Reaction products of N,N'-ethane-1,2-diylbis(1,3-propanediamine), cyclohexane, peroxidized 4-butylamino-2,2,6,6-tetramethylpiperidine and 2,4,6-trichloro-1,3,5-triazine

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 22.01.2014 to 28.02.2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP study according to current OECD guideline.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

but with no impact on the study results: In the definitive test the temperature was in a range of 19.2 – 21.7 ºC with a variation of 2.5 °C. However, the daily range was with a variation of up to 2.0 °C

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Control and the WAF of a loading of 10 mg/L (limit test).
- Sampling method: The content of the total organic carbon (TOC) was determined to characterize the exposure level of the test item at the time of renewing the solutions. The content of the TOC was determined using a validated method and the total organic carbon analyzer TOC-L.
- Sampling: In the definitive test the samples collected from the WAF of a loading of 10 mg/L and the control were: fresh solutions at exposure initiation and old solutions on day 2 (first renewal); fresh solutions on day 7 (third renewal) and old solutions on day 9 (fourth renewal); fresh solutions on day 14 (sixth renewal), and old solutions on day 16 (seventh renewal); old solutions at exposure termination. Old solutions were exposed in the test conditions without test organisms and without feeding them.

Vehicle:

no

Details on test solutions:

Control and the WAF of a loading of 10 mg/L (limit test).

DETAILS ON PRETREATMENT
PRELIMINARY TEST:
- Mixing: The prolonged mixing was to enable the highest solubility of the test item in the test conditions. Two portions of the test item were weighed separately and mixed with test medium. The mixtures of 10 mg/L were non-homogeneous with non-dissolved particles visible, therefore each mixture was sonicated for 30 min (ultrasonic bath, Sonic-10, Polsonic). After sonication, the mixtures were non-homogeneous with visible non-dissolved particles suspended in the liquid phase and therefore they were mechanically shaken at 60 rpm at room temperature. After 24 hours of mixing, the mixtures were visually unchanged.
- Filtration: One mixture was filtered through a conditioned nitrocellulose membrane filter (HAWG filter, pore size: 0.45 µm, Millipore) and the other was not. The filtrate WAF of a loading of 10 mg/L was transparent, colourless and visually homogeneous. The filtrate WAF of a loading of 10 mg/L and the other non-filtered mixture plus the control were used for exposure.

DEFINITIVE TEST:
The definitive test was performed with a single test item concentration as a limit test. Since the test item is of low solubility, the Water Accommodated Fraction of the test item in test medium was used for exposure.
-Mixing: The weighed amount of the test item was mixed with test medium to receive a mixture of a loading of 10 mg/L (analytical balance SBC-22, Scaltec Instruments GmbH). The mixture was nonhomogeneous with non-dissolved particles of the test item visible. The mixture was sonicated for 30 min (ultrasonic bath, Sonic-10, Polsonic). Then, it was mixed continuously at 60 rpm at room temperature for 24 h (mechanic shaker, JWElectronic).
- Filtration: After 24 h the mixture was non-homogeneous with visible non-dissolved particles, thus it was separated by a filtration through a conditioned nitrocellulose membrane filter (HAWG filter, pore size: 0.45 µm, Millipore).
The filtrate was transparent, colourless and visually homogeneous. The filtrates of the mixture and the control were used for exposure as the Water Accommodated Fraction, i.e. WAF of a loading of 10 mg/L and the control.

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
The content of the total organic carbon (TOC) was determined to characterize the exposure level of the test item at the time of renewing the solutions. Old solutions were exposed in the test conditions without test organisms and without feeding them.
The results are presented as the concentrations of TOC and were corrected by the value measured for the control sample in each analyzed series.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna Straus
- Source: neonates collected from the laboratory culture cultivated at the Institute of Industrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Laboratory of Aquatic Toxicology.
- Age of parental stock: 21 – 25 days old
- Feeding during test:
Preliminary test: fresh suspension of Pseudokircheriella subcapitata acquired from standard mass laboratory culture in AAP medium, concentrated by centrifugation and resuspended in Elendt M7 medium.
Definitive test: In the definitive test the test organisms were fed with algal suspension mixture: Pseudokirchneriella subcapitata : Desmodesmus subspicatus (2:1, volume per volume) acquired from separate standard mass laboratory cultures of these species. The cultures of these algal species were concentrated by centrifugation, resuspended in Elendt M7 medium and combined.

ACCLIMATION
- Acclimation period: 24 h
- Acclimation conditions:
- Temperature: 18 – 22 °C
- Light: dark cycle of 16 h/8 h
- Medium: The culture was maintained in Elendt M7 medium
- Type and amount of food: algal suspension mixture of Desmodesmus subspicatus originating from separate cultures in the Laboratory of Aquatic Toxicology.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

21 d

Hardness:

Total hardness determined in fresh and old solutions was in range of 199 – 211 mg CaCO3/L

Test temperature:

- Preliminary test: 20.2 - 22.6 °C
- Definitive test: 19.2 – 21.7 ºC with a variation of 2.5 °C. However, the daily range was with a variation of up to 2.0 °C with only one exception; on day 9 the daily range was 2.1 °C.

pH:

- Preliminary Test: 7.32 - 7.74
- Definitive Test:
Control: In fresh solutions of control measured pH values were in the range of 7.43 – 7.73. In old solutions of control measured pH values were in the range of 7.66 – 8.34
WAF Solutions: In fresh solutions of the WAF of a loading of 10 mg/L measured pH values were in the range of 7.48 – 7.77. In old solutions of the WAF of a loading of 10 mg/L measured pH values were in the range of 7.58 – 8.36

Dissolved oxygen:

- Preliminary test: 8.3 - 8.9 mg/L
- Definitive test:
Control: In fresh solutions of the control the dissolved oxygen concentration was in the range of 8.2 – 8.6 mg/L. In old solutions of control the dissolved oxygen concentration was in the range of 8.2 – 9.2 mg/L.
WAF solutions: In fresh solutions of the WAF of a loading of 10 mg/L the dissolved oxygen concentration was in the range of 8.1 – 8.4 mg/L. In old solutions of the WAF of a loading of 10 mg/L the dissolved oxygen concentration was in the range of 7.9 – 9.4 mg/L.

Salinity:

- Conductivity was in the range of 563 – 606 µS/cm

Nominal and measured concentrations:

- Control and the WAF of a loading of 10 mg/L (limit test).
- TOC results:
The Limit of quantification (LoQ) is 1.0 mg C/L for aqueous solutions.
At exposure initiation the sample of the filtrate of a loading of 10 mg/L was collected for analysis. The results were 0.944 mg C/L in the control and 0.391 mg C/L in the filtrate of a loading of 10 mg/L.

Details on test conditions:

TEST SYSTEM
- Test vessel: glass beakers of 150 mL capacity, containing 100 mL of each treatment per replicate and a complete renewal three times per week.
- Type: Test vessels were covered with a transparent plastic lid in order to minimize evaporation and to prevent accidental contamination.
- Aeration: no
- Renewal rate of test solution: Three renewals of each test concentration and the control per week were performed.
- No. of organisms per vessel: 1
- No. of vessels per concentration: 10 replicates per treatment each with one parent daphnid held individually.
- No. of vessels per control: 1

TEST MEDIUM / WATER PARAMETERS
- In both the preliminary test and the definitive test the Elendt M7 medium was used as the test medium.

OTHER TEST CONDITIONS
- Temperature: 19.2 – 21.7 ºC
- Photoperiod: 16 h light : 8 h darkness
- Light intensity: 1161 – 1392 lux

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material (WAF)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

>= 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material (WAF)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material (WAF)

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 10 mg/L

Nominal / measured:

nominal

Conc. based on:

other: test material (WAF)

Basis for effect:

reproduction

Details on results:

In the semi-static reproduction test on Daphnia magna the endpoint values were determined on the basis of the WAF prepared from the loading of test item.
The results of the limit semi-static test demonstrated no effects on reproductive output (the mean cumulative number of offspring per survivor), immobilization, final body size of parent daphnid and intrinsic rate of population growth and the age of the first reproduction; after 21 days of exposure in the WAF of a loading of 10 mg/L. The endpoint values therefore were based on nominal concentration of the test item expressed as the WAF of a loading of test item as follows: LOEC > 10 mg/L, NOEC ≥ 10 mg/L, 21 d-EC10 > 10 mg/L and 21-d EC50 > 10 mg/L.


The content of the total organic carbon (TOC) was determined to characterize the exposure level of the test item at the time of renewing the solutions. The results are presented as the concentrations of TOC and were corrected by the value measured for the control sample in each analyzed series. In fresh solutions of the WAF of a loading of 10 mg/L the determined TOC content was in the range of 0.549 – 0.611 mg C/L. In old solutions of the WAF of a loading of 10 mg/L the determined TOC content was in the range of 0.227 – 0.401 mg C/L.

Reported statistics and error estimates:

The statistical tests were performed with the age of the first reproduction data. The Levene’s Test on Variance Homogeneity (with Residuals) indicated homogeneity of variances. The Student-t test for Homogeneous Variances showed no significant difference between the WAF of a loading of 10 mg/L and the control. Therefore, the lowest test concentration causing changes in the age of the first reproduction LOEC is higher than WAF of a loading of 10 mg/L and the test item concentration not causing any effect on the age of the first reproduction NOEC is higher or equal the WAF of a loading of 10 mg/L.

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:

LC10

Effect concentration:

>= 10 mg/L

Additional information

The long-term toxicity to aquatic invertebrates was assessed with the test item in a key study according to OECD 211 (2012). In this semi-static reproduction test, young Daphnia magna (< 24 h old) were exposed to the test item for 21 days.

The test item is poorly water soluble, therefore a limit test with a single concentration at the limit of solubility was performed. The limit of solubility was reached by applying the Water Accommodated Fraction (WAF) method prepared from a loading of 10 mg/L with sonication, heating, prolonged mixing and sequential filtration to separate the not dissolved test item. The aim of a limit test was to demonstrate that the LOEC and NOEC values based on number of alive offspring, immobilization and final body size (body length) of parent daphnid were respectively higher than and higher than or equal to the solubility limit of the test item in test medium. The test was performed in glass beakers of 150 mL capacity, containing 100 mL of each treatment per replicate and a complete renewal three times per week. At each renewal the parent daphnids were transferred from old to fresh solutions. The offspring produced in each test vessel was counted and removed daily. The observations on immobilization of parent animals were reported daily. The body size of parent daphnids was measured at exposure termination.

 

Analytical measurements of the test item concentration were carried out by measuring the total organic carbon (TOC) content.

The results of the limit semi-static test demonstrated no effects on reproductive output (the mean cumulative number of offspring per survivor), immobilization, final body size of parent daphnid and intrinsic rate of population growth and the age of the first reproduction; after 21 days of exposure in the WAF of a loading of 10 mg/L. Since no immobilization or any other effects on parent daphnids were observed, the conclusions of the estimations based on the number of living offspring produced per parent Daphnia alive at exposure termination (i.e. the mean cumulative number of offspring per survivor) are the same as the conclusions of the estimations based on the total number of living offspring produced per parent Daphnia introduced at exposure initiation (excluding parental accidental and/or inadvertent immobilization from the analysis). The endpoint values based on the number of living offspring produced per parent Daphnia alive at exposure termination, immobilization of parent daphnid, the intrinsic rate of population growth, the final body size (body length) of parent daphnid and the age of the first reproduction are up to the solubility limit of the test item in test medium, i.e. up to a loading rate of 10 mg/L. Therefore following overall effect concentrations (mg/L) were obtained based on the WAF of a loading of 10 mg/L: LOEC > 10 mg/L, NOEC ≥ 10 mg/L, 21 d-EC10 > 10 mg/L and 21-d EC50 > 10 mg/L.