Environmental Toxicology and Pharmacology 57 (2018) 115–130

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Environmental Toxicology and Pharmacology

journal homepage: www.elsevier.com/locate/etap

Review or Mini-review

Antifouling processes and toxicity effects of antifouling paints on marine T

environment. A review
⁎
Intissar Amaraa, , Wafa Mileda, Rihab Ben Slamab, Neji Ladharic
a
Textile Engineering Laboratory, University of Monastir, Tunisia
b
Laboratory of Analysis, Treatment and Valorization of Pollutants of the Environment and Products, Faculty of Pharmacy, University of Monastir, Tunisia
c
Higher Institute of the Fashion Trades of Monastir, University of Monastir, Tunisia

A R T I C L E I N F O A B S T R A C T

Keywords: The production infrastructure in aquaculture invariably is a complex assortment of submerged components with
Antifouling agents cages, nets, floats and ropes. Cages are generally made from polyamide or high density polyethylene (PEHD). All
Biofouling of these structures serve as surfaces for biofouling. However, cage nets and supporting infrastructure offer
Fish fouling organisms thousands of square meters of multifilament netting. That's why, before immersing them in
Invertebrate
seawater, they should be coated with an antifouling agent. It helps to prevent net occlusion and to increase its
Crustacean
lifespan. Biofouling in marine aquaculture is a specific problem and has three main negative effects. It causes net
Algae
occlusion and so restricts water and oxygen exchange. Besides, the low dissolved oxygen levels from poor water
exchange increases the stress levels of fish, lowers immunity and increases vulnerability to disease. Also, the
extra weight imposed by fouling causes cage deformation and structural fatigue. The maintenance and loss of
equipment cause the increase of production costs for the industry. Biocides are chemical substances that can
prohibit or kill microorganisms responsible for biofouling. The expansion of the aquaculture industry requires
the use of more drugs, disinfectants and antifoulant compounds (biocides) to eliminate the microorganisms in
the aquaculture facilities. Unfortunately, the use of biocides in the aquatic environment has proved to be harmful
as it has toxic effects on the marine environment. The most commonly used biocides in antifouling paints are
Tributyltin (TBT), Chlorothalonil, Dichlofluanid, Sea-Nine 211, Diuron, Irgarol 1051 and Zinc Pyrithione.
Restrictions were imposed on the use of TBT, that's why organic booster biocides were recently introduced. The
replacement products are generally based on copper metal oxides and organic biocides. This paper provides an
overview of the effects of antifouling biocides on aquatic organisms. It will focus on the eight booster biocides in
common use, despite little data are available for some of them. Toxicity values and effects of these antifoulants
will also be mentioned for different species of fish, crustaceans, invertebrates and algae.

1. Introduction biocide in water, therefore inhibiting the development of fouling

Marine biological fouling, usually termed marine biofouling, can be
defined as the accumulation of microorganisms, plants, and aquatic
animals on artificial surfaces immersed in sea water (Yebra et al.,
2004). In the case of ships, biofouling is an unwanted phenomenon
which may cause several problems such as increased fuel consumption
due to water resistance, as well as increase in weight; in aquaculture,
reduction of water exchange through net mesh, occurs (Champ, 2000;
Cronin et al., 1999; Eckman et al., 2001; Phillippi et al., 2001).
To prevent the attachment of fouling organisms, antifouling paints
have been developed and used (Koutsaftis and Aoyama, 2007). They
contain chemical compounds (biocides), which are released from the
paint matrix, to provide a constant threshold concentration of the
communities (Boxall et al., 2000; Terlizzi et al., 2001). Tributyltin self-
polishing copolymer paints (TBT-SPC paints), such as tributyltin oxide
(TBTO: C24H54OSn2) and tributyltin fluoride (TBTF: C12H27SnF) are the
most successful compounds against biofouling. They belong to orga-
notin biocides which contain at least one tin-carbon bond (Ingham
et al., 1960; Omae, 2003). Unfortunately, TBT-SPC systems adversely
affect the environment (Yebra et al., 2004). Due to its high toxicity to
molluscs, fish reproduction and fish behavior at very low concentra-
tions (Alzieu et al., 1980; Antizar-Ladislao, 2008; Bao et al., 2011;
Dimitriou et al., 2003; Fent, 1991; Hongxia et al., 1998), the use of
tributyltin (TBT) has been restricted since the early 1990s and marine
paint companies have been developed alternatives to antifouling to
substitute TBT (Alzieu, 2000).

⁎
Corresponding author.
E-mail addresses: intissar.amara@hotmail.fr (I. Amara), w.miledbenltoufa@gmail.com (W. Miled), rbs_23@yahoo.fr (R.B. Slama), neji.ladhari@isetkh.rnu.tn (N. Ladhari).

https://doi.org/10.1016/j.etap.2017.12.001
Received 16 May 2017; Received in revised form 3 October 2017; Accepted 3 December 2017
Available online 08 December 2017
1382-6689/ © 2017 Elsevier B.V. All rights reserved.
I. Amara et al.
Currently, new alternatives to antifouling paints are based on
copper compounds such as cuprous oxide (Cu2O) and copper thiocya-
nate (CuSCN), with supplementation of booster biocides to control Cu
resistant fouling organisms (Guardiola et al., 2012; Voulvoulis, 2006).
Commercial boosters such as Irgarol 1051, Sea-Nine 211, Diuron,
Chlorothalonil, and other metallic compounds like zinc pyrithione
(ZnPT) and copper pyrithione (CuPT) are the most commonly used
booster biocides (Konstantinou and Albanis, 2004). These biocides are
intended to be environmentally less harmful compared to the organotin
biocides. However, the problem of toxicity on several marine species
remains (Bejarano et al., 2005; Braithwaite and Fletcher, 2005;
Jacobson and Willingham, 2000; Ma et al., 2002; Mochida et al., 2010;
Sherrard et al., 2003). In addition, the environmental effects such as
toxicity and persistence of these biocides are not understood, as they
have only been recently introduced (Maraldo and Dahllöf, 2004;
Terlizzi et al., 2001).
This review is a comparative study of acute toxicities among TBT,
copper and the six commonly used booster biocides (Chlorothalonil,
Sea-Nine 211, Irgarol 1051, Zinc Pyrithione, Dichlofluanid and Diuron)
on marine species essentially invertebrate, algae, fish and crustacean.
Toxicity will be evaluated with numerous concentrations such as LC50,
LD50, EC50, LOEC and NOEC. All these toxicological dose descriptors
will be detailed in the following sections.
2. Marine biofouling problems
When a pristine object is placed in seawater, it is not long before
fouling with vegetable and animal organisms becomes a significant
problem. In the case of ships, the adverse effects caused by this biolo-
gical settlement are well known. Firstly, it produces high frictional re-
sistance, combined with increased of weight, which reduces speed and
maneuverability, as well as increasing fuel consumption by up to 40%
(Champ, 2000).
In the case of aquaculture field, biofouling causes two problems:
firstly, fouling communities constrict net openings, and left unchecked,
this leads to a significant increase in the weight of netting (Phillippi
et al., 2001). In the other hand, it reduces the flow through fouled net
mesh or tray perforations so restricting nutrient exchange, removal of
waste products and oxygen supply (Cronin et al., 1999; Eckman et al.,
2001). All of these factors can affect the health of fish stock and also
impact the local environment (Folke et al., 1997).
3. Marine biofouling details
The colonization of a substratum in the aquatic realm has been
viewed as proceeding through a four- step process (Maki and Mitchell,
2003; Wahl, 1997):
– Primary film formation
– Biofilm formation
– Diatom and protozoan colonization
– Settlement of invertebrate larvae and algal spores
This scenario can be modeled as shown in Fig. 1:
3.1. Primary film formation
Biofouling usually begin by an abiotic surface conditioning, i.e. the
formation of a non −living chemical/biochemical film (Characklis,
1981; Marshall et al., 1971; Mitchell and Kirchman, 1984). This process
starts with a spontaneous and rapid adsorption of organic molecules
already present in the water, such as proteins, polysaccharides, nucleic
acids, humic acids and possibly inorganic compounds onto the sub-
strate. It will be achieved within minutes of exposure of surfaces to
seawater (Abarzua and Jakubowski, 1995; Callow and Fletcher, 1994).
Through the modification of the physicochemical properties of the
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Environmental Toxicology and Pharmacology 57 (2018) 115–130
surface, this film promotes bacterial adhesion on it by constituting a
source of nutrients and specific interaction between bacteria and or-
ganic molecules (Dunne, 2002; Walker and Marsh, 2004).
3.2. Biofilm formation
The figure below (Fig. 2) presents the different growth times of the
biofilm using the SEM (Fernández et al., 2008):
The formation of the biofilm is a process taking place in several
stages as shown in Fig. 3 (Brian-Jaisson, 2014):
– The first step is the transport of bacteria to the surface. Bacteria are
transported to the conditioned area due to many factors including
gravity, Brownian movement, diffusion, dynamics of fluids, elec-
trostatic interactions and cell mobility (Fig. 3-a) (Bos et al., 1999;
Carpentier and Cerf, 1993; Dunne, 2002; Harbron and Kent, 1988;
Palmer et al., 2007; Wahl, 1989).
– The second step is termed reversible adhesion. When the bacteria
approach a conditioned surface, weak interactions occur (Van Der
Waals attraction, electrostatic and hydrophobic interactions) be-
tween the bacteria and the support. These interactions lead to par-
tial immobilization of the bacteria on the surface, and it can be
detached simply by rinsing or by shear conditions (Fig. 3-b) (Dunne,
2002; Harbron and Kent, 1988).
– The third step is called irreversible adhesion. After the bacteria in-
itially interact with the primary film, permanently attachment oc-
curs in minutes through their production of extracellular polymeric
substances (EPS). The existence of these adhesive exudates and the
roughness of irregular microbial colonies help to trap more particles
and organisms (Fig. 3-c) (Wahl, 1989; Yebra et al., 2004).
– The fourth step follows in days, and is termed maturation and dis-
persion. This involves the settlement and the growth of larger
marine invertebrates together with the growth of macroalgae (sea-
weeds) (Yebra et al., 2004). Thanks to nutrients present in the
conditioned film and those present in the surrounding fluid, the
biofilm is developed by involving several mechanisms such as
binary division, mobility on the surface and co-adhesion (Fig. 3-d)
(Hall-Stoodley and Stoodley, 2002; Kumar and Anand, 1998). At an
advanced stage of maturation of the biofilm, individual cells or parts
of the biofilm can be separated from the heterogeneous mass due to
the decrease of nutrients, to the occurrence of anaerobic conditions,
or under the effect of shear forces or other environmental stress.
This step is illustrated in Fig. 3-e (Dunne, 2002; Walker and Marsh,
2004).
3.3. Diatom and protozoan colonization
The settlement of unicellular eukaryote typically begins within days
to weeks after immersion of new substratum. These cells are subjected
to the same physical forces as bacteria. But due to larger cell size and
higher motility, their contribution to the adsorption process relative to
behavioral aspects should be smaller. After contacting the substratum,
the cells attach with polysaccharide or protein glues to biofilm, con-
ditioning film or substratum surface (Wahl, 1997).
3.4. Settlement of invertebrate larvae and algal spores
This is the longest and final step in biofouling process. It needs
several days to weeks after biochemical conditioning. It involves the
settlement and the growth of larger marine invertebrates together with
the growth of macroalgae (Fig. 4). Additionally to the roughness of the
irregular microbial colonies, the existence of adhesive exudates (EPS),
such as polysaccharides, proteins, lipids and nucleic acids helps to trap
more particles and organisms. These are likely to include algal spores,
marine fungi and protozoa (Yebra et al., 2004).
I. Amara et al.
Fig. 1. Schematic representation of fouling formation sequences.
4. Toxicological dose descriptors
A dose descriptor is the term used to identify the relationship be-
tween a specific effect of a substance and the dose at which it takes
place. Dose descriptors are determined in the toxicological studies on
the hazards of the substance and are usually expressed as LC50, LD50,
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Environmental Toxicology and Pharmacology 57 (2018) 115–130
Fig. 2. Photographs of biofilm at different growth
times using the scanning electron microscopy (SEM).
Fig. 3. Schematic representation of formed biofilms.
NOEL, NOEC, EC50, etc. We will summarize the definition of common
toxicology dose descriptors (Brunton et al., 2011; Crane and Newman,
2000):
– Half Lethal Concentration (LC50) presents the concentration at
which half of the sample population of a specific test-animal dies, in
I. Amara et al.
a specified period from exposure via inhalation or respiration.
– Median Lethal Dose (LD50) presents the amount of a material, given
all at once, causing the death of 50% of a group of test animals.
– Median Effective Concentration (EC50) represents the concentration
of a compound where 50% of the population exhibits a response,
after specified exposure duration.
– No Observable Effect Concentration (NOEC) is the highest con-
centration in a test with a mean response. Statistically, this de-
scriptor does not differ significantly from the mean response of the
control.
– Low Observable Effect Concentration (LOEC) is the lowest test
concentration having a mean response that differs significantly from
that of the control.
5. Chemical antifouling methods in aquaculture field
Biofouling has been recognized as problematic for more than 2000
years, and many kinds of antifouling methods have been investigated
(Callow, 1990; Yebra et al., 2004). The early Phoenicians have been
credited with the first advance in the form of lead and copper sheets to
prevent biofouling on their wooden boats. By the late 18th and into the
19th centuries, coatings containing copper, arsenic and mercury were
increasingly applied to vessel hulls (Dafforn et al., 2011). Since the late
20th century, organotin compounds and their derivatives have been
widely used as antifouling coatings thanks to their effectiveness against
a wide range of fouling species. Organotin compounds are substances in
which at least one tin-carbon bond is present (Ingham et al., 1960). The
most popular ones used as antifoulants are tributyltin oxide (TBTO:
C24H54OSn2) and tributyltin fluoride (TBTF: C12H27SnF). These com-
pounds are powerful fungicides, and will completely inhibit the growth
of most fouling organisms at a very low concentration (Omae, 2003).
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Environmental Toxicology and Pharmacology 57 (2018) 115–130
Fig. 4. Photographs (taken at the fish farm “RAFAHA” located in
Tunisia) showing the settlement of algal spores on nets.
Fig. 5. Penis development in female gastropod
Ocenebra erinacea, as soon as the TBT concentration
in the water exceeds 1 ng/L.
5.1. The classical process: antifouling by organotin compounds
These coatings can be classified into three categories, based on the
chemical properties of the paint matrix and mechanisms involved in
releasing toxic compounds. For conventional antifouling paints, the
matrix is usually a water-soluble resin, and the toxic compound is
available at the coating surface. The biocides are oxides of lead, arsenic,
mercury or copper, and their lifespan is as short as 6–12 months. For
Long-life antifouling paints, the matrix must be insoluble in water. High
quantities of toxicants remain in the paint, but the concentration at the
surface falls below the effective level, and coatings have to be replaced
after 18–24 months. Finally, for Self-polishing antifouling paints, the
biocide, mostly TBT, is bound to the polymeric matrix and is released
by hydrolysis at the paint surface. The rate of release is constant but
depends on water movement. Moreover, its life usually ranges between
4 and 5 years (Terlizzi et al., 2001). However, TBT and its related
agents were found to be highly toxic because of their lipophilicity,
which allows them to penetrate the biological membranes (Champ and
Pugh, 1987). The impact of the organotins on marine organisms in-
duced many governments to restrict its use. For example, in 1982,
France has banned the application of TBT-based antifouling paints on
vessels less than 25 meter long, when several oyster farms experienced
major declines due to reduced oyster growth, anomalies in larval de-
velopment, and shell malformation affecting 80–100% of individual
oysters (Alzieu et al., 1986; Terlizzi et al., 2001). Intersex effects were
identified as another negative effect of TBT contamination (Gibbs and
Bryan, 1986). In Fig. 5, we can observe the development of a penis in
female gastropod Ocenebra erinacea at only 1 ng/L of TBT (Compère
and Quiniou, 2009).
The restriction on the use of TBT, mercury and arsenic is leading to
a renewed use of old-fashioned copper-based paints and/or to the use of
new paints incorporating high booster levels of copper (Terlizzi et al.,
2001).
I. Amara et al. Environmental Toxicology and Pharmacology 57 (2018) 115–130

5.2. Ecological alternatives: old fashioned paints and new biocides

(Dafforn et al., 2011; DeLorenzo and Fulton,

(Bao et al., 2008; Dafforn et al., 2011; Yebra
(Costello et al., 2001; Dafforn et al., 2011)

(Dafforn et al., 2011; Evans et al., 2000)

(Cima et al., 2008; Dafforn et al., 2011)

(Cima et al., 2008; Dafforn et al., 2011)

(Dafforn et al., 2011; Hall et al., 2009)
With the gradual elimination of organotin based formulations,
copper has become the principal biocide component of most antifouling
paints. It is usually used in the form of copper oxide (Cu2O) (Yebra
et al., 2004). Inorganic zinc is often used in combination with copper to
increase the overall toxicity of the formulation or to facilitate the
leaching process (Watermann et al., 2005).
In the food products industry, the use of copper and zinc as anti-

et al., 2004)
fouling compounds is unwelcomed for both health and marketing per-

References
spectives. The Dangerous Substances Directive, which is one of the

2012)
main European Union laws concerning chemical safety, listed these
metals as being toxic to aquatic organisms, with long-term adverse ef-
fects on the environment and that their release into the environment

Diuron is registered for pre- and post-emergent weed control in both crop and non-crop areas, as a preservative in paints and
stains to prevent mildew growth, as an algaecide in antifouling paints, and in commercial fish production and aquariums. It
fungicide in agriculture, silviculture, and urban settings. It is also employed as a preservative for paints and adhesives. It was
It is one of the most popular surrogate antifouling biocides. It has been widely used as algaecide, bactericide and fungicide.

It was introduced specifically as an antifoulant in 1996 by Rohm & Haas. It is usually present in commercial paints as a main
or booster biocide at a concentrations of 1–3% and is registered as highly toxic by U.S. EPA Pesticide Production Information
It is present in antifouling paints, mainly as a booster biocide and it has been first in use as a broad-spectrum organochlorine
It is relatively insoluble in water and it may have the potential to bioaccumulate by becoming associated with particulate
It is a slightly soluble and moderately lipophilic triazine herbicide used in concert with copper to control fouling on boat

commercially introduced in 1969 as a General Use Pesticide (GUP) classified by the U.S. EPA in the toxicity « class II −
requires control (Nikolaou et al., 2014). That’s why the use of un-
conventional biocides has received significant attention recently thanks
to their ecological and economic relevance. Many other formulations
have been developed in order to replace toxic antifouling. These new
environment-friendly biocides have been developed as tributyltin (TBT)
free coating alternatives, such as Irgarol 1051, Zinc pyrithione, Di-
chlofluanid, Chlorothalonil, and Sea Nine 211 (Thomas, 2001).
We will detail in the following tables the basic characteristics of

It is added as a thiocyanate, copper metal oxide or sulfide (resulting different colored paints).
these antifouling agents (Table 1).
The toxicity of the selected booster biocides has been reported in
many researchers as shown in table below (Table 2) (Fig. 6).

6. The toxicity of antifouling biocides

has a water solubility of 42 mg/L and an aqueous half-life of 33 days.

In this part, we will detail some of tests applied to evaluate toxic
effects of antifouling biocides and resume their toxicity on algae, in-
vertebrates, crustaceans and fishes.
The table below (Table 3) presents the most studied and tested

moderately toxic », due to its potential for eye irritation.

marine species and organisms.
Evaluation tests present some ways provided that help to evaluate
the toxicity of different biocides on many aquatic species:

– For algae, toxicity can be measured using the algal growth inhibition
test. This test can be performed with Algaltoxkit system which is a
72 hours assay based on growth inhibition of the freshwater green
algae Selenastrum Capricornutum, with calculation of the 72 h EC50.
The algal cells are cultured in test tubes using the medium provided
with the test kit. The initial number of algal cells is adjusted to
System since 2002.

106 cells/mL and the test tubes are incubated at 25 °C for 3 days
under continuous illumination. Inhibition of the algal growth re-
Application

lative to controls is determined by measurements of optical density

matter.

in a spectrophotometer at a wavelength of 670 nm. The 72 hours

hulls.

EC50 value in this test is calculated as the concentration of the test

substance which causes a 50% reduction in growth relative to the
Organometallic salt

Organometallic salt

control (Fernández-Alba et al., 2002).

Organochlorine

Organochlorine

– For crustaceans (such as Daphnia magna, Daphnia pulex and Artemia

Chemical class

Isothiazolone

Phenylurea
Description of booster biocides used in antifouling paints.

salina), the toxicity of the test chemicals is assessed using the

s-Triazine

TOXKITs: Daphtoxkit F magna, Daphtoxkit F pulex and Artox-kit M.

Young neonates born in 24 hours are used in the toxicity testing. All
tests are conducted in the dark at 20 °C (Okamura et al., 2000). The
CuSCN; Cu2O; Cu2S;

neonates are considered immobile, if after 24 or 48 hours of in-

Chemical structure

C9H11Cl2FN2O2S2
C10H8N2O2S2Zn

cubation with the toxicant they remain settled at the bottom of the
C11H17Cl2NOS

C9H10Cl2N2O

test container and doesn’t resume swimming within the 15 seconds

C11H19N5S

observation period. The median effective concentration (EC50) is

C8Cl4N2

determined as the concentration of the toxicant requires to im-

mobilize 50% of the daphnids (Daphnia magna, Ceriodaphnia dubia)
after 24 and 48 hours exposure (Fernández-Alba et al., 2002).
Commercial name

Zinc pyrithione

– For fishes, cytotoxicity test can be applied using cultured fish cells.
Chlorothalonil
Dichlofluanid

Sea Nine 211

Irgarol 1051

For this test, the suspension-cultured fish cells are prepared by an

Adhering cell Layer Produces Suspension cells (ALPS) culturing
Copper

Diuron
Table 1

system. The kit contains the fish cells and the media for both culture
and for toxicity tests. The cell culture and toxicity tests are carried

119
I. Amara et al. Environmental Toxicology and Pharmacology 57 (2018) 115–130

Table 2
Toxicity of selected booster biocides.

Antifouling agent Toxicity References

Copper Although the use of copper in antifouling products in aquaculture has its own (Alzieu et al., 1980; Calabrese et al., 1973; Mochida et al., 2006;
environmental problems, it is much less toxic than TBT, and since the banning of Priour, 1995)
organotin antifouling formulations, copper-based antifouling experienced a renaissance.
It is a poison for algae and mollusks, suitable for seawater, and not recommended for
freshwater and its effect is limited to about 5–12 months depending on its quality and
environmental conditions such as temperature, salinity, light, current, etc.
Irgarol 1051 It is highly effective against freshwater and marine algae. Irgarol 1051 has been (Arrhenius et al., 2006; Braithwaite and Fletcher, 2005; Fernández-
reported to disturb the electron transfer process within Photosystem-II and have fatal Alba et al., 2002; Moreland, 1980; Okamura et al., 2000)
effects on marine life.
Zinc pyrithione It was found to be highly toxic to aquatic plants and animals, but it was assumed to be (Bao et al., 2011; Koutsaftis and Aoyama, 2006; Mochida et al.,
environmentally neutral because it could easily photo-degrade to less toxic compounds. 2006; Turley et al., 2000, 2005; Yamada, 2006)
Dichlofluanid It has a lower toxicity compared with other antifouling agents, although some studies (Bellas, 2006; Guardiola et al., 2012; Xu et al., 2011)
have identified its toxic effects.
Chlorothalonil There are numerous toxicity studies for Chlorothalonil on marine species, such as (Bao et al., 2011; Bellas, 2006; Davies et al., 1994; Ernst et al.,
crustaceans and invertebrates. Chlorothalonil can be acutely toxic (LC50) to fish 1991; Fernández-Alba et al., 2002; Sherrard et al., 2003)
following 96 h exposures ranging from 8.2–110 μg/L, depending on the species and the
exposure conditions. It can accumulate in the tissue of fish. Bioaccumulation factors
have been reported to be 18 for willow shiner (Gnathopogon caerulescens) and 25 for carp
(Cyprinus carpio) following sub lethal exposures (1.1–1.4 μg/L).
Sea Nine 211 Sea-Nine is acutely toxic to a wide range of aquatic organisms although no chronic (Arrhenius et al., 2006; Bellas, 2006; Braithwaite and Fletcher,
toxicological effects have been observed in the extensive toxicology tests conducted on 2005; Cima et al., 2008; Wang et al., 2011; Xu et al., 2011;
it. Yamada, 2006)
Diuron Diuron has been proven to be very toxic for the reproduction of the green freshwater (Arrhenius et al., 2006; Fernández-Alba et al., 2002; Koutsaftis and
alga Scenedesmus vacuolatus. It has also been proven to affect planktonic and periphytic Aoyama, 2006; Ma et al., 2002)
microalgae by reducing the chlorophyll a levels. Moreover, it has been proven to be
toxic to certain bacterial species.

out in an incubator maintained at 20 °C under no-light conditions.
After inoculation, the micro plate is incubated for 24 hours to pro-
pagate the cells over the bottom of the well. At appropriate in-
cubation intervals (12, 24 and 48 hours), the plate is transferred to a
micro plate reader to measure absorbance at 570 and 600 nm. The
toxic effect of SDS (The surfactant sodium n-dodecyl sulphate) on
cells is evaluated at different exposure times and different incuba-
tion times with Alamar Blue (Okamura et al., 2002).
We have reviewed the known literature on the toxicity of numerous
antifouling biocides on algae, invertebrates, crustaceans and fishes
below.
6.1. Toxicity of TBT as a biofouling compound
Toxic effects of TBT were evaluated toward ecologically important
species such as algae, invertebrate, crustaceans and fishes. For all these
species, TBT was found to be severely toxic since it affects their growth

Table 3
Tested marine organism species.

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I. Amara et al. Environmental Toxicology and Pharmacology 57 (2018) 115–130

Table 4
Acute toxicity of TBT to aquatic organisms.

Species Effect Measured effect/concentration (μg/L) Refernces

Algae
Hormosira banksii Significant impairment to the germination after 48 h and 72 h EC50 = 0.16
EC50 = 0.2
Significant impairment to growth responses after 48 hours and EC50 = 0.34
72hours
EC50 = 0.32
Scenedesmus vacuolatus Inhibition of the periphyton photosynthesis activity EC50 = 14.75 Arrhenius et al. (2006)
Inhibition of the algal reproduction EC50 = 53.75
Selenastrum capricornutum Inhibition of photosynthesis activity at 72 h EC50 = 3 ± 0.4 Fernández-Alba et al. (2002)

Invertebrate
Crassostrea gigas
Mytilus galloprovincialis
Paracentrotus lividus
Ciona intestinalis
After 24 h, 30% died and 8% have an abnormal transformation 5 LC100 = 5 Alzieu et al. (1980)
Mortality after 48 h
Formation of abnormal oyster’s larvae and total destruction after 1
4–5 days
Formation of abnormal oyster’s larvae and total destruction after 1
4–5 days
Reduction by 50% of normal larvae EC50 = 0.309 Antizar-Ladislao (2008)
Reduction of percentages of normal larvae by 50% EC50 = 7.1 Antizar-Ladislao (2008)

Crustacean
Elasmopus rapax
Tigriopus japonicus
Palaemon serratus
Nitocra spinipes
Eurytemora affinis
Daphnia magna
50% of the population for juvenile died after 96 h LC50 = 9.4 Bao et al. (2011)
Death of half of the population for adult after 96 h LC50 = 18
Copepod mortality at 96 h LC50 = 0.15
Larval mortality at 24 h and 48 h LC50 = 22.3 Bellas (2006)
LC50 = 17.52
Copepod mortality at 96 h LC50 = 13 Karlsson et al. (2006)
Mortality at 48 h and 72 h LC50 = 2.2 and 0.6 Antizar-Ladislao (2008)
50% of the population exhibit a response after 48 h EC50 = 0.001 Hernando et al. (2003)

Fishes
Oryzias melastigma Reduction of survival larvae by 50% after 96 h LC50 = 25 Bao et al. 2011 and Hongxia et al.
(1998)
Tilapia Mortality at 96 h LC50 = 3.8
Phoxinus phoxinus 100% mortality in larvae after 96 h LC100 = 6.55–9.25 Fent (1991)
Sparus aurata Expiration of half of fertilized eggs after 24 h LC50 = 28.3 Dimitriou et al. (2003)

even at very low concentrations. Those effects are cited in Table 4: High (Antizar-Ladislao, 2008).
potency of TBT toxicity in multiple species.

6.1.1. TBT effect on algae 6.1.4. TBT effect on fishes

Tributyltin-oxide is an effective antifouling biocide for both
Hormosira banksii and Selenastrum capricornutum, as well as for per-
iphyton community (complex mixture of algae, cyanobacteria, hetero-
trophic microbes) photosynthesis and to the reproduction of the green
algae Scenedesmus vacuolatus. The NOEC for algal reproduction and for
periphyton communities are respectively 33.25 μg/L and 8 μg/L
(Arrhenius et al., 2006).
6.1.2. TBT effect on invertebrates
TBT acetate is also very toxic for both the Pacific oyster (Crassostrea
gigas) and mussel species such as Mytilus galloprovincialis. In the case of
the oysters, larvae formation is damaged leading to complete destruc-
tion after 4–5 days of exposure to only 1 μg/L. (Alzieu et al., 1980).
Moreover, TBT shows high toxicity for both Paracentrotus lividus and
Ciona intestinalis, as seen in Table 4.
6.1.3. TBT effect on crustaceans
Further tests for the acute toxicity of TBT on crustaceans have been
applied as shown in Table 4. TBT is very toxic for many crustacean
species such as Elasmopus rapax, Tigriopus japonicas, Palaemon serratus
and Daphnia magna. Additionally, further studies have reported 96 h
LC50 values for copepod (small crustacean) Nitocra spinipes of 13 μg/L
(Karlsson et al., 2006). This value is in accordance with other crusta-
cean species, but the copepod Tigriopus japonicas is less sensitive (Kwok
and Leung, 2005). The estuarine zooplankton Eurytemora affinis’s TBT
sensitivity is very high, as at only 0.1 μg/L, neonatal survival was
curtailed at less than half way through a 13 day chronic experiment
Toxicological data regarding the impact of TBT on fishes are lim-
ited. Studies showed that TBT has a very high toxicity to Oryzias mel-
astigma’s larvae, as well as to Tilapia and Phoxinus phoxinus. However,
Sparus aurata is less sensitive to TBT, comparing to other species, since
after only 24 h of exposure, half of its fertilized eggs and larva expired
respectively at 28.3 μg/L and 38.6 μg/L (Dimitriou et al., 2003).
However, with the evolution in the use of TBT-based paints, due to
their efficiency and versatility, disastrous effects on the marine en-
vironment have resulted. Organotins have also accumulated in mam-
mals and weakened fish immune systems (Almeida et al., 2007). In
addition, extreme toxicity of TBT to aquatic organisms in early life
stages has been observed. For example, fish larvae are very sensitive to
this antifoulant and often exhibit effects in low concentrations. More-
over, the danger posed by organotin compounds to humans depends on
the solubilization and on the possibility that they may degrade during
human digestion (Antizar-Ladislao, 2008). Consequently, in October
2001, when the International Maritime Organization (IMO) took stock
of the adverse effects of TBT on the marine environment, an order was
issued banning the use of this type of biocides in the manufacturing of
antifouling paints as from 1 st January 2003, and the presence of these
paints on ship surfaces as from 1 st January 2008 (Almeida et al., 2007).
6.2. Toxicity of biofouling compounds based on copper
The limited toxicity data for copper on aquatic organisms is re-
viewed below.

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6.2.1. Copper effect on algae
Copper was found to be toxic for some algae. It can inhibit growth of
Chlorella vulgaris and Dunaliella tertiolecta when concentration is about
4 μg/L and 600 μg/L respectively. Moreover, the photosynthetic activity
can be blocked from 50 μg/L for Chlorella pyrenoidosa and visible lesions
appear in macroalgae from 100 μg/L (Alzieu et al., 1980).
6.2.2. Copper effect on invertebrates
Molluscs are moderately sensible to copper oxides such as CuO and
Cu2O when concentrations are between 1 and 2 mg/L (Floch et al.,
1964). Also, whilst copper (II) chloride CuCl2 has no effect on the larvae
of C. gigas at the concentration of 10 μg/L, embryonic development is
totally disrupted at 50 μg/L (Alzieu et al., 1980). Besides, the LC50 and
LC100 for embryos of Crassostrea virginica are respectively 103 μg/L and
130 μg/L (Calabrese et al., 1973).
6.2.3. Copper effect on crustacean
Copper can kill 50% of the population of different crustacean spe-
cies after only 48 h of exposure. Larvae are more sensitive than adults
with respective LD50s 0.6 and 109 mg/L for crab Carcinus maenas, va-
lues of 0.3 and 29.5 mg/L were recorded and for shrimp Crangon
crangon and 0.1 and 0.3 mg/L for lobster larvae Homarus gammarus
(Alzieu et al., 1980). After 96 h of copper exposure, an LC50 of 113 μg/L
for Heptacarpus futilirostris (Mochida et al., 2006).
6.2.4. Copper effect on fishes
After 96 h of exposure, copper reduces survival of Pagrus major by
50% at a low concentration 84.4 μg/L (Mochida et al., 2006). Copper is
lipophilic and shows only a slight tendency towards bioaccumulation,
which explains why it has remained in the formulations of antifouling
paints for many years (Voulvoulis et al., 1999). Therefore, even though
there are some doubts concerning the effect of copper concentrations in
sea water on marine organisms (Alzieu et al., 1980; Mochida et al.,
2006), it is normally considered that the low bioavailability of copper
ions released by antifouling paints seems to present an environmental
profile that complies with current environmental quality standards
(Almeida et al., 2007).
6.3. Toxicity of Chlorothalonil as a biofouling compound
Chlorothalonil antifoulant has previously been reported to be highly
effective against several marine species such as crustaceans, in-
vertebrates and fishes. All information collected are quoted in this part.
6.3.1. Chlorothalonil effect on invertebrates
The toxicity of this pesticide to aquatic invertebrates is unclear.
Some studies show that chlorothalonil is the most toxic compound to
the early developmental stages of three species of marine invertebrates:
Paracentrotus lividus, Ciona intestinalis and Mytilus edulis. The con-
sequences of applying chlorothalonil on these invertebrates were noted
to be embryotoxicity, larval inhibition and mortality (Bellas, 2006).
Chlorothalonil affects also the shell deposition of Crassostrea virginica at
low concentrations (between 5 μg/L and 26 μg/L) (Bellas, 2006).
6.3.2. Chlorothalonil effect on crustaceans
Many crustacean species have been studied and have been found to
be very sensitive to Chlorothalonil such as Amphiascus tenuiremis,
Penaeus duorarum, Ceriodaphnia dubia and Daphnia magna. Furthermore,
for three life history stages (embryo, larvae, adult) of the grass shrimp
Palaemonetes pμgio, studies show that embryos are the least sensitive
and that larvae are the most sensitive to chlorothalonil exposure
(Fig. 6). Also, larvae exposed to a concentration between 250 μg/L and
500 μg/L become increasingly less active and remains motionless on the
bottom of the beakers until death (Key et al., 2003). And finally, for
daphnids (species unstated), the 48 h LC50 is equal to 70 μg/L (Jones
et al., 1983).
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Environmental Toxicology and Pharmacology 57 (2018) 115–130
Fig. 6. Survival of Palaemonetes pμgio larvae exposed to Chlorothalonil at different
concentrations.
6.3.3. Chlorothalonil effect on fish
Chlorothalonil can be acutely toxic (LC50) to fish following 96 h
exposure ranging from 8.2 to 76 μg/L, depending on the species and the
exposure conditions (Davies et al., 1994; Ernst et al., 1991). For three
species of salmoniform fish in the Galaxiidae family, studies show that
Chlorothalonil has a high toxicity for Galaxias auratus, Galaxias macu-
lates and Galaxias truttaceus. Moreover, other fish species show a high
sensitivity against this antifoulant as shown in Table 5. Pseudaphritis
urvillii is the most sensitive with a LC50 value of 8.2 μg/L. However,
Anguilla japonica and Oryzias melastigma are the least sensitive to with a
LC50 equals to 100 μg/L.
6.4. Toxicity of Sea Nine 211
Sea Nine 211 was introduced specifically as an antifouling com-
pound in 1996 (Cima et al., 2008). Its chemical structure is based on
sulfur, chlorine and nitrogen (C11H17Cl2NOS). There are numerous
studies that have investigated the toxicity and the effects of Sea Nine
211 on several marine species. These studies (detailed in Table 6) show
that algae, crustacean and fishes are very sensitive to this biocide since
survival begins to decrease even at low concentration (between 2 μg/L
and 4 μg/L).
6.4.1. Sea Nine 211 effect on algae
As shown in the table above, previously reported results demon-
strate excellent activity against a wide range of algae. For example, for
Enteromorpha intestinalis, Fucus serratus and Scenedesmus vacuolatus, Sea
Nine 211 shows high toxicity even at low concentration. For Hormosira
banksii, its spores (reproductive bodies of an organism, which are able
to give rise to new individual) appear to be similar in sensitivity to this
antifoulant as other algal species. Its EC50 values range from 340 μg/L
to 460 μg/L for both germination (beginning to develop and grow) and
growth responses of spores.
6.4.2. Sea Nine 211 effect on invertebrates
Sea-Nine 211 affects the embryonic development and larval growth
of both M. edulis and P. lividu at very low concentrations. Besides, recent
studies reported extreme toxicity of this antifoulant to the embry-
ogenesis (the process by which the embryo forms and develops) of the
sea-urchin Anthocidaris crassispina and for Balanus amphitrite larvae.
Furthermore, the EC50 of Sea-Nine 211 for mussel embryogenesis has
been reported at 2 μg/L, and the EC50 for oyster embryogenesis ranged
from 7 to 24 μg/L (Shade et al., 1993; Willingham and Jacobson, 1996).
The pre-exposure of S. Intermedius gametes for 1 h (sperm) and 4 h
(eggs) to five antifouling biocides including Sea Nine 211 results many
problems. This exposure causes significant decrease in the normal larva
rate, rise of embryonic and larval malformations and decrease in larval
growth (Wang et al., 2011).
I. Amara et al. Environmental Toxicology and Pharmacology 57 (2018) 115–130

Table 5
Acute toxicity of Chlorothalonil to aquatic organisms.

Species Effect Measured effect/concentration References

(μg/L)

Invertebrate Bellas (2006)

Paracentrotus lividus Inhibition of the embryonic development after 48 h EC10 = 4.3
EC50 = 6.6
Inhibition of the larval growth after 48 h EC10 = 0.5
Ciona intestinalis Inhibition of the embryonic development after 48 h EC10 = 12
EC50 = 33
Inhibition of larval settlement after 48 h EC10 = 27
EC50 = 42
Mytilus edulis Embryotoxicity after 48 h: 50% of its maximum response is observed EC10 = 4.5
EC50 = 8.8
Mortality at 96 h LC50 = 5940 Ernst et al. (1991)
Crassostrea virginica Shell deposition affected EC50 = 5–26 Bellas (2006)

Crustacean
Daphnia magna Mortality at 48 h LC50 = 130–200 Ernst et al. (1991) and Fernández-Alba
EC50 = 28 ± 0.5 et al. (2002)
Amphiascus tenuiremis Mortality at 96 h EC50 = 28 ± 0.5 Bejarano et al. (2005)
LC50 = 27–53
Penaeus duorarum Mortality at 48 h and 96 h LC50 = 320 Mayer (1987) and Montforts (1999)
LC50 = 162
Mortality of juvenile at 96 h LC50 = 165 Cima et al. (2008)
Cancer magister Larval mortality after 48 h LC50 = 560 Armstrong et al. (1976)
Palaemonetes pugio Mortality for embryos at 96 h LC50 = 396 Key et al. (2003)
Mortality for larvae at 96 h LC50 = 49.5
Mortality for adult at 96 h LC50 = 152.9
Larvae became less active and remained motionless on the bottom of Between 250 and 500
the beakers until death
Ceriodaphnia dubia Mortality with 0.94% mortality/μg/L LC50 = 156 Sherrard et al. (2003)
LC100 = 210
Artemia salina Mortality at 24 h LC50 = 1000 Koutsaftis and Aoyama (2007)

Fishes
Galaxias maculatus Mortality at 24 h and 96 h LC50 = 23.7 and 16.3 Davies and White (1985)
Galaxias trutaceus Mortality at 48 h and 96 h LC50 = 25,8 and 18.9
Galaxias auratus Mortality at 48 h and 96 h LC50 = 46.6 and 29.2
Oryzias melastigma Mortality for larvae at 96 h LC50 = 110 Bao et al. (2011)
Pimephales promelas 6.1% mortality/μg/L after 7 days LC50 = 22.6 Sherrard et al. (2003)
LC100 = 30.8
Pseudaphritis urvillii Half of the population of juvenile died in 10 days LC50 = 8.2 Davies et al. (1994)
Gasterosteus aculeatus Mortality at 96 h LC50 = 27 Ernst et al. (1991)
Cyprinodon variegatus Mortality LC50 = 33 Bellas (2006)
Leiostomus xanthurus Mortality at 48 h LC50 = 32 Mayer (1987)
Anguilla japonica Mortality at 24 h LC50 = 110

6.4.3. Sea Nine 211 effect on crustaceans
There is a lack of knowledge on toxicity of Sea-Nine 211 in crustacean
species. Data on chronic toxicity to mysid shrimp, Uca pugilator and Daphnia
magna show that Uca pugilator is the least sensitive and mysid shrimp is the
most sensitive. Moreover, and according to a technical report on the pre-
vention of marine pollution by antifouling paints (Japan 1999), Penaeus
japonicas and Tigriopus japonicus are, also, very sensitive (Yamada, 2006).
6.5.1. Irgarol effect on algae
Irgarol shows highly significant toxicity in tests against several
seaweeds. As shown in Table 7, Fucus serratus is the least sensitive and
that Chaetoceros Gracilis is the most sensitive to Irgarol. Also, the toxi-
city towards periphyton photosynthesis and for algal reproduction of S.
vacuolatus and for growth of Selenastrum capricornutum and En-
teromorpha Intestinalis.is particularly high.

6.4.4. Sea Nine 211 effect on fishes
Few studies show the effect of Sea-Nine 211 on fishes, but all data
suggest that its toxicity is extremely high for many fish such as: red sea
bream Pagrus major, mummichog Fundulus heteroclitus, Oncorhynchus
mykiss and Lepomis macrochirus.
Pesticide Ecotoxicity Database (2000) reported that Sea-Nine 211
reduces survival by 50% of Cyprinodon variegatus after 96 h of exposure
at very low concentration.
6.5. Toxicity of Irgarol as a biofouling compound
Irgarol is an herbicide used in concert with copper to control fouling
on boat hulls. Its chemical structure is based on sulfur and nitrogen
(C11H19N5S) (Hall et al., 2009). The toxicity of this pesticide to aquatic
species is largely studied. Its effects on algae, invertebrates, crustacean
and fishes are presented in Table 7.
6.5.2. Irgarol effect on invertebrates
Irgarol is the least toxic biocide on the embryonic development and
the larval growth development of M.edulis and P. lividus. Likewise,
Strongylocentrotus Intermedius sea urchin is less sensitive to Irgarol 1051
compared to other antifouling biocides as shown in Table 7.
6.5.3. Irgarol effect on crustaceans
Available studies investigated lethal and sub lethal effects of the
antifouling herbicide Irgarol 1051 on larval and adult grass shrimp
Palaemonetes pugio (Key et al., 2008). Mortality is not observed until
48 h after exposure. For the larval grass shrimp, there is 87% mortality
in the highest concentration of 3 mg/L after 96 h. Besides, for adult
grass shrimp, there is 73% mortality in the highest concentration of
8 mg/L after 24 h, and after 72 h, all shrimp in that highest con-
centration are dead (Key et al., 2008). Moreover, the toxicity of this
antifoulant on five other crustacean species has been investigated and

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Table 6
Acute toxicity of Sea Nine 211 to aquatic organisms.

Species Effect Measured effect/concentration References

(μg/L)

Algae
Hormosira banksii Significant impairment to the germination after 48 h and 72 h EC50 = 340
EC50 = 420
Significant impairment to growth responses after 48 h and EC50 = 430
72 h
EC50 = 460
Enteromorpha intestinalis Mortality for spore germination LD50 = 2 Jacobson and Willingham (2000)
Fucus serratus NOEC and LOEC for zygote germination NOEC = 8 Braithwaite and Fletcher (2005)
LOEC = 31
Zygote germination effected at 24 h EC50 = 19.4
Scenedesmus vacuolatus Inhibition of the periphyton photosynthesis activity EC50 = 137.25 Arrhenius et al. (2006)
NOEC = 44.5
Inhibition of the algal reproduction EC50 = 78.5
NOEC = 24

Invertebrates
Paracentrotus lividus Inhibition of the embryonic development after 48 h EC10 = 5.9 Bellas (2006)
EC50 = 12.1
Inhibition of the larval growth after 48 h EC10 = 1.7
EC50 = 25
Mytilus edulis Inhibition of the embryonic development after 48 h EC10 = 7.1
EC50 = 11
Balanus amphitrite Mortality for larvae LC50 = 340 Jacobson and Willingham (2000)
Strongylocentrotus intermedius Reduction of the embryogenesis success by 50% for the EC50 = 14.38 Wang et al. (2011)
embryos
Reduction of the embryogenesis success by 50% for treated EC50 = 31.69
eggs and sperm
Reduction of the embryogenesis success by 50% for treated EC50 = 57.52 Wang et al. (2011)
eggs
Reduction of the embryogenesis success by 50% for treated EC50 = 113.59
sperm
Glyptocidaris crenularis 50% of the population of embryos exhibit a response EC50 = 0.65 Xu et al. (2011)

Crustacean
Tigriopus japonicus 50% of the population exhibit a response after 24 h EC50 = 30 Yamada (2006)
LC50 = 77
Daphnia magna 50% of the population exhibit a response after 48 h EC50 = 4 ± 0.7 Hernando et al. (2003)
Mysid shrimp Mortality at 96 h LC50 = 4.7 Shade et al. (1993)
Uca pugilator Mortality at 96 h LC50 = 1312
Penaeus japonicus Mortality at 96 h LC50 = 12.6 Yamada (2006)

Fishes
Pagrus major
Fundulus heteroclitus
Cyprinodon variegatus
Oncorhynchus mykiss
Lepomis macrochirus
Mortality at 96 h LC50 = 5.1 Mochida et al. (2010)
Mortality at 96 h LC50 = 4.7
NOEC and LOEC for embryos NOEC = 1,1
LOEC = 2.8
Mortality at 96 h LC50 = 20.5 Cima et al. (2008)
Mortality at 96 h LC50 = 2.7 Okamura et al., 2002 and Shade et al.,
Mortality at 96 h LC50 = 14 1993

several studies show that it is not as toxic as other biocides for Daphnia
magna, Daphnia pulex, Thamnocepharus platyurus, Artemia salina and
Elasmopus rapax.
6.5.4. Irgarol effect on fish
The effect of Irgarol on different kinds of fishes has been reviewed in
several articles (Geigy, 1995; Hall et al., 1999; Key et al., 2009). As
shown in Table 7, Irgarol is not very toxic to mummichogs Fundulus
heteroclitus, Cyprinodon variegates, Menidia beryllina, Barachydanio rerio
and Lepomis macrochirus with a lethal concentration higher than
1.5 mg/L. Additionally, another study shows that Oncorhynchus mykiss
is the most sensitive with a LC50 equal to 790 μg/L, and that its NOEC at
60 days post-hatching is 4 μg/L in a flow-through system (Hall et al.,
1999).
The herbicide Irgarol 1051’s environmental effect has not been fully
assessed. It shows good effectiveness against freshwater and sea water
algae and less effectiveness against crustaceans, fishes and invertebrates,
with effective toxic concentrations being higher than 1 mg/L.
6.6. Toxicity of Zinc pyrithione
Zinc pyrithione (ZnPt) is one of the most popular surrogate anti-
fouling biocides that have been widely used as algaecide, bactericide
and fungicide. Its chemical structure is based on sulfur, zinc and ni-
trogen (C10H8N2O2S2Zn) (Bao et al., 2008; Yebra et al., 2004). For the
few presented studies, it has been found to be highly toxic to aquatic
plants and animals as shown in Table 8. However, ZnPt has been as-
sumed to be environmentally neutral because it can easily photo-de-
grade to less toxic compounds (Bao et al., 2008; Turley et al., 2005,
2000).
6.6.1. Zinc pyrithione effect on algae
Zinc pyrithione appears to be very toxic to Chaetoceros Gracilis be-
cause only 3.2 μg/L can inhibit its growth. Also, Selenastrum capri-
cornutum shows a sensitivity towards ZnPt, because after 5 days of ex-
posure to 28 μg/L, its growth is inhibited (Koutsaftis and Aoyama,
2006). Also, ZnPt shows significant toxicity for germination and for
growth responses of spores of Hormosira banksii.

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Table 7
Acute toxicity of Irgarol to aquatic organisms.

Species Effect Measured effect/concentration References

(μg/L)

Algae
Enteromorpha intestinalis
Fucus serratus
Scenedesmus vacuolatus
Chaetoceros gracilis
Selenastrum capricornutum
Inhibition at 72 h EC50 = 2.5 Scarlett et al. (1997)
NOEC and LOEC for zygote germination NOEC = 8 Braithwaite and Fletcher (2005)
LOEC = 31
Inhibition of the periphyton photosynthesis activity EC50 = 1.13 Arrhenius et al. (2006), Fernández-Alba et al.
NOEC = 0.45 (2002) and Okamura et al. (2000)
Inhibition of the algal reproduction EC50 = 6.21
NOEC = 0.5
92% reduction in photosynthetic activity 3.38–4.8 Fernández-Alba et al. (2002) and Okamura et al.
50% inhibition of growth at 72 h EC50 = 1.1 (2000)
50% inhibition of growth at 72 h EC50 = 1.6–10.8

Invertebrates
Paracentrotus lividus Inhibition of the embryonic development after 48 h EC10 = 2904 Bellas (2006)
EC50 = 4021
Inhibition of the larval growth after 48 h EC10 = 1868
EC50 = 6032
Mytilus edulis Inhibition of the embryonic development after 48 h EC10 = 797
EC50 = 1540
Strongylocentrotus intermedius Reduction of the embryogenesis success by 50% for EC50 = 5890 Wang et al. (2011)
the embryos
Reduction of the embryogenesis success by 50% for EC50 = 8100
treated eggs and sperm
Reduction of the embryogenesis success by 50% for EC50 = 1050
treated eggs
Reduction of the embryogenesis success by 50% for EC50 = 17270
treated sperm
Glyptocidaris crenularis 50% of the population of embryos exhibit a EC50 = 412.5 Xu et al. (2011)
response

Crustacean
Elasmopus rapax Mortality of juvenile at 96 h LC50 = 1000 Bao et al. (2011)
Palaemonetes pμgio Mortality of larvae at 96 h LC50 = 1520 Key et al. (2008)
Mortality of adult at 96 h LC50 = 2460
NOEC and LOEC for larvae NOEC = 1000
LOEC = 2000
NOEC and LOEC for adult NOEC = 500
LOEC = 103
Artemia salina Mortality at 24 h LC50 ≫ > 40.103 Okamura et al. (2000)
Daphnia magna Mortality at 24 h and 48 h LC50 = 16. 103 and 8,3.103
Daphnia pulex Mortality at 24 h LC50 = 5,7.103
Thamnocepharus platyurus Mortality at 24 h LC50 = 12.103

Fishes
Fundulus heteroclitus Mortality at 96 h NOEC = 1250 Key et al. (2009)
LOEC = 2500
LC50 = 3220
Oncorhynchus mykiss Mortality at 96 h LC50 = 790 Hall et al. (1999)
No effect on the hatchability or early life-stage NOEC = 184
survival
Cyprinodon variegates Mortality at 96 h LC50 = 3500
Menidia beryllina Mortality at 96 h LC50 = 1580
Lepomis macrochirus Mortality at 96 h LC50 = 2600
Barachydanio rerio Mortality at 96 h LC50 = 4000 Geigy (1995)

6.6.2. Zinc pyrithione effect on invertebrates
There is no current information on the toxicity of ZnPt on in-
vertebrates, hence the toxicity of other zinc salts was cited in the table
above. Previous studies considered that zinc chloride is toxic to the
oyster embryos of Crassostrea virginica when its concentration in the
area exceeds 75 μg/L (Calabrese et al., 1973). Besides, the percentage of
fixation of Crassostrea gigas larvae is significantly reduced (13.2%
against 43.5%) when it is exposed to 125 μg/L of zinc sulphate for
5 days. Toxic and reversible effects on larval growth are observed at a
concentration of 250 μg/L (Boyden et al., 1975). Moreover, for the flat
oyster Ostrea edulis, some evidence suggested that larval growth is
significantly reduced in the presence of 500 μg/L of zinc salts (Alzieu
et al., 1980).
6.6.3. Zinc pyrithione effect on crustaceans
In Table 8, ZnPt is toxic to juvenile (physiologically immature/
undeveloped) Elasmopus rapax and toy shrimps Heptacarpus futilirostris
even at low concentration. Furthermore, according to a technical report
on the prevention of marine pollution by antifouling paints (Japan
1999), many tests on the influence of ZnPt on crustacean have been
applied. It appears to be extremely toxic for the mysid shrimp and less
toxic for Daphnia magna and Tigriopus japonicas (Yamada, 2006). It is
also clear that the brine shrimp Artemia salina and Penaeus japonicus are
the least sensible to ZnPt with a lethal concentrations upper than 1 mg/
L.
6.6.4. Zinc pyrithione effect on fishes
There is a lack of data on the toxicity of ZnPT. However, fish are
impacted by this antifoulant even in low concentrations, although some
crustacean species are insensitive (concentrations higher than 1 mg/L
are lethal). However, ZnPT is toxic to Japanese medaka fish Oryzias
latipes and causes teratogenic effects, such as spinal cord deformities in

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Table 8
Acute toxicity of ZnPt to aquatic organisms.

Species Effect Measured effect/concentration References

(μg/L)

Algae
Hormosira banksii Significant impairment to the germination after 48 h and 72 h EC50 = 210
EC50 = 190
Significant impairment to growth responses after 48 h and 72 h EC50 = 310
EC50 = 240
Chaetoceros gracilis 50% growth inhibition at 72 h EC50 = 3.2 Koutsaftis and Aoyama (2006)
Selenastrum capricornutum 50% growth inhibition at 5 days EC50 = 28
NOEC = 7.8

Invertebrates
Crassostrea gigas Significant reduction of larvae percentage of fixation after 5 days 125 Boyden et al. (1975)
(zinc sulphate)
Crassostrea virginica Mortality of embryos at 48 h (zinc chloride) LC50 = 310 Calabrese et al. (1973)
LC100 = 500
Ostrea edulis Significant reduction of larval growth (zinc salts) 500 Alzieu et al. (1980)

Crustacean
Elasmopus rapax Mortality of juvenile at 96 h LC50 = 29 Bao et al., (2011) and Mochida et al.
Heptacarpus futilirostris Mortality at 96 h LC50 = 120 (2006)
Tigriopus japonicus Mortality at 24 h EC50 = 160 Yamada (2006)
LC50 ≫ > 500
Daphnia magna 50% of the population exhibit a response after 48 h EC50 = 29–34
Artemia salina Mortality at 24 h LC50 = 3170 Koutsaftis and Aoyama (2007)
Mysid shrimp Mortality at 96 h LC50 = 6.3 Yamada (2006)
Penaeus japonicus Mortality at 96 h LC50 = 1780

Fishes
Oryzias latipes
Brachydanio rerio
Oryzias melastigma
Pagrus major
Pimephales promelas
Oncorhynchus mykiss
Cyprinodon variegatus
50% of the population exhibit a response after 20 days EC50 = 5 Goka (1999)
Embryo toxicity EC50 = between 3 − 7
50% of the population exhibit a response after 7 days EC50 = 9 Goka (1999)
Embryo toxicity EC50 = between 8–10
Half of larvae population died after 96 h LC50 = 43 Bao et al. (2011)
The secondary lamellae of the gill filaments of this fish heavily LC50 = 98.2 Mochida et al. (2006)
damaged, causing death after 96 h
Mortality at 96 h LC50 = 2.6 Yamada (2006)
Mortality at 96 h LC50 = 3.2
Mortality at 96 h LC50 = 400

Fig. 7. (a) A 4-day-old zebra fish larva showing normal straight ver-
tebral column. (b) A 4-day-old zebra fish larva exposed to Znpt
(10 μg/L) continuously from the egg stage showing an inflexible and
extremely wavy structure of the vertebral column.

embryos and on larvae of zebra fish Brachydanio rerio at very low sub
lethal concentration as shown in the figure below (Fig. 7) (Goka, 1999;
Sánchez-Bayo and Goka, 2006).
Therefore, its embryo toxicity has been evaluated using fertilized
eggs of those fishes and has been reported to be very high for both of
them. It has been concluded that ZnPT can induce embryo toxicity in
fish species at relatively low concentrations (Goka, 1999). Moreover,
ZnPt is highly toxic to red sea bream Pagrus major since the secondary
lamellae of the gill filaments of this fish were heavily damaged after
exposure to this antifoulant. Hypoxemia was observed as a cause of
death after treatment with this agent (Mochida et al., 2006). Finally,
and according to Technical report on the prevention of marine pollution
by antifouling paints (Japan 1999), P.promelas, O. mykiss are the most
sensitive and Cyprinodon variegatus is the least sensitive to ZnPt
(Yamada, 2006).
6.7. Toxicity of Dichlofluanid
Dichlofluanid is widely used as a protective fungicide in agriculture
(Hernando et al., 2003). Its chemical structure is based on sulfur,
fluorine, chlorine and nitrogen (C9H11Cl2FN2O2S2). This antifoulant
compound has been found to be moderately toxic to invertebrates.
Unfortunately, there is lack of data about its toxicity for other aquatic
species.

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I. Amara et al. Environmental Toxicology and Pharmacology 57 (2018) 115–130

6.7.1. Dichlofluanid effect on invertebrates 6.8.3. Diuron effect on crustacean

The toxicity of Dichlofluanid on Strongylocentrotus Intermedius sea urchin
has been quantified in terms of the median effective concentration (EC50)
reducing the embryogenesis success by 50%. The corresponding concentra-
tions are cited below (Wang et al., 2011): these include 549.15 μg/L for the
exposed embryos; 754.16 μg/L for treated eggs and sperm;1112 μg/L for
treated eggs and 1472.3 μg/L for treated sperm. This study shows that the
pre-exposure of S. Intermedius gametes for 1 h (sperm) and for 4 h (eggs) to
five antifouling biocides including Dichlofluanid also results in (Wang et al.,
2011)a significant decrease of the normal larva rate; a rise to embryonic and
larval malformations; a decrease in larval growth; likewise, another study
investigates the acute toxicity of this biocide using the sea urchin embryos of
Glyptocidaris crenularis at six typical developmental stages. Dichlofluanid has
been found to be less toxic than Sea Nine 211 (Xu et al., 2011).
Furthermore, the EC10 and EC50 values of embryonic development
of P.lividus are 277 and 627 μg/L respectively, and the EC10 of larval
growth was 206 μg/L. Also, for embryonic development of M.edulis, its
EC10 valued of 52 and 49 μg/L and its EC50 valued of 81 and 74 μg/L,
respectively (Bellas, 2006).
6.8. Toxicity of Diuron
Diuron is applied as an algaecide in antifouling paints and as an
emergent weed control in different areas. Its chemical structure is based
on chlorine and nitrogen C9H10Cl2N2O (DeLorenzo and Fulton, 2012).
The published literature contains little data on the acute toxicity of
Diuron to aquatic organisms. It can be summarized as follows:
6.8.1. Diuron effect on algae
Diuron is very toxic to the growth of the marine algae such
Chaetoceros Gracilis, Selenastrum capricornutum and Chlorella vulgaris as
shown in Table 9. However, Hormosira banksii is less sensitive to Diuron
comparing with other algae, since the effective concentration exceeds
to 6 mg/L for both germination and growth responses of spores.
6.8.2. Diuron effect on invertebrates
Diuron is quite toxic to juvenile oysters Crassostrea virginica and for
embryos of Paracentrotus lividus. Whereas, for embryos and for sperms
stage of P. lividus, the NOEL are respectively 0.25 mg/L and 0.5 mg/L
(Manzo et al., 2006).
Diuron reduces survival of Nitocra spinipes by 50% after 96 h of
exposure at 4 mg/L, and according to Pesticide Ecotoxicology Database
(USEPA, 2000), Diuron has the same effect on Mysidopsis bahia at
1.1 mg/L (DeLorenzo and Fulton, 2012; Karlsson et al., 2006).
Moreover, brine shrimp Artemia salina is the less sensitive to this
antifoulant. Also, Diuron is not very toxic to the juvenile of Palaemon
serratus and of Daphnia magna with an effective concentration upper
than 3 mg/L as shown in Table 9.
6.8.4. Diuron effect on fish
As presented in the table above, half of the population of
Oncorhynchus kisutch and Mugil cephalus dies at a concentration between
2.4 mg/L and 6.3 mg/L. According to Pesticide Ecotoxicology Database
(USEPA, 2003) and Extension Toxicology Network (EXTOXNET, 2000),
Cyprinodon variegatus and Oncorhnchus mykiss are less sensible to this
antifoulant and Diuron is classified as moderately toxic to fish
(DeLorenzo and Fulton, 2012; Okamura et al., 2002). By comparing
Diuron with other antifouling agents, this compound presents the least
toxicity to aquatic species. To exhibit a response, invertebrates require
concentrations higher than 1 mg/L. For fishes, mortality isn’t observed
since concentration does not exceed 2.4 mg/L.
7. Conclusion
Biofouling is a natural phenomenon that appears on any immersed
structure in sea water. It causes several problems on both maritime and
aquaculture fields. Hence, many antifouling formulations have been
applied to control it such as TBT, cooper, Chlorothalonil, Sea Nine 211,
Irgarol 1051, Zinc Pyrithione, Dichlofluanid and Diuron. In this paper,
we collected all data found about these antifouling toxicities on several
marine species. We also detailed some tests applied to evaluate the
toxicity of different biocides on algae, crustaceans and fishes. Bans on
TBT were primarily based on the ecological impacts on species,
bioaccumulation by a range of organisms and potential human health
risks. Meanwhile, the toxic action of copper and TBT has led to the
development of other biocides with better ecological properties.
Among the currently available reinforcing biocides, reference may
be made to Irgarol 1051, Dichlofluanid, Chlorothalonil, Zinc
Pyrithione, Sea Nine 211 and Diuron. Although the lack of information

Table 9
Acute toxicity of Diuron to aquatic organisms.

Species Effect Measured effect/concentration (μg/L) References

Algae
Hormosira banksii Significant impairment to the germination after 48 h and 72 h EC50 = 6290
EC50 = 6820
Significant impairment to growth responses after 48 h and 72 h EC50 = 6750
EC50 = 7330
Chlorella vulgaris 50% growth inhibition at 96 h EC50 = 4.3 Ma et al. (2002)
Chaetoceros gracilis 50% growth inhibition at 72 h IC50 = 36 Koutsaftis and Aoyama (2006)
Selenastrum capricornutum 50% inhibition of growth at 72 h EC50 = 45 Fernández-Alba et al. (2002)

Invertebrates
Paracentrotus lividus Inhibition of the embryonic development after 48 h EC50 = 2390 ± 210 Manzo et al. (2006)
50% inhibition of growth for sperms stage at 48 h EC50 = 5090 ± 450
Crassostrea virginica 50% growth inhibition of juvenile at 96 h EC50 = 1800 Mayer (1987)

Crustacean
Palaemon serratus Mortality of juvenile at 48 h LC50 = 3044 Bellas (2006)
Nitocra spinipes Mortality at 96 h LC50 = 4000 Karlsson et al. (2006)
Daphnia magna 50% of the population exhibit a response after 48 h EC50 = (8.6 ± 1.3). 103 Hernando et al. (2003)
Artemia salina Mortality at 24 h LC50 = 12010 Koutsaftis and Aoyama (2007)
Mysidopsis bahia Mortality at 96 h LC50 = 1100 DeLorenzo and Fulton (2012)

Fishes
Oncorhynchus kisutch Mortality at 96 h LC50 = 2400 Mayer (1987)
Cyprinodon variegatus Mortality LC50 = 6700 DeLorenzo and Fulton (2012)
Mugil cephalus Mortality at 24 h LC50 = 6300 Mayer (1987)
Oncorhnchus mykiss Mortality at 96 h LC50 = 3500 Okamura et al. (2002)

127
I. Amara et al.
available on the effects of these compounds to the marine ecosystems,
we can conclude that Diuron and Irgarol 1051 seem to be the less
harmful to the environment, while Chlorothalonil and Sea Nine 211 are
seen to be more toxic.
Generally, marine aquatic species are sensitive to all these biocides,
so it is important to develop environment-friendly non-stick coatings to
prevent the adhesion of fouling organisms by providing an extremely
smooth surface, on which organisms have great difficulties in settling.
In addition, removal of those which do settle will be easier. Many so-
lutions and formulations have been proposed such as polyethylene
oxide, acrylic resins, silicones, etc. As an example, antifouling paints
based on silicones, specifically poly(dimethylsiloxane), have been de-
veloped and are now commercially available. Whilst they have been
proved to be significantly efficient for the protection of high speed ship
hulls, their application and fixing on textiles used in aquaculture (nets
and ropes) remains a challenge. However, such developments will
hopefully in the future prevent fouling without environmental toxicity.
Conflict of interest
None.
Acknowledgements
We wish to thank the fish farm “RAFAHA” for allowing us to take
some photographs.
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
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