The determination of 4-nonylphenols has been developed using HPLC with fluorescence detection method. 4-Nonylphenols in sea water samples were extracted using a liquid–liquid extraction. Acidification and adding salting-out agents (NaI) of water samples increased the extraction degree. The extraction of 4-nonylphenols was done twice with 20 mL of methylene chloride. The applied concentrated range was over 5–1000 ng/mL for the 4-nonylphenol. The recovery test ranged from 78.5 to 89.9% with relative standard deviations between 1.0 and 7.5% of 100 ng/mL of the standard phenols spiked with the water sample and the detection limit was 1 ng/mL. The proposed method was applied satisfactorily for the determination of 4-nonylphenole in sea water samples. Levels of 4-nonylphenol were determined in the sea waters of the Amur Bay. Water samples were collected every year from 2008 to 2015. Concentrations of 4-nonylphenol in the waters ranged from levels below the detection limits up to 1.24 μg/L.
4-Nonylphenol ; HPLC ; Sea waters ; Amur Bay
Alkylphenol compounds appear in waste waters of major industrial centers, and their content in coastal sea water areas may be rather high and influence living organisms negatively (Taylor and Harrison, 1999 ). One of the representatives of alkylphenol class is 4-nonylphenol not allowed to be dumped in the exclusive economic zone of the Russian Federation (http://base.garant.ru/12147594/ ). However there are some data about the presence of 4-nonylphenol and other compounds of alkylphenol class in sea waters (Kondakova et al., 2012 ).
4-Nonylphenol, as well as some other alkylphenols, has strongly marked xenoestrogen properties (Bernacka et al ., 2009 ; Peng et al ., 2006 ). For the first time estrogenic activity of phenols was noted in 1978 (Muller and Kim, 1978 ). Xenoestrogens (destroyers of endocrine system) are substances of anthropogenic origin, simulating, copying or blocking the effect of natural hormones synthesized by the endocrine glands; they interact with estrogen receptors and change a normal course of biochemical processes of the cell (Renner, 1997 ). Xenoestrogens influence the reproductive and endocrine systems of animals as well as estrogen, cause feminization of the population and have mutagenic and carcinogenic effects (Vazquez-Duhalt et al., 2005 ).
4-Nonylphenol formed in the environment by the degradation alkylphenolpolyethoxylates (AP) (Ike et al ., 2002 ; Jonkers et al ., 2001 ). AP are actively used in various industrial processes (Kneeper and Berna, 2003 ). Oil, mining, chemical, textile, plastic and pulp-and-paper industries are the major consumers of these products (Kneeper and Berna, 2003 ; Maguire, 1999 ; Soto et al ., 1992 ). Alkylphenol polyethoxylates are the most widespread non-ionogenic surface-active substances all over the world due to their high stability and excellent cleaning and emulsifying properties (Maguire, 1999 ; Berryman et al ., 2004 ).
Despite the fact that alkylphenolpolyethoxylates are not classified as highly toxic substances and can be effectively removed from waste waters by means of standard cleaning, they represent a class of highly environment-hazardous compounds (Canadian Council of the Ministers of the Environment, 2001 ). The reason for that is the fact that anaerobic condensation and biotransformation result in nonylphenol polyethoxylates transforming into stable short-chain metabolites, such as 4-nonylphenol which is considered as a potential destroyer of the endocrine system (Berryman et al., 2004 ).
In connection with marked lipophilic properties, 4-nonylphenol can be accumulated in the organic matter and lipids of solid matrices and animals, respectively. Bioaccumulation effects, which may be present in the next generations though may be of sharp-toxic nature as well (Peng et al., 2006 ).
Due to high toxicity, large production volumes and high stability in the environment, the worlds ecological and governmental organizations suggested to impose restrictions on the use of APs (Canadian Council of the Ministers of the Environment, 2001 ). Use of household detergents based on 4-nonylphenol is already forbidden or restricted in most European countries, for example, in Switzerland the ban to use polyethoxylates of 4-nonylphenol was imposed as far back as 1986. In some other countries risk reduction was achieved by voluntary agreement with detergent manufacturing companies (Ahel et al., 2000 ). According to the Russian legislation (Regulation of the Russian Federation Government no. 251 dated March 24, 2000) and MARPOL Convention 73/78, discharge of nonylphenol to the marine environment is forbidden in the exclusive economic zone of the Russian Federation. Nonylphenol is entered in the “Bulletin of the Russian Register of Potentially Dangerous Chemical and Biological Substances” (http://rpohv.ru ).
There are some data in the literature about 4-nonylphenol content in river and lake waters as well as in coastal sea water areas. 4-Nonylphenol was found in surface waters, discharged waters and bed silt of most developed countries: Canada, Great Britain, Spain, Japan, USA, Germany, Taiwan, Switzerland and Italy (Li et al ., 2004 ; Bester et al ., 2001 ; Ying et al ., 2002 ). Concentration of nonylphenol in the environmental objects is varied from below detection level values to rather high concentration of 644 μg/L (Bernacka et al., 2009 ). According to the classification given by Baronti (Soto et al., 1992 ), surface waters with content of nonylphenol less than 1 μg/L relate to poorly contaminated, 1–10 μg/L to contaminated, and more than 10 μg/L highly contaminated. According to a lot of researches, 4-nonylphenol is toxic for fishes (LC50 = 17–3000 μg/L). Invertebrate and marine algae are also sensitive to the effect of 4-nonylphenol (LC50 in intervals of 21–3000 and 27–2500 μg/L, accordingly) (Vazquez-Duhalt et al., 2005 ).
Analytical determination of alkylphenols is complicated due to their low concentration in the investigated matrices, dilution and frequently high saline background of the sample. Therefore, concentration of 4-nonylphenol is necessary prior to its determination.
Earlier, 4-nonylphenol content in coastal waters of the Russian Far East was not analyzed at all, that is why the purpose of this work was to determine 4-nonylphenol levels in the region most affected by anthropogenic influence — the south of Primorsky region.
Analytical standard of 4-nonylphenol (> 98% pure) was obtained from Alfa Aesar (Ward Hill, MA, USA). Sodium chloride, magnesium sulfate, magnesium chloride, calcium chloride, potassium chloride, sodium bicarbonate, potassium iodide, potassium bromide, sodium hydroxide, and anhydrous sodium sulfate, all > 99% pure, were from Neva-reaktiv (St. Petersburg, the Russian Federation). Hydrochloric acid solution (36% v./v. in water) was > 98% pure. To perform the extraction, pentanol, benzene, and hexane were used from Vekton (St. Petersburg, the Russian Federation), and methylene chloride, carbon tetrachloride were from Ekos-1 (Moscow, the Russian Federation). All employed organic solvents were of high purity grade.
In the preparation of the basic, working standard solutions and extracts for chromatographic separation, acetonitrile (class 0 for chromatography) was used from Kriohrom (St. Petersburg, the Russian Federation). Basic standard solution with a concentration of 1000 μg/mL was prepared by dissolution of precisely weighed portion of 4-nonylphenol in acetonitrile and stored at a temperature of − 18 °С for not longer than 10 days. Standard working solutions of 4-nonylphenol in acetonitrile with concentrations of 0.01, 0.05, 0.1, 0.5, 1.0 and 2.0 μg/mL were used for plotting the calibration curve (Fig. 1 ). Working standard solutions were prepared immediately before the analysis.
Artificial seawater prepared according to Krot (1966) was used as artificial matrix. For this purpose weighed portions of dry salts (sodium chloride, magnesium sulfate, magnesium chloride, calcium chloride, potassium chloride, sodium bicarbonate) were dissolved in 500 mL of distilled water. The derived mixture was put into an ultrasonic bath for 5 min and diluted with distilled water to 10 L. Salinity of the prepared artificial seawater was 35 ± 0.5‰. Artificial seawater was stored at a temperature from 0 to + 2 °С for not longer than 5 days.
Sonication processing was performed used Sonorex Super, RK 52 (Bandelin electronic GmbH & Co KG, Germany, 50 Hz, 220 V).
The sea water samples were taken at times from May to September every year from 2008 to 2015 (Fig. 2 ). Samples were collected manually. One liter of filtered water sample was collected in 1 L dark glass bottle with Teflon cap. It was acidified with 1 mL of conc. HCl (pH ~ 2) to protect it from biodegradation. Samples were stored at 1 °С. The water samples were analyzed within 3 days.
Location of sampling sites in the Amur Bay.
Stations no. 1 — estuary of the Ob'yasneniya River, no. 2 — the Zolotoy Rog Bight, no. 3 — Kuper Саре, no. 4 — estuary of the Kedrovaya River, nos. 5, 7 — central part of the Amur Bay, no. 6 — estuary of the Barabashevka River, no. 8 — Tokarevsky Cape, no. 9 — Staritsky Cape, no. 10 — Ushi i., no. 11 — Matveeva stone, no. 12 — Vasiliev Cape, no. 13 — the Stark str., no. 14 — Prokhodnoy Cape, no. 15 — Likander Cape, no. 16 — Lighthouse, no. 17 — De Vries Bay, no. 18 — Vladivostok Marine Station, no. 19 — estuary of the Razdolnaya River.
PH-meter “HANNA-210” was used for control of the environment acidity.
Extraction of phenols was performed using liquid–liquid extraction treatment. The extraction of total phenols from the water samples was performed with a total of 40 mL DCM (extracted twice with 20 mL of DCM), in the presence of sodium iodide and pH is adjusted between 2.0 and 3.0 with hydrochloric acid in separating funnel. After separation the methylene chloride layer was filtrated through anhydrous sodium sulfate. The obtained extracts were evaporated to 1 mL in a water bath (60 °C) on a rotary evaporator at reduced pressure. The solution is then concentrated with dried nitrogen gas to dry residue. The total volume is adjusted to 1 mL with acetonitrile. This solution is ready for HPLC analysis. The concentration of total phenols from environmental water samples was calculated by using “introduced–found” method.
Chromatographic measurement were taken using liquid chromatograph LC-10 ADvp (Shimadzu, Japan) with fluorescent detector RF-10AXL (excitation wave — 230 nm, emission wave — 320 nm). Separations were carried out on analytical column Discovery С18 (25 cm × 0.46 cm, 5 μm particle size), mobile phase:water–acetonitrile (90:10 v./v.), and isocratic mode. The flow rate was 0.5 mL/min.
Limit of detection was calculated as three times the signal of the background noise obtained in the analysis of water sample at the retention times of 4-nonylphenol.
Physical and chemical properties of the determined substance are important in the selection of conditions for chromatographic separation. Alkylphenols are usually divided using sorbents, modified by alkyl groups (С8 , С18 ). It makes it possible to make an analysis of highly polar compounds as well as to vary broadly polarity of mobile phase. Chromatographic determination of 4-nonylphenol was carried out in reversed-phased conditions.
When selecting the eluent, various mobile phases were studied: compounds of acetonitrile and ethanol with water. If water–alcohol solution is used, analytical definition is impossible due to asymmetry of 4-nonylphenol peak. Use of the phase acetonitrile–ethyl alcohol–water–acetic acid (50:30:19:1) did not bring any positive results as well due to the impossibility to detect the peak of 4-nonylphenol. Apparently, decrease of eluents рН value reduces a dissociation degree of 4-nonylphenol and will increase molecular form content, which in turn leads to the increase in retention time. If acidified eluents were used, efficiency got worse and the peaks became asymmetrical. An optimal variant was the use of the system acetonitrile–water (90:10), when the peak form was symmetric and retention parameters were optimal.
Limit of detection for 4-nonylphenol when fluorescent detector was used equaled 1 ng.
4-Nonylphenol extraction level was defined using various solvents. Artificial sweater was used as matrix. Benzene, hexane, pentanol, carbon tetrachloride, and methylene chloride were used as solvents. Fig. 3 represents a dependence of 4-nonylphenol recovery and polarity of the solvent used. Pentanol which has the highest dielectric permeability showed the most complete extraction. However pentanol forms poorly splitting emulsions and that makes the results of extraction to be non-reproducible. Methylene chloride was chosen as the most adequate solvent. It does not form any emulsions with water, and extraction level of a target component is close to the level of extraction with pentanol.
Influence of polarity (p′) on the recovery of 4-nonylphenol.
When studying the influence of acidity on the recovery of 4-nonylphenol, рН values were varied from 1 to 14 (Fig. 4 ). When pH values are from 1 to 11 the recovery is unchangeable. When рН value is more than 11 the recovery is decreasing. Undissociated molecules of 4-nonylphenol and its ions are unequally extracted with organic solvents out of water solutions. During extraction, undissociated molecules transfer into the organic phase, and ions hydrated with water molecules remain in water phase.
Influence of pH on the recovery of 4-nonylphenol.
Some discrepancy in the obtained dependence and theoretically calculated ionization degree is caused by an influence of the saline background of artificial seawater. The high recovery was when pH value is up to 3 since at this stage the 4-nonylphenol dissociation is almost completely suppressed and extraction is performed as effective as possible. However, when pH is less than 2, the scattered results of the test are obtained. Thus, the best рН value for 4-nonylphenol extraction is 2.
Addition of strong electrolytes into water solution of target compound may decrease or increase its solubility in water. The salting-out is explained by chemical interaction of salting-out agents and salting-out substances in the extraction systems. In result, compounds or complexes may be formed, which ultimately affect the value of the partition coefficient of the target component between the organic and aqueous phases. Potassium salts were used as the salting-out agents; the added quantity of the total sum of salts in the artificial matrix was 0.2 mol per 1 L (Helaleh et al., 2001 ). The salting-out agent ions with a small radius have the greater charge density in comparison with the ions with a larger radius. Therefore, the ions with a small radius are hydrated better, and their salting-out effect is less than the same of the large ions. Iodide ion has the largest radius among the salting-out anions being researched, and chloride ion has a minimal one. At that, the change of anion radius from 1.81 to 2.16 Å leads to an increase of 4-nonylphenol recovery in three fold, therefore the potassium iodide should be reasonably used as a salting-out agent.
Thus, the technique of the analysis of 4-nonylphenol content in natural waters using methylene chloride as a solvent is offered, at pH = 2 and in the presence of potassium iodide (0.2 mol per 1 L) as a salting-out agent with subsequent analysis applying the method of reverse-phase high performance liquid chromatography.
The offered method is applied to determine the content of 4-nonylphenol in sea waters of the Amur Bay, the Russian Federation. The least polluted with 4-nonylphenol are the central part (St 5, 7) and the western coast (St 4, 6) of the Amur Bay, and the most polluted are the Zolotoy Rog Bight (St 2, 8, 18) and the Ob'yasneniya River (St 1). The eastern coasts of the Amur Bay become polluted with insufficient waste water treatment from settlements and industrial enterprises (St 19). The main pollutants are electric power, municipal engineering, chemical industry, machinery and metal enterprises (Ogorodnikova, 2001 ). During a year, a lot of waste water are discharged into the waters of the Amur Bay and adjacent waters. According to the state statistics, 46,332,96 thousand m3 /year is discharged into Amur Bay, 23,480,27 thousand m3 /year into the Zolotoy Rog Bight, and among them 8222,83 thousand m3 /year into the Ob'yasneniya River. All discharged waste waters are classified as “polluted, not treated”; they include such polluting substances as: petrohydrocarbons (29.9 t/year), phenols (2.96 t/year), synthetic surface-active substances (86.37 t/year) and others (Chernyaev and Nigmatulina, 2013 ; Nigmatulina, 2007 ).
In result of the researches performed it was established that the content of 4-nonylphenol in the area of eastern coast of the Amur Bay (Kuper Саре, St 3) makes 0.44 μg/L. One of 4-nonylphenol income sources is the cardboard factory located in Ussuriysk constantly discharging the waste waters heavily contaminated with organic substances to the Razdolnaya River (St 19). Additional contamination sources may be industrial wastes from some other enterprises as well as domestic waste water. By results of the complex assessment in 2003, the waters of the Razdolnaya River are classified as “very dirty”, and maximum permissible concentration (MPC) of phenols was six fold increased: this river delivers 26–28 fold more contaminants to Amur Bay in comparison with all industrial and household city drains of Vladivostok. Existing currents in Amur Bay transfer water of the Razdolnaya River along the east coast, and it evidences the presence of 4-nonylphenol in the coastal waters of the islands (St 8–15). The concentration of 4-nonylphenol in the seawaters of these stations is less than 0.01 μg/L. In the central part of the Amur Bay and in area of the western coast (St 4–7), content of 4-nonylphenol is lower than the detection level. It is probably caused by the large area, depth of the Bay and system of surface and underwater circulating currents owing to which the significant dilution of the polluted waters takes place, and by significant remoteness of sources of 4-nonylphenol emission.
Content of 4-nonylphenol in the Zolotoy Rog Bight makes 1.22 μg/L (St 2). Specificity of this bay concerning the human impact is that the same is one of the old-developed areas and consequently is the most exposed to the city waste waters. City ports and ship repair yards discharging bilge waters, technical oils and fuel to Amur Bay water area exert a huge negative influence. The Ob'yasneniya River (St 1) which waters contain 1.24 μg/L of 4-nonylphenol makes a significant contribution to the bay pollution. Probably, one of the basic contamination sources is thermoelectric plant (CHP). A great quantity of waste water from cooling and blow-down waters from return water supply systems, frequently polluted by transformer fuel components, is discharged from the power plant without special treatment. It has been established that many components of transformer materials are made of 4-nonylphenol containing phenol formaldehyde resins. Replacement of transformers and their destruction probably leads to uncontrollable discharge of toxic components into the environment.
According to the above presented classification, waters of the Amur Bay may be referred to as “poorly contaminated waters”. The existing concentrations of 4-nonylphenol in Amur Bay waters are not capable to give any significant negative influence in the endocrine system of marine organisms and much less to cause their death. In the waters of the Zolotoy Rog Bight and the Ob'yasneniya River, content of 4-nonylphenol is at a subcritical level and is capable of causing some changes in the structure of marine organism communities. These waters are classified as “contaminated” (Baronti, 2000 ).
Marine organisms are one of the important links in a food chain when toxicants get into a human organism. Thus the existing situation predetermines the necessity of carrying out high-grade monitoring works, revealing and classifying sources of pollution by the substances showing xenoestrogenic effect.
The analysis of samples was performed with support from the Russian Science Foundation (agreement no. 14-50-00034 ).