Research Sample Paper on Toxicity Testing with Earthworms


In this experiment, the effects of nicotine and caffeine on the locomotion of Eisenia foetida were examined. Earthworms (Eisenia foetida) exhibited a distinctive thrashing motion in freshwater. The rate of their thrashing motions was determined as tail whips per second.  Caffeine affected the Eisenia foetida more than the nicotine. Hence, it is more toxic to the earthworms than the nicotine. The effects of both chemicals on the locomotion of Eisenia foetida depended on their concentrations. The thrashing motion increased with increase in concentrations of nicotine and caffeine.


Earthworms are suitable test-organisms in the ecotoxicological studies since they are widely found in soil and water bodies (Van Gestel, 1992). They represent about sixty to eighty percent of the population of soil and water animal or invertebrate biomass. Earthworms are usually in intimate contact with water and soil. They are the foundation of various food webs.  Further, earthworms accumulate a lot of different metals into their tissues when they come in contact with the polluted water and soil (Van Gestel, 1992). Earthworms play crucial roles in soil structure and fertility. They recycle nutrients, upsurge soil drainage, and aeration. Additionally, they constitute part of the diet of small mammals, birds, and reptiles (Van Gestel, 1992). Hence, earthworms are fundamental biological pointers of contaminants in the soil and water.

The toxicity tests with earthworms are presently used as a foundation for international regulatory rules in the European Union risk assessment. Earthworms have been used for toxicity testing for more than twenty years. Though, there are some hurdles. For instance, the Eisenia foetida is commonly used as test organism since it is more susceptible to pollutants as compared to other earthworms (Van Gestel, 1992). However, it is not frequently found in natural soils.

Earthworms respond differently to stimulants and depressants. In this research, nicotine and caffeine were used to determine their effects on the locomotion of Eisenia foetida. The objectives of the experiment were to expound Probit analysis, dose concept, E50 values, and determine the route exposure, distinguish between chronic and acute toxicity and identify the detoxification process. It was hypothesized that both caffeine and nicotine increase thrashing motion of Eisenia foetida in fresh water.


The experiment was conducted in two parts: the nicotine and caffeine tests.

Nicotine test

Serial dilution was prepared using a 2:1 serial dilution. The dilution range was from 0mg/ml to 0.0066mg/ml nicotine.  Earthworms were carefully removed from the pre-testing soil, followed by placing them in a diluted water bath to clean the worms. The cleaned worms were removed from diluted water bath one by one and placing them into the prepared exposure well. The locomotion characteristic of each worm was cautiously observed.  After 8 minutes each worm was removed from the exposure well and placed in distilled recovery water bath.  The worms were allowed to stay in the distilled recovery water bath for another 8 minutes.  They were then lifted to the post-test soil. The lifted worms were not tested again.

Caffeine test

0.25mg/ml caffeine stock solution was used as the caffeine exposure solution.  By using a caffeine stock solution the same procedures followed for nicotine test were repeated to determine the caffeine effects on the locomotion of Eisenia foetida.

Treatments Locomotion observation
1 Increased thrashing motion
2 Increased thrashing motion
3 Increased thrashing motion
4 Increased thrashing motion
5 Increased thrashing motion
Control No observable change

Table 1: Nicotine test

Treatments Locomotion observation
1 Increased thrashing motion
2 Increased thrashing motion
3 Increased thrashing motion
4 Increased thrashing motion
5 Increased thrashing motion
Control No observable change

Table 2: caffeine test





Figure 1: Earthworm behavioral endpoint

The locomotory inhibition endpoint for nicotine became apparent at 0.0066mg/ml, while for the caffeine it became apparent at 0.25mg/ml.

The estimate of the toxic range of nicotine was from 0.0042mg/ml to0.0066mg/ml. On the other hand, the toxic range of caffeine was from 0.22mg/ml to 0.25mg/ml.

C Inhb




(Motulsky, 2002)


N Inhb means Eisenia foetida locomotion influence in the presence of nicotine

C Inhb means Eisenia foetida locomotion influence in the presence of caffeine

Figure 2: Graph of means of EC50 values for nicotine and caffeine


Nicotine and caffeine acted as stimulants. They increased the thrashing motion of Eisenia foetida in freshwater environments.  According to the results (Figure 2), caffeine was more toxic to the Eisenia foetida as compared to the nicotine.  Nicotine and caffeine increased the thrashing motion while there was no observable change seen in the control (Tables 1 and 2). It was also observed that the nicotine and caffeine did not have a consistent influence on the Eisenia foetida locomotion. Their effects depended on the concentration. As the concentration of each chemical was increasing, the thrashing motion was also increasing. Hence, the toxicity of the caffeine and nicotine increase with the increase in their concentration. When the nicotine and caffeine are released to the aquatic environment, their effects on the aquatic organisms would be less the same as their effects on the Eisenia foetida in fresh water. The contaminated of the exposure wells might have influenced the results of the experiment. Additionally, change of the environment of the worms might have interfered with their movement, hence giving false results.


The nicotine and caffeine, both had effects on the locomotion of the Eisenia foetid. They acted as stimulants. This implies that the Eisenia foetida were moving away from these chemicals for their safety. The caffeine was more toxic than the nicotine as it increased the thrashing locomotion of the Eisenia foetida more than the nicotine. Therefore, both the caffeine and nicotine are apt for the toxicology research. They are a suitable model for determining the effects of the stimulants for the possible drug interactions.






Reference Lists

Motulsky, H. (2002). Fitting models to biological data using linear and nonlinear regression: a particular guide to curve fitting. Melbourne: Graphpad Software Inc, 1-53.

Van Gestel, CA.(1992). Validation of earthworm toxicity tests by comparison with field studies: a review of benomyl, carbendazim, and carbaryl. Ecotoxicol Environ Saf., 23(2), 221-236.