Andrew J. Harford

Independent cytotoxic and inflammatory responses to zinc oxide nanoparticles in human monocytes and macrophages

Abstract Significant public and scientific concerns remain for the use of nanoparticles (NPs) in commercial products, particularly those applied topically for skin care. There are currently a range of metal oxides formulated into many sunscreens that are present at the nanoscale. In this study, we sought to determine the effect of the size and dispersion of one type of these NPs (zinc oxide) on immune cell function and cytotoxicity for human macrophages and monocytes, which are key cells for particle and debris clearance in the skin. We have found that particle size and coating, but surprisingly, not agglomeration, are key determinates of nanoparticle cytotoxicity in an in vitro culture system of human immune cells. Most importantly, we found that this nanoparticle-induced cellular immune signalling, can be decoupled from cytotoxicity and surface coating, so that at an equivalent cytotoxic load, smaller particles induce a greater cellular response.

Effects of alumina refinery wastewater and signature metal constituents at the upper thermal tolerance of: 2. The early life stages of the coral Acropora tenuis.

The success of early life history transitions of the coral Acropora tenuis were used as endpoints to evaluate thermal stress and the effects of wastewater discharged to a tropical marine environment. The studies assessed the effects of: (i) temperature; (ii) three signature metals of the wastewater, aluminium (Al), vanadium (V) and gallium (Ga); and (iii) the wastewater (at 27°C and 32°C) on fertilisation and larval metamorphosis. The median inhibition temperatures for fertilisation and metamorphosis were 32.8°C and 33.0°C, respectively. Fertilisation IC(50)s for Al, V and Ga were 2997, 2884 and 3430 μg L(-1), respectively. Metamorphosis IC(50)s for Al, V and Ga were 1945, 675 and 3566 μg L(-1), respectively. The wastewater only affected fertilisation and metamorphosis at moderate concentrations (IC(50)s=63% and 67%, v/v, respectively, at 27°C), posing a low risk to this species in the field. The effects of wastewater and temperature on fertilisation and metamorphosis were additive.

Cytochemical characterisation of the leucocytes and thrombocytes from Murray cod (Maccullochella peelii peelii, Mitchell)

Cytochemistry has proven effective in differentiating specific cell lineages and elucidating their functional properties. This study utilised a range of cytochemical techniques to further investigate the leucocyte populations from Murray cod, an iconic Australian teleost fish species. This analysis provided clear insight into the structure and function of the leucocytes from this fish, which were found to be broadly similar to those of other fish species. However, some important differences were identified in Murray cod, such as the presence of naphthol AS chloroacetate esterase activity in the heterophil population, positive staining for periodic acid Schiffs, alkaline phosphatase and Sudan black B in the lymphocyte population, and a prevalent population of myeloid precursor cells.

Effect of in vitro and in vivo organotin exposures on the immune functions of Murray Cod (Maccullochella peelii peelii)

Murray cod (Maccullochella peelii peelii) is an iconic native Australian freshwater fish and an ideal species for ecotoxicological testing of environmental pollutants. The species is indigenous to the Murray-Darling basin, which is the largest river system in Australia but also the ultimate sink for many environmental pollutants. The organotins tributyltin (TBT) and dibutyltin (DBT) are common pollutants of both freshwater and marine environments and are also known for their immunotoxicity in both mammals and aquatic organisms. In this study, TBT and DBT were used as exemplar immunotoxins to assess the efficiency of immune function assays (i.e., mitogen-stimulated lymphoproliferation, phagocytosis in head kidney tissue, and serum lysozyme activity) and to compare the sensitivity of Murray cod to other fish species. The organotins were lethal to Murray cod at concentrations previously reported as sublethal in rainbow trout (i.e., intraperitoneal [i.p.] lethal dose to 75% of the Murray cod [LD75] = 2.5 mg/kg DBT and i.p. lethal dose to 100% of the Murray cod [LD100] = 12.5 mg/kg TBT and DBT). In vivo TBT exposure at 0.1 and 0.5 mg/kg stimulated the phagocytic function of Murray cod (F = 6.89, df = 18, p = 0.004), while the highest concentration of 2.5 mg/kg TBT decreased lymphocyte numbers (F = 7.92, df = 18, p = 0.02) and mitogenesis (F = 3.66, df = 18, p = 0.035). Dibutyltin was the more potent immunosuppressant in Murray cod, causing significant reductions in phagocytic activity (F = 5.34, df = 16, p = 0.013) and lymphocyte numbers (F = 10.63, df = 16, p = 0.001).

Toxicity and metal speciation characterisation of waste water from an abandoned gold mine in tropical northern Australia

The decommissioned Mount Todd gold mine, located in the wet-dry tropics of northern Australia, consists of a large waste water inventory and an acid rock drainage problem, which has the potential to impact upon freshwater ecosystems of the Edith River catchment. The toxicity of retention pond 1 (RP1) water was determined using six local freshwater species (duckweed, alga, cladoceran, snail, hydra and a fish). RP1 water was very toxic to all species, with the percentage dilution of RP1 water inhibiting 10% of organism response (IC10), or lethal to 5% of individuals (LC5), ranging from 0.007 to 0.088%. The percentage dilution of RP1 water inhibiting 50% of organism response (IC50), or lethal to 50% of individuals (LC50), ranged from 0.051% to 0.58%. Based on chemical analyses and geochemical speciation modelling of the test waters, Cu, Zn and Al were the most likely toxic components at acidic dilutions (i.e. > or =1%), while Cu and Zn were the most likely toxic components at 0.1% RP1 water, where pH was 6.5. Species sensitivity distributions (SSDs) were used to predict dilutions of RP1 water that would protect or unacceptably affect the downstream aquatic ecosystems. A dilution ratio of 1 part RP1 water to 20000 parts Edith River water (0.005% RP1 water) was calculated to be required for the protection of at least 95% of species. This information can be used in conjunction with field chemical and biological data to better predict the ecological risks of RP1 waste water downstream of the Mount Todd mine.

Aluminium, gallium, and molybdenum toxicity to the tropical marine microalga Isochrysis galbana

There is a shortage of established chronic toxicity test methods for assessing the toxicity of contaminants to tropical marine organisms. The authors tested the suitability of the tropical microalga Isochrysis galbana for use in routine ecotoxicology and assessed the effects of 72-h exposures to copper (Cu, a reference toxicant), aluminium (Al), gallium (Ga), and molybdenum (Mo), key metals of alumina refinery discharge, on the growth of I. galbana at 3 temperatures: 24 °C, 28 °C, and 31 °C. The sensitivity of both I. galbana and the test method was validated by the response to Cu exposure, with 10% and 50% effect concentrations (EC10 and EC50) of 2.5 μg/L and 18 μg/L, respectively. The EC10 and EC50 values for total Al at 28 °C were 640 μg/L and 3045 μg/L, respectively. The toxicity of both Cu and Al at 24 °C and 31 °C was similar to that at 28 °C. There was no measurable toxicity from dissolved Ga exposures of up to 6000 μg/L or exposures to dissolved Mo of up to 9500 μg/L. Solubility limits at 28 °C for the dissolved fractions (<10 kDa) of Al, Ga, and Mo were approximately 650 μg/L Al, >7000 μg/L Ga, and >6000 μg/L Mo. In test solutions containing >650 μg/L total Al, dissolved and precipitated forms of Al were present, with precipitated Al becoming more dominant as total Al increased. The test method proved suitable for routine ecotoxicology, with I. galbana showing sensitivity to Cu but Al, Ga, and Mo exhibiting little to no toxicity to this species.

Manganese toxicity to tropical freshwater species in low hardness water

Elevated manganese (Mn) is a common contaminant issue for mine water discharges, and previous studies have reported that its toxicity is ameliorated by H(+), Ca(2+), and Mg(2+) ions. In the present study, the toxicity of Mn was assessed in a high risk scenario, that is, the slightly acidic, soft waters of Magela Creek, Kakadu National Park, Northern Territory, Australia. Toxicity estimates were derived for 6 tropical freshwater species (Chlorella sp., Lemna aequinoctialis, Amerianna cumingi, Moinodaphnia macleayi, Hydra viridissima, and Mogurnda mogurnda). Low effect chronic inhibition concentration (IC10) and acute lethal concentration (LC05) values ranged between 140 μg L(-1) and 80,000 μg L(-1), with 3 of the species tested (M. macleayi, A. cumingi, and H. viridissima) being more sensitive to Mn than all but 1 species in the international literature (Hyalella azteca). A loss of Mn was observed on the final day for 2 of the H. viridissima toxicity tests, which may be a result of the complex speciation of Mn and biological oxidation. International data from toxicity tests conducted in natural water with a similar physicochemistry to Magela Creek water were combined with the present studys data to increase the sample size to produce a more reliable species sensitivity distribution. A 99% protection guideline value of 73 μg L(-1) (33-466 μg L(-1)) was derived; the low value of this guideline value reflects the higher toxicity of Mn in slightly acidic soft waters.

A novel bioassay using the barnacle Amphibalanus amphitrite to evaluate chronic effects of aluminium, gallium and molybdenum in tropical marine receiving environments

A need exists for appropriate tools to evaluate risk and monitor potential effects of contaminants in tropical marine environments, as currently impact assessments are conducted by non-representative approaches. Here, a novel bioassay is presented that allows for the estimation of the chronic toxicity of contaminants in receiving tropical marine environments. The bioassay is conducted using planktonic larvae of the barnacle Amphibalanus amphitrite and is targeted at generating environmentally relevant, chronic toxicity data for water quality guideline derivation or compliance testing. The developmental endpoint demonstrated a consistently high control performance, validated through the use of copper as a reference toxicant. In addition, the biological effects of aluminium, gallium and molybdenum were assessed. The endpoint expressed high sensitivity to copper and moderate sensitivity to aluminium, whereas gallium and molybdenum exhibited no discernible effects, even at high concentrations, providing valuable information on the toxicity of these elements in tropical marine waters.

A chronic toxicity test for the tropical marine snail Nassarius dorsatus to assess the toxicity of copper, aluminium, gallium, and molybdenum

Chronic toxicity test methods for assessing the toxicity of contaminants to tropical marine organisms are generally lacking. A 96-h chronic growth rate toxicity test was developed for the larval stage of the tropical dogwhelk, Nassarius dorsatus. Growth rates of N. dorsatus larvae were assessed following exposures to copper (Cu), aluminium (Al), gallium (Ga), and molybdenum (Mo). Exposure to Cu at 28 °C validated the sensitivity of the test method, with 10% (EC10) and 50% (EC50) effect concentrations of 4.2 μg/L and 7.3 μg/L Cu, respectively. The EC10 and EC50 values for Al (<0.45-μm filtered fraction) at 28 °C were 115 μg/L and 185 μg/L, respectively. The toxicity of Cu and Al was also assessed at 24 °C and 31 °C, representing average year-round water temperatures for subtropical and tropical Australian coastal environments. At 24 °C, the growth rate of control larvae was reduced by 52% compared with the growth rate at 28 °C and there was an increase in sensitivity to Cu (EC50 = 4.7 μg/L) but a similar sensitivity to Al (EC50 = 180 μg/L). At 31 °C the control growth rate increased by 35% from that measured at 28 °C and there was reduced sensitivity to both Cu and Al (EC50s = 8.5 μg/L and 642 μg/L, respectively). There was minimal toxicity resulting from Ga (EC50 = 4560 μg/L) and Mo (no effect at ≤7000 μg/L Mo). Environ Toxicol Chem 2016;35:1788-1795.

Assessing the chronic toxicity of copper and aluminium to the tropical sea anemone Exaiptasia pallida

The worlds most productive bauxite mines and alumina refineries are located in tropical or sub-tropical regions. The discharge water from alumina refineries can contain elevated aluminium (Al, <0.45µm fraction), from 30 to 1000μg/L. There is a need for additional information on the toxicity of Al to aquatic organisms to improve the environmental regulation and management of alumina refinery operations in tropical coastal regions. A 14-d chronic toxicity test was developed for the tropical sea anemone Exaiptasia pallida. Asexual reproduction and growth rates of E. pallida were assessed using the number of lacerates produced and oral disc diameter. The comparative sensitivity of E. pallida was assessed through exposure to a commonly-used reference toxicant, copper (Cu) at 28°C, with asexual reproduction toxicity estimates of 10% (EC10) and 50% (EC50) effect concentrations, calculated as 8.8µg/L (95% confidence limits (CL): 1-18µg/L) and 35µg/L Cu (95% CL: 30-39µg/L), respectively. Growth rate was a suitable additional endpoint (EC50=35µg/L Cu, 95% CL: 23-49µg/L). The EC10 and EC50 for Al (total fraction, based on reproduction) at 28°C were 817µg/L (95% CL: 440-1480µg/L) and 2270µg/L (95% CL: 1600-3900µg/L), respectively. The toxicity of Cu and Al was also assessed at 24°C and 31°C, representing average year-round water temperatures for sub-tropical and tropical Australian coastal environments. Changing the temperature from 28°C to 24°C or 31°C resulted in up to 45% less reproduction of anemones and increased their sensitivity to Cu (EC50s at 24°C=21µg/L, 95% CL: 17-26µg/L and at 31°C=23µg/L, 95% CL: 21-25µg/L). Sensitivity to Al was reduced at 24°C with an EC50 of 8870µg/L (95% CL: 6200-NC). An EC50 for Al at 31°C could not be calculated. This test is a reliable and sensitive addition to the suite of standardised tests currently developed for tropical marine species.