Environmental Pollution 82 (1993) 263 267
Cu(II) ACUTE TOXICITY TO THE ROTIFER
Brachionus calyciflorus, AS AFFECTED BY FULVIC
ACIDS OF FRESHWATER ORIGIN
Atilio A. Porta & Alicia E. Ronco*
Centro de Investigaeiones del Medio Ambiente ( CIMA ) , Facultad de Cieneias Exaetas,
Universidad Naeional de La Plata, Calle 47 esquina 115, 1900--La Plata, Argentina
(Received 29 January 1992; accepted 11 September 1992)
Abstract
Natural fulvic acids ( F A ) from the Rio Santiago, an
affluent o f the Rio de la Plata estuary, were isolated by
adsorption on XAD-2 at p H 2, and were then character-
ized by IR and fluorescence spectra, acidity and com-
plexing capacity to Cu(II). The effect o f the interaction
between FA and Cu(II) on the acute toxicity o f this
heavy metal was assayed with the rotifer Brachionus
calyciflorus in standardized in-vitro tests. A CuSO4.5H:O
concentration o f 0.080 ppm produced 89.2% mortality in
the absence of FA. A t FA/Cu ratios > 1 no mortality was
observed. Based on the values of the F A - C u binding
parameters and acute toxicity inhibition, it was possible
to predict the effect o f dissolved organic matter on the
toxicity o f copper in this surface freshwater body.
Keywords: copper toxicity, fulvic acids, Brachionus
calyciflorus, toxicity inhibition, complexation
INTRODUCTION
Heavy metals are natural components of aquatic
ecosystems, and some of them are essential for living
organisms. They are, however, among the top 10 pollu-
tants in rivers, lakes and reservoirs, (US Environmental
Protection Agency (USEPA), 1990), as a consequence
of human activity. Prediction of the ultimate fate and
possible effects of heavy metals on the aquatic biota
has been the focus of considerable research (e.g.
Demayo & Taylor, 1981; Petersen, 1982; Salomons &
FOrstner, 1984; Betti & Papoff, 1988). From the ecotoxi-
cological point of view, it is important to characterize
associations of heavy metals with various ligands
and/or particles present in the water (Salomons &
F6rstner, 1984; Mizuike, 1987; Sunda & Gessner,
1989). For example, dissolved organic matter regulates
the bioavailability of both essential and toxic heavy
metals such as copper and lead (Demayo & Taylor,
1981; Salomons & F6rstner, 1984; Newell & Sanders,
* To whom correspondence should be addressed.
Environ. Pollut. 0269-7491/93/$06.00 © 1993 Elsevier Science
Publishers Ltd, England. Printed in Great Britain
263
1986). Dissolved organic matter consists of various
compounds from natural sources, mainly large polydis-
persed molecules with multiple functional groups such
as polysaccharides, polypeptides, and dissolved humic
substances (DHS) (Salomons & FOrstner, 1984). The
content of dissolved organic matter in colored waters,
expressed as dissolved organic carbon (DOC), ranges
from 5 to > 50 mg liter i. The fraction of total DOC as
DHS varies considerably, reaching values as large as
80%. Typically, 90% of the DHS consists of fulvic acids
(FA) (Newell & Sanders, 1986; MacCarthy & Suffet,
1989).
In the present work, we studied the acute toxicity of
Cu(II), in the presence or absence of FA isolated from
the Rio Santiago (an affluent of the Rio de la Plata
estuary), using the widely distributed rotifer Brachionus
calyciflorus as the test organism. FA were chemically
characterized by their elemental composition, IR and
fluorescence spectra, carboxylic acidity and complexing
capacity. FA/Cu(II) molar concentration ratios used in
treatments reflected those seen in the Rio Santiago. The
effect of the FA content on the copper toxicity in the
aquatic system studied was calculated by means of the
measured FA-Cu binding parameters and inhibition of
acute toxicity.
MATERIALS AND METHODS
Study site, sampling and water analysis procedures
The Rio Santiago is an 8 km long tributary of the
Rio de la Plata estuary located near the city of La
Plata, Argentina. The environment of this highly indus-
trialized area is being degraded by contamination
from a large petrochemical and oil refinery complex, a
steel rolling mill and shipyards. Studies on water and
sediments in this region show high levels of pollution
from refractory organic compounds such as organo-
chloride pesticides (Colombo et al., 1990; Catoggio,
1991), polyaromatic hydrocarbons (Ronco et al., 1992)
and heavy metals (Catoggio, 1991; Ronco et al., 1992).
Water samples for FA isolation and characterization
were taken in October 1990 and April 1991 from the
ESE end of the Rio Santiago (Fig. 1), where the highest
264 A. A. Porta, A. E. Ronco

Table 1. Rio Santiago water quality parameters

URUGUAY
+ Parameter Units Whole
water
Filtered
water
(0.45/zm)
ARGENTINA L ,
pH 7.20 7.20
1 km
Temperature °C 19.0 --
50 km Dissolved oxygen mg liter ~ 3-4 --
Chemical oxygen demand mg liter ~ 29.7 18.5
DOC" mg liter l __ 7.2
Rio S a n t i a g o TOC" mg liter ~ 11-8 --
Solids
105°C mg liter 1 334.5 172.3
550°C mg liter 1 185-5 101.7
Suspended solids mg liter 1 22.3 --
Hardness (CaCO3) mg liter I 63.2 --
Sulphates mg liter ~ 51.0 --
CI + Br mg liter J 77-2 --
Conductivity txmhos liter ~ 560.0
OO i
Alkalinity (HCO3) mg liter ~ 72.8 --
Cu(II)b p~g liter ] 18.0 --
Fe total b txg liter ~ 86.1 --
FA concentrationc mg liter-I __ 15.5
• - petrochemical industries FA concentrationa mg liter ~ -- 26.1
- oil r e f i n e r y Complexing capacity
o - sulfuric acid Cu(lI) (anodic mol liter t -- 3 x 10 7
- steel rolling mill stripping voltametry)
• - shipyards
• water supply treatment plant
-

Calculated from the COD values (Kononova, 1982).

Fig. 1. Rio Santiago location.
D O C contents were observed. Samples were collected
in acid-washed, 20-liter polyethylene containers, sub-
merged below the water surface. Chemical analyses were
performed according to standard methods (American
Public Health Association (APHA), 1985).
Fulvic acid isolation and characterization
FA isolation was carried out at pH 2.0 (HCI), at which
humic acids are insoluble; the soluble sample fraction
was separated by filtration through a filter of 0.45-/xm
pore size followed by concentration in a column con-
taining a hydrophobic resin (Amberlite XAD-2) that
retains FA. Elution was performed with 0.1 M N a O H ,
and the eluate was chromatographed in a second
column with an ion exchange resin (Zeocarb 225) to
obtain F A in the acid form (Thurman & Malcom,
1981; Porta, 1991). The elemental composition of the
F A was determined by conventional direct methods
(Cheronis & Entrikin, 1957). The IR spectra of a dry
sample (70-80°C) in a matrix of KBr (1%, w/w)
(Gauthier et al., 1987; Malcolm & MacCarthy, 1989)
was also obtained, using a Perkin-Elmer 580 B IR
spectrophotometer.
Fluorescence spectra were obtained with a Perkin-
Elmer 2000 fluorescence spectrophotometer at an
incident wavelength of 364 nm, at 25°C, and with FA
concentrations of 10-20 mg liter -l at pH 2.0 (Ewald et
al., 1983; Liebezeit, 1988). Complexing capacity (CC)
was determined by potentiometric titration of the F A
with Cu(II) at a 7 mM constant ionic strength, at pH 7.40
and 25°C, with an Orion cupric ion selective electrode
(ISE-Cu), using an Orion 701A digital pH/mV meter
bAtomic absorption methods.
c Calculated in comparison with the absorption vs concentra-
tion calibration curve of the Suwannee FA standard at 254 nm
(Ryan & Weber, 1982).
d Calculated in comparison with the fluorescence vs concen-
tration calibration curve of the Suwannee FA standard (Ryan
& Weber, 1982; Liebezeit, 1988).
(Cabaniss & Shuman, 1988; Perdue, 1989). CC and
conditional concentration quotient (K*) calculations
were carried out using the computer program F G M I N
developed by Perdue & Parrish (1986). Carboxylic acidity
was determined by the acetate method (Kononova, 1982;
Bonn & Fish, 1991). The Baryta (Ba(OH)2) adsorption
method was employed for total acidity analysis
(Kononova, 1982; Bonn & Fish, 1991).
Toxicity tests
Acute toxicity tests were carried out with B. calyciflorus
kits (Toxkits), by measuring immobility under the micro-
scope as the end-point after 24 h of exposure (LCs0)
(Snell & Persoone, 1989; Persoone et al., 1990). Assays
were performed in multiwell plates using 10 B. calyci-
florus neonates (less than 2 h old) per treatment. These
organisms were obtained by hatching cysts in moder-
ately hard synthetic freshwater (96 mg NaHCO3, 60 mg
C a S O 4 . 2H20, 60 mg MgSO4, and 4 mg KC1 per liter
of deionized water, adjusted to p H 7-5) according to
Snell et al. (1991). Calibration of the assay with
CuSO4 . 5H20 (Mallinckrodt, analytical grade) was
carried out using five replicates and six dilutions under
the conditions of the protocol supplied with the kits.
LCs0 was estimated by means of the best-fit dose-
response curve with the Grandpro program (Hong et
al., 1988). Bioassays in the presence of F A were
performed in the same way as for those for the calibra-
 C u ( I I ) - F A acute toxicity to Brachionus calyciflorus 265

Table 2. Fulvic acids characterization

Parameter analyzed Method Rio Santiago FA Other FA reported results

Elemental Conventional % C = 45.7 % C = 5542"

composition direct methods % H = 4-1 % H = 3-5"
% N =2.3 % N =0-5-3 a
Acidity
Total Ba(OH) 2 6-54 mequiv g i 5.35-14.0 mequiv g Ib
Carboxylic Ca(Ac)2 3.48 mequiv g i 3.80-6.00 mequiv g ib
Complexing ISE-Cu 3.33 mmol g i 2.96-4.37 mmol g Ic
capacity FGMIN
Cu(II)-FA ISE-CU log K* = 7.44 5.90-7.85 c
association constant FGMIN
Fluorescence spectra Spectroscopy Aex 364 nm Aex" 350-360 nm d
Acm"447 nm Aem"445-455 nm d

A~. = excitation wavelength, Aem" = emission wavelength.

a Malcolm and MacCarthy (1986); Gauthier et al. (1987).
b Kononova (1982); Bonn and Fish (1991).
c Castetbon et al. (1986) Cabaniss and Shuman (1988); Perdue (1989).
aRyan and Weber (1982); Ewald et al. (1983); Liebezeit (1988).

tion experiments, by adding F A isolated from the Rio
Santiago at five different molar ratios of F A : C u ( I I )
(0:1, 0.25:1, 0.5:1, 1:1 and 2:1). All experiments
were performed at 25°C. Controls either in the presence
of a F A concentration corresponding to each given
ratio or with no F A were carried out. The arcsine
transformation (arcsin X/P, where p is the number of
dead organisms/total number of organisms) was applied
to each result, to ensure homoscedasticity before analysis
of variance (ANOVA). Treatments were compared by
the least significance differences test (LSD).
R E S U L T S AND D I S C U S S I O N
Sample water analysis
Water from the Rio Santiago (Table 1) shows a high
F A content characteristic of fresh surface colored
waters. This is shown by its CC, absorption at 254 nm
and fluorescence values. Taking into account, the high
K* for C u - F A (Table 2) and the CC of this water, a
low cupric ion toxicity could be expected.
Results of the parameter characterization of the F A
from the Rio Santiago (mean values for two sampling
dates) were compared with those reported in the litera-
ture and are shown in Table 2. I R absorption bands at
3400, 2920, 1720,and 1640 cm -~ from carboxylic groups
were present. The ratio of absorption at 1720 cm ~
to absorption at 1640 cm l was greater than one, as
expected. This ratio is used as a criterion of purity
and low ash content, and as an indication that SH
groups are in the acidic form (Gauthier et al., 1987;
MacCarthy & Suffet, 1989).
The cupric ion content of the water samples was also
10 times higher than 2 /xg liter J, the recommended
concentration limit for the protection of aquatic life
(Demayo & Taylor, 1981).
Toxicity tests with B. calyciflorus
Results from the calibration tests gave a mean LCs0 of
0.037 mg liter ~ CuSO4.5H20 (0.0094 mg liter 1 of
Cu(II)). The coefficient of variation was 10.1%. This
result is similar to that reported in the Toxkits inter-
calibration exercise (Persoone et al., 1990), in which the
mean LCs0 was 0-033 mg liter -~, with a coefficient of
variation of 48.5%.
Experiments with F A were carried out at a
CuSO4.5H20 concentration of 0.08 mg liter 1, at which
more than 80% of deaths would be expected if no F A
were present, as assessed by the calibration curve. FA
from the Rio Santiago greatly affected toxicity (Table 3).
N o toxic effects occurred when the ratio was greater
than one. Experimental results corresponding to various
treatments were studied by analysis of variance using a
random design. Means corresponding to each treatment
were compared by using the LSD test with a p -- 0-05.
N o significant differences were observed between

Table 3. Brachionus calyciflorus acute toxicity tests results as % mortality for various FA/Cu(II) concentration ratios

[FA] : [Cu]

0:1 0-25:1 0-5:1 1:1 2:1

2? sd ~T sd 2? sd a7 sd ~7 sd

Controls 7-5 5.0 6.7 5.8 3.3 5.8 6.7 5.8 -- --

0.080 ppm 89-2 0.5 65.8 11.6 53.0 21.0 6.7 5.8 6.7 5.8
CuSO 4 . 5H20

Results shown are mean values from three or four replications.

266 A. A. Porta, A. E. Roneo
controls and F A : C u 1:1 and 2:1. Treatments with
ratios 0.25:1 and 0-5:1 showed significant differences
from F A : C u ratios of 0: 1, 1 : 1, 2:1 and controls, but
no significant differences were found between them.
Implications for Cu toxicity in the aquatic ecosystem studied
Assuming that the FA content of Rio Santiago water
reaches an approximate concentration of 15 mg liter 1
(Table 1) and a CC of 3.33 mmol Cu(II) g~ FA (Table
2), and considering that no acute toxic effect occurred
at FA/Cu ratios > l, the m a x i m u m concentration of
cupric ion buffered by the system can be estimated as
follows:
3.33 m m o l Cu
x 15 mg FA liter -1 x 0.79
1000 mg FA
= 0.039 m m o l Cu(II)
The 0-039 m m o l Cu(II) is equivalent to 2-5 p p m Cu(II).
The factor 0.79 weighs the effect of other heavy metals
( H M ) competing for FA as a function of their respec-
tive concentrations and conditional concentration
quotients (K*) for the H M / F A equilibrium (Porta,
1991).
This analysis does not take into account variations in
water pH, salinity, hardness or temperature, nor the
equilibrium between dissolved and particulate matter.
It applies only to acute toxic effects on the very sensi-
tive species used in this test study.
Results of earlier studies on the attenuation of heavy
metal toxicity by dissolved organic matter and humic
substances agree with the data reported here in Table 3
(Sunda & Lewis, 1978; D e m a y o & Taylor, 1981; Newell
& Sanders, 1986; Stephenson & Wren, 1991; Garvey et
al., 1991). However, this approach considers FA
isolated from the water body under study and the CC
and K* associated with the inhibition of the toxic effect.
Water from the Rio Santiago could buffer fluctua-
tions in Cu input as large as 2.5 ppm, and therefore
only Cu concentrations above this value would affect
the rotifer B. calyciflorus. Reduction of toxicity would
suggest that Cu(II) is not bioavailable in the presence
of FA, at least under the exposure conditions used.
The bioassay technique, together with chemical
parameters related to speciation, predicts more accu-
rately the possible effects of copper inputs to freshwater
and estuarine systems. Further studies involving other
species, which would also consider the effect of salinity
on the copper binding capacity to D O C (Newells &
Sanders, 1986), as well as the influence of water hard-
ness and alkalinity on the copper toxicity ( D e m a y o &
Taylor, 1981), could be of interest for the development
of chemical speciation models in aquatic ecotoxicology.
ACKNOWLEDGEMENTS
The authors thank D r C. Blaise for the Toxkits,
D r R. L. Malcolm for the Suwannee FA standard
and D r E. M. Perdue for the F G M I N program. A. A.
Porta is a fellow of the Buenos Aires Province
Research Council (CICPBA), A. E. Ronco is a member
of the Research Career of the National Research
Council (CONICET). This project was supported by a
C O N I C E T grant.
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