Toxicity of gold nanoparticles for plants in experimental aquatic system

Increased production and use of nanomaterials can lead to new types of pollution of the environment, including aquatic ecosystems. Pollution of the aqueous environment with nanoparticles can be a new type of pollution of the environment. This requires a more detailed study of the biological effects during exposure of nanoparticles on aquatic organisms. The interactions of gold nanoparticles (Au) with aquatic macrophytes Ceratophyllum demersum have been studied. Aquatic microcosms with these plants were used. Gold nanoparticles (Au) were added to the aqueous medium of C. demersum macrophyte containing microcosms. The state of the plants was then analyzed. Phytotoxicity of Au nanoparticles for aquatic macrophytes was shown for the first time. A new method of phytotoxicity detection was suggested and successfully approved. Phytotoxicity at a concentration of Au (in the form of nanoparticles) of 6 × 10−6 M-1.8 × 10−5 M was shown.


INTRODUCTION
At present, the biosphere is affected by chemical pollution [1,2], which manifests itself in both terres trial and aquatic ecosystems.
Continuous entry of different chemical substances into aquatic ecosystems makes topical problems of ecological and ecotoxicological monitoring, hazard assessment of chemicals, and study of different aspects of the interactions between the chemicals entering the biosphere and organisms [1][2][3][4][5]. In these studies, new aspects of the interactions between pollutants with aquatic macrophytes under the conditions of labora tory microcosms were studied previously [6][7][8][9].
This work is devoted to the study of earlier unknown biological effects observed under the influ ence of gold nanoparticles on aquatic organisms (using macrophytes as the example). Gold (Au) is a heavy metal of the first and six periods of D.I. Men deleev's systems of elements with the atomic number 79 and atomic weight of 196.9665 ± 1. The biological effects of this element have been studied less than the influence of other heavy metals [10,11].
In connection with the development of the possi bilities of using gold nanoparticles (AuNPs) for medi cal purposes, including for diagnostic and therapy of cancer, Alzheimer's disease, arthritis, HIV, and tuber culosis [11], it is of interest to study the whole spec trum of possible biological effects caused by different Au preparations. Certain Au (I) preparations are toxic; they accumulate in the kidneys and, to a less degree, in the liver, spleen, and hypothalamus; accumulation in the kidneys can lead to diseases of the kidneys, as well as to dermatitis, stomatitis, and thrombocytopenia [10]. Scientific literature lacked the information about the interactions of Au nanoparticles with aquatic mac rophytes, as well as data whether gold nanoparticles can exert toxic action in terms of higher aquatic plants. The current problems in the study of ecotoxicology and chemical-biotic interactions [12][13][14][15][16][17][18] make it necessary to gain scientific information on the poten tial toxicity of a maximum broad range of chemical substances and products, including nanoparticles.
The aims of this study were to verify the hypothesis on the possible biological activity of gold nanoparti cles and reveal if they can exert toxic action on aquatic macrophytes Ceratophyllum demersum L.

MATERIALS AND METHODS
The experiments were carried out in freshwater microcosms. The microcosms were created using abundantly occurring freshwater organisms-aquatic plants Ceratophyllum demersum L. According to the previously worked out method of macrophyte keeping under laboratory conditions [6,8,9], aquatic macro phytes and settled tap water (STW) were applied to microcosms. The Ceratophyllum demersum plants were collected in a pond in a floodplain in the upper reaches of the Moscow River.
Macrophytes C. demersum were incubated in microcosms of transparent polymeric material under the conditions of natural photoperiodicity. Each microcosm contained 500 mL of water (STW) and macrophytes at a quantity corresponding to the biom ass of 2-4 g of the wet weight ( Table 1). The tempera ture of water was 20°C. Preparations of colloid nano sized gold particles of Au (AuNPs) were added to the microcosms. The size of the particles was 20 ± 5 nm. The AuNPs preparation contained 3 × 10 -4 M Au. The volume added to microcosm nos. 1 and 2 was 2 mL and that added to microcosm nos. 3 and 4 was 6 mL. The mode of AuNPs additions: 5 additions in each microcosm were made. The first addition was made at the beginning of incubation. Following additions were made on the third, eighth, 17th, and 25th days of incu bation. After 28 days, the incubation was terminated. The total application of Au into microcosm nos. 1, 2, 3, and 4 after the last fifth addition comprised: in microcosm nos. 1 and 2 after introduction of five addi tions of 2 mL-6 × 10 -6 M; in microcosm nos. 3 and 4 after introduction of five additions of 6 mL-1.8 × 10 -5 M. Microcosm nos. 5 and 6 were control sam ples-nanoparticles were not added.

RESULTS AND DISCUSSION
The state of macrophytes during incubation is characterized in Table 2. During incubation of macro phyte macrocosms in the first days, signs of AuNPs phytotoxicity were not observed.
It was shown that, under the conditions of experi ment after the total addition of AuNPs of 1.8 × 10 -5 M, marked phytotoxicity was observed after 24 days.
At the total addition of AuNPs of 6 × 10 -6 M, cer tain signs of phytotoxicity also manifested but to a lesser degree.
We note that, during influence of AuNPs along with death of a portion of shoots, sublethal effects were observed associated with localization of plants' shots in a column of water. During toxic sublethal influence, the shoots located in the column of water, on average, lower than in the control. In control, all the shoots floated in the column of water and did not touch the bottom and the shoots sank lower and certain shoots touched the bottom during action of AuNPs. We noted similar sublethal effects when observing macrophytes that were incubated in the presence of such heavy met als as Cu, Zn, Cd, and Pb, as well as during incubation of macrophytes in the presence of an organic pollutant (sodium dodecyl sulphate, SDS). This indicates that we found and used a new method for detection and characterization of sublethal manifestations of phyto toxicity during influence of pollutants on aquatic plants C. demersum.
These results contribute to the study of the interac tions of metals with aquatic plants [34,35], as well as to study of toxicity of nanomaterials for aquatic organ isms [36]. The accumulated facts on a potential toxic ity of nanomaterials are of interest because nanomate rials are proposed for use in decontamination of water [37].
The fields of possible use of the results are given in Table 4 [38,39].
This study leads to the following conclusions.  [33] Nanoparticles of copper oxides Aquatic macrophytes Elodea Candensis [24]  Inexpensive and efficient method for assessing the potential hazards of nanoparti cles was developed and approved Methodology of biotesting of poten tially toxic substances New, easy to use, practical method for estimating phytotoxicity of substances was developed and approved using aquatic macrophytes and microcosms Science of nanomaterials New type of nanomaterials exerting phytotoxicity was identified (gold nanoparti cles) and the database of metal nanoparticles was expanded Prediction of the properties of new substances and materials New data useful for predicting the properties of substances and materials with regard to their degree of dispersion were obtained. The range of specific examples was expanded where increased dispersion of nontoxic substances leads to the appear ance of a new property-toxicity Environmental education, environ mental safety

Seedlings of plants of Lens culinaris
Range of facts about substances that can act as toxic pollutants of the environment with harmful effects on living organisms was expanded. It can be used for a more complete assessment of environmental impacts (AEI) Vol. 69 No. 3 2014 exert phytotoxicity to aquatic environment. Data have been obtained for the first time that gold nanoparticles (Au) under certain conditions have a toxic influence on aquatic macrophytes.
(2) Phytotoxicity under the conditions of labora tory macrocosms was shown. Under the conditions of the experiment in microcosms, toxic influence of gold nanoparticles (Au) on macrophyte C. demersum was established.
(3) In the conditions of the experiments, the toxic influence of gold nanoparticles manifested after quite long term exposure during 17 days and more.
(4) Phytotoxicity was detected at a concentration of gold (in the form of nanoparticles) of 6 × 10 -6 M-1.8 × 10 -5 M. That phytotoxicity can also manifest at different concentrations can also not be excluded. When increasing the concentration of the Au nano sized particles (AuNPs) to 1.8 × 10 -5 M, phytotoxic effects manifested earlier than at the total concentra tion of 6 × 10 -6 M.
(5) This study expands the methodological arsenal of biotesting. A new efficient method for phytotoxicity assessment of water soluble or substances suspended in water was successfully approved in this study, which includes analysis of the location of macrophytes' shoots (using C. demersum as example) in a column of water. MOSCOW