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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.

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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:

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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.

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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.

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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.

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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.

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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

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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.

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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.

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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)

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(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,

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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.

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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).

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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.

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iv. Temperature

The highest surface water temperature was recorded in summer season

as 29.0oC followed by 21.0, 19.0 and 26.0oC on autumn, winter and spring

respectively, (Fig. 4). Average temperature was 23.5oC at the investigated

area of Suez Gulf.

Meshal (1967) found that the temperature at Suez was 28.4oC during

September 1967. Also, Hamed (1992) showed that the temperature was

17.5oC in winter and 28.5oC 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.

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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.

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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

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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

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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.

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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

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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



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


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 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

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 No. of dead mussels after 96 hours*

Mortality

(%) conc.


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

No. of dead mussels after 96 hours* Mortality

(%) conc.


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



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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