Nalco New Approach for Macrofouling

New Nalco approach for macrofouling control

________________________ ___________ _________________________ _______________________ ________________________ _________________ ____

MACROFOULING Advanced Control Strategy Introduction The term macrofouling is is used to differentiate from the normal fouling produced by microorganisms such as bacteria, molds, fungi, algae, etc. Macrofouling means the deposit and/or scale produced by some multi-cellular invertebrate organisms, that at the adult stage live strongly attached on the surfaces in contact with both seawater and river (lake) waters. Large production of the world’s major industry, such as power generation, petroleum refineries, steel manufactures and petrochemical production uses sea water or river water as cooling medium for their units. Mainly seawater, but also some fresh water in certain conditions, are the preferred habitat for invertebrates to growth and proliferate forming macrofouling. This process varies for each family of animal and depending from ambient conditions. Fouling by macro invertebrates is common in seawater and brackish cooling water systems. These organisms readily enter plant systems, foul intakes, plug heat transfer equipment, and reduce the overall efficiency and reliability of the plant. This fouling costs industry millions of dollars in increased maintenance, shorter equipment life, and lost efficiency and production. Many organisms are capable of causing macrofouling. Barnacles, bryozoans, hydroids, and mussels are common species in marine environments. Asiatic clams, Zebra mussels may found in fresh waters. All of these species can attach onto surfaces within the cooling system and cause problems while they are attached. Even after the organism dies or is physically removed from the surface, the shells and other materials may stay attached and encourage under deposit corrosion and pitting. Cost of the problem may be very high, depending from the severity of the infestation and the type of production. Just considering that only one day of forced shut down of the plant to clean the equipments may justify the treatment. This documentation has the purpose to present a new strategy to control macrofouling. We can now offer to the facilities that use sea or fresh or brackish water as refrigerant in once through cooling systems for their productions, a complete and efficient treatment to prevent the fouling formed by the invertebrates growth on the water intake, inside the pipes and heat transfer units. In Appendix 5 is also included the Nalco approach in US by using EVAC. This program is treated separately because it cannot be applied in Europe since it is not registered in the BSD. This document is a compendium of the most recent Nalco achievement in this field.

Nalco New Approach for macrofouling.doc

1/73

6/24/2014


________________________ ___________ _________________________ _______________________ ________________________ _________________ ____ Table of content 1.

2.

Status of macrofouling control st 1.1. 1 Generation for macrofouling control - Oxidants biocides nd 1.2. 2 Generation of macrofouling control – control – Bromination rd 1.3. 3 Generation of macrofouling control – control – Non-oxidant biocides Nalco Advanced Strategy Control 2.1. Problem description 2.2. Questions & answers 2.3. Description of new approach 2.4. Application targets 2.5. Treatment program 2.6. Program efficacy study 2.7. Stream speed to prevent settlement 2.8. Treatment application 2.9. Feeding 2.10. Monitoring

page 4 page 4 page 7 page 8 page 9 page 9 page 11 page 11 page 12 page 13 page 14 page 17 page 18 page 20 page 21

Appendixes 1)

2) 3) 4) 5) 6)

Product bulletins a) C-TREAT-6 b) MT200 Regulations and environmental impact Program efficacy and toxicity study – study – Experimental section Toxicity of MT-200 in plant application Monitoring protocol Analytical procedures

page 24

page 31 page 36 page 43 page 59 page 64

Attachments 1. 2. 3. 4. 5. 6.

UK, DOE: C-TREAT-6 Hong Kong Government – Government – Environmental Protection Department Hong Kong - Environmental Protection Agency Approval from UK, DOT C-TREAT-6 C-TREAT-6 – C-TREAT-6 – Environmental profile Compatibility of C-TREAT-6 with RO membranes


2/73

page 78 page 79 page 80 page 81 page 82 page 88

6/24/2014


________________________ ___________ _________________________ _______________________ ________________________ _________________ ____ Table of content 1.

2.

Status of macrofouling control st 1.1. 1 Generation for macrofouling control - Oxidants biocides nd 1.2. 2 Generation of macrofouling control – control – Bromination rd 1.3. 3 Generation of macrofouling control – control – Non-oxidant biocides Nalco Advanced Strategy Control 2.1. Problem description 2.2. Questions & answers 2.3. Description of new approach 2.4. Application targets 2.5. Treatment program 2.6. Program efficacy study 2.7. Stream speed to prevent settlement 2.8. Treatment application 2.9. Feeding 2.10. Monitoring

page 4 page 4 page 7 page 8 page 9 page 9 page 11 page 11 page 12 page 13 page 14 page 17 page 18 page 20 page 21

Appendixes 1)

2) 3) 4) 5) 6)

Product bulletins a) C-TREAT-6 b) MT200 Regulations and environmental impact Program efficacy and toxicity study – study – Experimental section Toxicity of MT-200 in plant application Monitoring protocol Analytical procedures

page 24

page 31 page 36 page 43 page 59 page 64

Attachments 1. 2. 3. 4. 5. 6.

UK, DOE: C-TREAT-6 Hong Kong Government – Government – Environmental Protection Department Hong Kong - Environmental Protection Agency Approval from UK, DOT C-TREAT-6 C-TREAT-6 – C-TREAT-6 – Environmental profile Compatibility of C-TREAT-6 with RO membranes


2/73

page 78 page 79 page 80 page 81 page 82 page 88

6/24/2014


________________________ ___________ _________________________ _______________________ ________________________ _________________ ____

1. Status of macrofouling control In addition to the mechanical removal of macrofouling, some physical and chemical methods have been historically tested to prevent the problem. -

Temperature elevation Addition of potassium salts, copper, zinc, aluminum Oxidant biocides Non-oxidant biocides

However, main difficulties encountered to control effectively the macrofouling are: -

Lack of convenient knowledge’s on macrofouling organism’s life cycles and dyna mic of population, in a specific site. Knowledge of the real efficacy of biocides on specific animals Interaction of the animals with the applied biocide

It is then clear that deep knowledge of these subjects is needed to manage effectively macrofouling problem.

1.1 - 1st Generation for macrofouling control - Oxidants biocides Historically, chemical control of macrofouling uses oxidants, such as chlorine gas, hypochlorite, and chlorine dioxide.

1. Chlo rine Chlorine is an effective microbiocide at low dose levels, and because it is widely available as a commodity chemical (as chlorine gas or sodium hypochlorite), it is the market standard for biological control in seawater systems. Chlorine is also produced by the electrolysis electrolysis of seawater (electro-chlorination). Some marine organisms such as mussels sense the presence of chlorine in seawater, and by closing their shells withstand high levels, living in an anoxic state for periods in excess of thirty days. For this reason continuous chlorination is generally preferred to intermittent addition. Chlorine, injected as bleach or chlorine gas, was an early-adopted control method since it was often widely used for disinfecting and biological control in municipal and industrial systems. However, chlorine was not a cure all. Chlorine requires a continuous feed of three to four weeks at a free halogen residual of 0.3 mg/liter to 0.5 mg/liter to achieve 100% mortality of adult zebra mussels. Feeding a significant amount of chlorine often requires detoxification with with sodium sulfite prior to discharge to face environmental restrictions. As with any chemical control program, regulatory requirements had to be met, which at times was a formidable barrier. Many other concerns lingered over the wide spread use of chlorine in these applications which ultimately limited its’ wide spread use. This approach has the main advantage on using low cost chemicals, but the effectiveness is limited to the need to apply high dosage that complies with environmental restrictions and increased water aggressiveness. Handling, storage and generation of these chemicals represented also a problem for the industry. Main disadvantage of using chlorine is the potential high consumption. In fact, the quantity of chlorine needed depends from the specific chlorine demand of the water that must first be satisfied to have some free chlorine available to be effective in the micro and macro fouling control. Seawater chlorine demand is in the range of 2 to 6 ppm, depending from the local severity of pollution. General reactions for bleach (1) and chlorine gas (2) in water are: 1)

NaClO + H2O ->

HClO + NaOH

2)

Cl2 + H2O + X

->


HClO + HCl

3/73

6/24/2014


________________________________________________________________ HClO + H

+

+ 2e

->

-

Cl

+ H2O

Concentration of free chlorine at the discharge point is restricted to limit the formation of AOX (Adsorbable Halo-organic on activated carbon) that may form by reaction of chlorine with organic matters. 3)

HClO + RH

->

RCl + H 2O

4)

Cl2 + RH

->

RCl + HCl

Particular consideration merits: 

The formation of chloroamines formed by reaction of chlorine with ammonia. Since the stoichiometric ratio is 5:1, it is clear that also trace of ammonia in the water will increase significantly the chlorine demand.



The formation of AOX (haloorganic compounds) that are highly restricted, specially in close waters (lagoons, lakes)

Use of chlorine gas is limited by the hazardous handling and, in some case, to the lowering pH of the water caused by the formation of hydrochloric acid as by-product that destroy the alkalinity. Max storage is normally 1000 kg and may represent a further problem to the handling, especially in large facilities. Four strategies of chlorine dosing are applied: 1) Continuous treatment with halogens Goal: Maintain a high level of residual oxidant to assure the settlement all time. This approach has the higher cost, but guarantee the prevention of problem. However, continuous treatment may have serious limitation on efficacy on all type of invertebrates and from environmental point of view. 2) Semi-continuous treatment with halogens + monitoring of larvae in water. Goal: Elimination of Veliger larvae to avoid totally the mollusks settlement. The semi-continuous treatment allows optimizing continuous treatment based on the result of Veliger larvae monitoring. The potential reproductive season for mussels is spring through fall when water temperature is greater than 12°C. There is no need to chlorinate during winter when the water temperature is too low for reproduction. However, even during the warm season, there may occur periods of a few weeks when no veligers are being released. Such periods without veligers offer an opportunity to cease chlorination without jeopardizing protection of the hydropower plant. Chlorination does not have to be resumed until 1 to 2 weeks after veligers reappear in samples. Even if difficult it is possible to design a monitoring at a distance that it allows this approach (Planktonic approach). 3) Intermittent treatment + monitoring of juveniles on artificial substrates Goal: Allow veliger settlement (and growth to size 300-500 µm) and to eliminate them at this size by intermittent treatment. Intermittent chemical use is designed to prevent initial mussel infestation at facilities that cannot tolerate macrofouling. Dosing at frequent intervals (e.g., 6, 12, 24 hr or more) destroys postveligers that have settled since the previous treatment. Post-veligers are more susceptible to oxidizing chemicals than are adults; thus, the concentration of the chemical and exposure times will be considerably less than if adults were the targets. Because post-veligers with shells about 250 um long can easily pass through the system, disposal and under-deposit corrosion is eliminated. 4) Periodic chemical treatment + monitoring of settled mussels


4/73

6/24/2014


________________________________________________________________ Goal: Eliminate the neo-settled young people (1-3 mm) by means of one long contact (>20 days) periodic chemical treatment. Periodic chemical treatment, like end-of-season treatment, is usually a reactive treatment (usually conducted on a regular basis, such as every 1 to 2 months) designed to eliminate adults that have accumulated since the previous application. Again, limited infestations must be tolerable, but when treatments are more frequent, infestations will be proportionally smaller. In all cases, successful of treatment depends totally from how effective is the monitoring. In the Appendix 1 is the print out of the excel sheet Contact time included in the workbook Nalco where is calculated the contact time needed to kill off 100% mussels using low N e w A p p r o a c h .x l s level continuous chlorination based on Europe and North America data. 2. Chlorin e dioxide

Chlorine dioxide is applied in large units from long time. It is a valid alternative to chlorine. It is produced on site by reaction of sodium chlorite with hydrochloric acid (5) or with chlorine (6): 5)

5 NaClO2 + 4 HCl

6)

2 NaClO2 + Cl2

-> ->

4 ClO2 + 5 NaCl + 2 H 2O

2 ClO2 + NaCl

Final by-product of chlorine dioxide in water is chlorides: +

ClO2 + 4H

+ 5e

-

->

Cl

+ 2H2O

Some chlorite ion, restricted to the discharge, is still present in the final product. Therefore, his concentration must be under control. Chlorine dioxide has the advantage that doesn’t react with ammonia and has been observed that AOX are not formed. As consequence, the amount of chlorine dioxide to satisfy the demand is significantly lower if compared with chlorine. Chlorine dioxide is generally applied continuously at 0.1 – 0.3 ppm on water flow rate. In the workbook N a lc o N e w A p p r o a c h . x l s is included also the calculation sheet “Chlorine dioxide” for estimation of consumption and cost. It is also foresee the contact time needed to kill off 100% mussels extrapolated from data in literature. nd

1.2 - 2 Generation of macrofouling control - Bromination 1) B r o m o - c h l o r i n a t i o n Total or partial bromination was introduced by Nalco to improve the effectiveness of chlorine base chemistry. Several plants are using this approach with success to prevent micro and macro fouling. This approach is valid particularly to prevent the macrofouling in plant using fresh water. However, although seawater contains about 70 ppm of bromides, use of Acti-Brom 3434, as promoter to convert chlorine into bromine, is often justified and effective. In fact, it is very difficult to predict if the chlorine added to the seawater may be available to react with the bromides present in the seawater. This because seawater may contains several pollutants that may consume first the added chlorine. Converting chlorine to bromine before the addition by reacting chlorine with ActiBrom at high concentration, 100% of bromine produced is then added to the water to make the job. The Acti-Brom approach was first reported in 1984 at the American Power Conference.3 This program consists of a bromide salt and biodispersant mixture that forms a more effective oxidizing biocide when added to chlorine or sodium hypochlorite. The chemistry is shown in equations 7 and 8. First the bromide ions are oxidized by hypochlorous acid (HOCl) to form hypobromous acid – (HOBr), which then dissociates to form the hypobromite (OBr ). The biocide system is converted from chlorine to more reactive bromine chemistry by these reactions. -

7)

HOCl + Br

–>

8)

HOBr

->


–

+

+ H

5/73

6/24/2014


________________________________________________________________ In addition, the patented Acti-Brom program contains a surface-active material, which works in conjunction with the more effective bromine chemistry. This surfactant material provides a critical component in the control of macrofoulant species. The surfactant in Acti-Brom is widely used in industrial cleaning solutions because it easily wets metal surfaces. Wettability can be measured by determining the contact angles of a material on various substrates. The surfactant also helps keep surfaces clean by re-moving the biofilm through its cleaning action. Most macrofouling experts believe that the biofilm plays a key role in the attachment of mussels to the surface. Lack of a biofilm reduces the possibility of attachment of macrofoulant species. All of these factors combine to produce a patented program , which helps to prevent macrofoulants attachment with intermittent treatments. Chloro-bromination application In the Appendix 1 is the print out of the excel file N a l c o N e w A p p r o a c h . x l s prepared to organize the treatment year schedule of chloro-bromination. 2. Peracetic acid

Peracetic acid has been recently tested for macrofouling control because it is environmentally accepted. In fact, the decomposition products of peracetic acid are: water, carbon dioxide and some acetic acid. No conclusive results have been achieved still now. However the dosage and global treatment cost are high (3.5 – 4 Euro/kg at 15% activ). The product is commercialized at 5 – 15% activ. Handling and transportation may be also harmful, depending from the concentration of the active and then must be considered. Degussa and Chimec propose Peracetic technology. Nalco has also this technology (LAZON - Nalco 74700) but not sufficient investigation has been made. The experience made in North Italy show that peracetic acid may be effective only with continuous treatment. With discontinuous treatment (3-4 hrs/day) is not effective on mussels. It is comparable to chlorine but at much higher cost. rd

1.3 - 3 Generation of macrofouling control – Non-oxidizing biocides About 15 years ago was introduced the use of QUAT non-oxidizing biocide to control m acrofouling. This base chemistry is very effective and has the main advantage to prevent the growth of mollusks with relatively few applications per year and it is easy to apply with limited storage and handling problems. The QUAT chemistry provides a cost-effective alternative to chlorine and reduces the total amount of chemical required. This approach has the disadvantages that it is not compatible with the environment at high dosages and then may need to be neutralized before the ultimate discharge. In addition, this method uses bentonite clay as adsorbent with complex adsorption/desorption mechanism and increasing suspended solids at the discharge. Several successful applications use this approach. However, the effectiveness of these chemistry on different family of invertebrates that may infest the cooling systems were not well investigated. Environmental impact is a matter of discussion for their acceptance from the final users. There are a number of advantages to use of non-oxidizing molluscicides relative to oxidizing molluscicides. They are more safely stored on site, are less hazardous to human being, may have greater toxicity to mussels than oxidizing molluscicides, may be less toxic to non-target organisms,


6/73

6/24/2014


________________________________________________________________ require application hardware that is generally inexpensive and easily installed, are readily deactivated in discharge waters, are not corrosive or damaging to metal or silicone/rubber seals, and do not produce carcinogenic by-products compared to some oxidizing molluscicides.

2. Nalco Advanced Strategy Control 2.1 Problem description SEA WATER Antifouling consolidated strategies often show themselves to be ineffective in comparison with the adaptability of a benthic population and with the alteration of environmental and ecological parameters. The lack of convenient knowledge’s on macrofouling organism’s life cycles and dynamic of population, in a specific site, can be the cause of negative results of an antifouling treatment. Knowledge of the specific effectiveness of the chemicals on different animals, relative toxicity and environmental impact, is essential to face successfully the macro-fouling problem in cooling water industrial loops. Monitoring for the presence of fouling organisms, both before and after treatment, is essential to a fouling control program. A wide range of organisms is found to colonize cooling water plants. At the lowest grade of organization microorganisms, especially bacteria, colonize heat exchangers and other surfaces. The resulting layer of slime reduces heat exchange efficiency thereby increasing generating costs. Corrosion is also increased in the anaerobic condition under layers of slime. Macrofouling usually colonizes the intake structures, cooling water intake tunnels and culverts, condenser tubes, plates and occasionally the discharge tunnels. A wide range of animals is found but the main culprits are mussels, barnacle and serpulids [Fig.1]. Problem arise the macro-invertebrates fouling in several ways; the development of an encrusting growth which may reach such a thickness that flow of water is reduced, pump head losses are increased and sloughing off of encrusting growth leading to screens or condenser tube plates. Detachment of mussel shells, which become lodged in condenser tubes, leads to long-term erosion-corrosion and hence to salt contamination of the inflowing water which in turn causes corrosion of boiler tubes and turbine blades. Shell filters largely avoid the problem of mussels shell reaching the condenser tubes, thought no specific comparison of efficiency of functioning against fouling organism debris has been undertaken. It often happens that


7/73

[Fig.1] - Main

organisms belonging to fouling

PLANKTON PHASE

TRICOFORA

LARVA CILIATA NAUPLII

VELIGER

PEDYVELIGER

MUSSELS

TROCOFORA

M. TROCOFORA

SERPULIDES

CYPRIS

BARNACLES

BENTHIC PHASE [Fig. 2] - Planktonic and benthic phase of the life cycle of mussels, serpulids and barnacle 6/24/2014


________________________________________________________________ changes of typology of treatment, modifications of environmental parameters or casual pauses of the plant can allow the settlement of fouling organisms even in a very short time. Generally, the life cycle of the main fouling organisms is always divided in two clearly distinct phases: planktonic and benthic [Fig.2]. During planktonic stage the swimming larvae grow, often trough a sequence of forms or larval stages, till arriving to the last stage, generally called “competent stage” [Fig.3] in which, by the metamorphosis, larvae change, after the settlement on the substratum, into adult organisms which, when they reach the sexual maturity, will give rise to a new larval generation.

Balani Barnacles

COMPETENTS LARVAE

Cypris

Mitili

Pediveliger Mussels

Obviously, the length of life cycle, the number of larval stages and the number Metatrocophora Serpulidi of annual cycles change from species to species and they are influenced by local Serpulids environmental conditions, however an LARVE COMPETENTI important site-specific characteristic [Fig. 3] - Competent stage of barnacle, mussels and keeps constant: the interaction between ser ulids the planktonic and the benthic phase. Adult organisms, which by their growing, cover every surface immersed on the sea, generally cause the fouling “damage”, however their presence is the direct consequence of the planktonic development of their life cycle. The density of adult macrofouling organisms present on a site is the results of the density, survival and staying in the site of the larvae of this important biological compartment. The lack in knowledge on the dynamic of macrofouling population of a specific site and on the variation of hydrological parameters is the main cause of negative results in some antifouling treatment. FRESH WATER Major infestation in fresh water is due to zebra mussels (Dreissena polymorpha). Other animals found to form macrofouling are: Anadonta anatine, This problem occurs mainly in North Europe and US lake regions. The New Nalco Approach may be applied also to control macrofouling occurring in fresh water. Main difference comes from the legislation that foresee different animals for standard toxicity tests. Before to apply the Nalco program, it is necessary to consider the local legislation concerning this specific aspect. For an exhaustive description of Zebra Mussels and the status of art to control the infestation, refer to the document included in the hard copy section of this package: “Zebra mussels in Ireland – An International workshop: to consider the economic and ecological impact of zebra mussels and their control”.


8/73

6/24/2014


________________________________________________________________ 2.2 Questions & answers. We tried to give an answer to the questions that arise from customers. -

W h a t d o e s a w a t er t r e at m e n t c o m p a n y w i t h r e g a r d t o M a c r o f o u l i n g ?

Prevent the macrofouling growth in the water system and, very important, without affecting the environment. All countries are now sensitive to this aspect. -

W h a t t e c h n i c a l s er v i c e s d o t h e s e c o m p a n i e s p r o v i d e a n d h o w d o t h e y a p p r o a c h t h e business?

Good expertise in field. -

Which is the value we provide?

Offering a global program that should includes:

-



Efficient treatment to eliminate production shut down



Minimize the maintenance (mechanical and manual cleaning)



Monitoring of treatment efficacy



Monitoring of environment impact



Guarantee that no risk for the environment at water discharge



Easy and reliable feeding system



Take the responsibility of the program (efficacy and environment)



Continue optimization



Analysis of residual biocide



Toxicity in the system at water discharge



Expertise assistance

W h a t e n v i r o n m e n t a l r e s t r i c t io n s a r e l i k el y t o a p p l y a t b o t h l o c a l l e v el a n d t h r o u g h o u t b u s i n e s s r eg i o n ?

The restrictions can be applied only to the oxidant program. In this case general limit is 0.2 ppm Cl2 and no AOX formation. In case of NON-OXIDANT programs, restrictions depend from the composition of the active, but in general, should be considered: total nitrogen ammonia, surfactant responding material, COD. Nevertheless, the real restriction is: do not kill fishes!

2.3 Description of new Nalco approach The purpose of this approach was: -

Understand the specificity of chemical additives on different animals. Test already in use chemistry Test new chemistry Prove potential synergism Run standard toxicity tests Check toxicity in real environment Organize an efficient monitoring strategy

Intensive studies in laboratory and industrial trials have been carried out to study the best costperformance-environmentally accepted (CEPA) treatment program to control macrofouling in cooling water industrial loops. The study has been made with cooperation of CNR-ISMAR - Institute of marine science department of Genoa, Italy . Aim of the study was mainly to analyze, through laboratory experiments, the efficacy of the products towards some representative fouling species, which can be generally found in industrial


9/73

6/24/2014


________________________________________________________________ plants using sea water for their cooling system, and to evaluate the consequent larval toxicity towards non target organisms. Moreover, it is suggested alternative method, valid for hydraulic smooth pipes, to prevent settlement without using biocides. These results have been complemented with field trials efficacy and toxicity data to demonstrate the validity of the new approach. This new approach is based on the following consolidated evidences that occurring macrofouling problem depends from: 1.

Water salinity (seawater or fresh water)

2.

Ambient conditions (temperature, pollutants)

3.

Periods of major growth of the infesting invertebrates may be different for each site and type of animal, but it is rare with temperature lower than 12-12°C.

4.

Most relevant type of invertebrate infesting the cooling system is specific for each site

5.

Mussels spawning and consequent attachment occurs mainly in two periods of the year (April – June and October-November). In the ocean (mitilus edulis) this phenomenon occurs mainly in the first period only. Attachment of mussels may be accepted up to some size (1-5 mm), depending from the equipment geometry. This because, once killed with chemical treatment, they detach with time from the surface without creating plugging problem.

6.

Barnacles may proliferate overall the year, if temperature is sufficient for their metabolism. Once attached to the surfaces, even if killed, will occurs long time for their detachment and a mechanical cleaning is necessary. This fact suggests that it is mandatory to prevent their attachment all time.

7.

Worms (serpulids) may also form big volume of encrustation but this happens only in the intake zones of seawater.
8.

Due to the biological nature of the phenomena, that is then different site by site, good monitoring is necessary to optimize the treatment in each location.

2.4 Application targets It is then evident that the strategy to control macrofouling needs a complete treatment program that provides:   



Control of microfouling (sessile bacteria) Limit growth of algae Control of macrofouling (invertebrates settlement): Treatment to control barnacles and worms - Applied with intermittent program o and relative high frequency. The frequency and dosage must be optimized for each site based on the monitoring of the phenomena. Treatment to control mussels - Applied, as it is necessary, according to the o monitoring evidence. Continuous and effective monitoring.


10/73

6/24/2014


________________________________________________________________ 2.5 Treatment program New approach is based on the laboratory studies and field experiences, exploiting the specificity of the tested products.. Products resulting to match the requirements are: MT-200 C-TREAT-6 Major evidences are:  MT-200 Shows high antibacterial activity (gram- and gram+) o Very effective to kill mussels (5 - 10 times > C-TREAT-6) o Effective to kill Barnacles but only if are newly settled o Has low toxicity on Artemia.S. naupli (non-target by law)) o Has high toxicity on Balanus.A.naupli (non-target) o  C-TREAT-6 Has low effect as antibacterial o Is highly effective to prevent the larvae settlement of Barnacles with non-toxic o mechanism Is effective to kill barnacles and Serpulids newly settled and adults (5 – 10 times > o MT-200) Has low toxicity on Balanus A. naupli (non-target) o Has high toxicity on Artemia S.naupli (non-target by law) o  The combination of the two products produces a synergic effect , evident at extremely low dosage, but only on inhibition of larvae cypris settlement Program treatment 

NALCO MT200 – Low dosage continuous/intermittent application to prevent bacteria slime formation.



NALCO CTREAT6 – Continuous/Intermittent application to prevent the biofilm formation and macrofouling encrustation (barnacles, worms)



NALCO MT200 – Discontinuous application (as needed) to clean the system from juvenilia mussels (0.5-5 mm).



NALCO MT200 – Slug application (as needed) to clean the system from adult mussels (0.5-5 cm).

Detailed description of the two products is given in the appendix 2. Since the two products may form some foam, must provide also the injection of an antifoam at the discharge point. So, in the program treatment is included also the product: NALCO 71101 – Antifoam to be applied at discharge point at 0.1 ppm. IMPORTANT NOTES: 

 



In case chlorination is applied, during the injection of the products MT-200 and CTREAT-6, chlorine feeding must be stopped. This because, in presence of chlorine the invertebrates close the shells and then do not filter the water and then the two anti-foulants cannot act. The treatment proposed in this document should not be used in sea inlet pipes used to supply feed water to reverse osmosis desalination plant (see attachment 8) Particular situation may occur with algae infestation. In such case, to avoid increasing the cost of treatment and minimizing the environmental impact using higher dosage of MT-200, may be necessary to apply a chlorination maintaining 0.5 1.0 ppm FRO for 1 to 2 weeks. In case that seawater is also used to feed seawater evaporator producing drinking water, only C-TREAT-6 has DOE “no objection” to be applied.


11/73

6/24/2014


________________________________________________________________ 2.6 Program efficacy study Has been demonstrated that combining use of Nalco CTREAT6 and MT200 together, exploiting their synergism, it is possible to prevent totally micro and macro fouling problems. Both products have a broad-spectrum antifoulant. They are effective to control bacteria including sulfate reducing species, shellfish attachment and detachment. The effective concentration of use is lower then the toxicity value (LC 50) determined in laboratory and in field on real case.

Efficacy summary In Figure 4 has been summarized the efficacy of the biocides C-TREAT-6 and MT-200 compared with an alternative product that resulted having low activity. EFFECT High

Medium

Low

Absent MT200

Gram +

C-TREAT-6 2594

Antibacterial activity (MIC) Gram -

Settlement inhibition (B . a m p h i t r i t e )

Efficacy

Non toxic mechanism Newly settled barnacle

Adult barnacle

Adult efficacy (mortality) Mussels

Serpulids

Environmentally friendly effect

B. amphitrite nauplii

A. salina nauplii

[Fig. 4] - Summarizing scheme of tested products performance

MT200 show high antibacterial activity and high efficacy on killing mussels. It is effective to kill Barnacles but only if are newly settled. The product has low toxicity on Artemia.S. naupli, but high toxicity on Balanus.A.naupli. C-TREAT-6 has low effect as antibacterial, but it is highly effective to prevent the larvae settlement of Barnacles with non-toxic mechanism. The product is effective to kill barnacles and Serpulids newly settled and adults. The product has low toxicity on Balanus A. naupli, but high toxicity on Artemia S.naupli. L a r v a l s e t t le m e n t i n h i b i t i o n

As regards larval settlement inhibition of the selected model organism ( B. amphitrite), C-TREAT-6 proved to be the most effective product; the toxicity test performed on the same larval stage (cyprid larva) highlights the non-toxic inhibiting mechanism of this product respect to MT200, which shows,


12/73

6/24/2014


________________________________________________________________ c ypris s ettlement (EC)

on the contrary, a toxic mechanism (Fig. 5). From the graph it is possible to note that LC50 and LC99 are always higher than the respective EC values, this means that the settlement inhibiting effect appears at definitely lower concentrations respect to those necessary to kill larvae. This characteristic could be linked to the filming properties of this ammine.

cypris mortality (LC)

50 45 40 35 30

EC-LC 50

EC-LC 99

EC-LC 50

EC-LC 99

MT200

MT200

C-TREAT-6

C-TREAT-6

m p 25 p

20 15 10

From the efficacy analysis (toxicity) on settled organisms, it comes out that the products efficacy is different depending on the considered species. As regards barnacles and serpulids, C-TREAT-6 shows a higher effect than MT200; for mussels, on the contrary, the specificity of MT200 for this taxon (molluscs) is confirmed.

5 0

[Fig. 5] - Cypris settlement vs. cypris mortality – evaluation of inhibition mechanism.

Adult settlement inhibition

As regards barnacles, it is evident that the efficacy can change in relation to the organisms’ age. MT200, in fact, shows a higher efficacy towards newly settled barnacles, while it has a lower effect than C-TREAT-6 towards adult organisms. Toxicity

Finally, from the products toxicity evaluation towards non-target organisms, it comes out how the choice of the reference organism (often imposed by legislative actions) can deeply influence the perception of environmental biocide’s performances. Comparing the lethal concentration towards not-target organisms (Fig.6) to the effective concentration able to inhibit cypris adhesion, it has been observed that MT200 was highly toxic towards B. amphitrite nauplii, while its effective concentration (EC) was lower than the lethal one (LC) towards A. salina nauplii. This was not observed for C-TREAT-6, which has an EC value lower than the LC one, not depending on considered organism. Moreover, toxicity towards not-target organisms is species-specific. In fact MT200 was the most toxic towards B. amphitrite nauplii, while C-TREAT-6 was the most toxic towards A. salina nauplii.

cypris settlement

tox artemia nauplii

tox barnacle nauplii

12 EC-LC 50

EC-LC 99

EC-LC 50

EC-LC 99

MT200

MT200

C-TREAT-6

C-TREAT-6

10 8 m p p

6 4 2 0

[Fig.6] - Cypris settlement vs. non target organisms mortality

Mytilus galloprovincialis Using adult organisms, 3 – 4 cm length. Animals were transferred in laboratory, cleaned from debris and epibionts, bissus thread was cut and they were allocated in tanks with aerated natural filtered (0.22 µm) seawater (NFSW) at 20°C for at least one week, in order to acclimate. T h e r es u l t s o f t h i s t e s t a r e t o b e c o n s i d e r ed o n l y f o r c o m p a r i s o n ; M . g al l o p r o v i n c i a l i s i s , i n fact, a big filter org anism (from 4 to 5 liter/hour) and this ch aracteristic pro duc es, in a static e x p e r i m e n t , a q u i c k r e d u c t i o n o f b i o c i d e c o n c e n t r a t io n , f o r t h i s r e a s o n , t h e o b t a i n e d L C v a l u e s a r e n o t c o m p a r a b l e w i t h t h o s e ( l o w e r ) t h at c o u l d r e s u l t s i n f i e l d c o n d i t i o n s .


13/73

6/24/2014


________________________________________________________________ The product MT200 gave the best results (being specific for mollusks), and its combination with CTREAT-6 doesn’t improve the efficacy. C-TREAT-6 does not show a good efficacy against adult mussels. The table shows the LC50 and LC99 for the two products and their mixing at 1:1 ratio.

Mytilus Galloprovincialis adult organism (3-4 cm)

C-TREAT-6 MT200 1:1 C-TREAT-6/MT200

Contact time (hrs) 96 96 96

LC50

LC99 ppm nc nc 4.56 nc nc nc

Finally, comparing effective concentrations able to remove adult organisms (except mussels) to the lethal concentrations towards not-target organisms (Fig. 7), it could comes out that a periodic cleaning treatments (24 hours), especially using MT200 at these concentrations, could be harmful towards not-target organisms but, this newly s ett led barn. adult barn. s erpulids evaluation is based only on laboratory tox barnacle nauplii tox artemia nauplii experimental data, which could be not 30 representatives of the real field EC-LC 50 conditions where, generally, a lot of the 25 active product is adsorbed inside the 20 plant before its discharged in open sea. A better characterization of this query m p 15 would need specific efficacy tests to be p performed in field, using native fouling 10 organisms, and toxicity tests towards nottarget organisms assessed using water 5 sampled in situ during the treatment. In conclusion, these results show as MT200 and C-TREAT-6 have remarkable differences in performances, could be considered suitable for different typologies of antifouling treatments.

0 MT200

C-TREAT-6

[Fig. 7] - Adult mortality vs. non target organisms mortality

The opportunity of using mixtures of these products, evidenced by the absence of antagonism phenomena in our tests, suggests the possibility of developing suitable treatment strategies (physical or temporal combination of the two products) depending on the specific requirements (technical, environmental or economical) of the prospective customers. In Appendix 4 is reported the experimental section of the study. In Appendix 5 is reported the study carried out in a plant to detect the efficacy and water discharge toxicity in a real case.


14/73

6/24/2014


________________________________________________________________ 2.7 Stream speed to prevent settlement This test has been executed to obtain a mathematical model able to provide an average pipe flow velocity that is effective against settlement of barnacle larvae, for different pipe diameters and shapes (circular or rectangular). The species used for experimentation, Balanus amphitrite, is very representative of fouling problem. Moreover, the application of an increase of 50% (security edge) allows to generalize the obtained results to other barnacles. The cyprid larva is more resistant to detachment than larvae of other infesting species, such as serpulid larvae. Several studies have demonstrated that cyprid settlement is influenced by the velocity gradient due to friction near pipe walls. Such gradient generates a tangential stress that may be so strong to rip out the larvae, thus avoiding their settlement. Therefore, the worked mathematical model considers physical variables related to friction, water viscosity and density, and flow conditions. The starting equations are the following (see Marchi E. – 1961 – Il moto uniforme delle correnti liquide nei condotti chiusi e aperti. “L’Energia Elettrica”: vol. XXXVIII, n.4): Results of the study are reported in the below graph (Fig. 8) that allows to quickly finding the average flow velocity as a function of the pipe radius (for circular sections) or depth (for rectangular sections). Together with the curves representing the limit velocity for settlement, two curves representing the security flow velocity USEC (increased of 50% with respect to the limit velocity for settlement) are plotted, corresponding to a hydraulic condition with a water flow surely effective.

[Fig.8] - Worked model to quickly determine minimum and security

values for flow velocity, depending on pipe radius or depth.


15/73

6/24/2014


________________________________________________________________ 2.8 Treatment application Although it is very difficult to suggest exact dosages and frequency, due to the variability typical of the biological type problem, the following should be considered as first indication to start. Further optimization during the first year of application is strongly recommended by applying efficient monitoring. Important note: each application must be designed specifically case by case. It is then strongly recommended to carry out a very good survey of the plant before the preparation of the proposal. The survey should be made with the help of the customer to clarify the following: 



Problem identification Type of fouling o Periods of infestation o Economic impact of macrofouling Actual treatment, type and cost o Production loss o Maintenance cost o

In the Support CD can be found a survey form that may facilitate the data collection. The print out is in the attachment 7. With the data collected in the survey, is then designed the treatment by filling the treatment program sheet as reported in appendix 2. Basic treatment approach The following suggestions take into account: -

Specific efficacy of each product on bacteria and invertebrates Specific standard toxicity of each product Expected spawning period

The strategy will include: 1.

Normal treatment – Prevention of biological fouling and settlement of invertebrates

2.

Upset treatment – Killing and removal of newly settled barnacles and/or serpulids

3.

Upset treatment – Killing of adult barnacles and/or serpulids

4.

Upset treatment – Killing and removal of newly settled mussels

5.

Upset treatment – Killing and removal of adult mussels

1. Normal treatment - Prevention To prevent the formation of micro and macro biological fouling, the treatment must be effective to kill bacteria, biological lime forming, and prevent settlement. In this way it is avoided the formation of bio-fouling and settlement of invertebrates maintaining clean the canals, pipes and heat transfer units. The program then foresee: 1.

Injection of MT200 at low concentration (2 ppm) and 2 to 8 times per month frequency with 1 to 2 hour contact time, depending from the period of the year and specific season to prevent the biological fouling.

2.

Injection of C-TREAT-6 at low concentration (2 to 4 ppm) to prevent barnacles and serpulids settlement.

3.

Injection of 2 times per year of long slug (24 – 36 hrs) of MT-200 to kill and remove the potential newly attached mussels


16/73

6/24/2014


________________________________________________________________ In Appendix 2 is reported the print out of the calculation sheet Nalco New Approach of the excel workbook Nalco New Approach available in the Support CD to prepare the yearly detailed treatment program. The example reported here may be considered as starting point but it needs to be optimized for each site based on the first year experience. In the sheet is considered also the water temperature. If the temperature is less than the minimum value entered on the top of the sheet, no treatment is applied. Since the temperature in the year is all time > 12°C considered as minimum to apply the treatment, in the specific example the treatment is applied all the year. General experience show that at temperature < 12°C the biological life is not significant. 2. Upset treatment – Killing and removal of newly settled barnacles and/or serpulids In case that newly settled barnacles and/or serpulids are attached to the surfaces, it is recommended to apply the following treatment to kill the animals that will slowly be detached. Product C-TREAT-6

Dosage ppm

Contact time Hours

Remarks

4 -8

36 – 12

Dosage and contact time is depending from the size and density of the animals

3. Upset treatment – Killing of adult barnacles and/or serpulids In case that adult settled barnacles and/or serpulids, big size (> 2 mm), are attached to the surfaces, a long slug treatment should be applied to kill the animals but removal will take long time after and depending from the size. Following treatment is suggested: Product C-TREAT-6

Dosage ppm 6 -12

Contact time Hours 96 - 24

Remarks Dosage and contact time is depending from the size and density of the animals

4. Upset treatment – Killing and removal of newly settled mussels In case that newly settled mussels, big size (< 3 mm), are attached to the surfaces, a long slug treatment should be applied to kill the animals that will be detached in relatively short time (1 to 2 weeks). Product MT-200

Dosage ppm 3-4

Contact time Hours 36 - 24


5. Upset treatment – Killing and removal of adult mussels In case that adult settled mussels, big size (> 3 mm), are attached to the surfaces, a long slug treatment should be applied to kill the animals that will be slowly be detached (weeks to months). Product MT-200

Dosage ppm 4-5

Contact time Hours 48 – 36



17/73

6/24/2014


________________________________________________________________ 2.9 Feeding Particular care needs the injection of the antifoulants to exploit their efficacy at maximum level and then spend money without the right pay back. Feeding system must be well dimensioned and reliable. It should include: Equipment Feeding pump Feeding pump Feeding pump Dilution pump Distribution system

Water flow 3 rate, m /h

Pump rate

Injection of Nalco MT200

100.000

500 L/h

Injection of Nalco C-TREAT-6

100.000

500 L/h

Injection of Nalco 71101

100.000

20 L/h

100.000

30 m /h

Target

Dilute the products and continuous washing feeding line (*) Optimize the distribution along the longitudinal flow direction (**)

Injection point Water intake – Dilution pipe Water intake – Dilution pipe Water discharge

3

Water intake

(*) Purpose of dilution pump is to optimize the distribution of the products. In [Fig. 9] is shown the basic of the feeding system. (**) Distribution system is particularly important to optimize the injection of the product to be in contact with the total flow section area of the water (canal, tunnel, pipe). The system should be designed in a way that the products are injected along the longitudinal direction of the water flow at the intake with different deep. In [Fig. 10] is given the basic scheme of the distribution system.

C-TREAT-6

MT-200

Dilution water

Fi . 9 – Feedin s stem

Fi.g. 10 - Distribution system


18/73

6/24/2014


________________________________________________________________ 2.10 Monitoring Monitoring is of great importance to optimize bio-fouling treatment. Three parameters should be monitored: (1) (2) (3) (4)

Microfouling Macrofouling. Level of residual products Residual toxicity

(1) Microfouling (bacteria and slime) This could be monitored very well with the Sessile Monitoring tools such as the BioBox. Install a BioBox and check the slides every 2 weeks during the "fouling season" (that is generally from April or May to October or November) (2) Macrofouling Nalco used to offer the Aquabox, which was a piece of equipment for monitoring macrofouling. This equipment is not sold anymore! It does not have a replacement. A reprint is included in the CD just to have a good reference on type of equipment that may be needed. However, Nalco can offer a macrofouling-monitoring program in cooperation with the National Research Centre (CNR) in Genova, Italy. The monitoring program could either include:     

Monitoring of arriving larvae density (in the water) Monitoring of settled juvenile mussels (on a substrate). Efficacy of treatment on substrates Efficacy of killing application for cleaning Residual toxicity at discharge

Technicians of our customers or of Nalco will be trained to collect a water sample through a special filter (for larvae monitoring) or to collect the (artificial) substrate on which the mussels have settled. They will then send this sample to Genova where the expertise from CNR will investigate it and detect the number of larvae / settled (juvenile) mollusks. They will report back to the customer / Nalco and with this feedback the treatment can be optimized. Monitoring of arriving larvae density (in the water) This type of monitoring has the purpose to individuate the pick period of spawning and type of larvae. In fact, during this or just after (days) it is expected that settlement will start. So, a specific treatment application may be considered to prevent the attachment of the invertebrates. Sampling Methodology Plankton is sampled with an immersion pump and then filtered through a net with suitable mesh [Fig. 11]. Fully automated sampling could also be envisaged with the installation of automatic or semi-automatic samplers allowing for sampling at regular intervals (at least one sampling a week), at the same depth and with the same quantity of samples. In this way operator’s involvement is much lower. These samplers feature an immersion pump (fixed or portable) and a kit for plankton filtration and (automated or manual) fixation with a preservative solution.

Fig. 11 – Planktonic sampling

Benthic Sampling Benthic sampling, while less difficult and complex from an operational point of view than plankton, is more complex from a strategic point of view. Different factors must be taken into account in order to organize an effective sampling.


19/73

6/24/2014


________________________________________________________________ Selection of Stations for Artificial Substrata Positioning Similarly to plankton sampling, the selection of sampling stations for settling organisms where artificial substrata shall be positioned depends on the plant features. Sometimes the very structures of the plant must be used as substrata. Furthermore, a detailed study on water flow dynamics inside the plant is required in order to know the direction, speed, and the site of stagnation of the larvae tran sported by current. At least a Lagrange analysis is recommended. Organisms/Substrata Affinity Substrata made with different materials and of different types depending on the life cycle of the organism to check can be used. For Barnacles and Serpulids, generally, “Cembonit” panels are used which are [Fig. 12] - Type of immersed with a special support structure. Conversely, for Mussels, substrata utilised for collectors for larvae are employed. They consist of support frames for benthic sampling ropes and nets in mixed fiber (synthetic and natural), which are very similar to those employed in mussel farming [Fig. 12]. In all these cases, the substrata must first of all be kept in natural seawater in order to allow microfouling growth, which is necessary to stimulate metamorphosis and settlement of larvae. [Fig.13] shows the preparation of a rope substrate and a simple method to monitor macrofouling on substrates by using a 1000 l container. The water enters on the bottom of the container at a good flow rate to change the volume in 30 minutes. In the containers have been inserted vertical and horizontal substrates (ropes for mussels and cembonit for barnacles).

[Fig. 13] - Type of substrata utilized for benthic sampling

Immersion Time The minimum time necessary to identify settled organisms on the immersed substrata varies depending on target organisms, the type of plant, season, as well as other factors. Usually, a method based on substrata replacement is applied, which allows, in each station, to observe the samples that have been immersed for different periods, from 15 days to 1-2-3-6-12 months. Sample Analysis Benthic samples will be sent to the laboratory for detailed analysis. A brief analysis can be made initially by sending a photo of surfaces via e-mail.


[Fig. 14] – Exposition of different t e of substrates to the real water

20/73

6/24/2014


________________________________________________________________ [Fig. 14] shows an example of simple direct exposition of different type of substrates to the real flow of the water in the plant. Monitoring of efficacy on adult animals on site This requires the collection of the representative animals (i.e. mussels, barnacles, serpulids, as shown in [Fig. 15] and simply putting them in a basket that may be positioned in the normal water flow to be in contact with and without the applied treatment in the plant. In [Fig. 16] is shown an example of how to expose the animals to the flow for efficacy test.

[Fig. 15] – Collection of invertebrates (mitilus galloprovincialis) by selecting different size

[Fig. 16] – Collection of invertebrates (mitilus galloprovincialis) to be exposed in the water flow for efficacy test

In Appendix 7 is reported a monitoring protocol that may be taken as reference to prepare one specific for your application.

////////////////////////////////////////////////


21/73

6/24/2014


________________________________________________________________

APPENDIX 1

Product Bulletins


22/73

6/24/2014


________________________________________________________________

Product Bulletin

NALCO C-TREAT-6

Cooling Water Chemicals MACROFOULING CONTROL TREATMENT

PRODUCT BENEFITS        

Environmentally acceptable Infrequent application, cost effective alternative to chlorination Anti-fouling properties keep surface clean, improve heat transfer and operating economies Safe to handle, easier to apply and less hazardous than chlorine Provides corrosion inhibition, extend plant life minimizes maintenance and downtime. Wide ranges of applications include once through cooling, thermal desalting and static water systems. No need for deactivation with clay or any other compound. No concerns over additional suspended solids in the discharge or associated equipment reliability and expense.

PRINCIPAL USES C-TREAT-6 has been specifically developed for use in seawater systems and provides an effective alternative to chlorination. C-TREAT-6 removes and inhibits marine growth such as mussels and barnacles. It is also effective in preventing fouling caused by bacterial slimes. When C-TREAT-6 IS used in static waters, such as fire hydrant systems and pipe lines under hydraulic pressure testing, it affords significant corrosion protection far carbon steel and copper alloys.

GENERAL DESCRIPTION   

C-TREAT-6 is a pale brown viscous liquid with slight odor. The product has been specifically developed for use in seawater systems and provides an effective alternative to chlorination. C-TREAT-6 has DOE (UK) approval as marine antifoulant to the inlet of evaporators producing potable water up to 8,0 ppm. C-TREAT-6 has DOT (UK) approval as an inhibitor of marine growth in evaporators producing drinking water up to 8,0 ppm.

DOSAGE Optimum application of C-TREAT-6 depends on plant operating conditions and the nature of the problems. A program designed to prevent mussel colonization would be different from one selected to quickly remove an existing mussel infestation. A typical range of dose levels, contact time, and addition frequency used for routine treatment is as f ollows:   

Dose rate: 2-8 mg/l. A cleanup program for established infestation would require 10-20 mg/1 Contact time: 1-8 hours. Frequency of addition: once a week to once a month

During the spring and summer months when marine activity (especially spawning) is at its greatest, dosage should be most frequent, whilst in the cooler winter months one addition per month is usually sufficient. Your NALCO representative will recommend the optimum dosage necessary to ensure maximum program performance according to your specific system parameters.

Mechanism of action: 





Shellfish Inhibition: C-TREAT-6 forms a non-wettable film on all surfaces within the system, which presents a hostile environment and prevents colonization by young shellfish such as mussels. Established adult animals continue feeding in the presence of C-TREAT-6 and are gradually removed. The time to removal is dependant on the application technique selected, and can vary from four weeks to twelve months. Bactericide: C-TREAT6 inhibits the transport of nutrients across the m embranes of microorganisms which disrupts the energy producing processes of the cell. Although C-TREAT-6 is a good broad-spectrum bactericide, it is particularly effective against sulfate reducing bacteria (SRB). The elimination of SRBs and other slime formers improves heat transfer efficiency and removes a specific potential for corrosion. Corrosion Inhibition: C-TREAT-6 functions as a typical amine and is chemisorbed into the metal surface film providing added stability and significantly reducing the oxygen corrosion process. Practical experience has shown that corrosion rate for carbon steel and copper alloys have been reduced by 30-70% by the regular use of C-TREAT-6. There are some systems where enhanced corrosion inhibition is desirable and an application technique is chosen to optimize this effect.


23/73

6/24/2014


________________________________________________________________ The level of C-TREAT-6 present in a seawater may be determined using a simple calorimetric method. See Technical Sheet TS 1000 for full details.

C-REAT-6 - A practical alternative Chlorine is an effective microbicide at low dose levels, and because it is widely available as a commodity chemical (as chlorine gas or sodium hypochlorite), it is the market standard for biological control in seawater systems. Chorine is also produced by the electrolysis of seawater (electro-chlorination). Some marine organisms such as mussels sense the presence of chlorine in seawater, and by closing their shells withstand high levels, living in an anoxic state for periods in excess of thirty days. For this reason continuous chlorination is generally preferred to intermittent addition. The graph below shows the typical in-use cost of C-TREAT-6 compared with the three basic forms of chlorination.

Hypochlorite Electro-chlorination

Cost/ year

Chlorine Gas

C-TREAT-6

Chlorine dose rate m /l

FEEDING  

Nalco C-TREAT-6 can be metered through a pump with PVC liquid end construction is recommended. It is also recommended that dilution water and the use of an injection quill be considered at the point of chemical addition. Materials: Storage and application equipment (pumps, lines) should be made of SS, PE, PP, PVC, and PTFE. Do not use carbon steel, copper, brass, aluminum or cast iron in contact with the neat product.

HANDLING – STORAGE - SHIPPING Potential hazards: irritating to eyes and skin. Sill procedures: flush to drain with large amounts of water, observe local disposal regulations; the relevant authorities should be consulted before disposing of large spills. Protective equipment: wear suitable protective clothing, gloves and eye/face protection. First aid:  Skin: wash immediately with soap and water. Seek medical attention. Eyes: rinse immediately with plenty of water and seek medical attention.  Ingestion: wash out mouth and drink plenty of water. DO NOT INDUCE VOMITING. Seek medical attention.  Environmental: when used as recommended, C-TREAT-6 has no adverse environmental impact when seawater containing the product is returned to coastal waters. BOD (g/O2/g substance) : Negligible COD (g/O2/g substance) : 1.73 TOC (g/O2/g substance) : 0.59 Biodegradability : 64% degraded in 8 days Eventual breakdown products :Ammonia and Carbon Dioxide Handling – Do not use this product in a m anner inconsistent with its labeling. Avoid splashing, spillage, eye and skin contact, extremes of heat and cold. Do not ingest. Replace caps securely after use. Do not breathe vapor or mist. Use with adequate ventilation. Remove contaminated clothing and wash before reuse. Wash thoroughly after handling. Keep container closed when not in use. Storage - Do not contaminate water, food, or feed by storage. Store in original container. As a standard precaution with all chemicals we recommend the use of protective equipment such as goggles and rubber gloves when handling. For detailed information and typical data please consult the MATERIAL SAFETY DATA SHEET as the only official source of safety information. The product can be stored for more than 6 months from date of shipping if kept in its original unopened containers and under normal warehouse conditions. Protect from freezing and from exposure to high temperature. Approvals: UK, DOE: C-TREAT-6 as a marine antifoulant to the inlet of evaporators producing potable water at 8 PPM.   UK, DOT: C-TREAT-6 as an inhibitor of marine growth in evaporators producing drinking water up to 8.0 ppm. IMO class: (Non-hazardous) 

PACKAGING NALCO C-TREAT-6 is available in non-returnable containers of different sizes. C-TREAT-6 registered trademark applied for.


24/73

6/24/2014


________________________________________________________________

Product Bulletin

NALCO MT-200

Cooling Water Chemicals MACROFOULING PREVENTION

PRODUCT BENEFITS - Prevents fouling caused by growth of invertebrates (mollusks) on piping and heat exchanging surfaces - Effective on a wide range of species - Protects plant productivity by preserving heat exchange efficiency - Effective also against microbio fouling - Environmentally acceptable - Can be used alone or integrated within an “oxidizing + non-oxidizing” approach for maximum flexibility.

PRINCIPAL USES NALCO MT-200 is applied in non-potable water systems subject to infestation from invertebrates. Most common applications are in the once-through cooling systems of power stations, refinery and petrochemical plants, steel mills.

GENERAL DESCRIPTION NALCO MT-200 is a liquid formulation that controls macrofouling in sea and fresh industrial water systems. It is effective on practically all invertebrates causing macrofouling including: - sea water mussels and species causing calcium carbonate tube-like (sea-worms) and cone-like (barnacles) incrustations, - fresh water invertebrates such as Asiatic Clam and Zebra Mussels.

DOSAGE The specific dosage of NALCO MT-200 will vary depending upon the operating characteristics of your system, the water chemistry, and the severity of problems encountered. Your Ondeo Nalco representative will recommend the optimum dosage necessary to ensure maximum program performance accor-ding to your specific system parameters.

FEEDING The injection is non-continuous and depends on several parameters such as: type of program chosen (MT-200 alone or in combination with “oxidizing approach”), temperature, spawning period, etc. Use neat product. If dilution in day-tank is desired, test the compatibility with the available water by mixing product and water at the desired ratio. If no precipitation occurs, which is the most likely situation, the water can be used. MATERIALS: Storage and application equipment (pumps, lines) should be made of Stainless Steel (304, 316), PE, PVC, PP, Hypalon, Teflon or Plexiglas.

HANDLING – STORAGE - SHIPPING As a standard precaution with all chemicals we recommend the use of protective equipment such as goggles and rubber gloves when handling. Consult the Material Safety Data Sheet as the only official source of Environmental and Safety information. The product can be stored for at least 12 months from date of shipping if kept in its original, unopened container and under normal warehouse conditions. Protect from freezing and from exposure to high temperature. NALCO MT-200 is available in non-returnable containers of different sizes.

05/02-GD/sl/3D


25/73

6/24/2014


________________________ ___________ _________________________ _______________________ ________________________ _________________ ____

APPENDIX 2

Regulations & Environmental Impact


26/73

6/24/2014


________________________ ___________ _________________________ _______________________ ________________________ _________________ ____ 1. Registrations General To use the biocides in the European Market it is necessary their notification in the list of the BSD (Bio Safe Directive): Directive 98/8/ 98/8/EC EC of the Euro pean Parliament Parliament and of the Cou ncil of 16 February February 1998 concerning th e placing of bio cidal pr odu cts o n the m arket- Official Jo urnal L 123 , 24/04/ 24/04/1998 1998 p. 0001 – 0063

The registration is not needed if the product is not acting as biocide, or killing the reference animals, as indicated in the standard ecotoxicological test. MT200 MT-200 is notified as a molluscicide in the European Biocide Directory. It is thus approved as a biocide that may combat macrofouling. The quaternary biocide MT-200 is notified within the European Biocide Product Directive for macrofouling control applications. Use of the product MT-200 in whole Europe is allowed now, and also will be allowed after 2008. Currently there is no European law regarding the discharge of biocides in surface water. In conclusion: there are no European regulations that forbid the use of MT-200, and there will not be any European regulations that forbid the use of MT-200 in the near future. The use of MT-200 for macrofouling control is allowed now and in the future by all existing European regulations. C-TREAT-6 C-TREAT-6 is not classified as biocide and then is not registered in the European Biocide directory. Has been demonstrated that the product, at the concentration of use is not toxic for the barnacles, but is effective to prevent their attachment. According to CTB (College voor de Toelating van Bestrijdingsmiddelen) if the effect of C-treat-6 is (just) by the formation of a hydrophobic layer on surfaces, it is not a biocide and would not need biocide registration in the Netherlands (i.e. CTB registration). (see below declaration in Dutch). We have some "hard" evidence (experimental results) that show that C-treat 6 at low dosages works only as a filmer and lots of "circumstantial evidence" to support our case. ________________________________________________________ ________________ Geachte Mevr. van Baal Op grond van de door u verstrekte gegevens trek ik de conclusie dat het product C-treat-6 een hydrofobe laag vormt op het oppervlak van wanden. U geeft daarbij aan dat dit puur een fysische werking is, nl dat de aanhechting van mossielen en pokken sterk verminderd wordt. U geeft tevens aan dat de stof geen chemische of biologische werking heeft op de organismen. Op grond van die gegevens concludeer ik dat het product uitsluitend werkt via fysische weg en niet via chemische of biologische weg. Dan is het geen biocide volgens de biocidenrichtlijn 98/8/EC en betekent dat dat geen toelating als biocide nodig is. Met vriendelijk groeten Ad Meijs _____________________________________ ___________________________________________________________ _________________________________________ _______________________________ ____________ Ir. A.W.H.M. Meijs Stafmedewerker Stafmedewerker strategische projecten CTB - College voor de Toelating van Bestrijdingsmiddelen Stadsbrink 5 - 6707 AA W ageningen - Postbus 217, 6700 AE W ageningen _____________________________________ ___________________________________________________________ _________________________________________ _____________________________________ ___________________ _


27/73

6/24/2014


________________________ ___________ _________________________ _______________________ ________________________ _________________ ____ 2. No Objection” Objection” declarations C-TREAT-6 gets the following No Objections declarations: 

UK, DOE: C-TREAT-6 DOE: C-TREAT-6 as a marine antifoulant to the inlet of evaporators producing potable water at 8 PPM. PPM. (See attachment 1)



UK, DOT: C-TREAT-6 as C-TREAT-6 as an inhibitor of marine growth in evaporators producing drinking water up to 8.0 ppm. (See attachment 2)



Hong Kong Government – Government – Environmental Environmental Protection Department (See attachment 3).



Hong Kong - Environmental Protection Agency. (See attachment 4)



Department of Trade – Trade – Marine Marine Division – Division – (See (See attachment 5)



IMO class: class: (Non-hazardous)

3. Environmental Product

C-TREAT-6 MT-200

Biodegradability (Japanese Food Research Laboratories) 64% degraded in 8 days 90% in 48 hrs

BOD gO2g substance

COD gO2g substance

TOC gO2g substance

Negligible

1.73

0.59

Negligible

1.66

NA

Surfactants % NA 35

It has also been shown that C-TREAT 6 and MT-200 does not bio-accumulate in the marine ecosystem.

4. Toxicity data In this section is given the toxicity data on non-target animals to foresee the environmental impact. Animals suggested in the European Directives are considered. Results of toxicity tests are reported as % of larvae mortality, estimated as the mean and standard error of the four replicates for each concentration. For the estimation of LC50 value (median dilution of mortality), a statistical program, based on probit method, was used: the Trimmed Sperman-Karber method. This method is a modification of Spearman-Karber, a nonparametric statistical procedure for estimating the LC50 and the associated 95% confidence interval. This procedure estimates the trimmed mean of the distribution of the log of the tolerance. If the log 10 tolerance distribution is symmetric, this estimate of the trimmed mean is equivalent to an estimate of the median of the log tolerance distribution. Artemia salina nauplii

Artemia salina is salina is a crustacean with an oloplanktonic life cycle, i.e. the whole life cycle is spent in plankton; it was used for testing at its larval stage called nauplius [Fig.1]. Mortality percentage was calculated as the mean of the four replicates for each concentration respect to the total number of individuals. Data were processed in order to calculate the lethal concentration for the 50% and 99% of the population (LC50 and LC99), according to EPA guidelines (EPA/600/490/027F).


28/73

[Fig. 1] - Artemia salina nauplius

6/24/2014


________________________________________________________________

C-TREAT-6

ppm

LC50

LC99

24 h

2.34 1.95 9.23 5.82

9.58 4.92 nc 9.92

48 h 24 h

MT200

48 h

Towards this organism MT200 result significantly less toxic respect to C-TREAT-6 . Balanus amph itrite Cypris larvae

Mortality percentage was calculated as the mean of the four replicates for each concentration respect to the mean of the total number of individuals. Larvae were considered dead when there was no movement and larval body was completely opaque. Data were processed in order to calculate the lethal concentration for the 50% and 99% of the population (LC50 and LC99), according to EPA guidelines (EPA/600/4-90/027F).

C-TREAT-6 Cypris larvae toxicity test (single products)

MT200

Contact time (hrs)

LC50

24h 48h 72h 24h 48h 72h

nc nc 9.89 17.35 5.87 2.14

LC99 ppm nc nc 47.6 nc 9.89 4.92

Rainbouw trout

The table below shows the LC 50 values of C-TREAT-6 and chlorine for Rainbow Trout (S. Gaidneri) Contact time hrs 24 48 72 96 168

C-TREAT-6 mg/l 1.98 1.24 1.05 0.85 -

MT-200 mg/l 4.4

Chlorine mg/l 0.28 0.13 0.07 0.06

Other aquatic toxicity Product

Type

C-TREAT-6 C-TREAT-6 C-TREAT-6 C-TREAT-6 MT-200 MT-200

Japanese Kilifish (Onyzias latipes) Japanese Kilifish (Onyzias latipes) Brown Shrimps (Grandon grandon) Harlequin Fish Catfish Dafnia Magna


29/73

Contact ime hrs 24 48 96 96 48

LC50 2 mg/l 2 mg/l 0.9 mg/l 2.5 mg/l 4.8 mg/l

6/24/2014


________________________________________________________________

Toxicity in effluent from MT-200-treated test site Acute toxicity of MT-200 biocide to non-target seawater aquatic organisms is moderate, while it is high to fresh non-target aquatic organisms in the above laboratory studies. However, in field studies, there is no toxicity to aquatic non-target organisms exposed to the effluent water. Recent bioassay results from 2003 field tests performed in Italy (ENICHEM Plant - Brindisi) are provided in Fig. 2. % Mortality 24 hours acute toxicity test of sample during treatment does not allow calculating the median lethal concentration (LC50). In fact tests with non-diluted wastewater show, in the water outlet basins, values lower than 50% (red line) This allows, referring to D.Lgs 152/99, to consider discharge non-toxic. The law in fact fixes as emission limit that sea discharges do not have to exceed 50% of mortality, in a 24 hours acute toxicity test with the model organisms ( Artemia salina).

100 Policentrica Nord

90 80 70 60 50 40 30 20 10 0 PT

T

DT 3

DT 6

DT 12

DT 24

[Fig.2] - Toxicity in Effluent from MT-200-Treated Test Sites

Toxicity in effluent from C-TREAT-6-treated test site Clearly, the acute toxicity of C-TREAT-6 to non-target seawater aquatic organisms is higher than MT-200, while it is low to target seawater aquatic organisms in the above laboratory studies. This fact confirm that C-TREAT-6 act with non-biocidal mechanism. However, in field studies, there is no toxicity to aquatic non-target organisms exposed to the effluent water. Because of the filming amine properties of this product, the toxic component in water is removed from solution as it moves through the cooling water system. Therefore, freshly prepared solutions are toxic to the targeted zebra mussels, but there is very little, if any, of the amine remaining in the water when it leaves the plant. Analysis results of residual concentration of C-TREAT-6 from 1993 field tests performed in Pacific Central Coast (MUNMORAH Power Station) were:   

Dosing rate was in the range of 6 to 10 ppm Residual product at the C.W. outlet was in most of the cases nil with only in some cases up to maximum 0.5 ppm In the canal outlet, the residual concentration was nil all time

//////////////////////////////////////////////////////////////////////////


30/73

6/24/2014


________________________________________________________________

APPENDIX 3 Program efficacy and toxicity study Experimental section


31/73

6/24/2014


________________________________________________________________

Program efficacy study Experimental Antimicrobial efficacy Table 1 shows the antimicrobial activity of C-TREAT –6 AND MT200 by disk diffusion method (see the study in appendix 2 for detailed description). Table 2 shows the MIC (Minimum Concentration Inhibition) of the two products.

Tab.1:. Antimicrobial activities of target compounds assayed against two Gram positive (Bacillus subtilis, Staphylococcus aureus) and five Gram negative bacteria (Pseudomonas putida , Pseudomonas perfectomarina

NCIMB 1141, Pseudoalteromonas atlantica ATCC 19262, Escherichia coli ATCC 25922, and Aeromonas sp.) (diameters of inhibition zones are in mm).

MT200

CTREAT6

12

0

Bacillus subtilis ATCC 10774

0

0

P s eu d o m o n a s p u t i d a B1

0

0

P s e u d o m o n a s p e r f e c t o m a r i n a NCIMB 1141

8

0

P s e u d o a l t e r o m o n a s a t l a n t i c a ATCC 19262

11

0

E s c h e r i c h i a c o l i ATCC 25922

10

0

0

0

Indicator lawns S t a p h y l o c o c c u s au r e u s ATCC 25923

A e r o m o n a s sp. CH34

Tab. 2: Values of MIC against Gram-positive and Gram-negative bacteria.

TEST ORGANISM Gram positive bacteria

MIC (mg/l) C-TREAT-6 MT200

S t a p h y l o c o c c u s a u r e u s - ATCC 25923

8

1

B a c i l l u s s u b t i l i s - ATCC 10774

16

0.25

4

1

P s e u d o a l t e r o m o n a s a t l a n t i c a - ATCC 19262

64

4

Pseudomonas perfectomarina - ATCC 14405

64

16

P s e u d o m o n a s p u t i d a B1

128

8

E s c h e r i c h i a c o l i - ATCC 25922

64

4

A e r o m o n a s s p . CH34

8

4

Enterococcus faecalis - ATCC 10741 Gram negative bacteria

MT200 has the highest efficacy, showing in vitro significant antibacterial activity against both Grampositive and Gram-negative bacteria. Activity of C-TREAT-6 against both Gram -positive and Gram-negative bacteria was just lower than MT200, but it provided efficient control of many bacterial species. The results of antibacterial assays for the test compounds are superior to those claimed for the latex paint industry’s most widely used in-can preservatives- formaldehyde releasers, and clearly demonstrated the broadspectrum antimicrobial efficacy of MT200 and C-TREAT-6 molecules. Based on these evaluation, MT200 and C-TREAT-6 could provided efficient control of bacterial invasion in cooling water systems; particularly sea water bacteria as well as potential pathogens are inhibited in their multiplication and diffusion by low quantity of these compounds.


32/73

6/24/2014


________________________________________________________________ Minimum inhibitory concentration (MIC) is the lowest level that a particular biocide is effective against an organism. The MIC value, however, is usually determined in nutrient broth using laboratory organisms. As a result, the reported MIC value may not necessarily be indicative of the level of biocide required to control organisms in a fluid. The type of fluid base stock, the nature of its components and the conditions under which it must operate affect the amount of biocide needed to control growth. Larval settlement inhibition activity of barnacles This efficacy test was carried out by using Balanus amphitrite cypris larvae [Fig.1]. Specimens of this organism were collected and then maintained and reared in the laboratory, following the standard methodology. Settlement percentage was calculated as the mean of the four replicates for each concentration respect to the mean of the total number of individuals. Data were processed in order to calculate the inhibiting concentration for the 50% and 99% of the population (EC50 and EC99), according to EPA guidelines (EPA/600/4-90/027F). C-TREAT-6 and MT200 inhibit B. amphitrite cypris settlement at a low concentration, in particular C-TREAT-6 acts at concentration which are one order of magnitude lower than MT200. The combination of the two products (MT200 and C-TREAT-6) produces a synergic effect , evident at extremely low dosage. C-TREAT-6/ MT200

100

[Fig. 1] - Cyprid larva of Balanus am hitrite. C-TREAT-6

100 t 80 n e m60 e l t t e s 40 %

20 0 0

0.01

0.05

0.1

0.5

1

5

10

50 ppm

1

5

10

50 ppm

MT200

100 80

t 80 n e m60 e l t t e 40 s %

t n e m60 e l t t e40 s %

20

20

0 0

0.005/0.005 0.025/0.025

0.05/0.05

0.25/0.25

0.5/0.5

2.5/2.5

0

5/5

0

ppm

0.01

0.05

0.1

0.5

Cypris larvae toxicity test This toxicity test was carried out by using Balanus amphitrite cypris larvae. Mortality percentage was calculated as the mean of the four replicates for each concentration respect to the mean of the

total number of individuals. Larvae were considered dead when there was no movement and larval body was completely opaque. Data were processed in order to calculate the lethal concentration for the 50% and 99% of the population (LC50 and LC99), according to EPA guidelines (EPA/600/4-90/027F).


33/73

C-TREAT-6 100

80

y t i l 60 a t r o m40 % 20

0 0

0.01

0.05

0.1

0.5

1

5

10

50

6/24/2014

ppm


________________________________________________________________

100

- AQZ2010 - / MT200

MT200 100

80 80

y t i l a 60 t r o m %40

y t i l a60 t r o m 40 % 20

20

0

0 0

0 .0 05 /0 .0 05

0 .0 25 /0 .0 25

0 .0 5/ 0. 05

0 .2 5/ 0. 25

0 .5 /0 .5

2 .5 /2 .5

0

5 /5

0.01

0.05

0 .1

0.5

1

5

10

50

ppm

ppm

C-TREAT-6 shows an LC99 value, which is remarkably higher than its EC99 value; this means that this product is characterized by a good efficacy with a non-toxic mechanism on cypris larvae. As regards MT200, its EC99 and LC99 values are quite the same, demonstrating a toxic mechanism of settlement inhibition. In the table are compared the LC50 and EC50 for the two products and their mixing at 1:1 ratio.

C-TREAT-6 Balanus amphitrite cypris larvae

MT200 1:1 C-TREAT-6/MT200

Contact time (hrs) 24 48 72 24 48 72 24 48 72

LC50

LC99 ppm

nc nc 9.89 17.35 5.87 2.14 nc 12.52 4.86

nc nc 47.6 nc 9.89 4.92 nc 19.82 19.3

EC50 EC99 ppm 0.13 4.73 0.23 4.69 0.71 0.17 2.12 nc 2.29 nc 2.04 7.19 0.12 4.62 0.28 4.62 0.1 3.94

LC50 = Lethal dose to kill 50% of the animals LC99 = Lethal dose to kill 99% of the animals EC99 = Needed dose to prevent settling of 99% of the animals EC50 = Needed dose to prevent settling of 50% of the animals

Efficacy tests on settled adult barnacles organisms Neo-metamorphosed organisms (newly settled barnacles (about 1-2 mm) Mortality percentage was calculated as the mean of the four replicates for each concentration respect to the mean of the total number of individuals. Larvae were considered dead when there wasn’t any movement and the body was partially or completely extruded out from MT200 AQZ2010 2594 100 barnacles’ shell. Data were processed in order 90 to calculate the lethal concentration for the 50% 80 and 99% of the population (LC50 and LC99), y 70 t according to EPA guidelines (EPA/600/4 i l 60 a t 90/027F). r 50 The efficacy of C-TREAT-6 and MT200 is very similar towards this developmental stage. The mixture of C-TREAT-6 and MT200 does not increase the efficacy of the single products.

o m 40 %

30 20 10 0 0


34/73

0.5

1

5

10

6/24/2014

ppm


________________________________________________________________ Adult organisms (settled barnacles (about 0.5-2 cm) For this test adult organisms were used [Fig.2]. Before the beginning of the test, specimens were maintained in laboratory for at least two weeks, under controlled conditions. After this period of time, organisms measuring 0.5 – 2 cm were selected and used for test. Animals were considered dead if the body was partially or completely extruded from the shell. Data were processed in order to calculate the lethal concentration for the 50% and 99% of the population (LC50 and LC99), according to EPA guidelines (EPA/600/4-90/027F). Only the treatment with C-TREAT-6 shows good efficacy against adult specimens of Balanus amphitrite. MT200 proved to be effective only at high concentrations, and if mixed with C-TREAT6 seems to reduce its efficacy. 100

[Fig. 2] - Adult of Balanus am hitrite.

MT200

80 y t i l 60 a t r o m40 % 20 0

5

10 24

MT200 / AQZ2010 C-TREAT-6

100

20

80 y t i l 60 a t r o m 40 % 20

0

0

7.5/7.5 24

24+24

24+48

10/10 24+96

24+48

40

ppm

24+96

AQZ2010 C-TREAT-6

100

y 80 t i l a 60 t r o m 40 %

5/5

24+24

20

2.5

5

7.5

10

ppm 24

24+24

24+48

24+96

ppm

In the table are reported the LC50 and LC99 for the two products and their mixing at 1:1 ratio for different size of Balanus adult organisms. Efficacity tests on settled Balanus adult organisms (1-2 mm) (0.5-2 cm)




LC50

LC99

LC50

nc nc nc

7.2 28.4 15.32

ppm 8.06 7.55 17.4

35/73

LC99 ppm nc nc nc

6/24/2014


________________________________________________________________ Mytilus galloprovincialis Efficacy test was carried out by using adult organisms, 3 – 4 cm length [Fig.3], collected in Genoa harbor. Animals were transferred in laboratory, cleaned from debris and epibionts, bissus thread was cut and they were allocated in tanks with aerated natural filtered (0.22 µm) seawater (NFSW) at 20°C for at least one week, in order to acclimate. Animals were considered dead if shells were partially or completely opened. Data were processed in order to calculate the lethal concentration for the 50% and 99% of the population (LC50 and LC99), according to EPA guidelines (EPA/600/4-90/027F).

[Fig. 3] - Adults of Mytilus galloprovincialis.

MT200

100

The results of this test are to be considered only for comparison; M. gallopro vinc ialis is, in fact, a big filter o r g a n i s m ( fr o m 4 t o 5 l i t e r /h o u r ) a n d t h i s characteristic produces, in a static experiment, a quick reduction of biocide concentration, for this reason, the o b t a i n e d L C v a l u e s a r e n o t c o m p a r ab l e with th ose (lower) that could results in f i e ld c o n d i t i o n s .

The product MT200 gave the best results (being specific for mollusks), and its combination with C-TREAT-6 doesn’t improve the efficacy. C-TREAT-6 does not show a good efficacy against adult mussels.

80 y t i l a 60 t r o m 40 %

20 0

0.5

1

24

24+24

24+48

5

ppm

24+96

AQZ2010 -

100 80

y t i l 60 a t r o m 40 % 20 0

1 24

The table shows the LC50 and LC99 for the two products and their mixing at 1:1 ratio.

Mytilus Galloprovincialis adult organism (3-4 cm)


5


36/73


24+24

24+48

10 24+96

ppm

LC50

LC99 ppm nc nc 4.56 nc nc nc

6/24/2014


________________________________________________________________ Hydroides elegans (Worms)

Efficacy tests were carried out by using adult organisms, 1-2 cm length [Fig.4]. Animals were considered dead if the body was partially or completely out from the calcareous tube. Data were processed in order to calculate the lethal concentration for the 50% and 99% of the population (LC50 and LC99), according to EPA guidelines (EPA/600/4-90/027F). C-TREAT-6 is the most effective product against adult specimens of Hydroides elegans.

C-TREAT-6

[Fig. 4] - Adults of Hydroides elegans.

MT200

100

100

80

80

y t i l a 60 t r o m 40 %

y t i l 60 a t r o m 40 %

20

20

0

0 5

24

7.5

24+24

24+48

24+96

10

5

ppm

24

10

24+24

24+48

20

24+96

ppm

The table shows the LC50 and LC99 values for the two products and their mixing at 1:1 ratio.

Contact time Hydroides elegans adult organisms (1-2 cm)

C-TREAT-6 MT200

(hrs) 96 96

LC50

LC99 ppm 9.42 nc 14.68 nc

//////////////////////////////////////////////////////////////////////


37/73

6/24/2014


________________________________________________________________

APPENDIX 4 Toxicity of MT-200 in plant application


38/73

6/24/2014


________________________________________________________________

Toxicity of MT-200 in plant application A biological experimentation has been programmed and executed, using MT-200 by performing laboratory acute toxicity tests on larvae of marine organisms. Aim of this study was to quantification in laboratory the toxic effect of products as such through acute toxicity tests on larvae of Artemia salina, a non-target organism, as suggested from D.Lgs 152/99, for detecting environmental toxicity of industrial discharges in the sea-water. This Italian law resumes the concepts of the “European Water Framework Directive” and the directive 76/464/CEE. Results and statistical analysis Results of toxicity tests are reported as % of larvae mortality, estimated as the mean and standard error of the four replicates for each concentration. For the estimation of LC50 value (median dilution of mortality), a statistical program, based on probit method, was used: the Trimmed Sperman-Karber method. This method is a modification of Spearman-Karber, a nonparametric statistical procedure for estimating the LC50 and the associated 95% confidence interval. This procedure estimates the trimmed mean of the distribution of the log of the tolerance. If the log 10 tolerance distribution is symmetric, this estimate of the trimmed mean is equivalent to an estimate of the median of the log tolerance distribution.

LC50 values MT 200 at 20°C

Time 24 h 48 h

50 % Lethal Concentration LC50 = 12.17 mg/L 95% IC = (10.66- 13.89) LC50 = 7.31 mg/L 95% IC = (6.82 - 7.82)

MT 200 at 28°C

Time 24 h 48 h

50 % Lethal Concentration LC50 = 5.64 mg/L 95% IC = (4.95 - 6.41) LC50 = 3.98 mg/L 95% IC = (3.51 - 4.50)

MT-200 shows a toxicity trend that allows calculating the mean toxicity concentration (LC50), which seems to duplicate together with the increase of temperature from 20°C to 28°C, both after 24 and 48 hours of contact.


39/73

6/24/2014


________________________________________________________________

Acute toxicity of products on b a l a n u s am p h i t r i t e larvae 100

MT 200

90 80 24h

70

48h

y t i l 60 a t r 50 o m 40 %

30 20 10 0 0

1 5 10 mg/L Naupliar mortality percentage of B. amphitrite after 24 and 48 hours and [Fig. 1] - the values of LC50  confidence limits at 95% (IC).

Time 24 h 48 h

0,1

0,5

50 % Lethal Concentration LC50 = 2.13 mg/L 95% IC = (1.92 – 2.36) LC50 = 1.90 mg/L 95% IC = (1.74 – 2.08)

MT-200 has put in evidence a higher toxicity on this organism (LC50 48h: 1,90 mg/L) respect to that showed on Artemia salina at the same temperature (LC50 48h: 3,98 mg/L). This appears not surprising, as usually Balanus larvae are much more sensitive respect to Artemia salina larvae.

Environmental toxicity of water at the plant sea discharge During a first investigation on the plant, discharge basins going into seawater were located (Policentrica Nord e Policentrica Sud), where collection of water samples for acute toxicity tests had to be carried out (Fig.3). As expected from D.Lgs 152/99 and adjournments, the toxicological characterization of wastewaters and the verification of not-exceeding the discharge limits in seawater, was performed by using the crustacean Artemia salina sp. (II stage larvae) as a model organism for ecotoxicological tests. Tests were carried out by following the protocol IRSA-CNR (Guzzella, 1997), specific for acute toxicity tests of environmental samples with Artemia sp.. The protocol was partially changed because of the necessity of performing the tests directly in the plant.


40/73

6/24/2014


________________________________________________________________

[Fig 2] - Localization of terminal basins of waters sea discharges where

water samples for biological toxicity tests were collected.

TEST EXECUTION Collection and preparation of solutions - In this experimentation, all toxicity tests were carried out by using water samples (250 ml), collected at the discharge in the two different sites (P. Nord and P. Sud), in polystyrene bottles. Before performing the test, samples were acclimatized at the temperature reported in the protocol (25  1 °C), measuring pH, which resulted to be between 8,1 and 8,5. It wasn’t necessary to perform any alteration to salinity, as the value was in the range for survival of utilized organism. Cysts reactivation - Cysts reactivation has been carried out 24 hours before the execution of tests. About 500 mg of cysts have been introduced in a 700 ml beaker, adding 500 ml of seawater, not treated and previously filtered on a Millipore 0,45 µm membrane. Beakers, hermetically closed and aerated with pre-filtered air (0,22 µm), were incubated for 24 hours at 25°C, without photoperiod. Test execution - Different procedures can be followed, depending on the fact that the range of concentration that includes the toxic effect of analyzed wastewater is known (standard test) or not (preliminary test). Preliminary test - When the toxicity of analyzed sample is unknown, it is necessary to test a wide range of dilutions or, as in this case, a range of concentrations of the product. Together with the control solution, six concentrations were tested: 0,1- 0,5-1-5-10-50-100 mg/L. The test was performed preliminarily in laboratory with Artemia and the product, diluted in natural sea water, to locate the median lethal concentration (LC 50) that, for MT200 resulted to be:


41/73

6/24/2014


________________________________________________________________ LC50 = 5.64 mg/L

95% IC = (4.95 - 6.41).

This procedure has allowed us to select the range of dilutions to be used during the definitive test, performed at 24 hours of contact. 24 hours definitive test - The concentration of MT200 used during seawater treatment in the plant was 3 ppm. This has led us to select, following results of preliminary test, the following dilutions (as % of treated water): 100%, 50%, 25%, 12,5%, 6,25%, 3,125%, 1,56%, 0%. These dilutions have been selected in logarithmic sequence in order to allow a simple rectification of the eventual toxicity curve to calculate the median toxicity dilution. In both cases, for each test, multi-well plates (25 wells) have been used. In the first column there was the control solution (not treated water), and in the following the different dilutions in increasing order, arriving to pure treated water. In each well about 30 individuals were added, each dilution was repeated in four replicates, for each water sample collected in the two discharge sites, following this temporal scheme:

HOURS

0

CT

BEGINNING OF TREATMENT

END OF TREATMENT

6

24

PT

T

48

72

DT 3

DT 6

DT 12

DT 24



CTR = water sampling in a non-treated site



PT = water sampling at the discharge before treatment.



T = water sampling at the discharge during treatment.



DT 3 = water sampling at the discharge after three hours from the end of treatment.



DT 6 = water sampling at the discharge after six hours from the end of treatment.



DT 12 = water sampling at the discharge after twelve hours from the end of treatment



DT 24 = water sampling at the discharge after twenty-four hours from the end of treatment

Toxicity tests on artemia, performed at 25  1°C, have been carried out after 24 hours of exposure, by exposing different environmental samples, collected at the two discharge basins before the treatment (PT), during the treatment with MT200 (T), after 3 hours (DT3), 6 hours (DT6), 12 hours (DT12) and 24 hours (DT24) from the end of treatment, to a sequence of logarithmic dilutions. Multi-well plates, hermetically closed with Para film and lid, were incubated at dark for 24 hours. After this period of time they were observed at binocular, in order to count the number of dead organisms respect to the total number of individuals. Larvae were considered dead when there was completely no movement for 10 seconds.


42/73

6/24/2014


________________________________________________________________ RESULTS AND STATISTICAL ANALYSIS Results of toxicity tests with Artemia salina are reported as % of mortality, estimated as the mean and standard error of the four replicates for each dilution. For the estimation of LC50 value (median dilution of mortality), a statistical program, based on probit method, was used: the Trimmed Sperman-Karber method. This method is a modification of Spearman-Karber, a nonparametric statistical procedure for estimating the LC50 and the associated 95% confidence interval. This procedure estimates the trimmed mean of the distribution of the log of the tolerance. If the log10 tolerance distribution is symmetric, this estimate of the trimmed mean is equivalent to an estimate of the median of the log tolerance distribution.

Toxicity before treatment ( PT )

% mortalità (24 ore)

100

Policentrica

90 80 70 60 50 40 30 20 10 0

100

50

25

12.5

6.25

3.12

1.5

0

% Dilution

% mortality (24 ore) Policentrica Sud

100 90 80 70 60 50 40 30 20 10 0

100

50

25

12.5

6.25

3.12

1.5

0

% Dilution

Trend of Artemia salina mortality percentage after 24 hours of contact at t he [Fig. 3] - different % of dilutions of water sampled in the two sites before treatment.


43/73

6/24/2014


________________________________________________________________ Toxicity during treatment

(T)

% mortality (24 ore)

100 90

Policentrica Nord

80 70 60 50 40 30 20 10 0 100

50

25

12.5

6.25

3.12

1.5

0

% Dilution

% mortality (24 ore) 100 90

Policentrica Sud

80 70 60 50 40 30 20 10 0 100

50

25

12.5

6.25

3.12

1.5

0

% Dilution

[Fig. 4] - Trend of Artemia salina mortality percentage after 24 hours of contact at the

different % of dilutions of water sampled in the two sites during treatment.


44/73

6/24/2014


________________________________________________________________ TOXICITY AFTER THREE HOURS FROM THE END OF TREATMENT

( DT 3 )


Policentrica Nord

100 90 80 70 60 50 40 30 20 10 0

100

50

25

12.5 6.25 % Dilution

3.12

1.5

0


Policentrica Sud

100 90 80 70 60 50 40 30 20 10 0

100

50

25


3.12

1.5

0

[Fig. 5] - Trend of Artemia salina mortality percentage after 24 hours of contact at the different %

of dilutions of water sampled in the two sites after three hours from the end of treatment.


45/73

6/24/2014


________________________________________________________________ TOXICITY AFTER SIX HOURS FROM THE END OF TREATMENT

( DT 6 )

% mortalità 24 ore)

100 90

Policentrica Nord

80 70 60 50 40 30 20 10 0 100

50

25


3.12

1.5

0

% mortality (24 ore) 100

Policentrica Sud

90 80 70 60 50 40 30 20 10 0

100

50

25


3.12

1.5

0


of dilutions of water sampled in the two sites after six hours from the end of treatment.


46/73

6/24/2014


________________________________________________________________ TOXICITY AFTER TWELVE HOURS FROM THE END OF TREATMENT

( DT 12) % mortalit

24 ore

Policentrica Nord

100 90 80 70 60 50 40 30 20 10 0

100

50

25

12.5 6.25 % dilution

3.12

1.5

0


Policentrica Sud

100 90 80 70 60 50 40 30 20 10 0

100

50

25

12.5 6.25 % dilution

3.12

1.5

0


of dilutions of water sampled in the two sites after twelve hours from the end of treatment.


47/73

6/24/2014


________________________________________________________________ TOXICITY AFTER TWENTY-FOUR HOURS FROM THE END OF TREATMENT % Mortality (24 ore)

( DT 24 )

100 90

Policentrica Nord

80 70 60 50 40 30 20 10 0 100

50

25


3.12

1.5

0

% Mortality (24 ore)

100 Policentrica Sud

90 80 70 60 50 40 30 20 10 0

100

50

25

12.5

6.25

3.12

1.5

0

% Dilution

[Fig. 8] - Trend of Artemia salina mortality percentage after 24 hours of contact at the different % of

dilutions of water sampled in the two sites after twenty-four hours from the end of treatment.

From the analysis of toxicity data, it is possible to understand immediately that these wastewater samples are not much toxic. Graphs show a low toxicity during treatment (Fig. 4) and, after 3 hours from the end of treatment, mortality values are statistically comparable to those of non-treated water sample (Fig. 5). The toxic effect noticed during the treatment puts in evidence a different toxicity between the two basins. An analysis of mortality trend (Fig. 4) shows that only non -diluted sample (100%) of water collected in Policentrica Nord presents a significant difference respect to control. The sample collected in Policentrica Sud shows a quantitatively higher (about 10%) toxicity respect to the Nord if not-diluted


48/73

6/24/2014


________________________________________________________________ (100%), and evidences significant toxicity differences respect to control also at 50 and 25% dilutions. 24 hours acute toxicity test of sample T (during treatment) does not allow in any case for the two sites to calculate the median lethal concentration (LC50). In fact tests with non-diluted wastewater show, in both basins, values lower than 50% (Fig. 9, red line).

% Mortality 100 Policentrica Nord

90 80 70 60 50 40 30 20 10 0 PT

T

DT 3

DT 6

DT 12

DT 24

% Mortality 100 90

Policentrica Sud

80 70 60 50 40 30 20 10 0 PT

T

DT 3

DT 6

DT 12

DT 24

Trend of mortality percentage of Artemia salina after 24 hours of the non-diluted [Fig. 9] - sample for water sampled in the two sites of discharge during all sampling times.

This is an extremely important data, as it allows, referring to D.Lgs 152/99, to consider both discharges non-toxic. The law in fact fixes as emission limit that sea discharges do not have to exceed 50% of mortality, in a 24 hours acute toxicity test with the model organisms ( Artemia salina).


49/73

6/24/2014


________________________________________________________________ CONCLUSIONS FOR BOTH THE TESTS As regards toxicity tests on biocides performed with Artemia salina and Balanus amphitrite larvae, results have put in evidence a different behavior of active biocides even if we must remember that the law considers at present the use of the Artemia salina like organism for the toxicological tests in seawater. Results of mortality test with MT-200 on Artemia sp. are consistent, and are also confirmed by field experimentation. A good correlation between LC50 values derived from laboratory and field experimentation allows resolving any doubt as regards the performance of this biocide. Toxicity values obtained during laboratory tests with Artemia salina at different biocide concentrations have put in evidence, at both tested temperatures, LC50 values higher than 5 ppm. This obviously led us to suppose that toxicity limit imposed by the law 152/99 was not reached (the water sample at the discharge cannot exceed 50% of mortality after 24 hours) if during a plant treatment the product is used at a concentration lower than 5 ppm. The opportunity to monitor, during the month of September, waste water toxicity in the course of a treatment at 3 ppm carried out at Polimeri Europa in Brindisi has confirmed this hypothesis. In fact acute toxicity tests with larvae of Artemia salina show that toxicity detected during treatment is compatible with limits imposed by D.Lgs 152/99.

//////////////////////////////////////////////////////////////////////////


50/73

6/24/2014


________________________________________________________________

APPENDIX 5 MONITORING PROTOCOL


51/73

6/24/2014


________________________________________________________________

MONITORING PROTOCOL Microfouling (bacteria and slime) There are a variety of side-stream monitoring tools. All measure the tendency of your customer’s plant to form or clean off soft deposits. A BioBox installed side-stream will provide a good visual indicator. Swabbing the BioBox slides and submitting the swab for Surface Differential Microbio Analysis (DMA) will provide a quantitative record.

Swab Coupon or Slide

Sterile Buffer

Macrofouling (Invertebrates) [Fig.1] – BioBOX & swabbing method During the first year of plant’s functioning, monitoring protocol foresees a series of weekly and monthly biological samples (planktonic and benthic), in order to identify the periods with the higher organisms settlement, to plan the periodic treatment with MT200 and C-TREAT-6. After a suitable preservation treatment, these samples will be sent to the laboratory where skilled personnel will execute suitable analyses, in order to detect weekly the larval density, and monthly the number of young settled metamorphosized individuals.

In this way, during the year, possible periods with a high settlement risk will be identified, so that treatments with MT200/C-TREAT-6 will be carried out. Treatment efficacy (Adult Organisms Mortality) during the application will be verified by baskets, which contains mussels, exposed to the real water flow. Besides this monitoring, which aim is to prevent mussels’ settlement, it is necessary to perform a study of macrofouling existing in the site, in order to preserve the plant from settlement of other possible organisms (Serpulids and Barnacles) that in certain periods of the year could represent a serious problem. For this reason up to 3 annual visits of specialists, after 3, 6 and 12 months from the beginning of monitoring may be foreseen. During these visits the structures (panels, ropes and nets), permanently kept in the sampling sites, will be analysed. Analyses (non-destructive) will be performed directly on the spot. Will be also monitored and quantified the fouling community, particularly observing barnacles and serpulids, or other possible abundant and harmful species. Subsequently, the same structures will be submerged again in the sampling sites. This will allow having a complete and dynamic view of the main fouling organisms in the plant. A training will be necessary (at least during the first visit), to plan and to explain to a reference operator, the techniques of counting the percentage of mortality during and after the treatment phase. A site for a permanent control site should be also identified as control comparison.


52/73

6/24/2014


________________________________________________________________ Sampling and analysis plan Sampling sites and structures [Fig. 2,3,4] During the first investigation on the spot in the plant, an easily accessible and suitable for the immersion of the structures area should be identified (i.e.: a collection tank (OWC type) located between the point of treatment injection and the pump room (Sampling Station 1). For this station the installation of one vertical structure, called “A”, two vertical structures, called “B”, and one horizontal structure called “C”, may be foreseen. Structures “B” are submerged in two replicates, in case of damages during the year, but for the analyses and collections only one structure will be utilized. Structure “A” is used for monthly sampling; therefore substrates for macrofouling settlement (ropes, nets and panels) have to be replaced every 30 days. Structure “B” is permanent and will be submerged for 12 months. Analyses will be carried out on the spot, using not destructive techniques, and the structure will be immediately submerged again.

[Fig.2] - Location of sampling stations

Structure “C” is made of two components: on one side (left) there are substrates (2 nets alternated with 2 ropes), which have to be replaced monthly, on the other side (right) identical permanent substrates (2 nets alternated with 2 ropes), to be analysed on the spot.

Plan of vertical benthic sampling [Fig.3] -

At the sampling station 3 should be taken the sample to determine the residual products and residual toxicity.

[Fig.4] - Plan of horizontal benthic sampling


53/73

6/24/2014


________________________________________________________________ a) Weekly Planktonic Sampling Planktonic sampling may be performed directly in the pump room, using a tap placed next to the pumps. This will obviously indicate the larval density inside the plant’s pipes 3

A weekly collection of 1-2 m of seawater, filtered with a column filter Veliger Filter ZVF001 (63 µm nylon mesh filter), will be performed from a single sampling station. Sampling has to be carried out not at the same time of chemical treatments. This material will be fixed and sent to the laboratory every week for the analysis of larval density. Analysis of Veliger and Pediveliger density Samples will be split up in representative sub-samples and density of Veliger and Pediveliger will 3 be calculated (number of organisms x m ). As samples are made of dead organisms, the only possibility of detecting the stages is that of measuring the size of larvae. It means that for each sub-sample (at least 10) it is necessary to identify, count and measure the larvae. b) Monthly benthic Sampling Monthly collection Every month substrates (nets, ropes and panels) in A (vertical structure), at the 3 different depths (d1, d2, d3) and the 2 ropes and 2 nets in the horizontal structure C, have to be replaced. Total surfaces to be analysed: Structure A:   

3 panels (20 x 30 cm) 3 nets (20 x 30 cm) 3 structures for the ropes (30-60 ropes of 15-30 cm length)

Structure C:  

2 ropes of 1 m length 2 nets (1 m)

This material will be fixed and sent to the laboratory every month for the analysis of density of adult 2 neo-metamorphosized individuals (number of organisms x dm ). c) Periodic Observation of Macrofouling I n s i t u an a l y s i s o f m a i n f o u l i n g o r g a n i s m s

Non-destructive analyses, performed directly on the spot, of settlement of main fouling organisms on structures B and C every 3, 6 and 12 months. O b s e r v a t io n i n t h e p e r i o d s o f 3 , 6 an d 1 2 m o n t h s

Substrates (ropes, nets and panels) of the 3 different depths (d1, d2 ,d3) of structure B and the 2 ropes and 2 nets of horizontal structure C, are observed in vivo. Subsequently the percentage of 2 covering, or number of individuals x dm , of main fouling organisms is estimated. The structures are then placed in the stations again for the following monitoring. Total surfaces to be analysed in vivo: Structure B:   

3 panels (20 x 30 cm) 3 nets (20 x 30 cm) 3 structures for the ropes (30-60 ropes of 15-30 cm length)

Structure C:  

2 ropes of 1 m length 2 nets (1 m)


54/73

6/24/2014


________________________________________________________________ M o n i t o r i n g d u r i n g t h e t r e at m e n t

In case of treatment application for inver tebrate’s infestation, monitoring of efficacy can be easily performed: 1) Collecting a number of 30-100 individual (i.e. mussels) of the infesting animals. Putting them in a basket at least 1 week before the application. Checking their vitality just before the treatment and mortality after 3-6 days from the end of the treatment injection. 2) Checking the mortality of the infesting animals settled on the plant surfaces. 3) Determination of residual concentration of the injected product(s) Mortality of the animals may be simply verified:  

Mussels – Checking if touching them their shells remain open Barnacles/Serpulids – Observing their vitality

In case, one week before and/or after the end of the treatment application, a visit of a specialist may be foreseen to check:     

Acclimatization of the organisms Calculation of pre-treatment vitality Creation of a control site without treatment Monitoring of mortality of different species Monitoring the residual toxicity at discharge

In the below figures is shown an example of monitoring sampling points and installation of the substrates. Residual concentration of products Determination of the concentration of active of antifoulants should be carried out at discharge point to verify the quantity of products that are released to the environment. To determine the products are given the methods in Appendix 8:     

Determination Determination Determination Determination Determination

of MT-200 of C-TREAT-6 – Spectrophotometer of C-TREAT-6 – Lovibond comparator High range (0 - 20 ppm) of C-TREAT-6 – Lovibond comparator Low range (0 - 4 ppm) of C-TREAT-6 – Lovibond comparator Very low range (0 – 0.8 ppm)

Residual toxicity TRA-CIDE is an invaluable tool to measure the residual toxicity at discharge point. If standard toxicity test is required, this can be requested to CNR-ISMAR. Following a specific sampling method, send the water samples to their laboratory to quantify the toxic effect of products as such through acute toxicity tests on larvae of Artemia salina, a non-target organism, as suggested from D.Lgs 152/99, for detecting environmental toxicity of industrial discharges in the sea-water. This Italian law resumes the concepts of the “European Water Framework Directive” and the directive 76/464/CEE. In the Support CD can be found the offer from CNR for each specific intervention.

/////////////////////////////////////////////////////////////////////////


55/73

6/24/2014


________________________________________________________________

APPENDIX 6 Analytical procedures Determination residual MT-200 Determination residual C-TREAT-6

NOTE: All reported methods read both products. Cannot be possible to distinguish the two products with these methods. When both products are fed, the readings should be expressed as sum of both and expressed as product of reference.


56/73

6/24/2014


________________________________________________________________

Determination of MT-200 High/Low Range, 0 – 4 ppm - Spectrophotometer ANALYTICAL PROCEDURE AP-97E - Quaternary Am ine Test – MT-200 SPECIFICATIONS Method: Acid Orange Method Range: (0.1-4.0 mg/L) Testing Time: 2 Hours DESCRIPTION In this procedure, the dye in the Orange II Buffer solution complexes with the active quaternary amine. This complex is extracted into methylene chloride. The organic layer containing the complex is separated from the aqueous layer. The organic layer with the complexed quaternary amine will be present in the bottom layer of the flask. The aqueous (water) layer will be at the top of the funnel. The colour intensity of the methylene chloride layer is then measured in a spectrophotometer at 485 nm. The level of the colour intensity correlates to the level of quaternary amine in the sample. ORDERING INFORMATION Order as Test Kit for QUAT determination. Order all replacement parts and reagents by their part numbers. To be sure this product is compatible with your treatment program, contact your local Nalco representative. Items marked with an “*” are included with the kit or set. To place your order, please contact your local Customer Service Department. REPLACEMENT PARTS AND REAGENTS Description Methylene chloride NaCl Solution (20% required) Glacial Acetic Acid Isopropanol Orange II reagent Methanol Distilled Water

Size Not available from Applied Services 500 g 500 ml 4L 25 g 500 ml 500 ml

Part No S0820-84 S0821-74 S0495-08 S0822-80 S496A-74 S0700-74

REQUIRED APPARATUS S p e c t r o p h o t o m e t e r ( w i t h v i s i b l e l am p ) i n c l u d i n g 1 a n d 5 c m c u v e t t e s

Curve Preparation Stock Solution

Flask, Volumetric, 1 L Pipette, 1 ml

Low Level Curve

Flask, Volumetric, 1 L (5 required) Pipette, 1, 2, 5 ml

High Level Curve

Flask, Volumetric, 1 L (5 required) Pipette, 10 ml Graduated Cylinder, 100 ml

Solution Preparation Orange II Solution

Balance Cylinder, Graduated, 100 ml

Orange II Buffer Solution

Cylinder, Graduated, 100 ml Cylinder, Graduated, 250 ml Flask, Volumetric, 500 ml


57/73

6/24/2014


________________________________________________________________ Extraction

Centrifuge or Filter Apparatus (for samples with particulate matter) Cylinder, Graduated, 250 ml Funnel, Separatory, 500 ml Pipette, 8 ml or 20 ml Pipette, 30 ml Pipette, 1 ml Flask, 25 ml Pipette, 25 ml Spectrophotometer Cells, Optical, 1 or 5 cm

Chlorine Interference

0.1 N Sodium Thiosulphate Pipette, Dropper

SAFETY PRECAUTIONS 

Methylene chloride is a suspected carcinogen—testing must be performed under a hood.



When carrying out extractions with methylene chloride in separator funnels, make sure to vent the funnels after each inversion—pressure buildup is immediate.



Methylene chloride is a priority pollutant and a specifically listed RCRA-regulated material subject to specific disposal restrictions and/or prohibitions. For this reason, all used methylene chloride should be segregated from other waste streams. Dispose of waste methylene chloride in an approved manner (e.g., lab packing or incineration).

Read the Material Safety Data Sheet for Methylene chloride for complete handling and safety information before using it. SUPPORT If you have any questions regarding this procedure, please contact your local Nalco rep... STANDARD CALIBRATION Curve preparation 1) Two standard calibration curves must first be generated for use when doing testing during treatment, one curve for low levels (0.1 to 0.5 mg/ L active quaternary amine) and one curve for high levels (0.6 to 4.0 mg/L active quaternary amine). Water samples collected at outfall should be tested using the low calibration curve; water samples collected at the feed point or from the area of mussels’ infestation should be tested using the high standard curve. Table 1 provides dilution information to make desired quaternary amine concentrations. 2) Prepare a 100-mg/L quaternary amine stock solution by adding 1.0 ml of MACROTROL™ 9210 into a 1-L glass, volumetric flask containing about 500 ml of deionised water. Swirl to mix. Dilute to volume using deionised water. Mix thoroughly. This is the 0.01% stock solution of quaternary amine referred to in Table 1. 3) To generate the low-level standard curve, pipette 1.0, 1.5, 2.0, 3.0, and 5.0 ml of the stock solution 4) Into five separate 1-L volumetric flasks. Dilute to volume using deionised or distilled water. These are the standard solutions used in preparing the low-level calibration curve. You now have 5 volumetric flasks with quaternary amine concentrations of 0.10, 0.15, 0.20, 0.30, and 0.50 ppm. 5) Follow the General Procedure on page 4 to prepare the low-level standard curve (plot absorbance readings on the Y-axis and quaternary amine values on the X-axis). 6) To generate the high-level standard curve, pipette 6.0, 10, 20, 30, and 40 ml of the stock solution into five separate 1-L volumetric flask. Dilute to volume using deionised or distilled water. These are the standard solutions used in preparing the high-level calibration curve. You now have 5 volumetric flasks with quaternary amine concentrations of 0.60, 1.0, 2.0, 3.0, and 4.0 ppm.


58/73

6/24/2014


________________________________________________________________ 7) Follow the General Procedure on page 4 to prepare the high-level standard curve (plot absorbance readings on the Y-axis and quaternary amine values on the X-axis). 8) Determine the absorbance of a blank solution using distilled (or deionised) water. This blank is subtracted from the sample absorbance or used to zero the spectrophotometer so that the calibration curve goes through the origin. The calibration curve should be linear over the indicated ranges. Table 1 – Dilutions for calibration curve preparation based on a final solution volume of 1 L*

Low level curve

High level curve

Desired quaternary amine concentration (ppm) 0.10 0.15 0.20 0.30 0.50 0.60 1.0 2.0 3.0 4.0

ml of quaternary amine stock solution (0.01% ) added to 1 L to make desired quaternary amine concentration, ppm 1.00 1.50 2.00 3.00 5.00 6.00 10.0 20.0 30.0 40.0

* Dilutions should be made using demonized or distilled water.

Table 2 – Suggested volumes for various levels of quaternary amine a

Range Volume Volume Volume Volume Quaternary Orange II Methylene Isopropanol Water Amine (mg/L) Buffer (ml) chloride (ml) (ml) Sample (ml) 0.5-10.00 8 30 1 100 0.1-0.50 20 30 1 250 a The lower limit given in the range represents the minimum detectable limit for the test con ditions listed.

Optical Cell Size 1.0 cm 5.0 cm

b Five-centimeter cells are not available for use with all spectrophotometers. Many laboratory spectrophotometers require an adapter to accommodate 5-cm cells. Check with the instrument manufacturer for details.

GENERAL PROCEDURE Two solutions need to be prepared—an Orange II solution and an Orange II buffer solution. The Orange II solution is used to prepare the Orange II buffer solution. Preparation of Orange II Solution: To make 100 ml of 0.5% Orange II solution, add 0.5 grams of Orange II reagent (S0822) to 100 ml of deionised water. This solution will be used to prepare the Orange II buffer solution. Note: Make sure this solution is mixed well. The Orange II solution is stable for 5 days after preparation. Preparation of Orange II Buffer Solution To prepare the Orange II buffer solution, combine 60 ml of Orange II solution (prepared above) to 145 ml of glacial acetic acid (S0821), and 295 ml of 20% NaCl solution in a 500-ml volumetric flask. The Orange II buffer solution is stable for 5 days after preparation. Note: Prepare 20% NaCl—Dissolve 80g NaCl (S0820) in deionised water and dilute to 400 ml. Performing the Extraction 1) If the water being treated has a lot of particulate matter (=100 Ntu), it may be necessary to centrifuge or filter the sample. Centrifuging the sample will be the quickest method; if centrifuge is not available, the sample should be filtered. 2) Refer to Table 2 for the appropriate range and volumes to use in this procedure (e.g., if you think the quaternary amine concentration is around 5.0 ppm, then the water sample should be 100 ml). Transfer this volume of water sample to a separatory funnel.


59/73

6/24/2014


________________________________________________________________ 3) Add Orange II buffer solution (refer to Table 2 for correct volume) to the sample and shake for one minute. Note: Be sure to vent funnel after first few inversions of funnel. 4) Using a pipette, add 30 ml methylene chloride to the separatory funnel. Never pipette by mouth. 5. Insert the stopper in the separatory funnel. 5) (Invert the funnel and quickly remove the stopper to vent any pressure build-up.) Initial pressure build-up is significant. When venting the funnel, point the mouth of the funnel away from yourself and others (see Notes 4 and 5). 6) Shake the funnel moderately for 2 minutes, vent the funnel, then allow it to stand for 5 minutes (but no longer than 15 minutes). 7) Add one ml of isopropanol (S0495) to a 25-ml volumetric flask. 8. With a pipette inserted through the mouth of the separatory funnel and through the orange layer, collect the lower layer (methylene chloride) extract from the separatory funnel. Bring the 25-ml volumetric flask to volume with this methylene chloride extract. 8) Set the spectrophotometer at 485 (vis) nm and zero with methylene chloride. Measure and record the absorbance of the sample (see Note 6). 9) The sample absorbance is used to determine the concentration of quaternary amine in the sample. From the prepared calibration curve, determine the quaternary amine concentration in the sample (see Calibration Curve Preparation on page 2). Samples ranging from 0.1 to 0.5 ppm active quaternary amine should be read in a five-cm cell; samples ranging from 0.5 to 10.0 ppm should be read in a one-cm cell. 10) Clean the cells after each measurement (see Notes 7 and 8).

NOTES 2) For maximum accuracy, the calibration curve should be checked by every operator using this test and should be verified a minimum of twice per month using a freshly prepared quaternary amine standard. 3) A blank measurement (the blank should be a sample of the system water prior to MACROTROL treatment) must be recorded for each set of samples (i.e., each sample point tested). The blank reading may vary slightly; however, the absolute difference between the sample and the blank remains relatively constant. 4) Chlorine causes a negative interference in the test. This can be eliminated by adding 0.1 N Sodium Thiosulphate to the water sample before running the test. The amount added is based on the concentration of chlorine in the system. For a 100-ml water sample containing 0.3 mg/L chlorine, add 10 drops of 0.1 N Sodium Thiosulphate to remove the interference. 5) A slight emulsion may form when using natural water samples. When this happens, vary Step 5 of the procedure. Shake the funnel for 30 seconds, vent it, then allow it to stand for 5 minutes. Gently invert the funnel once, then allow the funnel to stand for 5 minutes. 6) It is important to vent the separatory funnel both before and after shaking it. Otherwise, pressure will build up in the funnel that can cause the stopper to be forced out of the top of the funnel. Always point the mouth of the funnel away from yourself and others. 7) Use caution when inserting or removing the sample cell in the spectrophotometer. The methylene chloride can damage the cell compartment. 8) It is imperative that the sample cells are kept clean during the running of the test. It is recommended that the cells are cleaned after each measurement using the following procedure: a) Rinse the cell three times with methanol (S496A). b) Rinse the cell three times with methylene chloride to remove methanol from the cell.


60/73

6/24/2014


________________________________________________________________ 9) Separatory funnels must be cleaned following each test. Use the following cleaning procedure: a) Drain the aqueous layer out of the separatory funnel. b) Rinse the funnel with methanol. (Make sure no traces of yellow are left in the funnel.) c)

Rinse the funnel with deionised water. (Small amounts of water may remain in the funnel. This will have no effect on the test.)

10) Turbidity can interfere with this test procedure. Turbidity may: a) Create an emulsion in the methylene chloride layer that does not separate after standing for 10 minutes when the funnel is shaken. b) Create a positive interference. (A yellow color is extracted into the methylene chloride layer.) 11) Do not change test conditions (i.e., use different volumes than those given in Table 1). Contact Research in Leiden for assistance if you experience any difficulties running this test. 12) This method is a modification of Wang and Langley. Wang, L.K. and D.F. Langley. Ind. Eng. Chem., Prod. Res. Dev., Vol. 14, No. 3, 1975.

///////////////////////////////////////////////////////////////////////


61/73

6/24/2014


________________________________________________________________

Determination of C-TREAT-6 High Range, 0 – 20 ppm - Spectrophotometer Principle of Method C-TREAT-6 reacts with sulphonphtalein to form a yellow complex. The complex can be extracted from the aqueous solution with chloroform., the intensity of the color is directly proportional to the concentration of C-TREAT-6 present. This is measured by photometer and compared against a standard graph or table. AS C-TREAT-6 contains amines which film out on glass and metal surfaces (resulting in product loss during the test), all glassware, which come into contact with the product, should be treated with “Repelcote” so as to avoid this adsorption process. It is advisable to clean glassware and recoat every ten tests or when glass wetting become evident. Sampling procedure Care should be taken when sampling to ensure that “representative” samples are taken. Sampling points should be valved, short in length and have access to the bulk flow of water. Water samples should be collected in Repelcoated glass bottles. These should be thoroughly rinsed out with fresh water from their respective sample points before an actual sample is taken. Samples should be analyzed immediately and not stored. AS well as the samples to be analyzed, a blank water sample should be taken which does not contain any product.

Apparatus required for one determination The apparatus listed below is for a single C-TREAT-6 determination. 2 x 250 ml Repelcoated sampling containers 2 x Pasteur pipettes 2 x 5 ml pipettes 2 x rubber suction valves 1 x spectrophotometer 2 x glass cuvettes

2 x funnels (100 ml) with PTFE taps 2 x measuring cylinders (50 ml) 1 x pH meter Assorted beakers Retort stands and clamps

Reagent required Buffer solution:

Potassium dihydrogen orthophosphate 68 g Distilled water to 500 ml Add ortho-phosphoric acid to pH 3.5 (approx. 5 ml)

Indicator solution:

Bromocresol green (water soluble) 0.05 g Distilled water to 100 ml

Chloroform:

For amine extraction process

“Repelcoate - Silicone Emulsion:

For glassware (*)

Experimental procedure The sample and blank are prepared in the same way, at the same time: 1.

Measure out 50 ml of the sample and the blank into the stoppered separation funnels

2.

To each, add 5 ml of buffer solution, followed by 5 ml of indicator solution. Check the pH value using pH meter and adjust if necessary to within the range 3.0 – 3.5. Dilute (approx. N/10) acid or alkali may be used to adjust the pH value. (The colour of the shaken solution should be yellow, but if blue, add 5 ml more of buffer solution more to obtain the yellow colour).


62/73

6/24/2014


________________________________________________________________ 3.

Extract each sample two times with 10 ml of chloroform. Shaking the funnel and contents for one minute and then allowing standing for two minutes should carry out each extraction. The lower chloroform portion containing the extracted amine should be collected in 50 ml measuring cylinder.

4.

Dilute the combined extracts to 25 ml with chloroform and ensure that they are thoroughly mixed.

5.

Measure the optical density or optical transmission of the chloroform extract in a 1 cm glass cell at 417 nm which is the optimum wavelength using a photometer, or colorimeter with a suitable color filter.

6.

Record the difference in optical density between the sample and the blank. Use the figure to determine the concentration of C-TREAT-6 in the sample from a previously prepared calibration table or graph.

NB: 

If the intensity of the color of the chloroform extract is too great to measure accurately using the calibrated instrument, then the C-TREAT-6 sample should be diluted by a known factor with untreated water and the test carried out on the diluted solution.



Samples containing high levels of suspended solids may appear cloudy and lead to erroneous readings on the spectrophotometer. Consequently samples may need to be centrifuged before this whole process is undertaken. Filtration is not considered suitable because of product adsorption.

Calibration graph

Calibration C-TREAT-6

Using the above procedure, determine the optical densities of a range of standard C-TREAT-6 solutions made up in the real water. A good range would be 2.5, 5, 10, 20, and 30 ppm. Plot a graph of C-TREAT-6 concentration against optical density or optical transmission. Note: The graph constructed will be specific for the instrument used. Should a different instrument be used, a new calibration graph will have to be used. The graph shown is for a Palintest 5000 photometer using the 410 nm filter.

120

100

e 80 c n a t t i m 60 s a r T % 40

20

0 0

5

10

15

20

25

30

C-TREAT-6, ppm

Clean up procedure Wash all glassware immediately and thoroughly after use to avoid any interference with the next series of tests. “Repelcote” can be removed by soaking glassware in chromic acid. It is important to ensure that spectrophotometer cells are clean and discoloration can be removed with isopropylalcohol. The analysis technique described above has been developed specifically for determining the amount of C-TREAT-6 present in seawater. An operator, familiar with the technique could expect an accuracy of +/- 5%. Note: If greater sensitiv ity is requ ired, take high er water volum e sample (i.e. to increase 10 times the sensiti vity, measure 500 ml of w ater into 1000 ml sto ppered separation f u n n e l s , an d t h e n f o l l o w t h e n o r m a l p r o c e d u r e f r o m s t e p 2 . (*) The stoppered cylinders can be Silicone Treated by completely filling them with Silicone Emulsion and stoppering the cylinders. After 5 minutes drain the solution and rinse with water, preferably deionised or distilled water. The coating will last about 6 months. Re-treatment will be necessary when a normal water meniscus reappears.


63/73

6/24/2014


________________________________________________________________

Determination of C-TREAT-6 Low Range, 0 – 20 ppm – LOVIBOND Comparator Reagents and Apparatus Amine Indicator Tablets Silicone Emulsion 2 x 20 ml LOVIBOND Comparator Tubes 10 ml Measuring Cylinder Chloroform LOVIBOND Comparator LOVIBOND Comparator Disc 3/64 2 x 100 Stoppered Separating Funnels, Silicone Treated (*)

Principle of the method This test has been developed primarily for the determination of traces of long-chain aliphatic amines in water. Primary, secondary, tertiary and quaternary amines (quaternary ammonium compounds – QAC) react with sulphonphtaleins in acid solution, to form a yellow complex. This complex is extracted from the aqueous solution by shaking with chloroform. The intensity of the yellow colour is the lower (chloroform) layer after the extraction is proportional to the concentration of the amine ì, and it estimated by comparison with Lovibond permanent glass colour standards. This test, although primarily developed for long-chain amines, can be applied to the determination of other amines provided that the disc comparator is recalibrated in terms of the particular amine being estimated.

Method 7.

To one of the Silicone Treated 100 ml Separating Funnels, add 20 ml of water to be tested. To the other, add 20 ml of untreated water (the blank). 8. To both Separating Funnels, add 1 Amine Indicator Tablet and crush to disintegrated, then shake to mix. The solution should be yellow, but if blue, add 1 more Amine Indicator Tablet to obtain the yellow colour. 9. Add 10 ml of Chloroform to both cylinders, shake constantly for at least one minute, and allow standing until the two phases separate. (If amine is present, the chloroform layer will be yellow – ignore any yellow colour in the aqueous layer). Discard as much of the upper (aqueous) layer as possible. 10. Transfer the lower (chloroform) layer in each 1000 ml Separating Funnel to each of the two Lovibond Tubes, (i.e. one for the blank and one for the sample). 11. Repeat (3-4) above (to extract any remaining C-TREAT-6 from the aqueous solution) 12. Place the “sample” in the right -hand compartment and the “blank” in the left-hand compartment of the Comparator and read the amine level from the matching colour on the Comparator disc.

Calculation The concentration of C-TREAT-6 = Comparator Reading x 10

(*) The stoppered cylinders can be Silicone Treated by completely filling them with Silicone Emulsion and stoppering the cylinders. After 5 minutes drain the solution and rinse with water, preferably deionised or distilled water. The coating will last about 6 months. Re-treatment will be necessary when a normal water meniscus reappears.


64/73

6/24/2014


________________________________________________________________

Determination of C-TREAT-6 Low Range, 0 – 4 ppm – LOVIBOND Comparator Reagents and Apparatus Amine Indicator Tablets Silicone Emulsion 2 x 20 ml LOVIBOND Comparator Tubes 10 ml Measuring Cylinder Chloroform LOVIBOND Comparator LOVIBOND Comparator Disc 3/64 2 x 250 Stoppered Separating Funnels, Silicone Treated (*)


Method 13. To one of the Silicone Treated 250 ml Separating Funnels, add 100 ml of water to be tested. To the other, add 100 ml of untreated water (the blank). 14. To both Separating Funnels, add 1 Amine Indicator Tablet and crush to disintegrated, then shake to mix. The solution should be yellow, but if blue, add 1 more Amine Indicator Tablet to obtain the yellow colour. 15. Add 10 ml of Chloroform to both cylinders, shake constantly for at least one minute, and allow standing until the two phases separate. (If amine is present, the chloroform layer will be yellow – ignore any yellow colour in the aqueous layer). Discard as much of the upper (aqueous) layer as possible. 16. Transfer the lower (chloroform) layer in each 1000 ml Separating Funnel to each of the two Lovibond Tubes, (i.e. one for the blank and one for the sample). 17. Repeat (3-4) above (to extract any remaining C-TREAT-6 from the aqueous solution) 18. Place the “sample” in the right -hand compartment and the “blank” in the left-hand compartment of the Comparator and read the amine level from the match ing colour on the Comparator disc.

Calculation The concentration of C-TREAT-6 = Comparator Reading x 2 (*) The stoppered cylinders can be Silicone Treated by completely filling them with Silicone Emulsion and stoppering the cylinders. After 5 minutes drain the solution and rinse with water, preferably deionised or distilled water. The coating will last about 6 months. Re-treatment will be necessary when a normal water meniscus reappears.


65/73

6/24/2014


________________________________________________________________

Determination of C-TREAT-6 Very Low Range, 0 – 0.8 ppm – LOVIBOND Comparator Reagents and Apparatus Amine Indicator Tablets Silicone Emulsion 2 x 20 ml LOVIBOND Comparator Tubes 10 ml Measuring Cylinder Chloroform LOVIBOND Comparator LOVIBOND Comparator Disc 3/64 2 x 1000 Stoppered Separating Funnels, Silicone Treated (*)


Method 19. To one of the Silicone Treated 1000 ml Separating Funnels, add 500 ml of water to be tested. To the other, add 500 ml of untreated water (the blank). 20. To both Separating Funnels, add 1 Amine Indicator Tablet and crush to disintegrated, then shake to mix. The solution should be yellow, but if blue, add 1 more Amine Indicator Tablet to obtain the yellow colour. 21. Add 10 ml of Chloroform to both cylinders, shake constantly for at least one minute, and allow standing until the two phases separate. (If amine is present, the chloroform layer will be yellow – ignore any yellow colour in the aqueous layer). Discard as much of the upper (aqueous) layer as possible. 22. Transfer the lower (chloroform) layer in each 1000 ml Separating Funnel to each of the two Lovibond Tubes, (i.e. one for the blank and one for the sample). 23. Repeat (3-4) above (to extract any remaining C-TREAT-6 from the aqueous solution) 24. Place the “sample” in the right -hand compartment and the “blank” in the left-hand compartment of the Comparator and read the amine level from the match ing colour on the Comparator disc.

Calculation The concentration of C-TREAT-6 = Comparator Reading x 0.4 (*) The stoppered cylinders can be Silicone Treated by completely filling them with Silicone Emulsion and stoppering the cylinders. After 5 minutes drain the solution and rinse with water, preferably deionised or distilled water. The coating will last about 6 months. Re-treatment will be necessary when a normal water meniscus reappears. ////////////////////////////////////////////////////////////////////////////////


66/73

6/24/2014


________________________________________________________________

APPENDIX 7 Nalco EVAC Non-Oxidant Macrofouling control


67/73

6/24/2014


________________________________________________________________

Attachment 1


68/73

6/24/2014


________________________________________________________________

Attachment 2


69/73

6/24/2014


________________________________________________________________

Attachment 3


70/73

6/24/2014


________________________________________________________________

Attachment 4

C-TREAT-6


71/73

6/24/2014


________________________________________________________________

Attachment 5


72/73

6/24/2014

Nalco New Approach for Macrofouling

Recommend Documents