全 文 ：热带业热带植物学报 2006，14(1)：卜6
Journd q Tropical and Subtropical Bot(my
柳树对亚铁氰化物的吸收、代谢及其毒性研究
于晓章 ， 周朴华b，唐雅雯a，彭晓英b
(湖南农业大学，a．环境科学系；b．生物技术系，长沙 410128)
摘要：为探明业铁氰化物在植物体内的迁移、转化及对植物的毒性作用，以长出新根须和嫩叶的垂柳(Sa!ix babylonicn
L．)枝条为材料，在白行设计的 250 ml牛物反应器中生长192 h，培养温度为24．0xl℃，业铁氰化物水溶液的浓度分)jU
为 52．99，105．98，211．95和 317．93 mg CN L。结果表明：(1)低浓度实验组(52．99 mg CN L )水溶液中 10．85％的业铁氰
化物被植物吸收，随着浓度的升高吸收到植物体内的业铁氰化物的比例(％)依次递减，但是统汁学分析硅示各实验
组单位体重(湿重)的植物吸收亚铁氰化物的最无娃著性差异：(2)在植物的各个部位都能检测到微量的亚铁氰化物，表
明 铁氰化物通过植物的蒸腾作用在植物体内的迁移。由于没有检测到1乍气态下的总氰化物，表明植物的蒸腾作_L}j没
有将、】 铁氰化物释放到人气中；(3) 管植物吸收到体内的亚铁氰化物足有限的，但物质平衡实验 明其仵植物体内
迁移的过程【I1超过96％的郜能被植物自’效转化；(4)所_L}j的4种亚铁氰化物浓度在 192 h内没有刑柳树产 毒性作
用。凼此 认为：依据亚铁氰化物在水溶液一植物一中气系统内的迁移和转化，亚铁氰化物的植物修复足可能的。
关键词：氰化物；业铁氰化物；代谢；植物修复：毒性；迁移；柳树
中图分类号：Q945．1 文献标识码：A 文章编号：1005—3395(2006)01—0001—06
Uptake，Metabolism，and Toxicity of Iron
Cyanide Complex in W eeping W illows
YU Xiao—zhang ， ZHOU Pu—hua ， TANG Va—wen ， PENG Xiao—Ying
(a．Department Environmental Sciem：e，Humm Agricultural University
b．Department ’Biotechnology,Hunan Agricultural University，Changsha 410128，China)
Abstract：Uptake，metabolism and toxicity of iron cyanide complex in trees were investigated
． Pre—rooted weeping
wilows(Solix babylonica L．)were exposed to hydroponic solution spiked with ferocyanide at 24．0~1℃for 1 92 h．
Four different treatment concentrations offerrocyanide were used(52．99，105．98，21 1．95 and 317．93 mg CN L。)
．
Cyanide in water， in tissues of aerial part of plants and in air was analyzed spectrophotometrically． Results from
this study indicated less than 1 0．85％ reduction of the applied iron cyanide complex was detected in hydroponic
solution in the presence of plants．Little amounts of cyanide were found in all parts of plant tissues
， indicating the
passage of ferrocyanide through the plants．Mass balance studies showed that iron cyanide complex moving
into plants from hydroponic solution can be metabolized during transport．Phytotoxic efects were not found in all
treatment groups，even at high doses of ferrocyanide within a 1 92-h exposure period．in conclusion，transport and
metabolism of ferrocyanide in plants is most likely to happen and phytoremediation of this iron cyanide complex
in field application may be possible．
Key words：Cyanide；Ferrocyanide；Metabolism；Phytoremediation；Toxicity；Transport；Willows
Cyanide is the commonly used reagent for gold
and silver extraction． The annual production of
cyanide hydrogen is about 1．4 million tons；more than
1 00 000 tons of cyanide disperse to the environment
Recieved：2005——08—·29 Accepted：2005-10——3 1
Foundation item：Research foundation from the Hunan Agricultural University，China for scientists ：03YJ05)
Corresponding author
维普资讯 http://www.cqvip.com
2 热带业热带植物学
annuallyl“．It is therefore not surprising that problems
and catastrophic accidents occur repeatedly， mainly
associated with gold mining． Although free cyanide
(CN。， HCN) is one of the most toxic chemicals to
wildlife and human health， a number of plants
investigated so far，were found to posses enzymes that
can detoxify cyanide I ’ ．Cyanide in plants is rapidly
metabolized by the enzyme 3-cyanoalanine synthase
(CAN)I4， ．Asparagine was the only metabolic product
detected in experiments with’4C．1abeled CN for both
cyanogenic and non—cyanogenic plants[f1_．Cyanide in
environment can be present as free or simple cyanide
or as cyanates and thiocyanatcs f71．Most reacts with
metal cations， forming a variety of metal cyanide
complexes．Among them，iron cyanides(Fe(CN)6*)are
the most common and stable species， which are
frequently found in contaminated aqueous environ-
mental matrices r ．Degradation by microorganisms of
these compounds was confined to a limited number of
bacterial and fungal strainst · ．The dccolnposition of
complexed cyanide to free cyanide under thc exposure
of light was reported by Kjeldsen 。．Phytoremediation
of cyanide has been carefully studied in a number of
plants from three diffcrent continents and climate
zonest2·3·7 ll一 i No work has been found concerning the
phytoremediation of ferrocyanide to terrestrial plants
from China． Beavis and Vercesi【】 ] described a
mitochondrial anion channel capable of transporting
ferocyanide． Therefore， we assume that plants may
utilize iron cyanide complexes as a substrate during
the plant metabolism． In this study， the uptake，
transport， metabolism and toxicity of iron cyanide
complex were determined for weeping willows
growing in hydroponic solution． These results are
笫 14替
suggestive of phytoremediation of fbrrocyanide in field
application．
1 Material s and Methods
1．1 Tree and exposure regimes
Weeping willow (Salix babyloniC(1 L．)was
sampled from nature at the campus of the Hunan
Agricultural University， China． Forty—cm long tree
cuttings were removcd from mature specimens of a
single tree． After two．months of growth in buckets
with tap water，pre-rooted cuttings were transferred to
a 250 ml Erlenmeyer flask filled with approximately
200 ml modified ISO 8692 nutrient solution(Table 1 1．
The flasks were all sealed with cork stoppers and play
dough to prevent thc escape of water or chemicals，and
wrapped with aluminum foil to inhibit algae growth．
The flasks were put in a climate chamber with a
constant tempcrature of 24．0±l℃ under continuous
artificia1 light．Thc plants remained there 48 h to allow
them to adapt to their new living environment． Then，
the weight of the plant system was measured．Twenty—
four hours later，the flasks with the trees were weighed
again．By this，the transpiration was determ ined．Trees
with similar transpiration were selected for the tests．
For each treatmcnt concentration， six replicatcs were
mcasured． The nutrient solution of these trees was
exchanged to ferrocyanide—spiked solution，except for
controls． Five diferent treatment concentrations of。
fbrocyanide were used(0，52．99，l05．98，2 l1．95 and
3 l 7．93 mg CN L’)．Note that 1 mg K4Fe(CN)~equals
to 0．423 9 mg CN．At the end of the experiment
(1 92 h1， the water，roots， leaves and stems were al1
analyzed for~ ocyanide or the total cyanide．A mass
Table 1 Composition of nutrient solution used in plant uptake experiments
维普资讯 http://www.cqvip.com
第 1川 r晓节等：柳树对业铁氰化物的吸收、代f『j及其毒性研究
balance was computed to assess whether ferrocyanide
lost from water could be recovered in plant biomass or
whether metabolism of this iron cyanide complex had
occurred．
1-2 Chemical analysis
The concentration of ferocyanide in water was
analyzed by a standard method provided by State
Environmental Protection Administration of China[15]．
Results were expressed as mg CN L～． The detection
limit of this method was determined from blank plus
three standard deviation of l 0 replicates to be
0．08 mg CN L ．
Total cyanide is the sum of easy l iberatable
cyanide and complexed cyanide．Sample pretreatment
was prepared in the following way． Ten milliliters of
sodium hydroxide of l％ was poured in the absorption
vessel of the distillation unit．Fresh plant biomass f2．0
tO 1()g FW ． depending on the harvested weight of
plant materials) was cut into pieces and placed in a
500 1ill round botom flask． and then 200 ml of
distilled water was added． Then l 0 ml of ethy1．
enediamine tetraacetic acid disodium salt with the
concentration of 1 0％ (V／M)and l 0 ml of phosphoric
acid er andysis， in China： ≥85％ purity) were
added betbre heating and mixing． Approximately
1 00 ml distilled solution containing cyanide from
plant tissues were collected，quantitatively transferred
to a l 00 ml volumetric flask and made up to volume
with water．The solution was stored at below 6℃ until
the concentration of cyanide was determined
．
The
samples were all analyzed with a maximum hold time
of 4 hours． Cyanide in distilled solution was also
analyzed by a standard method provided by State
Environmental Protection Administration of China t~51
．
The detection limit of this method was determ ined to
be between 0．004 and 0．25 mg CN L ．
1．3 Cyanide transpired by plants
Fcrrocyanide transpired was measured using a
refined test chamber(Fig． 1)．Treated plants were
prepared as described above and placed into a glass
chamber f20 cm ×20 tin ×50 cm1 with airflowing
through at 24。C．Tubing from the outflow of the vessel
is connected to a gas trap tube containing 5 ml sodium
hydroxide of 1％ that would trap any airborne cyanide．
The gas trap tube was wrapped with aluminum foil
and changed daily， after which all gas tubes were
analyzed for the total cyanide and free cyanide． The
duration of this test was 1 92 h
Fig 1 Test chamber system for measuring ferrocyanidc transpired in air
2 Results and Discussion
2．1 Iron cyanide complex uptake from hydroponic
solution by willows
Fig．2 shows the mass reduction(％)of ferro．
cyanide in hydroponic solution with diferent
treatment concentrations after 1 92 h exposure． In the
controls in the absence of plants， no change of
ferocyanide concentrations was detected over the
entire period of exposure(data not shown)，indicating
the disappearance of ferocyanide in water can be
accounted to the uptake by willows． Amounts of
ferocyanide in hydroponic solution were reduced in
all treatments， ranging from 1．21％ to 1 0
．85％ of
initial mass． Results showed amounts of applied
~rocyanide moving into plant tissues， but predo—
minantly remaining in the hydroponic solutions
．
Ebbs et a1． used wilows(Salix eriocephala L．var．
michaux) to quantify the plant uptake，transport and
metabolism of potassium cyanide and potassium
ferocyanide labeled with N． Approximately 8％ of
the initial ferrocyanide was reduced in water during
the presence of plants． Results from the study of
Samiotakis and Ebbs also imply barely fHorde um
vulgare L．)， oat vena sativa L．) and wild cane
(Sorghum bicolor L．)can extract ferrocyanide from the
contaminated compartments．
维普资讯 http://www.cqvip.com
4 热带业热带植物学报 第 l4卷
52 99 1 0 98 l1 9 5 31j 93
Fer~oeyan c0nceⅡefatio1+{lug CN L。’)
Fig 2 Ferocyanide reduction f％1 in hydroponic solutions with
di竹_erent treatment concentrations
Bars=Standard errors ofthe means(n=6)
2．2 The mass balance of cyanide
The water， roots， leaves and stems were al
analyzed for ferrocyanide or total cyanide after 1 92 h
exposure． Fig．3 shows the concentration of tota1
cyanide in plant tissues (mg CN kg‘。FW)． Cyanide
was found in all parts of plant materials in all
treatment concentrations，confirming passage of ferro—
cyanide through the plants．Significant concentrations
of cyanide in roots were found in all treatment groups．
The same results were also found in the studies of
Ebbs et a1．[71 and Samiotakis and Ebbs[I6_．It is also
shown from Fig．3 that the measured concentrations in
plant materials are increased with the exposed doses
of ferrocyanide．
Due to the leafy portion being exposed to the air，
ferrocyanide in water may have transpired through the
5
4
3
2
l
0
20
15
lO
5
O
} 优
I 厂
} r÷1 l l ； I l
f { l l l } l
￡⋯上一⋯ — ⋯ L— 一— 一— 一⋯ 三一— ～ —L ——L
52．99 lO5．98 2l l+95 317．93
Ferocyanide concentrations(mg CN L )
Fig 3 Measured total cyanide concentrations(mg CN kg F w)in the
tissues ofplants exposed to diferent concentrations offerocyanide
Values are means of six replicates， Bars：Standard errors of the
means(n=6)．
1eaves without metabolism． In this study， no cyanide
(the concentration may below the limit of detection)
transpired by plant leaves was trapped over a 1 92一h
test period using the test chamber． The SalTle was al so
reported in the study of Ebbs et a1．_7】．Therefore．a mass
balance for ferrocyanide within the planted system
was calculated using total cyanide in plant tissues and
Table 2 Mass balance of ferrocyanide
Values are means of six replicates
加 ∞ 蚰 ∞ 如 O 6
一≥‰l- Zu 崔一I王0一篁 # u^ 0u 0 一I王蜘
_ ， 一 ．n b ，， ．¨U
； 盎 葛 至1 j
维普资讯 http://www.cqvip.com
第1期 十晓亭等：柳树对业铁氰化物的吸收、代谢及其毒性研究
the solution total cyanide data． Table 1 gives the
amounts of recovered mass from plant tissues and
from the solution． W hen wilows were exposed to a
low dose of ferrocyanide (52．99 mg CN L )， only
3．33±0．489％ (n=6)of the total reduction ferocyanide
in hydroponic solution was detected in the plant
materials． W ith increasing the exposed doses of
ferrocyanide， the initial mass remaining in the plant
tissues increased in a range from 1 0．46±3．087％ to
14．3 1±3．908％ ．The mass balance studies fdata shown
in Table 2) showed that the majority of the total
reduction ferrocyanide in water was metabolized
during transport through wilows． Results from the
mass balance studies also indicated the difference
between the metabolism rates was comparably smal1．
The mean of the four values of metabolism rates was
0．2 1 mg CN kg- h‘ and the standard deviation was
0．034 mg CN kg。 h～． implying the velocity of
fe~ocyanide metabolism in wilows was independent
of the substrate concentrations and probably limited
by enzyme capacity．
Results presented here and additional data l6J
suggest that uptake， transport and metabolism of
ferrocyanide for plants are most likely to happen
．
Then． it is of interest to discuss the mechanism of
ferrocyanide transport from hydroponic solution to
plants． The structure that enables plants to extract
water and nutrient from different environmental
compartments alows chemical to enter the plants and
to be subsequently transported inside．However．plant
cuticles are the limiting barrier in the uptake of a wide
range of chemicals． Probably there are two pathways
for water soluble compounds moving into the plant
tissues， passive transport (uptake with water and
translocation upwards in the xylem and the diflusion
in water phase into roots)and active transport (via a
special carier)． So which pathway causes the
ferocyanide moving into plant tissues? Ferrocyanide
has long been considered membrane impermeable[17]
．
In this case， weeping willows almost extracted the
same amount of applied ferocyanide from the
hydroponic solution in al treatment groups。 This
implies that active transport might be the pathway
causing ~rocyanide moving into root sym plasm．
Therefore， we postulate that there may have a special
carier or anion channel in the root membrane
responsible for the transport of ferocyanide． If this
iron cyanide complex can be transported across one
membrane， then there may be mechanisms that allow
for its transport across other membranes as wel1．Since
trace amounts of initial mass was al so found in al1
parts of plant tissues， mass balance studies provided
additional evidence to confirm the transport of
ferocyanide through the different tissues of plants．
The assimilation of free cyanide into asparagine
through cyanoalanine pathway has been widely found
in a number of plants L2-61 yet it is not clear whether
cyanoalanine synthase can use~rocyanide directly as
a substrate in plants． Further comprehensive studies
are needed to determ ine the detail mechanism of this
iron cyanide complex transport and metabolism．
2．3 Phytotoxicity of iron cyanide complex in
weeping willows
The toxic effect was quantified by measuring the
transpiration of the trees．The transpiration of plants is
coupled to the photosynthesis， and an inhibition of
transpiration is a reliable and fast measurement of
toxic effects Fig．4 shows the relative transpiration
of willows exposed to different doses of~rocyanide．
The relative transpiration was expressed by the ratio of
the transpiration of plants exposed to toxicant to the
tran spiration of plants exposed to nutrient solution
without toxicant． A higher relative transpiration
(1．26士0．1 05， n=6) was observed in treated plants
exposed to ferocyanide of 52．99 mg CN L一 in
comparison with untreated plants(1．08±0．165，nTM 6)，
implying this dose may stimulate the growth of plants．
A slight diference in the relative transpiration
between treated and untreated plants was found，
probably stemming from plants(No atempt was made
to select homogeneous plant materials in this study)．
The mean of the relative transpiration of four
treatment groups is 1．09±0．1 32．The relative trans—
piration of control is 1．08+0．t 65(n=6)，indicating the
doses offerocyanide to be used in this study did not
cause toxic efects on weeping willows． Symptoms of
维普资讯 http://www.cqvip.com
6 热带业热带植物学报
chlorosis in leaves were not found in all plants over
the entire period of exposure． There are also no
significances in the growth of plants between treated
and untreated plants(data not shown)，giving the con-
clusion that the plant can keep up their physiological
functioning within the plant systems spiked with
ferrocyanide over the entire period of exposure．
1．6
1．4
I+2
1．O
O．8
O．6
0．4
O．2
0 O 52
．99 l05．98 2l 1．95 317．93
Ferroeyanide eoncentrations(mg CN L’)
Fig 4 Relative transpiration ofplants exposed to di ferent
treatment concentrations of ferrocyanidc
Bars=Standard c~ors of the means(1=6)．
3 Conclusions
It was found in this study that weeping wilows
can extract， transport and metabolize ~wocyanide
without phytotoxicity．Less than 1 0．85％ of the applied
iron cyanide complex was detected to move into plant
materials from hydroponic solution． Amounts of
fe~ocyanide were found in all parts of plant tissues，
indicating passage of ferrocyanide through the plants．
Mass balance studies showed that initial~ ocyanide
moving into plant materials can be metabolized during
transport． Phytotoxic effects were not found in all
treatment groups， even at high doses of ferrocyanide．
This gives the conclusion that phytoremediation of this
iron cyanide complex is most likely to happen．
Acknowledgments This work was supported by
research foundation from the Hunan Agricultural
University，China，for scientists(No：03YJ05)．Thanks
to CHEN Liang and YANG Yong—miao for their
engaged help．
References
⋯I Muddcr T，Botz M．A guide to cyanide[J]．Mining Environ Manag
第 14卷
2001．9：8一l2．
[2】 Yu X Z，Trapp S，Zhou P H，ct a1．Metabolism of cyanide by
Chinese vegetation lJ]．Chemosphere，2004，56：l2l一126
[3] Larsen M，Trapp S，Pirandclo A Removal of cyanide by woody
plants[J1l Chemosphere，2004，54：325—333．
[4] Miler J M，Conn E E．Metabolism of bydrogen cyanide by higher
plants⋯ Plant Physiol，1980，65：Il99 1202．
[5] Maruyama A，Saito K，Ishizawam K B—cyanoalanine synthase and
cysteine synthase from potato： molecular cloning， biochemical
characterization，and spatial and hormonal regulation ⋯．Plant
Mol Bio1．200 1．46：749—760．
【6] Manning K．Detoxifcation of cyanide by plants and hormone
action fA]In：Ciba Foundation．Cyanide Compounds in Biology
fM]Clfichcstcr，UK：John Wiley＆Sons，1988．92-l1O
[7] Ebbs S D，Bushcy J，Poston S，et a1．Transport and metabolism of
free cyanide and iron cyanide complexes by willow [JIl Plant Cel
Environ．2003．26：l467 l478
[8J Barclay M，Tctt V，Knowles C J．Metabolism and enzymology of
cyanidc／metallocyanjdc biodegradation by Fusarium solani under
neutral and acidic conditions【JJ．Enzy Microb Techn，1 998，23：
321 331)+
[9]Dursun A Y，Calik A，Aksu z．Degradation of fe~ous(I)cyanide
complex ions by 19~et,Iontonas ㈨ “e，¨ 【J] Proc Biochem，
1999．34：901—908．
【1 0】Kjeldscn P．Behaviour of cyanides in soil and groundwater：a
rcvicw『J]．Water Air Soil Pollut，l 999，Il 5：279 307
[11】 Trapp S，Christiansen l I．Phytoremediation ofcyanide—poluted
soils【A]In：McCutcheon S C，Schnoor J L_Phytorcmcdiation：
Transfbrmation and Control ofContaminants[M]Iloboken，USA：
John W iley＆ Sons 2003．829 862．
[12]Yu X Z，Trapp S，Zhou P H，ct a1．The effbct oftemperature on the
rate ofcyanide metabolism oftwo woody plants【JJ．Chemosphcrc，
2005．59：1099一Il04．
[1 3】Yu x z，Trapp S，Zhou P H．Phytoloxicity of cyanide to weeping
willows【J]Environ Sci Polut Rcs，2005，1 2：l09一l13．
[14]Bcavis A D，Vcrcesi A E．Anion uniport in plant mitochondria is
mediated by a Mg2-insensitive inncr membt。ant anion channel⋯
J Biol Chem．1 992．267：3079—3087
[1 5 J State Environmental Protection Administration of China．Analysis
Method for Water and Wastewater【M】．3rd ed．Bcijing：Environ
mental Science Press，China，l 989 145 l 54+(in Chinese1
【I 6】Samiotakis M，Ebbs S D Possible evidence for transpol I ot、al iron
cyanide complcx by plants⋯ ．Environ Polut，2004，l 27：1 n9
173
[1 7]Fedcrico R，Oiartosio C E．A【ransplasmamembranc electron trans—
port system in maize⋯．Plant Physiol，l983，73：l82—184．
【l8]Trapp S，Zambrano K C，Kusk K O，ct a1 A phytotoxicity test
using transpiration of wilows lJ]Arch Environ Contain Toxicol
2000．39：l 54—160．
S 一甚 置∞§ 苗l。
维普资讯 http://www.cqvip.com