ECOPET Prevention Systems S.L.

BUREAU OF ENVIRONMENT

The company that ECOPET represents in Europe, has 20 years experience in cleaning the

different oil and gas fields and they are inventors of the products. Their last action was the

successful cleaning of the contaminated fields in Sahalin Island.

We have three main products within this process of cleaning the oil. The first is a product that

homogenizes the oil, the second product is a pump with a filter which cleans the oil which has been homogenized, and the final product is a bio product which cleans the last residues and

also cleans the entire area and water affected including beach, reeds etc. More detailed

specifics of each product are as follows:

- The product which homogenizes the oil comes in the form of a granulate and is

commercially sold in kilograms. This product has to be mixed with water which will

give us a concentrated solution. This solution will need to be diluted with water prior

to spreading over the oil. The ratio, quantities and concentration of the solution will

be determined on the spot when our specialist assesses how much pollution there is.

- The pump with the filter sucks the dirty oil out of the water and filters out clean

oil. The experience shows that the Russian pumps are very reliable in the duration of

working and performing much better than other pumps.

- The bio product is an enzyme bacteria which comes in granulate packs of 1

kilogram as has to be dissolved in the water as well also on the spot (the action of

dissolving with water releases the bacteria and from that moment has a duration of

life of 2 weeks). The concentration of the solution will be decided on the spot. This

bacteria is our invention and as far as we know we are the only ones at the moment

who have this product.

Lastly we would also like to inform you very recently that the Ministry of Industry

and Catastrophic affairs in Russia approached us and offered to supply us with an airplane which is specially constructed to spread our biological cleaning product. As far as we know

also, this is a unique type of plane which does not exist in other countries.

Furthermore we would like to inform you that we will provide you with the prices and the costs in

our future meeting and/ or correspondence. The price will be separated into two categories,

one for the product and the pumps and the other for the cost of the consultation and direct

expertise on the field of our team of specialists.

SURFACE – ACTIVE SUBSTANCE OF BIOLOGICAL ORIGIN

«UNI-REM E-7»

Specification

SPEC 2483-001-19572435-09

Moscow 2009

SPEC 2483-001-19572435-09

The present specifications extend on surface-active substance of biological origin «UNI-REM E-7»

(further – bio PAW, «UNI-REM E-7») which is applied as an emulsifying preparation for clearing

spread oil and mineral oil from soil or a water surface.

«UNI-REM E-7» represents a mixture of hydrogenated non ionogenic surface-active

substances.

«UNI-REM E-7» is a viscous transparent liquid. An insignificant stratification is supposed at a

long-term storage. Before the use the product is recommended to be mixed.

«UNI-REM E-7» is well dissolved in water and low spirits.

Under normal storage conditions of bio PAW are not subject to destruction,

polymerization, oxidation or hydrolysis.

The example of production identification marking at its order: Bio PAW «UNI-REM E-7»

1. Technical requirements

1.1. Bio PAW should be issued according to the requirements of the present specifications

under the compounding and production schedules confirmed when due hereunder.

1.2. The basic physical and chemical indicators of a bio PAW should correspond to the

values specified in table 1.

1.3. Packing.

1.3.1. Package of bio PAW should correspond to the requirements of SSS 6732.3.

1.3.2. Bio PAW is to be packed into plastic containers of capacity 160 dm3; made of steel,

type II according to Spec 6-25570365.071-98, with a double polyethylene bag-shell according to

Spec 6-52-18-90.

1.3.3. Product net weight is according to a container capacity. Boundary deviations from

normal weight can be according to SSS 6732.3.

1.3.4. By agreement with the consumer packing of bio PAW in others types of packaging

which provide leak tightness and product preservation is supposed.

Degree of container filling should not exceed 98 % of its capacity.

2. Safety requirements

2.1. Bio PAW consists of a mixture non ionogenic PAW: Neonol, Sintanol and

polyethylene glycol. The basic indicators of safety are defined proceeding from the properties

of components. Bio PAW is slow reacting. According to the stability (degree of biological

dissociation) refers to the 3d class – are moderately stable (according to T.M. Krasovskiy)

2.2. Neonol. Has an irritating effect on the intact skin, does not resorb through a skin,

and does not possess sensobilizing properties. The cumulating factor at introduction on a Lim’s

method is equal to 9, 6, cumulative properties are not expressed. If reaching stomach renders

slow irritating impact on mucous membranes of a gastro enteric path, causes insignificant

disorders in hypo dynamics of parenchymatic organs. Reaching eyes causes a sharp

keratoconjunctivitis. The product is non volatile, is safe in the inhalation respect, is not

normalized in air of a working zone. Maximum concentration limit in waters of economic-

household using reservoirs is 0, 3 mg/dm (Spec 2483-077-05766801-98). Fire danger indicators:

flash temperature in opened tigl is - 243 °С, in closed - 218 °С; an ignition point is no lower than -

255 °С,; self-ignition temperature is no lower than - 409 °С.

2.3. Sintanol. Low-toxic, refers to the VI th hazardous class, IV th group, LD5о=3, 9±О, 6

g/kg - white rats, intragastric (PAW Ref. edited by A.A. Abramson). The product is non volatile, is

safe in the inhalation respect, is not normalized in air of a working zone. Maximum

concentration limit in atmosphere is 0,005 mg/m3 (PAW Ref. edited by A.A. Abramson).

2.4. Polyethylene glycol. Does not render irritating and sensobilizing impact. The product

is non volatile; is safe in the inhalation respect, is normalized in air of a working zone (on etan-

1,2-diol) maximum concentration limit is 5 mg/m.c., a hazardous class is 3, a modular condition

p+а (PAW Ref. edited by A.A. Abramson, HN 2/2/5686).

2.5. Protection of respiratory organs in accordance with SSS 12.4.034. At spill and in the

case of dispersion of a product in the form of an aerosol in conditions of production apply

respirator FG-2 with a box A, BKF; at spill of products in the conditions of consumption – dust

masks of FA type, SHB - "Petal", respirator RPG-67, RU-60М with a cartridge of mark A.

2.6. By manufacture, test and application of materials requirements of fire safety SSS

12.1.004, Spec 12.4.009 and industrial sanitation in accordance with SSS 12.3.005 should be

observed.

2.7. All works with materials should be conducted in the premises supplied with

mechanical general forced-air and exhaust ventilation, providing a condition of a working zone

air and atmosphere according to SSS 12.1.005, GN 2.2.5.686, GN 2.2.5.687, GN 2.1.6.695, GN

2.1.6.696, San Insp. 2.1.6.1032 and fire extinguishing means (foamy carbon dioxide fire

extinguisher cylinders, asbestos coating, sand;

fine-air water, air-mechanical or chemical foam - for suppression of fire of a product on the

considerable area).

2.8. Industrial and laboratory premises on PAW manufacturing and use should meet the

requirements SNiP 2.4, SNiP 2.04.01, SNiP 2.04.02, SNiP 2.04.03, SNiP 2.04.05, SSS 12.4.021, potable

water provision in accordance with SSS 2874.

2.9. All workers occupied in manufacture, should pass regular medical check ups

according to the requirements to the order № 90 of MPH of the Russian Federation.

2.10. A shift-time damp cleaning of premises should be fulfilled.

2.11.At sampling, tests and use of bio PAW it is necessary to use means of an individual

defense from hit on a skin and mucous membranes of eyes, into the respiratory and digestion

organs according to the typical industrial norms of free delivery of overalls, protective boots

and other means of individual protection of the personnel, confirmed when due hereunder:

goggles in accordance with SSS 12.4.013; protective mittens in accordance with SSS 3-88, SSS

12.4.010, SSS 20010; overalls in accordance with SSS 27651, SSS 27653, protective boots in

accordance with SSS 12.4.072, SSS 5375, SSS 6410. It is necessary to follow the rules of personal

hygiene. Bio Paw removal from a skin and mucous membranes is made by warm water with

soap.

3. Environmental protection

3.1. Bio PAW manufacturing technique can be referred to almost waste-free.

3.2. In the course of manufacture, storage and use of bio PAW secondary hazardous

joints are not formed. The product includes insignificant quantities of a polyethylene glycol

impurity, water and acetic sodium which do not evaporate and do not form volatile

substances at storage.

3.3. The control over observance of maximum-permissible discharges (MPD) into the

atmosphere, confirmed when due hereunder, should be conducted according to SSS 17.2.3.02.

3.4. Washing waters from periodic cleaning of devices and pipes are directed to the

local installation BHO. All chemically soiled sewage is utilized and is partially directed to the

additional cleaning by the system of biological treatment facilities.

3.5. At mismatch of indicators of a product to the requirements of these technical

requirements bio PAW is used as an active basis for technical washing-up liquids.

3.6. At considerable spills bio PAW is covered with sand, gather in containers and send

for a burial. The burial place of toxic production wastes of bio PAW should be made according

to the SP 3183.

4. Acceptance procedures

4.1. Acceptance of bio PAW is made in accordance with SSS 6732.1.

4.2. Sampling - in accordance with SSS 6732.2.

The weight of average laboratory test should be not less than 500 g.

5. Test methods.

5.1. Detection of kinematic viscosity.

Detection of kinematic viscosity «UNI-REM E-7» at temperature t=40 °C is made in accordance

with SSS 33 or on Heppler’s ball viscosimeter by the manual that is applied for the device.

5.2. Detection of active hydrogen ions in a water solution with a mass fraction of bio

PAW of 5% is defined in accordance with SSS 22567.5. For preparation of a water solution with a

mass fraction a bio PAW of 5 % 5,00 г kta is placed in a glass and a solution of the water

released from a carbonic acid.

5.3. Detection of turbidity temperature of bio PAW of concentration Jug/dm

5.3.1. Equipment, reactants and solutions.

Laboratory scales in accordance with SSS 24104, 2nd class of accuracy with border of

weighing 200 g, an error ± 0,75 mg or similar. The temperature indicator in accordance with SSS

28498 of the 2nd class of accuracy with a range of temperature from 0 °С to 100 °С, by division

0,2 °С and an admissible error ± 0,1 °С.

Flask P-2-250-34 THS or similar in accordance with SSS 25336. A flask of Kn-2-250-34 ТHS or

similar in accordance with SSS 25336.

Test tube П2Т-31-115 THS in accordance with SSS 25336 or similar, made of heat resistant

glass, diameter 31-33 mm, height 110-120 mm with the reamed end.

Plate is electric with the closed coil of any design in accordance with SSS 14919. Water

is distilled in accordance with SSS 6709. Glycerin is distilled in accordance with SSS 6824. The

cylinder is 1-50 in accordance with SSS 1770 or similar.

5.3.2. Testing. 30 sm of a water solution of a product is poured in a test tube which is

closed in a flask with two holes. A temperature indicator is inserted into the central hole of a

flask for the bottom of a mercury cylinder to be at a distance of 10 mm from a test tube

bottom. A ring mixer is inserted into another hole. A test tube is placed into a flat-bottomed flask

with capacity of 250sm3, which is half filled with glycerin. Thus the test tube should be kept by

sides on a flask neck. The distance from a test tube bottom to the bottom of flask should be 5-

10 mm.

Installation is put on the electric plate covered with a metal cover, and a flask is

heated at periodic mixing of contents of a test tube to a solution sharp turbidity. The

temperature at which water solution turbidity was observed is taken for turbidity temperature.

An arithmetic average of two parallel tests, absolute range between which does not exceed admissible

range, equal to ± 0, 8 °С is taken or result. Admissible absolute total error of test result is ± 0,8 °С atР=0,95.

5.4. Use of other methods of measurements with metrological characteristics and the equipment is not worse with reactants not lower specified in technical conditions is conceded.

6. Transportation and storage.

6.1. Transportation of bio PAW is fulfilled in accordance with SSS 6732.5.

Bio PAW is transported by all kinds of transport according to the freight traffic requirements

operating on the given kind of transport.

6.2. Bio PAW is stored in the closed container in covered warehouses.

7. Guarantees of the manufacturer.

7.1. The manufacturer guarantees conformity a bio PAW to the requirements of these

technical conditions at observance the requirements of the established rules of transportation and

storage by the consumer.

7.2. A warranty period of bio PAW storage is one year from the moment of

manufacturing.

7.3. Expiration date of bio PAW is one year from the moment of manufacturing, after

which necessary consumer properties are checked and product expiration date is defined.

8. Application methods.

8.1. It is applied at liquidation of emergency spills of oil and mineral oil on a water

surface. Flow rate is from 0, 01 to 0, 05 %.

8.2. It is applied at liquidation of emergency spills of oil and mineral oil on a ground. Flow

rate is from 0, 02 to 0, 07 %.

8.3. It is applied at liquidation of soil pollution with oil as a result of economic activities of

the previous years. Flow rate is from 0,05 to 0,1 %.

8.4. Dilution with water (1: 3) at temperature 15-25 °С is also possible.

8.5. The information for the consumer according to SSS P 51121 is given in appendixes.

Spec 2483-001-19572435-09

Appendix А

(Library reference) Reference normative documents

Title of the document

Identification of the referred document

Number of section,

subsection, point, sub

point,

enumerations,

appendixes of the SSS 3-88. Surgical rubbery gloves. 2.13

SSS 12.1.004-91. SSBT. Fire safety. General requirements. 2.6

SSS 12.1.005-88. SSBT. General sanitary-hygienic requirements to the

air of a working zone.

2.7

SSS 12.1.007-76. SSBT. Harmful substances. Classification and

general safety requirements.

Section 2

SSS 12.4.009-83. SSBT. Fire engineering for objects protection.

Principal types. Placement and service.

2.6

SSS 12.4.010-75. SSBT. Individual protection means. Special mittens. 2.13

SSS 12.4.011-89. SSBT. Protection means for workers. General

requirements and classification.

Section 2

SSS 12.4.013-85Е. SSBT. Goggles. General technical requirements. 2.13

SSS 12.4.021-75. SSBT. Ventilating systems. General requirements. 2.8

SSS 12.4.028-76. SSBT. Respirators SHB-1 "Petal". Section 2

SSS 12.4.034 2.5

SSS 12.1.072-79. SSBT. Special rubber form boots, protecting from

water, oil and mechanical impacts.

2.13

SSS 12.4.103-83. SSBT. Special protective clothes, individual defense

means for feet and hands. Classification.

Section 2

SSS 17.2.3.02-78. Environmental protection. Atmosphere. Rules for

admissible hazardous substances emissions by the industrial

3.3

SSS 33-82. Oil products. A method of kinematic and calculation of

dynamic viscosity.

5.1

SSS 1770-74Е. Laboratory glass measures. Cylinders, beakers, flasks,

test tubes.

5.3.1

SSS 2874-82. Potable water. Hygienic requirements and quality

control.

2.8

SSS 5375-79. Rubber form boots. 2.13

SSS 6709-72. Distilled water. 5.3.1

SSS 6732.1-5-89. Organic dyes, intermediate dyes, textile-auxiliary substances. Acceptance. Sampling methods. Packing. Identification mark. Transportation.

1.3.1, 1.3.2,1.3.3,1.4.1,

4.1,4.2,6.1

SSS 6824-96. Distilled glycerin. 5.3.1

SSS 14192-77. Marking of goods. 1.4.2

SSS 14919-85Е. Electric plates, electro cookers and home electro cabinet boilers. 5.3.1

SSS 20010-93. Rubber technical mittens. 2.13

SSS 22567.5-93. Synthetic washing means. A method of hydrogen ions concentration

determination. 5.2

SSS 24104-88Е. Laboratory scales of general purpose and standard. 5.3.1

SSS 25336-82Е. Glass laboratory ware and equipment. Types, main parameters and

sizes. 5.3.1

SSS 27651-88. Women suits for protection from mechanical effects, water and alkali.

2.13

SSS 27653-88. Men suits for protection from mechanical effects, water and alkali. 2.13

SSS 28498-90. Glass liquid thermometers. General technical requirements. Test

methods. 5.3.1

SSS Р 51121-97. Information for the consumer. 8.5

GN 2.2.5.686. Maximum contaminants concentrations in the working zone air. 2.7

GN 2.2.5.687. Approximate safe impact levels (ASIL) of contaminants in the working

zone air. 2.7

GN 2.1.6.695-98. Maximum contaminants concentrations (MCC) in the atmosphere air

of community settlements.

2.7

GN 2.1.6.696-98. Approximate safe levels of contaminants impact (ASLCI) in

atmospheric air of community settlements. 2.7

Order of RF MH № 90 dated on 14.03.96. Concerning order of preliminary and periodic

medical checkups of workers and medical regulations for the permit to work.

2.9

SanPin 2.1.6.1032-01. Hygienic requirements to the maintenance of atmospheric air quality of the community settlements.

2.7

SNiP 2.4-79. Daylight and artificial lighting. 2.8

SNiP 2.04.01-85. Internal water supply and sewage systems. 2.8

SNiP 2.04.02-84. Water supply. External networks and constructions. 2.8

SNiP 2.04.03-85. Sewage system. External networks and constructions. 2.8

SNiP 2.04.05-86. Heating, ventilation and air-conditioning. 2.8

SP 3183-84. Procedure of accumulation, transportation, deactivation and burial of

toxic industrial wastes. 3.6

SP №1042-73. Sanitary requirements of production processes and hygienic

requirements to the industrial equipment. 2.11

Appendix B

(reference)

Label text

1. Name: UNI REM E-7.

2. Country- Manufacturer: Russia.

3. Manufacturer: CJC «Bureau of ecological problems “Tibet”.

4. Purpose: Is applied as surface – active substance of biological origin under the liquidation of

oil and mineral products spills.

5. Application method: In accordance with section 8.

6. General consumer properties: in accordance with table 1

7. Net weight.

8. Material composition: In accordance with consumption norms

9. Trade mark of the manufacturer.

10. Production date.

11. Keeping date - 12 months.

12. Expiration date - 12 months.

13. Normative document – Spec 2483-001-19572435-09.

14. Obligatory certification information: is not liable to obligatory certification.

15. Facultative certification information: if any

16. Production strip code.

17. Manufacturer’s address

18. Additional information on labels, booklets and shells - at the discretion of the manufacturer

and seller.

Recommendations for application of surface-active substance of biological origin

«UNI-REM E-7»

The surface-active substance of SAW "UNI-REM E-7», forming a component in

biochemical preparations of "UNI-REM" brand, is intended for clearing a ground and

water from oil and mineral oil. The product represents a difficult mixture of block

copolymers on the base of oxiethylized fat acids.

Concentration of the process solution SAW "UNI-REM E-7» is supposed to increase

from 0,01% with the further rise up to an optimum level in industrial use.

Concentration is up to 1,0 % and more is supposed to be used during the

refinement of bed silt or processing polymerized asphalt like sediments in a heel of tanks

or capacities for mineral oil storage. Thus a separate tank from which the solution will

move on cleared surfaces will be used for preparation process solution. Modes of concentration and solution feed rate are specified as a result of experimental-industrial

works.

At the first stage of works it is supposed to use water having temperature

85 - 90 °C. In the course of probes, at enough high efficiency of cleanup a possibility of a

process solution temperature drop will be determined for the purpose to decrease power

inputs of the process.

A direct washout of heavy bed polymerized silts with the raised concentration of

SAW up to 2 % (probably with the ultrasonic or other impact use) is possible. Higher SAW

concentrations promote formation of steady emulsions of mineral oil in a process solution.

The possibility of desorption from mechanical fallouts (a scale with an impurity of

the firm weighed substances), soiled by mineral oil had been studied by us in the

laboratory environment. SAW high concentration allow to essentially lower the mineral oil

maintenance, but it is necessary to use vibrodesorptions or ultrasonic influence on a

processed solution and a firm sediment for speed increase and wash-up quality.

On the basis of our experience of SAW "UNI-REM" application, direct

decomposition (division) of the three-phase system formed as a result of a thermo

sediment of waters after washing occurs in a SAW process solution approximately 20 % -

30 % faster.

The content of mineral oil in an average part of system - liquid as a result of desalt

process does not fall lower than 15-20 mg/l. Data on washing petrosoiled soils by hot

water at SAW presence testifies the possibility of processing temperatures drop from 86 –

90 C degrees to 50 - 60 C almost without any loss of process efficiency.

Influence of a process solution water temperature fall is compensated by an increase in

mineral oil mobility under SAW impact.

We most thoroughly investigated:

possibility of solutions working capacity in the field of low concentration as under large-

scale projects, even insignificant excess of concentration over the optimum leads to substantial

growth of reagent quantity at insignificant increase of process efficiency;

possibility of waters disinfecting after washing at a place with the subsequent dump in

the system of city sewage system.

According to the information available for us, water for tanks cleaning is used on the

closed cycle (but periodically demands water refreshing). Saturation of used water by readily

soluble mineral oil to concentration of 10-20 mg/l occurs quickly enough.

While tanks clearing from crude oil, first of all there is water saturation with easy

hydrocarbons up to С12. Even so small concentration of easy fractions promotes increase of

clearing efficiency from heavy mineral oil (black oil etc.).

Use of a SAW process solution has proved to be positive while clearing works of

surface layer from mineral oil at the areal pollution. Efficiency increase of mineral oil washout

increases with the rise in temperature of a process solution under the pressure of a liquid at a

pumping installation trunk from 0,25 to 0,4 MF. Recommended concentration of a process

solution is from 0,02 to 0,1 %. The quantity of the process solution spent for the unit of an area,

essentially depends on the type of mineral oil and age of oil pollution.

While hydrocarbon products processing on a soil and water surface occurs active

hydrophilization and emulsification of molecules. As a result hydrocarbons lose the toxicity and

subsequently easily decay by the hydrocarbon oxide microorganisms (natural and a part of

biological products).

Consumer’s Protection and Human Welfare Federal Inspectorate Service

SANITARY-HYGIENIC CONCLUSION

№ 77.99.21.248.Д.013373.11.09 dated on 12.11.2009

The present sanitary-hygienic conclusion certifies that the production

BIO PAW “UNI REM E-7”

Produced in accordance with SPEC 2483-204-17032122-209 “Surface Active Substance of

Biological Origin UNI REM E-7”

Corresponds to the sanitary norms

GN 2.1.6. 2309-07 “Approximately safe levels of contaminants impact (ASLI) in the atmospheric

air of the human communities”

The reason for production certification, corresponding (not corresponding) to the sanitary norms

are (enumerate the examined analysis protocols, the name of the establishment, conducting

the analysis, other examined documentation):

Expert conclusion № 2/10 and analysis protocol № 1700 C dated on 08.10.2009 IlTS ANO “An

Independent Institution of Expertise and Certification” (att. Akkr. № POCC RU 0001, 513042,

GSEN RU TSOA 159)

№ 0075480

BIOPREPARATION «UNI-REM E-7» for clearing soil and water from oil

SPEC 9291-206-17032122-2008

The present specification spreads to the microbiological preparation “UNI REM” obtained in laboratory and

industrial conditions by means of cultivation by deep fermentation on liquid nourishing environments with

the subsequent spray-type drying of natural oil oxidizing bacteria distracted from the oil polluted soils

distinguished by a destruction specifics of hydrocarbon and heterocyclic oil components.

A preparation is intended for the bio destruction of oil pollutions in soils generated in the result of

emergency on oil fields, oil pipelines in oil and oil products warehouses.

1. Technical conditions

1.1. “UNI REM” preparation should meet the requirements of the present technical conditions and fulfilled

according to the technological regalement, approved in the established order procedure with the

adhearance to the sanitary norms and rules MH of RF. The preparation “UNI REM” depending on a composition and properties of oil which should be cleared off

from soil includes bacteria of the following kinds: Pseudomonas sp, Rhodococcus cryslopottis, Rhodotorula

glutinis.

1.2. Raw and auxiliary materials.

For the production of “UNI REM” series the following raw materials are necessary:

Melinis minutiflora SSS 5833-75

KH2PO4 SSS 4198-75

MgSO4 SSS 4523-77

KCI SSS 4234-77

NaNo3 SSS 4168-79 Yeasty autolysate Spec 10P 306-69

Alkanet (C14-18)

1.3. Main parameters and properties. According to the organoleptic, physical, biological characteristics and properties UNI REM should

correspond to the requirements and norms indicated in Table 1.

Factor name Characteristics and norms Tests methods

1 2 3

Appearance and color Powder of light brown and (or)

pink-brown color

Acc. to sec.5.1

Moisture mass fraction 7 Acc. to sec.5.2

A number of viable cells in the

preparation mlgd/ml, not less than

1-3,5 Acc. to sec.5.3

Up to the end of storage

guarantee period mlgd/ml, not

less than

0,5-1 Acc. to sec.5.3

Presence of extraneous micro flora mln/g, not more than

20 Acc. to sec.5.4

Strain purity, % of character

morph types, not less than

95 Acc. to sec.5.4

Density, p/cm3 1,428

2. Safety requirements.

Preparation “UNI REM” is non toxic for human being, endothermal animals and plants in the

norms recommended by the instruction for use.

For microorganism impact prevention under preparation production and use requirements

of the biological safety of work with microorganisms SSS 12.1.007-76 and safety general

requirements according to SSS 12.1.007-76, 12.1.012-78, 12.1.003-83 should be fulfilled.

The production equipment in the process of its operation should provide the fulfillment of

labor conditions according to SSS 12.2.003-74.

The working areas air should correspond to SSS 12.1.005-88.

Working with the preparation it is necessary to use overalls (overall or one-piece suit) and

personal protection equipment:

for hands protection – mittens acc. to SSS 12.4.103-83

for respiratory organs protection - a gauze bandage or respirator TTTB-1 “Petal”.

Persons with respiratory organs, skin and face inflammatory disease are not permitted to

work.

Meals and overalls should be held in special premises.

When works with the preparation “UNI REM” are done, it is necessary to wash hands with a

soap.

3. Acceptance procedure.

Acceptance is fulfilled by lots according to SSS 59.04.070. 23-83. A lot is considered to be a

preparation made at one enterprise, homogeneous by characteristics, arranged in one type of

package, drawn up in one quality document.

A document should include:

The name of manufacturer

The name of preparation

A lot number

A lot net weight

Preparation due date

Quality document issue date

A number of package units in a lot

Storage conditions

Guarantee storage time

Factors data stipulated in the present technical conditions

Position and signature of a person, responsible for preparation issue.

Present technical requirements marking

4. Sampling methods.

1 gr. average sampling is taken from every lot with a sterile palette knife and is mixed with

10 g of water in separate sterile test tube.

4.1. A mixed sample is divided into two parts, is put into two sterile test tubes closed by

ground-in stoppers. One test tube is sent to the laboratory for quality analysis; another one is

kept for the case of the repeated analysis during a guarantee time in a fridge under

temperature of +4+6 C.

4.2. Samples are sealed and supplied with the documentation with indications:

The name of preparation

Preparation due date

A lot number

Guarantee storage time

Sampling date

Position and signature of a person making sampling.

Present technical requirements marking

5. Tests methods.

5.1. Appearance and color detection

Appearance and color should be detected visually for every package unit; a sampling

should be placed on flat clean white surface and examined under daylight comparing it

with standard.

5.2. Moisture mass fraction

Moisture mass fraction is fulfilled according to SSS 14870-77 by sample drying out up to

constant mass under the temperature 103+ 2C in a drying oven.

Equipment and reagents.

A drying oven of ShS - 40M model with temperature accuracy +2 C or of analogous types.

Laboratory balance of the second class accuracy with small weighting limits of 200 gr

according to SSS 24104-80E.

Temperature indicator: glass, mercury on 150 C according to SSS 2823-73.

Boxes: glass according to SSS 2393-2 + 79E.

Preparation for tests.

A drying oven should be ready for work in accordance with maintenance and care

guidance.

Test operation.

5 ml of biomass are put into glass boxes preliminary dried out up to constant weight and

are put into a drying oven with a temperature + 103 +2 C. Drying out is made up to a

constant weight which is considered to be achieved if a difference between hour

weightings doesn’t exceed 0,05 gr.

Results observation.

5.3. Viable cells number count.

A method is based on a count of cells sowed by a method of ultimate rearing on Chapki-

Petri with a sterile agarized environment of Chapek-Dox.

Analysis operation:

Medium composition of Chapek-Dox, tap water:

Sucrose - 30

Agar agar - 20

Magnesium sulfuric – 0,5

Muriate – 0,5

Potassium dehydrogenate phosphate – 1,0

Sodium nitrite – 2,0

Autolyzed yeast – 0,01

Sterilization – 1,5 atm/1,5 h

Ware:

Test tubes biological glass SSS 23932-79

Pipette black 1,2,5 and 100 sm3 – SSS 20272 – 74

Test tube rack laboratory

Chishki Petri SSS 25336 – 82

Spirit lamp laboratory SSS 23932-79

In sterile box after 30 minutes long ultraviolet lamp illumination a Chipek’s hot sterilized

agarized medium is poured into burnt under temperature 150 C and chilled Petri’s cups. A

series rearing of the analyzed sampling is prepared as follows:

9 mg of sterile distilled water is poured into 10 sterile test tubes by a sterile pipette. An

analyzed sample is taken by a sterile pipette over a spirit lamp flame and transferred to the

first test tube with water. The received dilution, being mixed with a pipette, is transferred in

the amount of 1 mg to the following test tube with sterile water. The further procedure is

fulfilled with the remained test tubes with sterile water in the same order.

10 Petri’s cups with chilled agarized nutrient medium are marked from 1 till 10 and put on

the table near spirit lamps. 1 mg of solution is sterilely taken from a test tube with a tenfold

rearing by a clean sterile pipette and transferred to 10 Petri’s cups. A drop in agar is evenly

distributed along the whole agar’s surface by a cup staggering from one side to another

without lid opening. A procedure with 9 and further test tubes is fulfilled by the same test

tube, transferring the received suspension to the Petri’s cup test tube, corresponding to the

number. The cups are placed into a thermostat under the temperature of 22C and kept for

3 days and nights after which a calculation of the grown colony is fulfilled. One grown

colony corresponds to one living cell in the analyzed rearing. Colonies are counted in a

cup with those rearing where gown colony forming units (CFU) do not pour together and

are liable to the calculation. A titer of a ready preparation is equal to a number of counted

colonies in one from cups and multiplied by ten in a degree equal to a cup number.

5.4 Extraneous micro flora content detection.

Under a calculation of micro flora number on Petri’s cups a qualitative composition of the

grown colony forming units is estimated. When a procedure of titer calculation is over,

species, morphologically referring to the preparation components, are visually detected,

and with the colonies detection produce the definition of the extraneous view. The

extraneous micro flora is not permitted in the ready preparation, pathogenic for human

being, animals and plants.

6. Packing, marking, transportation and storage.

6.1. Packing.

A preparation is packed into plastic bags with a mass of 1-5 kg and sealed thermally from

all the sides.

6.2. Marking.

Every packing unit is provided with a label, characterizing a production: a name of

organization, which includes, for example, an enterprise-manufacturer. Holding

‘Selkhozhimia”, Russian Academy of Sciences, the name of the enterprise-manufacturer,

name of the production stamps, net weight, due date, a lot number, a guarantee storage

date, storage conditions, packing units number, the marking of the present technical

conditions. A preparation application instruction is supplied with every production lot.

6.3. Transportation.

6.3.1. A preparation transportation should be operated according to SSS 59. 04.070, 24-83.

6.3.2. A preparation is transported by all kinds of transport with its protection from the

temperatures higher + 28C and lower 0C.

6.4. Storage.

6.4.1. Storage should be operated in accordance with SSS 59.04.070.24-83 under the

temperature from 4 to 15C.

7. Manufacturer’s guarantees.

A manufacturer guarantees the correspondence of the preparation “UNU REM” to the

requirements of the present technical conditions under the consumer’s adherence to the terms

and conditions of transportation and storage, stated by the present technical conditions. Under the distribution of the preparation to the consumer it should be checked on the

correspondence to the technical conditions.

A guarantee storage date of preparation is six months from a due date.

8. Application guidance.

A preparation should be applied in accordance with “The guidance for the preparation

“UNI REM” application for the struggle against oil pollution.

To work without personal defense means is forbidden.

It is necessary to use rubber mittens (SSS 12.04. 103-83) for hands protection while suspension

is prepared.

It is necessary to work in an overall, one-piece suit, rubber boots while the preparation of a

working suspension, its filling into the spraying machine and spraying itself.

People working with the preparation “UNI REM” should follow personal hygiene rules.

Smoking, meals, drinking water, putting off personal defense means are forbidden while

working.

It is necessary to put off overalls, to wash hands and face with a soap, to gargle a mouth,

to clean a nose, to take a hygiene shower after a work with a preparation “UNI REM”.

Persons with chronic diseases of respiratory organs, allergic, pregnant women, nursing mothers,

and teenagers till 18 are not permitted to work with the preparation “UNI REM”.

A list of referred documents

Indication Name

SSS 12.2. 003-74 Equipment industrial. General safety requirements.

SSS 12.2. 003-83 SSBT. A special protective clothes. Hands and feet protective means.

MSH USSR “An instruction of safety engineering under storage, transportation and

appliance of pesticides in agriculture”

№ 700 19.06. 84 “An order of USSR MOH about periodical medical checkups”

SSS 12.1. 008-76 Requirements of biological safety

SSS 12.1. 007-75 SSBT. Hazardous substances. Classification and general safety

requirements.

SSS 12.1. 012-78 SSBT. Vibration. General safety requirements.

SSS 12.1. 003-83 SSBT. Noise. General safety requirements.

SSS 12.1. 005-88 SSBT. Working area air. General sanitary-hygienic requirements.

SSS 24104- 80 E Balance laboratory of 2-3 class of accuracy.

SSS 25336 – 82 E Ware laboratory glass.

SSS 2823-73 Temperature indicator technical glass mercury.

SSS 8284-78 Microscope BIOLAM L 211

Spec 64-1-816-77 Gorjaev Chamber

SSS 6672-75 Cover Glass 24/24

SSS 12026-76 Filter paper laboratory

SSS 6709-72 Distilled water

SSS 4142-77 Potassium nitrate

SSS 20292-74E Saccharometer C2

SSS 17206-84 Agar micro biologic

SSS 17206-84 Potable water

SSS 2874-82 Ethyl alcohol technical

A list of specialized literature on analysis methods

What section

referred to

Methodology and original source

Sec. 5.3. Detection and quantitative calculation of microorganisms on firm nutrient

mediums: V.V. Anikiev, K.A. Lukomskaya. A guidance to the practical

lessons on microbiology. M, 1983

Sec. 5.2. ‘A moisture level detection: methods of general bacteriology’ edited by F.

Gerhardt, 1984

Sec.5.4. ‘Extraneous micro flora content detection methods of general

bacteriology’ edited by F. Gerhardt, 1984

Sec. 5.4. ‘Extraneous micro flora identification. Berge’s bacteria identification’ in 2

vol. M, 1997

Sec. 5.4. ‘Extraneous pathogen micro flora identification: Medical microbiology’

edited by V.I. Pokrovskiy, O.K.Pozdeeva. M, 1999.