Reaction products of N6,N6'-hexane-1,6-diylbis[N2,N4-dibutyl-N2,N4,N6-tris(2,2,6,6-tetramethylpiperidin-4-yl)-1,3,5-triazine-2,4,6-triamine] and allylbromide, subsequently reacted with ethaneperoxoic acid, hydrogenated

Long-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method C.20 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

no

Remarks:

A reliable analytical method for the quantitative determination of the test substance in the test media could not be developed since the test substance is a multi-constituent material and the solutions were prepared as WAF.

Vehicle:

no

Details on test solutions:

The test substance is a mixture of sparingly water soluble components. Therefore, the test solution was prepared following general guidance provided in OECD 23 in order to achieve a water accommodated fraction (WAF) of the test substance. The test solution (10 mg/L) was prepared separately by directly adding 10 mg test substance to 1 L test medium and the treatment was stirred for about 2 days. Undissolved test substance was removed by filtration with a membrane filter (Whatman, pore width 0.2 μm). The filter was conditioned with approx. 50-100 mL test solution and the filtrate was discarded. The following filtrate was used in the test. The exposure was started after separation of the undissolved material. Fresh test solutions were prepared daily. All test solutions were visibly colorless and clear throughout each renewal period.

Test organisms (species):

Daphnia magna

Details on test organisms:

Test species : Daphnia magna STRAUS
Reason for selection of the test species: Recommended species in the test guidelines
Origin: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978. From this date on this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.

Culture conditions: Daphnia brood stock are kept in mass cultures consisting of approx. 20–30 individuals for a maximum of 4 weeks. All individuals in the mass culture originate from a single female. After approximately 14 days the adults have produced at least 3 broods and the young can be used in tests. Offspring are removed from the mass cultures at least once daily during the normal work week to ensure that young daphnia are <24-h old (first instar) at test initiation. Detailed records are kept (in test facility archives) to monitor the health of Daphnia brood stock cultures including observations of young production, mortality, ephippia, and measurement of water chemistry parameters. Only young from healthy cultures without signs of stress are used for testing. Acclimatization: The Daphnia are cultured under the identical conditions as the test including test media (Elendt M4), water quality, temperature (20 ±1°C), and diet.
Age at start of exposure: <24 h

Reference substance testing:
In order to verify that the Daphnia magna culture is responding normally to toxic stress, acute toxicity tests with a reference substance are conducted monthly. Reference substance tests are conducted generally according to the OECD 202 guideline and in accordance with GLP, but without a GLP status. The EC50(48 h) of the reference substance sodium chloride (NaCl) was 4.46 g/L (experiment date: 12 Oct 2016, project number: 50E0789/12E060). This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

2.38 - 2.47 mmol/L

Test temperature:

19 - 20 ºC

pH:

7.6 - 8.0

Dissolved oxygen:

7.8 - 8.7 mg/L

Salinity:

n/a

Nominal and measured concentrations:

nominal: 10 mg/L

Details on test conditions:

Test medium: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes.
The general properties of this medium are as follows.
Total hardness: 2.20–3.20 mmol/L
Acid capacity up to pH 4.3: 0.80–1.00 mmol/L
Molar ratio Ca:Mg: about 4:1
pH value: 7.5–8.5
Conductivity: 550–650 μS/cm
Total organic carbon: <2 mg/L

Dissolved oxygen: Must remain ≥3mg/L during the test. To assure optimal dissolved oxygen levels, the M4 medium is aerated for approximately 24 h prior to use.

Test vessels : Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation.
Test volume: 50 mL
Biological loading: 1 animal/test vessel (0.02 animals/mL)
Light intensity / Photo period: 644–708 lux at a wave length of 400–750 nm. The light intensity will not exceed 1000–1500 lux for cool white light (equivalent to 15–20 μE•m-2•s-1 according to the test guideline. 16 hours light : 8 hours darkness
Aeration: none
Test solution renewal: Semi-static, daily renewal
Diet: During the test daphnids were fed daily a diet of live green algae Desmodesmus subspicatus, cultured in a synthetic medium. The algae were separated from their culture medium by centrifugation, resuspended in daphnid's medium (M4) corresponding to concentrations of 1470 mg and 1680 mg TOC*/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤136 μL) of the concentrate to reach the amount of food defined in the table below. The algae were stored in a refrigerator (dark, about 4-8°C) for maximum 21 days. By adding the algal concentrate, the test solution was slightly diluted. During a 24 h test interval with 1 feeding, a total of 0.136 mL are added to 50 mL test volume resulting in a maximal dilution of 0.27%.
Test groups: 0 (control), 10 mg/L as nominal (loading) concentrations based on test substance.
Replicates/test group: 10 replicates (1 parent animal each)
Reason for the selection of the test concentrations: In a preliminary test (experimental conduct in accordance with GLP but without a GLP status), after 14 days exposure to the test substance, no significant effect on reproduction was observed at 10 mg/L. Based on the this result and according to OECD-guideline, this chronic study was conducted as a limit test at the recommended chronic limit concentration of 10 mg/L. The raw data of the range finding test are archived together with the raw data of this study. Each test group initially consisted of 10 replicates containing one juvenile Daphnia (<24 h old) each.
Concentration control analyses are not performed, because a reliable method for analyses in the required concentration range could not be developed. However, since all reasonable efforts were taken to produce a saturated solution of the test substance in test media, following the guidance in OECD 23, the test results should be based on loading concentration. The daphnids were exposed to the entire loaded mass of test substance over each renewal period.
At start of exposure 1 neonate (<24 h old) Daphnia magna was distributing impartially into each of the 10 replicate test vessels per test group. Parent mortality, abnormal effects, and numbers of live and dead offspring were assessed every day throughout the experiment. Reproductive success was measured by counting and discarding the offspring produced by each replicate for the duration of the study. Separating neonates from adults was accomplished by gently removing adult daphnids from each chamber by means of a pipet and pouring remaining water and young daphnids through a fine mesh screen. The young collected on the screen were placed in a petri plate and counted on a light table before being discarded. On each day of the test the parent daphnids were returned to their test vessels with fresh test solution. On day 21 of exposure, the surviving adults were removed from the test vessels and euthanized by immersion in 70% isopropanol. Individuals were then isolated on a labeled glass slide. The body length of each adult daphnid was measured from the apex of the helmet to the base of the posterior spine (i.e. excluding the anal spine and protruding second antennae) with a binocular dissecting microscope (Leitz Diavert) and a calibrated eyepiece micrometer which was checked with a standardized slide. Throughout the test, the appearance of the test solutions and dissolution behavior of the test substance was observed and recorded daily. The chemical and physical parameters of the test medium (total hardness, acid capacity, pH, conductivity and total organic carbon) were determined after aeration and prior to use in the test and were within acceptable ranges. Dissolved oxygen, pH and temperature were measured in the old and in the freshly prepared test solution in replicate 1 of each test group for one interval per week. In addition, temperature was measured continuously during the whole exposure period in a separate vessel filled with water proximal to the test vessels. Hardness was measured in the freshly prepared test solution in an additional replicate of each concentration for one interval per week and in the old test solution from the combined replicates of each test group for one interval per week.

Reference substance (positive control):

yes

Remarks:

sodium chloride

Key result

Duration:

21 d

Dose descriptor:

NOELR

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

LOELR

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

reproduction

Details on results:

In a 21-day chronic toxicity study, Daphnia magna were exposed to the test substance at loading rates of 0 (control) and 10 mg/L under semi-static renewal conditions in accordance with the OECD 211 guideline. The water pH, temperature and dissolved oxygen were within acceptable guideline specifications. Mortality, reproduction and sublethal effects were observed daily. The following overall effect concentrations (mg/L) are based on reproduction (living offspring per surviving adult) as the primary population relevant response and were obtained based on loading rate (nominal concentration): LOEC/LOELR >10 mg/L, NOEC/NOELR ≥10 mg/L
The data were not sufficient to calculate ECx values for reproduction or mortality. The test substance had no observable chronic effect on Daphnia magna up to its saturation limit in test media and under test conditions. The toxicity results presented here are consistent with the results from preliminary tests. A reliable analytical method for the quantitative determination of the test substance in the test media at the required test concentration could not be developed since the test substance is a multi-constituent material and the solutions were prepared as water accommodated fractions. According to OECD 23, for tests with chemicals that cannot be quantified by analytical methods at the concentrations causing effects, the effect concentration can be expressed based on the nominal concentrations or loading rate (for mixtures). The results in this study are consistent with all validity criteria and the test is valid according to the guidelines of this study. No deviations from test guidelines or other incidents occurred during the course of the reported test which may have influenced the results.

Results with reference substance (positive control):

The EC50(48 h) of the reference substance sodium chloride (NaCl) was 4.46 g/L (experiment date: 12 Oct 2016). This result is within the range of 3.88–7.22 g/L, which represents ±2 standard deviations from the published EC50(48 h) of 5.55 g/L and indicates that the culture of Daphnia magna used in this study is responding normally to toxic stress.

Validity criteria fulfilled:

yes

Description of key information

Based on long-term (chronic) toxicity study data, the product is very likely not harmful to aquatic invertebrates.

Additional information

The long-term toxicity to aquatic invertebrates was assessed in a long-term toxicity study conducted according to OECD 211 under consideration of GLP. The test was conducted was semi-static test with a single test concentration of 10 mg/L (limit test). A reliable analytical method for the quantitative determination of the test substance in the test media at the required test concentration could not be developed since the test substance is a multi-constituent material and the solutions were prepared as water accomodated fractions. The effect concentrations are therefore based on the loading rate of 10 mg/L. After an exposure time of 21d the test substance had no observable chronic effect on Daphnia magna up to its saturation limit. The NOELR was determined to be >= 10 mg/L.