MOF, Alm.del - 2016-17 - Endeligt svar på spørgsmål 983: Spm. om ministeren vil oversende den supplerende redegørelse fra Mærsk vedrørende deres ulovlige udledning af kemikalier, som Miljøstyrelsen har anmodet om inden den 7. august 2017, jf. http://www.dr.dk/nyheder/indland/maersk-redegoerelse-om-ulovlig-udledning-er-mangelfuld, til miljø- og fødevareministeren

Miljø- og Fødevareudvalget 2016-17
MOF Alm.del
Offentligt

INVESTIGATION REPORT FOR

MAERSK OIL

Supply of SCAVTREAT 7103

JULY 31, 2017

CLARIANT OIL SERVICES SCANDINAVIA AS

Bergen, Norway

CONFIDENTIAL

MOF, Alm.del - 2016-17 - Endeligt svar på spørgsmål 983: Spm. om ministeren vil oversende den supplerende redegørelse fra Mærsk vedrørende deres ulovlige udledning af kemikalier, som Miljøstyrelsen har anmodet om inden den 7. august 2017, jf. http://www.dr.dk/nyheder/indland/maersk-redegoerelse-om-ulovlig-udledning-er-mangelfuld, til miljø- og fødevareministeren

ATE:

SUMMA

Contents

Executive Summary ....................................................................................................................................... 2

Introduction and Background ....................................................................................................................... 3

Supply of SCAVTREAT 7103 ......................................................................................................................... 4

Corrective Actions .......................................................................................................................................... 5

Ecotoxicology Testing ................................................................................................................................. 8

Root Cause Analysis ....................................................................................................................................... 9

Preventive Actions ....................................................................................................................................... 10

Appendix A

–

Letters to Maersk Oil and Danish Product Registry

Appendix B

–

Ecotoxicology Test Results

Appendix C

–

Other Test Results

SUMMARYTITLE

Page 1 of 10

CONFIDENTIAL

Executive Summary

SCAVTREAT 7103 is a Clariant hydrogen sulfide scavenger of a type commonly deployed in Oil & Gas

production operations. The material is manufactured on behalf of Clariant by Synthite Ltd.

An unauthorised modification was made to the formulation of SCAVTREAT 7103 during 2005, in an

attempt to meet a customer’s technical specification for the material. The modified formulation had 0.1%

active benzyl-C12-16-alkyldimethyl, chlorides

(“BAC”)

added as 0.2% of a 50% solution (“BAC

50”).

This modification was not internally approved and was not communicated to any customer.

Consequently, HOCNF documentation was not prepared as required and environmental testing was not

undertaken. This resulted in the inclusion of a minor component that was classified as

“red”

to a material

that was otherwise classified

as “yellow”.

Clariant became aware of the situation when one customer using SCAVTREAT 7103 detected some

quaternary amine in the condensed water from a gas treatment system. The customer asked Clariant to

confirm whether SCAVTREAT 7103 contained quaternary amine. Following consultation with Synthite,

it was discovered that SCAVTREAT 7103 contained low levels of BAC.

Immediate action was taken to inform stakeholders, as well as concurrently eliminating BAC from

produced material. Clariant conducted detailed technical evaluations to validate alternative solutions

including the elimination of BAC from SCAVTREAT 7103.

A transition to supply of material free of undisclosed components to Maersk Oil was completed by 12

June 2017.

Clariant conducted extensive root cause analysis, including review of historic technical records and

correspondence, on site visits and discussions with Synthite and review of contract documentation.

It was identified that there were two primary causes for this occurrence:



Unauthorised instruction was given by an individual to a third party toll manufacturer. This

bypassed established control systems which requires approval from four individuals for any

product formulation changes.

Lack of a contractual requirement with this manufacturer that documents changes to product

formulations and respective communications to toll manufacturers

according to Clariant’s

standard format, including multiple levels of approval.

Preventive actions have been formulated and are being rapidly implemented to eliminate the risk of a

similar occurrence in the future. Full details of the change in Management Systems to ensure no re-

occurrence takes place can be found in the Preventive Actions chapter on page 10.

Page 2 of 10

CONFIDENTIAL

Introduction and Background

The product SCAVTREAT 7103 is manufactured for Clariant Oil Services Scandinavia by a 3

party toll

manufacturer in the UK, Synthite. Between 2003

–

2017 the product has been supplied to customers in

Scandinavia.

SCAVTREAT 7103 is a hydrogen sulfide (H

S) scavenger, supplied as an aqueous solution of 30-60%

2,2',2"-(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol,

commonly referred to as “MEA triazine”. It is

applied in oil and gas production systems to reduce levels of H

S, which is a highly toxic and corrosive

gas that is naturally present in some oil and gas reservoirs, and thereby contributes to safety and

environment protection.

On 22

March 2017, Clariant Oil Services Scandinavia was contacted by a customer regarding the

SCAVTREAT 7103 composition. It was questioned whether the product contained a surfactant, because

the customer had detected some quaternary amine in the condensed water from a gas treatment system.

The immediate response from Clariant sales personnel was that the product only contained MEA

triazine, based on the

records in Clariant’s

product database and Harmonised Offshore Chemical

Notification Format (HOCNF) documentation.

The customer asked Clariant to re-check the composition, and come back with a written confirmation.

Clariant contacted Synthite by e-mail, and asked whether there was any quaternary amine added to

SCAVTREAT 7103. Synthite informed Clariant that SCAVTREAT 7103 contains 0.1% active quaternary

ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides (commonly known as benzalkonium

chloride or

“BAC”) added

as 0.2% of a 50% solution (“BAC 50”). This was confirmed by Synthite to

Clariant in writing on 28

March 2017.

Synthite further informed Clariant that BAC had been added to SCAVTREAT 7103 in an attempt to meet

technical specifications from Maersk Oil, namely to control the levels of formic acid and N-(2-

hydroxyethyl) formamide (”NHEF”) generated during normal product application when SCAVTREAT

7103 is exposed to temperatures in Maersk Oil process systems which can be up to 170°C.

Synthite explained that they had received instructions from an individual at Clariant Oil Services

Scandinavia in 2005 to add BAC to SCAVTREAT 7103. The individual also requested that this

information must not be disclosed to Maersk Oil, ostensibly to protect Clariant proprietary formulation

information.

In a meeting on 31

March 2017 with the customer who informed Clariant about the quaternary amine,

Clariant was informed that the customer had performed an analysis of SCAVTREAT 7103 that confirmed

that the product contained quaternary amine.

The level of addition of BAC was so low that, even if Clariant has been aware of its presence, Clariant

would have not been required to show this component on the material Safety Data Sheet (SDS).

However, as the component was deliberately added it should have been declared in the HOCNF, which

did not happen.

As the addition of BAC to SCAVTREAT 7103 was unauthorised in Clariant’s management systems,

investigations and internal discussions were initiated to determine:

Page 3 of 10

CONFIDENTIAL



The history of when the addition of BAC began and the technical justification for this addition

The reason that this addition was not properly approved

in Clariant’s systems

Possible solutions for continued supply to Clariant customers, including understanding the

technical consequences of removing BAC from SCAVTREAT 7103

Supply of SCAVTREAT 7103

SCAVTREAT 7103 is an aqueous solution of 30-60% 2,2',2"-(hexahydro-1,3,5-triazine-1,3,5-triyl)

triethanol,

commonly referred to as “MEA triazine”. This

product is an industry standard treatment used

globally in the Oil & Gas industry to remove hydrogen sulfide

(“H

S”) from process streams. H

S is a

highly toxic and corrosive gas that is naturally occurring in some oil and gas reservoirs. The

sequestration of hydrogen sulfide by scavengers, such as SCAVTREAT 7103, greatly improves the safety

of oil and gas operations and helps to protect the environment.

Supply of SCAVTREAT 7103 to Maersk Oil began in October 2003. Subsequent discussions with Maersk

Oil indicated that NHEF levels were higher than desired. On the instruction of the same individual that

later gave direction to change the formulation of SCAVTREAT 7103, testing of samples from Synthite

was conducted by the Danish Technological Institute (DTI). This testing evaluated modifications to

SCAVTREAT 7103 which would lower levels of formic acid and NHEF generated from SCAVTREAT 7103

under field temperatures. BAC was identified as helping to address this issue. No other Clariant

employees received test results.

Beginning in January 2005, BAC was added to the SCAVTREAT 7103 formulation at a rate of 0.1%. Since

12th June 2017 SCAVTREAT 7103 is free of BAC.

Page 4 of 10

CONFIDENTIAL

Corrective Actions

When we learned of the unauthorised addition of BAC, Clariant identified that SCAVTREAT 7103 was

being supplied to three customers in Scandinavia. Based on historic information provided by Synthite,

it was also noted that the addition of BAC to SCAVTREAT 7103 was initiated for technical reasons related

to application of the product by Maersk Oil, which required detailed discussion and investigation.

Following an internal discussion on 6

April 2017 between Clariant specialists from Commercial,

Technical, Product Stewardship, Procurement and Environmental functions, a number of actions were

initiated. These included the following:



Requested samples of SCAVTREAT 7103 with and without BAC to be mobilised from Synthite

to Clariant labs in Aberdeen, UK and Bergen, Norway, for the required testing

Requested samples of SCAVTREAT 7103 with and without BAC to be mobilised from Synthite

to DTI in Denmark for testing of formic acid and NHEF levels according to Maersk Oil

specifications

Determined availability of alternative manufacturing sources of SCAVTREAT 7103, in case

Synthite produced material without BAC added would not meet customer technical

specifications

Investigated manufacture of material by Synthite via a different process to determine effect on

levels of formic acid and NHEF in SCAVTREAT 7103

Reviewed contract documentation between Clariant, Maersk Oil and Synthite to fully

understand responsibilities

Initial discussions within Clariant focussed on finding a technically acceptable product to propose to

Maersk Oil, based on their specific requirements and specifications, as the addition of BAC to

SCAVTREAT 7103 had originally been carried out to address these specifications. It became clear that

this was likely to be a complex task, and that detailed technical discussions with Maersk Oil were the

best way to expedite a solution.

Maersk Oil in Denmark were informed by Clariant on 11

April 2017; a copy of this letter is included in

Appendix A. On 18

April 2017, Clariant received a reply from Maersk Oil informing Clariant that

Maersk Oil would inform the Danish authorities, and advising Clariant to also engage with the

authorities. On 20

April 2017, Clariant informed the Danish Product Registry about the undocumented

addition of BAC to SCAVTREAT 7103. A copy of this letter is included in Appendix A.

In order to seek a solution to address Maersk Oil’s technical requirements and provide a product which

was compliant with the applicable regulations, Clariant mobilised the airfreight of the necessary product

samples and initiated testing at an independent laboratory, the Danish Technological Institute (DTI).

This testing evaluated the generation of formic acid and NHEF under Maersk

Oil’s

field temperatures

by:



SCAVTREAT 7103; manufactured by Synthite; containing BAC (benchmark)

SCAVTREAT 7103; manufactured by Synthite; without BAC

Page 5 of 10

CONFIDENTIAL



SCAVTREAT 7103; manufactured by Synthite by alternative manufacturing method; without

BAC

SCACTREAT 7103; manufactured by an alternative supplier, without BAC

Results were compared to Maersk Oil specification, and are summarised in the table below. Results

highlighted in green are within the specification limits defined by Maersk Oil. Results highlighted in

blue are outside of the specification limits and deemed unacceptable by Maersk Oil.

Measured content (%)

SCAVTREAT 7103

alternative

Specification

SCAVTREAT 7103 SCAVTREAT 7103

Method

production

(%)

with BAC, batch: without BAC,

method; without

17D025

batch: 17D037

BAC. Batch

RD282

Formic acid

%w/w Formic acid (as received)

%w/w Formic acid (reflux at

SCAVTREAT 7103

alternative

supplier;

without BAC

0.1 max

0.2 max

Dionex

Column

Dionex

Column

Dionex

Column

Dionex

Column

0.014

0.046

0.017

0.011

0.1

0.021

0.17

0.23

0.19

0.0067

0.14

0.0076

140 C for 10 min)

%w/w Formic acid (aerated for 7

0.1 max

days)

%w/w Formic acid (aerated for 7

days, then reflux at 140

c for 10 0.2 max

min.)

N-(2-hydroxyethyl) formamide

('NHEF')

%w/w NHEF (as received)

0.25 max

%w/w NHEF (reflux at 140 C for

2.00 max

10 min)

%w/w NHEF (aerated for 7 days) 1.00 max

%w/w NHEF (aerated for 7 days,

2.00 max

then reflux at 140

c for 10 min.)

0.084

0.036

0.23

0.15

GC-MS

<0.1

0.36

<0.1

1.7

<0.1

1.2

<0.1

2.0

0.12

2.0

0.13

2.0

<0.1

0.53

<0.1

1.6

The full test results reported by DTI are included in Appendix C.

These results confirm that:



SCAVTREAT 7103 manufactured by Synthite meets the Maersk Oil specification, with and

without the addition of BAC

SCAVTREAT 7103 produced by an alternative manufacturing process (without the addition of

BAC) does not meet the Maersk Oil specification

SCAVTREAT 7103 produced by an alternative manufacturer (without the addition of BAC) does

meet the Maersk Oil specification

Page 6 of 10

CONFIDENTIAL

These results give a positive indication that removal of BAC from SCAVTREAT 7103 is a viable solution

for continued supply to Maersk Oil, and that an additional source of compliant material is available.

Clariant conducted further testing to evaluate the corrosivity of SCAVTREAT 7103 with and without

BAC. This testing was requested by Maersk Oil. Maersk

Oil’s technical specifications for formic acid and

NHEF are designed to reduce the risk of corrosion during application of the product in field process

systems, and this testing was requested as an additional confirmation of the technical suitability of

SCAVTREAT 7103 without BAC.

Clariant mobilised the transatlantic airfreight of samples of SCAVTREAT 7103 with and without BAC to

our Global Technology Centre at the business unit headquarters in The Woodlands, near Houston,

Texas. Testing in the corrosion lab was prioritised over all other work in progress, and all of the necessary

resources dedicated to rapid completion of this testing.

The testing conducted was a Rotating Cylinder Autoclave (RCA) test. In order to provide a high degree

of accuracy and confidence in the test results, individual tests were conducted in triplicate. Testing was

conducted under different temperatures and gas compositions, to reflect field conditions in different

parts of Maersk Oil’s field operations.

Workscopes and methodology were agreed with Maersk Oil prior

to testing.

Results are shown in the graph below:

Page 7 of 10

CONFIDENTIAL

These results confirm that the addition of BAC to SCAVTREAT 7103 does not have a significant impact

on the corrosivity of SCAVTREAT 7103 under Maersk Oil field conditions. There is not a consistent

increase or decrease in corrosion rates caused by the addition of BAC to the product.

The test results detailed above, showing the impact of BAC on formic acid and NHEF levels, as well as

the corrosivity of SCAVTREAT 7103 without BAC, satisfied Maersk Oil that BAC could be removed from

SCAVTREAT 7103 without undue risk to their process or operations.

Following agreement with Maersk Oil, supply of SCAVTREAT 7103 to Maersk Oil in Denmark has been

free of BAC since 12

June 2017.

Ecotoxicology Testing

As Clariant was unaware of the addition of BAC to SCAVTREAT 7103, and had hence not submitted a

HOCNF showing this component, the ecotoxicology data was incorrect. Clariant initiated ecotoxicology

testing of BAC in

order that this data could be retrospectively updated and submitted to Clariant’s

customers and the concerned regulatory authorities.

The evaluation of the partition coefficient, log Pow, was determined by testing in the Clariant

laboratories in Frankfurt, Germany. The other ecotoxicology testing was conducted by an external

specialist company, Chemex Environmental International in Cambridge, UK. Samples of BAC 50 were

mobilised by Clariant to both Frankfurt and Cambridge to facilitate this testing.

The complete test reports are available in Appendix B.

The mandatory ecotoxicological tests performed on a sample of BAC 50, as specified in the OSPAR

HMCS (Harmonised Mandatory Control System), and subsequently evaluated using the OSPAR

Recommendation 2016/4 Harmonised Pre-screening Scheme, confirmed the expected classification of

this substance as a candidate for substitution ("red" in Denmark).

* Note:

“OSPAR”

(stands for Oslo and Paris) refers to The Convention for the Protection of the Marine Environment of the North-

East Atlantic

(the ‘OSPAR Convention'). More details are available at www.ospar.org

Page 8 of 10

CONFIDENTIAL

Root Cause Analysis

An investigation team was formed to conduct an investigation into the root cause of the occurrence. This

was led by Clariant’s

QHSE Manager for Scandinavia, and included Clariant specialists from QHSE,

Procurement, Technical, Regulatory and Commercial functions.

A full document review was conducted, as well as a visit to the Synthite plant in the UK.

A root cause analysis based on the known information was performed to determine why SCAVTREAT

7103 contained an unauthorised and undocumented component.

The investigation reveals:



That the decision to add BAC at a low concentration to SCAVTREAT 7103 was made based on

data indicating that this would control levels of formic acid and NHEF generated by

SCAVTREAT 7103 during product application.

This decision to add BAC to SCAVTREAT 7103 was made by one individual.

An unauthorised change requirement was communicated to Synthite.

There was no formal requirement for Synthite to confirm process changes to Clariant.

The change to the formulation of SCAVTREAT 7103

was not recorded in Clariant’s

systems. In

doing so Clariant’s internal approval

process was bypassed. Consequently, a HOCNF was not

prepared and environmental testing of BAC was not undertaken.

The customers were not informed of the new formulation with BAC.

Synthite Ltd were audited on three occasions by Clariant Oil Services Scandinavia AS (formerly

Clariant (Norge) AS). On all occasions the audit team included the individual who had requested

the addition of BAC to SCAVTREAT 7103.

The level of BAC present in SCAVTREAT 7103 was too low to be detectable by standard quality

control (QC) tests designed to verify compliance with product specifications agreed with

customers.



In summary, this occurrence was an isolated incident, as four highly exceptional things came together.

The change to the product was (1) unauthorized, (2) undocumented, (3) implemented by a single

individual and (4) so slight that it could not be detected by the usual quality control tests.

Page 9 of 10

CONFIDENTIAL

Preventive Actions

The following measures will be implemented to eliminate the possibility of a single individual instructing

a third party toll manufacturer to make unauthorised changes to a formulation.



All recipes and manufacturing instructions to third party toll manufacturers must be supplied

in writing by Clariant on an approved Specification and Manufacturing Instruction Sheet. This

requires separate approval from Technical, Commercial, Manufacturing and Management

functions.

Any changes to recipes or manufacturing instructions for existing products must be

communicated on an updated Specification and Manufacturing Instruction Sheet (approved as

above). Only changes detailed on the updated sheet will be implemented by the third party.

Upon receipt of an updated instruction, the Third Party must confirm in writing to Clariant that

they have received and understood the required change(s), along with the implementation date.

Third party manufacturers will be required to periodically confirm that product supplied has

been manufactured in line with the Manufacturing Instructions provided by Clariant.

The above points are to be included in any contract with a Third Party manufacturer.

These records are to be retained by Clariant for the duration of the contract plus a suitable

period (to be defined in consultation with Legal Services).

The frequency of auditing Third Party manufacturers is to be increased to every two years.

Audits of the Third Party manufacturers must not be done solely by the Clariant Business Unit

organisation. They must involve local, regional or global corporate support functions

responsible for Quality, Health, Safety and Environmental (QHSE) affairs.



Page 10 of 10

CONFIDENTIAL

APPENDIX A

CLARIANT OIL SERVICES

SCANDINAVIA AS

Thormøhlensgate 53D

N-5006 Bergen

Norway

CLARIANT OIL SERVICES SCANDINAVIA AS - N-5006 BERGEN -

NORWAY

Confidential

Mærsk Olie og Gas A/S

Britanniavej 10

6700 Esbjerg

Denmark

FAO: Mr. Peter Christensen, Production Chemistry Lead

JOHN JEX

General Manager

Phone +47 55 36 34 90

Fax

+47 55 36 34 8

[email protected]

11.04.2017

SCAVTREAT 7103

Dear Peter,

Clariant Oil Services offers with SCAVTREAT products a suite of water-soluble and oil-soluble

scavengers for removing sulfide species from both gases and liquids, as well as a range of batch and

continuously injected products designed to remove FeS or treat the cause of its formation.

SCAVTREAT 7103 is an aqueous solution of 30-60% 2,2',2"-(hexahydro-1,3,5-triazine-1,3,5-

triyl)triethanol, which is currently used by Maersk in Denmark. Clariant discloses in our safety data

sheet information on our products that correspond to the present state of our knowledge. This

knowledge is well documented in our SAP EH&S system, which is the base of all our Product

Stewardship relevant information, including all product compositions.

Very recently it came to our knowledge that SCAVTREAT 7103 contains between 0.06 to 0.12%

(typically < 0.1%) quaternary ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides, EC

939-350-2. The result of the evaluation of this modified composition is as follows: no change in

Classification, Labelling and Packaging (CLP) and thus no impact on workers due to the presence of

quaternary ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides.

As we were not aware of this ingredient we failed to meet the requirements of the Harmonised

Offshore Chemicals Notification Format (HOCNF) for this component. This is very much to our

regret. We decided to immediately take care that this component will be removed from our

preparation, and we will work closely with Maersk to support the transition and field application of

the product following the removal of this component.

We will conduct the necessary testing under Good Laboratory Practice in order to generate the data

to confirm the environmental classification of SCAVTREAT 7103 containing the additional

component. Based on our current knowledge, we expect that the classification will be “red”.

In addition we routinely updated our Safety Data Sheet to align with the classification of 2,2',2"-

(hexahydro-1,3,5-triazine-1,3,5-triyl)triethanol as disseminated by ECHA:

Page 1 of 2

CONFIDENTIAL

From:

•

Acute Tox 2

–

Inhalation

Acute Tox 4

–

Oral

Skin Sens 1

STOT 1

–

Resp.

To:

•

Acute Tox 2

–

Inhalation

Acute Tox 4

–

Oral

Skin Sens 1

STOT 1

–

Resp.

Eye Irr 2

The changes to the Safety Data Sheet are not related to the discovery of the additional component in

the product.

Both myself and our senior management are committed to addressing this incident promptly and

fully to the satisfaction of Maersk. We are happy to attend meetings with Maersk s staff as required

and to fully discuss and resolve this issue.

Yours sincerely,

John Jex

General Manager

Page 2 of 2

CLARIANT OIL SERVICES

SCANDINAVIA AS

Thormøhlensgate 53D

N-5006 Bergen

Norway

CLARIANT OIL SERVICES SCANDINAVIA AS - N-5006 BERGEN -

NORWAY

Confidential

Maersk Olie og Gas A/S

Britanniavej 10

6700 Esbjerg

Denmark

FAO: Mr. Peter Christensen, Production Chemistry Lead

JOHN JEX

General Manager

Phone +47 55 36 34 90

Fax

+47 55 36 34 8

[email protected]

20.04.2017

SCAVTREAT 7103

Dear Peter,

Further to your letter of 18.04.2017, we would like to update you on the actions taken by Clariant as

requested in your letter.

Clariant has today informed in writing the Danish Product Registry of our recent discovery that

SCAVTREAT 7103 contains between 0.06 to 0.12% (typically < 0.1%) Quaternary ammonium

compounds, benzyl-C12-16-alkyldimethyl, chlorides, EC 939-350-2.

As this occurrence was a unique set of circumstances relating to supply of a material from a third

party, we do not believe that there is a significant risk of similar situations with other products

supplied by Clariant. However, we are reviewing all products supplied to the Danish oil industry to

ensure that there are no additional components added to any product that we are not currently aware

of. As most products are manufactured by Clariant, we have full control of all components added to

the products, but where necessary we will seek assurances from third party suppliers. We do not

expect any changes, but will if required update HOCNF accordingly.

We are also reviewing all MSDS for material supplied to the Danish oil industry. Again, we do not

expect any changes to be required but are doing this to ensure full transparency and compliance.

As we have previously explained, we are conducting the necessary 3

party lab testing to determine

the environmental rating of SCAVTREAT 7103 based on the revised understanding of the

composition. Once this work is completed, which is expected to take 10

–

12 weeks, we will submit a

revised HOCNF to the Danish Environmental Protection Agency based on the updated environmental

data.

We are also conducting test work to determine the impact of the removal of the Quaternary

ammonium compounds, benzyl-C12-16-alkyldimethyl, chlorides component from the SCAVTREAT

7103 on the specification of the product, in particular the levels of formic acid and formamide. We

Page 1 of 2

CONFIDENTIAL

will communicate with you further on this subject once the testing is completed. We anticipate that

this will take approximately 2 weeks.

As previously expressed, both myself and our senior management are committed to addressing this

incident promptly and fully to the satisfaction of Maersk Olie og Gas. We are happy to attend meetings

with Maersk staff as required and to fully discuss and resolve this issue.

Yours sincerely,

John Jex

General Manager

Page 2 of 2

CLARIANT OIL SERVICES

SCANDINAVIA AS

Thormøhlensgate 53D

N-5006 Bergen

Norway

CLARIANT OIL SERVICES SCANDINAVIA AS - N-5006 BERGEN -

NORWAY

Confidential

Mærsk Olie og Gas A/S

Britanniavej 10

6700 Esbjerg

Denmark

FAO: Mr. Peter Christensen, Production Chemistry Lead

JOHN JEX

General Manager

Phone +47 55 36 34 90

Fax

+47 55 36 34 8

[email protected]

03.05.2017

SCAVTREAT 7103

Dear Peter,

Further to our telephone meeting on 28

April 2017, we would like to provide you with an update

regarding the actions we have already taken plus the action plan going forward to resolve the issues

related to the supply of our triazine based product SCAVTREAT 7103.



The Danish Product Registry has been advised of the new knowledge that has emerged which

affects the HOCNF for SCAVTREAT 7103. The Product Registry has acknowledged receipt of

our letter.

We have sent a sample of SCAVTREAT 7103 for environmental testing to obtain the correct

data to support the HOCNF application for this product.

Samples of SCAVTREAT 7103 with and without the BAC 50 have been sent to the DTI to

assess the formamide and formic acid content.

We believe that the BAC 50 component was included in the product to reduce the formamide

and formic acid levels which to meet the specification requirements for Maersk.

The formamide and formic acid components which are in the product are a result of the

specific raw materials used in the triazine manufacturing process. We are working with our

existing supplier to evaluate an alternative triazine manufacturing method which does not

result in high levels of formamide and formic acid. Samples of the finished product from this

method have been sent to the DTI to determine the formamide and formic acid content.

We have also identified an alternative source of material from a second reputable supplier,

and based on earlier testing at DTI we believe that this will offer an alternative with

acceptable levels of formamide and formic acid. In the short term however, we do not believe

that this supplier can meet 100% of the volume needs for Maersk’s requirements, but may

form part of a future supply solution.

Once all of the results from the DTI are available we will be able compare with the Maersk

specifications and advise the best material going forward.

An investigation team has been brought together from different parts of our organisation and

they will review all of the circumstances which has led to the current situation. We are

committed to ensure any measures identified to prevent reoccurrence are implemented.



Page 1 of 2

CONFIDENTIAL



Our suppliers have been contacted to confirm that no additional components have been

added to any of the other products supplied to Maersk.

We are in the process of commercialising a replacement product called SCAVTREAT 15211

and we will work with Maersk to ensure all product qualification requirements are captured

and approved by Maersk prior to supply.

I trust the activities described above meet with your satisfaction and reiterate our commitment to

resolve this issue in a timely manner. We will be able to provide a regular updates on progress and

attend meetings as required by Maersk.

Yours sincerely,

John Jex

General Manager

Page 2 of 2

CLARIANT OIL SERVICES UK LTD.

Howe Moss Place, Kirkhill

Industrial Estate, Dyce

AB21 0GS Aberdeen

United Kingdom

CLARIANT OIL SERVICES UK LTD. - AB21 0GS ABERDEEN - UNITED

KINGDOM

Confidential

F.A.O.

Michelle Aspelin

Arbejdstilsynet

Produktregistret

Postboks 850

2100 København Ø

Danmark

LAURA GORDON

Head of Product Stewardship UK/IE

Phone +44 (0) 1224 797436

Fax

+44 (0) 7968 108553

[email protected]

20.04.2017

SCAVTREAT 7103

Dear Michelle,

Clariant Oil Services UK Ltd. and Clariant Oil Services Scandinavia AS

(hereinafter together “Clariant

Oil Services”) are committed to supporting its customers to ensure compliance with all aspects of

global Regulatory compliance, by ensuring that any products that are placed on the market are both

sustainable and in regulatory compliance.

However, it has recently came to the knowledge of Clariant Oil Services that SCAVTREAT 7103, as

supplied to Maersk, contains between 0.06 to 0.12% (typically < 0.1%) Quaternary ammonium

compounds, benzyl-C12-16-alkyldimethyl, chlorides, EC 939-350-2.

Regrettably, as we were not aware of this ingredient in our product, we have failed to meet our

obligations under the Harmonised Offshore Notification Format (HOCNF).

Clariant Oil Services has sanctioned the immediate removal of the Quaternary ammonium compound

from our product, and aim to fully co-operate with the Danish authorities to fully understand how

this situation came to be, and ensure that any remedial actions are promptly implemented.

In order to comply with the regulations governing offshore use, Clariant Oil Services has submitted a

sample of the Quaternary ammonium compound to a third party environmental testing facility,

Chemex. It is anticipated that the new environmental data will be available within 10 - 12 weeks and

upon receipt of the information, an amended HOCNF for SCAVTREAT 7103 will be submitted.

We are working with our customer to find a suitable product replacement.

In the meantime, if you have any queries or require anything further from us then please do not

hesitate to contact me in the first instance.

Yours faithfully,

Page 1 of 2

CONFIDENTIAL

Clariant Production UK Ltd.

On behalf of the selling Clariant group companies

Laura Gordon

Head of Product Stewardship, UK & Ireland

Page 2 of 2

APPENDIX B

Clariant Produkte (Deutschland) GmbH

CLAS R&D Analytics CIC

G860 Physics, Dr. Michael Schopferer

Phone.: 069 - 305 39450

Date: 19.07.2017

Page:1/4

REPORT

Sample:

Project:

Account:

Customer:

CLAS Order No.:

Empigen BAC 50

logP slow stirring /surf. tens. method

200318804 - COS - 4504702535/10

Phil McWilliams, Clariant Oil Services Scandinavia AS

17-011734

Summary

The logP was measured with the slow stirring / surface tension method

Apparent logP values at 1 g/L:

1,56

No corrected for micellisation effects is needed as the cmc is much higher (> 0,25 g/L) as the

concentrations in aqueous phase.

Details: see following sheets.

Electronic document valid without signature

Attachments:

Distribution:

[email protected]

Clariant Produkte (Deutschland) GmbH

CLAS R&D Analytics CIC

G860 Physics, Dr. Michael Schopferer

Phone.: 069 - 305 39450

Date: 19.07.2017

Page:2/4

LogP WITH SLOW STIRRING / SURFACE TENSION METHOD

Sample: Empigen BAC 50 (analog Dodigen 1611) WS = 50 %

Method

Equilibration

Aqueous solution of surfactant (1 g/L) was equilibrated with octanol under the following

conditions:

100 mL aq. soln + 100 mL (82g) 1-octanol in a 300 mL conical flask.

Stirring bar: 4cm long, 7.5 mm diameter

Stirring speed: 100 ± 7 rpm

Time: 8h stirring + stand overnight

Replicates: Experiment carried out in duplicate with the same batch of surfactant solution.

The experiment was performed at room temperature (approx 23 °C).

Determination of surfactant concentration

10 mL aqueous phase was removed and pipetted into a tensiometer glass. It was heated at 130°C for

30 min to remove water and octanol. The residue was redissolved in 50 mL 0.2 M KCl solution.

Then the surface tension was measured and the concentration determined by comparison with a

calibration curve. Two samples of each experiment were taken.

Surface tension measurements

The surface tension was measured with a Krüss K100 tensiometer using the Pt plate. Due to slow

equilibration at these low surfactant concentrations, the measurement was allowed to run for 15

min. The lowest surface tension achieved during the 15 minute period was used for the calculations.

Micellisation effects

This correction is only required if c

> cmc

oct

which is not the case:

< 0.03 g/L; cmc

oct

> 0.25 g/L

Results

The sample forms a clear solution in both demin. water and 0.2 M KCl at 1 g/L.

A satisfactory calibration curve was obtained (Fig. 1).

Clariant Produkte (Deutschland) GmbH

CLAS R&D Analytics CIC

G860 Physics, Dr. Michael Schopferer

Phone.: 069 - 305 39450

Date: 19.07.2017

Page:3/4

Dodigen 1611 in 0.2 mol KCl

in mN/m

0,001

0,01

0,1

concentration in g/L

Fig. 1 Calibration curve in 0.2 M KCl

After the evaporation/redissolution procedure, the surface tensions of the diluted aqueous phases

were in the steep part of the curve, which is a requirement for satisfactory evaluation. Numerical

data are shown in Table 1.

Table 1: results. From each equilibration expt, A and B, four samples of the aq. layer were analysed. The

concentration is calculated from the surface tension via the calibration curve. The concentration in the aq.

layer is obtained from the sample concentration by multiplying with the dilution factor.

initial concentration 0.5 g/L

sample

dil factor surf. tens.

mN/m

51,15

50,76

52,63

50,38

Mean

conc. g/L

sample

0,00547

0,00591

0,00399

0,00637

in aq layer

0,0274

0,0296

0,0200

0,0319

0,0272

Apparent logP

at 0.5 g/L

1,55

1,52

1,69

1,48

1,56

The critical micelle concentration in the presence of octanol was determined by diluting stock

solution containing 0.5 g/L Empigen BAC 50 and 0.5 g/L octanol. The result of this measurement

was used as a rough estimate of the cmc in octanol-saturated solution.

Clariant Produkte (Deutschland) GmbH

CLAS R&D Analytics CIC

G860 Physics, Dr. Michael Schopferer

Phone.: 069 - 305 39450

Date: 19.07.2017

Page:4/4

Empigen BAC 50 + Octanol cmc

Ofs (mN/m)

rough estimate of

cmc in the presence of

octanol: 0.25 g/L

1E-3

0,01

0,1

Konz (g/l)

Fig. 1 cmc in the presence of octanol. Concentration axis: surfactant only (i.e. excluding octanol)

The cmc in octanol-saturated solution, cmc

oct,

> 0.25 g/L.

Conclusions

Apparent logP values at 1 g/L:

no corrections for micellisation effects necessary

Empigen BAC 50

1.56

Commercial-in-Confidence

Chemex reference: ENV 11446/ECO 170408

The toxicity of Empigen BAC 50 to the

marine alga

Phaeodactylum

tricornutum

over a 72-hour exposure

period

Report for

Clariant Oil Services Scandinavia AS

Report issued by:

Chemex Environmental International Limited

Unit J

Broad Lane Industrial Estate

Cottenham

Cambridge CB24 8SW

Sponsor:

Clariant Oil Services Scandinavia AS

Post box 6054 BS

5892 Bergen

Norway

June 2017

ENV 11446 /ECO 170408

Page 1 of 20

Contents

Page

Compliance with Good Laboratory Practice standards....................................................................3

Quality Assurance Statement...........................................................................................................4

Summary..........................................................................................................................................5

1. Introduction..................................................................................................................................6

2. Materials and methods.................................................................................................................6

2.1 Test material...............................................................................................................................6

2.2 Test organism.............................................................................................................................7

2.3 Dilution water............................................................................................................................7

2.4 Preparation of test solutions.......................................................................................................7

2.5 Test methods and conditions......................................................................................................8

2.6 Study Plan Deviations..............................................................................................................11

3. Results........................................................................................................................................12

3.1 Cell density measurements......................................................................................................12

3.2 Test observations......................................................................................................................13

3.3 Percent inhibition.....................................................................................................................13

3.4 EC(r)

, EC(r)

values and NOEC(r).......................................................................................13

3.5 Test validity criteria.................................................................................................................14

4. Discussion..................................................................................................................................15

5. References..................................................................................................................................15

Graph 1 Growth curves for control and test concentrations..........................................................16

Graph 2 Determination of 0 to 72 hours EC(r)

values and NOEC(r)...........................................17

Graph 3 0 to 72 hours dose-response plot......................................................................................18

Appendix 1 – Test Conditions.......................................................................................................19

Appendix 2 Culture media.............................................................................................................20

ENV 11446 /ECO 170408

Page 2 of 20

Summary

This section summarises aquatic toxicity test results obtained by Chemex Environmental

International Limited on a sample as detailed below:

Test commissioned by:

Substance under test:

Chemex reference:

Test species:

Test type:

Registration guideline:

Clariant Oil Services Scandinavia AS

Empigen BAC 50

Sample: ECO170408

Study: ENV 11446

Phaeodactylum tricornutum,

strain CCAP 1052/1A.

Acute toxicity: 72-hour EC

50.

Static test conditions according to SOP E209 based

on ISO 10253:2006 “Water quality – Marine Algal

Growth Inhibition Test with

Skeletonema costatum

and

Phaeodactylum tricornutum”.

Range finding test: 11 to 15 May 2017

Definitive test 1: 23 to 26 May 2017

Definitive test 2: 30 May to 02 June 2017

0 (control), 0.27, 0.35, 0.46, 0.59, 0.77, 1.0mg/l

Chemex Environmental International Limited

Unit J, Broad Lane Industrial Estate

Cottenham

Cambridge

CB24 8SW

Experimental period:

Test concentrations:

Test performed at:

Results: All study validity criteria were met. The results are summarised in the table below.

Exposure period

(hours)

0 to 48

0 to 72

NOEC(r)

(0-72h)

EC(r)

and NOEC results (95% confidence limits)

EC(r)

0.305mg/l

(0.249 – 0.338mg/l)

0.384mg/l

(0.327 – 0.414mg/l)

EC(r)

0.420mg/l

(0.392 – 0.448mg/l)

0.485mg/l

(0.462 – 0.511mg/l)

0.27mg/l

(Determined by Bonferroni t Test (1-Tail, 0.05)

Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit

Following Shapiro-Wilk’s Test for normality of distribution and Bartlett’s Test which indicated equal variances

All concentrations of the test substance are reported as nominal as received.

ENV 11446 /ECO 170408

Page 5 of 20

1. Introduction

This report contains a description of the methods used and the results obtained during a study to

investigate the growth inhibition of Empigen BAC 50 to the marine algae

Phaeodactylum

tricornutum.

The objective of this study was to determine the 72-hour effective concentration of

Empigen BAC 50 and the NOEC based on specific growth rate relative to the controls.

Specifically, the results to be determined if possible are the 72-h EC(r)

, 72-h EC(r)

and the 72-

h NOEC(r) at 20 ± 2°C. The guideline followed was “Water quality - Marine algal growth

inhibition test with

Skeletonema costatum

and

Phaeodactylum tricornutum"

(ISO 10253:2006).

2. Materials and methods

Unless otherwise specified all methods mentioned in this report are according to Chemex

Environmental International Limited standard procedures.

All records of measurements and observations made during this test will be collated and held in

the Chemex Environmental International Limited archives at Unit J, Broad Lane Industrial

Estate, Cottenham, Cambridge CB24 8SW, UK.

2.1

Test material

Empigen BAC 50

120417SAM1

Multi-Constituent Substance

50% Active in water

Quaternary ammonium compounds, benzyl-C12-

14(even numbered)-alkyldimethyl, chlorides 30-

60% (MSDS)

ECO 170408

0.99g/cm

0.9878g/cm

Straw Coloured Liquid

Soluble in water

Homogeneous

Stable

Not known

Aquatic Half Life: Freshwater 365 days (MSDS)

Ambient. No Protection from light

Ambient 15 ± 10°C

Huntsman Holland BV

Merseyweg 10

3197 KG Botlek-Rotterdam

The Netherlands

Identification:

Name as supplied:

Batch number:

Type of Substance:

Activity:

Purity / Composition:

Chemex reference:

Density (specified):

Density (observed):

Appearance (specified):

Appearance (observed):

Solubility (specified):

Homogeneity (specified):

Homogeneity (observed):

Stability in container:

Stability in water:

Required storage conditions:

Actual storage conditions:

Source of supply:

Expiry date:

27 April 2018

All quoted information is taken from TSDS (Test Substance Data Sheet) unless otherwise stated.

ENV 11446 /ECO 170408

Page 6 of 20

2.2

Test organism

Phaeodactylum tricornutum

strain: CCAP 1052/1A

received 10 January 2017

Culture Collection of Algae and Protozoa

SAMS Research Services Ltd

Scottish Marine Institute

OBAN

Argyll PA37 1QA

Scotland, United Kingdom

Temperature: 20.9 – 21.6

Illumination: 6290 – 7270 lux continuous white

light.

Orbiting: set to 200 rpm.

Natural seawater with added nutrients

according to the ISO 10253 standard (see

Appendix 2).

Species:

Source:

Culture conditions:

Culture media:

2.3

Dilution water

The stocks of algal culture were maintained, and the tests performed, in nutrient growth medium

(ISO10253) prepared from natural seawater collected from the CEFAS laboratories at Lowestoft.

The seawater was collected via a pipeline directly from the estuary. The sand acts as the first stage

of filtration. After collection, the water was stored in the dark at approximately 15 ± 2°C within

the testing facility. It was then filtered and sterilised by autoclaving at 120°C for 15 minutes.

To make nutrient growth medium, appropriate amounts of nutrient stocks (Appendix 2) were added

to sterilised seawater at a pH of 8.14.

2.4

Preparation of test solutions

Information provided by the Sponsor indicated that the sample was soluble in water.

Stock solutions were prepared for both the range-finding and definitive tests by addition of the

test sample directly to nutrient growth media in a volumetric flask of adequate volume.

Calculated volumes of the stock solution were added to nutrient growth media and made to

volume to prepare the test concentrations and provide enough solution for testing and subsequent

water quality measurements.

Two definitive tests were performed, the first initial definitive test performed at 0.0032, 0.01,

0.032, 0.1, 0.32 and 1.0mg/l did not provide sufficient data to calculate an EC50 with 100%

inhibition at 1.0mg/l with no intermediate response seen from 0.32 to 0.0032mg/l. The test was

repeated at concentrations 0.27, 0.35, 0.46, 0.59, 0.77 and 1.0 mg/l and has been reported in full.

ENV 11446 /ECO 170408

Page 7 of 20

2.5

Test methods and conditions

Static test conditions according to "Water quality - Marine algal growth inhibition test with

Skeletonema costatum

Phaeodactylum tricornutum"

(ISO 10253:2006).

Chemex SOP reference:

E209 “Marine Algal Growth Inhibition test with

Skeletonema costatum

Phaeodactylum

tricornutum”

A preliminary (range-finding) test was conducted at

concentrations of 0 (Control), 1, 10, 100 and

1000mg/l. The duration of the preliminary study was

72 ± 2 hours. There was a single replicate at each

concentration.

Data from the preliminary test identified the 72

hour EC

as being under 1.0mg/l (by growth rate)

Nominal

concentration

(mg/l)

100

1000

Percent inhibition

by growth rate

0 - 72 hours

100

Preliminary test method:

Preliminary test results:

Note: Negative numbers indicate an increase in growth

compared to control data.

All concentrations of the test substance are reported as nominal

as received.

Definitive test 1 results:

Nominal

concentration

(mg/l)

0.0032

0.0100

0.0320

0.1000

0.3200

1.0000

Percent inhibition

by growth rate

0 - 72 hours

100

Note: Negative numbers indicate an increase in growth

compared to control data.

All concentrations of the test substance are reported as nominal

as received.

ENV 11446 /ECO 170408

Page 8 of 20

Definitive Test Method:

Test period:

Test duration:

Test volume:

Test vessel:

Number of replicates:

Test concentrations:

Composition of test medium:

30 May to 02 June 2017

72 ± 2 hours

200ml

250ml conical flask

Six control flasks, three replicates at each test

concentration.

0 (control), 0.27, 0.35, 0.46, 0.59, 0.77 and 1.0 mg/l

Nutrient medium prepared according to the ISO 10253

(see Appendix 2) in natural seawater (Section 2.3). The

final salinity of the medium used was 33‰.

From a pre-culture growing under conditions described

in 2.2 above. Inoculum level adjusted to give an initial

cell density of 1 x 10

cells/ml.

Initial pH of at 0 hours: 8.14

(Required: 8.0±0.2)

pH range in control and test concentrations throughout

test: 7.96 – 8.60

Temperature range within incubator throughout test:

20.1 – 21.6°C

(Required: 20±1°C)

Illumination range within incubator throughout test:

6240 - 7480 Lux

(Required: 6x10

-10x10

Lux)

Orbital shaking: 200rpm

(Required: 200-250rpm)

Water quality measurements:

The temperature (to 0.1

C) and light intensity (lux)

within the incubator was recorded at the beginning of

the study, after 24, 48 hours and at the end of the 72-

hour test period. The pH (to 0.01) and temperature (to

0.1

C) were recorded for each test and control solution

at the beginning of the test and on the pooled replicates

at the end of the 72-hour test period.

Cell densities were measured microscopically by direct

cell counts on each test and control replicate in

triplicate at 24, 48 and 72 hours (±2h). The cell counts

were made using a haemocytometer and microscope.

The Sponsor did not request analytical confirmation of

exposure concentrations. All effect concentrations have,

therefore, been calculated from nominal concentrations.

The average specific growth rate was calculated for

each test and control culture using equation 1:

(1)

= (lnN

– lnN

)/ (t

–

)

where:

Page 9 of 20

Algal test inoculum:

Test conditions:

Observations/frequency:

Analysis of test substance:

Calculation of results:

ENV 11446 /ECO 170408

time of test start

= time of test termination or the time of the last

measurement within the exponential growth period in

the control

= the nominal initial cell density

= the measured cell density at time

The percentage inhibition for each individual test flask

was calculated from equation 2:

(2)

µi

= ((µ

–

) * 100

where:

µi

= percentage inhibition (growth rate) for flask

= growth rate for flask I

= mean growth rate for the control

Growth curves for each test concentration were plotted

as the logarithm of the mean cell density against time.

Where possible, the EC(r)

and EC(r)

values with

95% confidence limits and NOEC(r) were calculated

according to the statistical methods of ToxCalc™

Version 5.0 “Comprehensive Toxicity Data Analysis

Reference substance:

A separate reference study (ENV11398) was run from

20 to 23 January 2017 using 3,5-Dichlorophenol as a

means of checking the test procedure and sensitivity of

the test species. The 72-hour EC(r)

was 2.6mg/l, which

was within the acceptable range (2.3 to 3.1mg/l) quoted

in ISO 10253:2006 guideline. In addition, at 2.7mg/l a

percentage inhibition, for growth rate at 72-hours, of

56% relative to the controls was seen. This was within

the range (20 to 80%) required for OSPAR compliance

(OSPAR Guidelines 2005).

ENV 11446 /ECO 170408

Page 10 of 20

2.6

Study Plan Deviations

1.) The study plan stated (Section 9.4 ) vessels are placed in a temperature controlled incubator

set at 20±1°C and temperature of incubation should not vary by more than 2°C. The temperatures

recorded during the study were within this 2°C variability. However the temperatures recorded at

test initiation and the maximum temperature recorded at the 24 hour time point were 21.3°C and

21.6°C (taken from manual temperature records of incubator) respectively. The temperature

returned to within 20±1°C for remainder of the study.

It was considered that this slight deviation outside of the permissible range stated in the study

plan would not have affected the integrity of the study. The growth parameters of the algae in the

control vessels were achieved and no abnormalities were recorded for cell observations.

2.) The study plan (Section 9.2) stated that the dilution water is stored at 15±2°C within the

facility until use when it would be filtered and sterilised. During storage and before use on the

study the temperature exceeded the range specified in the study plan on two occasions, the

maximum temperature recorded for each occasion respectively was 26.3°C and 17.1°C. On both

occasions the temperature exceeded the range for a period of 24 hours and then returned to

within acceptable limits.

The principle behind storing under cool conditions is to limit any biological activity from

organisms that may be present in the water should there be a need to use unsterilised water.

However in this instance whereby all water was sterilised prior to use any increase in biological

activity that may have been caused by the higher storage temperatures would have been negated

due to the sterilisation process. Therefore this deviation was considered not to have affected the

integrity of the study.

ENV 11446 /ECO 170408

Page 11 of 20

3. Results

Cell densities were measured microscopically by direct cell counts on each test and control

replicate in triplicate. Average results of the cell density determinations are given in 3.1. All

results in this study are calculated from the measured cell densities. All concentrations of the test

substance are reported as nominal as received.

3.1 Cell density measurements

Mean initial cell density: Approximately 1 x 10

cells/ml based upon inoculation volume, not

counted microscopically.

Nominal

Concentration

(mg/l)

Replicate

Cell density measurements

(cells/ml x 10

)

24 hours

48 hours

72 hours

6.0

2.0

7.0

2.7

1.3

5.3

4.1

2.0

1.3

1.7

2.3

1.7

1.9

1.0

2.0

1.7

1.3

2.0

1.0

1.4

0.3

1.3

0.3

0.6

0.0

17.7

14.3

24.3

18.0

16.7

20.3

18.6

10.3

14.3

16.0

13.5

7.0

10.7

11.0

9.6

2.7

4.0

3.0

3.2

0.7

0.3

0.7

0.6

0.3

0.0

0.2

0.0

86.3

78.0

106.7

115.0

71.3

90.7

91.3

52.0

59.3

68.3

59.9

34.7

41.0

36.3

37.3

19.7

23.3

22.3

21.8

0.0

2.7

1.0

1.2

0.0

0 (control)

Mean

0.27

0.35

0.46

0.59

0.77

1.0

Growth curves of logarithm of cell density against time are shown in Graph 1.

ENV 11446 /ECO 170408

Page 12 of 20

3.2

Test observations

The algal cells were examined microscopically during the determination of the cell density, all cells

within the control and all test concentrations where applicable appeared normal, no morphological

abnormalities were observed. Solution appearance in the control progressed from clear colourless

to very little brown colouration to brown by 72 hours. Test concentrations 0.27 to 0.46mg/l were

clear colourless at 0 and 24 hours, had very little brown colouration at 48 hours to a little brown

colouration at 72 hours. 0.59 to 1.0mg/l appeared clear colourless throughout.

3.3

Percent inhibition

Nominal Concentration

(mg/l)

0.27

0.35

0.46

0.59

0.77

1.0

Percent inhibition by specific growth rate

48 hours

100

72 hours

100

3.4 EC(r)

, EC(r)

values and NOEC(r)

Exposure

period

(hours)

0 to 48

0 to 72

NOEC(r)

(0-72h)

EC(r)

and NOEC results (95% confidence limits)

EC(r)

0.305mg/l

(0.249 – 0.338mg/l)

0.384mg/l

(0.327 – 0.414mg/l)

EC(r)

0.420mg/l

(0.392 – 0.448mg/l)

0.485mg/l

(0.462 – 0.511mg/l)

0.27mg/l

(Determined by Bonferroni t Test (1-Tail, 0.05)

Statistical methods used in ToxCalc v5.0: Maximum Likelihood-Logit

Following Shapiro-Wilk’s Test for normality of distribution and Bartlett’s Test which indicated equal variances

All concentrations of the test substance are reported as nominal as received.

ENV 11446 /ECO 170408

Page 13 of 20

3.5

Test validity criteria

The control cell density should increase by a factor of more than 16 in 72 hours, corresponding to

a specific growth rate of 0.9 d

-1

. The measured control cell density increase was recorded as 91.3,

corresponding to a specific growth rate of 1.505 d

-1

The pH of the control media should not increase by more than 1.0 during the test. The pH was

8.14 at the beginning of the test and 8.60 at the end of the test.

The coefficient of variation of the control specific growth rate should not exceed 7%. The table

below shows the calculation for variation coefficient of the control replicates for this study.

Control growth rate data coefficient of variation calculations.

Control

replicate

no.

Cell density (1.0 x 10

cells/ml)

0 hr

1.0

24 hr

6.0

2.0

7.0

2.7

1.3

5.3

48 hr

17.7

14.3

24.3

18.0

16.7

20.3

72 hr

86.3

78.0

106.7

115.0

71.3

90.7

Mean:

Standard deviation:

Coefficient of variation:

0-72 hr average

specific growth

rates

1.49

1.45

1.56

1.58

1.42

1.50

0.0616

4.11

Note: 0-hour cell density based upon inoculation volume and not counted microscopically.

ENV 11446 /ECO 170408

Page 14 of 20

4. Discussion

The definitive test conducted from 30 May to 02 June 2017 was performed according to the ISO

10253:2006 guideline.

The growth curves illustrated in Graph 1 demonstrate that the algae in the control were in

logarithmic growth for the duration of the study.

The 48-hour EC(r)

and 72-hour EC(r)

of Empigen BAC 50 to

Phaeodactylum tricornutum

were 0.420mg/l (determined by Maximum Likelihood-Logit) and 0.485mg/l (determined by

Maximum Likelihood-Logit) respectively. Graphical representations of the 0 to 72 hours EC(r)

values and NOEC(r) and 0-72 hour dose-response plot are given in Graphs 2 and 3, respectively.

The 0 to 72-hour NOEC(r) and LOEC(r) were 0.27mg/l and 0.35mg/l respectively (determined

by Bonferroni t Test (1-Tail, 0.05).

The algal cells were examined microscopically during the determination of the cell density, all cells

within the control and all test concentrations where applicable appeared normal, no morphological

abnormalities were observed. Solution appearance in the control progressed from clear colourless

to very little brown colouration to brown by 72 hours. Test concentrations 0.27 to 0.46mg/l were

clear colourless at 0 and 24 hours, had very little brown colouration at 48 hours to a little brown

colouration at 72 hours. 0.59 to 1.0mg/l appeared clear colourless throughout.(see section 3.2).

All validity criteria for the definitive test were met (see section 3.5).

No analytical confirmation of the test concentrations was performed. The dissolved

concentrations were likely to be as stated in this report as Empigen BAC 50 was soluble in water.

The water quality measurements and incubation conditions are given in the summary tables in

Appendix 1 and were within accepted limits.

5. References

ISO 10253:2006 “Water quality – Marine Algal Growth Inhibition Test with

Skeletonema

costatum

and

Phaeodactylum tricornutum”.

OSPAR – Guidelines for Toxicity Testing of Substances and Preparations Used and

Discharged Offshore (Reference number: 2005-12)

ToxCalc™ Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”,

ENV 11446 /ECO 170408

Page 15 of 20

Graph 1 Growth curves for control and test concentrations

ENV 11446 /ECO 170408

Page 16 of 20

Graph 2

Determination of 0 to 72 hours EC(r)

values and NOEC(r)

–----

Indicates 95% confidence limits

Indicates actual data plot

Statistical data

EC(r)

value

EC(r)

value

0.384mg/l (0.327 – 0.414mg/l)

0.485mg/l (0.462 – 0.511mg/l)

Determined by Maximum Likelihood-Logit (P = 0.58), using ToxCalc v5.0.

NOEC(r)

LOEC(r)

0.27mg/l

0.35mg/l

Determined by Bonferroni t Test (1-Tail, 0.05), following tests for normality of distribution and

equal variances, using ToxCalc v5.0.

ENV 11446 /ECO 170408

Page 17 of 20

Graph 3

0 to 72 hours dose-response plot

* = location of statistically significant difference (1-tail, P=0.05)

ENV 11446 /ECO 170408

Page 18 of 20

Appendix 1 – Test Conditions

pH values and temperatures of the test solutions:

0 hours

8.14

7.96

8.04

8.08

8.10

8.13

8.12

Temp

(°C)

21.9

21.7

21.6

21.7

21.8

8.60

8.40

8.33

8.21

8.05

8.03

8.01

72 hours

Temp

(°C)

20.2

20.5

20.1

20.3

19.9

20.0

20.5

Concentration

(mg/l)

0 (Control)

0.27

0.35

0.46

0.59

0.77

1.0

Note: Water quality was determined on pooled replicates for the test and control solution at the

end of the 72-hour test period and on excess solution at the start of the study.

Temperature and light intensity measurements recorded in test incubator:

Within

incubator

Current

Minimum

Maximum

Temperature (°C)

0 hours

21.3

24 hours

20.6

20.3

21.6

48 hours

20.8

20.3

21.0

72 hours

20.8

20.1

21.0

Within

incubator

Minimum

Maximum

Light intensity (lux)

0 hours

6370

7270

24 hours

6300

7480

48 hours

6240

7320

72 hours

6330

7350

ENV 11446 /ECO 170408

Page 19 of 20

Appendix 2

Culture media

Culture medium:

Seawater:

Method of preparation:

Prepared in natural seawater according to the ISO 10253

standard.

Filtered, and sterilised by autoclaving at 120°C for 15

minutes.

Sterile nutrient stock solutions were prepared and added to

the seawater to obtain the culture medium. The pH was 8.14

at test initiation.

Nutrient

FeCl

MnCl

ZnSO

.7H

CuSO

.5H

CoCl

.6H

EDTA.2H

Thiamin hydrochloride

Biotin

(cyanocobalamin)

NaNO

SiO

.9H

Final concentration in test

solution ± 5%

149µg/l (Fe)

605µg/l (Mn)

150µg/l (Zn)

0.6µg/l(Cu)

1.5µg/l (Co)

3.0mg/l (B)

15mg/l (as Na

EDTA)

25µg/l

0.005µg/l

0.05µg/l

0.438mg/l (P)

8.24mg/l (N)

1.97mg/l (Si)

Composition of culture medium:

ENV 11446 /ECO 170408

Page 20 of 20

Commercial-in-Confidence

Chemex reference: ENV 11447 /ECO 170408

The acute toxicity of Empigen BAC 50

Acartia tonsa

over a 48-hour exposure

period

Report for

Clariant Oil Services Scandinavia AS

Report issued by:

Chemex Environmental International Limited

Unit J

Broad Lane Industrial Estate

Cottenham

Cambridge CB24 8SW

Sponsor:

Clariant Oil Services Scandinavia AS

Post box 6054 BS

5892 Bergen

Norway

July 2017

ENV 11447/ECO 170408

Page 1 of 14

Contents

Page

Compliance with Good Laboratory Practice Standards...................................................................3

Quality Assurance Statement............................................................................................................4

Summary..........................................................................................................................................5

1. Introduction.................................................................................................................................6

2. Materials and methods.................................................................................................................6

2.1 Test material............................................................................................................................6

2.2 Test organism..........................................................................................................................7

2.3 Dilution water.........................................................................................................................7

2.4 Preparation of test solutions...................................................................................................7

2.5 Test methods and conditions...................................................................................................8

2.6 Study Plan Deviation..............................................................................................................9

3. Results........................................................................................................................................10

3.1 Cumulative mortalities..........................................................................................................10

3.2 LC

10,

and NOEC values................................................................................................10

4. Discussion..................................................................................................................................11

5. References..................................................................................................................................11

Graph 1 Determination of 48-hour LC

values..............................................................................12

Graph 2 Dose-response plot for 48-hours exposure......................................................................13

Appendix 1 Water qualities and incubation conditions...................................................................14

ENV 11447/ECO 170408

Page 2 of 14

Summary

This section summarises aquatic toxicity test results obtained by Chemex Environmental

International Limited on a sample as detailed below:

Test commissioned by:

Substance under test:

Chemex reference:

Test species:

Test type:

Registration guideline:

Experimental period:

Clariant Oil Services Scandinavia AS

Empigen BAC 50

Sample: ECO 170408

Study: ENV 11447

Acartia tonsa

Acute toxicity: 48-hour EC

ISO 14669:1999 “Water quality - Determination of acute

lethal toxicity to marine copepods (Copepoda, Crustacea)”

Range finding test 1: 16 to 18 May 2017

Range finding test 2: 17 to 19 May 2017

Definitive test 1: 23 to 25 May 2017

Definitive test 2: 15 to 17 June 2017

0 (control), 0.63, 1.25, 2.5, 5 and 10 mg/l

Chemex Environmental International Limited

Unit J, Broad Lane Industrial Estate

Cottenham

Cambridge

CB24 8SW

Test concentrations:

Test performed at:

Results: All study validity criteria were met. The results are summarised in the table below.

Exposure

Period

(hours)

NOEC

(48 hours)

and NOEC values mg/l

(95% confidence limits)

1.09mg/l

(0.4473 – 1.519mg/l)

2.775mg/l

(2.371 – 3.247mg/l)

1.989mg/l

(1.373 – 2.520mg/l)

1.25 mg/l determined by Dunnett’s Test (1-tail , 0.05)

Statistical methods used in ToxCalc v5.0: Trimmed Spearman Karber 0% Trim (24 hours), Maximum

Likelihood Probit (48 hours).

* Not possible to determine with Trimmed Spearman Karber

Following Shapiro-Wilk’s Test indicating normal distribution (equality of variance could not be determined)

All concentrations of the test substance are reported as nominal as received.

ENV 11447/ECO 170408

Page 5 of 14

1. Introduction

This report contains a description of the methods used and the results obtained during a study to

investigate the acute toxicity of Empigen BAC 50 to

Acartia tonsa.

The objective of this study

was to determine the 48-hour lethal concentrations of Empigen BAC 50 specifically, the 24 and

48-hour LC

and the NOEC according to ISO 14669:1999.

2. Materials and methods

Unless otherwise specified, all methods mentioned in this report are according to Chemex

Environmental International Limited standard procedures.

All records of measurements and observations made during this test will be collated and held in

the Chemex Environmental International Limited archives at Unit J, Broad Lane Industrial Estate,

Cottenham, Cambridge CB24 8SW, UK.

2.1

Test material

Empigen BAC 50

120417SAM1

Multi-Constituent Substance

50% Active in water

Quaternary ammonium compounds, benzyl-C12-14(even

numbered)-alkyldimethyl, chlorides 30-60%

(MSDS)

ECO 170408

0.99g/cm

(SDS)

0.9878g/cm

Straw Coloured Liquid

Soluble in water

Homogeneous

Stable

Not Known

Aquatic Half Life: Freshwater 365 days (MSDS)

Ambient. No Protection from light

Ambient 15 ± 10°C

Huntsman Holland BV

Merseyweg 10

3197 KG Botlek-Rotterdam

The Netherlands

27 April 2018

Page 6 of 14

All quoted information is taken from TSDS (Test Substance Data Sheet) unless otherwise stated.

Identification:

Name as supplied:

Batch number:

Type of Substance:

Activity:

Purity / Composition:

Chemex reference:

Density (specified):

Density (observed):

Appearance (specified):

Appearance (observed):

Solubility (specified):

Homogeneity (specified):

Homogeneity (observed):

Stability in container:

Stability in water:

Required storage conditions:

Actual storage conditions:

Source of supply:

Expiry date:

ENV 11447/ECO 170408

2.2

Test organism

Acartia tonsa

Guernsey Sea Farms

15 June 2017

12-14 days old

Adult

Species:

Source:

Date

Acartia

delivered to laboratory:

Age of copepods at start of test:

Life stage:

2.3

Dilution water

The test organisms were maintained, and tests performed in natural seawater collected from CEFAS

Laboratories, Lowestoft. The seawater was collected via pipeline directly from the estuary. The

sand acting as the first stage of filtration. After collection, the water was stored in the dark at

approximately 15 ± 2°C within the testing facility. Prior to use it was filtered through a Whatman 54

filter and sterilised by autoclaving at 120°C for 15 minutes.

The pH of dilution water was 8.17.

2.4

Preparation of test solutions

Information provided by the Sponsor indicated that the sample was soluble in water.

Both the preliminary range-finding test and the definitive test were prepared by direct addition of

weighed amounts of test substance to dilution water made to volume.

For the preliminary (range-finding) tests initially a 1000mg/l stock (±5% by weight) of test

substance was prepared. 0.5000g was dissolved in 500ml of dilution water. Preparation of the

remaining concentrations were by dilution of this 1000mg/l test concentration. 0.25, 2.5 and 25ml

were made to 250ml volume with dilution water to prepare 1, 10 and 100mg/l respectively.

The second preliminary (range-finding) was performed between 0.001 and 1 mg/l. A 20mg/l stock

solution was prepared by weighing 0.0201g of test substance and making to 1000ml volume.

1mg/l was prepared by the addition of 12.5 ml of this stock solution and made to 250 ml volume

with dilution water. The remaining test concentrations were prepared by serial dilution.

Two definitive tests were performed, the first failed the control validity criteria with 20%

mortality recorded after 48 hours. The second successful definitive test has been reported in full.

The definitive test concentrations were prepared by weighing 0.0200g (±5% by weight) and

dissolving in 2000ml of dilution water to prepare a 10mg/l stock solution. This stock solution was

used to prepare the nominal test concentrations. 15.75, 31.25, 62.5 and 125 ml of the 10 mg/l

stock were diluted individually to 250ml with dilution water to prepare 0.63, 1.25, 2.5 and 5 mg/l

respectively. Enough solution was prepared for testing and subsequent water quality

measurements.

ENV 11447/ECO 170408

Page 7 of 14

2.5

Test methods and conditions

Static test conditions according “Water quality - Determination of acute lethal toxicity to marine

copepods (Copepoda, Crustacea)” ISO 14669:1999.

Chemex SOP reference:

Preliminary test method:

E207 “Testing of the marine copepod,

Acartia

tonsa”

Preliminary (range-finding) tests were conducted

at concentrations of 0 (Control), 1, 10, 100 and

1000mg/l and 0.001, 0.01, 0.1 and 1mg/l. The

duration of the preliminary tests were 48 ± 1

hour. There was a single replicate at each

concentration with five animals per replicate.

Data from the preliminary tests identified the 48-

hour LC

as likely being between 1 and 10mg/l.

1.) Control (0%), 1mg/l (20%), 10mg/l (100%),

100mg/l (100%) and 1000mg/l (100%).

2.) Control (0%), 0.001mg/l (0%), 0.01mg/l

(40%), 0.1mg/l (40%) and 1.0mg/l (40%).

Definitive test results:

The first definitive test was repeated as the

control validity criteria were not achieved, the

mortality results of this test were as follows:

Control (20%), 0.63mg/l (30%), 1.25mg/l (50%),

2.5mg/l (80%) 5mg/l (100%) and 10mg/l (100%).

Definitive Test Method:

Test period:

Test duration:

Test volume:

Test vessel:

Number of replicates:

Test concentrations:

Test Media:

15 to 17 June 2017

48 ± 1 hours

25ml

40ml glass dishes.

Four replicates at each concentration.

0 (control), 0.63, 1.25, 2.5, 5 and 10mg/l

Natural Seawater

(Batch Number: ECO170308).

Salinity at start of exposure: 34ppt

Five

Acartia

(Batch No: A22b) were transferred

to each control and test concentration vessel.

Static

Initial pH at 0 Hours: 7.97

(Required: 8±0.3)

pH range in control and test concentrations

throughout test: 7.95 – 8.15

Initial Dissolved Oxygen at 0 Hours: 98.6% ASV

(equivalent to approx. 7.13mg/l)

DO range in control and test concentrations

Page 8 of 14

Preliminary test results:

Test organism:

Replacement regime:

Test conditions:

ENV 11447/ECO 170408

throughout test: 7.01 – 7.59mg/l

(Required: ≥4mg/l)

Temperature range within incubator throughout

test: 18.9 – 19.7°C

(Required: 20±2°C)

Illumination: 16 Hours Light, 8 Hours Dark

Water quality measurements:

The temperature (to 0.1°C) within the incubator

was recorded at the beginning of the study, after

24 hours and at the end of the 48-hour test

period. The pH (to 0.01), temperature (to 0.1°C),

dissolved oxygen (%ASV 0.1 and mg/l to 0.01)

and salinity (to 1‰) were recorded for each test

and control solution at the start of the test and on

the pooled replicates at the end of the 48-hour

test period.

The number of dead and alive

Acartia

was

recorded after 24 and 48-hour (±1h) exposure

periods.

The Sponsor did not request analytical

confirmation of exposure concentrations. All

effect concentrations have, therefore, been

calculated from nominal loading rates.

Where possible the LC

values are shown

graphically and calculated with 95% confidence

limits following Abbott's correction for

background response if required.

LOEC and NOEC are also calculated, following

tests to determine normality of distribution and

equality of variance, using ToxCalc™ Version 5.0

“Comprehensive Toxicity Data Analysis and

Reference Substance:

A separate reference study (ENV11397) was

conducted from 17 to19 January 2017 using 3,5

Dichlorophenol as a means of checking the test

procedure. The 48-hour LC

was 0.72mg/l which

is within the acceptable range (0.5 - 1.5mg/l)

quoted in ISO 14669:1999 guideline.

Observations/frequency:

Analysis of test substance:

Calculation of results:

2.6

Study Plan Deviation

1.) The Study Plan stated that the seawater is stored in the dark at approximately 15±2°C within

the testing facility. The seawater batch used in the study deviated from the required temperature

on one occasion during storage. The temperature recorded was 26.3°C and returned to within the

required range the following day and for the remainder of the time it was stored prior to it being

sterilised.

This deviation in temperature was for a short period of time and its effects were considered to be

minimal. Any effects the high temperature may have had on biological activity in the water would

have been reduced once sterilised prior to use therefore this deviation has not impacted the

integrity of the study.

ENV 11447/ECO 170408

Page 9 of 14

3. Results

3.1

Cumulative mortalities

Nominal concentration

(mg/l)

Control

0.63

1.25

2.5

Number dead

24 hours

48 hours

% mortality

24 hours

100

48 hours

100

Graph 1 illustrates the estimation of the 48-h LC

values and Graph 2 shows the dose-response

curve at 48-hours.

Test solutions appeared clear and colourless throughout the study.

3.2

10,

and NOEC values

Exposure

Period

(hours)

NOEC

(48 hours)

and NOEC values mg/l

(95% confidence limits)

1.09mg/l

(0.4473 – 1.519mg/l)

2.775mg/l

(2.371 – 3.247mg/l)

1.989mg/l

(1.373 – 2.520mg/l)

1.25 mg/l determined by Dunnett’s Test (1-tail , 0.05)

Statistical methods used in ToxCalc v5.0: Trimmed Spearman Karber 0% Trim (24 hours), Maximum

Likelihood Probit (48 hours).

* Not possible to determine with Trimmed Spearman Karber

Following Shapiro-Wilk’s Test indicating normal distribution (equality of variance could not be determined)

All concentrations of the test substance are reported as nominal as received.

ENV 11447/ECO 170408

Page 10 of 14

4. Discussion

The definitive test conducted from 15 to 17 June 2017 was performed according to the ISO

14669:1999 guideline and met all validity criteria.

The 24-hour and 48-hour LC

of Empigen BAC 50 to

Acartia tonsa

were determined to be

2.77mg/l and 1.989mg/l respectively (determined by Trimmed Spearman Karber and Maximum

Likelihood Probit). Graphical representations of the 48-h LC

values and the 48-hour dose-

response curve are given in Graphs 1 and 2.

The 48-hour NOEC and LOEC were 1.25mg/l and 2.5mg/l respectively (Determined by

Dunnett’s, 1-tail, P=0.05).

The highest concentration tested (10mg/l) resulted in 100% mortality. No mortalities were

observed at the lowest concentration tested (0.63mg/l).

Test solutions appeared clear and colourless throughout the study.

Two (10%) of the twenty control copepods died during the study. This indicates a satisfactory

level of "health" of organisms maintained under test conditions according to the test guideline

allowing for 10% mortality within control vessels.

The dissolved oxygen concentration remained ≥7.01mg/l throughout the test. The toxicity of the

reference chemical was within the range specified in the guideline (see point 2.5).

No analytical confirmation of the test concentrations was performed. The dissolved

concentrations were likely to be similar to those stated in this report as Empigen BAC 50 was

soluble in water.

The water quality measurements of the test solutions and incubation conditions are given in

Appendix 1.

5. References

(1)

(2)

(3)

ISO 14669:1999 “Water quality - Determination of acute lethal toxicity to marine

copepods (Copepoda, Crustacea)”.

OSPAR Guidelines for Toxicity Testing of Substances and Preparations Used and

Discharged Offshore (Ref 2005-12).

ToxCalc

Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”,

ENV 11447/ECO 170408

Page 11 of 14

Graph 1

Determination of 48-hour LC

values

----------

Indicates 95% Confidence Limits

Indicates actual data plot

Number of

Acartia tonsa

per test concentration = 20

Statistical data

value

(1)

value

(1)

value

(1)

1.090mg/l (0.4473 – 1.519mg/l)

1.989mg/l (1.373 – 2.520mg/l)

3.631mg/l (2.830 – 6.224mg/l)

Determined by Maximum Likelihood Probit using ToxCalc v5.0.

NOEC

1.25mg/l

LOEC

2.5mg/l

Determined by Dunnett’s Test, 1-tail, P=0.05, following tests for normality (equality of variance

could not be determined), using ToxCalc v5.0.

ENV 11447/ECO 170408

Page 12 of 14

Graph 2

Dose-response plot for 48-hours exposure

ENV 11447/ECO 170408

Page 13 of 14

Appendix

Water qualities and incubation conditions

Concentration

(mg/l)

Control

0.63

1.25

2.5

0 hours

Temp

(°C)

%ASV

48 hours

Salinity

‰

Temp

(°C)

(mg/l)

Salinity

‰

7.97

8.08

8.13

8.11

8.14

8.15

21.6

98.6

7.13 mg/l

97.1

7.01 mg/l

99.0

7.16 mg/l

98.9

7.15 mg/l

98.1

7.09 mg/l

99.5

7.19 mg/l

7.95

8.04

8.05

8.08

8.11

8.09

20.9

21.1

21.3

21.4

21.6

21.2

7.14

7.48

7.36

7.52

7.59

7.39

Note: Water quality was determined on pooled replicates for the test and control solution at the

end of the 48-hour test period and on excess solution at the start of the study.

Summary of temperature values (°C) within incubator

Within

incubator

Current

Minimum

Maximum

Temperature (°C)

0 hours

19.5

24 hours

19.0

19.7

48 hours

19.0

18.9

19.6

ENV 11447/ECO 170408

Page 14 of 14

Commercial-in-Confidence

Chemex reference: ENV 11450/ECO

170408

The toxicity of Empigen BAC 50 to

Corophium

sp over a 10-day exposure

period.

Report for

Clariant Oil Services Scandinavia AS

Report issued by:

Chemex Environmental International Limited

Unit J

Broad Lane Industrial Estate

Cottenham

Cambridge,

CB24 8SW

Sponsor:

Clariant Oil Services Scandinavia AS

Post box 6054 BS

5892 Bergen

Norway

June 2017

ENV 11450/ECO 170408

Page 1 of 10

Contents

Page

Compliance

with Good Laboratory Practice standards.................................................................3

Quality Assurance Statement.........................................................................................................4

Summary.......................................................................................................................................5

1. Introduction...............................................................................................................................6

2. Materials and Methods..............................................................................................................6

2.1 Test substance......................................................................................................................6

2.2 Test organism......................................................................................................................7

2.3 Sediment.............................................................................................................................7

2.4 Dilution water.....................................................................................................................7

2.5 Test procedure.....................................................................................................................7

3. Results.......................................................................................................................................9

3.1 Mortality after 10-days exposure........................................................................................9

3.2 LC

50,

NOEC and LOEC values after 10-days exposure......................................................9

3.3 Water quality data................................................................................................................9

4. Discussion.................................................................................................................................10

5. References.................................................................................................................................10

ENV 11450/ECO 170408

Page 2 of 10

Summary

This section summarises aquatic toxicity test results obtained by Chemex Environmental

International Limited on a sample as detailed below:

Test commissioned by:

Substance under test:

Chemex reference:

Test species:

Test type:

Registration guideline:

Clariant Oil Services Scandinavia AS

Empigen BAC 50

Sample: ECO 170408

Study: ENV 11450

Corophium

volutator

Toxicity: 10-day LC

Static test conditions according to SOP E211

based on OSPAR / PARCOM Protocols on

Methods for the Testing of Chemicals Used in the

Offshore Industry 2006 Part A: A sediment

bioassay using an amphipod

Corophium

sp.

Definitive test: 10 to 22 May 2017

Temperature:

Dissolved Oxygen:

pH value

Salinity:

Photoperiod:

14.1 – 14.7°C

>85.3% ASV

8.16 – 8.36

35-36ppt

16 hours light, 8 hours dark.

Experimental period:

Test conditions range:

Test performed at:

Chemex Environmental International Limited

Unit J, Broad Lane Industrial Estate

Cottenham

Cambridge CB24 8SW

All study validity criteria were met. The results are summarised below.

Result:

10-day LC

50(1)

95% C.L.

NOEC

(2)

LOEC

(2)

306.5 mg/kg dry sediment

232.9 – 403.4 mg/kg dry sediment

125mg/kg dry sediment

401mg/kg dry sediment

(1)

(2)

calculated by Trimmed Spearman-Karber (Trim Level 5.3%) using ToxCalc v5.0.

calculated by Fisher's Exact Test using ToxCalc v5.0.

ENV 11450/ECO 170408

Page 5 of 10

1. Introduction

This report contains a description of the methods used and the results obtained during a study to

investigate the toxicity of Empigen BAC 50 to

Corophium

volutator.

The objective of this study

was to determine the 10-day LC

, which is defined as the concentration that kills 50% of the test

animals after a 10-day exposure period according to OSPAR/PARCOM 2006 Protocol

(1)

2. Materials and Methods

Unless otherwise specified, all methods mentioned in this report have been carried out according

to Chemex Environmental International Limited standard operating procedures.

All records of measurements and observations made during this test will be collated and held in

the Chemex Environmental International Limited archives at Unit J, Broad Lane Industrial

Estate, Cottenham, Cambridge CB24 8SW, UK.

2.1

Test substance

Empigen BAC 50

120417SAM1

Multi-Constituent Substance

50% active in water

Quaternary ammonium compounds, benzyl-C12-

14(even numbered)-alkyldimethyl, chlorides 30-60%

(MSDS)

ECO 170408

0.99g/cm

0.9878g/cm

Straw Coloured Liquid

Soluble in water

Homogeneous

Identification:

Name as supplied:

Batch number:

Type of Substance:

Activity:

Purity / Composition:

Chemex reference:

Density (specified):

Density (observed):

Appearance (specified):

Appearance (observed):

Solubility (specified):

Homogeneity (specified):

Homogeneity (observed):

ENV 11450/ECO 170408

Page 6 of 10

Stability in container

Stability in water:

Stable

Not

Known

Aquatic Half Life: Freshwater 365 days (MSDS)

Ambient. No Protection from light

Ambient 15 ± 10°C

Huntsman Holland BV

Merseyweg 10

3197 KG Botlek-Rotterdam

The Netherlands

27 April 2018

Required storage conditions:

Actual storage conditions:

Source of supply:

Expiry date:

All quoted information is taken from TSDS (Test Substance Data Sheet) provided by the sponsor

unless otherwise stated.

2.2

Test organism

Corophium

volutator

Todd Fish Tech Ltd. Dalgety Bay, Fife, Scotland

19/04/17

Temperature: 13.9 – 14.4°C

Dissolved oxygen: 92.4 – 98.9% ASV

pH: 7.87 – 8.07

Salinity: 32 - 34‰

Test species:

Source:

Date of receipt:

Holding conditions:

The stock of

Corophium volutator

used in this study was received from Todd Fish Tech Ltd of

Dalgety Bay, Fife, Scotland. The animals were sieved from the sediment and transported in

natural seawater. The animals were then maintained in the laboratory under static conditions in

reconstituted seawater, in the presence of a small amount of detrital material, until the start of the

test. During holding they were fed with homogenized Aqua Care Tropical

fish food. They were

not fed during the test.

The lengths of a sample of the control animals were measured at the end of the test and the mean

was 11.6mm with a range of 10 - 13mm.

2.3

Sediment

Sediment from Snettisham Beach, Norfolk collected on 21 March 2017 was used for the test.

This site is known to be populated by

Corophium,

and to the best of our knowledge free from

significant contamination. The aerobic layer (top 5 to 10cm) of sediment was collected, sieved to

500µm, washed, settled and stored refrigerated (4± 2°C) in the dark until the start of the test.

The sediment was thoroughly homogenised and a small sample dried at 97°C for >24 hours to

determine the dry matter weight. From this it was determined that the water content of the

sediment was 23.93%.

ENV 11450/ECO 170408

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2.4

Dilution water

The stock of test organisms was maintained, and the tests performed, in artificial seawater. The

seawater was prepared using dechlorinated mains water with artificial sea salt (Tropic Marin

) to

give a salinity of approximately 34‰. The dilution water was stored in a constant temperature

room set at 15 ± 2°C and at low light level within the test facility.

2.5

Test procedure

The toxicity test was carried out according to Chemex SOP E211 and the procedures given

below, based on the guidelines produced by the OSPAR / PARCOM Protocols on Methods for the

Testing of Chemicals Used in the Offshore Industry 2006 Part A: A sediment bioassay using an

amphipod

Corophium

sp.

Information supplied from the Sponsor indicated the test substance was soluble in water.

The test concentrations were therefore prepared by weighing out appropriate quantities of test

substance and washing into a 2-litre conical flask containing 500g wet sediment with 300ml of

artificial seawater.

The following test concentrations were used: 0 (control), 95, 305, 977, 3125 and 10000mg/kg

expressed as the amount of test substance as received per kg wet sediment.

The 2-litre conical flasks containing the test substance/sediment/seawater mixtures were then

placed on an orbital shaker and the slurries were mixed at approximately 150rpm for 3 hours.

The sediment/seawater slurry was then divided between two replicate 2-litre beakers. These were

the test vessels. The beakers were left overnight to allow the sediment to settle before carefully

adding seawater to bring the total volume up to the 1400ml graduation mark. A plastic disc was

placed above the sediment to avoid disturbance of sediment during this process. This resulted in

a total depth of approximately 133 - 136mm and sediment layer depth of 23 - 28mm in each

beaker.

After a further period of settling, gentle aeration was applied to each beaker until the dissolved

oxygen content reached a minimum of 80% air saturation value (ASV). The pH value (to 0.01),

dissolved oxygen (to 0.1% ASV), temperature (to 0.1°C) and salinity (to 1‰) were measured in

each test beaker immediately prior to initiating the test, and then at intervals during the test

period.

Individual

Corophium

of >5mm length were selected from the stock using a wide bore Pasteur

pipette. Batches of 10 animals were transferred to plastic pots containing 50ml seawater. The

exposure period was started on addition of 10

Corophium

to each test vessel.

Records were made of the numbers of animals observed alive and dead on the surface of the

sediment as well as any showing abnormal behaviour on days 3, 5 and 10. At the end of the 10-

day exposure period, water quality measurements were made, and each of the test and control

sediments were sieved to determine the number of animals still alive. As dead animals may

decompose or be consumed, any missing animals were counted as dead.

Where possible the 10-day LC

was determined, with 95% confidence limits, together with the

highest no observed effect concentration (NOEC) and the lowest observed effect concentration

(LOEC) for mortality, using ToxCalc version 5.0 “Comprehensive Toxicity Data Analysis and

Database Software”. The Abbotts correction for control response was applied where necessary.

ENV 11450/ECO 170408

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The 10-day LC

, NOEC and LOEC were determined, where possible, using test concentrations

expressed as mg/kg dry sediment.

3. Results

3.1

Mortality after 10-days exposure

Total

number

dead

Nominal concentration

Number alive

(1)

(mg/kg)

Wet

Dry

Replicate 1

Replicate 2

sediment

0 (control)

125

401

305

1285

977

4107

3125

13141

10000

(1)

Number exposed in each replicate = 10.

Percent

mortality

100

Abnormal behaviour among the

Corophium

was recorded on day 5 and 10 at 125mg/kg dry

sediment, no abnormal behaviour was recorded for the other other test concentrations for the

duration of the study.

3.2

50,

NOEC and LOEC values after 10-days exposure

95% confidence limits

(mg/kg dry sediment)

232.9 – 403.4

NOEC value

(mg/kg dry

sediment)

125

(2)

LOEC value

(mg/kg dry

sediment)

401

(2)

10-day LC

value

(mg/kg dry sediment)

306.5

(1)

calculated by Trimmed Spearman-Karber (Trim 5.3%) using ToxCalc v5.0.

(2)

calculated by Fisher's Exact Test using ToxCalc v5.0.

3.3

Water quality data

Range of values

Dissolved

Temperature

oxygen

(°C)

(% ASV)

85.4 – 92.6

14.1 – 14.7

85.3 – 95.1

14.1 – 14.5

86.0 – 96.4

14.2 – 14.6

86.3 – 96.5

14.2 – 14.6

86.2 – 97.8

14.1 – 14.6

85.9 – 98.3

14.2 – 14.7

Nominal concentration

(mg/kg)

Wet

Dry

sediment

0 (control)

305

977

3125

10000

0 (control)

125

401

1285

4107

13141

8.16 – 8.28

8.19 – 8.30

8.20 – 8.31

8.20 – 8.34

8.20 – 8.36

8.20 – 8.33

Salinity

(‰)

35 -36

ENV 11450/ECO 170408

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4. Discussion

The 10-day LC

of Empigen BAC 50 to

Corophium

volutator

was determined to be 306.5mg/kg

dry sediment (232.9 – 403.4 mg/kg dry sediment 95% C.L).

The highest no-observed effect concentration (NOEC) after 10-days exposure was 125mg/kg dry

sediment and the lowest observed effect concentration (LOEC) was 401mg/kg dry sediment.

Abnormal behaviour among the

Corophium

was recorded on day 5 and 10 at 125mg/kg dry

sediment, no abnormal behaviour was recorded for the other other test concentrations for the

duration of the study.

The water quality measurements of the test solutions are given in 3.3 and were within the ranges

specified in the test guideline which were for temperature: 13-17

C and within a range of 2

The dissolved oxygen was maintained at ≥85.3% ASV. The salinity remained at 35-36‰

throughout the test and this was acceptable.

One (5%) of the twenty control

Corophium

died during the study and this represents an

acceptable level of health of the test organisms maintained under test conditions.

5. References

(1)

(2)

OSPAR / PARCOM Protocols on Methods for the Testing of Chemicals Used in the

Offshore Industry 2006 Part A: A sediment bioassay using an amphipod

Corophium

sp.

ToxCalc

Version 5.0 “Comprehensive Toxicity Data Analysis and Database Software”,

ENV 11450/ECO 170408

Page 10 of 10

APPENDIX C

Teknologiparken

Kongsvang Allé 29

Clariant Oil Services Scandinavia AS

Attn.: Frode Bekkestad

P.O. Box 6054 BS

NO-5892 Bergen

Norway

DK-8000 Aarhus C

Tel. +45 72 20 20 00

Fax +45 72 20 10 19

[email protected]

www.teknologisk.dk

Analysis Report No. 716974

Assignment:

Analysis of MEA-Triazin 51%

Purchase order 4504539049

Sampling by:

The client

Sample(s)

received:

19 September 2016

Test performed:

19 September

–

14 October 2016

Test results:

The results of the analysis and the method(s) used

concern only the sample(s) analysed or the subsample(s)

selected for analysis.

This analysis was carried out in accordance with Danish Technological Institute’s General Terms and

Conditions Regarding Commissioned Work Accepted by Danish Technological Institute. This analysis

report may be quoted in extract only if the Laboratory for Chemistry and Microbiology has approved

of the extract in writing.

The Laboratory for Chemistry and Microbiology

Inge Bondgaard Nielsen

Consultant

Ulla Christensen

Team Manager

2016.10.10

Report No.:

716974

No. of encl.: 0

Page 1 of 2

Sample preparation

The sample of H

S scavenger was divided into four subsamples:

Subsample I was analysed directly.

Subsample II was heated to 140°C by reflux for 10 minutes and then cooled to

room temperature with running water.

Atmospheric air was blown through subsample III for seven days.

Subsample IV was aerated for seven days and then heated to 140°C.

After the treatment, all subsamples were analysed for content of formic acid

(HCOOH) and N-(2-hydroxyethyl) formamide (NHEF).

Analytical methods

Formic acid

Formic acid were determined by Ion chromatography (DIONEX AS 14).

N-(2-hydroxyethyl) formamide

N-(2-hydroxyethyl) formamide were determined by extraction with solvent added

internal standard and analyses by gas chromatography with mas selective detection

(GC-MS)

Results

Substance

Subsample

Formic acid

%w/v

0.0067

0.14

0.0076



4% rel.

0.15

N-(2-hydroxyethyl)-formamide

%w/w

<0.1

0.53

<0.1



25% rel.

1.6

III

Precision:

2016.10.10

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716974

No. of encl.: 0

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