Levels of seven urinary phthalate metabolites in a human reference population

Using a novel and highly selective technique, we measured monoester metabolites of seven commonly used phthalates in urine samples from a reference population of 289 adult humans. This analytical approach allowed us to directly measure the individual phthalate metabolites responsible for the animal reproductive and developmental toxicity while avoiding contamination from the ubiquitous parent compounds. The monoesters with the highest urinary levels found were monoethyl phthalate (95th percentile, 3,750 ppb, 2,610 microg/g creatinine), monobutyl phthalate (95th percentile, 294 ppb, 162 microg/g creatinine), and monobenzyl phthalate (95th percentile, 137 ppb, 92 microg/g creatinine), reflecting exposure to diethyl phthalate, dibutyl phthalate, and benzyl butyl phthalate. Women of reproductive age (20-40 years) were found to have significantly higher levels of monobutyl phthalate, a reproductive and developmental toxicant in rodents, than other age/gender groups (p < 0.005). Current scientific and regulatory attention on phthalates has focused almost exclusively on health risks from exposure to only two phthalates, di-(2-ethylhexyl) phthalate and di-isononyl phthalate. Our findings strongly suggest that health-risk assessments for phthalate exposure in humans should include diethyl, dibutyl, and benzyl butyl phthalates.     

Mortality during heat waves in New York City July, 1972 and August and September, 1973.

Twelve phthalic acid esters, including those commonly used as plasticizers for biomedical devices, were subjected to various biological tests for acute, short-term, and chronic toxicity. Certain aspects of subtle toxicity were investigated as well as overt toxic manifestations. The acute ip LD₅₀ for these compounds in mice ranged from 3.22 to more than 100 g/kg. A comparison of acute LD₅₀ to chronic LD₅₀ reveals that most of these phthalates are 2–4 times more toxic chronically. However, the chronic toxicity of di-n-octyl phthalate was 21.74 times greater than its acute toxicity, while for di-2-ethylhexyl phthalate the chronic toxicity was 27.99 times greater. Most of these compounds produced a prolongation of pentobarbital sleeping time in mice pretreated with a phthalate ester.     

发光菌评价3种药物与个人护理品的毒性研究

目的 评价3种药物与个人护理品(pharmaceuticals and personal care products, PPCPs),即红霉素、乙酰螺旋霉素和邻苯二甲酸二丁酯对海洋发光菌的毒性。方法 根据预试验的结果将红霉素、乙酰螺旋霉素和邻苯二甲酸二丁酯分别配制成不同浓度，以3%NaCl溶液作为空白对照，加入明亮发光杆菌T3变种，分别用生物毒性测试仪测试样品和空白对照的发光度，计算发光损失率，数据用SPSS 21.0软件进行统计学分析。结果 样品发光度值分别随着红霉素、乙酰螺旋霉素和邻苯二甲酸二丁酯浓度的增加而降低，而发光损失率则分别随着3种PPCPs浓度的增加而增高，发光菌的发光损失率分别与3种PPCPs的浓度间呈线性回归关系(P<0.001),拟合的红霉素毒性回归方程为Y=13.25+47.38X(R=0.933),乙酰螺旋霉素的毒性回归方程为Y=6.24+80.33X(R=0.956),邻苯二甲酸二丁酯毒性回归方程为Y=6.32+38.18X(R=0.952),3种PPCPs对发光菌的毒性各不相同，红霉素、乙酰螺旋霉素和邻苯二甲酸二丁酯的EC₅₀值分...     

Effects of di(2-ethylhexyl) phthalate and dibutyl phthalate on the collembolan Folsomia fimetaria

Lethal and sublethal effects of di(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) on adult individuals of the collembolan Folsomia fimetaria were investigated in the laboratory by the use of small microcosms. Effects of DEHP and DBP were also tested on newly hatched collembolans in a multidish system. The endpoints were juvenile mortality, growth, and development. When exposed to DEHP, adults and juveniles were unaffected at all test concentrations, that is, up to 5,000 mg/kg. However, DBP caused increased adult mortality at 250 mg/kg and juvenile mortality at 25 mg/kg. For DBP, adult reproduction was a more sensitive endpoint than was survival, with an EC10 and EC50 of 14 and 68 mg/kg, respectively. Juvenile molting frequency seems to be a sensitive parameter, because number of cuticles produced by young springtails was reduced at 1 mg/kg. Toxicity was reduced when soil spiked with DBP was stored at 20 degrees C for a period of up to 28 d before adding the animals. Reduction in toxicity of DBP may be due a combination of degradation, evaporation, and adsorption of DBP to soil material. This was confirmed by chemical analyses, which showed a rapid initial disappearance followed by a much slower disappearance. Our results lead to the overall conclusion that significant adverse effects of phthalates on collembolans are not likely to occur as a result of normal sewage sludge application.     

Identification of Trace Organic Pollutants in Drinking Water and the Associated Human Health Risks in Jiangsu Province,China

Organochlorine pesticides (OCPs), polycyclic aromatic hydrocarbons (PAHs), dibutyl phthalate (DNBP) and di-2-ethylhexyl phthalate (DEHP) were all detectable in surface water in Jiangsu Province, China. Concentrations of OCPs ranged from 5.13 to 8.15 ng/L. PAHs were found ranging from 14.7 to 24.5 ng/L. Concentrations of DNBP and DEHP ranged from 16 to 5,857.5 ng/L and 556 to 15,670.7 ng/L, respectively. Greater than 70 % of chemicals were removed in water treatment processes. The carcinogenic risks posed by trace organic pollutants through tap water ingestion were lower than 10(-6), and the noncarcinogenic risks were less than 10(-5).     

Environmental phthalate exposure in relation to reproductive outcomes and other health endpoints in humans

After briefly discussing human exposure to phthalates—diesters of 1,2-benzenedicarboxylic acid (phthalic acid)—this article first presents recent findings from the Study for Future Families, a multi-center pregnancy study in which the human analogue of the phthalate syndrome was first identified. This is one of an increasing number of studies that have investigated human endpoints in relation to environmental exposure to these ubiquitous compounds. This literature, which includes a range of human health endpoints following prenatal, neonatal, childhood, and adult exposures, is then summarized. At least one significant association has been reported for urinary metabolites of di-n-butyl phthalate (DBP), butylbenzyl phthalate (BzBP), diethyl phthlate (DEP), and di-isononyl phthalate (DINP) and for three of the urinary metabolites of di(2-ethylhexyl) phthalate (DEHP). Many of the findings reported in humans—most of which have been in males—are consistent with the anti-androgenic action that has been demonstrated for several phthalates. Replication of the results described here and further mechanistic studies are needed to strengthen links between phthalates and adverse health outcomes.     

An assessment of the toxicity of phthalate esters to freshwater benthos. 1. Aqueous exposures

Tests were performed with the freshwater invertebrates Hyalella azteca, Chironomus tentans, and Lumbriculus variegatus to determine the acute toxicity of six phthalate esters, including dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), butylbenzyl phthalate (BBP), di-n-hexyl phthalate (DHP), and di-2-ethylhexyl phthalate (DEHP). It was possible to derive 10-d LC50 (lethal concentration for 50% of the population) values only for the four lower molecular weight esters (DMP, DEP, DBP, and BBP), for which toxicity increased with increasing octanol-water partition coefficient (Kow) and decreasing water solubility. The LC50 values for DMP, DEP, DBP, and BBP were 28.1, 4.21, 0.63, and 0.46 mg/L for H. azteca; 68.2, 31.0, 2.64, and > 1.76 mg/L for C. tentans; and 246, 102, 2.48, and 1.23 mg/L for L. variegatus, respectively. No significant survival reductions were observed when the three species were exposed to either DHP or DEHP at concentrations approximating their water solubilities.     

Characterization of phthalate exposure among pregnant women assessed by repeat air and urine samples

BACKGROUND: Although urinary concentrations of phthalate metabolites are frequently used as biomarkers in epidemiologic studies, variability during pregnancy has not been characterized. METHODS: We measured phthalate metabolite concentrations in spot urine samples collected from 246 pregnant Dominican and African-American women. Twenty-eight women had repeat urine samples collected over a 6-week period. We also analyzed 48-hr personal air samples (n = 96 women) and repeated indoor air samples (n = 32 homes) for five phthalate diesters. Mixed-effects models were fit to evaluate reproducibility via intraclass correlation coefficients (ICC). We evaluated the sensitivity and specificity of using a single specimen versus repeat samples to classify a woman's exposure in the low or high category. RESULTS: Phthalates were detected in 85-100% of air and urine samples. ICCs for the unadjusted urinary metabolite concentrations ranged from 0.30 for mono-ethyl phthalate to 0.66 for monobenzyl phthalate. For indoor air, ICCs ranged from 0.48 [di-2-ethylhexyl phthalate (DEHP)] to 0.83 [butylbenzyl phthalate (BBzP)]. Air levels of phthalate diesters correlated with their respective urinary metabolite concentrations for BBzP (r = 0.71), di-isobutyl phthalate (r = 0.44), and diethyl phthalate (DEP; r = 0.39). In women sampled late in pregnancy, specific gravity appeared to be more effective than creatinine in adjusting for urine dilution. CONCLUSIONS: Urinary concentrations of DEP and DEHP metabolites in pregnant women showed lower reproducibility than metabolites for di-n-butyl phthalate and BBzP. A single indoor air sample may be sufficient to characterize phthalate exposure in the home, whereas urinary phthalate biomarkers should be sampled longitudinally during pregnancy to minimize exposure misclassification.     

Toxicity, residue dynamics, and reproductive effects of phthalate esters in aquatic invertebrates

Aquatic invertebrates were exposed to di-n-butyl and di-2-ethylhexyl phthalate esters in water to determine toxicity, accumulation, and reproductive effects of these compounds. The acute toxicities were low and ranged from 2.1 mg/liter to greater than 32 mg/liter. Residue accumulation was rapid resulting in body residues 70-13,600 times that of the water concentration. Phthalate residues were essentially gone after 10 days in fresh water. A reproductive impairment of 60% occurred in Daphnia magna exposed continuously to 3 μg/liter of di-2-ethylhexyl phthalate.     

Phthalate monoesters in association with uterine leiomyomata in Shanghai

Phthalates are ubiquitous environmental pollutants because of the broad use of plastics. We conducted a case-control study to determine whether uterine leiomyomata were related to exposure to phthalates. Urine specimens and questionnaires were collected from 61 cases and 61 age-matched controls. Nine phthalate monoesters were determined by ultra performance liquid chromatography coupled with tandem mass spectroscopy. Cases had significantly higher levels of creatinine-adjusted mono-iso-butyl phthalate (MiBP), mono-n-butyl phthalate (MnBP), mono-2-ethylhexyl phthalate (MEHP), mono-2-ethyl-5-oxohexyl phthalate, mono-2-ethyl-5-hydroxyhexyl phthalate (MEHHP), mono(2-ethyl-5-carboxypentyl) phthalate (MECPP), total di(2-ethylhexyl) phthalate metabolites (∑DEHP_met), and total dibutyl phthalate metabolites (∑DBP_met) than controls. After adjusting for potential confounders, logistic regression analyses demonstrated that leiomyomata were positively associated with MiBP, MnBP, MEHP, MEHHP, MECPP, ∑DEHP_met, and ∑DBP_met. In summary, our data support the hypothesis that uterine leiomyomata are related to phthalate exposure.     

Comparative Aquatic Toxicity Evaluation of 2-(Thiocyanomethylthio)benzothiazole and Selected Degradation Products Using Ceriodaphnia dubia

2-(Thiocyanomethylthio)benzothiazole (TCMTB) is a biocide used in the leather, pulp and paper, and water-treatment industries. TCMTB may enter aquatic ecosystems during its manufacture and use. TCMTB is environmentally unstable; therefore, it is important to evaluate the toxicity of the more persistent degradation products. This study compared the toxicity of TCMTB with its degradation products 2-mercaptobenzothiazole (2-MBT), 2-(methylthio)benzothiazole (MTBT), benzothiazole (BT), and 2-hydroxybenzothiazole (HOBT). Toxicity was determined using Ceriodaphnia dubia 48-hour acute and 7-day chronic test protocols. TCMTB was the most toxic compound evaluated in both the acute and chronic tests with EC50s of 15.3 and 9.64 g/L, respectively. 2-MBT, the first degradation product, was the second most toxic compound with acute and chronic EC50s of 4.19 and 1.25 mg/L, respectively. The toxicity of MTBT and HOBT were similar with acute EC50s of 12.7 and 15.1 mg/L and chronic EC50s of 6.36 and 8.31 mg/L, respectively. The least toxic compound was BT with acute and chronic EC50s of 24.6 and 54.9 mg/L, respectively. TCMTB was orders of magnitude more toxic than its degradation products. Toxicity data on these benzothiazole degradation products is important because of concerns regarding their release, degradation, persistence, and non–target organism effects in aquatic ecosystems.     

Bioaccumulation and metabolism of phthalate esters by oysters,brown shrimp,and sheepshead minnows

The bioaccumulation and metabolic fate of three phthalate esters, dimethylphthalate (DMP), dibutylphthalate (DBP) and di(2-ethylhexyl)phthalate (DEHP), were studied in three species. The species used, the oyster Crassostrea virginica, the brown shrimp Penaeus aztecus, and the sheepshead minnow Cyprinodon variegatus, were chosen for this study as previous studies indicated that these organisms possessed different capacities for detoxifying xenobiotics. The effects of concentration as well as ester chain length and species were studied. Bioaccumulation did not vary significantly among the three species, possibly due to high levels of individual variation. A trend to higher accumulation in oysters was noted. Bioaccumulation was significantly affected by chain length, with DBP accumulating to a greater extent than DMP or DEHP, and by the concentration of phthalate. Biodegradative indices, on the other hand, were not significantly affected by the ester used or by concentration, but did vary significantly with species. Oysters were less efficient in the biodegradation of the esters than fish or shrimp. The major metabolites found were the monoesters, phthalic acid, and a group of unidentified polar metabolites, probably conjugates. These latter metabolites did not appear in oysters, but were present in significant amounts in fish and shrimp.     

Assessment of the toxicity of triasulfuron and its photoproducts using aquatic organisms

The toxicological effects of the sulfonylurea herbicide triasulfuron and its photoproducts were assessed on four aquatic organisms. Toxicity varied with tested organism and with triasulfuron irradiation time. Triasulfuron and its photoproducts had no significant effects on the crustacean (Cladocera) Daphnia magna (causing 50% effective concentration [EC50] [48 h] = 49 +/- 1 mg/L) and the marine bacteria Vibrio fischeri (EC50 [30 min] > 100 mg/L). In contrast, primary producers (the duckweed Lemna minor, the microalgae Pseudokirchneriella subcapitata, and Chlorella vulgaris) were very sensitive to triasulfuron (EC50s < 11 microg/L). For these organisms, triasulfuron photoproducts were less toxic than the parent compound but the residual toxicity observed still represented a potential environmental hazard.     

Occurrence and daily variation of phthalate metabolites in the urine of an adult population

Phthalates like di-(2-ethylhexyl) phthalate (DEHP) are commonly used as plasticizers and their metabolites are suspect of especially reproductive toxicity. The aim of our study was to assess phthalate exposure in adults by measuring urinary phthalate metabolite levels and to explore individual temporal variability. Urine samples were collected by 27 women and 23 men aged 14-60 years during 8 consecutive days. We quantified four monoesters, four oxidative DEHP metabolites, and two secondary metabolites of di-isononyl phthalate (DiNP) by a LC/LC-MS/MS method. If we analyzed all 399 available samples independent of classification, the highest median values of primary metabolites in this study were found for mono-n-butyl phthalate (MnBP: 49.6 microg/l), followed by mono-isobutyl phthalate (MiBP: 44.9 microg/l), mono-benzyl phthalate (MBzP: 7.2 microg/l), and mono-2-ethylhexyl phthalate (MEHP: 4.9 microg/l). The median concentrations of the oxidized metabolites of DEHP were 8.3 microg/l for mono-(2-carboxymethylhexyl) phthalate (2cx-MMHP), 19.2 microg/l for mono-(2-ethyl-5-hydroxyhexyl) phthalate (5OH-MEHP), 14.7 microg/l for mono-(2-ethyl-5-oxohexyl) phthalate (5oxo-MEHP), and 26.2 microg/l for mono-(2-ethyl-5-carboxypentyl) phthalate (5cx-MEPP). The concentrations of the two DiNP secondary metabolites mono (oxoisononyl) phthalate (oxo-MiNP) and mono(hydroxyisononyl) phthalate (OH-MiNP) ranged from 相似文献   

Structure-activity relationships for induction of peroxisomal enzyme activities by phthalate monoesters in primary rat hepatocyte cultures

Rat hepatocytes were cultured for 70 hours with a series of four isomeric octyl and five isomeric hexyl phthalate monoesters, and their effects on peroxisomal fatty acid beta-oxidation (palmitoyl-CoA oxidation) and carnitine acetyltransferase activities determined. All nine monoesters produced dose-related increases in enzyme activities and marked quantitative compound potency differences were observed. Generally octyl isomers were more potent than hexyl isomers and 2- and 3-ethyl substituted isomers were more potent than their straight chain and 1-ethyl substituted analogs. For example, mono(2-ethylhexyl)phthalate was more potent than mono(1-ethylhexyl)phthalate, and this was also observed after oral administration of the two isomers to rats for seven days. The cell culture data for induction of palmitoyl-CoA oxidation were used to generate quantitative structure-activity relationships. Relatively poor correlations were observed between biological activity and simple hydrophobic parameters, but a good correlation was obtained when compound electronic structural parameters, obtained by molecular orbital calculations, were employed. These studies demonstrate relationships between biological activity and chemical structure for a series of phthalate monoesters and indicate the potential usefulness of primary rat hepatocyte cultures to screen compounds for peroxisome proliferation.     

An assessment of the toxicity of phthalate esters to freshwater benthos. 2. Sediment exposures

Seven phthalate esters were evaluated for their 10-d toxicity to the freshwater invertebrates Hyalella azteca and Chironomus tentans in sediment. The esters were diethyl phthalate (DEP), di-n-butyl phthalate (DBP), di-n-hexyl phthalate (DHP), di-(2-ethylhexyl) phthalate (DEHP), diisononyl phthalate (DINP), diisodecyl phthalate (DIDP), and a commercial mixture of C7, C9, and C11 isophthalate esters (711P). All seven esters were tested in a sediment containing 4.80% total organic carbon (TOC), and DBP alone was tested in two additional sediments with 2.45 and 14.1% TOC. Sediment spiking concentrations for DEP and DBP were based on LC50 (lethal concentration for 50% of the population) values from water-only toxicity tests, sediment organic carbon concentration, and equilibrium partitioning (EqP) theory. The five higher molecular weight phthalate esters (DHP, DEHP, DINP, DIDP, 711P), two of which were tested and found to be nontoxic in water-only tests (i.e., DHP and DEHP), were tested at single concentrations between 2,100 and 3,200 mg/kg dry weight. Preliminary spiking studies were performed to assess phthalate ester stability under test conditions. The five higher molecular weight phthalate esters in sediment had no effect on survival or growth of either C. tentans or H. azteca, consistent with predictions based on water-only tests and EqP theory. The 10-d LC50 values for DBP and H. azteca were >17,400, >29,500, and >71,900 mg/kg dry weight for the low, medium, and high TOC sediments, respectively. These values are more than 30x greater than predicted by EqP theory and may reflect the fact that H. azteca is an epibenthic species and not an obligative burrower. The 10-d LC50 values for DBP and C. tentans were 826, 1,664, and 4.730 mg/kg dry weight for the low, medium, and high TOC sediments, respectively. These values are within a factor of two of the values predicted by EqP theory. Pore-water 10-d LC50 values for DBP (dissolved fraction) and C. tentans in the three sediments were 0.65, 0.89, and 0.66 of the water-only LC50 value of 2.64 mg/L, thereby agreeing with EqP theory predictions to within a factor of 1.5. The LC50 value for DEP and C. tentans was >3,100 mg/kg dry weight, which is approximately 10x that predicted by EqP theory. It is postulated that test chemical loss and reduced organism exposure to pore water may have accounted for the observed discrepancies with EqP calculations for DEP     

Determination of Five Phthalate Monoesters in Human Urine Using Gas Chromatography-Mass Spectrometry

We have developed a gas chromatography-mass spectrometry (GC–MS) method to determine five phthalate monoesters (monoethyl phthalate (MEP), mono-n-butyl phthalate (MBP), mono-(2-ethylhexyl) phthalate (MEHP), monoisononyl phthalate (MINP) and monobenzyl phthalate (MBz)) in human urine. Human urine samples were subjected to enzymatic deconjugation of the glucuronides followed by extraction with hexane. The extracted phthalate monoesters were methylated with diazomethane, purified on a Florisil column and then subjected to GC–MS analysis. The recoveries from urine spiked with five phthalate monoesters were 86.3%–119% with coefficients of variation of 0.6%–6.1%. We measured phthalate monoester levels in human urine by analyzing 36 samples from volunteers. MBP and MEP were detected in all samples, and their median concentrations were 60.0 and 10.7 ng/mL, respectively. MBzP and MEHP were found in 75% and 56% of samples, and their median concentrations were 10.9 and 5.75 ng/mL, respectively. MINPs were not detected in most samples (6% detectable). Women had significantly (p < 0.05) higher mean concentrations of MBP and MEP than men. The estimated daily exposure levels for the four parent phthalates excluding diisononyl phthalate ranged from 0.27 to 5.69 μg/kg/day (median).     

Distribution of Phthalate Esters in Soil of E-Waste Recycling Sites from Taizhou City in China

In recent years, increasing concern has surrounded the consequences of improper electric and electronic waste (e-waste) disposal. In this paper, Phthalate esters (PAEs) including dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), Di-(2-ethylhexyl) phthalate (DEHP) and di-n-octyl phthalate (DnOP) in the e-waste soils were collected and analyzed from sites Fengjiang, Nanshan and Meishu in Taizhou city. The result showed that the total PAEs concentrations ranged from 12.566 to 46.669 mg/kg in these three sites. DEHP, DBP and DEP were the major phthalates accounting for more than 94% of total phthalates studied. Comparing to the results from other studies, the e-waste soils from Taizhou city were severely contaminated with PAEs.     

Reactions between fulvic acid,a soil humic material,and dialkyl phthalates

Summary Fulvic acid, a water-soluble soil humic material that occurs widely in soils and waters, can complex hydrophobic dialkyl phthalates and make them soluble in water. The extent of the reaction depends on the type of phthalate. Thus, one number-average molecular weight of FA can solubilize four moles of bis(2-ethylhexyl) phthalate but only one mole of dibutyl phthalate, while 2 number-average molecular weights of FA can interact with three moles of dicyclohexyl phthalate. IR spectroscopy failed to provide evidence for the occurrence of chemical reactions between FA and the phthalates. The latter appeared to be adsorbed on the surface of the FA molecule.Contribution No. 362.     

Assessment of lead toxicity to the marine bacterium, Vibrio fischeri, and to a heterogeneous population of microorganisms derived from the Pearl River in Jackson, Mississippi, USA.

Microorganisms are known to be excellent test organisms because of the relative ease for handling and suitability for analysis related to their small size, large number and convenient growing conditions. In this research, we tested the toxic effects of lead against a marine bacterium (Vibrio fischeri), and a heterogeneous population of bacteria derived from the Pearl River in Jackson, Mississippi. Using the level of bioluminescence in the Microtox Assay (V. fischeri), and the kinetics of dissolved oxygen uptake and growth (mixed bacterial population) as measures of toxicity, lead concentrations effecting a 50% reduction in these parameters (EC50) were determined as the toxic end-points. The activity quotients were also computed to determine the degrees of toxicity. Optical density (measure of growth) and oxygen uptake were measured over an extended period of time (20 h). EC50 values of 0.34 +/- 0.03, 3.10 +/- 0.01, and 3.80 +/- 0.02 mg/L were recorded for bioluminescence, growth, and oxygen uptake, respectively. As expected, the results indicated that the sensitivity to lead toxicity of V. fischeri was about one order of magnitude (10 times) greater than that of the mixed population of Pearl River microorganisms. Reductions in bioluminescence, growth, and oxygen uptake were directly correlated to lead concentrations, with toxic levels ranging from slightly toxic in lower concentrations to extremely toxic in higher concentrations. Upon 20 h of exposure, the times required to produce 50% reduction in dissolved oxygen uptake were (TD50S) 8.01 +/- 0.44, 9.60 +/- 0.46, 11.29 +/- 0.46, 13.03 +/- 0.57, 17.32 +/- 0.95, and 20.00 +/- 0.00 h in 0, 1, 2, 3, 4, 5, and 6 mg/L of lead, respectively, indicating a time-response relationship with respect to lead toxicity.