Coastal Circulation and Sediment 

Dynamics in Hanalei Bay, Kauai 

PART III: 

Studies of Sediment Toxicity 

U.S. Department of the Interior 

U.S. Geological Survey 

Open-File Report 2006-1147 

 

 

 QuickBird satellite image of Hanalei Bay
 

Coastal Circulation and Sediment 

Dynamics in Hanalei Bay, Kauai 

PART III: Studies of Sediment Toxicity 

 

Robert S. Carr 1, Marion Nipper 2, Michael Field 3, and James M. 

Biedenbach 1

 

1

 U.S. Geological Survey, Marine Ecotoxicology Research Station, Corpus Christi, Tex. 2 Texas A&M University-Corpus Christi, Center for Coastal Studies, Corpus Christi, Tex. 

3 U.S. Geological Survey, Pacific Science Center, Santa Cruz, Calif. 

U.S. Geological Survey 

Open-File Report 2006-1147 

Corpus Christi, Texas 

April 2006 
TABLE OF CONTENTS 

 

INTRODUCTION .......................................................................................................................................... 1 

 Project Objectives .......................................................................................................................... 1 

 Study Areas ..................................................................................................................................... 1 

 

MATERIALS AND METHODS................................................................................................................... 1 

 Scientific Personnel.......................................................................................................................2 

 Sample Collection and Tracking ..................................................................................................2 

 Toxicity Testing ...............................................................................................................................2 

 

RESULTS AND DISCUSSION...................................................................................................................3 

 

ACKNOWLEDGEMENTS............................................................................................................................4 

 

LITERATURE CITED ....................................................................................................................................4 

TABLES 

Table 1. Station locations and site descriptions......................................................................... 6 

Table 2. Scientific personnel.......................................................................................................... 6 

Table 3. Water quality parameters after salinity adjustment and original salinity of 

sediment porewater samples from Hanalei Bay, Kauai, Hawaii in 2005 ................ 7 

Table 4. Sea urchin fertilization test raw data and mean values for sediment porewater 

samples from Hanalei Bay, Kauai, Hawaii. Asterisks denote statistically

significant differences (Dunnett’s t-test) and detectable significance criteria

(DSC) between test and control station 6 (* alpha < 0.05, ** alpha < 0.01) ............ 8 

Table 5. Sea urchin embryological development test raw data and means for sediment 

 porewater samples from Hanalei Bay, Kauai, Hawaii. Asterisks denote 

 statistically significant differences (Dunnett’s t-test) and detectable significance 

criteria between test and control station 6 (* alpha < 0.05, ** alpha < 0.01) ......... 10 

FIGURE 

Figure 1. Toxicity test results for samples taken in Hanalei Bay, Kauai, Hawaii. Color 

 differentiation of the circles indicates the level of toxicity observed in the sea 

 urchin fertilization test. There were no toxic samples observed in the sea 

 urchin embryological development test ....................................................................... 12 

i
 APPENDICES 

 

Appendix 1. (SOP F10.24) Extraction and Storage of Porewater Samples Using In Situ Devices. 

Appendix 2. (SOP F10.12) Water Quality Adjustment of Samples 

Appendix 3. (SOP F10.6) Sea Urchin Fertilization Toxicity Test 

Appendix 4. (SOP F10.7) Sea Urchin Embryological Development Toxicity Test 

 

ADDITIONAL DIGITAL INFORMATION 

 

For an online PDF version of this report, please see: 

http://pubs.usgs.gov/of/2006/1147/

For more information on the U.S. Geological Survey Marine Ecotoxicology Research Station 

please see: 

http://sci.tamucc.edu/mers/index.htm

For more information on the U.S. Geological Survey Western Coastal and Marine Geology 

Team, please see: 

http://walrus.wr.usgs.gov/

For more information on the U.S. Geological Survey’s Coral Reef Project, please see: 

http://coralreefs.wr.usgs.gov/

DIRECT CONTACT INFORMATION 

 

General Project Information 

Dr. Michael E. Field (Project Chief): mfield@usgs.gov

Regarding this Report: 

Dr. Robert. S. Carr (Station Leader) scott_carr@usgs.gov

SUGGESTED CITATION 

Carr, R.S., Nipper, M., Field, M.E. and Biedenbach, J.M., 2006, Coastal circulation and sediment 

dynamics in Hanalei Bay–studies of sediment toxicity: U.S. Geological Survey Open-File Report 

2006-1147, 12 p. plus 4 appendices. 

 

Any use of trade, product, or firm names is for descriptive purposes only and does not imply 

endorsement by the U.S. Government.

ii
INTRODUCTION 

Toxicity tests are commonly conducted as a measure of the bioavailability of toxic 

chemicals to biota in an environment. Chemical analyses alone are insufficient to determine 

whether contaminants pose a threat to biota (Carr and Nipper, 2003). Porewater toxicity tests 

are extremely sensitive to a broad range of contaminants in marine environments and provide 

ecologically relevant data on sensitive life stages. The inclusion of porewater toxicity testing as 

an additional indicator of sediment quality provides a more comprehensive picture of 

contaminant effects in these sensitive habitats. 

In this study purple-spined sea urchin (Arbacia punctulata) fertilization and 

embryological development porewater toxicity tests were used to evaluate the sediments 

collected from the coastal environment around Hanalei Bay, Kauai, Hawaii. These tests have 

been used previously to assess the bioavailability of contaminants associated with sediments in 

the vicinity of coral reefs (Carr and Nipper, 1998, Carr et al. 2001; Nipper and Carr, 2000, 2001). 

 

Project Objectives 

• Collect pore water in situ from 11 sites in Hanalei Bay and from a reference site at KN’ N

Beach. 

• Measure water quality parameters (salinity, dissolved oxygen, pH, temperature, and 

ammonia) of thawed porewater samples prior to testing and adjust salinity, temperature 

and dissolved oxygen, if necessary. 

• Conduct the fertilization and embryological development toxicity tests with pore water 

using sea urchin gametes. 

• Quality control assays with reference pore water, dilution blanks and a positive control 

dilution series with sodium dodecyl sulfate (SDS) in conjunction with each test 

• Make statistical comparisons between test and reference stations. 

Study Area 

 The study area is located in Hanalei Bay, on the north coast of Kauai, Hawaii. Most of 

the samples were collected near the central and east coasts of the bay, with some collected in 

the central part of the bay. One reference site was collected west of the bay at KN’ N Beach. 

Table 1 details the geographic coordinates of the stations, dates of sampling and physical 

descriptions of the sites. 

MATERIALS AND METHODS 

This section details information about the personnel, equipment, and procedures used in 

the collection, handling, and testing of sediment pore water. Table 2 provides the list of 

personnel involved in this study and appendices 1 through 4 detail the equipment and precise 

1
methodology used in the collection, adjustment, measurement and toxicity testing of the sample 

pore water. 

Scientific personnel 

 Collection of the pore water in the field was performed by two scientists, one from the 

USGS Marine Ecotoxicology Research Station (MERS) and one from the Texas A&M Corpus 

Christi Center for Coastal Studies. A third scientist, also from MERS, was responsible for 

processing and testing the water samples once they were returned to Corpus Christi. 

Sample Collection and Tracking 

Collection of samples took place on August 25, 26 and 27 of 2005. Six of the samples 

were collected near shore in a water depth of approximately 2 m. Five samples were taken in 

deeper water from a boat at depths ranging from 12 to 15 m. One reference seawater sample 

was taken by submerging two precleaned acid washed 290-ml polycarbonate centrifuge tubes 

to a depth of 1 m and removing the lids. All porewater samples were collected in situ, by 

vacuum extraction (see Appendix 1, SOP F10.24), using a device that consisted of a groundglass airstone attached to a presoaked 60-ml polypropylene catheter type syringe. All vacuum 

extraction device sampling materials were presoaked for 1 week in filtered seawater (5μm) and 

4 weeks in deionized water with water exchanges every 2 to 3 days. Seawater samples from 

the vacuum extraction devices were tested for latent toxicity following the soaking procedure 

and found to be nontoxic. Following collection, pore water collected in the syringes was 

composited in acid washed 290-ml polycarbonate centrifuge tubes, capped and kept on ice or 

refrigerated until they were transported back to MERS. Samples were received at MERS on 

August 30, 2005, where they were immediately centrifuged at 1200 x g for 20 minutes. The 

supernatant for each sample site was composited and then divided among 250-ml precleaned 

glass bottles and frozen at -20°C until testing. 

Toxicity Testing 

 Two days before conducting the toxicity test, the samples were moved from the freezer 

to a refrigerator set to 4°C. One day prior to testing, samples were thawed completely in a tepid 

(20°C) water bath. Temperature of the sample was brought up to 20°C + 1°C. Sample salinity 

was measured and adjusted to 30 + 1‰, if necessary using purified deionized water or 

concentrated brine (see Appendix 2, SOP F10.12). Following salinity adjustments, the samples 

were returned to the refrigerator set to 4°C. They were brought up to 20°C + 1°C before the 

start of the toxicity testing the next day. 

 On the day of testing, after temperature had been returned to 20°C + 1°C, further water 

quality measurements were made on the samples. Dissolved oxygen (DO) was measured with a 

Yellow Springs Instrument meter; salinity was measured with a Reichert® temperature 

compensated refractometer; and pH and total ammonia expressed as nitrogen (TAN) were 

measured with Orion® meters and their respective probes. Un-ionized ammonia expressed as 

nitrogen (UAN) was calculated for each sample using the respective salinity, temperature, pH 

2
and TAN values. Any samples containing less than 80 percent DO saturation were gently 

aerated by stirring the sample on a magnetic stir plate. 

 Toxicity of the sediment pore water, controls, and reference toxicants was determined 

using the fertilization and embryological development tests with the purple-spined sea urchin 

following the procedures outlined in SOP F10.6 and SOP F10.7 (appendices 3 and 4). Purplespined sea urchins were obtained from the Marine Biological Laboratory in Woods Hole, Mass. 

Each of the 13 samples was tested in a dilution series design at 100, 50, and 25 percent of the 

water quality adjusted sample with 5 replicates per treatment. Dilutions were made with 0.45 

μm filtered seawater. A reference porewater sample collected from Aransas Bay, Texas was 

included with each toxicity test as a negative control. This site is far removed from any known 

sources of contamination and has been used previously as a reference site (Carr and Nipper, 

1998). In addition, a dilution water blank of filtered seawater was also included in each test. A 

dilution series test with sodium dodecyl sulfate (SDS) was also included as a positive control in 

each assay. 

 For both sea urchin assays, SAS (SAS Institute Inc., 1989) was used to make statistical 

comparisons among treatments. Analysis of variance (ANOVA) and Dunnett's one-tailed t-test 

(which controls the experimentwise error rate) were used on the arcsine square root 

transformed data. The trimmed Spearman-Karber method (Hamilton and others, 1977; 

Environmental Science and Technology, 1978) with Abbott's correction (Morgan, 1992) was 

used to calculate EC50 (50% effective concentration) values for dilution series tests. Prior to 

statistical analysis, the transformed data sets were screened for equal variance using 

SAS/LAB®

 Software (SAS Institute Inc., 1992). The SAS/LAB® software performs a Levene’s test 

for equal variance. If a one way ANOVA (performed on the absolute deviations of the 

observations from their respective group mean values) revealed any statistical evidence of 

unequal variances, additional data transformations were performed and outliers removed if 

necessary. Outliers were detected by comparing the studentized residuals to a critical value 

from a t-distribution chosen using a Bonferroni-type adjustment. The adjustment is based on 

the number of observations, n, so that the overall probability of a type I error is at most 5%. The 

critical value, cv, is given by the following equation: cv = t(dfError , 0.05/(2 x n)). 

 A second criterion was also used to compare test mean values to reference mean values. 

Detectable significance criteria (DSC) were developed to determine the 95 percent confidence 

value based on a power analysis of similar tests performed by our lab (Carr and Biedenbach, 

1999). This value is the percent minimum significant difference from the reference necessary to 

accurately detect a difference from the reference. The DSC value for the sea urchin fertilization 

assay at α = 0.05 is 15.5 percent. At α = 0.01, the DSC value is 19 percent. The DSC value for the 

sea urchin embryological development assay at α = 0.05 is 16.4 percent. At α = 0.01, the DSC 

value is 20.6 percent. 

RESULTS AND DISCUSSION 

 This section reviews the data collected from the water quality measurements as well as the 

results of the toxicity tests and discusses their possible implications. Water quality 

measurements can be found in Table 3, fertilization test raw data and statistical comparison 

3
results in Table 4 and embryological development test raw data and statistical comparison 

results in Table 5. 

 To satisfy the test’s salinity requirement of 30 + 1 ‰, all 12 samples required salinity 

adjustment using Milli-Q® purified water. The Texas reference pore water required a salinity 

adjustment using concentrated brine. The initial dissolved oxygen was > 80 percent in all 

samples and therefore did not required aeration. The pH of test samples ranged from 7.843 to 

8.112 with the reference seawater collected on site slightly higher at 8.283 and the Texas 

reference pore water slightly lower at 7.647. Total ammonia as nitrogen (TAN) was low for all 

samples and ranged from < 0.1 to 0.912 mg/L with the un-ionized fraction (the most toxic 

fraction) never rising above 20 μg/L. The un-ionized ammonia concentrations did not approach 

the lowest observed effect concentration (LOEC) for the fertilization nor the embryological 

development assays at 800 μg/L and 90 μg/L, respectively. 

 Three porewater samples in the fertilization test were found to be toxic when compared to 

the reference station (sample 6) from Ke ́e Beach. Stations 8, 10 and 12 were all found to be 

toxic at the 100 percent water quality adjusted concentration with toxicity also observed in the 

50% dilution from Station 10. Figure 1 identifies with colored dots the locations of the stations 

found to be toxic. No porewater samples were found to be toxic in the embryological 

development test. Reference toxicant controls were acceptable for both tests with EC50 values 

of 5.63 mg/L (5.27-6.00) for the fertilization test and 3.79 mg/L (3.59-4.00) for the embryological 

development test. Both of these results fall within the control charts for this laboratory. The 

control chart mean for the fertilization test is 5.54 mg/L (3.32-7.76). The control chart mean for 

the embryological development test is 4.20 mg/L (1.82-6.58). 

ACKNOWLEDGEMENTS 

 

 We would like to acknowledge the logistical support provided by Josh Logan during the 

offshore field sampling. We are also indebted to Capt. Earl Loder who provided and piloted the 

research vessel. Dr. Carl Berg provided insightful information to add in the selection of 

sampling stations. 

LITERATURE CITED 

Carr, R.S., and Biedenbach, J.M., 1999, Use of power analysis to develop detectable 

significance criteria for sea urchin toxicity tests: Aquatic Ecosystem and Health 

Management, v. 2, p. 413-418. 

Carr, R.S., and Nipper, M.G., 1998, Preliminary survey of sediment toxicity in the vicinity of 

Honolulu, Hawaii: Menlo Park, Calif., U.S. Geological Survey, 18 p., 4 attachments. 

Carr, R.S., and Nipper, M.G., eds., 2003, Porewater toxicity testing: biological, chemical, and 

ecological considerations: Pensacola, Fl, USA, Society of Environmental Toxicology and 

Chemistry (SETAC), 346 p. 

4
5

Carr, R.S., Nipper, M.G., and Plumlee, G.S., 2001, A preliminary survey of marine contamination 

from mining-related activities on Marinduque Island, Philippines–porewater toxicity and 

chemistry, results from a field trip October 14-19, 2000: U.S. Geological Survey Open-File 

Report 01-441, 19 p., 3 appendices, http://pubs.usgs.gov/of/2001/ofr-01-0441/

Environmental Science and Technology, 1978, Trimmed Spearman-Karber method for estimating 

median lethal concentrations in toxicity bioassays [correction of original]: Environmental 

Science and Technology, v. 12, p. 417. 

Hamilton, M.A., Russo, R.C., and Thurston, R.V., 1977, Trimmed Spearman-Karber method for 

estimating median lethal concentrations in toxicity bioassays: Environmental Science 

and Technology, v. 11, p. 714-719; Correction 12:417 (1978). 

Morgan, B.J.T., 1992, Analysis of Quantal Response Data: London, England, Chapman and Hall, 

511 p. 

Nipper, M.G. and Carr, R.S., 2000, Toxicity testing of sediment pore water from the Flower 

Garden Banks, Gulf of Mexico: Corpus Christi, Tex., Texas A&M University–Corpus 

Christi, 7 p. and 3 attachments. 

Nipper, M.G., and Carr, R.S., 2001, Porewater toxicity testing–a novel approach for assessing 

 contaminant impacts in the vicinity of coral reefs: Bulletin of Marine Science, v. 69, p. 

407-420. 

SAS Institute Inc., 1989, SAS/STAT® user's guide, version 6, volume 2 (4th ed.): Cary, N.C., SAS 

Institute Inc., 846 p. 

SAS Institute Inc., 1992, SAS/LAB®

 software–user's guide, version 6 (1st ed.): Cary, N.C., SAS 

Institute Inc., 291 p. 
6

TABLE 1. Station locations and site descriptions.

Station # Date Sampled Location Latitude Longitude Physical description1 8/25/2005 Hanalei River Transect 22.213766 -159.4972 ~100 m offshore of coordinates, 5 m water depth, obvious 

2 8/25/2005 Hanalei River Mouth 22.201266 -159.49775 ~ 50 m offshore in mixing zone, approximately 0.5m water 3 8/25/2005 Hanalei Pavilion Beach Park 22.2073 -159.498433 ~100 m offshore of coordinates, 2 m water depth, sandy b

4 8/25/2005 Waioli Beach Park 22.203033 -159.505733 ~100 m offshore of coordinates, 2.3 m water depth, fine sa

5 8/25/2005 Foamy River Beach Park-Waipa Beach 22.2055 -159.514966 ~ 30 m offshore from coordinates, 2 m water depth, samp

6 8/25/2005 KN'N Beach-Haena State Park 22.223216 -159.5816 ~40 m offshore from coordinates, 2 m water depth, end of 7 8/26/2005 Outer Wall 22.2149477 -159.5032211 10 m west of wall, 13.2 m water depth, medium sand8 8/26/2005 Black Hole (study designation) 22.2121235 -159.5014578 inside boat mooring area, 15.2 m water depth, flocky neph

9 8/26/2005 CRAMP Monitoring Site 22.2139833 -159.5127388 near wall on western side of bay, 13.3 m water depth, cor

10 8/26/2005 Inner Wall 22.2133682 -159.5007138 along the wall out of visual range, flocky bottom, mixing z

11 8/26/2005 Mid-Bay Deep 22.2125964 -159.5083736 middle of the bay opposite Waouli Beach Park, 12 m wate12 8/27/2005 Princeville Resort Beach 22.217916 -159.498516 ~100 m offshore of coordinates, 2 m water depth, sand gr

Ref. SW 8/25/2005 KN'N Beach-Haena State Park 22.223216 -159.5816 ~40 m offshore from coordinates, 2 m water depth, sampl

TABLE 2. Scientific personnel. Person Affiliation Responsibilities Robert S. Carr USGS Principal Investigator, Collection of pore water

Marion Nipper TAMU-CC/CCS Co-Principal Investigator, Collection of pore waterMichael Field UGSS Project LeaderJames M. Biedenbach USGS Pore water processing, toxicity testing
 sediment porewater samples from Hanalei Bay, Kauai, Hawaii in 2005.

Salinity1 DO 2 % TAN 4 UAN 5 % o

/oo (mg/L) DO 3 (mg/L) (ug/L) OUS 6

1 35 8.7 100.4 7.948 < 0.1 < 2.8 85.7

2 32 8.78 101.0 7.988 0.258 7.9 93.8

3 35 8.91 102.6 8.078 0.0947 3.5 85.7

4 35.5 8.83 101.5 8.112 < 0.1 < 18.0 84.7

5 35 8.72 100.3 7.987 0.115 3.5 85.7

 6 7 35 8.85 101.7 7.905 0.27 6.8 85.7

7 35 8.88 102.3 7.843 0.912 20.1 85.7

8 35.5 8.42 96.6 7.853 0.179 4.0 84.7

9 35.5 8.76 100.1 7.898 < 0.1 < 2.5 84.7

10 35.5 8.81 100.6 7.912 0.137 3.5 84.7

11 35 8.29 94.7 7.944 < 0.1 < 2.8 85.7

12 35 8.35 95.6 7.877 < 0.1 < 2.4 85.7

Tex. Ref 8 24 8.24 95.9 7.647 0.204 2.9 92.3

REFSW 9 35.5 8.01 93.1 8.283 < 0.1 < 5.8 84.7

MFS 10 35 7.58 88.4 8.1 < 0.1 < 3.9 85.7

2

 Dissolved oxygen

3

 Percent saturation of dissolved oxygen

4

 Total ammonia as nitrogen

TABLE 3. Water quality parameters after salinity adjustment and original salinity of 

10 Millipore filtered seawater diluent

9 Reference seawater collected in Hanalei Bay, Kauai, Hawaii

7 Porewater sample designated as the reference site

6

 Percent of original sample after salinty adjustment

Station pH

1

 Salinty of sample prior to adjustment. Sample adjusted to 30 + 1‰.

8

 Reference pore water extracted from sediment collected in Aransas Bay, Texas

5 Un-ionized ammonia

7
% % of

WQAS 1 Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Reference

100 71 68 68 71 65 68.6 2.51 94.0

50 68 79 69 62 71 69.8 6.14 95.4

25 72 68 78 67 76 72.2 4.82 105.4

100 69 65 65 75 70 68.8 4.15 94.2

50 76 72 87 72 77 76.8 6.14 104.9

25 68 83 64 63 76 70.8 8.53 103.4

100 82 81 80 70 85 79.6 5.68 109.0

50 68 78 83 81 70 76.0 6.67 103.8

25 81 68 81 72 68 74.0 6.60 108.0

100 73 80 76 76 72 75.4 3.13 103.3

50 72 76 72 69 75 72.8 2.77 99.5

25 77 75 70 76 75 74.6 2.70 108.9

100 64 70 75 61 78 69.6 7.16 95.3

50 62 74 78 69 73 71.2 6.06 97.3

25 78 76 62 68 69 70.6 6.47 103.1

100 75 75 73 71 71 73.0 2.00 100.0

50 68 77 73 71 77 73.2 3.90 100.0

25 69 73 90 5 64 68 68.5 3.70 100.0

100 68 60 57 72 68 65.0 6.24 89.0

50 73 64 71 65 71 68.8 4.02 94.0

25 75 71 69 68 69 70.4 2.79 102.8

100 26 27 24 30 35 28.4 4.28 ** 38.9

50 77 81 73 75 65 74.2 5.93 101.4

25 82 79 78 73 70 76.4 4.83 111.5

100 80 78 80 79 80 79.4 0.89 108.8

50 81 83 74 68 72 75.6 6.27 103.3

25 76 68 73 65 76 71.6 4.93 104.5

100 39 27 34 43 38 36.2 6.06 ** 49.6

50 52 45 58 52 50 51.4 4.67 ** 70.2

25 68 61 66 61 58 62.8 4.09 91.7

100 69 80 80 72 83 76.8 5.97 105.2

50 66 75 68 69 72 70.0 3.54 95.6

25 82 73 70 65 76 73.2 6.38 106.9

TABLE 4. Sea urchin fertilization test raw data and mean values for sediment porewater samples

 from Hanalei Bay, Kauai, Hawaii. Asterisks denote statistically significant differences (DSC)

 (Dunnett's t -test) and detectable significance criteria between test and control 

 station 6 (*alpha < 0.05, **alpha < 0.01). 

Sig.2

3

4

5

6 3

Station Mean SD % Fertilized

1

2

7

8

11

9

10

8
% % of

WQAS1 Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Reference

100 42 45 47 44 44 44.4 1.82 ** 60.8

50 77 84 81 72 78 78.4 4.51 107.1

25 71 78 77 73 69 73.6 3.85 107.4

100 84 83 82 77 76 80.4 3.65 110.1

50 70 72 62 80 73 71.4 6.47 97.5

25 71 74 81 69 77 74.4 4.77 108.6

100 69 76 71 74 69 71.8 3.11 98.4

50 78 73 72 72 68 72.6 3.58 99.2

25 74 74 80 71 72 74.2 3.49 108.3

MFS 7 100 76 63 75 68 69 70.2 5.36 96.2

10 0 0 0 0 0 0.0 0.00 ** 0.0

5 41 53 39 58 48 47.8 7.98 ** 65.5

2.5 89 91 89 91 94 90.8 2.05 124.4

1.25 79 78 75 74 77 77.0 2.16 105.5

SD Sig. Mean 2

TABLE 4. Continued.

SDS 8

Station % Fertilized

Ref. SW 6

12

Tex. Ref. 4

1

 Percent of water quality adjusted pore water tested

2 Significant difference from reference denoted as asterisks or plus signs

3 Sample #6, reference porewater collected from Hanalei Bay, Kauai, Hawaii.

4

 Reference pore water extracted from sediment collected in Aransas Bay, Texas

5 Statistical outlier removed from analysis.

6 Reference seawater collected in Hanalei Bay, Kauai, Hawaii.

7 Millipore filtered seawater diluent

8

 Sodium Dodecyl Sulfate positive reference control

9
% % of

WQAS 1 Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Reference

100 99 89 93 96 91 93.6 3.97 102.6

50 90 94 90 100 96 94.0 4.24 101.5

25 93 96 92 89 95 93.0 2.74 101.1

100 98 94 92 95 93 94.4 2.30 103.5

50 97 93 91 93 95 93.8 2.28 101.3

25 97 93 91 93 95 93.8 2.28 102.0

100 96 91 95 100 94 95.2 3.27 104.4

50 92 94 96 98 96 95.2 2.28 102.8

25 94 92 99 89 90 92.8 3.96 100.9

100 98 95 91 94 95 94.6 2.51 103.7

50 95 89 93 90 89 91.2 2.68 98.5

25 92 89 92 92 94 91.8 1.79 99.8

100 98 95 94 94 95 95.2 1.64 104.4

50 87 96 92 95 93 92.6 3.51 100.0

25 95 99 93 93 82 92.4 6.31 100.4

100 93 90 89 94 90 91.2 2.17 100.0

50 93 90 94 96 90 92.6 2.61 100.0

25 95 91 90 89 93 92.0 2.58 100.0

100 94 97 98 94 98 96.2 2.05 105.5

50 96 95 98 96 97 96.4 1.14 104.1

25 94 93 95 93 91 93.2 1.48 101.3

100 88 93 89 85 90 89.0 2.92 97.6

50 84 88 94 82 93 88.2 5.31 95.2

25 96 88 91 85 91 90.2 4.09 98.0

100 94 87 98 92 84 91.0 5.57 99.8

50 91 87 88 96 91 90.6 3.51 97.8

25 85 92 96 90 93 91.2 4.09 99.1

100 86 94 94 97 95 93.2 4.21 102.2

50 91 94 91 91 94 92.2 1.64 99.6

25 91 94 94 95 95 93.8 1.64 102.0

100 86 91 93 93 93 91.2 3.03 100.0

50 91 91 90 90 92 90.8 0.84 98.1

25 91 91 91 94 94 92.2 1.64 100.2

Percent Fertilized Mean SD

1

2

Sig.2

3

4

5

6 3

Station

11

9

10

TABLE 5. Sea urchin embryological development test raw data and mean values for sediment 

 porewater samples from Hanalei Bay, Kauai, Hawaii. Asterisks denote statistical

 between test and control station 6 (*alpha < 0.05, **alpha < 0.01). 

 significant differences (Dunnett's t-test) and detectable significance criteria (DSC) 

7

8

10
% % of

WQAS 2 Rep 1 Rep 2 Rep 3 Rep 4 Rep 5 Reference

100 97 96 97 96 97 96.6 0.55 105.9

50 96 95 97 86 93 93.4 4.39 100.9

25 91 96 93 97 91 93.6 2.79 101.7

100 95 91 92 97 90 93.0 2.92 102.0

50 89 92 89 93 94 91.4 2.30 98.7

25 98 95 92 96 93 94.8 2.39 103.0

100 95 95 94 96 94 94.8 0.84 103.9

50 95 97 91 94 85 92.4 4.67 99.8

25 95 93 88 89 91 91.2 2.86 99.1

MFS 6 100 88 93 94 90 91 91.2 2.39 100.0

10 0 0 0 0 0 0.0 0.00 ** 0.0

5 16 11 21 11 6 13.0 5.70 ** 14.3

2.5 84 92 84 86 88 86.8 3.35 95.2

1.25 91 94 94 90 90 91.3 1.89 100.1

4

 Reference pore water extracted from sediment collected in Aransas Bay, Texas

5 Reference seawater collected in Hanalei Bay, Kauai, Hawaii.

6 Millipore filtered seawater diluent

Sig. Mean SD 2

Tex. Ref. 4

TABLE 5. Continued.

12

SDS 7

Station % Fertilized

Ref. SW 5

1

 Percent of water quality adjusted pore water tested

2 Significant difference from reference denoted as asterisks or plus signs

3 Sample #6, reference porewater collected from Hanalei Bay, Kauai, Hawaii.

7

 Sodium Dodecyl Sulfate positive reference control

11
12
Corpus Christi SOP: F10.24 Appendix 1 Page 1 of 7 pages 

Date Prepared: October 1, 2001 

Date Revised: October 11, 2001 

EXTRACTION AND STORAGE OF 

POREWATER SAMPLES USING IN SITU DEVICES 

1.0 OBJECTIVE

This protocol describes procedures for extracting pore water samples by vacuum from 

marine, estuarine, or freshwater sediments, and for processing and storing pore waters 

for use in toxicity testing. A suction device is used to extract the pore water from 

sediment samples. This procedure may be performed in situ, or in the laboratory on 

samples collected with sediment coring devices. 

2.0 PREPARATION 

2.1 Description of the Porewater Extraction System 

The extraction device consists of a filtration medium attached to a 60 mL disposable 

syringe (Winger and Lasier, 1991). A ground glass aquarium air stone is usually used 

as the filtration medium. Stainless steel cone-shaped filters with 250 μm mesh have 

also been used successfully as filtration media. In order to prevent breakage when 

inserted into the sediment, the stainless steel filters can be placed inside a 100 and 

1000 μl disposable pipette tips with small holes bored throughout their surface. 

Figure 1 shows extraction devices with both filtration media. All sampling material is 

pre-soaked in deionized water for a minimum of 24 hours prior to use in the field. 

Figure 1. Porewater extraction devices with two types of filtration media. 
Corpus Christi SOP: F10.24 Appendix 1 Page 2 of 7 pages 

2.2 Equipment List

 Supplies and equipment needed are listed in Attachment 1. 

3.0 PROCEDURE 

3.1 In Situ Procedures

In situ pore water and sediment extractions are performed by divers. 

3.1.1 In situ porewater extraction

 This procedure was developed for use at sites with coarse sands, typically in 

the vicinity of coral reefs. Eight to ten 60 mL syringes are usually sufficient to 

obtain the necessary amount of pore water for toxicity testing and water 

quality measurements. The syringes with attached connectors and air stones 

attached to a short length (~ 0.75 cm) of aquarium air tubing are placed in a 

mesh bag or other suitable container for transport to the sampling location by 

divers. Once at the sampling location, one air stone (or other selected 

filtration medium) is attached to each syringe. The device is gently inserted 

into the sediment until at least the whole filtration medium and connecting 

parts are buried in the sediment, ideally until the bottom 10 or 20 mL of the 

syringe are also buried. The total buried depth should approximate 10 cm. 

Once this is achieved, vacuum is applied through the syringe, promoting the 

suction of pore water into the syringe. When the syringe is full, the filtration 

medium should be carefully removed from the sediment, the whole device 

placed in the bag, and taken to the surface. Should the air stone be broken or 

lost in the sediment, the extracted pore water will still remain in the syringe 

and can be taken to the surface and transferred to appropriate jars as 

described below. If necessary, the same air stone can be removed from a full 

syringe and attached to an empty one for further porewater extraction. 

 Once on board of the research vessel, the filtration medium is removed from 

the tip of the syringe and the pore water is gently transferred into clean, prelabeled polycarbonate centrifuge bottles. This procedure should be done 

very slowly to avoid excessive aeration of the sample, which may promote 

alterations of the equilibrium between water, particles, and contaminants. 

Samples must then be stored on ice until arrival at the laboratory. Pore water 

should be centrifuged as soon as possible after extraction (section 3.2). 

 

3.1.2 Sediment coring 

 This procedure should be used when fine grain sediments are encountered 

and in situ porewater extraction is not practicable. The upper 10 cm of 
Corpus Christi SOP: F10.24 Appendix 1 Page 3 of 7 pages 

sediment are collected by multiple cores at each sampling station. Prior to 

leaving the research vessel, the coring tubes may be placed inside a bucket

with screw cap and holes in the lid to allow water intake and easy 

submersion. Once the divers reach the sampling location, the lexaneTM

coring tubes are inserted in the sediment to a depth of approximately 10 cm. 

Once the insertion is complete, the stoppers are placed into the upper part of 

the tube and the core is extracted from the surrounding sediment. The cap is 

placed in the lower part of the tube as soon as it emerges from the sediment. 

The filled cores are placed in the bucket and taken back to the research 

vessel. 

 One liter of sediment will typically provide 100-200 mL pore water. However, a 

larger volume of coarse sediments may be required since they contain less 

water. Nine cores will provide approximately four liters of sediment, which 

should be sufficient to obtain the amount of pore water necessary for toxicity 

testing and water quality measurements. Once on board of the research 

vessel, the overlying water is drained from the cores and the sediment from 

each core is placed in a suitable pre-labeled container (e.g., clean high 

density polyethylene containers or Zip-Lock®

 bags), thoroughly shaken for 

sediment homogenization and stored on ice. 

 Pore water should be extracted from the samples as soon as possible, 

because the toxicity of sediments in storage may change over time. 

Porewater extraction is performed using the same devices described for the 

in situ extraction (section 3.1.1). A plastic or wooden rod with the length of the 

syringe may be used to prop up the piston while vacuum is being applied. 

Once the syringes are filled with pore water, they may be removed from the 

sediment and the pore water gently transferred to polycarbonate centrifuge 

bottles. Samples should be centrifuged as soon as possible after extraction 

(section 3.2). 

 A sample tracking system should be maintained for each sediment sample 

collected and porewater sample extracted. All manipulations made on 

samples should be recorded on the Sample History Data Form (Attachment 2). 

3.2 Centrifugation of Porewater Samples

 Porewater samples used for toxicity testing at the Marine Ecotoxicology Research 

Station are usually stored frozen until tested. The porewater samples should be 

centrifuged shortly after they are collected and before they are frozen. 
Corpus Christi SOP: F10.24 Appendix 1 Page 4 of 7 pages 

 1. After collection, keep the porewater samples refrigerated or chilled on ice until 

they are centrifuged. 

 2. Place the polycarbonate bottle in the centrifuge and spin at •1200 g for 20 minutes. 

Decant the supernatant from the centrifuged sample to a pre-cleaned and labeled 

jar, taking care not to disturb any material that may have settled on the 

bottom/sides of the centrifuge bottle. 

 3. If multiple jars of pore water were collected from a single sediment sample, they 

should be composited after centrifugation and redistributed to replicate sample 

jars before testing or storage. 

3.3 Storage of Porewater Samples

If the porewater samples are not to be used on the day of collection, they should be 

frozen for storage. Sufficient room for freeze expansion should be left in the jars (for 

example, 200 mL maximum sample in a 250 mL jar). If the volume needed for testing is 

known in advance, it is prudent to allocate only that specific volume plus a little 

excess (~10 mL) to each jar in order to conserve pore water (once thawed, the pore 

water cannot be refrozen and reused), and to simplify the volume measurements 

required for Water Quality Adjustment of Samples (SOP F10.12) performed the day 

prior to testing. Frozen porewater samples may be shipped with dry ice. 

4.0 QUALITY CONTROL

A sample tracking system is maintained for each sediment sample collected and 

porewater sample extracted. All actions taken with that respective sample are recorded 

on the Sample History Data Form (Attachment 2). This information includes, at a minimum: 

a) the date of collection or receipt, b) the date of porewater extraction, c) the date of 

porewater centrifugation and relevant information about this procedure, d) the volume or 

number of jars of pore water collected, e) date frozen and location (freezer number or 

other relevant information), and f) date and jar number thawed and used in which test. 

The Sample History Forms are kept in a three-ring binder at the same location where the 

samples are stored. 

5.0 TRAINING

Persons who will perform this procedure should read this SOP and operate under the 

supervision of an experienced individual for at least one series of extractions. 
Corpus Christi SOP: F10.24 Appendix 1 Page 5 of 7 pages 

6.0 SAFETY

The sediment and porewater samples handled may contain contaminants. Care should be 

taken to avoid contact with the samples. Protective gloves, glasses and clothing may be 

worn. Waste sediment should be properly disposed.

7.0 ATTACHMENTS 

Attachment 1. Required Equipment and Materials 

Attachment 2. Sample History Form 

8.0 REFERENCES

Carr, R.S., M. Nipper, and G.S. Plumlee. 2001. A Preliminary Survey of Marine 

Contamination from Mining-related Activities on Marinduque Island, Philippines: 

Porewater Toxicity and Chemistry: Results from a Field Trip - October 14-19, 2000. 19 

pp. + 3 attachments. 

Nipper, M., and R.S. Carr. 2000. Toxicity testing of sediment pore water from the Flower 

Garden Banks, Gulf of Mexico. Prepared for the Center for Coastal Studies, Texas A & 

M University - Corpus Christi, 5 pp. + 3 tables, 3 figures, and 3 attachments. 

 

Winger PV, Lasier PJ. 1991. A vacuum-operated pore-water extractor for estuarine and 

freshwater sediments. Arch. Environ. Contam. Toxicol. 21:321-324. 

Prepared by: 

 Marion Nipper 

 Senior Research Scientist 

Approved by: ________________________ 

 R. Scott Carr 

 Field Station Leader 

 

 ________________________ 

 Laverne Cleveland 

 Chief, Field Research Division 
Corpus Christi SOP: F10.24 Appendix 1 Page 6 of 7 pages 

Attachment 1

REQUIRED EQUIPMENT AND MATERIALS

For porewater extraction: 

60 mL disposable syringes 

Connectors 

Standard aquarium air tubing 

Ground glass aquarium air stone or stainless steel conic filters with 250 μm mesh 

100 and 1000 μL disposable pipette tips 

 

For sediment coring: 

Lexane® cores with 5 cm inner diameter 

Plastic caps for lower side of corer 

Rubber stoppers for upper side of corer 

Other required supplies/equipment: 

Sediment sample containers or bags 

Centrifuge bottles 

Coolers with ice 

Pore water sample jars 

Sample labels or labeling tape 

Beakers 

Deionized water (DI) 

Wash bottles, 500 mL 

Protective gloves, glasses, clothing 

Pens, pencils, markers 

Centrifuge and centrifugation materials 

Refrigerator 

Freezer 
Corpus Christi SOP: F10.24 Appendix 1 Page 7 of 7 pages 

Attachment 2

SAMPLE HISTORY DATA FORM

Sample Designation: Study Protocol: Initials: 

Date of acquisition: Sample type: 

How acquired (refer to sample site data sheet number, if appropriate): 

 

 Initials Date Action Taken 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 
Corpus Christi SOP: F10.12 Appendix 2 Page 1 of 6 pages 

Date Prepared: March 14, 1991 

Date Revised: May 17, 1994 

WATER QUALITY ADJUSTMENT OF SAMPLES

1.0 OBJECTIVE

In order to perform toxicity tests with saline samples, all test and reference samples should 

be similar in salinity so that salinity is not a factor in survival of test organisms. Additionally, 

dissolved oxygen (DO) concentrations should be sufficiently high to ensure that low DO is 

not a source of stress to the test organisms. At the Corpus Christi field station, toxicity tests 

are performed using a variety of marine and estuarine organisms, including the sea urchin 

Arbacia punctulata, the polychaete Dinophilus gyrociliatus, the harpacticoid copepod 

Longipedia sp., and the red drum Sciaenops ocellatus. The aqueous samples tested may 

be pore water, different kinds of discharges and effluents, surface microlayer, or 

subsurface water samples that may range in salinity from 0-36o

/oo. Although from test to 

test salinities used in the different toxicity tests may vary, the individual toxicity tests 

performed on a particular day are run at a single target salinity. Since initial salinities of 

the porewater or water samples to be tested commonly vary, they will require salinity 

adjustment to within 1o/oo of the target salinity. Additionally, DO should normally be $80% 

saturation in all samples tested. 

2.0 PREPARATION

2.1 Equipment and Labware

The supplies and equipment needed are listed in Attachment 1. 

2.2 Source of Dilution Water

For samples lower in salinity than target salinity, concentrated brine (-100o

/oo) is added 

to increase salinity. Concentrated brine is prepared by heating (to 35-40EC) and gently 

aerating filtered natural seawater (1 μm) to concentrate the salts by evaporation. For 

samples higher in salinity than target salinity, HPLC ultrapure sterile water (J.T. 

Baker7 Cat. #JT4218-2) is added to decrease salinity. 

3.0 PROCEDURES

 The following describes the procedures required for the adjustment and determination of 

 specific water quality parameters of a sample. 
Corpus Christi SOP: F10.12 Appendix 2 Page 2 of 6 pages 

3.1 Preparation for Salinity Adjustment

1. Although fresh samples are routinely tested at the Corpus Christi field station, most 

of the samples tested are stored frozen in amber I-Chem7 jars. If frozen, remove 

samples from freezer and allow them to thaw at room temperature or immerse 

them in a tepid water bath to thaw, ensuring that sample temperature does not 

exceed 25EC. The samples may be thawed the day of water quality adjustment 

(WQA) or may be transferred from the freezer to a refrigerator (4EC) the day before 

WQA and then completely thawed the following day. After thawing, allow the 

samples to come to room temperature. Generally, the samples should be 

maintained at the same temperature required for the toxicity test that will be 

conducted. The temperature requirement for most toxicity tests performed at this 

field station is 20"1EC, and room temperature should be maintained accordingly. 

2. Turn bottled sample end over end a few times to mix thoroughly before measuring 

salinity. Using a salinity refractometer, measure salinity and record on Water 

Quality Adjustment Data Form (Attachment 2). 

3. In order to make calculations for the salinity adjustment, the volume of the sample 

must be known. When porewater or other water samples are collected and 

transferred to amber jars for storage, they are commonly measured to an 

approximate volume (-110 mL, for example) prior to freezing. On the day of WQA, 

this volume should be recorded on the WQA data form for the respective samples. 

If the volume is unknown at this point, it should be measured using a graduated 

cylinder of appropriate size, and recorded on the data sheet. 

3.2 Salinity Adjustment

3.21 Reducing the salinity of aqueous samples

Refer to the formulas below to calculate the volume of HPLC water needed to 

reduce the initial sample salinity to the target salinity. Add the volume 

calculated, mix the bottle thoroughly, check the salinity with a refractometer, 

and record the volume of HPLC water added as well as the final salinity. 

 (i) (target o

/oo ) sample o/oo) H sample vol. in mL = A 

 (ii) sample vol. ! A = B 

 (iii) sample vol. ) A = C 

 (iv) B H C = volume of HPLC water to add 
Corpus Christi SOP: F10.12 Appendix 2 Page 3 of 6 pages 

3.22 Increasing the salinity of aqueous samples

Refer to the formula below to calculate the volume of concentrated brine 

needed to increase the initial sample salinity to the target salinity. Add the 

volume calculated, mix the bottle thoroughly, check the salinity with a 

refractometer, and record the volume of brine added as well as the final salinity. 

(i) ((target o/oo ! sample o/oo) H sample vol. in mL) ) (brine o/oo ! target o/oo) = vol. of 

brine to add 

3.3 Dissolved Oxygen Adjustment

Measure and record DO and percent DO saturation of sample (SOP F10.13). 

Occasionally, a sample will have DO of less than 80% saturation. Any such samples 

should be gently stirred on a magnetic stirrer to increase the DO level above 80%. 

Record initial DO, the elapsed mixing time, and final DO in the comments section of 

the Water Quality Adjustment Data Form. (On the following day, DO should be 

rechecked and brought to >80% by stirring again if necessary before the toxicity test 

is performed.) 

3.4 Other Water Quality Determinations

1. Measure pH (SOP F10.21) and record on the Water Quality Adjustment Data Form. 

2. Measure and record ammonia concentration (SOP F10.4). 

3. Measure and record sulfide concentration if required. 

4.0 DATA COLLECTION

All raw data are entered on one standardized form, the Water Quality Adjustment Data 

Form (see Attachment 2) at the time the determinations or adjustments are made. 

5.0 QUALITY CONTROL

A data form (Attachment 2) will be used to document all sample handling procedures for 

each sample. The person(s) recording data on the sheet will initial each sheet. Original 

data forms after completion will be stored in a three-ring file in the possession of the field 

station leader. Copies will be kept in the lab. 
Corpus Christi SOP: F10.12 Appendix 2 Page 4 of 6 pages 

6.0 TRAINING

Personnel who will perform this task should first read this protocol and then operate under 

supervision during the preparation of at least two samples. 

 

7.0 SAFETY

The NaOH solution used in the ammonia determination procedure is a highly caustic liquid. 

Care should be taken to avoid its contact with skin or clothing. Should such contact occur, 

quickly flush affected with water. A sink is present along the west wall of the dry lab, 

another is present along the east wall of the wet lab, and an eye flushing station is present 

in the northwest corner of the wet lab near the entrance door. The samples handled may 

be pore water, effluent, discharges, or other water samples that may contain contaminants. 

Care should be taken to avoid contact with the samples. 

8.0 ATTACHMENTS

Attachment 1. Equipment List for Water Quality Adjustment 

Attachment 2. Water Quality Adjustment Data Form 

Prepared by: 

 Duane C. Chapman 

 Fishery Biologist 

 

Approved by: _____________________ 

 R. Scott Carr 

 Field Station Leader 

 ______________________ 

 Anne E. Kinsinger 

 Chief, Field Research Division 

 _______________________ 

 Joseph B. Hunn 

 Quality Assurance Officer 
Corpus Christi SOP: F10.12 Appendix 2 Page 5 of 6 pages 

ATTACHMENT 1

EQUIPMENT LIST FOR WATER QUALITY ADJUSTMENT

Graduated cylinders 

Pipetters 

Latex gloves 

Magnetic stirrer and stir bars 

10 M NaOH 

Concentrated brine (See section 2.2 for preparation) 

HPLC ultrapure sterile water (J.T. Baker7 #JT4218-2) 

Salinity refractometer 

Dissolved oxygen meter 

pH electrode, buffer solutions, and meter 

Ammonia electrode, standard solutions, and meter 

Sulfide electrode, standard solutions, and meter 

Data sheets 

Hand calculator 
Corpus Christi SOP: F10.12 Appendix 2 Page 6 of 6 pages 

 Attachment 2 

 WATER QUALITY ADJUSTMENT DATA FORM 

 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

 

A. Salinity Adjustment: 

Initial volume (mL) _______________________ 

Initial salinity (o/oo) _______________________ 

Vol. Milli-Q water added (mL) _______________________ 

Vol. o/oo brine added (mL) _______________________ 

% of original sample 

(initial vol./final vol. x 100) ________________________ 

B. Character of Sample (after salinity adjustment): 

Volume (mL) ________________________ 

Salinity (o/oo) ________________________ 

pH ________________________ 

Dissolved oxygen (mg/L) _________________________ 

DO saturation (%) _________________________ 

Total ammonia (mg/L) _________________________ 

Sulfide (mg/L) _________________________ 

COMMENTS ___________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

___ __ 
Corpus Christi SOP: F10.6 Appendix 3 Page 1 of 17 pages

Date Prepared : April 10, 1990 

Date Revised: March 10, 1995 

SEA URCHIN FERTILIZATION TOXICITY TEST

1.0 OBJECTIVE

The purpose of the fertilization toxicity test with the sea urchin, Arbacia punctulata, is to 

determine if a sea water, pore water, sea surface microlayer, or other sample reduces 

fertilization of exposed gametes relative to that of gametes exposed to a reference sample. 

The test may also be used to determine the concentration of a test substance which reduces 

fertilization. Test results are reported as treatment (or concentration) which produces 

statistically significant reduced fertilization or as concentration of test substance which 

reduces fertilization by 50 percent (EC50). This test can be performed concurrently with Sea 

Urchin Embryological Development Toxicity Test (SOP 10.7) and/or Sea Urchin 

Genotoxicity/Teratogenicity Test (SOP 10.8), using the same pretest and sperm and egg 

collection. 

2.0 TEST PREPARATION

2.1 Test Animals

Gametes from the sea urchin, Arbacia punctulata are used in the sea urchin fertilization 

toxicity test. Animals can be collected in the field or obtained from a commercial supplier. 

A. punctulata can be differentiated from other species of urchins which are found in 

Texas by the five plates surrounding the anal opening, and by round sharp spines on the 

dorsal surface of the test and flattened spines surrounding the Aristotle's lantern. 

Urchins can be maintained easily in aquaria or other tanks with running seawater or an 

aquarium filter. Urchins will eat a wide variety of marine vegetation. A good diet may be 

provided by placing rocks from jetties (which have been colonized by diatoms and 

macroalgae) into the tank with the urchins or romaine lettuce may be provided as a 

substitute. Temperature manipulations of the cultures will prolong the useful life of the 

urchins. Cultures are maintained at 16 " 1EC when gametes are not required. 

Temperature is gradually increased to 19 " 1EC at least one week prior to gamete 

collection and subsequently decreased if no further tests are planned. Photoperiod is 

maintained at 16 hours of light per day. Water quality parameters should be monitored 
Corpus Christi SOP: F10.6 Appendix 3 Page 2 of 17 pages

weekly and salinity maintained at 30 " 3 o

/oo. Males and females should be kept in 

separate tanks. 

2.2 Dilution Water

HPLC reagent grade purified water or concentrated seawater brine is used to adjust samples 

to 30 o

/oo as described in Water Quality Adjustment of Samples (SOP 10.12). Concentrated 

seawater brine (90-110 o

/oo) is made in large batches by heating seawater to 40°C or less in 

large tanks with aeration for 3-4 weeks. Brine quality will remain constant over long periods 

with no refrigeration. At the time of salinity adjustment, pH, ammonia, and dissolved oxygen 

are also measured. Salinity adjustment and water quality data are recorded on prepared 

data forms. 

Filtered (0.45 μm) seawater adjusted to 30 o/oo is used to wash eggs and is also used for sperm 

and egg dilutions. The acronym MFS (for Millipore7 filtered seawater) is used for this filtered 

and salinity adjusted seawater. 

2.3 Test System: Equipment 

When testing samples for potential toxicity, five replicates per treatment are 

recommended. One replicate is a 5 mL volume of sample in a disposable glass scintillation 

vial. When conducting a dilution series test, fifty percent serial dilutions may be made in 

the test vials, using MFS as the diluent. 

2.3.1 Equipment 

A list of equipment necessary for conducting this test is given in Attachment 1 

(Equipment List for Fertilization Toxicity Test). 

2.3.2 Solutions

10% Buffered Formalin: 

1,620 mL sea water 

620 mL formaldehyde 

6.48 g NaH2

P04 or KH2PO4 (mono) 

10.5 g Na2HPO4 or K2HPO4 (dibasic) 

0.75 mL needed for each replicate. Fill the dispenser. 
Corpus Christi SOP: F10.6 Appendix 3 Page 3 of 17 pages

2.4 Collection and Preparation of Gametes

 Quality gametes must first be collected, and then diluted to the appropriate concentration 

 for addition to the test vials. 

2.4.1 Selection of Urchins to be Used in Toxicity Test.

 1. Take two or three females and place in shallow bowl, barely covering tests with 

seawater. 

 2. Stimulate release of eggs from gonopores of a female by touching test with 

electrodes from a 12V transformer. 

 3. Collect a few eggs from between spines using a 10 mL disposable syringe with a 

large gauge blunt-tipped needle attached. Discard the first small quantity of eggs 

expelled from each gonopore and continue collecting. Place a 2 to 5 drops of 

eggs onto a scintillation vial containing 10ml of filtered seawater. Rinse syringe 

and repeat for each female. 

 4. Select females which have round, well developed eggs, and which do not release 

 clumps of eggs or undeveloped ovarian tissue. 

 5. Place 2-4 males in shallow bowl(s) with a small amount of seawater, leaving the 

upper 1

/2 to 1/3 of the animals uncovered. 

 6. Stimulate release of sperm from gonopores by touching test with electrodes from 

12V transformer (about 30 seconds each time). If sperm is watery, reject the 

animal and choose another. Sperm should be the consistency of condensed milk. 

Collect sperm using a pastuere pipette with a rubber bulb attached. 

 

Generally, a gamete check is performed in order to ensure that both the male and 

the female urchins used in the test have gametes with a high degree of viability. If 

the gamete check is performed, two to five females (depending on confidence in the 

proportion of urchins in the holding facility in good reproductive status) and at least 

two males should be selected using the above procedures. The check is performed 

by adding 5 to 7 drops of a concentrated dilution of sperm to the eggs in the 

scintillation vials ( collected as described above) and observing the eggs under the 

microscope after 10 minutes. The concentrated dilution of sperm is usually made by 

diluting 20-50μl of sperm in 10 ml of filtered seawater. If the proportion of eggs 

fertilized is high (95-100%), that female and male may be used in the pretest and 

test. Sperm from a number of males or females may be combined in the beginning if 
Corpus Christi SOP: F10.6 Appendix 3 Page 4 of 17 pages

the gamete check reveals a number of high quality animals or the confidence is high 

in the quality of the gametes Once a good male and female are selected a pretest 

can be conducted to determine the correct dilution of sperm to use in the test 

(Attachment 2). 

2.4.2 Obtain Eggs 

 1. Place selected female in large Carolina dish and add enough water to cover the 

urchin's test with approximately 1 cm of seawater. Stimulate release of eggs 

from female with 12V transformer. 

 2. Collect eggs as above using the 10 mL syringe. Remove needle before dispensing 

eggs into a disposable shell vial or other clean container capable of holding 25-50 

mL. Collect enough eggs for pretest and test. If female stops giving eggs readily 

or starts giving chunky material, cease stimulation and collection of eggs from 

that female. 

 3. Add MFS to fill shell vials, gently mixing eggs. Allow eggs to settle to bottom of 

vial. Remove water with a pipette. Replace water, again gently mixing the eggs. 

 4. Repeat washing procedure. 

2.4.3 Prepare Appropriate Egg Concentration 

 

 1. Put approximately 100 mL of 30 o

/oo MFS in a 250 mL beaker, and add enough washed 

eggs to bring the egg density to approximately 10,000 per mL . If more than 400 total 

replicates (27 treatments) are to be tested, a larger amount of water and a 

correspondingly larger amount of eggs should be used. Two hundred μL of this egg 

solution will be used per replicate, and it is easier to maintain proper mixing and 

uniform egg density if there is an excess of at least 50%. 

 2. Check egg density and adjust to within approximately 9000 to 11,000 eggs per mL, as 

follows. Gently swirl egg solution until evenly mixed. Using a pipette, add 1 mL of 

the solution to a vial containing nine mL seawater. Mix and transfer 1 mL of this 

diluted solution to a second vial containing 4 mL of seawater. Again, mix and 

transfer 1 mL of this diluted solution to a counting slide such as a Sedgewick-Rafter 

slide. 

 3. Using a microscope (either a compound microscope with a 10x objective or a 

dissecting scope may be used here), count the number of eggs on the slide. If the 
Corpus Christi SOP: F10.6 Appendix 3 Page 5 of 17 pages

number is not between 180 and 220, then adjust by adding eggs or water. If egg 

count is > 220 use the following formula to calculate the amount of water to add: 

 ("egg count" - 200/200) x Current Volume of Eggs = Volume seawater to add 

 to stock (mLs) 

 

 If egg count < 200 add a small amount of eggs. Since it is less arbitrary and more 

 likely to arrive at an acceptable count when using the water addition formula, it is 

 better to originally overestimate the amount of eggs to add to the 100 mL of water. 

 4. Repeat steps 2 and 3 until an acceptable egg count (between 180 and 220) is 

obtained. 

 2.4.4 Obtain Sperm 

Place selected male urchin in a large Carolina dish containing 1-2 cm of water. About 

half of test should be above water level. Stimulate male with 12V transformer, and 

collect about 0.5 mL of unwetted sperm from between spines using a pasteur pipette. 

Place sperm into a plastic microcentrifuge tube. Keep on ice until used. Be careful not 

to add any water or sperm which has contacted water to the vials. High quality sperm 

collected dry and kept on ice will last at least eight hours without measurable decline 

in viability. 

2.4.5 Prepare Appropriate Sperm Dilution 

It is desirable for control fertilization to be within 60-90%. Although controls outside 

these bounds do not automatically disqualify a test, particularly if a valuable dose 

response is generated, the sensitivity of the test is reduced by fertilization rates greater 

than 90% and good dose responses may be difficult to obtain with less than 60% 

fertilization in controls. Density of sperm in the sperm solution should be determined 

with this goal in mind. Condition of the animals and length of acclimation to the 

aquarium may effect the chosen sperm density. The pretest (Attachment 2) may be 

used to calculate an appropriate sperm dilution. Generally, a dilution of between 

1:10,000 and 1:2500 will result in desirable fertilization rates, if the animals are in good 

condition. 

For example, if a sperm dilution of 1:5000 is required (as determined from the pretest), 

add 20 μL sperm to 10 mL MFS. Mix thoroughly, then add 1 mL of this solution to 9 mL 

MFS. Sperm should not be wetted until just before starting the test. Sperm wetted 

more than 30 minutes before the test has begun, including sperm dilutions used in any 

pretest, should be discarded and a new dilution made from sperm kept on ice. 
Corpus Christi SOP: F10.6 Appendix 3 Page 6 of 17 pages

 

3.0 TEST PROCEDURES

1. Add 50 μL appropriately diluted sperm to each vial. Record time of sperm addition. Sperm 

should be used within 30 minutes of wetting. 

2. Incubate all test vials at 20 " 2°C for 30 minutes. At this point it is useful to set a timer for 

five to ten minutes prior to the end of the incubation period. This will notify the worker 

early enough to be ready to start the next step exactly on time. 

3. While gently swirling the egg solution to maintain even mixing of eggs, use a 200 μL 

pipetter to add 200 μL diluted egg suspension to each vial. Pipette tips are cut back using 

a clean razor blade to prevent crushing the eggs during pipetting. Record time of egg 

addition. 

4. Incubate for 30 minutes at 20 " 2°C. The timer may be used again at this point. 

5. Using the dispenser, add 1 mL of 10% buffered formalin to each sample. 

6. Vials may now be capped and stored overnight or for several days until evaluated. 

Fertilization membranes are easiest to see while eggs are fairly fresh, so evaluation within 

two to three days may decrease the time required for evaluation. 

7. If it is not possible to make the evaluations within several days or the membranes are 

difficult to discern, an optional technique may be employed. Make up a 200 o

/oo NaCl 

solution (pickling salt) and add 2 to 4 drops of the solution to a 1 mL egg sample on a 

microscope slide. This solution causes the egg, but not the membrane, to shrink briefly 

thereby making the membrane easier to see. The effect only lasts for a short time (~5 

min.) so the observations must be made immediately after the NaCl solution is added. If 

this optional technique is employed, it must be used on all samples in that test series. 

4.0 DATA COLLECTION AND TABULATION

1. Transfer approximately 1 mL eggs and water from bottom of test vials to counting slide. 

Observe eggs using compound microscope under 100X magnification. Dark field viewing 

is useful here in identifying fertilization membranes. 
Corpus Christi SOP: F10.6 Appendix 3 Page 7 of 17 pages

2. Count 100 eggs/sample using hand counter with multiple keys (such as a blood cell 

counter), using one key to indicate fertilized eggs and another to indicate unfertilized 

eggs. Fertilization is defined by the presence of fertilization membrane surrounding egg. 

3. Calculate fertilization percentage for each replicate test: 

 Total No. Eggs - No. Eggs Unfertilized x 100 = Percent Eggs Fertilized 

5.0 DATA ANALYSIS

Data are recorded on standardized data sheets (See Attachments 3-7). Normally, percent 

fertilization in each treatment is compared to an appropriate reference treatment (seawater, 

pore water or sea surface microlayer from an uncontaminated environment). Statistical 

comparisons are made using analysis of variance (ANOVA) and Dunnett's t-test (Sokal and 

Rohlf 1981) on the arc sine square root transformed data. For multiple comparisons among 

treatments, Ryan's Q test (Day and Quinn 1989) with the arc sine square root transformed 

data is recommended. The trimmed Spearman-Karber method with Abbott's correction is 

recommended to calculate EC50 values for dilution series tests (Hamilton et al. 1977) 

6.0 QUALITY CONTROL 

Quality control tests may be run using both positive and negative controls with multiple 

replicates (as many as desired). Typically, a reference toxicant dilution series (sodium 

dodecyl sulfate) is tested with each test to evaluate the effectiveness of the sperm dilution 

chosen. Negative controls may include a reference porewater, filtered seawater, and/or a 

reconstituted brine. 

7.0 TRAINING

A trainee will conduct the test with supervision initially. Determining egg concentrations and 

fertilization counts are test specific activities. These functions can be performed 

independently after a trainee has demonstrated he or she can accurately reproduce the test. 

8.0 SAFETY

The sea urchin fertilization toxicity test poses little risk to those performing it. Care should be 

taken when making and dispensing the 10% buffered formalin solution; use a hood if 
Corpus Christi SOP: F10.6 Appendix 3 Page 8 of 17 pages

available, but make sure the test area is well ventilated. Protective gloves can be worn when 

pipetting or dispensing formalin or potentially toxic samples. 

Care should be taken when collecting or otherwise handling sea urchins. Urchin spines are 

sharp and fragile and may puncture the skin and break off if handled roughly. First aid similar 

to treatment of wood splinters is effective in this case (removal of spine and treatment with 

antiseptic). Collection of sea urchins by snorkeling should not be done alone. 

9.0 ATTACHMENTS

 Attachment l. Equipment List for Fertilization Toxicity Test 

 Attachment 2. Pretest to Insure Selection of Quality Gametes 

 Attachment 3. Water Quality Adjustment Data Form 

 Attachment 4. Sea Urchin Pretest Data Sheet 

 Attachment 5. Sea Urchin Pretest Continuation Data Sheet 

 Attachment 6. Sea Urchin Fertilization/Embryological Development Toxicity Test Gamete 

 Data Sheet 

 Attachment 7. Sea Urchin Fertilization Toxicity Test Fertilization Data Sheet 

10.0 REFERENCES

 Day, R.W. and G.P. Quinn. 1989. Comparisons of treatments after an analysis of variance 

 in ecology. Ecol. Monogr. 59:433-463. 

 Hamilton, M.A., R.C. Russo, and R.V. Thurston. 1977. Trimmed Spearman-Karber method 

 for estimating median lethal concentrations in toxicity bioassays. Environ. Sci. 

 Technol. 11(7):714-719; Correction 12(4):417 (1978) 

 Sokal, R.R., and F.J. Rohlf. 1981. Biometry. 2nd edition. W.H. Freeman and Company, 

 San Francisco, CA 859 pp. 
Corpus Christi SOP: F10.6 Appendix 3 Page 9 of 17 pages

 ______________________ 

Revised by: James M. Biedenbach 

 Biologist 

Prepared by: _________ 

Duane Chapman 

Fishery Biologist 

Approved by: ______________________ 

R. Scott Carr 

Field Station Leader 

 _______________________ 

Anne E. Kinsinger 

Chief, Field Research Division 

 ________________________ 

Joseph B. Hunn 

 Quality Assurance Officer 
Corpus Christi SOP: F10.6 Appendix 3 Page 10 of 17 pages

Attachment 1 

EQUIPMENT LIST FOR FERTILIZATION TOXICITY TEST 

Large Carolina dishes (at least 2) 

20 mL Wheaton scintillation vials (These should be type shipped with caps with polyseal cone 

liners. If other brand or type is used, the vials should be tested for toxicity prior to use.) 

400 mL beaker or wide-mouthed thermos for holding vials of sperm 

250 mL beakers (4) 

Pasteur pipettes and latex bulbs 

plastic microcentrifuge tubes 

25 mL shell vials or equivalent 

Test tube rack (to hold shell vials) 

12V transformer with pencil type electrodes 

Styrofoam (or something to hold electrode tips) 

10 cc syringe with large diameter blunt ended needle (make by grinding sharp point off the needle 

with a grinding stone) 

Marking pens 

Ice 

10-100 μL pipetter 

50-200 μL pipetter 

5 mL pipetters (2) 

Counting slide such as Sedgewick-Rafter chamber 

Compound microscope with 10x objective and dark field capability 

Hand tally counter 

Calculator 

Timer for exposure / incubation periods 

Buffered formalin and dispenser 

Filtered (0.45 μm) seawater, adjusted to 30 o/oo

Data sheets 

Baker reagent grade water 

Approximately 100 o

/oo concentrated brine 
Corpus Christi SOP: F10.6 Appendix 3 Page 11 of 17 pages

Attachment 2 

PRETEST TO INSURE SELECTION OF QUALITY GAMETES 

1. Using the procedure in section 2.4.1, select 2 to 5 females and at least 2 male urchins to be 

used in the pretest. 

2. Fill pretest vials with five mL of reference water. There should be at least two vials for 

each combination of male, female, and pretest sperm concentration (step 4 below). For 

example, in a pretest with two females, one male, and six pretest sperm concentrations, 

24 vials (2 X 2 X 6) would be needed. Arrange and mark vials accordingly in a rack. 

3. Perform steps 2.4.2 (egg collection) and 2.4.3 (egg dilution) for each female urchin. Make 

enough volume of the egg suspension to perform the pretest and the test. 

4. Perform step 2.4.4 (sperm collection) for each male urchin or male combination. Prepare a 

dilution series of sperm concentrations which will bracket the 60-90% fertilization rate in 

the test. Sperm dilution will depend on the health and reproductive status of the male 

urchin, but in most cases the following "standard dilution" should be used: 

 1: 250 (20 μL dry sperm added to 5 mL MFS. This concentration is used only as 

 stock solution to make up the rest of the dilution series and is not used full 

strength in the pretest.) 

 1: 1250 (1 mL of 1:250 and 4 mL MFS) 

 1: 2500 (1 mL of 1:250 and 9 mL MFS) 

 1: 5000 (2 mL of 1:2500 and 2 mL MFS) 

 1: 7500 (2 mL of 1:2500 and 4 mL MFS) 

 1:10000 (3 mL of 1:7500 and 1 mL MFS) 

 1:12500 (1 mL of 1:2500 and 4 mL MFS) 

Sperm must be used within 30 minutes of dilution. Leave undiluted sperm on ice and 

retain, because a new sperm dilution of the concentration determined in this pretest will 

be needed for the toxicity test. Sperm diluted for use in the pretest may not be used in the 

toxicity test, because the time elapsed since the addition of water is too great. 

5. As in section 3.0 add 50 μL of the diluted sperm to each pretest vial. Incubate for 30 

minutes at approximately 20°C, and add 200 μL of the egg suspension. Incubate for 

another 30 minutes, then fix with 1 mL of the buffered formalin solution. 

6. As in section 4.0, obtain a fertilization rate for the vials. There is no need to count all vials, 

enough vials should be counted to determine a good male/female combination, and an 

appropriate sperm dilution factor. If more than one male/female combination is 

acceptable, this is a good opportunity to choose a female which exhibits easily visible 

fertilization membranes or in cases where there are many samples, to combine eggs from 

different females . The appearance of the fertilization membranes may vary among female 

urchins, and presence of easily visible membranes facilitates counting. 
Corpus Christi SOP: F10.6 Appendix 3 Page 12 of 17 pages

 Attachment 3 

WATER QUALITY ADJUSTMENT DATA FORM 

 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

 

A. Salinity Adjustment: 

Initial volume (mL) _______________________ 

Initial salinity (o/oo) _______________________ 

Vol. Milli-Q water added (mL) _______________________ 

Vol. o/oo brine added (mL) _______________________ 

% of original sample 

(initial vol./final vol. x 100) ________________________ 

B. Character of Sample (after salinity adjustment): 

Volume (mL) ________________________ 

Salinity (o/oo) ________________________ 

pH ________________________ 

Dissolved oxygen (mg/L) _________________________ 

DO saturation (%) _________________________ 

Total ammonia (mg/L) _________________________ 

Sulfide (mg/L) _________________________ 

COMMENTS ___________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________ 
Corpus Christi SOP: F10.6 Appendix 3 Page 13 of 17 pages

Attachment 4 

SEAURCHIN PRETEST DATA SHEET 

 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

EGGS 

Female number: _ _ _ _____ 

Collection time: _ _ _ _____ 

Count: _____ _____ _____ _____ 

 

SPERM 

Male number: _____ 

Collection time: _____ 

Dilution start time: _____ 

TEST TIMES 

Sperm in: Eggs in: Formalin in: ________________ 

SPERM DILUTION: _____________________________________________________________

COMMENTS: _________________________________________________________________ 

______________________________________________________________________________

% FERTILIZATION Reference sample designation: ______________________ 

 Female # Male #___________ 

Sperm Dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

 % FERTILIZATION Reference sample designation: ________________________ 

 Female # Male #________________

 Sperm dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 
Corpus Christi SOP: F10.6 Appendix 3 Page 14 of 17 pages

 

Attachment 5 

SEA URCHIN PRETEST CONTINUATION DATA SHEET 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

% FERTILIZATION Reference sample designation: _______________________ 

 Female # Male #____________ 

 Sperm Dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

% FERTILIZATION Reference sample designation: ______________________ 

 Female # Male #____________ 

 Sperm dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

% FERTILIZATION Reference sample designation: ______________________ 

 Female # Male #____________ 

 Sperm Dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

 % FERTILIZATION Reference sample designation: _______________________ 

 Female # Male #_____________ 

 Sperm dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 
Corpus Christi SOP: F10.6 Appendix 3 Page 15 of 17 pages

Attachment 6 

SEA URCHIN FERTILIZATION/EMBRYOLOGICAL DEVELOPMENT 

TOXICITY TEST GAMETE DATA SHEET 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

EGGS 

Collection time: ______________________________________________________________

Initial count/volume: ____________________________________________________________ 

Final count: ____________________________________________________________________

SPERM 

Collection time: Dilution start time: _____________________ 

Sperm dilution: ________________________________________________________________

 ________________________________________________

Test start temperature: 

 TEST TIMES

Box # Sperm in: Eggs in: Formalin in:

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

COMMENTS 

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________ 
Corpus Christi SOP: F10.6 Appendix 3 Page 16 of 17 pages

 Attachment 7 

SEA URCHIN FERTILIZATION TOXICITY TEST 

FERTILIZATION DATA SHEET 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

PERCENT FERTILIZED 

 Replicate 

Treatment 1 2 3 4 5 Mean"SD Unfert. 

Comments:_____________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________ 
Corpus Christi SOP F10.7 Appendix 4 Page 1 of 18 pages

Date Prepared : April 10, 1990 

Date Revised: February 29, 2000 

SEA URCHIN EMBRYOLOGICAL DEVELOPMENT TOXICITY TEST

1.0 OBJECTIVE

The purpose of the embryological development toxicity test with the sea urchin, Arbacia 

punctulata, is to determine if a sea water, pore water, sea surface microlayer, or other 

sample affects development of exposed embryos (development arrested at an early stage or 

a developmental abnormality) relative to that of embryos exposed to a reference sample. The 

test may also be used to determine the concentration of a test substance which affects 

development. Test results are reported as treatment (or concentration) which produces 

statistically significant developmental effect. This test can be performed concurrently with 

Sea Urchin Fertilization Toxicity Test (SOP 10.6) and/or Sea Urchin 

Genotoxicity/Teratogenicity Test (SOP 10.8), using the same pretest and sperm and egg 

collection. 

2.0 TEST PREPARATION

2.1 Test Animals

Gametes from the sea urchin, Arbacia punctulata are used in the sea urchin 

embryological development toxicity test. Animals can be collected in the field or obtained 

from a commercial supplier. A. punctulata can be differentiated from other species of 

urchins which are found in Texas by the five plates surrounding the anal opening, and by 

round sharp spines on the dorsal surface of the test and flattened spines surrounding the 

Aristotle's lantern. Urchins can be maintained easily in aquaria or other tanks with 

running seawater or an aquarium filter. Urchins will eat a wide variety of marine 

vegetation. A good diet may be provided by placing rocks from jetties (which have been 

colonized by diatoms and macroalgae) into the tank with the urchins or romaine lettuce 

may be provided as a substitute. Temperature manipulations of the cultures will prolong 

the useful life of the urchins. Cultures are maintained at 16 " 1EC when gametes are not 

required. Temperature is gradually increased to 19 " 1EC at least one week prior to 

gamete collection and subsequently decreased if no further tests are planned. 

Photoperiod is maintained at 16 hours of light per day. Water quality parameters should be 

monitored weekly and salinity maintained at 30 " 3 o

/oo. Males and females should be kept 

in separate tanks. 
Corpus Christi SOP F10.7 Appendix 4 Page 2 of 18 pages

2.2 Dilution Water 

HPLC reagent grade purified water or concentrated seawater brine is used to adjust 

samples to 30 o

/oo as described in Water Quality Adjustment of Samples (SOP 10.12). 

Concentrated seawater brine (90-110 o

/oo) is made in large batches by heating seawater to 

40°C or less in large tanks with aeration for 3-4 weeks. Brine quality will remain constant 

over long periods with no refrigeration. At the time of salinity adjustment, pH, ammonia, 

and dissolved oxygen are also measured. Salinity adjustment and water quality data are 

recorded on prepared data forms. 

Filtered (0.45 μm) seawater adjusted to 30 o/oo is used to wash eggs and is also used for 

sperm and egg dilutions. The acronym MFS (for Millipore7 filtered seawater) is used for 

this filtered and salinity adjusted seawater. 

2.3 Test System: Equipment 

When testing samples for potential toxicity, five replicates per treatment are 

recommended. One replicate is a 5 mL volume of sample in a disposable glass 

scintillation vial. When conducting a dilution series test, fifty percent serial dilutions may 

be made in the test vials, using MFS as the diluent. 

2.3.1 Equipment 

A list of equipment necessary for conducting this test is given in Attachment 1 

(Equipment List for Embryological Development Toxicity Test). 

2.3.2 Solutions

10% Buffered Formalin: 

1,620 mL sea water 

620 mL formaldehyde 

6.48 g NaH2

P04 or KH2PO4 (mono) 

10.5 g Na2HPO4 or K2HPO4 (dibasic) 

 

0.75 mL needed for each replicate. Fill the dispenser. 
Corpus Christi SOP F10.7 Appendix 4 Page 3 of 18 pages

2.4 Collection and Preparation of Gametes

 Quality gametes must first be collected, and then diluted to the appropriate concentration 

 for addition to the test vials. 

2.4.1 Selection of Urchins to be Used in Toxicity Test.

 1. Take two or three females and place in shallow bowl, barely covering tests with 

seawater. 

 2. Stimulate release of eggs from gonopores of a female by touching test with 

electrodes from a 12V transformer. 

 3. Collect a few eggs from between spines using a 10 mL disposable syringe with a 

large gauge blunt-tipped needle attached. Discard the first small quantity of 

eggs expelled from each gonopore and continue collecting. Place a 2 to 5 drops 

of eggs onto a scintillation vial containing 10mL of filtered seawater. Rinse 

syringe and repeat for each female. 

 4. Select females which have round, well developed eggs, and which do not 

release clumps of eggs or undeveloped ovarian tissue. 

 5. Place 2-4 males in shallow bowl(s) with a small amount of seawater, leaving the 

upper 1/2 to 1/3 of the animals uncovered. 

 6. Stimulate release of sperm from gonopores by touching test with electrodes from 

12V transformer (about 30 seconds each time). If sperm is watery, reject the 

animal and choose another. Sperm should be the consistency of condensed 

milk. Collect sperm using a pastuere pipette with a rubber bulb attached. 

 

Generally, a gamete check is performed in order to ensure that both the male and 

the female urchins used in the test have gametes with a high degree of viability. If 

the gamete check is performed, two to five females and at least two males should 

be selected using the above procedures. The check is performed by adding 5 to 7 

drops of a concentrated dilution of sperm to the eggs in the scintillation vials ( 

collected as described above) and observing the eggs under the microscope after 

10 minutes. The concentrated dilution of sperm is usually made by diluting 20-50μL 

of sperm in 10 mL of filtered seawater. If the proportion of eggs fertilized is high 

(95-100%), that female and male may be used in the pretest and test. Sperm from a 
Corpus Christi SOP F10.7 Appendix 4 Page 4 of 18 pages

number of males or eggs of females may be combined if the gamete check reveals a 

number of high quality animals or the confidence is high in the quality of the 

gametes Once a good male and female are selected a pretest can be conducted to 

determine the correct dilution of sperm to use in the test (Attachment 2). 

2.4.2 Obtain Eggs 

 1. Place selected female in large Carolina dish and add enough water to cover the 

urchin's test with approximately 1 cm of seawater. Stimulate release of eggs 

from female with 12V transformer. 

 2. Collect eggs as above using the 10 mL syringe. Remove needle before dispensing 

eggs into a disposable shell vial or other clean container capable of holding 25-

50 mL. Collect enough eggs for pretest and test. If female stops giving eggs 

readily or starts giving chunky material, cease stimulation and collection of eggs 

from that female. 

 3. Add MFS to fill shell vials, gently mixing eggs. Allow eggs to settle to bottom of 

vial. Remove water with a pipette. Replace water, again gently mixing the eggs. 

 4. Repeat washing procedure. 

2.4.3 Prepare Appropriate Egg Concentration 

 1. Put approximately 100 mL of 30 o/oo MFS in a 250 mL beaker, and add enough washed 

eggs to bring the egg density to approximately 10,000 per mL . If more than 400 total 

replicates (27 treatments) are to be tested, a larger amount of water and a 

correspondingly larger amount of eggs should be used. Two hundred μL of this egg 

solution will be used per replicate, and it is easier to maintain proper mixing and 

uniform egg density if there is an excess of at least 50%. 

 2. Check egg density and adjust to within approximately 9000 to 11,000 eggs per mL, as 

follows. Gently swirl egg solution until evenly mixed. Using a pipette, add 1 mL of 

the solution to a vial containing nine mL seawater. Mix and transfer 1 mL of this 

diluted solution to a second vial containing 4 mL of seawater. Again, mix and 

transfer 1 mL of this diluted solution to a counting slide such as a Sedgewick-Rafter 

slide. 

 3. Using a microscope (either a compound microscope with a 10x objective or a 

dissecting scope may be used here), count the number of eggs on the slide. If the 

number is not between 180 and 220, then adjust by adding eggs or water. If egg 
Corpus Christi SOP F10.7 Appendix 4 Page 5 of 18 pages

 count is > 220 use the following formula to calculate the amount of water to add: 

("egg count" - 200/200) x Current Volume of Eggs = Volume seawater to add to 

stock. If egg count < 200 add a small amount of eggs. Since it is less arbitrary 

and more likely to arrive at an acceptable count when using the water addition 

formula, it is better to originally overestimate the amount of eggs to add to the 

100 mL of water. 

 4. Repeat steps 2 and 3 until an acceptable egg count (between 180 and 220) is 

obtained. 

 5. Just before the eggs are to be used, add 2 mL of a penicillin-G stock solution 

(5000 units/mL) per 100 mL of eggs in the egg suspension. The addition of 

penicillin to the embryological development test has been shown to be beneficial 

in evalution of the stages of development by inhibiting bacterial growth which 

can cause the embryos to disintegrate before the test is terminated. 

 The penicillin stock solution is prepare by diluting 296 mg of Penicillin-G sodium 

 salt (1690 units/mg) in 100 mL of MFS and mixing until dissolved. The addition of 

 2 mL/100 mL of eggs will result in a final concentration of 4 units/mL in each 

 replicate. The number of units of penicillin per mg of penicillin-G sodium salt is 

 variable with each lot. Thus, the quantity added to the stock will change in order 

 to keep the final concentration at 4 units/mL. 

2.4.4 Obtain Sperm 

Place selected male urchin in a large Carolina dish containing 1-2 cm of water. 

About half of test should be above water level. Stimulate male with 12V transformer, 

and collect about 0.5 mL of unwetted sperm from between spines using a pasteur 

pipette. Place sperm into a plastic microcentrifuge tube. Keep on ice until used. Be 

careful not to add any water or sperm which has contacted water to the vials. High 

quality sperm collected dry and kept on ice will last at least eight hours without 

measurable decline in viability. 

2.4.5 Prepare Appropriate Sperm Dilution 

As in the Sea Urchin Fertilization Test, it is desirable for control fertilization to be 

70-90%. Although controls outside these bounds do not automatically disqualify a 

test, particularly if a valuable dose response is generated, the chance of inducing 

polyspermy is increased with increased concentrations of sperm, and good dose 

responses may be difficult to obtain with less than 70% normal pluteus in controls. 
Corpus Christi SOP F10.7 Appendix 4 Page 6 of 18 pages

Density of sperm in the sperm solution should be determined with this goal in 

mind. Condition of the animals and length of acclimation to the aquarium may 

effect the chosen sperm density. The pretest (Attachment 2) may be used to 

calculate an appropriate sperm dilution. Generally, a dilution of between 1:1250 

and 1:7500 will result in desirable fertilization rates, if the animals are in good 

condition. 

For example, if a sperm dilution of 1:5000 is required (as determined from the 

pretest), add 20 μL sperm to 10 mL MFS. Mix thoroughly, then add 1 mL of this 

solution to 9 mL MFS. Sperm should not be wetted until just before starting the 

test. Sperm wetted more than 30 minutes before the test has begun, including 

sperm dilutions used in any pretest, should be discarded and a new dilution made 

from sperm kept on ice. The quantity of sperm to be added to the egg dilution is 

calculated by dividing the total volume of eggs by five and adding 50 μL of sperm 

dilution per that number. Sperm should be allowed to incubate with the eggs for 

10 minutes to allow fertilization to take place. After 10 minutes, eggs should be 

evaluated under 100 X magnification for fertilization membranes. If 70-90% of the 

eggs are fertilized, the embryos can be pipetted into the test vials. If the 

percentage is lower than 70%, additional sperm may be added and/or more time 

allowed for fertilization. If the fertilization does not increase above 70% after 30 

minutes, the embryos should be discarded and new gametes selected for use. 

Embryos should not be allowed to undergo division before pipetting them into the 

test vials. 

3.0 TEST PROCEDURES

1. While gently swirling the embryo solution to maintain even mixing, use a 200 μL pipetter to 

add 200 μL diluted embryo suspension to each vial. Record time of embryo addition. 

2. Incubate all test vials at 20 " 1°C for 48 hours. 

3. Using the dispenser, add 0.75 mL 10% buffered formalin to each vial. 

4. Vials may now be capped and stored overnight or for several days until evaluated. 

4.0 DATA COLLECTION AND TABULATION

1. Transfer approximately 1 mL embryos and water from bottom of test vials to counting 

slide. Observe embryos using a compound microscope under 100X magnification. 
Corpus Christi SOP F10.7 Appendix 4 Page 7 of 18 pages

2. Count 100 embryos/sample using hand counter with multiple keys (such as a blood cell 

counter), using one key to indicate normally developed pluteus larvae and others to 

indicate unfertilized eggs, embryos arrested in earlier developmental stages, and other 

abnormalities or for more efficient data collection, stages other than pleuteus and 

abnormalities may be lumped together and counted on one key. Attachment 3 has a list of 

developmental stages and drawings of each. 

3. Calculate the proportion of normal plutei for each replicate test: 

Number normal plutei X 100 = Percent normal plutei 

 Total no. eggs/embryos 

5.0 DATA ANALYSIS

Data are recorded on standardized data sheets (See Attachments 4-9). Normally, percent 

normal development (normal plutei) in each treatment is compared to an appropriate 

reference treatment (seawater, pore water or sea surface microlayer from an 

uncontaminated environment). Statistical comparisons are made using analysis of variance 

(ANOVA) and Dunnett's t-test (Sokal and Rohlf 1981) on the arc sine square root transformed 

data. For multiple comparisons among treatments, Ryan's Q test (Day and Quinn 1989) with 

the arc sine square root transformed data is recommended. The trimmed Spearman-Karber 

method with Abbott's correction is recommended to calculate EC50 values for dilution series 

tests (Hamilton et al. 1977) 

6.0 QUALITY CONTROL 

Quality control tests may be run using both positive and negative controls with multiple 

replicates (as many as desired). Typically, a reference toxicant dilution series (sodium 

dodecyl sulfate) is tested with each test to evaluate the gametes chosen. Negative controls 

may include a reference porewater, filtered seawater, and/or a reconstituted brine. 

7.0 TRAINING

A trainee will conduct the test with supervision initially. Determining egg concentrations, 

embryological stages and counts are test specific activities. These functions can be 

performed independently after a trainee has demonstrated he or she can accurately 

reproduce the test. 
Corpus Christi SOP F10.7 Appendix 4 Page 8 of 18 pages

8.0 SAFETY

The sea urchin embryological development toxicity test poses little risk to those performing it. 

Care should be taken when making and dispensing the 10% buffered formalin solution; use a 

hood if available, but make sure the test area is well ventilated. Protective gloves can be 

worn when pipetting or dispensing formalin or potentially toxic samples. 

Care should be taken when collecting or otherwise handling sea urchins. Urchin spines are 

sharp and fragile and may puncture the skin and break off if handled roughly. First aid similar 

to treatment of wood splinters is effective in this case (removal of spine and treatment with 

antiseptic). Collection of sea urchins by snorkeling should not be done alone. 

9.0 ATTACHMENTS

Attachment l. Equipment List for Embryological Development Toxicity Test 

Attachment 2. Pretest to Insure Selection of Quality Gametes 

Attachment 3. Development of Sea Urchin Eggs to Pluteus Larvae 

Attachment 4. Water Quality Adjustment Data Form 

Attachment 5. Sea Urchin Pretest Data Sheet 

Attachment 6. Sea Urchin Pretest Continuation Data Sheet 

Attachment 7. Sea Urchin Fertilization/Embryological Development Toxicity Test Gamete 

 Data Sheet 

Attachment 8. Sea Urchin Embryological Development Test Data Sheet 

10.0 REFERENCES 

 Day, R.W. and G.P. Quinn. 1989. Comparisons of treatments after an analysis of variance 

 in ecology. Ecol. Monogr. 59:433-463. 

 Hamilton, M.A., R.C. Russo, and R.V. Thurston. 1977. Trimmed Spearman-Karber method for 

 estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 

11(7):714-719; Correction 12(4):417 (1978) 

 Sokal, R.R., and F.J. Rohlf. 1981. Biometry. 2nd edition. W.H. Freeman and Company, 

 San Francisco, CA 859 pp. 
Corpus Christi SOP F10.7 Appendix 4 Page 9 of 18 pages

Revised by: ___________________ 

 James M. Biedenbach 

 Biologist 

Prepared by: ____ 

Duane Chapman 

Fishery Biologist 

Approved by: __________________ 

R. Scott Carr 

Field Station Leader 

____________________ 

Anne E. Kinsinger 

Chief, Field Research Division 

 ______________________

Joseph B. Hunn 

 Quality Assurance Officer 

 
Corpus Christi SOP F10.7 Appendix 4 Page 10 of 18 pages

Attachment 1 

 EQUIPMENT LIST FOR EMBRYOLOGICAL DEVELOPMENT TOXICITY TEST 

Large Carolina dishes (at least 2) 

20 mL Wheaton scintillation vials (These should be type shipped with caps with polycone liners. 

If other brand or type is used, the vials should be tested for toxicity prior to use.) 

400 mL beaker or wide-mouthed thermos for holding vials of sperm 

250 mL beakers (4) 

Pasteur pipettes and latex bulbs 

plastic microcentrifuge tubes 

25 mL shell vials or equivalent 

Test tube rack (to hold shell vials) 

12V transformer with pencil type electrodes 

Styrofoam (or something to hold electrode tips) 

10 cc syringe with large diameter blunt ended needle (make by grinding sharp point off the needle 

with a grinding stone) 

Marking pens 

Ice 

10-100 μL pipetter 

50-200 μL pipetter 

5 mL pipetters (2) 

Counting slide such as Sedgewick-Rafter chamber 

Compound microscope with 10x objective and dark field capability 

Hand tally counter 

Calculator 

Timer for exposure / incubation periods 

Buffered formalin and dispenser 

Filtered (0.45 μm) seawater, adjusted to 30 o/oo

Data sheets 

Baker reagent grade water 

Approximately 100 o

/oo concentrated brine 

 
Corpus Christi SOP F10.7 Appendix 4 Page 11 of 18 pages

Attachment 2 

PRETEST TO INSURE SELECTION OF QUALITY GAMETES 

1. Using the procedure in section 2.4.1, select 2 to 5 females and at least 2 male urchins to be 

used in the pretest. 

2. Fill pretest vials with five mL of reference water. There should be at least two vials for 

each combination of male, female, and pretest sperm concentration (step 4 below). For 

example, in a pretest with two females, one male, and six pretest sperm concentrations, 

24 vials (2 X 2 X 6) would be needed. Arrange and mark vials accordingly in a rack. 

3. Perform steps 2.4.2 (egg collection) and 2.4.3 (egg dilution) for each female urchin. Make 

enough volume of the egg suspension to perform the pretest and the test. 

4. Perform step 2.4.4 (sperm collection) for each male urchin or male combination. Prepare a 

dilution series of sperm concentrations which will bracket the 60-90% fertilization rate in 

the test. Sperm dilution will depend on the health and reproductive status of the male 

urchin, but in most cases the following "standard dilution" should be used: 

 1: 250 (20 μL dry sperm added to 5 mL MFS. This concentration is used only as 

 stock solution to make up the rest of the dilution series and is not used full strength in the 

pretest.) 

 1: 1250 (1 mL of 1:250 and 4 mL MFS) 

 1: 2500 (1 mL of 1:250 and 9 mL MFS) 

 1: 5000 (2 mL of 1:2500 and 2 mL MFS) 

 1: 7500 (2 mL of 1:2500 and 4 mL MFS) 

 1:10000 (3 mL of 1:7500 and 1 mL MFS) 

 1:12500 (1 mL of 1:2500 and 4 mL MFS) 

Sperm must be used within 30 minutes of dilution. Leave undiluted sperm on ice and 

retain, because a new sperm dilution of the concentration determined in this pretest will 

be needed for the toxicity test. Sperm diluted for use in the pretest may not be used in the 

toxicity test, because the time elapsed since the addition of water is too great. 

5. As in section 3.0 add 50 μL of the diluted sperm to each pretest vial. Incubate for 30 

minutes at approximately 20°C, and add 200 μL of the egg suspension. Incubate for 

another 30 minutes, then fix with 1 mL of the buffered formalin solution. 

6. As in section 4.0, obtain a fertilization rate for the vials. There is no need to count all vials, 

enough vials should be counted to determine a good male/female combination, and an 

appropriate sperm dilution factor. If more than one male/female combination is 
Corpus Christi SOP F10.7 Appendix 4 Page 12 of 18 pages

 acceptable, this is a good opportunity to choose a female which exhibits easily visible 

fertilization membranes or in cases where there are many samples, to combine eggs from 

different females . The appearance of the fertilization membranes may vary among female 

urchins, and presence of easily visible membranes facilitates counting. 
Corpus Christi SOP F10.7 Appendix 4 Page 13 of 18 pages

Attachment 3 

DEVELOPMENT OF SEA URCHIN EGGS TO PLUTEUS LARVAE 

The development of sea urchin eggs from fertilization to pluteus larvae normally occurs in 

approximately 48 hours. Although development is a continuous process of mitosis and cellular 

differentiation, developmental biology defines distinct stages of development by gross 

morphological characteristics. For the purpose of the Sea Urchin Embryological Development 

Test, six stages are defined and used in the characterization of embryos (Drawings on following 

page). 

1. Unfertilized egg - single cell which appears dense and lacks a fertilization membrane. 

2. Fertilized egg - egg with a distinct fertilization membrane which appears as a thin band lying 

slightly away from the central egg. The early stages of cell division are included in this 

group. 

3. Blastula - spherical, "hollow-ball" stage which is ciliated and becomes free-swimming by 

breaking out of the fertilization membrane. 

4. Early gastrula - beginnings of invagination of the blastula wall are evident. Cells move inward 

(invaginate) to form a central cavity (archenteron). Early gastrula includes embryos with the 

earliest stages of invagination and continues until the archenteron reaches approximately 

two-thirds of the diameter of the embryo. 

5. Late gastrula - gastrula in which archenteron has developed in length to two-thirds of the 

embryo diameter and has begun to differentiate and bend towards and break through the 

embryo wall. Included are the later stages (prism) with primitive gut (complete digestive 

system), early skeletal rod development, and beginnings of deltoid shape formation. 

6. Pluteus - deltoid-shaped larval stage with complete digestive system, skeletal rods, and 

growth of projecting arms. 
Corpus Christi SOP F10.7 Appendix 4 Page 14 of 18 pages

 Attachment 4 

WATER QUALITY ADJUSTMENT DATA FORM 

 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

 

A. Salinity Adjustment: 

Initial volume (mL) _______________________ 

Initial salinity (o/oo) _______________________ 

Vol. Milli-Q water added (mL) _______________________ 

Vol. o/oo brine added (mL) _______________________ 

% of original sample 

(initial vol./final vol. x 100) ________________________ 

B. Character of Sample (after salinity adjustment): 

Volume (mL) ________________________ 

Salinity (o/oo) ________________________ 

pH ________________________ 

Dissolved oxygen (mg/L) _________________________ 

DO saturation (%) _________________________ 

Total ammonia (mg/L) _________________________ 

Sulfide (mg/L) _________________________ 

COMMENTS ___________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________ 
Corpus Christi SOP F10.7 Appendix 4 Page 15 of 18 pages

Attachment 5 

SEAURCHIN PRETEST DATA SHEET 

 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

EGGS 

Female number: _ _ _ _____ 

Collection time: _ _ _ _____ 

Count: _____ _____ _____ _____ 

 

SPERM 

Male number: _____ 

Collection time: _____ 

Dilution start time: _____ 

TEST TIMES 

Sperm in: Eggs in: Formalin in: ________________ 

SPERM DILUTION: _____________________________________________________________

COMMENTS: _________________________________________________________________ 

______________________________________________________________________________

% FERTILIZATION Reference sample designation: ______________________ 

 Female # Male #___________ 

Sperm Dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

 % FERTILIZATION Reference sample designation: ________________________ 

 Female # Male #________________

 Sperm dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 
Corpus Christi SOP F10.7 Appendix 4 Page 16 of 18 pages

Attachment 6 

SEA URCHIN PRETEST CONTINUATION DATA SHEET 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

% FERTILIZATION Reference sample designation: _______________________ 

 Female # Male #_________ 

 Sperm Dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

% FERTILIZATION Reference sample designation: ______________________ 

 Female # Male #____________ 

 Sperm dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

% FERTILIZATION Reference sample designation: ______________________ 

 Female # Male #____________ 

Sperm Dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 

 % FERTILIZATION Reference sample designation: _______________________ 

 Female # Male #_____________ 

 Sperm dilution REP 1 REP 2 REP 3 REP 4

 ____ 

 ____ 

 ____ 

 ____ 
Corpus Christi SOP F10.7 Appendix 4 Page 17 of 18 pages

Attachment 7 

SEA URCHIN FERTILIZATION/EMBRYOLOGICAL DEVELOPMENT 

TOXICITY TEST GAMETE DATA SHEET 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

EGGS 

Collection time: ______________________________________________________________

Initial count/volume: ____________________________________________________________ 

Final count: ____________________________________________________________________

SPERM 

Collection time: Dilution start time: _____________________ 

Sperm dilution: ________________________________________________________________

 ________________________________________________

Test start temperature: 

 TEST TIMES

Box # Sperm in: Eggs in: Formalin in:

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

 _________ 

COMMENTS 

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________ 
Corpus Christi SOP F10.7 Appendix 4 Page 18 of 18 pages

Attachment 8 

SEA URCHIN EMBRYOLOGICAL DEVELOPMENT TOXICITY TEST 

DATA SHEET 

STUDY PROTOCOL INITIALS _______________________ 

SAMPLE DESIGNATION DATE _______________________ 

PERCENT NORMAL PLUTEUS 

 Replicate 

Treatment 1 2 3 4 5 Mean"SD % Not Plut. 

Comments:_____________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________

______________________________________________________________________________