Bioaccumulation and Male Reproductive Toxicity of the New Brominated Flame Retardant Tetrabromobisphenol A-Bis(2,3-dibromo-2-methylpropyl ether) in Comparison with Hexabromocyclododecane.

Tetrabromobisphenol A-bis(2,3-dibromo-2-methylpropyl ether) (TBBPA-DBMPE) has come into use as an alternative to hexabromocyclododecane (HBCD), but it is unclear whether TBBPA-DBMPE has less hazard than HBCD. Here, we compared the bioaccumulation and male reproductive toxicity between TBBPA-DBMPE and HBCD in mice following long-term oral exposure after birth. We found that the concentrations of TBBPA-DBMPE in livers significantly increased with time, exhibiting a bioaccumulation potency not substantially different from HBCD. Lactational exposure to 1000 µg/kg/d TBBPA-DBMPE as well as 50 µg/kg/d HBCD inhibited testis development in suckling pups, and extended exposure up to adulthood resulted in significant molecular and cellular alterations in testes, with slighter effects of 50 µg/kg/d TBBPA-DBMPE. When exposure was extended to 8 month age, severe reproductive impairments including reduced sperm count, increased abnormal sperm, and subfertility occurred in all treated animals, although 50 µg/kg/d TBBPA-DBMPE exerted lower effects than 50 µg/kg/d HBCD. Altogether, all data led us to conclude that TBBPA-DBMPE exerted weaker male reproductive toxicity than HBCD at the same doses but exhibited bioaccumulation potential roughly equivalent to HBCD. Our study fills the data gap regarding the bioaccumulation and toxicity of TBBPA-DBMPE and raises concerns about its use as an alternative to HBCD.

Addition of low concentration of cholesterol-loaded cyclodextrin (CLC) has a positive effect on cryopreserved canine spermatozoa evaluated by andrological and biophysical methods.

BACKGROUND: This study was conducted to find the best concentration of cholesterol-loaded cyclodextrin (CLC) which has a positive impact on canine post thaw semen quality. Three different concentrations of CLC (0.83 mg/ml; 1.66 mg/ml; 3.32 mg/ml) and 2-hydroxylpropyl-beta-cyclodextrin (HBCD) (1.66 mg/ml) were used in addition to cryopreservation extender and compared with the control after thawing. Samples were assessed using computer-assisted semen analyzer (CASA), flow cytometry, fluorimeter by measuring the fluorescence anisotropy (ANISO) and determining the generalized membrane polarization (GP). RESULTS: An addition of 0.83 mg/ml CLC significantly increased the percentage of progressive motile (PROG) and rapid spermatozoa (RAP) (P < 0.05). 1.66 mg/ml HBCD decreased progressive motility of spermatozoa and population with rapid movement relative to the control (P < 0.05). Furthermore, the groups with an addition of 1.66 mg/ml and 3.32 mg/ml of CLC, as well as the group with only cyclodextrin, increased percentage of dead spermatozoa without lipid peroxidation and decreased percentage of viable spermatozoa without LPO which was lower in these groups than in the control (P < 0.05). Other sperm parameters assessed on flow cytometer were not significantly different. The addition of CLC at 0.83 mg/ml and 3.32 mg/ml concentrations and 1.66 mg/ml of HBCD caused an increase in ANISO measured at 23 ºC (P < 0.05). CONCLUSIONS: In conclusion, the results suggest that increasing cholesterol in the plasma membrane of canine spermatozoa can improve their freezability. However, only low concentrations of CLC may improve semen quality after thawing without adversely affecting other parameters.

Bioavailability of non-aromatic brominated flame retardants in rats from dust and oil vehicles.

Hexabromocyclododecane (HBCD) is a brominated flame retardant (BFR) labeled by the Stockholm Convention as a persistent organic pollutant (POP) and exists primarily as three stereoisomers, i.e. α-, ß-, and γ. One of the major routes of human exposure to HBCD is dust found in homes, offices, and cars and dust may be the most important route of HBCD exposure in young children. A study was conducted to determine the oral bioavailability of HBCD from household dust in rats over a 21-d feeding period relative to HBCD bioavailability from a corn oil matrix. Twenty-four hours after the last exposure, rats were sacrificed, and various tissues were collected. HBCD diastereomers were detected in adipose, blood, and liver of both dose groups, suggesting HBCD is bioavailable from both oil and dust. ß-HBCD concentrations were below the limit of detection in all tissues, but α-HBCD was detected in the brain of oil-dose rats and in adipose and liver of both dose groups. γ-HBCD was the dominant diastereomer in adipose, blood, and liver samples regardless of dosing matrix. Except for γ-HBCD in muscle of the oil-dosed group, muscle did not contain measurable HBCDs. Adipose tissue accumulated HBCD to a greater extent than muscle or liver, having bioaccumulation factors greater than 1.

A Review of Distribution and Profiles of HBCD in Different Environmental Media of China.

Hexabromocyclododecane (HBCD) is the most important flame retardant that has been used in Expanded Polystyrene foam and Extruded Polystyrene foam in the past forty years across the world. China was the major producer and user of HBCD, and the total HBCD production was about 0.3 million tons. Although HBCD was completely banned in China in 2021 because of its long-range transport, bioaccumulation and toxicity, there is still a lot of residue in the environment. Therefore, we reviewed multiple studies concerning the distribution of HBCD in diverse environmental matrices, such as in the air, dust, soil, water, sediment, and biota. Results revealed that HBCD levels in different environments in China present geographical variation and were at a high level compared with other countries. In all environmental media, relatively high HBCD concentrations have been found in industrial and urban areas. Industrialization and urbanization are two important factors that influence the concentration and distribution of HBCD in the environment. In terms of isomer, γ-HBCD was the dominant isomer in soil, water, and sediment, while in the biota α-HBCD was the predominant isomer.

Levels, distribution and risk assessment of hexabromocyclododecane (HBCD) in fish in Xiamen, China.

In this study, hexabromocyclododecane (HBCD) was detected in 114 fish samples collected from 6 administrative regions of Xiamen city, China. HBCD amounts ranged between ND (not detected) and 2.216 ng g-1 ww (mean, 0.127 ± 0.318 ng g-1 ww). Besides, α-HBCD was the main diastereoisomer in these fish specimens, followed by ß-HBCD. Meanwhile, γ-HBCD was not detected in any of the samples. Significant differences were recorded among fish species. The results indicated that the levels and detection rates of HBCD were higher in Trachinotus ovatus compared with other aquatic organisms. Therefore, Trachinotus ovatus could be used as a marine biological indicator of HBCD. Within the regions investigated, Siming was significantly different from Jimei, Haicang, and Xiang'an. The spatial distribution of HBCD concentrations indicated higher mean levels in samples collected from Haicang, Jimei, and Xiang'an, respectively, with the highest detection rates in Jimei and Xiang'an, which might be related to geographical location and intense industrial and urban activities. Estimation of daily HBCD intake was performed according to fish consumption in Xiamen residents. The medium bound HBCD amounts in fish were approximately 0.073 and 0.088 ng kg bw-1d-1 for male and female residents of Xiamen, respectively. Exposure doses of HBCD indicated no health concern for Xiamen residents.

A PBPK model describing the pharmacokinetics of γ-HBCD exposure in mice.

The brominated flame retardant, hexabromocyclododecane (HBCD), is added-but not bound-to consumer products and is eventually found in the environment and human tissues. Commercial-grade HBCD mixtures contain three major stereoisomers, alpha (α), beta (ß), and gamma (γ), that are typically at a ratio of 12%:6%:82%, respectively. Although HBCD is widely used, the toxicological effects from its exposure in humans are not clearly understood. Using a physiologically based pharmacokinetic (PBPK) model could help improve our understanding of the toxicity of HBCD. The aim of this work was to develop a PBPK model, consisting of five permeability limited compartments (i.e., brain, liver, adipose tissue, blood, and rest of the body), to evaluate the pharmacokinetics of γ-HBCD in C57BL/6 mice. Physiological parameters related to body size, organ weights, and blood flow were taken from the literature. All partition coefficients were calculated based on the log Kow. The elimination in urine and feces was optimized to reflect the percent dose eliminated, as published in the literature. Compared with data from the literature for brain, liver, blood, and adipose tissue, the model simulations accurately described the mouse data set within 1.5-fold of the data points. Also, two examples showing the utility of the PBPK model supplement the information regarding the internal dose that caused the health effects observed during these studies. Although this version of the PBPK model expressly describes γ-HBCD, more efforts are needed to clarify and improve the model to discriminate between the α, ß, and γ stereoisomers.

Determination of Hexabromocyclododecane in Expanded Polystyrene and Extruded Polystyrene Foam by Gas Chromatography-Mass Spectrometry.

A gas chromatography-mass spectrometry (GC/MS) method for the determination of hexabromocyclododecane (HBCD) in expanded polystyrene and extruded polystyrene foam (EPS/XPS) was developed. The EPS/XPS samples were ultrasonically extracted with acetone and the extracts were purified by filtration through a microporous membrane (0.22 µm) and solid-phase extraction. The samples were analyzed using a GC/MS using the selected ion monitoring mode. The ions 157, 319 and 401 were selected as the qualitative ions, while ion 239 was chosen as the quantitative ion. An HBCD standard working solution with a concentration range of 1.0-50.0 mg/L showed good linearity. The detection limit of HBCD was 0.5 mg/kg, meeting the LPC limit (<100 or 1000 mg/kg). Six laboratories were selected to verify the accuracy of the method, and 10 samples were tested. The interlaboratory relative standard deviation range was 3.68-9.80%. This method could play an important role in controlling HBCD contamination in EPS/XPS.

Temporal-spatial distribution and diastereoisomer pattern of hexabromocyclododecane in the vicinity of a chemical plant.

Hexabromocyclododecane (HBCD) is an effective brominated flame-retardant additive, which is mainly produced in the coastal area of China. This study collected soil samples from a HBCD production plant and its surrounding area in Weifang, Shandong Province, China, and analyzed the temporal-spatial distribution of HBCD and its diastereoisomers in soil. The analysis results showed that the concentration of HBCD in soil near the plant was much higher than normal values, with an annual average concentration reaching 5405 ng/g. Soils 1, 2 and 4 km away from the plant were also analyzed, showing that the concentration of HBCD in soil decreased accordingly with the distance from the pollution sources. In order to investigate the effect of the season on HBCD content, the soil samples were collected in all four seasons of the year 2017-2018. According to variations in the wind direction, the concentration of HBCD in soil was also changed. The distribution trend showed that the concentration of HBCD in soil in the downwind direction of the prevailing wind was higher than that in the upwind direction. In addition, this work analyzed the distribution of HBCD in vertical soil sections. It was found that the concentration of HBCD decreased with depth in the soil vertical profile. Finally, the various diastereoisomer patterns in the soil compartments were examined, finding that α-HBCD and γ-HBCD were the predominant diastereoisomers in the soil of the study area.

Bioconcentration and effects of hexabromocyclododecane exposure in crucian carp (Carassius auratus).

As a cycloaliphatic brominated flame retardant, hexabromocyclododecane (HBCD) has been widely used in building thermal insulation and fireproof materials. However, there is little information on the bioconcentration as well as effects with respect to HBCD exposure in the aquatic environment. To investigate the bioconcentration of HBCD in tissues (muscle and liver) and its biochemical and behavioural effects, juvenile crucian carp (Carassius auratus) were exposed to different concentrations of technical HBCD (nominal concentrations, 2, 20, 200 µg/L) for 7 days, using a flow-through exposure system. HBCD was found to concentrate in the liver and muscle with a terminal concentration of 0.60 ± 0.22 µg/g lw (lipid weight) and 0.18 ± 0.02 µg/g lw, respectively, at an environmentally-relevant concentration (2 µg/L). The total thyroxine and total triiodothyronine in the fish plasma were lowered as a result of exposure to the HBCD. Acetylcholinesterase activity in the brain was increased, while swimming activity was inhibited and shoaling inclination was enhanced after exposure to 200 µg/L HBCD. Feeding rate was suppressed in the 20 and 200 µg/L treatment groups. In summary, HBCD concentrations 10-100× higher than the current environmentally-relevant exposures induced adverse effects in the fish species tested in this study. These results suggest that increasing environmental concentrations and/or species with higher sensitivity than carp might be adversely affected by HBCD.

Solvent effects on quantitative analysis of brominated flame retardants with Soxhlet extraction.

Reliable quantifications of brominated flame retardants (BFRs) not only ensure compliance with laws and regulations on the use of BFRs in commercial products, but also is key for accurate risk assessments of BFRs. Acetone is a common solvent widely used in the analytical procedure of BFRs, but our recent study found that acetone can react with some BFRs. It is highly likely that such reactions can negatively affect the quantifications of BFRs in environmental samples. In this study, the effects of acetone on the extraction yields of three representative BFRs [i.e., decabrominated diphenyl ether (decaBDE), hexabromocyclododecane (HBCD) and tetrabromobisphenol A (TBBPA)] were evaluated in the Soxhlet extraction (SE) system. The results showed that acetone-based SE procedure had no measureable effect for the recovery efficiencies of decaBDE but could substantially lower the extraction yields for both TBBPA and HBCD. After 24 h of extraction, the recovery efficiencies of TBBPA and HBCD by SE were 93 and 78% with acetone, 47 and 70% with 3:1 acetone:n-hexane, and 82 and 94% with 1:1 acetone:n-hexane, respectively. After 72 h of extraction, the extraction efficiencies of TBBPA and HBCD decreased to 68 and 55% with acetone, 0 and 5% with 3:1 acetone/n-hexane mixtures, and 0 and 13% with 1:1 acetone/n-hexane mixtures, respectively. The study suggested that the use of acetone alone or acetone-based mixtures should be restricted in the quantitative analysis of HBCD and TBBPA. We further evaluated nine alternative solvents for the extraction of the three BFRs. The result showed that diethyl ether might be reactive with HBCD and may not be considered as the alternative to acetone used solvents for the extraction of HBCD.

Bioaccumulation and Distribution of Hexabromocyclododecane Isomers in Duck Tissues.

Eighty ducks (Sheldrake, Anas platyrhynchos) from Zhejiang Province, China were fed capsules containing commercialized hexabromocyclododecanes (HBCDs) at low (0.8 mg/kg/day) or high (1.6 mg/kg/day) exposure level, or only maize starch for 21 days. Then the next 21 days was set as depuration period. Ducks were euthanizated at 0, 7, 14 and 21 days after last dose and ten duck tissues including skin, tongue, intestines, heart, gizzard, muscle, liver, lung, brain and blood were sampled, separately. Three HBCDs including α-HBCD, ß-HBCD and γ-HBCD in duck tissue samples were analyzed. At the end of depuration period, the total HBCDs concentration in skin was significantly higher than those in the other tissues (p < 0.05). The elimination rates of the three isomers in skin, tongue, intestines, heart, gizzard and brain were in the order ß- > γ- > α-HBCD. The enantioselectivity of three HBCDs enantiomers was also studied in ten duck tissues. It was shown that the EF (enrichment factor) for two γ-HBCD enantiomers was significant lower than 0.5 (p < 0.05) in gizzard, heart, muscle, tongue, intestinal and liver at the end of depuration day, showing a selective accumulation of (+)-γ-HBCD in these tissues. This study provided a reference for evaluation on the accumulation of the persistent contamination of HBCDs in edible poultry.

Demographic and temporal trends of hexabromocyclododecanes (HBCDD) in an Australian population.

Hexabromocyclododecanes (HBCDD) were measured in 67 pooled serum samples collected between 2002 and 2015 in South East Queensland, Australia. These data are the first report of HBCDD in Australian human serum. Temporal and demographic (age and gender) trends were investigated. HBCDD were detected in measurable concentrations in 69% of samples. The average ∑HBCDD concentration was 3.1ng/g lipid, whilst the range was <0.5 to 36ng/g lipid. α-HBCDD was the dominant stereoisomer making up an average of 60% of ∑HBCDD. The remainder was made up by γ-HBCDD. In contrast to another group of brominated flame retardants (BFRs, (polybrominated diphenyl ethers (PBDEs)), HBCDD were found in the lowest concentrations in pools from children aged 0-4 years. This could be attributed to differences in exposure, usage, and/ or the much lower half-life of HBCDD in the human body compared to PBDEs. HBCDD concentrations appear to be significantly higher in females than in males, however the reasons for this are unclear.

Neuroprotection by Taurine on HBCD-Induced Apoptosis in PC12 Cells.

Hexabromocyclododecane (HBCD) is a widely used brominated flame retardant (BFR). Because of their presence in human issues, including brain tissue, concern has been raised on their possible neurotoxicity. Presently, we explored the neuroprotection of taurine against HBCD-induced apoptotic damages in PC12 cells. Cells were pre-treated with taurine before HBCD exposure and the viability was assayed via the methyl-thiazolyl-tetrazolium (MTT) method. Apoptotic features were observed with Hoechst 33342 staining. Apoptotic ratio was measured using flow cytometry with Annexin V-FITC coupled propidium iodide (PI) double staining. The changes in the levels of Bcl-2 and Bax proteins were quantitated by the western blot. The activity of caspase-3 was tested and the results revealed that presence of HBCD decreased cell survival and led to apoptosis in the tested cells. Further, exposure of HBCD reduced protein expression of Bcl-2, increased expression in Bax protein and activity of caspase-3. Taurine attenuated HBCD-induced cell viability loss and cell apoptosis. Moreover, taurine significantly prevented from reducing Bcl-2 protein expression and elevating Bax protein expression and caspase-3 activity induced by HBCD. These results demonstrated that taurine can alleviate HBCD-induced apoptosis by altering Bcl-2 expression and Bax protein and Caspase-3 activity in PC12.

Taurine Alleviate Hexabromocyclododecane-Induced Cytotoxicity in PC12 Cells via Inhibiting Oxidative Stress.

Hexabromocyclododecane (HBCD) is a widely used brominated flame retardant. Its adverse effects on brain had been observed. Taurine, a sulfur amino acid, take part in many brain physiological functions and exhibits protective effects on a variety of detrimental situations. In this paper, we explored the protections of taurine on cytotoxicity induced by HBCD in PC12 cells. PC12 cells were pretreated with taurine (1 mM, 3 mM and 9 mM) for 30 min before 10 µM HBCD treatment for 24 h. Then, the cell survival was assayed by the lactate dehydrogenase (LDH) release and trypan blue dyeing method. The formation of reactive oxygen species (ROS) and a collapse of mitochondrial membrane potential (MMP) were evaluated with a fluorescence microplate reader using the non-fluorescent probe 2'7'-dichlorofluorescin diacetate (DCFH-DA) and the fluorescent cationic dyestuff Rhodamine 123 (Rh 123), respectively. Further, the activity of many antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and the content of glutathione (GSH) were tested by kits. Our results displayed that taurine significantly decreased the cell death induced by HBCD, prevented ROS production and disruption of mitochondrial membrane potential, and reversed the decline of SOD, CAT, GPx activity and GSH content induced by HBCD. These results suggested that taurine could alleviate cytotoxicity induced by HBCD in PC12 cells through inhibition of oxidative stress.

Effects of Taurine on Alterations of Neurobehavior and Neurodevelopment Key Proteins Expression in Infant Rats by Exposure to Hexabromocyclododecane.

Hexabromocyclododecanes (HBCDs) is a widely used flame retardant. Studies have found that HBCDs has toxic effects on endocrine and neural development, leading to adverse effects on behavior, learning and memory. This study aimed to investigate the protective effects of taurine on cognitive function, neurotrophic factors expression of infant rats exposured to HBCDs. Sprague-Dawley rats of 10-days old were oral gavaged of different doses (0.3, 3 and 30 mg/kg) of HBCDs and 30 mg/kg HBCDs with 300 mg/kg taurine for 60 consecutive days. Rat cognitive function was detected by the method of Morris water maze test. The protein expressions of brain derived neurotrophic factor (BDNF), nerve growth factor (NGF) and fibroblast growth factor (FGF) were assayed by Western-blotting. Results showed that rats exposed to HBCDs significantly declined rats spatial learning and memory ability by increasing the latency time of seeking the platform (P < 0.05), decreasing the numbers that each rat had crossed the non-exits and the time spent in the target quadrant as compared with those in control rats (P < 0.05). Taurine treatment significantly reversed the effects of HBCDs. Western-blotting results showed that expression of BDNF, NGF and FGF proteins in the low dose group were obviously increased compared with those in control rats (P < 0.01), and middle-dose and high dose groups significantly decreased. Taurine treatment increased BDNF and NGF expression as compared with high dose groups while Taurine seemed to have no effects on FGF. These result suggested that higher doses of HBCDs early exposure in the developing rats could decrease neurotrophic factors including BDNF, NGF, FGF, which have an impact on neural development, damage on learning and memory.

Rate of hexabromocyclododecane decomposition and production of brominated polycyclic aromatic hydrocarbons during combustion in a pilot-scale incinerator.

Here, we examined the incineration of extruded polystyrene containing hexabromocyclododecane (HBCD) in a pilot-scale incinerator under various combustion temperatures (800-950°C) and flue gas residence times (2-8sec). Rates of HBCD decomposition ranged from 99.996% (800°C, 2sec) to 99.9999% (950°C, 8sec); the decomposition of HBCD, except during the initial stage of combustion (flue gas residence time<2sec), followed a pseudo-first-order kinetics model. An Arrhenius plot revealed that the activation energy and frequency factor of the decomposition of HBCD by combustion were 14.2kJ/mol and 1.69sec-1, respectively. During combustion, 11 brominated polycyclic aromatic hydrocarbons (BrPAHs) were detected as unintentional by-products. Of the 11 BrPAHs detected, 2-bromoanthracene and 1-bromopyrene were detected at the highest concentrations. The mutagenic and carcinogenic BrPAHs 1,5-dibromoanthracene and 1-bromopyrene were most frequently detected in the flue gases analyzed. The total concentration of BrPAHs exponentially increased (range, 87.8-2,040,000ng/m3) with increasing flue gas residence time. Results from a qualitative analysis using gas chromatography/high-resolution mass spectrometry suggest that bromofluorene and bromopyrene (or fluoranthene) congeners were also produced during the combustion.

Accumulation and phytotoxicity of technical hexabromocyclododecane in maize.

To investigate the accumulation and phytotoxicity of technical hexabromocyclododecane (HBCD) in maize, young seedlings were exposed to solutions of technical HBCD at different concentrations. The uptake kinetics showed that the HBCD concentration reached an apparent equilibrium within 96hr, and the accumulation was much higher in roots than in shoots. HBCD accumulation in maize had a positive linear correlation with the exposure concentration. The accumulation of different diastereoisomers followed the order γ-HBCD>ß-HBCD>α-HBCD. Compared with their proportions in the technical HBCD exposure solution, the diastereoisomer contribution increased for ß-HBCD and decreased for γ-HBCD in both maize roots and shoots with exposure time, whereas the contribution of α-HBCD increased in roots and decreased in shoots throughout the experimental period. These results suggest the diastereomer-specific accumulation and translocation of HBCD in maize. Inhibitory effects of HBCD on the early development of maize followed the order of germination rate>root biomass≥root elongation>shoot biomass≥shoot elongation. Hydroxyl radical (OH) and histone H2AX phosphorylation (γ-H2AX) were induced in maize by HBCD exposure, indicative of the generation of oxidative stress and DNA double-strand breaks in maize. An OH scavenger inhibited the expression of γ-H2AX foci in both maize roots and shoots, which suggests the involvement of OH generation in the HBCD-induced DNA damage. The results of this study will offer useful information for a more comprehensive assessment of the environmental behavior and toxicity of technical HBCD.

Destruction of the flame retardant hexabromocyclododecane in a full-scale municipal solid waste incinerator.

Hexabromocyclododecane containing polystyrene foam obtained from the building and construction market has been co-incinerated in a full-scale waste incineration plant. The co-feeding of 1 and 2 wt% of polystyrene foam had no influence on the operation of the plant. The bromine content increased the raw gas hydrogen bromide concentration slightly. The air emission, including that of dioxins and bromine, was not altered and so was the quality of the solid residues. The hexabromocyclododecane concentrations in the solid residues were almost identical, regardless of whether or not and how much polystyrene foam was added. The obtained destruction efficiency was >99.999% independent of the amount of added polystyrene foam. This finding indicates a virtually total destruction of hexabromocyclododecane.