GC-ECD Analysis Research Articles

Biodegradation of γ-hexachlorocyclohexane (Lindane) by a non-white rot fungus conidiobolus 03-1-56 isolated from litter

Biodegradation of chlorinated pesticide γ-hexachlorocyclohexane (lindane) by a nonwhite rot fungus Conidiobolus 03-1-56 is reported for the first time. Conidiobolus 03-1-56, a phycomyceteous fungus isolated from litter, completely degraded lindane on the 5th day of incubation in the culture medium, and GC-ECD studies confirmed that lindane removal did not occur via adsorption on the fungal biomass. Degradation studies using different medium compositions showed that nitrogen/carbon limiting conditions (stress conditions) and presence of veratryl alcohol, induced the secretion of extracellular oxidative enzymes, which enhanced the rate of lindance biodegradation. Under optimum nutrient-limiting conditions, GC-ECD and GC-MS analysis showed complete absence of any degradation metabolite, indicating that lindane was completely mineralized. Assays for tannic acid utilization and lignin peroxidase showed similar enzymatic profiles between Conidiobolus 03-1-56 and standard white rot fungi Pleurotus ostreatus 1200 and Trametes versicolor 1086. Although Conidiobolus 03-1-56 showed a reduced enzyme activity compared to white rot fungi, preliminary evidence indicates that enzymes responsible for lignin degradation by white rots play a key role in lindane degradation by Conidiobolus 03-1-56.

Influence of chlorobenzoic acids on the growth and degradation potentials of PCB-degrading microorganisms

The biodegradation of polychlorinated biphenyls (PCBs) by diverse bacteria including those utilized in this study is often incomplete, a concomitant accumulation of chlorobenzoic acids (CBAs) are released as dead-end products. The build-up of these metabolites in the growth medium may result in feed-back inhibition and impede PCB biotransformation. In this investigation using GC-ECD and HPLC analyses, we confirmed that CBAs inhibit growth and PCB biodegradation potentials of five tropical bacteria namely, Pseudomonas aeruginosa SA-1, Enterobacter sp. SA-2, Ralstonia sp. SA-3, Ralstonia sp. SA-5 and Pseudomonas sp. SA-6. Among the four CBAs (2-CBA, 3-CBA, 4-CBA acids and 2,3-diCBA), 3-CBA was the strongest inhibitor followed by 4-CBA. Furthermore, we found that 3-CBA heavily inhibited growth of SA-3 and SA-6 on monochlorobiphenyls by 82–90% while elimination rate was inhibited by 71–88%. In the case of 2,3-diCBA, inhibition was generally less than 60%. However, effects of both acids were stronger in SA-3 than SA-6. We also found that 3-CBA and 2,3-diCBA completely inhibited carbon-chloride cleavage of 2-CB and 3-CB since cultivation in the absence of the acids resulted in recovery of 23–50% chloride in the culture fluids of organisms. These findings may therefore, have practical and ecological significance and are useful for improving the efficiency and the stability of some biological treatment processes.

GC–ECD analysis of S-metolachlor (Dual Gold) in cotton plant and soil in trial field

The analytical method of S-metolachlor residue and its degradation in cotton and soil in trial field were investigated. S-metolachlor EC (96% w/w) was applied as pre-emergence at dosages of 1,500 and 2,250 ml ha(-1) 3 days after sowing of the cottonseeds in the field. The soil and the plant samples were collected at different intervals and the residues of S-metolachlor were analyzed by GC-ECD. The results showed that the degradation of S-metolachlor in cotton leaves in Beijing and Nanjing coincides with C = 0.1113e(-0.1050t) and C = 0.1177e(-0.1580t), respectively; the half-lives were about 6.6 and 4.4 days. The degradation of S-metolachlor in soil in Beijing and Nanjing coincides with C = 1.0621e(-0.0475) (t), and C = 0.9212e(-0.0548) (t), respectively; the half-lives were about 14.6 and 12.6 days,. At harvest time, the S-metolachlor in cotton seeds and soil samples were detected by GC-ECD and confirmed by GC/MS. The results showed that the residues in cottonseeds were lower than the USA EPA's maximum residue limit of 0.1 mg kg(-1) in cottonseed. It could be considered as safe to human beings and environment.

PYROLYSIS OF WASTE ELECTRICAL AND ELECTRONIC EQUIPMENT: EFFECT OF ANTINOMY TRIOXIDE ON THE PYROLYSIS OF STYRENIC POLYMERS

This work has investigated the effect that antimony trioxide has on the pyrolysis of styrenic polymers and the effect that different types of brominated flame retardants used in plastics have on the composition of the pyrolysis products. Brominated high impact polystyrene (Br-HIPS) which contained either 5% or 0% antimony trioxide and either decabromodiphenyl oxide (DDO) or decabromodiphenyl ethane (DDE) was pyrolysed in a fixed bed reactor at 430°C. Some experiments on the fixed bed reactor involved mixing the Br-HIPS with polystyrene. The gaseous products were analysed by GC-FID and GC-TCD and it was found that antimony trioxide caused an increase in the proportion of ethane and ethene and suppressed the proportion of butane and butene. When DDE was the flame retardant increased proportions of ethane and ethene were found in the pyrolysis gas compared to when DDO used. When polystyrene was mixed with the Br-HIPS it suppressed the trends observed in the gas composition during the pyrolysis of Br-HIPS. The pyrolysis oils were characterised using FT-IR, GC-MS, GC-FID, and GC-ECD. It was found that the plastic which did not contain antimony trioxide pyrolysed to form mainly toluene, ethylbenzene, styrene, cumene, and alpha-methylstyrene. The oils produced from the pyrolysis of the plastic that contained antimony trioxide did not contain any styrene or alpha-methylstyrene, but instead contained greater concentrations of ethylbenzene and cumene. The absence of styrene and alpha-methylstyrene from the pyrolysis oil occurred even when the Br-HIPS was mixed with polystyrene. GC-ECD analysis of the oils showed that the plastics which did not contain antimony trioxide pyrolysed to form (1-bromoethyl)benzene, which was totally absent from the pyrolysis oils when antimony trioxide was present in the plastic.

Dynamic hollow fiber protected liquid phase microextraction and quantification using gas chromatography combined with electron capture detection of organochlorine pesticides in green tea leaves and ready-to-drink tea

The dynamic hollow fiber protected liquid phase microextraction (DHFP-LPME) technique was evaluated for the extraction of organochlorine pesticides (OCPs) in green tea leaves and ready-to-drink tea prior to gas chromatography combined-electron capture detection (GC–ECD) analysis. A conventional microsyringe with a 1.5 cm length of hollow fiber attached to its needle was connected to a syringe pump to perform the extraction. The microsyringe was used as both the microextraction device and the sample introduction device for GC–ECD analysis. In this work, the organochlorine pesticides were extracted and condensed to a volume of 3 μl of organic extracting solvent (1-octanol) confined within a 1.5 cm length of hollow fiber. The effects of extraction solvent, extraction time, sample agitation, plunger speed, and extraction temperature and salt concentration content on the extraction performance were also investigated. Good enrichments were achieved (34–297-fold) with this method, and good repeatabilities of extraction were obtained, with full name (RSDs) below 12.57%. Detection limits were much below 1 μg l −1 for ready-to-drink tea and much below 1 μg g −1 for green tea leaves.

Dechlorination of DDT Mediated by Bimetallic Systems

DDT dechlorination efficiencies of bimetallic systems, namely, Mg°-Zn, Mg°-Ni, and Mg°-Co were compared. All the systems transformed DDT with efficiencies exceeding 98% within 24 h. Based on GC-ECD and GC-MS analyses a step-wise and sequential dechlorination of DDT by Mg°-Zn system in 1:1 water acetone phase via 1,1 dichloro, 2,2- bis (p-chlorophenyl) ethane (DDD), 1, chloro, 2,2 bis (p-chlorophenyl) ethane (DDMS) to 2,2 bis (p-chlorophenyl) ethane (DDNS) was noted. Accumulation of DDNS as the end product indicates that all three alkyl chlorine atoms of DDT were removed by Mg°-Zn system. Mg°-Co also showed almost complete removal of DDT in water-acetone phase that was accompanied by the accumulation of DDD followed by a decline in its concentration as a function of time. On the other hand, Mg°-Ni system exhibited inefficient removal of DDT in water-acetone phase. In pure acetone phase, Mg°-Co system and Mg°-Ni dechlorinated DDT with the accumulation of DDE and DDMU as end products following 24 h of reaction. The presence of surfactants or organic solvents is required to ensure solubilization of DDT. Also addition of acid is essential to provide sufficient protons for efficient reductive dechlorination via hydrogenation. Advantage of Mg° based dehalogenation reactions is that they occur at high rates under ambient temperatures, and pressure and oxygen need not be excluded in the reaction phase. Our studies revealed that Mg°-Zn is the best option among all tested systems due to its high reactivity and low cost, and may be used to treat DDT contaminated water.

Displacement imprinted polymer receptor analysis (DIPRA) for chlorophenolic contaminants in drinking water and packaging materials

The preparation of a molecularly imprinted polymer (MIP) for pentachlorophenol is described together with two alternative reporter derivatives for use in a displacement imprinted polymer receptor analysis (DIPRA) format procedure. In this procedure, alternative reporter molecules were rebound to the synthetic receptor sites and their displacement by the target analyte was employed as the basis of a simple procedure for the measurement of chlorophenols in water and packaging material samples. Water samples were extracted using the standard procedure (EPA 528) and a detection limit of 0.5 microg l(-1) was achieved using the DIPRA detection method, with good agreement between the displacement technique and GC-ECD analysis. A variety of packaging materials, extracted using a buffered detergent solution were also analysed using the DIPRA procedure and showed good agreement with GC results. In addition, investigation of the cross-reactivity of a range of pesticides and materials commonly encountered in environmental analysis indicated the procedure gave good discrimination between pesticides bearing a chlorophenolic moiety and other materials. The procedure is considered highly suitable for use as a rapid field-test method or for incorporation into a test kit device.

Microwave-assisted extraction of pyrethroid insecticides from soil

An analytical method has been developed for the microwave-assisted extraction of synthetic pyrethroid insecticide residues in soils and their determination by gas chromatography with electron capture detector (ECD) and mass spectrometry (MS) detector. Clean soil samples were spiked with nine pyrethroids: tetramethrin, cyfluthrin, flucythrinate, deltamethrin, bifenthrin, permethrin, cypermethrin and fluvalinate were used for this study. Concentration levels were 50 ng g −1 for all the compounds studied except to sumitrin for which a 10 μg g −1 was evaluated. Two grams of sample were treated in a closed PTFE reactor with 10 mL toluene and 1 mL water and irradiated at 700 W during 9 min. The toluene extract was evaporated and reconstituted in 2 mL hexane and copper wires added in order to remove sulphur interferences. Clean-up with 2 g of florisil and elution with 20 mL ethyl acetate:hexane 33% (v/v) was performed. Best chromatographic conditions were established for GC-ECD analysis for routine analysis and GC-NCI-MS for confirmation. Recovery yields of spiked soils were from 97 to 106% for eight of the pyrethroid and 86% for the tetramethrin, with relative standard deviations from 1 to 7%. Results were comparable with those found by ultrasonic extraction in the analysis of real samples and limit of detection values obtained were in the range from 1 to 200 ng g −1 for ECD and from 0.3 to 2 ng g −1 for NCI-MS.

A Review of Instrumentation and Measurement Techniques for Ground-Based and Airborne Field Studies of Gas-Phase Tropospheric Chemistry

ABSTRACT The development, applications and intercomparisons of instrumentation and measurement techniques for ground-based and airborne field studies of gas-phase tropospheric chemistry are reviewed. Filter radiometry, chemical actinometry and scanning spectroradiometry for j-NO2 and j-(O1D) are described. Detection of OH using L-POAS/DOAS, MOAS, LIF/FAGE and CIMS is discussed. Observations of NO3 using DOAS, MIESR and CRDS, and of HO2 and RO2 with CA, CIMS/IMR-MS, MIESR, and LIF/FAGE are also reviewed. GC-FID, GC-ECD and GC-MS analyses of NMHCs and alkyl, peroxyacyl and bi-functional organic nitrates are described, together with applications of CIMS/PTR-MS, DOAS and LIF. CO measurements utilising GFC, GC-HgO/UV, VUV-RF and TDLAS are presented. Measurements of O3 using UV photometry, chemiluminescence, electrochemistry, LIDAR/DIAL and DOAS are discussed. Chemiluminescence and LIF detection methods for NO with photochemical and thermal convertors for NO2 and NOy are also discussed, as are TDLAS, MIESR, DOAS, CRDS and other approaches for NO2. HONO measurements using DOAS, CIMS, CRDS, TDLAS, denuder systems, chemiluminescence and on-line analyses of NO2 − are described. For HONO2, filter packs, denuder systems, chemiluminescence methods, mist chambers, TDLAS, CIMS and LIF are presented. Direct and indirect fluorimetric, chromatographic and spectroscopic detection techniques are discussed for CH2O and higher carbonyls. Observations of H2O2 and ROOH utilising colorimetry, chemiluminescence, fluorescence, HPLC and TDLAS are also described.

Assessment of matrix effects in gas chromatography electron capture pesticide-residue analysis

The analysis of pesticide residues in vegetable samples leads in most cases to different results when solvent or matrix-matched calibration is used for quantitation. Matrix effects in GC-ECD analysis of pesticides in vegetable samples have been assessed by comparing calibration curves prepared in solvent and in blank matrix extracts. Eight different vegetables have been considered among the most common commodities in routine pesticide residue laboratories.

Trace analysis of trichlorobenzenes in fish by microwave-assisted extraction and gas chromatography–electron-capture detection

An analytical method consisting of extraction, clean-up, and analysis by gas chromatography–electron-capture detection (GC–ECD) was developed for the determination of trichlorobenzenes (TCBs) in fish samples. Two extraction methods, saponification and liquid–liquid extraction (S-LLE), and microwave-assisted extraction (MAE), were evaluated. In both cases, n-pentane was used as the extraction solvent. For S-LLE, the recoveries ranged from 66.6±9.1% for 1-bromo-4-chlorobenzene (4-BCB) to 93.5±4.9% for 1,2,4-trichlorobenzene (1,2,4-TCB). The recoveries were significantly lower, between 31.0±3.9% for 1,2,3-trichlorobenzene (1,2,3-TCB) and 52.3±3.0% for 1,3,5-trichlorobenzene (1,3,5-TCB), in the absence of fish. Proteins and glycerides of the fish tissue seemed to compete with TCBs for the base, and hence decreased their decomposition rate. In the case of MAE, the recoveries were highly dependent on the pressure applied during extraction. At 5 bar, much higher recoveries were obtained, from 66.7±15.6% for 4-BCB to 79.9±13.6% for 1,2,4-TCB, than at 1 bar. Sulfur formation was, however, observed at 5 bar, and interfered with the GC–ECD analysis of TCBs. Sulfur was adequately removed by copper powder treatment, which was shown not to affect the recovery of analytes. The recoveries of target analytes by S-LLE and MAE did not differ statistically ( t-test, α=0.01). Both methods were appropriate for the detection of TCBs at concentration levels typically observed in marine biota, i.e. ∼1 ng/g. S-LLE was, however, more time consuming, and required larger volumes of high-purity organic solvents than MAE.

Improved analytical procedure for determination of chlorinated pesticide residues in human serum using solid phase disc extraction (SPDE), single-step clean-up and gas chromatography

An improved analytical methodology based on solid-phase disc extraction (SPDE) and a single-step clean-up on Florisil is proposed for a large number of organochlorine pesticide residues in serum. Extraction was performed following denaturation of proteins with formic acid after it was shown that it has no degradation effect on targeted analytes (α, β, γ-HCH isomers, HCB, DDT with its 5 analogues, endrin, aldrin, dieldrin, alachlor; heptachlor, heptachlorepaxide, α, β-endosulphan, endosulphansulphate, methoxychlor and mirex). Determination and quantification were by GC-ECD and GC-MS on two different, analytical capillary-columns using PCNB (pentachlonitrobenzene) and PCB 190 internal standards. Recoveries and limits of detection determined on pooled serum ranged 54–102% (for medium spiking level) and 10–50 pg ml−1 serum respectively. Twenty-one individuals serum samples from the University Hospital of Antwerp were analysed and results were related to the ages of the donors. For compounds not detected by GC-MS, eventual coelution with PCBs in GC-ECD analysis was studied.

Selected concentration techniques for gas chromatographic analysis of environmental samples

The paper describes the role and tasks of environmental analysis and monitoring. It emphasises the leading role played by gas chromatography, especially when coupled with suitable sample preparation. This is demonstrated by some literature evidence and mainly by the author's original work on the design of sample preparation apparatus and development of GC-based methods of determination of a wide variety of organic pollutants in environmental samples. A typical example is the physical speciation of PAHs and PCBs in river environment with the use of a home-made filtration vessel coupled with SPE cartridges to prepare samples for GC-MS analysis; thin layer head space with self-generation of aqueous concentrate to prepare humane urine samples for DAI (direct aqueous injection)-GC-ECD analysis for the content of volatile chloro-organic compounds; studies on capillary denuders in sampling organic air pollutants; passive dosimeters in indoor air quality measurements; design and application of thermal desorbers for the introduction of analytes into GC columns and modification of a speciation analyser for organometalic environmental pollutants.

GC analysis of trichloroacetic acid in water samples by large-volume injection and thermal decarboxylation in a programmed-temperature vaporizer

AGC method has been developed for the analysis of tricholoroacetic acid (TCA) in water samples. It entails large-volume injection (LVI) and programmed-temperature vaporization (PTV) of water samples, thermal decarboxylation of TCA to chloroform in the injector, and GC-ECD analysis of the decarboxylation product. Conditions such as final injector temperature, splitless time, and injection volume were optimized. The limit of detection is approximately 0.1 μg L−1 Several real samples were analyzed. The method presented is an easy means of determination of TCA and eliminates sample-preparation steps such as extraction and derivatization.

Analysis of pesticides volatilised from plants and soil by headspace solid-phase microextraction and gas chromatography

Pesticides volatilised from plants and soil were determined by headspace SPME and gas chromatography with electron-capture and mass spectrometric detection. SPME analyses were carried out with polydimethylsiloxane (PDMS) and polyacrylate (PA) fibres. A mixture of endosulfan, chlorpyrifos, lindane and pendimethalin was used in these assays. Fortified soil and plant samples were maintained at 30 °C, for 2 h and 1 h, respectively, before SPME sampling. Pesticides were extracted by SPME by inserting the sheathed fibre through the cap and exposing it to the headspace for 1 h. GC-ECD analysis was performed after thermal desorption of pesticides from the fibre for 5 minutes. Pesticide identity was confirmed by mass spectrometry operating in the electron impact mode (El). The SPME fibres assayed gave good results, the response obtained with the PDMS fibre being somewhat better in the concentration range studied. Detection limits for the compounds studied varied from 1 to 8 ng L -1 .

Induction of the P450 reporter gene system bioassay by polycyclic aromatic hydrocarbons in Ulsan Bay (South Korea) sediments

Polycyclic aromatic hydrocarbons (PAHs) and induction of the P450 reporter gene system (RGS) for 6- and 16-h exposure periods were determined in organic extracts of Ulsan Bay (South Korea) sediments to assess the utility of this bioassay as a screening tool for PAH contamination. The sum of the concentrations of 23 individual PAHs in 30 sediment samples (ΣPAH) based on GC–MS analysis ranged from 0.05 to 6.1 μg/g dry wt. P450 RGS fold induction ranged from 4.0 to 320 μg/g based on benzo[ a]pyrene toxic equivalents (BaPEq). P450 RGS BaPEq and the ‘chemical BaPEq’, defined as the sum of the products of individual PAH concentrations and pre-determined toxic equivalency factors, exhibited very strong positive correlations with ΣPAH ( r 2>0.90; P<0.001). Fold induction did not increase (and in some cases decreased) after the optimal incubation period (6 h) for PAHs, indicating that other compounds known to induce the P450 RGS (e.g. chlorinated organics) were not present at levels effecting significant induction. This was supported by GC–ECD analysis where non-ortho and mono-ortho polychlorinated biphenyls (PCBs) known to be strong P450 RGS inducers were found to be at very low or non-detectable levels in samples with the highest P450 RGS responses. The profound difference in PAH profiles for the two most contaminated sites suggested that this assay is especially sensitive for selected PAHs with greater than four rings. Combined with previous results, the P450 RGS shows promise as a useful screening tool for predicting deleterious biological effects resulting from CYP1A1-inducing, sediment-associated chemicals, particularly high molecular weight PAHs.

Use of stable tracer studies to evaluate pesticide photolysis at elevated temperatures.

New methods were developed to determine photolysis rates of medium-weight pesticides in the gas phase using elevated air temperatures and solid-phase microextraction (SPME). A 57-L glass chamber was constructed that utilized collimated xenon arc irradiation that could heat chamber air to increase the amount of pesticide in the gas phase. Gas-phase photolysis rates were determined at various air temperatures by comparing the rate of loss of each of the tested pesticides to a photochemically stable tracer, hexachlorobenzene. Interval sampling of gas-phase constituents was performed using SPME immediately followed by GC-ECD or GC-MSD analysis. The two pesticides under examination were the dinitroaniline herbicide trifluralin and the organophosphorus insecticide chlorpyrifos. The gas-phase photolysis for trifluralin was found to be rapid with half-lives of 22-24 min corrected for sunlight. These results were comparable to photochemical lifetime estimates from other investigators under sunlight conditions. Elevating temperatures from 60 to 80 degrees C did not affect photolysis rates, and these rates could be extrapolated to environmental temperatures. From 60 to 80 degrees C, gas-phase chlorpyrifos photolysis lifetimes were observed to range from 1.4 to 2.2 h corrected for sunlight and will thus be important together with hydroxyl radical reactions for removing this substance from the atmosphere. At these elevated temperatures, pesticides and tracer compounds were found to be substantially in the gas phase, and possible effects on reaction rates from wall interactions were minimized.

A New Method for the Measurement of Airborne Formaldehyde Using Derivatization with 3,5-Bis(Trifluoromethyl) Phenylhydrazine and Analysis by GC-ECD and GC-MS/SIM

A new method was developed and described for the measurement of airborne formaldehyde using derivatization with 3,5-bis(trifluoromethyl)phenylhydrazine (TFMPH) coated onto silica solid phase extraction cartridges. Analysis by GC-ECD provides a detection limit of 74 ng formaldehyde per sample. A field study was conducted to compare the use of TFMPH to 2,4-dinitrophenylhydrazine (DNPH) and NIOSH method 3500 (chromotropic acid, CTA). Samples were collected from indoor and outdoor environments known or suspected to contain formaldehyde. Use of TFMPH with GC-ECD analysis correlates well with both methods (R2=0.93, slope=1.07 vs. DNPH; R2=0.99, slope=1.06 vs. CTA). Spiked samples were shown to be stable at least 7 days when stored at –20 °C. Analysis of samples by GC-MS with selected ion monitoring (GC-MS/SIM) also proved feasible. Laboratory and field results show the use of TFMPH to be viable for quantifying airborne formaldehyde in occupational and environmental samples.

フェントン処理によるテトラクロロエチレン分解過程における炭素と塩素収支

This paper presents applicability of Fenton treatment to highly tetrachloroethylene (PCE)-contaminated groundwater. We performed flask-scale experiment in order to elucidate reaction mechanism in Fenton treatment of PCE in aqueous phase. Fenton treatment of PCE resulted in more than 95% of dechlorination and mineralization within 24 hours. It was found that the decomposition of PCE by Fenton treatment followed pseudo first order and its reaction coefficient was 0.17 h-1. During 24 hours in the reaction experiment the concentration of chloride ion, total inorganic carbon, and PCE in the vials were determined. These results showed that more than 98% of PCE decomposed by Fenton treatment were compensated for the increase in chloride ion and CO2 through the experiment and mineralization rates derived from the increase of CO2 are almost same to dechlorination rates from the increase of chloride ion. Detected trichloroacetic acid from GC-MS and GC-ECD analysis explained only 1% of decomposed PCE in the reaction experiment. This study suggested that both processes of dechlorination and mineralization proceeded almost simultaneously in the PCE decomposition by Fenton treatment, not via intermediates such as acetic acid and dichloroacetic acid.

Analytical method for assessing exposure of greenhouse applicators to procymidone by gas chromatography and whole body dosimetry

An analytical method for the determination of the potential and actual exposure of agricultural workers to procymidone has been developed. The methodology is based on the whole body technique, using Tyvek Pro-Tech and Sontara as sampling media, and uses hexane extraction, GC-ECD analysis and GC-MSD confirmation. The validation of the analytical method has been carried out incorporating the matrix of coverall in all steps when calculating parameters such as retention time window, limits of detection and quantification, linear ranges and precision. A field sampling strategy has also been developed and the method has been applied to the evaluation of the potential and actual dermal exposure of an applicator and an assistant in normal working conditions.