Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite

doi:10.4236/jep.2011.28131

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Journal of Environmental Protection, 2011, 2, 1127-1133

doi:10.4236/jep.2011.28131 Published Online October 2011 (http://www.scirp.org/journal/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.

Email: [email protected]

Received July 12th, 2011; revised August 23rd, 2011; accepted September 27th, 2011.

ABSTRACT

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,

8].

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

1128

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

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

analysis.

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-

Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite

1130

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

Development of the Sea Urchin Arbacia Punctulata in the Presence of the Environmental Toxin Sodium Hypochlorite

1132

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

morphologies.

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.

Welsh.

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