Toxicity of Herbicide Mixtures to Tropical Freshwater Microalgae Using a Multispecies Test.

Agriculture within the Great Barrier Reef catchment area has contributed to pesticide contamination of adjacent freshwater ecosystems that flow into the Great Barrier Reef World Heritage Area. A novel multispecies toxicity test was used to assess the toxicity of diuron and hexazinone, 2 herbicides commonly detected within the Great Barrier Reef catchment area, to a community of 3 tropical freshwater microalgae: Monoraphidium arcuatum, Nannochloropsis-like sp., and Pediastrum duplex. Diuron was the most toxic herbicide, with 10% inhibition concentration (IC10) values of 4.3, 7.1, and 29 µg/L for P. duplex, M. arcuatum, and Nannochloropsis-like sp., respectively, followed by hexazinone, with IC10 values of 15, 18, and 450 µg/L, respectively Toxicity testing on 2 commercial formulations (Barrage, 13.2% hexazinone and 48.6% diuron; Diurex, 90% diuron) showed that additives in the commercial formulations did not significantly increase the toxicity of diuron. Direct toxicity assessments were carried out on water samples from the herbicide-contaminated Sandy Creek, which discharges to the Great Barrier Reef lagoon, and a clean reference site, Tully Gorge in the Tully River. Toxicity was observed in several Sandy Creek samples. Artificial herbicide mixtures were assessed in synthetic soft water and natural freshwaters, with toxic responses being observed at environmentally relevant concentrations. The present study successfully applied a novel multispecies tropical microalgal toxicity test, indicating that it is an effective tool for the assessment of herbicide toxicity in both natural and synthetic freshwaters. Environ Toxicol Chem 2021;40:473-486. © 2020 SETAC.

Development and application of a multispecies toxicity test with tropical freshwater microalgae.

Microalgae are commonly used in ecotoxicity testing due to their ease of culturing and rapid cell division rates. These tests generally utilise a single species of algae; however, microalgae occur in the environment as complex communities of multiple species. To date, routine multispecies toxicity tests using tropical microalgae have not been available. This study investigated four tropical freshwater microalgal species for use in a chronic multispecies toxicity test based on the population growth (cell division) rate: Pediastrum duplex, Monoraphidium arcuatum, Nannochloropsis-like sp. and Chlorella sp. 12. Flow cytometric analysis identified the different fluorescence and light scattering properties of each algal species and quantified each species within multispecies mixtures. Following optimisation of test media nutrients and pH, a toxicity testing protocol was developed with P. duplex, M. arcuatum and Nannochloropsis-like sp. There were no significant differences in growth rates of each alga when tested over 72 h as single species or in multispecies mixtures. Atrazine and imazapic, two herbicides with different modes of action, were used to assess the sensitivity of the multispecies toxicity test. Atrazine was toxic to all species with 72-h IC10 values of 7.2, 63 and 280 µg/L for P. duplex, M. arcuatum and Nannochloropsis-like sp. respectively, while imazapic was not toxic to any species at concentrations up to 1100 µg/L. The toxicity of atrazine and imazapic to each microalgal species in the multispecies toxicity test was the same as that determined from single-species toxicity tests indicating that the presence of these microalgae in a mixture did not affect the toxicity of these two herbicides. This study is the first to develop a multispecies tropical microalgal toxicity test for application in freshwaters. This time- and cost-effective tool can be utilised to generate data to assist environmental decision making and to undertake risk assessments of contaminants in tropical freshwater environments.

Revisions to the derivation of the Australian and New Zealand guidelines for toxicants in fresh and marine waters.

The Australian and New Zealand Guidelines for Fresh and Marine Water Quality are a key document in the Australian National Water Quality Management Strategy. These guidelines released in 2000 are currently being reviewed and updated. The revision is being co-ordinated by the Australian Department of Sustainability, Environment, Water, Population and Communities, while technical matters are dealt with by a series of Working Groups. The revision will be evolutionary in nature reflecting the latest scientific developments and a range of stakeholder desires. Key changes will be: increasing the types and sources of data that can be used; working collaboratively with industry to permit the use of commercial-in-confidence data; increasing the minimum data requirements; including a measure of the uncertainty of the trigger value; improving the software used to calculate trigger values; increasing the rigour of site-specific trigger values; improving the method for assessing the reliability of the trigger values; and providing guidance of measures of toxicity and toxicological endpoints that may, in the near future, be appropriate for trigger value derivation. These changes will markedly improve the number and quality of the trigger values that can be derived and will increase end-users' ability to understand and implement the guidelines in a scientifically rigorous manner.

Reanalysis of uranium toxicity data for selected freshwater organisms and the influence of dissolved organic carbon.

The present study reanalyzed 46 existing uranium (U) chronic toxicity datasets for four freshwater species to generate consistent toxicity measures and explore relationships between U toxicity and key physicochemical variables. Dissolved organic carbon (DOC) was consistently the best predictor of U toxicity based on 10% inhibitory concentration (IC10) and median inhibitory concentration (IC50) values, with water hardness also being a significant co-predictor of IC50 concentrations for one species. The influence of DOC on acute and chronic U toxicity was further characterized using existing data for five species, and was found to vary depending on species, DOC source, and exposure duration (acute vs chronic). The slopes of the relationships between DOC and (normalized) acute and chronic U toxicity were modeled using cumulative probability distributions. From these, slopes were selected for which to correct acute or chronic U toxicity values or hazard estimates based on the aquatic DOC concentration. The fifth percentiles of these cumulative probability distributions for acute and chronic exposure data were 0.064 and 0.090, respectively, corresponding to a 6.4 and 9.0% reduction in U toxicity relative to the toxicity at the base DOC concentration for each 1 mg/L increase in DOC concentration (over the DOC range 0-30 mg/L). Algorithms were developed to enable the adjustment of U toxicity values and U hazard estimates, depending on DOC concentrations. These algorithms will significantly enhance the environmental relevance of water quality/risk assessments for U in fresh surface waters.

Effects of alumina refinery wastewater and signature metal constituents at the upper thermal tolerance of: 1. The tropical diatom Nitzschia closterium.

Ecotoxicological studies, using the tropical marine diatom, Nitzschia closterium (72-h growth rate), were undertaken to assess potential issues relating to the discharge from an alumina refinery in northern Australia. The studies assessed: (i) the species' upper thermal tolerance; (ii) the effects of three signature metals, aluminium (Al), vanadium (V) and gallium (Ga) (at 32°C); and (iii) the effects of wastewater (at 27 and 32°C). The critical thermal maximum and median inhibition temperature for N. closterium were 32.7°C and 33.1°C, respectively. Single metal toxicity tests found that N. closterium was more sensitive to Al compared to Ga and V, with IC(50)s (95% confidence limits) of 190 (140-280), 19,640 (11,600-25,200) and 42,000 (32,770-56,000) µg L(-1), respectively. The undiluted wastewater samples were of low toxicity to N. closterium (IC(50)s>100% wastewater). Environmental chemistry data suggested that the key metals and discharge are a very low risk to this species.

Development of a toxicity identification evaluation protocol using chlorophyll-a fluorescence in a marine microalga.

Growth inhibition bioassays with the microalga Nitzschia closterium have recently been applied in marine Toxicity Identification Evaluation (TIE) testing. However, the 48-h test duration can result in substantial loss of toxicants over time, which might lead to an underestimation of the sample toxicity. Although shorter-term microalgal bioassays can minimize such losses, there are few bioassays available and none are adapted for marine TIE testing. The acute (5-min) chlorophyll-a fluorescence bioassay is one alternative; however, this bioassay was developed for detecting herbicides in freshwater aquatic systems and its suitability for marine TIE testing was not known. In this study, a chlorophyll-a fluorescence bioassay using the marine microalga Isochrysis galbana was able to detect contaminants other than herbicides at environmentally relevant concentrations and tolerated the physical and chemical manipulations needed for a Phase I TIE. Phase I TIE procedures were successfully developed using this chlorophyll-a fluorescence bioassay and used to identify all classes of contaminants present in a synthetic mixture of known chemical composition. In addition, TIEs with both the acute fluorescence bioassay and the standard growth inhibition bioassay identified the same classes of toxicants in a sample of an unknown complex effluent. Even though the acute chlorophyll-a fluorescence end point was less sensitive than the chronic cell division end point, TIEs with the chlorophyll-a fluorescence bioassay provided a rapid and attractive alternative to longer-duration bioassays.

Acute toxicity and bioaccumulation of aqueous and sediment-bound metals in the estuarine amphipod Melita plumulosa.

The sensitivities of juvenile and adult amphipods to metals (Cd, Cu, Ni, Pb, and Zn) in whole sediment and water-only exposures were compared using a newly developed acute test with the estuarine species Melita plumulosa. Endpoints included survival and bioaccumulation in adults, and survival and growth (body length) in juveniles over 96 h water-only and 10 day sediment exposures. Juveniles were more sensitive than adults to metals, either bound to sediments or in the aqueous phase. Although LOEC values for copper and zinc in juvenile whole-sediment tests (820 and 2290 mg/kg dry weight, respectively) were high in comparison with interim sediment quality guideline values for individual metals (270 and 410 for copper and zinc respectively), they were generally within the range of concentrations found in contaminated sediments in local estuaries. Accumulation of metals, together with the low porewater metal concentrations in whole-sediment tests, indicated that the ingestion of sediment is an important source of zinc and copper and cause of toxicity in this species.

Rapid flow cytometric method for the assessment of toxic dinoflagellate cyst viability.

The inadvertent transfer and dispersal of non-indigenous marine species via shipping ballast water is of increasing environmental concern. Despite a major global effort to develop new ballast water treatment technologies, their acceptance has been hampered by the lack of suitable indicator species for assessing treatment effectiveness. Resistant dinoflagellate cysts are one proposed test organism, however their use has been limited due to difficulties in assessing their viability after treatment. The paper describes the development of a rapid method to determine the viability of cysts of the dinoflagellate Alexandrium catenella using staining with SYTOX Green and flow-cytometric analysis. The viability of A. catenella cysts was inversely proportional to their ability to take up the stain. There was excellent agreement between cysts measured as viable/non-viable using flow cytometry and cyst viability determined in standard long-term germination tests. Advantages of the flow-cytometric method include high test precision and rapid testing times of < 2 days, compared to > 4 weeks using existing germination methods.

Copper bioavailability in a coastal environment of Northern Chile: comparison of bioassay and analytical speciation approaches.

An integrated approach including chemical speciation analyses and microalgal bioassays was used to assess the impact of copper from copper mining on a coastal area in Northern Chile. Dissolved copper ranged from <1 microg l(-1) at reference sites to 48 microgl(-1) at sites close to the mine discharge. Dissolved copper at sites closest to the discharge always exceeded seawater complexing capacities determined by anodic stripping voltammetry (ASV), and consequently labile copper was always detected (1-37 microg l(-1)). Agreement between ASV-labile copper and copper retained by cation exchange (ALSA) columns was excellent. Measured labile copper also accurately predicted bioavailable copper determined by growth inhibition of Nitzschia closterium and enzyme inhibition in Dunaliella tertiolecta. Seawater from Caleta La Lancha had the highest dissolved and labile copper and was the most toxic to micro-algal growth and enzyme activity. Previous studies at this site confirmed it had the lowest level of biodiversity, suggesting that copper may have both direct and indirect effects on these communities.

The development of marine Toxicity Identification Evaluation (TIE) procedures using the unicellular alga Nitzschia closterium.

Unicellular algae are highly sensitive to a wide range of toxicants and have been used extensively in ecotoxicological testing. This, along with their ability to grow in very small test volumes over short test durations, make them ideal test organisms for use in Toxicity Identification Evaluations (TIEs). Despite this, microalgae have not previously been used in marine TIE studies. In this study, the marine diatom Nitzschia closterium was shown to be a highly suitable test organism after modification of the standard test protocol to reduce test volumes to 6 mL and test duration to 48 h. The alga was tolerant to the chemicals used in phase I of the standard USEPA TIE protocol, and physical TIE manipulations had no effect on algal growth. The cation exchange procedure, however, inhibited algal growth, while the anion exchange procedure stimulated growth, making these two procedures unsuitable for use with this species. Of the buffers trialed for the graduated pH procedure, 0.01 M PIPES buffer was found to be suitable for buffering at pH 7 because it maintained the required pH over the duration of the test and did not affect the growth or sensitivity of the algae to one reference toxicant (copper). A trial TIE on a secondary-treated sewage effluent for discharge into coastal waters showed that the developed protocols could successfully be used to identify ammonia as the major toxicant in the effluent.

Hardness corrections for copper are inappropriate for protecting sensitive freshwater biota.

Toxicity testing using a freshwater alga (Chlorella sp.), a bacterium (Erwinnia sp.) and a cladoceran (Ceriodaphnia cf. dubia) exposed to copper in synthetic and natural freshwaters of varying hardness (44-375 mg CaCO3/l), with constant alkalinity, pH and dissolved organic carbon concentration, demonstrated negligible hardness effects in the pH range 6.1-7.8. Therefore, the use of a generic hardness-correction algorithm, developed as part of national water quality guidelines for protecting freshwater biota, is not recommended for assessing the toxicity of copper to these, and other, sensitive freshwater species. Use of the algorithm for these sensitive species will be underprotective because the calculated concentrations of copper in water that cause a toxic effect will be higher.

Development of an improved rapid enzyme inhibition bioassay with marine and freshwater microalgae using flow cytometry.

A rapid toxicity test based on inhibition of esterase activity in marine and freshwater microalgae (Selenastrum capricornutum, Chlorella sp., Dunaliella tertiolecta, Phaeodactylum tricornutum, Tetraselmis sp., Entomoneis cf. punctulata, Nitzschia cf. paleacea) was developed using flow cytometry. Uptake of fluorescein diacetate (FDA) was optimized for each species by varying the substrate concentration, incubation time, and media pH. Propidium iodide (PI) was utilized to assess membrane integrity. The optimized FDA/PI staining procedure was then used to assess the toxicity of copper in short-term exposures (1-24 h). Esterase activity was a sensitive indicator of copper toxicity in S. capricornutum and E. cf. punctulata. As copper concentrations increased, esterase activity decreased in a concentration-dependent manner. The 3- and 24-h EC50 values (based on mean activity states) were 112 microg Cu L(-1) (95% confidence limits 88-143) and 51 microg Cu L(-1) (95% confidence limits 38-70) for S. capricornutum and 47 microg Cu L(-1) (95% confidence limits 43-51) and 9.1 microg Cu L(-1) (95% confidence limits 7.6-11) for E. cf. punctulata, respectively. This enzyme inhibition endpoint showed similar sensitivity to chronic growth rate inhibition in E. cf. punctulata (48-h and 72-h EC50 values of 17 and 18 microg L(-1), respectively) but was less sensitive compared to growth for S. capricornutum (48-h and 72-h EC50 values of 4.9 and 7.5 microg L(-1), respectively). For the other five species tested, inhibition of FDA fluorescence was relatively insensitive to copper, even at copper concentrations that severely inhibited cell division rate. These short-term bioassays that detect sublethal endpoints may provide a more rapid and cost-effective way of monitoring contaminant impacts in natural waters.

Development of flow cytometry-based algal bioassays for assessing toxicity of copper in natural waters.

Copper toxicity to the freshwater algae Selenastrum capricornutum and Chlorella sp. and the marine algae Phaeodactylum tricornutum and Dunaliella tertiolecta was investigated using different parameters measured by flow cytometry: cell division rate inhibition, chlorophyll a fluorescence, cell size (i.e., light-scattering), and enzyme activity. These parameters were assessed regarding their usefulness as alternative endpoints for acute (1-24 h) and chronic (48-72 h) toxicity tests. At copper concentrations of 10 micrograms/L or less, significant inhibition (50%) of the cell division rate was observed after 48- and 72-h exposures for Chlorella sp., S. capricornutum, and P. tricornutum. Bioassays based on increases in algal cell size were also sensitive for Chlorella sp. and P. tricornutum. Copper caused both chlorophyll a fluorescence stimulation (48-h EC50 of 10 +/- 1 micrograms Cu/L for P. tricornutum) and inhibition (48-h EC50 of 14 +/- 6 micrograms Cu/L for S. capricornutum). For acute toxicity over short exposure periods, esterase activity in S. capricornutum using fluorescein diacetate offered a rapid alternative (3-h EC50 of 90 +/- 40 micrograms Cu/L) to growth inhibition tests for monitoring copper toxicity in mine-impacted waters. For all the effect parameters measured, D. tertiolecta was tolerant to copper at concentrations up to its solubility limit in seawater. These results demonstrate that flow cytometry is a useful technique for toxicity testing with microalgae and provide additional information regarding the general mode of action of copper (II) to algal species.

Plant and algal interference in bacterial beta-D-galactosidase and beta-D-glucuronidase assays.

Several commonly occurring freshwater and marine plants and algae were screened for beta-D-galactosidase and beta-D-glucuronidase activities by using a 60-min enzyme assay based on the hydrolysis by these enzymes of 4-methylumbelliferyl-beta-D-galactoside and 4-methylumbelliferyl- beta-glucuronide, respectively. All freshwater plant extracts tested showed beta-D-galactosidase activity several at relatively high levels, and a number also showed beta-D-glucuronidase activity. A number of the macroalgae showed no activity of either enzyme, but those showing beta-D-galactosidase activity also showed beta-D-glucuronidase activity. The majority of microalgae showed some beta-D-galactosidase activity, but few showed beta-D-glucuronidase activity. Further studies, using the commercial Colilert test and the marine water formulation of Colilert, revealed that 2 of 11 of the microalgal species and several of the plant extracts tested caused positive reactions. It was concluded that several plant extracts and algae could significantly interfere with the detection of coliform bacteria and Escherichia coli with the use of rapid assays, on the basis of their production of beta-D-galactosidase and beta-D-glucuronidase, respectively. The significance of the plant and algal interferences in tests such as Colilert is dependent on the levels of enzymes released under natural conditions, the dilution which they may undergo, and the numbers of algal cells present. This also applies to interferences in rapid enzyme assays.(ABSTRACT TRUNCATED AT 250 WORDS)

Toxicity of nickel ores to marine organisms.

Queensland Nickel proposes to import New Caledonian (Ballande) and Indonesian (Gebe) nickel ores, one option being ship-to-barge transfer in Halifax Bay, North Queensland. Because small amounts of ore may be split during the unloading and transfer operations, it was important to investigate the potential impact of the spilt ore on the ecological health of the Bay. Long-term leaching of the ores with seawater showed that only nickel and chromium (VI) were released from the ores in sufficient concentrations to cause toxicity to a range of marine organisms. The soluble fractions of nickel and chromium (VI) were released from the ores within a few days. Nickel, chromium (VI) and the ore leachates showed similar toxicity to the juvenile banana prawn Penaeus merguiensis, the amphipod Allorchestes compressa and both temperature (22 degrees C) and tropical (27 degrees C) strains of the unicellular marine alga Nitzschia closterium. In a series of 30-day sub-chronic microcosm experiments, juvenile leader prawns Penaeus monodon, polychaete worms Galeolaria caespitosa and the tropical gastropod Nerita chamaeleon were all very resistant to the nickel ores, with mortality unaffected by 700 g ore per 50 l seawater. The growth rate of the leader prawns was, however, lower than that of the controls. From these data, a conservative maximum safe concentration of the nickel ores in seawater is 0.1 g l-1. The nickel ore was not highly toxic and if spilt in the quantities predicted, would not have a significant impact on the ecological health of the Bay.

Percutaneous absorption of inorganic lead compounds.

In vivo experiments with the stable lead isotope, 204Pb, have confirmed that inorganic lead compounds can be absorbed through the skin. Three different analytical techniques--thermal ionization mass spectrometry, inductively coupled plasma mass spectrometry and anodic stripping voltammetry--showed that lead, as lead nitrate or lead acetate, was rapidly absorbed through the skin and detectable in sweat, blood and urine within 6 h of skin application. Of the 4.4 mg of lead applied to the skin in one experiment, 1.3 mg was absorbed within 24 h. Initial rapid uptake was probably via sweat glands and hair follicles, followed by slower absorption via the transepidermal route. While increases in 204Pb concentration and abundance were observed, no increase in total lead in blood or urine was found. It is possible that the physicochemical form of skin-absorbed lead partitions strongly into extracellular fluid, but has a low affinity for erythrocytes. There was no significant difference in uptake of lead into erythrocytes (in vitro) from normal saline, synthetic sweat or sauna sweat. Ultrafiltration of sweat showed that up to 70% of lead in sweat was associated with > 30,000 MW particles. It is possible that percutaneous absorption of lead could contribute significantly to lead body burden, particularly from occupational exposure to lead in dust. Moreover, because lead absorbed through the skin was only just detectable in blood, and blood lead is the main criterion by which industry determines exposure, skin-absorbed lead may remain undetected.

Determination of trace element speciation and the role of speciation in aquatic toxicity.

Knowledge of trace element speciation in waters is essential to an understanding of aquatic toxicity and bioaccumulation, as well as to the partitioning of elements between water and colloidal and particulate phases. In natural waters, only very small percentages of the dissolved heavy metals, such as copper, lead cadmium or zinc, are present as free (aquo) metal ion; most of the metal is adsorbed to colloidal particles or combined in complexes. For aquatic toxicity studies, the aim of the speciation measurement is to determine the fraction of total dissolved metal (the 'toxic fraction') that will react with, and be transported across, a biological membrane such as a fish gill. In this review, a range of trace element speciation techniques is discussed and compared. A simple anodic stripping voltammetric method is recommended for the measurement of the fraction of electroactive metal in a sample, i.e. the fraction of total dissolved metal that can be deposited into a mercury electrode at the natural pH of the sample. The electroactive fraction is believed to approximate the toxic fraction. A rapid ion exchange method, suitable for field use, is proposed for the determination of the toxic fraction of copper in waters.

Manganese in scalp hair: problems of exogenous manganese and implications for manganese monitoring in Groote Eylandt Aborigines.

The use of scalp hair to monitor manganese was studied as part of an investigation of manganese intoxication amongst a group of Aborigines living on manganese-rich soil on Groote Eylandt, in the Northern Territory of Australia. High scalp-hair manganese values were due largely to manganese from exogenous sources. Manganese (IV) dioxide in dust, trapped in hair, was reduced by the components of sweat, leading to the diffusion of manganese (II) into the hair shaft. At least 15 micrograms Mn g-1 hair could be incorporated into hair via this exogenous route. To overcome the problems of manganese contamination, the ability of a number of leaching agents to remove exogenous manganese selectively from hair was tested. Measurements of manganese along the length of hair strands were extrapolated back to zero length to estimate the amount of manganese in the hair as it emerged from the scalp. Using this extrapolation technique, Aborigines on Groote Eylandt had a mean scalp-hair manganese of 16 ppm. Aborigines in non-manganese areas had 2 ppm manganese in hair. Caucasians living in the same manganese-rich area had 2.5 ppm manganese in hair, compared to 0.5 ppm manganese in non-manganese areas. Measurements of manganese in hair and blood of Groote Eylandt Aborigines showed that the population had a high exposure to manganese, but did not distinguish between those individuals affected/unaffected by the neurological condition, Groote Eylandt Syndrome.

Manganese catalysis of dopamine oxidation.

Manganese catalysis of the oxidation of dopamine by air was studied as part of an investigation of possible manganese intoxication amongst a group of Aborigines living on manganese-rich soil on Groote Eylandt, in the Northern Territory of Australia. Manganese significantly increased the oxidation rate of dopamine, and the manganese complexes with some purines were especially efficient catalysts. An oxidation mechanism, involving a manganese(II)/(III) redox couple and a semiquinone free radical intermediate, is proposed. Stoichiometric hydrogen peroxide was produced by the oxidation, and the oxidation products of dopamine were highly toxic to the marine diatom Nitzschia closterium. Hydrogen peroxide and the superoxide radical did not oxidize dopamine at physiological pH. Some electrophilic compounds, including ascorbic acid, dehydroascorbic acid and thiamine, effectively inhibited dopamine oxidation. The Groote Eylandt Aborigines are likely to be deficient in ascorbic acid (vitamin C) and thiamine (vitamin B1), and these deficiencies, as well as their lifestyle, may predispose them to manganese intoxication.