Determination of free and total S-phenylmercapturic acid by HPLC/MS/MS in the biological monitoring of benzene exposure

Abstract

Urinary S-phenylmercapturic acid (SPMA) is a biomarker suggested by the American Conference of Governmental Industrial Hygienists (ACGIH) for assessing occupational exposure to benzene. A possible cause of the miscorrelation between environmental monitoring and biological monitoring for benzene exposure, which many authors complain about, is the existence of a urinary metabolite that turns into SPMA by acid hydrolysis. Forty urine samples were tested to determine which concentration value would correspond to the ACGIH Biological Exposure Index (BEI) of 25 µg g^?1 creatinine if exposure assessment was based on the determination of SPMA after quantitative hydrolysis of its precursor. An aliquot of each sample was hydrolysed with 9 M H₂SO₄, a second one was brought to pH 2 and a third one was used as it was (free SPMA). SPMA was determined by high-performance liquid chromatography/tandem mass spectrometric technique (HPLC/MS/MS) using an internal standard. The analytical method was validated in the range 0.5–50 µg l^?1. The average SPMA in pH 2 samples is 45–60% of the total, while free SPMA varies from 1% to 66%. The hydrolysis of pre-SPMA reduces the likelihood of variability in the results by reducing pH differences in urine samples and increasing the amount of measured SPMA. The BEI limit value would be about 50 µg g^?1 creatinine.     

Comparison of hydrolysis and HPLC/MS/MS procedure with ELISA assay for the determination of S-phenylmercapturic acid as a biomarker of benzene exposure in human urine

The present study compared three methods for the determination of S-phenylmercapturic acid (S-PMA), a metabolite of benzene, in human urine: a HPLC/MS/MS technique with two different sample treatments (strong and partial hydrolysis) and a commercial assay based on anti-S-PMA monoclonal antibodies with chemiluminescence detection. Biological monitoring was done on 126 volunteers and the results were compared for the three methods and also with benzene exposure levels (range <3.0–592.5 μg/m³). The correlation between environmental monitoring data and S-PMA levels in non-smokers (n = 73) was highly significant (p < 0.0001, Student's t-test) for both HPLC/MS/MS methods (r = 0.65 both for strong acidic hydrolysis of the urine and hydrolysis at pH 2) but not for the immunoassay, which overestimated the S-PMA levels by about 8 μg/g creatinine (creat.). Therefore the immunoassay is only useful as a semiquantitative screening test, but quantitative results need to be confirmed by a more accurate method like HPLC/MS/MS. The HPLC/MS/MS procedure with strong acid hydrolysis led to a recovery of S-PMA about double that using pH 2 hydrolysis, giving more accurate results. The difference between the results with the two methods makes it difficult to compare the strong acidic hydrolysis data with the ACGIH BEI value of 25 μg/g creat. since the BEI^® documentation is based on data collected in pH conditions that were not always controlled, which may underestimate the true S-PMA concentration. Besides, as levels of benzene exposure were high, smoking was not considered a confounding factor. The BEI for S-PMA in end of shift urine samples could be reconsidered when sufficient data are available from studies where the analyses are carried out in comparable conditions of hydrolysis and monitoring only non-smoking subjects.     

A note on urinary trans,trans-muconic acid level among Thai press workers

Benzene is a common toxic volatile substance associated with many industrial processes. Benzene exposure is of particular concern because recent research indicates that it can result in chronic toxicity and thousands of workers in industrial plants experience ongoing exposure. Therefore, the determination and control of benzene exposure among at-risk workers is very important. Urinary trans,trans-muconic acid (ttMA) determination is a helpful test for monitoring groups of at-risk workers for exposure to benzene. In this study, 103 urine samples were obtained from 60 controls and 43 occupational exposed press workers in a press factory in Bangkok. All samples were analysed for ttMA using a previously reported method. The average urinary ttMA levels for the control and exposed groups were 0.08±0.03 mg g^-1 creatinine and 0.56±0.65 mg g^-1 creatinine, respectively. Significantly higher urinary ttMA levels were observed among the press workers (p=0.03). The introduction of public health policies concerning the prevention of exposure to benzene among at-risk workers is recommended, and more widespread use of biological monitoring for the assessment and control of occupational exposure to industrial chemicals is encouraged.     

Biological monitoring of exposure to low concentrations of benzene in workers at a metallurgical coke production plant: new insights into S-phenylmercapturic acid and urinary benzene

Context: Urinary S-phenylmercapturic acid (SPMA) and benzene (U-Ben) are usually measured at the end of the work shift (ES), although their kinetic of elimination is not clearly known.

Objective: To investigate SPMA and U-Ben elimination 16?h after the ES, in 93 coke production workers exposed to low benzene concentrations.

Materials and methods: Airborne benzene (A-Ben) was measured by passive samplings, while SPMA, U-Ben, methyl-tert-butyl ether (U-MTBE), cotinine (U-Cot) and creatinine were determined on urine samples collected at ES and before the beginning of the next work shift (next BS).

Results: Median A-Ben concentrations were 17.2?µg/m³ in the personal and 34.7?µg/m³ in the stationary samplings. SPMA was always detectable, whereas U-Ben was below the limit of quantification in 26.7% of the ES and 35.6% of the next BS samples, and U-MTBE in more than the 80.0% of the samples. At both the sampling times, SPMA and U-Ben showed a positive dependence on personal A-Ben, as well as on creatinine and U-Cot values.

Discussion and conclusion: SPMA and U-Ben at the next BS were dependent on the exposure to low benzene concentrations suffered in the previous work shift, prompting a reconsideration of the urine sampling time recommended by the American Conference Governmental Industrial Hygienists (ACGIH).     

Enrichment and properties of urinary pre-S-phenylmercapturic acid (pre-SPMA)

In benzene metabolism, pre-S-phenylmercapturic acid (pre-SPMA) is the precursor to S-phenylmercapturic acid (SPMA). Urinary pre-SPMA/SPMA ratios are variable. For the determination of urinary SPMA as a biomarker of exposure to benzene it is essential to completely convert pre-SPMA to SPMA. We developed a procedure for the enrichment and determination of urinary pre-SPMA by LC–MS/MS which allowed us to trace the conversion of pre-SPMA to SPMA. Complete conversion was found upon treatment of urine with HCl (37%) at pH 1.1. Previously reported treatment of urine with concentrated H₂SO₄ was found to yield SPMA levels higher than after HCl treatment. The origin of that extra SPMA amount is unknown. In conclusion, our findings suggest that pre-treatment of urine with HCl to adjust the pH to 0.5–1 is essential for complete conversion of pre-SPMA to SPMA and should be applied prior to analysis of SPMA in urine.     

Development and validation of a competitive immunoassay for urinary S-phenylmercapturic acid and its application in benzene biological monitoring

An immunoassay that quantifies urinary S-phenylmercapturic acid (PMA), a benzene-specific biomarker, has been developed and its potential usefulness as a screening tool for monitoring occupational exposure to benzene has been demonstrated. Analytical reliability has been confirmed by correlation of results with gas chromatography-mass spectrometry (GC/MS) data (R = 0.92). The assay has been configured as a competitive enzyme-linked immunosorbent assay (ELISA) to facilitate rapid throughput of samples. The ELISA has a working range of 40-1200 nmol l^-1 urinary PMA and appears to be unaffected by the presence of structurally related urinary metabolites. Background levels of 0-1.9µmol PMA/mol creatinine (mean 0.9 µmol mol^-1, n = 32) were measured in non-smoking control subjects. Recent exposures to benzene (8 h time-weighted averages-TWA), during diverse industrial processes, over the range 0-4.8ppm were identified by application of the assay in biological monitoring programmes.     

Augmentation of Soil with Sporangia of Actinoplanes spp. for Biological Control of Pythium Damping-off

A method was developed for applying strains of Actinoplanes spp. that are hyper-parasites of oospores of Pythium ultimum to soil for reducing Pythium damping-off of plants. The method is based on the augmentation of soil with sporangia of a strain of Actinoplanes spp. borne on clay granules. In vitro sporulation of strains K30, W57, W257 and 25844 was: (1) greater for most strains on dilute Czapek-Dox agar than on four other agar media; (2) inhibited by continuous exposure to fluorescent light of intensity 4-150 μEm^-2s^-1, but not by exposure to 1 μEm^-2s^-1 or darkness; (3) greater at 20-307deg;C than at 10°C;and (4) greater at pH 6-7 than at pH 5 or 8. On solid carriers treated with dilute Czapek-Dox broth (pH 7) and incubated in the dark at 30°C for 3 weeks, strains sporulated poorly or not at all on vermiculite, perlite and rice hulls, but sporulated abundantly (10⁷-10⁹ colony-forming units (CFU) g^-1 of granules) on montmorillonite clay granules. When strains 25844, W57 and W257 were applied as granules (4 10⁷ - 4 × 10⁸ CFU g^-1) at 5% (w/w) to field plots infested with 750-1000 oospores of P. ultimum g^-1 of soil, only strain 25844 consistently increased emergence and reduced root rot of table beets 8- 1 at 24-28 days after planting compared with controls. Strain 25844 (10⁸ CFU g^-1 of granules) at 1% (w/w) also increased the emergence of bush beans at 28 days after planting in P. ultimum-infested plots, but lower rates were ineffective. The inoculum viability of strain 25844 on clay granules declined 100-fold during 2 months of storage at 5-35°C, but thereafter remained stable for another 4 months. Strain 25844 on 6-month-old granules retained a high degree of hyper-parasitic activity toward oospores of P. ultimum. Augmentation of field soil with sporangia of Actinoplanes spp. is a valid approach to the biological control of pythium damping-off.     

Analysis of S-phenyl-L-cysteine in globin as a marker of benzene exposure

An assay has been developed to determine S-phenylcysteine (SPC) in globin as a potential biomarker for exposure to benzene. The sensitivity of the assay is less than 20 pmol SPC g^-1 globin. Following acidic hydrolysis of the protein, the modified amino acid is purified by reverse phase cartridge chromatography and HPLC, prior to conversion to the tert-butyldimethylsilyl derivative and GC-MS selected ion recording. Quantitation is achieved using the internal standard [²H₅]-SPC, and calibration lines were established using a synthetic peptide Leu-His-SPC-Asp-Lys. Control human globin was found to contain ca 30 pmol SPC g^-1 globin in two populations. The source of the apparent background level of SPC is unknown.     

Biological monitoring among benzene-exposed workers in Bangalore city, India.

Environmental and biological monitoring was carried out in the winter season of 2004 for 30 gasoline station workers (study subjects) and 30 office workers (controls) of Bangalore city, India. Personal air sampling was carried out in the breathing zone of workers using an Anasorb CSC sorbent tube (SKC 226-01) fitted to the low-flow personal samplers (PCXR4 and pocket pump Model No. 210-1002) at a flow rate of 200 ml min(-1) during the shift work of 8 h. The benzene content adsorbed in the sorbent tube (SKC 226-01) was desorbed with 1 ml of benzene-free carbon disulfide on a developing vibrator and later analysed by Trace GC fitted with MXT-624 column and flame ionization detector. The mean time weighted average benzene concentration found among study and controls was 1.10+/-1.08 and 0.070+/-0.035 mg m(-3), respectively. Biological monitoring for benzene exposure was performed by measuring trans,trans muconic acid (t,t-MA) in the end shift urine samples using HPLC-UV technique. End-shift urine samples (1 ml) were adjusted to pH 7-9 with phosphate buffer pH 7.4 passed through the preconditioned Q-SAX anion-exchange cartridge and the (t,t-MA) is extracted with 10% acetic acid and later analysed by HPLC-UV detection The mean t,t-MA found among study and controls were 563.16+/-281.81 and 266.88+/-110.65 microg g(-1) creatinine. About 50% of the study subjects (15) have higher t,t-MA values than the biological exposure index of the American Conference of Government Industrial Hygienist (ACGIH). Correlation is significant at 5% level (p<0.05) between personal air benzene concentration and urinary t,t-MA in the study group. Based on these findings, the t,t-MA can be used as a biomarker for benzene exposure.     

New ultrasensitive 32P-postlabelling method for the analysis of 3,N4-etheno-2'-deoxycytidine in human urine

Etheno-DNA adducts are generated from exogenous carcinogens such as vinyl chloride and urethane and also from endogenous lipid peroxidation products such as trans-4-hydroxy-2-nonenal (HNE). The present authors and others have established that 1,N⁶-ethenodeoxyadenosine (εdA) and 3,N⁴-ethenodeoxycytidine (εdC) are present in human urine and could be explored as biomarkers for monitoring whole-body oxidative stress. The present study reports on a new ultrasensitive ³²P-postlabelling/thin-layer chromatography (TLC) method for the analysis of εdC as deoxynucleoside in human urine. The urine samples were purified and enriched on a solid-phase silica C-18 column followed by a semi-preparative reverse-phase high-performance liquid chromatography. The purified sample was labelled with a multisubstrate deoxyribonucleoside kinase from Drosophila melanogaster (Dm-dNK) in the presence of 5'-bromo-2'-deoxyuridine (BrdU) as internal standard. The absolute sensitivity of the method was 0.1 fmol εdC detectable in 500 µl of human urine. The analysis of human urine samples from 15 healthy volunteers revealed a mean εdC level of 2.49±1.76 (SD) fmol µmol^-1 creatinine (range 0.66-6.42). By this non-invasive method, εdC in human urine could be explored as a biomarker for oxidative stress-related human diseases.     

Urinary hydroxylated metabolites of polycyclic aromatic hydrocarbons as biomarkers of exposure in asphalt workers

Background. Fumes and vapours released during laying of hot asphalt mix have been recognised as a major source of exposure for asphalt workers. Objectives. We investigated the relationships between inhalation exposure to asphalt emissions and urinary biomarkers of polycyclic aromatic hydrocarbons (PAHs) in asphalt workers (AW, n=75) and in ground construction workers (CW, n=37). Methods. Total polyaromatic compounds (PAC) and 15 priority PAHs in inhaled air were measured by personal sampling. Hydroxylated PAH metabolites (OH-PAHs) (2-naphthol, 2-hydroxyfluorene, 3-hydroxyphenanthrene, 1-hydroxypyrene, 6-hydroxychrysene and 3-hydroxybenzo[a]pyrene) were determined in urine spot samples collected in three different times during the work week. Results. Median vapour-phase PAC (5.5 µg m^-3), PAHs (≤50 ng m^-3) and OH-PAHs (0.08-1.11 µg l^-1) were significantly higher in AW than in CW, except in the cases of air naphthalene and 2-naphthol. Airborne levels of particle-phase contaminants were similar in the two groups and much lower than vapour-phase levels; metabolites of particulate PAHs were never found in quantifiable amounts. An appreciable increase in OH-PAH levels during the work day and work week was found in AW; median levels for 2-hydroxyfluorene, 3-hydroxyphenanthrene and 1-hydroxypyrene were, respectively, 0.29, 0.08 and 0.18 at baseline; 0.50, 0.18 and 0.29, pre-shift; 1.11, 0.44 and 0.44 µg l^-1, post-shift. Each OH-PAH exhibited a characteristic profile of increase, reflecting differences in half-lives of the parent compounds. In non-smoking subjects, positive correlations were found between vapour-phase PAC or PAHs and OH-PAHs both in pre- and post-shift samples (0.34 ≤ r≤69). Smokers exhibited 2-5-fold higher OH-PAHs than non-smokers, at any time and at both workplaces. Conclusions. Our results suggest that OH-PAHs are useful biomarkers for monitoring exposure to asphalt emissions. The work-related exposure to PAC and PAHs was low in all AW, but urinary metabolites reflected exposure satisfactorily.     

Biological monitoring of exposure to n-heptane by gas chromatographic mass spectrometric determination of its metabolites

Urine samples from 10 workers that had been exposed to n-heptane were analysed by the GC/MS technique to verify the concentrations and the relative abundances of its metabolites. The procedure of sample preparation has undergone some modifications with respect to the Perbellini method and the mass spectrometric detection was carried out in selected ions monitoring conditions. The analyses of samples collected during three different workshifts showed that 2 heptanol was not the main metabolite and that the remains of 2 heptanone, valerolactone and 2,5 heptanedione were present at the beginning of the successive work week at 12, 34 and 39 of the average values found at the end of the previous week. Overall, a very slow excretion rate was detected for the last metabolite. The main and significant metabolite at the end of the two workshifts was 2 heptanone which was detected in urine at average values of 413 and 238 μg g^-1 creatinine. This urinary ketone correlated better than other metabolites with respect to the airborne n-heptane at the end of both the workshift and work week. These preliminary data suggest that further studies should be carried out to confirm whether 2 heptanone is really useful as an n-heptane marker in biological monitoring.     

Hsp70 instability and induction by a pesticide in Folsomia candida

The heat shock protein Hsp70 has been shown to be a promising biomarker in aquatic and terrestrial organisms. However, its analysis in the soil insect Folsomia candida (Collembola) poses many problems as the protein is particularly unstable in this species. Western blotting has shown that the principal degradation fragment has a size of 48 kDa. We have developed a Western blot method that avoids the degradation of Hsp70 and was successful in detecting the protein in the springtail F. candida after a heat shock (12, 18 and 24 h at 32°C). In the second part of the study the organisms were exposed to artificial compressed soil contaminated with the dinitrophenol dinoseb (10, 15 and 20 µg g^-1 dry weight [DW]). Hsp70 was analysed in pooled samples (40 to 150 collembola according to age) after 1, 2, 3, 4, 5, 6, 7, 11 and 14 days. The only significant induction was observed after 5 days at 20 µg g^-1 DW of dinoseb. The induction patterns over time were dissimilar for the different concentrations and a relatively high variability between the replicates was observed. Our results show that we must be cautious when interpreting biomarker results, especially those for Hsp70.     

Methodological issues in the biological monitoring of urinary benzene and S-phenylmercapturic acid at low exposure levels

Biological monitoring of low level exposure to pollutants is a very challenging analytical activity, and the quality of results is difficult to assess, especially when a certified reference material is unavailable. The aim of this work was to evaluate the reliability of the assays used to measure urinary benzene (Benz-U) and S-phenylmercapturic acid (SPMA), by applying an internal quality control protocol. Urine spot samples from 705 subjects who were either members of the general urban population, gasoline station attendants, or refinery plant workers were assayed for Benz-U and SPMA, using GC/MS and LC/MS/MS, with quantification limits of 15 ng/L and 0.10 μg/L. The median Benz-U concentration was 263 ng/L (60-2789 ng/L, 5th-95th percentile), and the median SPMA concentration was 0.19 μg/L (<0.1-2.5 μg/L, 5th-95th percentile). Linearity of both assays was good, but a less-than-proportional response was found for SPMA concentrations below 1 μg/L. Between-run precision and accuracy for Benz-U concentration determination were assessed using quality controls at 120 ng/L and 1000 ng/L and were 10.3% and 4.8%, and 104.8% and 98.9%, respectively; while the precision and accuracy for SPMA concentration determination at 0.3 μg/L, 2.5 μg/L, and 20 μg/L were 40.3%, 6.2%, and 6.2%, and 48.3%, 96.3%, and 98.8%, respectively. Precision, estimated using duplicates of unknown samples, was 13.4% for Benz-U and 26.5% for SPMA analyses. Control charts for the means of the slope of the linear calibration curve of Benz-U showed good stability of the means over a five-year period. For SPMA, a two-laboratory comparison revealed acceptable agreement between ln-transformed data pairs, with a slope of the linear regression of 0.863 (confidence interval 0.774-0.952), null intercept, and a Pearson's r value of 0.844. Reliable results were obtained for Benz-U analyses over the entire concentration range, and for high and medium SPMA levels. However, the determination of SPMA concentrations at levels close to the limit of quantification was less reliable.     

Effects of Oxygenation on Hydrocarbon Biodegradation in a Hypoxic Environment

In 1984, an underground storage tank leaked approximately 41,000 L of gasoline into the ground water at the Naval Construction Battalion Command in Port Hueneme, CA (USA). Benzene, toluene, ethylbenzene, and xylenes (BTEX) contamination stimulated remedial action. In 1995, a ground water circulation well (GCW) and network of surrounding monitoring wells were installed. After year of operation, dissolved oxygen and nitrate concentrations remained low in all monitoring wells. Benzene utilization (the sum of respiration, uptake, and conversion to polar compounds) ranged from 0.03 to 4.6 µg L^-1 h^-1, and toluene utilization ranged from 0.01 to 5.2 µg L^-1 h^-1. Heterotrophic bacterial productivity (total carbon assimilation) increased dramatically in the GCW, although benzene and toluene utilization decreased markedly relative to surrounding wells. Benzene and toluene uptake accounted for a significant proportion (mean=22%) of the heterotrophic bacterial productivity except within the GCW, indicating other fuel contaminant or indigenous organic carbon and not BTEX compounds served as primary carbon source. The GCW effectively air-stripped BTEX compounds, but failed to stimulate benzene and toluene biodegradation and thus would not be effective for stimulating BTEX bioremediation under current deployment parameters. Air stripping was three orders of magnitude more effective than biodegradation for removing benzene and toluene in the GCW.     

Immobilization of soybean seed coat peroxidase on polyaniline: Synthesis optimization and catalytic properties

Soybean seed coat peroxidase (SBP) was immobilized on various polyaniline-based polymers (PANI), activated with glutaraldehyde. The most reduced polymer (PANIG₂) showed the highest immobilization capacity (8.2 mg SBP g^-1 PANIG₂). The optimum pH for immobilization was 6.0 and the maximum retention was achieved after a 6-h reaction period. The efficiency of enzyme activity retention was 82%. When stored at 4°C, the immobilized enzyme retained 80% of its activity for 15 weeks as evidenced by tests performed at 2-week intervals. The immobilized SBP showed the same pH-activity profile as that of the free SBP for pyrogallol oxidation but the optimum temperature (55°C) was 10°C below that of the free enzyme. Kinetic analysis show that the K_m was conserved while the specific V_max dropped from 14.6 to 11.4 µmol min^-1 µg^-1, in agreement with the immobilization efficiency. Substrate specificity was practically the same for both enzymes. Immobilized SBP showed a greatly improved tolerance to different organic solvents; while free SBP lost around 90% of its activity at a 50% organic solvent concentration, immobilized SBP underwent only 30% inactivation at a concentration of 70% acetonitrile. Taking into account that immobilized HRP loses more than 40% of its activity at a 20% organic solvent concentration, immobilized SBP performed much better than its widely used counterpart HRP.     

Hydrogen peroxide degradation by immobilized cells of alkaliphilic Bacillus halodurans

Whole cells of Bacillus halodurans LBK 261 were used as a source of catalase for degradation of hydrogen peroxide. The organism, B. halodurans grown at 55°C and pH 10, yielded a maximum catalase activity of 275 U g^-1 (wet wt.) cells. The catalase in the whole cells was active over a broad range of pH with a maximum at pH 8-9. The enzyme was optimally active at 55°C, but had low stability above 40°C. The whole cell biocatalyst exhibited a K_m of 6.6 mM for H₂O₂ and V_max of 707 mM H₂O₂ min^-1 g^-1 wet wt. cells, and showed saturation kinetics at 50 mM H₂O₂. The cells were entrapped in calcium alginate and used for H₂O₂ degradation at pH 9 in batch and continuous mode. In the batch process, the immobilized preparation containing 1.5 g (wet wt.) cells could be recycled at least four times for complete degradation of the peroxide in 50 mL solution at 25°C. An excess of immobilized biocatalyst could be used in a continuous stirred tank reactor for an average of 9 days at temperatures upto 55°C, and in a packed bed reactor (PBR) for 5 days before the beads started to deform.     

The Reaction of no With Superoxide

The rate constant for the reaction of NO with ·O₂^- was determined to be (6.7 ± 0.9) × 10⁹ 1 mol^-1 s^-1, considerably higher than previously reported. Rate measurements were made from pH 5.6 to 12.5 both by monitoring the loss of ·O₂^- and the formation of the product ^-OONO. The decay rate of ^-OONO, in the presence of 0.1 moll^-1 formate, ranges from 1.2s^-1 at pH 5 to about 0.2s^-1 in strong base, the latter value probably reflecting catalysis by formate.     

The reaction between the superoxide anion radical and cytochrome c

1. 1. The superoxide anion radical (O₂⁻) reacts with ferricytochrome c to form ferrocytochrome c. No intermediate complexes are observable. No reaction could be detected between O₂⁻ and ferrocytochrome c.

2. 2. At 20 °C the rate constant for the reaction at pH 4.7 to 6.7 is 1.4 · 10⁶ M⁻¹ · s⁻¹ and as the pH increases above 6.7 the rate constant steadily decreases. The dependence on pH is the same for tuna heart and horse heart cytochrome c. No reaction could be demonstrated between O₂⁻ and the form of cytochrome c which exists above pH ≈ 9.2. The dependence of the rate constant on pH can be explained if cytochrome c has pKs of 7.45 and 9.2, and O₂⁻ reacts with the form present below pH 7.45 with k = 1.4 · 10⁶ M⁻¹ · s⁻¹, the form above pH 7.45 with k = 3.0 · 10⁵ M⁻¹ · s⁻¹, and the form present above pH 9.2 with k = 0.

3. 3. The reaction has an activation energy of 20 kJ mol⁻¹ and an enthalpy of activation at 25 °C of 18 kJ mol⁻¹ both above and below pH 7.45. It is suggested that O₂⁻ may reduce cytochrome c through a track composed of aromatic amino acids, and that little protein rearrangement is required for the formation of the activated complex.

4. 4. No reduction of ferricytochrome c by HO₂ radicals could be demonstrated at pH 1.2–6.2 but at pH 5.3, HO₂ radicals oxidize ferrocytochrome c with a rate constant of about 5 · 10⁵–5 · 10⁶ M⁻¹ · s⁻¹

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Waste Fe/Cr sludge as flocculant for the treatment of dairy wastewater

Variation in the characteristics of dairy wastewater with storage time is shown. The efficiency of Fe³⁺/Cr³⁺ sludge, a waste material from the wastewater treatment in the fertilizer industry, is compared with the conventional flocculants, ferric chloride and ferrous sulphate, in the treatment of dairy wastewater. The percentage removals of turbidity, BOD, COD, oil and grease and total phosphate were 81 ± 5, 68 ± 5, 70 ± 5, 52 ± 5 and 70 ± 5 respectively, by 1·043 g dosage of sludge per litre of the effluent, and the pH of the treated water was 5·7.