Tiêu chuẩn iso 06341 2012

© ISO 2012 Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test Qualité de l’eau — Détermination de l’inhibition de la m[.]

Special precautions for sampling, transportation, storage and treatment of water, effluent, or

Sampling, transportation and storage of the samples should be performed as specified in ISO 5667-16.

It is essential to perform toxicity testing promptly, ideally within 12 hours of sample collection If immediate testing is not possible, cooling the sample to 0–5 °C allows testing within 24 hours For delays exceeding 72 hours, samples should be frozen at temperatures below –18 °C and stored for up to 2 months, ensuring accurate results in accordance with ISO 5667-16:1998, Clause 5.

Immediately test the frozen samples after complete thawing, e.g in a water bath at a maximum temperature of

30 °C Do not use a microwave for thawing the samples.

Before testing, homogenize the sample by shaking manually to ensure uniformity High concentrations of suspended solids, whether inorganic or organic, can harm filter-feeding D magna; thus, turbidity treatment is necessary to mitigate interference If needed, allow the sample to settle for up to 2 hours, then carefully draw the supernatant with a pipette, positioning the pipette tip mid-section between the sediment surface and the liquid surface To prevent residual suspended matter, protozoa, or microorganisms from affecting the test, centrifuge the sample at 5,000 g for 10 minutes or filter it, and then test the residual toxicity of the supernatant The choice of filter should be verified by performing a control test with medium passed through the filter to ensure compatibility.

Certain filters and apparatus may introduce measurable toxicity, often due to wetting agents used in filters However, filter paper can also help eliminate toxic substances by absorbing them from the sample filtrate, contributing to safer sample analysis.

The sample obtained by either of these methods is the sample submitted to testing.

Aeration of the sample or prepared test solutions is typically unnecessary; however, if the dissolved oxygen levels fall below 40% saturation, pre-aeration for up to 20 minutes using methods such as aeration or stirring is recommended Any supersaturation of dissolved oxygen should be corrected to ensure accurate test results and reliable environmental assessments.

Measure the pH (as specified in ISO 10523) and the dissolved oxygen concentration (as specified in ISO 5814) and record these values in the test report.

Report any pre-aeration of test solutions or sample.

Tests shall be carried out without pH adjustment of the test sample.

The pH of test batches (3.6) is measured at the beginning and at the end of the test and reported.

The final pH of a test solution can significantly differ from the original pH of the test sample due to the selected concentration range and the buffer capacity of the dilution water or test sample Variations in buffer capacity and dilution factors influence the accuracy of pH measurements, making it essential to consider these factors during testing Proper selection of concentration ranges ensures more reliable pH results, minimizing discrepancies between the initial sample pH and final test solution pH.

If toxic effects are observed at concentrations where the pH falls outside the 6.0 to 9.0 range, indicating incompatibility with organism survival, the tests should be repeated with pH adjustment of the test sample to ensure accurate toxicity assessment.

IMPORTANT — Adjustment of the pH can alter the nature of the sample.

To ensure proper pH adjustment, it is recommended to modify the solution to match the pH of the dilution water, which is 6.3 When adding hydrochloric acid or sodium hydroxide, choose concentrations that limit the volume fraction to no more than 5%, ensuring safe and effective pH regulation. -**Sponsor**Looking to optimize your article and make it SEO-friendly? [Blogify](https://pollinations.ai/redirect-nexad/NdcT3sqC) can help you transform your existing content, like this excerpt about pH adjustment, into engaging and optimized blog posts Blogify uses AI to extract key sentences and create coherent paragraphs that comply with SEO rules, ensuring your content ranks higher With Blogify, you can effortlessly convert various formats into high-quality articles, saving you time and boosting your online visibility, especially when dealing with technical topics like maintaining specific pH levels in solutions using hydrochloric acid or sodium hydroxide.

When pH adjustment causes issues with suspended matter, it should be separated from the sample following ISO 5667-16 guidelines, and all pH adjustments must be documented in the test report Additionally, the pretreated sample's temperature should be adjusted to the specified test temperature to ensure accurate results.

Copyright International Organization for Standardization

Preparation of solutions of substances to be tested

Prepare the stock solution of the substance to be tested by dissolving a known quantity of the substance in a specified volume of dilution water at the time of use If the stock solution remains stable under specific conditions, it can be prepared in advance and stored accordingly, ensuring consistent and reliable test results.

For substances sparingly soluble in the test medium, refer to the specifications given in ISO 5667-16.

Avoid using solvents, emulsifiers, or dispersants whenever possible, but they may be necessary for preparing concentrated stock solutions Refer to guidance in Reference [4] for suitable solvents, emulsifiers, and dispersants Additionally, special considerations in test design and data evaluation are essential, as outlined in ISO/TS 20281 [2].

Prepare the test solution (see 9.1) by adding the stock solutions (8.2.1) to the dilution water (6.3) in specified quantities.

To ensure accurate analysis, the test should include at least five different sample concentrations, selected within a geometric series The dilution factors should be tailored to the sample’s nature—such as chemical substances, effluents, waters, or extracts—and the specific type of assay being performed, whether range finding or definitive Proper selection of concentration levels enhances the reliability and precision of the test results.

For the range finding test with chemical substances, the separation factor for the serial dilutions is usually 10 (one order of magnitude difference between two successive dilutions).

For treated or untreated waste water, fresh water, pore water or extracts, a separation factor of2 between dilutions is usually performed (i.e dilution of the previous dilution by half).

The preparation of the dilution series for lowest ineffective dilution (LID) determinations is outlined in Annex F Depending on the testing purpose and statistical requirements, alternative dilution schemes using geometric or logarithmic series may also be suitable.

Dilution series for the definitive test on chemical substances are prepared with a separation factor not exceeding 3,2.

If steep concentration–response curves are expected, it is recommended that a separation factor not exceeding 2,2 be used.

Each dilution is preferably carried out in four replicates with a control (3.1) also in four replicates.

Substances which are poorly soluble in water may be solubilized or dispersed directly in pure water or dilution water by suitable means using ultrasonic devices or solvents of low toxicity to D magna Solvents should be used only when the EC50 is greater than the solubility of the test substance If a solvent is used, the concentration of the solvent in the final test solution shall not exceed 0,1 ml/l, and two control solutions, one with no solvent, the other with the maximum concentration of solvent, shall be included in the test Consider special requirements concerning test design for chemicals with solvents, e.g additional solvent-control and statistical evaluations according to ISO/TS 20281 [2]

General

Prepare a dilution series with the test solution (8.2.2) and the dilution water (6.3).

Combine increasing volumes of the test solution (8.2.2) with the dilution water (6.3), so as to obtain the desired concentrations for the test and transfer to the test containers.

To obtain a test and solution temperature of (20 ± 2) °C, for example, place the containers in a temperature- controlled room.

Once the target temperature is reached, carefully introduce D magna into the test containers using a pipette, ensuring minimal addition of hatching medium Gently release the crustaceans just below the water surface to ensure optimal conditions for hatching and development.

For each test concentration and control, at least 20 animals should be used, divided into four groups of five animals each A minimum of 2 ml of test solution should be provided per animal, ensuring sufficient volume for accurate assessment For example, administering 10 ml of solution to five daphnia per test container guarantees adequate exposure Properly distributing animals and test volumes is essential for reliable toxicity testing results.

To ensure accurate test results, prepare a control with a volume of dilution water (6.3) equal to that of the test solutions, containing the same number of D magna as in the test samples If solvents are used to dissolve or disperse substances, include a second control with dilution water containing the maximum solvent concentration used, not exceeding 0.1 ml/l This approach helps distinguish the effects of the test substance from the solvent and ensures the reliability of the toxicity testing process.

During testing, animals must not be fed, and all test containers should be kept in a temperature-controlled environment maintained at (20 ± 2) °C, as specified in section 7.1 Observations of the test organism’s responses are conducted at the end of the designated exposure period to ensure accurate and consistent results.

After the 24 or 48-hour test period, count the immobile D magna in each container Individuals that cannot swim following gentle agitation of the liquid for 15 seconds are classified as immobilized, even if they can still move their antennae This procedure helps assess the toxicity or health status of D magna in environmental testing.

Determine the concentration range giving 0 % to 100 % immobilization and note anomalies (e.g lethargy, floating on the surface, abnormal rotating or circling) in the behaviour of the D magna.

Preliminary test

This test determines the effective concentration range for the definitive assessment, utilizing a single series of concentrations typically in a geometric progression from the stock solution or sample Five D magna should be exposed to each test concentration without the need for replicates Depending on the test's objectives and statistical requirements, alternative dilution designs using geometric or logarithmic series may also be suitable, with an example provided in Annex B.

Definitive test

This test determines the percentage of D magna which are immobilized by different concentrations, the

24 h EC 50 or 48 h EC 50 , or a LID value (see Annex F).

For accurate EC₅₀ determination, it is essential to select a concentration range that produces at least three immobilization percentages between 10% and 90% Proper range selection ensures reliable and meaningful results in bioassays Examples of suitable concentration ranges are provided in Annex B to guide experimental design and optimize data accuracy.

For each concentration and each control, use a minimum of 20 D magna, preferably divided into four replicates, with five animals per test container.

Immediately after counting the immobilized D magna, measure the dissolved oxygen concentration (see

In accordance with ISO 5814, testing involves using test containers with both the control batch (3.1) and the most concentrated test batch (3.6) If needed, combine the contents of all containers at this concentration into a single container, ensuring careful precautions are taken to maintain the dissolved oxygen content This process helps ensure accurate and reliable test results while adhering to standardized procedures.

If the dissolved oxygen concentration in the most concentrated test batch drops below 2 mg/l, additional test batches must be measured to verify they meet the minimum requirement Test batches with dissolved oxygen levels below 2 mg/l should be excluded from final calculations to ensure accurate compliance with the specified standards Maintaining a dissolved oxygen concentration of at least 2 mg/l is essential for reliable test results and quality assurance.

Check of the sensitivity of the Daphnia magna and conformity with the procedure

Within one month of conducting the tests, the 24-hour EC50 of potassium dichromate (6.4) should be determined using dilution water (6.3) to assess the sensitivity of Daphnia magna This approach ensures accurate measurement of toxicity levels and compliance with standardized testing protocols.

When documenting toxicity data, record the 24-hour EC₅₀ in the test report, noting that it reflects only the toxicity of the specific compound tested and does not indicate D magna's sensitivity to other substances.

Carry out the check as described in 9.3 If the 24 h EC50 of the potassium dichromate falls outside the range 0,6 mg/l to 2,1 mg/l, verify that the test procedure has been strictly applied.

Limit test

The limit test (see Clause 4 and Annex F) is carried out with 20 D magna using the procedure described in 9.1

If the percentage of immobilization exceeds 10 % at the end of the test, a full study shall be conducted Any observed abnormal behaviour shall be recorded.

10 Interpretation and validity of the results

Estimation of the EC 50

At the conclusion of the 24-hour or 48-hour test, calculate the percentage immobilization for each concentration relative to the total number of D magna used to assess toxicity Determine the 24h EC50 or 48h EC50 values using appropriate statistical methods such as probit analysis, moving average, binomial techniques, or graphical estimation on a Gaussian logarithmic diagram These methods ensure accurate and reliable interpretation of the toxicity data for environmental risk assessments.

Accurate measurement of the test substance concentration is essential, requiring assessment at the highest and lowest test concentrations at both the start and end of the test It is advisable to base study results on measured concentrations to ensure data accuracy However, if there is sufficient evidence that the test substance concentration remained within ±20% of the nominal or measured initial concentration throughout the testing period, results can be confidently derived from the nominal or initial measured values.

When data are insufficient or EC50 calculation is unnecessary, report the minimum concentration that achieves 100% immobilization and the maximum concentration that results in 0% immobilization Additionally, record the mean percentage immobilization observed in the control group and at each tested concentration to ensure accurate interpretation of results.

Validity criteria

For the test results to be considered valid, two conditions must be met: first, the control group's percentage immobilization should be 10% or less, indicating minimal mortality in the control sample; second, the 24-hour EC50 value for potassium dichromate must fall within the range of 0.6 mg/l to 2.1 mg/l, ensuring the test's reliability and accuracy.

Express the EC50, and the values corresponding to 0 % and 100 % immobilization:

— as a percentage, in the case of effluents, waters, eluates or extracts;

— in milligrams per litre, in the case of chemical substances.

NOTE The data can be reported in other units.

If determined, report the LID value (see Annex F).

This test report shall contain at least the following information: a) the test method used, together with a reference to this International Standard (ISO 6341:2012);

ISO 6341:2012(E) b) all information required for the complete identification of the original sample (before treatment) or of the chemical substance under test; c) the methods of preparation of the samples:

For effluents, waters, eluates, and extracts, it is essential to document the sample collection method, storage duration, pH level, and dissolved oxygen concentration of the original sample Additionally, details regarding the procedures such as decantation, filtration, or centrifugation, including any pH adjustments made, should be recorded to ensure accurate analysis and reproducibility.

This article outlines the essential information required for conducting toxicity tests on D magna, including the method of preparation for chemical stock and test solutions, and comprehensive biological, chemical, and physical data such as the origin and age of the stock culture It emphasizes the importance of reporting test results like the 24 or 48-hour EC50 values, methods of calculation, and 95% confidence limits when possible The article also specifies documenting the limit test outcomes, minimum and maximum tested concentrations related to immobilization rates, and any abnormal behaviors exhibited by D magna during testing Additionally, it mandates recording operational details, incidents affecting results, reference chemical test results with dates, validation data ensuring criteria are met, and detailed information about the laboratory, personnel involved, and report approval, ensuring adherence to ISO standards.

Preparation of the Elendt M4 medium

This annex gives one option for the preparation of the M4 medium using stock solutions.

Prepare individual stock solutions in pure water for each trace element Then, combine these to create a comprehensive second stock solution containing all 13 trace elements listed in Table A.1 This method ensures accurate preparation of trace element solutions essential for precise scientific analysis.

Prepare the M4 medium using stock solution II, the macro-nutrients and vitamins in accordance with Table A.2.

Prepare the combined vitamin stock solution by adding the three vitamins to 1 l of pure water (6.2), as described in Table A.3.

Store the combined vitamin stock frozen in small aliquots Add the vitamins to the medium shortly before use.

To avoid any precipitation of salts when preparing the complete medium, add the aliquots of the stock solutions to about 500 ml to 800 ml of pure water (6.2), then dilute to 1 l.

Table A.1 — Stock solutions I and II for the Elendt M4 medium

(in relation to the M4 medium)

To prepare the combined stock solution II, add the following volume of stock solution I to pure water

FeSO4⋅7H2O a 1 991 2 000 fold a Both Na 2 EDTA and FeSO 4 solutions are prepared individually, then poured together and immediately autoclaved This gives:

To prepare a 1,000-fold Fe-EDTA solution, dissolve 5,000 mg of Na₂EDTA·2H₂O in pure water and dilute to 500 ml Separately, dissolve 1,991 mg of FeSO₄·7H₂O in pure water and make up to 500 ml Combine both solutions immediately and autoclave to ensure sterilization Store the resulting Fe-EDTA solution in a dark place to maintain stability.

Table A.2 — Preparation of the M4 medium using stock solution II, the macro-nutrients and vitamins

(in relation to the M4 medium)

Volume of stock solution added to prepare the M4 medium mg/l ml/l

Macro-nutrient stock solutions (single substance)

Table A.3 — Composition of the vitamin stock solution

(in relation to the M4 medium) mg/l

Example of graphical determination of the inhibition of mobility of

Daphnia magna by an effluent or stock solution of a substance at a concentration of 1 000 mg/l

NOTE The example relates to the procedure using test tubes.

Table B.1 — Result of the preliminary test

The range of concentrations over which the definitive test is to be carried out is therefore 0,35 % to 1 %

Table B.2 — Result of the definitive test

Number of mobile Daphnia magna in tube No.

1,0 0 1 0 0 1 95 a Number of mobile D magna at each concentration at the end of the test. b Percentage of immobilized D magna at each concentration.

For effluents, this is expressed as:

For a chemical substance, this is expressed as:

50 = × From the graph, the values of EC 50 , EC 16 and EC 84 can be obtained by interpolation The lower confidence limit (95 %), T 0 , may then be estimated as:

EC and the upper confidence limit (95 %), T 1, as:

T 1 small> 50 f EC 50 where the multiplication/division factor for obtaining confidence intervals, f EC 50 , is given by f EC 50 =S ( 2 77 , √ Ν ′ ) in which

S is the slope factor, given by:

N′ is the total number of organisms exposed in the interval 16 % to 84 %.

General recommendations for stock culturing

Loading of stock culture should be 25 to 50 animals per litre They should be kept in mass stocks.

NOTE A loading of stock culture similar to the loading to be employed in the test is recommended For example, a stock loading of 25 animals per litre would be suitable for test regimes employing replicates of five animals in approximately

Stock culture of daphnia should be fed by freshly prepared unicellular green algae from laboratory culture Food algae should be in the exponential growth phase Algae cultures can be used as long as they are growing without showing degradation effects Harvested algae should not be stored at room temperature for a long time, because then degradation processes occur They can be stored in a refrigerator in the dark or can even be frozen as long as daphnia cultures fed with these algae are still healthy and in a good reproduction status Food should not be overdosed It is recommended that daphnia stocks be fed with carbon levels at 0,1 mg per organism day to 0,2 mg per organism day To avoid the transfer of algae growth medium to stock culture of daphnia it is recommended that the algae be separated from the algae growth medium and resuspended in daphnia culture medium For stock culture a regular maintenance is necessary Change the medium at least

Perform water changes 2 to 3 times per week to maintain optimal conditions Carefully transfer adult specimens to a clean container with fresh medium, while separating the neonates from the main stock for proper growth management Regularly remove exuviae, dead or discolored animals, and feeding residues to ensure a healthy aquatic environment that promotes the well-being of your aquatic organisms.

Stock culturing of D magna should be conducted under a 16-hour light and 8-hour dark cycle using diffuse daylight or artificial lighting, ensuring optimal conditions The cultivation environment must maintain a temperature of (20 ± 2) °C and be free from toxic vapors or dusts that could harm D magna For detailed culture methods and best practices, refer to Sources [8]–[12].

Culturing of Daphnia magna for production of dormant eggs

D.1 Life cycle of Daphnia magna (see Figure D.1)

In nature, D magna can reproduce asexually as well as sexually.

In well-maintained stock cultures, Daphnia reproduce exclusively through asexual parthenogenesis, producing diploid female offspring Adult Daphnia lay parthenogenetic eggs that develop in the dorsal brood chamber, resulting in live, genetically identical offspring This reproductive method ensures rapid population growth and is key to understanding Daphnia's reproductive strategies.

The young daphnia grow through successive juvenile instars before they start to produce eggs.

Under specific environmental stressors, daphnia switch from asexual to sexual reproduction, producing haploid males and eggs that require fertilization This reproductive shift enables their adaptation and survival under changing environmental conditions Understanding these stressors provides insight into daphnia's reproductive strategies and ecological resilience.

The females subsequently produce a protective dorsal shell (called an ephippium) in which two fertilized eggs (called dormant eggs) are deposited.

Figure D.1 — Life cycle of Daphnia magna (Reference [7], reproduced with permission of the copyright holder)

The ephippium is cast off at the next moult of the female and usually sinks.

Dormant eggs typically undergo a "refractory phase" lasting several months, during which they are unresponsive to stimuli necessary for embryonic development This refractory period prevents the eggs from progressing to the next stage until conditions are suitable for hatching and neonate birth Understanding this dormancy process is crucial for effective management and incubation strategies in reproductive cycles.

D.2 Culturing of Daphnia magna for production of dormant eggs

Laboratory simulation of specific environmental conditions can induce D magna’s sexual reproduction, leading to the formation of dormant eggs known as ephippia These ephippia can be carefully stored and later hatched when needed, providing live biological material essential for conducting toxicity tests and environmental assessments.

Daphnia cultures to produce dormant eggs can be set up in any type of test container and in any volume, depending on the number of ephippia required.

The organisms are cultured and fed in exactly the same way as for laboratory stock cultures The same daphnia strains can be used for production of dormant eggs.

Cultures can be started with a population density of 100 daphnia/l to 200 daphnia/l, with a light cycle of 12L:12D at ambient temperature.

Provided there is enough food, the population density in the cultures gradually increases and can attain

The shift from asexual to sexual reproduction is mainly triggered by two variables: population density (crowding) and shortage of food.

The precise values for these two variables and the duration required to transition to sexual reproduction are highly dependent on specific culturing conditions These factors must be determined through experimental investigation to ensure accurate and reliable results.

Regular microscopic observations of the cultures reveal the onset of sexual reproduction, marked by the appearance of smaller males and females carrying ephippia After females release their ephippia, these protective cases can be collected from the bottom of the culture container, indicating successful reproduction.

The ephippia shall be stored in the refrigerator (at 4 °C) in darkness, in tubes with either tap water or a reconstituted natural water.

Taking into account the “refractory phase” (indicated in D.1.), the dormant eggs have to be stored for about

3 months after harvesting to obtain successful hatching Viable neonates can even be obtained after 1 to 2 years if the dormant eggs are stored properly.

Tiêu đề	Water quality — Determination of the inhibition of the mobility of Daphnia magna Straus (Cladocera, Crustacea) — Acute toxicity test
Trường học	University of Alberta
Thể loại	tiêu chuẩn
Năm xuất bản	2012
Thành phố	Switzerland

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Số trang	30
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