Effects of waterborne iron on growth, reproduction, survival and haemoglobin in Daphnia magna

The effects of waterborne iron (FeCl3 X 6H2O) on growth, reproduction, survival and haemoglobin content in Daphnia magna were studied from subnormal to toxic concentrations in hard reconstituted water. Low concentrations of iron stimulated reproduction and haemoglobin synthesis after chronic exposure for 21 days. Maximum reproduction occurred between 0.1 and 1 microgram Fe 1(-1). Juvenile growth was not stimulated by iron but was slightly inhibited between 1 and 8 micrograms Fe 1(-1) and above 128 micrograms Fe 1(-1). A slight inhibition of growth persisted for 21 days. Total haemoglobin content was above the control with no waterborne iron at all but one concentration (512 micrograms Fe 1(-1]. The highest value (3.8 X control value) was found at 2 micrograms Fe 1(-1). The haemoglobin content decreased between 64 and 512 micrograms Fe 1(-1) and increased at higher concentrations. The decrease coincided with an inhibited reproduction. The increase was found in non reproductive survivors. A comparison with a previous study in D. magna suggests that ambient conditions (hardness and pH) and ageing of the water are important for the effects of waterborne iron. At a hardness of 250 mg 1(-1) as CaCO3 and a pH range of 7.0-8.0 the ZEP (Zero Equivalent Point) for reproduction was 158 micrograms Fe 1(-1). Continuous exposure to higher concentrations is expected to lead to extinction of a D. magna population.     

Effects of fluoride on growth, reproduction and survival in Daphnia magna

The effects of waterborne fluoride (NaF) on growth, reproduction and survival in Daphnia magna were studied from subnormal to toxic concentrations in hard reconstituted water. The 24- and 48-hr EC50S for immobilization were 205 and 98 mg F (fluoride) 1(-1). Median survival times for fed and unfed Daphnia were reduced at concentrations of F above 8.9 and 10 mg F1(-1), respectively. Growth, determined as body length after 7 and 21 days, was partially inhibited at all concentrations above 3.7 mg F1(-1). Parthenogenetic reproduction was stimulated by all concentrations (dilution factor 0.5) between 0.45 X 10(-3) mg F1(-1) and 3.7 mg F1(-1) and inhibited by all concentrations above 3.7 mg F1(-1), compared to the control with no waterborne fluoride. The highest concentration with a reproduction (number of live progeny/live female) equivalent to the control after 21 days was 4.4 mg F1(-1). However, a progressive decline in reproduction between 14 and 21 days indicates a slight long-term inhibition above 0.58 mg F1(-1). The "safe" concentration equivalent to the geometric mean of NOEC or MATC for D. magna in hard water is 4.4 mg F1(-1), derived as ZEP, the Zero Equivalent Point, for reproduction after 21 days.     

The effects of cadmium on ALA-D activity, growth and haemoglobin content in the water flea, Daphnia magna

The ALA-D activity, haemoglobin content and growth was studied in the water flea, Daphnia magna, exposed to 0, 0.1, 0.2, 0.4, 0.8 and 1.6 micrograms Cd/l. The ALA-D activity in water fleas exposed to 0.2-1.6 micrograms Cd/l fluctuated around the control value. The activity in animals exposed to 0.1 micrograms Cd/l decreased during the entire experiment. After 16 days exposure to cadmium the haemoglobin content in water fleas ranged between 80 and 31% of control value. In animals exposed to 0.8 and 1.6 micrograms Cd/l the haemoglobin content decreased progressively during the experiment. Growth was not affected by cadmium at these concentrations.     

Hormone-stimulated secretion of luteinization factor in porcine granulosa cells.

Luteinization stimulator (LS) is an intrafollicular compound which was shown to be released by granulosa cells under in vitro conditions with stimulatory effects on immature granulosa cell differentiation. This study was undertaken to determine the effects of various endocrine agents which are involved in the regulation of ovarian function on LS secretion by porcine granulosa cells isolated from 5-8-mm follicles (LGC). Cell conditioned media (CM) obtained after the 4-day culture of LGC were tested in the culture of immature (small) granulosa cells (SGC). The activity of LS released into the LGC conditioned medium was estimated by measuring progesterone (P4) produced by SGC in the presence of CM. Stimulation of P4 secretion was observed after addition of media from cultures treated by LHRH (10(-4) mol.l-1), epinephrine (10(-5) mol.l-1), LH (1 microgram.ml-1), dbcAMP (0.5 and 2.0 micrograms.ml-1) or insulin (1.0-5.0 micrograms.ml-1). Norepinephrine (10(-5) and 10(-7) mol.l-1), estradiol (0.1 and 1.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml-1) did not change steroidogenic activity of CM. Epinephrine and norepinephrine (10(-5) and 10(-7) mol.l-1), LH (1 microgram.ml-1), dbcAMP (2.0 microgram.ml-1) and estradiol (1 microgram.ml-1) alone enhanced P4 production by SGC, whereas LHRH (10(-3) and 10(-4) mol.l-1), insulin (1.0-5.0 microgram.ml-1) and prolactin (0.1 and 1.0 microgram.ml.-1) did not have any effect. These observations suggest that the process of LS secretion in developing follicles is subject to a specific hormonal control.     

Acclimation of Daphnia magna to environmentally realistic copper concentrations

It may be hypothesised that as the bioavailable background concentration of an essential metal increases (within natural limits), the natural tolerance (to the metal) of the acclimated/adapted organisms and communities will increase. In this study the influence of acclimation to different copper concentrations on the sensitivity of the freshwater cladoceran Daphnia magna Straus was investigated. D. magna was acclimated over three generations to environmentally relevant copper concentrations ranging from 0.5 to 100 microg Cu/l (copper activity: 7.18 x 10(-15) to 3700 x 10(-12) M Cu2+). A modified standard test medium was used as culture and test medium. Medium modifications were: reduced hardness (lowered to 180 mg CaCO3/l) and addition of Aldrich humic acid at a concentration of 5 mg DOC/l (instead of EDTA). The effects of acclimation on these organisms were monitored using acute mortality assays and long-term assays in which life table parameters, copper body concentrations and energy reserves were used as test endpoints. Our results showed a two-fold increase in acute copper tolerance with increasing acclimation concentration for second and third generation organisms. Copper acclimation concentrations up to 35 microg Cu/l (80 pM Cu2+) did not affect the net reproduction and the intrinsic growth rate. The energy reserves of the acclimated daphnids revealed an Optimal Concentration range (OCEE) and concentrations between 5 and 12 microg Cu/l (0.5-4.1 pM Cu2+) and 1 and 35 microg Cu/l (0.023-80 pM Cu2+) seemed to be optimal for first and third generation daphnids, respectively. Lower and higher copper concentrations resulted in deficiency and toxicity responses. It was also demonstrated that up to 35 microg Cu/l, third generation daphnids were able to regulate their total copper body concentration. These results clearly indicate that bioavailable background copper concentrations present in culture media have to be considered in the evaluation of toxicity test results, especially when the toxicity data are used for water quality guideline derivation and/or ecological risk assessment for metals.     

Notes on the reproduction and survival of Moina hutchinsoni Brehm, 1937 (Moinidae: Anomopoda) grown in media of varying salinity

Moina hutchinsoni, a North American species distributed in saline and alkaline waters, was isolated from temporary puddles in the former Texcoco Lake basin near Mexico City. Its growth under laboratory conditions was assayed at different salinity values. Two culture media were tested: reconstituted hard-water plus sea salts and diluted natural brine. The alga Ankistrodesmus falcatus was supplied as food at a concentration of 0.4 x 10⁶ cells ml^-1. Salinity levels ranged from 2 to 20. For all experiments, twenty replicates, consisting of one neonate female individually placed in a glass container with 80 ml culture volume, were used. All vessels were reviewed daily and the progeny counted and removed. Culture media and food were renewed each day. Data on the survival and reproduction of M. hutchinsoni were recorded during its entire life cycle. The high average longevity (22.6) days, number of clutches (17.8), and average total progeny (138.9 neonates). were recorded at a salinity of 10 of natural brine water. In the synthetic medium reconstituted hard water plus sea salts, the highest average longevity 9.2 days, one of the highest number of clutches (9.6), and the highest average total progeny (29.5) neonates. were recorded at a salinity of 4. Reproduction and survival was affected by salinity and the culture medium, with the best results recorded in diluted natural brine. The information presented here is the first biological data surveyed for this euryhaline species in experimental conditions.     

Waterborne metals impair inducible defences in Daphnia pulex: morphology, life-history traits and encounters with predators

1.  Inducible defences may be temporary and favoured where predation is intermittent and have been demonstrated in several invertebrates and vertebrates when prey detect chemical cues (kairomones) released by predators. Daphnia pulex (a water flea) exposed to Chaoborus (midge larvae) kairomones produce small neckteeth on the dorsal surface of the head as a defence against this gape-limited predator and survive better in the presence of Chaoborus . Recent studies have shown that waterborne copper (Cu) impairs the induction of neckteeth which could lead to lower survival.
2.  Here, we examined the effects of Cu on morphological changes and shifts in life-history traits in D. pulex exposed to kairomone from Chaoborus americanus . We exposed D. pulex mothers to chemical cues of C. americanus fed on either D. pulex neonates or on brine shrimp Artemia salina , the same Chaoborus cues combined with an environmentally relevant concentration of copper (10 μg L⁻¹), or dechlorinated tap water. We examined several morphological characteristics of neonates and life-historical characteristics of adults as well as assessing survival of neonates by staging encounters with predators.
3.  Neonates from mothers exposed to kairomone plus copper had fewer and shorter neckteeth than neonates from mothers exposed to kairomone alone. Moreover, neonates exposed to Cu had lower survival during encounters with predators than neonates exposed to kairomone without Cu.
4.  Adult female Daphnia exposed to kairomones released more neonates within the first 24 h of brood release and emptied their brood pouches quicker than mothers not exposed to kairomones, irrespective of the presence of Cu.
5.  Impairment by metals of morphological defences in zooplankton could lead to a decline in population density and alter community structure.     

Calcium accumulation and regulation in Daphnia magna: Links with feeding, growth and reproduction

Calcium is involved in a wide variety of biological processes and has an important structural role in crustaceans. The present study aimed at exploring the possible link between Ca body concentrations and the ingestion rate and the role of soft tissue vs. total tissue Ca accumulation in Daphnia magna. D. magna was cultured for 21 days at different water Ca concentrations ranging from 3.4 to 32.5 mg/L. Every week Ca body concentrations (soft and total tissues), ingestion rate, growth, survival and reproduction were measured. Daily, algal food that was not deficient in Ca was supplied. Ca in the soft tissues represented 8 to 26% of the total Ca body concentrations. The ratio Ca in soft tissue/Ca in total tissue was generally not influenced by the Ca exposure concentration but decreased with time, i.e., age (from an average of 0.24 at day 7 to 0.09 at day 21). During week 1, a 54% decrease in Ca body concentrations was observed in daphnids exposed in medium with 3.4 mg/L Ca compared to those exposed to 32.5 mg/L. The concurrent decrease in ingestion rate was 14%. No significant differences among Ca treatments were observed during week 2 for ingestion rate and week 3 for calcium body concentrations. Also, no effects on growth and reproduction were observed, although these were expected at the lowest Ca concentration tested. It is hypothesised that Ca absorption from food in combination with an increased ingestion rate is used to maintain Ca homeostasis under Ca limiting conditions.     

Copper removal by dry and re-hydrated biomass of Spirulina platensis

Dried and re-hydrated biomass of Spirulina platensis was employed as a sorbent in tests of copper removal from water. Biomass re-hydrated for 24 h before use exhibited a shorter adsorption time as well as an increased percentage removal when compared with simply dried biomass. The combined effects of the concentrations of re-hydrated biomass (from 1.0 to 4.0 g l-1) and copper (from 0.1 to 0.4 g l-1) were then investigated. Copper was almost entirely removed (91% removal) at relatively high biomass levels (X0>or=2.0 gDM l-1), while 1.0 gDM l-1 removed only 81% of copper present initially, suggesting a situation of excess metal with respect to the adsorption capacity of biomass. Additional tests performed with biomass re-hydrated for variable time demonstrated that no less than 48 h of this treatment are needed to ensure a satisfactory copper removal, while no significant improvement was detected using biomass re-hydrated for longer times.     

Copper status of muskoxen: a comparison of wild and captive populations

We compared wild muskoxen (Ovibos moschatus) on Banks Island (Northwest Territories, Canada) with captive animals maintained on grass (Bromus sp.) hay and supplemental minerals. We measured copper (Cu) in liver, whole serum, and deproteinated serum (unbound Cu) as well as serum activity of the Cu-enzyme ceruloplasmin. Unbound serum Cu concentrations did not change with season in captive animals (n = 53). Ceruloplasmin activity was similar between seasons in females but elevated in males during breeding in autumn. Increasing concentrations of Cu in whole sera were mainly associated with protein whereas unbound Cu predominated at low concentrations of whole serum Cu. Ceruloplasmin activity and serum Cu concentration were linearly related to liver Cu in female muskoxen. Measures of copper status in females were lower in the wild (n = 19) than in captivity (n = 16): 8 vs. 160 ug Cu.g-1 of whole liver; 0.67 vs. 1.15 microgram unbound Cu.ml-1 whole serum and; 22 vs. 33 IU.l-1 ceruloplasmin activity. Bioavailability of Cu may limit the population on Banks Island especially when density of animals is high. The wide range of hepatic Cu concentrations in muskoxen indicated accumulation of Cu without apparent ill effect in captive animals. Hepatic storage of Cu may allow wild muskoxen to contend with low and fluctuating availability of Cu in small foraging areas at high latitudes.     

Effect of food type and concentration on the survival,longevity, and reproduction of Daphnia magna

The freshwater microalgae Ankistrodesmus falcatus and Scenedesmus incrassatulus, at three concentrations (6, 12 and 18 mg l⁻¹ t, dry weight), were tested as diets for Daphnia magna cultured in reconstituted hard water, at 19 ± 1 °C. Effects on survival, and reproductive characteristics were evaluated during a complete life-cycle. Test animals fed with the highest food concentration had the lowest survival, longevity, number of clutches, and average inter-brood times, but the total offspring was similar to that obtained with the lowest food concentration, for the same species of alga. Average longevity ranged from 40 to 85 days, with a minimum of 24 and a maximum of 119 days. The average inter-brood time ranged from 4.2 to 5.8 days, the highest values being observed at the lowest food concentration. The average total offspring was maximum for the intermediate concentrations of both algae. The maximum number of clutches ranged from 9 to 23, with the lowest values being observed at the highest food concentration. Even though both microalgae had similar effects on survival and reproduction, and the greatest differences observed were related to food concentration, it seems that S. incrassalulus is a comparatively better food for D. magna. The most suitable food level should be determined prior to carrying out chronic bioassays, and it is also an important factor in cultures for obtaining neonates for toxicological tests.     

Refinement and field validation of a biotic ligand model predicting acute copper toxicity to Daphnia magna

A previously developed biotic ligand model (BLM) was validated for its capacity to predict acute 48-h EC(50) values of copper to Daphnia magna in 25 reconstituted media with different pH values and concentrations of artificial dissolved organic carbon, Ca, Mg and Na. Before the BLM validation, fitting of measured (with a copper ion-selective electrode) and calculated (with the BLM) Cu(2+)-activity was performed by adjusting the WHAM model V (i.e. the metal-organic speciation part of the BLM) copper-proton exchange constant to pK(MHA)=1.9. Using this value, the 48-h EC(50) values observed agreed very well with BLM-predicted EC(50) values for tests performed at pH<8, but not at all for tests performed at pH>8. Additional experiments demonstrated that this was due to toxicity of the CuCO(3) complex, which is the most abundant inorganic copper species at pH>8. This was incorporated into the initial BLM by allowing the binding of CuCO(3) (next to Cu(2+) and CuOH(+)) to the biotic ligand of D. magna. The affinity of CuOH(+) and CuCO(3) for the biotic ligand was approximately five- and 10-fold lower than that of Cu(2+), respectively. With the refined BLM, 48-h EC(50) values could be accurately predicted within a factor of two not only in all 25 reconstituted media, but also in 19 natural waters. This validated and refined BLM could support efforts to improve the ecological relevance of risk assessment procedures applied at present.     

Levels of protein, RNA, DNA, glycogen and lipid during growth and development of Daphnia magna Straus (Crustacea: Cladocera)

1. Protein, RNA, DNA, glycogen and lipid content were determined in Daphnia magna on days 0, 2, 4, 6, 8 and 21 of growth and development. The composition of D. magna as percentage of reconstituted dry weight was similar to other zooplankton with the exception of DNA content, which was lower than values previously reported.
2. The relative content of protein, RNA, DNA and reconstituted dry weight changed during the 21-day growth period, and these changes were related to growth rate and total growth of D. magna . RNA:protein, RNA: reconstituted dry weight, and protein:RNA : DNA ratios were highly correlated to relative growth rate and total growth as measured by protein content or reconstituted dry weight.
3. Addition of progeny biomass to adult biomass increased correlations between biochemical ratios and absolute growth rate, but had little effect on relationships involving relative growth rate or total growth.
4. The relationship between biomolecule ratios and growth established for D. magna grown under optimal conditions was not successful in predicting growth of D. magna reared under crowded conditions.
5. These data indicate that variation in biochemical ratios among life, stages of D. magna may be used to predict growth of organisms grown under similar conditions, but may not be extended to other situations. It is suggested, however, that variation in biochemical ratios in a particular life stage of a zooplankton species may be related to the productivity for that species.     

Effects of endosulfan on survival,growth and reproduction of Daphnia magna

1. Acute and chronic toxicity tests with endosulfan were conducted on Daphnia magna. The 24-hr static lc₅₀ was 0.62 mg/l with a coefficient of variation of 14.2%.2. The sublethal effects of 0.12, 0.15, 0.20, 0.25 and 0.31 mg/l endosulfan on the survival, reproduction and growth of D. magna were monitored for 21 days.3. The parameters used to determined the effect of the pesticide on reproduction were: mean total young per female, maximum number of broods, mean brood size, mean number of broods, mean time to first reproduction and intrinsic rate of natural increase (r).4. Growth, as measured by body length, was depressed significantly at all endosulfan concentrations tested. The highest concentrations used (0.20, 0.25 and 0.31 mg/l) caused a decrease in survival and mean total young per female, and an increase in the time to first reproduction. The intrinsic rate of natural increase (r) and the mean number of broods were decreased significantly at all the sublethal concentrations of endosulfan tested.     

Life in the extreme environment at a hydrothermal vent: haemoglobin in a deep-sea copepod.

This is the first study, to my knowledge, quantifying the respiratory pigment haemoglobin discovered in a deep-sea copepod. Haemoglobin in copepods has previously been documented in only one other species from the deep water of an Italian lake. Specimens of the siphonostomatoid Scotoecetes introrsus Humes were collected during submersible dives at 2500 m depth near a hydrothermal vent at the East Pacific Rise (9 degrees N). The haemoglobin content in the copepods' haemolymph was 4.3 +/- 0.6 micrograms per individual female (n = 6) and 1.8 +/- 0.1 micrograms per individual male (n = 6). Weight-specific concentrations of haemoglobin were identical for females and males (0.25 +/- 0.04 and 0.26 +/- 0.02 microgram per microgram dry weight, respectively). These haemoglobin concentrations are higher than those found in other small crustaceans. Activity of the electron transport system indicated that the respiration rates in S. introrsus (13.7 +/- 7.7 microliters O2 per milligram dry weight per hour) were similar to those in the shallow-water copepod Acartia tonsa (9.1 +/- 1.3 microliters O2 per milligram dry weight per hour). It was concluded that the possession of highly concentrated haemoglobin allows S. introrsus to colonize a geologically young, thermally active site such as the vicinity of a hydrothermal vent, despite the prevailing oxygen depletion.     

Effects of iron,copper, zinc,calcium, and magnesium on human lymphocytes in culture

The effects of simultaneous changes of calcium, magnesium, iron, copper, and zinc concentrations were evaluated in normal human T and B lymphocytes, cultured in cation-depleted media. Optimal concentrations for thymidine incorporation (TI) in both cell populations were Fe and Zn 15 μM and Cu 5 μM; for t cells Ca 2 mM and Mg 4 mM; for B cells Ca 4 mM and Mg 6 mM. TI decreased with increasing molarity of cations and the decrease was particularly apparent with Cu. Minimal amounts of Ca and Mg (0.5 mM) were necessary for growth, even in presence of optimal concentrations of Fe, Cu, and Zn. Fe and Cu showed synergistic stimulatory effects at low concentrations and synergistic inhibitory effects at high concentrations. Antagonism between Fe and Zn, Cu and Zn, and Ca and Zn was also demonstrated. CD4/CD8 increased with PHA stimulation in presence of Zn, and decreased with ConA stimulation in presence of Zn or Fe. The results demonstrate: (1) the relationship and interdependence of Fe, Cu, and Zn concentrations in modulating the growth of normal lymphocytes; (2) the stimulatory effects of Fe on B cells and Zn on CD8 positive cells; (3) the inhibitory effect of Cu at concentrations lower than those of Fe and Zn; (4) the requirement of Ca and Mg in certain concentration and ratio for the action of the other cations; and (5) the Ca and Mg requirement for the growth of B cells higher than T cells.     

Effects of nisin and temperature on survival, growth, and enterotoxin production characteristics of psychrotrophic Bacillus cereus in beef gravy.

The presence of psychrotrophic enterotoxigenic Bacillus cereus in ready-to-serve meats and meat products that have not been subjected to sterilization treatment is a public health concern. A study was undertaken to determine the survival, growth, and diarrheal enterotoxin production characteristics of four strains of psychrotrophic B. cereus in brain heart infusion (BHI) broth and beef gravy as affected by temperature and supplementation with nisin. A portion of unheated vegetative cells from 24-h BHI broth cultures was sensitive to nisin as evidenced by an inability to form colonies on BHI agar containing 10 micrograms of nisin/ml. Heat-stressed cells exhibited increased sensitivity to nisin. At concentrations as low as 1 microgram/ml, nisin was lethal to B. cereus, the effect being more pronounced in BHI broth than in beef gravy. The inhibitory effect of nisin (1 microgram/ml) was greater on vegetative cells than on spores inoculated into beef gravy and was more pronounced at 8 degrees C than at 15 degrees C. Nisin, at a concentration of 5 or 50 micrograms/ml, inhibited growth in gravy inoculated with vegetative cells and stored at 8 or 15 degrees C, respectively, for 14 days. Growth of vegetative cells and spores of B. cereus after an initial period of inhibition is attributed to loss of activity of nisin. One of two test strains produced diarrheal enterotoxin in gravy stored at 8 or 15 degrees C within 9 or 3 days, respectively. Enterotoxin production was inhibited in gravy supplemented with 1 microgram of nisin/ml and stored at 8 degrees C for 14 days; 5 micrograms of nisin/ml was required for inhibition at 15 degrees C. Enterotoxin was not detected in gravy in which less than 5.85 log10 CFU of B. cereus/ml had grown. Results indicate that as little as 1 microgram of nisin/ml may be effective in inhibiting or retarding growth of and diarrheal enterotoxin production by vegetative cells and spores of psychrotrophic B. cereus in beef gravy at 8 degrees C, a temperature exceeding that recommended for storage or for most unpasteurized, ready-to-serve meat products.     

Multigenerational Effects of Heavy Metals on Feeding,Growth, Initial Reproduction and Antioxidants in Caenorhabditis elegans

Earlier studies showed that toxicities of excessive metals lasted over generations. Yet, these studies mainly employed one-generation exposure, and the effects of multigenerational challenges need further studies. Presently, Caenorhabditis elegans were exposed to cadmium, copper, lead and zinc for four consecutive generations (G1 to G4) at environmental concentrations. The feeding, growth, initial reproduction, superoxide dismutase (SOD) and catalase (CAT) were determined. All data were represented in the percentage of that in control (POC), and POC in the control was normalized to 100%. In G1 and G2, the POC values in feeding, growth and initial reproduction were generally within 10% of the control (100%), indicating non-significant effects. The POC values in SOD and CAT were significantly higher than 100%, showing stimulatory effects. In G3 and G4, the POC values in feeding, growth and initial reproduction were significantly lower than 100%, showing inhibitory effects which were more severe in G4 than in G3. Meanwhile, SOD and CAT continuously showed stimulatory effects, and the stimulatory effects on SOD increased from G1 to G4. The effects with multigenerational challenges were different from those in one-generation exposure. The effects in later generations demonstrated the importance of multigenerational challenges in judging long-term influences of metals.     

Effects of 1,25 dihydroxyvitamin d3 on the morphology and alkaline phosphatase activity of ROS 17/2,8 osteoblastic cells: influence of time and dose

Osteosarcoma-derived osteoblastic cells were exposed to 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) either to 0.001-1,000 nmol.l-1 for 4 days or to 10 nmol.-1 for 1-21 days. Between 0.01 and 10 nmol.l-1, a dose-dependent increase in alkaline phosphatase (AP) activity was found, which rose to a maximum level at 10 nmol.l-1 (+55%). At higher doses (100 and 1,000 nmol.l-1), 1,25(OH)2D3 induced a decrease in AP activity of 40%. After 1 day at 10 nmol.l-1 a slight increase in AP activity was noted (+20%) which augmented with the duration of exposure. This stimulatory effect was highest after 8 days (+130%). In contrast, after 15 and 21 days, AP activity decreased by 30%. Under 1,25(OH)2D3, microtubules were observed mainly in the perinuclear region.