IPW Report Chironomus Acute NaCN - Europa · Study Report 2nd of 5 Chironomid acute toxicity test...

Study Report 2nd of 5 Chironomid acute toxicity test Semi-static exposure Acute Toxicity of sodium cyanide on Chironomus riparius GLP compliant study following OECD 202 “Daphnia sp., Acute Immobilization Test”, adopted April 13, 2004 and considerations of “The Chironomid Acute Toxicity Test: Development of a New test System”, Weltje, L. et al. (2009) and OECD Draft Test Guideline for an Acute Chironomid Test (version of 11.03.2011, ENV/JM/TG (2011)16)

GLP-Code of Test Facility: IPW-001/4-36/R

REG-No.: 2010-0230-DGO

Principal Sponsor CEFIC Cyanide Sector Group Avenue E. van Nieuwenhuyse 4 1160 Brussels, Belgium

Study Monitor Dr. Daniel Bertow Industriepark Wolfgang GmbH Rodenbacher Chaussee 4 63457 Hanau-Wolfgang, Germany

Test facility Fraunhofer-Institute for Molecular Biology and Applied Ecology (IME) 57377 Schmallenberg Germany

Test facility management

Dr. Christoph Schäfers

Study director

Dr. Markus Simon August 23, 2011 of 40

Study Report: Chironomid acute immobilization test; Sodium cyanide - page 2/40 - Test Institute: Fraunhofer-Institute IME GLP-Code: IPW-001/4-36/R

This page was intentionally left blank for statements of the sponsor or submitter


Contents page

Statement of GLP-Compliance 3 Quality assurance statement 4 Contents 5 Distribution list 5 Definitions 6 List of tables 7 List of figures 7 1. Title 9 2. GLP-Code 9 3. Sponsor 9 4. Monitor 9 5. Test facility 9 6. Schedule 10 7. Test and reference items 11 8. Test principle 13 9. Details concerning the test 13 10. Treatment of the results 16 11. Results 17 12. Validity 20 13. References 21 14. Archiving 22 Annex 23

A.1 Annex 1: Details on test data 23 A.2 Annex 2: Evaluation of test data 25 A.3 Annex 3: Analytical Report - Details of method and results 31 A.4 Annex 4: Inspection certificate of the test item according to EN 10204 3.1 38 A.5 Annex 5: GLP certificate 39

Distribution list

GLP-archive: 1 original (1st of 5)

Principal Sponsor: 4 originals (2nd, 3rd, 4th, 5th, of 5) and pdf

Study director: 1 copy


Definitions

LOEC: (lowest observed effect concentration) is the lowest concentration tested at which the measured parameter shows significant increased immobilization relative to the control.

NOEC: (no observed effect concentration) is the highest concentration tested at which the measured parameter shows no significant increased immobilization relative to the control.

EC10/20/50: (effective concentration) is the concentration of the test item, which results in a 10, 20 or 50 per cent increased immobilization relative to the control.


List of tables page Table 1: Concentrations of free cyanide. Mean measured concentrations [µg CN-/L]

and percentage of nominal [%]. ............................................................................. 17

Table 2: Weighted average concentrations of free cyanide [µg CN-/L]. .............................. 18

Table 3: Mobile larvae after 24 h and 48 h. Sum of all 4 replicates, introduced specimens = 20. .................................................................................................... 19

Table 4: Immobilisation after 24 h and 48 h. Mean values [%]. ........................................... 19

Table 5: NOEC and EC50 values for immobilisation at test end [µg CN-/L]. ....................... 19

Table 6: Oxygen content of the test media. Single values [% air saturation]. ...................... 23

Table 7: pH-values of freshly prepared and aged test solutions .......................................... 23

Table 8: Temperature [°C] and light intensity [lx]. Measured in the fume hood. .................. 23

Table 9: Dilution water, chemical properties. ....................................................................... 24

Table 10: Mobile larvae after 24 h and 48 h. Number of specimens per replicate = 5 ........... 24

Table 11: Immobilisation after 24 h and 48 h. Single values of the replicates [%]. ................ 24

Table 12: Calculation of elimination rate ke for 50 µg CN-/L ................................................. 34

Table 13: Calculation of elimination rate ke for 500 µg CN-/L ............................................... 35

Table 14: Analysed concentrations [µg CN-/L] and recovery rates [% of nominal] of free cyanide in the test media at start of the exposure intervals. .................................. 36

Table 15: Analysed concentrations [µg CN-/L] of free cyanide in the aged test media at the end of the exposure intervals. ......................................................................... 37

List of figures page

Figure 1: Effects on mobility of the introduced C. riparius as observed after A) 24 h and

B) 48 h. .................................................................................................................. 20

Figure 2: Calibration function of cyanide ............................................................................... 33

Figure 3: Dissipation of cyanide during 24 hour in medium with 50 µg CN-/L ...................... 34

Figure 4: Dissipation of cyanide during 24 hour in medium with 500 µg CN-/L .................... 35


Summary

The influence of Sodium Cyanide on immobilisation of first instar larvae of the freshwater dipteran Chironomus riparius was investigated over an exposure period of 48 h at the Fraunhofer-Institute for Molecular Biology and Applied Ecology. The larvae were placed in water containing concentrations of 5.31, 13.3, 33.2, 82.8 and 208 µg CN-/L (nominal). The test was conducted under semi-static conditions. After 24 and 48 hours of exposure, immobilization was observed.

The concentrations of free cyanide in the acute chironomid test were analysed photometrically (Merck Spectroquant® 14800) at start and end of each exposure period (LOQ = 7.4 µg CN-/L).

The measured concentrations of free cyanide in freshly prepared solutions were 65 % - 113 % of nominal concentrations. The measured concentrations in aged solutions were below LOQ. Due to a decreasing concentrations of the test item over the course of the study and measured concentrations outside a range of 80 – 120 % of nominal, the calculations for the determination of NOEC and ECx- values were related to Time Weighted Average (TWA) measured concentrations.

No significantly increased immobility (NOEC) up to and including a concentration of 6.9 µg free CN-/L (TWA) was detected after 48 hours of exposure when compared to the control specimens. The 24h-EC50 for the tested species was estimated at 32.5 µg (95% CL = 21.4 – 75.6) free CN-/L (TWA). The 48h-EC50 for the tested species was estimated at 12.4 µg (95% CL = 6.4 – 23.0) free CN-/L (TWA).

Conclusion: Exposure to Sodium Cyanide resulted in a NOEC of 6.9 µg free CN-/L (TWA) for acute immobilization of larvae of Chironomus riparius under the chosen test conditions. The 24h- EC50 is 32.5 µg free CN-/L (TWA), the 48h-EC50 is 12.4 µg free CN-/L (TWA).


1. Title Chironomid acute toxicity test

Semi-static exposure Test item: Sodium cyanide 2. GLP-Code IPW – 001 / 4 – 36 / R

3. Sponsor Principal Sponsor CEFIC Cyanide Sector Group

Avenue E. van Nieuwenhuyse 4 1160 Brussels, Belgium

Co-Sponsors

CONCAWE Boulevard du Souverain 165 1160 Brussels, Belgium

EUROFER Avenue Ariane 5 1200 Brussels, Belgium

EUROMINES Ave de Broqueville 12 1150 Brussels, Belgium

4. Monitor Dr. Daniel Bertow Industriepark Wolfgang GmbH Rodenbacher Chaussee 4 63457 Hanau-Wolfgang, Germany

5. Test facility Fraunhofer-Institute for Molecular Biology and Applied Ecology (IME)

Postfach 1260 57377 Schmallenberg

Division Applied Ecology Test facility Management: Dr. Christoph Schäfers

Department: Ecotoxicology

Study director: Dr. Markus Simon Deputy: Dr. Andrea Wenzel


Technical staff, Biology: Katja Mock, Rainer Peters Quality Assurance Unit: Dr. Gerd Wasmus Dr. Ursula Wahle Karin Fink Cornelia Bernhardt

The study was performed without sub-contracting

6. Schedule Test start: Mai 17th, 2011 Test end: Mai 19th, 2011


7. Test and reference items The test item and certificates of analyses were delivered by the principal sponsor before the start of the study. Test item information is given by the principal sponsor. By his signature of the study plan the principal sponsor accepts that the chemical identity and purity of test and reference item were not checked analytically by the test facility. Test item which will not be needed for testing and for archiving will be returned to the principal sponsor or any other storage location proposed by the principal sponsor. 7.1 Test item (Data supplied by the sponsor)

7.1.1 Trade name: Sodium cyanide

7.1.2 CAS name: Sodium cyanide

7.1.3 CAS-Number: 143-33-9

7.1.4 Lot Number: 23.10.10

7.1.5 Purity: > 98 %

7.1.6 State of matter and appearance: White, crystalline powder

7.1.7 Water solubility (20 °C): 370 g/L

7.1.8 Vapour pressure (20 °C): None

7.1.9 Relative density (20 °C): 1.595 g/mL

7.1.10 Partition coefficient (logPow, 20 °C): Not applicable

7.1.11 Chemical stability (Water/Light): Stable under normal temperatures. Formation of HCN in aqueous solution.

7.1.12 Waste disposal: Via halogen-free, organic/aqueous wastes; pre-treatment with H2O2 35% for 24 hours

7.1.13 Origin of the test item: Principal Sponsor

7.1.14 Date of receipt: November 24, 2010

7.1.15 Expiry date: October 23, 2015

7.1.16 Required amount for testing and archiving: 5 g


7.1.17 Storage conditions: Store in a cool, dry place. Store in a

tightly closed container. Poison room locked. Keep away from acids. Do not store in metal containers. Do not store in aluminium containers.

7.1.18 Storage stability: Stable (see 7.1.11)

7.1.19 Safety data sheet available? Available

7.1.20 General preventive measures: Non-flammable protective clothing, gloves, eye protector


8. Test principle

First instar larvae of the chironomid C. riparius were exposed to the test substance dissolved in water at a range of 5 concentrations for 48 hours. At the end of the test, the total number of mobile specimens was assessed. The test was performed in consideration of the OECD guideline 202 and taking into account Weltje et al. 2009 and OECD Draft Test Guideline for an Acute Chironomid Test (version of 11.03.2011, ENV/JM/TG (2011)16). Using appropriate statistical methods statistical significant difference in immobilisation between the treatments and the controls (NOEC, EC50) were determined. 9. Details concerning the test 9.1 Test organisms:

Chironomus riparius (Insecta, Diptera) is a freshwater dipteran abundant in ponds and small lakes. It is a representative of the widely distributed group of aquatic insect larvae.

Young specimens of C. riparius of similar age (first instar) originally obtained from Bayer Crop Science AG, 40789 Monheim, Germany, and bred in the laboratory of the Fraunhofer-Institut for many generations, were used in the experiments.

Chironomids were derived from the same healthy stock (no signs of stress, i.e. high mortality or discoloured animals). These animals were maintained under similar conditions as used in the test. During this time the dipterans were fed daily with powder of TetraMin® Hauptfutter (Tetra Werke, Melle, Germany).

Seven days before adding the test organisms to the test vessels, egg masses (<24 h) were taken from the cultures and placed in small aerated vessels (Petri dishes) with test water (9.2) at about 20°C. Larvae were fed until start of exposure. First instar larvae (3 days post hatching) were used in the test. Only apparently healthy organisms were used in the study. Test specimens were removed by wide-bore pipette (to avoid damage) and isolated in fresh dilution water for at least 1 hour prior to being exposed to the appropriate test or control media.

9.2 Holding- and dilution-water:

Purified drinking water was used according to the OECD-Guideline 202. The purification included filtration with activated charcoal, passage through a lime-stone column and aeration. To avoid copper contamination, plastic water pipes are used for the test facilities. The water was aerated for seven days to the point of oxygen saturation to assure a valid test run. The following water chemistry data are recorded regularly in the testing facility and are reported: pH, conductivity, dissolved oxygen content, content of nitrate, nitrite, ammonium (NH4

+), chloride, phosphate, calcium, magnesium, total hardness, alkalinity, DOC content, content of metals (copper, iron, manganese and zinc). For details see Table 9.


9.3 Food:

The larvae were not fed during the test.

9.4 Test item: The test item was sodium cyanide. The nominal test concentration were 10, 25, 62.5, 156, 391 µg test item per liter, corresponding to 5.31, 13.3, 33.2, 82.8 and 208 µg free CN-/L. Concentrations applied were based on a non-GLP range finding test. Four replicates per concentration with 5 specimens per replicate were used for testing.

9.5 Control: The control consisted of dilution water only. Four replicates with 5 specimens per replicate were used for testing.

9.6 Introduction of the test item: The test item was distributed to the replicate beakers using double concentrated aqueous application solutions. The application solutions were prepared in dilution water. For the individual double concentrated application solutions, 0.4 mL, 1.0 mL, 2.5 mL, 6.24 mL, and 15.64 mL of an aqueous stock solution (10 mg/L) of the test item were dissolved in 200 mL dilution water. The individual test solutions were prepared by adding the double concentrated application solutions into the test vessels (1:1 dilution).

9.7 Test procedure: First instar larvae of C. riparius of similar age (3 days post hatching) were exposed to five concentrations of the test item under semi-static conditions for a period of 48 h. After 20 mL of dilution water was filled in the test vessels, test specimens were added (released under the control or test media surface). Subsequently, 20 mL of the double concentrated application solutions were added and mixed carefully by using a pipette. The larvae were exposed without aeration in 40 mL of test solution in 50 mL glass beaker. After 24 h, 20 mL of the aged test solutions were removed and replaced by fresh double concentrated application solution. Apparently dead animals were removed at the daily checks. For each test medium, pH value (pH-Meter, e.g. WTW 535), oxygen concentration (e.g. WTW Digital-Sauerstoff-Meßgerät Oxi Digi 550), and temperature (e.g. Digitalthermometer, Roth) were checked daily in a representative beaker.

9.8 Test conditions: Incubation took place in a fume hood. The beakers were covered with glass panes and subjected to a light/dark cycle of 16/8 hours. The test temperature during the test was 20 ± 2°C and was constant within ± 1°C during the test.


9.9 Observation and biological measurements:

The numbers of immobile animals were visually determined daily and dead specimens (if occurred) were removed. Any abnormalities (if occurred) in appearance and behaviour were recorded. Larvae were regarded as immobile if no movement was observed after gentle touching within 15 seconds.

9.10 Analytical measurement:

Concentrations of the test substance At test start samples of the control and all double concentrated application solutions were taken just before distributing it to the replicates and measured. The double concentrated solutions were applied for measurement to ensure adequate results also in the lowest treatment to demonstrate accurate concentrations at start and renewal. At media renewal, samples of the final test solutions were measured to show the real concentration right after introduction and mixing. Aged media were sampled out of each replicate and pooled per concentration for measurement. Samples were analyzed immediately after sampling.

Analytical method The determination of free cyanide was carried out by photometric measurement using the Merck Spectroquant® 1.14800 kit. For details see Annex 4.

Physical chemical parameters of the dilution water Oxygen concentration, pH value, and temperature were checked daily. The pH value varied not more than 1.5 units during the test. The following water chemistry data are recorded regularly in the testing facility and are reported: pH, conductivity, dissolved oxygen content, content of nitrate, nitrite, ammonium (NH4

+), chloride, phosphate, calcium, magnesium, total hardness, alkalinity, DOC content, content of copper, iron, manganese and zinc.

9.11 Evaluation:

Immobile larvae were determined after 24 h and at the end of the test (48 h). Due to analytical values outside the concentration range of 80 – 120 % of nominal concentrations and strongly decreased concentrations in aged media, the calculations for the determination of NOEC and ECx- values were related to Time Weighted Average (TWA) measured concentrations.

9.12 Deviations: Deviating from the study plan, double concentrated application solutions and not test solutions were measured at test start. This was due to technical reasons and without influence on the integrity of the study.


10. Treatment of the results

Data evaluation: Numerical values in this report are frequently rounded to a smaller degree of precision (number of digits) than were used in the actual calculation. Minor differences in results obtained from calculations with such rounded values in comparison to those obtained with higher precision values are possible. They are, however, well within the limits of the experimental accuracy and thus of no practical concern.

Statistical calculations: For immobilisation, the NOEC, LOEC, and the EC50, EC20 and EC10 were determined. Calculations were performed with the computer software ToxRat Professional version 2.10.05 (release 20.02.2010) by ToxRat® Solutions GmbH. A NOEC was calculated by using Fisher`s Exact Binomial Test suggested by the ToxRat program. When the test results showed a concentration-response relationship, the data were analysed by regression to determine the EC50 including the 95% confidence interval as well as the EC20 and EC10 using Probit-analysis assuming log-normal distribution of the values. The immobility of specimens in the test item treatments was corrected for control immobility according to Abbott (1925) (part of the computer based statistical evaluation). Results are presented in section 11 and Annex 1.


11. Results

Test conditions: The water temperature was maintained at 20.0 - 20.5°C throughout the test and thus within the range of 20 ± 2°C and was constant within ± 1°C during the test. The oxygen content was between 98.4 % and 102.0 % of air saturation, representing concentrations of > 8 mg/L. The pH values throughout the test were within a range of 8.4 – 8.6 at all treatment levels. The light intensity was 238 lx measured using an illuminance meter (MINOLTA) with photometric sensor. Thus, all water quality criteria mentioned in the guideline (13.1) were met. For raw data please refer to Annex 1.

Test item concentrations:

The measured concentrations of free cyanide in the freshly prepared test solutions were between 65 % and 113 % of nominal concentrations; measured values of the aged test concentrations were all below the limit of quantification (LOQ = 7.4 µg CN-/L).

Analyses of fresh and aged exposure media revealed a significant decrease of the test item concentrations within a 1-day interval (Table 1).

Table 1: Concentrations of free cyanide. Mean measured concentrations [µg CN-/L] and percentage

of nominal [%].

Nominal [µg CN-/L]

new 0 h [µg CN-/L]

% of nominal

aged 24 h[µg CN-/L]

% of nominal

new 24 h[µg CN-/L]

% of nominal

aged 48 h [µg CN-/L]

% of nominal

Control < LOQ < LOQ < LOQ < LOQ

5.31 5.44 102.4 < LOQ n.d. 4.43 83.5 < LOQ n.d.

13.3 15.0 113.2 < LOQ n.d. 8.6 64.9 < LOQ n.d.

33.2 33.8 101.8 < LOQ n.d. 28.3 85.4 < LOQ n.d.

82.8 75.4 91.1 < LOQ n.d. 64.1 77.4 < LOQ n.d.

208 201 97.0 < LOQ n.d. 176 84.7 < LOQ n.d.

* LOQ (Limit of quantification) = 7.4 µg/L. n.d. = not determined due to inappropriate data Due to the decrease of the exposure concentrations, the (no) effect concentrations were based on time weighted average (TWA) measured concentrations of free cyanide. For concentrations below the LOQ, a value half of the LOQ was set as specified in the OECD Guidance Document for Difficult Substances and Mixtures (12.2000). For raw data see Annex 3, Table 14 and Table 15.


The following formula was used for the calculation of the area under the concentration curve:

daysconcconc

concconc

1ln0ln10

= Area

where conc 0 = concentrations at start of treatment period conc 1 = concentrations at end of treatment period days = duration of treatment period The time-weighted mean is the total area divided by the total days. Table 2: Weighted average concentrations of free cyanide [µg CN-/L].

Nominal [µg CN-/L] Treatment period Area

Time weighted average concentration mean [µg CN-/L]

5.31 Start- 48 h n.d. 3.70*

13.3 Start- 48 h 13.90 6.95

33.2 Start- 48 h 25.70 12.9

82.8 Start- 48 h 44.97 22.5

208 Start- 48 h 94.03 47.0

n.d. = not determined due to inappropriate data * For concentrations below the LOQ (7.4 µg/L), a value half of the LOQ was set Immobilisation: The values for mobile specimens per treatment level are listed in Table 3. The NOEC and EC values are summarised in Table 5 (for details see Annex 1). The results for immobilisation are presented as total and percentage values (Table 3 and Table 4). For single values of the replicates see Annex 1, Table 10 and Table 11. Distinct effects occurred after 24 h. Immobilisation ≥ 40 % were detected starting at 22.5 µg CN-/L. A concentration response relationship could be observed. The 24h-EC50 was estimated at 32.5 µg CN-/L (TWA). The 24h-NOEC was found to be 12.9 µg CN-/L (TWA). Also at test end (48 h) a distinct concentration response relationship occurred. Immobilisation in the treatments was between 40 and 80 %. The 48h-EC50 was calculated at 12.4 µg CN-/L (TWA). The 48h-NOEC was found to be 6.95 µg CN-/L (TWA).

Physical/Pathological symptoms and changes in behaviour: Neither any physical or pathological symptoms nor changes in behaviour were observed for the surviving test specimens depending on test item concentration. All specimens were of healthy condition.


Table 3: Mobile larvae after 24 h and 48 h. Sum of all 4 replicates, introduced specimens = 20.

Concentrations given as TWA concentrations.

Incubation Control 3.70 µg CN-/L 6.95 µg CN-/L 12.9 µg CN-/L 22.5 µg CN-/L 47.0 µg CN-/L

24 h 17 16 16 12 7 9

48 h 17 11 12 9 6 4

Table 4: Immobilisation after 24 h and 48 h. Mean values [%]. Concentrations given as TWA concentrations.

*: significant when compared with control

Incubation Control 3.70 µg CN-/L 6.95 µg CN-/L 12.9 µg CN-/L 22.5 µg CN-/L 47.0 µg CN-/L

24 h 15 20 20 40 65 * 55 *

48 h 15 45 40 55 * 70 * 80 *

Table 5: NOEC and EC50 values for immobilisation at test end [µg CN-/L]. CL = Confidence level; n.d. = not determined due to mathematical reasons; Effect

concentrations given as TWA concentrations.

24 h 48 h

NOEC 12.9 6.95

LOEC 22.5 12.9

EC10 (95% CL) 5.3 (1.6 – 8.7) 0.9 (0.0 – 2.7)

EC20 (95% CL) 9.8 (4.8 – 14.7) 2.3 (0.2 – 4.9)

EC50 (95% CL) 32.5 (21.4 – 75.6) 12.4 (6.4 – 23.0)

Effect concentration calculation for the chironomid C. riparius after the test period of 24 h, based on TWA measured concentrations (Figure 1), results in EC10 and EC20 values of 5.3 µg free CN-/L (95 % CL = 1.6 – 8.7 µg CN-/L) and 9.8 µg free CN-/L (95 % CL = 4.8 – 14.7 µg CN-/L), respectively. The NOEC was found to be 12.9 µg free CN-/L (Table 5). The EC50 value was estimated at 32.5 µg free CN-/L (95 % CL = 21.4 – 75.6 µg CN-/L).

Effect concentration calculation for the chironomid C. riparius after the test period of 48 h, based on TWA measured concentrations (Figure 1), results in EC10 and EC20 values of 0.9 µg free CN-/L (95 % CL = 0.0 – 2.7 µg CN-/L) and 2.3 µg free CN-/L (95 % CL = 0.2 – 4.9 µg CN-/L), respectively. This was confirmed by NOEC calculation. The NOEC was found to be 6.95 µg free CN-/L (Table 5). The EC50 value was estimated at 12.4 µg free CN-/L (95 % CL = 6.4 – 23.0 µg CN-/L).

Due to EC10 and EC20 values below the lowest test concentration applied, these calculated results have to be handled with care. These values are not suitable to be taken as a real NOEC substitution. However, for an estimation of a possible 10 – 20 % effect range the results can be taken.


A B

Figure 1: Effects on mobility of the introduced C. riparius as observed after A) 24 h and B) 48 h. Concentration-effect curves.

Conclusion: Exposure to Sodium Cyanide resulted in a NOEC of 6.95 µg free CN-/L (TWA) concerning acute immobilization of larvae of Chironomus riparius under the chosen test conditions. The 24h-EC50 is 32.5 µg free CN-/L (TWA) and the 48h-EC50 is 12.4 µg free CN-/L (TWA).

12. Validity

Since Chironomus riparius is – until now – no standard test organism, no validity criteria for an acute toxicity or immobilization test exist. However, validity criteria are already indicated in the OECD Draft Test Guideline for an Acute Chironomid Test (version of 11.03.2011, ENV/JM/TG (2011)16). The test is considered to be valid, since:

- With 15 % immobility in the control is < 20 % as demanded in the study plan and equal to the threshold value of 15 % indicated in the OECD Draft Test Guideline

- the control specimens did not exhibit pathological symptoms and abnormal behavior

- oxygen content of the test media was above 3 mg/L during the whole test duration as demanded in the study plan and in the OECD Draft Test Guideline


13. References

13.1 OECD 202 (13.04.2004): Guideline for Testing of Chemicals - Daphnia sp., Acute Immobilization Test.

13.2 Weltje, Lennart; Rufli, Hans; Heimbach, Fred; Wheeler, James; Vervliet-Scheebaum, Marco; Hamer, Michael (2009): The Chironomid Acute Toxicity Test: Development of a New Test System. Integrated Environmental Assessment and Management Vol 6, nr. 2, pp. 301 - 307. SETAC.

13.3 OECD Draft Test Guideline for an Acute Chironomid Test (version of 11.03.2011, ENV/JM/TG (2011)16): OECD Guideline for Testing of Chemicals - Chironomus sp., Acute Immobilisation Test.

13.4 Directive 2004/10/EC of the European Parliament and of the Council of 11 February 2004 on the harmonisation of laws, regulations and administrative provisions relating to the application of the principles of good laboratory practice and the verification of their applications for tests on chemical substances (codified version).

13.5 OECD (1998): Principles of Good Laboratory Practice, adopted by the Council on 26th November 1997; Environment Directorate, Organisation for Economic Co-operation and Development, Paris.

13.6 Verordnung zur Neufassung der Gefahrstoffverordnung und zur Änderung sprengstoffrechtlicher Verordnungen vom 30. November 2010 (BGBl. I S 1643).

13.7 Gesetz zum Schutz vor gefährlichen Stoffen (Chemikaliengestz - ChemG) in der Neufassung vom 02. Juli 2008 (BGBl. I S. 1146), geändert am 11.08.2010 (BGBL. I S. 1163) (Anhang 1 (zu § 19a Abs. 1) Grundsätze der Guten Laborpraxis (GLP))

13.8 ToxRat® Professional 2.09. ToxRat® Solutions GmbH. Dr. Monika M. Ratte, Naheweg 15, 52477 Alsdorf, Germany.

13.9 OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures (12.2000). OECD Series on Testing and Assessment Number 23.


14. Archiving An aliquot of the test item, all raw data and all records necessary to reconstruct the study will be archived, following internal SOPs, in the GLP-archive of the Fraunhofer-Institute for Molecular Biology and Applied Ecology, 57377 Schmallenberg, Germany, according with the Principles of Good Laboratory Practice (for references see chapter 13). An aliquot of the test item will be archived for at least 2 years. List of records that will be archived for 15 years:

Data specifying the test item Data concerning test species and substrate (origin, species) List of correspondence between study director and monitor Because no GLP deviations occurred, no list is presented Original raw data of test Original study plan and one amendment Original final report

After the respective period is expired, the sponsor will be asked regarding further proceeding (destruction, further storage, or consignment of the test item/records). If no decision from the sponsor will be receipt within 3 month after contacting, test item/records will be destroyed.


Annex

A.1 Annex 1: Details on test data

A.1.1 Test conditions

Table 6: Oxygen content of the test media. Single values [% air saturation]. Replicates were measured twice. Concentrations given as TWA concentrations.

Start new 24 h aged 24 h new 48 h aged

Control 101 102

99 100

101 100

101 100

3.70 µg CN-/L 101 102

98 100

101 101

100 100

6.95 µg CN-/L 102 102

100 99

100 101

101 101

12.9 µg CN-/L 101 101

100 100

101 101

100 100

22.5 µg CN-/L 102 101

101 101

101 101

100 101

47.0 µg CN-/L 101 101

100 100

101 100

100 100

Table 7: pH-values of freshly prepared and aged test solutions Replicates were measured twice. Concentrations given as TWA concentrations.


Control 8.4 8.4

8.5 8.5

8.5 8.5

8.5 8.5

3.70 µg CN-/L 8.4 8.4

8.4 8.4

8.5 8.5

8.5 8.5

6.95 µg CN-/L 8.4 8.4

8.4 8.5

8.5 8.5

8.5 8.5

12.9 µg CN-/L 8.4 8.4

8.5 8.5

8.5 8.5

8.5 8.5

22.5 µg CN-/L 8.4 8.4

8.5 8.5

8.5 8.5

8.5 8.5

47.0 µg CN-/L 8.4 8.5

8.5 8.5

8.5 8.5

8.6 8.5

Table 8: Temperature [°C] and light intensity [lx]. Measured in the fume hood.


temperature 20.0 20.5 20.5 20.5

light intensity 238


Table 9: Dilution water, chemical properties. Cu-free tap water, Fraunhofer IME; Holding water identical to dilution water

Conductivity (µS/cm)

Alkalinity (mmol/l)

Tot. hardness(mmol/l)

Ca-hardness(mmol/l)

Mg-hardness(mmol/l)

DOC (mg/L)

NO3 (mg/L)

273 2.4 1.1 1.1 0.0 0.6 3.3

NO2 (mg/L) NH4 (mg/L) PO4 (mg/L) Cl (mg/L) Cd (µg/L) Cr (µg/L) Cu (µg/L)

0.033 < 0.01 0.28 < 0.02 < 1.02 < 1.99 < 4.45

Fe (µg/L) Mn (µg/L) Ni (µg/L) Pb (µg/L) Zn (µg/L)

< 66.0 < 2.63 < 4.21 < 7.8 < 15.2

A.1.2 Biological endpoints Table 10: Mobile larvae after 24 h and 48 h. Number of specimens per replicate = 5 Concentrations given as TWA concentrations.

replicate Control 3.70 µg CN-/L

6.95 µg CN-/L

12.9 µg CN-/L

22.5 µg CN-/L

47.0 µg CN-/L

24 h

1 5 4 3 3 1 2

2 4 4 4 3 2 1

3 4 3 5 3 2 3

4 4 5 4 3 2 3

48 h

1 5 2 3 2 0 1

2 4 4 3 2 2 1

3 4 2 3 3 2 1

4 4 3 3 2 2 1

Table 11: Immobilisation after 24 h and 48 h. Single values of the replicates [%]. Concentrations given as TWA concentrations.

replicate Control 3.70 µg CN-/L

6.95 µg CN-/L

12.9 µg CN-/L

22.5 µg CN-/L

47.0 µg CN-/L

24 h

1 0 20 40 40 80 60

2 20 20 20 40 60 80

3 20 40 0 40 60 40

4 20 100 20 40 60 40

48 h

1 0 60 40 60 100 80

2 20 20 40 60 60 80

3 20 60 40 40 60 80

4 20 40 40 60 60 80


A.2 Annex 2: Evaluation of test data

A.2.1 Relation of Chironomus riparius Endpoints on Concentration

A.2.1.1 Summary of Results for all Endpoints at the End of Exposure Period

Summary of Results for all Endpoints at the End of Exposure Period: Critical effects as observed at end of experimental time; EC: Effective concentration for xx% reduction; 95%-CL: 95% Confidence limits; LOEC: Lowest observed effect concentration; NOEC: No observed effect concentration

Critical Conc.s [µg/L] 0-24 h 0-48 h Mobility EC10 5.3 0.9 95%-CL lower 1.6 0.0 upper 8.7 2.7 EC20 9.8 2.3 95%-CL lower 4.8 0.2 upper 14.7 4.9 EC50 32.5 12.4 95%-CL lower 21.4 6.4 upper 75.6 23.0 Mobility LOEC 22.5 12.9 NOEC 12.9 6.95 n.d.: not determined due to mathematical reasons

A.2.1.2 Mobility of Chironomus riparius as Dependent on Concentration

Mobility of Chironomus riparius as dependent on concentration of the test item; Mean: arithmetic mean; Std.Dev.: standard deviation; n: number of replicates; CV: coefficient of variation (from InputRawData)

Treatm. [µg/L] Control 3.70 6.95 12.9 22.5 47.0 0 h 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Total Introduced: 20 20 20 20 20 20 n: 4 4 4 4 4 4 24 h 0 1 2 2 4 3 1 1 1 2 3 4 1 2 0 2 3 2 1 0 1 2 3 2 Total Immobile: 3 4 4 8 13 11 n: 4 4 4 4 4 4 48 h 0 3 2 3 5 4 1 1 2 3 3 4 1 3 2 2 3 4 1 2 2 3 3 4 Total Immobile: 3 9 8 11 14 16 n: 4 4 4 4 4 4


A.2.1.3 Overview Mobility after 24 h

% Immobility caused by the test item at 24 h. Treatm.[µg/L] Introduced Mobile Immobile % Immobility Control 20 17 3 15.0 3.70 20 16 4 20.0 6.95 20 16 4 20.0 12.9 20 12 8 40.0 22.5 20 7 13 65.0 47.0 20 9 11 55.0 The control mortality of 15.0% at 24 h will be compensated using Abbott`s formula.

A.2.1.4 Effective Concentrations (ECx) for Mobility at 24 h

Probit analysis using linear max. likelihood regression: Determination of the concentration/response function; data is shown which entered the probit analysis; Log(x): logarithm of the concentration; n: number of organisms; Emp. Probit: empirical probit; Reg. Probit: calculated probit for the final function.

Treatm. [µg/L] Log(x) % Immobility n Emp. Probit Weight Reg. Probit Control 0.00 20 excluded 3.70 0.568 5.88 20 -1.1059 5.204 -1.528 6.95 0.842 5.88 20 -1.1059 8.200 -1.085 12.9 1.109 29.41 20 -0.5161 10.888 -0.653 22.5 1.352 58.82 20 0.2212 12.424 -0.259 47.0 1.672 47.06 20 -0.0737 12.426 0.259 excluded: value not in line with the chosen function Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively.

Parameters of the probit analysis: Results of the regression analysis Parameter Value Computation runs: 5 Slope b: 1.61817 Intercept a: -2.44711 Variance of b: 0.15948 Goodness of Fit Chi²: 5.70195 Degrees of freedom: 3 p(Chi²): 0.12705 Log EC50: 1.51227 SE Log EC50: 0.11532 g-Criterion: 0.23397 F: 8.639 p(F) (df: 1;3): 0.061 Chi² is a goodness of fit measure. If the probability, p(Chi²), is lower or equal than 0.001, data is much scattering round the computed dose/response function. In this case and with quantal data, confidence limits are corrected for heterogeneity (= are made wider; so, check whether these results are reasonable!).


Results of the probit analysis: Selected effective concentrations (ECx) of the test item and their 95%- and 99%-

confidence limits Parameter EC10 EC20 EC50 Value [µg/L] 5.3 9.8 32.5 lower 95%-cl 1.6 4.8 21.4 upper 95%-cl 8.7 14.7 75.6 n.d.: not determined due to mathematical reasons Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively. The p(F) is greater than 0.05; i.e. the slope was not significantly different from zero. The effect parameters and confidence limits can be meaningless. Slope function after Litchfield and Wilcoxon: 4.149 (The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b); please note that small values refer to a steep concentration/response relation and large ones to a flat relation.)

Fig.: Concentration-effect curve showing the influence of the test item on mobility of the introduced

Chironomus riparius as observed after 24 h.


A.2.1.5 Overview Mobility after 48 h

% Immobility caused by the test item at 96 h. Treatm.[µg/L] Introduced Mobile Immobile % Immobility Control 20 17 3 15.0 3.70 20 11 9 45.0 6.95 20 12 8 40.0 12.9 20 9 11 55.0 22.5 20 6 14 70.0 47.0 20 4 16 80.0 The control mortality of 15.0% at 48 h will be compensated using Abbott`s formula.

A.2.1.6 Effective Concentrations (ECx) for Mobility at 48 h

Probit analysis using linear max. likelihood regression: Determination of the concentration/response function; data is shown which entered the probit analysis; Log(x): logarithm of the concentration; n: number of organisms; Emp. Probit: empirical probit; Reg. Probit: calculated probit for the final function.

Treatm. [µg/L] Log(x) % Immobility n Emp. Probit Weight Reg. Probit Control 0.00 20 excluded 3.70 0.568 35.29 20 -0.3686 11.173 -0.597 6.95 0.842 29.41 20 -0.5161 12.362 -0.285 12.9 1.109 47.06 20 -0.0737 12.731 0.020 22.5 1.352 64.71 20 0.3686 12.331 0.297 47.0 1.672 76.47 20 0.6635 10.840 0.662 excluded: value not in line with the chosen function Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively.

Parameters of the probit analysis: Results of the regression analysis Parameter Value Computation runs: 4 Slope b: 1.13998 Intercept a: -1.24455 Variance of b: 0.12027 Goodness of Fit Chi²: 1.57175 Degrees of freedom: 3 p(Chi²): 0.66581 Log EC50: 1.09173 SE Log EC50: 0.11385 g-Criterion: 0.35554 F: 20.624 p(F) (df: 1;3): 0.020 Chi² is a goodness of fit measure. If the probability, p(Chi²), is lower or equal than 0.001, data is much scattering round the computed dose/response function. In this case and with quantal data, confidence limits are corrected for heterogeneity (= are made wider; so, check whether these results are reasonable!).


Results of the probit analysis: Selected effective concentrations (ECx) of the test item and their 95%-confidence

limits (according to Fieller`s theorem). Parameter EC10 EC20 EC50 Value [µg/L] 0.9 2.3 12.4 lower 95%-cl 0.0 0.2 6.4 upper 95%-cl 2.7 4.9 23.0 n.d.: not determined due to mathematical reasons or inappropriate data Inhibitions lower equal 0% or greater equal 100% were replaced by 0.100 and 99.900, respectively. The p(F) is greater than 0.05; i.e. the slope was not significantly different from zero. The effect parameters and confidence limits can be meaningless. Slope function after Litchfield and Wilcoxon: 7.537 (The slope function is derived from the slope, b, of the linearized probit function and computes as S = 10^(1/b); please note that small values refer to a steep concentration/response relation and large ones to a flat relation.)

Fig.: Concentration-effect curve showing the influence of the test item on mobility of the introduced

Chironomus riparius as observed after 48 h.


A.2.1.7 No Observed Effect Concentrations (NOEC) for Mobility at 24 h

Fisher`s Exact Binomial Test with Bonferroni Correction: Pair-wise comparisons between treatment and control on the multiple significance level (alpha is 0.05; one-sided greater). Pair-wise comparisons are performed sequentially using the adjusted Alpha* (= alpha/(k-1); k: number of comparisons (after Holm 1979)); Ho (no effect)is accepted, if the probability p > Alpha*.

Treatm.[µg/L] Introduced Mobile Immobile % Immobility p alpha* sign. Control 20 17 3 15.0 3.70 20 16 4 20.0 0.500 0.050 - 6.95 20 16 4 20.0 0.500 0.025 - 12.9 20 12 8 40.0 0.078 0.017 - 22.5 20 7 13 65.0 0.002 0.010 + 47.0 20 9 11 55.0 0.009 0.013 + +: significant; -: non-significant A NOEC of 12.9 µg/L is suggested by the program.

A.2.1.8 No Observed Effect Concentrations (NOEC) for Mobility at 48 h

Fisher`s Exact Binomial Test with Bonferroni Correction: Pair-wise comparisons between treatment and control on the multiple significance level (alpha is 0.05; one-sided greater). Pair-wise comparisons are performed sequentially using the adjusted Alpha* (= alpha/(k-1); k: number of comparisons (after Holm 1979)); Ho (no effect)is accepted, if the probability p > Alpha*.

Treatm.[µg/L] Introduced Mobile Immobile % Immobility p alpha* sign. Control 20 17 3 15.0 3.70 20 11 9 45.0 0.041 0.025 - 6.95 20 12 8 40.0 0.078 0.050 - 12.9 20 9 11 55.0 0.009 0.017 + 22.5 20 6 14 70.0 0.001 0.013 + 47.0 20 4 16 80.0 <0.001 0.010 + +: significant; -: non-significant A NOEC of 6.95 µg/L is suggested by the program.


A.3 Annex 3: Analytical Report - Details of method and results

A.3.1 Preface and Scope

The concentrations of free cyanide in the Chironomid acute test were determined with a photometric method using the Merck Spectroquant® Testkit (No. 14800). The principle of the method is that cyanide ions react with a chlorinating agent to form cyanogen chloride, which in turn reacts with 1,3-dimethylbarbituric acid in the presence of pyridine to form a violet dye (König reaction). This dye is determined photometrically at 588 nm. The method is analogous to EPA 335.2, ISO 6703, and DIN 38405 D13 + 14. The test measures only cyanide ions (free cyanide). To assess the stability of free cyanide during the 24 hour exposure period the kinetic of the dissipation of the cyanide ions was determined in a preceding non-GLP study (see A.3.5.2).

A.3.2 Chemicals and equipment

Sodium cyanide (Merck) Package contents of Merck Spectroquant®:

1 bottle of reagent CN-1 1 bottle of reagent CN-2 1 bottle of reagent CN-3

Purified water, produced with purification system PURELAB® Ultra (ELGA LabWater)

15 mL plastic test tubes with screw-cap Spectroquant Photometer NOVA 400

A.3.3 Sample preparation and measurement

Sample aliquots between 2 and 10 mL of the aqueous test medium were pipetted into the test tubes and filled up with dilution water to 10 mL, if necessary. - Addition of reagent CN-1: 2 level green microspoons (in the cap of the bottle) were

added and shaken vigorously until the reagent was completely dissolved. - Addition of reagent CN-2: 2 level green microspoons (in the cap of the bottle) were

added and shaken vigorously until the reagent was completely dissolved. The pH was checked with pH strips (pH must be between 3.0 and 3.5).

- Addition of reagent CN-3: 6 drops of the reagent were added, the test tubes were closed and mixed.

The tubes were left to stand for 5 minutes (reaction time). Then the measurement sample was filled into the cuvette and the absorbance was measured in the photometer at 588 nm. Dilution water was used for zero position.


A.3.4 Calibration

A stock solution of sodium cyanide (purity >95 %) was prepared by adding 47.09 mg test item in 100 mL (250 mg CN/L). The stock solution was diluted to obtain the calibration standard solutions in a concentration range from 8.30 to 133 µg/L. 10 mL of the calibration solutions were measured as described in the previous chapter.

Concentration [µg CN/L] Absorbance 588 nm

132.72 2.1630

100.00 1.8860

80.00 1.2855

66.36 0.9490

60.00 0.9005

40.00 0.6295

33.18 0.3750

20.00 0.2730

16.59 0.0965

10.00 0.1130

8.30 0.0355

The basic calibration function was set up by plotting the measured absorbance against the used analyte concentrations. With the calibration data a linear regression analysis was carried out.


A.3.5 Results

A.3.5.1 Calibration

The calibration function is shown in Figure 2. As the calculated correlation coefficient (r2) was very close to 1, the linearity of the calibration function was accepted. The calibration function was calculated by linear regression analysis using the respective function of Microsoft Office Excel 2003.

y = 1.7934E-02x - 1.3319E-01

R2 = 9.8258E-01

0

0.5

1

1.5

2

2.5

0 10 20 30 40 50 60 70 80 90 100 110 120 130 140

Concentration [CN µg/L]

Ab

so

rba

nc

e 5

88

nm

Figure 2: Calibration function of cyanide

A.3.5.2 Stability of free cyanide during the exposure – non-GLP pre-test

50 and 500 µg CN/L were incubated under test condition. The concentrations of free cyanide were analysed immediately after test media preparation (0 h), after 2, 5, 7 and 24 hours. The measured values and the calculations of the elimination rates are summarised in the tables below.


Table 12: Calculation of elimination rate ke for 50 µg CN-/L

50 µg/L free cyanide nominal Calculated conc.

1 comp. fit[µg CN-/L]

Time h

Time d

Measured values [µg CN-/L] R/S

Means measured [µg CN-/L]

ln(y) means measured

0 0.0 45.986

0 0.0 46.675 1.41 46.330 3.84 42.931

2 0.08 38.060

2 0.08 42.138 1.41 40.099 3.69 37.753

5 0.21 34.039

5 0.21 35.073 1.41 34.556 3.54 31.133

7* 0.29 18.000*

7* 0.29 22.400* 1.41 20.200 3.01 27.378

23.5 0.98 8.500

23.5 0.98 11.700 1.41 10.100 2.31 9.481

R/s crit 2.43 mean 32.93 3.36

* considered as outlier, not included in the evaluation, see next Figure

Fit statistics: One compartment: ln-linear fit: c=c0 exp(-ke t) Intercept: 3.834 slope ke :1.490 t time [d] r²: 0.999 n: 4

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

45.0

50.0

0.0 6.0 12.0 18.0 24.0

Time [h]

Co

nc

. [

µg

/L]

data Conc.

means

Exponentiell (data Conc.)

outlier

Figure 3: Dissipation of cyanide during 24 hour in medium with 50 µg CN-/L


Table 13: Calculation of elimination rate ke for 500 µg CN-/L

500 µg/L free cyanide nominal Calculated conc.

1 comp. fit[µg CN-/L]

Time h

Time d

Measured values [µg CN-/L] R/S

Means measured [µg CN-/L]

ln(y) means measured

0 0.0 467.325

0 0.0 508.104 1.41 487.715 6.19 486.107

2 0.08 381.747

2 0.08 371.983 1.41 376.865 5.93 382.515

5 0.21 238.160

5 0.21 338.671 1.41 288.415 5.66 267.007

7 0.29 190.359

7 0.29 200.744 1.41 195.551 5.28 210.106

23.5 0.98 20.714

23.5 0.98 37.830 1.41 29.272 3.38 29.090

R/s crit 2.43 mean 275.56 5.29

Fit statistics: One compartment: ln-linear fit: c=c0 exp(-ke t) Intercept: 6.186 slope ke: 2.876 t time [d] r²: 0.998 n: 5

0

100

200

300

400

500

600

0.0 6.0 12.0 18.0 24.0

Time [h]

C [

µg

/L]

data C

means

Exponentiell (means)

Figure 4: Dissipation of cyanide during 24 hour in medium with 500 µg CN-/L


It can be seen, that 1st order equations show a good fit (r² 0.999). Therefore, a simple geometric mean or time weighted average (TWA) calculation can be applied to the results. It can be seen that the measured concentrations in the aged test media of all test concentrations in the chironomid acute test were lower than the LOQ. According to the OECD Guidance Document for difficult substances, for concentrations <LOQ (7.4 µg CN-/L) the half LOQ (3.7 µg CN-/L) was used for the calculation of the TWA measured concentration (Table 14).

A.3.5.3 Results of the analysed test solutions

Based on the calibration function the concentration was calculated using the equation x=(y+0.13319)/0.017411. The results of the analysed test samples are listed in Table 14 (start of exposure interval), Table 15 (end of exposure interval) and Table 2 (TWA values, main report).

Table 14: Analysed concentrations [µg CN-/L] and recovery rates [% of nominal] of free cyanide in the test media at start of the exposure intervals.

DF = Dilution factor of the measurement sample LOQ = 7.4 µg CN-/L < LOQ: for calculation of the mean measured concentration ½ LOQ was used (3.70 µg CN-/L). * At test start the double concentrated application solutions were measured. # Values in brackets represent the theoretical concentrations in the test vessels

Nominal concentration

Absorbance Conc. in sample

Final concentration

(Blank corrected)

% of nominal

[µg CN-/L] 1 2 Mean [µg CN-/L] DF [µg CN-/L] [µg CN-/L] [%] Day 0 fresh * (#) 415.16 1.325 1.358 1.342 82.23 5 411.14 402.6 (201.3) 97.0 165.64 1.309 1.284 1.297 79.72 2 159.44 150.9 (75.4) 91.1 66.36 1.193 1.271 1.232 76.12 1 76.12 67.6 (33.8) 101.8 26.54 0.544 0.574 0.559 38.60 1 38.60 30.1 (15.0) 113.2 10.62 0.228 0.202 0.215 19.42 1 19.42 10.9 (5.4) 102.4 Blank 0.022 0.018 0.020 8.54 1 8.54

Day 1 fresh 207.58 1.246 1.138 1.192 73.89 2.5 184.73 175.9 84.7 82.82 1.197 1.154 1.176 72.97 1 72.97 64.1 77.4 33.18 0.496 0.572 0.534 37.20 1 37.20 28.3 85.4 13.27 0.169 0.192 0.181 17.49 1 17.49 8.6 64.9 5.31 0.107 0.104 0.106 13.31 1 13.31 < LOQ - Blank 0.032 0.02 0.026 8.88 1 8.88


Table 15: Analysed concentrations [µg CN-/L] of free cyanide in the aged test media at the end of the

exposure intervals.

DF = Dilution factor of the measurement sample LOQ = 7.4 µg CN-/L < LOQ: for calculation of the mean measured concentration ½ LOQ was used (3.70 µg CN-/L). n.d. = Not detectable due to impropriate data

Nominal concentration

Absorbance Conc. in sample

Final concentration

(Blank corrected)

% of nominal

[µg CN-/L] 1 2 Mean [µg CN-/L] DF [µg CN-/L] [µg CN-/L] [%] Day 1 aged

207.58 (rep. 1) 0.073 0.087 0.08 11.89 1 11.89 <LOQ n.d. 207.58 (rep. 2) 0.056 0.098 0.08 11.72 1 11.72 <LOQ n.d. 207.58 (rep. 3) 0.071 0.078 0.07 11.58 1 11.58 <LOQ n.d. 207.58 (rep. 4) 0.037 0.039 0.04 9.55 1 9.55 <LOQ n.d. 207.58 (Mean) 11.18 <LOQ n.d. 82.82 0.048 0.062 0.06 10.49 1 10.49 <LOQ n.d. 33.18 0.049 0.037 0.04 9.82 1 9.82 <LOQ n.d. 13.27 0.075 0.061 0.07 11.22 1 11.22 <LOQ n.d. 5.31 0.079 0.051 0.07 11.05 1 11.05 <LOQ n.d. Blank 0.044 0.052 0.05 10.10 1 10.10

Day 2 aged

207.58 (rep. 1) 0.085 0.082 0.08 12.08 1 12.08 <LOQ n.d. 207.58 (rep. 2) 0.304 0.296 0.3 24.15 1 24.15 14.1 6.8 207.58 (rep. 3) 0.106 0.121 0.11 13.76 1 13.76 <LOQ n.d. 207.58 (rep. 4) 0.024 0.016 0.02 8.54 1 8.54 <LOQ n.d. 207.58 (Mean) 14.63 <LOQ n.d. 82.82 0.048 0.019 0.03 9.29 1 9.29 <LOQ n.d. 33.18 0.033 0.049 0.04 9.71 1 9.71 <LOQ n.d. 13.27 0.02 0.019 0.02 8.51 1 8.51 <LOQ n.d. 5.31 0.006 -0.005 0.00 7.45 1 7.45 <LOQ n.d. Blank 0.012 0.024 0.02 8.43 1 8.43

A.3.5.4 Discussion of the results of the analysed fresh test solutions

To ensure measured values for the start concentrations of all five treatments at least one time, at test start the double concentrated application solutions were measured. For TWA calculation, the results were halved. With recovery rates of 91 – 113 % of nominal, it was shown that chironomid larvae were exposed at date of application with the aspired concentrations. At renewal, new test solutions were measured after intensive mixing with a pipette out of an additional analytical replicate. Recovery rates of 65 – 85 % of nominal give a hint that during mixing the cyanide already begins to leave the test solutions. However, even when the chironomid larvae were exposed from the date of application (and not after mixing), for TWA calculation the measured values were applied.


A.4 Annex 4: Inspection certificate of the test item according to EN 10204 3.1


A.5 Annex 5: GLP certificate

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