Rev.
Mus. Argentino Cienc. Nat., n.s.
6111: 191-1 95,2004 Rue,~os Aires, ISSN 1514-5158ecute
C u
toxicity, metal bioaccumulation and ion loss in the
Neotropical cyprinodontiform
Jenynsia multidentata
Carlos A. VILLAR
',
Sergio E. GOMEZ Cristina A. BENTOS'
'
Muilrisro Argentino de Ciericias N a i w d e s *Brrnardino Rivadn\ia~>, Av. A . Gallardo 470, Cl405DJR Biieiios Ai- res, Argc~itini*. zIi~vestigador del CONICET sego.omoz@ii~ovi.coiii.up.Abstract: T h e aciitc toxicity or CII, metal bioacci~niulai,ion and iori lose in ihe cypriiiodontifo~-1x1 Jenynsin ,ni~ltider~loin, a highly eurihdinc Neotropicul f~oshwater fish of wide distribution in t h e Rio de la Plata ha.; -m, was cstabliahod during a 96 h st,atic exposure. The ineilian letlid conceiitraiiun a i 96 1) was 2 2 9 , ~ g C i l i'. The cKecl of CuS0,5H20 was tested in natural freshwater in two-liter Pyrex glass chmnbers at controllerl temperature, wit11 nat.ur.td light and artificial acriilion. During t,he First iiours of exposinre to conceritraiioris above 130 p,g Cu i', uii
irrcreaac iii aquatic snrfulcc respiration, air bulping aid erratic s w i i m i n g wcre observed, showing cvidcnce ol'rbe iiSrcct.ion of t,iie respiratory ~ y s i e m . The concci~tration ofCu, Na a ~ d K i n the wliole body brirdeii at t h e e i ~ d oreach experiment showed by i:orrel,?tion iindysio Cu bioacmmiulatioil anit loss of Na- and K'. T h e concentral,ion oi'Cu in water was posit,ively mrrelated with its bioacauninlation (r=0.79, p=0.06), and ncgaiabivcly correlated with the wliolc body burden orK7 (r=-0.84, p=0.037). K- loss was positively correlated with Na- loss (r=0.88, p=0.02). Cu I,C5O for this <:ypririodor~tiforni seems riiiher low when aompnred to Fiiihes often utilized in tonicity tests.
Key words: coppor toxiriiy, bioacc~mmlution, ion loss, Jenynsia rni~ltzdentnta, Ncotropicnl fish
With a n area of 3 x 10Qkm%nd a mean dis-
! charge of 25000 m? s:, the Rio de la Plata basin is
the second largest hydrographic systeui in South America, after the Amazoil. Large urbanizations and huge industrial settlements a r e particularly intense in the losver basin, leading to metal con- centrations close to or above levels t h a t could af-
fect biota (Boltovskoy et al., 1997; et nl.,
1998).
The Neotropical region has more than 2500 fish species (Lowe-McConnel, 1975) in coinparison with the around 700 of Holarctic America (Moyle
6: Cech, 1996). In sFijte of its el;ident relevance to environmental impact assessmnent, information on metal toxicity on Neotropical fish is scarce, and e x p e r i m e n t a l l a b o r a t o r y d a t a o n m e t a l bioaccumulation remairis unreported.
Cyprinodontids have long been used in toxic- ity bioassays (Jones, 1964; Heath, 1995). Jenyrrsia
~ n ~ c l l i d e ~ ~ t n t n , a widely distributed endemic small fish representative of Neotropical America is the southernmost cyprinodontid of the world, reach- ing 39" 20' S (Ferriz & L6pez, 1987). This species is observed in a t least four countries of Meridi- onal America, s u g g e s t i n g i t a s a possible bioindicator organisin for t h e region. G6mez (1996) assessed its thermal, salinity, and osmotic
pressure resistance, stating t,hat i t fulfils re-
quested conditions for laboratory bioassays. J.
,nr~ltident<cla is highly eurihaline, toleratinglong-
term exposure to seawater of 35 %o (Thormhalen
de Gil, 1949).
In the research presented here we investigated the acute effects of Cu on the survival of wild
caught specimens of J. rnultzder~tatii by means of
static acute bioassays performed in natural fresh- water, and we discussed metal bioaccumulation, ion loss, and t h e response of this eurihaline fish to Cu toxicity.
MATERIALS AND METHODS
Experimental design
Tests were conducted using 70 adults of J.
rr~rcllidei'tala (not sexed) captured in "La Salada de Monasterio" (35' 47'5, 57' 52'W), a small la- guna without any point source contamination. Fishes were acclimated in the laboratory for four weeks prior to experimental use.Water tempera-
ture ranged 24-26°C and pH 7.2-8.0. Composition
of the water used in the hioassaysresembled mean water characteristics of natural environments
populated by J. ~nullidenlata, having a conduc-
192 Rel~ista del Mususeo Argentino de Ciencias Nr~tumles, n. s. 6 ( I ) , 2004
tions of 1.6 mg dissolved organic carbon (DOC) I-', 26.7 mg Ca" il, 4.4 mg Mg2+1.', 35.0 mg Xa+
ti,
5.3 mg K+ I-', 28.6 mg C i 1-', 62.2 mg HCO; 1.'and 66.3 mgS0,2- 1'. The concentration of Cu was
below 4 p g 1-I.
P u r i n g acclimation animals were fed with a daily ration of ca. 3 %body weighi, ofcomniercial fisb food Tetra ~ e r k e @ . Acute static bioassays
were performed following Ward & Parrish (1982)
and S p r a w e (1990). Tile effect of CIISO,SH,O on seven experimental fish groups was tested in two- liter Pyrex glasschambers a t controlled tempera- ture, witllnatural light and artificial aeration. The test was performed with one control chamber and 6 different Cu doses: 2500, 1250, 600, 300, 150
and 5 p g 1.' nominal concentrations. Each cham-
ber contained a group of 10 specimens. Cu con- centration a t each chamber was attained by
pipetting from a stock solution of 1 g Cu 1.'. Total
Cu concentrations were measured a t the begin- ningand a t the end ofthe bioassays. The number of fish alive was registered twice a day during 96
b. All t h e fishes were measured (standard length)
and weighed (wet and dry weight a t 60°C) a t the end of the bioassays. >4verage fish load a t each
chamber was 0.47
k
0.13 g 1-I (wet weight). Dur-ing the experiment fish were not fed.
Analytical procedures
Water p H (Orion 250 pH meter), conductivity (Luftman conductance meter), and HCO; (Gran
titration), C1- (silver nitrate titration), SO: (tur-
bidimetry), Ca" and MgZ+ (flame atomic absorp- tion) concentrations were determined a t the be- ginning of t h e test (APHA, 1985). DOC was de- termined after Golterman et al. (1978).
Water samples were prepared for metal analy- sis a s follows: 100 mi of water were combined with 3 mi concentratedHNO,, refluxed in 250 ml flasks until volume was reduced to 10 ml and diluted to 100 mi. Samples with Cu concentrations lower
than 40 p g 1-I were concentrated in a cation ex-
change resin (Arnberlite IRCSO), and later eluted back with 2 M HNO,. Cu determinations (atomic absorption spectrometry, Buck 200A) were per-
formed following Bettinelli el al. (1989).Calibra-
tion curves and traceability of the method were assessed using commercially available certified stock solutions. The limit of detection of Cu for water samples was 4 p,g li-.
Fish tissue samples were prepared a s follows: a composite sainple was digested i n 250 ml flasks with the addition of 2 mi II,SO, and 5 mi of HNO, until total digestion of organic rnatter was oh- served. Cu (atomic absorption spectrometry, Buck 200A), Na and K (flame photometry, Buck 200A) d e t e r m i n a t i o n s were performed following
Bettinelli el al. (1989). Traceability of Cu, Na and
K results were assessed by the use of bovine liver
(SRM 1577b, NIST). The results obtained were in close agreement with t h e certified values.
Statistical methods
The median lethal concentration LC50 a t 24,
48, 72 and 96 h were calculated using the LC50
Calculation Program (Harrass, 1986). Statistical analysis was performed on the average measured concentrations. Thisprograrri calculates an LC50 estimate with confidence limits based on dat,a from toxicity tests using three methods: Probit analysis, Moving Average, a n d t r i m m e d Spearman-Karber. The arcsine transforrriation is used with tile first two methods. LC50 concen- trations obtained by Probit analysis were related with time by means of a decreasing function. Pearson correlation coefficients were calculated between Cu concentrations in water and Cu, K and Na content in the fish tissue.
Table 1 shows the average total Cu coricen-
trations in water and the % mortality a t different
tirnes. Initial Cu concentrations decreased on av-
erage 11 O/o by the end of the bioassays. No mor-
tality was observed a t the control and <4jig Cu i-
'
dose. The bioassay with Cu concentration below4 p g 1.' was assigned a value of 2 p g i-' for the sta-
tistical analysis. Table 2 shows the LC50 values
a s a function of exposure time. The average LC50 obtained by tile three methods declined during t h e 96 h test, and was related with time by means of a negative multiplicative function (Fig.1) (time in hours and L,CSO in p g Cu 1.'):
Along the 96 h acute toxicity test t h e behav-
ioral responses of J. ~nultiderzlatii were observed
and compared with normal activity. During the first hours of exposure to concentrations above 1 3 0 ~ g Cu il, a n increase in aquatic surface respi- ration, air gulping and erratic swimming were observed. After this initial phase, fish behaviour returned to normality.
Tlieconcentrationsof Cu, Na and Kin the whole body burden a t the end of each experiment showed
Cu accumulation and loss of Naf and K+ (Table 3).
The amourit of cation loss was related with the Cu dose. Cu concentration in water was positively cor- related with Cu fr=0.79, p=0.06) and negatively with IF ( r = -0.93, p=0.01) content in fish tissue.
K+ content in fish tissue was positively correlated
T'zller et a1 CIA to~icgty a n d b~oaccumulatior~ in J e n y n s ~ a multidentata 193
Table 1. Average Cu concentrations in water, water temperature, average standard length
and average weight of fish, and mortality
(M)
a t different times (b) in each Cu dose assayed.Cu conc. T Standardlength Weight M(24) M(48) M(72) M(96)
p g
ti
"C mm g % % 9% 9%2123 23.0-26.8 17.2 0.06 90 100 100 100
1252 24.0-25.7 18.6 0.07 70 100 100 100
536 22.9-26.8 16.4 0.04 0 40 70 80
250 22.9-26.8 16.3 0.04 0 10 30 40
131 22.7-26.3 18.3 0.05 0 10 20 30
<4 24.3-26.0 16.8 0.04 0 0 0 0
control 24.0-26.0 16.3 0.06 0 0 0 0
Table 2. LC50 of Cu @,g 1.') a t different exposure
times, calculated by three different methods. In each case lower and upper confidence limits are
indicated (LL and UL).
-
Time (hi 24 48 72 96
Probit
LC50 11.36 528 313 248
LL 797 359 203 144
UL 1470 774 460 369
Moving average
LC50 1092 417 291 205
I Spearman-Karber
LC50 1018 858 379 234
LI, bI>
Average 1082 601 328 229
Standard dewatioil 60 229 46 22
Table 3. Content of Cu, Na and K (DW) and Na'K
molar r a t i o of t h e whole body b u r d e n of J. 1,~ultr,ie7&tiLa a t the end of t h e bioassay
Cu conc. Cu Na K Na'K
p g l l m g k g ' g k g ' g k g '
2123 73.1 2.64 4.3 1.35
1252 16.9 3.03 7.8 0.63
536 30.8 4.24 8.2 0.88
250 29.8 4.37 10.0 0.74
131 21.4 4.04 9.3 0.74
<4 13.8 7.19 12.2 1.0
control 13 7 7.20 12.1 1.0
tively correlated with Cu bioaccumulation (r= -0.84, p= 0.04). Cii uptake by the fish was rapid, increas- ing 5.3 times in less than 24 h in the higher Cu concentration assayed. The molar ratio of Na and K i n fishes not exposed to Cu was 1.0. This rela- tion decreased in fishes exposed to Cu, excepting in the highest concertration assayed. Care must
be taken when comparing Cu, Na and X in the
whole body burden as a function of time because exposure time was not similar for each Cu con- centration.
DISCUSSION
The time-related development of the LC50 val-
ues (24,48, 72, and 96 h) did not compietely reach a stabilizing concentration within the 96 h of the
bioassay. LC50 of J. multidintala was 26 %: lower
a t 96 than a t 72 h, show~ng that acute mortality
had not reached an expected asymptotic value yet, a s is most generally observed withi119G h (Ward &
Parrish, 1982). LC50 24 h of this fish was 3.6 times
higher than that of C~~esterodon decemmaculahLs,
another sympatric cyprinodontid t h a t shares a similar life histow (V~llar et al., 2000). but the dif- ference between both species decreased multipli- catively with time
(RZ=
0.998), being only 1.6 tiines higher for J. rnullidentata a t 96 h (Fig.1). Higherresistance ofJ. rr~ullideictatu during the first hours
of exposition may be related with t h e enhanced capacity of osmoregiilation of eurihaline fishes, which have the ability to reverse the active move- ment of Na+ and C1- across the gill tissue, to change tile amount and composition ofurine excreted, and to alter epithelial permeability, all within a matter of hours. Nevertheless, Cu toxicity seems to im- pair t h i s preliminary enhanced tolerance of
Jncultider~tatu t.0 acute Cu exposition, leading to
194 Reriista del Museo Argentilzo de Ciencias N o t ~ ~ r u l e s , n. s. 5 (I), 2004
1500 Noen~acheili~s rupicola showed a LC50 96 h of230
p g Cu 1-"Joslli & Prakash-Semwal, 1990), and
-
Acl.ossocl~eilr~s paru.doxus a LC50 96 h of 26 p gCu 1-', being the sensitivity of the last species si~ni-
0
a
-
lar to that of Srrlrno sp. (Chen &Yuan, 1994.).The sensitivity to Cu of J. rnultide~~toto was
"
Y
'00l o o
kH
.
-
-.
.
.
-. . .in
used (U.S.EPA) a range US Environmental standard comparable test to organisnis t h a t Protection of the maintained commonly Agency in
0 c o n t i n u o u s c u l t u r e : D a p h n i a m a g n o a n d
0 24 48 72 96 Ceriodaphnia d i ~ h i a 48 h EC5O 27 and 52 pg Cu
I-', respectively; Seler~mtrrrirr <;apricornulum 96 h
Time (h)
EC50 40 p g Cu
i-';
Mysidopsis bahir~ 96 h LC50Fig. 1. LC50 values calculated by Probit analysis 160pg CU 1~'; andPimephalesproinelus 96 11 LC50
f o r J. m l r t i i l e r ~ l a t a (filled l i n e ) a n d , C. 480pgCu 1.' (Shedd etal., 1999). decemrr~,uculatrrs (dotted line, from V~llar et al.,
2000) and confidence limits (vertical bars) plot- ted a s a function of exposare time (hr).
This work was supported by grants from IFS, CONICET and Pablo Cassara Foilndation.
Cu causes a comparatively large upset in os- moreplation in freshwater fishes; exposed fish show a rather rapid decrease in plasma electro- lytes and/or osmolality, coincident with the ob-
served loss of Na and K in the body burden in the
present research. The variation of the Na/K ratio observed suggested that the processes r e l a t ~ d with Na+ regulation were more affected than those of K+. The mechanism of the Cu effect on osmoregu- lation in freshwater fishes appears to be a n inhi- bition of Na+ and C1- uptake by the gills probably
due to a n inhibition of 6he enzyme Nai,
K+
AT-Pase in t h e gill tissue. High doses of Cu cause death by the combination of hypoxemia due to impaired oxygen uptake by the gills, and dysfunc- tions in ionoregulation (Heath, 1995).
Because Cu toxicity in water is influenced by
water hardness, alkalinity, pH, and dissolved or- ganic matter, and because experiments on fish toxicity are carried out a t a wide range of differ- ent experimental conditions, caution should be taken when comparing LC50 data from the lit-
erature. Cu LC50 for J. rni~ltin'er~tuta seems rather
low when compared to fishes often utilized in tox- icity tests. Sorerlsen (1991) compiled LC50 96 11
of 11 fish species, ranging between GOpg Cu 1-' for
Oncorl~yrcchus Kisutciz a n d 2400 p g Cu 1 ' for Lepornis mn..rochiri~s, with a n average value of
561 p g Cu 1-' (SE = 144). I n the upper range of
t h e l i t e r a t u r e d a t a , fingerlings of I c t a l u r r ~ s punctatzls (catfish) showed a LC50 96 h of 730 , ~ p
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