Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

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:

1 November 1995 to 22 November 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was performed to a guideline and used GLP.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1996

Report date:

1996

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

other: OECD 202

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

EC Number:

421-660-1

EC Name:

Bis(dimethyl-(2-hydroxyethyl)ammonium) 1,2-ethanediyl-bis(2-hexadecenylsuccinate)

Molecular formula:

Hill formula: C50 H96 N2 O10 CAS formula: C42 H74 O8. 2(C4 H11 N O)

IUPAC Name:

bis((2-hydroxyethyl)dimethylazanium) (4E)-3-{[2-({3-carboxylato-3-[(2E)-hexadec-2-en-2-yl]propanoyl}oxy)ethoxy]carbonyl}-4-methyloctadec-4-enoate

Constituent 2

Reference substance name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

IUPAC Name:

BIS(DIMETHYL-(2-HYDROXYETHYL)AMMONIUM) 1,2-ETHANEDIYL-BIS (2-HEXADECENYLSUCCINATE)

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: The concentration and stability of the test material in the test solutions were verified by chemical analysis on days 0 (fresh media), 2, 5, 7, 9, 12, 14, 16, 19 and 21 (expired media). Chemical analysis was also carried out on the stock solutions used to prepare the test series on days 0, 2, 5, 7, 9, 12, 14, 16 and 19.
- Sampling method: Water samples were taken from the solvent control and the test groups (replicates pooled) for quantitative analysis.
- Sample storage conditions before analysis: NDA

Test solutions

Vehicle:

no

Details on test solutions:

Identity and concentration of auxiliary solvent for dispersal: No solvent or cosolvent was used. There were no mechanical
methods of dispersion employed.

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: The test material was prepared by a direct dispersion in water. An amount of test material (200 mg) was dispered in dechlorinated tap water with the aid of ultrasonic disruption and the volume adjusted to 2 L to give a 100 mg/L stock solution. Dilutions were then prepared from this solution. Aliquots of the 3 stock solutions were each separately dispersed in dechlorinated tap water to give the appropriate test concentrations.
- Eluate: NDA
- Differential loading: NDA
- Controls: The control group was maintained under identical conditions but not exposed to the test material.
- Chemical name of vehicle (organic solvent, emulsifier or dispersant): N/A
- Concentration of vehicle in test medium (stock solution and final test solution(s) including control(s)): N/A
- Evidence of undissolved material (e.g. precipitate, surface film, etc): NDA

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Daphnia magna
- Strain/clone: 1st instar
- Justification for species other than prescribed by test guideline: N/A
- Source: Laboratory culture from a strain supplied by the Institut National de Recherche Chimique Appliquee (I.R.CH.A), France
- Age of parental stock (mean and range, SD): NDA
- Feeding during test
- Food type: Mixed algae (predominantly Chlorella spp.)
- Amount: Feeding was at a level to maintain a green tinge in the test solutions. Equal amounts of food were given to each daphnid.
- Frequency: Food available contiuously


ACCLIMATION
- Acclimation period: NDA
- Acclimation conditions (same as test or not): Same as test conditions - adult Daphnia were maintained in polypropylene vessels containing approximately 2 L of dechlorinated tap water at a temperature of 21 °C. The lighting cycle was controlled to give a 16 h light and 8 h darkness cycle. Culture conditions ensured that reproduction was by parthenogenesis. Gravid adults were isolated 24 hours prior to the initiation of the test, the young daphnids produced overnight were then removed for testing.
- Type and amount of food: Mixed algae (predominantly Chlorella spp.)
- Feeding frequency: Daily
- Health during acclimation (any mortality observed): NDA


QUARANTINE (wild caught)
- Duration: N/A
- Health/mortality: N/A


METHOD FOR PREPARATION AND COLLECTION OF EARLY INSTARS OR OTHER LIFE STAGES: NDA

Study design

Test type:

semi-static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

21 d

Post exposure observation period:

N/A

Test conditions

Hardness:

100 mg/L as CaCO3

Test temperature:

21 °C

pH:

7.5-7.8

Dissolved oxygen:

7.8-8.5 mg/L O2

Salinity:

NDA

Nominal and measured concentrations:

Nominal concentration: 0.05, 0.16, 0.5, 1.6 and 5.0 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel:
- Type (delete if not applicable): Closed
- Material, size, headspace, fill volume: For each concentration, 10 daphnids were placed in 400 mL of the prepared test solutions in 500 mL glass flasks.
- Aeration: The diluent water only was aerated prior to use.
- Type of flow-through (e.g. peristaltic or proportional diluter): N/A
- Renewal rate of test solution (frequency/flow rate): The test solutions were renewed 3 times per week on days 0, 2, 5, 7, 9, 12, 14, 16 and 19. The adult daphnia were transferred to fresh media before the contents of each vessel were passed through a fine mesh. Young daphnia (live and dead) and any unhatched eggs were collected on the mesh and counted using a stero microscope before being discarded.
- No. of organisms per vessel: 10 daphnids
- No. of vessels per concentration (replicates): 4 vessels
- No. of vessels per control (replicates): 4 vessels
- No. of vessels per vehicle control (replicates): N/A
- Biomass loading rate: Each daphnia received approximately 5-10 µL of a mixed unicellular algal culture (equivalent to approximately 2.83x10^9 cells/mL) daily.



TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Dechlorinated tap water
- Total organic carbon: 1.0 mg C/L
- Particulate matter: NDA
- Metals: Various
- Pesticides: <0.08 µg/L
- Chlorine: 0.22 mg/L total chlorine, 0.17 mg/L free chlorine
- Alkalinity: pH 7.4
- Ca/mg ratio: 43 mg Ca/L, 7 mg Mg/L
- Conductivity: 382 M1CSM
- Culture medium different from test medium: The test water used for the studies was the same as that used to maintain the stock animals.
- Intervals of water quality measurement: Temperature of the test solutions was recorded daily. Dissolved oxygen concentrations, pH and temperature were recorded before and after each test media renewal.


OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: 16 hours light and 8 hours darkness
- Light intensity: NDA


EFFECT PARAMETERS MEASURED: The numbers of live and dead Daphnia of the parental (P1) generation were counted daily. An assessment of the general condition and size of the Daphnia as compared with the controls and the numbers of Daphnia with eggs or young in the brood pouch was determined at each test media renewal together with the numbers of live and dead filial (F1) Daphnia. The number of discarded unhatched eggs was also determined at this time.
Young daphnids were considered to be dead if no sign of movement was apparent during microscopic examination. Adult Daphnia which were unable to swim for approximately 15 seconds after gentle agitation (i.e. immobile) were considered to be dead. An immobilisation criterion for the young daphnids was considered to be inappropriate due to the large numbers of off-spring produced in the flasks.


VEHICLE CONTROL PERFORMED: No


RANGE-FINDING STUDY
- Test concentrations: N/A
- Results used to determine the conditions for the definitive study: N/A

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 2

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 3

Duration:

96 h

Dose descriptor:

EC50

Effect conc.:

> 5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Effect concentrations 4

Duration:

7 d

Dose descriptor:

EC50

Effect conc.:

2.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95 % CL: 2.3-2.8 mg/L

Effect concentrations 5

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95 % CL: 2.2-2.8 mg/L

Effect concentrations 6

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

2.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Remarks on result:

other: 95 % CL: 2.2-2.8 mg/L

Effect concentrations 7

Duration:

14 d

Dose descriptor:

EC50

Effect conc.:

> 0.5 - < 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 8

Duration:

21 d

Dose descriptor:

EC50

Effect conc.:

> 0.5 - < 1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 9

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

0.5 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

reproduction

Effect concentrations 10

Duration:

21 d

Dose descriptor:

NOEC

Effect conc.:

1.6 mg/L

Nominal / measured:

nominal

Conc. based on:

test mat.

Basis for effect:

immobilisation

Details on results:

- Mortality of parent animals: Mortality (immobilisation) occurred at the highest test concentration of 5.0 mg/L resulting in 33 % after 48 h and 100 % mortality by day 5. No further significant mortalities occurred at the test concentrations of 0.05, 0.16, 0.50 and 1.6 mg/L throughout the duration of the study. There was a significant toxic effect on the adult generation resulting in 100 % mortality in the 5.0 mg/L test group by day 5.
- No. of offspring produced per day per female: After both 14 and 21 days there were no statistically significant differences between the control and the 0.05, 0.16 and 0.5 mg/L test groups in terms of the number of young produced per adult. The 1.6 mg/L test group showed a statistically significant difference from the control on days 14 and 21 in terms of the numbers of young produced per adult. The 5.0 mg/L test group produced no young due to the acute side effects of exposure to the test material eliminating the daphnids prior to maturation and production of offspring.
The effect on reproduction appears to have been the result of prolonged exposure to the test material.
- Body length and weight of parent animals: There was a significant effect on the size and colour of the daphnids as a result of exposure to OS 114451A in that the surviving daphnids at the test concentration of 5.0 mg/L were markedly paler in colour than the control animals on day 2 and the surviving daphnids at the test concentration of 1.6 mg/L were observed to be markedly paler in colour than the control animals on day 5 and smaller in size and paler in colour from day 7 until the end of the study. The daphnids in the 0.05, 0.16 and 0.50 mg/L test groups were observed to be the same size and colour as the control animals.
- Type and number of morphological abnormalities: NDA
- Type and number of behavioural abnormalities: NDA
- Number of males and females (parental): NDA
- Time to first brood release or time to hatch: NDA
- Egg development time: NDA
- Brood size: NDA
- Time to sexual maturity: NDA
- Type and magnitude of biochemical changes: NDA
- Other biological observations: NDA
- Effect concentrations exceeding solubility of substance in test medium: NDA

Information on the effects of OS114451A on the filial (F1) generation is limited, since, by study design the young are removed soon after liberation from the brooding pouch. Numbers of unhatched eggs and dead young were low in all control and treatment groups surviving to maturation.

The results of the analyses of the test preparations from day 14 onwards showed the measured concentrations to be less than the LOQ of the analytical method. This effect is considered to be due to one or a combination of the following factors:
1). Deficiencies in the analytical method employed
2).Biological degradation
3). Adsorption to the algal cells given as food for the daphnids and/or the glassware used in the study.

Given that insufficient data were obtained from the analysis of the test preparations in order to calculate representative mean concentrations, the EC50 values have been calculated on nominal test concentrations only.

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

The EC50 (immobilisation) values and associated confidence limits were calculated by the moving average method of Thompson (1947).
The numbers of young produced per adult for the control and each test group were compared using ANOVA incorporating an Fmax test for homogeneity and the Williams test for differences between treatment means when several dose levels are compared with a zero dose control.

Any other information on results incl. tables

Table 1 Summary of findings following the exposure of Daphnia magna for 21 days to OS 114451A

Nominal concentration (mg/L)	% survival of P1	No. live young		No. dead young		No. unhatched eggs
		Total	Per female (cumulative)	Total	Per female (cumulative)	Total	Per female (cumulative)
Control	100	2062	52	0	0	5	<1
0.05	100	2040	51	0	0	0	0
0.16	100	1936	48	0	0	3	<1
0.50	100	1870	47	0	0	6	<1
1.6	90	349	10	8	<1	4	<1
5.0	0	0	0	0	0	0	0

The numbers of live and dead young and unhatched eggs are cumulative totals and have been calculated from the numbers of live and dead young and unhatched eggs produced per female observed at each test media renewal period.

 

Table 2 Number of young produced per adult* (non-cumulative)

Nominal concentration (mg/L)	Day
	0	2	5	7	9	12	14	16	19	21
Control	0	0	0	0	4	13	8	8	10	8
0.05	0	0	0	0	3	13	8	7	10	9
0.16	0	0	0	0	3	12	8	8	9	8
0.50	0	0	0	0	1	11	9	8	9	9
1.6	0	0	0	0	0	1	2	3	2	2
5.0	0	0	A/D	-	-	-	-	-	-	-

* number determined at test media renewal and termination only

A/D: all daphnids dead

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

Exposure of Daphnia magna to OS 114451A resulted in a toxic effect at the test concentration of 5.0 mg/L. No young were produced at this test concentration as a consequence of the mortality of the parent animals. A significant impairment of reproduction in terms of both the numbers of young produced per adult and the total numbers of young produced occurred at the test concentration of 1.6 mg/L. No impairment of reproduction was observed at the test concentrations of 0.05, 0.16 and 0.5 mg/L.

Executive summary:

The 21-day-chronic toxicity of OS 114451A toDaphnia Magnawas studied under semi-static renewal conditions. Daphnidswere exposed to nominal concentrations of 0.050, 0.16, 0.50, 1.6 and 5.0 mg a.i./L.  The 21 day EC₅₀(reproduction) based on mortality/sublethal effect was estimated to lie between 0.50 and 1.6 mg a.i./L.  The 21-day NOEC based on immobilisation was 1.6 mg a.i./L.  Production of offsprings in the treated groups indicated that OS114451A had an effect on the reproduction at concentrations greater than 1.6 mg a.i./L.