2. LITERATURE REVIEW
2.2 Nanoparticles
It is difficult to estimate how many nano-based products or a product group exists nowadays because the number is increasing continuously. The use of nanotechnology in the field of consumer products to grow on rapid on the basis of consistency (Figure 3). According to the PEN (Project on Emerging Nanotechnologies) over 1,300 manufactured identified, nano based products, have been introduced to the world commercial market. PEN Director David Rejeski says, “When we launched the inventory in March 2006 it contained 212 products. If the current trend continues, the number of products could reach 3,400 by 2020”(Nanotech project 2017).
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Figure 3.Production of nanomaterials last few years and potential of future projections. a) Total products listed; b) Product categories (From Serrano et al. 2014).
Nowadays nanotechnology is a highly promising and exciting molecular based technology that has produced the vast application in all sectors as medicine and consumer products (Moore 2006, Krysanov et al. 2006). These nanotechnologies based on nanoparticles that have unique properties. Due to industrial uses of nanoparticles for manufacturing products, they pose a risk to the environment (Pakarinen 2013). Nano particles can be composed a different kinds of materials. They can be classified according to their origin (engineered, incidental, natural, man-made), structure, composition or manufacture, etc. There are several types of classification given below as a representative by a figure 4.
Figure 4. Classification of nanoparticles on a several basis (Pakarinen 2013, Siddiqi and Husen 2016).
Recently, the main focus has been on biosysthesis of nano-particles in different field of application representative by a figure 5 (Siddiqi and Husen 2016).
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Figure 5. Application of nanoparticles for several fields (Siddiqi and Husen 2016).
2.3 Does Nanomaterial’s Properties necessary a New Toxicological Science?
One of the main properties of nanoparticles is size, which is between individual atoms or molecules and bulk materials (Zhu 2006, Wollenberger 2000).This character can modify the physicochemical properties of the materials and also provide greater opportunity for uptake and interaction with biological tissues. These properties require novel approaches to assess their hazard potential particle. In the field of toxicology, particle size and surface have been shown to be among the main characteristics. If a size of particles is increased, surface area decrease and vice versa (Nel et al. 2006, Krysanov et al. 2006, Figure 6).
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Figure 6. Inverse relationship between particle size and surface area (Nel et al. 2006).
For example in particle size 30nm, about molecules, 10% are expressed on a surface. Similarly at 10nm and 3nm size the surface increases 20% and 50%. This is the key aspects of chemical and biological properties of nanomaterial.
2.4 Releases of nanomaterials to the environment
Nowadays, reason and without any reason nanoparticles will be released and dispersed in environmental ecosystems due to the mass production and huge number of applications. Because of It has been very critical to determine to their potential bioaccumulation by ecological receptors (Petersen 2009). Nanomaterials are released to the environment point sources and non- point sources for example, landfills, wet deposition from the atmosphere, storm runoff, and waste waters. Humans are exposed to nano materials by inhalation of nanomaterial from the atmosphere by ingestion drinking water and fish (Wiesner et al. 2006).
Nanomaterials are deposited in the environment also by activities such as, use of fossil fuels, mining and automobile traffic etc (Figure 7). Traffic and fossil fuel combustion to produce nanoparticle or carbon black with diameter <100nm. Especially natural gas burning has produced carbon nanoparticles including carbon nanotube and fullerenes (Sustainable Nano 2014). On the other hand, mining and metal refinery operations generate metal and metal oxide nanoparticles.
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Figure 7. Pathways of nanomaterial release to the environment by human activities (Sustainable Nano 2014).
2.5 Fullerene
Fullerene is an allotrope of carbon It consists of 60 carbon atoms that from 20 hexagons, and 12 pentagons. It has the diameter of 0.72nm.They are also differently shaped carbon nanomaterias such as graphite; grapheme and diamonds are repeating atomic cage (Kroto et al., 1985).
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Figure 8. Structures of carbon allotropes: (A) Diamond, (B) Graphite, (C) Amorphous carbon, (D) Single-walled carbon nanotube, (E) C60 Fullerene (Pakarinen 2013, Kumar 2017, Reserchgate.net).
Recent studies have shown that in aquatic animals the main routes of entry are directly through gills and epithelia. In addition, fullerene can be taken by internalized routes by different fish species (Moore 2006). In terrestrial organisms are main routes of entry are inhalation or ingestion. Humans are exposed to fullerenes through ingestion of food and drinking water with fullerene and fullerene containing products (Moore 2006, Takahashi et al. 2012).
According to Arndt (2014), core structure and surface chemistry influence nanomaterial toxicity to daphnia”. Fullerene gammacyclodextrin complex (C60-GCD) introduce 100%
mortality daphnia 17days of exposure at 5 ppm. On the other hand, fullerene also introduce negative impacts to daphnia reproduction, growth rate, adult size at a concentration of 10ppm or 50ppm (Arndt 2014).
Multigenerational impacts on mortality and reproduction in D. magna have been shown for several particles and single walled carbon nanotubes functionalized with carboxy amides (SWCNT-CONH2) in the F0, F1 and F2 generations (Arndt 2014).
3. OBJECTIVES OF THE STUDY
The aim of the study was to investigate the acute and chronic toxicity of organotin with fullerenes on aquatic organism D. magna. First acute tests were used to find suitable concentration ranges for the chronic tests. The end points used to indicate the joint and chronic effects were (number of neonates, mother size, percentage of males and number of ephippia) over two generations.
4. MATERIALS AND METHODS 4.1Chemicals
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Figure 9. Triphenyltin chloride
Triphenyl Tin chloride (CAS 639-58-7, 95%) was purchased from Aldrich. Triphenyltin chloride is an organotin compound with formula )3SnCl. It is a colorless solid and dissolved in organic solvents. Carbon nanoparticles, crystalline fullerene ( ) purity (99.5%) was purchased from Sigma- Aldrich (USA). Impurities were checked by plasma emission spectrometer and thermal gravimetric analysis (Waissi-Leinonen et al. 2012).
4.2 Test Organism
Daphnia was cultured in artificial freshwater (Ca+Mg hardness 2.5mM pH 6.5 to 7.1) under a photoperiod of 16:8 h light: dark at 20-22˚C. It is easy to maintain in a laboratory and also maintain University of Eastern Finland, department of Environmental and Biological Sciences culture room. Daphnia was fed three times a week with green algae (Scenedesmus sp). Water from algae containers were taken with a beaker and quality must be checked every time. It should be light green and without larger particles which could block filter apparatus of daphnids.
13 Compound eye with 22 ommatidia and black pigment basic vision and orientation
Figure 10. The Anatomy of Daphnia. This picture shows an adult female daphnia with parthenogenetic embryos in her brood chamber (photo taken Risto Pöhö).
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Algae water is a mixture of Scenedesmus sp, Monoraphidium contortum and Selenastrum capricornutum. The dominate species investigated by light microscopy. The main species were Scenedesmus sp (figure 11). In algae containers; there are two crucian carps to ease nutrient cycling. Carps were fed three times a week with Tetramin fish food.
International standard tests were applied in the acute tests following international Organization for Standarzation (ISO, ISO6341:2012) and the Organization for Economic Co-operation and Development(OECD 2007).
Figure 11. Scenedesmus sp.
4.3 Preparation of artificial fresh water
Artificial fresh water was used the media basis of fullerene exposure for D. magna and also control alternative of natural water. It was prepared by adding analytical grade salts ( 58.8mg/L, 24.7 mg/L, 12.95 mg/L, KCl 1.2mg/L;
hardness ] + [ ] =0.5m M) to Millipore water and adjusting pH to 7.2. This water was very simple compared to natural waters. This low hardness also helped minimize precipitation and it also corresponds to that of Finnish freshwaters.
15 4.4 Preparation of new Suspension
The stirring method was used to prepare the fullerene suspensions. Stock suspensions were made by weighing 50mg crystalline fullerene ( ) and adding 500ml artificial fresh water on a dark glass bottle and leaving it to a magnetic stirrer at 1000rpm for two weeks at 20±2˚C.
Figure 12. Stirring method for Suspension. A) Fullerene powder. B) Magnetic stirring of 1000 rpm started. C) Colloidal of Suspended for two weeks.
After stirring the fullerene suspension was filtered with glass fibers (0.45 µm membrane, Membrane filters, lot: R7NN075, Ireland) to remove the larger fullerene agglomerates because of agglomerates different in size, shape and composition. The suspension was protected from light with aluminum foil to prevent light reactions of fullerenes.
Figure13. Remove the larger particles by glass fiber (0.45 µm membrane).
16 4.5 Concentration measurement of filtered n
Four replicates test tubes with1.5 ml of filtered suspension and 1.5 ml 2% of NaCl (Sigma- Aldrich, Denmark) stock solution was mixed with 3 ml of toluene and closed with a cap. Then tubes were vortexed (VORTEX GENIE-2, scientific industries)) about 15 s and extracted in ultrasonication bath Sonorex (Bandelin sonorex, super, PK-106) for 5 min. The concentrations were determind by measuring the absorbance of the toluene phase at 335 nm by spectrophotometer (50 Bio UV- Spectrophotometer, Varian).
Fullerenes absorb ultraviolet and visible light (VIS). The highest and strongest absorption peak of fullerenes in toluene is at 335 nm, a smaller peak at 407nm and the weakest absorption band between 415 to 680 mm. The concentrations were determined four samples by measuring the absorbance of the toluene phase-VIS at 335 nm by a spectrophotometer and using a standard curve (Figure 14).
Figure14. UV-VIS spectrum for fullerenes in toluene. Insert 1: a standard curve at 335 nm for concentrations between 0.675 and 25 mg/L. Insert 2: Purple solution of C60 in
toluene, extracted from AFW (Pakarinen, 2013).
4.6 Daphnia Toxicity tests for fresh water
The Daphtoxkit F Magna contains all the materials, including the test species D. magna in the form of “dormant eggs (Ephippia)” to perform at least 6 complete acute toxicity tests according to internationally accepted standard Method (e.g. OECD and ISO). This test makes use of the
“neonate” which hatches about 3 days from the eggs.
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Then preparation of fresh water as a hatching and dilution medium. 2-liter volumetric flask was filled with approximately one-liter deionized water and of concentrated salt solutions. Then add water up to 2000ml mark and shake to homogenize the medium. Two-liter stander Freshwater it will complete 3 bioassays. Then hatching of the ephippia should be initiated 3 days prior to a star of the toxicity test. Pour the contents of one vial of ephippia into the microssive and rinse with tap water to eliminate all traces of storage medium. Then transfer the ephippia into hatching petri dish in 15 ml standard freshwater prepared by air bubbling. Cover the petri dish and incubate for 3 days, at 20-22˚c, under continuous illumination of min 6000 Lux (figure15).
Figure 15. A) Petri dish, B) Illumination system.
The highest percentage of hatching occurred 72h and 80h of incubation. Standard testing procedures indicate that the neonates should not older than 24h also young daphnid must be collected 90h after the start of the incubation. On the other hand daphnids had a poor period and thus it was not easy to have enough less than 24h old neonates for tox tests. Then decided to try the kit. Nevertheless, the kit was not successful either, and finally it was found enough neonates from our culture room.
Then the preparation of toxicant dilution for the test, minimum volume of 50 ml was needed for each toxicant dilution. When exposure period is prolonged from 24 h to 48h, it has happened starvation to death. Pre –feeding of the neonates prior to the test by fill one of the tubes containing Spirulina powder with standard freshwater and shake to homogenize. It was prior 2h
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to collecting the neonates for the toxicity test. Swirl the Petri gently to distribute the algal food evenly. Then transfer of the neonates to the test wells.
The life cycle of D. magna occurs during the growth season by asexual mode of reproduction. A female produce a huge amount of parthenogenetic eggs. These eggs are placed in brood chamber figure 16. Development of eggs direct but some eggs are remaining in the brood chamber for further development. After 3 days in the brood chamber the young daphnia was released by the mother of ventral flexion post-abdomen. The newborn daphnia looks like adult daphnia except brood chamber not yet developed. In most of species daphnia at first time produce four to six neonates. At 5-10 days first neonates are deposit brood chamber at the same time an adult D.
magna also produces egg every 3 to 4 days until her death. In laboratory females may be live more than two months.
Figure16. Parthenogenetic life cycle of D. magna.
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This figure shows that the sexual and the asexual life cycle of a Daphnia. During the parthenogenetic cycle, females produce diploid eggs the develop directly into a daughter that same time female produces diploid asexual eggs to develop sons. But sometimes produce male under environmental control. On the other hand, the same females produce haploid eggs that were required fertilizer by males. These eggs are enclosed by a shell means ephippia and need to undergo a diapause before female offspring will hatch from them (Ebert 2005).
4.7 Acute exposure
The acute test was performed in accordance with the standard protocol for D. magna acute test.
Approximately 20 neonates aged less than 24 h, divided into four groups, and were exposed to each concentration for 48h in a static test (Figure 17).
Figure17. Acute exposure experiment
The test containers were filled with 10 ml fill with a solution. Then 5 neonates were transferred to each jar and the jars were covered with aluminum foil. Number of mobile and immobile neonates were counted after 24h and 48h. These series were continued until mobilized all neonates. It was determined 5 toxicity test in different series, 300 µg/l, 60 µg/l, 12 µg/l, 2.4 µg/l, 0.5 µg/l, 0.02 µg/l, 0.1 µg/l. For example tox test 01: (Triphenylitin chloride, 0.5 mM AFW)
.
Stock TPhTCl 9.1 mg/l, dilutions 1:10 (910 µg/l), 1:100 (91 µg/l) in experimental volume 10 ml, each jar dpahnds 5 in 4 replicates/ concentration. At 0.1µg/l all neonates were alive.
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Table 1. Recorded for immobilized of neonates. “0” means all alive.
Tox test 01:
Concentration(µg/l) 24h immobilized 48h immobilized
a b c d a b c d
Concentration(µg/l) 24h immobilized 48h immobilized
A b c d a b c d
Concentration(µg/l) 24h immobilized 48h immobilized
A b c d a b c d
21 Tox test 04:
Concentration(µg/l) 24h immobilized 48h immobilized
A b c d a b c d
0 0 0 0 0 0 1 0 0
~0.1 0 0 0(4) 0 1 0 0(4) 1
0.49 ~0.5 0 0 0 1 0 0 1 0
Tox test 05:
Concentration(µg/l) 24h immobilized 48h immobilized
A b c a b c
~0.1 0 0 0 0 0 0
0.49 ~0.5 0 0 0 0 1 0
5. Chronic Exposure
After the acute toxicity test complete, chronic exposure tests were conducted following the standard protocol for Daphnia magna Reproduction Test (Guideline 202: Daphnia magna Reproduction Test, adopted 13 April 2004). The daphnids were less than 24h old in the beginning of the test and exposed for a period of 28 days in different treatment groups (Control, Fullerene, Tin, Tin+Fullerene). Each replicate consisted of 200 ml beakers in150ml of water and 8 individuls per beaker. The same maintaining process, including feeding and water renew to use in culture room, for example feeding Monday, Wednesday, and Friday in each beaker. Only Friday change solution, it was 50% per beaker. An everyday collection of neonates, determination of female and male until last day of the experiment. Light microscopy (Dialux 20, Leitz Wetzlar) 100x magnification in normal condition all D. magna are females It was the environment for the experiment photoperiod of 16:8 h light: dark at 20-22˚C.
5.1 Quantification method of accumulated by D. magna
In the beginning of the test, the number of adult daphnids (f0) was transferred to the test jars with 13.2 ml , Triphenoltin chloride 212 µl and control with clean water to produce F1 generation. F1 generations were built up with 8 organisms as 2 replicates (figure 18) such as (Control, Fullerene, Tin, Tin+ Fullerene).
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Figure18. Each group 2 replicates.(Control, Fullrene,Tin, Tin+Fullerene).
Each generation was continuously exposed under similar conditions used (8 organisms/200 ml, 2 replicates, 13.2 ml n , Triphenol tin chloride 212 µl).
Figure 19. Each group (Cont, Ful, Tin, Tin+Ful)
2×8 neonates for example Control (1&2) from 1 to 8.
Group of 2×8 neonate (figure 19) were transferred new jars and produce to the next generation.
These processes were used until F2 generation. We can use this formula for the measurement of the real length of D. magna.
23 Real Length =
For example, measurement of total length 10.6 mm by 3mag from head to tail. So real length of D. magna
Real length =
Here,
= Total length : 10.6mm =3.533 mm mag×3 : 3
For quantitative analyses of fullerene in daphnid, four replicates were the test of each group including of control. After exposure, the organisms were removed fullerene spiked, tin component, and rinsed of clean Millipore TM to remove fullerene that had been attached to the carapace and antennae.
Then Daphnia (Neonates and mothers) was carefully dried on blotting paper. Neonates sample was measured 1-2 mg and mothers sample were 10mg of organism mass wet weight by microbalance (Sartorius gmbh Gottingen, type m3p fabr-nr.39070013, Germany) and placed in 10 ml glass tubes. After 2% NaCl solution (1.5ml) was added each glass tube for homogenizing media. Samples were homogenized with a probe tip sonicator (Vibra Cell, Sonics &Materials) for 2 min. After homogenization samples were extracted with 1.5ml of toluene by vortex shaking for 15s and then bath sonication for 5 min. Then spectra of toluene were recorded by ultraviolet-vis spectrophotometry. Fullerene peaks were identified 335 nm to 407 nm.The extraction of control organism cannot find any peak in 335 nm to 407 nm or any other spectrum. Total procedure represent by flow chart given bellow (figure 20).
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Figure 20. Quantification method of nC60 accumulated by D. magna at the flow chart.
6. Data processing
All preliminary calculations were done in Microsoft Excel Worksheet ( MS Excel 2016). IBM SPSS Statistics 23.0 was used to analyse the data and Graph Pad Prism 7.02 software (GraphPad Software, Inc. La Jolla, California 92037, USA) was used to create graphs. Values were determined by The Mann-Whitney U test for two independent samples. Differences were considered statistically significant when p <0.05. Values were determined by one-way ANOVA.
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. RESULTS
5.1 Toxicity test
0 .1 0 .5
2 .4 1 2 6 0 3 00
0 2 0 4 0 6 0 8 0 1 0 0
T o x i c it y a s im m o b il iz e p e r c e n t a g e
E x p o s u r e c o n c ( µ g /l )
Immobilized percentage
2 4 h i m m o b i li z e d 4 8 h i m m o b i li z e d
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Figure 21. Toxicity as immobilized percentage in Tin suspended AFW in daphnia at 24 and 48 h. Data was evaluated by Mann-Whitney U test for two independent samples. Data are statistically significant p <0.05.
First of all, acute toxicity of TPT was tested with Daphnia magna. Six concentrations were used to find out suitable concentration for the chronic tests. The exposure concentration (µg/l) series from higher to lower was 300 µg/l, 60 µg/l, 12 µg/l, 2.4 µg/l, 0.5 µg/l and 0.1 µg/l was tested. At the highest exposure concentration 300µg/l all (24 and 48h) Daphnia immobile. Also 60µg/l 24h 75% and 48h 85% Daphnia died and 2.4µg/l 24h 50%, 48h up to 70% On the other hand lowest exposure concentration was found 0.1µg/l 24h 0% and 48h less than 20% Daphnia died whereas in control 90% of Daphnia was mobilized.
5.2 Body size of adults
5.2.1 Light microscopy 3x magnification
According to Arndt (2014), change of body size depends on some environmental conditions such as environmental stress. Similarly to, our experiment results body size was higher in F1 and F2 daphnids. As a result, this could support a life strategy shift to produce fewer neonates and larger neonate size. This body size increase sensitivity to other contaminates e.g. heavy metals. Body size also reflects predator species which favor a specific prey size.
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Figure 22. Body size of Daphnia magna measurement by Light microscopy 3x mag.
Figure 23. Length measurement of D. magna (From Rosenkranz 2010). It was measured from base of the spina to top of the head above eye complex. Red arrow indicate the real length of D. magna.
27 Comparison F1 Vs F2 (Length).Here F1generation average length <F2 generation average length. Stars (*) indicate that significant level between two groups, one controlled another one (Ful,Tin+Ful, Tin).*significant compared to control with p
<0.05,*** means p<0.001.
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F2 generation, found that treatment group data were statistically significant in Ful vs. Control group is significant by t-test with p <0.05 and Mann- Whitney U=54.The Mann- Whitney U- Test is a version of the independent samples t- test that can be performed on ordinary (ranked data). Tin+Ful vs. control group is significant by t-test with p <0.05 and Mann – Whitney U=18.Also determined Tin component vs. control group, (p <0.05, Mann- Whitney U=18). F1 generation mother samples showed increased body size in fullerene exposure (Figure 24 A).
Instead, TPT body size were bit smaller than a control group. In addition, exposure TBT had also effects on the next generation.
Similarly, body sizes were a bit higher in generation F2 especially in the fullerene treatment, but body size also high all treatment groups. Compared to F 1 generation the body size was high in the F2 generation. In the case of F2 generation most of the fullerene and TPT treatments were statistically significant, but the F1 generation was less statistical significance compared to F2 generation. Because of TPT had negative effects on daphnia on next genation. Most of the daphnids were body size less 4mm but F2 generation body size upper 4mm.
5.3 Determination of sex ratio
Determination of sex was done by microscopic observation. Male daphnia are smaller, larger antennas also modified post abdomen even legs, which were armed also hook used clasping.
Determination of sex was done by microscopic observation. Male daphnia are smaller, larger antennas also modified post abdomen even legs, which were armed also hook used clasping.