An investigation on the inhibitory effects of physical and chemical sunscreens on the growth of bio indicators Allium cepa and Lepidium sativum.

Transcription

1 An investigation on the inhibitory effects of physical and chemical sunscreens on the growth of bio indicators Allium cepa and Lepidium sativum. By Victor Abrahamsson Extended Essay: Biology HL Session number: Session: May 2013 IBDP2, Katedralskolan, Lund, Sweden Supervisor: Annette Carlström Word Count: 3983

2 Abstract The aim of this investigation was to study environmental effects of 4 sunscreens. Hence the following research questions were addressed. Which category of sunscreens, physical or chemical UV-filters, containing active substances Titanium Dioxide, and Butyl Methoxydibenzoylmethane and Octorcrylene respectively has greatest inhibitory effects on mass of Allium cepa roots, furthermore which of the 2 physical and which of the 2 chemical sunscreens have greatest inhibitory effects Which of Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise and Vichy Capital Soleil have the least Minimum Inhibitory Concentrations affecting Lepidium sativum plant length and which decrease germinability of Lepidium sativum seedlings the most Solutions containing the different sunscreens were prepared in which Allium cepa and Lepidium sativum were grown 5 days. Allium cepa grew in concentrations of 2cm 3 /dm 3 sunscreen per dm 3 water, Lepidium sativum was grown in a dilution series of 2cm 3 /dm 3, 0.2cm 3 /dm 3, 0.02cm 3 /dm 3 and 0.002cm 3 /dm 3 sunscreen per dm 3 water. The mass of 5 Allium cepa roots for each Allium cepa was recorded to find the mass percentage decrease compared to the control to notify inhibitory effects. Plant lengths from root to tip of Lepidium sativum were recorded to determine the Minimum Inhibitory Concentrations. Compared to the control, Nivea Sun, containing Paraffinum Liquidum and Titanium Dioxide had the greatest inhibitory effects on root mass (83.2%). Apoteket Hjärtats Sollotion and L Oreal Solar Expertise containing Butyl Methoxydibenzoylmethane and Octorcrylene had inhibitory effects of 75.5% and Vichy Capital Soleil containing Titanium Dioxide had the least inhibitory effect (57.1%). MIC occurred at lowest concentrations for Nivea Sun and L Oreal Solar Expertise (0.02cm 3 /dm 3 to 0.002cm 3 /dm 3 sunscreen per dm 3 water). MIC for Apoteket Hjärtats Sollotion and Vichy Capital Soleil occurred later (0.02cm 3 /dm 3 and 0.2cm 3 /dm 3 sunscreen per liter water) suggesting Paraffinum Liquidum was most inhibitory followed by Butyl Methoxydibenzoylmethane and Octorcrylene. Titanium Dioxide had the least inhibitory effects. Word count: 300

3 Table of Contents Introduction... 1 Hypothesis... 5 Method... 6 Experiment Experiment Results... 8 Experiment Qualitative Observations... 9 Experiment Qualitative Observations Discussion and Conclusion Experiment Experiment Evaluation Acknowledgements Bibliography Appendix Appendix 1: Materials Bioindicators and Sunscreens Equipment for experiment Equipment for experiment Appendix 2 Method Method for Allium Test: Method for Lepidium sativum test: Appendix 3 Raw Data Experiment Experiment Appendix 4 Calculations Appendix 5 Ingredients of the sunscreen products... 51

4 Introduction Aim The aim of this investigation was to study environmental effects of a selection of sunscreens using 2 plant bioindicators Allium cepa and Lepidium sativum. The sunscreens investigated were chosen to represent a range of commonly used sunscreen products containing substances Butyl Methoxydibenzoylmethane, Octorcrylene, Paraffinum Liquidum and Titanium Dioxide. The sunscreens investigated were chosen to be Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise, and Vichy Capital Soleil Consequently, following research questions were investigated. Which category of sunscreens, physical UV-filters containing Titanium Dioxide or chemical UV-filters, containing Butyl Methoxydibenzoylmethane and Octorcrylene will have the greatest inhibitory effects on growth in mass of Allium cepa roots furthermore, which of the 2 physical and which of the 2 chemical sunscreens have greatest inhibitory effect Which of Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise and Vichy Capital Soleil have the least Minimum Inhibitory Concentrations affecting Lepidium sativum plant length and which decrease germinability of Lepidium sativum seedlings the most I read an investigation carried out by the Swedish environmental organization Naturskyddsföreningen who investigated the presence of environmentally damaging substances in UVA and UVB repellant lotions, the investigation also mentioned there had been a great increase in use of UVA and UVB repellant lotions 1. The substances investigated by Naturskyddsföreningen were known to be particularly harmful to aquatic environments. Sunscreen lotions are used to block radiation from the sun which in larger quantities can cause irritated skin, sunburn and cancer 2. 1

5 Because sunscreens are frequently used near beaches or shores and the use of sunscreens has increased lately it is important to study the effects of the sunscreens available and determine how damaging runoffs of these sunscreens are and at which concentrations the runoffs start to affect the environment. Sunscreens are PPCP substances, short for pharmaceuticals and personal care products 3. The environmental implications of PPCP s are very speculative and little research has been done to outline the environmental effects of PPCP s. Much research that has been done has investigated increased estrogen levels in fish leading to more hermaphroditic fish, said to be a result of wastes from hormonal pharmaceutics from sewages into lakes and seashores 4. However a research in Switzerland found evidence that substances commonly found in sunscreens were present in measurable quantities in freshwater fish 5. Other studies have confirmed that at least one of those substances increase estrogenic hormone production in animals 6, 7. Because little research has been done on the effects of sunscreen runoffs in the environment, there is need to further investigate. To carry out an investigation that represented a wide variation of the products available on the Swedish sunscreen market (this investigation occurred in Sweden), the environmental effects of following sunscreens were investigated using bioindicators Allium cepa (onion) and Lepidium sativum (cress): Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise and Vichy Capital Soleil. Investigating the environmental effects of 4 sunscreens allowed for thorough investigations and repeated trials to produce more trustworthy results than if there had been a larger variety of sunscreens but lower repeats per sunscreen. In year 2007 Läkemedelsverket, approved 28 UV-filters for use in sunscreens 8. Some of the most popular UV-filters are Butyl Methoxydibenzoylmethane, Octorcrylene and Titanium Dioxide 1. The sunscreens chosen in the investigation contain either one or more of these substances and are therefore a worthy set of representatives. 2

6 Apoteket Hjärtats Sollotion contains substances Butyl Methoxydibenzoylmethane and Octorcrylene. The UV-filters are commonly used together to cover the entire UVA and UVB wave span (wavelengths nm) and to prevent degrading of Butyl Methoxydibenzoylmethane 9, 10. Octorcrylene is considered a stable substance and works as photostablilizer for Butyl Methoxydibenzoylmethane 9, 10 which otherwise during high frequencies of radiation degrades into allergenic compounds, known to affect several organisms 10, 7. The sunscreen is a crème, it is applied by smearing onto the skin and it dissolves in water after some mixing. L Oreal Solar Expertise also contains Butyl Methoxydibenzoylmethane and Octorcrylene. Because it is a spray it mixes better in water than Apoteket Hjärtats Sollotion giving reason for a comparison between spray sunscreens and viscous sunscreens. Because many sunscreens are in the form of sprays it was important to include one in the investigation to yield a good representation of Swedish sunscreens. The sunscreens were chosen because the UV-filters in the sunscreens are important to study. Butyl Methoxydibenzoylmethane and Octorcrylene are both PBT- classified chemicals 1, (persistent, bio accumulative and toxic). Research has been done to outline effects of these chemicals on the environment and humans. Investigations have shown sunscreens containing Butyl Methoxydibenzoylmethane have environmental effects already at low concentrations 11. Investigations in Suisse lakes have detected Octocrylene in aquatic plants and fish, the chemical is said to have serious effects on the aquatic life 5. Octocrylene can, when applied to the surface of an organism, pass through epidermis and increase creation of free radicals when exposed to light 9. Free radicals are known to damage DNA 12 Choosing the same substances in the sunscreens allowed the comparison of different effects of the sunscreens depending on how well they dissolve in water. Because the substances are common in chemical sunscreens in Sweden, choosing the same substances for both chemical sunscreens in the investigation still provides a representative image of the environmental effects of chemical sunscreens available in Sweden. Since the other sunscreens are based on physical UV-filters good comparisons between the environmental effects of chemical and physical UV-filters can be made, making it possible to deduce based on the data from the investigation which sort of sunscreen has the least effects on the environment. 3

7 Sunscreens are categorized into two subgroups, chemical UV-filters and physical UV-filters. Chemical UV-filters protects skin from sunlight by absorbing sunrays while physical UVfilters repel sunrays away from the skin 13. Apoteket Hjärtats Sollotion and L Oreal Solar Expertise are chemical sunscreens while Nivea Sun and Vichy Capital Soleil are physical UVfilters. Vichy Capital Soleil contains the physical UV-filter Titanium Dioxide. It is regarded as nontoxic in its ordinary state 13. It is a viscous crème which dissolves in water after some mixing and was chosen to represent physical UV-filters which are approximately equally represented in Sweden as chemical UV-filters. Physical UV-filters have been suggested less dangerous for both the environment and humans which is why it is frequently used in children s sunscreens. Because Titanium Dioxide is the only physical UV-filter used in Sweden (in 2009 Zink Oxide was banned by Läkemedelsverket 14 ) it was important to include a Titanium Dioxide sunscreen in the investigation to represent the sunscreens available in Sweden. Nivea Sun contains Titanium Dioxide, it also contains Paraffinum Liquidum. It was chosen because it contained Paraffinum Liquidum, or mineral oil. Mineral oils are insoluble in water and used in 98% of all cosmetics worldwide 15. Because mineral oils don t dissolve in water, sunscreens containing mineral oils are often used when sunbathing near water. Mineral oils are believed to affect mineral and water uptake in plants 15. Inhibition of mineral and water uptake into plants will thus lead to decreased levels of growth. Because there is a large representation of sunscreens containing mineral oils it was important to include a sunscreen containing mineral oil in the investigation. Choosing the same active substances in the physical UV-filters, the investigation can deduce whether sunscreens containing mineral oils have greater effects on the environment than sunscreens which contain no mineral oils. In order to test inhibitory effects of the selected sunscreens, two methods using biological indicators were used. By recording growth of bioindicators, scientific conclusions could be drawn regarding toxicity of the substances tested. 4

8 The bioindicators used in this study to test the inhibition of the selected sunscreens were Allium cepa (onion) and Lepidium sativum (garden cress). They were chosen due to their rapid growth, common use as bioindicators and sensitivity to toxic compounds 16, 17. Test methods used in this investigation were the Allium test 16 measuring root growths in mass of Allium bulbs and a method referred to as the Lepidium test which measured the plant length from root to tip for Lepidium sativum. Hypothesis Based on information presented the introduction, experimental hypothesis (H 1 ) concerning first research question would predict greater inhibitory effects from chemical sunscreens than physical sunscreens, the prediction is based on results from earlier studies suggesting that the chemical sunscreens tested will have greater inhibitory effects than the physical sunscreen tested as Titanium Dioxide is said to be non-toxic, not as bio accumulative and not as persisting as the chemical UV-filters. Moreover, the hypothesis would predict larger inhibitory effects of L Oreal Solar Expertise than Apoteket Hjärtats Sollotion as it dissolves better in water. While Apoteket Hjärtats Sollotion is viscous and forms a sediment on the bottom of the container L Oreal Solar Expertise dissolves completely, contributing to lower concentrations of Apoteket Hjärtats Sollotion in the upper sections of the solution from which the roots of Lepidium sativum absorbs water, compared to L Oreal Solar Expertise Conclusively the hypothesis predicts that Nivea Sun will have higher inhibitory effects than Vichy Capital Soleil as it contains mineral oils and Vichy Capital Soleil does not. 5

9 Experimental hypothesis 2 (H 2 ) would predict that lowest MIC would occur in Nivea Sun as it consists of mineral oils, because they inhibit mineral and water uptake of the roots of plants Nivea Sun is likely to have lowest MIC being the only sunscreen containing mineral oils. Moreover chemical sunscreens L Oreal Solar Expertise and Apoteket Hjärtats Sollotion are likely to have intermediate Minimum Inhibitory Concentrations compared to the other sunscreens as they are chemical sunscreens with suggested greater inhibitory effects of plant lengths of Lepidium sativum seedlings. It is thus believed that Vichy Capital Soleil has highest Minimum Inhibitory Concentration as it is a physical sunscreen based on Titanium Dioxide, said to be non-toxic that does not contain any mineral oils. The germination percentage of Lepidium sativum seedlings grown in different solutions of sunscreens are believed to follow the suggested order in which the sunscreens will have their Minimum Inhibitory Concentrations because greater inhibitory effects will lead to lower germination percentages. The null hypothesis (H 0 ) states there will be no inhibitory effect on growth of Allium cepa roots or Lepidium sativum seedlings from the sunscreen products tested compared to the control. As such, research questions 1 and 2 are invalid if H 0 is proven correct as they depend on the presence of inhibitory effects. Method The aim of this study was to investigate inhibitory effects of the active substances in 4 sunscreens and to determine their Minimum Inhibitory Concentration and germination percentage. The Allium test and the Lepidium test were used on bioindicators Allium cepa and Lepidium sativum. Both methods needed a variety of materials and procedures (presented in Appendices 1 & 2). Experiment 1 Allium test was used to test whether the sunscreens had inhibitory effects on Allium cepa roots in terms of mass. 6

10 Peeled Allium cepa bulbs of sizes 1cm-2cm in diameter were placed on polystyrene boards floating in plastic containers of dimensions 13cm x 10cm x 5cm filled with solutions of the different sunscreens. Growths of the 5 largest roots of each Allium cepa were compared to a control grown in tap water. Each container was filled with 500cm 3 tap water and 1cm 3 sunscreen or 500cm 3 of tap water and no sunscreen (control). 5 Allium cepa were fitted into holes previously prepared in Polystyrene boards in such a way that all Allium cepa had equal access to water. The Allium cepa bulbs were pushed into holes of the Polystyrene boards so that approximately 0.5cm of the lower end of the Allium cepa bulb accessed the solution. After 5 days of growth at temperature 21 C and equal access to light and humidity, the Allium cepa were collected and the 5 largest roots on each Allium cepa were recorded for mass. The procedure was repeated 5 times and resulted in totally 25 recordings of the 5 longest roots of each Allium cepa in each solution. Detailed descriptions of the material list and experimental procedure are present in Appendix 1.2 and Appendix 2.1. Experiment 2 The Lepidium test was used to find MIC and germination percentage of the sunscreens on Lepidium sativum. A dilution series of 2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water was used to determine the range in which MIC occurred. Containers were filled with 250cm 3 of each of those concentrations, cotton pads approximately 70cm 2 were placed in the solutions and 25 Lepidium sativum seeds were spread over the cotton pads. After 5 days of growth at temperature 20 C and equal access to light and humidity, the Lepidium sativum seedlings were recorded for plant length from tip of root to top of cotyledon leaves (cm) and germination percentage (%) and compared to a control grown in tap water. Plant length was recorded to deduce the MIC of the sunscreens and germination percentage was recorded to further indicate inhibitory effects of sunscreens. The procedure was repeated 4 times and resulted in 100 recordings of plant length (cm) for the 16 solutions of sunscreen and 200 recordings of plant length (cm) for the control. Detailed descriptions of the material list and experimental procedure are present in Appendix 1.3 and Appendix

11 Results Experiment 1 Table 1. The table shows the mean mass (g) and standard deviation (g) of the 5 largest Allium cepa roots on an Allium cepa. Furthermore it shows the coefficient of variation (%), the percentage change in mass (%) compared to the control group. The table also shows the probability that the difference between the mean masses of the Allium cepa roots grown in sunscreen solutions and the mean mass of the control is due to chance (%). Each value in the table is based on the mass (g) of the 5 largest roots of 25 Allium cepa in each of the sunscreens (Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise and Vichy Capital Soleil) Statistical analysis of the inhibition on mass growth of Allium cepa in sunscreen solutions Mean Coefficient mass/g of variation / % Standard Deviation of mass/g Percentage change in mass as compared to water/ % P-value (from an unpaired t- test) / % Sunscreen Product Apoteket Hjärtats Sollotion <0.01 Nivea Sun <0.01 L Oreal Solar Expertise <0.01 Vichy Capital Soleil <0.01 Water

12 Mean mass of Allium cepa in sunscreens / g Victor Abrahamsson 0,060 0,050 Mean mass of Allium cepa roots grown in different solutions of sunscreens. Water 0,040 0,030 0,020 Apoteket Hjärtats Sollotion Nivea Sun L Oreal Solar Expertise Vichy Capital Soleil 0,049 0,010 0,000 0,012 0,021 0,008 0,012 Sunscreen Products 2cm 3 /dm 3 water Figure 1. The bar chart denotes the means of the mass of 5 Allium cepa roots for each of the sunscreens tested for inhibitory effect (Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise & Vichy Capital Soleil) along with their respective standard deviations (g). The values are based on the mean mass of the 5 largest onion roots collected from each of the 25 Allium cepa collected from the 4 different sunscreens and water solutions. The data presented in Figure 1 was obtained from Table 1. Qualitative Observations Allium cepa roots grown in the solution containing Nivea Sun were deformed while Allium cepa roots of the control grown in water were not. Allium cepa roots grown in Apoteket Hjärtats Sollotion and L Oreal Solar Expertise solutions appeared slightly less deformed. Allium cepa roots grown in Vichy Capital Soleil had almost no deformities. 9

13 Germination percentage of Lepidium sativum / % Victor Abrahamsson Experiment 2 Table 2. The table shows the germination percentage and the range of the plant length of the Lepidium sativum. The data is based on the recording of the germination and lengths of the 100 Lepidium sativum seedlings planted in each of the concentrations of sunscreen solution investigated during Experiment 2 (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water ), (Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise and Vichy Capital Soleil) Germination percentage of Lepidium Range of growth/cm Concentration of sunscreen solution / % Apoteket Hjärtats Sollotion sativum seedlings / % L Oreal Nivea Solar Sun Expertise Vichy Capital Soleil Apoteket Hjärtats Sollotion Nivea Sun L Oreal Solar Expertise Vichy Capital Soleil Germination Percentage of Lepidium sativum grown in different concentrations of sunscreen Apoteket Hjärtats Sollotion in water Apoteket Hjärtats Sollotion 2cm 3 /dm 3 water Apoteket Hjärtats Sollotion 0.2cm 3 /dm 3 water Apoteket Hjärtats Sollotion 0.02cm 3 /dm 3 water Apoteket Hjärtats Sollotion 0.002cm 3 /dm 3 water Apoteket Hjärtats Sollotion Figure 2. The bar chart illustrates the Germination Percentage of Lepidium sativum seedlings (%) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreens per dm 3 water) of sunscreen Apoteket Hjärtats Sollotion dissolved in water for 5 days, the value of the bars (%) are derived from data from Table 2. 10

14 Germination Percentage of Lepidium sativum/ % Victor Abrahamsson Germination Percentage of Lepidium sativum grown in different concentrations of sunscreen L Oreal Solar Expretise and water L Oreal Solar Expertise 2cm3/dm3 93 L Oreal Solar Expertise 0.2cm3/dm3 87 L Oreal Solar Expertise 0.02cm3/dm3 93 L Oreal Solar Expertise 0.002cm3/dm L Oreal Solar Expretise Figure 3. The bar chart illustrates the Germination Percentage of Lepidium sativum seedlings (%) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water) of sunscreen L Oreal Solar Expertise dissolved in water for 5 days, the value of the bars (%) are derived from data from Table 2. 11

15 Germination Percentage of Lepidium sativum / % Victor Abrahamsson Germination Percentage of Lepidium sativum grown in different concentrations of sunscreen Nivea Sun and water Nivea Sun 2cm3/dm3 water Nivea Sun 0.2cm3/dm 3 water Nivea Sun 0.02cm3/dm 3 water Nivea Sun 0.002cm3/dm3 water Nivea Sun Figure 4. The bar chart illustrates the Germination Percentage of Lepidium sativum seedlings (%) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water) of sunscreen Nivea Sun dissolved in water for 5 days, the value of the bars (%) are derived from data from Table 2. 12

16 Germination PErcentage of Lepidium astivum / % Victor Abrahamsson 100 Germination Percentage of Lepidium sativum grown in different concentrations of sunscreen Vichy Capital Soleil in water Vichy Capital Soleil 2cm3/dm3 Vichy Capital Soleil 0.2cm3/dm3 water Vichy Capital Soleil 0.02cm3/dm3 Vichy Capital Soleil 0.002cm3/dm3 water Vichy Capital Soleil Figure 5. The bar chart illustrates the Germination Percentage of Lepidium sativum seedlings (%) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water) of sunscreen Vichy Capital Soleil dissolved in water for 5 days, the value of the bars (%) are derived from data from Table 2. Table 3. The table shows the mean plant length (cm) of Lepidium sativum seedlings The data is based on the recording of the plant lengths of the Lepidium sativum seedlings in each of the concentrations of sunscreen solution investigated during Experiment 2 (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water), (Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise and Vichy Capital Soleil) Mean plant length of Lepidium sativum grown in solutions of Concentration of sunscreen in solution /% Apoteket Hjärtats Sollotion sunscreens / cm Nivea Sun L Oreal Solar Expertise Vichy Capital Soleil

17 Table 4. The table shows the standard deviations (cm) and coefficients of variation (%) of the mean plant lengths of the Lepidium sativum. The data is based on recordings of the plant lengths of the Lepidium sativum seedlings in each of the concentrations of sunscreen solution investigated during Experiment 2 (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water), (Apoteket Hjärtats Sollotion, Nivea Sun, L Oreal Solar Expertise and Vichy Capital Soleil) Concentration of sunscreen in solution / % Apoteket Hjärtats Sollotion Statistical analysis* of plant length of Lepidium sativum Nivea Sun L Oreal Solar Expertise Vichy Capital Soleil SD/cm CV / % SD/cm CV / % SD/cm CV / % SD/cm CV / % *SD= Standard Deviation, CV= coefficient of variation Table 5. The table presents the P-values from an unpaired t-test, comparing the probability (%) that the difference between the mean plant lengths (cm) of the Lepidium sativum grown in sunscreen solutions and the mean plant length (cm) of the Lepidium sativum grown in water is due to chance. The level of significance was set to 5%. All values were based on the number of germinated Lepidium sativum seedlings (as presented in Appendix 3.2, Table 11) received from Experiment 2. Concentration of sunscreen in solution / % Apoteket Hjärtats Sollotion P-value/ % (from an unpaired t-test) Nivea Sun L Oreal Solar Expertise Vichy Capital Soleil 100 <0.01 <0.01 <0.01 < <0.01 <0.01 <

18 Mean plant length of Lepidium sativum / cm Victor Abrahamsson Qualitative Observations Lepidium sativum seedlings grown in the most concentrated solution containing Nivea Sun were bent shaped while Allium cepa roots of the control grown in water did not. The most concentrated solutions of Apoteket Hjärtats Sollotion and L Oreal Solar Expertise had less bent Lepidium sativum seedlings. The most concentrated solution of Vichy Capital Soleil had the fewest bent Lepidium sativum seedlings. As concentrations fell to a tenth of original concentrations, the number of crooked and bent seedlings fell correspondingly. For a hundredth and a thousandth of the original concentrations the number of bent seedlings was approximately the same as those grown in the control. 18,0 16,0 14,0 12,0 10,0 8,0 Mean plant length of Lepidium sativum grown in different concentrations of sunscreen Apoteket Hjärtats Sollotion Hjärtats Sollotion 2cm 3 /dm 3 water Hjärtats Sollotion 0.2cm 3 /dm 3 water Hjärtats Sollotion 0.02cm 3 /dm 3 water Hjärtats Sollotion 0.002cm 3 /dm 3 water Water 6,0 4,0 8,6 9,9 11,3 11,1 11,0 2,0 0,0 Apoteket Hjärtats Sollotion Figure 6. The bar chart illustrates the mean plant length of Lepidium sativum (cm) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water) of sunscreen Apoteket Hjärtats Sollotion dissolved in water, the value of the bars (cm) are derived from data from Table 3, and the standard deviations (cm) are derived from data from Table 4. All values were based on the number of germinated Lepidium sativum seedlings (as presented in Appendix 3.2, Table 11) received from Experiment 2. 15

19 Mean plant length of Lepidium sativum / cm Victor Abrahamsson Mean plant length of Lepidium sativum grown in different concentrations of sunscreen Nivea Sun 18,0 16,0 14,0 12,0 10,0 Nivea 2cm 3 /dm 3 water Nivea 0.2cm 3 /dm 3 water Nivea 0.02cm 3 /dm 3 water Nivea 0.002cm 3 /dm 3 water Water 8,0 6,0 4,0 6,7 8,7 10,2 11,7 11,0 2,0 0,0 Nivea Sun Figure 7. The bar chart illustrates the mean plant length of Lepidium sativum (cm) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water) of sunscreen Nivea Children s Sun Lotion dissolved in water, the value of the bars (cm) are derived from data from Table 3, and the standard deviations (cm) are derived from data from Table 4. All values were based on the number of germinated Lepidium sativum seedlings (as presented in Appendix 3.2, Table 11) received from Experiment 2. 16

20 Mean plant length of Lepidium sativum / cm Victor Abrahamsson Mean plant length of Lepidium sativum grown in different concentrations of sunscreen L Oreal Solar Expertise 18,0 16,0 14,0 12,0 10,0 L Oreal Solar Expertise 2cm 3 /dm 3 water L Oreal Solar Expertise 0.2cm 3 /dm 3 water L Oreal Solar Expertise 0.02cm 3 /dm 3 water L Oreal Solar Expertise 0.002cm 3 /dm 3 water Water 8,0 6,0 4,0 8,3 8,3 9,7 11,6 11,0 2,0 0,0 L Oreal Solar Expretise Figure 8. The bar chart illustrates the mean plant length of Lepidium sativum (cm) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water) of sunscreen L Oreal Solar Expertise dissolved in water, the value of the bars (cm) are derived from data from Table 3, and the standard deviations (cm) are derived from data from Table 4. All values were based on the number of germinated Lepidium sativum seedlings (as presented in Appendix 3.2, Table 11) received from Experiment 2. 17

21 Mean plant length of Lepidium sativum / cm Victor Abrahamsson Mean plant length of Lepidium sativum grown in different concentrations of sunscreen Vichy Capital Soleil 18,0 16,0 14,0 12,0 Vichy Capital Soleil 2cm 3 /dm 3 water Vichy Capital Soleil 0.2cm 3 /dm 3 water Vichy Capital Soleil 0.02cm 3 /dm 3 water Vichy Capital Soleil 0.002cm 3 /dm 3 water Water 10,0 8,0 6,0 4,0 8,3 9,4 10,6 11,1 11,0 2,0 0,0 Vichy Capital Soleil Figure 9. The bar chart illustrates the mean plant length of Lepidium sativum (cm) when grown in the different concentrations (2cm 3, 0.2cm 3, 0.02cm 3 and 0.002cm 3 sunscreen per dm 3 water) of sunscreen Vichy Capital Soleil dissolved in water, the value of the bars (cm) are derived from data from Table 3, and the standard deviations (cm) are derived from data from Table 4. All values were based on the number of germinated Lepidium sativum seedlings (as presented in Appendix 3.2, Table 11) received from Experiment 2. 18

22 Discussion and Conclusion There were two main research questions addressed in this investigation in order to determine 1) which category of sunscreens, physical sunscreens containing Titanium Dioxide or chemical sunscreens containing active substances Butyl Methoxydibenzoylmethane and Octorcrylene had greatest inhibitory effect on mass of Allium cepa roots, and moreover deducing which of the 2 physical and the 2 chemical sunscreens had greatest inhibitory effect, and 2) finding which of the sunscreens had lowest Minimum Inhibitory Concentration and germination percentage. Experiment 1 Results show that all sunscreens tested inhibited the mean root growth of Allium cepa, varying from 83.7% (Nivea Sun containing Paraffinum Liquidum and Titanium Dioxide) to 57.1% (Vichy Capital Soleil containing Titanium Dioxide) decrease in mass compared to the mean of the control. The data supports hypothesis 1, that Nivea Sun, has greatest inhibitory effect. This was expected as mineral oils are known to affect water and mineral uptake of plants while the other active substances investigated have no or little research was available. Moreover, data received from experiment 1 concludes that parts of hypothesis 1 were incorrect predicting larger inhibitory effects caused by L Oreal Solar Expertise than Apoteket Hjärtats Sollotion (both containing Butyl Methoxydibenzoylmethane and Octocrylene) given its ability to dissolve better in water as reason. The inhibitions on mass of Allium cepa roots caused by Apoteket Hjärtats Sollotion and L Oreal Solar Expertise were both found to be 75.5% suggesting water-solubility of the sunscreens has no effect on inhibition of Allium cepa roots. The data received from experiment 1 supports hypothesis 1 stating that chemical sunscreens have greater inhibitory effects than physical sunscreens, comparing the inhibitory effects of Vichy Capital Soleil with those of L Oreal Solar Expertise and Apoteket Hjärtats Sollotion suggests physical sunscreens are less inhibitory than chemical sunscreens. 19

23 Results can be further supported as the data achieved for the 4 sunscreens showed very significant differences between their means and the mean of the control (P<0.01%), qualitative data suggested crooked roots of Allium cepa in the 4 solutions were due to sunscreens and the large sample of raw data collected, 125 sets of 5 Allium cepa roots. The controlling of the temperatures, light and humidity further contributed to reliability of the data. In addition to qualitative data, method and results, standard deviations were of acceptable sizes ranging from 25.0% for Nivea Sun and Apoteket Hjärtats Sollotion to 16,7% and 14,3% for L Oreal Solar Expertise and Vichy Capital Soleil respectively. One reason for relatively low standard deviations could be the collecting of only the 5 largest roots from each Allium cepa. Choosing only the largest roots auxiliary data is better avoided, providing lower standard deviations. However beneficial for the standard deviations, this could be seen as a weakness in the method as more general views on the growth of the roots is acquired by collecting all roots. Experiment 2 Results show that Minimum Inhibitory Concentrations tested on Lepidium sativum occurred in range 0.02cm 3 /dm 3 to 0.002cm 3 /dm 3 sunscreen per dm 3 water for Nivea Sun and L Oreal Solar Expertise where mean plant lengths were 10.2cm and 9.7cm as compared to that of the control which was 11.1cm while MIC for Apoteket Hjärtats Sollotion and Vichy Capital Soleil occurred in range 0.2cm 3 /dm 3 to 0.02cm 3 /dm 3 sunscreen per dm 3 water where mean plant lengths were 9.9cm and 9.4cm as compared to that of the control which was 11.1cm. Germination percentage of Lepidium sativum seeds grown in the most concentrated solutions of sunscreens were 89% for Apoteket Hjärtats Sollotion, 90% for Nivea Sun, 92% for Vichy Capital Soleil and 93% for L Oreal Solar Expertise. The data does thus partly support hypothesis 2 stating MIC was to occur in lowest concentrations for Nivea Sun and in highest concentrations in Vichy Capital Soleil. MIC for L Oreal Solar Expertise occurred before Apoteket Hjärtats Sollotion as it dissolved better in water, because Apoteket Hjärtats Sollotion formed sediment on the bottom of the plastic container and the other did not, the volume of sunscreen added to concentrations 0.02cm 3 /dm 3 and 0.002cm 3 /dm 3 sunscreen per dm 3 water was in Apoteket Hjärtats Sollotion than L Oreal Solar Expertise. 20

24 Germination percentage does not follow the hypothesis stating that germination percentage should follow the same order as the occurrence of MIC. Looking at germination percentages of the lower concentrations of sunscreen one can suspect that the sunscreens played a lesser role on germination percentage as percentages of the lower concentrations of sunscreen were almost identical to, and in some cases lesser than those of the most concentrated solutions. Instead, what probably caused the germination percentage were damaged Lepidium sativum seeds, being small, it is hard to deduce whether or not they are healthy and hence, presence of non-growing seeds should be expected. Moreover, standard deviations were of acceptable sizes ranging from 30% to 21%. Results could be supported by the fact that the data achieved for the sunscreens all showed very significant differences between their means and the mean of the control (P<0.01%) for the most concentrated solutions. Also for the concentrations diluted to a tenth of original concentration, differences between their means and the mean of the control were very significant (P=0.24% for Apoteket Hjärtats Sollotion, P<0.01% for remainders). For the concentrations diluted to a hundredth of the original concentration, differences between their means and the mean of the control were significant for Nivea Sun and L Oreal Solar Expertise (P=3.81% and P=0.03% respectively) while differences between the means of Apoteket Hjärtats Sollotion and Vichy Capital Soleil and the mean of the control were not significant (P= 31.58% and P=44.79% respectively), concluding the possibility that differences in means of plant lengths at concentration 0.02cm 3 /dm 3 sunscreen per dm 3 water were due to chance were small enough for sunscreens Nivea Sun and L Oreal Solar Expertise to be counted as significantly different from the mean of the control, meaning they have inhibitory effects at that concentration. The possibility that differences in means of plant lengths at concentration 0.02cm 3 /dm 3 sunscreen per dm 3 water were due to chance are too high to consider Apoteket Hjärtats Sollotion and Vichy Capital Soleil as inhibitory at this concentration. Eventually, at concentration 0.002cm 3 /dm 3 sunscreen per dm 3 water, also Nivea Sun and L Oreal Solar Expertise were not significantly different when compared to the control (P=5.96% and P=9.14% respectively). 21

25 The results are further strengthened by: qualitative data suggesting crooked and bent Lepidium sativum seedlings in the most concentrated solutions and the concentrations diluted to a tenth were due to the sunscreens and furthermore, the large sample of raw data collected, 1800 Lepidium sativum seeds were seeded. In addition, the controlling of the temperatures, access to light and humidity further contributed to reliability of the data. The null hypothesis can thus be rejected as there was an inhibitory effect asserted from all sunscreens tested. Evaluation Although the method provided reliable results, many variables were left uncontrolled. Where appropriate, I have included in my conclusion any new or unresolved questions that arose during my investigation. The most apparent and arguably most affecting uncontrolled variable is the presence of other chemicals in the sunscreens, (see Appendix 5). Despite discussing only active substances and mineral oils, the sunscreens tested contain no more than 5% Butyl Methoxydibenzoylmethane, 10% Octocrylene and 25% Titanium Dioxide 8, for mineral oils, there is no limit to how much is allowed 8. Thus it is likely that other chemicals also pose an effect on the mass of Allium cepa roots and plant length of Lepidium sativum seedlings. Many chemicals in the sunscreens have only had brief or no testing on their effect on the environment 1 Some of the chemicals contained in the sunscreens have documented effects on the environment, like Cyclopentasiloxane 1. In order to outline environmental inhibitory effects of the substances investigated more accurately; there is a need to investigate inhibitory effects of the other substances in the sunscreens. Allium cepa and Lepidium sativum used in the investigation did not represent a wide gene pool as all Allium cepa and Lepidium sativum were bought from two companies (see Appendix 1.1). As all samples of Allium cepa and Lepidium sativum were collected from one location, differences in the area where the plants have been grown were eliminated since any abnormalities in the location of growth or any odd genetic factors occurring in the plants then affected all the Allium cepa and Lepidium sativum. However, more spread gene-pools would yield results more general to the species than samples taken from one site. 22

26 Another problem in connection to use of top-quality produced Allium cepa and Lepidium sativum is that these have been chosen to be able to grow in large varieties of environments; these plants might not represent typical tolerances to toxics but rather more tolerant variants of the species. Allium cepa of different sizes ranging from 1cm-2cm in diameter were used, possibly resulting in different growth patterns of the roots of Allium cepa. Larger Allium cepa had tendencies to produce larger roots, thus there were variations in length of roots in each container after 5 days of growth. Possibly fractions of this variation of growth were due to size differences of Allium cepa. Conclusively chemical sunscreens have greater inhibitory effects than physical sunscreens, the sunscreens can be listed in terms of inhibitory effects in the decreasing order: Nivea Sun, L Oreal Solar Expertise and Apoteket Hjärtats Sollotion being equally inhibitory and Vichy Capital Soleil. The inhibitory order of the active chemicals tested can be listed in decreasing order: Paraffinum Liquidum, Butyl Methoxydibenzoylmethane and Octocrylene without particular order and Titanium Dioxide. The earliest occurring MIC occurs between 0.02cm 3 /dm 3 and 0.002cm 3 /dm 3 sunscreen per dm 3 water for Nivea Sun and L Oreal Solar Expertise and between 0.2cm 3 /dm 3 and 0.02cm 3 /dm 3 for Apoteket Hjärtats Sollotion and Vichy Capital Soleil. This is valuable information as it shows that presence of sunscreen chemicals in water not only will affect aquatic environments if present in larger volumes but also plant life and possibly even land living animals in the area. However the investigation does not provide enough evidence to point out which of the chemicals is the most hazardous to the environment which is why relevant further investigations would be on the effect of single chemicals frequently present in sunscreens. 23

27 Acknowledgements I would like to sincerely express my gratitude to following people for providing me with much appreciated support, motivation and help throughout the process. Annette Carlström Thank you Annette, my Biology teacher, Extended Essay supervisor and Mentor, for your excellent support and encouragement throughout my workings Family Thank you my family, for your never ending support and your encouraging thoughts and reflections. Friends Thank you my dear friends, for your constant support, for your sympathy and understanding, allowing me to stay home on weekends and work on my extended essay without losing touch with me. 24

28 Bibliography 1. Environmental toxins in sunscreens, Naturskyddsföreningen, published 2008, [Cited ] < 2. Health effects of UV-radiation, World Health Organization (WHO), published 2012, [Cited ] < 3. Pharmaceuticals and personal care products (PPCP s), US Environmental Protection Agency, published 2010, [Cited ] < 4. Effects of PPCP s on aquatic ecosystems, Environment Canada, published 2009, [Cited ] < 1&offset=9&toc=show> 5. Occurence of Organic UV-Filter compounds BP-3, 4-MBC, EHMC & OC in wastewater, surface water and in fish from Swiss lakes, M. E. Balmer, H.R. Buser, M.D Müller & T. Poiger, published 2004, [Cited ] < 6. Effects of environmental contaminants, J. Axelsson, published 2008, [Cited ] < 7. Comparison of In Vitro and In Vivo Estrogenic activity of UV-filters in fish, P.Y. Kunz, H.F. Galicia, K. Fent, published 2005, [Cited ] < 8. Läkemedelsverkets författningssamling, Läkemedelsverket, published 2007, [Cited ] < 9. Chemical UVB+UVA sunscreens: Octocrylene, G. Todorov, published 2012, [cited ] < 10. Chemical UVA sunscreen : Avobenzone, G. Todorov, published 2012, [cited ] < > 25

29 11. Sunscreens cause coral bleaching by promoting viral infections, Environmental Health Perspectives (R. Danovaro, L. Bongiorni, c. Corinaldesi, D. Giovannelli, E. Diamani, P. Astolfi, L. Greci & A. Pusceddu), published 2008, [Cited ] < 12. Octocrylene, Environmental Working Group, published 2012, [Cited ] < 13. Physical vs. chemical sunscreen, Skinacea, published 2012, [cited ] < 14. Banning of sunscreens containing Zinc Oxide as UV-filter, Läkemedelsverket, published 2009, [Cited ] < 15. Green tip; Mineral oil exposed, Pure and Green Organics, published 2010, [Cited ] < 16. Allium Test, G. Fiskesjo, [Cited ] < 17. Accumulation of Cesium in Leaves of Lepidium sativum and its influence on Photosynthesis and Transpiration, G. Ystrzejewska-Piotrowska, P.L. Urban, published 2003, [Cited ] < 18. Graphpad Software, Graphpad, last updated 2012, [accessed ] < 26

30 Appendix Appendix 1: Materials 1.1 Bioindicators and Sunscreens Sunscreen lotions: Apoteket Hjärtats Sollotion Nivea Sun L Oreal Solar Expretise Vichy Capital Soleil Inhibitory Indicators (plants): Allium cepa (Lord Nelson ) Lepidium sativum (Gar10land ) 1.2 Equipment for experiment 1 5 Plastic containers of dimensions 13cm x 10cm x 5cm (650cm 3 ) Knife Polystyrene boards of dimensions 10cm x 7cm (70cm 2 ) with 5 holes for Allium cepa growing sites 125 Allium cepa bulbs Scalpel Permanent marker Tap water Acetone0.1mol/dm 3 Apoteket Hjärtats Sollotion Nivea Sun L Oreal Solar Expretise Vichy Capital Soleil Analytical balance ±0.001gram 27

31 Glass rod 500 cm 3 volumetric flask ± 1cm 3 Balance ±0.01gram Ruler measuring centimeters ± 0.1cm 3 Syringe 1cm 3 Thermometer (±1 C) Mechanical Watch 1.3 Equipment for experiment 2 18 Plastic containers of dimensions 13cm x 10cm x 5cm (650cm 3 ) Cotton 1800 Lepidium sativum seedlings Permanent marker Tap water Orange A4 papers Apoteket Hjärtats Sollotion Nivea Sun L Oreal Solar Expertise Vichy Capital Soleil Ruler measuring centimeters (±0.1cm) Glass rod 250cm 3 measuring cylinder (±5cm 3 ) Scissor Thermometer (±1 C) Syringes 0.5cm 3 and 2cm 3 (±0.01cm 3 ) and (±0.1cm 3 ) respectively Mechanical watch 28

32 Appendix 2 Method 2.1- Method for Allium Test: See Appendix 1 for equipment 1. 5 pieces of 13cm x 10cm x 5cm (650cm 3 ) plastic containers were marked with a Permanent marker either A, B, C, D or Water (Where A represents a solution of water and Apoteket Hjärtats Sollotion, B represents a solution of water and Nivea Sun, C represents a solution of water and L Oreal Solar Expertise and D represents a solution of water and Vichy Capital Soleil. Water meant there was only water in the solution in which the Allium cepa grew). Each of the 5 plastic containers was marked differently so that there was only one solution of each type. 2. From a larger Polystyrene board, 5 Polystyrene boards of dimensions 7cm x 10cm (70cm 2 ) were cut out 3. Using 0.1mol/dm 3 concentrated Acetone to corrode 5 holes on each 70cm 2 Polystyrene board, the boards were readied for carrying 5 Allium cepa each (holes depended on Allium cepa size) 4. The densities of each of the sunscreens tested were measured weighing and recording the mass of 1cm 3 of each sunscreen. This was done by first recording the mass of a 5cm 3 measuring cylinder and then filling the 5cm 3 measuring cylinder with 1cm 3 of each of the sunscreens using a syringe measuring 1cm 3. The mass of the measuring cylinder containing the sunscreen was then recorded with the same balance. By subtracting the mass of the measuring cylinder one could find the mass of 1cm 3 of each sunscreen. 5. Each container was then put on a balance (±0.01grams) the balance was zeroed so that it said 0.00g when the plastic container was placed upon it. 29

33 6. In each of the containers marked either A,B,C or D, 1 cm 3 of the corresponding sunscreen was added to the plastic container by adding the mass of 1 cm 3 of the sunscreen specific for the plastic container, once the balance showed the mass of 1cm 3 for the specific sunscreen recorded earlier one could be certain that 1cm 3 sunscreen had been added to the plastic container. 7. A graduated cylinder measuring 500cm 3 was used to measure up 500cm 3 tap water. Each of the 5 plastic containers was then filled with 500cm 3 of tap water and then mixed with the sunscreens for 10 seconds using a glass rod healthy looking Allium cepa bulbs of approximate size 1-2cm in diameter were peeled by hand and arranged into 5 groups with 5 Allium cepa on each Polystyrene board. The Allium cepa were arranged into groups of 5 randomly and without any particular order to avoid any unconscious manipulation of the data. 9. The Allium cepa were securely and carefully squeezed into the holes of the Polystyrene boards to make sure that they were stuck but not damaged from the handling and then placed in the 5 branded containers containing 5 different solutions to float on the surface of the water in order to allow equal contact to the water for each of the Allium cepa. 10. The Allium cepa were left to grow in an inside environment for 5 consecutive days, room temperature was at a constant 20 C, since it was an indoor environment humidity was also rather stable and sunlight was equal for each of the Allium cepa making certain that conditions for the Allium cepa in the different containers were identical but for the solutions 11. After 5 days the Allium cepa were taken out of the solution. Only the 5 longest roots were picked for recording to decrease the range of the data. The roots were collected using a scalpel. The 5 roots collected were then weighed in an analytical balance (±0.001g). Mass was recorded for the 5 largest roots of every Allium cepa. 12. Procedure 4-10 was repeated 5 times to provide the replicates of the investigation. 30