Physiological adaptations to low temperature and brine exposure in the circumpolar amphipod Gammarus wilkitzkii

Summary The amphipod Gammarus wilkitzkii does not survive being frozen totally into solid sea ice. When the animals are cooled in air or freezing seawater, they will freeze and die at a temperature of about-4° C. However, during sea ice growth, the amphipods may tolerate to stay in the vicinity of the ice by conforming to the ambient brine in a salinity range of 34 ppt to about 60 ppt. A passive relationship between the concentrations of the haemolymph and seawater Na⁺ and Cl^-, lowers the melting point of the body fluids of the animals, thus preventing internal ice formation at low temperatures.     

Behavioral aspects of cold tolerance in blackchin tilapia,Sarotherodon melanotheron,at different salinities

Synopsis Cold tolerance and behavioral responses of blackchin tilapia, Sarotherodon melanotheron, to rapidly decreasing temperatures were investigated at salinities of 5, 15, and 35 parts per thousand (ppt). Cold tolerance did not significantly differ with salinity or social rank. Mean temperatures were 10.7° C for beginning loss of equilibrium, 9.6° C for complete loss of equilibrium, and 6.9° C for death at all salinities. Behavioral activity declined with decreasing temperature and ceased between 10–12° C. Certain behavioral actions were significantly more frequent at 15 or 35 ppt salinity than at 5 ppt. The northward range expansion by introduced populations of the blackchin tilapia in the United States probably will be limited by its lower lethal temperature limits, but may also be affected by temperatures at which social behavior becomes disrupted.     

Life history divergence of sympatric diploid and polyploid populations of brine shrimp Artemia parthenogenetica

In order to study how polyploidy affects life history patterns in animals, we have examined sympatric diploid and polyploid brine shrimp (Artemia parthenogenetica) from China, Italy and Spain under laboratory conditions. At optimal temperature and salinity (25°C and 90 ppt), diploids from the three populations had much higher intrinsic rates of increase, higher fecundity, faster developmental rates, and larger brood sizes than their sympatric polyploids. The Chinese and Italian populations were selected for further analysis to determine the life history responses of diploids and polyploids to temperature and salinity changes. Under intermediate and high salinities, Chinese and Italian polyploids produced most of their offspring as dormant cysts while their sympatric diploids produced most of their offspring as nauplii. This relationship is reversed in the Spanish diploid-polyploid complex. For the Chinese population at 25° C, pentaploid clones had higher developmental rates than diploid clones at 35 ppt; at 90 ppt, diploid clones had higher developmental rates than the pentaploids. Italian diploids and tetraploids had different responses to variation in both temperature (25° C and 31° C) and salinity (30 ppt and 180 ppt). Our results demonstrate that relative fitness of the two cytotypes is a function of environmental conditions and that sympatric diploids and polyploids respond differently to environmental changes. Chinese and Italian polyploids are expected to have lower fitness than their sympatric diploids when the physical environment is not stressful and when intraspecific competition is important. However, polyploids may have advantages over sympatric diploids in stressful habitats or when they encounter short-term lethal temperatures. These results suggest that polyploid Artemia have evolved a suite of life-history characteristics adapting them to environments that contrast to those of their sympatric diploids.     

Effects of temperature, salinity, and air exposure on development of the estuarine pulmonate gastropod Amphibola crenata

The primitive pulmonate snail Amphibola crenata embeds embryos within a smooth mud collar on exposed estuarine mudflats in New Zealand. Development through hatching of free-swimming veliger larvae was monitored at 15 salinity and temperature combinations covering the range of 2-30 ppt salinity and 15-25 °C. The effect of exposure to air on developmental rate was also assessed. There were approximately 18,000 embryos in each egg collar. The total number of veligers released from standard-sized egg collar fragments varied with both temperature and salinity: embryonic survival was generally higher at 15 and 20 °C than at 25 °C; moreover, survival was generally highest at intermediate salinities, and greatly reduced at 2 ppt salinity regardless of temperature. Even at 2 ppt salinity, however, about one-third of embryos were able to develop successfully to hatching. Embryonic tolerance to low salinity was apparently a property of the embryos themselves, or of the surrounding egg capsules; there was no indication that the egg collars protected embryos from exposure to environmental stress. Mean hatching times ranged between 7 and 22 days, with reduced developmental rates both at lower temperature and lower salinity. At each salinity tested, developmental rate to hatching was similar at 20 and 25 °C. At 15 °C, time to hatching was approximately double that recorded at the two higher exposure temperatures. Exposing the egg collars to air for 6-9 h each day at 20 °C (20 ppt salinity) accelerated hatching by about 24 h, suggesting that developmental rate in this species is limited by the rates at which oxygen or wastes can diffuse into and from intact collars, respectively. Similarly, veligers from egg capsules that were artificially separated from egg collars at 20 °C developed faster than those within intact egg collars. The remarkable ability of embryos of A. crenata to hatch over such a wide range of temperatures and salinities, and to tolerate a considerable degree of exposure to air, explains the successful colonization of this species far up into New Zealand estuaries.     

Abundance and growth response of microalgae at Megalon Embolon solar saltworks in northern Greece: An aquaculture prospect

There is continuous interest in many countries in maintaining and manipulating the rich ecological value of hypersaline ecosystems for aquaculture. The Megalon Embolon solar saltworks (northern Greece) were studied in sites of increasing salinity of 60–144 ppt to evaluate Dunaliella salina abundance and microalgal composition, in relation to physical and chemical parameters. Cluster and ordination analyses were performed based on the biotic and abiotic data matrices. Using fresh aliquots from 60 and 140 ppt salinity waters, phytoplankton performance was appraised with flask cultures in the laboratory by varying the inorganic PO₄-P concentration at 23 °C and 30 °C. At the saltworks, among the most abundant microalgae identified were species of the genera Dunaliella, Chlamydomonas, Amphora, Navicula, and Nitzschia. Dunaliella salina populations were predominant comprising 5–22% of the total microalgal assemblages during spring, but only 0.3–1.0% during the summer, when grazing by Artemia parthenogenetica and Fabrea salina was intense. D. salina cell density in April–July was in the range of 0.4–12.5 × 10⁶ L⁻¹ with typical densities of 1.5–4.5 × 10⁶ L⁻¹. Overall, microalgal densities were high in salinities of ≥100 ppt when inorganic-P concentrations were ≥0.20 mg L⁻¹ within saltworks waters. Multivariate analysis of species abundance showed that algal growth responses were primarily related to variation in salinity and inorganic-P concentrations, but also to NO₃-N concentration. In the laboratory, experiments indicated effective fertilization and denser microalgal growth under high inorganic PO₄-P applications (4.0 and 8.0 mg L⁻¹) at 60 ppt salinity and 23 °C. The lower PO₄-P applications (0.6–2.0 mg L⁻¹) were more effective at 60 ppt salinity and 30 °C. At 140 ppt salinity, microalgal growth response was less obvious at any of the corresponding phosphorus concentrations or temperatures. In both salinity experiments, Dunaliella salina bloomed easily and was predominant among the microalgae. Our observations indicate that Dunaliella salina populations and the overall rich microalgal profile of the saltworks, along with their performance in laboratory mono–and mixed cultures hold promise for mass cultivation within the M. Embolon saltworks basins.     

Salinity tolerance and osmoregulation of the arctic marine amphipods Onisimus litoralis (Kroyer) and Anonyx nugax (Phipps)

Summary The gammarid amphipod Onisimus litoralis, which inhabits arctic and subarctic intertidal and under-ice habitats, is a euryhaline hyperosmotic regulator. It survives 10 d exposures to salinities from 5 to 55 ppt. It hyperregulates its hemolymph osmolality during 3 h exposures to dilutions of 33 ppt seawater and remains hyperosmotic for at least 2 w. The hemolymph is isosmotic to the medium after 12 h exposures to salinities higher than 33 ppt. The gammarid amphipod Anonyx nugax, which inhabits arctic and subarctic subtidal areas, tolerates salinities from 23 to 45 ppt with little mortality. Unlike Onisimus, however, it is an osmoconformer and its hemolymph becomes isosmotic to all dilute salinities within its tolerance range after 12 h and to concentrated media after 3 h. The salinity tolerances and osmoregulatory abilities of both species are reflected in their distributions in the field.     

The combined influence of sub-optimal temperature and salinity on the in vitro viability of Perkinsus marinus, a protistan parasite of the eastern oyster Crassostrea virginica

Perkinsus marinus is a major cause of mortality in eastern oysters along the Gulf of Mexico and Atlantic coasts. It is also well documented that temperature and salinity are the primary environmental factors affecting P. marinus viability and proliferation. However, little is known about the effects of combined sub-optimal temperatures and salinities on P. marinus viability. This in vitro study examined those effects by acclimating P. marinus at three salinities (7, 15, 25 ppt) to 10 °C to represent the lowest temperatures generally reached in the Gulf of Mexico, and to 2 °C to represent the lowest temperatures reached along the mid-Atlantic coasts and by measuring changes in cell viability and density on days 1, 30, 60 and 90 following acclimation. Cell viability and density were also measured in 7 ppt cultures acclimated to each temperature and then transferred to 3.5 ppt. The largest decreases in cell viability occurred only with combined low temperature and salinity, indicating that there is clearly a synergistic effect. The largest decreases in cell viability occurred only with both low temperature and salinity after 30 days (3.5 ppt, 2 °C: 0% viability), 60 days (3.5 ppt, 10 °C: 0% viability) and 90 days (7 ppt, 2 °C: 0.6 ± 0.7%; 7 ppt, 10 °C: 0.2 ± 0.2%).     

Characterization of an antifreeze protein from the polar diatom Fragilariopsis cylindrus and its relevance in sea ice

Antifreeze proteins (AFPs), characterized by their ability to separate the melting and growth temperatures of ice and to inhibit ice recrystallization, play an important role in cold adaptation of several polar and cold-tolerant organisms. Recently, a multigene family of AFP genes was found in the diatom Fragilariopsis cylindrus, a dominant species within polar sea ice assemblages. This study presents the AFP from F. cylindrus set in a molecular and crystallographic frame. Differential protein expression after exposure of the diatoms to environmentally relevant conditions underlined the importance of certain AFP isoforms in response to cold. Analyses of the recombinant AFP showed freezing point depression comparable to the activity of other moderate AFPs and further enhanced by salt (up to 0.9 °C in low salinity buffer, 2.5 °C at high salinity). However, unlike other moderate AFPs, its fastest growth direction is perpendicular to the c-axis. The protein also caused strong inhibition of recrystallization at concentrations of 1.2 and 0.12 μM at low and high salinity, respectively. Observations of crystal habit modifications and pitting activity suggested binding of AFPs to multiple faces of the ice crystals. Further analyses showed striations caused by AFPs, interpreted as inclusion in the ice. We suggest that the influence on ice microstructure is the main characteristic of these AFPs in sea ice.     

Effect of acute pH depression on the survival of the freshwater amphipod Hyalella azteca at variable temperatures: field and laboratory studies

Field observations on temperature and pH of a small pond showed that a amphipod population of Hyalella azteca was exposed to variable seasonal pH between 5.10–5.85, and water temperatures between 2–21 °C. Laboratory experiments were designed to simulate seasonal temperatures and field pHs of a small pond habitat. Laboratory bioassay experiments were conducted to determine the survival of Hyalella azteca at pHs 4, 5, 6 and 7, and varying temperatures of 5°, 10°, 15°, 20° and 25 °C.The LT₁₀₀ at pH 4 and 25 °C was 5.7 ± 0.47 days, compared to 47.3 ± 2.49 days at 5 °C. An Analysis of Variance (ANOVA) showed temperature was a significant (p > 0.0001) source of variation in the acute lethality of pH to H. azteca. A Duncans Multiple Range Test (DMRT) further showed that in laboratory experiments at pH 4, there was a significant difference ( = 0.01) between the LT_100s at 5°, 10°, 15° and 20 °C, but not between temperatures 20° and 25 °C.     

Aerial and aquatic oxygen uptake by freely-diving snapping turtles (Chelydra serpentina)

Summary Aerial oxygen consumption of unrestrained, freely-diving warm-and cold-acclimated snapping turtles, Chelydra serpentina, was measured at 10, 20, and 30°C. Also, simultaneous determinations of aerial and aquatic oxygen uptake by voluntarilydiving animals were made at 4 and 20°C. The standard rates of aerial oxygen consumption are equivalent in cold-and warm-acclimated animals in water and in cold-acclimated ones in air; these rates are all lower than those of warm-acclimated animals in air. Thus either cold acclimation or voluntary submergence reduces the standard metabolic rate of snapping turtles but the effects are not additive. Aquatic oxygen uptake during voluntary submergence is more important at low than at moderate temperatures and probably contributes significantly to gas exchange in these animals as they overwinter beneath the ice of ponds and streams.     

Factors affecting low temperature preservation of the marine rotifer Brachionus rotundiformis Tschugunoff

Experiments were performed to determine suitable conditions for low temperature preservation of small S (Fukuoka) and ultra-small SS (Thai) strains of B. rotundiformis. For this, single rotifers (an adult bearing one egg or a 4-h neonate) were incubated for 10 days in 1 ml seawater (22 ppt salinity). The highest survival was achieved at 10 and 12 °C for S-strain and 12 °C for SS-strain. The effect of salinity, change of culture medium and feeding regime were further tested on rotifers (300 ind. ml^–1) cultured in vials containing 10 ml seawater and microalgae at 12 °C. Survival of S-strain was highest (55.5±0.8%) at 35 ppt, while SS-strain survived best (43.1±2.6%) at 17 ppt. Survival was suppressed by changing the culture medium every 4 days. Feeding rotifers every 2 days yielded better survival (66.2±6.6%: S-strains, cultured at 35 ppt and 81.8±5.2%, SS-strains cultured at 17 ppt) than feeding them only at the beginning of the experiment or at 4-day intervals. An acclimation at 20 °C for 24 h before transferring them from their usual culture temperature (28 °C) to 12 °C resulted in higher survival of SS-strain. For S-strain, however, no significant improvement resulted from acclimation. SS-strain was more susceptible to lower temperature and higher salinity than S-strain.     

Microalgae for use in tropical aquaculture II: Effect of salinity on growth,gross chemical composition and fatty acid composition of three species of marine microalgae

The influence of salinity on the growth, gross chemical composition and fatty acid composition of three species of marine microalgae,Isochrysis sp.,Nannochloropsis oculata andNitzschia (frustulum), was investigated. There was no significant change in growth rate ofIsochrysis sp. andN. (frustulum) over the experimental range of salinity (10–35 ppt), whileN. oculata had a significantly slower growth rate only at 35 ppt. The ash content of all three species increased with increasing salinity. Two species,Isochrysis sp. andN. oculata, showed significant linear increases in total lipid content with increasing salinity over the range 10 to 35 ppt.N. (frustulum) showed significant linear decrease in total lipids, with the highest percentage at low salinity within the range 10–15 ppt. Variation in salinity had only a slight effect on the total protein, the soluble carbohydrate and chlorophylla content of all species. All species responded to change in salinity by modifying their cellular fatty acid compositions. Significant positive correlations were observed between increase in salinity and increase in the percentage ofcis-9-hexadecenoic acid [16:1 (n-7)] over the entire experimental range inN. (frustulum) and between 25–35 ppt inN. oculata. There were curved relationships between salinity and percentage of hexadecanoic acid [16:0] inN. oculata andN. (frustulum), with maxima within the range 25–30 ppt for both species. A curved relationship was found between salinity and percentage of eicosapentaenoic acid [20–5(n-3)], forN. (frustulum), with lowest percentages of the fatty acid within the range 25–30 ppt. There was no consistent pattern in the percentages of other major fatty acids as functions of salinity. The Northern Territory isolateN. (frustulum) was unusual in having a substantial increase in total fatty acids with decreasing salinity (85 mg g^–1 dry wt at 10 ppt compared with 33 mg g^–1 at 35 ppt). The optimum salinities for the production of maximum amount of lipids and the essential fatty acids 20:5(n-3) and/or 22:6(n-3) were as follows:25 ppt forIsochrysis sp. [22:6(n-3)]; 20–30 ppt forN. oculata [20:5(n-3)]; 10–15 ppt forN. (frustulum) [20:5(n-3) and 22:6(n-3)].Author for correspondence     

Effects of the multiple stressors high temperature and reduced salinity on the photosynthesis of the hermatypic coral Galaxea fascicularis

This study investigates the physiological responses in the hermatypic coral Galaxea fascicularis exposed to salinity stress (from 37 ppt to 15 ppt) for 12 h, combined effects of reduced salinity (from 37 ppt to 20 ppt) and two temperatures (26 °C and 32 °C) for 12 h and combined effects of reduced salinity (from 37 ppt to 25 ppt) and two temperatures (26 °C and 29.5 °C) for 10 d. The results demonstrate that the coral is tolerant to 12 h exposure to extremely low salinity (15 ppt). The study also shows that combined effects of temperature and low salinity aggravate the damage on the photosynthesis of the symbiotic dinoflagellates in 12 h exposure to 20 ppt sea water. This study suggests that high temperature (29.5 °C) aggravates the damage of trivially low salinity (30 ppt) on the holobiont (the coral and its symbiotic dinoflagellates) in 10 d exposure. However, high temperature (29.5 °C) may have an antagonistic effect between temperature and low salinity (25 ppt) on metabolism of the holobiont. Based on the above results, we suggest that (1) the true mechanism of corals exposed to combined effects of low salinity and high temperature is complicated. This calls for more studies on different corals. Future studies should aim at investigating long-term low-level stress in order to simulate in situ conditions more accurately; (2) when corals exposed to extremely severe combined stressors for short-term or trivially severe stressors for relative long-term, the combined effects of two stressors (such as low salinity and high temperature) may be negative, otherwise, the effects may be additive.     

Meteorology and hydrography of a shoaling reef flat on the Caribbean coast of Panama

We describe the seasonal patterns and frequency distributions of meteorological and hydrographic conditions on a windward, shoaling reef flat at Punta Galeta, Panama (9° 24 N lat.), between 1974 and 1985. The factors monitored were wind speed, wind direction, air temperature, rainfall, solar radiation, water level, water temperature, and salinity. All conditions showed strong seasonal periodicity; however, the timing, duration, and amplitude of the seasonal fluctuations differed among years. The greatest variation occurred in 1981 and 1982, leading into an El Niño event. Emergence of the reef flat and extremes of water temperature were the most apparent physiological stresses; both were dependent on mean water levels. The seasonal pattern of emergence time was inversely related to mean water level. Extreme water temperatures only occurred during low water tevels, ranging between 22° to >37°C in depths <15 cm, but staying between 25°C and 30.9°C in depths <35 cm. Water temperatures averaged 1.5°C higher than air temperatures. Although rainfall was 200 to 400 cm year^-1, salinity remained between 24 and 36 ppt, with more than 98% of the records 30 ppt. The relationship of water temperature to depth is consistent with the hypothesis that the physical environment becomes more stressful when a reef reaches sea level and forms a shoaling platform. Mailing address: Smithsonian Tropical Research Institute, APO Miami, Florida 34002-0011, USA     

The environmental effects of salinity and temperature on the oxygen consumption and total body osmolality of the marine flatworm Procerodes littoralis

The present study examined the effect of salinity and temperature on the rate of oxygen consumption and total body osmolality of the triclad turbellarian Procerodes littoralis, a common marine flatworm normally found in areas where freshwater streams run out over intertidal areas. Extremes in environmental factors encountered by P. littoralis were recorded at the study site. These were salinity (0-44 psu), temperature (2.7-24.9 °C) and oxygen concentration (2.8-16.1 mg l⁻¹). Respirometry experiments showed minimal oxygen consumption rates at the salinity extremes encountered by the study species (0 and 40 psu). Further experiments showed relatively constant oxygen consumption rates over the temperature range 5-20 °C and elevated consumption rates at temperatures above 25 °C. Total body osmolality of P. littoralis increased with increasing salinity. The study illustrates how a marine flatworm uses integrated physiological and behavioural mechanisms to successfully inhabit an environment that is predominantly freshwater for up to 75% of the tidal cycle.     

Effects of temperature and salinity on survival of toxigenicVibrio cholerae O1 in seawater

In 1991 and 1992, the Latin American epidemic strain of Vibrio cholerae O1 was isolated from ballast water, bilge water, and sewage taken from cargo ships docked in Mobile Bay, Alabama. The findings raised questions regarding the organism's ability to survive long-term aboard ships and to withstand the exchange of ballast at sea. The effects of temperature (6, 18, and 30°C) and salinity (8, 16, and 32 ppt) on survival of V. cholerae O1 strains C6706 and C6707 and a ballast water isolate in sterile seawater were determined. The ballast water isolate, which had a D-value (number of days required to produce a 1 log₁₀ reduction in colony-forming units per milliliter) of 240 days at 18°C, 32 ppt salinity, had the longest survival time. The range of D-values was 36–240 days at 18°C, 60–120 days at 30°C, and 5–20 days at 6°C. In sterile seawater short-term survival was temperature dependent, whereas long-term survival was salinity dependent. In raw seawater, survival time of the ballast water isolate was reduced to 12–27 days, implying the existence of biological influences. As also shown in our previous work, the organism appeared to be able to survive for several months under relatively stable conditions in ballast water aboard ships; however, viability may be reduced to only a few weeks after the organism is introduced into estuarine or marine environments. Correspondence to: Susan A. McCarthy.     

Cold hardiness of the eggs of the plaice,Pleuronectes platessa

Summary The pelagic eggs of the plaice, Pleuronectes platessa, at the stage of first heart beat, can tolerate freezing into solid sea ice at temperatures down to-6°C. A long term freezing period of 5 days, at sublethal temperatures of-3 to-4°C, had only a minor effect on the eggs. When the ice was absent from the medium, the eggs supercooled readily to temperatures around-15° to-20°C. However, no thermal hysteresis agents were present in the eggs at low temperature and the cold hardiness therefore seem to rely on a high tolerance to brine solutions at low temperatures.     

The effect of temperature,irradiance and animal size on incorporation rates of Simocephalus vetulus

Carbon incorporation rates of Simocephalus vetulus were measured to study the effects of the physical state of the animals, size of the animal, varying temperature and light conditions. Physical state of the animal showed little effect on incorporation rates after the first hour. Incorporation rates increased in proportion to the third power of animal size. Experimental animals collected at temperatures of 12, 20 or 25°C fed maximally at 10, 15 and 25°C respectively, when subjected to a feeding temperature range of 5 to 30°C. We have interpreted this as an indication that S. vetulus is able to acclimate and incorporate maximally at various temperatures after prolonged exposure to that temperature. When fed over an irradiation range of 0 to 4.68 × 10^–3 cal cm^–2 s^–1 incorporation rates were indirectly proportional to irradiance. This suggests a response to decreased irradiance in the weedy, littoral habitat of these animals.     

Physiological constraints on the life cycle and distribution of the Antarctic fairy shrimp <Emphasis Type="Italic">Branchinecta gaini</Emphasis>

Maturation to adulthood and successful reproduction in the Antarctic fairy shrimp, Branchinecta gaini, must be completed within a physiologically challenging temporal window of ca. 2.5 months in the southern Antarctic Peninsula. Although adults show considerable metabolic opportunism at positive temperatures, little is known of their tolerance of two physiological insults potentially typical to pool life in the maritime Antarctic: sub-zero temperatures and salinity. B. gaini are freeze-avoiding crustaceans with temperatures of crystallisation (T _cs) of −5°C. No antifreeze proteins were detected in the haemolymph. Adults osmoregulate in relation to temperature, but rapid mortality in saline solutions of even low concentration, indicate they cannot osmoregulate in relation to salinity. Survival of ice encasement at temperatures above their T _c was found to be pressure but not time dependent: at severe inoculative ice pressures, there was little immediate survival and none survived after 48 h below −2°C; at mild inoculative ice pressures, immediate survival was ca. 100% at −3°C, but <20% after 48 h. There was no significant difference in survival after 1 and 6 h encasement at −3°C. Observations of ventilation suggest that it is not low temperature per se, but ice that represents the primary cryo-stress, with ventilatory appendages physically handcuffed below the freezing point of pool water. Both sub-zero temperatures and salinity represent real physiological constraints on adult fairy shrimp.     

Feeding kinetics of Brachionus plicatilis fed Isochrysis galbana

Clearance and ingestion rates of Brachionus plicatilis were measured using ¹⁴C-labeled Isochrysis galbana Tahiti. Experiments were conducted at 20–22 °C, 20 ppt salinity, and algal concentrations ranging from 0.13–64 mg C 1^–1. Clearance rates were constant and maximal at concentrations <2 mg C 1^–1, with maximum rates ranging from 3.4–6.9 µl ind.^–1 hr^–1. The ingestion rate varied with food concentration, and was described by a rectilinear model. The maximum ingestion rate varied considerably, and was dependent on the growth rate of the rotifers. Depending on the pre-conditions, B. plicatilis ingested about 0.5 to 2 times its body carbon per day at saturating food concentrations.