Reaction product of "Fatty acids, C16-18 (even numbered) and C18-unsaturated" and "reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol

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:

April 29 - June 03, 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 211 (Daphnia magna Reproduction Test)

Deviations:

yes

Remarks:

The 100 mg/l loading rates that were stirred for 2 days were not left for a 2-4 hour settlement as originally planned in the protocol. Evaluation: Settlement did not result in any separation of the undissolved fraction. Undissolved material remained ’floa

Principles of method if other than guideline:

In addition, the procedures were designed to meet the test methods prescribed by the following guidelines and guidance document:
• Commission Regulation (EC) No 440/2008 of 30 May 2008, Part C: Methods for the determination of ecotoxicity, Publication No. L142, C.20. "Daphnia magna Reproduction Test".
• ISO International Standard 10706: “Determination of long term toxicity of substances to Daphnia magna Straus (Cladocera, Crustacea)”, 2000-03-30.
• Guidance document on aquatic toxicity testing of difficult substances and mixtures, OECD series on testing and assessment number 23, December 14, 2000.

GLP compliance:

yes

Analytical monitoring:

yes

Details on sampling:

- Concentrations: Samples for Total Organic Carbon (TOC) analysis were taken from all freshly prepared test concentrations and the control.
- Frequency: At nominal days 0, 7, 14 and 19.
- Volume: 40 ml
- Sampling method:
- Sample storage conditions before analysis: Samples were stored in a refrigerator until analysis.

Vehicle:

no

Details on test solutions:

The standard test procedures required generation of test solutions, which should contain completely dissolved test substance concentrations or stable and homogeneous mixtures or dispersions. The testing of concentrations that disturb the test system should be prevented (e.g. precipitate or a film of the test substance on the water surface).

The batch of Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol tested was a yellow to amber coloured viscous liquid with a purity of 100%. The test substance was not completely soluble in test medium at the concentrations tested.

Preparation of test solutions started with a loading rate of 100 mg/l that was magnetically stirred in test medium for two days. This resulted in a hazy dispersion with undissolved particles and a floating layer. The middle fraction of the dispersion was then siphoned over a glass fibre filter (Whatman GF92) to remove the majority of undissolved particles. The resulting filtrate was still slightly hazy. The lower test concentrations were prepared by subsequent dilution of the filtrate in test medium.

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: Crustacea, Cladocera
- Source: In-house laboratory culture with a known history.
- Age of parental stock (mean and range, SD): For the test selection of young daphnia with an age of < 24 hours, from parental daphnids of more than two weeks old.
- Feeding during test: Yes
- Food type: A suspension of fresh water algae (Chlorella pyrenoidosa)
- Frequency: Daily

METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES:
Three times a week the young are counted and the parental daphnids are transferred to new media.

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Hardness:

Between 179 and 196 mg calcium carbonate per litre

Test temperature:

19.5 to 20.6°C

pH:

7.3 to 8.1

Dissolved oxygen:

4.7 to 9.2 mg/l

Nominal and measured concentrations:

The Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol range tested based on mean measured TOC concentrations in freshly prepared solutions was <0.14, 0.6, 2.3, 7.9 and 26 mg/l (Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol carbon content is 70%). Hence, based on a given limit of solubility below 1 mg/l the conclusion is that at least the start concentrations in 10, 32 and 100% filtrate were expected to be above the actual solubility in water.

Details on test conditions:

TEST SYSTEM
- Test vessel: Unknown
- Type (delete if not applicable): closed
- Material, size, headspace, fill volume: 50 ml test medium
- Aeration: No
- Type of flow-through (e.g. peristaltic or proportional diluter): Not applicable
- Renewal rate of test solution (frequency/flow rate): Three times a week
- No. of organisms per vessel: 1
- No. of vessels per concentration (replicates): 10
- No. of vessels per control (replicates): 20
- No. of vessels per vehicle control (replicates): not applicable
- Biomass loading rate: 100 mg/l

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: M7 medium
- Composition of medium M7:
Adjusted ISO medium: the following chemicals (analytical grade) are dissolved in tap water purified by Reverse Osmosis (RO-water, GEON Waterbehandeling, Berkel-Enschot, The Netherlands):

Macro salts: CaCl2.2H2O 211.5 mg/l
MgSO4.7H2O 88.8 mg/l
NaHCO3 46.7 mg/l
KCl 4.2 mg/l

Medium M7: trace elements, macro nutrients and vitamins are added to freshly prepared ISO medium to reach the following concentrations:

Trace elements: B 0.125 mg/l
Fe 0.05 mg/l
Mn 0.025 mg/l
Li, Rb and Sr 0.0125 mg/l
Mo 0.0063 mg/l
Br 0.0025 mg/l
Cu 0.0016 mg/l
Zn 0.0063 mg/l
Co and I 0.0025 mg/l
Se 0.0010 mg/l
V 0.0003 mg/l
Na2EDTA.2H2O 2.5 mg/l

Macro nutrients: Na2SiO3. 9H2O 10.0 mg/l
NaNO3 0.27 mg/l
KH2PO4 0.14 mg/l
K2HPO4 0.18 mg/l

Vitamins: Thiamine 75.0 µg/l
B12 1.0 µg/l
Biotin 0.75 µg/l

The hardness: 180 mg/l expressed as CaCO3 and the pH: 7.7 ± 0.3.

- Culture medium different from test medium: No

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 h photoperiod daily
- Light intensity:
intensity at the start: 631-716 lux (9.2-11.0 µE.m-2.s-1)
intensity at the end: 655-723 lux (10.0-11.2 µE.m-2.s-1)


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :

Parental daphnids:

Condition: Every workday, the number of living, immobile and dead parental daphnids was recorded. Dead daphnids were removed when observed.
Presence of eggs in the brood pouch: Every workday.
Body length: At the end of the test.

F1 generation:

Appearance first brood: When observed.
Newborn daphnids: Every workday, the number of newborn young was counted and the condition of the young recorded. Thereafter, the young were removed.
Presence of unhatched eggs: When observed.
Incidence of immobility: When observed.

Test medium:

Temperature, oxygen and pH: At the start of the test and just before and after each renewal in one of the vessels of each test group with surviving daphnids.
Hardness: Once a week in fresh and old media from the control and the highest test concentration
Light: At the start and the end of the test


VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: No data were available from an acute toxicity test. Based on the expected low solubility it was decided to start the project with a limit test at the maximum solubility by testing two test groups: a control and a glass fibre filtered solution prepared at a loading rate of 100 mg/l. Test procedure and conditions were similar to those applied in the final reproduction test.
- Results used to determine the conditions for the definitive study: After 5 days of exposure a total of 14 of the 20 daphnia exposed had died in the test group, while no mortality was observed at the same time in the control group. Test conditions were maintained within the limits prescribed by the protocol. Based on this result it was decided to continue the project with a range of lower test concentrations.

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: organic carbon

Basis for effect:

mortality

Remarks:

parental

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: organic carbon

Basis for effect:

reproduction

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

7.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: organic carbon

Basis for effect:

other: body length

Key result

Duration:

21 d

Dose descriptor:

other: Overall NOEC

Effect conc.:

7.9 mg/L

Nominal / measured:

meas. (not specified)

Conc. based on:

other: organic carbon

Details on results:

- Mortality of parent animals: See table 1 in section any other information on results incl. tables
- No. of offspring produced per day per female: See table 2 in section any other information on results incl. tables
- Body length of parent animals: See table 3 in section any other information on results incl. tables
- Type and number of morphological abnormalities: not examined
- Type and number of behavioural abnormalities: not examined
- Number of males and females (parental): unknown
- Time to first brood release or time to hatch: In the controls, the presence of eggs in the brood pouch was recorded for the first time on day 5 or 6 and the first brood generally appeared on day 8. The first recording of the presence of eggs in the brood pouch and the first recording of young daphnia was similar to the control in treated solutions up to and including 32% of the filtrate. In the undiluted filtrate only one parent survived the test period. This daphnid produced eggs after 9 days and a few young of which the first were observed after 14 days. This daphnia remained very small and pale during the entire test period.
- Egg development time: unknown
- Brood size: unknown
- Time to sexual maturity: unknown
- Type and magnitude of biochemical changes: unknown
- Other biological observations: none
- Effect concentrations exceeding solubility of substance in test medium: 32 and 100% filtrate

Results with reference substance (positive control):

Not applicable

Reported statistics and error estimates:

See attached document 490033 Statistics in section attached background material

Table 1 Cumulative mortality of parental daphnids during the 21-day exposure period.

Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol test group (% filtrate)	Number exposed	Cumulative number of dead parental daphnids on day:												Mortality %
		5	6	7	8	9	10	12	14	16	19	20	21
Control	10	0	0	0	0	0	0	0	0	0	1	1	1	10
1.0	10	0	0	0	0	0	0	0	1	3	3	4	4	40
3.2	10	0	0	0	0	0	0	0	0	0	1	1	1	10
10	10	0	0	0	0	0	0	0	0	0	0	0	0	0
32	10	0	0	0	0	0	0	0	0	0	1	1	1	10
100	10	0	5	7	7	8	8	9	9	9	9	9	9	90

 

Table 2 Cumulative mean number of living young per parent at the various Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol test groups (% filtrate) during the 21-day test period.

Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol test group (% filtrate)
	7	8	9	12	13	14	15	16	19	20	21
Control	0.0	8.0	12.2	35.6	35.6	62.6	73.0	73.0	107.6	129.7	145.4
1.0	0.0	10.6	15.2	36.5	35.0	58.0	72.6	72.6	107.7	119.6	138.8
3.2	0.0	13.3	15.3	39.5	43.9	75.8	81.7	81.7	113.1	150.8	151.3
10	3.8	13.1	16.8	45.6	60.1	79.8	93.1	101.4	136.6	154.2	164.1
32	0.0	6.5	17.4	40.0	45.8	62.0	73.6	76.2	107.1	130.0	139.7
100	0.0	0.0	0.0	0.0	0.0	2.0	7.0	7.0	7.0	15.0	15.0

Table3 Average body lengths (mm) of parental daphnids with the standard deviation (SD) per concentration measured at the end of the test and the percentage reduction of body length relative to the control.

Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol test group (% filtrate)	Average length (mm)	SD	% Reduction
Control	4.34	0.29	-
1.0	4.31	0.05	1
3.2	4.41	0.12	-2
10	4.40	0.19	-1
32	4.30	0.18	1
100	3.29	-	24

Validity criteria fulfilled:

yes

Conclusions:

Under the conditions of the present study Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol did not affect reproduction, growth or survival of Daphnia magna in a test group containing 32% of a glass fibre filtered solution prepared at a loading rate of 100 mg/l after 21 days of exposure (NOEC). Based on measurement of organic carbon the actual mean Reaction products of Fatty acids, C16-18 and C18-unsatd. and reaction mass of 1,3-alkanediol, 2-(hydroxymethyl)-2-[(methoxymethoxy)methyl]- and 1,3-heteromonocycle-5,5-dimethanol concentration in freshly prepared solutions corresponded to 7.9 mg/l, which exceeded the given limit of solubility in water (<1 mg/l).

It is therefore concluded that effects in the present reproduction study due to testing above the limit of solubility cannot be excluded.

Description of key information

The study was performed according to OECD Guideline 211 and GLP prinicples.

Key value for chemical safety assessment

Fresh water invertebrates

Fresh water invertebrates

Effect concentration:

7.9 mg/L

Additional information

The 21 -day reproduction test with daphnia magna was performed according to OECD test guideline 211. The test was performed in a semi-static system, in which daphnia magna were exposed to a glass fibre filtered solution of Radia 7853 prepared at a loading rate of 100 mg/l and dilutions containing 1.0, 3.2, 10 and 32% of the filtrate. The condition of the parental daphnids, the number of living offspring, immobile young and appearance of unhatched (aborted) eggs were recorded. At the end of the test the lengths of the surviving parental daphnids were measured. Based on mean measured TOC concentrations in freshly prepared solutions, <0.14, 0.6, 2.3, 7.9 and 26 mg/l Radia 7853 is tested. Under the conditions of the present study the substance did not affect reproduction, growth or survival of Daphnia magna up to 32% filtrate. In the highest dose group, 100% filtrate, 90% of the daphnia died during the test period. Based on measurement of organic carbon, the actual mean measured concentration of test substance for the 32% filtrate (no effects) corresponded to 7.9 mg/l, which exceeded the given limit of solubility in water (<1 mg/l). The NOEC was therefore determined to be 7.9 mg/l.