Text

Final Rept Chronic Toxicity of Betz Clam-Trol CT-1 Cyprinodon Variegatus
	ML17223B310
Person / Time
Site:	Saint Lucie
Issue date:	04/17/1991
From:	; AQUA SURVEY, INC.
To:	;
Shared Package
ML17223B307	List: L-91-299, Forwards Copy of NPDES Permit Notification Request Sent to EPA on 911016,per Section 3.2.4 of Environ Protection Plan; ML17223B306; ML17223B308; ML17223B309; ML17223B310; ML17223B311;
References
	91-089C, 91-89C, BR91-1513C, NUDOCS 9111140236
	Download: ML17223B310 (44)
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p4 Cg ATTACHMENT6 RECUR SURVEY, INC.

FINAL REPORT THE CHRONIC TOXICITY OF BETZ CLAM-TROL+ CT-1 APRIL 17, 1991 BR91-1513C JOB

$91- 089C 499 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (201) 788-8700 FAX (201) 788-9165 9fiii40236 91iiOi PDR AGOCK 05000335

- P PDR

RQUR SURVEY INC Final Report, The Chronic Bioassay of EPA Data Requirement:

Author:

Study Director Date Report Date:

April 17, 1991 Performing Laboratory:

Aqua Survey, Inc 499 Point Breeze Road Flemington, New Jersey 08822 For:

Betz Laboratories, Inc.

4636 Somerton Road

Trevose, PA 19047 ASI Project Number:

91-089C 1 of 12 0

499 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (908) 788-8700 FAX (908) 788-9165

RECUR SURVEY, INC.

STATEMENT OF NO CONFIDENTIALITY CLAIMS No claim of confidentiality is made for any information contained in this study on the basis of its falling within the scope of FIFRA Section 10 (d)

(1)

(A),

(B) or (C).

BETZ LABORATORIES, INC.

4636 Somerton Road

Trevose, PA 19047 Date ASI Project Number:

91-089C 2 of 12 CLfl 499 Point Breeze Road

~ Flemington. New Jersey 08822

~ Telephone (908) 788-8700 FAX(908) 788-9165

RECUR SURVEY, INC.

THE CHRONIC TOXICITY BIOASSAY OF CLAM-TROL, CT-1 This study meets the requirements for 40 CFR Part 160.

Submitter:

Signature:

Betz laboratories, Inc.

4636 Somerton Road

Trevose, PA 19047 Date Sponsor:

Signature:

Betz Laboratories, Inc.

4636 Somerton Road

Trevose, PA 19047 Date QA Officer:

K rl Ceraso~

Date 3 of 12 499 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (908) 788-8700 FAX (908) 788-9l65

RECUR SURVCY, lNC.

Summary A sample of Betz Clam-Trol~, CT-l, received from Betz Laboratories, Inc., Somerton

Road, Trevose, PA 19047 on March 21,

1991, was tested for potential chronic toxicity to conducted under static conditions with daily renewal of the test solutions.

Reported final test endpoints are a Nominal No Observed Effects Concentration (NOEC) of 5.0

PPM, a

Nominal Lowest Observed Effects Concentration (LOEC) of 10.0 PPM and a

168 hour0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months Lethal Concentration (LC) 50 of 9.48 PPM.

4 of 12 CLfl rt99 Point 8reeze Road

~ Flernington, New Jersey 08822

~ TelePhone (908) 788-8700 FAX (908) 788-9!88

RQUFl SURVEY, INC.

I.

Objective The objective of this study was to determine the chronic toxic effects of Betz CT-1 in a marine/estuarine aquatic environment with regard to finfish.

The measure of potency is that concentration, in water, which produces a significant toxic response in the exposed animals after 168 hours0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months (7 days).

II.

Test material Source:

Name:

Density:

4 Solubility in Water:

Betz Laboratories, Inc.

Somerton Road

Trevose, PA 19047 Clam-Trol, (CT-1) 100 Date Received:

March 21, 1991 Amount Received:

1 pint III. Materials and Methods A.

Method The method employed was a modification of the method outlined by The United States Environmental Protection

Agency, Environmental Monitoring Systems Laboratory, EPA-600/4-87/027 May 1988:

II" e-page 1 and the SOP on page B-1 in Appendix.)

B.

Test Organisms

~Sec ice The test species, representing a typical temperate

~

5 of 12 499 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (908) 788-8700 FAX (908) 788-9l65

FIQUR 5UFIVCV, INC.

Size A e sica Co itio Animals used for this test were <24-hour old sheepshead minnows. Animals appeared to be healthy and in good physical condition.

Source Accli t'o All animals utilized in this test were from Aquatic BioSystems, Inc. of Colorado and were received by this laboratory on March 29, 1991.

The animals were acclimated to filtered (0.45 microns)

Manasquan Inlet Water (natural seawater) for 6.5 hours5.787037e-5 days 0.00139 hours 8.267196e-6 weeks 1.9025e-6 months at a moderate changeover rate.

C.

Test System Source of Dilutio Water Water utilized for this test was filtered (0.45 microns) natural seawater obtained from Manasquan River Inlet, Manasquan, New Jersey.

The test temperature was 25 +/-

14 C

Test vessels The test, vessels were 1 liter, 10 x 14 cm glass beakers containing 500 ml of test solution at a

depth of 7.5 cm.

Photo eriod A 16-hour light/8-hour dark photoperiod with a 30 minute transitional period was utilized.

(This constitues a variation from the protocol, which states that a 14-hour light/10 hour dark photoperiod was to be utilized.

~Laadin Each test chamber contained ten organism in 500 ml of test solution.

6 of 12 CL/l rt99 Point Breeze Road

~ Flemington. New Jersey 08822

~ TelePhone (908) 788-8700 FAX (908) 788-9168

RECUR SURV~V, INC.

D.

Test Design Test Levels A range finding test was conducted separately.

Four concentrations of the test material were evaluated using 10 sheepshead minnows per treatment.

An LC50 determination with five concentrations spanning the appropriate range was evaluated using one replicate of ten organisms each per treatment.

The definitive test was conducted using 10 organisms in each of 4 replicates at each of five treatment levels.

Control A control treatment was run concurrently with the test substance treatments using the same dilution water and the same number of organisms as per the test concentrations.

Test Initiatio Testing was conducted under static conditions with daily renewal of the test solutions.

An initial test material stock solution of 1,000 ppm was prepared with distilled water.

Test solutions were prepared from the stock solution and then dispensed. into the test chambers.

The test was initiated when, within 30 minutes of test solution preparation, the test organisms were transferred to the test chambers.

The range-finding test spanned 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months and was conducted using 10 organisms at each of five treatment levels:

0.1, 1.0, 10 and 100 ppm.

Test results indicated a toxic response between 10.0 and 100 ppm.

Exposure at the lower concentration level resulted in no mortality after 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months , while exposure at the higher level resulted in 100% mortality after 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months .

The 24-hour LC50 determination was conducted using 10 organisms in one replicate at each of 5 treatment levels:

6.25, 12.5, 25.0, 50.0 and 100 ppm.

Final test data were used to generate a

24-hour LC50 of 14.64ppm by the Binomial Method.

Results from this test were used to determine the treatment levels for the definitive test, the highest exposure level to be approximately 70% of the generated LC50 value.

7 of 12 CUl A99 Point Breeze Road

~ Flernington, New Jersey 08822

~ Telephone (908) 788 8700 FAX(908) 7889168

R(j)UR SURVEY, INC.

The definitive test was of 168 hours0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months (7 days) duration and was conducted using four (4) replicates of ten organisms each at each of five treatment levels, 0.625, 1.25, 2.5, 5.0 and 10.0 pm, determined based upon 24-hour LC50 data.

Test vessels were examined at each 24 hour2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months interval from test initiation through test termination.

Survival/mortality determinations were recorded for each chamber.

Dead animals were removed and discarded.

C emical Anal ses Concentrations were validated at TO, T23, T73,

T95, T145 and T168 using Clam-Trol, CT-1 methyl orange method (supplied by Betz Lab) in,the range of 0.2 3.0 mg/l.

A Hack Spectrophotometer (DR/2000) or Bausch

& Lomb Spectronic 21 was used for the absorbance readings.

(see appendix, page C-1.)

Ph sical C e ical Parameters The temperature, Ph, dissolved oxygen (DO) concentration and salinity were measured and recorded for each treatment level at the beginning and end of each 24 hour2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months test interval.

Dr in and Wei i At termination of the test, all surviving organisms (by group) were preserved in 70% ethyl alcohol for several

days, at which time each group of organisms from each test chamber were transferred to tared weighing boats and dried at 100' for approximately 14 hours1.62037e-4 days 0.00389 hours 2.314815e-5 weeks 5.327e-6 months .

The organisms were allowed to cool before weighing.

The weight of each group of organisms were divided by the number of surviving organisms for the mean dry weight for each group.

8 of 12 CN rtrzr7 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (F708) 788.8700 FAX (O08) 788->> (55

RECUR SURVEY, INC.

IV.

Results The definitive test resulted in significant mortality at the 10.0 ppm level after 168 hours0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months .

However, there was no significant reduction in mean dry weight observed in any treatment level after 168 hours0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months .

(See Table I and Appendix page D-l).

The test solution temperature was maintained at 25 +/- 1 C.

(See Table II).

Dissolved oxygen was maintained between 4.2 and 6.6 mg/l.

(See Table II).

The range and median values of the pH, DO, temperature and salinity are presented in Table II.

The mean concentration validation before each renewal period at TO, T73, T145 and at the end of each renewal period at T23, T95 and T168 can be seen in Table III.

V.

Source of Documentation All original data documentation is being maintained at Aqua Survey, Inc.,

499 Point Breeze

Road, Flemington, NJ 08822, in Betz Laboratories Notebook 002, pages 3-36.

9 of 12 Clll 499 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (908) 788-8700 FAX (908) 788-9io8

RQUFI SURVEY, INC.

Table I:

Summar Sheet For Shee shead Mi now Larval Su ival a d Growth Test Test Conc Conc.

m 0.0 0.625 1.25 2.5 5.0 10.0 Percent of e l cate Su 100 100 100 100 100 100 100 90 100 90 100 100 100

. 100 100 100 90 100 100 100 50 50 30 50 Mean Survival 100.0 97.5 97.5 100.0 97.5 45.0 GROWT D T Test Conc Conc.

m Average Dry Weight (mg)

Mean I

Re

'cate E

osures Wei ht m

0.0

0. 625 1.25 2.5 5.0 10 '

1.20 1.32 1.28 1.13 1.24 1.21 1.06 1.06 1.50 1e12 1.26 1.53 1.49 1.31 1.17 2.02 1.28 1.22 1.29 1.47 1.12 0.90 1.13 0'8 1'3 1.14 1.35 1.48 1.32 1.03 10 of 12 CLll A99 Point Breeze Road

~ Flemington, New Jersey 08822

~ TelePhone (908) 788-8700 BAx (908) 788.>>

='o

,I s

RQUFI SURVEY, IN(.

Table XI:

Summar of Ph sica Chemical a ameters Conc.

ppm 0.0 (mean)

(range)

Tem erature 24.9 24.5-26.0 H

8.1 7.9-8.2 6.0 5.3-6.6 30.4 30.0-31.0 0.625 (mean)

(range) 24.9 24.5-26.0 8.1 7'-8 '

5.9 5.2-6.6

30. 3 29.5-31.0 1.25 2.50 5.00 (mean)

(range)

(mean)

(range)

(mean)

(range) 24.9 24.5-26.0 24.9 24.5-26.0 24.9 24 '-26.0 8.1 7'-8 '

8.1 7.8-8.2 8.0 7.8-8.2 5.9 5.2-6.6 5.8 5.0-6.6 5.7 4.8-6.6 30.3 30.0-31.0

30. 3 30.0-31.0 30.3 29.5-31.0 10.00 (mean)

(range) 24 '

24.5-26.0 8.0 7'-8 '

5 ~ 6 4.2-6.6 30.3 29.5-31.0 11 of 12 CL/I 499 Point Breeze Road

~ Ftemington, New Jersey 08S22

~ Telephone (908) 788-8700 FAX (908> 7SB-'?'oS

RECUR SURVEY, INC.

Table III Concentration Validation Befo e Renewal Conc.

ppm 0.0 TO X73

~Tl 5 Mean m((ml 0.625 1'5 2.5 5.0 10.0 0.788 1.202 2.353 5.474 11.19 0.559 1.105 2.468 5.239

0. 640

1. 357 2.579 4.936 11.650 0.662 1.221 2.467 5.216 11.420

Sample lost during preparation Concentration Validation After Renewal Conc.

ppm 0.0 T23 T95 T168 Mean m~1 0.625 1.25 2.5 5.0

10. 0

0. 403 1.070 1.858 5.353 9.978 0.256 0.983 1.074 1.785 3.166 0.226 0.357 0.569 0.569 0.913

0. 295 0.803 1.167 2.569 4.686 Summary of Chronic Test Endpoints NOEC LOEC

- 168-HR LC50 5.0 ppm 10.0 ppm 9.49 ppm

~* determined by the Moving Average Method 12 of 12 CLll 499 Point Breeze (todd

~ Flemington. New Jersey 08822

~ Telephone (908) 788-8700 FAX (908) 788-9

-'8

A P

N I

RECUR SURVEY, INt.

PROTOCOL ASX NOs 9100.01 February 21, 1991 CHRONXC TOXXCXTY BXOASSAY FOR SHEEPSHEAD MXNNOlif SPONSOR:

et aborator'es c.

4636 omerton Road T evose A 19047 STUDY NUMBER:

9 -0 9C TESTING FACILITY:

Aqua Survey, Znc.

(ASI) 499 Point Breeze Road Flemington, New Zersey 08822 United States of America PROPOSED START DATE:

March 25 1991 PROPOSED COMPLETION DATE:

A ril 12 1991 STUDY DIRECTOR QA OFFICER:

SPONSOR APPROVAL:

Signature Signa ure

<m (F'j Date J az!0/

Date Date 0/I 499 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (908) 788-8700 FAX (908) 788-91

RECUR SURVEY, INC.

'PURPOSE To estimate the safe or no effect concentration of the test article, using newly hatched larvae in a seven-day, static-renewal test.

TEST ARTICLE:

The test article is

'hich was received from et bs c.

on the test article is clea (description).

The sample is labeled with an ASI number 91-089 Any test article remaining after the test is completed will be disposed of a'ccording to instructions from the sponsor or returned to the sponsor.

TEST ARTICLE CHARACTERIZATION:'ata concerning stability, uniformity, composition and additional chemical/physical characteristics of the test article will be provided by the sponsor on the Test Substance Form (supplied by ASI).

Chemical analyses will be the responsibility of the sponsor unless prior arrangements have been made for ASI to assume that responsibility.

CONTROL ARTICLE None required.

i TEST SYSTEM representing an estuarine/marine finfish.

JUSTIFICATION Sheepshead Minnows are considered the appropriate test system as they are a characteristic system in which the response to toxicological agents can be evaluated.

DXLUTZON WATER Whenever possible, reconstituted sea water will be used.

Reconstituted water will be aged one or two weeks and intensely aerated prior to use.

If use of reconstituted water is not possible, an alternative dilution

water, uncontaminated and of constant quality, will be used.

2 of 7 499 Point Breeze Road

~ Flemington, New Jersey 08822

~ Telephone (908) 788-8700 FAX (908) 788-9>~

RECUR SURVEY, INC SOLVENTS Whenever possible, the toxicant will be introduced into the test solution without the use of solvents other than water.

If alternative solvents are necessary, they will be used sparingly, not to exceed 0.5 ml/1 in a solution.

TEST MATERIAL:

The technical grade, end-use product or both will be used in this study.

The brand name or chemical names and a lot number are used to define a

test material.

Mixtures are defined by ingredient and percent of each ingredient, where possible.

TEST ORGANZSMS for this test.

Newly hatched larvae (less than 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months old) will be used to initiate the test.

The age of the fish will be ascertained from the supplier along with any disease treatment schedule.

I I

I EXPOSURE CHAMBER One (1) liter glass beakers or other appropriate chambers will be used.

I RANGE-PINDING TEST:

I I If the toxicity of the test substance is not already

known, a range finding test will be performed to determine the range of concentrations to be used in the definitive test.

I s

DEPINITIVE TEST:

A minimum of 40 fish per concentration will be exposed to five or more concentrations of the chemical.

Each designated treatment group will be exposed to a concentration of toxicant that is at least 504 of the next highest, concentration, starting at the LC10 for 24 hour2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months range finding test and at the LC50 for 96 hours0.00111 days 0.0267 hours 1.587302e-4 weeks 3.6528e-5 months range finding test.

Test organisms will be divided into four chambers per treatment group (10 fish each).

All organisms will be randomly assigned to test vessels.

3 of 7 CVI 499 Point Breeze Road

~ Flemington. New Jersey 08822

~ Telephone (908) 7888700 FAX (908) 7889'l

RECUR SVRVEY, INC TEST PROCEDURES

'n preparation for the test, the chambers are filled with appropriate volumes of test solution.

The test is started by randomly introducing fish acclimated in accordance with the test design into the test chambers within 1 hour1.157407e-5 days 2.777778e-4 hours 1.653439e-6 weeks 3.805e-7 months after the addition of the test solution.

Fish in the test chambers are observed periodically during the test, the dead fish removed and the findings recorded.

Dissolved oxygen concentration, pH, temperature, and other water quality characteristics are measured at specified intervals in test chambers.

The test solution is renewed daily.

The larvae are fed newly-hatched

~Artemi

~au >~1' daily.

The test is terminated after seven days of exposure.

At termination, dead larvae are removed and surviving larvae in each test chamber are counted and immediate ly prepared for drying and weighing, or are preserved as a group in 704 ethanol and dried and weighed at a later date.

ACCLIMATION:

When. possible, fish will be hatched in diultion water.

If this is not possible the fish will be acclimated to the dilution water over as long a period of time as possible.

The organisms will be observed prior to testing for signs of disease,

stress, physical damage and mortality.

Injured dead and abnormal individuals will be disca'rded Organisms will not be used if they appear to be diseased or stressed or if more than 3 4 die during the 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months immediately prior to.testing.

TEST PARAMETERS e CONCENTRATION MEASUREMENT:

Analytical measurements of the test substance will be made for each concentration at the beginning and end of days 1,

4 and 7 of the test by a method validated before beginning the test by appropriate laboratory practices

~

TEMPERATURE:

The test temperature is 25 + 1'C and will be measured at the end of each 24 hour2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months exposure period in one test chamber at each test concentration and in the contro 1 ~

pH:

The pH will be measured at the end of each 24 hour2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months exposure period in one test chamber at each test concentration and in the control

~

4 of 7 CVI 499 Point 8reeze Road

~ Flemington. New Jersey 08822

~ Telephone (908) "888700 FAX (908) 7889)

RECUR SURVEY, INC SA'LZNZTY'he test salinity will be between 20 and 32 ppt.

The salinity should vary by no more than

+

2 ppt among the chambers on a given day and will be measured at the end of each 24 hour2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months exposure period in one test chamber at each test concentration and in the control.

DZSSOLVED OXYGBNe The dissolved oxygen concentration is measured at the beginning and end of each 24 hour2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months period in one test chamber at each test concentration and in the control.

LZVE COUNT The number of live larvae in each test chamber are recorded daily, and the dead larvae are discarded.

LOADZNG:

The size of the test vessels will be such that the loading factor is no greater than 0.5 g/l.

PHOTOPBRZOD:

A 14 hour1.62037e-4 days 0.00389 hours 2.314815e-5 weeks 5.327e-6 months light and 10 hour1.157407e-4 days 0.00278 hours 1.653439e-5 weeks 3.805e-6 months dark photoperiod with a 15 to 30 minute transition period will be employed.

TEST SOLUTZON RENEWAL The test solutions are renewed daily using freshly prepared solution, immediately after cleaning the test chambers.

For test solution

renewal, the water level in each chamber is lowered to a depth of 7 to 10 mm, which leaves 15 to 204 of the test solution.

New test solur'on should be added slowly by pouring down the side of the chamber to avoid excessive turbulence and possible injury to the larvae.

AERATZON:

Aeration may affect the toxicity of the test solution and will be used only as a last resort to maintain a satisfactory dissolved oxygen concentration.

5 of 7

CIrII 499 Point Breeze goon

~ Rernington. New Jersey 08822

~ Telephone (908) 88-8700 F*x (908) 788-9):

RECUR SURVEY, INC.

PEEDIHG The larvae are fed nettly hatched (less than 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months old)

~Arte la

~u ills once a day from hatch Day 0 through Day 6.

The larvae are not fed on Day 7.

DRYIHG MD WEIGHING At termination, each test chamber is counted and immediately prepared for drying and weighing.

The group of larvae from each test chamber is transferred to a Cared weighing boat and dried at 60'C for 24 hours2.777778e-4 days 0.00667 hours 3.968254e-5 weeks 9.132e-6 months or at 105'C for a minimum of 6 hours6.944444e-5 days 0.00167 hours 9.920635e-6 weeks 2.283e-6 months .

The weight of the group of larvae will be recorded to the nearest 0.01 mg.

ACCEPTABILITY OP TEST RESULTS The test is acceptable if:

(1)

The average survival of control larvae is equal to or exceeds 80%;

(2)

The average dry weight of unpreserved control larvae is equal to or greater than 0.60 mg; or, (3)

The average dry weight of preserved control larvae is equal to or greater than 0.50 mg.

EPPECTS MEASURED:

I The end points of toxicity are based on the adverse effects in survival and growth.

Point estimates, such as

LC1, LC5, LC10 and LC50 are calculated using probit analysis.

LOEC and NOEC values, for survival and growth, are obtained using a hypothesis test approach such as Dunnett's Procedure or Steel's Many-One Rank Test.

REPORT:

The following are included in the final report:

Test Material - Zf technical grade product is tested, the

source, batch and exact purity are reported.

When end-use product is tested, the exact percent of active ingredient and the type of formulation (i.e. granular, wettable powder) are reported.

2 ~

Dilution Water Dilution water source, its chemical/physical characteristics, and pretreatment are reported.

6 of 7

Clll r(99 Point 8reeze Road

~ Ftemington. New Jerzey08822

~ Telephone (908) 88-8700 FAX (908) 788-91

Fl@UR SURVEY. INC Exposure Chambers - The volume, diluent depth, and container construction material are reported.

Test Organisms - Source, quarantine, holding and acclimatization, inclusive of feeding schedules and disease treatment procedures, are reported.

The age and scientific names are reported.

5.

Range Pinding Test - Procedures and results are reported for the range finding study.

Included at a minimum are sample size, concentrations tested and mortality data.

6 ~

Definitive Test - Procedures used to prepare toxicant stock solutions are described.

The method of dosing is reported.

The criteria for determining effects are defined-:

At 6, 24, and 48

hours, raw data or percentage of deaths/effects at each treatment level along with the number of organisms exposed at each level is reported.

Toxic symptoms, both physical and behavioral, observed during the test are reported.

7.

Temperature/DO/pa/salinity

- Temperature, dissolved

oxygen, pH and salinity measurements are reported along with the range and mean of temperature.

8 ~

9.

Chemical Analyses When chemical analyses are made to validate toxicant concentrations, the analytical method is reported as well as the results.

Residue observed in exposure chambers is described in the report.

Testing Protocols This protocol is referenced in the report.

Deviations Deviations from protocol or standard operating procedures are reported.

Certification The Study Director and the QA Manager certify the final report.

RECORDS:

All raw data, data manipulations, drat" reports and final reports will be retained by Aqua Survey, Inc. for a period of five years from the date of completion of testing.

Records will be surrendered to the sponsor upon their written request.

QUALITY ASSURANCE This study will be conducted in accordance with the EPA Good Laboratory Practice Regulations (Federal Register, Volume 48, November 29, 1983) and Short-Term Methods for Estimating the Chronic Toxicity of Effluents and Receiving Water to marine and Estuarine organisms EPA/600/4-87/028 May 1988.

7 of 7 CLll 499 Point Breeze rzoad

~ Ftemington. ivew Jersey 08822

~ Telephone <908) "88-8700 FAx (908> 788-91

STANDARD OPERATING PROCEDURE ASZ

$ 311 PC 4/17/91 K. Cerasoli CHRONIC STATIC DAILY RENEWAL TOXICITY TEST WITH MARINE/ESTUARINE OBJECTIVE:

The objective of the chronic static daily renewal bioassay described in this procedure is to determine the lethal and sublethal toxicity of an 2KUUIIE~

F 1

other marine and estuarine finfish species.

MATERIALS a ~b.

c ~d.

e.f.

goh.

1 ~

k.l.

m.

- )C.

24 1-liter glass beakers 6 4-liter or 6-liter glass mixing chambers 2 2000-ml graduated cylindars magnetic stir plate and stir bars 6 250-ml readings cups 1 30-ml organism transfer cup 1 6-liter organism holding chamber wide-bore disposable pipets graduated glass pipets 1/4 inch inside diameter polyvinyl tubing 1/4 inch outside diameter rigid air tubing 1 thumb clamp n.

glass culture dishes PROCEDURES:

1.

Test Media Preparation A dilution series is chosen based on either a rangefinding tes or on historical data.

A typical concentration schedule for ch onic bioassays is based on a dilution factor of 0.5.

The test solution and diluent temperatures are determined before any further preparations are made.

Temperatures are adjusted as necessary using a water bath or a suitable heater.

The exposure chambers are labeled accoring to concentration and replicate and then rinsed with the appropriate test solution.

Mix'ng chambers are also appropriately labeled and rinsed.

~I SOP 0 311 PC The test treatments are prepared from lowest to highest concentration in accordance with the calculated dilution schedule.

Quantities of test substance and diluent are measured into the appropriate mixing chambers using the pipets graduated cylindars.

Each treatment is agitated to insure complete mixing, and dissolved oxygen is measured at each treatment level.

Test solutions are then dispensed into the appropriate exposure chambers.

2.

Test Organisms In general, larval finfish are hatched into the test diluent and are received from the culturing facility in a holding chamber.

If it becomes necessary to acquire organisms from an outside source, the fish are acclimated to the test diluent using a slow drip method over as long a period of time as is practical.

Fish are transferred to the test chambers with a 30 ml cup or a wide bore pipet, randomly dispensing them in groups of two or three until each chamber has 10 organisms.

Care is taken to insure that the fish are handled gently so as to prevent any undue stress or injury.

Fish that are injured during handling, or those that appear unhealthy or exhibit unusual

behavior, are removed and replaced prior to the initiation of the test.

Test initiation occurs when all organisms have been assigned to the test chambers.

3.

Feeding Test organisms are fed once daily with <24 hour old Artemia nauplii.

Each test chamber is given approximately 0.10 g wet weight of Artemia for days zero through two of the test, and 0.15 g wet weight of Ar"emia for days three through six.

No food is administered on the final day of testing.

5.

Daily Readings At the end of every 24-hour exposure

period, a fifty ml alliquot of test medium is drawn from each replicate at a given concentration level and combined into the appropriate readings cup.

Physical and chemical parameters - temperature, pH, dissolved oxygen and salinity for each concentration level are measured in the readings cups.

B-2

JLSX SOP g 311.PC 6.

Dai3.y Renewals Test renewal is accomplished using either of two methods, as follows:

Method One:

A syphon is made of flushed 1/4-inch air tubing fitted on the forward end with a short length of rigid air tubing.

A thumb clamp is situated along the syphon to adjust the flow rate.

Working from the control treatment

upward, each chamber is drawn down to a final depth of approximately 1/2 inch while thoroughly cleaning the chambers of all detritus and any dead organisms.

Great care must be exercised so as to avoid catching up any of the test organisms with the syphon.

Should an organism become caught up in the syphon, it is carefully returned to the test chamber and a notation of the occurrence and of the organism's condition is made on the bench sheet.

Any organism mortality resulting from the renewal procedure must be duly recorded as such.

Survival/mortality counts and behavioral and morphological obse~at'ons are made at this time.

New test solution is dispensed by carefully pouring the fresh solu"'ons into the exposure chambers so that the solution runs down the

'nne chamber walls.

Method Two:

Test chambers are carefully cleaned of all det itus and dead organisms using a disposable pipet or a syphon.

Each chambe

's then gently decanted into a clean culture dish until approximate'y

/2 inch of liquid remains in the chamber, taking care not to pour oz any of the test organisms.

If an organism is accidently decanted off, it 's carefully replaced into the test chamber and a notation of the occurrence is made on =he bench sheet.

Counts, observations and test solution additions are accomplished as Method One, above.

7.

Test Termination The test is terminated at the end of the seven day exposure pe 'od.

Final physical and chemical measurements are made, the test solu"'ons are drawn down to a minimum volume and final survival/mortality counts are recorded.

The organisms in each test chamber are sacrificed and prepared as a group for dry weight determination or are preserved or later analysis in a 704 ethanol solution.

B-3

ASX SOP

$ 311 PC 8.

Drying and Weighing Immediately prior to dry weight analysis, each group of organisms is

~

transferred to a

tared weighing boat and dried at 100 degrees centigrade for a minimum of six hours.

Upon removal from the drying oven, the weighing boats are placed in a dessicator until they are to be weighed.

All weights are determined to the nearest 0.01 mg.

Xf the test organisms are preserved prior to analysis, they must be weighed within two weeks of test termination.

CLAM-THOL CT-5 METHYLORANGE METHOD APPARATUS REQUIRED Beaker, glass, 50 mL (2 required)

Code Cylinder, graduated, 25 mL 2622 Funnel Rack, separatory 936 Funnel, separatory, with a Teflon stopcock, 250 mL i~

(2 required)

Glass Rod 114 Optical Cell, (2 required)

Spectrophotometer GENERAL APPARATUS i Code 917 CHEMICALS REQUIRED 1, 2-Dichloroethane (reagent) grade or equivalent)

Code 1666 Cylinder, graduated, 100 mL Cylinder, graduated, 250 mL Flask, volumetric, 1 L, glass (4 required) 935 Pipet, glass, graduated.

1 mL 140 Pipet, glass, volumetric, 1 mL 866 Pipet, glass, volumetric, 3 mL Plpet, glass, volumetric, 5 mL 124 Plpet, glass, volumetric, 10 mL 123 Plpet, glass, volumetric, 15 mL 861 Pipet, glass, volumetric, 20 mL Pipet, glass, volumetric, 25 mL 117 Plpet, glass, volumetric, 30 mL The general apparatus required for the test Is deter-mined by the specific test procedure used.

~~ Apparatus not available through Betz Lab Supply should be obtained through a local supplier.

CT-1 Buffer Reagent Methanol (reagent grade or equivalent)

Drying Reagent, with a plastic dipper

SUMMARY

OF METHOD 1591 1271 GENERAL PROCEDURE Use a well-ventilated or hooded area to run the test.

Always use a safety bulb when pipetting liquids.

1,2 - Dlchloroethane (also known as Ethylene Dichlo-ride) is a priority pollutant and a specifically-listed RCRA<egulatedmaterial subject to specific disposal re-strictions and/or prohibitions. For this reason, all used 1,2 - dichloroethane should be segregated from other waste streams. Dispose ofwaste 1,2-dlchloroethane in an approved manner (e.g., labpacking or incineration).

ln this procedure the dye in the CT-1 BufferReagent com-plexes with the active ingredients in Clam-Trol CT-1. This complex is extracted into 1, 2 - dichloroethane. The or-ganic layer containing the complex is separated fromthe aqueous layer and driedwith a drying reagent containing anhydrous sodium sulfate. The color intensity of the 1, 2 dichloroethane layer is then measured in a spectropho-tometer at 415 nm.

This method must be customized to each specific appli-cation. Vary the volumes of sample, CT-1 BufferReagent, and

1. 2 - dichloroethane according to the test range (see Table 1). Ifa higher absorbance is needed. increase the volume of sample or decrease the volume of 1, 2-dICnloraethane. When increasing the sample volume it may oe necessary to increase the volume of CT-1 Buffer Reagent used. For samples <150mL use 10mL of CT-1 Buffer Reagent; for samples between 150 and 300 mL use 15 mL of CT-1 Buffer Reagent.

Make sure that enough 1, 2 - dichloroethane is used to leave a small plug of solvent ln the separatory funnel when the bottom layer of solvent is removed and to fill the optical cell properly.

AP 368 9003

~1990 BETZ LABORATOAIES.INC. ALLRIGHTS RESERVED.

C-1

1.0 - 25.0 0.2-1.0 10 15

'Mle 1. Suggested Volumes for Various Ranges of CT-1.

Range Volume Volume CT-1 (mg/L)

CT-t Buffer Dlchloroethane (mL)

(mL) 0.2-3.0 15 10 Volume Sample (mL)

Optical Cell Size 1.0 cm 2.5 cm>>>>

50cm'>>>>

The1.0-cm cell (Code 1312) can be used with Hach spectrophotometers using a I~ cell adapter (Code 2776C).

>> The 2Am cell is the standard Hach 1-in. cell (Betz Code 2601).

>>*>> Five centimeter cells are not available for use with the Hach photometers. Many laboratory spectrophotometers require an adapter to accommodate &cm cells. Check with the instrument manufacturer.

1.

Transfer an aliquot of the water sample to a separa-tory funnel (the sample). Transfer the same volume of distilled (or deionized) water to a second separa-tory funnel (the blank). Run the blank once for each set of samples tested (see Notes 1, 2, and 3).

2.

Add CT-1 Buffer Reagent to both the sample and the blank.

3.

Using a pipet, add 1, 2-dlchloroethane to both se-paratory funnels.

4.

Insert the stoppers In each ofthe separatory funnels.

Invert and briefly open the stopcock to vent the fun-nels (see Notes 4 and 5). When venting the funnels, point the tip of the funnel away from yourself and others.

5.

Shake the funnels moderately for 30 sec, vent the funnels, then allow them to stand for 10 min (but no longer than 15 min).

6.

Collect the lower layer (1, 2 - dlchloroethane) from each funnel in RHnL beakers leaving about 1-2 mL in the funnel. This willprevent significant removal of water.

7.

Using the plastic dipper, add 2 scoops of Drying Re-agent to eachbeaker and stir with a glass rod for 15 sec (but no longer than 30 sec).

8.

Wait approximately 1 to 2 min (but not more than 5 min). Then carefully decant the extract offof the dry-ing reagent into an optical cell.

9.

Set the spectrophotometer at 415 nm and zero with 1, 2-dlchloroethane. Measure andrecord the absor-bance of the blank and the sample (see Note 6).

10. The sample absorbance minus the blank absor-bance is used to determine the concentration of CT-1 in the sample.

From a prepared calibration curve, determine the CT-1 concentration in the sam-ple (see Calibration Cwve Preparation).

11. Clean the cells after each measurement (see Note 7).

CAUBRATIONCURVE PREPARATION 1.

Prepare a 1000 mg/L CT-1 stock solution by accul rately weighing 1.00 g of CT-1 into 1 Lof distilled (or deionized) water.

2.

Pipet designated volumes of the stock so!ution into t-Lvolumetric flasks. These are the stanaaro solu-tions used in preparing a calibration curve. Use Table 2 to make appropriate dllutions of ee stock solution for each specific app! Ication.

3.

Followthe General Procedure using the specific so-lution volumes that have been determined tor the application and prepare a calibration curve. Oeter-mine the absorbance of a blank solution using dis-tilled (or deionized) water. Th!s blank can oe sub-tracted from the sample absorbance or used to zero the spectrophotometer so that the calibration curve goes through the origin. The calibration ~ive should be linear over the indicated ranges.

NOTES 1.

For maximum accuracy the calibration curve snould be checked by every operator using inis esi and should be verified a minimum of twice per mone us-ing a freshly prepared CT-1 standard.

2.

Ablank measurementmustberecorded for eacn set of samples.

The blank reading may vary siigntly; however, the absolute difference between ee sam-ple and the blank remains relatively constant.

3.

Chlorine causes a negative inter',erence in the test.

This can be eliminated by adding 0.1 N Soaium Thiosulfate (Code 235) to the water sampie oetore running the test. The amount added be is based on the concentration of chlorine in the system.

For a I0&mLwater sample containing 0.3 mg/L chlorine, add 10 drops of 0.1 N Sodium Thiosulfate to remove the interference.

C-'2 AP 368 9003

Concentration CT-1 Desired (mg/L)

CT-1 Stock Solu-tion Added to Make 1 L (mL) 0.2 0.4 0.6 0.8 1.0 5.0 10.0 15.0 OZ 0.4 0.6 0.8 1.0 5.0 10.0 15.0

'thhte 2.

DHutfons for CaGbratfon Curve Prepara-tion Based on a Final Solution Volume of1L 5.

6.

7.

procedure. Shake the funnel for30 sec, vent It, then allow It to stand for 5 min. Gently Invert the funnel once then allow the funnel to stand for 5 min.

It ls Important to vent the separatory funnel both be-fore and after shaking it. Otherwise, a pressure will build up ln the funnel that can cause the stopper to be forced out of the top of the funnel.

Use caution when inserting or removing the sample cell In the photometer. The 1, 2-dfchloroethane can damage the cell compartment.

lt ls imperative that the sample cells are kept clean during the tunning ofthe test. ItIs recommendedthat the cells are cleaned after each measurement using the followingprocedure:

a)

Rinse the cell three times with distilled (or deionized) water.

b)

Rinse the cell three times with methanol.

20.0 25.0 20.0 25.0 c)

Rinse the cell three times with 1, 2 - dlchloro-ethane to remove methanol from the cell.

4.

Aslight emulsionmay formwhenusing natural water samples.

When this happens, vaIy step 5 of the 8.

Thlsmethodlsbasedupon Wang, L K.; Langly, D.F.

Ind. Eng. Chem., Prod. Rea dev.,

1975, 14, 3, 210-212.

AP MS 9003

~1990 BETZ LA8ORATQRIES,INC. AIJ. RIGHTS RESERVED.

C-3

91-089c Betz C. variegatus, survival 1

Pile: 91-089c.cvs Transform:

ARC SINE(SQUARE ROOT(Y) )

SUMMARY

STATISTICS ON TRANSFORMED DATA TABLE 1 of 2 IDENTIFICATION N

control 0.625 1'5 2 '5.0 10 '

MZN 1.412 1.249 1.249 1.412 1.249 0 '80

1. 412

1. 412 1 ~ 412 1.412 0.785 1 ~ 412

1. 371 1-371 1.412 1'71 0.734 91-089c Betz C. variegatus, survival File: 91-089c.cvs Transform:

ARC SINE(SQUARE ROOT(Y) )

SUMMtMY STATISTICS ON TRANSFORMED DATA TABLE 2 of 2 GRP IDENTIFICATION control 0.625 1'5 2.5 5.0 10.0 VARIANCE 0.000 0.007 0.007 0.000 0.007 0 011 SD 0.000 0 F 081 0.081 0.000 0.081 0.103 0.000 0 '41 0 '41 0.000 0 '41 0.051

91-OR9c Betz C. variegatus, survival Pile: 91-089c cvs Transform:

ARC SINE(SQUARE ROOT(p))

Shapiro Wilks test for normality 0 '92 0.712 Critical W (P = 0.05)

(n = 24)

= 0.916 "ritical W (P = 0.01)

(n = 24)

= 0.884 Data FAIL normality test.

Try another transformation.

Warning

The two homogeneity tests are sensitive to non-normal data and should not be performed.

91-089c Betz C. variegatus, survival File: 91-089c. cvs Transform:

ARC SINE(SQUARE ROOT(Y) )

Hartley test for homogeneity of variance Bartletts test for homogeneity of variance e two tests can not be performed because at least one group has variance.

Da a FAIL to meet homogeneity of variance assumption.

Additional transformations are useless.

D-2

91-089c Betz C. variegatus, survival Filei 91-089c.cvs Transform:

ARC SINE(SQUARE ROOT(Y))

STEELS MANY-ONE RANK TEST Ho: Control<Treatment 1

2 3

4 5

6 P

IDENTIFICATION control 0.625

1.25 2.5 5.0 10 '

TRANSFORMED MEAN

1. 412

1. 371

1. 412 1 '71 0.734 16.00 16.00 18.00 16.00 10.00 CRIT.

VALUE 10 F 00 10.00 10.00 10.00 10.00 4.00 4.00 4.00 4.00 4.00 SIG Critical values use k = 5, are 1 tailed, and alpha

= 0.05 D-3

SAMPLE.ZD: 91-089c Betz C. variegatus 168 hour0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months LC50 NTRATION 10.0000 5.0000 2.5000 1.2500 0 '250 NUMBER EXPOSED 40 40 40 40 40 NUMBER AFFECTED 22 1

0 1

1 PERCENT AFFECTED 55.0000000 2.5000000 0.0000000 2.5000000 2.5000000 BINOMIAL PROBABILITY

(~)

31.7914000 0 0000000 0 0000000 0.0000000 0.0000000 RESULTS METHOD SPAN EC50 95 PERCENT CONFIDENCE LIMITS BINOMIAL"'.487959 5.000000

0.000000 MOVING AVERAGE PROBZT PROBZT 0.114173 5.428608

8. 334561 -

11. 651390 9.487959

(........................NOT VALID.....................)

INTERPRETATION OF STATISTICS The order of preference of results is:

BEST:

Probit Method BETTER: Moving Average GOOD:

Binomial GOOD:

Graphic Interpolation (Graph drawn by hand) assuming all are statistically valid.

To Determine if a method has given valid results:

GRAPHICAL:

Always valid BINOMIAL:

The program will tell you if this is not valid.

MOVING AVERAGE: The number of spans needs to be no less than one lower than the number of concentrations tested (excluding control).

PROBIT:

The program will tell you if this is not valid D-4

EPA PROBIT ANALYSIS PROGRAM USED FOR CALCULATING EC VALUES Version 1.4 9

89c Betz C. variegatus 168 hour0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months LC50 Number Conc.

Exposed Number Resp.

Observed Proportion Responding Adjusted Proportion Responding Predicted Proportion Responding 0.6250 1.2500 2.5000 5.0000 10.0000 40 40 40 40 40 1

1 0

1 22 0.0250 0.0250 0.0000 0.0250 0.5500 0 0250 0.0250 0.0000 0.0250 0.5500 0.0009 0.0083 0.0469 0.1689 0.4047 Chi Square Heterogeneity

=

37.595 WARNING Significant heterogeneity exists.

The results reported for this data set may not be valid.

The results should be interpreted with appropriate caution.

4*****************************************************

NOTE Slope not significantly different from zero.

EC fiducial limits cannot be computed.

Mu Sigma Parameter 1 ~ 101140 0.419530 Estimate Std. Err.

954 Confidence Limits Intercept Slope 2.375300 1.399709

(

-2.078572, 2.383621 1 743007

(

-3.162628, 6.829173) 7.929870)

Theoretical Spontaneous

Response

Rate

= 0.0000 D-5

91-089c Beta C. variegatus 168 hour0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months LC50 Estimated EC Values and Confidence Limits EC 1.00 EC 5.00 EC10.00 EC15.00 EC50.00 EC85.00 EC90.00 EC95.00 EC99.00 Conc.

1. 3341 2.5766 3.6599 4.6381 12.6223 34.3511 43.5321 61.8336 119 '241 Lower Upper 954 Confidence Limits D-6

91-0&9c Betz C. variegatus 168 hour0.00194 days 0.0467 hours 2.777778e-4 weeks 6.3924e-5 months LC50 PLOT'OF ADJUSTED PROBITS AND PREDICTED REGRESSION LINE P

it 9+

~

0 3+o 0

EC01

+

<<<<<<<<<<+

<<<<+

<<<<<<+

~

+<<<<+

EC10 EC25 EC50 EC75 EC90 EC99 D-7

91-089c Betz C. variegatus dry weight File: 91-089c.cvw Transform:

NO TRANSFORMATION

SUMMARY

STATISTICS ON TRANSFORMED DATA TABLE 1 of 2 1

2 3

4 5

IDENTIFICATION N

control 0 '25 1.25 2.5 5.0 MIN

1. 130 1.060 1.120 1'70 1.220 1.320 1.240 1'30 2 '20 1.470

1. 233

1. 142 1.353 1'48 1.315 91-089c Betz C. variegatus dry weight File: 91-089c.cvw Transform:

NO TRANSFORMATION

SUMMARY

STATISTICS ON TRANSFORMED DATA TABLE 2 of 2 GRP IDENTIFICATION control 0.625 1.25 2.5 5.0 VARIANCE

0. 007 0.009 0.039 0.123 0.012 SD

0. 085

0. 096

0. 197 0.351 0.108 SEM 0.042 0.048 0.098 0 ~ 176 0.054 D-8

91-089c Betz C. variegatus dry weight

.File: 91-089c.cvw Trans form:

NO TRANSFORMATION Shapiro Wilks test for normality 0.570 W =

0 '31 Critical W (P = 0.05)

(n = 20)

= 0.905 Critical W (P = 0.01)

(n = 20) 0.868 Data PASS normality test at P=0.01 level. Continue analysis.

91-089c Betz C. variegatus dry weight File: 91-089c.cvw Transform:

NO TRANSFORMATION Bartletts test for homogeneity of variance Calculated B statistic

=

8.15 Table Chi-square value

=

13.28 (alpha

= 0.01)

Table Chi-square value =

9.49 (alpha

= 0.05) rage df used in calculation

==>

df (avg n - 1) for Chi-square table value==>

df (regroups-1) 3.00 4

Data PASS homogeneity test at 0.01 level. Continue analysis.

NOTE: If groups have unequal replicate sizes the average replicate size s

used to calculate the B statistic (see above).

91-089c Betz C. variegatus dry weight Pile: 91-089c. cvw Transform:

NO TRANSFORMATION ANOVA TABLE NcE Between Within (Error)

Zotal DF 15 19 SS 0.368 0.570 0.938 0.092 0.038 Critical F value =

3.06 (0.05,4,15)

Since F ( Critical F FAIL TO REJECT Ho:All groups equal D-10

~l

<~ 91-089c Betz C. variegatus dry weight

'.ile:

~ 91-089c. cvw Trans form:

NO TRANSFORMATION DUNNETTS TEST

TABLE 1 OF,2 Ho:Control<Treatment G

P IDENTIFICATION TRANSFORMED MEAN MEAN CALCULATED IN ORIGINAL UNITS T STAT SIG 1

2 3

4 5

control 0.625 1.25 2.5 5.0

1. 233 1.142 1.353 1.548 1.315 1.233 1.142 1.353 1.548

1. 315 0.653

-0.871

-2.285

-0.599 Dunnett table value =

2.36 (1 Tailed Value, P=0.05, df=15,4) 91-089c Betz C. variegatus dry weight File: 91-089c.cvw Transform:

NO TRANSFORMATION DUNNETTS TEST

TABLE 2 OF 2

Ho: Control<Treatment GROUP IDENTIFICATION NUM OF REPS Minimum Sig Diff 4 of DIFFERENCE (IN ORIG.

UNITS)

CONTROL FROM CONTROL control 0.625 1'5 2.5 5.0 0.325 0.325 0.325 0 '25 26.4 26.4 26.4 26.4 0.090

-0.120

-0.315

-0.083

ML17223B310

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ML17223B310

Text

SUMMARY

SUMMARY

Response

SUMMARY

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