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Long-term toxicity to aquatic invertebrates

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Reference
Endpoint:
long-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 211 (Daphnia magna Reproduction Test)
Version / remarks:
2012
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
- Lot No: S019341917
- Batch No: MK 188/19
- Composition type: UVCB
- Purity: UVCB with 35.2% active substance in water
- Physical state: clear liquid
- Colour: colourless
- Storage conditions: Keep tightly closed, store at room temperature in a dry place, protect from sunlight and frost.
- pH: 5.8 (100 g/L, 20°C) in water
- Density: 1.01 g/cm³ (20°C)
Analytical monitoring:
yes
Details on sampling:
- Sampling method:
Samples of fresh test solutions were taken from the test solution preparations before distributing it to the 10 replicates. For aged test solutions, respective samples were taken from a representative test vessel per treatment. The representative test vessel for the sample of the aged test solution was chosen randomly at each sampling. Sampling was performed three times per week in fresh and aged test media. Duplicate samples of 5 mL were taken and 5 mL methanol was added to each sample for stabilising.
- Sample storage conditions before analysis:
Chemical analysis for one set of the processed samples was applied immediately after sampling. The second set of samples was stored as retain samples. If it turns that samples cannot be measured within 24 h after sampling, they were immediately stored at -18 °C until analysis. All samples were discarded after finalisation of the study.
Details on test solutions:
PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
For preparations of test item concentrations, approximately 31.6 mg test item were weighed and transferred to 1000 mL test medium and afterwards stirred for 2 hours at about 300 rpm at room temperature, yielding a final concentration of 31.6 mg test item/L, corresponding to 11.1 mg active substance/L. This solution served as highest test concentration and was serially diluted with test medium to prepare the lower test concentrations.
Once per week, new test vessels were saturated with fresh test solution. On these days, 1500 mL test medium were prepared by weighing in approximately 47.4 mg test item. The test solutions were freshly prepared each day immediately before renewal.
Test organisms (species):
Daphnia magna
Details on test organisms:
TEST ORGANISM
- Common name: Daphnia magna
- Age at study initiation (mean and range, SD): 4-24h old
- Source: German Federal Environment Agency, Institut für Wasser-, Boden- und Lufthygiene. Specimens used in the test were bred in the laboratory of the Fraunhofer IME.
- Feeding during test: The daphnids were fed during the test with suspensions of the unicellular alga Desmodesmus subspicatus and JBL ArtemioFluid in a ratio of 9 : 1 (vol/vol). The content of food in the test suspensions, measured at 585 nm, was kept constant at 0.2 mg C/Daphnia * Day.

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES, INCLUDING CULTURING CONDITIONS:
Pre-Treatment:
Adult Daphnia, at least 3 weeks old, were separated from the stock population by sieving. Batches of 30 to 50 animals were held at room temperature in ca. 1.8 L dilution water for one week. During this week the daphnids were fed daily with an algal suspension (Desmodesmus subspicatus) and ArtemioFluid (JBL GmbH & Co. KG). Algae growing in the log-phase were centrifuged and the pellet was re-suspended in a few mL of medium. 5 mL of this suspension was given to about 1.8 L Daphnia medium. The water was changed three times per week. Newborn D. magna were separated by sieving, the first generation was discarded. Individuals applied in the test were transferred with a bore Pasteur pipette a few hours after sieving to ensure applying only healthy specimens.
Test type:
semi-static
Water media type:
freshwater
Limit test:
no
Total exposure duration:
21 d
Hardness:
250-350 mg CaCO3/L
Test temperature:
18.7-19.6°C
pH:
7.08-9.00 (7.25-8.42 in new medium, 7.08-9.00 in aged medium)
Dissolved oxygen:
6.02-11.9 mg/L
Nominal and measured concentrations:
Nominal concentrations: Control, 0.316, 1.00, 3.16, 10.0 and 31.6 mg test item/L (equivalent to 0.111, 0.352, 1.11, 3.52 and 11.1 mg active substance/L)

Measured concentrations: The TWM measured concentrations of initial and 24h aged test solutions were 0.184, 0.675, 2.33, 6.96 and 28.6 mg test item/L, corresponding to 0.065, 0.238, 0.822, 2.45 and 10.1 mg a.s./L (58.1 – 90.4 % of the nominal concentrations)




Details on test conditions:
TEST SYSTEM
- Test vessel: Numbered glass beakers (50 mL nominal volume) were used as test containers. The beakers were pre-saturated with the test item for at least 30 minutes. Two sets of glass vessels were saturated before test start. Test beakers with aged test medium were not emptied until the next renewal of test medium. This way it was ensured that the test beakers did not dry out and the glass walls remained saturated. Before the media renewal, the aged test solution was discarded and the beakers were cleaned by flushing with test medium once. The beakers were filled with test medium of the same test concentration again. The containers were filled up with about 50 mL test solution. The beakers were covered with glass panes to prevent from evaporation as much as possible, but also permit gaseous exchange between the medium and the atmosphere and access of light. A new set of glass vessels was saturated and used every new week.
- Renewal rate of test solution (frequency/flow rate): A non-GLP analytical pre-test was performed prior the Daphnia magna, Reproduction Test, in order to investigate the solubility and stability of the test item and its homologues (alkyl amido amine oxide constituents with alkyl C-chain lengths of C8-C18) under test conditions. For this, two test concentrations of 0.1 and 10.0 mg test item /L were prepared. The test solutions were filled into glass beakers (50 mL) and incubated over 72 hours under test conditions of a chronic Daphnia magna, reproduction test. Analytical measurements of the different homologues of the test item (alkyl amido amine oxide constituents with alkyl C-chain lengths of C8 – C18) were performed at test start (0h) and after 24h, 48h and 72h. The time period of 72 hours was chosen as this reflects the longest incubation period in a chronic Daphnia study with a standard approach of three medium renewals per week.
Based on the results of the analytical pre-test, which showed a fast decrease of the concentrations of the homologues C12-C18, it was decided to perform the definitive test with a daily media renewal.
- No. of organisms per vessel: 1 young female Daphnia per vial
- No. of vessels per concentration (replicates): The test was performed in ten replicates per concentration with one individual specimen per replicate, respectively, resulting in ten specimens tested per concentration.
- No. of vessels per control (replicates): The control consisted of dilution water only. Ten replicates with one individual specimen each were conducted.

TEST MEDIUM / WATER PARAMETERS
- Holding and dilution water: Purified, Cu-reduced drinking water was used as holding and dilution water. The purification included filtration with activated charcoal and aeration. To avoid copper contamination, plastic water pipes were used for the testing facilities.
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, phosphate, calcium, magnesium, total hardness, alkalinity, DOC content (or NPOC, as appropriate), content of metals (cadmium, chrome, copper, iron, lead, manganese, nickel and zinc).
Hardness of the Cu-reduced water was below the recommended range (>140 mg/L as CaCO3) for Daphnia magna indicated in the guideline. Therefore, the Cu-reduced dilution water was enriched with an appropriate amount of CaCl2 to increase the hardness, resulting in values of about 250 – 350 mg CaCO3/L.
- Frequency of measurements: Oxygen concentration, pH value, and temperature of the test solutions were checked directly before adding the animals and at each water renewal in new and aged test solutions. The pH did not vary by more than 1.5 units.

OTHER TEST CONDITIONS
- Photoperiod: light/dark cycle of 16/8 hours.
- Light intensity: The light intensity did not exceed 15 μE/(m2*s) or 1125 lux.

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
The numbers of immobile daphnids were visually determined daily and the immobile daphnids were removed. Any abnormalities in appearance and behavior were recorded. The newborn daphnids per beaker were counted and removed daily. Abnormalities in condition (including male sex of adults) or presence of winter eggs was checked and recorded, if occurred. At study termination, length of the adults were measured by digital photography and image analysis and compared with the equally measured control animals.

TEST CONCENTRATIONS
The concentrations to be tested in the definitive test were based on available data of a Daphnia magna, acute Immobilization test (OECD 202) (EC50 = 55.5 mg test item/L corresponding to 19.5 mg a.s./L). The nominal test concentrations were arranged in a geometric series and spaced by a factor of 3.16.
The nominal test concentrations were: Control, 0.316, 1.00, 3.16, 10.0 and 31.6 mg test item/L,
Considering the purity of 35.2%, the test item concentrations were equivalent to 0.111, 0.352, 1.11, 3.52 and 11.1 mg active substance/L.

Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
2.04 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.82 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
immobilisation
Key result
Duration:
21 d
Dose descriptor:
EC10
Effect conc.:
1.33 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Key result
Duration:
21 d
Dose descriptor:
NOEC
Effect conc.:
0.82 mg/L
Nominal / measured:
meas. (TWA)
Conc. based on:
act. ingr.
Basis for effect:
reproduction
Details on results:
Offspring per introduced parent:
EC10 = 4.62 mg test item/L , 1.63 mg active substance/L
NOEC = 2.33 mg test item/L, 0.82 mg active substance/L
Offspring per survived parent:
EC10 = 3.79 mg test item/L, 1.33 mg active substance/L
Immobility:
EC10 = 5.80 mg test item/L, 2.04 mg active substance/L
NOEC = 2.33 mg test item/L, 0.82 mg active substance/L
Length:
EC10 = 2.86 mg test item/L, 1.01 mg active substance/L
NOEC = 2.33 mg test item/L, 0.82 mg active substance/L
Age of first reproduction:
NOEC = 2.33 mg test item/L, 0.82 mg active substance/L
Development rate:
EC10 = 6.77 mg test item/L, 2.38 mg active substance/L
NOEC = 2.33 mg test item/L, 0.82 mg active substance/L
Intrinsic rate:
EC10 = 6.24 mg test item/L, 2.20 mg active substance/L
NOEC = 2.33 mg test item/L, 0.82 mg active substance/L

Test concentrations:
The test item consists of a series of alkyl amido-amine oxide homologues. The individual alkyl amido amine oxide constituents were measured by LC-MS/MS at the start of the test and three times per week at media renewal. The sum of the measured constituents for the test concentrations showed mean recovery values between 96.7 and 106 % of nominal concentrations in freshly prepared test solutions. During the time interval until renewal of the test solution, test item concentrations decreased to 34.6 – 83.4 % of nominal (35.0 – 85.4 % of initial concentrations). Since recovery rates did not remain in a range of ± 20% of nominal concentrations throughout the test, the evaluation of biological effects was performed based on the time weighted mean (TWM) measured test concentrations, which were calculated for the evaluation of the biological parameters and determination of LOEC/NOEC.
Reported statistics and error estimates:
For each endpoint, the NOEC, LOEC, and the EC50 and EC10 were determined. A LOEC was calculated by using ANOVA followed by Williams’ t-test or an appropriate non-parametric test (e.g. Cochran-Armitage test, Bonferroni U-test or step-down Jonckheere-Terpstra). The test results showed a concentration-response relationship, therefore, the data was analyzed by regression to determine the EC50, EC20 and EC10 including the 95% confidence interval using Probit-analysis assuming log-normal distribution of the values.
The computer software ToxRat® [12] was used for statistical evaluations.
The evaluation of the concentration-effect-relationships and the calculation of effect concentrations were based on the mean measured concentrations of the sum of the measured constituents (time weighted mean, TWM) as the measured concentrations deviated by more than 20% from the nominal concentrations throughout the test.
Validity criteria fulfilled:
yes
Conclusions:
The 21-day toxicity of Amides, C12-C18 (even numbered), N-[3-(dimethylamino)propyl], N’-
oxides to the test organism Daphnia magna was determined in a semi-static system (OECD
211) exposed to nominal concentrations of 0.316, 1.00, 3.16, 10.0 and 31.6 mg test item/L
with a daily renewal of test solutions. The deviations of the measured test item concentrations from the nominal test concentrations were greater than 20 % and thus, the evaluation of the results was based on the time weighted mean (TWM) measured concentrations of the test item. During the 21-day exposure period significant effects of the test item on the reproduction of
introduced parent daphnids, immobilisation of parent daphnids, age of first reproduction,
length, development rate and intrinsic rate of population increase r of the test organisms
Daphnia magna were observed. For all considered endpoints, a NOEC of 2.33 mg test item/L and a LOEC of 6.96 mg test item/L were determined, equivalent to 0.822 and 2.45 mg a.s./L. In addition, an EC10 of 4.62 mg test item/L (1.63 mg a.s./L) and 3.79 mg test item/L (1.33 mg a.s./L) was calculated for reproduction of introduced and survived parent, respectively. For immobilization an EC10 of 5.80 mg test item/L (2.04 mg a.s./L) was calculated.

Description of key information

The study of Brüggemann (2020) is considered as key study for endpoint coverage. The study investigates the chronic toxicity of the substance to Daphnia magna according to OECD guideline 211, under semi-static conditions in freshwater and with an exposure period of 21 days. The 21d-NOEC was determined to be 0.82 mg a.i./L. The study is given a Klimisch score of 1 and was conducted under GLP.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates
Dose descriptor:
NOEC
Effect concentration:
0.82 mg/L

Additional information

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