Tributyl-2-methoxypropylphosphonium salt with 4,4'-[2,2,2-trifluoro-1-(trifluoromethyl)ethylidene]bis[phenol]

Toxicity to aquatic algae and cyanobacteria

Administrative data

Endpoint:

toxicity to aquatic algae and cyanobacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18 Jul 2011 - 18 Aug 2011

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study under GLP with analytical determination of concentrations

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 201 (Alga, Growth Inhibition Test)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method C.3 (Algal Inhibition test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

ISO 8692 (Water Quality - Fresh Water Algal Growth Inhibition Test with Scenedesmus subspicatus and Selenastrum capricornutum)

Deviations:

no

GLP compliance:

yes

Test material

Test material information

Constituent 1

Reference substance name:

MTDID 3285

IUPAC Name:

MTDID 3285

Details on test material:

- Name of test material (as cited in study report): MTDID 3285, CAS# 126049-00-1
- Substance type: Technical grade substance
- Physical state: Pink waxy solid with lumps
- Analytical purity: 86.67%
- Lot/batch no.: DZ-2200 - lot 30069
- Expiration date of the lot/batch: 31 May 2014
- Stability under test conditions: Stable
- Storage condition of test material: At room temperature in the dark

Sampling and analysis

Analytical monitoring:

yes

Details on sampling:

- Concentrations: WSFs prepared at loading rates of 1.0, 10 and 100 mg/L for the rangefinding test, and 1.0, 3.2 and 10 mg/L for the definitive test.

- Sampling method: Aliquots (2 mL) were taken from dedicated test bottles at times 0, 24 hours and 72 hours. Parallel samples were taken of an uninoculated test bottle having growth medium prepared at the 10 mg/L loading rate to verify test substance stability in the medium.

At the end of the test, replicates were pooled at each concentration before sampling.

- Sample storage conditions before analysis: Samples were stored frozen (≤-15 °C) until the day before analysis, thawed, diluted with an equal volume of methanol, and held overnight at room temperature before analysis.

Test solutions

Vehicle:

no

Details on test solutions:

PREPARATION AND APPLICATION OF TEST SOLUTION (especially for difficult test substances)
- Method: Water soluble fraction. Loading rates of 1.0, 3.2 and 10 mg/L were sonicated for ten minutes and then were stirred for three days. Undissolved material present in the test solutions was removed by filtration through 0.45 µm membranes. Portions of the solution loaded at 1.0 mg/L was then further diluted to produce 10% and 32% dilution. The final test solutions were all clear and colourless.
- Controls: Blank
- Evidence of undissolved material (e.g. precipitate, surface film, etc): None after the filtration step

Test organisms

Test organisms (species):

Raphidocelis subcapitata (previous names: Pseudokirchneriella subcapitata, Selenastrum capricornutum)

Details on test organisms:

TEST ORGANISM
- Common name: Green algae
- Strain: NIVA CHL 1
- Source (laboratory, culture collection): In-house laboratory culture
- Age of inoculum (at test initiation): 3 days prior to test initiation, cells from algal stock culture were inoculated in culture medium at a cell density of 10,000 cells/mL. Cells counts were obtained using a microscope and counting chamber.
- Method of cultivation: In M1 growth medium, continuously aerated and exposed to 60- 120 µE/(m²∙s) in the wavelength range of 400 to 700 nm at 21 - 24 °C.

ACCLIMATION
- Acclimation period: 3 days in M2 pre-culture medium
- Culturing media and conditions (same as test or not): Same during acclimation period
- Any deformed or abnormal cells observed: None noted

Study design

Test type:

static

Water media type:

freshwater

Limit test:

no

Total exposure duration:

72 h

Test conditions

Hardness:

24 mg CaCO3 / L

Test temperature:

22.0 °C - 23.4 °C

pH:

8.0 - 8.3

Dissolved oxygen:

NA

Salinity:

NA

Nominal and measured concentrations:

Nominal concentrations: Based on WSF loading rates: Control (blank), 0.10* mg/L, 0.32* mg/L, 1.0 mg/L, 3.2 mg/L, 10 mg/L.
* denotes solutions made as 10% and 32% dilutions of the 1.0 mg/L loading rate

Mean measured concentrations: <0.002 mg/L (blank), 0.08 mg/L, 0.23 mg/L, 0.78 mg/L, 2.60 mg/L, 8.60 mg/L

Details on test conditions:

TEST SYSTEM
- Test vessel: 100-mL glass vessels filled to 50 mL.
- Aeration: Continuous shaking.
- No. of organisms per vessel: initial cell density was 1E+04 cells/mL (fresh dilution of 3 day old culture).
- No. of vessels per concentration (replicates): three
- No. of vessels per control (replicates): six
- Extra replicates for sampling: yes, inoculated bottles of each concentration for sampling, plus one uninoculated bottle at 10 mg/L for sampling..

TEST MEDIUM / WATER PARAMETERS
- Source/preparation of dilution water: Water used to make growth medium was tap water purified by reverse osmosis.
- Culture medium different from test medium: yes, propagation in M1, test in M2 (OECD TG201 medium).

OTHER TEST CONDITIONS
- Adjustment of pH: No
- Photoperiod: continuous illumination using 30 Watt 'Cool-white' TLD-Iamps. Light intensity within the range of 78 to 91 µE/(m²∙s)

EFFECT PARAMETERS MEASURED (with observation intervals if applicable) :
-Cell counts by microscopy with counting chamber at start of test. Cell counts at 24, 48 and 72 hours determined by spectrophotometric measurement at 720 nm using a Shimadzu UV-1800 spectrophotometer with immersion probe. Microscopic observation of cells in highest test concentration showing < 20% effect, at end of test only.

TEST CONCENTRATIONS
- Spacing factor for test concentrations: 3.2 (0.5 log unit)
- Range finding study: Yes
- Test concentrations: WSFs based on 1.0 mg/L, 10 mg/L and 100 mg/L loading rates, plus 10% dilution of 1.0 mg/L WSF
- Results used to determine the conditions for the definitive study: Yes. EC50 between 1.0 and 10 mg/L loading rate.

Reference substance (positive control):

yes

Remarks:

Potassium dichromate, reference test done 7 weeks prior to the definitive MTDID 3285 test

Results and discussion

Effect concentrations

Duration:

72 h

Dose descriptor:

EC50

Effect conc.:

1.3 mg/L

Nominal / measured:

meas. (TWA)

Conc. based on:

act. ingr.

Remarks:

anionic component

Basis for effect:

growth rate

Remarks on result:

other: 95% CI: 1.2-1.4 mg/L; NOEC: 0.23 mg/L; EC10: 0.75 mg/L (95% CI: 0.69-0.81 mg/L)

Details on results:

Measured concentrations in the definitive test are shown in Table 1. Concentrations of both the anionic and cationic portions of the substance were measured. Because Time-weighted average concentrations were somewhat lower for the anion, EC50 is calculated using based on anion measurements. The average concentration during the test was calculated using:

Concentration = 24/72*sqrt(C0 x C24) + 48/72*sqrt(C24 x C72)

where C0, C24, C48 and C72 are the concentrations at 0, 24, 48 and 72 and hours.

In the definitive test, the control group had >16-fold growth by 72 hours. The mean CV for section-by-section specific growth rate was 15% over this period, with variation in average specific growth rates of 1% (Tables 2, 3).

- Any observations (e.g. precipitation) that might cause a difference between measured and nominal values: No. Test solutions were clear and colorless after filtration. There was no indication that the standards contained any insoluble material

- Effect concentrations exceeding solubility of substance in test medium: no

Results with reference substance (positive control):

In a test of potassium dichromate conducted 7 weeks prior to the definitive test, a 72-h EC50 of 1.9 mg/L was observed. This EC50 is within the range of values for reference tests of P. subcapitata performed by the testing lab.

Any other information on results incl. tables

Table 1, Concentrations of MTDID 3285 in the test medium based on anion concentration
Time of sampling (hour)	Concentration (mg/L)		Percent of initial concentration
	Loading rate	Analysed
0	0	<0.002	─
	10% dilution	0.0928	─
	32% dilution	0.282	─
	1.0	0.913	─
	3.2	2.48	─
	10	9.46	─
	10¹	9.70	─
24	0	<0.002	─
	10% dilution	0.0899	97
	32% dilution	0.269	96
	1.0	0.880	96
	3.2	2.68	108
	10	9.71	103
	10¹	9.51	98
72	0	<0.002	─
	10% dilution	0.0621	67
	32% dilution	0.163	58
	1.0	0.597	65
	3.2	2.59	105
	10	7.19	76
	10¹	7.99	82
1, Test bottle prepared without algae

Table 2, Individual cell densities in test bottles during the algae tests
Concentration (mg/L)¹	Vessel number	Cell density (x 1E+04) at exposure time:
		0 hours	24 hours	48 hours	72 hours
Control	1	1.00	6.93	40.94	172.73
	2	1.00	7.44	40.53	173.81
	3	1.00	7.68	42.92	165.18
	4	1.00	7.34	40.37	170.64
	5	1.00	7.67	40.79	168.97
	6	1.00	7.80	39.87	162.19
	1	1.00	8.08	42.20	171.55
0.08	2	1.00	7.30	37.55	161.34
	3	1.00	6.73	38.34	168.91
	1	1.00	6.94	31.84	140.21
0.23	2	1.00	7.48	37.76	150.45
	3	1.00	8.21	44.80	185.52
	1	1.00	7.37	27.53	96.20
0.78	2	1.00	6.41	23.91	80.34
	3	1.00	6.35	23.85	87.14
	1	1.00	2.01	2.59	1.00
2.60	2	1.00	2.37	3.19	1.17
	3	1.00	1.00	2.30	1.00
	1	1.00	1.07	1.84	1.00
8.80	2	1.00	1.54	2.50	1.00
	3	1.00	1.00	1.77	1.00
1, Time weighted average concentration based on anion concentration

Table 3, Specific growth rates and Percent growth inhibition during the algae tests
Time-weighted average conc. (mg/L)	Vessel number	Growth rate (/hour) during section:				Percent growth inhibition by section (h):
		0-24 h	24-48 h	48-72 h	0-72 h	0-24	24-48	48-72	0-72
Control (<0.002)	1	0.08066	0.07401	0.05998	0.07155	─	─	─	─
	2	0.08361	0.07064	0.06066	0.07164	─	─	─	─
	3	0.08496	0.07168	0.05615	0.07093	─	─	─	─
	4	0.08306	0.07103	0.06006	0.07138	─	─	─	─
	5	0.08490	0.06961	0.05922	0.07125	─	─	─	─
	6	0.08556	0.06800	0.05847	0.07068	─	─	─	─
	mean	0.08379	0.07083	0.05909	0.07124	─	─	─	─
	CV	2%	3%	3%	1%
Mean CV for section-by-section specific growth rate: 15%
	1	0.08707	0.06887	0.05843	0.07146	-4	3	1	0
0.08	2	0.08280	0.06827	0.06075	0.07060	1	4	-3	1
	3	0.07943	0.07251	0.06179	0.07124	5	-2	-5	0
	1	0.08069	0.06350	0.06177	0.06865	4	10	-5	4
0.23	2	0.08383	0.06746	0.05760	0.06963	0	5	3	2
	3	0.08772	0.07070	0.05921	0.07254	-5	0	0	-2
	1	0.08320	0.05493	0.05213	0.06342	1	22	12	11
0.78	2	0.07740	0.05485	0.05051	0.06092	8	23	15	14
	3	0.07700	0.05516	0.05399	0.06205	8	22	9	13
	1	0.02909	0.01055	0.00000	0.00000	65	85	100	100
2.60	2	0.03602	0.01228	0.00000	0.00217	57	83	100	97
	3	0.00000	0.03465	0.00000	0.00000	100	51	100	100
	1	0.00262	0.02269	0.00000	0.00000	97	68	100	100
8.8	2	0.01810	0.02015	0.00000	0.00000	78	72	100	100
	3	0.00000	0.02379	0.00000	0.00000	100	66	100	100

Applicant's summary and conclusion

Validity criteria fulfilled:

yes

Remarks:

16-fold growth in controls, <1% Coefficient of Variation (COV) for average specific growth rate, 15% COV for section specific growth rates

Conclusions:

The 72-hour EC50 for MTDID 3285 to the green algae Pseudokirchneriella subcapitata is 1.3 mg/L

Executive summary:

The toxicity of MTDID 3285 to the green algae Pseudokirchneriella subcapitata was assessed in a 72-hour test. Water-soluble fractions (WSFs) were made using loading rates of 1.0, 3.2, and 10 mg/L, and the 1.0 mg/L loading rate was additionally diluted to 10% and 32% to cover a lower concentration range. Analytically measured concentrations were similar to loading rates. Time weighted average concentrations for both the cationic and anion portions of the substance were determined using analytical results, with the anion concentrations providing the more conservative result. The 72-hour EC50 of MTDID 3285 to Pseudokirchneriella subcapitata was 1.3 mg/L based on growth rate. The 72-hour NOEC was 0.23 mg/L. The 72-hour EC10 was 0.75 mg/L.

The study was conducted under accepted guidelines and according to GLP criteria. The test material contained an unspecified amount of insoluble material which was removed by filtration. Also, anion concentrations declined during the test period whereas the cation remained stable. However, analytically determined concentrations on a time-weighted basis were similar to nominal concentrations. There was no indication that the standards, which were made in 50% (v/v) methanol, contained any undissolved material. Therefore, the study is classified as reliable and is suitable for use in classification and labelling.

Results Synopsis

Test Type: Static

EC50: 1.3 mg/L                         95% C.I.:  1.2-1.4 mg/L

NOEC: 0.23 mg/L                  Probit Slope:  NA