Journal of Environmental Protection, 2011, 2, 1127-1133
doi:10.4236/jep.2011.28131 Published Online October 2011 (
Copyright © 2011 SciRes. JEP
Development of the Sea Urchin Arbacia Punctulata
in the Presence of the Environmental Toxin
Sodium Hypochlorite
Meghan O. Rock1, Elizabeth C. Davis-Berg1, Brittan A. Wilson2
1Department of Science and Mathematics, Columbia College Chicago, Chicago, USA; 2Department of Biology and Chemistry, Texas
A&M International University, Laredo, USA.
Received July 12th, 2011; revised August 23rd, 2011; accepted September 27th, 2011.
Sodium hypochlorite (NaOCl) or bleach, found in effluent from wastewater treatment plants, can act as an environ-
mental toxin. The sea urchin Arbacia punctulata is a common subject of embr yological toxicity tests due to its sensitiv-
ity to environmental pollu tants. Using con cen tration s of NaOCl that mimic those found in treated wastewa ter (0.11 ppm,
0.06 ppm, and 0.03 ppm) we observed minimal affects on early larval development, though most larvae took longer to
develop at higher NaOCl concentration. There was a significant difference in the percentage of non-normal plutei
based on concentration (P = 0.038) and significant interaction between the percent of each morphology and NaOCl
concentration (P = 0.0027). The most significant change in non-normal plutei was in the retarded (shortened skeletal
rods) malformation which increased in frequency with NaOCl concentration (P = 0.001). There was a significant re-
duction in skeletal length in both normal and retarded plutei (P < 0.05) as NaOCl increased.
Keywords: Arbacia, Urchin, Hypochlorite, Development, Toxin
1. Introduction
Sea urchins are a useful indicator species for environ-
mental contamination due to the fact that their sperm,
embryos, and larvae are very sensitive to toxins in the
water [1-5]. Sea urchins also make an excellent research
species because spawning and gamete collection is rela-
tively simple, literature on echinoid embryological de-
velopment is plentiful, the larvae develop quickly, ani-
mals are available year round and are easily maintained
under laboratory conditions [1,3,6]. The sea urchin, Ar-
bacia punctulata (Lamark, 1816), is readily available and
has well documented early development stages as well as
established EPA embryological toxicology methods [1,7,
The effects of heavy metals, butylins and other envi-
ronmental toxins on sea urchin embryos have been well
researched. Bioaccumulation [9,10], development and
embryotoxicity [11-13], and more recently genotoxicity
and genetic mutation [14-16] are the primary areas of
concern for these toxins. These toxins can have potent
effects on embryos: concentrations of 250 mg·Pb·l–1 can
cause accumulation of 3 mg·Pb·g–1 dry weight [9].
Relatively little work has been done to determine the
effects of sodium hypochlorite on sea urchin embryo
development. Sodium hypochlorite (NaOCl), (commonly
found in household bleach) has wide applications in sci-
ence, medicine, and especially sewage treatment, where
it is used to reduce the number of viable bacteria in ef-
fluents [17]. In tests against the budding yeast, Sac-
charomyces cerevisiae, sodium hypochlorite causes in-
duced genotoxic effects [18].
Human activities directly impact the ocean and in par-
ticular estuarine environments [19]. With the growing
population on our coastlines, estuaries are becoming
primary receiving waters for treated wastewater effluent
from coastal communities. Therefore, the presence of
NaOCl in wastewater, used in the terminal process to kill
bacteria, has the potential to affect both geochemical
cycles and resident organisms in the estuarine environ-
ment [20].
One previous study focused on the effects of waste-
water NaOCl on sea urchin fertilization [20]. Even at
trace amounts, 0.025 - 0.125 ppm NaOCl, it was found
that NaOCl negatively affected sea urchin fertilization
success by reducing viability of sperm [20]. Chlorinated
Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite
sewage, in comparison to unchlorinated sewage, was a
significant and potent fertilization inhibitor though sperm
showed sensitivity to either kind of sewage [20].
This study focuses on determining what, if any, de-
velopmental effects sodium hypochlorite has on Arbacia
punctulata embryos and larvae. We tested the effects of
sodium hypochlorite exposure at concentrations of 0.00,
0.03, 0.06 and 0.11 ppm NaOCl on sea urchin larvae.
From this we were able to determine if there were dif-
ferences in the pluteus morphology (Figure 1), skeletal
lengths and the ratio of normal and abnormal sea urchin
larvae at each concentration.
2. Materials and Methods
These two experiments were performed in the spring of
2009 and repeated in spring of 2010. A pilot experiment
showed that developmental timing was sensitive to low
or variable laboratory temperatures causing delays (past
36 h) in pluteus formation. In the work reported below,
all samples were monitored for temperature throughout
the experiments reported here and were maintained at
20˚C ± 2˚C.
Sodium hypochlorite solutions were prepared using
Clorox bleach containing 6.15% sodium hypochlorite or
293 ppm [21]. Solutions were prepared with artificial
seawater (originally Instant Ocean) from an established
cycling marine aquarium (36 ppt NaCl) filtered through a
Whatman Grade 1 Qualitative filter (11 microns), to re-
duce biologic sources of contamination. The concentra-
tions made were 0.11 ppm, 0.06 ppm, and 0.03 ppm
NaOCl mimicking the wastewater hypochlorite concen-
trations reported by Muchmore and Epel [20]. We tested
Figure 1. Major characters used to classify pluteus mor-
phologies and length calculations. This illustration is based
on a related urchin, Strongylocentrotus purpuratus, at ~five
days old.
these concentrations after the experiment was complete
using Varian ICP-OES and found that they were accurate
to 0.005 ppm.
Using standard procedure [1,7-9,20], sea urchins (or-
dered from Gulf Specimen Marine Lab: Panacea, FL)
were spawned with a 0.5 ml intracoelomic injection of
0.5 M KCl. The eggs were collected in sea water and the
sperm were stored dry in an ice bath until use. To make a
standard sperm suspension, 1 ml of sperm was added to
10 ml of seawater. This was used to fertilize the eggs at a
ratio of 2 drops of standard sperm suspension per every
10 ml of eggs and seawater. This was repeated again
after 10 min to ensure fertilization.
The egg/sperm mixture was kept at room temperature
(20˚C ± 2˚C); at 40 min the eggs were checked for de-
velopment, then were checked at 10 min intervals until
first cleavage was observed in the majority of the cells in
the sample (60 min). For each replicate one ml of fer-
tilized egg solution was placed in a sterile 50 ml cell
culture bottle, and then 9 ml of the respective treated
seawater were added. The final NaOCl concentrations
were 0.11 ppm, 0.06 ppm and 0.03 ppm NaOCl respec-
tively. The bottles were sealed with parafilm, capped
lightly, and left to develop at room temperature in low
light. Organisms reached the pluteus developmental stage
by 45 h for experiment 1 and 50 h for experiment 2. For
each of the four concentrations of NaOCl (0.00, 0.03,
0.06 and 0.11), three replicates were prepared.
When the majority of larvae reached pluteus stage,
samples were preserved by adding 1 ml of 37% buffered
formaldehyde to each culture bottle and then placing the
samples in a 4˚C refrigerator for 24 h. After 24 h in 3%
buffered formaldehyde, the samples were rinsed twice
with de-ionized water to remove formaldehyde and re-
duce its degrading effects, then the fluid was serially
replaced with ethanol in the following concentrations:
30%, 40%, 50%, 60% and 70%. Between each ethanol
rinse the samples stood 10 min, and after the final 70%
ethanol rinse the preserved larvae were stored in a 4˚C
refrigerator to slow cellular decay.
Counting was performed with a Sedgewick-Rafter
counting cell on a Fisher Scientific Micromaster micro-
scope (model CK) at 200x magnification. For each
treatment, three individual 1 ml aliquots were tallied un-
til 400 larvae were counted. There were four counting
morphological categories used: normal development (N),
reduced skeletal rod length or retarded growth (R), mal-
formed plutei (M) and pre-pluteus larvae (P) based on
Kobayashi and Okamura [22]. An embryo was counted
as Normal (N) when spicule length was longer than one
body length (accounting for normal larvae that were
shorter) and lacking any obvious physical deformity
(Figure 2(a), (f), Figure 3). An embryo was counted as
opyright © 2011 SciRes. JEP
Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite1129
Figure 2. Different kinds of pluteus larvae seen in the ex-
periment (from one 0.11 ppm NaOCl sample in experiment
2): (a) Normal larva; (b) Retarded larva; (c) Malformed
(Toothless embryo with torsion); (d) top larva Arabesque
malformation, bottom two Normal; (e) upper larva Re-
tarded, lower larva Malformed (Toothless, slightly Vam-
pire); f) Normal larva with labels.
retarded (R) if the skeletal rods length was smaller than a
body length (Figure 2(b), Figure 3). For a malformed
(M) count, any physical deformities present were noted,
and when possible, photographed (Figure 2(c)-(e)). An
embryo was considered pre-pluteus (P) when it did not
have any skeletal rods or any of the internal structure
expected of a larva at pluteus stage.
Measurements were taken with a marked eyepiece
calibrated with a micrometer. Larvae were only meas-
ured when it was clear the whole larva was on a parallel
plane to the lens and slide, so that the measurements were
not distorted by foreshortening. The larva was measured
along its whole length from the apical end to the end of
one skeletal rod (Figure 3).
We used the program PASW Statistics (Version 17)
for statistical analysis of the count and length data. Raw
count data and length data were analyzed using the gen-
eralized linear model (GLM), as well as post hoc Tukey’s
tests. The count data was normalized using a logit func-
tion, ln(count + 1) (Figure 3). The +1 was added in order
to assess instances where there was not a specific mor-
phology of sea urchin malformation found. The GLM
used for count data was as follows: NaOCl was coded as
a random factor, to determine if there were differences
between treatments in total counts for each malformed
morphology. In addition, the average percentage of each
type of pluteus (normal, malformed, pre-pluteal, and
retarded) was analyzed to better determine how concen-
tration altered the presence of each type using an
ANOVA followed by the post hoc Tukey’s analysis. The
total length of the normal and retarded plutei were ana-
lyzed using an ANOVA followed by the post hoc Tukey’s
3. Results
Four malformed morphologies were noted during count-
ing. The first were primarily skeletal rods with most of
the cell-tissue missing, these larvae were classified as
“Wishbone malformations”, and were prevalent in the first
experiment and seem to be related to pre-formaldehyde
larvae death. The second abnormal morphology, more
common in the first experiment than second, was the
“Vampire malformation” (Figure 2(e)). This occurred
when the two front skeletal arms of the larva lacked tis-
sue on the spicule, making them appear as if they were
fangs. The lack of tissue while a skeletal rod remains
may be an indicator of larval stress. The third notable
malformed morphology, and the most common in both
experiments, was the “Toothless malformation” (Figure
2(c) and (e)) where an larva’s smaller skeletal rods were
greatly shortened or missing on one or both sides beyond
what is expected due to the organism’s natural asymme-
try. Lastly, the “Arabesque larvae” were noted in the
second experiment only; larvae had arms (usually pos-
toral arms) that did not lie in the same plane, were bent at
odd angles relative to the body and other arms, or miss-
ing altogether (Figure 2(d)). Abnormal morphologies
were not counted separately, but samples with many of a
single malformed morphology were noted. Some forms,
especially the toothless malformation, appear similar to
those found in centrifuged embryos [23] and embryos
exposed to styrene derivatives [24]
We used a general linear model (GLM) to test for any
significant differences between experiments; none were
found. There was a significant difference in the percent-
age of each morphology present based on concentration
(P = 0.038) and evidence for a significant interaction
between the percent for each morphology found and
NaOCl concentration (P = 0.0027). This data suggests an
increase in the percent of non-normal morphology plu-
teus found with an increase in the concentration of so-
dium hypochlorite. We then compared the percent of
each larval type present to determine which was contri-
Copyright © 2011 SciRes. JEP
Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite
Copyright © 2011 SciRes. JEP
Figure 3. Larval length measurements between (a) Normal larva and (b) Retarded larva. While the total overall length is
close, retarded larvae were determined via the body to arm ratio.
buting to the differences found (Figure 4). There were
no significant differences in the percent of larvae that
were normal, malformed or pre-pluteal due to concentra-
tion (P > 0.05). There was a significant increase in the
number of retarded larvae with an increase in NaOCl
concentration, both 0.06 and 0.11 ppm (P < 0.01), (Fig-
ure 4).
Given the most significant change in the pluteal mor-
phology was found in the shortening of the skeletal rods
(retarded type plutei) body length of the normal plutei
and retarded plutei were assessed by concentration and
type (Figure 5). Total body length is significantly shorter
in retarded larvae for all treatments (P < 0.001). Total
body length, though proportional, was significantly re-
duced in normal larvae at 0.11 ppm NaOCl (P < 0.001).
There is suggestive evidence that the body length is
shorter in normal larvae in 0.06 ppm NaOCl (P = 0.06).
The two lower concentrations of NaOCl produced aver-
age larval lengths ~25 µm longer than the average length
of the larvae in the highest concentration. The increased
frequency of retarded plutei resulted in the 0.03 ppm
NaOCl treatment having average larval lengths ~30 µm
longer than the average larval length of the larvae in the
0.11 ppm NaOCl treatment.
4. Discussion
We found significant effects due to the concentration of
sodium hypochlorite on both the observed larval mor-
phologies and on the lengths of normal and retarded plu-
tei. Count data shows that there is a significant difference
between concentrations and larval morphologies.
At the highest concentrations of sodium hypochlorite
solution there were more developed larvae present. How-
ever, in comparison to larvae in the lowest concentration
and the control groups, these larvae had a significantly
shorter total length, even in normally developed plutei. It
Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite1131
Figure 4. Differences in the average percent of each type of pluteal formation (shown with standard deviation error bars)
scored for replicate counts during each of the 2 experiments; (a) normal, (b) malformed, (c) pre-pluteal, and (d) retarded.
Averages were calculated by averaging the percentage of each type noted in each trial, not by combining the total counts and
recalculating a pooled average.
Figure 5. Pooled average larvae lengths in the Normal and
Retarded categories with standard error bars. The retarded
larvae are significantly shorter than normal morphology
for all treatments. There is suggestive evidence that normal
larvae exposed to 0.06 ppm are shorter than controls and
those exposed to 0.03 ppm NaOCl (P < 0.06). Total body
length, though proportional, was significantly reduced in
normal larvae to 0.11 ppm NaOCl (P < 0.001). This suggests
that the presence of NaOCl may be affecting the overall
growth of plutei.
is possible that, while the larvae were able to develop in
the 0.11 ppm NaOCl, their growth was affected by the
presence of sodium hypochlorite. These observations
may be an indication that development can occur even in
higher concentrations of NaOCl, though Muchmore and
Epel [20] reported that fertilization at these concentra-
tions was negatively impacted.
There was also a noticeable developmental delay at
room temperature (16˚C - 20˚C) for our samples. During
the pilot study for this project (data not reported) a delay
in development due to temperature was noted. Larvae
were preserved after checking for development but be-
cause we did not scan all samples, we did not know the
extent of the developmental delay. In subsequent experi-
ments samples were checked periodically at and after the
36-h mark using the control treatment to determine if the
majority of the larvae were at pluteus stage.
5. Conclusions
We originally hypothesized that there would be fewer
developed larvae in the samples with higher levels of
sodium hypochlorite and the data is inconclusive to that
end, though our results show that there is a difference
Copyright © 2011 SciRes. JEP
Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite
between samples by concentration levels, especially at
the highest concentrations of NaOCl. At higher levels of
NaOCl exposure, A. punctulata larvae have shorter
skeletal lengths than those larvae in lower levels of
NaOCl. The significant increase in retarded type plutei
exposed to NaOCl and overall shortening of body length
even in normal type plutei, suggests that NaOCl may be
impacting the development of the skeletal rods. Further
investigation into the physiological mechanisms behind
this finding needs to be addressed. Development may be
slowed by the presence of NaOCl as noted by the in-
creased number of pre-pluteus larvae present in the 0.11
ppm NaOCl treatment. Further research is necessary to
determine the impact NaOCl has on observed larval
6. Acknowledgements
This study would not have been possible without the help
of many people. Very special thanks to the Science and
Math department at Columbia College Chicago for their
continued financial support and encouragement through-
out the course of this experiment, in particular G. Adams,
B. Budy, O. Carnate, D. Jordan, M. & R. Rock, and M.
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