I:\MEPC\57\2-5.doc For reasons of economy, this document is pr inted in a limited number. Delegates are kindly asked to bring their copies to mee tings and not to request additional copies. INTERNATIONAL MARITIME ORGANIZATION

IMO 

# E

MARINE ENVIRONMENT PROTECTION COMMITTEE 57th session Agenda item 2 MEPC 57/2/5 7 September 2007 Original: ENGLISH

HARMFUL AQUATIC ORGANI SMS IN BALLAST WATER

Application for Final Approval of a ballast water management system using Active Substances

Submitted by Germany

SUMMARY

Executive summary: This document contains an applica tion for Final Appr oval of a ballast water management system under the Procedure for approval of ballast water management systems that make use of Active Substances (G9) adopted by resolution MEPC.126(53)

Action to be taken: Paragraph 6

Related documents: BWM/CONF/36; MEPC 53/24/A dd.1; MEPC 54/21; BLG 11/4/11, BLG 11/4/15, and BWM.2/Circ.10

1 Regulation D-3.2 of the International Conve ntion for the Control and Management of Shipsí Ballast Water and Sediments stipulates th at ballast water management systems that make use of Active Substances to comply with the Convention shall be appr oved by the Organization.

2 Germany herewith submits an application for Final Approval accord ing to the Procedure for approval of ballast water management system s that make use of Active Substances (G9). This Procedure stipulates the required informatio n (MEPC 53/24/Add.1, anne x 4, paragraph 4.2.1) which, according to section 6, should be evaluated by the Organization. In accordance with BWM.2/Circ.10, the document at the annex, contains the non-confidential part of the manufacturerís applicat ion dossier including:

- general report with summaries of studies carried out with PERACLEAN Æ Ocean and ballast water treated with the SEDNA Æ System according to Guidelines (G8) and Procedure (G9);

- reference list;

- attachments:

.1 Germanischer Lloyd (GL) certificat e confirming appropriate safety management of the installed SEDNA Æ system as required by GESAMP-BWWG in document MEPC 54/2/12;MEPC 57/2/5 - 2 -

I:\MEPC\57\2-5.doc .2 GL certificate confirming compatibili ty, i.e. non-corrosi vity, to ballast water tank coating;

.3 extraction of QAPP for full scale type approval testing according to Guidelines for approval of ballast wa ter management systems (G8) and the Procedures (G9); and

.4 assessment report of full scale land-based testing

− BSH.

The complete dossier will be made available to the experts of the GESAMP-BWWG with the understanding of conf idential treatment.

3 It should be noted that this report is stru ctured in accordance with the headlines in the ìProcedure for approval of ballast water mana gement systems that make use of Active Substances (G9)î (numbers in pare nthesis next to the headlines re fer to relevant paragraphs in Procedure (G9)). The approval tests performed, acco rding to Guidelines (G8) and Procedure (G9) for this report, show that the SEDNA Æ System meets the D-2 standard and there are no significant toxic effects or residuals of PERACLEAN Æ Ocean.

4 Basic Approval has been gr anted at MEPC 54 (MEPC 54 /21, paragraph 2.8) and the competent authority took into account all the recommendations contained in paragraph 9 of annex 5 of the Report of the first meeting of the GESAMP-BWWG ( document MEPC 54/2/12).

5 The receiving competent authority in Germa ny has verified the application dossier and believes it to satisfy the data requirements of the Procedure (G 9) adopted by resolution MEPC.126(53).

Action requested of the Committee

6 The Committee is invited to consider the pr oposal for approval and decide as appropriate.

***MEPC 57/2/5

I:\MEPC\57\2-5.doc ANNEX

TECHNICAL SUMMARY OF DOSSIERS BY DEGUSSA GMBH FOR THE FINAL EVALUATION OF PERACLEAN Æ OCEAN AS AN ACTIVE SUBSTANCE FOR BALLAST WATER TREATMENT

Table of Contents

0 Introduction

1 Identification (4.1)

2 Additional Data on effects of Activ e Substances on aquatic organisms (4.2.1.1, 5.3.9)

2.1 Additional data on PERACLEAN(r) Ocean

2.1.1 Acute aquatic toxicity of PERACLEAN(r) Ocean

2.1.2 Chronic aquatic toxicity

2.1.3 Conclusion

2.2 Endocrine disruption

2.3 Sediment toxicity

2.4 Bioavailability/biomagnification/bioc oncentration/Food web population effects

2.5 Aquatic toxicity of Hydrogen peroxide

2.5.1 Acute aquatic toxicity of Hydrogen peroxide

2.5.2 Chronic aquatic toxicity of Hydrogen peroxide

3 Data on mammalian toxicity (4.2.1.2, 5.3.12)

3.1 Toxicokinetics

3.2 Acute toxicity

3.3 Effects on skin and eyes

3.4 Repeated dose toxic ity (chronic and long term)

3.5 Developmental and reproductive toxicit

3.6 CarcinogenicityMEPC 57/2/5 ANNEX Page 2

I:\MEPC\57\2-5.doc 3.7 Mutagenicity

4 Data on environmental fate and effect under aerobic and anaerobic conditions (4.2.1.3, 5.3.4, 5.3.5)

4.1 Modes of degradation

4.1.1 Degradation of PERACLEAN(r) Ocean

4.2 Bioaccumulation, partition coefficient, octanol/water coefficient

4.3 Persistence and identification of the main metabolites in relevant media (marine and freshwater)

4.4 Reaction with organic matter

4.5 Potential physical effects on w ildlife and benthnic habitats

4.6 Potential residues in seafood

4.7 Known interactive effects

5 Physical and chemical properties (4.2.1.4)

5.1 General physical properties

5.2 Corrosion

6 Treated ballast water ñ Pre-trials (5.2)

6.1 Toxicity test on Pacifi c herring (Clupea pallasi)

6.2 Toxicity testing in Fresh a nd Seawater (Environment Canada)

7 Land-based testing with the SE DNA(r) System and PERACLEAN(r) Ocean

7.1 SEDNA(r) Test system with PERACLEAN(r) Ocean

7.2 Test site

7.3 Test methods

7.3.1 Determination of degradation of Peracetic acid and Hydrogen peroxide

7.3.2 Determination of water parameters and by-productsMEPC 57/2/5 ANNEX

Page 3

I:\MEPC\57\2-5.doc 7.3.3 Ecotoxicological Tests

7.4 Results of the land-based testing

7.4.1 Fulfilment of the chemi cal water quality criteria

7.4.2 Fulfilment of the biological water quality criteria

7.4.3 Biological Efficacy

7.4.4 Dissolved organic carbon (DOC)

7.4.5 Influence on pH level

7.4.6 Content of dissolved oxygen

7.4.7 Degradation of PERACLEAN(r) Ocean

7.4.8 Halogenated by-products

7.4.9 Aquatic toxicity of ballast water

7.4.9.1 Low Salinity

7.4.9.2 High Salinity

7.4.9.2.1 Bacterial Screening assays

7.2.9.2.2 Acute Fish test

7.4.9.2.3 Oyster larvae development test

7.4.9.2.4 Algal growth inhibition test

7.4.9.2.5 Long term fish test

7.4.9.2.6 Conclusion

8 Analytical methods at environmentally relevant concentr ations (4.2.1.5, 5.3.15)

8.1 Specific Analysis of Peracetic Acid (LC-MS/MS)

8.1.1 Method

8.2 Specific Analysis of Hydrogen Peroxide

8.2.1 MethodMEPC 57/2/5 ANNEX Page 4

I:\MEPC\57\2-5.doc 9 Exposure assessment

9.1 Environmental exposure assessment (5.2.7)

9.2 Human exposure assessment

10 Risk characterization (5.3)

10.1 Screening for persistency, bioaccumulation and toxicity (5.1)

Persistence (P)

Bioaccumulation (B)

Toxicity (T)

10.2 Assessment of possible reaction with organic matter (5.3.3)

10.3 Rate of abiotic and biotic degrada tion and degradation products (5.3.4, 5.3.5)

10.4 Partition coefficients (5.3.6)

10.5 Bioaccumulation potential and seco ndary poisoning potential (5.3.7, 5.3.10)

10.6 Assessment of sediment species (5.3.11)

10.7 Comparison of the results of the effect assessment with the resu lts of the discharge toxicity testing (5.3.14)

10.8 Human health risk assessment

References AppendicesMEPC 57/2/5 ANNEX

Page 5

I:\MEPC\57\2-5.doc 0 INTRODUCTION

Since the Active Substance PERACLEAN Æ Ocean, produced by Degussa GmbH in Germany, has been granted Basic Approval by IMOís Marine Environment Protection Committee on 24 March 2006, additional testing according to the appropriate IMO Guidelines has been carried out in order to provide comprehensive in formation needed to apply for Final Approval.

Questions raised by the GESAMP-BWWG in document MEPC 54/2/12 concerning:

• Certification confirming an appropriate safety management of the shipboard installation;

• Information on realistic occupational e xposure scenarios required in order to characterize human health ri sk of use and handling;

• Justification for use of scenarios for modelling environmental exposure concentrations and assumptions and details of the modelling;

• Tests with treated ballast water on more species, including long-term data;

• Justification for non-submission of data in cases where the data requirements of G9 are not fulfilled; and

• Documentation of the quality-control/quality assurance programme as requested in Procedure (G9), paragraph 4.2.4;

were addressed in the relevant chap ters of this applica tion. Copies of certific ates and test reports are provided in full length by the applicant to the GESAMP-BWWG.

Focussing on ecotoxicological aspects, some re sults of tests alread y provided in document MEPC 53/2/12 have been repeated thus enabli ng expert judgement to consider whether the residual toxicity of the discha rge conforms to the evaluation undertaken for Basic Approval.

1 IDENTIFICATION (4.1)

PERACLEAN Æ Ocean is a proprietary formulation based on Peroxygen chemistry. The main components are:

15% Peracetic acid (PAA) 14.3% Hydrogen peroxide (H 2 O 2 ) 26.5% Acetic acid < 1% stabilizer (in to tal), e.g. phosphonic acid

• Peracetic acid is produced by reacting Hydrogen peroxide (H 2 O 2 ) with Acetic acid in aqueous solution. In this process, Peracetic acid is not obtained as a pure substance but in the form of aqueous equilibrium solu tions containing peracetic acid, acetic acid, hydrogen peroxide and water:

CH 3 COOH + H 2 O 2 CH 3 CO(O 2 )H + H 2 O

The amount of Peracetic acid in the equi librium solution can range from 0.25 to 40% depending on the targeted use. The equilibrium solution is the biocidal product which is placed on the market.

Peracetic acid is a biocidal Active Substan ce with strong bacteric idal, fungicidal, and virucidal activity.MEPC 57/2/5 ANNEX Page 6

I:\MEPC\57\2-5.doc • Hydrogen peroxide (H 2 O 2 ) is commonly viewed as an ìenvironmentally friendlyî or ìgreenî chemical. This is particularly due to the physicochemical properties of this small inorganic water-soluble molecule. H 2 O 2 is non-persistent, has innocuous or even beneficial degradation products , and does not accumulate.

Hydrogen peroxide is produced naturally by many living organisms either within the organism itself or in the surrounding medi um. For example, blue-green algae produce H 2 O 2 under light irradiation at a ra te of 1 ñ 50 μg/l per hour (Zepp et al. 1987).

• Acetic acid and its salts, acetates, are natu rally occurring in the environment and in organisms. They are rapidly biodegradable and used as a Carbon-source in metabolic reactions.

2 ADDITIONAL DATA ON EFFECTS OF ACTIVE SUBSTANCES ON AQUATIC ORGANISMS (4.2.1.1, 5.3.9)

2.1 Additional data on PERACLEAN Æ Ocean

2.1.1 Acute aquatic toxicity of PERACLEAN Æ Ocean

The existing data for marine and freshwater orga nisms were given in th e application for Basic Approval (MEPC 53/2/12).

2.1.1.1 Freshwater species

Additional studies for freshwater species were pe rformed by Environment Canada using different concentrations of PERACLEAN Æ Ocean. The results are summarized in Table 1.MEPC 57/2/5 ANNEX

Page 7

I:\MEPC\57\2-5.doc Table 1: Acute toxicity of PERACLEAN Æ Ocean in different freshwater species (DeLafontaine and Despatie 2006)

Result EC 50 [mg PAA/L] 1 Trophic level Species Endpoint 100 ppm
PERACLEAN Æ
Ocean 200 ppm
PERACLEAN Æ
Ocean 300 ppm
PERACLEAN Æ
Ocean Product 0.74 0.46 0.42 Bacteria Vibrio fischeri light inhibition (15 min) Microtox TM PAA 0.111 0.069 0.063

Product 1.4 1.5 < 1.5 Algae Selenastrum capricornutum Growth inhibition (72 h) PAA 0.21 0.225 < 0.225

Product 7.1 7.0 7.8 Cladoceran Daphnia

magna Immobilization (48 h) PAA 1.065 1.17 1.05

Trophic level Species Endpoint 100 ppm
PERACLEAN Æ
Ocean 200 ppm
PERACLEAN Æ
Ocean 300 ppm
PERACLEAN Æ
Ocean Product < 0.78 1.22 < 2.34 Microcrusta-

cea Thamnocephal us platyurus Acute mortality (24 h) PAA < 0.112 0.183 < 0.351

Product > 12.5 38.4 31.8 Hydra Hydra

attenuata Acute mortality/Submortality PAA > 1.5 5.76 3.96

Product 16.2 14.2 13.2 Fish Oncorhynchus mykiss Acute mortality
PAA 2.43 2.16 1.98

1 These values were calculated on the basis of the values given in Table 7 for the 2 h treated ballast water

.

The results of the toxicity tests for different fr eshwater species show di fferences in sensitivity. Small organisms are more sensitive towards PERACLEAN Æ Ocean than higher organisms, except for the Microcrustacea, which was also found very sensitive. This ranking is not surprising since the active compound Peracetic aci d is a well known bactericide. Comparing the results of daphnia, fish and algae with recent findings (ECETOC 2001), the now presented values correspond very well.MEPC 57/2/5 ANNEX Page 8

I:\MEPC\57\2-5.doc 2.1.1.2 Marine species

Additional studies for marine water species, requested by a nd conducted under supervision of Washington State Department of Ecol ogy, USA, are summarized in Table 2.

Table 2: Acute toxicity of PERACLEAN Æ Ocean in different seawater species

Trophic level Species Method Endpoint Results EC 50 ( PERACLEAN Æ

Ocean ) Results EC 50 (PAA) Reference

Algae Macrocystis pyrifera EPA/600/ R-95/136 static EC 50 Germination

EC 50 Germ tube

length 48 h 7.91 mg/L

4 mg/L 1.19 mg/L

0.6 mg/L (Degussa 2005a)

Algae Skeletonem

a costatum E1218- 97a static EC 50 Growth inhibition, 96 h > 16 mg/L > 2.4 mg/L (Degussa 2005a)

Fish 16 PSU Clupea pallasi Not statedSemistatic EC 50 96 h > 16 mg/L > 2.4 mg/L (Degussa 2005b)

All tests were performed according to guidelines and protocols of Washington State Department of Ecology without GLP.

The acute effects of PERACLEAN Æ Ocean were investigated in two marine algae species, Macroystis pyrifera and Skeletonema costatum . The EC 50 values (48 h) in Macroystis pyrifera were 7.91 mg PERACLEAN Æ Ocean /L for germination and 10.9 mg PERACLEAN Æ Ocean /L for germ tube length, corresponding to 1.19 and 1.64 mg PAA/L, respectively. The species Skeletonema costatum , exposed for 96 h, was found less sensitive with an EC 50 value of EC 50 > 16 mg PERACLEAN Æ Ocean/L, corresponding to 2.4 mg PAA/L (Degussa 2005a).

In an additional acute toxi city test with PERACLEAN Æ Ocean (test duration 96 h, water renewal after 48 h), using th e pacific herring ( Clupea pallasi ), no toxic effects we re observed up to the highest test concentrat ion of 16 mg PERACLEAN Æ Ocean/L. Therefore, the LC 50 is > 16 mg PERACLEAN Æ Ocean/L, which corresponds to > 2.4 mg PAA/L. (Degussa 2005b).MEPC 57/2/5 ANNEX

Page 9

I:\MEPC\57\2-5.doc 2.1.1.3 Conclusions

All findings are in the same range of previous data already presen ted in the application for Basic Approval (MEPC 53/2/12). Compared to the results obtained in freshwater, the sensitivity of marine fish is similar.

Comparing available results obtained for th e three trophic levels (not restricted to PERACLEAN Æ Ocean), fish was found less sensitive (lowest acute LC 50 is 1.6 mg PAA/L). For invertebrates the lowest EC 50 was obtained for Daphnia magn a, with 0.5 mg PAA/L. The acute toxicity appears to be relate d to the size of the test organism.

Embryonic stages of oysters and mussels were found most sensitive with 48 h EC 50 values of 0.27 to 0.54 mg PAA/L.

2.1.2 Chronic aquatic toxicity

Further new reports on chronic aquatic toxicity of PERACLEAN Æ Ocean were obtained which are summarized in Table 3.

Table 3: Long term/chronic aquatic toxicity of PERACLEAN Æ Ocean in fresh and seawater

Trophic level Species Method Endpoint Results (NOEC) ( PERACLEAN Æ

Ocean ) Results (NOEC) (PAA) Reference

Freshwate r Fish Danio rerio OECD 210 Flow through conditions (GLP) Hatch

success, Development

Mortality 150 μg/L

150 μg/L

15 μg/L 22.5 μg/L

22.5 μg/L

2.25 μg/L (Degussa 2007j)

Seawater Fish (16 PSU) Clupea pallasi Not stated Semi-tatic Hatching

success 16 days 1.9 mg/L 29 mg/L (Degussa 2005b)

Algae Macrocystis pyrifera EPA/600/ R-95/136 static Germ tube length 48 h 4 mg/L 6 mg/L (Degussa 2005a)

Algae Skeletonem

a costatum E1218-97a static Growth inhibition, 96 h 8 mg/L 2 mg/L (Degussa 2005a)

A fish early life stage test with the freshwater fish Danio rerio was performed according to OECD 210. Neither effects on hatching succ ess nor behavioural an d morphological changes were observed. Surprisingly, the only effect wa s a reduced survival in the two highest concentrations (50 and 150 μg/L PERACLEAN Æ Ocean) in a time range 5 to 12 days post hatching which is considered to be a rather insensitive life stage of fish larvae. No dose dependency was observed. The overall NOEC is considered to be 2.25 μg PAA/L (Degussa 2007j).MEPC 57/2/5 ANNEX Page 10

I:\MEPC\57\2-5.doc Based on a specific stu dy protocol (non-GLP), recommende d by Washington State Department of Ecology, a 16 day fish embryo devel opment test using pacific herring ( Clupea pallasi ) gametes with PERACLEAN Æ Ocean was performed. The NOEC (endpoint: percentage of hatched larvae) was 1.9 mg PERACLEAN Æ Ocean /L (0.29 mg PAA /L) (Degussa 2005b).

The NOEC for Macroystis pyrifera ( germination tube length) was 4 mg PERACLEAN Æ Ocean /L (0.6 mg PAA/L). The species Skeletonema costatum was found less sensitive with a NOEC of 8 mg PERACLEAN Æ Ocean/L (1.2 mg PAA/L) (Degussa 2005a).

2.1.3 Conclusion

The results for marine algae indicate that mari ne algae are less sensitive towards PERACLEAN Æ Ocean than freshwater species (r efer to MEPC 53/2/12). All men tioned studies indicate a very steep dose response curve. However, fish larvae were much more sensitive than Daphnia magna (long-term NOEC of 0.05 mg/L) leading to an overall NOEC of 2.25 μg PAA/L based on the results of the fish test. Using an assessment f actor of 10, a PNEC of 0. 225 μg/L can be derived corresponding to a PNEC of 1.5 μg PERACLEAN Æ Ocean/L.

2.2 Endocrine disruption

No new information (refer to MEPC 53/2/12).

2.3 Sediment toxicity

No new information (refer to MEPC 53/2/12 ).

2.4 Bioavailability/biomagnification/biocon centration/food web population effects

No new information (refer to MEPC 53/2/12).

2.5 Aquatic toxicity of Hydrogen peroxide

The aquatic toxicity data of H ydrogen peroxide were already summarized in the application for Basic Approval (MEPC 53/2/12).

2.5.1 Acute aquatic toxicity of Hydrogen peroxide

2.5.1.1 Freshwater species

No new information (refer to MEPC 53/2/12)MEPC 57/2/5 ANNEX Page 11

I:\MEPC\57\2-5.doc 2.5.1.2 Seawater species

Based on the intended use, some additional mari ne species were tested and the corresponding results on the effects of H 2 O 2 are summarized in Table 4.

Table 4: Acute toxic effects of Hy drogen peroxide in marine species Trophic level Species Guideline Endpoint Result [mg/L] Reference

Rotifier Brachionus plicatilis Rotox Æ Manual EC 50 (24 h), static, mortality 4.8 (Degussa 2007k) Bacteria Vibrio fischeri. MICROTOX basic manual EC 50 (15 min), luminescence 20.6 (Degussa 2007k) Fish Solea solea larvae OECD 203 LC 50 (96 h), static, mortality > 151 < 268 (Degussa 2007k) Fish Solea solea juvenile OECD 203 LC 50 (96 h), static, mortality > 84 < 150 (Degussa 2007b) Oyster Crassostrea gigas EPA/600/R95/136 EC 50 (48 h), static development 3.2 (Degussa 2007k) Chaetocaros gracilis OECD 201 EC 50 (72 h) growth

rate 3.2 (Degussa 2007k) Algae

Skeletonema

costatum OECD 201 EC 50 (72 h) growth

rate 2.7 ñ 3.4 (Degussa 2007m)

In the ROTOX Æ test kit, the EC 50 value was 4.8 mg/L. In the MICROTOX basic assay the EC 50 found was 20.6 mg/L (Degussa 2007k).

In an acute toxicity study in fish larv ae, no mortality occurred up to 151 mg/L H 2 O 2 . But with a concentration of 268 mg/L, all larvae were found dead (Degussa 2007k). The juvenile fish ( Solea solea) was found more sensitive under same conditions with a LC 50 between 84 and 150 mg H 2 O 2 /L (Degussa 2007b).

Hydrogen peroxide did not affect the developm ent of oyster larvae in concentrations up to 2.8 mg/L (NOEC). In higher concentrations, th e development into D-larvae was inhibited with a corresponding EC 50 value of 3.2 mg H 2 O 2 /L (Degussa 2007k).

The two algal species ñ Chaetocaros gracilis and Skeletonema costatum ñ showed comparable sensitivity towards Hydrogen peroxide, resulting in an EC 50 value of appr. 3 mg/L (Degussa 2007k; Degussa 2007m).

2.5.2 Chronic aquatic toxicity of Hydrogen peroxide

In a 21-day continuous exposure study on Daphnia magna conducted by the USGS (United States Geological Survey, USA), th e chronic no observable eff ect concentration (NOEC) for reproduction was 0.63 mg H 2 O 2 /L and for mortal ity it was 1.25 mg H 2 O 2 /L (Meinertz et al. 2007; Vaughn 2006).MEPC 57/2/5 ANNEX Page 12

I:\MEPC\57\2-5.doc The results of toxicity tests pe rformed in different marine species are summarized in Table 5.

Table 5: Chronic toxic effects of Hy drogen peroxide in marine species Trophic level Species Guideline Endpoint Results NOEC [mg/L] Reference

Oyster Crassostrea gigas EPA/600/R95/136 NOEC (48 h), static development 2.8 (Degussa 2007k)

Chaetocaros gracilis OECD 201 72 h chronic study, growth rate 1.9 (Degussa 2007k) Algae

Skeletonema

costatum OECD 201 72 h chronic study growth rate 1 (Degussa 2007m)

Fish Solea solea larvae OECD 210 (adapted) 18 days, semistatic mortality, egg hatch, development 48 (Degussa 2007l)

Hydrogen peroxide did not affect the developm ent of oyster larvae, in concentrations up to 2.8 mg/L (NOEC) (Degussa 2007k).

Of the two tested algal species ñ Chaetocaros gracilis and Skeletonema costatum ñ the latter showed sensitivity slightly towards Hydrogen pe roxide, resulting in an NOEC of 1 mg/L (Degussa 2007k; Degussa 2007m).

In an 18-day fish embryo development test (Solea solea) with water renewal every 2 days, mortality, behaviour, hatching succ ess and development were observed during the course of the test. In the highest concentration of 148 mg/L, no egg survived (day 6). At concentrations of 98 mg/L, hatching was not affected, but only 25% of the larvae survived into free-swimming stage (day 6 to 10) and nearly all larvae died until day 18. Lower concentrati ons of Hydrogen peroxide did not show any effects. Therefore, the NOEC can be set to 48 mg/L under conditions tested (Degussa 2007l).

2.5.3 Conclusion

Algae were found as the most sensitive species towards Hydrogen peroxide. These findings confirm previous studies (e.g. EU Risk Assessmen t, 2003). The toxicity of the oyster larvae was slightly lower than that of algae. This is in accordance with the results of freshwater invertebrates, already discusse d in the application for Basic Approval (MEPC 53/2/12). With a view to the test kits used, the sensitivity of the ROTOX Æ assay is more comparable to that of the oyster larvae and algae than that of the MICRO TOX basic assay. The fish larvae and juveniles were the less sensitive species, as expect ed, even in the long term study performed.

In the EU Risk Assessment (2003), a PNEC of 10 μg/L for Hydrogen peroxide was derived. However, this value appears to be very conser vative and is related to continuous long-term exposures. When deriving a tolerable level for H ydrogen peroxide in ballast water applications, some considerations about the chemistry and oc currence of the substanc e should be taken into account, already discussed in the applica tion for Basic Approval (MEPC 53/2/12). H 2 O 2 acts as an oxidant and, therefore, its a dverse effects are only local and s hort term. Hydrogen peroxide is a normal metabolite in nearly all organisms and naturally produced by sunlight in water.MEPC 57/2/5 ANNEX Page 13

I:\MEPC\57\2-5.doc Therefore, almost all organisms are particularly well equipped in defence mechanisms against Hydrogen peroxide and othe r Active Oxygen species.

In seawater, the available natural concentr ations ñ ranged from 0.5 to 14 μg Hydrogen peroxide/L, in lakes values > 100 μg/L ñ were reported.

3 DATA ON MAMMALIAN TO XICITY (4.2.1.2, 5.3.12)

No new information (refer to MEPC 53/2/12).

3.1 Toxicokinetics

The systemic absorption of Peracetic acid and Hydrogen peroxide is limited by their rapid enzymatic degradation in cells of mucous membra nes lining the gastrointe stinal and respiratory tract and to certain extend also in skin. Tissue damage, for example, on contact with severely irritant concentrations of Per acetic acid, can lead to absorption into the circulation. In blood Peracetic acid and Hydrogen per oxide are very rapidly degrad ed yielding Oxygen and Acetic acid or Oxygen and Water, respectively. The re maining Acetic acid is a physiological substance that is included into physiological metabol ic pathways or excr eted via the urine.

The rapid degradation limits the systemic avai lability of both Peracetic acid and Hydrogen peroxide. It is, therefore, unlikel y that organs beyond the site of first contact will be affected.

3.2 Acute toxicity

No new information (refer to MEPC 53/2/12).

The predominant effect in all acute toxicity studies is local irritation at the site of contact. The extent depends on the concentration and the composition of the applied test solution.

3.3 Effects on skin and eyes

No new information (refer to MEPC 53/2/12).

PERACLEAN Æ Ocean should be considered severely co rrosive to skin and eyes and severely irritating to mucous membranes of the respiratory tract. The RD 50 for an aerosol generated from a product with a similar composition was 21.5 to 24.1 mg/m 3

.

No skin sensitization was observed in two B ̧hler te sts in guinea pigs with different formulations of PAA. These studies are cons idered relevant for PERACLEAN Æ Ocean.

In document MEPC 54/2/12, it was recommended to perform a skin penetration study with Peracetic acid. Due to the results of the toxicoki netics, repeated dose toxicity and the corrosive properties of PERACLEAN Æ Ocean, no further data are consid ered to be required for risk assessment and recommendation of appropr iate risk management measures.

3.4 Repeated dose toxicity (chronic and long term)

Repeated dose studies with diluted equilibrium PAA solutions are relevant for PERACLEAN Æ Ocean. Likewise, the test substance has to be diluted to tolerable concentrations before the administration to the test animals.MEPC 57/2/5 ANNEX Page 14

I:\MEPC\57\2-5.doc No further repeated toxicity studies ar e considered necessary with PERACLEAN Æ Ocean for the following reasons:

The available studies indicate no specific system ic toxicity of equilibrium Peracetic acid;

These findings are in accordance with th e toxicokinetic data (see chapter 2.2.1, MEPC 53/2/12) and support that Peracetic acid will not be systemically available beyond the site of first contact; and

The effects at the site of contact are determ ined by the acute irritation potential of the compound and its ability to release Oxygen. It is not expected that this mode of action and the accompanying no effect levels will va ry considerably between species and with the duration of the study.

In document MEPC 54/2/12, it was recommended to perform a chronic inhalation study in rats. PERACLEAN Æ Ocean is used on ships in a closed and automated system and contact of the ship crew or personnel will be re stricted to very rare acut e dermal exposure (see also 0). Therefore, it is considered, that from that point, no additi onal chronic inhalation study will be necessary which was also confirmed in the registration pro cess of Peracetic acid as a biocide.

The nature of the toxicological e ffects (local irritation or corrosion and secondary effects thereof) can be deduced from results from acute and su bacute inhalation studie s on Peracetic acid and from the subchronic oral study. Sufficient data are now available for risk assessment.

3.5 Developmental and reproductive toxicity

In a developmental toxicity study ñ according to OECD 414 performed with a 35% Peracetic acid solution, administered in drinking water ñ no treatment related teratogenic effects were found in the litters observed up to the highesttested dose of 700 mg PAA /L (M ̧ller 2005).

3.6 Carcinogenicity

Due to the toxicokinetics, ma king it unlikely th at PERACLEAN Æ Ocean is systemically available and the absence of a genotoxic pot ential in the majority of the genotoxicity studies, it is unlikely that the product has a relevant carcinogenic potential for huma ns, and further testing is not warranted.

3.7 Mutagenicity

The weight of evidence, in particul ar in the more recent studies in ì in vitroî experiments in mammalian cell cultures, indicates that equilibri um Peracetic acid does not have a significant genotoxic potential and does not cause gene muta tions and chromosomal ab errations both in the presence and absence of a metabolic activatio n system. Only in one chromosomal aberration assay in vitro a positive result was obtained at highly cytotoxic concentra tions. However, a recent in vitro chromosomal aberration assay did not show such an effect even at cytotoxic concentrations suggesting that the earlier findings may have b een an artefact. P AA did not induce micronuclei in ì in vivoî mouse micronucleus tests when ad ministered by gavage up to the maximum tolerated dose levels and did not indu ce unscheduled DNA synthesi s in two recent rat in vivo/ex vivo UDS assays when administered by gavage up to the maximum tolerated dose level. Therefore, it can be concluded that PERACLEAN Æ Ocean is unlikely to have a genotoxic or mutagenic potential.MEPC 57/2/5 ANNEX Page 15

I:\MEPC\57\2-5.doc 4 DATA ON ENVIRONMENTAL FATE AND EFFECT UNDER AEROBIC AND ANAEROBIC CONDITIONS (4.2.1.3, 5.3.4, 5.3.5)

4.1 Modes of degradation It has been already shown and discussed in th e application for Basic Approval (MEPC 53/2/12), that both active ingr edients of PERACLEAN Æ Ocean, Peracetic acid and Hydrogen peroxide rapidly degrade under abiotic as well as under biot ic conditions. Therefor e, only some recently performed studies will be discussed here.

4.1.1 Degradation of PERACLEAN Æ Ocean

The primary and ultimate biodegradability in seawater in the Shake Flask Test (OECD Guideline 306) was determined for PERACLEAN Æ Ocean over a period of 28 days. Beginning of biodegradation (10% level) was obse rved after 10 days of adaptation time and the pass level (>50%) was reached after 12 days. Af ter 21 days, there was an almost complete mineralization. The elimination/primary degradation of Peracetic acid was rapid (after two days below limit of detection) corres ponding with an elimination rate of >94%. The elimination of Hydrogen peroxide reached 71% after 7 days an d came to >88% (limit of detection) on day 14. Therefore, PERACLEAN Æ Ocean is considered as ultimately biodegradable in seawater (Degussa 2007i).

In a non-guideline study performed by Environment Canada, degradation of Peracetic acid and Hydrogen peroxide over 7 days at 6°C was an alytically investigated in dependence of salinity (0 ñ 27 PSU) and presence or absence of se diment taken from a ballast tank aboard the ship Canadian Provider. This sediment contained large amounts of rust particles and debris from ballast tank corrosion. In unfiltered freshw ater, the concentration of PAA and H 2 O 2 was reduced by 30% and 12.5% after 7 days.

In the presence of sediment, PAA was degraded by 91% and H 2 O 2 by 62% after 7 days, respectively. In salt water (> 7 PSU), PAA and H 2 O 2 were degraded by 92 and 52%, respectively. In the presence of sediments, PAA became unde tectable (< 0.5 mg/L) after 3 days and the degradation rate of H 2 O 2 was 93%. (DeLafontaine and Despatie 2006).

Large-volume (4.5 m 3 ) experiments with PERACLAEN Æ Ocean were also conducted at very cold water temperatures (1 ñ 2°C) in salt a nd freshwater. In the fres hwater experiment, PAA degraded gradually down to 51 to 66% over the cour se of the test. In salt water, the detection limit was very high (10 mg/L) and degradation could not been properly monitored. However, only at concentration of 300 ppm PERACLEAN Æ Ocean, levels above 10 mg/L were observed. Levels of H 2 O 2 remained more or less stable in fresh and salt water over time until catalase was added which dropped the H 2 O 2 level down to zero under both, fr esh and salt water conditions. The levels found in freshwater we re always higher than in salt water (DeLaf ontaine and Despatie 2006).

After addition of PERACLEAN Æ Ocean, the average pH immediately dropped from 7.5 (freshwater control) to 6.7, 6.16 and 5.56 in the 100, 200 and 300 pp m treated tanks, respectively. The average pH in salt water dropped from 8.2 (control tank) to 7.3, 6.75 and 6.2 with the addition of 100, 200 and 400 ppm PERACLEAN Æ Ocean. The pH of the treated waters remained stable over time and did not vary significantly. Very high levels of dissolved Oxygen were noted in all treated tanks across both experiments. Furt hermore, a concentration dependent increase of dissolved organic Carbon (DOC) was found, which can be ascribed to the Carbon introduced by Peracetic acid and Acetic acid. However, that increase was found less in salt water than in freshwater at the 100 and 200 ppm treatment level (DeLafontaine and Despatie 2006).MEPC 57/2/5 ANNEX Page 16

I:\MEPC\57\2-5.doc Gregg and Hallegraeff investigated the degradation of PERACLEAN Æ Ocean in filtered seawater (28.6 PSU and 23.7 PSU) at 17°C storag e temperature using an indirect method by testing the effectiveness against vegetative microalgae in different seawater samples and different light conditions. At concentr ations of 200 ppm PERACLEAN Æ Ocean, PERACLEAN Æ Ocean samples were found to degrade to a level non-toxi c to marine microalgae within 2-6 weeks. In the dark, degradation rate wa s lower even in presence of sedi ment (Gregg and Hallegraeff 2007).

Actually, the viability of the microalgae re-incr eased up to >90% within one (high salinity) or two weeks (lower salinity) post tr eatment. With sediment, the c ontrol level (100% viability) was achieved within one week. The performance of the degradation test is not usual and questionable because the authors did not measur e the actual content of PAA and H 2 O 2 , e.g. by photometric determination. Therefore, it is not sure whether PAA and H 2 O 2 were actually still present or whether the effects found were ca used by other influences, e.g. the pH-level, the presence of nutrients or bacteria or the Oxygen content.

4.1.2 Conclusion

PERACLEAN Æ Ocean is readily (bio)degradable unde r conditions according to the OECD guidelines. Peracetic acid showed a rapid degradation especially in salt water and presence of sediment and light. Hydrogen peroxide degraded slower but, in presen ce of sediment and salt water, degradation significantly increased.

In freshwater, both components are more stable es pecially under extremely cold conditions. Also, the presence of Peracetic acid can be observed ove r a longer period of time. The presence of sediments facilitates degradation in freshwater, too.

Under realistic condi tions, it can be assumed that sediment , which is usually present in shipsí ballast water tanks, enhances degradation. When the ballast water is discharged, photodegradation additionally occu rs. Therefore, the actual envi ronmental concentrations are most probably lower than measur ed under laborato ry conditions.

4.2 Bioaccumulation, partition coefficient, octanol/water coefficient

From the Octanol-Water partition coefficients of the Active Substances PAA and Hydrogen peroxide, the organic carbon nor malised distribution coefficien t can be derived using the following equations according to the EU technical guidance document on risk assessment (EC, 2003).

For organic acids, the follo wing equation can be used:

log Koc = 0.6 log Kow + 0.32

For Peracetic acid with a log Kow of -0.26 at pH 7 (Degussa 2007h), a log Koc of 0.164 (Koc = 1.45 L/kg) is derived.

The following equation for non-hydrophobics ma y be used for Hydrogen peroxide:

log Koc = 0.52 log Kow + 1.02

Using that equation, a log Koc of 0.2 (Koc = 1.58 L/kg) was derived for Hydrogen peroxide (EU, 2003).MEPC 57/2/5 ANNEX Page 17

I:\MEPC\57\2-5.doc Both Koc values indicate a negligible absorption to organic matter which would also indicate a very low adsorption potential to soil and sediment. This is confirmed by the Mackay level equilibrium distribution (see document for Basic Approval, MEPC 53/2/12).

Furthermore, it can be assumed that bioaccumulation for PERACLEAN Æ Ocean is negligible.

4.3 Persistence and identification of the main metabolites in relevant media (marine and freshwater)

No new information (MEPC 53/2/12).

4.4 Reaction with organic matter

In addition to the presented st udies on disinfectant by -products of Perace tic acid in water treatment (see MEPC 53/2/12), Nurizzo et al . (2005) has published inve stigations of effluents from municipal wastewater treatm ent plant and surface water.

Only under extreme high bromide content (0.9 mg /L) and a high content of oxidizable organic carbon (3.2 - 4.9 mg/L), a distinct amount of aldehydes was observed. No formation of total tribrommethanes was observed under any condition (Nurizzo et al. 2005).

Additional laboratory findings indicate that no brominated and chlorinated phenol will be formed during waste water disinfection with Peracetic acid (DellíErba et al. 2007; Kitis 2004).

For that reason, Peracetic acid is gaining more and mo re interest as an alternative to replace other Active Substances in municipal waste water treatment plants.

4.5 Potential physical effects on wildlife and benthic habitats

No new information (MEPC 53/2/12).

4.6 Potential residues in seafood

No new information (MEPC 53/2/12).

4.7 Known interactive effects

None.

5 PHYSICAL AND CHEMICAL PROPERTIES (4.2.1.4)

5.1 General physical properties

The relevant physical chemical properties were already presented in the application for Basic Approval (see MEPC 53/2/12). Some ne w data were determined in the meantime, summarized in Table 6.MEPC 57/2/5 ANNEX Page 18

I:\MEPC\57\2-5.doc Table 6: Physical chemic al properties of PERACLEAN Æ Ocean

Property Value Reference/comment pH value (23°C) 0.39 (Degussa 2006a) relative density 1.122 (20°C) (Degussa 2006a) Kinematic viscosity at 20°C 1.77 mm 2 /s (Degussa 2006a) Kinematic viscosity at 40°C 1.13 mm 2 /s (Degussa 2006a)

5.2 Corrosion

In addition to the discussed studies on corrosion (MEPC 53/2/12), a mayor supplier of ballast tank coatings conducted tests in the own laboratory and confir med that the common Epoxy-based coatings are compatible with PERACLEAN Æ Ocean (Jotun 2005).

A new extensive study has been performed by Un iversity of S ̧dwestfalen, Iserlohn, Germany, under supervision of Germanischer Lloyd, Hamburg. The tests included the determination of resistance against liquids according to ISO 2812-2 a nd the determination of extent of blistering according to ISO 4628-2. Duration was 120 days . Further tests were done on Cathodic Disbonding according to ASTM G8-96 (duration 30 days) (Degussa 2007g). After assessment of the report, Germanischer Lloyd certified PERACLEAN Æ Ocean as compatible with Epoxy-based ballast tank coatings (Germanischer Lloyd, 2007). A dditional tests at Univer sity of S ̧dwestfalen confirmed that PERACLEAN Æ Ocean does not significantly increase the corrosion of galvanized steel used for piping (Degussa 2007f).

6 TREATED BALLAST WATE R ñ PRE-TRIALS (5.2)

Additionally to the presented pre-tests simulating ballast water treatment (refer to MEPC 53/2/12), further tests were performed under similar conditions.

6.1 Toxicity test on Pacifi c herring (Clupea pallasi)

Fertilized Pacific herring eggs were treated for 16 days with a fresh 150 mg/L solution of PERACLAEN Æ Ocean (150 ppm) and compared with that solution which was stored for 48 h prior to test initiation at 8°C simulating conditions as in a ballast water tank.

The NOEC of the unstored sample was 1.9 mg/L, corresponding to a 1.2% solution of PERACLEAN Æ Ocean (150 ppm), After 48 h storage of the solution prior testing, no toxicity occurred up to the highest solution (1:1 d ilution of mother solu tion) (Degussa 2005b).

The results indicate a significan t reduction of the toxicity (40- fold) during the 48 h storage. Therefore, neither any remaining ingredie nt nor any degradation product of stored PERACLAEN Æ Ocean causes significant effects on the mo st sensitive life stage on the Pacific herring.

6.2 Toxicity testing in Fresh a nd Seawater (Environment Canada)

Large-volume (4.5 m 3 ) experiments with PERACLAEN Æ Ocean were also conducted at very cold water temperatures (1 - 2 ° C) in freshwater and seawater (32 PSU).MEPC 57/2/5 ANNEX Page 19

I:\MEPC\57\2-5.doc The water was stored in tanks for 5 days simu lating ballast water treatment. Seventy-two hours after treatment, catalase was added. After 2 h, 71 h and 96 h, samples were taken, diluted with non-stored water and EC 50 values were determined (DeLafontaine and Despatie 2006).

The test conditions and resu lts are given in Table 7.

Table 7: Conditions and Results of the toxicity testing with ballast water treated with PERACLEAN Æ Ocean, 100, 200 and 300 ppm in ice-cold freshwater and salt water Result (EC 50 ) [%

TBW] Trophic level Species Endpoint 1 Storage of the water (% TBW) 100 ppm 200 ppm 300 ppm Freshwater

Bacteria Vibrio fischeri light inhibition (15 min) Microtox TM 2 h 71 h 96 h +Cat 0.74 0.43 0.6 0.23 0.21 0.29 0.14 0.24 0.29 Algae Selenastrum capricornutum Growth inhibition (72 h) 2 h 71 h 96 h +Cat 1.4 1.31 2.6 0.75 0.67 1.08 < 0.5 0.5 0.75 Crustacea Daphnia

magna Immobilization (48 h) 2 h 71 h 96 h +Cat 7.1 7.1 10 3.5 3.2 4.7 2.6 2.1 3.7Microcrustacea Thamnocephal us platyurus Acute mortality (24 h) 2 h 71 h 96 h +Cat < 0.78 2.93 4.17 0.61 1.62 1.69 < 0.78 0.91 1.33 Hydra Hydra

attenuata Acute mortality/ Submortality 2 h 71 h 96 h +Cat >10 >30 >30 19.2 22.5 >30 8.8 10.6 10.6 Fish Oncorhynchus mykiss Acute mortality 2 h 71 h 96 h +Cat 16.2 13.6 17.7 7.2 5.1 8.1 4.4 8.8 4.3 Salt water 100

ppm 200 ppm 300 ppm

Bacteria Vibrio fischeri light inhibition (15 min) Microtox TM 1 h

71 h

96 h +Cat Horm.s

H orm.

orm. 20.2

H orm.

H orm. 1.1

>50

Horm.

1 Tests were performed according to Canadian national guidelines

. Horm. = Hormesis

Under ice-cold freshwater conditions, the tr eated water showed a c oncentration-dependent toxicity (see also Table 1). Th e bacteria were found most sens itive and the hydra and fish less sensitive. However, after addition of catalase, destroying Hydrogen per oxide completely, the toxic effects decreased only in Hydra, Microcrustacea and algae at 200 or 100 ppm PERACLEAN Æ Ocean.

No samples after 96 h storage without catalase were investigated; therefore it is not clear whether the effects can be ascribed to the loss of Hydr ogen peroxide or degradat ion of Peracetic acid.MEPC 57/2/5 ANNEX Page 20

I:\MEPC\57\2-5.doc In salt water, fluorescence in the Microtox test increased indi cating a growth of fluorescent bacteria

.

7 LAND-BASED TESTING WITH THE SEDNA Æ

SYSTEM AND PERACLEAN Æ

OCEAN

7.1 SEDNA Æ Test system with PERACLEAN Æ Ocean

The SEDNA Æ system operates in-line duri ng the uptake of the ballast water. It is based on a modular concept which includes the two-step physical separation as well as the secondary treatment with PERACLEAN Æ Ocean, an oxidizing disinfecting agent.

The first step of the physical separation is a newly developed hydro cyclone that was specially designed for ballast water applications. The second step of the physical separation is a compact, self-cleaning filter with only 50μm meshes. The back-flushing of the filter is triggered by the differential pressure (max. 100 mbar), and the filter elements are cleaned one by one with seawater without addition of any cleaning substances.

Any concentrates from the hydro cyclones (mainly se diments) and any rejects from the fine filter remain at their point of origin and no new wast e lines are produced, when the system is operated on a ship.

The secondary treatment is the chlori ne-free disinfection with PERACLEAN Æ Ocean. Though Peracetic acid is mainly used as a bactericide, f ungicide or virucide, it is also effective against amoeba and algae. PERACLEAN Æ Ocean is dosed automatically to the ballast water after the physical treatment. The dosage is permanently and automatically controlled and adjusted according to the actual flow rate of the ballast water pump.

7.2 Test site

The test runs were carried out at The Royal Netherlands Institute for Sea Research (NIOZ) on the island of Texel from April to July 2007 (NIOZ 2007a).

All tests were conducted according to the Guidelin es for approval of ballast water management systems (G8), adopted on 22 July 2005 by Resolution MEPC.125(53).

The NIOZ test site is equippe d with three coated concrete tanks of 300 m3 volume each to simulate the ballast water tanks of the ship. Fo r each test run, one tank was filled as reference with untreated ballast water (UBW). The water filled into the other tank was treated by the so-called SEDNA Æ system, including mechanical pre-sepa ration of solids and the addition of the Active Substance PERACLEAN Æ Ocean (treated ballast water, TBW).

In the test runs 1 to 6, freshwater was ad ded during uptake to re duce the salinity from approx. 26 PSU (natural level) to 22 PSU in or der to fulfil the requirements given in the Guidelines (G8)

. In the test runs 7 to 12, a salinity of >32 PSU was adjusted. This was done by the injection of a salt brine (14 ñ 18%), made from harbour water and ar tificial sea salt for aq uarium purposes (Instant Ocean Æ ), into the test water upon uptake. Water samp les were taken at least three times per day to monitor the degradation of PERACLEAN Æ Ocean. On day-5 of each test run, the tanks were emptied. For ecotoxicological tests, the required amounts of untreated ballast water (UBW) and treated ballast water (TBW) were taken from the system and transfe rred to the TNO Imares test facility in Den Helder.MEPC 57/2/5 ANNEX Page 21

I:\MEPC\57\2-5.doc 7.3 Test methods

7.3.1 Determination of degradation of Peracetic acid and Hydrogen peroxide

Sampling procedures

Water samples were taken during each filling of the treated tank (to get the initial concentrations) and during the following days until the concentrations were below the detection level.

At least three samples per day were taken in order to get sufficient data for a degradation curve for each test run. The samples were taken at the exit of the SEDNA Æ unit (during tank filling) and from the sampling point at the treated tank.

Analytical measurements

The amount of Peracetic acid and Hydroge n peroxide was measured by photometric determination. The detection limit for both compounds was as low as 0.5 ppm. For each individual sample, three measurements were done and the average value was calculated.

7.3.2 Determination of water parameters and by-products

- Dissolved organic carbon (DOC): measur ed according to Benner and Strom, 1993;

- pH level: measured using a calibrated pH-meter;

- Salinity: measured at the end of each te st cycle using a refractometer (Atago) calibrated against 0 ñ 33 PSU standard seawater;

- Dissolved Oxygen: spectrophotometric met hod of the Winkler method (Benner and Strom 1993); and

- Halogenated by-products: according to DIN EN ISO/IEC 17025, AOX, EOX, chlorophenols, highly volat ile halogenated hydrocarbons.

7.3.3 Ecotoxicological Tests

- Acute toxicity in rotifier (Brachionus plicat ilis): according to the manual of test kit of the ROTOXÆ Assay (according to the manual of test kit);

- Acute toxicity in Bacteria ( Vibrio fischeri) : according to the manua l of the test kit MICROTOX basic assay with 5 minutesí incubation time;

- Algae growth inhibition test ( Skeletonema costatum ): according to the OECD guideline 201, adapted for saline species;

- Static conditions, growth inhibition over 72 h, the UBW was filtered prior the test to exclude algae from the UBW;

- Oyster larvae development test ( Crassostrea gigas ): according to Dutch guidelines for testing sediment elutriates (Schipper et al. 1999) and EPA/600/R-95/136;MEPC 57/2/5 ANNEX Page 22

I:\MEPC\57\2-5.doc - Static conditions, development into D-larvae , UBW was filtered (30 μ) prior the test;

- Acute fish test ( Solea solea ) : according to OECD guideline 203;

- Early life stage test in fish ( Solea solea ) : according to OECD guideline 210; and

- 18 days, semistatic conditions (water rene wal every two days, batch renewal once a week), hatching success, development, mo rtality, UBW and TBW filtered prior the test (0.2 μ).

7.4 Results of the land-based testing

All results described in chapters 7.4.1 ñ 7.4.7 ar e taken from the report of NIOZ (NIOZ 2007b).

7.4.1 Fulfilment of the chemic al water quality criteria

All relevant environmental water pa rameters are summarized in Table 8.

Table 8: Chemical water quality criteria of the inlet water for the test runs 2-12

Low salinity (test run 2 ñ 6) High salinity (test run 7 ñ 12) Average Salinity 21.7 33.4 Total Suspended Solids (TSS) >50 mg/L >1 mg/L

Particulate Organic Carbon (POC) > 5 mg/L > 1mg/L

Dissolved Organic Carbon (DOC) > 5 mg/L > 1 mg/L

The quality criteria as described in the Guidelines (G8) were fulfilled.

7.4.2 Fulfilment of the biolog ical water quality criteria

Test run 1 did not meet the criteria for the number of organisms in the intake water. This test run was declared invalid and will not be taken into further considerations. The other test runs (2 ñ 12) fulfilled the G8 requirements for the wate r quality required for land-based testing.MEPC 57/2/5 ANNEX Page 23

I:\MEPC\57\2-5.doc Table 9: Biological water quality criteria of the test water (low and high salinity)

Low Salinity High Salinity Variety Number Variety Number Organisms > 50 μm > 5 species, > 3 phyla > 100,000/m 3 10 different species, 4-5 different phyla > 5 species, > 3 phyla

Organisms 10-50 μm (flow cytometer) > 5 species, > 3 phyla > 1000 /mL > 5 species, > 3 phyla > 1000 /mL

Bacteria E. coli and total enterococci < 0.1/mL E. coli and total enterococci < 0.1/mL

Colony forming heterotrophic bacteria < 10 ñ 60/mL 10 ñ 200 /mL

1 In the treated tank, Acetic acid stimulated the growth of heterotrophic bacteria resulting in colony numbers <10 to 10,000 mL -1 for low saline treated ballast water and 5,000 to >10,000 mL -1 for high saline water, respectively.

Since for all tests natural plankt on was used, the required diversity of organisms (5 species of at least 3 different phyla) was fulfilled.

In the test runs with low salinity, plankton conditions were typical for a spring bloom of plankton, while the high salinity runs were con ducted after the spring peak in organisms and towards the more clear water phase. Moreover, testing at high tide also implied an increase in the contribution of water from the ope n North Sea with an organism composition different from that of the inner Wadden Sea.

Both factors resulted in a shift towards lower numbers of the larger organisms and an increase of smaller sized plankton. Nevertheless, G8 requirements were always fulfilled.

During the test runs with high salinit y, traces of human pathogens (total E. coli ; <0.1 to 50/ml and total enterococci; < 0.1 to 40/ml) were detected in the Wadden Sea. In the reference and treated tank, however, the numbe r of these human pathogens remained below the detection limit (<0.1 mL -1 ).

Numbers of colony forming heterotrophic bacter ia revealed colony numbers ranging from 10 to 200 per ml for Wadden Sea but remained low in the control tank (<10 to 10 ml -1 ).

In the treated tank, the presence of Acetic acid s timulated growth of, in particular, heterotrophic bacteria resulting in colony nu mbers ranging from 5,000 to >10,000 ml -1 for the six test runs.

Total bacteria counts as measured flow cytometr ic showed that in all tanks and in the Wadden Sea/North Sea initial numbers were >200,000 ml -1 . After the incubation period of five days, some increase was observed in the total bacteria numbers in the control but in the treated tank there was an increase by a factor >100.MEPC 57/2/5 ANNEX Page 24

I:\MEPC\57\2-5.doc 7.4.3 Biological Efficacy

At the time of submission of this document, the pr actical tests were comple ted, but in particular for the biological data some replicate samples still had to be processed for additional and complementary analysis. Nevertheless, based on the present results, it is already justified to conclude that the SEDNA Æ system, including PERACLEAN Æ Ocean as Active Substance, tested at NIOZ from April to June 2007 was in comp liance with regulation D-2 of the Convention.

PERACLEAN Æ Ocean almost instantaneous ly killed all planktonic organisms exposed to the concentration of 150 ppm. With the exception of some diatom shells, the application of PERACLEAN Æ Ocean completely dissolved all orga nisms. Of all 36 analysed samples on discharge (12 test runs, triplica te sampling) combined, in less than 10 cases identifiable, remains of dead planktonic organisms other than the me ntioned diatoms could be found after five days.

7.4.4 Dissolved organic carbon (DOC)

The average DOC contents of the untreated a nd treated ballast water are given in Table 10.

Table 10: Average DOC content of the treated (TBW) and untreated (UBW) ballast water

DOC in the UBW [mg/L] DOC in the TBW [mg/L] Salinity Day 0 Day 5 Day 0 Day 5

Low (test run 2 - 6) 5.1 4.1 21.3 20.5

High (test run 7 - 11) 3.0 2.8 19.5 17.4

The addition of PERACLEAN Æ Ocean resulted in an immediate increase of the DOC at the intake of the tanks from about 5.1 to 21.3 and from 3.0 to 19.5 mg/L, respectively.

Pure PERACLEAN Æ Ocean contains as potential Carbon source 15% Peracetic acid (PAA) and 26.5% Acetic acid. Theoretica lly, a dosage of 150 ppm PERACLEAN Æ Ocean introduces 58.5 mg/L Acetic aci d and increases the DOC content by 23 mg/L. Thus, it can be assumed that the higher DOC content in the treated tank was caused by addition of PERACLEAN Æ Ocean itself rather than other organic ma tter. In previous tests, similar findings were also obtained in both, fresh and sa ltwater (deLafontaine and Despatie, 2006).

Acetic acid is readily biode gradable under aerobic and anoxi c conditions. E.g., 95% of the substance was degraded during 5 days in a Za hn-Wellens test (OECD 302B) with non-adapted activated sludge (IUCLID 2000).

During storage in the dark subterranean tank, the DOC content slightly decreased over five days (see Figure 1).

Treated ballast water of test run 7 was additio nally stored for another eight days in a 700 L container to determine the DOC daily. A stea dy reduction of the DOC was observed which can be related to the degrada tion of the Acetic acid.MEPC 57/2/5 ANNEX Page 25

I:\MEPC\57\2-5.doc Figure 1: Results on DOC content ov er 13 days (test run 7, high salinity)

10,7

3,1 19,7

9,3

2,6 16,8

8,1
6,0

0 4 8 12 16 20 24

reference tank treated tank post storage of treated water DOC (mg/L)

da y 0 day 0

da y 5 day 5

day 7 day 9 day 11 day 13

In conclusion, the components of PERACLEAN Æ Ocean, Peracetic acid and Acetic acid, mainly contributes to the DOC in the treated ballast wate r, but they degrade over a period of two weeks significantly, even without any diluti on or addition of fresh bacteria.

The entry of additional DOC caused by ballast water treatment with PERACLEAN Æ Ocean is considered to be acceptable, because Perace tic acid is degraded very rapidly (see 0) and Acetic acid is consumed and degraded by marine organisms. It can be assumed that under environmental conditions, the degradation will be even faster than presented.

7.4.5 Influence on pH level

The addition of PERACLEAN Æ Ocean, including Peracetic aci d and Acetic acid as acidic components, caused a significant drop of the pH level of 1.9 on average.

Table 11: pH level and temperatures in trea ted and untreated (reference) ballast water

Test run Reference tank Treated tank Temperature (°C) Day 1 Day 5 Day 1 Day 5 Day 1 D ay 5 Low salinity 2 8.42 8.4 6.56 6.76 13 14 3 8.54 8.48 6.66 6.76 15 15 4 8.18 8.14 6.3 6.38 16 16 5 8.24 8.2 6.25 6.29 18 16 6 8.19 8.15 6.31 6.39 16 17 average 8.31 8.27 6.42 6.52 High salinity Test run Reference tank Treated tank Temperature (°C) Day

1 Day 5 Day 1 Day 5 Day 1 Day 5

7 8.17 8.11 6.34 6.39 18 18 8 8.16 8.08 6.32 6.38 18 18 9 8.03 8.01 6.14 6.23 20 20 10 8.15 8.11 6.28 6.23 21 20 11 8.14 8.08 6.31 6.32 21 19 12 8.15 8.10 6.3 6.31 21 19 average 8.13 8.08 6.28 6.31MEPC 57/2/5 ANNEX Page 26

I:\MEPC\57\2-5.doc During the five days storage insi de the subterranean tanks, the di fference of pH values between untreated and treated wa ter was still 1.8 on average. Moreove r, no difference in buffer capacity was observed among both salinities. However, d ilution tests performed af ter the test runs 4 and 10 showed that as soon as the treated wa ter was mixed with fresh natural seawater, the pH level started to recover considerably.

At a dilution rate of 1 part TBW and 5 parts ha rbour water, it took about 4 days to reach the level of pH = 8.0. It took about 7 days to reach the initial le vel of the untreated water of pH = 8.3 (NIOZ 2007b). It can be assumed that bacteria from the harbour water in combination with sunlight might accelerate the de gradation of Acetic ac id. With the dilution rate after discharging of ballast water, the pH will recover completely. In conclusion, there is no sustainable pH drop caused by the treatment with PERACLEAN Æ Ocean.

7.4.6 Content of dis solved Oxygen

The addition of PERACLEAN Æ Ocean significantly increased the content of dissolved oxygen (see Table 12).

Table 12: Content of dissolved Oxygen in treated and untreated ballast water

O 2 content [mg/L] Test run Day Ballast pump Reference tank Treated tank Low salinity (22 PSU) 0 13.8 12.2 28.3 2 5 8.5 18.9 0 13.6 10.5 26.3 3 5 5.3 17.7 0 9.0 7.9 24.5 4 5 5.8 18.7 0 7.6 7.4 23.9 5 5 4.5 20.5 0 7.7 7.8 23.3 6 5 5.9 15.5 High salinity (33 PSU) 0 8.2 8.5. 24.9 7 5 6.2 7.8 0 7.8 7.6 24.7 8 5 6.7 6.8 0 7.9 8.2 24.9 9 5 6.4 6.8 0 7.2 7.0 23.5 10 5 7.4 7.4 0 8.0 8.9 28.8 11 5 6.8 8.1

At lower salinity (test runs 2 - 6), the Oxygen content decreased only sl ightly in the treated ballast water and was still high after five days although the concentrations of Hydrogen peroxide and Peracetic acid were below the detection limit.

Under high saline conditi ons (test runs 7 - 12), Oxygen decreas ed drastically ove r five days to a level comparable to the content found in the untreated water. This might be due to the observed re-growth of heterotrophic bacteria.MEPC 57/2/5 ANNEX Page 27

I:\MEPC\57\2-5.doc 7.4.7 Degradation of PERACLEAN Æ Ocean

PERACLEAN Æ Ocean degraded very rapidly under conditi ons tested. In all test runs, Peracetic acid (PAA) was completely degraded within less than 20 h. In brackish water, the half-life was about 4.5 h, in high saline water 3.5 h, respectivel y (see attached degradation curves and examples in Figure 2)

Figure 2: Degradation of PERACLEAN Æ Ocean components in the land-based testing at NIOZ. Left figure: salinity 22 PSU, right figure: salinity 33 PSU

Landbased Testing at NIOZ / Texel, testrun 5, start on 10th of May 2007

0 2 4 6 8 10 12 14 16 18 20 22 24

0 12 24 36 48 60 72 84 96 108 120 132 144

time ( h )content (ppm) PAA

H2O2

detection limit Landbased Testing at NIOZ / Texel, testrun 10, start on 14th of June 2007

0 2 4 6 8 10 12 14 16 18 20 22 24

0 12 24 36 48 60 72 84 96 108 120 132 144

time ( h )content (ppm) PAA

H2O2

detection limit

Beside the main component Peracetic acid, also Hydrogen peroxide was an alysed. It was found to degrade slower, but in all test runs there was no H 2 O 2 detectable on day 5 (detection limit 0.5 ppm). In the test runs 2 to 6, the Hydrogen peroxide degradation was slightly slower, probably caused by the lower temperature and the lower salinity. It took on average four days (96 hrs) until Hydrogen peroxide had disappeared.

In the test runs 7 to 12, the detection limit of H 2 O 2 was reached after about three days. The estimated half-lifes found were 24 h in br ackish water and 19.5 h in high saline water, respectively (NIOZ 2007b).

7.4.8 Halogenated by-products

Untreated and treated ballast wa ter from testrun 11 were additio nally analysed for AOX, EOX, Chlorophenols and highly volat ile halogenated hydrocarbons. Results are summarized in Table 13 .MEPC 57/2/5 ANNEX Page 28

I:\MEPC\57\2-5.doc Table 13: Concentration of AOX, EOX, Ch lorophenols and Trihalomethanes in untreated (UBW) and treated (TBW) ballast water (Degussa, 2007k) UBW [μg/L] TBW [μg/L] AOX n.d. n.d.AOX solid phase extraction (SPEAOX) < 20 28

EOX < 0.01 <0.01

UBW [μg/L] TBW [μg/L]

Chlorophenols 2,3,4,5-Tetrachlorophenol <0.1 <0.1 Phenol <0.1 <0.1 Pentachlorophenol <0.1 <0.1 4-Chlorophenol <0.1 <0.1 3-Chlorophenol <0.1 <0.1 3,5- Dichlorophenol <0.1 <0.1 3,4-Dichlorophenol <0.1 <0.1 3,4,5-Trichlorophenol <0.1 <0.1 2-Chlorophenol <0.1 <0.1 2,6-Dichlorophenol <0.1 <0.1 2,4- u. 2,5-Dichlorophenol <0.1 <0.1 2,4,6-Trichlorophenol <0.1 <0.1 2,4,5-Trichlorophenol <0.1 <0.1 2,3-Dichlorophenol <0.1 <0.1 2,3,6-Trichlorophenol <0.1 <0.1 2,3,5-Trichlorophenol <0.1 <0.1 2,3,5,6-Tetrachlorophenol <0.1 <0.1 2,3,4-Trichlorophenol <0.1 <0.1 2,3,4,6-Tetrachlorphenol <0.1 <0.1

Highly volatile halogenated hydrocarbons Trichloromethane <0.3 <0.3 Dibromochloromethane <0.1 0.2 Bromodichloromethane <0.1 <0.1 Tribromomethane <0.2 1.5 Total of determinated LHKW -/- 1.7 n.d.: Not determined (if amount of chloride is > 15 g/L, the SPE-AOX has to be determined).

From the tested compounds, only slightly increa sed amounts of AOX (solid phase extraction), bromoform and dibromochloromethane could be detected in the ballast water treated with PERACLEAN Æ Ocean.(Degussa 2007e) These results ar e contradictory to previous findings showing that trihalomethanes were not formed even under extreme conditions (see 0). Nevertheless, since these values are potencies be low recent limit values in drinking water, they can be considered as negligible.

7.4.9 Aquatic toxicity of ballast water

The results of de gradation (see 0) indicate, that already one day after the application of PERACLEANÆ Ocean, no Peracetic acid could be detected any more in the treated water. Furthermore, Hydrogen peroxide was degraded by approximately 50% within one day ofMEPC 57/2/5 ANNEX Page 29

I:\MEPC\57\2-5.doc treatment. After 3-5 days retention time in the ballast tanks, even Hydrogen peroxide was not present any more. (NIOZ 2007b)

7.4.9.1 Low Salinity

The results on the toxicity of ballast water of low salinity (22 PSU) ar e summarized in Table 14.

Table 14: Toxic effects of treated ballast water in the land-based testing (22 PSU) Trophic level Species Endpoint storage in the ballast water tank [days] Result [% TBW 1 ] Reference

Rotifier Brachionus plicatilis 24 h EC 50 , static, mortality 5 Undiluted: no effects (Degussa 2007d)

Bacteria Vibrio fischeri. 5 min EC 50 , luminescence 5 17.94% inhibition with 50% TBW 2 (Degussa 2007c)

Algae Skeletonema

costatum 72 h chronic study growth rate (limit test) 5 Undiluted: no effects (Degussa 2007m)

Oyster Crassostrea gigas 48 h EC 50 , static development 5 Restrictedly usable (Sneekes 2007)

1 TBW: treated ballast water.

2 Based on the given method of the Microtox basic test, the highest test concentration is 50% (1:1 dilution)

. Under low saline conditions, onl y the two test kits (ROTOX Æ , Microtox basic test) and the most sensitive species (algae and oyster larvae) were i nvestigated using day-5 ballast water batches. All tests performed at low salinity showed no adverse effects related to the treated ballast water. Only in the MICROTOX basic as say, some inhibitory effects (17.94%) were detected at the highest TBW solution (Degussa 2007c; Degu ssa 2007d; Degussa 2007m). The oyster larvae, which were also investigated, showed a very low development rate in the control and the test is considered as restrictedly usable. But with respec t to the observed effects it can be assumed, that ballast water up to 31.6% had even positive effects to the devel opment of larvae. In undiluted TBW, the development was again sim ilar to the contro l (Sneekes 2007).

An acute fish test was not performed once agai n due to the low sensitivity towards Hydrogen peroxide and treated ballast water found in prior tests (see 0 and Table 15).MEPC 57/2/5 ANNEX Page 30

I:\MEPC\57\2-5.doc 7.4.9.2 High salinity The effects of treated ballast water with hi gh salinity (>31 PSU) are described in Table 15.

Table 15: Toxic effects of treated balla st water in the land-based testing (high salinity, 33 PSU) Trophic level Species storage in the ballast water tank [days] Endpoint Result [% TBW 1 ] Reference

Acute Rotifier Brachionus plicatilis 1

5 EC 50 (24 h), static, mortality 58.8%

Undiluted: no effects (Degussa 2007d)

Bacteria Vibrio fischeri 1

5 EC 50 (5 min), luminescence 11.95% inhibition with 50% TBW 2 Hormesis (Degussa 2007c)

Fish Solea solea, juvenile 5 LC 50 (96 h), static, mortality Undiluted: no effects (Degussa 2007b) Oyster Crassostrea gigas 1 EC 50 (48 h), static development aerated: 36.0% unaerated: 35.7% (Degussa 2007a)

Oyster Crassostrea gigas 5 EC 50 (48 h), static development 41.5% (Degussa 2007a)

Algae Skeletonema

costatum 1 2 3 3

5 EC 50 (72 h) growth rate 31.6% 35.4% Undiluted: no effects Undiluted: no effects (Degussa 2007m)

Chronic Oyster Crassostrea gigas 1 NOEC (48 h), development static aerated: 31.6% unaerated: 31.6% Degussa, 2007f

Oyster Crassostrea gigas 5 NOEC (48 h), development static 31.6 (Degussa 2007a)

Algae Skeletonema

costatum 1 2 3 3

5 NOEC (72 h) growth rate 17.8% 17.8% Undiluted: no effects Undiluted: no effects (Degussa 2007m)

Fish Solea solea 5 NOEC (6 days) Egg hatch success Mortality 31.6%

31.6% (Degussa 2007l)

1 TBW: Treated ballast water.

2 On the given method of the Microtox basic test, the highest test concentration is 50% (1:1 dilution)

. 3. Control growth was very low, therefore thes e results might be only restrictively usable.MEPC 57/2/5 ANNEX Page 31

I:\MEPC\57\2-5.doc 7.4.9.2.1 Bacterial Screening assays

The EC 50 value found in the ROTOX Æ test kit of the day-1 TBW was 58.8% TBW, whereas the day-5 TBW did not show increas ed mortality, compared to th e UBW control. (Degussa 2007d). Peracetic acid was degraded comp letely. Therefore, the toxicity of day-1 TBW correlates to the Hydrogen peroxide concentration found (see 0).

In the MICROTOX basic assay, no EC 50 values could be calculated. While for the day-1 TBW the highest TBW solutions showed slight inhib itory effects (11.95%), incubation of the day-5 water resulted in increasing hor mesis effect with increased am ounts TBW compared to the NaCl control or UBW controls, respectively (Degussa 2007c).

7.4.9.2.2 Acute fish test

In an acute toxicity study in juvenile fish (96 h, static conditions) with day-5 TBW, no mortality occurred up 100% TBW. (Degussa 2007b)

7.4.9.2.3 Oyster larvae development test

As a rapid decrease of Oxygen in tests with oysters was measured in former tests, some of the test vessels were additionally aerated during incubation.

Day-1 TBW did not affect the development of oys ter larvae in concentra tions up to 31.6% TBW (NOEC), regardless whether aeration was perfor med or not. In higher TBW concentrations, the development of larvae was influenced resulting in EC 50 values of 36.0% TBW for aerated samples and 35.7% for the unaerated samples, respectively. With a concentration of 5.9 mg H 2 O 2 /L found in the TBW sample at test start, the EC 50 and NOEC of TBW correspond to 2.1 and 1.86 mg H 2 O 2 , respectively. These values are in the range of the effect and no effect concentrations of pure Hydrogen peroxide solutions determined in parallel as toxicant control (EC 50 2.6 mg H 2 O 2 /L; NOEC: < 2.4 mg/L).

The day-5 TBW showed inhibitory effect s in similar concentration range (EC 50 41.5%, NOEC: 31.6%). (Degussa 2007a)

Since the Peracetic acid and H ydrogen peroxide levels were below the detection limit in the 5-day TBW, the toxicity observed must have been caused by other factors than PERACLEAN Æ Ocean. Oyster larvae are know n to be very sensitive to wards even slight changes of environmental conditions like pH or temperature or other water parameters (Douillet and Langdon 1993). It ca n not be excluded, that the observed bacteria re-growth (see also 0 and NIOZ 2007b) might also play a role in the development of D-Larvae. Therefore, results got from the performed tests with ballast water and oyster larvae might be questionable.

7 .4.9.2.4 Algal growth inhibition test

The algae Skeletonema costatum was used for toxicity tes ting of TBW day-1, -2, -3 and -5. Day-1 and day-2 TBW did not c ontain Peracetic aci d any more (see 0) and had a measurable amount of Hydrogen peroxide (5.9 and 6.0 mg H 2 O 2 /L). The TBW related EC 50 values for both TBWs were quite similar ( 31.6 and 35.4% TBW, respectively) as well as the NOECs (17.8% TBW for both). These values correlate well with the Hydrogen peroxide concentration in the test samples

.MEPC 57/2/5 ANNEX Page 32

I:\MEPC\57\2-5.doc Day-3 and -5 TBW (no Peracetic acid and Hydrogen peroxide detectable), did not show any growth inhibition compared to the control (Degussa 2007m).

Therefore, it can be assumed, that the main t oxicity of TBW is due to Hydrogen peroxide.

7.4.9.2.5 Long-term Fish test

A performed fish early life stag e test, accordin g to OECD 210 adapted for Sole, did not meet valid criteria because the UBW control had a mort ality of 100% after 18 days. This might have happened due to the aeration system installed during the test. Desp ite of that, during the egg and early larvae stage, the su rvival rate (up to test day 6) was high enough that some observations can be used for further hazard assessment. Furtherm ore, unaerated controls were also tested (UBW and undiluted TBW) and in this case, th e survival rate of the UBW control was 77.5% after 18 days, the TBW-treated fish eggs died up to test day 6.

The egg hatch success and early mortality of the aerated samples were only affected above 31.6% TBW (NOEC) (Degussa 2007l).

Considering this result together with previous find ings in a long-term test on sensitivity of fish in a laboratory-treated water test (refer to 6.1 for more details), it might be concluded that fish is not the most sensitive species.

7.4.9.2.6 Conclusion

Treated ballast water was tested in various sp ecies. The observed toxici ty of the day-1 and day-2 TBW correlates well with the Hydrogen per oxide level in the water sample, observed for Rotifier, algae and oyster, leading to an overall NOEC of 17.8% TBW.

In the algae, the toxicity decreased with increasing storage time of the ballast water indicating that no secondary toxicity is present.

However, TBW on day-5 caused some toxic effects towards oyster and fish larvae suggesting that either bacteria re-growth (see also 0), a well known phenomenon (Douillet and Langdon 1993; Vaughn 2006), was responsible for the toxicity or other water parameters like pH or Oxygen leve l disturbing oyster larv ae and fish eggs in their development.

The most sensitive species found were th e oyster and fish larvae yielding a NOEC of 31.6% TBW.

No adverse effects were observed in the low saline environment under tested conditions

, but this might be restricted due to the limited number of results. Because of the extreme algae bloom in May, no further tests could be performed under low saline conditions.MEPC 57/2/5 ANNEX Page 33

I:\MEPC\57\2-5.doc 8 ANALYTICAL METHODS AT E NVIRONMENTALLY RELEVANT CONCENTRATIONS (4.2.1.5, 5.3.15)

8.1 Specific Analysis of Peracetic Acid (LC-MS/MS)

8.1.1 Method

The quantitative reaction of perace tic acid (PAA) with methyl ptol yl sulphide (M TS) yields the corresponding sulfoxide (MTSO). To prevent th e reaction of the acti ve ingredient hydrogen peroxide (H 2 O 2 ) with MTS, triphenylphosphine (TPP) is added. Analytical evaluation of MTSO is carried out via LC-MS/MS on a reversed-phase column (C8) using a cetic acid/methanol as mobile phase in gradient mode. Detection is carried out with an electrospray tandem mass spectrometer in posi tive mode using an external standard.

8.2 Specific Analysis of Hydrogen Peroxide

8.2.1 Method

The quantitative reaction of Hydrogen Peroxide (H 2 O 2 ) with potassium bis (oxalato) oxotitanate (IV) yields a yellowish peroxidetitan-comple x with a maximum of absorption at 385 nm. Analytical evaluation of the complex is done sp ectrophotometrically usi ng external standard (Degussa 2007i).

9 EXPOSURE ASSESSMENT

9.1 Environmental exposure assessment (5.2.7) Release of PERACLEAN Æ Ocean into the aquatic environmen t during ballast water treatment is considered unlikely due to the short degradatio n time. Peracetic acid degrades within 12 - 24 h. Therefore, it is unlikely that considerable am ounts will attain to the aquatic environment if PERACLEAN Æ Ocean is used in recommended manner. Even if the ship has to discharge all ballast water immediately afte r treatment, there will be no bui ld up of this component. However, measurable Hydrogen peroxide might be released into the environment, when ballast water is discharged 24 to 96 h after treatment (see 0).

If released into the environment, residual Hydrogen peroxide is further diluted to an extent that rapid degradation by the various routes will occur in natural processes.

After confirmation by the Ballast Water Review Group at MEPC 56 that none of the software currently available meets the requirements for mode lling an Emission Scenario, calculations were carried out based upon information provided by documents BLG 11/4/11 and BLG 11/4/15.

Calculations disregarding any mean s of degradation at the time of release demonstrated that even if instantaneously discharged at berth th e concentration of the Active Substance under consideration is 3 orders of magnitude lower than the acute NOEC of the most sensitive species.

In order to consider possible l ong-term effects arising from con tinous release of ballast water discharge, a monitoring throughout one year with the full initial dose of PERACLEAN Æ Ocean was simulated. The half-life time of 24 h, as derived from the land -based testing, was used to calculate the possible build up of Hydrogen peroxide. It could be demonstrated that even after continuous release of ballast water immedi ately after treatment with PERACLEAN Æ Ocean fromMEPC 57/2/5 ANNEX Page 34

I:\MEPC\57\2-5.doc all incoming ships after 365 days, the PEC value in the receiving waters remained one order of magnitude below the PNEC. Thus the calcula ted PEC/PNEC ratio reaches a steady state of 0,0090 after 7 days of continuous discharge. If degradation during sea passage, pump out etc. were taken into account, the value would be mu ch lower. After discharge, the concentration would even in an extended worst case not exc eed the level of naturally formed and depleted Hydrogen peroxide.

Since ships do not enter the port with full ballast water tanks but usually start to deballast when approaching the coast line it might be worth me ntioning that a study carried out by the US EPA measured dilution ranging between 200,000 and 650,000 : 1 at a speed betw een 9 to 19 knots and different pump rates.

The penetration of the substance under cons ideration was measured down to 18 m depth whilst the draft to the lowe st screw tip was 7.5 m. The study can be downloaded from www.epa.gov/owow/oceans/cruise_shi ps/plumerpt2002/plumereport.pdf.

9.2 Human exposure assessment

When using the recommended operating and mainte nance instructions, wo rkers and ship crew will not be directly exposed to PERACLEAN Æ Ocean during the regular procedure of ballast water treatment, since the SEDNA Æ system runs in a separated room or compartment via an automated closed dosing system (Degussa, 2007n).

During the refilling process of the PERACLEAN Æ Ocean tank, a possible vinegar smell (= Acetic acid) is removed by a ventilation extraction unit.

Refilling of the PERACLEAN Æ Ocean storage tank on board is the same or a similar procedure to picking up other consumables such as bunkers or lubr icants. PERACLEAN Æ Ocean is delivered as a typical bulk chemical. The pr oduct is pumped for maximum 2 hours, e.g., from a large storage tank on the pier or from a tank tr uck, to a smaller storage tank on board the ship. Leakage-free couplings are used for hose connections. After filling of the PERACLEAN Æ Ocean tank, residual product in the hose is removed by a pressured air device or other appropriate equipment.

Incidental exposure can only occur for a short ti me during connecting and disconnecting the hose pipe, in case of spillage or for maintenance works.

In these cases, the recommended personal protec tion equipment consists of coverall, gloves, goggles, boots and, if require d, respiratory protection.

Safety training is offered by the producer of PERACLEAN Æ Ocean.

The SEDNA Æ system including the PERACLEAN Æ Ocean dosing system and the storage tank on board a newly built container vessel have been checked regarding safety aspects and accident prevention and received approval by Germanisch er Lloyd (Germanischer Lloyd 2006) and by the Federal Maritime and Hydrographic Agency (BSH 2006).

The estimated exposure is considered to be negligible, since the TLV of 0.6 mg/m 3 is reached to at maximum 7% during pouring of the liquid (TnG, 2002, Model 7).MEPC 57/2/5 ANNEX Page 35

I:\MEPC\57\2-5.doc 10 RISK CHARACTERIZATION (5.3)

10.1 Screening for persistency, bioaccu mulation and toxicity (5.1)

The active components of PERACLEAN Æ Ocean do not fulfil the PB T criteria as outlined in Methodology (MEPC 56/2/2, annex 4) and Pr ocedure (G9) (MEPC 53/24/Add.1, annex 4):

Criterion PBT criteria

P Half-life > 60 d in marine water or > 40 d in freshwater*or half-life > 180 d in marine sediment or > 120 d in freshwater sediment B Experimentally determined BCF > 2,000 or, if no experimentally BCF has been determined, log Pow> 3 T Chronic NOEC < 0.01 mg/l

Persistence (P)

Due to the ready biodegradability and the abio tic degradation of the active components of PERACLEAN Æ Ocean, it can be concluded that it is not persistent in the environment. Environmental half-lifes are in the order of hours or a few days at the maximum.

Bioaccumulation (B)

Due to the high water solubility and the low octa nol water partition coeffi cients of the active components of PERACLEAN Æ Ocean, there is no concern for a possible bioaccumulation potential of the produc t. (log Kow below 0).

Toxicity (T)

One active component of PERACLEAN Æ Ocean has a NOEC level below 0.01 mg/L. However, no indications for relevant e ffects concerning carcinogenicity, mutagenicity or reproductive toxicity as well endocr ine disruption are found.

10.2 Assessment of possible reactio n with organic matter (5.3.3)

PERACLEAN Æ Ocean is a chlorine-free oxidizer. The little amounts of trihalomethanes found are contradictive to previous findings with Per acetic acid and might be due to special water qualities (addition of synthetic sea salt) a nd can be considered as negligible.

10.3 Rate of abiotic and biotic degradatio n and degradation products (5.3.4, 5.3.5)

The active ingredients of PERACLEAN Æ Ocean, PAA and Hydrogen pe roxide degrade rapidly in the environment.

Abiotic degradation half-lifes in seawater are in the range of several minutes for Peracetic acid, and up to 48 h in freshwater at pH 4 to 7. For Hydrogen peroxide half-lifes of several hours up to 5 days were predicted under environmental conditions.

Under clean laborato ry conditions, using filtered water and in the dark, the degradation will be rather slow. The degradation will be more rapid at elevated temperature or in the presence ofMEPC 57/2/5 ANNEX Page 36

I:\MEPC\57\2-5.doc transitions metals, metal oxides and hydroxides (ru st) or suspended solids. Sunlight induces a photochemical decomposition.

However it should also be consid ered that Hydrogen peroxide is also generated photochemically in water systems. In seawater, the limited amo unt of available natural concentrations ranged from 0.5 to 14 μg Hydrogen peroxide/L, in lakes values > 100 μg/L were reported.

Both, PAA and Hydrogen peroxide are readily bi odegradable with half-lifes with sewage treatment plant inoculum of 2 to 3 minutes, if the toxicity levels are not superseded.

Degradation of PERACLEAN Æ Ocean results only in the formation of Oxygen and water as well as Acetic acid that can be readily metabolis ed and consumed by bacteria. No persistent metabolites are formed.

10.4 Partition coefficients (5.3.6)

Due to the low log Kow and Koc values obtained for bot h, Peracetic acid and Hydrogen peroxide (see 0), adsorption to organic matter or so il as well as in tissues is unlikely.

10.5 Bioaccumulation potential and seconda ry poisoning potential (5.3.7, 5.3.10)

Due to the low Octanol-water partition coefficients of the active ingredients (log Kow of PAA = -0.26, of Hydrogen per oxide -1.5) PERACLEANÆ Ocean has no bioaccumulation potential and thus an assessment of secondary poisoning is redundant.

10.6 Assessment of sediment species (5.3.11)

As the Koc values of the active ingredients PAA and Hydrog en peroxide are very low (1.45 L/kg and 1.58 L/kg), an assessment of sediment poisoning is redundant.

10.7 Comparison of the results of the effect a ssessment with the results of the discharge toxicity testing. (5.3.14)

After 24 h treatment of balla st water with PERACLEAN Æ Ocean, the lowest NOEC of the treated ballast water was 17.8% TBW, corre sponding to an initial PERACLEAN Æ Ocean concentration of 26.7 ppm. The toxicity completely disappeared af ter 3 - 5 days of storage in the ballast water tank. Peracetic acid was degraded completely and the residual toxi city after one day of treatment could be completely attributed to residuals of Hydrogen peroxide, which was present in the ballast water tank until day 3 to 4 of treatment.

The toxicity of PERACLEAN Æ Ocean observed with the oyster larvae decreased >50-fold when the ballast water was stored for one day. Again, the residual toxicity was mainly due to the Hydrogen peroxide left in the TB W. However, the toxicity did not decrease with a longer storage time indicating, that not PERACLEAN Æ Ocean itself but other pa rameters (e.g. bacterial re-growth) caused adverse effects in the sensitive test system.

Nevertheless, when TBW will be released into the environment residual Hydrogen peroxide is further diluted and also expected to be rapidly degraded by the various routes of degradation.

The effects observed after extended storage time of TBW are considered to be negligible under realistic scenario.MEPC 57/2/5 ANNEX Page 37

I:\MEPC\57\2-5.doc 10.8 Human health risk assessment

Direct contact during this operation is minimi zed by the recommended handling procedures that include the appropriate personal protective equipm ent, gloves, goggles and, if needed, respiratory protection. On ship, neither additi onal risk for workers nor for sh ip personnel is considered to arise from ballast water treatment with PERACLEAN Æ Ocean used by the SEDNA Æ system.

Furthermore, there is no concern fo r mutagenicity or carcinogenicity.

After all, the product can be handled safely in this met hod of ballast water treatment.MEPC 57/2/5 ANNEX Page 38

I:\MEPC\57\2-5.doc Reference List

Benner R, Strom M (1993): A critical evaluation of the analytical blank associated with DOC measurements by high-temperature catalytic oxidation. Marine Chemistry 41, 153-160. BSH (2006): Statement on ship installation. Degussa (1982a) Degussa GmbH, unpublished report: On the Toxicological Testing of Peracetic Acid 15% Following Single Oral Admini stration on the Rat. Degussa GmbH REG-No. 82-0098-DKT. Degussa (1982b) Degussa GmbH, unpublished report: On the Test of Lo cal Irritant Action of Peracetic Acid 15% Following Single Applicati on on the Skin of the Rabbit (Patch Test). Degussa GmbH REG-No. 82-0101-DKT. Degussa (2002) Degussa GmbH, unpublished re port: Measurement of unscheduled DNA synthesis (UDS) in rat hepatocytes using an in vivo procedure with acide peracetique 5%. Degussa GmbH REG-No. 2002-0674-DGM. Degussa (2005a) Degussa GmbH, unpublished re port: Algal Toxicity Testing using PERACLEAN Æ Ocean. Degussa GmbH REG-No. 2005-0370-DKO. Degussa (2005b) Degussa GmbH, unpublished report: Pacific Herring Toxicity Testing using PERACLEAN Æ Ocean. Degussa GmbH REG-No. 2005-0344-DKO. Degussa (2005c) Degussa GmbH, unpublished report: Toxicological Evaluation of PERACLEAN Æ Ocean. Degradation of Toxicity. Degussa GmbH REG-No.: 2005-0346-DKO. Degussa (2006a) Degussa GmbH, unpublishe d report: Final Report AN-ASB 0340 ñ Determination of Physical-che mical Properties of PERACLEAN Æ Ocean. Degussa GmbH REG-No. 2006-0002-DGA. Degussa (2007a) Degussa GmbH, unpub lished report: Acute Toxicity of Ballast water treated with PERACLEAN Æ Ocean to embryo-larvae of the marine mollusc Crassostrea gigas . Degussa GmbH REG-No. 2006-0188-DKO. Degussa (2007b) Degussa GmbH, unpublished report: Acute Toxicity of Ballast water treated with PERACLEAN Æ Ocean to juvenile marine fish, Sole (Solea solea) . Degussa GmbH REG-No. 2006-0190-DKO. Degussa (2007c) Degussa GmbH, unpub lished report: Acute Toxicity of Ballast water treated with PERACLEAN Æ Ocean to marine bacteria Vibrio fischeri using the Microtox Basic test. Degussa GmbH REG-No. 2006-0182-DKO. Degussa (2007d) Degussa GmbH, unpublished report: Acute Toxicity of Ballast water treated with PERACLEAN Æ Ocean to neonates of the marine rotifer (Brachionus plicatilis) using ROTOXKIT M Æ . Degussa GmbH REG-No. 2006-0186-DKO. Degussa (2007e) AQura Test Report No. A 070017296 according to DIN EN ISO/IEC 17025. Degussa (2007f) Degussa GmbH, unpublished report: Corrosion experiments on the resistance of galvanized steel against the wa ter treatment with PERACLEAN Æ Ocean. Degussa GmbH REG-No. 2007-0114-DKO. Degussa (2007g) Degussa GmbH, unpublished repor t: Corrosion expermiments on the resistance of tank coatings against the water treatment with PERACLEAN Æ Ocean. Degussa GmbH REG-No. 2007-0112-DKO. Degussa (2007h) Degussa GmbH, unpublished report: Estimation of the Partition coefficient (n-Octanol/Water) of Perace tic acid CAS-No.: 79-21-0 in dependence of pH by Q uantitative S tructure A ctivity R elationship (QSAR-Method = Ca lculation). Degussa GmbH REG-No. 2007-0094-DKB. Degussa (2007i) Degussa GmbH, unpublished report: Peracetic acid 15%. Biodegradability in Seawater in the Shake Flask Test . Degussa GmbH REG-No. 2005-336-DGO.MEPC 57/2/5 ANNEX Page 39

I:\MEPC\57\2-5.doc Degussa (2007j) Degussa GmbH, unpublished report: Peracetic ac id 15%. Early-Life Stage Toxicity Test with Zebrafish (Danio rerio) under Flow-Through Conditions. Degussa GmbH REG-No. 2005-0338-DGO. Degussa (2007k) Degussa GmbH, unpublished repor t: Range finding test w ith hydrogen peroxide for the assessment of the sensitivity of serveral test species. Degussa GmbH REG-No. 2006-0236-DKO.

Degussa (2007l) Degussa GmbH, unpublished report: Toxicity of Ballast Water treated with PERACLEAN Æ Ocean to Early Life Stages of the Marine Fish Solea solea . Degussa GmbH REG-No. 2007-0096-DKO. Degussa (2007m) Degussa GmbH, unpublished report: Toxicity of Ballast water treated with PERACLEAN Æ Ocean to the marine algae (Skeletonema costatum) in an algal growth inhibition test. Degussa GmbH REG-No. 2006-0184-DKO. Degussa (2007n) Degussa GmbH, unpublished report: Concept for Storage and Application of PERACLEAN Æ Ocean on Ships. DeLafontaine Y, Despatie S (2006) Degussa GmbH, unpublished report: Effectiveness and Potential Toxicological Impact of the PERACLEAN Æ Ocean Ballast Water Treatment Technology. AEP-TN07-001. Dell'Erba A, Falsanisi L, Liberti L, Notarnic ola M, Santaro D (2007): Disinfection by-products formation during wastewater disinfection with peracetic acid. Desalin ation 215 (10), 177-186. Douillet P, Langdon CJ (1993): Effects of marine bacteria on the cultue of axenic oyster Crassostrea gigas (Thunberg) Larvae. Biol.Bull. 184, 36-51. ECETOC Peracetic acid (CAS No. 79 -21-0) and its Equilibrium So lutions. [JACC No. 41]. 2001. Germanischer Lloyd (2006): Review on ship installation. Germanischer Lloyd (2007a) Degussa GmbH, un published report:Technical Report on Corrosion. Germanischer Lloyd (2007b): Certificate on Corrosion.MEPC 57/2/5 ANNEX Page 40

I:\MEPC\57\2-5.doc Gregg MD, Hallegraeff GM (2007): Efficacy of three commercially available ballast water biocides against vegetative microalgae, di noflagellate cysts and bacteria. Harmful Algae 6, 567-584. Howarth, J. Decay (2003): Kinetics of Pero xyacetic Acid (PAA) and Hydrogen Peroxide (PERASAN (R), EPA #63838-2) in a Variety of Water Matrices. 2003. Enviro Tech Chemical Services, Inc. IUCLID Dataset (2000), Acetic Acid, European Commission - European Chemicals Bureau. Jotun (2005): Statement on corrosion. Kitis M (2004): Disinfection of wastewater w ith peracetic acid: a review. Environment International 30 (9), 47-55. Meinertz, J. R., Greseth, S. L., and Schreier, T. M. (2007): Chronic toxic ity of hydrogen peroxide to Daphnia magna in a flow through continous exposure test system. Aquaculture America 2005 WAS Meeting Abstract 71. 2007. Monarca S, Feretti D, Zerbini I (2002): Studies on mutagenicity and disinfection by-products in river drinking water disinfected with peracetic acid or sodium hypochlorite. Water Science and Technology 2 (3), 199-204. M ̧ller W (2005) Degussa GmbH, unpublished report: Prenatal Development Toxicity study with Wofasteril in the rat using oral (drink ing water) Administration. Degussa GmbH REG-No. 2005-0444-FGR. NIOZ (2007a) Degussa GmbH, unpublished report: Quality Assurance Project Plan for the full-scale testing of the SEDNA Æ ballast water treatment system including the Active Substance PERACLEAN Æ Ocean. NIOZ (2007b) Degussa AG , unpublishe d report: Preliminary Report on the full scale testing of the SEDNA Æ ballast water treatment system incl uding the Active Substance PERACLEAN Æ

Ocean. Nurizzo U, Antonelli M, Profai zer M, Romele L (2005): By-products in surface and reclaimed water disinfected with various ag ents. Desalination 176 (4), 567-571. Pinkernell U, L ̧ke H-J, Karst U (1997): Selective Photometric Determination of Peroxycarboxylic Acids in the Presence of Hydrogen Peroxide. Analyst 122, 567-571. Schipper, C. A., Burgess, R. M., van den Di kkenberg, B., Kater, B. J, and Stronkhorst, J. Standard operating procedure spec ie 05 Marine Oyster crassostre a gigas embryo-larvae mortality and development sediment toxicity test. RIKZ /AB-99. 1999. National Inst itute for coastal and marine Management/ RIKZ, Neth erlands. Ministry of transp ort, public works and water management. Sneekes, A. RE: Antwort: RE: Oyster test R4D5. Ed. S Sch‰fer. 13-7-2007. Vaughn, S. Finding of no significant impact for Hydr ogen Peroxide for Cont rol of Mortality in Freshwater Finfish and Finfish Eggs Due to External Fungal and Bacterial Diseases. HFA-305. 2006.MEPC 57/2/5 ANNEX Page 41

I:\MEPC\57\2-5.doc Veldhuis MJW, Fuhr F, Boon JP, Hallers-Tjabbes CC ten (2006): Treatmen t of ballast water; how to test a system with a modular c oncept? Environmental Technology 27:909 - 921.

Wagner R, Ruck W (1984) : Die Bestimmung von Wasse rstoffperoxid und anderen Peroxyverbindungen. Wasser-Abwaseer-Forsch. 17 (6), 262-267. Wallat Dr (1984a) Degussa GmbH, unpublished report: Testing for Mutagenicity with the Ames Test. Degussa GmbH REG-No. 1984-0392-FKM. Wallat Dr (1984b) Degussa GmbH, unpublished report: Test for Mutagenicity in the Micronucleus Test In Vivo. Degu ssa GmbH REG-No. 84-0390-FKM. Zepp RG, Skurlatov YI, Pierce JT (1987): Alga l-induced Decay and Formation of Hydrogen peroxide in Water: Its Possible Role in Oxidation of Anilines by Algae. Phytochemistry of Environmental Aquati c Systems 327, 215-224.

* * *MEPC 57/2/5 ANNEX Page 42

I:\MEPC\57\2-5.doc APPENDICES

- Certificate of the SEDNA Safety Management System

- Certificate of the Compatibility of PERACLEANÆ Ocean with Tank Coatings

- Content of the QAPP for the Full Scale Testing of SEDNA at NIOZ

- Assessment of the Land-based Test Site at NIOZMEPC 57/2/5 ANNEX Page 43

I:\MEPC\57\2-5.docMEPC 57/2/5 ANNEX Page 44

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I:\MEPC\57\2-5.docMEPC 57/2/5 ANNEX Page 48

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