### University of Maryland

Wye Research and Education Center

Queenstown, MD 21658



## Final R eport



# Short

- term Chronic Toxicity Testing of a De

- oxygenation

Ballast Water Treatment to Receiving Water Organisms

From: D.J

. Fisher, D.T. Burton, L.T. Yonkos, G. Ziegler, S.T. Turley, M. Osborne , E. Friedel

University of Maryland, Wye R esearch and E ducation Center

Queenstown, MD 21658

To: Peter McNulty

NEI Treatment Systems, LLC

3530 Wilshire Blvd, Suite 1465

Los Angeles, CA 90010

Date: March 27 , 20 09

### 2

Abstract

Ballast water treatment has become a major issue in the last decade due to the problem of

invasive species transported and released by the uptake and discharge of ballast water for

shipping operations. Two important issues must be address ed considering ballast water

treatment: 1) Is the treatment effective in eliminating aquatic species carried from port to port in

ballast water?; and 2) Is the treated ballast water, once discharged, safe to the aquatic

environment? NEI has developed a d e

- oxygenation ballast water treatment using a ventur i unit

to inject a N 2 

### /CO 2 

### gas mixture into ballast water , reduce dissolved oxygen and thus kill invasive

species . Prior to discharge to the environment the ballast water is then reaerated.

Standard US Environmental Protection Agency test methods were conducted in the

laboratory to determine the short

- term chronic toxicity of this reaerated N 2 

### /CO 2 

### treated ballast

water. Short

- term chronic toxicity tests were conducted with a freshwater fish ( Pimephales

promelas ), a freshwater crustacean ( Ceriodaphnia dubia ), a saltwater fish ( Cyprinodon

variegatus ) and a saltwater crustacean ( Americamysis bahia ). The reaerated N 2 

### /CO 2 

### treated

ballast water was not chronically toxic to any of the species tested.

### 3

INTRO DUCTION

Ballast water treatment has become a major issue in the last decade due to the problem of

invasive species transported and released by the uptake and discharge of ballast water for

shipping operations. Two important issues must be addressed consi dering ballast water

treatment: 1) Is the treatment effective in eliminating aquatic species carried from port to port in

ballast water ? ; and 2) Is the treated ballast water, once discharged, safe to the aquatic

environment? Our labo ratory was contract ed by NEI to determine the environmental safety of

their d e

- oxygenation ballast water treatment process based on short

- term chronic toxicity stud ies

with aquatic organisms.

METHODS

A lab

- scale NEI venturi unit was used to generate the freshwater and salt water test solutions.

The unit was first used in the ballast water treatment step by passing a N 2 

### /CO 2 

### gas mixture

through 40L of either freshwater or saltwater (25‰) to reduce dissolved oxygen (DO) to

treatment levels. Once DO was reduced the ventur i inj ector unit was then used to draw air

through the treated mixture as will be done prior to discharge to the aquatic environment. This

reaerated mixture was then used to start the freshwater or saltwater toxicity tests. A volume of

this reaerated solution (saltwater and freshwater) was hel d throughout the test period at the test

conditions to use for the daily renewals for each test. These renewal solutions were lightly

aerated during the holding period to reduce the possibility of a reduction in dissolved oxygen

during holding.

Four aquatic species were used to test the short

- term chronic toxicity of this ballast water

treatment. Species included t wo freshwater organisms (a fish, the fathead minnow Pimephales

promelas ; and a crustacean

, the daphnid Cer iodaphnia dubia ) and two saltwater organisms (a

fish, the sheepshead minnow Cyprinodon variegat us ; and a crustacean the mysid Americamysis

bahia )

. The saltwater crustacean was formerly known as Mysidopsis bahia and is identified as

such in the U.S Environ me n tal Protection Agencies short

- term chronic test methods (US EPA,

2002 b ). The test method summaries are given in Tables 1

- 4 . These test method s are the

standard methods used by the U.S. Environmental Protection Agency to conduct effluent and

receivin g water short

- term chronic toxicity tests on freshwater (US EPA, 2002 a ) and saltwater

species (USEPA, 2002b)

. Our laboratory has used these test methods in the past starting in the

mid 1980s. Under the direction of Dr. Fisher

, the University of Maryland’ s Wye Research and

Education Center conducted all of the acute and chronic effluent testing for the State of

Maryland’s Department of the Environment NPDES bioassay program using these species.

Treatments tested in this study included a 100% re aerated sam ple of the final test solution and a

dilution series of 50% and 25% of this re aerated solution. The endpoint s for the fish and mysid

tests w ere proportion survival in each replicate and biomass ( growth (dry weight )/# of organisms

alive at the start of the test in each replicate ) )

. The endpoints in the C. dubia test were proportion

survival in each treatment and the number of young produced in each replicate. This test is a

true full life cycle reproduction toxicity test.

### 4

DATA ANALYSIS

Data were analy zed using US EPA (2002a, 2002b) statistical procedures. All proportion survival

data was arc sine transf

orm ed prior to analysis. Data were analyzed for normality using a Chi

Square Test and for homogeneity of variance using a Bartlett’s Test

. All data w ere found to be

normal and homogeneous

. The survival and growth data were then analyzed using a one

- tailed

analysis of variance (ANOVA) followed by a Dunnett’s mean comparison t est . All statistical

tests were conducted using the statistical package TOXST AT 3.5 (Gulley, 1996). Since there

was 100% survival in the control and test treatment s in the sheepshead minnow test

, no statistical

comparison with control data w as necessary. The alpha was 0.01 for the normality and

homogeneity tests and 0.05 for the ANOVA and Dunnett’s Test.

RESULTS AND DISCUSSION

Water quality data are presented for the dilution water, either fresh and salt, and the treated water

and reaerated water at the beginning of the test in Table 5

. It took between five to 10 minutes to

treat the test water with the N 2 

### /CO 2 

### mixture and also to reaerate the treated mixture. DO and pH

w ere reduced in both water types after treatment with N 2 

### /CO 2



### . After reaeration using the ventur i

unit, the DO and pH w ere raised in both the freshwater and s altwater test solutions although not

to pretreatment levels

.

Over the toxicity test period, the salinity remained consistent at 25 ‰ 

### in the 100% saltwater

reaerated test solution. In the 100% freshwater reaerated test solution t he condu ctivity ranged

from 700 to 800 μmhos throughout the test period

. T he DO and pH in test beakers slowly

returned to values consistent with the values measured before treatment (Tables 6 and 7 )

.

All of the chronic toxicity tests were completed succe ssfully. All test acceptability criteria given

for these tests in Tables 1 through 4 were met (Tables 8 through 11)

. Survival in all of the

control treatments for every species was equal to or greater than 80%. Average control dry

weights in the Pimepha les promelas

, Cyprinodon variegatus and Americamysis bahia tests were

0.45, 0.61 and 0.26 mg, respectively. In the Ceriodaphnia dubia reproduction test, the

organisms in the control treatment produced an average of 22.2 young per survivor and 90 % of

the s urviving control organisms produce d three broods

. There were no survival, growth or

reproductive chronic effects due to the reaerated N 2 

### /CO 2 

### ballast water in any of the species

tested, including the 100% ballast water treatments (Tables 8 through 11).

CONCLUSION

The NEI de

- oxygenation ballast water treatment, once reaerated, was not chronically toxic to the

saltwater or freshwater fish or crustaceans tested ( Pimephales promelas

, Ceriodaphnia dubia

, Cyprinodon variegatus and Americamysis bahia )

. Thi s finding is consistent with the lack of

acute toxicity found in earlier work conducted on this ballast water treatment (Burton et al.,

2008)

### 5

R EFERENCES

Burton, DT, Fisher, DJ, Yonkos, LT, Ziegler, G., Turley, ST and Osborne, M. 2008. Short

- term

Toxic ity Testing of a De

- oxygenation Ballast Water Treatment to Receiving Water Organisms

. Final Report. NEI Treatment Systems, LLC., Los Angeles, CA. 16 pp.

Gully, D. 1996. TOXSTAT Version 3.5. Western Ecosystem Technology, Cheyenne, WY.

USEPA. 2002a. Short

- term Methods for Estimating the Chronic Toxicity of Effluents and

Receiving Waters to Freshwater Organisms . Fourth Edition. EPA

- 821

- R

- 02

- 013

. U.S.

Environmental Protection Agency

, Office of Water

, Washington, DC . 350 pp.

USEPA. 2002b. Short

- Term Met hods For Estimating the Chronic Toxicity of Effluents and

Receiving Water to Marine and Estuarine Organisms . Third Edition. EPA

- 821

- R

- 02

- 014

. U.S. Environmental Protection Agency

, Office of Water

, Washington, DC . 486 pp.

### 6

Table 1. Summary of Test Condition s and Acceptability Criteria for the Fathead Minnow

Pimephales promelas 7

- day Larval Survival and Growth Chronic Toxicity Test

Test type: Static renewal

Test Duration: 7 d

Temperature: 25 oC (± 1oC)

Lighting: Fluorescent

Photoperiod: 16 h light, 8 h dark

Test chamber size: 500 mL

Test solution volume: 250 mL

Renewal of test solutions: Daily

Age of test organisms: Newly hatched larvae <24 hours old

No. larvae per test chamber: 10

No. replicate chambers per concentration: 4

No. larvae per test co ncentration: 40

Feeding regime: Artemia nauplii (<24 h old). On days 0

- 6, feed

0.15g wet weight newly hatched (<24 hours

old) brine shrimp nauplii twice daily at 6

- hours

intervals (at the beginning of the work day

prior to renewal, and at the end of the work day

following renewal). Sufficient nauplii are

added to provide an excess

Cleaning: Siphon daily, immediately before test solution

renewal

Aeration: None

Dilution water: Moderately hard synthetic water

Test concentrations: 100%, 50%, 25%, control

Dilution factor: 0.5

Test duration: 7 days

Endpoints: Survival and biomass (dry weight)

Test acceptability criteria: 80% or greater survival in controls; average

dry weight per surviving organism in control

chamber equals or exceeds 0.25 mg

### 7

Table 2. Summary of Test Conditions and Acceptability Criteria for the Cladoceran

Ceriodaphnia dubia 7

- d ay Survival and Reproduction Chronic Toxicity Tests

Test type: Static renewal

Test duration: 7 d

Temperature: 25oC (±1oC)

Lighting: Fluorescent

Photope riod: 16 h light, 8 h dark

Test chamber size: 30 mL beaker

Test solution volume: 15 mL

Renewal of test solutions: 24 hours

Age of test organisms: Less than 24 h

, all released within a 8

- h period

No. neonates per test chamber: 1

No. replicate chambers per concentration: 10

No. neonates per test concentration: 10

Feeding regime: 0.1 mL S. capricornutum suspension and 0.24

mL digested Cerophyl ® 

### per test chamber daily

Cleaning: Use new plastic cups daily

Aeration: None

Dilution water: Moderately hard synthetic water

Test concentrations: 100%, 50%, 25%, control

Dilution series: 0.5

Endpoint: Survival and reproduction

Test acceptability criteria: 80% or greater survival of all control animals;

average of 15 of more young per surviving

female in the control solutions; and 60% of

surviving control females must produce three

broods

### 8

Table 3. Summary of Test Conditions and Acceptability Criteria for the Sheepshead Minnow

Cyprinodon variegatus 7

- d Larval Survival and Growth Chronic Toxicity T est

Test type: Static renewal

Test Duration: 7 d

Temperature: 25 oC (± 1oC)

Lighting: Fluorescent

Photoperiod: 16 h light, 8 h dark

Test chamber size: 500 mL

Test solution volume: 250 mL

Renewal of test solutions: Daily

Age of test organisms: Newly hatched larvae <24 hours old

No. larvae per test chamber: 10

No. replicate chambers per concentration: 4

No. larvae per test concentration: 40

Feeding regime: Artemia nauplii (<24 h old). On days 0

- 2, feed

0.10g wet weight newly hatched (<24 hours

old) brine shrimp nauplii daily. On days 3

- 6,

feed 0.15g wet weight newly hatched (<24

hours old) brine shrimp nauplii daily.

Cleaning: Siphon daily, immediately before test solution

renewal

Aeration: None

Dilution water: 1 μm/UV f iltered Wye River water adjusted to

2 5 ‰ with Crystal Marine Bioassay Mix ®



### Test concentrations: 100%, 50%, 25%, control

Dilution factor: 0.5

Endpoint: Survival and growth (dry weight)

Test acceptability criteria: 80% or greater survival in controls; average

dry weight per surviving o rganism in control

chamber equals or exceeds 0.60 mg

### 9

Table 4. Summary of Test Conditions and Acceptability Criteria for the Mysid Americamysis

bahia 7

- day Larval Survival and Growth Toxicity Test

Test type: Static renewal

Test Duration: 7 d

Temperatu re: 2 5 oC (± 1oC)

Lighting: Fluorescent

Photoperiod: 16 h light, 8 h dark

Test chamber size: 400 mL

Test solution volume: 150 mL

Renewal of test solutions: Daily

Age of test organisms: 7 d

No. organisms per test chamber: 5

No. replicate chambers per concentration: 8

No. organisms per test concentration: 40

Feeding regime: Feed 150 <24 h old Artemia nauplii daily, half

after test solution renewal and half after 8

- 12 h.

Cleaning: Siphon daily immediately before test solution

renewal and feeding.

Ae ration: None

Dilution water: 1 μm/UV f iltered Wye River water adjusted to

2 5 ‰ with Crystal Marine Bioassay Mix ®



### Test concentrations: 100%, 50%, 25%, control

Dilution factor: 0.5

Endpoint: Survival, biomass

Test acceptability criteria: 80% or greater survival in controls; average

dry weight per surviving organism in control

chamber equals or exceeds 0.20 mg

### 10

Table 5

. Water chemistry for test treatment preparation. Pre/Initial = test water prior to

treatment; Treated = test water after N/CO 2 

### treatme nt; Post/ Reaerated = Test solution

after reaeration. Post/ Reaerated is the solution used in the toxicity tests.

Freshwater Preparation

DO pH

Pre/Initial 7.60 8.1 9

Treated 0.53 5. 49

Post/ Reae r ated 5.31 5. 82

Saltwater Preparation (25 ‰ )

DO pH

Pre/Initial 7. 21 8.11

Treated 0.98 6.18

Post/ Reaerated 5.15 6. 3 3

### 11

Table 6

. Water quality data during freshwater toxicity tests (2/24/09

– 3/3/09)

. 24 h indicates

the measurement at the end of the 24 h exposure period while New indicates the

measurem ent of the test renewal water for that day. Measurements taken in a different

replicate of each treatment each day.

Fish ( Pimephales promelas ) Invertebrate ( Ceriodaphnia dubia )

Freshwater Rep 24 h

DO New

DO 24 h

pH New

pH Rep 24 h

DO New

DO 24 h

p H New

pH

Day 1 DWC A 7.5 8.4 6.94 7.32 A 8 8.4 6.95 7.32

25% 7.7 8.4 7.07 7.45 7.7 8.4 7.13 7.45

50% 7.8 8.5 6.85 7.31 7.9 8.5 7.24 7.31

100% 7.6 8.5 6.44 7.25 7.9 8.5 7.15 7.25

Day 2 DWC B 6.6 7.1 7.09 7.37 B 6.4 7.1 7 7.37

25% 6.5 7.2 6.99 7.3 6.1 7.2 7.06 7.3

50% 6.5 7.3 7.09 7.36 6.4 7.3 7.07 7.36

100% 6.4 7.2 7.1 7.4 6.3 7.2 7 7.4

Day 3 DWC C 7.5 7.4 6.93 7.32 C 6.5 7.4 7.76 7.32

25% 7.3 7.6 7.07 7.58 6.4 7.6 7.81 7.58

50% 7 7.7 7 7.53 6.6 7.7 7.85 7.53

100% 7.2 7.9 7.01 7.49 6.4 7.9 7.84 7.49

Day 4 DWC D 7.1 7.5 7.7 7.37 D 7.3 7.5 7.09 7.37

25% 7.2 7.5 7.43 7.4 7.1 7.5 7.34 7.4

50% 7.1 7.5 7.28 7.47 7 7.5 7.33 7.47

100% 7.1 7.4 7.25 7.53 7.5 7.4 7.36 7.53

Day 5 DWC A 7.2 7.6 7.09 7.33 A 7.2 7.6 7.92 7.33

25% 7.2 7.4 7.24 7.44 7.2 7.4 7.49 7.44

50% 7.2 7.5 7.28 7.51 7.2 7.5 7.48 7.51

100% 7.3 7.7 7.27 7.62 7.1 7.7 7.49 7.62

Day 6 DWC B 6.5 7.4 6.83 7.07 B 7.2 7.4 7.53 7.07

25% 6.1 7.4 7.03 7.49 7.1 7.4 7.40 7.49

50% 6.4 7.5 7.09 7.6 7.3 7.5 7.35 7.6

100% 6.8 7.7 7.16 7.6 7.2 7.7 7.45 7.6

Day 7 DWC C 5.5 6.96 C 6.7 7.93

25% 4.1 6.88 6.5 7.58

50% 4.8 6.95 6 .3 7.51

100% 5.9 7.04 6.3 7.53

### 12

Table 7

. Water quality data during saltwater toxicity tests (2/24/09

– 3/3/09). 24 h indicates the

measurement at the end of the 24 h exposure period while New indicates the

measurement of the test renewal w ater for that day. Measurements taken in a different

replicate of each treatment each day.

Fish ( Cyprinodon variegatus ) Invertebrate ( Americamysis bahia )

Saltwater Rep 24 h

DO New

DO 24 h

pH New

pH Rep 24 h

DO New

DO 24 h

pH New

pH

Day 1 D WC A 7.6 7.8 8 8.03 A 7.8 7.8 8.16 8.03

25% 7.6 7.8 7.59 7.82 7.7 7.8 8.14 7.82

50% 7.6 7.6 7.53 7.71 7.9 7.6 8.15 7.71

100% 7.6 7.9 7.47 7.36 7.8 8.15 7.36

Day 2 DWC B 6.3 7.1 8.03 8.22 B 6.8 7.1 8.2 8.22

25% 6.3 7 7.92 8.1 8 6.6 7 8.19 8.18

50% 6 7 7.93 8.14 6.7 7 8.17 8.14

100% 6.4 7 7.94 7.96 6.7 7 8.2 7.96

Day 3 DWC C 7.4 7 8.03 8.08 C 7.4 7 8.14 8.08

25% 7.6 7 8.08 8.1 7.1 7 8.14 8.1

50% 7.7 7 8.07 8.1 7.1 7 8.15 8.1

100% 7.3 7 8.03 8 .11 7.3 7 8.18 8.11

Day 4 DWC D 7 7.2 8.01 8.06 D 7.4 7.2 8.14 8.06

25% 7 7 8.03 8.05 7.2 7 8.15 8.05

50% 7 7 8.02 8.07 7.1 7 8.15 8.07

100% 6.8 6.7 8.06 8.13 7 6.7 8.18 8.13

Day 5 DWC A 7.1 7 7.94 8.12 E 7 7 8.12 8 .12

25% 7.1 7.1 7.92 8.2 7.1 7.1 7.99 8.2

50% 7.2 7 7.82 8.32 7 7.1 7.98 8.32

100% 6.9 7.1 7.95 8.35 7.1 7.1 8.04 8.35

Day 6 DWC B 6.6 7.7 8.01 8.64 F 7.1 7.7 8.1 8.64

25% 6.4 7.8 8.11 8.64 7.2 7.8 8.11 8.64

50% 6.4 7.7 8 .14 8.54 6.9 7.7 8.12 8.54

100% 6.7 7.7 8.17 7.78 7.2 7.7 8.17 7.78

Day 7 DWC C 5 8.06 G 7.1 8.2

25% 5.1 8.14 6.6 8.16

50% 5 8.09 6.8 8.19

100% 5.3 7.98 6.4 8.08

### 13

Table 8 . Growth and surv ival of Pimephales promelas in NEI chronic toxicity test (2/24/09

– 3/3/09). * indicates endpoint mean significantly different from control mean.

# Organisms

%

Survival (S.D.)

Treatment

% Survival Wt./org.

mg Biomass

mg (S.D.)

Treatm ent

Biomass Treatment Rep Start End

Control A 10 8 80

80.0 (8.16) 0.48 0.38

35.8 (0.032)

B 10 8 80 0.49 0.39

C 10 9 90 0.37 0.33

D 10 7 70 0.47 0.33

25% A 10 9 90

82.5 (5.00) 0.41 0.37

36.5 (0.034)

B 10 8 80 0.51 0.41

C 10 8 80 0.42 0.33

D 10 8 80 0.44 0.35

50% A 10 10 100

82.5

(20.62) 0.27 0.27

35.5 (0.113) B 10 10 100 0.52 0.52

C 10 7 70 0.48 0.33

D 10 6 60 0.51 0.3

100% A 10 7 70

80.0 (8.16) 0.42 0.29

34.0 (0.036)

B 10 8 80 0.43 0.34

C 10 9 9 0 0.4 0.36

D 10 8 80 0.46 0.37

### 14

Table 9

. Survival and r eproduction of Ceriodaphnia dubia in the NEI chronic toxicity test

(2/24/09

– 3/3/09). * indicates endpoint mean significantly different from control

mean.

T rea tment (A)live or

(D)ead #

Neonates (S.D) Treatment

% Survival (S.D) Treatment

Reproduction

(# Neonates)

Control A A 2 0

Control B A 26

Control C A 2 5

Control D A 22

Control E A 11 100 (0.0) 22.2 (4.80)

Control F A 23

Control G A 2 2

Control H A 2 0

Control I A 24

Control J A 2 9

25% A A 11

25% B A 16

25% C A 27

25% D A 19

25% E A 16 100 (0.0) 21.0 (7.18)

25% F A 33

25% G A 24

25% H A 15

25% I A 19

25% J A 30

50% A A 17

50% B A 21

50% C A 29

50% D A 27

50% E A 2 3 100 (0.0) 23.9 (4.12)

50% F A 24

50% G A 23

50% H A 29

50% I A 27

50% J A 19

### 15

Table 9 . Continued

.

Treatment (A)live or

(D)ead #

Neonates (S.D) Treatment

% Survival Treatment (S.D.)

Reproduction

(# Neonates)

100% A A 14

100.0 100% B A 18

100% C A 26

100% D A 20

100% E A 11 100 (0.0) 19.8 (5.12)

100% F A 18

100% G A 20

100% H A 27

100% I A 19

100% J A 25

### 16

Table 10 . Growth and survival of Cyprinodon variegatus in the NEI chronic toxicity test

(2/24/09

– 3/3/09). * indicates endpoint mean significantly different from control

mean.

# Organisms

% (S.D.)

Treatment

% Survival Wt./org.

mg Biomass

mg (S.D.)

Treatment

Biomass Treatment Rep Start End Survival

Control A 10 10 100

100 (0.0) 0.55 0.55

0.61 (0.043)

B 10 10 100 0.61 0.61

C 10 10 100 0.63 0.63

D 10 10 100 0.65 0.65

25% A 10 10 100

100 (0.0) 0.61 0.61

0.61 ( 0.033)

B 10 10 100 0.59 0.59

C 10 10 100 0.59 0.59

D 10 10 100 0.66 0.66

50% A 10 10 100

100 (0.0) 0.65 0.65

0.66 (0.038)

B 10 10 100 0.67 0.67

C 10 10 100 0.62 0.62

D 10 10 100 0.71 0.71

100% A 10 10 100

100 (0.0) 0.7 0.7

0 .71 (0.041)

B 10 10 100 0.68 0.68

C 10 10 100 0.77 0.77

D 10 10 100 0.69 0.69

### 17

Table 11

. Growth and survival of Americamysis bahia in the NEI chronic toxicity test (2/24/09

– 3/3/09). * indicates endpoint mean significantly different from control mean.

# Organisms

% (S.D.)

Treatment

% Survival Wt./org.

mg Biomass

mg (S.D.)

Treatment

Biomass Treatment Rep Start End Survival

Control A 5 4 80

80.0 (18.52) 0.282 0.226

0.21 (0.068)

B 5 4 80 0.378 0.302

C 5 4 80 0.241 0.193

D 5 2 40 0.203 0.081

E 5 5 100 0.263 0.263

F 5 5 100 0.241 0.241

G 5 4 80 0.199 0.159

H 5 4 80 0.241 0.193

25% A 5 4 80

90.0 (15.12) 0.314 0.251

0.26 (0.059)

B 5 5 100 0.242 0.242

C 5 5 100 0.221 0.221

D 5 3 60 0.342 0.205

E 5 5 100 0.222 0.222

F 6 6 100 0.322 0.322

G 5 4 80 0.383 0.306

H 5 5 100 0.271 0.271

50% A 5 4 80

95.0 (9.26) 0.205 0.164

0.20 (0.028)

B 5 5 100 0.217 0.217

C 5 5 100 0.207 0.207

D 5 5 100 0.183 0.1 83

E 5 5 100 0.216 0.216

F 5 4 80 0.217 0.174

G 5 5 100 0.235 0.235

H 5 5 100 0.242 0.242

100% A 5 4 80

89.4 (11.48) 0.302 0.242

0.22 (0.049)

B 5 5 100 0.312 0.312

C 5 5 100 0.246 0.246

D 4 3 75 0.238 0.143

E 5 4 80 0.281 0.225

F 5 5 100 0.222 0.222

G 4 4 100 0.241 0.193

H 5 4 80 0.244 0.196