Acute and chronic toxicity of manganese to tropical adult coral (Acropora millepora) to support the derivation of marine manganese water quality guideline values.

Adult corals are among the most sensitive marine organisms to dissolved manganese and experience tissue sloughing without bleaching (i.e., no loss of Symbiodinium spp.) but there are no chronic toxicity data for this sensitive endpoint. We exposed adult Acropora millepora to manganese in 2-d acute and 14-d chronic experiments using tissue sloughing as the toxicity endpoint. The acute tissue sloughing median effect concentration (EC50) was 2560 µg Mn/L. There was no chronic toxicity to A. millepora at concentrations up to and including the highest concentration of 1090 µg Mn/L i.e., the chronic no observed effect concentration (NOEC). A coral-specific acute-to-chronic ratio (ACR) (EC50/NOEC) of 2.3 was derived. These data were combined with chronic toxicity data for other marine organisms in a species sensitivity distribution (SSD). Marine manganese guidelines were 190, 300, 390 and 570 µg Mn/L to provide long-term protection of 99, 95, 90, and 80 % of marine species, respectively.

Adult Corals Are Uniquely More Sensitive to Manganese Than Coral Early-Life Stages.

Manganese (Mn) is an essential element and is generally considered to be one of the least toxic metals to aquatic organisms, with chronic effects rarely seen at concentrations below 1000 µg/L. Anthropogenic activities lead to elevated concentrations of Mn in tropical marine waters. Limited data suggest that Mn is more acutely toxic to adults than to early life stages of scleractinian corals in static renewal tests. However, to enable the inclusion of sufficient sensitive coral data in species sensitivity distributions to derive water quality guideline values for Mn, we determined the acute toxicity of Mn to the adult scleractinian coral, Acropora muricata, in flow-through exposures. The 48-h median effective concentration was 824 µg Mn/L (based on time-weighted average, measured, dissolved Mn). The endpoint was tissue sloughing, a lethal process by which coral tissue detaches from the coral skeleton. Tissue sloughing was unrelated to superoxidase dismutase activity in coral tissue, and occurred in the absence of bleaching, that is, toxic effects were observed for the coral host, but not for algal symbionts. We confirm that adult scleractinian corals are uniquely sensitive to Mn in acute exposures at concentrations 10-340 times lower than those reported to cause acute or chronic toxicity to coral early life stages, challenging the traditional notion that early life stages are more sensitive than mature organisms. Environ Toxicol Chem 2023;42:1359-1370. © 2023 Commonwealth Scientific and Industrial Research Organisation. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Evidence for de novo acquisition of microalgal symbionts by bleached adult corals.

Early life stages of most coral species acquire microalgal endosymbionts (Symbiodiniaceae) from the environment, but whether exogenous symbiont uptake is possible in the adult life stage is unclear. Deep sequencing of the Symbiodiniaceae ITS2 genetic marker has revealed novel symbionts in adult corals following bleaching; however these strains may have already been present at densities below detection limits. To test whether acquisition of symbionts from the environment occurs, we subjected adult fragments of corals (six species in four families) to a chemical bleaching treatment (menthol and DCMU). The treatment reduced the native microalgal symbiont abundance to below 2% of their starting densities. The bleached corals were then inoculated with a cultured Cladocopium C1acro strain. Genotyping of the Symbiodiniaceae communities before bleaching and after reinoculation showed that fragments of all six coral species acquired the Cladocopium C1acro strain used for inoculation. Our results provide strong evidence for the uptake of Symbiodiniaceae from the environment by adult corals. We also demonstrate the feasibility of chemical bleaching followed by reinoculation to manipulate the Symbiodiniaceae communities of adult corals, providing an innovative approach to establish new symbioses between adult corals and heat-evolved microalgal symbionts, which could prove highly relevant to coral reef restoration efforts.

Automated flow control of a multi-lane swimming chamber for small fishes indicates species-specific sensitivity to experimental protocols.

In fishes, swimming performance is considered an important metric to measure fitness, dispersal and migratory abilities. The swimming performance of individual larval fishes is often integrated into models to make inferences on how environmental parameters affect population-level dynamics (e.g. connectivity). However, little information exists regarding how experimental protocols affect the swimming performance of marine fish larvae. In addition, the technical setups used to measure larval fish swimming performance often lack automation and accurate control of water quality parameters and flow velocity. In this study, we automated the control of multi-lane swimming chambers for small fishes by developing an open-source algorithm. This automation allowed us to execute repeatable flow scenarios and reduce operator interference and inaccuracies in flow velocity typically associated with manual control. Furthermore, we made structural modifications to a prior design to reduce the areas of lower flow velocity. We then validated the flow dynamics of the new chambers using computational fluid dynamics and particle-tracking software. The algorithm provided an accurate alignment between the set and measured flow velocities and we used it to test whether faster critical swimming speed (U crit) protocols (i.e. shorter time intervals and higher velocity increments) would increase U crit of early life stages of two tropical fish species [4-10-mm standard length (SL)]. The U crit of barramundi (Lates calcarifer) and cinnamon anemonefish (Amphiprion melanopus) increased linearly with fish length, but in cinnamon anemonefish, U crit started to decrease upon metamorphosis. Swimming protocols using longer time intervals (more than 2.5 times increase) negatively affected U crit in cinnamon anemonefish but not in barramundi. These species-specific differences in swimming performance highlight the importance of testing suitable U crit protocols prior to experimentation. The automated control of flow velocity will create more accurate and repeatable data on swimming performance of larval fishes. Integrating refined measurements into individual-based models will support future research on the effects of environmental change.

Effects of dissolved nickel and nickel-contaminated suspended sediment on the scleractinian coral, Acropora muricata.

Intensification of lateritic nickel mining in Southeast Asia and Melanesia potentially threatens coastal ecosystems from increased exposure to nickel and suspended sediment. This study investigated the response of Acropora muricata when exposed to either dissolved nickel, clean suspended sediment or nickel-contaminated suspended sediment for 7 days, followed by a 7-d recovery period. Significant bleaching and accumulation of nickel in coral tissue was observed only after exposure to high dissolved nickel concentrations and nickel-spiked suspended sediment. No effect on A. muricata was observed from exposure to a particulate-bound nickel concentration of 60 mg/kg acid-extractable nickel at a suspended sediment concentration of 30 mg/L TSS. This study demonstrates that bioavailability of nickel associated with suspended sediment exposure plays a key role in influencing nickel toxicity to corals. These findings assist in assessments of risk posed by increasing nickel mining activities on tropical marine ecosystems.

The effect of dissolved nickel and copper on the adult coral Acropora muricata and its microbiome.

The potential impacts of mining activities on tropical coastal ecosystems are poorly understood. In particular, limited information is available on the effects of metals on scleractinian corals which are foundation species that form vital structural habitats supporting other biota. This study investigated the effects of dissolved nickel and copper on the coral Acropora muricata and its associated microbiota. Corals collected from the Great Barrier Reef were exposed to dissolved nickel (45, 90, 470, 900 and 9050 µg Ni/L) or copper (4, 11, 32 and 65 µg Cu/L) in flow through chambers at the National Sea Simulator, Townsville, Qld, Australia. After a 96-h exposure DNA metabarcoding (16S rDNA and 18S rDNA) was undertaken on all samples to detect changes in the structure of the coral microbiome. The controls remained healthy throughout the study period. After 36 h, bleaching was only observed in corals exposed to 32 and 65 µg Cu/L and very high nickel concentrations (9050 µg Ni/L). At 96 h, significant discolouration of corals was only observed in 470 and 900 µg Ni/L treatments, the highest concentrations tested. While high concentrations of nickel caused bleaching, no changes in the composition of their microbiome communities were observed. In contrast, exposure to copper not only resulted in bleaching, but altered the composition of both the eukaryote and bacterial communities of the coral's microbiomes. Our findings showed that these effects were only evident at relatively high concentrations of nickel and copper, reflecting concentrations observed only in extremely polluted environments. Elevated metal concentrations have the capacity to alter the microbiomes which are inherently linked to coral health.

Comparing the capacity of five different dietary treatments to optimise growth and nutritional composition in two scleractinian corals.

Developing an optimal heterotrophic feeding regime has the potential to improve captive coral growth and health. This study evaluated the efficacy of three exogenous diets: Artemia nauplii (ART), a commercially available coral diet (Reef Roids) (RR), and a novel, micro-bound diet (ATF), against a comparatively natural, unfiltered seawater treatment (RAW), and an unfed, ultra-filtered seawater treatment (CTL), in adult Acropora millepora and Pocillopora acuta nubbins. After 90 days, both species showed significantly positive weight gain in response to one treatment (A. millepora-RAW, P. acuta-ART), and comparatively low growth in response to another (A. millepora-ATF, P. acuta-RR). The results highlighted substantial differences in the nutritional requirements between species. The nutritional composition of A. millepora in the best performing treatment was dominated by high-energy materials such as storage lipids and saturated and monounsaturated fatty acids. In contrast, the P. acuta nutritional profile in the superior treatment showed a predominance of structural materials, including protein, phospholipids, and polyunsaturated fatty acids. This study demonstrates that Artemia nauplii can successfully replace a natural feeding regime for captive P. acuta, yet highlights the considerable work still required to optimise supplementary feeding regimes for A. millepora.

Intra-colonial diversity in the scleractinian coral, Acropora millepora: identifying the nutritional gradients underlying physiological integration and compartmentalised functioning.

Scleractinian corals are colonial organisms comprising multiple physiologically integrated polyps and branches. Colonialism in corals is highly beneficial, and allows a single colony to undergo several life processes at once through physiological integration and compartmentalised functioning. Elucidating differences in the biochemical composition of intra-colonial branch positions will provide valuable insight into the nutritional reserves underlying different regions in individual coral colonies. This will also ascertain prudent harvesting strategies of wild donor-colonies to generate coral stock with high survival and vigour prospects for reef-rehabilitation efforts and captive husbandry. This study examined the effects of colony branch position on the nutritional profile of two different colony sizes of the common scleractinian, Acropora millepora. For smaller colonies, branches were sampled at three locations: the colony centre (S-centre), 50% of the longitudinal radius length (LRL) (S-50), and the colony edge (S-edge). For larger colonies, four locations were sampled: the colony centre (L-centre), 33.3% of the LRL (L-33), 66.6% of the LRL (L-66), and the edge (L-edge). Results demonstrate significant branch position effects, with the edge regions containing higher protein, likely due to increased tissue synthesis and calcification. Meanwhile, storage lipid and total fatty acid concentrations were lower at the edges, possibly reflecting catabolism of high-energy nutrients to support proliferating cells. Results also showed a significant effect of colony size in the two classes examined. While the major protein and structural lipid sink was exhibited at the edge for both sizes, the major sink for high-energy lipids and fatty acids appeared to be the L-66 position of the larger colonies and the S-centre and S-50 positions for the smaller colonies. These results confirm that the scleractinian coral colony is not nutritionally homogeneous, and while different regions of the coral colony are functionally specialised, so too are their nutritional profiles geared toward meeting specific energetic demands.

Functional genomic analysis of corals from natural CO2 -seeps reveals core molecular responses involved in acclimatization to ocean acidification.

Little is known about the potential for acclimatization or adaptation of corals to ocean acidification and even less about the molecular mechanisms underpinning these processes. Here, we examine global gene expression patterns in corals and their intracellular algal symbionts from two replicate population pairs in Papua New Guinea that have undergone long-term acclimatization to natural variation in pCO2 . In the coral host, only 61 genes were differentially expressed in response to pCO2 environment, but the pattern of change was highly consistent between replicate populations, likely reflecting the core expression homeostasis response to ocean acidification. Functional annotations highlight lipid metabolism and a change in the stress response capacity of corals as key parts of this process. Specifically, constitutive downregulation of molecular chaperones was observed, which may impact response to combined climate change-related stressors. Elevated CO2 has been hypothesized to benefit photosynthetic organisms but expression changes of in hospite Symbiodinium in response to acidification were greater and less consistent among reef populations. This population-specific response suggests hosts may need to adapt not only to an acidified environment, but also to changes in their Symbiodinium populations that may not be consistent among environments, adding another challenging dimension to the physiological process of coping with climate change.

Influence of different feeding regimes on the survival, growth, and biochemical composition of Acropora coral recruits.

Heterotrophic feeding in newly-settled coral planulae can potentially improve survivorship and accelerate early development in some species; however, an optimal diet to facilitate this does not currently exist. This study evaluated the efficacy of three heterotrophic feeding regimes (enriched rotifers, unfiltered seawater, and a novel, particulate diet), against a wholly-phototrophic treatment on Acropora hyacinthus, A. loripes, A. millepora, and A. tenuis recruits, over 93 days post-settlement. The unfiltered seawater treatment recorded maximum survival for all species (A. hyacinthus 95.9±8.0%, A. loripes: 74.3±11.5%, A. millepora: 67±12.7%, A. tenuis: 53.2±11.3%), although not significant. Growth (% surface area gain) was also greatest in the unfiltered seawater, and this was significant for A. millepora (870±307%) and A. tenuis (693±91.8%) (p<0.05). Although total lipid concentration was relatively stable across treatments, the lipid class composition exhibited species-specific responses to each treatment. Lower saturated and higher polyunsaturated fatty acids appeared beneficial to recruit performance, particularly in the unfiltered seawater, which generally contained the highest levels of 20:5n-3 (EPA), 22:6n-3 (DHA), and 20:4n-6 (ARA). The present study demonstrates the capacity of a nutritionally adequate and readily accepted heterotrophic feeding regime to increase coral recruit survival, growth, and health, which can greatly reduce the time required in cost- and labour-intensive culture.

Diel CO2 cycles reduce severity of behavioural abnormalities in coral reef fish under ocean acidification.

Elevated CO2 levels associated with ocean acidification (OA) have been shown to alter behavioural responses in coral reef fishes. However, all studies to date have used stable pCO2 treatments, not considering the substantial diel pCO2 variation that occurs in shallow reef habitats. Here, we reared juvenile damselfish, Acanthochromis polyacanthus, and clownfish, Amphiprion percula, at stable and diel cycling pCO2 treatments in two experiments. As expected, absolute lateralization of A. polyacanthus and response to predator cue of Am. percula were negatively affected in fish reared at stable, elevated pCO2 in both experiments. However, diel pCO2 fluctuations reduced the negative effects of OA on behaviour. Importantly, in experiment two, behavioural abnormalities that were present in fish reared at stable 750 µatm CO2 were largely absent in fish reared at 750 ± 300 µatm CO2. Overall, we show that diel pCO2 cycles can substantially reduce the severity of behavioural abnormalities caused by elevated CO2. Thus, past studies may have over-estimated the impacts of OA on the behavioural performance of coral reef fishes. Furthermore, our results suggest that diel pCO2 cycles will delay the onset of behavioural abnormalities in natural populations.

Expression of calcification and metabolism-related genes in response to elevated pCO2 and temperature in the reef-building coral Acropora millepora.

Declining health of scleractinian corals in response to deteriorating environmental conditions is widely acknowledged, however links between physiological and functional genomic responses of corals are less well understood. Here we explore growth and the expression of 20 target genes with putative roles in metabolism and calcification in the branching coral, Acropora millepora, in two separate experiments: 1) elevated pCO2 (464, 822, 1187 and 1638 µatm) and ambient temperature (27°C), and 2) elevated pCO2 (490 and 822 µatm) and temperature (28 and 31 °C). After 14 days of exposure to elevated pCO2 and ambient temperatures, no evidence of differential expression of either calcification or metabolism genes was detected between control and elevated pCO2 treatments. After 37 days of exposure to control and elevated pCO2, Ubiquinol-Cytochrome-C Reductase Subunit 2 gene (QCR2; a gene involved in complex III of the electron chain transport within the mitochondria and critical for generation of ATP) was significantly down-regulated in the elevated pCO2 treatment in both ambient and elevated temperature treatments. Overall, the general absence of a strong response to elevated pCO2 and temperature by the other 19 targeted calcification and metabolism genes suggests that corals may not be affected by these stressors on longer time scales (37 days). These results also highlight the potential for QCR2 to act as a biomarker of coral genomic responses to changing environments.

Natural volcanic CO2 seeps reveal future trajectories for host-microbial associations in corals and sponges.

Atmospheric carbon dioxide (CO2) levels are rapidly rising causing an increase in the partial pressure of CO2 (pCO2) in the ocean and a reduction in pH known as ocean acidification (OA). Natural volcanic seeps in Papua New Guinea expel 99% pure CO2 and thereby offer a unique opportunity to explore the effects of OA in situ. The corals Acropora millepora and Porites cylindrica were less abundant and hosted significantly different microbial communities at the CO2 seep than at nearby control sites <500 m away. A primary driver of microbial differences in A. millepora was a 50% reduction of symbiotic Endozoicomonas. This loss of symbiotic taxa from corals at the CO2 seep highlights a potential hurdle for corals to overcome if they are to adapt to and survive OA. In contrast, the two sponges Coelocarteria singaporensis and Cinachyra sp. were ∼ 40-fold more abundant at the seep and hosted a significantly higher relative abundance of Synechococcus than sponges at control sites. The increase in photosynthetic microbes at the seep potentially provides these species with a nutritional benefit and enhanced scope for growth under future climate scenarios (thus, flexibility in symbiosis may lead to a larger niche breadth). The microbial community in the apparently pCO2-sensitive sponge species S. massa was not significantly different between sites. These data show that responses to elevated pCO2 are species-specific and that the stability and flexibility of microbial partnerships may have an important role in shaping and contributing to the fitness and success of some hosts.

Symbiodinium community composition in scleractinian corals is not affected by life-long exposure to elevated carbon dioxide.

Ocean acidification (OA) is expected to negatively affect coral reefs, however little is known about how OA will change the coral-algal symbiosis on which reefs ultimately depend. This study investigated whether there would be differences in coral Symbiodinium types in response to OA, potentially improving coral performance. We used denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer 2 (ITS2) region of ribosomal DNA to investigate the dominant types of Symbiodinium associating with six species of scleractinian coral that were exposed to elevated partial pressures of carbon dioxide (pCO2) in situ from settlement and throughout their lives. The study was conducted at three naturally occurring volcanic CO2 seeps (pCO2 ∼500 to 900 ppm, pHTotal 7.8 - 7.9) and adjacent control areas (pCO2 ∼390 ppm, pHTotal ∼8.0 - 8.05) in Papua New Guinea. The Symbiodinium associated with corals living in an extreme seep site (pCO2 >1000 ppm) were also examined. Ten clade C types and three clade D types dominated the 443 coral samples. Symbiodinium types strongly contrasted between coral species, however, no differences were observed due to CO2 exposure. Within five species, 85 - 95% of samples exhibited the same Symbiodinium type across all sites, with remaining rare types having no patterns attributable to CO2 exposure. The sixth species of coral displayed site specific differences in Symbiodinium types, unrelated to CO2 exposure. Symbiodinium types from the coral inhabiting the extreme CO2 seep site were found commonly throughout the moderate seeps and control areas. Our finding that symbiotic associations did not change in response to CO2 exposure suggest that, within the six coral hosts, none of the investigated 13 clade C and D Symbiodinium types had a selective advantage at high pCO2. Acclimatisation through changing symbiotic association therefore does not seem to be an option for Indo-Pacific corals to deal with future OA.

Does trophic status enhance or reduce the thermal tolerance of scleractinian corals? A review, experiment and conceptual framework.

Global warming, and nutrient and sediment runoff from coastal development, both exert increasing pressures on coastal coral reefs. The objective of this study was to resolve the question of whether coastal eutrophication may protect corals from thermal stress by improving their nutritional status, or rather diminish their thermal tolerance through the synergy of dual stressors. A review of previous studies on the topic of combined trophic status and heat exposure on the thermal tolerance of corals reveals a broad range of outcomes, including synergistic, additive and antagonistic effects. We conducted a 90-day long experiment exposing corals to realistic levels of elevated nutrients and sediments, and heat stress. Colonies of two common scleractinian corals (Acropora millepora and Montipora tuberculosa) were kept in coastal seawater, or coastal seawater that was further organically and nutrient enriched (OE), and/or enriched with nitrate. Batches of OE were created daily, facilitating nutrient uptake, plankton succession and organic enrichment as observed in coastal waters. After 10 days of acclimation, 67% of the colonies had their temperature gradually increased from 27° to 31.2°C. After 3-7 weeks of heat stress, colonies of both species had significantly greater reductions in fluorescence yields and lower survival in OE than without addition of OE. Furthermore, photophysiological recovery was incomplete 31-38 days after ending the heat stress only in the OE treatments. Nitrate alone had no measurable effect on survival, bleaching and recovery in either species. Skeletal growth rates were reduced by 45% in heat-stressed A. millepora and by 24% in OE-exposed M. tuberculosa. We propose a conceptual trophic framework that resolves some of the apparently contradictory outcomes revealed by the review. Our study shows that management actions to reduce coastal eutrophication can improve the resistance and resilience of vulnerable coastal coral reefs to warming temperatures.

A bioindicator system for water quality on inshore coral reefs of the Great Barrier Reef.

Responses of bioindicator candidates for water quality were quantified in two studies on inshore coral reefs of the Great Barrier Reef (GBR). In Study 1, 33 of the 38 investigated candidate indicators (including coral physiology, benthos composition, coral recruitment, macrobioeroder densities and FORAM index) showed significant relationships with a composite index of 13 water quality variables. These relationships were confirmed in Study 2 along four other water quality gradients (turbidity and chlorophyll). Changes in water quality led to multi-faceted shifts from phototrophic to heterotrophic benthic communities, and from diverse coral dominated communities to low-diversity communities dominated by macroalgae. Turbidity was the best predictor of biota; hence turbidity measurements remain essential to directly monitor water quality on the GBR, potentially complemented by our final calibrated 12 bioindicators. In combination, this bioindicator system may be used to assess changes in water quality, especially where direct water quality data are unavailable.

Chemical and physical environmental conditions underneath mat- and canopy-forming macroalgae, and their effects on understorey corals.

Disturbed coral reefs are often dominated by dense mat- or canopy-forming assemblages of macroalgae. This study investigated how such dense macroalgal assemblages change the chemical and physical microenvironment for understorey corals, and how the altered environmental conditions affect the physiological performance of corals. Field measurements were conducted on macroalgal-dominated inshore reefs in the Great Barrier Reef in quadrats with macroalgal biomass ranging from 235 to 1029 g DW m(-2) dry weight. Underneath mat-forming assemblages, the mean concentration of dissolved oxygen was reduced by 26% and irradiance by 96% compared with conditions above the mat, while concentrations of dissolved organic carbon and soluble reactive phosphorous increased by 26% and 267%, respectively. The difference was significant but less pronounced under canopy-forming assemblages. Dissolved oxygen declined and dissolved inorganic carbon and alkalinity increased with increasing algal biomass underneath mat-forming but not under canopy-forming assemblages. The responses of corals to conditions similar to those found underneath algal assemblages were investigated in an aquarium experiment. Coral nubbins of the species Acropora millepora showed reduced photosynthetic yields and increased RNA/DNA ratios when exposed to conditions simulating those underneath assemblages (pre-incubating seawater with macroalgae, and shading). The magnitude of these stress responses increased with increasing proportion of pre-incubated algal water. Our study shows that mat-forming and, to a lesser extent, canopy-forming macroalgal assemblages alter the physical and chemical microenvironment sufficiently to directly and detrimentally affect the metabolism of corals, potentially impeding reef recovery from algal to coral-dominated states after disturbance. Macroalgal dominance on coral reefs therefore simultaneously represents a consequence and cause of coral reef degradation.

Investigation of the mud crab (Scylla serrata) as a potential bio-monitoring species for tropical coastal marine environments of Australia.

Mud crabs, Scylla serrata, were sampled from four estuaries (the Normanby, Herbert, Burdekin and Fitzroy Rivers) along the coast of northern Queensland, Australia, representing a pollution gradient from low to high contamination based upon previous chemical monitoring. Four biomarkers; glutathione-S-transferase (GST) activity, cholinesterase (ChE) inhibition and the urinary metabolite concentrations of naphthalene (NPH) and benzo-a-pyrene (BaP) were evaluated in S. serrata hepatopancreas, haemolymph and urine. Site-specific evidence of elevated GST activity and BaP metabolite concentrations and significant ChE inhibition was detected. Biomarker responses from this field study provide evidence of contaminant exposure of S. serrata from river estuaries along the coast of northern Queensland and indicate that further investigation is warranted. Based on the current results, and with further work on characterising the dose-response and seasonal variation in this species, mud crabs have great potential as indicator species for water quality and ecosystem monitoring programs across tropical coastal regions of Australia.

Effects of the herbicide diuron on the early life history stages of coral.

The effects of the herbicide diuron on the early life history stages of broadcast spawning and brooding corals were examined in laboratory experiments. Fertilisation of Acropora millepora and Montipora aequituberculata oocytes were not inhibited at diuron concentrations of up to 1000 microg l(-1). Metamorphosis of symbiont-free A. millepora larvae was only significantly inhibited at 300 microg l(-1) diuron. Pocillopora damicornis larvae, which contain symbiotic dinoflagellates, were able to undergo metamorphosis after 24 h exposure to diuron at 1000 microg l(-1). Two-week old P. damicornis recruits on the other hand were as susceptible to diuron as adult colonies, with expulsion of symbiotic dinoflagellates (bleaching) evident at 10 microg l(-1) diuron after 96 h exposure. Reversible metamorphosis was observed at high diuron concentrations, with fully bleached polyps escaping from their skeletons. Pulse amplitude modulation (PAM) chlorophyll fluorescence techniques demonstrated a reduction in photosynthetic efficiency (Delta F/F(m)') in illuminated P. damicornis recruits after a 2 h exposure to 1 microg l(-1) diuron. The dark-adapted quantum yields (F(v)/F(m)) also declined, indicating chronic photoinhibition and damage to photosystem II.

Validation of an ELISA test kit for the detection of total aflatoxins in grain and grain products by comparison with HPLC.

An ELISA Microtiter Plate, Total Aflatoxin Test called AgraQuant was validated to measure total aflatoxins in a range from 4 to 40 ppb in corn, corn meal, corn gluten feed, corn gluten meal, corn germ meal, corn/soy blend, popcorn, sorghum, wheat, milled rice, soybeans, peanuts and cottonseed. The test is performed as a solid phase direct competitive ELISA using a horseradish peroxidase conjugate as the competing, measurable entity. For the test method, aflatoxins are extracted from ground samples with 70% methanol and sample extracts plus conjugate are mixed and then added to the antibody-coated microwells. After 15 min incubation at room temperature, the plate is washed and enzyme substrate is added and allowed to incubate for an additional 5 min. Stop solution is then added and the intensity of the resulting yellow color is measured optically with a microplate reader at 450 nm. Results obtained from internal validation studies assessing accelerated stability indicate a minimum of 1 year shelf life for the kits; accuracy and precision are comparable to HPLC in the range of 0-320 ppb and limit of detection in corn is 2.5 ppb. Comparison of the method to HPLC, ability to detect individual aflatoxins and ruggedness of the test kits at 18-30 degrees C determined this test to be rugged, sensitive, accurate, precise and effective comparable to HPLC for measuring total aflatoxins ranging from 4 to 40 ppb in the commodities evaluated.