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The neutral red lysosomal retention assay and Comet assay on haemolymph cells from mussels (Mytilus edulis) and fish (Symphodus melops) exposed to styrene.

Despite the extensive transport of chemicals at sea, there is current lack of knowledge of the fate and effects of many of them on the marine biota. The current regulation that follows the GESAMP-MARPOL classification is mainly based on ecotoxicity assessment from fresh water based studies. Repetitive spills in marine coastal environment from tanker ship loaded with several thousand tonnes of chemicals raised concern about whether the existing freshwater data location can be used to predict the behaviour and the environmental effects of contaminants in marine surroundings. There is a general lack of information of the fate of chemicals at sea. A deviating pattern in marine environment from that in freshwater may have significant consequences for the counteracting actions taken to fight the spill, on staff working on the site of spill as well as on marine life present in the vicinity of the accident. In the present article, an environmental effect study of styrene was conducted as part of the ECOPEL program. We report some biological effects of styrene in laboratory-exposed marine organisms. Styrene was continuously supplied at a nominal concentration of 2mg L(-1) over 7 days to both mussels (Mytilus edulis) and fish (Symphodus mellops). At the end of this period, DNA damage was assessed by the Comet assay performed on blood (fish) and haemolymph (mussel) cells. In mussels, the lysosomal membrane stability was additionally assessed by the neutral red retention time assay (NRRT). Significant biological responses were observed over the studied period in both organisms with these two tests. Hence, the results favour the use of a biomarker-based approach to assess the health conditions in case of spill.

Animals↗

Biodegradation of styrene laden waste gas stream using a compost-based biofilter.

Biological treatment of waste gas styrene vapor was investigated in a three-stage bench-scale biofilter. Yard waste compost mixed with shredded hard plastics in a 25:75 v/v ratio of plastics:compost was inoculated with thickened municipal activated sludge. Microbial acclimation to styrene was achieved by exposing the system to an inlet concentration (C(In)) of 0.25 gm(-3) styrene and an empty bed retention time (EBRT) of 360 s for 30 days. Under steady-state conditions, maximum elimination capacity (EC) obtained was 45 gm(-3)h(-1) at a loading rate (L) of 60 gm(-3)h(-1) (C(In) of 2 gm(-3) and EBRT of 120 s). Reduction of retention time adversely impacted the performance resulting in the maximum EC of 39 and 27 gm(-3)h(-1) for EBRT of 60 and 30 s, respectively. Evaluation of the concentration profile along the bed height indicated dominance of first-order kinetics at C(In) < or = 0.45 gm(-3) and zero-order for higher concentrations.

Air Pollutants↗

Adsorption of branched alkylbenzene sulfonate onto styrene and acrylic ester resins.

In this study, a series of acrylic ester resins were prepared and characterized. The adsorption behaviors of sodium 6-dodecyl benzenesulfonate (6-NaDBS) on styrene resin XAD-4 and acrylic ester resins have been studied. As compared to commercial acrylic ester resins Amberlite XAD-7 and Diaion HP2MG, YWB-38, one of the as-prepared resins, exhibits better adsorption properties toward 6-NaDBS. The experimental equilibrium data were fitted to the Langmuir and double-Langmuir models. The two models provide very good fit for all acrylic ester resins over the temperature range studied, whereas fitted results for styrene resin XAD-4 are less accurate. The investigation indicates that the adsorbate molecules are directly adsorbed on acrylic ester resins as a bi-layer, while the molecules are adsorbed on XAD-4 as monolayer formation followed by the establishment of bi-layer. Adsorption kinetics and elution processes were also investigated for further exploring the adsorption behavior and mechanism on styrene resin XAD-4 and YWB-38.

Acrylates↗

Ocular toxicity of styrene.

We completed histories and ocular examinations of 345 workers in a styrene plant in order to determine the ocular toxicity of styrene. Despite previous case reports of retrobulbar neuritis and central retinal vein occlusion (associated with a toxic hematopoietic disorder), no such conditions were found in these workers. Conjunctival irritation from styrene was found in 22% of the workers and correlated with intensity of exposure, thus confirming previous reports.

Conjunctivitis↗

Styrene-catabolism regulation in Pseudomonas fluorescens ST: phosphorylation of StyR induces dimerization and cooperative DNA-binding.

Styrene is an important chemical extensively used in the petrochemical and polymer industries. In Pseudomonas fluorescens ST, styrene metabolism is controlled by a two-component regulatory system, very uncommon in the degradation of aromatic compounds. The two-component regulatory proteins StyS and StyR regulate the expression of the styABCD operon, which codes for styrene degradation. StyS corresponds to the sensor kinase and StyR to the response regulator, which is essential for the activation of PstyA, the promoter of the catabolic operon. In two-component systems, the response regulator is phosphorylated by the cognate sensor kinase. Phosphorylation activates the response regulator, inducing DNA-binding. The mechanism underlying this activation has been reported only for a very few response regulators. Here, the effect of phosphorylation on the oligomeric state and on the DNA-binding properties of StyR has been investigated. Phosphorylation induces dimerization of StyR, the affinity of dimeric StyR for the target DNA is higher than that of the monomer, moreover dimeric StyR binding to the DNA target is cooperative. Furthermore, StyR oligomerization may be driven by the DNA target. This is the first direct demonstration that StyR response regulator binds to the PstyA promoter.

Bacterial Proteins↗

A study on toxic organic emissions from batch combustion of styrene.

Results from a laboratory-scale investigation on batch combustion of styrene are reported herein. Limited quantities of waste styrene monomer are incinerated, however this monomer is, also, the primary pyrolyzate during combustion of waste polystyrene, the second most abundant polymer produced worldwide. Thus, its combustion-generated emissions are of importance to the operation of hazardous waste incinerators and municipal waste-to-energy powerplants. This work focuses on emissions of polycyclic aromatic hydrocarbons (PAHs), particulates, as well as carbon monoxide. To investigate methods for minimizing such emissions, batch combustion of the monomer was conducted in a two-stage muffle furnace. An additional air mixing chamber was installed between the two stages. Small quantities of the liquid monomer were inserted in the primary furnace which served as a gasifier/burner. The furnace temperature was in the range of 300-1000 degrees C and diffusion flames were formed under most conditions. Upon mixing with additional air, combustion of unburned gaseous fuel and primary reaction products continued in the secondary furnace (afterburner), which was kept at a constant temperature of either 1000 or 800 degrees C. Using this technique, conditions that minimize emissions were explored and theoretical investigations on the fate of pollutants in the secondary furnace were undertaken. Results revealed that combustion of styrene, which is a highly volatile fuel, occurred with the formation of flames that were often non-anchored, unsteady and unstable. Emissions of organic pollutants, soot and CO were more intense than in the case of the polystyrene combustion, studied previously under identical conditions, due to the additional depolymerization/pyrolysis steps therein. The emissions from the secondary furnace exceeded those of the primary furnace, consistent with the fact that a very significant fraction of the fuel conversion occurred in the secondary chamber. Clear trends in the emissions of PAHs and soot, products of incomplete combustion, with the temperature of the primary furnace (gasifier) were observed. Emissions were drastically reduced with lowering the gasifier temperature. While final cumulative emissions of PAHs and soot accounted for more than one third of the mass of the fuel at high temperatures, their concentrations at the exit of the afterburner were negligible when the primary furnace was operated at 300 degrees C under pyrolytic conditions. In the latter case air was added to the afterburner. Numerical modeling based on a complex reaction network was used for the description of the primary furnace as well as of the afterburner. Kinetic analysis showed acetylene and benzene to be key species in the growth of PAHs. Formation of PAHs in the afterburner, found experimentally, was reproduced by the model using a plug-flow assumption.

Air↗

Construction of a bacterial biosensor for styrene.

A new bacterial biosensor for styrene has been developed and characterized. A translational fusion of the lacZ gene to the sty promoter of Pseudomonas sp. strain Y2 has been inserted into miniTn5. Transposition of the recombinant transposon to the chromosome of Pseudomonas sp. strain Y2 resulted in a whole-cell biosensor able to detect and degrade styrene. In this biosensor, the endogenous StyS/StyR system detects the presence of styrene and turns on the expression of the exogenous reporter gene from the transferred construction. Other compounds such as toluene, epoxystyrene, phenylacetaldehyde and 2-phenylethanol also induced expression of beta-galactosidase although quantitative differences in their effect are clearly detected. Non-inducing compounds affect differently the sensitivity to inducing compounds when present in a mixture.

Biosensing Techniques↗

Endocrine-disrupting effects of styrene oligomers that migrated from polystyrene containers into food.

The endocrine-disrupting effects of styrene dimers (SD: NSD-01, -08 and -09) and styrene trimers (ST: NST -01, -03 and -12), which migrated from polystyrene (PS) containers into instant food, were investigated together with styrene monomer (SM) using in vitro and in vivo assays. In the estrogen (ER) and androgen receptor (AR) binding assay, SM, SD and ST showed no binding activity at concentration of 10(-10)-10(-5) mol/l. In order to evaluate the estrogenic activity in vivo, the uterotrophic assay was conducted. When prepubertal and ovariectomized adult rats were dosed with SM, SD and ST for 3 days by subcutaneous injection, these compounds did not induce significant increase in uterine weight. Additionally, to evaluate anti-androgen activity in vivo, the Hershberger assay for anti-androgenic activity in the presence of testosterone treatment was conducted. When castrated, testosterone-treated immature male rats were dosed SM, SD and ST for 7 days by oral gavage, these compounds did not induce a decrease in the seminal vesicle, ventral prostate and levator ani plus bulbocavernosus muscle weights. To evaluate the effects on hormones other than sex hormones, the thyroid hormone receptor (TR) binding assay and rat serum prolactin (PRL) was conducted. In the TR binding assay, SM, SD and ST showed no binding activity at a concentration of 10(-5) mol/l. When ovariectomized rats were dosed with SM, SD and ST for 3 days by sc injection, the results showed there was no change in rat serum PRL. From the above these results, we concluded that SM, SD and ST exhibit no apparent estrogenic, androgenic, anti-androgenic and thyroid activity.

Animals↗

Estimation of a possible tumorigenic risk of styrene from daily intake via food and ambient air.

Concerns of a tumorigenic risk of styrene (ST) originate from the findings that styrene (ST) is metabolized to the genotoxic intermediate styrene-7,8-oxide (SO). Therefore, it was hypothesized that results of animal long-term studies with ST and SO together with the SO tissue burden are sufficient for conducting a 'worst case' estimate of the tumorigenic risk of ST. On this basis we predicted the excess human lifetime risk for lung tumors (p(EXL)) and the highest possible risk for other systemic tumors (p(HPS)) resulting from daily intake of ST via food and ambient air. As measures for p(EXL) the mean lifetime concentration of SO in the transitional zone of the lung and for p(HPS) the mean lifetime concentration of SO in blood were calculated using a physiological toxicokinetic model. For a daily oral intake of 12 microST, p(EXL) was obtained to be between 5x10(-9) and 2x10(-8) and p(HPS) to be between 7x10(-9) and 2x10(-8). Lifetime risks calculated for continuous exposure to 3 microg/m(3) ST in ambient air were between 8x10(-7) and 3x10(-6) (p(EXL)) and between 2x10(-8) and 4x10(-8) (p(HPS)). Although these values indicate very low risks, the actual risks are expected to be even by far smaller. This is discussed in detail for lung tumorigenesis.

Adult↗

Simultaneous high-performance liquid chromatographic determination of urinary mandelic and phenylglyoxylic acids as indirect evaluation of styrene exposure.

Styrene is rapidly metabolised to mandelic acid (MA) and phenylglyoxylic acid (PGA), which are excreted in urine. In this work, we have developed a simple, sensitive and specific high-performance liquid chromatographic method with minor sample preparation procedures for the simultaneous determination of MA and PGA in urine of workers exposed to styrene. Moreover, urine samples from workers of two plastic factories were analysed, styrene exposure levels of the workers were estimated and data obtained from the two factories were compared.

Chromatography, High Pressure Liquid↗

Enzyme-mediated free radical polymerization of styrene.

Horseradish peroxidase-mediated polymerization of styrene at ambient temperature is reported. Molecular weight and yield of polystyrene were influenced by solvent, concentration of hydrogen peroxide, and initiator (beta-diketones, coumarin). THF:H2O (v/v) and hydrogen peroxide (0.082 mol/L) provided maximum yield of polymer (21.2% weight conversion of styrene to polystyrene) with 2,4-pentanedione as initiator. 1,3-Cyclopentanedione and dibenzoylmethane as initiators resulted in higher yield of polymer (approximately 60%) and a higher molecular weight (Mn = 96,504, polydispersity = 2.16), respectively. This enzymatic strategy was also used for the synthesis of polymers from styrene derivatives, 4-methylstyrene and 2-vinylnaphthalene, the latter resulting in a > 90% yield of polymer. The presence of the initiators in the polymer chains is reported.

Free Radicals↗

Polymorphism of xenobiotic-metabolizing enzymes and excretion of styrene-specific mercapturic acids.

The role of polymorphic xenobiotic-metabolizing enzymes in the interindividual variability of phenylhydroxyethyl mercapturic acids (PHEMAs) was investigated in 56 styrene-exposed workers. Ambient monitoring was carried out using passive personal samplers (geometric mean, 157 mg/m3 8-h time-weighted average; geometric standard deviation, 2.90). Biomonitoring was based on mandelic acid and phenylglyoxylic acid in urine spot samples collected at the end of the work shift ("end-of-shift") and prior to the subsequent shift ("next morning"). Four PHEMA diastereoisomers, namely (R,R)-M1, (S,R)-M1, (S,R)-M2, and (R,R)-M2, were determined by HPLC/tandem mass spectrometry. The genotypes of glutathione S-transferases M1-1 (GSTM1), T1-1 (GSTT1) and P1-1 (GSTP1), and microsomal epoxide hydrolase (EPHX) were characterized by PCR-based methods. Workers bearing the GSTM1pos genotype showed PHEMA concentrations five and six times higher (in end-of-shift and next-morning samples, respectively) as compared to GSTM1null people. In GSTM1pos subjects, (R,R)-M1 was the main mercapturate affected by the GSTM1 status, accounting for 54 and 68% of total PHEMAs in end-of-shift and next-morning samples, respectively. Compared to GSTM1null, GSTM1pos subjects excreted more -M1 than -M2 and more (R,R)-M1 and (S,R)-M2 than (S,R)-M1 and (R,R)-M2 diastereoisomers. Thus, GSTM1-1 is the main isoenzyme catalyzing GSH-conjugation of styrene-7,8-oxide in humans and it seems to act in a regio- and stereoselective way. PHEMAs cannot be recommended as biomarkers of exposure to styrene, unless the GSTM1 genotype is considered in data interpretation. Their role as biomarkers of susceptibility deserves further studies.

Acetylcysteine↗

Enhancement of styrene removal by Pseudomonas sp. SR-5 in mixed culture with a benzoic acid-degrading bacterium in biofilter.

The growth of a styrene-degrading bacterium, Pseudomonas sp. SR-5, was inhibited by benzoic acid (BA), one of the styrene degradation intermediates, in liquid culture. A benzoic acid-degrading microorganism, Raoultella sp. strain A, was isolated from a peat biofilter inoculated with a wastewater. The styrene removal efficiencies of the two laboratory-scale biofilters inoculated with only strain SR-5 and a mixed culture of strains SR-5 and A were compared using a mixed packing material of peat and ceramic (1:1) for 45 days. The biofilter with the mixed culture showed a higher removal efficiency than that with a single culture of SR-5. The maximum elimination capacities of the biofilters with the mixed culture and the single culture were 141 g m(-3)h(-1) and 106 g m(-3)h(-1), respectively. In the biofilter with the single culture, 136 g of benzoic acid (m3 of dry packing material)(-1) was accumulated at the end of the experiment. However, no accumulation of benzoic acid was observed in the biofilter with the mixed culture.

Air Pollutants↗

Influence of selected inhibitors on the metabolism of the styrene metabolite 4-vinylphenol in wild-type and CYP2E1 knockout mice.

4-Vinylphenol (4-VP), a minor metabolite of styrene, is a more potent hepato- and pneumotoxicant than either styrene or styrene oxide. In CD-1 mice 4-VP is metabolized primarily by cytochrome P-450 (CYP) 2E1 and CYP2F2. However, there is no difference in the rate of metabolism of 4-VP between wild-type and CYP2E1 knockout mice, indicating that other cytochromes P-450 play an important role. To understand the role of various cytochromes P-450, the in vitro metabolism of 4-VP was measured in the presence of selected inhibitors. Chemical inhibitors used to ascertain the contributions made by various cytochromes P450 were imipramine for CYP2C, alpha-methylbenzylaminobenzotriazole (MBA) for CYP2B, alpha-naphthoflavone (ANF) for CYP1A, 5-phenyl-1-pentyne (5P1P) for CYP2F2, and diethyldithiocarbamate (DTTC) for CYP2E1. Imipramine, MBA, and ANF produced significant inhibition in both the wild-type and CYP2E1 knockout mouse liver with minimal effects in lung. 5P1P significantly inhibited enzymic activity in both tissues, but to a greater extent in lung. The greatest inhibition was observed with DDTC even in the knockout mice, suggesting that it must also inhibit cytochromes P-450 in addition to CYP2E1. The results show little difference between the wild-type and knockout mice with respect to the contributions made by the cytochromes P-450 in the metabolism of 4-VP.

Alkynes↗

Worker exposure to styrene monomer in the reinforced plastic boat-making industry.

The National Institute for Occupational Safety and Health (NIOSH) conducted an industrial hygiene study of workers exposed to styrene monomer in the fiberglass reinforced plastic boat industry. This industry was chosen for study because of its length of operation and the high styrene monomer exposures experienced by these workers. The objective of the study was to provide extent of exposure data for specific jobs by including plants representing a cross-section of the industry. Industrial hygiene surveys were conducted at seven plants. Overall exposure to styrene monomer ranged from 2 parts per million (ppm) to 183 ppm. Mean exposures for the four primary job categories of hull lamination, deck lamination, small parts lamination and gel coating were found to be 78 ppm, 67 ppm, 44 ppm and 48 ppm respectively.

Air Pollutants↗

A hematology survey of workers at a styrene-butadiene synthetic rubber manufacturing plant.

A previous epidemiologic study of the U.S. rubber industry indicated that there has been an excess of leukemia and lymphoma mortality among hourly workers at one styrene-butadiene rubber manufacturing plant. This investigation was a combined industrial hygiene and hematology cross-sectional survey at the same plant. The objectives of the survey were to quantify exposure levels for styrene, butadiene, benzene and toluene, and to relate these levels to hematologic variation. Personal air samples and blood specimens were obtained from 157 production workers. All exposure levels for the four chemicals assayed were well below recommended standards. The higher mean styrene (13.67 ppm) and butadiene (20.03 ppm) concentrations were found in the Tank Farm area; in all other departments the mean levels for the four chemicals were less than 2 ppm. The Tank Farm workers had slightly lower levels of circulating erythrocytes, hemoglobin, platelets and neutrophils, and slightly higher mean corpuscular red cell volumes and neutrophil band counts than the other workers. Overall, in this population there was no pronounced evidence of hematologic abnormality, as determined from examination of peripheral blood.

Adult↗

Should styrene be sampled on the left or right shoulder?--An important question in employee self-assessment.

A self-operated personal sampling technique called 'self assessment of exposure' (SAE) has been suggested as an easy method for collecting inhalation exposure data, as the workers themselves are performing the sampling. Employers and employees have raised the question of whether a different estimate of the air concentration is likely to be obtained depending on whether the sampler is fastened at the left or the right shoulder. In order to answer this question, the exposure to styrene vapour in two different small enterprises within the reinforced plastics industry was measured. Seven workers participated and the air sampling was performed by diffusive sampling. We observed no statistically significant difference in the determined air concentration of styrene between the left and right shoulder (P = 0.878). The results strongly indicate that the fastening of a sampler on the left or right shoulder does not produce a difference in the estimation of the inhalation exposure. SAE can thus be used to collect reliable exposure data of styrene vapour. The reliability of SAE will most certainly inspire occupational hygienists, physicians and other experts to involve the workers in repeated exposure measurements. Taking the exposure variability into account, repeated measurements are crucial when evaluating acute and chronic health effects following inhalation exposure to gases and vapours from chemical hazards.

Air Pollutants, Occupational↗

Persistence of O6-guanine DNA adducts in styrene-exposed lamination workers determined by 32P-postlabelling.

A modified 32P-postlabelling method was used for the detection of styrene-specific DNA adducts in lamination workers. The persistence of O6-styrene DNA adducts was studied in DNA from lymphocytes and granulocytes of an exposed and a control group. We compared O6-adduct levels obtained from a sampling prior to vacation, after 2 weeks of vacation and after an additional 1 month of work. In granulocytes, there was no significant difference in adduct levels between the control and the exposed groups in any individual samplings. In lymphocytes of laminators the detected adduct levels were significantly higher (5.4 adducts/10(8) nucleotides) than those in the controls (1.0 adduct/10(8) nucleotides). The 2 week interruption of exposure did not influence the total O6-adduct level (4.9 adducts/10(8) nucleotides in the first sampling versus 5.1 adducts/10(8) nucleotides in the second), indicating very slow removal of the specific O6-styrene adducts from DNA.

Adult↗