Ghekiere, A.

Open Marine Archive

  
 
 
 
 
 
 
 
AN GHEKIERE 
 
 
 
 
 
 
 
STUDY OF INVERTEBRATE-SPECIFIC EFFECTS OF ENDOCRINE DISRUPTING 
CHEMICALS IN THE ESTUARINE MYSID NEOMYSIS INTEGER (LEACH, 1814)  
 
 
 
 
 
 
 
 
 
 
 
 
Thesis submitted in fulfillment of the requirements 
For the degree of Doctor (PhD) in Applied Biological Sciences 
 
 Dutch translation of the title: 
Studie van invertebraat-specifieke effecten van endocrien-verstorende stoffen in de estuariene 
aasgarnaal Neomysis integer (Leach, 1814) 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
ISBN 90-5989-105-8 
 
 
 
 
 
 
 
 
 
 
 
The author and the promotor give the authorisation to consult and to copy parts of this work 
for personal use only. Every other use is subject to the copyright laws. Permission to 
reproduce any material contained in this work should be obtained from the author. 
 
 
 
 
CHAPTER 7 
 
 
 
 
 
EFFECT OF NONYLPHENOL AND ESTRONE ON 
MARSUPIAL DEVELOPMENT OF NEOMYSIS INTEGER  
 
 
 
 
 
 
 
 
 
 
EMBRYOGENESIS AND EXPOSURE 
 85
CHAPTER 7 
 
 
EFFECT OF NONYLPHENOL AND ESTRONE ON MARSUPIAL 
DEVELOPMENT OF NEOMYSIS INTEGER 
 
 
 
ABSTRACT---------------------------------------------------------------------------------------------------- 
Recently, marsupial development of Neomysis integer (Crustacea: Mysidacea) was described 
in detail by Fockedey et al. (2005a). Subsequently, we reported on the use of mysid marsupial 
development as a sensitive assay to evaluate endocrine disruption by exposure to the juvenile 
hormone analog methoprene. The present study further evaluates the potential use of this 
assay by exposing mysid embryos to the alkylphenol degradation product nonylphenol and 
the natural hormone estrone. Stage I embryos were exposed to 0.01, 1, and 100 µg 
nonylphenol, and 10, 100, and 1000 ng/l estrone, until hatching to a free-living juvenile. 
Duration of the different developmental stages, survival and hatching succes were recorded. 
Nonylphenol exposure had no effect on the duration of the different developmental stages but 
did significantly reduce survival and hatching at the highest test concentration. Estrone 
affected hatching at the highest exposure concentration, but had no effects on embryonic 
development at lower concentrations.  
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7.1 INTRODUCTION 
 
Embryonic development is generally regarded as a sensitive time window for toxic effects 
during an organism’s life history. Neomysis integer, like other mysids, carries its embryos in a 
marsupium where the entire larval development takes place from oviposition to the release of 
free-living juveniles (Wittmann, 1984). The marsupial development of N. integer was recently 
described in detail by Fockedey et al. (2005a). Marsupial development in mysids was 
subsequently shown to be a sensitive endpoint to evaluate the effects of the insecticide, 
CHAPTER 7 
 86 
methoprene (Chapter 6). Our ongoing and future efforts are to further explore mysid early 
embryonic development as a sensitive target to assess endocrine disruption. 
ENDIS-RISKS is a multidisciplinary project that has studied the occurrence, distribution and 
potential effects of endocrine disruptors on mysids in the Scheldt estuary (Belgium/The 
Netherlands), one of the most polluted estuaries in the world (ENDIS-RISKS project, 
http://www.vliz.be/projects/endis). We earlier reported high levels of organotins, surfactants 
and flame retardants in mysids of the Scheldt estuary (Verslycke et al., 2005). More recent 
studies found significant levels of estrone in water of the Scheldt estuary (Noppe et al., 2005). 
The effect of nonylphenol and estrone on the vitellogenesis of N. integer are described in 
more detail in Chapter 4 (§ 4.1). This chapter describes the effects of nonylphenol and estrone 
on marsupial development of N. integer. 
 
7.2 MATERIAL AND METHODS 
 
7.2.1. Chemicals 
 
Estrone was obtained from Sigma-Aldrich (Bornem, Belgium) and nonylphenol from Acros 
Organics (Geel, Belgium). Stock solutions of the test compounds were prepared in absolute 
ethanol. The ethanol concentration in the solvent control and in the different test 
concentrations was 0.01%. 
 
7.2.2. Test organisms 
 
Mysid crustaceans, Neomysis integer, were collected in March 2005 by handnet in the 
Braakman, a brackish water (10 psu) near the Scheldt estuary in Hoek (The Netherlands). 
Mysids were taken to the laboratory and cultured as described in Chapter 2 (§ 2.2.1.). 
 
7.2.3. Test design 
 
A detailed description of the marsupial development assay in N. integer is described in 
Chapter 6 (§ 6.2.3). In short, stage I embryos were transferred at random to each of the wells 
of a 12-cell plate each containing 4 ml of the different exposure concentrations of 
nonylphenol and estrone (dilution water is artificial seawater of 15 psu). Embryos were 
exposed to 0, 0.01, 1, and 100 µg nonylphenol/l, and 0, 10, 100, and 1000 ng estrone/l. All 
EMBRYOGENESIS AND EXPOSURE 
 87
concentrations reported in this study are nominal, based on dilutions of the stock solutions. 
Fifteen replicates per concentration, and a minimum of 6 embryos per replica, were used. 
Multiwells containing the embryos were placed on an orbital shaker (80 rpm), and survival, 
developmental stage and hatching were recorded daily. Concurrently, dead embryos were 
removed and 75% of the medium was replaced. 
 
The different developmental stages of the mysid embryo were distinguished as described in 
Chapter 6 (§ 6.2.4.). Stage I embryos are spherical and stage I ends with hatching from the 
egg membrane (Fig 7.1). Stage IIa embryos are dorsally bent and have a comma-like 
appearance. In stage IIb, the body is further extended and the thoracic appendages become 
more elongated. Stage II ends with molting of the embryo. Stage III embryos have stalked 
eyes and a developed telson and uropods. Stage III terminates with a final molt, leading to 
free-living juveniles. 
 
Figure 7.1: Different developmental stages of Neomysis integer embryo. Scale bar = 250 µm. 
 
7.2.4. Statistics 
 
All data were checked for normality and homogeneity of variance using Kolmogorov-
Smirnov and Levene’s test respectively, with an α = 0.05. The effect of the treatment was 
tested for significance using a one-way analysis of variance (Dunnett’s test; Statistica™, 
Statsoft, Tulsa, OK, USA). All box-plots were created with Statistica™ and show the mean 
(small square), standard error (box), and the standard deviation (whisker). 
 
 
 
 
 
CHAPTER 7 
 88 
7.3. RESULTS 
 
7.3.1. Survival 
 
Percentage embryo survival was calculated each day for the different exposure concentrations 
(Fig. 7.2). Survival was highest during stage I for both chemicals. Exposure to all test 
concentrations of estrone had no significant effect on survival in all three different 
developmental stages of the embryo. Exposure to the highest concentration nonylphenol (100 
µg/l), however, resulted in  significant mortality on embryos of stage II and III. 
 
7.3.2. Duration of the different developmental stages 
 
Figure 7.3 shows the duration of the different developmental stages of the embryos exposed 
to nonylphenol and estrone. 
 
Duration of stage I was approximately 3 days, while stage II and III lasted about 6 and 3.5 
days, respectively. The total embryonic development time of N. integer embryos development 
was around 12.5 days. Nonylphenol and estrone had no effect on the duration of the different 
developmental stages and the total embryonic development time of N. integer. 
 
7.3.3. Hatching 
 
Exposure to 100 µg nonylphenol/l, and 1000 ng estrone/l resulted in significantly lower 
hatching rates compared to the control (Fig. 7.4). Average hatching percentages were 81.4 ± 
19.4 %, 78.1 ± 14.5 %, 82.1 ± 13.8 % and 11.1 ± 16.2 % for the control, 0.01, 1 and 100 µg 
nonylphenol/l treatments, respectively. For estrone, average hatching percentages were 81.1 ± 
15.2 %, 75.5 ± 19.8 %, 76.2 ± 16.9 % and 64.2 ± 11.0 % for the control, 10, 100, 1000 ng 
estrone/l treatments, respectively. 
 
 
 
 
 
 
EMBRYOGENESIS AND EXPOSURE 
 89
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 7.2: Percentage survival of Neomysis integer embryos during the different 
developmental stages exposed to 0.01, 1, 100 µg nonylphenol/l (left), and 10, 100, 1000 ng 
estrone/l (right).(Anova, Dunnett; *p<0.05, significance from control). 
 
 
control 10 100 1000
estrone (ng/l)
0
20
40
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120
su
rv
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%
 (S
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 II
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control 0.01 1 100
nonylphenol (µg/l)
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 (S
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control 10 100 1000
estrone (ng/l)
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 I)
control 10 100 1000
estrone (ng/l)
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control 0.01 1 100
nonylphenol (µg/l)
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control 0.01 1 100
nonylphenol (µg/l)
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CHAPTER 7 
 90 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 7.3: Duration of the different developmental stages of Neomysis integer embryos 
(days) exposed to 0.01, 1, 100 µg nonylphenol/l (above), and 10, 100, 1000 ng estrone/l 
(below). Total= duration of the total development time.  
 
 
I II III total
stage
2
4
6
8
10
12
14
ag
e 
(d
ay
s)
 control
 0.01
 1
 100 µg nonylphenol/l
I II III total
stage
2
4
6
8
10
12
14
ag
e 
(d
ay
s)
 control
 10
 100
 1000 ng estrone/l
□  control 
◊   0.01 
Δ  1 
•  100 µg nonylphenol/l 
□  control 
◊   10 
Δ  100 
•  1000 ng estrone/l 
EMBRYOGENESIS AND EXPOSURE 
 91
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 7.4: Percentage hatching of Neomysis integer embryos exposed to 0.01, 1, 100 µg 
nonylphenol/l (left), and 10, 100, 1000 ng estrone/l (right) from oviposition till free-living 
juveniles. (Anova, Dunnett; *p<0.05, significance from control) 
 
7.4 DISCUSSION 
 
In this doctoral study, we described the development of three physiological processes in 
Neomysis integer that are regulated by invertebrate-specific hormones, the ecdysteroids 
(Chapter 1). These three processes, vitellogenesis (Chapter 4), molting (Chapter 5), and 
embryogenesis (Chapter 6) were subsequently evaluated as endpoints to assess endocrine 
disruption through exposure with the test compound methoprene. These experiments 
demonstrated that methoprene lowers vitellin levels in N. integer, but this effect was not 
statistically significant. Methoprene did affect mysid molting at 100 µg/l, and embryogenesis 
at 1 µg/l. These results indicate that embryogenesis was the most sensitive endpoint at 
detecting the endocrine-disruptive effects of methoprene.  
In this chapter we evaluated the potential effects of nonylphenol and estrone on the 
embryogenesis of N. integer. Estrone is frequently detected in water of the Scheldt estuary at 
concentrations of up to 8 ng/l (Noppe et al., 2005, Vethaak et al., 2002). Nonylphenol 
concentrations in the Scheldt estuary are similar to levels found in U.K. rivers and are around 
10 µg/l (Verslycke et al., 2005; Vethaak et al., 2002). Nonylphenol and estrone did not affect 
embryogenesis of N. integer at environmentally relevant concentrations. However, 
control 10 100 1000
estrone (ng/l)
0
20
40
60
80
100
120
ha
tc
hi
ng
 %
control 0.01 1 100
nonylphenol (µg/l)
-20
0
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ha
tc
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ng
%
*
*
CHAPTER 7 
 92 
nonylphenol did significantly reduce survival and hatching at 100 µg/l when compared to the 
control. Estrone significantly lowered hatching at 1000 ng/l. Estrone was previously shown to 
affect mysid vitellogenesis at 1000 ng/l by lowering vitellin levels in females (Chapter 4). In 
the same study, nonylphenol increased vitellin levels at 0.01 µg/l.  
We also observed that hatched embryos exposed to 1 and 100 µg nonylphenol/l were more 
lethargic, and higher mortality was observed at these concentrations during the first two days 
after final embryonic molt to free-living juveniles. Behavioural changes will be examined in 
future studies and might provide a sensitive way for evaluating endocrine disruption. Forget-
Leray et al. (2005) found that nonylphenol significantly reduced molting success in the 
copepod Eurytemora affinis (LOEC of 15 µg/l). Molting from one stage to another was often 
incomplete. Animals from unsuccesfull molt were deformed and displayed a mix of naupliar 
and copepodid characteristics, and were also unable to move. 
Alkylphenols, natural hormones and synthetic hormones, have been suggested as the most 
likely candidate chemicals responsible for the ‘feminization’ detected in male fish (Desbrow 
et al., 1998; Vos et al., 2000). Effects of these vertebrate (xeno-) estrogens on crustaceans, 
however, remains controversial and poorly understood giving the current knowledge on the 
presence of vertebrate-type steroids, such as estrogens, in these animals (Billinghurst et al., 
2000; Sanders et al., 2005; Tsukimura, 2001). As such, existing studies on the effects of 
(xeno-)estrogens on crustacean vitellogenesis remain fragmented and contradictory (Chapter 
4, § 4.4). However, the present study further adds to the weight-of-evidence that 
(xeno)estrogens appear to be less effective in causing endocrine disruption in crustaceans than 
in oviparous vertebrates. This also supports the growing consensus that invertebrate-specific 
approaches are needed for a relevant assessment of the risk of endocrine disrupting chemicals 
to invertebrates as they are most likely affected through hormonal pathways that are different 
from those of vertebrates. 
 
 
 


Effect of nonylphenol and estrone on marsupial development of <i>Neomysis integer</i>

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