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Full Text - Radboud Repository
PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/16732 Please be advised that this information was generated on 2016-09-29 and may be subject to change. 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. References Bansal, S.K., R.C. M u rth y and S.V. C h a n d ra (1985) T he effects o f some divalent metals on cardiac and branchial C a :+-ATPase in a fresh w ater fish Saccobranchusfossilis. Ecotoxicol. Environ. Saf. 9, 373-377. Brown, D.W. (1979) A dsorption o f lead from solution on the q u a rtz and feldspar-containing silt fraction o f a natural stream bed sediment. In: Chemical Modelling in A queous System: Speciation, Sorption, Solubility and Kinetics. Am. Chem oc. Symp. Ser. 93, 237-260. Dave, G. and R. Xiu (1991) Toxicity o f mercury, copper, nickel, lead, and cobalt to em bryos and larvae of zebrafish, Bracliydanio rerio. Arch. Environ. C o n ta m . Toxicol. 21, 126-134. Eddy, F.B. an d C. Talbot (1985) Sodium balance in eggs and dech orion ated em bryos o f the Atlantic salmon (Salmo salar L.) exposed to zinc, alum inium and acid waters. C om p. Biochem. Physiol. 81C, 259-266. Flik. G., J.A. van der Velden, K.J. Dechering, P.M. Verbost, T.J.M . Schoenm akers, Z.I. K lar and S.E. W endelaar Bonga (1993) C a :+ and M g 2+ tran sp o rt in gills and gut o f tilapia Oreochromis mossambicus : a review. J. Exp. Zool. 265, 356-365. Hagenm ayer, H.E. (1974) T he hatching process o f fish em bryos - IV. the enzymological properties o f a highly purified enzyme (chorionase) from the hatching fluid o f the rainbow trout, Salmo gairdneri Rich. C om p. Biochem. Physiol. 49B, 313-324. H odson, P.V., B.R. Blunt and D.J. Spry (1979) C hronic toxicity o f w ater-borne lead to rainbow trout, Salmo gairdneri. in lake O ntario water. W ater Res. 12, 869-878. Holcom be, G.W.. D.A. Benoit, E.N. Leonard and J.M . M cK im (1976) Long-term effects o f lead exposure on three generations of brook trout (Salvelinus fontinalis). J. Fish. Res. B oard Can. 33, 1731-1741. H w ang, P.P. and P. H iran o (1985) Effects o f environm ental salinity on intercellular organization and junctional structure o f chloride cells in early stages o f teleost development. J. Exp. Biol. 236, 115-126. Janus, J.A., J.A. A nnem a, J .M .M . Aben and W. Slooff (1991) Evaluation do cum ent lead. R IV M . Bilthoven. T he N etherlands, 119 pp. Macek, K.J. and B.H. Sleight (1977) Utility o f toxicity tests with em bryos a n d fry o f fish in evaluating hazards associated with the chronic toxicity o f chemicals to fishes. In: A quatic Toxicology and H azard Evaluation, edited by F.L. M ayer and J.L. Hamelink. A ST M STP 634, Am erican Society for Testing and Materials, Philadelphia, PA, pp. 137-146. M cK im . J.M . (1985) Early life stage toxicity tests. In: F u n d a m e n tals o f A quatic Toxicology, edited by G .M . R and and S.R. Petrocelli. Hemisphere. W ashington, D C, pp. 58-95. Marshall, W.S. (1985) Paracellular ion tran sp o rt in trout opercular epithelium models osm oregulatory effects o f acid precipitation. Can. J. Zool. 63, 1816-1822. M cCom ish, M.F. and J.H . O ng (1988) Trace metals. In: Environm ental Inorganic Chemistry, edited by I. Bodek, W.J. Lyman, W.F. Reehl and D .H . Rosenblatt. Pergam on, New York. M cD o n ald , D .G . (1983) T he interaction o f environm ental calcium and low pH on the physiology o f the rainbow trout, Salmo gairdneri. I. Branchial and renal net ion and H + fluxes. J. Exp. Biol. 102, 123-140. M cD o n ald , D .G ., R.L. Walker and P.R.H. Wilkes (1983) T he interaction o f environm ental calcium and low water pH on the physiology o f the rainbow trout, Salmo gairdneri. II. Branchial ionoregulatory mechanisms. J. Exp. Biol. 102, 141-155. A.J.H.X. Stouthart et al. I Aquatic Toxicology 30 (1994) 137-151 151 >han, C.V., N .R . M cnon an d T.R .C . G u p ta (1983) Effects o f cadm ium on the early developm ental stages of Cirrhina mrigala (H am ). M ysore J. Agric. Sci. 17, 378-381. xnmsen, T.R and RJ. Walsh (1988) Vitellogenesis and oocyte assembly. In: Fish Physiology XI-A, •dited by W.S. H o a r and D.J. Randall. Academic Press, L ondon, pp. 348-395. mgu, J.O. (1979. G lobal inventory o f natural and an thropogenic emissions o f trace metals to the a tm o s phere. N a tu re 279, 409-411. en, F.G.F., L .E .C .M .N . C a m p s and S.E. W endelaar Bonga (1991) Effects o f acid stress on the e m b ry o nic developm ent o f the c o m m o n carp ( Cyprinus carpio). A quat. Toxicol. 19, 1-12. en, F.G.F. (1993. A lum inium and w ater acidity. Effects on early developm ent and juveniles o f the carp, Cyprinus carpio. Ph.D. thesis, Univ. Nijmegen, T he N etherlands. rry, S.F. and G. Flik (1988) C haracterization o f branchial transepithelial calcium fluxes in fresh water trout, Salmo Gairdneri. Am. J. Physiol. 254, 491-498. rry, S.F., G .G . G oss and J.C. Fenwick (1992) Interrelationships between gill chloride cell m orphology .ind calcium up take in fresh water teleosts. Fish Physiol. Biochem. 10(4), 327-337. lerson, R .H . and D.J. M a rtin -R o b ic h a u d (1983) E m bryo m ovem ents o f Atlantic salm on (Salmo salar) as influenced by pH , tem perature, and state o f developm ent. Can. J. Fish. A quat. Sci. 40, 777-782. terson, R .H . and D.J. M a rtin -R o b ic h a u d (1986) Perivitelline and vitelline potentials in teleost eggs as influenced by am bient ionic strength, natal salinity, an d electrode electrolyte; and the influence o f these potentials on cadm ium dynam ics within the egg. Can. J. Fish. A quat. Sci. 43, 1445-1450. oader, J.P., N .C . Everall, M .D .J. Sayer and R. M orris (1989) T he effects o f eight trace metals in acid soft water on survival, mineral u ptak e and skeletal calcium deposition in yolk-sac fry o f brow n trout, Salmo trutta L. J. Fish Biol. 35, 187-198. ickard, D.T. a n d J.O. N riagu (1978) A q ueo us environm ental chemistry o f lead. In: T he Biogeochemistry of Lead in the E nvironm ent 1A, edited by J.O. Nriagu. Elsevier/N orth-H olland Biomedical Press, A m sterdam, pp. 219-284. ilomons, W. and U. F o rstn e r (1984) M etals in the Hydrocycle. Springer, Berlin, 349 pp. lyer, M .D .J., J.P. R eader and R. M orris (1991) E m bryonic and larval developm ent o f brow n trout, Salmo trutta L.: Exposure to alum inium , copper, lead o r zinc in soft, acid water. J. Fish Biol. 38, 431-455. hen, A.C.Y. and J.F. Leatherland (1978a) Effects o f am bient salinity on ionic a n d osmotic regulation o f eggs, larvae, and alevins o f rainbow tro u t (Salmo gairdneri). C an. J. Zool. 56, 571-577. hephard, K. and K. Simkiss (1978) T he effects o f heavy metal ions on C a 2+-ATPase extracted from fish gills. C om p. Biochem. Physiol. 61B, 69-72. urner, D .R ., M. Whitefield an d A .G . Dickson (1981) The equilibrium speciation o f dissolved com ponents in freshwater and seawater at 25°C and 1 atm. pressure. G eochem . C osm ochim . Acta 45, 855-881. an der Velden, J.A., F.A.T. Spanings, G. Flik an d S.E. W endelaar Bonga (1991) Early life stages o f carp (Cyprinus carpio L.) depend on am bient m agnesium for their development. J. Exp. Biol. 158, 431-438.