Toxicity of emerging antifouling biocides to non-target freshwater organisms from three trophic levels.

Antifouling (AF) systems provide the most cost-effective protection against biofouling. Several AF biocides have, however, caused deleterious effects in the environment. Subsequently, new compounds have emerged that claim to be more environment-friendly, but studies on their toxicity and environmental risk are necessary in order to ensure safety. This work aimed to assess the toxicity of three emerging AF biocides, tralopyril, triphenylborane pyridine (TPBP) and capsaicin, towards non-target freshwater organisms representing three trophic levels: algae (Chlamydomonas reinhardtii), crustacean (Daphnia magna) and fish (Danio rerio). From the three tested biocides, tralopyril had the strongest inhibitory effect on C. reinhardtii growth, effective quantum yield and adenosine triphosphate (ATP) content. TPBP caused sub-lethal effects at high concentrations (100 and 250µgL-1), and capsaicin had no significant effects on algae. In the D. magna acute immobilisation test, the most toxic compound was TPBP. However, tralopyril has a short half-life and quickly degrades in water. With exposure solution renewals, tralopyril's toxicity was similar to TPBP. Capsaicin did not cause any effects on daphnids. In the zebrafish embryo toxicity test (zFET) the most toxic compound was tralopyril with a 120h - LC50 of 5µgL-1. TPBP's 120h - LC50 was 447.5µgL-1. Capsaicin did not cause mortality in zebrafish up to 1mgL-1. Sub-lethal effects on the proteome of zebrafish embryos were analysed for tralopyril and TPBP. Both general stress-related and compound-specific protein changes were observed. Five proteins involved in energy metabolism, eye structure and cell differentiation were commonly regulated by both compounds. Tralopyril specifically induced the upregulation of 6 proteins implicated in energy metabolism, cytoskeleton, cell division and mRNA splicing whilst TPBP lead to the upregulation of 3 proteins involved in cytoskeleton, cell growth and protein folding. An ecological risk characterization was performed for a hypothetical freshwater marina. This analysis identified capsaicin as an environment-friendly compound while tralopyril and TPBP seem to pose a risk to freshwater ecosystems. Noneless, more studies on the characterization of the toxicity, behaviour and fate of these AF biocides in the environment are necessary since this information directly affects the outcome of the risk assessment.

Acute toxicity of tralopyril, capsaicin and triphenylborane pyridine to marine invertebrates.

A need for environmentally acceptable alternative antifouling (AF) biocides has arisen through restrictions in the use of many common biocides in the European Union through the Biocidal Product Regulation (Regulation EU No. 528/2012). Three such alternatives are triphenylborane pyridine (TPBP), tralopyril and capsaicin. This study aims at extending the available information on the toxicity of these three emerging AF biocides to key marine invertebrates. Here we investigate the toxicity of tralopyril and capsaicin to the early life stages of the mussel Mytilus galloprovincialis and the sea urchin Paracentrotus lividus and also of tralopyril, capsaicin and TPBP to the early life stages of the copepod Tisbe battagliai. The EC50 that causes abnormal development of mussel's D-veliger larvae and impairs the growth of sea urchin pluteus larvae are respectively 3.1 and 3.0 µg/L for tralopyril and 3,868 and 5,248 µg/L for capsaicin. Regarding the copepod T. battagliai, the LC50 was 0.9 µg/L for tralopyril, 1,252 µg/L for capsaicin and 14 µg/L for TPBP. The results obtained for the three substances are compared to a reference AF biocide, tributyltin (TBT), and their ecological risk evaluated. These compounds pose a lower environmental risk than TBT but still, our results suggest that tralopyril and TPBP may represent a considerable threat to the ecosystems.

Toxicity of degradation products of the antifouling biocide pyridine triphenylborane to marine organisms.

We evaluated the acute toxicities of the main degradation products of pyridine triphenylborane (PTPB), namely, diphenylborane hydroxide (DPB), phenylborane dihydroxide (MPB), phenol, and biphenyl, to the alga Skeletonema costatum, the crustacean Tigriopus japonicus, and two teleosts, the red sea bream Pagrus major and the mummichog Fundulus heteroclitus. DPB was the most toxic of the degradation products to all four organisms. The acute toxicity values of DPB for S. costatum, T. japonicus, red sea bream, and mummichog were 55, 70, 100, and 200-310 µg/L, respectively. The degradation products were less toxic than PTPB to S. costatum and T. japonicus; however, the toxicities of DPB and PTPB to the fish species were similar. We also examined changes in the inhibition of growth rate of S. costatum as well as the percentage of immobilization of T. japonicus as end points of toxicity of PTPB after irradiation of PTPB with 432 ± 45 W/m(2) of 290-700 nm wavelength light. After 7 days of irradiation with this light, the concentration of PTPB in the test solutions decreased markedly. A decrease in toxic effects closely coincided with the decrease in the concentration of PTPB caused by the irradiation. PTPB probably accounted for most of the toxicity in the irradiation test solutions. Because the concentrations of PTPB that were acutely toxic to S. costatum and T. japonicus were <10 % of the corresponding concentrations of its degradation products, PTPB probably accounted for most of the toxicity in the irradiation test solutions.

Effects of five antifouling biocides on settlement and growth of zoospores from the marine macroalga Ulva lactuca L.

Antifouling biocides are found in the marine ecosystem were they can affect non-target organisms. In this study the effects of five antifouling biocides on the settlement and growth of Ulva lactuca zoospores were investigated. The biocides investigated were copper (Cu(2+)), 4,5-dichloro-2-n-octyl-3(2H)-isothiazolone (DCOIT), triphenylborane pyridine (TPBP), tolylfluanid and medetomidine. Full concentration-response curves where determined for each compound. EC50 values were determined for copper, DCOIT, TPBP and tolylfluanid, all of which inhibited settlement and growth in a concentration dependent manner with the following toxicity ranking; tolylfluanid (EC50 80 nmol L(-1)) ~ DCOIT (EC50 83 nmol L(-1)) > TPBP (EC50 400 nmol L(-1)) > Cu(2+) (EC50 2,000 nmol L(-1)). Medetomidine inhibited settlement and growth only at the extreme concentration of 100,000 nmol L(-1) (93% effect). The low toxicity is possibly a consequence of a lack of receptors that medetomidine can bind to in the U. lactuca zoospores.

An assessment of the genetic toxicology of novel boron-containing therapeutic agents.

Boron-containing compounds are being studied as potential therapeutic agents. As part of the safety assessment of these therapeutic agents, a battery of genetic toxicology studies was conducted. The battery included a bacterial reverse mutation (Ames) assay, an in vitro chromosome aberration assay in peripheral human lymphocytes, and an in vivo rat micronucleus study. The following compounds represent some of the boron-containing compounds that have been advanced to human clinical trials in various therapeutic areas. The borinic picolinate, AN0128, is an antibacterial compound with anti-inflammatory activity that has been studied in clinical trials for acne and the treatment of mild to moderate atopic dermatitis. AN2690 (tavaborole) is a benzoxaborole in Phase 3 clinical trials for the topical treatment of onychomycosis, a fungal infection of the toenails and fingernails. Another benzoxaborole derivative, AN2728, a phosphodiesterase-4 (PDE4) inhibitor, is in Phase 2 clinical trials for the treatment of atopic dermatitis. AN2898, also a PDE4 inhibitor, has been studied in clinical trials for atopic dermatitis and psoriasis. AN3365 is a leucyl-tRNA synthetase inhibitor that has been in clinical development for the treatment of various Gram-negative bacterial infections. These five representative compounds were negative in the three genotoxicity assays. Furthermore, AN2690 has been studied in mouse and rat 2-year bioassays and was not found to have any carcinogenic potential. These results demonstrate that it is possible to design boron-based therapeutic agents with no genetic toxicology liabilities.

Use of species sensitivity distributions to predict no-effect concentrations of an antifouling biocide, pyridine triphenylborane, for marine organisms.

We used species sensitivity distributions (SSDs) and a Bayesian statistical model to carry out a primary risk assessment for pyridine triphenylborane (PTPB) in Hiroshima Bay, Japan. We used SSDs derived from toxicity values, such as EC50 and LC50, obtained from this study and previous work to calculate hazardous concentrations that should protect 95% and 99% of species (HC5 and HC1) and demonstrated that the medians of the HC5 and HC1 were 0.78 and 0.17 µg/L, respectively. We also used liquid chromatography/mass spectrometry to investigate the occurrence of PTPB in seawater from several coastal sites of Hiroshima Bay and detected PTPB at concentrations of 4.8-21 pg/L. Comparison of environmental concentrations to the HC values suggests that the current ecological risk posed by PTPB in Hiroshima Bay is low. This is the first report of the detection of PTPB in the natural marine environment.

Intensely fluorescent azobenzenes: synthesis, crystal structures, effects of substituents, and application to fluorescent vital stain.

2-[Bis(pentafluorophenyl)boryl]azobenzenes bearing hydrogen, methoxy, dimethylamino, trifluoromethyl, fluoro, n-butyl, and tert-butyldimethylsiloxy groups at the 4'-position or methoxy and bromo groups at the 4-position have been synthesized. The 4-bromo group of the 2-boryl-4-bromoazobenzene derivative was converted to phenyl and diphenylamino groups by palladium-catalyzed reactions. The absorption and fluorescence properties have been investigated using UV/Vis and fluorescence spectroscopy. The 2-borylazobenzenes emitted an intense green, yellow, and orange fluorescence, in marked contrast to the usual azobenzene fluorescence. The 4'-siloxy derivative showed the highest fluorescence quantum yield (0.90) among those reported for azobenzenes to date. The correlation between the substituent and the fluorescence properties was elucidated by studying the effect of the substituent on the relaxation process and from DFT and TD-DFT calculations. An electron-donating group at the 4'-position was found to be important for an intense emission. Application of fluorescent azobenzenes as a fluorescent vital stain for the visualization of living tissues was also investigated by microinjection into Xenopus embryos, suggesting these compounds are nontoxic towards embryos.

Ecotoxicity of the degradation products of triphenylborane pyridine (TPBP) antifouling agent.

Triphenylborane pyridine (TPBP) is an alternative to organotin antifouling compounds. This work aimed to identify the unknown Peak #1, and to evaluate the ecotoxicity of TPBP and its degradation products. Peak #1 was produced from TPBP dissolved in acetonitrile under UV-A photolysis using a high-pressure mercury lamp. The Peak #1 fraction was purified using two-step column chromatography from a TPBP-acetonitrile solution. The major compound of the fraction was identified as being biphenyl from the 1H NMR and 13C NMR spectra. The ecotoxicity of four degradation products (diphenylborane hydroxide, phenylborane dihydroxide, phenol, and biphenyl) and TPBP towards two marine planktons were assessed. The 48 h LC(50) values of the crustacean, Artemia salina, were 0.13 mg L(-1) for TPBP, 14 mg L(-1) for biphenyl, 17 mg L(-1) for phenol, and > 50 mg L(-1) for the other degradation products. The 72 h EC(50) values of the diatom, Skeletonema costatum, were 0.0022 mg L(-1) for TPBP, 1.2 mg L(-1) for biphenyl, and > 2 mg L(-1) for the other degradation products. Thus, the ecotoxicity of biphenyl and the other degradation products were not high compared to the parent compound, TPBP.

Respiratory irritation associated with inhalation of boron trifluoride and fluorosulfonic acid.

The objectives of this study were to examine the respiratory irritancy of boron trifluoride (BF(3)) and fluorosulfonic acid (FSA) following acute inhalation exposure. Testing was conducted using groups of 10 male and 10 female rats (BF(3)) or groups of 6 male rats (FSA). Rats were exposed for a single 4-h period (BF(3)) or a single 1-h period (FSA) and necropsied 1 or 14 days after exposure (BF(3)) or 14 days after exposure (FSA). Measurements consisted of clinical signs, body weight, kidney and lung weight, histopathology (BF(3)), and breathing parameters (FSA) and were used to evaluate the possible irritating effects of these compounds. The results indicated treatment-related findings in the larynx and trachea in the rats exposed to 74.4 mg/m(3) BF(3), consisting of ventral cartilage necrosis, hemorrhage, and an increase in ventral epithelial hyperplasia and ventral inflammatory cell inflammation 24 h postexposure. In the animals sacrificed 14 days postexposure, the only notable observation was ventral cartilage necrosis, present in 2 animals. The next lower level tested, 24.6 mg/m(3) BF, was considered a no-observed-adverse-effects level (NOAEL). A concentration of 4125 mg/m(3) FSA resulted in a clearly decreased breathing rate during and shortly after exposure with 67% (4/6) mortality on days 5-9 after exposure. A concentration of 845 mg/m(3) FSA resulted in only minor signs of irritation, consisting of slight changes in breathing pattern shorlty after exposure. The results of the present 4-h inhalation study with BF(3) indicated that respiratory irritation was present at a level of 74.4 mg/m(3) whereas 24.6 mg/m(3) was a NOAEL. A single 1-h exposure to 845 mg/m(3) FSA resulted in only minor signs of respiratory irritation, indicating that on a mass basis FSA is no more toxic or irritating than hydrogen fluoride (HF) or sulfuric acid.

Effect of occupational safety measures on micronucleus frequency in semiconductor workers.

OBJECTIVES: To examine whether semiconductor workers exposed to complex mixtures of chemical waste show an increase in genotoxic effects, and, if so, whether occupational safety measures protect these workers. METHODS: To assess chemical exposure in the workplace, air monitoring of boron trifluoride and boron trichloride was performed and urinary concentrations of fluoride were measured. The cytokinesis-block micronucleus test on isolated lymphocytes was used for the detection of genotoxic effects. Two series of monitoring have been performed in order to assess the effect of implemented protection measures. RESULTS: We found a significantly higher mean frequency of micronuclei in exposed workers than in controls, whereas air monitoring and measurement of urinary fluoride failed to detect chemical exposure of these workers. Twelve years after implementation of protective measures, the mean level of micronuclei in exposed individuals was found to be as low as those from controls. CONCLUSIONS: These findings indicate that exposed workers in the semiconductor industry may have an increased risk of genotoxic effects from complex mixtures of chemical waste products. The decline of the mean level of micronuclei in exposed workers down to the base level of controls after implementation of protective measures points to the significance of adequate safety standards to protect against chromosomal damage in semiconductor personnel.

Synthesis and comparative toxicology of a series of polyhedral borane anion-substituted tetraphenyl porphyrins.

Three structurally similar tetraphenylporphyrins bearing polyhedral borane anions have been synthesized and their toxicological profiles obtained in rats. These conjugates were found to have quite different acute toxicities as manifested at the maximum tolerated dose (MTD). When given at the MTD and observed over 28 days, the most acutely toxic porphyrin was found to be devoid of toxicity, as measured by blood chemistry panels. The remaining two less acutely toxic compounds both elicited significant changes, characterized by moderate to severe thrombocytopenia, failure to gain weight normally and changes in liver enzymes indicative of mild hepatotoxicity. All toxic effects were transient, with platelets rebounding to above normal levels at day 28. We conclude that thrombocytopenia is the dose limiting toxicity for boronated porphyrins in mammals and suggest that these effects may be due to the porphyrin, not the borane or carborane.

Synthesis and antifungal activity of a novel series of alkyldimethylamine cyanoboranes and their derivatives.

A series of new amine cyanoborane derivatives were synthesized and exhibited antifungal activity. A long alkyl chain attached to the nitrogen of the amine cyanoboranes and carboxyboranes enhances antifungal activity. An enhanced activity was also obtained upon the halogenation of the amine cyanoboranes as well as in the presence of C=C double bond at the end of the N-alkyl group. The lead compounds were dimethylundecylamine cyanoborane (C11H23N(CH3)2BH2CN), 9, and its dibromo derivative dimethylundecylamine dibromocyanoborane (C11H23N(CH3)2BBr2CN), 11. The MIC values for the lead compounds against the most important human pathogenic fungi ranged from 16.25 to 32.5 micromol/L and from 10.05 to 79 micromol/L, respectively. Both compounds were found to be relatively safe in intravenous injections to mice, (MTD = 121.9 and 73.1 micromol/kg, respectively) and active against strains that are resistant to fluconazole (a conventional antifungal medicine). These data indicate their potential to become antifungal agents.

Azanonaboranes containing imidazole derivatives for boron neutron capture therapy: synthesis, characterization, and in vitro toxicity evaluation.

A number of azanonaboranes containing imidazole derivatives have been synthesized by a ligand-exchange reaction. The exo-NH(2)R group of the azanonaborane of the type [(RH(2)N)B(8)H(11)NHR] can be exchanged by one hetero-nitrogen atom of the imidazole ring. In the case of histamine, the exchange takes place on the aliphatic amino group, the hetero-nitrogen atom of the imidazole ring or both of them. The products were confirmed by NMR, IR spectroscopy, elemental analysis, and mass spectrometry. The electron-withdrawing effect of the nitro group in 2-nitroimidazole is the main hindrance to achieve the exchange reaction. In vitro experiments were performed with B16 melanoma cells. A comparison of the biological properties of the products in which the B(8)N cluster is connected to the hetero-nitrogen atom of imidazole ring or the aliphatic NH(2) group showed that incorporation of B(8)N cluster unit into primary amino group increases the compound's toxicity. In contrast, this specificity for cytotoxicity effect was not observed in the case of histamine containing two B(8)N clusters which was relatively nontoxic and did not inhibit colony formation up to concentrations of 2 mM.

Unilamellar liposomes with enhanced boron content.

A new type of boron-rich, DSPC-free, unilamellar liposomes was formed using the novel dual-chain, ionic, nido-carborane lipid, K[nido-7-(C16H33OCH2)2CHOCH2-7,8-C2B9H11] (DAC-16), and cholesterol for encapsulation of an aqueous buffer core. Since DSPC was not necessary for the formation of stable DAC-16 liposomes, the boron concentration of these vesicles was increased dramatically to approximately 8.8 wt % in the dry lipid; these liposomes had a high bilayer boron incorporation efficiency of 98%. DSPC-free liposomes exhibited a size distribution pattern of 40-60 nm, which was in the range normally associated with selective tumor uptake. This size distribution was maintained throughout storage at room temperature for several months. Additionally, optimized liposome formulations incorporating DAC-16, DSPC, and cholesterol were identified having stable size distribution patterns after storage for more than two months at a variety of temperatures. Although animal studies indicate that DAC-16 liposomes are toxic, this new ionic nido-carborane lipid allows the formation of liposomes of high boron content for in vitro applications that require the delivery of large amounts of boron.

Case report: the clinical toxicity of dimethylamine borane.

CONTEXT: Dimethylamine borane (DMAB) is a reducing agent used in nonelectric plating of semiconductors. Exposures are usually through occupational contact. We report here four cases of people who suffered from work-related exposure to DMAB. CASE PRESENTATION: Three patients exposed to DMAB decontaminated immediately by drinking a lot of water; they reported dizziness, nausea, diarrhea 6-8 hr later. The other patient did not decontaminate at once, and he suffered from more severe symptoms, including dizziness, nausea, limb numbness, slurred speech, irritable mood, and ataxia 13 hr later. Magnetic resonance imaging showed symmetric lesions with hyperintensity on T2WI and FLAIR in bilateral cerebellar dantate nuclei. This patient was readmitted to the hospital due to difficulty in walking and climbing 18 days after exposure. Lower leg weakness and drop foot were found bilaterally. A nerve conduction study revealed polyneuropathy with motor-predominant axonal degeneration. This patient receives regular outpatient followups and still walks with a clumsy gait and has difficulty with hand-grasping activity. DISCUSSION: This case study demonstrates that DMAB is highly toxic to humans through any route of exposure, and dermal absorption is the major route of neurotoxicity. DMAB induces acute cortical and cerebellar injuries and delayed peripheral neuropathy. RELEVANCE: Further investigation of the toxic mechanism of DMAB is warranted. Early decontamination with copious water is the best current treatment for exposure to DMAB.

Synthesis and cellular studies of porphyrin-cobaltacarborane conjugates.

The total syntheses of five new porphyrin-cobaltacarborane conjugates (1-5) have been achieved in 88-98% yields in a single-step reaction between a nucleophilic meso-pyridyl-containing porphyrin and zwitterionic cobaltacarborane [3,3'-Co(8-C(4)H(8)O(2)-1,2-C(2)B(9)H(10))(1',2'-C(2)B(9)H(11))]. These unique zwitterionic compounds have one to four cobaltabisdicarbollide anions conjugated to the porphyrin macrocycle via (CH(2)CH(2)O)(2) chains. The X-ray structure of one of these conjugates (1) is presented and discussed. The cellular uptake, cytotoxicity, and subcellular localization of cobaltacarboraneporphyrins 1-5 were investigated in human HEp2 cells. The number and distribution of cobaltacarborane residues linked to the porphyrin macrocycle has a significant effect on the cellular uptake of the conjugates.

Synthesis of (aminoalkylamine)-N-aminoalkyl)azanonaborane(11) derivatives for boron neutron capture therapy.

New boron-containing polyamine have been synthesized: (aminoalkylamine)-N-(aminoalkyl)azanonaborane(11) derivatives [H(2)N(CH(2))(n)H(2)NB(8)H(11)NH(CH(2))(n)NH(2)], where n = 4-6 and 12, and [H(2)N(CH(2))(3)H(2)NB(8)H(11)NH(CH(2))(4)NH(2)]. (4-Aminobutylamine)-N-(4-aminobutyl)azanonaborane and (3-aminopropylamine)-N-(4-aminobutyl)azanonaborane were less toxic in vitro (LD(50) of approximately 700 and approximately 1100 microM, respectively) than spermine, while (4-aminobutylamine)-N-isopropylazanonaborane with its hydrophobic isopropyl group and those with n = 5, 6, and 12 were already toxic under similar conditions (LD(50) << 500 microM). These compounds may be useful as delivery agents for boron neutron capture therapy.

Comparative in vitro evaluation of dequalinium B, a new boron carrier for neutron capture therapy (NCT).

A boronated derivative of dequalinium, a delocalized lipophilic cation (DLC), was synthesized as a potential boron carrier for the selective targeting of mitochondria in malignant versus benign cells for boron neutron capture therapy (BNCT), a binary modality for the treatment of cancer. This agent, designated DEQ-B, was taken up and retained in vitro in the KB, F98, and C6 tumor cell lines but not in the normal epithelial cell line CV1. DEQ-B was also less toxic in the latter cell line at lower exposure concentrations The uptake, retention, and toxicity profiles of DEQ-B are comparable to those of the non-boronated DLCs, dequalinium, MKT 077, RH 123, and tetraphenylphosphonium chloride. Our results suggest that the synthesis and further evaluation of boronated DLCs as potential delivery agents for BNCT is warranted.

Uptake, toxicity and radiation effects of the boron compounds DAAC-1 and DAC-1 in cultured human glioma cells.

PURPOSE: To study the uptake, toxicity and radiation effects in vitro of a diol-amino acid-carborane (DAAC-1) and make comparisons with the previously studied diol-amine-carborane (DAC-1). MATERIALS AND METHODS: Toxicity and radiation effects were studied with clonogenic survival, uptake by measuring the cellular boron content and the subcellular distribution was investigated after organelle separation with centrifugation. The studied cell line was human glioma U343. RESULTS: DAAC-1 showed an accumulation of 1-1.5 times, compared with the culture medium, and was non-toxic up to 47 microg boron/ml. The accumulation of DAC-1 was about 90 times, but toxic effects were detectable already at the concentration 5 microg boron/ml. None of the compounds was localized in the cell nucleus. Following irradiation with thermal neutrons, DAC-1 was about 2.5 times more effective than DAAC-1 and about 4.9 times more effective than neutrons alone, at the survival level 0.2. The dose modifying factors, when compared with the neutron beam alone, were for both DAAC-1 and DAC-1 about 1.5 and about 5 when compared with 60Co-gamma-radiation. CONCLUSIONS: DAAC-1 was less toxic than DAC-1 but gave less accumulation of boron. Both substances gave significant boron-dependent cell inactivation when the test cells were exposed to thermal neutrons.