Lead toxicity at different life stages of the giant prawn (Macrobrachium rosenbergii,de Man): considerations of osmoregulatory capacity and histological changes in adult gills

This study evaluated the acute toxicity of lead in different life stages of the freshwater prawn Macrobrachium rosenbergii, determined the effect of sublethal Pb concentrations on osmoregulatory capacity (OC), measured the Pb level in gills, and investigated the effect of Pb on the structure of the gills of adult prawns. The 24-, 48-, and 96-h LC₅₀ values for Pb to M. rosenbergii increased progressively with increasing life stage, from post-larvae (PL), juvenile to adult. The 24- and 48-h LC₅₀ values for post-larvae ranged from 0.01- to 0.09-mg Pb/L. The 24-, 48-, and 96-h LC₅₀ values for Pb were lower at 12 ppt than those at 0 ppt for either the juveniles or the adults. At 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.47-, 0.58-, and 2.03-mg Pb/L, respectively. Meanwhile, at 12 ppt, the 96-h LC₅₀ values in PL11, juvenile and adult were 0.63-, 4.44-, and 7.98-mg Pb/L, respectively. In adults, the OC values of controls and prawns exposed to 2- and 4-mg Pb/L at 0 ppt were not significantly different. The OC of prawns exposed to and 2-mg Pb/L at 12 ppt increased by 72 and 109% from the OC of the control prawns. At media 12 ppt, the OC value of prawns exposed to 1-mg Pb/L was significantly different from that of prawns exposed to 2-mg Pb/L. The concentrations of Pb in gill tissues increased significantly in Pb exposed prawns both at 0 and 12 ppt. The level of Pb in gills of prawns exposed to 2-mg Pb/L at 12 ppt was not significantly different from those exposed at 0 ppt. The severe toxic actions of Pb were noted in gills of prawns exposed in media 12 ppt. Hyperplasia and necrosis were observed in gill lamellae, resulting in abnormal gill tips after Pb exposure at media 12 ppt. Since the effect of Pb is more pronounced in higher salinity (12 ppt) than in freshwater (0 ppt) it is clear that aquaculture of M. rosenbergii should be conducted in freshwater ponds.     

Effect of cadmium on survival,osmoregulation and gill structure of the Sunda prawn,Macrobrachium sintangense (de Man), at different salinities

The prawn Macrobrachium sintangense is likely to be subjected to occasional exposure to combined metal and saline stressors in its natural environment. This research evaluated the acute toxicity (96?h LC₅₀) of cadmium (Cd) on the prawn M. sintangense, with respect to the osmoregulatory capacity (OC) of prawns and to document histological changes in the gills after exposure to sublethal Cd concentrations at different salinities. The 96?h LC₅₀ of Cd to M. sintangense decreased with increasing salinity. The 96?h LC₅₀ values were 89.12 (72.53–109.50), 681.26 (554.20–837.46) and 825.37 (676.99–1006.27) μg CdL^?1 at 0, 10 and 20 ppt, respectively. The OC of prawns exposed to 30?μg?CdL^?1 at 0 ppt and to 300?μg?CdL^?1 at10 ppt decreased significantly compared with that of control prawns exposed to 0 and 10 ppt respectively. Swelling, hyperplasia and necrosis of gill lamellae resulting in the loss of marginal canals were observed in the gills of prawns exposed to 30?μg?CdL^?1 at 0 ppt and to 300?μg?CdL^?1 at 10 ppt for 7?days.     

Acute toxic impacts of three heavy metals (copper,zinc, and cadmium) on <Emphasis Type="Italic">Diaphanosoma brachyurum</Emphasis> (Cladocera: Sididae)

Diaphanosoma brachyurum (Cladocera: Sididae) is a common limnetic species in summer-temperate and tropical water bodies. Few studies have investigated the sensitivity of D. brachyurum to toxic chemicals despite this species often being dominant in natural lakes and ponds. We performed acute toxicity tests of three heavy metals, copper (Cu), zinc (Zn), and cadmium (Cd), to D. brachyurum. For D. brachyurum, the lethal concentration (LC)₅₀ values of Cu (24-h LC₅₀ = 16.4 μg/L, 48-h LC₅₀ = 10.4 μg/L) and Zn (24-h LC₅₀ = 253.4 μg/L, 48-h LC₅₀ = 174.1 μg/L) were lower than those for D. magna, one of the most used test organisms for toxic chemicals. On the other hand, for D. brachyurum the 24-h LC₅₀ of Cd (166.4 μg/L) was much greater than that for D. magna, and the 48-h LC₅₀ of Cd (69.8 μg/L) was comparable. Our results indicate that D. brachyurum may be more strongly influenced by Zn and Cu than is D. magna. It is likely that the summer plankton community in which Diaphanosoma species is dominant is more sensitive to heavy metals than a community in which Daphnia species are dominant.     

Effect of avermectin (AVM) on the expression of γ‐aminobutyric acid A receptor (GABAAR) in Carassius gibelio (Bloch, 1782)

This study describes the effect of avermectin (AVM) on the expression of γ‐aminobutyric acid A receptor (GABA_AR) in Carassius gibelio. To assess the specific expression of GABA_AR in the brain, gonads, liver, kidneys, heart, muscles, and skin of C. gibelio, the expression of GABA_AR α1 subunit (GABA_ARα1) was measured by Western blotting. To study the effects of AVM on the expression of GABA_AR, the median lethal concentration (LC₅₀) at 24, 48, and 96 h of AVM was determined and the expression of GABA_AR in the brain, liver, and kidneys of the corresponding C. gibelio evaluated by Western blotting and immunohistochemistry. The results show that GABA_AR was expressed in the brain, gonads, liver, kidneys, heart, intestines, muscles, and skin, while primarily distributed in the central nervous system and moderately distributed in peripheral tissues. The expression of GABA_AR in the brain, liver, and kidney tissues of C. gibelio was increased with the treatment of AVM at 24 h LC₅₀, but attenuated by the treatment of AVM at 48 h LC₅₀ and 96 h LC₅₀. This suggests a threshold effect of AVM.     

Distribution and Toxic Effects of Cadmium and Lead on Maize Roots

Two-day-old seedlings of maize (Zea mays L.) were incubated on Cd and Pb nitrate solutions at the concentrations that inhibited root growth approximately by 50% after two-day-long incubation (LC₅₀; 10^–4 and 10^–3 M, respectively) or completely terminated growth of the primary root after one-day-long incubation (LC; 5 × 10^–4 and 10^–2 M, respectively). Cd and Pb contents were measured using an anodic inversion voltammetric technique in a flow injection system and a histochemical method. At LC₅₀, Cd and Pb were discerned, by histochemical techniques, in all root apical tissues, whereas in the root hair zone, the heavy metals were primarily accumulated in the apoplast of the rhizodermis and cortex and to a lesser extent, in the vascular tissues and parenchyma cells surrounding the metaxylem vessels. Insignificant accumulation of Cd and Pb in the pericycle probably explains why root branching was tolerant to these agents. At LC, Cd and Pb were found in the apoplast of all root tissues, in accordance with the practically complete inhibition of root growth and branching. Irrespectively of Cd and Pb concentrations in the external solution, the metal contents in the root apex exceeded those in the basal region. Procion dyes were used to assess cell death inflicted by Cd and Pb. At LC, the root cap and meristematic cells perished, together with the rhizodermal cells and the outer cortical cells of the root apex, whereas only the rhizodermal cells in the root apical region died at LC₅₀. The evidence that Cd and Pb cross the endodermal barrier at LC presumes that, at lower metal concentrations, the Casparian strip and plasmalemma of the endodermis regulate the transport of these metals into the central cylinder. The authors conclude that the identical barriers control Cd and Pb transport in root tissues.     

The effects of salinity on acute and chronic nickel toxicity and bioaccumulation in two euryhaline crustaceans: Litopenaeus vannamei and Excirolana armata

We investigated the influence of salinity (5 ppt versus 25 ppt) on acute (96-h LC₅₀) and chronic toxicity (15–30 day LC₅₀) of Ni in two euryhaline crustaceans, the shrimp (Litopenaeus vannamei) and the isopod (Excirolana armata). 96-h LC50 values were 41 μmol L⁻¹ and 362 μmol L⁻¹ for L. vannamei and 278 μmol L⁻¹ and > 1000 μmol L⁻¹ for E. armata at 5 ppt and 25 ppt, respectively. Speciation analysis demonstrated that complexation with anions such as SO₄²⁻, HCO₃⁻ and Cl⁻ at 25 ppt had a negligible effect on reducing the free Ni²⁺ ion component in comparison to 5 ppt. The salinity-dependent differences in acute Ni toxicity could not be explained by differences in Ni bioaccumulation. Therefore, differences in physiology of the organisms at the two salinities may be the most likely factor contributing to differences in acute Ni toxicity. Chronic LC₅₀ values (2.7–23.2 μmol L⁻¹) were similar in the two species, but salinity had no significant effect, indicating that water chemistry and osmoregulatory strategy do not influence chronic toxicity. However chronic (15-day) mortality in both species could be predicted by acute (96-h) Ni bioaccumulation patterns.     

Changes of photosynthetic activities of maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) seedlings in response to cadmium stress

Maize (Zea mays L.) seedlings were grown in nutrient solution culture containing 0, 5, and 20 μM cadmium (Cd) and the effects on various aspects of photosynthesis were investigated after 24, 48, 96 and 168 h of Cd treatments. Photosynthetic rate (P _N) decreased after 48 h of 20 μM Cd and 96 h of 5μM Cd addition, respectively. Chl a and total Chl content in leaves declined under 48 h of Cd exposure. Chl b content decreased on extending the period of Cd exposure to 96 h. The maximum quantum efficiency and potential photosynthetic capacity of PSII, indicated by F_v/F_m and F_v/F_o, respectively, were depressed after 96 h onset of Cd exposure. After 48 h of 5μM Cd and 24 h of 20 μM Cd treatments, the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco, EC 4.1.39) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) in the leaves started to decrease, respectively. We found that the limitation of photosynthetic capacity in Cd stressed maize leaves was associated with Cd toxicity on the light and the dark stages. However, Cd stress initially reduced the activities of Rubisco and PEPC and subsequently affected the PSII electron transfer, suggesting that the Calvin cycle reactions in maize plants are the primary target of the Cd toxic effect rather than PSII.     

Screening the Toxicity of Ni,Cd, Cu,Ivermectin, and Imidacloprid in a Short-Term Automated Behavioral Toxicity Test with Tubifex tubifex (Müller 1774) (Oligochaeta)

A new automated online toxicity test for screening of short-term effects of chemicals is presented using the freshwater oligochaete Tubifex tubifex in the Multispecies Freshwater Biomonitor? (MFB). Survival and locomotory behavior of the worms were observed during 24 h of exposure to metals (Cd, Cu, Ni), pesticides (Imidacloprid), and pharmaceuticals (Ivermectin). The LC₅₀ values revealed increasing toxicity in the following order: Ni (> 100 mg/l) < Cu (15.2 mg/l) < Cd (4.9 mg/l) < Ivermectin (1.8 mg/l) < Imidacloprid (0.3 mg/l). The EC₅₀ for locomotion showed a similar order of increasing toxicity: Ni (86 mg/l) < Cu (3.8 mg/l) < Ivermectin (2.0 mg/l) < Cd (1.1 mg/l) < Imidacloprid (0.09 mg/l). Toxicity was dependent on both concentration and exposure time. This could be demonstrated in 3d response models and proven in the statistical analysis showing a significant interaction term (C × T) for the experiments with Cu and Ni. T. tubifex proved to be very tolerant, but even then behavioral responses were more sensitive than mortality for Cu, Cd, and Imidacloprid.     

Acute toxicity tests on three species of the genus Lecane (Rotifera: Monogononta)

Three rotifer species, Lecane hamata L. luna, and L. quadridentata, were submitted to acute toxicity tests to compare their susceptibility to 11 toxicants. In acute tests with 48-h exposure of neonates of less than 24 h old, copper was most toxic with LC₅₀ values in the range of 0.06–0.33 mg l^–1, while acetone was the least toxic with LC₅₀ values in the range of 5000–7000 mg l^–1. Differences in LC₅₀ value of up to 22-fold were found in the susceptibility to lead between the three species. These data indicate large differences in toxicity among members of the same genus, and point out that it is necessary to submit several species to toxicity tests in order to assess the potential effects of toxicants to rotifers. The commonly used Brachionus calyciflorus cannot be considered representative of all freshwater rotifers in this respect.     

Acute toxicity of a household bleach,sodium hypochlorite,to a whirligig beetle Orectogyrus alluaudi Régimbart, 1889 (Coleoptera: Gyrinidae)

Acute response of Orectogyrus alluaudi Régimbart, 1889 to sodium hypochlorite was reported after a 48-h acute toxicity test in an imitated field condition. O. alluaudi belongs to the water beetle family Gyrinidae (whirligig beetles) which includes more than 700 species. The test organisms were exposed to various concentrations of sodium hypochlorite ranging from 10 to 160 mg/l along with the control, and median lethal concentration (LC₅₀) values at 24 and 48 h of exposure were assessed. Mortality increased with the concentration of exposure. The 24 and 48 h LC₅₀ values estimated for sodium hypochlorite were 87.30 (73.21–107.06) and 72.32 (62.24–84.53) mg/l, respectively. A significant mortality (P < 0.001) was observed at 40, 80 and 160 mg/l of sodium hypochlorite when compared with the control. Addition of sodium hypochlorite altered the alkalinity and pH of the test water. Behavioural responses such as agitation and disorientation were also observed. The finding showed that indiscriminate use of the household bleach would have serious impact on the bionomics of this species.     

Impact of environmental pollution on cyanobacterial proline content

This study describes the toxic effects of different prominent aquatic pollutants—heavy metals (Cd & Pb), pesticides (alphamethrin and deltamethrin) and salt (NaCl)—on the intracellular proline content in the cyanobacterium, Westiellopsis prolifica–Janet strain–NCCU331. Despite a reduction in growth (measured as chlorophyll a content), the intracellular proline content increased in the presence of heavy metals, pesticides and high salt concentration. The intracellular cyanobacterial proline accumulation was more pronounced under salt stress than in the presence of pesticides and heavy metals. We have also compared whether or not anionic components influence heavy metal toxicity. It was found that the chlorides of Cd and Pb were more toxic than the NO₃ and the order of toxicity was CdCl₂ > PbCl₂ > Cd (NO₃)₂ > Pb (NO₃)₂. Among pyrethroids, deltamethrin was more toxic than alphamethrin. Presented at the 6th Meeting of the Asian Pacific Society of Applied Phycology, Manila, Philippines.     

Effects of triflumuron on the two-spotted spider mite,Tetranychus urticae (Acari: Tetranichydae)

The effects of triflumuron on the mortality, fecundity, and fertility of the two-spotted spider mite, Tetranychus urticae, were evaluated in the laboratory. No differences in toxicity for larvae, protonymphs and deutonymphs were observed, but immature stages were 3.8-times more susceptible than adults at the LC₅₀. The compound exhibited a direct contact ovicidal activity influenced by eggs age. 48–72 hold eggs were significantly more sensitive than eggs of the other age classes. No hatch inhibition was observed in eggs laid by treated adult females using a sublethal dose at two physiological times (<12 and 48–72h old). However, fecundity decreased in younger treated females, but it increased in the older-ones. The toxicity for immatures and eggs, and the sublethal effects described suggest that triflumuron could be an interesting incorporation in integrated pest programs of T. urticae. This revised version was published online in July 2006 with corrections to the Cover Date.     

Acute toxicity of tributyltin to encapsulated embryos of a marine gastropod

The marine gastropod Buccinanops globulosus is known to have high imposex incidence in areas moderately polluted by tributyltin (TBT). Acute toxicity was previously studied in adults but no information is known about embryonic intracapsular development. To estimate the potential effects of organotin pollution on the progeny of B. globulosus, acute toxicity tests were conducted on encapsulated and excapsulated pre-hatching embryos. The lethal median concentration estimated for 96?h (LC₅₀ 96?h) in B. globulosus excapsulated embryos was 196.70?µg?TBTCl?L^?1, while in encapsulated embryos it was 2951.28?µg?TBTCl?L^?1. The LC₅₀ 96?h was 15-fold higher for encapsulated embryos compared to excapsulated embryos, denoting egg capsule protection against pollutants from the external environment. Our results show that TBT pollution can have significant effects in molluscs other than the chronic effect of imposex.     

镉和十二烷基硫酸钠对克氏原螯虾的单一和联合毒性

采用静水生物测试法,研究了十二烷基硫酸钠(SDS)和Cd2+对克氏原螯虾的单一和联合毒性效应。结果表明,SDS对克氏原螯虾24、48、72和96h的半致死浓度(LC50)分别为1365.60、1171.72、1128.43和1120.43mg·L-1,安全浓度(SC)为258.79mg·L-1;Cd2+对克氏原螯虾24、48、72和96h的LC50分别为1197.09、142.06、90.85和82.64mg·L-1,SC值为0.6mg·L-1。SDS和Cd2+对克氏原螯虾的毒性分别为低毒和高毒,毒性大小为Cd2+>SDS。联合毒性试验表明,二者毒性比为1∶1,暴露时间为24、48、72和96h时,相加指数(AI)分别为0.335、0.017、0.030和0.032,联合作用结果均为毒性增强的协同作用。     

Toxicity of the new pyrethroid insecticide,deltamethrin, to Daphnia magna

The toxicity of deltamethrin, a synthetic pyrethroid insecticide, was determined under standardized conditions (ISO, 1982) in neonates and juveniles of Daphnia magna. Neonates (6 to 24 h old) were more sensitive than juveniles (48 to 72 h old). The 24- and 48-h EC₅₀s (immobilization) in neonates were 0.113 and 0.031 µg l^–1, respectively. The toxicity of deltamethrin was highly toxic. The 96-h EC₅₀ was in the ppt (µg l^–1) range. Toxicity tests with Daphnia may be used to detect toxic residues in water and sediment in areas treated with deltamethrin and other highly toxic pyrethroid pesticides.     

Effect of Azo Dyes on Survivorship,Oxygen Consumption Rate,and Filtration Rate of the Freshwater Cladoceran Moina macrocopa

Toxicity of the azo dyes Procion Red MX-5B (PR), Procion Yellow HE-4R (PY), and Congo Red (CR) on the freshwater cladoceran Moina macrocopa was studied. The 4-day LC₅₀ values for PR, PY, and CR were 59.0, 9.50, and 0.16 mg/L, respectively. Reproduction was a more sensitive endpoint than mortality. The onset of reproduction was delayed at azo dye concentrations ≥ 0.01 mg/L. The total number of young produced over 7 days was reduced by 49.8% in PR, 44.5% in PY, and 69.0% in CR. No reproduction was recorded at PY concentrations ≥ 100 mg/L and CR concentrations ≥ 1.0 mg/L. A significant decrease in oxygen consumption rate was observed after 4 days of exposure to azo dyes at concentration equivalent to 25% of the 4-day LC₅₀. The effect of CR on oxygen consumption rate was observed after 4 days of exposure to a concentration equivalent to 10% of the 4-day LC₅₀. Filtration rate was a more sensitive endpoint than oxygen consumption rate. For all three dyes, filtration rate was reduced after 4 days of exposure to concentrations equivalent to 10% of the 4-day LC₅₀. Based on mortality, reproduction, oxygen consumption, and filtration, the order of toxicity was CR > PY > PR.     

Acacia nilotica L. Bark Removes Toxic Elements from Solution: Corroboration from Toxicity Bioassay Using Salix viminalis L. in Hydroponic System

The present investigation deals with the advantages and potential of the Acacia nilotica bark as an adsorbent of toxic metals. Bark (1 g) when added to 100 ml of aqueous solution containing 10 μg ml^-1 metal solution exhibited different metal adsorption values for different metals. The order of metal adsorption being Cr ≥ Ni > Cu > Cd > As > Pb. A similar trend of metal adsorption was observed when the bark is reused (1^st recycle) Cr> Ni > Cu > Cd > Pb and also in the column sorption. In order to verify the metal removal property of A. nilotica bark, toxicity bioassay with Salix viminalis stem cuttings in hydroponic system augmented with Cd, Cr, and Pb together with A. nilotica bark powder was carried out. The results of toxicity bioassay confirmed the metal adsorption property of the bark powder. The functions of toxicity studies include leaf area, root length and number of new root primordia produced per stump. The leaf area, root length, and the number of new root primordia increased considerably in the presence of A. nilotica bark. The order of metal toxicity for leaf area and new root primordial is Cd > Cr > Pb. However, for root length the order of metal toxicity is Cr > Cd > Pb. The metal budgets of the leaf and root confirmed that the bark powder had adsorbed substantial amount of toxic metals and thus alleviates the toxicity imposed by the various tested elements. Hence, the utility of A. nilotica bark in developing and designing innovative technology for the clean up of toxic elements in aqueous solutions and possible scope for its use in phytoremediation are discussed.     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

Partial inactivation of the mosquitocidal activity of Clostridium bifermentans serovar malaysia by extracellular proteinases

Summary Clostridium bifermentans serovar malaysia is toxic to mosquito larvae. During large-scale preparation in a fermentor, the bacteria enter the sporulation stage after 5 h culture, whereupon high larvicidal activity is obtained (LC₅₀ 48 h on Anopheles stephensi = 3.1 × 10^–5). The toxicity becomes maximal around 3–5 h later (LC₅₀ 48 h = 1.3 × 10^–5) and remains unchanged until sporangium lysis. An important loss of toxicity is then observed when the cells lyse. This loss appears to be due to the fact that C. bifermentans serovar malaysia synthesizes and excretes, mainly during vegetative growth, metallo- and/or cystein-proteinases, which are active between pH 6.0 and pH 8.0. Extracellular proteinases are most likely responsible in large part for the decrease in toxic activity concomitant with cell lysis. Lysis is however prevented by addition of 10 mM ethylenediaminetetraacetic acid to the culture medium before forespore formation, and under these conditions the larvicidal activity can be maximized. Offprint requests to: L. Nicolas     

Photosynthetic and population growth response of the test alga Selenastrum capricornutum Printz to zinc, cadmium and suspended sediment elutriates

Short-term ¹⁴C-fixation (4 h) Selenastrum capricornutum algal toxicity tests were conducted with Cd (n=8), Zn (n=9) and suspended sediment aqueous elutriates (n=28) and the results were compared to those obtained in a 48 h population growth test. In order to provide more realistic experimental conditions, toxicity tests were carried out in prefiltered nutrient-spiked Lake Geneva water. The population growth inhibition test was significantly more sensitive than the¹⁴ C-fixation test for Cd (median EC50-4h and EC50-48h values of 600 and 118 μg L^-1, respectively) whereas no significant difference was measured for Zn toxicity (median EC50-4h and EC50-48h values of 97 and 96 μg L^-1, respectively). With suspended sediment aqueous elutriates, the relative sensitivity of the two different end points is sample dependent, with ratios of the EC25 for the¹⁴ C-fixation: population growth test ranging from <0.26 to >53.3. Elutriate toxicity shows no apparent relationship between the acute and chronic test, indicating that population growth inhibition cannot be derived directly or predicted from¹⁴ C-fixation. Both tests with their specific advantages and limitations provide valuable complementary information to measure the impact of single toxicants or complex mixtures on aquatic plants. This revised version was published online in June 2006 with corrections to the Cover Date.