1.1. The selection of a suitable test species
Toxicity tests are designed to determine the specific concentrations of chemicals that induce a measured effect on a target organism. However, the potency of any toxicant can be influenced by the characteristics of the chemical, as well as environmental (temperature, ph, water chemistry, salinity) and species (life stage, sensitivity, pre-exposure) specific factors. Environmental factors can either modify the toxicant itself or the immediate environment of an organism, increasing or decreasing the effects of the toxicants. Species specific factors can alter the organism/toxicant interaction by modifying the rate of uptake, distribution, elimination and detoxification pathways. Therefore when conducting toxicity tests, it is important to have controlled environmental conditions and select suitable test species to ensure reliable, relevant, reproducible, defensible and ecologically significant results. The selection of a suitable test species can be based on several criteria:
the species should be widely available
they should be easily maintained under laboratory conditions and provide sufficient numbers of an appropriate size and age
the genetics, genetic composition and history of the organisms should be known
they should the most sensitive species in the environment.
should be recreationally, ecologically and commercially important
organisms should be in good physiological condition
it should be indigenous or representative of the eco-region being studies
Since the first toxicity tests were performed over 60 years ago, fish and invertebrates continue to be the most popular test species because there exist a significant knowledge base on their physiology, biochemistry, behavior, reproduction, life cycle and ecological importance. However there continues to be an ongoing debate as to whether certain life stages are more sensitive to toxicants than others. The general assumption in toxicology has been to utilize the ‘most sensitive’ life stage which toxicologists assume to be the earliest life stages for a given species. Selecting the most sensitive life stage provides a quick, relatively easy and sensitive toxicity test with the added advantage of having a low cost and test duration. In fact, many standardized test protocols often specify the preferred life stage to be used for testing. For example, US Environmental Protection Agency (US EPA) [EPA-600/4-80/001, EPA-812-R-02-012], and American Society for Testing and Materials (ASTM) [ASTM E1192-97(2008), ASTM E1267-03(2008) and ASTM E724-98(2004)] test protocols recommend the use of early life stages; first instar of daphnia, juvenile mysids, juvenile fish or embryos of mollusks as the most suitable for toxicity tests. The choice of early life stages has been based on the premise that they are the most susceptible to toxicants and that toxicity data using the most vulnerable life stage would offer protection to all life stages in the natural environment.
2. Early life stages in toxicity tests
Numerous studies have reported that the early life stages of fish and invertebrates were more sensitive to toxicants than the adult organisms (Herkovits et al. 1997, Schmieder et al. 2000, Hutchinson et al. 1998, Mohammed et al. 2009). Schmieder et al. (2000) reported that for medake, embryo-larval stages showed 50% mortality when 2,3,7,8-Tetrachlorodibenzo
3. Early life stages as the most sensitive stage
Variations in sensitivity between life stages have been reported for organisms such as
Other studies have reported that early life stages such as first instar of daphnia, juvenile mysids, juvenile fish and embryos are more susceptible than adults following exposure to toxicants such as heavy metal (Bodar et al. 1989; Gopalakrishnan 2008; Hoang and Klaine 2007; Green et al. 1986; Verriopoulos and Morai'tou-Apostolopoulou 1982). For example, George et al. (1996) reported that the larva (yolk sac stage) of
4. Probable causes for variations in sensitivity between life stages
The apparent variability in sensitivity between early life stages and adults may be due to several factors; surface area/volume ratio (particularly with young fish); the greater likelihood that juveniles may have accumulated less fat than adults thus having less capacity to store lipophilic substances; greater uptake of toxicant from the environment; under developed homeostatic mechanism to deal with the toxicants; immature immune systems and under developed organs (liver and kidney) which has an important role in detoxification and elimination of toxicants.
There are various ideas that seek to explain why early life stages are more sensitive than adults. These often take into consideration specific behavioral, morphological, physiological and biochemical characteristics which may be different between life stages. Some of the main ideas include:
Organ systems may become sensitive to the effects of toxicant at certain periods during early development but once developed, they may no longer be vulnerable (Ozoh 1979, Bentivena and Piatkowski 1998).
The time taken for toxicants to reach target sites may be shorter in early life stages, because of their smaller size when compared to later stages and adults.
Most embryo and larval forms may have poorly developed organs such as gills, liver and kidneys. They also have permeable skin which, in early life stages, is the primary means of ionic regulation. The skin presents a larger surface area for the uptake of toxicants, resulting in increased susceptibility of the larva when compared to the adults.
In crustaceans research has shown that metals can concentrate in the body covering thus reducing their entry into the body. The completion of body covering diminishes the entry of metal into the body, thereby increasing the resistance of older forms.
Some toxicants may be sequestered in fat tissue or specific proteins preventing them from reaching target organs.
The increased susceptibility of early life stages may also be related to other factors such as; differential rates of absorption/uptake distribution or detoxification. Organisms have also evolved intricate regulatory (physiological, immunological and biochemical) mechanisms which allow them to survive, grow and reproduce. Some of these mechanisms are also important in the elimination, detoxification or reduction of the effects of toxicant. For example, kidneys and liver may be involved in the elimination of toxicants, while various proteins and enzymes such as Metallothioneins and Mixed Function Oxygenases are induced following exposure to metals and hydrocarbons. It is often suggested that the difference in the development of these mechanism and immature detoxification pathways in early life stages can also be a basis for the apparent increased sensitivity of juveniles exposed to toxicants. A few specific factors for increased sensitivity will me discussed below.
4.1. Avoidance strategies
The higher sensitivity of early life stages may be explained by behavioral, morphological, physiological or biochemical changes. Free swimming species are able to avoid toxicants, while some sessile species such as bivalves may close their valves to avoid contact with the toxicants. Disruption in behavioral responses may include:
impaired feeding ability resulting in poor diet, which can cause reduced growth and longevity;
altered predator - avoidance behavior;
impaired schooling leading to increased mortality and/or altered reproductive function,
movement away from the source of the toxicant, or
as in the case of bivalves, closure of the valves for varying periods of time in order to reduce exposure (Weis, 2005).
Behavioral responses can have greater impacts on earlier life stages, which show significantly less physiological and morphological development than adults. Kennedy et al. (2006) showed that adult
4.2. Morphological characteristics
Avoidance strategies undoubtedly result in decreased exposure, but it is unlikely that chemical avoidance alone can account for the difference in sensitivity between adults and early life stages. In some species such as
In many fish species, larval forms are generally more sensitive than juveniles and adult forms. Newly hatched larvae constitute a particularly critical and sensitive life stage, because at hatching the embryos lose their protective membrane and are fully exposed to potential toxicants (Arufe et al. 2004). A significant characteristic of most larval stages is the fast changing morphology giving rise to the adult forms. Organ systems may become sensitive to the effects of toxicant at certain periods during early development but once developed, they may no longer be vulnerable (Ozoh 1979, Bentivena and Piatkowski 1998). Middaugh and Dean (1977) reported that cadmium was more toxic to the 7-day-old larvae (LC50 = 12 mg Cd/L) of
4.3. Detoxification mechanisms
Toxicants may induce synthesis of specific proteins specific proteins such as Mix Function Oxygenases and Metallotheinions which may detoxify or sequester toxicants, thus reducing their toxic effects. However, early life stages may lack fully-expressed enzyme systems for efficient detoxification and elimination of toxicants because of slow organ development. In most adult organism detoxification and elimination processes follow one of two pathways depending on whether the toxicant is a metal or organic compound (Figure 1). As previously stated, difference in the development of these mechanism and immature detoxification pathways in early life stages can also be a basis for the apparent increased sensitivity of juveniles exposed to toxicants.
Metallothioneins are non-enzymatic cysteine rich, low molecular weight proteins of about 7 kDa and apparent molecular weight of 13 kDa. The metallothioneins pool is made up of various isoforms each having different physiological roles and different induction pathways and are important in homeostasis of metals such as copper and zinc, and detoxification of heavy metal (Butler and Roesijadi, 2001). Mason and Jenkins (1995) proposed two roles for metallothioneins in the regulation of metals in organisms.
They may comprise a non-toxic zinc and copper reservoir available for the synthesis of metalloenzymes, allowing the homeostasis of many cellular processes (Brouwer et al. 1989; Viarengo and Nott 1993; Roesijadi 1996).
The induction of metallothioneins confers metal tolerance to organisms (Klaasseen et al. 1999) due to their ability to bind and sequester some heavy metals. However, the ability of metallothioneins to reduce metal toxicity can vary with the age of organism.
The sequestration of metal ions by metallothionein is considered to be one of the most common detoxification pathways for metals in adult organisms. Its presence in organisms can therefore also be used to help explain the variable susceptibility of different life stage to metals. Synthesis of metallothionein is strongly induced by transcriptional activation of metallothionein gene expression following exposure to metals (George et al. 1992, 1996; George and Olsson, 1994; Zafarullah et al. 1989). Laville (1988) showed that in mice, metallothionein mRNA in liver depended on the age at which exposure to cadmium occurred. Exposure to 2mg Cd/kg resulted in a small increase (two- to threefold) in levels of metallothionein mRNA in livers of 7- and 14-day-old mice. However, cadmium treatment of 28- and 56-day-old mice resulted in 12- to 19-fold increases in levels of metallothionein mRNA in liver. George et al. (1996) used metallothionein gene expression (mRNA) to map changes in protein expression during development of
All organism have at least some ability to metabolize organic compounds. These often involve some enzyme mediate detoxification pathway requiring one or more enzymes such as cytochrome P450 monooxygenase, epoxide hydrolase and other conjugating enzymes (Figure 1) associated with the liver or kidney. In most adult organisms, these pathways are well developed (Shailaja and D’Silva 2003; Tuvikene 1995; Eisler, 1987). In juveniles, induction of these may also occur, once organ systems are fully functional. Oikari et al. (2002) was able to show that in juvenile rainbow trout, exposure to contaminated sediments significantly induced trout liver CYP1A activity. However Sassi et al. (2012) reported that for gilthead sea bream larvae were unable to show transcription of Gpx in following exposure to cadmium. Gpx is responsible for the break down hydrogen peroxide as in adult organisms.
The greater sensitivity of early life stages when compared to adults can therefore be explained by a number of physiological, morphological, behavioral and biochemical characteristics. It may appear that in early life stages these responses are either underdeveloped or have not yet developed fully thus contributing to the increased sensitivity of these early life stages when compared to the adults.