1. Introduction
Diesters of
o-phthalic acid (PAEs) are of particular importance among organic micropollutants in the environment as they income from both biotic and abiotic sources. The PAEs are used as plasticizers and added to polymer materials in order to obtain required characteristics of plastics. Absence of chemical links between a polymer mesh and a plasticizer results in gradual migration of PAEs from plastic things into the environment at their exploitation and utilization. Traces of PAEs are found in the atmospheric aerosol and surrounding air, in drinking, marine, lacustrine waters and in bottom sediments [
1,
2,
3,
4,
5,
6,
7,
8]. Recent studies suggest a probable biosynthesis of PAEs by different plants, freshwater algae, cyanobacteria and fungi [
9]. Therefore, PAEs found in surface water at trace levels are evidently not to be related exclusively to anthropogenic pollutants as they can income both from biogenic and abiogenic sources, the contributions of which may be comparable by their amount.
PAEs are biologically active and manifest hepo-, neuro-, cytotoxicity. Toxic effect of PAEs, such as disturbance of endocrine and reproductive functions is found out not only at humans but also at on various types of wildlife – mollusks, crustaceans, fishes and invertebrates [
10,
11,
12]. Taking into account biological properties of PAEs and polymer materials production volume – up to 390 million tons per year [
13], PAEs, as industrial pollutants, are included into the list of permanent organic pollutants (POPs) [
14]. Six PAEs: di-methyl phthalate (DMP), di-ethyl phthalate (DEP), di-
n-butyl phthalate (D
nBP), benzyl butyl phthalate (BBP), di-(2-ethylhexyl) phthalate (DEHP) and di-
n-octyl phthalate (DOP
) are considered as priority ones and are to be permanently controlled in the environment. It is shown at the same time that PAEs possess an allopathic activity, antimicrobial and insecticide properties, which increase competitiveness of plants, algae and microorganisms [
15].
PAEs income from stationary sources (waste waters from urban agglomerations purification facilities, emissions of industrial enterprises, areas of stock and treatment of industrial wastes and household plastic) determines a high level of these pollutants in surface and underground waters at local sites. The amount of found PAEs can reach 15-16 congeners, their ratio and concentration depend on sampling area and sources nature. In Lake Taihu, China 16 PAEs congeners in the range of summary concentration (Ʃ
16PAEs) from 0.02 to 16 µg/L were found, dominant among them were D
nBP, DEHP and di-
i-butyl phthalate (D
iBP) [
16]; in the water of Kaveri River, India six PAEs congeners in the range of summary concentration (Ʃ
6PAEs) from 0.31 to 4.6 µg/L were found, dominant congeners were DEHP, DEP and D
nBP [
4].
Water bodies pollution with household plastic is considered as one of probable PAEs sources in aquatic environment. However, as it was shown in model experiments [
15], colonies of microorganisms, which recycle PAEs incoming from plastic develop in water bodies on plastic surfaces. As rates of PAEs diffusion and biodegradation are comparable, only a part of their amount diffuses into water.
Atmospheric transfer of polluted air masses and POPs precipitation from the atmosphere is main PAEs source in background areas including waters in Central Arctic and near Norwegian shore [
17], high mountains in China [
18]. In the atmosphere, PAEs congeners are distributed among aerosol particles and gas phase of surrounding air, the gas phase includes congeners with short alkyl chains, while congeners with long ones are adsorbed on solid aerosol particles. From the atmosphere, PAEs income onto a spreading surface – land or water by dry precipitation, or with atmospheric precipitations including aerosols particles with adsorbed hydrophobic substances [
6]. In surface waters, at background level of pollution in PAEs fraction, D
nBP and DEHP congeners determine a dominant group of PAEs, and change of their source influence drastically both its composition and its concentration.
In the present paper, we chose Lake Baikal as a model for studies of PAEs in surface waters with background pollution level. Choice of Lake Baikal is due to a huge volume of its water mass – up to 23600 km3, to water surface area of 31700 km2, to the depth up to 1642 m, to sharply continental climate of East Siberia and to availability of potential POPs sources on the shore. The studies tasks included: i) identification and assessment of PAEs concentration level in Lake Baikal water at present; ii) revealing of main factors influencing PAEs concentrations level; iii) distribution of PAEs in the water area and in Lake Baikal deep horizons; iv) identification of sources of PAEs in the water of Lake Baikal; v) assessment of environmental risk of PAEs in the surface waters with background pollution. In order to resolve these tasks, we monitored PAEs during 2015-2022 in the upper water layer in the pelagic site, at deep horizons, in near-shore zone and in bays of the lake, in water of South Baikal tributaries and performed a comparative statistical analysis of monitoring results.
2. Materials and Methods
2.1. Reagents and standards
Extraction of PAEs congeners from samples of water and preparation of standard solutions for GC-MS analyses were done using n-hexane (HPLC grade, Cryochrom, Russia) and acetone (reagent grade, EKOS-1, Russia). The content of PAEs in organic solvents was controlled by GC-MS prior analysis according to the signal-to-noise ratio (S/N) of the analytes, when S/N ≥ 3, the solvents were distilled before use. Glassware was sequentially washed with a solution of K2Cr2O7 in anhydrous sulfuric acid, then with distilled water, hermetically sealed with aluminum foil stoppers. EPA 606-M Phthalate Esters Mix (Supelco, Germany) were used as reference standards, deuterated phthalates: dimethyl phthalate (DMP-d4), dipropyl phthalate (DPP-d4) and dihexyl phthalate (DHP-d4) (Witega, Germany) – as surrogate internal standards.
2.2. Sampling
Water samples were collected after disappearance of ice cover on the lake (late May – early June) and after summer season (September) in 2015-2022. From pelagic site and deep horizons of the lake (
Figure 1), samples were collected using an SBE-32 cassette sampler (Carousel Water Sampler, Sea-Bird Electronics, Bellevue, WA, USA). Water samples from lake tributaries were collected in river mouths of South Baikal, upper water layer (0 m). In near-shore zone, water samples were collected from upper water layer (0 m) at the distance of at least 50 m from a shore and at the depth of water layer not more than 20 m. At each station, two samples (1 L water) were taken in 1 L glass bottles, and to which 0.5 mL of a 1 M aqueous solution of sodium azide (Merck, Germany) was added as a preserving agent. Water bottles were closed using a lid with an aluminium foil gasket and stored at 5 °C until laboratory analysis.
2.3. Sample Processing
The content of PAEs in water samples was estimated by a method [
19] including LLE of PAEs into
n-hexane and direct analysis of extract aliquots by GC-MS-SIM. Taking into account minimal content of suspended organic matter and POPs in Baicalein water [
20,
21,
22], in order to decrease prepare stage while determining PAEs and in order to assess biota habitat, we analyzed non-filtered water. Before extraction deuterated phthalates: DMP-d
4, DPP-d
4 and DHP-d
4 were added in water samples for quantitation.
2.4. GC-MS-SIM Analysis
Aliquots of extract samples were analyzed using an Agilent Technologies 7890B GC System 7000C GC–MS Triple Quad chromatography–mass spectrometer with an HT- 8, SCE Analytical Science capillary column (30 m × 0.25 mm × 0.25 μm). The program for the GC oven was initiated at 50 °C, increased to 300 °C at a rate of 25 °C/min, and finally held for 5 min. The sample injector was set at 280 °C, 2.0 μL of sample solution was injected using spitless mode. The temperatures of the ion source and quadrupole were maintained at 230 °C and 150 °C, respectively. The electron impact ionization energy was 70 eV. A constant pressure at 25 psi of ultra-pure helium (99.999%) was used for a carrier gas.
PAE peaks were recorded using the SIM mode and references ions с m/z: 149, 153, 163, 167 and identified by relative retention times. PAE congeners were quantitated by the method of internal standards. The GC-MS was calibrated in the range of expected PAEs concentrations in water from 0.01 to 10 µg/L. Taking into account a simple matrix of the samples (surface waters in the background regions), causing no matrix effect, the calibration was carried out using calibration solutions prepared in a solvent used as an extractant. The solutions were prepared twice by mixing corresponding volumes of the certified reference standards PAEs mixture and surrogate standards. The reliability of approximation of calibration dependences Sa/Sst = k(ma/mst) were corresponded to the condition R2 ≥ 0.99. The concentration of PAEs in water samples was measured as the average value of the results for two parallel samples. The secondary contamination of the analyzed samples by PAE congeners from the laboratory background was evaluated by the procedure of a blank experiment, the obtained concentrations were subtracted from the results of analyses. The limit of determination of PAE congeners was estimated as 0.07 – 0.10 µg/L, relative standard deviation (RSDRl) for the determination procedure was 10-16 % for individual congeners.
2.5. HPLC-HRMS-TOF Analysis
The ratio of stable carbon isotopes
13С/
12С in the composition of DEHP from surface water at a trace concentration level was estimated by a method proposed in [
23]. It is based on the concentration of hydrophobic components of water using an analytical reversed phase HPLC column followed by their gradient separation and detection of eluted PAEs using a high-resolution time-of-flight mass spectrometer (HRMS-TOF) in the form of molecular ions. The ratio of stable carbon isotopes
13С/
12C in DEHP congener is calculated as a ratio of the peak areas of the monoisotopic masses [M+1+H]
+ and [M+H]
+. Ratio values of
12C/
13C (Δ
13C value
1) are calculated relatively to the
13C/
12C ratio in commercial DEHP congener. Borderline values in the scale of Δ
13C were established on the base of Δ
13С values in DEHP of deliberately biogenic or anthropogenic genesis. DEHP found in Baicalein phytoplankton (Δ
13С –46‰), in cells of biomass of
Aconitum baicalense Turcz ex Rapaics 1907 (Δ
13С –50‰) is assessed as a congener of biogenic origin. DEHP present in just fallen snow on an urban territory (Δ
13С +5.2‰), in waste waters after their purification at purification facilities (Δ
13С +0.2‰) is estimated as a congener incoming from anthropogenic sources. The minimal concentration of congener DEHP in water required for a reliable determination of
13С/
12C value is estimated at the level of ca. 0.2 µg/L.
2.6. Environmental risk assessment
In order to assess a potential environmental risk from PAEs found in Lake Baikal water, we used a method of risk quotient (RQ), according to the European technical guidance document on risk assessment [
24]. Values of RQ were calculated from the average concentration of PAE congeners in the water (C
PAE, µg/L) and the predicted no effect concentration (PNEC), as in Equation (1):
In this work, we used values of PMEC selected before for baicalein biota [
25] and from [
26].
2.7. Statistical Methods
Pairwise correlations between two replicates of measurements and the average concentration of PAEs over the entire concentration range for priority congeners in samples of Baikal water were estimated with Spearman’s r correlation coefficient. P values for the correlation coefficients were calculated using Spearman’s «W» statistics. Unreliable values of correlation coefficients (P values > 0.05) were replaced with 0 values. Pairwise correlations were visualized with a heat map generated using «gplots» in R [
27]. Lines and columns in the correlation matrix were clustered and grouped in order of similarity (i.e., Euclidean distance metric and the complete-link clustering method).
For further analysis, these missing data were replaced with averages for these congeners according to the recommendations [
28]. Water samples that contained concentrations of congeners (at least one congener) outside the limits of three differences between the first (Q1) and third (Q3) quartiles from the median concentration values were considered outliers and excluded from the analysis. PERMANOVA used Euclidean distance metric and 1000 permutations for P values calculations. Before PERMANOVA analysis, all concentration values в were transformed to eliminate the physical dimensions by ranging from 0 to 1.
Confidence intervals for the mean values of the concentrations PAEs, grouped according to the explanatory parameter (year, season (spring, autumn), ecotope (pelagic, сoastal, bay, river, sampling site (pelagic or other), basin (southern, central, northern), sample points), were estimated using the bootstrap method in the «boot» package and the R programming language (1000 bootstrap replicas).