Influence of Different Redox Conditions on the Biodegradation of the Pesticide Metabolites Phenol and Chlorophenols

Abstract

The fate and behaviour of phenol and monochlorophenols during bankfiltration and underground passage with variable redox conditions were investigated. A model ecosystem was used consisting in laboratory filter columns filled with natural underground material and operated with natural aerobic and anaerobic groundwater to create different redox situations.

The test substances (phenol, 2-chlorophenol, 3-chlorophenol, 4-chlorophenol) were added continuously to the infiltrating water and their concentration in the filter effluents determined. Beside the redox conditions other factors known to affect microbial degradation processes like the substrate concentration and the underground material were varied stepwise.

Phenol was degraded under both, aerobic and anaerobic conditions. The presence of oxygen is more favourable to degradation; no lag phase was observed under aerobic conditions. In a sulfate reducing environment, phenol could only be degraded after microbial adaptation. The length of the lag phase was strongly influenced by the substrate concentration and the undergroundmaterial. Prior contact with phenol resulted in a shorter lag phase.

Monochlorophenols behaved almost persistent in the model system. Degradation could only be observed in a test filter that provided a more active microbial population due to prior adaptation to phenol and a more favourable underground material.     

Degradation and metabolism of imazapyr in soils under aerobic and anaerobic conditions

The degradation of imazapyr in four soils was investigated under laboratory aerobic and anaerobic conditions. Under aerobic conditions, imazapyr degraded faster in yellow–red soil than in other soils, and its persistence decreased depending on soil pH in the order coastal soil (pH 8.8)?>?silt-loamy paddy soil (pH 7.9)?>?fluvio-marine yellow loamy soil (pH 7.1)?>?Yellow–red soil (pH 5.3). However, soil pH did not affect imazapyr degradation under anaerobic conditions. The half-lives of imazapyr in soils under aerobic conditions were in the range of 26–44 days estimated by the first-order kinetics model, while 3–10 days calculated by two-stage model under anaerobic conditions. The preceding results demonstrated that anaerobic conditions contributed to imazapyr disappearance in soils. Based on the spectral data of APCI-MS, ¹H NMR and IR, structures of the following metabolites: 2,3-pyridinedicarboxamide, 2,3-pyridinedicarboxylic anhydride and 2,3-pyridinedicarboximide for aerobic treatments; 2,3-pyridinedicarboxylic anhydride and 2-(4-hydroxy-5-oxo-2-imdazolin-2-yl) nicotinic acid for anaerobic treatments, were identified. Degradation mechanism under the different conditions was also discussed.     

Degradation of Alkylphenols by White Rot Fungus Irpex lacteus and Its Manganese Peroxidase

Alkylphenols are common endocrine disrupters that are produced from the degradation of widely used surfactants. Since they cause various harmful effects on aquatic life and in humans, they should be removed from the environments being contaminated. White rot fungus Irpex lacteus can completely degrade 100?mg/L of octylphenol, nonylphenol, and phenylphenol during 1?day of incubation in the complex YMG medium, which was the highest degrading capability among nine strains of white rot fungi tested. In the N-limited Kirk??s basal salts medium, I. lacteus could degrade almost 100?% of 100?mg/L octylphenol and nonylphenol in 1?h, and exhibited a high activity of manganese peroxidase (MnP; 1,790?U/L). MnP of I. lacteus was purified by ion exchange chromatography, and this degraded 99?% of 50?mg/L octylphenol and removed 80?% of estrogenic activity in 2?hours. In addition, the purified MnP (10?U/mL) degraded over 90?% of 50?mg/L nonylphenol in 1?h.     

Degradation of substituted phenols with different oxygen sources catalyzed by Co(II) and Cu(II) phthalocyanine complexes

Research on substituted phenol degradations has received substantial attention. In this work, effective Co(II) and Cu(II) phthalocyanine complexes as catalysts were studied to degrade toxic phenols to harmless products. The effect of various process parameters, such as initial concentration of phenol, catalyst, oxygen sources, and temperature on the degradation reaction was investigated to achieve maximum degradation efficiency. The catalytic activities of Co(II) and Cu(II) phthalocyanines were evaluated for oxidation of phenolic compounds such as p-nitrophenol, o-chlorophenol, 2,3-dichlorophenol, and m-methoxyphenol. Co(II) phthalocyanine displayed good catalytic performance in degradation of 2,3-dichlorophenol to 2,3-dichlorobenzaldehyde and 2,3-dichloro-1,4-benzoquinone with the highest TON and TOF values within 3?h at 50?°C. The fate of catalyst during the degradation process was followed by UV–Vis spectroscopy.     

Transformation of 2,4-dichlorophenol by H2O2/UV-C, Fenton and photo-Fenton processes: Oxidation products and toxicity evolution

In the present study, H₂O₂/UV-C, Fenton and photo-Fenton treatment of 2,4-dichlorophenol was compared in terms of oxidation products and acute toxicity. The oxidation products were identified by gas chromatography-mass spectroscopy, high performance liquid chromatography and ion chromatography, whereas changes in acute toxicity were evaluated by the Vibrio fischeri luminescence inhibition assay. H₂O₂/UV-C and photo-Fenton processes ensured complete 2,4-dichlorophenolremoval, detoxification and significant mineralization. Hydroquinone and formic acid were identified as the common oxidation products of the studied advanced oxidation processes investigated. 3,5-dichloro-2-hydroxybenzaldehyde, phenol, 4-chlorophenol and 2,5-dichlorohydroquinone were identified as the additional H₂O₂/UV-C oxidation products of 2,4-dichlorophenol. Acute toxicity decreased with decreasing 2,4-dichlorophenol and increasing chloride release.     

Aquatic degradation of triclosan and formation of toxic chlorophenols in presence of low concentrations of free chlorine

The degradation of 2-(2,4-dichlorophenoxy)-5-chlorophenol (triclosan) in chlorinated water samples was investigated. Sensitive determination of the parent compound and its transformation products was achieved by gas chromatography with mass spectrometry detection after sample concentration, using a solid-phase extraction sorbent and silylation of the target compounds. Experiments were accomplished using ultrapure water spiked with chlorine and triclosan concentrations in the low mg/l and ng/ml ranges respectively. Chlorination of the phenolic ring and cleavage of the ether bond were identified as the main triclosan degradation pathways. Both processes led to the production of two tetra- and a penta-chlorinated hydroxylated diphenyl ether, as well as 2,4-dichlorophenol. The formation of 2,3,4-trichlorophenol was not detected in any experiment; however, significant amounts of 2,4,6-trichlorophenol were noticed. All of these five compounds were also identified when triclosan was added to tap-water samples with free chlorine concentrations below 1 mg/l. Minor amounts of three di-hydroxylated phenols, containing from one to three atoms of chlorine in their structures, were also identified as unstable triclosan chlorination by-products. The analysis of several raw wastewater samples showed the co-existence of important concentrations of triclosan and its most stable by-products (2,4-dichlorophenol and 2,4,6-trichlorophenol), reinforcing the potential occurrence of the described transformations when products containing triclosan are mixed with chlorinated tap water.     

Pathways for Degrading TNT by Thu-Z: a Pantoea sp. Strain

2,4,6-Trinitrotoluene (TNT), an extensively used and versatile explosive, is harmful in soil and water. In the present study, four bacterial strains capable of degrading TNT have been isolated from contaminated sites and named as Thu-A, Thu-B, Thu-C, and Thu-Z. Thu-Z, which gave the highest degradation efficiency compared to the others, was assigned to the genus Pantoea according to its 16S rRNA gene. Similarities in both biochemical properties and morphology suggested that Thu-Z was a Pantoea sp. strain. Thu-Z was proved to be capable of using TNT as a sole nitrogen source by cleaving NO₂ from the nitroaromatic ring by direct aromatic ring reduction. Under nitrogen-limited conditions, 96.6?%?N of TNT was consumed by Thu-Z for growth, which was determined in terms of NaNO₂. Trace nitro reduction metabolites such as 2,4-diamino-6-nitrotoluene (24Dam) and 2,6-diamino-4-nitrotoluene (26Dam) were identified in the presence of (NH₄)₂SO₄. On the other hand, 4,4??,6,6??-tetranitro-2,2??-azoxytoluene (22Azo) and 2,2??,6,6??-tetranitro-4,4??-azoxytoluene (44Azo) were detected in the absence of (NH₄)₂SO₄. These indicated the existence of a dual pathway for Thu-Z, while the direct aromatic ring reduction was predominant. Addition of a nitrogen source ((NH₄)₂SO₄) after inoculation stimulated the growth of Thu-Z and accelerated TNT degradation.     

Photolytic degradation of triclosan in the presence of surfactants

The formation of 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) in the photolytic degradation of triclosan has evoked a great concern for its safety and environmental fate. The photochemical behaviour of triclosan in daily-used chemical products, in which triclosan is present in relatively high concentrations and coexists with surfactants, was, however, addressed less frequently. The present work is focused on the mechanistic aspects of triclosan photodegradation in an aqueous medium with a relatively high concentration (≥ 30 mg L⁻¹) and on the influence of pH (8.7 and 10.5) and surfactants (Triton X100, SDS, and CTMAB) on this process. The results demonstrated that photodegradation was strongly affected by the pH and the presence of surfactants. Photodegradation products, including 2,4-dichlorophenol (2,4-DCP), 5-chloro-2-(4-chlorophenoxyl)-phenol, 2,8-DCDD, dimers, trimers, and other intermediates, were identified. Based on the analysis of photoproducts, homolytic scission of ether bond, dechlorination, ring closure, and photo-polymerisation were proposed as the main routes of triclosan photodegradation.     

Anaerobic-anoxic-aerobic sequential degradation of synthetic wastewaters

This study was conducted in a continuous three-stage system of anaerobic (R1)-anoxic(R2)-aerobic (R3) reactors with synthetic wastewater containing phenol (1000 mg/L), chemical oxygen demand (COD) (3000 mg/L), CN⁻ (30 mg/L), SCN⁻(400 mg/L), and NH ₄ ⁺ -N (600 mg/L) as principal pollutants and well-acclimated heterogeneous microbial cultures. The final effluent was partially returned to R2 with a recycle ratio of 1. Anaerobic stage served to detoxify the feed by removing up to 80% of cyanide. Complete SCN⁻ removal and denitrification could be achieved in the anoxic stage by utilizing phenol as an internal source of carbon. Nitrification efficiency of 93% was obtained in the aerobic reactor. The results demonstrated that the three-stage system can give the desired final treated effluent quality (0 mg/L of phenol, 0.2 mg/L of CN⁻, 210 mg/L of COD, and 20 mg/L of NH ₄ ⁺ -N) and that the NO ₃ ⁻ -N concentration can be lowered by a higher recycle ratio.     

Visible-light-induced photocatalytic degradation of 4-chlorophenol and phenolic compounds in aqueous suspension of pure titania: demonstrating the existence of a surface-complex-mediated path

The visible-light-induced degradation reaction of 4-chlorophenol (4-CP) was investigated in aqueous suspension of pure TiO2. Contrary to common expectations, 4-CP could be degraded under visible illumination (lambda > 420 nm), generating chlorides and CO2 concomitantly. The observed visible reactivity was not due to the presence of trace UV light since the visible-light-induced reactions exhibited behaviors distinguished from those of UV-induced reactions. Dichloroacetate could not be degraded under visible light, whereas it degraded with a much faster rate than 4-CP under UV irradiation. The addition of tert-butyl alcohol, a common OH radical scavenger, did not affect the visible reactivity of 4-CP, which indicates that OH radicals are not involved. Other phenolic compounds such as phenol and 2,4-dichlorophenol were similarly degraded under visible light. The surface complexation between phenolic compounds and TiO2 appears to be responsible for the visible light reactivity. Diffuse reflectance UV-vis spectra showed that 4-CP adsorbed on TiO2 powder induced visible light absorption. The visible light reactivity among several TiO2 samples was apparently correlated with the surface area of TiO2. The visible-light-induced photocurrents on a TiO2 electrode could be obtained only in the presence of 4-CP. It is proposed that a direct electron transfer from surface-complexed phenol to the conduction band of TiO2 upon absorbing visible light (through ligand-to-metal charge transfer) initiates the oxidative degradation of phenolic compounds. When the surface complex formation was hindered by surface fluorination, surface platinization, and high pH, the visible-light-induced degradation of 4-CP was inhibited. The evidence of visible-light-induced reactions and the experimental conditions affecting the visible reactivity were discussed in detail.     

Sonochemical oxidation of phenol and three of its intermediate products in aqueous media: Catechol,hydroquinone, and benzoquinone. Kinetic and mechanistic aspects

The sonochemical oxidation of phenol has been examined in airequilibrated aqueous media at various pH’s and at various insonation powers. Its disappearance follows zero-order kinetics at [phenol]_initial ~ 30 to 70 μM Three principal intermediate species formed at pH 3: catechol (CC), hydroquinone (HQ), and p-benzoquinone (BQ); at natural pH (5.4–5.7) only catechol and hydroquinone formed. No intermediate species were detected at pH 12 under the conditions used. The sonochemical fate of CC, HQ, and BQ was also examined at pH 3 and at natural pH’s. At pH 3, BQ is the major species formed during insonation of HQ, while HQ is produced during insonation of BQ. In both cases, an additional intermediate formed in trace quantities that is identified as hydroxy-p-benzoquinone. These same intermediate species have been identified in the heterogeneous photocatalyzed oxidation of phenol in irradiated titania suspensions. The present results confirm the important role of ’OH radicals in degradation processes. Although CO₂ is the ultimate product in heterogenous photocatalysis, irradiation of a phenolic aqueous solution by ultrasounds showed no loss of total organic carbon (TOC) after several hours, even though the aromatic substrate and the intermediates had degraded. A simple kinetic model/scheme is described to account for the events in the conversion of the substrates to products. It is concluded that the hydrophobic benzoquinone reacts with ¹OH and H¹ radicals at the hydrophobic gas bubble/liquid interface, while the hydrophilic species (phenol, CC, and HQ) react, to a large extent, with the ¹ OH radicals in the solution bulk.     

Toxicity identification of gamma-ray treated phenol and chlorophenols

Ionizing radiation, such as gamma-rays and electron-beams, has been applied to modify toxicity of refractory pollutants and industrial wastewaters, however, very few studies reported the cause of toxicity changes by radiation treatment. In this work, degradation of phenol and chlorophenols (5·10⁻⁴M) by gamma-ray treatment and consequent toxicity changes were evaluated. Toxicity of 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP) was gradually decreased with increasing absorbed dose of gamma-radiation. However, in the case of phenol and monochlorophenols (2-, 3-, and 4-CPs), toxicity was dramatically increased particularly, for a dose of as low as 1 kGy. Hydroquinone, benzoquinone, catechol, chlorohydroquinone, and 4-chlorocatechol were identified to be main by-products of gamma-ray treatment. From the solid phase extraction (SPE) fractionation study, toxicity-causing by-products were found to be hydroquinone, benzoquinone, chlorohydroquinone, and/or 4-chlorocatechol.     

In situ photoproduction of dichlorodibenzo-p-dioxin from non-ionic triclosan isolated in solid argon

The infrared spectrum of triclosan [or 5-chloro-2-(2,4-dichlorophenoxy)phenol] isolated in a low temperature (～15 K) argon matrix has been recorded and assigned with help of DFT claculations undertaken with the B3LYP functional and the 6-311++G(d,p) basis set. The obtained spectrum doubtlessly exhibits the characteristic vibrational signature of the neutral (phenol) form of the compound, which exists in two different conformations (forms I and II) in the matrix, in a I: II population ratio of ca. 0.75. Upon broadband UV irradiation of the matrix-isolated triclosan with unfiltered light provided by a xenon arc lamp, formation of 2,8-dichlorodibenzo-p-dioxin (2,8-DCDD) was observed, together with HCl. The reaction seems to occur through initial photoproduction of the triclosan phenol radical derivative, and involve in the initial step participation of dissociative (πσ¹) excited states along the OH stretching coordinate, as observed previously for other phenol derivatives. The photochemically detached hydrogen atom derived from triclosan may then react with the closest located chlorine atom in the triclosan molecule to yield HCl and a biradical species, which can subsequently undergo a ring-closure reaction by intramolecular recombination, leading to the observed 2,8-DCDD.     

Toxicity of butyltin,phenyltin and inorganic tin compounds to sulfate‐reducing bacteria isolated from anoxic marine sediments

The toxicity of butyltin, phenyltin and inorganic tin compounds to three pure strains of sulfate‐reducing bacteria (SRB), isolated from a tributyltin (TBT)‐polluted sediment, was determined. The isolated strains were identified as belonging to the genus Desulfovibrio. A new toxicological index (GR₂₅) was developed to assay the toxicity of organotin compounds. Deleterious effects on suspended anaerobic cell cultures were observed for concentrations ranging between 500 and 600 µM for tin tetrachloride, 55 and 260 µM for triorganotins, 30 and 90 µM for diorganotins, and 1 and 6 µM for mono‐organotins. Whereas the number of substituents influenced the toxicity of organotins, the type of substituent (butyl or phenyl) proved to have little or no impact. Trisubstituted compounds (tributyl‐ and triphenyl‐tin) were less toxic to these strains of SRB than the monosubstituted forms (monobutyl‐ and monophenyl‐tin). This is the opposite trend to that currently reported for aerobic organisms. Under the given anoxic conditions, the toxicity of organotin compounds obtained yielded a significant negative correlation with the total surface area (TSA) of the tested molecules. Comparison of the TBT toxicity data observed for different microbial groups suggests that the tolerance of bacteria to organotin compounds might be related to organotin–cell wall interactions as well as to aerobic or anaerobic metabolise pathways. Copyright © 2000 John Wiley & Sons, Ltd.     

Rapid Isolation of a Facultative Anaerobic Electrochemically Active Bacterium Capable of Oxidizing Acetate for Electrogenesis and Azo Dyes Reduction

In this study, 27 strains of electrochemically active bacteria (EAB) were rapidly isolated and their capabilities of extracellular electron transfer were identified using a photometric method based on WO₃ nanoclusters. These strains caused color change of WO₃ from white to blue in a 24-well agar plate within 40 h. Most of the isolated EAB strains belonged to the genera of Aeromonas and Shewanella. One isolate, Pantoea agglomerans S5-44, was identified as an EAB that can utilize acetate as the carbon source to produce electricity and reduce azo dyes under anaerobic conditions. The results confirmed the capability of P. agglomerans S5-44 for extracellular electron transfer. The isolation of this acetate-utilizing, facultative EBA reveals the metabolic diversity of environmental bacteria. Such strains have great potential for environmental applications, especially at interfaces of aerobic and anaerobic environments, where acetate is the main available carbon source.     

Enzymatic degradation of highly phenolic lignin-based polymers (lignophenols)

Enzymatic degradation of two lignin-based polymers (lignophenols), lignocatechol and lignocresol, prepared by selectively grafting catechol and p-cresol to C_α positions of lignin, respectively, were carried out in aqueous organic solvents. Both lignophenols showed high reactivity in the peroxidase-catalyzed oxidation. Structural analyses by NMR spectroscopies revealed that the degraded lignophenols contained aliphatic chain content, which might be mainly formed in the reduction of the intermediate initially generated by the aromatic ring cleavage. Lower amount of aromatic units in the lignophenols after degraded by peroxidase also indicted the cleavage of aromatic rings. Due to the substitution of phenols at C_α positions of lignin, the degraded lignophenols did not have carbonyl structure, which was abundant in the biodegradation products of native lignin. The two lignophenols were also degraded by Rhus vernicifera laccase. But the degree of degradation was lower than that of the degradation by peroxidase, which might be due to the low activity of laccase on the lignin moieties in lignophenols.     

Mineralization and Kinetics of Reactive Brilliant Red X-3B by a Combined Anaerobic–Aerobic Bioprocess Inoculated with the Coculture of Fungus and Bacterium

Mineralization of Reactive Brilliant Red X-3B by a combined anaerobic–aerobic process which was inoculated with the co-culture of Penicillium sp. QQ and Exiguobacterium sp. TL was studied. The optimal conditions of decolorization were investigated by response surface methodology as follows: 132.67 g/L of strain QQ wet spores, 1.09 g/L of strain TL wet cells, 2.25 g/L of glucose, 2.10 g/L of yeast extract, the initial dye concentration of 235.14 mg/L, pH 6.5, and 33 °C. The maximal decolorization rate was about 96 % within 12 h under the above conditions. According to the Haldane kinetic equation, the maximal specific decolorization rate was 89.629 mg/g˙h. It was suggested that in the anaerobic–aerobic combined process, decolorization occurred in the anaerobic unit and chemical oxygen demand (COD) was mainly removed in the aerobic one. Inoculation of fungus QQ in the anaerobic unit was important for mineralization of X-3B. Besides, the divided anaerobic–aerobic process showed better performance of COD removal than the integrated one. It was suggested that the combined anaerobic–aerobic process which was inoculated with co-culture was potentially useful for the field application.     

Stable partial nitritation of mature landfill leachate in a continuous flow bioreactor: Long-term performance,microbial community evolution,and mechanisms

A continuous flow bioreactor was operated for 300 days to investigate partial nitritation (PN) of mature landfill leachate, establishing the long-term performance of the system in terms of the microbial community composition, evolution, and interactions. The stable operation phase (31–300 d) began after a 30 days of start-up period, reaching an average nitrite accumulation ratio (NAR) of 94.43% and a ratio of nitrite nitrogen to ammonia nitrogen (NO₂⁻-N/NH₄⁺-N) of 1.16. Some fulvic-like and humic-like compounds and proteins were effectively degraded in anaerobic and anoxic tanks, which was consistent with the corresponding abundance of methanogens and syntrophic bacteria in the anaerobic tank, and organic matter degrading bacteria in the anoxic tank. The ammonia-oxidizing bacteria (AOB) Nitrosomonas was found to be the key functional bacteria, exhibiting an increase in abundance from 0.27% to 6.38%, due to its collaborative interactions with organic matter degrading bacteria. In-situ inhibition of nitrite-oxidizing bacteria (NOB) was achieved using a combination of free ammonia (FA) and free nitrous acid (FNA), low dissolved oxygen (DO) with fewer bioavailable organics conditions were employed to maintain stable PN and a specific ratio of NO₂⁻-N/NH₄⁺-N, without an adverse impact on AOB. The synergistic relationships between AOB and both denitrifying bacteria and organic matter degrading bacteria, were found to contribute to the enhanced PN performance and microbial community structure stability. These findings provide a theoretical guidance for the effective application of PN-Anammox for mature landfill leachate treatment.     

Enhanced photocatalytic activities of ZnO thin films: a comparative study of hybrid semiconductor nanomaterials

Nanostructure single ZnO, SnO₂, In₂O₃ and composite ZnO/SnO₂, ZnO/In₂O₃ and ZnO/SnO₂/In₂O₃ films were prepared using sol?Cgel method. The obtained composite films were characterized with X-ray diffraction (XRD), scanning electron microscopy (SEM), and UV?CVis spectroscopy. The photocatalytic activities of composite films were investigated using phenol (P), 2,4-dichlorophenol (2,4-DCP), 4-chlorophenol (4-CP) and 4-aminophenol (4-AP) as a model organic compounds under UV light irradiation. Hybrid semiconductor thin films showed a higher photocatalytic activity than single component ZnO, SnO₂ and In₂O₃ films. The substituted phenols degrade faster than phenol. The ease of degradation of phenols is different for each catalyst and the order of catalytic efficiency is also different for each phenol. The use of multiple components offered a higher control of their properties by varying the composition of the materials and related parameters such as morphology and interface. It was also found that the photocatalytic degradation of phenolic compounds on the composite films and single films followed pseudo-first order kinetics.     

Biodegradation of Mixed Phenolic Compounds Under High Salt Conditions and Salinity Fluctuations by <Emphasis Type="Italic">Arthrobacter</Emphasis> sp. W1

High salt concentration and salinity fluctuations in wastewater challenge the efficiency of microbial strains used for cleanup of pollutants. In this study, it was investigated that the new isolated Arthrobacter sp. W1 degraded mixed phenolic compounds under complex salt conditions. The results showed that Arthrobacter sp. W1 was able to utilize various phenolic compounds as carbon source under high salt conditions. It can degrade phenol and p-cresol mixture at 10% NaCl, although rates of degradation and cell growth were lower compared to 5% NaCl. The presence of trace p-cresol significantly inhibited phenol biodegradation. When salinity fluctuations were between 1% and 10% NaCl, strain W1 was able to degrade substrates and survived. It was also suggested that the presence of salts (i.e., NaCl, KCl, Na₂SO₄, and K₂SO₄) had almost no effects on the microbial growth and biodegradation process. Therefore, Arthrobacter sp. W1 would be a promising candidate for bioremediation of phenolic compounds under complex salt conditions.