| Fluoride ions(F-)in natural water bodies partly exceed the China national water standard for fisheries,meanwhile,they caused the major endemic of China-skeletal fluorosis.However,the effects of waterborne F-to aquatic animals are relatively less reported.Sturgeon species are noted as cartilage composed most of their endoskeleton. Siberian sturgeon(Acipenser baerii)is not only an endangered species,but also one of the sturgeon species widely raised in fish farms in China and around the world.We adopted the principle of ecotoxicology to explore the toxicity of F-on Siberian sturgeon.The determination of F-effects were made at different development stages,for which morphological,biochemical,ionic,behavioral and growth endpoints were selected and quantified,aiming to better understand F-metabolism/accumulation mechanism in fish and the adaptation of fish to F-exposure.As results,we got the sensitive biomarkers of F-toxicity to fish,suggested the F-related safe water/feed criteria to fish protection,tentatively evaluated the ecological effects of F-in natural water of China,suggested the possibility of fish meal substitution by high F-krill meal,meanwhile we first studied F-accumulation in cartilage.1.Effects of fluoride on embryo development of Siberian sturgeonThe aim of this study was to investigate the effect of F-on embryo development of Siberian sturgeon Acipenser baeri.In semi-static toxicity bioassays,Siberian sturgeon embryos were exposed to six different concentrations of waterborne fluoride (100mg/L-600mg/L)at 17±1℃.Fluoride resulted in increased embryo mortality, decreased hatching rate,and increased deformed rate of larva.A hatching delay of up to 9-22 hours was noted.144h median lethal concentration(LC50)was 447.6 lmg/L for Siberian sturgeon embryo,Safety Concentration(SC)of hatching were 4.476mg/L, median effective concentration(EC50)of larva deformed were 536mg/L.SC of embryo Siberian sturgeon is lower than that of some natural water,it reminds that some embryo fish might adversely affected by water F-.Meanwhile,we investigated susceptibility of several endpoints and their applicability as toxicity indicators in Siberian embryo-larval assay.Mortality, hatching median time,and abnormality all concentration-related effects,and they showed similar sensitivity to F-toxicity.Larval abnormality which was defined with binary responses constituted major ones such as cyrtosis,cyst and minor ones such as short tail,eye hemorrhaging.Pericardial edema was the most sensitive mark among the deforming characteristics.After larval were moved out of F-,delaying effects were observed.Larval body length was the most sensitive mark compared to larval mortality,body weight and specific growth rate.Taken together these data indicated that total larval deforming rate and delaying body length were most sensitive among whole selected endpoints,while pericardial edema was the second.We suggest total larval deforming rate and delaying body length to be the endpoints indicating F-toxicity to embryo Siberian sturgeon.2.Acute toxicity and safety assessment of fluoride to larval Siberian sturgeonSemi-static acute toxicity trial on larval Siberian sturgeon was carried out to explore the safe criteria of fluoride on fish.Behavior changes in larval Siberian sturgeon included abnormal motility,loss of equilibrium,and violation in gill ventilation.The mortality rate of larval Siberian sturgeon increased with exposure concentration and exposure time.The 48,72 and 96h LC50of F-in Siberian sturgeon were 1014.10,288.28 and 181.18mg/L,respectively.SC of fluoride in water for larval Siberian sturgeon was 1.81mg/L.Compared to F-concentration in water bodies of China,our result highlights the need of management about F-in water to protect aquatic animals.3.Acute toxicity of F-in Siberian sturgeon96h acute toxicity tests were conducted to explore the LC50of Siberian sturgeon exposed to waterborne F-.Two different sizes of Siberian sturgeon(body length 10.60±0.05cm,body weight 8.01±0.51g and body length 17.21±0.91cm,body weight 32.35±3.73g)were tested,resulting behavior reactions such as initial excitement, faster swimming rate,back to normal cruising speed for low dosage F-group,and first excitement,then slower swimming rate,finally dying for high dosage F-group.The LC50for these fish in two sizes were 125 and 200mg/L,respectively,while SC was at 1.25 and 2.00mg/L,respectively,indicating size influences on F-toxicity.Sublethal acute F-toxicity test on juvenile Siberian sturgeon(body length 17.69±1.11cm,body weight 42.04±7.73g)showed no significant effect on serum Na+, K+,Ca2+,Mg2+and P in 10mg/L and 62.5mg/L group compared with control. However,125mg F-/L significantly affected the above ions,and Ca2+is the most sensitive ionic indicator showing F-toxicity.4.The definition of sensitive developmental stage and effects of chloride and hardness on F-toxicity to Siberian sturgeon based on sensitive stages12h short-term acute toxicity tests with at distinct stages of Siberian sturgeon were conducted to determine the most sensitive stage to F-toxicity.LC50of embryos exposed to F-was＞600mg/L,cleavage was the most sensitive stage during embryo development based on hatching success,while cleavage and blastula were the most equally sensitive stage based on latent growth.After hatching,the sensitivity of fish post hatching first declined and then increased,as 20-30d larval was the most sensitive stage with 96h LC50at 107.31-112.77mg/L,while the least was 1d post hatching with 96h LC50＞600mg/L.We concluded that the 12h acute toxicity performed well telling the mode of action,and F-toxicity was most effective at 20-30d for all life stage while that was during embryo development.Based on above results,we conducted a new test exploring the effects of chloride and hardness on F-toxicity.Hatching success and larval survival showed positive concentration-response relationships with chloride and hardness,while F-concentration of embryo and larval showed negative ones.Results testified that chloride and hardness alleviates F-toxicity.5.The influences of waterborne F exposure in Siberian sturgeonA 90-day growth trial was conducted to determine the growth inhibition,blood biochemical and ionic disturbance and accumulation of F-to juvenile Siberian sturgeon.Fish growth was inhibited when exposed to 7.8,18.7,and 51.8 mg F-/L in comparison with control.Among plasma biochemical parameters,ALP activity, UREA and CREA concentrations were significantly affected when water F-≥7.8 mg/L.ALP changes by F-was considered to be functional interference of bone metabolism,while the recovery of CREA and UREA concentration in the late period of the present experiment denote that function of gill and kidney was initially suffered from F-,but became normal gradually.Blood Na+,K+ were not influenced by F-.After exposed to F-for 30 d,concentrations of Ca2+and Mg2+declined,while P concentration increased,till 90 d.Significant increases in F-concentration were observed in bone,cartilage,skin and gill of Siberian sturgeon exposed to or above 3.1mg F-/L,with F-content in bone was the highest(3204.4mg/kg),followed by cartilage(1401.2mg/kg),gill(389.4mg/kg),skin(100.1mg/kg).Muscle,liver,gut and pylorus did not show any accumulation with increasing F-levels in the water,and liver,gut,pylorus showed higher F-concentration compared to that of muscle.The LOEC(lowest-observed-effect concentration)and NOEC(no-observed-effect concentration)was 7.8mg F-/L and 3.1mg F-/L,respectively.It is summarized that F-accumulates mainly in bone and cartilage in Siberian sturgeon,with 3.1 mg F-/L was the LOEC,and NOEC should be＜3.1mg F-/L.F-functions through blood biochemical and ionic interference,among which ALP activity,UREA,CREA,Ca2+, Mg2+and P indicate F-toxicity well.6.F-effects after dietary fluoride exposure in Siberian sturgeonThis study was carried out to evaluate the possible uptake of F-in several tissues of juvenile Siberian sturgeon.Fish were fed diets added F-in triplicate for 12 weeks. Growth was inhibited in fish fed diets with 710.2,1478.3 mg F-/kg,but not in fish fed diets with 162.6,360.8 mg F-/kg.F-content in bone,cartilage,skin and gill progressively increased as the F-concentration in diets increased,with the highest levels found in bone(7721.3mg F-/kg),followed by cartilage(2324.2),gill(744.7), skin(269.2).Muscle,liver,gut,and pylorus did not show any accumulation with increasing F-levels in the diets.Results indicate that(1)F-can be included in the Siberian sturgeon diet up to 360.8 mg F-/kg without adverse effect on growth performance or survival,and thus krill meal has a promising prospect to be an alternate protein source to sturgeon aquaculture in the perspective of F-effects;(2) bone and cartilage of Siberian sturgeon accumulated F-concentration when diet containing≥162.6 mg F-/kg,and F-accumulation in cartilage is an alarm to human food safety.In conclusion,SC of F-to Siberian sturgeon is 1.25 mg F-/L according to F-toxicity test in embryo,larval,and juvenile Siberian sturgeon.The adverse effects of F-to aquatic animals should be paid attention to,as SC is lower than natural water F-concentration in part of China and globe.The most sensitive life stage to F-toxicity is 20-30d post hatching based on the utility of 12h toxicity test which is a new methodology for toxicity screens.F-concentrations≤3.1mg F-/L in water or≤360.8mg F-/kg in diet do not inhibit growth of Siberian sturgeon.The mode of F-action functions through interference of blood biochemistry and ion balance.The F-accumulation in cartilage is an alarm to human food safety.Based on the present study and the actual water F-concentration(0.27-0.85mg/L)in estuary of Yangtse River during March,2007 to March,2008,and the actual water F-concentration (≤0.92mg/L)in some aquarium,we hypothesize that water F-is not a risk for Chinese sturgeon(Acipenser sinensis G.),and water F-is not the reason for sturgeon deformation in fish farms.Further chronic testing will be required to provide more detailed information regarding the adverse effects on different species under different environmental conditions,for ecological risk assessment purposes and economic benefit. |
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