Legislation, pollution parameters and biologically active substance concentrations: evaluating origin of sapropel for pharmaceautical production

Background Development trends need the necessity for wider use of the local resources and available natural materials are one of the priorities around the world. Freshwater sapropel is a common material in the water basement of the lakes in Latvia, but still not sufficiently explored. The main goal of the project to start detailed and systematic research on the medical properties of sapropel to be obtained in Latvia, promote its scientifically based use in balneology, develop new medical procedures and services, and promote development of new exportable products. Results The results include the survey, sampling depths and processing, evaluation of external signs, physical, chemical and biochemical parameters and evaluation of microbiological indicators. Active components from the sapropel samples extracted using the alkaline method. Sapropel extracts were characterized by organic carbon content, humic and fulvic acid concentrations, total phenolic content, trace metal and pesticide concentrations, total antioxidant status and microbiological flora. (AO) fulvic acid a concentration of carbon fraction (FA-C) till 700 mg/L. AO activity has been measured by a various methods such as Total phenolic concentration (TPC), Total Antioxidant Status (TAS) level, 2,2’-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid (ABTS), and DPPH radical scavenging assays. Results revealed that the AO activity is dependent on the concentration of carbon fraction in FA because lower concentration was not sufficient to scavenge free radicals due to its low TAS level. One tendency is that one of the lakes – Dunaklu – gives considerably lower both AO and humic and fulvic acids levels. However, Ivusku Lake with the lowest AO levels is high at the FA level. The concentration of humic acid and FA and the AO levels varies strongly between different lakes. It was found that AO level is considerably higher in organic sapropel extracts from the lakes Audzelu, Mazais Kivdalovas, and Zeilu. The total AO level is almost threshold between the highest and lowest values. The difference in HA (max. 167.8 mg/ml, min. 113.1 mg/ml) levels between different lakes much or less pronounced than the difference in the FA (max. 76.1 mg/ml, min. 44.5 mg/ml) and AO levels


Background
Sapropel medical importance. Sapropel might seem something mysterious and incomprehensible that can be found somewhere deep in the water and is sunlight inaccessible. However, an extremely interesting and useful material has long been a major success for health improvement and treatment.
Sapropel used in medicine for a long time and is widely used in various health sectors, but still not sufficiently explored. This amazing natural material found in the climatic zone of Europe and Asia.
Sapropel is a common material in the water basement of the lakes in Latvia (Stankevica 2012 Sapropel is a pasty mass of light grey, pink, brown, brownish olive or almost black. Sapropel's deposits in swamps and lakes only occurred on post-ice age, which took place in the Baltic States 12-15 thousand years ago. Medical mud formed by complex biological transformations of Holocene sediments. The composition of the therapeutic mud depends on the location of the acquisition -freshwater, saltwater or thermal springs. Sapropel sludge is classified as inorganic sediment sludge, river or lake mud, organic sediment sludge, freshwater and saltwater lake mud, peat sludge, mixed sludge, volcanic sludge and artificial sludge (Sigl 2007;Batzias 2014).
In ancient times, people considered that sapropel can cure almost any disorder, even improve the long-term effects on the skin. Even today it is attributed that sapropel is marvellous material for wide range applications.
People are always concerned about maintaining beauty and health, and illness treating. Already in the 5th century B.C. ancient Greek scientist, Herodotus developed a method of using mineral waters for health improvement (Erfurt 2011).
Hippocrates also wrote about the healing properties of the seawater and saltwater. However, only in the 19th Century the science direction, that explores the use of various mineral and thermal waters as well as sludge or sapropel for health maintenance and treatment of various diseases progressed (Cirillo 1994). This type of treatment called balneology. Despite the fact, balneology as mud therapy became popular in Europe in the 17th Century already. Balneology developed in Germany, France, Italy, Austria, and Romania. Various experiments with mineral water and sludge helped to understand the healing properties. Sludge began to use in cosmetics and cosmetic surgery to promote skin firmness and skin rejuvenation after surgical manipulations and skin damages (Van Tubergen 2002).
To improve health quickly, in the 20th Century, hydrotherapy began to use to supplement other therapies, such as peloid therapy or mud therapy, massage, ionophoresis, phonopheresis, physiotherapy and gymnastics (Constantin 2011;Andrade 2011).
Balneotherapy has proven its healing abilities in the treatment of various rheumatologic (connective tissue) diseases -osteoarthritis, fibromyalgia, spondylitis, rheumatoid arthritis and other skeletal muscle diseases (Celso de Sousa Figueiredo Gomes 2018).
Sapropel is a multifunctional and widely used medical treatment, and believed to be useful for lymphatic and circulatory enhancement, vascular strengthening, skin structure, cellulite and subcutaneous fat reduction. It has a pronounced antibacterial effect and enriches the body with calcium, magnesium, bromine, iodine, potassium, and amino acids. Sapropel has an antioxidant effect that improves skin structure, smoothens wrinkles and prevents new wrinkles, removes swelling, strengthens nails and hair, normalizes sebaceous gland secretion, helps hair loss. The therapeutic effect of sapropel helps to restore immunity, maintain the cellular structure of various skin diseasesdermatitis, seborrhea, acne, and other rashes and other skin diseases. However, today, sapropel preparations are most widely used in balneotherapy and cosmetology, especially in the treatment of The multifunctional effect of sapropel on the whole organism explained by its complex of chemical and biological structure. The bioactivity of sapropel determines by its humic acids, fulvic acids and heratomelic acids, various vitamins and microorganisms that release antibiotics. Previously, sapropel commonly used in raw form and there is no standard methods for sapropel extraction, generally.
Currently, there are few extraction methods for getting bioactive components from raw sapropel (Klavina 2019). Latvian freshwater sapropel could be used as raw material for getting sapropel extract and use it as remedy. All mentioned above brings us to the main question for sapropel usage in medicine, balneology and pharmacy "how to develop quality criteria for raw sapropel and its extracts". The quality criteria should include minimum requirements for pollution levels (heavy metals, pesticides), biologically active substance concentration, pH values, antioxidants as well as physical characteristics (Obuka, 2018; Sánchez-Espejo 2014).
Sapropel legislation. It is important to monitor and inspect sapropel extraction sites to assess the level of contamination and the environmental impact of anthropogenic activity. Sediment contamination is considered to be a major environmental issue because sediment acts as a reservoir for pollution. Sediments are an integral part of the aquatic ecosystem, which provides food and habitat for various aquatic species.

Production of sapropel in the industrial scale in Latvia is regulated by several Laws and Cabinet
Regulations. One of them is the Environmental Protection Law (2013), which is the main normative act in the field of environmental protection. The purpose of the law is to ensure the preservation of the quality of the environment and the sustainable use of natural resources.
The Law on Environmental Impact Assessment (2017) defines the activities that require environmental impact assessment. The need for an environmental impact assessment procedure for the extraction of sapropel in lakes is governed by Chapter IV Section I point 1 and point 25 of Annex 1.
Obtaining Sapropel must also comply with the On Pollution Law (2018). Pollution, purpose is to prevent or reduce damage to human health, property and the environment caused by pollution. The law sets out the procedures and guidelines that must be taken into account when performing polluting activities to minimize the impact on natural resources such as soil, air, and water. The planned extraction of minerals should take into account the emissions of water, and air pollutants. With regard to the extraction of sapropel, it is necessary to assess its impact on lake water quality; to ensure that the environmental quality standards for priority and hazardous substances in surface waters and the priority substances in the lake biota are not exceeded during the extraction process

Material And Methods
An important step is to determine the potential sources of sapropel accumulating within the depositional setting. Another consideration for site selection is to ensure that access granted from the relevant landowner and permission sought from the relevant agency if the site is designated as protected. It is vital to ensure that there is no risk of damaging any subsurface utilities (gas, electricity etc.) (Klavina 2019).
In most of the Latvian lakes, there is sapropel, in many swamps and under the peat layers, it also found. Major stocks of this mineral resource are around 300 million cubic meters, mostly located in Latgale districts, eastern Latvia (the blue region on a map, Fig. 1).
Official geological survey of Latvia lakes, from Latvian lake database (ezeri.lv), used in the selection of the area of the exploration.
The main selection criteria were the sapropel deposits depth, hydrological regime, the history of agriculture next to the lake and the potential exposure to industrial waste. One hundred and five sapropel samples obtained from five lakes (Audzelu -A, Dunakla -D, Ivusku -I, Zielu -Z and Mazais Kivdalova -K) during the wintertime.
Since the sapropel accumulates in the lake, there may be differences, depending on inflowing brooks and trenches in the lake, which may bring pollutants that are deposited closer to estuaries and also on the age/depth of the sapropel layer (therefore, there can be differences in the concentration of potential pollutants).
Prior to the sample collection, the thickness of the proper sediment layer was determined and the depth of sapropel deposit established for each of the lakes as well as within each of the lakes by taking probes. Well-composed sapropel layer for further laboratory analyses taken on the three different depths of sapropel sediment at each extraction point (7-11) through the lake coordinates ( Fig. 2).
The location of the lakes influenced by several external and internal factors. The soil conditions, climatic conditions, and access facilities to the main road and to the fields applied to mainly natural external factors. Internal factors depend on the type of business enterprises around, farmstead and the relative position of its different buildings. Among general principles that must be taken into account is the availability of transportation between buildings and driveway to the lakes.
To better explore the possibilities of using Latvian sapropel, Riga Stradins university researchers have launched a three-year study to test and standardize a composition, properties, storage options and therapeutic effects of the sapropel.
There are main characteristics of the sapropel samples. Organoleptic properties -the color ranges from pale yellow to black, depending on the type of sapropel and the site of exploration. The texture is determined based on the initial description of the site. The smell is neutral (if any changes in the smell observed, the storage conditions of the samples should be checked). Sapropel must be homogeneous inconsistency, with no inclusions or excess water. Another one characteristic of sapropel is dry matter content. The sapropel is dried and weight loss compared to samples of a recognized sapropel site. This is mainly to determine if the series of raw materials are not obtained too shallow at the top of the sapropel layer. In addition, the physical examination of sapropel.
Sapropel is divided into four main types -organic, silica-containing, carbonate and mixed type sapropel. There are several kinds (peat, carbonated, iron-rich, mixed, silicate with increased ashes contain, etc.) of each type of sapropel, the main type being determined by the biological and oxide Each sample identified by specifying the exact location of the site in the lake and the depth of extraction from the surface of the water and the beginning of the sludge layer.
All sapropel samples kept in closed plastic containers (Fig. 3a) without oxygen access in order to prevent oxidation of the sapropel and its active components. The sediments were refrigerated and kept at 4 °C (temperature closer to the natural water temperature at the bottom of the lake) and then, stored.
For the extraction of active components from the sapropel samples the alkaline method was selected ( Fig. 3c) (ISO 14502-1:2005).
Sapropel extracts were characterized by total organic carbon content (TOC), humic acid (HA) and fulvic acid (FA) concentrations by the use of the spectrometric method.
The sample pH level was determined using distilled water (volumetric ration sample: water − 1:2.5).
Sapropel samples were analyzed for organic matter and carbonate content using the loss-on-ignition (LOI) method Loss on ignition. The dried sapropel sample was heated for 4 h at 550 °C and 2 h at 900 °C, after each heating the sample weighed and calculated by assuming that all organic matter in the sample is burned at 550 °C, and at the next temperature (900 °C). An important parameter of sapropel is the amount of organic matter. It can be determined whether organic substances mineralize by releasing their nutrients or accumulate in sediment and their mineralization process is slow. The amount of organic matter in the sediments of the lake can vary (from 20-90%), depending on the productivity of the lake and the type of land use in the catchment area. The carbonate content (from 1 to 15%), in turn, depends on the amount of carbonate soils in the catchment area, as well as on benthic organisms in mollusks whose shell may contain carbonates (Heiri 2001).
Trace metal concentrations were determined in sapropel samples by electrothermal atomic absorption spectrometry with Zeeman background correction. Before starting the analysis, sapropel samples were dried at 105 °C and finely ground with a mortar and pestle. Sampling was carried out in a closed container with microwaves in the digested system using nitric acid and hydrogen peroxide.
The contents of the vessels were quantitatively transferred to 20 mL graduated polypropylene flasks and filled to mark with Milli-Q water (ISO 14502- 1:2005).
The content of the total phenolic content of the extract was expressed as gallic acid (GA) equivalents.
The gallic acid was used to set up a standard curve. An aliquot of 500 µl of an extract was mixed with 2.5 ml of Folin-Ciocalteu phenol reagent (10x dilution) and allowed to react for 5 min. Then 2 ml of 7.5% Na 2 CO 3 solution was added and allowed to stand for 1 h before the absorbance of the reaction mixture was read at 765 nm. All tests were performed six times. The total polyphenol contents of the extract were evaluated from the gallic acid standard curve and expressed as mg of gallic acid per

Results & Discussion
The main goal of the project is to carry out detailed and systematic research on the medical properties of sapropel to be obtained in Latvia, to promote its scientifically based use in balneology, to develop new medical procedures and services, and to promote the development of new exportable products. So far, studies have been basically focused on sapropel for other purposes, such as agriculture or cosmetology, thus without using sapropel and its acquired mud biomedical and biopharmaceutical potential.
Sapropel is a jelly-like homogeneous mass, its texture in upper layers is close to cream-like, and in the lower layers, it becomes denser. The sediments are odorless except for separate types that smell of hydrogen sulfide (Table 1). Sapropel color depends on organic substance and mineral additions and it refers to caustobiolites (Leontiev 2017). The temperature of 4 o C without exposure to light and oxygen were sufficient for preserving sapropel. Samples from different lakes and depths have different organoleptic characteristic and must be checked on color, texture, visual consistency, impurities and uniformity, as well as smell. Organoleptically sapropel samples found from greenishyellow to almost black ( Table 2). High silica content usually relates to green and yellow colored sapropel and could be found in moraine lakes. The high organic matter relates to black colored sapropel and could be found in the lakes with low mineral content.
Sapropel consists of a sludge solution, a skeleton, and a colloidal complex. The sludge solution contains water and dissolved substances that mean mineral salts, low molecular weight organic substances, vitamins, and enzymes.
Brown and dark green sapropel affiliate the mixed type of sapropel and its origin comes from lake's plankton, plants and sometimes connected with peat existence. This type of sapropel mostly can be found in Latvian lakes. Sapropel sample pH level is around 7-8 it means that these sapropel sediments have high mineral content (Table 3).  Heavy metals are one of the most widespread and significant contaminants in sediment, causing serious environmental effects due to their toxicity, persistence, and bioaccumulation.
Lake sediments can be polluted in many ways, mainly man-made pollution, such as sewage disposal, runoff from agricultural land, lakes and transport from nearby roads. However, the accumulation of heavy metals in lake sediments is not always associated with anthropogenic pollution, and the sediment may be naturally "enriched" with various metals influenced by the local geochemical Results revealed that the AO activity is dependent on the concentration of carbon fraction in FA because lower concentration was not sufficient to scavenge free radicals due to its low TAS level. One tendency is that one of the lakes -Dunaklu -gives considerably lower both AO and humic and fulvic acids levels. However, Ivusku Lake with the lowest AO levels is high at the FA level. The concentration of humic acid and FA and the AO levels varies strongly between different lakes. It was found that AO level is considerably higher in organic sapropel extracts from the lakes Audzelu, Mazais Kivdalovas, and Zeilu. The total AO level is almost threshold between the highest and lowest values. The difference in HA (max. 167.8 mg/ml, min. 113.1 mg/ml) levels between different lakes much or less pronounced than the difference in the FA (max. 76.1 mg/ml, min. 44.5 mg/ml) and AO levels ( Table 3). These guidelines may apply to sapropel as the topical application to the skin or in the form of various gels, creams, shampoos and other products for external use. EU guidelines state that cosmetic products must not contain microbial pathogens and the total aerobic microorganisms must be low.
For cosmetic products intended for paediatric or use near to eye zone (Category 1), the CFU or if the material used is of low toxicity or non-toxic and not in high volume, is to dilute it with large amounts of water and then ensure that it is discharged into the sewage system, prevent entry into natural waterways (WHO 1999). If preservatives or other compounds are added to the material used before or after application, it should be considered their occurrence in nature, effects on flora, fauna, and potential degradation time.

Conclusions
The use of organic-rich lake sediment like sapropel considered a solution because of the necessity for wider use of the local resources and available natural materials and due to previously insufficient research of sapropels for pharmaceutical needs.
The appropriate sapropel layer found from 2.0 to 9.0 m (actual layer -0.9-11.4 m) from the surface of the sediment layer. If the depth is less than 1.5 m from the surface of the sediment layer, sapropel sediments are not fully developed.
Sapropel is a jelly-like homogeneous mass, its texture in upper layers is close to cream-like, and in lower layers, it becomes denser. The sediments are odorless except the separate types that smell of H 2 S. Organoleptically sapropel found from greenish-yellow (with high silica content) to almost black (with high organic matter and low mineral content Also in very high concentrations, Ni has low potential mobility and low ecological risk. The major route of exposure expected to be via the skin, although the potential for absorption of heavy metals through the skin is relatively low. Some water and sapropel at different depth levels showed the presence of DDT pesticide and its decomposition product DDE. The concentrations of DDE/DDT found in surface water from lakes were in general lower than those found in sapropel. DDE/DDT was found in all depth of Mazais Kivdalova and Zeilu and in the 2nd extraction site of Audzlu lake and the amount was below the limit of quantification. The antioxidant activity was determined in fulvic acid with the concentration of carbon fraction till 700 mg/L. Results revealed that the total AO level is almost threshold between the highest and the lowest values. The difference in HA (max. 167.8 mg/ml, min. 113.1 mg/ml) levels between different lakes much or less pronounced than the difference in the FA (max. 76.1 mg/ml, min. 44.5 mg/ml) and AO levels.
Despite the fact that in raw sapropel samples no active pathogens identified, CFU exceeds the limit allowed by tenfold or more in all of sapropel samples. It is necessary to reduce CFU/g in the raw sapropel by sterilization or by adding preservatives before using it in cosmetic or medical applications.
It is important to evaluate the utilization of sapropel material after use in therapeutic or cosmetic applications to prevent organic and microbial contamination of the environment.
In the framework of this study, the Guidelines for the extraction of sapropel have been developed, thus, practically all the activities that the sapropel industrial miners would have to perform. As a result, these guidelines will outline the steps to be taken to ensure that the sapropel samples obtained retain their original properties.

Declarations
• Ethics approval and consent to participate Not applicable • Consent for publication The authors whose names listed immediately below certify that they all have seen and approved the final version of the manuscript being submitted. They warrant that the article is the authors' original work, have not received prior publication and is not under consideration for publication elsewhere.
• Availability of data and materials • Acknowledgements The authors are thankful to Riga Stradins University for providing necessary facilities to carry out this research.

• Authors' information
In the manuscript, our team presented the findings about sapropel as raw material -it`s properties and potential usage in medicine. This manuscript builds on our findings (stability, pH level, pesticide and heavy metal pollution, content of biological active substances, etc.) and looks deeper into sapropel quality and safety. We also described and characterized freshwater sapropel types and developed guidelines for extraction of raw sapropel.  Figure 1 Sapropel extraction in Latgale District.

Figure 2
Overview of sapropel samples' drillholes coordinates in the lakes. Storage of sapropel in the: a -closed plastic containers, b -removed from plastic containers and c -selected samples for the extraction of active components.