Aquaculture 291 (2009) 6573

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Aquaculture
j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / a q u a - o n l i n e

The accumulation of substances in Recirculating Aquaculture Systems (RAS) affects

embryonic and larval development in common carp Cyprinus carpio
Catarina I.M. Martins , Marco G. Pistrin, Stephan S.W. Ende, Ep H. Eding, Johan A.J. Verreth
Aquaculture and Fisheries Group, Wageningen University, P.O. Box 338, 6700 AH, Wageningen, The Netherlands

a r t i c l e

i n f o

Article history:
Received 1 July 2008
Received in revised form 26 February 2009
Accepted 2 March 2009
Keywords:
Recirculating Aquaculture Systems (RAS)
Early-life stages
Ortho-phosphate-P
Nitrate
Heavy metals
Growth retardation

a b s t r a c t
The accumulation of substances in Recirculating Aquaculture Systems (RAS) may impair the growth and
welfare of sh. To test the severity of contaminants accumulated in RAS, early-life stages of sh were used.
Ultraltered water from two Recirculating Aquaculture Systems (RAS), one RAS with a high accumulation of
substances (water exchange rate 30 L/kg feed/day) and one RAS with a low accumulation of substances (water
exchange rate 1500 L/kg feed/day), was used to incubate eggs and rear larvae of common carp Cyprinus carpio.
A broad range of read-out parameters was used to determine the effect of accumulation level on the
development of the early-life stages; from hatching dynamics to larvae length and dry weight. The water
quality (temperature, pH, dissolved O2, conductivity, total bicarbonate, ortho-phosphate-P, TAN, NO
2 N,
NO
3 N and minerals) was compared between the 2 treatments. Carp eggs developing in the highaccumulation water had higher mortality percentages (both for eggs and larvae), reduced hatching
percentages, delayed hatching dynamics and reduced larvae length and body weight. However, these larvae
exhibited fewer deformities than larvae incubated in the low-accumulation water. Furthermore, an
accelerated development both of the embryo (appearance of heart beat, pectoral n bud and tail movement)
and yolk-sac larvae (depletion of the yolk sac) was observed in the high-accumulation water. The high
accumulation water had signicantly lower pH and higher conductivity, NO
2 N, NO3 N and orthophosphate-P. Most of the minerals (As, Cu, Mn, Ni, Zn, K, Mg, Na, P and S) including heavy metals, were
present at a higher concentration in the high-accumulation water. The inuence of these parameters on the
embryonic and larval development of sh is discussed. It is suggested that in the high-accumulation water,
the concentration of ortho-phosphate-P, nitrate and of the heavy metals arsenic and copper is likely to have
impaired the embryonic and larval development and therefore deserves further research as potential growth
inhibiting factors in RAS.
2009 Elsevier B.V. All rights reserved.

1. Introduction
Recirculating Aquaculture Systems (RAS) are used to reduce water
consumption and waste discharge in land-based aquaculture. The
trend in the way these RAS are operated is to decrease the level of
water consumption per kg feed and therefore to increase the recycling
percentage (Eding et al., 2006). On one hand such trends offer further
environmental advantages, but on the other hand it raises concern
that accumulation of substances not treated sufciently by conventional water treatment units that may impair sh growth performance
and welfare. In fact, impaired growth performance, i.e. growth
retardation, has already been reported in some species cultured in
RAS as compared with ow-through systems, e.g. sea bass Dicentrarchus labrax (Deviller et al., 2005). The basis behind the concept of
growth retardation is currently unknown, although several hypotheses have been raised such as the accumulation of sh metabolites
(e.g. steroids), system-produced products (e.g. quantity and composi Corresponding author. Tel.: +31 317485157; fax: +31 317483937.
E-mail address: catarina.martins@wur.nl (C.I.M. Martins).
0044-8486/$ see front matter 2009 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquaculture.2009.03.001

tion of bacteria, bacteria metabolites related with the age of the

biolter) and feed-related substances (e.g. heavy metals) (Martins
et al., 2007). Considering the present state of knowledge on this topic,
the only solution to this problem may be to use high water exchange
rates per kg feed per day. This however, is not a real sustainable
solution when considering all the limitations for freshwater-use in the
future. Therefore, it becomes crucial to devise a sustainable application of RAS in order to understand which growth inhibiting factors
(GIF) are being accumulated, how they affect sh performance, and
how accumulation of these substances in RAS can be prevented.
To detect potential GIF, bioassays can be used. Bioassays using sh
eggs and larvae have been frequently used to test many types and
varying degrees of aquatic pollution as they are the most sensitive
stages of the sh life-cycle (e.g. Rieckhoff and Nellen, 1993; Skinner
et al., 1999; Dumas et al., 2007). One may expect that if recirculated
water from RAS affects the growth of juvenile sh due to a possible
accumulation of GIF, then its effect on early-life stage development
should be even more signicant. Such a bioassay, when developed,
may provide a tool to test how far farmers can increase their waterrecycling percentage. This study is intended to be the rst step towards

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C.I.M. Martins et al. / Aquaculture 291 (2009) 6573

the development of such a bioassay. Therefore, the goal of this study is

to investigate the effect of substances accumulated in RAS on early-lifestage development of common carp Cyprinus carpio. Because these
substances may originate from the sh, biolter and/or the feed, it was
our intention to operate RAS in which all possible sources were used.
This was achieved by operating two RAS differing in stocking density,
age of the biolter, feed load and water exchange level. The water from
these RAS was then used to incubate eggs and grow larvae of common
carp. By differing on the level of water exchange it is expected that the
level of substances accumulated and water quality will differ between
the treatments. It, however, was not our intention to identify the factor
responsible for hampering embryonic and larval development. Such a
single-factor experiment goes beyond the scope of this study where
commercial conditions were mimicked, and in which several parameters vary simultaneously (Colt, 2006). A broad range of read-out
parameters was used, from embryonic development, until larvae

length and dry weight. Common carp was chosen as our bioassay
species as its embryonic and larval development are well documented
(Oyen et al., 1991) and its egg incubation and larval rearing can be
easily controlled. Furthermore, the carp chorion is very clear, thus
facilitating the identication of embryonic developmental stages.
2. Material and methods
2.1. Recirculating Aquaculture Systems
The water used for egg and larval development originated from 2
RAS differing in the level of water exchange per kg feed and thus on
substances accumulated (Fig. 1). As it is not known whether these
substances arise from the accumulation of sh and/or system and/or
feed-related substances, a combination of all these potential sources
was assumed to create the differences in the experimental treatments.

Fig. 1. Scheme of RAS 1 (high-accumulation water) and 2 (low-accumulation water) used as a water source for the bioassay. In RAS 1 water ows from culture tanks and experimental
tanksdrum ltersump 1trickling ltersump 2oxygen conesh tanks. One side ow across the denitrication reactor using only fecal carbon as energy source ows from the
drum lterbuffer tankdenitrifying reactordrum lter. In RAS 2 water ows from sh tankstube settlersump 1trickling ltersump 2sh tanks.

C.I.M. Martins et al. / Aquaculture 291 (2009) 6573

Therefore, a high accumulation of substances was created in RAS 1

(high-accumulation water, total system volume was 4.1 m3) by
stocking all-male Nile tilapia (Swansea silver strain, TilAqua, The
Netherlands) at high densities, at a high feed load, using an old
biolter of more than 5 years old and a low water exchange rate (30 L/
kg feed/day). RAS 2 (low-accumulation water, total system volume
was 3.0 m3) functioned as a control system with a low accumulation of
substances using a low stocking density, a low feed load, a young
biolter (6 months old) and a high water exchange rate (1500 L/kg
feed/day, Table 1). The high stocking densities in the high-accumulation system were realized by using 4 tanks of 450 L with group housed
sh, each containing between 67 and 75 sh (start stocking density:
30.0 12.3 kg/m3; end stocking density: 52.9 11.0 kg/m3). As it is
not known if sh-related substances that accumulate in RAS are
produced by sh of a certain size class, 2 size classes were used in the
group housed sh, one size class with a start weight of 60 g (2 tanks)
and one size class of 300 g (2 tanks). These sh were fed restrictively
at a ration of 2.2% of body weight/day with automatic feeding belts.
Fish were fed with commercial tilapia feed (3 mm oating pellets; 44%
crude protein, 10% fat, 25% carbohydrates, 11.5% ash; Skretting, France).
The low-accumulation system contained only 24 tanks of 40 L with
individually housed tilapia (fed ad libitum), thus low stocking density
and low feed load. Both RAS were kept undisturbed (no sampling
procedures) during the bioassay.
Daily water exchange was performed according to a standard
procedure consisting of calculating the water exchange based on the
feed load of the previous day and refreshing either 30 L/kg feed/day
(high-accumulation water) or 1500 L/kg feed/day (low-accumulation
water) from sump 1 (see Fig. 1) of both RAS. The volume of water to
be discharged was then ltered using an ultraltration membrane (XFlow ultraltration 2005, Triqua, Wageningen, The Netherlands) with
a pore size of 0.03 m which is enough to lter-out all ultra-ne
particulates and suspended solids, bacteria, fungi and protists before
being used for egg incubation. Before the water was introduced into
the incubation unit, the temperature of the ltered water was adjusted
to 24 C.
2.2. Egg incubation and larval rearing
Eggs and milt from common carp (C. carpio) were obtained as
previously described by Komen et al. (1988) from broodstock kept at
De Haar Vissen, Wageningen University. Articial fertilisation and egg
incubation were done following standard procedures (Komen et al.,
1988). Samples of 150180 eggs were mixed with milt (fertilisation
time, t = 0) and were spread on the screen bottom (mesh size of
1 mm) of a 10 cm diameter PVC basket (wall height of 5 cm). Carp eggs
are sticky and easily attach to the mesh bottom. The PVC baskets were
placed in a thermo-regulated (24 C) incubation unit (total volume of
65 L) which consisted of an incubation unit (35 L) and a buffer tank
(30 L). Two incubation units were used, one per treatment. The
incubation tank contained the egg incubators, larvae tanks and a
digital thermostat to keep the temperature constant throughout the
entire experimental period. Egg incubators and larvae tanks were
Table 1
Differences between RAS differing on the level of substances accumulated.

Fishsh contact
Biolter age
Water exchange
Initial biomass (kg)
Final biomass (kg)a
Mean feed intake (kg/d)a
Cumulative feed burden (mg/L)a,b
a

High accumulation

Low accumulation (control)

High
N5 years
Low (30 L/kg feed)
57.98
100.37
1.15
33,323

Not present
6 months
High (1500 L/kg feed)
4.27
5.48
0.04
660

Based on 58 days of growth period.

Calculated as mg of feed per day divided by liters of water discharge per day
(adapted from Colt et al., 2006).
b

67

supported on a perforated submerged bench. Daily, the entire volume

of the buffer tank was replaced by new ltered water from the RAS.
This was done to expose eggs, on a daily basis, to actual concentrations
of substances accumulated in each of the two RAS.
Fifteen egg incubators were used per treatment water. The owrate over each egg incubator was checked daily and kept constant at a
level of 92.4 0.9 ml/min.
Eight hours post-fertilisation (hpf) the number of fertilised eggs
(unfertilised eggs were opaque in colour) was counted and this was
the number used for the calculation of mortality, hatching percentage
and hatching dynamics (i.e. hatching percentage over time). After
hatching, larvae were transferred to 2 L rectangular plastic tanks
equipped with two outow holes covered with mesh (1 mm) to
prevent larvae from escaping. Carp larvae were fed Artemia nauplii
after the depletion of the yolk sac (23 days after fertilisation, dpf)
until the end of the experiment (10 dpf) following Zhang (1994).
2.3. Measurements
Water quality was checked daily for temperature, conductivity, pH
and dissolved oxygen using portable meters. At 1, 5 and 10 dpf, 10 ml
of ultraltered water was collected and analyzed for total ammonia
nitrogen (TAN) (TAN = NH3N + NH+
4 N, mg/L, Skalar protocol
number 155006; based on Clesceri et al., 1998), nitrite (NO
2 N,
mg/L, Skalar protocol number 467-033; based on Clesceri et al., 1998),
nitrate NO
3 N, mg/L, Skalar protocol number 461-318; based on
Clesceri et al., 1998), ortho-phosphate-P (mg/L, Skalar protocol
number 503-317; based on Clesceri et al., 1998), total bicarbonate
(mg/L, Skalar protocol number 185-312; based on Clesceri et al., 1998)
with an autoanalyser (SAN, Skalar, The Netherlands). TAN was used to
calculate un-ionized ammonia based on Emerson et al. (1975).
Heavy metal and macro mineral's composition (Al, As, Cd, Cr, Cu,
Mn, Ni, Pb, Zn and Ca, K, Mg, Na, Fe, P and S) of the ultraltered water
were determined at the start (1 dpf) and end (10 dpf) of the bioassay
using an inductively coupled plasma mass spectrophotometer (ICPOES, Iris Advantage 2000, Thermo, protocol E1304, Chemisch
Biologisch Laboratorium, Wageningen, The Netherlands).
Embryonic development was determined following the description of Oyen et al. (1991).
Five out of fteen replicates (egg incubators) were used for
determination of egg-mortality, hatching percentage, deformity
percentage and hatching dynamics. The egg-mortality and hatching
percentage were calculated as the ratio of the number of dead eggs to
the number of fertilised eggs and the number of yolk-sac larvae to the
number of fertilised eggs, respectively. The deformity percentage was
expressed as the ratio of the number of deformed larvae to the total
number of larvae hatched. The number of dead eggs (white or
partially white in appearance) was determined at 24, 48, 72 and
70 hpf. At 50, 53, 56, 59, 62, 65, 68, 72, 74, 76, 77 and 79 hpf, the
number of hatched and deformed (e.g. skeletal deformities, duplication of heads/tails) larvae was determined. The hatched larvae were
not used for further measurements.
Five other egg incubators were used for determination of
embryonic development (dissection microscope, 1.6 20 magnication) tted with a RGB Kappa Microscope Camera, type CF 15/4 MCC.
Video recordings were done using a Sony TimeLapse recorder, type
SVT-S3000P, a Colour Monitor, S-VHS VCR, and S-VHS-tape. Ten
randomly selected eggs per egg incubator were video recorded for 10 s
in order to determine the appearance of eye pigmentation and
pectoral n bud and observation of heart beat and tail movement. In
the case of the heart beat and tail movement the frequency of beats
(beats/min) and tail movements (movement/min) was also registered. The hatched larvae were not used for further measurements.
The remaining 5 replicates were used to measure on larvae the
yolk-sac area, length and dry weight. Hatched larvae were gently
transferred to 5 larvae tanks using a plastic 5 ml pipette. The number

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C.I.M. Martins et al. / Aquaculture 291 (2009) 6573

Table 2
Water quality parameters of the ultraltered water from the high- and lowaccumulation treatments, used to incubate eggs and rear larvae of common carp
(N = 10 for temperate, pH, conductivity and dissolved oxygen; N = 3 for TAN, NO
2 N,
NO
3 N, ortho-phosphate-P and total bicarbonate; N = 2 for minerals).

movements) were tested with a chi-square test. Statistical signicance

was taken at P b 0.05.

Water quality parameter

High accumulation

Low accumulation

P-value

24.0 0.0
7.27.3
1304.2 10.1
8.5 0.0
0.15 0.04
0.001 0.0
0.1 0.0
65.3 1.0
19.5 1.0
18.5 1.8

24.0 0.0
8.58.6
586.9 9.6
8.5 0.0
0.02 0.01
0.004 0.002
0.0 0.0
14.8 0.1
0.51 0.0
169.4 1.5

N 0.05
0.00
0.00
N 0.05
0.09
N 0.05
0.01
0.00
0.00
0.00

3.1. Water quality

Temperature (C)
pH
Conductivity (S/cm)
Dissolved oxygen (mg/L)
TAN (mg/L)
NH3N (mg/L)
NO
2 N (mg/L)
NO
3 N (mg/L)
Ortho-phosphate-P (mg/L)
Total bicarbonate (mg/L)
Minerals
(g/L)
Aluminum (Al)
Arsenic (As)
Cadmium (Cd)
Chromium (Cr)
Copper (Cu)
Manganese (Mn)
Nickel (Ni)
Lead (Pb)
Zinc (Zn)
(mg/L)
Calcium (Ca)
Potassium (K)
Magnesium (Mg)
Sodium (Na)
Iron (Fe)
Phosphorus (P)
Sulphur (S)

b 30
19.00 0.00
b 0.19
b 1.30

b 30
8.00 0.00
b 0.19
b 1.30

59.00 1.00
5.00 1.00
9.30 0.40
b 10
145.50 3.50

13.50 3.50
1.50 0.050
2.20 0.40
b 10
84.50 44.50

0.00
0.00
0.00

45.00 2.30
112.50 4.50
20.70 0.80
56.85 0.95
b 0.01
17.00 0.30
38.85 1.65

75.45 5.85
6.30 1.30
7.46 0.87
23.15 3.75
b 0.01
0.61 0.11
7.92 1.09

0.00
0.00
0.00
0.00

0.00

0.00

3. Results

The comparison of the water quality between the 2 treatments is

presented in Table 2. Temperature, dissolved oxygen and ammonia did
not differ signicantly between the treatments. The incubation unit
using high-accumulation water had signicantly lower pH, lower
bicarbonate and higher nitrite-N, nitrate-N, ortho-phosphate-P and
conductivity levels than the control incubation unit. The concentration of heavy metals in the water was signicantly higher in highaccumulation water as opposed to low-accumulation water. Likewise,
the concentration of macro minerals in the high-accumulation water
was signicantly higher than in the low-accumulation water, with the
exception of calcium.
3.2. Egg mortality, hatching percentage, dynamics and deformities
Carp eggs incubated in the high-accumulation water exhibited
a higher percentage of mortality at 24 (independent t-test, df = 8,
P = 0.015), 48 (df = 8, P = 0.007), 72 (df = 8, P = 0.005) and 79 hpf

0.00
0.00

Below detection limit.

of larvae per 2 L tank varied between 122 and 154. Yolk-sac area was
determined on 50 larvae (10 per egg incubator) by video recording
randomly selected larvae at 72 and 96 hpf. These time points were
used to determine the rate of yolk-sac depletion. Videos were
analyzed using the image analysis software from Olympus-SoftImaging to determine yolk-sac area and the total larvae area (mm2).
Relative yolk-sac area (yolk-sac area/larvae area) was used to
compare the possible effect of recirculated water type on the rate of
yolk-sac depletion. Total length (mm) and dry weight (mg) were
determined on 50 larvae (10 per replicate) 10 dpf. For total length
measurement, each larva was placed on top of a millimeter paper. Dry
weight was determined in ten sub-samples of 30 individuals each, per
replicate, by drying the samples at 70 C overnight followed by 5 h at
103 C. Finally dried samples were weighed on a micro-balance
(Mettler AE 160, accuracy of 10 g). During the period of exogenous
feeding the larvae mortality was determined once everyday.
2.4. Statistics
The results are expressed as means standard error. Statistical
analyses were performed using SPSS (version 12.0). Percentage data
were arcsine transformed before further analysis. Homogeneity of
variance was tested using Levene's F-test (Field, 2000). Possible
differences in water quality (except for minerals) and in all
measurements of embryonic and larvae development between the
two treatments were tested using a t-test for independent samples.
Mineral data were analyzed using the non-parametric MannWhitney
U test. The effect of time on hatching percentage and egg and larvae
mortality was tested using repeated measures analysis. Mauchly's test
was performed to assess the assumption of sphericity (Field, 2000).
Categorical data concerning embryonic development (i.e. presence of
heart beating, appearance of eye pigmentation, presence of blood
circulation, appearance of pectoral n bud and presence of tail

Fig. 2. Comparison of egg mortality (A), hatching (B) and larvae deformities (C) over
time between the incubation units using water from the high- and low-accumulation
treatments (N = 5 replicates per water type tested). hpf means hours post-fertilisation.

C.I.M. Martins et al. / Aquaculture 291 (2009) 6573

69

(df = 8, P = 0.015) as compared with eggs incubated in the lowaccumulation water (Fig. 2).
Eggs incubated in the high-accumulation water exhibited signicantly lower hatching percentage (85 1.4%) than eggs incubated
in the low-accumulation water (91 1.1%, t-test, independent
samples, df = 8, P = 0.013, Fig. 2A). The inuence of recirculated
water on the hatching dynamics is depicted in Fig. 2B. There was a
signicant interaction between time and the treatment effect
(repeated measures ANOVA, F11.88 = 40.9, P b 0.001) suggesting that
the hatching dynamics is inuenced by the type of water. Carp eggs
hatched faster in the low-accumulation water than in the high-

Fig. 4. Relative yolk sac of common carp larvae at 72 and 96 hpf (hours postfertilisation) in the high- and low-accumulation water treatments. N = 5 replicates per
water type tested (10 yolk-sac larvae per replicate). indicates P b 0.05.

accumulation water. However, the percentage of deformities was

higher in the low-accumulation water (24.0 1.1%) when compared
to the high-accumulation water (9.4 1.5%, t-test, independent
samples, df = 8, P b 0.001). This observation was also reected over
time as shown in Fig. 2C (repeated measures ANOVA, F11.88 = 16.0,
P b 0.001).
3.3. Embryonic development
Carp eggs incubated in the high-accumulation water developed
faster in terms of heart beat (Fig. 3A, chi-square = 5.83, df = 1,
P = 0.014), pectoral n bud (Fig. 3C, chi-square = 5.0, df = 1,
P = 0.021) and tail movement (Fig. 3D, chi-square = 11.11, df = 1,
P = 0.001) than eggs incubated in the low-accumulation water.
However, when the continuous data for heart beat (number of beats
per minute, high-accumulation water: 81.6 2.10, low-accumulation
water: 87.5 3.82) and tail movement (number of movements per
minute, high-accumulation water: 21.0 1.62, low-accumulation
water: 19.2 1.95) were analyzed, no signicant differences between
treatments were found.
3.4. Yolk-sac area and depletion
The relative yolk-sac area of larvae raised in the high- and lowaccumulation water is depicted in Fig. 4. Both measurement points (72
and 96 hpf) were signicantly different with larvae raised in the highaccumulation water showing larger yolk-sac areas (t-test, independent
samples, df = 8, P = 0.000 for 72 hpf and P = 0.01 for 96 hpf). Also
when the depletion of the yolk sac is considered, larvae in the highaccumulation water exhibited a faster depletion of the yolk sac than
the control low-accumulation water (high-accumulation water: 0.12

Fig. 3. Embryonic development at 26, 30, 34 and 50 hpf (hours post-fertilisation) of

common carp raised in two RAS differing on the level of substances accumulated. N = 5
replicates per water type tested (10 eggs per replicate). indicates P b 0.05.

Fig. 5. Mortality (07 days after hatch) of carp larvae raised in RAS water with a high
and low level of substances accumulated. N = 5 replicates per water type tested.

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C.I.M. Martins et al. / Aquaculture 291 (2009) 6573

Fig. 6. Total length and dry weight of carp larvae raised in RAS water with a high and low
level of substances accumulated. N = 5 replicates per water type tested (total length: 10
larvae per replicate; dry weight: per replicate 10 sub-samples of 30 individuals each).

0.01 mm2 in 24 h and low-accumulation water: 0.07 0.01 mm2 in

24 h, t-test, independent samples, df = 8, P = 0.004).
3.5. Larvae mortality, length and weight
On day 1 (t-test, independent samples, df = 8, P = 0.028), 5
(P = 0.007), 6 (P = 0.000) and 7 (P = 0.000) after hatching, the
number of dead larvae was higher in the high-accumulation water
than in the low-accumulation water (Fig. 5). On days 2 and 4 after
hatching there was a trend for higher mortality percentages in the
high-accumulation water. Interesting to note is the stabilization of
mortality from day 3 after hatching onwards in the low-accumulation
water while in the high-accumulation water the mortality still
increased on day 7.
Larvae of common carp in the high-accumulation water exhibited
signicantly smaller total lengths (11.93 0.4 mm) and dry weights
(2.09 0.09 mg) as compared with larvae in the low-accumulation
water (total length: 13.46 0.3 mm, t-test, independent samples,
df = 8, P = 0.015; dry weight: 3.01 0.07 mg, P = 0.000; Fig. 6).
4. Discussion
This study showed that embryonic and larval development of
common carp are affected by the level of substances accumulated in a
recirculating aquaculture system (RAS). Water from a RAS operated at
30 L/kg feed/day (high-accumulation water), induced higher mortalities both during the embryonic and larval development. This was
also reected in lower hatching percentages in the high-accumulation
water than in the low-accumulation water. However, the hatched
larvae in the low-accumulation water had signicantly higher
percentage of deformities than larvae in the high-accumulation
water. Whether the earlier hatching in the low-accumulation treatment is a cause for the higher deformity percentages is not clear.
Certain development stages during embryonic development appeared
earlier in embryos incubated in the high-accumulation water than
embryos incubated in the low-accumulation water. This faster
development was also followed by faster yolk-sac depletion. Despite
this faster development in the high-accumulation water, the length
and dry weight of larvae, 7 days after the start of exogenous feeding,
were higher in the low-accumulation water than in the high-

accumulation water. These results seem somehow contradictory as

on one hand high-accumulation water leads to higher egg and larvae
mortalities, lower hatching percentages and lower larvae length and
body weight but on the other hand leads to lower percentage of
deformities and to a faster embryonic and yolk-sac larvae development. Despite the percentage of deformities was lower in the highaccumulation water than in the low-accumulation water the number
of dead eggs was higher in the high-accumulation water. We
hypothesize that the negative inuence of the high-accumulation
water on the embryonic development was such that it did not allow the
embryos to develop further, probably due to an initiation of deformity
when the larvae is still inside the egg sac. To clearly understand this
hypothesis one would need to evaluate the accumulation of substances
originated from the RAS water inside the egg environment.
The faster embryonic and yolk-sac larvae development observed in
the high-accumulation water may have been a consequence of an
increase metabolic rate. Luckenbach et al. (2003) showed an
accelerated development on brown trout early-life stages exposed to
xenobiotics. In their case the accelerated development was followed
by a premature hatching and increased heart beat rates which were
not the case in our study. A key question to this apparent contradiction
is to understand whether a potential initial increase in metabolic rate
was paralleled with metabolic stress that may have nally affected
hatching percentage. Weis and Weis (1989) also suggested that
there could be a basis for accelerated development in polluted
environments.
Possible explanations for the observed differences in embryonic
and larvae development could be related to the differences in water
quality parameters shown in Table 2. pH was signicantly lower in the
high-accumulation water (~7.2) than in the low-accumulation water
(~8.5). This difference was a consequence of 1) the higher total NH3N
excretion, 2) the lower alkalinity supply by the make-up water supply,
per unit of feed and 3) insufcient alkalinity supply by the
denitrication unit to compensate for the alkalinity consumption
due to nitrication in the high-accumulation water. Oyen et al. (1991)
tested acid stress on the embryonic development of common carp and
showed that critical levels hampering the normal development were
between 5.15 and 4.75. In their study, the higher pH level tested was
7.5 which corresponds to approximately the minimum level of pH
achieved in the current study. The question remains whether small
differences in pH levels, despite within the range for a normal
development, could explain the observed differences in development.
The enzyme responsible for hatching, chorionase, has its optimum
activity at pH 8.5 (Hagenmaier, 1974) which is the pH of the lowaccumulation water. Therefore, the fact that the low-accumulation
water had a pH closer to the optimum of the hatching enzyme could
explain the observed differences in hatching dynamics and percentage
between the treatments.
Conductivity also differed signicantly between the treatments
being approximately two fold higher in the high-accumulation water
than in the low-accumulation water. Lam and Sharma (1985) tested
different salinity levels on larval survival, growth and development of
common carp. They showed that increasing salinity (0.3, 1.5 and 3
corresponding to approximately 500, 2500 and 5500 S/cm at 25 C)
gave increasing rates of larval survival, growth and development.
However, the hatchability of the eggs did not differ between the
treatments. The authors hypothesize that the salinity effect may be
related to a reduction in the osmotic and ionic gradients between the
internal and external uids and therefore less energy is needed for
osmotic and ionic regulation and for maintenance of neutral buoyancy. In our study, the high-accumulation water had conductivity
levels of ~ 1300 S/cm as compared with 600 S/cm in the lowaccumulation water. The higher conductivity levels in the highaccumulation water did not correspond to higher larvae survival or
growth. It could be that the difference in conductivity was not high
enough to allow a signicant positive effect that would overcome the

C.I.M. Martins et al. / Aquaculture 291 (2009) 6573

negative inuence of other parameters in the high-accumulation

water.
Un-ionized ammonia is considered highly toxic to sh. The level of
un-ionized ammonia did not differ signicantly between the treatments. The highest NH3N value found (0.007 mg/L) remained 10
times lower than the lowest value reported in the literature to affect
early-life stages development (0.07 mg/L, Fairchild et al., 2005).
NitriteN was also signicantly different between the treatments but
just like for ammonia the levels found in the present study are far
below the values reported to affect embryonic development (14 mg/L
NO2N, Williams and Eddy, 1989).
In the present study, nitrate was 14.8 0.07 mg NO3N/L and 65.3
0.94 mg NO3N/L in the low- and high-accumulation treatment,
respectively. These values seem to be above the tolerance limits for
freshwater early-life stages. Nitrate levels, 1.14.5 mg NO3N/L, are
considered mildly toxic to early-life stages of rainbow trout Oncorhynchus mykiss, Chinook salmon Oncorhynchus tshawytscha, Steelhead
trout Salmo gairdneri and cutthroat trout Salmo clarki (Kincheloe et al.,
1979). McGurk et al. (2006) showed that the acute (96-h) median lethal
concentration (LC50) for swim-up fry was 1.1 mg NO3N/L for lake
trout Salvelinus namaycush and 1.9 mg NO3N/L for lake whitesh Coregonus clupeaformis. The mechanism behind the inuence of the high
nitrate levels found in this study on the embryonic and larvae
development is still not clear. Although the toxicity effect of nitrate
has been attributed to osmoregulation problems (Colt, 2006), the
nitrate appears to be taken to the plasma passively, with plasma
concentrations remaining below ambient nitrate concentrations, after
8 days exposure (Stormer et al., 1996). These authors suggested that
the limited uptake of nitrate did not inuence electrolyte balance or
haematology in juvenile rainbow trout. One could however expect a
greater inuence when early-life stages of sh would be subjected to
the same nitrate levels. In addition, Guillette and Edwards (2005)
suggested that nitrate could alter steroidogenesis in aquatic vertebrates, including sh and therefore inuence their development
pathways. The question remains whether this effect is already possible at the early-life stages of sh.
It is worth to mention that despite the possible inuence of the
nitrate levels found in this study on early-life stage development, such
nitrate levels are still below the tolerance of freshwater juvenile sh
(e.g. Siberian sturgeon Acipenser baeri of 66.9 g exhibited 96 h LC50 at
601 mg/L NO3N; Hamlin, 2006). However, it is unknown what the
effect of these lower levels is when exposure is continuous as in the
case of RAS. Nevertheless, considering the higher sensitivity of earlylife stages of sh to nitrate it is advisable to use water from separate
systems operated at high exchange rates and not water from the RAS
used to grow juveniles.
Phosphate levels also differed signicantly between RAS (highaccumulation water: 19.45 0.89 mg/L; low-accumulation water:
0.51 0.01 mg/L). Toor et al. (1983) showed that levels of N0.12 mg/L
resulted in decreased hatching and increased incidence of larval
deformities in carp. These results partly corroborate the results found
in this study as a decreased hatching but not an increased incidence of
deformities was observed in the incubation unit using water from the
high-accumulation water as compared with the low-accumulation
water.
Total bicarbonate levels were also signicantly different between
the high-accumulation water (18.5 1.8 mg/L, ~3 mg/L free CO2 at pH
7.2) and low-accumulation water (169.4 1.5 mg/L, ~ 0 mg/L free CO2
at pH 8.5). Elevation of ambient PCO2, beyond the capacity for pH
internal regulation, can quickly increase H+ concentrations of both
extra and intracellular uids, decreasing their pH and nally affecting
cellular metabolic pathways (Heisler, 1989). Despite the lack of
scientic support for the carbon dioxide limits in freshwater
aquaculture, an upper limit of 1520 mg/L CO2 has been recommended as steady state maximum for nsh (Timmons and Ebeling,
2007). The values obtained in both RAS are well below the suggested

71

limit. Despite the lack of studies on the effect of hypercapnia on earlylife stages the values obtained in this study also seem to be negligible
to embryonic and larvae development. Kikkawa et al. (2003) for
example showed that PCO2 of 1.0 kPa (~18 ppm CO2) did not affect
hatching and survival percentage for four marine teleosts embryos
and larvae within 24 h. Also Brownell (1980) showed that free CO2
concentrations as high as 28 mg/L did not affect rst feeding of cape
sole Heferonycferis capersis. Long-term effects of sublethal CO2 levels
have been described by Fivelstad et al. (2003) using smolts of Atlantic
salmons. These authors showed that 4 weeks after exposure to 6 mg/L
CO2 was enough to induce nephrocalcinosis in Atlantic salmon smolts.
In the present study, the highest free CO2 levels that the early-life
stages of common carp could have been exposed were 3 mg/L and for
a maximum period of 10 days. Therefore it seems unlikely that the CO2
levels found in the high-accumulation water could have contributed to
the observed results.
In recirculating systems with low water exchange (high accumulation of substances), such as RAS 1 used in this study, there is a
potential to build up minerals, including heavy metals originated by
the feed as part of the vitamin premix (Colt, 2006). As, Cu, Ni and Zn
were signicantly higher in the highly RAS. Nayak et al. (2007)
showed that arsenic levels of 2 and 10 g/L (both considered safe
levels in drinking water) affect the immune response of zebrash.
Therefore the values found in the present study (19 and 8 g/L in
high- and low-accumulation treatments, respectively) could have
interfered with the normal embryonic development of common carp.
Also the copper values measured in the high- and low-accumulation
treatments (59.0 1.0 g/L and 13.5 3.5 g/L, respectively) are well
above the values shown to impair hatching in zebrash (0.05 g/L,
Dave and Xiu, 1991). Since copper toxicity is hardness dependent, one
should consider the water hardness of both treatments to calculate the
critical maximum concentration (CMC) as suggested by the environmental protection agency (EPA) for freshwater aquatic communities.
Water hardness in our treatments can be estimated based on Mebane
(2006) giving values of 198 and 219 mg/L as calcium carbonate for the
high- and low-accumulation treatments, respectively. At these water
hardness the CMC for copper is 26 g/L and 28 g/L in the high- and
low-accumulation waters, respectively (based on EPA, 2006). The Cu
levels measured in the high-accumulation water are two times higher
than the CMC suggesting its inuence on hampering normal
embryonic/larval development. Comparing the previous values one
could have expected a stronger effect on the embryonic and larval
development of common carp when raised in the high-accumulation
water. Whether copper was bioavailable in the high-accumulation
water to explain the observed differences is not yet clear. A more
detailed study on the effect of water alkalinity, hardness and the
complexing effect of humic acids on minerals bioavailability should be
considered. Also the combined effects of different minerals worth
further studies.
It is worth to mention that also in the low-accumulation treatment,
the concentration of As and Cu is likely to have impaired the
development. This could explain why 100% of hatchability was not
achieved in the low-accumulation water.
Despite the signicant difference in Ni concentration between
both treatments, the levels found seem unlikely to have interfered
with the normal embryonic development as Blaylock and Frank (1979)
showed that concentrations of up to 4000 g/L had no effect on the
hatchability of eggs of common carp. Nickel toxicity is also hardness
dependent. However, considering the hardness of our waters, the CMC
is 835 and 909 g/L for the high- and low-accumulation waters,
respectively. The values obtained in this study are, therefore, well
below the CMC suggested by EPA. Likewise, the zinc concentrations
found in both treatments seem unlikely to have played a major role on
the results observed. Williams and Holdway (2000) studied the effect
of pulse-exposed zinc on embryo hatchability, larval development and
survival of an Australian rainbow trout, Melanotaenia uviatilis.

72

C.I.M. Martins et al. / Aquaculture 291 (2009) 6573

They found that the minimum concentration of zinc affecting

embryonic development (spinal deformities) was at 330 g/L and
the 96 h LC50 for 910 days old larvae was at 270 g/L. Also, the
CMC for Zn (209 and 228 g/L for the high- and low-accumulation
waters, respectively) is higher than the values measured in this study
(145.5 and 84.5 g/L for the high- and low-accumulation waters,
respectively).
The effect of RAS-water-borne macro minerals (Ca, K, Mg, Na, P and
S) on the development of early-life stages of sh is still poorly
understood despite the large number of studies done on mineral
requirements of sh (for a review see Lall, 2002). These studies have
been done with juvenile/adult sh and only a few have focused on the
requirements of water-borne minerals at the early-life stages of sh.
Examples of these studies are Van der Velden et al. (1991) and Chen
et al. (2003) who showed that that embryos and larvae of sh can
regulate the uptake of minerals from the water and that low
concentrations are often the limiting factor. The levels of magnesium
and calcium found in the present study seem to be above the limiting
values that could hamper a normal embryonic development in sh
(see Van der Velden et al., 1991 for Mg and Chen et al., 2003 for Ca).
The challenge remains to understand the potential effect of the other
macro minerals and their combined effects. The accumulation of
macro (micro) minerals in RAS deserves more attention as the energy
spent by sh on the active uptake of minerals from the water to
maintain normal plasma levels could be reduced. It would also be very
interesting to investigate how sh, once they start feeding, balance the
uptake of minerals from the water and from the diet. Alternatively,
one may also wonder what are the threshold macro (micro) mineral
levels above which they become toxic by interfering with normal
cellular ion transport mechanisms.
Besides the water quality parameters mentioned above there are
probably others that could have interfered with our results. As
mentioned by Colt (2006) in intensive RAS, the species of interest is
cultured in a kind of a soup of chemical, physical and biological
factors that are interrelated in a complex series of reactions. The
water used in the bioassay differed in several water quality
parameters. This makes the interpretation of the results difcult as
it is not possible to discriminate the effect of each parameter. This
however, was not the goal of this study as in practice RAS operated at
different refreshment rates will also differ in several parameters.
Such bioassay gave an insight about the impact of recirculated water
on the embryonic and larvae development and raised several
hypotheses on possible causes for such impact. Interesting to
mention is that the mortality and deformity percentages obtained
in the present study are relatively low as compared with other
studies testing single environmental parameters on the development of early-life stages of common carp (e.g. deformity percentage
up to 96% and egg and larvae mortality up to 100%, Oyen et al., 1991;
Van der Velden et al., 1991). Such observation may be a consequence
of the synergistic effect of the different water quality parameters
that may neutralize potential negative effects of single parameters.
Furthermore, RAS with high accumulation of substances are often
characterized by a high accumulation of humic acids which are
known to complex other substances such as steroids (Hubbard et al.,
2002). The question remains if the observed differences reect
what will happen during the on-growing stage of sh produced in
highly recirculated water (a study that is currently on-going in our
facilities). In this way, a more simplied version of the bioassay
presented in this study could be used to guide farmers in how far
they could go in closing the RAS.
Acknowledgement
This research was funded by the Dutch Ministry of Agriculture,
Nature Conservation and Food Quality (LNV bestek Duurzame
viskweek Ond/2005/08/01).

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