1,3,5-triazine-2,4,6(1H,3H,5H)-trione, compound with 1,3,5-triazine-2,4,6-triamine (1:1)

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:

from 2016-03-30 to 2016-06-03

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:

- Batch number: 17321797V0
- Expiry date: 27 September 2041

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentrations of the stock solutions were analytically determined during the study (without GLP status) and used as a basis to prepare the test solution. Aliquots of the samples were filled into into vials and then the aliquots were directly measured. Aliquots therefrom were further diluted with water (concentration range: 0.5 mg/L to 20.72 mg/L).
- Sampling method: The concentration of the test substance was determined in one representative test interval per week in each freshly prepared test solution (test vessels without daphnids) and in the 48 h or 72 h old test solutions before renewal (combined sample from test vessels with daphnids.
- Sample storage conditions before analysis: The samples were stored under ambient conditions.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: A WAF was prepared. The test concentration was prepared by dissolving the test substance in 2 L of test medium (10 mg/L) and stirring for approx. 3 days. After this time, undissolved test substance was removed by filtration. The exposure was started after separation of the undissolved material. The aqueous fraction of the test solution, after separation of the undissolved material, is considered 100 % saturated in test media.
- Stability and homogeneity: The stability of the test substance as a solution in test media and under testing conditions was determined by concentration control analysis. The stability of the test substance in the test media at room temperature over a period of 72 hours was demonstrated (without a GLP status). The test solutions were visibly homogeneous.
- Controls:
Blank Control: Control performed with the test solution without test substance
Positive control: Performed with the reference substance sodium chloride
- Evidence of undissolved material: All test solutions were visibly colorless and clear throughout each renewal period.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna STRAUS
- Source: The clone of Daphnia magna STRAUS 1820 used was supplied by the Institut National de Recherche Chimique Appliquée, France, in 1978.
- Age at study initiation: < 24 hours
- Method of breeding: Since 1978 this clone was cultured and bred continuously in the Ecotoxicology Laboratory of Experimental Toxicology and Ecology, BASF SE, Ludwigshafen Germany.
- Feeding during test: Yes
- Food type: During the test daphnids were fed with 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 1340 mg and 1350 mg TOC/L (respectively) in the algal concentrates used. The daphnids were fed a defined volume (≤149 μL) of the concentrate to reach the amount of food defined. 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 72 h test interval with 3 feedings, 0.149 mL each, a total of 0.447 mL are added to 50 mL test volume resulting in a maximal dilution of 0.9%.
- Amount: The daphnids were fed a defined volume (≤149 μL) of the concentrate to reach the amount of food defined. 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 72 h test interval with 3 feedings, 0.149 mL each, a total of 0.447 mL are added to 50 mL test volume resulting in a maximal dilution of 0.9 %.
- Frequency: daily

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

22 d

Remarks on exposure duration:

None

Post exposure observation period:

None

Hardness:

2.20 – 3.20 mmol/L

Test temperature:

21 - 22 °C

pH:

7.5 – 8.5

Dissolved oxygen:

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

Salinity:

Not applicable

Conductivity:

550 - 650 μS/cm

Nominal and measured concentrations:

- Nominal concentration: 0 (control) mg/L and 10 mg/L (Limit test)
- Measured concentrations: The measured concentration after 22 days was determined to be 7.64 mg/L.
The analytically measured concentrations of the test substance in the test water varied in the range of 61-94 % of the nominal concentration. Although variability was high, all reasonable efforts were taken to produce a saturated solution of the test substance in test media following the guidance in OECD 23. The measured concentrations do confirm exposure to the test substance, and the geometric mean measured concentrations were considered to be an accurate representation of exposure levels throughout the exposure period.

Details on test conditions:

TEST SYSTEM
Test vessel: Numbered glass beakers (nominal volume 100 mL), covered with glass Petri plates to slow evaporation.
- Test volume: 50 mL
- Material, size, headspace, fill volume: 50 mL
- Aeration: Yes. Dissolved oxygen: Must remain ≥3 mg/L during the test. To assure optimal dissolved
oxygen levels, the M4 medium is aerated for approximately 24 h prior to use.
- Renewal rate of test solution (frequency/flow rate): 22 hours
- No. of organisms per vessel: 1 animal/test vessel
- No. of vessels per concentration (replicates): 10 replicates (1 parent animal each)
- No. of vessels per control (replicates): 2
- Biomass loading rate: 1 animal/test vessel (0.02 animals/mL)

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: A synthetic fresh water (Elendt M4) is used as media for culture and test purposes according to OECD 211.
- Total organic carbon: < 2 mg/L
- Alkalinity:
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
- Ca/mg ratio: about 4 : 1
- Conductivity: 550 - 650 µS/cm
- Culture medium different from test medium: No
- Intervals of water quality measurement: 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 according to the OECD. 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.

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light : 8 hours darkness
- Light intensity: 696–758 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.


EFFECT PARAMETERS MEASURED: The objective of the study was to assess the effect of chronic exposure to the test substance on the reproduction (live offspring production) and mortality (immobilization) of Daphnia magna STRAUS over a 22-day exposure period. In addition, observed substance related effects on other endpoints (i.e. growth) are reported. 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 parent for the duration of the study. 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 protunding second antennae) with a binocular dissectiong microscope (Leitz Diavert) and a calibrated eyepiece micrometer which was checked with a standardized slide.

TEST CONCENTRATIONS
- Range finding study: In a preliminary test (experimental conduct in accordance with GLP but without a GLP status), after 16 days exposure to the test substance, no significant effect on reproduction was o bserved at 10 mg/L. According to OECD-guideline, the highest suggested test concentration for a limit test is 10 mg/L or the saturation limit in test medium whichever is lower.

- Test concentrations:
- Nominal Concentration: 0 (control) and 10 mg/L (Limit test)
- Measured Concentration: n.d (control) and 7.64 mg/L. The analytically measured concentrations of the test substance in the test water were within the range of 61-94% of the nominal concentration.
- Reason for the selection of the test concentrations: In a preliminary test (experimental conduct in accordance with GLP but without a GLP status), after 16 days exposure to the test substance, no significant effect on reproduction was observed at 10 mg/L.
- Results used to determine the conditions for the definitive study: According to OECD-guideline, the highest suggested test concentration for a limit test is 10 mg/L or the saturation limit in test medium whichever is lower.

Reference substance (positive control):

yes

Remarks:

sodium chlorid

Duration:

22 d

Dose descriptor:

LOEC

Effect conc.:

> 7.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Duration:

22 d

Dose descriptor:

NOEC

Effect conc.:

>= 7.64 mg/L

Nominal / measured:

meas. (geom. mean)

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

22 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Key result

Duration:

22 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Details on results:

There was no significant adverse effect on survival or reproduction in the treatment group after 22 days. The data were not sufficient to calculate ECx values for reproduction. 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 geometric mean measured concentrations: LOEC was determined to be greater than 7.64 mg/L and the NOEC was determined to be 7.64 mg/L.

Results with reference substance (positive control):

The EC50(48 h) of the reference substance sodium chloride (NaCl) was 5.30 g/L. 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.

Reported statistics and error estimates:

Statistical evaluations were performed to determine LOEC and NOEC and (if possible) effect concentrations (ECx). The following parameters and statistical procedures were used to evaluate significant differences to the control and determine the LOEC and NOEC:
- Reproduction as number of living young (Wilcoxon’s test, one sided analysis)
- Growth as length in mm (Student´s test, one sided analysis)
- Time to first brood (Wilcoxon’s test, one sided analysis)
- % aborted eggs (Wilcoxon’s test, one sided analysis)
- % immobile young (Wilcoxon’s test, one sided analysis)
The results of the statistical analyses were used to aid in the determination of the NOEC and LOEC. However, scientific judgment was used to determine if statistical differences were biologically meaningful. In this limit test with one test concentration, the NOEC was defined as greater than or equal to the saturation concentration under test conditions when no significant treatment-related adverse effects on survival, reproduction or growth are observed. The LOEC was defined as greater than the NOEC when no significant treatment related effects on survival, reproduction or growth are observed. Effect concentrations (ECx) could not be calculated since there were no effects at the tested concentrations. ECx values, where reported, are estimated based on best professional judgment.

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.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Dose descriptor:

NOEC

Effect concentration:

>= 10 mg/L

Additional information

In the Daphnia Reproduction toxicity test of the test substance (BASF SE, 51E0834/09E040, 2016) was conducted for a period of 22 days, following the OECD 211 (2012). The LOEC was determined to be > 10 mg/L nominal (> 7.64 mg/L measured) and the NOEC was determined to be ≥10 mg/L nominal ( ≥ 7.64 mg/L measured).

 

In addition two studies conducted with melamine (CAS 108-78-1) did not reveal regulatorily relevant effects.