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

By R. Scott Carr1, Marion Nipper2, and Geoffrey S. Plumlee3 

1

U.S. Geological Survey, Marine Ecotoxicology Research Station, TAMU-CC, NRC Suite 3200,

6300 Ocean Drive, Corpus Christi, TX 78412

2

Texas A & M University – Corpus Christi, Center for Coastal Studies, NRC Suite 3200, 6300 Ocean

Drive, Corpus Christi, TX 78412

3

U.S. Geological Survey, MS964 Federal Center, Denver, CO 80225

Open File Report 01-441 

TAMU-CC-0105-CCS 

The content of this document has not been reviewed for conformity with U.S.Geological 

Survey editorial standards. The use of firm, trade, and brand names is for identification 

purposes only and does not constitute endorsement by the U.S. Government. 

This report is available online at: http://geology.cr.usgs.gov/pub/open-file-reports/ofr-01-0441 

U.S.DEPARTMENT OF THE INTERIOR 

U.S.GEOLOGICAL SURVEY 
1 

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

By R. Scott Carr, Marion Nipper, and Geoffrey S. Plumlee 

Executive Summary 

As a follow-up of an initial overview of 

environmental problems caused by mining 

activities on Marinduque Island, Philippines, 

USGS and TAMU-CC scientists went to 

Marinduque in October 2000 to do a 

preliminary assessment of potential impacts 

of mining-related activities on the marine 

environment. Like the previous visit in May 

2000, the marine assessment was conducted 

at the invitation of Philippine Congressman 

Edmund O. Reyes. 

In this report we present the results of 

sediment porewater toxicity tests and 

chemical analyses. Toxicity tests consist of 

laboratory analyses for the assessment of 

adverse effects caused by environmental 

contaminants to animals or plants. 

Sediments (sand or mud) are known to 

accumulate contaminants (e.g., copper and 

other heavy metals). Therefore, it is 

common to perform toxicity tests using 

different phases of the sedimentary 

environment in order to analyze adverse 

effects of contaminants accumulated in the 

sediment. Sediment pore water (or 

interstitial water, i.e., the water distributed 

among the sediment grains) is a sedimentary 

phase which controls the bioavailability of 

contaminants to bottom dwelling aquatic 

organisms (both plants and animals). 

There are several different kinds of 

organisms with which toxicity tests can be 

performed. Among those, tests with sea 

urchin early life stages (gametes and 

embryos) are very common due to their high 

sensitivity to contaminants, ease of 

maintenance under laboratory conditions, 

and ecological importance, particularly in 

coral reefs. The basis of these tests is the 

exposure of gametes or embryos to the pore 

water to be analyzed for toxicity. If the pore 

water contains contaminants in levels that 

can adversely affect a number of marine 

species, fertilization and/or embryological 

development of sea urchins is inhibited. 

Chemical analyses provide additional 

information and aid in the interpretation of 

the toxicity test results. For the current 

study, chemical analyses were performed for 

the measurement of porewater 

concentrations of several heavy metals 

associated with copper mining activities. 

Pore waters for toxicological and 

chemical analyses were collected at several 

stations on the coast of Marinduque, near the 

mouths of the Boac and Mogpog rivers, and 

near the causeways formed by mine tailings 

disposal. Porewater samples were also 

collected at the Tres Reyes Marine Reserve, 

so that these non-contaminated samples 

could serve as a reference for test 

performance. 

Sea urchin embryological development 

and fertilization were only significantly 

impaired by two porewater samples, 

suggesting the presence of contaminants in 

toxic amounts at those stations. The toxic 

samples were collected near the up current 

side of the Calancan (Marcopper) mine 

tailings causeway (stations 2 and 3 – see 

figure 10). The pore water from station 2 

also had the highest levels of heavy metals, 

particularly cadmium, cobalt, copper, nickel, 

lead and zinc (Table 5). The concentrations 

of cobalt, nickel and zinc were also elevated 
2

at station 3. Copper concentrations were 

also elevated at the two river mouth stations 

(8 and 9) and near the CMI tailings 

causeway (station 7). 

Visual observations also indicated 

biological degradation due to heavy siltation 

and smothered coral at a gradient off the 

Calancan causeway, suggesting that siltation 

might also be causing a physical impact. 

This preliminary survey suggests that 

effects related to past mining activities are 

still evident and warrant a more 

comprehensive study to assess their severity 

and areal extent. 

Introduction 

In May 2000 a team of environmental 

and health scientists from the U.S. 

Geological Survey (USGS) and the U.S. 

Armed Forces Institute of Pathology 

(USAFIP) visited the Island of Marinduque, 

Philippines, for an overview of 

environmental problems created by a 1996 

tailings spill from the Marcopper open-pit 

copper mine. As a follow-up of that initial 

overview, USGS and TAMU-CC scientists 

visited Marinduque in October 2000, with 

the objective of collecting environmental 

samples for a preliminary assessment of 

potential impacts of mining-related activities 

on the marine environment. Like the 

previous visit, in May 2000, the marine 

assessment visit was at the invitation of 

Philippine Congressman Edmund O. Reyes. 

In this report we present the results of 

sediment porewater toxicity tests and 

chemical analyses. However, due to the 

limited number of samples, the results 

presented in this report should be considered 

preliminary. A more extensive survey is 

recommended, particularly in the areas 

where elevated levels of heavy metals and 

toxic samples were collected, in order to 

assess the spatial extent of the affected 

areas. 

Summary of potential environmental 

concerns due to mining-related 

activities in Marinduque’s marine 

environment 

Impact of mine-tailings spill on the biota at 

the mouth of the Mogpog and Boac rivers 

Concern with metals contamination at 

the mouth of the Boac and Mogpog rivers 

was expressed by the staff of Congressman 

Edmund Reyes and Governor of 

Marinduque, Carmencita Reyes, due to the 

transport of mine-tailings down river and 

into the estuarine and marine environments 

off the mouths of these rivers. Porewater 

samples were collected at these sites to 

assess potential biological effects. 

Impact of mine-tailings causeways on 

surrounding environment 

Two mine tailings causeways extend 

into the ocean on Marinduque Island (Fig. 

1). One of these has been built with tailings 

from the CMI copper mine, which operated 

in the 1970’s. The other one, more recent 

and located in Calancan Bay, is composed of 

CMI 

causeway 

Tres Reyes 

marine reserve 

Calancan tailings 

causeway 

Calancan 

Bay 

CMI Pit 

San Antonio Pit 

Tapian Pit 

Boac River Makulapnit 

River 

Bol River 

Mogpog River 

0 1500 

Meters 

N 

Figure 1. Map of Marinduque showing 

mine-tailings causeways 
3

mine tailings from the Marcopper Mine 

deposited from 1975 through 1990. Both are 

cause for concern because of the possibility 

of copper leachates spreading throughout the 

surrounding marine waters and sediments. 

USGS – TAMU-CC October, 2000 Trip 

Objectives 

Based on the information gathered by a 

team of USGS and USAFIP environmental 

and health scientists on a previous trip 

(Plumlee et al. 2000), a second trip to 

Marinduque was undertaken by marine 

ecotoxicologists, Drs. R. Scott Carr (USGS) 

and Marion Nipper (TAMU-CC), with the 

purpose of assessing the bioavailability and 

potential environmental impact of 

contaminants introduced into the marine 

environment by copper mining activities on 

the island. A meeting with local citizens 

and administrators also allowed the 

presentation of information on our activities 

and their objectives (Fig. 2). 

Figure 2. Dr. Scott Carr discussing the 

objectives of our field trip on Marinduque to 

the local community, including 

representatives of non-government 

organizations, church and city 

administration. 

USGS – TAMU-CC October, 2000 Trip 

Itinerary and Observations 

October 13 (Friday) 

• Flew into Manila, arriving in the 

evening, and met with Congressman 

Edmund O. Reyes and Governor 

Carmencita Reyes. Took ferry to 

Marinduque later that night, arriving on 

the morning of the next day. 

October 14 (Saturday) 

• Were received by the Governor’s staff, 

including Dindo Asuncion, Joven Lilles 

Ton Monteagudo, and colleagues, who 

accompanied us at all times during our 

stay in Marinduque and provided for all 

the field trip-related needs. 

• After a brief stop at the hotel for 

breakfast, an exploratory field trip was 

undertaken, with guidance of the 

Marinduque field team, for the 

inspection of potential sampling sites 

and analysis of local maps. During this 

initial field trip the following sites were 

visited: mouths of the Boac and Mogpog 

rivers, and the two mine tailings 

causeways protruding into the ocean at 

Calancan Bay (Marcopper) and North of 

the Mogpog River (CMI). We returned 

to those sites on the next days to collect 

sediments and/or pore waters. All 

sampling sites are presented on Figure 3. 

October 15 (Sunday) 

• Collected samples from six selected 

stations in the vicinity of the Marcopper 

mine tailings causeway (Fig. 4), 

Calancan Bay, to analyze the spatial 

extent of contamination and biological 

effects, if any (Fig. 3 - Stations 1-6). 

October 16 (Monday) 

• Collected samples from 3 stations: 

Northern side of the CMI mine tailings 

causeway (Fig. 5) (Station 7 on Fig. 3); 

mouth of the Mogpog River (Fig. 6, 
causeway arcopp

s

4 

Station 8 on Fig. 3); and mouth of the pollution sources (Fig. 8). 

Boac River (Fig. 7; Station 9 on Fig. 3). • Met with local citizens, including 

representatives from NGOs, the city 

October 17 (Tuesday) administration and the church. 

• • Collected samples from three reference October 18 (Wednesday) 

sites off Gaspar Island, in the marine 

sanctuary of Tres Reyes, distant from • Flew back to Manila, conditioned 

Calancan tailings 

(M er) 

Mogpog River 

CMI tailings 

Boac River 

1 

4 5 6 

10 

11 

12 

7 

9 

8 

2 

3 

Gaspar Island 

Hakupan 

I land 

Figure 3. Satellite view of Marinduque Island with sampling stations (Landsat image taken in August 

2000). 
5 

collected porewater and overlying water ice, and shipped them by Federal 

samples in a cooler with dry ice and Express to the laboratory in the USA. 

sediment samples in a cooler with blue 

a 

2 

b 

6 

Figure 4. a) Marcopper mine tailings 

causeway in Calancan Bay, showing 

sampling station 2; b) Station 6, the 

farthest removed from the Marcopper 

tailings causeway in the spatial sampling 

gradient. 

d 

b 

CMI Tailings causeway 

7 

Fish trap 

a 

Location of fish trap 

7 

c 

Figure 5. View of CMI mine tailings causeway showing: a) Sampling station number 7, 

with an adjacent fish trap; b) Northern side of causeway, as seen from station 7; c) View of 

station 7 and adjacent fish trap from the northern side of the causeway. Notice housing on 

causeway, visible on b and c; d) Detail of causeway soil with visible copper-bearing 

nuggets. 
6

a b 

c 

8 

Figure 6. Mouth of Mogpog River: a) 

With good weather; b) After rain, showing 

considerable amount of sediment being 

carried down the river; c) After rain, 

showing sampling station. 

b 

9 

a 

Figure 7. Mouth of Boac River: a) Detail of c

the river mouth meeting the ocean; b) Dr. 

Carr taking GPS coordinates at sampling 

station 9 and Marinduque field team on the 

river’s edge helping with sampling gear; c) 

Drs. Carr and Nipper collecting sediment 

for porewater extraction. 
7

Figure 8. View of some reference 

sampling sites at Gaspar Island, in the 

marine reserve of Tres Reyes, and Drs. 

Carr and Nipper processing porewater 

samples on board an outrigger. 

Materials and Methods 

Sampling sites and collection methods 

Pore water and/or sediment and 

overlying water samples were collected 

from 12 stations off Marinduque Island (Fig. 

3) in October 2000. Sampling site 

designation, details and coordinates are 

presented in Table 1. Sediment samples and 

water overlying the sediment surface were 

collected at several stations (Table 1) for 

chemical analyses. Pore water for toxicity 

testing and chemical analyses was collected 

in situ, by divers, at stations 1-6 and 10-12. 

Weather and sea conditions did not permit in 

situ sampling by diving at stations 7, 8 and 

9. Therefore, sediment cores were collected 

at knee depth at these three stations and pore 

water was extracted on shore. 

Sediment and overlying water were 

collected directly into pre-cleaned 250 ml 

high-density polyethylene containers with 

screw caps and kept on ice for up to three 

days, until arrival in Manila and shipping to 

the USA. Pore water was extracted by 

vacuum, using a device that consisted of a 

ground glass aquarium air stone as the 

filtration medium, attached to a 60-ml high 

density polyethylene syringe (Fig. 9). All 

sampling material was pre-soaked in 

distilled water for a minimum of 24 hours 

prior to use, and syringes were acid-washed. 

Samples were removed from the syringes 

and placed in pre-cleaned 500 ml high-
8

density polyethylene containers, and kept in 

coolers on ice until arrival at the hotel. 

Porewater samples were then filtered 

through acid washed, binder free 

borosilicate glass fiber filters with pore size 

of 0.7 μm. Samples to be used for toxicity 

testing were placed in pre-cleaned 250 ml 

high density polyethylene jars with Teflon 

lined screw caps, and an aliquot of the 

filtered pore waters for chemical analyses 

was placed in 50 ml high density 

polyethylene containers. The filters used for 

porewater filtration were of the kind 

routinely used in chemistry laboratories for 

the filtration of samples to be analyzed for 

metals. These filters may have removed 

other categories of contaminants from the 

samples, if they were present, but since the 

present survey was conducted to determine 

environmental effects caused by metals 

contamination from the mine tailings, 

removal of other contaminants by the 

filtration procedure was deemed acceptable. 

Filtered samples were kept in coolers on ice 

for up to 3 days, until arrival in Manila, 

whereupon they were shipped to the USA by 

Federal Express, accompanied by sample 

tracking sheets. Water samples for toxicity 

testing were shipped on dry ice, whereas 

water and sediment samples for chemical 

analyses were shipped on blue ice. Upon 

arrival at the USGS toxicity testing 

laboratory in Corpus Christi, Texas, the 

samples were logged into the laboratory 

sample tracking systems. The samples for 

toxicity testing were placed in a freezer at -

20°C, and those for chemical analyses were 

shipped on blue ice to the USGS chemistry 

laboratory in Denver, Colorado. 

Table 1. Description of sampling sites and related information

Site Site Latitude/Longitude Site Description Depth Type of sample 

No. Designation (m) collected 

Hakupan 13E33.08N/121E56.66 ~200 m S of Hakupan Island 15.0 Pore water (PW) 

Island E 

Calancan 13E33.70N/121E58.30 ~200 m W of tip of Calancan 2.0 PW, sediment (S), 

Causeway E Causeway overlying water (OW) 

Calancan 13E32.84N/121E57.58 ~200 m W of Calancan 7.5 PW 

Cove E Causeway and ~400 m N of 

mainland 

Botilao 13E32.65N/121E56.20 ~300 m N of mainland and 9.0 PW

E directly S of Hakupan Island

Dinahunan 13E32.26N/121E55.62 ~300 m W of shore and ~500 6.5 PW 

E NW of oil tank and pier 

Guisian 13E32.83N/121E53.56 ~100 m from shore – 7.6 PW, S, OW 

E believed to be “unimpacted” 

area in aerial photo (Fig. 11 

in Plumlee et al., 2000) 

CMI 13E30.04N/121E50.66 ~200 m N of CMI causeway, 1.5 PW, S 

Causeway E ~20m from shore 

8 Mogpog River 13E29.05N/121E50.31 South side of river mouth 0.5 PW, S 

E 

9 Boac River 13E27.04N/121E48.70 North side of mouth of 0.2 PW 

E northern tributary 

10 Gaspar Island- 13E14.72N/121E51.82 South side of Gaspar Island 12.0 PW 

south E ~50 m from shore 

11 Gaspar Island- 13E14.95N/121E50.95 West side of Gaspar Island 12.0 PW, S, OW 

west E ~50 m from shore 

12 Gaspar Island- 13E15.20N/121E51.67 North side of Gaspar Island 12.0 PW 

north E ~50 m from shore 

1 

2 

3 

4 

5 

6 

7 
9

Figure 9. Porewater sampling device 

Porewater Quality Measurement and 

Adjustment 

Two days before conducting the toxicity 

tests, the samples were moved from the 

freezer to a refrigerator at 4°C. One day 

prior to testing samples were thawed in a 

tepid (20°C) water bath. Temperature of the 

samples was maintained at 20 ± 1°C. 

Sample salinity was measured and adjusted 

to 30 ± 1 o/oo using purified deionized water 

or concentrated brine (see SOP F10.12, 

Appendix 1). Other water quality 

measurements (dissolved oxygen, pH, 

sulfide, and ammonia concentration) were 

made. Dissolved oxygen (DO) was 

measured with an YSI  meter, salinity was 

measured with a Reichert  refractometer, 

and pH, and total ammonia (expressed as 

nitrogen; NH4) were measured with an 

Orion  meter and the respective probes. 

Unionized ammonia (expressed as nitrogen; 

NH3) concentrations were calculated for 

each sample using the respective salinity, 

temperature, pH, and NH4 values. 

Following water quality measurements and 

adjustments, the samples were stored 

overnight at 4°C but returned to 20 ± 1°C 

before the start of the toxicity tests. 

Toxicity Testing with Sea Urchin Gametes 

and Embryos 

Toxicity of the pore water was 

determined using the fertilization and 

embryological development tests with the 

sea urchin Arbacia punctulata following the 

procedures outlined in SOP F10.6 and F10.7 

(Appendices 2 and 3). 

Arbacia punctulata urchins used in this 

study were obtained from Gulf Specimen 

Company, Inc. (Panacea, Florida). A 

standard reference porewater sample 

collected from Redfish Bay, Texas, which 

had been extracted by pneumatic pressure 

and thereafter handled identically to the test 

samples, was included with the toxicity tests 

as a negative control, in addition to the 

reference samples from Gaspar Island, 

Philippines. The standard site for reference 

porewater collection in Redfish Bay is far 

removed from any known sources of 

contamination and has been used 

extensively as a reference site (Carr and 

Chapman, 1992; National Biological 

Survey, 1993, 1994; National Biological 

Service 1995a, b; Carr et al., 1996a, b, 2000; 

U.S. Geological Survey, 1997, 1998, 1999; 

Nipper and Carr, 2001). In addition, 

dilution blanks of filtered seawater and brine 

controls (purified deionized water with brine 

added to reach a 30 o

/oo salinity) were also 

included. A dilution series test with a 

reference toxicant, sodium dodecyl sulfate 

(SDS), was included as a positive control. 

Reference toxicant test results were 

compared to a control chart with the results 

of the previous tests with this reference 

toxicant, in order to ensure that the 

sensitivity of the test organisms was within 

the standard range (Environment Canada, 

1990). 

Toxicity Test Data Analysis 

The standard statistical analyses for 

these toxicity tests involve comparisons 

among treatments using ANOVA and 
10

Dunnett's test. Prior to statistical analysis, 

the transformed data sets were screened for 

outliers (SAS Institute Inc., 1992) 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 , .05/(2 x 

n)). No outliers were detected. The 

transformed data sets were tested for 

normality and for homogeneity of variance 

using SAS/LAB  Software (SAS Institute 

Inc., 1992). Reference toxicant test results 

were analyzed by the trimmed SpearmanKarber method (Hamilton et al., 1977) with 

Abbott's correction (Morgan, 1992) to 

calculate EC50 (effective concentration to 

50% of the test organisms) values. 

A second criterion was also applied to 

compare test means to reference means. 

Detectable significance criteria (DSC) were 

developed to determine the 95% confidence 

value based on power analysis of all similar 

tests performed by our laboratory (Carr and 

Biedenbach, 1999). This value is the 

percent minimum difference from the 

reference that is necessary to detect a 

significant effect while minimizing type I 

errors. The DSC value for the sea urchin 

fertilization assay is 15.5% at _ #0.05, and 

19% at _# 0.01. For the embryological 

development test the DSC values at _ 

#0.05 and _# 0.01 are 16.4 and 20.6%, 

respectively. 

Chemical analyses of pore waters and 

sediments 

Pore waters were analyzed for major 

cations and selected trace metals and 

metalloids using inductively coupled 

plasma-mass spectrometry. A detailed 

description of the methods applied for the 

chemical analyses and quality assurance 

procedures is described in Lamothe et al. 

(1999). It is believed that chloride 

interference with the copper analyses caused 

an increase of approximately 10 μg/L in the 

measured concentrations. Therefore, this 

was accounted for in the interpretation of 

results. However, the copper values 

measured at stations 8 and 9 are expected to 

be accurate due to their low salinity (Table 

2), thus lacking chloride interference. 

Results and Discussion 

Pore Water Quality Measurements 

Water quality measurements for all 

porewater samples, including the reference 

pore water from Redfish Bay, Texas, as well 

as the 0.45 μm filtered seawater control, are 

presented on Table 2. Salinity was the most 

variable of the measured porewater quality 

parameters, ranging from 2 to 38 ppt. All 

samples required salinity adjustment prior to 

toxicity testing in order to satisfy the test 

salinity requirement of 30 ± 1 ppt. The 

percentage of pore water in the original test 

samples (%OS) after salinity adjustment 

(Table 2) ranged from 75 to 96%. Dissolved 

oxygen was above 80% saturation in all 

samples, and pH ranged from 7.43 and 8.25. 

Unionized ammonia (NH3) was less than 5.4 

μg/L in all samples from Marinduque. The 

Redfish Bay reference pore water had a 

somewhat higher pH (8.38), as well as 

higher NH3 concentration, of 57.8 μg/L, 

which is still below toxic levels to A. 

punctulata fertilization and embryological 

development (Carr et al., 1996a). 

Sea Urchin Toxicity Tests 

Raw data and means from the 

fertilization test are given in Table 3, and for 

the embryological development test, in 

Table 4. Only the two porewater samples 

nearest to the Calancan mine tailings 

causeway were significantly toxic to sea 

urchin (A. punctulata) early-life stages. The 
11

sample from station 2, closest to the 

causeway, was the most toxic overall, 

causing significant adverse effects in both 

tests even at a dilution of 25%, whereas the 

sample from station 3 was toxic in both tests 

at the 50% dilution (see Fig. 10). The 

gradient sampling performed in the area of 

the Calancan causeway was in the 

predominately up-current direction. Based 

on the toxicity of the two samples collected 

closest to the causeway, it is likely that 

environmental effects caused by the tailings 

would be more widespread in the downcurrent direction. The EC50 values for the 

SDS tests (Tables 3 and 4) were within the 

normal range based on the control chart with 

the results of previous tests performed in our 

laboratory with this reference toxicant, 

indicating that the sensitivity of the test 

organisms was within the acceptable 

standard. 

In previous surveys, pore water from 

sandy sediment collected in the vicinity of 

coral reefs in southeastern Mexico and in 

Honolulu, Hawaii, exhibited toxicity, 

showing that these kinds of tests are useful 

indicators of contaminant accumulation in 

toxic amounts around coral reefs, and could 

serve as early warning signals of potential 

biological effects to the reef itself (Nipper 

and Carr, 2001). The results obtained in the 

current study suggest that the area adjacent 

to the Calancan causeway is adversely 

affected by the mine tailings disposal. 

Visual observations at the sites also 

indicated biological degradation, with only 

one species of small fish observed at station 

2 and heavy siltation and smothered coral 

seen at station 3 (see next section). Pore 

water from stations 4 and 5 was not toxic to 

sea urchin early life stages, but a large 

amount of silt covering corals was still 

observed at those sites, suggesting that 

although there was no evidence of biological 

degradation caused by chemical 

contamination, a physical impact might be 

caused by siltation due to erosion of fine 

particulate matter from land. 

Table 2. Water quality measurements of samples used for toxicity testing

Sample Original Adjusted OUS1 DO2 DO pH NH4 

3 NH3 4 Sulfide 

Salinity Salinity 

(ppt) (ppt) (%) (mg/L) % sat (mg/L) ( g/L) (mg/L) 

Redfish Bay Ref. 40 30 75 6.69 90.7 8.38 0.803 57.8 <0.01 

1 34.5 30 88 6.67 90.2 7.61 <0.1 <1.3 <0.01 

2 34 30 88 6.93 93.1 7.52 <0.1 <1.1 <0.01 

3 38 30 75 7.15 95.9 7.49 <0.1 <1.1 <0.01 

4 26 30 96 6.79 91.9 7.72 <0.1 <1.7 <0.01 

5 34 30 88 6.86 92.2 7.550 <0.1 <1.1 <0.01 

6 35 30 86 6.87 92.3 7.43 <0.1 <0.9 <0.01 

7 32 30 94 6.92 92.8 7.60 0.137 1.7 <0.01 

8 2 30 75 7.00 94.00 8.25 <0.1 <5.4 <0.01 

9 2 30 75 6.94 92.9 8.09 <0.1 <3.8 <0.01 

10 35 30 87 6.75 91.0 8.05 <0.1 <3.5 <0.01 

11 35 30 87 6.87 92.5 7.51 0.119 1.2 <0.01 

12 35 30 86 6.77 90.9 7.49 <0.1 <1.0 <0.01 

Brine control 0 30 75 6.36 87.8 8.41 <0.1 <7.7 <0.01 1

Percent of original undiluted sample after salinity adjustment; 2Dissolved oxygen; 3Total ammonia; 4

Unionized ammonia. 
12

Figure 10. Map of Marinduque showing porewater toxicity data for sea urchin fertilization and

embryological development.

CMI

causeway

10

12

11

9

7

8

5

6

1

4

2

3

Tres Reyes

marine reserve

Calancan

tailings

causeway

Calancan

Bay

CMI

Pit

San Antonio Pit

Tapian Pit

Boac River

Makulapnit

River

Bol River

Mogpog River

0 150

Meters

N

Not toxic

Toxic at>25%, < 0.01

Toxic at>50%, < 0.01
13 

Table 3. Mean (n=5) percent fertilization of the sea urchin, A. punctulata, in porewater samples 

from Marinduque, reference and control samples, and in the reference toxicant test with SDS 

Sample % Dilution Mean Fert. Standard EC50 Significant 

(%) Deviation (95% CI)1 Effect2 

Redfish Bay 100 99.6 0.70 

Redfish Bay 50 99.8 0.42 

Redfish Bay 25 99.8 0.42 

1 100 99.0 0.71 

1 50 98.6 1.67 >100 

1 25 98.6 0.55 

2 100 0.8 1.30 ** 

2 50 0.2 0.45 <25 ** 

2 25 0.2 0.45 ** 

3 100 19.6 4.39 ** 

3 50 52.8 8.47 53.45 ** 

3 25 87.4 4.16 (46.99-60.80) 

4 100 99.4 0.55 

4 50 99.2 1.30 >100 

4 25 99.4 0.89 

5 100 100.0 0.00 

5 50 100.0 0.00 >100 

5 25 99.8 0.45 

6 100 98.2 1.48 

6 50 98.0 1.00 >100 

6 25 97.2 2.49 

7 100 96.8 1.48 

7 50 97.2 1.79 >100 

7 25 99.2 0.45 

8 100 86.4 6.07 

8 50 91.6 2.07 >100 

8 25 93.0 3.54 

9 100 100.0 0.00 

9 50 99.4 0.89 >100 

9 25 99.8 0.45 

10 100 100.0 0.00 

10 50 99.4 1.34 >100 

10 25 100.0 0.00 

11 100 98.8 2.68 

11 50 99.2 0.45 >100 

11 25 99.2 0.45 

12 100 99.4 0.89 

12 50 99.4 0.89 >100 

12 25 99.2 0.84 

Seawater Contr. 100 99.2 1.03 

Brine control 100 96.6 0.55
14 

Table 3. Continued 

SDS (mg/L) 20 0.0 0.00 

SDS (mg/L) 10 0.4 0.89 

SDS (mg/L) 5 43.0 6.40 4.76 

SDS (mg/L) 2.5 100.0 0.00 (4.45-5.10) 

SDS (mg/L) 1.25 99.8 0.45 1

95% confidence interval in parenthesis; 

2

Significantly different from the reference and below DSC at α≤0.01. 

Table 4. Mean (n=5) percent normal embryological development of the sea urchin, A. 

punctulata, in porewater samples from Marinduque, reference and control samples, and in the 

reference toxicant test with SDS. 

Sample % Dilution Mean Normal Standard EC50 Significant 

Larvae (%) Deviation (95% CI)1 Effect2 

Redfish Bay 100 96.1 3.35 

Redfish Bay 50 97.9 2.13 

Redfish Bay 25 99.0 1.05 

1 100 93.2 2.39 

1 50 98.2 1.48 >100 

1 25 98.6 0.89 

2 100 0.0 0.00 ** 

2 50 0.0 0.00 <25 ** 

2 25 0.0 0.00 ** 

3 100 0.0 0.00 ** 

3 50 44.8 11.99 48.05 ** 

3 25 93.6 0.55 (44.60-51.76) 

4 100 94.0 1.58 

4 50 96.4 1.14 >100 

4 25 97.6 1.52 

5 100 96.4 1.34 

5 50 96.2 2.17 >100 

5 25 99.0 0.71 

6 100 95.6 1.14 

6 50 95.4 2.41 >100 

6 25 96.4 2.61 

7 100 94.4 2.30 

7 50 96.6 2.30 >100 

7 25 96.6 1.52 

8 100 93.2 2.49 

8 50 93.8 2.77 >100 

8 25 98.2 0.84 

9 100 96.8 1.64 

9 50 96.2 1.48 >100 

9 25 99.2 0.84 
15 

Table 4. Continued 

10 100 96.2 2.77 

10 50 95.6 2.30 >100 

10 25 98.0 0.71 

11 100 98.2 1.30 

11 50 98.0 0.71 >100 

11 25 98.2 1.64 

12 100 94.6 2.07 

12 50 96.2 1.48 >100 

12 25 98.6 0.89 

Seawater Contr. 100 97.7 2.41

SDS (mg/L) 20 0.0 0.00 

SDS (mg/L) 10 0.0 0.00 3.46 

SDS (mg/L) 5 0.2 0.45 (3.38-3.55) 

SDS (mg/L) 2.5 93.6 3.21 

SDS (mg/L) 1.25 98.0 1.87 1

95% confidence interval in parenthesis; 

2

Significantly different from the reference and below DSC at α≤0.01 

Visual observations of sampling stations 

Station 1: Healthy appearance, with 

small reef patches exhibiting small stag horn 

coral, gorgonians, large basket sponges, 

crinoids, starfish, and a variety of fish. 

Station 2: Coarse sand. The only visible 

life was one species of small burrowing fish. 

Station 3: Heavy siltation, with large 

amount of smothered hard coral. Sabellids, 

crinoids and echinoids were observed, and 

some small reef fish were seen in the area. 

Station 4: Siltation was still observed, 

but not as strongly as at station 3, although a 

large amount of coral debris was present. 

There were a large variety of hard corals, 

crinoids, starfish, nudibranch mollusks and 

reef fish. 

Station 5: Heavy siltation and coral 

suffocation with only a few reef fish seen. 

Station 6: Healthier appearance, with 

wide variety of corals, fish, and crinoids. 

Large groups of one species of sea urchin 

were observed, as well as starfish, large 

bivalves, basket sponges, and sabellid 

polychaetes. 

Stations 7, 8 and 9: Sampling was 

performed in shallow waters without diving, 

for safety reasons, due to inclement weather. 

Water was turbid and no visual observations 

were made. The sediment was mostly 

composed of coarse pebbles. 

Stations 10, 11 and 12: These were the 

reference stations at the marine reserve, off 

Gaspar Island. They presented a healthy 

appearance, with large biological diversity, 

although little hard coral was present in the 

area. There were many different species of 

reef fish not seen at any other stations, giant 

clams (Tridacna sp.) and nudibranch 

mollusks, basket sponges, anemones and 

gorgonians, tunicates, crustaceans, and a 

variety of echinoderms, including 

ophiuroids and sea urchins. Six different 

species of sea urchins were observed at 

station 12. 

Chemical analyses 

Chemical analyses of the porewater 

samples exhibited elevated concentrations of 

several metals at the toxic stations, 

particularly Cd, Co, Cu, Ni, Pb and Zn at 

station 2, and Co, Ni and Zn at station 3 

(Table 5). In a comparison of the 

concentrations of some selected metals with 

the USEPA acute and chronic saltwater 

criteria, it can be observed that criteria for 
1

2

3

4

5

6

7

8

9

10

11

12

1

2

3

4

5

6

7

8

9

10

11

12

16

copper were exceeded at stations 2, 7, 8, and 

9 in the vicinity of the CMI tailings 

causeway and at the mouth of the Mogpog 

river (Table 6). Acute and chronic criteria 

for zinc were exceeded at stations 2 and 3, 

and the chronic criteria for lead and nickel 

were exceeded at station 2, which also had 

the most toxic porewater sample. 

Additional chemical and mineralogical 

analyses of sediments collected at several 

sites are still in progress and will be reported 

in subsequent publications. 

Table 5. Concentration of metals in pore waters of Marinduque Island. 

Station Ag Al Ba Be Ca Cd Co Cr Cu1 Fe K 

(μg/L) mg/L (μg/L) 

<5 450 30 <0.5 460 <0.2 <0.2 <10 10 890 460,000 

<5 120 78 <0.5 480 2 5 <10 300 930 470,000 

<5 56 62 <0.5 500 <0.2 3 <10 10 850 480,000 

<5 43 52 <0.5 490 <0.2 <0.2 <10 10 770 480,000 

<5 49 50 <0.5 490 <0.2 0.4 <10 10 710 480,000 

<5 50 72 <0.5 480 <0.2 0.5 <10 10 660 470,000 

<5 74 72 <0.5 450 <0.2 0.8 <10 20 620 410,000 

<5 520 50 <0.5 13 <0.2 <0.2 <10 30 350 11,000 

<5 170 40 <0.5 41 <0.2 <0.2 <10 8 140 11,000 

<5 83 66 <0.5 470 <0.2 <0.2 <10 10 630 460,000 

<5 75 57 <0.5 490 <0.2 0.4 <10 10 650 470,000 

<5 230 40 <0.5 480 <0.2 0.4 <10 10 630 470,000 

Table 5. Continued

Station Mg Mn Na Ni Pb Sb Tl V Zn 

(mg/L) (μg/L) mg/L (μg/L) 

1,500 5.8 >3000 <1 <0.5 0.5 <0.5 2 20 

1,500 54 >3000 10 18 0.4 <0.5 <2 320 

1,500 17 >3000 6 0.7 0.4 <0.5 <2 100 

1,400 17 >3000 <1 <0.5 0.5 <0.5 5 10 

1,400 22 >3000 <1 <0.5 0.3 <0.5 4 20 

1,400 14 >3000 1 <0.5 0.3 <0.5 3 20 

1,200 160 >3000 <1 <0.5 <0.2 <0.5 <2 30 

18 7 190 <1 1 <0.2 <0.5 <2 10 

34 3.2 230 <1 <0.5 <0.2 <0.5 <2 9 

1,400 7 >3000 <1 <0.5 0.2 <0.5 <2 20 

1,400 13 >3000 1 <0.5 <0.2 <0.5 <2 10 

1,400 17 >3000 <1 <0.5 0.2 <0.5 <2 20 1

Copper concentrations are likely to be 10 μg/L above true levels, due to chloride interference with the 

measurements, except for stations 8 and 9, with 2 ppt salinity (see Table 2). 
17 

Table 6. U.S.EPA acute (short-term exposure) and chronic (long-term exposure) water quality criteria for selected 

metals (in μg/L) in saltwater (USEPA, 1998), and station numbers at which criterion concentrations were exceeded 

in the filtered samples. Criteria are not available for other metals analyzed. 

Metal Acute Chronic Acute criteria exceedances Chronic criteria exceedances 

Criteria Criteria Stations Stations 

Cadmium 42 9.3 

Zinc 90 81 2, 3 2, 3 

Copper1 4.8 3.1 2, 7, 8 2, 7, 8 

Lead 210 8.1 2 

Silver 1.9 NA2 

Nickel 74 8.2 2 1

10μg/L were subtracted from the measured values, to account for suspected chloride interference in the

measurements.

2

NA: Not Applicable. The method detection level was above the acute water quality criterion for silver.

Summary and Conclusions 

High toxicity was observed and elevated 

levels of metals were measured in the pore 

waters collected at the two stations nearest 

to the Calancan tailings causeway, although 

those sites were in the predominately upcurrent direction. Visual observations also 

indicated biological degradation, with heavy 

siltation and smothered coral at stations 2 

through 5, suggesting that although samples 

from stations 4 and 5 were not toxic to sea 

urchin early life stages and did not present 

particularly elevated levels of metals, a 

physical impact might be caused by 

siltation. 

The toxicity data and the visual 

observations suggest that the reestablishment of the benthic community, as 

well as of coral reefs and their associated 

fauna and flora, is unlikely to occur in the 

vicinity of the Calancan causeway. The 

elevated concentration of copper in the pore 

water from the station 2, closest to the 

causeway, suggests that this causeway is a 

constant source of copper contamination to 

the surrounding environment and is 

impairing the re-colonization of the area by 

the local natural biota. 

This preliminary survey suggests that 

effects related to past mining activities are 

still evident and warrants a more 

comprehensive study to assess the severity 

and areal extent both up and down current of 

the tailings causeways. 

Acknowledgements 

We thank Congressman Edmund O. 

Reyes for the invitation to conduct this 

survey on Marinduque, and we are grateful 

for the warm welcome and continued 

logistical assistance given to us by the staff 

of Governor Carmencita Reyes during our 

stay in Marinduque. We are indebted to the 

expert technical assistance of Jim 

Biedenbach, Russell Hooten, and Linda May 

who processed the samples in the laboratory 

and conducted the toxicity tests, and to Al 

Meier and Peter Theodorakos for 

performing the chemical analyses. We 

thank Drs. Jack Medlin and Tom May for 

their helpful reviews of this report. 
18

Literature Cited 

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

criteria for sea urchin toxicity tests. Aquat. Ecosys. Health Manage. 2:413-418. 

Carr, R.S. and D.C. Chapman. 1992. Comparison of solid-phase and pore-water approaches for 

assessing the quality of marine and estuarine sediments. Chem. Ecol. 7:19-30. 

Carr, R.S., D.C. Chapman, C.L. Howard, J.M. Biedenbach. 1996a. Sediment quality triad 

assessment survey of the Galveston Bay, Texas system. Ecotoxicology 5:341-364. 

Carr, R.S., E.R. Long, H.L. Windom, D.C. Chapman, G. Thursby, G.M. Sloane, D.A. Wolfe. 

1996b. Sediment quality assessment studies of Tampa Bay, Florida. Environ. Toxicol. Chem. 

15:1218-1231. 

Carr, R.S., P.A. Montagna, J.M. Biedenbach, R. Kalke, M.C. Kennicutt, R. Hooten, G. Cripe. 

2000. Impact of storm-water outfalls on sediment quality in Corpus Christi Bay, Texas, 

USA. Environ. Toxicol. Chem. 19:561-574. 

Environment Canada. 1990. Guidance document on control of toxicity test precision using 

reference toxicants. Environmental Protection Series, Report EPS 1/RM/12. Environment 

Canada, Ottawa, Ontario, Canada. 

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

estimating median lethal concentrations in toxicity bioassays. Environ. Sci. Technol. 11:714-

719; Correction 12:417 (1978). 

Lamothe, P.J., A.L. Meier, S. Wilson. 1999. The Determination of Forty Four Elements in 

Aqueous Samples by Inductively Coupled Plasma - Mass Spectrometry. U.S. Geological 

Survey, Open-File Report 99-151, Denver, CO, 14 p. 

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

511 pp. 

National Biological Service (NBS). 1995a. Toxicity testing of sediments from Biscayne Bay and 

Surrounding Areas. Report submitted by the National Biological Service to the National 

Oceanic and Atmospheric Administration, Coastal Monitoring and Bioeffects Division, 

Seattle, WA, 11 pp. + 17 tables, 11 figures and 4 attachments. 

National Biological Service (NBS). 1995b. Toxicity testing of sediments from western Florida 

and coastal South Carolina and Georgia. Report submitted by the National Biological 

Service to the National Oceanic and Atmospheric Administration, Coastal Monitoring and 

Bioeffects Division, Seattle, WA, 14 pp. + 35 tables, 10 figures and 4 attachments. 

National Biological Survey (NBS). 1993. Toxicity testing of sediments from Charleston Harbor, 

South Carolina and vicinity. Report submitted by the National Biological Survey to the 

National Oceanic and Atmospheric Administration, Ocean Assessment Division, Seattle, 

WA, 7 pp. + 16 tables and 4 attachments. 

National Biological Survey (NBS). 1994. Survey of sediment toxicity in Pensacola Bay and St. 

Andrew Bay, Florida. Report submitted by the National Biological Survey to the National 

Oceanic and Atmospheric Administration, Ocean Assessment Division, Seattle, WA, 12 pp. 

+ 24 tables and 5 attachments. 

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

contaminant impacts in the vicinity of coral reefs. Bull Mar Sci 68 (in press). 
19 

Plumlee, G.S., R.A. Morton, T.P. Boyle, J.H. Medlin, J. Ceteno. 2000. An Overview of Miningrelated Environmental Issues, Marinduque Island, Philippines: Observations from a Joint 

U.S. Geological Survey – Armed Forces Institute of Pathology Reconnaissance Field 

Evaluation, May 12-19, 2000. Denver, Colorado: U.S. Geological Survey, Open-File Report 

00-397. 36 p. http://geology.cr.usgs.gov/pub/open-file-reports/ofr-00-0397/ 

SAS Institute Inc. 1992. SAS/LAB Software: User's Guide, Version 6, First Edition, Cary, NC: 

SAS Institute Inc., 291 pp. 

U.S. Environmental Protection Agency (USEPA). 1998. National recommended water quality 

criteria; republication. Federal Register, Part IV, v. 63, p. 68354-68364. 

U.S. Fish and Wildlife Service (USFSW). 1992. Amphipod solid-phase and sea urchin porewater 

toxicity tests of Tampa Bay, Florida sediments. Report submitted by the U.S. Fish and 

Wildlife Service to National Oceanic and Atmospheric Administration, Ocean Assessment 

Division, Seattle, WA, 9 pp. + 19 appendices. 

U. S. Geological Survey (USGS). 1997. Toxicity testing of sediments from Biscayne Bay, 

Florida and surrounding areas - Phase II. Report submitted by the USGS to National 

Oceanic and Atmospheric Administration, Coastal Monitoring and Biological Assessment 

Division, Seattle, WA, 10 pp. + 8 tables, 10 figures and 4 attachments. 

Sampling team left to right: Joven Lilles, Dr. Scott Carr, Dindo Asuncion, unidentified 

driver, driver Dan, Ton Monteagudo, Erwin Penafiel, and Dr. Marion Nipper (the photographer). 
APPENDIX 1

SOP F10.12

WATER QUALITY ADJUSTMENT OF SAMPLES
Corpus Christi SOP: F10.12 Page 1 of 6 

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 35-40°C) 

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. Baker® 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 Page 2 of 6 

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-Chem® 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 25°C. The samples may be thawed the day of water quality adjustment 

(WQA) or may be transferred from the freezer to a refrigerator (4°C) 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±1°C, 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) _ sample vol. in mL = A 

(ii) sample vol ÷ A = B 

(iii) sample vol. ÷ A = C 

(iv) B _ C = volume of HPLC water to add 

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. 
Corpus Christi SOP: F10.12 Page 3 of 6 

(i) ((target o/oo _ sample o/oo) _ sample vol. in mL) ÷ (brine o/oo _ targeto/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. 

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. 
Corpus Christi SOP: F10.12 Page 4 of 6 

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 Page 5 of 6 

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. Baker® #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 Page 6 of 6 

ATTACHMENT 2

WATER QUALITY ADJUSTMENT DATA FORM

STUDY PROTOCOL INITIALS _________ 

SAMPLE DESIGNATION DATE 

A. Salinity Adjustment: 

Initial volume (mL) 

Initial salinity (o

/oo) 

Vol. Baker® HPLC 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): 

Final Volume (mL) 

Final Salinity (o

/oo) 

pH 

Dissolved oxygen (mg/L) 

DO saturation (%) 

Total ammonia (mg/L) 

Sulfide (mg/L) 

COMMENTS 
APPENDIX 2

SOP F10.6

SEA URCHIN FERTILIZATION TOXICITY TEST
Corpus Christi SOP: F10.6 Page 1 of 15 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 weekly and salinity maintained at 30 ± 3 o

/oo. Males 

and females should be kept in separate tanks. 
Corpus Christi SOP: F10.6 Page 2 of 15 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 Millipore® 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 NaH2P04 or KH2PO4 (mono)

10.5 g Na2HPO4 or K2HPO4 (dibasic)

1 mL needed for each replicate. Fill the dispenser.

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 vial. 
Corpus Christi SOP: F10.6 Page 3 of 15 pages 

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 pasteure 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 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. 
Corpus Christi SOP: F10.6 Page 4 of 15 pages 

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 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 
Corpus Christi SOP: F10.6 Page 5 of 15 pages 

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 affect 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. 

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. 
Corpus Christi SOP: F10.6 Page 6 of 15 pages 

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. 

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 

Total No. Eggs 

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. 
Corpus Christi SOP: F10.6 Page 7 of 15 pages 

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 

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 Page 8 of 15 pages 

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 Page 9 of 15 pages 

Attachment 1 

EQUIPMENT LIST FOR FERTILIZATION TOXICITY TEST 

Large Carolina dishes (at least 2)

20 mL KIMBLE scintillation vials (These should be type shipped with caps off, and without cap

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 Page 10 of 15 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 Page 11 of 15 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 Page 12 of 15 pages 

Attachment 4 

SEA URCHIN PRETEST DATA SHEET 

TEST ID INITIALS 

STUDY PROTOCOL 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 Page 13 of 15 pages 

Attachment 5 

SEA URCHIN PRETEST CONTINUATION DATA SHEET 

TEST ID INITIALS 

STUDY PROTOCOL 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 Page 14 of 15 pages 

Attachment 6 

SEA URCHIN FERTILIZATION/EMBRYOLOGICAL DEVELOPMENT 

TOXICITY TEST GAMETE DATA SHEET 

TEST ID INITIALS 

STUDY PROTOCOL 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: 

COMMENTS 

Eggs in: Formalin in:
Corpus Christi SOP: F10.6 Page 15 of 15 pages 

Attachment 7 

SEA URCHIN FERTILIZATION TOXICITY TEST 

FERTILIZATION DATA SHEET 

TEST ID INITIALS 

STUDY PROTOCOL DATE 

PERCENT FERTILIZED 

Replicate 

Treatment 1 2 3 4 5 Mean±SD Unfert. 

COMMENTS 
APPENDIX 3 

SOP F10.7 

SEA URCHIN EMBRYOLOGICAL DEVELOPMENT TOXICITY TEST 
Corpus Christi SOP: F10.7 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 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 40EC 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 Millipore® 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 NaH2P04 or KH2PO4 (mono)

10.5 g Na2HPO4 or K2HPO4 (dibasic)

0.75 mL needed for each replicate. Fill the dispenser.

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. 
Corpus Christi SOP: F10.7 Page3 of 18 pages 

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 20-50μ L 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 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. 
Corpus Christi SOP: F10.7 Page 4 of 18 pages 

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 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 (mL) 

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. 
Corpus Christi SOP: F10.7 Page 5 of 18 pages 

The penicillin stock solution is prepared 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. 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. 
Corpus Christi SOP: F10.7 Page 6 of 18 pages 

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 ± 1EC 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. 

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 pluteus 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). 
Corpus Christi SOP: F10.7 Page 7 of 18 pages 

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. 

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 

Attachment 9. Sea Urchin Embryological Development Test Abridged Data Sheet 
Corpus Christi SOP: F10.7 Page 8 of 18 pages 

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 Page 9 of 18 pages 

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 Page 10 of 18 pages 

Attachment 1 

EQUIPMENT LIST FOR EMBRYOLOGICAL DEVELOPMENT TOXICITY TEST 

Large Carolina dishes (at least 2)

20 mL KIMBLE scintillation vials (These should be type shipped with caps off, and without cap

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 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 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 Page 12 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 Page 13 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 Page 14 of 18 pages

Attachment 5

SEA URCHIN PRETEST DATA SHEET

TEST ID INITIALS 

STUDY PROTOCOL 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 Page 15 of 18 pages

Attachment 6

SEA URCHIN PRETEST CONTINUATION DATA SHEET

TEST ID INITIALS 

STUDY PROTOCOL 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 Page 16 of 18 pages

Attachment 7

SEA URCHIN FERTILIZATION/EMBRYOLOGICAL DEVELOPMENT

TOXICITY TEST GAMETE DATA SHEET

TEST ID INITIALS 

STUDY PROTOCOL 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 Page 17 of 18 pages

Attachment 8

SEA URCHIN EMBRYOLOGICAL DEVELOPMENT TEST DATA SHEET

TEST ID INITIALS 

STUDY PROTOCOL DATE 

 Early Late % Normal %NonTreatment Rep. Eggs Blastula Gastrula Gastrula PluteusDevelopment Norm

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Comments 

 
Corpus Christi SOP: F10.7 Page 18 of 18 pages

Attachment 9

SEA URCHIN EMBRYOLOGICAL DEVELOPMENT TOXICITY TEST DATA SHEET

TEST ID INITIALS 

STUDY PROTOCOL DATE 

PERCENT NORMAL PLUTEI

 Replicate

Treatment 1 2 3 4 5 Mean±SD

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Comments