LDJ\.. \..UIJLJUJ 1\IUIJIUt:::l 

359495 ,...nrr ,... __ 4--1 1\r .. _ .... __ 

RECF i \'ED ')~";'1"' t'nrc r f vD. 

20 I~ ftPR I 5 Mi II : 13 

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.com 
II 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 EC50, 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 

Final report and data inspection completed: 04/04/14 

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 EL50 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 of26 
the 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 EL50 (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 * 

K ey t o a bb revm . f IOns 

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 mill 
The 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 EL50 

1.0 

0.9 

0.8 

0.7 

c 

.9 

...... 0.6 :s 

:= 

c 

..... 

"; 0.5 

c 

.9 

..... 

-

0 0.4 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.> 

Q,l 100 .... 

~ 

= 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 

Na2Mo04.2HzO 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 

Na2Se03 0.0438 1000 1.0 

~vo3 0.0115 1000 1.0 

Na2EDT A.2H20* 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.2H20 146.90 500 1.0 

MgS04.7HzO 123.30 500 0.5 

NaHC03 32.40 500 1.0 

KCl 14.50 250 0.1 

NazSi03.9HzO 12.50 250 0.2 

NaN03 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 mill 
Appendix 2 

Environmental parameters 

pH values- Dissolved oxygen (mg/1) 

Concentration Test duration in days from date of initiation 

Control (0) 0 3 5 7 10 12 14 17 

i 

pH fresh 8.01 8.11 6.92 7.28 6.86 7.33 7.23 8.11 

aged - 7.02 7.39 7.11 7.29 7.37 7.26 7.85 

DO fresh 7.85 6.81 5.13 6.63 4.97 6.10 7.99 7.05 

mgll aged - 7.12 6.02 6.22 6.14 8.55 8.11 8.93 

Concentration Test duration in days from date of initiation 

0.5mg/l 0 3 5 7 10 12 14 17 

pH fresh 7.07 7.17 7.04 7.43 7.06 7.45 7.45 7.77 

aged - 7.25 7.43 7.70 7.34 7.50 7.35 7.63 

DO fresh 7.33 7.16 5.34 7.06 5.43 6.42 7.88 7.56 

mg/1 aged - 6.19 6.01 6.13 5.57 6.58 7.95 7.54 

Concentration Test duration in days from date of initiation 

1.3mgll 0 3 5 7 10 12 14 17 

pH fresh 7.11 7.28 7.18 7.47 7.14 7.62 7.44 7.75 

aged - 7.36 7.58 7.69 7.39 7.52 7.37 7.65 

DO fresh 7.45 7.01 5.10 7.01 5.26 6.35 7.84 7.20 

mg/l aged - 7.55 6.24 6.89 5.55 6.32 8.71 7.12 

Concentration Test duration in days from date of initiation 

3.2mgll 0 3 5 7 10 12 14 17 

pH fresh 7.14 7.42 7.22 7.55 7.17 7.73 7.52 7.57 

aged - 7.43 7.60 7.70 7.43 7.97 7.34 7.52 

DO fresh 7.11 6.56 5.24 7.02 .· 5.16 6.60 7.82 7.58 

mg/1 aged - 8.25 5.85 6.09 5.35 6.92 7.66 5.85 

E}fV10259/130506 Page21 of26 
Concentration Test duration in days from date of initiation 

8mg/l 0 3 5 7 10 12 14 17 

pH fresh 7.15 7.41 7.25 7.57 7.18 7.66 7.43 7.58 

aged - 7.46 7.65 7.32 7.48 7.55 7.76 7.60 

DO fresh 7.46 8.03 5.40 6.98 4.84 7.12 7.09 8.20 

mg/1 aged - 8.27 6.27 6.11 5.33 6.73 8.00 8.12 

Concentration Test duration in days from date of initiation 

20mg/l 0 3 5 7 10 12 14 17 

pH fresh 7.19 7.43 7.25 . 7.53 7.13 7.62 7.44 7.49 

aged - 7.46 7.68 7.41 7.45 7.73 7.53 7.47 

DO fresh 7:89 6.93 5.26 6.83 4.04 7.26 8.50 7.76 

mgll aged - 8.04 = 6.10 6.00 5.27 6.23 8.05 7.61 

Temperature ("C) within incubator 

Test 

duration 

in days 0 1 2 3 4 5 6 7 8 9 10 ·U 12 13 14 15 16 17 

from date 

of 

initiation 

Current 20.9 20.7 20.4 20.5 19.8 19.9 20.4 20.0 20.0 20.1 2(U 20.1 20.4 19.6 19.8 19.8 19.8 19.8 

Min 20.1 - - 20.2 19.8 19:8 19.9 19.9 - - 19.9 20.0 20.4 19.5 19.8 - - 19.7 

Max 21.1 - - 21.1 20.7 20.1 20.4 20.5 - - 20.3 20.4 20.5 19.6 20.5 - - 20.1 

- Minimum and maximum temperature not taken at the weekend 

E}fV10259/130506 Page 22 of26 
Appendix 3 

Number of offspring per individual parent Daphnia 

I Control 11234567 Test duration in days from date of initiation 

8 9 10 11 12 13 14 15 16 17 18 19 20 2

1 0 0 0 0 0 0 0 14 0 0 0 23 0 24 0 0 0 40 0 0 3

2 0 0 0 0 0 0 0 22 0 0 0 12 0 0 18 0 0 44 1 0 2

:... 

0 0 0 0 0 0 0 17 0 0 0 28 0 25 0 0 0 4I I 0 4

.. 3 

..Q 

e 4 0 0 :::: 0 0 0 0 0 10 0 0 32 0 0 I8 0 0 30 0 1 38 

= 5 0 0 0 0 0 0 0 10 0 32 0 0 0 21 0 0 27 0 0 20 

.. 

.... 

0: 6 0 0 0 0 0 0 0 11 0 7 0 25 0 0 35 0 0 45 0 0 2

7 0 0 0 0 0 0 0 12 0 0 34 0 0 30 0 0 39 0 0 46 

-a 

.. 8 0 0 0 0 0 0 0 10 0 7 34 0 0 16 0 0 38 0 1 55 

9 0 0 0 0 0 0 0 9 0 5 33 0 0 20 0 0 29 0 0 44 

10 0 0 0 0 0 0 0 0 29 0 0 0 20 0 0 33 0 0 50 0 

Cumulative 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 

mortalities 

I 0.5mg/l 11 Test duration in days from date of initiation 

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 2

1 0 0 0 0 0 0 0 0 19 0 0 24 0 0 0 13 0 22 0 0 2

2 0 0 0 0 0 0 0 0 19 0 0 19 10 0 0 17 0 0 26 0 

:... 

.. 3 0 0 0 0 M 0 0 

..Q 

e 4 0 0 0 0 0 0 0 0 15 0 0 21 10 0 0 12 0 0 26 0 

:::: 

= 5 0 0 0 0 0 0 0 0 17 0 0 28 0 0 18 0 0 33 0 0 3

.. 

.... 

0: 6 0 0 0 0 0 0 0 0 12 0 0 15 0 9 0 0 10 0 0 4 1

-a 7 0 0 0 0 0 0 0 0 18 0 0 18 0 1 18 0 0 24 0 0 3

.. 

8 0 0 0 0 0 0 0 0 16 0 0 25 10 0 0 17 0 1 34 0 

9 0 0 0 0 0 0 0 0 0 0 18 0 17 0 0 30 0 0 49 0 

10 0 0 0 0 0 0 0 0 0 0 16 0 0 10 0 0 19 0 0 29 

Cumulative 0 0 0 0 1 1 f 1 1 1 1 1 1 1 1 1 1 I 1 1 

mortalities 

-

M :- indicates mortality of adult Daphnia 

E}fV10259/130506 Page 23 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 2004/10/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 ~--
Number of offspring per individual parent Daphnia continued. 

8mgll Test duration in days from date of initiation, 

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17· 18 19 20 2

1 0 0 0 0 0 .0 11 0 0 . 

2 0 0 0 0 0 0 0 15 0 

.... 

a. 3 0 0 6 0 0 4 0 14 0 

.c 

s 4 M 

= c 5 0 0 M •' 

a. 

.... 6 0 0 0 0 0 4 0 . 16 -0 

7 0 0 0 0 0 _.() 7 1 0 

"i5.. 

a. 8 M .... 

9 0 0 0 0 0 0 0 11 . 0 

10 0 0 0 0 -- 0 ? 0 -. 16 0. 

Cumulative .. 3 -; s 

mortalities 0 0 1 1 1 1 1 1 1 2 2 2 2 3 . 3 3 3,; 3 

.. . -

·:.:::-: 

Test duration in days from date of initiation . 

20m gil 1 2 3 4 5 6 7 8 9 10 11 12 13 14 lS 16 17 .. . 18 19 29; 

1 M - .__. .. 

-· 

2 M " .. 

.... 3 M ... 

. . 

a. 

.c 

s 4 M - 

= c 5 M .. 

a. 

.... 6 M 

7 M 

"i5.. 

a. 8 M 

9 0 0 0 0 0. 0 0 0 0 

10 M . 

-

Cumulative ... 

mortalities 0 0 2 6 6 7 8 8 8 9 9 9 9 9 9 9 9 9 9. 9 

.. 

M :- indicates mortality of adult Daphnia 

E}fV10259/130506 Page 25 of26 
AT'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