### A-1 O ctober 2011

Document No. 12699-001-500 

# A ppendix A

Select photographs of different phases of the brown trout reproductive study

### Document No. 12699-001-500 A

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### 1 December 2008

Photo 1: Egg cups used for hatching of brown trout eggs; 10 replicates with 60 (target) eggs / replicate.

Ph oto 2: Egg cup with eggs at test initiation. Photo includes egg pickers, container of remaining eggs for analytical, and counter.

### Document No. 12699-001-500 A

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### 2 December 2008

Photo 3: Eggs in egg cups for CC-350-008 brown trout eggs; 10 replicates with 60 eggs / replicate.

Ph oto 4: Close up of eggs in egg cup at test initiation.

### Document No. 12699-001-500 A

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### 3 December 2008

Photo 5: Water bath covered with black curtain to keep brown trout eggs in the dark during initial stage.

Ph oto 6: Test chambers in water bath for brown trout reproductive study during swim-up stage.

### Document No. 12699-001-500 A

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### 4 December 2008

Photo 7: Dilutor panel (one of two) used to feed adjusted Horsetooth water to each testing chamber.

Ph oto 8: Separation of water bath into light conditions for swimup fry and dark for pre swimup yolk sac fry.

### Document No. 12699-001-500 A

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### 5 December 2008

Photo 9: Brown trout fry in test chamber at swim-up (prior to thinning).

Ph oto 10: Brown trout fry in test chamber at swim-up (after thinning to 100 organisms).

### B-1 O ctober 2011

Document No. 12699-001-500 

# A ppendix B

Wet weight estimation for adult fish from Saratoga hatchery and raw data for adult fishData from Brown trout Parental Study, 12699-001
filename: adult data.xls

T reatment Total #

eggs Adult fish

length (mm) Adult fish weight (g) - CAS Adult fish weight (g) Predicted

adult fish wt (g) Se - wholebody (mg/kg dw) Se Egg (mg/kg dwt) SC -001 4,173 498 1,393 1 855.4 3.6 0.76 SC-002 4,005 420 826 1 088.9 4.1 0.94 SC-003 5,120 520 1,553 2 071.6 3.7 0.83 SC-004 1,248 562 2,500 3 350.1 4.3 0.92 SC-005 5,448 558 2,187 2 927.5 3 1.2 SC-006 3,176 439 842 1 111.0 3.1 1.2 SC-007 3,224 449 1,175 1 560.9 2.7 1 SC-008 4,005 494 1,446 1 926.9 2.5 0.96

SPC-001 0.73 SPC-002 0.73 SPC-003 0.73 SPC-004 0.73 SPC-005 0.73 SPC-006 0.73

High LSV2C-002 1,096 304 221 280 271.2 8.9 12.8 L SV2C-003 474 300 217 260 266.6 13.8 40.3 LSV2C-004 766 290 219 260 268.6 17.9 36 LSV2C-005 476 294 217 250 265.6 13.6 26.8 LSV2C-006 3 46 335 420 424.9 17.2 26.9 LSV2C-007 773 315 243 290 301.8 6.7 18.6 L SV2C-008 372 296 194 230 234.8 9.6 17.7 L SV2C-010 161 311 278 314 348.9 22.6 38.8 LSV2C-012 1,031 360 341 4 33.6 7.2 13.2 LSV2C-016 826 300 198 260 239.8 9.2 13.4 L SV2C-017 447 341 275 310 344.3 13.2 20.5 L SV2C-019 693 330 282 364 354.0 8.6 12.5 L SV2C-020 525 280 198 241 241.0 11.3 11.2 L SV2C-021 1,208 307 246 317 305.4 20 28.1

Bkd CC-150-009 1,215 324 520 315 * 675.3 8.4 12.8 C C-150-011 488 342 303 351 382.4 5.6 8.4 C C-150-012 556 317 232 269 286.1 6.7 8.5 C C-150-013 1,234 332 283 376 355.1 5.9 8.4 C C-150-015 1,003 313 213 281 260.1 6 9.1 C C-150-016 1,658 391 468 621 604.7 7 7.5 C C-150-017 414 265 150 178 175.6 5.6 6.6 C C-150-018 959 308 224 279 275.0 4.7 6.9 C C-150-020 1,332 310 238 318 294.9 7.2 6.2

Low CC-350-006 1,154 370 373 475 477.0 9.2 14 C C-350-007 1,174 350 321 416 407.3 5.5 6.9 C C-350-008 922 335 254 341 315.7 8.5 9.5

m in (field) 161 265 150 178 176 4.7 6.2 max (field) 1658 391 520 621 675 22.6 40.3

* value not included in developing wet weight estimate s for SC hatchery fishRelationship between wet wt data of field fish from CAS and from field measurements.y = 1.351x - 27.062R2= 0.96301002003004005006007000 100200300400500600700wt (CAS)

wt (field)Note: dashed line represents a slopeof 1.0

### C -1 October 2011

Document No. 12699-001-500 

# A ppendix C

Egg counts for Saratoga hatchery fish (SC) and field collected fish

### Estimation of Brown trout eggs numeration using volume-estimating technique, 12699-001

October 25, 2007 Hatchery Eggs from Saratoga National Fish Hatchery (SNFH, Saratoga, WY)



### The number of eggs placed into each study was counted manually. For all of the treatments, the remaining

n umber of eggs was estimated using a volume technique to develop a #egg/volume ratio for that particular female. The technique consisted of counting the number of eggs that filled a graduated cylinder to a particular volume (e.g., 50 mL) to determine the # of eggs per mL for that female. Initially we conducted two separate counts by two different staff personnel. Based on the agreement of these counted numbers for the particular volume, we subsequently only conducted counts once per female. Using this ratio, we then calculated the total number of remaining eggs for the total volume of eggs measured in a graduated cylinder. The total number of eggs used to initiate the studies (i.e., 600) was then added to the estimated number of remaining eggs to determine the total number of eggs for that particular female trout.

# Eggs placed # eggs counted Avg # eggs Vol of eggs Avg # Total vol (ml) Total 

### Treatment in study Count# to est. #eggs/vol counted counted (ml) eggs/mL of remaining eggs # eggs 

### SC-001 6 00 1 1058 1051 100 10.51 340 4,173 

### 2 1 044 100 SC-002 600 1 570 567.5 50 11.35 300 4,005 

### 2 5 65 50 SC-003 600 1 452 452 50 9.04 500 5,120 

### SC-004 6 00 1 506 506 50 10.12 64 1,248 

### SC-005 6 00 1 480 480 50 9.6 505 5,448 

### SC-006 6 00 1 477 477 50 9.54 270 3,176 

### SC-007 6 00 1 495 495 50 9.9 265 3,224 

### SC-008 6 00 1 532 532 50 10.64 320 4,005 Avg 3,800 Geomean 3,525 SD 1,305 CV 34%

### Estimation of Brown trout eggs numeration using direct counts and volume-estimation technique, 12699-001 November 16 & 17, 2007 Treatment eggs from background, low, and high treatment areas (ID)



### The number of eggs placed into each study was counted manually. For almost all of the treatments, the

rem aining eggs were also counted manually. When there were too many eggs to count, the number of remaining eggs were estimated using a volume technique to develop a #eggs/volume ratio for that particular female. The technique consisted of counting the number of eggs that fill a graduated cylinder to a particular volume (e.g., 50 mL) to determine the # eggs per mL for that female. Using this ratio, we then calculated the total number of remaining eggs for the total volume of eggs measured in the graduated cylinder. The total number of eggs used to initiate the studies (e.g., 600) was then added to this estimated number of remaining eggs to determine the total number of eggs for that particular female trout.

# Eggs placed # Remaining # eggs counted Vol of eggs Avg # Total vol (ml) Total 

### Treatment in study eggs counted to est. #eggs/vol counted (ml) eggs/mL of remaining eggs # eggs High 

### LSV2C-002 600 496 1,096 

### LSV2C-003 400 74 474 

### LSV2C-004 500 266 766 

### LSV2C-005 300 176 476 

### LSV2C-006 eggs dead / dying upon arrival (not counted) LSV2C -007 500 273 773 

### LSV2C-008 300 72 372 

### LSV2C-010 100 61 161 

### LSV2C-012 600 431 1,031 

### LSV2C-016 600 226 826 

### LSV2C-017 300 147 447 

### LSV2C-019 500 193 693 

### LSV2C-020 400 125 525 Avg 

### LSV2C-021 600 608 1,208 681

Bkd 

### CC-150-009 600 615 1,215 

### CC-150-011 300 188 488 

### CC-150-012 350 206 556 

### CC-150-013 600 634 1,234 

### CC-150-015 600 403 1,003 

### CC-150-016 600 460 50 9.2 115 1,658 

### CC-150-017 250 164 414 

### CC-150-018 600 359 959 Avg 

### CC-150-020 600 732 1,332 984

Low 

### CC-350-006 600 554 1,154 

### CC-350-007 600 574 1,174 Avg 

### CC-350-008 600 322 922 1,083

### D -1 October 2011

Document No. 12699-001-500 

# A ppendix D

Summary of fungal treatment methods for SC Hatchery eggs and field collected eggs

### 1



### T reatment of brown trout eggs for fungal control. Study # 12699-001

Different methods were employed in order to help control fungal growth and improve overall success rate of the brown trout eggs in the reproductive study. As mentioned in the report, this primarily affected only the SC hatchery eggs because they were the first batch to get in. And additional methods were employed to reduce fungal growth with the field collected eggs (initiated Nov. 16 & 17, 2008) and the second set of hatchery eggs (initiated Dec. 4, 2007).

November 2, 2007 : 1 % salt (as NaCl) solution was added to SC-002 and SC-004 chambers (i.e., 50 g of NaCl was mixed with small amount of water from each test chamber until all salt was dissolved and added). Measured chloride concentration at 5,620 mg/L. Time = 1700 hrs.

Conductivity check at 1920 hrs: SC-004 @ 12,800 μS/cm 2 

### SC-002 @ 11,440 μS/cm 2 

### Background conductivity @ 123 μS/cm 2



### November 3, 2007 : C onductivity check in chambers @ 0550 hrs SC-004 @ 1,770 μS/cm 2 

### SC-002 @ 2,670 μS/cm 2 

### Background conductivity @ 118 μS/cm 2



### 1% salt (as NaCl) solution was added to SC-003 (@0630 hrs) and SC-001 (@0700 hrs) chambers (i.e., 50 g of NaCl was mixed with small amount of water from each test chamber until all salt was dissolved and added).

Conductivity check in chambers @ 1620 hrs SC-004 @ 343 μS/cm 2 

### SC-002 @ 602 μS/cm 2 

### SC-001 @ 3,660 μS/cm 2 

### SC-003 @ 3,830 μS/cm 2



### November 6, 2007 :

I nstalled small UV disinfection system on HT water line prior to going into 5-gallon head tank bucket above dilutor panel. This treats all water going into the study. Because there was algal / fungal growth in 5-gallon head tank, treated head tank with NaCl (189 g mixed in 5 gallons). Salt solution was allowed to flush through the dilutor and into the test chambers (SC test chambers only).

November 9, 2007 :

A dded 50 g of NaCl to each SC chamber because still seeing fungus ( Saprolegnia ?).

November 12, 2007 :

N ow that eggs were eyed up, salt treatment consisted of removing egg cups from test chamber (SC treatments) and soaking in 3% salt treatment for 1 hour, and then transferring

### 2



### b ack to cleaned test chamber. Treated drip lines with (~1,700 ppm) formalin. The formalin solution did not go into test chambers with eggs.

November 16, 2007 :

A 1.5% NaCl solution (75 g NaCl) was added to 5 L water in test container for SC hatchery eggs.

November 24, 2007 :

S aw some dead eggs in newly received field egg treatments. Add 190 g of NaCl to 5 gal head tank. Let it flush through dilutor and into all chambers.

November 25, 2007 :

M easured conductivity @ 0800 hrs, ~200 μS/cm 2 

### . Moved all SC hatchery eggs into new egg cups. Let soak in 3% NaCl solution (60 g / 2 L) for ~ 1 h while being transferred into new egg cups. Some eggs were very covered by fungus. SC005 treatment appeared to have a lot of unfertilized (non-eyed up) eggs. Cleaned (soap and water wash) SC test chambers while eggs were in NaCl soak (all SC treatments).

Treated all test chambers in bath #2 (ENSR water bath #3) with 1.5% NaCl, except SC chambers. Mixed 75 g NaCl with deionized water (Milli-Q) and poured into each chamber. 15 test chambers in all for bath #2.

Consisted of: CC-350-(LOW)-007 CC-150-(BKD)-009, -012, -015, & -016 LSV2C-(HIGH)-004, -005, -007, -008, -017, -019, & -020 SPC-(2 ND 

### HATCHERY)-002, -003, & -006

November 26, 2007 :

T reated all test chambers in bath #1 (ENSR water bath #4) with 1.5% NaCl, except SC chambers. Mixed 75 g NaCl with deionized water (Milli-Q) and poured into each chamber. 16 test chambers in all for bath #1.

Consisted of: CC-350-(LOW)-006 & -008 CC-150-(BKD)-011, -013, -017, -018, & -020 LSV2C-(HIGH)-002, -003, -010, -012, -016, & -021 SPC-(2 ND 

### HATCHERY)-001, -004, & -005

Drained and cleaned large circular (trout tank) hardness mixing tank. Chambers were static for ~2 hours.

November 28, 2007 :

S oaked SC treatments by placing egg cups in 3% NaCl for one hour (120 g / 4 L) in separate chamber. Removed and placed back into test chambers. Cleaned as necessary.

### 3



### N ovember 29, 2007 :

A fter discussions with S. Covington and hatchery personnel, decided to use formalin to help minimize fungal growth on SC eggs. Treated SC-001 eggs with formalin (~1700 ppm, 6.8 ml in 4 L), by placing egg cup in separate chamber with formalin (next to original test chamber in water bath). From 1054 hrs to 1115 hrs.

Also treated with following test chambers with formalin in a similar way.

SC-002 (1124 hrs to 1140 hrs) SC-003 (1142 hrs to 1157 hrs)

LSV2C-(HIGH)-007 (1720 hrs to 1738 hrs).

Noticed a lot of dead / dying eggs in the LSV2C-(HIGH)-007 treatment at the end of the day so decided to treat with formalin, even though it did not appear that there was fungus on these eggs 1



### . November 30, 2007 :

T reated the following egg treatments with formalin (~1700 ppm, as 7 ml in 4 L). Performed in secondary chamber as before, soaked for ~30 min, and returned to original chamber. Started soak at 1520 hrs, finish at 1550 hrs.

SC-001, SC-005, SC-007, SC-008 (bath #1) SC-004, SC-002, SC-003, SC-006 (bath #2)

December 1, 2007 :

P laced egg cup for SC-001 in old formalin solution (prepared on Nov. 30, but still in water bath) for 15 minutes. Returned to original test chamber.

December 2, 2007 :

T reat SC-001 with formalin by mixing 8.5 ml in 200 ml water (i.e., cold hardness adjusted Horsetooth water, similar as all preps before). Poured this mixture directly into test chamber (assumes ~5L so target is ~1700 ppm). After 15 min, pur 4 L HT water into test chamber to flush out formalin.

Still seeing fungus on control eggs, try a 4% NaCl solution on 1 SC batch for 1 hour. Prepared by dissolving 200 g NaCl in 1 L of water and pouring directly into test chamber (assumes 5 L of water in chamber). Added to SC-005 @ 1340 hrs.

Treated the following test chambers with formalin (8.5 ml into test chamber for 15 minutes, followed by 4-5L flush): SC-004, SC-002, SC-006

December 3, 2007 :

O bserved first hatched out alevin in SC-004 and 1 egg that appeared to explode (termed ‘dead while hatching’).

1 

### None of these eggs appeared to be fertilized and all eventually died.

### 4



### N o more treatments for SC test chambers.

December 5, 2007 :

A dded 1% NaCl (50 g NaCl, assuming 5 L water in each chamber) to all test chambers except SC treatments (-001 through -008), and SPC-002, and SPC-004 (small SPC test chambers, i.e., ~ 2.5 L chambers).

December 7, 2007 :

C lean out test chambers for LSV2C-008 and -019 with soap and water and replaced in bath.

December 8, 2007 :

C leaned egg cups by brushing outside surface with toothbrush. Did not disturb eggs. After cleaning egg cup, placed egg cup into a cleaned test chamber. This was done for the following treatments.

LSV2C-(HIGH)-017, -020, -007 (transferred few remaining eggs to a new egg cup), -005, -004, -010, -002, & -016

December 9, 2007 :

C leaned egg cups by brushing outside surface with toothbrush. Did not disturb eggs. After cleaning egg cup, placed egg cup into a cleaned test chamber. Also replaced aeration pipettes. This was done for the following treatments.

LSV2C-(HIGH)-021, -012, & -003

December 10, 2007 :

C leaned egg cups by brushing outside surface with toothbrush. Did not disturb eggs. After cleaning egg cup, placed egg cup into a cleaned test chamber. Also replaced aeration pipettes. This was done for the following treatments.

CC-350-(LOW)-006, -007, & -008 CC-150-(BKD)-009, -015, & -016

December 7 – 18, 2007 :

N oticed that flows on some treatments were reduced due to fouling of tubing lines (from splitter box to test chamber). Flushed out lines with hydrogen peroxide (1.5%). Test chambers were static during the flush out and either overnight or during the day (~ 8 hr) while water was allowed to flush through the tubing to waste. Also blew biofilm out of some lines, but this did not work for all chambers. This was done over a period of time because there were only a few extra tubing lines that did not directly feed a treatment. Therefore, a few were cleaned each day and allowed to flush prior to using for a test chamber.

December 31, 2007 :

E gg cup for LSV2C-(HIGH)-019 was very dirty so changed out with clean egg cup.

### 5



### Ja nuary 1, 2008 :

E gg cups for the following treatments were very dirty so changed out with clean egg cups:

LSV2C-(HIGH)-021, -004 & -012 CC-150-(BKD)-013 & -012 CC-350-(LOW)-006

January 2, 2008 :

C leaned dilutor panels (scrubbing only) to remove biofilm buildup. Test chambers were static for ~ 1 hr during this time.

January 18, 2008 :

E xchanged test chamber for LSV2C-(HIGH)-003 with clean one due to fungal growth and high mortality.

### E-1 O ctober 2011

Document No. 12699-001-500 

# A ppendix E

Summary of water quality data selenium and sulfate analysis in water and selenium concentrations in eggs measure during reproductive studyAverage Daily Chamber temp0.02.04.06.08.010.012.014.05-Oct-07 25-Oct-07 14-Nov-07 4-Dec-07 24-Dec-07 13-Jan-08 2-Feb-08 22-Feb-08Time

Avg chamber temp (C)Bath 3Bath 4Water temperature in test chambers for Brown trout studies

12699-001 filename: water qual.xls S C treatments SPC treatments CC-150 (BKD) treatments CC- 350 (LOW) treatments LSV2C (HIGH) treatments Water temperature ( °

C) Water temperature ( ° C) Water temperature ( ° C) Water temperature ( ° C) Water temperature ( ° C) Water Water W ater Water Water Water Water Water Water Water Daily Avg Temp ( ° C) Date Test Day bath #3 bath #4 Test Day bath #3 bath #4 Test Day bath #3 bath #4 Test Day bath #3 bath #4 Test Day bath #3 bath #4 bath #3 bath #4 25-Oct-07 0 2 6-Oct-07 1 27-Oct-07 2 11.3 9.9 11.3 9.9 28-Oct-07 3 10.5 9.1 10.5 9.1 29-Oct-07 4 11.3 9.8 11.3 9.8 30-Oct-07 5 11.9 10.0 11.9 10.0 31-Oct-07 6 10.8 9.3 10.8 9.3 1-Nov-07 7 9.5 10.4 9.5 10.4 2-Nov-07 8 9.1 9.8 9.1 9.8 3-Nov-07 9 9.3 9.1 9.3 9.1 4-Nov-07 10 10.3 10.0 10.3 10.0 5-Nov-07 11 10.3 9.7 10.3 9.7 6-Nov-07 12 9.3 9.1 9.3 9.1 7-Nov-07 13 10.2 9.7 10.2 9.7 8-Nov-07 14 10.0 9.5 10.0 9.5 9-Nov-07 15 11.2 10.9 11.2 10.9 10-Nov-07 16 10.0 9.5 10.0 9.5 11-Nov-07 17 9.7 9.5 9.7 9.5 12-Nov-07 18 9.8 9.7 9.8 9.7 13-Nov-07 19 9.7 9.7 9.7 9.7 14-Nov-07 20 8.9 9.1 8.9 9.1 15-Nov-07 21 8.5 9.0 8.5 9.0 16-Nov-07 22 10.3 9.6 0 10.3 9.6 10.3 9.6 17-Nov-07 23 9.7 9.5 0 0 1 9.7 9.5 18-Nov-07 24 9.8 9.6 1 1 2 9.8 9.6 19-Nov-07 25 10.6 9.7 2 10.3 10.3 2 10.8 10.6 3 10.5 9.7 10.6 10.1 20-Nov-07 26 10.2 9.7 3 10.3 9.2 3 9.9 10.3 4 10.1 10.2 10.1 9.9 21-Nov-07 27 9.4 9.1 4 9.7 9.5 4 9.9 10.2 5 9.7 9.3 9.7 9.5 22-Nov-07 28 9.3 9.4 5 9.4 10.1 5 9.6 9.4 6 9.6 10.4 9.5 9.8 23-Nov-07 29 9.8 9.3 6 9 9.8 6 9.8 10.3 7 9.3 8.8 9.5 9.6 24-Nov-07 30 9.7 9.3 7 9.1 9.6 7 9.8 10.4 8 9.8 10.6 9.6 10.0 25-Nov-07 31 10.0 9.3 8 10.7 10.2 8 10.4 9.6 9 10.5 10.2 10.4 9.8 26-Nov-07 32 9.8 9.6 9 9.8 9.5 9 10.1 10.4 10 10.4 10.1 10.0 9.9 27-Nov-07 33 9.7 9.3 10 9.2 9.4 10 9.6 9.3 11 10.2 10 9.7 9.5 28-Nov-07 34 10.3 9.6 11 9.8 10.1 11 10.1 10.5 12 9.8 9.4 10.0 9.9 29-Nov-07 35 10.1 9.5 12 9.9 9.7 12 10.3 10.4 13 10.4 9.5 10.2 9.8 30-Nov-07 36 9.2 9.4 13 10.3 9.5 13 9.3 9.7 14 9.8 9.8 9.7 9.6 1-Dec-07 37 9.4 8.7 14 8.7 9.8 14 9.1 9.5 15 9.1 9.2 9.1 9.3 2-Dec-07 38 9.1 9.3 15 8.8 9.1 15 9.5 10.1 16 9.6 10.4 9.3 9.7 3-Dec-07 39 9.9 9.6 16 10.4 9.9 16 10 9.4 17 9.9 9.4 10.1 9.6 4-Dec-07 40 11.2 10.2 0 11.3 10.1 17 10.3 10.4 17 10.8 10.1 18 11 11.2 10.9 10.4 5-Dec-07 41 10.2 9.4 1 10.2 10.1 18 10.7 9.5 18 10.4 10.3 19 10.4 10.1 10.4 9.9 6-Dec-07 42 9.6 9.7 2 9.4 9.4 19 9.8 9.3 19 10.1 9.5 20 10.1 9.3 9.8 9.4 7-Dec-07 43 10.0 9.3 3 10.5 9.3 20 9.7 9.3 20 10.3 10.1 21 9.5 9.2 10.0 9.4 8-Dec-07 44 10.1 9.8 4 9.3 10 21 9.1 9.8 21 9.8 9.6 22 9.8 10.1 9.6 9.9 9-Dec-07 45 9.7 9.4 5 9.3 9.7 22 9.7 10.1 22 9.7 9.8 23 9.6 9.8 9.6 9.8 10-Dec-07 46 9.7 9.3 6 9.8 9.5 23 9.9 9.5 23 9.6 9.4 24 9.1 9.4 9.6 9.4 11-Dec-07 47 9.8 9.4 7 9.2 9.3 24 8.9 9.3 24 9.1 9.8 25 9.3 10.1 9.3 9.6 12-Dec-07 48 8.5 9.1 8 8.6 9 25 8.9 9.5 25 8.8 9.1 26 8.7 9.1 8.7 9.2 13-Dec-07 49 10.0 9.6 9 10 9.7 26 10.5 9.9 26 10.4 9.5 27 10.4 9.6 10.3 9.7 14-Dec-07 50 10.7 10.0 10 10 10.2 27 10.4 10.1 27 10.3 11.1 28 10.7 10.5 10.4 10.4 15-Dec-07 51 10.5 10.3 11 9.8 10 28 9.7 10.1 28 10.4 10 29 10.5 10.2 10.2 10.1 16-Dec-07 52 10.1 10.5 12 10.7 10.1 29 10.3 10 29 10.4 11.3 30 10.5 9.8 10.4 10.3 17-Dec-07 53 11.3 10.4 13 10.8 10.7 30 11.3 11.1 30 11 10.4 31 10.9 12.1 11.1 10.9 18-Dec-07 54 11.4 10.1 14 10.2 10.3 31 10.7 10.3 31 11 11.5 32 11 10.9 10.9 10.6 19-Dec-07 55 11.8 10.2 15 11.2 10.3 32 9.7 10.3 32 11.1 10.9 33 10.7 10.9 10.9 10.5 20-Dec-07 56 11.3 10.6 16 10.9 10.6 33 11.3 11.2 33 11.3 10.4 34 10.7 10.2 11.1 10.6 21-Dec-07 57 10.6 10.6 17 10.1 10.4 34 11.2 10.6 34 10.9 11.4 35 10.6 10.4 10.7 10.7 22-Dec-07 58 10.3 10.5 18 10.1 11 35 10.9 10.6 35 10.9 11 36 10.1 9.9 10.5 10.6 23-Dec-07 59 10.0 10.5 19 11.1 10.3 36 10.9 10 36 10.9 10.1 37 10.7 10.3 10.7 10.2 24-Dec-07 60 10.9 10.2 20 10.6 10.4 37 10.9 11.2 37 10.9 11.5 38 10.7 11.4 10.8 10.9 25-Dec-07 61 10.2 10.1 21 10.9 10.7 38 10.9 10.1 38 10.8 10.2 39 10.2 10.1 10.6 10.2 26-Dec-07 62 22 39 39 40 27-Dec-07 63 9.8 10.5 23 10.4 10.3 40 10.2 10.5 40 10.3 11.2 41 10.7 10.9 10.3 10.7 28-Dec-07 64 9.7 10.4 24 9.7 9.9 41 10.2 10.3 41 9.9 9.7 42 10 9.9 9.9 10.0 29-Dec-07 65 10.7 10.7 25 9.5 10.4 42 9.9 10.4 42 10.1 10.8 43 10 10.7 10.0 10.6 30-Dec-07 66 10.5 11.1 26 10.4 10 43 10.2 10.5 43 10.3 10.2 44 9.9 10.7 10.3 10.5 31-Dec-07 67 10.5 10.8 27 9.9 11.1 44 10.3 9.6 44 10.5 11 45 10.3 9.8 10.3 10.5 1-Jan-08 68 11.1 10.8 28 10.3 10.5 45 10.3 9.7 45 10.1 10.1 46 9.5 10.5 10.3 10.3 2-Jan-08 69 11.1 10.5 29 9.8 9.8 46 9.9 9.9 46 10.2 10.7 47 9.7 10.4 10.1 10.3 3-Jan-08 70 30 47 47 48 4-Jan-08 71 10.7 10.6 31 11.1 10.6 48 10.6 10.5 48 10.5 9.8 49 10.5 10.4 10.7 10.4 5-Jan-08 72 10.9 11.0 32 10.9 10.6 49 10.3 10.4 49 10.2 10 50 10.6 10.6 10.6 10.5 6-Jan-08 73 11.1 10.6 33 10.8 10.9 50 10.9 10.4 50 10.3 9.8 51 10.4 9.8 10.7 10.3 7-Jan-08 74 10.8 10.5 34 10.6 10.5 51 10 9.7 51 10.2 9.9 52 10.3 10.3 10.4 10.2 8-Jan-08 75 10.7 10.9 35 10.9 10.6 52 10.3 9.5 52 10.2 9.9 53 9.5 10.3 10.3 10.2 9-Jan-08 76 11.1 11.2 36 11.1 11.3 53 10.2 11 53 10.3 10.1 54 10.7 10 10.7 10.7 10-Jan-08 77 11.4 11.1 37 11.1 10.9 54 10.7 10.5 54 10.7 10.3 55 10.1 10.3 10.8 10.6 11-Jan-08 78 11.3 11.3 38 11.4 11.1 55 10.6 10.6 55 10.8 11.1 56 10.9 11.2 11.0 11.1 12-Jan-08 79 10.9 11.4 39 11.3 11.4 56 10.1 11 56 10.5 10.2 57 10.5 10.2 10.7 10.8 13-Jan-08 80 11.2 11.3 40 11.3 10.7 57 10.3 10.7 57 10.5 10.3 58 10.6 10.9 10.8 10.8 14-Jan-08 81 10.1 10.9 41 9.9 10 58 9.9 10.2 58 9.7 10 59 10.1 10.5 9.9 10.3 15-Jan-08 82 11.3 10.9 42 11.3 11.1 59 10.9 10.3 59 10.7 10.3 60 10.9 10.7 11.0 10.7 16-Jan-08 83 11.4 11.1 43 11.6 11.6 60 11.4 10.7 60 11.3 11.3 61 11.3 10.9 11.4 11.1 17-Jan-08 84 10.8 11.1 44 11 10.7 61 10.3 10 61 10.8 10 62 10.1 9.7 10.6 10.3 18-Jan-08 45 11.7 11.6 62 10.5 11.7 62 11.5 11.4 63 11.1 10.5 11.2 11.3 19-Jan-08 46 11 10.6 63 11 10.7 63 11 10.1 64 10.5 10.1 10.9 10.4 20-Jan-08 47 11.5 11.9 64 11.7 11.8 64 11.5 11.7 65 11.5 11.7 11.6 11.8 21-Jan-08 48 11.2 10.7 65 10.4 10.3 65 11.1 10.7 66 10.9 10.9 10.9 10.7 22-Jan-08 49 10.5 10.3 66 10.3 10.3 66 10.4 10.2 67 10.7 9.8 10.5 10.2 23-Jan-08 67 11.1 11.2 67 11.7 11.4 68 11.1 11.9 11.3 11.5 24-Jan-08 68 11.6 11.4 68 11.7 11.7 69 11.6 10.7 11.6 11.3 25-Jan-08 69 10.6 10.7 69 11.5 11.1 70 11.3 11.4 11.1 11.1 26-Jan-08 70 11 11.5 70 11.6 11.9 71 11.2 11.1 11.3 11.5 27-Jan-08 71 10.9 11.2 71 11.2 11 72 11.8 11.7 11.3 11.3 28-Jan-08 72 11.5 12.4 72 12.2 11.6 73 11.5 12.4 11.7 12.1 29-Jan-08 73 11.3 10.6 73 11.4 11.6 74 10.9 10.1 11.2 10.8 30-Jan-08 74 11 11.1 74 11.8 11.3 75 11.5 11.9 11.4 11.4 31-Jan-08 75 11.2 11.3 75 12 12 76 11.9 12.3 11.7 11.9 1-Feb-08 76 11.8 11.6 76 12.1 11.9 77 11.4 12.1 11.8 11.9 2-Feb-08 77 10.9 11.2 77 11.5 11.7 78 10.7 11.3 11.0 11.4 3-Feb-08 78 11 11.1 78 11.4 11.4 79 10.9 11 11.1 11.2 4-Feb-08 79 11.8 11.1 79 12.2 12.1 80 11.8 10.8 11.9 11.3 5-Feb-08 80 11.5 11.2 80 11.5 11.3 81 10.7 11.7 11.2 11.4 6-Feb-08 81 11.3 11.7 81 11.7 11.3 82 11.5 11.7 11.5 11.6 7-Feb-08 82 12.1 11.7 82 12.1 83 12.1 11.8 12.1 11.9 8-Feb-08 83 11.8 11.1 83 11.6 84 12.1 11.9 12.0 11.5 9-Feb-08 84 12.5 12.3 84 11.8 85 12.5 11.7 12.5 11.9 10-Feb-08 85 11.4 10.9 85 11.5 86 11.8 12.1 11.6 11.5 11-Feb-08 86 12.1 11.5 86 11.8 87 12.2 11.7 12.2 11.7 12-Feb-08 87 11.9 11.9 87 11.4 88 12 11.7 12.0 11.7

Avg 10.1 10.5 10.5 10.6 10.6 Std 0.733 0.680 0.798 0.782 0.822 Min 8.5 8.6 8.7 8.8 8.7 Max 11.9 11.9 12.5 12.2 12.5Water hardness measured in dilution water during BT study0102030405060705-Oct-07 25-Oct-07 14-Nov-07 4-Dec-07 24-Dec-07 13-Jan-08 2-Feb-08 22-Feb-08Time

Hardness (mg/L as CaCO3)4 of 5

Water hardness (mg/L) measurements in Brown trout study (12699-001) filename: water qual.xls

W ater hardness Da te Test Day (mg/L)
25-Oct-07 D 0
26-Oct-07 D1
27-Oct-07 D2
28-Oct-07 D3
29-Oct-07 D4 50
30-Oct-07 D5 54
31-Oct-07 D6 50
1-Nov-07 D7 48
2-Nov-07 D8 52
3-Nov-07 D9 54
4-Nov-07 D10 48
5-Nov-07 D11 58
6-Nov-07 D12 50
7-Nov-07 D13 50
8-Nov-07 D14 50
9-Nov-07 D15 46
10-Nov-07 D16 48
11-Nov-07 D17 54
12-Nov-07 D18 52
13-Nov-07 D19 48
14-Nov-07 D20 52
15-Nov-07 D21 48
16-Nov-07 D22 50
17-Nov-07 D23 48
18-Nov-07 D24 54
19-Nov-07 D25 54
20-Nov-07 D26
21-Nov-07 D27 46
22-Nov-07 D28 50
23-Nov-07 D29 50
24-Nov-07 D30 46
25-Nov-07 D31 48
26-Nov-07 D32 50
27-Nov-07 D33 50
28-Nov-07 D34 46
29-Nov-07 D35 50
30-Nov-07 D36 46
1-Dec-07 D37 48
2-Dec-07 D38 50
3-Dec-07 D39
4-Dec-07 D40 50
5-Dec-07 D41 54
6-Dec-07 D42 50
7-Dec-07 D43 48
8-Dec-07 D44 50
9-Dec-07 D45 48
10-Dec-07 D46 46
11-Dec-07 D47 52
12-Dec-07 D48 54
13-Dec-07 D49 50
14-Dec-07 D50 50
15-Dec-07 D51 48
16-Dec-07 D52 50
17-Dec-07 D53 48
18-Dec-07 D54 48
19-Dec-07 D55 50
20-Dec-07 D56 46
21-Dec-07 D57 50
22-Dec-07 D58 50
23-Dec-07 D59 52
24-Dec-07 D60 54
25-Dec-07 D61 525 of 5

Water hardness (mg/L) measurements in Brown trout study (12699-001) filename: water qual.xls

W ater hardness Da te Test Day (mg/L)
26-Dec-07 D 62
27-Dec-07 D63 50
28-Dec-07 D64 50
29-Dec-07 D65 54
30-Dec-07 D66 50
31-Dec-07 D67 48
1-Jan-08 D68 50
2-Jan-08 D69 52
3-Jan-08 D70 50
4-Jan-08 D71 44
5-Jan-08 D72 42
6-Jan-08 D73 54
7-Jan-08 D74 48
8-Jan-08 D75 48
9-Jan-08 D76 50
10-Jan-08 D77 44
11-Jan-08 D78 44
12-Jan-08 D79 44
13-Jan-08 D80 44
14-Jan-08 D81
15-Jan-08 D82 40 SC fish (Oct. 25 - Jan. 17)
16-Jan-08 D 83 44 Avg StDev Min Max
17-Jan-08 D 84 50 49.32 3.19 40 58
18-Jan-08 44
19-Jan-08 42
20-Jan-08 44 SPC fish (Dec. 4 - Jan. 22)
21-Jan-08 5 2 Avg StDev Min Max
22-Jan-08 5 2 48.63 3.55 40 54
23-Jan-08 46
24-Jan-08 54
25-Jan-08 50
26-Jan-08 44
27-Jan-08 38
28-Jan-08 48
29-Jan-08 44
30-Jan-08 48
31-Jan-08 46
1-Feb-08 48
2-Feb-08 42
3-Feb-08 48
4-Feb-08 46
5-Feb-08 48
6-Feb-08 46
7-Feb-08 54
8-Feb-08 50
9-Feb-08 52
10-Feb-08 48 CC-150 & CC-350 fish (Nov. 17- Feb. 12)
11-Feb-08 Av g StDev Min Max
12-Feb-08 4 4 48.36 3.51 38 54 Av erage (overall) 48.78 StDev 3.506 LSV2C fish (Nov. 16- Feb. 12) Min 38 Avg StDev Min Max Max 58 48.38 3.49 38 54