| | l IK asean, ASEAN-Canada Cooperalive Programme ‘on Marine Science ~ Phase It Prepared for: Prepared by: tc 7 Canada swoi7AiC: 3lu% CANADIAN INTERNATIONAL, DEVELOPMENT AGENCY CANADA ASEAN CENTRE PROJECT STEERING COMMITTEE CANADIAN EXECUTING AGENCY‘TABLE OF CONTENTS Page No. Title Page i Table of Contents i List of Tables : ii List of Figures it 1.0 INTRODUCTION «2... beste : 1 20 ‘TERMINOLOGY AND DEFINITIONS. feteeeeee . 1 3.0 QUALITY ASSURANCE/QUALITY CONTROL... sees 6 SAMPLE COLLECTION AND HANDLING . . 8 Sample Cotlection . . > a Sample Shipment and Storage... 9 Chain-of-Custedy and Sign-in Procedures 2 5.0 TEST SYSTEM : 10 Ba Facilities and Equipment |...” 10 52 Water Supply 02... 1" 53 Lighting... " 5.4 Temperature... 12 55 Cleaning Procedures... 2 56 Test Containers... 1. 12 87 Aeration . nt 13 6.0 TEST ORGANISMS ..., $s 6a Species eee. 14 62 Souree 2200000 II 16 63 Acclimation and Handling... ma 14 84 Feeding... se... fee ta ‘20 TEST PROCEDURES , 18, A Experimental Design . 18 72, Test Conditions... 22. 18 ' ds ‘Test Solution Preparation . 7 : 74 Beginning the Test... 20 ‘zs Dally Observations ||. 1.) 2.23 76 Test Termination... fees 23 80 DATA ANALYSIS... theese beeeeeees 25 2.0 REPORTING AND RECORD KEEPING . cite 26 10.0 REFERENCES . cteeeees we +. 29 APPENDICES APPENDIX A Datasheets for acute toxlctty tests, APPENDIX B Outline for specles-specttic lest detalls, sestous pian omnes soa aeUST OF TABLES Page No. Table 1. ‘Summary of recommended test conditions for ASEAN acute toxicity tests. 16 > List oF FiGuREs : Figure 1. ‘Sample datasheet for summarizing test data. 2 Figure 2. Sample datasheet for recording acute toxicity test data. 2 | Figure 8. Example ealuiaton of 9h LCS0 valu by grapiea! metiod 28 : Figure 4, Example calculation of 96-n LCS0 value by computer program. 2 saseuscona ome few ies SS1.0 INTRODUCTION ‘The purpose of this document isto provide a detailed methodology for performance ol static acute toxicity tests with marine organisms. The purpose of these tests is to determine 96h LCSO values (ine Concentration of material 1hat fs lethal to 50% of the test population). The generic protocol will serve as 2 framework for method development in the ASEAN region, Specific details pertinent to individual test species will be provided in separate appendices, This document is based on information and methodologies previously established by other agencies in other jurisdictions (e.9., Environment Canada 1990; EPA, 19912; Relsh and Oshida, 1986; ASTM, 1992; APHA, 1992; UNEP, 1988). itis intended thar Unis document wil evolve ané be adopted as a standard method for appication within the ASEAN region, ‘This protocol is intended tor use In testing effluents, receiving waters, chemicals and chemica formutations using marine organisms, . 20 ‘TERMINOLOGY AND DEFINITIONS WORDING ‘Certain words (‘must, ‘should, ‘may’, “cant and ‘might’ used in this protocol have specific meanings. ‘The following datintions are used (ASTM, 1992): ‘aust’ fs Used 10 exproes an absolute requirement, that is, to state that the test ought to 2 designed to satisty the spectied condition, uniess the purpose of the test requires a 90% saturation ater should be analysed periocicaly for ammonia, total organic carbon, suspended solids, Pesticides and any ether contaminants of concern, !naddition to hot and cold run fequired for use in clean: ‘The quality of this water ring water, an adequate supply of cistiled of deionized ing glassware and equipment, and Should also be monitored regulary, water will also be '0 preparation of chemical stock solutionsaR 12 {or testing within ASEAN. 5.4 Temperature Constant temperature conditions must be maintained throughout the toxicity tests, either by use of water baths or constant environment chambers, The test temperature should be 27 = 4"C, There should be Some means of monitoring temperature fluctuations over a 24-h period, either by use of @ maximum. minimum thermemeter, or with a continuously recording thenmograph, Accimating test organisms shouts Rot be subjected to more than a S*C/day temperature change, and should be held at ine test temperature for at least 48 h prior 16 Dagianing a toxicly test 85 Cleaning Procedures All glass and plasticware which will come in contact with test organisms or test solutions must be thoroughly cleaned prior to use. New plastieware should be soaked in clean seawater for at least 24 n Prior to use, All glassware must be cleaned by the following procedure (EPA, 19812) prior to use: + sea 18 min int naterand wasn wih trent + tinse twice with tap water + rinse once with 10% nitric acid ((0 remove scale, metals, bases) + finse twice with deionized water + finsa ones with ful-strength, pesticide-grade acetone (to remove organics) + finse thee times with detonized water. Rinse all equipment in dilution water just prior to use. 5.6 ‘Test Containers The exact shape of the test container is net important, but all containers used lor a test must be identical The container dimensions must provide adequate test solution depth, Glass is the preferred material lor fest containers, Size of the test organisms will determing the appropriate size of test containers. Various secommonded for these tests. sassann meg bare dont,13 87 Aeration sade Test solutions should not be aerated lnles the dissolved oxy gen concentration falls below 40% saturation Guring the eSt, Air should be delivered by means of cib{cee pumps: this may iavoNe the use of one large Compressor or many small aquarium pumps, A disposable glass pipette lied to an aitine should ue used to provide gentle aeration to each test Contdiner. The pipettes are replaced aker each test. The Si Supply should be adjusted so that the flow of alr does not disturb the test organisms, If aeration ie ‘quired. K must be provided to all treatments for @ particular test and must continue for ine Guration ot the test.4 6.0 TEST ORGANISMS 64 Species ‘This Generic protocols intended for use with juvenite Stages of fish and invenebrate species, Proposed Species for inital testing are jwvenite seabass (Lares Celcaries) and juvenile pray (Peneaus monodon). Species-specitic requirements are Provided in separate appendices. 62 Source Test organisms may be eutured Ia the laboratory, collected com its Dalcheries. A consistent, ciable source is essential ara the Collection site location must be known, If Mortality should not exceed 10% duri Holding containers and transfer equipment must be Kept clean, and shouig be bateh of organisms is received,15 1.0 ‘TEST PROCEDURES: TA Experimental Design When no information is available about the expected toxicity of a sample, a rangelinder test is ‘ecommended, This is especially appropriate for testing chemical formulations, The rangetinder test Usually uses fewer organisms and smaller test volumes than a definitive test and may not run tor the full 95h duration. A series of 4 - 5 concentrations is generally prepared, each ditfering by a factor of 10. 4 Second rangefincing test working within 1 to 2 orders of magnitude may be required for resaarch-oriented Projects where a precise LCSO estimate is required. The purpose of the rangefinder testis to determine ‘Re upper and lower limits of concentrations to test when perorming the detiniive toxicity test. ‘Where preliminary information fs required for a large number of samples, or where a sample has Previously Been shown to be non-toxic tothe test organisms, a simple screening test volving a single test concentration may.be adequate, For effluents or receiving waters, this is usually 100% (vat al) Definitve tests use a series of at least five concentrations f test material to determine the 98-h LCSO value. The LCSO value Is the concentration of material that digits @ lethal response in $0% of the test Population, To determine the LOS0 value, at least one concenttation should demonstrate more than a 65% response and atleast one other should demonstrate less than a 35% response. Selecting a concentration ‘ange that provides two or more partiat responses (nol 0 oF 100%) is desirable, The series of dilutions should be geometric (2.9., 6.25, 12.8, 25.0, 50.0 and 100.0 oF 1.0, 1.8, 82, 5.6 and 10.0), so that ihe Concentrations are evenly spaced when ploted on a logarithmic scale, Unis of concentvation may range from percent to mg/L or lower. . ‘The test end-point(s) must be identified, as Part of the experiment’s hypothesis, before the ‘study is intiated, Death is the adverse etfect most often used in acute toxicity tests with aquatic organisms. The Stealer deat are usualy ERRNO speci the SERMEMRERDIMEEY movements in ish and shrimp, andfSERBISESEVOHRS ‘Other elfecis can de used as end-points, but these ‘must be well delined and reported. 72 Test Conditions Recommended test conditions are summarized in Tabte 1.Table 1 1 Test type Static “| 2 Satiny 302 2 ppt ie) 9 Temperature aero Light quatity Ambient laboratory itumination 5. Photoperiog 14:10 h light:darkness & Test chamber size 20 glass aquaria 7 Test solution volume wOL 8 Renewal of test concentrations Rone Aae/size of test organisms Juventes; 2 em tong 10. Larvaeftest chamber and control 10 "1. Replicate chambers/cdiccenvation 3 (minimum 2) . 12 Feeding regime none during test (depends on species fequirements) 13. Aeration None, unless D0 is <40% of saturation, then deat® all chambers. Rate should be less than 100 bubbies/min, 14, Dilution water Clean seawater 18. Test concentrations Atleast § and a control 18. Dilution tactor 85 dition series of logarithmic series (10, 1.8, 3.2, 86, 10.9) 17. Test duration 96h 18. Effects measured Survival 19:__Test acceptabitiy criterion 390% control survival17 73 ‘Test Solution Preparation General Requirements Prepare test solutions immediately prior to test intaton, 2 Have enough diution water available (at test temperature) to prepare all the controls and “realments for @ panicular test series, All treatments in atest en where, ©, = intial concentration of sample or stock, solution 1 = volume of sample or stock solution to ada Cx = Gesirad test solution concentration Vz = test solution volume Example 1 wo oaDMeD fet tock3. 4 Te prepare a :L volume ofthe 28.0 mai. Cd treatment fom a 1.000 mg/L. Cd stock solution, the “equation would be: it the sample is stored in more than one Container, and has not previously been composited, Composte the entre sample before preparing test soktione Gently stir the sample to distibute Sny material vnhich has setled. Aker composting, unusea ‘sample may be returned to the storage containers, Measure and record the dissolved oxygen (00) Soncentration, pH and salinty of the sample, "the DO ls <40% or > 100% saturation, brielly aerate the sample (maximum 30. min) until the DO Concentration reaches this acceptable range, ‘adusied sample, Adjust the pH by addition of 1N Hor or NaOH. eeu nem ome fone_ o water, Preparation of Chemical Formulations 1 Chemicals must be in solution, or evenly dispersed, Uniess very high concentrations of a Chemical re being tested, the use of stock solutions is recommended rather than addition of the pre-welghed chemical directly to the test container, Prepare stock solutions with deionized water, rather than dilution water. Pre-weighing the Chemical, using an analytical balance, and adding it to a known volume in a volumetiic flask is the most accurate way to prepare a stock solution; the concentration | then expressed on a weight-per-votume basis (a.g,, mg/).20 ‘brought to volume with deionized water to make the stock solution. I! a solvent is required, an Additional solvent control must be tested concurrently. The solvent control consists of di water plus an amount of solvent equivalent to that in the highest test concentration, 14 Beginning the Test ‘Sesgiable all equipment required to conduct the test, Pian to begin tests earry in the day to allow time to make early observations of survival and monitor possible changes in dissolved oxygen concentration. Confirm test organism haalth, abundance and size before preparing test solutions: test volumes must meet loading density requirements for the test organism. Prepare datasheets, Examples of datasheets used to record and summarize data recorded ‘uring @ 96-h LCSO test are provided in Figures 1 and 2. Blank versions of these datasheets are provided In Appendix A, Calibrate water quality instruments, according to manutacturer's instructions. Rinse pre-cleaned test containers in dilution water and tabel them as to sample identification, treatment and replicate, Prepare solutions as described in Section 7.3. Distribute the test solutions to each test container and then randomize the positions of the containers. Record the container size and test solution volume on the datasheets, Measure temperature, dissolved oxygen, pli and sainity in each test container and record this: Information on the datasheets, ~ Randomly disvibute 10 organisms to each test container. This should be done by adding one © two organisms to each container, and then repeating this process until each holds 10 organisms. Discard any organisms which do not appear healthy, of which are accidentally ‘dropped or touch dry surfaces during transfer. Size of the test organisms should be as uniform as possible, The largest organism should not be more than twice the length of the smallest one: ‘Contitm that each container has exactly 10 animals in k. Se Sach conteiner has exactly 10 animals in | Set aside an addtional 10 randomly selected organisms for lengthfweight measurement to contiem mean test organism size, ©} Do not feed the organisms during the test,at ACUTE TOXICITY TEST OATA SUMMARY heen ‘Analystis) Ant Project LsSs * O/.11 (iad) . ‘work Oraer # #90067 Test Type z Te Test initiation Date zy 1, 1999 SAMPLE Ienitieation ammount faceied 208 wf ate Cotected abiceenlec 38. 194§ ate Received stdin BL MER pH 3 DissoWves Onygen fait) gS onduesvey emhesiemy =) — Omnar ———_____ ‘SILUTION AND CONTROL MEDIUN TEST SPECIES INFORMATION . Source Mubdwwate: Leb 2 orguvn — Paoroden eH 22 Sowee Atel, Ase Dissolved Oxygen mgey a Colection Date/Baich _Regnsbar 10,1958 Sati (op) anton ony Other = _ Parca toxicant “Bu 80 ‘Curent Raterance Presrenmment igs Mbalincaad ‘organt Resut Ay agll ta ht theitgalinn Laboratory Range tor Relerence Tbxicart (mean = 250) 502 0.29 ‘TEST coNDImioNS. Temperature Range (10) ——st____ PH Range 3t-F0 Dissaived Oxygen Range (mg/L), FO-2-6 ‘Satinily Range (ppt) 30-3) Awation et sepa Pactopeiod 9:0 5 ee ‘No, Orgaismayvohine 102/06. Other Figure 1. Sample datasheet for summarkzing test data, Sassari ones soery same 4 moxmnvuvs TT orm 9 wrocus san Cp “sms Ry umn VaVG 1831 ALOKOL ALTVHLT BLNOY ‘Sample datasheet for recording acute toxicity test data, Sesser W129 oan os sere Figure 2.10. n 12, 75 78 23 Mt possible, cheek ies at 1, 2 and 4 h intervals alter test initiation. Organisms are Considered dead iLthey shew no signs of movement and do not respond to gentle prodeing, Movement of test chambers and prodding must be done very genlly. so as nol to stress live Organisms. Because of the potential for cannibalism, alvays report monalities in terms of the percentage of survivors remaining in each container, Uithe dissolved onygen concentration is going to decrease below a crtcal level. this wil usually happen uring the first few hours of the test. Check the dissolved oxygen ater several hours; intlate gentle aerationit it crops below 40% saturation. I aeration is requiced, it must be provided (0 allteatments and continued for the duration of the test. Record the time aeration was iated. A positive (toxic) control test should be initiated with a standard reference toxicant compound, to assess the relative health and sensitivity of the batch of test organisms. The test procedure is the same as that used to test the sample, Dally Observations ‘Temperature, dissolved oxygen and pH In each treatment must be measured and recorded every 24h. For a static test, itis only necessary to measure salinity at 0 and 96 h. ‘The number of mortalities in each test container must be recorded every 24 h, or more otven i appropriate. Monalities must be repaced minimized, Observations of unusual behaviour by the lest organisms should be recorded, These ‘observations should include such things as loss of equilibrium, erratic ‘swimming, toss of reflex, exctabilty, dlscoloration, changes in behaviour, hyperventiation, haemorthagi 19. motting or cannibalism. 2ch day. Disturbance of the remaining live organisms should be ore Irthe dissolved orygen concentration in any treatment als below 40% saturation, gentle aeration Imus Be inated in all eatments forthe duration ofthe test. The time that aeration was inate must be recorded. ‘Test Termination For the test to be considered vaid, mean survival in the negative contol must be 290%, Water24 Quality measurements (pH, temperature, dissolved oxygen, salinity) should remain within acceptable timits. 2 After 96 , record survival in each test container. For dark er opaque solutions, it may be necessary to carefully pour the contents of the test container through a net to contirm the number of surviving animals. 3 Record temperature, pH, dissolved oxygen and salinity in each test container, Dispose ol test organisms and solutions by appropriate means. Surviving test organisms must be sacrificed; they are never re-used. watson waa ome Aen we8.0 DATA ANALYSIS Fit a straight line by eye, with greatest RRMA on the points betwer Ira! BPPrOach arrows tor 0 and 100% mortal Rta shoule ‘not pass through them. if there is doubt about Other Methods Sawired, tho use of software packages designed to calculate LCSOs is recommended. Among these methods are parametric (probit and inethods), non-parametic (Spearmankaiber anc immed Spearman-Karber) and numerical interpolation (moving average and binomial Gistibution) techniques, An example of some of these methods, ‘sing survival data trom Figure 2, is shown in Figure 4. This is requited it parametric lalresponses, but the fut range {rom 0% to 100% monafity does need to be covered, The binomial t estimate the LCSO when there are no partial fesponses, ~Hitenusne26 eek) —Bhugtt g Ca errr g et s@ bs = 2 z (7/ ay wovenusaueg wesver Figure 3. Example calculation of 96-h LCSO value by graphicat method, spss % Survivaliteneegd speasas-ihasze ANALYSTS] PELE: §/555-02.21 (332-21 yest: Pheao! «g/t. DATE: acwary 2, L859 Species: P.monodon (10-a old) DURATION: $6-h CHENECAL: Pheno’ (CARRIER cor: 1 conc t mg/L} REREOSED «MORTALITY 319.600 10 é 4 is.000 10 3 32°00 10 3 56.000 20 ' 1001000, 10 20 Spearman-Karber trin: 9.000 Spearsan-Karber est LCS0: . 26.347 98t Lover contidence 22.360 958 Upper Confidence: 371820 Lesosstepeei’ EAT VERS 1.0 1 Rercent —Bingatat ' dead Probipeccent) 100.0 010897 89: Slasar G0 37.6353 30:0 00 neo a ‘Te Binon test shova that 20.000 and 100.000 can be o used as scatistically sound at $5 cont since the actual cont Level associated with these linits Le An approx Uc50 1s © 26.477 -RESUUTS USING THE HOVING AVERAGE HETHOD--- \ Tete Arcot tieies ' o.224608 “aid ahas or2i4aa3 20.790 42353 0780250) Baa 736 a35a sintialey zjREswurs usinG Tam FnoerT wETKon . : ikerations “g Pigood. of thei . 3 0.2015 2.000 0.546 slopes 3.860 35 eontidence Lintese 2.327 and 5.895 AeS0e 28.537 95 contigence Laiess 20.807 and 30.532 dels 2a : . 95 contigence Limits» 2.225 and 1.663 Figure 4. Example calculation of 96-h LCs0 value by computer program. fest nan oan28 9.0 REPORTING AND RECORD KEEPING ‘Tne test report should include a statement ol the study objectives, a complete description of the materials 2nd methods, a summary and ciscussion of tha tast results and copies of the test datasheet 306 aay Statistical calculations. Any information not included in the repont needs to be recorded and archived in such a way that it can easily be retrieved at a later date, Records for a particular toxicity test need to contain enough information to allow a person untemiia: with the test to completely reconstruct it. This includes information about the test material, test organisms and the test apparatus. Information mey be recorded directly on datasheets or in laboratory notebooks, These notebooks should be bound and have numbered pages, to prevent loss of information. Notebooks or logbooks are recommended for documenting *SAGoing" activites, such as instrument calibration, temperature control records of dateits of test organism holding/acelimation. . ‘The following information should be recorded for all tests: 1. Name and location of the laboratory, dates of testing and personnel involved in the study. Information about the sample, Including source, type, description, physical properties, collection ‘method, dates and times of collection and receipt by laboratory, identification, transpor, storage. 3. Information about the test organisms, Including scientific and common names, age, lite stage, size, source, collection/euture methods, food type, feeding frequency, acclimation conditions, pre- test morality, reference toxicant data, 4. A description of the methods used including relerences for test protocols, source and characterization of dituion/controt water, test container size and construction, test solution volume, Preparation of lest solutions, test conditions and frequency of observations. Data on temperatures, salinity, pH and dissolved oxygen levels in test solutions, and a description of photoperiod and aeration, 6 Observations of test organism mortality and behaviour, and any changes in test solution appearance. Criteria for determination of test acceptabiliy, 96-h LCSO values (including 95% confidence limits) Any deviations from the protocol not addressed in protocol amendments, together with a discussion of their impact on the study.Ey 10.0 REFERENCES APHA. 1992. Standard Meinods for the Examination of Water and Wastewater. 18h edition, American Public Health Association, American Water Works Association and Watzr Pollution Controt Federation, Washington, D.C. ASTM, 1992, Standard guide for conductiig acute toxicity tests with fishes, macroinventebrates, and amphibians, Method €729-88a, In: Annual Book of ASTM Standards, Volume 11.05, Amarican Society lor Testing and Materials, Philadelphia, Pennsylvania. Environment Canada. 1990. Biological test method: acute lethalily test using rainbow trout Environmental Protection Series, Report EPS 1/RM/9, July, 1990. EPA 1991a, Methods for maasuting the acute toxicity of effluents to freshwater and marine organisms. 4td edition, C.. Weber (ed). Environmental Monitoring and Support Leboratory. US. Environmentat Protection Agency, Cincinnati, Ohio. EPA/600/4-80/027, EPA, 1991b, Recommended guidelines for conducting laboratory bioassays on Puget Sound sedimenis. Prepared by PT! Environmental Services for U.S, Environmental Protection Agency, Region 10, Seattle, Washington. Interim Final Report, July 1991. Reich, O.J. and P.S, Oshida. 1986. Manual of methods in aquatic environment research. Part 10. Shon term static bioassays. FAO Fish. Tech. Paper 247, 62 p. Stephan, CE. 1977. Methods for calculating an LCSO. pp. 65-84, In: Aquatic Toxicity and Hazard Evaluation. F.L Mayer and J.L Hamelink (eds). ASTM STP 634, American Society for Testing ‘and Materials, Philadelphia, Pennsylvania. UNEP, 1989. Test of the acute lethal toxicity of pollutants to mazine fish and Invenebrates. Reference Methods for Marine Pollution Studies No. 43. United Nations Environment Programme, September 1999,- Maisou aig une soa inesACUTE TOXICITY TEST DATA SUMMARY ent Analyst(s) ‘oject # ‘otk Order # AMPLE, Jentitication mount Received late Collected rate Received 4 Jssolved Oxygen (mg/L) Sonductivty Gimhos/em) wher | pILUTION AND CONTROL MEDIUM ‘TEST SPECIES INFORMATION Source Organism oH . Source dissolved Oxygen (mgh) Collection Date/Batch Salinity (Ppt) ‘Age (on Day 0) Other Relerence Toxicant Current Reference Toxicant Resut Laboratory Range for Reference Toxicant {mean * 28D) Pre-treatment il ‘TEST CONDITIONS ‘Temperature Range (10) PH Range Dissolved Oxygen Range (mg/t) Salinity Range (pp!) Aerat Photoperiod {L:0 h) No. Organisms/olume Aoading Density (gf) j Other ll Toxicity Test Results Etrities By:al | iL oF sypapeyTeTeye;ale Tel]! |e STEpOpTe ale yey yet ‘war Toro 96 OF) Aus He (0) suruvuRena {6u) n304x0 oanTOSSIO “WAIN IN3OERS NoNoo Wwonva # Z0un0S nnn -0) 21m onsriseevau0 snes ss Bravo sa “oN 193084 ————_ ator ava oO ‘BRIN LOBOS ‘VAVO 1831 ALIOIXOL ALITHIT SLAW ‘aa arvsassent (sa Danes fot[A GENERIC PROTOCOL FOR CONDUCTING ACUTE TOXICITY TES™ WITH FISH AND INVERTEBRATES TLINE FOR APPENDICES Commercial, ecological and/or economic importance Details of life-history and life-cycle Test organisms . + seasonal availabilty of larvaland juvenile stages + potential sources + methods for collection and transport + methods for laboratory culture + diseases or parasites, and methods of treatment + evel of effort required - collection, culture, acclimation + methods of handling organisms (i.e, transfer between containe + feeding - food type and frequency (for different life stages) + recommended age, and corresponding size, for testing: + growth rate " + range of acceptable environmental conditions - temperature, pH “Y + dissolved oxygen requirements + fight requirements - intensity and duration +. acclimation to test conditions - duration, tolerance to ch=7S i environmental conditions ‘Toxicity test specifications test duration (96 h is proposed) test container size number of replicates + loading density - test solution volume, organisms per containe! + need for feeding during test . . daily observations (other than mortality and water quality meas' events) Acceptable control survival Sensitivity to contaminants References and other sources of information