), chloridazon (CHD) (PSII-inhibitor) and their mixtures to floating plants and planktonic algae. Toxicity of MCPA (4-chloro-2-methylphenoxyacetic acid) and CHD (5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone) was first assessed in two growth inhibition tests with Lemna minor (ISO/DIS 20079) and Desmodesmus subspicatus (ISO 8692). Next, herbicide mixtures at concentrations corresponding to the EC values were used to assess their interactive effects, and the biomarkers were: for duckweed fresh weight, frond area, chlorophyll content and number of fronds, and for algae cell count and cell volume. The 3d EC₁₀ and EC₅₀ values using cell counts of D. subspicatus were 142.7 and 529.1 mg/L for MCPA and 1.7 and 5.1 mg/L for CHD. The 7d EC₁₀ and EC₅₀ values using frond number of L. minor amounted to 0.8 and 5.4 mg/L for MCPA and 0.7 and 10.4 mg/L for CHD. Higher sensitivity of reproductive (number of cells/fronds) than growth processes (cell volume/frond area) to herbicides applied individually and in mixtures was especially pronounced in the responses of Desmodesmus. Herbicide interactions were assessed by the two-way ANOVA and Abbott's formula. Generally, an antagonistic interaction with Lemna was revealed by MCPA and chloridazon, whereas additive effect of both herbicides was observed for Desmodesmus. A significant stimulation of SOD and APX activity by binary mixtures was noted in algal cells mainly after 24 and 48 hours of exposure. The extremely high stimulation of the activity of both enzymes was induced by the combination EC₁₀CHD + EC₅₀MCPA (48 h). Presumably due to oxidative stress, the treatment with CHD at concentration EC₅₀ after 72h was lethal for algae grown in aerated cultures, in contrast to standardized test conditions. Taking into account the consequences of risk assessment for herbicide mixtures we can state that a relatively low toxicity, as well as the lack of significant synergy between MCPA and CHD to non-target plants appears to be the most important result.     

Biodegradability and ecotoxicity of amine oxide based surfactants

The aerobic and anaerobic biodegradability as well as the aquatic toxicity of two fatty amine oxides and one fatty amido amine oxide were investigated. Aerobic biodegradation was evaluated using the CO(2) headspace test (ISO 14593) and biodegradation under anaerobic conditions was assessed employing a standardised batch test. The three amine oxide based surfactants tested were readily biodegradable under aerobic conditions but only the alkyl amido amine oxide was found to be easily biodegradable under anaerobic conditions. Toxicity to Photobacterium phosphoreum and Daphnia magna was evaluated. Bacteria (EC(50) from 0.11 to 11 mg l(-1)) proved to be more sensitive to the toxic effects of the amine oxide based surfactants than crustacea (IC(50) from 6.8 to 45 mg l(-1)). The fatty amido amine oxide showed the lowest aquatic toxicity.     

Anaerobic degradation and toxicity of commercial cationic surfactants in anaerobic screening tests

Anaerobic biodegradability and toxicity on anaerobic bacteria of di(hydrogenated tallow) dimethyl ammonium chloride (DHTDMAC) and two esterquats have been investigated. A batch test system containing municipal digester solids as a source of anaerobic bacteria, based on the method proposed by the ECETOC, has been applied. To evaluate the potential toxicity of such surfactants on anaerobic sludge, a co-substrate, an easily biodegradable compound in anaerobic conditions, has been added to the samples to test and the effects on biogas production have been determined. For the esterquats studied high biodegradation levels were obtained and no toxic effects on anaerobic bacteria were observed even at the highest concentrations tested, 100 and 200 mg C/l, respectively. On the contrary, DHTDMAC was not degradated at the same test conditions. However, no inhibitory effects on the biogas production were detected for this surfactant at concentrations <100 mg C/l.     

Evaluation of the OECD activated sludge,respiration inhibition test

The OECD Activated Sludge, Respiration Inhibition Test was evaluated as a method for assessing the potential impact of chemicals on wastewater treatment systems. Reproducibility of the test method was examined with respect to variation in oxygen consumption rates measured for a series of reactions set under identical conditions, and the variability in IC50 values estimated for three reference compounds. Several statistical procedures were also evaluated for analyzing activated sludge inhibition data. The test method was subsequently used to examine the effects of a wide variety of inorganic and organic chemicals on activated sludge respiration rates.     

Sequential UV-biological degradation of chlorophenols

The sequential UV-biological degradation of a mixture of 4-chlorophenol (CP), 2,4-dichlorophenol (DCP), 2,4,6-trichlorophenol (TCP), and pentachlorophenol (PCP) was first tested with each pollutant supplied at an initial concentration of 50 mg l(-1). Under these conditions, the chlorophenols were photodegraded in the following order of removal rate: PCP>TCP>DCP>CP with only CP and DCP remaining after 40 h of irradiation. The remaining CP (41 mg l(-1)) and DCP (13 mg l(-1)) were then completely removed by biological treatment with an activated sludge mixed culture. Biodegradation did not occur in similar tests conducted with a non-irradiated mixture due to the high microbial toxicity of the solution. UV treatment lead to a significant reduction of the phytotoxicity to Lipedium sativum but no further reduction of phytotoxicity was observed after biological treatment. Evidence was found that the pollutants were partially photodegraded into toxic and non-biodegradable products. When the pollutants were tested individually (initial concentration of 50 mg l(-1)), PCP, TCP, DCP, 4-CP were photodegraded according to first order kinetic model (r2>99) with half-lives of 2.2, 3.3, 5.7, and 54 h, respectively. The photoproducts were subsequently biodegraded. This study illustrates the potential of UV as pre-treatment for biological treatment in order to remove toxicity and enhance the biodegradability of organic contaminants. However, it also shows that UV treatment must be carefully optimized to avoid the formation of toxic and/or recalcitrant photoproducts and results from studies conducted on single contaminants cannot be extrapolated to mixtures.     

Strategies to evaluate biodegradability: application to chlorinated herbicides

The biodegradability of nitrochlorinated (diuron and atrazine) and chlorophenoxy herbicides (2,4-D and MCPA) has been studied through several bioassays using different testing times and biomass/substrate ratios. A fast biodegradability test using unacclimated activated sludge yielded no biodegradation of the herbicides in 24 h. The inherent biodegradability test gave degradation percentages of around 20–30 % for the nitrochlorinated herbicides and almost complete removal of the chlorophenoxy compounds. Long-term biodegradability assays were performed using sequencing batch reactor (SBR) and sequencing batch membrane bioreactor (SB-MBR). Fixed concentrations of each herbicide below the corresponding EC₅₀ value for activated sludge were used (30 mg L^?1 for diuron and atrazine and 50 mg L^?1 for 2,4-D and MCPA). No signs of herbicide degradation appeared before 35 days in the case of diuron and atrazine and 21 days for 2,4-D, whereas MCPA was partially degraded since the early stages. Around 25–36 % degradation of the nitrochlorinated herbicides and 53–77 % of the chlorophenoxy ones was achieved after 180 and 135 days, respectively, in SBR, whereas complete disappearance of 2,4-D was reached after 80 days in SB-MBR.     

Mixture Toxicity of the Antifouling Compound Irgarol to the Marine Phytoplankton Species Dunaliella tertiolecta

This study examined the toxicity of irgarol, individually and in binary mixtures with three other pesticides (the fungicide chlorothalonil, and the herbicides atrazine and 2,4-D), to the marine phytoplankton species Dunaliella tertiolecta. Standard 96-h static algal bioassays were used to determine pesticide effects on population growth rate. Irgarol significantly inhibited D. tertiolecta growth rate at concentrations ≥ 0.27 μ g/L. Irgarol was significantly more toxic to D. tertiolecta than the other pesticides tested (irgarol 96 h EC₅₀ = 0.7 μ g/L; chlorothalonil 96 h EC₅₀ = 64 μ g/L; atrazine 96 h EC₅₀ = 69 μ g/L; 2,4-D 96 h EC₅₀ = 45,000 μ g/L). Irgarol in mixture with chlorothalonil exhibited synergistic toxicity to D. tertiolecta, with the mixture being approximately 1.5 times more toxic than the individual compounds. Irgarol and atrazine, both triazine herbicides, were additive in mixture. The toxicity threshold of 2,4-D was much greater than typical environmental levels and would not be expected to influence irgarol toxicity. Based on these interactions, overlap of certain pesticide applications in the coastal zone may increase the toxicological risk to resident phytoplankton populations.     

Inhibition of heavy metals on fermentative hydrogen production by granular sludge

This study was conducted to investigate the toxicity of six electroplating metals on the H(2)-producing activity of a granular sludge sampled from an H(2)-producing upflow reactor treating sucrose-containing wastewater. The H(2) production activities of the sludge were measured in serum vials using wastewater containing not just sucrose and proper nutrient, but also individual heavy metals at concentrations ranging 0-5000 mg l(-1). The relative toxicity to H(2) production was found in the following order: Cu (most toxic)>Ni approximately Zn>Cr>Cd>Pb (least toxic). The C(I,50) values, at which the bioactivity of the sludge was reduced to 50% of the control, for individual heavy metals were Cu 30 mg l(-1), Ni and Zn 1600 mg l(-1), Cr 3000 mg l(-1), Cd 3500 mg l(-1), and Pb >5000 mg l(-1). Compared with the literature data, H(2)-producing sludge exhibited in general higher resistance to metal toxicity than methanogenic granular sludge.