A Preliminary Survey of Marine Contamination from Mining-related

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

1

, Marion Nipper

2

, and Geoffrey S. Plumlee

3

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

T

res Reyes

marine reserve

Calancan tailings

causeway

Calancan

Bay

C

MI Pit

Sa

n Antonio Pit

T

apian Pit

Boac River

Mak

ulapnit

River

Bo

l 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

C

alancan tailings

(M er)

M

ogpog River

C

MI tailings

B

oac River

1

4

5

6

1

0

1

2

7

9

8

2

3

Gaspar Island

H

akupan

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

c

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

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; NH

4

) were measured with an

Orion



meter and the respective probes.

Unionized ammonia (expressed as nitrogen;

NH

3

) concentrations were calculated for

each sample using the respective salinity,

temperature, pH, and NH

4

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(df

Error

, .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 Spearman-

Karber method (Hamilton et al., 1977) with

Abbott's correction (Morgan, 1992) to

calculate EC

50

(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 (NH

3

) 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 NH

3

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 down-

current direction. The EC

50

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 OUS

1

DO

2

DO pH NH

4

3

NH

3

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;

2

Dissolved oxygen;

3

Total 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

<

0.01

Toxic at

>

25%,

<

0.01

Toxic at

>

50%,

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 EC

50

Significant

(%) Deviation (95% CI)

1

Effect

2

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 EC

50

Significant

Larvae (%) Deviation (95% CI)

1

Effect

2

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 Cu

1

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

Copper

1

4.8 3.1

2, 7, 8 2, 7, 8

Lead 210 8.1

2

Silver 1.9

NA

2

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 up-

current 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 re-

establishment 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 Mining-

related 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 0-36

o

/

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 1

o

/

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 (_100

o

/

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

_ target

o

/

oo

) =

vol. of brine to add

3.3 Dissolved Oxygen Adjustment

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

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

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

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

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

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

is performed.)

3.4 Other Water Quality Determinations

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

Form.

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

3. Measure and record sulfide concentration if required.

4.0 DATA COLLECTION

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

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

5.0 QUALITY CONTROL

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

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

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

station leader. Copies will be kept in the lab.

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 (EC

50

). 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 NaH

2

P0

4

or KH

2

PO

4

(mono)

10.5 g Na

2

HPO

4

or K

2

HPO

4

(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 EC

50

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. 2

nd

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 NaH

2

P0

4

or KH

2

PO

4

(mono)

10.5 g Na

2

HPO

4

or K

2

HPO

4

(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