3,3'-methylenebis[5-methyloxazolidine]

Long-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

long-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study according to guideline (OECD 211) in compliance with GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Institut Fresenius

Test material

Test material information

Constituent 1

Reference substance name:

3,3'-methylenebis[5-methyloxazolidine]

EC Number:

266-235-8

EC Name:

3,3'-methylenebis[5-methyloxazolidine]

Cas Number:

66204-44-2

Molecular formula:

C9H18N2O2

IUPAC Name:

3,3'-methylenebis[5-methyloxazolidine]

Details on test material:

- Test material: Reaction products of paraformaldehyde and 2-hydroxypropylamine (ratio of 3:2)
- Lot-No. 1116148
- Specification: “Grotan Ox”, 3,3’-Methylenbis(5-methyloxazolidin)
- Colourless to yellowish liquid with an aminic odour, density 1.060 g/cm3
- Purity: Not applicable (reaction product)
- Composition of Product: Not applicable
- Further relevant properties: The stability of the test item under test conditions was tested during this study. The test item dissociates in the aqueous phase to release formaldehyde.

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Chemical analyses performed with the test solutions at the start of incubation and after 48 h (four times during the test duration).

Test solutions

Details on test solutions:

- The test solutions were prepared freshly every 2 days

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

- Strain / Clone: Daphnia magna STRAUS (clone 5)
- Source: Laboratory breed originally derived from BASF AG, Germany
- Age: < 24 h
- Breeding method: Animals were transferred in new test medium at an interval of 1 to 3 days followed by feeding
- Kind of food: Desmodesmus subspicatus- and/or Chlorella-cells
- Amount of food: 0.1-0.2 mg C/Daphnia
- Feeding frequency: Every 1 to 3 days
- Pretreatment: Daphnids transferred into fresh medium 1 day before starti of the test
- Feeding of animals during test: Yes (Desmodesmus subspicatus)
- Handling of offspring: Offspring animals were counted daily and were then eliminated from treatment.

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Test conditions

Test temperature:

19.6 – 22.6°C (mean 21.5°C)

pH:

7.51 - 9.49

Dissolved oxygen:

7.2 – 8.8 mg/L

Nominal and measured concentrations:

- Nominal concentrations: 50.0, 20.0, 8.0, 3.2, 1.28, 0.512 mg/L + control
- Concentrations ranging from 0.512 to 20.0 mg/L were monitored. Recovery values of formaldehyde are given in the table below.

Details on test conditions:

TEST SYSTEM
- Renewal of test solution: every 2 days
- Volume of test vessels: 90 mL
- Volume/animal: 90 mL
- Number of animals/vessel: 1
- Number of vessels/ concentration: 13

TEST MEDIUM / WATER PARAMETERS
-Source: Elendt M4 medium, according to Guideline
- Salinity: Not applicable
- Ca / Mg ratio: according to Guideline
- Na / K ratio: according to Guideline
- Holding water different from dilution water: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Quality/Intensity of irradiation: Mean intensity 0.93 klx ≈ 14.0 µE/m2s
- Photoperiod: Day/night : 16 h / 8 h

TEST PARAMETER
- Mortality
- Number of living offspring produced per parent animal
- Intrinsic rate of population growth
- Age at first reproduction
- Size of the parent animals, measured in control and at 20 mg/L only at test end

SAMPLING AND EXAMINATION SCHEDULE
- Daily examination of biological parameters, except size of animals (at end of test)
- pH and oxygen content measured in freshly prepared test solutions and after 48 h

STATISTICS
- The statistical calculations were done using the commercial computer program ToxRatPro Version2.09 (08.11.2006)

Reference substance (positive control):

yes

Remarks:

potassium dichromate (tested prior beginning of the test)

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

related to cumulative offspring of survivors

Effect concentrations 2

Duration:

21 d

Dose descriptor:

LOEC

Effect conc.:

3.2 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

related to cumulative offspring of survivors

Effect concentrations 3

Duration:

21 d

Dose descriptor:

EC10

Effect conc.:

1.1 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

related to cumulative offspring of survivors

Remarks on result:

other: 95% CI: 0.0-3.0

Effect concentrations 4

Duration:

21 d

Dose descriptor:

other: EC20

Effect conc.:

3.3 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

related to cumulative offspring of survivors

Remarks on result:

other: 95% CI: 0.2-7.0

Effect concentrations 5

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

26.4 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

mortality

Remarks:

related to cumulative offspring of survivors

Remarks on result:

other: 95% CI: 11.6-1608

Details on results:

- For effects related to mobility, age of first reproduction, intrinsic rate of population growth, and size measurement see tables below.
- In the high-dose group (50 mg/L) all parental animals have died after day 4

Results with reference substance (positive control):

EC50 (mobility, 24h)=1.26 mg/L

Any other information on results incl. tables

Nominal Conc. [mg/L]	No. offspring / living parent	Dead parent animals
control	64.5	0
0.512	63.6	0
1.28	61.5	0
3.20	47.8	0
8.00	41.8	0
20.0	37.1	2 (after 6 d)
50.0	0	13 (after 4 d)

Effect values, related to mobility (95% CI): 

NOEC	8.0 mg/L
LOEC	20.0 mg/L
EC10	11.4 mg/L (8.4-15.6)
EC20	14.9 mg/L (11.3-19.7)
EC50	24.9 mg/L (18.8-32.9)

Effect values, related to age of first reproduction:

NOEC	≥ 20 mg/L
LOEC	> 20 mg/L

Effect values, related tointrinsic rate of population growth :

NOEC	≥ 50 mg/L
LOEC	> 50 mg/L

Size measurements of parental animals at test end as percental difference between 20 mg/L and non-treated control (negative value indicates, that animals are larger):
3.17% (length)
-2.14% (diameter)

In conclusion, no test item related effects were observed for the additional parameters intrinsic rate of poupulation growth, age at first reproduction, and size of parantel animals at test end.

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The substance was found to be harmful towards daphnids reproduction.

Executive summary:

The substance was found to be harmful towards daphnids reproduction.The numerical results are given below; all results refer to the nominal concentrations since the formaldehyde content was shown to be stable within the incubation period.

A clear concentration-response relationship can be established from the study results. While at 1.3 mg/L no significant effectswere observed,reproduction was inhibited by >10% at 3.2 mg/L.

Test item related effects were found for the additional endpoints mobility (NOEC = 8.0 mg/L), intrinsic rate of population growth (NOEC > 50 mg/L), and age at first reproduction (NOEC > 20 mg/L). Length and diameter of the parent animals were not affected at 20 mg/L (determined after termination of exposure).

The constituents of the reaction product (substance) are not volatile from aqueous solutions, therefore this removal mechanism could not influence the test results.It is not expected that thethe low adsorptivity of the test substance influenced test substance concentrations. Possible substance losses due to biodegradation are unlikely in the semistatic system.

The constituents of the reaction product (substance) hydrolyse completely in concentrations which are expected to occur in waste waters and surface waters. Also in the media of toxicity tests, complete hydrolysis of the substance is expected, therefore the observed effects are assumed to be caused by a mixture of hydrolysis products.

Analysis of the formaldehyde content in the test solutions proved the test item to remain stable at >80% of the initial values over the respective 48 h inbubation period. For this reason, the results of this test are based on the nominal concentrations tested.