### Marrowstone Sodium Hypochlorite Mesocosm September 2004

The first test of the sodium hypochlorite gene rator/filtration treatmen t system was started on September 3, 2004. The experiment include d a chlorine/no filtration treatment and chlorine with filtration treatment. The target chlorination level was 4 mg/L. Initial chlorine levels for the filtered treatment we re 3.5 mg/L and 2.95 mg/L without filtration (Table 1). There were 4 mesocosms (tanks) per treatment and 4 cont rol mesocosms. The mesocosms were analyzed for Total Residual Oxidant (Cl 2 

### mg/L), culturable heterotrophic bacteria, chlorophyll a , and zooplankton at 5, 24, 48, 120, and 240 hours following treatment.

Methods
Total Residual Oxidant (TRO) was measured as Cl 2 

### using a Hach Spectrophotometer and Hach Colorimeter. DPD powder pillows were used for the analysis. TRO measurements were after the initial fill and at a ll of the time points listed above. The 1500-gallon tank of raw seawater was tested for bacteria leve ls before the start of the experiment. Water samples taken at the 5 time poi nts were also tested for bacteria levels. Bacteria colonies were cultured from know n volumes of water to calculate colony forming units (CFU) per liter of water. Col onies were cultured on petri dishes containing a growth medium suited for marine heterotroph ic bacteria. The inoculated medium was analyzed for colony formation after a few days at room temperature incubation. Water samples were also taken for chlorophyll a analysis. Chlorophyll a is an indicator for the presence of live phytoplankton. A known volume of sample water was filtered through glass fiber filters w ith a pore size small enough to retain phytoplankton cells. The filters were frozen for later analysis in our Seattle laboratory. Chlorophyll a was extracted from the filters using acetone and then analyzed for fluorescence to determine the concentration in μg/L. Mesozooplankton were collected in Myster y Bay, using a 110μm mesh net 1 meter diameter the morning of the test. Enough we re collected to achieve approximately 150 mesozooplankton per liter. A Stempel pipe tte was used three times to collect a randomized 5 ml sample of the mesozooplankt on “soup”. The densi ties counted using a dissecting scope were used to calculate how much of the “soup” was needed for the 1500-gallon tanks to achieve 150 mesozooplankton per liter. The calculated amount of soup was poured into the tank and allowed to se ttle for at least an hour to let zooplankton acclimate. Three preliminary samples were collected out of the 1500-gallon tank after mixing. These were used to check calcula tion and to see effects of pumps. Sampling periods for mesozooplankton were 5,24,48,120, a nd 240 hours. Samples were collected out of the 72-gallon mesocosms using a 1-l iter Nalgene bottle afte r thoroughly mixing the contents of the mesocosm. Liter sample was filtered through a 73μm sieve and placed in counting tray. Mesozooplankton counted were placed into one of eight generic

### categories and then by state; live, dead (a bsolutely no response to poking) or moribund (internal movement and no flight response to needle poke).

Results
Total Residual Oxidant (TRO) levels declined steadily for the durati on of the experiment (Figure 1). TRO in the nonfiltered test tanks dr opped more than the filtered test tanks in the first 5 hours and then the TRO dissipate d equally for the remaining time points. Bacteria were greatly reduced in both tr eatments and showed minimal rebound over the 10 days of the experiment (Figure 2). Ther e was a slight rebound of bacteria in the treatment without filtration. Chlorophyll a is an indicator of phytoplankt on. In the treated seawater, Chlorophyll a levels were at or below the detection limit starting with the 5-hour time point and continued to drop for both of the treatments (Figure 3.B, Fi gure 3.C). In the control tanks, chlorophyll a levels dec lined over the duration of the experiment, likely due to the absence of light since al l of the mesocosms were covered (Figure 3.A).

After the first treatment trial it was conclude d that no statistical difference could be found between the two treatments, at all time points and states (F igure 4A-4C). The chlorine plus filter 5 hour time point ha d one live polychaeta larvae in two of the replicates Figure 4.E. This was the only time that live organi sms were found in any of the treatments and time points. The difference between the contro l and treatments was highly significant. Some mesozooplankton were still able to get through the 50μm filter, all were dead except for the 2 mentioned above.

### Heterotrophic Bacteria Marrowstone Sodium Hy pochlorite Mesocosom September 2004
Marrowstone Sodium Hypochlorite Mesocosm Total Residual Oxidant Cl 2 9-3-04

0.00 0.50 1.00 1.50 2.00 2.50 3.00 3.50 4.00

0 50 100 150 200 250 Time (hrs)TRO Cl2(ppm) Control Cl w/ filter Cl w/o filter

Marrowstone Sodium Hypochlorite Mesocosm Heterotrophic Bacteria 9-3-04

1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 1.00E+08 1.00E+09 1.00E+10

0 50 100 150 200 250 Time (hrs)Colony Forming Units / liter

Control Cl w/ filter Cl w/o filter 

### Figure 1

Figure 2

### Chlorophyll a (μg/L) Marrowstone Sodium Hy pochlorite Mesocosom September 2004

Control

0 2 4 6 8 10 12 14

0 20 40 60 80 100 12 0 140 160 180 200 220 240 Time (hours)chl a (ug/L) Control Pretreatment



### Chlorine with Filter

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

0.1

0 20 40 60 80 10 0 12 0 14 0 16 0 18 0 20 0 22 0 24 0 Time (hours)chl a (ug/L)



### Chlorine with no Filter

0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09

0.1

0 20 40 60 80 10 0 12 0 14 0 16 0 18 0 20 0 22 0 24 0 Time (hours)chl a (ug/L) 

### Figure 3.A

Figure 3.B

Figure 3.C

### Zooplankton analysis Marrowstone Sodium Hy pochlorite Mesocosm September 2004 Live Mesozooplankton

0 50 100 150

5 24 48 120 240 Time(hours)Zooplankton/L



### Pre-treatment Control Chlorine Filter Chlorine no-filter

Total Mesozooplankton

0 50 100 150 200

5 24 48 120 240 Time(hours)Zooplankton/L



### Pre-treatment Control Chlorine Filte r Chlorine no-filte r

Percent Live Mesozooplankton

0% 20% 40% 60% 80% 100%

5 24 48 120 240 time(hours)percent live



### Control Chlorine Filter Chlorine no-filter

### Control Treatment

0% 20% 40% 60% 80% 100%

5 24 48 120 240 Time(hours)PercentMesozooplankton 

# Dead Moriund Live

Chlorine Filter Treatment

0% 20% 40% 60% 80% 100%

5 24 48 120 240 Time(hours)PercentMesozooplankton
Dead Moriund Live

Chlorine Non-filter Treatment

0% 20% 40% 60% 80% 100%

5 24 48 120 240 Time(hours)PercentMesozooplankton Dead Moriund Live