1、STD-BSI BS bObB-5.24-ENGL 1777 1bZibbS 0808775 b95 BRITISH STANDARD Water quality - Part 5: Biological methods - Section 5.24: Determination of acute lethal toxicity to marine copepods (Copepoda, Crustacea) ICs 13.060.70 BS IS0 14669:1999 6068-5 -24: 1999 U- NO COPYING WITHOUT BSI PERMISSION EXCEPT
2、AS PERMITTED BY COPYRIGHT LAW STDOBSI BS bObB-5.24-ENGL 1777 lb24bb9 080897b 521 been prepared under the Environment Sector Committee, was published under the authority of the Standards Committee and comes into effect on 15 September 1999 direction of the Health and Amd. No. Date O BSI 09-1999 ISBN
3、O 680 32660 4 BS 6068-5.24 :1999 Comments National foreword This British Standard reproduces verbatim IS0 14669:1999 and implements it as the UK national standard. The UK participation in its prepamtion was enmsted to Technical Committee Em, Water quality, which has the responsibility to: - aid enqu
4、irers to understand the text; - present to the responsible inte b) industrial or sewage eff luents, treated or untreated, afer decantation, filtration or centrifugation if necessary; c) marine or estuarine waters. 2 Normative references The following normative documents contain provisions which, thr
5、ough reference in this text, constitute provisions of this International Standard. For dated references, subsequent amendments to, or revisions of, any of these publications do not apply. However, parties to agreements based on this International Standard are encouraged to investigate the possibilit
6、y of applying the most recent editions of the normative documents indicated below. For undated references, the latest edition of the normative document referred to applies. Members of IS0 and IEC maintain registers of currently valid International Standards. IS0 5667-2, Water Qualiy - Sampling - Par
7、t 2: Guidance on sampling techniques. IS0 5667-16, Water Quality - Sampling - Part 16: Guidance on biotesting of samples. 3 Principle Copepods are exposed to a range of concentrations in seawater of a chemical substance, effluent or water sample. Mortality of the copepods is recorded afer 24 h and 4
8、8 h. The concentration which, in 48 h, kills 50% of exposed copepods under the conditions defined in this International Standard is determined. This concentration, known as the median lethal concentration, is designated 48 h LC50. NOTE If possible, the concentration which kills 50% of the exposed co
9、pepods in 24 h is also determined. This concentration is designated 24 h LC50. It may be appropriate for certain purposes to extend the exposure period to 96 h and to determine the 96 h LC50. An indication of the lowest concentration tested which kills all the copepods and the highest concentration
10、tested which does not kill any of the copepods is desirable and provides useful information in cases where the 48 h LC50 cannot be determined. The test is carried out in one or two stages: - a preliminary test which determines the range of concentrations to be tested in the definitive test and gives
11、 an approximate value of the 48 h LC50 (and where appropriate, the 24 h LC50). 1 STD-BSI BS bObB-5-24-ENGL 1999 Lb2Libb7 0808781 779 BS 6068-5.24:1999 - a detinitive test, conducted when the approximate value given by the preliminary test is not sufficient, which permits calculation of the 48 h LC50
12、 (and where appropriate, the 24 h LC50) and determines concentrations corresponding to O and 100% mortality. If the method described in this International Standard is used for chemical substances, a limit test can be performed at 100 mgA or at a lower concentration which is the maximum at which the
13、substance is soluble or is in stable dispersion under the conditions of the test. 4 Test environment The procedure described in this International Standard shall be carried out in a room, incubator or water-bath controlled at 20 OC * 2 OC and under a 16 W8 h lightldark photoperiod. The atmosphere sh
14、all be free from vapours or dusts toxic to copepods. 5 Reagents and materials 5.1 Test organism, one of the following species of marine copepod: a) Acattia tonsa Dana; b) Tisbe battagliai Volkmann-Rocco; c) Nitocra spinipes Weck. Obtain the test organisms from laboratory cultures. Guidance on identi
15、fication and culture methods for each species are given in Annex B. After hatching of eggs, the lifecycle of copepods consists of naupliar, copepodid and adult stages. The age and lifestage at the start of the test shall be indicated in the test report and are as follows: - Acattia tonsa: large cope
16、podids (Stage 5) or adults; - Tisbe battagliai: copepodids 6 I 2 days old; - Nitocra spinipes: adults 3 to 4 weeks old. 5.2 Dilution water. A natural or a synthetic seawater may be used as the dilution water. If natural seawater is used, it shall be collected from a location as distant as possible f
17、rom known sources of pollution and filtered to remove indigenous organisms. If synthetic seawater is used, it shall be prepared by dissolving reagents of recognized analytical grade, or a commercially available formulation, in distilled or deionized water. However, for these copepod species, there i
18、s insufficient information on the use of synthetic seawater to allow a particular example to be recommended. The saliniy of the dilution water shall be between 29 x 1W and 36 x 10-3. The use of a lower salinity, which may be more appropriate for tests concerning estuarine or brackish water situation
19、s, shall be justified in the test report. Nitmm spinipes can be used at salinities down to 1 x 1W and Tisbe battagliai can be used at salinities down to 20 x 10-3. Whichever salinity is employed, the test organisms shall be cultured or maintained at the same salinity (I 3 x 103) for at least 7 days
20、before the start of the test. The dilution water shall have a dissolved oxygen concentration above 80 % of the air saturation value, and a pH of 8.0 i 0,3 before being used to prepare the test solutions. The dilution water shall permit survival of the copepods for at least 48 h and should be from th
21、e same source as water that has been found to support culture of the organisms through at least two generations. 5.3 Reference chemical toxicant, e.g. 3,s-dichlorophenol or a suitable alternative (8.5), of recognized analytical grade. 2 6 Apparatus Ordinary laboratory apparatus, and in particular: 6
22、.1 Apparatus for measuring dissolved oxygen, salinity and pH. 62 Low-power stereo microscope, preferably with darkfield illumination. 6.3 Ultrasonic device or other apparatus for the preparation of stock solutions of poorly soluble substances (7.2.1). 6.4 Test containers, of chemically inert materia
23、l and of sufficient capacity (for example glass beakers or disposable rigid plastic tissue-culture well-plates). Loose-fitting lids or covers are recommended to minimize evaporation of the test solutions. Containers which are suitable for low-power microscopical observation may be necessary for naup
24、lii or copepodid stages. Before use, the test containers shall be carefully washed then rinsed, first with water and then with the dilution water (5.2). 7 Sampling, treatment and preparation of samples 7.1 Special precautions for sampling and transportation of samples of water or effluent Sampling o
25、f water or effluent shall be carried out in accordance with the general procedure specified in IS0 5667-2. Bottles shall be completely filled to exclude air. The preservation and storage of water or effluent samples shall be carried out in accordance with IS0 5667-16; the following is only a summary
26、. The toxiclty test should be carried out as soon as possible, ideally within 12 h of collection. If this time intewal cannot be observed, cool the sample (O OC to 4 OC) and test the sample within 48 h. If testing cannot be carried out within 48 h, the sample may be frozen (below -18 OC) for testing
27、 within 2 months of collection. 7.2 Preparation of solutions of substances to be tested 7.2.1 Preparation of stock solutions Prepare stock solutions of the substance to be tested by dissolving or diluting a known quantity of the substance in a known volume of dilution water, deionized water or disti
28、lled water in a glass container. They shall be prepared at the moment of use unless the substance is known to be stable in solution, in which case the stock solution may be prepared up to two days in advance. For substances which are poorly soluble in water, ultrasonic or other suitable devices may
29、be used in the preparation of the stock solutions to aid solubilization or dispersion of the substance. Organic solvents of low toxicity to copepods (for example acetone) may be used provided that the concentration of the solvent in the final test solution does not exceed 0,l mVI and two series of c
30、ontrol tests, one with no solvent, the other with the maximum concentration of solvent, are carried out at the same time as the test. No single procedure for the preparation of stock solutions of poorly soluble substances can be recommended due to the differing nature of chemicals. 7.29 Preparation
31、of test solutions Prepare the test solutions by adding the stock solutions (7.2.1) or effluent (7.1) to the dilution water (5.2) in specified quantities so as to obtain the concentrations selected (8.1, 8.2) for the test. If the stock solutions are prepared in deionized or distilled water, all the s
32、olutions, including the control, shall receive the same quantity of distilled or deionized water and the final salinity shall be within the range specified for the test (5.2). 3 STD=BSI BS bOb8-5-2!-ENGL 1997 Lb2Libb9 0808983 7bL BS 6068-6.24:1999 It is recommended that the volume of test solution p
33、repared be sufficient to allow determination of the dissolved oxygen concentration and pH at the start of the test (8.3) using the excess remaining after filling the test containers. 8 Procedure 8.1 Preliminary test This test provides an approximate value of the 48 h LC50 and enables, if necessary,
34、a range of concentrations to be selected for use in the definitive test. For this purpose, a wide range of concentrations (generally chosen in geometric progression) of the chemical substance, effluent or water sample is tested. Typically, a factor of 10 or 3,2 between concentrations and a minimum o
35、f five animals per concentration, without replication. is appropriate. An example is given in annex A. 8.2 Definitive test This test enables determination of the percentages of copepods which are killed by different concentrations and determination of the 24 h and 48 h LC50. Select a range of concen
36、trations, based on the results of the preliminary test (8.1). but employing a smaller factor (typically 1,8 or 2) between concentrations. It is desirable that the concentrations selected result in two or three percentages of mortality between 10 Oh and 90 %. An example of the selection of a range of
37、 concentrations is given in annex A. For each concentration and each control, use a minimum of 20 copepods (for example, four replicates each containing five copepods). Replicate containers are recommended in order to facilitate counting of the copepods. 8.3 General procedure Place equal volumes of
38、the test solutions (7.2.2) into a series of test containers (6.4). The volume per container shall be such that, with the required number of copepods (8.1,8.2), the density of copepods does not exceed 1 per 0,5 ml of solution. For Acartia tonsa, the recommended maximum density is 1 copepod per 5 ml o
39、f solution. For each series of tests, prepare control containers each having a volume of dilution water (5.2) equal to the volume of the test solutions. If a solvent is used to solubilize or disperse the substance, prepare a second set of control containers with the dilution water (5.2) containing t
40、he solvent at the maximum concentration used. Before the start of the test determine, as a minimum, the dissolved oxygen concentration and pH of the dilution water (or control solution) and the pH of test solutions corresponding with the lowest and highest concentrations being tested. Place the requ
41、ired number of copepods in each test container. It is recommended that the copepods be transferred to the test solutions using a pipette of sufficiently wide bore to avoid damage to the organisms. Minimize the quantity of water transferred to the test solutions. The copepods shall not be fed during
42、the test. During the test, keep the vessels at a temperature of 20 OC I 2 OC and under a 16 h/8 h lightldark photoperiod. After 24 h and 48 h, count the surviving copepods in each container. It is recommended that a low-power microscope (6.2) is used to aid observation. Those which are showing no sw
43、imming or appendage movements within an observation period of 10 s are considered to be dead. Record any abnormal appearance or behaviour of the copepods in the test concentrations, compared with the control animals. Ater counting the surviving copepods at 48 h, measure the dissolved oxygen concentr
44、ation and pH of the solution in at least one test container for each concentration and control (if necessary, pour into one container the contents of the containers corresponding to this concentration, taking suitable precautions so as not to modify the dissolved oxygen content). 4 STD*BSI BS bOb-5-
45、24-ENGL 1994 M Lb24bb9 OBO74 bTB M BS 6068-6.24:1999 Species 8.4 Limit test 3,5-dichlorophenol The limit test (see clause 3) is carried out with 20 copepods at a single concentration of 100 mq/l or at a lower concentration which is the maximum at which the substance is soluble or is in stable disper
46、sion under the conditions of the test. Acattia tonsa 7st.w battagliai Niocra Sp“lipes 8.5 Check of sensitivity of copepods and conformity with the procedure msn 48 h LC50 Concn. for 20 Yo to 80 % mottali 0,5 to 1,5 1 ,O 1,l to 33 23 1,9 to 5,7 33 Periodically, determine the 48 h LC50 (9.1) of an app
47、ropriate reference chemical, in order to verify the sensitivity of copepods representative of the animais used for testing other samples. The recommended reference toxicant is 3,5dichlorophenol (5.3), but other chemicals (for example potassium dichromate) may be employed if a suitable historical dat
48、abase exists. Report the recent 48 h LC50 in the test report (bearing in mind that it represents the toxicity of this compound only and is not representative of the sensitivity of the copepod species to other substances). If the 48 h LC50 of the reference chemical falls outside the range given in Ta
49、ble 1, verify the strict application of the test procedure, manner of breeding the copepods and, if necessary, use a new culture of the copepod species. An alternative procedure, suitable for checking the sensitivity of the copepods at more frequent intervals, is to determine the mortality at a single concentration of the reference chemical after 48 h. If the mortality is between 20 % and 80 % at the concentration given in Table 1, the sensitivity of the copepods is acceptable. If the mortality is not within this range, perform the full check described above. The LC50 of chemicals
