### 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 Re search 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 issu e in the last decade due to the problem of invasive species transported and released by th e 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 elimin ating aquatic species carrie d 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-oxygenati on ballast water treatment using a venturi unit to inject a gas mixture into ballast water, redu ce dissolved oxygen and thus kill invasive species. Prior to discharge to the environment the ballast water is then reaerated. Standard US Environmental Protection Ag ency 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 fr eshwater green alga test with Selenastrum capricornutum was conducted to assess the effects of the ballast water discharg e 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 eff ect on green algae population growth.

2

### INTRODUCTION

Ballast water treatment has become a major issu e in the last decade due to the problem of invasive species transported and released by th e 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 elimin ating aquatic species carrie d 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 us ed to generate the freshwater and saltwater test solutions.

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

### /CO 2 

### gas mixture through 40L of either a freshwater or saltwat er (25‰) to reduce di ssolved oxygen (DO) to

treatment levels. Once DO was reduced the ventur e 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 sp ecies (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 Enviro nmental 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. Environm ental 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 populat ion level toxicity test due to the rapid algal population increases measured by the method (US EP A, 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 Educa tion Center has conducted all of the acute and chronic effluent testing for th e State of Maryland’s Department of the Environment NPDES bioassay program using these species.

Treatments tested in this study in cluded 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 test s other than the algae was proportion (percent) survival. The algal endpoint was ce ll density at the end of 96 h.

DATA ANALYSIS

Data were analyzed using US EPA (2002a, 2002b) statistical proce dures. All proportion survival data was arc sine transformed prior to an alysis. 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 trea tment survival. Tests were conducted using the statistical package TOXS TAT 3.5 (Gulley, 1996). Where there was 100%

survival in a test treatment no statistical co mparison with control da ta was necessary.

RESULTS AND DISCUSSION

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

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

the N 2 

### /CO 2 

### mixture and also to reaerate the treated mixture. DO was reduced in both water types

after treatment with N 2 

### /CO 2 

### . An initial dissolved oxygen read ing 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 N 2 

### /CO 2 

### treated freshwater is no t known for certain, although

saltwater when treated for the same amount of tim e had a DO of 1.0 mg/L. Both the freshwater and saltwater treated waters had lower pHs than the untreated water. Af ter reaeration using the venturi unit, the DO was raised in both the freshw ater 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 (Tab les 7 – 9). Salinity in the saltwater sheepshead

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

increased from 25‰ at test initiati on to 30‰ at test end due to ev aporation. This is consistent with results for this test in the past using a static test method. Because this species is cannibalistic more replic ates were used and fewer organi sms (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 N 2 

### /CO 2 

### ballast water treatment (Tables 10 and 11). All test acceptability cr iteria given for these tests in Tables 1 through 5 were met. The undiluted

reaerated ballast water treatme nt 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 10 6 

### cells/mL) was slightly, but not significantly, greater than the cell density in the control treatment algae (9.4 X 10 6 

### cells/mL). All other statis tical 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, th e treatment was not toxic to the green algal species tested. The green algae test method is a sensitive ch ronic toxicity population growth test for single cell plants.

4

### REFERENCES

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

US EPA. 2002a. Methods for Measuring the Acute Toxicity of Effluents and Receiving Waters to Freshwater and Marine Organisms. Fift h Edition. EPA-821-R-02-012. U.S. Environmental Protection Agency, Office of Wa ter, Washington, DC. 275 pp.

US EPA. 2002b. Short-term Methods for Estim ating the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms. Fourth Edition. EPA-821-R-02-013. U.S. Environmental Protection Agency, Offi ce 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 crite rion: 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 screen ed 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 crite rion: 90% or greater survival in controls

7

### Table 3. Summary of Test Condi tions 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 Erlenm eyer 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 te st chambers: 10,000 cells/mL

Shaking rate: 100 rpm continuous on a mechanical shaker

Aeration: None

Dilution water: 2X AAP algal nutri ents 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 10 6 

### cells/mL in the controls; and variability (CV%) among control replicates less th an or equal to 20%

8

### Table 4. Summary of the Test Conditions and Te st 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 crite rion: 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 crite rion: 90% or greater survival in controls

10

### Table 6. Water chemistry for te st treatment preparation. Pre/ Initial = test water prior to treatment; Treated = test water after N/CO 2 

### treatment; Post/Air = Test solution after

reaeration. Post/air is the soluti on 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



### 11

### 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 speci es. 100Air represents the aerated test solution. The designation of A or B represent the repl icate 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



### 12

### 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



### 13

### Table 9. Sheepshead minnow ( Cyprinodon variegates) and mysid ( Americamysis bahia ) test water quality data. 100Air re presents 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



### 14

### 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/CO 2 

### 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 (x10 6 /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/CO 2 

### treatment. 100Air represents the test solution continuously aerated. The de signation 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