Reaction mass of 3-phenyl-6-[(3-phenyl-3,4-dihydro-2H-1,3-benzoxazin-8-yl)methyl]-3,4-dihydro-2H-1,3-benzoxazine and 6,6'-methanediylbis(3-phenyl-3,4-dihydro-2H-1,3-benzoxazine)

Short-term toxicity to aquatic invertebrates

Link to relevant study record(s)

Reference

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

Experimental starting date: 11 July 2008 and Experimental completion date: 05 August 2008

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please refer to the Read-across justification document enclosed in chapter 13 for more details.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Analytical monitoring:

yes

Details on sampling:

For the characterization of the WAFs, at least duplicate samples were taken from each treatment before the start of the test and at the end of the test after 48 hours.
For the 48-hour stability samples, additional flasks with adequate volumes of the freshly prepared test media of all test concentrations and of the control were incubated during the test period under the same conditions as in the actual test (but without daphnids). Sampling from the test beakers with daphnids was not possible because the test medium volume per beaker (50 mL) was too small for the analytical requirements (500 mL per sample).
All samples were stored deep-frozen (at about -20 °C) immediately after sampling. Based on pre-experiments for investigation of the storage stability (without GLP), the test item was found to be sufficiently stable in the test water under these storage conditions.
The concentration of the test item was analytically measured in the duplicate test medium samples of the two highest loading rate of 32 and 100 mg/L from both sampling times (0 and 48 hours).
The samples from the lower loading rates were not analyzed since the concentrations were below the 48-hour NOEC determined in this test.
From the control, only one of the duplicate samples was analyzed from each of the sampling times.

Vehicle:

no

Details on test solutions:

Test Water
Reconstituted test water according to ISO 6341 [1] was used in the study. It consisted of analytical grade salts dissolved in purified water at the following nominal concentrations:

- CaCl2 × 2H2O: 2.0 mmol/L and 294 mg/L
- MgSO4 × 7H2O: 0.5 mmol/L and 123 mg/L
- NaHCO3: 0.75 mmol/L and 65 mg/L
- KCl: 0.075 mmol/L and 5.8 mg/L
- Water Hardness: 2.5 mmol/L and 250 as CaCO3 mg/L
- Alkalinity: 0.8 mmol/L

The ratio of Ca:Mg and Na:K was 4:1 and 10:1, respectively, based on molarity.
The test water was aerated prior to the start of the study until oxygen saturation was reached. During the test period, the test water was not aerated.

Test organisms (species):

other: Daphnia magna straus

Details on test organisms:

The study was performed with young daphnids of the species Daphnia magna Straus. A clone of this species (defined by the supplier as clone 5) was originally supplied by the University of Sheffield / UK in 1992. Since that time, the clone has been bred in the laboratories of RCC in reconstituted water of the quality identical to the water quality used in the tests (in respect to pH, main ions, and total hardness) and under temperature and light conditions identical to those of the tests (see below).
At the start of the test, the organisms used in the test were 6-24 hours old and were not first brood progeny.

The test was performed in a temperature-controlled room with continuous monitoring of the room temperature. The water temperature was maintained at 20 °C.
A 16-hour light to 8-hour dark cycle with a 30 minute transition period was used. Light intensity during the light period was between approximately 500 to 630 Lux.
The daphnids were not fed during the test.

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Post exposure observation period:

Not applicable

Hardness:

250 mg/L as CaCO3

Test temperature:

20°C

pH:

8.0

Dissolved oxygen:

8.3-8.4 mg/L

Salinity:

Not applicable

Nominal and measured concentrations:

Nominal concentration: 1.0/3.2/10/32/100 mg/L
Mean measured test item concentration: --/--/--/0.29/0.35 mg/L respectively.
--: Not analysed.

Details on test conditions:

Material
The test was performed in 100-mL glass beakers filled with 50 mL of test medium. The test vessels were covered with glass plates to reduce the loss of water by evaporation and to avoid the entry of dust into the solutions.
The test vessels were labeled with the RCC study number and all necessary additional information to ensure unique identification.

Study Design
A static test without test medium renewal was performed.
In order to assess the toxicity to daphnids of the test item containing different components, water accommodated fractions (WAFs) with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control was tested in parallel (test water without test item). WAFs with loading rates above 100 mg/L were not tested in accordance with the test guidelines.
The selection of the loading rates was based on the results of a range-finding test and on results of a pre-experiment to determine the solubility of the test item in the test water (without GLP).
For each treatment, 20 daphnids were used divided into four replicates of five daphnids each. The volume of test solution provided for each daphnia was 10 mL. Thus, the requirement of the test guidelines for the minimum volume of 2 mL test medium per daphnia was fulfilled. The daphnids were randomly distributed to the test vessels at initiation of the test.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC0

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

24 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC0

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

32 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Duration:

48 h

Dose descriptor:

EC100

Effect conc.:

> 100 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mobility

Details on results:

Since water accommodated fractions of the test item were tested, all reported biological results were based on the loading rates of the test item.

During the first 24 hours of the test, no immobilized test organisms were determined in the control and up to and including the loading rate of 32 mg/L. At the loading rate of 100 mg/L two daphnids were found to be immobile. The 24-hour EC50 was clearly higher than the loading rate of 100 mg/L .
After 48 hours of exposure, no immobilized test organisms were determined in the control and up to and including the loading rate of 32 mg/L. At the highest loading rate of 100 mg/L, five organisms were found to be immobile.
The 48-hour NOEC and the 48-hour EC0 of the substance to Daphnia magna were 32 mg/L. The 48-hour EC50 and the 48 hour EC100 were clearly higher than the loading rate of 100 mg/L. These values could not be quantified due to low toxicity of the test item up to the solubility limit of the test item.
No remarkable observations were made concerning the appearance of the test media. All test media were clear solutions throughout the entire test duration.
At the beginning and end of the test period, the dissolved oxygen concentrations in the test media and control were at least 8.3 mg/L, the pH value of the test media was in the range of 7.9 to 8.0, and the water temperature during the test was 20 °C.

Results with reference substance (positive control):

For evaluation of the quality of the daphnia clone and the experimental conditions, potassium dichromate is tested as a positive control twice a year. The result of the latest positive control test in March 2008 (48-hour EC50: 0.73 mg/L, RCC Study No. B83744) indicated that the sensitivity of the test organisms was within the historical range of the RCC laboratory (48-hour EC50 from 1996 to 2008: 0.53-1.1 mg/L).
The test method and the test species are recommended by the international test guidelines.

Reported statistics and error estimates:

No data

Effect of the test item on the Mobility of Daphnia magna

Loading rate (mg/L)	N° of daphnids tested	Immobilized daphnids after 24 hours		Immobilized daphnids after 48 hours
		N°	%	N°	%
Control	20	0	0	0	0
1.0	20	0	0	0	0
3.2	20	0	0	0	0
10	20	0	0	0	0
32	20	0	0	0	0
100	20	2	10	5	25

Appearance of the Test Media during the Test Period

Abbreviations:

0: no remarkable observations, clear test medium

1: homogeneous dispersion in the water, turbidity observable

2: noticeable turbidity caused by the test item

3: noticeable coloration caused by the test item

4: inhomogeneous dispersion of the test item

5: precipitation of the test item

6: test item at the surface

7: test item lying at the bottom of the test beaker

Loading rate	Exposure time
mg/L	0 hours	24 hours	48 hours
1.0	0	0	0
3.2	0	0	0
10	0	0	0
32	0	0	0
100	0	0	0

Dissolved Oxygen Concentrations, pH Values, and Temperature in the Treatments

	Start (0 hour)			End (48 hours)
Loading rate (mg/L)	pH	Oxygen (mg/L)	Temperature (°C)	pH	Oxygen (mg/L)	Temperature (°C)
Control	7.9	8.5	20	8.0	8.4	20
1.0	7.9	8.5	20	8.0	8.4	20
3.2	7.9	8.4	20	8.0	8.3	20
10	7.9	8.3	20	8.0	8.3	20
32	7.9	8.5	20	8.0	8.3	20
100	7.9	8.3	20	8.0	8.4	20

Validity criteria fulfilled:

yes

Conclusions:

Since water accommodated fractions of the test item were tested, the following biological results were based on the loading rates of the test item:
– 24-hour EC50: >100 (loading rate in mg/L)
– 24-hour EC0: 32 (loading rate in mg/L)
– 24-hour EC100: >100 (loading rate in mg/L)
– 48-hour EC50: >100 b(loading rate in mg/L)
– 48-hour EC0 and 48-hour NOEC: 32 (loading rate in mg/L)
– 48-hour EC100: >100 (loading rate in mg/L)

Executive summary:

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

In order to assess the toxicity of the substance containing different components to daphnids, water accommodated fractions (WAFs) with the loading rates of 1.0, 3.2, 10, 32 and 100 mg/L were tested. Additionally, a control (test water without test item) was tested in parallel. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

For preparation of the WAFs, individual dispersions of the test item with the loading rates as mentioned above were prepared. The dispersions were stirred for 96 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters (0.45 μm) and the undiluted filtrates were tested as WAFs. Due to technical reasons, the WAF with the lowest loading rate of 1.0 mg/L was prepared as a dilution of the WAF with the loading rate of 3.2 mg/L.

The measured test item concentrations in the test media at the start of the test were 0.48 and 0.46 mg/L for loading rates of 32 and 100 mg/L, respectively. During the test period of 48 hours, a decrease of test item concentration in the test media was determined. At the end of the test 0.17 and 0.27 mg/L were found. The mean measured test concentrations (calculated as the geometric means of the concentrations measured at the start and the end of the test) for loading rates of 32 and 100 mg/L were 0.29 und 0.35 mg/L, respectively.

Since water accommodated fractions of the test item were tested, the following biological results were based on the loading rates of the test item:

– 24-hour EC50: >100 (loading rate in mg/L)

– 24-hour EC0: 32 (loading rate in mg/L)

– 24-hour EC100: >100 (loading rate in mg/L)

– 48-hour EC50: >100 b(loading rate in mg/L)

– 48-hour EC0 and 48-hour NOEC: 32 (loading rate in mg/L)

– 48-hour EC100: >100 (loading rate in mg/L)

Description of key information

The acute toxicity of the test item to Daphnia magna was determined in a 48-hour static test according to the Commission Regulation (EC) No 440/2008, Part C.2 and the OECD Guideline for Testing of Chemicals, No. 202 (2004).

This data is coming from a read across ARALDITE MT 35600 (SOURCE) to ARALDITE 35700 (TARGET).

In order to assess the toxicity of the substance containing different components to daphnids, water accommodated fractions (WAFs). Additionally, a control (test water without test item) was tested in parallel. The test method was based on the OECD Guidance Document on Aquatic Toxicity Testing of Difficult Substances and Mixtures, 2000.

For preparation of the WAFs, individual dispersions of the test item were prepared. The dispersions were stirred for 96 hours to dissolve a maximum amount of the different compounds of the test item in the dispersion. Then, the dispersions were filtered through membrane filters and the undiluted filtrates were tested as WAFs.

During the test period of 48 hours, a decrease of test item concentration in the test media was determined.

Since water accommodated fractions of the test item were tested, the following biological results were based on the loading rates of the test item:

– 48-hour EC0 and 48-hour NOEC: 32 (loading rate in mg/L)

– 48-hour EC50: >100 (loading rate in mg/L)

– 48-hour EC100: >100 (loading rate in mg/L)

According to the results, the test item is not toxic for Daphnia magna under the conditions of the test.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

100 mg/L