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 fish

Administrative data

Endpoint:

fish early-life stage toxicity

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

other:

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
In Annex IX of Regulation (EC) No 1907/2006, it is laid down that long-term toxicity testing to fish shall be proposed by the registrant if the chemical safety assessment indicates the need to investigate further the effects on aquatic vertebrates. According to Annex I of this regulation, the chemical safety assessment triggers further action when the substance or the preparation meets the criteria for classification as dangerous according to Directive 67/548/EEC or Directive 1999/45/EC or is assessed to be a PBT or vPvB. The hazard assessment of the present substance reveals neither a need to classify the substance as dangerous to the environment, nor is it a PBT or vPvB substance, nor are there any further indications that the substance may be hazardous to the environment. In Annex XI of Regulation (EC) No 1907/2006 the general rules for adaptation of the standard testing regime set out in Annexes VII to X are listed. According to this Annex the general rules can be adapted if testing does not appear scientifically necessary. In the present case, no toxic effects could be observed in any of the available studies (acute and chronic). Furthermore, the substance is not expected to accumulate in organisms due to its molecular size. Thus, its bioavailability at the biological target is negligible and no molecular interaction is expected to occur. Therefore, an effect towards fish is not expected. In line with Annex XI of Regulation (EC) No 1907/2006 testing does not appear scientifically necessary. Therefore, and for reasons of animal welfare a long-term toxicity test in fish is not provided.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2012-04-20 to 2012-04-27 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples for chemical analysis were collected after thorough mixing of the test concentration. The filtrates of the collected samples were analyzed within 24 h from admission in the subcontractor laboratory. Each test sample was analyzed in triplicate. The results were corrected by the value measured for blank sample in each series. The test samples were diluted with deionized water, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: Preliminary test: Each test concentration was prepared by weighing (analytical balance SBC22, Scaltec) the grinded test item and transferring into flask by multiple washing with test medium. The mixtures were sonicated for 5 min at 40°C (Ultrasonic bath, Sonic-10, Polsonic) and shaken at room temperature for 120 h (mechanic shaker, WL-2000, JW Electronic). After shaking the non-dissolved part of each mixture was separated by filtration through a conditioned nitrocellulose membrane filter (0.2μm pores, Sartorius). These Water Accommodated Fractions (WAF) were used for exposure.
Definitive study: In the definitive test the test item was used in one concentration of 100.0 mg/L WAF with one replicate of 7 fish (limit test). For the test concentration the amount of the grinded test item was weighed into a glass cristallizer and quantitatively transferred into a glass flask by multiple washing with the test medium. The glass flask was filled up to 5 L volume. The mixture was sonicated for 30 min at 40°C (Ultrasonic bath, Sonic-10, Polsonic) and shaken at room temperature for 72 h (mechanic shaker, WL-2000, JW Electronic). After shaking the non-dissolved part of the mixture was separated by filtration through a conditioned nitrocellulose membrane filter (0.45 μm pores, Millipore) [4]. This filtrate was the Water Accommodated Fraction (WAF) used for exposure. The water for the control was shaken for 72 h (mechanic shaker, WL-2000, JW Electronic). After shaking the control was filtrated through a conditioned nitrocellulose membrane filter (0.45 μm pores, Millipore) [4]. Samples for chemical analysis were collected after thorough mixing of the test concentration. 7 fish were introduced into each aquarium. A total number of 14 fish was used in the test, i.e. seven fish in the test concentration and the control.

Test organisms (species):

Danio rerio (previous name: Brachydanio rerio)

Details on test organisms:

TEST ORGANISM
- Common name: Zebrafish
- Source: ‘The Culture of fish in Goczałkowice’ (Hodowla ryb w Goczałkowicach), Poland.
- Age at study initiation: 5 months
- Average body length: 2.7 cm ± 0.2 cm (min 2.4 cm, max 3.0 cm)
- Average weight at study initiation: 0.26 g
- Feeding during test: none

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

22.6 – 23.7 ºC

pH:

7.69 - 7.83

Dissolved oxygen:

91 - 99%

Nominal and measured concentrations:

Nominal concentrations: 0, 100 mg/L
Measured concentrations: Based on the result of non-specific analysis of TOC content it cannot be excluded that only a very small part of the test item (some components) were dissolved in test medium.

Details on test conditions:

TEST SYSTEM
- Test vessel: 5L glass aquaria
- Aeration: yes
- No. of organisms per vessel: 7
- No. of vessels per concentration: 1
- No. of vessels per control: 1
- Biomass loading rate: 0.36 g/L

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline
- Intervals of water quality measurement: daily

OTHER TEST CONDITIONS
- Photoperiod: day:night ratio of 16:8 h
- Light: fluorescent light source

EFFECT PARAMETERS MEASURED: Mortality, symptoms

TEST CONCENTRATIONS
- Range finding study: yes, test vessels used were aquaria of 3L capacity. ln each aquarium five fish were exposed for 96 hours.
- Test concentrations: 1.0; 10.0 and 100.0 mg/L.
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3,4-dichloroaniline

Duration:

96 h

Dose descriptor:

LC0

Effect conc.:

> 100 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Water accommodated fraction

Duration:

96 h

Dose descriptor:

LC50

Effect conc.:

> 100 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality (fish)

Remarks on result:

other: Water accommodated fraction

Details on results:

- Behavioural abnormalities: none
- Mortality of control: none
- Other adverse effects control: none
- Abnormal responses: none

Results with reference substance (positive control):

- Test conditions: The test was performed April 24 – April 27, 2012. The reagent used for test was 99.9% pure and provided by Sigma-Aldrich. The reference substance was used in one concentration of 8.0 mg/L.
- LC0 >= 8 mg/L, LC100 > 8 mg/L

Validity criteria fulfilled:

yes

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term toxicity to fish

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 203 (Fish, Acute Toxicity Test)

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

For the analysis of the actual test substance concentration, 2ml of test solution was taken from each test vessel at 0, 3, 24, 48, 72 and 96 hours after test initiation.

Vehicle:

no

Details on test solutions:

The test substance was added into the test water and stirred (via magnetic stirrer) in the dark for 24h. The vortex depth was about 1/10 of the solution depth. The solution was drawn off from the middle of the vessel after 1 hours setting, and then filtrated by using a 0.45μm nitrocellulose membrane. The first 50ml of filtrate was discarded. The filtration was interrupted for approx.15 minutes to saturate the filter material. The next 50ml of filtrate was also discarded. The following filtrate was used in the test. During filtration the filter was always kept covered.

Test organisms (species):

other: Gobiocypris rarus

Details on test organisms:

The test was performed with Gobiocypris rarus which were bred in Bioassay and Safety Assessment Lab, Shanghai Academy of Public Measurement. This freshwater fish species is suitable for fish toxicity testing because of their ease of culturing and handling, sensitivity to a variety of chemical substances, and the extensive data base for this species. This species complies with "Ministry of environmental protection of the people's Republic of China, The Guidelines for the Testing of Chemicals-Effects on Biotic System, 2013, 203 Fish, Acute Toxicity Test", "OECD, Guidelines for the Testing of Chemicals. 203 Fish, Acute Toxicity Test, 1992" and "GB/T 29763-2013 Chemicals-Rare Minnow (Gobiocypris rarus) acute toxicity test". For evaluation of the fish quality and experimental conditions, potassium dichromate was tested as a positive control to demonstrate satisfactory test conditions once a year (recently performed on 2016.8.1-8.5). The 96h LC50 value of the test substance to Gobiocypris rarus was 243mg/L (95% confidence limits: 194-297mg/L), which was within the ± 2SD range of the QC chart in our lab (81.6-307mg/L. Before being used in the test, all fish were acclimatized in the following test conditions for more than 14 days.

Test type:

semi-static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

96 h

Test temperature:

22.1 -22.8 deg. C

pH:

8.07 - 8.44

Dissolved oxygen:

60.3%-91.1 % ASV (air saturation volume)

Salinity:

n/a

Conductivity:

n/a

Nominal and measured concentrations:

nominal: 100 mg/L

Details on test conditions:

The test substance has no unique and identified molecular structure and is poorly soluble in water. The test solutions were prepared as water-soluble fractions (WSFs). The test substance was added into the test water and stirred (via magnetic stirrer) in the dark for 24h. The vortex depth was about 1/10 of the solution depth. The solution was drawn off from the middle of the vessel after 1 hours setting, and then filtrated by using a 0.45μm nitrocellulose membrane. The first 50ml of filtrate was discarded. The filtration was interrupted for approx.15 minutes to saturate the filter material. The next 50ml of filtrate was also discarded. The following filtrate was used in the test. During filtration the filter was always kept covered. The test was performed with semi-static with 24 hour renewal periods, since the test substance is a mixture of multiple unidentified components and the stability of the test substance under test conditions cannot thus be demonstrated analytically. Therefore all means were used to maximize the test concentration during the exposure period.

Reference substance (positive control):

yes

Remarks:

potassium dichromate

Key result

Duration:

96 h

Dose descriptor:

LL50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat. (dissolved fraction)

Basis for effect:

mortality (fish)

Details on results:

In the limit test, the measured concentrations were below LOD2, LOD2=0.422mg/L. There were no mortalities and abnormalities observed in the fish of the control group and the treatment group during the test.

Validity criteria fulfilled:

yes

Cross-reference 3

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-20 to 2012-04-25 (experimental phase)

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: Samples for chemical analysis were collected after thorough mixing of the test concentration. The filtrates of the collected samples were analyzed within 24 h from admission in the subcontractor laboratory. Each test sample was analyzed in triplicate. The results were corrected by the value measured for blank sample in each series. The test samples were diluted with deionized water, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item is not soluble in water and after addition to test medium the test item sticks to the bottom and walls of the vessel. After shaking and sonication the grinded test item in the test medium was visually a heterogeneous suspension. Since the test item is a mixture of components the composition of the soluble part of the test item may be different from the original composition and may change with the concentration. The following way of preparing the test concentration results in a homogeneous solution i.e. filtrate named Water Accommodated Fraction (WAF) which was used for exposure. The test concentration was prepared separately by weighing the amount of the test item grinded in mortar to a powder of yellowish colour and mixing it with the test medium. The mixture was sonicated for 30 min at 40°C (ultrasonic bath, Sonic-10, Polsonic) and then shaken at room temperature for 72 h at 70 rpm. After 72 h of shaking the control and the test concentration were filtered through a conditioned nitrocellulose membrane filter (0.45 µm-pore-size, Millipore).

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: water flea
- Source: Standard Iabaratory culture maintained at the Institute of lndustrial Organic Chemistry, Branch Pszczyna, Department of Ecotoxicology, Labaratory of Aquatic Toxicology
- Age at study initiation: Up to 24 hours
- Method of breeding: Parthenogenetical reproduction
- Feeding during test: none
- Type and amount of food during culturing: The daphnids were fed with Pseudokirchneriella subcapitata algae suspension originating from the culture at the Labaratory of Aquatic Toxicology, Group B vitamins and micronutrients necessary for proper growth were supplied with lyophilized Spirulin sp. suspension.

Test type:

static

Water media type:

freshwater

Limit test:

yes

Total exposure duration:

48 h

Test temperature:

20.0 – 21.2°C

pH:

7.79 – 7.77

Dissolved oxygen:

8.0 - 8.1 mg/L

Nominal and measured concentrations:

Nominal concentration: 100 mg/L
Measured concentrations: Based on the result of non-specific analysis of TOC content it cannot be excluded that only a very small part of the test item (some components) were dissolved in test medium.

Details on test conditions:

TEST SYSTEM
- Test vessel: Glass beakers of 150 mL capacity covered with transparent Iids in order to minimize evaporation and to prevent accidental contamination
- Fill volume: 400 mL of the test concentration and the control was divided into four replicates of 100 mL.
- Aeration: none
- No. of organisms per vessel: 5
- No. of vessels per concentration (replicates): 4
- No. of vessels per control (replicates): 4

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline
- Intervals of water quality measurement: at test start and at test termination

OTHER TEST CONDITIONS
- Photoperiod: light: dark ratio of 16 h: 8 h

EFFECT PARAMETERS MEASURED: Mobility

TEST CONCENTRATIONS
- Range finding studies: 3
- Concentrations in the first range finding study: 0.2, 2 and 20 mg/L
- Concentrations in the second range finding study: 0.1, 1, 10 and 100 mg/L
- Concentrations in the third range finding study: 0.8; 4; 20 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

>= 100 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Water accommodated fraction

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Remarks on result:

other: Water accommodated fraction

Details on results:

- Mortality of control: none

Results with reference substance (positive control):

- Results with reference substance valid? Yes
- EC50/LC50: The EC50 (24 h) value was 0.97 mg/L and the EC50 (48 h) value was 0.53 mg/L

Validity criteria fulfilled:

yes

Cross-reference 4

Reason / purpose for cross-reference:

data waiving: supporting information

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

Cross-reference 5

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-04-17 to 2012-04-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study with analytical monitoring

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

- Sampling method: The fresh solutions at test initiation and the solutions of pooled replicates after test termination were analysed for TOC. Samples from the additional replicates for background measurements without algae were analysed for TOC at test termination. All samples for chemical analysis were filtered through a conditioned nitrocellulose membrane filter (0.45 µm-poresize, Millipore) and transferred to subcontractor laboratory.
- Sample preparation: The filtrates of the collected samples were analyzed within 24 h from admission in the subcontractor laboratory. Each test sample was analyzed in triplicate. The results were corrected by the value measured for blank sample in each series. The test samples were diluted with deionized water, if necessary.

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION
- Method: The test item is not soluble in water and after addition to test medium the test item sticks to the bottom and walls of the vessel. After shaking and sonication the grinded test item in the test medium was visually a heterogeneous suspension. Since the test item is a mixture of components the soluble part of the test item may be different from the original composition and may change with the concentration. The following way of preparing the test concentrations results in homogeneous solutions i.e. filtrates Water Accommodated Fraction (WAF) which were used for exposure. Each test concentration was prepared separately by weighing the amount of the test item grinded in mortar to powder of yellowish colour and mixing it with the test medium. Each mixture was sonicated for 30 min at 40°C (ultrasonic bath, Sonic-10, Polsonic) and then shaken at room temperature for 72 h at 70 rpm. After 72 h of shaking the control and the test concentrations were filtered through a conditionednitrocellulose membrane filter (0.45 µm-pore-size, Millipore). The filtrates were used for exposure.

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: SAG 61.81
- Source: Algae Collection University of Göttingen, Germany
- Method of cultivation: Algae were transferred from agar bevels to the fresh medium in 250 mL Erlenmeyer flasks and incubated in 21- 24 °C in constant illumination. The algae culture was renewed (inoculated to a new medium) twice a week in sterile conditions A pre-culture with cell density of 1 x 10E4 per mL was renewed four days before the definitive test. The pre-culture was used for inoculation of the test concentrations and the control.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test temperature:

22.2 – 22.6ºC

pH:

pH of control: 7.31 – 7.89

Nominal and measured concentrations:

Nominal concentrations: Control, 3.2, 10, 32, 100, 320 and 1000 mg/L WAF
Measured concentrations: Based on the result of non-specific analysis of TOC content it cannot be excluded that only a very small part of the test item (some components) were dissolved in test medium.

Details on test conditions:

TEST SYSTEM
- Test vessel: 250 mL glass flasks
- Fill volume: 100 mL
- Initial cells density: 10E4 cells/mL
- Control end cells density: 2.521E6 cells/mL
- No. of vessels per concentration: 3
- No. of vessels per control: 6

GROWTH MEDIUM
- Standard medium used: yes

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: According to guideline
- Intervals of water quality measurement: at test start and at test end

OTHER TEST CONDITIONS
- Photoperiod: constant illumination
- Light intensity: 6850 – 7580 lx

EFFECT PARAMETERS MEASURED
- Determination of cell concentrations: The absorbance of algae suspension was measured at wavelength 670 nm and recalculated to cell numbers based on a standard curve. The standard curve is the regression curve of algae cell numbers versus measured absorbance. The standard curve is based on results of algae cells counting in a Burker chamber by microscopic observation (Microscope CX-40 Rf-200. Olympus Optical Co. Ltd.). The spectrophotometric measurements were performed at 24, 48 and 72 hours of exposure (Model 6300, Jenway Ltd.). At test termination microscopic observations of algae cell morphology were performed. The exposure time was 72 hours.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2
- Range finding study: yes
- Test concentrations: 0.1, 1.0, 10 and 100 mg/L
- Results used to determine the conditions for the definitive study: yes

Reference substance (positive control):

yes

Remarks:

3.5 dichlorophenol

Duration:

72 h

Dose descriptor:

NOEC

Effect conc.:

> 1 000 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: growth rate, yield

Remarks on result:

other: Water accommodated fraction

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

> 1 000 other: mg/L WAF

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

other: Growth rate, yield

Remarks on result:

other: Water accommodated fraction

Details on results:

- Exponential growth in the control (for algal test): yes
- Observation of abnormalities (for algal test): none
- Any stimulation of growth found in any treatment: no

Results with reference substance (positive control):

- ErC50 = 2.09 mg/L (1.89-2.33)

Validity criteria fulfilled:

yes