E:11
This paper not to be eited without prior referenee to the authors
1~5.
146.
I.
INTRODUCTION
Up to now, the most widely used groups of marine invertebrates for bio-
Comparison of demographie (life- and feeundity table analysis) and
biochemical (ATP and AEC) eharaeteristies as sublethai pollution indices in the marine nematode MOYlhiI~te!la fujuYlIUa.
assay tests are bivalves, erustaceans and polyehaetes.
Undoubtely, the eeo-
nomic importance of 'erustaceans and bivalves was the main reason for their "election.
.
Despite their eeological importance (Heip et Gi.,1979 ; Warwiek & Price,I979 ;
K.
Verschrae~en,
G. Vranken, D. Van Gansbeke, C. Heil' and A. Boffe
Marine Iliolo~y Seetion, Zllology Institute, State Vniv('rsity
Belgium
0'
1I"i" rt
ae.,
1985), marine free-living nematodes have only recently been t1SI'<1
.," l'c's t organ i sms in
11 fl'W
s ttl<1 i eR (ßogllert et a
e.,
19111,
lIowell, I'JR4 ;
et Gi., 1984a, 1985, 1986). The aim of the present
Tietjen, 1984 ; Vranken
study was to research the sublethai effects of cadmium and nickel on the frecliving marine nematode
MOYlhy~te'r.a.
fujuYlc.ta. in laboratory conditions.
MOYlh1j6te!llt d.i.J.,juYlcta is previously used as test orr,llnism in sever"l bio-
assays, by Vranken and co-workers.
Sublethai effects of cadmium and nickel on demographie charaeteristics
and adenylate metabolism of
MOYlhy~teJta
fujuYllUa., a marine free-living
nematode. were studied during chronic exposure in eultllre conditions.
Mortality is a rather insensitive criterium to predict the environmental
impact of pollutants.
MEC (minimal effect eoncentration) values based
on development rate and daily egg produetion were the most sensitive
eriteria : values were up to two orders of magnitude less than the eorresponding LC50 (96h).
EC50's, effeetive coneentrations resulting in
a 50% inhibitory effeet.on either the intrinsic rate of natural inerease
(rm) or on net-fecundity (Ro ) were, on the one hand, less sensitiv!'
than MEC's based on development and egg production, but on the other hand
Vranken
et ai.,(1984) found that the juve-
nile stage was the most sensitive life-stage, when expuscd to thrcc diffcr.,lIt
mercury compounds.
Short-term aeute tests with cadmium as toxicant showed
that LC50 values are very time-dependent and that MEC (minimal effect concentrations) values, based either on mortality or on a developmental assay in
which success in attaining the adult stage was tested, are probably ecologically more meaningful than LC50 values (Vranken et Gi., 1985).
Finally, a
comparison between mortality, developmental rate and fecundity as toxieityindiees was made (Vranken et ai.,1986).
Based on a large data-base (seven
heavy metals, pentachlorophenol and ohexaehlorocyclohexane were tested),
~C
fecundity turned out to be the most sensitive criterion, though,
values
remained substantially high.
more than one order of magnitude less than LC50 values.
::i}~lliri,·.llIf 1!I'I'rl':lul't1
nr
ATP
l'lIllll'rH Wl'n' ohtH1rvl'.) ;11
consiJcrahJy IC:iS lhoill1 lhc l.C;'O'S.
l'IIIIJ'I'J)l'r;)'
illl)~j
lIowcvcr, cUlIIl'an'd \~i t h litt' " .. 'l1Ip,.."OI-
phic charaeteristics studied. this criterion is less sensitive.
It is
argued that neither ATP concentrations, nor AEC measurements ean give
ecological relevant information about detrimental effects caused by
long-term exposure to
sublethai concentrations of the metals tt'sted.
In this study , we tested for sub-Iethal changes in demographie and
biochemical characteristies.
The demographie eharaeteristies studicd are
mortality as a function of age, generation time and fecundity.
From these
I igurcs wc ealculatcu the intrinsie rate of natural i.ll('rc.lsc (rm)
net-reproductivity (R o )'
öll1d
L1,c
Several authors proposed to uetermine EC50 (rm)
values, the concentration which has a 50% inhibitory effeet on population
growth (Hummon
&
Hummon, 1975 ; Sabatini
&
marcotte, 1983).
They bel icvc
that such EC50 values are more reliable criteria than LC50 values,
t0
deter-
mine so-ealled treshold-eoncentrations with regard to safe-guarding communides of organisms in the environment.
The exact determination of ,these
parameters is tedious since at eaeh level of intoxication, complete life
and fecundity tables (lx and mx ) have to be eonstructed. In our study we
tested how the ECSO (rm) relates to the LC50 (96h) and the so-called MEC
value (Vranken
et at.,1985). The latter variables are mueh easier to de-
141.
148.
3.
2.
determine, but it is generally feared that lethai eoneentrations in shortterm experiments) may seriously underestimate the influence of pollutants
on a population.
Aoenylate energy charge as a measure of pollution stress
i~ demographie eharaeteristics as net-reproductivity Ro and intrinsic rate of natural increase r m• Tbis would considerably reduce time necessary to evaluate sublethai effects.
changes
~L\TERIAL
AND
~lliTHODS
In the late 1960s, Atkinson & Wal ton (1967) proposed the adenylate
energy charge (AEC) as a means of expressing the metabolic energy status of
an
or~anism.
Tcstspecies - culturetechniques
It is given by :
AEC • (AT1'
+
and varies between 0 and I.
Yz.
.lfollhy5.te'Ul. di5junc..ta was sampled in the Suice dock of Ostend, a marine
An1' ) I (AT1'
+
An1'
+
A111')
1:lgoon near the Belgian coast.
Tbe observation that its value decreases in
response to stress, irrespective of the type of stress, led Ivanovici &
Wiebe (1981) to propose AEC as a general biochemical index of sublethai
M.~juac.ta
tode with a cosmopolitan distribution.
.is a marine bacterivorous nema-
Adults have a mean length of ± 0.85 mm.
The procedures for isolation a:d cultivC1tion are described at full lenght by
Vranken et aL (l984a,b) and Vranken ct al.(l985).
stress: in optimal conditions, AEC ranges between 0.8 - 0.9, values between
0.5 - 0.7 point'to suboptimal but still viable eonditions whereas at values
below 0.5 viability is lost (Ivanoviei, 1980).
food.
The experiments were
tures.
In several bio-assay studies, AEC determinations were used to assess
Stockcultures were main-
tained on 0.57. baeto-agar (Difco) plates with a mixed bacterial cul ture as
eond~cted
A monospecific bacterial
in
isolat~
co~~letely
controlled monoxenie cul-
(belonging to the
aUe.tc'I':OHC.5
IUtlo-
"sublethai" effeets of man-made pollutants on marine organisms (Bakke & Skjol-
1_,e,111I:ti5 rR.'lA branch) was added as food in a ring-formed excavation in agarplates (see Vranken et al., 1985). In order to avoid pH-fluctuations, Tris
dal, 1979 ; Zaroogian et al., 1982 ; Neuhoff, 1983 ; Haya et al., 1983).
buffer (5r.J'1) was added to the culture mt!dium (Vranken et aL, 1986).
A major drawback, however, is the fact that no information is available in
multicellular organisms on how a decrease of AEC is related to ecologieal
relevant parameters as growth, reproduction, etc ••• (Livingstone, 1985).
Without such information, the predictive power of AEC as an early warning
indicator of unfavarouble environmental conditions is limited.
If a decrease
of AEC is correlated with for example low growth rates and/or impaired reproduction. than AEC would represent one of the most sensitive bioche",ical
Toxicity tests
Nickel and cadmium were added as NiCIZ.6H 20 (Merck) 'and CdClZ.ZYzHZO
(Baker Chemieals BV Holland), both of analytical grade. Tbe final concentrations tested ranged between 0.5 anJ 10 mg/l Cd and 1 and 35 mg/l Ni.
for each concentration, 3 replicates were studied.
All cultures were kept
in the dark at a constant temperature (17"C) and salinity (30%).
indices of stress available at present.
Another drawback of AEC measurements eoneerns the reliability of thc "'ethodology used.
Verschraegen et al.(1985) described a reliable assay for AT1',
AD1' and A}W in two polychaetes (Ne~ei6 diven4~coto~ and Nephty~ sp.).
this study we tried to adapt this method.
In
The main problem was the very
small biomass of thc nemtodes (t 0.5 ~1l\ adult fresh.'eight for !.fv1"1:Y5tVra
d~juncta
against a mean of about 0.3 g for the two polychaetes.
The demographie characteristics studied are mortality as a function
of age, generation time and fecundity.
fcm~les
For each concentration, 10 gravid
were allowcd to deposit cggs during 24h (48h for 35 mg/l Ni).
mortality, juvenile and total preadult mortality.
AT1'-eoncentrations as indicator for pollution stress
De-
velopment was daily followed to calculate minimal generation time Tmin, egg
Tbe minimum generation
time Tmin is estimated as the period between identieal stages of successive generations - this is almost equal to the development time (Vranken &
Duc to these problems, we mainly concentrated on the determination of
the AT1'-eoncentrations in the nematodes.
ATP-concentrations were determined
in different life-stages of the nematodes under Ni as weIl as under Cd intoxieation.
in
The objective was to establish a possible eorrelation between changes
ATP-concentrations in an early stage of chronic exposure experiments and
Heip, 1983).
Criteria for death were inactivity and the lack of movement
even after prodding with the tip of a needle.
1,11en fem.l!es be<"".~ ;d'llt (~ftcr aperiod Tmin) , eggproduction of 5
adult 'f'f together with 3 d"d'; was counted every 2 days for each test con-
I·B.
150.
4.
centration.
Every 4 days the adults were ·transferred to a new culture.
To test for adult survival, observations were done every Z or 3 days on
40
6J and 40
S~
per concentration.
5.
bioluminescence reagent. For a detailed description of the reagents and
equipment used, see Versehraegen et al., 1985.
Every 6 days, surviving adults were
transferred to fresh cultures to distinguish between parents and offsnring.
Dead organisms were eleminated from the cultures.
PrelitJinary experiments rc\"ealed that the results impro l7 ed when the
nematodes in the extraets wer~ fraetionated by sonication. It appeared
that by doing so, the variar.(c b~tween the results deereased considerably.
The ~?;:~?~~~.:~;~.~!.?~;~:~~.~~~:~~~~.:~
Lotka equation.
max age
is calculated with the Euler -
Lx=O
x
Ix
=
pivotal age, age of the females in the age-interval (X,X+I)
age-specific survival rate, probability to survive from the egg-
= age-specific
tll gene ra te waves of ZO k!:: (po\,'er IOQ\n during 60 sec.
in ATP measurements.
Prolongation of the extraetion time from 1 min to 10
fecundity, number of female offspring produced per
r."01I'"rillllll of 7 cJiffpTpnt ('xtr.1"tillf'. "rpntll
...........................................
For ea~h teehnique, 4 or 5 replieate extractions were made in Eppendorf
Test Tubes, using adult nematodes from stoekeultures.
neration with age x and p is the proportion of females in the adult popula-
iee-cooled. except otherwise mentioned.
tion.
determination of the adenylate coneentrations.
The ~~;::~~:?~~~;~~~;~.~9' the multiplication rate per generation, is ealeulated as
max age
max age
Ux =
x=O
x=O
TCA: 10 nematodes were transferred into 50 ~l TCA extraetion medium (O.5M trichloroacetie acid C13CCOOH and 0.Z5 NaZHPO~).
After neutralization with 50 pI NaOH (0.5N) and dilution to I ml
with Tris-HCl buffer (Tris 0.OZ!1 ; pH 7.75), the extraets were stored at -ZO·C. Immediate before the ATP determination, extraets
were sonicated during 60 sec.
EXTR 2
H2S04: Extraetion as in EXTR I, but with H2S04 (0.5N) instead of
TCA extraction medium.
EXTR 3
Formic acid: 10 ;ndividuals were transferred in 50 pI 10% formic acid (HCOOH). Extraets were lyophilised (Chriss, Delta Ils)
to remove acids, and ciluted to 1 ml with Tris-HCl.
PCA: Extraets eonsisted of 10 nematödes in 50 ~l 6% PCA (perehlorie acid). Upon neutralisation with Z5 ,,1 KZC0 3 (5N), the extracts
were eentrifuged. The supernatans was dlluted to I rol wi th TrlsHCl
net-feeunJity, the realized number of female offspring per female
(X,X+I)
The mean generation time T is estimated as T
......................
EXTR 4
The tests were executed simultaneously with the demographie assay.
The organisms were harvested from two of the three replicates of each test-
EXTR 5
Boiling Tris : 10 nematodes were transferred into 10 pI artifieial seawater (Dietrich & Kalle, 1957). 490,,1 boiling Tris-HCl
was added ; after 30 sec extraets were eooled-down, sonieated
and stored at -ZO·C.
EXTR (,
n" i 1 i Ilf~
EXTR 7
NRB/NRS (Lumac) : The extraeting medium was a mixture of 25 ~l
NRB and 25 pI NRS (NRB is an extraetant for baeteria, NRS for somatic cells ; eomposit;n~ is not mentioned by the manufaeturer).
Proeedures to determine ATP, ADP and AMP were based
on the method used for N~~ div~leo!04 ~nd Nephty~ sp. in previous studies ( Verschraegen et al., 1985).
bioluminescence reaction.
This method is based on the firefly
The (ATP dependent) light emission of the
luciferin-luciferase substrate-enZVMe eomplex was measured with the integration method.
A Constant Light Signal (CLS) reagent was used as
The media used were
Extracts were stored at -ZO'C until
EXTR J
2:
cf the preceeding generation, with the latter in the age-interval
eoncentration studied.
Furthe rmore ,
pH 7.75) as
mueh as possible to reduee possible interfering factors (Karl & LaRoek, 1975)
The age-specific fecundity mx is estimated from the egg-counts as mx = Nex'p
<:l,ere Ne x is the number of eggs produced by a female of the parental ge-
Ux
0.02~,
min did not significantly affeet the results.
female alive in the age interval (X,X+I)'
2:
To avoid warminl!
up of the extraets, the test tubes were placed in an iee-bath.
extraets had to be diluted (with Tris-HCl buffer : Tris
stage onwards until age x
mx
A sonicator (Brown, Labsonic 1510) with a needle probe (, ~ ': 4mm) was used
I' th.1n" J : same' proeC'cJlITp .111 in F:XTR 5 hllt wi th hili J i nr. 1'thanol 'instead of Tris-HCl, and with 10-fold dilution just prior
to ATP-measurement.
•
151.
6.
950 ~l Hepes buffer ( 4(2hydroethyl) Ipiperazin - ethansulfon
acid) was added.
152.
7.
ATP-content is expressed on freshweight-base.
For each concentra-
tion, maximal length and width of 25 fixed individuals (4% formalin ;
for each extract, light emission is
Extracts were thawed on ice
measured of :
~l
extract + 100
~l
Tris + 200 pI CLS
I)
100
2)
as in I) but 100 pI internal standard (10- 8M ATP) is added in stead
3)
Blank: 200
80·C) was measured.
(Andrassy, 1956) :
a
Freshweight is calculated with Andrassy's formula
2
ab
16öäööO
= freshweight (ug)
maximal length (um)
b = maximal width (um)
of Tris
~l
Tris + 200 pI CLS
RESULTS
Extracts for the actual assay were made in TCA extraction medium (EXTR I)
but with 30 nematodes per extract.
plicate extractions were made.
For each testconcentration, 4 or 5 re-
ATP was measured in juveniles of about 3d
old and in nematodes of about 8.5d old - this is when females became adult
Minim~~
generation time
.......................
Tables 1 and 2 show that minimum generation times increased with in-
in the blank.
('Il',";illl; "(JIII'I'lltration9 of Co uno Ni.
c. Ee~eEmin~tio~~f_ AT~..!. 3EP_..!.
_A!!!_
and 15 mg/l Ni.
Extractions were performed according to EXTR 1 (TCA) but with 100 nematodes per extract.
Preliminary experiments revealed that an extra amount
Tldll wall vl'ry ,,1 ..,"' ;Jt 2.'.
'"lo./l
Cli
AGames & Howe11 test (Sokai & Rohlf, 1981) revealed a
MEC value (minimal effect concentration) of I mg/l (P=0.05) for both Cd
and Ni.
of ATP, ADP and A}W had to be added to enhance the transformation of ADP
In the whole experiment, males developed a little faster than females.
end A}W into ATP.
For Cd, the sex ratio (measured as the percentage females in the adult po-
The procedure is summarized in fig I.
The incubation mixture con-
sisted of 50 pI extract and 25 pI Tris (containing, where necessary, the
ipternal standard), added to 100 ul TrisA (Tris buffer + Mg++ and K+),
Iris b (Tris A + Phosphenolpyruvatc, pyruvate kinase and co-factors Mg++
and K+, for transformation of ADP into ATP) or Tris C (Tris B + myokinase
ta transform ADP and AMP into ATP) for the determination of ATP, ADP or
To each mixture, 25 1'1 ATP, ADP or AMP (each I .10-7~1)
.
.
nucleotide.
d
T0 determlne
.
AMP , an ex t ra
was added, accord1ng
to Wh1Ch
was
determ1ne.
7
amount of 25 pI ATP (5,10- M) was added. For ADP and AMP measurements,
ANP respectively.
pulation) increased with increasing concentration (except for 0.5 mg/l Cd)
while for the Ni-assay, an opposite trend was observed.
~!orL1li ty HS
a function of age
..............................
For both Cd and Ni, mortality during the egg stage increased only
sh1wly with increasing concentrations (table 1
&
2).
The increase was
very steep between 25 and 35 mg/l Ni, the 1atter concentration causing
10m: mortality.
Concerning the juvenile mortality, aG-test (Sokai
&
Rohlf, 1981)
the mixt ure was incubated for 30 min at,30·C, 10 pI TCA and 5 ,,1 pepsin
showed that juvenile mortality was significant1y influenced by the amount
were added, and incubation continued for 60 min at 35·C.
of metal added (P(O,OOI :
After neutrali-
zation with 10 pI NaOH (0.5N), the solution is diluted to 3001'1 with
Tris-HCI buffer.
For each nucleotide, 3 measurements were done
an internal standard, and one blank.
~/q
=1186 for Cd ;
~/q
= 509 for Ni). The
~lliC
values (P=0.05) are 2.5 mg/1 for Cd and 5 mg/1 Ni.
The preadlll t mortality, which campiles both egr,- and juvenile morta-
one wi th. 'and one wi thout
J ily, UbUWl·J alJlJut tbe Ullllle pattern au tbc juvenile lIIul'lality.
The adult surviva1 of the females is represented in fir.. 2.
adult female
lon~evity
The mean
in both the Ni and Cd assay was not sienificantly
different (P=O.05) from the control ( Games & Howell test: Sokal & Rohlf,
1981) •
153.
154.
8.
9.
acidic extraction techniques (EXTR S,6,7) were significantly different
Total eggproduction decreased with increasing Cd and Ni concentrations ; the mean curnulative eggproduction per fernale alive is a linear
function of time (fig. 3).
from EXTR land 2.
these with formic acid (EXTR 4). EXTR 6 and 7 gave the lowest ATP-yield.
All regressions were significant ; the para-
ATP concentration in the bio-assay
.
meters of the regressions are given in table 3.
The slope b represents the mean daily egg production of a female
alive.
It dropped significantly (in comparison to the control) at I mg/l
for Cd and 2.S mg/l Ni (P(O.OOI) ; these values are minimal effect con-
From this group, results with EXTR 5 approximated
.
The ATP-content of 3d old juveniles decreased allometrically with
metal concentration (fig. 6 and 7).
A similar relationship was found b,,-
tween freshweight and Cd (Ni) concentration (fig. 6 and 7).
centrations for this criterion.
Analysis of variance showed that the weight-specific ATP content was
significantly influenced by Cd (F s = 4.717 ; 0.001< P <0.01) and by Ni
(F s = 6.395 ; O.OOI<P<O.OI). COIT.prison limits (P=0.05) were calcula-
From the results above, several demographic parameters were calculated (table 4). Both the net-reproductivity Ro and the intrinsic rate of
natural increaser m were clearly depressed by metal intoxication, mean generation time T was prolonged compared to the control. (table 4). The values calculated for O.S mg/l Cd are exceptional due to the very low sex
(44.2~
ratio
females against 81% for J mg/l Cd) observed.
For both metals, ECSO(Ro ) and ECSO(rm) were calculated. These are
concentrations at which Ro ' respectively r m, are depressed with 50% compared to the control.
For Cd there was no clear correlation between Ro
This is due to the aberrant va-
(rm) and the concentration of the metal.
lue of Ro (rm) at 0.5 mg/l Cd Üig. 4a).
the control and 2.S mg/l Cd gave :
Linear interpolation between
EC50(Ro ) = 0.64 mg/l Cd
An exponential correlation was found between r m (Ro ) and Ni concentration
(table 5, fig. 4b). From the regressions, EC50 values were calculated as
=
at 0.5 and 2.5 mg/l Cd.
For Ni, a (marginal) significant difference
existed between 15 mg/l Ni and J mg/l Ni.
Of more importance is that none
of the measured values differed significantly from the values in the control (P=O.OS).
Für 8.Sd old organisms, Cd and Ni aga in affected the weight-specific
ATP content measured
significantly (F s = 43.988 , O.OOJ<P<O.OI for Cd
and Fs = 64.674 • P< 0.001 for Ni). Comparison limits (Gabriel test,
P=0.05) showed a significant decrease compared to the control at the high-
est concentrations of both metals tested (fig. S)
At these concentrations, significantly higher values (P=O.OS) were
calculated.
ADP and/or AMP were not always measurable.
12.29 mg/l Ni
in table 7.
3.48 mg/l Ni
At the lowest concen-
tration, this difference was not significant except at 1 mg/l and 2.5 mg/l
Cd.
EC50(rm) = 1.37 mg/l Cd
EC50(rm)
EC50(Ro )
ted with the Gabriel-test (Sokai & Rehlf, 1981) and given in fig 4.
Values for 10 mg/l Cd were (marginally) significantly different of values
A few results are given
The AEC was higher in "healthy" organisms than in starved
organisms (After S to 6 days starvation, the nematodes were barely alive).
DISCUSSlON
Extraction procedures test
..........................
Results were expressed on freshweight base and are represented in table 6.
A Bartlett's chi-square test showed that variances were heteroge-
neous.
Therefore, the results were analysed with the Garnes & Howell test.
This revealed that there were no significant differences between the four
acidic methods (P=O.OS).
EXTR 1 gave the highest ATP-yield.
All non-
Several studies, field studies as weIl as laboratory experiments,
show that nematodes cornmonly exhibit relative high resistance to pollutants.
LC50 values recorded after intoxication with inorganic and orga-
nic xenobiotics are regularly among the highest values noted for other
taxa, or even higher.
V~anken
•
et al.(1986) studied the aeute toxieity of seven heavy me-
tals, PCP and "~HCH on Monh!!-&teJta. d.iAjunc:ta.
For Ni and Cd, they found
156.
155.
11.
10.
Cd to stop growth of Pan.a.gkellu-&
(Haight et at., 1982)mentioned 507. mortality in the J2 juvenile
stage of Panag~ettuJ -&~u-&~ae after 48h intoxieation with 15.1 mg/l Cd
In a mixed population of ?anagketeuJ and Rh~b~t.iA
and 105 mg/l Ni.
whereas at 15 mg!l Cd, 507. of
A eomparison with the results reported by Vranken et at.(1986) re-
for the J2 juvenile stage a LC50 (96h) of 103 mg/l Ni and 37 mg/l Cd.
Oth~rs
-&~u-&~e
the J2 juvenile stage died.
veale1
th~t,
althoubh
t~e sa~e
test org3nism and an identical culture
teehnique was used, our MEC values, based on juvenile mortality, development rate and feeundity, are eonsistently less.This ean be explained
thC' LC50(48h) value ranged between 35 and 40 mg/l Cd (Feldmesser & Re-
by I) differences in exposure time used in the experimental design:
bois, 1965).
Vranken and co-workers studied these criteria during apre-set period of
In this study, no signifieant differenee was found neither
in adult survival, nor in mean adult longevity of M. ~juncta, at the
time (96h) whereas in this study physiologieal standards (development
In the juvenile stage, whieh
time and total lifespan) were used as time duration of the experiment,
ia Illore sensitive than the adult stage (Vranken et al., 1984a), r.lortality
and by 2) a higher susceptibility of the smallest juveniles as freshly
inereased signifieantly at 2.5 mg/l Cd and 5 mg/l Ni.
hatched juveniles (2.5 - 3d old) were exeluded from their experiments
different Cd and Ni eoneentrations studied.
In table B, ·a sununary of the effeets o[ Cd and Ni on the demographie
criteria studied is given.
(they started with 4.5d old juveniles).
In the Cd-assay, there was a steep decrease of both the intrinsie
Obviously, the LC50 (96h) values reported
by Vranken et at.(J986) are mueh higher (almost one to two orders of magnitude) than all demographie eiteria studied here.
Also, the minimal
rate of natural increase (rm) and the net-reproduetivity (Ro ) with increasing coneentrations. In the niekel-assay, the decrease of both pa-
effeet eoneentration (MEC) for mortality during the juvenile stage, al-
rameters is less pronounced.
Ro can be eonsidered as the most sensitive
EC50's based on Ro are 1.64 mg/l Cd and 3.48
though less than the LC50, is higher than MEC's based on Tmin' daily egg
life history parameter.
produc tion, ne t reprodue tivi ty(Ro ) and population growth (rm).
~~en eompared with the LC50 values, development rate and daily egg
with 507. when compared to the control.
produetion are the most sensitive eriteria.
2.37 mg!l for Cd and 12.29 mg/l for Ni.
differenee amounts to a faetor 37.
In the Cd-assay, this
In the Ni-assay, treshold levels as
measured by the daily eggproduetion and development rate are 41 and 103
til11"S less when eompared with the LC50.
Sil11ilar results are repcrted in the literature.
Reish & Carr (1978) and
mg!l Ni.
At these concentrations the produetion of female progeny drops
EC50 values based on r m are
Consequently the effective eon-
centrations based on r m and Ro are higher than MEC's based on development
time and fecundity.
Biochemical characteristics in pollution studies
.................................................
Petrieh & Reish (1979) found a signifieant reduetion of feeundity in po-
For a particular biochemical response to be acceptable as an index of
lychaetes, exposed to a variety uf heavy metals at levels almost two
biological effeet, it must fulfill two important eriteria (Livingstone,
orders of magnitude less than the corresponding LC50(96h).
toM ral1a!l~('Ull-&
For the nema-
!l('div.i.vu-&, Samol1off et aL (1980) reported a difference
flr Ihr,·,> "r,I"rli "r
magnitude hl'twC'l'n thl' Co,1 li'Vl' 1 Rllrrrl'lIsinf; f",'.mclitv
and the MEC as measured by juvenile mortality.
Furthermore they showed
that growth inhibition in this species is a more sensitive toxieity index than mortality.
For V.i.plola1mella spec I, Vranken & Heip (in press)
found differenees of two orders of magnitude (Cu & Pb) and 1.5 (H~) between development time and the corresponding LC50. Other observations
are however in variance with these findings.
Vranken et al.(1984a) found
1985) : I. The measurable change in biochemical processes must resuIt
f'"Clm. nr hp 01
7. 11
'"il'''
r(,Rp~nRe
to, a change in the environmentn.l conditions.
I,,' l'"fltlih11' tn IlrmnnRtrntr thnt Ihr rhol1l-:l' il1 hiorh,'mil'"l
proeess(es) will have, either direct or indireet, adetrimental effect
on growth, reproduction or survival of the organism.
Concerning the
use of the adenylate energy charge in multicellullar organisms, only
the first criterium is fulfilled implying that its use is limited."
"ep to now, only a few papers have reported on the ATP eontent of
marine nematodes.
Only Ernst (1970) and Goereke & Ernst (1975) made
no effeet on the development rate of some speeimens of MoMy6te}r.C!. cLü-
measurements to study the relationship
juncta, whereas for most individuals the mereury eoneentration tested was
lethai. Haight et al .(1982) needed eoneentrations of 100 mg!l for e.g.
Expressed in percentage of the total amount of organic carbon. Ernst
(1970) found 2.3% for
ATP
Pana.g'LellLL~ ke~v.i.vLL~,
- biomass.
while for Anopl06toma
151.
158.
12.
13.
v~v~p~um
and Adoncho!aimU6
tha!a4~ophyga6
to 1.3% (Coercke & Ernst, 1975).
in
MOJlhlj~telLa. ~juncta.
values ranged from 0.9%
In this study, the mean ATP content
was 2.4% to 3.6% of the total amount of organic
the aberrations are a consequence of an inadequate biomass determination.
In fact, the determination of the mean body masswas based on its measure-
carbon (calculated with an approximate conversion factor Corg = 112 fresh-
ment in o:lly 25 individu11s, whieh exhibited
Io/eight) •
at ,the time 'of the observations (8.5d).
The above mentioned authors used boiling Tris buffer as extracting fluid.
after 8.5 days \oI?srelatively high (and higher than at higher metal eon-
Our experience is, however, that' the cuticle of Monhy~telLa. ~juncto. re-
eentratio:ls where growth stopped almost completely - results not shololn) so
mained intact after boiling, probably resulting in less homogeneous and
that it became
less stable extracts.
ture, poss,ibly Jea.Hng to
Dur results improved when the nematodes were
fractionated by sonication.
Nevertheless, the ATP-yield remained below
03
very steep exponential grolo/th
The variance of the body weight
difficult to pick the nematodes at random from the culerrC~(;CLS
occur whe:l body mass and ATP
~ere
estimates.
SOlch errors would not
measured in the same organism.
In
the values obtained with the four acid extraction procedures tested.
this study, this was not p(>s3il.>le as Mcnhy~telLa. di.6junc.ta is too small
TCA was selected as extracting fluid as it gave the highest ATP-yield,
(±
0.5 Pb freshweight per
Sum~arizing
although it was not significantly different from the three other acid
~Jult).
\oie can say that : within the range of metal concentra-
tions tested, the ATP turn-over rcmained eonstant in an early stage (ju-
extrac tions.
veniles), and also at the lowest Cd and Ni concentrations in a later
ATP as a measure of pollution stress in bio assay tests
.......................................................
stage of the chronic exposure experiment.
The objective was to establish a possible correlation between chanees in
ATP concentrations in'an early stage of the experiment and changes in deIn juveniles (3d old), no significant alteration'of weight-specific
AlP content was observed at each level of Cd and Ni intoxication compared
Yet, at this stage of the experiment, the impairement of
grolo/th and development could be observed even at the 10lo/est concentrations
tested.
delayed and impaired reproduction al ready occured at metal concentrations
below those affecting the ATP concentration.
A possible explanation is that with increasing metal concentration and
mographic characteristics.
to the control.
2) developmental inhibition,
In a later stage of the experiment (8.5d old organisms), ATP con-
tent decreased significantly at the highest metal concentrations.
At
probably with increasing exposure time, an increasing amount of energy
(potentially available in the adenylate system) is used in processes
related to adaptive responses such as avoidance reactions and active detoxification.
As a consequence,less energy will be available for growth
and reproduction.
For Cd, the level at which growth ceased completely
was observed at 5 mg/l Cd (the organims are moribund, wonrt survive till
lower concentrations, at which significant effects on Ro and rm were measured, the ATP concentration retained the same level as in the control
adulthood and consequently won't reproduce) and for Ni it is above
(fig. 4 & 5), except at ) and 2.5 mg/l Cd.
in the Cd-assay as at the highest concentrations (5 and 15 mg/l Ni)
At these concentrations"a
15 mg/l Ni.
The situation in the Ni-assay seems to be different from
(only rnarginally) significantly higher weight-specific ATP content was
the organisms still grew and reproduced despite the significant decrease
:Jlculated.
of the mean weight-specific f.TP content measured in 8.5d old organims.
We believe, however, that these two values are erroneous and
that the mean weight-specific ATP content should remain constant up to
2.5
m~/l
Cd inclusive, for two reasons.
Firstly, it is very unlikely
that ATP concentrations would increase when organims live in stressful
conditions.
Changes of ATP concentration as
03
consequence of harmful
~e
bclievc howcvcr, that the ATP content in reproducing adults (although
less reproducing) may have remained constant and that the decrease may
have been a reflection of the proportion moribund animals (see the ~n­
creased juvenile mortality) at the time of (at random) sampling.
conditions are more than once reported, but the change is always in the
reverse direction.
Secondly, the mean ATP content per individual, which
is based on the measurement in 120 to 150 nematodes per concentration,
remained constant up to and inclusive 2.5 mg/l Cd.
It is possible that
\~e
cr:eountered ma:lY problems in determining the AEC in Monhlj~teJLct
fujllllC.t.1, using thc same method as previously described for t\olO polychaet~3
(Verscraegen et al., 1985).
The major problem was, aßain, the
159.
160.
14.
low biomass of the nematode and consequently the very low concentrations
of the adenylates.
Even with 100 nematodes per extract (which were mani-
pulated one by one with a needle), transformation appeared to be too low
to pe measurable.
In all different adaptations of the method tried, AlP
was the least reliable faetor.
In
spite of it, we determined AEC's in starved nematodes and in. organisms
Results showed that AEC in Monhy~te~ ~juncta
actually drops with increasing stress.
&
IJaiwood (1983) summarized four possible ways in which the
adenylate energy metabolism can be altered during sublethai intoxieation
with xenobiotlics.
These are:
I)
AEC deereases due to an alteration
in relative proportions of the adenine nueleotides while the level of
total adenylates remains eonstant.
2)
level of total adenylates deereases.
ereasc.
I.)
AEC remains eonstant while the
3)
AEC and total adenylates de-
Total atlenylatcs anti ACe, remain eonslant, hul l'l"ccursors ,Ir
Applied to Dur own data, this means that if a decrease of AEC could have
been measured, this would only have been possible in organisms of 8.5d
old, at the highest metal concentrations tested (at 5 and 10 mg!l Cd and
Indeed, if AEC were de-
creased at coneentrations where the ATP concentration
is eonstant, this
Such a respons type has never bcen
In conelusion, we think that neither ATP coneentration or AEC measurements can give an eeologieal relevant idea about harmful effeets caused
Both
eriteria are less sensitive than· the demographic eharacteristics studied.
These findings strengthen our previous idea that the usefulness of AEC
as an index in pollution monitoring in the field is questionable
(Verschraegen
et at., 1985). The hypothesis was that the maintenanee of
a stable population is impossible when the individuals constantly have
low AEC's, so that in polluted stations only pollutant-resistant speeies will be found with normal AEC·s.
of Seienee) 2 : 1-15.
in metabolie rep,ulation : rat liver citrate cleavage enzyme.
Biol. ehern. 242 : 3239-3241.
.T.
Effects of toluene on the survival, res-
pirati"I., Jr.d adenylate system of a marine isopod.
13 : J 11-1 i5.
Bogaen, T., t'I.R.' Samoiloff an<1 G. P"rs(>one 1984.
Mar. PoIl. Bull.
Determination of the
toxicity of four heavy metal compounds and three carcinogens using
two ma ri ne nematode specie s,
metfoide~ b~uc{~.
environrr.ent,
'\!"Y'h~~ te 'La.
nJ.icJrophthatma and VipfofaJ.-
In: Eeotoxicologieal testing for the marine
pp 21-30.
Ed. bv
G.
Persoone, E. Jaspers and C. Claus
State Vniv. Ghent ar.d Inst. Xar. Scient. Res. Bredene 1984.
DietriCh, G. & K. Kalle 19"7. All gelneine,
'I~eres k·unde. Eine
.
..
Einfuhrung
J
Ernst, W. 1970.
•
Gebrüder Borntraeg?r, Berlin, Nikolassee
AlP als Indikator fiir die Biomasse mariner Sedimente.
Oecologia (Beul), 5 : 56-60.
Feldmesser, J. & R.V. Rebois 1966.
Nematocidal effeets of several cad-
t\t~matologica 12 :91.
rnLtt:Tl compounis.
Goerke, H. & W. Ernst 1975.
"lrerrr-~".
L.i. by
observed (Haya & Waiwood, 1983).
by long-term exposure to sublethai coneentrations of heavy metals.
A~.ldcmy
Waltnn 1967. Adenosine tri phosphate conservation
AlP eontent of estuarine nematodes
eontribution to the determination of meiofauna biomass by ATP mea-
would imply that, the more the individuals were stressed, the larger
their total adcnylate p001 w0uld be.
& G.1I.
in die Ozeanographie.
endproducts of adenylate energy metabolism are altered.
possibly but not likely at 5 and 15 mg!l Ni).
The ddermination of volwne and weight of nematodes.
'/,.,-.!. (1I",.g:Hian
1,'LI
Bakke, T. & H.R. Skjoldal 1979.
from old neglected stock cultures (old nematodes in overcrowded and
hypersaline conditions).
Andrassy, 1. 1956.
Atkinscn, D.E.
In the long run, the procedure became
sufficiently complex, and still results were not always consistent.
Haya
REFERfTCES
:1.
"roe. 9th Europ. mar. Biol. Symp •• 1975, pp 683-691.
b..iI"Iu::s;
An~rc..1e~n
Cniversity Press.
Haight, M., T. Hudry & J. Pasternak 1982.
c., r~;:'::~1CU~l..) -!JLll.4!JLa.L
as an
Haya, K.
~
tv
ü!
; tli~ efficacy of the frBe-living nematode
v<vo toxicological bio-assay.
B.A. Waiwoo1 1983.
1"
:ential
bi"~h,rri(''11 jrJi·:~tors
Ed by J. O. ilriagu.
Nematologica 26 : 1-11.
Adenylate energy charge and AlP-ase aetivi-
Ly pollutnnts in aquatic animals.
33:'.
Toxicity of seven heavy metals
of sublethai effects eaused
In: Aquatic Toxicology, PP 307-
l;ew 'fork : Wiley.
Haya, K., B.A. Waiwood & D.W. Johnston 1983.
Adenylate enerey charge and
ATP-ase actbity of lobster (Ho"Ja!l1H amcJL.ieanu.6)during sublethai ex-
posure to zine.
Aquatie Toxicology 3 : 115-126.
Heip, C.,R. Herman, G. Bisschop, J.C.R. Govaere, M. Holvoet, D. Vandamme,
C. T~?aEos~dcl, K.R. h'illems & Lu\.P. De Coninck 1979. Benthic studies of the Southern Bight of the North Sea and its adjacent eonti-
162.
161.
nental estuaries.
port I : 133-163.
Geconc. onderzoeksacties Ocean.
Heip, C., M. Vincx & G. Vranken 1985.
Progress Re-
Tbe ecology of marine nematodes.
Howell,R. 1984 : Acute toxicity of heavy metals to two species of marine
nematodcs.
Humman, IJ.D.
&
!'.ar. Environ. Res. 11 : 153-161.
M.R. Hunnnon ,\975.
periments.
Use of life table da ta in'tolerance ex-
Adenylate energy charge : an evaluation of appli-
cability to assessment of pollution effects snd direction for future research.
Ivanovid, A.M.
&
Rapp.P.-v.Reun.Cons.int.Explor. Mer \79 : 23-28.
W.J. Wiebe 1981.
Bru~sel,
D. Van
C. Ileip
&
D. Hermans 1934a.
free~living
Ita ~juncta (Bastian, 1865).
In: Ecotoxicological testine for
marine nematode
/.~ol'llt'l~te­
the marine environment, Val 2, pp.271-291. Ed by G. Persoone, E.
rine scientific research Bredene (Belgium).
Vranken, G., D. Van Brussel, R. Vanderhaeghen & C. Heip 1984b.
Research
on the development of a standardized ecotoxicological test on mari-
Towards.a working definition of "stress"
ne nematodes.
I. Culturing conditions and criteria for two monhy-
A review and critique. In : Stress in natural ecosystems. Ed. by
s te rids, /.!anhY6tV..a ~jul'lcta and /.fonhY6teJU m-i.C!topltthalma. In :
G.W. Barret & R. Rosenberg. Wiley.
Ecotoxicological testing for marine environment. "01 2, PI'. 159-184
Karl, D.M. & P.A. LaRock \975.
sediments.
Adenosine triphosphate measurements in
J.Fish.Res. Board Can. 3\ : 599-607.
Livingstone,D.R. 1985.
Ed. by B.L. Bayne et al.
Synergestic physiological effects of low copper and
vadous oxygen concentrations on Macoma ba.tt:hica.
91-99
Petrich, S.M. & D.J. Reish 1979.
Reish, D.J.' & R.S. Carr 1978.
Mar.Biol. 54 :
Bull.environ.Contam.
The'effects of heavy metals on the survival,
chaetous annelids.
Mar.Poll.Bull. 9 : 24-27.
Sabatini, G. & B.M. Marcotte 1983.
Water pollution
Toxicity of cadmium to
free-living marine and brackish water nematodes (MonhY6teita m-i.C!toph-
~ssay
nematode Panaglte111L1J ltecüv-i.vILIJ.
Sokal, R.R. & F.J. Rohlf 1981.
A ra;>id
for aquatic contaminants using the
Can.J.Fish.Aquat.Sci. 37 : 1167-1174.
Biometry (2nd ed.),
W.H. Freeman & Co.,
859 pp.
Verschraegen, K., P.M.J. Herman, D. Van Gansbeke & A. Braeckman 1985.
Measurement of the adenylate energy charge in
Neit~~ div~-i.cololt and
Nephty~ sp. (Polychaeta : Annelida) : Evaluation of. the usefulness snd
AEC in pollution monitoring.
Vranken, G., R. Vanderhaeghen, K. Verschraegen & C. Heip 1986.
juncta.
Toxicity
MOl'lh!l~telta ~­
A comparison between development rate, fecundity and r.lor-
tality as toxicity-indices. (This volume).
Ecological and metabolie studies on free
living nematodes from an estuarine mudflat.
9 : 257-271.
1982.
Estuar.Coast.mar.Sci.
Application of adenine nucleotide measurements for the eva-
luation of stress in M!ftUM edutU, and
:."iloff, M.R., S. Schulz, Y. Jordan, K. Denich & E. Arnott 1980.
toxicity
Deseases of
Aquatic Organisms I : 49-58.
Zaroogian, G.E., J.H. Gentile; ,J.F. Heltshe, M. Johnson & A~M. Ivanovici
a view from ecology.
}la=.Poll.Bull. 14 : 254-256.
San Fransisco.
Vranken, G., R. Vanderhaeghen & C. Heip 1985.
Warwiek, R.M. & R. Price 1979.
reproduction, development and life cycles for two species of poly
long~term
C. Claus, State University of
of environmental toxicants on the marine nematode
Effects of aluminium and nickel on sur-
vival and reproduction in polychaetous annelids.
Toxicol. 23 : 698-702.
simple
&
thalma, MOl'lhY6teita ~jUl'lcta, PelUocU..tu., mM-ina).
Praeger New York.
Neuhoff. H.G. 1982.
Ed. by G. Persoone, E., Jaspers
Ghent and Institute ofmarine scientific research, Bredene
Biochemical measurements. In : The effects of stress
and pollution on marine animals, pp 81-115.
•
Va"n('rha~~hen,
Tbe toxicity of mercury on the
Jaspers & C. Claus, State University of Ghent and Institute of ma-
Cah. Biol. Mar. 16 : 743 : 749.
Ivanovici, A.M. 1980.
Calculation of the intrinsic rate öf natu-
ral increase r m with Rhab~ maJt-i.na (Bastian, 1865) (Nematoda).
Nematologica 29 : 468~477.
Vranken, G., R.
Oceanogr. Mar: Biol. Ann. Rev. 23 : 399-489.
..
Vranken, G. & C. Heip 1983.
Mar.Biol. 86, 233-240.
Comp.Biochem.Physiol. 71B : 643-649.
Clta.6606.t'tea v-i.ltg-i.n-i.ca •
163.
e
50
TriSA~
100 pI
Tris
1
Cd
EXTRACT
ADU~T
Tris-He1
25 pI
B
164.
:.::.
1S
SURVIVAL
9~
. .
Cd (1I\g/l)
Tris C
(~
lD.a.1. t SE
95:C1)
control
)j
m
ATP(lO·.7M
ADP(10- 7 1-_ _2_5_11_1_ _~
11 ...
AMP( 10-7 )'
..--.........
M
0.5
2_5.....;..11_
1 _ _ ATP (5.10-711)
20.64! 1.84 (! 2.12)
(~
21.96. 0.91
....-
i
(on1y for AMP)
i
2.5
1.86)
21.26! 0.98 (! 1.8:)
I
:.:: 1
30 min at 30·C
(not for ATP)
I
TCA
ä
a
I
~
-,
l
1
!
45 min at 3S·C
(not for ATP)
o
20
10
3C
time (cays)
10~1
. . - - - - - - - - - - NaOH (O.SN)
''''')'8
J 75 111 Tri s-;JCl
Co<
C.2: 1
JO pI
5 pI
pepsine
(ISO "
I
Ni ADULT SURVIVAL
~~
p
Ni <mgli)
i
contral
I
O.S
I
~~
,
"" •• _
I
J.
<.
95ICl)
22.90! 0.90 <! 1.84)
19.03. 1.39 <. 2.84)
19.13 ! I.OS <, 2.14)
1
2.5
200 111
m••• 1. • SE
19.511: 1.16
(~
:.38}
~------------I
,S
~.
15.82. I.B5 C, 3.99)
I
25 1
PHOTmIETER
I
time (Cays)
I
Fig. 2 : Female adult surviva1 at different Cd (upper) and Ni (lower) concentraticns.
Protoco1 for ATP-, ADP-, and
1S = interna1 standard
A}~-determination.
P 15 survival in proportions, m.a.1. is the mean adult longe-
vity (in days), SE is standard error. 95%C1 between brackets.
e
165.
'"
...
.
'"
J
'>,
N
z'"
."
<>
<>
on
'"
U
0
0
0
"
0
'"
lil
N~
N
~
on
~
~
'""
"
~
z'"
z
•. - - - - - - - - - - - .,
0
0
0
.,
0
0
on
..
Pl
N
.~"
.
Fig 4b
WR o' Wl'm' gATf'.lO-4/ g>iwt
4
WR o' wr m• gATf' .10- /gwwt
1;1; I
lL.;J'
u
~
40
T
h
T
T
1
I
~
~
"u
0
...
~on
'"
.....
rn
Fig 4a
~o ~
~
0'"
0'"
E
In
.,
N_
O
N.!!
'~
::::
on
N_
166.
~
0
0
~
"
.~
'"
.
...
."
C
on
on
"'NI;
0
.........
",
..
;.
"
~,
.....
'"
,.,"
~
z'"
-----
.
0
0
'"<>on
u
..,"
0
~
.."
-'"
0
N
!!l
" ..........
............
"
z
0
0
~
0
"on
0
..,n
0
0
N
~
~
..
,~
."
.
0
.~
"
u
....
0
on
on
N_
N-
o
·.
•
~
In
0
..
.,<>
0
0
C>
U
0
0
Pl
0
0
N
0
u
E
lf)
N
z
<>
Cl
.,
'" "- '" "
0
N
---0
. ---
~
..,
"
0
N
0
0
.
~~
....
lC_
on
0'"
0'"
!!l
~
~
~
I\I~
N~
-120
-120
0.0
2.5
5.0
10.0
7.5
Cd (mg/ll
0
5
10
15
Ni (mg!IJ
Fig. 4 : Comparison of demographic parameters and weight-speciHc ATP
content of j uveni le (3d old) at different Cd (Hg. 4a) and Ni (Hg. 4b)
concentra dons (mg/I). Ordinate : W, H tness relative to the the control;
.
r m intrinsic rate of ngtural increase ; 0 Ra net-reproduetivity
4
mean ATP content (in g ATP 10- ) per g freshweight with eomparison limits (P=0.05 ; Gabriel tes t) .
Fig 5il
Fig 5b
wn o' Nr'ß!' qHf'. IO-4/ gWrlt
WR u' Wr m• gA TP .10-4/gwwt
40
~tI
I
100 .. "
40
I
I
~I
I
leU
I
"-
"
,
e"'"
'"'"
.~
a
u
a
U
."
~
0
0
..
:l
~
"
.....rn
<>
<>
on
U
"ue,
.......
N~
~
"
u
"
~
".
.~
.:;
~
.,..
.
..,
"
..---_._----0
Z
i".,
!!l
.2
-'
0
::::rn
"
-!!..
:=z
~
0
··•
•
a
N~
".........
'!l
...............
~
u
0'"
N~
~-.................
::::
.2
0'"
I
..,u
~
"'-
..
0
0
""-
2
u
~
.........
.....
In
"
0'"
N~
'.,
!!l
.,
~-
...
0'"
,"
z
z'"
0
0
on
.
0
0
..,
0
0
0
0
N
""
~
-."I
0.0
2.5
5.0
10.0
7.5
Cd (mg/11
-."I
0
5
10
15
Ni (mg/11
of demographie parameters (rm, Ro ) and weight-specific
5a) and Ni
I.. TP C0C:C:lt in neI:l:l L .. )"-~.:: , 0: r; . .5 <! !)~d 1 ~ ~Lff'2r~i1t Cd (fig.
(Hg. :b) concentrations (mg/I) • Abbrevia dons and symbols as in Hg. 4.
Fig. 5 :
Compar.l..~vn
'0
'...."
::l
ro
;3
0>
rt
0
0-
ro
....::>
;'~r:
:>-
H
'"tl
c::
~
0>
::>
0-
....Z
n
0
..
::l
n
::>
rt
....
.......
Dl
0
::>
'"'
........
":}
...
---
..
-
00
N
S'
0.
Z
e ....n
~
()Q
ro
'0
>-
-<
W~
0>
0-
....Z
n
0
::>
n
::>
.
....
....
Dl
rt
....
0
::>
~
....... .
_0
0
\0
....
.,::>
0-
0'
::l
()Q
~
-N
-111
0'
Dl
~
::>
.:
ro
'"
0
0
'"tl
00
U\
::l
:>
0
I
0
:1
::l
rt
'"
::l
rt
'0
-
_
0
~C1
......
0
U\-
i
-
:::>
Q.
(I>
n
........,
0-
n
0
::>
n
::>
..
0
n
....
='"
....
....
n
::l
::l
.....
'"'
0
.......,
......:
::l
0-
...,
..
....
H
''::>""
:>
0
•
(I>
.:
....'"
'".... '"
:T
()Q
()Q
:T
rt
B
0
0
0
0
U\
0
I
0
0
?~
~
0
I
_.
:::>
0.
."
rt
""
'0
u:
0
n
e
I
e"1
=
-"3
.....
......
'"
==c
::l
:T
rt
0
0-
01
/18<
0
0'
...
'..."
.,....... ---., n""::>
.... ::>
::l
::l
'"
0' ...
'"'
....
'
"
.... .: ....
'"
~
,. ::>''"" .....
--- ~ ~
::l
"
::> '....
n
0.
~:; 1
::>
Dl
S'
~~t
_u:
0
rt
~191
-<
W~
KH
!
u:i
rt
..."....
0
L-
Dl
9~
=
'" '.,;3"
n
rt
-
--
0-
0-
0
zl1l
::: ~ 1
..a
::>
>-
N
n..:;
0
I--l
07
Dl
Dl
-
0
clI
-oe....
~
n
0-
rt
;3
.:
c::
. --
...'.:" ro
''::>"" ....."c
..<
'::l" ::l.........
..., '"
.... ::l
:T
....::>
~
N
Dl
rt
n
~
..
....
....
rt
...,
'"
rt
0
0-
0
...
0
'"tl
ro
n
.....
~
::l
::l
.....
~
::l
~.
()Q
0>
-''''..
..
0
1-"~
::> I~
.
co
< :> '"
ro
......... ... ..n
. eI~
.....C....
ro
c
~
rt
0
0-
ro
rt
;:;::
:"
C,'.'
control
0.5 mg/l
I mfl/l
233
223
331
304
206
176
e«)
6.01
6.28
6.04
6.25
7.30
14.18
j ( +)
2.28
6.22
5.14
61.40
100
100
P (+)
8.15
12.10
10.80
63.80
100
100
102
245
92
~1It:H
---Ne
N
143
'f'f
2.5 mg/l
T,nin ~~
8.14 + 0.086
(± 0~173)
8.59 :!: 0.105
(± 0.209)
8.86 :!: 0.088
(±0.176)
16.05 ± 9.234
(± 0.467)
lrJ'
71
129
56
II
8.01 ± 0.099
(± 0.199)
7.98 ± 0.065
(± 0.129)
8.84 ± 0.192
(:!: 0.384
13.73 ± 0.407
(:!:0.906)
N
Tmin
C!./
sex ratio
Table 1 :
66.8
44.2
81
5 mg/l
10 mfl/l
e
89
CADMIUM: Minimum generation time Tmin ± standard error
(wit~
95% CI between brackets).
egg mortality e(+). juvenile mortality j(+) and preadult mortality p(+) at different Cd concentrations (mg/I).
Ne is the number cf eg&6 studied, N ~~ (~~ the number of adults and sex is the pro-
portion females in the adult porulation.
""
ca
.i
•
..
e
110.
169.
a (± 95% CI)
b (± 99% CI)
-8.14 (i 24.40)
-):'.02 (± 28.89)
32.30 (:!: 3.79)
3S.77
5.23)
1
4.80 (± 24.62)
2.5
4.28 (± 16.61)
:HCKEL
(mg/I)
a (r. 95% CI)
b (i 99% CI)
-8.14 (± 24.40)
32.30 (± 3.79)
-6.71 (>; 25.23)
32.28 (± 5.49)
2.5
-10.42 (± 25.14)
25.36 (± 3.91)
5
-10.64 (i 14.49)
15
-1.22 (i 31.97)
CADMIUM
r2
Fs
n
0.992
888
9
0.991
33
8
25.92 (± 3.35)
0.988
67
10
17.54 (± 3.62)
0.987
38
7
(~g/l)
....
-...
00
a
11
control
....
f<
'" ""
0
0
0
0
0
0.5.
0
l/l
M
<1l
....
-...
00
..,...
a
l/l
c::
0
0
0
0
0
0
0
....>
<1l
'"
GI
N
....
-...
00
S
'"
""
O'
O'
.....
0
N~
0
'" '"
er.
N
'"..;"" ....
0
'"
N
0'"
+1 '"
0
N
....'"
"
.D
~
.....
·co
oco
+1 0
N
N
~~
M
. +1
O'
""
control
.;
c::
.....,0
'-'
N
M
<1l
H
lJ
....
-...
00
a
'"
'"
N
.....
N
0
er.
..., 0...,
N
-
er.
-
0
....
M
+1
0
O'
GI
-:co
0 ....
-~
""
""
c::
....
....
....
""
u
0
~.:!J
~.:!:!
co
'"
'"-:~
N
N
O'
'"
'"
...: ..;
"" '"
0
....co
0'"
+1
N
.... 0
O'
888
9
562
7
0.987
516
9
23.03 (± 2.25)
0.995
1281
9
16.37 (± 19.])
0.973
centrations (mg/l) : parameters of the regression
72~
4
ON
N
'"
.... Ö '"
+1
ma~ge
Nex=a+b.time (d)
xwith b the mean daily eggproduction per female.
n is the number of observations, r 2 the coefficient of determination, Fs the
...."GI
~.t!
co
~~
0.992
0.991
Table 3 : Egg production (cumulative) per female. at different Cd (Ni) con-
...c::
........"'0
-~
.",
....
n
u
....z
GI
...,
O'
Fs
c::
'".... ...,+1 Ö '"
M
r2
0
N
....
-...
00
a
(±
F-statistic of the linear regression (P<O.OOI ;
...
ll:'
0.01<1'<0.05)
<1l
"'0
...."'0
GI
.,:l
l/l
-. ........,
l/l
N
....
-...
00
a
N
....
0
....
'"co '"co
'" N
.0
O'
.0
....
N
+1 0
GI
O'
.,":l
O~
...,
....
.N
oco
+1 0
co
~.:!:!
co
~~
O'
N
'"
....
CADMIUM
(mg/I)
~
...
....
0
control
-
c::
0
u
o~
o~
""
M
N
co
0
N
.0
N
'" .......,
oÖ
• O'
.M
0
....
...."" ö
I
'"'"
m
'"
....
.
0'"
....
0
~~
co
-:.:!.
co
co
.0
'"
'">4
....u
z
GI
z
.....+
.
'-'
0+
0+
~
~
..:!:...,
.:!:.
Po
0+
0+
Z
.....c:
H
e
::g
~
.....c:
.......
"
Z
H
a
<1l
X
GI
'l/l
0.422
13.53
194
0.376
12.48
0.403
14.32
2.5
72
0.199
21.40
control
302
0.422
13.53
1
165
0.354
14.43
-
2.5
176
0.333
15.52
5
154
0.328
15.36
.D
15
21
0.182
16.85
,11
............'" ....UJ
.. ..."
....<1l
GI
,..:I
!:lu
....
z
0
,..:I
302
321
l/l
....
0
.,"
T (d)
GI
0.5
,r1
A
rm(d- I )
Ro
<1l
NICKEL
(mg/I)
+<
r
.......
GI
<1l
.D
c:
~ .~
Table 4 :
DCiThJ{;raphic p..irileeters
.J.>::
different Cd (Ni) concentrations (mg/!)
Ro is the net-reproductivity. r m is the intrinsic rate of natural increase
(per day) and T the mean generation time (in days).
171.
NICKEL
Table 5
a
b
n
172.
CRITERIUH
267
-0.164
0.953
61.1
5
LC50 (96h)iIi
0.398
-0.052
0.965
81.5
5
MEC'J(+)
Nickel:
Ro (respeetively r m) as a funetion of Ni eoneentration
(mg/I). Parameters of the regression Y=ae b [N~ with a and b eonstants, Y=R
o
(respeet. Y=rm) and [Nlj in mg/I. Abbreviations as in table 3., O.OOI<P<O.OI
Cd
Ni
37
103
2.5
5
HEC Tmin
}IEC Ne x
HEC m.a.l.
•
2.5
EC50 (Ra)
1.64
3.48
EC50 (rm)
2.37
12.29
Compiled table of demographie eriteria studied in ~wnhy~t~
Extraetion proeedure
n
ATP/gwwt ± SE
Table 8:
EXTR 1 (TCA)
5
EC = effeetive eoneentration with 507. inhibitory effeet, J(+) = juvenile
EXTR 2 (H2 S0 4)
Eilu\ .) (PCA)
5
26.771 ± J. 785
22.782 ± 1.255
5
19.599
EXTR 4 (Formie acid)
4
EXTR 5 (NRB/NRS)
5
12.115 ± 3.170
1I. 303 :I: 0.589
EXTR 6 (Tris 100·C)
4
EXTR 7 (Ethanol :l:80·C)
3
d[~junc.ta
Table 6:
1.415
3.559 :!: 0.295
0.334 :I: 0.143
Extraetion proeedures test: mean ATP eontent (in g ATP 10- 4 )
per g wet weight ± standard error.
Table 7:
:I:
n i8 the number of replieate extractions
Artifieial stress induetion : AEC in old nematodes (in over- .
erowded and hypersaline conditions) and in starved organisms.
under Cd (Ni) intoxieation.
llEC = minimal effeet coneentration,
mortality, Tmin = minimal generation time, Ne x = mean daily egg production,
m.a.l.
= mean
adult lonr.evity, Ro = net-feeundity, r m = intrinsie rate of
~ = data from Vranken et at., 1986.
natural inerease.
eoneentrations in mg/I.