University of Maryland

Wye Research and Education Center

Queenstown, MD 21658

Final Report

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 Research and Education Center

Queenstown, MD 21658

To: Peter McNulty

NEI Treatment Systems, LLC

3530 Wilshire Blvd, Suite 1465

Los Angeles, CA 90010

Date: March 27, 2009
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 addressed 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 de-oxygenation ballast water treatment using a venturi unit

to inject a N2/CO2 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 N2/CO2 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 N2/CO2 treated

ballast water was not chronically toxic to any of the species tested. 
3

INTRODUCTION

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 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? Our laboratory was contracted by NEI to determine the environmental safety of 

their de-oxygenation ballast water treatment process based on short-term chronic toxicity studies

with aquatic organisms.

METHODS

A lab-scale NEI venturi unit was used to generate the freshwater and saltwater test solutions. 

The unit was first used in the ballast water treatment step by passing a N2/CO2 gas mixture 

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

treatment levels. Once DO was reduced the venturi injector 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 held 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 two freshwater organisms (a fish, the fathead minnow Pimephales 

promelas; and a crustacean, the daphnid Ceriodaphnia dubia) and two saltwater organisms (a 

fish, the sheepshead minnow Cyprinodon variegatus; 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 Environmental Protection Agencies short-term chronic test methods (US EPA, 

2002b). The test method summaries are given in Tables 1 - 4. These test methods are the 

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

receiving water short-term chronic toxicity tests on freshwater (US EPA, 2002a) 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% reaerated sample of the final test solution and a 

dilution series of 50% and 25% of this reaerated solution. The endpoints for the fish and mysid

tests were 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 analyzed using US EPA (2002a, 2002b) statistical procedures. All proportion survival 

data was arc sine transformed prior to analysis. Data were analyzed for normality using a ChiSquare Test and for homogeneity of variance using a Bartlett’s Test. All data were 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 test. All statistical 

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

was 100% survival in the control and test treatments in the sheepshead minnow test, no statistical 

comparison with control data was 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 N2/CO2 mixture and also to reaerate the treated mixture. DO and pH 

were reduced in both water types after treatment with N2/CO2. After reaeration using the venturi

unit, the DO and pH were raised in both the freshwater and saltwater 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 the conductivity ranged 

from 700 to 800 μmhos throughout the test period. The 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 successfully. 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 Pimephales 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 surviving control organisms produced three broods. There were no survival, growth or 

reproductive chronic effects due to the reaerated N2/CO2 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). This finding is consistent with the lack of 

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

2008)
5

REFERENCES

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

Toxicity 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 Methods 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 Conditions 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 concentration: 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-day Survival and Reproduction Chronic Toxicity Tests

Test type: Static renewal

Test duration: 7 d 

Temperature: 25oC (±1oC)

Lighting: Fluorescent

Photoperiod: 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 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 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 filtered Wye River water adjusted to 

25‰ 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 organism 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

Temperature: 25oC (± 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.

Aeration: None

Dilution water: 1 μm/UV filtered Wye River water adjusted to 

25‰ 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/CO2 treatment; Post/Reaerated = Test solution 

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

Freshwater Preparation

DO pH

Pre/Initial 7.60 8.19

Treated 0.53 5.49

Post/Reaerated 5.31 5.82

Saltwater Preparation (25 ‰)

DO pH

Pre/Initial 7.21 8.11

Treated 0.98 6.18

Post/ Reaerated 5.15 6.33
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 

measurement 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 

pH

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 water 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 DWC 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 8.18 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 survival 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.) 

Treatment

Treatment Rep Start End Biomass

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 90 0.4 0.36

D 10 8 80 0.46 0.37
14

Table 9. Survival and reproduction of Ceriodaphnia dubia in the NEI chronic toxicity test 

(2/24/09 – 3/3/09). * indicates endpoint mean significantly different from control 

mean. 

Treatment (A)live or

(D)ead

#

Neonates

 (S.D) Treatment

% Survival

 (S.D) Treatment

Reproduction

(# Neonates)

Control A A 20

Control B A 26

Control C A 25

Control D A 22

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

Control F A 23

Control G A 22

Control H A 20

Control I A 24

Control J A 29

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 23 100 (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

Treatment Rep Start End Survival Biomass

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

Treatment Rep Start End Survival Biomass

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 0.183

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