3,5,8-Trioxa-4-phosphaundec-10-en-1-aminium, 4-hydroxy-N,N,N,10-tetramethyl-9-oxo-, inner salt, 4-oxide

Short-term toxicity to aquatic invertebrates

Administrative data

Endpoint:

short-term toxicity to aquatic invertebrates

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 November 2008 to 19 March 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: This study was conducted according to international guidelines, and the study report presents all information required to confirm validity of the test results and allow reproduceability.

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

reference to same study

Cross-reference 2

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 202 (Daphnia sp. Acute Immobilisation Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.2 (Acute Toxicity for Daphnia)

Deviations:

no

Principles of method if other than guideline:

N/A

GLP compliance:

yes (incl. QA statement)

Remarks:

UK GLP Regulations (1999)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

417-560-0

EC Name:

-

Cas Number:

67881-98-5

Molecular formula:

C11H22NO6P

IUPAC Name:

trimethyl[2-({2-[(2-methylprop-2-enoyl)oxy]ethyl phosphonato}oxy)ethyl]azanium

Details on test material:

- Name of test material (as cited in study report): HEMA-Pc
- Substance type: Pure Active Substance
- Physical state: Solid (White Powder)
- Analytical purity: 99 %
- Purity test date: 29 September 2008
- Lot/batch No.: SS148076
- Expiration date of the lot/batch: 27 August 2009 (Assigned by Testing Laboratory)
- Stability under test conditions: Stable
- Storage condition of test material: ca -20 degrees celsius in the dark under nitrogen.

Specific details on test material used for the study:

Details on properties of test surrogate or analogue material (migrated information):
N/A

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: All concentrations were monitored over the duration of the study (0, 6.25, 12.5, 25, 50 and 100 mg/L)
- Sampling method: Duplicate aliquots (ca 20 ml) were removed from each prepared concentrations at the commencement of the test as 0 hour samples.
At test termination, an aliquot (ca 20 ml) was removed from each replicate vessel and combined for each concentration. Duplicate aliquots (ca 20 ml) were removed from the combined solutions as 48 h samples.

- Sample storage conditions before analysis: Not reported

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method:
Based on the previous experimental work carried out on HEMA-Pc. the test was conducted at nominal HEMA-Pc concentrations of 6.25, 12.5, 25, 50 and 100 mg HEMAPc/L. together with a control.
Due to the hygroscopic nature of HEMA-Pc all dose preparation procedures were carried out using an Atmos bag. This allowed for the preparation of a controlled reduced moisture environment using Nitrogen. All apparatus required for the preparation of the dose solutions was placed within the Atmos bag and the bag then sealed. Any existing air within the bag was removed using a vacuum pump and the bag inflated using moisture reduced Nitrogen.

Test solutions were prepared by serial dilution of a stock solution of HEMA-Pc prepared in 1 L of Elendt M4. Refer to Table 1 for stock solution preparation.

Four vessels each containing 100 ml of treated or control media, as appropriate, were prepared for each treatment group. Five D. magna were added to each vessel within 30 minutes of preparation of the test vessels.

- Eluate: Elendt M4 Medium
- Controls: Elendt M4 Medium only control
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None

Test organisms

Test organisms (species):

Daphnia magna

Details on test organisms:

TEST ORGANISM
- Common name: Water Flea
- Source: In-house culture
- Age at study initiation: <24 h
- Method of breeding: acyclical parthenogenesis
- Feeding during test: Daphnia not fed during test


ACCLIMATION
- Acclimation period: N/A (In-house culture)
- Acclimation conditions (same as test or not): Same as test conditions
- Type and amount of food: Chlorella vulgaris (Strain 211/12/ CCAP, Oban, UK), amount not reported
- Feeding frequency: Not reported

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

48 h

Remarks on exposure duration:

NOT_SPECIFIED

Post exposure observation period:

N/A

Test conditions

Hardness:

Hardness of Elendt M4 Medium Stock used for test = 254 mg CaCO3/L

Test temperature:

Refer to Table 2.

pH:

Refer to Table 2.

Dissolved oxygen:

Refer to Table 2.

Salinity:

Conductivity = 562 μS

Nominal and measured concentrations:

Nominal Concentrations: 6.25, 12.5, 25, 50 and 100 mg/L with an untreated control.
For data on measured concentrations refer to Table 3.

Details on test conditions:

TEST SYSTEM
- Test vessel: 200 ml glass crystallising dishes
- Type (delete if not applicable): open but with perspex cover to prevent dust contamination and to minimise evaporation
- 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: Reconstituted freshwater (Elendt M4)
- Conductivity: 562 μS
- Culture medium different from test medium: N/A - Same as test medium
- Intervals of water quality measurement: 0 h and 48 h


OTHER TEST CONDITIONS
- Adjustment of pH: N/A
- Photoperiod: 16h Light: 8 h Dark
- Light intensity: 733 lux


EFFECT PARAMETERS MEASURED (with observation intervals if applicable) : immobilisation


TEST CONCENTRATIONS
- Spacing factor for test concentrations: 2
- Justification for using less concentrations than requested by guideline: N/A
- Range finding study: Not conducted

Reference substance (positive control):

no

Results and discussion

Effect concentrations

Effect concentrations 1

Duration:

24 h

Dose descriptor:

EC50

Effect conc.:

> 103 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Remarks on result:

other: N/A

Effect concentrations 2

Duration:

24 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Remarks on result:

other: N/A

Effect concentrations 3

Duration:

48 h

Dose descriptor:

EC50

Effect conc.:

> 103 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Remarks on result:

other: N/A

Effect concentrations 4

Duration:

48 h

Dose descriptor:

NOEC

Effect conc.:

25 mg/L

Nominal / measured:

meas. (arithm. mean)

Conc. based on:

test mat.

Basis for effect:

other: Immobilisation

Remarks on result:

other: N/A

Details on results:

- Behavioural abnormalities: None
- Observations on body length and weight: None
- Other biological observations: None
- Mortality of control: None
- Other adverse effects control: None
- Abnormal responses: None
- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: N/A
- Effect concentrations exceeding solubility of substance in test medium: No

Results with reference substance (positive control):

N/A

Reported statistics and error estimates:

No statistics were carried out as there was insufficient immobilisation of daphnid neonates to calculate any effects concentrations. The NOEC was the lowest tested concentration with no immobilisation of daphnids (25 mg/L), therefore no statistical analysis was required to determine this value.

Any other information on results incl. tables

Table 2. Test Solution Water Quality Parameters Recorded During the Definitive Test

Time/Parameter	Overall Mean Measured Concentration of HEMA-Pc 0-48h (mg/L)
0h	Control	6.18	12.7	25.5	49.5	103
pH	7.96	7.89	7.90	7.80	7.79	7.83
Temperature (^oC)	21.1	21.1	20.9	20.9	20.9	20.9
Dissolved Oxygen (% ASV)	96.0	89.0	89.4	90.3	87.9	90.3
48 h	Control	6.18	12.7	25.5	49.5	103
pH	7.99	7.79	7.74	7.73	7.73	7.72
Temperature (^oC)	20.8	20.8	20.8	20.8	20.6	20.7
Dissolved Oxygen (% ASV)	78.4	81.7	86.4	87.6	85.1	86.3

Table 3. Measured Concentrations of HEMA-Pc at 0 and 48 hours During the Definitive Test.

Nominal Concentration of HEMA-Pc (mg/L)	Time (h)	Mean Measured Concentration of HEMA-Pc (mg/L)	Deviation of 48 h Measured Concentration from 0 hour Measured Concentration (%)	Overall Mean Measured Concentration of HEMA-Pc (mg/L)
Control	0	ND	NC	NA
	48	ND
6.25	0	6.36	-5.7	6.18
	48	6.00
12.5	0	13.0	-4.6	12.7
	48	12.4
25	0	25.2	+2.0	25.5
	48	25.7
50	0	50.2	-2.8	49.5
	48	48.8
100	0 48	101 104	+3.0	103

Limit of Quantification: 0.905 mg/L

ND: not detected (limit of detection = 0.272 mg/L)

NC: not calculable

NA: not applicable

Table 4. Cumulative Immobilisation During the Definitive Test

Time (h)	Replicate	Overall Mean Measured Concentration of HEMA-Pc 0-48 h (mg/L)
		Control	6.18	12.7	25.5	49.5	103
24	1	0	0	0	0	0	2
	2	0	0	0	0	0	2
	3	0	0	0	0	1	0
	4	0	0	0	0	0	2
	% Immobile D. magna	0	0	0	0	5	30
48	1	0	0	0	0	0	2
	2	0	0	0	0	0	2
	3	0	0	0	0	2	1
	4	0	0	0	0	1	4
	% Immobile D. magna	0	0	0	0	15	45

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Conclusions:

The 48 hour EC50 for HEMA-Pc to D. magna was estimated to be > 103 mg/L based on overall mean measured concentrations of HEMA-Pc.

The 48 h NOEC for immobilisation was 25 mg/L

Executive summary:

This study was designed to estimate the median effective concentration (EC50) for immobilisation of Daphnia magna exposed to HEMA-Pc. The study was conducted over a 48 hour period, under static conditions. Four replicate vessels, each containing five D. magna neonates, were prepared for each treatment group in the test. The study was performed to meet the requirements of OECD (2004) Guideline 202, EC (1992) Guideline C2 and to satisfy the requirements of Regulation EC 1907/2006.

Based on preliminary work, the test was conducted with nominal concentrations of 6.25, 12.5, 25, 50 and 100 mg HEMA-Pc/L, together with an untreated control. The corresponding mean measured (0 and 48 h) concentrations of HEMA-Pc were 6.18, 12.7, 25.5, 49.5 and 103 mg/L, respectively. Chemical analysis of test samples taken at 0 and 48 hours indicated that concentrations of HEMA-Pc were maintained over the 48 hour test period, with concentrations in the range -5.7 % to 3 % of the initial measured values.

The cumulative number of immobile D. magna recorded at 48 h were 3 (15 %) at 50 mg/L and 9 (45 %) at 103 mg/L. No immobile D. magna were recorded in the control 6.25, 12.5, and 25 mg/L concentration throughout the test period.

The EC50 could not be estimated, as it was greater than the maximum concentration of HEMA-Pc tested. The no observed effect concentration (NOEC) is reported for immobilisation. The results are based on overall mean measured concentrations of HEMA-Pc.

The 48 hour EC50 for HEMA-Pc to D. magna was estimated to be >103 mg/L based on overall mean measured concentrations of HEMA-Pc. The 48 hour NOEC for immobilisation was 25 mg/L.

During the test, test solution quality parameteras were within the following ranges, pH: 7.72 -7.99; temperature: 20.6 -21.1 degrees celsius, and dissolved oxygen concentration: 78.4 -96.0 % of air saturation value.