349-+81 

RECEiVED 

OPPT CBIC 

2012 OCT 25 Af110: 06 

3676 Davis Road N.W. • Dover, Ohio 44622 • (330) 343-7711 • 1-800-321-8805 • Fax: (330) 364-9626 

October 18, 2012 

Document Control Office (DCO) (74707M) 

Office of Pollution Control and Toxics (OPPT) 

U.S. Environmental Protection Agency (EPA) 

EPA East Building, Room E6428 (202-564-8930) 

Washington, D.C. 20460 

Attn: TSCA Section 4 

40 CFR 790 (Docket# EPA-HQ-OPPT-2007-0531) 

Re: Test Results 

Dear Sir or Madam: 

This letter serves as Dover Chemical Corporation's completion of analytical testing under the above 

TSCA Section 4 Test Rule covering CAS # 4 719-04-4. Under this rule we are providing the attached 

test results following the Tier 1 test requirements. 

If you have any questions, please contact me at (330-365-3610) 

Regards, 

?~ 

Tim Nowicki 

Manager Product Compliance 

Dover Chemical Corporation 

tnowicki@doverchem.com 

cc: MCD 

\IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIII IIIII 1111111111111 

4 0 1 3 0 0 0 0 0 0 6 

CONTAINS NO CB! 
3676 Davis Road N.W • Dover, Ohio 44622 • (330) 343-7711 • 1-800-321-8805 • Fax: (330) 364-9626 

July 20, 2011 

Document Control Office (DCO) (74707M) 

Office of Pollution Control and Toxics (OPPT) 

U.S. Environmental Protection Agency (EPA) 

EPA East Building, Room E6428 (202-564-8930) 

Washington, D.C. 20460 

Attn: TSCA Section 4 

40 CFR 790 (Docket# EPA-HQ-OPPT-2007-0531) 

Re: Notice oflntent to Test 

Dear Sir or Madam: 

This letter serves as Dover Chemical Corporation's notice of intention to test under the above TSCA 

Section 4 Test Rule covering CAS # 4 719-04-4. Under this rule we are providing the following 

required information: 

1. Identification ofTest Rule-76 Fed. Reg. 1067 (Jan 7, 2011) 

2. Name, Address, Telephone of Sponsoring Firm3. Name, Address, Telephone of Individual ContactDover Chemical Corporation 

3676 Davis Road N.W. 

Dover, Ohio 44622 

Melissa Clark-Dross 

3676 Davis Road N.W. 

Dover, Ohio 44622 

(330)-365-3628 

4. Signatory Authority for Testing Consortium Members- N/ A 

5. Testing Requirements- Tier I Testing 

6. Testing of Representative Substances- N/A 

Regards, 

Melissa Clark-Dross 

Corporate EHS & Regulatory Manager 
Commercial-in-Confidence 

Chemex reference: ENV9857/051201 

Report issued by: 

The toxicity ofHexahydro-1,3,5,-

tris(2-hydroethyl)s-triazine to the 

alga Pseudokirchneriella subcapitata 

over a 72-hour exposure period 

Report for 

Dover Chemical Company 

Sponsor: 

Chemex Environmental International Limited 

UnitJ 

Dover Chemical Company 

3676 Davis Road NW 

Broad Lane Industrial Estate Dover, OHIO 

Cottenham 

Cambridge CB24 SSW 

UK 

October 2012 

ENV9857/051201 

USA 

44622 

Page 1 of26 
Contents 

Page 

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

Quality Assurance audit statement ......................................................................................... .4 

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

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

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

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

2.2 Reference substance .................................................................................................... 6 

2.3 Test organism .............................................................................................................. 7 

2.4 Test and culture medium ............................................................................................. 7 

2.5 Test methods and conditions ....................................................................................... 7 

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

3.1 Cell density measurements ......................................................................................... 1 0 

3.2 Observations on cell morphology ............................................................................... 11 

3.3 Percent inhibition at each test concentration compared to the control.. ..................... 11 

3.4 ECx and NOEC values by yield (EyCx) and growth rate (ErCx) ................................. 11 

3.5 Control validity results ............................................................................................... 12 

4. Discussion .................................................................................................................. 13 

5. References .................................................................................................................. 13 

Graphs 

1. Growth curves for control and test concentrations ..................................................... 14 

2. Estimation of 0 to 48 hours EyCx values .................................................................... 15 

3. Estimation ofO to 72 hours EyCx values .................................................................... 16 

4. Estimation of 0 to 48 hours ErCx values ..................................................................... 17 

5. Estimation ofO to 72 hour ErCx values ...................................................................... 18 

Appendices 

1. Analytical confirmation of test concentrations .......................................................... 19 

2. Culture media ............................................................................................................. 23 

3. Water quality data ...................................................................................................... 24 

4. Reference test ............................................................................................................. 25 

5. GLP Certificate ........................................................................................................... 26 

ENV9857 /051201 Page 2 of26 
Compliance with Good Laboratory Practice standards 

I, the undersigned, hereby declare that the study described in this report was performed under my 

supervision, and that the final report fully and accurately reflects the raw data generated during 

the conduct of the study, in compliance with international codes of Good Laboratory Practice 

based on: 

• Section II of Annex 1 to the European Parliament and council Directive 2004/1 0/EC and 

Annex 1 to the European Parliament and council Directive 2004/9/EC (Official Journal 

No. L 50) and embodied within: 

• The UK Good Laboratory Practice Regulations 1999 (The United Kingdom GLP 

Regulations 1999, Statutory /nstrument3106) as amended by: 

• The UK Good Laboratory Practice (Codification Amendments Etc.) Regulations 2004 

(Statutory Instrument No 994) 

These principles are in accordance with the OECD Principles of Good Laboratory Practice, 

revised 1997 (ENV IMC/CHEM(98) 17). 

The analytical confirmation work (page 9, analytical confirmation of exposure concentrations 

and Appendix 1) was outside ofthe GLP compliant scope ofthe study and compliance is not 

claimed . 

........... !:\~~······················· ...... 8. ... C:l:JclX?t ..... 2.012 ..................... . Heather Beattie Date 

Study Director 

Key personnel 

Other key personnel at Chemex involved in this study were: 

Agnes Grabczewska 

Sravanthi Chitikeshi 

ENV9857 /051201 Page 3 of26 
Quality Assurance audit statement 

The Quality Assurance unit inspects all operating procedures as part of a program of process and 

study specific based audits. 

Annual routine facility-based inspections are also conducted, which cover the general facilities 

and activities within the test facility. 

The inspections applicable to this study are detailed below. The dates are given as dd/mm/yy. 

Audit Date of audit Date reported to Study 

Director I Manaaement 

Study plan 18/06/12 18/06/12 

Process based audits 

Aquatic plant or algal test set up 23/04/12 30/04/12 

Detennination of algal cell density 23/04112 30/04112 

Equipment calibration 26/04/12 28/06/12 

Inoculation with algae 23/04112 30/04112 

Labelling and paperwork 23/04/12 30/04/12 

Preparation of growth medium 28/06/12 28/06112 

Preparation of solutions 23/05112 29/05/12 

Taking and recording readings 26/04/12 30/04/12 

Weighing out test or reference materials 23/05/12 29/05/12 

This report has been audited by the undersigned and, as far as can be reasonably established, the 

methods described and the results incorporated into this report accurately reflect the raw data 

generated during this study. 

Final report audit started: 15/08112 

Final report audit completed: 08/10112 

Signed: ·····~····································· Rasa Cooke 

Quality Assurance Manager 

Date: ............................................................... OJ Od~ ~o/2--

ENV9857/051201 Page4 of26 
Summary 

This section summarises aquatic toxicity test results obtained by Chemex Environmental 

International Limited on a sample as detailed below: 

Test commissioned by: Dover Chemical Company 

Substance under test: Hexahydro-1 ,3,5,-tris(2-hydroethyl)s-triazine 

Chemex reference: 

Test species: 

Test type: 

Test conditions: 

Test period: 

Test carried out at: 

Results: 

Period of 

Sample: 

Study: 

EC0051201 

ENV9857 

Pseudokirchneriella subcapitata, strain CCAP 278/4 

Growth inhibition: 72 hour EC5o 

Test conditions according to SOP E203 based upon OECD 

Guideline for testing of chemicals, Freshwater Alga and 

Cyanobacteria, Growth Inhibition Test reference 201 (2011) 

(I) 

Council Regulation (EC) No 440/2008 of 30 May 2008(2l. 

From 16 to 19 July 2012 

Chemex Environmental International Limited 

Unit J 

Broad Lane Industrial Estate 

Cottenham 

Cambridge CB24 8SW 

UK 

ECx and NOEC values by yield (EyCx) and growth rate (ErCx) 

EyCx value ErCx value 

exposure (95% confidence limits) (95% confidence limits) 

(hours) EvCIO EyC2o EyC50 ErCIO ErC2o ErC50 

0.05mgll 0.146mgll 1.14mg/l 1.8lmg/l 3.13mgll 8.93mgll 

0 to 48 (0.000- (0.000- (0.00- (0.139- (0.516- (4.54-

0.405mg/l) 0.749m_g/ll 3.25mgll) 3.82mg/l) 5.71mg/l) 17.2m_g/l) 

O.lOlmg/1 0.293mgll 2.24mg/l 6.76mg/l 8.85mgll 14.8mg/l 

0 to 72 (0.000- (0.000- (0.069- (2.86- (4.84- (11.2-

0.63Imwn 1.21mgll) 6.94mgll) 9.39mg/l) I1.7mwn 20.9mg/l) 

NOEC <lmg/1 (0-72 hours) <lmg/1 (0-72 hours) 

by yield by growth rate 

ENV9857 /05120 I Page 5 of26 
1. Introduction 

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

investigate the growth inhibition of Hexahydro-1 ,3,5,-tris(2-hydroethyl)s-triazine to 

Pseudokirchneriella subcapitata. The objective of this study was to determine the 72 hour EC5o, 

which is defined as the concentration that inhibits the growth of the algae by 50% after a 72 hour 

exposure period at 21-24°C. 

2. Materials and methods 

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

Environmental International Limited standard procedures. 

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

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

Estate, Cottenham, Cambridge CB24 SSW. 

2.1 Test substance 

Name of test substance: 

Lot number: 

Supplied by: 

Chemex reference: 

Date of receipt: 

Active ingredients: 

Required storage conditions: 

Actual storage conditions: 

Quoted stability: 

Quoted appearance: 

Observed appearance: 

Quoted odour: 

Quoted solubility in water: 

2.2 Reference substance 

Identity: 

Purity: 

Source: 

Hexahydro-1 ,3,5,-tris(2-hydroethyl)s-triazine 

None 

Dover Chemical Company 

ECO 051201 

23/04/12 

CAS# 4719-04-4, 78.5% 

Keep container tightly closed. Store away from 

incompatible substances in a cool, dry, well ventilated 

area. 

Room temperature 

Stable under normal temperatures and pressures 

Water white to pale yellow liquid 

Water white to pale yellow liquid 

Mild characteristic odor 

Very high 

Potassium dichromate (K2Cr20 7) 

>99.9% 

Fisher Chemicals Ltd 

Reference substance used in a separate in-house study in order to check test procedure. 

ENV9857 /05120 I Page 6 of26 
2.3 Test organism 

Test species: 

Source: 

Culture conditions: 

Pseudokirchneriel/a subcapitata strain: CCAP 278/4 

Culture Collection of Algae and Protozoa 

SAMS Research Services Ltd 

Scottish Marine institute 

OBAN 

Argyll PA37 IQA 

Scotland, United Kingdom 

Temperature: 

Illumination: 

Shaking: 

20.2- 21.9°C 

6000 - 7000 lux continuous white light 

Orbital set to 200rpm 

2.4 Test and culture medium 

The tests were performed in deionised water with added nutrients. Before use the water was 

sterilised by autoclaving at l20°C for I5 minutes. Sterile nutrient stock solutions were then 

added and the pH value adjusted to 8.0 ± 0.2 to obtain the culture medium (see Appendix 2). 

2.5 Test methods and conditions 

The test was carried out according to the procedures given below, based on the guidelines 

produced by OECD 20I: Alga Growth Inhibition Test<') and Council Regulation (EC) No 

440/2008 of 30 May 2008 (2). 

Preliminary test method: 

Preliminary test results: 

Nominal 

concentration 

A preliminary study was conducted at concentrations of 0, 

O.I, I, I 0 and 1 OOmg/1. The duration of the preliminary 

study was 72 hours. There was a single replicate at each 

concentration. 

Data from the preliminary study identified the 72 hour ECso 

as being between I and I Omg/1 by yield and I 0 and I OOmg/1 

by growth rate. 

Percent inhibition Percent inhibition 

by yield integral by growth rate 

(m_g/1) 0-48 hours 0-72 hours 0-48 hours 0-72 hours 

0.1 22 22 II 6 

I 0 -24 0 -6 

10 95 62 87 25 

IOO IOO 88 100 53 

Note: Negative inhibition values indicate an increase in growth compared with controls. 

ENV9857/05I201 Page 7 of26 
Definitive test method: 

Test period: 

Test volume: 

Test vessel 

Number of replicates 

Test concentrations: 

Test organism: 

Test conditions: 

Water quality measurements: 

Observations/frequency: 

Calculation and expression 

of results: 

ENV9857/051201 

From 16 to 19 July 2012 

100ml 

250ml conical flask. 

6 control flasks, 3 replicates at each test concentration. 

After discussion with the client, definitive test concentrations 

were prepared as 0 (Control), 1, 3, 10, 30, 100 and 200mgll. 

A stock solution of 200m gil was prepared and stirred well to 

produce a clear colourless solution. The test concentrations 

were prepared by addition of the appropriate volume of the 

stock solution using pipettors to test media. They were then 

made up to the mark in volumetric flasks to give the required 

concentrations for the test. 

From a pre-culture growing in exponential phase under 

conditions described in 2.3 above. Inoculum level calculated 

to give an initial cell density of 1 x 104 cells/mi. 

Temperature: 

Illuminati on: 

Shaking: 

20.1 - 22.0°C 

approximately 6,500 to 8,500 lux 

continuous white light 

Orbital set to 200 rpm 

The pH (to 0.1) and temperature (to 0.1 °C) were measured. 

Water qualities determinations on pooled replicates for each 

test and control solution were carried out immediately prior 

to initiating the test and at the end of the 72 hour test period. 

The cell density was determined after 24, 48 and 72 hour 

exposure periods on a small volume removed from each 

replicate flask. The cell counts were made using a 

haemocytometer and microscope. In determining the cell 

density, cells showing morphological abnormalities were 

included in the count. If such abnormalities were observed, 

they were recorded in the observations section of the report. 

Growth curves for each test concentration were plotted 

as 

logarithm of the mean cell density against time. The 

inhibition of growth was calculated using both the biomass 

(area under the growth curve) and the growth rate method. 

Where possible, the ECso values were estimated graphically 

and 95% confidence limits calculated according to the 

method of ToxCalcTM Version 5.0 "Comprehensive Toxicity 

Data Analysis and Database Software", copyright 1994-

1996(3). 

Page 8 of26 
Analytical confirmation of 

exposure concentrations: 

ENV9857 /051201 

A method for analytical confirmation of test concentrations 

was researched, firstly using the GC/FID. Concentrations of 

20, 200 and 500mgll were analysed. No peak could be 

identified at 20mgll and the estimated limit of detection was 

found to be approximately I OOmg/1, therefore the method of 

analysis was deemed to be inappropriate to the study with the 

EC50 expected to be <I OOmg/1 and no further attempts were 

made to improve the performance of the method. 

Therefore a HPLC method was looked at, although a peak 

could be seen without the use of a column, a column could 

not be found that could separate the compound peak from the 

mobile phase peak. As a suitably sensitive method could not 

be found it was decided, after communication with the study 

monitor, to report results as nominal concentrations. 

Page 9 of26 
3. Results 

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

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

results in this study are calculated from the measured cell densities. 

3.1 Cell density measurements 

Mean initial cell density: Approximately 1 x 104 cells/m1 based upon inoculation volume, 

not counted microscopically. 

Nominal Cell density measurements (cells/ml x 104

) 

concentration Replicate 24 hours 48 hours 72 hours (mg/1) 

1 7.7 52.0 156.7 

2 10.0 46.7 152.3 

3 12.7 49.0 117.3 

0 4 11.3 50.7 153.7 

5 11.0 51.7 146.3 

6 13.0 47.7 131.3 

Mean 11.0 49.6 142.9 

1 8.7 23.3 41.7 

1 2 8.3 21.3 67.7 

3 7.0 17.7 67.7 

Mean 8.0 20.8 59.0 

1 9.0 23.7 102.7 

3 2 10.3 28.7 107.0 

3 8.3 28.7 112.3 

Mean 9.2 27.0 107.3 

1 2.0 6.0 33.7 

10 2 1.7 9.0 40.3 

3 1.3 11.3 47.3 

Mean 1.7 8.8 40.4 

1 3.7 0.3 2.3 

30 2 1.3 1.7 2.0 

3 2.3 0.3 1.3 

Mean 2.4 0.8 1.9 

1 0.3 0.7 1.0 

100 2 1.0 2.0 1.0 

3 0.7 0.0 1.3 

Mean 0.7 0.9 1.1 

1 0.0 0.7 1.0 

200 2 0.0 0.3 1.0 

3 0.0 0.0 1.3 

Mean 0.0 0.3 1.1 

ENV9857/051201 Page 10 of26 
3.2 Observations on cell morphology 

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

appeared normal, no cells were observed that showed any morphological abnormalities. 

3.3 Percent inhibition at each test concentration compared to the control 

Nominal Percent inhibition Percent inhibition 

concentration by' ield by 2rowth rate 

(mg/1) 0-48 hours 0-72 hours 0-48 hours 0-72 hours 

1 59 59 22 18 

3 46 25 16 6 

10 84 72 45 26 

30 100 99 100 88 

100 100 100 97 98 

200 100 100 100 98 

Graph 1 illustrates the growth curves for the control and all test concentrations. 

Graphs 2 to 5 illustrate the estimation of the EyCx and ErCx values. 

3.4 ECx and NOEC values by yield (EyCx) and growth rate (ErCx) 

Period of EyCx value ErCx value 

exposure (95% confidence limits) (95% confidence limits) 

(hours) Ey_Cto EyC2o Ey_Cso ErCto ErC2o ErCso 

0.05mgll 0.146mg!l 1.14mg/l 1.81mg/l 3.13mg!l 8.93mg!l 

0 to 48 (0.000- (0.000- (0.00- (0.139- (0.516- (4.54-

0.405mg!l) 0.749mg/l) 3.25mg!l) 3.82mg/l) 5.71mg/l) 17.2mg/l) 

0.101mg!l 0.293mg!l 2.24mg/l 6.76mg/l 8.85mg!l 14.8mg!l 

0 to 72 (0.000- (0.000- (0.069- (2.86- (4.84- (11.2-

0.631mg/l) 1.21mgll) 6.94mg!l) 9.39mgill_ 11.7mg/1)_ 20.9mg/l) 

NOEC <1mg/l (0-72 hours) <1mg!l (0-72 hours) 

by yield by growth rate 

ENV9857/051201 Page 11 of26 
3.5 Control validity results 

Control growth rate data coefficient of variation calculations 

Control Cell density (1.0 x 104 cells/ml) 

replicate 

no. 0 hr 24 hr 48 hr 72 hr 

I 1.0 7.7 52.0 I56.7 

2 1.0 IO.O 46.7 I52.3 

3 1.0 I2.7 49.0 II7.3 

4 1.0 Il.3 50.7 I53.7 

5 1.0 Il.O 51.7 I46.3 

6 1.0 13.0 47.7 131.3 

Mean: 

Standard deviation: 

Coefficient of variation: 

0-72 hr average 

specific growth 

rates 

1.68 

1.68 

1.59 

1.68 

1.66 

1.63 

1.65 

0.0367 

2.22 

Note: 0 hr cell density based upon inoculation volume, not counted microscopically. 

Control Day-by-day specific growth rates Coefficient of replicate variation 

no. 0-24 hr 24-48 hr 48-72 hr 

I 2.04 1.9I 1.10 30.26 

2 2.30 1.54 1.18 34.17 

3 2.54 1.35 0.87 54.19 

4 2.42 1.50 1.11 40.I2 

5 2.40 1.55 1.04 41.31 

6 2.56 1.30 1.01 50.76 

Coefficient of variation: 22.I4 

ENV9857/051201 Page 12 of26 
4. Discussion 

The EC50 values (by yield) of Hexahydro-1 ,3,5,-tris(2-hydroethyl)s-triazine to 

Pseudokirchneriella subcapitata were estimated as 1.14mgll at 48 hours and 2.24mg/l at 72 

hours. The EC50 value (by growth rate) was estimated as 8.93mgll from 0 to 48 hours and 

14.8mg/l from 0 to 72 hours. 

The NOECr (0-72h) (growth rate) was estimated as <lmg/1. The NOECy (0-72h) (yield) was 

estimated as <lmg/1. Both were determined by the Bonferroni T test. 

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

all cells appeared normal, no cells were observed that showed any morphological abnormalities. 

The full water quality measurements are given in the summary tables in Appendix 3 and were 

within acceptable limits. 

The cell density of the control replicates increased by a factor of 143, the coefficient of variation 

for the control replicates was less than 35% for each of the 24 hour time periods and less than 

7% for the whole time period, 0- 72 hours. This fulfils the validity criteria of the study and it is 

therefore compliant with the OECD Guideline for Testing of Chemicals reference 201 Alga, 

Freshwater Alga and Cyanobacteria, Growth Inhibition Test 2011 guideline. There were no 

deviations to the protocol that affected the results ofthe study. 

There were a number of deviations from the protocol; the temperature during the culturing 

period was low, the temperature during the test period was low and the light was high. As the 

control validity criteria were met it does not appear that these deviations were significant. 

5. References 

1. OECD Guideline for Testing of Chemicals reference 201 Alga, Freshwater Alga and 

Cyanobacteria, Growth Inhibition Test reference 20 1 (20 11 ). 

2. Council Regulation (EC) No 440/2008 of 30 May 2008. 

3. ToxCalcTM Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software", 

copyright 1994-1996. 

ENV9857 /051201 Page 13 of26 
Graph 1 

Growth curves for control and test concentrations 

16.00 

::-

e 14.00 ~ 

-~ 

CJ 

- 12.00 ~ 

·-~ 

= 

~ 10.00 

"0 

--

~ 

CJ 8.00 

= 

= ~ 

= 6.00 ""' Q 

CIJ 

.s 4.00 

-; 

a. 

.... = 2.00 

z = 

0.00 

Statistical data 

\ ,£3". .-

I 

\ I 

\ I 

\ I 

\ I 

\ I 

0 24 48 

Exposure period (hours) 

Control _ ... _ 10.0 

- {3- 200.0 

Concentration in mg/1 

• 1.0 

--30.0 

--3.0 

• 100.0 

NOECy (yield)= <lmg/1 

NOECr (growth rate) = <lmg/l 

As determined by the Bonferroni T test, based on the 0-72 hour values. 

ENV9857 /05120 I Page 14 of26 

72 
Graph 2 

Estimation of 0 to 48 hours EyCx values 

1.0 

0.9 

0.8 

0.7 

= .s 0.6 .... 

:c 

:= 

... = 

'; 0.5 

= Q 

t 

Q 0.4 c. 

Q 

... 

~ 

0.3 

0.2 

0.1 

0.0 

0.000001 0.0001 0.01 100 10000 

Concentration mg/L 

Indicates 95% confidence limits +-t --+• Indicates averaged data plot 

Statistical data 

EyC10 value 

EyC2o value 

EyCso value 

ENV9857/051201 

0.050mgll (0.00 to 0.405) 

0.146mgll (0.00 to 0.749) 

1.14mg/l (0.00 to 3.25) 

(as determined by the Maximum Likelihood-Probit Method) 

Page 15 of26 
Graph 3 

Estimation of 0 to 72 hours EyCx values 

1.0 

0.9 

0.8 

0.7 

= .s 

... 0.6 :c 

:a 

.... = 

-; 0.5 

= .s 

t: 

= 0.4 c. 

.. = 

=- 0.3 

0.2 

0.1 

0.0 

0.0001 0.001 O.ot 0.1 10 100 1000 10000 

Concentration mg/L 

Indicates 95% confidence limits +-• --+• Indicates averaged data plot 

Statistical data 

EyCIO value 

EyC2o value 

EyCso value 

ENV9857/051201 

0.10 I mg/1 (0.00 to 0.631) 

0.293mgll (0.00 to 1.21) 

2.24mg/l (0.069 to 6.94) 

(as determined by the Maximum Likelihood-Probit Method) 

Page 16 of26 
Graph 4 

Estimation of 0 to 48 hours ErCx values 

1.0 

• 

0.9 

0.8 

0.7 

= .s 

.. 0.6 :c 

:= 

.... = 

-; 0.5 

= Q 

t 

Q 0.4 c. 

Q 

~ "' 

0.3 

0.2 

0.1 

0.0 

0.01 0.1 10 100 1000 

Concentration mg/L 

Indicates 95% confidence limits +-• --+• Indicates averaged data plot 

Statistical data 

ErCJO value 

ErC2o value 

ErCso value 

ENV9857/051201 

1.81mg/l (0.139 to 3.82) 

3.13mg/l (0.516 to 5.71) 

8.93mg/l (4.54 to 17.2) 

(as determined by the Maximum Likelihood-Probit Method) 

Page 17 of26 
Graph 5 

Estimation of 0 to 72 hour ErCx values 

1.0 • • 

0.9 

0.8 

0.7 

= .5! 0.6 .... 

:c 

:= 

... = 

-; 0.5 

= Q 

t: 

Q 0.4 c. 

Q 

~ ""' 

0.3 

0.2 

0.1 

0.0 

0.1 10 100 1000 

Concentration mg/L 

Indicates 95% confidence limits ••---+• Indicates averaged data plot 

Statistical data 

ErCIO value 

ErC2o value 

ErCso value 

ENV9857/051201 

6.76mg/l (2.86 to 9.39) 

8.85mgll (4.84 to 11.7) 

14.8mgll (11.2 to 20.9) 

(as determined by the Maximum Likelihood-Probit Method) 

Page 18 of26 
Appendix 1 

Analytical confirmation of exposure concentrations 

GC/FID method development: On 22 May 2012 a calibration standard of 200mg/l was prepared 

by adding 0.0200g ofHexahydro-1,3,5,-tris(2-hydroethyl)s-triazine to 100ml of water. This was 

analysed used on the following dates and conditions using the GC/FID 

23rd and 241h May 2012 

Phenomenex Chromatography Capillary Column: 

Zebron ZB5 (column phase: 5%-phenyl-95%-dimethylpolysiloxane) 

Part No: 7HM-C002-22 

30m x 1.0um film thickness x 0.32mm Internal Diameter. 

Various different temperature programs were attempted to see where the compound eluted from 

the system. The peak was not observed. 

25th May 2012 

J & W Chromatography Megabore Capillary Column: 

DB-624 (column phase: 6% Cyanopropylphenyl-94%-dimethylpolysiloxane) 

Part No: 125-1334 

30m x 3.0um film thickness 0.53mm internal Diameter. 

Carrier gas: Nitrogen 

Detector: Flame Ionisation Detector (FID) 

Injection port temperature: 250 C 

Detector temperature: 300 C 

Column temperature: JOOC isothermal 

Total analysis time: 6 minutes 

Sample volume: lui (Direct Aqueous Injection) 

A peak believed to be Hexahydro-1 ,3,5,-tris(2-hydroethyl)s-triazine was observed and 

successfully resolved from the solvent front. Peak eluted after approximately 1.1 minutes. From 

this point it was decided that this was to be the best capillary column for validation purposes. 

The peak appeared to be reproducible in terms of retention time and peak intensity. 

In order to determine that the peak identified was the sample, two more calibration standards 

were prepared on the 28 May 2012, a 503mg/l standard was prepared by 0.0503g ofHexahydro1 ,3,5,-tris(2-hydroethyl)s-triazine being added to 1 OOml of deionised water, a 20mg/l standard 

was prepared by adding 4ml of the 503mg/l stock to 1 OOml of deionised water. 

11th June 2012 

Using the conditions above (25th May 2012). The calibration standard (@20mg/l) was analysed 

to determine the approximate limit of detection. No peak was observed. The two further 

ENV9857/051201 Page 19 of26 
standards of 200mg/l and 500mgll were analysed. On each occasion peaks were observed. (with 

peak areas consistent with the concentration of the standard). 

With an estimated limit of detection at approximately I OOmgll, the method of analysis was 

deemed to be inappropriate to the study and no further attempts were made to improve the 

performance of the method. 

HPLC method development: On I2 June 20I2 a stock solution of IOOmg/l was prepared by 

0.0 I OOg of Hexahydro-I ,3,5,-tris(2-hydroethyi)s-triazine being added to I OOml of deionised 

water. 

25 June 2012 

Used a cyano column in normal phase with a mobile phase of 45:4:I (hexane:DCM:H20) at 

225nm isocratic 

1 OOmgll Triazine identical to water result (one small peak at about 3 mins) 

26 June 2012 

Used short CIS column (150mm) with 3:I MeCN:H20 at I95nm 

I OOmgll Triazine similar to water result with some small additional unresolved peaks at around 

1.5 mins 

27 June I2 

I95nm 3:I MeCN:H20 

I) Chiral column 

Some small peaks co-eluting with water peaks. Retention times less than 2 mins 

2) SAX column 

Some small chromatography at around 5 minutes 

3) No column 

Sample and solvent both eluted very quickly with sample peak about 5 times bigger in height 

(decent sized peak) 

4) NH2 column 

Some small peaks with extra small sample peak at about 3.5 mins 

5) Phenyl column 

ENV9857/05I20I Page 20 of26 
No difference between sample and solvent chromatography with two small peaks noted at 

around 1.2 and 2.5 mins 

On 28 June 2012 a fresh stock solution was prepared by adding 0.0105g of Hexahydro-1,3,5,-

tris(2-hydroethyl)s-triazine to 1 OOml of deionised water. 

28 June 2012 

195nm 3:1 MeCN:H20 

1) Different Cyano column 

Some small peaks in both acetonitrile and sample- small peaks at 2.6 and 3.7 mins 

2) Nocolumn 

Peak area of 100mg/l triazine is about 11 times bigger than acetonitrile (1188297 and 105435). 

Eluted straight away 

3) Another cyano column 

Small sample peak at about 0.3 mins 

4) Another p}1enyl column 

Sample and acetonitrile both hardly retained (0.25 mins)- no separation 

5) Another C 18 column 

Very small peaks for both sample and acetonitrile- no difference 

29 June 2012 

1) Gemini-NX C18 195nm 

Reasonably busy chromatograms with some small unresolved peaks eluting around 1.2 to 1.6 

minutes. 

2) Gemini-NX C18 225nm 

Not as busy at this wavelength with larger unresolved sample peaks at 1.3 and 1.6 mins (still too 

small) 

3) Hypersil C12 195nm 

Some small number of peaks with a small additional resolved sample peak at 0.6mins 

ENV9857/051201 Page 21 of26 
4) Gemini-NX Cl8 225nm 

Similar to 195nm with a small resolved sample peak at 0.6 mins 

3 July 2012 

Gradient method with 15min gradient from 100% H20 to 100% MeCN, return to 100% H20 

over a minute and equilibration for a further 4 minutes. 

225nm with a very short C 18 column. 

Busy baseline with additional, random, peaks. Sorted out random peaks but no peaks noted for 

sample which weren't in acetonitrile. 

In all this work, whilst some small peaks were noted for the various column/mobile 

combinations, they were too small as this was for a I OOmg/1 standard. When the column was 

removed, a much larger peak was noted (even after removing the solvent area) but the peak or 

peaks noted with all the various attempts were not as large suggesting that the sample was not 

being eluted (ie retained by the various columns). 

ENV9857 /051201 Page 22 of26 
Culture medium: 

Dilution water: 

Method of preparation: 

Composition of culture medium: 

ENV9857/051201 

Appendix 2 

Culture media 

According to OECD guidelines. 

De-ionised, filtered and sterilised by autoclaving at l20°C for 

15 minutes. 

Sterile nutrient stock solutions were prepared and added to 

the dilution water to obtain the culture medium. 

Nutrient Final concentration in 

culture medium (mWJ) 

NRtCl 15 

MgCh.6H20 12 

CaCh.2H20 18 

MgS04.?H20 15 

KH2P04 1.6 

FeCh.6H20 0.064 

N a2EDT A.2H20 0.1 

H3B03 0.185 

MnChAH20 0.415 

ZnCh 0.003 

CoCh.6H20 0.0015 

CuCh2H20 10-5 

Na2Mo04.2H20 0.007 

NaHC03 50 

Page 23 of26 
Appendix 3 

Water quality data 

Nominal pH Temperature 

concentration (OC) 

(mg/1) Start of test End of test 0 Hours 24 48 72 

Hours Hours Hours 

0 7.8 7.3 

I 7.8 7.3 

3 8.1 7.2 

10 8.7 7.3 21.1 20.3 20.3 20.4 

30 9.2 7.4 

100 9.7 7.7 

200 10.0 7.8 

Note: Water qualities determinations on pooled replicates for each test and control solution 

were carried out immediately prior to initiating the test and at the end of the 72 hour test 

period. 

ENV9857/051201 Page 24 of26 
Appendix4 

Reference test 

This section summarises aquatic toxicity test results obtained by Chemex International 

Environmental Ltd. on a sample as detailed below: 

Test commissioned by: Chemex Environmental International Ltd 

Substance under test: Potassium Dichromate 

Chemex reference: 

Test species: 

Test type: 

Test conditions: 

Test period: 

Test carried out at: 

Results: 

Period of 

exposure 

(hours) 

Oto 48 

0 to 72 

NOEC 

Historical Data: 

Mean: 

Standard deviation 

ENV9857 /051201 

Sample: 

Study: 

EC0110706 

ENV9820 

Pseudokirchneriella subcapitata, strain CCAP 278/4 

Growth inhibition: 72 hour EC50 

Test conditions according to SOP E203 based upon OECD 

Guideline for testing of chemicals, Alga Growth Inhibition 

Test reference 201 (2006) Ol. 

Council Regulation (EC) No 440/2008 of30 May 2008(2). 

From 5 to 8 March 20 12 

Chemex Environmental International Limited 

Unit J 

Broad Lane Industrial Estate 

Cottenham 

Cambridge CB24 8SW 

UK 

ECx and NOEC values by yield (EyCx) and growth rate (ErCx) 

ErCx value 

95% confidence limits 

EyCso (0 to 72 hours) 0.53mg/l 

ErCso (0 to 72 hours) 0.86mg/l 

EyCso (0 to 72 hours) 0.11 

ErCso (0 to 72 hours) 0.17 

Page 25 of26 
Appendix 5 

GLP Certificate 

f o-;;, Department 

C ofHealth 

THE DEPARTMENT OF REALm OF THE GOVERNMENT 

OF THE UNITED KINGDOM 

GOOD LABORATORY PRACI'ICE 

STATEMENT OF COMPLIANCE 

IN ACCOitDANCE WITH DIRI'.CTIVE ~ 

TEST FACILITY 

a.-.~ ............ Ltd 

UllltJ 

..... ._ ........... l:lale 

()!tteettam 

Culbl. 

CBJ48SW 

DATE OF INSI'I!C110N 

25N.-ber2t10 

A general iaspection for compliance with the Principles of Good Labol'lllory Practice 

was carried out at the above test facility as pan of the UK GLPCompliance Programme. 

AI the time of inspection no deviations were found of sufficient magnitude to affect 

the validity of IJOD.Ciinical studies performed at these facilities . 

Dr. Alldrew J, c..., 

H.d, UK GLPMeaitoriJI& Alldlorily • 

ENV9857/051201 Page 26 of26 
Chemical 

Corporation 

A Subsidi•ry of ICC lndustrlfl Inc 

West Fifteenth & Davis • _e; ' ! 19 • Dover, Ohio 44622-0040