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



### April 7, 2014

Document Control Office (DCO) (7407M) Office of Pollution Prevention and Toxics (OPPT) Environmental Protection Agency 1200 Pennsylvania Avenue, NW Washington, DC 20460

ATTN: TSCA Section 4 40 CFR 799.5089; Docket ID Number EPA-HQ-OPPT-2009-0112 Dover Chemical Study Plan for CAS# 72162-15-3

To whom it may concern:

Dover Chemical Corporation is submitting the attached Chronic Toxicity to Daphnia report based the EPA's HPV3 TEST PLAN AND DATA ADEQUACY REVIEW (Contract No. EP-W-09-027).

The Combined Repeat Dose Toxicity with Reproduction/Developmental Tox Screening (Reproductive Toxicity Developmental Toxicity) study is complete but under review before finalization and will be forwarded as soon as possible.

Should you have any questions, please contact me.
Sincerely,

Tim Nowicki Manager Product Compliance Dover Chemical Corporation 330-365-3610 tnowicki@doverchem.comII m 11

### Commercial-in-Confidence



### Chemex reference: 10259/130506

The effect of Sulfurated Decene on the reproductive output of 

### Daphnia magna

over a 21 day exposure period

Report issued by: 

### Report for Dover Chemical

Chemex Environmental International Limited UnitJ Broad Lane Industrial Estate Cottenham Cambridge CB24 SSW UK

April2014

E}fV10259/130506 Page 1 of26 

### Sponsor:

Dover Chemical 3676 Davis Rd N.W. Dover OHIO USA 44622

### Contents

. r,

. 

### Page Compliance withGood.Laboratory**Prac~ice** 

### standards ........•....•............................... , ............... 3

. Quality Assurance Statement

.................................................................................................. 4
Summary .00·.·.·····.-··········'······,····················,······,,.·::·········:·:··········::············:························:·············5
·1.

2.

2.1 

### 2.2 2.3 2.4


### 3.

3.1 3.2 

### 3.3 3.4

... ·'3.5·



### 4.


### 5. Intr,oduction

.. · .... ·

..... :

... ;

............... • ::.:

.... ·' ·'::

.. . :

.. ::

... ....... : •......... :·

.. :

... .'

. ..•. ::·

............ ;

. .• :

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

.......................................................... :

.. :

.............. :

......... :

......... 6

Test substance

............ ,., .... ····:·

... 000 ....• ,. .. 00

... , .. ,.**~·.·.-** 

### ... :.,ooooo ........•.... .'

.. 000000

.. 00

.... 00

...... 0000

........ 00

.... 6 Test species

.. ooooOoooo

..**ooooooooo.oooo.ooooo.oooooOooooooooo.•••o:•····oo,ooo~oooooooooooooo,ooooooOOOOOOOOOooooOOOooooooooooooooooo6** 

### Dilution water

... 0000········· 00000000·····

........ oooo·····ooooOoooooo··········

...... 000000········· oo•·····oo•oo••·oooo•· 7 Test methods and conditions •;·········,··oo···:oo·;oo

.. ·::·oo·-_···,:··oo

....... oooooooo •..... , ...... ooooooooooo'····· 7
Results

...... oo

... ooooooo•······•oooooooo············•oooooooooooooOoooo: •... ::

.. oo.oo·

... oo.:

........ oo

.......... oo .....••.. oooo09
Number of offspring per replica,te lilUd percent inhibition ofreproduction,

... oooooo00oooo.9 Summary of information on reprodt!ctio'il and mortalities 00

.. :·00. 

### 000000 00 00 >0000 0000

..**00~00.:** 

### 0000 00 9 ; 21-day EC 50 , 

### NOEL and LOEL values 000000000000000000

..**~::000000.:000000~0000000000000000000000000000000:.9** 

### Environmental parameters

...................... , .................................................................... 1 0 Physico-chemical properties t>f'dil uti on water

..... :

... .',: 00

..... 00

....... : 00 00

........ 00 00

.. 00 00 00

.. 00 0011

Discussion ······oo······oooo

........... oooo········oooo····ooooooooooooo•········ oo•··•oooooo•·······•oooooooooooo•·····oo··12 · References

.................................................................................................................. 12
Graphs



### 1 

### Estimation of the 21-day reproduction inhibition EC50 0000000000000000000000000000000000000000000013 2 Plot of total number ofliving offspring per concentrationOOOOOOOOOOOOOOooOOOooooOOOOooooooooo;

.. oo14 3 Plot of total number ofliving·offspring per surviving adult Dapliliiaoooooooooooooooooooooooo'I5 4 Reproductive rate curve

........**oo····''·oooooo••••oooo•··········•oooooooo•··········oooooooo•······oo:.~,** 

### .. ::

....... l6 5 Intrinsic growth rates according to Euler-Lotka equation (means and 95% confidence limits)

........ 000

.... 0000

... 0000.00

............. 000

......... 00

.. 00

.. 00000

....... 00000000

............ 000000

... 17 6 Plot of cumulative adult mortality rateOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOooOOOOooooOOOOOOooOOoooooooooo018 7 Plot ofweight of adults per concentration.at21 days .:oooo;oo;oooooooooooooooo

.... oooo0000oooo0000oo19

Appendices



### 1. Dilution water composition

............... oooooo···oo·························oooooo•·······:oooooo•····oo·········20

. 2. Environmental parameters

.. oooo·······oooooo···•oooo•············oo············oooo•······•oooooo•···········oo·

... 2i · 3. Number of offspring per individual parent Daphniaoo00oo00oo000000oooooooooooooooooooooooooooooooooo23 

### 4. Certificate of Analysis

.. oooo

......... oooo

........ oo

....... oo

........ oooooooo•···········oo········oo············oo····26

ENV1 0259/130506 Page 2 of26

II m II I-

### 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 I to the European Parliament and council Directive 2004/1 0/EC and Annex I 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 Instrument 3106) 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 /MC/CHEM(98) I7).

The analytical method development was performed outside of this study, no GLP compliance is claimed

.



# ........**\:\.k~** 

# ............................. . . ....... !±:

....**A~.ci\.** 

# ..**,~D.\.1!:** 

# .......................... . Heather Beattie Study Director Date



### Other key personnel at Chemex involved in this study were:

Sravanthi Chitikeshi Jadwiga Jonkisz Agnes Grabczewska

ENVt 0259/130506 Page 3 of26

## Quality Assurance Statement

The Quality Assurance unit inspects the final report to confirm that the methods, procedures and observations are accurately and completely described, and that the reported results accurately and completely reflect the raw data of a regulatory study.

This is achieved by conducting routine annual facility and system inspections at approximately I 2 monthly intervals. In addition, internal process-based audit programme is also adhered to at 3 monthly intervals. Where required, study specific inspections are also conduced. All study plans and amendments are verified by the QA unit to confirm compliance with GLP.

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

Study Number: ENV 10259 Study Title: The effect of Sulfurated decene on the reproductive output of Daphnia
magna 

### over a 21 day exposure period



### Procedures and Processes Type Date of inspection Date reported to Study Director I



### Mana2ement Aquatic crustacean test set up 

### p 09/12/13 17/12/13 Reproduction test set up 

### p 09/12/13 17/12/13 Weighing out test or reference materials 

### p 13/ll/13 10/12/13 Equipment calibration 

### p 13/ll/13 10/12/13 Labelling and paperwork 

### p 09/12/13 17/12/13 Organism stock records 

### p 09/12/13 17/12/13 Preparation of media/ solutions 

### I WAF 

### p 09/12/13 17/12/13 Taking and recording readings 

### p 09/12/13 17/12113 Key: P- Process-based, S- Study spec1fic, 0- other mspect1on type.



### This report has been inspected by the undersigned and, as far as can be reasonably established, the methods, procedures and observations are accurately and completely described and the results incorporated into this report accurately and completely reflect the raw data generated during this study.



### Final report and data inspection started: 13/03/14 04/04/14 

### Final report and data inspection completed:

Signed:



# ........

# ~:·· ............................... . Rasa Cooke 

### Quality Assurance Manager

Date:



# ......**9.~** 

# .. 

# l:Jp..{

..**~P!.t** 

# ........................... .

ENVI0259/130506 Page 4 of26

II m II I -I

### Summary

This section summarises aquatic. toxicity test ·results obtained by Cherriex Eriviroinnental International Ltd. on a sample as detailed below:'···

Test commissioned by: ·' Dover Chemical
Substance

### u.nd~r test:
Chemex reference:

Test species:

Test type:
Test guideline:

Test concentrations:

### Expo~ure conditions:

Preliminary phase: 

### 1st :Definitive phase: 2nd Definitive phase:

'festcarried out at: •**~** , I ; Sulfurated decene Sample:

. ·EC0130506,. ·,, , . Stud;r: ENV10259

.,i, 

### Daphnia magna 21-Day Reproduction test
OECD Guideline 

### for Testing' of Chemicals, 

### Daphnia magna, Reproduction Test, reference 211, OECD 2012. Expressed as WAF nominal concentrations = 0,. 0.5; 1.3, 3.2, 8 and 20mg/l as Sulfurllied decene. •'.>
<24h juveniles

### expos~d one per 1 OOml test solutions, 10 replicates for controls; lOreplicates per test concentration, located in jncubator set at 20°C with 16h light:8h dark cycle,

### semi~static. (3 times 'weekly) solution**replacem~nt.** . . .·. · ·' ·

6 to 23 N ovember:20 13 30 November to 20 December 2013 (not reported; see poirit 

### 2.4) OS to 

### 31. January 2014 (reported)
Chei:nex Environmental International ·Limited, Unit J, Broad Lane

•. Industrial Estate, 'Cotteriham, Cambridge, CB24 SSW, UK · ·


### Summary ,of results: ,21-cJ.ay 

### ELso, NOEL and LOEL**·c~** .
·' , , 

### ELso value in mg/1 

### P;arameter (95% 

### confid(mce'limits) . NOEL in mg/1 

### LOELin mg/1

Fecundity 0.830 (0.397 to 1.29) <0.5 0.5 Weight (mg) 1.14 (0.952 to 3.97) <0.5' 

### 0.5
Parent survival · 12.0 (6.93 to 15.2) 8

.. 20
Intrinsic rate of 3.81 (2.00 to 13.3) <0.5 0.5 natural increase

These are expressed as loading rates • of' the 'test material as supplied, as no analytical confirmation was perforrried.

Results were calculated using the Toxcalc v5.0(2) statistics program.

ENV1 0259/130506 Page 5 of26

### 1. 

### Introduction

This report contains a description of the methods used and the results obtained during a study to investigate the effect of Sulfurated decene on the reproductive output of 

### Daphnia magna according to OECD Test Guideline 211 

### 'Daphnia magna Reproduction Test' 2012 and Chemex SOP E277. The objective of this study was to compare the reproductive output of the animals exposed to the test substance compared to that of the controls in order to determine EL 50 and the lowest observed effect concentration (LOEL) and hence the no observed effect concentration (NOEL).

It was not possible to conduct analysis to confirm exposure concentrations. Attempts were made outside of this study including the use of GC/MS but a combination of the low solubility and Sulfurated decene containing multiple constituents meant that these attempts were unsuccessful.



### 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, UK.


### 2.1 Test substance

Identification:

Name as supplied: Supplied by:

Date of receipt: Expiry date:

Batch/Lot number:

CAS number: Purity & composition

Chemex reference: Required storage conditions:

Actual storage conditions:
Quoted appearance:
Observed appearance: Homogeneity: Sulfurated decene Mayco Base 1540

Dover Chemical 09May 2013

02 May 2015 (Sponsor's e-mail)

S-31343 (Certificate of Analysis)

72162-15-3
90-100%, 1-Decene, sulfurized (MSDS)

EC0130506

Ambient temperature

Room temperature, in the dark

Dark brown oily liquid

Dark brown oily liquid Homogeneous Quoted stability in container after opening: Stable
Quoted stability in water: Quoted solubility in water: Stable Negligible

Unless otherwise indicated the information was taken from the TSDS.

ENV1 0259/130506 Page 6 of26

II Ill II I

### 2.2 Test species

Test organism:

Source:

Date of gravid isolation: Age of Daphnia 

### at start of test:

Culture conditions:

Feeding regime:



### · 2.3 · Dilution water Daphnia magna


### Astra Zeneca (Brixham Environmental Laboratory) and Smithers Viscient

9 January 2014

Less than 24 hours
Temperature: 20 ± 

### 2°C Dissolved oxygen: >80% ASV Photoperiod: 16 hours light and 8 hours dark Every 1-2 working days, a suspension of Chiarella vulgaris 

### plus YCT at a rate of 1mg organic carbon/litre of culture water. ·

The stocks of animals were cultured, and the tests performed, in Elendt M4 media. The water

was adjusted to 18 to 22°C, aerated until the dissolved oxygen concentration had reached a minimum of 60% air saturation and the pH stabilised between 6 and 9 (Appendix 1 ).



### 2.4 Test methods and conditions

The toxicity test was carried out according to the procedures given below, based on the guidelines produced by OECD entitled Guideline for Testing of Chemicals, Daphnia magna 

### Reproduction Test, reference 211, OECD 2012. Preliminary phase
The preliminary test was run between 6 and 23 November 2013 at concentrations of 0 (control), 0.01, 0.1, 1 and 10mg/l nominal as Sulfurated Decene. There were two replicates of 100ml for each control and concentration. The ELso for the preliminary test was determined to be between 0.1 and 1mg/l nominal as Sulfurated Decene.

Definitive toxicity procedure

The first reproduction test was run between 30 November to 20 December 2013 at concentrations of 0 (control), 0.2, 0.5, 1.3, 3.2 and 8mg/l nominal as Sulfurated Decene. This
test was terminated as too few neonates in control were produced and there was less· inhibition than expected in the top concentration of 8mg/l. The data collected during this test was not reported however it was included in this study data pack.

The second reproduction test was run between 08 to 31 January 2014. The following concentrations were used in the second reproduction test: 0 (control), 0.5, 1.3, 3.2, 8 and 20'mg/l nominal as Sulfurated Decene.

For each test concentration, a wide bore glass tube was used to transfer one Daphnia, 

### in the minimum amount of culture water (<0.5ml), into ten replicate 120ml glass bottles. Each bottle contained 100ml of the test sample concentration. Ten control vessels were prepared with 1 OOml of dilution water, each containing a single Daphnia. 

### The test vessels were then placed into an incubator at a temperature of 19.5 to 21.1 °C with a light cycle of sixteen hours light and eight hours dark with a light intensity of 1000-1400 Lux. The test vessels were not aerated during the test period. To limit aerial contamination of

ENV10259/130506 Page 7 of26the test vessels and reduce evaporative losses, a transparent perspex sheet was placed over the tops of the beakers. There was no adjustment of the pH during the study.

The stock solutions were prepared in dilution water (Elendt M4 daphnia media) three times weekly. A stock of20mg/l was prepared by adding O.lg of sample into 5000ml of dilution water. This was prepared as a Water Accomodated Fraction (WAF) by stirring for 48 ± 3 hours and being left to settle for a minimum of 4 hours. This was diluted to give loading rates of 0.5, 1.3, 3.2, 8 and 20mg/l nominal as Sulfurated decene by adding 50, 130, 320 and 800ml of the appropriate stock to the corresponding 2 litres volumetric flask and making the volume with Elendt M4 daphnia media.

The pH value and dissolved oxygen were measured immediately after initiating the test in the control and each test concentration. The above measurements were also made in the new control and test concentrations and in pooled replicates of the old solutions, after transfer of the parent animals, for every solution renewal. The temperature (to an accuracy of ±0.1 

## oq of the incubator

was also recorded daily. The full water quality measurements are given in Appendix 2.

The mortality of the parent Daphnia and the number of offspring (neonates) produced by each parent animal were recorded every day. The presence, if any, of aborted eggs or dead offspring

were also recorded and removed along with the live neonates.
The parent Daphnia were fed daily with 0.5ml of a unicellular algal suspension (Chiarella vulgaris), which equates to 0.14mg organic carbon per litre of test solution.

Toxcalc v5.0( 2 ) statistics program was used to determine the EL 50 (the concentration resulting in 50% effect among the test population compared to the controls), LOEL (lowest observed effect concentration) and NOEL (no observed effect concentration) for each of the following four endpoints: 1) adult mortality after 21-days exposure, 2) adult growth as wet weights of surviving adults measured on day 21, 3) fecundity (numbers of juveniles produced per surviving adult over the 21-day exposure period) and 4) intrinsic

. rates of natural increase, this is a measure of population growth (calculated using the Euler-Lotka equation). Fecundity, growth and intrinsic rates of natural increase were determined, following tests for normality of distribution and equality of variance, using the Maximum Likelihood-Probit-test since the numbers of replicates differed between treatments. The parent survival used linear interpolation as the numbers of replicates were the same between treatments. LOEL and NOEL for adult survival were calculated using the Dunnett's test with the top concentration of 20mg/l removed as there was only replicate remaining and more than one replicate is required.

Deviations

There were a number deviations from the protocol;

The hardness of the Elendt M4 media was not measured and the calculated value was relied on. The control validity criteria were met and this deviation is thought to be insignificant.

The range finding study did not have a solvent control, the replacement days differed and a number of dates/time and signatures were not recorded on the lab book. These are thought to be administrative errors and would not have an impact on the outcome of the study.

Some of the Water Accommodated Fractions were only allowed to settle for 3 hours and 45 minutes rather than the minimum of 4 hours specified. The impact of these 15 minutes is thought
to be minimal due to the nature of the sample that stuck to the glassware and floated on the surface.**E~10259/130506** Page 8 of26

II m II I - I

### 3. Results

3.1 Number of offspring per replicate and percent inhibition of reproduction
Rep Control 

### (0)1 0.5 

# I 1.3 

# I 3.2 

# I 8 

# I 20

1 136 103 38 27 28 M 2 125 91 24 41 15 M 

### 3 152 M 36 42 24 M 4 129 84 38 12 M M 5 110 132 41 18 M M 6 150 68 39 36 48 M 

### 7 161 110 M 7 28 M 

### 8 161 103 41 39 M M 9 140 114 35 38 37 10 10 132 74 M 21 19 M
Total 1396 879 292 281 199 10
Adults surviving 10 9 8 10 7 1 Neonates/adult 140 97.7 36.5 28.1 28.4 10.00 

### % inhibition of 0.00 30.0 73.9 79.9 79.6 92.8

reproduction

M -mortality of daphnia

3.2 21-day EL50, NOEL and LOEL

Parameter 

### ELso value in mgll (95% confidence limits) NOEL in mg/1 LOEL in mg/l

Fecundity 0.830 (0.397 to 1.29) <0.5 0.5 Weight (mg) 1.14 (0.952 to 3.97) <0.5 0.5
Parent survival 12.0 (6.93 to 15.2) 8 20
Intrinsic rate of 3.81 (2.00 to 13.3) <0.5 0.5 natural increase

These are expressed as loading rates of the test material as supplied as no analytical confirmation

was performed.

ENV1 0259/130506 Page 9 of26

### 3.3 Summary of information on reproduction and mortalities

Concentration (mg/1) Control.(O) 

### 0.5 1.3 3.2 8 

### 20

Number of adult mortalities 0 1 2 0 3 9

Total number of neonates 1396 879 292 281 199 10 Mean number of broods per 5 4.44 2.86 2.40 2.14 1.00 adult Mean number offspring per 27.9 22.0 12.7 11.7 13.3 10 brood Number of days to first brood 8-9 9-11 13-14 12-18 14-19 21

Number of days to second 10-13 12-14 14-19 14-21 17-21 No second brood brood Number of aborted broods 0 0 0 0 0 0 Number of males 0 0 0 0 0 0 Mean intrinsic Rates of 0.473 0.446 0.284 0.226 0.214 0.110 Natural Increase Parental Daphnids: mean wet 8.03 5.14 3.71 3.76 3.09 2.10 weight (mg)

Where number of offspring in brood = 3 or less, not counted as a full brood for determination of the mean number of broods.

For full data on numbers of offspring and date produced, see Appendix 3.

3.4 Intrinsic rates of Natural Increase (IR) determined using the Euler-Lotka equation 

### l:(eA*IR)*Sx*Jx = 1

Replicate

### -----------~-q!!~~I}~.I:~~}QI_l~-~r-~_':.I!W.~~s-~J~~~!I_l_g**!"~!~-~~-~~!f!J:t.:!l_~~~P..~~~!l-~-----------** no. Control 

### (0) 0.5 1.3 3.2 8 

### 20

1 0.425 0.469 0.286 0.190 0.208 M 

### 2 0.512 0.470 0.273 0.222 0.191 M 

### 3 0.473 M 0.279 0.239 0.260 M
4 0.467 0.457 0.278 0.180 M M 

### 5 0.490 0.468 0.289 0.206 M M
6 0.464 0.433 0.293 0.250 0.229 M
7 0.480 0.463 M 0.227 0.197 M
8 0.472 0.465 0.302 0.235 M M
9 0.454 0.408 0.270 0.292 0.200 0.110
10 0.493 0.381 M 0.215 0.211 M
MeaniR 0.473 0.446 0.284 0.226 0.214 0.110
SD 0.024 0.032 0.011 0.032 0.024 *
CV% 5.00 7.18 3.80 14.1 11.1 *
ey o a revm IOns K t bb

. f
IR = Intrinsic rate of natural increase lSD = Standard deviation 

## cv = coefficient of variation IM =mortality of parental mortality 

## * = could not be determined as only one daphnia remaining 

### 3.5 Environmental parameters

ENV1 0259/130506 Page 10 of26

II millThe environmental parameters, dissolved oxygen and pH of the test were recorded at the start of the study, before and after replacement of test solutions and at the end of the study, and are detailed in Appendix 2. Temperature ( 0 C) of the incubator was monitored and recorded daily.



### 3.6 Physico-chemical properties of dilution water

The composition of the dilution water is given Appendix 1. The dilution water was aerated until the dissolved oxygen equalled the air saturation value.

The dilution water has a standard hardness of 199.88mg CaC03/l (calculated from the chemical composition ofElendt M4 media).

ENV1 0259/130506 Page 11 of26

### 4. Discussion

Using the parameter of fecundity, that is live offspring per parent Daphnia 

### alive at the end of the
test the 21-day reproduction the ELso value of Sulfurated Decene to Daphnia magna 

### was determined to be 0.830mg/l by loading rate. The highest no observed effect concentration (NOEL) after 21 days was determined to be <O.Smg/1 as Sulfurated Decene by loading rate. The lowest observed effect concentration (LOEL) after 21 days was O.Smg/1 as Sulfurated Decene by loading rate.

The 21-day NOEL for parent Daphnia 

### survival was determined to be 8mg/l as Sulfurated Decene by loading rate. The 21-day LOEL for parent Daphnia 

### survival was determined to be 20mg/l as Sulfurated Decene by loading rate,

None (0%) of the twenty control parent Daphnia 

### died during the study and no Daphnia 

### were trapped at the surface of the water. The number of live offspring produced per parent animal surviving to the end of the test in the control vessels was 110 to 161, with a mean value of 139.6. The coefficient of variation for control fecundity was calculated as 11.8%. This fulfils the validity criteria of the study which states that adult control mortality must not exceed 20% at the end of the test, that the average fecundity per parent Daphnia 

### surviving at the end of the test will be equal. to or greater than 60 and that the coefficient of variation around the mean number of living offspring produced per parent Daphnia 

### would be gs%. The full record of mortalities and live offspring per parent Daphnia 

### for the controls and at each test concentration are given in Appendix 3.



### 5. References

1. OECD Guideline for Testing of Chemicals, "Daphnia magna, 

### Reproduction Test" reference 211, OECD 2012.

2. ToxCalcTM Version 5.0 "Comprehensive Toxicity Data Analysis and Database Software", copyright 1994-1996.

ENV10259/130506 Page 12 of26

II mill

### Graph 1

Estimation of the 21-day reproduction inhibition EL 50

1.0

0.9

0.8

0.7

c 

### .9

...... 0.6 

# :s 

# := c



### ..... "; 0.5
c 

### .9



### .....


# - 0.4 0 Q. 0



# -**~** 0.3

0.2

0.1

0.0

0.0001 0.001 O.Ql 0.1 10

Concentration mg!L

Number of Daphnia magna per test vessel= 1 Number of replicates per controls= 10 Number of replicates per test concentration= 10 Concentrations are as a loading rate of Sulfurated Decene

Statistical data- Fecundity

21 day ELsa value 0.830mg/l 100 1000

(as determined by Maximum Likelihood-Probit)

95% confidence limits 0.397 to 1.29mg/l

NOEL LOEL <O.Smg/1 by Dunnett's Test O.Smg/1 by Dunnett's Test 10000

Shapiro- Wilk's test indicates normal distribution (p>O.Ol), Bartlett's Test indicates equal variances (p = 0.03).

E}fV10259/130506 Page 13 of26

### Graph 2

Plot of total number of living offspring per concentration

1000



### " 0 '-1:1



# .. l:: !l 800 " 

## " 0 " 1il 

### "'**~** 

# .. " 0 600 " 

## " ]
0 E-<

400

0 0.5 1.3 3.2 20
Loading concentration (mgfl)

Loading rate of Sulfurated Decene (mg/1)
Control 

### 0.5 1.3 3.2 8 20 (0)

Number of adults alive 

### 10 9 8 10 7 1

Total number of

offspring per 

### 1396 879 292 281 199 10 concentration

ENV10259/130506 Page 14 of26

II mill

### Graph 3

Plot of total number of living offspring per surviving adult 

### Daphnia

;::: 140 

# = "0 llil**~** 

# = 120 

### ·;;: 

## .E



# =**~** '"' 100 cu c.. 

## !**~** 

### 80 0 cu 

## =

..... 0 '"' 60 cu ,.Q


# = z 40

20

0
0 0.5 1.3 3.2 8 

### 20 Loading rate (mg/1)

The graph above displays the number of offspring produced for each adult Daphnia alive at the end of the 21-day test period. The table below shows the number of adults alive and the mean number of offspring per adult produced for each test concentration. For full details of offspring produced per replicate, see Appendix 3.



### Loading rates of Sulfurated Decene (mg/1) Control 

### (0) 0.5 1.3 3.2 8 

### 20 Number of 

### 10 9 8 

### 10 7 

### 1 adults alive Mean number of offspring per 140 97.7 36.5 28.1 28.4 10.0 

### surviving adult

E}fV10259/130506 Page 15 of26

### Graph 4
Reproductive rate curve

160



## .... :; "0 140**~** ell .5 

### .e; > 120 ;.... 

# = 17.> ;.... Q,l 0..

17.> 100 Q,l



## ....**~** 

# = 0 Q,l 

# = 80



## .... 0 ;.... Q,l ..c 

## e 60 

# = 

# = =**~** Q,l 

## e 40 Q,l 

### .e;



## ....**~** 

### :; 

## e 20 

# = u

0 

# /·-

/ 

# ____-I

• 

# / /



# . 

# --· •



# __ / //

....---· 

# /+ .--· .-·


# ___ ./

# /~·------· 

# --·**//·/~** 

# 1-:--·- /.-A"**·~"1'**

.. .. "

# .~:~--~·~--~**~~~/** /.**1--:e:-,--:•~:·~·~·~·~·~•--r-•--r-•--r-•** 0-10 11 12 13 14 15 16 17 18 19 20 21


### Time (Days) 

# 1-•- Control (0) -•- 0.5 -•- 1.3 -x- 3.2

### ---i~ 8 -•- 20 

# I

Cumulative mean number of offspring per surviving adult 

### Daphnia Days 

### 0-10 11 

### 12 13 

### 14 15 16 17 18 19 20 21
Control 19.5 32.8 41.6 43.6 59.0 64.3 67.6 83.9 101 106 127 140 

### (0)
0.5 12.9 16.7 33.3 38.6 40.8 44.8 54.7 57.9 66.8 81.8 85.4 97.7


### 1.3 0 0 0 2.00 8.75 8.75 8.75 17.3 17.9 27.3 34.0 36.5


### 3.2 0 0 0.3 0.3 1.9 1.9 1.9 5 9.3 15.3 17 28.1



### 8 0 0 0 0 0.86 0.86 0.86 7.14 5 15.4 15.4 28.4


### 20 0 0 0 0 0 0 0 0 0 0 0 10

ENV1 0259/130506 Page 16 of26

II m II I

### Graph 5

Intrinsic growth rates according to Euler-Lotka equation (means and 95% confidence limits)

1.0

0.9

0.8

0.7



## = 0**~** 0.6 

# :.c :a = '"" '"; 0.5 

## = .s



# - .. 0.4 0 c. 0 

# ..**~** 0.3

0.2

0.1

0.0

0.0001 0.01 1 100 10000 1000000

Concentration mg!L

Loading rates of Sulfurated Decene (mg/1)_ Control (0) 0.5 1.3 3.2 8 20 Intrinsic 0.473 0.446 0.282 0.226 0.214 0.110 2rowth rates 

## % inhibition 0.00 5.71 39.96 52.22 54.76 76.74



### E}fV10259/130506 Page 17 of26

### Graph 6

Plot of cumulative adult mortality rate

10

9 

# I 

# !

- 

# j 7 

# 1 

# I I j 

# I 

# !I - 

# I 2

0 

# f!

2 4 6 7 9 10 11 12 13 14 15 16 17 18 19 20 21 22

Number of days

1-+-contro1(0mg/1) --e-o.5mg/1_._1.3mg/1**~3.2mg/l** --+-Smg/1 --.-20mg/ll



### Cumulative number of adult mortalities at each day after test initiation
Days 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21


### Control 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

### (0) 0.5 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 

### 1.3 0 0 0 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 

### 3.2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

8 0 0 0 1 1 1 1 1 1 2 2 2 2 2 3 3 3 3 3 3 3 3 

### 20 0 0 0 2 6 6 8 8 9 9 9 9 9 9 9 9 9 9 9 9 9 9

ENV1 0259/130506 Page 18 of26

II mill

### Graph 7

Plot of weight of adults per concentration at 21 days

90



### .... 

# \ \ 80

70 

# \ 

# "**~~** T

# ~**~**

.·. ,· .·.

# ~ 20

10

0
0 0.5 1.3 3.2 8 20
Loading concentration (mg/1)

Loadin2 rates of Sulfurated Decene (m2/l) Control 

### (0) 0.5 1.3 3.2 8 

### 20 Average weight of surviving 8.03 5.14 3.71 3.76 3.09 2.10

adult (mg)
% inhibition 0 35.9 53.8 53.2 61.6 73.9

ENV10259/130506 Page19 of26

### Appendix 1

Dilution water composition

Elendt M4:

Table 1 Preparation of Stock 2

Composition Weight required (g) Volume (ml) Volume in Stock 2 H3B03 5.719 100 1.0 MnClz 0.457 100 1.0

LiCl 0.612 100 1.0 RbCl 0.142 100 1.0 SrClz_6HzO 0.304 100 1.0 NaBr 0.032 100 1.0 Na 2 Mo04.2HzO 0.123 100 1.0

CuClz.2HzO 0.0335 100 1.0 ZnClz 0.0260 100 1.0 CoClz.6HzO 0.02 100 1.0 KI 0.065 1000 1.0 Na 2 Se03 0.0438 1000 1.0**~vo3** 0.0115 1000 1.0 Na 2 EDT A.2H 2 0* 0.5 100 20.0

FeS04.7HzO* 0.1191 100

*these are prepared as above then once dissolved poured together and autoclaved immediately.

Table 2 Preparation of test medium

Each nutrient is prepared individually and then added to the test media as described below as well as 50ml ofstock2 (description of preparation illustrated in above table).

Composition Weight required (g) Volume (ml) Volume in lL of test media CaClz.2H 2 0 146.90 500 1.0 MgS04.7HzO 123.30 500 0.5

NaHC0 3 32.40 500 1.0

KCl 14.50 250 0.1 NazSi03.9HzO 12.50 250 0.2 NaN0 3 0.6850 250 0.1 KHzP04 0.3575 250 0.1 KzHP04 0.4600 250 0.1 Combined vitamin 0.1 stock 

### - -

Table 3 Preparation of Combined vitamin stock

The combined vitamin stock is prepared by adding the following vitamins to 1L of deionised water. Composition Weight required (g) Thiamine hydrochloride 0.75 Cyanocobalamine (B12) 0.01 Biotin 0.0075

ENV10259/130506 Page 20 of26

II millAppendix2EnvironmentalparameterspHvalues-Dissolvedoxygen(mg/1)ConcentrationTestdurationindaysfromdateofinitiationControl(0)0357101214171921ipHfresh8.018.116.92 7.286.867.33 7.238.117.34-aged-7.02 7.397.117.297.37 7.267.85 7.457.23DOfresh7.856.815.136.634.976.107.997.055.15-mgllaged-7.126.026.22 6.148.558.118.936.405.67ConcentrationTestdurationindaysfromdateofinitiation0.5mg/l0357101214171921pHfresh7.077.177.047.437.067.45 7.457.777.61-aged-7.257.437.707.347.50 7.357.637.607.49DOfresh7.337.165.347.065.436.427.887.567.13cmg/1aged-6.196.016.135.576.587.95 7.547.625.59ConcentrationTestdurationindaysfromdateofinitiation1.3mgll0357101214171921pHfresh7.117.28 7.187.47 7.147.627.447.757.60-aged-7.36 7.58 7.69 7.397.527.377.657.547.51DOfresh7.457.015.107.015.266.357.847.207.41-mg/laged-7.556.246.895.556.328.717.126.936.37ConcentrationTestdurationindaysfromdateofinitiation3.2mgll0357101214171921pHfresh7.147.42 7.227.557.177.737.527.577.52-aged-7.437.60 7.707.437.977.347.527.487.52DOfresh7.116.565.247.02.·5.166.60 7.827.58 6.90-mg/1aged-8.255.856.095.356.927.665.856.736.69E}fV10259/130506Page21of26ConcentrationTestdurationindaysfromdateofinitiation8mg/l0357101214171921pHfresh7.157.417.257.577.18 7.66 7.437.587.53-aged-7.46 7.657.327.48 7.557.767.607.46 7.75DOfresh7.468.035.406.984.847.127.098.206.73-mg/1aged-8.276.276.115.336.738.008.126.03ConcentrationTestdurationindaysfromdateofinitiation20mg/l0357101214171921pHfresh7.197.437.25.7.537.137.627.44 7.497.45-aged-7.46 7.687.417.457.73 7.537.477.337.49DOfresh7:896.935.266.834.047.268.507.769.18-mgllaged-8.04=6.106.005.276.238.057.616.797.85Temperature("C)withinincubatorTestdurationindays01 2 345 678910·U12131415 161718 192021fromdateofinitiationCurrent20.920.7 20.4 20.519.819.920.420.020.020.12(U20.120.419.619.819.8 19.8 19.819.719.919.8Min20.1- -20.219.819:819.9 19.9- -19.920.020.419.519.8- -19.7 19.719.519.719.8Max21.1- -21.120.720.120.420.5- -20.320.420.519.620.5--20.120.220.020.020.1-MinimumandmaximumtemperaturenottakenattheweekendE}fV10259/130506Page22of26Appendix3NumberofoffspringperindividualparentDaphniaIControl11234567TestdurationindaysfromdateofinitiationTotalWeight8 9101112 1314 15 161718192021offspring(mg)1000000014000230240004000351366.62000000022000120018004410281258.I:...000000017000280250004II040..31526.3..Qe400::::0000010003200I80030013801298.I=5000000010032000210027002001107.3......6000000011070250035004500271508.70:7000000012003400300039004601618.4-a..8000000010073400160038015501619900000009053300200029004401409.21000000000290002000330050001328.6Cumulative000000000000000000000Meanmortalities139.68.03I0.5mg/l11TestdurationindaysfromdateofinitiationTotalWeight23 456 7 89101112 131415 161718192021offspring(mg)1000000001900240001302200251034.2200000000190019100017002600915.2:...30000M00MeM....Qe400000000150021100012002600844.7::::=5000000001700280018003300361325.I......60000000012001509001000418684.50:700000000180018011800240031110-a4.8..800000000160025100017013400I036900000000001801700300049001146.6100000000000160010001900290745.2Cumulative000011f1 111 1 1 1 11 1I11 1Meanmortalities97.7I5.I4-M:-indicatesmortalityofadultDaphniaE}fV10259/130506Page23of26

## 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 2004/10/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 Instrument 3106) 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 /MC/CHEM(98) 17).

The analytical method development was performed outside ofthis study, no GLP compliance is claimed

.



# ........**\:\.k~** 

# ............................. . Heather Beattie Study Director 

# . ....... 4.:

.... Af.ci\

....**;bO.\.~** 

## .......................... . Date

Other key personnel at Chemex involved in this study were:

Sravanthi Chitikeshi Jadwiga Jonkisz Agnes Grabczewska

ENV 1 0259/130506 Page 3 of26

II m II I**~--**NumberofoffspringperindividualparentDaphniacontinued.8mgllTestdurationindaysfromdateofinitiation,TotalWeight123 4 5 6 7 891011121314 151617·18192021offspring(mg)100000.01100.17283.3200000001500152.5....30060040140024-·3.3a..c6s4M=00c5M•'0a.I....60000040.16-028482.7'!700000_.()71020284.1"i5..a.8M....09000000011.02631.2.4.100000--0?0-.160.0..1'93.3Cumulative..3-;sMeanmortalities0011 1 1 1 1 12 22 23 .3 33,;33'28:433.09...-·:.:::-:Testdurationindaysfromdateofinitiation.TotalWe\ght20mgil1 2 345 6 7 8910 111213 14lS1617...181929;21offsprjng(m,g)1M-.__....0..-·2M"..-....3M......0:..a..cs4M-'··O=c5M..0a.....6M07M0"i5..a.8M0·•900000.000010102;110M.-0Cumulative...Meanmortalities0026678 8 8 9 99 999 9 9 99.9 910__l2.1..M:-indicatesmortalityofadultDaphniaE}fV10259/130506Page25of26AT'I'EN'l'ION CUSTOMER CUSTOMER ORDER# OHW NUMBER SHIPPING DATE CONTAINER NUMBER FAX NOI4BER

PRODUCT: MANUFACTURED: QUANTITY: BATCH NUMBER:

TEST NAME
SULFUR, 't coc SG/60F SUS/100F 

### Appendix 6

Certificate of Analysis

DOVER CHEMICAL-HAMMOND WORKS 3000 SHEFFIELD AVE. HAMMOND, IN. 46320 (219) 331 2630 CERTIFICATE OF ANALYSIS

MAYCO BASE 1540 CODE: MFG SITE: HAMMOND,

S31343

LOWER OPPER RESULT FOR SPEC SPEC THIS LOT

37.0 39.0 38.1 360.0 N/A

.. 365.0 1.055 1.075 1.070 400.0 700.0 496.0

ANALYZED BY: QC LA130RATORY CERTIFIED BY: DAN ATTEBERRY MANAGER, QUALITY INDIANA

TEST METHOD

CNTM096 ASTM 092 ASTM D1475 ASTM D445

This product is supplied·under·an IS09001:2008 certified process. This certificate of Analysis represents the results of testing for the quaHty of this shipment.

ENVl 0259/130506 Page 26 of26

II m II