This investigation was designed to study the effect of low concentrations of tetracycline on population growth rate of Lemna minor. Lemna fronds were cultured in solutions made up of Sach nutrient solution of fixed volume and tetracycline of variable concentrations at 0%, 0.00125%, 0.0025%, 0.005%, 0.01% and 0.02% for seven days under the same temperature and light intensity. On the seventh day, frond number in each Petri dish was recorded and growth rate was determined using formula. Results indicated that the higher tetracycline's concentration, the lower the growth rate. At 0.02%, growth was totally inhibited. Pearson Product-Moment Correlation Coefficient analysis proved a statistically significant negative linear relationship between tetracycline concentration and population growth rate at 5 % significant level, supporting the experimental hypothesis. Hence it can be concluded that Lemna minor population growth rate decreases as tetracycline concentration increases.
Key words: Tetracycline; Lemna minor; growth rate
Research and Rationale
This investigation aims to perform a bioassay to study the relationship between low concentrations of tetracycline and population growth rate of Lemna minor.
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Bioassay refers to a scientific procedure to measure the extent of toxic effects of a specific chemical contaminant on a number of one specific sensitive species by exposing them to a range of concentrations of the chemical over a period of time .
Increasing number of scientific publications about the occurrence and impacts of pharmaceuticals in the aquatic environment  is reflecting concerns on these substances being important sources of chemical pollution in this modern age. Among all therapeutic classes, antibiotics have been actively studied owing to their relative significant amounts detected in the environment.
Figure Relative percentages of therapeutics detected in the environment. 
Antibiotics are therapeutic agents capable of killing or inhibiting growth of microorganisms including bacteria, fungi and protozoa . Extensive usage in aquaculture, human and veterinary medicine means that both excreted metabolites and unaltered parent compounds of antibiotics can easily enter the aquatic ecosystem through the discharge of human, livestock, hospital and pharmaceutical industry wastes as well as the disposal of unwanted or expired products. Despite of useful therapeutic application antibiotics offer, the same properties are often reported to post negative impacts on other non-target organisms in the ecosystem.
Figure Pathways of Pharmaceutical Contamination of Aquatic Environment 
Figure Chemical structure of tetracycline 
Tetracycline is a protein synthesis inhibitor with a basic structure of four fused 6-membered rings. Tetracyclines are broad range antibiotics which are effective against many aerobic and anaerobic gram-positive and gram-negative bacteria. They interfere with protein synthesis in bacteria by preventing aminoacyl-tRNA from binding to the acceptor (A) site on 30S ribosomes . This is done by changing the shapes of proteins and ribosomal RNA in the ribosomes so that aminoacyl-tRNAs are no longer compatible to the binding sites. Consequently, codons on messenger RNA cannot be translated into polypeptide sequences. Without proteins, bacteria are halted from growing and reproducing but not killed - tetracyclines are bacteriostatic.
In the USA, tetracyclines have been detected at a concentration of 110 ngl-1 in surface water . Although this concentration is still low to cause any significant effects on aquatic species at the present moment, tetracyclines' occurrence in the environment, like any other pharmaceuticals should be given considerable attention since their amount might increase in the future.
Previous works have reported some contradicting evidence about the effect of tetracyclines on Lemna species' growth. One reported stimulatory effect for concentration between 1 to 10 Î¼gl-1 and strong inhibitory effect at 1000 Î¼gl-1  while another study using a mixture of tetracyclines indicated no inhibitory effect on Lemna minor's close relative, Lemna gibba's growth rate .
Lemna minor is a member of family Lemnaceae (duckweed). This emergent species reproduces by vegetative budding - growing more leaves (fronds) and eventually separate into new individuals. Lemna minor is an ideal indicator species for this study due to several reasons. Firstly, its short life expectancy allows the experiment to be designed for a short period of time. Due to fast-growing property under nutrient-rich conditions and requirement of minimal space for growth, Lemna minor can be easily cultured in nutrient-rich solution in Petri dishes. More importantly, a previous study has found that Lemna minor is the most sensitive species towards pharmaceuticals in many cases in comparison to other test species, namely Daphnia Magma and Desmodesmus subspicatus .
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Figure Lemna minor structure from top view
There are varieties of methods in measuring the growth of Lemna minor including root number, plant number, frond number, root length, frond diameter, dry mass and wet mass. Frond (leaf-like structure) number was chosen as the parameter for this investigation since this method is simple, fast and non-destructive. Growth rate can be calculated using the following equation :
Where r = (population) growth rate per day
xt1 = final number of fronds
xt0 = initial number of fronds
t1 = final day of experiment
t0 = first day of experiment
To allow calculation of growth rate based on this formula, a species must be density independent, small-sized, develops rapidly, reproduce early, and has a short lifespan  . Lemna minor fulfil all these criteria.
Hence, this investigation is an attempt to apply the concept of indicator species to explore the side effect of antibiotics on non-target organism in the aquatic environment.
The higher the concentration of tetracycline, the lower the population growth rate of Lemna minor.
There is no correlation between different concentrations of tetracycline and population growth rate of Lemna minor.
This is to confirm tetracycline's inhibitory effect on Lemna minor's growth. Lemna minor fronds were cultured in two Petri dishes with the first containing mixture of pond water and tetracycline solution and the second containing only pond water for 5 days. The number of fronds was counted and recorded on a daily basis.
Result of Trial 1
Frond number on day
Pond water (control)
Pond water + 2 cm3 of 1% tetracycline
Tetracycline totally inhibited Lemna minor's growth and the fronds even turned brown on the very first day. Meanwhile in the control, Lemna minor continued growing new fronds and remained green throughout the 5-day-period.
The span of the experiment should be increased to better see the effect of tetracycline on the species' growth. To provide optimum nutrition condition for Lemna minor, pond water was decided to be replaced by Sach mineral solution.
This is to work out a suitable range of tetracycline concentrations to be used in the experiment as well as to determine suitable day for t1. Similar method as in Trial 1 was applied. Concentrations chosen were 0.1%, 0.075%, 0.05%, 0.025%, 0.0125% and 0%. For each concentration, 22.5 cm3 of Sach mineral solution was mixed with 2.5 cm3 tetracycline solution of 10 times the required concentration. Different concentrations of tetracycline were prepared by serial dilution method. Lemna minor were allowed to grow for 7 days.
Result of Trial 2
Initial frond number
Frond number on the 7th day
Growth rate (n day-1)
The population growth of Lemna minor was totally inhibited even at a concentration as low as 0.0250%. Hence it was decided that the range should be further lowered. Seven days was found to be a suitable span for the experiment. For the real experiment, 15 fronds would be used for each Petri dish in order to increase the statistical significance of data obtained.
Manipulated : Concentration of tetracycline (%)
Responding : Growth rate per day, r (n day-1)
Constant : Initial number of fronds (15 per Petri dish), initial size of fronds,
volume of Sacch's solution in each Petri dish (22.5 cm3), light intensity
and temperature (placed in same area outside the laboratory).
Petri dishes, 10 cm3 and 25 cm3 measuring cylinders, calibrated dropping pipette, forceps and beakers.
1% tetracycline solution, Sacch's nutrient solution, distilled water, Lemna minor plants and labelling stickers.
Six Petri dishes were labelled as 0%, 0.00125%, 0.0025%, 0.005%, 0.01% and 0.02%.
Serial dilution method was used to prepare tetracycline solutions of concentrations 0.0125%, 0.025%, 0.05%, 0.1% and 0.2% from 1% tetracycline.
25 cm3 measuring cylinder, 10 cm3 measuring cylinder and calibrated dropping pipette were used to measure out Sach nutrient solution and tetracycline solution into each Petri dish as follows:
Concentration required (%)
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Volume of Sach Nutrient Solution (cm3)
Concentration of tetracycline solution used (%)
Volume of tetracycline concentration used (cm3)
15 Lemna fronds of similar sizes were picked out from pond water, rinsed in a beaker of distilled water and placed in Petri dish labelled 0%.
Step 4 was repeated for concentrations 0.00125%, 0.0025%, 0.005%, 0.01% and 0.02%.
Steps 1 to 5 were repeated to produce another two set of replicates.
All Petri dishes were placed in a bright area and left for seven consecutive days.
Frond number was counted on the seventh day and recorded.
Figure Set up of experiment
Lids of Petri dishes were always put on except when transferring Lemna minor into the culture solutions. This is to prevent contamination of culture solutions.
Lemna minor fronds were rinsed in a beaker of distilled water before being placed in culture solution to prevent any unnoticed small buds sticked to them from creating false picture of new frond growth.
Gloves were worn when dealing with Lemna minor from pond water to prevent bacterial infection.
During frond counting, any visible protruding bud was counted as individual frond regardless of size to avoid bias. If any uncertainty arises, counting was repeated to obtain accurate results.
Initial frond number
Frond number on the 7th day
Mean frond number on the 7th day
Sample calculation of growth rate per day:
Pearson product-moment correlation coefficient (PMCC) was chosen to measure the extent of linear dependence between concentration of tetracycline and growth rate of Lemna minor.
Coefficient r indicates the strength of relationship which ranges from -1 to +1. 0 value means no correlation. The range between -1 and 0 represents negative correlation; the further the value lies to the left, the stronger is the negative correlation with -1 representing the perfect negative correlation value. The opposite is true for the positive range.
Table for calculation of PMCC value
*SS = Sum of Squares
Formula to find correlation coefficient, r
r = 0.8886 > 0.0811 (critical value) for 5% significant level
Analysis using PMCC showed a statistically significant negative linear relationship between concentration of tetracycline and Lemna's population growth rate since the calculated r value was greater than critical value at 5% significant level.
Therefore, null hypothesis can be rejected.
From Table 4 and Graph 1, it is clear that the general trend shows a decline in Lemna minor's growth rate per day as the concentration of tetracycline increased. This negative correlation is verified by statistical analysis using PMCC.
At 0%, Lemna minor recorded growth rate of 0.1169. This is the normal growth rate under nutrition-rich circumstances. The graph shows a steep decline in growth rate when tetracycline concentration increased to 0.00125% where growth was inhibited up to about 43%. This is followed by a slower and almost linear decrease in subsequent concentrations. At 0.02%, Lemna minor was totally inhibited from growing new fronds.
Although no noticeable anomalies were found in the data, the decrease in growth rate from 0.00125% to 0.0025% might not be necessarily revealing a true decline since the mean numbers of fronds between the two concentrations only differ by 1 and the percentage decrease in growth rate was only about 5%.
Several sources of errors that could give rise to inconsistencies in the set of data were noted. First of all, the concentrations of tetracycline solutions prepared might not be exact due to limitations in accuracies of measuring cylinder and dropping pipette. Besides, solutions evaporated faster in some Petri dishes and slower in others, leading to alterations in concentrations of tetracycline throughout the seven days.
The data does not reveal the region of stimulatory effect on growth as found in previous study. This is because the range of tetracycline concentration used is far above the concentrations corresponding to this region, i.e. 1-10 Î¼gl-1. Hence, it can be said that there might be hidden trends in the change in Lemna minor's growth rate between 0% and 0.00125%, the steep region of the graph. Apart from that, the decline in growth rate at higher concentrations supported inhibitory effect of tetracycline found in previous study. Hence the findings of this study could be considered valid.
Although previous studies have proven tetracycline's toxicity towards aquatic macrophytes like Lemna minor, the exact mechanism is still an uncertainty. It is interesting that receptors for tetracyclines have been identified in plants, suggesting an uptake mechanism . It is very likely that tetracycline inhibits Lemna minor's growth by the same mode of action against bacteria - protein synthesis inhibition, according to several published journals. For instance, the yellowing effect of tetracycline in some plants was related to the inhibition of translational activity of chloroplast . Chlorotetracycline, which is similar in structure to tetracycline, was found to be able to inhibit protein synthesis including chloroplast synthase . Furthermore, when tested with several photosynthetic organisms, tetracycline was found to be toxic . Another suggestion is that tetracycline complex mineral nutrients, preventing absorption by Lemna, thus causing mineral deficiency and inhibits growth . Tetracyclines are also known to influence the production of abscisic acid (ABA), a type of plant stress hormone involved in growth regulation .
An important limitation in this experiment was that no information could be obtained about the fate of tetracycline in culture solutions throughout the 7-day-period. There are findings stating that tetracycline is an unstable compound which is easily hydrolysed in water and broken down by sunlight ; this could be measured using HPLC-UV/VIS spectrophotometer . However, in another study the efficiency of degradation has been proven low, ranging from 10-20% . Another source stated that tetracycline is non-biodegradable in 28 days . If tetracycline did undergo degradation, the concentration of tetracycline would decrease over time. Nevertheless, this should not significantly affect the result since the span of the experiment was relatively short and the rate of degradation should be fairly uniform in all samples since they were subjected to the same temperature and light intensity.
Besides, although fronds chosen initially were of similar sizes and consisted of one leaf only, they were not all at the same level of maturity. Thus, new fronds could develop faster in some and slower in others. However this is compensated by enough number of replicates.
Frond counting method has a few disadvantages. To avoid bias, small buds that protrude were all counted as individual fronds. A small bud may be less than 5% the biomass of a healthy normal frond but both were considered equal when frond counting method was applied . Thus, frond number is not an accurate measure of growth in terms of biomass. Meanwhile, frond counting did not reflect the status of fronds - alive or dead .
Modification can be done to this experiment by increasing number of fronds used and measuring dry or wet mass of fronds on the 7th day. This is a more accurate means of determining growth rate. Excluding dead fronds in measurements should better indicate population growth since only living individuals are considered to be included in any population.
To further extend this investigation, similar set up can be applied to compare effects of same concentrations of different classes of antibiotics on Lemna minor, for example sulphonamides, macrolides, glycopeptides and quinolones . Different antibiotics may either stimulate or inhibit Lemna minor's population growth. In addition, mixtures of pharmaceuticals could be tested on Lemna minor. This gives a better picture of real situations in the aquatic ecosystem because a drug normally does not exist alone in the environment. Besides, different species such as algae and Daphnia magma could replace Lemna minor as the test species since different species have different sensitivities towards pharmaceuticals, although the use of Daphnia magma may raise ethical issues.
The study showed that as tetracycline concentration increases, Lemna minor's population growth rate decreases. PMCC statistical analysis has proven a statistically significant negative correlation between tetracycline concentration and growth rate. A 100% growth inhibition was achieved at 0.02%. Although the mechanism has not been confirmed, it is clear there are underlying physiological causes behind tetracycline's toxicity towards Lemna minor. Hence, the experimental hypothesis is accepted and the null hypothesis is rejected.
This individual investigation was greatly referred to a number of online scientific journals from Science Direct from subscribed by UiTM Library EZAccess service. Science Direct is a world-leading online database providing more than 2,500 peer-reviewed journals, more than 11,000 books and more than 9 million full-text articles. Thus it is a highly reliable source of journals. Source 5 from Science Direct has provided a great deal of information about antibiotics, their usage, occurrence and consequences these on the environment. Data summarised in this journal were also derived from sources 11 and 14, indicating that these journals are important references among the scientific community.
Source 16 summarised important criteria in selecting indicator species for ecological impact assessments, helping the process in choosing Lemna minor as the most suitable organism to be used in this investigation. These include abundance, biogeographic distribution, population stability, longevity, body size, ecological importance and sensitivity which cross-refer to key points highlighted by online source 13 which stated that macrophytic species such as Lemna are "important in oxygen production, nutrient cycling, control of water qualityâ€¦" (ecological importance) and "relevant to many aquatic environments including lakes, streamsâ€¦ Duckweed plants are widely distributed in the worldâ€¦" (biogeographic distribution).