Reaction mass of (4Z,8E)-dodeca-4,8,11-trienal and (4E,8Z)-dodeca-4,8,11-trienal

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

Test item Dodecatrienal
Batch number V15 Trienal-Dest. F.4-7+V16 Trienal-Dest.F4-8.
Test substance number 18/0072-2
Purity (certified) 91.6 area-% (GC, RTX-1701 capillary)
92.7 area-% (GC, DB-5 MS UI capillary)
Chemical name 4,8,11-Dodecatrienal
Molecular weight 178 g/mol
Appearance Colorless, clear liquid
Expiry date 2020-05-31
Recommended storage Keep container tightly closed in a cool and well-ventilated place at ambient conditions (room temperature).

Analytical monitoring:

yes

Details on sampling:

All concentration levels, the solvent control and the control were the test item analytically verified via GC-MS. Samples were taken in the fresh media at the start of the exposure and at the renewal of the test solutions (0 and 24 hours) as well as in 24-hours old media at the renewal and at the end of the exposure (24 and 48 hours). Due to 100% mortality after 24 hours, the highest test item concentration was analyzed at 0 and 24 hours only. Additionally, samples prepared with nitrogen-treated dilution water and incubated without daphnids were analyzed at the start and the end of the first exposure interval. The samples were analyzed with a GC-MS method, implemented under non-GLP and documented finally in the GLP raw data. The method was validated acc. to SANCO/3029/99 rev. 4 (2000) with satisfactory results respecting linearity, limit of quantification (LOQ), accuracy and specificity in a separate non-GLP study (Study-ID: 181108BH/CMV18470N). Single concentrations of the isomers were not determined, but all detectable signals were evaluated as one analyte. The composition of the analyte dodecatrienal composes of two signals with a response of approximately 60% signal 1 (cis,trans-Dodecatrienal) and 40% of signal 2 (trans,cis-Dodecatrienal), thus representing the composition of the isomers of dodecatrienal given in the final report. There was no specific signal for the third isomer, trans,trans-Dodecatrienal, either due to its low prevalence or since there is no chromatographic separation to the other isomers.
At the start of the exposure and at the renewal (0 and 24 hours), monitoring two samples per concentration level, solvent control and control were taken after preparation of all concentration levels and analyzed directly after sampling. At the renewal and at the end of the exposure (24 and 48 hours), two samples of the 24-hours old media per concentration level, solvent control and control were taken directly from the test vessels and analyzed directly after sampling.

Vehicle:

yes

Details on test solutions:

The test item is a mixture of 3 isomers and has a solubility of 43 mg/L test loadings in water. Since the limit of solubility for each isomer may be different, the composition of the test item in solution and the overall solubility may change with the loading of test item/L. Furthermore, the test item is potentially unstable in presence of oxygen. Thus, the test solutions were prepared with defined loading of the test item.
Solutions with defined loadings of the test item were prepared prior to the start of the exposure. The analytical results from the range finding test and the first definitive test show an extremely high variability of the measured concentrations at start of exposure when the concentrations were prepared by prolonged stirring. Additionally, the losses during the preparation increased with decreasing loading. During preparation losses caused by volatility could not be distinguished from losses by chemical instability, so that no loading could be defined for the start of exposure. To overcome these problems a solvent had to be used for the preparation of the test concentrations as last resort, to accelerate the dissolution of the soluble parts of the test item as much as possible. Accelerated dissolution allowed the application of sufficiently high test concentrations to reveal toxic effects of the parent compound.
For the preparation of the test loadings of the test item Dodecatrienal, appropriate stock solutions were prepared in DMF (solvent). The stock solutions were prepared in measuring flasks (5 mL capacity) by weighing out appropriate amounts of the test item and transferring it into the measuring glass flasks. Thereafter, the flasks were filled up with DMF to 5 mL. The measuring glass flasks containing the stock solutions prepared with DMF were closed and opened only for taking aliquots for preparation of the test loadings.
The test loadings were prepared by adding 0.05 mL/L of the appropriate stock solutions (see Table 3) to the dilution water (see Table 2) under powerful stirring. Thereafter, the stock solutions were overlaid with nitrogen, as the test item may be oxidized, and stirred for 1 hour with a magnetic stirrer (room temperature, approx. 1100 rpm). Additionally, for the first exposure interval two samples for each concentration were prepared with dilution water that was treated with nitrogen beforehand to reduce the oxygen content, as the test item may be oxidized. One sample was analyzed at the start of the exposure, the second one was incubated under test conditions without daphnids and was analyzed at the end of the exposure interval of 24 hours. These analyses were made to quantify the losses from oxidation during preparation of the test solutions.
After completion of stirring and a separation phase of 1 hour of standing, the test solutions were removed by siphoning (from the approximate bottom of the glass flask) via a glass tube (the glass tube was inserted into the dilution water after stirring and the glass flask was kept covered). The test item solutions were checked visually for undissolved test item (turbidity). All test item concentrations were visually clear throughout the exposure period.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test system Daphnia magna STRAUS, obtained from continuous laboratory cultures. The daphnids originated from clone 5 and were continuously cultured at the test facility over many years.
Reason for the selection of the test system Daphnia magna is the preferred species in accordance with the test guidelines and is bred at the test facility.
Origin Institut für Wasser-, Boden- und Lufthygiene (WaBoLu), 14195 Berlin, Germany
Breeder Noack Laboratorien GmbH, Käthe-Paulus-Str. 1, 31157 Sarstedt, Germany
Culture In glass vessels (2 - 3 L capacity) with approximately 1.8 L culture medium, at 20  2 °C, 16 hours illumination, light intensity of max. 1500 lx
Culture medium Elendt M4, according to OECD 202, Annex 3 (2004) was used. The composition of the culture medium is presented in Table 2.
Culture feeding The culture daphnids are fed at least 5 times per week ad libitum with a mix of unicellular green algae, e.g. Pseudokirchneriella subcapitata and Desmodesmus subspicatus. The algae are cultured at the test facility.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Hardness:

275-295 mg CaCO3/L

Test temperature:

20.0 - 20.4 °C

pH:

7.4-7.8

Dissolved oxygen:

8.15-8.80 mg/L

Nominal and measured concentrations:

Nominal: Control, solvent control, 22.2, 10.0, 4.54, 2.07, 0.939 and 0.427 mg/L
Measured: 13.5, 8.01, 3.33, 1.36, 0.490 and 0.156 mg/L (arithmetic mean initially measured)

Details on test conditions:

Test concentrations
Six concentration levels of the test item in a geometric series with a separation factor of 2.2 were tested as follows: 0.427 - 0.939 - 2.07 - 4.54 - 10.0- 22.0 mg/L
The test item concentrations were selected based on the results of non-GLP preliminary range finding tests. The study was repeated twice considering a comparable preparation procedure of the test solutions for the alga and daphnia study and the high variation of the recoveries in the 2nd definitive test.

Solvent control Additionally, a solvent control with a maximum of 0.05 mL DMF/L dilution water was prepared and tested under the same conditions as the test groups.
Solvent DMF (Merck, purity ≥ 99.8%, batch K48097253647, expiry date July 2019) was used as solvent to achieve the maximum solubility.
Control Dilution water without test item pretreated and incubated under the same conditions as the test groups.

Reference Test
A reference test with potassium dichromate was conducted as an acute immobilization test (acc. to AQS P 9/2 and OECD 202) in Elendt M4 medium under static conditions with a test duration of 24 hours once per month in order to prove the validity of the test system and test conditions at the test facility.

Test method
The study was performed under semi-static conditions (with a renewal of the test solutions after 24 hours), since the results of the preliminary tests demonstrated a marked decrease of the test concentrations over the exposure period (see Annex II). Due to the volatility of the test item, the study was carried out in closed test vessels without headspace to reduce contact with air and losses of the test item by evaporation.

Test duration 48 hours
Test vessels / volume sealed glass flasks (4.5 (ID) x 9.5 (H) cm) with screw caps were used and filled up to the top with test solutions. A test volume of approximately 130 mL was provided in each test vessel.
Number of daphnids and replicates 20 daphnids, divided into 4 replicates, each with 5 daphnids, were used for each concentration level, the solvent control and the control.
Age of the daphnids Less than 24 hours old daphnids from a healthy stock were used for at the start of the exposure the study. To achieve this juvenile daphnids were removed from the culture vessels at the latest 24 hours before the start of the exposure and discarded. The juveniles born within the following period were used for the test. No first brood progeny was used for the test.
Acclimatization Acclimatization of the daphnids was not necessary, because the composition of the dilution water (as specified in Table 2) was equivalent to the culture medium.
Application Approximately 130 mL test solution per replicate were filled into each test vessels. There was no headspace in the test vessels. The daphnids were inserted with a small amount of dilution water (start of the exposure) or test solution (water renewal) by pipette. Thereafter, the test vessels were closed immediately with screw caps.
Renewal of the test solutions The test solutions were renewed after 24 hours. For this purpose, a second set of test vessels were filled up to the top (approximately 130 mL) with the freshly prepared test solutions and the daphnids were transferred by pipette (see ‘Application’).
Test temperature (target) 18 – 22 °C, constant within ± 1 °C
Illumination (target) Diffuse light, light intensity of max. 1500 lx
Photoperiod (target) 16/8 hours light/dark cycle
Feeding The daphnids were not fed during the study.

Immobilization and other observations
Immobilization was determined in all groups after 24 and 48 hours. An animal was considered immobile, if it was not able to swim in the water phase within 15 seconds after gentle agitation of the test vessel. Observation of other adverse, sub-lethal effects (e.g. discoloration, abnormal behavior etc.) was done, but none occurred during the course of the study:

Dilution water
Prior to the start of the exposure (0 hours) and the renewal of the test solutions (24 hours), the water quality parameters (i.e. pH-value, dissolved oxygen concentration, total hardness, temperature and conductivity) of the dilution water were measured. Acid capacity and acidity of the dilution water are determined quarterly.

Test media
At the start of the exposure and at the renewal (0 and 24 hours), the water quality parameters of the fresh media (i.e. pH-value, dissolved oxygen concentration) were measured in one additional replicate (without daphnids) per concentration level, solvent control and control. At the renewal and at the end of the exposure (24 and 48 hours), the water quality parameters of the old media were measured in one appropriate replicate (containing daphnids) per concentration level, solvent control and control. The replicate with the highest immobilization rate per concentration level and control was measured.

Temperature
The incubator temperature (measured in air with a thermohygrograph) was recorded throughout the period of the test.

Criteria for the water quality parameters (target)
• The dissolved oxygen concentration during the exposure period should be ≥ 3 mg/L in all concentration levels, the solvent control and in the control.
• The pH should be in the range of 6 – 9.
• The deviation of the final pH-values (old media) from the initial values (fresh media) should not exceed 1.5 units

Reference substance (positive control):

yes

Key result

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

2.35 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

At the end of the preparation 65 – 80% of the nominal test concentration was detected in oxygen-free test medium without any concentration-related tendency. In test medium with oxygen 20 – 82% of the nominal concentration were measured at start of exposure. In conclusion, it cannot be excluded that the deviations from the nominal test item concentrations at the start of exposure were mainly caused by evaporation during the preparation. The arithmetic mean measured concentrations at start of the first and second exposure was considered to provide a conservative estimation of the loading of test item at the start of exposure.
The measured concentrations at the end of the two exposure intervals were in the range of 64 to 1% of the initially measured concentration. In the oxygen-free test medium the values were in the range 71 - 1% of the initially measured value. The decrease was obviously markedly higher in lower test concentrations. A markedly higher stability of comparable test concentrations was observed in the parallel algae test (Noack Study-ID: 181108BH / SPO18470) in oxygen-free test medium (measured concentration after 24h = 78 – 97% of the initially measured concentration). These observations indicate that the test item was instable in the daphnia test medium, however, it was less likely that the losses were caused by evaporation.
The effects may be caused by the test item and by its degradation products, which were not considered by analytical measurements.

Results with reference substance (positive control):

The percentage of immobility for the reference item potassium dichromate (SIGMA-ALDRICH, batch number MKBV0900V, purity 99.0%, CAS RN 7778-50-9) was determined after 24 hours from 2019 03-06 to 2019-03-07.
EC50 = 2.17 mg/L

Reported statistics and error estimates:

The EC10- and the EC50-values with 95% confidence limits after 24 and 48 hours of exposure were calculated by probit analysis with the software ToxRat Professional.

Validity criteria fulfilled:

yes

Conclusions:

≤ 10% of the daphnids in the control immobile or showing signs of disease or stress, e.g. discoloration or unusual behavior such as trapping on the surface of the water, during the 48-hour test period.
≥ 3 mg O2 /L during the exposure period in all concentration levels, the solvent control and in the control.

Based on the arithmetic mean initially measured concentrations of the test item Dodecatrienal (main components), the 48 hours-EC50 for Daphnia magna was 2.35 mg test item/L.

Description of key information

Acutely toxic for aquatic invertebrates.

Additional information

In a semi-static test conducted according to OECD TG 202 50 % of Daphnia magna were immobile at a substance concentration of 2.35 mg/l after 48 hours (BASF SE, 2018) based on the arithmetic mean initially measured concentrations of the test item.