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E L S E V IE R
A quatic Toxicology 30 (1994) 137-151
AQUATIC
TOXICOLOGY-
Effects of low water pH on lead toxicity to early life stages of
the common carp (Cyprinus carpio)
V.J.H.X. Stouthart*, F.A.T. Spanings, R.A.C. Lock, S.E. Wendelaar Bonga
Department o f Animal Physiology, Faculty o f Science, University o f Nijmegen, Toernooiveld,
6525 ED, Nijmegen, The Netherlands
(Received 21 D ecem ber 1993; revision received 29 M arch 1994; accepted 29 M arch 1994)
\bstract
Carp eggs were exposed immediately after fertilization to Pb concentrations of 0.12 to 0.96
¿nnol I-1 at water pH 7.5 and 5.6. At regular intervals mortality, the incidence of spinal cord
ieformation, heart rate, body movements, hatching success, and whole body concentration of
1C, Na, Mg, Ca, and Pb were assessed. At pH 7.5, Pb increased heart rate, and decreased body
movements, while at pH 5.6, Pb also reduced hatching success, caused spinal cord deformation,
decreased net Ca2+ uptake, and increased mortality of the larvae, in a concentration-dependent
manner. In controls of pH 5.6 no significant changes of any of the above parameters were
observed when compared to controls at pH 7.5.
Key words: Acid stress; Lead; Embryonic development; Carp; Cyprinus carpio
1. Introduction
Lead (Pb) is one o f the m ostly used industrial metals. T he an th ro p o g en ic release o f
Pb to the environm ent is the highest o f all heavy m etals (Pb > Ag > M o > Sb > Z n >
Cd > As > C r > C o > M n > Hg; S alom on and Forstner, 1984). A lth o u g h the release
o f Pb into the env iro nm en t has been greatly reduced in the past decade, e.g., via
in tro d u ctio n o f Pb-free gasoline, and recycling o f Pb, it is still used by fishermen and
hunters in large quantities. Indeed, hun tin g an d fishing activities are responsible for
86% o f the yearly Pb emission o f approxim ately 375 t into D u tch surface waters
(Janus et al., 1991).
In buffered and eutrophic w ater the bioavailability o f Pb is reduced because o f
com plexation with w ater-borne organic particles (N riagu, 1979). In contrast, the bi­
oavailability o f Pb is strongly enhanced at decreasing w ater p H (Brown, 1979), in­
creasing the toxicity o f Pb for fish.
C o r r e s p o n d i n g author.
0166-445X/94/S07.00 © 1994 Elsevier Science B.V. All rights reserved
S S D I 0 1 66-445X (94)00027-N
138
A.J.H.X. St out hart et al. / Aquatic Toxicology 30 (1994) 137-151
However, the effects o f this phenom enon on the early developm ental stages have
received little attention. Eggs and larvae, are considered m uch m ore sensitive to heavy
metals than adults (M acek et al., 1977; M cK im , 1985; H odson et al., 1979; M o h a n et
al., 1983; Dave and Xiu, 1991; Sayer et al., 1991). We have therefore examined the
effects o f varying Pb concentrations at w ater pH 5.6 in com parison to pH 7.5 on the
developm ent o f eggs and larvae o f the com m on carp ( Cyprinus carpio).
2. Materials and methods
In vitro fertilization and incubation o f eggs
C arp gametes were obtained after artificial fertilization o f carp eggs as described by
Oyen et al. (1991). C arp eggs ( 3 0 0 ^ 0 0 ) placed in petridishes were fertilized by sperm
from three different males resulting in three different batches o f eggs. Immediately
after fertilization different petridishes with the fertilized eggs stuck to the b o tto m were
placed in a 4-1 aq u ariu m (five per group; for details, see Oyen et al., 1991; Oyen, 1993),
containing the following m ain ion concentrations in demineralized water (in mmol
I"1): 0.06 KC1; 0.40 N a H C 0 3; 0.20 M g S 0 4; 0.80 C aC l2). M ortality o f eggs and larvae,
deform ation o f larvae and hatching success were examined. In each aq u ariu m one
petridish was specifically used to examine mineral content, heart rate and tail m ove­
ments o f the developing embryos.
Eggs were exposed, during a 12 h p h o to p eriod in w ater (8.7 ppm D O C ) o f 23°C o f
pH 7.5 ± 0.05 and pH 5.6 ± 0.07 (controls) and to Pb (as P b ( N 0 3)2) in a co n cen tra­
tion range o f 0.12, 0.24, 0.48 and 0,96 //m ol I"1. W ater p H was adjusted to 5.6 via
gradual addition o f 0.01 M sulfuric acid using p H -stat equipm ent. C o n sta n t Pb c o n ­
centrations were m aintained via a flow-through system resulting in a complete tu r­
nover o f the system ’s w ater content after 10 h. Desired Pb concentrations were within
5% o f the calculated values as verified by A tom ic A b so rp tio n S pectrophotom etric
(AAS) analyses.
Mortality and deformation o f the spinal cord
D ead eggs and larvae were counted at 6, 12, 24, 48, 58, 72, 96, 120, 144 and 170 h
after fertilization and immediately removed to prevent fungal growth. Eggs were
considered dead when parts o f the content turned o p aq u e and white, or when heart
beat had stopped.
The percentage o f deform ed larvae (including dead ones) was determ ined after
microscopic exam ination. D eform ed larvae tended to lay on the b o tto m o f the a q u a ­
ria.
Heart rate and tail movements
Shortly before hatching, heart rate (beats m in " 1) and rate o f tail m ovem ents (beats
m in -1) were determ ined for at least 20 em bryos per group.
Mineral content
To determ ine dry weight, 125 eggs or larvae from each group were collected at
A.J.H.X. Stouthart et al. I Aquatic Toxicology 30 (1994) 137-151
139
12, 24, 48, 58, 72, 96, 120, 144, and 170 h after fertilization. To discriminate
etween the a m o u n t o f Pb adsorbed to the chorion and the a m o u n t o f which had
ntered the em bryo, an extra 10 em bryos were stripped from their chorion just before
atching (50 h after fertilization). T he eggs, chorions, em bryos and larvae were freeze ried to co n stan t weight.
Total body K, N a, Mg, C a and Pb content o f eggs, chorions and larvae were
llculated on a dry weight basis. Tissues were dissolved for 24 h at 70°C in 65%
I N 0 3. The K and N a concentrations were m easured in a flame photom etric A uto
\nalyzer (Technicon); M g and C a were analyzed with Inductively C oupled Plasma
vtomic Emission Spectrom etry (Plasm a IL200, T h erm o Electron, USA). Pb was
etermined with an atom ic absorption spectrom eter (Video II, T h erm o Jarrell Ash,
iSA). All concentrations are expressed as //m ol g_1 dry weight.
latching success
H atching success was defined as the percentage o f larvae hatched every 3 h, from
i 1 h to 72 h after fertilization. H atching was defined as rupture o f the egg m em brane
)y the tail. Fully as well as partially hatched larvae were counted.
Stat isticaI analysis
D a ta are expressed as m eans ± SE (n = 6). A one-way analysis o f variance was
ised to assess differences between groups. Significance o f differences was subsequenty tested using the Student /-test. Significance was accepted for P < 0.05. Significance
s expressed at the level o f AP < 0.05; hP < 0.02; CP < 0.01; dP < 0.001 com pared to
control values.
3. Results
Mortality o f eggs and larvae and deformation rate
N o significant difference in m ortality o f eggs and larvae was found between the
groups exposed to Pb at pH 7.5 and the control g ro u p (Table 1). At p H 5.6 egg
mortality was not significantly increased by Pb. Exposure to p H 5.6 resulted in co n ­
centration-related m ortality o f larvae com pared to the control (Table 1). D efo rm a­
tion percentage was identical for all experimental groups at p H 7.5 (Table 2). H ow ev­
er, the Pb-exposed larvae exhibited a deviating swimming p attern com pared to c o n ­
trol larvae. M ost o f the Pb exposed larvae tended to lie dow n on the b o tto m whereas
most o f the control larvae were freely swimming (Fig. 1). Significant deform ation
occurred in all Pb exposed treatm ents at pH 5.6 (Table 2). A 100% deform ation with
increasing severity was observed at concentrations o f 0.48 and 0.96 //m ol I-1 Pb. The
different types o f d eform ation are shown in Fig. 2.
Heart rate and tail movements
H eart rate and tail m ovem ents were not significantly increased at pH 5.6 com pared
to pH 7.5. In the presence o f Pb, a concentration-related increase o f heart rate and tail
m ovem ents was observed (Table 3).
140
A.J.H.X. Stout hart et al. I Aquatic Toxicology 30 (1994) 137-151
Table 1
M ortality o f carp eggs and larvae during exposure to Pb at pH 7.5 and 5.6
[Pb]
M ortality o f eggs (%)
M ortality o f larvae {%)
//m ol r 1
pH 7.5
pH 5.6
pH 7.5
pH 5.6
n.d.
0.12
0.24
0.48
0.96
11.2
8.9
8.8
9.6
10.7
12.0
11.6
9.8
11.8
11.0
3.7
2.9
3.8
3.5
3.8
3.3
12.5
23.5
33.0
42.0
± 3 .5
± 2.4
± 2 .7
± 3 .1
± 4 .7
± 3.9
± 3.3
± 2 .5
± 2.5
± 3 .1
±
±
±
±
±
1.4
0.5
2.7
1.0
1.3
± 1.3
± 4 .2 b
± 2.8d
± 1.2d
± l.ld
Significance is expressed at the level o f P < 0.02; ''P < 0.01; dP < 0.001 c o m p ared to control values. M o r ta ­
lity is expressed as percentage o f total n u m b e r o f eggs an d larvae: m eans ± S.E. (// = 6) are given (n.d..
non-detectable: less than 1 x 1 0 6/m iol 1 ').
Table 2
Percentage o f deform ed larvae during exposure to Pb a t pH 7.5 an d 5.6
[Pb]
D eform ation o f larvae (%)
/vmol I-1
pH 7.5
pH 5.6
n.d.
0.12
0.24
0.48
0.96
11.7
13.2
10.6
10.4
13.7
12.2
20.5
26.7
100.0
100.0
± 3.8
± 2.3
± 4 .9
± 3 .8
± 2.8
± 4 .1
± 1.4“
± 2.4C
± 0 .0 d
± 0 .0 d
Significance is expressed at the level o f 'P < 0.05; CP < 0 .0 1 ;dP < 0.001 c o m p ared to control values. D efor­
m ation is expressed as percentage o f the total n u m b e r o f larvae; m eans ± S.E. (// = 6) are given (n.d., non
detectable: less than 1 x 1 0 6//m ol I ').
Table 3
H eart rate (beats min ') and the frequency o f tail m ovem ents (beats min ') o f c arp em bryos 50 h after
fertilization exposed to different am b ien t Pb levels at pH 7.5 and 5.6
[Pb]
H eart rate
//m ol r 1
pH 7.5
pH 5.6
pH 7.5
pH 5.6
n.d.
0.12
0.24
0.48
0.96
130
129
129
136
142
131
147
166
171
186
24
27
18
16
14
23
22
18
16
13
±4
± 3
± 2
± 3
± 3b
Tail m ovem ents
± 2
± 2d
± 3d
±4d
±4d
±3
± 3
± 2
± lb
± 3b
± 1
±3
± 3
±3
± ld
Significance is expressed at the level o f bP < 0.02; dP < 0.001 c o m p a re d to control values. D a ta are ex­
pressed as m eans ± S.E. (// = 6) (n.d., non-detectable: less than 1 x 10"6 /m iol I"1).
Mineral content
N o differences in mineral content were observed between controls at pH 7.5 and
pH 5.6. Also no significant differences were observed in whole body K, N a, M g and
A.J.H.X. Stouthart et a i l Aquatic Toxicology 30 (1994) 137-151
141
ig. 1. Representative view o f larvae 114 h reared at pH 7.5 ( l a ) and larvae 144 h exposed to U.96 //mol
1 Pb at pH 7.5 (lb ), showing the difference in swimming activity between both groups.
Ca content o f eggs and larvae between controls and groups exposed to 0.12, 0.24, 0.48
and 0.97 //m ol I ' 1 Pb at pH 7.5 (Fig. 3a, 3b, 3c and 3d). Similar results were found for
K, N a and M g content in eggs and larvae exposed to the same concentrations o f Pb
at pH 5.6 (Fig. 4a, 4b and 4c). A significant decrease was observed for the C a content
in larvae exposed to 0.24, 0.48 and 0.97 //m ol I“ 1 Pb (Fig. 4d).
Lead accumulation
Pb was recovered from the chorion in a concentration-related fashion (Fig. 5a and
5b). At pH 5.6 m ore Pb was associated with the chorion th an at pH 7.5. Only a small
a
Fig. 2. D e fo rm a tio n o f larvae 144 h reared at p H 5.6 a n d 0.96 //m ol 1 1 Pb. (2a) N o rm a l larvae, p H 5.6, 8 x
(2b)-(2d) D eform ed larvae, p H 5.6 and 0.96 //m ol I ' 1 Pb, 8 x .
A.J.H.X. Stouthart et al. / Aquatic Toxicology 30 ( ¡994) 137-151
142
H ours a f t e r
f e r t ili z a t io n
H ours a f t e r
fe rtiliz a tio n
O)
o
E
3
b
Fig. 3. W hole body K (a), N a (b), M g (c) a n d C a (d) c o n c e n tra tio n s (//mol g ” 1 dry weight) o f c arp
eggs/larvae exposed to Pb at p H 7.5. D a ta are expressed as m eans ± SE (/? = 6). O nly values o f control an d
o f highest Pb c o n cen tratio n are shown.
a m o u n t o f Pb entered the eggs and the em bryos kept at either pH . A t pH 7.5 and pH
5.6 respectively 92% and 84% o f the Pb was b o u n d by the chorion. A t pH 5.6 the Pb
A.J.H.X. Stouthart et al. I Aquatic Toxicology 30 (1994) 137-151
Mg]
<Mmol.g")
143
40
O
0
80
120
H ours a f t e r
f e r t ili z a t io n
160
200
350
(Cai
(M m ol.g-)
280
210
140
70
0
d
40
80
120
H o u rs a f t e r
f e r t ili z a t io n
160
200
Fig. 3. ( continued)
concentration found in the em bryo in all groups was twice (16%) the Pb co n cen tratio n
of em bryos from p H 7.5 (8%). A ccu m u latio n o f Pb in the em bryos was n o t influenced
by rising Pb concentrations. Im m ediately after hatching, larvae started to accum ulate
A.J.H.X. Stouthart et a!. I Aquatic Toxicology 30 (1994) 137-151
144
3
H ours a f t e r
f e r t ili z a t io n
b
H ours a f t e r
f e r t ili z a t io n
Fig. 4. W hole body K (4a), N a (4b), M g (4c) and C a (4d) co n ce n tra tio n s (/ymol g_l dry weight) o f carp
eggs/larvae exposed to Pb at pH 5.6. In Fig. 4d, significance is expressed at the level o f (a) 0.05, (b) 0.02,
(c) P < 0.01 and (d) P < 0.001, co m p ared to control values. D a ta are expressed as m eans ± SE (/; = 6). F o r
4a, 4b and 4c, only values o f controls and highest Pb co n ce n tra tio n are shown, and no significant differen­
ces were observed.
A.J.H.X. Stouthart et al. / Aquatic Toxicology 30 (1994) 137-151
145
o>
o
E
0»
0
40
C
80
120
Hours a f t e r
f e r t ili z a t io n
80
120
160
200
160
200
c*
o
E
3.
ro
O
0
d
40
H ours
a fte r
f e r t ili z a t io n
Fig. 4. (continued)
Pb. T he Pb co n ten t o f the larvae exposed to 0.97 //m ol 1 1 Pb at pH 7.5 was similar to
that o f the larvae exposed to 0.12 //m ol I"1 Pb at p H 5.6.
A.J.H.X. Stouthart et al. I Aquatic Toxicology 30 (1994) 137-151
146
1.50
hatching
1.20
o
control pH 7.5
0 .1 2 Aimol.r’ Pb
0 .2 4 ¿im ol.r' Pb
0 .4 8 /xm ol.r' Pb
0 .9 6 Mmol.l" Pb
V
-
pH
pH
pH
pH
7.5
7.5
7.5
7.5
0.90 -
E
A
a
0.60 -
0.30 -
0.00
0
a
80
120
H ours a f t e r
f e r t ili z a t io n
40
160
200
1.75
hatching
1.40
I?
o
=
□=
A =
o =
control pH 5.6
0 .1 2 mmol.l'1 Pb pH 5.6
0 .2 4 yumol.l-1 Pb pH 5.6
0 .4 8 A i m o l . r ’ Pb pH 5.6
1.05
E
3.
a
0.70
Cl
0.35
0.00
b
H ours a f t e r
fe rtiliz a tio n
Fig. 5. A ccum ulation o f Pb by carp eggs an d larvae exposed to Pb at indicated co ncentration at pH 7.5 (5a)
and p H 5.6 (5b). All values are significantly different c o m p a re d to control values. H atching, took- place
between 52 and 72 h. D a ta are expressed as m eans ± SE (n = 6).
A.J.H.X. Stouthart et al.l Aquatic Toxicology 30 (1994) 137-151
147
100
80
0 s
Q
>
C
JZ
u
60
i
<d
>
4 ->
ro
D
E
D
o
54
a
57
60
H ours a f t e r
63
f e r t ili z a t io n
ox
O
)
C
£
.
U
4 -'
ro
I
d)
>
4 ->
ro
D
E
D
u
48
b
51
54
57
60
H ours a f t e r
63
66
72
75
fe rtiliz a tio n
Fig. 6. C um ulative hatching success o f carp eggs exposed to Pb at p H 7.5 (6a) a n d p H 5.6 (6b). In Fig. 6b,
significance is expressed at the level o f (a) P < 0.05 and (c) P < 0.01, co m p are d to control values. D a ta are
expressed as m eans ± SE (// = 6). Only values o f controls and o f highest Pb con cen tratio n are shown.
148
A.J.H.X. S tout hart et al. I Aquatic Toxicology 30 ( 1994) 137-151
Hutching success
N o differences in hatching success were observed between controls at pH 7.5 and
pH 5.6. At pH 7.5 hatching success o f the em bryos was not significantly different
between controls and eggs exposed to Pb (Fig. 6a). Only at pH 5.6 hatching success
o f em bryos exposed to 0.97 //m ol I"1 Pb differed significantly from the controls (Fig.
6b).
4. Discussion
O u r results dem onstrate that the toxicity o f Pb for carp eggs is greatly enhanced at
low w ater pH . This phenom enon is likely due to increased bioavailability o f Pb to the
developing em bryo. At w ater pH 6-10, Pb-hydroxides and P b-carbonates are the
prevailing forms in the w ater (M cC om ish and Ong, 1988). However, at w ater pH < 6
Pb predom inates as P b “ , a form which is bioavailable (R ickard and N riagu, 1978).
F o r example T urn er et al. (1981) observed that when Pb-salts were added to w ater of
pH 9, the dissolved P b :+ concentration was only 1%, but it increased to 86% at water
o f pH 6. This increased occurrence o f P b 2+ at low w ater pH m ay explain the observed
high percentage o f e m b ry o ’s exhibiting a serious deform ation o f the spinal cord. O ur
results are in agreement with those o f H o d so n et al. (1979), who observed necrotic
tails (early sym ptom s indicative o f spinal deformities) in rainbow tro u t exposed to
0.11 /ymol I“1 Pb at pH 8.2. N o deform ed e m b ry o ’s were observed in eggs exposed to
the same Pb concentration at pH 7.5.
The question arises how P b 2+ exerts its toxic action on the developing em bryo. It is
generally accepted th a t the chorion acts as an effective barrier to protect the em bryo
from heavy metals. This was also confirmed by o u r experiments: Pb was mainly
located in the chorion (92% at pH 7.5 and 84% at pH 5.6), probably b ou n d by the
m ucopolysaccharides attached to the chorion. Similarly, H olcom be et al. (1976)
found that only the chorion o f brook tro u t eggs takes up Pb. In this connection it
should be m entioned that in the present experim ents exposure o f the eggs started
immediately after fertilization before hardening o f the egg m em brane. D uring this
process Pb may enter the chorion (8% and 16% was found in the em bryo at resp. pH
7.5 and pH 5.6), possibly changing its selective perm eability characteristics, leading to
a disturbed ion exchange capacity between the perivitelline fluid and the am bient
water. The perivitelline fluid contains a negatively charged colloid (Eddy and Talbot,
1985), which attracts cations from the am bient water and m aintains a perivitelline
potential (pvp) difference between the chorion and the w ater (Peterson and M artinR obichaud, 1986). The exchange o f cations like C a :+ and M g :+ is crucial for norm al
em bryonic developm ent (Van der Velden et al., 1991). Peterson and M artin -R o b ichaud (1986) showed that when the pvp is depolarized by low am bient pH , the ability
o f the perivitelline fluid to concentrate a divalent cation like C a 2+ is reduced as p re­
dicted by changes in the m agnitude o f the pvp. A heavy metal like P b 2+ could strongly
bind to acidic protein groups in the chorion and the em bryo. As a consequence
the passive diffusion increases com pared to the active process, because o f reduced
pvp. C o ncen tratio ns o f Pb in the chorion increase the probability o f entering the
^
i
A.J.H.X. Síouíliarí eí al. I Aquatic Toxicology 30 (1994) 137-151
149
rivitelline fluid by either passive diffusion or as a result o f exchange with other cations
the perivitelline fluid. In general, altered characteristics o f the chorion and pvp are
e main causes o f the adverse effects observed during em bryonic development. Fish
as initially contain all the N a, K, M g and Ca required for em bryonic developm ent
,d are highly resistant to ion loss unless the vitelline m em brane is irreversibly injured
lommsen and Walsh, 1988). In the beginning the em bryo mobilizes all ions from
e yolk sac. P b 2+ can occupy the binding sites o f C a 2+, interfering with C a 2+-m etabom necessary for developm ent o f the skeleton. R educed m obilisation o f C a 2+ from
he yolk sac could also limit the C a 2+ uptake by the em bryo.
After hatching, the larvae actively accum ulate ions required for growth and devel•ment. The likely sites o f active ion uptake in fish larvae are the chloride cells found
yolk sac epithelium and skin as well as in the developing gills (Shen and Leatherid, 1978a; H w ang and H irano, 1985; Oyen, 1993). In particular the gills are sensie to heavy metals because o f their intim ate contact with the water. Branchial upke o f w aterborne C a 2+ is m ediated by chloride cells (Perry et al., 1992) in a tra n sp o rt
ocess that is passive across the apical m em brane and active across the basolateral
em brane (Perry and Flik, 1988; Flik et al., 1993). H ydrogen ions are know n to act
ion the gill m em brane to displace C a 2+ from tight ju n ctio ns (M acD o n ald , 1983;
arshall, 1985) and block io n -tran sp o rt channels (M a c D o n a ld et al., 1983), thereby
■creasing net ion uptake. Similarly, Pb has been shown to interfere with C a 2+-trans>rt m echanism s (S hephard and Simkiss, 1978; Bansal et al., 1985). Reduced net
-»take o f C a 2+ was also found in o u r experim ent at pH 5.6. This was in accordance
ith results found by R eader et al. (1989) and Sayer et al. (1991) on early life stages
brow n trout. Therefore a com bination o f increased H + and P b 2+ could block the
a2+ uptake. M ortality o f the larvae at pH 5.6 in the presence o f Pb was concentraon-dependent. Significantly decreased hatching success was observed in larvae exosed to 0.97 //m ol I"1 at pH 5.6. Dave and Xiu (1991) found th a t at concentrations
f Pb < 2.3 //m ol P 1 hatching o f zebrafish ( Brachydanio revio) was slightly delayed,
I though no clear concentration-effect relationship was found. Delayed hatching by
b can also be attributed to an inhibition o f egg shell digesting enzyme, chorionase.
his enzyme has its o ptim u m at pH 8.5 (H agenm aier, 1974), and its activity is reduced
t a pH o f 5.2 to 10% o f the optim al rate. Peterson and M a rtin -R o b ic h a u d (1983) and
)yen (1993) have suggested that decreased tru n k m ovem ents o f em bryos reared at
ow pH contribute to delayed hatching through less efficient chorionase distribution,
causing delayed rupture o f the chorion.
An im p o rta n t question rem aining is w hether o u r results on the effect o f low w ater
'»H on the toxic action o f Pb on the early developm ent o f carp eggs have implications
or the kno effect levels1 o f Pb. In T he N etherlands the m axim um allowable concentraion o f Pb in surface waters is 0.05 //m ol I” 1 Pb (Janus et al., 1991). However, this
concentration has been based on waters with pH > 7. In recent years, in m any waters,
particularly those with low buffer capacity, the pH has gradually decreased to values
below pH 6.0. In these waters Pb concentrations up to 2.4 //m ol I"1 have been found
(Janus et al., 1991). T he lowest observed effect concentration (L O E C ) o f Pb in our
experiment was 0.12 //m o l.I“ 1 at pH 5.6, which is approxim ately twice the m axim al
allowable concentration (0.05 //m o l.I"1 Pb) for surface waters in The N etherlands.
150
A.J.H.X. Stouthart et al.l Aquatic Toxicology 30 (¡994) ¡37-151
O u r studies indicate that a larger safety-margin for Pb in water should seriously be
considered.
I
Acknowledgements
The technical assistance and helpful suggestions o f Mr. Jelle Eygensteyn and
Mr. Tony Coenen is gratefully acknowledged.
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