University of Maryland 

Wye Research and Education Center 

Queenstown, MD 21658

Final Report 

Short-term Toxicity Testing of a De-oxygenation Ballast 

Water Treatment to Receiving Water Organisms

From: D.T. Burton, D.J Fisher, L.T. Yonkos, G. Ziegler, S.T. Turley, M. Osborne 

 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: August 29, 2008 
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 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 toxicity of this released ballast water. Short-term acute toxicity tests 

were conducted with a freshwater fish (Pimephales promelas), a freshwater crustacean (Daphnia 

magna), a saltwater fish (Cyprinodon variegatus) and a saltwater crustacean (Mysidopsis bahia). 

In addition, a short-term chronic freshwater green alga test with Selenastrum capricornutum was 

conducted to assess the effects of the ballast water discharge on population growth of this 

species. The reaerated NEI ballast water treatment was not acutely toxic to any of the species 

tested. In addition, it had no effect on green algae population growth.

2
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 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 a freshwater or saltwater (25‰) to reduce dissolved oxygen (DO) to 

treatment levels. Once DO was reduced the venture 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 conduct the acute toxicity tests. 

Five aquatic species were used to test the short-term toxicity of this ballast water treatment. 

Species included three freshwater species (a fish, the fathead minnow Pimephales promelas; a 

crustacean, the daphnid Daphnia magna; and a single cell algae Selenastrum capricornutum) and 

two saltwater organisms (a fish, the sheepshead minnow Cyprinodon variegate; 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 acute test methods 

(US EPA, 2002a). The test method summaries are given in Tables 1 -5. These test methods are 

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

receiving water acute toxicity tests (US EPA, 2002a). The 96-h freshwater algae test is a shortterm chronic toxicity test and is a true population level toxicity test due to the rapid algal 

population increases measured by the method (US EPA, 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 has 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% non aerated sample of the final test solution and 

a dilution series of 50% and 25% of this non aerated solution. In addition, a 100% aerated test 

solution was tested. The endpoint for all tests other than the algae was proportion (percent) 

survival. The algal endpoint was cell density at the end of 96 h. 

DATA ANALYSIS

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

survival data was arc sine transformed prior to analysis. Cell density for the algal test was not 

transformed. Data were analyzed for normality using a Chi-Square Test and for homogeneity of 

3
variance using a F-test for Equality of Two Variances prior to analysis. A simple pairwise t-test 

was then conducted between the control survival and the treatment survival. Tests were 

conducted using the statistical package TOXSTAT 3.5 (Gulley, 1996). Where there was 100% 

survival in a test treatment no statistical comparison with control data was necessary. 

RESULTS AND DISCUSSION 

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

and reaerated water in Table 6. It took between five to 10 minutes to treat the 40 L of water with 

the N2/CO2 mixture and also to reaerate the treated mixture. DO was reduced in both water types 

after treatment with N2/CO2. An initial dissolved oxygen reading of 3.0 mg/L in the freshwater 

treatment was thought to be a DO probe problem. The probe was fixed for further experiments. 

Because of this, the final DO of the N2/CO2 treated freshwater is not known for certain, although 

saltwater when treated for the same amount of time had a DO of 1.0 mg/L. Both the freshwater 

and saltwater treated waters had lower pHs than the untreated water. After reaeration using the 

venturi unit, the DO was raised in both the freshwater and saltwater although not to pretreatment 

levels. This final reaerated treatment was used in the toxicity tests. 

Over the toxicity test period, the DO and pH in test beakers slowly returned to values consistent 

with the values measured before treatment (Tables 7 – 9). Salinity in the saltwater sheepshead 

minnow test remained consistent from test initiation to end. The salinity in the mysid test 

increased from 25‰ at test initiation to 30‰ at test end due to evaporation. This is consistent 

with results for this test in the past using a static test method. Because this species is 

cannibalistic more replicates were used and fewer organisms (5) were used per treatment 

replicate than in the sheepshead minnow test (10). The mysid test beakers were designed to 

spread the organism out in that the beakers were shorter and had a larger diameter. Thus, in this 

static test the test volume was reduced due to evaporation and the salinity therefore increased. 

There were no survival effects due to the N2/CO2 ballast water treatment (Tables 10 and 11). All 

test acceptability criteria given for these tests in Tables 1 through 5 were met. The undiluted 

reaerated ballast water treatment resulted in 100% survival for the freshwater fish Pimephales 

promelas, the freshwater daphnid Daphnia magna and saltwater fish Cyprinodon variegatus. 

Although there was a slight decrease in survival in the saltwater mysid Americamysis bahia test 

(95% survival), this survival was not significantly different from the control mysid survival. The 

mean cell density in the algae Selenastrum capricornutum undiluted reaerated ballast water 

treatment (9.743 X 106

 cells/mL) was slightly, but not significantly, greater than the cell density 

in the control treatment algae (9.4 X 106

 cells/mL). All other statistical comparisons indicated 

no significant decrease in organism survival or algal cell density for any treatment (100% 

continuously aerated; dilution series of non aerated). 

 

CONCLUSION 

The NEI de-oxygenation ballast water treatment, once reaerated, was not acutely toxic to the 

saltwater or freshwater fish or crustaceans tested. In addition, the treatment was not toxic to the 

green algal species tested. The green algae test method is a sensitive chronic toxicity population 

growth test for single cell plants. 

4
REFERENCES 

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

US EPA. 2002a. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters 

to Freshwater and Marine Organisms. Fifth Edition. EPA-821-R-02-012. U.S. Environmental 

Protection Agency, Office of Water, Washington, DC. 275 pp. 

US EPA. 2002b. 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.

5
Table 1. Summary of the Test Conditions and Test Acceptability Criteria for the Fathead 

 Minnow Pimephales promelas 96-Hour Acute Toxicity Test 

Test type: Static 

Test duration: 96 h 

Temperature: 25 oC (± 1oC) 

Lighting: Normal fluorescent laboratory lighting 

Photoperiod: 16 h light, 8 h dark 

Test chamber size: 1 L 

Test solution volume: 850 mL 

Renewal of test solutions: None 

Age of test organisms: 8 day; 24-h range in age 

No. organisms per test chamber: 10 

No. replicate chambers per concentration: 2 

No. organisms per concentration: 20 

Feeding regime: Artemia nauplii (<24 h old) during holding; 

Fed approximately 0.2 mL Artemia nauplii 

concentrate at 48 h. 

Test chamber cleaning: Cleaning prior to 48 h feeding 

Test chamber aeration: None except for 100% aerated treatment where 

rate was approximately 100 bubbles/min 

Dilution water: Deep unchlorinated well water

Test concentrations: 100%, 50%, 25% unaerated and 100% aerated 

Endpoint: Mortality 

Test acceptability criterion: 90% or greater survival in controls 

6
Table 2. Summary of the Test Conditions and Test Acceptability Criteria for the Daphnid 

(Daphnia magna) 48-Hour Acute Toxicity Tests 

Test type: Static 

Test duration: 48 h 

Temperature: 25 oC (± 1 oC) 

Lighting: Normal fluorescent laboratory lighting 

Photoperiod: 16 h light, 8 h dark 

Test chamber size: 100 mL screened baskets in fathead minnow 

test beakers 

Test solution volume: 50 mL in basket 

Renewal of test solutions: None 

Age of test organisms: <24 h neonates 

No. neonates per test chamber: 10 

No. replicate chambers per concentration: 2 

No. neonates per test concentration: 20 

Feeding regime: S. capricornutum and YCT 1-2 hours prior to 

use in test 

Cleaning: None 

Aeration: None except for 100% aerated treatment where 

rate was approximately 100 bubbles/min 

Dilution water: Deep unchlorinated well water 

Test concentrations: 100%, 50%, 25% unaerated and 100% aerated 

Endpoint: Survival 

Test acceptability criterion: 90% or greater survival in controls 

7
Table 3. Summary of Test Conditions and Test Acceptability Criteria for the Green Alga 

Selenastrum capricornutum Static Growth Test 

 

Test type: Static 

Test duration: 96 h 

Temperature: 25oC ± 1oC 

Lighting: Fluorescent 

Photoperiod: Continuous fluorescent illumination 

Test chamber size: 100 mL Erlenmeyer flasks with foam plugs 

Test solution volume: 50 mL 

Renewal of test solutions: None 

Age of test organisms: 4 to 7 d in log growth phase 

Initial cell density in test chambers: 10,000 cells/mL 

Shaking rate: 100 rpm continuous on a mechanical shaker 

Aeration: None 

Dilution water: 2X AAP algal nutrients added to each flask 

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

Endpoint: Growth (cell counts) 

Test acceptability criterion: Mean cell density of at least 1 x 106 cells/mL 

in the controls; and variability (CV%) among 

control replicates less than or equal to 20% 

8
Table 4. Summary of the Test Conditions and Test Acceptability Criteria for the Sheepshead 

 Minnow Cyprinodon variegatus 96-Hour Acute Toxicity Test 

Test type: Static 

Test duration: 96 h 

Temperature: 25 oC (± 1oC) 

Lighting: Normal fluorescent laboratory lighting 

Photoperiod: 16 h light, 8 h dark 

Test chamber size: 1 L 

Test solution volume: 850 mL 

Renewal of test solutions: None 

Age of test organisms: 8 d; 24-h range in age 

No. organisms per test chamber: 10 

No. replicate chambers per concentration: 2 

No. organisms per concentration: 20 

Feeding regime: Artemia nauplii (<24 h old) during holding; 

Fed approximately 0.2 mL Artemia nauplii 

concentrate at 48 h. 

Test chamber cleaning: Cleaning not required 

Test chamber aeration: None except for 100% aerated treatment where 

rate was approximately 100 bubbles/min 

Dilution water: 1 μm filtered Wye River water adjusted to 

25‰ with Crystal Sea Bioassay Laboratory 

Formulae® salt

Test concentrations: 100%, 50%, 25% unaerated and 100% aerated 

Endpoint: Mortality 

Test acceptability criterion: 90% or greater survival in controls 

9
Table 5. Summary of the Test Conditions and Test Acceptability Criteria for the Mysid 

Americamysis bahia (formerly Mysidopsis bahia) 96-Hour Acute Toxicity Tests 

Test type: Static 

Test duration: 96 h 

Temperature: 25 oC (± 1oC) 

Lighting: Normal fluorescent laboratory lighting 

Photoperiod: 16 h light, 8 h dark 

Test chamber size: 250 mL 

Test solution volume: 200 mL 

Renewal of test solutions: None 

Age of test organisms: 5 d; 24-h range in age 

No. organisms per test chamber: 5 

No. replicate chambers per concentration: 4 

No. organisms per concentration: 20 

Feeding regime: Artemia nauplii (<24 h old) during holding; 

Feed approximately 0.2 mL Artemia nauplii 

daily. 

Test chamber cleaning: Cleaning not required 

Test chamber aeration: None except for 100% aerated treatment where 

rate was approximately 100 bubbles/min 

Dilution water: 1 μm filtered Wye River water adjusted to 

25‰ with Crystal Sea Bioassay Laboratory 

Formulae® salt

Test concentrations: 100%, 50%, 25% unaerated and 100% aerated 

Endpoint: Mortality 

Test acceptability criterion: 90% or greater survival in controls 

10
Table 6. Water chemistry for test treatment preparation. Pre/Initial = test water prior to 

treatment; Treated = test water after N/CO2 treatment; Post/Air = Test solution after 

reaeration. Post/air is the solution used in the toxicity tests. 

Freshwater Preparation 

 DO pH 

Pre/Initial 8.4 8.12 

Treated 3.0* 5.64 

Post/Air 6.2 5.78 

* probe problem 

Saltwater Preparation (25 ‰) 

 DO pH 

Pre/Initial 7.7 7.98 

Treated 1.0 5.91 

Post/Air 5.7 6.23 

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Table 7. Fathead minnow (Pimephales promelas) and daphnid (Daphnia magna) test water 

quality data. Daphnid numbers are the same because we used baskets within the 

fathead minnow beakers to test this species. 100Air represents the aerated test 

solution. The designation of A or B represent the replicate from which the water 

quality measurements were taken. 

Fathead Minnow / Daphnia magna Assay 

0 hr 8/7/2008 

Treatment DO pH Cond Alkalinity Hardness 

ConA 8.3 7.68 310 105 60 

25A 

50A 

100A 8.1 6.45 490 125 80 

100AirA 

24 hr 8/8/2008

Treatment DO pH 

ConB 8.2 7.40

25B 7.9 7.22

50B 8.0 7.05

100B 7.4 6.75

100AirB 8.4 7.48

48 hr 8/9/2008

Treatment DO pH 

ConA 7.9 7.53

25A 7.7 7.69

50A 7.6 7.63

100A 7.1 7.45

100AirA 8.2 8.12

72 hr 8/10/2008

Treatment DO pH 

ConB 8.0 7.23

25B 7.7 7.45

50B 7.7 7.41

100B 7.6 7.32

100AirB 7.9 7.58

96 hr 8/11/2008

Treatment DO pH 

ConA 8.1 7.42

25A 8.1 7.64

50A 8.0 7.58

100A 8.1 7.82

100AirA 8.1 7.62

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Table 8. Algal (Selenastrum capricornutum) test water quality data. Only initial and final water 

quality data are taken in this test method. No aerated treatment was tested because the 

method does not allow aeration. 

Algal Assay 

Starting Water Chemistry 

pH DO

Control 7.95 8.2

25% 7.02 7.9

50% 6.67 7.7

100% 6.24 7.5

 

Final Water Chemistry (96 hr) 

pH DO

Control 9.41 8.5

25% 9.32 8.3

50% 9.21 8.1

100% 9.08 8.0

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Table 9. Sheepshead minnow (Cyprinodon variegates) and mysid (Americamysis bahia) test 

water quality data. 100Air represents the test solution continuously aerated. The 

designation of A or B for the fish and A - D for the mysids, represent the replicate 

from which the water quality measurements were taken. 

Sheepshead Minnow assay Americamysis bahia assay 

0 hr 8/7/2008 0 hr 8/7/2008

Treatment DO pH Salinity Treatment DO pH Salinity 

ConA 8.1 8.06 25 ConA 8.1 8.06 25

25A 25A 

50A 50A 

100A 7.0 6.43 25 100A 7.0 6.43 25

100AirA 100AirA 

24 hr 8/8/2008 24 hr 8/8/2008 

Treatment DO pH Treatment DO pH 

ConB 8.2 7.85 ConB 7.2 8.04 

25B 7.8 7.68 25B 7.3 7.80 

50B 7.8 7.44 50B 7.3 7.81 

100B 7.2 7.17 100B 7.3 8.04 

100AirB 8.2 8.01 100AirB 7.6 8.10 

48 hr 8/9/2008 48 hr 8/9/2008 

Treatment DO pH Treatment DO pH 

ConA 7.7 7.82 ConC 7.5 7.94 

25A 7.4 7.93 25C 7.4 8.06 

50A 7.3 7.86 50C 7.3 8.12 

100A 7.0 7.76 100C 7.2 8.17 

100AirA 8.0 8.19 100AirC 7.7 8.23 

72 hr 8/10/2008 72 hr 8/10/2008 

Treatment DO pH Treatment DO pH 

ConB 7.7 8.02 ConD 7.5 8.24 

25B 7.6 7.99 25D 7.5 8.24 

50B 7.5 7.98 50D 7.5 8.24 

100B 7.6 7.96 100D 7.7 8.27 

100AirB 7.8 8.17 100AirD 7.9 8.31 

96 hr 8/11/2008 96 hr 8/11/2008

Treatment DO pH Salinity Treatment DO pH Salinity 

ConA 7.8 8.02 25 ConD 7.0 8.19 30

25A 7.7 8.11 25 25D 6.9 8.21 30

50A 7.7 8.09 25 50D 7.0 8.23 30

100A 7.7 8.10 25 100D 7.0 8.25 30

100AirA 7.8 8.21 25 100AirD 7.0 8.31 30

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Table 10. Survival data for fathead minnow (Pimephales promels) and daphnid (Daphnia 

magna) and cell density data for the algae Selenastrum capricornutum exposed to 

reaerated N/CO2 treatment. 100Air represents the test solution continuously aerated. 

The designation of A or B for the fish, or A – C for the algae, represent the replicate 

data. 

Fathead Minnow 96 hr Assay

 Survival 

Treatment 24 hr 48 hr 72 hr 96 hr % Tot 

ConA 10 10 10 10 90 

ConB 10 10 10 8 

25A 10 10 9 9 95 

25B 10 10 10 10 

50A 9 9 9 8 90 

50B 10 10 10 10 

100A 10 10 10 10 100 

100B 10 10 10 10 

100AirA 10 10 9 7 80 

100AirB 10 10 9 9 

Daphnia magna 48 hr Assay 

 Survival 

Treatment 24 hr 48 hr % Tot

ConA 10 10 100

ConB 10 10 

25A 10 10 100

25B 10 10 

50A 10 10 100

50B 10 10 

100A 10 10 100

100B 10 10 

100AirA 10 10 100

100AirB 10 10 

Green Alga 96 hr Assay 

 Cell Density (raw count data) 

Treatment A B C Mean 

Control 184.7 191.0 188.3 188.0

25% 199.0 180.7 178.3 186.0

50% 201.3 200.3 183.0 194.9

100% 197.7 199.7 174.0 190.5

 

 Cell Density (x106

/mL) 

Treatment A B C Mean CV% 

Control 9.235 9.550 9.415 9.400 1.7 

25% 9.950 9.035 8.915 9.300 6.1 

50% 10.065 10.015 9.150 9.743 5.3 

100% 9.885 9.985 8.700 9.523 7.5 

 

Starting Density Mean 

23,520 2.09E+04 

18,620 

20,580 

15
Table 11. Survival data for sheepshead minnow (Cyprinodon variegates) and mysid 

(Americamysis bahia) exposed to reaerated N/CO2 treatment. 100Air represents the 

test solution continuously aerated. The designation of A or B and C or D for the 

mysids, represent the replicate data. 

Sheepshead Minnow 96 hr Assay 

 Survival 

Treatment 24 hr 48 hr 72 hr 96 hr

% 

Tot

ConA 10 10 10 10 100

ConB 10 10 10 10 

25A 10 10 10 10 100

25B 10 10 10 10 

50A 10 10 10 10 100

50B 10 10 10 10 

100A 10 10 10 10 100

100B 10 10 10 10 

100AirA 10 10 10 10 100

100AirB 10 10 10 10 

Americamysis bahia 96 hr Assay 

 Survival 

Treatment 24 hr 48 hr 72 hr 96 hr

% 

Tot

ConA 5 5 5 5 100

ConB 5 5 5 5 

ConC 5 5 5 5 

ConD 5 5 5 5 

25A 5 5 5 5 100

25B 5 5 5 5 

25C 5 5 5 5 

25D 5 5 5 5 

50A 5 5 5 5 85

50B 5 5 5 5 

50C 5 5 3 3 

50D 5 5 5 4 

100A 5 5 5 5 95

100B 4 4 4 4 

100C 5 5 5 5 

100D 5 5 5 5 

100AirA 5 5 4 4 85

100AirB 5 5 4 4 

100AirC 5 5 4 4 

100AirD 5 5 5 5 

16