3. THE THESIS TEXT

1
INTRODUCTION
Bivalvia constitute the second largest class of Mollusca. They have a
great economics importance (Abbott, 1952). Many of them are edible,
while some bivalves act as intermediate hosts of several trematodes
(Malek, 1962). Marine species represent about two thirds of this class,
while fresh-water ones forms the remaining third. Many marine species are
distributed from intertidal areas to great water depths. The majority of this
group lives in sand and mud bottoms (Sharabati, 1984).
In general, fouling is defined as the formation of deposits on surfaces
of heat exchangers and processing equipments which impede the transfer of
heat and increase the resistance of water flow. The growth of these deposits
causes thermal and hydrodynamic performance of heat transfer equipment
to decline with time. Fouling affects the energy of industrial processes and
decides the amount of material employed in the construction of these
equipments. However, it is necessary to provide extra heat transfer area to
compensate the effects of fouling (Somerscales, 1979).
According to Epstein (1979), fouling was classified into six
distinguished categories:
i. Precipitation Fouling
Deposition of a solid layer on heat transfer surface mainly
resulting from the existing dissolved inorganic salts in the flowing
solution which become supersaturated under the process
conditions.
ii. Particulate Fouling
Accumulation of solid particles suspended in a fluid onto a heat
transfer surface leads to fouling.

2
iii. Chemical Reaction Fouling
In this type deposits that are formed as a result of chemical
reactions in which heat exchanger surface material does not react
itself but it may act as a catalyst.
iv. Corrosive Fouling
Fouling is due to corrosion deposits from a chemical reaction
between the heat transfer surface and the heat transfer medium.
v. Freezing Fouling
This is developed as a result of partial solidification of the heat
transfer medium on a subcooled heat transfer surface.
vi. Biological Fouling
This category of fouling requires deposition of a biofilm on the
heat transfer surface due to bacteria, fungi and algae that is called
microbial fouling. Also macrobial fouling that is attachment and
growth of other macro-organisms such as barnacles, clams and
mussels.
Biological fouling is a common problem in chemical industry and
particularly in petroleum refineries. Many species of mussels are known to
be causative agents of biofouling such as Brachidontes variabilis and
Modiolus barbatus (Ghobashy and El-Komy, 1981), Corbicula fluminea
(Lyons et al., 1988), Dreissena polymorpha and D. bugensis (Ackerman et
al., 1994), B. striatulus (Rajagopal et al., 1997), Mytilus edulis and M.
galloprovincialis (Khalanski, 1998), and Perna viridis (Masilamon et al.,
2002b).
B. variabilis Kraus, 1848 (Feinberg, 1979) (Phylum: Mollusca; Class:
Bivalvia; Subclass: Lamellibranchia; Super family: Mytilacea; Family:

3
Mytilidae), the subject of the present work, is controlled through
application of an appropertiate biocide (Epstein, 1979; Hare, 2000) for
instance pentachlorophenol and 2-nitrophenol (Borcherding, 1992),
chlorination (Ackerman et al., 1994; Rajagopal et al., 1997; Masilamon et
al., 2002b), dodecyldimethylammonium chloride (Bargar and Fisher,
1997), butylated hydroxyanisole [BHA] (Cope et al., 1997), bacterial
products (Armstrong et al., 2000), copper compounds (Nicholson, 2001),
carbamate and gluteraldehyde (Pereira et al., 2001), or by the use of
physical parameters including temperature (Masilamon et al., 2002a;
Gunasingh et al., 2002).
Aim of the Work
The present work aims to study:
i. Some ecological parameters such as pH, salinity, dissolved oxygen,
temperature and some elements such as magnesium, potassium,
calcium, nickel, zinc and lead at Suez Gulf.
ii. Macro and microanatomy of some organs of the mussel Brachidontes
variabilis.
iii. Effect of some physicochemical parameters (pH, salinity), some
elements (calcium, nickel, zinc and lead) and some molluscicides
(gesapax, uccmaluscide, cetyl trimethylammonium chloride and
copper sulfate) on survival of B. variabilis.
iv. The histological changes of gills, digestive gland and ovary after
exposure to the above mentioned parameters.
On the other hand, successful control of the biological fouling must
rely, in the first place, on the deep knowledge of the biology and the
histology of pests causing it.

4
HISTORICAL REVIEW
Mytilidae have been the subject of investigation of many authors.
Concerning Brachidontes variabilis, it could be stated that their scientific
information is rather sporadic and not integrated. However, Macpherson
and Gabriel (1962) and Davis (1980) described the shell of B. variabilis,
while Achille and DiGeronimo (1978) made a biometric study of the same
species. Feinberg (1979) studied the habitat and distribution of B.
variabilis.
Different marine bivalves were subjected to different values of
salinities. It was found that salinity tolerance for a given species was not
constant but varied with season (Castagna and Chanely, 1973). In addition,
Shumway (1977) exposed eight species of bivalves to both gradual and
abrupt salinity fluctuations. In seven of the tested species the water content
of the muscles varied by only a small amount. Also, he concluded that the
amplitude of change in tissue water content was greater in low salinity-
accimilated animals than in high salinity ones. Moreover, the effects of
temperature and salinity on metabolism and byssal formation of
B. variabilis were studied by Stern and Achituv (1978). They also stated
that mortality and survival were modified by salinity regimen. On the other
hand, influence of lowering salinity on the respiratory rate of B. solisianus
and Perna perna was studied by Fontes and Sonia (1981). They recorded
that on diluting sea water B. solisianus appeared to be more resistant.
Westerbom et al., (2002) studied the effect of lowering salinity on the
growth rate of Mytilus edulis. Their results showed a marked decline in
mean mussel size and biomass as salinity decreased.

5
Concerning the effect of pH value on the bivalves Mercenaria
mercenaria and Crassostrea virginicia, Calabrese and Davis (1966) found
that the optimum pH range for growth was 7.50-8.00 and 8.25-8.50
respectively. Calabrese and Davis (1969) determined the minimum and
maximum pH levels for spawning of C. virginicia, these were 6.00 and
10.00, respectively.
Regarding the heavy metals in seawater of the Gulf of Suez, Abd-El
Salam (1981) evaluated the range of concentrations of some heavy metals.
It was found that Pb = 1.00, Cu = 1.60-9.60 and Zn = 1.62-29.22ppb.
Moreover, the concentrations detected by El-Moselhy (1953) for Cd, Pb,
Cu and Zn were 0.11, 1.11, 7.31 and 2.55ppb respectively in Suez Bay.
Mohamed (1996) found that the concentrations of the same elements were
0.01-4.00, 0.10-21.60, 0.05-13.10 and 0.08-34.2ppb respectively in the
same region. However, Yassien (1998) reported that the average
concentrations of Cd, Pb, Cu and Zn were in the order 0.20, 1.95, 1.59 and
11.27ppb in Suez Bay.
On the other hand, little information was known about the biological
effects of heavy metals on marine bivalves. A number of studies was
conducted to determine the levels of metals concentrated by bivalves.
Calabrese and Nelson (1974) studied the toxicity of some heavy metals as
metallic salts including nickel as nickel chloride, zinc as zinc chloride and
lead as lead nitrate on the subsequent development of Mercenaria
mercenaria. It was found that LC50 was 0.31ppm for Ni, 0.17ppm for Zn
and 0.78ppm for lead. Brereton et al., (1973) reported that Zn caused total
mortality to Crassostrea gigas and Ostrea edulis at doses 0.10 and
0.50ppm respecively.

6
Concerning biocides, Cremyln (1978) reported that simazine and
atrazine were initially introduced as triazine-based herbicides. Later on,
deNoyelles et al., (1982); Thurman et al., (1992) and Pereira and
Hostetler (1993) approved that atrazine was extensively used as herbicide.
In other words, the triazine herbicides were not regarded as molluscicides.
The effect of other biocides such as bayluscide (a commercial formula
of niclosamide amine salt) on the mussel Dreissena polymorpha was
examined by Hoestlandt (1971). He tested its toxicity as compared to other
biocides such as Frescon (insecticide). It was found that bayluscide was 4
times toxic. Moreover, Fisher and Dabrowska (1994) developed methods
for measuring the toxicity of Bayer 73 (a formula of niclosamide amine
salt) for several stages of D. polymorpha. They evaluated the toxicity of
this biocide after 24hours static tests, where they found that the sensitivity
of zebra mussel varied as the life stages varied, whereas the adults were
less sensitive.
In addition, quaternary ammonium compounds were used to control
the biofouling mussel D. polymorpha (Lyons et al. 1988 and Martin et al.,
1993). Zebra mussel was also controlled by quaternary ammonium
compositions (1:2 mixt. of poly (dimethyl diallylammonium chloride and
didecyl dimethylammonium chloride) within 72hours (Muia and Donlan,
1990). D. polymorpha were statically exposed to various concentrations
(0.5, 1.0, 2.0, 4.0 and 8.0ppm) of a polyquaternary ammonium compound
was killed at all tested concentrations (McMahon et al. 1990). Fellers et
al., (1990) totally controlled these mussels using 5ppm of aliphatic
quaternary ammonium compound (Duomeen C) after 4days of exposure.

7
On the other hand, copper sulfate was found to possess molluscicidal
properties, and it was used in many parts of the world, especially in Egypt
and Middle East (Malek and Cheng, 1974). However, Vyskebets et al.,
(1976) studied controlling biological fouling formed in industrial water-
supply system applying a copper compound (copper tetramine sulfate) at
5-10ppm. Moreover, Calabrese et al. (1977) investigated the toxicity of
copper to Mercenaria mercenaria and Crassostrea virginica. The obtained
LC50 was 16.4ppb and 32.8ppb, respectively. Further studies were carried
out by Nelson et al. (1988), they examined the toxicity of copper against
some bivalves including Mytilus edulis after 96hours, where LC50 was
0.122ppm. However, Blume and Fitzgerald (1990) inhibited the biofouling
zebra mussel in seawater and piping systems by using electrolytic
dissolution of copper.
Morphological and anatomical studies on bivalves were investigated
by Morton (1969 and 1973); Reid and Peter (1974); Gabal (1982) and
Kenk and Wilson (1985). Besides, a comparative anatomical study of eight
species of clams was performed by Norton and Jones (1992).
Concerning histopathological studies, Armstrong and Millemann
(1974) reported the histopathology of gills of Macoma nasuta after
exposure to the insecticide sevin for 96hours. They observed that the gills
were the most severely affected organs. Epithelial cells of the gill filaments
bearing the frontal, laterofrontal and lateral cilia were sloughed after
24hours. Furthermore, Greig et al. (1982) studied the gills of Cancer
irroratus treated with niclosamide. The data obtained revealed some
pathological effects in the gill filament, cell nodules, swelling of the gill
filaments with coagulated hemolymph and focal ercosis. The chronic effect

8
of copper on gills of M. edulis was described by Sunila (1986 and 1988). It
was observed that several abnormalities occurred in the form of fusion of
parts of the gill filaments.
On the other hand, Calabrese et al. (1984) studied the changes
occurred in the digestive tubules of M. edulis exposed to different
concentrations of copper. The mussels subjected to 5ppb of Cu showed an
extensive vaculation of the cytoplasm of the digestive cells, besides, the
digestive tubules became dilated. Genthner et al. (1997) tested the
histological changes resulted in the digestive tubules of D. polymorpha
previously treated by a molluscicidal strain of bacteria. The mussels
exposed for 24hours to whole bacterial cultures showed extensive abnormal
vacuolization in digestive tubule epithelia. After exposure for 36hours, the
mussels had disrupted apical cytoplasm and sloughed tissue. In addition,
exposure for 48hours caused extensive vacuolization, whereas the vacuoles
are large in size and filled most of the cells.

9
MATERIALS AND METHODS
i. Experimental Animals
Adult marine mussels Brachidontes variabilis were collected from the
area of Ataqah Mountain at Gulf of Suez. They were obtained by
scratching the surface of the off-shore rocks. These mussels were put in a
large clean tank contained seawater and transferred to the laboratory. They
were reared in glass aquaria of the dimensions 70X40X40cm filled with
seawater, which was continuously aerated using air compressor to supply
adequate air for oxygenation. The water was changed twice a week and the
dead bivalves were removed continuously. Animals of size 2.0-2.5cm were
used in all experiments.
ii. Physicochemical Analysis of Water
Some physicochemical parameters were determined in the study area
including:
1. pH value using a Pracitronic-MV870 pH meter.
2. Salinity using a yellow Spring SCT-33 salino-meter.
3. Dissolved oxygen using a Jenway-M9070 oxygen meter.
4. Some selected elements in seawater (potassium, magnesium,
calcium, nickel, zinc and lead) using a Unicam 939/959 atomic
absorption spectrophotometer.
iii. Effect of some Physicochemical Parameters
Biological experiments were carried out to study the effect of certain
parameters including pH, salinity, selected elements and biocides on the
mortalities of the mussels under investigation.

10
In these experiments, mussels were taken out from the laboratory
stock and kept in a separate aquaria filled with aerated seawater and they
were reared for one week before starting the experiments.
1. Effect of pH value
Twenty mussels were kept for 96 hours in 1200ml seawater supplied
with air via a small air compressor at room temperature. Different pH
values of seawater were applied (8.5, 9.0, 9.5, 10.0 and 10.5). The
corresponding pH values were adjusted by adding the appropriate amount
of dilute HCl or NaOH solutions. Moreover, each pH value was re-adjusted
every 12hours (Calabrese and Davis, 1966). Another group of 10 mussels
in 1200ml normal seawater (pH = 8.17) was taken as control and the test
was replicated four times. Mortality was recorded every 24hours and the
calculated mortality after 96hours was corrected according to Abott’s
formula (Finney, 1971;Stephan, 1977) as follows:
C = 100 (O-X)/100-X
where:
O = percentage of observed mortality from experimental samples
X = percentage of dead animals from control samples.
LC50 after 96hours was determined by Probit method using graphical
analysis. Plotting percentage mortality against log concentration gave a
direct relation.
2. Effect of salinity
Adult mussels (20) in seawater (1200ml) provided with air introduced
by a small compressor were kept for 96hours at room temperature.
Different salinities of water were adjusted by dilution successively with tap
water (40%o, 35%o, 30%o, 25%o, 20%o, 15%o, 10%o, 5%o and 2%o)

11
(Castagna and Chanely, 1973). The test was replicated 4times and 10
mussels in seawater (1200ml) without dilution (salinity = 42.4%o) was
taken as a control. Mortality was recorded daily and calculated after 96
hours according to Abott’s formula.
3. Effect of some elements
a. Effect of calcium
Twenty adult mussels were reared in aerated seawater (1200ml) at
room temperature for 96 hours. Different concentrations of calcium
chloride were obtained by successive dilution of a stock solution (5%) (4.0,
6.0, 8.0, 10.0, 20.0, 40.0 and 80.0ppm). The test was replicated 4 times and
other 10 mussels were kept in seawater (1200ml) without calcium chloride
as control. Mortality was observed daily, calculated after 96hours and
corrected according to Abott’s formula.
b. Effect of heavy metals (nickel, zinc and lead)
Twenty adult mussels were reared in aerated seawater (1200ml) at
room temperature for 96 hours. The concentrations 4.0, 6.0, 8.0, 10.0, 20.0,
40.0 and 80.0ppm were obtained from a stock solution (5%) of nickel
sulfate, zinc oxide or lead nitrate. The test was replicated four times and a
group of ten adult mussels in 1200ml seawater without metal salt was
considered as control. Mortality was observed each 24 hours, calculated
after 96 hours and corrected according to Abott’s formula.
4. Effect of Molluscicides
a. Effect of gesapax (a commercial formula of ametryn)
Twenty adult mussels (B. variabilis) were reared in seawater (1200ml)
supplied with a stream of air and were kept at room temperature for
48hours. Different concentrations of gesapax were applied (40.0, 80.0,

12
100.0, 120.0, 140.0, 160.0, 180.0, 200.0 and 250.0ppm respectively). The
corresponding ametryn concentration was obtained by serial dilution of a
stock solution (5%). The test was replicated four times and the control
sample was using 10 adult mussels in seawater (1200ml) without a
molluscicide. Mortality was determined after 48 hours and the correction
equation was applied.
b. Effect of uccmaluscide (a commercial formula of niclosamide
monoethanolamine salt)
Different concentrations of uccmaluscide (commercial formula
containing 83.1% niclosamide amine salt) (0.5, 1.0, 1.5, 2.0, 2.5, 3.0 and
3.5ppm) were prepared. Mortality was determined from treated and control
groups were calculated according to Abott’s formula.
c. Effect of cetyl trimethylammonium chloride
The previous procedure was repeated but the active ingredient used
was cetyl trimethylammonium chloride in different concentrations (25.0,
50.0, 100.0, 150.0, 200.0 and 250.0ppm).
d. Effect of copper sulfate
Different concentrations of copper sulfate (1.0, 1.5, 2.0, 2.5, 3.0, 3.5,
4.0, 4.5 and 5.0ppm) were tested applying the forgoing procedure as in
gesapax.
iv. Anatomical Studies
Fresh and live specimens were dissected with fine tools. The soft parts
were examined under sterobinocular microscope with the aid of natural
dyes namely methylene blue using Camera Lucida.

13
v. Histological Studies
The soft parts of normal and treated specimens were dissected out of
the shells and they were immediately fixed in Bouin’s fluid for 48hours.
Then the tissues were dehydrated in ascending series of ethyl alcohol from
70% to 100% and cleared in terpinol for 72 hours. Tissues were embedded
in paraffin wax and transverse sections were cut at 5 thickness. Sections
were stained with Mayer’s haematoxylin and eosin and prepared for
microscopical examination. Sections were photographed using Carl Zeiss
Camera.
Histological changes were investigated in gills, digestive gland and
ovary due to exposure of B. variabilis to sub-lethal doses of different tested
parameters (pH, salinity, the elements Ca, Ni, Zn and Pb and molluscicides
including ametryn, niclosamide, cetyl trimethylammonium chloride and
copper sulfate). In addition, a test under some combined parameters
conditions (sub-lethal values of pH, salinity and Ca) for one week was
evaluated. The test was carried out to study the combined effects of these
parameters which were considered to be less toxic and more effective on
the histology of the three organs namely gills, digestive gland and ovary.
Twenty adult mussels were kept in 1200ml aerated seawater at room
temperature for one week. Sub-lethal values of the tested parameters were
introduced (pH=8.5; salinity = 15%o; Ca = 15ppm).

14
RESULTS AND DISCUSSION
I. PHYSICOCHEMICAL ANALYSIS OF WATER
i. pH Value
The pH value of water at the investigated area at Suez Gulf was found
to lie on the alkaline side, it seems to vary within narrow limits. The
recorded pH values at different seasons were 8.11, 8.12, 8.17 and 8.16 in
summer, autumn, winter and spring respectively. Therefore, the average pH
value along the whole year was 8.14 (Fig. 1).
The obtained result (average) was close to that determined by Yassien
(1998) (8.13), but different from that observed by Hamed (1996) (8.05).
The latter two values were recorded at Suez Bay.
ii. Salinity
The average water salinity was 42.75%o. Regarding salinity of each
season, it was 43.2, 43.0, 42.4 and 42.4% in summer, autumn, winter and
spring respectively (Fig. 2). Increase of salinity during summer could be
attributed to increase of evaporation of water.
On the other hand, Ghobashy and El-Komy (1981) and El-Sabh and
Beltagy (1983) found that the average salinity was 43.0%o and from
40.14%o at south to 42.85%o at north respectively at Suez Gulf.
iii. Dissolved Oxygen
The level of dissolved oxygen in the investigated area varied from
season to another. Thus, it was 4.1, 4.4, 4.6 and 4.2 in summer, autumn,
winter and spring giving an average value of 4.35ppm during the whole
year (Fig. 3).
However, Hamed (1996) reported that the average value was 4.77ppm at
Suez Bay.

15
iv. Temperature
The highest surface water temperature was recorded in summer season
as 29.0o
C followed by 21.0, 19.0 and 26.0o
C on autumn, winter and spring
respectively, (Fig. 4). Average temperature was 23.5o
C at the investigated
area of Suez Gulf.
Meshal (1967) found that the temperature at Suez was 28.4o
C during
September 1967. Also, Hamed (1992) showed that the temperature was
17.5o
C in winter and 28.5o
C in summer at Suez Bay.
v. Some Selective Elements in Seawater
Atomic absorption measurements showed that the average
concentration of magnesium, potassium, calcium, nickel, zinc and lead
were 1584.00, 5500.00, 776.60, 0.009, 0.029 and 0.096ppm respectively.
The concentrations of zinc and lead were determined by Abdel-Salam
(1981) as 1.62-29.22 and 1.00ppb at Suez Gulf.

20
II. MORPHOLOGICAL STUDY
The shell of B. variabilis (Figs. 5 and 6) is medium in size (2inches).
It varies from reddish brown to dark green in color, which become more
pale towards the umbo. It is composed of two equivalves triangular in
shape. The sculpture consists of numerous ribs, which produce further
branched radial ribs and extend over the whole surface of the valve. The
ligament is external and posterior to the umbo.
However, Moore (1969) considered the presence of such ligament as
distinguishing feature of the superfamily Mytilacea.
The valve margins are crenulated and the points of insertion of the
shell and byssal mussels were discriminated on the inner surface of the
shell valves. The scar of the posterior adductor muscle is oval and situated
at the postero-dorsal region of the valve occupying the largest area in
comparison with other muscle scars. This scar is differentiated into
subequal areas, one antero-ventral and the other postero-dorsal. The
anterior adductor scar is narrow, elongated in shape and extends close to
the antero-ventral part of the inner pallial line. There is an oblong area
anterior to the posterior adductor scar representing the scar of the posterior
byssal retractor muscle scar. It has a V-shape appearance. The anterior
byssal retractor muscle scar is oval in shape and is located at the anterior
roof of the deepest inner area of the shell valve behind the level of the
umbo.
Feinberg (1979) and Awad (1999) reported similar descriptions of
some bivalves.

21
III. ANATOMICAL STUDY
The soft parts of B. variabilis (Figs. 7 and 8) are bilaterally symmetrical
and laterally compressed. The most important constituents are:
i. The Pallial Lobes
The visceral mass is covered with two lateral pallial lobes fused
together dorsally and posterioly between the two siphons and anteriorly
beneath the anterior adductor muscle. On the outer surface of each pallial
lobe, the pallial muscles and the shell muscles connecting the mantle lobe
with the corresponding shell valve are discriminated.
ii. The Siphons (Fig. 8)
The exhalant siphon is an elongate oval slit formed between the
posterior free edges of the two pallial lobes. It is limited dorsally by the end
of the floor of the posterior dorsal groove and extends ventrally to become
separated from the inhalant siphon by a narrow region of the fusion of the
two inner folds of the mantle lobes. The internal diaphragm lies within the
excurrent siphon which resembles that of Mytilus (White, 1937)
The inhalant siphon is not separated from the pedal/byssal aperture by
any partition. Therefore, they form a common aperture.
This common aperture resembles that described by Lithophage
(Wilson, 1979), Aboul-Dahab (1983) for Modiolus auriculatus, Botula
(Wilson and Tait, 1984) and Bathymodiolus (Kenk and Wilson, 1985).
iii. The Byssus
The byssus (Fig. 7) is an organ for attachment to the substratum. It is
stalked brush-like structure that projecting from the mid-ventral side of the
visceral mass. It is composed mainly of the byssal stalk, sheath, threads and
gland. The byssal stalk is a solid wedge-shaped rode, which is

22
differentiated, into a proximal and an upper unsheathed one. The proximal
end of the stalk is the broadest part and attached to the posterior byssal
retractor muscle fibers. The unsheathed part of the stalk lies close to the
base of the foot. It carries a large number of irregular silky threads, forming
a brush-like structure.
The solid stalk of B. variabilis differs from that present in the byssal
system of the mussel Lithophaga which is described by Gohar and
Soliman (1963a).
iv. The Foot
The foot (Fig. 7) is tongue shaped, dorsoventrally compressed
muscular organ which slightly tapers towards its free ends. It protrudes
from the ventral side of the visceral mass at a point very close to the
anterior side of the byssal sheath opening. It is differentiated into a terminal
part and a basal one. Along its mid-ventral side there is a narrow groove
extending from its anterior most free tip till it becomes continuous with the
byssal sheath opening.
However, this description of the reduced foot is typical in most
mytilids as in Modiolus auriculatus (Aboul-Dahab, 1983).
v. Musculature (Fig. 7)
The extrinsic muscles can be divided according to their functions into
the following groups:
1. The adductor muscles.
2. The retractor muscles.
1. The adductor muscles
The anterior and posterior adductor muscles are distinct. The anterior
adductor is in the form of a flat bright plate. It is located transversely on the

23
antero-ventral surface of the visceral mass connecting the two pallial lobes
together. The anterior adductor muscle of B. variabilis resembles that of
many other mytilids such as Mytilus edulis (White, 1937), Modiolus
modiolus and M. demissus (Stanely, 1972), Limnoperna forteni (Morton,
1973), Musculista senhausia (Morton, 1974), M. auriculatus (Aboul-
Dahab, 1983) and Bathymodiolus thermophilus (Kenk and Wilson, 1985).
The posterior adductor muscle is cylindrical in shape and larger in size than
the anterior one. It is located in the postero-dorsal part of the visceral mass
connecting the two mantle lobes and shell valves together.
Moreover, the posterior adductor muscle is translucent which is
typical for all Mytilacea (Morton, 1977).
However, the presence of a small anterior adductor muscle and a
comparatively large posterior one is considered to be one of the
characteristic features of the superfamily Mytilacea (Moore, 1969).
2. The retractor muscles
a. The byssal retractor muscles
The byssal retractors are mainly used for fixation and retraction of the
byssal sheath and stalk.
The anterior byssal retractor muscles of each side raised from the
anterior part of the byssal sheath and extends anteriorly to become inserted
into the corresponding shell valve at a part on its internal surface just
behind the umbo in the form of a cylindrical rod. It becomes differentiated
into anterior and posterior group of muscle fibers. The former consists of
four blocks while the latter is formed of two blocks.
Soot-Ryen (1955) considered such structures as distinguishing
characters of the species Modiolus.

24
The posterior byssal retractor muscles on both sides have a V-shape
appearance. They arise from the dorsal tip of the byssal stalk and the two
lateral walls of the byssal sheath as a mass of muscle fibers which bifurcate
to form a right muscle component and a left one. The posterior byssal
muscle of each side passes posterodorsally along the visceral mass till its
dorsal side to become attached to the inner surface of the corresponding shell
valve.
Aboul-Dahab (1983) reported similar structure of muscle fibers of the
posterior byssal retractor of M. auriculatus
b. The pedal retractor muscle
The posterior pedal retractor muscle comprises a right small band of
muscle fibers and another similar left one. The anterior pedal muscle is
absent. The right band of the posterior part of the foot passes posterodorsally
to become close to the upper most portion of the anterior part of the pedal
retractor muscle of the same side. The left band follows a similar course in
the left side of the visceral mass.
Such observations resemble those described by Aboul-Dahab (1983)
for Modiolus auriculatus.
c. Siphonal retractor muscles
Siphonal retractor muscles are strong and formed of amalgamated
strands originating in inner mantle folds in region of excurrent siphon.
The present investigation is similar to that found by Kenk and Wilson
(1985) in the mytilid Bathymodiolus thermophilus.
vi. Ctenidia
There are two pairs of ctenidia, each one consisting of an inner and an
outer demibranchs. Each demibranch is composed of descending and

25
ascending lamellae forming W-shaped gill typical of mytilids.
Demibranchs are equal-sized and they end anteriorly. Ctenidia are of the
filibranchiate type.
Such observations resemble those of some bivalves (Feinberg, 1979;
Awad, 1999).
vii. Digestive System
Digestive system of B. variabilis consists of the digestive tract and the
digestive gland
A. The digestive tract
1. The mouth and labial palps: (Fig. 8)
The mouth lies at the antero-ventral side of the visceral mass which
is transverse and slit-like. On each side of the mouth opening, there are
two adjacent distinct labial palps; one in front of the mouth and the other
behind it.
2. The oesophagus: (Fig. 8)
The mouth opening leads directly to the oesophogus. It passes
slightly upwards and backwards till it joins the anterior end of the
stomach.
3. The stomach: (Fig. 8)
The oesophogus leads directly to an elongated stomach. It lies
under the posterior part of the ligament. The stomach is completely
covered by the digestive gland except in a small anteromid-dorsal area.
The stomach is divided into anterior and posterior chambers. Three pairs
of digestive ducts enter the stomach laterally, one pair into the anterior
chamber and two pairs into the posterior chamber.

26
4. The intestine: (Fig. 8)
The intestine begins at the posterior end of the stomach and extends
posterioly between the two components of the posterior byssal retractor
muscle, till they reach the posterior adductor muscle.
Rectum extends posterioly to enter pericardium and ventricle from
below, then it turns downwards to the anus on the posterior side of the
posterior adductor muscle.
Concerning the foregoing structures, the opening of the rectum
within the ventricular lumen is similar to that found in certain mytilid
species as Mytilus edulis and Modiolus squamosus (Pierce, 1973) and
Modiolus auriculatus (Aboul-Dahab, 1983).
Besides, the structure of this digestive tract resembles that described
in Bathymodilus thermophilus (Kenk and Wilson, 1985).
B. The digestive gland
The digestive gland is a reddish brown mass. It consists of
numerous tubules which are connected medially with the stomach. It lies
in the antero-dorsal region of the visceral mass.
This is typical for bivalves as appeared in Tridacna and Hippopus
(Norton and Jones, 1992).
viii. The Female Reproductive System
It consists mainly of an ovary and two oviducts
1. The ovary
It consists of a large number of follicles occupying most of the dorsal
and ventral surfaces of the visceral mass. Each follicle has rounded
elongate and oval outlines.

27
2. The oviducts
The female gametes are collected from the ovary in the anterodorsal
region of the body by small two oviducts which in turns open in outer
larger one. The oviducts are then joining the common female reproductive
duct which extends posteriorly before it bends downwards to open by an
oval reproductive opening, very close to the anus.
Structure of the female reproductive system resembles that of most
bivalves as in Modiolus auriculatus (Aboul-Dahab, 1983), Bathymodiolus
thermophilus (Kenk and Wilson, 1985) and Corbicula fluminea (Awad,
1999).

28
IV. HISTOLOGICAL STUDY
i. Gills:
There are two gills located on both sides of the body. Each one
consists of two V-shaped demibranchs and each demibranch being
composed of two ctenidial lamella. The cetnitial lamella is formed of a
large number of thin filaments. These filaments are frontally and laterally
ciliated. The adjacent filaments of each lamella are united by a group of
large interlocking cilia, placed at regular intervals constituting the
interfilamentar ciliary junctions. A branchial vein runs through each
filament. The epithelium of each filament is composed of four types of
cells: the frontal, the laterofrontal, the endothelial and the abfrontal cells
depending on their positions (Fig. 9).
Similar epithelial cells were observed in the gill filaments of Mytilus
edulis (Sunila, 1986).
ii. Digestive Gland:
The digestive gland of B. variabilis, as any other bivalves, is formed
of a large number of more or less similar tubules separated from each other
by a thin sheet of vascularly pigmented connective tissue. Each tubule is
lined by two main cell types, the secretory and the excretory cells.
Secretory cells are tall columnar cells with round apices and flat bases.
They form the major constituents of the cellular lining of the digestive
gland tubules. The excretory cells are present in smaller numbers than the
secretory cells. They are pyramidal in shape and their cytoplasm is usually
crowded with a variable number of granules and globules (Fig. 10).
This structure is typical in most molluscs as in Corbicula flaminea
(Awad, 1999) and Lymnaea caillaudi and Bulinus truncatus (Saad, 1986).

29
iii. The Ovary
The ovary is composed of a large number of oogenic follicles
occupying most of the dorsal and ventral portion of the visceral mass above
the foot. The female follicles have irregular size and shape and they are
connecting together by a connective tissue. The follicles have different
oogenia. They are usually found in groups and they appear rounded in
outline with distinctly acidophilic cytoplasm. The nucleus is spherical and
relatively large occupying a central position in the oogonium, but it
sometimes takes different eccentric positions. The chromatin material is
usually found lumped into large irregular masses arranged along the inner
surface of the nuclear membrane.
The primary oocytes are ovoid and have relatively spherical nuclei.
The secondary oocytes have a large ovoid outline and each has a large
central nucleus.
The mature ova stain more intensely than the oocytes due to the
accumulation of yolk material in their cytoplasm. The nucleus of the ovum
is considerably large.

30
Figure (5): Diagrammatic drawing of a dorsal view of the left shell
valve of Brachidontes variabilis showing the external
features of the shell [anterior side, a.s; posterior side, p.s.;
lines of growth, lg; umbo, u].
lg
a.s. p.s.
u
0.1mm

31
Figure (6): Diagrammatic drawing of ventral view of the left valve of
B. variabilis showing the internal features of the shell [anterior
adductor muscle insertion, aa; anterior byssal retractor muscle,
abr; anterior retractor muscle insertion, ari; ligament, l;
posterior adductor muscle insertion, pai; posterior byssal
retractor muscle insertion, pbri; pallial line, pl; siphonal
retractor muscle insertion, sri; umbo, u].
0.1mm
abri
u
aai
sri
pl
pbri
pai
ari
l

32
ar ppr abr pbr
pa
l sr
u
aa
f b
Figure (7): Diagrammatic drawing of the soft parts of B. variabilis
after removal of left valve, mantle lobe and ctenidia to
show musculature [anterior adductor muscle, aa; anterior
retractor muscle, ar; byssus, b; foot, f; ligament, l;
posterior adductor muscle, pa; posterior byssal retractor
muscle, pbr; posterior pedal retractor muscle, ppr;
siphonal retractor muscle, sr; umbo, u].
0.1mm

33
i r an
st id
l exs
o bs
mth
u vsm
lp
bpg al dl
Figure (8): Diagrammatic drawing of the soft parts of B. variabilis
after removal of left valve and mantle lobe to show the
ctenida and labial palp [ascending lamella, al; anus, an;
byssal-pedal gland, bpg; branchial septum, bs;
descending lamella, dl; excurrent siphon, exs; internal
diaphragm, id; intestine, i; ligament, l; labial palp, lp;
mouth, mth; oesophagus, o; rectum, r; stomach, st;
umbo, u].
0.1mm

34
Fig. 9 photomicrograph of T.S. of the gills of B.
variabilis showing gill filaments (gf), frontal
cells (fc), frontal cilia (fci), laterofrontal cells
(lfc), endothelial cells (ec) and abfrontal cells
(afc). (Bouin, Hx and E; X825).
Fig. 10 photomicrograph of T.S. of the digestive
glands of B. variabilis showing digestive
tubules (dt), the secretory cells (sc), excretory
cells (exc), lumen (l), the basement membrane
(bm) and the intertubular connective tissue
(ct).. (Bouin, Hx and E; X825).
Fig. 11 photomicrograph of T.S. of the ovary of B.
variabilis showing oogenic follicles (of),
primary oocytes (oo1), secondary oocytes
(oo2) and mature ova (ov). (Bouin, Hx and E;
X660).

36
V. EFFECT OF SOME PHYSICOCHEMICAL PARAMETERS
The mussel Brachidontes variabilis is considered as the principal
fouling agent in seawater at the investigated area at Suez Gulf (Ghobashy
and El-Komy, 1981). However, this mussel is recorded as one of the
inhabitants of both eastern and western coasts of Africa. Regarding the
eastern coast, its distribution extends northwards till Ismailia city, Egypt
(Feinberg, 1979). The present work is concerned with seawater used in
cooling towers in petroleum refineries at Suez city, Egypt. The target of
this study is to combat this mussel physically and chemically by controlling
pH, salinity of water and concentration of some metals (Ca, Ni, Zn and Pb).
Besides, the chemical controlling by application of the appropriate biocide
including gesapax (commercial formula of ametryn), uccmaluscide
(commercial formula of niclosamide), cetyl trimethylammonium chloride
or copper sulfate is carried out. The histological study of the gills, digestive
gland and ovary of the mussel was achieved. It was convenient to evaluate
this study at sub-lethal doses. All investigations were carried out on the
adult B. variabilis since it was expected to be the more resistant stage.
i. Effect of pH
Average number of dead mussels of B. variabilis was determined at
different pH values of seawater. As shown from table (1), the percentage of
mortality was 40, 50, 70, 90 and 100% for pH 8.5, 9.0, 9.5, 10.0 and 10.5
respectively, control pH value was 8.17. The observed mortality was
plotted against the corresponding log pH value and LC50 was elucidated at
pH 9.0 (Fig. 12).
Calabrese and Davis (1966) proved similar results. The recorded pH
value potent to Mercenaria mercenaria was 9.0.

37
A transverse section in the gills of the adult mussel B. variabilis after
treatment with sub-lethal pH value (8.5) for one week revealed remarkable
deformation. A pronounced loss of some frontal cilia and slight disruption
in the epithelial cells of some gill filaments were observed (Fig. 13).
The digestive gland was highly affected which appeared in the
presence of extensive vaculation in cytoplasm of digestive tubule epithelia,
severe disruption in the basement membrane of some digestive tubules and
rupture in the intertubular connective tissue (Fig. 14).
Marked deformation was developed in most of the primary oocytes
while mature ova were slightly affected (Fig. 15).
It is to be emphasized that pH variations were directed towards the
basic medium to avoid corrosion problems normally encountered in cooling
water.
A significant disruption in the embryonic development of Saccostrea
commercials occurred when pH was adjusted to < 6.5 (Wilson and Hyne,
1997).

38
Table 1: Mortality (%) of B. variabilis at different pH values
pH log pH
No. of dead mussels after 96 hours*
Mortality
(%)Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
8.17 (control)
8.5
9.0
9.5
10.0
10.5
0.91
0.93
0.95
0.98
1.00
1.02
---
6
9
14
16
20
---
8
11
14
17
20
---
8
10
14
19
20
---
10
10
14
20
20
---
8
10
14
18
20
---
40%
50%
70%
90%
100%
* 20 mussels were used for each replicate.

40
Fig. 13 Photomicrograph of T.S. of the gills of B.
variabilis treated with sub-lethal pH value (8.5)
for one week, showing loss of some frontal
cilia (fci), slight disruption of epithelial cells
(ec), (Bouin, Hx and E; X1030).
Fig. 14 Photomicrograph of T.S. of the digestive
gland of B. variabilis treated with sub-lethal pH
value (8.5) for one week, showing extensive
vaculation of epithelial cells (ec), severe
disruption in the basement membrane (bm) and
rupture in the intertubular connective tissue
(ct). (Bouin, Hx and E; X825)
Fig. 15 Photomicrograph of T.S. of the ovary of B.
variabilis treated with sub-lethal pH value (8.5)
for one week, showing deformation of primary
oocytes (oo1) and the mature ova (ov) are
slightly affected. (Bouin, Hx and E; X660)

42
ii. Effect of Salinity
Percentage of mortality was determined at different salinities. The
data obtained was 10%, 15%, 20%, 30%, 35%, 50%, 70% and 100% for
salinities 40.0%o, 35.0%o, 30.0%o, 25.0%o, 20.0%o, 15.0%o, 10.0%o,
5.0%o and 2.0%o , (Table 2). The salinity of the control group was 42.4%o.
The percentage of mortality was plotted against the corresponding log
salinity and LC50 was calculated at salinity 10%o (Fig. 16). It was observed
that mortality increased as the dilution increased.
Allen (1960) proved that 95% mortality of B. recturvus occurred at
salinities below 4.5%o after 19days.
Histopathological examination of the adult mussel of B. variabilis
exposed to a sub-lethal salinity (15%o) for one week showed deformation
in gill filaments. These were loss of some of the frontal cilia and
pronounced degeneration of the epithelial cells and dilatation of the
branchial veins (Fig. 17).
The cytoplasm of digestive tubule epithelia was markedly vaculated,
the digestive tubules appeared dilated, while the intertubular connective
tissue was ruptured (Fig. 18).
The ovary was affected as shown by deformation of most oogonia
(Fig. 19).

43
Table 2: Mortality (%) of B. variabilis at different salinities
Salinity No. of dead mussels after 96 hours*
Mortality
(%)salinity (%o)
log
salinity
Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
42.4 (control)
40.0
35.0
30.0
25.0
20.0
15.0
10.0
5.0
2.0
1.63
1.60
1.54
1.47
1.39
1.30
1.17
1.00
0.70
0.30
---
---
2
4
5
6
6
10
14
20
---
2
2
4
5
6
5
8
14
20
---
2
4
4
7
6
9
10
41
20
---
4
4
4
7
6
8
12
14
20
---
2
3
4
6
6
7
10
14
20
---
10%
15%
20%
30%
30%
35%
50%
70%
100%

45
variabilis exposed to sub-lethal salinity (15%o)
for one week, showing loss of some frontal cilia
(fci), pronounced degeneration of epithelial cells
(ec) and dilatation of branchial veins (bv).
(Bouing, Hx and E; X1030)
Fig. 18 photomicrograph of T.S. of the digestive gland
of B. variabilis exposed to sub-lethal salinity
(15%o) for one week, showing marked vaculation
of secretory cells (sc) and dilatation of digestive
tubules (dt). (Bouin, Hx and E; X1030).
variabilis exposed to sub-lethal salinity (15%o)
for one week, showing deformation of most
primary oocytes (oo1), secondary oocytes (oo2)
and mature ova (ov). (Bouin, Hx and E; X660).

47
iii. Effect of some Elements
1. Effect of calcium
The efficacy of different concentrations of calcium chloride on B.
variabilis was evaluated. Mortality (%) was determined together with the
logarithmic value of calcium chloride concentration. Percentage of
mortality was 10, 20, 30, 50, 70 and 100% with respect to different
concentrations 4.0, 6.0, 8.0, 10.0, 20.0, 40.0 and 80.0ppm (Table 3). The
resulting LC50 was 20.0ppm CaCl2 = 7.3ppm Ca (Fig. 20). It is worth
noting that control sample bears additional 776.6ppm Ca.
A transverse section in adult mussel of B. variabilis subjected to sub-
lethal dose of calcium chloride (15ppm) proclaimed loss of some of the
frontal cilia, necrosis of the endothelial cells together with some rupture in
the basement membranes of the gill filaments and dilatation of the
branchial veins (Fig. 21).
Digestive gland exhibited vaculation in cytoplasm of some epithelial
cells while other cells were sloughed. The intertubular connective tissue
was pronouncedly degenerated (Fig. 22).
Oogenic follicles were deformed. Thus, most of the primary and
secondary oocytes and the mature ova were degenerated, (Fig. 23).
However, the intercellular reserve of calcium in the present specimens
of B. variabilis was 173.0 and 4.27ppm for untreated mussles and those
treated with sub-lethal doses of calcium chloride respectively. This could
be attributed to extrusion of calcium reserve of B. variabilis, i.e. decrease
of calcium content. This in favour with that finding obtained by Stricker

48
(1999) where development of some mammals proceeded abnormally as
calcium level decreased.
Table 3: Mortality (%) of B. variabilis at different calcium chloride
concentrations
Calcium chloride
concentration
No. of dead mussels after 96 hours* Mortality
(%)
conc. (ppm) log conc. Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
Control
4.0
6.0
8.0
10.0
20.0
40.0
80.0
0.60
0.77
0.90
1.00
1.30
1.60
1.90
---
---
1
4
6
9
14
20
--
--
2
4
5
11
14
20
--
--
2
4
5
10
14
20
--
--
3
4
8
10
14
20
--
--
2
4
6
10
14
20
--
--
10%
20%
30%
50%
70%
100%

50
variabilis exposed to sub-lethal dose of calcium
chloride (15ppm), proclaiming loss of some
frontal cilia (fci), necrosis of endothelial cells
(ec) and dilatation of the branchial veins (bv).
(Bouin, Hx and E; X1030)
gland of B. variabilis exposed to sub-lethal
dose of calcium chloride (15ppm), showing
vaculation of some secretory cells (sc) and
degeneration of intertubular connective tissue
(ct). (Bouin, Hx and E; X825)
variabilis exposed to sub-lethal dose of calcium
chloride (15ppm), showing degeneration of
most of the primary oocytes (oo1), secondary
oocytes (oo2) and the mature ova (ov). (Bouin,
Hx and E; X660).

52
2. Effect of nickel
Toxicity of nickel sulfate to B. variabilis was studied at different
concentrations (4.0, 6.0, 8.0, 10.0, 20.0, 40.0 and 80.0ppm). The
corresponding moralities were 10, 30, 40, 50, 70, 90 and 100% (Table 4).
The data obtained showed that the calculated LC50 was 10.0ppm (Fig. 24).
The control sample initially has 0.009ppm Ni.
Calabrese and Nelson (1974) found that LC50 of nickel chloride after
48hours of the oyster Crassostrea virginica was 1.2ppm, while for the clam
Mercenaria mecenaria it was 5.7ppm after 48hours as recorded by
Calabrese et al. (1977).
Exposure of adult mussels of B. variabilis to sub-lethal dose of nickel
sulfate (8ppm) for one week produced loss of some of the frontal cilia and
destortion of the gill filaments (Fig. 25).
The digestive gland appeared more affected as shown by the destorted
epithelial cells and degenerated intertubular connective tissues. (Fig. 26).
Moreover, deformation of most of the primary oocytes of the ovary
was observed (Fig. 27).
Internal abnormalities including extrusion of tissues from the shells of
Crassostrea virginica and Mercenaria mercenaria, initially treated with
sub-lethal doses of nickel chloride were reported by Calabres et al. (1977).

53
Table 4: Mortality (%) of B. variabilis at different nickel sulfate
concentrations
Nickel sulfate
concentration
(%)
conc. (ppm) log conc. Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
Control
4.0
6.0
8.0
10.0
20.0
40.0
80.0
--
0.60
0.77
0.90
1.00
1.30
1.60
1.90
--
--
4
8
10
14
18
20
--
1
4
8
8
13
18
20
--
1
7
8
10
14
18
20
--
6
9
8
12
15
18
20
--
2
6
8
10
14
18
20
--
10%
30%
40%
50%
70%
90%
100%

55
variabilis treated with sub-lethal dose of
nickel sulfate (8ppm) for one week, showing
loss of some frontal cilia (fci) and destortion
of the gill filaments (gf). (Bouin, Hx and E;
X1030).
gland of B. variabilis treated with sub-lethal
dose of nickel sulfate (8ppm) for one week,
showing destorted epithelial cells (ec) and
degenerated intertubular connective tissue
(ct). (Bouin, Hx and E; X825).
variabilis treated with sub-lethal dose of
nickel sulfate (8ppm) for one week, showing
deformation of most primary oocytes (oo1).
(Bouin, Hx and E; X660).

57
3. Effect of zinc
Specimens of B. variabilis applying different concentrations of zinc
oxide were considered. The obtained mortality (%), zinc oxide
concentration (ppm) was 20%, 4.0ppm; 5%, 6.0ppm; 50%, 8.0ppm; 70%,
10.0ppm; 85%, 20.0ppm; 95%, 40.0ppm and 100%, 80.0ppm, (Table 5).
LC50 of zinc oxide was 8.0ppm (Fig. 28). Besides, normal seawater
contains 0.029ppm Zn.
Studies of Nelson et al. (1988) showed that LC50 of zinc chloride on
Spisula solidissima was 2.95ppm after 96 hours.
Adult mussels of B. variabilis exposed to sub-lethal dose of zinc oxide
(6ppm) for one week exhibited pronounced alteration in gills as observed in
loss of some of the frontal cilia, the epithelial cells were sloughed and
atrophy was observed in the branchial veins (Fig. 29).
Effect on digestive gland was in the form of vaculation in the cytoplasm
of digestive tubule cells together with marked dilatation of the digestive
tubules and the intertubular connective tissue was ruptured (Fig. 30).
In addition, different oogenic follicles of the ovary were extensively
deformed (Fig. 31).
On the other hand, Tolba et al. (1991) found a reduction in total protein
content due to toxication by Cd and Zn in the marine isopod Sphaeroma
serratum. They also suggested that the reduction brought about by heavy
metals could result in disturbance in the functioning of the internal organs as a
consequence of structural damage. Yan et al. (1996) reported that sub-lethal
concentrations of Cd, Zn and Hg inhibited the mean enzyme activities of the

58
mussel Perna viridis which is considered as biofouling mussel. Thus, the
resulted abnormalities in gills, digestive gland and ovary of B. variabilis
could be attributed to a similar structural damage caused by zinc.
Table 5: Mortality (%) of B. variabilis at different zinc oxide
concentrations
Zinc oxide concentration No. of dead mussels after 96 hours* Mortality
(%)conc. (ppm) log conc. Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
Control
4.0
6.0
8.0
10.0
20.0
40.0
80.0
0.60
0.77
0.90
1.00
1.30
1.60
1.90
--
4
7
11
13
17
20
20
--
5
7
9
14
16
18
20
--
2
7
9
15
17
18
20
--
5
7
11
14
18
20
20
--
4
7
10
14
17
19
20
--
20%
35%
50%
70%
85%
95%
100%

60
variabilis exposed to sub-lethal dose of zinc
oxide (6ppm) for one week, illustrating loss of
some frontal cilia (fci) and sloughed epithelial
cells (ec) and atrophy of branchial veins (bv).
(Bouin, Hx and E; X1030)
dose of zinc oxide (6ppm) for one week,
showing vaculation of epithelial cells (ec) and
dilatation of digestive tubules (dt). (Bouin, Hx
and E; X825).
variabilis exposed to sub-lethal dose of zinc
oxide (6ppm) for one week, indicating
extensive deformation of oogenic follicles
(of). (Bouin, Hx and E; X660).

62
4. Effect of lead
Mortality (%) of B. variabilis was determined at different lead nitrate
concentrations. Mortality (%), lead nitrate concentration (ppm) were 10%,
4.0ppm; 25%, 6.0ppm; 40%, 8.0ppm; 50%, 10.0ppm; 80%, 20.0ppm; 90%,
40.0ppm and 100%, 80.0ppm. (Table 6). This data indicated that the
elucidated LC50 of lead nitrate was 10.0ppm (Fig. 32). It is to be mentioned
that control sample includes 0.096ppm Pb.
Results of Calabrese et al. (1974) found that LC50 of lead nitrate on
Mercenaria mercenaria was 0.78ppm after 48hours. Moreover, Awad
(1999) showed that LC50 of lead nitrate on Corbicula fluminea was
32.0ppm after one week.
Histologically, the gills of B. variabilis exposed to a sub-lethal
concentration of lead nitrate (8ppm) for one week are slightly affected.
This resulted in loss of the frontal cilia, rupture in some frontal cells and
slight dilatation of the branchial veins (Fig. 33).
Similar observations were achieved by Sunila (1988) after subjecting
Mytilus edulis to lead at a concentration of 5ppm for two weeks.
Decay of both the digestive tubules and the intertubular connective
tissue was observed in the digestive gland (Fig. 34).
Besides, increased destortion of a marked number of mature ova
occurred in the ovary (Fig. 35).
Effect of Pb on growth of another fouling agent namely Corbicula
fluminea was studied by Awad (1999). Thus, exposure of this clam for one
week using a dose of 16ppm (1/2LC50) caused a moderate vaculation in the

63
cytoplasm of the secretory cells, while vaculation was slight in cytoplasm
of the oogenic stages.
Table 6: Mortality (%) of B. variabilis at different lead nitrate
concentrations
Lead nitrate
concentration
Mortality
(%)conc.
(ppm)
log conc. Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
Control
4.0
6.0
8.0
10.0
20.0
40.0
80.0
0.60
0.77
0.90
1.00
1.30
1.60
1.90
--
--
4
7
9
16
19
20
--
--
5
7
10
16
19
20
1
2
5
9
11
16
19
20
--
6
6
9
10
16
19
20
--
2
5
8
10
16
19
20
--
10%
25%
40%
50%
80%
95%
100%

65
variabilis subjected to sub-lethal dose of lead
nitrate (8ppm) for one week, showing loss of
some frontal cilia (fci) rupture in some frontal
cells (fc) and slight dilatation of branchial
veins (bv). (Bouin, Hx and E; X1030).
gland of B. variabilis subjected to sub-lethal
dose of lead nitrate (8ppm) for one week,
showing decayed digestive tubules (dt) and
intertubular connective tissue (ct). (Bouin, Hx
and E; X825).
variabilis subjected to sub-lethal dose of lead
nitrate (8ppm) for one week, indicating
destortion of mature ova (ov). (Bouin, Hx and
E; X660).

67
iv. Effect of Molluscicides
1. Effect of gesapax (ametryn)
The activity of ametryn (formulated as gesapax) against B. variabilis
was evaluated. The mortality (%) applying different doses was 10%,
80.0ppm; 25%, 100.0ppm; 40%, 120.0ppm; 50%, 140ppm; 80%,
160.0ppm; 90%, 180.0ppm; 95%, 200.0ppm and 100%, 250.0ppm (Table
7). Plotting percentage of mortality against log concentration of ametryn
indicated that LC50 was 140.0ppm (Fig. 36).
Exposing adult mussels of B. variabilis to 1/2LC50 of ametryn for one
week revealed loss of some of the frontal cilia, disruption in some
endothelial cells and marked atrophy in the branchial veins of different gill
filaments (Fig. 37).
On the other hand, extensive vaculation in cytoplasm of digestive
tubule cells, slight degeneration in the basement membranes and ruptures in
the intertubular connective tissue were produced in the digestive gland
(Fig. 38).
Moreover, deformation in a marked number of primary oocytes
appeared in the ovary (Fig. 39).

68
Table 7: Mortality (%) of B. variabilis at different concentrations of
ametryn (gesapax)
Ametryn concentration No. of dead mussels after 96 hours* Mortality
1
Replicate
2
Replicate
3
Replicate
4
average
Control
80.0
100.0
120.0
140.0
160.0
180.0
200.0
250.0
--
1.90
2.00
2.07
2.12
2.220
2.25
2.30
2.39
--
--
6
8
10
16
17
18
20
--
2
4
8
10
15
18
19
20
--
2
4
8
10
16
18
19
20
--
4
6
8
10
17
19
20
20
--
2
5
8
10
16
18
19
20
--
10%
25%
40%
50%
80%
90%
95%
100%

70
variabilis treated with 1/2LC50 of ametryn
for one week, illustrating loss of some frontal
cilia (fci), disruption in some endothelial
cells (e) and atrophy of branchial veins (bv).
gland of B. variabilis treated with 1/2LC50 of
ametryn for one week, showing extensive
vaculation in epithelial cells (ec), slight
degeneration in the basement membrane
(bm) and rupture of the intertubular
connective tissue (ct). (Bouin, Hx and E;
X1030).
variabilis treated with 1/2LC50 of ametryn
for one week, showing deformation of
primary oocytes (oo1). (Bouin, Hx and E;
X660).

72
2. Effect of uccmaluscide (niclosamide monoethanolamine salt)
The average number of dead mussels was determined after exposure
to different concentrations of uccmaluscide. Mortality (%), uccmaluscide
concentration (ppm) were 30%, 1.0ppm; 35%, 1.5ppm; 50%, 2.0ppm; 75%,
2.5ppm; 80%, 3.0ppm and 100ppm, 3.5ppm (Table 8). Lethal dose (LC50)
of uccmaluscide was 1.8ppm (Fig. 40).
Abdel-Rahman et al. (1988) reported that LC50 of bayluscide (another
commercial formula of niclosamide amine salt) on Physa acuta was 0.8ppm.
This biocide was extensively used for combating schistozomiasis against
Biomphalaria alexandrina and Bulinus truncatus (Nabih and Metri, 1973;
Emara, 1994). In fact, niclosamide monoethanolamine salt was not evaluated
for its molluscicidal efficacy against fouling. In addition, biodegradation of
this compound through 48hours (Muir and Yavechewski, 1982) renders it
advantageous to other molluscicides in particular when applied in seawater.
Adult mussels of B. variabilis treated with 1/2LC50 of niclosamide
monoethanolamine salt (uccmaluscide) for one week indicated that gills
were extensively affected, this appeared in loss of nearly all the frontal cilia
and the gill filaments were severely deformed losing their normal
architecture (Fig. 41).
Digestive gland showed slight vaculation in cytoplasm of the
secretory cells and rupture in the intertubular connective tissue (Fig. 42).
Treatment of Lymnaea glabra with niclosamide revealed different
results as the necrosis of epithelial cells of the digestive gland (Rondelaud
and Dreyfuss, 1996).

73
On the other hand, severe decay of the oogenic follicles of the ovary
took place (Fig. 43).
niclosamide monoethanolamine salt (uccmaluscide)
Uccmaluscide
concentration
Mortality
(%)
conc.
(ppm)
log conc. Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
Control
1.00
1.50
2.00
2.50
3.00
3.50
0.00
0.18
0.30
0.40
0.48
0.60
--
6
6
15
14
14
20
--
5
6
17
15
15
20
--
7
8
15
15
17
20
--
6
8
1
16
18
20
--
6
7
15
15
16
20
--
30%
35%
75%
75%
80%
100%

75
variabilis subjected to sub-lethal dose of
uccmaluscide (1/2LC50) for one week,
showing loss of some frontal cilia (fci) and
severe deformation of gill filaments (gf)
losing its architecture. (Bouin, Hx and E;
X1030).
gland of B. variabilis subjected to sub-lethal
dose of uccmaluscide (1/2LC50) for one
week, indicating slight vaculation in
secretory cells (sc) and rupture of the
intertubular connective tissue (ct). (Bouin,
Hx and E; X1030).
variabilis subjected to sub-lethal dose of
uccmaluscide (1/2LC50) for one week,
showing severe decay of oogenic follicles
(of). (Bouin, Hx and E; X660).

77
3. Effect of cetyl trimethylammonium chloride
The percentage of mortality of B. variabilis obtained when applying
different doses of cetyl trimethylammonium chloride were in the order
10%, 25.0ppm; 15%, 50.0ppm; 30%, 100ppm; 70%, 150.0ppm; 80%,
200.0ppm and 100%, 250.0ppm (Table 9). The elucidated LC50 was
140.0ppm (Fig. 44).
However, Fellers et al. (1992) reported that LC50 of Dumen C
(a quaternary ammonium compound) against Dreissena polymopha was
100% at 5ppm after 4days.
Examinations of adult mussels of B. variabilis after exposure to
1/2LC50 of cetyl trimethylammonium chloride for one week illustrated that
some frontal cilia were lost and necrosis of the endothelial cells was
produced (Fig. 45).
Sections of digestive gland revealed some vaculation in digestive
tubule epithelia and disruption in the intertubular connective tissue
(Fig. 46).
The ovary was pronouncedly affected that is destortion of a marked
number of secondary oocytes and mature ova was observed (Fig. 47).

78
cetyltrimethylammonium chloride
Cetyltrimethyl-
ammonium chloride
concentration
Mortality
(%)conc.
(ppm)
log conc. Replicate
1
Replicate
2
Replicate
3
Replicate
4
average
control
25.0
50.0
100.0
150.0
200.0
250.0
1.39
1.69
2.00
2.17
2.30
2.39
---
2
2
6
15
17
20
---
2
2
6
14
16
20
---
2
4
6
13
16
20
---
2
4
6
14
15
20
---
2
3
6
14
16
20
---
10%
15%
30%
70%
80%
100%

80
variabilis exposed to sub-lethal concentrations
of cetyl trimethylammonium chloride, (1/2LC50)
for one week, showing loss of some frontal cilia
(fci) and necrosis of endothelial cells (ec).
concentrations of cetyl trimethylammonium
chloride, (1/2LC50) for one week, showing
vaculation in epithelial cells (ec) and
disruption in the intertubular connective tissue
(ct). (Bouin, Hx and E; X1030).
variabilis exposed to sub-lethal concentrations of
cetyl trimethylammonium chloride, (1/2LC50) for
one week, indicating destortion of secondary
oocytes (oo2) and mature ova (ov). (Bouin, Hx
and E; X1030).

82
4. Effect of copper sulfate
Data obtained (Table 10) showed that mortality (%) together with the
corresponding copper sulfate concentration (ppm) were 20%, 1.0ppm;
50%, 1.5ppm; 55%, 2.0ppm; 60%, 2.5ppm; 65%, 3.0ppm; 80%, 3.5ppm;
85%, 4.0ppm; 90%, 4.5ppm and 100%, 5.0ppm. LC50 of copper sulfate was
1.5ppm (Fig. 48).
In Mytilus edulis, LC50 of copper had different values: 0.122, 0.25 and
22.3ppm (Wisely and Blick, 1967; Davenport, 1977; Nelson et al. 1988)
respectively. Portman (1972) determined LC50 of copper in Cardium edula
as 1.0ppm while in Caelatura teretiusculua it was 10.00ppm (Saad and
Emam, 1998).
Treating the adult mussels of B. variabilis with ½ LC50 of copper
sulfate for one week showed that gills were slightly affected where some of
the frontal cilia were lost with vaculation in the cytoplasm of epithelial
cells and dilatation of the branchial veins (Fig. 49).
Atkins (1931a) reported rupture of epithelial cells on exposing M.
edulis to copper. Other findings were attained including swollen epithelial
cells (Engel and Fowler, 1979), cellular disruption (Sunila, 1981; Pickwell
and Steinert, 1984). While, Sunila (1986) reported loss of frontal cilia and
vaculation in epithelial cells of the same species after exposure to copper.
Digestive gland was slightly affected, a slight rupture in some lining
epithelia of digestive tubules and appearance of dilated digestive tubules
were observed (Fig. 50).

83
These results agree with those of Calabrese et al. (1984) on M. edulis,
but varied from those of Fujiya (1960) and Martin (1971) where necrosis
and sloughing of epithelial cells of digestive gland of the same species took
place.
The ovary was severely affected where the majority of mature ova
were extensively deformed (Fig. 51).
Calabrese et al. (1984) observed that copper (as cupric chloride) led
to a little follicular development, both in size and number in the ovary of
M. edulis.

84
copper sulfate
Copper sulfate
concentration
1
Replicate
2
Replicate
3
Replicate
4
average
Control
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
0.00
0.17
0.30
0.39
0.47
0.54
0.60
0.65
0.71
---
2
9
11
1
12
16
14
17
20
---
2
10
11
12
15
16
17
17
20
---
2
10
11
12
13
16
18
19
20
---
2
11
11
12
12
16
18
19
20
---
4
10
11
12
13
16
17
18
20
---
20%
50%
55%
60%
65%
80%
85%
90%
100%

86
of copper sulfate, (1/2LC50) for one week,
showing loss of some frontal cilia (fci),
vaculation in the epithelial cells (ec) and
dilatation of the branchial veins (bv). (Bouin,
Hx and E; X825).
concentrations of copper sulfate, (1/2LC50) for
one week, showing slight rupture in epithelial
cells (ec) and dilatation of digestive tubules
(dt). (Bouin, Hx and E; X825).
of copper sulfate, (1/2LC50) for one week,
showing deformation of majority of mature
ova (ov). (Bouin, Hx and E; X660).

88
v. Test of some combined parameters
Sub-lethal values of some physical parameters (pH, salinity and Ca)
were used in combination to evaluate its toxicity and the related
histopathological alterations of the tested organs of B. variabilis. The
applied pH value was 8.5 which are basic since the acidic values would not
be used to avoid their corrosive effect on the cooling towers in petroleum
refineries. Salinity was 15%o and calcium chloride dose was 15ppm.
Calcium was chosen due to its lower toxicity to the non-target organisms in
comparison with the rest of the selected elements (Ni, Zn and Pb).
Regarding these viewpoints, a combination between the three mentioned
parameters was designed to obtain the task of the present work that is to
control physically with least toxicity. Thus, sub-lethal doses of the selected
parameters caused 70% mortality after 96 hours. It is to be noticed that
each of the tested parameters (pH, salinity and calcium) was individually
less toxic (pH, 40%; salinity, 35%; calcium, 40%). This could be attributed
to synergism between the different parameters.
Histological examination of B. variabilis subjected to sub-lethal doses
of pH, salinity and calcium for one week resulted in the expected effects on
gills, digestive gland and ovary. Loss of frontal cilia, degeneration of some
endothelial cells and destortion of the branchial veins occurred in gills
(Fig. 52). The lining epithelia of digestive tubules were vaculated (Fig. 53).
Besides, most oogenic stages of the ovary were deformed (Fig. 54).

89
In general, combination of the three parameters enhanced
deterioration of the gills and ovary while the digestive gland was relatively
antagonized the combined efficacy of the three parameters.
variabilis subjected to some combined
parameters (pH, 8.5; salinity, 15%o; Ca,
15ppm) for one week, showing loss of some
frontal cilia (fci), degeneration of some
endothelial cells (ec) and dilatation of the
branchial veins (bv). (Bouin, Hx and E;
X1030).
gland of B. variabilis subjected to some
combined parameters for one week,
illustrating vaculation of secretory cells (sc).
variabilis exposed to some combined

90
parameters for one week, showing increased
deformation of primary oocytes (oo1),
secondary oocytes (002) and mature ova (ov).

91
However, Histological study (Table 11) proved that gills showed severe
deformation with nickel, zinc and uccmaluscide (sub-lethal doses). Thus,
loss of some cilia was generally remarked except with uccmaluscide most of
cilia disappeared. Alterations in branchial veins graded from dilatation to
atrophy.
Severe alterations in the digestive gland occurred when sub-lethal
treatments of pH, Ni and Pb were applied. In other words, the lining
epithelia of the digestive tubules sufferd extensive vaculation in case of pH,
Ni and Pb, also Ca and gesapax (sub-lethal treatments). The intertubular
connective tissue was nearly sloughed on exposure to sub-lethal
concentrations of pH, Ni and Pb also Ca and gesapax. The oogenic stages
revealed severe deformation with sub-lethal doses of Ca, Zn and
uccmaluscide and copper sulfate.
In fact, histopathological evaluation of the effect of the different
physicochemical parameters on B. variabilis proved that none of these
parameters had a pronounced toxicity on the three organs. Thus, sub-lethal
concentration of each parameter resulted in the following generalizations:
1. pH value: destortion of digestive gland as compared to gill and ovary.
2. Salinity: marked deformation of ovary while gill filaments and
digestive tubules were moderately affected.
3. Calcium: oogenic follicles showed advanced deformation whereas,
gill filaments and digestive tubules exhibited marked deformation.

92
4. Nickel: severe destortion of gill filaments and digestive tubules while
ovary developed mild alterations.
5. Zinc: sharp disforming of gill filaments and oogenic follicles, on the
other hand, the digestive tubules were not markedly affected.
6. Lead: severe necrosis of digestive tubule epithelia whilst oogenic
follicles and gill filaments were clearly disformed.
7. Gesapax: legible decay in digestive tubules and gill filaments was
proclaimed, whereas about half the oogenic stages in the follicle were
destorted.
8. Uccmaluscide: normal architecture was lost in gill filaments and
oogenic follicles. Digestive tubules were slightly affected.
9. Cetyl trimethylammonium chloride: mild disforming of gills,
digestive gland and ovary.
10. Copper sulfate: gill filaments were disnatured, digestive tubule was
slightly affected, whereas the ovary was markedly destorted.
11. Test under some combined parameters: the expected lesion due to
combination of the different optimum conditions was not realized.
This may be similar to antagonism of biocides. Thus, a pronounced
disformation was observed in gills and ovary while digestive gland
was slightly disformed.
In general, the foregoing data have confirmed that uccmaluscide,
nickel and zinc shared the first rank when regarding their efficacy against
the investigated organs of B. variabilis. Similarly, Walne (1970) and
Brereton et al. (1970) found that zinc inhibited growth and developed
remarked abnormalities in Ostrea edulis. Also, Calabrese et al. (1977)
reported extreme reduction of growth and even tissue extrusion in

93
Mercenaria mercenaria. Application of sub-lethal doses of niclosamide
(active ingredient in uccmaluscide) resulted in tissue lesions in digestive
gland and gonads of Lymnae glabra (Rondeland and Dreyfuss, 1996).

95
However, seawater has a fundamental task in cooling water systems.
Thus, it is worthnoting to emphasize that the acute toxicity of nickel and
zinc should be carefully considered. This renders the moderate toxicity of
pH acquires a special interest in order to keep the environmental balance,
i.e. growth of the mussel B. variabilis is expected to be controlled
physically via adjustment of pH of seawater. Besides, uccmaluscide should
be taken in consideration on the basis of its higher toxicity to B. variabilis,
in addition to its biodegradability.

96
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Physicochemical parameters Physicochemical parameters
Physicochemical parameters
physicochemical parameters

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